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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