L170 4 Report of Dr. avery (assisted by Drs. McCarty and Taylor) Studies on the transformation of types of pneumococcus (Avery and MeCarty). During the past year, studies have been continued on the predict- able und heritable changes induced in R variants of pneumococci by active desoxyribonucleic acid fractions derived from encapsulated pneumococcal cells. Three papers comprising the work presented in the last report have recently been published. The isolation and purification by Dr. McCarty of the onzyme, desoxyribonucloawe (1) has provided a biochemical tool, the use of which has furnished confirmatory evidenes of tho chemical identity of the transforming substance (”). In addition, the fact that the destructive action of this enzyme on decoxyribonucleic acid is completely inhibited by citrate, has led to the development of an improved metho! for obtaining much larger yields of the transforming oubstance from lysates of living pneumo- cocci of several ditforent types (3). Moreover, a@& will be pointed out later in the present report, the enzyme also provices a means of determining the optimal conditions and length of time required for the uptake of the specific nucleic acid by the # cells in the transforming reaction. Before discussing the newer work now in progress concerning ‘the nature and biochemical properties of. certain environmental factors essential in transformation, it may be well to recall bricfly the present status of our knowledge of the transforming substance itself. Accumulated evidence based on the results of innumerable tests of the specificity and biological activity of various preparations, together with data obtained by chemical, enzymatic and serological analysis of the active material, has established beyond reasonabls doubt thit the active gubstence responsible for transfor- mation is e specific nucloic acid of the desoxyribose type. Bik Gnade ce anager Ep SE nea de ke r'§ 171 Biochemical, studies of onvironmentel factors ossential in trans- formetion of pneumococcal types (Avery and McCarty). In ths historical development of the problem it is of interest to recall that Griffith, who originally describec the transiormetion phenomenon in vivo, was unibls to obtuin positive results in vitro. The first successful domonstretion of the reaction in the test tube was cerriea out Sy Lawson anc Sia in nutrient broth containing anti-R rabbit serum. Prom that time on, ivrum or Lerous fluic of one surt or another hrs alvzys been usec, ond hee bee shown to be an eusentinl factor, since in its absence it is jmpossidl: to incuce btrens- formition. However, the function of serun is not merely one of enrichment, ‘ cinc: the nutrient broth itself contains adequat« amount: of accessory grovth factors required for tho initiation and maintenence of growth. The next advanes, which, if successfully accomplirhed, would undoubtedly throw con- a sicerable light on the mechanism of transformation, rest: upon fanaing the solution to the following questions: Why is the presonce of serum or serous I fluid in the medium essential? Why are some sera capable of supporting transformation, while others utterly fail? What components function as essenticl factors, how do they act, and what is the biochemical nature of : their action in respect to the cellular changes evokea by the specific if pneumococcal nucleic acid? Studies currently in progress indicate that serum factor is conm- posed of at least three essential constituents. These aro 1) the R-antibody, oo iy entnaie which causes agglutination of unencepsulated K pneumococci; 2) a dialyzable constituent; and 3) an additional protein factor occurring in the globulin fraction of the serum. The evidence for assuming that the serum factor depends upon the collective action of the three components is summarized below, together with a description of certain experiments designed te 172 elucidete the function of each, and the mechanism of their combined effect. Finally, a statement is made of the current working hypothesis of the nature of this mechanism. 1) Reantibody. When unencapsnlated R-pneumococci are grown in the presence of R-antibody, large aggregates are formed which settle to the bottom of the tube. The supernatant broth is thus teft. clear, so that if transformation occurs and encapsulated S cells aro formed, the changs is readily apparent, since the nowly formed $ cells, not being atfected by the Keantibody, grow diffusely throughout the culture. While this phenomenon has been useful in the technique of the transforming test, the K-antibody appears to do more than merely provide visible evidence of transformation. In the usucl fluid media it has not deen possible to induce trans- formation unless fe-antibody is present. ilowever, uncer special conditions, results have been obtained which give some indication of the role of R- antibody. In 2 semi-solid medium containing « low concentration of agar (0.2 °/o), pneumococci grow in colonies rather then diffusely, and loose aggregates are formed not unlike those that result from antibody agglut- ination, although they do not settle to the bottom of the tube. Transforma- tion of type has been obtained in semi-solid medium containing normal rabbit serum, but wholly lacking in R-antibody. These experiments suggest that the type of colonial growth produced by anti-R is an important factor, and that when this type of growth is simulated by other means, the anti-R can be dispensed with. Although it cannot be stated with certainty why colonial growth is required, it is possible that local reducing conditions arising in the aggregated cells are of primary importance. This thesis is supported by the results of experiments in which the medium is placed in « shallow layer = arreaeey «ep 173 not exceeding 1-2 mm. in depth. In the shallow layer, oxidizing conditions are promoted, and even in the usual serum medium containing R-antibody, anifost transformation does not occur. Attempts to reverse the effect of the shallow layer by the addition of reducing agents have not yielded con- sistent results, but on one occasion transformation was obtained in a group of flasks in which glutathione had been adizd to the usual serum medium. There is, then, cortain evidence that reducing conditions are essential in some phase of the transforming reaction. To sunmarize, the R-antibody serves the purpose of causing an apparently cssential colonial aggregation of R-pneumococci, which in turn results in local. conditions, possibly reducing in character, that are required in the transformation reaction. 2) Dialyzable constituent. Early attempts at salt fractionation i of serum factor by th: classical methods of protein chemistry yielded totally inactive fractions. Some light has been thrown cn these results Lo by the discovery that a dialyzable component of serum is essential. When an active serum is dialyzed against physiological saline, there is a pro- gressive decrease in its efficacy in the transforming system, and if dialysis is sufficiently prolonged, the serum becomes completely inactive. Under these conditicns, however, the R-antibody is unimpaired, and no denaturation of protein is apparent. Serum which has been inactivated by dialysis can be reactivated for use in the transforming system by two procedures which differ in certain rie tant ibis AE aoa le ciate. niles Seen ee rm important respects. In the first place, if inorganic phosphate is added to the dialyzed serum, and the mixture incubated 1-2 hours, the serum regains its ability to support transformation. The period of incubation of the serum with phosphate is essential. The interaction between phosphate and 174 the serum appears to be prevented by the presence of nutrient broth, for if the latter is added at the same time as the phosphate, or after a short period of incubation, no reactivation is achieved. In contrast to the reactivation by phosphate, which requires time, immediate reactivation cen be achieved by adding to the scrum such materials as unheated neopeptone, or tryptic digest of casein. Nutrient broth does not interfere with this effect. The nature of the substence responsible for immediate reactivation has not been determined. However, it has been shown to be a dialyzable substance, and to be precipitable by alcohol. Tho aveilabls det. suggest that phosphate brings about reactivation of dialyzed serum by promoting ». chemical or enzymatic reaction, which re- sults in synthesis of the dis lyzabli constituent of serum factor. On the other hand, the neopeptone, or cusein digest, provides a preformed source of the dialyzabie constituent, or some related substence which is able to replace it. It is of interest that globulin fractions of serum obtained by ammonium sulfate fractionation, that hitherto were found to be ineffec- tive in the transforming system, can be rendered effective by the addition of unheated neopeptone as source of the dialyzable factor. R-antibody plus unheated neopeptone does not support transformation, however, and this fact is one piece of evidence for the existence of an essential pro~ tein constituent other than anti-R. 3) Globulin factor. It has long been recognized that the titer of R-antibody does not parallel the efficacy of the serum in the transforn- ing systen. Indeed, some of the most potent sera in terms of ability to support transformation, have been shown to have the lowest titers of anti-R. This fact, together with the evidence cited in the preceding paragraph, has led to the assumption that another essential constituent is present in effective sera. The results of fractionation experiments in which are 175 used salts such as ammonium sulfate and organic protein precipitants such as elcohol, have estublished that this additional constituent, as well as Reantibody, occurs in the globulin fraction of the serum. For the purpose of orienting further research, the possibility as been considerve that the globulin factor aay be an enzyme. If this is indeed the casc, it Svems highly probable on gonural grounds that Sune organ of the animal body contains the enzyme in much higher concentration than does th: seruny, and wuld serve es @ more favorable source for possible purification and identification of the enzyme. To test this assumption, & preliminary survey wag made -f several rabbit organs by proparing simple saline extracts und testing them for the presence of the globulin factor. The procedure used consisted of n.dding the extract to broth containing 4 small emount of concentrated rabbit R-antibody and unheated neopeptone as na source of the dianlyzable constituent. The broth containing these added components was tested for its ability to support transformation. Positive results indicate that the organ extract has supplied the missing constituent {globulin factor), since the Reantibody «nd dialyzable factor alone are ynable to support transformation. Rabbit spleen proved to be a good source of the globulin factor. To provide larger organs a8 source material, extracts of calf spleen and calf thymus werc then tried, and it was found that thymus is superior not only to spleen, but to the best sera available. Fractionation experiments with thymus extracts are now in progress, in an effort to determine whether isolation and purification of the active globulin component can be achieved. The principal interest in purification 4g the possibility of determining the nature of the substance and its rdle in the transforming reaction. The results of preliminary studies with organ extracts lend some support to the hypothesis on which the experiments were based, 1.¢-, that the globulin feetor of the sorum moy be en onzyme. 176 Muchanism of action of serum factor. A series of experiments which were designed to provide a more intimate knowledge of the interaction between the specific transforming substance (pneumococcal. desoxyribonucleic aci’) and th. susceptible pneumococcal cells proved to have an important bearing on the problem of the role of serum factor. The customary procedure in demonstrating the phenomcnon of transformation is to add the specific desoxyribonucleic acid to the serum mediun and ty inceul:te with a suscep- tible strain of R-pneumococcus. Trensformation becomes apparent after 16 to 20 hours! incubaticn, but little is known of the course of events during this period of incubation. the purificd enzyme, desoxyribonucleise, which specifically inactivates the transforming substence, has been usec as a tuvl in an uttempt to study certain phases of this problem. By adding desuxyribonuclease to the transforming system at various intervals after inoculation, in a concentration known to give almost immedc- jate inactivaticn of the specific transforming substance, it is possible to determine the length of time required for the transforming substance to be taken up or "fixed" by the susceptible cells. The addition of the enzyme at any time up to 3 or 4 hours after inoculation interferes with the reaction, so that transformation does not occur, and it is, therefore, likely that throughout this period the transforming substance is readily accessible to the desoxyribonuclease. After 4 to 5 hours, on the other hand, the addition of aesoxyribonuclease has no observable effect on the course of the reaction. Consequently it appears that growth of the R cells in serum medium for 3 to 5 hours is required before the specific desoxyribonucleic acid is taken up by the cells, and thus protected from enzymatic destruction. A striking confirmation of the foregoing is provided by experiments in which the R cells are grow in serum medium in the absence of the specific 177 transforming substance. After 4 to 5 hours! growth under these conditions, the cells are so "sensitized" that when transferred to a mecium containing the transforming substance, the latter is taken up in as short a time as 15 minutes. If the transfer is mace after shorter periods of growth in serun medium, c.g. 2 to 3 hours, the “sensitization” has appurently not taken place and rapid fixation of the transforming substance camot be “enon- strated. Thus, the events that occur in the first four hours of growth of K eclls in the transforming system ure independent of the presence of the specific transforming substance. It must be concluded that growth uncer these conditions alters the cell in some way, or provites suitable environ- mental conditions so that the interaction betwoun the cell end the trans- forming substance can tuke place. The relation of the above cxperiments to the role of serum factor becomes apparent from the fact that growth of the cells in plain broth, in the presence of R-antibody, or in serun inactivated by dialysis, fails in each case to "sensitize" the cells. Thus, the hypothesis is suggested that the major part played by serum in the transforming system is concerned with a modification of the cell so that the specific transforming substance can be taken up. A tentative hypothesis of the mechanism of serum factor action. The evidence available at present favors the view expressed above that the r§le of serum factor is concerned with some action on the surface of the susceptible R cell which permits interacticn between the cell and the specific transforming substance. A plausible and perhaps useful hypothesis with regard to the mechanism of the serum effect can be based on the assumption that the action is enzymatic in nature. In this view, the enzyme is present in the globulin fractions of active sera, as well as in 178 extracts of certain mammalian orgens. The R-antibody, as a result of the colonizl growth of the cells, provides the required conditions for the action of the enzyme. It is suggested that the reducing cunditions resulting from this type of growth muy be of significance. The enzymatic system as outlinec is not more complex than some already known. Recent work un muscle hexokinase in the laboratory of Dr. Carl T. Cori, provides a rather striking analogy. Muscle extracts lose hexokinase activity upon dialysis, anc, as in the case of serum factor, activity can be restored by two moans: 1) by incubation with phosphate, or 2) immediately by adding the preformec co-factor, which in this cuse proves to be guanine. In addition to guenine as co-fector, muscle hexokinase also requires the presence of reduced co-enzyme I (dihydrocozyiase). This lattor fact is suggestive, in view of the possible réle of reducing conditions in the action of serum factor. Although the hypothesis outlined above is admittedly only a tentative one, it hes made possible an experimental approach to the problem of defining the significance anc essential role of the so-called "serum factor" in the transforming system. While the interpretation may be moci-. fied as knowledge increases, the facts thus far obtained indicate that transformation consists of two phases:~ 1) an initial phase in which, as the result of the combined action of the serum components, the R cells are rendered receptive anc become capable of taking up the transforming substance; and 2) a phase in which a chin of biochemicel renctions is initiated within the cells that culminates in synthesis of the capsular polysac- charide, the chemical identity and type specificity of which cun be pre- determined, depending on type of encapsulated cells used as source of the transforming substance. cine SERED te ENRON A PETE MR LENNIE Si dt 179 Cytochemical, studics on localization of desoxyribonucleic acid in pneumococcal cells (Taylor). Genetic anc cytological studies of animals and plants have for the most part localized the hereditary units of the cell in the nucleus. Cytochemiccl. studies have shown that the most charac- teristic component cf the nucleuc is Cesoxyribonucleic acid. The active substance inducing transformation of pneumococcal types and determining the specificity of the changes has been founc to be a nucleic acid of the des- oxyribose type. Sinee transformation may be Cascribed as a change in the heredity of the pneumococcal cell, it is of interest to know whether pneumo- coccal desoxyribonucleic acic occurs in a formec structure comparable to the nucleus of higher organisms. Under controlleé cundivions the Feulgen nuclear reaction appears to be specific for desoxyribonucleic acid. When the Feulgen reaction is carried out on pneumococcal cells fixec with osmium tetroxide vapor, a deeply staining red eranule can readily be observed within each cell. This suggests thet cesoxyribonucleic acid is not evenly distributed throughout the cell, but is partially or entirely Localized. In a medium containing anti-R serum, unencapsulated pneumococci grow in long chains of diplococci. Comparison of the Feulgen positive granules in the individual diplococci of these chains indicates that the granules undergo enlargement and duplication in the growing cells. The data thus far obtained do not warrant the conclusion that Pneumococcus is a nucleated cell, particularly in view of the questioned specificity of the Feulgen reaction. It has been observed in this laboratory that the Feulgen positive material in the nuclei isolated from animal tissues is rapidly removed by the specific action of highly purified preparations of desoxyribonuclease. Adaptation of this technique to the study of the cytology of pneumococcal cells is being made, since the enzyme affords a more specific method for identifying the nature of the central granules than do any of the know staining methods available at present. Publications McCarty, M. Purificaticn and propertics of Gesoxyribonuclerase isolated from beef pancreas, J. Gen. Physiol., 1946, 29, 1¢3. McCarty, M. and Avery, O. T. Studies on the chemical nature of the sub- stance inducing trunsformation of pneumococcal types. II Effect uf desoxyribonuclense on the biological activity uf the transforn- ing substance, J. Exp. Med., 1946, 83, 89. McCarty, M. and Avery, 0. T. Studies on the chemical nature of the sub- stunce inducing transformation of pneumococcal types. III An improved method fur the isslation of the transforming substance anc its application to Pneumococcus Types TI, III and VI. J. Exp. Med., 1946, 83, 97.