170 Ltudies on th3 trarlsformation of types or *~eumococcus -.---- - -. (Avery anil thz transforming sub:;t::ncc (2). In addi-tion, ?,h;: i'act. t}Fit the destructive i:ction 0:' this enz,vm!' on (~,:~oxS~ibonuzleic ai:id is ccmp1.5t,c.Ly,, irihibit3d hy largi?r ;{ic' Ld. s of th<: tr::.nsforming :,uGst;lncn from lyS3t'dl: 01.' living pneumo- cocci of sevt:rsl ci.i~f':rc.:nt t.brp-32 (3) . i'nortiov3r a~ will be poin?,,?d out I ? I later in the prosont rc?port, the enqymo t&30 proviueo a means 0.f determining the optimal conditions and length of time required for thi: qtnke of the , specific nucleic acid by the 11 cell 8 in thi: transforming rcnction. :I Bafors discussing tho newor work now in progress concerning the i ', ! nature and biochemical properties of certain environmental factors assentiul 4 in transformation, it may be well to recall briefly the present status of 4 our knowledge of thz transforming substnncs itsttlf. Accurnulatzd evidence based on th;, results of innumerable tests of the specificity and biological activity of various propnrations, togathcr with data obtained by chemical, enzymatic nnd serological anelysis of the nctive materisl, has established beyond roasorxtblr: doubt ttiit the act,iv~ :;ubstznc<: rcasponsibl.-! for trc;nsPor- mation is c? specific nuclc:ic acid of th; d>zoxfri.bosc typo. 171 !Xoch--nit,-23. studi;:~~ of -~nvironm2ntzl 0, f'nctors osr;onti;il .in tran%- forirk. tion of pzut;ioCoCCai type: (Avery anti McZsrty) . 1~1 the: historical , I d2V,:lOpncIlt of the problorn it is of intarost, to rccz1.1 th::t Grifi'ith, who 0riginallJ ti.e:;cribw the tr~n~:Yorncrtion flh2norn::non in vivo, was un:lbl:.: to obt.-:ir? i>ositivr: rczl.LLts in vitro. 'Th-n first tucccssful dw!orititrY t,ion of -- ths rcactiori in the test 1,ubk VU. ci,:'ricti out 2; Lawson (;I'!c~ Lia in riutri?!nt brotil contt,ining iznti-fi r:;bSit, :;cr~. i'r.om t,!xi.t -time on, : :2r'urr or serous fluid in the medium essential? Khy we some s2ra capable of sqqorting trtinsformation, while others utterly. fail? What components function as essentiel factors, how do they act, and what is the biochemical nature of their action in respect to th2 cellular changes evoked by the specific pn8umococcdl nucleic acid? Studies currently in progress indicate that serum factor is com- , posed of at least three essential constituont3. These are 1) the R-antibody, t i which causes agglutination of unencapsultited h. pneumococci; 2) a dialyzable constituent; and 3) an additional protein factor occurring in t,he globulin fraction of the serum. The evidence for assuming that tlw bcrum factor depends upon the collective action of the three comyonentz is mmrnarimi below, together with a description of' certain experiment:. &xigned to 172 elucidate the function of each, and the mechanism of th;:ir combined effect. Finally, a statement is made of the current working hypothesis of the nature of this mechanism. 1) R-antibody. Men unencapsulated Ii-pneumoco(+ci are grof#n in the presence of &antibody, large aggregates arc formed which settle to ihe bottom of' the tube, The sup?rnztant broth is thus ief'i. clear, so that if transformation occur3 end br~c::-ApsliiEt8d S cells ar'3 formed, tI\C chingo is readily apparent, since: thi: nzwQ formcci S toll:;, not bziil,r: r.l'fected by thr: li-cmtiboci;r, groa dif'i'u:;eLy t,hrop;hout t?le culture. Vhi'le this phenomenon has been useful in thy t,~>ci~ni~;uc ot' t!.p? trnnuforming tzE;t., th:? fr-antibody appears to do more t.hl;n mer*el,f provide iri:;ihle oTritIencc oi' transformation. In t,he usuc?l fluid m&i:1 it h;:s :lot Sz?t?n poesib~lr, to induce izens- formation unless A-a.r:tibody is pr!?:ji-'nt. tloxwer) utltfer Spccinl conditions, results hav: been obtained which give some: indic;,tior! of the role of II- entibodjr L In u semi-solid medium containing a :Low concentration of agar (0.2 O/o), pnsumococci grow+ in colonies rather tiu?n 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. Transforrna- tion of Qpe 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. witah certainty why cdonial 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 z:. shallow layer 173 not exceeding 1-2 mm. in depth. In the shallow layer, oxidizing conditions are promoted, and evon in the usual serum medium containing R-antibody, manifest transformation does not occur. Attempts to reverse the effect of the shallow layer by the addition of reducing agents have not yielded con- sistent result- &,, but on one occasion trmsformntim was obtained in n group of flasks in which glutathionc had br:en addled to the uswl swum medium. Thwi` is, then, cortzin cvidoncc that reducing conditions :wr? csnsntial in some pha SC: of the tr:msforming reaction. To s~lmerizo, the R-antibody serves the purpose of ccusing EII nppar~ntly i:ssi:ntir.l colonial aggregation of R-pncumococci, which in turn results in loca7. conditions, possibly reducing in charoctm*, that me rquired in the transformation rcsction. 2) Diolymblc constituent. FArYLy attempts at salt' fractionation of serum factor by th: clnssicnl m&hods of protein ahemictry yielded totally inactive fractions. Some light has been thrown on these results by the discovery that a dialyeable component of serum is essential. When an active serum is dialyzed against physiological saline, th-sre 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 conditions, however, the R-antibody is unimpaired, and no dcnaturation 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 important respects; In the first place, if inorganic phosphate is added to the dialyzed serum, and the mixture incubated l-2 hours, the serum regains its ability to support transformation. The period of incubation of the serum with phosphate is essential. The interaction betvfecn phosphate and the serum appears to be prevented by the presence of nutrient broth, for if the latter is added at the same time as the phos;:hate, or after a short period of incubation, no reactivation is achieved. In contrast to the reactivation by phosphate, which requires time, immediate reactivation can be achieved by adding to the sorum such materials as unheated neopeptono, or tryptic digast of cascin. Nutrient broth doe:, not interfere with this effect. The nature of the substznco responsible for immediate reactivation has not been determinod. tioviever, it has boron shown to bo a dislyznble substance, and to be prccipitable by elcohoi. Tk avt.flabl~: dr:t::. suggest that phosphntc brings about reactivation of dialyzed scrufn b-j promoting 1' chemical or cnzymutic renction, which re- sults in syntkzsis of thti dizlyzabl:. constituent of serum f?rctor. On the other hand, the neopeptonc, or c;&.sein digest, provides a preformed source of the dinlyzable constituent, or some related substcnce 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 dialyaable 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 `b.nBfoITk ing system. 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 used salts :-Jch as ammonium sulfate and organic protein przcipitants such as alcohol, have ust;-blishod that this additional constituent, as well a.5 R-antibody, occurs in the globulin fraction of the serum. For the IJwpOse of orienting further research, the possibility has been consider& that the globulin ft?ctor ;Xiy k an enzyme. If this is indeed the CELS~, it S.:WIS highly probable on g~nr:ral grounds that wme organ of the anim;il body contilins the enzyrnc in much higher concentration than does th,, serum, and asuM s~rv:: his z morz fL:vorablf2 s3urco for possible purificution sn3 iwntification of the enzyme. TO test this nssumption, c, prolininiry survey was ;~,;!c ,.f sevr:rc~l ribbit L>rgans by preparing simple saline cxtri~cts r;.nrl tzsting thorn for t'ne pr- csence of the globulin factor. 'Ihe procedure u s'3d c.Jnsistxd .jf Ading the extract tu broth containing a srnii1-1 emount of concentratad rabtit R-antibody wd unheated noopeptone as n source crf the diOaljxablo cowtituent,. 'Ihc broth wnt3ining those added components was tes?~d for its ability tc: support tr~nsformatiN.2n. Positive results indicate that ttso organ extract has supplisd the: missing constituent (globulin fwtor), since the R-antibody End dialyzablc factor alone are unable to support transformation. Rabbit spleen proved to be a good source of the globulin factor. TG provide larger organs as source material, extracts of calf spleen and calf thymus were then tried, and it was found that tkymus is suparior not only to spleen, but to the best sera available. Fractionation experiments with tmus 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 is the possibility of determining the nature of the substance and its r&e in the transforming reaction. The rvalts of preliminary studies with organ extracts lend some support to the &pothesi.s onwhich the experiments were bc.sed, i.e., th:;t thy ir,l~buliri f::(:t& of t;h~ r::r~m mzy be zn enzyme. 176 Muchanism of Fiction of serum factor. A series of axp2riments Yihich were? dcsigncd to provide n more intincte !=nonlsdgc of the inlzrcction betv,r::en thti specific transforming substance (pneumococccl desoxyribonuclcic nci6Y) anti the susceptible pneumococcnl cells proved to have an important bearing on tho problem of thn r$le of swum fxtor. The customary procdure in demur&rating the phenomcnon'of transformati:)n is to add the specific dc;?oxyribonuclai.c ticid tn thz sew medium end tr, inccul:~te with R SUSCOF- tible strain of R-pnoumococcus. Transformation becomes apparent nfter 16 to 20 hours' incubatia, but little is knovlrn of the course of events during this period of incubation. 'ihe purified enzyme, dcsoxyribonucle:lse, which specificall;r inactivates th<: transforming subst::nce, b:; been US& i1G n tuol in an attempt to stur1.y certain phases of this problem. By adding dcsvxyribonuclosue to the transforming system at various intervals after inoculation, in a concentrati:Jrl known to givs almost immsr1- Lztc: inactivntisn of the specific transforming sub:;tancc, it is possible t;, determine the length of time requirr!d for the trsnsforming substance ta be taken up or "fixed" by the susceptible cells. The aciditi:,n 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 substincc is readily accessible to the desoxyribonuclcase. After 4 to 5 hours, on the other hand, the arIc?ition of desoxyribonuclease has nci observable effect on the course of the reaction. Consequently it appears that growth of the R cello in serum medium for 3 to 5 hours is required before the specific dcsoxyribonucleic acid is t&en up by the cells, and thus protected from enzymatic destruction. A striking wnfirmatir,n of the foregoing is provided by experiments in which the K calls arc gra?m in serum medium in the absance of the specific 177 transforming substance. After 4 to 5 hours' growth under these conditions, the cells arc so "sensitized 1' that when transfr:r.r:xl to a medium containing the transforming substance, the latter is taken up in as short a time as 15 minutx. If the transfer ia mar!e after short'Jr ~xziod:s of growth in sarum medium, c.g. 2 to 3 hours, the 11~~n:5tization~1 h:~s qxrcntly not taken ph?Ce :;ni! r:i)Jid fixation of tht> trzinsforming substance ciinuot be G.cmon- strater:. Thus, the avant:; that occur in that first, four htiurs of @o&h of H cells in the transforming systljm :ux indqxxxl~;nt of the prxxncc of the specific transforming substr:nce. It must ba conclucM that growth unclcr thcsc conditions alters the cell in some way, or provi!!co ouitablc onviron- mental conditions so t`n:t the intxhction b:;t,v;::r;n the.: cell xxi the trans- forming substance cm t,:k<: pl.acz. The rclatiori of the: abovc. .3xpcrimc;nts to th;: r$Lk of: 3i:rum fzctoz becomes apprrent from th? fact thrrt growth of the cells in p1:li.n broth, in the prosonce of R-antibody, or in semm inactiv,atcd by dis.Lysia, fails in each case to 'lsensitizel' the cells. Thus, tha hypothesis is suggssixd that tha major part played bf serum in the transforming systam is concernad 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 &e of serum factor is concerned with some action on the surface of the susceptible R cell which permits interaction between the cell c.nd the specific transforming substxncc. A plausibla and pnrhaps useful hypothesis with regard to the mechanism of tha serum effect can be bassd on the assumption that the action is enzymatic in nature. In this view, the enzyme is present in the globulin fractions of active sora, as well cs in 178 extracts of certain mammalian organs. The R-antibody, as a rdsu3.t of the colonial gronth of the cc:Lls, provides the required conditions for the action .,f the mzpc. It is suggosteil %bJtt the reducing ctinditions rc.ouLting from this type of grov:th may be of significance. The enzymatic sjrstcm ~1s outlirictl is not mcrc cumplex than some already known. f¢ work tin au~cl~ hex*akbilss in thz laboratory of Dr. Carl '1. Cori, provides a rather striking; analogy. Muscle extract:; lose hexokinase activity upon diulysi:;, and, a=; in the case of serum factor, activity can bu restored 'by two means: 1) by inculbation with phosphate, or 2) immodiatelg by adding the preformed co-factor, which in this cast! prov,>s to bc guaninti. In addition to guzninc: as co-fL:ctor, muscle hexokinzJe also requires the pr`>-:a c.9..nce of IC:~UXXL co-enzyme I (tlihyi!rocozy.;aoe). This latter fact is swzestlvi::, in vic:w of the possible r&c of reducing conditions in the action of serum factor. Although the hypothesis outlinu; abov:: is admittedly only a tentative one, it h&s rmk? pOSSibh an cxp.zimental approach to the problem of defining the significance ant' essential role of the so-called ltsarum factor" in the transforming system. While the interpretation may be mod-- fied as knowledge increases, the facts thus far obtained indicate that transformation consists of trro phases:- 1) on initial phase in which, as the result of the combined action of the serum components, the R cells are rendered receptiv e anl: become capable of taking up the transforming substnnce; and 2) a phase in which a chain of biochemical rettctions is initiated within the cells that culminates in synthesis of the cnpsti;r polysac- charidc, the chemical identity and type specificity of which can be pre- determined, depending on type of encspsulated ceLLs used as source of the transforming substance. 174, C-?tochomical studies on localization of desoxflibonucleic acid in pneumococcal cells (Taylor). Genetic cnil cytological studies of animals and plants have for the most p?rt localized the hcrcctitary units of the cell in the nucleus. Cytochcmicc;l stullies have shown that the most charac- teristic component of the nucleus is ~~csoxyribonucleio acid. The active substance in2ucing trcsnsfornntion of pneumococcal types end determining the spccif icity of the Ch;:~2C has Soen found to he a nucleic ucic? of the des- oxyribosc: type. Since trtinsformntion may be S~~cribcd as a change in the heredity of the pneumococcnl cell, it is of interest to know whether pneumo- coccr;l dcso>ryribonucleic XciC occurs in a form&. structure comparable to t!le nucleus of hik:hcr orgflnismc. under ccJntrollk!c ctindiiicns t,he Fculf;cn nuclear reactJon appears to te specific for desoxyribonuclcic acid. Ghcn the Feulcen reXAii.,n is CWried {Jut On pneUI'nOcOcct21 Cells fixed with osmium tetroxidc vapor, a deeply staining ro,L; qanule can rcacli.Lg 7 be observed ai.tMn each cell. This suggests that desoqwibonucleic aci< is not cvcnly distributed throughout the cell, but is partially or entirely localized. In a medium containing anti-8 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 Seen 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 prep:lrati.ons of r~esoxyribonuclcnse. 180 Adnptation of this technique to th2 study of the cytology of pncumococcal cells is Laing i32c!e, since the enzymo affords ;L wrc specific method for Lientifying the nature of thr! central granules than do any of the known staining m&hods :Ivailnblo at present. Publicctions IlcCnrty, M. PurificatL:n onci proportias of clcs;oxyrihonucfe~r:c isolnterl from beaf pancreas, J. Gcn. Ph..siol., 1946, 2, 123. McCarty, 2:. and ILvvc;ry, 0. T. Studi2s on thu chemiwl natur;: of the su1e stance inducing: transformation of pneumococcal types. II Effect uf dcsoxyribonuc%wso 011 the biological activity uf tht transform- ix-q! substance, 11. E&p. Med., 1946, Q, 89. McCarty, M. end Avery, 0. T. Studies on ths chemical nature of the sub- stincc inducing tr:lnuformuti.un of pncum~coccnl ti/pcs. III An improved metho- A fur thr: ispjlation of the trwsforming suSstc;ncc snd its opplic;tLn to Pneumucoccus Types II, III and VI. J. Exp. Med., l946, &3, 97. I