IN CELEBRATION OF
THE 50~~ ANNIVERSARY
of the publication of theexperiment that transformed biology andshowed that genes are made of DNA
The Rockefeller University is pleased
to reprint the original 1944 paper by
Oswald T. Avery, Colin M. MacLeod,and Maclyn McCarty

STUDIESON THE CHEMICALTHE SUBSTANCEINDUCING

OF PNEUMOCOCCALNATUREOF

TRANSFORMATION
TYPES

1944-1994

MaclyMcCarty-FIFTY YEARS AGO

t h’is F eb ruary, The]ournal of Experimental
Medicine published an account by Oswald Avery, Cohn MacLeod and Maclyn McCarty ofwhat the Nobel Prize-winning British immunologist Peter Medawar has called “the mostinteresting and portentous biological experiment of the 20th century.” Reading this revolutionary paper today, it is difficult to recover the state of ignorance that then existed
regarding the ultimate underpinnings of life processes. Before this paper’s publication in
1944, the gene was an entity without substance. The position of genes on chromosomes infruit flies and maize could be mapped by classical genetic methods; but unlocking thechemical identity of the gene lay beyond the reach of geneticists working on these higherorganisms. Thus the revolutionary character of Avery, MacLeod and McCarty’s conclusion: the chemical agent causing a genetic transformation of pneumococcal bacteria isdeoxyribonucleic acid, known today by its famous acronym, DNA. By showing for the
first time that DNA was a carrier of genetic information, Avery, MacLeod and McCarty
opened the gateway to the modern era of the life sciences.
Oswald T. Avery, the senior author of the 1944 paper, was recruited to the
Hospital of The Rockefeller Institute for Medical Research (now The Rockefeller
University) in 1913 by the Hospital’s first director, Rufus Cole. The goal of Avery’s
research for the next 35 years was to understand the pneumococcus bacteria and design
therapies for lobar pneumonia. Cohn MacLeod joined the Avery laboratory in 1934.
Maclyn McCarty joined the laboratory in 1941. Like Avery, both were medical scientists,
not geneticists or biochemists. How did three medical scientists studying the microbe
responsible for lobar pneumonia, which was called “captain of the men of death” by thelate 19th century physician William Osler, discover the secret of life? Rene Dubos, whose
studies in the Avery laboratory helped launch the antibiotic era, wrote that “the investigations carried out by Avery and his school between 1913 and 1940 have provided the pattern, the master plan, used by our generation for the immunochemical study of infectious
processes.” Throughout this period, Avery’s laboratory focused on the major factor underlying the virulence of pneumococci: the polysaccharide capsules that surround different
pneumococci strains, enabling these bacteria to evade phagocytosis by the body’s immune
cells. It was Avery and his colleagues who determined the chemical identity of these capsules, setting the stage for the diagnosis of lobar pneumonia, the application of specificserum therapies, and the design of specific antibiotics and novel vaccines. All of these diagnostic and therapeutic procedures depended on understanding the chemical specificity ofthe different protective capsules that shroud different pneumococcal types. It was this sameobsession with understanding the chemical specificity of microbial pathogenesis that drovePthe Avery lab to discover the stuff genes are made of.
In 1928, the British medical researcher Fred Griffith discovered that if virulent,
encapsulated Type III pneumococci were killed and injected with living, non-encapsulated(and thus harmless) Type II pneumococci into a laboratory mouse, the mouse died. But
why? From the mouse’s body Griffith recovered living, virulent Type III pneumococci.
The killed bacteria had provided some chemical substance that enabled the naked type IIbacteria to grow a lethal type III capsule. One type of bacteria had become transformed
into another.
At first, Avery refused to believe Griffith’s results. Avery’s research career, after
all, had centered on the analysis of the distinct types of pneumococcal capsules. Griffith’s
claim that these types could become transformed one into the other challenged the verybasis of Avery’s research program. But Griffith’s results were soon confirmed and extended within Avery’s laboratory by Martin Dawson and Lionel Alloway. Through Alloway’s
efforts, a crude extract was prepared that caused transformation in a test tube. Within thisextract was some mysterious “transforming principle”-- a substance that could cause a heritable change of bacterial cells. The task of Avery, Colin MacLeod and Maclyn McCarty
was to purify this transforming substance--this genetic needle within the bacterial haystack.
“The crude extract (type III) is full of capsular polysaccharide,” Avery wrote to his
brother Roy in 1943, “ C (somatic) carbohydrate, nucleoproteins, free nucleic acids of boththe yeast [RNA] and thymus [DNA] types, lipids and other cell constituents. Try to find inthat complex mixture the active principle..11 Try to isolate and chemically identify the particular [transforming] substance....1 Some job--full of heartaches and heartbreaks. But atlast perhaps we have it....” The specific substance was DNA--a direct challenge to thethen-current dogma that only proteins could exist in the multitude of forms needed to store
all an organism’s genetic information. As Avery remarked to his brother: “Who could haveguessed it?”In the years that followed, the conclusion that genetic information was carried by
DNA, and not proteins, was greatly bolstered by the work of McCarty and Rollin
Hotchkiss at The Rockefeller University, and by the bacteriophage workers Al Hershey andMartha Chase at Cold Spring Harbor Laboratories. But the most perplexing aspect of the1944 discovery remained. DNA was widely assumed to be a “stupid molecule,” a polymer
whose four chemical bases of adenine, thymine, guanine, and cytosine were repeated without variation in the chromosomes of all species. This “tetranucleotide hypothesis” wasimpossible to reconcile with the requirement that different genes have different chemicalcompositions. As Avery, MacLeod and McCarty concluded in their 1944 paper: “If theresults of the present study on the chemical nature of the transforming principle are confirmed, the nucleic acids must be regarded as possessing biological specificity the chemicalbasis of which is as yet undetermined.”
The biochemist Erwin Chargaff, working at the College of Physicians andSurgeons at Columbia University, has recalled his response to the 1944 paper and its conclusion. “ . . ..I saw in dark contours the beginning of a grammar of biology.....Avery gave
us the first text of a new language, or rather he showed us where to look for it. I resolved
to search for this text.” Chargaffs pioneering chemical analysis of DNA from different
organisms showed definitively that the base compositions of DNA varied from species tospecies; a genetic code could be written with sequences of DNA’s four bases. Chargaff’s
studies also revealed a puzzling surprise: while the base composition varied among species,in the DNA from all species examined the amount of thymine equaled that of adenine, andthe amount of cytosine that of guanine.
The puzzle of these base ratios, or “Chargaff’s rules”, was not pieced together
until a Saturday morning in 1953, when a young phage worker named James Watson, thenworking in the Cavendish Laboratory at Cambridge, began shifting cardboard cut-outs of
DNA bases in out of various pairing possibilities. “Suddenly,” Watson recalled,“I became aware that an adenine-thymine pair held together by two hydrogen bonds was
identical in shape to a guanine-cytosine pair....” The two sets of equally-sized base pairs
could form the steps of the spiral staircase turning within the core of DNA’s double-helicalbackbone. As Watson wrote: “Chargaffs rules then suddenly stood out as a consequenceof a double-helical structure for DNA. Even more exciting, this type of double helix suggested a [genetic] replication scheme...Always pairing adenine with thymine and guaninewith cytosine meant that the base sequences of the two intertwined chains were complementary to each other. Given the base sequence of one chain, that of its partner was automatically determined.” With Watson and Crick’s determination of DNA’s three-dimensional structure, the great mystery of how genetic information was replicated from cell tocell and from parent to offspring was essentially solved. By 1966 DNA’s genetic code forsynthesizing proteins had been deciphered as well.
The effect of this revolution in biology unleashed by Avery, MacLeod and
McCarty’s paper has been incalculable. It is only more recently, however, that our knowledge of DNA’s genetic role has circled back to the concerns of these three medical scientists--the diagnosis and treatment of human disease. The Human Genome Project;prospects for gene therapy; the production of new drugs and vaccines by recombinant
DNA technology; discoveries uncovering the genetic basis of cancer; the role of “naturaltransformation” in the transfer of genes for antibiotic-resistance to virulent bacteria such as
Avery’s pneumococcus--all these advances promise the kind of rational medical science
only glimpsed by Avery and his colleagues at The Rockefeller University Hospital.
Oswald T. Avery retired from Rockefeller in 1948, and moved to Nashville,
Tennessee to live near his beloved brother Roy. He died there in 1955, at the age of 76.
Colin MacLeod left Rockefeller in 1941 to begin a distinguished career as professor of
microbiology at New York University Medical School; he died in London in 1972. The
paper’s junior author, Maclyn McCarty, has remained a vita1 member of the Rockefeller
campus since his arrival in 1941,as John D. Rockefeller Jr. Professor, vice president for
medical affairs, physician-in-chief of The Rockefeller University Hospital, and now as aprofessor emeritus who still comes in daily to his office on campus. Like Avery, Mac is aquiet and modest man, one who would readily echo a favorite saying of his mentor’s:“Apply your brakes when tempted to blow your own horn.” One of the great pleasures ofour 50th anniversary celebration of this historic paper’s publication is to be able to see
Mac’s face light up when we sound the trumpet for him, and for Oswald Avery and Colin
MacLeod.

Torsten Wiesel
President, The Rockefeller University

Suggested Reading
Rene Dubos
The Professor, the Institute and DNA
Maclyn McCarty
The Transforming Principle
JamesWatson The Double Helix
Robert Olby The Path to the Double Helix
Horace Freeland Judson The Eighth Day of Creation

[Reprinted from TEE JOURNAL OF EXPERIIQWTAL MEDICZNE,February 1,1944,
Vol. 79, No. 2, pp. 137-1581

STUDIES ON THE CHEMICALINDUCING
TRANSFORMATIONINDUCTION

NATURE OF THE SUBSTANCE
OF PNEUMOCOCCAL TYPES

OF TRANSFORMATION B Y A DESOXYRIJSONUCLEIC ACID FRACTIONISOLATED FROM PNE~AXOCOCCUSTYPE III

B Y OSWALD

T. AVERY, M.D., COLIN M. MAcLEOD,
MACLYN MCCARTY,* M.D.

M.D.,

AND

(From the Hospital of The Rockefeller Institute for Medical Research)
PLATE 1

(Received for publication, November 1, 1943)

Biologists have long attempted by chemical means to induce in higherorganisms predictable and specific changes which thereafter could be transmitted

in series as hereditarycharacters.

Among

microorganisms

the most

striking example of inheritable and specific alterations in cell structure andfunction that can be experimentally induced and are reproducible under welldefined and adequately controlled conditions is the transformation of specificThis phenomenon was first described by Griffith (1)types of Pneumococcus.
who succeeded in transforming
an attenuatedand non-encapsulated(R)variant derived from one specific type into fully encapsulated and virulent (S)cells of a heterologous specific type.
A typical instance will suffice to illustrate
the techniques originally used and serve to indicate the wide variety of transformations that are possible within the limits of this bacterial species.
GrifIith found that mice injected subcutaneously with a small amount of a living
R culture derived from Pneumococcus Type II together with a large inoculum ofheat-killed Type III (S) cells frequently succumbed to infection, and that the heart’s
blood of these animals yielded Type III pneumococci in pure culture. The fact that
the R strain was avirulent and incapable by itself of causing fatal bacteremia and the
additional fact that the heated suspension of Type III cells contained no viable organisms brought convincing evidence that the R forms growing under these conditions had newly acquired the capsular structure and biological specificity of Type III
pneumococci.
The original observations of Griffith were later confirmed by Neufeld and LevinSubsethal (2), and by Baurhenn (3) abroad, and by Dawson (4) in this laboratory.
quently Dawson and Sia (5) succeeded in inducing transformation in vitro. Thii
they accomplished by growing R cells in a fluid medium containing anti-R serum andheat-killed encapsulated S cells. They showed that in the test tube as in the animal
body transformation can be selectively induced, depending on the type specificityof the S cells used in the reaction system. Later, Alloway (6) was able to cause* Work done in part as Fellow in the Medical Sciences of the National ResearchCouncil.
137

138

TRANSFORMATIONOF PNEUMOCOCCAL

TYPES

specific transformation in vitro using sterile extracts of S cells from which all formed
elements and cellular debris had been removed by Berkefeld filtration. He thus
showed that crude extracts containing active transforming material in soluble form
are as effective in inducing specific transformation as are the intact cells from which
the extracts were prepared.
Another example of transformation which is analogousto the interconvertibility ofpneumococcaltypes lies in the field of viruses. Berry and Dedrick (7) succeededinchanging the virus of rabbit fibroma (Shope) into that of infectious myxoma (Sanarelli). These investigators inoculated rabbits with a mixture of active fibroma virus
together with a suspensionof heat-inactivated myxoma virus and produced in the
animals the symptoms and pathological lesions characteristic of infectious myxomatosis. On subsequent animal passagethe transformed virus was transmissible andinduced myxomatous infection typical of the naturally occurring disease. Later
Berry (8) was successfulin inducing the same transformation using a heat-inactivated suspensionof washed elementary hodies of myxoma virus. In the caseof theseviruses the methods employed were similar in principle to those used by Gri&th in
the transformation of pneumococcaltypes. These observations have subsequently
been con6rrned by other investigators (9).
The present paper is concerned with a more detailed analysis of the phenomenon of transformation of specific types of Pneumococcus. The major interest
has centered in attempts to isolate the active principle from crude bacterial
extracts and to identify if possible its chemical nature or at least to characterize it sufficiently to place it in a general group of known chemical substances.
For purposes of study, the typical example of transformation chosen as aworking model was the one with which we have had most experience and whichconsequently seemed best suited for analysis. This particular example represents the transformation of a non-encapsulated R variant of Pneumococcus
Type II to Pneumococcus Type III.
EXPERIMENTAL

Transformation of pneumococcal types in z&o requires that certain cultural
conditions be fulfilled before it is possible to demonstrate the reaction even in
the presence of a potent extract. Not only must the broth medium be optimalfor growth but it must be supplemented by the addition of serum or serous
fluid known to possess certain special properties. Moreover, the R variant,
as will be shown later, must be in the reactive phase in which it has the capacity
to respond to the transforming stimulus. For purposes of convenience theseseveral components as combined in the transforming test will be referred toas the reaction syslem. Each constituent of this system presented problems
which required clarification before it was possible to obtain consistent andreproducible results. The various components of the system will be describedin the following order: (1) nutrient broth, (2) serum or serous fluid, (3) strainof R Pneumococcus, and (4) extraction, purification, and chemical nature of
the transforming principle.

aOSWALDT.

AVERY,COLIN

M.

MAcLEOD,AND

MACLYNMCCARTY

139

1. Nutrient Broth.-Beef heart infusion broth containing 1 per cent neopeptonewith no added dextrose and adjusted to an initial pH of 7.6-1.8 is used as the basic
medium. Individual lots of broth show marked and unpredictable variations in theproperty of supporting transformation.
It has been found, however, that charcoal
adsorption, according to the method described by MacLeod and Mirick (10) for
removal of sulfonamide inhibitors, eliminates to a large extent these variations; consequently this procedure is used as routine in the preparation of consistently effective
broth for titrating the transforming activity of extracts.
2. Serum or Serous Fluid.-In
the first successful experiments on the induction oftransformation in vitro, Dawson and Sia (5) found that it was essential to add serum
to the medium. Anti-R pneumococcal rabbit serum was used because of the observation that reversion of an R pneumococcus to the homologous S form can be induced
by growth in a medium containing anti-R serum. Alloway (6) later found that ascitic or chest fluid and normal swine serum, all of which contain R antibodies, arecapable of replacing antipneumococcal rabbit serum in the reaction system. Someform of serum is essential, and to our knowledge transformation k vitro has neverbeen effected in the absence of serum or serous fluid.
In the present study human pleural or ascitic fluid has been used almost exclusively.
It became apparent, however, that the effectiveness of different lots of serum varied
and that the differences observed were not necessarily dependent upon the content
of R antibodies, since many sera of high titer were found to be incapable of supporting transformation.
This fact suggested that factors other than R antibodies are
involved.
It has been found that sera from various animal species, irrespective of theirimmune properties, contain an enzyme capable of destroying the transforming principle in potent extracts. The nature of this enzyme and the specific substrate on
which it acts will be referred to later in this paper. This enzyme is inactivated byheating the serum at 60”-65”C., and sera heated at temperatures known to destroy
the enzyme are often rendered effective in the transforming system. Further analysis has shown that certain sera in which R antibodies are present and in which theThisenzyme has been inactivated may nevertheless fail to support transformation.
fact suggests that still another factor in the serum is essential. The content of thisfactor varies in different sera, and at present its identity is unknown.
There are at present no criteria which can be used as a guide in the selection ofsuitable sera or serous fluids except that of actually testing their capacity to support
Fortunately, the requisite properties are stable and remain unimtransformation.
paired over long periods of time; and sera that have been stored in the refrigerator
for many months have been found on retesting to have lost little or none of their
original effectivenessin supporting transformation.

The recognition of these various factors in serum and their r81e in the reaction
system has greatly

facilitatedthe standardizationof the cultural

conditions

required for obtaining consistent and reproducible results.
3. The R Struin (ZWA).-The
unencapsulated R strain used in the present
study was derived from a virulent ‘5” culture of Pneumococcus Type II.
It will be recalled that irrespective of type derivation all “R” variants ofPneumococcus are characterized by the lack of capsule formation and the

140

TRANSFORMATIONOF PNElJbtOCOCCAL

TYPES

consequent loss of both type specificity and the capacity to produce infectionin the animal body. The designation of these variants as R forms has beenused to refer merely to the fact that on artificial media the colony surface is“rough” in contrast to the smooth, glistening surface of colonies of encapsulated
S cells.
The R strain referred to above as R36A was derived by growing the parent S
culture of PneumococcusType II in broth containing Type II antipneumococcus
rabbit serum for 36 serial passagesand isolating the variant thus induced. The
strain R36A has lost all the specific and distinguishing characteristics of the parent S
organisms and consists only of attenuated and non-encapsulatedR variants. ThechangeS + R is often a reversible one provided the R cells are not too far “degraded.”
The reversion of the R form to its original specifictype can frequently be accomplishedby successiveanimal passagesor by repeated serial subculture in anti-R serum. Whenreversion occurs under these conditions, however, the R culture invariably reverts tothe encapsulatedform of the samespecifictype as that from which it was derived (11).
Strain R36A has becomerelatively fixed in the R phaseand has never spontaneously
reverted to the Type II S form. Moreover, repeated attempts to causeit to revert
under the conditions just mentioned have in all instances been unsuccessful.
The reversible conversion of S$R within the limits of a single type is quite
different from the transformation of one specific type of Pneumococcus intoanother specific type through the R form. Transformation of types has neverbeen observed to occur spontaneously and has been induced experimentally
only by the special techniques outlined earlier in this paper. Under these
conditions, the enzymatic synthesis of a chemically and immunologically
difIerent capsular polysaccharide is specifically oriented and selectively determined by the specific type of S cells used as source of the transforming agent.
In the course of the present study it was noted that the stock culture of R36 on
serial transfers in blood broth undergoes spontaneous dissociation giving rise to anumber of other R variants which can be distinguished one from another by colony
form. The significance of this in the present instance lies in the fact that of four
different variants isolated from the parent R culture only one (R36A) is susceptibleto
the transforming action of potent extracts, while the others fail to respond and arewholly inactive in this regard. The fact that differences exist in the responsivenessof different R variants to the same specific stimulus enphasizesthe care that must be
exercised in the selection of a suitable R variant for use in experiments on transformation. The capacity of this R strain (R36A) to respond to a variety of different
transforming agents is shown by the readinesswith which it can be transformed toTypes I, III, VI, or XIV, as well as to its original type (Type II), to which, as pointed
out, it has never spontaneouslyreverted.
Although the significance of the following fact will become apparent later on, it
must be mentioned here that pneumococcalcells possessan enzyme capable of destroying the activity of the transforming principle. Indeed, this enzyme has been

OSWALDT.

AVERY,COLIN

hf. MACLEOD,AND

MACLYNMCCARTY

142

found to be present and highly active in the autolysates of a number of different
strains. The fact that this intracellular enzyme is released during autolysis may
explain, in part at least, the observation of Dawson and Sia (5) that it is essential inbringing about transformation in the test tube to use a small inoculum of young andactively growing R cells. The irregularity of the results and often the failure toinduce transformation when large inocula are used may be attributable to the releasefrom autolyzing cells of an amount of this enzyme sufficient to destroy the transforming principle in the reaction system.
In order to obtain consistent and reproducible results, two facts must beborne in mind: first, that an R culture can undergo spontaneous dissociationand give rise to other variants which have lost the capacity to respond to thetransforming
stimulus; and secondly, that pneumococcal cells contain an
intracellularenzyme which when released destroys the activity of the transforming principle.
Consequently, it is important to select a responsive strain
and to prevent as far as possible the destructive changes associated withautolysis.
Method of Titration of Transforming Activity.-In
the isolation and purification of the active principle from crude extracts of pneumococcal cells it isdesirable to have a method for determining quantitatively
the transforming
activity of various fractions.
The experimental procedure used is as follows: Sterilization of the material to betested for activity is accomplished by the use of alcohol since it has been found that
this reagent has no effect on activity.
A measured volume of extract is precipitatedin a sterile centrifuge tube by the addition of 4 to 5 volumes of absolute ethyl alcohol,
and the mixture is allowed to stand 8 or more hours in the refrigerator in order to effectsterilization.
The alcohol precipitated material is centrifuged, the supernatantdiscarded, and the tube containing the precipitate is allowed to drain for a few minutes
in the inverted position to remove excess alcohol. The mouth of the tube is thencarefully flamed and a dry, sterile cotton plug is inserted. The precipitate is redissolved in the original volume of saline. Sterilization of active material by thistechnique has invariably proved effective. This procedure avoids the loss of active
substance which may occur when the solution is passed through a Berkefeld filter oris heated at the high temperatures required for sterilization.
To the charcoal-adsorbed broth described above is added 10 per cent of the sterile
ascitic or pleural fluid which has previously been heated at 60°C. for 30 minutes, inorder to destroy the enzyme known to inactivate the transforming principle. The
enriched medium is distributed under aseptic conditions in 2.0 cc. amounts in steriletubes measuring 15’ X 100 mm. The sterilized extract is diluted serially in saline
neutralized to pH 7.2-7.6 by addition of 0.1 N NaOH, or it may be similarly diluted
in aa/ phosphate buffer, pH 7.4. 0.2 cc. of each dilution is added to at least 3 or 4
tubes of the serum medium. The tubes are then seeded with a 5 to 8 hour blood
broth culture of R36A. 0.05 cc. of a 1O-4 dilution of this culture is added to eachtube, and the cultures are incubated at 37°C. for 18 to 24 hours.

142

TRANSFORMATIONOF PNEUMOCOCCALTYPESThe anti-R properties of the serum in the medium cause the R cells toagglutinate during growth, and clumps of the agglutinated cells settle to thebottom of the tube leaving a clear supernatant. When transformation occurs,
the encapsulated S cells, not being affected by these antibodies, grow diffusely
throughout the medium.
On the other hand, in the absence of transformation
the supernatant remains clear, and only sedimented growth of R organisms

occurs.

This difference in the character of growth makes it possible by inspec-tion alone to distinguish tentatively
between Positive and negative results.
As routine all the cultures are plated on blood agar for confirmation and further
bacteriological identification.
Since the extracts used in the present study
were derived from Pneumococcus Type III, the differentiation
between thecolonies of the original R organism and those of the transformed S cells is
especially striking, the latter being large, glistening, mucoid colonies typical of

Pneumococcus Type III.
Figs. 1 and 2 illustrate these differences in colonyform.
A typical protocol of a titration of the transforming activity of a highly
purifiedpreparation

is given in Table IV.

Preparative Methods
Source Mule&l.--In
the present investigation a stock laboratory strain of Pneumococcus Type III (A66) has been used as source material for obtaining the active
principle. Mass cultures of these organisms are grown in 50 to 75 liter lots of plain
beef heart infusion broth. After 16 to 18 hours’ incubation at 37°C. the bacterial
cells are collected in a steam-driven sterilizable Sharples centrifuge. The centrifuge
is equipped with cooling coils immersed in ice water so that the culture fluid is thoroughly chilled before flowing into the machine. This procedure retards autolysis
during the course of centrifugation.
The sedimented bacteria are removed from thecollecting cylinder and resuspended in approximately 150 cc. of chilled saline (0.85
per cent NaCl), and care is taken that all clumps are thoroughly emulsified. The
glass vessel containing the thick, creamy suspension of cells is immersed in a waterbath, and the temperature of the suspension rapidly raised to 65°C. During theheating process the material is constantly stirred, and the temperature maintained
at 65°C. for 30 minutes. Heating at this temperature inactivates the intracellularenzyme known to destroy the transforming principle.
Extraction of Heat-Killed Cells.-Althoughvarious procedures have been used,
only that which has been found most satisfactory will be described here. The heatkilled cells are washed with saline 3 times. The chief value of the washing process
is to remove a large excess of capsular polysaccharide together with much of the protein, ribonucleic acid, and somatic “C” polysaccharide. Quantitative titrations oftransforming activity have shown that not more than 10 to 15 per cent of the active
material is lost in the washing, a loss which is small in comparison to the amount ofinert substances which are removed by this procedure.
After the final washing, the cells are extracted in 150 cc. of saline containing sodium
desoxycholate in final concentration of 0.5 per cent by shaking the mixture me-

OSWALD

T. AVERY,

COLIN

hf.

UcLEOD,AND

MACLYNMCCARTY

143

chanically 30 to 60 minutes. The cells are separated by centrifugation, and the extraction process is repeated 2 or 3 times. The desoxycholate extracts prepared inthis manner are clear and colorless. These extracts are combined and precipitatedby the addition of 3 to 4 volumes of absolute ethyl alcohol. The sodium desoxycholate being soluble in alcohol remains in the supematant and is thus removed at thisstep. The precipitate forms a fibrous mass which floats to the surface of the alcoholand can be removed directly by lifting it out with a spatula. The excess alcohol isdrained from the precipitate which is then redissolved in about 50 cc. of saline. The
solution obtained is usually viscous, opalescent, and somewhat cloudy,
Deproteinization and Removal of Capsular Polysaccharide.-The solution is thendeproteinized by the chloroform method described by Sevag (12). The procedure isrepeated 2 or 3 times until the solution becomes clear. After this preliminary treatment the material is reprecipitated in 3 to 4 volumes of alcohol. The precipitate
obtained is dissolved in a larger volume of saline (150 cc.) to which is added 3 to 5
mg. of a purified preparation of the bacterial enzyme capable of hydrolyzing theType III capsular polysaccharide (13). The mixture is incubated at 37’C., and thedestruction of the capsular polysaccharide is determined by serological tests withType III antibody solution prepared by dissociation of immune precipitate according to the method described by Liu and Wu (14). The advantages of using theantibody solution for this purpose are that it does not react with other serologically
active substances in the extract and that it selectively detects the presence of the capsular polysaccharide in dilutions as high as 1: 6,000,OOO. The enzymatic breakdownof the polysaccharide is usually complete within 4 to 6 hours, as evidenced by the lossof serological reactivity.
The digest is then precipitated in 3 to 4 volumes of ethyl
alcohol, and the precipitate is redissolved in 50 cc. of saline. Deproteinization by thechloroform process is again used to remove the added enzyme protein and remaining
traces of pneumococcal protein. The procedure is repeated until no further film ofprotein-chloroform gel is visible at the interface.
Alcohol FractionaLion.-Following
deproteinization and enzymatic digestion of thecapsular polysaccharide, the material is repeatedly fractionated in ethyl alcohol asfollows. Absolute ethyl alcohol is added dropwise to the solution with constantstirring. At a critical concentration varying from 0.8 to 1.0 volume of alcohol theactive material separates out in the form of fibrous strands that wind themselves
around the stirring rod. This precipitate is removed on the rod and washed in a 50
per cent mixture of alcohol and saline. Although the bulk of active material is removed by fractionation at the critical concentration, a small but appreciable amountremains in solution. However, upon increasing the concentration of alcohol to 3
volumes, the residual fraction is thrown down together with inert material in the formof a flocculent precipitate. This flocculent precipitate is taken up in a small volume
of saline (5 to 10 cc.) and the solution again fractionated by the addition of 0.8 to 1.0
volume of alcohol. Additional fibrous material is obtained which is combined withthat recovered from the original solution. Alcoholic fractionation is repeated 4 to 5
times. The yield of fibrous material obtained by this method varies from 10 to 25
mg. per 75 liters of culture and represents the major portion of active material present
in the original crude extract.
Eject of Temperature.-As a routine procedure all steps in purification were carried144

TRANSFORMATIONOF PNEUMOCOCCAL

TYPES

out at room temperature unless specifically stated otherwise. Becauseof the theoretical advantage of working at low temperature in the preparation of biologically
active material, the purification of one lot (preparation 44) was carried out in the cold.
In this instance all the above procedures with the exception of desoxycholate exextraction and enzyme treatment were conducted in a cold room maintained at O-PC.
This preparation proved to have significantly higher activity than did material similarly prepared at room temperature.
Desoxycholate extraction of the heat-hilled cells at low temperature is less eflicientand yields smaller amounts of the active fraction. It has been demonstrated thathigher temperatures facilitate extraction of the active principle, although activity
best preserved at low temperatures.

Analysis of Pwijed
General Properties.-Saline

solutionsisTransforming Material
containing

0.5 to 1.0 mg. per cc. of thepurified substance are colorless and clear in diffuse light. However, in strongtransmitted light the solution is not entirely clear and when stirred exhibits asilky sheen. Solutions at these concentrations are highly viscous.
Purified material dissolved in physiological salt solution and stored at 24’C.
retains its activity in undiminished titer for at least 3 months. However, when
dissolved in distilled water, it rapidly decreases in activity and becomes completely inert within a few days. Saline solutions stored in the frozen state in a
CO* ice box (-70°C.) retain full potency for several months. Similarly,
material precipitated from saline solution by alcohol and stored under thesupernatant remains active over a long period of time. Partially purified
material can be preserved by drying from the frozen state in the lyophileapparatus. However, when the same procedure is used for the preservationof the highly purified substance, it is found that the material undergoes changes
resulting in decrease in solubility and loss of activity.
The activity of the transforming principle in crude extracts withstands
heating for 30 to 60 minutes at 65°C. Highly purified preparations of active
material are less stable, and some loss of activity occurs at this temperature.
A quantitative study of the effect of heating purified material at higher temperatures has not as yet been made. Alloway (6), using crude extracts prepared from Type III pneumococcal cells, found that occasionally activity could
still be demonstrated after 10 minutes’ exposure in the water bath to temperatures as high as 90°C.
The procedures mentioned above were carried out with solutions adjusted
to neutral reaction, since it has been shown that hydrogen ion concentrationsin the acid range result in progressive loss of activity.
Inactivation occurs
rapidly at pH 5 and below.
Qualitative Chemical Tests.-The purified material in concentrated solution
gives negative biuret and Millon tests. These tests have been done directly
on dry material with negative results. The Dische diphenylamine reaction

OSWALDT.

AVERY,COLIN

hf.

MAcLEOD,AND

MACLYN

145

MtXARTY

for desoxyribonucleic acid is strongly positive. The orcinol test (Bial) for
ribonucleic acid is weakly positive. However, it has been found that in similar
concentrations pure preparations of desoxyribonucleic acid of animal origin
prepared by different methods give a Bial reaction of corresponding intensity.
Although no specific tests for the presence of lipid in the purified material
have been made, it has been found that crude material can be repeatedly extracted with alcohol and ether at - 12’C. without loss of activity.
In addition,
as will be noted in the preparative procedures, repeated alcohol precipitationand treatment with chloroform result in no decrease in biological activity.
Elementary
Chmical Analysis.LFour
purified preparations were analyzed
for content of nitrogen, phosphorus, carbon, and hydrogen. The results arepresented in Table I. The nitrogen-phosphorus ratios vary from 1.58 to 1.75
with an average value of 1.67 which is in close agreement with that calculated
TABLE

Elementary ChcmicaEAnalysis of Purijed
Preparation No.

37
38B
42
4%
Theory for sodium
desoxyribonucleate

I

Preparations of tke Transforming Substams

Ca1hOll

Hydrogen

NitrogenPhosphorus

fin Ccrrl

pn cm&l)LI Ceml

pn cml

34.27
35.50
-

3.89
3.76
-

14.21
15.93
15.36
13.40

8.57
9.09
9.04
8.45

34.20

3.21

15.32

I

9.05

NIP ratio

1.66
1.75
1.69
1.58

I

1.69

on the basis of the theoretical structure of sodium desoxyribonucleate (tetranucleotide). The analytical figures by themselves do not establish that thesubstance isolated is a pure chemical entity. However, on the basis of thenitrogen-phosphorus ratio, it would appear that little protein or other substances containing nitrogen or phosphorus are present as impurities since ifthey were this ratio would be considerably altered.
Enzymatic Analysis.--Various crude and crystalline enzymes2 have beentested for their capacity to destroy the biological activity of potent bacterial
extracts. Extracts buffered at the optimal pH, to which were added crystalline
trypsin and chymotrypsin or combinations of both, suffered no loss in activity
following treatment with these enzymes. Pepsin could not be tested because
1 The elementary chemical analyseswere made by Dr. A. Elek of The Rockefeller
Institute.
2 The authors are indebted to Dr. John H. Northrop and Dr. M. Kunitz of The
Rockefeller Institute for Medical Research, Princeton, N. J., for the samples ofcrystalline trypsin, chymotrypsin, and ribonuclease used in this work.

146

TRANSFORMATIONOF PNETJ?.fOCOCCAL.

TYPES

extracts are rapidly inactivated at the low pH required for its use. Prolonged
treatment with crystalline ribonuclease under optimal conditions caused no
demonstrable decrease in transforming activity.
The fact that trypsin,chymotrypsin, and ribonuclease had no effect on the transforming principle isfurther evidence that this substance is not ribonucleic acid or a protein susceptible to the action of tryptic enzymes.
In addition to the crystalline enzymes, sera and preparations of enzymes
obtained from the organs of various animals were tested to determine theireffect on transforming activity.
Certain of these were found to be capable ofcompletely destroying biological activity.
The various enzyme preparations
tested included highly active phosphatases obtained from rabbit bone by themethod of Martland and Robison (15) and from swine kidney as described by
TABLE IIThe Inactivation of Transforming Prindple

I
Dog intestinal mucosa....................
Rabbit bonephosphatase.................
Swinekidney“.................
Pneumocaccus
autolysates................
Normal dog and rabbit serum.............

by Crude Enzyme Preparations
Enzymatic activity

+
+
+
-l-

+
+
+
+

+
+
+

+
+
+

H. and E. Albers (16). In addition, a preparation made from the intestinal
mucosa of dogs by Levene and Dillon (17) and containing a polynucleotidase
for thymus nucleic acid was used. Pneumococcal autolysates and a commercial preparation of pancreatin were also tested. The alkaline phosphataseactivity of these preparations was determined by their action on &glycerophosphate and phenyl phosphate, and the esterase activity by their capacity
to split tributyrin.
Since the highly purified transforming material isolated
from pneumococcal extracts was found to contain desoxyribonucleic acid,these same enzymes were tested for depolymerase activity on known samples
of desoxyribonucleic acid isolated by Mirskys from fish sperm and mammaliantissues. The results are summarized in Table II in which the phosphatase,esterase, and nucleodepolymerase activity of these enzymes is compared with
their capacity to destroy the transforming principle. Analysis of these resultsshows that irrespective of the presence of phosphatase or esterase only those
3 The authors express their thanks to Dr. A. E. Mirsky of the Hospital of The
Rockefeller Institute for these preparations of desoxyribonucleicacid.

OSWALDT.

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147

preparations shown to contain an enzyme capable of depolymerizing authentic
samples of desoxyribonucleic acid were found to inactivate the transforming
principle.
Greenstein and Jenrette (18) have shown that tissue extracts, as well as themilk and serum of several mammalian species, contain an enzyme system whichcauses depolymerization of desoxyribonucleic acid. To this enzyme system
Greenstein has later given the name desoxyribonucleodepolymerase (19).
These investigators determined depolymerase activity by following the reduction in viscosity of solutions of sodium desoxyribonucleate. The nucleateand enzyme were mixed in the viscosimeter and viscosity measurements madeat intervals during incubation at 30°C. In the present study this method wasused in the measurement of depolymerase activity except that incubation was
carried out at 37°C. and, in addition to the reduction of viscosity, the actionof the enzyme was further tested by the progressive decrease in acid precipitability of the nucleate during enzymatic breakdown.
The effect of fresh normal dog and rabbit serum on the activity of thetransforming substance is shown in the following experiment.
Sera obtained from a normal dog and normal rabbit were diluted with an equalvolume of physiological saline. The diluted serum was divided into three equal
portions. One part was heated at WC. for 30 minutes, another at 60°C. for 30
minutes, and the third was used unheated as control. A partially purified preparation of transforming material which had previously been dried in the lyophile apparatus was dissolvedin saline in a concentration of 3.7 mg. per cc. 1.0 cc. of this solution
was mixed with 0.5 cc. of the various samplesof heated and unheated diluted sera,
and the mixtures at pH 7.4 were incubated at 37°C. for 2 hours. After the serum had
been allowed to act on the transforming material for this period, all tubes were heatedat 65’C. for 30 minutes to stop enzymatic action. Serial dilutions were then made insaline and tested in triplicate for transforming activity according to the proceduredescribed under Method of titration. The results given in Table III illustrate thedifferential heat inactivation of the enzymesin dog and rabbit serum which destroy
the transforming principle.
From the data presented in Table III it is evident that both dog and rabbit
serum in the unheated state are capable of completely destroying transforming
activity.
On the other hand, when samples of dog serum which have beenheated either at 60°C. or at 65°C. for 30 minutes are used, there is no loss oftransforming activity.
Thus, in this species the serum enzyme responsiblefor destruction of the transforming principle is completely inactivated at
6O’C. In contrast to these results, exposure to 6X. for 30 minutes was
required for complete destruction of the corresponding enzyme in rabbit serum.
The same samples of dog and rabbit serum used in the preceding experimentwere also tested for their depolymerase activity on a preparation of sodium
desoxyribonucleate isolated by Mirsky from shad sperm.

148

TRANSFORMATIONOF PNEUMOCOCCAL

TYPES

A highly viscous solution of the nucleate in distilled water in a concentration of 1
mg. per cc. was used. 1.0 cc. amounts of heated and unheated sera diluted in saline
as shown in the preceding protocol were mixed in Ostwald viscosimeters with 4.0 cc.
TABLE III
Real Inactivation of Enzymes in Dog and Rabbit Serum Which Destroy theTransjorming SubstanceBfferential--

Triplicate tests

Dilution*-.-

Unheated

Undiluted
1:s
1:25

60°C. for 30
min.

Undiluted
1:s
1:25

I

Dog serum

6YC. for 30
min.
-_

Unheated-- - 1
CfilZY
3~'s
"M
fg
,
-- -

+
t
t

Rabbit serum

1

60°C. for 30
min.
6S’C. for 30
min.

-.

Control (nc,serum)-

None

R only
R “
R “
SIII
SIII
SIII

+
+
+

SIII
+
SIII
+
SIII
+
-- - -R only
Undiluted
R “
1:s
Undiluted
1:s
1:25

R

-

+
+
+
-_-_
Undiluted
+
1:s
+
1:25
+
- -Undiluted
1:s
1:25

3

2

.- - 72
113 COlOllY
lo
form
!&
0!G
ca- I- .R only
R only
R “
R “
R “
R “

Undiluted
1:s
1:25

1:25

-

“

R only
R “
R “

+
+
+

SIII
SIII
SIII

SIII
+
SIII
+
SIII
+
- -R only
R I‘
R “

+
+
+
-

SIII
SIII
SIII

-

R onlyR “
R “-

R only
R “
R “

SIT1
SIII
SIII

+
+
+
- _
+
+
+

SIII
SIII
SIT1

+
+
+
- -SD1
+
SIII
t
SIII
t
- - -

SIII
SIII
SIII

SIII
SIII
SIII

SIII
SIII
SIII

_

R only
R “
R “

-

SIII
SIII
SIII

* Dilution of the digest mixture of serum and transforming substance.
of the aqueous solution of the nucleate. Determinations of viscosity were madeimmediately and at intervals over a period of 24 hours during incubation at 37%

The results of this experiment are graphically presented in Chart 1. In thecase of unheated serum of both dog and rabbit, the viscosity fell to that ofwater in 5 to 7 hours. Dog serum heated at 60°C. for 30 minutes brought about

OSWALD

T. AVERY,

COLIN

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?!+UcLEOD,AND

MACLYNMCCARTY

149

no significant reduction in viscosity after 22 hours. On the other hand, heating
rabbit serum at 60°C. merely reduced the rate of depolymerase action, andafter 24 hours the viscosity was brought to the same level as with the unheatedserum. Heating at 65’C., however, completely destoyed the rabbit serum
depolymerase .
Thus, in the case of dog and rabbit sera there is a striking parallelism between
the temperature of inactivation of the depolymerase and that of the enzyme
which destroys the activity of the transforming principle. The fact that thisdifference in temperature of inactivation is not merely a general property of all
enzymes in the sera is evident from experiments on the heat inactivation oftributyrin esterase in the same samples of serum. In the latter instance, theresults are the reverse of those observed with depolymerase since the esteraseof rabbit serum is almost completely inactivated at 60°C. while that in dog
serum is only slightly affected by exposure to this temperature.
Of a number of substances tested for their capacity to inhibit the actionof the enzyme known to destroy the transforming principle, only sodium fluoride
has been found to have a significant inhibitory effect. Regardless of whether
this enzyme is derived from pneumococcal cells, dog intestinal mucosa, pancreatin, or normal sera its activity is inhibited by fluoride. Similarly it has
been found that fluoride in the same concentration also inhibits the enzymatic
depolymerization of desoxyribonucleic acid.
The fact that transforming activity is destroyed only by those preparations
containiig depolyrnerase for desoxyribonucleic acid and the further fact that

150

TRANSFORMATIONOF PNEUMOCOCCAL

TYPES

in both instances the enzymes concerned are inactivated at the same temperature and inhibited by fluoride provide additional evidence for the belief thatthe active principle is a nucleic acid of the desoxyribose type.
Serological Analysis.-In
the course of chemical isolation of the active
material it was found that as crude extracts were purified, their serologicalactivity in Type III antiserum progressively decreased without corresponding
loss in biological activity.
Solutions of the highly purified substance itself
gave only faint trace reactions in precipitin tests with high titer Type III
antipneumococcus rabbit serum.4 It is well known that pneumococcal protein
can be detected by serological methods in dilutions as high as 1: 50,000 and thecapsular as well as the somatic polysaccharide in dilutions of at least 1: 5,000,000.
In view of these facts, the loss of serological reactivity indicates that these cell
constituents have been almost completely removed from the final preparations.
The fact that the transforming substance in purified state exhibits little or no
serological reactivity is in striking contrast to its biological specificity ininducing pneumococcal transformation.
Physicochemical Studies.5-A purified and active preparation of the transforming substance (preparation 44) was examined in the analytical ultracentrigue. The material gave a single and unusually sharp boundary
indicating that the substance was homogeneous and that the molecules were
uniform in size and very asymmetric. Biological activity was found to be
sedimented at the same rate as the optically observed boundary, showingthat activity could not be due to the presence of an entity much different
in size. The molecular weight cannot be accurately determined until measurements of the diffusion constant and partial specific volume have beenmade. However, Tennent and Vilbrandt (20) have determined the diffusion
constant of several preparations of thymus nucleic acid the sedimentation rateof which is in close agreement with the values observed in the present study.
Assuming that the asymmetry of the molecules is the same in both instances,
it is estimated that the molecular weight of the pneumococcal preparation is of
the order of 500,000.
Examination of the same active preparation was carried out by electrophoresis in the Tiselius apparatus and revealed only a single electrophoretic component of relatively high mobility comparable to that of a nucleic acid. Transforming activity was associated with the fast moving component giving the
4 The Type III antipneumococcusrabbit serum employed in this study was furnished through the courtesy of Dr. Jules T. Freund, Bureau of Laboratories, Department of Health, City of New York.
s Studies on sedimentation in the ultracentrifuge were carried out by Dr. A.
Rothen; the electrophoretic analyseswere made by Dr. T. Shedlovsky, and the ultraviolet absorption curves by Dr. G. I. Lavin. The authors gratefully acknowledge
their indebtednessto these members of the staff of The Rockefeller Institute.

OSWALDT.

AVERY,COLIN?d. MACLEOD,AND

MACLYN151

MCCARTY

optically visible boundary. Thus in both the electrical and centrifugal fields,
the behavior of the purified substance is consistent with the concept that biological activity is a property of the highly polymerized nucleic acid.
Ultraviolet absorption curves showed maxima in the region of 2600 A andminima in the region of 2350 A. These findings are characteristic of nucleic
acids.
Quantitative Determination oj Biological Activity.-In its highly purified state
the material as isolated has been found to be capable of inducing transformationin amounts ranging from 0.02 to 0.003 pg. Preparation 44, the purification ofwhich was carried out at low temperature and which had a nitrogen-phosphorus
TABLE

IV

Titration of Transforming Activity of Preparation 44
Quadruplicate tests

Transfonnin~ principle
Preparation 44’

1

T

2
-7

Dilution%E

Diffusegrowth

3

4

-7

ii!%

DiffusegrowthDiffusegrowth

I
g
-

4%.

10-Z
1(-J-2.6 A:;
10-a0.1
10-s.’
0.03
10-4
0.01
10-4.6
0.003
10-s
0.001
Control
None

+
+
+
+
+
-

SIII
SIII
SIII
SIII
SIII
R only
R “
R “

+
+
+
+
+
+
-

-

SIII
SIII
SIII
SIII
SIII
SIII
R only
R “

+
+
+
+
+
+-

SIII
SIII
SIII
SIII
SIII
R only
R “
R “

SIII
SIII
SIII
SIII
SIII
SIII
R onlyR “-* Solution from which dilutions were made contained 0.5 mg. per cc. of pursed material.
0.2 cc. of each dilution added to quadruplicate tubes containing 2.0 cc. of standard serum
broth. 0.05 cc. of a lo-’ dilution of a blood broth culture of R36A is added to each tube.

ratio of 1.58, exhibited high transforming activity.
of this preparation is given in Table IV.

Titration of the activity

A solution containing 0.5 mg. per cc. was serially diluted as shown in the protocol.
0.2 cc. of each of these dilutions was added to quadruplicate tubes containing 2.0 cc.
of standard serum broth. All tubes were then inoculated with 0.05 cc. of a lOA
dilution of a 5 to 8 hour blood broth culture of R36A. Transforming activity wasdetermined by the procedure described under Method of titration.
The data presented in Table IV show that on the basis of dry weight 0.003
pg. of the active material brought about transformation.
Since the reaction
system containing the 0.003 pg. has a volume of 2.25 cc., this represents a final
concentration of the purified substance of 1 part in 600,000,000.

152

TRANSFORMATIONOF PNEUMOCOCCALTYPESDISCUSSION

The present study deals with the results of an attempt to determine the chemical nature of the substance inducing specific transformation of pneumococcal
types. A desoxyribonucleic acid fraction has been isolated from Type III
pneumococci which is capable of transforming unencapsulated R variants
derived from Pneumococcus Type II into fully encapsulated Type III cells.
Thompson and Dubos (21) have isolated from pneumococci a nucleic acid of theribose type. So far as the writers are aware, however, a nucleic acid of thedesoxyribose type has not heretofore been recovered from pneumococci nor has
specific transformation been experimentally induced in vitro by a chemicallydefined substance.
Although the observations are limited to a single example, they acquire
broader significance from the work of earlier investigators who demonstrated
the interconvertibilityof various pneumococcal types and showed that the
specificity of the changes induced is in each instance determined by the particular type of encapsulated cells used to evoke the reaction. From the pointof view of the phenomenon in general, therefore, it is of special interest that in
the example studied, highly purified and protein-free material consisting largely,
if not exclusively, of desoxyribonucleic acid is capable of stimulating unencapsulated R variants of Pneumococcus Type II to produce a capsular polysaccharide identical in type specificity with that of the ceils from which theinducing substance was isolated. Equally striking is the fact that the substance evoking the reaction and the capsular substance produced in response
to it are chemically distinct, each belonging to a wholly different class of chemical compounds.
The inducing substance, on the basis of its chemical and physical properties,
appears to be a highly polymerized and viscous form of sodium desoxyribonucleate. On the other hand, the Type III capsular substance, the synthesisof which is evoked by this transforming agent, consists chiefly of a non-nitrogenous polysaccharide constituted of glucose-glucuronic acid units linked inglycosidic union (22). The presence of the newly formed capsule containing
this type-specific polysaccharide confers on the transformed cells all the distinguishing characteristics of Pneumococcus Type III.
Thus, it is evident
that the inducing substance and the substance produced in turn are chemicallydistinct and biologically specific in their action and that both are requisite indetermining the type specificity of the cell of which they form a part.
The experimental data presented in this paper strongly suggest that nucleic
acids, at least those of the desoxyribose type, possess different specificities asevidenced by the selective action of the transforming principle. Indeed, thepossibility of the existence of specific differences in biological behavior of nucleic
acids has previously been suggested (23, 24) but has never been experimentallydemonstrated owing in part at least to the lack of suitable biological methods.

OSWALDT.

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MACLYNMCCARTY

153

The techniques used in the study of transformation appear to afford a sensitivemeans of testing the validity of this hypothesis, and the results thus far obtained add supporting evidence in favor of this point of view.
If it is ultimately proved beyond reasonable doubt that the transforming
activity of the material 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. At first glance, immunological methods would appear to offer theideal means of determining the differential specificity of thisgroupof biologicallyimportant substances. Although the constituent units and general patternof the nucleic acid molecule have been defined, there is as yet relatively little
known of the possible effect that subtle differences in molecular configuration
may exert on the biological specificity of these substances. However, since
nucleic acids free or combined with histones or protamines are not known tofunction antigenically, one would not anticipate that such differences would be
revealed by immunological techniques. Consequently, it is perhaps not surprising that highly purified and protein-free preparations of desoxyribonucleic
acid, although extremely active in inducing transformation, showed only fainttrace reactions in precipitin tests with potent Type III antipneumococcus
rabbit sera.
From these limited observations it would be unwise to draw any conclusion
concerning the immunological significance of the nucleic acids until further
knowledge on this phase of the problem is available. Recent observations by
Lackman and his collaborators (25) have shown that nucleic acids of both theyeast and thymus type derived from hemolytic streptococci and from animaland plant sources precipitate with certain antipneumococcal sera. The reactions varied with different lots of immune serum and occurred more frequently
in antipneumococcal horse serum than in corresponding sera of immune rabbits. The irregularity and broad cross reactions encountered led these investigators to express some doubt as to the immunological significance of theresults. Unless special immunochemical methods can be devised similar to
those so successfully used in demonstrating the serological specificity of simple
non-antigenic substances, it appears that the techniques employed in the studyof transformation are the only ones available at present for testing possible
differences in the biological behavior of nucleic acids.
Admittedly there are many phases of the problem of transformation that
require further study and many questions that remain unanswered largely
because of technical difficulties. For example, it would be of interest to know
the relation between rate of reaction and concentration of the transforming
substance; the proportion of cells transformed to those that remain unaffectedin the reaction system. However, from a bacteriological point of view, numerical estimations based on colony counts might prove more misleading thanenlightening because of the aggregation and sedimentation of the R cells ag-

154

TRANSFORMATIONOF PNEUMOCOCCAL

TYPES

glutinated by the antiserum in the medium. Attempts to induce transformation in suspensions of resting cells held under conditions inhibiting growth andmultiplication have thus far proved unsuccessful, and it seems probable thattransformation occurs only during active reproduction of the cells. Importantin this connection is the fact that the R cells, as well as those that have undergone transformation, presumably also all other variants and types of pneumococci, contain an intracellular enzyme which is released during autolysis
and in the free state is capable of rapidly and completely destroying the activityof the transforming agent. It would appear, therefore, that during the logarithmic phase of growth when cell division is most active and autolysis least
apparent, the cultural conditions are optimal for the maintenance of the balance
between maximal reactivity of the R cell and minimal destruction of the transforming agent through the release of autolytic ferments.
In the present state of knowledge any interpretation of the mechanism involved in transformation must of necessity be purely theoretical. The biochemical events underlying the phenomenon suggest that the transforming
principle interacts with the R cell giving rise to a coordinated series of enzymatic
reactions that culminate in the synthesis of the Type III capsular antigen.
The experimental findings have clearly demonstrated that the induced alterations are not random changes but are predictable, always corresponding intype specificity to that of the encapsulated cells from which the transforming
substance was isolated. Once transformation has occurred, the newly acquired
characteristics are thereafter transmitted in series through innumerable transfers in artificial media without any further addition of the transforming agent.
Moreover, from the transformed cells themselves, a substance of identicalactivity can again be recovered in amounts far in excess of that originally added
to induce the change. It is evident, therefore, that not only is the capsularmaterial reproduced in successive generations but that the primary factor,
which controls the occurrence and specificity of capsular development, is also
reduplicated in the daughter cells. The induced changes are not temporary
modifications but are permanent alterations which persist provided the cultural conditions are favorable for the maintenance of capsule formation. The
transformed cells can be readily distinguished from the parent R forms notalone by serological reactions but by the presence of a newly formed and visible
capsule which is the immunological unit of type specificity and the accessory
structure essential in determining the infective capacity of the microorganismin the animal body.
It is particularly significant in the case of pneumococci that the experimentally induced alterations are definitely correlated with the development of a
new morphological structure and the consequent acquisition of new antigenic
and invasive properties. Equally if not more significant is the fact that thesechanges are predictable, type-specific, and heritable.

OSWALDT.

AVERY,COLIN

M.

MAcLEOD,AND

MACLYNMCCARTY

1.55

Various hypotheses have been advanced in explanation of the nature of thechanges induced. In his original description of the phenomenon Griffith (1)suggested that the dead bacteria in the inoculum might furnish some specificprotein that serves as a “pabulum” and enables the R form to manufacture acapsular carbohydrate.
More recently the phenomenon has been interpreted from a genetic point ofview (26, 27). The inducing substance has been likened to a gene, and thecapsular antigen which is produced in response to it has been regarded as a geneproduct. In discussing the phenomenon of transformation Dobzhansky (27)
has stated that “If this transformation is described as a genetic mutation-and
it is difficult to avoid so describing it-we are dealing with authentic cases of
induction of specific mutations by specific treatments. . . .”Another interpretation of the phenomenon has been suggested by Stanley(28) who has drawn the analogy between the activity of the transforming agent
and that of a virus. On the other hand, Murphy (29) has compared the causative agents of fowl tumors with the transforming principle of Pneumococcus.
He has suggested that both these groups of agents be termed “transmissible
mutagens” in order to differentiate them from the virus group. Whatever
may prove to be the correct interpretation, these differences in viewpoint indicate the implications of the phenomenon of transformation in relation to similar
problems in the fields of genetics, virology, and cancer research.
It is, of course, possible that the biological activity of the substance describedis not an inherent property of the nucleic acid but is due to minute amounts
of some other substance adsorbed to it or so intimately associated with it as toescape detection. If, however, the biologically active substance isolated inhighly purified form as the sodium salt of desoxyribonucleic acid actually proves
to be the transforming principle, as the available evidence strongly suggests,
then nucleic acids of this type must be regarded not merely as structurally
important but as functionally active in determining the biochemical activities
and specific characteristics of pneumococcal cells. Assuming that the sodium
desoxyribonucleate and the active principle are one and the same substance,then the transformation described represents a change that is chemically induced and specifically directed by a known chemical compound. If the resultsof the present study on the chemical nature of the transforming principle areconfirmed, then nucleic acids must be regarded as possessing biological
specificity the chemical basis of which is as yet undetermined.
SUMMARY

1. From Type III pneumococci a biologically active fraction has been isolated
in highly purified form which in exceedingly minute amounts is capable underappropriate cultural conditions of inducing the transformation of unencapsulated R variants of Pneumococcus Type II into fully encapsulated cells of the1.56

TRANSFORMATIONOF PNETJMOCOCCAL

TYPES

same specific type as that of the heat-killed microorganisms from which theinducing material was recovered.
2. Methods for the isolation and purification of the active transforming material are described.
3. The data obtained by chemical, enzymatic, and serological analyses
together with the results of preliminary studies by electrophoresis, ultracentrifugation, and ultraviolet spectroscopy indicate that, within the limits of themethods, the active fraction contains no demonstrable protein, unbound lipid,
or serologically reactive polysaccharide and consists principally, if not solely, ofa 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 inseries. The various hypotheses that have been advanced concerning thenature of these changes are reviewed.
CONcLUSION

The evidence presented supports the belief that a nucleic acid of the desoxyribose type is the fundamental unit of the transforming principle of Pneumococcus Type III.
BIBLIOGRAPHY
1. Griffith, F., J. Hyg., Cambridge, Eng., 1928, 27, 113.
2. Neufeld, F., and Levinthal, W., 2. Zmmuniliitsjmsch., 1928, 66, 324.
3. Baurhenn, W., Centr. Bakt., 1. Abt., Orig., 1932, 126, 68.
4. Dawson, M. H., Z. Exp. Med., 1930,61, 123.
5. Dawson, M. H., and Sia, R. H. P., J. Exp. Med., 1931,64,681.
6. Alloway, J. L., J. Exp. Med., 1932, 66, 91; 1933, 67, 265.
7. Berry, G. P., and Dedrick, H. M., J. Bacf., 1936, 31, 50.
8. Berry, G. P., Arch. Path., 1937, 24, 533.
9. Hurst, E. W., Bril. J. Exp. Palh., 1937, 18, 23. Hoffstadt, R. E., and Pilcher,
K. S., J. Infect. Dis., 1941, 68, 67. G ar d ner, R. E., and Hyde, R. R., J.
Infect. Dis., 1942, 7l, 47. Houlihan, R. B., Proc. Sot. Exp. Biol. and Med.,

1942,61,259.
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TRANSFORMATIONOF PNEUMOCOCCALEXPLANATIONTYPESOF PLATE 1

The photograph was made by Mr. Joseph B. Haulenbeek.
FIG. 1. Colonies of the R variant (R36A) derived from Pneumococcus Type II.
Plated on blood agar from a culture grown in serum broth in the absence of thetransforming substance. X 3.5.
FIG. 2. Colonies on blood agar of the same cells after induction of transformation
during growth in the same medium with the addition of active transforming principle isolated from Type III pneumococci. The smooth, glistening, mucoid colonies
shown are characteristic of Pneumococcus Type III and readily distinguishable fromthe small, rough colonies of the parent R strain illustrated in Fig. 1. X3.5.

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