CC 1993 Else\ icr Scmce Publ~~hcrs B.V. .A11 rights I-esmed. 037% I 1 19 93 `SO6.00

GENE 07414

Co-chairman's remarks: before the double helix*

(Proteins; DNA: Avery: Watson: Crick)

Max F. Perutz

Received by G. Bernardi: 19 hiay 1993: Accepted: 3 June 1993: Received at publishers: 16 July 1993

SUMMARY

In the nineteen-thirties and -forties, genes were universally believed to be made of protein. Biochemists met Avery,
MacLeod and McCarty's discovery, that the transforming factor of pneumococci consists of DNA, with disbelief, and
the notion that this was true of genes took a long time to be generally accepted even after Watson and Crick's discovery
of its double helical structure. Until Watson's arrival. Kendrew. Crick and I were interested mainly in solving the
structure of proteins, but Watson made us think about the structure of genes which determine protein structure.

In 1936, I left my hometown Vienna, Austria, for
Cambridge, England, to seek the Great Sage. I asked him:
`How can I solve the secret of life? He replied: `The secret
of life lies in the structure of proteins, and X-ray crystal-
lography is the only way to solve it'. The Sage was John
Desmond Bernal, a flamboyant Irishman who headed the





tances (Bernal and Crowfoot, 1934). We really called him
Crystallographic Department of the Cavendish





Sage, because he knew everything. During a discourse at
the Royal Institution in London Bernal said: `The struc-
Laboratory and who had been the first to discover that






ture of proteins is the major unsolved problem at the
boundary of chemistry and biology today. It is difficult
protein crystals give detailed X-ray diffraction patterns







to exaggerate the importance of this study to many
branches of science. The protein is the key unit in bio-
extending to spacings of the order of interatomic dis-









chemistry and physiology. . . . All protein molecules that

Corrr.spo,irl~,~?~,~ 10: Dr. M.F. Perutz, M RC Laboratory of Molecular
Biology. Cambridge CB2 ZQH, UK. Tel. (44-223) 24801 I: Fax (44-
223) 213556.
*Presented at the COGENE'Symposium. `From the Double Helix to
the Human Gcnome: 40 Years of Molecular Genetics'. UNESCO. Paris,
21-23 April 1993.

we know now have been made by other protein mole-
cules, and these in turn by others' (Bernal, 1939). When
Bernal advanced this argument in a later BBC discussion,
the physicist W.H. Bragg asked him where the first pro-
tein had come from. Instead of replying `I don't know',






What had attracted me to Cambridge as an under-
Bernal skilfully sidestepped Bragg's awkward question.






graduate in Vienna was Gowland Hopkins' work on vita-
mins and enzymes. He was the founder of Cambridge
Inspired by Bernal's enthusiasm, I became a crystallogra-







biochemistry. In the nineteen-thirties he still had had to
battle against vitalism to get acceptance for his then he-
pher and began to work on the structure of haemoglobin,









retical view that `The living cell, at one definite level of
its organisation, admitting that higher levels may be su-
because it was the protein that was most abundant and









perimposed, is to be pictured as the seat of diverse but
organised chemical reactions, in which substances identi-
easiest to crystallise.









fiable by chemical methods undergo changes which can
be followed by chemical methods. The molecules of these
substances are activated and their reactions directed in
space and time by the catalytic agencies which are com-
monly known as intracellular enzymes. The influence of


10

these differs in no essentials from that of catalysts in non-
living systems save that it is displayed in relations which
are exceptionally specific' (Hopkins, 1932). Even today
some philosophers would reject Hopkins' views as
reductionist.

Hopkins was the professor of biochemistry. The reader
was J.B.S. Haldane, a de\,oted communist in the guise of
an English squire, and one of the most imaginative scien-
tists of his generation. He pointed out that many enzy-
matic reactions as well as blood groups are genetically
controlled.

`Two possibilities are now open. The gene is a catalyst
making a particular antigen, or the antigen is simply the
gene or part of it let loose from its connection with the
chromosome. The gene has two properties. It intervenes
in metabolism. sometimes at least by making a definite
substance. And it reproduces itself. The gene. considered
as a molecule, must be spread out in a layer one building
block deep. Otherjvise it could not be copied. The most
likely method of copying is by a process analogous to
crystallization, a second similar layer of building block
being laid down on the first. But we could conceiae of a
process analogous to the copying of a grawoplrone record
by the intemediation of u "negative" perhaps related to
the original as an antihod~ ro all autigen` (Haldane, 1937)
(author's italics).

Haldane dismissed the idea that genes might be made
of nucleic acids and asserted that the most likely sub-
stances are the histones, which are proteins.

Three years later Pauling and Delbriick published a
seminal paper. It was an attack on the German theoreti-
cian Pascual Jordan, who had advanced the idea that
there exists a quantum-mechanical stabilizing interaction,
operating preferentially between identical and near-
identical molecules, which governs biological processes
such as the reproduction of genes (Jordan, 1938). Pauling
and Delbriick pointed out that interactions between
molecules were now rather well understood to give sta-
bility to two molecules of cor,~p/e,tlrrlt~~~, structure in jux-
taposition, rather than two molecules with necessarily
iderlticwl structures (Pauling and Delbriick, 1940).

In 1944 the almost uni\,ersally held view that genes
consist of proteins was overturned by Avery, MacLeod
and McCarty's discovery that the transforming principle
of pneumococci is made of DNA. Oswald Avery, who
took the first step as a young man and persevered until
his retirement, was ;tn even more reluctant revolutionary
in biology than Max Planck had been in physics earlier
in the century. He was born in Halifax. Nova Scotia in
1877, the son of a Baptist hlinister who had emigrated
there from England and later became pastor of a Baptist
church in a poor district of New York.

Motivated by his family's Christian missionary back-

ground Oswald Avery became a doctor, but soon aban-
doned practice for research. This remained humdrum
until. aged 36. he was appointed bacteriologist to the
hospital of the Rockefeller Institute for Medical Research
in New York. At that time lumbar pneumonia killed
50000 people a year in the United States. Avery's mother
had died of it. Avery wondered why it killed some people,
ivhile others recovered.

The first pointers came in 1916. Avery's friend A.R.
Dochez discovered in the filtrate of a pneumococcus cul-
ture a speci'c soluble substance that flocculated in an
antiserum against the type of pneumococcus growing in
the culture. Dochez and Avery then found that same sub-
stance in the blood and urine of their patients and con-
cluded, wrongly as it turned out, that it was a protein.
In succeeding years Avery convinced himself that it was
the same substance which formed the bacterial capsule
originally described by Neufeld in Robert Koch's labora-
tory in Berlin.

Avery sensed that this specijic soluble substance played
a vital part in the disease and wanted to find out more
about it. When the organic chemist Michael Heidelberger
joined the Institute, he used to agitate a tube of it in front
of him saying: `Michael, the whole secret of bacterial spec-
ificity is in this tube. When can you work on it?' When
Heidelberger finally did take it up, he found it to consist,
not of protein, but of polysaccharide (Avery and
Heidelberger, 1923).

The previous year Fred Griffith at the laboratory of
the Ministry of Health in London had discovered the
nature of the difference between virulent and non-virulent
pneumococci that Avery had sought for so long. He found
that the virulent cocci were encapsulated and the non-
virulent ones were not. Griffith called the encapsulated
ones smooth and the others rough. So the specific soluble
substcn,ce of the capsule consisted of polysaccharide and
was associated with virulence.

Heidelberger and Avery tried to make it produce an
antiserum. They failed, but Avery did make an effective
antiserum by immunizing horses with virulent pneumo-
cocci, and this saved many lives.

The next great advance came in 1928 with a startling
disco\,ery by Griffith. He injected into mice a mixture of
live rough pneumococci of type 1 and heat-killed smooth
pneumococci of type II. This mixture killed the mice, even
though the rough pneumococci were non-virulent. The
reason became apparent when Griffith isolated from their
blood live smooth pneumococci of type II. Their capsular
shell contained polysaccharide of type II, sho\ving that
the live rough pneumococci of type I had been trans-
formed into live ones of type II! (Griffith, 1929). Griffith
interpretated the transformation as a Lamarckian kind
of adaptation. `When the R form of either t!pe is fur-


nished under suitable experimental conditions with a
mass of the S form of the other type, it appears to utilize
that antigen as a pabulum (food) from which to build up
a similar antigen and thus it develops into an S strain of
that type' (Griffith, 1928). He thought that the recipient
rough cocci had retained the power to synthesize the
capsular polysaccharides of several serological types and
needed only the specific stimulus of the killed encapsu-
lated cells to adapt themselves and make the polysaccha-
ride capsule again. Apparently he never thought of
mutations, perhaps because bacteria were then believed
not to exhibit genetics.

According to his colleague Renl Dubos, Avery refused
to believe Griffith's results until another member of the
Institute, Henry Dawson, repeated them while Avery was
away sick (Dubos, 1976). Dawson and Sia did away with
the mice and transformed rough pneumococci into
smooth ones in cultures in beef broth. After this Avery
was convinced and used to ask persistently, day after day,
year after year: `What is the substance responsible for the
transformation?`, but he was handicapped by being a bac-
teriologist rather than a biochemist.
After Dawson had left the Institute in 1930, Avery en-
couraged his successor J.L. Alloway to pursue the prob-
lem, He dissolved the virulent cocci in deoxycholate,
filtered off the cellular debris and found the solution to
be active. The activity came down as a thick, syrupy pre-
cipitate on addition of alcohol and could be redissolved
in water without loss of activity but, unbelievably, it took
another 13 years to find out what this precipitate was
made of. Not even Colin MacLeod's pregnant observa-
tion, made in 1936, that the transforming activity was
destroyed by ultraviolet light gave the clue.
The slow progress was due partly to the very inconsis-
tent yields of transforming activity extracted from the
pneumococci, due to its variable destruction by bacterial
enzymes. It was not until 1941 that Avery surmounted
this trouble by heating the transformed cocci to 65oC for
30 min before lysing them with deoxycholate. This inacti-
vated the bacterial enzymes that had destroyed the activ-
ity, and left the transforming principle intact.
The final attack started in September 1941 with the
arrival of Maclyn McCarty, a young medical doctor with
a good biochemical training. It then seemed most likely
that the transforming principle was a protein, whence
enzymes that digest proteins should have destroyed it.
Nowadays such enzymes can be bought off the shelf, but
111 those days McCarty was lucky to be given some tryp-
sin and chymotrypsin by Northrop at the Rockefeller
Institute in Princeton who had been the first to isolate
and crystallise them in pure form.
Northrop's enzymes left the transforming activity
intact. Could it reside in RNA'? Northrop's colleague

11

Moses Kunitz had just crystallised ribonuclease and gave
Avery some of his crystals. Ribonuclease also left the
transforming activity intact. The same held for enzymes
that hydrolyse polysaccharides. Avery and McCarty's
therefore set out to rid an extract of the virulent bacteria
of protein, RNA and polysaccharides without destroying
the transforming activity, and then isolating that activity
in pure form.

After a lot of hard work McCarty obtained a gooey
precipitate of ivhite fibres which took up a stain charac-
teristic of DNA and showed properties similar to the
DNA that another member of the Rockefeller Institute,
Alfred Mirsky, had isolated from calf thymus. Now came
the crucial test. Would the transforming activity be de-
stroyed by deoxyribonuclease? NC-one could provide
this. McCarty had to isolate it laboriously from dog intes-
tinal mucosa, or swine kidneys, or rabbit blood. He made
sure that it degraded neither protein, nor RNA, nor poly-
saccharide, but it did destroy the transforming activity
(McCarty, 198.5). Avery and McCarty now piled proof
upon proof to convince themselves of their finding, yet it
was so revolutionary that it took Avery a long time before
he finally summoned the courage to publish it. The paper
is absolutely rigorous and leaves no shadow of doubt
that the transforming factor consists of DNA and nothing
but DNA.

Did Avery comprehend the full significance of his dis-
covery? Rollin Hotchkiss who worked with him, has testi-
fied that Avery `was well aware of the implications of
DNA transforming agents for genetics and infections'.
McFarlane Burnett. who visited Avery in 1943, wrote
home to his wife that Avery `had just made an extremely
exciting discovery which, put rather crudely, is nothing
less than the isolation of a pure gene in the form of DNA
(Olby, 1974). It was a revolutionary finding, but Avery
was no revolutionary. He was a small. delicate monkish
bachelor who lived only for his science, and for his life's
aim to find the cause and cure of virulent pneumonia.
He wore pince-nez, was fastidious with his words, ever
cautious in his public utterances, never went on lecture
tours, wrote no books and never travelled. He never co-
authored any paper on research to which he had not
actively contributed. did not patent his discovery and
never became rich.

In 1944 genes were still almost universally believed to
be made of protein. Aaron Levene. a chemist at the
Rockefeller Institute, had proposed that all DNAs are
made up of regular sequences of the same four nucleo-
tides. whence they could not carry information. Alfred
Mirsky, who had spent years working on DNA without
realizing its significance, was sure that the transforming
activity was carried by protein impurities in Avery's
DNA. As late as 1947. Mirsky said at Cold Spring


12

Harbor: `In the present state of knowledge, it would be
going beyond the experimental facts to assert that the
specific agent in transforming bacterial types is DNA
(McCarty, 1985). Seeing that as little as 3 ng of DNA
had been sufficient to transform the cocci in half an as-
sembly of infected test tubes, the idea that transformation
could have been effected by a protein impurity seems far
fetched. Perhaps because of Mirsky's continued smear
campaign, Avery. MacLeod and McCarty never received
the Nobel Prize for one of the century's greatest discover-
ies. Yet to its credit. the Royal Society recognised the
discovery by making Avery a Foreign Member and
awarding him their highest honour, the Copley Medal.
Sir Henry Dale, the President, said in his citation: `Here
surely is a change to which, if we were dealing with higher
organisms, we should accord the status of a genetic varia-
tion; and the substance inducing it -the gene in solution,
one is tempted to call it - appears to be a nucleic acid of
the deoxyribose type' (Dale, 1946).
Robert Olby concludes his authoritative account of the
history of the transforming factor with the words: `With
the passage of time the work of Avery. MacLeod and
McCarty looks, if anything, more significant than in 1953;
perhaps it was the most important discovery in the path
to the double helix' (Olby, 1974), but most people re-
mained sceptical for many years afterwards. Some were
convinced when Hotchkiss (1951) transferred penicillin
resistance to a non-resistant strain of pneumococci with
DNA from a resistant strain. Others dropped their
doubts when Alfred Hershey and Martha Chase (1952)
demonstrated that on phage infection only the phage
DNA and not the phage protein enters E. co/i. References
to the transforming principle in successive editions of J.N.
Davidson's textbook on nucleic acids illustrate the
scepticism that persisted for a long time even after that.
`If the active DNA is in fact protein-free. we have here
an example of a specific biological property, the ability
to induce the synthesis of a characteristic immunological
polysaccharide, which is peculiar to one form of DNA
and no other. Not only is this the first good example of
a biological activity attributable to a nucleic acid per se;
it indicates that there may be important differences be-
tween one specimen of DNA and another \vhich may not
be detectable by chemical means' (Davidson, 1950; 1953).
`It seems reasonable therefore to interpret bacterial
transformation as indicating that DNA is the active mate-
rial of the gene: .,. It has proved a matter of some diffi-
culty to find evidence in confirmation of that hypothesis'
(Davidson, 1960; 1963).

Davidson did not specify the nature of that difficulty.
I was still submerged in haemoglobin when one day in
September 1951 a strange young head \vith a crew-cut
and bulging eyes popped through my door and asked,

without saying as much as MIO. *Can l cl,,,lc .;,~~:   (
here?' He was Jim Watson. who \\.anted to join ,hc 2,,,.:,,
team of etltllusiasts for molecular biology ;,t ,hc
Cavendish Laboratory in Cambridge \vhich 1 led,
My colleagues were John Kendren. a chemist like
myself, and Francis Crick and Hugh Huxley. both ph!>i-
cists. We shared the belief that the nature of life could bc
understood only by getting to know the atomic structure
of living matter, and that physics and chemistry would
open the way. if only we could find it.
In his best-selling book `The Double Helix' Watson
mirrors himself as a brash western cowboy entering our
genteel circle, but this is a caricature. Watson's arrival
had an electrifying effect on us because he made us look
at our problems from the genetic point of view. He asked
not just: what is the atomic structure of living matter?
but, foremost, what is the structure of the gene that deter-
mines it? Watson found an echo in Crick who had begun
to think along similar lines. Crick was 34, a more than
mature graduate student due to years lost by the war;
Watson was 22, a whiz-kid from Chicago who had en-
tered university aged 15, and got his Ph.D. in genetics
at 20.

They shared the sublime arrogance of men who had
rarely met their intellectual equals. Crick was tall, fair,
dandyishly dressed and talked volubly, each phrase in his
King's English strongly accented and punctuated by
eruptions ofjovial laughter that reverberated through the
laboratory. To emphasise the contrast, Watson went
around like a tramp, making a show of not cleaning his
one pair of shoes for an entire term (an eccentricity in
those days), and dropped his sporadic nasal utterances
in a low monotone that faded before the end of each
sentence, and was followed by a snort.

To say that they did not suffer fools gladly would be
an understatement: Crick's comments would hit out like
daggers at uou sequirurs and Watson demonstratively un-
folded his newspaper at seminars that bored him. Watson
had put Crick's mind to the structure of DNA, yet their
relationship was something of teacher and pupil because
there was little that Watson could teach Crick, but much
that Crick could teach Watson. Crick had a profound
understanding of that hardest of the sciences, physics,
without which the structure of DNA would never have
been solved. This crucial fact is obscured in Watson's
`Double Helix'. Yet Watson had an intuitive knowledge
of the features that DNA ought to have if it were to make
genetic sense.

At some stage there was much argument as to whether
genes consist of two or three chains of DNA wound
around each other. Watson lodged with a lady retired
from the stage who kept a boarding house for youn??
girls. One day she noticed him pacing restlessly and mul-


13

tering to himselI: `There must be two . . . there must be
two . ..' She guessed that this referred to matters of the
heart. But we knew better. He reasoned on genetic
grounds that genes must be made of two chains of DNA,
and he was right.

Like Leonardo, Crick and Watson often achieved most
when they seemed to be working least. They did an im-
mense amount of hard work. studying hidden away, often
at night, but when you saw them they were more likely
engaged in argument and apparently idle. This was their
way of attacking a problem that could be solved only by
a tremendous leap of the imagination, supported by pro-
found knowledge. Imagination comes first in both artistic
and scientific creations. But in science Nature al\vays
looks over your shoulder. To paraphrase \f'inston
Churchill: `In science you don't need to be polite, you
only have to be right'.

REFERENCES

Avery, O.T. and Heidelberger, M.: Immunological relationships of cell
constituents of pneumococcus. J. Exp. Med. 38 (1923) 81-85.
Avery, O.T., MacLeod, CM. and McCarty, M.: Studies on the Chemical
nature of the substance inducing transformation of pneumococcal
types. Induction of transformation by a desoxyribonucleic acid frac-
tion isolated from pneumococcus type III. J. Exp. Med. 79 (1944)
  137--158.

Bernal. J.D. and Crowfoot, D.: X-ray photographs of crystalline pepsin.
Nature 133 11931) 791-795.

Crick. F.: What ?vlad Pursuit. Basic Books Inc.. New York. 1988.
Dale. H.: Presidential address. Proc. Roy. Sot. A 155 (1946) 127-243.
Davidson, J.N.: The Biochemistry of the Nucleic Acids. Methuen.
  London. 1950 and 1953.

Davidson. J.N.: The Biochemistry of the Nucleic Acids. Methuen,
London 1960 and 1963.

Dubos. R.: The Professor. The Institute and DNA. Rockefeller
University Press. New York. 1976.
GriHith. F.:  The signiticance of pneumococcal types. J. Hyg.
(Cambridge. UK) 27 (1918) 113-159.
Haldanc. J.B.S.: The biochemistry of the individual. In: Needham. J.
and Green. D.E. (Eds.). Perspectives in Biochemistry. Cambridge
University Press. Cambridge. 1937. pp. l-10.
Hcrshcy. A.D. and Chnse. hl.: Independent functions of viral protein
and nucleic acid in grol\th of bacteriophage. J. Gen. Physiol. 36
(1952) 39-52.

Hopkins. F.G.: Some aspects of biochemistr!: lhe organising capacities
of specific catalysts. Second Purser Xlemorial Lecture 1933. In:
Needhom. J. and Baldwin. E. lEds.). Hopkins and Biochemistry. W.
Heffer, Cambridge. 1949. pp. 226-242.
Hotchkiss. R.D.: Transfer of penicillin resistance in pneumococci by the
deoxyribonucleate derived from resistant cultures. Cold Spring
Harbor Symp. Quant. Biol. 16 (1951) 457-460.
Jordan. P.: Zur Frage einer spezifischen Anziehung zwischen den
Molekiilen. Phys. Z. 39 (1938) 711-714.
McCarty, M.: The Transforming Principle. W.W. Norton, New York.
  1985.

Olby. R.: The Path to the Double Helix. MacMillan, London, 1974.
Pauling. L. and Dclbriick. M.: The nature of the intermolecular forces
operative in biological systems. Science 92 ( 1940) 77-79.