CIA-RDP79-00999A000200010001-4

VRESS'
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AFSLAPB
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nature
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Vol 251 No 5476 October 18 1974
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5.
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almost astonishment to all readers of his work.
Fig. 5 shows a buzzard saddled with the machinery
which, by means of the two tubes running downwards
from it, transmits the vertical and horizontal movements
of its wing to the recording apparatus, which is not repre-
sented. In the study of the more intricate points the
BURGUN
necessary instruments are so heavy that the whole bird
has to be partially supported. This is done by attaching
it to the extremity of a long lever which revolves, with
scarcely any friction, on a pivot.
This is found not
seriously to interfere with the normal flight of the bird.
-
Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4

Co. Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4
18 1974
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1
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Nature.1 Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4
Vol. 251 No. 5476 October 18, 1974
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· October 18, 1974
Cover Picture
A hundred years ago Nature was
reviewing. E. J. Marey's Animal
Mechanism (page 518, October 29,
1874). These cumbersome mechanisms
were soon to be replaced by Muy-
'bridge's zoopraxiscope camera. On
'page 567 we look at a Muybridge
sequence and-a century later-what
happens when the light is switched on.
nature
Volume 252
October 18, 1974 .
Investigating the paranormal
559
For those in peril on the factory floor.
560
INTERNATIONAL NEWS
562
NEWS AND VIEWS
569
ARTICLES
Human, reproduction and' family planning: research strategies in developing countries --
A. Kessler and C. C. Standley .
577
Compositional variation in recent Icelandic tholeiites and the Kverkfjoll hot spot-
G. E. Sigvaldason, S. Steinthorsson, N. Oskarsson and P. Imsland .
579
Climatic significance of deuterium abundance. in growth rings of Picea-W. E. Shiegl
582
Properties of hybrids between Salmonella phage P22 and coliphage.\-
D. Botstein and 1: Herskowitz
584
LETTERS TO NATURE-Physical Sciences
Distance to Cygnus X-1-C .- C. Cheng, K. J. H. Phillips and A. M. Wilson.
589
High energy radiation from white holes-J. V .. Narlikar, K. M. V. . Appa Rao and
`N: Dadhich
590
.
Spectrum of the cosmic background radiation, between 3.mm and .800 um-
E. I. Robson, D. G. Vickers, J. S. Huizinga, J. E. Beckman and. P. E. Clegg
591
A new. solar-terrestrial. relationship . G: M. Brown
592
Rainfall, drought and the solar cycle .- C. A. Wood and R. R. Lovett
594
Dynamic implications of mantle hotspots-M. A. Khan
.596
A-type doubling in the CH molecule-R. E. Hammersley and W. G. Richards
597
Drag-reducing polymers and liquid-column oscillations-W .. D. McComb.
598
1/f noise with a low frequency white noise limit --- K. L .. Schick and A, A. Verveen
599
. Second Law of Thermodynamics-D. R. Wilkie.
601
Information transmission under conditions of sensory shielding .- R. Targ and H. Puthoff 602
LETTERS TO NATURE-Biological Sciences
The stability of a feasible random ecosystem-A. Roberts
607
'Objective evaluation of auditory evoked EEG responses-B. McA. Sayers and.
:
H. A. Beagley
608
Imprinting and exploration of slight novelty in chicks-P. S. Jackson and .
P. P. G. Bateson
609
·Microbial activation of prophenoloxidase from immune insect larvae -. A. E. Pye
610
Elevation of total serum IgE in rats following helminth parasite infection-
E. Jarrett and H. Bazin
612
Alternative route for nitrogen assimilation in higher plants-P. J. Lea and
· B. J: Miflin
614
Evolution of cell senescence, atherosclerosis and benign tumours :D. Dykhuizen
616
Insulin stimulates myogenesis in a rat myoblast line-J .- L. Mandel and
· M. L. Pearson . .
618
Sickle cell resistance to in vivo hypoxia -- O. Castro, S. C. Finch and G. Osbaldistone
620
Expression of the dystrophia muscularis (dy) recessive gene in mice -- R. Parsons
621
Growth of human muscle spindles in vitro-B. J. Elliott and D. G. F. Harriman
622
Multiple control mechanisms underlie initiation of growth in animal cells-
.
L. J. de Asua and E: Rozengurt .
624
Control of cell division in yeast using the ionophore, A23187 with calcium and
magnesium- J. H. Duffus and L. J. Patterson
626
Antigen of mouse bile capillaries and cuticle of intestinal mucosa-
N. I. Khramkova and T. D. Beloshapkina
627
Ultrastructural analysis of toxin binding and entry. into mammalian cells-
G. L. Nicolson
628
Serum dopamine ß-hydroxylase activity in developing hypertensive rats-T. Nagatsu;
T. Kato, Y. Numata (Sudo), K. Ikuta; H. Umezawa, M. Matsuzaki and T. Takeuchi
630
Enzymatic synthesis of acetylcholine by a serotonin-containing neurone from Helix-
M. R. Hanley, G. A. Cottrell, P. C. Emson and.F. Fonnum
631
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Assignment of the gene for galactokinase to human chromosome 17 and its regional
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Growth of rust fungi of wheat and flax on chemically-defined media-A. Bose and
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New inhibitor of reagin-mediated anaphylaxis-B. J. Broughton, P. Chaplen,
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Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4 59
nature
October 18, 1974
Volume 251
Investigating
the paranormal
WE publish this week a paper by Drs R. Targ and H.
Puthoff (page 602) which is bound to create something of
a stir in the scientific community. The claim is made that
information can be transferred by some channel whose
characteristics appear to fall "outside the range of known
perceptual modalities". Or, more bluntly, some people can
read thoughts or see things remotely.
Such a claim is, of course, bound to be greeted with a
preconditioned reaction amongst many scientists. To some
it simply confirms what they have always known or
believed. To others it is beyond the laws of science and
therefore necessarily unacceptable. But to a few-though
perhaps to more than is realised-the questions are still
unanswered, and any evidence of high quality is worth a
critical examination.
The issue, then, is whether the evidence is of sufficient
quality to be taken seriously. In trying to answer this, we
have been fortunate in having the help of three indepen-
dent referees who have done their utmost to see the paper
as a potentially important scientific communication and
not as a challenge to or confirmation of prejudices. We
thank them for the considerable effort they have put in to
helping us, and we also thank Dr Christopher Evans of the
National Physical Laboratory whose continued advice on the
subject is reflected in the content of this leading article.
A general indication of the referees' comments may be
helpful to readers in reaching their own assessment of the
paper. Of the three, one believed we should not publish,
one did not feel strongly either way and the third was
guardedly in favour of publication. We first summarise the
arguments against the paper.
(1) There was agreement that the paper was weak in
design and presentation, to the extent that details given as
to the precise way in which the experiment was carried out
were disconcertingly vague. The referees felt that insuf-
ficient account had been taken of the established method-
ology of experimental psychology and that in the form
originally submitted the paper would be unlikely to be
accepted for publication in a psychological journal on these
grounds alone. Two referees also felt that the authors had
not taken into account the lessons learnt in the past by
parapsychologists researching this tricky and complicated
area.
(2) The three referees were particularly critical of the
method of target selection used, pointing out that the
choice of a target by "opening a dictionary at random" is
a naive, vague and unnecessarily controversial approach to
randomisation. Parapsychologists have long rejected such
methods of target selection and, as one referee put it,
weaknesses of this kind reveal "a lack of skill in their
experiments, which might have caused them to make some
other mistake which is less evident from their writing".
(3) All the referees felt that the details given of various
safeguards and precautions introduced against the pos-
sibility of conscious or unconscious fraud on the part of
one or other of the subjects were "uncomfortably vague"
(to use one phrase). This in itself might be sufficient to
raise doubt that the experiments have demonstrated the
existence of a new channel of communication which does
not involve the use of the senses.
(4) Two of the referees felt that it was a pity that the
paper, instead of concentrating in detail and with meti-
culous care on one particular approach to extra-sensory
phenomena, produced a mixture of different experiments,
using different subjects in unconnected circumstances and
with only a tenuous overall theme. At the best these were
more "a series of pilot studies . . . than a report of a
completed experiment".
On their own these highly critical comments could be
grounds for rejection of the paper, but it was felt that
other points needed to be taken into account before a final
decision could be made.
(1) Despite its shortcomings, the paper is presented as a
scientific document by two qualified scientists, writing
from a major research establishment apparently with the
unqualified backing of the research institute itself.
(2) The authors have clearly attempted to investigate
under laboratory conditions phenomena which, while
highly implausible to many scientists, would nevertheless
seem to be worthy of investigation even if, in the final
analysis, negative findings are revealed. If scientists dispute
and debate the reality of extra-sensory perception, then
the subject is clearly a matter for scientific study and
reportage.
(3) Very considerable advance publicity-it is fair to
say not generated by the authors or their institute-has
preceded the presentation of this report. As a result many
scientists and very large numbers of non-scientists believe,
as the result of anecdote and hearsay, that the Stanford
Research Institute (SRI) was engaged in a major research
programme into parapsychological matters and had even
been the scene of a remarkable breakthrough in this field.
The publication of this paper, with its muted claims, sug-
gestions of a limited research programme, and modest data,
is, we believe. likely to put the whole matter in more reason-
able perspective.
(4) The claims that have been made by, or on behalf of,
one of the subjects, Mr Uri Geller, have been hailed pub-
licly as indicating total acceptance by the SRI of allegedly
sensational powers and may also perhaps now be seen in
true perspective. It must be a matter of interest to scientists
to note that, contrary to very widespread rumour, the
paper does not present any evidence whatsoever for
Geller's alleged abilities to bend metal rods by stroking
them, influence magnets at a distance, make watches stop
or start by some psychokinetic force and so on. The publi-
cation of the paper would be justified on the grounds of
allowing scientists the opportunity to discriminate between
the cautious, limited and still highly debatable experi-
mental data, and extravagant rumour, fed in recent days
by inaccurate attempts in some newspapers at precognition
of the contents of the paper.
(5) Two of the referees also felt that the paper should
be published because it would allow parapsychologists, and
all other scientists interested in researching -this arguable
field, to gauge the quality of the Stanford research and
assess how much it is contributing to parapsychology.
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5€ Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4 . 18 1974
(6) Nature, although seen by me as one of the world's
most respected journals cannot afford to live on respect-
ability. We believe that our readers expect us to be a home
for the occasional 'high-risk' type of paper. This is hardly to
assert that we regularly fly in the face of referees' recom-
mendations (we always consider the possibility of publishing,
as in this case, a summary of their objections). It is to
say that the unusual must now and then be allowed a
toe-hold in the literature, sometimes to flourish, more
often to be forgotten within a year or two.
The critical comments above were sent to the authors
who have modified their manuscript in response to them.
We have also corresponded informally with the authors on
one or two issues such as whether the targets could have
been forced by standard magical tricks, and are convinced
that this is not the case. As a result of these exchanges
and the above considerations we have decided to publish
in the belief that, however flawed the experimental pro-
cedure and however difficult the process of distilling the
essence of a complex series of events into a scientific
manuscript, it was on balance preferable to publish and
maybe stimulate and advance the controversy rather than
keep it out of circulation for a further period.
Publishing in a scientific journal is not a process of
receiving a seal of approval from the establishment; rather
it is the serving of notice on the community that there is
something worthy of their attention and scrutiny. And this
scrutiny is bound to take the form of a desire amongst
some to repeat the experiments with even more caution.
To this end the New Scientist does a service by publishing
this week the results of Dr Joe Hanlon's own investiga-
tions into a wide range of phenomena surrounding Mr
Geller. If the subject is to be investigated further-and no
scientist is likely to accept more than that the SRI experi-
ments provide a prima facie case for more investigations-
the experimental technique will have to take account of
Dr Hanlon's strictures, those of our own referees and those,
doubtless, of others who will be looking for alternative
explanations.
Perhaps the most important issue raised by the circum-
stances surrounding the publication of this paper is whether
science has yet developed the competence to confront
claims of the paranormal. Supposedly paranormal events
frequently cannot be investigated in the calm, controlled
and meticulous way that scientists are expected to work,
and so there is always a danger that the investigator, swept
up in the confusion that surrounds many experiments,
abandons his initial intentions in order to go along with his
subject's desires. It may be that all experiments of this sort
should be exactly prescribed beforehand by one group, done
by another unassociated group and evaluated in terms of
performance by the first group. Only by increasing austerity
of approach by scientists will there be any major progress
in this field.
For those in peril on the factory floor
In this article Peter J. Smith argues
that a greater commitment (in deed
as well as word) to community
science by the Scientific Establish-
ment might help the world of science
regain some of the public respect it
has lost.
THE question of who speaks, or should
speak, on behalf of the scientific com-
munity has been debated on many oc-
casions, most often without result. On
the face of it, such lack of resolution
is hardly unexpected, for scientists and
scientific institutions are not noted for
their ready ability to achieve con-
sensus. Yet there is no doubt that they
can put up a pretty collective front
when they feel so moved. The one.
famous occasion on which a near con-
sensus was reached was when the
scientific community saw itself put at
risk financially by the Rothschild pro-
posals. Then individuals and institu-
tions miraculously found a common
cause of self-preservation.
But when it comes to the defence of
less privileged groups it is quite a dif-
ferent story; the voice of the British
scientific community is seldom to be
heard, whether taking a moral stance,
exerting humanitarian pressure, supply-
ing expertise or even simply providing
information. A good case in point is
provided by a new Socialist Worker
pamphlet entitled Asbestos: The Dust
that Kills in the Name of Profit. As
the title hints, the object of Socialist
Worker is nothing less than the com-
plete overthrow of the capitalist system;
and one of the ways of achieving this
aim, it seems, is to give strident publi-
city to defects in the capitalist-indus-
trial system. Fortunately, one can easily
avoid a sharp turn to the left and
still admit that what some British
workers have been subjected to in the
name of asbestos production is beyond
the limit of acceptability in a humani-
tarian society.
For what clearly emerges from the
rhetoric of the pamphlet in question is
a picture of men and women reacting
in some bewilderment to the long-term
ill effects of a technological activity.
The chief consequence is, of course,
asbestosis-a killing disease acquired
by breathing in asbestos fibres. The
bulk of the pamphlet is devoted to
case histories of men to whom asbes-
tosis has come as a shock after a
decade or so in the industry. But more
instructively, there is also a short ac-
ccount of the fight for safety put up
by a small group of the 7/162 Glasgow
insulation workers' branch of the
Transport and General Workers Union
against the obstruction of the asbestos
companies, the indifference of politi-
cians, the weakness of the Factory
Inspectorate, the silence of much of
the press, the impotence of health
authorities, the equivocal official stance
of unions in general, and, last but not
least, apathy among many of the
asbestos workers themselves.
And there is certainly something
to fight about. According to Patrick
Kinnersly (The Hazards of Work: How
to Fight Them, Pluto Press, 1973),
asbestosis is taking an increasing toll:
64 are known to have died in 1965,
107 in 1970 and 113 in 1971. The
number of new cases diagnosed rose
from 82 in 1965 to 153 in 1970.
Moreover, asbestosis is only one of
the asbestos-induced diseases. Lung
cancer appears to require a smaller
exposure to asbestos. There is also
another form of cancer known as
mesothelioma which involves growths
in the linings of the lungs and stomach.
Almost all mesotheliomas are caused
by asbestos; but no one knows how
many workers in Britain are killed by
them, partly because they take so long
to develop and partly because they are
not always identified. The TUC Cen-
tenary Institute of Occupational Health
has suggested that, 30 years after first
exposure, about one in 200 will be
found to have died of mesothelioma;
but Dr Irving J. Selikoff of Mount
Sinai Hospital in New York is ap-
parently more pessimistic. He has
recently been quoted as saying that,
for every 100,000 workers entering the
asbestos industry under the safety
standards obtaining in the United
States as recently as 1971, he would
expect 20,000 to die of lung cancer,
7,000 of mesothelioma and 7,000 of
other cancers and asbestosis.
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Nature \ Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4 61
The point of drawing attention to
the asbestos problem is not to elevate
it into some sort of special case; any
one of a hundred examples could just
as easily be used for illustration. Nor is
it to argue that the whole asbestos
industry should be closed down forth-
with. Such a course of action would
be inconceivable in the face of the
fact that asbestos currently finds some
3,000 uses in almost every sphere of
material living (a testimony to its
unique combination of fibrosity and
imperviousness to heat). Asbestos pro-
ducts (and thousands of other equally
or more dangerous substances) are
here to stay.
.
But under what conditions are they
to stay? And who is to influence those
conditions? The most striking thing to
emerge from the story of the asbestos
workers of Glasgow and elsewhere is
that no organisation remotely con-
nected with the world of established
science has played any part in it. The
British Association has been silent. The
Science and Natural Environment Re-
search Councils have been silent. The
Royal Institution has been silent. The
University Grants Committee has been
silent. The Association of University
Teachers has been silent. In this and
many other comparable cases a few
and
individual
scientists
science
journalists have spoken and acted; but
what has been completely lacking is
the evidence of any sense of collective
responsibility on behalf of the whole,
or even a significant part, of the British
scientific community.
It is true of course, that the Medical
Research Council and the recipients of
their grants are responsible for a great
deal of basic biological and medical
research into this and other industrial
diseases; and my remarks should not be
construed as criticism of that organisa-
tion, the individuals associated with it or
the valuable work they do. The point
I make is much more general-that
even when the adverse side-effects of
science and technology directly involve
health (and they do not always do so),
their mitigation requires much more
than fundamental research into the
nature of the disease itself.
Here, with particular reference to the
asbestos case but with general applic-
ability to many other situations, are
some specific things which the scientific
community could (and should) have
done and could still do.
· Admit moral responsibility for tak-
ing a clear lead in seeing that the ill
effects of science and technology are
eliminated or at least mitigated. As Dr.
Bernard Dixon (What is Science for?,
Collins, 1973) has said: "When an un-
expected, unforeseen calamity does
happen, the scientist is more, not less
or equally, responsible compared with
others in doing all he can to fight
A· worker displays symptoms of asbestosis
against it". In practical terms, taking
a moral lead would probably first mean
overseeing the organisation of a suit-
able team to map out the full extent of
the problem and identify gaps in exist-
ing knowledge. Between them, the
organisations mentioned above encom-
pass the elite (albeit often self-elected),
if not most participants, of British
science, and form a body with an almost
infinite variety of expertise and an
immense potential for „influencing
public and government opinion. That
these powers are so often allowed to
lie dormant is, to say the least, aston-
ishing.
. Ensure that the outstanding ques-
tions and problems identified by the
investigating
team
are
followed
through. In almost all cases it is prob-
able that more research is needed, in
which case the distinguished bodies of
science should regard it as their duty
to, create the necessary political, finan-
cial and organisational climate in
which such research can be carried
out. Is monitoring equipment highly
enough developed? Are safety levels
adequate and are they formulated in the
right way? To what extent is the gen-
eral public, as well as factory workers,
at risk? These and many other ques-
tions covering a variety of disciplines
can be investigated but they require
effort. Again, the principal scientific
organisations have it in their power to
encourage the mobilisation of scientists
for this purpose. Will they use this
influence?
· Exert pressure to ensure that the
high principles so proudly proclaimed
by the scientific community are ac-
tually put into effect. For example, it
is quite plain that one of the greatest
difficulties encountered by industrial
workers and others exposed to risk is
that of securing the scientific data (for
example, from monitoring) from which
a proper assessment risk may be made.
Up to July of this year, Factory Ins-
pectors divulging such information to
employees or others without the em-
ployer's permission were actually sub-
ject to imprisonment. The Health and
Safety at Work Act 1974 has mitigated
these harsh conditions, although as the
Trade Union Research Unit at Oxford
has pointed out (Health and Safety at
Work Legislation: A Critical Study,
1974), disclosure of information is still
left largely to the Factory Inspector's
discretion. It is difficult to understand
how a community which puts such a
high value on the freedom of scienti-
fic information can reasonably con-
tinue to allow the principle to be
treated with contempt. Is there any
respectable reason why human health
should be less privileged in this respect
than the activity of pure science? Of
course, employers frequently attempt
to justify their refusal to disclose vital
information on the grounds of commer-
cial secrecy-a ploy which is sometimes
legitimate, sometimes not. The scien-
tific community has a unique role to
play here, for a group of independent
scientific experts of integrity is the
only body able to make a proper assess-
ment of what is truly confidential and
what is unreasonably claimed as such.
Why has the scientific community
as a whole failed in this respect? The
reason seems to be that, in the past,
scientists have found it adequate to
justify widespread public support of
science on the grounds of the supposed
cultural value of the activity and of
its supposed role as the progenitor of
technology. But what they have failed
to grasp is that the first of these
grounds was never very convincing to
the wider world (at least as justifica-
tion for a high level of public support).
and that the second is rapidly losing
its credibility.
In fact, science has a third role,
which is neither "science for its own
sake" nor science conceived as a means
to a materialistic end. This is science
for human welfare-call it "com-
munity science" if you like. It is prac-
tised in a few university departments;
it is practised with great effect by, for
example, the staff of the Greater
London Council's Scientific Adviser.
It is regarded as second rate by
most university scientists,
as
a
threat by industry, and with indiffer-
ence by our major scientific institu-
tions. But it is just possible that a
greater commitment to it by the scien-
tific community might go some way
towards regaining for science the
public respect (not to mention the
public money) it has so patently lost
in recent years.
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1

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5 Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4 , 18 1974
international news
Frameworks for
energy in the US .. .
by Colin Norman, Washington
ON THE eve of its recess for
the
November elections, the US Congress
approved what could turn out to be the
most significant piece of energy-related
legislation to reach the lawbooks since
the oil crisis began to bite.
Minutes before President Ford ap-
peared before a joint session of Con-
gress to deliver his economic pep talk,
a House-Senate Conference committee
reached agreement on a bill which will
abolish the Atomic Energy Commis-
sion and replace it with a Nuclear
Regulatory Commission (NRC) and an
independent Energy
Research and
Development Administration (ERDA).
Two days later, the bill was sitting on
Ford's desk awaiting his signature.
Until last week, the bill had been mak-
ing glacial passage through the Con-
gressional mill, and there were fears
that no agreement would be reached
on the measure before the end of the
session.
The importance of the legislation is
that it consolidates energy research
and development programmes which
are now spread out over a host of
federal departments and agencies into
a single agency. Furthermore, it re-
moves a long standing complaint that
the Atomic Energy Commission suffers
from a conflict of interest by both pro-
moting and regulating nuclear energy,
because it splits those functions into
separate agencies. Furthermore, the
bill considerably elevates the status of
research programmes concerned with
nuclear safety and the safeguarding of
nuclear materials.
The bill, which was originally pro-
posed by former President Nixon's
Administration, sets up the NRC to
take over virtually all of the regulatory
functions that are now performed by
the Atomic Energy Commission, but
it also completely alters the present
bureaucratic structure. The NRC will
consist of three co-equal offices, each
of which will report to a 5-member
commission. The Office of Nuclear
Reactor Regulation will be concerned
with licensing nuclear reactors. The
Office of Material Safety and Safe-
guards will license reprocessing facilities
and facilities associated with trans-
porting nuclear materials, and it will
also make sure that nuclear materials
are adequately safeguarded.
.
The third, the Office of Nuclear
Regulatory Research, will perform
backup research for evaluating licence
applications to ensure that safety and
safeguards criteria are met.
As for ERDA, it will take, over vir-
tually all the energy research and
development activities performed by
the federal government, using the
laboratories of the Atomic Energy
Commission as the base of its opera-
tions. It will be split into six divisions,
each of which will be headed by an
Assistant
Administrator, concerned
with fossil fuels; nuclear energy; envir-
onmental safety; conservation, solar,
geothermal, and advanced energy
systems; and nuclear security.
ERDA will carry out all the energy
programmes related to Project Inde-
pendence-the much vaunted drive to
make the United States self-sufficient
in energy supplies-and it will be
armed with a huge budget, amounting
at present to about $2,000 million a
year. Nuclear energy will soak up
nearly half of the ERDA funds, with
the breeder reactor getting the biggest
single slice. But it will also take over
such programmes as the solar energy
research effort which was recently
launched by Congress (see Nature, 251,
368, 1974), as well as Congressionally
inspired research and development
programs concerned with geothermal
energy and other longer-term energy
options.
. .
and in Holland
from Arie de Kool, Amsterdam
THE Netherlands are going nuclear,
but slowly, and with a maximum of
government control. In fact, the gov-
ernment will increase its influence in
the whole of the energy field consider-
ably-if possible, together with other
European countries, if not, alone.
Its proposals are set out in a long-
awaited White Book on Energy.
Several of these are concerned with
getting the government a measure of
control in the privately owned energy
supply sector. As far as nuclear power
is concerned, the government proposes
to establish a monopoly for the ex-
ploitation of nuclear power stations
and promises to undertake studies on
overall nuclear safety within two years.
It plans to build three 1,000 MW nuc-
lear power stations, to be on stream
by 1985 or when the establishment of
the government nuclear monopoly and
the safety studies permit.
The
government also
wants to
negotiate a say in the managerial con-
ditions of energy supply corporations,
and by bringing the planning of all
types of power stations under govern-
ment responsibility, ensure that elec-
trical power supply is discussed in
parliament. To stimulate the use of
coal for electricity generation the
government will stipulate that all new
power plants shall be fitted for oil and
coal firing. The Dutch will also con-
tinue to participate in the German-
Belgian-Dutch
prototype
sodium-
cooled fast breeder reactor at Kalkar
in Germany, although the government
is strongly considering pulling out of
' the next phase, the construction of a
1,000 MW demonstration reactor.
Looking outwards, the government
plans to take a 40% share in all nat-
ural gas and oil exploitations on the
Dutch part of the continental shelf,
with the condition that the govern-
ment share in the profit may rise as
high as 80% if conditions allow "with-
out impeding the attractiveness to the
oil companies". The Dutch government
also plans to build up a "strategic
reserve" of natural gas by stimulating
exploration and exploitation of smaller
fields in order to have the major
source at Slochteren available in case
of another oil crisis.
It is no secret, that the cabinet has
been highly divided, especially over
the use of nuclear energy. A couple
of days before the White Book was
officially released, it leaked out that
the government proposed a consider-
able delay in the construction of nuc-
lear power plants. This was the inter-
pretation the 'progressive' members
of the cabinet were giving (and were
supposed to give) to the delay that is
bound to result from the establish-
ment of the government monopoly
and the requirements of an overall
safety report.
However, Dr Lubbers, Minister of
Economic Affairs, maintained at a
press conference, that the three plants
should be ready, by 1985. The safety
reports might lead to a change in
reactor type, a change of site, even
to a postponement of the start of the
first reactor, but not to the goal of
3,500 MW of nuclear energy in 1985.
Naturally, the opponents of nuclear
energy are not very happy with the
White Book. Some 10,000 people
biked and bussed to Kalkar in protest,
three days after the White Book was
published.
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Nature : Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4 63
The chemistry
of profits
from Nechemia Meyers, Rehovot
DETAILED plans are now being made
for an investment of some £240 mil-
lion in Israel's chemical industry.
Roughly half this sum will be used to
promote the exploitation of inorganic
Negev chemicals, with the other half
being used to expand the Haifa Bay
complex of petrochemical firms.
The investment, enormous in Israeli
terms, is a natural outcome of the
chemical industry's growing success,
both in supplying vital products to the
home market and in the export sphere.
Overseas sales of Israeli chemicals
already account for 20% of her indus-
trial exports (excluding diamonds),
and there is no apparent limit to
potential customers.
The 393-square-mile Dead Sea, most
saline of the world's lakes, has long
been the major focus of Israel's chemi-
cal industry in the 20th century. Plans
for exploiting its potash and other
mineral resources were drawn up in
1911 by Moshe Novomeysky (an im-
migrant from Siberia) and the first
plant, run by Novomeysky, began
operation in 1931. For many years,
however, the Dead Sea Works were
usually in the red, causing cynics to
suggest that "the only thing that can
sink in the Dead Sea is money".
It sinks no longer. On the contrary,
Dead Sea potash, bromine and mag-
nesium oxide-extracted from a 50-
square-mile area of evaporation pans
at the southern end of the saline lake
whose waters initially contain only 1%
(by weight) of potassium chloride-are
today sold at a handsome profit to
customers in Europe, Africa and the
Far East.
Other Negev enterprises are also
profitable, in large measure because
of rising prices. Israel's phosphate
mines-part
of the Mediterranean
phosphate belt that stretches from
Morocco in the west to Jordan in the
east and Turkey in the north-are a
striking case in point.
In 1971 Government experts, dis-
mayed by 20 years of losses, suggested
that they be closed down. Fortunately
they were not, and as a result profits
are now rolling in to the State Treasury.
Ironically, they largely reflect Arab
Morocoo's successful exploitation of
the world-wide fertiliser shortage to
push up phosphate prices from $11 to
$60 a ton in just one year.
Copper prices are now lower than
they were at their peak, but they are
still high enough to ensure the profit-
ability of the modern copper mines at
Timna (which are very near the Chal-
colithic ones). However, the copper-
1
WOMEN who graduated from Ameri-
can universities this year with a degree
in chemistry or chemical engineering
commanded higher salaries than their
male counterparts, according to a
study carried out by the American
Chemical Society. It is the first time
since the society began keeping track
of such matters that women graduates
have earned more than men.
The ACS survey showed that, on
average, the starting salary for women
chemists this year was 5% higher than
that for men, whereas in. 1964, newly
graduated women chemists could ex-
pect to earn only 68% of the starting
salary for men. While this seems to
indicate that employers are attempting
to recruit more women, the survey
also turned up evidence that the battle
for equal employment opportunities
still has some way to go. For one
thing, women and minority groups
had a higher unemployment rate than
the average for all ACS members. The
overall rate of 1.4% was exceeded by
American Indians (6.3%), Orientals
(3.1%), Spanish surnamed and black
chemists (1.5%), and women (3.5%).
But at least that's an improvement on
previous years, when the unemploy-
ment rate among women chemists was
typically running at about three times
that for men.
bearing strata that were worked in
ancient times are not the same as
those mined today. Formerly copper
nodules associated with fossilised trees
within the Middle White Sandstone
were exploited. This layer appears
near Timna about 330 feet above the
copper-bearing Cambrian rocks that
are now worked (by both underground
`and open-cast mining).
The only major failure in this
sphere has been with a controversial
plant, near the Judean Desert town of
Arad, for the manufacture of phos-
phoric acid by means of hydro-
chloric acid produced from residual
Dead Sea brines. An American firm
whose fluidised refining process was
used in the plant, the Madera Com-
pany, is being sued in an attempt to
recover at least part of the £25 million
loss, and alternative operating methods
are now being studied.
When the current expansion plan is
completed, potash production will rise
from one million to 1.5 million tons a
year, phosphate production from one
million to 2.8 million tons a year and
bromine production from 20,000 to
50.000 tons annually. Profits from the
mining and processing of Negev
chemicals, this year expected to total
£15 million, will presumably also rise
in at least the same measure.
The scheduled £240 million invest-
ment in the petrochemical industry
will meet local needs for petro-
chemicals (including raw materials for
plastics) and also give a boost to ex-
ports. Items whose production is to
rise include polyethylene, styrene, poly-
styrene, phenolics and PVC. In addi-
tion, much larger quantities of potas-
sium nitrate - a highly regarded,
Israeli-developed chemical fertilizer --
will be produced in the Haifa area.
While the cost of the oil used as a
base for these petrochemical products
has obviously risen steeply, Israel is no
worse off in this respect than her com-
netitors, and looks ahead to profits
from petrochemicals in addition to
those she is already earning from in-
organic chemicals.
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5€ Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4 · 18 1974
ICI puts money on
genetic engineering
by Miranda Robertson
THE news
that Britain's Imperial
Chemical Industries Ltd (ICI) is to
invest £40,000 over the next three
years in research on genetic engineer-
ing initiated by University of Edin-
burgh biologists seems to bring the
more optimistic speculations on the
potential benefits of these controversial
techniques a step closer to reality. One
of the most widely cited possibilities has
been the production of insulin from a
mammalian gene inserted into
a
bacterial phage or plasmid. Yet projects
aimed at this kind of application, in-
cluding the Edinburgh-ICI collabora-
tion, are still very much at the research,
and short of the development, stage.
The outstanding problem takes the
form of a fundamental question in
molecular biology : will the synthetic
machinery of a bacterium lend itself to
the production of a protein specified
by a mammalian (or any other eukary-
otic) gene? Morrow and his colleagues
in the United States have succeeded in
demonstrating 'that bacteria can tran-
scribe eukaryotic DNA into RNA
(Proc. natn. Acad. Sci., 71, 1743; 1974);
but whether they can achieve the next
step-the translation of the RNA into
protein-is still not known. Further-
more, this is much the trickier step of
the two, for while the genetic code is
universal, the software for translating
it into protein is not. Optimists believe
that the first eukaryotic protein will be
emerging from bacterial cells within the
next year or two. Equally distinguished
sceptics foresee that the highly specific
molecular
recognition
and control
systems governing protein synthesis will
present serious problems.
Dr Kenneth Murray, whose work
with his wife Noreen on phage lambda
(Nature, 251, 476; 1974) led to the ICI
project, suspects that bacteria can be
made to translate mRNA specified by
eukaryotic genes, though perhaps rather
inefficiently. One way in which he can
conceive of overcoming
specificity
problems is by reducing the inserted
DNA to the bare structural gene, as
free as possible from regulatory
sequences that would be unrecognisable
to a bacterial synthetic system.
That raises the further technical
problem of accurate excision of single
genes from the donor DNA. The
current burst of genetic tailoring took
off from the isolation in 1972 of the
bacterial restriction enzyme EcoRI,
whose peculiar properties make it par-
ticularly suitable for the cleavage and
reconstitution of heterologous DNA.
But the very property of specific cleav-
age that makes the enzyme so useful
also makes it inflexible : it will only cut
a molecule where it wants to, and not
necessarily where you want it to. Con-
sequently one of the first requirements
will be a range of enzymes with differ-
ent specific cleavage sites. Researchers
at Edinburgh and elsewhere are already
at work on the accumulation of such a
versatile set of molecular scalpels, but
no-one is yet ready even to think in
concrete terms about operating on such
exotica as insulin genes.
With the recent emphasis on the
possible hazards of genetic engineering
research, safety has become a prominent
issue. In fact, the experiments on phage
lambda fall outside the forbidden cate-
gories I and II of the US National
Academy of Sciences' cautionary state-
ment, into the more amorphous cate-
gory of experiments which "should not
be undertaken lightly". One of the
principal points which Murray has
stressed in connection with the phage
lambda system is its relative intrinsic
safety. It does not carry drug resistance
factors and is therefore susceptible to
control, and has only been used so far
with strains of E. coli to be found in
the human gut. If the system comes
into use on a commercial scale, Dr
Murray is willing to envisage the
THE bacterial enginering argument
so far has been given most promin-
ence in the United States and Brit-
ain, and it is likely that these two
countries will be the first to formu-
late a set of guidelines, either as
legal constraints or as informal codes
of practice.
But the problem is worldwide, and
the International Association of
Microbiological Societies has rec-
ently set up an ad hoc committee to
advise it on the hazards inherent in
recently developed techniques of
genetic engineering.
This committee will not attempt
to duplicate the activities of either
Professor Paul Berg and his com-
mittee in the United States or Lord
Ashby's Working Party in the
United Kingdom, according to the
chairman, Professor S. W. Glover of
the University of Newcastle-upon-
Tyne. Rather it hopes to evaluate
any recommendations made by these
committees or at the international
meeting planned to take place in
California next February, and if
necessary, to arrive at a set of con-
straints or guidelines which it can
recommend to the IAMS for world-
wide communication through the
network of
national
societies
affiliated to the IAMS.
Apart from Professor Glover, the
committee consists of Dr E. Woll-
man (France), Dr A. Demain (US)
and Professor G. Terui (Japan).
development of bacterial protein factor-
ies custom-built to be incapable of
infecting a human host.
In the meantime, precautions at
Edinburgh include scrupulous sterilis-
ation procedures. So far, the Murrays
themselves have been the only people
to have handled doctored phage par-
ticles. Not that they really believe that
phage presents a serious threat to
health; with the courage of their con-
viction, each Murray swallowed an
experimental dose of 109 labelled phage
-without either immediate ill effects,
or traces of surviving organisms in the
gut flora thereafter.
The injection of ICI funds will now,
of course, make it possible to adapt a
laboratory designed to contain patho-
gens; while at the same time the expan-
sion of the project will mean the
involvement of more personnel. Proper
training of laboratory personnel will be
emphasised since structural safeguards
such as air-locks and protective hoods
-both on the agenda-offer no pro-
tection from human carelessness.
The
obvious
advantage
of an
academic-industrial collaboration such
as that between Edinburgh University
and ICI is the pooling of intellectual
resources not normally available to
industry and practical resources not
normally available to universities. There
is one university department, however,
which is uniquely placed from both
points of view. The Department of
Biochemistry at Imperial College Lon-
don possesses a pilot plant for growing
up to 60 kg of bacteria, as well as facili-
ties for purifying large quantities of
enzymes, and is now under the chair-
manship of Professor Brian Hartley.
Hartley and his collaborators have been
working for the past few years on
enzyme adaptations in bacteria (see for
example Nature, 251, 200; 1974). The
most recent development in this work
has been the use of genetic engineering
techniques to transfer the genes specify-
ing the enzymes between bacterial
species. Hartley's plans for the bio-
chemistry department include the
recruitment of young molecular gene-
ticists with a view to developing and
exploiting these sophisticated techniques
in the production of commercial quanti-
ties of enzymes tailor-made to the
purposes of medicine and industry.
His approach differs from that of
the Edinburgh team in a number of
ways. For one thing, their project is
based principally on the use of phage
lambda as the vehicle for the trans-
planted genes, whereas Hartley's will
involve both phage (where that seems
advantageous) and bacterial plasmids.
(This will bring the Imperial College
project into NAS category I, so that
out of respect for the moratorium it
will have to be deferred, though prob-
ably not for long.) But there are also
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Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4 565
Nature vol. 2JI VULOUCI 10
197-
FACED with two proposals for dealing owns when it comes to a vote among plans. The 600 employees of Cam-
with their company's liquidity crisis, shareholders. If he decides to go along bridge Scientific Instruments voted in
employees of George Kent Ltd have with the Brown Boveri plan it seems favour of the GEC plan to a man be-
voted in favour of an arrangement with almost certain to be the one that will cause they regard an independent
the Swiss company Brown Boveri which win the day.
would give Kent more cash to the tune
instrument company as a doubtful
Brown Boveri originally proposed starter because of its small size. Over
of £6 million but leave effective control that it should buy 53% of the Kent George Kent as a whole, however, 75%
of the company in the United King-
dom. The name Kent on scientific
instruments is well known in its own
right in research laboratories, as is that
of a company which it owns-Cam-
bridge Scientific Instruments.
of the 7,300 employees said yes to
Brown Boveri.
This desire to run into the arms of
of a Swiss-based multinational is almost
Business report
by Roger Woodham
certainly based on fears that 'ration-
alisation' after a takeover by GEC
The result of this exercise in indus-
trial democracy flew in the face of Mr
would threaten job security. By con-
trast there is little overlap between
Anthony Benn, Secretary of State for. shares but it has now decided to be the activities of Kent and Brown
Industry, who preferred an arrangement content with less than 50%, thus as- Boveri, and the latter sees Kent as a
whereby George Kent would be taken suaging fears that control of George useful marketing base in the United
over by GEC, Sir Arnold Weinstock's Kent would be lost to a foreign com- Kingdom. Indeed the prospect of access
company. But Mr Benn was also keen pany. It also envisages Kent's scientific to France, Germany and Switzerland,
that Kent's employees should have a and medical instruments business being where. Brown Boveri sells some £780
chance to make their views known and set up as a separate company owned million worth of equipment and where
he now has to decide, in the light of by the mooted Brown Boveri Kent Ltd, neither Kent nor GEC is particularly
the ballot, how to use the 24% of the and this is where some Kent employees strong, is an added attraction from
Kent shares which the government part company with the Brown Boveri Kent's point of view.
i-
major differences in both methods and
aims. With his background in enzyme
evolution and bacterial adaptation,
Hartley expects to rely very much more
heavily than the Murrays on the adap-
tive potential of the bacteria themselves.
This might be used, for example, to
produce enzymes stable enough to sur-
vive long periods of storage at room
temperature. The first step would be
to introduce the gene into Bacillus
stearothermophilus, which thrives at
high temperatures. If an environment
could then be devised in which the
bacterium's survival would be greatly
enhanced by the production of large
quantities of the new enzyme, the
bacterium would be under considerable
selective pressure not only to make the
enzyme, but to evolve a structure for it
that would be stable at high tempera-
tures.
In a collaborative effort with the
Microbial Research Establishment at
Porton, Hartley has already achieved
the purification to homogeneity of 20
enzymes from thermophilic bacteria,
in quantities of 50 kg. The facilities are
to be moved to Imperial College in
January 1975, and the intention is
eventually to produce enzymes for sale
to industry. There is also the possibility
of selling enzymes for research to other
laboratories. Imperial College has at its
disposal a potential wealth of restriction
enzymes in the vast range of organisms
grown there for antibiotic research.
Isolation and purification of these en-
zymes will be an important part of the
research programme.
The problem of maximising bacterial
production of a particular enzyme for
either commercial or research purposes
may be another case in which the
answer lies in the versatility of the
bacteria themselves. In the case of
phage lambda, there is a well known
promotor system which it may be
possible to commandeer. This enzyme
system, together with the product of
another phage gene, can override the
controls which normally serve to pre-
vent or limit the transcription of a given
gene. Up to half the cell's synthetic
capacity may in consequence be re-
cruited to the production of a single
type of protein molecule. In bacteria,
however, it may actually be possible to
increase the number of genes from
which transcription of a given mRNA
taken place. Hartley and his colleagues
have recently demonstrated (Nature,
251, 200; 1974) that bacteria often
respond to metabolic demands for large
quantities of a particular enzyme by
duplication of the genes which code for
it. Again, of course, this technique
would involve manipulation of the en-
vironment and not the bacterium.
Hartley draws a categorical distinc-
tion between the prospect of getting
bacteria to mass-produce enzymes to
human specifications (science), and that
of inducing them to make mammalian
hormones such as insulin (science
fiction). This is partly because of his
own emphasis on bacterial evolution-
ary potential in the implementation of
human designs. While it may not be
too difficult to devise environmental
exigencies that would force a bacterium
into an unnatural need for an outré
enzyme, it is extremely difficult to
imagine how one might provide it with
an
incentive to produce insulin-
although with elaborate genetic manipu-
lation it may be possible. Hartley is not,
however, among those who think trans-
lation will be a major problem, though
he concedes it is the rate-limiting step.
But in view of the extensive use already
made in industry of microbial enzymes,
not to mention long-term plans for
solving the global food problem with
microbial proteins, bacterial enzyme
production may prove quite enough to
be getting on with.
Little Red
greenery book
from our Soviet Correspondent
THE all-Union Botanical Society of the
USSR has completed compilation of its
"Red Book" of flora, listing some
20,000 forms in need of governmental
protection, some 600 of which are in
immediate danger of extinction.
In commenting on the report, Pravda
predictably pays prime attention to
those species which provide "irreplace-
able" raw material, such as the Siberian
and Korean cedars, and the Caucasian
ironwood, and notes that such valuable
species as the waterfall poplar, Vavilov's
almond, and the Kolyma currant are
apparently, already, irretrievably lost.
Nevertheless, the red book also in-
cludes plants which have so far found
no' economic use, including certain
weeds "against which mankind has
waged an agelong struggle". This
would appear, at first glance, contrary
to Soviet utilitarian theories, but, the
commentator explains, the "dialectics
of 'harm' and 'use' is in this case com-
plicated and contradictory", and quotes
the classic case of penicillin in order to
justify the presence of the weeds.
2
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Musical chairs
on Capitol Hill
by Colin Norman, Washington
FOR six days early in October the US
House of Representatives put on a
display of squabbling and infighting
which would almost have done justice
to Gladstone's Parliament during the
stormy days of Irish 'reform'. One
venerable Representative announced at
one point that she would "fight to the
death" to get her way, while another
remarked that the House had lost its
collective sanity. In the end, however,
there were no open fisticuffs on the
floor, as there were in Gladstone's day,
and the whole affair got scant attention
from the general press. But for science
and technology, the titanic struggle on
Capitol Hill holds some important
implications.
The basis of the squabbling was a
bold attempt to reform the House's
antiquated committee structure-not
exactly a heart-stopping issue, which is
probably why it did not grab too many
headlines-and the outcome can best be
described as a standoff between the
reformers and those who were hoping
to preserve the status quo. But it will,
at least, change the way in which the
House handles a good deal of legisla-
tion involving research and develop-
ment.
In short, the committee on Science
and Astronautics, which has never
exactly been a giant on the Congres-
sional landscape and whose influence
has been shrinking recently along with
the decline in the space programme, is
set to pick up some important new
responsibility.
In addition to its present authority,
which is essentially limited to the pro-
grammes of NASA, the National
Science Foundation, the National
Bureau of Standards and general
science policy deliberations, the com-
mittee will pick up jurisdiction over
all non-nuclear energy research and
development, environmental research
and development, civil aviation re-
search and development and the
Weather Service. In line with its new
roles, the committee also gets a new
title-the Committee on Science and
Technology.
Certainly, it can be argued that the
committee still cannot be called the
focal point for scientific matters in the
House since the really big spenders
remain outside its jurisdictional patch.
Defence research and development
stays under the jurisdiction of the
Armed Services Committee, for ex-
ample, the Commerce Committee re-
tains control over biomedical research,
and the Joint Committee on Atomic
Energy has lost none of its authority
over nuclear matters. But the changes
could turn out to be significant, par-
ticularly in regard to energy research
and development.
First, by way of background, some
remarks are in order on the functions
of Congressional committees and the
need for reform.
Committees in the House resemble a
collection of mediaeval fiefdoms pre-
sided over by extremely powerful-
and usually elderly-chairmen who
have risen slowly through the ranks
during their years of residence on
Capitol Hill. The so-called seniority
system requires that committee chair-
manships (and the chairmanships of
subcommittees) go to the longest-serv-
ing-and not necessarily the most able
-committee members who belong to
the political party which holds the
majority of House seats.
Each committee has its own area of
responsibility-thus, for example, the
Armed Services Committee deals with
legislation concerning programmes of
the Department of Defense and the
Agriculture Committee deals with
agricultural legislation. When a bill is
introduced into the House, it is re-
ferred to the appropriate committee,
which usually assigns it to one of its
subcommittees. Hearings are held on
the bill, it usually gets amended or
completely rewritten by the subcom-
mittee before being passed on to the
main committee, which can further
amend it before it moves on to the
full House for further amendment and
final passage.
The committee therefore represent
the centres of power since they shape
the legislation which reaches the floor.
Moreover, committee chairmen (and
to some extent subcommittee chair-
men) wield a tremendous amount of
power over the system since they
control the appointment of committee
staff, who deal with the nuts and bolts
of the committee work and frequently
write the legislation themselves. And,
equally important, the chairmen can
kill off legislation with which they dis-
agree simply by keeping it bottled up
in their committees, never allowing it
to be brought to a vote on the floor of
the House.
One problem with the system is
that it has been more than a quarter
of a century since any serious attempt
was made to alter committee jurisdic-
tions, and areas of authority have con-
sequently become considerably blurred.
Take energy research and develop-
ment, for example. The Committee on
Science and Astronautics is deep into
that subject since it has jurisdiction
over the programmes of the National
Science Foundation, but so is the
Interior Committee, the Public Works
Committee, the Commerce Committee
and the Joint Committee on Atomic
LYRIC VUI. 201 VCIOVER 18 1974
Energy. The net result is that there
has been considerable jurisdictional
turf fighting between committee mem-
bers for control of energy research
legislation.
Another effect of confused jurisdic-
tions is that when a member of the
Committee on Science and Astronau-
tics, for example, writes a bill aimed
at setting up a programme of, say,
solar energy research, he shapes the
legislation to give the programme to
the National Science Foundation or
NASA so that his committee gets
jurisdiction over it. The result is that
programmes don't always get assigned
to the most suitable agencies.
Then there is the problem that
Members of Congress often serve on
several committees, which consider-
ably dilutes their effectiveness.
So, all-in-all, the House of Repre-
sentatives is not a terribly efficient
place, and that's why two years ago a
special bipartisan committee, headed
by Richard Bolling, a skilled legislator
from
Missouri,
was appointed
to
recommend some reforms.
Almost a year ago, the Bolling Com-
mittee came up with a batch of re-
commendations
which
immediately
threw most of the committee chairmen
into fits of apoplexy. It suggested a
fundamental realignment of committee
jurisdictions, that members could only
serve on one major committee, and
that the powers of the Speaker should
be elevated.
But by far the most controversial
provisions it contained were the com-
mittee realignments.
For a start, the Bolling proposals
would have stripped one of the most
powerful committees-the Ways and
Means Committee-of many of its
functions, it would have split the
Education and Labour Committee in
two, and it would have abolished the
Post Office Committee, the Internal
Security Committee, and-to all in-
tents and purposes - the Merchant
Marine Committee.
Not surprisingly, a good number of
committee chairmen who had reached
the pinnacle of their political careers
were not altogether happy about having
their power suddenly stripped from
them, and they persuaded the Demo-
cratic Caucus to appoint another com-
mittee to come up with some counter
proposals.
It was those recommendations which
were adopted last week, with a few
minor modifications, after six days of
bitter debate which at times left the
House leadership entirely confused.
As far as science and technology are
concerned, the Bolling Committee had
recommended that the Committee on
Science and Astronautics should be
given authority over all energy re-
search and development, including
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Fast workers
EADWEARD MUYBRIDGE (he thought
the Anglo Saxon version more strik-
ing than plain Edward Muggeridge)
was a Victorian photographer who
left Kingston-on-Thames in 1852 to
sail for America. There he was run
over by a stage coach and tried for
the murder of his wife's lover. He was
declared insane and acquitted after a
former employee had testified: "He
was most eccentric in his work; he
would not take a picture unless the
view suited him." On the credit side,
he made a stereoscopic record of the
Modoc Indian war and toured Cen-
tral America (as Edwardo Santiago
Muybridge), later selling collections
of 120 prints at 100 dollars the set,
which was a close call to skinning the
market at 1876 exchange rates. More
importantly, Muybridge made photo-
graphic studies of movement which
laid the foundations of a completely
new high speed photographic indus-
try (not to mention a completely new
movement in European art). In 1880
he became the first person to photo-
graph movement (using "the famous
zoopraxiscope camera") and resynthe-
sise the movement on a screen. A
strip from this original sequence
(above) was featured in an exhibition
accompanying the eleventh interna-
tional congress on high speed photo-
graphy at Imperial College, London.
The same congress was marked by an
exhibition of applied photography at
the
Royal
Photographic Society,
where the very latest achievements of
the high speed photographic industry
included the series reproduced below.
It shows the development of a con-
vective flow in the gas filling of an
electric bulb as the current through
the filament is switched on. The
photographs are from eight three-
dimensional holographic images re-
constructed from a sequence of double
exposure holograms recorded on a
single plate with a pulsed ruby laser.
The pulse length was about 1 ms and
the interval between pulses was 20
ms. The pictures were produced by
the National Physical Laboratory,
with whose permission they are re-
produced. The Muybridge series
appears by courtesy of the John
Judkyn Memorial.
nuclear energy. But the powerful Joint
Committee on Atomic Energy objected
at such an intrusion on its patch, and
the Science and Astronautics Com-
mittee eventually wound up with only
non-nuclear energy research and deve-
lopment. But that is a distinct im-
provement over the present arrange-
ment in which authority is split over
a number of committees, and it also
means that the new Science and
Technology Committee will get juris-
diction over the non-nuclear pro-
grammes of the umbrella Energy
Research and Development Adminis-
tration.
The Bolling Committee had also
recommended that the science com-
mittee be given "special oversight"
over military R & D, which would have
given it authority to make studies and
recommendations on the Pentagon's
programmes, but it wouldn't have had
the power to write laws in that area.
But Edward Hébert. the 74-year-old
chairman of the Armed Services Com-
mittee would have none of that, and
his monopoly over the Defense Dep-
artment
programmes remains
un
broken.
Finally, the Bolling proposals would
.
have given the Science and Astronau-
tics Committee jurisdiction over vir-
tually all the programmes of the
National Oceanic and Atmospheric
Administration.
But Mrs Lenore Sullivan, Chair-
man of the Committee on Merchant
Marine and Fisheries didn't relish
the idea of her committee disappear-
ing from under her, and the upshot
is a grotesque split with some atmos-
pheric programmes going to the new
Science and Technology Committee,
while oceanography stays with the
Committee on Merchant Marine. But
at least it was agreed during the floor
debate last week that the two commit-
tees will work together on oceanic and
atmospheric matters.
As for environmental research and
development, both the Bolling Com-
mittee and the Democratic Caucus
committee recommended that the new
Science and Technology Committee
would gain responsibility
for that
entire area, and so it will.
The upshot, then, is that many
science and technology matters in the
House will now be consolidated into
one committee, which should make
for smoother operations in some areas.
But it should be pointed out that the
seniority system has survived com-
pletely untouched, and nobody had the
temerity to alter the workings of per-
haps the most powerful committee of
all-the House Appropriations Com-
mittee, which deliberates on the
budgets of the executive departments
and agencies.
Bolling, at least, remains philo-
sophical about the outcome of his
attempts at+ reform. "It is a good
start", he said last week, and indicated
that he will try again next session.
Meanwhile, the Senate has for some
time been making noises about taking
a look at its own hopelessly confused
committee structure, but so far has
shown no signs of doing anything
about it. It is far from clear at this
stage, for example, which committees
will get jurisdiction over the Energy
Research and Development Adminis-
tration, and science affairs are strung
out over a whole range of Senate
committees.
Since all bills must be approved by
both the House and the Senate before
they become law, the House reforms
are clearly only half-way measures, no
matter how good they are.
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STUKTV TVI. 201 VLIVVET 18 1974
news and views
Fluctuations in climate
THROUGH most of the first part of this century it was
widely taken for granted that climate is essentially constant,
apart from short-term fluctuations, some of which might
involve shadowy cyclic changes. In fact, the global climate
was at that time changing-a rather general warming and
increasing moisture in continental interiors (apart from the
Americas)-in ways that made life easier for most people
in most places. Hence, there was little investigation of the
phenomenon. Now, however, the decades of neglect have
given place to widespread concern over climatic change.
This is partly because there is some evidence of a global
cooling, and a change in the rainfall trends also, setting in
from the 1950s onwards: There is also a more pressing
alarm over the many signs in recent years of an increased
range of variability of climate from one year (or short group
of years) to another. The world population is already so
large that there is no margin for even occasional bad years
-- bad, that is, in the sense of lowered harvest yields in
several of the world's principal grain-producing areas.
Indeed, the world's grain reserves have been reduced each
year since 1970: both 1972 and 1974 will rank as bad years
in the sense just mentioned, but even in a good year, such
as 1971, the reserves fell, and are now under a quarter of
what they were before this decade.
It was with facts such as these in mind that US Secretary
of State Henry Kissinger told the United Nations General
Assembly in April 1974:
"The poorest nations, already beset by man-made
disasters, have been threatened by a natural one: the
possibility of climatic changes in the monsoon belt and
perhaps throughout the world. The implications for
global food and population policies are ominous. The
United States proposes that the International Council of
Scientific Unions and the World Meteorological. Organi-
sation urgently investigate this problem .
And, in similar vein, Lord Rothschild wrote recently:
". . . there are several subjects in which I regret that
the Think Tank has not so far taken an interest: one
of these is the effect of the possible changes in our
climate on the life of the inhabitants of this island. It
would, I believe, repay study."
Also, the Climatic Research Unit at the University of East
Anglia in Norwich, of which I am director, whose financial
plight over the first 22 years of its existence has several
times been mentioned in Nature, now has the funds needed
to start its work-thanks to the generosity of the Wolfson
and Nuffield Foundations in this country and the Rocke-
feller Foundation in the United States.
This issue of Nature carries three more in the series of
contributions to knowledge on the variations and variability
of climate which it has been publishing in recent years.
Drs Wood and Lovett report on page 594 on the rainfall
variations measured in Ethiopia over the last 70 years, and
the records of major drought years since AD 1540, analysed
in relation to the 11-year sunspot cycles. Their result high-
lights the great range of variation of the annual rainfall in
that country within each 10 or 11-year period as likely to
have more impact than the longer-term trends, though
possibly made more serious by these. On page 592 Brown
reports a new link between variations in the Earth's
magnetic field at the time of sunspot minimum and the
strength of the subsequent sunspot maximum; together with
the climatic evidence such as that presented by Wood and
Lovett this raises the possibility of the use of the Earth's
magnetic field to forecast changing weather patterns five
or six years ahead.
The third contribution, on page 582, is concerned with
what can be learnt of the longer record of climatic
behaviour from tree rings, in this case the year-rings in
spruce (Picea) growing in southern Germany. Dr Schiegl
uses deuterium measurements on the tree rings to show
how an indication of a long history of annual mean tem-
perature might be derived. This technique is one example
of a rapidly increasing number of types and uses of 'proxy'
data to extend and corroborate the climatic record for
periods before the invention of most meteorological
instruments.
H. H. LAMB
Mass extinctions in the fossil record
THE problem of what caused the extinction of particular
groups of fossils continues to intrigue both palaeontologist
and layman. Every schoolboy learns about the dramatic and
relatively sudden extinction of the dinosaurs at the end of
the Mesozoic Era, and many are the more or less ingenious
hypotheses put forward to account for it. My own favourite
relates the extinction to the relative decline of the gymno-
sperms or naked seed plants in favour of the flowering
plants during the Cretaceous period. The surviving conifer
and cycad representatives include many producing oils with
renowned purgative properties, from which one is drawn
ineluctably to the conclusion that the poor dinosaurs died of
constipation! The trouble with all such hypotheses is their
ad hoc character, devoted specifically to the dinosaurs.
Viewed in a broader context, the dinosaurs are seen as but
one of a whole series of animal groups, both terrestrial and
marine, which died out at the end of the Cretaceous about
65 million years ago. An even more spectacular phase of
mass extinction, affecting a majority of invertebrate and .
vertebrate classes both on land and in the sea, took place
towards or at the end of the Permian some 160 million years
earlier. It is no coincidence that the three faunally defined
eras of Phanerozoic time, the Palaeozoic, Mesozoic and
Cainozoic, are divided by these two so-called crises in the
history of life.
Attempts to account for these, and lesser, phases of mass
extinction can be grouped into two broad categories, involv-
ing phenomena either extrinsic or intrinsic to our planet.
The extraterrestrial explanations have usually centred
around the deleterious effect of high levels of cosmic
radiation, leading to widespread destruction of organisms.
either directly or by damaging genes and thereby preventing
successful reproduction. Some have argued for episodic
pulses of increased radiation compared with that operating
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at present, for which there is no independent evidence;
others have postulated that at times when the geomagnetic
field was reversing the 'magnetic protection' of the Earth
diminished for period of perhaps as much as a few thousand
years, allowing more radiation to penetrate to the surface
of the continents and oceans. Explanations such as these
have fallen out of favour in the last few years for a variety
of reasons. For example, they have not been able to explain
why marine organisms have been affected at the era bound-
aries more than terrestrial ones, especially plants. Further-
more, in the ocean the intensity of radiation diminishes to
a small fraction of its atmospheric value only a few metres
below the surface.
Attention has now turned to intrinsic factors. Following
the pioneer work of Valentine and Moores (J. Geol., 80,
167; 1972) who were the first to seek an explanation for the
Permian extinctions by bringing together concepts derived
from plate tectonics and modern ecological theory,
attempts adopting a similar approach have been made
recently to account for the 'crises' at the close of the Meso-
zoic and Palaeozoic.
Hays and Pitman (Nature, 246, 18; 1973) have speculated
along the following lines. In the late Cretaceous there was
a huge marine transgression over as much as a third of
the continents, when sea level was raised hundreds of
metres as a result of uplift of the mid-oceanic ridges. This
was a consequence of an acceleration in the rate of seafloor
spreading for which there is apparently good independent
evidence. The transgression was instrumental in inducing
a more stable, equable world climate, which led to significant
diversification of many animal groups including the dino-
saurs and, in the marine realm, reef corals, rudistid bi-
valves and planktonic foraminifera. Highly diverse, steno-
topic organisms in stable environments are vulnerable,
however, to even slight environmental change. The pro-
nounced marine regression at the end of the Cretaceous,
related by Hays and Pitman to deceleration of seafloor
spreading rates, induced an episode of increased thermal
gradients, seasonal temperature contrasts and storminess,
together with a significant change in oceanic circulation
patterns. This sudden decrease in environmental stability
was sufficient to cause widespread extinction, affecting the
groups cited above and many others. The dinosaurs' un-
satisfied need was not so much for laxatives as for winter
woollies!
A significant relationship between the late Permian ex-
tinctions and fall of sea level is also inferred by Schopf (J.
Geol., 82, 129; 1974). His survey disclosed that the number
or marine invertebrate families halved from the beginning
to the end of the period while at the same time the area
of shallow epicontinental seas diminished by rather more
than half. The lowering of sea level was probably a con-
sequence of the creation of the supercontinent Pangaea
as separate continents collided. When this happened the
seafloor spreading rate diminished as a 'brake' was in-
evitably applied to plate movement. There is no satisfactory
explanation of why sea level began to rise again in the
early Triassic, although Pangaea did not begin to disinte-
grate, with the creation of a new spreading axis between
Africa and North America, until the early Jurassic. In the
earlier hypothesis of Valentine and Moores, the extensive
late Permian extinctions were attributed partly to increased
interfaunal competition as formerly isolated continental
shelves were joined together, and partly to a reduction in
environmental stability consequent upon the creation of
one large continent from several smaller ones (thus,
seasonal climatic contrasts would have increased). Schopf
draws attention to another factor.
-
Ecologists recognise an exponential relationship between
species number and area of the habitat occupied. Modern
theory has it that animal populations in a given region are
in a condition of dynamic equilibrium, with rate of im-
migration being balanced by rate of extinction. The theory
has been applied with great success to oceanic islands,
which form an excellent laboratory for ecologists. Small
islands cannot be held to possess 'impoverished' faunas, as
formerly thought; they can only support small populations
and consequently extinction rates are higher. It is a big
jump from species abundance in tiny islands to familial
abundance in seas bordering whole continents, with rate of
immigration being replaced by rate of origination through
evolution, but Schopf is prepared to take this bold step.
He can at least claim the support of a leading researcher
in the new ecology (D. Simberloff, J. Geol., 82, 267; 1974),
who has erected a model for the Permian diversity
reductions and extinctions based on reasonable biological
assumptions, which shows a good accord with Schopf's data.
Thus merely reducing the area of the Permian shelf seas
might be sufficient in itself to explain the increased rate of
extinction, reaching a maximum at the end of the period.
An attractive feature of the two hypotheses outlined
above is that they account for changes in organisms inhabit-
ing a wide variety of habitats in a way which is consistent
with modern ecological theory, and provide a satisfying
correlation with independently deduced geological events
of great significance. In both cases the fundamental control
on the extinctions is likely to have been variation in heat
flow from the mantle, affecting the density, volume and
spreading rates of oceanic ridge systems. Though many
important questions remain unanswered, an explanation
along these lines is much less of a Deus ex machina than
that involving variations in cosmic radiation.
A. HALLAM
Neural hypothesis
of muscular dystrophy
is flourishing
MANY workers in the field of neuromuscular diseases will
feel that there is more evidence in favour of the neural
hypothesis of muscular dystrophy than Professor Bradley's
article1 would imply. Although it is true that Harris and
Marshall2 did not find evidence of functional denervation in
dystrophic mouse muscle, the earlier and contrary observa-
tions of McComas and Mrozek3 have now been confirmed
by Law and Atwood4. Fibrillation potentials, usually re-
garded as a sign of denervation, are also a feature of murine
dystrophy5. Possible ultrastructural correlates of synaptic
dysfunction have been found in the axon terminals of
dystrophic mice in studies from two laboratories6,7. Even if
Harris and Marshall were correct, these authors demons-
trated nonetheless that the neuromuscular junctions of
dystrophic mice were unusually susceptible to adverse con-
ditions within the experimental milieu.
Dr Salafsky8 was one of the first to transplant minced
muscle between normal and dystrophic mice and, by con-
trolled experiments encouraging reinnervation of the trans-
plants, was able to study their contractile properties. The
explanation offered by Dr Bradley is insufficient to account
for all his findings. Of those muscle transplantation studies
in which care was taken to permit reinnervation, two9,10
have largely confirmed Salafsky's work and one11 has not.
'In the cases of human Duchenne dystrophy the evidence
is mixed. Fibrillation potentials certainly occur12 and the
work of Desmedt and Borenstein13 is cited as indicative that
denervated muscle fibres become reinnervated. In contrast,
a careful electron-microscopical study by Dr Andrew Engel
and his associates14 has not yielded any evidence of struc-
tural abnormality in the motor axon terminals. This result,
and the finding of normal numbers of motoneurones at
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autopsy15, does not contradict the
neural hypothesis as originally prop-
osed16. Indeed, recent studies with A.
Upton and P. Jorgensen, as yet un-
published, strongly suggest that it is
possible for motoneurones to have
'silent' synapses. In patients with
unequivocally neuropathic disorders it
now seems that neuromuscular junc-
tions may become inexcitable but still
capable of transmitting a neuro-
trophic influence to the muscle fibres.
If this interpretation is correct, it is
not difficult to conceive of more
severe dysfunction in which the syn-
apses, although present, can no longer
subserve a trophic action.
So far as the motor unit counting
results are concerned, the necessary
electrophysiological
technique
was
first applied by McComas, Sica and
Currie17 to the extensor digitorum
brevis (EDB) muscle. The choice of
muscle was subsequently criticised on
the grounds that the motor nerve was
vulnerable to trauma, particularly in
disabled patients such as those with
dystrophy18,19. This criticism would
seem
unfounded
the
if
recent
reports20,21 of normal numbers of
motor units in Duchenne dystrophy
are correct. Our own solution to the
problem of trauma was to study pat-
ients with very early stages of the
disease and to extend the technique to
muscles other than EDB. These last
results22 are in keeping with the earlier
ones. Since the full papers by Panayio-
topoulos et al.23 and by Ballantyne and
Hansen24 have yet to appear, it is diffi-
cult to be certain that these workers
have achieved the refinements of the
counting
technique
reported by
Bradley.
Scarpalezos
and Panayio-
topoulos20 in their brief description
claim to be able to detect very small
muscle responses "overlapping at noise
level" on single, rather than super-
imposed, oscilloscope sweeps. Not only
is this feat a logical impossibility, but
the value of the superimposition tech-
nique (employed by ourselves) in
detecting very small evoked biological
signals has been accepted ever since
the pioneering experiments of Daw-
son25. As practised by our own group,
the motor unit counting technique
has proved itself as a sensitive and
reliable means of detecting neuro-
pathic disorders and it is gratifying
that Ballantyne and Hansen24 have
obtained almost identical values for
healthy subjects. It is important to
add that the technique has recently
been employed in a 'blind' study of 17
possible cases of malignant hyperther-
mia (unpublished work with B. A.
Britt and W. Kalow) and has so far
been completely accurate in predicting
the status of the subjects.
I feel that the most direct evidence
against a neural aetiology of dystophy
is the parabiotic cross-innervation
experiment of Douglas and Cosmos26
in mice. The strongest evidence against
a myopathic aetiology (though not
necessarily for a neural one) is the
exceedingly elegant chimaera study of
Peterson27. At first sight contradictory,
these obseravtions may be reconciled
by taking into account the stage of
development of the animal at which
the foreign nerve was introduced to
the muscle. Thus, it is possible that
the very first contact between a geno-
typically dystrophic motoneurone and
a genotypically normal muscle fibre in
the embryo will commit the latter to a
dystrophic growth thereafter; if so,
any later experimental manipulations
with innervation would be ineffective.
Regardless of the true state of play,
the neural hypothesis has successfully
drawn attention to the possibility of
deranged trophic mechanisms being
present in neuromuscular disease.
Whether or not the hypothesis is
eventually shown to be correct, it will
have served a useful function if it
advances
our
understanding
of
dystrophy or brings a cure one step
closer.
A. J. MCCOMAS
1 Bradley, W. G., Nature, 250, 285-286
(1974).
2 Harris, J. B., and Marshall, M. W.,
Exp. Neurol .. 41, 331-344 (1973).
3
McComas, A. J., and Mrozek, K., J.
neurol.
neurosurg. Psychiat., 30,
525-530 (1967).
4 Law, P. K., and Atwood, H. L.,
Experientia, 30, 155-156 (1974).
5
McIntyre, A. R., Bennett, A. L., and
Brodkey, J. S.,
Archs
neurol.
Psychiat .. 81, 678-683 (1959).
6 Ragab, A. H. M. F., Lancet, ii, 815-816
1971).
7 Gilbert, J. J., Steinberg, M., and
Banker, B. Q., J. Neuropathol. exp.
Neurol., 32, 345-364 (1973).
Salafsky, B., Nature, 229, 270-272
(1971).
9
Dubowitz, V., J. Physiol., Lond., 231,
59P (1973).
10 Hironaka, T., and Miyata, Y .. Nature
new Biol., 137, 221-223. (1973).
11 Cosmos, E., Physiologist .. 16, 167-177
(1973).
12 Norris. F. H., and Chatfield, P. O.,
Electroenceph. clin. neurophysiol., 7,
391-397 (1955).
13
Desmedt, J. E .. and Borenstein, S.,
Nature, 246, 500-501 (1973).
14
Jerusalem, G., Engel, A. G., and
Gomez, M. R., Brain, 97, 123-130
(1974).
15
Tomlinson. B. E., Walton, J. N., and
Irving, D., J. neurol. Sci., 22, 305-
327 (1974).
16
McComas, A. J., Sica, R. E. P., and
Campbell, M. J., Lancet, i, 321-325
(1971).
17
McComas, A. J., Sica, R. E. P., and
Currie, S., Nature, 226, 1263-1264
(1970).
18
Engel, W. K., and Warmolts, J. R., in
New Developments in Electromyo-
graphy and Clinical Neurophysio-
logy (edit. by Desmedt. J. E.), 1, 141-
177 (Karger, Basel, 1973).
19
Roselle, N., and Stevens, A., in New
Developments in Electromyography
and Clinical Neurophysiology (edit.
by Desmedt, J. E.), 1, 69-71 (1973).
20
Scarpelezos, S., and Panayiotopoulos,
C. P., Lancet, ii, 458 (1973).
21
Ballantyne, J. P., and Hansen, S.,
Lancet, i, 1060 (1974).
22
McComas, A. J., Sica, R. E. P., and
Upton, A. R. M., Archs Neurol., 30,
249-251 (1974).
23
Panayiotopoulos, C. P., Scarpalezos,
S., Papapetropoulos, T. A., and
Caronis, A., J. neurol. Sci. (in the
press).
24
Ballantyne, J. P., and Hansen, S., J.
neurol. neurosurg. Psychiat. (in the
press).
25
Dawson, G. D., J. neurol. neurosurg.
Psychiat., 10, 137-140 (1947).
26
Douglas, W. B., and Cosmos, E., in
Exploratory Concepts in Muscular
Dystrophy (edit. by Milhorat, A. T.)
2, (Excerpta Medica, Amsterdam,
in the press).
27 Peterson, A. C., Nature, 248, 561-564
(1974).
Drilling in
the Antarctic
from Peter J. Smith
So much attention is given to the large-
scale geological programmes, such as
the Deep Sea Drilling Project, that there
is a tendency to overlook the more
modest investigations which face tech-
nical and logistic problems scarcely less
formidable. A good case in point is the
three-nation programme of drilling in
the McMurdo Sound region of Antarc-
tica. By the late 1960s it was clear that
if significant progress were to be made
in investigating
the geology, geo-
chemistry,
glaciology,
lake-bottom
stratigraphy and thermal characteristics
of the dry valleys west of McMurdo
Sound, it would be necessary to supple-
ment surface studies with borehole
data. Accordingly, several drilling pro-
posals were submitted to the National
Science Foundation which coordinated
all acceptable proposals into a single
programme with participating scientists
from the United States, Japan and New
Zealand. Thus was born the Dry
Valley Drilling Project (DVDP), since
expanded to include Ross Island and
McMurdo Sound itself.
The original aims of the DVDP as
described by McGinnis et al. (Antarctic
J., 7, 53; 1972) included studies of pal-
aeontological and volcanic evolution
during the poleward migration of An-
tarctica, palaeomagnetic reversals, the
geochemistry of polar desert soils and
permafrost, heat flow, the hydrogeology
of ice cap margins and the global
tectonic significance of the McMurdo
volcanics, although it is already clear
that the first of these objectives will not
be met. An initial exploratory season
was envisaged, followed by three drill-
ing seasons involving a minimum of 10
holes. During the exploration in the
1971-1972 summer season electrical
depth soundings were taken to deter-
mine permafrost thickness and the
nature of the material beneath the
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571
Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4
Nature
permafrost, seismic refraction profiles
were run in lake basins to establish the
relief and structure of bedrock and
overburden thicknesses, and a regional
aeromagnetic survey was made to pro-
vide structural control. In addition,
environmental monitoring (physical and
microbiological) was begun and will
be continued throughout the project.
Drilling began during the 1972-1973
season with two holes through the
volcanic rocks of Ross Island; and
preliminary geological and geochemical
results from these sites (boreholes 1
and 2) were described about a year ago
(Antarctic J., 8, 157; 1973). During the
1973-1974 summer, seven more holes
were drilled in the Ross Island volcanics
(borehole 3 adjacent to hole 2) and in
the dry valleys at Lake Vanda (bore-
holes 4 to 9); and a series of reports
on this work has just been published
(Antarctic J., 9, 125; 1974).
According to Kyle and Treves the
cores from holes 1, 2 and 3 reveal that
the geological history of Hut Point
Peninsula (Ross Island) is much more
complex than the surface geology
suggests,
which is itself sufficient
vindication of drilling. The oldest unit
penetrated by holes 2 and 3 is a 200+
m thick pile of hyaloclastite represent-
ing early eruptive events that took
place below ice or water; in other
words, an early stage of marine vol-
canism involved the construction of a
hyaloclastite pedestal which may have
impinged on a thick ice shelf covering
the Ross Sea more than 1.2 million
years ago. The higher lavas, on the
other hand, are apparently subaerial
flows and pyroclastic units which
represent a single differentiation series
starting with olivine-augite basalts,
working through
augite-kaersutite
basalts and ending with kaersutite
hawaiites, although the phonolites on
an adjacent hill may well be more
extreme differentiates of the same
magma chamber.
The cores from the dry valleys not
unexpectedly
comprise glacial and
marine sediments, although two of the
holes also penetrated the crystalline
basement.
Both
sedimentary
and
igneous cores are still under laboratory
investigation so few geochemical and
mineralogical results are available. But
Tarii reports that stable isotope studies
have already revealed the sources of
core ice; in Lake Vanda, for example,
most of the present water apparently
originates as fresh water whereas
deeper sedimentary layers are still under
the influence of sea water. Also Gumb-
ley et al. have begun to use the upper
few metres of sediment from Lake
Vanda to trace the lake's Late Quarter-
nary history.
The DVDP can already be credited
with the resolution of at least one long-
standing disagreement. Over a decade
ago, Armitage and House (Limnol.
Oceanog., 7, 36; 1962) discovered that
although Lake Vanda lies in a region
where the mean air temperature is
-18° C, it has a bottom water temper-
ature of +25° C. This led Armitage
and House, and later Angino et al.
(Sci. Bull., 45, 1097; 1964), to suggest
that below the lake there are either
high geothermal gradients or hot
springs.
Wilson and Wellman (Nature, 196,
1171; 1962) ruled out hot springs on the
grounds that the measured isotherms
in the lake are nearly horizontal. Not
only are hot springs unlikely in Antarc-
tica because the great thickness of
frozen
ground precludes abundant
groundwater; they argued that the
entrance of springwater into the lake in
conjunction with any possible hot spring
would produce a much more compli-
cated thermal pattern. Instead, they
developed a theory of solar heating in
which solar energy penetrates the lake's
ice cover (found to be extremely trans-
parent) and is absorbed in the water
below. In support of this view, Wilson
and Wellman pointed to the extreme
clarity of the water and to the decrease
in temperature gradient with depth
(which implies a heat 'source' in the
water itself). Heat flow measurements
in the upper 30 cm of lake sediment
also seemed to show that heat is flow-
ing from the water to the sediment.
But Ragotzkie and Likens (Limnol.
Oceanog., 9, 412, 1964) produced pre-
cisely the opposite result from similar
measurements and therefore attributed
the high bottom temperature to a com-
bination of solar heating and high
geothermal gradient.
Wilson et al. have now resolved this
question by making thermal measure-
ments in DVDP hole 4 which pene-
trated the crystalline basement below
Lake Vanda. The temperature in the
basement 15.5 m below the lake bottom
is consistently 0.48° C lower than that
in the sediment 0.5 m below the lake
bottom. The corresponding temperature
gradient (average 0.032° C m-1), com-
bined with estimates of thermal con-
ductivity, shows that Lake Vanda is
losing heat downwards at a rate of
0.5-1.0 cal cm~2s-1, thus convincingly
supporting the view that geothermal
heat is not the reason for the lake's
high temperature.
Corvine cannibalism
from our Animal Ecology Correspondent
ARGUMENTS have been raised for years
about the functions and consequences
of territories to animals. Since the
ultimate restraint to population in-
crease is availability of food, one might
expect the relationship of territory size
to food to be both positive and linear.
For some species, mostly herbivores,
Eltanin bailed out
To oceanographers the name
Eltanin probably stands second only
to Glomar Challenger. From 1962
to 1972 this vessel carried out geo-
logical, geophysical, geochemical,
biological and meteorological re-
searches which covered some 80
per cent of the southern ocean be-
tween 35° S and Antarctica. Then
a $1.5 million budget cut in the US
Antarctic Research Program ended
its active work.
But now the Eltanin is about to
begin a new five-year programme
on Antarctic research as a result of
an agreement between the United
States and Argentina. The ship, re-
named Islas Orcadas and operated
from Buenos Aires by the Argentine
navy, will carry out joint scientific
expeditions with support from the
National Science Foundation and
the Argentine National Antarctic
Directorate.
this may be true or nearly so. But often
for both carnivores and herbivores
territory size is unrelated to food
supply (Watson and Moss, in Animal
Populations in Relation to their Food
Resources, 167, Blackwell, Oxford,
1970). Hinde points out that territories
have complex functions with conse-
quences both harmful and advan-
tageous to an individual's chance of
breeding success (Ibis, 98, 340; 1956).
Simple answers cannot be expected to
complex questions.
In a well designed series of field ex-
periments on carrion crows in north-
east Scotland, Yom-Tov added extra
food to the environment to ascertain
if there was a direct relationship be-
tween territory size, food supply and
breeding success (J. Anim.Ecol., 43,
479; 1974). There was circumstantial
evidence that there was no absolute
food shortage during the breeding
season. Food in the form of hens' eggs
and dead hens' chicks was placed near
to artificial trees both within and with-
out established crow territories. This
treatment failed to increase the breed-
ing density of adults. But one egg and
five chicks offered daily to a group of
fourteen breeding pairs in the close
proximity of their nests resulted in a
significantly higher survival rate of
nestlings although there was no differ-
ence in clutch size. At fledging an
average of 2.3 young had survived in
the experimental nests compared with
1.1 in the controls.
The other effect of food added daily
from the start of the year was to signi-
ficantly shift the date of the start of
laying, bringing it forward by 5 days.
Earlier layed clutches were almost
twice the size of late clutches, but the
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5 Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4
number of fledglings produced per nest
was about half. So increased winter
food affected date of laying but not,
per se, the number of fledglings pro-
duced per nest.
How does extra food increase the
survival rates of nestling crows? Preda-
tion of the eggs and desertion by the
adults were the main factors respon-
sible. Krebs has pointed out that arti-
fically increased winter food has no
effect on the number of territorial
breeding pairs of great tits during the
following spring (Ecology, 52, 2; 1971).
Those nests that were closer together
than 45 m suffered 12% more preda-
tion (mostly by weasels) than did those
nests spaced over 45 m apart. Carrion
crows are their own chief predators-
they prey to a significant extent upon
the eggs and nestlings of their own
kind. With cannibalism as the regula-
tory mechanism, the ultimate limiting
factor for the number of nestlings
reared is the dispersion of food within
the territory. An abundance of food
close to the nest means that the clutch
is left unattended for shorter intervals
than if the food is widely scattered. To
remain near the nest breeding crows
limit their territory size. The lower
limit to territory size depends upon the
amount of food available and the
upper limit depends upon its dispersion
and the ability of the pair to defend
its nest.
Cyclic AMP and
pattern formation
from Paul Epstein
BY staining with fluorescent antibody
specific for cyclic AMP, Pan et al.
(Proc. natn. Acad. Sci. U.S.A., 71, 1623;
1974) have been able to observe the
distribution of cyclic AMP in both the
unicellular amoebae and the multicel-
lular pseudoplasmodium of Dictyostel-
ium discoideum and several related
species of cellular slime mould. Cyclic
AMP was first shown to be important
in the life cycle of D. discoideum when
it was isolated as the naturally occur-
ring acrasin, or chemotactic agent by
which the vegetative amoebae signal
each other to aggregate (Barkley,
Science, 165, 1133; 1969). Now the
studies by Pan et al. suggest that cyclic
AMP is important not only for aggre-
gation, but for determination, or
pattern formation, as well.
In the cellular slime moulds, once
the aggregation process is completed,
the amoebae form a pseudoplasmodium,
or grex, which, depending on environ-
mental conditions, may migrate for an
indefinite period before beginning to
differentiate into two morphologically
and functionally distinct cell types-
spore cells and stalk cells. By staining
with vital dyes, Bonner (The Cellular
Slime Molds, second ed., Princeton
University Press, Princeton, New Jer-
sey, 1967) showed that the cells in the
anterior third of the grex become stalk
cells, whereas the cells in the remaining
posterior portion become spore cells.
The cells in the grex are not pre-
determined, however, since a grex which
is sliced transversely can form two
normal fruiting bodies, containing the
normal proportions of spore and stalk
cells (Raper, J. Elisha Mitchell scient.
Soc., 56, 241; 1940). This observation
indicates that the cells can recognise
their position within the grex, and
somehow alter their developmental
fate accordingly.
How do the cells know their position
in a field? One theory is that they
respond to gradient(s) of concentration
of small molecule(s) (Wolpert, J.
theoret. Biol., 25, 1; 1969). More
recently, McMahon developed a theory
of
D.
pattern formation
using
discoideum as a model, which holds
that a sharp concentration boundary of
a small molecule can develop through
its regulation by contact-sensing mole-
cules on the plasma membranes of cells
in the field (McMahon, Proc. natn.
Acad. Sci. U.S.A., 70, 2396; 1973).
These regulatory molecules would be
the macromolecules which synthesise
and degrade the small molecule. In this
case, the polarity of the morphogenetic
field is determined by the boundary of
the small molecule, which in turn is
determined by the distribution of
contact-sensing molecules.
Although
one theory invokes a
gradient and the other a boundary, both
models require non-uniform distribution
of a small molecule to determine the
pattern of development. The results of
Pan et al. suggest that cyclic AMP
might be one such . small molecule
which determines pattern formation in
D. discoideum. Pan et al. showed that
as the pseudoplasmodium was allowed
Migrating grex of Dictyostelium dis-
coideum seen from the side and from
above. From Developmental Biology
by Nelson Spratt.
to migrate, a distribution of cyclic
AMP arose such that the higher con-
centration was found in the anterior,
pre-stalk region. In some cases, they
observed an abrupt boundary of cyclic
AMP concentration between the anter-
ior and posterior portions of the grex,
as theorised by McMahon.
It is interesting to consider two
earlier observations which bear on pat-
tern formation and differentiation in
D. discoideum, and to see how they fit
with the idea that pattern formation in
the grex is determined by the distri-
bution of cyclic AMP. Bonner (Proc.
natn. Acad. Sçi. U.S.A., 65, 110; 1970)
subjected isolated amoebae to high con-
centrations of cyclic AMP and found
that they developed into stalk cells.
From this observation, he concluded
that cyclic AMP functions as an
inducer of stalk cell differentiation. The
observation of Pan et al. isconsistent
with this conclusion.
The other observation was made by
Raper (J. Elisha Mitchell scient. Soc.,
241, 1940), who sliced a pseudoplas-
modium transversely into four sections.
Section one, the anterior tip, could be
made to migrate for various times. If
induced to begin fruiting body forma-
tion immediately, without any resump-
tion of migration, this section developed
into an extremely abnormal fruiting
body, bearing very few or no spores,
and an excessively heavy stalk. If 3-6
h of migration were allowed before
fruiting formation began, the resulting
structure was less abnormal but still
bore fewer spores and more stalk. If 24
h of migration were allowed, the tip
gave rise to a completely normal fruit-
ing body, with the correct proportion of
spore and stalk cells. Hence, Raper
demonstrated that the cells at the
anterior tip of a pseudoplasmodium
are determined to become stalk cells,
and that this determined state is altered
progressively as the tip migrates.
In accord with this observation, Pan
et al. found that the distribution of
cyclic AMP in the grex becomes more
apparent as it migrates. Likewise, Mc-
Mahon, in his theoretical treatment.
showed the boundary of cyclic AMP
concentration arising near one end of
the field, and moving along the field
with time. McMahon calculated that by
about 3-5 h the boundary becomes
stabilised at a position one-third of the
way down the field: that is, at the posi-
tion which would give rise to the normal
proportion of spore and stalk cells.
Although
substantial
proof
is
required before one can state that
cyclic AMP is responsible for pattern
formation in D. discoideum, the results
of Pan et al. at least suggest such a
function. If further studies provide
proof, this would represent the first
identification of a small molecule res-
ponsible for pattern formation. .
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Nature Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4 573
Clustered genes
and non-transcribed
spacers
from Benjamin Lewin
MOST eukaryotic genes are probably
present in only one copy for each
haploid genome. Two notable ex-
ceptions to this rule are provided by
the genes coding for histone proteins
and those which specify ribosomal
RNAs: here many genes, apparently
identical for ribosomal RNA genes and
with little variation in the histone genes,
are organised in a cluster in which each
transcribed
sequence seems to be
separated from the next by a. non-
transcribed spacer region. The structure
and function of the spacer are pre-
sumably relevant to the control of
gene activity, and perhaps to the prob-
lem of the suppression of variation in
the multiple copies of each transcribed
sequence.
During oogenesis in Xenopus laevis
the nucleoli which contain ribosomal
DNA are amplified to become a major
cell component. The ribosomal DNA
can thus be isolated in bulk and has
been the subject of much work on
sequence organisation. This DNA was
recently used as a substrate for the
EcoRI endonuclease of Escherichia
coli by Morrow et al. (Proc. natn. Acad.
Sci. U.S.A. 71, 1743-1747; 1974) who
found that each repeating unit (tran-
scribed plus non-transcribed) suffers two
cuts. When X. laevis rDNA molecules
of about 50x106 daltons were treated
with excess EcoRI enzyme the principal
fragments had weights of 3.0×106 and
4.2×106 daltons; prominent among the
minor fragments were two of 3.9×106
and 4.8×106 daltons. These products
were annealed with DNA of the plasmid
pSC101 (which is cleaved at a single
site, generating a linear molecule from
its circular genome) to give plasmid-
rDNA recombinant molecules that were
then replicated by growth in E. coli.
When the recombinant plasmid
DNAs were isolated from several
bacterial cell lines and analysed by
cleavage with the EcoRI restriction
enzyme, each recombinant plasmid gave
fragments with sizes typical of the
linear molecule of pSC101 and of the
cleavage products of Xenopus rDNA.
By hybridisation with 18S and 28S
Xenopus rRNA, Morrow et al. showed
that the hybrid plasmid CD4, which
contains both .the 3.0×106 and 4.2×106
cleavage fragments, anneals equally
well with both rRNAs; the CD18 plas-
mid containing only the 3.0×106
dalton fragment anneals principally
with 28S rRNA; while the DNAs of
plasmids CD30 and CD42, which con-
tain the 3.9×106 and 4.2×106 dalton
fragments respectively, react mostly
with 18S rRNA. These results suggest
that the tandem repeats in the rDNA
are heterogeneous, each consisting of
the 3.0×106 dalton sequence (which
contains the 28S rRNA sequence) and
the 3.9×106 or the 4.2×106 dalton
sequence (containing the 18S rRNA
sequence and presumably differing by
the presence or absence of an extra
sequence; other variations in this region
are suggested by the existence of other
size fragments).
Since the transcribed region of
Xenopus rDNA takes only one form,
heterogeneity in sequence must be
ascribed to the non-transcribed spacer
region, a conclusion supported by the
report of Wellauer et al. in the Pro-
ceedings of the US National Academy
of Sciences (71, 2823-2827; 1974). By
cleaving Xenopus rDNA with the
EcoRI enzyme, they were able to
isolate two classes of fragment on
agarose gel electrophoresis. Every pre-
paration contains a prominent band of
3.0×106 daltons and several bands are
present in lesser amounts which vary
from 4.0 to 5.8×106 daltons. To decide
whether the heterogeneity of the larger
bands exists within single genomes or
reflects differences between animals,
they examined the rDNA from a single
nucleolus organiser; this contained at
least three of the larger size classes as
well as the 3.0×106 dalton band. The
repeat length of transcribed plus non-
transcribed Xenopus rDNA is about
8×106 daltons, clearly implying that
the repeats must be heterogenous in
length (since otherwise the total length
of fragments would equal rather than
exceed the repeat length). When the
duplex lengths of the fragments were
measured by electron microscopy, the
smallest proved to have a homogenous
size of 3.0×106 daltons and the number
of these small fragments equalled the
sum of the number of the larger frag-
ments. Each repeat therefore seems to
comprise one 3 × 106 dalton region and
one of the larger regions.
Secondary structure mapping has
previously been used by Wellauer and
Dawid (Proc. natn. Acad. Sci .. U.S.A.
70, 2827-2831; 1973) to order the
sequences for ribosomal RNA in the
precursor of the mature rRNA (see
Lewin, Nature, 250, 619-621; 1974).
When RNA is spread for electron
microscopy, hairpin loops may form
between short complementary sequen-
ces and the position of each loop along
the linear molecule provides a map of
it. The 28S rRNA has a characteristic
map present at the 5' end of the pre-
cursor and the 18S rRNA is character-
ised by an absence of loops. Wellauer
et al. now extend this technique to
DNA which has been denatured to
single strands. The small 3.0×106
dalton fragment cleaved from Xenopus
rDNA
by the EcoRI enzyme is homo-
geneous in structure; it seems to result
from cuts made by the enzyme about
0.3×106 daltons into the 28S rRNA
sequence and about 0.2×106 daltons
into the 18S rRNA sequence; this
fragment thus represents the starting
region of the rRNA precursor, lacking
the immediate 5' end. The other frag-
ment generated by these two cuts has
one end devoid of structure, presumably
corresponding
to the 18S rRNA
sequence, with the other end displaying
the structure typical of the very begin-
ning of the 28S rRNA structure.
40S
28S
18S
Nontranscribed
Spacer .
=
3.0 × 106
4.0-5.9 X 106
Summary by Wellauer et al. of struc-
ture of an rDNA repeat unit.
The central regions of all larger frag-
ments show a related structure, but
vary in length. It is therefore the non-
transcribed spacer region in which the
heterogeneity resides and the formation
of heteroduplexes suggests that the
shorter
large
fragments represent
deletions of sequences present in the
longer large fragments. How this
heterogeneity relates to the functions
of the nucleolar rDNA is not yet
known; one reservation about the
interpretation of these results, however,
is that the source of the rDNA is the
amplified material of the oocyte and it
remains to be proven whether the same
situation is found in the somatic cell.
Analysis of histone genes is at a less
advanced stage than that of ribosomal
RNA genes. Each of the five histone
proteins seems to constitute a unique
amino acid sequence in any species; and
the hybridisation analysis of Weinberg
et al. (Nature, 240, 225-228; 1972) sug-
gested that their messenger RNAs are
coded by genes repeated some 500-
1,000 times in the genome of the sea
urchin Psammechinus milaris. Because
of the multiple codons representing each
amino acid, identity of protein
sequences need not imply identity of
the repeated nucleic acid sequences
coding for them, but the hybridisation
analysis suggests only limited hetero-
geneity. The histone messengers of
another sea urchin, Lytenichus pictus,
have been separated by gel electro-
phoresis into a small number of groups
by Grunstein et al. (Cold Spring Harb.
Symp. quant. Biol., 38, 717-724; 1973)
and a fingerprint analysis of one of
these messengers is consistent with the
idea that its sequences are fairly
uniform.
The histone genes of Psammechinus
milaris band on CsCl gradients at a
density only slightly greater than that of
bulk DNA, but Birnsteil et al. (Proc.
1
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57 Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4 18 1974
natn. Acad Sci. U.S.A., 71, 2900 2904;
1974) now report that they can be iso-
lated by their ability to bind prefer-
entially to actinomycin CI which causes
them to form a low density band. The
band isolated in this way hybridises well
with a preparation that contains the
messengers for four histones (mRNA
activity for histone Fi has not yet been
demonstrated); since each of the four
histone messengers shows a virtually
identical reaction with the fractions of
DNA separated on the CsCl-actino-
mycin gradient, it is likely that the
different histone genes are intermingled
rather than clustered in four individual
groups.
Comparison of G+C content of
histone DNA and RNA, and the results
of shearing and of melting histone
DNA all suggest that the histone gene
cluster also contains other sequences
that are poorer in G+C content. The
release of the histone-coding from the
other sequences only by shearing sug-
gests that both components are integral
parts of the gene cluster. The melting
curve of renatured DNA from the
preparation implies the presence of
some heterogeneity in sequence, pro-
bably in the sequences which do not
code for histones. Whether the com-
ponent poor in G+C represents a non-
transcribed spacer region analogous to
that of the rDNA gene cluster is not
of course revealed by present data
but this is one of the functions that
can be imagined for it.
Two by two
from D. H. Jennings
IT is rare these days to attend a con-
ference covering the whole spectrum
of biology. But the Society for Experi-
mental Biology mounted such a con-
ference-on symbiosis-at Bristol on
September 2-6. By retaining the
original de Bary definition, namely
that symbiosis is the association of two
different organisms, it was possible to
have all shades of interest from mole-
cular to whole organism biology rep-
resented. Thus, ecologists, though not
contributing directly, could find much
to interest them: Indeed, two contribu-
tions were particularly seminal. for
ecological studies. Fricke (Max-Planck-
Institut, Seeiweesen) indicated how
behavioural studies of organisms in
the Red Sea can provide an under-
standing of animal interactions in
nature thus adding flesh to the bare
bones of numerical population studies.
Cox (Kings College, London) discus-
sing his work on intraerythrocytic
parasites, pointed out that population
biologists cannot continue to think
about particular species without con-
sidering their parasites. He showed
how susceptibility to various virus
diseases can be dramatically modified
by the presence of parasites.
There was much emphasis on how
the invading symbiont avoids attack
by its potential host. Terry (Brunel
University) and Smithers (National
Institute for Medical Research, Mill
Hill) presented their elegant studies on
the evasion of the immune response by
Schistosoma which can survive by its
ability to acquire host molecules. Mus-
catine (University of California, Los
Angeles) provided data indicating the
recognition by Hydra of the appro-
priate Chlorella cells takes place after
engulfment at the stage when the algal
cells move to the base of the digestive
cells. Studies on symbiosis in Para-
mecium are now also beginning to
yield relevant information with respect
to bacteria (Preer, University
of
Indiana, Bloomington) and Chlorella
(Karakashian, Max - Planck - Institut,
Wilhelmshaven).
In spite of the decision to use the
de Bary definition, there was much
discussion of what is meant by the
term
'symbiosis'.
Mortimer Starr
(University of California, Davis) pro-
duced a new classificatory scheme
which has the virtue of both clarifying
thinking about the phenomenon and
indicating where further experimental
work is required-to decide, for ex-
ample, whether a symbiotic association
is mutualistic or obligately parasitic.
In this respect, Smith (University of
Bristol) put those interested in lichens
further in his debt by his careful
analysis of the present data in the
field which indicate that there is no
detectable benefit to the algal partner.
Also Coffey (Trinity College, Dublin)
presented further information about
the rust fungi, once thought to be the
classic example of obligate pathogens
in plants but which can now be grown
in culture.
Many problems are emerging for
the biochemists, particularly on how
a symbiont affects the physiology of
the host which it has invaded. Bio-
chemists should be impressed by
Aplysia which does a far better job
than they of isolating chloroplasts.
There is no doubt, as shown by
Trench (Yale University), that these
chloroplasts
are
fully
functional,
though the relationship between these
organelles and the animal cytoplasm
in which they reside is not the same
as that which exists in the intact cell
from
which
the chloroplasts
are
extracted.
But biochemists and molecular bio-
logists have other reasons for being in-
terested in symbiosis. Chloroplast sym-
biosis is, in a number of ways, the
living expression of how eukaryotic
organelles are believed to have arisen,
namely by successful invasion of a
primitive glycolytic prokaryote by oxy-
gen utilising (to give mitochondria) and
photosynthesising (to give chloroplasts)
prokaryotes. Though the theory has a
respectable age, Lynn Margulis (Uni-
versity of Boston) has done the most
recently to bring it up to date. In a
debate on the theory she propounded
her views with infectious enthusiasm.
But though most of the audience were
believers, Raff (University of Indiana,
Bloomington) in a careful comparison
of bacteria and mitochondria showed
that their faith may be blind.
Five hundred
million years ago
in Wales
from 1. Strachan
ABOUT half of those attending the
symposium organised by the Palaeonto-
logical Association of London on the
Ordovician System (September 17-20)
had spent the previous week touring
Wales looking at the classic geological
sites of Arenig, Llandeilo, Caradoc and
Bala.
Much of the material presented at
the meeting in Birmingham was fac-
tual and included new information as
well as regional summaries, but there
was also a considerable questioning
of current dogmas. F. J. Fitch (Birk-
beck College, London) and others
speaking on Ordovician geochrono-
logy pointed out the possible sources
of error inherent in radio-isotopic
dating, particularly in Lower Palaeo-
zoic rocks, and stressed the need for
the use of standard reference scales
(such as that produced by the Geologi-
cal Society of London in 1964) so that
data from different sources could be
accurately compared. They concluded
that a length of 65 Myr (between 510
and 445 Myr ago) was reasonable for
the Ordovician, compared with 70 Myr
for the preceding Cambrian and 40
Myr for the succeeding Silurian.
Given this broad time span, it was
not surprising that the details of the
regional pictures presented by later
speakers should show some differences.
The biostratigraphical problems were
analysed in various groups of fossils.
W. B. N. Berry (University of Cali-
fornia) related his graptolite faunas to
warm and cool water masses using a
palaeogeographical reconstruction with
the land all on one hemisphere. M.
Lindström (University of Marburg)
felt. that the conodont faunas agreed
in provincialism with the graptolites
but C. R. C. Paul (University of Liver-
pool) found distinct North American,
Baltic and Mediterranean cystid faunas
only up to the lower part of the
Caradocian, after which time provincial
boundaries became fuzzy. This he
related to closing up of ocean basins
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Nature Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4 575
during late Ordovician plate move-
ments.
He also discussed the functional
aspects of respiration in cystids to
critically low oxygen levels in shallow
tropical seas. This linked the palaeo-
geographic approach to the climatic
one which was discussed by N.
Spjeldnaes (University of
Aarhus,
Denmark). The now well known gla-
cial deposits of the Moroccan Anti-
Atlas were beautifully illustrated by J.
Destombes (Service géologique, Rabat)
and other speakers referred to climatic
cycles in the Ordovician which influ-
enced the occurrence of various groups
of fossils. Changes in faunas could be
caused as much by climatic changes
as by migration between continental
plate margins and the disentangling of
plate movements through the whole
length of the Ordovician needs much
further work.
None of the formal papers included
any discussion of the boundaries of the
Ordovician, most speakers making it
clear whether they. regarded the Tre-
madocian ás Cambrian (as in English
usage) or Ordovician. But the large
gathering of experts from so many
countries, including the Soviet Union, .
provided the opportunity for meetings
of the Commission on Stratigraphy of
the International Union of Geological
Sciences. Several recently formed sub-
commissions and working groups of
the commission will have a great deal
to do with correlation of strata be-
tween countries and continents. Dif-
ferences of interpretation were much
in evidence during the closing general
discussion in Birmingham, particularly
between those based on groups of
fossils traditionally used, such as bra-
chiopods and trilobites, and those
which have come into prominence in
the last few years, such as conodonts.
Some of the ideas were certainly pro-
vocative and augur well for the future
liveliness of the subject. As an eminent
worker on Palaeozoic gastropods put
it to close the meeting-a snail never
gets anywhere unless he sticks his neck
out.
Plant conifers
and lose water
from Peter D. Moore
A KNOWLEDGE of the rate of water Ioss
from a forest is essential both for
forest water budget calculations and in
the management of water catchments.
Many comparative studies have been
undertaken to provide such informa-
tion, on forest stands and on other
vegetation types. Differences in water
loss from various types of vegetation
are explicable in terms of transpiration
rates, in the effect of vegetation upon
evaporation direct from the soil by
Expanding wings in moths
from our Insect Physiology Correspondent
EVERY lepidopterist
knows
that
emerging butterflies and moths go
through a characteristic sequence
of wing-folding movements, with
wing expansion and general harden-
ing of the cuticle, before settling
down into their normal resting pos-
ture; and that the force for wing
expansion is the hydrostatic pres-
sure of the blood generated by
strong contraction of the abdomen.
Truman and Endo (J. exp. Biol., 61,
47; 1974) have now elucidated the
complex
of
physiological
fac-
tors engaged
in
this
familiar
phenomenon.
It was shown by Fraenkel many
years ago that if the newly moulted
blowfly is obliged to continue bur-
rowing through the soil, it remains
soft with the wings and body un-
expanded for many hours. Only
when it makes its escape is the
cuticle expanded, hardened and
darkened. It was generally assumed
that this hardening process must be
controlled by a hormone, but that
was demonstrated by Cottrell only
in quite recent years and confirmed
by Fraenkel and Hsiao, who named
the hormone in question 'bursicon'.
It is liberated from the brain and
other ganglia of the nervous
system.
Truman and Endo found that the
emerging adult of the tobacco
hawkmoth Manduca behaves in the
same way. It likewise pupates in
the soil, and so long as the emerg-
ent moth is confined it shows in-
tense digging behaviour, and the
spreading of the wings is sup-
pressed, sometimes for 24 hours.
The stimulus which maintains the
inhibition comes from the head:
decapitated moths spread
their
wings immediately, regardless of
whether they are confined. Moths .
decapitated at the very moment of
eclosion, however, show neither
wing inflation nor wing folding. But
if decapitation is delayed until five
seconds after emergence, the full
wing-spreading behaviour occurs.
This effect of the brain is not
dependent on the nerve supply; it
is the result of the neurosecretory
'eclosion hormone': a brain re-
moved from the pupa and im-
planted into the abdomen of a
decapitated pupa is wholly effective
in evoking complete wing spread-
ing. The suboesophageal ganglion
is also necessary for wing spread-
ing; but this seems to be a neural
effect on movement. The hormone
bursicon, set free from the abdo-
minal nerve cord, will cause neither
wing inflation nor wing folding; it
has two important functions: it
serves to plasticise the cuticle (as
was shown by Cottrell in the blow-
fly) and it sets in motion the har-
dening of the cuticle which, in
turn, apparently dictates by feed-
back to the central nervous system
the timing of the wing-folding
movements.
In brief the eclosion hormone,
discovered by Truman a few years
ago, not only initiates emergence,
but also wing spreading, which is
brought about by a central motor
programme involving neural and
hormonal stimuli and some degree
of sensory feedback.
providing insulating layers of varying
thickness and spatial structure, and in
the interception of precipitation and its
subsequent evaporation directly from
leaf surfaces.
The quantification of these para-
meters, however, is extremely difficult.
One of the most useful approaches has
been the development of detailed
models which attempt to account for
all of the microclimatic and spatial
variables, each of which can be deter-
mined individually (for example, Mon-
teith, Symp Soc. exp. Biol., 19, 205;
1965). Various attempts have been
made to compare water losses from
deciduous and coniferous stands by
observational rather than theoretical
methods, but most of these have shown
negligible differences during the summer
months (for example Zahner, Forest
Sci., 1, 258; 1955); no reliable data are
available for longer periods covering
the winter season, when evergreens may
continue to transpire and also to inter-
cept more rainfall than deciduous trees.
Comparative data between forest stands
on different sites will always be some-
what suspect because of possible dif-
ferences in the soil water storage capa-
cities, hence long term studies on a
single site where deciduous trees have
been replaced by conifers seems to be
the most hopeful approach to this
problem.
The results of just such a study have
now been published by Swank and
Douglass (Science, 185, 857; 1974).
Their study area is in the Appalachian
Mountains of the south-western region
of North Carolina, originally bearing
oak-hickory forest. Streamflow studies
were carried out on selected watersheds
within the area over a period of ten
7
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57
Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4 18 1974
years, which provided data sufficient for
the prediction of stream flow under any
given conditions of precipitation. In
1956 a 16 hectare watershed was clear-
felled and planted with eastern white
pine (Pinus strobus). For the next six
years the streamflow was greater than
that predicted for the forest stand, in
the initial period by as much as 15%.
Subsequently streamflow fell below pre-
dicted annual values and for the past
6 yr it has been between 15 and
20 cm (equivalent to 15-20%) below
that expected from a hardwood stand
on the same site.
Monthly streamflow values over the
course of a year are particularly inter-
esting because they show that between
June and October the monthly stream-
flow is less than 1.5 cm below the
expected value, whereas in November,
December, April and May streamflow
is over 2.0 cm below expected. Thus the
additional losses of water from a coni-
fer stand take place mainly in winter
and spring; the possibility of this being
the case had already been postulated by
Penman (in Forest Hydrology, edit. by
W. E. Sopper and H. W. Lull, Per-
gamon Press, Oxford, 373; 1967) on the
basis of very little observational evi-
dence.
The difference is likely to be caused
by both the increased interception and
the higher transpiration rates in the
conifers in winter and spring. The leaf
area index of hardwoods in winter is
low (<1) in comparison with white pine
(9.9), which indicates the much larger
surface area with pine available for the
interception of precipitation.
This provides one more good reason
for caution in replacing hardwood
forests with conifers, especially in water
catchment sites.
Field effect
seen in glassy
semiconductors
from Andrew Holmes-Siedle
AT the start of the solid-state age,
Bardeen and colleagues attempted to
modulate the conductivity of a slab of
germanium by putting a metal plate
near to the surface and applying a high
electric field. Unexpectedly, the conduc-
tivity of the slab did not change and,
because of this, the idea of the 'field-
effect transistor' lay dormant for over
ten years while the reasons for failure
were explored. Meanwhile, in order to
meet the urgent pressure for a working
solid-state amplifier, the device makers
(Sockley and colleagues) went on to
develop the less elegant point-contact
and diffused bipolar transistor prin-
ciples.
The field-induced 'accumulation' or
Table 1 Estimated densities of localised states in chalcogenide glasses deduced from field-effect
experiments
Fritzsche
Egerton
Typical composition
AS 35 Te2 8S22Ge15
Te 5 0Si 12 Ge10AS10
Tick et al.
Te2As Si
Form
Evaporated
Evaporated
Melted and drawn
Bulk states (eV-1 cm -3)
Surface states (eV-1 cm -2)
6×1019
1019-1020
3× 1017- 2×1019
not estimated
2 ×1013.
2 ×1012- 3×1016
'inversion' of current carriers in the
Tick and co-workers used a thin
filament of chalcogenide with an un-
filament was produced by melting and
drawing down a sample of chalcogenide
powder enclosed in a special silica glass
jacket. The result was a thin silica glass
rod of diameter 7 mm with a very thin
central filament of chalcogenide, of dia-
meter only 0.01 mm. When this was
sliced, one could gain contact to the
ends of the filament with probes while
the glass disk contributed support for
an annular metal field plate as well as
providing
surface
passivation and
mechanical strength. For normal semi-
conductors and high fields, the surface
space-charge regions are of the order
of micrometres, so with these dimen-
sions there is a chance that the field will
influence a fair fraction of the filament
volume.
germanium did not take place as pre-
dicted because immobile charge was . usual form of surface passivation. The
generated in surface states. The sheet
of charge 'screened' the interior of the
semiconductor and prevented the re-
quired bending of energy bands. Now-
adays, surface states in crystalline semi-
conductors are under fair control and
the
metal-oxide-silicon
field-effect
transistor is a standard component in
computer circuits. It might now be said
that we are at the start of the 'glassy
solid state' age; it is perhaps a good
omen that, despite a slow start, the field
effect experiment can apparently be
performed successfully in some glassy
semiconductors. This is fortunate be-
cause the penetration of a semiconduc-
tor by a field provides a probe of band
structure which is particularly appro-
priate for the glassy semiconductors.
Spear and le Comber (J. non-cryst.
Solids, 8-10, 727; 1972) gained some in-
formation on localised states within the
band gap of amorphous silicon, but H.
Fritzsche failed to find any effect in
the 'switching' materials, of chalco-
genide composition, despite painstaking
attempts (Ann Rev. Mater. Sci., 2, 697;
1972). R. F. Egerton (Appl. Phys.
Lett., 19, 203; 1971) reported definite
field effects which were, however, too
small to be useful in determining the
nature of the localised states. Now, by
special methods of preparation, Tick
and Watson of Corning Research and
Development labs and Hindley of the
University of Birmingham (J. non-cryst.
Solids, 13, 229-242; 1974) have pre-
pared samples which have lower appar-
ent densities of localised states and
which thus give a higher response to
the field effect. There is, therefore, some
hope of using the field effect to deter-
mine the distribution with energy of
the localised energy states in this very
important group of materials.
The fabrication technique used was
unusual. It is a fair assumption that the
evaporated glass layers used by many
investigators are more disordered than
glasses cast and cooled from the melt.
But a field-effect experiment on a size-
able cast block of glass would be hope-
lessly swamped by the conduction
currents in the bulk of the sample. In
order to make the bulk volume of the
sample commensurate with the thin
field-modulated region on the surface,
In fact, negative fields of the order
of 106 V cm-1 modulated the bulk
conduction by about 5%, though
greater fields produced little further
effect; this could be termed a saturating
dependence on field. Positive fields
(metal positive) gave a much smaller
effect. The simple analysis which best
fits the saturating characteristic is that
of a density distribution of localised
states which is uniform with energy all
across the band gap. The field depend-
ence of current predicted for a sharply
peaked distribution of localised states,
such as that called for in the Davis-
Mott model for chalcogenides (see for
example Mott and Davis Electronic
Processes in Non-crystalline Materials,
Clarendon Press, Oxford, 1971) is of a
very different form from that observed.
The model proposed also postulates a
significant density of surface states. The
figures for density are compared with
the estimates made by Egerton and by
Fritzsche in Table 1. The values given
for certain samples in this latest work
are seen to be considerably lower than
in the earlier efforts-possibly a hope-
ful sign that we are learning better
control of the preparation of glassy
semiconductors.
These results, while not constituting
a definitive disproof of the commonly
accepted peaked energy distribution of
the Davis-Mott model show that the
field-effect method is a tool which, with
perseverance, may provide definitive
evidence on this question.
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Nature
Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4 177
articles
Human reproduction and family planning:
research strategies in developing countries
Alexander Kessler & C. C. Standley
Human Reproduction Unit, World Health Organization, Geneva, Switzerland
The difficulties of providing family planning care on a
world-wide scale have led to considerable emphasis on
research in human reproduction. How much of this
research should, and can, be carried out in developing
countries raises many issues in research administration.
.
RESEARCH into human reproduction and family planning
has expanded considerably in the past 15 years, and is now
in progress in many institutions both in developed and
developing countries. Rarely, however, has sufficient effort
gone into formulating appropriate national policies for
such research.
Developing countries raise some of the severest problems;
frequently, esoteric research done in developed countries
is repeated, while opportunities for local clinical investiga-
tions which are closer to the health needs of the countries
are ignored. The nature of research training may be partly
to blame; trainees, may be exposed to a technique rather
than being provided with an approach to research and a
broader background in reproduction. The same incoherence
is often found in the location of facilities: new institutes
are created where expansion of existing ones would have
been sounder and more economic. Funds are at times
monopolised by small groups of scientists, and a contribu-
tory factor has been the multiplicity of funding sources,
foreign ones often being the most important.
The urgency of this research and the relatively ready
availability of funds means that policy issues require serious
consideration. The World Health Organization recently con-
vened a workshop for this purpose-research administrators
from Argentina, India, Pakistan, Thailand, Turkey and the
United States met to pool experiences. Their problems
can be discussed under the following heads :
· Should research be done?
. How should priorities be determined?
· What administrative structure is necessary?
Should research be done?
Increasingly, policy makers are asking for clear orientation
of research towards specific problems. Stephen Toulmin, in
Encounter, has described this process as ". . . calling in
the implied technological IOUs previously issued by the
scientists". Policy makers also ask whether better use of
existing knowledge and resources, or a simple increase
in services would not better solve problems than would
more research. Do methods of fertility control now
available (whether by drugs, devices or surgical procedures)
not meet the needs of the great majority of couples? Is not
the need now to make these methods more widely available?
We believe there is still a need for research. The low
initial acceptor rates and high discontinuation rates for
present methods in almost all family planning programmes
of developing countries are undoubtedly due in part to a
lack of services; often, however, they are due to the wrong
service approach.At the same time contraceptive technology
still leaves room for improvement. Although more methods
are now available, they remain relatively crude. At times
they are associated with undesirable side effects, and they
often fail to meet the needs posed by differences in culture,
education, and service-structure. Moreover, many forms
of contraception are still not available: a male pill, a
post-coital pill, a once-a-month pill, a vaccine against
pregnancy and so on.
Fertility regulation presents special problems for research :
techniques require an assessment well beyond that required
for other therapeutic modalities because of their extremely
wide, long term and frequently unsupervised use. Further,
expected users differ in genetic constitution and environ-
ment. In most cases return of fertility must be allowed
and methods are usually self selected, not prescribed. Even
conceding the need for more research, the role of develop-
ing countries in such research must still be clarified :
For established methods, dosages appropriate for women
of a high level of nutrition may not be applicable. Side
effects of concern in the West may not manifest themselves,
while new side effects may appear arising from disease and
deficiency states in developing countries.
· For new methods, developing countries should be in-
volved at an early stage of the research to determine
acceptability. They must also be involved in testing at all
stages of development so that safety and effectiveness data
can be acquired rapidly for different populations.
As far as services are concerned, the argument that
family planning programmes can go forward on a common
sense basis, without any service research, does not stand
scrutiny. We have no precedent for providing health services
on a continuing basis to most couples of reproductive age
in a community. Sensible studies and small experiments
are needed on the major components of family planning
activities in a local context : information, education, and
provision of clinical services. For instance, inappropriate
presentation of educational material may spoil an other-
wise well-designed programme. Local research is also
essential in determining the right location of facilities,
personnel, frequency and timing of clinics, schedules for
follow-up care and provision of methods.
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57. Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4 18 1974
How should priorities be determined?
Data are frequently lacking on how to identify specific
problems, how to rate their relative importance, how to
determine their susceptibility to solution through research,
what manpower and facilities are needed, what is the
potential pay-off, and what is the likely duration and cost.
On the other hand, fundamental questions of strategy
have to be settled, such as the balance between goal-
oriented and non-directed research, between research and
research training, and between research in universities,
government institutes and industry.
The usual guidelines for setting priorities in biomedical
research are the levels of unresolved morbidity and mor-
tality, and indices of general welfare. The same criteria
hold here. Initially, broad problem areas must be identified
and ranked in importance : pregnancy, labour, development,
family planning and sterility. Then specific topics for study
and research approaches must be ranked. For instance,
should the emphasis be on development of new therapies or
on service research?
The contribution of research to reducing morbidity and
mortality has already been demonstrated in some fields,
such as pregnancy and labour; beginnings have been made
in research on the biology and health of the foetus and the
foeto-maternal complex. This research has generally been
in developed countries. Malnutrition and infectious diseases
in developing countries are likely, however, to lead to
great incidence of pathology and different forms of
conditions associated with pregnancy, labour and foetal
development.
In ranking family planning research, account must be
taken of indices both of morbidity and mortality and of
'welfare'. Birth control prevents death and disease that
result from unwanted pregnancy, short birth intervals, or
illegally induced abortions, whilst the advantages of deter-
mining family size need little elaboration at the family level
and will accord, in many communities, with population
policies.
Problems of local importance undoubtedly have first
priority in developing countries. The epidemiological,
clinical, and service research required clearly cannot be
carried out elsewhere. Amongst such problems are the
effects of hormonal contraceptives in women infected with
Schistosoma haematobium, foetal development in com-
munities with a high incidence of thalassaemia and prenatal
care in protein-deficient pregnant women.
More controversial are the priorities for problems that
could be investigated in a developed country, for example,
development of new contraceptives. Ideally, as we have
said, any new drug should be investigated in developing
and developed countries simultaneously, using a common
protocol. This, however, demands highly specialised man-
power and equipment. Should scarce resources be expended
on the earlier stages of development, such as those that
precede testing on human subjects, for instance, identifica-
tion of reproductive processes susceptible to regulation,
synthesis or isolation of suitable chemicals, screening for
effectiveness and toxicological studies in animals?
Several arguments can be made for not automatically
excluding from developing countries any aspect of research
needed to resolve important problems. One is that it would
make little sense and detract from the intellectual chal-
lenge if there were arbitrary cutoff points to research.
Another is that the whole range of scientific expertise must
be available in a developing country, since scientists and
institutions in developed countries are often not interested
in the problems of the developing country. An example is
the lack of research on injectable contraceptives-a
modality that meets many of the needs of developing coun-
tries. An artificial limitation would further deprive the
scientific community of talent and would preclude scientists
in these countries from pursuing freely their choice.
Finally, university teaching is improved by the presence of
an active research programme.
Should public funds support research that is left entirely
to the choice of the individual? A comparison of non-
directed with directed research is difficult to make, and
the level of funding for non-directed research cannot yet
be determined on the basis of general principles.
Feasibility of research on priority problems
A problem may be important and it may be seen to be
amenable to solution through research, but it will have to
be ranked in terms of the manpower and facilities that it
would tie up. Work can sometimes start immediately on
a problem not of the highest priority, while a more
important one must await the necessary resources. A
10-20 yr plan, regularly reviewed, is needed.
A representative ranking of research areas in many
developing countries could take the following form. First,
the health rationale for family planning (for example,
studies of effects on health of different patterns of family
formation, identification of women particularly requiring
family planning care because of their health status);
second, clinical trials of contraceptives at the community
level; third, the effects of widely-used contraceptives in
the presence of conditions such as malnutrition, diseases,
use of other drugs; fourth, the acceptability and service
implications of different methods of contraception; fifth,
sterility, and, sixth, the in-depth, pharmacological studies
in humans of new contraceptives.
.
Some developing countries may be able to start rapidly
on the first item as they may already have manpower in
the required disciplines such as obstetrics and gynaecology,
maternal and child health, sociology, biostatistics. Spe-
cialised technicians may need to be trained for field work,
computer analysis, and clinical laboratory measurements.
This might take about three years. If centred around one or
two good investigators, a research nucleus would have been
created which could then, if required, move into research
projects in almost all of the priority areas.
In contrast, item six demands manpower and facilities
unlikely to be present in many countries. Several years
may be needed to train specialists with skills in steroid
assays, peptide chemistry, radioisotope use, pharmacology,
metabolic studies and so on. Technicians are also essential
as is the availability of specialised clinical facilities.
One issue that repeatedly comes up is the proportion of
funds to be spent on defined projects as opposed to longer-
term more
comprehensive
'institution-building'
ones.
Assurance of financial support over a number of years is
of importance to all investigators, but particularly so in the
case of institution-building.
A shortage of qualified manpower may make it difficult
to bring together experts, other than the applicants them-
selves, to review research proposals for scientific merit, so
foreign assistance may be necessary. The low level of
literacy in a population may pose problems in ensuring
that 'informed consent' has been obtained.
Should research training be restricted, for instance, to
clinical trial methodology, or widely based in clinical phar-
macology? The narrow approach quickly produces an in-
vestigator able to apply himself to an important problem;
the more broadly educated scientist is potentially more
versatile. During his training, however, he may find his
interests shifting away from clinical trials. Western research
training for scientists from developing countries has been
problematical: we may mention the 'brain drain' and the
lack of orientation of the research training either to
problems or conditions the scientist will encounter on his
return home. Failure to provide a research position for the
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Nature
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investigator on completion of his training, to ensure attrac-
tive career opportunities regularly leads to loss of talent to
the developed countries; some countries are now trying to
remedy these shortcomings.
Keeping up with rapid advances presents a technical and
financial problem. Airmail subscriptions to journals, access
to computer retrieval of bibliographies, participation in
international meetings go some way to help. Less attention
has been given to ensuring that research results rapidly
reach policy-makers, administrators and service personnel
in a form appropriate to their needs.
What administrative structure is necessary?
The number of agencies with interests in research in this
field is probably greater than in most other areas of
biomedical research. The concerns of such bodies as the
Ministry of Health; the national Family Planning Autho-
rity; the Ministry of Education; the Medical Research
Council (MRC); the Council for Science and Technology;
the government offices responsible for the national drug
industry; the national drug regulatory agency and so on,
may be difficult to unite in a common policy. It may be
even harder to bring in the private drug industry whose
orientation is primarily commercial. Yet another factor is
the aggressive attitude sometimes taken to family plan-
ning, including its research aspects, by foreign aid
assistance agencies, whose priorities may not necessarily
coincide with national ones.
The national MRC sometimes offers a plausible focus:
it has experience in formulating biomedical research policy,
could place research in reproduction and family planning
within the overall context of the country's biomedical
research programme, and could use its existing mechanisms
for administration. In many developing countries, however,
there may not be an MRC capable of assuming these res-
ponsibilities; a ready focus may then exist in the Ministry
of Education in whose institutions much of the research
will be carried out anyway. Another approach creates a
body within the national family planning authority for the
administration of research. This should ensure a research
policy directed to the problems encountered in the family
planning programme and ensure rapid transmission of
research results to field workers; unfortunately, such bodies
may have little tradition of research, encounter resistance
from older established ministries, and restrict themselves
to a narrow and short term view of research.
External aid has often been forthcoming, but on occa-
sions not so much to support research efforts as to in-
fluence family planning policies, or even to gain access to
subjects to test drugs under less stringent regulatory
conditions than those prevailing in the industrialised
countries. The magnitude of the funds offered has at times
led to this assistance being accepted in spite of the dis-
tortion it brought to nationally selected priorities. Some
countries now place rigid embargos on accepting assistance
from any other than multilateral sources.
The most neglected use of foreign assistance has been
in the areas of policy and research organisation. Few
donors have wished to support activities that have little
visibility and no quick returns, and few recipients have
insisted on using research funds for this preliminary ground
work.
Compositional variation in recent Icelandic
tholeiites and the Kverkfjöll hot spot
G. E. Sigvaldason, S. Steinthorsson, N. Oskarsson & P. Imsland
Science Institute, University of Iceland, Geoscience Building, Reykjavik, Iceland
Geochemical studies of recent igneous rocks from Iceland
lend support to the 'hot spot' theory. There is a geo-
graphical variation in basaltic composition between
Iceland and the submerged parts of the Reykjanes Ridge
and Mid-Atlantic Ridge, which suggests that the single-
source model for basalts of this region may be correct.
THE puristic mantle plume hypothesis1-3 holds that all ocean
ridge volcanism is fed by the plumes. The plate systems
drift passively across the Earth's surface relative to the
plumes, as shown by the universal symmetry of magnetic
anomaly patterns about the ocean ridges. Thus, the posi-
tion of the ridges is not fixed by subcrustal agencies such
as convection cells in the mantle. Outside the mantle
plumes there are no heat sources other than the plume
overflows. All the heat flow in the ocean bottom can be
accounted for by the loss of heat from the cooling plates
as they drift away from the ridges4.
The single-source theory has been challenged by Schilling'
on geochemical grounds, particularly with reference to Sr-
isotope variation6. We propose to show that geochemical
variations in Iceland and the North Atlantic may be con-
sistent with the one-source model, both as regards petro-
chemistry and isotope geochemistry.
We have collected and analysed 182 samples that are
considered to cover volcanic activity in the tholeiitic rift
zones of Iceland' during the last 10' yr. The geological for-
mations represented are the 'grey dolerites' from the last
interglacial, pillow lavas and breccias of the subglacial mo-
berg (palagonite) formation, and postglacial lava flows. The
volumes of the individual lavas vary greatly: monogenetic
shield volcanoes range from 1-15 km3, whereas lavas pro-
duced on fissures and isolated scoria cones are much
smaller-generally less than 1 km3.
The samples have been divided into three groups on a
geographical basis (Fig. 1) representing the western rift
zone (WRZ, 126 samples), the northern part of the eastern
volcanic zone8 (NEZ, 26 samples), and the Kverkfjöll area
(30 samples), a relatively small area in the vicinity of the
Kverkfjöll volcanic complex in central Iceland (K. Albert-
son, unpublished).
The Kverkfjoll area is the hot spot of the North Atlantic.
Away from that area the maximum content of potassium
in basalts on the volcanic rift zones decreases continuously
with distance (Fig. 2). The minimum K2O content remains
constant, at about 0.05-0.1%, in the Kverkfjöll area
and probably along the entire adjoining ridges. A pro-
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58
Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4 18 1974
Skagı
Mývatn
Snaefellsnes
Askja.
Snaefell
SFZ.
Landjokull
, Vatna-65
, Jokull Ri
Reykjanes
Oraefajokull
0
50
100 km
Ridge
Surtsey.
Heimacy
Fig. 1 Neovolcanic areas in Iceland. The Snaefellsnes and
the southern part of the eastern zones are alkaline in char-
acter, the others tholeiitic. WRZ, Western Rift Zone; EVZ,
Eastern Volcanic Zone; SFZ, Snaefellsnes Fracture Zone.
nounced positive correlation exists between K2O, TiO2 and
P2O, whereas KO and Rb show insignificant covariation.
In central Iceland basalts with a high Rb content exist side
by side with basalts highly depleted in Rb. Away from the
Kverkfjöll area the basalts show gradual depletion of Rb
towards the ocean ridges together with increasingly nar-
rower spreads in K2O values. At a distance of 500-600 km
from central Iceland the K2O content of the basalts reaches
a value of 0.1%, with very little spread around that figure.
The samples from the NEZ have been discussed in detail
by Sigvaldason8. A model was proposed to explain the large
compositional spectrum in Icelandic basalts as compared
with the narrow range observed in the bulk of ocean ridge
basalts. The analyses presented here corroborate those
results. In the model it is assumed that the Icelandic
mantle plume feeds all volcanism in the North Atlantic.
The thermal gradient within the plume centre increases
continuously to great depths in the mantle, providing a
thermal environment for partial melting over a large depth
range. Conversely, as the plume overflow spreads below
the crust along the volcanic rift zones in Iceland and the
adjoining ridges, the thermal gradient goes through a tem-
perature maximum at which partial melting occurs. With
increasing distance from the plume centre, the thermal
maximum becomes better defined at a depth which happens
to be appropriate for the formation of ocean tholeiites.
As that represents relatively low-pressure partial melting,
0.6} KFA
Iceland
Reykjanes Ridge
K2O (weight ":)
NEZ- I WRZ
0.4
MIL |
0.2
-
0
100
200
300
400
500
600
700
Distance from K verkfjöll (km)
Fig. 2 Range of K2O values plotted against linear distance
from Kverfjöll, central Iceland. Each column represents the
range obtained in a 25 km sector. KFA, Kverfjöll area;
NEZ, northern part of Eastern Volcanic Zone; MIL; Mid
Iceland; WRZ, Western Rift Zone; (see Fig. 1).
all chemical types formed by partial melting at higher pres-
sure gradually disappear (Fig. 2). Thus, two processes are
in evidence: partial melting at varying depths, and pro-
gressive depletion of dispersed elements at the source.
The Icelandic tholeiites span almost the entire spectrum
of observed compositions of oceanic tholeiite. To illustrate
this is a plot of MgO against iron (Fig. 3) compares the Ice-
landic tholeiites to analyses of oceanic tholeiites9-11. In
spite of this great compositional span, however, the bulk
of the Icelandic analyses tends to fall on the iron-rich side
of the oceanic analyses. This is in keeping with the fact
that, as regards volume and frequency, Icelandic tholeiites
are distinguished by high alkalis and iron magnesium
ratios12 relative to oceanic basalts. The Juan de Fuca11
analyses resemble the Icelandic suite most (Fig. 3), which
is in accord with the idea that the Juan de Fuca ridge is
the site of a mantle plume2.
Within the Icelandic group the enrichment of iron and
potassium', together with other related chemical variations,
12
JDF -- >
10
MgO (weight ";)
-MAR 45 _
MAR
30
8
"%-AU-
EPG-
EPR
·- EPG
EPR
... Au-
Pac
6
MAR 30
6
8
10
12
14
16
Total Fe as FeO (weight ";)
Fig. 3 MgO plotted against total iron in the samples,
showing the relatively iron rich nature of Icelandic tho-
leiites. The range of values observed in oceanic tholeiites is
shown for comparison. Atl, Atlantic Ocean9; Pac, Pacific
Ocean®, EPR, East Pacific Rise11; EPG, Gorda ridge
(41º N)11; MAR 45°, Mid-Atlantic Ridge (45° N)11; MAR 30°,
Mid-Atlantic Ridge (30°N)11; JDF, Juan de Fuca Ridge
(44°N)11;
A, Kverkfjoll Area;
", North-eastern Zone;
., Western Rift Zone.
seems to bear an inverse relationship to lava volume8. This
effect could be attributed to fractional crystallisation11. The
Ti concentration, however, which has been shown to be
sensitive to pressure13, shows close positive covariation with
K, and could be explained equally well by a degree of
partial melting (compare with Fig. 2). The basaltic liquids
may have formed. over a relatively large depth range in
the mantle. Frequent eruptions of small batches of lavas
which have undergone only a low degree of partial melting,
and which have been derived from a considerable depth,
result in large total volumes of high alkali, high iron/
magnesium tholeiites, relative to low potassium tholeiites
which are produced at shallower depths by a higher degree
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WRZ .
Hek, .TTTTT
Eldeja
Reykjanes
TTr
Kalla
Laki Kar
EVZ
Mil
·K verkfjoll
.
.Pac:
JDF
.

581
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Nature Vol. 251 October 18 1974
-
$1.535
1.200
-
-
1.000
1
800 -
K/Rb
600
0
1
400
200 -
-0
0
0.01
0.02
0.05
0.1
0.2
0.5
1.0
K ("%)
.
Fig. 4 The K/Rb ratio plotted against log K. Lower part
of the field of oceanic tholeiites11,16 enclosed by dashed line.
a, Rb=2 p.p.m .; b, Rb=10 p:p.m .; c, K =0.5%; 0, NEZ
shield volcanoes and table mountains; O, WRZ shield
volcanoes and table mountains; , NEZ fissures and minor
eruptions; 0, WRZ fissures and minor eruptions; A, KFA
fissures and minor eruptions. The Rb values were obtained
by XRF analysis. Although this has a distinct disadvantage
at low Rb values this was overcome partially by adding a
fixed amount of 20 p.p.m. Rb to the samples. The precision
is considered to be better than ± 1 p.p.m.
of partial melting. The volume relationships between pro-
ducts of partial melting of high and low degree in Iceland
are, therefore, entirely different from those of the ocean
ridges. Along the Reykjanes ridge, in a direction away
from Iceland, the low degree partial melting products dis-
appear, giving an impression of an increase in the degree
of partial melting at the same time as volcanic productivity
decreases. This seems to explain adequately the apparent
decrease in light rare earth elements south of Iceland5.
Figure 4 shows the K/Rb ratio plotted against K for
some of the samples, with the field of ocean ridge basalts11,16
shown for comparison.
Several features of the diagram are worthy of discussion.
The K/Rb values are generally lower than previously ob-
served for tholeiites11. Analyses of plagioclase separates
from porphyritic basalts show K/Rb ratios in the range
50-300. There is no consistent relationship between the
K/Rb ratios of phenocrysts and groundmass. Furthermore,
REE analyses of Icelandic tholeiites14,15 do not show Eu
anomalies, suggesting that there has been no plagioclase
differentation. These low ratios, therefore, may be derived
from the mantle and could indicate a higher content of Rb
(phlogopite for example) in the plume than has been
assumed previously17.
The K/Rb-K field is defined by three curves (Fig. 4). its
low K side is bounded by the curve Rb=2 p.p.m., whereas
for the high K side, at K/Rb ratios below 400, the curve
Rb=10 applies, and for higher values the line K=0.5%
(Fig. 4). The values for the shield volcanoes tend to fall on
the diagram to the left of those of other type of volcanoes.
As a group the shield volcanoes show the lowest K and Rb
values in Iceland even if the K content may be higher, and
the Rb content is much higher, than in the depleted ocean
ridge basalts. The voluminous shield eruptions occurred
in the specific volcano-tectonic environment found during
the isostatic rebound of Iceland immediately after the
Pleistocene glaciation. The prevailing conditions of pres-
sure and temperature were suddenly disturbed towards
lower pressures, resulting in the melting of large batches
of mantle material that had already gone through an earlier
stage of melting. They are the most depleted basalts in
Iceland.
Schilling', chiefly on the basis of Sr isotopes6, has ad-
vocated two primary magma sources in the North Atlantic.
Magmas derived from the Icelandic plume have high
(~ 0.7032) 87Sr/88Sr ratios6,15,18, show an undepleted REE
pattern, and have generally high contents of K, Ti, P, and
high Fe/Mg ratios. The oceanic source yields lavas with a
much more depleted chemistry and lower (0.7026-0.7028)
Sr isotope ratios6. The gradient in composition, from
Iceland along the ridge, results, according to that theory,
from the mixing of the two magmas. The observed varia-
tion in Sr isotopes can possibly be accounted for in terms
of the single source model. At the depth at which magma
segregation is supposed to take place the upper mantle
consists of olivine, two pyroxenes, plagioclase and minor
phases, including phlogopite19 and amphibole20,21. The K
and Rb enrichment of the basalts in central Iceland results
from the important contribution of phlogopite to the early
partial melts of the plume. Phlogopite contains about 7%
K, 250 p.p.m. Rb, and 18 p.p.m. Sr (ref. 17). The other
components in the upper mantle contain between 1 p.p.m.
and 0.1 p.p.m. Rb. The high Rb content of the phlogopite
results in an increased 87Sr/86Sr ratio relative to the rest
of the mantle. Using the values of Rb and Sr already given
for phlogopite the 87Sr/$6Sr ratio would increase by 7x 10-5
every million years. Conversely, the highest Rb/Sr
ratios observed in Iceland tholeiites increase by 2×10-6
every million years, whereas the Sr isotopic ratio of the
depleted ocean tholeiites has very little capacity for change
with time and probably reflects that of the bulk of the
upper mantle.
The implication of possible isotope disequilibrium be-
tween mineral phases in the upper mantle may be far
reaching as regards the interpretation of isotopes in partial
melts. As long as a given mineral assemblage remains
stable, however, each mineral may develop its own isotope
ratio-which is the basis for the isochron method of radio-
metric dating22. Unless recrystallisation occurs there seems
little impetus for isotope re-equilibration between phlogo-
pite and the mantle phases with a low Sr content. In
general, because Sr is produced in phlogopite by radio-
active decay it should tend to migrate one way only -.
away from the phlogopite.
There is, as yet, insufficient documentation of the rela-
tionship between precisely measured Sr isotope ratios, and
the petrochemistry of Icelandic rocks. Preliminary work6,15
indicates, however, that, in general, rocks from the alkalic
provinces in Iceland possess slightly higher Sr isotope ratios
than the tholeiites. That is consistent with their supposed .
nature as lower degree partial melts19.
On the basis of the available data we therefore conclude
that there is no well established geochemical evidence to
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1
i
-

5L.
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Nature Vol. 251 October 18 1974
contradict the single-source model for the North Atlantic.
There is a continuous gradational variation between basalts
erupted in Iceland and basalts on the submerged part of
the Reykjanes and Mid-Atlantic Ridges. This compositional
gradient probably results from the combination of a chang-
ing environment of pressure and temperature in an up-
welling and radially spreading mantle plume system, and
from a progressive depletion in the low temperature melt-
ing fraction at the source.
Note added in proof: Unknown to us at the time of
writing, similar ideas on isotope disequilibrium in the
mantle have been published23.
Received December 31, 1973; revised May 2, 1974.
1
Morgan, W. J., Nature, 230, 42 (1971).
2
Morgan, W. J., Mem. geol. Soc. Am. (in the press).
3
Vogt, P. R., Earth planet. Sci. Lett., 13, 153 (1971).
4
Pálmason, G., Geophys. J. R. astr. Soc., 33, 451 (1973).
5
Schilling, J .- G., Nature, 242, 565 (1973).
6 Hart, S. R., Schilling, J .- G., and Powell, J. L., Nature phys.
Sci., 246, 104 (1973).
7
Jakobsson, S. P., Lithos, 5, 356 (1972).
8
Sigvaldason, G. E., J. Petrology (in the press).
9
Engel, A. E. J., Engel, C. G., and Havens, R. G., Bull. geol.
Soc. Am., 76, 719 (1965).
10
Aumento, F., Can. J. Earth Sci., 5, 1 (1968).
11
Kay, R., Hubbard, N. J., and Gast, P. W., J. geophys. Res.,
75, 1585 (1970).
12
Sigvaldason, G. E., Contr. Miner. Petrol., 18, 1 (1968).
13
MacGregor, I. D., Yb. Carnegie Inst. Wash., 64, 135 (1965).
14
Shimokawa, T., and Masuda, A., Contr. Miner. Petrol., 37, 39
(1972).
15
O'Nions, R. K., and Gronvold, K., Earth planet. Sci. Lett.,
19, 397 (1973).
16
Gast, P. W., Geochim. cosmochim. Acta, 32, 1057 (1968).
17
Griffin, W. L., and Murthy, V., Geochim. cosmochim. Acta,
33, 1389 (1969).
18
Moorbath, S., and Walker, G. P. L., Nature, 207, 837 (1965).
19
Green, D. H., and Ringwood, A. E., Contr. Miner. Petrol.,
15, 103 (1967).
20
Oxburgh, E. R., Geol. Mag., 101, 1 (1964).
21
Hill, R. E. T., and Boettcher, A. L., Science, 167, 980 (1970).
22
Nicolaysen, L. O., Ann. NY Acad. Sci., 91, 198 (1961).
23
O'Nions and Pankhurst, Earth planet. Sci. Lett., 21, 13 (1973).
Climatic significance of deuterium
abundance in growth rings of Picea
W. E. Schiegl
Institut für Radiohydrometrie der Gesellschaft für Strahlen- und Umweltforschung GmbH, Munich, Germany
Deuterium variations in growth rings of a Picea from southern
Germany are essentially a function of annual air temperature.
Short term isotope variations are partly influenced by
changes of relative humidity, but it is the long term annual
temperature fluctuations which are mainly responsible for
the deuterium variations in the rings. Long term deuterium
variations in Picea record climatic changes of the past
and also reflect the deuterium content of the annual
precipitation.
NATURAL, stable isotopes are used more and more frequently
in the field of palaeoclimatic research. Apart from 180 thermo-
- metry1,2 on sea cores, measurement of the deuterium content
of plants opens new aspects for palaeoclimatic investigations.
The basic idea of the deuterium method is that the natural
deuterium content of chemically bound hydrogen in plants is
correlated with that in precipitation3. As the deuterium content
of precipitation in middle and high latitudes depends mainly
on the climate and decreases with annual mean temperature4,
the deuterium content in plants is closely related to climate5,
and tree rings should therefore bear information on past
climates.
Sample preparation and mass spectrometric deuterium
determinations have already been described3. The overall
measuring accuracy is ± 2%, and the results are reported in
per mille deviation from Standard Mean Ocean Water
(SMOW)6.
During evapotranspiration of the leaves, complicated isotopic
interactions occur between atmospheric vapour and water which
is replenished from the soil. The hydrogen component of the
water is transformed by photochemical processes into woody
matter and so evapotranspiration is a process which is import-
ant. in the development of the deuterium content of plant
material.
Isotopic equilibria in these interactions can be described
mathematically ?:
81 = 85 (1-h)+h8,+&p+8k (1-h),
(1)
where 81 is the deuterium content of the leaf sap, 85 that of
soil water, and 8, that of atmospheric vapour. Ep is the equili-
brium separation factor which increases with decreasing
temperature, Ek is the kinetic separation factor, and h the
relative humidity. This formula describes the isotopic processes
during evapotranspiration in leaves to a good degree of
approximation8.
The average deuterium concentration in leaves from a Picea
was calculated for the period 1881-1970. For the calculations
I used meteorological data from the Hohenpeissenberg Observa-
tory (unpublished information and ref. 9), which is 16 km east
of Sachsenried, the place where the tree grew. Measurements
of the deuterium content of precipitation and ground water of
this area indicated that 8; ~ 70%%. Because atmospheric
vapour in Central Europe, contains on average, 75%% less
deuterium than does precipitation: 8 ~~ 85-75 (ref. 10).
Ep is temperature dependent and is +93%, at 15° C (ref. 11),
and Ex (= 16%%) is independent of temperature. The relative
humidity in the area around Sachsenried is about 75%%, and
Table 1 Correlation coefficients between the deuterium content of
Picea and meteorological and other parameters
1785-1910 1910-1970 1785-1970 1785-1970
5-yr means
10-20-yr
means
.
0.4
0.6
0.33
0.54
Summer temperature
0.035
0.52
0.04
0.41
Annual temperature
0.51
0.74
0.47
0.83
Humidity
0.47*
0.056*
0.34+
Leaf sap
0.57
0.43*
8 18 ice
0.61
* r20 = 0.49, t r2g = 0.7 (beginning in 1881).
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583
-25
ÖD(" 06)
1-30
-70
-75
Relative humidity
1-80
-65 +
-70
OD(%)
Picea
-75
4-80
-14
-1.3. 1
Summer temperature
("C)
-12
1.7
(ºC)
6
-5
-
1800
1850
1900
1950
ad
Fig. 1 Comparison bet-
ween summer . tempera-
ture
(mean
May-
September), annual mean
temperature, deuterium
content of Picea, relative
humidity (mean May-
September) and the
calculated
deuterium
content of the leaf sap all
in the 5-yr means. Dashed
lines indicate the trends
in the histograms.
so equation (1) shows that the influence of the atmospheric
vapour is more significant than the influence of the water which
the plants take up.
Implications of a deuterium content
From equation (1) it follows that the deuterium content should
depend essentially upon the temperature and relative humidity
during the growing season. In order to test this conclusion the
deuterium content of growth rings of a Picea from Sachsenried
(46° 50'N, 10° 50'E), with an age of about 200 yr was determined.
For the period 1910-1970 each ring was analysed twice for
deuterium to diminish any errors which may have arisen from
the analysis of an incomplete ring. Growth rings formed between
1785-1910 were analysed. In each case a block of rings formed.
in five consecutive years was analysed and care was taken to
burn the same amount of wood from each block, so that a
weighted mean was not obtained.
The deuterium content of each block has been compared with
the summer temperature (mean May-September), the annual
mean temperature, the relative humidity (mean May-Septem-
ber) (Fig. 1), and the calculated deuterium concentration in the
sap of leaves (for the same summer period) using equation (1).
In order to be able to compare the results for the various
possible combinations in a statistically meaningful manner the
correlation coefficients for the various periods with different
measuring accuracies have been calculated (Table 1). The
maximum error of the correlation coefficient for the 2o limit
is given as r20 in Table 1.
In. the 5-yr mean, there is a reasonable correlation between
the deuterium content and the summer temperature (r = 0.52;
r20 = 0.6) in contrast to the poor correlation with mean annual
values. In particular, the deuterium trend is distinctly indicated
by the trend of the temperature curve. A perfect correlation
cannot be expected in any case, because the meteorological
parameters are averaged continuously over five months-that
is not the case for the deuterium content of wood. On overcast
days there is hardly any assimilation because of insufficient
intensity of solar radiation. Furthermore, the humidity still
shows some fluctuations in the 5-yr mean, so some degree of
influence of the humidity on the deuterium content is evident.
On the long term mean the humidity variations level off and
the trend of the deuterium curve follows more perfectly that
of the temperature curve (Fig. 1).
The measuring accuracy is best for the period 1910-1970,
13
12-
Summer temperature
( C) .
7
6
Annual temperature
5
-65|
SD(",00)
Picca
-28-
BIS(",06) 29
lee core
-30-
1800
1850
1900
1950 ad
Fig. 2 Comparison be-
tween the 180 content of
an ice core from Camp
Century on Greenland
and
the deuterium
content of the growth
rings of a Picea from
Sachsenried. The upper
graphs show the summer
and the annual mean
temperatures from the
Hohenpeissenberg, Met-
eorological Observatory
near Sachsenried.
i
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-
Leaf sap (calculated)
(%)
Annual temperature
-70F

58 Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4
Nature Vol. 251 October 18 1974
because the average of 10 single measurements is used. In the
period 1785-1910 the accuracy is poorer because only two
single analyses are averaged (Table 1) and the time scale for
the older year rings becomes increasingly inaccurate because
some rings are missing.
The deuterium variation of the leaf sap is smaller than that
of the growth rings. Presumably, that is because the calculation
is based on a constant correlation between the deuterium
content of atmospheric vapour and the atmospheric temperature
(3%% ° C-1) for Central Europe. Such a relationship, however,
is an idealisation of the natural conditions, because at any one
temperature, depending on the meteorological conditions,
substantial variations in the deuterium content of atmospheric
vapour can still occur.
The growing period comprises hardly half a year, and so it
must be considered whether the climatic processes during that
period really reflect, to any degree, the general climatic trend
of the entire year. A comparison between summer and annual
temperatures shows that similar trends do, in fact, occur. The
low temperatures of the 19th century, which caused a consider-
able advance of the mountain glaciers, in temperature latitudes,
are indicated clearly. In addition to that, a comparison of the
correlation coefficients for summer and annual temperatures
(Table 1-5-yr means) shows that the annual temperature
correlates much better with the deuterium content of the
growth rings (r = 0.47; r20 = 0.33) than does the summer
temperature (r = 0.04; r20 = 0.33). That observation is
further supported by the comparison in Fig. 2. In the lower
graphs the deuterium content of Picea is compared with the
180 result from an ice core from Camp Century on the Green-
land ice sheet12. There is a good relationship, although the ice
reflects the isotopic composition of the annual precipitation
(r = 0.61; r20 = 0.54). On the long term mean the relationship
between the deuterium content again seems better with the
annual termperature (r = 0.83; r20 = 0.54) than with the
summer temperature (r = 0.41; r20 = 0.54). I therefore con-
clude that the deuterium concentration in the atmospheric
vapour is a function of the climatic history of the whole year
and influences the deuterium content of the leaf sap so that, even
though only a relatively short growth period is measured, the
results reflect throughout a whole year. That is important for
all palaeoclimatic investigations which use deuterium variations
in biogenic material. Finally, as two localities thousands of
kilometres apart show similar isotopic trends, it follows that
essentially global climatic changes must be responsible for the
deuterium variations in Picea.
I thank Oberforstmeister Porsch from Sachsenried, Dr. A.
Aniol (Meteorological Observatory Hohenpeissenberg) and
Professor H. Moser of the Institut für Radiohydrometrie in
München.
Received February, 21; revised May 30, 1974.
1'Urey, H. C., Epstein, S.L., Mckinney, Cl, and McCrea, I., Bull.
geol. Soc. Am., 62, 1359 (1948).
2 Emiliani, C., J. Geol., 63, 538 (1955).
3 Schiegl, W. E., and Vogel, J. C., Earth planet. Sci. Lett., 7, 307
(1970).
4 Zimmermann, U., thesis, Univ. Heidelberg (1969).
5 Schiegl, W. E., Science, 175, 512 (1972).
6 Craig, H., Science, 133, 1833 (1961).
7 Craig, H., Proc. Spoleto Conf., Pisa (1965).
8 Schiegl, W. E., thesis, Univ. Pretoria (1970).
9 Berichte des Deutschen Wetterdienstes No. 36, 5 (Bad Kissingen,
1957).
10 Knott, K., thesis, Univ. Heidelberg (1964).
11 Rennow, M., Staatsexamensarbeit Heidelberg (1970).
12 Johnsen, S. J., Dansgaard, W., Clausen, H. B., and Langway,
C. C., Nature, 227, 482 (1970).
Properties of hybrids between
Salmonella phage P22 and coliphage 2
David Botstein
Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
Ira Herskowitz
Department of Biology, University of Oregon, Eugene, Oregon 97403
The temperate bacteriophages P22 of Salmonella
typhimurium and ^ of Escherichia coli form viable
hybrids in which the immunity, early control and DNA
replication genes of P22 are substituted for the analogous
À genes. The specificity of the early control and DNA
replication genes differs between P22 and »; the im-
munity of the hybrid is identical with that of the
lambdoid coliphage 21. Implications for the evolution
of viruses are discussed.
THE temperate bacteriophages P22 of Salmonella typhi-
murium and » of Escherichia coli are very similar in
functional organisation1,2. In particular, the regions con-
cerned with control, recombination, integration, DNA
replication and lysis functions of both phages are arranged
in the same order on their genetic maps (Fig. 1). The phages
share DNA sequence homology, as measured by DNA-DNA
hybridisation3, most of which occurs in the right arm of A
DNA1, where the functions listed above are located. These
similarities are counterbalanced by profound differences
between the two phages. (1) P22 DNA is circularly per-
muted and terminally repetitious, whereas A DNA is not
permuted and has single-stranded cohesive ends3,6; (2) The
P22 genetic map is circular, whereas the map of A is
linear7,8; (3) P22 particles have only a short baseplate struc-
ture whereas À has an elongated tail; (4) P22 adsorbs only to
Salmonella typhimurium whereas À adsorbs only to E. coli;
(5) Control of lysogeny differs : P22 has two regions essential
for immunity and repression whereas » has but one11,12; (6)
P22 lysogens exclude superinfecting phages in several ways
beyond the immunity and repression system whereas À lyso-
gens display solely the immunity provided by the single
repressor13,14.
These considerations led us to inquire whether hybrid
phages containing DNA from both P22 and À could be
constructed, and, if so, which blocks of P22 genes were
interchangeable with analogous blocks from A. We show
here that P22 DNA can be substituted in the right arm of
A to yield hybrid phages with distinctive properties. The
construction of P22/X hybrid phages was accomplished in-
dependently by a different method by Gemski et al.15. Studies
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Nature Vol. 251 October 18 19/4
-
heads
1
8
2
5
A -F
10
3
26
20
R
- 19
r
16 -
S
lysis
A
Q
G - J
`23
P
control
mnt
imm 434
0
12
control
and
9
att
immunity
18
CI
int
red CM N
CI
DNA
synthesis
int
erf
C3 24 C2
.
integration
recombination
[imm C]
Fig. 1 Comparison of the genetic maps of P22 and 2. Inside
the map are the A gene designations; outside are the P22
gene designations .. One-to-one correspondence means great
similiarity in mutant phenotype. The heavy bars outside in-
dicate imml and immC, the heavy bars inside indicate the
extent of non-homology between X and Nimm21 and Nimm-
434. The map is redrawn from Botstein et al.1.
with these hybrid phages indicate that P22 and the E. coli
lambdoid phage 21 (ref. 16) have repressors and operators
with apparently identical specificity, indicating common
ancestry; on the other hand, the DNA replication and early
gene control systems of. P22 and the lambdoid phages are
analogous but have significantly different properties.
The cross
Phage P22 does not adsorb to E. coli and phage A does
not adsorb to S. typhimurium. To get both phage DNAs into
the same cytoplasm so that they might recombine, we took
advantage of the ability of E. coli episomes to be transferred
between the two species of bacteria. Roth and Hoppe17
showed that the F'lac+pro+ episome of E. coli carries a
prophage attachment site for phage P22. Consequently, P22
was inserted into this episome by infection of an F'lac+ pro+
Salmonella strain which was deleted for the P22 prophage
attachment site on the chromosome. The P22 carried on this
episome has a temperature-sensitive repressor (c2 gene
product); hence, the prophage is induced at high temper-
ature in Salmonella strains. The F'lac+pro+str$ (P22c2-ts)
Salmonella strain was mated with an E. coli lac str" strain,
and E. coli lac+str" recombinants selected .. Most of these
recombinants were temperature-sensitive for growth and
produced 20-50 P22 phage per cell at high temperature.
Therefore, it appears that all host components required for
P22 growth are present in wild type E. coli. In contrast, it
was found that A+ will not grow under similar circumstances
in S. typhimurium1.
The hybrid phages described here are derived from an
E. coli lysogen-carrying P22/2ts30 prophage; when this
lysogenic strain was infected with a marked A (X_c160) and
the culture was shifted to 41º C, a small yield -of ) and P22
(about 20 of each) was produced. Among the progeny were
recombinants (about 0.01%) with the host range of A
(that is, adsorption to E. coli), but which had acquired a
new immunity (that is, growth in E. coli lysogenic for A).
In a parallel infection, nonlysogenic but otherwise identical
F'lac+ pro+ E. coli strain was infected with > cI60 at
41° C; no phages able to grow on E. coli lysogenic for X
were obtained.
Structure of NimmP22
(a) The immunity region derives from P22. Since the
hybrid phages were selected for their ability to grow on a
À lysogen of E. coli, it was expected that the hybrids had a
new immunity, presumably from the P22 prophage. This
idea was confirmed by several observations. (1) The hybrid
.phages all have a temperature-sensitive clear plaque
morphology, unlike that of the A parent, but like that of
the P22 prophage. As expected if the ts-clear mutation
affects the P22 c2 gene, E. coli lysogens carrying these
hybrids as prophages are induced when shifted to 41º C. (2)
A phages grow on the E. coli strain lysogenic for P22. The
hybrid phages do not grow on these lysogens, indicating that
they have immunity of P22. The hybrid phages grow
normally on the F'lac+pro+ E. coli strain which does not
carry a P22 prophage (Table 3) (3) In other experiments,
hybrids were constructed between 1 and P22 prophages
which carry amber or ts mutations in the genes near the c2
gene; in these cases, the hybrid phages inherit the amber
or ts phenotype (S. Hilliker, unpublished work).
These bybrid phages-obtained from the cross of AcI60
with P22c2ts30 are called NimmP22 by analogy with
Nimm434 and Nimm21, A hybrids with the immunity specifi-
city of the lambdoid phages 434 and 21 (refs 19 and 20).
(b) Tail genes derive from A. Since the hybrid phages can
grow on E. coli strains, the genes determining host range
must be inherited from the A parent. As anticipated, the
hybrid phages are as sensitive as A wild type to antibodies
raised against A wild type phage particles. Furthermore,
mutants of E. coli lacking the A receptors for adsorbtion
are also resistant to the NimmP22 hybrids (data not shown).
NimmP22 hybrids, therefore, have tail genes from A.
(c) Prophage insertion specificity derives from A. The
hybrid phages can integrate into an F'gal att) episome and
be tranferred into another strain by conjugation. NimmP22
hybrids, therefore, have the insertion specificity (attach-
ment site and int gene) of À (S. Hilliker, unpublished work).
(d) The source of other genes in the hybrids is as follows.
Marker rescue tests were done by plating » amber mutants
on su (nonpermissive) lysogens of NimmP22 hybrids. For
comparison, phage were also plated on lysogens of Nimm434
(known to substitute none of the essential A genes tested)
and Nimm21 (known to substitute only gene N)21. Recom-
binants with immunity of A and wild type gene function
Table 1 Efficiency of plating (marker rescue experiment)
No prophage
Nimm434
Lysogen
Nimm21
++
++
NimmP22
2.Bam1
++
2Jam6
++
2Nam7
2× 10-7
1×10-5
3× 10-7
4× 10-7
2Nam53
1×10-6
2× 10-4
1× 10-6
1×10-6
10am29
1× 10-6
5×10-3
1×10-3
2× 10-6
2Pam3
3×10-8
4×10-1
1×10-2
1×10-7
2.Pam80
5× 10-7
2× 10-1
7×10-3
7×10-7
2 Qam73Sami7
<10-8
3×10-2
2× 10-2
8×10-3
2.Sam7
++
++
++
Ram5
++
++
Lysogens were constructed in strain 594 which is strongly nonper-
missive for amber mutants. Phage were plated either for efficiency
(numbers) or as a serial-dilution spot test. Plates were incubated at
:30° C since: NimmP22 carries a cats allele. Nimm21 and Nimm434
were described by Herskowitz and Signer21.
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ant
imm21 com
[imm Il
tails
attP22
1
control and immunity
++
++
+13
sieg

580
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Nature Vol. 251 October 18 1974
Table 2 Lefthand endpoint of nonhomology region
cIII
N
cI
*
imm434
imm21
immP22
bio243
bio10
.
bio252
Frequency of recombinants
immP22
Nimm21
Nimm434
Abio243
<2×10-7
<2x10-5
2 x10-2
Abio10
2×10-3
4 x10-4
Abio252
1.x10-2
6x10-3
Crosses were done as standard ) crosses without ultraviolet-
irradiation as described by Signer and Weil33. Recombinants were
scored by their ability to grow on E. coli lysogenic both for P2 (which
selects bio) and 2 (which selects against immÃ). None of the parental
phages grown in parallel produced progeny capable of growth on the
test dilysogen at a frequency in excess of 2X10-5. In the diagram,
solid line indicates 2 DNA replaced.
1
were scored. Table 1 shows that functional alleles of muta-
tions in A genes Q, S, R, B and J can be rescued, whereas
the functional alleles of mutations in A genes N, O and P
cannot.
The appearance of nondefective immà recombinants
in the cases of Q, S, R, B and J mutants indicates that the
NimmP22 prophage contains either (1) the wild type allele
from its A parent, or (2) an analogous and functionally
compatible allele from the P22 parent which is separable
from immP22.
Failure to find functional imm» recombinants in the cases
of N, O and P mutants indicates that the hybrid phage
does not carry these genes from the A parent, and that the
analogous P22 genes in the hybrid phage cannot be
separated from the P22 immunity region of the hybrid
phage.
These results suggest that in NimmP22 the immunity
region of A has been replaced by an analogous region from
phage P22 (Fig. 1). The region of nonhomology (or non-
compatible function) between NimmP22 and A in the hybrid
apparently includes genes N to P on the A genetic map.
The analogous region in P22 (immC and its flanking genes)
has been found to contain genes with functions analogous
to those of X (gene control and DNA replication in addition
to immunity specificity)1,22,23
We have performed additional crosses to determine in
detail the lefthand endpoint of the nonhomology between
phages AimmP22 and λ. Abio10immP22 but not Abio243-
immP22 recombinants can be formed, indicating that the
region of nonhomology extends to between the endpoints
of these deletions. Table 2 shows that the lefthand endpoint
of nonhomology of phage Nimm21 falls within same region.
Since the imm21 substitution replaces the \ N gene, these
crosses provide independent evidence that the immP22
substitution also replaces À gene N.
Functional comparisons of 2/P22 hybrids
(a) NimmP22 has the same immunity specificity as Nimm21.
NimmP22 hybrids establish and maintain lysogeny
normally; less than 1% of the cells in a lysogenic culture
are cured. These lysogens are immune to superinfecting
phage of the same genotype. We tested various lambdoid
phages for the ability to grow in NimmP22 lysogens. We
also tested the ability of NimmP22 to grow in lysogens of
these lambdoid phages. As Table 3 shows, NimmP22 grows
normally in all lysogens except those carrying prophages
with the immunity of phage 21 or P22. Furthermore,
NimmP22 lysogens are immune to those phages carrying
the immunity region of phage 21 or P22. This result implies
that the immC repressor and operators of phage P22 are
functionally equivalent to the repressor and operators of
phage 21 and different from those of all other lambdoid
phages tested. It should be emphasised that the immC
region of P22 is sufficient to control the hybrid phage
NimmP22, even though two immunity regions (imml and
immC) control .wild type P22. This result means that immC
is probably the primary regulator of repression and im-
munity in P22.
(b) We have obtained >/P22 hybrids with substitutions of
regions other than immunity. To construct hybrids different
from NimmP22, the F'lac+pro+(P22) strain was infected
with various defective À mutants. For example, this strain
was infected with A carrying amber mutations in both
genes Q and S. amber+ recombinants with the host range of
A were selected by plating the progeny phage on su nonlyso-
genic E. coli; recombinants were obtained at frequencies be-
tween 10-3 and 10 .- 4. These recombinants have less DNA than
the Q"S" parent, as judged from their ability to grow in the
presence of chelating agents25. Most of these phages were
able to grow in NimmP22 or Nimm21 but not in A lysogens,
indicating that they had retained the immunity specificity
of the A parent. If these hybrid phages were formed by the
minimal recombination event (a double crossover), they
should carry the P22 region analogous to the Q-S region
of X. Since both phages in this region confer the specificity
of late gene expression1,25, these hybrids should contain
the head and tail genes of X and the late gene control
region from P22. Preliminary studies (P. Toothman and
Table 3 Immunity of 2immP22
F'lac (P22)
F'lac
No prophage
AimmP22
Nimm21 (b5)
Nimm434
2+
AimmP22 (3)
0
Aimm21cI
0
Nimm21cIIh80
0
Nimm21cIbio252
0
+
0
Aimm21 (bs)p4
0
+
0
0
Aimm434
$80
82
381
424
does not adsorb
100000. +++++
0
0
++++++ +++
0
+0+++++ ++++
1
+++++
+++++++ +++++
1
1
0
0
0
0
0
++++
These experiments represent serial-dilution spot tests. + indicate an efficiency of plating of near 1 and 0 represents no plaques seen, to a
sensitivity of less than 1 in 105. The F'lac strains are the same as those used to isolate NimmP22; the other lysogens are in strain 594. The Nimm21
phages are derived from 21hycl (ref. 20) or from 2 b5, where indicated
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Nature vol. 201 October 10 1974
587
a
Fig. 2 Electron micrographs of DNA heteroduplexes pre-
pared by the formamide method34 from purified phage
DNA. The ba deletion serves as, a position and' informal
calibration marker. a, Ab2/NimmP22 heteroduplex molecule;
b, }/Nimm21b2 heteroduplex molecule; c, Nimm21 b2/
NimmP22 heteroduplex molecule.
S. Hilliker, unpublished work) indicate that the A late gene
positive regulator (Q gene product) can turn on late genes
from such prophages. The Q genes of A and P22 may, there-
fore, have the same specificity.
Physical mapping of 1immP22
The genetic tests presented above indicate that the
NimmP22 chromosome is essentially a X chromosome in
which the region from the left of gene N to some point
beyond gene P of À has been substituted by DNA encod-
ing the analogous functions of phage P22. In addition,
NimmP22 and phage 21 have the same immunity specificity,
suggesting that 21 and P22 share sequence homology in
their immunity regions. To test these views directly, we
constructed DNA heteroduplexes between all combinations
of wild type >, Nimm21 and NimmP22. Each pair of DNAs'
differed also by a deletion (b2, in the central region of X
DNA) in order to orient the heteroduplex molecules and
to provide an internal calibration for measurements. As
Figs 2 and 3 show, the heteroduplexes confirm the genetic
results; clearly NimmP22 has a DNA substitution in the
right arm of ^; A DNA sequences are replaced by non-
homologous sequences from P22.
The regions of nonhomology between the three pairs
of phages (λ/Ximm21, λ/immP22 and himm21/dimmP22)
all begin at about the same point at the left (14.1-14.6
units from the b2 deletion). The imm21 and immP22 sub- .
stitutions therefore have approximately the same lefthand
endpoint of nonhomology with respect to A DNA. Since
the imm21 substitution replaces the N gene of , the
immP22 substitution must likewise remove the N gene of
A. These conclusions confirm the results of recombinational
studies shown above (Table 2).
The region of nonhomology between A and Nimm21 is
known to end to the left of A genes O and P (ref. 26). The
righthand endpoint of the major region of nonhomology
between AimmP22 and either X or himm21 extends well
beyond the point of nonhomology between 1 and Nimm21
(Figs 2 and 3). Comparison with the physical map of David-
son and Szybalski26 indicates that NimmP22 must have DNA
nonhomologous with A extending to the right of A gene P.
Therefore, as suggested by earlier genetic results, NimmP22
has a substitution covering the À DNA replication genes
and origin of replication. We show below that NimmP22
behaves as if it has a P gene different from that of A:
Three small regions of nonhomology between NimmP22
and either A or Nimm21 are found at the far right of the DNA
heteroduplexes. The last of these is about 10.0 2 units from
the right end of the DNA. If NimmP22 were formed by only
two crossovers between > and P22 DNA, then the cross-
over on the right must have occurred somewhere to the
right of this point. If this be the case, the existence of
homology to the left of this point (between the small
bubbles) suggests that there is natural homology between
P22 and A DNA in this region. Alternatively, NimmP22
may have been formed by multiple crossovers between A
and P22 DNAs, such that only the three small nonhomo-
logous regions are from P22. We favour the view that
NimmP22 resulted from two crossovers, since DNA-DNA
hybridisation measurements4 indicate that ) and P22 DNAs
share 18% homology in the right arm.
Whereas the heteroduplex between ) and NimmP22 shows
a large region of nonhomology beginning at A gene N, the
heteroduplex between NimmP22 and Nimm21 shows only a
short region of nonhomology. To the right of this region
is an equally short region of homology followed by another
large region of nonhomology. The existence of a homo-
logous region implies that the cI region of Nimm21 and the
. c2 region of NimmP22 are not only functionally similar,
but are also coded by homologous sequences in the DNA.
In support of this placement of the homology to the re-
pressor genes of the two phages, recombinants have been
obtained with crossovers between the operators (S. Hilliker,.
personal communication). The repressor gene sequence
homology suggests that the repressors of phages P22 and .21
have a common ancestry.
Control and replication genes
Physical and genetic mapping indicates that the immP22
substitution replaces A genes N, O and P with analogous
genes (24, 18 and 12) of P22 (Fig. 1.). This phage can, there-
fore, be used to study the behaviour of these P22 genes in
an E. coli host. We have found that NimmP22 hybrids grow
normally in certain E. coli mutants (groP-, groN-, and
nus ) which fail to support ) growth (Table 4).
GroP- mutants of E. coli have alterations of a host repli-
cation function (dnaB) which apparently must interact with
the X P gene product for successful > replication27. Since
NimmP22 grows in these mutants, the P22 gene product
analogous to P gene product either interacts with dnaB
in a different manner or perhaps not at all. It should be
noted that groPA and groPE mutations, known to lie in the
scale (% X )
0
10
20
60
70
80
90
100
À gene locations
A- J
b2
N cicHOP
Q SR
. heteroduplexes
48.4
146
9.7
103 +
x b2 / > imm P22
10.5 -
/x imm 21b2
46.1
1020
22
47.8
14.5
2.
-48.
10.0 -+
1 imm 21by/ximmP22
Os
+36
Fig. 3 Summary of measurements of DNA heteroduplexes.
All the numbers are averages of at least eight molecules
and are normalised to the total length of A DNA. The
locations of the A genes are taken from Davidson and
Szybalski26.
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8.5
7.6
21.7
3.5
10.7-

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Table 4 Efficiency of plating on bacterial mutants unable to support
growth of 2
Host
2immP22
Nimm21
gro+
(1)
(1)
(1)
groP A15 (dnaB)
2×10-6
1.1
5×10-6
groP B560 (dnaB)
2×10-6
0.7
3×10-6
groP AB756
2×10-6
0.07
3×10-5
groN 785
2×10-6
1.1
0.9
nus+ (42° C)
(1)
(1)
(1)
nus (42° C)
2×10-6
1.0
1.0
nus (30° ℃)
1.0
0.7
0.7
The groP and groN mutants are described in refs 27 and 29.
nus strains are described by Friedman 30. Figures in parentheses
indicate normalisation to this value. All strains are nearly isogenic
with either gro+ or nust. Two independent NimmP22 hybrids were
tested.
dnaB gene28, do not affect growth of NimmP.22, whereas
the groPAB mutation, which is known to lie outside dnaB.
(I.H., unpublished result), might have some effect on growth
of the hybrid phages.
E. coli mutations groN- or nus-29,30 block early & tran-
scription, probably by affecting the function of the early
gene positive regulator, N protein. Nimm21 is able to grow
in these bacteria (Table 4); NimmP22, likewise, grows
normally in groN" and nus mutants under conditions
completely restrictive for A growth, indicating that the early
genes are regulated differently from 2.
From the behaviour in these E. coli mutants, it is clear
that the early gene control and DNA replication genes
of P22 have specificities distinctly different from As when
assayed in the same genetic background.
Implications for virus evolution
The formation of viable hybrids between coliphage A
and the Salmonella phage P22 shows that the similarity
between these two phages in the arrangement of functions
on their genetic maps is the result of a substantial degree
of common evolutionary ancestry. We imagine that the
hybrid phages were formed by phage-specified or bacterial
recombination rather than "illegitimate recombination"31
because of the relatively high frequency with which they
are formed. Not only are viable hybrids formed, but the
immunity specificity of P22 and phage 21 are identical,
both in function and, to the resolution afforded by hetero-
duplex mapping, in nucleotide sequence. Similarly, the
Q gene of À and the analogous gene (gene 23) of P22
seem to be functionally identical.
,
A second striking fact about these hybrids is the extent
to which nonhomologous genes controlling DNA replica-
tion, regulation and immunity of phage P22 remain fully
functional when inserted into the chromosome of phage A.
Also notable is the retention of regions of homology flank-
ing nonhomologous segments of DNA which contain non-
identical genes such as those controlling DNA replication.
The conclusion which emerges is that evolution of the
lambdoid phages (which must now include P22) appears to
have proceeded within regions of function which have
been selected for the ability to form viable combinations
with functional segments from other members of the
lambdoid phage group. Since P.22 and À have different
bacterial hosts, selection must have maintained this func-
tional and recombinational compatibility for at least the
time in evolution since E. coli and S. typhimurium diverged.
It should be noted that Salmonella and E. coli can exchange
genetic material and have similar genetic organisation35.
Perhaps the enteric bacteria have themselves evolved by
a mechanism similar to that suggested for bacteriophages.
Our view of bacteriophage evolution depends on the
clustering of related functions on the genetic map31-34. The
idea that such clustering might be selected through hybrid
formation has been advanced as well. But, the idea that
the lambdoid phage "races were derived from still other
races by re-assortment of unlike segments" the conco-
mitant idea that "presumably both the diversity [of the
functional segments] and the capacity to form hybrid re-
combinants have served a joint evolutionary purpose" was
first clearly formulated by Hershey32. This idea must now
be taken to extend across the species lines of the enteric
bacteria.
Heteroduplex studies of the DNA of the lambdoid
phages36,37
and of the lytic T series of coliphages38,39
support this view of bacteriophage evolution. One can see
in the heteroduplexes the whole range of evolutionary
divergence: at some points, entire regions of function are
nonhomologous and at other points the region of non-
homology may involve only a single gene or even a part
of a gene". Although the principle of exchange of func-
tional regions might apply to all bacteriophages, not all
phages seem to be in the same group. For example, the
temperate coliphages P2 and 186 form viable hybrids with
each other (unpublished results of B Egan, H. Young-
husband and R. B. Inman) but not, thus far, with the
lambdoid phages; similarly, the lytic coliphages T3 and T7
appear to belong to a group separate from that containing
phages T2, T4 and T6.
In conclusion, we suggest that evolution proceeds by
diversification within segments encoding particular func-
tions: each segment retains functional compatibility and
recombinational homology so that reassortment among
them is easy and frequent. We imagine that this idea might
apply quite generally to bacteriophages, and, indeed, to all
viruses, including those of mammals. One might expect
therefore that functional hybrids among viruses of quite
divergent host species might frequently- be encountered.
We thank Sandra Hilliker and Penny Toothman for
allowing us to use unpublished results, Bik-Kwoon Tye for
initiating work on the heteroduplex, David Friedman and
Bill Dove for phage and bacterial strains and Van Jarvik
for technical assistance. D. B. holds a career development
award from the National Institutes of Health. This work
was supported in part by grants from the National Insti-
tutes of Health. D. B. is also partially supported by a grant
from the American Cancer Society.
Received May 9; revised June 24, 1974.
1 Botstein, D., Chan, R. K., and Waddell, C. H., Virology, 49,
268-282 (1972).
Dove, W. F., in The bacteriophage lambda (edit. by Hershey,
2
A. D.). 297-312 (Cold Spring Harbor Laboratory, New
York, 1971).
3
Cowie. D. B., and Szafranski, P., Biophys. J., 7, 567-584
(1967).
4
Skalka, A., and Hanson. P., J. Virol., 9, 583-593 (1972).
5
Rhoades, M., MacHattie. L. A .. and Thomas, C. A., jun.,
J. molec. Biol., 37, 21-40 (1968).
6 Hershey. A. D., Burgi, E., and Ingraham, L., Biophys. J., 2,
423-431 (1962).
7
Gough, M., and Levine, M., Genetics, 58, 161-169 (1968).
8
Campbell, A., in The bacteriophage lambda (edit. by Hershey,
A. D.), 13-44 (Cold Spring Harbor Laboratory, New York,
1971).
9
Anderson, T. F., Proc. Eur. Reg. Conf. Electron Microsc., 2,
1008-1011 (Delft, 1960).
10
Kellenberger, E .. and Edgar, R. S., in The bacteriophage
lambda (edit. by Hershey. A. D.). 271-295 (Cold Spring
Harbor Laboratory, New York, 1971).
11
Chan, R. K., and Botstein, D., Virology, 49, 257-268 (1972).
12
Hopkins. N .. and Ptashne. M., in The bacteriophage lambda
(edit. by Hershey. A. D.), 571-574 (Cold Spring Harbor
Laboratory, New York, 1971).
13
Rao, R. N .. J. molec. Biol., 35, 607-622 (1968).
14 Susskind. M. M., Wright, A., and Botstein, D., Virology, 45,
638-652 (1971).
15
Gemski, jun., P., Baron, L. S., and Yamamoto, N., Proc.
natn. Acad. Sci. U.S.A., 69, 3110-3114 (1972).
16
Jacob, F., and Wollman, E. L., Sexuality and the genetics of
bacteria (Academic Press, New York, 1961).
17
Roth, J. R., and Hoppe, I., Genetics, 71, 553 (1972).
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Nature Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4 589
18 Baron, L. S., Ryman, I. R., Johnson, E. M., and Gemski,
jun., P., J. Bact., 110, 1022-1031 (1972).
19
Kaiser, A. D., and Jacob, F., Virology, 4, 509-521 . (1957).
20 Liedke-Kulke, M., and Kaiser, A. D., Virology, 32, 475-481
(1967).
21 Herskowitz, I., and Signer, E. R., Cold Spring Harbor Symp.
quant. Biol., 35, 355-368 (1970).
22
Levine, M :; Curr. top. Microbiol. Immun., 58, 135-156 (1972).
23
Bronson, M. J., and Levine, M., J. Virol., 7, 559-568 (1971).
24
Parkinson, J. S., and Huskey, R. J., J. molec. Biol., 56, 369-
384 (1971).
25
Herskowitz, I., and Signer, E. R., J. molec. Biol., 47, 545-556
(1970).
26
Davidson, N., and Szybalski, W., in The bacteriophage
lambda (edit. by Hershey, A.D. 45-82), (Cold Spring Har-
bor Laboratory, New York, 1971).
27
Georgopoulos, C. P., and Herskowitz, I., in The bacterio-
phage lambda (edit. by Hershey, A. D.), 553-564 (Cold
Spring Harbor Laboratory, New York, 1971).
28
Herskowitz, I., thesis, Massachusetts Institute of Technology
(1971).
29
Georgopoulos, C. P., in The bacteriophage lambda (edit, by
Hershey, A. D.), 639-646 (Cold Spring Harbor Laboratory,
New York, 1971).
30
Friedman, D., in The bacteriophage lambda (edit. by Hershey,
A. D.), 733-738 (Cold Spring Harbor Laboratory, New
York, 1971).
31
Franklin, N., in The bacteriophage lambda (edit. by Hershey;
A. D.), 621-638 (Cold Spring Harbor Laboratory, New
York, 1971).
32
Hershey, A. D., in Carnegie Institution of . Washington Year
Book 1970, 3-7 (1971).
34
33
Signer, E. R., and Weil, J., J. molec. Biol .; 34, 261-271 (1968).
Davis, R. W., Simon, M., and Davidson, N., in Methods in
enzymology (edit. by Grossman, L. and Moldave, K.), 21,
part D, 413-418 (1971):
.
35
Sanderson, K. E., Bact. Rev., 36, 558-586.
36
Simon, M. N., Davis, R. W., and Davidson, N., in The
bacteriophage lambda (edit. by Hershey, A. D.), 313-328
(Cold Spring Harbor Laboratory, New York, 1971).
37 Fiandt, M., Hradecna, Z., Lozeron, H., and Szybalski, W.,
in The bacteriophage. lambda (edit. by Hershey, A. D.),
329-354 (Cold Spring Harbor Laboratory, New York, 1971).
38
Hyman, R. W., Brunovskis, I., and Summers, W. C., Virology,
57, 189-206 (1974).
39
Kim, J. S., and Davidson, N., Virology, 57, 93-111 (1974).
40
Beckendorf, S. K., Kim, J. S., and Lielausis, I., J. molec.
Biol., 73, 17-35 (1973).
LETTERS TO NATURE
Distance to Cygnus X-1
THERE has been much discussion concerning the X-ray
object
Cygnus
X-1 and its optical counterpart,
HDE226868, a highly reddened star of type 09.7 Iab (ref.
1). The O star is a single-lined spectroscopic binary, and
various assignments of the minimum mass of the sec-
ondary, taken to be the X-ray object, suggest that the
latter is a black hole. These conclusions are dependent
on the mass of the O star being ~ 20-30 MO, as would
be the case if the star were 'normal' for its luminosity
class. But the possibility of heating of the primary by the
nearby X-ray object, and the spectroscopic indications of
mass loss2, indicate that such an assumption is unsafe. In
order to determine the mass of HDE226868, a more
complete spectroscopic analysis, in which the effective
temperature and surface gravity are deduced, and a deter-
mination of luminosity (from the distance to the object)
are necessary. The distance to the object is crucial to the
problem, because once the effective temperature and
gravity of the O star are known, the mass will vary as the
square of the distance. An error in the distance by a factor
of about two means that the model for the X-ray source
need not include a black hole.
· Accurate estimates of the distance to HDE226868 have
recently been claimed by Margon et al.3 and Bregman
et al.4 who give. 2.5 kpc. Such a large distance favours the
black hole model. Both works used the reddening pro-
duced by interstellar dust as a distance indicator. A con-
sistent distance-reddening relationship has been assumed
for this region of the sky. The evidence for this needs to
be examined closely.
Interstellar reddening is particularly irregular in the
Cygnus region. The absorbing clouds of dust collectively
form the Cygnus Rift, in which there are many highly
reddened
OB
stars. Bregman et al. showed that
HDE226868 is highly reddened compared to the objects-
mostly F stars-in their small field of view. Apart from
the fact that HDE226868 is located extremely near, or
actually within, the Great Rift, it is clear that it has
photometric properties very similar to reddened O stars
in this same region surveyed by Johnson and Morgan5.
This, of course, does not necessarily imply that the object
is a normal O star close to the main sequence. Plotting
the colours of HDE226868 measured by Bregman et al.
(B-V=+0.81, U-B =- 0.30) on a reproduction of
Johnson and Morgan's two-colour diagram (Fig. 1), shows
that it is not anomalous: . there are many O stars in this
area which are more highly reddened.
The relationship between total extinction, Av (com-
monly taken to be 3E(B-V)), and distance for fields in
the vicinity of the Milky Way has been investigated by
Neckel6. Neckel was unable to deduce any relationship
between A, and distance within a region centred on !I=
73°; b"I == 0°, which is extremely close to HDE226868. That
survey is, however, considered by Margon et al. to be
inapplicable because Neckel's fields were wide enough to
include regions where large changes of absorption could
occur over small angular scales. Margon et al. attempted
to overcome this difficulty by selecting stars within 50' of
1.2
1.0
0.8
-
0.6
0.4
U-B
-0.2
0.0
+0.2
0.4
0.6
0.8
-0.4 . 0.2 0:0+0.20.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 +2.4
B -- V
Fig. 1 Two-colour plot for reddened O stars in Cygnus
from Johnson and Morgan5.
, unreddened main sequence
stars (from types B1 to K3); O, O stars in the Cygnus Rift
Region; x HDE226868, from the colours of Bregman
et al.1.
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590 Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4 18 1974
HDE226868. This procedure was clearly not successful,
because even at distances below 1 kpc there is an ex-
tremely large scatter of points in their plot. The extra-
polation of such data to greater distances is thus hardly
justified. The same applies to the plot of E(B-V) against
distance for the survey of stars' within 30' of HDE226868.
In this case, an independent distance check is provided by
the Cepheid V547 Cyg, for which d=6.6 kpc, about two
and a half times greater than would have been estimated
from the assumed E(B-V) against d relationship.
We therefore conclude that this region is extremely
patchy over angular scales as small as 1°. This is con-
firmed by Lynds" survey of dark nebulae which shows not
only general patchiness but also the presence of extremely
small dark nebulae. The space density of these tiny
nebulae may, moreover, be underestimated because of the
lack of sufficient background illumination. If these objects
are representative of the familiar 'Bok globules', then
many may have linear dimensions of only 0.05 pc (ref. 8),
and would therefore subtend only 10" at a distance of
1 kpc. As the reddening produced by the globules is as
high as Av ~ 5 magnitudes, large extinction changes can
occur over areas of sky which are very much smaller than
those covered by either Margon et al. or Bregman et al.
Because of the extremely complex nature of the Cygnus
region, and the fact that the only independent distance
check is so widely in error, the quotation of a distance
to ± 16% by Margon et al. cannot be taken seriously.
At present we consider that it is over-optimistic to expect
reddening measurements to be able to provide the distance
of HDE226868 to the required accuracy. Until there is a
far more accurate determination of the distance of this
object it seems premature to suggest that the X-ray source
is a black hole.
Naval Research Laboratory,
Washington, DC 20375
CHUNG-CHIEH CHENG
K. J. H. PHILLIPS
A. M. WILSON
Laboratory for Solar Physics,
Goddard Space Flight Center,
Greenbelt, Maryland 20771
Received April 8, 1974.
1. Walborn, N., Astrophys. J. Lett., 179, L123 (1973).
Smith, H. E., Margon, B., and Conti, P. S., Astrophys. J. Lett.,
179, L125 (1973).
3 Margon, B., Bowyer, S., and Stone, R. P. S., Astrophys. J.
Lett., 185, L113 (1973).
‘ Bregman, J., Butler, D., Kemper, E., Kosti, A., Kraft, R. P.,
and Stone, R. P. S., Astrophys. J. Lett., 185, L117 (1973).
'Johnson, H. L., and Morgan, W. W., Astrophys. J., 122, 142
(1955).
8
Neckel, T., Landessternwarte Heidelberg-Konigstuhl Verof-
fentlichungen, 19 (1967).
Lynds, B. T., Astrophys. J. Suppl. Ser., 7, 1 (1962).
Allen, C. W., Astrophysical Quantities (University of London
Press, London, 1962).
High energy radiation from white holes
COMPARED to black holes, their time-reversed versions, white
holes have attracted little attention from theoreticians. Here we
point out possible ways in which white holes may be useful to
high energy astrophysics.
Basically a white hole is an object exploding from a highly
dense or a singular state. It may be a case of delayed big bang1
in a Friedman universe, or it may be an instance of a collapsing
object reversing implosion to explosion. For the latter pos-
sibility unconventional equations of state2 or negative energy
fields3 are required if the theoretical framework is that of
general relativity4. We will take the white hole to be an object
exploding from a singularity and obeying Einstein's equations
of gravitation subsequent to the singular event. Before turning
to the astrophysical implications we calculate the spectral
features to be expected from a white hole.
For simplicity we make the following assumptions: (1) The
white hole emerges from the singularity as a spherical object of
uniform density and zero pressure in the comoving frame of the
outward moving particles; (2) the light emitted by the white
hole is monochromatic and is being emitted radially outwards
from the surface at a uniform rate in the comoving frame of
reference; (3) the space-time exterior to the object is empty.
The comoving frame of reference in the interior will be
denoted by coordinates (r, 0, $, t) in terms of which the line
element within the object is given by
ds2 = c2 dt2 -- S2 (t) [dr2/(1-ar2)+r2 (de2+sin20d2)] (1)
where c = speed of light, S(t) is the expansion factor and a a
parameter related to the mass M and the comoving radius rb
of the object by
2GM = arb3 c2
(2)
The similarity of equation (1) to the Robertson-Walker line
element of cosmology is well known. Also, if we change t to -t,
equation (1) represents a freely collapsing ball of dust.
For convenience we will measure t from the instant of
explosion so that S(0) = 0. For t> 0, S(t) satisfies the equation
Ss2 = ac2 (1-S)
(3)
so that it attains its maximum value S = 1 at
t = to = T/(2cva)
(4)
We will investigate light emission from the white hole in the
interval 0<t<to.
The space exterior to the white hole is described by the
Schwarzschild line element
ds2 = [c2-(2GM|R)] dT2-dR2/[1-(2GM|c2R)] --
- R2 (d02-+-sin20d@2)
(5)
A typical Schwarzschild observer has R = constant, 0 =
constant, + = constant. We wish to calculate the spectrum of
radiation from the white hole as seen by a Schwarzschild
observer with R = R1 > 2GM/c2. In accordance with our
assumption (2) we will take the luminosity spectrum of the
white hole as L 8(v-Vo), where L = constant.
To calculate the spectrum received at R1, define
S = sin20, 0≤ 0≤1/2
(6)
Then 0 as a function of comoving time coordinate t is
given by
t = (2to/Tt) (0-sin 0 cos 0)
(7)
The white hole bursts out of the Schwarzschild radius at
t = t., 0 = 0., where
sin 0c = (arb2)!
(8)
Suppose two successive light signals are sent out from the
surface at comoving instants t and t+dt and are received by the
observer at R1 at instants T and T+dT measured in the
Schwarzschild coordinate. Then a straightforward calculation
shows that
dT/dt = sin 0/sin(0+0.)
(9)
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591
So an electromagnetic wave of frequency vo emitted from the
surface appears to the receiver to have the frequency
V = VO [sin(0+0℃)/sin0]
(10)
A result of this type but in different forms had been obtained
earlier by others5,6 but the above form is suitable for working
out the spectrum of the radiation as seen by the Schwarzschild.
observer. Under our assumption (2), L/hvo photons of
frequency vo are being emitted per unit t-time from the surface.
The number emitted in the interval [t, t+dt] is therefore
Ldt/hvg. The same number must be received in the interval
[T, T+dT], but with frequencies in the range (v, v+dv) where
dv is related to dt through equations (7) and (10).
A simple calculation gives
dt = (4toV03 sin30c dv)/(It (v2+v02-2vvo cos})2)
(11)
Writing E = hv, Eo = hv0, the number of photons in the range
[E, E+dE] received from the white hole per unit area at
R = R1 is given by
N(E)dE =(Lto/Tt2R12)E,2sin30.dE/(E2+E,2 -- 2EE(cosfc)2
(12)
For E> E.
N(E) dE = Lt0 E,2 (sin30c/m2 R12) dE|E4
(13)
The energy spectrum I(E) is given by
I(E) = EN(E) ℃ E-3
(14)
This is the spectrum at the high energy end under the simplify-
ing assumptions made here. More general (and perhaps more
realistic) assumptions can lead to different types of spectra
which we have also worked out. We now wish to indicate
possible fields in high energy astrophysics where white holes
may find applications.
(i) The hard electromagnetic radiation from white holes
situated at the centres of, say Seyfert galaxies, can be a source
of background X and gamma radiation. The energy spectrum
(14) seems, at first sight to be too steep compared to the
observed? spectrum o E-1.2. But absorption effects in the gas
present in the nuclei surrounding the white hole tend to flatten
the spectrum given by equation (14). Detailed calculation with
available data8,9 shows that these absorption effects can in fact
flatten the E-3 spectrum to ~ E-1 form in the range 0.2 ke.V
to 1 keV. At lower energies, the ultraviolet radiation seems to be
of the right order of magnitude to account for the infrared
emission of ~ 1045 erg s-1 through the dust grain heating
mechanism10.
(ii) The transient nature of X-ray and gamma-ray bursts11
suggests a white hole origin. The shape of the spectrum at the
emitting end is likely to be more complicated than the very
simple form assumed in the above example. In general, however,
the spectrum should soften with time.
(iii) Although we have worked out the spectrum of photons, it
is not difficult to see that similar conclusions will apply to
particles of non-zero restmass provided they have very high
energy. It is possible therefore to think of white holes in the
Galaxy on the scale of supernovae, yielding high energy cosmic
rays right up to the highest energy observed.
J.V.N. is grateful to the Jawaharlal Nehru Memorial Fund
for the award of a fellowship. N. D. thanks the University Grants
Commission for a travel grant and the Tata Institute of Funda-
mental Research for hospitality.
J. V. NARLIKAR
K. M. V. APPA RAO .
Tata Institute of Fundamental Research,
Bombay 400 005, India
N. DADHICH
Department of Mathematics and Statistics
University of Poona, Poona 411 007, India
Received June. 17, 1974.
1 Neeman, Y., Astrophys. J., 141, 1303 (1965).
2 Novikov, I. D., and Zeldovich, Y. B .; Ann. Rev. Astro. Astrophys.,
11, 387 (1973).
3 Narlikar, J. V., Pramana, 2, 158 (1974).
4 Hawking, S. W., and Penrose, R., Proc. R. Soc., A314, 187 (1970).
5 Faulkner, J., Hoyle, F., and Narlikar, J. V., Astrophys. J., 140,
1100 (1964).
6 Nariai, H., and Tomita, K., Prog. Theor. Phys., 34, 1046 (1965).
7 -Silk, J. I., Ann. Rev. Astr. Astrophys., 11, 269 (1973).
8 Burbidge, G. R., and Stein, W. A., Astrophys. J., 160, 573 (1970).
9 Brown, K. L., and Gould, R. J., Phys. Rev. D, 1, 2252 (1970).
10 Rees, M. J., Silk, J. I., Werner, M. W., and Wickramasinghe, N. C.,
Nature, 223, 788 (1969).
11 Proc. Los Alamos Conf. on Transient Cosmic Gamma and X-Ray
Sources (edit. by Strong, I. B.) (Los Alamos, 1973).
Spectrum of the cosmic background
radiation between 3 mm and 800 pm
WE report the successful flight of an experiment designed to
measure the spectrum of the cosmic background radiation
from ~3 mm to wavelengths shorter than the Planck peak
for a temperature of 2.7 K. The experiment was launched, to an
altitude of 40 km, on March 13, 1974, from Palestine, Texas.
Because of an on-site computer fault, the complete data were
not available to us until May 7, 1974. The instrument has
been described previously1,2 and will be discussed in detail
in a future paper; we present here the results of preliminary
analysis of the flight data.
10-9
Specific intensity W cm-2 sr-1 (cm-1) :- 1
10-10
10-11
10- 12
1
10
100
Wavenumber cm - 1
Fig. 1
Cosmic background spectrum at 1 cm-1 resoluton:
0 --- , Observed spectrum;
-
- - , predicted window
emission;
window emission using the results of
Chantry et al.4. Smooth solid line denotes 2.7 K minus 1.4'K curve.
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592 Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4 18 1974
The instrument consists of a polarising Michelson inter-
ferometer3 cooled, by a liquid helium bath, to a temperature
of 1.4 K. The processed output of the interferometer is the
difference between the spectrum of radiation entering the
cryostat and the spectrum of the radiation within the cryostat
cavity. The interferometer received radiation at a zenith distance
of 50° within a cone of semi-angle 3º; the zenith distance of the
extremity of the balloon, as seen from the package, was 27°.
The interferometer operated in vacuo, separated from the
atmosphere by a 50 um thick window of polyethelene; the
measured spectrum should therefore consist of the cosmic
background radiation plus emission from the atmosphere and
the window. The designed (unapodised) spectral resolution
was 0.25 cm-1 (Rayleigh criterion). The instrument was
calibrated in the laboratory using simulated black body
sources at room, liquid nitrogen, liquid hydrogen and liquid
helium temperatures; for in flight calibration, we decided to
use the emission from the window. The efficiency of the instru-
ment, as a function of frequency, was deduced from the
observed room-temperature laboratory spectrum.
One hour of useful time was achieved at float altitude
(reached at 0145 CDT), giving 16 interferograms. Total integra-
tion time for each sampled point was 19 s. The spectra obtained
from these interferograms were averaged at 1 cm-1 resolution;
the result was divided by the instrumental efficiency function
and the high frequency part fitted to the expected window
emission between 20 and 30 cm-1 to establish a vertical scale.
The fitted spectrum is shown in Fig. 1. Also shown is the
spectrum of à 2.7 K black body (representing the supposed
cosmic background) minus the spectrum of a 1.4 K blackbody
(representing the interior of the interferometer cavity).
It is not clear why, below 20 cm-1, the observed spectrum of
emission from the window departs radically from that pre-
dicted. The prediction of emission, however, is difficult because
of the problems of measuring absorption in thin samples. Esti-
mates of absorption vary between experimenters; for example,
we show in Fig. 1 the predicted window emission using the
absorption figures of Chantry et al.4. We believe, however, that
the measured values (E. I. Robson and B. Carli, to be published)
used for fitting are correct to (+20, -60)%. It is seen that, if the
predicted emission were decreased appreciably, the measured
low frequency spectrum would fall below that of 2.7 K minus
1.4 K blackbodies. We have tried the effect of assuming higher
temperatures for the interferometer cavity, but to obtain any
significant reduction in the height of the predicted background
curve requires that the peak emission be displaced to such high
frequencies as to exceed the measured values. We are therefore
satisfied that the calibration is substantially correct.
Our measured spectrum is a factor less than ~2 higher than
that of a 2.7 K background at frequencies below 10 cm-1.
A temperature ~ 21/3 higher than 2.7 K is ruled out, however, by
the position of the peak. The excess may well be due to unresolved
atmospheric emission5. In any case, this first direct spectral
measurement shows very clearly the sharp decline in the
spectral intensity above 6 cm-1, and it would be very difficult
to reconcile our observation with a background temperature
higher than ~2.9 K.
We thank our colleagues at Queen Mary College who have
contributed to this experiment and the staff of the National
Center for Atmospheric Research Balloon Facility for their
expert assistance. The project was supported by a grant from
the Science Research Council. E.I.R. and J.S.H. acknowledge
an SRC Associateship and Studentship respectively.
E. I. ROBSON
D. G. VICKERS
J. S. HUIZINGA*
J. E. BECKMANt
P. E. CLEGG
Received July 17, 1974.
*Present address: 3M Research, Ltd, Harlow, UK.
¡Present address : ESTEC, Noordwijk, Holland.
1 Beckman, J. E., and Robson, E. I. Infrared Detection Techniques for
Space Research (edit. by Manno and Ring), 63-72 (Reidel, 1972).
Robson, E. I., Beckmann, J. E., Clegg, P. E., Vickers, D. G., and
Huizinga, J. S., Proc. Internat. Conf. on Submillimetre Waves
and Their Applications, 199-201 (IEEE/OSA, Atlanta, Georgia,
1974).
3
Martin, D. H., and Puplett, E., Infrared Phys., 10, 105-109 (1969).
4
Chantry, G. W., Fleming, J. W., Smith, P. M., Cudby, M., and
Willis, H. A., Chem. Phys. Lett., 10, 473-477 (1971).
Muehlner, D. J., and Weiss, R., Phys. Rev. Lett., 30, 757-760 (1973).
A new solar -terrestrial relationship
RECENT suggestions of interrelationships between solar activity,
the Earth's magnetic field and the weather1-3 make more
urgent the need to have a reliable method of predicting sunspot
activity.
Most attempts to develop prediction techniques have been
associated with empirical relationships derived from the
preceding behaviour of many sunspot cycles, but none of these
has been really successful over any extended period. Wood4
has re-opened consideration of the possibility5 that solar
activity may be controlled by the tidal influence of the planets.
In the course of a detailed study6 of 25 yr of geomagnetic data
it was found that the phase of the solar diurnal variation of the
horizontal field on quiet days, Sq(H), exhibits a marked solar
control: the variability of the phase is most marked in winter
and at sunspot minimum epochs. The solar cycle behaviour
has now been studied over an 89-yr period from 1884, the
earliest year for which International Quiet Days have been
designated, and a consistent tendency has been found for the
magnetic data at sunspot minimum to anticipate the magnitude
of the subsequent sunspot maximum.
Both the amplitude and phase of the Sq variation vary
considerably from day to day, even on very quiet days. The
variability in the amplitude has been well studied, but relatively
little work has been published on the variability in the phase.
Here, the phase variability is discussed. Figure 1 shows the
diurnal variation of the occurrence of the time of daily minimum
horizontal field for all the international quiet days in the
period 1884-1972. Although the most common time of H-min
is between 1030 and 1130 LT, there is a wide range of appropriate
times. Days for which H-min occurred within 2} h of 1100 LT
16
=
125
8
1.8
-----
4
10.4
1
1
: NOD.
0
04
08
12
16
20
GMT
Fig. 1 Diurnal variation of the percentage number of inter-
national quiet days of the period 1884-72 for which the daily
minimum horizontal magnetic field occurred within each
half-hour of the day at Greenwich/Abinger/Hartland. Dashed
curve is for night-time data plotted on a x 10 ordinate scale,
indicated on the right-hand side.
Queen Mary College,
University of London, UK
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30
20-
it (%)
10-
U
0.
40
80
120
R
Fig. 2 Average percentage occurrence of AQDs (n) as a function
of average sunspot number (R) derived from annual mean 3-yr
smoothed data averaged over increments of 20 in R. Data are
the same as in Fig. 1, subdivided into two periods: moderate
activity, 1885-1936 (squares); strong activity, 1937-71 (circles).
are styled 'normal quiet days' (see ref. 6). These are included
within the shaded region marked NQD in Fig. 1. All days
outside this region are defined as 'abnormal quiet days' (AQD).
The dashed curve in Fig. 1 represents a replot of the points
for the night-time period on a x 10 ordinate scale; it seems that
within the AQD category there is a subsidiary maximum
occurrence of near-midnight minima in H. At such times the
' conductivity of the E region of the ionosphere is so low that
essentially no electric current flow can occur, so that the
reduction in the horizontal magnetic field cannot be ascribed
merely to positional changes of the atmospheric Sq current
loops. Evidence is presented elsewhere? for a connection
between the occurrence of the more moderate AQDs and
changes in the structure of the terrestrial atmosphere and the
orientation of the Sq current loops, but it seems likely that the
more extreme types of AQD have an extraterrestrial (probably
solar) cause.
frequency of AQDs with decrease in solar activity, such that the
· occurrence is primarily confined to times near solar minimum.
Extrapolating the Fig. 2 data shows that the mean occurrence
- frequency of AQDs for 'zero sunspots' for the 1885-1936 cycles
is 16.5%, and for the 1937-71 cycles it is 29%. These differences
probably emphasise the relative crudity of the sunspot.
number as an index of solar activity. The earlier group is for a
period of relatively low sunspot maxima and low minima;
the later group has high sunspot maxima and relatively high.
sunspot minima, and it seems likely that the bodily displacement
of the lines in Fig. 2 is conditioned by varying thresholds of
solar activity which are not apparent in the values of sunspot
numbers themselves. The 'AQD count' parameter is interesting
since it is an index which maximises at sunspot minimum, and
it is conceivable that it thereby provides a rather sensitive
measurement of the 'depth' of solar minimum not evident
in the conventional sunspot number.
Further insight into the solar cycle control of AQD occurrence
is provided by an examination of the long-term behaviour over
the eight separate solar cycles investigated. Figure 3 shows a
3-yr running mean of the average quarterly values of AQD
count (solid curve) and sunspot number (dotted curve). The
inverse correlation between the two curves corroborates the
result of Fig. 2; a new feature is the striking variation in the
amplitude of the maxima of the AQD count (at sunspot
minima) such that the change from one cycle to the next is
always in the same sense as the change in amplitude of the
subsequent sunspot maxima. This effect is better illustrated in
Fig. 4 which shows 3-yr running mean curves of the annual
values of both parameters, plotted so that the time scale for
sunspot number is 6 yr ahead of that for the AQD count. The
effect is consistently observed over the ups and downs of the
past eight solar cycle maxima, and is particularly evident over
the period 1920-72 when after three consecutive increases in
the size of the AQD count and the subsequent solar maxima
(culminating in the record IGY maximum) the following
maximum was significantly smaller for both parameters.
It is now possible to rationalise the two lines obtained in
Fig. 2 when concurrent values of AQD count and sunspot
number are plotted against each other. Figure 5 shows a plot
of the amplitudes of the cycles in each parameter, read from
Fig. 4, using both the ascending and descending parts of each
cycle, where each AQD count amplitude is associated with the
corresponding sunspot number amplitude one half-cycle ahead.
The approximately linear relationship shows that each change
in AQD count is approximately mirrored by a proportional
change in sunspot number during the corresponding part of the
subsequent cycle.
35
200
30
150
25
100
20 =
15
50F
10
0
15
1885
1900
1915
1930
1945
1960
`1972
Fig. 3 Three-yearly run-
ning mean of the quarterly
occurrence of AQDs (n,
solid curve) and of sunspot
number (R, dotted curve)
for the data as in Fig. 1.
It was previously found that the occurrence of AQDs is
primarily a winter phenomenon, and examination of the
detailed seasonal variation over the period 1884-1972 shows
that the most extreme AQDs occur exclusively in winter.
Confirmation of this inverse relationship to solar radiation is
provided by the negative correlation between AQD occurrence
and sunspot number, illustrated in Fig. 2. In this diagram the
data have been smoothed over 3 yr and the mean AQD count
determined for each successive interval of 20 in sunspot
number.
For each group of data in Fig. 2 (1885-1936 and 1937-71)
there is a remarkably linear increase in the occurrence
If this relationship proves valid it implies that the Sun
'breathes' with an 11-yr period, such that the size of a solar
activity maximum is determined at the very beginning of a
cycle, or perhaps the very end of the preceding cycle, from the
'depth' of the solar minimum. This quantity is essentially
unmeasurable by the conventional sunspot number, and
may be insensitive of detection by other indices of solar
activity which decrease at this time. The AQD count
index, based on a simple analysis of the variation of the
Earth's magnetic field on quiet days in winter, seems
to provide a highly sensitive index of the extent of solar
minima. Detailed examination of the various cycles in
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594 Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4 18 1974
1885 91 97 1903 09 15 21 27 33 39 45 51 57 63 69
.R
Year scale for.n
200
130
25
120
120
+15
40
410
0
Cycle 13
14
15
16
17
18
19
20
15
1891 97 1903 09 15 21 27 33 39 45 51 57 63 6
Year scale for R
Fig. 4 Three-yearly running mean of the annual occurrence of
AQDs (n, dashed curve, upper abscissa scale) and of sunspot
number (R, solid curve, lower abscissa scale) for the data as in
Fig. 1. Note the 6-yr displacement of the time scales.
.
Fig. 3 reinforces the conclusion that an apparently close
relationship exists between the two parameters separated
by an interval of 5-6 yr. For example, there is a marked
tendency for some maxima in AQD count to be double-humped,
with peak separations of about 2 yr. Double maxima are a
feature8 of the solar cycle (although not always evident in
sunspot number) and have been interpreted as the result of the
superposition of two processes having different physical
properties. It is tempting to suggest that the extra large and
single-humped sunspot maximum of 1957-58 arose from a
coincidence of the two peaks, and it is noteworthy that the
preceding AQD count maximum in 1953-54 also exhibits a
single sharp peak ..
There are obvious difficulties in accounting for a time
constant as long as 5-6 yr from the solar side. But it is now
evident that the solar cycle is a much more complicated
periodicity than that indicated by the variation of any one
index of activity. The cycles in solar wind intensity and velocity,
coronal green line intensity and coronal shape, and sunspot
number all differ in phase from one another, with a maximum
difference of at least 3 yr (ref. 9). One model10 of the Sun's
magnetic field proposes that the magnetic polarity of solar polar
regions changes sometime during the decline from sunspot
maximum to sunspot minimum, and a solar activity centre is
considered to have a 'history' related to solar happenings during
the preceding years. The interplanetary field near the Earth is
largely controlled by the solar polar field and, in keeping with
the above result, the phase of the annual variation of the
interplanetary field changes about 2.7 yr after sunspot maxi-
mum11.
22-
18.
1%DuV
14.
10-
0
50
90
130
170
AR
Fig. 5 Amplitudes of the cycles in AQD occurrence (An) and
sunspot number (AR) one half-cycle ahead, read from Fig. 4.
(Open circle is for the ascending part of cycle 13 and involves an
estimate of the value of An, since no magnetic data are available
for the time of the AQD minimum.)
From the terrestrial magnetic side it is difficult to speculate on
possible mechanisms while the essential cause of the AQD
phenomenon remains obscure. Comparison of Sq data for
observatories spaced 1,000 km apart has shown6 that 80% of
the AQDs are common at the two sites in winter and at sunspot
minimum; the agreement falls to 35% in summer and sunspot
maximum. This suggests that when the most variability is
encountered it influences larger areas of the Earth. Thus,
although the AQD index, as presented, is derived from data
for one station only, it reflects a regional or planetary
phenomenon. The unusual minima in the horizontal magnetic
field at a station tend to occur around 0000 UT and are
generally additional to the normal minima around 1100 LT
which are associated with the ionospheric Sq current system12.
So it seems very likely that the cause of the AQD incidence is
extraterrestrial, and is probably solar in origin. A likely line of
communication between the Sun and Earth seems to exist
in the interplanetary field. The varying 'sector structure' of this
field, which is related to the solar magnetic field, can be seen
clearly in the behaviour of the geomagnetic field at high
latitudes13. It has also been proposed14. that during quiet days
the magnitude of the total interplanetary magnetic field is
partly responsible for the day-to-day variability of Sq (range
rather than phase) at low latitudes. Furthermore, the sector
structure of the interplanetary field shows a solar cycle
variation15, and exhibits a particularly stable structure at sun-
spot minimum16.
There remains the possibility that the relationship reported
here is fortuitous, albeit being obeyed for a tantalisingly long
period. The ultimate test must await much more extensive data,
or the realisation of a sound scientific basis for such a con-
nection along the lines suggested above. Meanwhile, the index
could probably be refined considerably by basing it on magnetic
data from several geographically separated stations, and by
defining the incidence of an AQD more precisely. It seems
(Fig. 4) that there is a long term secular trend in AQD
occurrence, evidenced by the almost continual increase in the
size of the minimum count of each cycle over the period covered.
G. M. BROWN
Department of Physics,
University College of Wales,
Penglais, Aberystwyth, UK
Received June 17; revised July 31, 1974.
1
King, J. W., Nature, 245, 443-446 (1973).
2
King, J. W., Nature, 247, 131-134 (1974).
3
Stringfellow, M. F., Nature, 249, 332-333 (1974).
4
Wood, K. D., Nature, 240, 91-93 (1972).
5
Brown, E. W., Mon. Not. R. astr. Soc., 60, 599-606 (1900).
6
Brown, G. M., and Williams, W. R., Planet. Space Sci., 17,
455-470 (1969).
7 Brown, G. M., J. atmos. Terr. Phys. (in the press).
8
Gnevyshev, M. N., Solar Phys., 1, 107-120 (1967).
9
Hirshberg, J., Astrophys. Space Sci., 20, 473-481 (1973).
10 Babcock, H. W., Astrophys. J., 133, 572-587 (1961).
11
Wilcox, J. M., and Scherrer, P. H., J. geophys. Res., 77, 5385-5388
(1972).
12 Mizzi, C., and Schlapp, D. M., Planet. Space Sci., 19, 273-274
(1971).
13 Svalgaard, L., Geophys. Papers, R-6, (Danish Met. Inst.,
Charlottenlund, 1968).
14 Cane, K. P., Nature phys. Sci., 232, 21-22 (1971).
15
Svalgaard, L., J. geophys. Res., 77, 4027-4034 (1972).
16
Abdel-Wahab, S. and Goned, A., Planet. Space Sci., 22, 537-544
(1974).
Rainfall, drought and the solar cycle
THE present drought devastating the Sahel, India and Ethiopia
has been ascribed to long term climatic changes. Winstanley1
and Lamb2 suggest that the general circulation of the atmos-
phere is slowly changing, resulting in an equatorward shift of
the principal climatic belts. If these shifts are part of a 200-yr
cycle, as Lamb suggests, severe droughts should become more
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595
common during the next 60 yr. But at Addis Ababa the 11-yr
solar cycle seems to have a greater influence on rainfall
variations and so the timing of droughts, than does the long
term cycle.
- In Fig. 1 we compare the variation of annual rainfall recorded
at Addis Ababa during the past 72 yr (refs 3-5 and unpublished
data from Addis Ababa Geographical Observatory) with the
Zurich sunspot number. Both sets of data have been smoothed
by plotting 3-yr running means. The rainfall clearly follows a
cyclic pattern in which rainfall peaks and troughs precede the
sunspot peaks and troughs by a few years. This agreement is not
caused by the smoothing technique used: comparing each
year's unsmoothed rainfall and sunspot values the correlation
coefficient, r, is 0.21 (significant near the 5% level). If each
year's sunspot value is compared with the previous year's
rainfall, r=0.29 (significant near the 1 % level) and if compared
with the rainfall 2 yr before, r=0.33 (significant at better than
the 1% level). Thus, there is a strong statistical correlation
between Addis Ababa rainfall and sunspot number, the rainfall
peaks leading the sunspot peaks by an average of 1.3 yr; the
actual values for the 7 cycles being: 1, 2, 3, 2, 1, 1, 0 yr. It is
noteworthy that, in a reported correlation between atmospheric
ozone content and sunspot values6, the ozone troughs preceded
the sunspot peaks by 1.5 to 2 yr.
1,600;
Rainfall (mm)
100
1.200
Sunspot number
800
1910
1930
1950
1970
Fig. 1 Three-year running means of. Addis Ababa rainfall
() and three-year running means of Zurich sunspot
numbers ( -- - ).
., Known Ethiopian drought years. In
cases where discrepancies occur between rainfall data from
refs 3 and 5 preference has been given to ref. 3.
The phase relationship of the two curves of Fig. 1 is well
maintained, but the amplitude relationship is more complicated.
There is a tendency for the magnitude of the rainfall peak
to be inversely related to the magnitude of the sunspot peak
(Fig. 2a). A similar relationship appears for the trough values
(Fig. 2b). But since two of the data points on each graph
disagree with this trend there is not sufficient evidence to state
this relationship definitely.
If this rainfall/sunspot cycle relationship continues then,,
using King-Hele's? prediction of 1978 as the peak year of the
next solar cycle, we would expect Addis Ababa rainfall to
increase until about 1976-77 and then decline to a minimum
about 5 yr later. Further, if the relationship shown by Fig. 2a
holds, then the predicted value of 110 for the next sunspot
number peak7 gives a value of about 1,600 mm for the next
rainfall peak.
It is important to find whether such a relationship applies
for Ethiopia as a whole, and for other drought afflicted areas of
Africa. Instrumental rainfall records have been obtained only
recently in most areas of Ethiopia, but a 70-yr record exists for
Asmara. A preliminary study of this shows little correlation
with solar cycle before 1930, but a negative correlation after that
date.
To support the scanty instrumental data on rainfall, an
indication of past rainfall troughs can be obtained from
historical records of drought, and drought-induced famines.
To be recorded by travellers or historians a drought would
have to have been severe enough, and lengthy enough, to
affect seriously the life of the country, and not have been
merely an isolated year of poor rainfall. Figure 3 is a histogram
of presently known Ethiopian drought years, since 1540 AD,
a
b
1,150|
Peak rainfall (mm)
1,700
1,500
Trough rainfall (mm)
1,050
1,300}
0
0
950
20
Peak sunspot number
Trough sunspot number
Fig. 2 a, Rainfall cycle peak values against corresponding
sunspot cycle peak values. The line is a least squares fit to the
solid points only. b, As for a but for corresponding trough
values.
as a function of years from sunspot minimum. Droughts have
occurred in each of the years of the sunspot cycle, but 14 of
the 27 drought years occurred in the 3 yr centred on sunspot
minimum. Six more occurred in the years following minimum,
possibly representing droughts caused by an accumulation of
bad years (the effects of a long deficiency of rain persist during
the first years of improved rainfall). Thus, there is some evidence
that the rainfall/solar cycle relationships observed at Addis
· Ababa has applied for the past few hundred years over highland
Ethiopia as a whole.
It may be significant that the early 1930s coincided with
several changes in climate/solar cycle correlations. From this
time the Asmara rainfall shows some solar correlation, and
for Addis Ababa, the time lag between rainfall and sunspot
peaks decreased. There was, at the same time, a phase change
in the relationship between sunspot values and 8 m depth earth
temperatures in the UK, and also phase changes in equatorial
pressures and temperatures". Additionally, the level of Lake
Victoria showed high solar cycle correlation from 1890 to 1930,
but the relationship broke down after that time8. These changes
may be related to the proposed 200 and 700-yr climatic cycles
which peaked at that time1.
3
6F
st
-
4-
Number .
3
2
-6
-5
-4
-3
-2
-|
Min. +1 +2 +3
Years from sunspot minima
Fig. 3 Distribution of presently known Ethiopian drought years,
1540 to 1974, relative to sunspot minima.
An intensive study of the Indian monsoon rains9, based upon
the 50-yr records of many stations, has found significant
. correlations with solar cycles. In regions of orographic rainfall
near the Himalayas, strong, positive correlations are found,
whereas in some other regions there are negative correlations.
Zones of positive and negative correlation are separated by
areas of little correlation. The Indian data illustrate that the
solar influence on rainfall is spatially modulated, in unknown
ways, resulting in systematic variations of the sign and degree
of correlation. Similar solar cycle influences should be looked
for in the rainfall records of the Sahel and elsewhere.
If this sunspot-rainfall relationship is substantiated by
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0
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data from other stations in the Sanel and India, governments
and relief organisations will have a valuable warning of periods
of greatest drought danger.
CHARLES A. WOOD
RAYMOND R. LOVETT
Geophysical Observatory,
Haile Sellassie I University,
Addis Ababa, Ethiopia
Received July 4, 1974.
1
Winstanley, D., Nature, 245, 190-194 (1973).
2
Lamb, H. H., Drought in Africa, (edit. by Dalby, D., and Harrison
Church, R. J.), 27-28 (School of Oriental and African Studies,
London, 1973).
3 Fantoli, A., Contributo alla Climatologia dell' Etiopia, 39, (Ministeri
degli Afferi Esteri, Rome, 1965).
Giffiths, J. F., Climates of Africa, 375, (Elsevier, Amsterdam, 1972).
4
5 Civil Aviation Dept., Monthly Summaries of Precipitations, 2nd
Edition, (Climatological Institute, Addis Ababa, 1963).
6 Willett, H. C., J. geophys. Res., 67, 661-670 (1962).
7
King-Hele, D. G., Nature, 209, 285-286 (1966).
8
Lawrence, E. N., Weather, 20, 334-343 (1965).
9
Jagannathan, P., and Bhalme, H. N., Monthly Weather Review,
101, 691-700 (1973).
Dynamic implications of mantle hotspots
MOVEMENT of lithosphere plates over hotspots which are
fixed relative to the mantle could explain the origin of island
chains and aseismic ridges1-4. Observations of phase velocities
against epicentral distances for Tonga-Samoa earthquakes5
show anomalies which can be interpreted as a radial and lateral
inhomogeneity in the lower mantle beneath Hawaii. That
indicates the possible existence of deep mantle plumes.
Here, I consider some implications of the mantle plume
hypothesis on the basis of a simplified flow model. I represent
the flow of the plume by viscous flow through a pipe. The
Navier-Stokes equations, assuming steady state, fluid incom-
pressibility and uniform viscosity, reduce to a simple form for
the case of pipe flow. The maximum flow velocity, V2, given by
the exact solution of these equations, is
Vz == (dp/dz) (a2/4n)
(1)
where a is the radius of the plume, the flow is along z axis and
n is the coefficient of viscosity.
The volume flux == (na2/2) V2
(2)
The associated energy equation, based on the same
assumptions as for Navier-Stoke equations, also yields an
exact solution for pipe flow, giving the temperature ST,
generated by the flow:
δT = n V 2/4K
(3)
where K is the coefficient of thermal conductivity.
Table 1 lists the temperature differences and the volume flux
for a range of velocities of plume flow.
The values used in computing Table 1 are: n == 3 × 1021
g cm-1 s-1; K = 10-2 calorie ℃-1 cm-1 s-1; and the plume
radius, a, = 75 km. The most frequently quoted2 vertical
velocities of plume flow are 2 myr-1. It is clear that such velocities
will generate unacceptably high temperatures if the frictional
viscosity is of the same order of magnitude as present estimates
of the mantle viscosity.
With a speed of flow of 2 m yr-1 each plume would bring up
about 18 km3 yr-1 of mantle material. Twenty plumes will
bring up a total of 360 km3 yr-1. That would be the total flux
of the material moving away from the rise crests. Lithosphere
is created at a rate of about 170 km3 yr-1, which is the flux of
counterflow towards the ridge crests. The resultant drag
exerted by the flow on the lithosphere will be in the direction
of plate motion, that is, away from the rise crests2. The drag
can be increased by increasing either the number of plumes, or
their vertical flow speeds or dimensions.
If, however, the flow speed is reduced to, for instance,
3 cm yr-1, temperatures would become reasonable. But the
total flux from 20 plumes would be reduced to about 6 km3 yr-1.
Then, because the total flux of flow towards the rise axes
would be many times the flux away from them, the net stress
on the plates would tend to close the plates rather than move
them apart. Thus, as well as the necessity for a source to
provide adequate motive forces for plate motions it becomes
necessary to find additional stresses to overcome the closing
trend. That adds unnecessary complexity to the problem.
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75
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Fig. 1 Relationship between proposed hotspot localities and
gravity anomalies (mGal).
Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4
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597
Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4
Nature rul. LJI
Table 1 Typical temperature differences and volume flux for a
range of flow velocities
Flow velocity
.(cm yr-1)
Temperature difference
(º℃)
Volume flux per plume
(km3 yr-1)
200
6.75 × 104
18
100
1.69 × 104
9
10
1.69 × 102
9 × 10-1
3
: ~ 15
2.7 × 10-1
1
~ 2
9 × 10-2
Therefore, either an alternative way of keeping down tempera-
tures must be found or the idea that mantle plumes have any
significant role in plate tectonics must be rejected.
It may be possible to introduce variations in viscosity and
conductivity, although I have assumed uniform viscosity and
conductivity. Both, however, are temperature dependent.
Therefore, the temperature, ST, generated by the flow, will be
affected, but it is uncertain whether the effect would be
sufficient to reduce the temperatures to a reasonable level.
Thus, assuming that the results of Table 1 are correct to an
order of magnitude then, if the temperatures are to be kept
reasonable, some other parameter in equation (3) must be
adjusted. The only reasonable possibility at this stage is a
reduction in the velocity of the vertical flow of the plume,
perhaps accompanied by some increase in its diameter.
Unfortunately, that makes the mantle plume hypothesis much
less attractive.
It has been contended? that the viscous drag exerted on the
bottom of the lithosphere by a typical plume, will result in the
doming of lithosphere, producing a regional topographic high
and a gravity excess over the area. Most hotspots do indeed lie
over areas of positive gravity, though very few lie directly over
gravity maxima; Fig 1 shows the probable locations of the
hotspots, superimposed on isostatic gravity anomalies based on
gravity Model GEM 4 (Khan, M. A., and Lerch, F., et al., .
unpublished). A significant number of hotspots, however,
show association with negative gravity; Galapagos, Juan de
Fuca, St Helena, Macdonald and Salay Gomez. That contra-
dicts the general claim that plume locations are always
associated with positive gravity.
M. A. KHAN*
Hawaii Institute of Geophysics,
University of Hawaii,
Honolulu, Hawaii 96822
Received November 21, 1973.
* Work completed at: Geodynamics Program Division, Goddard
Space Flight Center, Greenbelt, Maryland 20770, now at Computer
Sciences Corporation, 8728 Colesville Road, Silver Springs,
Maryland 20910.
1 Wilson, J. T., Phil. Trans. R. Soc., A258, 145-167 (1965).
2 Morgan, W. J., Bull. Am. Ass. Petrol. Geol., 56, 203-213 (1972).
3
Duncan, R. A., et al., Nature, 239, 82-86 (1972).
4 Francheteau, J., et al., J. geophys. Res., 75, 2035-61 (1970).
5 Kanasewich, E. R., et al., Nature phys. Sci., 239, 99-100 (1972).
A-type doubling in the CH molecule
ALMOST a year ago we set out to calculate an accurate value of
the A-doubling in the lowest rotational level of the X2II,
state of CH, to assist the radioastronomers, who had been
searching for this species in interstellar space for some years.
The value we have obtained, which is reported here, represents
a substantial improvement on the results of terrestrial experi-
ments1,2, but its significance is reduced by the astronomical
observation of the species, which was reported during the course
of this work3. The result of the latter experiment indicates just
how close our calculation comes to predicting the actual
splitting and clearly underlines the power of the methods now
available in theoretical chemistry.
Van Vleck4 has shown that the A-doubling of the rotational
levels of a 2II state may be represented as a perturbation by
2E states. To second order, the splitting may be expressed in
terms of two parameters, (}p+q) and q. For the lowest level of
a regular 2II state, for which J = = {, the doubling is simply
2(p+q), where
P = 4
<2[ | H 50 | 2>> <? I| B(L+ +L-) |2Σ>
En-Ez
and q = 2
W
En-Ez
where the summation extends over all vibrational levels of all
2E states, the contribution from 22- being taken as negative.
Matrix elements are taken over both electronic and vibrational
wave functions. Hs. is the. spin-orbit coupling operator, and
B is the operator h/8m2ur2.
An ab initio calculation of the parameters p and q was per-
formed for CH by Hinkley et al." It was assumed that the
electronic matrix elements were independent of internuclear
distance, so that vibronic matrix elements could be factorised
into electronic and vibrational integrals. The Hartree-Fock
orbitals calculated for the X2II state by Cade and Huo6 were
used; the invarient orbital approximation was made, so that
Slater's rules might be applied to the matrix elements. Vibra-
tional wavefunctions were calculated numerically from Morse
curves. Only the contributions of the B22- and C2E+ states
were considered. The results are shown in Table 1; they are
some 20 % too low.
In the present work, the calculation of Hinkley et al. was
modified in several ways. Herzberg and Johns7 have shown
experimentally that both 2E states have maxima in their
potential curves, so that the latter hardly resemble Morse
curves. Vibrational wave functions were therefore calculated
from the extended configuration interaction potential curves
of Lie et al.8, which represent the states rather well. The
functions were calculated for the hypothetical case where
J = 0, and the effects of predissociation on excited vibrational
levels were ignored. The vibrational integrals obtained from
these wave functions were combined with the electronic matrix
elements of Hinkley et al. to produce the result labelled (a) in
Table 1.
Table 1 A-type doubling in the X2Il; (v = 0, J = }) state of CH
৳p+q (cm-1) q (cm-1) Doubling (MHz)
Hinkley et al.5
0.0447
0.0254
2,680
This work: (a)
0.0430
0.0245
2,576
(b)
0.0447
0.0274
2,680
(c)
0.0563
0.0386
3,374
(d)
0.0552
0.0379
3,311
Terrestrial experiment1,2
Astronomicalexperiment3
0.0563
0.038
3,374 ± 20
0.0556
3,335.47 ± 0.0!
The error introduced by the invariant orbital approximation
was then investigated. A separate set of SCF orbitals was
constructed for each of the 2E states from the basis set of
Cade and Huo, using the ALCHEMY program9. Electronic
matrix elements were then calculated using a program written
by J. A. Hall10. The values now obtained for the A-doubling
parameters (b) show that further improvements were necessary.
The electronic wave functions were further modified by con-
figuration interaction; six configurations were included for the
X2II and B2E- states, while eight were used for the C22+ state.
Electronic matrix elements were again calculated by the Hall
method; the result (c) was in close agreement with the experi-
ment.
Finally, the C.I. wave functions were calculated over a range
of internuclear distances. The electronic matrix elements were
then integrated numerically over the vibrational wave functions.
The results (d) are within 1% of the experimental values.
Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4
<? I | B(L+ +L-) [2X)2

598 Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4
18 1974
The A-doubling is predicted more accurately by this calcula-
tion than by terrestrial experimental estimates.
R. E. HAMMERSLEY
W. G. RICHARDS
Physical Chemistry Laboratory,
University of Oxford,
South Parks Road, Oxford OX1 3QX, UK
Received June 12; revised August 8, 1974.
1 Douglas, A. E., and Elliot, G. A., Can. J. Phys., 43, 496 (1965).
2 Baird, K. M., and Bredohl, H., Astrophys. J. Lett., 169, L83
(1971).
3 Rybeck, O. E. H., Ellder, I., and Irvine, W. M., Nature, 246, 466
(1973).
4 Van Vleck, J. H., Phys. Rev., 33, 467 (1929).
5 Hinkley, R. K., Hall, J. A., Walker, T. E. H., and Richards,
W .. G., J. Phys. B., S, 204 (1972).
6 Cade, P. E., and Huo, W. M., J. Chem. Phys., 47, 614 (1967).
7 Herzberg, G., and Johns, J. W. C., Astrophys. J., 158, 399 (1969).
8 Lie. G. C., Hinze, J., and Liu, B., J. Chem. Phys., 59, 1872 (1973).
9
Bagus, P. E., in: Proc. Seminar Selected Topics in Mol. Phys.
(Ludwigburg, Germany, IBM Germany).
10 Hall, J. A., Schamps, J., Robbe, J. M., and Lefèbvre-Brion, H.,
J. Chem. Phys., 59, 3271 (1973).
Drag-reducing polymers
and liquid-column oscillations
WE report here on one of many anomalies which occur
when drag-reducing polymers are dissolved. in water1-4:
the damping of manometer-type oscillations.
Our apparatus was made from clear PVC tubing (nominal
bore 1.3 cm) mounted vertically on a wooden board. The
tube was partially filled with liquid, thus creating a
rudimentary manometer. The basic experiment consisted
of giving the liquid column an initial displacement (25 cm
in all cases) from equilibrium and then counting the subse-
quent oscillations. Graphs of decaying amplitude of oscil-
lation against time were obtained by filming the process
at a speed of 18 frames per second. Two pipe configurations
were studied: a semicircle, of radius 29 cm; and a con-
ventional U-tube with the bend having a radius of curva-
ture of 4.5 cm. The length of the liquid column (88 cm) was
the same in both cases.
In each case the experiment was performed first with
15
·. Amplitude (cm)
10
--
---
S
00
2
4
10
T2
Number of periods
Fig. 1 Amplitude of oscillation against time for Polyox
WSR 301 in a semicircular tube. x, Water; O, 10 p.p.m.
WSR 301; 4, 25 p.p.m. WSR 301;
., 250 p.p.m. WSR 301;
4, 500 p.p.m WSR 301. Each period =1.6 s.
water and then with increasing concentrations of drag-
reducing polymer in aqueous solution. The additives used
were polyethylene oxide (Polyox WSR 301 and WSR N3000)
and polyacrylamide (Separan AP 273 and MG 200).
.
We found that both grades of polyethylene oxide reduced
the damping (relative to water) at quite small concentra-
tions; whereas the polyacrylamides, although effective
drag-reducers in turbulent flow, gave no measurable re-
duction ..
For small additive concentrations (Polyox WSR 301),
a marked reduction in damping (relative to water alone)
was evident after the first few oscillations (Fig. 1).
The behaviour at larger concentrations was particularly
interesting. For the first few oscillations the damping
actually increased (presumably reflecting the increase in
solution viscosity) but for the smaller amplitude motions
a reduction in damping again occurred (Fig. 1). These re-
sults suggest that, during the first few oscillations, the flow
is controlled by the viscosity of the solution alone.
The corresponding results for the U-tube were very
similar; there is little apparent difference between the
two flow configurations (Fig. 1).
Counting the total number of oscillations does, however,
reveal that the additives have surprising quantitative effects
on the damping. If the total number of oscillations with
160
120
O
100(Nip-Nw)/Nw
40
.
A. A
4
0
0
10.
20
30
40
30
60
70
80 90
Concentration (p.p.m.)
Fig. 2 Variation of damping reduction
(assessed by
observing the percentage increase in the total number of
oscillations) . with concentration of Polyox WSR 301.
O, Semicircular tube; 4, U-tube.
water is Nw and with polymer solution Np, the percentage
increase in the number of oscillations can be defined as
100 (Np-Nw)/Nw
Results for the semicircular tube (Fig. 2) show that
the total number of oscillations is more than doubled in
the concentration range 10-30 p.p.m. On the other hand,
the U-tube (Fig. 2) shows a much smaller peak enhance-
ment of about 30-40%, over the broader range of 25-65
p.p.m.
Polyethylene oxide tends to adhere to the pipe wall.
In an attempt to assess the importance of this wall coating,
a tube which had previously been used with solution con-
centrations of up to 500 p.p.m. was rinsed out, to remove
any remaining drops of polymer solution and the tests
were then repeated, beginning with water. It was found
that, when water was added to a coated tube, there was a
considerable degree of enhancement (relative to water
in a clean tube). To exclude the unlikely possibility that a
portion of the polymer coating had dissolved in the fresh
.
.
·
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-

Nature F Vi. LJI VUIUUUI
Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4 599
water, the sample was decanted after a marked reduction
had been observed. It was then placed in a clean PVC
tube and the experiment was repeated and no significant
reduction in damping was observed. It is, therefore, difficult.
to avoid the conclusion that observed reductions in damp-
ing occur because of an adsorbed layer of polymer additive
on the; tube wall, rather than because of the viscoelastic
properties of the actual solution.
Our results indicate that very small amounts of polyethy-
lene oxide can produce quite large reductions in the degree
of damping of manometer-type oscillations. The failure of
polyacrylamide to show any enhancement may result from
the fact that it does not seem to coat the tube, or it may
conceivably be a matter of time scales. Unfortunately, as
the period of. oscillation is proportional to the square root
of the length of liquid in the tube, an investigation of
much smaller time scales would have its difficulties.
The much greater reduction in the degree of damping .
found with the semicircular tube is presumably because
the tube is entirely curved, whereas the U-tube is only
partially so. This would be consistent with previous results"
for steady flow in curved pipes, in which polymer additives
resulted in marked reductions in drag.
I thank Dr M. Driels for discussion, and Mr J. Allan for
assistance with the experimental work.
W. D. McCOMB
School of Engineering Science,
Edinburgh University,
Mayfield Road, Edinburgh \EH9 3JL, UK
Received July 1; revised July 29, 1974.
1
Giles, W. B., Nature, 224, 584 (1969).
2 Morgan, D. T. G., Nature phys. Sci., 231, 62 (1971).
3
Barnes, H. A., Townsend, P., and Walters, K., Nature, 224,
586 (1965) ..
4 Balakirshnan, C., and Gordon, R. J., Nature phys. Sci., 231,
177 (1971).
" Barnes, H. A., and Walters, K., Proc. R. Soc., A314, 85 (1969).
1/f noise with a low
frequency white noise limit
ONE of us (A.A.V.), who had recently been studying channel
models for electrical fluctuations caused by potassium transport
through nerve membrane, noticed a possible analogy with the
flow pattern of grain through an hourglass of unusual shape
designed by F. Vaudan, Paris. The hourglass was filled with
small grains (0.1 to 1.0 mm diameter) of coloured glass and
differed from ordinary hourglasses in the length of the pore
connecting the two compartments which was drawn out to
about 25 cm; about 80 times the smallest inner diameter (3 mm).
When the hourglass was turned, the grain did not flow
steadily but exhibited slow irregular density fluctuations
(Fig. 1). With a view to the analogy with potassium channels
in excitable membranes, we decided to examine comparative
power spectra1-3.
In a series of experiments to measure spectral properties of
the grain flow, a laser beam (Spectra-Physics model 155) was
sent through the stem of the hourlgass and then through a
small hole (0.2 mm diameter) placed in front of and close to a
photosensitive cell and a de amplifier.
Light-beam switching resulted in square-waves with a resolu-
tion time of about 10-4 seconds for the system of cell and
amplifier. Another test using a white-noise modulated light
source with a white band between 1 and 5,000 Hz showed that
the system response was flat within the available range.
In the resulting pulse sequence, most of the pulses had com-
parable shapes (about triangular, or triangular with clipped
peaks when the grains completely blocked the beam during part
of their passage) and durations which varied from about 10-3 s
to 10-2 s. After amplification, the power spectrum was calculated
(Fig. 2, curve A). Its shape did not change for measurements
through different levels of the stem (higher up, where the stream
is dense and the light shines through occasionally, or lower
down, where individual particles pass through the beam).
The mechanism responsible for the spectral shape must be the
interaction between particles and wall and between the particles
themselves, while a third friction coefficient is given by the
counterflow of air. Clustering is known to occur in bins in the
absence of a counterflow of air. The actual values of the friction
coefficients (between particles and wall and between the particles
themselves) are not of primary importance4, although they
may influence the detailed structure of the spectrum, such as
the corner frequencies and the actual slope of the 1/f part.
We therefore also examined the spectrum associated with free
fall flow out of an open compartment through a gradually
narrowing pore (3 mm final diameter) (Fig. 2, curve B). Irregu-
larly shaped, sharp edged steel grit (0.2 mm mean diameter) was
used. In both systems the spectrum was white at low frequencies
and changed to 1/f at higher frequencies. At still higher fre-
quencies the spectrum shape is sensitive to several system
parameters and is rather complex.
Fig. 1 Flow through hourglass exhibiting clustering (exposure
time 1/1000 s).
This is to our knowledge the first report on a physical system
with 1/f noise which changes at lower frequencies into a different
and finite power spectrum. No special tricks need to be invoked,
therefore, to circumvent the famous 1/f paradox with its explo-
sion of power for frequencies approaching zero5. The mecha-
nism at work here might, for this reason, be of general interest,
since noise power spectra are investigated in a diversity of fields
such as magnetism, superconductivity, semiconductors, artificial
and biological membranes and synapses.
Furthermore, the transition from a 1/f spectral density into
some other spectral density is not the kind predicted by
McWhorter6; there one must invoke a Poisson process and a
,distribution of pulse time constants.
It is not very difficult to explain some of the major features of
the measured spectra7. These include the low frequency form of
the power spectrum (white) and the bandwidth over which the
spectrum is essentially 1/f. Further, it is possible to show, in
general, the conditions under which one may expect deviations
from a white spectrum and from this obtain important addi-
tional insights into possible source mechanisms.
In order to demonstrate these ideas, let us7, following
Heiden8, view a noise sequence as a sequence of unitary events
(pulses) which may represent current, voltage, magnetic lines,
and so on. Each individual pulse has its own time constant,
t, amplitude, h, and shape y(t,t). In addition, characterising
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60( Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4
10.000
$1,000
Intensity (arbitrary units)
A
100
B
10/
0.1
0.5
]
5 10
50 100
500 1,000 5,000.
Frequency (Hz)
Fig. 2 Power spectra associated with particle flow through
hourglass (curve A) and through a narrow pore out of an open
compartment (curve B). Line between curves indicates slope of .
(ideal) ]/f noise.
each pulse is the interval o between its beginning and the
beginning of the next pulse. The simplified sequence can be
drawn as in Fig. 3.
The interval distribution function u (q) may be Poisson or
non-Poisson. The sequence can be written as
I (t) == h1y(t,"t1) + hay(t - (1, 12)
+ h3y(t - 01 - 02, T3) + ...
For those sequences in which q, t, and h are functionally
independent of one another, the power spectrum is7
S(f)=N<h2><|F(f,t)(>2
1+
2<h>2 |‹F(f,t)>|2
Re
2.
where < > indicates average value, N is the mean number of
pulses per unit time, F(f,t) is the Fourier transform of the
individual pulse form y(t,t), and
v = 100 H(D) exp(2xif()do.
For convenience, we can write S(f) = NA(f)P(f) where
A(f) = < h2>{IF(ft)}>2 and P(f) is the expression in the
brackets.
For Poisson distributions, u(q) = Nexp(-No) and Re
(w/(1-v)) is zero. In these cases, then, the form of the power
spectrum is determined by F(f,t) and the distribution of pulse
time constants, v (t).
In general,
F(f,t) = |
+00
y(t, t)exp(-2tift) dt.
At low frequencies (2uft
00
< 1), however,
F(f,t) =
e
+00
Hence the Fourier transform is frequency independent and the
low frequency portion of the spectrum is white9.
If there is a distribution of time constants, v(t), among the
pulses, then, physically, there exists a maximum t, Um, such that
v(t) is small for t > Im. Now
(F(ft)= = F(ft) V(t)dt,
so at frequencies (2mftm< 1), <F(f,t)> is also frequency
independent and represents a white spectrum. This is also true
for <|F(f,t)|>. Hence, for a sequence of pulses with a
distribution of time constants, A(f) is white at low enough
frequencies so long as there is no coupling among the para-
meters of a single pulse.
A similar conclusion obtains for the factor P(f). which
incorporates the effect on the power spectrum of deviations
from a Poisson sequence.
1
0
Fig. 3 Pulse sequence parameters.
We have
v = H(D)exp(2TifQ) do.
00
Physically there exists a maximum o, say Om, beyond which
u(q) is arbitrarily small. Hence, for low enough frequencies,
(2mf(m<1), y is frequency independent and, therefore, so is
Re (w/(1-)).
We therefore have a theorem which says that the power
spectrum of any pulse sequence, Poisson or non-Poisson, even
with a distribution of pulse time constants, is white at low
enough frequencies provided that there is no coupling among the
parameters of a single pulse.
There are at least two immediate and useful consequences.
First, if there is not too much overlap between pulses, simple
visual inspection of the sequence will permit estimates of tm and
Om. This in turn allows an approximate determination of the low
frequency bandwidths over which A(f) and P(f) respectively
are white. Hence, under these conditions, visual inspection can
tell us whether deviations from a white spectrum are due to
A(f) or P(f) over a particular bandwidth. This is particularly
easy to do in those cases where om > Um. Secondly, if one
should find that at low frequencies (2mf < the smaller of
a-1 or (m-1) the spectrum is not white, then the implication
is that coupling must exist among the parameters of a single pulse.
E
The first consequence permits direct comparison between the
experimentally determined form of the power spectrum and the
theoretical frequency range over which A(f) and P(f) should be
white. Since one now knows over what bandwidth the form of
the spectrum is due to the shape of the individual pulses, we
have a direct way of determining if the sequence is non-Poisson
(via P(f)). The second allows one to deduce that functional
relations (for example, q = (constant)t or h = constant/o) exist
between the pulse parameters. We note for example that o =
(constant)t is a kind of inhibition where the presence of a
pulse with a long time constant tends to delay the appearance of
a next pulse while h = constant/o is a kind of facilitation where
a large pulse tends to encourage the appearance of the sub-
sequent pulse. Detailed analysis of the low frequency deviations
from a white spectrum will make it possible, in some cases, to
select the precise form of the pulse coupling and thereby give
further insight into the physical origins of the noise.
If the hourglass flow is examined at different angles with
respect to the vertical, the formation of unstable vaults can easily
be seen. Clearly, the formation of unstable vaults of different
lifetimes generates the clustering effects (Fig. 1) in the flow. In
an extensive study (published in Dutch) Peschl4 investigated the
flow of particles from bin openings. A key parameter is the
ratio of the opening to the grain diameter. In an hourglass the
ratio threshold, 4, is exceeded.
Only unstable vaults occur in normal use and their lifetimes
have an upper limit. Hence, the distribution of the intervals
between successive particles flowing through a plane must have
an upper time limit. In the case of the flow of steel grit through a
pore, in our experiments, for example, no intervals longer than
200 ms were found during a period of 10 min with a mean flow of
1,250 grains s-1 through the laser beam (0.2 mm diameter).
Generally, the individual pulse time constants were considerably
less than 20 ms. According to the theorem developed earlier in
the paper, the low frequency spectrum should be white for
frequencies below about 1/0.2 = 5 Hz as has been found (Fig.
2). The 1/f portion of the spectrum is due to the non-Poisson
character (clustering) of the individual particles. At higher
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<h2>
<|F(f,t)|>2
1-₩
.

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Nature , 01. 221
frequencies a change from 1/f to some other spectrum is pre-
dicted due to pulse shape, time constant and photocell response.
This occurs, as expected, at frequencies > 100 Hz.
It is tempting to conjecture that in some systems of molecular
dimensions with barriers, particles and pores analogous situa-
tions might prevail. Molecular size vaults might be formed and
thermal motion function as the agent for the introduction of the
instabilities. These systems would exhibit power spectra with
strong low frequency contributions (perhaps l/f in form) but,
yet, at the very lowest frequencies their spectra would always
turn white.
K. L. SCHICK*
A. A. VERVEEN
Department of Physiology,
University of Leiden,
Leiden,
Netherlands
Received August 24, 1973.
*Present address: Department of Physics, Union College, Schenec-
tady, New York 12308.
1 Verveen, A. A., and Derksen, H. S., Proc. Instn elect. Engrs, 56,
906 (1968).
2. Siebenga, E., and Meyer, A. W. A., Pflugers Arch., 343, 165 (1973).
3 Siebenga, E., and Verveen, A. A., Proc. first Europe. Biophys.
Congs., 5, 219 (1971).
4 Peschl, I. A. S. Z., thesis, Technische Hogeschool Eindhoven (1969).
5 Flinn, I., Nature, 219, 1356 (1968).
6 McWhorther, A. L., Semiconductor Surface Physics, 207 (University
of Pennsylvania Press, Philadelphia, 1956).
7 Schick, K. L., Acta biotheoretica (in the press).
8
Heiden, C., Phys. Rev., 188, 319 (1969).
9 Rice, S. O., Bell Syst. tech. J., 23 and 24, 1 (1944).
Second Law of Thermodynamics
IT is widely realised that there is no single and uniquely cor-
rect statement of the Second Law of Thermodynamics but
rather that there exist a number of different and mutually
compatible, correct statements1 (Everett2 mentions "two or
three dozen") each of which illuminates a different facet of
what H. A. Bent (private communication) has called
the "Second Law type of behaviour observed in nature".
There is no brief statement from which '
. all of the
thermodynamic relationships .. . "3 can be deduced without
further knowledge. In considering a particular problem one
particular statement is generally more apposite than another,
and for those who are concerned with the mechanisms
that produce work, a statement of the Law that stresses
the significance of work has distinct advantages. One such
statement of mine4 has been criticised by Legon3. Un-
fortunately the revised article containing the basis for
some of my statements has only recently appeared' though
it was submitted long before the other". This paper' should
have forestalled many of Legon's objections. For example,
its first page deals with the question of "maximum work"
and its equations (5) to (7) and (9) to (13) deal with equi-
libria and with entropy creation respectively.
Legon's objections are numerous and diverse but I will
try to deal systematically with them, as summarised in his
last paragraph.
Lack of originality. I had not seen the passage quoted
from Butler6 (page 32) when I wrote my paper4. Legon
writes of my statement: "All it says is that something
that is happening" (that is, that can proceed spontaneously)
"can be made to do useful work". Exactly so-it is very
simple. Taken with its corollary, that unless a proposed
change can be made to do useful work it cannot happen
spontaneously, the statement specifies conditions both neces-
sary and sufficient for predicting which changes can happen
spontaneously and which cannot. There is no logical neces-
sity to add any statement about "maximum work".
Readers of my two articles',' can surely be in little doubt
that I understand at least as well as does Legon the
fundamental conclusion of Carnot7 that to every specified
change (isothermal or not) there must correspond a
certain definite maximum output of work when going in
one direction and an equally definite minimum input of
work when going in the opposite one: these transfers of
work would be realised under reversible conditions; they
may or may not be equal depending on the particular
system under consideration.
I made no claim that my statement of the Second Law
sums up the whole of thermodynamics in one sentence :
neither for that matter does Legon's italicised quotation
from Planck8 (page 103) do so. It is necessary to know
more of the subject in order to comprehend either state-
ment, and the two can then be seen as not contradictory,
but complementary.
To understand the 'work' view of the subject in relation
to the 'entropy' view of it, one must understand the fact
(explicitly stated in refs 4 and 5) that in order to obtain work
from a spontaneous process a machine of appropriate de-
sign must be introduced, in imagination at least, into the
situation. This machine constrains the process in such a
way that it can occur only if at the same time work is
delivered; the machine must also contain a device (for
example, a weight) for storing this work since many types
of system are not, in themselves, capable of storing work.
All machines consist of several phases and possess geometri-
cal features, and far from being a mere artifice, the nature
of such machines and their limitations are the main topics
of interest that thermodynamics has had to offer to many
scientists, from the Carnots onwards. As Joule® (who was
interested in muscles as well as in galvanic cells and heat
Table 1 Range of spontaneous processes
Completely irreversible
Completely reversible
Resulting change in Universe
.Maximum entropy creation
Zero entropy creation
Zero increase in stored work
Max. increase in stored work
Max. inc. in thermal energy
Min. inc. in thermal energy
engines) wrote in 1853: "Perhaps the most important appli-
cations of dynamical theory are those which refer to the
production of motive power from chemical and other
actions." The matter could not be more succinctly ex-
pressed.
Failure to apply to isolated systems. I did not in fact
claim that the same equation relating work to entropy
creation applied to isolated systems, merely that the "argu-
ment can be easily extended" to them, by imagining a
machine to be introduced into the system itself. This basic
idea is far from novel10,11. A simple example that illustrates
the point well is provided by an isolated system in two parts
that are initially at different temperatures. Heat is allowed
to flow either by ordinary conduction or through a heat
engine. For a given change of state'it is found that the
entropy created ASer is always a single-valued function of
the work wasted. If one part of the system (e) is much larger
than the other. the equations approach as a limit the simple
form given in my paper (ref. 5, equation (10)):
AScr= [wasted work] /Te
Legon's preoccupation with semantics has led him to
make heavy weather out of a simple situation. Work
can certainly be stored in an isolated system-by lifting
a weight within the system or in various other ways.
Revealingly enough, the one form of energy that cannot
be used for this purpose is the very one suggested by Legon,
that is, thermal energy. The fundamental distinction be-
tween thermal energy (for definition, see ref. 12, page 11)
and other types of energy is absolutely basic.
------ -
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IVI AVI VVIVULI
18 1974
D. R. WILKIE
Legon poses, as a challenge, the problem of obtaining
work from the mixing of two ideal gases in an isolated
system of constant total volume. It is elementary that if
the mixture is allowed to form by merely withdrawing a
partition between the gases we have a good example of
a completely irreversible process with maximal entropy
creation (+11.53 J K-1 if we started with 1 mol of each
at 300 K) and no performance or storage of work. On the
other hand, by introducing into the system a suitable
machine, the uniform mixture could be allowed to form
in such a way that a weight within the system was raised.
(The machine described by Planck (ref. 8, page 219) may
be readily adapted for this purpose.) At the end of the
latter mixing process the isolated system would accordingly
contain more mechanical energy than it did at the begin-
ning. From the First Law it follows that the system must
necessarily contain less thermal energy; that is, its tempera-
ture must have fallen. In the limit, where the mixing
was reversible, the maximum possible work would have
been performed and transferred to the weight (2,769 J if
the gases were monatomic) and the temperature would
have fallen to 189 K. In this reversible case the change
in entropy arising from mixing (+11.53 J K-1) is exactly
counterbalanced by that attributable to cooling (-11.53 J
K-1): no entropy is created.
At this point it might be objected that the change in the
gases is not exactly the same as if they had mixed
irreversibly, because their thermal energy and temperature
have decreased. This is a simple consequence of the First
Law which applies equally no matter whether one is
considering an isolated system, a non-isolated one or the
whole Universe. If a change is conducted in such a way
that a weight is lifted then all the other bodies involved
cannot possibly end up in the same state as if the weight
had not been lifted.
Failure to apply to nonisothermal systems. Legon ex-
presses doubts about the validity of the equation for
entropy creation (refs 3 and 4) save for "the trivial
case for which the temperature Te of the environment is
equal to the temperature T of the system throughout the
process"3. On what grounds are these doubts based? Legon
does not discuss, let alone dismiss, any of the sources
quoted in my article5. Other relevant sources which should
be considered are Keenan, and Hatsopoulos13 and the classic
accounts by Maxwell10 and by Gouy14.
Legon's quotation from Planck (ref. 8, page 104) con-
cerning "dissipated energy" deserves close consideration.
It seems to state that the maximum work is a definite
quantity only for isothermal processes. If true this would
directly contradict the views of Thomson15 (later Lord
Kelvin) "On a universal tendency in Nature to the dissi-
pation of mechanical energy". On pages 113-117 of ref. 8,
however, Planck discusses his own statement (ref. 8, page
104) and we see that there is in fact no contradiction. What
Planck demonstrates is that although the change in Helm-
holtz free energy, -dA =- d(U-TS), measures Wmax under
isothermal conditions, it cannot conveniently be used
to determine Wmax under nonisothermal conditions because
the term S dT that then appears is frequently indeterminate.
The same point has already been made in a footnote by
Gouy (ref. 15, page 506) who had also given the correct
equation for determining Wmax under nonisothermal con-
ditions. Accordingly I find no substance in Legon's objec-
tions under this heading.
If it is thought that there is conflict between the 'work'
view of thermodynamics and the 'entropy' view it is high
time that the idea was abandoned. The two views are
different, but symmetrical, aspects of the same reality.
Spontaneous processes of all kinds fall somewhere within
the pattern shown in Table 1, their position depending
on the efficiency of the machinery used for the extraction
of work.
Department of Physiology,
University College London,
Gower Street,
London WCIE 6BT, UK
Received December 3, 1973; revised June 4, 1974.
1 Bridgman, P. W., The Nature of Thermodynamics, 116 -
(Harvard University Press, Cambridge, Massachusetts,
1943).
2
Everett, D. H., Chemical Thermodynamics, 216 (Longman,
London, 1971).
3
Legon, A. C., Nature, 244, 431 (1973).
4
Wilkie, D. R., Nature, 242, 606 (1973).
5
Wilkie, D. R., Nature, 245, 457 (1973).
6
Butler, J. A. V., Chemical Thermodynamics, fourth ed.
(Macmillan, 1955).
7 Carnot, S., Reflections on the motive power of fire (1824),
translation (Dover, New York, 1960).
8
Planck, M., Treatise on Thermodynamics, third ed., trans.
from seventh German ed., 1922 (Dover, New York, 1958).
9
Joule, J. P., Phil. Mag., Series 4, 5, 1 (1853).
10
Maxwell, J. C., Theory of Heat, fifth ed., chapter XII (Long-
mans Green, London, 1877).
11
Thomson, W., Phil. Mag., Series 4, 5, 102 (1853).
12
Guggenheim, E. A., Thermodynamics, third ed. (North Hol-
land, Amsterdam, 1957).
13
Keenan, J. H., and Hatsopoulos, G. N., Principles of General
Thermodynamics (Wiley, New York, 1965).
14
Gouy, M., J. de Phys., 2ª serie, t.VIII (Novembre 1889).
15 Thomson, W., Phil. Mag., Series 4, 4, 304 (1852); corrections
in ibid, 5, viii.
Information transmission under
conditions of sensory shielding
WE present results of experiments suggesting the existence of
one or more perceptual modalities through which individuals
obtain information about their environment, although this
information is not presented to any known sense. The litera-
ture1-3 and our observations lead us to conclude that such
abilities can be studied under laboratory conditions.
We have investigated the ability of certain people to describe
graphical material or remote scenes shielded against ordinary
perception. In addition, we performed pilot studies to determine
if electroencephalographic (EEG) recordings might indicate
perception of remote happenings even in the absence of correct
overt responses.
We concentrated on what we consider to be our primary
responsibility-to resolve under conditions as unambiguous
as possible the basic issue of whether a certain class of para-
normal perception phenomena exists. So we conducted our
experiments with sufficient control, utilising visual, acoustic
and electrical shielding, to ensure that all conventional paths of
sensory input were blocked. At all times we took measures to
prevent sensory leakage and to prevent deception, whether
intentional or unintentional.
Our goal is not just to catalogue interesting events, but to
uncover patterns of cause-effect relationships that lend them-
selves to analysis and hypothesis in the forms with which
we are familiar in scientific study. The results presented here
constitute a first step towards that goal; we have established
under known conditions a data base from which departures as a
function of physical and psychological variables can be studied
in future work.
REMOTE PERCEPTION OF GRAPHIC MATERIAL
First, we conducted experiments with Mr Uri Geller in
which we examined his ability, while located in an electrically
shielded room, to reproduce target pictures drawn by experi-
menters located at remote locations. Second, we conducted
double-blind experiments with Mr Pat Price, in which we
measured his ability to describe remote outdoor scenes many
miles from his physical location. Finally, we conducted pre-
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603
liminary tests using EEGs, in which subjects were asked to
perceive whether a remote light was flashing, and to determine
whether a subject could perceive the presence of the light,
even if only at a noncognitive level of awareness.
In preliminary testing Geller apparently demonstrated an
ability to reproduce simple pictures (line drawings) which had
been drawn and placed in opaque sealed envelopes which he
was not permitted to handle. But since each of the targets was
known to at least one experimenter in the room with Geller,
it was not possible on the basis of the preliminary testing to
discriminate between Geller's direct perception of envelope
contents and perception through some mechanism involving
the experimenters, whether paranormal or subliminal.
So we examined the phenomenon under conditions designed
to eliminate all conventional information channels, overt or
subliminal. Geller was separated from both the target material
and anyone knowledgeable of the material, as in the experiments
of ref. 4.
In the first part of the study a series of 13 separate drawing
experiments were carried out over. 7 days. No experiments
are deleted from the results presented here.
At the beginning of the experiment either Geller or the
experimenters entered a shielded room so that from that time
forward Geller was at all times visually, acoustically and
electrically shielded from personnel and material at the target
location. Only following Geller's isolation from the experi-
menters was a target chosen and drawn, a procedure designed
to eliminate pre-experiment cueing. Furthermore, to eliminate
the possibility of pre-experiment target forcing, Geller was kept
ignorant as to the identity of the person selecting the target
and as to the method of target selection. This was accomplished
by the use of three different techniques: (1) pseudo-random
technique of opening a dictionary arbitrarily and choosing the
first word that could be drawn (Experiments 1-4); (2) targets,
blind to experimenters and subject, prepared independently by
SRI scientists outside the experimental group (following
Geller's isolation) and provided to the experimenters during
the course of the experiment (Experiments 5-7, 11-13); and (3)
arbitrary selection from a target pool decided upon in advance
of daily experimentation and designed to provide data concern-
ing information content for use in testing specific hypotheses
(Experiments 8-10). Geller's task was to reproduce with pen
on paper the line drawing generated at the target location.
Following a period of effort ranging from a few minutes to
half an hour, Geller either passed (when he did not feel con-
fident) or indicated he was ready to submit a drawing to the
experimenters, in which case the drawing was collected before
Geller was permitted to see the target.
To prevent sensory cueing of the target information, Experiments
1 through 10 were carried out using a shielded room in SRI's facility
for EEG research. The acoustic and visual isolation is provided
by a double-walled steel room, locked by means of an inner and
outer door, each of which is secured with a refrigerator-type locking
mechanism. Following target selection when Geller was inside
the room, a one-way audio monitor, operating only from the inside
to the outside, was activated to monitor Geller during his efforts.
The target picture was never discussed by the experimenters after the
picture was drawn and brought near the shielded room. In our
detailed examination of the shielded room and the protocol used in
these experiments, no sensory leakage has been found.
The conditions and results for the 10 experiments carried out in the
shielded room are displayed in Table 1 and Fig. 1. All experiments
except 4 and 5, were conducted with Geller inside the shielded room.
'In Experiments 4 and 5, the procedure was reversed. For those
experiments in which Geller was inside the shielded room, the target
location was in an adjacent room at a distance of about 4 m, except
for Experiments 3 and 8, in which the target locations were, respec-
tively, an office at a distance of 475 m and a room at a distance of
about 7 m.
A response was obtained in all experiments except Numbers
5-7, In Experiment 5, the person-to-person link was eliminated
by arranging for a scientist outside the usual experimental
group to draw a picture, lock it in the shielded room before
Geller's arrival at SRI, and leave the area. Geller was then led
0
DEVIL
TARGET
RESPONSE 1
63
10
o
RESPONSE 2
TARGET
RESPONSE
TARGET
RESPONSE 1
RESPONSE 2
RESPONSE 3
a
b
Sun
1
1
.€
Solar System
0
TARGET
1
TARGET
BP.IDGE
1
0)
:
URI GELLER:
7/2/2016
RESPONSE
RESPONSE
RESPONSE
TARGET
RESPONSE
d
e
4
g
Fig. 1 Target pictures and responses drawn by Uri Geller under shielded conditions.
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TARGET
!

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ITWWIL F VI. AJI VLIVUEI 18 1974
Table 1 Remote perception of graphic material
Experiment
Date
Geller Location
Target Location
Target
Figure
(month, day, year)
1
8/4/73
Shielded room 1*
Adjacent room (4.1 m);
Firecracker
1a
2
8/4/73
Shielded room 1
Adjacent room (4.1 m)
Grapes
1b
3
8/5/73
Shielded room 1
Office (475 m)
Devil
1c
4
8/5/73
Room adjacent to
Shielded room 1
Solar system
1d
5
8/6/73
Room adjacent to
Shielded room 1
(3.2 m)
Rabbit
No drawing
6
8/7/73
Shielded room 1
Adjacent. room (4.1 m)
Tree .
7
8/7/73
Shielded room 1
Adjacent room (4.1 m)
Envelope
No drawing
No drawing
8
8/8/73
Shielded room 1
Remote room (6.75 m)
Camel
1e
9
8/8/73
Shielded room 1
Adjacent room (4.1 m)
Bridge
1 f
11
8/9/73
Shielded room 2}
Computer (54 m)
Kite (computer CRT)
2a
12
8/10/73
Shielded room 2
Computer (54 m)
Church (computer memory)
2b
13
8/10/73
Shielded room 2
Computer (54 m)
Arrow through heart
2c
(computer CRT, zero
intensity)
*EEG Facility shielded room (see text).
tPerceiver-target distances measured in metres.
#SRI Radio Systems Laboratory shielded room (see text).
by the experimenters to the shielded room and asked to draw
the picture located inside the room. He said that he got no clear
impression and therefore did not submit a drawing. The elimina-
tion of the person-to-person link was examined further in the
second series of experiments with this subject.
Experiments 6 and 7 were carried out while we attempted to
record Geller's EEG during his efforts to perceive the target
pictures. The target pictures were, respectively, a tree and an
envelope. He found it difficult to hold adequately still for good
EEG records, said that he experienced difficulty in getting
impressions of the targets and again submitted no drawings.
Experiments 11 through 13 were carried out in SRI's Engin-
eering Building, to make use of the computer facilities available
there. For these experimenters, Geller was secured in a double-
walled, copper-screen Faraday cage 54 m down the hall and
around the corner from the computer room. The Faraday cage
provides 120 dB-attenuation for plane wave radio frequency
radiation over a range of 15 kHz to 1 GHz. For magnetic fields
the attenuation is 68 dB at 15 kHz and decreases to 3 dB at
60 Hz. Following Geller's isolation, the targets for these
experiments were chosen by computer laboratory personnel
not otherwise associated with either the experiment or Geller,
and the experimenters and subject were kept blind as to the
contents of the target pool.
.
.
For Experiment 11, a picture of a kite was drawn on the face
of a cathode ray tube display screen, driven by the computer's
graphics program. For Experiment 12, a picture of a church
was drawn and stored in the memory of the computer. In
Experiment 13, the target drawing, an arrow through a heart
(Fig. 2c), was drawn on the face of the cathode ray tube and
then the display intensity was turned off so that no picture
was visible.
.
To obtain an independent evaluation of the correlation be-
tween target and response data, the experimenters submitted
the data for judging on a 'blind' basis by two SRI scientists
who were not otherwise associated with the research. For the
10 cases in which Geller provided a response, the judges were
asked to match the response data with the corresponding
target data (without replacement). In those cases in which
Geller made more than one drawing as his response to the
target, all the drawings were combined as a set for judging.
The two judges each matched the target data to the response
data with no error. For either judge such a correspondence has
an a priori probability, under the null hypothesis of no in-
formation channel, of P = (10!)-1 = 3 x 10-7.
A second series of experiments was carried out to determine
whether direct perception of envelope contents was possible
without some person knowing of the target picture.
One hundred target pictures of everyday objects were drawn
by an SRI artist and sealed by other SRI personnel in double
envelopes containing black cardboard. The hundred targets
were divided randomly into groups of 20 for use in each of the
three days' experiments.
On each of the three days of these experiments, Geller passed.
That is, he declined to associate any envelope with a drawing
that he made, expressing dissatisfaction with the existence of
such a large target pool. On each day he made approximately 12
recognisable drawings, which he felt were associated with the
entire target pool of 100. On each of the three days, two of his
drawings could reasonably be associated with two of the 20
daily targets. On the third day, two of his drawings were very
close replications of two of that day's target pictures. The
drawings resulting from this experiment do not depart signific-
antly from what would be expected by chance.
In a simpler experiment Geller was successful in obtaining
information under conditions in which no persons were know-
ledgeable of the target. A double-blind experiment was per-
formed in which a single 3/4 inch die was placed in a 3 x 4 x 5
inch steel box. The box was then vigorously shaken by one of the
experimenters and placed on the table, a technique found in
control runs to produce a distribution of die faces differing non-
significantly from chance. The orientation of the die within the
box was unknown to the experimenters at that time. Geller
would then write down which die face was uppermost. The
target, pool was known, but the targets were individually pre-
pared in a manner blind to all persons involved in the experi-
ment. This experiment was performed ten times, with Geller
passing twice and giving a response eight times. In the eight
times in which he gave a response, he was correct each time.
The distribution of responses consisted of three 2s, one 4, two
5s, and two 6s. The probability of this occurring by chance is
approximately one in 106.
In certain situations significant information transmission can
take place under shielded conditions. Factors which appear to
be important and therefore candidates for future investigation
include whether the subject knows the set of targets in the target
pool, the actual number of targets in the target pool at any
given time, and whether the target is known by any of the
experimenters.
It has been widely reported that Geller has demonstrated the
ability to bend metal by paranormal means. Although metal
bending by Geller has been observed in our laboratory, we have
not been able to combine such observations with adequately
controlled experiments to obtain data sufficient to support the
paranormal hypothesis.
REMOTE VIEWING OF NATURAL TARGETS
A study by Osis" led us to determine whether a subject could
describe randomly chosen geographical sites located several
miles from the subject's position and demarcated by some
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appropriate means (remote viewing). This experiment carried
out with Price, a former California police commissioner and
city councilman, consisted of a series of double-blind, demon-
stration-of-ability tests involving local targets in the San
Francisco Bay area which could be documented by several inde-
pendent judges. We planned the experiment considering that
natural geographical places or man-made sites that have
existed for a long time are more potent targets for paranormal
perception experiments than are artificial targets prepared in the
laboratory. This is based on subject opinions that the use of
artificial targets involves a 'trivialisation of the ability' as com-
päred with natural pre-existing targets.
In each of nine experiments involving Price as subject and
SRI experimenters as a target demarcation team, a remote
location was chosen in a double-blind protocol. Price, who
remained at SRI, was asked to describe this remote location, as
well as whatever activities might be going on there.
Several descriptions yielded significantly correct data per-
taining to and descriptive of the target location.
In the experiments a set of twelve target locations clearly
differentiated from each other and within 30 min driving time
from SRI had been chosen from a target-rich environment (more
than 100 targets of the type used in the experimental series)
prior to the experimental series by an individual in SRI manage-
ment, the' director of the Information Science and Engineering
Division, not otherwise associated with the experiment. Both
the experimenters and the subject were kept blind as to the
contents of the target pool, which were used without replace-
ment.
An experimenter was closeted with Price at SRI to. wait 30 min to
begin the narrative description of the remote location. The SRI
locations from which the subject viewed the remote locations con-
sisted of an outdoor park (Experiments 1, 2), the double-walled
copper-screen Faraday cage discussed earlier (Experiments 3, 4, and
6-9), and an office (Experiment 5). A second experimenter would then
obtain a target location from the Division Director from a set of
travelling orders previously prepared and randomised by the Director
and kept under his control. The target demarcation team (two to
four SRI experimenters) then proceeded directly to the target by
automobile without communicating with the subject or experimenter
remaining behind. Since the experimenter remaining with the subject
at SRI was in ignorance both as to the particular target and as to
the target pool, he was free to question Price to clarify his descrip-
tions. The demarcation team then remained at the target site for
30 min after the 30 min allotted for travel. During the observation
period, the remote-viewing subject would describe his impressions of
the target site into a tape recorder. A comparison was then made
when the demarcation team returned.
Price's ability to describe correctly buildings, docks, roads,
gardens and so on, including structural materials, colour,
ambience and activity, sometimes in great detail, indicated the
functioning of a remote perceptual ability. But the descriptions
contained inaccuracies as well as correct statements. To obtain
a numerical evaluation of the accuracy of the remote viewing
experiment, the experimental results were subjected to inde-
pendent judging on a blind basis by five SRI scientists who were
TARGET
TARGET
1
-
TARGET
RESPONSE 1
RESPONSE 1
1
1
.
RESPONSE
RESPONSE 2
RESPONSE 2
a
b
C
Fig. 2 Computer drawings and responses drawn by Uri Geller. a, Computer drawing stored on video display; b, computer drawing stored in
computer memory only; c, computer drawing stored on video display with zero intensity.
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Table 2 Distribution of correct selections by judges A, B, C, D, and E in remote viewing experiments
Descriptions chosen by judges
Places visited by judges
Hoover Tower
İ
ABCDE
1
2
3
4
5
6
7
8
9
Baylands Nature Preserve
2
ABC
E
Radio Telescope
3
ACD
BE
Redwood City Marina
4
CD
ABDE
E
Bridge Toll Plaza
5
ABD
DCE
Drive-In Theatre
6
B
A
C
ABCE
AB
Rinconada Park
9
CE
AB
Of the 45 selections (5 judges, 9 choices), 24 were correct. Bold type indicates the description chosen most often for each place visited. Correct
choices lie on the main diagonal. The number of correct matches by Judges A through E is 7, 6, 5, 3, and 3, respectively. The expected number
of correct matches from the five judges was five; in the experiment 24 such matches were obtained. The a priori probability of such an occurrence
by chance, conservatively assuming assignment without replacement on the part of the judges, is P = 8.10-10.
not otherwise associated with the research. The judges were
asked to match the nine locations, which they independently
visited, against the typed manuscripts of the tape-recorded nar-
ratives of the remote viewer. The transcripts were unlabelled
and presented in random order. The judges were asked to find a
narrative which they would consider the best match for each
of the places they visited. A given narrative could be assigned
to more than one target location. A correct match requires that
the transcript of a given date be associated with the target of
that date. Table 2 shows the distribution of the judges' choices.
Among all possible analyses, the most conservative is a per-
mutation analysis of the plurality vote of the judges' selections
assuming assignment without replacement, an approach inde-
pendent of the number of judges. By plurality vote, six of the
nine descriptions and locations were correctly matched. Under
the null hypothesis (no remote viewing and a random selection
of descriptions without replacement), this outcome has an a
priori probability of P = 5.6 x 10-4, since, among all possible
permutations of the integers one through nine, the probability
of six or more being in their natural position in the list has that
value. Therefore, although Price's descriptions contain in-
accuracies, the descriptions are sufficiently accurate to permit
the judges to differentiate among the various targets to the
degree indicated.
EEG EXPERIMENTS
An experiment was undertaken to determine whether a
physiological measure such as EEG activity could be used as an
indicator of information transmission between an isolated
subject and a remote stimulus. We hypothesised that perception
could be indicated by such a measure even in the absence of
verbal or other overt indicators.6.7.
It was assumed that the application of remote stimuli would
result in responses similar to those obtained under conditions
of direct stimulation. For example, when normal subjects are
stimulated with a flashing light, their EEG typically shows a
decrease in the amplitude of the resting rhythm and a driving
of the brain waves at the frequency of the flashes8. We hypothe-
sised that if we stimulated one subject in this manner (a sender),
the EEG of another subject in a remote room with no flash
present (a receiver), might show changes in alpha (9-11 Hz)
activity, and possibly EEG driving similar to that of the sender.
We informed our subject that at certain times a light was to
be flashed in a sender's eyes in a distant room, and if the subject
perceived that event, consciously or unconsciously, it might be
evident from changes in his EEG output. The receiver was
seated in the visually opaque, acoustically and electrically
shielded double-walled steel room previously described. The
sender was seated in a room about 7 m from the receiver.
To find subjects who were responsive to such a remote
stimulus, we initially worked with four female and two male
volunteer subjects, all of whom believed that success in the
experimental situation might be possible. These were designated
'receivers'. The senders were either other subjects or the
experimenters. We decided beforehand to run one or two
sessions of 36 trials each with each subject in this selection
procedure, and to do a more extensive study with any subject
whose results were positive.
A Grass PS-2 photostimulator placed about 1 m in front of the
sender was used to present flash trains of 10 s duration. The receiver's
EEG activity from the occipital region (Oz), referenced to linked
mastoids, was amplified with a Grass 5P-1 preamplifier and associated
driver amplifier with a bandpass of 1-120 Hz. The EEG data were
recorded on magnetic tape with an Ampex SP 300 recorder.
On each trial, a tone burst of fixed frequency was presented to both
sender and receiver and was followed in one second by either a 10 s
train of flashes or a null flash interval presented to the sender. Thirty-
six such trials were given in an experimental session, consisting of 12
null trials-no flashes following the tone-12 trials of flashes at 6 f.p.s.
and 12 trials of flashes at 16 f.p.s., all randomly intermixed, deter-
mined by entries from a table of random numbers. Each of the trials
generated an 11-s EEG epoch. The last 4 s of the epoch was selected
for analysis to minimise the desynchronising action of the warning
cue. This 4-s segment was subjected to Fourier analysis on a LINC 8
computer.
Spectrum analyses gave no evidence of EEG driving in any receiver,
although in control runs the receivers did exhibit driving when
physically stimulated with the flashes. But of the six subjects studied
initially, one subject (H. H.) showed a consistent alpha blocking effect.
We therefore undertook further study with this subject.
Data from seven sets of 36 trials each were collected from this
subject on three separate days. This comprises all the data collected
to date with this subject under the test conditions described above.
The alpha band was identified from average spectra, then scores of
average power and peak power were obtained from individual trials
and subjected to statistical analysis.
Of our six subjects, H. H. had by far the most monochromatic
EEG spectrum. Figure 3 shows an overlay of the three averaged
spectra from one of this subject's 36-trial runs, displaying
changes in her alpha activity for the three stimulus conditions.
Mean values for the average power and peak power for each
Table 3 EEG data for H.H. showing average power and peak power
in the 9-11 Hz band, as a function of flash frequency and sender
Flash
Frequency
0
6
16
0
6
16
Sender
Average Power
Peak Power
J.L.
94.8 84.1 76.8
357.7 329.2 289.6
R.T.
41.3 45.5 37.0
160.7 161.0 125.0
No sender
(subject
informed)
25.1 35.7 28.2
87.5
95.7
81.7
J.L.
54.2 55.3. 44.8
191.4 170.5 149.3
J.L.
56.8 50.9 32.8
240.6 178.0 104.6
R.T.
39.8 24.9 30.3
145.2
74.2 122.1
No sender
(subject
not
86.0 53.0 52.1
318.1 180.6 202.3
informed)
56.8 49.9 43.1
214.5 169.8 153.5
Averages
-12% -24%(P<0.04)
-21%-28%(P<0.03)
Each entry is an average over 12 trials
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of the seven experimental sets are giver nt Table 3. The power
measures were less in the 16 f.p.s. case than in the 0 f.p.s. in all
seven peak power measures and in six out of seven average
power measures. Note also the reduced effect in the case in
which the subject was informed that no sender was present
(Run 3). It seems that overall alpha production was reduced
for this run in conjunction with the subject's expressed appre-
hension about conducting the experiment without a sender.
This is in contrast to the case (Run 7) in which the subject was
not informed.
Siegel's two-tailed t approximation to the nonparametric randomi-
sation test9 was applied to the data from all sets, which included two
sessions in which the sender was removed. Average power on trials
associated with the occurrence of 16 f.p.s. was significantly less than
when there were no flashes (t = 2.09, d.f. = 118, P<0.04). The
second measure, peak power, was also significantly less in the 16 f.p.s.
conditions than in the null condition (t = 2.16, d.f. = 118, P<0.03).
The average response in the 6 f.p.s. condition was in the same direc-
tion as that associated with 16 f.p.s., but the effect was not statistically
significant.
Spectrum analyses of control recordings made from saline with a
12 kQ resistance in place of the subject with and without the addi-
tion of a 10 Hz, 50 uV test signal applied to the saline solution,
revealed no indications of flash frequencies, nor perturbations of the
10 Hz signal. These controls suggest that the results were not due to
system artefacts. Further tests also gave no evidence of radio fre-
quency energy associated with the stimulus.
Subjects were asked to indicate their conscious assessment for each
trial as to which stimulus was generated. They made their guesses
known to the experimenter via one-way telegraphic communication.
An analysis of these guesses has shown them to be at chance, in-
dicating the absence of any supraliminal cueing, so arousal as evid-
enced by significant alpha blocking occurred only at the noncognitive
level of awareness.
We hypothesise that the protocol described here may prove to be .
useful as a screening procedure for latent remote perceptual ability
in the general population.
0
Potential (arbitrary units)
6
16
5 Hz
10 Hz
15 Hz
Fig. 3 Occipital EEG spectra, 0-20 Hz, for one subject (H. H.)
acting as receiver, showing amplitude changes in the 9-11 Hz
band as a function of strobe frequency. Three cases: 0, 6, and 16
f.p.s. (12 trial averages).
CONCLUSION
From these experiments we conclude that:
.A channel exists whereby information about a remote location
can be obtained by means of an as yet unidentified perceptual
modality.
.As with all biological systems, the information channel
appears to be imperfect, containing noise along with the
signal.
. While a quantitative signal-to-noise ratio in the information-
theoretical sense cannot as yet be determined, the results of
our experiments indicate that the functioning is at the level
of useful information transfer.
It may be that remote perceptual ability is widely distributed
in the general population, but because the perception is generally
below an individual's level of awareness, it is repressed or not
noticed. For example, two of our subjects (H. H. and P. P.) had
not considered themselves to have unusual perceptual ability
before their participation in these experiments.
Our observation of the phenomena leads us to conclude that
experiments in the area of s stalled paranormal phenomena can
be scientifically conducted, and it is our hope that other
laboratories will initiate additional research to attempt to
replicate these findings.
This research was sponsored by The Foundation for Parasen-
sory Investigation, New York City. We thank Mrs Judith
Skutch, Dr Edgar D. Mitchell of the Institute of Noetic
Sciences-as well as our SRI associates, Mr Bonnar Cox,
Mr Earle Jones and Dr Dean Brown-for support and
encouragement. Constructive suggestions by Mrs Jean Mayo,
Dr Charles Tart, University of California, and Dr Robert
Ornstein and Dr David Galin of the Langley Porter Neuro-
psychiatric Institute are acknowledged.
RUSSELL TARG
HAROLD PUTHOFF
Electronics and Bioengineering Laboratory,
Stanford Research Institute,
Menlo Park, California 94025
Received March 11; revised July 8, 1974.
1 Pratt, J., Rhine, J. B., Stuart, C., and Greenwood, J., Extra
Sensory Perception after Sixty Years (Henry Holt, New
York, 1940).
Soal, S., and Bateman, F., Modern Experiments in Telepathy
(Faber and Faber, London, 1954).
Vasilliev, L. L., Experiments in Mental Suggestion (ISMI Pub-
lications, Hampshire, England, 1963).
4 Musso, J. R., and Granero, M., J. Parapsychology, 37, 13-37
(1973).
5
Osis, K., ASPR Newsletter, No. 14 (1972).
6
Tart, C. T., Physiological Correlates of Psi Cognition, Int. J.
Parapsychology, V, No. 4 (1963).
7 Dean, E. D., Int. J. Neuropsychiatry, 2 (1966).
8
Hill, D., and Parr, G., Electroencephalography : A Symposium
on its Various Aspects (Macmillan, New York, 1963).
9
Siegel, S., Nonparametric Statistics for the Behavioral
Sciences, 152-156 (McGraw-Hill, New York, 1956).
The stability of a feasible
random ecosystem
THE weight of the evidence, and the beliefs of most biologists,
seem to support the view1 that ecosystems tend to be more
stable, the larger the number of interacting species they con-
tain. It is puzzling, therefore, that a variety of mathematical
models of complex ecosystems appear to give the contrary
answer: that complexity makes for instability2.
Prominent among such models is the complex system with
random interactions, studied in various forms by Gardner and
Ashby3 and May4; but their results cannot be applied.as they
stand to ecological systems. In an ecosystem, the interacting
variables are species populations (or species biomass) which
cannot take on negative values. Thus, for example, the equili-
brium population values must be positive, and it is convenient
to denote this necessary property of an ecosystem model by
saying that it must be 'feasible'.
The work referred to imposed no such constraint on equili-
brium populations in the samples considered. It is of some
interest, therefore, to examine the stability of a random model
capable of representing ecosystems, by imposing the restriction
that the sample be feasible.
I report here the results of computer calculations on such a
model. The interaction equations were of the well-known
quasi-linear type, in which the rate of fractional increase of a
species population is a linear function of the current populations
in all T'species. That is, the number Na in the ith species obeys
dNidt = Νi (bi + Ej arj N;).
All birth rates b; were taken as 1, and the self-regulating
coefficients au as -1. The feasibility requirement was that the
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100-
FT=16
Percentage of feasible samples stable
T=10
T=7
90-
80-
70-
0
10
20
30
40
100
Percentage feasible of all samples
Fig. 1 Stability of feasible samples.
solutions for the equilibrium populations N*i, obtained by
equating the dNi/dr to zero, should be strictly positive. The
stability matrix S is given by Sij = N *¿ au; S is unstable if it
has an eigenvalue with positive real part. The interaction
coefficients were chosen to have a fixed magnitude z but
random sign (+ or - equally likely), with all T species
uniformly self regulated. A minimum of 2,000 samples were
examined for each value of T and z.
When all samples were accepted-whether feasible or not-
the results for T = 7, 10 and 16 agreed qualitatively with the
related (though not identical) models of refs 3 and 4. That is,
for z small, essentially all samples were stable, but the stable
fraction tended to zero as z approached T-+.
On examining the stability of the feasible samples only,
however, a significantly different behaviour emerges. We see
from Fig. 1 that feasible samples are generally stable, the stable
percentage departing from close to 100% only when the occur-
rence of a feasible sample is in any case rare (that is, when z is
large). Moreover, this tendency to stability increases with the
number T of interacting species. (It should be noted that the
curves in Fig. 1 contain no extrapolated portions, but are
constructed by interpolating between computed points of
negligible Monte Carlo error.)
The results displayed suggest the conclusion that, for this
model type, almost all feasible systems are stable when the
number of interacting species increases without limit. (The curve
for T = 16 is already barely distinguishable from such a step
function.)
The results confirm previous findings (both numerical3 and
analytical4), in showing that both feasibility and stability are
rare when z > T-+. It is not unreal, however, to expect average
interaction strength to decrease as the number of interactions
increases.
It is easy to see why the general conclusions emerging differ
from those of refs 3 and 4, when these are specialised to eco-
systems. The latter studies worked directly on the stability
matrix, and thus could not impose any feasibility requirement
(this must be derived from the solutions of the original inter-
action equations, which do not appear in their approach). Now,
unfeasible samples contain at least one species with a negative
equilibrium population; this simply means, biologically, that
after a time the species becomes extinct. But, if this extinction
is ignored, such 'ghost species' are being implicitly retained in
the stability examination and exert a profoundly destabilising
effect. In the quasi-linear model above, for example, they 'live
on' as an unregulated, exponentially increasing component
of the system. The diagonal element of the stability matrix is
-N+1, which is positive when N *: < 0. Thus the ghost species
figures in it like a real species (that is, one with a positive
number at equilibrium) with au = +1, that is, with an eventual
population increase proportional to N22.
The results cited awe've show that, when these 'mathematical
ghosts' are excluded, the model shows an actual increase of
stability in the larger systems.
ALAN ROBERTS
Physics Department,
Monash University,
Clayton, 3168,
Victoria, Australia
Received March 28; revised June 22, 1974.
1
May, R. M., Math. Biosc., 12, 59 (1971).
2
May, R. M., Math. Biosc., 12, 74 (1971).
3
Gardner, M. R., and Ashby, W. R., Nature, 228, 784 (1970).
May, R. M., Nature, 238, 413 (1972).
Objective evaluation of auditory
evoked EEG responses
THE averaging of often very small responses is widely used
in physiology, psychology and clinical medicine to evaluate
stimulus threshold levels. It is sometimes difficult to
recognise responses in the average waveform because of
residual activity, so a means of improving sensitivity and
objectively recognising positive responses would be useful.
We have previously found' that the averaged responses
(ERA) evoked in the electroencephalogram (EEG) of a
subject by auditory stimuli were produced by imposing a
phase constraint on the different Fourier harmonics of
the spontaneous EEG, without necessarily influencing their
amplitudes.
Merely imposing the phase-spectrum characteristic of a
response to high-level stimuli on length of unstimulated
spontaneous EEG from the same or another individual,
creates a waveform similar to and highly correlated
with a true average of responses to high-level stimuli
and a similar result is produced by imposing the appro-
priate phase spectrum on lengths of pseudo-random white
Gaussian noise instead of on spontaneous EEG. These
observations provide a convenient basis for the objective
detection of the existence of positive responses to given
stimuli and for the determination of auditory threshold
levels.
Our previous findings concerned the average, rather
than the individual, evoked response but clearly must
reflect phenomena in the separate lengths of EEG follow-
ing individual stimuli. If so, it should be possible to demon-
strate a phase-constraint operative on the various har-
monic components of the individual post-stimulus lengths
of EEG and such a demonstration would be useful in sug-
gesting the basis of an objective assessment of the occur-
rence of response. Unfortunately, phase spectra do present
difficulty of interpretation and in the present context, may
be liable to some error if unnecessarily long record lengths
are used. One problem is that phase values are restricted
to the range -180° to +180°, and as a result of the
repeated recurrence of the principal range of angles that
can occur, averaging phase-spectral values for any given
harmonic component, in successive sweeps, is usually
unsatisfactory. We have therefore investigated instead
whether the ensemble distribution of phase values for each
harmonic component shows any aggregation, or if it shows
the uniform distribution characteristic of uncontrolled
phases as seen in unstimulated records.
Phase values have been calculated for each of the first
ten Fourier harmonic components and classified into four
equal-size groups covering the full 360° range of possible
values. The null hypothesis leads to a uniform distribution
of phase values so that for 60 sweeps, the expected number
of values is 15 in each class. A x2 test with d.f. = 3 can
then be applied to test the null hypothesis (that is, no
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phase control), for each harmonic porately. The results
show that for high-level stimuli, the null hypothesis must
be rejected on the basis of one or more harmonics
(P<0.01), but for stimuli that are evidently sub-
threshold according to both subjective judgment and
appearance of the averaged response waveform, the null
hypothesis holds. Intermediate levels lead to x2 values
that broadly decrease in significance as stimulus level
decreases and the 5% level occurs near the subjective
threshold. For high-level stimuli which produce an evident
response, most of the harmonics exhibit the phase con-
straint, but as the stimulus level is reduced towards
threshold, significant aggregation of phase values is con-
fined only to a few harmonics. The effects reported here
have been studied for increasing lengths of post stimulus
record, starting with about 0.32 s and ultimately reaching
the full length of our records (0.94 s post stimulus). The
existence of a phase constraint is increasing evident with
increasing record length; the effect often fails altogether,
however, when the record length is allowed to exceed
. about 0.8 s. This observation suggests that after about
0.75 s any phase control of spontaneous EEG activity
diminishes, allowing fully spontaneous conditions to be
generally restored beyond this point. If the record used for
analysis did include, for this reason, a segment of spon-
taneous activity, the appearance of the phase constraint
would then be lost.
These findings apply in each of the trials with different
tonal or click stimuli for all the six subjects (three adults,
three children) reported previously1, and similarly to each
of the records from three further subjects that we have so
far examined. Thus, a non-subjective and quantitative
system of auditory assessment would seem to be feasible,
utilising phase-spectral measurements alone. This approach,
however, implicitly utilises some information about the
amplitude spectrum of the signal, in that low amplitude
Fourier components are neglected (by restricting analysis
to the first ten or fewer harmonics), and we would not rule
out the possibility of utilising further amplitude spectral
information to supplement the indications provided by a
phase analysis.
We would also remark that the characterisation of these
signals in phase-spectral terms is an abstraction to describe
a mechanism that is perhaps more realistically envisaged
as a synchronisation process in the time domain, rather
than as a phase-constraint in the frequency domain.
We acknowledge the support of the Medical Research
Council, the Spastics Society and the Ewing Foundation.
B. McA. SAYERS
Engineering in Medicine Laboratory,
Department of Electrical Engineering,
Imperial College of Science and Technology,
London SW7 2BT, UK
H. A. BEAGLEY
Institute of Laryngology and Otology,
330-336 Gray's Inn Road,
London WCIX 8EE, UK
Received May 10; revised July 16, 1974.
Sayers, B. McA., Beagley, H. A., and Henshall, W. R., Nature,
247, 481 (1974).
Imprinting and exploration
of slight novelty in chicks
SHORTLY after hatching, domestic chicks will respond
socially to a wide range of moving or conspicuous objects.
As a result of 'imprinting', their social behaviour is increas-
ingly limited to objects with which they have had visual
experience. In general, the longer that chicks have been
exposed to an object, the, more strongly they prefer it when
given a choice between it and something else1-5. Some
evidence, however, suggests that in the early stages of im-
printing, the trend towards a highly restricted preference
is temporarily reversed and some birds may even prefer
the unfamiliar object6,7.
Here we report that day-old chicks will even work to
expose themselves to novel stimulation at an early stage
in imprinting. At that stage, by changing the characteristics
of a visual stimulus when a chick stepped onto a pedal,
it was possible to increase the likelihood that it would step
on to the pedal again.
Bateson and Reese® found that day-old chicks hatched
and reared in the dark would rapidly learn to press a pedal
which turned on a flashing rotating light. We have used a
similar technique in this study but with two important
differences: first, the chicks had been exposed to a flashing,
rotating light for varying amounts of time before the ex-
periment; second, the familiar stimulus was turned on when
a bird was not on the pedal. As soon as the chick stepped
on the pedal the colour of the stimulus was changed. In
other words, the bird was exposed to a novel stimulus for
as long as it stood on the pedal.
The apparatus consisted of a chamber measuring 37.5
by 34.5 by 33 cm. Two of the walls were made of hard-
board and the front and back walls of 1.27-cm wire mesh.
Two pedals were set into the floor; each measured 4.8 cm
square and was placed 2.0 cm from the wire mesh front and
2.6 cm from one of the sides. All the apparatus was painted
matt black except for the pedals which were matt white.
The temperature 5 cm above the floor of the apparatus
was 23.5-26.5° C. The stimulus was placed 60 cm in front
of the wire mesh and midway between the two pedals, It
N
32
24
24
d
70
b
C
4
H
4
Percentage ratio
50
-
-
-
301-
N
15
30
60
Time of initial exposure (min)
Fig. 1 Means and s.e. for the total presses by day-old
chicks of a pedal altering a flashing light from a familiar to
unfamiliar colour. The pedal presses are expressed as
percentages of the total presses of that pedal and another
symetrically placed pedal having no effect on the light.
Open bars are percentage durations of pressing and solid
bars are percentage frequencies of pressing. The chicks
were previously exposed to the familiar light for 15, 30
or 60 min. Departures from the chance level of 50% are
indicated as follows: a, t= 3.29, P < 0.01; b, t = 2.56,
P < 0.02; c, t = 2.14, P < 0.05.
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consisted of a translucent cylinder 15.3 cm in diameter and
16 cm in length rotating at 45 r.p.m. on a horizontal axis.
The cylinder was covered with a chequerboard pattern
(2-cm squares). A primary red and primary yellow filter
were fitted inside the cylinder, each over a 45 W bulb. The
bulbs could be individually lit to make the whole cylinder
appear as either red or yellow checks; the background was
matt black. When one pedal was pressed the colour of the
cylinder changed; the other pedal had no effect on the
stimulus and was used to measure any general changes in
activity.
Eighty Ross 'Chunky' chicks were hatched in the
laboratory and individually exposed to constant white light
for 50 min. When they were 14-22 h old, half were indivi-
dually exposed in activity wheels' to a flashing rotating light
like the red stimulus in the operant conditioning situation
and half were exposed to one like the yellow stimulus. The
chicks were exposed for 15, 30 or 60 min and 3 min later
were separately placed in the operant conditioning box.
The experimental design was balanced so that for half the
chicks the pedal changing the colour of the stimulus was
on the right hand side and for the other half it was on the
left hand side. Each chick was left in the apparatus for
50 min.
The results are shown in Fig. 1. The presses of the pedal
changing the colour of the light are expressed as a per-
centage of the total presses of both pedals; if they were
responding at the chance level the ratio would have been
50%. The absolute measures of pedal pressing were the
same for all groups. In Fig. 1 percentages are given for both
the number of presses in the 50-min tests and the total time
spent pressing the pedals. It can be seen that after 15 min
previous experience with the familiar stimulus the chicks
pressed the pedal which changed the colour of the light
more frequently and for longer than they did the other
pedal. The red-trained birds did not differ from the yellow-
trained birds in the extent to which they worked for change.
In the red-trained chicks exposed for 30 min, the effect
was much less clearly marked and by 60 min the birds in
both groups were responding at the chance level. The over-
all decline in the ratios is statistically significant for both
measures (for response number Pearson r =- 0.242, P<0.05,
for response duration, r =- 0.240, P<0.05).
Differential pressing of the two pedals cannot be attributed
to an initial preference for one stimulus over the
other regardless of previous experience because the ex-
perimental design was balanced and the subgroups did not
differ. Similarly their behaviour cannot be explained in
terms of a preference for the position of one pedal over
the other. We believe that the results unequivocally support
earlier suggestions that chicks prefer novel objects at an
early stage in imprinting6. Moreover, they show that the
birds will actively work to expose themselves to slightly
novel stimulation. The biological advantages of such be-
haviour may lie in the very complexity of what the young
bird has to learn under natural conditions. The ability to
recognise a parent from all likely angles almost certainly
requires more information than could possibly be acquired
as the result of learning the characteristics of a single view
of that parent. The transformation from front view to side
view and from side view to back view could not easily be
accomplished by mechanisms ordinarily understood as being
involved in perceptual constancy7. Therefore, if a young
bird must recognise its parent from a variety of angles it
must have first seen its parent from all those angles. This
would in part be accomplished by rotations of the parent
as it went about its business; but the process of attachment
would be accelerated if, in addition, the young moved round
the parent after familiarising themselves with one view.
Such a process of working for slight novelty balanced
against a growing restriction of preference for what is
familiar would not only make good biological sense, it
would account for the birds preferring the unfamiliar object
only in the early stages of imprinting10.
This work was supported by a grant from the Science
. Research Council to P.P.G.B. We thank Joan Stevenson-
Hinde, N. K. Humphrey and S. E. G. Lea for their com-
ments on the manuscript.
P. S. JACKSON
P. P. G. BATESON
Sub-Department of Animal Behaviour,
University of Cambridge,
Madingley, Cambridge CB3 8AA, UK
Received June 7, 1974.
1
Bateson, P. P. G., J. comp. physiol. Psychol., 86, 586 (1974).
Connolly, K., Br. J. Psychol. 59, 453 (1968).
3
Hess, E. H., Science, 130, 133 (1959).
'Sluckin, W., and Salzen, E. A., Q. Jl exp. Psychol., 13, 65,
(1961).
$ Zajonc, R. B., Reimer, D. J. and Hausser, D., J. comp.
physiol. Psychol., 83, 434 (1973).
Bateson, P. P. G., in Constraints on Learning (edit. by Hinde,
R. A., and Stevenson-Hinde, J.) 101, (Academic Press, Lon-
don, 1973).
7
Bateson, P. P. G., Scient. Am. (in the press).
8
Bateson, P. P. G., and Reese, E. P., Anim. Behav. 17, 692
(1969).
9
Bateson, P. P. G., and Wainwright, A. A. P., Behaviour 42,
279 (1972).
10 Bateson, P. P. G., J. theor. Biol., 41, 249 (1973).
Microbial activation of prophenoloxidase
from immune insect larvae
INTEREST in the infection of insects by parasites has been
generated by the pathogenic potential of the parasite to the
insect itself, or to a vertebrate host to which the parasite
may be transmitted. For example, because of the effects.
of malaria on man, the relationships between Plasmodia
and various mosquito species have received much atten-
tion1.2. An important part of the host-parasite relationship
is the defence system of the host. In insects, phenoloxi-
dase (o-diphenol: O2 oxidoreductase, EC 1.10.3.1.) has
been suggested to be at least partially responsible for insect
immunity and parasite resistance3-5.
The enzyme produces quinones which can react with
proteins6 and with the thiol and amino groups of other
compounds6-8. Quinones can be bactericidal9. When qui-
nones are present in excess, the black pigment melanin
is formed6. A layer of melanin is frequently observed
deposited on parasites by insects as part of their resistance.
The exact reactions of quinones with parasite tissues have
not been determined, but by blocking quinone formation
with phenylthiourea an insect's parasite resistance can be
suppressed5. Melanin deposition is often associated with
insects' cellular response5, but can also be a strictly humoral
response1º. In some cases of unsuccessful malaria transmis-
sion by mosquitoes there is an apparent melanisation
manifest in Ross black spores and degenerate oocysts1.2. For
quinones to be involved in resistance to an insect parasite,
however, control of phenoloxidase activity would be essen-
tial. Quinones randomly reacting with the insect host would
be ineffective as antiparasitic agents. Furthermore, large
amounts of active phenoloxidase can kill insects11. As a
contribution to this problem, I describe here the activation
of prophenoloxidase in the plasma of immune12,13 Galleria
mellonella larvae by microbial products.
For immunisation, larvae (each larger than 200 mg) were
injected with 1.0-ug doses of Shigella flexneri lipopoly-
saccharide B (Difco)13 and incubated at 23° C. Control
larvae were uninjected. Haemolymph was collected 18-24 h
after immunisation and pooled in a CO2 environment; at
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Table i"Enzyme activity in (NH4),SO, fractions of immune plasma
Protein
Phenoloxidaseț
Lysozyme
Zymosan activation
a-Chymotrypsin activation
(NH),SO4
fraction*
mg g-1
lyophilisatet
U g-1
lyophilisate
Specific
activity§
U g-1
lyophilisate
Specific
activity§
Ug-1
lyophilisate ||
0-40% ppt., fraction A
10
0
0
43,950
4,395
0
40-50% ppt.
85
0
0
21,850
257
0
50-60% ppt.
140
0
0
50
0
300
60% Supernantant, fraction B
Whole plasma T
558
144,045
258
153,745
276
* Plasma was prepared from reconstituted haemolymph as described in the text. Fractions were prepared by the addition of solid salt18 (100%
saturation = 70.6 g per 100 ml). Precipitates were dissolved in 0.01 M KPO4-2, pH 6.0 (the pH and ionic concentration-as determined by
freezing point depression-of the plasma). Before testing enzymatic activities all fractions were dialysed against this buffer to remove the salt.
t Assayed by the method of Lowry et al.19.
# 0.20 ml of a fraction was mixed with 0.10 ml of activator in water: a chymotrypsin, 6 mg ml-1: zymosan, 1 mg ml-1. Mixtures were incuba-
ted for 5 min at 30° C, then assayed for phenoloxidase. Values for water controls have been subtracted.
§ U mg-1 protein.
| AA450; == - 0.001 per min at 30° C of a suspension of Micrococcus lysodeikticus was one lysozyme unit20.
T No salt added, no dialysis ..
least 20 larvae were used for each pool. Care was taken
to avoid gross contamination of haemolymph with fat body
tissue. To quantitate properly phenoloxidase activity per
Prophenoloxidase activation (U per 0.20 ml immune plasma)
400} P>0.15
Cont.
P<0.01
Imm.
Imm.
300
P<0.01
Imm.
200
100
Cont.
Cont.
79
381
33
225
393
361
90 139
329:333
425
32 | 287
280 313
13
353
23
217
616
263
174
339
130
211
.a Chymo- Zymosan
Pseudomonas
aeruginosa
trypsin
Activator
Fig. 1 Activation of prophenoloxidase in control (Cont)
and immune (Imm) plasmas by various materials. The values
indicated by the bars are the average amounts of pro-
phenoloxidase found in five different determinations. A
determination used 0.20 ml of plasma from a different pool
of control or immune haemolymph mixed with 0.01 ml of
activator solution or suspension, incubated for 5 min at
30° C, then assayed for phenoloxidase. (See text for assay
technique.) Results for individual determinations are listed
within each bar. With no activator materials present-water
controls-the phenoloxidase activities of plasmas ranged
from zero to two units; these values have been subtracted.
Statistical comparisons are between control and immune
plasmas. «-Chymotrypsin (Nutritional Biochemicals Corp.,
three times crystalline) 6 mg. ml-1 water. Zymosan16 (General
Biochemicals) 1 mg ml-1 water. Pseudomonas aeruginosa
P11-112 was grown in nutrient broth +1% glucose and
collected at 24 h by centrifugation, 8,000g for 15 min. Cells
were washed with 0.85% NaCl, then brought to A350=1.1,
a concentration of about 5 x 109 bacteria per ml and frozen.
The cell suspension was thawed, resuspended in water and
refrozen. It was rethawed and heated at 100° C for 30 min.
After cooling, 0.10 ml aliquots were used to activate pro-
phenoloxidase. None of these activator materials had
inherent phenoloxidase activity.
amount of haemolymph and to facilitate handling, pooled
haemolymph was quick frozen with acetone-dry ice, lyophi-
lised and stored at -20° C. Haemolymph from control
larvae was collected and stored in an identical manner.
My preliminary experiments and those of Evans14 have
shown that haemolymph phenoloxidase occurs as the
proenzyme in plasma and can be activated by material
from haemocytes. To test for other types of activation,
lyophilised haemolymph was reconstituted with distilled
water to a concentration of 10 mg solids per ml. This was
1/17 to 1/18 the usual concentration of solids in fresh
haemolymph, and its purpose was to slow spontaneous
activation of prophenoloxidase by haemocytes, so that
plasma could be collected before activation. The plasma
and cellular fractions were separated by centrifuging whole
reconstituted haemolymphs at 650g for 15 min at 4º C.
The supernatant fluids-the plasmas-were removed and
used immediately. When water was added to the haemo-
cytes in the lyophilised material no lysis was observed
microscopically. Also, haemocyte concentrations were cal-
culated, with correction factors for dilutions, to be.
18,590 mm-3 for immune and 40,040 mm-3 for control
reconstituted haemolymph, slightly higher than those
reported by Stephens15 for immune and control fresh
haemolymph. These observations tend to preclude massive
haemolysis during handling although the release of some
haemocyte materials into the plasmas was possible.
Phenoloxidase activity was measured by spectrophoto-
metric determinations of initial reaction rates with a Gilford
recording spectrophotometer. For a standard assay, 0.30 ml
of test material was mixed with 4.7 ml of 0.1 M KPO,2",
pH 6.4, containing 20. umol of 4-methylcatechol and 40
umol of 4-hydroxyproline ethyl ester. 4-Methylcatechol was
enzymatically oxidised to its quinone. The quinone reacted
nonenzymatically with 4-hydroxyproline ethyl ester to form
a stable product, 4-(4'-hydroxy-2'-carbethoxy-1'-pyrrolidyl)-
5-methyl-o-benzoquinone, with, a Amax at 520 nm (ref. 7).
In the system used, with excess substrates, AA520 was
directly proportional to enzymatic activity. One phenoloxi-
dase unit was defined as AA 520= +0.001 per min at 30° C;
the maximum rate observed during the first 2 min after the
initiation of the reaction was used. By measuring the
phenoloxidase activity of plasmas incubated with and with-
out activator materials, prophenoloxidase activation was
quantitated.
Immune plasmas showed significantly more prophenoloxi-
dase activation than control plasmas when both were in-
cubated with either zymosan (a yeast polysaccharide)16 or a
preparation of damaged Pseudomonas aeruginosa (Fig. 1).
After treatment with @ chymotrypsin (EC 3.4.4.5.) both
control and immune plasmas showed a high degree of
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14,300
66
0
0
0
241
64
82
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prophenoloxidase activation, similar to that in immune
plasmas treated with the microbial products.
The activator(s) associated with zymosan and P., aeru-
ginosa did not have esterase activity towards benzoyl-L-
tyrosine ethyl ester when tested at pH 6.0 (the pH of the
plasmas) or at pH 7.8. The activator(s) in zymosan suspen-
sions was associated not only with particles; but also with
the particle-free solution. It (they) passed through a 0.22-
um filter, but not through a .dialysis membrane. The
zymosan activator(s) was stable at 100° C for 3 h, but after
3 h in 0.2 M H2SO4 at 100° C and then neutralisation with
Ba(OH)2 it was not found when assayed. Activation asso-
ciated with P. aeruginosa was not manifest unless the cells
were damaged by physical means or by pretreatment with
egg white lysozyme (EC 3.2.1.17.) (Fig. 2). Egg white
lysozyme does not lyse or kill P. aeruginosa, but alters its
cell wall17.
Immune plasma was fractionated with (NH4)2SO4 (Table
1). The material precipitated with 40% saturation (fraction
A) contained most of the prophenoloxidase. The material
still in solution after 60% saturation (fraction B) had most
of the lysozyme activity. Prophenoloxidase in fraction A
was activated by « chymotrypsin, but not by zymosan
alone; however, the prophenoloxidase in fraction A could
be activated by zymosan if fraction B was also added. If
egg white lysozyme was substituted for fraction B, activa-
tion did not occur. The Kms of phenoloxidase activated by
zymosan and « chymotrypsin for 4-methylcatechol were
similar, 1.20 and 1.07 mM respectively. Figure 3 shows
that when prophenoloxidase was diluted before activation
80}
Phenoloxidase (U)
60
40
20
1
1/4
1/2
3/4
1/1
Dilution
Fig. 3 Effect of prophenoloxidase concentration in the
zymosan activation mixture on the resulting phenoloxidase
activity. , Prophenoloxidase diluted before activation
(y =81x2): fraction. A was brought to various dilutions with
0.01 M KPO.2-, pH 6.0. 0.10 ml of a dilution was mixed
with 0.10 ml of fraction B, and 0.10 ml of zymosan suspen-
sion, 1 mg ml-1 water. Each of these mixtures was incubated
for 5 min at 30° C, then assayed. for phenoloxidase.
., Phenoloxidase diluted after activation (y = 81x): fraction
A, fraction B, and zymosan suspension were mixed 1:1:1
(v : v: v), then incubated at 30° C. After 5 min, various
dilutions of this mixture were made using a mixture of
0.01 M KPO42-, pH 6.0, and zymosan suspension, 2: 1 (v : v).
Immediately after all dilutions had been prepared, 0.30 ml
aliquots were assayed for phenoloxidase activity. These
assays were completed within 15 min.
.
by zymosan there was an exponential relationship between
proenzyme concentration and activity, while dilution of
the enzyme after activation gave an almost linear relation-
ship.
..
Activation of prophenoloxidases is associated with the
aggregation of enzyme subunits18,21. An aggregation mech-
anism is consistent with the concentration effect shown in
Fig. 3. Haemolymph lysozyme in fraction B may partially
digest zymosan material which then acts as a site for the
adsorption, aggregation and activation of prophenoloxidase
subunits. This would explain the differences observed be-
tween control and immune plasmas in Fig. 1, since immune
haemolymph contains larger concentrations of lysozyme22.
Another hypothesis is that zymosan activates a proteolytic
enzyme in fraction B, as it does in complement23. The
proteolytic enzyme would in turn activate the prophen-
oloxidase.
I thank Dr J. D. Briggs for help and Dr V. Raghavan
for the use of a spectrophotometer. I thank Dr H. G.
Boman for useful discussions and for help in manuscript
preparation. Financial support for these investigations came
in part from the World Health Organization International
Reference Centre for Diseased Vectors.
ALBERT E. PYE*
Department of Entomology,
Ohio State University,
Columbus, Ohio 43210
Received September 4, 1973; revised July 12, 1974.
*Present address: Department of Microbiology, University of
Umea, S-901 87 Umea, Sweden.
1 Garnham, P. C. C., in Host-parasite relationships in inverte-
brate hosts (edit. by Taylor; A. E. R.), 33 (Blackwell
Scientific Publications; Oxford, 1964):
2
Bafort, J. M., Ann. Soc. Belge Med. Trop., 51, 1 (1971).
3
Eckstein, F., Anzeiger für Schadlangskunde, 7, 49 (1931).
4
Taylor, R., J. Invertebrate Pathol., 14, 427 (1969).
5.
Nappi, A. J., Parasitology, 66, 23 (1973).
6
Mason, H. S., in Pigment cell biology (edit. by Gordon, M.),
563 (Academic Press, New York, 1959) ..
7 Jackson, H., and Kendal, L. P., Biochem. J., 44, 447 (1949).
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Prophenoloxidase activation (U per 0.20 ml immune plasma)
400
. 300
P <0.01
200-
P<0.05
100
8
217
6
88
23
478
16
357
209
293
465
80
Frozen
in saline
Frozen
in water
Lysozyme .
Treatment of Pseudomonas aeruginosa
Fig. 2. Manifestation of prophenoloxidase activator(s) in
Pseudomonas aeruginosa after various treatments. The acti-
vator(s) was determined by the activation of prophenol-
oxidase in immune plasmas. The assay procedures were the
same as for Fig. 1. P. aeruginosa cells frozen in 0.85%. NaCl
saline (see Fig. 1), resuspended in water; but not refrozen
or heated, served as controls. Statistical comparisons are
between these cells and those submitted to other treatments.
The cell suspension frozen in water was the P. aeruginosa
preparation used for Fig. 1: Cells treated with lysozyme
were frozen in saline, resuspended in 0.1 M KPO.2-, pH 7.0,
with 10 mg lysozyme (Sigma, from egg white, three times
crystallised) ml-1, incubated 20 min at 30° C, and then re-
suspended in water. Bacterial concentrations were the same
for all three suspensions.
85
30
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613
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Nature VOL. ZJI VCIVUCI
8
Mason, H. S., and Peterson, E. W., Biochem. biophys. Acta,
111, 134 (1965).
9
Thalhimer, W., and Palmer, B., J. Infect. Dis., 9, 181 (1911).
10
Poinar, G. O., and Leutenegger, R., J. Ultrastr. Res., 36, 149
(1971).
11
Zlotkin, E., Gurevitz, M., and Shulov, A., J. Insect Physiol.,
19, 1057 (1973).
12
Stephens, J. M., Can. J. Microbiol., 8, 491 (1962).
13
Chadwick, J. S., and Vilk, E., J. Invertebrate Pathol., 13, 410
(1969).
14
Evans, J. J. T., J. Insect Physiol., 14, 107 (1968).
15
Stephens, J. M., J. Insect Pathol., 5, 152 (1963).
16
Pillemer, L., and Ecker, E. E., J. biol. Chem., 137, 139 (1941).
17
Cox, S. T., and Egon, R. G., Can. J. Microbiol., 14, 913 (1968).
18
Ashida, M., and Ohnishi, E., Arch. Biochem. Biophys., 122,
411 (1967).
19
Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall,
R. L., J. biol. Chem., 193, 265 (1951).
20
Worthington enzyme manual. Enzymes, enzyme reagents,
related biochemicals (Worthington Biochemicals Corp.,
Freehold, New Jersey, 1972).
21
Munn, E. A., and Bufton, S. F., Eur. J. Biochem., 35, 3 (1973).
22
Chadwick, J. S., J: Invertebrate Pathol., 15, 455 (1969).
23
Davis, B. D., Dulbecco, R., Eisen, H. N., Ginsberg, H. S.,
and Wood, W. B., Microbiology, second ed. (Harper and
Row, Hagerstown, Maryland, 1973).
Elevation of total serum IgE in rats
following helminth parasite infection
HELMINTH parasites provide the most potent stimulus
known for the biosynthesis of IgE. In every species which
has been studied, infections with parasitic worms lead to
the production of high levels of reaginic or IgE antibodies
against parasite antigens1,2 and, in man, greatly elevated
levels of total IgE occur in most individuals infected with
any one of a variety of helminths including Ascaris
lumbricoides3, Capillaria philipinensis4 and ancylostomes5.
Experiments in the rat have indicated how the IgE
stimulating effect of helminth parasites may operate. In this
species, infections with Nippostrongylus brasiliensis or
Fasciola hepatica, as well as stimulating parasite specific IgE
antibodies, cause nonspecific potentiation of unrelated IgE
responses to such antigens as egg albumen, keyhole limpet
haemocyanin, and house dust6-8. Present evidence indicates
that live worms produce a factor which, acting via T cells9,
has the capacity to stimulate those B cells which have been
programmed for IgE production by previous immunisation.
In so far as IgE responses against different antigens may be
simultaneously potentiated* it seems likely that all the
existing IgE responses of the animal are affected.
Here we now present evidence which supports this. We
have compared the level of total serum IgE in rats follow-
ing conventional immunisation with that of rats whose
response has been potentiated by subsequent infection with
helminth parasites. In the latter, the amount of total serum
IgE is much greater, and far more than can be ascribed to
IgE antibodies specific for the immunising antigen or the
parasite.
Until recently it has not been possible to estimate total
serum IgE in the rat, or indeed in any other species but
man, for lack of specific antiserum to this immunoglobulin.
The recent discovery of a strain of rats bearing IgE immuno-
cytomas10,11 has overcome this difficulty since they provide
a source of myeloma protein from which purified IgE can
be isolated in relatively large amounts. Such a purified IgE
preparation has been used to induce specific antiserum to
rat IgE and also as a reference preparation in an assay for
rat total serum IgE using the radioactive single-radial-
diffusion technique of Rowe12.
The specific immunological reagents were prepared as
follows: The purification of rat IgE from the serum or rats
bearing IgE secreting immunocytomas was as previously
described10 with the following modifications. Ammonium
sulphate precipitation was performed at 50% final concen-
tration, and as a supplementary step preparative agarose
electrophoresis was done in 1% agarose equilibriated
with 63 mM sodium barbiturate, 15 m.M Barbital, 1.8
mM calcium lactate buffer, adjusted to pH 8.6 with HCI.
The electrophoresis was carried out for 1 h at 20 V cm-1.
After electrophoresis, the position of the monoclonal IgE
was located by comparison with a fixed and stained agarose
gel electrophoretic pattern of the same IgE preparation. The
agarose-containing myeloma IgE was cut out and removed
from the slide and the proteins were extracted from the gel
by pressure. Goat antiserum to rat IgE was obtained by
immunisation with purified antigen (IR2 monoclonal
protein) emulsified in complete Freunds adjuvant. Antisera
were absorbed with a solid immunoabsorbant13 made of
serum from germ-free rats. Antiserum to goat IgG was
prepared by immunisation of rabbits with goat IgG (pre-
pared by the caprylic acid method)14 in complete Freunds
adjuvant. The antiserum was also purified by the caprylic
acid technique and the resultant IgG fraction was labelled
with 125I by the method of McConahey and Dixon15.
Dilutions of anti-rat IgE in the agar from 1:100 to
1 :4,000 could be used. The last dilution gave the greatest
sensitivity enabling estimation in the range of 0.35-22.24 µg
IgE ml-1. The reference preparation (IR162 Temoin) con-
tained 5.7 mg ml-1 of rat IgE.
Outbred female Hooded Lister rats weighing 150-200 g
were used in the experiments described here. Twenty normal
rats (Group 1) were bled before treatment and were then
Table 1 Reaginic antibody titre and total serum IgE (mean ± s.e.) in serum from normal, immunised
infected and potentiated rats
Passive cutaneous anaphylaxis*
Group
Treatment of rats
titre
N. brasiliensis
Total serum IgE
(µg ml-1)
most rats <0.35
1
Normal rats no treatment
2
Primary response to 1 µg EAt
92 ± 36
1.33 ± 0.25
3
Secondary response to 10 ng EA
1,228 ± 231
1.80 ± 0.39
4a
Potentiated reagin response to EA 12 d after N. brasiliensist
1,316 ± 270
288 ± 47
4b
Not potentiated reagin response to EA 12 d after N. brasiliensis
(pool of 12 sera)
4,096
493
5
Unimmunised rats 12 d after N. brasiliensis infection
247 ± 42
6
Unimmunised rats 18 d after N. brasiliensis infection
640 + 109
104 ± 8
*To estimate specific reaginic antibodies by the passive cutaneous anaphylaxis technique17, 0.1 ml. quantities of saline dilutions of the test
sera were injected intradermally in Hooded Lister recipient rats each injection being duplicated on a different animal. 48-72 h later, the
animals were injected intravenously with 2.5 mg of EA or 0.5 ml N. brasiliensis antigen (saline extract of 500 homogenised worms) together
with 0.5 ml of 1% Evans blue which acts as an indicator of vasodilation. The skin reactions were examined after 20 min and the titres
recorded above are the greatest dilutions of serum which gave reactions larger than 5 mm in diameter.
tEgg albumen.
#The method of culture of N. brasiliensis and preparation of the infective dose have been previously described18,19.
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Ivature vol. 201 October 18 1974
immunised with 1 µg egg albumen (Sigma grade V) injected
intraperitoneally together with 101º B. pertussis organisms
(Wellcome Biological Reagents). They were bled again 20 d
after immunisation (Group 2) and were then divided into
two groups and treated as follows: Group 3 rats received a
booster injection of 1 µg egg albumen and were bled 4 d
later, while Group 4a rats were infected by subcutaneous
inoculation of 4,000 N. brasiliensis larvae and were bled 12
d later. These times of bleeding were determined by previous
observations of the timing of peak secondary and potentiated
reagin responses respectively6,16
.
Table 1 shows the specific reaginic antibody levels and
total serum IgE in the serum of these animals. It is evident
that the second injection of egg albumen although resulting
in a good secondary antigen-specific IgE response (Group 3)
did not have a marked effect on total serum IgE. Infection
with N. brasiliensis on the other hand (Group 4a), as well as
causing a similar elevation of egg-albumen specific IgE
resulted in a most dramatic elevation of total serum IgE
from 1.33-288 µg ml-1. This level is well in excess of that
which, by comparison of the egg-albumen reagin titres of
the two groups, can be accounted for by egg-albumen
reagins alone. Equally the parasite-specific response cannot
be implicated at this time after infection since anti-N.
brasiliensis reagins do not normally appear until later in the
infection and no parasite specific reagins could be detected
in these areas. A pool of serum from another group of
rats with a very marked potentiated reagin response to
egg albumen (Group 4b) showed even greater levels of total
serum IgE.
The remaining two groups shown in Table 1 were 10
normal animals infected with N. brasiliensis without pre-
vious immunisation. Group 5 were bled 12 d after infection
with 4,000 larvae at which time no parasite specific reagins
were present. Greatly elevated levels of total serum IgE
occurred in this group in the absence of IgE antibody
responses to any known antigens. Group 6 were bled 18 d
after infection when parasite specific IgE was present, but
when the level of total serum IgE had actually declined.
The results following infection with the parasite Fasciola
hepatica have been essentially similar to those we report
here.
We have also estimated total serum IgE in Wistar rats
and find that animals of this strain do not produce levels as
high as those of the Hooded Lister strain. For instance, 12 d
after N. brasiliensis infection the IgE level in Wistar serum
(five rats) was 25.3+5.6 µg ml-1. It has been reported that
Hooded Lister rats have an exceptional ability to produce
IgE antibodies, in that prolonged responses are induced by
immunisation with small doses of conventional antigen and
that marked secondary responses can occur16. It is interest-
ing that this facility is also reflected in very high levels of
total serum IgE (up to 493 µg ml-1) following helminth
infection in rats of this strain.
The main points which emerge from these results are that
infection of the rat with helminth parasites leads to a
great elevation of total serum IgE and that the bulk of
this IgE is surplus to levels of IgE antibody against known
antigens. It would seem reasonable to assume that this IgE
consists at least in part of a number of potentiated reagin
responses to miscellaneous unknown antigens to which the
animal has previously become sensitised. By analogy it is
of course tempting to suppose that the high levels of total
serum IgE seen in parasite infected human beings consist
not exclusively of antiparasite IgE responses to unrelated
antigens.
This work was supported by the Medical Research Coun-
cil and the Fonds Cancerologique of the Caisse Generale
Generale d'Epargne et de Retraite (Brussels). E. J. is in
receipt of a fellowship from the Asthma Research Council.
H.B. is a staff member of Euratom Biology Division. We
thank David Haig and Jenny Naze-deMets, Bernadette
Plateau and Jean Pierre Kints for their assistance.
ELLEN JARRETT
Wellcome Laboratories for Experimental Parasitology,
University of Glasgow,
Veterinary Hospital,
Bearsden, Glasgow G61 1QH
and
University Department of Bacteriology and Immunology,
Western Infirmary,
Glasgow GI1 6NT, UK
HERVÉ BAZIN
Faculty of Medicine,
University of Louvain,
Clos Chapelle-aux-Champs 4,
B.1200 Brussels, Belgium
Received July 4, 1974.
1 Ogilvie, B. M., and Jones, V. E., Cellular and Humoral
Mechanisms in Anaphylaxis and Allergy, 13-22 (Karger
Basel, 1969).
Sadun, E. H., in Immunity to Animal Parasites (edit. by
Soulsby, E. J. L.), 97-129 (Academic Press, 1972).
3 Johansson, S. G. O., Melbin, T., and Vahlquist, B., Lancet, i,
1118-1121 (1968).
4 ]
Rosenberg, E. G., Whalen, G. E., Bennich, H., and Johansson,
S. G. O., New Engl. Jl Med., 238, 1148-1149 (1970).
Bennich, H., and Johansson, S. G. O., Adv. Immun., 13, 1-55
(1971).
Orr, T. S. C., and Blair, A. M. J. N., Life Sci., 8, part III,
1073-1077 (1969).
7 Jarrett, E. E. E., Immunology, 22, 1099-1101 (1971).
8
Jarrett, E. E. E., and Stewart, D. C., Immunology, 23, 749-
755 (1972).
Jarrett, E. E. E., and Ferguson, A., Nature, 250, 420-422
(1974).
10
Bazin, H., Beckers, A., Deckers, C., and Moriame, M., J.
natn. Cancer Inst., 51. 1351-1361 (1973).
11
Bazin, H., Querinjean, P., Beckers, A., Heremans, J. P., and
Dessy. F., Immunology, 26, 713-723 (1974).
Rowe, D. S., Bull. Wld Hlth Org., 40, 613-616 (1969).
13
Avrameas, S., and Ternynck, J., Immunochemistry, 6, 53-66
(1969).
14
Steinbuch, N., and Audran, R., Arch. Biochem. Biophys., 134,
279-284 (1969).
15
McConahey, P., and Dixon, F. J., Int. Arch. Allergy, 29, 185-
189 (1969).
16
Jarrett, E. E. E., and Stewart, D. C., Immunology, 27, 365-
381 (1974).
17 Ovary, Z., Immunological Methods (edit. by Ackroyd, J. F.),
259-283 (Blackwell, Oxford. 1964).
18
Jennings, F. W., Mulligan, W., and Urquhart, G. M., Exp.
Parasit., 13, 367-373 (1963).
19 Jarrett, E. E. E., Jarrett, W. F. H., and Urquhart, G. M.,
Parasitol., 58, 625-639 (1968).
Alternative route for nitrogen
assimilation in higher plants
IT is generally considered that glutamate dehydrogenase
(EC.1.4.1.3) is the enzyme that is chiefly responsible for
the incorporation of nitrogen into the «-amino group of
amino acids :
@-oxoglutarate + NH:+NAD(P)H+H+
=L-glutamate + H.O+NAD(P)+
(1)
We suggest that this is not the case in the leaves of higher
plants.
There is considerable evidence that chloroplasts are a
major site of «-amino nitrogen production1-5 but, although
the chloroplast contains glutamate dehydrogenase, the
level of activity is low and the enzyme has a high Km
for ammonia5, greater in fact than the concentration of
ammonia required to uncouple chloroplasts. Chloroplasts
also contain glutamine synthetase which has a much lower
Km for ammonia and a much higher activity4,6,7. If gluta-
mine synthetase is the route of entry for ammonia, how-
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615
Table 1 Glutamate formation by intact chloroplasts in the presence
of various amino donors in the light
Nitrogen donor
Acceptor
Glutamate
formed
Glutamine
lost
(pmol per mg chlorophyll h-1)
None
a-oxoglutarate
0.4
Glutamine
a-oxoglutarate
6.9
3.6
NH 4CI
a-oxoglutarate
0.5
Asparagine
a-oxoglutarate
3.5
Aspartate
a-oxoglutarate
4.8
Glutamine
None
0.7
0
The reaction mixture consisted of intact chloroplasts containing
about 100 µg chlorophyll, 0.5 umol a-oxoglutarate and 0.5 umol
of amino donor except asparagine which was 1.25. umol, in a
final volume of 0.3 ml of 0.33 M Sorbitol, 5 mM potassium chloride
and 10 mM sodium pyrophosphate, pH 7.5.
ever, some way must exist to transfer the amide group of
glutamine to the amino group of glutamate. Such an enzyme,
glutamate synthase (EC.2.6.1.53), also known as GOGAT, L-
glutamine: «-oxoglutarate aminotransferase (NADPH-
oxidising) (equation (2)), has been shown to be present in
bacteria® and yeasts9, particularly when grown under
limiting concentrations of ammonia, and in nitrogen-fixing
bacteria and the bacteroids of nodulated higher plants10,11.
L-glutamate+ «-oxoglutarate+NADPH+H+
=2 L-glutamate+ NADP+
(2)
This pyridine nucleotide-linked enzyme has not been
reported in the tissues of higher plants in spite of attempts
by ourselves and others7,11 to find it. Here we present
evidence to show that chloroplasts can perform a light-
dependent synthesis of glutamate from «-oxoglutarate and
glutamine and that extracts of chloroplasts contain a
ferredoxin-dependent glutamate synthase.
Chloroplasts from 10-15-d-old Pisum sativum (var.
Meteor) plants were extracted by a method based on that
of Walker12 in a medium containing 0.33 M sorbitol,
1 mM MgCl2, 2 mM EDTA, 1 mM mercaptoethanol, 10 mM
sodium pyrophosphate, pH 7.0. The chloroplast pellet was
washed in 0.33 M Sorbitol, 5 mM KCI, 10 mM sodium
pyrophosphate, pH 7.5 and then resuspended in the same
medium. Aliquots containing 100-150 µg chlorophyll were
incubated with substrates for 20 min at 30° C. The reaction
was stopped by the addition of ethanol. Amino acids were
separated by paper chromatography in 75% (w/w) phenol,
in the presence of ammonia vapour, and quantified by the
method of Atfield and Morris13.
The results (Table 1) show that chloroplasts are capable
of converting a-oxoglutarate to glutamate in the light,
confirming the findings of earlier workers14. They also
confirm that this reaction is not stimulated by ammonia-
the substrate for glutamate dehydrogenase. When glutamine
is supplied, however, there is a seventeenfold stimulation
in the rate of glutamate formation and the reaction is
absolutely light dependent. The only reaction shown in
Table 1 that occurred in the dark was between aspartate
and «-oxoglutarate, which was at the same rate as de-
termined in the light. Asparagine also markedly stimulates
the conversion of a-oxoglutarate to glutamine. The light
dependence of the reaction, compared with the light
independence of transaminase activity, and the inability of
ammonia to act as an amino group donor would suggest
that asparagine is not being cleaved by asparaginase to give
aspartate and ammonia which could be subsequently in-
corporated.
The results with glutamine are best explained by the
presence of glutamate synthase. To test this, chloroplasts
prepared and washed as described above were resuspended
in 0.1 M tricine, pH 7.5. After standing for 30 min the
ruptured chloroplast debris was centrifuged off and the
supernatant passed over a Sephadex G-75 column pre-
equilibrated with 0.1 M tricine pH 7.5. The chloroplast
extract was eluted with the same buffer. Aliquots of the
extract were incubated with various substrates for 20 min at
30° C. The reaction was stopped and the amino acids
separated and determined as described above.
Preliminary studies with a spectrophotometric assay con-
firmed that these extracts had no NADPH dependent
glutamate synthase and only a trace of glutamate dehydro-
genase activity. But experiments using methyl viologen and
dithionite as an electron donating system showed the pres-
ence of a glutamine and «-oxoglutarate dependent synthesis
of glutamate (Table 2). Subsequent studies showed that
ferredoxin is an extremely effective donor; none of the
other donors tried was capable of supporting the reaction.
The stoichiometry of the reaction is two molecules of
glutamate formed for each glutamine utilised, as pre-
dicted by equation (2). The rates of glutamate synthesis
expressed on a chlorophyll basis are of the same order
of magnitude as those in the intact chloroplasts and are
also similar to those of nitrite reduction and «-amino nitro-
gen production measured in other studies2,4.
The demonstration of a ferredoxin-dependent glutamate
synthase in the chloroplasts completes a chain of reactions
for the incorporation of nitrate into «-amino nitrogen
(Fig. 1). Several considerations suggest this is the major
Table 2 Glutamate formation by chloroplast extracts under various conditions
Electron donor
Nitrogen donor
Acceptor
Glutamate formed
Glutamine lost
Experiment 1
Fd + DIT
None
a-oxoglutarate
0*
Fd + DIT
Glutamine
a-oxoglutarate
23.2
10.8
Fd + DIT
NHẠCI
a-oxoglutarate
0
Fd + DIT
Asparagine
a-oxoglutarate
0
Fd + DIT
Aspartate
a-oxoglutarate
2.08
None
Aspartate
a-oxoglutarate
1.55
Fd + DIT
Glutamine
None
0
0
None
Glutamine
a-oxoglutarate
0
0
Fd + DIT.
Glutamine
a-oxoglutarate
9.6
4.9
MV + DIT
Glutamine
a-oxoglutarate
2.8
1.3
NADPH
Glutamine
a-oxoglutarate
0
0
NADH
Glutamine
a-oxoglutarate
0
0
FMN + DIT
Glutamine
a-oxoglutarate
0
0
Fd
Glutamine
a-oxoglutarate
0.5
0.1
DIT
Glutamine
a-oxoglutarate
0.5
0.2
The reaction mixture consisted of 70 umol tricine, pH 7.5, chloroplast extract equivalent to approximately 100 µg chlorophyll, 3.5 umol
a-oxoglutarate and the electron donors and nitrogen donors as indicated. The final volume of the reaction mixture was 0.7 ml. The amounts
of electron donors in the reaction mixture was as follows: ferredoxin (Fd) 0.1 mg, sodium dithionite + NaHCO3 (DIT) 1.6 mg each, FMN
0.2 mg, methyl viologen (MV) 0.1 mg, NAD(P)H 0.4 mg. Amino donors were present at 2 mM concentration except asparagine which was at
5 mM. Ferredoxin was derived from Spirolina maxima.
*No visible glutamate spot on the chromatogram.
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:
.
,
Experiment 2
(umol per mg chlorophyll h-1)

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Nature Vol. 231 October 18 1974
LIGHT
CHLOROPLAST
O2
요.
·H2O
ATP ADP
CYTOSOL
ATP ADP
Fd
NOS
+ NO 2
NH3
GLUTAMINE
a
GLUTAMATE
Fd
(1)
GLUTAMATE
u-OXOGLUTARATE
u KETO-ACID
AMINO
ACID
Fig. 1 The proposed route of entry of nitrate into amino acids
in leaves. The enzymes are, a, nitrate reductase ; b, nitrite reductase ;
c, glutamine synthetase; d, glutamate synthase and e, trans-
aminase.
route of entry of nitrogen into the organic form in the
leaves of higher plants. The leaf normally receives its
inorganic nitrogen in the form of nitrate. This nitrate is
metabolised through nitrate and nitrite reductases to am-
monia. Both of these reactions are strongly light stimu-
lated2,3 as is the incorporation of "NO3, 15NO2, and
15NH3 into «-amino nitrogen15. Nitrite reductase is located
in the chloroplasts and the presence of glutamine synthetase
with a low Km for ammonia ensures that the ammonia
formed can be incorporated without building up to levels
which would uncouple the chloroplasts. The presence of
glutamate synthase does not preclude a role for glutamate
dehydrogenase in ammonia assimilation, particularly under
conditions of high ammonia availability. These are most
likely to occur in roots. Since ammonia is not normally a
major nitrogen constituent of the xylem sap16 it is unlikely
that it occurs in any concentration in the leaves unless
it is produced by deamination of transported amines and
amino acids.
The operation of the route in Fig. 1 would result in the
same pattern of incorporation of the nitrogen of NO3,
glutamate and the amide group of glutamine into all the
amino acids. The results of Lewis and Pate17 with 15N
feeding studies confirm that this occurs in leaves. This
is distinct from the traditional idea of incorporation of
glutamine into relatively few amino acids, as suggested
for yeast by Sims and Folkes18. The 15N studies of Baker
and Thompson19 with Chlorella are also consistent with
Fig. 1.
The results we present suggest that asparagine is meta-
bolised by chloroplasts although not by the same enzyme
as glutamine. It is probable that the amide is transferred
to another acceptor molecule (such as, pyruvate, phos-
phoenol pyruvate or oxoaloacetate) in the presence of a
reductant or to glutamate to form glutamine and pass
into the chain. The strong light dependence of glutamate
synthesis from asparagine is consistent with the rapid
disappearance of asparagine from leaves in the light.
We suggest that the leaves of higher plants contain a
glutamate synthase which plays a crucial role in nitrogen
assimilation. This enzyme differs from those previously
described in being ferredoxin dependent. Its presence helps
to explain a major anomaly of intermediary nitrogen
metabolism in higher plants, namely the unsuitability of
glutamate dehydrogenase as a major point of entry of
nitrogen into organic combinations in chloroplasts.
We thank Mrs Shirley Burgess for technical assistance
and Dr D. O. Hall of Kings College, London for a gift of
ferredoxin. P. J. L. was supported by a Pickering Research
Fellowship.
Note added in proof: Since this paper was submitted we
have become aware of a report20 on NAD(P)H-dependent
glutamate synthase type activity in extracts of carrot cell
cultures.
P. J. LEA
B. J. MIFLIN
Rothamsted Experimental Station,
Harpenden,
Hertfordshire AL5 2JQ, UK
Received June 24, 1974.
1
Givan, C. V., and Leech, R. M., Biol. Rev., 46, 409-428 (1971).
2
Mifflin, B. J., Planta, 116, 207-224 (1974); Planta, 105, 225-233
(1972).
3
Beevers, L., and Hageman, R. H., Ann. Rev. Plant Physiol.,
20, 495-522 (1969).
4 Mifflin, B. J., Plant Physiol., Lancaster (in the press).
5 Lea. P. J., and Thurman, D. A., J. exp. Biol., 23, 440-449
(1972).
6 O'Neal, D., and Joy, K. W., Nature new Biol., 246, 61-62
(1973); Plant Physiol., Lancaster (in the press).
7 Haystead, A., Planta, 111, 271-274 (1973).
8
Tempest, D. W., Meers, J. L., and Brown, C. M., in Enzymes
of Glutamine Metabolism (edit. by Prusiner, S., and
Stadtman, E. R.), 167 (Academic Press, New York, 1973).
9
Brown, C. M., Burn, V. J., and Johnson, B., Nature new
Biol., 246, 115-116 (1973).
10
Nagatani, H., Shimizu, M., and Valentine, P. C., Arch.
Mikrobiol., 79, 164-175 (1971).
11
Sloger, C., Plant Physiol., Lancaster, 51, 34s (1973).
12
Walker, D. A., Biochem. J., 92, 22c (1964).
13
Atfield, G. N., and Morris, C. J. O. R., Biochem. J., 81,
606-614 (1961).
14
Givan, C. V., Givan, A. L., and Leech, R. M., Plant Physiol.,
Lancaster, 45, 624-630 (1970).
15
Canvin, D. T., and Atkins, C. A., Planta, 116, 207-224 (1974).
16
Bollard, E. G., Ann. Rev. Plant Physiol., 11, 141-166 (1960).
Lewis, O. A. M., and Pate, J. S., J. exp. Bot .; 24, 596-606
(1973).
18
Sims, A. P., and Folkés, B. F., Proc. R. Soc., B159, 479-502
(1964)
19
Baker, J. E., and Thompson, J. F., Plant Physiol., Lancaster,
36, 208-212 (1964).
20
Dougall, D. K., Biochem. Biophys. Res. Commun., 58, 639
(1974).
Evolution of cell senescence,
atherosclerosis and benign tumours
IT has been proposed that cell senescence is the cause
of organismic senescence1-3 and that senescence is under
evolutionary control4. Orgels has argued that cell sene-
scence could not be under evolutionary control because it
is difficult to imagine how senescence of the organism has
a positive evolutionary advantage.
I propose that cell senescence itself, rather than sene-
scence of the organism, is the genetically controlled and
programmed event selected by evolution and that any effects
on somatic senescence is a pleiotropic effect of this
function. The advantage of programmed cell senescence to
the organism is that it stops cells which have escaped from
normal control from dividing indefinitely. If a cell with
potential for further division gets out of place in the body,
it is possible for it to settle in a new location where the
normal environmental signal inhibiting division is lacking.
Were these now uninhibited cells capable of unlimited
growth, they would divide to fill the entire space available.
A limited number of cell divisions, however, would limit
the size of the growth, thus saving the organism.
In vitro normal human fibroblasts are limited in the num-
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Nature vol. 201 October 10 19/4
ber of divisions1,2. The life of a culture of cells from a
tissue explant can be divided into three phases1. Phase I
ends with the formation of the first monolayer; phase II
refers to a period when rapid growth occurs in proper
culture conditions, while phase III refers to the period of
senescence of the culture. Not only is the percentage of
non-dividing cells higher in phase III cells than phase II
cells6, but the growth rate of dividing cells is is reduced™ : so
all cells of the culture are affected by senescence. Phase II
cells, however, are unaffected by being mixed with phase III
cells2.
Phase II cells can be held for long periods in a non-
dividing state without affecting the number of divisions
the cells undergo before senescence8. Thus, the metabolic
age of a culture can be separated from the doubling age.
The onset of senescence is shown to be dependent on
the doubling age and the doubling age of a culture can be
estimated9.
Growth of a clone which arose from a single cell is
restricted to the edge. The cells in the centre are inhibited
by contact inhibition. Thus, the cells in the centre are at
a younger divisional age than the cells near the edge10.
As the cells go out from the centre, they become pro-
gressively older and growth stops at the outer edge of the
colony when the cells there have divided the maximum num-
ber of times and senesce.
The evidence also indicates that cells senesce in vivo.
'With increasing age of the human donor there is a re-
duction in the number of cell doublings before senescence
in culture11. Serial transplants of normal tissue into young
inbred animals die out12-14 and the death of the tissue is
related to number of cell divisions rather than to its meta-
bolic age15. In contrast, transformed cells (malignant cells)
which do not show contact inhibition and do not senesce
in tissue culture, can be transferred in vivo indefinitely12.
Hayflick postulated that organisms age because their cells
age2,3 and this relationship has been supported by studies
on Werner's syndrome, a genetic disease characterised by
the development in early adulthood of a wide variety of
degenerative features similar to those found in old age.
Cells from sufferers of this syndrome are already senescent,
doubling less than ten times in tissue culture11, while cells
of normal people of similar age double 35 times. Cell
senescence is not necessarily the only cause of organismic
senescence since humans aged 70-90 yr are senescent; but
fibroblast cultures from them are not senescent, doubling
at least twenty times in tissue culture11, which is over 40%
of the doublings found in human embryo cultures.
Atherosclerotic lesions consist of 'plaques' of fibroblasts
lining the interior of the great arteries. Limitation in the
size of these plaques provides a strong selection for cell
senescence.
Benditt and Benditt16 present the following evidence that
a single plaque is monoclonal; that is, all of the cells of a
plaque are derived from a single dividing precursor cell.
They looked at plaques from the three females who were
heterozygous for two electrophoretic variants of the X-
linked glucose-6-phosphate dehydrogenase. There is ran-
dom inactivation of one or the other of the X chromosomes
at about the 1,000 cell stage of embryo development, con-
sequently each heterozygous female has a mixture of cells
with one or the other X chromosome inactivated17. When
small pieces (0.1 mm3) were taken from the aorta wall,
both enzyme types were present. But when larger pieces
from plaques (0.5 mm3 to 20-30 mm3) were taken, most of
them had only one isozyme present. It is unlikely that these
plaques are scars18 formed where the artery had been
injured, since hypertrophic scar tissue is polyclonal16.
Cells escaping into the blood stream from points where
there is injury to the vascular system would be carried
away from the wound by blood flow and could settle on
the inner wall of arteries. Possibly they have trouble
settling or dividing except where there are fatty streaks,
thus giving the correlation between fats and athero-
sclerosis. The fibroblasts, now living in a new place in the
body, exposed to a high velocity flow of nutrient rich
blood, would no longer be contact inhibited19 and would
start dividing. As the single cell forms a clone, the cells
in the centre away from the flow of blood, will cease
dividing, while the cells on the edge will continue to
divide. If these cells were able to continue dividing they
would form quite a large growth and constrict the artery.
Since they are limited to a certain number of divisions,
however, the size of the plaque is limited in the same way
as the size of a colony arising from a single cell in tissue
culture10.
I am postulating that atherosclerotic plaques arise from
cells which escape into the arteries, settle and start growing.
To keep these plaques at a minimum size, evolution has
selected for a 'clock' which limits the number of cell divi-
sions. This number is set by the conflicting demands of
the large number of divisions required for wound repair
and cell replacement and the restriction in the number of
cell divisions needed to control atherosclerotic plaques
and other abnormal growths.
The theory does make several predictions which can be
tested. The first is that plaques will grow quickly and then
stop when the cells on the outer surface senesce. Second,
with fully grown but relatively young plaques, the cells
at the centre of the plaque should be able to divide in
tissue culture and the ones at the edge should not. Third,
the number of divisions the cells from the centre of a
plaque can go through should be correlated with the size
of the plaque, given that the plaque started from a single
cell; and last, the median final size of a plaque should be
correlated with the age of the person at the time the
cells were released into the blood stream.
Limitation in the size of some benign tumours could
also provide strong selection for cell senescence. It has
been shown that leiomyomas are monoclonal20. Many leio-
myomas seem to originate near arteries21 and thus may
have arisen from cells from arterial walls which have
escaped into the lyometrium of the uterus. Similar pre-
dictions as those made for atherosclerotic plaques apply
to such benign tumours. Other benign tumours particularly
the large ones, could originate from other causes, such as
by a mutation which destroys the system providing con-
tact inhibition. In this case, all the cells, not just the ones
on the edge, would go through the maximum number
of divisions.
So far no mechanism has been proposed as to how cell
senescence takes place. The theory presented above explains
how natural selection can select for cell senescence and
implies that cell senescence is under genetic control. Views
on cell senescence may be divided into two groups: one
favours an evolutionary-genetic basis, as in this paper,
and the other believes that senescence is caused by random
events which create disturbances within the cell which are
not fully correctable and that this accumulation of errors
eventually leads to death. Any random error theory, such
as the 'error catastrophe theory", must be able to ex-
plain why cultures of transformed cells are not severely
enough affected by the errors to senesce and why normal
cells can not evolve the state of perfection enjoyed by
transformed cells. Moreover, any random error theory
needs to explain why germ line cells are less affected by the
errors than the somatic cells. The simplest explanation to
these problems is that senescence is under genetic control
and that induction of senescence takes place during differen-
tiation leaving the germ line unaffected.
Any mechanism, such as that proposed by the 'error
catastrophe theory', could be used by evolution as the
means to induce senescence. But a special compound could
be produced whose sole function is to induce senescence.
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618
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Milo22 has presented evidence that a glycoprotein extracted
from the cell wall of senescent phase III cells causes almost
immediate senescence when put on phase II cells.
I thank Dr P. Morrow, Dr B. Richardson, Dr J.
Campbell and Professor W. Hayes for helpful criticism.
DANIEL DYKHUIZEN
Department of Genetics,
Research School of Biological Sciences,
The Australian National University,
PO Box 47, Canberra, ACT 2601,
Australia
Received May 21; revised July 16, 1974.
1 Hayflick, L., and Moorhead, P. S., Expl Cell Res., 25, 585-621
(1961).
2 Hayflick, L., Expl Cell Res., 37, 614-636 (1965).
3 Hayflick, L., Expl Geront., 5, 291-303 (1970).
* Comfort, A., Ageing, The Biology of Senescence (Routledge
and Kegan Paul, London, 1964).
Orgel, L. E., Nature, 243, 441-445 (1973).
6
Merz, G. S., and Ross, J. D., J. Cell Physiol., 74, 219-222
(1969) .:
7
Macieira-Coelho, A., Ponten, J., and Philipson, L., Expl Cell
Res., 42, 673-684 (1966).
8
Dell'orco, R. T., Martin, J. G., and Kruse, P. F., jun., Expl
Cell Res., 77, 356-360 (1973).
9
Cristofalo, V. J., and Sharf, B. B., Expl Cell Res., 76, 419-427
(1973).
10
Brunk, U., Esicsson, J. L. E., Pontén, J., and Westermark, B.,
Expl Cell Res., 79, 1-14 (1973).
11
Martin, G. M., Sprague, C. A., and Epstein, C. J., Lab. Invest.,
23, 86-92 (1970).
12 Daniel, C. W., DeOme, K. B., Young, J. T., Blair, P. B., and
Faulkin, L. J., jun., Proc. natn. Acad. Sci. U.S.A., 61, 53-60
(1968).
. 13
Krohn, P. L., Proc. R. Soc., B157, 128-147 (1962).
14 Williamson, A. R., and Askonas, B. A., Nature, 238, 337-339
(1972).
15
Daniel, C. W., and Young, L. J. T., Expl Cell Res., 65, 27-32
(1972).
16 Benditt. E. P., and Benditt, J. M., Proc. natn. Acad. Sci.
U.S.A., 70, 1753-1756 (1973).
17 Ohno, S., Ann Rev. Gen., 3, 495-524 (1969).
18
Timiras, P. S., Developmental Physiology and Ageing, ch. 25
(Collier-MacMillan Ltd., London, 1972).
19
Stoker, M. G. P., Nature, 246, 200-202 (1973).
2º Linder, D., and Gartler, S. M., Science, 150, 67-69 (1965).
21
Anderson, W. A. D., Pathology (2nd ed.), 1089 (C. V. Mos Co.,
St Louis, 1953).
22 Milo, G. E., jun., Expl Cell Res., 79, 143-151 (1973).
Insulin stimulates myogenesis
in a rat myoblast line
THE clonal rat myoblast cell line Lo isolated by Yaffe1 under-
goes myogenesis when the mononucleated myoblasts become
confluent and fuse into multinucleated myotubes. This
morphological differentiation is correlated with the synthesis
of characteristic muscle proteins such as myosin and creatine
kinase2. When Le myoblasts fuse the specific activity of
adenylate cyclase3 decreases and cyclic AMP, dibutyryl
cyclic AMP and theophylline reversibly inhibit the differen-
tiation process4. In primary cultures of rat muscle cells, a
decrease in cyclic AMP has been reported at the time of
fusion'. These facts support the hypothesis that low levels
of cyclic AMP correlate with myoblast differentiation, while
high levels inhibit fusion. In view of the relationship
between the action of insulin and the biosynthesis of cyclic
nucleotides® and the effects of the hormone on muscle
metabolism7, we have examined the influence of insulin on
myogenesis of cultured LG myoblasts. The hormone replaces
partially the serum requirement for morphological differen-
tiation of primary cultures of chick embryo myoblasts8,9,,
and now we have found that it stimulates fusion and the
specific activity of creatine kinase in Lo myoblasts in the
presence or absence of serum.
Nature Vol. 251 October 18 1974
In an attempt to separate the effects of insulin on myo-
genesis from those on metabolism, we examined the effect
of the hormone on myoblast fusion in the presence of
different concentrations of serum (Fig. 1). In serum-free
medium almost no myotubes were observed in the absence
· of insulin and many cells died (Fig. 1a). In 1% serum,
myotubes formed after 2 d and in 10% serum they appeared
after 4 d (Fig. 1c and e). Addition of insulin (1 µg ml-1) to
the medium stimulated both the rate and extent of fusion at
all serum concentrations tested (Fig. 1b, d and f), most of
the nuclei being incorporated into myotubes within 24 h of
the onset of fusion. Myotubes formed in the presence of
insulin were much larger than those formed in its absence,
-
Fig. 1 Effect of insulin and serum on myoblast fusion.
Rat myoblast Le cells (from Dr S. Heinemann, Salk Institute)
were grown at 37° C in a humidified incubator under a 5%
CO: 95% air atmosphere in complete medium (Dulbecco's
modified Eagle's medium supplemented with 10% foetal
calf serum, Grand Island Biological Co.). To study fusion,
cells were plated in complete medium at 8 x 103 cell cm-2 in
35 mm dishes. This medium was removed 24 h later, the
dishes were rinsed with phosphate-buffered saline, and re-
incubated under the following conditions. a and b, Serum-
free medium containing 1.5 mg ml-1 bovine serum albumin
(BSA); c and d, medium containing 1% foetal calf serum;
e and f, complete medium (10% foetal calf serum). Insulin
(1 µg ml-1, zinc-free, from Dr C. Yip, University of
Toronto) was added when the medium was changed for b. d.
and f and the dishes were stained with methylene blue 3 d
later.
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--

iii
Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4
Nature Uciover 10 1717
Les Euphausiacés du Pacifique equatorial et sud tropical
EUPHAUSIIDS OF THE EQUATORIAL AND SOUTH TROPICAL PACIFIC OCEAN
C. Roger
Col. Mémoires ORSTOM nº 71
1974-21×27, 266. p., 98 fig. 77 tabl., bibliogr., English abstract, ISBN 2-7099-0335-0. £9.00*
Quantitative zoogeography (600 samples, 34 species). Ecological groups. Biological cycles of the main mesopelagic
species. Trophic levels and feeding behaviour. Vertical distribution, which decide upon what category of predators
they will be linked with: this analysis brings some light on the causality and structure of pelagic food webs.
L'architecture de la forêt guyanaise
THE ARCHITECTURE OF THE GUIANESE FOREST
R. A. A. Oldeman
Col. Mémoires ORSTOM nº 73
1974-21X27, 206 p., 113 fig., photogr., index alphabétique, bibliogr., ISBN 2-7099-0346-6. £7.50*
Based on analysis of the architecture of the trees, according to the. method of the aborescent methods (HALLE and
OLDEMAN, 1970), and introducing the idea of reiteration of these models from meristems the study of the equatorial
forest architecture is given, as a basis, the objective criterion of form. The criteria of biomass and energy are in
addition to the form function. This, the forest stratification may be defined as a functional phenomenon-the stratified
production of the biomass-taking place during the known stages of the sylvigenesis. Its agents, the trees, are
divided into present, future and past forest groups. Simple morphological markers define each stage of the sylvigenesis
thanks to the morphological and ecological inversion surfaces.
Aspects du contact forêt-savane dans le centre et l'ouest de la Côte d'ivoire. Etude descriptive
ASPECTS OF THE FOREST SAVANNA CONNECTION IN THE WEST AND CENTER OF THE IVORY COAST.
A descriptive study
J. M. Avenard, J. Bonvallot, M. Latham, M. Renard-Dugerdil, J. Richard
Col. Travaux et Documents de l'ORSTOM nº 35
1974-254 p., 69 fig., bibliogr. ISBN 2-7099-0345-8 £7.25*
An investigation concerning geomorphology-soils-vegetation relations has been undertaken in order to determine
correlations between distribution of vegetable formations and environmental factors. At the same time, human investi-
gations attempted to define man's relationship with the natural environment. Various observations seem to prove the
preponderance of geomorphological evolution and soils over the distribution of vegetable formations.
L'aluminium dans les sols
ALUMINIUM IN SOILS
P. Segalen
Col. Initiations/Documentations Techniques nº 22
1973-21×27, 281 p., 33 fig., bibliogr. 1,500 réf. ISBN 2-7099-0314-8. £9.00*
Aluminium is the third most abundant elements in soils. It is responsible for many soil properties and its know-
ledge greatly helps to understand the development of soils. This book consists of two main parts: the aluminium
constituent of the soils and their dynamics starting with the weathering of rock minerals and ending with the concen-
tration of hydrovides. Finally different unsolved problems are considered.
"Monographies hydrologiques" dealing with the big hydrographical basins and with specific regions, like islands.
This monography includes a complete and historical inventory of the hydrometeorological information: rain fall data,
water levels and discharges, etc. . .. A critical analysis and an homogeneization of this information are followed by
the presentation of the basic numerical data concerning the hydrological regime. Statistical analysis allow to set up
an optimal state of the knowledge of the available water resources.
Recently issued:
Le bassin du fleuve Sénégal
THE SENEGAL RIVER BASIN
C. Rochette
nº 1-1974-21X27, 440 p., 3 cart. annex. (noir)
ISBN 2-7099-0344-X. £14.50*
-
Le bassin du fleuve Chari
THE CHARI RIVER BASIN
B. Billon, J. Guiscafre, J. Herbaud, G. Oberlin
nº 2-1974-21×27, 450 p., 5 cart. annex. (noir)
ISBN 2-7099-0329-6. £14.50*
Végétation du Vietnam -- Le massif sud-annamitique et les régions limitrophes
THE SOUTH ANNAMITIC MASSIF AND THE LIMITROPHE REGIONS
M. Schmid
Col. Mémoires de l'ORSTOM nº 74
1974-21X27, about 290 p., 15 cart. et fig., 16 pl. photos, index noms botaniques, bibliogr. in preparation ISBN 2-7099
-0349-0. £8.75*
"Vegetation de la partie méridionale du Centre Viet-Nam" is a plant ecological study of an area of about 40,000
square kilometers consisting, north of the delta plain of the Mekong-Donnai, of basalt plateaux and eruptive moun-
tains reaching about 2,400 m. The Indo-malayan and Sino-himalayan floras overlap in this region forming very varied
associations whose composition, structure, distribution and finally dynamics are considered in relation to climatic
and edaphic factors. The work is illustrated by six original maps and some twenty plates.
*Postage extra
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L'OFFICE DE LA RECHERCHE-SCIENTIFQUE
ET TECHNIQUE OUTRE-MER (ORSTOM/N1)
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ORSTY
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chemical Centres
We concentrate on being a truly international operation;
our two associate companies and 30 distributors form the
largest and most widespread radiochemical network in the
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2932

vi Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4
ivuire vciover 18 1974
Success
for the competitive yachtsman must in some part
depend upon the quality and reliability of the equipment he uses.
Likewise for you , the researcher, these virtues must apply to your reagents
for them to contribute to your success.
To broaden your Biochemical Research 'horizon' Boehringer Mannheim
·offer the following new preparations.
396 11
Reverse transcriptase (AMV) Cat.No. 1564 Adenosine-5'-0-(3-thiotriphosphate) .
(RNA-dependent DNA-polymerase)
(ATP.Y .S)
Cat.No. 15623
Purified by chromatography on DEAE-cellulose,
phosphocellulose and SephadexRG-100.
Supplied at -20°C in 50% glycerol, 1mM
dithiothreitol and 0.3M potassium phosphate;
pH approx. 8.0. Stabilised with bovine serum
albumin (0.5mg/ml).
With oligo-dT stimulated AMV-RNA the enzyme
preparation performs well.
The latest in a line of nucleotide analogues of
particular interest to anybody working on ATP-
dependent enzyme systems. Supplied as tetra-
lithium salt to facilitate solubilisation combined
with stability.
Typical analysis: ATP- Y-S Li4 85%, H2O 15%.
Application: Inhibition of phosphatases and
ATPases; investigation of muscle
myosin; 'affinity labelling' of
proteins; labelling of ATP+S
binding enzymes prior to X-ray
2'-0-Succinyl-A-3:5-MP
Cat.No.15663
Introduced to satisfy the growing demand for an'
ultra-sensitive radio-immuno assay for A-3:5MP *.
Techniques currently being developed make it
possible to measure A-3:5-MP in the p-gram
range with antibodies produced from Succinyl-
A-3:5-MP linked to albumin.
.
structural analysis or electron
microscopy (ATP-Y-S has an affinity
for mercury compounds).
References: Nachr.“Chern. Techn. 20, 494 (1972)
*First described by A.L.STEINER et.al.,
Proc. Nat. Acad. Sci. 64, 367 (1969).
Cholesterol oxidase
Cat.No. 15626
Phospholipase C
Cat.No 15636
from-Bacillus cereus
E.C. 3.1.4.3.
Specific activity : approx. 20U/mg
Only 0.1U of enzyme is needed for assay of
approx. 5mMoles/litre cholesterol.
Of particular interest to food analysts, id
researchers and research clinical (Decgi!
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Geological Journal
Per volume (2 parts) £6-60 (SU.S. 19-00)
Publishes original papers in the whole field of geology. It is published in. two
parts per year. The current part is volume 9, part 2.
SPECIAL ISSUES
No. 2 Mechanism of Igneous Intrusion
Ed. Newall and Rast, 1970
£7.00 (SU.S. 19-60)
Contains 21 contributions related to the mechanism of magmatic penetration
in the crust, ranging from theoretical and experimental to geochemical and field
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380 pp
No. 3 Trace Fossils
. Ed. Crimes and Harper, 1970
£8.00 (SU.S. 22-20)
Contains 35 contributions on many aspects of stratigraphic usage, taxonomy
and palaeontology of ichnofossils from many parts of the world. The volume is of.
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students. Plates, figures, trace fossil index, and subject index.
547 pp
No. 4 Faunal Provinces in Space and Time
Ed. Middlemiss, Rawson and Newall, 1971
£7-00 (SU.S. 19-60)
Contains 13 contributions dealing with aspects of faunal provinces through
the whole of Phanerozoic time. This volume should encourage the application
of new rigour to future studies and also encourage the palaeontologist to
apply his findings to wider problems related to global tectonics. The book is of
value as a modern compendium work for many grades of students. Plates, figures,
subject and name indices.
236 pp
No. 5 The Boreal Lower Cretaceous
Ed. Casey and Rawson, 1973
£9-75 ($U.S. 27-00)
Contains 26 contributions from international scientists, combining to form a
unique synthesis of knowledge of the Lower Cretaceous of northern regions.
The papers review geographical areas from Alaska eastwards to Siberia and
deal with the wider problems of palaeobiogeography, chronostratigraphy and
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index.
448 pp
No. 6 Ice Ages: Ancient and Modern
Ed. Wright and Moseley
Approx. £9-75 (SU.S. 27.00)
In press. Expected January 1975.
This is the first symposium volume to give a review comparing the features
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ary field where palaeoclimatology, palaeoecology, sedimentary petrology,
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The authors have brought their various specialities to bear on the one subject of
the recognition of Ice Ages.
Approx. 400 pp
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Serl
Prices include sea mail postage. These
publications and journal back issues are
available, cash with order, from:
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.
Biochemica et Biophysica Acta 276,
155-161 (1972)
from Nocardia erythropolis
E.C. 1.1.3.6.
When elucidating the type and arrangement of
membrane phospholipid structures in erythro. .
cytes the enzyme from B. cereus has the
advantage over other sources of being free from
proteases, hydrolases neuraminidase and has
negligible haemolytic activity.
Reference: J.SHILOACH et al., Biotechnol.
" and Bioengen. XV. 551.11973)
farther defans contact

Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4
Nature Vol. 231 October 16 19/4
619
with nuclei located more centrally. They also tended to
degenerate more rapidly8.
We then found that in the presence of insulin the specific
activity of creatine kinase increased markedly at three
serum concentrations (Fig. 2). The increase coincided with
the onset of fusion (data not shown). The highest specific
activity was reached in cultures grown in 1% serum in the
presence of insulin, and under these conditions the maxi-
mum was reached in only 48 h (Fig. 2b). The increase in
creatine kinase activity (as well as in morphological fusion)
was delayed by about 24 h in cultures containing 10% serum
(Fig. 2c). By changing the insulin-containing medium at 4
d, we obtained a further stimulation of creatine kinase levels
in the presence of 10% serum, but not 1% serum (dashed
lines, Fig. 2b and c). In other experiments the glycogen
phosphorylase content of myotubes, detected histochem-
ically2, also increased after addition of insulin. It is inter-
esting that de la Haba et al. found no accumulation of the
characteristic muscle enzyme glycogen synthetase in pri-
mary cultures of myoblasts fused in the presence of
insulin in serum-free medium9. Since under similar con-
ditions with the L6 myoblast line we observed a marked
stimulation of creatine kinase activity by insulin, either
there are differences in the response to insulin of primary .
cultures and L6 cells, or glycogen synthetase and creatine
kinase levels are controlled independently during myo-
genesis.
All the above experiments were carried out using 1 µg
ml-1 of insulin. When we used 10-2 µg ml-1 insulin (Fig. 3a)
we observed a significant stimulation of morphological
fusion and creatine kinase activity: this concentration is
about one order of magnitude higher than that found in
vivo. When the concentration of serum was low, the maxi-
a
25-
Creatine kinase specific activity
(pmol per 20 min per mg protein)
20
15
-
101
VI
.
0
2
4
0
2
4
6 0
2
4
6
8
Time in culture (d)
Fig. 2 Effect of insulin and serum on specific activity of
creatine kinase. Cells were plated as described in the legend
to Fig. 1, and the medium was replaced by medium con-
taining various concentrations of serum in the presence or
absence of insulin (1 µg ml+1). At the indicated times after
the medium change, the medium was removed, the cells were
rinsed quickly with buffer A (100 mM Tris-acetate, pH 6.8
(22° C), 25 mM magnesium acetate, 10%, v/v, glycerol),
drained, and kept frozen at -35° C. After thawing, cells
were scraped into 0.35 ml buffer A supplemented with 0.2%,
v/v, Nonidet P-40, and 0.1 mM phenymethylsulphonylfluo-
ride (Sigma) and subjected to two cycles of freezing and
thawing. Aliquots from duplicate plates were assayed for
creatine kinase10 and protein11. Variations in specific activity
were usually less than 15% for values greater than 1 umol
per 20 min per mg protein. a, Serum-free medium containing
1.5 mg ml-1 BSA; b, medium containing 1% foetal calf
serum; c, complete medium (10% foetal calf serum). A,
Minus insulin;
., 1 µg ml-1 insulin. The open symbols
indicate values obtained after replacing the medium on day
4 (arrows) with fresh medium with (O), or without insulin
(△).
a
b
Stimulation of creatine kinase .
9
specific activity
7
0
5
3
1
1
.
0 10-3 10-2 10-1
1
5
0
1
2
3
4
Insulin (µg ml-1)
Time in insulin(d)
Fig. 3 Characteristics of the stimulation of creatine kinase
by insulin. Cells were handled as described in the legend
to Fig. 1, insulin was added at the time of the medium
change, and cells were frozen 4 d later for determination of
specific activities as described in the legend to Fig. 2. Re-
sults are expressed as relative stimulation over the control
incubated without insulin. a, Insulin concentration depen-
dence. Control specific activities were 1.0, 1.0 and 0.6 umol
per 20 min per mg protein in 0, 1% and 10% serum respec-
tively. b, Dependence on time of exposure to insulin. Insulin
(1 µg ml-1) was added at time 0 for various times after
which the medium was removed and replaced by medium
at the same serum concentrations but lacking insulin. Initial
specific activities without insulin were 1.3 and 0.6 umol per
20 min per mg protein in 1% and 10% serum respectively.
O, No serum; 4, 1% serum;
0, 10% serum.
mum stimulation was obtained with 1 µg ml-1 insulin, while
in 10% serum no plateau was apparent even with 5 µg ml-1
insulin. The requirement for high concentrations of insulin
for maximum stimulation of creatine kinase may be due in
part to inactivation of the insulin, especially in 10% serum
(compare Fig. 2c). It may also indicate that a continuous
and prolonged exposure to insulin is needed for the increase
in fusion and creatine kinase activity. This possibility was
tested by exposing cultures to insulin for various times, up
to 4 d, removing the hormone, then measuring creatine
kinase activity at 4 d. Figure 3b shows that insulin had to
be present for more than 24 h to stimulate significantly
creatine kinase specific activity, and longer exposure
resulted in greater enzyme activity.
Insulin stimulates many cellular activities, especially in
muscle cells7, and its observed effect on myogenesis in both
L6 cells and primary cultures® may not be a specific effect on
differentiation, but an indirect result of a general stimulation
of cellular metabolism, especially in serum-free medium.
For example, in fibroblasts serum starvation results in a
reduction in RNA and protein synthesis, and these activities
can be restored by the addition of insulin12. In the case of
L6 myoblasts, however, cell density and protein synthesis are
higher in 10% serum minus insulin than in serum-free
medium plus insulin, yet myogenesis is increased in the latter
case. (We have also found that insulin does not increase
RNA or protein synthesis in the first 30 h after its addition
to cells grown in 10% serum; a twofold stimulation was
obtained later at 48 h during an 8 h label period.) Thus,
although further experiments are needed, we believe it
unlikely that the stimulation of the specific activity of
creatine kinase and the morphological changes caused by
insulin are due to nonspecific stimulation of protein
synthesis.
Alternatively, the insulin stimulation of myogenesis may
be a consequence of alterations in cyclic nucleotide meta-
bolism, since insulin is known to affect cyclic AMP syn-
thesis13 and degradation14 and cyclic GMP levels® in
different cell types. Therefore it is attractive to suppose that
such effects account for the stimulation of myogenesis in L6
myoblasts reported here, and perhaps for the insulin-
promoted differentiation of mammary gland in organ
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620
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Nature Vol. 251 October 18 1974
culture15, and of primary cultures of lens epithelium16. In
the case of LG myoblasts, it is worth noting that insulin
improves both the reproducibility and synchrony of fusion,
and allows myogenesis to be studied in a defined serum-free
medium.
This work was supported by grants from the National
Cancer Institute of Canada, the Medical Research Council
of Canada and the National Cancer Institute, National
Institutes of Health.
JEAN-LOUIS MANDEL
MARK L. PEARSON
Department of Medical Genetics,
University of Toronto,
Toronto, Canada M5S 1A8
Received June 12; revised July 12, 1974.
1 Yaffe, D., Proc. natn. Acad. Sci. U.S.A., 61, 477-483 (1968).
2
Shainberg, A., Yagil, G., and Yaffe, D., Devl Biol., 25, 1-29
(1971).
3
Wahrmann, J. P., Luzzati, D., and Winand, R., Biochem.
biophys. Res. Commun., 52, 576-581 (1973).
4 Wahrmann, J. P., Winand, R., and Luzzati, D., Nature new
Biol., 245, 112-113 (1973).
" Reporter, M., Biochem. biophys. Res. Commun., 48, 598-604
(1972).
6 Illiano, G., Tell, G. P. E., Siegel, M. I., and Cuatrecasas, P.,
Proc. natn. Acad. Sci. U.S.A., 70, 2443-2447 (1973).
7 Manchester, K. L., in Biochemical actions of hormones (edit.
by Litwack, G.), 1, 267-320 (Academic Press, N.Y., 1972).
8 de la Haba, G., Cooper, G. W., and Elting, V., Proc. natn.
Acad. Sci. U.S.A., 56, 1719-1723 (1966).
9 de la Haba, G., Cooper, G. W., and Elting, V., J. cell. Physiol.,
72, 21-28 (1968).
10 Zalin, R., Expl Cell Res., 78, 152-158 (1973).
11 Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall,
R. J., J. biol. Chem., 193, 265-272 (1951).
12 Hershko, A., Mamont, P., Shields, R., and Tomkins, G. M.,
Nature new Biol., 232, 206-211 (1971).
13
Illiano, G., and Cuatrecasas, P., Science, 175, 906-908 (1972).
14
Woo, Y .- T., and Manery, J. F., Arch. Biochem. Biophys., 154,
510-519 (1973).
15
Vonderhaar, B. K., Owens, I. S., and Topper, Y. J., J. biol.
Chem., 248. 467-471 (1973).
16 Piatigorsky, J., Devl Biol., 30, 214-216 (1973).
Sickle cell resistance to in vivo hypoxia
THE rapid disappearance from circulation of human erythrocytes
transfused to rats can be prevented if the animals are pretreated
with ethyl palmitate (EP) and a cobra venom factor (CVF)1.
The improvement in human erythrocyte survival in these animals
is due to EP-induced impairment of reticuloendothelial
function2 and to suppression of intravascular haemolysis
through CVF inhibition of the third component of complement3.
The halflife of normal human erythrocytes in the blood of
untreated rats is less than 15 min, but is about 30 h in the EP
and CVF treated rats4.
.
Application of this animal system to the study of human
sickle cell anaemia erythroctyes (sickle cells) has verified many
of their known survival characteristics in man5-7. These include
a short intravascular survival, an improved survival with in
vivo hyperoxygenation and an improved survival following in
vitro cyanate treatment8,9. Human sickle cells transfused into
rats exposed to widely divergent concentrations of oxygen
seem to consist of two populations with regard to their in vivo
sensitivity to oxygen changes. The circulation of a minor
population of sickle cells is dependent on the concentration
of oxygen the rats breathe, as shown by rapid increases and
decreases in circulating cells with increases and decreases in
the amount of oxygen administered to the rats (O2-sensitive
cells)8,10. In contrast, the majority of sickle cells seems to be
relatively insensitive to the levels of hypoxia attainable in. vivo.
They continue to circulate after the initial rapid removal of the
oxygen-sensitive cells in spite of exposure to hypoxic atmospheres
barely compatible with life. The proportion of sickle cells which
is oxygen sensitive is not influenced by the total number of
sickle cells transfused (O.C., S.C.F., and G.O., unpublished).
Oxygen-sensitive sickle cells are most readily demonstrated when
the animals breathe 100% O2 followed by 7-8% O2 in short
alternating periods. In these conditions the 51Cr survival curve
of the transfused sickle cells assumes a characteristic 'saw
tooth' pattern with peaks during hyperoxia and valleys during
hypoxia10.
Here we report that separation of hypoxia-resistant sickle
cells is possible if blood from a rat previously transfused with
sickle cells is removed during hypoxia and then retransfused
into another rat. Presumably all of the human sickle cells in
circulation in the blood of donor animals during hypoxia are
insensitive to a low pO2 after the sensitive population has been
sequestered. Figure 1 shows that in the recipient animals the
110
100/
901
a
80-
(2)
70|
60
15Cr RBC survival (%)
110
100
90
b
80|-
701
(5)
60
110
100-
C
(8)
901-
0
1
2
3
4
Time (h)
Fig. 1 Human sickle cell resistance to O2-dependent circulatory
changes after their removal from hypoxic rats and retransfusion
into separate animals. Each rat was injected with 0.5 g kg-1 of
EP and with 10 units of CVF 2 h before transfusion. Three donor
animals then were transfused with 4 ml of a 50% saline suspension
of 51Cr-labelled. erythrocytes (RBCs) from one of three patients
with sickle cell anaemia. After a 20-60 min exposure of the donor
animals to 7.5 % O2 in N 2 5 ml of rat blood-containing transfused
51Cr human sickle cells-were removed by cardiac puncture.
The RBCs were washed with physiological saline, adjusted to a
50% suspension in saline and then 1-3 ml were retransfused into
recipient animals. Following this, all rats were exposed to a
minimum of three hyperoxia-hypoxia cycles each consisting of
15 min of 100% O2 followed by 15 min of 7.5% O2 in N2. The
ordinate measures 51Cr RBC survival expressed as % activity
found in the rats' blood immediately before exposure to the
hyperoxia-hypoxia cycles. The abscissa measures time (h) after
start of the first hyperoxia-hypoxia cycle. Points in the graph
represent means and bars + 1 s.d. of the values for each patient
study. The number of rats used in each group is shown in paren-
theses. a, Transfused sickle cells showing that the sickle cells
remaining in the two donor animals that survived cardiac punc-
ture exhibited characteristic O2-dependent circulation during
each hyperoxia-hypoxia exposure. b, Retransfused sickle cells
(previously obtained from hypoxic donor animals) did not show
this behaviour initially. After the third cycle, an O2-sensitive
population developed in the retransfused sickle cells. c, The
survival in the rats' blood of 51Cr-labelled control human RBCs
obtained from two individuals without haematological abnor-
malities was not influenced by oxygen changes. Stippled areas,
hypoxia; cross-hatched areas; hyperoxia.
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621
survival curves of retransfused sickle cells obtained from hypoxic
donor animals do not exhibit the saw tooth pattern during the
initial alternating periods of hyperoxia and hypoxia. The sickle
cells remaining in the donor animal (transfused sickle cells)
continue to show this response. Sickle cells removed from
donor animals while they breathe 100% O2 and retransfused
into recipient animals (not shown in the figure) retain an O2
sensitive population. Continuation of the hyperoxia-hypoxia
cycles in the recipient rats with O2 insensitive retransfused
sickle cells eventually results in a survival pattern consistent
with the development of a population of sickle cells which is
oxygen sensitive (Fig. 1). The proportion of O2-sensitive sickle
cells to total sickle cells is similar to that in the original popula-
tion. Wide changes in O2 tension seem to be necessary for the
appearance of an O2-sensitive population of sickle cells in the
recipient animals. This statement is based on the observation
(O.C., S.C.F., and G.O., unpublished) that the O2-insensitive
retransfused sickle cells in rats maintained on room air will
remain resistant to O2 changes until they have been subjected
to several hyperoxia-hypoxia cycles.
The physical separation of hypoxia resistant sickle cells from
the total population confirms the heterogeneity of human
sickle cells with regard to their oxygen dependent circulation
in this animal model. The temporary loss of O2-sensitive sickle
cells from the animals' circulation during exposure to low O2
tension is best explained by in vivo sickling and trapping of
sickled erythrocytes in the animals' microvasculature. This
explanation is supported by our finding in this animal model of
hypoxia sensitive erythrocytes not only from patients with
sickle cell anaemia but also from patients with sickle-
thalassaemia and with sickle-Hb C disease4. In contrast, none
of eight individuals with sickle trait studied thus far has shown
this type of response11.
The observations reported here demonstrate that a proportion
of O2-insensitive sickle cells can be transformed into an oxygen
sensitive population when challenged with repeated hyperoxia-
hypoxia cycles. Our findings suggest that repeated sickling
may be an important factor in the production of cells that do not
circulate during hypoxia. Direct extrapolation of animal data
to the human situation must be viewed with caution. Neverthe-
less, it is possible that vaso-occlusive episodes in patients with
sickle cell anaemia may be a function of the relative number of
sickle cells that are oxygen sensitive with regard to their
capacity to circulate. The isolation and study of any oxygen
resistant population may help to determine those factors that
protect sickle cells from sequestration during in vivo hypoxia.
OSWALDO CASTRO
STUART C. FINCH
Department of Internal Medicine,
GEORGE
OSBALDISTON
Division of Health Science Resources,
Section of Laboratory Animal Science,
Yale University School of Medicine,
New Haven, Connecticut
Received June 12; revised August 12, 1974.
1 Wright, M. C., Nelson, R. A., jun., and Finch, S. C., Yale J. Biol.
Med., 43, 173-176 (1970).
2 Stuart, A. E., Lancet, ii, 896-897 (1960).
3 Nelson, R. A., jun., Surv. Ophthal., 11, 498-505 (1966).
4 Finch, S. C., Castro, O., Orlin, J., Rosen, M. W., and Aponte, L.,
Conn. Med., 38, 338-341 (1974).
5 Bensinger, T. A. and Gillette, P. N., Arch. Int. Med., 133, 624-631
(1974).
6 Callender, T. E., Nickel, J. F., Moore, C. V., and Powell, E. O.,
J. lab. clin. Med., 34, 90-104 (1949).
7 Cerami, A., and Manning, J. M., Proc. natn. Acad. Sci. U.S.A.,
68, 1180-1183 (1971).
8 Castro, O., Orlin, J., Rosen, M. W., and Finch, S. C., Proc.
natn. Acad. Sci. U.S.A., 70, 2356-2359 (1973).
9 Castro, O., Orlin, J., and Finch, S. C., Yale J. Biol. Med., 47,
55-60 (1974).
10 Castro, O., Orlin, J., Rosen, M. W., and Finch, S. C., Ann. Int.
Med. (in the press).
'' Orlin, J., Castro, O., and Finch, S. C., Clin. Res., 21, 969 (1973).
Expression of the dystrophia
muscularis (dy) recessive gene in mice
STUDIES in vitro of mutant gene expression in murine mus-
cular dystrophy now seem to have reached an impasse. For
15 years there was only one species of dystrophic mouse1, the
Bar Harbor 129 ReJ strain (denoted dy/dy). Then in 1970,
a second strain BH WK ReJ (denoted dy2J/dy2J) was
reported2 which was described as being similar in all histo-
pathological respects to the allelic dy/dy mutant except that
it showed a slower progression of muscle necrosis. The new
mutant gene was transferred to another mouse strain
C57B1/6J for commercial retail. The new strain, offering
many advantages over the original strain, has been readily
adopted for study by research workers; certain findings
obtained with this strain have added confusion to a difficult
problem simply because it is not the same as the original
strain.
Over the past four years I have been growing dystrophic
(dy/dy) muscle in vitro, and have noticed that muscle
explants from crush lesions in dystrophic (dy/dy) muscle did
not form multinucleate myotubes, whereas muscle explants
from crush lesions in normal muscle readily did so. I report
here some brief details of this work (previously described
in refs 3 and 4), and include the results of some preliminary
experiments using dystrophic (dy2/dyJ2) mice which suggest
that allelic mutant genes do not have the same phenotypic
expression.
Crush lesions were made in the triceps and quadriceps
muscles of normal Bar Harbor 129 ReJ+/+, dystrophic
129 ReJ dy/dy, and dystrophic C57B1/6J dy2J/dy2J mice
under pentobarbital anaesthesia. Four days after the lesion
was inflicted the mouse was killed by cervical dislocation
and the lesion zone was removed under sterile conditions for
tissue culture. The excised regions were dealt with separ-
ately. The tissue was chopped and the explants set up in
coded Pulvertaft5 chambers or 50-mm Petri dishes. The
culture medium was 10% EE50, 10% horse serum and 80%
Eagle's MEM with streptomycin and penicillin.
Each day, over a period of 8 d, all the coded cultures
were assessed microscopically using differential interference
optics. The assessment was carried out blind and the results
are therefore unbiased. The total number of cultures was
in excess of 100 for normal and dy/dy, and 40 for dy2J/dy2J
mice.
Days 1, 2 and 3 showed essentially the same pattern of
growth in all cultures. Proliferation and explant spreading
were similar, and occasional cells with up to five nuclei were
found. From day 4 to day 8 proliferation continued until
the cultures became overgrown and degenerated. During
this second period all normal cultures (Fig. 1a) and all
dystrophic dy2J/dy2J cultures (Fig. 1b, d) developed multi-
nucleate myotubes of considerable size. There was an
indication, which could not be quantified, that explants of
dy2J /dy2J quadriceps showed less multinucleation than
triceps cultures from the same animal. All dystophic dy/dy
cultures (Fig. 1c) showed associations of cells which have
been named pseudostraps4. These are muscle strap-like
or myotube-like aggregates of myoblasts which do not fuse.
There were no multinucleate structures of any kind to be
found. I believe that these results clearly demonstrate an
abnormality of early myogenesis caused by the expression
of the dy/dy gene, but no indication of any abnormality
caused by the expression of the dy2/dy2 gene.
The results clarify some of the conflicting evidence
already produced. Three groups6-8 have used organotypic
methods of tissue culture combining foetal spinal cord
sections with adult muscle. Paul et al.7 were very careful
not to draw any far-reaching conclusions from their work,
recognising the genetic background on which it was based.
But they did state that adult myogenic cells were in some
way affected by the dystrophy (dy/dy) and were incapable of
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Nature Vol. 251 October 18 1974
Fig. 1 Unstained, living cultures seen using differential
interference optics (Reichert). They are representative of
muscle formation in vitro from crush lesion explants. a, Part
of a large multinucleate myotube showing a chain of central
nuclei (arrows), normal +/+. b, Part of a myotube from
dystrophic dy23 /dy2 quadriceps. c, Pseudostraps from
dystrophic dy /dy muscle. Note the close associations of cells
which always remain discrete. d, A clear multinucleate
myotube from a dystrophic dy2J /dy2J triceps culture. All
photographs were taken at 7 d in vitro, under identical
conditions.
regeneration. Hamburgh et al.8 came to the conclusion that
muscle from dystrophic (dy2J /dy2J) mice exhibited a capa-
city to regenerate similar to normal muscle. Gallup et al.6,
using both strains of mice, concluded that the regeneration
of normal and dystrophic muscle is dependent on the geno-
type of the cord section with which it is coupled. Peterson9,
using mouse chimeras (SWV+C57B1/6J dy2J/dy2J), was led
to suggest that the presence of dystrophic nuclei did not
affect myogenesis in any way. My results are essentially
similar to those of Paul et al.7 and Hamburgh et al.8 I
cannot, however, agree with any of the findings or conclu-
sions of Gallup et al.6. Finally, I am in agreement with
Peterson9 that dy2J/dy2J muscle behaves in the same way as
normal muscle with regard to myogenesis.
Therefore I would submit that the recent study of murine
dystrophy has been based on the false assumption that
allelic mutant genes have the same phenotypic expression.
Consequently, if researchers into muscular dystrophy are
to continue to use the mouse as an experimental animal
they must take care to specify the strain of animal used
before drawing up conclusions relating to the patho-
genesis. These findings suggest that the causal lesion of the
dy/dy dystrophic mutant lies in the musole cell; this seems
unlikely to be the case in the so-called (dy2J/dy2J) dystrophic
strain.
I thank Professor J. N. Walton and Dr J. B. Harris for
discussion and criticism of the manuscript. The work was
supported by The Medical Research Council, The Muscular
Dystrophy Group of Great Britain, and The Muscular
Dystrophy Associations of America.
ROGER PARSONS
Muscular Dystrophy Group,
Newcastle General Hospital,
Westgate Road,
Newcastle-upon-Tyne NE4 6BE, UK
Received June 10; revised July 15, 1974.
Michelson, A. M., Russell, E. S., and Harman, P. J., Proc.
1
natn. Acad. Sci. U.S.A., 41, 1079-1084 (1955).
2 Meier, H., and Southard, J. L., Life Sci., 9 Part III, 137-144
(1970).
3
Parsons, R., thesis Univ. Newcastle-upon-Tyne (1971).
4
Moore, M. J., thesis Univ. Newcastle-upon-Tyne (1973).
5
Pulvertaft, R. J. V., in Modern Trends in Pathology (edit. by
Collins, D. H.), 2 (Butterworth, London, 1959).
6
Gallup, B., and Dubowitz, V., Nature, 243, 287-289 (1973).
7 Paul, C. V., and Powell, J. A., J. neurol. Sci., 21, 365-379
(1974).
8 Hamburgh, M., Bornstein, M. B., Peterson, E. R., Crain,
S. M., Masurovsky, E. B., and Kirk, C., Progr. Brain Res.,
40, 497-508 (1973).
9 Peterson, A. C., Nature, 248, 561-564 (1974).
Growth of human muscle spindles in vitro
STRIKING histological, histochemical and physiological
differences between intrafusal and extrafusal muscle have
been observed in a large number of species. Enzyme pro-
files, for instance, of intrafusal muscle not only fail to
reflect the extrafusal pattern but show considerable inter-
species variation1,2.
The precise relationship of genetic, neural and environ-
mental influences and their role in determining the peculiar
properties of intrafusal muscle, is uncertain. There is strong
evidence that innervation is essential for the normal onto-
genetic development of spindles3 and neural control of the
histochemical properties of fully differentiated extrafusal
muscle has been shown to be considerable". Spindles de-
nervated at maturity, however, are remarkably resistant
to change.
We considered that an investigation of the growth and
differentiation of intrafusal muscle in vitro, thus isolated
from its normal neural and environmental influences, might
elucidate the role of genetic factors in determining its
characteristics in vivo. It was of particular interest to
examine the behaviour of human tissue in this context in
view of the large number of genetically determined muscle
disorders and the relative lack of information concerning
the pathological role of the spindle.
The regenerative capacity of skeletal muscle following
injury, disease, or experimentally-induced trauma is con-
siderables," and is, to some extent, reflected by the growth
and differentiation of muscle explants in culture. Normal
and diseased adult human muscle has been successfully
grown in vitro by several workers7,9, but none has speci-
fically investigated the growth of intrafusal muscle.
Whether spindles possess a similar regenerative potential
and to what extent intrafusal muscle retains its identity
in the simplified conditions of tissue culture is as yet un-
known.
The varied findings of other workers in the field indicate
that the culture employed may itself influence results and,
for valid comparisons to be made between the behaviour
of different tissues in vitro, must be a controlled factor.
For this reason, in our study, explants of both extrafusal
and intrafusal muscle from each specimen were cultured
in identical conditions.
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623
Fig. 1 A multinucleate myoblast from a culture of intra-
fusal muscle after 36 d in vitro ( x 280.80).
Muscle specimens (commonly of palmaris longus) were
obtained from patients undergoing diagnostic, motor-point
muscle biopsy. Adjacent specimens in each case were
examined by routine histological and histochemical pro-
cedures and cases in which the results of these investiga-
tions were found to be within normal limits were used in
this study.
The specimen, approximately 2 cm×0.5 cm was placed
in a Petri dish containing Eagles minimum essential medium
(MEM) with 100 U ml-1 penicillin and streptomycin. Any
spindles present were dissected out from the surrounding
tissue under a microscope, care being taken to isolate the
spindle completely from the extrafusal fibres. The outer
layers of the spindle capsule were removed and the spindle
cut transversally to form explants approximately 1 mm
long. The explants were placed on coverslips coated with
reconstituted rat tail collagen and covered with a thin clot
of chicken plasma. They were then placed in test tubes
with 3 ml of a nutrient medium consisting of 80% MEM,
10% horse serum and 10% chick embryo extract. Each
tube was sealed with a silicone rubber bung, placed on a
tube roller and incubated at 37º C. Small pieces of extra-
fusal muscle (approximately 1 mm cube) were set up in a
similar manner. The medium was changed once a week
and living cultures were examined from time to time using
the Maximow slide system and phase contrast microscopy.
All the above procedures were carried out aseptically. At
intervals between 20 and 70 d after setting up, suitable
cultures were fixed and stained with PTAH.
Fifteen stained cultures of normal intrafusal muscle have
so far been obtained, these being derived from nine
different patients and representing less than half the
number of normal intrafusal cultures initially set up, the
remainder have failed to grow. In all cases parallel extra-
fusal cultures were also obtained.
.
. ..
i
Fig. 2 A myotube from a culture of intrafusal muscle after
55 d in vitro ( x 176.80).
-
-
1
The first growth of intrafusal muscle is observed between
10 and 25 d in vitro and may precede signs of growth in
parallel extrafusal cultures by several days. Microscopic
examination at this early stage reveals, however, only
mononucleate cells and it is likely that a large proportion
of these are fibroblasts derived from the relatively large
amount of capsular connective tissue inevitably left within
the explant.
Immediately following the initial proliferation of mono-
nucleate cells, typically elongated and spindle-shaped in ap-
pearance, the majority of cultures show in addition a vari-
able number of multinucleate cells. Figure 1 shows a
multinucleate myoblast in an intrafusal culture at 36 d;
such cells are visible within the first few days of growth
following the lag period and continue to be found in cul-
tures 70 d old. The cells show extreme variability of size,
shape and number of nuclei and in older cultures there is,
in addition, an increasing proportion of myotube forms,
elongated syncitia in which some or all of the nuclei be-
come separated or aligned in chains (Fig. 2). At a later
stage peripherally situated nuclei are seen in many of these
cells.
Fig. 3 Cross striations in a myoblast from a culture of
intrafusal muscle after 70 d in vitro ( x 728).
Cross striations were observed in a small minority of
myoblasts and myotubes in both intrafusal and extra-
fusal cultures (Fig. 3). They were seen in both mono-
nucleate and multinucleate cells and when fully differen-
tiated Z lines: A and I bands and occasionally H zones
were visible, but at no time during the observation of
any of our living cultures was spontaneous contraction
observed.
Owing to the small number of cultures so far obtained
and the widely varying appearance of the growing cells,
statistical examination of results has not yet been under-
taken, though some measurements of size have been made.
In a culture of intrafusal muscle at 31 d in which the lag
period was 20 d (that is, after 11 d growth) myotubes
reached a maximum length of 720 um. This value became
800 um in another culture after 18 d growth. (These cells
were less than 20 um in width.) Another intrafusal culture
after 35 d growth produced many shorter, .broader myo-
blasts, commonly 100 um long and 50-60 um wide, contain-
ing numerous densely packed nuclei. The longest cell
reached only 400 um.
In parallel extrafusal cultures, cells also showed extreme
variation of form, but in all cases measured, the maximum
length of myotube exceeded the value for intrafusal cul-
tures, . for example up to 1,100 um after 18 d `growth.
This observation, however, may only reflect the consider-
ably larger number of extrafusal explants obtainable from
any specimen and their relatively greater mass.
It is apparent that intrafusal muscle possesses a consider-
able regenerative capacity under tissue culture conditions
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and there is little doubt that regeneration of muscle is a
function of satellite cells, of which there are many within
muscle spindles9. No ultrastructural differences can be dis-
cerned between extrafusal and intrafusal satellite cells and
our preliminary observations indicate that the growth and
differentiation of intrafusal muscle in vitro closely parallels
the behaviour of extrafusal muscle. If confirmed by further
investigation, for instance of the histochemical and ultra-
structural properties of the growing cells, it would suggest
that a critical factor for the differentiation of muscle
spindles is trophic rather than genetic and is absent under
these conditions. A strong possibility is that such a factor
derives from the neural influence and it is interesting to
consider to what extent this could be supplied in a tissue
culture system and how much its precise nature may be
analysed by this means.
This work was supported by the West Riding Medical
Research Trust.
B. J. ELLIOTT
D. G. F .: HARRIMAN
Department of Neuropathology,
University of Leeds, UK
Received June 11, 1974.
1
Spiro, A. J., and Beilin, R. L., Arch. Neurol., 20, 271-275
(1969).
2 James, N. T., Histochem. J., 3, 457-462 (1971).
3
Zelena, J., J. Embryol. exp. Morph., 5, 283-292 (1957).
4
Romanul, F. C. A., and Van der Meulen, J. P., Nature, 212,
1369-1370 (1966).
' Gilbert, R. K., and Hazard, J. B., J. Path. Bact., 89, 503-512
(1965).
6 Pogogeff, I. A., and Murray, M. R., Anat. Rec., 95, 321-329
(1946).
7 Bishop, A., Gallup, B., Skeate, Y., and Dubowitz, V., J.
neurol. Sci., 13, 333-350 (1971).
Kakulas, B. A., Papadimitriou, J. M., Knight, J. O., and
Mastaglia, F. L., Proc. Aust. Ass. Neurol., 5, 79-85 (1968).
9 Muir, A. R., in Regeneration of striated muscle and myo-
genesis (edit. by Mauro, A., Shafiq, S. A., and Milhorat,
A. T.), 199, 91-100 (International Congress Service,
Excerpta Medica, Amsterdam, 1970).
Multiple control mechanisms underlie
initiation of growth in animal cells
THE understanding of the control of cell proliferation and
differentiation requires the unravelling of the early events
that trigger the initiation of growth. An in vitro system to
study the initiation of growth is provided by the mouse 3T3
cell line. These cells exist in two alternative growth states:
either reversibly arrested in the Go phase of the cell cycle
or in active proliferation. When resting 3T3 cells are ex-
posed to fresh serum they recommence DNA synthesis and
cell division1. Functional membrane changes are among
the earliest events associated with the reinitiation of
growth2. Within minutes of serum addition, the rate of
transport of inorganic phosphate, nucleosides and glucose
is increased several fold2-6, while cyclic AMP, which has
been implicated in the regulation of growth of cultured
fibroblasts, shows an opposite change7-11.
The coordinated expression of these membrane changes
raises the possibility that some of these events are cause-
effect related .to each other12,13. Two different hypothesis
concerning this important problem have evolved. A unify-
ing view holds that cyclic AMP acts as a pleiotypic modu-
lator that regulates the overall rates of all the membrane
transport systems affected by changes in cell growth5.14.
The alternative view proposes that cyclic AMP might
control some specific changes in membrane transport but
not a general change in cell permeability 10,11. The first
hypothesis predicts that there should be a high degree of
ivature vol. 201 October. 18 1974
coupling between the various biochemical changes stimu-
lated by serum or insulin while the second envisages the
changes as dissociable, since they are supposed to be con-
trolled by several distinct mechanisms.
-
The experiments presented here were conducted to dif-
ferentiate between these alternative possibilities. Our
results substantially support the hypothesis of specific
changes and suggest that at least three distinct mechanisms
of control of membrane transport operate when growth is
initiated.
Uridine and phosphate transport systems respond to
separate control mechanisms as shown by the differential
effects produced by the addition of ouabain, cycloheximide
and cyclic AMP elevating agents. Typical experiments
illustrating this point are shown in Fig. 1. Ouabain, an
Phosphate
Uridine
With serum
With scrum
6
0
0
b
12
3
16
0
0
Uptake rate (c.p.m. x 10 3)
0
Id
0
0
0
0
-
00
0
0
Fig. 1 Differential effects of. a, 1 mM ouabain; b, 10 µg
ml -! cycloheximide; c, 1.5 mM theophylline; d, 0.2 mM
SQ 20006 and e, 40 µg ml-1 PGE, on uridine and phosphate
uptake rates. Swiss 3T3 fibroblasts were maintained in
Dulbecco's modified Eagle's medium supplemented with
10% foetal calf serum, 100 units ml-1 penicillin and 100 mg
ml-1 streptomycin in Nunc Petri dishes. Transport was
measured on cells attached to 30 mm dishes. Confluent
cultures were used usually 3 d after the last change of
medium. First, the cells were washed with phosphate-free
medium and incubated in this medium with (hatched bars)
or without the above mentioned compounds at the concen.
trations indicated. After 10 min, dialysed foetal calf serum
(10% final concentration) was added to some dishes and the
culture incubated for additional 20 min. Then, the cells
were labelled with
32 P-phosphate (75 Ci umol-1)
3.75 uCi ml-1) and 3H-uridine (29 mCi umol-1, 2.5 }Ci ml-1)
for 5 min. Uptake into acid-soluble pools and all other
materials and methods were measured as described
previously10-11. Each bar represents the average of two
transport determinations. Each experiment was repeated at
least three times. The treatments that produced effects were
shown with dark bars.
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0

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inhibitor of the (Na+, K+) APTase, significantly decreases
the activation of phosphate transport produced by serum
but has no effect either on the increase of uridine uptake or
on cyclic AMP levels. Conversely, elevation of the intra-
cellular levels of cyclic AMP by prostaglandin E1 (PGE1),
theophylline or 1-ethyl-4-hydrazino-1H-pyrazolo-(3,4-b)-pyri-
dine-5-carboxylic acid, ethyl ester, hydrochloride (SQ 20006)
(ref. 15), depresses the activation of uridine transport by
serum while the stimulation of phosphate uptake is un-
affected. In the absence of serum, PGE, stimulates the
uptake of phosphate but not that of uridine. Further,
cycloheximide added in the absence of serum activates
uridine uptake, in agreement with earlier results5,12 but it
does not modify the basal uptake level of phosphate. In
addition, when the uridine transport rates determined in
the presence of different compounds are plotted as a func-
tion of cyclic AMP levels measured in parallel cultures, an
inverse correlation is obtained. On the other hand, no cor-
relation is obtained when phosphate transport rates are
plotted as a function of cyclic AMP concentration (data not
shown). All these results show that the early transport
changes of uridine and phosphate are dissociable.
In addition to stimulating phosphate and uridine trans-
port, serum brings about a large increase in 2-deoxyglucose
uptake in resting 3T3 cells4-6. A third level of transport
control was identified when this phenomenon was carefully
examined. The activation curve as a function of time is
clearly biphasic (Fig. 2). The early phase was unaffected
by the presence of cycloheximide while the second was
completely prevented by this inhibitor. Other experiments
reveal that the kinetic parameter modified as a function
of time is the maximum velocity (L. J. de A., and E. R., un-
published). Thus, in addition to changing rapidly the uptake
of 2-deoxyglucose, serum seems to induce also the syn-
thesis of new carrier molecules. For comparison, uridine
and phosphate transport rates were measured in similar
conditions (Fig. 2). A biphasic activation curve with a
cycloheximide-sensitive second phase was also seen with
phosphate uptake but not with uridine up to 5 h after
serum addition.
The induction of the second phase of glucose transport
is not triggered by a drop in cyclic AMP since SQ 20006 does
not block it (Table 1) and insulin which decreased cyclic
AMP in resting cells7,8,10 and increases cyclic GMP in dif-
ferent tissues16 does not induce it. Cyclic AMP, however,
could modulate the activity of the transport system when
it is already inserted in the membrane (Table 1) which may
account for the rapid phase stimulated by serum.
The possibility of eliciting or inhibiting selectively some
early biochemical events by using a variety of compounds
clearly shows that these membrane changes are dissociable.
All these results support the hypothesis that conceives dis-
Table 1 Effect of SQ 20006 on the uptake of 2-deoxyglucose stimulated
by serum*
Uptake rate
Additions
Serum SQ 20006 (c.p.m. per dish)
A
320
890
.
B
280
7,751
++
4,256
5,179
+ 8
7,002
* Resting cultures of 3T3 cells were exposed to 15% foetal calf
serum for 40 min (A) or 4 h 30 min (B). Then, the uptake of
2-deoxyglucose was measured as described in the legend to Fig. 3.
+ Added at time 0.
# Added 4 h after serum.
§ Added at time 0 and removed 4 h later by washing four times
with prewarmed medium.
2-Deoxyglucose
Uridine
Phosphate
10-
10
10-
Uptake rate (c.p.m. x 10 .- 3)
a
5
a
5.
5
5
5
b
0
2
4
0
2
4
4
Time (h)
Fig. 2 Kinetics of the changes in 2-deoxyglucose, uridine
and phosphate transport produced by the addition of fresh
serum to resting cultures of 3T3 cells in the absence or
presence of cycloheximide (10 µg ml-1). The inhibitor was
added at time 0. The uptake of 3H-2-deoxyglucose was
determined as follows: the cells were washed four times
with prewarmed serum-free medium minus glucose and
incubated for 10 min in this medium with orwithout
dialysed serum or other additions. The cells were then
exposed to labelled substrate (3 uCi ml-1; 5 x 10-5 M) for
10 min. Uptake into the 5% TCA soluble fraction was linear
for 15 min and was measured as described earlier1f-11. The
other determinations were carried out as indicated in the
legend to Fig. 1. a, Serum; b, serum + cycloheximide; c,
serum-free medium.
tinct mechanisms of control switched on when growth is
initiated and seem incompatible with a pleiotypic role
for cyclic AMP in the control of all the changes in mem-
brane transport produced by serum. In fact from the pre-
sent and previous findings10,11 it is possible to define at least
three distinct mechanisms of control, that can be dif-
ferentiated on the basis of temporal expression, dependence
on cyclic AMP levels and on (Na+, K+) ATPase activity and
requirements for protein synthesis.
The various metabolic changes studied here might be
produced independently by different factors present in
serum or by different chemical signals synthesised by the
cells when exposed to a single factor .. Another important
question that can now be asked is whether or not all the
biochemical changes stimulated by serum are required for
the initiation of cell division. Our studies offer experimental
basis for attacking these problems, the clarification of
which will contribute substantially to our understanding
of the initiation of growth.
L. J. de A. is a Fellow of the Leukemia Society of
America, Inc. We thank Drs R. Burk, R. Dulbecco and M.
Stoker for critical discussions and Miss H. Wiltshire for
technical assistance. PGE1 and SQ 20006, a new inhibitor
of cyclic 3',5'-nucleotide phosphodiesterase activity were
gifts of Dr Pike, Upjohn, and Dr Chasin, Squibb,
respectively.
LUIS JIMENEZ DE ASUA
ENRIQUE ROZENGURT
Imperial Cancer Research Fund Laboratories,
Lincoln's Inn Fields,
London WC2A 3PX, UK
Received June 11; revised July 19, 1974.
Todaro, G. J., Matsuija, Y., Bloom, S., Robbins, A., and
1
Green, H., in Growth Regulating Substances for Animal
Cells in Culture (edit. by Defendi, V., and Stoker, M.),
87-98 (Wistar Institute Press, 1967).
2 Pardee. A. B., and Rozengurt, E., in Biochemistry of Cell
Walls and Membranes (edit. by Fox, C. F.), (Medical and
Technical Publishing Company. London, in the press).
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++
520
1++++
1
1
-

626 Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4 8 1974
3 Cunningham, D. D., and Pardee, I. s., Proc. natn. Acad. Sci.
U.S.A., 64, 1049-1056 (1969),
4 Grimes, W. J., and Schroeder, J. L., J. Cell Biol., 56, 487-491
(1973).
5 Kram, R., Mamont, P., and Tomkins, G. M., Proc. natn.
Acad. Sci. U.S.A., 70, 1432-1436 (1973).
6 Bradley, W. E. C., and Culp, L. A., Expl Cell Res., 84, 335-
350 (1974).
7 Otten, J., Johnson, G. S., and Pastan, I., J. biol. Chem., 247,
7082-7087 (1972).
&
Sheppard, J. R., Nature new Biol., 236, 14-16 (1972).
9
Burger, M. M., Bombik, B. M., Breckenridge, B. M., and
Sheppard, J. R., Nature new Biol., 239, 161-163 (1972).
10
Rozengurt, E., and Jimenez de Asua, L., Proc. natn. Acad.
Sci. U.S.A., 70, 3609-3612 (1973).
11
Jimenez de Asua, L., Rozengurt, E., and Dulbecco, R., Proc ..
natn. Acad. Sci. U.S.A., 71, 96-98 (1974).
12
Hershko, A., Hamont, P., Shields, R., and Tomkins, G. M.,
Nature new Biol., 232, 206-211 (1971).
13
Pardee, A. B., Jimenez de Asua, L., and Rozengurt, E., in
Control of Proliferation in Animal Cells (edit. by Clarkson,
B., and Baserga, R.), 547-561 (Cold Spring Harbor Labora-
tory, 1974).
14 Kram, R., and Tomkins, G. M., Proc. natn. Acad. Sci. U.S.A.,
70, 1659-1663 (1973).
15 Chasin; M., Harris, D. N., Phillips, M. B., Hess, S. M.,
Biochem. Pharmac., 21, 2443-2450 (1972).
16
Illiano, G., Tell, G. P. E., Siegel, M. I., and Cuatrecasas, P.,
Proc. natn. Acad. Sci. U.S.A., 70, 2443-2447 (1973).
Control of cell division in yeast
using the ionophore, A23187
with calcium and magnesium
A23187 is an ionophore which exhibits specificity for divalent
cations and promotes their passive transport across biological
membranes1. It therefore effects equilibration of divalent ions
between the cell sap and the surrounding medium and counter-
acts any active accumulation of ions within the cell. Evidence,
mainly from studies on sea urchin eggs, supports the hypo-
thesis that calcium ions play an important part in bringing
about cell division2-4. We have found that division in the
fission yeast, Schizosaccharomyces pombe, is associated with
a doubling in magnesium concentration within the cells5. It,
therefore, seems likely that, if accumulation of divalent ions
within the cell were an essential prerequisite of cell division,
this should be inhibited by A23187 and cell division should
not take place, and so we have investigated this possibility.
S. pombe, strain 132, was grown in the defined medium,
EMM2 (ref. 6). Once the cells were growing exponentially
and had reached a concentration of 106 cells ml-1 or more,
1.0 ml of the stock solution of A23187 was thoroughly mixed
with 100 ml of culture. The stock solution was prepared by
dissolving 0.5 mg A23187 in 0.05 ml of Analar acetone and
adding 1 ml of absolute ethanol7. Subsequently, cell division
was inhibited quite rapidly, only 10-15% of the cells dividing
before inhibition was complete. The ethanol/acetone mixture
on its own had no effect.
If cells which had been left in growth medium with A23187
as above for 3 h at 30° C were collected and immediately
inoculated into fresh medium, synchronous division (cell plate
index = 25-30%) ensued about 30 to 40 min after transfer.
The next division was also synchronous. DNA measurements
showed that the cells had been inhibited by A23187 in the G2
phase of the cycle. Following staining with Giemsa8, up to
47% of the cells appeared to be binucleate. Cells with a complete
cell plate, but in which division had not started as indicated
by formation of a 'waist' at the cell plate, were taken as one
cell. Thus, slightly more than half of the binucleate cells con-
tained cell plates. Occasionally a cell was observed containing
two cell plates close together and, in between, a piece of cyto-
plasm undergoing autolysis.
To define more precisely the point at which inhibition of
division was occurring, a synchronous culture of S. pombe
Table 1 Change in calcium and magnesium content of cells following
addition of 5 µg ml-1 A23187 and incubation at 30° C for 3 h
Ca
Before addition of A23187
(g per cell)
Mg
(g per cell)
Supernatant liquid
Pellet
3.0×10-14
0.5×10-14
37× 10-14
1.3×10-14
After addition of A23187 and incubation
Supernatant liquid
Pellet
5.0×10-14
0.9×10-14
5.0×10-14
0.6×10-14
was established using selection of newly divided cells from a
sucrose gradient6. Samples were taken from this culture at
suitable intervals and A23187 added to give the same final.
concentration as above. These samples were left for 3 h at
30° C and the cell number measured. In the absence of inhibi-
tion, division should have occurred within this time. This was
verified using controls without A23187 but with the ethanol/
acetone solvent. Even with a sample of cells taken only 10 min
before division could be detected, A23187 completely prevented
division taking place. Thus the block to cell division must
occur very close to the end of the cell cycle. Possibly the initial
blockage is at a stage just before nuclear division, whereas any
cell at a point after nuclear division and before cell division
is blocked at other stages which are particularly sensitive to
the changes brought about by the ionophore.
Since A23187 is supposed to make membranes permeable
to divalent ions, it was decided to investigate in general terms
what happened to calcium and magnesium within the cell.
Samples of cells were taken before and after A23187 treatment
in EMM2 for 3 h as described above. These samples were
quickly washed twice in glass-distilled water and homogenised
in 5 ml glass-distilled water in an Eaton press9. The homo-
genate was centrifuged at 40,000g (Rav) for 30 min. The super-
natant liquid was analysed without further treatment, using
atomic absorption spectrophotometry to measure calcium and
magnesium. The pellet was solubilised by digestion overnight
at 40° C with 3 ml of a 1:2 (v/v) mixture of 60% perchloric
acid and 30% hydrogen peroxide. The resultant solution was
diluted to 10 ml with glass-distilled water and analysed
for calcium and magnesium in the same way as the
supernatant. The results obtained are shown in Table 1. It is
clear that, following A23187 treatment, calcium in the pellet
has dropped dramatically (sevenfold) while magnesium has
dropped by half. The supernatant values have increased, but
to a lesser extent. Since the pellet almost certainly contains
mitochondrial vesicles, and there is evidence from other eukary-
otic cells that mitochondria accumulate calcium and, to a
must lesser extent, magnesium10, this may reflect leakage of
ions from the mitochondria brought about by A23187. The
supernatant levels would then reflect altered steady state
concentrations in the cytosol. Overall, the total calcium content
of the treated cells has dropped to 25% of that of untreated
cells while the magnesium content has dropped to 80%. This
probably reflects the differential gradients for these ions be-
tween the cell and the medium where the calcium concentra-
tion is 0.07 mM and the magnesium concentration 2.5 mM.
Finally, we attempted to reverse the effects of A23187 by
adding calcium and magnesium to cells which had been treated
with A23187 in EMM2 as previously described for 3 h. Increas-
ing the calcium concentration of the medium tenfold (to 0.7 mM)
and the magnesium concentration likewise (to 25 mM) enabled
division of about 90% of the cells to occur between 15 and 20
min after adding the ions as their chloride salts. This is somewhat
faster than the recovery when the cells are transferred to fresh
medium (see above) but no further divisions ensue whereas the
cells in fresh medium recover completely.
Similar results to the above have been obtained for the
budding yeast, Kluyveromyces fragilis (Penman, unpublished).
We therefore conclude that a pool of calcium and magnesium
is essential for cell division in yeast. It may be required to
promote the breakdown of microtubules involved in nuclear
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division and cell plate formation11 and
1
ris possibility is being
investigated.
This work was supported by a grant from the Science Research
Council. A23187 was a gift from Eli Lilly and Company.
J. H. DUFFUS
LINDA J. PATTERSON
Department of Brewing and Biological Sciences,
Heriot-Watt University,
Chambers Street,
Edinburgh EH1 1HX, UK
Received July 12; revised August 7, 1974.
1 Reed, P. W., and Lardy, H. A., in The Role of Membranes in
Metabolic Regulation (edit. by Mehlman, M.A., and Hanson,
R. W.), 111-131 (Academic Press, New York, 1972).
2
Mazia, D., J. cell. comp. Physiol., 10, 291-304 (1937).
3
Heilbrunn, L. V., in Dynamics of Living Protoplasm (Academic
Press, New York, 1956).
4 Clothier, G., and Timourian, H., Expl Cell Res., 75, 105-110
(1972).
5 Duffus, J. H., and Paterson, L. T., Z. Allg. Mikrobiol., 14,
(1974), in press.
6 Mitchison, J. M., in Methods in Cell Physiology (edit. by Prescott,
D. M.), 4, 131-165 (Academic Press, New York, 1970).
7 Prince, W. T., Rasmussen, H., and Berridge, M. J., Biochim.
biophys. Acta, 329, 98-107 (1973).
8
Ganesan, A. T., and Swaminathan, M. S., Stain Technol., 33,
115-121 (1958).
9 Eaton, N. R., J. Bact., 83, 1359-1360 (1962).
10 Lehninger, A. L., Carafoli, E., and Rossi, C. S., Adv. Enzymol.
Relat. Areas molec. Biol., 29, 259-320 (1967).
11 Borisy, G. G., Olmsted, J. B., Marcum, J. M., and Allen, C.,
Fedn Proc., 33, 167-174 (1974).
Antigen of mouse bile capillaries
and cuticle of intestinal mucosa
CELL surface antigens have been isolated by solubilisation and
fractionation of membranes1-3. Here we present data on the
membrane antigens of mouse liver cells soluble in the nonionic
detergent Triton X-100.
Mouse liver cell ghosts were obtained as in ref. 4. Triton
fractions (TF) from ghosts and from whole mouse liver cells
were obtained as in ref. 5. TFs were also prepared from mouse
kidney, intestine, spleen and the mouse ascitic hepatoma
22-a.
Liver TFs were analysed by gel double diffusion6 and by
immunoelectrophoresis7 using rabbit antisera to liver cell
ghosts. These sera do not react with soluble antigens of the liver
cell and in immunofluorescent studies they stain predominantly
the plasma membranes of parenchymal cells8 (Fig. 1a). Before
analysis all the TFs were concentrated 10-20 times and anti-
ghost sera were concentrated four times. Each TF of mouse
liver was checked by the precipitin reaction with 40 samples of
rabbit anti-ghost sera obtained from different animals and in
different cycles of immunisation.
In all TFs of mouse liver cells or ghosts studied, six antigens
were found which were precipitated by the analysed rabbit anti-
ghost sera. These antigens were presumably membrane antigens.
One of them, called here BCCA (bile capillary-cuticular antigen)
was regularly found in each liver TF whereas the other five
antigens were detected in only some of the TFs. The latter five
antigens are probably present in too small amounts to be de-
tected consistently. It seems however, that modifications in the
method of preparation of TF will eventually overcome these
variations. Using immunoprecipitation and indirect immuno-
autoradiography the distribution of BCCA in different mouse
organs and in the ascitic hepatoma 22-a was studied. BCCA is
the dominant antigenic component of the TFs from the liver
cells and ghosts and it is stable during storage. It is detected in
considerable amounts in TFs of the mouse kidney. In TFs of the
ascitic hepatoma 22-a it is determined only by immunoauto-
radiography. It is not detected in concentrated saline extracts
of mouse liver, kidney pleen or intestine by either the agar
precipitation test or immunoautoradiography.
BCCA was partly purified by agar-gel preparative electro-
phoresis7. The combined and concentrated TFs of the mouse
liver were electrophoresed in buffered 1% agar (0.5 N barbital
buffer, pH 8.6). The electrophoretic fractions (in the region of ß1-
globulin) with maximal BCCA concentration and minimal
amounts of other antigens were collected and combined.
This partially purified BCCA and the anti-ghost serum
containing antibodies predominantly to BCCA were used for
the preparation of monospecific antibodies to BCCA10.
The cell localisation of the BCCA was studied by indirect
immunofluorescence. The acetone-fixed cryostat sections of
mouse organs were treated with monospecific antibodies to the
BCCA and then by FITC-labelled purified donkey antibodies to
rabbit IgG. The specificity of the fluorescence was checked by
fluorescence inhibition.
Distinct, bright fluorescence was found on the liver cell surface
surrounding the bile capillaries between two hepatocytes (Fig. 1b).
The Kupfer cells, the epithelium of the bile duct and the vascular
endothelium were not stained. The fluorescence of bile capilla-
ries disappeared after the absorption of the monospecific anti-
bodies to the BCCA with the BCCA-containing electrophoretic
fraction. The native anti-ghost serum used for the preparation of
the BCCA monospecific antibodies also selectively lacked the
ability to stain bile capillaries after the absorption with the
electrophoretic fraction containing BCCA (Fig. 1c).
In the sections of intestine (Fig. 1d) the monospecific anti-
bodies to the BCCA stained the cuticle of the mucosa epithelial
cells of villi and crypts. This distinct cuticular fluorescence, at
different intensities, was observed for the whole length of the
intestine from the duodenum to the rectum. The other
morphological structures in the sections of the intestine were
not stained. The cuticular fluorescence disappeared after the
absorption of the monospecific antibodies to BCCA with TFs
of the liver or intestine.
h
Fig. 1 a, Mouse liver section treated with native antiserum
to liver cell ghosts. x 162. b, Mouse liver sections treated with
monospecific antibodies to the BCCA. x 72. c, Mouse liver
section treated with antiserum to liver cell ghosts absorbed by
the partially purified BCCA (electrophoretic fraction). x 162.
d, Sections of mouse intestine and e, stomach treated with mono-
specific antibodies to BCCA. x 162.
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The monospecific antibodies to Be did not stain sections
of the mouse oesophagus, stomach, kidney, spleen, pancreas and
sections of the solid variant of the mouse ascitic hepatoma 22-a
(the ascitic hepatoma, inoculated subcutaneously) (Fig. 1e).
So, BCCA is specifically localised in the bile capillaries of the
' mouse liver and the cuticle of the intestinal mucosa epithelial
cells. A small amount of BCCA (or an antigen with a similar
determinant) is also found on the plasma membrane of each
liver cell. The absence of BCCA in the cytoplasm of liver cells
suggests that it is synthesised on the cell surface.
The specific antigen of rat liver bile capillaries was described
by Sulitzeanu et al.12. They also found it in kidney proximal
tubule cells. Another antigen of rat bile capillaries has been
reported13. The authors considered this antigen to be organo-
specific but they did not demonstrate its absence in the intestine
and other rat organs.
We thank O. A. Gavrilova for technical assistance.
N. I. KHRAMKOVA
T. D. BELOSHAPKINA
Laboratory of Tumour Immunochemistry,
N. F. Gamaleya Institute for Epidemiology and Microbiology,
Moscow, USSR
Received September 26, 1973; revised April 16, 1974.
1
Chavin, S. I., FEBS Lett., 14, 269-280 (1971).
Winzler, R. J., Int. Rev. of Cytol., 29, 77-114 (1970).
3 Bustin, M., Eshhar, Z., and Sela, M., Eur. J. Biochem., 31, 541-553
(1972).
4 Bruemmer, N. C., and Thomas, L. E., Expl Cell Res., 13, 103
(1957).
5 Harris, T. M., Ocburn, C. A., and Harris, A., Transplantation, 6,
427-436 (1968).
6 Khramkova, N. I., Immunochemical analysis, 143-160 (Medicine,
Moscow, 1968).
7 Tzvetkov, V. S., Immunochemical analysis, 120-140 (Medicine,
Moscow, 1968).
8
Beloshapkina, T. D., and Khramkova, N. I., Folia biol. (Praha),
13, 335-342 (1967).
9 Elgort, D. A., and Abelev, T. I., Byull. eksp. Biol. Med., 2, 118-120
(1971).
10
Avrameas, S., and Ternycnk, F., Immunochemistry, 6, 53-66 (1969),
11
Engelgardt, N. B., Byull. eksp. Biol., 57, 1, 67 (1964).
12
Sulitzeanu, D., and Slavin, M., Clin. exp. Immun., 3, 1005-1008
(1968).
13 Sheffield, J. B., and Emmelot, P., Expl Cell. Res., 71,97-105(1972).
Ultrastructural analysis of toxin binding
and entry into mammalian cells
VERY low concentrations of phytotoxins can kill mammalian
cells, and some of them, such as ricin and abrin, isolated from
Ricinus communis and Abrus precatorius, have been reported
to suppress protein synthesis in cells 1-3 and cell-free systems4,5.
Ricin inhibits peptide chain elongation by catalytically inac-
tivating the 60S subunits of ribosomes5-7. It is a galactose-
binding glycoprotein of molecular weight 60,000, containing
two nonidentical subunits linked by disulphide bridges8-11
and seems to be identical to the lectin R. communis agglutinin
II (RCAn) isolated by affinity chromatography8-12. Ricin,
or. RCAII, has been used to suppress the growth of ascites
tumour cells in vivo13, and as a cell surface probe for
oligosaccharide receptors14-17 containing terminal D-galactose
or N-acetyl-D-galactosamine-like saccharides11,14,18,19. I have
found that RCAn enters mammalian cells by endocytosis
(similar to the findings of Oliver et al.20) and is then released
into the cytoplasm, where it apparently acts directly on
protein synthesis.
Although 125I-labelled RCAn binds rapidly to mammalian
cell surfaces14,21 (saturation <10 min at 5° C)14, there is a
significant lag before protein synthesis is inhibited (>30 min
at 37° C)21, 22. This suggests that events subsequent to cell binding
are essential for ROZTI toxicity21,22. Also, the toxic action of
RCAn can be prevented by addition of lactose21 or antitoxin22
within 15-30 min. In cell-free systems RCAn suppresses protein
synthesis almost immediately (~1-5 min)4,6,21, and this
action is not sensitive to RCAn saccharide inhibitors21. To
investigate the ultrastructural events subsequent to cell
binding I have used an electron-dense ferritin-conjugate of
RCAII (Fer-RCA1) 23, 24
Fer-RCAI was synthesised as described before23-25 and
purified by affinity chromatography on Biogel A-1.5 m24.
After elution of column-bound Fer-RCA1 by 0.2 M D-galactose,
the conjugate was dialysed extensively. Affinity purified
Fer-RCAII was active in suppressing murine 3T3 fibroblast
protein synthesis, and the inactivation required much more
time than required for simple binding to the cell surface
(Table 1). The Fer-RCAn conjugate was not as active in
suppressing protein synthesis as native RCAn; this might
reflect some inactivation during the coupling process or a
steric interference of ferritin with the enzymatic properties of
RCAII. But, as with RCAni, the conjugate was blocked by the
addition of D-galactose to the incubation medium (Table 1).
To study the ultrastructural interaction of Fer-RCAn with cells,
the conjugate was added to 0.02% EDTA-dissociated, wash-
ed, confluently grown murine 3T3 cells suspended in Dulbecco's
modified Eagle's minimal essential medium26 (DMEM)
containing 5% calf serum for various times at 4°, 20°
or 37° C. Alternatively, cells were pulsed with Fer-RCAII
for 5-15 min at 4° C, washed by centrifugation and suspended
in DMEM and then incubated further at 37º C for different
times. Controls were labelled identically escept that 0.1M
D-galactose was present in the labelling and wash solutions.
In some of the samples D-galactose was added at specific
times during Fer-RCAn labelling. Incubations were terminated
by washing the cells in fresh DMEM and then adding 1.5%
glutaraldehyde in sodium phosphate-buffered saline (pH 7.2;
310 mosmol). Fixation proceeded for at least 1 h, the first
30 min at the incubation temperature. The cells were washed
again and postfixed in 1% osmium tetroxide in 0.1 M sodium
phosphate buffer, pH 7.2, for 1 h at 20° C, washed, dehydrated
in ethanol-propylene oxide and embedded in Epon 812. Thin
sections were stained with 1% uranyl acetate and observed
in a Hitachi HU-12 electron microscope.
3T3 cells labelled with Fer-RCAj for 5-30 min at 0°-4° C
or 5-10 min at 20°-37° C before fixation showed continuous
labelling of RCAn receptors, exclusively at the cell surface
(Fig. 1a). Labelling was specific as D-galactose inhibits con-
jugate binding (Fig. 1b), as previously demonstrated24.
Labelling at higher temperatures (20°-37º C) resulted in
eventual lectin-induced receptor clustering of some but not all
RCAn receptors and subsequent endocytosis. At 37° C this
sequence of events was more rapid than at 20° C, and sig-
nificant endocytosis was seen within 60 min (Fig. 1c). With
times greater than 60 min at 37° C most Fer-RCAn molecules
were localised inside intact or broken endocytotic vesicles and
free in the cell cytoplasm (Fig. 1d).
To determine the sequence of Fer-RCA1-receptor interaction
and endocytosis, pulse labelling experiments were performed.
Cells were labelled with Fer-RCAn for 5-10 min at 0°-4º C,
washed by centrifugation, and resuspended in DMEM at
37º C for various times. To duplicate samples, D-galactose
was added immediately after the Fer-RCAn pulse at 0°-4º C,
or after 5, 15, 30 and 60 min subsequent incubation at 37° C.
After the 5 or 10 min pulse of Fer-RCAn at 0°-4º C, the
ferritin-lectin molecules located at the cell periphery could be
completely or almost completely removed from the cell surface
by addition of D-galactose (0.05 M final). After shifting
Fer-RCAn-labelled cells to 37° C following labelling and then
washing at 0°-4° C, progressively less Fer-RCAI could be
removed from cells with time by addition of D-galactose. Also,
the location of Fer-RCAn changed from exclusively extra-
cellular in a dispersed distribution (0-5 min after shifting to
37° C), to both extracellular and intracellular (clustered at the
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Table 1 Inhibition of cen protein synthesis by ferritin-conjugated
Ricinus communis II agglutinin
Pretreatment*
Time
(min)
3H-Leu into protein c.p.m.t .
None
0
111,700
#RCA11
.0
102,100
~10
103,200
60
2,400
#RCA11 + §D-galactose:
¡Fer-RCAII
60
91,400
- 0
105,100
10
103,300
60
5,100
"Fer-RCAn.+: §D-galactose
60.
85,700
** Confluently grown 3T3 cells were removed from substrate with
0.02% EDTA in TC-PBS and washed twice in DMEM containing 5%
calf serum. The cells were adjusted to a concentration of 5x 106 per
ml and 0.5 ml aliquots were incubated with or without Fer-RCAn
. (~100 µg ml-1 total protein) for various times in DMEM at 37º C in
a CO2 incubator:
t Cells were treated with Fer-RCA11 and washed in DMEM minus
leucine and incubated in DMEM plus 3H-leucine (3 uC ml-1) for 60 .
min at 37° C in a CO2.incubator. The cells were washed three times in
TC-PBS and dissolved in 1 mlof 1M sodium hydroxide containing 0.1%
bovine serum albumin. Protein was precipitated by addition of 4-5 ml
cold 10% trichloroacetic acid. After a 60 min incubation at 4º C,
the contents of each tube were filtered through Whatman G.F/C giass
filters with vacuum. The filters were washed twice with 5% trichloro-
acetic acid and once with 95% ethanol and dried. The dried filters were
placed in scintillation fluid and counted using a Beckman Model
LS200 scintillation counter:
# Final concentration, 1 µg ml-1
§ Final concentration, 0.05 M
" Final concentration (total protein), approximately 100 jug ml-1
Fer-RCAu that had moved into. cells by endocytosis was not
susceptible to removal by addition of D-galactose.
Results consistent with these have been obtained with
'125I-RCAn (ref. 21). After a 10 min pulse (1 ug ml -1-125]-RCA11)
at 4° C, approximately 95% of the cell-bound 125I-RCAII
could be removed by lactose, but less than 20% could be
removed after a 60 min subsequent incubation at 37°, C (ref.
.21)., In parallel experiments less than 5% inhibition of protein,
synthesis. was obtained after the 10 min pulse of RCAni at
0°-4° C. By 30 min after shifting to 37° C, inhibition. was
approximately 10-20%, and by 60-90 min, it was > 80%,
. similar to the results obtained with ferritin-RCAIi (Table 1).
¡ In cell-free protein synthesis systems inhibition was almost
complete within 1-2 min under these conditions21. These
studies also stress the importance of performing quantitative
lectin labelling experiments for short times at low temperatures
to preclude endocytosis of the surface bound lectin molec-
ules14-16. 27, 28
Refsnes et al.22 proposed a two-step model for the toxic action
of abrin15,22,29 and ricin on mammalian cells: in the first step.
the toxin binds to the cell surface, and in the second step it (or
portion of it) is transported into the cytoplasm. My results are .
consistent with this model, and further suggest that lectin-
induced surface aggregation30-33 and endocytosis20,21,22 are
important in the entry of toxin into mammalian cells .. The.
ultrastructural fate of Fer-RCAn after endocytosis, suggests
that the lectin molecules are released from endocytotic vesicles,
perhaps by breakdown. Lysosomes do not appear to be in-
volved in this process, because I did not observe fusion of
lysosomes with the endocytotic vesicles; however, this cannot
be ruled out for all vesicles. The fact that simple RCAn binding
to cells is not sufficient to cause inhibition of protein synthesis
suggests that the endocytosis and release into the cell cyto-
plasm of: RCAI where it can directly interact with poly-
ribosomes is the natural mode of action of this toxin. Other
toxins such as diphtheria -toxin may enter the cell to act in
similar fashion 34,35
This work was supported by a grant from the Human Cell:
Biology Programme of the US National Science Foundation, a
contract from the Tumor Immunology Program of the .
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b
C
Fig. 1 Fer-RCA. binding to murine 3T3 cells at 37º C. a, After
a· 10 min incubation, ferritin was localised exclusively at the cell
surface more or less randomly distributed. Note that the vesicle to
the left is unlabelled. b,.Labelling is specific as D-galactose abol-
ished Fer-RCAn binding activity. c, Between 30 and 60 min of
incubation Fer-RCA11 clustered at the cell surface and endocy-
tosis has begun. d, After 60-90 min incubation ferritin. was
localised inside the cell in intact or broken endocytotic vesicles
and free in the cell cytoplasm. All magnifications 68,000; bars
equal 0.1 um.
cell surface and also inside endocytotic vesicles) 30-60 min
after shifting to 37° C, to predominantly intracellular (inside
endocytotic vesicles and free in the . cell cytoplasm), at times
greater than 60 min after shifting: to. 37° C. Generally, the
Incorporation of
Additions

630
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National Cancer Institute and a g
search Institute, Inc.
from the Cancer Re-
GARTH L. NICOLSON
Cancer Council and Electron Microscopy Laboratories,
The Salk Institute for Biological Studies,
San Diego, California 92112
Received June 3; revised July 1, 1974.
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Nicolson, G. L., and Blaustein, J., Biochim. biophys. Acta, 266,
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19
VanWauwe, J. P., Loontiens, F. G., and De Bruyne, Biochim.
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21 Nicolson, G. L., Lacorbiere, M., and Hunter, T. R. (in the press).
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Refsnes, K., Olsnes, S., and Pihl, A., J. biol. Chem., 249, 3557-3562
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Nicolson, G. L., and Singer, S. J., Proc. natn. Acad. Sci. U.S.A., 68,
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Nicolson, G. L., Nature new Biol., 239, 193-197 (1972).
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28
Nicolson, G. L., J. natn. Cancer Inst., 50, 1443-1451 (1973).
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Chem., 248, 3845-3850 (1973).
Serum dopamine ß-hydroxylase activity
in developing hypertensive rats
SPONTANEOUSLY hypertensive (SH) rats1 have increased2,3
tyrosine hydroxylase and dopamine ß-hydroxylase (DBH)
activities in the adrenal glands. They also show a pro-
nounced hypotensive reaction2,3 to the specific microbial
inhibitors of these enzymes4. The results suggest some in-
volvement of the aurenergic nervous system in the hyper-
tension. But the activity of DBH in serum of SH rats,
which is released from the sympathetically innervated
organs and adrenals5, does not differ significantly from that
of normotensive Wistar rats6.
We have now compared the developmental change in
serum DBH activity in SH rats with that in normotensive
Wistar-Kyoto rats. It was from rats of this strain that SH
rats were bred, so we felt that they would be better con-
trols than the previously used normotensive Wistar rats.
We found that the serum DBH activity in young rats was
higher in the SH than in the Wistar-Kyoto strain. We also
examined the effect of NaCl administration on serum DBH
activities. We found that in 10-week-old rats it caused the
enzyme activity to increase simultaneously with a rapid
increase in blood pressure, indicating activation of the
sympathetic nerves.
SH rats and Wistar-Kyoto rats of the same age were
raised in the same conditions. Blood pressure was
measured in unanaesthetised animals using the indirect
plethysmograph1. Blood samples were obtained from rats
by decapitating and exsanguinating them into a test tube
kept in ice, and serum was removed after centrifuging at
10,000g for 10 min. The enzyme activity was assayed by
sensitive dual-wavelength spectrophotometry using tyra-
mine as substrate7. This method permits the assay of the
maximum velocity at a saturated substrate concentration
(20 mM) under the optimum pH (5.0). The incubation
mixture (total volume 1.00 ml) contained: 100 ul of serum
diluted to 400 ul with water, 200 ul of 1 M sodium acetate
buffer, pH 5.0, 100 pl of 0.1 M N-ethylmaleimide, 50 ul of
20 AM CuSO4, 50 ul of 0.2 M sodium fumarate, 50 ul of
0.2 M ascorbic acid, 50 ul of 20 mM pargyline, 1,500 units
of catalase in 50 ul of aqueous solution (1 mg ml-1), and
50 ul of 0.4 M tyramine. A sample of boiled enzyme prepa-
ration was used as control. Incubation was carried out for
45 min at 37° C. The product, octopamine, was isolated by
a microcolumn (0.5 x 10 cm) of Dowex-50-H+ (packed
volume, 0.2 ml), and converted by NaIO4 (10ul of 2%
aqueous solution) to p-hydroxybenzaldehyde which was then
isolated by successive extractions with ether and ammonia,
and finally measured by a dual-wavelength spectrophoto-
meter at 333 nm and 360 nm. Octopamine (2.00 nmol) was
added to a control incubation as internal standard. The .
activity was expressed as nmol octopamine formed per min
per ml serum.
Table 1 shows that serum DBH activity of both SH and
Wistar-Kyoto rats decreased markedly with age after 6
week. This contrasts with the situation in which humans
DBH activity increases with age, especially after an erect
posture is assumed®. The decrease in serum DBH activity
could not result from formation of endogenous inhibitors in
serum during development, since recovery of pure DBH
from bovine adrenal glands added to a rat serum sample was
similar (95%) from rats of various ages. One possibility is
that the rate of development of the sympathetic nerves in
rats may be slower than the increase of blood volume, so
that the DBH activity in a volume of serum may decrease
with age. The difference between humans and rats in this
respect may have the same explanation as the observation
that human serum contains a much higher enzyme activity
than sera from various animals9.
The enzyme activity in SH rats after 6 weeks of age is
not significantly different from those in Wistar-Kyoto rats.
This agrees with our previous result on serum DBH activi-
ties of SH rats and normotensive Wistar rats at 16 weeks
of age6. But at 3 weeks of age, serum DBH activity of SH
rats was about two times higher than that of Wistar-Kyoto
rats, indicating that the sympathetic nerve activity in SH
rats may be increased only when SH rats are very young.
The loss of difference between serum DBH activities of
SH rats and Wistar-Kyoto rats during development may
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1
Table 1 Serum dopamine ß-hydroxylase activities of developing
spontaneously hypertensive (SH) rats
Age
(weeks)
DBH activity
(nmol min-1 ml-1 serum ± s.e.m.)
Wistar-Kyoto rats
SH rats
3
1.18 ± 0.03
2.23 ± 0.09*
6
0.44 + 0.02
0.33 ± 0.01
(4)
12
0.22 ± 0.01
14
0.19 ± 0.04
0.19 ± 0.03
(6)
(6)
0.30 ± 0.07+
16
0.17 ± 0.03
53
0.20 ± 0.02
0.19 ± 0.03
(4)
*Differs from control (Wistar-Kyoto rats), P<0.01.
tNaCl was administered from 10 weeks of age for 4 weeks:
Differs from control (SH rats without NaCl administration),
P <0.05. Numbers in parentheses refer to no. of rats.
be due to a secondary compensatory mechanism in the
course of the development of hypertension.
In order to determine the reactivity of the sympathetic
nervous system of SH rats, we administered NaCl by sup-
plying 1% NaCl-aqueous solution for drinking instead of
tap water after 10 weeks of age for 4 weeks. The blood
pressure of control SH rats without NaCl administration
was increased from 170± 3 mm Hg (at 10 weeks) to 189 +
3 mm Hg (at 14 weeks), whereas that of SH rats with NaCl
administration increased from 173 ± 2 mm Hg (at 10 weeks)
to 204 + 5 mm Hg (at 14 weeks). Thus NaCl administra-
tion to SH rats increased blood pressure more pro-
nouncedly. After NaCl administration serum DBH activi-
ties were also increased significantly as shown in Table 1.
The results suggest that the sympathetic nerve activity in
SH rats may be increased only when they are very young
and decreased by a compensatory mechanism during the
development of hypertension, but may be easily activated if
blood pressure is raised acutely.
The elevated serum DBH levels when SH rats are very
young and the rapid increase in serum DBH activity during
the rapid increase of blood pressure by NaCl administration
suggest some role for the sympathetic nervous system in
the onset and maintenance of hypertension in SH rats.
We thank Professor K. Okamoto and Dr Y. Yamori
(Kyoto University, Kyoto) for spontaneously hypertensive
rats and Wistar-Kyoto rats and Miss N. Yamada and Miss
M. Suzuki for technical assistance. This work was supported
by a grant from Science and Technology Agency, Japan.
TOSHIHARU NAGATSU
TAKESHI KATO
YUKIKO NUMATA (SUDO)
KEIKO IKUTA
Department of Biochemistry,
School of Dentistry,
Aichi-Gakuin University,
Chikusa-ku, Nagoya, Japan
HAMAO UMEZAWA
MEIKI MATSUZAKI .
TOMIO TAKEUCHI
Institute of Microbial Chemistry,
Shinagawa-ku, Tokyo, Japan
Received May 24; revised July 5, 1974.
2
1
Okamoto, K., and Aoki, K., Jap. Circ. J., 27, 282-293 (1963).
Nagatsu, I., Nagatsu, T., Mizutani, K., Umezawa, H., Mat-
suzaki, M., and Takeuchi, T., Nature, 230, 381-382 (1971).
3 Nagatsu, T., Mizutani, K., Nagatsu, I., Umezawa, H., Mat-
suzaki, M., and Takeuchi, T., Molec. Cell. Biochem., 1, 107-
113 (1973).
4 Umezawa, H., Enzyme
Thibitors of Microbial Origin, 57-106
(Tokyo University Press, Tokyo, 1972).
5
Axelrod, J., Pharmac. Rev., 24, 233-243 (1972).
6
Nagatsu, T., Kato, T., Kuzuya, H., Umezawa, H., Matsuzaki,
M., and Takeuchi, T., Experientia, 28, 905-906 (1972).
7 Kato, T., Kuzuya, H., and Nagatsu, T., Biochem. Med., 10,
320-328 (1974).
8 Freedman, L. S., Ohuchi, T., Goldstein, M., Axelrod, F., Fish,
I., and Dancis, J., Nature, 236, 310-311 (1972).
9
Nagatsu, T., and Udenfriend, S., Clin. Chem., 18, 980-983
(1972).
Enzymatic synthesis of acetylcholine by a
serotonin-containing neurone from Helix
ACETYLCHOLINE (ACh) and serotonin are strong neurotrans-
mitter candidates in the snail species Helix aspersa and Helix
pomatia1,2. Experiments1 have indicated that the enzymes
. responsible for their biosynthesis, choline acetyltransferase
(ChAc, EC 2.3.1.6.) and L-aromatic amino acid decarboxylase
(AAD, EC 4.1.1.26.), were specifically localised to separate
populations of neurones in H. aspersa, and similarly for
H. pomatia (M.R.H., unpublished). Out of 150 neurones, 36
were found to contain ChAc only (detection limit 2 pmol h-1)
and 20 were found to contain AAD only (detection limit 8
pmol h-1). This clear biochemical differentiation between pre-
sumably 'cholinergic' neurones and presumably 'serotonergic'
neurones has been previously noted in other invertebrates3,4.
It was therefore surprising when, in two laboratories (St
Andrews and Kjeller), we independently found that the two
homologous giant metacerebral neurones, which contain
serotonin5 and are thought to use it as a neurotransmitter6,
could also enzymatically synthesise ACh. The coincidence of
these two enzymatic activities in the giant serotonin cell (GSC)
is a unique and therefore intriguing situation among character-
ised invertebrate nerve cells.
In experiments at Kjeller, ChAc was assayed as described by
Fonnum? with modifications in the incubation medium to yield
optimal activity for Helix8. In experiments at St Andrews,
ChAc was assayed by the same method as modified by Cog-
geshall et al.º using the following incubation medium (final
concentration): 200 uM 14C-acetyl-CoA, 20 mM choline chlo-
ride, 300 mM NaCl, 1 mM EDTA. 2H2O, 80 mM sodium
phosphate buffer (pH 7.4), 0.1 mM eserine, and 0.5% (v/v)
Triton X-100. The cells were incubated for 1 h at 37º C (to give
maximal in vitro activity), and the blanks were 1.5-2.0 pmol.
In one series of experiments, both ChAc and AAD were assayed
in the same incubation, using the following medium: 0.45
mM 3H-acetyl-CoA, 0.2 mM D,L-1-14C-DOPA, 8 mM
choline bromide, 250 mM NaCl, 20 mM sodium phosphate
Table 1 ChAc activity of the giant metacerebral cells (GSCs) after
different treatments
Treatment
Activity
H. aspersa
22.3±6.3(4)
(pmol cell-1 h-1)
H. pomatia
20.6±2.0(8)
Incubation medium + AChE-
eserine
<2 (3)
<2 (3)
Incubation medium-choline
<2 (3)
<2 (3)
Incubation medium +
Acryloylcholine (1.25 mM)
<2 (3)
<2 (5)
Chloroacetylcholine (0.125 mM)
<2 (3)
<2 (5)
Incubation medium +
CoA (100 mM)
10.8±3.7(3)
10.4±2.2(3)
Incubation medium +
ACh (20 mM)
13.0±2.9(3)
11.4±2.5(3)
Incubation medium +
8.9±4.2(3)
11.2±2.6(3)
Incubation medium +
CaCl2 (100 mM)
3.6±1.7(3)
5.5±2.3(3)
Heat denaturation (1 h at 90° C)
<2 (3)
<2 (7)
TCA precipitation
<2 (3)
<2 (7)
Results are expressed as means ± s.e.m. The numbers in parentheses
refer to no. of values used for mean.
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(5)
(4)
(4)
(4)
(10)
(4)
(4)
Control
Incubation medium +
MgCl2 (100 mM)

632
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buffer (pH 7.4), 0.1 mM eserine, 0.1 .. . A pyridoxal phosphate,
0.125 mM nialamide, 11.5 mM EDTA, and 0.2% (v/v) Triton
X-100. The assay was based on the absorption in hyamine of the
evolved 14C-CO2 from DOPA, and the extraction of 3H-ACh
by Kalignost as before7. Animals were maintained in artificial
hibernation by storage in a humid environment at 10° C. The
dissection was performed as described elsewhere1. In some
experiments, the removal of single cells was expedited by con-
ducting the dissection under 70% ethylene glycol (-20° C).
Enzyme activities were found to be unaffected by the choice of
dissection solution, as noted earlier4. All experiments were
conducted in late spring and summer.
The identification of the extractable radioactivity as acetyl-
choline was based on the following observations (see Table 1).
First, electrophoresis of the extract gave a single radioactive
product comigrating with standard ACh (Fig. 1) in two sets of
conditions4,10; second, no activity was found in the absence of
choline; third, no activity was found in the presence of acetyl-
cholinesterase and the absence of cholinesterase inhibitor and,
fourth, Kalignost extraction completely separated ACh from
acetylcarnitine and other possible acetylated contaminants11.
Synthesis of ACh was abolished on boiling the cells or after
trichloroacetic acid (TCA) precipitation (Table 1), indicating
that it is enzymatically mediated. The enzymatic ACh synthesis
was inhibited by the presence of MgCl2, CaCl2, ACh, CoA,
acryloylcholine, and chloroacetylcholine, in concentrations
known to similarly affect the partially purified ChAc from whole
snail brain8. The latter two substances should be regarded as
fairly specific ChAc inhibitors, and inhibited the GSC enzyme
to the same degree as they did other choline acetyltransferase-
containing neurones.
The level of ACh synthesis by the GSC was highly reprodu-
cible in this assay. It was lower than that of a neurone found on
the posterio-dorsal surface of the visceral ganglion (known as
cell no. 21 (ref. 1) on cell no. 4 (ref: 12)), but comparable to that
of the medial buccal cell (cell no. 4 (ref. 1)), which was considered
to have a relatively low ChAc activity. Other cells known to
contain large amounts of serotonin or AAD, such as the one
localised near the right parietal ganglion in the posterio-dorsal
surface of the visceral ganglion (known as cell no. 11 (ref. 1) on
cell no. 4 (ref. 13)), contained insignificant ChAc activity. These
cells served as controls that the observed ACh synthesis in the
GSC was not in any way associated with serotonin-metabo-
lising enzymes. Using the double assay, the uniqueness of the
GSC was reaffirmed as both enzymes could indeed be measured
simultaneously, whereas in control cells, either one activity or
none was observed (Table 2). A control extract of 0.5 ul dis-
secting medium taken from the area of the GSC dissection did
not reveal any background activity liberated by tissue disruption
(Table 2).
Examination of related species having GSCs5 revealed that
they also share this dual-enzyme characteristic in these neurones,
albeit the activity varies somewhat (M.R.H., G.A.C., P.C.E.,
and F.F., unpublished).
+
rigin
ACh
Ch
-
- a
- 1
d
e
Fig. 1 Electrophoresis of the choline acetyltransferase assay
product. Samples were electrophoresed for 50 min in the condi-
tions of Potter and Murphy9, and subsequently scanned for
radioactivity in a Nuclear Chicago Actigraph III (960 V high
voltage, I s counting, chart speed of 240 cm h-1, 6 mm colli-
mator slit width). Scans a and b represent the results of assays
using a 0.8 ul extract of snail brain homogenate as the source of
enzyme. In a, 50 ul of Kalignost extraction of the completed
incubation was applied. In b, 2 ul of the unextracted, completed
incubation was applied. In c, six GSCs were used as the enzyme
source, and in d, one cell 21 neurone. Scan e is the control to which
no enzyme was added. Acetylcholine (ACh) and choline (Ch)
mobilities were determined by staining added standards with
iodine vapour.
There is consequently strong evidence that acetylcholine can
be synthesised in a known serotonin-containing neurone by a
choline acetyltransferase.
Assuming that the ChAc serves a physiological role of ACh
production in the GSC, it is worthwhile considering the func-
tions of ACh and serotonin within the neurone. Both may serve
as neurotransmitters, in which case the long-held dogma of each
neurone making and utilising a single neurotransmitter must be
abandoned14. In this regard, it should be noted that the GSC
ChAc is more active than the base levels we quote here during
those months when it is difficult to elicit responses to serotonin
from the follower cells6. Far more likely is the possibility that
either the ACh or serotonin (or both) serves as alternative
cellular or hormonal function besides classical neurotrans-
mission, perhaps modulating metabolic activity of innervated
regions. Last, it should be considered that the ChAc activity
might arise from contamination, either by invaginating glia or
adhering microneurones. This can never be excluded when work -
Table 2 Enzyme activities of single neurones using both single and double assays for ChAc and AAD in Helix aspersa
Neurone
Location
ChAc activity
pmol cell-1 h-1
ChAc activity
pmol cell-1 h-1
AAD
activity
GSC
Ventral surface of metacerebral ganglion, near input of
inner lip nerve
22.3±6.3(4)*
19.8±9.0(6)
26.0±14.3(6)
Medial buccal cell
Lateral edge of dorsal surface of buccal ganglia.
Medial of group of three giant neurones
21.0±5.0(4)
20.3±10.2(3)
<8 (3)
Cell 21
Posterio-dorsal surface of visceral ganglion, between
65.6±7.9(5)
56.3±12.0(3)
<8 (3)
Cell 11
inputs of cutaneous pallial nerve and visceral nerve
Posterio-dorsal surface of visceral ganglion, bordering right
parietal ganglion
<2 (5)
<2 (3)
56.4±7.5(3)
Posterior buccal cell
As medial buccal cell. Posterior of group of three giant
neurones
<2 (5)
<2 (3)
<8 (3)
Control extract from area of GSC dissection (0.5 ul)
<2 (8)
Double assay
* Numbers in parentheses refer to number of determinations used to derive figure.
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633
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Nature FUI. 201
ing with hand-dissected preparations15; but the specific localisa-
tion of the enzymes to particular neurones and the absence of
ChAc activity from the dissection area do not support this
objection.
The possibility that the ACh in the conditions of the assay, is
synthesised by an enzyme other than ChAc cannot be excluded.
It seems very unlikely, however, in view of the effect of the
ChAc inhibitors. Carnitine acetyltransferase (EC 2.3.1.7.) and
aryl amine N-acetyltransferase (EC 2.3.1.5.) have been dis-
counted on the basis of their hundredfold lower activities and
different kinetic parameters (P.C.E., and M.R.H., unpublished).
Further work will focus on the direct measure of ACh and its
functional role in the GSC.
We thank Dr. A. Butler of the Chemistry Department of the
University of St Andrews for the use of the liquid scintillation
spectrometer, and Dr Catherine Hebb for advice. P.C.E. is a
Beit Memorial Fellow.
MICHAEL R. HANLEY*
G. A. COTTRELL
Wellcome Laboratories of Pharmacology,
Gatty Marine Laboratory,
The University of St. Andrews,
Fife KY16 8LB, UK
PIERS C. EMSON
MRC Brain Metabolism Unit,
Department of Pharmacology,
University of Edinburgh,
Edinburgh, UK
F. FONNUM
Norwegian Defence Research Establishment,
Division for Toxicology,
PO Box 25-N-2007 Kjeller,
Norway
Received June 13; revised August 8, 1974.
*Permanent address: Department of Molecular Biology, University
of California, Berkeley, California 94720.
1 Emson, P. C., and Fonnum, F., J. Neurochem., 22, 1079 (1974).
2 Gerschenfeld, H., Physiol. Rev., 53, 1 (1973).
3
Weinreich, D., McCaman, M. W., McCaman, R. E., and
Vaughn, J. E., J. Neurochem., 20, 969 (1973).
4 Giller, E., and Schwartz, J. H., J. Neurophysiol., 34, 93 (1971).
5
Cottrell, G. A., and Osborne, N. N., Nature, 225, 471 (1970).
6 Cottrell, G. A., and Macon, J. B., J. Physiol., 236, 435 (1974).
7 Fonnum, F., Biochem. J., 115, 465 (1969).
8
Emson, P. C., Malthe-Sorenssen, D., and Fonnum, F., J. Neuro-
chem., 22, 1089 (1974).
Coggeshall, R., Dewhurst, S. A., Weinreich, D., and McCaman,
R. E., J. Neurobiol., 3, 259 (1972).
10
Potter, L. T., and Murphy, W., Biochem. Pharmac., 16, 1386 (1967).
11 Fonnum, F., J. Neurochem., (in the press).
12
Kerkut, G. A., French, M. C., Walker, R. J., Comp. Biochem.
Physiol., 32, 681 (1970).
13
Osbornel, N. N., Szczepaniak, A. C., and Neuhoff, V., Int. J.
Neurosci., 5, 125 (1973).
14 Kandel, E. R., in Electrophysiological Studies in Neuropharma-
cology, (edit. by Koella W.), 385-398, (Thomas, Springfield,
Illinois, 1967).
15 Osborne, N. N., and Neuhoff, V., Naturwissenschaften, 59, 371
(1973).
Assignment of the gene for galactokinase
to human chromosome 17 and
its regional localisation to band q21-22
PROGRESS in somatic cell genetics now allows more rapid and
precise localisation of genes within the human genome. A large
number of mouse x human hybrid cell lines are available,
permitting the investigator to choose, for phenotype assay,
lines with a particular reduced human chromosome comple-
ment. An increasingly large number of hybrid lines carrying
rearranged chromosomes are also becoming available for
regional localisation studies. We have used such hybrid cell
lines to assign a gene coding for galactokinase (EC 2.7.1.6)
to human chromosome 17 and to further localise the gene to
band 21-22 on the long arm of the chromosome. Results from
this study have provided new information regarding the
functioning of the genes coding for the Leloir pathway enzymes
in man1, and have revealed a relatively close and potentially
useful linkage between the genes for galactokinase and for
thymidine kinase (EC 2.7.1.21).
The 70 mouse x. human cell lines used in this analysis were
produced, selected and propagated by methods reviewed
elsewhere2. Hybrid series 41 is composed of 14 primary cell
lines, 11 of known independent origin, isolated after fusion of
mouse temperature sensitive line ts-1 (ref. 3) with GM 126,
human skin fibroblasts. Series 50c consists of 5 primary lines,
4 of known independent origin, isolated after fusion of mouse
line TA3 (ref. 4) with human peripheral blood leukocytes. The
AIM series consists of 11 primary lines, 9 of known independent
origin, isolated after fusion of mouse line A9 (ref. 5) with
human skin fibroblasts. The YA series consists of 20 primary
lines, 8 of known independent origin, isolated after fusion of
A9 with human skin fibroblasts.
We also used a panel of unrelated hybrid lines chosen from a
collection of mouse x human hybrids on the basis of their
specific partial human karyotypes. Only a few different human
chromosomes are represented within a given hybrid line, the
same chromosomes being present in many or most of the
individual cells. The specific human chromosomes represented
varied widely between hybrids of the panel. Analysis of this
set of cell lines, then, generally allows the rapid and un-
equivocal correlation of a given human phenotype with a
particular human chromosome. Hybrids which composed the
panel were five subclones of JFA-14a and six of JFA-16a (two
independent hybrid lines resulting from a fusion of A9 with
human skin fibroblasts6), WA-Ia-D66 (A9 x WI-387, human
embryonic lung fibroblasts) and IL-II-58 (mouse L cell line
LM(TK-)9 x IMR 32, human neuroblastoma).
For regional localisation studies we used two hybrid lines
from the WL series10, WL-24a-2-A and WL-24a-5-C, plus five
subclones of WL-24a-2-A isolated after treatment of the hybrid
line with adenovirus-12 (ref. 11). WL hybrids originated from
a fusion of LM(TK -) with WI-38. They were maintained in
HAT selective medium12 in which cells retaining the human
thymidine kinase gene complementing the mouse deficiency
survive. Neither of these two hybrids, however, retained an
intact human chromosome 17, the chromosome to which the
cytosol form of thymidine kinase has been assigned13-15. In
WL-24a-5-C a portion of the long arm of 17 persists as a
marker chromosome, t(11; 17) (q22; q21), which arose
Table 1 Enzymatic activities assayed in hybrids and their respective
human chromosome assignments
Chromosome
Enzyme®
1
Pep C
peptidase C (EC 3.4.3 .- )
2
IDH-1
isocitrate dehydrogenase (EC 1.1.1.42)
2
MDH-1
(NAD)-malate dehydrogenase (EC 1.1.1.37)
6
ME-1
(NADP)-malic enzyme (EC 1.1.1.40)
7
MPI
mannose phosphate isomerase (EC 5.3.1.8)
10
GOT
glutamate oxaloacetate transaminase
(EC 2.6.1.1)
11
LDH A
lactate dehydrogenase (EC 1.1.1.27)
12
Pep B
peptidase B (EC 3.4.3 .- )
14
NP
purine nucleoside phosphorylase (EC 2.4.2.1)
16
APRT
adenine phosphoribosyltransferase
(EC 2.4.2.7)
18
Pep A
peptidase A (EC 3.4.3 .- )
19
GPI
glucose phosphate isomerase (EC 5.3.1.9)
20
ADA
adenosine deaminase (EC 3.5.4.2)
21
SOD-1
cytoplasmic indophenol oxidase or super-
X
G6PD
glucose-6-phosphate dehydrogenase
X
PGK
oxide dismutase-1
(EC 1.1.1.49)
phosphoglycerate kinase (EC 2.7.2.3)
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FUL. LJI VULVUCI 18 1974
Table 2 Number of hybrids showing presence (+) or absence (-) of
the two given human enzymatic activities
+/+
+/-
-1+
-1-
Total
GaK/IDH-1
5
18
3
24
50
GaK/MDH-1
6
17
7
20
50
GaK/Pep A
17
6
10
17
50
GaK/TK
16
2
0
20
38
Hybrids assayed for IDH, MDH-1 and Pep A are from series 41,
50c, AIM and YA. Hybrids assayed for TK are from series AIM, YA
and WL.
spontaneously in the hybrid16. In WL-24a-2-A the entire long
arm of 17 has been translocated to a mouse chromosome,
(M; 17q). In both cases, the translocated product represents
the sole identifiable human chromosome element in the hybrid.
Treatment of WL-24a-2-A with adenovirus-12 induced breaks
in the translocation chromosome (M; 17q) specifically in the
region 17q21-22. In subclones A12/1B and A12/1D, the break
occurred in 17q21. In subclones A12/2A, A12/2C, and A12/2D,
a break occurred in 17q22. Terminal deletions occurred distal
to these breaks. These lines have been used previously to assign
the gene coding for thymidine kinase to human chromosome
band 17q21-22 (ref. 11).
Extracts of the hybrid lines were assayed by vertical starch
gel electrophoresis for the presence of human electrophoretic
forms of 16 enzymes. Genes coding for these enzymes have
previously been mapped to specific human chromosomes,
thus the presence of the human isozyme is considered to be
strong evidence for the presence of the corresponding human
chromosome in the hybrid line. The enzymes used as markers
in this study are listed in Table 1 with their chromosome
assignments. Methods of assay have been described elsewhere17.
Thymidine kinase activity was assayed on Cellogel (Chemetron,
Milan, Italy) using the method of Migeon et al.18. Electro-
phoresis was carried out at 4º C for 4 h at 30 V cm-1 using a
buffer of 50mM TES (N-tris(hydroxy-methyl)methyl-2-amino-
ethane sulphonic acid) adjusted to pH 6.2 with triethanolamine.
Our method used to assay galactokinase activity on vertical
starch gels has been described19, (Fig. 1).
Enzymatic analysis of the hybrids from series 41, 50c, AIM
and YA indicated that galactokinase (GaK) activity segregated
discordinately with the 16 enzymes used as chromosome
markers. A representative data sample given in Table 2 shows
discordant segregation of GaK with IDH-1 and MDH-1, two
markers on chromosome 2 and with Pep A, a marker for
chromosome 18. These two chromosomes plus 12 others
corresponding to the remaining 13 enzymes could thus be
eliminated from consideration in attempts to assign the gene
coding for GaK activity to a particular human chromosome.
When the panel of well-karyotyped hybrid lines were assayed
or GaK activity (Table 3) and conversely the 11 AIM hybrids
were karyotyped this conclusion was further substantiated.
The 14 chromosomes which were eliminated from consideration
on the basis of the enzyme assays could also be eliminated on
the basis of chromosome analysis of the hybrids. Nine additional
chromosomes which either carried no easily assayed enzyme
marker or carried markers which were not included in our
assays could also be eliminated from consideration on the
basis of chromosome analysis. It then became clear that only
chromosome 17 segregated concordantly without exception
with GaK activity. The gene for GaK was assigned on this
basis to human chromosome 17. The assignment was reinforced
by thymidine kinase (TK) assay of the AIM, YA, and WL
hybrids. With only two exceptions where human GaK appeared
to be present in a hybrid and TK not, the two enzymatic
activities segregated concordantly (Table 2).
More precise localisation of the GaK gene along the length
of chromosome 17 was made possible by analysis of the WL
hybrids. Cell line WL-24a-2-A carrying the (M; 17q) trans-
location chromosome retained human GaK activity restricting
the map position of the gene to the long arm of the chromo-
some. Hybrid WL-24a-5-C which carried the t(11; 17) (q22; q21)
marker likewise showed human GaK activity further restricting
the gene to a position in or distal to 17q21. WL-24a-2-A
subclones A12/1B and A12/1D which had lost the chromosome
portion distal to a break in 17q21 had also lost GaK activity.
Subclones A12/2A, A12/2C and A12/2D in which the chromo-
some loss involved only regions distal to the break in 17q22
retained GaK activity. The GaK gene, from this evidence, must
be located between the two breakpoints, and we have
accordingly assigned the gene to human chromosome 17 band
q21-22 (Fig. 2).
Analysis, then, of the chromosome and enzyme complement
of a large number of independent hybrid cell lines of diverse
mouse x human parentage has yielded compelling evidence
for the assignment of the galactokinase gene to human chromo-
some 17. GaK activity segregated discordantly with 16 marker
enzymes and concordantly with only chromosome 17 and TK
activity. Data from two WL subclones which were functionally
back-selected for loss of TK activity indicate that GaK activity
is lost concordantly with the loss of a portion of chromosome 17.
Information obtained from the WL hybrids allowed us to
further restrict the map position of the galactokinase gene to
band q21-22. The discrepant data which we have obtained,
that regarding discordant segregation of TK with GaK in two
YA hybrids, could be explained either by the difficulty in
assaying TK or by a possible breakage and rearrangement of
chromosome 17. We have not been able to identify an intact 17
in one of the discrepant hybrids.
Only chromosome 17 is consistently present in hybrid lines
which show human GaK activity as substantiated by the
chromosome data (Table 3) and the complementary results
Table 3 Human GaK activity and human chromosomes present in hybrid cell lines
Hybrid
Human
GAK
1
2
3 4
5
6 7
8
9
10 11
12 X 13 *14 15 16 17
18 19 20 21 *22 Y
JFA-16a-5
JFA-16a-8
1 + 1
+
+
+
JFA-16a-15b
JFA-16a-19
1
1
JFA-16a-21
JFA-14a-5
JFA-14a-10c
+
+
1
+
++
JFA-14a-13
+
JFA-14a-23b
+
JFA-14a-6b
1
WA-Ia-D6
+
+
+ +
+
+
+
+ +
IL-II-5
*t(14;22).
+
+
=++++++
+
+
-
+
+
+
JFA-14a-12
+
+
.
+ ++++
+++++ ++++++
+
-
++
+
Karyotypes of hybrids were analysed using air dried preparations24 stained with quinacrine mustard or Atebrin to produce fluorescent banding
of the chromosomes25 and stained subsequently with Giemsa to reveal constitutive heterochromatin26. At least 20 cells from each hybrid line
were analysed for the presence of human chromosomes. In all cases specific chromosomes were scored as present in the hybrid if they were
found in at least 10% of the cells examined.
Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4
Human chromosomes
1

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Nature Pe. De.
origin
1 2 3 . 4 5 6 7 8 +
.
Fig. 1 Galactokinase assay of five WL-24a-2-A subclones. After
electrophoresis in citrate phosphate buffer at pH 6.8, the gel was
incubated in the reaction mixture of Mayes and Guthrie27.
14C-galactose-1-phosphate produced by the enzyme was then
precipitated in the gel with lanthanum chloride and subsequently
detected by autoradiography28. Omission of ATP from the
reaction mixture prevented the appearance of the reaction spots.
Addition of 10 mM D-glucose did not. Mouse control, LM(TK -),
was run in channel 1. Human control, KB, is shown in channel 2.
Channels 4, 6 and 8 represent extracts of subclones A12/2C,
A12/2A and A12/2D, respectively, all of which show both mouse
and human forms of the enzyme. Channels 3 and 7 represent
extracts of A12/1B and channel 5 represents A12/1D, neither of
which shows the human activity band.
from enzyme analysis (Table 2). In addition, hybrid WL-24a-
2-A which shows human GaK activity carries the long arm of 17
as its sole human chromosomal element. For this reason we
feel that the genetic locus which we have mapped codes for
a structural element of the enzyme. In this respect it might be
noted that galactokinase purified from human red blood cells
is reported to consist of two subunits of approximately equal
size with only the dimeric form showing enzymatic activity20.
We find only one spot of activity for human galactokinase by
our assay method and might suppose that it represents this
dimer. It is surprising .then that no heteropolymer of inter-
mediate electrophoretic mobility forms between human and
mouse isozymes in the hybrids as has been observed with
numerous other multimeric enzymes17.
3
1
2
1
1
-
2
1
TK, GOK
2
2
3
4
5
Fig. 2 Chromosome 17 as given by the Paris Conference (1971)
(ref. 29) indicating the assigned map position for GaK.
Galactokinase is the
hurst of three enzymes of the Leloir
pathway. Since the structural genes for these enzymes map
within the same operon in Escherichia coli21 and are closely
linked also in yeast, Saccharomyces cerevisiae22, it is interesting
to know whether the corresponding genes are linked in man.
Human galactose-1-phosphate uridyltransferase (EC 2.7.7.12)
has been assigned in two conflicting reports to chromosome 2
(ref. 23) and 3 (personal communication from T. A. Tedesco),
and we assign galactokinase to 17. It seems, then, that the
genes coding for these two enzymes of the pathway in man are
probably not linked.
Linkage of the gene coding for galactokinase with that for
thymidine kinase on the other hand, is one of the closest
linkages to be detected using the techniques of somatic cell
genetics. Since thymidine kinase is a widely used selective
marker, these two genes form an especially useful gene pair.
For example, it will be of interest to test the possible coordinate
transfer of these markers in gene transfer experiments.
This work was supported by a grant from the National
Institute of Health. S.M.E. is an NIH graduate trainee. R.K.
is a Damon Runyan cancer research fellow. K.W. was sup-
ported by a fellowship from the German Research Association.
We thank Mae Reger and Suzie Chen for technical assistance:
S. M. ELSEVIER
R. S. KUCHERLAPATI
E. A. NICHOLS
R. P. CREAGAN
R. E. GILES
F. H. RUDDLE
Department of Biology,
Yale University,
New Haven, Connecticut 06520
Institute für Genetik,
5 Köln 41,
Wyertal 121, West Germany
J. K. McDOUGALL
Department of Cancer Studies,
University of Birmingham,
Birmingham B15 2TJ, UK
Received May 27; revised September 3, 1974."
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2
Ruddle, F. H., Nature, 242, 165-169 (1973).
3
Thompson, L. H., Mankovitz, R., Baker, R. M., Wright, J. A.,
Till, J. E., Sminovitch, L., and Whitmore, G. F., J. Cell
Physiol., 78, 431-439 (1971).
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McMorris, F. A., thesis, Yale Univ. (1972).
9
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10
Boone, C., Chen, T .- R., and Ruddle, F. H., Proc. natn. Acad.
Sci. U.S.A., 69, 510-514 (1972).
11 McDougall, J. K., Kucherlapati, R., and Ruddle, F. H.,
Nature new Biol., 245, 172-175 (1973).
12
Littlefield, J. W., Science, 145, 709-710 (1964).
13
Miller, O. J., Allerdice, P. W., Miller, D. A., Breg, W. R., and
Migeon, B. R., Science, 173, 244-245 (1971).
14
Munyon, W., Buchsbaum, R:, Paoletti, E., Mann, J., Kraisel-
burd, E., and Davis, D., Virology, 49, 683-689 (1972).
15
Kit, S., Leung, W .- C., and Kaplan, L. A., Europ. J. Biochem.,
39, 43-48 (1973).
16
Kucherlapati, R., McDougall, J. K., and Ruddle, F. H., New
Haven Conference (1973): First International Workshop on
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Series, X: 3, 108-110 (National Foundation, New York,
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17
Nichols, E. A., and Ruddle, F. H., J. Histochem. Cytochem.,
21, 1066-1081 (1973).
18
Migeon, B. R., Smith, S. W., and Leddy, C. L., Biochem
Genet., 3, 583-590 (1969).
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[
.
K. WILLECKE
·

636 Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4
1
19
Nichols, E. A., Elsevier, S. M., and Ruddle, F. H., Cytogenet.
Cell Genet., 13, 275-278 (1974).
20
Blume, K .- G., and Beutler, E., J. biol. Chem., 246, 6507-6510
(1971).
21
Adhya, S. L., and Shapiro, J. A., Genetics, 62, 231-247 (1961).
22
Bassel, J., and Mortimer, R., J. Bact., 108, 179-183 (1971).
23
Sun, N. C., Chang, C. C., and Chu, E. H. Y., Proc. natn.
Acad. Sci. U.S.A., 71, 404-407 (1974).
24
Moorhead, P. S., Nowell, P. C., Mellman, W. J., Battips,
D. M., and Hungerford, D. A., Exp Cell Res., 20, 613-616
(1960).
25 Caspersson, T., Zech, L., Johansson, C., and Modest, B. J.,
Chromosoma, 30, 215-227 (1970).
6 Chen, T .- R., and Ruddle, F. H., Chromosoma, 34, 51-72
(1971).
27 Mayes, J. S., and Guthrie, R., Biochem. Genet., 2, 219-230
(1962).
28
Tischfield, J. A., Bernhard, H .- P., and Ruddle, F. H., Anal.
Biochem., 53, 545-554 (1973).
29
Paris Conference (1971): Standardization in Human Cyto-
genetics. Birth Defects: Original Article Series VIII: 7,
18-19 (National Foundation, New York, 1972).
5-Methylcytosine localised in
mammalian constitutive heterochromatin
DEOXY-5-METHYLCYTIDYLIC acid (5-MeC) is an almost
universal, although minor component of DNA in plants
and animals. It makes up 4-7% of the bases in plants1, no
more than 1.5% of the bases in the mouse2 and even less
in the human3. 5-MeC itself is not incorporated directly into
DNA. Instead, specific deoxycytidylic acid residues in
preformed DNA are methylated enzymatically by a highly
specific DNA methylase, which transfers a methyl group
from S-adenosyl methionine to specific sites in DNA‘. In
the mouse, as in other mammals, virtually all of the 5-MeC
is in the 5-MeCpG doublet", which is distributed non-
randomly in the DNA. Mouse satellite DNA contains about
3% 5-MeC in comparison to about 1.3% in main band
DNA2 and is located at the centromeric end of nearly every
chromosome®.
Antisera to 5-methylcytosine (anti-5-MeC) are available
(ref. 7 and Sawicki, Erlanger and Beiser, unpublished) and
these can react with 5-MeC residues in single-stranded
DNA. Earlier we showed that antibodies specific for
particular purine or pyrimidine bases®, which react with
denatured DNA but not-native DNA', can bind to fixed
· metaphase chromosomes. The major sites of binding can
be localised by immunofluorescence10,11. These antibodies
can therefore serve as probes of mammalian chromosome
organisation. After ultraviolet irradiation, for example, of
fixed mouse or human chromosomes, anti-adenosine (anti-A)
and anti-thymidine antibodies bound intensely to the AT-
rich constitutive heterochromatin12.
We have now investigated the chromosomal location of
5-MeC residues in both the mouse and the human. Chromo-
some preparations which had been either ultraviolet
irradiated or heated to 85° C in 0.07 M Sorensen's buffer at
pH 6.5 for 15 or 30 min and chilled suddenly to prevent
reassociation, were exposed to one or other of two different
anti-5-MeC antisera. These antisera showed no cross
reaction with thymidine or with cytidine after absorption
with an excess of cytidine conjugated to rabbit serum
albumin. The immunofluorescent banding of chromosomes
with these two antisera was thus a result of specific binding
to 5-MeC residues in the DNA and could be totally abolished
by competition with added 5-MeC.
In the mouse, anti-5-MeC showed preferential binding to
the C-band regions of centromeric heterochromatin (Fig. 1).
The fluorescent regions were very small on a few of the
larger chromosomes. These appeared to be the same
chromosomes (1 and 3) which have very little centromeric
heterochromatin in the AKR strain of mouse used in these
experiments13. The intense fluorescence of the C-band
regions partly reflects the greater concentration of 5-MeC
in mouse satellite DNA than in main band DNA2, but also
the greater degree of ultraviolet-induced strand separation
in the very AT-rich satellite DNA, which contains sequences
of up to four thymine residues in a row in its light strand,
and up to five in its heavy strand14.
The presence of 5-MeC in mouse satellite DNA indicates
that the repeating nucleotide sequence in this DNA contains
the 5-MeCpG doublet. The amount of 5-MeC, 3 mol%
(ref. 2), sets a lower limit on the length of the repeating
sequence in the satellite DNA. Assuming that each strand
is methylated equally, and taking into account the presence
of a small proportion of non-identical sequences, this
repeating sequence must be about 30 nucleotides long or
some multiple of this. This fits rather well with the estimates
of Hutton and Wetmur15 and Sutton and McCallum16.
Sutton's estimate of 8-13 nucleotides17 is clearly too short
unless the repeating sequence contains even shorter repeating
sequences, some of them not containing 5-methylcytosine.
In the human, the pattern of anti-5-MeC binding was
virtually identical whether strand separation was achieved
by ultraviolet irradiation or by heating to 85° C. In both
cases anti-5-MeC showed preferential binding to the C-band
regions of constitutive heterochromatin of chromosomes 1,
9. and 16, the centromere-short arm region of chromo-
somes 15, and the distal part of the Y (Fig. 2). The C-band
region of chromosomes 10, 17, 22 and Y sometimes showed
a small region of enhanced anti-5-MeC binding. The
observed pattern of binding, and the absence of comparable
binding of anti-5-MeC to the C-band regions of most of the
chromosomes suggests that methylation occurs only in
certain clases of repetitive DNA. For example, the strongly
positive regions in chromosomes 1 and 16 are the chief sites
Fig. 1 Metaphase spread from an AKR strain mouse
showing sites of anti-5-MeC binding. Note the bright fluor-
escence at the centromeric end of most chromosomes and
the low level of fluorescence of the remainder of all the
chromosomes. Mitotic cells from a primary embryonic cul-
ture were expanded with 0.075 M KCI hypotonic solution,
fixed in three changes of methanol : glacial acetic acid (3: 1).
and air dried. The spreads were ultraviolet irradiated for
16 h (30 cm from a germicidal lamp) either dry or in
Sorenson's buffer, incubated for 30 min with anti-5-MeC at
room temperature, washed thoroughly with phosphate
buffered saline (PBS: 20 g NaCl, 85 ml 0.25 M Na2HPO.,
15 ml 0.25 M KH2PO4 in 2,400 ml distilled water H2O,
pH 7.2-7.4), incubated for 30 min with@fluorescein-tagged
anti-rabbit . gammaglobulin prepared in sheep, washed
thoroughly with PBS, mounted in PBS and analysed by
fluorescence microscopy using transmitted light from an
HBO 200 W high pressure mercury lamp delivered through
a cardioid condenser, BG-12 exciter filter and K530 barrier
filter. The photograph was taken on Kodak Plus X film
with a 2 min exposure, developed with Acufine and printed
on Ilford No. 4 grade paper.
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Nature rvi. LUI VUIVUCI 10 1917
637
1
Shapiro, H. S., and Chargaff, E., Biochem. biophys. Acta,
39, 68-82 (1960).
Salomon, R., Kaye, A. M., and Herzberg, M., J. molec. Biol.,
43, 581-592 (1969).
3
Lawley, P. D., Crathorn, A. R., Shah, S. A., and Smith, B. A.,
Biochem. J., 128, 133-138 (1972).
4
Sheid, B., Srinivasan, P. R., and Borek, E., Biochemistry, 7,
280-285 (1968).
5
Sinsheimer, R. L., J. biol. Chem., 215, 579-583 (1955).
6
Pardue, M. L., and Gall, J. G., Science, 168, 1356-1358
(1970).
7 Levine, L., Van Vunakis, H., and Gallo, R. C., Biochemistry,
10, 2009-2013 (1971).
Erlanger, B. F., Senitzer, D., Miller, O. J., and Beiser, S. M.,
in Trans. fifth Karolinska Symp. on research methods in
reproductive endocrinology,
Stockholm, 1972. Acta
Endocr. (Copenhagen), Suppl., 168, 206-221 (1972).
Erlanger, B. F., and Beiser, S. M., Proc. natn. Acad. Sci.
U.S.A., 52, 68-74 (1964).
10
Dev, V. G., Warburton, D., Miller, O. J., Miller, D. A.,
Erlanger, B. F., and Beiser, S. M., Expl Cell Res., 74, 288-
293 (1972).
11
Schreck, R. R., Warburton, D., Miller, O. J., Beiser, S. M.,
and Erlanger, B. F., Proc. natn. Acad. Sci. U.S.A., 70,
804-807 (1973).
12
Schreck, R. R., Miller, O. J., and Erlanger, B. F., Expl Cell
Res. (in the press).
13
Dev, V. G., Miller, D. A., and Miller, O. J., Genetics, 75,
663-670 (1973).
14
Harbers, K., and Spencer, J. H., Biochemistry, 13, 1094-1101
(1974).
15
Hutton, J. R., and Wetmur, J. G., Biochem. biophys. Res.
Commun., 52, 1148-1155 (1973).
16
Sutton, W. D., and McCallum, M., Nature, 232, 83-85 (1971).
Southern, E. M., Nature, 227, 794-798 (1970).
17
18
Jones, K. W., and Corneo, G., Nature new Biol., 233, 268-
271 (1971).
19
Jones, K. W., Prosser, J., Corneo, G., and Ginelli, E.,
Chromosoma, 42, 445-451 (1973).
Structure of DNA in DNA
replication mutants of yeast
As in higher eukaryotes1,2, the nuclear DNA of the simple
eukaryote Saccharomyces cerevisiae is replicated from
multiple internal initiation sites along the chromosome3-5.
The very small size of the chromosomal DNA in yeast3,6,7
and the availability of temperature-sensitive DNA synthesis
mutants®,9 make a detailed study of DNA replication easier
in yeast than in most other eukaryotic systems. In this
report, nuclear DNA molecules from several DNA synthesis
mutants which had been classified as DNA initiation or
DNA propagation mutants on the basis of incorporation
kinetics were examined with the electron microscope. Our
results provide independent criteria for the classification of
DNA initiation and propagation mutants on the basis of
the structure of the nuclear DNA at the restrictive tempera-
ture.
Three temperature-sensitive DNA replication mutants
were used in these studies: cdc4, cdc7 and cdc8 (refs 8,9).
If synchronous cultures of cells carrying mutations in cdc4
or cdc7 are shifted to the restrictive temperature imme-
diately after initiation of DNA synthesis has occurred, ap-
proximately a complete round of DNA is made before
synthesis stops8,9. It is been suggested, therefore, that the
gene functions cdc4 and cdc7 are required for the initiation
of nuclear DNA synthesis8,9. The gene function cdc8 is
necessary for DNA propagation since cells carrying this
mutation abruptly shut off nuclear DNA synthesis when
shifted to the restrictive temperature8.
When DNA molecules from cdc4 at the restrictive tem-
perature were examined in the electron microscope, all
molecules (126/126) were simple linear structures, contain-
ing neither replication 'bubbles' ('eye-forms') nor Y-shaped
replication forks. In DNA isolated from cdc7 at the restric-
tive temperature (36° C), 144/150 molecules were simple
linear structures. Six of the molecules contained replication
forks, possibly the result of some leakiness of DNA syn-
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1
2
3
4
5
10
11
12
7
8
6
*
14
15
16
17
18
13
19
20
21
22
x
Fig. 2 Karyotype of a human cell showing sites of anti-
5-MeC binding. Note the intensely bright fluorescent regions
on chromosomes 1, 9, 15, 16 and the Y, and the difference
between homologues in each case in the size of this region.
Smaller bright centromeric dots are present on one or both
copies of chromosomes 14, 17, 22 and Y. Technique as in
Fig. 1. Chromosomes were identified by restaining the cell
with quinacrine mustard and rephotographing, using the
same fluorescence microscopy system and film but with an
exposure of only 9 s instead of the 2 min necessary with
the antibody.
of human satellite II DNA as revealed by in situ DNA-
RNA hybridisation18, whereas the corresponding 5-MeC-
positive region of chromosome 9 contains both satellite
II DNA18 and satellite III DNA19. Our results suggest that
satellite II, and perhaps satellite III, contain 5-MeC. We
are unaware of a direct chemical analysis of the 5-MeC
content of the various human satellite DNAs. Since neither
satellite 1I nor satellite III is abundant in chromosome 15
or the Y, these chromosomes must contain different types
of highly repetitious, methylated DNA.
The immunofluorescence approach described here can be
used to locate classes of highly repetitive DNA which contain
5-MeC. It is already apparent that these classes show
remarkable quantitative variation between homologues and
among individuals. These differences provide chromosome
markers which can be used in genetic linkage studies or in
the analysis of structurally altered chromosomes. This might
be particularly useful in identifying a structurally altered
chromosome 15, since its proximal portion can now be more
specifically identified than before. Striking differences have
been noted in comparing anti-5-MeC binding to chromo-
somes of gorilla, chimpanzee and orang utan (unpublished
data); and the technique seems to offer a promising
approach to the evolution of satellite or other classes of
highly repetitive DNA.
This work was supported by grants from the National
Foundation-March of Dimes and the United States
National Institutes of Health. O.J.M. is a Career Scientist of
the Health Research Council of the City of New York.
W.S. is a Fulbright-Hays Research Scholar on leave from
the University of Vienna. We should like to thank Dr
Konrad C. Hsu for making fluorescein-tagged antisera
available to us.
ORLANDO J. MILLER
WOLFGANG SCHNEDL
JULIAN ALLEN
BERNARD F. ERLANGER
Departments of Human Genetics and Development,
Obstetrics and Gynecology and Microbiology,
College of Physicians and Surgeons,
Columbia University,
New York, New York 10032
Received April 17, 1974.
9

638
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Ivature. rui. Lor. Uciover 18 1974
Fig. 1 . Small DNA replication bubble in DNA isolated
from a temperature-sensitive DNA. propagation mutant
(cdc8) at the restrictive temperature. A culture of cdc8 (strain
198-1) was grown: at 23° C in 10 ml of Y-minimal media6 ..
supplemented with 0:2 g glucose, 5 mg yeast extract, 200 µg
uracil, 200 ug adenine, 500 µg tyrosine, 500 µg histidine and
500 µg lysine. The doubling time of the culture under these
conditions is approximately 3 h. At a cell density of 2 x 106
cells ml-1, the culture was shifted to 36° C for 5 h. Cells
were then collected and spheroplasted as described pre-
viously6. 0.2 M hydroxyurea (Matheson, Coleman and Bell,
Norwood, Ohio) was added to the spheroplasting solutions.
Nuclear DNA was separated from other cellular components
by centrifugation in sucrose gradients3. Methods for spread-
ing DNA for electron microscopy have been described3.
Each branch of the bubble in the photographed molecule is
0.4 um in length; arrows show the forks. Nuclear DNA was
prepared from cdc4 (strain 135.1.1.) and cdc7 (strain 4008).
as described above. For both initiation mutants all the DNA
molecules observed in the experiments should come from cells
arrested in the cell cycle at the beginning of the S period8.º.
For cdc8, however, although most of the molecules of DNA
should come from cells blocked near the beginning of the
S period, some DNA molecules should come from cells
within the S period. Since only 3-5% of the DNA molecules .
in an asynchronous culture are replicating at any given
time3, most of the DNA molecules observed in the cdc8
experiment should contain no replication structures before
the shift to the restrictive temperature.
thesis at the restrictive temperature. This allele of cdc7
(4008) is somewhat leaky at 36° (ref. 9) synthesising nuclear
DNA at approximately 10% the normal rate (C.S.N., un-
published observations). When the experiment is done at
38° C with cdc7, 149/150 DNA molecules were simple linear
structures4,3.
.Hartwell1º has pointed out the difficulties in distinguishing
true DNA initiation mutants from mutants which have a
leaky replication block, a replication block with a delayed
'onset of expression or a replication block early in the pro-
cess of DNA replication. The observation that very few of
the DNA molecules observed in DNA isolated from the
initiation mutants cdc4 and cdc7 contain DNA replication
intermediates argues that the cells are blocked before the
onset of DNA replication. These data and the incorpora-
tion data from previous studies8,9 indicate that cdc4 and
cdc7 may be true DNA initiation mutants.
The findings with cdc8, the DNA propagation mutant,
were quite different. Approximately half (114/240) of the
DNA molecules isolated from cdc8 at the restrictive tem-
perature contained one or more replication bubbles. Often
these bubbles were very small, less than 2 um in size
·(Fig. 1). Figure 2 is a histogram of bubble sizes: The average
bubble (3 pm) observed in this experiment is less than that
found in replicating molecules isolated from a synchronous
early S-phase population of cells4.3. Figure 3 is a histogram
of centre-to-centre distances between bubbles in molecules
which contain more than one bubble.
The high proporation of DNA molecules with replication
bubbles found in experiments with cdc8 indicate that this
mutant can initiate DNA synthesis at the restrictive tem-
perature. The spacings between adjacent bubbles. (Fig. 3)
'are similar to those observed during normal DNA replica -:
tion4,3 suggesting that DNA synthesis may be initiating in
cdc8 at the restrictive temperature at the same sites as
during normal DNA replication. The small size of the re-
plication bubbles probably reflects a much reduced rate of
chain propagation at the restrictive temperature. The- rate
of nuclear DNA synthesis in cdc8 at the restrictive tempera-
ture is less than 1% the normal rate (C.S.N., unpublished
observations). An alternative explanation for the small
replication bubbles. is that the cdc8 gene function is not .
required for replication of a small amount of DNA at the
initiation site but. is required for subsequent chain propaga-
tion. This explanation seems somewhat unlikely but cannot
yet 'be ruled out. The small proportion of molecules with
large replication bubbles may be molecules which contained
replication intermediates at the time of the shift to the
restrictive temperature (as discussed in the legend to Fig. 1).
Therefore, the, large number of DNA molecules in cdc8 at .
the restrictive temperature which contain small replication
bubbles confirms the classification of cdc8 as a DNA pro-
pagation mutant. Hartwell® has recently classified another .
cell cycle mutant, cdc21, as a possible DNA propagation
mutant on the basis of incorporation kinetics. Preliminary
20
Number .
10
.
5
10
15
> 15
Bubble size (um)
Fig. 2 . Histogram of bubble sizes from DNA molecules
isolated from cdc8 at the restrictive temperature. Photo-
graphs were printed at a final magnification of 10,000 to
25,000 and contour measurements were made with a Keuffel
and Esser map measurer.
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Nature voi. 201 VLIVVel 10 1914
639
4
Number
1
2
1
0
10
20
30
.40
Centre-to-centre distance (um)
Fig. 3 Distribution of centre-to-centre distances between
bubbles on molecules containing more than one bubble.
Measurements were made on DNA molecules isolated from
cdc8 at the restrictive temperature.
experiments with cdc21 at the restrictive temperature have
shown that these DNA molecules also contain small re-
plication bubbles (T.D.P., unpublished observations).
The appearance of small replication bubbles in the
elongation mutant cdc8 but not in the initiation mutants
cdc4 and cdc7 cannot be explained by suggesting that
cdc8 is more leaky at the restrictive temperature than
cdc4 or cdc7. As already discussed, at 36° C, cdc7 is
considerably more leaky than cdc 8. These results, there-
fore, also imply a qualitative difference in the leaki-
ness of cdc7 and cdc8 (ref. 10). In cdc8 at the restrictive
temperature, a high proportion of cells are apparently syn-
thesising DNA at a slow rate of chain propagation (since
many of the DNA molecules contain small bubbles). In
cdc7 at the restrictive temperature, the leakiness of syn-
thesis may be the result of a small proportion of cells which
escape the initiation block and replicate their DNA at a
normal or nearly normal rate of chain propagation (since
very few of the DNA molecules contain DNA replication
intermediates).
This work was supported by a grant to Dr W. L. Fang-
man, University of Washington from the US National
Institutes of Health. T.D.P. was supported by a US National
Institutes of Health genetics training grant at the University
of Washington. C.S.N. was a recipient of an American
Cancer Society postdoctoral fellowship. We thank Dr W. L.
Fangman for support and encouragement and Drs D. H.
Williamson and L. H. Hartwell for suggestions concerning
the manuscript and Dr Hartwell also for providing the
three mutants.
THOMAS D. PETES*
CAROL SHAW NEWLONt
Department of Genetics,
University of Washington,
Seattle, Washington 98195
* Present address: Division of Microbiology, National Institute
for Medical Research, Mill Hill, London NW7 1AA.
t Present address: Department of Zoology, University of Iowa,
Iowa City, Iowa 52242.
Received June 4, 1974.
1 Huberman, J. A., and Riggs, A. D., J. molec. Biol., 32, 327-
341 (1968).
2 Callan, H. G., Proc. R. Soc., Lond., B181, 19-41 (1972).
3 Petes, T. D., Byers, B., and Fangman, W. L., Proc. natn. Acad.
Sci. U.S.A., 70, 3072-3076 (1973).
' Newlon, C. S., Petes, T. D., Hereford, L. M., and Fangman,
W. L., Nature, 247, 32-35 (1974).
5 Petes, T. D., Newlon, C. S., Byers, B., and Fangman, W. L.,
Cold Spring Harb. Symp. quant. Biol., 38, 9-16 (1973).
6 Petes, T. D., and Fangman, W. L., Proc. natn. Acad. Sci.
U.S.A., 69, 1188-1191 (1972).
7
Blamire, J., Cryer, D. R., Finkelstein, D. B., and Marmur, J.,
J. molec. Biol., 67, 11-24 (1972).
8
Hartwell, L. H., J. molec. Biol., 59, 183-194 (1971).
9
Hartwell, L. H., J. Bact., 115, 966-974 (1973).
10
Hartwell, L. H., Bact. Rev. (in the press).
Radiological mapping of the ribosomal
RNA transcription unit in E. coli
THE ribosomal RNA genes of Escherichia coli are arranged
in tandem behind a common promotor in the order of 16S,
23S, 5S (for review see ref. 1). In this report we redetermine the
positions of the 16S (0.55 x 106 dalton), 23S (1.10 x 106 dalton),
and 5S rRNA (0.04×106 dalton) cistrons on their unit of
transcription (scripton) by the ultraviolet light mapping
technique2-9 principally to demonstrate the applicability of this
technique to the determination of the transcriptional linkage of
genes in E. coli.
The ultraviolet light technique for mapping transcriptional
linkage is based on the observation that certain lesions induced
in DNA by ultraviolet light cause premature termination of
transcription at the site of the lesion and that no reinitiation
occurs beyond the lesion up to the next promotor5,6,10,11.
Therefore, the expression of promotor distal genes is more
sensitive to ultraviolet irradiation than that of promotor
proximal ones.
Rates of synthesis of 23S, 16S and 5S rRNA after irradiation
of E. coli with various doses of ultraviolet light were determined
by 3H-uridine pulses and quantitation of the RNA species after
polyacrylamide gel electrophoresis. Errors originating with the
isolation procedure were minimised by providing internal
standards through prelabelling of the mature rRNAs with
14C-uridine. In Fig. 1 (left and right panels) frames (a) display
rRNAs synthesised in the unirradiated sample, frames (b) to
(f) (left panel) and (b) to (d) (right panel) the synthesis in
ultraviolet-light-irradiated cultures in the order of increasing
dose. As Fig. 1 shows, synthesis of 23S rRNA was reduced
more by ultraviolet light irradiation than was the synthesis
of 16S rRNA. Synthesis of 5S rRNA was very sensitive to
ultraviolet light. A greater reduction in 23S rRNA synthesis
compared with 16S rRNA synthesis after ultraviolet irradiation
has been observed previously12-14.
Evaluation of a series of such experiments leads to the dose-
effect curves in Fig. 2. The straight line parts of the curves,
which extrapolate to 160 and 170% at the ordinate, yield
inactivation coefficients of -3.7x 10-2 s-1, -0.12 s-1, and
-- 0.12 s-1 for synthesis of 16S, 23S and 5S FRNA, respectively.
The ratio of 16S versus 23S rRNA synthesis as a function of
dose is given in the insert of Fig. 2. We have utilised this RNA
ratio curve in determining the extent of photoreactivation of
RNA chain terminating lesions.
Inactivation curves for 16S, 23S and 5S rRNA synthesis all
have shoulders in a semi-log plot, but with ultraviolet doses
greater than 10 s they all enter into a straight line portion
(Fig. 2). From this it can be concluded that all six rRNA trans-
scription units1 in a single E. coli chromosome are structurally
identical.
We suggest that the shoulders of the survival curves are
caused by the following effect. Not all ribosomal RNA scriptons
are transcribed at the maximal rate in the unirradiated cell.
As the number of unhit scriptons decreases within a cell with
increasing ultraviolet dose, dormant rRNA scriptons become
activated, or the remaining rRNA scriptons are utilised better.
When cells are irradiated for more than 10 s the rates of
rRNA synthesis are exclusively determined by the number of
unhit cistrons for the mature rRNA species.
16S, 23S and 5S rRNA can be positioned fairly accurately
on p30, the precursor RNA containing the mature rRNA
species, if the inactivation coefficients and molecular weights of
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640 Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4
ivature vol. 2JI vctover 18 1974
23 16
23 16
23 16
0.
10
4
8
5S
5S
3H,1+C c.p.m. (x 10-3)
2
6
4
2
16
2
14
V
12
12
10
10
7
(
5S
5S
0
6
2)
4
4
2
4
8
4
8
1
8
1
4
8
Distance from origin (em)
Fig. 1 Left panel: tritiated uridine labelling of 16S and 23S
rRNA as a function of ultraviolet irradiation dose. A 20 ml
culture of the ultraviolet-light-sensitive strain E. coli Bs_1 (ref.
24) was grown in M-9 medium (M-9 plus 5% (w/v) casamino acids)
at 37° C. At a concentration of 0.5 x 108 cells per ml, 14C-uridine
(54 mCi mmol-1, Amersham-Searle) was added (0.01 µCi ml-1)
and cells were grown for approximately two more generations,
about 90 min. This prelabelled culture was chilled on ice, centri-
fuged (5,100g, 15 min), resuspended in 30 ml fresh M-9 medium
(approximately 108 cells ml-1) and incubated for another 45 min
at 37° C. The culture was then chilled on ice, split into six equal
5 ml portions and all but one irradiated at 0° C with a GE
G4T4/1 low pressure mercury lamp for various times (incident
dose rate at the position of the sample was 14 erg mm-2 s-1,
the effective dose rate was 10.5 erg min-1 s-1). The unirradiated
and five irradiated samples were transferred to culture tubes
and incubated with vigorous shaking for 5 min. Tritiated uridine
(42 Ci mmol-1, Amersham-Searle) was added to a concentration
of 1 µCi ml-1' to each culture. At 12.5 min after 3H-uridine
addition, the cultures were poured into ice cold centrifuge tubes,
the cells were pelleted, resuspended in 2 ml 3mM EDTA (pH 7.2)
and lysed by addition of 0.2 ml 10% Na dodecyl sulphate
(SDS, Biorad) in a 1 min, 80° C incubation. RNA was extracted
from the lysates with H2O-saturated phenol, precipitated and
resuspended in 0.30 ml 3 mM EDTA. To these RNA preparations
sucrose was added to 15% and SDS to 1%. Samples of 0.10 ml
of each RNA preparation were layered onto 15 cm 2.6%
acrylamide gels and electrophoresed at 70 V for 5.5h; the gels
were sliced into 2 mm fractions, digested in peroxide and
counted in a liquid scintillation counter. The counts in the
3H-pulse-labelled RNA and 14C-prelabelled RNA were
corrected for interchannel spillover. Right panel: tritiated uridine
labelling of 5S and 4S RNA as a function of ultraviolet irradia-
tion dose. Samples of 0.10 ml of the same RNA preparations
used in the left panel were layered on to 8.0% polyacrylamide
gels and electrophoresed at 75 V for 6 h in order to resolve the
5S and 4S RNAs. The major peak to the right of 5S rRNA is
the 4S tRNA fraction. In both panels the ultraviolet irradiation
times were (a) 0 s; (b) 8 s; (c) 15 s; (d) 23 s; (e) 31 s; (f) 38 s. No
tritium counts in the 5S region were detectable at irradiation
times in excess of 23 s (data not shown). . , 3H c.p.m .; O,
14C c.p.m.
the rRNAs are known. Since synthesis of the 23S is 3.25 times
more sensitive than that of 16S rRNA, its 3' terminus must be
located at least 3.25× 0.55× 106 daltons = 1.80x 106 daltons
away from the p30 promotor. Since the molecular weight of the
5S rRNA is 0.04 × 106 and since 5S synthesis has essentially the
same inactivation coefficient as 23S (Fig. 3), 5S must follow
the 23S very closely on the transcriptional map. Thus, by
converting ultraviolet-light-inactivation coefficients into molecu-
lar weights, using the molecular weight of 16S rRNA as a
standard, and assuming the 16S RNA cistron to be adjacent to
the promotor locus, we project a molecular weight of about
1.85 × 10€ for p30. This size is close to previous determinations
of the p30 molecular weight between 1.75 and 106 (ref. 15) and
1.85×106 (ref. 16). If this size for p30 is correct, our data
would locate the 16S, 23S and 5S rRNA as shown in Fig. 3a,
with the 3' terminus of p30 at about 1.85x 106 daltons. If the
molecular weight of p30 were about 2.1x106 (ref. 17) the
16S rRNA could be preceded by approximately 0.09 x 106
daltons of non-conserved spacer RNA and the second spacer
situated between 16S and 23S would be about 0.30x 106
daltons (Fig. 3b). Alternatively, the additional 0.25 x 106
daltons of non-conserved RNA could be located beyond the
3' end of the map as drawn (dashed line) in Fig. 3a. Figure
3a and b gives the extreme positions of 16S, 23S and 5S rRNA
on p30. Any intermediate position that places the 3' end of
the 23S at 3.25 times the distance of the 16S 3' end from the
promotor would be consistent with our data ..
From the inactivation coefficient for the synthesis of 16S
rRNA, the dose required to produce 1 transcription terminating
200
100
50
8
10
% of control
0
0
5
0
O
0
1
0.5
10
18
6
4
.
T
RNA ratio
2
10
20
30
: 40
50
-
Dose (s)
Fig. 2 Reduction of synthesis of rRNA species as a function of
ultraviolet light dose. Data are taken from several experiments
of the type shown in Fig. 1. The relative amount of 23S, 16S
(converted from 17S values) and 5S rRNA can be calculated
accurately, using the 14C-prelabelled rRNA as an internal stan-
% of control synthesis =
L
14C c.p.m. RNAx
dard, from the expression:
1
3H c.p.m. RNAx
14℃ c.p.m. RNAx
d
3H c.p.m. RNAx
control
where x refers to 'the species of rRNA under considera-
tion, the RNA in the brackets subscribed with a d is
from a culture irradiated with a dose d, and the RNA in the
brackets subscripted with 'control' is from the unirradiated
culture. Background radioactivity due to non-rRNA was
subtracted from the total radioactivity in the peak fractions
for 23S, 17S and 5S rRNA. The exponential slopes, determined
from least squares analyses of all points greater than 10 s,
were K16 = - 3.7.x 10-2 s-1, K23 = K; =- 0.120 s-1. The
ordinate intercepts of the exponential portions of the RNA
synthesis dose-effect curves for 16S and 23S rRNA were 160%
and 170% respectively. . , 16S rRNA; O, 23S rRNA; x,
5S rRNA. The inset shows the increase in 16$ rRNA synthesis
relative to 23S rRNA synthesis (the RNA ratio) with increasing
ultraviolet light dose.
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d
d
1
0

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Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4 641
5S
-16S
-23S
b
.
5S
-16S
23S
1
0
0.5
1.0
1.5
2.0
5
Daltons ( x 10-0)
3'
Fig. 3 Map of the E. coli ribosomal RNA transcription unit
determined by radiological mapping. Two possibilities for the
arrangement of the rRNAs (hatched areas) and non-conserved
spacer RNAs (solid or dashed lines) are shown ; model a assumes
the molecular weight of p30 is 1.85x106 (refs 15 and 16) and
model b assumes the molecular weight is 2.1 x 106 (ref. 17):
lesion per 1,000 DNA base pairs in the p30 scripton was
determined to be between 550 and 640 erg mm -2.
From the extent to which ultraviolet-light-induced transcrip-
tion terminating lesions in DNA can be eliminated by photo-
reactivation, one can estimate what fraction of these lesions
comprises pyrimidine dimers. After maximal photoreactivation;
70% of the transcription terminating lesions were repaired as
determined from the shift in the ratios of 16S to 23S rRNA
(ref. 7 and inset, Fig. 2). This suggests that at least 70% of the
transcription terminating lesions comprises pyrimidine dimers18.
Photoreactivation of ultraviolet-light-induced lesions which
impair RNA synthesis in general has been reported earlier19.
Actinomycin D has been used in studies of transcriptional
linkage20,21 with the same rationale as we use in the ultraviolet
light mapping technique. The ribosomal genes of E. coli,
however, were incorrectly assigned to separate transcription-
units through the actinomycin D technique20, and the tran-
scriptional linkage of the ß-galactosidase and transacetylase
genes of the lac 'operon of E. coli (ref. 22) was determined
correctly only through an error in evaluating the experimental
data. The transcriptional arrangement of the rRNAs in mouse
L cells was incorrectly inferred from actinomycin D inhibition
of RNA synthesis (see ref. 7). Thus far, actinomycin D has
failed as an agent for transcription mapping in both prokaryotes
and eukaryotes7, whereas ultraviolet light mapping has been
used successfully in both systems (for review see ref. 23).
. This research was supported by grants from the United States
National Science Foundation and the National Institutes of
Health.
Department of Biophysics and Genetics,
University of Colorado Medical Center,
4200 East Ninth Avenue,
Denver, Colarado 80220
Received March 18; revised July 29, 1974.
1
Pace, N. R., Bact: Rev., 37, 562 (1973).
2
Starlinger, P., and Kölsch, E., Biochem. Biophys. Res. Commun.,
17, 508 (1964).
3
van den Bos, R. C., Rètel, J. and Planta, R. J., Biochim. biophys.
Acta; 232, 494 (1971).
4
Herrlich, P., Rahmsdorf, H. J., Pai, S. H., and Schweiger, M.,.
Proc. natn. Acad. Sci. U.S.A., 71, 1088 (19.74).
5
Bräutigam; A., and Sauerbier, W., J. Virol., 12, 882 (1973).
6
Bräutigam, A .; and Sauerbier, W., J. Virol., 13, 1110 (1974).
7
Hackett, P. B., thesis, Univ. Colarado (1974).
8
Hercules, K., and Sauerbier, W., J. Virol., 12, 872 (1973).
9
Hercules, K., and Sauerbier, W., J. Virol,, 12; 341 (1974).
10
Michalke, H. and Bremer, H., J. molec. Biol., 41, 1 (1969).
11
Sauerbier, W., Millette, R., and Hackett, P., Biochim. biophys.
Acta, 209, 368 (1970).
12
Sibatani, A., and Mizuno, N., Biochim. biophys. Acta, 76, 188
(1963).
13
Kroes, H. H., Schepman, A .- M., and Rorsch, A., Biochim.
biophys. Acta, 76, 201 (1963).
14 Matsuzaki, K., and Nozu, K., Biochim. biophys. Acta, 186, 85
(1969).
15 . Kossman, C. R., thesis; Univ. Colorado (1973).
16
Dunn, J. J., and Studier, F. W., Proc. natn. Acad. Aci. U.S.A., 70,
3296 (1973).
17 Nikolaev, N., Silengo, L., and Schlessinger, D., Proc. ,natn.
.
Acad. Sci. U.S.A., 70, 3361 (1973).
18
Setlow, J. K., Boling, M. E .; and Bollum, F. J:, Proc. natn. Acad.
Sci. U.S.A., 53, 1430 (1965).
19 Brdar, B., Drakulic, M., and Kos, E., Biochim. biophys. Acta, 119,
362 (1966).
20 Bleyman, M., Kondo, M., Hecht, N., and Woese, C., J. Bact., 99,
535 (1969).
21
Bleymän, M., and Woese, C., Proc. natn. Acad. Sci. U.S.A., 63,
532 (1969).
22
Webb, K., and Bleyman, M., Nature new Biol., 237, 79 (1972).
23
Sauerbier, W., Adv. Radiat. Biol. (in the press). -
24 Hill, R. F., Biochim. biophys. Acta., 30, 636 (1958).
Distribution of DNA in
dividing spinach chloroplasts
SPINACH chloroplasts, like other higher plant chloroplasts,
contain DNA1 and divide2. It has been estimated3-3 that
chloroplasts contain many copies of DNA, but in higher
plants, which contain large numbers of chloroplasts per
cell2,6 it is not known when the DNA of chloroplasts repli-
cates and how it is distributed when they divide. In the
unicellular alga, Ochromonas, which contains a single
chloroplast, 3H-thymidine is . readily incorporated into
chloroplast DNA and is distributed equally among the
progeny chloroplasts3. On the other hand, in Acetabularia,
which contains large numbers of chloroplasts per cell, DNA
could not be detected in 65-80% of the plastids, but could
easily be detected in the remainder7.
. Disks of spinach leaves grown in sterile culture were used
to study the distribution of DNA in dividing chloroplasts in
both pulse and pulse/chase experiments. In this system
chloroplast number per cell increases 5-10-fold over a 7 d
culture period in the light2. There is virtually no cell divi-
sion during this time2.
Using ?H-thymidine and autoradiography on EDTA-
separated cells dried on to glass slides (Fig. 1) we found that
in spinach; leaf discs, both chloroplast and nuclear DNA
synthesis continues during. cell expansion. The 'specificity
of incorporation into DNA was established using DNase,
RNase and acid extractions8.
The distribution of label amongst daughter chloroplasts
was followed in freshly excised spinach disks (2 mm dia-
Fig. 1 Autoradiograph of leaf cell showing 3H-thymidine
incorporation. Tissue was incubated in 3H-thymidine for 24 h
and chased 4 d as indicated in Table 1 ( x 400).
Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4
:
PERRY B. HACKETT
WALTER SAUERBIER
?
.

642 Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4
18 1974
1
Table 1 3H-Thymidine incorporation into spinach chloroplasts
End of pulse
End of chase
23.9
4.5
100%
99%
23
148
Spinach disks 2 mm in diameter were excised from the base of
leaves 2 cm long and grown on sterile agar4. 1 ml of 50 uCi
6-3H-thymidine (27 Ci mM-1, The Radiochemical Centre,
Amersham, UK) was added under sterile conditions to the 20 ml
of medium in 9 cm Petri dishes. The tissue was incubated for
24 h (14 h d-1, 20,000 1 x ) in a growth cabinet. After incubation,
nine disks were fixed in 3% glutaraldehyde after a rinse in
2:07 mM cold thymidine. The other nine disks were transferred
to fresh medium (see text), containing approximately a 1,000
times excess of cold thymidine. After 4 d in the fresh medium,
the nine remaining disks were fixed in 3% glutaraldehyde. The
disks from both the pulse and chase were treated with 0.05 M
EDTA (pH 9) overnight at 60° C to separate the cells. The cells
were spread on to slides, air dried and dipped in Ilford L4
nuclear emulsion. The slides were exposed 13 d at 5° C before
developing and mounting as permanent slides. The data for the
pulse was obtained from 10 cells with all plastids scored as
labelled or unlabelled, and 10 plastids from each cell scored for
grain number. The chase data was obtained in the same way
except that 20 plastids per cell were scored for grain number.
Independent counts were made by two observers and only chloro-
plasts clearly separated from each other were scored. Background
corrections were made; these varied from zero to two grains per
chloroplast.
meter) incubated in 3H-thymidine for 24 h. At the end of
the incubation period, one half of the disks were collected
for autoradiography, and the other half rinsed with sterile
water and transferred to fresh medium containing an excess
of cold thymidine for a further 4 d growth period.
At the end of the 3H-thymidine pulse there were 23
plastids per cell and this increased to 148 at the end of the
chase (Table 1). This represents approximately 2.7 genera-
tions. Almost all plastids were labelled both at the end of
the pulse and at the end of the chase (Table 1). These re-
sults indicate that the label is distributed to virtually all
daughter chloroplasts. The mean number of grains de-
creased from 23.9 at the end of the pulse to 4.5 at the end
of the chase (Table 1), which is close to the 3.7 expected
if the grain number was halved at each generation. This
is consistent with the chloroplast DNA being conserved
during the chase period. The nuclear DNA also appears to
20}
Frequency
10-
4-7
12-15
20-23
28-31
36-39
> 43
30-
h
20
Frequency.
10
0 2 4 6 8 10 12
1 3. 5 7 9 11 13
Silver grains per chloroplast
Fig. 2 Frequency distribution histograms of silver grains
over a 100 chloroplasts after, a, a 24-h pulse in 3H-thymidine,
and, b, a 4-d chase as indicated in Table 1. The observed
distribution of the pulse differs from that of the chase. The
distribution of the chase does not differ significantly from a
Poisson distribution having the same mean (chi-square test:
P > 0.05). An autoradiograph of a cell after the chase is
shown in Fig. 1.
be largely conserved as there were 44% labelled nuclei at
the end of the pulse and 36% at the end of the chase. We
have also found a fourfold increase in the total DNA of
discs, over a 6 d growth period, even though there is no
significant cell division during this time.
The distribution of silver grains at the end of the pulse
probably reflects the different times at which plastids com-
pleted their last division (Fig. 2). No highly labelled chloro-
plasts were found at the end of the chase and the labelling
pattern corresponds closely to a Poisson distribution. This
is good evidence that the label distributes almost equally
amongst daughter chloroplasts
as
is
the
case in
Ochromonas3.
Our results differ from those of Woodcock and Bogorad"
with Acetabularia where there are large variations in the
DNA content of chloroplasts. Herrmann9,10 has found, us-
ing chloroplast preparations, isolated from whole or parts
of Beta vulgaris leaves, that large chloroplasts contain more
DNA than small chloroplasts. He did not look at variations
in the DNA content of chloroplasts in single cells. Some of
the variation in DNA amongst chloroplasts of different sizes
can be related to developmental and nuclear genotype
changes. We consider these differences are unrelated to the
segregation of DNA that occurs in a rapidly dividing
chloroplast population which is concomitantly synthesising
DNA.
The data we have obtained establish that there is active
chloroplast DNA synthesis in expanding leaf cells of
spinach and that chloroplast DNA segregates to all
daughter chloroplasts during chloroplast replication. The
data further suggest that all chloroplasts of the cell divide
rather than a small subpopulation as has been proposed
from chloroplast size and observations6.
We thank Mrs J. Nikandrow for technical assistance.
R. J. ROSE
D. G. CRAN
J. V. POSSINGHAM
CSIRO,
Division of Horticultural Research,
PO Box 350, Adelaide,
South Australia, 5001
Received July 10, 1974.
1 Whitfield. P. R .. and Spencer, D., Biochim. biophys. Acta,
157, 333-343 (1968).
2 Possingham, J. V., and Smith, J. W., J. exp. Bot., 23, 1050-
1059 (1972).
3
Gibbs. S. P .. and Poole. R. J .. Cell Biol., 59, 318-328 (1973).
4 Tewari, K. K .. and Wildman, S. G., Symp. Soc. exp. Biol., 24,
147-179 (1970).
5
Wells, R., and Birnstiel, M., Biochem. J., 112, 777-786 (1969).
6
Honda, S. I .. Hongladarom-Honda. T .. Kwanyuen, P., and
Wildman, S. G., Planta, 97, 1-15 (1971).
Woodcock, C. L. F., and Bogorad, L., Cell Biol., 44, 361-375
(1970).
8 Ruthmann, A., Methods in Cell Research (Bell, F., and Sons
Ltd, London, 1970).
9 Herrmann, R. G., Planta, 90, 80-96 (1970).
10
Herrmann, R. G., and Kowallik, K. V., Protoplasma, 69,
365-372 (1970).
Friend virus release and induction of
haemoglobin synthesis in erythroleukaemic
cells respond differently to interferon
EXPERIMENTS with intact cells have provided the firmest
evidence that the synthesis of viral proteins is the main
function inhibited in cells treated with interferon. Changes
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8-11
16-19
24-27
32-35
40 -43
Grains per chloroplast
Labelled chloroplasts
Chloroplasts per cell

Natur
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SFFV ( x 10 3 per 106 cells)
16 B
.12
O
8
X
()
-X
3
4
6
Time (d)
b
c.p.m. (x 10 3)
+
2
0
1
X
12
16
20
Fraction no.
SFFV ( x 10-3 per 10" cells)
Interferon
No interferon.
.
+
0
-0
0)
7
7
5
7
10
Time (d)
Fig. 1 Effect of interferon on Friend virus synthesis in
unstimulated FSD1/F4 cells. Mouse interferon was pre-
pared by the infection of L-929 cells with Newcastle disease
virus (NDV-California strain). Cell supernatants were dia-
lysed against 0.1 M glycin-HCI buffer (pH 2.0) for 5 d.
and further purified by gel chromatography15. The highly
purified samples of mouse interferon (107 mouse reference
research standard units per mg protein) were assayed
by a plaque reduction test of vesicular stomatitis
virus in mouse L-929 cells15. FSD1/F4 cells have been
cultured as described previously6 at a density of 1.0 x 106
cells ml-1. Medium was removed by centrifugation and
cells transferred to the same volume of either fresh
medium or fresh medium containing 300 or 900 U ml-1
mouse interferon, respectively. Cells were transferred in daily
intervals to fresh medium with or without interferon. After
removal of the cells, aliquots of cell supernatants were
assayed for SFFV in DBA-2 mice15. Total synthesis of
C-type particles was analysed by measurement of 3H-uridine
incorporation into cell supernatant particles banding at
1.16-1.18 g cm-3. Cells were exposed to either 600 U ml-1
mouse interferon or 500 U ml-' chicken interferon from
0-48 h and 25 „Ci ml-1 5,6 3H-uridine ( Amersham; specific
activity, 50 Ci mmol-1) from 24-48 h. Cell supernatants were
cleared from debris at 8,000g, 10 min and centrifuged at
60,000g 1 h 4° C. Pellets were resuspended in a buffer con-
taining 0.1 M NaCl, 0.01 M Tris-CI, 0.001 M EDTA,
pH=7.4, layered on top of a 15-60% (w/v) sucrose gradient
in the same buffer and centrifuged for 75 min in a Spinco
SW 50-1 rotor at 42,000 r.p.m. Acid precipitable radioactivity
was determined in each fraction. a, Reduction of Friend
virus (FV) release by interferon. Spleen focus formation of
supernatant particles. O, 300 U ml-1 interferon; x, 900
U ml-1 interferon. b, Reduction of FV release by interferon,
uridine incorporation into viral particles, density 1.16-1.18
g cm-3 ( + ), sucrose gradient. O, Untreated cells; x, mouse
interferon 600 U ml-1; 4, chicken interferon 500 U ml-1.
c, Aftereffect of interferon treatment. 900 U ml-1 treatment
as in a. Interferon-free medium from day 5 on.
in an interferon-treated cell which lead to the development
of resistance may be reflected also in a cell-free system
for protein synthesis. Thus, polyribosomes of interferon-
treated L cells were found unable to translate either
mengovirus mRNA or haemoglobin mRNA-in contrast
to the endogenous mRNA synthesis which was not im-
paired'. A similar inhibition of the translation of exogenous
viral or cellular mRNAs was reported for cell-free extracts
from interferon-treated Ehrlich ascites cells2. These obser-
vations raise the question of whether the expression of
some cellular functions is as equally affected as that of
viruses. The inhibition of haemopoietic colony growth and
of phytohaemagglutinin stimulation of lymphocytes by in-
terferon seems to indicate that interferon does have such
a dual effect3,4. We show here, however, that interferon
inhibits virus release or replication but that it does not
interfere with DMSO-induced differentiation.
Friend virus-transformed erythroleukaemic spleen cells
can be maintained in culture. They can be induced to
differentiate5 and to synthesis haemoglobin6. Some of the
erythroleukaemic cell lines (clone FSD1/F4) liberate bio-
logically active Friend virus (FV), that is, spleen focus
forming virus (SFFV) (ref. 7) and lymphatic leukaemia
10º
SFFV .10° cells
0
105
104
1031
- 102
10'
10º
80
Ox
60
40 -
20-
O
()
1
3
1
5
Time (d)
Fig. 2 Effect of exogenous interferon on the production of
FV (SFFV) and on haemoglobin synthesis during dimethyl-
sulphoxide (DMSO)-induced differentiation of FSD1/F4
cells. Stimulation of FSD1/F4.cell cultures by DMSO treat-
ment has been described6. Interferon was added to the
culture medium at a concentration of 900 U ml-1. During
the course of stimulation, cells were transferred daily to
fresh medium containing 1.5% DMSO with or without inter-
feron. The production of SFFV was tested at various inter-
vals during stimulation as described previously16. Erythroid
differentiation of cells was estimated by staining cells with
benzidine. a, Effect of interferon on virus synthesis in DMSO
stimulated cells. O, DMSO 1.5%, no interferon; x, DMSO
1.5%, 900 U ml-1 mouse interferon. b, Lack of effect of
interferon on haemoglobin synthesis. The % of benzidine-
positive cells is plotted. O, DMSO 1.5%, no interferon;
x, DMSO 1.5%, 900 U ml-1 mouse interferon, .
Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4
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!
i
+
16
12
",, Haemoglobin synthesising cells
T

644 Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4
18 1974
virus (LLV). LLV acts as a helper in spleen focus forma-
tion. This cell line can therefore be used to study the effect
of exogenous homologous (mouse) interferon on the re-
lease of C-type viruses and on the induction of erythroid
differentiation (haemoglobin synthesis).
Interferon inhibits tumour formation by RNA tumour
viruses8,9. In some instances, however, interferon can be
used to enhance neoplastic development1º. Interferon also
interferes with the transformation of fibroblasts by sarcoma
viruses11. Mice with Friend erythroleukaemia have been
reported to display a reduced capacity to respond to inducers
of interferon12,13. No interferon could be detected in the
supernatants of FSD1/F4 cells or two other lines of SFFV-
transformed cells when proliferation was either in the
absence or presence of dimethylsulphoxide (DMSO), indi-
cating that FV does not trigger the interferon response
in these cells. Although interferon production can be in-
duced in these cells by an exogenous viral inducer (New-
castle disease virus) no response to poly(I) · poly(C) (ref. 12)
was observed (P.S. and W.O., unpublished). Similar ob-
servations on differential inducibility of interferon in trans-
formed mouse cells were made by others (F. Ruddle,
unpublished).
The effect of interferon on FV release in unstimulated
cells was demonstrated by the addition of 300-900 mouse
reference research standard units (U) interferon ml-1 to
cell clone FSD1/F4. We find a more than 100-fold reduction
both of spleen focus formation with cellular supernatants
and of uridine label in C-type particles. (Fig. 1a, b).
Heterologous chicken interferon does not reduce the virus
yield (Fig. 1b). The recovery of virus production after
interferon treatment for 5 d and then the omission of inter-
feron is slow and possibly incomplete. Five days after the
omission of interferon only 10% of the normal virus
level is obtained (Fig. 1c). The growth rate of the interferon
treated cells was only slightly reduced.
When FSD1/F4 cells are exposed to growth medium
which contains 1-1.5% DMSO, not only the onset of
erythroid differentiation but also a 5-10-fold increase in
released virus particles during the first and second day
of differentiation can be observed. The titre of released
virus increases 10-100-fold as assayed either by the XC-test
10,000
8,000
c.p.m.
6,000
4,000
2,000
100
150
200
Fractions
Fig. 3 Stimulation of globin synthesis with 1.5% DMSO;
Effect of interferon. Cells were pretreated with interferon
900 U ml-1 for 3 d. DMSO (1.5%) was then added and
incubation with DMSO+interferon continued for another
4 d. Medium was replaced daily. Cells were labelled with
3H- or 14C-leucine in leucine-free medium for 16 h. Extrac-
tion of NP40 soluble cytoplasmic proteins and separation
'of globin chains on carboxy methylcellulose columns was
done as described previously6.
DMSO 1.5% no
interferon, 1+C-leucine;
DMSO 1.5% 900 U ml-1
mouse interferon, 3H-leucine.
(LLV) or by the spleen focus forming test (LLV+SFFV)'.
Interferon, however, depresses the virus synthesis below
background level (Fig. 2a). Differentiation as measured by
globin synthesis is only slightly inhibited by 300-900 U ml-1
interferon. If interferon is added 3 d before the onset of
DMSO induced differentiation, perfectly normal differentia-
tion is observed. The course of differentiation, that is the
number of benzidine positive cells after adding DMSO
or DMSO and interferon, remains unchanged (Fig. 2b).
The amount of globin synthesised after 4 d stimulation
with DMSO is not reduced by adding 300-900 U ml-1
interferon (Fig. 3).
As the degree of purity of interferon often reflects on
the meaningfulness of results we confirmed our data ob-
tained with partially purified interferon (1.5×10' U. mg-1
protein), by using a highly purified interferon preparation
(107 U mg 1 protein). We found that within our limits
the degree of purity had no effect on the interferon action.
The data above make possible, for the first time, the
differentiation between the effect of exogenous interferon
on C-type virus release in transformed cells and the effect
on induced differentiated cellular functions in the same
cell. While virus synthesis is strongly inhibited, differentia-
tion remains unaffected. Interferon neither changes the
time course nor the amount of haemoglobin synthesis. It
does not change the number of cells involved in haemo-
globin synthesis either. We conclude that the release of
Friend virus is not required for in vitro erythroid differentia-
tion of these cells.
We thank Mrs H. Wocelka, A. Rohmann, B. Neu and
Miss W. Domenig for assistance. The work was supported
in part by a grant of the Deutsche Forschungsgemeinschaft.
We thank Dr Bodo for supplying some of the interferon.
P. SWETLY
Ernst-Boehringer-Institut für Arzneimittelforschung
1121 Wien, Austria
W. OSTERTAG
Max-Planck-Institut für experimentelle Medizin,
Abt. Molekulare Biologie 34 Göttingen, West Germany
Received April 4; revised August 2, 1974.
Falcoff, E., Falcoff, R., Lebleu, B., and Revel, M., Nature
new Biol., 240, 145-147 (1972).
2 Gupta, S. L., Sopori, M. L., and Lengyel, P., Biochem. Bio-
phys. Res. Commun., 54, 777-783 (1973).
3 McNeill, T. A., Gresser, 1., Nature new Biol., 244, 173-174
(1973).
" Lindahl-Magnusson, P., Leary, P., and Gresser, I., Nature
new Biol., 237, 120-121 (1972).
5 Friend, C., Scher, W., Holland, J. G., and Sato, T., Proc.
natn. Acad. Sci. U.S.A., 68, 378-382 (1971).
6 Ostertag, W., Melderis, H., Steinheider, G., Kluge, N., and
Dube, S., Nature new Biol., 239, 231-234 (1972).
7 Ostertag, W., et al., in Differentiation and Control of Malig-
nancy of Tumor Cells (University of Tokyo Press, in the
press).
8
Wheelock, F., and Larke, R. P. B., Proc. Soc, exp. Biol. Med.,
127, 230-238 (1968).
9
Liebermann, M., Merigan, T. C., and Kaplan, H. S., Proc.
Soc. exp. Biol. Med., 138, 575-578 (1971).
10
Gazdar, A. F., Sims, H., Spahn, G. J., and Baron, S., Nature
new Biol., 245. 77-78 (1973).
11
Sarma, P. S., Shiu, G., Baron, S., and Huebner, R. J., Nature,
223, 845-846 (1969).
12
De Maeyer-Guignard, J., Science, 177, 797-799 (1972).
13
Wheelock, E. F., Proc. natn. Acad. Sci. U.S.A., 55, 774-780
(1966).
14 Ostertag, W., Crozier, T., Kluge, N., Melderis, H., and Dube,
S. K., Nature new Biol., 243, 203-205 (19.73).
15 Bodo, G., Palese, P., and Lindner, J., Proc. Soc. exp. Biol.
Med., 137, 1392-1395 (1971).
16
Axelrad, A. A., and Steeves, R. A., Virology, 24, 513-518
(1964).
Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4

Nature
Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4 645
Erythropoietin responsiveness of
differentiating Friend leukaemia cells
INCLUSION of dimethylsulphoxide (DMSO) in the media of
suspension cultures of Friend leukaemia cells (FLC) results
in the erythroid differentiation of more than 50% of the
cells1. This differentiation is demonstrated by morphological
and biochemical alterations which are characteristic of
normal erythroid maturation2-5. We report here that the
differentiation of these leukaemic cells is accompanied by
the development of responsiveness to erythropoietin (Ep),
the physiological regulator of erythropoiesis.
Friend leukaemia cells (clone 745A) were grown in sus-
pension culture in media consisting of basal Eagle's medium
with Earl's balanced salts (GIBCO), made 15% with foetal
calf serum (GIBCO) at 37° C in a humidified atmosphere of
5% CO2 and 95% room air. The cultures were passaged at
3 or 4 d intervals at a seeding concentration of 1×10' cells
ml-1. DMSO (Fisher Sci.) was added (2% v/v) where appro-
priate at the time of seeding the cultures. Erythropoietin
(human urinary erythropoietin 75.4 IU mg-1) was added to
the suspension cultures at the times indicated.
To assess the rate of haem synthesis, 0.5 uCi of mouse
transferrin-bound "9Fe was added to culture plates contain-
ing 3 ml of the suspension culture and after 4 h further
incubation the cells were collected, washed twice with
isotonic saline and the haem extracted with cyclohexanone
according to the method of Krantz6, and counted. This
procedure provides an accurate assessment of the rate of
haem synthesis by Friend leukemia cells7. We have already
shown that during DMSO-induced differentiation of Friend
erythroleukaemia cells, increased iron incorporation into
haem was coordinated with increases in the rate of synthesis
of globin, haemoglobin, and globin mRNA8. To assess the
rates of DNA, RNA and protein synthesis, 5 uCi of 3H-
thymidine, 3H-uridine or 3H-leucine (New England Nuclear
Co.) were added to appropriate culture plates containing
4 ml of suspension cell and after 40 min of incuba-
tion the cells were collected, washed twice, and the rate of
precursor incorporation determined according to the
method of Schneider as discussed by Shatkin9. All radio-
active assays were determined using three plates for each
experimental point and each experiment was carried out at
least three times.
Ep was added to suspension cultures of erythroleukaemia
cells which had previously been grown in the presence of
DMSO for 3 d (Table 1). This significantly stimulated the
rate of haem synthesis of the cells. . Ep had no significant
effect on the rate of increase in cell numbers in the culture
(which increased from 0.95+0.03×106 cells ml-1 on day 3
to 1.24 ±0.04×106 cells ml-1 on day 4) nor did it result in
an increase in the proportion of benzidine-positive cells in
the culture. Ep had no effect on the rate of haem synthesis
by cells which had not been cultured in the presence of
DMSO (Table 2).
The stimulatory effect of Ep on DMSO-treated cells was
not limited to haem synthesis. The effects of Ep on the rate
of precursor incorporation into DNA, RNA and protein
are given in Table 2. The increases in leucine and uridine
incorporation were quantitatively similar to that observed
for "9Fe; the rate of DNA synthesis also increased. These
effects of Ep on the rates of macromolecular synthesis by
these differentiating leukaemic cells were similar to those
reported for normal rat bone marrow cells10 and foetal
liver erythroid cells11. Although Ep had no effect on the
rates of haem, RNA or protein synthesis by cells which
had not been cultured in the presence of DMSO, the
addition of Ep to suspension cultures of these cells resulted
in a decrease in the rate of thymidine incorporation into
DNA.
During cell growth and replication, Ep responsiveness
could be reliably detected when Ep was added on the
third day of culture and haem synthesis assayed 1 d later.
The addition of Ep earlier during culture with assay of haem
synthesis 24 h after addition produced no significant effect.
The addition of Ep to cultures which had been growing in
the presence of DMSO for more than 3 d also resulted in an
increase in the rate of haem synthesis. By contrast, cultures
to which Ep was added at the time of seeding in the
presence of DMSO synthesised less haem on the fourth
day of culture than cultures also seeded in the presence of
DMSO but to which Ep was not added. In six such experi-
ments the rate of haem synthesis (mean c.p.m. per plate ±
s.e. for 17 degrees of freedom) was 16+4.8% less than
cultures seeded without Ep.
Table 1 Effect of Ep on erythroleukaemic cells grown in DMSO
medium
Parameter
Mean % stimulation
. s.e.m.
No.
experiments
(1) Haem synthesis
a CPM-59 Fe per plate
+ 29 ±4.6
29
b CPM-59 Fe per 106 cells
+ 29 ± 4.1
29
(2) Cell number
+ 3 ± 2.4
29
(3) % Benzidine-positive cells
+6 ± 4.7
20
Tissue culture plates were set up containing 4 ml of suspension
culture which was made 2% (v/v) with DMSO at the time of seeding.
After 3 d of culture Ep (0.06 IU ml-1) was added to appropriate
plates and 24 h later, after removal of 1 ml from each plate for cell
counts and morphological studies, 59Fe was added and 4 h later the
haem extracted. For morphological studies, slides were made with a
cytocentrifuge, were benzidine stained13, and counterstained with
Wright-Giemsa. The proportion of benzidine-positive cells was
determined by a single observer in a single-blinded position. The
following equation was used to determine % stimulation of each
parameter:
(Parameter Ep culture) - (Parameter no Ep culture) × 100
(Parameter no Ep culture)
The % stimulation was determined for each individual experiment
and the data so derived pooled and used to calculate the grant mean
and s.e.m. for each parameter.
Ep was an effective stimulant of haem synthesis within
a broad range of concentrations. A comparison of the
stimulatory effects of 0.04 IU ml-1 and 0.2 IU ml-1 on the
rate of haem synthesis revealed no significant difference
(34.5±6.4% stimulation for 0.04 IU ml-1 compared with
44 ±6.6% stimulation for 0.2 IU ml-1).
We have tested three different human urinary Ep and
three different Step III sheep plasma Ep and have found no
significant differences in their effects.
In a preliminary communication we reported that Ep
stimulated the rate of haem synthesis by Friend leukaemia
cells which had previously been cultured in the presence of
DMSO12. In those studies, however, the condition of culture
were such that the cells were not replicating and the rate of
haem synthesis in all the cultures was declining. The
studies reported here demonstrate that under conditions of
growth the erythroid differentiation of these leukaemic cells
is accompanied by the development of responsiveness to Ep.
It is the process of differentiation per se and not the
penetrant carrier properties of DMSO, that is responsible
for the responsiveness to Ep, since Ep stimulated the rate
of haem synthesis when the differentiating cells had been
transferred to fresh medium before adding Ep12. It is of
interest in this regard that under the conditions of culture
reported here, the appearance of responsiveness to Ep
correlated with a major increase in the proportion of
recognisable differentiating cells in the culture (the per-
centage of benzidine-positive cells on days 1 and 2 of
culture were 1% and 5%, whereas on the third day it was
50%). This suggests that under these conditions Ep affects
cells which have differentiated to the point of having
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64 Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4
18 1974
synthesised enough haem to be recognisable by benzidine
staining. The effects of Ep on normal erythroid pre-
cursor cells which are more differentiated than the com-
mitted erythroid stem cell have been reported by others 13-15.
The apparent inhibitory effects of Ep on DNA synthesis by
control cells and on the rate of haem synthesis by DMSO-
treated cells when Ep was added at the time of seeding
was unexpected and may have been due to other sub-
stances present in the preparation of human urinary Ep.
The resolution of this question awaits the availability of
pure Ep.
Table 2 Effects of Ep on synthesis of DNA, RNA and protein
Macromolecule
Ep
DMSO-containing
cultures
Mean c.p.m.
per 106 cells ± s.e.
Control
cultures
Mean.c.p.m.
per 106 cells L s.e.
(a) DNA
0
6,073 ± 1182
3,928 ± 158
(b) RNA
0
1,737 ±
145
5,018 + 254
(c) Protein
0
4,408 ±
84
15,687 ± 310
(d) Haem
0
1,169
+ 128
7
1
1,796 士
103
7
Suspension cultures were set up in bottles containing 40 ml
each. After 3 d of culture Ep was added to appropriate bottles
(0.06 IU ml-1) and after 24 h of additional culture, the suspension
cultures were distributed into triplicate plates for determination of the
rate of macromolecular synthesis. The differences between the rates
of macromolecular synthesis between Ep and non-Ep containing
cultures were statistically significant (P < 0.05) for all of the DMSO-
containing cultures and for the decrease in the rate of DNA synthesis
of the control cultures.
All determinations were done in triplicate.
These observations suggest that erythroleukaemic cells,
and perhaps leukaemic cells in general, possess the genetic
information needed for the development of responsiveness
to normal physiological regulators and that under appro-
priate conditions this information is expressed. Respon-
siveness to Ep by a continuous suspension culture cell line
promises of facilitate studies of the mechanism of action of
this hormone.
HARVEY D. PREISLER*
MIRIAM GILADI
Mount Sinai School of Medicine,
New York, New York 10029
Received April 26; revised August 16, 1974.
* Present address: Department of Medicine A, Roswell Park
Memorial Institute, Buffalo, New York 14203.
1 Friend, C., Scher, W., Holland, J. G., and Sato, T., Proc.
natn. Acad. Sci. U.S.A., 68, 378-382 (1971).
2 Boyer, S. H., Wuu, K. D., Noyes, A. N., et al., Blood, 40,
823-835 (1972).
3
Ostertag, W., Melderis, H., Steinheider, G., Kluge, N., and
Dube, S., Nature new Biol., 239, 231-234 (1972).
4 Ross, J., Ikawa, Y., and Leder, P., Proc. natn. Acad. Sci.
U.S.A., 69, 3620-3623 (1972).
5 Preisler, H. D., Scher, W., and Friend, C., Differentiation,
1, 27-37 (1973).
6
Krantz, S. B., Life Sci., 4, 2393-2397 (1965).
7
Scher, W., Holland, J. G., and Friend, C., Blood, 37, 428-
437 (1971).
9
Shatkin, A. J., in Fundamental Techniques in . Virology (edit.
by Habel, K., and Salzman, N. P.), 118-140 (Academic
Press, New York, 1969).
10 Krantz, S. B., and Jacobson, L. D., in Erythropoietin and the
Regulation of Erythropoiesis, 118-140 (University of
Chicago Press, Chicago, 1970).
Paul, J., and Hunter, J. A .. J. molec. Biol., 42, 31-41 (1969).
11
12
Preisler, H. D., and Zanjani, E. D., in Proc. Am. Soc.
Hematol. 16th Annual Meeting, Chicago, Abst. 122, 1973.
13
Stephenson, J. R., Axelrod, A. A., McLeod, D. L., and
Shreeve, M. M., Proc. natn. Acad. Sci. U.S.A., 68, 1544-
1546 (1971).
14 Necheles, T. F., Sheehan, R. G., and Meyer, H. J., Ann. N.Y.
Acad. Sci., 149, 449-455 (1968).
15
Glass, J., Lavidor, L., and Robinson, S. H., in Proc. Am.
Soc. Hematol. 16th Annual Meeting, Chicago, Abst. 121,
1973.
Growth of rust fungi of wheat and flax
on chemically-defined media
THE rust fungi cause. serious economic losses of wheat
and other crops. In nature, these organisms complete their
life cycles only on living tissues of their hosts and, until
recently1, they have been regarded as obligate parasites
due to their failure to grow in axenic culture on non-living
substrates. We have now obtained highly reproducible
vegetative growth and sporulation of the flax rust fungus,
Melampsora lini (Ehrenb.) Lév. (race 3) and vegetative
growth of two North American races (15B-4 and 56) of
the wheat stem rust fungus, Puccinia graminis f. sp. tritici
(Erikss. and Henn.) on chemically defined liquid media,
sterilised by millipore filtration and seeded with uncontamin-
ated uredospores. Vegetative growth and some sporula-
tion were also obtained with an Australian isolate of wheat
rust, race 126-ANZ 6, 7.
Williams et al.2 reported the first successful establishment
of axenic colonies of the wheat stem rust fungus, race
126-ANZ 6,7, starting with uncontaminated uredospores
seeded on to an agar medium containing glucose, mineral
salts, Evans's peptone and yeast extract. Subsequently much
progress has been made in the axenic culture of a number
of rust fungi belonging to the Pucciniaceae4-7 and Melamp-
soraceae8-10. Nevertheless the results obtained suffer from
a number of defects. Most experimenters have employed
agar media containing complex organic substances like
peptone yeast extract and bovine serum albumin. Only
a few attempts11-13 have been made to grow rusts on
chemically defined media containing amino acids and
growth on such media has not been equivalent to that
obtained in the presence of peptone, yeast extract or bovine
serum albumin. Moreover, all the results reported thus far
indicate that sporulation in axenic culture is erratic, and
there are only two reports in which uredospores produced
in axenic culture have been shown to be capable of reinfect-
ing the host3,6. Finally, the use of agar based media, steri-
lised by autoclaving, is itself a disadvantage, since the exact
composition of the sterilised medium is not known. Our
objectives were to develop chemically defined media (see
Table 1 legend) in order to examine the factors con-
trolling sporulation and to compare the amino acid re-
quirements of the wheat and flax rust fungi.
Uncontaminated uredospores of both rusts were raised
on susceptible hosts as described elsewhere14. All media
were sterilised, after the addition of amino acids, glutamine
and glutathione as indicated in Table 1, by millipore filtra-
tion (pore size 0.22 um). No agar was employed. The pH
of all media was adjusted to 5.0. Uredospores (1,000-
2,000 mm-2) were floated on the liquid surface (10 ml) in
50 ml flasks. For each treatment five replicate flasks were
incubated without shaking at 17° C in the dark. The growth
and development of the rust colonies were observed over
a period of two months.
The chief results are summarised in Table 1. Luxuriant
growth of M. lini was obtained on medium A with 45 mM
aspartic or glutamic acid, plus cysteine (4.6 mM) and reduced
glutathione (3.3 mM), or with 45 mM aspartic or glutamic
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1
7,474 +
161
1,501 ± 34
+
2,556 士
174
5,381 ± 137
+
6,653 +
557
15,553 ± 428
HHHHHHHHH
8
Friend, C., Scher, W., and Preisler, H. D., Ann. N.Y. Acad.
Sci. (in the press).

Nature Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4 647
2
Table 1 Comparative behaviour of flax and wheat stem rust fungi in vitro
Fungus
Medium
Aspartic acid*
Cysteine
Glutathione
Growth
Sporulation
Melampsora lini
Race 3
A
45
0
4.6 (or 2.3) .
0
Luxuriant
Frequent
A
0
42
4.6
0
Luxuriant
Nil
Puccinia graminis tritici
Race 126-ANZ 6, 7
B
45
0
4.6
3.3
Luxuriant
Infrequent
C
45
0
4.6 (or 2.3)
0
Luxuriant
Infrequent
C
0
42
4.6
0
Luxuriant
Nil
C
0
6
4.6
0
Poor .
Nil
Race 15B-4 and Race 56
B
45
0
4.6
3.3
Nil
Nil
C
45
0
4.6 (or 2.3)
0
Nil
Nil
C
0
42
4.6
0
Luxuriant
Nil
C
22.5
21
4.6
0
Luxuriant
Nil
C
0
42 (Asp 45)
4.6
0
Luxuriant
Nil
*Aspartic acid can be replaced by equimolar glutamic acid with identical results. Asp 45 denotes that after 3 weeks' growth 42 mM glutamine
was replaced by 45 mM aspartic acid. Basic components of media (1-1): medium A : Sucrose, 50 g; Ca(NO3)2 . 4H2O, 2.0 g; KNO 3, 0.25 g;
MgSO4 . 7H2O, 0.25 g; KH2PO4, 0.25 g; K2HPO4, 0.75 g; NH4NO 3, 20 mg; 0.8 ml micronutrient solution (containing 13% NaFe (Geigy),
10 g; MnSO4 . 7H2O, 447 mg; KI, 10 mg; NiCl2 . 6H2O, 18 mg; Ti(SO4)2 . 9H2O, 42 mg; ZnSO4, 35 mg; CuSO4 . 5H2O, 15 mg; BeSO4,
20 mg; H3PO4 (85%), 10 mg; H2SO4 (concentrated), 0.2 ml; all in 200 ml glass distilled water). Medium B: glucose, 30 g; NaNO 3, 2.0 g;
KCI, 0.5 g; MgSO4 . 7H2O, 0.5 g; KH2PO4, 1.0 g; Ca(NO3)2 · 4H2O, 2 to 6 g; FeSO4 . 7H2O, 10 mg, plus 0.8 ml micronutrient solution as
in Medium A. Medium C: Major components as in Medium B, plus sodium citrate, 1.5 g; no micronutrients.
b
Fig. 1 Growth and development of M. lini (race 3) on
liquid medium A (inorganic salts, sucrose, aspartic acid and
cysteine). a, Fully developed vegetative colonies (1 month
old) on liquid surface (scale bar, 0.5 cm); b, sporulating
colony, 6 week old. The entire colony has developed into
a uredosorus (scale bar, 0.5 cm); c, Uredospores (oblong
cells) from colony like that in h (scale bar, 50 um); d,
teliospores from 2 month old colony (scale bar 50 um); e, 1
month old vegetative colonies of P. graminis (race 15B-4) on
liquid medium C (inorganic salts, glucose, glutamine and
cysteine) (scale bar, 1 cm).
acid plus cysteine (2.3 or 4.6 mM). Sixty to seventy vegeta-
tive colonies per flask developed in 90-100% of the flasks
within 4 weeks (primary axenic colonies). Two weeks later
typical orange uredopustules were formed on the surfaces
of most colonies (Fig. 1a and b) and dark brown teliosori
appeared on some of them after 8 weeks growth. The
uredospores and teliospores and their supporting hyphae
were apparently closely similar to those found in nature
(Fig: 1c and d). When uredospores produced in vitro
were used to inoculate intact week-old flax cotyledons,
infection and development of sporulating pustules occurred
in a normal manner. When seeded on fresh medium, flax
rust uredospores developed in vitro grew into secondary
axenic colonies. These developed and sporulated in a manner
indistinguishable from the primary axenic colonies. Uredo-
spores from the secondary colonies also reinfected intact
flax cotyledons. Decreases in the levels of aspartic or
glutamic acid (22.5 mM) and cysteine (2.3 mM) resulted
in poor growth and a low frequency of initiation of colonies,
which failed to sporulate; glutathione was not essential
for growth or sporulation. Replacement of aspartic acid
with a high level of glutamine (42 mM) gave a high rate
of colony initiation and excellent vegetative growth. These
colonies were white and fluffy and had a yellowish stroma
on the under surface, but did not sporulate and finally died.
An Australian isolate of P. graminis tritici, race 126-
ANZ 6,7, developed large colonies on medium B plus
high levels of aspartic acid (45 mM), cysteine (4.6 mM)
and glutathione (3.3 mM); small areas of uredospores and
teliospores developed on a few colonies in about 50% of
the inoculated flasks. When uredospores formed by these
colonies were placed on exposed mesophyll tissue they pro-
duced typical infections of seedling leaves of wheat14.
Identical results were obtained on medium C plus 45 mM
aspartic acid and 4.6 mM cysteine, so that glutathione and
micronutrients were evidently not essential for growth. Of
the three races of wheat stem rust, 126-ANZ 6,7 therefore
most closely resembles race 3 of M. lini in its requirement
for amino acids. Two North American isolates, races
15B-4 and 56, behaved in an entirely different manner and
completely failed to grow on these media (Table 1).
Races 126-ANZ 6,7, 15B-4 and 56 all formed large
vegetative colonies on medium C plus high levels of gluta-
mine (42 mM) and cysteine (4.6 mM), but no sporulation
occurred. Races 15B-4 and 56 also formed luxuriant
vegetative colonies but did not sporulate on medium C
plus aspartic acid, glutamine and cysteine (Fig. le and
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1
Organic nitrogen source (mM)
Glutamine
4.6
3.3
Luxuriant
Frequent
A
45
0
:

648 Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4 18 1974
Table 1). When glutamine was replaced with aspartic
acid after 3 weeks' growth, vegetative growth continued
and the stromata of individual colonies merged with each
other, but there was no sporulation. Apparently aspartic
acid will support vegetative growth of the wheat rust
once it has started, but will not serve as a source of amino
nitrogen for the initiation of colonies.
These results show that chemically-defined media can
support luxuriant growth of wheat rust and both growth
and, sporulation of flax rust without the addition of com-
plex organic substances like peptone or yeast extract.
Further work will be required to discover the key to con-
trol of sporulation by the wheat rust fungus.
We thank Miss J. Johnson for technical assistance.
This work was supported by grants (to M. S.) from the
National Research Council of Canada and the Canada
Department of Agriculture.
AMITAVA BOSE
MICHAEL SHAW
Department of Plant Science,
Faculty of Agricultural Sciences,
University of British Columbia,
Vancouver, BC, Canada V6T 1W5
Received July 9, 1974.
1
Scott, K. J., and Maclean, D. J., Ann. Rev. Phytopathol., 6,
123-146 (1969).
2
Williams, P. G., Scott. K. J., and Kuhl, J. L., Phytopathology,
56. 1418-1419 (1966).
3
Williams, P. G., Scott, K. J., Kuhl, J. L., and Maclean, D. J.,
Phytopathology, 57, 326-327 (1967).
4 Bushnell, W. R., Phytopathology, 58, 526-527 (1968).
5 Coffey, M. D., Bose, A., and Shaw, M., Can. J. Bot., 47,
1291-1293 (1969).
6
Bose, A., jand Shaw, M., Can. J. Bot:, 49, 1961-1964 (1971).
7
Coffey, M. D., and Allen, P. J., Trans. Br. mycol. Soc., 60,
245-260 (1973).
8
Turel, F. L. M., Can. J. Bot., 47, 821-823 (1969).
9
Coffey, M. D., Bose, A., and Shaw, M., Can. J. Bot., 48,
773-776 (1970).
10
Turel, F. L. M., Can. J. Bot., 49, 1993-1997 (1971).
11
Turel, F. L. M., Can. J. Bot., 51, 131-134 (1973).
12
Coffey, M. D., and Shaw, M., Physiol. Pl. Pathol., 2, 37-47
(1972).
13
Howes. N. K., and Scott, K. J., Can. J. Bot., 50, 1165-1170
(1972).
14 Bose, A., and Shaw, M., Can. J: Bot., 52,. 1183-1196 (1974).
Anti-receptor antibody and resistance to
graft-versus-host disease
GRAFT-versus-host (GvH) disease is a complex syndrome
initiated by the reaction of donor lymphoid cells against
histocompatibility antigens of the host. Although newborn
hybrid animals injected with parental-strain lymphoid cells
develop fatal GvH disease, adult hybrid animals receiving
weight-adjusted doses of such cells develop no clinically
evident disease1,2. This resistance to GvH disease is age-
dependent and radiosensitive; resistance develops at 3-4
weeks of age and is abolished by sublethal total-body
irradiation1. Immunologically specific suppression of GvH
reactions has been achieved by immunisation of F1 hybrid
host to produce antibody reactive with receptors on donor
lymphoid cells for host antigens3,4.
The GvH reactivity of lymphoid cells from the appro-
priate parental strain was suppressed in F1 hybrids produc-
ing such anti-receptor antibody (ARA)3.4. This finding led
to the suggestion that ARA could suppress GvH reactivity
by blocking receptors on aggressor lymphocytes. We have
investigated whether the natural resistance of adult F,
hybrids to GvH disease is associated with the production
of. ARA. We found ARA in rats not developing the
disease, while those rendered susceptible to GvH by irra-
diation did not produce ARA.
The radiosensitivity of the processes responsible for the
resistance of adult rats to fatal GvH disease was confirmed
as follows: groups of five adult female Lewis x Brown
Norway (LBN) F1 hybrid rats each either received 400
rad of total-body X irradiation or were left untreated.
Twenty-four hours later each group received from 2.7X
106 to 2×108 normal adult female Lewis spleen cells in-
travenously. Animals were weighed three times weekly and
deaths were recorded daily (Fig. 1). All non-irradiated
animals receiving Lewis spleen cells survived; none lost
weight or had clinically evident disease. All irradiated
hybrid rats, however, receiving more than 107 Lewis spleen
cells died. Weight loss of up to 30% initial body weight
preceded death. Control animals receiving 400 rad only
had no clinical ill effects. Thus, non-irradiated LBN rats
were resistant to fatal GvH disease when injected with 100
times more Lewis cells than were required to induce sig-
nificant and often fatal GvH disease in those LBN rats
pretreated with 400 rad of X irradiation.
Similar results were obtained when spleen cells from the
Brown Norway parental, strain were injected in analogous
experiments. All unirradiated LBN rats receiving BN
spleen cells in doses as high as 2×108 survived. All
irradiated rats receiving more than 1× 107 BN spleen cells
died of GvH disease.
Sera from irradiated and nonirradiated adult hybrid rats
receiving parental-strain spleen cells were assayed for ARA
by two methods. One method involves a complicated bio-
assay in which ARA inhibits the formation of a chemo-
tactic factor (product of antigen recognition or PAR)
generated when parental-strain lymphoid cells are cultured
briefly with hybrid lymphoid cells3. The other method
involves measurement of the interaction between the
specific alloantibody used for immunisation and serum
from the immunised hybrid animal3,6. Such interaction can
be detected by precipitin reactions in gel3. In a more
sensitive assay, anti-receptor antibody can be detected and
quantified by agglutination of sheep erythrocytes to which
specific alloantibody has been coupled. ARA produced by
immunisation of hybrid rats with alloantiserum and ARA
produced by immunisation with parental-strain spleen cells
have similar reactivity in all these assays.
To determine whether production of ARA correlated
with the resistance of LBN animals to GvH disease,
samples of serum were obtained from adult female LBN
rats that had been injected with normal Lewis spleen
cells. Groups of five LBN F1 hybrid rats were given 400
rad of total-body X irradiation or were left untreated.
Twenty-four hours later, 5 × 107 or 1 × 107 Lewis spleen
100
. 80-
",, Survival
60
40-
1
20-
O
1
5
10
15
20
25
30
35
Days
Fig. 1 Survival of adult female LBN F1 rats after intravenous
injection of normal adult female Lewis spleen cells. Groups of
5 F1 rats each either received 400 rad of total body x radiation
(open symbols) or were not irradiated (closed symbols). Twenty-
four hours later, the rats were injected intravenously with either
2 × 108 (O or .), 5 x 107 (4 or A), 1.2 x 107 (I
or
or
2.8 x 106 (open and solid hexagons) normal Lewis spleen cells.
No F1 rats receiving 400 rad only (not shown) died.
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Nature Vol. 231 October 18 1974
649
Table 1 Development of ARA in F1 hybrid rats receiving parental
strain normal Lewis spleen cells
ARA titre*
Days after parental strain spleen cells
7
14
.
21
'28+
35
42
0
0
0
64
4
0
0
0
32
512
128
64
0
0
16
256
32
0
0
0
32
64
0
0
-
0
0
8
16
0
0
1 × 107 Lewis
spleen cells
0
0
128
256
Not done Not done
0
0
32
64
0
0
0
0
0
0
8
4
0
0
256
64
400 rad plus
0
0
Dead
5 × 107 Lewis
0
0
Dead
spleen cells
0
0
0
Dead
0
0
Dead
0
0
Dead
400 rad plus
0
0
0
Dead
1 × 107 Lewis
0
0
Dead
spleen cells
0
0
0
Dead
0
0
0
0
·Dead
0
0
0
Dead
* Reciprocal of the highest serum dilution which causes agglutina-
tion of sheep erythrocytes coated with the IgG fraction of Lewis
anti-BN serum.
t These sera failed to agglutinate SRBCs coated with IgG fractions
of Lewis anti-DA serum, Fischer anti-BN serum or BN anti-Lewis
serum.
cells were injected intravenously and serum samples were
obtained at weekly intervals. A haemagglutinin assay, in-
volving sheep erythrocytes to which IgG antibody was
coupled, was used to measure anti-receptor antibody. Lewis
anti-BN antibody was purified by ammonium, sulphate pre-
cipitation and elution from diethylaminoethyl-cellulose
with 0.04 M phosphate buffer, pH 8.0. After dialysis
100
80-
". Survival
60-
40-
20-
5
10
15
20
25
30
35
52
Days
Fig. 2 Protection of irradiated LBN F1 rats from lethal GvH
disease by adaptive transfer of F1 spleen cells. Irradiated LBN
rats receiving 1 x 107 normal Lewis spleen cells only(
all died of GvH disease. Irradiated LBN rats receiving 1 x 107
normal Lewis spleen cells and 5 x 107 spleen cells from F1 rats
resistant to GvH disease (- - -- ) suffered no'mortality or weight
loss. The group of irradiated LBN rats receiving 1 x 107 normal
Lewis spleen cells and 5 x 107 normal LBN spleen cells
( -----
-) had an intermediate mortality: LBN rats receiving
400 rad of X radiation only (- - ) all survived. No
additional mortality occurred in any group during the interval
of 52-120 d.
against saline, the IgG antibody was coupled to sheep
erythrocytes by means of chromic chloride7. Serial dilu-
tions of serum were incubated with sensitised erythrocytes
for at least 3 h and the settling pattern was observed (Table
1). Serum samples were also titered with normal sheep
erythrocytes, and with sheep erythrocytes to which normal
Lewis IgG, Lewis anti-DA IgG, or BN anti-Lewis IgG
had been coupled.
As observed previously, all irradiated hybrid rats receiv-
ing 5 × 107 Lewis spleen cells and four or five irradiated
rats receiving 1 × 107 Lewis spleen cells were dead within
four weeks after cell injection. ARA could not be detected
in any serum sample from the irradiated rats. None of the
nonirradiated hybrid rats receiving Lewis spleen cells
. developed GvH disease. By 4 weeks after injection, 9 of
10 nonirradiated hybrid rats receiving Lewis spleen cells
had measurable serum ARA. This antibody activity was
first noted 3 weeks after injection and in most animals
was no longer detectable 3 weeks later. The specificity of
the ARA reactivity is indicated by the failure of serum
samples obtained at 28 d to agglutinate erythrocytes coated
with Lewis anti-DA IgG or with BN anti-Lewis IgG. The
following observations indicate that ARA detected in the
serum of hybrid rats receiving parental-strain lymphoid
cells is an anti-idiotypic antibody and not a soluble com-
plex of anti-BN antibody and BN antigen formed in anti-
gen excess. Such a complex would be expected to aggluti-
nate sheep erythrocytes coated with anti-BN antibody of
any idiotype, whereas anti-idiotypic antibody would agglu-
tinate only erythrocytes coated with anti-BN antibody of
the appropriate idiotype. Fischer anti-BN and Lewis anti-
BN IgG fractions possessing equivalent titres of anti-BN
haemagglutinin antibody were used to coat sheep erythro-
cytes. The two preparations of coated erythrocytes agglu-
tinated to the same extent with a rabbit anti-rat immuno-
globulin serum. Serum from hybrid rats obtained 28 d
after injection of Lewis spleen cells agglutinated Lewis
anti-BN coated cells (Table 1), but did not agglutinate
erythrocytes coated with Fischer anti-BN antibody. This
pattern of agglutination indicates the anti-idiotypic nature
of ARA.
Thus, ARA is detected in the serum of hybrid rats that
do not develop GvH disease. Irradiation renders hybrid
rats susceptible to fatal GvH disease; irradiated rats do
not produce detectable amounts of ARA. Production of
ARA may be a part of the radiosensitive process by
which hybrid animals resist fatal GvH disease. If ARA
plays such a protective role, lymphoid cells from hybrid rats
producing ARA should enable irradiated hybrids to resist
fatal GvH disease. To test this hypothesis, we gave LBN
rats 400 rad of total-body X radiation. Twenty-four hours
later, they received one of the following: 107 Lewis spleen
cells; 107 Lewis spleen cells plus 5 x 107 normal LBN
spleen cells, or 107 Lewis spleen cells plus 5 × 107 spleen
cells from LBN rats that had previously received two
injections of Lewis spleen cells. All the previously injected
LBN rats had ARA in their serum when their spleen cells
were taken. Irradiated LBN rats given spleen cells from
the LBN rats that had resisted fatal GyH disease were
protected from the lethal effects of injection of parental
spleen cells (Fig. 2). Normal LBN spleen cells were some-
what less effective in conferring protection to irradiated
hybrid rats.
Thymus-derived (T) lymphocytes must be present in the
responder populations to produce GvH reactions. Recent
evidence obtained from GvH reactions in vivo9 and mixed
lymphocyte cultures in vitro10 indicates that genetic disparity
at one or more lymphocyte-defined (LD) loci closely linked
to the serologically defined (SD) major histocompatibility
complex is essential for a strong proliferative response. The
generation of cytotoxic effector cells appears to require in
addition a disparity at one or more of the SD loci. Cyto-
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5 x 107 Lewis
spleen cells

656
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toxic effector cells, like the cells responsive to the LD
loci, are believed to be T lymphocytes11.
The generation of effector cells in culture requires DNA
synthesis12. Cultures treated so as to eliminate cells that
have undergone DNA synthesis do not yield cytotoxic
effector cells13. Since treatment to eliminate cells that had
undergone DNA synthesis could be delayed until placing
the cells into the effector cell assay, the cytotoxic effector
cells must have replicated during the sensitisation process.
Thus it seems that proliferation of both LD responsive and
SD responsive lymphocytes is required for full cytotoxic
expression.
Several groups have suggested a model of cellular coope-
ration involving two populations of T lymphocytes to
explain the apparent requirement for both LD and SD
responsive T cells. Hayry and Andersson14 suggest that the
effector cell responsive to SD antigen requires cooperative
interactions of an LD responsive T lymphocyte (and per-
haps an adherent cell as well) before it can divide and
cause significant target cell lysis.
Local GvH reactions may involve predominantly the
recognition and proliferative phases of the immune res-
ponse. The syndrome of GvH disease, however, probably
includes an additional component of cell-mediated injury
initiated by effector lymphocytes8. If such effector lym-
phocytes react with the SD antigens of the host through
an
immunologically specific receptor molecule, then
blockade of that receptor may lead to a blunting or inhibi-
tion of the killer cell potential of these lymphocytes. One
immunologically specific receptor molecule able selectively
to bind SD antigen is antibody. We suggest that the anti-
idiotypic antibody demonstrable in the serum of F1 hybrid
rats after injection of parental strain cells may function
by blocking or eliminating those parental lymphoid cells
which display receptors for the serologically defined histo-
compatibility antigens of the host.
This work was supported by grants from the US Public
Health Service. Franklin McLean Memorial Research
Institute is operated by the University of Chicago for the
US Atomic Energy Commission.
T. J. McKEARN
Y. HAMADA
F. P. STUART
F. W. FITCH
Departments of Pathology and Surgery and
Franklin McLean Memorial Research Institute,
University of Chicago,
Chicago, Illinois 60637.
Received May 13; revised July 1, 1974.
1 Fiscus, W., Morris, B., Session, J., and Trentin, J., Ann. N.Y.
Acad. Sci., 99, 355 (1962).
2 Field, E., and Gibbs., J., Transplantation, 3, 634 (1965).
McKearn, T., Science, 183, 94 (1974).
Binz, H., Lindenmann, J., and Wigzell, H., Nature, 246, 146
(1973).
" Ramseier, H., and Lindenmann,, J., J. exp. Med., 134, 1083
(1971).
6 Binz, H., and Lindenmann, J., J. exp. Med., 135, 872 (1972).
7 Vyas, G., Fudenberg, H., Pretty, H., and Gold, E., J. Immun.,
100, 274 (1968).
8
Elkins, W., Prog. Allergy, 15, 78 (1971).
9
Livnat, S., Klein, J., and Bach, F., Nature new Biol., 243, 42
(1973).
10 Bach, F., Widmer, M., Bach, M., and Klein, J., J. exp. Med.,
136, 1430 (1972).
11 Cerottini, J., Nordin, A., and Brunner, K., Nature, 228, 1308
(1970).
12 Bach, M., Alter. B., Lightbody, J., and Bach, F., Transplantn
Proc., 4, 169 (1972).
13 Hayry, P., Andersson, L., Nordling, S., and Virolainen, M.,
Transplantn Rev., 12, 91 (1972).
14
Hayry, P., and Andersson, L., Transplantn Proc., 5, 1697
(1973).
New inhibitor of reagin-mediated anaphylaxis
METHYLXANTHINES, such as theophylline and caffeine, inhibit
the antigen-induced release of histamine and of slow reacting
substance of anaphylaxis (SRS-A) from passively sensitised
human lung and human basophilic leukocytes1,2. We have
shown that these methylxanthines also inhibit a passive
cutaneous anaphylactic (PCA) reaction mediated by reaginic
antibodies in the rat. The methylxanthines are, however, from
20 to 50 times less potent than disodium cromoglycate. Because
of the relatively low potency of theophylline and because of its
other pharmacological actions, notably as a bronchodilator, it
is not clear to what extent inhibition of reagin-mediated
anaphylaxis may be important in its use for the treatment of
allergic bronchial asthma.
By progressive modification of the chemical structure of
methylxanthines, we have found that certain 2-phenyl-8-
azapurin-6-ones3 possess high and selective anti-allergic activity.
In particular, 2-o-propoxyphenyl-8-azapurin-6-one, M&B 22,948
(1), is from 20 to 50 times more potent than disodium cromo-
glycate in inhibiting reagin-mediated anaphylaxis in a number
of test systems and is also active by oral administration.
H
0
1
N
HN
NH,
2
N
N
N
NH
2
OC_H
OC_H.
37
3"7
(1)
(2)
2-o-Propoxyphenyl-8-azapurin-6-one is a white crystalline solid,
melting point 241° C (decomp.). It is less than 0.1 % w/v soluble
in water but readily forms salts, for example the triethanolamine
salt has a melting point of 138-139º C. The azapurinone was
prepared by nitrous acid treatment of the diamine (2) which
itself was prepared from o-propoxybenzamidine by conventional
methods.
M&B 22,948 has been compared with disodium cromoglycate
in three experimental models involving reagin-mediated ana-
phylaxis. These were first, the inhibition of the allergen induced
release of histamine and SRS-A from passively sensitised human
lung in vitro4, second, the inhibition of a reagin-mediated PCA
reaction in the rat5 and third, the inhibition of reagin-mediated
anaphylactic bronchospasm in the guinea pig. In experiments
involving aqueous solutions, M&B 22,948 was used as its
triethanolamine salt. Doses refer to the parent azapurinone.
M&B 22,948 was more potent than disodium cromoglycate
in inhibiting the anaphylactic release of both histamine and
SRS-A from human lung tissue (Fig. 1). The effective concentra-
tion range for each drug was fairly narrow. The optimum
concentration for inhibition of mediator release determined in
at least seven separate experiments was in the range of 1-3 ug
ml-1 for M&B 22,948 and 30-100 ug ml-1 for disodium
cromoglycate.
The intravenous dose of M &B 22,948 causing 100% inhibition
of the rat PCA reaction was from 35-50 times less than that of
disodium cromoglycate. Thus, complete inhibition of the rat
PCA reaction was consistently obtained with intravenous doses
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Nature Vol. 231 October 18 1974
C
HN

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Nature Vol. 201 October 18 19/4
651
",, inhibition of meditator release
80
1
60
0
40-
20-
O
0 &
A
0.03 0.1
0.3
1.0
3.0
10.0
30.0 100.0 300.0
Drug concentration (µg ml - 1)
Fig. 1 Inhibition of allergen-induced release of histamine and
SRS-A from passively sensitised human lung tissue by M&B
22,948 and disodium cromoglycate in two separate experiments.
Using M&B 22,948: , histamine; O, SRS-A. Using disodium
cromoglycate: , histamine; 4, SRS-A. Chopped human lung
was incubated for 18 h with serum from asthmatic subjects.
Aliquots were challenged with extracts of Dermatophagoides
farinae (500 µg ml-1) immediately after the addition of graded
concentrations of the test compounds. Histamine was assayed
fluorometrically. SRS-A was assayed on the mepyraminised
guinea pig ileum. Points refer to single determinations from
three pooled samples at each drug concentration.
of 0.1 mg kg-1 of M&B 22,948 and 2-5 mg kg-1 of disodium
cromoglycate. Intravenous theophylline had approximately
one-fiftieth the potency of disodium cromoglycate.
When administered orally to rats 15 min before allergen
challenge, M&B 22,948 was effective in inhibiting the PCA
reaction. The dose response curve was 'bell shaped' (Fig. 2) and
complete inhibition of the PCA reaction was rarely obtained.
Maximal inhibition was obtained with oral doses of between
0.5-2.0 mg kg-1. Disodium cromoglycate showed poor to
negligible activity; an oral dose of 500 mg kg-1 produced less
than 30% inhibition when administered either 15 or 40 min
before allergen challenge. Theophylline showed some oral
activity with 80% inhibition of the PCA reaction occurring with
a dose of 100 mg kg-1.
Anaphylactic bronchospasm was produced in the guinea pig
passively, systemically sensitised with Trichinella spiralis anti-
serum and premedicated with propranolol (5 mg kg-1, intra-
peritoneally). The mean preconvulsion time of groups of a
minimum of six sensitised guinea pigs exposed to an aerosol of
Table 1 Inhibition of reagin-mediated anaphylactic bronchospasm
in the guinea pig by M&B 22,948 administered intravenously, orally
or by the inhalation of an aqueous aerosol
Route of drug
Dose
administration (mg kg-1)
Time (min) between
dose and bronchial .
allergen challenge
Mean
% protection
5
1
19 (4)}
Intravenous
10
1
50 (4)
20
1
69 (4)
Oral
200
60
48 (6)
120
43 (3)
Inhalation
50
1
34(11)
The % protection was 100 [1-(C/T)] where C and T were the pre-
convulsion times of control and treated groups.
*Concentration of aerosol generating solution. Animals exposed
to aerosol for 2 min.
+Figures in parentheses refer to the number of experiments.
allergen was increased by 19%, 50% and 69% following intra-
venous doses of 5, 10 and 20 mg kg-1 of M&B 22,948 (Table 1).
Disodium cromoglycate (100 mg kg-1 intravenously) produced
a protection comparable to that of 10 mg kg-1 of intravenous
M&B 22,948. An oral dose of 200 mg kg-1 M&B 22,948 pro-
duced a significant protective action. Maximum protection
was afforded when the animals were challenged 15 min after
receiving 200 mg kg-1 M&B 22,948 orally but a significant
increase in the preconvulsion time was also seen when challenge
was carried out 4 h after drug administration (Table 1). No
reduction in the severity of anaphylactic bronchospasm was
produced with oral doses of up to 200 mg kg-1 of disodium
cromoglycate, administered 15 or 40 min before antigen
challenge. Theophylline has not been studied in these tests.
M&B 22,948 antagonised some of the pharmacological
effects of the mediators of anaphylaxis. Thus, concentrations of
M&B 22,948 of 1-30 µg ml-1, which inhibit the anaphylactic
release of mediators from human lung tissue, also antagonised
the spasmogenic effects of histamine, SRS-A and PGF2a on
isolated human bronchial muscle. In the rat, intravenous doses
of 10-25 mg kg-1 of M&B 22,948 were required to reduce the
increase in capillary permeability produced by intradermal
histamine and 5-HT. These dose levels are considerably higher
than that of M&B 22,948 required to inhibit the reagin-mediated
PCA reaction (0.1 mg kg -1 intravenously).
=1004
", inhibition of PCA reaction-
r
(8)
ㅎ 50%
(2)
(9)
0.1
1
10
Dose (mg kg- "orally)
Fig. 2 Inhibition by oral M.&B 22,948 of reagin-mediated PCA
reactions in the rat. Rats were sensitised by intradermal injection
of Nippostrongylus brasiliensis antiserum. 48 h later they were
challenged intravenously with N. brasiliensis extract. M&B
22,948 was administered orally 15 min before challenge. Each
point is the mean percentage inhibition of the PCA reaction
determined in the number of animals indicated in parentheses
± standard error ..
The concentrations of M&B 22,948 (µg ml-1) required to
cause a 50% reduction in the heights of the contractions of
superfused guinea pig ileum produced by various agonists were
as follows : histamine, 32; SRS-A, 7; acetylcholine, 1; PGE2, 15;
PGF24, 2.5; 5-HT, 6.6. All these values were within the range
of concentrations which inhibit the reagin-mediated anaphylactic
release of histamine and SRS-A from human lung tissue. As the
histamine released from the human lung tissue was determined
fluorimetrically, the antihistamine activity of M&B 22,948
would not have interfered in the assays. M&B 22,948 showed
no significant activity, either intravenously or orally, in protect-
ing conscious guinea pigs from histamine-induced broncho-
spasm. Similarly, in experiments carried out in the anaesthetised
guinea pig, using either the Konzett-Rössler or the Dixon-
Brodie method of recording bronchial tone, no indication was
obtained of bronchodilator action or of a specific antihistamine
or anti-5-HT action of M&B 22,948.
Certain 2-phenyl-8-azapurin-6-ones, and in particular M&B
22,948, have been shown to have high activity in inhibiting the
release of the pharmacological mediators of anaphylaxis from
isolated human lung tissue in vitro, in a reagin-mediated PCA
reaction in the rat, and in reagin-mediated anaphylactic broncho-
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-
15
62 (4)
40
54 (2)
240
42 (3)
(mg ml-1)

652
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Ivature vol. 201 October 18 1974
spasm in the guinea pig. The potency of M&B 22,948 was up
to 50 times that of disodium cromoglycate, depending on the
test system and the method of drug administration. M&B 22,948
may be of interest in the prophylactic treatment of allergic
bronchial asthma. Since this work was carried out, we have
learnt of the oral activity of an oxoxanthene derivative
(AH 7725) in inhibiting an allergen induced immediate-type
asthmatic response in man6.
BARBARA J. BROUGHTON
P. CHAPLEN
P. KNOWLES
E. LUNT
D. L. PAIN
K. R. H. WOOLDRIDGE
Chemical Research Group,
R. FORD
S. MARSHALL
JOYCE L. WALKER
D. R. MAXWELL
Pharmacological Research Group,
May and Baker Ltd,
Dagenham,
Essex, UK
Received May 6, 1974.
1 Orange, R. P., Kaliner, M. A., Laraia, P. J., and Austen, K. F.,
Fedn Proc., 30, 1725-1729 (1971).
2 Lichtenstein, L. M., and Margolis, S., Science, 161, 902-903 (1968).
3 British Patent 1338235 (1973).
4 Augustin, R., Handbook of Experimental Immunology, (edit. by
Weir, D.M.) 1076-1086 (Blackwell Scientific Publications,
Oxford, Edinburgh, 1967).
" Ogilvie, B., Nature, 204, 91-92 (1964).
6 Assem, E. S. K., Evans, J. A., and McAllen, M., Br. med. J.,
2, 93-95 (1974).
Significance of immunofluorescent
staining of lymphocytes with
antisera to IgM immunoglobulins
IMMUNOLOGICAL reactions depend for their completeness
on at least two different populations of lymphocytes, one
derived from the bone marrow and the other from the
thymus (B and T cells). B lymphocytes, precursors of the.
antibody-forming cells of vertebrates, are recognised in
vitro by two characteristic reactions: surface fluorescence
with antisera to immunoglobulins' and rosette formation
with red cells sensitised with some components of com-
plement2. Since the cells that show surface immunofluo-
rescence are retained on columns of beads coated with
antibodies to immunoglobulins3, surface fluorescence is
generally interpreted to mean that immunoglobulins are
present on the surface of B lymphocytes4.
Various means have been devised to prove this point
primarily by using high resolution optical devices' or speci-
fic antisera6. Although antisera to IgM immunoglobulins
bind specifically to structures on the surface of lymphocytes,
in only two cases to our knowledge have IgM immuno-
globulins been separated from lymphocytes7,8 and in such
small quantities that the authors had to rely on measure-
ments of radioactivity of subcellular fragments obtained
by specific coprecipitations to support their assertions. It
is generally believed as a result of these and other experi-
ments, that lymphocytes indeed bear immunoglobulins and
in particular IgM immunoglubulin, on their surface. We
were therefore surprised to find that it was possible to
.
B
M
C)
.
-
B.
9
Fig. 1 Double diffusion in agar of. IgM immunoglobulin
(A) with anti-CHO (C), anti-p (M), and anti-IgM (B). The
reaction shows complete immunological identity of the three
antisera with IgM immunoglobulin. Purified IgM immuno-
globulin was prepared by gel filtration of 1.4 1 of normal
human serum on columns of Biogel P300 resin. The fraction
eluting in the exclusion volume of the resin was concentrated
over Ficoll and applied to a column of ethanolysed cellulose
in a Tris-borate buffer of pH 9.4. The colloids were electro-
phoresed at a potential of 240 V for 20 h at 4° C and
eluted from the column at a rate of 10 ml h-1. The fractions
reacting with an antiserum to human IgM immunoglobulin
were pooled, dialysed against 0.15 M NaCI, and re-electro-
phoresed. a-macroglobulin could be separated from the IgM
in this way. Fractions containing IgM immunoglobulin were
chromatographed twice on Sephadex G200 resin to a single
homogeneous chromatographic peak (0.330 g). By electro-
phoresis, sedimentation and precipitation with an antiserum
to human IgM immunoglobulin over 98% of the protein
contained in this fraction was shown to be IgM immuno-
globulin. At no time during this preparation were the solu-
ted containing IgM immunoglobulin frozen. The purified
IgM was digested first with trypsin (1% w/w) at pH 8.0 in
0.65 M Tris HCI containing 0.01 M CaCl2 for 24 h at 37° C,
followed by pronase (1% w/w) under the same conditions
for an additional 24 h at 37° C. The digested protein was
dialysed against distilled water. The dialysate was concentra-
ted in vacuo and applied to a column of Sephadex G25
(fine) resin (0.8 x 200 cm) in 1% ammonium bicarbonate.
Materials eluting from the Sephadex resin were analysed
for carbohydrate by the Mohlish reaction. and the Mohlish-
positive materials were rechromatographed on Sephadex
G25 to a single symmetrical peak. The materials within this
peak were concentrated in
vacuo and separated by
paper chromatography (butanol-ethanol-acetic acid-water
8:2: 1 : 3). Five oligosaccharides were separated in this way.
The anti-CHO antiserum (C above) is specific for these five
oligopeptides.
.
quantify IgG separable from human B cells and that
human B lymphocytes could be induced to synthesise IgG
in vitro10; but we were unable either to quantify IgM or
to show synthesis of IgM in these cells even though they
fluoresced with antisera to IgM immunoglobulin. Some
infants with severe combined immunodeficiency possess
in their serum a monoclonal IgG; we have shown that
lymphocytes from the serum of one of these children could
be induced to synthesise in vitro the monoclonal IgG that
was in the child's serum11. Yet all of his lymphocytes
showed surface fluorescence with an antiserum to IgM.
We decided to study this apparent discrepancy. We wish
to present evidence which will show that surface immuno-
fluorescence of lymphocytes with antisera to IgM is not
caused by the presence of IgM on the surface of these
lymphocytes.
Human IgM was prepared by conventional physico-
chemical procedures from normal human serum. Over
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Nature Vol. 251 October 18 19/4
653
94% of the protein in this preparation sedimented in the
centrifuge as a single boundary with a sedimentation coeffi-
cient of 19S. It precipitated quantitatively with an anti-
serum to human IgM.
Proteolysis of IgM with trypsin and pronase has been
shown to degrade extensively both u and L chains of IgM12.
Five glycopeptides containing a minimum of 5 and a maxi-
mum of 13 sugar residues each, together with six to nine
amino acids, represent the only material separable in the
exclusion volume of a Sephadex G25 resin when a trypsin-
pronase digest of IgM is analysed. Each oligopeptide con-
tains N-acetyl glucosamine as the reducing carbohydrate
end group, and a fucose (oligopeptide 1 and 2) or a mannose
(oligopeptide 3 to 5) in each case bound to an aspartic acid
of the polypeptide chain, representing the non-reducing
carbohydrate ends. These five glycopeptides comprise all
the carbohydrate of the u chain13. Purified IgM was digested
with trypsin followed by pronase. The glycopeptides
obtained after hydrolysis were separated on Sephadex into
Mohlish-positive materials that on paper chromatography
(butanol-ethanol-acetic acid-water 8 :2: 1 : 3) were shown to
be composed of five chemical entities each containing
carbohydrate residues. We obtained 10 mg of these glyco-
peptides (dry weight).
An immunoadsorbent resin was prepared by a modifica-
tion of the method of Axen and Ernback14. To assure that
the carbohydrate moiety of the glycopeptides would bind
to the immunoadsorbent, Sepharose 4B activated with
CNBr was coupled to L-lysine. CNBr-activated IgM gly-
copeptides were in turn, coupled to the Sepharose-lysine
adduct. Over 84% of the carbohydrate was bound to the
Sepharose in these conditions.
IgG globulin was separated (DEAE cellulose) from a
rabbit antiserum to normal human IgM globulins. The
antiserum was rendered monospecific for the u chains of
IgM by passage through immunoadsorbents made by cou-
pling human IgG and IgA immunoglobulins and BenceJones
proteins to Sepharose 4B. This rabbit IgG globulin (1 g)
was incubated at 37° C for 2 h with the immunoabsorbent
containing the glycopeptides of IgM in phosphate-buffered
saline (PBS) at pH 7.2. The unbound globulin was eluted
at room temperature, and the absorbent washed with PBS.
Table 1 Percentage of cells staining with fluorescein-labelled anti-
sera to human IgG and Igm Immunoglobulins and the glycopeptides
(CHO) or polypeptide chains (u) of IgM
Pre-incubation of
Cell Cells with Antisera with
type antisera to
Fluorescein-labelled antibody
(% of cells staining)
IgM
CHO
μ
IgG
Bİ*
94(91-99) 95(91-99)
<2
<2
B1
20(12-32) 20(12-32)
B1
CHO
JgM
22(10-33)
16( 8-26)
B1
μ
91(90-96) 94(90-98)
B2+
55(45-70)
56(50-62)
<2
20
B2
CHO
IgM
17( 9-23)
14( 8-21)
B2
54(42-66)
56(46-69)
40§
10
40§
BM±
BM
IgM
10
10
<2
BM
CHO
40
<2
40
Fluorescent antisera were prepared by dialysing the protein to be
labelled (1 ml of a 1% solution) against 300 ml of a solution con-
taining fluorescein isothiocyanate (0.5 mg ml-1) buffered at pH 9.0 for
72 h in the dark. Labelled antisera were dialysed extensively against
PBS and stored in the dark. 0.1 ml of fluorescein-conjugated antisera
was added to an equal volume of a suspension containing 2 x 106
cells ml-1 in medium 199 supplemented with 5% foetal calf serum
and 10-2 mol 1-1 sodium azide. Cells were incubated for 45 min in
the cold, washed three times in medium 199, and transferred to a glass
slide in a drop of glycerol:PBS (9:1). Slides were examined, and the
percentage of fluorescent cells recorded.
* Six determinations.
t Four determinations.
# Bone marrow from a patient with Waldenström's macroglobulin-
emia (cne determination).
§ Cytoplasmic staining (all others were surface stained).
Bound antibodies were eluted with a glycine/HCI buffer,
pH 2.0 (0.2 M). After five adsorption cycles, no antibodies
remained bound to the immunoadsorbent. A total of 3%
of the rabbit IgG fraction was eluted as specific antibody
to the glycopeptides (anti-CHO). The remainder was de-
signated anti-u even though antibodies to u determinants
of IgM were not specifically isolated. Both fractions (anti-
CHO and anti-p) reacted with human IgM as shown in
Fig. 1. Both antisera gave reactions of complete fusion
against IgM. Immunodiffusion in agarose (1.5%) rather
than agar did not alter the pattern of precipitation. Neither
the anti-CHO nor the anti-u precipitated in agar with the
glycopeptides isolated from IgM as described above, show-
ing that the glycopeptide fraction did not contain large
u-chain fragments that could react with either the frac-
tionated or non-fractionated antiserum. The anti-CHO
and anti-u antibodies had sedimentation coefficients of 6.8
and 6.6, respectively, indicating that neither antibody con-
tained aggregated y globulin. Fluorescent antisera were
prepared by dialysing the proteins against fluorescein
isothiocyanate buffered at pH 9.0 (ref. 15). Fluorescein-
labelled anti-IgM, anti-CHO, and anti-u were reacted with
B cells. The results are summarised in Table 1.
B cells were separated from peripheral blood lymphocytes
or from tonsillar lymphocytes by density centrifugation on
gradients of bovine serum albumin16. Two different popula-
tions of B cells were obtained from the gradients, both of
which possessed a complement receptor indicated by rosette
formation of these cells with EAC1423. One cell population
comprised large cells that sedimented in the upper region
of the gradient (17-21% albumin, B1). These could not be
induced to form IgG in vitro. The second cell population
comprised small cells with a high density which sedimented
toward the bottom of the gradient (29-31% albumin, B2),
and these could be induced to form IgG immunoglobulins
in vitro. Over 95% of BI cells reacted with fluorescein-
labelled anti-IgM or anti-CHO, but only 55% of B2 cells
did so. Less than 2% of either B1 or B2 cells reacted with
anti-u. For comparison, less than 2% of B1 and 20% of
B2 cells reacted with fluoresceinated anti-IgG. Upwards of
80% of the fluorescence of B1 cells and 70% of B2 could
be inhibited when the cells were preincubated with unlabelled
anti-CHO or anti-IgM, but the fluorescence remained un-
altered when either cell was preincubated with anti-u. The
specificity of the antisera was tested by incubating the bone
marrow of a patient with Waldenström macroglobulinemia
with fluorescein-labelled anti-IgM, anti-u and anti-CHO.
Two types of fluorescent pattern were visible; cytoplas-
mic fluorescence and surface fluorescence. The cytoplasmic
fluorescence of the plasmacytoid cells of the marrow syn-
thesising IgM was revealed both with the anti-IgM and
anti-p antisera. This experiment (positive control for anti-p)
indicated that the anti-u antiserum had activity against the
u chain determinants of IgM. This activity could be in-
hibited by preincubating the antiserum with IgM but not
with the glycopeptides of IgM, showing specificity of this
antiserum for the polypeptide chains of IgM. A small
amount of B1 cells were present in the marrow (10%), and
presumably it was these cells that reacted with the antiserum
to IgM or to the glycopeptides in a pattern showing
surface fluorescence. This activity could be inhibited
by preincubating the antiserum with the glycopeptides
but not with IgM showing specificity of the antiserum
for the glycopeptides of IgM but not for the polypep-
tide chains of lgM. Comparable results were obtained
when the following experiments were done. Anti-
IgM antiserum (5 ml) was adsorbed 10 times with 107 B1
cells each time at 37º C for 1 h. The adsorbed antiserum
showed unimpaired reactivity with IgM by double diffusion
in agar. It failed, however, when preincubated with either
B1 or B2 cells to inhibit the fluorescence of the anti-CHO
antiserum with both B1 and B2 cell populations.
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19(12-27)
15(10-22)
B2
antigen

654
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Nature Vol. 251 October 18 1974
Immunoglobulins are glycoproteins. Over 10% of the
mass of IgM is carbohydrate so that antisera to IgM should
contain antibodies directed to the carbohydrate, as well as
to the polypeptides of the immunoglobulin chains. The
data presented here show that immunofluorescence of B
lymphocytes with antisera to IgM is not caused by IgM
immunoglobulin present on the surface of B lymphocytes.
It is rather caused by interaction of antisera to IgM im-
munoglobulin with carbohydrate on the surface of the
lymphocyte. This carbohydrate must be cross reactive and,
therefore, structurally similar to the carbohydrate of IgM
immunoglobulin.
We thank Dr Chester Alper for the gift of the anti-IgM
antiserum and Dr Harvey Colten for providing the sheep
red cell intermediate. This investigation was supported by
a United States Public Health Service Grant. J.G. is the
recipient of a Queen Elizabeth II grant from the Canadian
Government.
E. MERLER
J. GATIEN
G. DEWILDE
Immunology Division,
Children's Hospital Medical Center,
300 Longwood Avenue,
Boston, Massachusetts 02115
Received June 6, 1974.
1 Pernis, B., Forni, L., and Amante, L., J. exp. Med., 132,
1001-1018 (1970).
2 Bianco, C., Patrick, R., and Nussenzweig, V., J. exp. Med.,
132, 702-720 (1970).
3
Wigzell, H., and Andersson, B., J. exp. Med., 129, 23-36
(1969).
4 Froland, S., Natvig, J. B., and Berdal, P., Nature new Biol.,
234, 251-252 (1971).
" Hämmerling, V., and Rajewsky, K., Eur. J. Immun., 1, 447-
452 (1971).
6
Pernis, B., Chiappino, G., Kelus, A. S., and Gell, P. G. H.,
J. exp. Med., 122, 853-876 (1965).
7
Vitetta, E. S., Baur, S., and Uhr, J. W., J. exp. Med., 134,
242-264 (1971).
8
Marchalonis, J. J., Cone, R. E., and Atwell, J. L., J. exp. Med.,
135, 956-971 (1972).
9
Merler, E., and Silberschmidt, M., Immunology, 22, 821-831
(1972).
10
Geha, R. S., Schneeberger, E., Rosen, F. S., and Merler, E.,
J. exp. Med., 138, 1230-1247 (1973).
11
Geha, R. S., Schneeberger, E., Gatien, J., Rosen, F. S., and
Merler, E., New Engl. J. Med., 290, 726-728 (1974).
12 Hurst, M. M., Niedermeier, W., Zikan, J., and Bennett, J. C.,
J. Immun., 110, 840-847 (1973).
13
Putnam, F. W., Florent, G., Paul, C., Shinoda, T., and
Shimiza, A., Science, 182, 287-291 (1973).
14
Axen, R., and Ernback, S., Eur. J. Biochem., 18, 351-360
(1963).
15
Clark, H. F., and Shephard, C. C., Virology, 20, 642-645
(1963).
16 Dicke, K. A., Tridente, G., and Van Bekkum, D. W., Trans-
plantation, 8, 422-434 (1969).
Cellular immune response to a drug-treated
L5178Y lymphoma subline
TUMOUR-specific transplantation antigens (TSTA), have been
detected on a wide range of experimental tumour cells and
elicit weak immunogenic activity. As a rule, little immune
response of the host to autocthonous tumours or to implanted
syngeneic tumours has been detected. Furthermore, immunologi-
cal techniques, such as active immunisation, passive transfer of
antibodies or adoptive transfer of immune lymphocytes, have
not been entirely satisfactory. Attempts have also been made
to increase the immunogenic properties of tumour cells by in
vitro enzymatic treatments1 or in vitro coating of antigenic
determinants on cancer cells2.
Some studies have shown that it is possible to increase the
immunogenicity of tumour cells3. In our laboratory immuno-
genic L1210 sublines were induced by prolonged in vivo treat-
ment with the drug 5-(3,3-dimethyl-triazeno)imidazole-4
carboxamide (DIC)4. Mice compatible with the untreated
L1210 leukaemia were resistant to a heavy challenge of L1210/
DIC cells, whereas host resistance could be abrogated by
immunodepression. The alteration of antigenicity acquired by
L1210 DIC-treated cells (L1210/DIC) was found to be herit-
able4,5.
We present evidence that a subline of L5178Y tumour,
transformed by DIC. in vivo (L5178Y/DIC) (manuscript in
preparation) implanted into mice compatible with the parental
leukaemia, stimulated a specific cell-mediated cytotoxic
response.
L5178Y/DIC was maintained by serial intraperitoneal (i.p.)
passages in immunosuppressed 200 mg kg-1 (cyclophosphamide
given intraperitoneally 24 h before tumour challenge) hybrid
CDF1 mice. Other tumour lines used in this study were main-
tained by serial i.p. passages in compatible inbred mice.
The cellular immune response was evaluated by the in vitro
51Cr release assay6, modified by Canty7.
A sample of 107 spleen cells (attackers) from normal (control)
or immune mice were suspended in a plastic Petri dish (Falcon,
· 35 x 10 mm) in 1 ml Medium 199 (DIFCO) supplemented
with 10% heat inactivated foetal calf serum (GIBCO) and
incubated for 4 h with 5 x 104 (50 ul) 51Cr-labelled cells (targets)
on a rocking platform at 37° C. in a moist atmosphere of 10%
CO2-90% air. Radioactivity released in the supernatant by
labelled target cells was counted in an automatic scintillation
counter.
In each experiment quadruplicated samples were used for
each experimental group and the data reported are from one
out of three similar experiments. The percentage cytotoxicity
for each group was calculated as follows:
c.p.m. 51Cr released from incubated cells
c.p.m. 51Cr released from frozen-thawed cells (4 times)
× 100
Experimental results were expressed as (% cytotoxicity in
experimental samples)-(cytotoxicity in control samples). The
51Cr release in control samples was 12 ± 3%.
Spleen cells from mice immune to L5178Y/DIC tumour cells
were highly cytotoxic in vitro to 51Cr-labelled L5178/DIC cells.
Parental radiolabelled L5178Y cells and other tumour and thy-
mus cells syngeneic or allogeneic with CDF1 mice, did not
release the isotope after incubation with anti-L5178Y/DIC
attacker cells. The specific cell-mediated 51Cr release indicates
50-
.-
.51Cr release (±s.e.)
40-
30-
20-
10-
F
10
20
30
40
50
60
Days after immunisation
Fig. 1 Kinetics of immune spleen cell activity. - , Direct assay;
--- , assay 24 h after incubation. Attacker cells were from CDF1
mice immunised with 107 L5178Y/DIC viable cells injected i.p.
and assayed against 51Cr-labelled L5178Y/DIC cells. In these
and the following assays the ratio of attacker cells to labelled
target cells was 200:1.
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Nature vol. 231 October 10 1914
655
cantly. The failure to detect L5178Y/DIC antigens on foreign
cells provides strong evidence of the specificity of L5178Y/DIC
induced antigen(s).
Immune cells were assayed immediately or 4 h after X-ray
treatments. The activity of the attacker cells was only partially
reduced even by heavy doses of X rays (2.00-4.00 r.), in accord-
ance with the known resistance of cytolytic cells (immune
lymphocytes and macrophages) to X irradiation. (Table 3)9.
Our studies indicate that L5178Y/DIC lymphoid cells carry
new antigen(s) not detectable on parental cells. The mechanism,
by which DIC alters the immunological properties of L1210
and L5178Y lymphomas is unknown. The drug-induced
antigen(s) were not found on normal lymphocytes or on cancer
Table 3 Cytotoxic activity of X-irradiated immunised spleen cells
X-ray dose
% lysis (± s.e.)
% Reduction in lysis
relative. to non-
irradiated cells
500 r.
19.4 (0.8)
5
1,000 r.
18.5 (0.7)
9
2,000 r.
12.4 (0.5)
39
4,000 r.
8.1 (1.1)
61
After 4 h assay
18 (0.7)
500 r.
17.1 (0.9)
5
1,000 r.
17.1 (1)
5
2,000 г.
14.3 (0.6)
21
4,000 r.
10.3 (0.8)
43
Attacker cells were from CDF1 mice challenged i.p. with 10?
L5178Y/DIC viable cells and assayed after 10 d against 51Cr-labelled
L5178Y/DIC cells. Attacker cells were X irradiated (Securix Compact
-CGR, 200 kV; 12 mA ; filter Cu 0.5 mm, Al 0.5 mm, 23 cm distance,
dose rate 98.2 r. min-1) at 4º C. and the activity tested immediately or
after 4 h incubation at 4° C.
..
cells. But, the strong transplantation antigen(s) seems to be a
permanent genetic feature, as the L5178Y/DIC cells maintained
the acquired antigen(s) even when the drug treatment was
discontinued over a long period.
This work was supported in part by the Centro di Farmaco-
logia delle Infrastrutture of the Consiglio Nazionale delle
Ricerche, Rome.
ANGELO NICOLIN
ALFREDO BINI
EMANUELA CORONETTI
Institute of Pharmacology,
School of Medicine,
University of Milan, 20129 Milan, Italy
ABRAHAM GOLDIN
1
National Cancer Institute,
Bethesda, Maryland 20014
Received May 2; revised August 5, 1974.
1
Weiss, L., J. natn.Cancer Inst., 50, 3-19 (1973).
2
Martin, J. W., Wunderlich, J. R., Fletcher, F., and Inman, J. K.,
Proc. natn. Acad. Sci. U.S.A., 68, 469-472 (1971).
3 Mihich, E., Cancer Res., 29, 2345-2350 (1969).
Bonmassar, E., Bonmassar, A., Vadlamudi, S., and Goldin, A.,
Proc. natn. Acad. Sci. U.S.A., 66, 1089-1095 (1970).
5
Nicolin, A., Franco, P., Bonmassar, E., and Goldin, A., Proc.
Am. Ass. Cancer Res. Abst., 162 (1973).
6 Wigzell, H., Transplantation, 3, 423-431 (1965).
7 Canty, T. G., Wunderlich, J. R., and Fletcher, F. J. Immun., 106,
200-211 (1971).
MSV
12 (14.3/16.1)
4 (15.4/16.1)
GLV
16 (13.5/16.1)
0 (16.1/16.1)
Spleen DBA/2 Cr
-11 (21/18.8)
0 (18.8/18.8)
CDF1
0 (18.8/18.8)
2 (19.1/18.8)
BALB/c
8 (23.5/25.5)
3 (24.7/25.5)
C57BL/10 ScCr
4 (18/18,8)
8 (17.2/18.8)
C3H
8 (23.5/25.5)
4 (24.5/25.5)
. 1.
Attacker cells were from CDF, mice i.p. challenged with 10?
L5178Y/DIC viable cells and assayed after 10 d against 51Cr labelled
L5178Y/DIC cells.
% lysis without inhibitors.
% Reduction in lysis*
5 × 105
105
L5178Y/DIC
68 (7.8/24.9)
L5178Y
6 (15.6/16.6)
39 (15.3/24.9)
-6 (17.6/16.6)
L1210
8 (22.9/24.9)
4 (24/24.9)
EdG2
-2 (39/38)
-5 (40/38)
ELA
13 (22.7/26.4)
13 (22.7/26.4)
8
Ortiz de Landazury, M., and Herberman, R. B., J. exp. Med., 136,
969-983 (1972).
9 Ortiz de Landazury, M., and Herberman, R. B., J. natn. Cancer
Inst., 49, 147-153 (1973).
"A lymphocyte-inhibiting factor isolated
from normal human liver
WHILE we were looking for cell-mediated immunoreactions in
patients with active chronic hepatitis (ACH), non-reproducible
results with the leukocyte migration inhibition test1, using
Attacker cells
Target cells
51Cr release ( +s.e.)
EL,
14.1 (0.4)
SELA
L5178Y
3.4 (0.4)
L5178Y/DIC
2.8 (0.6)
E Gs
16.6 (0.8)
SEỞ G2
L5178Y
3.4 (1)
L5178Y/DIC
-- 2 (0.9)
C3H
42.8 (0.7)
SC 2H
L5178Y
3.3 (0.9)
L5178Y/DIC
-1.8 (0.9)
C57BL10/Sc Cr
12.4 (1)
SC57BL/10 Se Cr
L5178Y
2 (0.6)
L5178Y/DIC
3.5 (0.8)
Attacker cells (S) were spleen cells from CFD1 mice 10 d after
immunisation with an i.p. inoculum of 107 tumour cells (EL,-EdG2)
or a Sc implant of 1 mm3 skin tissue pieces (C3H-C57BL/10 Sc Cr).
L5178Y and L1210 were from Microbiological Ass. Bethesda.
EL4 and EG2 were obtained from Dr. G. Della Porta, National
Cancer Institution Milan. Moloney Sarcoma (MSV) and Gross
leukaemia (GLV) were virus-induced by Dr A. Di Marco, National
Cancer Institution, Milan.
that DIC treatment can induce transplantation antigen(s) on
L5178Y/DIC cells, not detectable on the original L5178Y
lymphoma and on other tumour, or normal thymus cells.
The cytolytic activity of spleen cells from CDF1 mice assayed
at different days after administration of the immunising
inoculum is shown in Fig. 1. The activity reached a peak at
10 d after tumour cell inoculation and could not be detected
after 13 d. 24 h in vitro incubation fully reactivated the cytotoxic
activity and it was still evident at 60 days following immunisa-
tion. The immune cell reactivity, its disappearance a few days
after the peak was reached as the result of a block by some still
unknown factor and its reactivation by 24 h incubation, agree
with previous observations involving the 51Cr release assay
technique8.
Immune spleen cells from CDF1 mice sensitised to allogeneic
antigens were cytotoxic to 51Cr-labelled tumour cells bearing
the corresponding alloantigens (Table 1). The spleen cells failed
to release the radiolabel from either the original L5178Y or the
L5178Y/DIC suggesting that the cells of the subline had not
acquired some nonspecific immunosensitivity. Evidently neither
L5178Y or L5178Y/DIC associated antigens can react with
H-2b or H-2k associated antigens.
The specificity of L5178Y/DIC antigen(s) was determined by
using an assay for inhibition of cytotoxicity involving addition
of unlabelled cells. Addition of unlabelled cells to the mixture
of immune lymphocytes and labelled L5178Y/DIC target cells
would result in inhibition of 51Cr release if the unlabelled cells
shared some common antigen(s) with L5178/DIC cells. Only
unlabelled L5178Y/DIC cells elicited marked inhibitory activity
(Table 2). All other normal or tumour cells added, including
the parental L5178Y cells, did not modify 51Cr release signifi-
Table 2 Inhibition of immune spleen cell-mediated cytotoxicity by
addition of unlabelled cells
Added inhibitor cells
Table 1
Cytotoxic activity of spleen cells immunised against
allogeneic antigens
* % lysis with inhibitor cells.
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Immediate assay
20.3 (0.9)

656
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Nature Vol. 251 October 18 1974
Table 1 Dose-dependent effect of inhibitor on PHA- and MLC-stimulated human lymphocytes and Hela cells
Inhibitor
(pg/0.2 ml)
Lymphocytes
Inhibitor
(ug/0.2 ml)
Lymphocytes
HeLa
HeLa
(Ly-control)
(c.p.m.)
(Ly-control)
(c.p.m.)
(c.p.m.)
(c.p.m.)
0
8,442 (59)
0
7,319 (312)
0
77,000
42,000
12
2,923
12.5
1,590
12.5
80,200
34,000
.25
26
25
288
25
61,300
27,000
50
39
n.d.
50
n.d.
22,000
100
28
n.d.
100
n.d.
12,000
different preparations of liver antigen from human liver, led us
to attempt to prepare a better defined antigen from human
liver-already proposed2 for this test system. Besides a liver
specific protein as previously described11, our preparation
yielded a soluble fraction, which inhibited the peripheral
leukocytes of patients with active chronic hepatitis, but also
inhibited the leukocyte migration of controls. The migration
inhibition in these cases was therefore not due to a cell-mediated
immune reaction against liver antigen, but was caused by a
previously unknown inhibiting substance, extracted from
human liver. Here we report some properties of this inhibitor.
The inhibitor was isolated from the soluble supernatant after
homogenising and centrifugation of normal human liver at
100,000g for 90 min in Hanks solution, pH 7.2. The supernatant
was transferred to a Sephadex G-100 column, equilibrated with
0.01 M Tris-HCI buffer, pH 8.0. The inhibiting factor was
eluted in the second (II) and third (III) fraction. Rechromato-
graphy of these active fractions yielded a homogeneous peak,
but no pure substance. An ion exchange chromatography
procedure was therefore added, and we obtained a pure
substance with inhibiting properties.
This inhibitor is a protein. The molecular weight, estimated
by gel filtration, is about 65 x 103-8 x 103. The electrophoretic
mobility is slow, similar to the B-y-globulins.
The functional properties of this protein are characterised by
a potent inhibition of lymphocyte transformation after stimula-
tion by phytohaemagglutin (PHA); purified protein derivative
(PPD) and by cellular antigens or transplantation antigens
(mixed lymphocyte culture (MLC)) (Fig. 1). Besides, the
metabolism of in vivo activated lymphocytes3,4 and the migra-
tion of the macrophages are inhibited also. After PHA stimula-
tion, most of all the de novo synthesis of RNA is inhibited.
The specificity of lymphocyte metabolism inhibition seems
to be very high. Doses which cause a 100% inhibition of lym-
phocyte's metabolism lead only to a very slight reduction of the
metabolic rate of Hela cells (Table 1). On the other hand, no
species specificity of the inhibitor could be found, since a
preparation from rabbit liver has about 40% of the inhibiting
potency on human lymphocytes compared with inhibitor
prepared from human liver.
..
The inhibitor reported here is not identical with the immuno-
10,000 *
-PHA
3H-Thymidine (c.p.m.)
1,000
100
Ly control
5 10
20
30
40
50
60
70
80
90
100
ug liver cytoplasm per 0.2 mi
Fig. 1 Dose-dependent inhibition of PHA-stimulated lympho-
cytes by fractions (Sephadex G100) from soluble liver cytoplasm.
O - - -
O.
Fraction 1;
X
-. . X, fraction II; V - - - V,
fraction III; X
-X, fraction IV; A · - · A, fraction V;
., liver cytoplasm.
suppressive a globulins from human serum6,7, because the
inhibitor from human liver has an electrophoretic mobility of a
B or y globulin. We have isolated this inhibitor from the
pooled serum of normal people also (by gel-filtration) but the
inhibiting strength of this preparation was only 10% of that
prepared from liver. We therefore suppose that the inhibitor
is released from the liver to the serum.
Whether or not the prolonged survival time of skin allografts
with simultaneous liver transplants on Diemictylus viridescens?
was due to the immunosuppressive effect of the lymphocyte
inhibitor from liver is unknown. The clear cut time dependency
of liver implantation and immunosuppressive effect and the
survival of the skin allograft does not exclude this possibility.
The inhibitor described here has some similarity with the
inhibitor of protein synthesis in polyribosomes, reported by
von der Decken8. The fact that the polyribosomes were
prepared from rat liver, however, makes it unlikely that the
inhibitor of protein synthesis in polyribosomes and our inhibitor
are identical or similar. The inhibitor reported here seems to be
lymphocyte specific.
The inhibitor is not a chalone9, because chalones are defined
as non species-specific inhibitors, present in and produced by
the tissue, the proliferation of which they specifically inhibit10.
The physiological significance of this inhibitor remains to be
elucidated, especially the mode of action and the question
whether or not this substance has something to do with regula-
tion or repair mechanisms. Studies on the inhibitory effect on
T or B lymphocytes are in progress.
This work was supported by Deutsche Forschungsgemein-
schaft, Bonn.
KURT SCHUMACHER
GISEALA MAERKER-ALZER
URSEL WEHMER
Medical University Clinic,
5 Köln 41, .
Germany
Received May 2, 1974.
1
Soborg, M., and Bendixen, G., Acta med. scand., 181, 247; (1967).
2
Smith, M. G. M., Golding, P. L., Eddleston, A. L. W. F.,
Mitchell, C. G., Kemp, A., and Williams, R., Br. med. J.,
7, 527 (1972).
3
Schumacher, K., and Alzer, G., Die chronische Hepatitis (edit. by
Lindner, H.), (Gerhard Witzstrock, Baden-Baden-Brüssel)
4
Alzer, G., Schumacher, K., and Gross, R., Klin. Wschr., -52,
190 (1974).
5
Cooperband, S. R., Boudevik, H., Schmid, K., and Mannick,
J. A., Science, 159, 1243 (1968).
6
Yachnin, St., J. Immun., 108, 845 (1972).
7
Baldwin, W. M., and Cohen, M., Am. J. Zool., 9, 1131 (1969).
8
Decken, A. von der, Hoppe-Seyler's Z. physiol. Chem., 353, 1405
(1972).
9
Bullough, W. S., and Rytomaa, T., Nature, 205, 573 (1965).
10
Mathe, G., Eur. J. clin. biol. Res .; 17, 548 (1972).
11
Meyer zum Buschenfelde, K. H., and Miecher, P. A., Clin. exp.
Immunol., 10, 89 (1972).
Two types of resistance to polyene
antibiotics in Candida albicans
STUDIES reported during the past 15 years have led to the
widely accepted conclusion that polyene antibiotics function
Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4
6.2
78,000
33,000
16
102
8
5,116
Inhibitor
(ug/0.2 ml)
3H-Thymidine 3H-Thymidine
PHA (3H-Thymidine Mikro)
MLC (3H-Thymidine Mikro)

Nature Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4 657
(Ax20 per 5 x 107 CFU) Survival CFU (ml-1)
b
10%
107
-0
10º
105
10+
.
51.0
Phosphate release
0.8
0.6
0.4
.
0.2
16
0
2
4
0
2 4 0
2 4 60 2 4 6 0 2 4 6
Time (h)
.
Fig. 1 The effect of polyene on the viability (CFU ml-1) and on
the release of phosphate (A820 nm per 5 x 107 CFU) from
mutants A1 (a), A2 (b), A3 (c), A4 (d) and A7 (e) at room
temperature. Only exponentially growing cells were suspended
in saline. After incubation with polyene for the periods. shown,
viability was determined by plate-counting and the release of
phosphate was quantitated colorimetrically21 on the supernatant
fluid after centrifugation: The amount of release in the blank
control samples remained negligible. . , 10 µg amphotericin
B ml-1; O, 40 µg amphotericin B ml-1; 4, 100 µg nystatin ml-1.
by binding with the sterol molecules in susceptible cells to
cause permeability change and,eventual death of the cell1-3. In
recent reports4-6 based on liposomal model membrane studies,
it has been suggested that susceptibility to polyene antibiotics
is determined by organisation rather than by a single component
of the membrane, namely sterol. Here, through study of
polyene-resistant mutants, we further propose that, at least in
some cases, permeability alteration may not be the specific
critical event in the fungicidal action of polyene antibiotics.
Five mutagen-induced mutants were derived from a clinical
isolate of Candida albicans (E139-A, wild type) as described by
Hamilton-Miller7. Their levels of resistance vary with inoculum
size and with the medium (Table 1). For example, mutant A2
(colony forming units ml-1)
108
107
Viability.
10°
105
10+
103
1
A
10
. 6 4260 material release
8
6
4
2
1
F
0
İ
2 3 4 0
1
2 3 4
Time (h)
Fig. 2 The effect of polyene on the viability (CFU ml-1) and
on the release of ultraviolet-absorbing substances (260 nm)
from the wild type E139-A (A) and from mutant strain A4 (O).
Exponentially growing cells were used. In (a) amphotericin B
was present at 20 µg ml-1; 'in (b) nystatin was present at
100 µg ml-1; in (b) nystatin was present at 100 µg ml-1.
Experimental conditions were identical to those described for
Fig. 1. The maximal extractable ultraviolet-absorbing material
from the cells was determined by treatment with 1.2 N perchloric
acid17 and taken as 100% release.
grew in 100. µg ml-1 nystatin on solid medium but was killed in
saline. Compared with E139-A, the mutants grew more slowly,
responded differently in the Liebermann-Burchard colorimetric
reaction (Table 1) and, in accordance with previous reports8,9,
contained proportionally more sterol (Table 2). In other
laboratories, differences in the sterol pattern of polyene-
resistant mutants have been observed8-13, and the major
sterols have been characterised completely in one set of
mutants14. Polyene resistance was attributed to altered patterns
of sterols. In several studies10,11,13, decrease in ergosterol
content has been singled out as the direct cause of polyene
resistance. For comparison, the ultraviolet absorption spectra
of the non-saponifiable fraction extracted from E139-A and
our polyene-resistant mutants were examined10. In all cases,
5,7-diene sterols (for example, ergosterol and 24(28)-de-
hydroergosterol) were detected but not as major components.
This was confirmed by analysis of the sterol composition with
",, Maximum marker release.
100
80
A
60
O
0
40
-
20
0
.0
'S
10
15
20
0
20
40
60 80 100
(ug ml- 1).
Fig. 3 Polyene (a, amphotericin B; b, nystatin) susceptibility of
),
liposomes derived from total lipid extracts of 139 (
, A1 (
Ą2 (), A3(A), A4(O) and A7 (4). Liposomes with the test
agents, as well as the blank control, were incubated at room
temperature for 30 min. The percentage maximum glucose
released was calculated from the expression: (glucose released in
the presence of polyene - blank control)/(total amount of
glucose trapped - blank control)
× 100, as described
previously4.6.
thin-layer chromatography (Table 3). A similar situation has
been reported in a strain of Saccharomyces cerevisiae in which
ergosterol constituted only 10% of the total sterol15.
We have found that effects on viability and cell leakage
depend on temperature and phase of growth. At room tempera-
ture (23°-24° C), nystatin and amphotericin B caused extensive
killing and leakage of phosphate and ultraviolet-absorbing
material from E139-A. At 2° C, however, no cells were killed
and we observed no leakage of phosphate and ultraviolet-
absorbing material from them. Similarly, cells in stationary
%, Maximum marker release
6
100!
80
60
40
201-
A
0
2.5 5.0 7.5 10.0 0 10 20 30 40 50
(µg ml-1)
Fig. 4 Polyene (a, amphotericin B; b, nystatin) susceptibility of
mixed lipid liposomes. Phospholipid and neutral lipid derived
from one strain were mixed, respectively, with the neutral lipid
and phospholipid derived either from the same strain (control
liposome) or from a different strain (mixed lipid liposome).
Experimental conditions were identical to those described for
Fig. 3. Neutral lipids of: , E139-A; , E139-A; A, E139-A;
V, A1; O, A1; [
, A3; 4, A3. Phospholipids of: O, A1;
, A3; A, E139-A control; V, E139-A; O, Al control;
,
E139-A; A, A3 control.
Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4
i
:
-
R
0

658 Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4 18 1974
1
Table 1 Characterisation of polyene-resistant mutants of C. albicans
Polyene added
(µg ml-1) Strain E139-A
A1
A2
A3
A4
A7
0.0
++
+
+ +
+
+
40.0
20.0
10.0
+
2.5
0.5
+
Nystatin
100.0
+
+
25.0
10.0
* ++
+
*
*
*
*
Colour reactionț
Greenish
blue
65
Bright
yellow
85
70
97
135
* Log phase cells were adjusted to approximately 5 x 104 colony forming units per ml in fresh growth medium; 2 ml of this dilution was
added to tubes containing the given polyenes. Dimethyl sulphoxide was present to 1% in all samples including the blank control. After over-
night incubation at 37° C, the turbidity was recorded.
t A single colony of the given strain was streaked out on plate containing the given polyenes, and the growth was checked after 24 h of
incubation at 37° C. The nystatin (4530 U ml-1) and the amphotericin B (876 ug ml-1) were gifts from the Squibb Institute of Medical Research.
# Liebermann-Burchard reaction used. Total lipid extract of whole cells were used for assay. Preparations from both log and stationary
phase cells gave identical results.
§ Growth was monitored turbidimetrically with a Klett-Summerson photoelectric colorimeter (green filter).
phase were affected far less than those in the logarithmic phase.
(Unless otherwise stated, only exponentially growing cells
were used subsequently.) These temperature and growth phase
effects might be explained in three ways: (1) changes of
chemical composition in the membrane; (2) changes of effective
pore size cell wall (a molecular sieving effect16); (3) a tempera-
ture-dependent metabolic process, operative only in growing
Table 2 Incorporation of sodium acetate-1-14C into lipid
fraction of wild type and mutant strains
% Distribution of
phospholipids
Ratio of
phospholipid/
neutral lipid*
E139-A,
73.8
20.9
5.3
2.41
A1
60.6
28.0
11.4
1.74
A2
69.5
22.1
8.3
1.62
A3
68.7
19.9
11.3
1.63
A4
70.7 .
22.4
7.0
1.92
A7
76.5
13.1
10.4
1.46
Total lipid extract was subject to thin-layer chromatography on
silica gel G plates. with system CHCI3/CH3COOH/H2O/AcOH
(65:43:3:1, v/v). Percentage distribution of phospholipids was
calculated from the expression: [(c.p.m. of given phospholipid)
(total c.p.m. in phospholipid fraction)] x 100. PC, phosphatidyl-
choline; PE, phosphatidylethanolamine; PS, phosphatidylserine;
PI, phosphatidylinositol.
* Represent ratios of c.p.m. in phospholipid fraction to c.p.m. in
neutral lipid fraction. Preliminary experiments indicated that about
90% of the counts in neutral lipid fraction was associated with free .
sterols.
cells, may be involved in the fungicidal action. No decisive
evidence favours any of these suggestions, and temperature
dependence and growth phase dependence could be unrelated.
But, we have observed no appreciable difference in sterol
patterns nor in the polyene-sensitivity of liposomes prepared
from growing and stationary cells. Furthermore, protamine
(molecular weight approximately 18,500) can release ultraviolet-
absorbing material from a strain of C. utilis at both 30° C and
0° C (ref. 17).
Figure 1 shows the relationship of viability and leakage in
mutants. The release of cellular constituents did not result in
or parallel extensive killing in mutants A4 (Fig. 1d) and A7
(Fig. 1e). The latter phenomenon is illustrated more clearly
in Fig. 2. In the presence of amphotericin B and nystatin, the
viability of A4 was maintained whereas E139-A was soon
killed; the release of ultraviolet-absorbing material from both
strains remained comparable. If the alteration in permeability
is a direct measure of membrane-polyene interaction, the data
in Figs. 1 and 2 suggest that there may be two mechanisms of
polyene-resistance in yeast. With type 1 mutants, Al and A3,
resistance arises from a reduced interaction between the cell
membrane and the antibiotics as indicated by the low level of
leakage. With type 2 mutants, A2, A4 and A7, the antibiotics
interact with the membranes, as evidenced by the altered
permeability, but some critical step in the fungicidal effect fails
to occur.
The categorisation of mutants based on the loss of cell
constituents can be reproduced in liposomal membrane systems
derived from the total lipid extract of each strain (Fig. 3).
Liposomes prepared from mutants A2, A4 and A7 leaked
glucose in a manner comparable with the wild type. The
apparent decrease in glucose release at high concentrations of
amphotericin B was due to the interference of the antibiotic
molecule at the absorption wavelength used in our assay.
Mixed liposomes were also prepared after fractionating total
lipid extracts into phospholipid and neutral lipid components
by silicic acid column chromatography18. Figure 4 shows
that polyene-induced permeability of E139-A was associated
Table 3 Mobility and percentage distribution of major sterol classes
of wild type and mutant strains on thin-layer chromatography
RF values
<0.14
0.32
0.48
0.55
0.67
0.72
E139-Ą
30.6
18.1
11.7
29.7
10.0
A1
38.8
8.8
18,2
22.4
11.8
A2
24.2
5.7
12.4
23.6
25.9
8.3
A3
45.2
7.4
15.5
21.2
10.8
A4
36.9
10.8
17.2
25.5
9.7
A7
24:4
13.3
22.3
27.4
12.7
Non-saponifiable sterol extracts were subject to thin-layer chroma-
tography on AgNO3-impregnated silica gel G plates. The plates were
developed half-way with benzene-ethyl acetate (2:1, v/v), dried under
N2 and redeveloped in the same direction the full distance with
benzene-ethyl acetate (5:1, v/v). Figures given were calculated from
the expression: [(c.p.m. of a given spot)/(total c.p.m. in sterol
fraction)] x 100.
* As references, the RF values of ergosterol and cholesterol are
0.32 and 0.55, respectively. Sterols with polarities (as measured by
their RF values on silica gel) greater than ergosterol were not well
resolved in this system.
Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4
-
- ...
*
++++
+
+ ++++*
+
+
Nystatin
100.0
Growth on
Nystatin*
100.0
solid mediumt
Amphotericin B
20.0
Purplish
blue
Bright
yellow
Greyish
blue
90
Greyish
blue
Doubling time (min)§
50.0
+
+
2.5
+
+++++++++++*
+
+
*
Growth in
liquid medium*
Blank control
Amphotericin B
PC
PE
PS:+ PI
!

659
Natur Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4
with the neutral lipid fraction (shown to be predominantly
free sterol in preliminary experiments). Similarly the in-
sensitivity of A1 and A3 to polyene-induced permeability
changes was conferred on mixed liposomes by the neutral lipid
fraction of these organisms. Our results clearly show that, in
type 1 mutants, resistance arises from changed sterol patterns.
Structural characterisation of major sterols in all strains is
needed before this type of resistance can be discussed more
specifically.
The existence of type 2 mutants indicates that leakage does
not always result in killing. The discovery of nystatin-dependent
mutants19 suggests that this antibiotic could play a role in cell
physiological function. The lethal action may involve polyene-
induced malfunction of an essential physiological process or
processes. When such a process is not operative, for example,
at 2° C or when cells enter stationary phase or when a specific
mutation occurs, its susceptibility to the fungicidal effect of
polyenes may diminish. It has been reported20 that iodoacetate
and dinitrophenol reduce the fungicidal action of the polyenes
on yeast.
This work was supported in part by a grant from the National
Institute of Allergy and Infectious Diseases. C.C.H. was
supported by a cancer training grant from the National Cancer
Institute.
C. C. HSUCHEN
DAVID S. FEINGOLD
Departments of Medicine,
Beth Israel Hospital,
and Harvard Medical School,
Boston, Massachusetts 02215
Received May 28; revised August 12, 1974.
1
Lampen, J. O., Am. J. clin. Pathol., 52, 138-146 (1969).
2
Kinsky, S. C., Ann. Rev. Pharmac., 10, 119-142 (1970).
3
Hamilton-Miller, J. M. T., Bact. Rev., 37, 166-196 (1973).
4
HsuChen, C. C., and Feingold, D. S., Biochem. biophys. Res.
Commun., 51, 972-978 (1973).
5 Abramson, M. B., and Ockman, N., J. Colloid Interface Sci., 43,
530-538 (1973).
6 HsuChen, C. C., and Feingold, D. S., Antimicrob. Ag. Chemother.,
4, 309-315 (1973).
5
Hamilton-Miller, J. M. T., J. med. Microbiol., 5, 425-440 (1972).
8
Molzahn, S., and Woods, R. A., J. gen. Microbiol., 72, 339-348
(1972).
9
Hamilton-Miller, J. M. T., J. gen. Microbiol., 73, 201-203 (1972).
10
Woods, R. A., J. Bact., 108, 69-73 (1971).
11
Bard, M., J. Bact., 111, 649-657 (1972).
12 Athar, M. A., and Winner, H. 1., J. med. Microbiol., 4, 505-517
(1971).
13 Woods, R. A., Bard, M., Jackson, I. E., and Drutz, D. S., J.
infect. Dis., 129, 53-58 (1974).
14 Barton, D. H. R., Corrie, J. E. T., Widdowson, D. A., Bard, M.,
and Woods, R. A., J. Chem. Soc Chem. Comm., 30-31 (1974).
15
Gollub, E. G., Trocha, P., Liu, P. K., and Sprinson, D. B.,
Biochem. biophys. Res. Commun., 56, 471-477 (1974).
16 Scherrer, R., Louden, L., and Gerhardt, P., J. Bact., 118, 534-540
(1974).
17 Yphantis, D. A., Dainko, J. L., and Schlenk, F., J. Bact., 94,
1509-1515 (1967).
18
Owens, K., and Hughes, B. P., J. Lipid Res., 11, 486-495 (1970).
19
Woods, R. A., and Ahmed, K. A., Nature, 218, 369-370 (1968).
20 Gale, E. F., J. gen. Microbiol., 80, 451-465 (1974).
21
Ames, B. N., in Methods of enzymology, 8 (edit. by Neufeld, E. F.,
and Ginsburg, V.), 115-118 (Academic Press, New York, 1966).
.
Crystallisation of a modified fibrinogen
Mosr fibrous proteins do not crystallise. An artful way to
promote protein crystallisation is to modify the structure
by proteolytic attack. For example, papain cleavage of
y-globulin yields the homogeneous Fc fragment which
readily crystallises1. Following a similar approach, we have
crystallised fibrinogen.
Native fibrinogen precipitated at low ionic strength forms
fibrous aggregates which
are aperiodic in the electron
microscope. After these precipitates had been stored in the
cold for several months, we observed unusual rod-shaped
structures which showed microcrystalline order in the
electron microscope2. We had previously noticed that such
preparations were often contaminated with bacteria, and it
seemed likely that a bacterial protease had modified the
native molecule so that it formed ordered aggregates. We
cultured strains of Pseudomonas aeruginosa from such
preparations and prepared a crude protease extract. This
protease cleaved fibrinogen to a form that yielded highly
ordered microcrystalline arrays3. We summarise here our
studies on the effect of the protease digestion on fibrinogen
and show that more extensive controlled cleavage leads to
a modified molecule which forms crystals suitable for X-ray
diffraction analysis.
The protease used in these studies was isolated from the
growth medium of a strain of Pseudomonas aeruginosa
grown on trypticase soy. The crude enzyme was salted out
with ammonium sulphate. The protease was incubated at
31° C with native fibrinogen (5 mg ml-1 in 0.3M NaCl
buffered to about pH 7) for various times. The reaction
was terminated by addition of 10-3-10-4M N-a-Tosyl-L-
lysine chloromethyl ketone (TLCK), and the fibrinogen was
dialysed to low ionic strength (5×10-3M KSCN, 0.01M
2-(N-morpholino) ethane sulphonic acid (MES), pH 6.2).
Samples were stored in the presence of the inhibitor and
chloroform.
The progressive modification of the molecule was fol-
lowed by measuring the percentage of mass lost as small
trichloroacetic acid (TCA)-soluble peptides, and by observ-
ing the type and amount of ordered aggregates formed at
low ionic strength. Four stages of digestion were distin-
guished. In the very early phase, corresponding to less than
about 2% mass loss, most of the material precipitated out
of solution at low ionic strength and consisted chiefly of
aperiodic fibres, similar to native fibrinogen. With further
digestion (corresponding to a loss of 4-6% of the mass
as TCA-soluble material) the modified fibrinogen was more
soluble, but within about 3 d as much as 85% of the
material precipitated as microcrystals with a 450 Å axial
repeat3. In the narrow digestion range between about 6
and 8% mass loss, precipitation was even slower, and both
microcrystals and large birefringent crystals appeared; the
proportion of crystals could be very high. On further diges-
tion (about 10% mass loss) some amorphous precipitate
formed, but most protein remained in solution.
The extent of protease digestion could be correlated with
the appearance of the sodium dodecyl sulphate (SDS) gel
electrophoresis patterns run in the presence of reducing
agents4. The native molecule has a molecular weight of
about 340,000 and consists of three pairs of chains design-
ated A« (~70,000 daltons), BB (~60,000 daltons) and y
(50,000 daltons)5,6 (Fig. 1c). The Aa chain was attacked
first. Microcrystalline samples, digested to about 4% mass
loss, gave gel patterns with most of the Ad band and part
of the BB band missing. The gel patterns of crystals showed
a single heavily staining broad band with the same mobility
as the y chain of the native molecule (Fig. 1c). The clot-
ability by thrombin of the molecules from the crystals was
about 50% to 80% of that of native fibrinogen.
The crystals we obtained were thin birefringent plates
with the longest dimension up to 1 mm (Fig. 1a). They were
very fragile and characteristically cleaved in a direction
perpendicular to the long axis to give rod-shaped fragments.
Preliminary X-ray diffraction patterns of single crystals
taken at room temperature show reflections out to about
3 Å (Fig. 1b). The space group is P21. The unit cell dimen-
sions are a=134 Å, b=97.3Å, c=174Å. B=92°20'. The
density of the crystals is in the range 1.135-1.140 g cm-3
as determined by flotation of individual crystals in xylene-
bromobenzene mixtures. Assuming that the partial specific
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660 Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4 18 1974
a
b
b
b
C
d
1
2
2
Fig. 1 a, Crystal of proteolytically modified fibrinogen. The
b-axis is parallel to the long (vertical) axis of the crystal.
Note small fragments cleaved perpendicular to this direction.
(Approximately x 80.) b, A 42º Precession photograph of
of the Okl plane of the reciprocal lattice. The systematic
absence of the OkO reflections with k odd indicates a two-
fold screw along the b-axis (vertical direction). c, SDS poly-
acrylamide gel electrophoresis
patterns
of (1) native
fibrinogen; (2) modified fibrinogen from redissolved crystals.
Both reduced with mercaptoethanol. d, Electron micrograph
of crystal fragments showing (1) a net-like region: (2) fine
diagonal, lateral and longitudinal striations spaced at about
45 Å, 60 Å and 35 Å, respectively. The sides of the mesh are
about 105 Å by 115 Å; the acute angle is about 65°. Samples
negatively contrasted with 1% uranyl acetate. (x 190,000.)
volume of the protein is 0.727, the water content of the
crystals is about 65%. The molecular weight of the protein
in the asymmetric unit was therefore calculated to be
330,000, with an uncertainty of about 6%.
Crystals were washed, fragmented and negatively con-
trasted with 1% uranyl acetate for electron microscopy.
Commonly observed images showed fine diagonal and
longitudinal striations spaced at about 45 Å and 35 Å.
More rarely, a net-like form was seen (Fig. 1d). The beaded
strands of the mesh were about 30 Å in diameter, with the
edges about 115 Å by 105 Å in length, defining an acute
angle of about 65°. The negatively stained crystal frag-
ments showed no direct correspondence with images of
the microcrystalline forms which have a 450 Å axial
repeat3.
The unit cell dimensions and density of the crystals
indicate a molecular weight for the modified protein of
about 330,000 comparable with the native molecule. The
estimate of mass lost from modified fibrinogen which
crystallises is about 6-8% by measurement of the TCA-
soluble peptides released during digestion. SDS gels of the
crystals run in reducing conditions, where polypeptide
chains separate by molecular weight, show material only
in the y chain region. Since no other large chain fragments
are seen, the cleavage may split off relatively small frag-
ments from the Aa and BB chains, reducing them to the
size of the y chain. The y chain has a molecular weight of
about 50,000; thus the weight inferred for the modified
molecule from these gels is again about 300,000. Migration
of the whole modified molecule on SDS gels without reduc-
ing agents present also gives a rough estimate of molecular
weight close to 300,000. The chemical and crystallographic
estimates of the mass of the modified molecule thus agree,
within experimental uncertainties. This preliminary. evi-
dence on the modified molecule forming the crystal indi-
cates that in spite of the cleavage it may be largely intact.
Redissolved crystals show a clotability about 50-80%
of that of native fibrinogen. The fibres formed appear very
similar to those of native fibrin in the electron microscope.
This indicates that the modified molecule has retained the
critical sites which interact to form the fibrin clot.
Some inferences about molecular shape and packing can
be made at this stage, although the crystallographic data
do not provide strong constraints. The modified fibrinogen
crystallises in the monoclinic space group P21, and the unit
cell contains two molecules of weight about 300,000. The
molecule is a dimer of elongated shape, but a twofold axis
is not a symmetry element of the crystal. From the sym-
metry of the crystal, these rod-shaped molecules must be
arranged with two different orientations. Since the crystals
readily cleave along the plane perpendicular to the b axis,
the forces are weak between arrays of molecules related
by the twofold screw. Only about 35% of the unit cell
volume is occupied by protein, so that the molecules must
form a fairly open meshwork of cross-connected rods.
--
The body diagonal of the unit cell in the crystal is about
half the 450 Å length inferred for the fibrinogen molecule
from studies of the microcrystals3. The dimensions of the
unit cell in the crystal lattice need not, however, be related
simply to the molecular dimensions. As in the assembly
of other fibrous proteins8-11 the rod-shaped molecules may
pack with extensive side-to-side overlaps and end-to-end
gaps, so that the observed periods may be only a fraction
of the molecular lengths. The microcrystals of the modified
fibrinogen molecule display a 450 Å axial period, and a
specific staggering of arrays with this period can produce
the 225 Å axial period and density distribution seen in the
fibrin clot3. Although these large periodicities are not
evident in the crystal lattice, it is likely that the modified
fibrinogen in these crystals will show interactions similar
to those in the microcrystals and in fibrin.
This work was supported by grants from the US Public
Health Service and the National Science Foundation. We
thank Dr Stephen Harrison for taking the X-ray photo-
graphs and for discussions. We thank Dr D. L. D. Caspar
for discussions, Isa Bernardini for technical aid and Paul
Norton for electron microscopy.
CAROLYN COHEN
Laboratory of Structural Biology,
Rosenstiel Basic Medical Sciences Research Center,
Brandeis University,
Waltham, Massachusetts 02154
NANCY M. TOONEY
Department of Chemistry,
Polytechnic Institute of Brooklyn,
Brooklyn, New York 11201
Received May 2; revised August 2, 1974.
Cohen. S., and Porter, R. R., Adv. Immun., 4, 287 (1964).
Cohen, C., Slayter. H., Goldstein, L., Kucera, J., and Hall, G.,
J. molec. Biol., 22, 385 (1966).
3
Tooney. N. M., and Cohen, C., Nature, 237, 23 (1972).
Weber, K., and Osborn, M., J. biol. Chem., 244, 4406 (1969).
5
.Henschen, A., Arkiv. Kemi., 22, 1 (1964).
6
McKee, P. A., Rogers, L. A .. Marler. E., and Hill, R. L.,
Arch. Biochem. Biophys .. 116, 271 (1966).
Scheraga. H. A .. and Laskowski, M., Adv. Protein Chem., 12,
1 (1957).
8
Hodge, A. J .. and Schmitt, F. O., Proc. natn. Acad. Sci.
U.S.A .. 46, 186 (1960).
9
Cohen, C .. Szent-Gyorgyi .. A. G., and Kendrick-Jones, J.,
J. molec. Biol .. 56, 223 (1971).
Cohen. C .. Lowey. S., Harrison, R. G., Kendrick-Jones, J.,
10
and Szent-Gyorgyi, A. G., J. molec. Biol., 47, 605 (1970).
11
Squire, J. M., J. molec. Biol., 77, 291 (1973).
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The Gunn Effect
G. S. Hobson
This book uses simple physical models to describe the
Gunn effect and the electronic devices based on it. Its
account of the phenomenon is developed logically from the
origins and limitations of the transferred-electron effect
and the effect of boundaries to the use of the phenomenon
is oscillators and amplifiers. The construction, technology,
and operation of device chips and circuits are described,
and an outline is given of desirable and undesirable
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Numerical Solution of
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Edited by L. M. Delves and J. Walsh
This book provides an up-to-date introduction to the
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Also discussed are non-linear problems, and some
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Introduction to Control
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O. L. R. Jacobs
.
This book gives a uniform presentation of the main
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£6.75 Oxford Applied Mathematics and Computing Science
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Upland Kenya Wild
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A. D. Q. Agnew
Here is a concise work on the wild flowers and ferns of an
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Essentials of Biology
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A. E. Brooks
576pp Price £6.05 June 1974
Hardback binding ISBN 0 03 089447 6
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640pp Price £6.95 March 1974
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¥ Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4 18 1974
WILEY
INTERSCIENCE
CONSERVATION IN PRACTICE
Edited by A. Warren and F. B. Goldsmith, both of University
College London.
As Britain and Europe wake up to the necessity for conservation it is
clear that more trained graduates will be needed .to understand
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This book describes the ways in which scientists and managers can
study resources, determine their capacity, and plan their exploitation
and management. Most of the authors are contributors to the
successful post-graduate course in conservation at University
College London and the editors are full-time lecturers to the course.
They have selected topics to cover the whole field of Conservation
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conservation in planning, economics and politics. Many of the
authors are senior staff in the Nature Conservancy, Country
Commission, or Local Planning Authorities and are especially
competent to talk about conservation in the real world-in practice.
September 1974
532 pages
£9.50 (cloth)
September 1974
532 pages
£4.50 (paper)
REGISTRY OF MASS SPECTRAL DATA, Volumes 1-4
Edited by the late Einer Stenhagen and Sixten Abrahamsson,
both of the University of Goteborg, and Frederick W. McLafferty,
Cornell University.
An updated and expanded version of the ATLAS OF MASS
SPECTRAL DATA. Contains the mass spectral data for 18,806
different compounds in bar graph form. Every effort has been made
to choose the best available spectrum for each compound and to
eliminate all errors from the spectra. Bar graph format provides easy
visualization of the data and conserves space in presentation of the
complete spectrum. A new computer tape containing the same
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9 track, with 200,556, or 800 bits per inch.
September 1974
3378 pages
£125.00
BIOMEDICAL SCIENCES INSTRUMENTATION, Volume 10
Edited by Richard D. Barnett, U.S. Air Force Academy.
The book comprises proceedings of the 11th Annual Rocky
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national ISA Biomedical Sciences Instrumentation Symposium.
The joint symposium, conducted April, 1974, in Colorado Springs,
Colorado, was programmed by ISA's Biomedical Sciences Division,
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in Medicine and Biology, and RMBS, Inc.
September 1974
194 pages
£6.75
Published by Instrument Society of America and distributed by
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CLOUD PHYSICS AND WEATHER MODIFICATION
Edited by Yu. S. Sedunov, Main Administration of the Hydro-
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The papers in this collection review various trends in cloud physics
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specialists interested in behaviour of disperse system. (Translated
from the Russian.)
September 1974
110 pages
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Published by Israel Program for Scientific Translations Ltd., and
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AMNIOTIC FLUID : Physiology, Biochemistry, and
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A compendium of current information and opinions on the clinical
import of amniotic fluid. Examines the present state of knowledge
on the composition of amniotic fluid under normal and abnormal
conditions in order to aid the diagnosis and prognosis of the
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September 1974
404 pages
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CLOUD PHYSICS EXPERIMENTATION INVESTIGATIONS
Edited by O. A. Volkovitskii, Institute of Experimental
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A collection of papers dealing with the experimental study of aerosol
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and fog modification by condensation nyclei, the distribution of
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(Translated from the Russian.)
126 pages
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September 1974
Published by Israel Program for Scientific Translations Ltd., and
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WEATHER AND CLIMATE MODIFICATION
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The first collection of current thought on weather modification.
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only the physical science of weather modification but also the social
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September 1974
858 pages
£15.00
A STEREOTAXIC ATLAS OF THE MONGOLIAN GERBIL
BRAIN
By William J. Loskota, Ph.D., Peter Lomax, M.D., D.Sc.,
M. Anthony Verity, M.D., Department of Pathology and Brain
Research Institute, University of California at Los Angeles.
This Atlas is a compilation of selected coronal and saggital sections
of the Mongolian gerbil brain presented sequentially from rostral
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August 1974
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Invertebrate Zoology
By R. D. Barnes, PhD, Professor of Biology, Gettysburg College.
This leading text in the field, has now been updated by Dr. Barnes. In an effort to make his
presentations easier to assimilate, he has de-emphasised the morphological material and
placed greater stress on ecological behavioural ideas.
Approx 775pp Approx 50 illust Approx £6.65 August 74 Hardback binding
Fundamental Experiments of Microbiology
By K. T. Crabtree, PhD, Department of Biology, University of Wisconsin, Wausau and R. D. Hindsill, PhD,
Department of Bacteriology, University of Wisconsin, Madison.
Designed for students with some knowledge of the subject, this laboratory manual serves
as introductory courses in microbiology. It is an excellent teaching tool, guiding the student
through numerous experiments involving non-medical, applied microbiology and microbial
ecology. Particular attention is paid to the intracacies of soil and water microbiology-and
an ingenious experiment on bacterial conjugation helps the student grasp the genetic
concepts involved. Developed by two authors of the ninth edition of FUNDAMENTALS
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biology laboratory.
Approx 200pp Approx 50 Illust Approx £2.60 August 74 Paperback binding
Abnormal Haemoglobins
(Clinics in Haematology Vol 3 No 2)
Edited by D. J. Weatherall, MD, FRCP, MRCpath, Professor, Department of Haematology, University of
Liverpool.
This latest issue of Clinics in Haematology demonstrates the impact of molecular biology
on clinical medicine. In it an international team of clinicians, protein chemists and geneticists
highlights the haemoglobin disorders, particularly the sickling diseases and the thalassaemia.
250pp Illustrated Price £4.50 June 74
Environmental Science
By A. Turk, PhD, Department of Chemistry, CCNY, New York.
J. Turk, PhD, Naturalist.
J. T. Wittes, PhD, Division of Epidemiology, Columbia University School of Public Health, New York.
R. E. Wittes, M.D., Department of Medicine, Memorial Sloan Kettering Cancer Centre, New York.
This is a highly illustrated, general textbook on environmental science, with special emphasis
on pedagogic excellence. Various topics are developed in careful sequences, whereby the
student's knowledge can build upon itself. Economic and social aspects are integrated, and
new terms carefully defined. In addition, biological, physical, chemical, geological and
engineering considerations are covered.
563pp 187 Illust-9 colour plates Price £6.20 Hardback binding April 74
W. B. Saunders Company Ltd.,
12 Dyott Street, London WC1A 1DB.
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> Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4 18 1974
Stereochemistry
Two Volumes
In Memory of van't Hoff
Topics in Current Chemistry/Fortschritte der chemischen Forschung
Managing Editor: F. Boschke
Volume 47
Stereochemistry I
40 figures. VI, 156 pages. 1974
Cloth DM 58, -; US $23.70
ISBN 3-540-06648-9
Prices are subject to change wihtout notice
Volume 48
Stereochemistry II
15 figures. VI, 129 pages. 1974
Cloth DM 48, -; US $19.60
ISBN 3-540-06682-9
On the centenary of the publication in September 1874 of the work that laid
the foundations of stereochemistry: "La Chimie dans l'Espace" by van't Hoff
I Prospectus on request
Volume I
;
K. Mislow, D. Gust, P. Finocchiaro,
R. J: Boettcher:
Stereochemical Correspondence Among
Molecular Propellers
To be identified as a molecular propeller,
a molecule must possess two or more
subunits which can be considered as
"blades" radiating from an axis of
rotation. Furthermore, each blade
must be twisted in the same sense so as
to impart a helical conformation to the
.
molecules. (47 references)
J. H. Brewster:
On the Helicity of Variously Twisted
Chains of Atoms
It is important to consider in some
detail the geometric attributes
of twisted chains of atoms regarded as
irregular helices, because this is one
of the significant ways in which the
chirality of asymmetric atoms finds
structural expression and becomes
manifest. (18 references)
.
H. H. Wasserman, G. C. Clark, P. C. Turley :.
Recent Aspects of Cyclopropanone
Chemistry
Cyclopropanones are highly reactive
1
organic systems. They represent valu-
able substrates for studying theoretical
aspects of the chemistry of small strained
ring systems and are of special interest
in synthesis because of the variety of
transformations in which they take part.
(133 references)
Volume II
J. Gasteiger, P. D. Gillespie,
D. Marquarding, I. Ugi:
From van't Hoff to Unified Perspectives
in Molecular Structure and Computer
Oriented Representation
Observable properties of molecules are
determined from equilibrated ensembles
of species differing in geometry and.
energy. This demands a representation
invariant against changes of the latter
features. Thus, it is necessary to use
problem-oriented models for molecules,
neglecting any properties that are irrele-
vant for a given problem. Such models
represent equivalence classes of mole-
cules with a common characteristic
feature. (35 references)
B. Vennesland:
Stereospecificity in Biology
Virtually all biological reactions are
stereospecific: (101 references)
-
F. Vogtle, P. Neumann:
[2.2]Paracyclophanes, Structure and
Dynamics
Non-planar benzene rings, anomalous
bond lengths and angles, and electronic
interactions between parallel benzene
nuclei are common features of the [2.2]
paracyclophane system. Chirally sub-
stituted [2.2]paracyclophanes offer inter-
resting parallels to the stereochemistry
of the metallocenes. (132 references)
W
S
ACH
825
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Nature
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(1407)
SCARBOROUGH COLLEGE
UNIVERSITY OF TORONTO
CHAIRMAN-LIFE SCIENCE
Applications are invited for the position of
Chairman of the Life Science Group at Scar-
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July 1. 1975. The Life Science Group is made up
of about 25 faculty in Biochemistry, Botany, .Psy-
chology and . Zoology. Applicants should have
established reputations in scholarship in any of
these fields, and be eligible for full professorship
rank at the University of Toronto. A letter of
application . together with a detailed curriculum
vitae and the names of three persons who have
agreed to act as referees, should be directed to
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University of Toronto, 1265 Military Trail, West
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(1411)
ROYAL POSTDOCTORAL MEDICAL
SCHOOL
POSTDOCTORAL BIOCHEMIST.
required to study the enzymology of carboxylases
both from animal tissues and children with inborn
errors of metabolism affecting mitochondrial car-
boxylases. This position is available for 1 year
in the first instance with the possibility of exten-
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Applications including full curriculum vitae to
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pital, DuCane Road. London W12 OHS, quoting
ref. no. 2/474.N.
(1485)
CSIRO
AUSTRALIA
DIVISION OF LAND USE
RESEARCH
CANBERRA, A.C.T.
RESEARCH
SCIENTIST
The Commonwealth Scientific and Industrial Research Organization has a broad charter
for research into primary and secondary industry areas. The Organization has approxi-
mately 6,500 employees-2,100 of whom are research and professional scientists-
located in Divisions and Sections throughout Australia.
FIELD
OPERATIONS
RESEARCH
GENERAL The Division of Land Use Research is currently developing research pro-
grammes in both socio-economic and biophysical aspects of land use planning, utilising
geographic data base systems. The Division is seeking an Operations Research Scientist
capable of development and innovative work on optimization problems associated
with patterns of regional land use allocation. The following are topics of current interest:
mathematical programming algorithms recognizing multiple goals, both tangible and
intangible; combinatorial allocation under proximity constraints; simultaneous
optimization of land use and infrastructure networks.
DUTIES: To initiate research in these topics in an inter-disciplinary team working on
land use problems on the South Coast of N.S.W. Scope exists for a later extension of
research into broader aspects of system optimization."
QUALIFICATIONS: Ph.D. or equivalent research experience in a relevant discipline
together with demonstrable research ability.
SALARY: Appointment will be made within the salary ranges of Research Scientist or
Senior Research Scientist: $A10,448 to $A15,456 p.a.
TENURE: Either an indefinite or a fixed term appointment may be negotiated. An
indefinite appointment carries Australian Government Superannuation benefits.
Applications stating full personal and professional details, the names of at least two
professional referees and quoting Reference Number: 618/387, should reach:
The Personnel Officer,
Australian Scientific Liaison Office,
64-78, Kingsway, LONDON, WC2B 6BD
by the 30th November, 1974
Applications in U.S.A. and Canada should be sent to
The Counsellor (Scientific),
Embassy of Australia, 1601 Massachusetts Avenue, N.W.,
WASHINGTON, D.C. 20036, U.S.A.
(1504)
ELECTRONICS TECHNICIAN
(Grade 5)
required for small research group in Zoology &
Comparative Physiology Department; investigating
ultrasonic communication and echolocation systems
of animals. Responsibilities will include maintenance
of various advanced instruments and the develop-
'ment of new equipment and techniques for in-
vestigating ultrasound in air. The appointment is
temporary for one year in the first instance. Salary
scale £2.007 by £75 to .£2,382 p.a. plus £228 p.a.
London Weighting and current .Threshold payment.
Five weeks (in total) annual leave. Five day week.
Letters only to Assistant Secretary (Establishment)
FT/Z. Queen Mary College. Mile End Road, EI
4NS stating age, qualifications and experience.
(1471)
-
·MEMORIAL UNIVERSITY OF
NEWFOUNDLAND
FACULTY OF MEDICINE
Pre and postdoctoral positions are available for
studies on the protein synthesizing system with
special emphasis on ribosomes-topography, bio-
genesis and function-under various. conditions of
growth and development. Teaching opportunities
available. Reply with resume to:
Professor Bruce H. Sells
Laboratories of Molecular Biology
Faculty of Medicine
Memorial University of Newfoundland
St John's, Newfoundland, Canada
(1478)
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xii
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AMERIC VI. VI VLIVET 18 1974
Tropical Products Institute
BIOLOGIST
(Plant Sciences)
. ... for applied research and development work in a multidisciplinary
team concerned with the post-harvest. biology and technology of fruit,
vegetable and root crops in the tropics.
Candidates (normally aged under 30) must hold a good honours
degree, or equivalent, in an agricultural or biological subject and,
preferably, have postgraduate training or experience and a special
interest in plant pathology or physiology. Much time will be spent
working in developing countries, and the ability to work independently
under tropical field conditions is important.
Appointment will be as Higher Scientific Officer (over £2,800 to around
£3,750). Starting salary will depend on qualifications and experience,
and there is a non-contributory pension scheme.
For further details and an application form (to be returned by ]
November 1974) write to Miss C. A. Hall, Administrative Secretariat,
Tropical Products Institute, 127 Clerkenwell Road, London ECIR 5DB.
Please quote reference SA/36/JD.
(1497)
AARHUS UNIVERSITY
BIOPHYSICS INSTITUTE
Applications are invited for the
position of lecturer from February
1, 1975.
The Institute's research- work is
concerned with the investigation of
the molecular mechanism of muscle
contraction.
The Institute's teaching responsi-
bilities include a course in medical
physics and part of a combined bio-
physics-physiology course. The Insti-
tute is also planning courses in
physics/physiology, instrumentation,
clinical physics and physical methods.
Preference will be given to appli-
cants having a knowledge of low-
angle X-ray diffraction techniques
and experience with computer-gener-
ated models.
Salary is according to age and ex-
perience.
Applications (5 copies) should be
sent to the Administration, Aarhus
University, Ndr. Ringgade 1, DK-
8000 Aarhus C, Denmark, before .
November 4, 1974.
(1491)
1
Papua New Guinea
Botanist
Applications are invited from interested per-
sonnel for the post of Botanist with the
Department of Forests. Candidates should be
graduates with major studies in appropriate
subjects and, ideally, research experience in
botanical taxonomy. The ability to prepare
research material for publication is essential.
A salary of $A10075 p.a. is offered together
with a gratuity of $A1950 p.a. ($A1.77=
£1.00 approx.): Period of engagement is for
two years (renewable in most instances).
General entitlements are very attractive and
include an education allowance for dependent
children attending school overseas, return air
passages with personal effects and baggage
allowance, low cost married and single ac-
commodation and generous leave conditions.
.
Please write for an application form and full
details of the post.
The Papua New Guinea Public Service Board
Representative
22 Garrick Street
London, W.C.2
Telephone: 01-240 1780
Papua New Guinea
apua New Guine
(1475)
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VLIVUVI
Nature
SCOTTISH HORTICULTURAL
RESEARCH INSTITUTE
West of Scotland Unit,
Auchincruive, Ayr. KA6 5AE.
HIGHER SCIENTIFIC OFFICER
An H.S.O. is required to assist
with the strawberry breeding pro-
gramme. Duties include screening
seedlings for disease resistance and
fruit characteristics plus practical
work associated with the develop-
ment of new varieties.
Qualifications :
A
pass
degree,
H.N.C. or equivalent and at least
five years' appropriate experience
since qualifying.
Salary: £2,461 to £3,371.
The post is pensionable; a new
non-contributory scheme
is being
devised.
Further particulars may be ob-
tained from the Officer-in-Charge,
with whom applications, naming two
referees and stating age, education
and experience, should be lodged by
November 8, 1974.
(1473)
RESEARCH TECHNICIAN
required to work in the Microbiology laboratories
at Queen Mary College. Experience in general
bacteriological techniques essential.
Salary on
Grade 3 scale £1,650 by £54 to £1.920 p.a.'plus
£228 p.a. London Weighting and current Threshold
payments. Five day week. Five weeks (in total)
annual leave. Letters only to Assistant Secretary
(Establishment) RT/PB, Queen Mary College,
University of London. Mile End Road, E1 4NS
stating age, qualifications and experience.
(1469)
TECHNICIAN (GRADE 3)
required to assist with research in animal genetics.
and in the teaching laboratories in the School of
Biological Sciences. Present research is concerned
with'a study of the genetic regulation of enzyme
activity in the mouse. Salary scale £1.650 by £54
to £1.920 p.a. plus £228 p.a. London Weighting
and current Threshold payments. Five week (in
total) annual leave. Pension Scheme. Five day
week. Letters only to Assistant Secretary, (Es-
tablishment) DMH, Queen Mary College, Mile
End Road. London El 4NS stating age, experience
and qualifications.
(1470)
SYNECOLOGIST
Assistant or Associate Professor for September
1. 1975. Interests in insular and tropical syneco-
logy. Emphasis in research and graduate instruction.
Send résumé and names of 3 referees by November
30. to Dr N. P. Kefford, Chairman, Department
of Botany, 3190 Maile Way. University of Hawaii
at Manoa, Honolulu, Hawaii 96822.
(1490)
Portsmouth Polytechnic
BIOPHYSICS LABORATORIES
The post of
RESEARCH ASSISTANT
is available in the above laboratories to join
a large research team under Dr E. M.
Bradbury. working on the problem of the
structure of the chromosome. A wide range
of physical and biochemical techniques are
employed, including X-ray
and
neutron
diffraction, electron microscopy. electropho-
resis and ultracentrifugation. The successful
candidate will probably have an honours
degree in either chemistry, biochemistry or
physics, together with a lively interest in
the control of fundamental biological pro-
cesses at the molecular level.
The appointment is for a period of three
years and registration for a higher degree
is expected.
Salary scale: £1.320 to £1.420 per annum,
plus cost of living payment.
Application forms and further particulars
may be obtained from the Staff Officer.
Portsmouth Polytechnic. Alexandra House.
Museum Road, Portsmouth POI 2QQ to
whom completed application forms should
be returned by November 1, 1974. Please
quote ref: S15.
(1476)
UNIVERSITY OF TROMSØ NORTH NORWAY
FISHERY BIOLOGY RESEARCH GROUP
Applications are invited for the following positions:
TWO PROFESSORSHIPS AND ONE LECTURESHIP
One Professorship in Fisheries Biology for candidates with appropriate qualifications
and experience in the fields of Population Dynamics and/or Systems modelling. Duties
will include development of courses and teaching of fishery students in these topics plus
research in an integrated programme on fjord fish populations and their ecosystem.
One Professorship and one Lectureship in Agriculture. There is much interest in
agricultural developments and possibilities in North Norway, in which the successful
candidate will have ample opportunity to participate. It is expected that those who are
appointed contribute to the fjord research programme. Though experience in agricultural
techniques is required it is also hoped that the persons appointed will have a broad
biological experience so that in both teaching and research he will be able to stress the
more fundamental aspects of this subject.
The University is now considering long-range plans for expansion of its teaching and
research capability in Fisheries Biology. A major programme of fishery and environmental
research of fjords is being developed by a group of biologists leaded by Professor A. H.
Weatherley. Other fishery research programmes are being planned.
Tormsø is situated in a very large northern commercial fishing region and fishery
biology is one of the areas of study being emphasised in the development of the
University. The work of the fishery group is being conducted in conjunction with marine
biologists, fishery economists, sociologists and technologists, and fishery biology will be
offered at both undergraduate and graduate levels. English may be used for teaching until
Norwegian is learned.
The University is new and expanding rapidly, and those who obtain posts will be
expected to participate in planning activities. The fishery group is part of the Institute
of Biology and Geology and is housed in a new building well equipped for various kinds
of teaching and research.
A new highly sophisticated 112 ft. fishery research vessel will be available by 1975.
The University has a computer centre featuring a 64-K Nor, computer, there is a DCT
2000 Simulator connected to a UNIVAC 1110 at the University of Bergen. and DCT 1000
Simulator is under construction.
Tromsø lies north of the Arctic Circle (lat. 69"), but has a mild climate because of
its coastal position. There are two months of midnight sun and two with darkness. There
are fine opportunities for outdoor life.
Salaries: Professor 104,780; Lecturer 69,760 to 87,510 N.Kr. per annum. The University
will help with housing and other problems. Applications should be sent by November 14,
1974. including five copies of curriculum vitae and three sets of relevant publications.
Formal applications should be addressed to Universitetet i Tromsø, Postboks 635,
9001 Tromsø, Norway, Further enquiries should be addressed to Professor A. H.
Weatherley, same address.
(1492)
Overseas
Development
Ethiopia
Technical Records Officer (Mining)
At the Department of Mines, Addis Ababa, to be responsible for organising and
establishing a comprehensive record system, and the training of local staff.
Applicants must have a degree or equivalent qualification in Geology or Mining
Engineering. Subsequent experience of the mineral industry, including the use of
data processing methods essential. Appointment for I year.
Salary in range £4,400 to £5,200 pa plus a tax free overseas allowance in range
£895€ to 1,995 pa.
Other benefits include free family passages, paid leave, children's educational
allowances, and free accommodation and medical attention. Superannuation rights
may be safeguarded and all emoluments are paid by the British Government.
Applicants should normally be citizens of, and permanently resident in, the
United Kingdom.
For full details and an application form please apply giving age and details of
qualifications and experience to:
Appointments Officer
Ministry of
Overseas Development
Room 301
Eland House
Stag Place
London SWIE 5DH
(1466)
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FOOD
MICROBIOLOGIST
The Union International Co. Ltd. has a vacancy for a Graduate Food
Microbiologist at the St. Albans Research Centre.
The work is varied and interesting, and will include investigations into
· vacuum packed and cured meats.
Preference will be given to candidates who have some practical experience
in meat microbiology.
A certain amount of travelling will be involved, visiting plants in the U.K.
Applications giving full personal and career details' should be sent to the
Staff Manager (AD 5849), 14 West Smithfield, London EC1A 9JN.
(1493)
BIOCHEMIST
A Research Assistant is required to investigate the microbial metabolism
of detergent products, in particular, the metabolic pathways used during
their biodegradation in the environment.
This post would be suitable for a recent graduate Biochemist with an
interest in mircobiology and some concern for environmental issues.
The Laboratory is situated on the Wirral Peninsula, a few miles from the
sea and convenient for Chester, Liverpool and North Wales.
Interested persons should write for an Application Form quoting reference
No. PS467AM to Personnel Department, Unilever Research, Port Sunlight
Laboratory, Port Sunlight, Merseyside L62 4XN.
(1532)
Micro-Biologist
A Micro-Biologist, graduate level, to join a small
team servicing international product research on
proprietary medicines, food, toiletry and cosmetic
products. Vitamin bio-assay experience an advantage.
Good opportunities for a young graduate with
management capacity.
Benefits include free pension and life assurance .
schemes.
Please apply in writing quoting qualifications and
brief career details to :
Personnel Manager
Beecham Products Research Dept.,
Randalls Road, Leatherhead, Surrey.
B
Beecham Products
(1483)
UNIVERSITY OF OTAGO
DUNEDIN, NEW ZEALAND
CHAIR OF HUMAN NUTRITION
The University Council invites applications from
medical or science graduates for the foundation
Chair of Human Nutrition.
Applications will be welcomed from candidates
with interests in any field of Human Nutrition.
Professorial salaries are fixed at various points
within the present range of NZ$15,111 per annum
to NZ$19.233 per annum, and are regularly re-
viewed.
Further particulars are available from the Secre-
tary-General. Association of Commonwealth Uni-
versities (Appts). 36 Gordon Square, London
WCIH OPF, or from the Registrar of the Univer-
sity.
Applications close in New Zealand and London
on October 31, 1974.
(1488)
ACADEMIC POSITION
One immediate opening for a Ph.D. chemist,
preferably in the area of analytical chemistry with
research interest in biomedical applications. Duties
include graduate and undergraduate teaching and
establishing a vigorous research programme. Rank
open. This position has been created through a
grant from the Minority Schools Biomedical Sup-
port Programme of the National Institutes of
Health. Successful applicants will be required to
interact strongly with minority students in this pro-
gramme. Send résumé to: Dr Ralph G. Wilkins,
Head, Department of Chemistry, Box 3C, New
Mexico State University. Las Cruces, New Mexico
88003. New Mexico State University is an equal
opportunity and affirmative action employer.
(1489)
UNIVERSITY OF LEEDS
DEPARTMENT OF FOOD
AND LEATHER SCIENCE .
Applications are invited for the post of
RESEARCH ASSISTANT to take part in a
basic research programme on the relation
between the properties, structure and method
of manufacture of leather.
An interest in materials science, especially
in mechanical behaviour, with an appropriate
degree qualification is required but no pre-
vious experience with leather is necessary.
The post is for one year in the first instance
renewable for a further two years. Salary
within the range £1.638 to £2,007. Applica-
tions giving the' names of three referees
should be sent to Professor A. G. Ward,
Procter Department of Food and Leather
Science, University of Leeds. LS2 9JT by
October 25.
(1479)
UNIVERSITY OF SYDNEY
PROFESSOR/ASSOCIATE
PROFESSOR IN BEHAVIOURAL
AND SOCIAL SCIENCES
(In Relation to Medicine)
Applications are invited for the position of
Professor / Associate Professor in Behavioural and
Social Sciences (in relation to Medicine).
The
appointee will be responsible for the direction of
a new course, introduced in 1974, designed to give
medical undergraduates a substantial orientation to
the behavioural and social sciences as they relate
to health, disease and medical practice.
The salary for. a Professor is SA19,614 per
annum and for an Associate Professor is $A16.389
per annum.
A statement of Conditions of Appointment and
Information for Candidates may be obtained either
from the Secretary-General. Association of Com-
monwealth Universities (Appts). 36 Gordon Square.
London WC1H . OPF or from the Registrar, Uni-
versity of Sydney. N.S.W. 2006. with whom appli-
cations close on November 22, 1974.
(1487)
TRANSLATORS
. to prepare English summaries of
German and French texts required
full time by Woolcott & Company,
93 Chancery Lane, London . WC2.
Salary £2,500 p.a. plus according to
age and experience
(1480)
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Nature
PLANT BREEDING INSTITUTE
CAMBRIDGE
CONTROLLED ENVIRONMENT
ENGINEER
. An engineer is required to take charge of
the running and maintenance of controlled
environment installations at the Institute. He
will also be required to operate the installa-
tions to maintain the environmental regimes
needed for crop research. He will be respon-
sible to the Head of the Physiology Depart-
ment.
Candidates should have experience of
environmental engineering, air conditioning,
and electrical circuits. They must be able
to keep detailed records of maintenance
schedules and environmental data.
Candidates should have a degree in a
scientific or engineering subject; degree-
standard membership of a professional institu-
tion; a Higher National Certificate or Higher
National Diploma: or qualifications equivalent
to at least H.N.C.
The post will be filled at the grade of
Scientific Officer (£1,592 to £2,675 per annum)
or Higher Scientific Officer (£2.461 to £3,371
per annum). ,Grading and starting salary will
be determined in relation to qualifications and
experience. Non-contributory pension scheme.
Candidate for the higher grade should have
had at least five years appropriate experience
since qualifying.
Further particulars may be obtained from
the Assistant Secretary (Establishment). Plant
Breeding Institute. Maris Lane, . Trumpington,
Cambridge CB2 2LQ. Applications including
curriculum vitae and the names and addresses
of three referees should reach him not later
than October 31, 1974.
(1506)
AUSTRALIAN NATIONAL
UNIVERSITY
FACULTY OF SCIENCE
CHAIR OF FORESTRY
The Chair of Forestry will become vacant in
late October 1974 when Professor J. D. Ovington,
the foundation Professor, resigns in order to take
up a position with the Australian Government.
The appointee would for an initial period of
some years be Head of the Department. The Uni-
versity would regard as essential the appointment
of a person who would preserve the current em-
phasis on the multi-disciplinary nature of the
Department's interests and who would maintain
rapport with the forest services and with industry.
The salary for professor is $A19,614 p.a.
OTHER CONDITIONS-Tenure of Professor is
to retiring age (65 years).
Reasonable travel expenses are paid and assist-
ance with housing is provided for an appointee
from outside Canberra. Superannuation is on the
F.S.S. U. pattern with supplementary benefits.
The University reserves the right not to make
an appointment or to make an appointment by in-
vitation at any time.
Prospective applicants should write to the Asso-
ciation of Commonwealth Universities (Appts),
36 Gordon Square, London WC1H OPF for further
particuars before appying.
Closing Date: November 18, 1974.
(1486)
PALEOSERVICES LTD.
requires a
PALYNOLOGIST
with Tertiary and Mesozoic interest (two to
three years postgraduate experience desirable).
Apply to: Paleoservices Ltd., Paramount
Industrial Estate. Sandown Road. Watford,
Herts. WD2 4XA. Tel: Watford 25678.
(1503)
· CHELSEA COLLEGE
University of London
BIOPHYSICS LABORATORY
GRADE 3 TECHNICIAN
required to assist in research and teaching activi-
ties within the Biophysics Section of the Basic
Medical Sciences Group. Main duties will be as-
sociated with research into photobiological pro-
cesses and artificial membrane systems. Experience
of biophysical and/or
biochemical techniques
would be an advantage. Salary Scale: £1.878 to
£2,148 (including £228 London Allowance) plus
Threshold Payments. Enquiries and application
forms from Dr W. P. Williams, Biophysics Labo-
ratory N. Chelsea College. Manresa Road, Lon-
don. SW3 6LX (01-352 6421).
(1484)
SCOPE/ICSU
MONITORING CENTRE
CHELSEA COLLEGE
UNIVERSITY OF LONDON
SCIENTIFIC
PROGRAMME OFFICER
Applications are invited for a physical or biological
scientist with substantial post-graduate experience (5-8 years)
who is prepared to accept administrative, executive and
liaison responsibilities within an international research
. group. The group is concerned with Environmental Monitor-
ing and Assessment and forms part of the activities of the
Scientific Committee on Problems of the. Environment
(International Council of Scientific Unions).
The appointment is for 1 year renewable for up to 2 years
and will be within the University of London at the upper
end of the Lecturer Scale or within the Senior Lecturer/
Reader level (£2,580 to £5,976 plus £213 London allowance),
depending upon age, qualifications and experience. Duties
will include liaison with international scientific groups, the
organisation of scientific workshops and symposia, co-
ordination of the research group activities and provision of
administrative assistance to the director of the group.
Fluency in French would be an advantage.
Further particulars of the above post and conditions of
appointment can be obtained on request from the Appoint-
ments Officer, Chelsea College, Manresa Road, London
SW3 6LX, to whom applications including names of two
referees should be sent by November 11.
(1481)
UNIVERSITY OF MALAYA
FACULTY OF ENGINEERING
Applications are invited for the posts of
LECTURER/ASSISTANT LECTURER
in the fields listed below in the Faculty of En-
gineering. Candidates should have at least a good
Honours degree in Engineering, Mathematics or
Physics, teaching at degree level would be an
advantage.
Subject to suitable academic qualifications and
experience, preference will be given to candidates
who are proficient in Bahasa Malaysia (Malay)
but this requirement is not applicable to overseas
staff. If selected, overseas candidates may be
offered a short-term contract subject to the possi-
bility of renewal by mutual agreement.
Salary scales (approx. stg. equivs.): Lecturer
£1.524 to £2,441; Assistant Lecturer £1,330 to
£1,460 p.a. In addition the following allowances
are payable: Variable Allowance £243 min. £649
max. p.a. calculated at 35% of basic salary. Sup-
plementary Housing Allowance £503
p.a. and
medical benefits.
Further particulars and application forms are
obtainable from the Association of Commonwealth
Universities (Appts), 36 Gordon Square, London
WCIH 0PF.
The closing date for the receipt of applications
is November 14, 1974.
(1512)
UNIVERSITY OF THE
WITWATERSRAND
Johannesburg, South Africa
SENIOR LECTURER/LECTURER
IN GENETICS
Applications are invited for appointment to the
above post in the newly established Department of
Genetics. The ability to teach Population Genetics
and a research interest in Molecular Genetics will
be a recommendation.
The salary attached to the post presently falls
within the following ranges:
Senior Lecturer
R7,245 to R9,315
Lecturer
R5,520 to R7,935
Improved salary scales are to be introduced
shortly. Benefits include an annual bonus, pension
and medical aid facilities, and a housing subsidy
(where applicable).
Intending applicants should obtain the informa-
tion sheet relating to this post from the Registrar,
University of the Witwatersrand, Jan Smuts Avenue,
Johannesburg. South Africa, with whom applica-
tions should be lodged not later than November
20, 1974. U.K. applicants may obtain the informa-
tion sheet from the London Representative. Uni-
versity of the Witwatersrand, 278 High Holborn,
London, W.C.1 to whom a copy of the application
should be sent.
(1514)
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ivaure vol. 231 Octover 18 1974
THE UNIVERSITY OF NEW SOUTH WALES
WOLLONGONG UNIVERSITY COLLEGE
To become the UNIVERSITY OF WOLLONGONG
1st January 1975
PROJECT SCIENTIST
Applications are invited from Ph.Ds. with a background in Quantum
Chemistry for the position of Project Scientist in the Department
of Chemistry. The successful applicant will be expected to work
for Dr P. G. Burton on a project involving studies of the influence
of the environment on the chemical reactivity in molecules in con-
densed media. New theoretical methods of electronic structure
computation will be used and a detailed working experience with
large Flortran programmes will be an advantage. Of particular
interest are transition metal systems and the study may impinge
on areas of inorganic photochemistry and of functions of metallo-
proteins.
Appointment in the first instance will be for up to 14 months
beginning immediately with initial salary Aus.$9,002.
Further details of the project are obtainable from Dr Burton and
applications should be made immediately to the College Bursar,
Wollongong University College, P.O. Box 1144, Wollongong, N.S.W.
2500, Australia by November 22, 1974.
Applicants should request two referees to write immediately to
the College Bursar.
( 1502)
Recently Qualified
Veterinarian
... to join a team of research scientists involved in the screening and
identification of new compounds active in the field of growth promotion,
feed utilisation and parasitology. He will be responsible for a section of
research work in the growth promotion field and will also have general
responsibility for the health of both research and farm animals.
The Thurgarton Research Station is located in rural surroundings between
Nottingham and Southwell and has excellent field and laboratory facilities
alongside the Boots farms which provide opportunity for extensive field
testing. Later stages of research also involve trials throughout the U.K. and
in overseas locations.
Terms and conditions of employment are attractive and include profit
sharing and help with re-location. There is ample scope for career
development in both the scientific and commercial sectors.
Further information on the position involved can be provided on request.
Interested applicants should apply with details of their career to date to :
J. F. Pattison, Employment Manager,
Boots
The Boots Company Ltd.,
Station Street, Nottingham NG23AA.
Tel: Nottingham 56255 Ext. 243.
(1510)
NATIONAL INSTITUTE OF
TECHNOLOGY, MALAYSIA
Kuala Lumpur
Appointments of
LECTURERS/SENIOR LECTURERS
in the following disciplines:
Engineering
(a) Mechanical
(b) Electrical
(c) Civil
Surveying
(a) Photogrammetry
(b) Geodesy
(c) Cartography
(d) Estate Management
(e) Hydrographical
Applications are invited for the above appoint-
ments tenable as' from May 1, 1975. Applicants
for the above appointments should possess high
academic qualifications and have
considerable
teaching experience in the academic disciplines
concerned and for Engineering, in the fields as
follows:
Mechanical Engineering
Production Engineering: machine tools; metro-
logy Thermodynamics: Heat and Mass transfer
Control Engineering: Mechanical bias Auto-
motive Engineering: Structures and dynamics
Mechanics of
Materials
and
metallurgy;
Mechanical Design
Electrical Engineering
High Voltage; control;
electrical machines:
computer; communications: transmission and
broadcasting; Industrial Electronics
Civil Engineering
Structural Design and Analysis
Soil and Foundation Engineering
Hydraulics and Hydrology
Environmental Engineering
SALARY: Senior Lecturer-(M)$1,405 by
55
to 1.735/RP/1.800 (Basic). Lecturer-(M)$940 by
45 to 1.210/1.255 by 50 to 1.505 (Basic).
Points of entry will depend on qualifications
and experience.
OTHER PRIVILEGES will include variable
allowance calculated at 35% of basic salary subject
to certain maxima, superannuation benefits on the
basis of contribution of 5% of basic salary by
appointee and 15% of basic salary by the Institute,
leave, medical benefits, housing allowance at
(M)$310 per mensem, and, in the case of appoin-
tees on overseas terms, air passages for the
appointee. his wife and dependent children on
appointment and on completion of contract. Can-
didates, if appointed, will be on contract for 36
months in the first instance.
All applications must be made in writing giving
full personal details and should also include the
names of two referees.
Further information on the terms and conditions
of service may be sought from the Association of
Commonwealth Universities (Appts), 36 Gordon
Square, London WCIH OPF, or obtained directly
from:
The Registrar.
National Institute of Technology.
Jalan Gurney,
Kuala Lumpur (15-02).
. Malaysia.
(1494)
THE MIDLAND CENTRE FOR
NEUROSURGERY AND
NEUROLOGY
Holly Lane, Smethwick.
Warley, Worcs. B67 7JX
DEPARTMENT OF NEUROPATHOLOGY
TECHNICIAN IN HISTOLOGY
Applications are invited for the post
of
Technician in Histology. The
vacancy offers excellent opportunities
for further study and post H.N.C./
/ A.I.M.L.T. experience in all aspects
of histology and is particularly suit-
able for an Associate studying for the
Special examination in histological
technique. The Department is well
equipped and a wide range of tech-
niques are utilised including histo-
chemistry and electron microscopy.
Candidates should possess A.I.M.L.T.
or H.N.C. but consideration will be
given to holders of O.N.C. Labora-
tory may be viewed by appointment.
Applications with the names and
addresses of 2 referees to the Hos-
pital Secretary at the above address.
(1529)
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Nature vol. 201 October 10 1914
Young Graduates
for Research
In the Royal Dutch/Shell Exploration and Production
Laboratory at Rijswijk, near The Hague, research is
carried out in connection with the exploration for and
recovery of oil and natural gas. Research work is directed
towards improving techniques and methods applied in
exploration, drilling and production, which calls for
active co-operation with operating companies in solving
their field problems. Work is done in project teams
which are often made up of people of different disci-
plines.
Due to increased activity we need additional scientific
staff, male or female, with preferably a PhD, to fill
vacancies in the following fields :.
General Research and Mathematics
Fundamental research in the rheology of rocks and
granular materials, interfacial chemistry of liquid/liquid
and liquid/solid contacts. Development of mathematical
models for field development, particularly in deep water.
Mathematical and computational service projects in
support of research in other departments.
We require a classical experimental physicist, preferably
in the field of solid and fluid mechanics, a colloid
chemist and an applied mathematician with a back-
ground in operations research.
Geophysics
Developing and improving techniques for seismic ex-
ploration. Signal processing with the aid of computers,
and interpretation of results. Study. of the relation
between seismic reflection and the actual geology of the
area.
We require a physicist with a keen interest in digital
processing and filtering techniques. Acoustics, inform-
ation theory, signal theory and electronics are relevant
fields of interest.
Petrophysics
Improvement and innovation of physical formation
evaluation techniques and their interpretation in terms
of formation characteristics, such as lithology, grain and
pore-size distribution, pore fillers, cementation etc.
Establishment of relations between production qualities.
of the reservoir and formation characteristics.
A good background in mathematical physics, both
classical and modern, together with some experimental
bias is required.
Production Geology
Geological studies of oil and gas reservoirs e.g. sedi-
mentology, diagenesis, depositional tectonics. Analysis of
petrophysical, mineralogical and sedimentological data
to translate these into measurable parameters that will
lead to better evaluation of reservoir character and dis-
tribution.
We require a general geologist and a geologist with ex-
perience in the physical chemistry of organic shales.
Production Chemistry
Studying problems of a chemical and physico-chemical
nature encountered in drilling and completion of oil,
water and gas wells. In particular, investigations of
chemical methods of controlling sand influx into oil and
gas wells, laboratory investigations and interpretation of .
results. Designing and organising field tests in co-
operation with drilling and production experts. Study of
technological problems in commissioning wells and in
oil and gas production.
Chemists or chemical engineers are required who have a
sound knowledge of physical and inorganic chemistry
and are capable of making an engineering approach to
field problem solving. A good understanding of the
rheology of non-Newtonian fluids would be an advan-
tage.
Oil and Gas Reservoir Engineering
Improvement of recovery from oil and gas reservoirs,
involving fluid flow in porous media, interfacial phe-
nomena in gas/liquid/solid systems, heat and mass
transfer, thermodynamics of gas/liquid systems. Studies
are experimental and/or are made with the aid of
numerical simulators.
Both physicists with an interest in mathematical models
and mathematicians to work on the development and
extension of such models are required.
We offer training opportunities within and outside your
professional field, flexible working hours to meet your
personal preference and the possibility of overseas
assignments.
Travelling and other reasonable expenses will be paid
for interviews in London and The Hague. Financial
assistance will be given for the removal of household and
personal effects to The Netherlands.
Please write, with full -
details, to Shell
International Petroleum
Company Limited,
· Recruitment Division (A),
PNEL/31, Shell Centre,
London SE1 7NA.
Shell
(1525)
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XVIII
Nature Vol. 251 October 18 1974
OXFORD POLYTECHNIC
Applications are invited for the post of
Lecturer II in Mammalian Physiology
The successful applicant will be expected to
contribute to the teaching of Mammalian
Physiology in the Modular (CNAA) degree
course and HNC courses. An ability to teach the
Cell Physiology and/or Pharmaecology could be
an advantage.
Further details and application forms from
Dr R. E. Yorke,
Department of Science,
Oxford Polytechnic,
Oxford. OX3 OBP.
(1467)
ANGLIAN WATER AUTHORITY
WELLAND AND NENE RIVER DIVISION
BIOLOGIST
Salary: Grade AP IV/V (£2,394 to £3,018)
There is a vacancy for a Biologist within the existing establishment
of the Welland and Nene River Division of the Anglian Water
Authority. This appointment will be based initially upon the
existing laboratory at Oundle, and a major responsibility of the
post will be water quality control for the Empingham Pumped
Storage Scheme-involving intensive hydrobiological studies of the
reservoir and supply rivers.
The successful applicant will be a university graduate and/or a
member of the Institute of Biology and previous experience in this
field is desirable. An essential car user allowance will be paid.
Applications giving details of age, qualifications, training and
experience, together with names and addresses of two referees
should reach the Divisional Manager, Welland and Nene River
Division, Anglian Water Authority, North Street, Oundle,
Peterborough, by November 11, 1974.
(1537)
CHIEF TECHNICIAN
Applications are invited for the post of
Chief Technician to be in charge of the
serological reagents and standards section.
An extensive knowledge of all aspects of
blood group serology
is essential and
experience of automation would be an ad-
vantage. Salary on Whitley Council Scales.
Applications and enquiries to the ' Director,
North London Blood Transfusion Centre,
Deansbrook Road, Edgware, Middlesex.
(1538)
POSTDOCTORAL RESEARCH
Mass Spectrometry ion source research on ionisa-
tion of non-volatile organic and biochemical com-
pounds from a liquid surface. Research includes
instrumentation, parametric studies and applica-
tions development. Related experience in mass
spectrometry, analytical instrumentation, AEI MS-9,
mini-computers, and/or MS of organic or bio-
chemical compounds considered important.' Salary
$11,000 to $11.500. Two-year appointment possible.
Contact C. A. Evans, Jr., Materials Research
Laboratory. University of Illinois, Urbana, Illinois
61801 U.S.A.
(1521)
IMPERIAL COLLEGE
Postdoctoral Research Assistant
in Precambrian Stratigraphy
Applications are invited for the above post which
is tenable in the Geology Department under the
direction of Dr M. D. Muir and is supported by
. the metal mining industry. The work will involve
the application of geological and micropalaconto-
logical correlation methods to mid-precambrian
stratigraphy and mineral exploration.
Candidates should hold a Ph.D. or have appro-
priate industrial or research experience in earth
or life sciences.
The post (starting salary £2,118 per annum plus
£213 London allowance. with F.S.S.U.) is tenable
for up to three years. Threshold payments at the
authorised rate apply to this post.
Further details may be obtained from Dr M. D.
Muir, Department of Geology. Royal School of
Mines, Prince Consort Road. London SW7. Appli-
cations with the names of two referees should
reach Dr Muir at the above address not later than
November 1, 1974.
(1495)
IMPERIAL COLLEGE
DEPARTMENT OF CHEMISTRY
Applications invited for S.R.C. Postdoctoral Re-
search Assistantship with Dr W. P. Griffith on
interaction of Osmium tetroxide with opel and
actual biological tissue and membrane materials.
Tenable for one year. Salary up to £2.460 includ-
ing London allowance, plus threshold payments
and with F.S.S.U. Commencing preferably on
December 13, 1974. Previous experience in pre-
parative organic or organometallic chemistry or
biochemistry is desirable. Applications (with curri-
culum vitae and names of two referees) to Dr
W. P. Griffith, Chemistry Department, Imperial
College, London SW7 2AY.
(1465)
UNIVERSITY COLLEGE
GALWAY
DEPARTMENT OF MICROBIOLOGY
POSTGRADUATE
RESEARCH ASSISTANT
Applications are invited for the
post of postgraduate research assist-
ant to participate in a Euratom
sponsored project on the effects of
radiation on blue-green algae. Suitable
candidates should be qualified in
Microbiology or a related field and
may register for a higher degree. Full
particulars from Dr J. A. Houghton,
Department of Microbiology.
(1517)
TECHNICIAN
required in the Department of Plant Biology and
Microbiology to assist in research and teaching in
the Microbial Genetics laboratories. Previous ex-
perience together with I.S.T. or C & G Science
Laboratory
Technicians
Advanced
Certificate.
H.N.C. or similar qualification. The post will be
at Grade 5 salary scale £2.007 by £75 to £2,382
p.a. but if candidate does not have the appropriate
experience and qualifications appointment would
be on the Grade 3 salary scale £1.650 by £54 to
£1.920 p.a. In both cases in addition £228 p.a.
London Weighting and current threshold payment.
Five day week. Five weeks (in total) annual leave.
Pension Scheme. Letters only to Assistant Secretary
(Establishment) E/PB. Queen Mary College. Mile
End Road. London E1 4NS, stating age. qualifi-
cations and experience.
(1468)
NATIONAL INSTITUTE FOR
RESEARCH IN DAIRYING
(University of Reading)
A Scientific Officer is required in the Chemical
Microbiology Department to work on biological
problems
connected with milk processing.
A
candidate with ambition is needed who prefer-
ably also has an interest in physical or colloid
chemistry.
within the scale £1,592 to £2,675 p.a. The post
Minimum qualification is H.N.C. or equivalent.
Salary according to qualifications and experience
is .pensionable.
Apply on forms obtainable' from Secretary,
N.I.R.D., Shinfield, Reading RG2 9AT. Quote
reference 74/35.
(1498)
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xix
Government of Malawi
Ministry of Agriculture and Natural Resources
Fisheries
Officer
Salary up to £4,446 + Tax Free Gratuity
To be responsible for the control and administration of a Fisheries Area in
Malawi. The work will involve the management of the fresh water fisheries
development and extension programme; mechanised fishing; fish marketing;
fisheries training; fish farming and boat building.
Candidates, aged 25-50, should hold a degree in Biology, Fisheries Management
or related natural science subject, preferably with some postgraduate training
or experience in Fisheries work. They should be able to plan and execute
development programmes and be competent to handle small craft. They must
be capable of controlling staff. Some administration experience would be an
advantage.
Generous paid leave with free passages and baggage allowance. Education
allowances and subsidised housing. Loan for the purchase of a car. Free
medical attention.
Please apply to MALAWI BUYING AND TRADE AGENTS, Recruitment
Section, c/o Berners Hotel, Berners Street, London WIA 3BE, for application
form and further details quoting reference 793/D.
(1535)
Opportunities for
Graduate Organic Chemists
If you have a degree in Chemistry, we can offer you an
opportunity to work as a member of an interdisciplinary
team attacking challenging problems in pharmaceutical
research. The work is concerned with the synthesis of organic
compounds with novel structures for evaluation
as
therapeutics by our biologists.
Our laboratories at Welwyn Garden City provide excellent
accommodation and facilities for innovative research and
you would be encouraged to keep abreast of current
developments by attending seminars and making use of our
library and information services.
Excellent conditions of service and fringe benefits are offered,
including a generous contributory pension scheme and, where
appropriate, assistance with relocation would be available.
If you would like to know more about these posts and the
Company generally, please write, quoting reference R/ART
to the Welwyn Personnel Manager.
ROCHE
Roche Products Limited
Welwyn Garden City Hertfordshire AL7 3AY
(1528)
HEAD OF
PHARMACOLOGICAL
DEPARTMENT
interested in CARDIO-VASCULAR
research is wanted by large European
pharmaceutical . company
for
its
Brussels research Centre.
Applicants should be graduate in
pharmacology, preferably M.D. and
member. of the British Pharmaco-
logical Society. The successful. appli-
cant will have the responsibility of
conducting : cardiovascular research
on new substances with the help of
well trained technicians. Possibility to
extend the responsibilities to research
in general pharmacology.
Our modern and well equipped
research laboratories are located in
the suburbs of Brussels. Domiciliation
in Brussels is required. Knowledge of .
French is highly desirable.
A good starting salary will be
offered and reviewed regularly.
For application, please send .com-
plete résumé to: Dr . F. Binon,
LABAZ, 1 avenue de Béjar, 1120;
Brussels, Belgium.
(1520)
THE MIDDLESEX HOSPITAL
MEDICAL SCHOOL
(University of London)
COURTAULD INSTITUTE OF
BIOCHEMISTRY
Clinical Biochemist (Research Assistant Grade)
required in the Clinical Chemistry Laboratory. The
post is suitable for a recent graduate to train in
Clinical Chemistry . with a view to taking the
M.C.B. Salary according to age and experience
within the range £1,560 to £2,265 (inclusive of
London allowance) plus
superannuation
with
F.S.S.U. Applications, including a curriculum vitae
and the names and addresses of two referees to
the Director, Courtauld Institute of Biochemistry,
The Middlesex Hospital Medical School, London
WIP SPR; by November 15, 1974.
(1499)
KENNEDY INSTITUTE OF
RHEUMATOLOGY
CHIEF TECHNICIAN
required for the Division of Experimental Path-
ology. The work involved is mainly of a research
nature and histology autoradiography, and electron
. miscroscopy services are provided for other divi-
sions in the Institute.
This is a well equipped, modern laboratory
investigating lymphoid tissue function. All . the
usual techniques required to study cells and tissues
are employed. A suitable candidate would be ex-
pected to have the relevant background experience
as well as to be prepared to take on administrative
responsibilities. Written applications giving full
curriculum vitae and naming two referees to:
Laboratory Superintendent, Kennedy Institute of
Rheumatology, Bute Gardens, London W6 7DW.
(X1507)
UNIVERSITY OF ABERDEEN
SENIOR LECTURESHIP IN
MEDICAL PHYSICS
Applications are invited from experienced radio-
isotope hospital physicists to be. responsible for
team of physicists, technicians and ancillary staff
engaged in wide range of research activities and
providing nuclear medicine service to the Grampian
'Area Hospital Board. The sucecssful candidate
will also be responsible for team providing D.M.R.
physics course, and join in M.Sc. (Medical Physics)
teaching. Salary on scale £4,707 to £5,844 with
initial placing according to qualifications and
experience.
Further particulars from The Secretary, The
University, Aberdeen, with whom applications (2
copies) should be lodged by November 9, 1974.
(1509)
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18 1974
-- --- -
Ministry of Agriculture,
Fisheries and Food
Food Science Division, London
Food Scientists/
Chemists
Advise on manufacture and composition
of foods and beverages i Liaise with Govern-
ment departments, industry and research
organisations
Participate in current
awareness studies.
Ist/2nd hons degree or equivalent in food
science/technology, chemistry or a related science
4 years' postgraduate experience in food industry
or research
Age under 32 [ Appointment as
Senior Scientific Officer (over £3700 - around
£5000)
Ref: SB/II/AE.
Food Chemists/
Biochemists/
Analytical
Chemists
:
Advise on manufacture and composition
of foods, particularly the functions and uses
of food additives and processing aids in food
manufacturing processes and their specifica-
tions and analytical determination.
] Ist/2nd hons degree or equivalent in chemistry
or biochemistry
4 years' experience in food in-
dustry or research [ Knowledge of metals and
other contaminates of food, together with an
understanding of relationships between chemical
. constitution and biological activity an advantage
Age under 32 [] Appointment as Senior Scientific
Officer (around £3550 - over {4800) {
Ref:
SB/12/AE.
Application forms (for return by 7 November
1974), from Civil Service Commission, Alencon
Link, Basingstoke, Hants RG21 IJB, telephone
Basingstoke 29222 ext. 500 or, (for 24 hour
answering service), London 01-839 1992.
Ministry of Agriculture,
Fisheries and Food
Royal Botanic Gardens, Kew
Botanists
(Ref: SB/11/AH). To work in Mycological
Section on taxonomy of Ascomycetes
Assist in curating Herbarium collection
..
Answer routine enquiries.
Ist/2nd hons degree in Botany [ Age under 32
Appointment as Senior Scientific. Officer
(over £3400 - around £4700), Higher Scientific
Officer ({2700 - over {3600), or Scientific
Officer (around £1850 - over {2900), depending
on age, qualifications and experience.
(Ref: SB/12/AH). In Herbarium and
library " Scan current literature for ref-
erences to be included in indexes of plant
names and bibliographies | Assist in routine
naming and curation in the Herbarium
.
Undertake taxonomic research in the
Herbarium.
7 Degree, HNC or equivalent in Botany []
Interest in literature on natural history and
geography
Age under 27 [ Appointment as
Scientific Officer (around £1850 - over {2900).
Application forms (for return by 6 November
1974), from Civil Service Commission, Alencon
Link, Basingstoke, Hants RG21 IJB, telephone
Basingstoke 29222 ext. 500 or, (for 24 hour
answering service), London 01-839 1992.
Ministry of Agriculture,
Fisheries and Food
Fish Diseases Laboratory,
Weymouth
Pathologist/
Histopathologist
Undertake research and diagnostic work
into diseases of marine and freshwater fish
in fish farms # Develop knowledge in little
known field of biology.
,
1.Ist/2nd hons degree in appropriate subject, or
veterinary qualification
Postgraduate exper-
ience in pathology and histopathology essential []
Age under 32
Appointment as Senior Scientific
Officer (£3300 -around £4600) [
Ref: SB/18/AD.
] Application forms (for return by 8 November
1974), from Civil Service Commission, Alencon
Link, Basingstoke, Hants RG21 IJB, telephone
Basingstoke 29222 ext. 500 or, (for 24 hour
answering service), London 01-839 1992.
Science
group
CIVIL SERVICE
.
(1541)
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.
1
:
1
i
1
.
1

xxi
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Nature
KING'S COLLEGE HOSPITAL
MEDICAL SCHOOL
(University of London)
DENMARK HILL, LONDON SE5 8RX
Graduate required to work on the development
of radioimmunoassay systems for the determination
of steroids, prostaglandins and their synthetic
analogues. Applications to the Secretary of the
Medical School not later than November 1, 1974.
(1500)
BRUNEL UNIVERSITY
PHYSICS DEPARTMENT
RESEARCH ASSISTANT
(Salary scale £1,539 to £1,983 per annum. A
London Allowance of £213 is payable).
Applications are invited for the above post
which is connected with a research project for the
study' of Thin Film Thermocouples and .Heat
Transfer gauges for use in aerodynamic investiga-
tions. Vacuum evaporation and sputtering tech-
niques will be required. The appointment is ini-
tially for one year, but may be extended.
Postcard for. application form and further
details from the Assistant Secretary (Establishment),
Brunel University. Uxbridge, Middlesex or tele-
phone UXBRIDGE 37188 extension 49. Closing
date: November 1, 1974.
(1511)
UNIVERSITY OF THE
WITWATERSRAND
Johannesburg, South Africa
FACULTY OF MEDICINE AND
SCHOOL OF GENETICS
CHAIR IN HUMAN GENETICS
This is a new post in the Faculty of Medicine
as well as in the School of Genetics. The Chair
is not attached to a specific department, though,
for administrative purposes, the appointee will be
allocated to one of the Medical School Depart-
ments, in accordance with the sucecssful applicant's
background and wishes, and the needs of the
University. Although this arrangement will obtain
initially, it is envisaged that ultimately a separate
Division of Human Genetics will be established
within the existing School of Genetics. The suc-
cessful applicant should be medically qualified. He
will be expected to teach medical students, science
and honours students majoring in subjects taught
at the Medical School, and general science students
at the Milner Park campus. Experience in heredity
counselling will be an advantage, and the encum-
bent will be expected to co-ordinate existing coun-
selling services.
Salary will be determined according to qualifica-
tions and experience and will be either R13,800
(fixed) or within the range R11,040 to R12,420.
Interested persons should obtain a copy of the
information sheet relating to this post from the
Registrar, University of the Witwatersrand, Jan
Smuts Avenue, Johannesburg, South Africa with
whom applications should be lodged not later than
November 30, 1974.
U.K. Applicants may obtain the information
sheet from the London Representative, University
of the Witwatersrand, 278 High Holborn, London,
W.C.1 to whom a copy of the application should
be sent.
(1513)
MEDICAL RESEARCH COUNCIL
Demyelinating Diseases Unit
SENIOR
VIROLOGIST
To join a team working on an inter-
disciplinary approach to the study of multiple
sclerosis and related neurological conditions.
Applicants should be age 27 or over, and
have wide postgraduate experience in virus
"rescue" methodology and an interest in
neuropathic viruses especially measles, dis-
temper, para-influenza, herpes and visna.
The appointment will be for not less than
5 years initially.
Applications with curriculum vitae, list of
publications and the names of two referees,
should be sent to The Director (Dr H. M.
Wisniewski), MRC Demyelinating Diseases
Unit, Newcastle General Hospital, Westgate
Road, Newcastle upon Tyne NE4 6BE.
(AK 1545)
Ministry of Agriculture, Fisheries and Food
Fisheries Laboratory, Burnham-on-Crouch
Chemist
In Marine Environment Protection Division " Develop and
improve analytical techniques for petroleum hydrocarbons
.
Examine samples of water, sediment, fish and other marine organ-
isms Support laboratory and field experiments and monitor
results.
Degree, HNC or equivalent in chemistry
Experience of GLC analysis
essential and of oil analysis and Mass Spectrometry an advantage
Age
under 27
Appointment as Scientific Officer (around £1750 - over {2800)
Ref: SB/15/AD.
Effluent Control Officer
Inspect waste disposal operations " Liaise with industry and other
bodies on waste disposal Assess physical and other effects of
wastes dumped at sea.
HND, degree or equivalent professional qualification in relevant subject
Experience of water or effluent quality control desirable
The appointee
must be prepared to spend periods away from station and will be expected to
drive
Age under 30
Appointment as Higher Scientific Officer (£2600 -
over £3500)
Ref: SB/17/AD.
Ministry of Agriculture, Fisheries and Food
Fisheries Laboratories, Burnham-on-Crouch
Sedimentologist
In Marine Environment Protection Division Examine samples of
marine sediments from estuarine, coastal and offshore areas | Carry
out sediment surveys with an ecological team Develop and improve
methods of sediment analysis I Advise on sedimentological aspects
of fauna distribution, waste disposal and gravel extraction.
] Degree, or equivalent in geology or related subject
Interest or experience
in marine sedimentology desirable
Some biological training an advantage
Age under 27 [
] Appointment as Scientific Officer (around £1750 - over
£2800) [] Ref: SB/16/AD.
Application forms (for return by 8 November 1974), from Civil Service
Commission, Alencon Link, Basingstoke, Hants RG21 IJB, telephone
Basingstoke 29222 ext. 500 or, (for 24 hour answering service), London
OI-839 1992.
Science
jįgroup
CIVIL SERVICE
(1541)
UNIVERSITY OF CAMBRIDGE
DEPARTMENT OF ZOOLOGY
A position exists immediately for a TECHNICAL
ASSISTANT in cell culture. Applicants should have
a first degree in a biological science, H.N.C. or
equivalent experience. Some experience in tissue
culture would be useful. The successful applicant
will take over the maintenance of tissue culture
facilities and will assist in research projects. The
post is funded by the Medical Research Council,
Salary £1,500 to £2,163 according to age and
experience.
Application forms can be obtained from Dr R. T.
Johnson, Department of Zoology, Downing Street.
Cambridge CB2 3EJ.
(1519)
UNIVERSITY OF OXFORD
PHYSIOLOGIST or BIOCHEMIST
Research assistant required for the investigation
of control of long- and short-term gonadal changes
in mammals. Techniques will include radioimmuno-
assay. Project aided by a grant from the Medical
Research Council. Post tenable for 3 years. Initial
salary up to £2,118 p.a. Applications including
curriculum vitae and names of two referees to be
sent to Dr J. R. Clarke, Department of Agricul-
tural Science, Oxford University, Parks Road,
Oxford OX1 3PF.
(1523)
RHODES UNIVERSITY
GRAHAMSTOWN
SOUTH AFRICA
Applications are invited for the post of
SENIOR LECTURER/LECTURER
IN PHARMACOLOGY
in the School of Pharmaceutical Science from
January 1, 1975, or as soon as possible thereafter.
The salary scales are: LECTURER-R4,800x300
to R6,900 per annum; SENIOR LECTURER-
R6,300x300 to R8,100 per annum (These scales
are presently under review). (Note: £1 =approxi-
mately R1.60.)
A supplement of 15% on the above scales and
a vacation savings bonus are also payable. The
successful applicant will become a member of the
University's pension and medical aid schemes.
Preference will be given to applicants who hold
a
Master's degree or higher qualification in
Pharmacology.
Full particulars relating to the post and staff
benefits together with application forms, may be
obtained from the Registrar, Rhodes University,.
Grahamstown 6140, South Africa, to whom com-
pleted applications with copies of recent testimonials
and a photograph, should be sent by November 16.
1974.
(1544)
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18 1974
nature
A vacancy will arise soon in the Washington office of Nature for the important
post of Assistant Editor responsible for supervising the refereeing of biological
manuscripts submitted to the Washington office. A period of training in the London
office under the supervision of the biological manuscripts editor will precede the
posting to Washington. The successful applicant may expect to be in Washington
for not less than two years.
This job is an outstanding opportunity for a recently qualified British or American
Ph.D who has a strong interest in ensuring the high quality of the biological papers
we publish.
Apply with curriculum vitae and the names of two referees to :-
The Editor,
Nature,
Macmillan Journals Ltd.,
4 Little Essex Street,
London, WC2R 3LF
by November 15th
Queensland Institute of Technology
QUOTE: V.95/74
HEAD OF
DEPARTMENT OF PHYSICS
The Department of Physics is one of five
academic departments within the School of
Applied Science. The Department is responsible
for :
(i) a Bachelor of Applied Science Degree
course in Physics,
(ii)
Associate Diploma courses in Diagnostic
and Therapeutic Radiography, and
(iii)
a Master of Applied Science degree course
in Medical Physics which is intended for
introduction in 1975.
Service physics subjects are taught to other
degree students within the School of Applied
Science and to Engineering School students.
There are currently. 20 full-time academic staff
within the Department of Physics.
Applicants should possess appropriate qualifica-
tions in physics and have relevant experience
within tertiary educational establishments. Appli-
cations would also be welcomed from persons
with experience in industrial or applied research
areas.
Salary : $16.389 per annum.
Further information can be obtained from the
Registrar, Queensland Institute of Technology,
P.O. Box 246, North Quay, 4000, with whom
applications close on October 26, 1974. Applica-
tions should contain the names of three referees.
(1539)
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Nature
UNIVERSITY OF THE
WEST INDIES - TRINIDAD
Applications are invited for:
(a) SENIOR LECTURESHIP
or
(b) LECTURESHIP IN
AGRICULTURAL ECONOMICS
in the Department of Agricultural Economics and
Farm Management. Preference will be given to a
person with particular interest in economic theory,
and agricultural policy and development. Salary
scales: (a) TT$18,108 to TT$26,730
p.a .;
TT$13,200 to TT$20,904 p.a. (£1 sterling=TT$4.8).
(b)
F.S.S.U. Unfurnished accommodation for maximum
of three years at 10% of salary; thereafter 20%
of salary payable in lieu of housing. Family
passages; study leave. Detailed applications (6
copies), including a curriculum vitae and naming
3 referees, should be sent by airmail as soon as
possible to the Secretary, University of the West
Indies, St Augustine, Trinidad. Further particulars
will be sent to all applicants.
(1516)
FOURAH BAY COLLEGE
UNIVERSITY OF SIERRA LEONE
Applications are invited for LECTURESHIP IN
GEOGRAPHY. Preference will be given to appli-
cants with teaching
and research interest
in
Geomorphology. Appointee will be expected to
teach both General and Honours Degree students
and conduct research. Salary scale (under review):
Le2.400 to Le4,740 p.a. (£1 sterling=Le2). The
British Government may supplement salary in range
£950 to £1.300 p.a. (sterling) for married appointee
or £350 to £700 p.a. (sterling) for single appointee
(normally free of all tax) and provide children's
education allowances and holiday visit passages.
F.S.S.U. Family passages; various allowances;
regular overseas leave. Detailed applications (2
copies), including a curriculum vitae and naming
3 referees, should be sent by airmail, not later
than November 18, 1974, to the Secretary, Uni-
versity of Sierra Leone, Private Mail Bag, Free-
town, Sierra Leone. Applicants resident in U.K.
should also send 1 copy to Inter-University Council,
90/91 Tottenham Court Road, London WIP ODT.
Further particulars may be obtained from either
address.
(1526)
ROYAL POSTGRADUATE
MEDICAL SCHOOL
POSTDOCTORAL CHEMIST
OR BIOCHEMIST
required for Department of Clinical Pharmacology
to undertake studies of drug metabolism in ani-
mals and man. Applicant should be conversant
with metabolite separation and identification tech-
niques; experience in the use of gas chromatography
and mass spectrometry is particularly desirable.
Post available for two years. Salary according
to age and experience, on scale up to £2,781 per
annum.
Applications to the Personnel Officer, R.P.M.S.,
Hammersmith Hospital, Du Cane Road, London
W12 OHS, quoting Ref. No. 20/487/N.
(1515)
UNIVERSITY OF READING
WOLFSON FOUNDATION
OILSEED RESEARCH PROJECT
Applications are invited for two Research Asso-
ciates to join the University's Research Group
working on the breeding. agronomy and chemistry
of temperate oilseed crops.
The programme is financed over a 5 year period
by the Wolfson Foundation Capital, and the suc-
cessful candidates will be attached either to the
Department of Agriculture or of Agricultural
Botany according to research and experience.
Candidates should have postgraduate experience
preferably a Ph.D. in Applied Plant Genetics,
plant breeding or experimental agronomy, and
must have a special concern for the practical
application of the research programme.
Salary in range £2,050 to £2,602 including
F.S.S.U. The appointment will be initially for
three years with possibility . of extension to a
maximum of 5 years.
Further details may be obtained from Professor
Watkin Williams. Plant Science Laboratories, Uni-
versity of Reading. Whiteknights. Reading RG6
2AS. to whom application including the names
of two referees should be sent.
(1533)
ISLE OF MAN
DEPARTMENT OF MARINE BIOLOGY
Port Erin
(University of Liverpool)
SENIOR
LABORATORY TECHNICIAN
Candidates will be expected to have a good
knowledge of biological laboratory procedures and
supervise some junior technicians. An interest in
marine organisms. and a willingness to organise the
collection of such material on the shore and from
the research vessel would be an advantage.
Candidates must possess an HNC or Final City
and Guilds in an appropriate subject. and have
several years of laboratory experience. Initial salary
within a range up to £2,163 per annum (under
review) plus threshold payments. Further particulars
may be obtained by writing to Professor E. Naylor,
Marine Biological Station, Port Erin, Isle of Man.
Application forms may be obtained from the
Registrar, The University, P.O. Box 147, Liverpool
L69 8BX. Quote ref RV/277/N.
(1542)
THE NEW YORK STATE
VETERINARY COLLEGE
A STATUTORY COLLEGE OF THE STATE
UNIVERSITY AT CORNELL UNIVERSITY
is seeking a
CHAIRMAN OF
THE DEPARTMENT OF PATHOLOGY
Applications and nominations are invited for
Department
1975.
the
position
of Chairman,
of
Pathology, available February
1,
The
applicant should have a veterinary degree with
advanced training and experience in veterinary
pathology. The Chairman will be responsible for
the leadership of a large and . diversified depart-
ment. Salary and rank are negotiable and to be
commensurate
with training and
experience.
Applicants should direct their correspondence to
Dr James H. Gillespie, Chairman Search Com-
mittee. New York State Veterinary College, Cornell
University, Ithaca, NY 14853.
An
Equal
Opportunity / Affirmative
Action
Employer.
(1543)
Senior CNS Pharmacologist
Lilly Research Centre
Surrey
We seek a broadly-based Neurophysiologist or Neuro-
pharmacologist to lead a newly created team of eight
graduate and technical staff conducting fundamental
research on neuroleptic and antidepressant drugs.
The Lilly Research Centre is one of the most modern and
well equipped establishments of its kind in Europe, and is
attractively situated in its own wooded grounds near
Windlesham in Surrey.
Over 100 graduate scientists work at the Centre and major
research projects are directed towards the discovery and
development of potential new medicines for the treatment
of allergy, inflammatory disease and disorders of the central
nervous system. The CNS research programme has particular
emphasis on behavioural pharmacology, CNS biochemistry
and neuroelectrophysiology.
The person appointed will be one of three pharmacology
team leaders and will be responsible for :
Planning and executing a soundly-based CNS research
programme.
Promoting interdisciplinary co-operation with other
research teams at the Centre and liaising with outside
workers.
Keeping abreast of new developments in his field and
striving to improve the model systems and methodologies
he uses.
Leadership, guidance, encouragement and development
of his own team.
He will spend the greater part of his time in active research,
and will be encouraged to publish important findings.
The ideal candidate for this appointment will have at least
three years' post-doctoral research experience in neuro-
physiology or neuropharmacology. He (or she) will be aged
about 28-35, and will currently be working on an appropriate
research topic in an academic or industrial environment.
He should be capable of considerable further development
as a research scientist and be able to bring ideas and
leadership skills to the position.
The starting salary will be negotiable within wide limits, as
it is our client's intention to attract a first rate research
scientist. Other benefits include non-contributory superann-
uation, free life assurance, heavily subsidised membership
of BUPA, an excellent income protection scheme, and
generous relocation expenses.
Please write in complete confidence for further information
and an application form, or 'phone if you would like to discuss
the position:
J.A. Edwards, Talentmark Limited,
King House, 11 Westbourne Grove,
London W2 4UA. Tel : 01-229 2266.
Talentmark
Biomedical and Scientific Consultants
(1548)
Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4

XX
Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4 18 1974
PHARMACOLOGISTS
We are a major international pharmaceutical company and we are
seeking enthusiastic and competent graduates in either phar-
macology or a related discipline to . join our Pharmacology
Department. An interest in either anti-inflammatory or central
nervous system pharmacology would be desirable, though not
essential.
We offer salaries based on qualifications and experience com-
mensurate with the responsibilities of the positions; conditions of
employment are in keeping with the best modern practice.
Assistance with relocation expenses will be available in appropriate
cases.
Organon
Write with details of age, qualifications
and career to date to R. W. Pollock,
Personnel Manager, Organon Laboratories
Limited,
Newhouse,
Motherwell,
Lanarkshire.
(1508)
WESTFIELD COLLEGE
(University of London)
CHEMISTRY DEPARTMENT
Applications are invited for the following posts
on
the
Medical
Research
Council's
Steroid
Reference Collection.
JUNIOR
EXPERIMENTAL OFFICER
This post offers varied and interesting work
suitable for a young lady with . A-level or com-
parable qualifications, preferably but not necessarily
including chemistry. Salary within the range of
£1,211 to £1,679 (under review) plus threshold,
depending upon qualifications and experience.
POSTDOCTORAL or GRADUATE
. RESEARCH ASSISTANT
to engage in synthesis of steroids for the Collec-
tion. Much of this work involves research into
new methods of synthesis, or the synthesis of
novel steroids. Suitably qualified graduates may be
permitted to undertake postgraduate studies for the
M.Phil. or Ph.D. degree. Salaries: Postdoctoral
scale £2,118 to £2,412 plus threshold and London
Weighting (under review) plus threshold; Post-
graduate student, equivalent to an S.R.C. student-
ship (under review).
Applications, including the names of two referees,
should be addressed to Dr D. N. Kirk, Chemistry
Department, Westfield College N, Kidderpore
Avenue, Hampstead. London NW3 7ST. (1534)
UNIVERSITY OF PAPUA
NEW GUINEA
(Port Moresby)
Applications are invited for the post of CHIEF
CARTOGRAPHER in the Department of Geogra-
phy. Applicants must be experienced Cartographers.
Appointee will be responsible for the supervision
of junior cartographers, and will be required to
prepare maps for research, publications and classes.
A knowledge of photographic and lithograhpic
reproduction as well as basic photogrammetric pro-
cedures would be an advantage. The Department
of Geography possesses a standard cartographical
equipment plus a phototypesetter. The Chief Car-
tographer's office and the work laboratory are
air-conditioned. The level of appointment will be
determined by qualifications and experience. Salary
scale: A$9,099 to AS10,432 p.a. plus A$500 p.a.
dependants allowance, if married or A$200 p.a.
dependants allowance if single. (These salaries in-
clude an overseas allowance). (£1 sterling=A$1.78).
Contracts are usually for three years but a two-
year appointment would be negotiable. Conditions
include provision of housing. F.S.S.U. and leave
fares. Further details about the University and the
conditions of appointment may be obtained from
the Secretary. Applications should be in duplicate
and include particulars of age, nationality, marital
status, qualifications and experience, names and
addresses of three referees, a recent small photo-
graph and an indication of estimated starting date.
Applications should be sent to K. R. Long, Secre-
tary, P.O. Box. 4820, University, Papua New
Guinea. Closing date: November 13, 1974. (1527)
THE UNIVERSITY OF LIVERPOOL
SUB-DEPARTMENT OF IMMUNOLOGY
RESEARCH TECHNICIAN
(Grade 5)
Required to assist with M.R.C. supported project
on transplantation.
Applicants should have much laboratory experi-
ence, preferably in immunological techniques.
Apply, giving details of qualifications and ex-
perience and the names of two referees, to:
Dr John Bradley,
Director,
Sub-department of Immunology,
University of Liverpool,
1st Floor Nuffield Wing,
Medical School,
Crown Street,
Liverpool L69 3BX.
(1523A)
INFORMATION SCIENTIST
Senior management in the Malaysian natural
rubber industry needs to be kept informed of
techno-economic and statistical trends in rubber
consuming countries. An abstracts-based informa-
tion bulletin has been started as the core of a
new information service for management, and a
computerised
retrieval
system
is
now
being
developed.
An information scientist is required to develop.
and operate these services at the Malaysian Rubber
Producers' Research Association.
Please apply to:
The Director,
The Malaysian Rubber Producers' Research
Association,
Brickendonbury,
Hertford SG13 8NP.
(1524)
THE YOUNGER RESEARCH
WORKERS INTERCHANGE
SCHEME 1974
Applications are invited from British re-
search workers in the Physical Biological and
Social sciences and in all fields of technology
for short visits to centres working on similar
programmes in any of the following coun-
tries :- Austria, Belgium, Denmark, Finland,
West Germany, The Netherlands. Norway,
Poland, Spain, Sweden, Switzerland. Details
and application forms have been sent to
University and Polytechnic Registrars for cir-
culation. The completed forms should be
endorsed by the appropriate Head of De-
partment and be returned to the address
below by November 12, 1974: Higher Edu-
cation Department, The British Council, 10
Spring Gardens, London SW1A 2BN.
(1554)
EAST BIRMINGHAM HEALTH DISTRICT
CYTOGENETICS
Cytogenetist required (salary as for top grade Biochemist, presently £5190 - £5919) to take charge of a
new Regional Cytogenetics Laboratory within the East Birmingham Hospital Laboratory system. New
building under construction. Candidates should have higher degree (Ph.D. or equivalent) and wide
experience of Cytogenetics.
The work of the department will include elucidation of chromosome abnormalities, anti-natal diagnosis
of chromosomal defects, post graduate teaching and research. The Director of the laboratory will also
have duties in diagnostic Cytogenetics in the Department of Human Genetics in the Infant Development
Unit at the Birmingham Maternity Hospital adjoining the University. The successful candidate may be
accorded honorary status in the University of Birmingham.
Candidates are invited to visit the Laboratory by direct arrangement with the Regional Virus
Laboratory (Tel: 021-772 4311 Ext. 675).
Applications (1 copy), must include the names of three referees, age and relevant experience and must
be received by the District Administrator, East Birmingham Health District, 45 Bordesley Green East,
Birmingham B9 5ST by 2nd November 1974.
-
..
(1549)
Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4

XXV
Nature Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4
THE POLYTECHNIC OF
CENTRAL LONDON
SCHOOL OF ENGINEERING SCIENCE
READER IN
LIFE SCIENCES
£4,842 to £5,175 plus Threshold
Agreement
Applications are invited for this newly
established post in Life Sciences. The success-
ful candidate will be expected to strengthen
and extend existing fields of research by his
expertise, his experience in supervising re-
search students, his ability to attract exter-
nally sponsored research and his links with
private industry and public agencies. Research
in Life Sciences will continue to be concen-
trated in three areas (1) Bio-organic Studies
(2) Aquatic pollution (3) Neurophysiology
and behaviour.
The Life Sciences area has a major teach-
ing commitment in the PCL's Modular
BSc. Degree (C.N.A.A.) and is also actively
involved in the development of a number of
postgraduate courses. The Reader will be
expected to contribute to project teaching
and to participate in the planning of new
courses.
Details and application form from . The
Establishment
Officer, PCL, 309 Regent
Street, London WIR 8AL. 01-580 2020 Ext.
212. Closing date November 4, 1974.
(1553)
FELLOWSHIPS AND
STUDENTSHIPS
UNIVERSITY OF WARWICK
RESEARCH FELLOWSHIP
in
THEORETICAL PHYSICS
Applications are invited for an appointment as
a Postdoctoral Research Fellow in the Theoreti-
cal Physics Group. The successful candidate will
work on the theory of hopping conductivity in
amorphous materials with Professor P. N. Butcher.
Salary in the range £2,118 to £2,580 p.a. plus
threshold payments and participation in FSSU.
Applications as soon as possible to the Academic
Registrar, University of Warwick,
Coventry
·CV4 7AL, quoting Ref. No: 10/A/74.
(1496)
UNIVERSITY
OF DUBLIN
Trinity College
PROTEIN CHEMIST
There is a vacancy under a contract
with the E.E.C. Environmental Re-
search Programme for a Postdoctoral
Fellow to analyse the amino acid
sequence in regions of the polypep-
tide coded by the trpA gene of
Salmonella typhimurium. The Fellow
will be part of a team designing tests
to evaluate the likelihood that various
micropollutants induce mutations in
man. Parallel test systems are being
developed in bacteria and in mice.
The work is intellectually challenging
and socially important. Applicants
must have had experience of protein
purification and amino acid sequence-
ing. The salary is negotiable.
Applications should be sent to:
Professor Dawson,
Department of Genetics,
Trinity College, Dublin 2.
by November 2, 1974.
(1403)
University of Cambridge
Department of Physical Chemistry
POST-DOCTORAL
RESEARCH FELLOWSHIP
-
Applications are invited for a post-doctoral
fellowship in the research group of Dr. I. W. M.
Smith. The successful applicant will determine the
rates at which vibrational energy is transferred
from HF and DF to other molecular species, in
processes which are important in chemical lasers.
Previous experience in gas kinetics, energy transfer,
gas-lasers or flow-discharge systems would be an
advantage but is not essential. The salary will
depend on age and experience but is likely to be in
the range £2,100 to £2,600 per annum plus F.S.S.U.
Applications, naming 2 referees, or requests for
further details should be sent to Dr. I. W. M. Smith,
Department of Physical Chemistry, University
Chemical Laboratories, Lensfield Road, Cambridge
CB2 1EP.
(1477)
NEWNHAM COLLEGE, CAMBRIDGE
proposes to elect to one or more
RESEARCH FELLOWSHIPS
tenable for 3 years from October 1, 1975. Applications are invited from women graduates
of any University in Great Britain or the Commonwealth for research in any .subject.
Applications are also invited for the SARAH SMITHSON RESEARCH FELLOWSHIP
which is restricted to students of Philosophy or Psychology but is . open to women
graduates of any University.
Unless she receives other emoluments, a Research Fellow who has not yet obtained a
Ph.D. will receive £992 a year and free board and residence: or. if she chooses to be
non-resident, she will receive £1,230 a year. For a Research Fellow who has already
obtained a Ph.D. degree, the emoluments are increased by £157 a year. The Research
Fellow may choose to be pensionable under the F.S.S.U.
Full particulars may be obtained from the Principal and the closing date for the receipt
of completed applications is January 11. 1975.
(1536)
GIRTON COLLEGE
CAMBRIDGE
-
A Clothworkers' Company Visiting Fellowship is available for a senior
woman scientist for the academical year 1975/76. The stipend will be
£2,500 per annum. Residence and commons are free. Scientists wishing
their names to be considered should write to the Secretary to the Council,
Girton College, by November 15, 1974.
(1505)
Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4
-----

XX
Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4 18 1974
LEICESTER POLYTECHNIC
SCHOOL OF CHEMISTRY
Applications are invited for:
(1) An S.R.C. postdoctoral research assistantship
with experience in organic synthesis to col-
laborate with -- Dr R. H. Dahm on a project
concerned with developing a novel small
rechargeable battery containing solely organic
constituents. A specialised knowledge of
electrochemistry is not required. Post would
interest organic chemists who desire to widen
their field of interest. Normal S.R.C. salary.
and conditions apply. Post is tenable for up
to 20 months.
(2) An S.R.C. CASE studentship to study ionic
conduction and diffusion in a range of un-
usual compounds. Candidates will read for
a C.N.A.A. Ph.D. The academic supervisors
are Dr J. M. Pollock and Dr R. G. Linford
and the project involves close collaboration
with the Royal Radar Establishment at
Malvern. Candidates should have either a first
or an upper second class honours degree, a
masters degree or a lower second degree with
several years relevant industrial experience.
The grant is £880 p.a. tax free; this would
be higher in the case of students with de-
pendants or with industrial experience.
Apply for further particulars and application forms
to the Staffing Officer, Leicester Polytechnic, P.O.
Box 143, Leicester LE1 9BH.
(1472)
UNIVERSITY OF READING
NATIONAL COLLEGE OF
FOOD TECHNOLOGY
Applications, are invited for a 3 year S.R.C.
(C.A.S.E.) Research Studentship to investigate the
use of fungi in the treatment of liquid wastes aris-
ing from the processing of biological materials.
The emphasis will be on developing an economically
viable process and will include a study of the
isolation of enzymes and an assessment of fungal
mycelium as a source of protein for animal food.
For part of the programme the student will work
under Dr R. A. Grant, Tasman Vaccine Labora-
tories, Poole, Dorset. Graduates in Science or
Technology with a biological background, with
some knowledge of Microciology and Biochemistry
preferred. Apply stating the names of two referees
to Dr J. T. Worgan, N.C.F.T., St George's
Avenue, Weybridge, Surrey. (Ref: MN 50).
(1518)
UNIVERSITY OF NOTTINGHAM
MEDICAL SCHOOL
DEPARTMENT OF PHYSIOLOGY
Autonomic Nervous Control of the
Cardiovascular System
Applications are invited from honours graduates
in physiology, pharmacology or a related discipline,
for a studentship for training in research methods
tenable from November 1974. The holder will be
eligible for registration as a Ph.D. student of the
University. The value of the award and conditions
of appointment will be those of M.R.C. Student-
ships.
Facilities are available for the postgraduate
student to join a group working on several aspects
of autonomic nervous control of the cardiovascular
system in animals and in man. Applicants should
write as soon as possible to Dr P. H. Fentem
giving a brief curriculum vitae and the names and
addresses of two referees, at the Department of
Physiology, The Medical School. University
of
Nottingham, Nottingham NG7 2RD.
(1531)
UNIVERSITY OF CALGARY
DEPARTMENT OF CHEMISTRY
BIOCHEMISTRY GROUP
Applications are invited for the position of
POSTDOCTORAL
RESEARCH FELLOW
commencing early in January 1975. Research will
be concerned with the structure-function relation-
ship of
(1) proteolytic enzymes from Anthozoa; or
(2) extracellular enzymes produced by thermophilic
fungi.
The initial. appointment would be for one year
with provision for a second year. Salary according
to NRC regulations ($8,700 per annum).
Application with curriculum vitae and names of
two referees to:
Dr K. J. Stevenson
Department of Chemistry
Biochemistry Group.
University of Calgary
Calgary, Alberta, Canada
T2N IN4.
(1540)
SENIOR NICHOLAS FELLOW
required, with several years postdoctoral experience
in cell biology to lead small team to investigate the
nature of naturally occurring lymphocyte sup-
pressors. Salary on University Lecturer scale plus
London Allowance and F.S.S.U. benefits. Apply
to Dr D. L. Dewey, Gray Laboratory, Mount
Vernon Hospital, Northwood, Middlesex HA6 2RN.
(1501)
LECTURES AND COURSES
UNIVERSITY OF LEICESTER
DEPARTMENT OF CHEMISTRY AND
SCHOOL OF EDUCATION
PH.D. IN CHEMISTRY WITH
GRADUATE CERTIFICATE
OF EDUCATION
Applications are invited from persons holding,
or expecting to be awarded during 1975, a degree
with First or Second Class Honours (Upper divi-
sion). or equivalent qualifications, in Chemistry,
Combined Sciences. Chemical Engineering, Physics.
etc., who wish to pursue a 3-year course of research
and teacher training.
The course may attract a limited number of
Science Research Council quota awards, principally
for new graduates, but candidates from industry or
teaching may be eligible to apply for awards under
other S.R.C. schemes.
For further information and application forms
please write to Professor R. D. Peacock, Depart-
ment of Chemistry, University of Leicester LE1
7RH
The closing date for applications is January 31,
1975.
(1530)
GRANTS & SCHOLARSHIPS
FRENCH GOVERNMENT
SCHOLARSHIPS
for further study and postgraduate
research in
France in Pure and Applied Science,
Technology,
Medicine, Social Sciences, Economics, Geography
and Agriculture are offered for the academic year
1975-1976. Candidates, who must be United King-
dom citizens, should be university graduates or
have equivalent qualifications. Closing date: Decem-
ber 31. 1974: Details and application forms from
Scientific Department, French Embassy, 41 Park-
side. Knightsbridge, London SW1X 7JP.
Citizens of the Commonwealth and of other
countries should apply to French Embassies in
their respective countries.
(1522)
BUSINESS OPPORTUNITIES
TOP COMMISSION
SOLE AGENTS
A Professional Electronic Company
producing laboratory stabilised power
supplies of advanced design requires
Agents in several parts of the world:
Individuals or companies of excep-
tional technical and market know-
ledge capable of exploiting the profit
potential of a high commission line
are invited to contact Box No. 1397,
Nature, T. G. Scott & Son Ltd., 1
Clement's Inn, London WC2A 2ED.
(1397)
FOR SALE AND WANTED
ELECTRONIC
SAVING UP TO
CALCULATORS
VERY LATEST NEW MODELS MFR'S GUARANTEE
HAND
DESK
PRINTERS
ADLER 805
€31,50
ADLER BOC
£24.00 ADLER 1204P
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ADLER 815
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ADLER 80S
£30.00 ADLER 1214P
£128.00
ADLER 1205
£50.50
ADLER 81
£35.00 ADLER 1414P
£150.00
ADLER 1215
£58.00
ADLER 81S
£36,50
CBM 121P
£68.50
ADLER 1215S
£79.50
CBM MM6
£ 9.00 CBM 152P
£125.00
CBM CB
£25.00
CBM MM3R
£16,50
LOGOS 58
LOGOS 59
CBM E12
£36.00
CBM MM3PR
£21,00
OLYMPIA CD201
£56.50
CBM MM3MRT
OLYMPIA CD60
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LOGOS 68
OLYMPIA CD301
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DIVISUMMA 28
OLYMPIA CD520
£71.00
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CBM MM3SR
£21.00
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CBM MM3MC
ADLER 815AD
£54.00
CBM -MM2SR
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OLYMPIA CD100
£45.00
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OLYMPIA CD101
£47.50
£130.00
All prices include P. & P. Add 8% VAT
Buy it with Access
C.W.O. or C.O.D. Specification lists ch request.
Goods sent by return. Callers welcome.
MOUNTAINDENE LTD.
308 Grays Inn Road, London, WC1
Tel: 01-837 8695
(1458
Always wanted :
BACK RUNS OF JOURNALS
IN SCIENCE AND THE HUMANITIES
WM. DAWSON & SONS LTD.
Back Issues Department,
Cannon House, Park Farm Road,
Folkestone,, Kent, England.
Tel .: Folkestone 57421.
(1)
SYMPOSIUM
UNIVERSITY OF TEHRAN
TEHRAN, IRAN
ANNOUNCES AN INTERNATIONAL SYMPOSIUM
to be held
IN TEHRAN on MAY 5, 6 and 7, 1975
Sponsored by
THE INTERNATIONAL UNION OF BIOCHEMISTRY,
THE INTERNATIONAL UNION OF
PURE AND APPLIED BIOPHYSICS ON
The Structural Basis of Membrane Function
with sessions on
Membrane Structure, Transport in Membranes, Energy Trans-
duction in Membranes, Regulatory Functions of Membranes,
Excitable Membranes, and Microsomal Membranes.
Information regarding Symposium Registration, Hotel Reservation,
and Sight-seeing Excursion on May 8 and 9, etc., may be
obtained from :
DR. K. JAVAHERIAN
POST OFFICE BOX 14-1700
UNIVERSITY OF TEHRAN
TEHRAN, IRAN
(1474)
Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4
£60.00
OLYMPIA CP140
£148.00
ADLER 6BT
OLYMPIA CD701
£172,00
ADLER 805AD
£93.50
£30.00 OLYMPIA CP550
OLYMPIA CP141
£23.90
DIVISUMMA 18
£130.00
OLYMPIA CD401
£115.00
DIVISUMMA 18
50%
£205.00
£235.00
£285.00

Na ....
Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4
.. Cover 3
CROPICO
D. C. NULL DETECTOR
(microvoltmeter)
10 RANGES
RECORDER OUTPUT
BATTERY OPERATED
0.3/4V FULL SCALE
LOG and LINEAR RANGES
Input Impedance 24,0002
CROYDON PRECISION INSTRUMENT COMPANY
HAMPTON ROAD, CROYDON CR9 2RU
Telephone: 01-684 4025 & 4094
Radioactive
Poly A & Poly U
from stock
Polyadenylic acid, ammonium salt
[adenylate-2, 8-3H]
>500mCi/mmole Current Lot: 1 Ci/mmole
Lyophilized solid in screw-cap bottle, shipped in dry ice.
NET-450
$105/25/Ci
$325/100/Ci
Polyuridylic acid, ammonium salt
[uridylate-5, 6-3H]
>500mCi/mmole Current Lot: 13 Ci/mmole
Lyophilized solid in screw-cap bottle, shipped in dry ice.
NET-436
$105/25/Ci
$325/100uCi
Molecular hybridization with 3H-labeled Polyuridylic
acid (Poly U) has been used 1-2 to detect Poly A
sequences in viral RNA.
1. D. Gillespie, S. Marshall, and R.C. Gallo, Nature 236, 227 (1972).
2. J. Schlom, D. Colcher, S. Speigelman, S. Gillespie, and D. Gillespie,
Science 179, 697 (1973).
NEN New England Nuclear
575 Albany Street, Boston, Mass. 02118
Customer service 617-482-9595
NEN Canada Ltd., Dorval, Quebec; NEN Chemicals GmbH, Dreieichenhain, W. Germany
NEW ... Portable Area Meter ... Model LI-3000
Measures leaf area on living plants or detached
leaves in the field or laboratory.
· Measures 500 leaves per hour on living
plants.
· Accumulate 1 mm2 to 999,999.99 cm2.
· Less than ±1% error (detached leaves).
· Less than ±2% error on living plants.
LABORATORY CONVEYER
ATTACHMENT AVAILABLE
(not shown)
· Cost of portable unit with
conveyer attachment is
less than conveyer-type
instruments now selling.
Patented
The Instrument utilizes a unique microcircuit flying spot scanner developed by LI-COR and Collins Radio Co.
Collins has vast experience with high reliability aerospace microcircuitry.
Applications Include:
Evaluating insect damage (illustrated).
Record individual plant leaf development or LAI throughout the
growing season.
Measuring leaf area as affected by pollution.
Development and testing were performed in cooperation with the University of Nebraska,
College of Agriculture.
For more information and complete price listings, write to:
T. & J. CRUMP,
44 Mount Road, Wickford, Essex
Phone (03744) 4163
LAMBDA Instruments Corp.
LI.COR
P.O. Box 4425 / Lincoln, Nebraska 68504 U.S.A.
Phone (402) 467-3576
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Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4
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Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4
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353 South
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Declassified and Approved For Release 2014/01/09 : CIA-RDP79-00999A000200010001-4
251 559-660 October 18, 1974