The Avery–MacLeod–McCarty experiment was an experimental demonstrat...
This work builds upon Griffith's 1928 experiment in which mice were...
While the work of Griffith and his successors showed a transforming...
This section describes the preparation of bacterial DNA in reproduc...
From a modern perspective, this section highlights some fairly mund...
As per the Griffith experiment, the transforming principle must be ...
Both Biuret and Millon tests are used to identify proteins in a sol...
These elegant experiments use enzymes known to digest protein (tryp...
This sentence is the crux of the paper; they isolated DNA from the ...
The authors prophecy the discovery of genes. Taking a step back to ...
At the time, it was known that proteins could take almost infinite ...
Given the limited understanding of molecular genetics at the time, ...
Short of calling DNA the hereditary molecule, they recognize that t...
At this point, a "gene" is simply a hereditary unit. The term itsel...
Here is the most anodyne statement of fact in all of biology, folks.
STUDIES ON THE CHEMICAL NATURE OF THE SUBSTANCE
INDUCING TRANSFORMATION OF PNEUMOCOCCAL TYPES
INDUCTION OF TRANS]~ORMATION BY A DESOXYRIBONUCLEIC
ACID FRACTION
ISOLATED I~RO~¢ PNEUMOCOCCUS TYPE
III
BY OSWALD T. AVERY, M.D., COLIN M. MACLEOD, M.D., AND
MACLYN McCARTY,* M.D.
(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 higher
organisms predictable and specific changes which thereafter could be trans-
mitted in series as hereditary characters. Among microSrganisms the most
striking example of inheritable and specific alterations in cell structure and
function that can be experimentally induced and are reproducible under well
defined and adequately controlled conditions is the transformation of specific
types of Pneumococcus. This phenomenon was first described by Gri~th (1)
who succeeded in transforming an attenuated and 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 trans-
formations that are possible within the limits of this bacterial species.
Gri~th found that mice injected subcutaneously with a small amount of a living
1~ culture deri, ed from Pneumococcus Type H together with a large inoculum of
heat-killed Type III (S) cells frequently succumbed to infection, and that the heart's
blood of these animals yielded Type HI pneumococci in pure culture. The fact that
the P~ strain was avirulent and incapable by itself of causing fatal bacteremia and the
additional fact that the heated suspension of Type HI cells eoataincd no viable or-
ganisms brought convincing evidence that the 1~ forms growing under these condi-
tions had newly acquired the capsular structure and biological specificity of Type III
pneumococci.
The original observations of Griffith were later confirmed by Neufeld and Levin-
thal (2), and by Banrherm (3) abroad, and by Dawson (4) in this laboratory. Subse-
quently Dawson and Sia (5) succeeded in inducing transformation in ~tro. This
they accomplished by growing R cells in a fluid medium containing anti-R serum and
heat-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 specificity
of 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 Research
Council.
137
138
TRA.NS~ORMATION O:F PNEITMOCOCCAL TYPES
specific transformation in vilro using sterile extracts of S cells from which all formed
elements and celiular 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 analogous to the interconvertibility of
pneumococcal types lies in the field of viruses. Berry and Dedrick (7) succeeded in
changing the virus of rabbit fibroma (Shope) into that of infectious myxoma (San-
arelli). These investigators inoculated rabbits with a mixture of active fibroma virus
together with a suspension of heat-inactivated myxoma virus and produced in the
animals the symptoms and pathological lesions characteristic of infectious myxoma-
tosis. On subsequent animal passage the transformed virus was transmissible and
induced myxomatous infection typical of the naturally occurring disease. Later
Berry (8) was successful in inducing the same transformation using a heat-inacti-
vated suspension of washed elementary bodies of myxoma virus. In the case of these
viruses the methods employed were similar in principle to those used by Griffith in
the transformation of pneumococcal types. These observations have subsequently
been confirmed by other investigators (9).
The present paper is concerned with a more detailed analysis of the phenome-
non 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 charac-
terize it sufficiently to place it in a general group of known chemical substances.
For purposes of study, the typical example of transformation chosen as a
working model was the one with which we have had most expenence and which
consequently seemed best suited for analysis. This particular example repre-
sents the transformation of a non-encapsulated R variant of Pneumococcus
Type II to Pneumococcus Type III.
EXPEJ~I~E.NTAL
Transformation of pneumococcal types in ritro 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 optimal
for 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 these
several components as combined in the transforming test will be referred to
as the reaction system. Each constituent of this system presented problems
which required clarification before it was possible to obtain consistent and
reproducible results. The various components of the system will be described
in the following order: (1) nutrient broth, (2) serum or serous fluid, (3) strain
of R Pneumococcus, and (4) extraction, purification, and chemical nature of
the transforming principle.
OSWALD T. AVERY, COLIN M. M.AcLEOD~ AND ~T.ACLYlq McCARTY 139
1. Nutrient Broth.--Beef heart infusion broth containing 1 per cent neopeptone
with no added dextrose and adjusted to an initial pH of 7.6--7.8 is used as the basic
medium. Individual lots of broth show marked and unpredictable variations in the
property 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; conse-
quently 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 of
transformation 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 observa-
tion 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 as-
citic or chest fluid and normal swine serum, all of which contain R antibodies, are
capable of replacing antipneumococcal rabbit serum in the reaction system. Some
form of serum is essential, and to our knowledge transformation in vitro has never
been 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 support-
ing transformation. This fact suggested that factors other than R antibodies are
involved.
It has been found that sera from various animal species, irrespective of their
immune properties, contain an enzyme capable of destroying the transforming prin-
ciple 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 by
heating 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 an-
alysis has shown that certain sera in which R antibodies are present and in which the
enzyme has been inactivated may nevertheless fail to support transformation. This
fact suggests that still another factor in the serum is essential. The content of this
factor 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 of
suitable sera or serous fluids except that of actually testing their capacity to support
transformation. Fortunately, the requisite properties are stable and remain unim-
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 effectiveness in supporting transformation.
The recognition of these various factors in serum and their r61e in the reaction
system has greatly facilitated the standardization of the cultural conditions
required for obtaining consistent and reproducible results.
3. The R Strain (R36A).--The unencapsulated R strain used in the present
study was derived from a virulent "S" culture of Pneumococcus Type II.
It will be recalled that irrespective of type derivation all "R" variants of
Pneumococcus are characterized by the lack of capsule formation and the
140
TRANSFORMATION OF PNED'MOCOCCAL TYPES
consequent loss of both type specificity and the capacity to produce in/ection
in the animal body. The designation of these variants as R forms has been
used 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 Pneumococcus Type II in broth containing Type II antipneumococcus
rabbit serum for 36 serial passages and 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-encapsulated R variants. The
change S -~ R is often a reversible one provided the g cells are not too far "degraded."
The reversion of the R form to its original specific type can frequently be accomplished
by successive animal passages or by repeated serial subculture in anti-R serum. When
reversion occurs under thes e conditions, however, the g culture invariably reverts to
the encapsulated form of the same specific type as that from which it was derived (11).
Strain R36A has become relatively fixed in the R phase and has never spon~neously
reverted to the Type II S form. Moreover, repeated attempts to cause it 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 into
another specific type through the R form. Transformation of types has never
been 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
different capsular polysaccharide is specifically oriented and selectively de-
termined 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 K36 on
serial transfers in blood broth undergoes spontaneous dissociation giving rise to a
number of other P-. 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 susceptible to
the transforming action of potent extracts, while the others fail to respond and are
whoUy inactive in this regard. The fact that differences exist in the responsiveness
of different R variants to the same specific stimulus enphasizes the care that must be
exercised in the selection of a suitable R variant for use in experiments on trans-
formation. The capacity of this R strain (R36A) to respond to a variety of different
transforming agents is shown by the readiness with which it can be transformed to
Types I, Ill, VI, or XIV, as well as to its original type (Type If), to which, as pointed
out, it has never spontaneously reverted.
Although the significance of the foUowing fact will become apparent later on, it
must be mentioned here that pneumococcal cells possess an enzyme capable of de-
stroying the activity of the transforming principle. Indeed, this enzyme has been
OSWALD T. AVERY, COLIN M. ~cLEOD, AND M.ACLYN McCARTY 141
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 in
bringing about transformation in the test tube to use a small inoculum of young and
actively growing R cells. The irregtflarity of the results and often the failure to
induce transformation when large inocula are used may be attributable to the release
from autolyzing cells of an amount of this enzyme sutScient to destroy the trans-
forming principle in the reaction system.
In order to obtain consistent and reproducible results, two facts must be
borne in mind: first, that an R culture can undergo spontaneous dissociation
and give rise to other variants which have lost the capacity to respond to the
transforming stimulus; and secondly, that pneumococcal cells contain an
intracellular enzyme which when released destroys the activity of the trans-
forming principle. Consequently, it is important to select a responsive strain
and to prevent as far as possible the destructive changes associated with
autolysis.
Method of Titration of Transforming Activity.pin
the isolation and purifica-
tion of the active principle from crude extracts of pneumococcal cells it is
desirable 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 be
tested 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 precipitated
in 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 effect
sterilization. The alcohol precipitated material is centrifuged, the supernatant
discarded, 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 then
carefully flamed and a dry, sterile cotton plug is inserted. The precipitate is redis-
solved in the original volume of saline. Sterilization of active material by this
technique has invariably proved effective. This procedure avoids the loss of active
substance which may occur when the solution is passed through a Berkefeld filter or
is 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 l~leural fluid which has previously been heated at 60°C. for 30 minutes, in
order to destroy the enzyme known to inactivate the transforming principle. The
enriched medium is distributed under aseptic conditions in 2.0 cc. amounts in sterile
tubes measuring 15 × 100 ram. The sterilized extract is diluted serially in saline
neutralized to pH 7.2-7.6 by addition of 0.1 r~ NaOH, Or it may be similarly diluted
in u/40 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. 0.0~ cc. of a 10 -4 dilution of this culture is added to each
tube, and the cultures are incubated at 37°C. for 18 to 24 hours.
142
TILa.-NS~'OI~ATION
O~
PN'EU~OCOCCAL TYPES
The anti-R properties of the serum in the medium cause the R cells to
agglutinate during growth, and clumps of the agglutinated cells settle to the
bottom 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 the
colonies of the original K 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 colony
form.
A typical protocol of a titration of the transforming activity of a highly
purified preparation is given in Table IV.
Preparative Methods
Source MateriaL--In the present investigation a stock laboratory strain of Pneu-
mococcus 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. Mter 16 to 18 hours' incubation at 37°C. the bacterial
ceils are collected in a steam-driven sterilizable Sharpies centrifuge. The centrifuge
is equipped with cooling coils immersed in ice water so that the culture fluid is
thor-
oughly chilled before flowing into the machine. This procedure retards autolysis
during the course of centrifugation. The sedimented bacteria are removed from the
collecting cylinder and resuspended in approximately !50 cc. of chilled ~aline (0,85
per cent NaC1), and care is taken that all clumps are thoroughly emulsified. The
glass vessel containing the thick, creamy suspension of cells is immersed in a water
bath, and the temperatur e of the suspension rapidly raised to 65°C. During the
heating process the material is Constantly stirred, and the temperature maintained
at 65°C. for 30 minutes. Heating at this temperature inactivates the intracellular
enzyme known to destroy the transforming principle.
Extraction of Heat-Killed Cdls.--Although various procedures have been used,
only that which has been found most satisfactory will be described here. The heat-
killed cells are washed with saline 3 times. The chief value of the washing process
is to remove a large excess of capsular poly~ccharide together with much of the pro-
tein, ribonucleic acid, and somatic "C" polysaccharide. Quantitatiye titrations of
transforming 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 of
inert substances which are removed by thi s procedure.
Mter 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 M. MACLEOD, AND MACLYN McCARTY 143
chanically 30 to 60 minutes. The cells are separated by centrifugation, an d the ex-
traction process is repeated 2 or 3 times. The desoxycholate extracts prepared in
this manner are clear and colorless. These extracts are combined and precipitated
by the addition of 3 to 4 volumes of absolute ethyl alcohol. The sodium desoxycho-
late being soluble in alcohol remains in the supernatant and is thus removed at this
step. The precipitate forms a fibrous mass which floats to the surface of the alcohol
and can be removed directly by lifting it out with a spatula. The excess alcohol is
drained from the precipitate which is then redissolved in about 50 ce. of saline. The
solution obtained is usually viscous, opalescent, and somewhat cloudy.
Deproteinization and Removal of Capsular Polysaccharide.--The
solution is then
deproteinized by the chloroform method described by Sevag (12). The procedure is
repeated 2 or 3 times until the solution becomes dear. After this preliminary treat-
ment 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
nag. of a purified preparation of the bacterial enzyme capable of hydrolyzing the
Type III capsular polysaccharide (13). The mixture is incubated at 37°C., and the
destruction of the capsular polysaccharide is determined by serological tests with
Type III antibody solution prepared by dissociation of immune precipitate accord-
ing to the method described by Lin and Wu (14). The advantages of using the
antibody 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 cap-
sular polysaccharid e in dilutions as high as 1 : 6,000,000. The enzymatic breakdown
of the polysaccharide is usually complete within 4 to 6 hours, as evidenced by the loss
of 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 the
chloroform process i s again used to remove the added enzyme protein and remaining
traces of pneumococcal protein. Th e procedure is repeated until no further film of
protein-chloroform gel is visible at the interface.
Alcohol Fractionation.--FoUowing
deprote'mization and enzymatic digestion of the
capsular polysaccharide, the material is repeatedly fractionated in ethyl alcohol as
follows. Absolute ethyl alcohol is added dropwis e to the solution with constant
stirring. At a critical concentration varying from 0.8 to 1.0 volume of alcohol the
active material separate s 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 re-
moved by fractionation at the critical concentration, a small but appreciable amount
remains in solution. However, upon increasing the concentration of alcohol to 3
volumes, the residual fraction is thrown down together with inert material in the form
of a flocculent precipitate. This flocculent precipitate is take n up in a small volume
of saline (5 to 10 cc.) and the solutio n again fractionated by the addition of 0.8 to 1.0
volume of alcoho L Additional fibrous material is obtained which is combined with
that 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
rag. per 75 liters of culture and represents the major portion of active material present
in the original crude extract.
Effect of Temperature.--As
a routine procedure all steps in purification were carried
144
TRANS]~OR.M.ATION OF PNEU3KOCOCCAL TYPES
out at room temperature unless specifically stated otherwise. Because of the theo-
retical 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 ex-
extraction and enzyme treatment were conducted in a cold room maintained at 0-4°C.
This preparation proved to have significantly higher activity than did material simi-
larly prepared at room temperature.
Desoxycholate extraction of the heat-killed cells at low temperature is less efficient
and yields smaUer amounts of the active fraction. It has been demonstrate d that
higher temperatures facilitate extraction of the active principle, although activity is
best preserved at low temperatures.
Analysis of Purified Transforming Material
General Properties.--Saline
solutions containing 0.5 to 1.0 rag. per co. of the
purified substance are colorless and clear in diffuse light. However, in strong
transmitted light the solution is not entirely clear and when stirred exhibits a
silky sheen. Solutions at these concentrations are highly viscous.
Purified material dissolved in physiological salt solution and stored at 2-4°C.
retains its activity in undiminished titer forat least 3 months. However, when
dissolved in distilled water, it rapidly decreases in activity and becomes com-
pletely inert within a few days. Saline solutions stored in the frozen state in a
CO2 ice box (--70°C.) retain full potency for several months. Similarly,
material precipitated from saline solution by alcohol and stored under the
supernatant remains active over a long period of time. Partially purified
material can be preserved by drying from the frozen state in the lyophile
apparatus. However, when the same procedure is used for the preservation
of 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 tem-
peratures has not as yet been made. Alloway (6), using crude extracts pre-
pared from Type III pneumococcal ceils, found that occasionally activity could
still be demonstrated after 10 minutes' exposure in the water bath to tem-
peratures 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 concentrations
in 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
OSWALD T. AVERY, COLIN M. MACLEOD, AND MACLYN McCARTY
145
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 Blal 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 ex-
tracted with alcohol and ether at -- 12°C. without loss of activity. In addition,
as will be noted in the preparative procedures, repeated alcohol precipitation
and treatment with chloroform result in no decrease in biological activity.
Elementary Chemical Analysis.L-Four purified preparations were analyzed
for content of nitrogen, phosphorus, carbon, and hydrogen. The results are
presented 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 I
Elementary Cieraical A nalysis of Purified Preparations of tie Transforming Substanc, e.
Preparation No.
37
38B
42
44
Carbon
34.27
35.50
Hydrogen
p~ G~t
3.89
3.76
Nitrogen
pet ~¢~
14.21
15.93
15.36
13.40
Phosphorus
~er c~
8.57
9.09
9.04
8.45
N/P ratio
1.66
1.75
1.69
1.58
Theory for sodium
desoxyribonucleate ..... 34.20 3.21 15.32 9.05 !. 69
on the basis of the theoretical structure of sodium desoxyribonucleate (tetra-
nucleotide). The analytical figures by themselves do not establish that the
substance isolated is a pure chemical entity. However, on the basis of the
nitrogen-phosphorus ratio, it would appear that little protein or other sub-
stances containing nitrogen or phosphorus are present as impurities since if
they were this ratio would be considerably altered.
Enzymatic Analysis.--Various crude and crystalline enzymes 2 have been
tested 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
t The elementary chemical analyses were made by Dr. A. Elek of The Rockefeller
Institute.
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 of
crystalline trypsin, chymotrypsin, and ribonuclease used in this work.
145
TRANS~'ORMATION
O~
PNEUMOCOCCAL 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 is
further evidence that this substance is not ribonucleic acid or a protein suscep-
tible 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 their
effect on transforming activity. Certain of these were found to be capable of
completely destroying biological activity. The various enzyme preparations
tested included highly active phosphatases obtained from rabbit bone by the
method of Martland and Robison (15) and from swine kidney as described by
TABLE II
The Inacti~ogion of Transforming Principle by Crude Enzyme Preparations
Crude enzyme preparations
Dog intestinal mucosa.
Rabbit bone phosphatase .................
Swine kidney
" .................
Pneumococcus autolysates
................
Normal dog and rabbit serum .............
Phosphatase
+
+
+
+
Enzymatic
activity
nepolymer-
Tributyrin ase for
esterase
desox.yribo-
nucleate
+ +
+
+ +
+ +
Inactivation
of
trans°
forming
prlnc~p~e
+
+
+
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 commer-
cial preparation of pancreatin were also tested. The alkaline phosphatase
activity of these preparations was determined by their action on ~-glycero-
phosphate 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 Mirsky s from fish sperm and mammalian
tissues. 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 results
shows 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 desoxyribonucleic acid.
OSWALD T. AVERY, COLIN ~r. MACLEOD~ AND MACLYN MCCARTY 147
preparations shown to contain an enzyme capable of depolymerizing authentic
samples of desoxyribonucleic acid were found to inactivate the transforming
principle.
Greenstein and J'enrette (18) have shown that tissue extracts, as well as the
milk and serum of several mammalian species, contain an enzyme system which
causes depolymerization of desoxyribonucleic acid. To this enzyme system
Greenstein has later given the name desoxyribonucleodepolymerase (19).
These investigators determined depolymerase activity by following the reduc-
tion in viscosity of solutions of sodium desoxyribonucleate. The nucleate
and enzyme were mixed in the viscosimeter and viscosity measurements made
at intervals during incubation at 30°C. In the present study this method was
used in the measurement of depolymerase activity except that incubation was
carried out at 37°C. and, in addition to the reduction of viscosity, the action
of the enzyme was further tested by the progressive decrease in acid precip-
itability of the nucleate during enzymatic breakdown.
The effect of fresh normal dog and rabbit serum on the activity of the
transforming substance is shown in the following experiment.
Sera obtained from a normal dog and normal rabbit were diluted with an equal
volume of physiological saline. The diluted serum was divided into three equal
portions. One part was heated at 65°C. for 30 minutes, another at 50°C. for 30
minutes, and the third was used unheated as control. A partially purified prepara-
tion of transforming material which had previously been dried in the lyophile appara-
tus was dissolved in saline in a concentration of 3.7 rag. per cc. 1.0 cc. of this solution
was mixed with 0.5 cc. of the various samples of 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 heated
at 65°C. for 30 minutes to stop enzymatic action. Serial dilutions were then made in
saline and tested in triplicate for transforming activity according to the procedure
described under Method of titration. The results given in Table III illustrate the
differential heat inactivation of the enzymes in 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 been
heated either at 60°C. or at 65°C. for 30 minutes are used, there is no loss of
transforming activity. Thus, in this species the serum enzyme responsible
for destruction of the transforming principle is completely inactivated at
60°C. In contrast to these results, exposure to 65°C. 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 experiment
were also tested for their depolymerase activity on a preparation of sodium
desoxyribonucleate isolated by Mirsky from shad sperm.
148
TRANSFORMATION 0~'
PN~'MOCOCCAL
TYPES
A
highly viscous solution
of
the nucleate in distilled water in a concentration of 1
rag. 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 HI
Differential Heat Inactivation of Enzymes in Dog and Rabbit Serum Wkic, k Destroy the
Transforming Substanze
Dog serum
Rabbit serum
Heat
treatment
of
serum
Unheated
60°C.
for 30
min.
65°C. for 30
m~.
Unheated
60°C.
for 30
min.
Dilution*
Triplicate tests
1 2
3
~I ~ Colony ~ Colony ~ ~ Colony
Undiluted -- ; R only -- R only -- R only
1:5 --I R " -- R " -- R "
1:25 --R " -- R " -- R "
Undiluted + SllI + Sill + Sill
1:5 + SllI + SIII + SIII
1:25 + Sill + Sill + Sill
Undiluted
+
SIII
+
Sill
+
Sill
1:5
+
SIII
+
Sill
+
SHI
1:25
+
Sill
+
Sill
+
Sill
Undiluted -- R only
1:5 -- R "
1:25 --I R "
--
R only
m l~ t~
Control
selqlm)
65°C. for 30
min.
(no
None
--
R only
Undiluted -- R only -- R only -- R only
1:5 -- R " -- R " -- R "
1:25 -- R " -- R " -- R "
I
Undiluted + ~ Sill + SIII + Sill
1:5 + ~ SIII "b Sill + SIII
1:25 + SIII + SllI + Sill
Undiluted + ' Sill + SllI [ + SIII
1:5 + Sill + SIII I + SIII
1:25 + SIII + SIII -b Sill
* Dilution of the digest mixture of serum and transforming substance.
of the aqueous solution of the nucleate. Determinations of viscosity were made
~mmediately and at intervals over a period of 24 hours during incubation at 37°C.
The results of this experiment are graphically presented in Chart 1. In the
case of unheated serum of both dog and rabbit, the viscosity fell to that of
water in 5 to 7 hours. Dog serum heated at 60°C, for 30 minutes brought about
OSWALD T. AVERY, COLIN M. MACI,EOD, AND MACLYN McCARTY
149
no significant reduction in viscosity after 22 hours. On the other band, heating
rabbit serum at 50°C. merely reduced the rate of depolymerase action, and
after 24 hours the viscosity was brought to the same level as with the unheated
serum. Heating at 65oc., however, completely destoyed the rabbit serum
depolymerase.
Thus, in the case of dog and rabbit sera there is a strl]dng 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 this
difference in temperature of inactivation is not merely a general property of all
enzymes in the sera is evident from experiments on the heat inactivation of
])iffe~ential Hea~
Ina~atAon
~[\ llleated 65° ~" SO'
3L_ - .~ ~ I-I~ed ~° fo~ 30 '
.................................
[~, -o.. i.ieat~L 60o f~, 30,
11- i i i ,
HI,#.
5 10
15 Z0
'l'imo
CHART I
tributyrin esterase in the same.samples of serum. In the latter instance, the
results are the reverse of those observed with depolymerase since the esterase
of 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 action
of 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, pan-
creatin, 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
containing depolymerase for desoxyribonucleic acid and the further fact that
150
TRANS]~OR3~ATION
0~'
PNEU3~OCOCCAL TYPES
in both instances the enzymes concerned are inactivated at the same tempera-
ture and inhibited by fluoride provide additional evidence for the belief that
the 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 serological
activity 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 the
capsular 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 in
inducing pneumococcal transformation.
Physicochemical Studies)--A
purified and active preparation of the trans-
forming substance (preparation 44) was examined in the analytical ultra-
centrigue. 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, showing
that activity could not be due to the presence of an entity much different
in size. The molecular weight cannot be accurately determined until meas-
urements of the diffusion constant and partial specific volume have been
made. However, Tennent and Vilbrandt (20) have determined the diffusion
constant of several preparations of thymus nucleic acid the sedimentation rate
of 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 thepneumococcal preparation is of
the order of 500,000.
Examination of the same active preparation was carried out by electropho-
resis in the Tiselius apparatus and revealed only a single electrophoretic compo-
nent of relatively high mobility comparable to that of a nucleic acid. Trans-
forming activity was associated with the fast moving component giving the
4 The Type III antipneumococcus rabbit serum employed in this study was fur-
nished through the courtesy of Dr. Jules T. Freund, Bureau of Laboratories, Depart-
ment of Health, City of New York.
5 Studies on sedimentation in the ultracentrifuge were carried out by Dr. A.
Rothen; the electrophoretic analyses were made by Dr. T. Shedlovsky, and the ultra-
violet absorption curves by Dr. G. I. Lavin. The authors gratefully acknowledge
their indebtedness to these members of the staff of The Rockefeller Institute.
OSWALD T. AVERY, COLIN M. MACLEOD, AND MACLYN MCCARTY
151
optically visible boundary. Thus in both the electrical and centrifugal fields,
the behavior of the purified substance is consistent with the concept that bio-
logical activity is a property of the highly polymerized nucleic acid.
Ultraviolet absorption curves showed maxima in the region of 2600/~ and
minima in the region of 2350/~. These findings are characteristic of nucleic
acids.
Q.uantitati~e Determination of Biological Act{dty.--In
its highly purified state
the material as isolated has been found to be capable of inducing transformation
in amounts ranging from 0.02 to 0.003/zg. Preparation s 44, the purification of
which was carried out at low temperature and which had a nitrogen-phosphorus
TABLE IV
Titration of Transforming Activity of Preparation 44
Transformln$ principle Quadruplicate
tests
Preparation
44*
1 2
Dilution ?kmotmt
added
/Jg.
10 -~ 1.0
I0 -~'s
0.3
10 "a
O.
1
10 -3"5 O. 03
10 ~ 0.01
10 4.5
0.003
10 -~ O. 001
Control None
Diffu~
growth __
+
'
Colony
sIII
SIII
SIII
SIII
SIII
R only
R "
R "
Diffuse
growth
Colony
Diffuse
form growth
SIII
SIII
SIII
SIII
SIII
-b
SIII
R only
R
"
3 4
Colony Diffuse Colony
form growth form
SIII ~ SIII
SIII SILl
SIII ~ SIII
SIII
SIII
SIII ~ SIH
R
only
SIII
R " R only
R " R
"
* Solution from which dilutions were made contained 0.S rag. per cc. of purified material.
0.2 cc. of each dilution added to quadruplicate tubes containing 2.0 cc. of standard serum
broth. 0.05 cc. of a 10 -'~ dilution of a blood broth culture of R36A is added to each tube.
ratio of 1.58, exhibited high transforming activity. Titration of the activity
of this preparation is given in Table IV.
A solution containing 0.5 rag. 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 ec. of a 10 -4
dilution of s 5 to 8 hour blood broth culture of R36A. Transforming activity was
determined by the procedure described under Method of titration.
The data presented in Table IV show that on the basis of dry weight 0.003
/~g. of the active material brought about transformation. Since the reaction
system containing the 0.003/~g. has a volume of 2.25 cc., this represents a final
concentration of the purified substance of 1 part in 600,000,000.
152
TRANSFORMATION O1 ~ PNEUMOCOCCAI, TYPES
DISCUSSION
The present study deals with the results of an attempt to determine the chem-
ical nature of the substance inducing specific transformation of pneumococcal
types. A desoxyribonucleic acid fraction has been isolated from Type III
pneumoeocci 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 pneumococei a nucleic acid of the
ribose type. 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 experimentally induced
irb vitro
by a chemically
defined substance.
Although the observations are limited to a single example, they acquire
broader significance from the work of earlier investigators who demonstrated
the interconvertibility of various pneumococcal types and showed that the
specificity of the changes induced is in each instance determined by the par-
ticular type of encapsulated cells used to evoke the reaction. From the point
of 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 unencap-
sulated R variants of Pneumococcus Type II to produce a capsular polysac-
charide identical in type specificity with that of the cells from which the
inducing substance was isolated. Equally striking is the fact that the sub-
stance evoking the reaction and the capsular substance produced in response
to it are chemically distinct, each belonging to a wholly different class of chem-
ical compounds.
The inducing substance, on the basis of its chemical and physical properties,
appears to be a highly polymerized and viscous form of sodium desoxyribo-
nucleate. On the other hand, the Type III capsular substance, the synthesis
of which is evoked by this transforming agent, consists chiefly of a non-nitrog-
enous polysaccharide constituted of glucose-glucuronic acid units linked in
glycosidic union (22). The presence of the newly formed capsule containing
this type-specific polysaccharide confers on the transformed cells all the dis-
tingnishing characteristics of Pneumococcus Type HI. Thus, it is evident
that the inducing substance and the substance produced in turn are chemically
distinct and biologically specific in their action and that both are requisite in
determining 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 as
evidenced by the selective action of the transforming principle. Indeed, the
possibility of the existence of specific differences in biological behavior of nucleic
acids has previously been suggested (23, 24) but has never been experimentally
demonstrated owing in part at least to the lack of suitable biological methods.
OSWALD T. AVERY, COLIN M. MAcLEOD, AND MACLYN McCARTY
153
The techniques used in the study of transformation appear to afford a sensitive
means of testing the validity of this hypothesis, and the results thus far ob-
tained 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 the
ideal means of determining the differential specificity of this group of biologically
important substances. Although the constituent units and general pattern
of 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 to
function antigenically, one would not anticipate that such differences would be
revealed by immunological techniques. Consequently, it is perhaps not sur-
prising that highly purified and protein-free preparations of desoxyribonucleic
acid, although extremely active in inducing transformation, showed only faint
trace 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
Lackrnan and his collaborators (25) have shown that nucleic acids of both the
yeast and thymus type derived from hemolytiC streptococci and from animal
and plant sources precipitate with certain antipneumococcal sera. The reac-
tions varied with different lots of immune serum and occurred more frequently
in antipneumococcal horse serum than in corresponding sera of immune rab-
bits. The irregularity and broad cross reactions encountered led these in-
vestigators to express some doubt as to the immunological significance of the
results. 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 study
of 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 di~culties. 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 unaffected
in the reaction system. However, from a bacteriological point of view, nu-
merical estimations based on colony counts might prove more misleading than
enlightening because of the aggregation and sedimentation of the R ceils ag-
154
TRANSFORMATION OF PN~OCOCCAL
TYPES
glutinated by the antiserum in the medium. Attempts to induce transforma-
tion in suspensions of resting ceils held under conditions inhibiting growth and
multiplication have thus far proved unsuccessful, and it seems probable that
transformation occurs only during active reproduction of the cells. Important
in this connection is the fact that the R ceils, as well as those that have under-
gone transformation, presumably also all other variants and types of pneu-
mococci, contain an intracellular enzyme which is released during autolysis
and in the free state is capable of rapidly and completely destroying the activity
of the transforming agent. It would appear, therefore, that during the loga-
rithmic 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 trans-
forming agent through the release of autolytic ferments.
In the present state of knowledge any interpretation of the mechanism in-
volved in transformation must of necessity be purely theoretical. The bio-
chemical 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 altera-
tions are not random changes but are predictable, always corresponding in
type 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 trans-
fers in artificial media without any further addition of the transforming agent.
Moreover, from the transformed cells themselves, a substance of identical
activity 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 capsular
material 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 cul-
tural conditions are favorable for the maintenance of capsule formation. The
transformed cells can be readily distinguished from the parent R forms not
alone 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 microorganism
in the animal body.
It is particularly significant in the case of pneumococci that the experi-
mentally 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 these
changes are predictable, type-specific, and heritable.
OSWALD T. AVERY, COLIN M. MACLEOD, AND MACLY'N McCARTY 15S
Various hypotheses have been advanced in explanation of the nature of the
changes induced. In his original description of the phenomenon Griffith (1)
suggested that the dead bacteria in the inoculum might furnish some specific
protein that serves as a "pabulum" and enables the R form to manufacture a
capsular carbohydrate.
More recently the phenomenon has been interpreted from a genetic point of
view (26, 27). The inducing substance has been likened to a gene, and the
capsular antigen which is produced in response to it has been regarded as a gene
product. 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 causa-
tive 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 indi-
cate 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 described
is 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 to
escape detection. If, however, the biologically active substance isolated in
highly 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 ceils. 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 in-
duced and specifically directed by a known chemical compound. If the results
of the present study on the chemical nature of the transforming principle are
confirmed, then nucleic acids must be regarded as possessing biological
specificity the chemical basis of which is as yet undetermined.
SUMMARY
I. From Type III
pneumococci
a biologically active fraction has been isolated
in highly puTified form which in exceedingly minute amounts is capable under
appropriate cultural conditions of inducing the transformation of unencapsu-
lated R variants of Pneumococcus Type II into fully encapsulated cells of the
156
TRANSFORMATION
OF
PNEUMOC~CAL TYPES
same specific type as that of the heat-killed microorganisms from which the
inducing material was recovered.
2. Methods for the isolation and purification of the active transforming ma-
terial are described.
3. The data obtained by chemical, enzymatic, and serological analyses
together with the results of preliminary studies by electrophoresis, ultracen-
trifugation, and ultraviolet spectroscopy indicate that, within the limits of the
methods, the active fraction contains no demonstrable 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.
CONCLUSION
The evidence presented supports the belief that a nucleic acid of thedesoxy-
ribose type is the fundamental unit of the transforming principle of Pneumo-
coccus Type III.
BIBLIOGRAPHY
1. Griifith, F., 3". Hyg., Cambridge, Eng., 1928, 27, 113.
2. Neufeld, F., and Levinthal, W., Z. Immunitgtsforsch., 1928, 5§, 324.
3. Baurhenn, W., Centr. Bakt., 1. Abt., Orig., 1932, 126, 68.
4. Dawson, M. H., J. Exp. Med., 1930, 51, 123.
5. Dawson, M. H., and Sia, R. H. P., Y. Exp. Med., 1931, 54, 681.
6. Alloway, J. L., Y. Exp. Med., 1932, 55, 91; 1933, ST, 265.
7. Berry, G. P., and Dedrick, H. M., Y. Baa., 1936, 31, 50.
8. Berry, G. P., Arch. Path., 1937, 24, 533.
9. Hurst, E. W., Brit. Y. Exp. Path., 1937, 18, 23. Hoffstadt, R. E., and Pilcher,
K. S., Y. Infect. Dis., 1941, 68, 67. Gardner, R. E., and Hyde, R. R., Y.
Infect. Dis., 1942, 71, 47. Houlihan, R. B., Proc. Soc. Exp. Biol. and Med.,
1942, 51,259.
10. MacLeod, C. M., and Mirick, G. S., Y. Baa., 1942, 44, 277.
11. Dawson, M. H., Y. Exp. Mecl., 1928, 47, 577; 1930, 51, 99.
12. Sevag, M. G., Biochem. Z., 1934, 273, 419. Sevag, M. G., Lackman, D. B.,
and Smolens, J., Y. Biol. Chem., 1938, 124, 425.
13. Dubos, R. J., and Avery, O. T., J. Exp. Med., 1931, 54, 51. Dubos, R. J.,
and Bauer, J. H., J. Exp. Mcd., 1935, 62, 271.
14. Liu, S., and Wu, H., Chinese J. Physiol., 1938, 13, 449.
15. Martland, M., and Robison, R., Biochem. J., 1929, 23, 237.
16. Albers, H., and Albers, E., Z. physiol. Chem., 1935, 232, 189.
17. Levene, P. A., and Dillon, R. T., J. Biol. Chem., 1933, 96, 461.
18. Greenstein, J. P., and Jenrette, W. Y., Y. Nat. Cancer Inst., 1940,1,845.
OSWALD T. AVERY, COLIN ~f. MACLFOD~ AND MACLYN McCARTY
157
19. Greenstein, J. P.,
J. 2Vat. Cancer Inst.,
1943, 4, 55.
20. Tennent~ H. G., and Vilbrandt,
C. F., J. Am. Chem. Soc.,
1943, 65, 424.
21. Thompson, R. H. S., and Dubos,
R. J., J. Biol. Chem.,
1938, 125, 65.
22. Reeves, R. E., and Goebd,
W. F., J. Biol. Chem.,
1941, 139, 511.
23. Schultz, J., in Genes and chromosomes. Structure and organization, Cold
Spring Harbor symposia on quantitative biology, Cold Spring Harbor, Long
Island Biological Association, 1941, 9, 55.
24. Mirsky, A. E., in Advances in enzymology and related subjects of biochemistry,
(F. F. Nord and C. H. Werkman, editors), New York, Interscience Publishers,
Inc., 1943, 3, 1.
25. Lackman, D., Mudd, S., Sevag, M. G., Smolens, ]., and Wiener,
M., J. Immunol.,
1941, 40, 1.
26. Gortner, R. A., Outlines of biochemistry, New York, Wiley, 2nd edition, 1938,
547.
27. Dobzhansky, T., Genetics and the origin of the species, New York, Columbia
University Press, 1941, 47.
28. Stanley, W. M., in Doerr, R., and Haliauer, C., Handbuch der Virusforschung,
Vienna, Julius Springer, 1938, 1, 491.
29. Murphy, J. B.,
Tr. Assn. Am. Physn.,
1931, 46, 182;
Bull. Johns Hopkins Hosp.,
1935, 56, 1.
158
TRANSFORMATION O~" PNEITMOCOCCAL TYPES
EXPLANATION OF PLATE
The photograph was made by Mr. Joseph B. Haulenbeek.
FIG. 1. Colonies of the R variant (R36A) derived from Pneumococcus Type n.
Plated on blood agar from a culture grown in serum broth in the absence of the
transforming substance. X 3.5.
FIO. 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 prin-
ciple isolated from Type nI pneumococci. The smooth, glistening, mucoid colonies
shown are characteristic of Pneumococcus Type In and readily distinguishable from
the small, rough colonies of the parent R strain illustrated in Fig. 1. X3.5.
TtIE JOURNAL OF EXPERIMENTAL MEDICINE VOL. 79 PLATE 1
(Avery
et al.:
Transformation of pneumococcaI types)
This sentence is the crux of the paper; they isolated DNA from the smooth strain and showed, through exhaustive experimentation, it to be both necessary and sufficient for transformation of the rough.
From a modern perspective, this section highlights some fairly mundane facts about DNA: it is clear and viscous in solution, stable in the refrigerator, and can be desiccated. Having benefited from seventy years of progress since its publication, reading a paper like this engenders a feeling akin to that of owning an ant farm.
Short of calling DNA the hereditary molecule, they recognize that the specific character of the transformed DNA is passed from one generation to the next. Indeed, while their results demonstrate DNA heritability, they do not by themselves show that DNA is the genetic molecule.
Given the limited understanding of molecular genetics at the time, this is a staggeringly accurate description of how DNA can ultimately produce a polysaccharide capsule; the details are not known, but it is apparent from their results that DNA produces "enzymatic reactions" which generate the capsule.
The authors prophecy the discovery of genes. Taking a step back to recall the difference between rough and smooth strains, we remember that a polysaccharide capsule surrounds the latter. Yet Avery et al. found the transformation from rough to smooth was mediated by nucleic acid, not sugar. As contemporary readers, we take for granted that DNA acts as a blueprint, not as a structural element, but at the time this finding was completely remarkable because it implies DNA somehow induces the formation of a polysaccharide capsule.
At the time, it was known that proteins could take almost infinite forms, which is why they appealed as a possible transforming principle. DNA, by contrast, has basically one form with no obvious means of providing specificity. This conundrum persisted until the 60s when work by Nirenberg, Matthaei, and Khorana elucidated how sequences of DNA can encode proteins.
At this point, a "gene" is simply a hereditary unit. The term itself had been around since the early 1900s, while the concept of discrete heredity dates back to Mendel's pea crosses in the 1850s. This paragraph is another example of the surprising lucidity with which genetics was understood at the time without the molecular underpinnings, which is to say, DNA.
Here is the most anodyne statement of fact in all of biology, folks.
The Avery–MacLeod–McCarty experiment was an experimental demonstration, reported in 1944 by Oswald Avery, Colin MacLeod, and Maclyn McCarty, that DNA is the substance that causes bacterial transformation, when it had been widely believed that it was proteins that served the function of carrying genetic information. This experiment has been responsible for the launch of molecular biology and thus modern biologic science as we know it.
This section describes the preparation of bacterial DNA in reproducible detail. In order to isolate DNA, the polysaccharide, lipid, and protein components of the cells are removed by chemical methods. DNA is then precipitated from the remaining "active fraction" by addition of ethyl alcohol. It is worth noting that this procedure is fairly similar to current protocols for genomic DNA extraction.
This work builds upon Griffith's 1928 experiment in which mice were injected with one of four variations of the bacteria Streptococcus pneumoniae: conditions:
1. Smooth strain - a strain of the bacteria encapsulated in polysaccharide, hiding its surface antigens from the host immune system and thus, preventing an immune response. Mice injected with this strain died.
2. Rough strain - a strain of the bacteria lacking the polysaccharide capsule that allows the smooth strain to evade the immune system. Mice injected with this strain survived.
3. Heath-killed smooth strain - mice injected with a culture of the virulent smooth strain heated at 60 ºC survived.
4. Rough strain and heat-killed smooth strain - mice injected with a combination of the nonvirulent rough strain combined with the inactivated virulent strain died, demonstrating that a component of the killed smooth strain was able to induce virulence in the rough strain. Griffith termed this the "transforming principle."
Until the Avery–MacLeod–McCarty paper, the transforming principle was assumed to be protein.
As per the Griffith experiment, the transforming principle must be able to withstand the temperature at which the cells were killed, about 60 ºC, in order to induce virulence in the rough strain. Denaturation, the process by which the strands of a DNA double helix separate, occurs at a range of temperatures depending on the chemical composition of a given strand, but can be reversible. As such, heating DNA to 90 ºC may not have an effect on its transformation ability.
Both Biuret and Millon tests are used to identify proteins in a solution. This test confirms that the isolated active component is not protein as previously thought. Additionally, it is "strongly positive" for DNA and weakly so for RNA. Due to its chemical similarity and abundance in the cell, RNA will slip into DNA preparations, which is why contemporary protocols include the addition of ribonuclease, an enzyme that cleaves RNA.
These elegant experiments use enzymes known to digest protein (trypsin and chymotrypsin) and ribonuclease to show that the absence of these molecules from the cellular extract does not impact transformation. They also lengthily demonstrate that extracts treated with deoxyribonuclease (DNase, a component of dog and rabbit sera) do not transform the rough strain while those heated to 60 ºC or 65 ºC do. This is taken to mean that the activity of DNase, cleavage of DNA, prevents transformation unless it is inactivated at higher temperatures. This provides further evidence that DNA is the transforming principle.
While the work of Griffith and his successors showed a transforming principle in bacteria and viruses, none clearly identified the responsible chemical. Avery, MacLeod, and McCarty effectively repeated the fourth condition of the Griffith experiment using cultures treated to pare down the possibilities. Specifically, they showed that cultures from which protein was eliminated still transformed the rough strain, meaning protein cannot be the transforming principle. However, cultures of the smooth strain treated with an enzyme that cleaves DNA did not induce virulence in the rough strain, indicating that DNA is responsible for transformation.