Reprinted from the Archives of Environmental Health

September 1970 Volume 21 “FEE ]
Copyright 1970, American Medical Association

256

The “Undiscovered”
Discovery

Wendell M. Stanley, PhD, ScD,
Berkeley, Calif

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Prologue

Tus STORY deals with the very impor-
tant discovery that DNA could possess and
transmit genetic information and some as-
pects of the scientific climate existing at the
time which probably were responsible for
the fact that the discovery was really “un-
discovered,” generally unappreciated, and
failed, for several years, to affect the trend
of science. It is written in honor of Thomas
Francis, Jr., MD, because, when Dr. Francis
came to the hospital of the Rockefeller Insti-
tute for Medical Research in the summer of
1928 to do some research under the supervi-
sion of Oswald T. Avery, MD, he became
interested in bacterial transformation. He
tried to obtain the transformation of a type
of pneumococcus in vitro but unfortunately
did not succeed. The time was not yet ripe,
for later when Alloway came to work with
Dr. Francis, he took on the problem and did
succeed as did Dawson and Sia later on. Dr.
Francis never returned to research on bacte-
rial transformation, but during the time he
was at the Rockefeller Institute he de-
veloped a close and lasting friendship with
Dr. Avery, the main architect of the DNA
discovery. They shared an active interest in
respiratory disease, Dr. Francis mainly with
influenza, and Dr. Avery, with pneumonia,
and they enjoyed getting together for late
afternoon social hours during which science
was often discussed. In this one respect it is
unfortunate that Dr. Francis left the insti-
tute to go to New York University in 1938
and then on to the University of Michigan
in 1941, for I know he would have greatly
enjoyed a continuation of his close associa-
tion and almost daily conversations with Dr.
Avery during the exciting years immedi-
ately preceding the publication about trans-
forming DNA by Avery, MacLeod, and
McCarty in 1944. Dr. Francis had a great
capacity for friendship.

Submitted for publication Dec 29, 1969; accepted
Jan 5, 1970.

From the Virus Laboratory and Department of
Molecular Biology, University of California, Berke-
ley.

Reprint requests to Virus Laboratory, University
of California, Berkeley, Calif 94720 (Dr. Stanley).

Arch Environ Health—Vol 21, Sept 1970
THE “UNDISCOVERED”

The Discovery

The first of a class of substances to be
called “nucleic acids” by Altman in 1889
was isolated by Miescher in 1869. As so
often happens with a discovery, Miescher
was somewhat uncertain about the nature of
the nonprotein, phosphorus-containing mate-
rial he had obtained from pus cells. He
called it a “nyclein’ and described its prop-
erties in a paper he submitted to Hoppe-
Seyler who was SO perplexed he held up
publication while he and his students re-
peated and extended Miescher’s experl-
ments. Hoppe-Seyler became satisfied that
Miescher’s results were correct, for in his
journal in 1871 appeared not only Miescher’s
paper but three from Hoppe-Seyler’s labora-
tory plus an additional one from Miescher,
all on this new class of substances. Miescher
extended his work to include fish sperm,
and, in 1879, Kossel, working in Hoppe-Sey-
ler’s laboratory, entered the field and gradu-
ally gained control. Kossel dominated the
field until the entrance of Levene about the
turn of the century.

The structural organic chemistry of the
nucleic acids was worked out in beautiful
detail by Levene and co-workers during the
next 35 years. Levene considered DNA to be
a tetranucleotide containing repeating units
of the bases adenine, guanine, cytosine, and
thymine. In the book N ucleic Acids by Lev-
ene and Bass published in 1931 they state
on p 262:

On the basis of the data discussed in previous
chapters, namely, on the basis of the isolation
of four bases in equimolecular proportions on
complete hydrolysis of ribodesose nucleic acid
and on the basis of the isolation of four nucleo-
sides on partial hydrolysis, it is warranted to
attribute to the substance 4 tetranucleatide
structure.

On pp 276 to 277, it is stated:

Thus the tetranucleotide structure of yeast
nucleic acid has been re-established by the
analytical method. It will be seen later that the
formulation of Levene has been confirmed by
the physicochemica! method.

Even at that time Levene was uncertain
about the molecular weight, for on P 989 the
following occurs:

On the other hand, it must be borne in mind
that the true molecular weight of nucleic acids
ig as yet not known. The tetranucleotide theory
is the minimum molecular weight and the nu-

Arch Environ Healt

pISCOVERY—STANLEY 257

cleic acid may as well be a multiple of it.

Probably because of the general accep-
tance of the tetranucleotide theory no at-
tempt seems to have been made to relate
biological activity to the nucleic acids. The
early piologists certainly connected the nu-
cleus of the cell to piological activity, but
because of the diversity displayed by Pro
tein, tended to believe that protein played
the central role in the transmission of genet-
ic properties. One of the first clear state-
ments was that of the great American cytol-
ogist Edmund B. Wilson who in 1895 stated,
Chromatin is known to be closely similar to, if
not identical with, a substance known as nu-
clein, which analysis shows to be a tolerably
definite chemical compound composed of nu-
cleic acid and albumin. And thus we reach the
remarkable conclusion that inheritance may,
perhaps, be effected by the physical transmis-
sion of a particular chemical compound from
parent to offspring.

The efiect of the Levene school of thought is
clearly evident for in 1925 Wilson wrote in
his book The Cell the following:

Apart from the characteristic differences be-
tween animals and plants, the nucleic acids of
the nucleus are on the whole remarlably
uniform, showing with present methods of anal-
ysis no differences in any degree commensurate
with those from the various species of cells
from which they are derived. In this respect
they show a remarkable contrast to the pro-
teins, which, whether simple OT compound,
seem to be of inexnaustible variety. It has been
suggested accordingly, that the differences be-
tween different “Chromatins” depend upon
their basic ot protein components and not upon
their nucleic acids.

There is little wonder that scientists in
the 1930’s considered proteins to be impor-
tant in genetic transmission and showed lit-
tle interest in the nucleic acids, apart, of
course, from their structural organic chemis-
try. A further indication of this lack of
interest is the fact that in their 900-page
volume Practical Physiological Chemistry
published in 1931, Hawk and Bergheim de-
voted only 12 pages to nucleic acids and
nucleoproteins. The discussion indicates that
the nucleic acids could be divided into two
classes—animal and plant—with the former
containing deoxy pentose rather than pentose
and thymine rather than uracil. Levene was
credited with the structural work supporting
the tetranucleotide theory. I am convinced

p—Vol 21. Sept 1970
258 THE “UNDISCOVERED”

that the general acceptance of this theory
was largely responsible for the reluctance on
the part of scientists to consider that nucleic
acids might carry biological and especially
genetic information. This was, therefore, the
scientific atmosphere surrounding the early
work on the transforming principle of the
pneumococcus. When Avery, who had been
working on and off on transformation since
1928, finally directed the work toward the
purification and isolation of the agent re-
sponsible for bacterial transformation, he
certainly did not expect to end up with
nucleic acid. As the experimental work con-
ducted with his two younger associates,
MacLeod and McCarty, proceeded it be-
came obvious that the transforming princi-
ple was in fact DNA. Finally a paper was
submitted on Nov 1, 1943, and published
Feb 1, 1944, in the Journal of Experimental
Medicine entitled “Studies on the Chemical
Nature of the Substance Inducing Transfor-
mation of Pneumococcal Types: Induction
of Transformation by a Desoxyribonucleic
Acid Fraction Isolated From Pneumococcus
Type III.”

One has only to read this paper in detail
to recognize the turmoil and mental anguish
these investigators must have gone through
as the work proceeded. Although mentioned
in the title, nucleic acid is not mentioned in
the first eight pages of the paper. On p 9, a
table showing the results of elementary
chemical analysis is presented with the
statement that while there was close agree-
ment with the figures for nucleic acid, the
analytical figures by themselves did not es-
tablish that the isolated substance was a
pure entity. Obviously, although the results
indicated otherwise, they were still thinking
about the possibility of a trace of highly
biologically active protein. They tested their
preparations extensively with known en-
zymes and tissue extracts since certain of
these were found to destroy the biological
activity. They found an enzyme that would
destroy the activity. This was the enzyme
reported from Greenstein’s laboratory in
1940 and called “deoxyribodepolymerase”
by Greenstein in 1943.

Finally after extensive enzymatic and ser-
ological analysis and some physicochemical
and quantitative studies, the tentative con-
clusion was reached on the 14th page of the

DISCOVERY—STANLEY

paper that:

The fact that transforrning activity is destroyed
only by those preparations containing depoly-
merase for desoxyribonucleic acid and the fur-
ther fact that in both instances the enzymes
concerned are inactivated at the same tempera-
ture and inhibited by fluoride provide addi-
tional evidence for the belief that the active
principle is a nucleic acid of the desoxyribose
tvpe....

Thus in both the electrical and centrifugal

fields, the behavior of the purified substance is
consistent with the concept that biological ac-
tivity is a property of the highly polymerized
nucleic acid. Ultraviolet absorption curves
showed maxima in the region of 2600 A and
minima in the region of 2350 A. These findings
are characteristic of nucleic acids.
Yet in the immediately following para-
graphs the terminology that was used was
“transforming principle” or ‘‘active materi-
al,” and not “nucleic acid.” The paper con-
tains an excellent discussion and the sum-
mary is as follows:

1. From Type III pneumococci a biologically
active fraction has been isolated in highly
purified form which in exceedingly minute
amounts is capable under appropriate cultural
conditions of inducing the transformation of
unencapsulated R variants of Pneumococcus
Type II into fully encapsulated cells of the
same specific type as that of the heat-killed
microorganisms from which the inducing ma-
terial was recovered.

2. Methods for the isolation and purification
of the active transforming material are de-
scribed.

3. Th> data obtained by chemical, enzymatic,
and serological analyses together with the re-
sults of preliminary studies by electrophoresis,
ultracentrifugation, and ultraviolet spectrosco-
py indicate that, within the limits of the
methods, the active fraction contains no demon-
strable protein, unbound lipid, or serologically
reactive polysaccharide and consists principally,
if not solely, of a highly polymerized, viscous
form of desoxyribonucleic acid.

4. Evidence is presented that the chemically
induced alterations in cellular structure and
function are predictable, type-specific, and
transmissible in series. The various hypotheses
that have been advanced concerning the nature
of these changes are reviewed.

The final conclusion in the paper is sim-
ply and clearly stated but in a manner
characteristic of a very conservative scientist
as follows: “The evidence presented sup-
ports the belief that a nucleic acid of the

Arch Environ Health—Vol 21, Sept 1970
THE “UNDISCOVERED” DISCOVERY—STANLEY

desoxyribose type is the fundamental unit of
the transforming principle of Pneumococcus
Type ITI.”

Clearly the evidence presented was sub-
stantial and the investigators recognized
that they had made a significant discovery.
Why, therefore, was this great discovery not
immediately recognized by the scientific
world and why did it not influence the
direction of biomedical research? Why did
not the discovery that nucleic acid could
carry and transmit genetic information re-
ceive the recognition that it so richly de-
served, for this was a major discovery, one
contrary to general thought, and hence one
that should have immediately affected scien-
tific thinking in several fields. I am con-
vinced that an unfortunate combination of
circumstances was responsible. Perhaps of
major importance was the fact that the dis-
covery was quite contrary to the dominant
thinking of many years and, hence, required
not only a vigorous presentation but also a
vigorous and continuing promotion for ac-
ceptance. This was not forthcoming. In fact,
although the authors made the correct con-
clusion based on the scientific evidence, they
were modest and somewhat cautious in their
presentation. In their paper, after the first
statement of their “belief that the active
principle is a nucleic acid,” they returned in
the very next and succeeding paragraphs to
terms such as “active material” and “‘trans-
forming substance.” In the discussion they
stated:

So far as the writers are aware, however, a
nucleic acid of the desoxyribose type has not
heretofore been recovered from pneumococci
nor has specific transformation been experi-
mentally induced in vitro by a chemically de-
fined substance,

thus expressing surprise not only in finding
DNA in pneumococci but in the biological ac-
tivity. Later on they said:

If it is ultimately proved beyond a reasonable
doubt that the transforming activity of the ma-
terial described is actually an inherent property
of the nucleic acid, one must still account on a
chemical basis for the biological specificity of
its action.

Similar hedges about the possibility of im-
purities and about the possibility of confirma-
tion of the results are in the summary. Thus
it would appear that they felt that they had
not proved the point “beyond a reasonable

259

doubt,” and thus did not tend to imbue the
reader with confidence in their results. Yet
today most scientists would regard their evi-
dence as impressive.

Another factor was Dr. Avery himself. At
the time of the writing of the paper he was
66 years of age and planning his retirement.
He was a warm and wonderful man of great
intelligence but nevertheless a cautious and
timid man. He had spent most of his scien-
tific effort on the pneumococcus and, although
he had made many important contributions,
his dream of conquering the dreaded acute
lobar pneumonia by means of specific sera
had just been punctured by the discovery
and development of the sulfa drugs. He had
encouraged work on the transforming prin-
ciple over many years. Perhaps the best pic-
ture of Dr. Avery at this time can be gained
from a letter he wrote to his brother, Roy
C. Avery, on May 13, 1943. This letter
should be published in full sometime but
some excerpts may suffice here. After relating
the ups and downs of the early work on
the transforming principle, Avery comments,
“Some job—full of heartaches and heart-
breaks. But at last, perhaps we have it.”
After describing some of the properties of
the nucleic acid Dr. Avery concluded, “This
does not leave much room for impurities—
but the evidence is not great enough yet.”
Later on he stated, “If we are right—of
course that’s not yet proven.” Near the end
of the long letter he wrote:

Sounds like a virus—may be a gene. But
with the mechanisms I am not now concerned
—one step at a time and the first step is, what
is the chemical nature of the transforming
principle? Someone else can work out the rest.
Of course, the problem breathes with implica-
tions. It touches the biochemistry of thymus
type of nucleic acids which are known to con-
tribute the major part of chromosomes but
have been thought to be alike regardless of
major species. It touches genetics, enzyme
chemistry, cell metabolism, and carbohydrate
synthesis, etc. But today it takes a lot of well
documented evidence to convince anyone that
the sodium salt of desoxyribose nucleic acid,
protein free, could possibly be endowed with
such biologically active specific properties and
this evidence we are now trying to get. It’s lots
of fun to blow bubbles but it's wiser to prick
them yourself before someone else tries to. So
there’s the story Roy—right or wrong its been
good fun and lots of work. This supplemented

Arch Environ Health—Vol 21, Sept 1970
260 THE “UNDISCOVERED”

by war work and general supervision of other
important problems in the lab has kept me
busy, as you can well understand. Talk it over
with Goodpasture but don’t shout it around—
until we’re quite sure or at least as sure as
present methods permit. It’s hazardous to go
off half-cocked and embarrassing to have to
retract later. I’m so tired and sleepy I’m afraid
I have not made this very clear—but I want
you to know-—and sure you will see that I
cannot well leave this problem until we’ve got
convincing evidence. Then I look forward and
hope we may all be together—God and the war
permitting and live out our days in peace.

It would appear that by the time the
paper was submitted for publication the fol-
lowing November, Dr. Avery, despite the
terminology used in the paper, had con-
vinced himself that the work and the conclu-
sion were correct and he was ready to retire,
leave the rest to others and to live in peace.
The war was still on, making especial de-
mands of the younger men. The interests of
his two younger physician associates had
changed, for MacLeod had left to accept a
position as professor at New York Universi-
ty School of Medicine in 1941 and McCarty
had accepted additional obligations by join-
ing the Rockefeller US Naval Medical Re-
search Unit in 1942. Although two addition-
al papers on the transforming substance
were published in 1946 by McCarty and
Avery, one on the effect of deoxyribonu-
clease and the other on an improved method
for isolation, Avery had no desire to argue
the merits of the discovery before the scien-
tific world at that time, a world that was
fully preoccupied with the war. I am sure
that he felt the pride of accomplishment
within himself and that sooner or later the
world would recognize that accomplishment.
But the fact remains that no one undertook
the task of describing the discovery and
arguing its merits and significance before
scientific audiences across the nation; hence,
several years passed before there was gen-
eral acceptance.

The Rockefeller Institute for Medical Re-
search, as an organization, seems to have
given scant official attention to the discov-
ery. In the 1944-1946 “Descriptive Pam-
phlet” of the institute there is the state-
ment:

More recently it has been found that desoxy-
ribonucleic acid is intimately associated with

DISCOVERY—STANLEY

the structural organization of pneumococci and,
indeed, that certain nucleic acid polymers of
the desoxyribose type possess the capacity, un-
der appropriate conditions, to induce transfor-
mation of the various types of pneumococci.
Thus, the nature of the capsular polysaccharide
appears to be dependent upon a metabolic
system which at some point is specifically ori-
ented by desoxyribonucleic acid.

The identical words appear in the 1946-1948
“Descriptive Pamphlet,” and there is no
evaluation whatsoever of the significance or
importance of the discovery. One would nev-
er guess that one of the really great discov-
eries within the history of the institute was
being discussed. Hotchkiss, then at the insti-
tute, did take up research on the transform-
ing DNA and made important contributions
as did Alexander, Leidy, and Zamenhof.

Still another factor that undoubtedly con-
tributed to the overlooking of the work on
the transforming principle was the series of
important scientific accomplishments that
took place in the early 1950’s. By this time
Chargaff had demonstrated quite convinc-
ingly that nucleic acid could not be a simple
repeat unit of the four nucleotides by show-
ing that the base composition of different
DNAs differed considerably, although A al-
ways equaled the T content and G the C
content. Ideas with respect to the structure
of the nucleic acids were beginning to
change. In 1951, Lederberg and Zinder dis-
covered transduction and Pauling described
the a-helix for peptides and proteins. In
1952, Hershey and Chase published their
famous experiment showing that the genetic
information of bacterial viruses was carried
in their DNA, work which shared recogni-
tion by the 1969 Nobel Prize in Medicine.
Needless to say this work was generally
regarded as supportive of the earlier work by
Avery and colleagues and did much to se-
cure acceptance of DNA as a transmitter of
genetic information, but even then the suspi-
cion remained strong that protein might be
involved. Also in 1952 Schachman, Pardee,
and Stanier discovered ribosomes and
showed that they contained RNA and not
DNA. In 1958, Watson and Crick revolu-
tionized scientific thought by their presenta-
tion of the double helix for the structure of
nucleic acid. And in 1956, Fraenkel-Conrat
and Gierer and Schramm made the impor-

Arch Environ Health—Vol 21, Sept 1970
THE “UNDISCOVERED”

tant discovery that the biological activity of
the tobacco mosaic virus nucleoprotein was
carried solely by the RNA of the virus, thus
proving for the first time that RNA could
also carry and transmit genetic information.
Truly an unbelievably rich harvest of scien-
tific discovery occurred during a five-year
period, and today Lederberg, Pauling, Her-
shey, Watson, and Crick are Nobel Laure-
ates as a result of accomplishments made
during that period. With so much to occuvy
their minds and so much scientific progress
to digest and assimilate, it is small wonder
that scientists continued to fail to give prop-
er recognition to the discovery of transform-
ing DNA and simply accepted it as a fact of
life. Perhaps full acceptance of the role
played by nucleic acids in the storage and
transmission of genetic information did not
come until the genetic code and the synthe-
sis of DNA, RNA, and proteins was under-
stood.

These, then, are the factors which I be-
lieve to be responsible for the failure of the
scientific world to accord proper recognition
to the discovery of the DNA nature of the
transforming principle of the pneumococcus.
It is dificult to assess which, if any, of these
factors was more important than the others.
Certainly the scientific atmosphere at the
time was very important. The general ac-
ceptance over many years of the “tetranu-
cleotide theory” as the basis for the structure
of DNA, with the great organic chemist Le-
vene as its proponent, certainly was a major
obstacle, for it was argued vigorously and
convincingly that DNA could not possibly
carry biological information. The work of
Brachet and Caspersson in the early 1940s,
relating DNA to protein synthesis within
cells, had little effect. The theory died a slow
and lingering death and the person most re-
sponsible for its demise, Chargaff wrote on p
368 in a book, Nucleic Acids, co-authored by
Davidson, published in 1955:

The tetranucleotide theory continues, for this
reason, to lead a stubborn existence. There
never were any but psychological reasons for its
formulation, as has been pointed out before;
but even in a very recent and massive treatise
there will be found the statement that thymus
nucleic acid is a large chain consisting of 500 to
1,000 tetramucleotide units and that each tet-
ranucleotide is formed by the combination of

DISCOVERY—STANLEY 261
four nucleotides containing adenine, cytosine,
guanine, and thymine, respectively.

If the true structure of DNA had only
been known and accepted, it is highly prob-
able that the discovery by Dr. Avery and his
colleagues and its significance would have
received ready recognition. Furthermore, in-
stead of a timid and unusually cautious
presentation the authors might have set
forth their conclusions with greater firmness
and confidence and this would have fostered
ready acceptance. If Dr. Avery or one of his
younger associates, all of whom were trained
as physicians, had been trained as a profes-
sional biochemist there might have been a
difference in acceptance of the discovery, as
well as a high probability that a person so
trained would have wanted to carry on the
work. The war was undoubtedly a major
factor but one which is difficult to evaluate.
Many persons felt tired and worn out by the
end of the war in 1945, but had Dr. Avery
been ten years younger, instead of looking
forward to retirement, things might have
been different. Many factors played a part
and with the rush of scientific discovery of
the early 1950’s it is easy to understand why
the discovery of transforming DNA did not
receive the public recognition that it so rich-
ly deserved. But of one thing we can be sure
—-Dr. Avery, at the time, recognized the
significance of the discovery and he was
inwardly completely happy and at ease with
himself. We have all come to realize the
greatness of the discovery and I am sure Dr.
Avery knew that this would happen, sooner
or later. He did not need public acclaim for
he was inwardly content because of his
knowledge of the discovery and the honor
accorded him by his close friends.

An Apology

In 1935, I reported the isolation of a
crystallizable protein possessing the proper-
ties of tobacco mosaic virus. By 1938, as a
result of discussions with Bawden and Pirie
in London in July 1936 and subsequent
work in their laboratory and in my labora-
tory, I knew this crystallizable material was
a ribonucleoprotein. I also was familiar with
the work that was going on in Dr. Avery’s
laboratory on the transforming principle and.
over River’s objections, I included a dis-
cussion of it in a chapter entitled ‘“Biochem-

Arch Environ Health—Vol 21, Sept 1970
262 THE “UNDISCOVERED”

istry and Biophysics of Viruses,” written
for Doerr and Hallauer’s Handbuch der Vi-
rusforschung, published in 1938. In this dis-
cussion, I commented: “This phenomenon is
virus-like, and it is because of this and the
fact that it may become important from the
standpoint of the chemistry of viruses that a
discussion is included here.” After describ-
ing the method of preparation and biological
properties of the purified material, I ended
the discussion with:

No chemical tests were made on these purified
preparations, hence nothing is known about the
nature of the active agent. It is to be hoped that
the study of this agent will be continued be-
cause of its virus-like nature.

I was also interested in the RNA of tobac-
co mosaic virus. In 1942, Cohen and I re-
ported the isolation of this RNA with an
unusually large molecular weight and we
reached the conclusion “that the nucleic
acid exists in thread-like molecules, the
length of which is that of the intact virus
molecule.” It is obvious that despite my
1938 writings, I was not impressed with the
significance of the 1944 discovery by Avery,
MacLeod, and McCarty or I would have pre-
pared high molecular weight tobacco mosaic
virus-RNA once again and tested it for virus

DISCOVERY—STANLEY

activity despite the fact that RNA was not
suspected to have genetic properties. It re-
mained for Fraenkel-Conrat to do this im-
portant experiment in my laboratory 14
years later. I have searched my memory and
have failed to find any really extenuating
circumstances for my failure to recognize
the full significance of the discovery of
transforming DNA. Some of the factors
mentioned in the body of this paper may
have had some influence, and with the out-
break of World War II in 1941 my labora-
tory effort was converted almost overnight to
the development of preventative vaccines for
our armed forces and this total effort contin-
ued until well past the end of the. war in
September 1945. But there should have been
time for me to accord proper early recogni-
tion to the discovery of transforming DNA
in 1944, and for my failure to do this I
apologize. I am pleased to be able to record
in this paper the fact that in 1947 Lasker
Awards in Basic Research were presented to
Drs. Avery and Francis.

This communication was developed from notes
which were used in connection with a speech I pre-
sented on the occasion of the dedication of the
Avery Memorial Gateway at the Rockefeller Insti-
tute in New York, Sept 29, 1965.

Arch Environ Health-——Vol 21, Sept 1970

Printed and Published in the United States of America