Avery, McLeod, McCarty

7/29/2019 Avery, McLeod, McCarty

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S T U D I E S O N T H E C H E M I C A L N A T U R E O F T H E S U B S T A N C EI N D U C I N G T R A N S F O R M A T I O N O F P N E U M O C O C C A L T Y P E S

INDUCTION OF TRANS]~ORMATION BY A DESOXYRIBONUCLEIC ACID FRACTIONISOLATED I~RO~ PNEUMOCOCCUS TY PE II I

BY OSWAL D T. AVERY, M .D., COLIN M. MACLEOD, M.D., ANDMACLYN McCARTY,* M.D.(From the Hospital o f The Rockefeller Institute for Medical Research)

PLATE 1(Received fo r publication, Novem ber 1, 1943)

Bio log i s t s have long a t t empted by chemica l means to induce in h ighe rorganisms predictable and specif ic changes which thereafter could be t rans-mi t t ed in s er i es a s he red i t a ry cha rac te rs . Among mic roSrganisms the mos ts t r iking example of inheri table and specif ic a l tera t ions in cel l s t ructure andfunc t ion tha t can be expe r imenta l ly induced and a re reproduc ib le under we l ldefined and adequately control led condi t ions is the t ransformat ion of specif ictypes of Pneumococcus . Thi s phenom enon was f i rs t desc r ibed by G r i~ th (1)who succeeded in t rans forming an a t t en ua ted and non-encapsu la t ed (R)va r i an t de r ived f rom one spec i fi c type in to fu l ly encapsu la t ed and v i ru l en t (S )cells of a heterolo gou s specific typ e. A typic al instance will suffice to il lustra tethe t echniques or ig ina lly used and s e rve to ind ica te the w ide va r i e ty o f t r ans -form at ions tha t are poss ible wi thin the l imits of this bacter ia l species.

Gri ~th found that mice injected subcutaneously with a small amo unt of a l iving1~ culture de ri, ed from Pneumococcus T yp e H together with a large inoculum o fheat-killed T yp e III (S) cells frequ ently succumbed to infection, and tha t th e hear t'sblood of these animals yielded Ty pe HI pneumococci in pure culture. Th e fact tha tthe P~ strain was avirulen t and incap able by itself of causing fatal b acteremia and theadditional fact that the heated suspension of Ty pe H I cells eoataincd no viable or-ganisms brought convincing evidence that the 1~ forms growing under these condi-tions had newly acquired the capsular stru cture an d biological specificity of Ty pe I IIpneumococci.Th e original observations of Griffith were later confirmed b y Neufeld an d L evin-thal (2), and by Banrherm (3) abroad, and by Dawson (4) in this laboratory. Subse-que ntly Dawson and Sia (5) succeeded in inducing transform ation in ~tro. Thisthe y accomplished by growing R cells in a fluid medium containing anti-R serum an dheat-killed encapsulated S c ells. Th ey showed tha t in the test tube as in the animalbod y transformation can be selectively induced, depending on the typ e specificityof the S cells used in the reaction system. Later , Alloway (6) was able to cause

* Wo rk done in pa rt as Fellow in the Medical Sciences of the Natio nal ResearchCouncil.13 7

Published February 1, 1944


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13 8 TRA.NS~ORMATION O:F PNEITMOCOCCALTYPESspecific transformation in vi lro using sterile ex tracts of S cells from w hich al l formedelements and celiular debr is h ad be en removed by Berkefeld f i l tra t ion. He thusshowed that crude ext racts containing act ive t ransforming mater ia l in soluble formare a s effective in inducing specific transfo rma tion as are the inta ct cells from whichthe ex t racts were prepared.Ano ther example of t ransformat ion which i s analogous to the interconver t ibi l i ty ofpneumococcal type s l ies in the field of viruses. Be rry and De drick (7) succeeded inchanging the vi rus of rabbi t f ibroma (Shope) into that of infect ious myxoma (San-arelli) . The se investigators inoculated rabbits with a mixture of active fibrom a virustogether wi th a suspension of heat - inact ivated myxoma vi rus and produced in theanimals the symptoms and pathological lesions characterist ic of infectious myxoma-tosis. On subsequent an imal passage the transform ed virus was transmissible andinduced myx omatou s infect ion typical of the natu ral ly occurr ing disease . Late rBerry (8) was successful in inducing the same transformation using a heat-inacti-vated suspension of washed elementary bodies of myx om a vi rus . In the case of theseviruses th e me thod s emplo yed were similar in principle to those used b y G riffi th inthe t ransformat ion of pneumococcal types . These observations hav e subsequent lybeen conf i rmed b y other invest igators (9) .

T h e p r e s e n t p a p e r i s c o n c e r n e d w i t h a m o r e d e t a i l e d a n a l y s is o f t h e p h e n o m e -n o n o f t r a n s f o r m a t i o n o f s p e c if ic t y p e s o f P n e u m o c o c c u s . T h e m a j o r i n t e r e s th a s c e n t e r e d i n a t t e m p t s t o i s o l a t e t h e a c t i v e p r i n c i p l e f r o m c r u d e b a c t e r i a le x t r a c t s a n d t o i d e n t i f y i f p o s s ib l e it s c h e m i c a l n a t u r e o r a t l e a s t t o c h a r a c -t e r i z e i t s u f fi c i en t l y t o p l a c e i t i n a g e n e r a l g r o u p o f k n o w n c h e m i c a l s u b s t a n c e s .F o r p u r p o s e s o f s t u d y , t h e t y p i c a l e x a m p l e o f t r a n s f o r m a t i o n c h o s e n a s aw o r k i n g m o d e l w a s t h e o n e w i t h w h i c h w e h a v e h a d m o s t e x p e n e n c e a n d w h i c hc o n s e q u e n t l y s e e m e d b e s t s u i t e d f o r a n a l y s i s. T h i s p a r t i c u l a r e x a m p l e r e p r e -s e n t s th e t r a n s f o r m a t i o n o f a n o n - e n c a p s u l a t e d R v a r i a n t o f P n e u m o c o c c u sT y p e I I t o P n e u m o c oc c u s T y p e I I I .

EXPEJ~I~E.NTALT r a n s f o r m a t i o n o f p n e u m o c o c c a l t y p e s i n ritro r e q u i r e s t h a t c e r t a i n c u l t u r a l

c o n d i t i o n s b e f u l f il le d b e f o r e i t i s p o s si b le t o d e m o n s t r a t e t h e r e a c t i o n e v e n i nt h e p re s e n c e o f a p o t e n t e x t ra c t . N o t o n l y m u s t t h e b r o t h m e d i u m b e o p t im a lf o r g r o w t h b u t i t m u s t b e s u p p l e m e n t e d b y t h e a d d i t i o n o f s er u m o r s e r o u sf l u id k n o w n t o p o s se s s c e r t a i n s p ec i a l p r o p e r t i e s . M o r e o v e r , t h e R v a r i a n t ,a s w i ll b e s h o w n l a t e r , m u s t b e i n t h e r e a c t i v e p h a s e i n w h i c h i t h a s t h e c a p a c i t yt o re s p o n d t o t h e t r a n s f o r m i n g s ti m u l u s . F o r p u r p o s e s o f c o n v e n i e n c e t h e s es e v e r a l c o m p o n e n t s a s c o m b i n e d i n t h e t r a n s f o r m i n g t e s t w i l l b e r e f e r r e d t oa s t h e react ion sys tem. E a c h c o n s t i t u e n t o f t h i s s y s t e m p r e s e n t e d p r o b l e m sw h i c h r e q u i r e d c l a r if i c a ti o n b e f o r e i t w a s p o s s ib l e t o o b t a i n c o n s i s t e n t a n dr e p r o d u c i b l e r e s u l ts . T h e v a r i o u s c o m p o n e n t s o f t h e s y s t e m w il l b e d e s c r i b e di n t h e f o l l o w i n g o r d e r : ( 1) n u t r i e n t b r o t h , ( 2 ) s e r u m o r s e r o u s f lu i d , (3 ) s t r a i no f R P n e u m o c o c c u s , a n d ( 4 ) e x t r a c t i o n , p u r i fi c a t io n , a n d c h e m i c a l n a t u r e o ft h e t r a n s f o r m i n g p r i n c i p l e .



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OSWALD T. AVE RY, COLIN M. M.AcLEOD~ AND ~T.ACLYlq McCARTY 13 91 . N u t r i e n t B r o t h . - - B e e f hear t in fus ion b ro th con ta in ing 1 pe r cen t neopep tone

wi th no a dded dex t rose and a d jus ted to an in i t i a l pH of 7 .6 - -7 .8 i s used as the bas icm e d i u m . I n d i v i d u a l lo t s o f b r o t h s h o w m a r k e d a n d u n p r e d i c ta b l e v a r i at i o n s in t h ep r o p e r t y o f s u p p o r t i n g tr a n s f o r m a t io n . I t h a s b e e n f o un d , h o w e v er , t h a t c h a r c o a la d s o r p ti o n , a cc o r d i n g t o t h e m e t h o d d e s c ri b e d b y M a c L e o d a n d M i r i c k ( 10 ) f o rremo va l o f su lfonamide inh ib i to r s , e l imina tes to a l a rge ex ten t these va r ia t ion s ; conse -quen t ly th i s p rocedure i s used as rou t ine in the p repara t ion o f cons i s ten t ly e f fec t iveb r o t h f o r t i t r a ti n g t h e t r a n s f o rm i n g a c t i v i t y o f e x t r a c ts .

2 . S e r u m o r S e r o u s F l u i d . - - I n the f i r s t successfu l exper iments on the induc t ion o ft r a n s f o r m a t i o n in vitro, Dawson and S ia (5 ) found tha t i t was essen t ia l to add se rumt o t h e m e d i u m . A n t i - R p n e u m o c o c c a l r a b b i t s e r u m w a s u se d b e c au s e o f t h e o b s e r v a -t i o n t h a t r e v er s io n o f a n R p n e u m o c o c c u s t o t h e h o m o l o g o u s S f o r m c a n b e i n d u c e db y g r o w t h i n a m e d i u m c o n t a i n in g a n t i - R s e r u m . A l l o w a y (6 ) l a t e r f o u n d t h a t a s -c i t i c o r ches t f lu id and normal swine se rum, a l l o f which con ta in R an t ibod ies , a recapab le o f r ep lac ing an t ipneum ococca l r abb i t se rum in the reac tion sys tem. Someform of se rum i s essen tial , and to our know ledge t r ans fo rmat ion in vitro h a s n e v e rbeen e f fec ted in the absence o f se rum or se rous f luid .

In the p resen t s tud y hum an p leura l o r a sc i t i c f lu id has been used a lmos t exc lus ive ly .I t becam e apparen t , however , th a t the e f fec tiveness o f d i f fe ren t lo ts o f se rum v ar iedand tha t the d i f fe rences obse rved were no t necessa r i ly dependen t upon the con ten to f R an t ibod ies , s ince many se ra o f h igh t i t e r were found to be incapab le o f suppor t -ing t r ans fo rmat ion . Th is f ac t sugges ted tha t f ac to r s o the r than R an t ibod ies a reinvo lved .

I t has been found tha t se ra f rom var ious an imal spec ies , i r r e spec t ive o f the i ri m m u n e p r o p e r ti e s , c o n t a i n a n e n z y m e c a p a b l e o f d e s t r o y in g t h e t r a n s f o r m i n g p r i n -c ip le in po te n t ex t rac t s . Th e na tu re o f th i s enzyme and the spec if ic subs t ra te onwhich i t ac t s wil l be re fe rred to l a te r in th i s paper . Th is enzy me is inac t iva ted byh e a t i n g t h e s e r u m a t 6 0 - 6 5 C . , a n d s e r a h e a t e d a t t e m p e r a t u r e s k n o w n t o d e s t r o ythe enzym e a re o f ten rendered e f fec t ive in the t r ans fo rming sys tem. Fur th e r an -a lys i s has shown tha t ce r ta in se ra in which R an t ibod ies a re p resen t and in which thee n z y m e h a s b e en i n a c t i v a te d m a y n e v e rt h e le s s fa i l t o s u p p o r t t r a n s fo r m a t i o n . T h i sfac t sugges t s tha t s t i ll ano the r f ac to r in the se rum i s e ssen tial . Th e con te n t o f th i sfac to r va r ies in d i f fe ren t se ra , and a t p resen t i t s iden t i ty i s unknown.

There a re a t p resen t no c r i t e r i a which can be used as a gu ide in the se lec t ion o fsu i tab le se ra o r se rous f lu ids excep t tha t o f ac tua l ly t e s t ing the i r capac i ty to suppor tt r a n s f o r m a t io n . F o r t u n a t e l y , t h e r e q u is i te p r o p e rt i e s a r e s ta b l e a n d r e m a i n u n i m -p a i r e d o v e r l o n g p e r io d s o f t i m e ; a n d s e r a t h a t h a v e b e e n s t o r e d i n t h e r e f r ig e r a t o rf o r m a n y m o n t h s h a v e b e e n f o u n d o n r e t e s t i n g t o h a v e l o s t l i t t l e o r n o n e o f t h e i ro r ig ina l e f fec t iveness in suppor t ing t r ans fo rmat ion .

T h e r e c o g n i t i o n o f th e s e v a r i o u s f a c t o r s i n s e r u m a n d t h e i r r 61 e i n t h e r e a c t i o ns y s t e m h a s g r e a t l y f a c i li t a te d t h e s t a n d a r d i z a t i o n o f t h e c u l t u r a l c o n d i ti o n sr e q u i r e d f o r o b t a i n i n g c o n s i s t e n t a n d r e p r o d u c i b l e r e s u lt s .

3 . T h e R S t r a i n ( R 3 6 A ) . - - T h e u n e n c a p s u l a t e d R s t r a i n u s e d i n t h e p r es e n ts t u d y w a s d e ri v e d f r o m a v i r u l e n t " S " c u l t u r e o f P n e u m o c o c c u s T y p e I I .I t w i ll b e re c a l le d t h a t i r re s p e c ti v e o f t y p e d e r i v a t i o n a l l " R " v a r i a n t s o fP n e u m o c o c c u s a r e c h a r a c t e r i z e d b y t h e l a c k o f c ap s u l e f o r m a t i o n a n d t h e



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14 0 TRANSFORMATION OF PNED'MOCOCCAL TYPE Sc o n s e q u e n t l o s s o f b o t h t y p e s p e c i f ic i t y a n d t h e c a p a c i t y t o p r o d u c e i n / e c t i o ni n th e a n i m a l b o d y . T h e d e s i g n a t i o n o f t h e s e v a r i a n t s a s R f o r m s h a s b e e nu s e d t o r e f e r m e r e l y t o t h e f a c t t h a t o n a r t i fi c i a l m e d i a t h e c o l o n y s u r fa c e i s" r o u g h " i n c o n t r a s t t o t h e s m o o t h , g l i s te n i n g s u r f a c e o f c o l o ni e s o f e n c a p s u l a t e dS cel ls .

The R s t ra in referred to above as R36A was der ived by g rowing the paren t Scu l tu re o f P n eu m o co ccu s T y p e I I i n b ro th co n tain in g T y p e I I an t ip n eu m o co ccu srabb i t serum fo r 36 ser ia l passages and iso la t ing the va r ian t thus induced . Thest ra in R 36A has los t a l l the speci fic and d is t ingu ish ing character is t ics o f the pa ren t Sorgan isms and consis ts on ly o f a t ten uate d and non-encapsu lated R var ian ts . Thechange S -~ R is o f ten a revers ib le one p rov ided the g cel ls are no t to o far "deg raded ."The revers ion o f the R fo rm to i t s o r ig inal specif ic type can f reque n t ly be accomplishedby success ive an ima l passages o r by repeated ser ia l subcu l tu re in an t i -R se rum. Wh enrevers ion occurs under thes e cond it ions , however , the g cu l tu re inva r iab ly rever ts tothe enc apsu lated fo rm of the sam e speci fic type a s that f rom which i t was der ived (11) .S t r a in R 3 6 A h as b eco m e r e l a t i v e ly f ix ed i n t h e R p h ase an d h as n ev e r sp o n ~ n eo u s lyrev e r t ed t o t h e T y p e I I S fo rm . M o reo v er , r ep ea t ed a t t em p t s t o cau se i t t o r ev e r tunder th e condi tions jus t m en t ioned ha ve in a l l in s tances been unsuccessfu l .

T h e r e v e r s i b l e c o n v e r s i o n o f S ,- ~- R w i t h i n t h e l i m i t s o f a s i n gl e t y p e i s q u i t ed i f f e r e n t f r o m t h e t r a n s f o r m a t i o n o f o n e s pe c i fi c t y p e o f P n e u m o c o c c u s i n t oa n o t h e r s p e c if ic t y p e t h r o u g h t h e R f o r m . T r a n s f o r m a t i o n o f t y p e s h a s n e v e rb e e n o b s e r v e d t o o c c u r s p o n t a n e o u s l y a n d h a s b e e n i n d u c e d e x p e r i m e n t a l l yo n l y b y t h e s p e c i a l t e c h n i q u e s o u t l i n e d e a r li e r i n t h i s p a p e r . U n d e r t h e s ec o n d i t i o n s , th e e n z y m a t i c s y n t h e s i s o f a c h e m i c a l l y a n d i m m u n o l o g i c a l l yd i f f e r e n t c a p s u l a r p o l y s a c c h a r i d e i s s p e c i f ic a l l y o r i e n t e d a n d s e l e c t i v e l y d e -t e r m i n e d b y t h e s p e c if i c t y p e o f S c e ll s u s e d a s s o u r c e o f t h e t r a n s f o r m i n g a g e n t .

In t h e co ur se o f t h e p resen t s t u d y i t w as n o t ed t h a t t h e s to ck cu l tu re o f K 3 6 o nserial transfers in blood broth undergoes spontaneous dissociation giving rise to anum ber o f o ther P -. var ia n ts which can b e d is tingu ished one f rom ano th er b y co lonyform. The s ign ificance o f th is in the p resen t ins tance lies in the fact tha t o f fourd i f feren t var ian ts i so la ted f rom the par en t R cu l tu re on ly one (R36A) is suscep t ib le tothe t ransfo rming act ion o f po ten t ex t racts , wh i le the o thers fa i l to respond and arewhoUy inact ive in th is regard . The fact tha t d i f ferences ex is t in the responsivenessof d if ferent R var ian ts to the s ame specif ic s timulus enphasizes the ca re that mu st beexercised in the select ion o f a su i tab le R var ian t fo r use in exper imen ts on t rans-fo rmat ion . The ca paci ty o f th is R s t ra in (R36A) to respond to a var ie ty o f d i f feren tt ransfo rming agen ts i s shown by the read iness wi th which i t can be t ransfo rmed toT y p e s I , I l l , V I , o r X IV , a s w e l l a s t o i t s o rigina l t y p e (T y p e I f ) , t o w h ich , a s p o in tedou t , i t has never spon taneously rever ted .

Al though the s ign ificance o f the foUowing fact w i ll become app aren t la ter on , i tmust be men t ioned here that pneumococcal cel ls possess an enzyme capab le o f de-s t roy ing the act iv i ty o f the t ransfo rming p rincip le . Indeed , th is enzyme has been



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OSWALD T. AVERY , COL IN M. ~c LE O D , AND M.ACLYN McCARTY 141f o u n d t o b e p r e s e n t a n d h i g h l y a c t i v e i n t h e a u t o l y s a t e s o f a n u m b e r o f d i f f e r e n ts t ra ins . Th e fac t tha t th i s in t race l lu la r enzy me is r e leased dur ing au to lys i s ma yexp la in , in pa r t a t l eas t , the obse rva t ion o f Daw son and S ia (5 ) tha t i t i s e ssen tia l inb r i n g i n g a b o u t t r a n s f o r m a t i o n i n t h e t e s t t u b e t o u s e a s m a l l i n o c u l u m o f y o u n g a n dac t ive ly g rowing R ce l ls . Th e i r reg t f l a ri ty o f the resu l t s and o f ten the fa i lu re toinduce t r ans fo rmat ion when la rge inocu la a re used may be a t t r ibu tab le to the re leasef r o m a u t o l y z i n g c e ll s o f a n a m o u n t o f t h i s e n z y m e s u t S ci e n t t o d e s t r o y t h e t r a n s -fo rm ing p rinc ip le in the reac t ion sys tem .

I n o r d e r t o o b t a i n c o n s i s t e n t a n d r e p r o d u c i b l e r e s u l ts , t w o f a c t s m u s t b eb o r n e i n m i n d : f ir s t, t h a t a n R c u l t u re c a n u n d e r g o s p o n t a n e o u s d i s s o ci a t io na n d g i v e r is e t o o t h e r v a r i a n t s w h i c h h a v e l o s t th e c a p a c i t y t o r e s p o n d t o t h et r a n s f o r m i n g s t i m u l u s ; a n d s e c o n d ly , t h a t p n e u m o c o c c a l c el ls c o n t a i n a ni n t r ac e l l u la r e n z y m e w h i c h w h e n r e le a s ed d e s t r o y s t h e a c t i v i t y o f t h e t r a n s -f o r m i n g p r i n c i p le . C o n s e q u e n t l y , it i s i m p o r t a n t t o s e l ec t a r e sp o n s i v e s t r a i na n d t o p r e v e n t a s f a r a s p o s si b l e t h e d e s t r u c t i v e c h a n g e s a s s o c i a t e d w i t ha u t o l y s i s .Method of Ti trat ion of Transforming Ac t ivi ty .pin t h e i s o l a t i o n a n d p u r i f i c a -t i o n o f t h e a c t i v e p r i n c i p l e f r o m c r u d e e x t r a c t s o f p n e u m o c o c c a l c el ls i t i sd e s ir a b le t o h a v e a m e t h o d f o r d e t e r m i n i n g q u a n t i t a t i v e l y t h e t r a n s f o r m i n ga c t i v i t y o f v a r i o u s f r a c t i o n s .

The exper imenta l p rocedure used i s a s fo l lows : S te r i l i za t ion o f the mate r ia l to betes ted fo r ac t iv i ty i s accompl i shed by the use o f a lcoho l s ince i t has been found tha tth i s r eagen t has no e ffec t on ac t iv i ty . A measured vo lume of ex t rac t i s p rec ip i t a tedin a s t e ri l e cen t r i fuge tube b y the add i t ion o f 4 to 5 vo lumes o f abso lu te e thy l a lcoho l,and the m ix tu re i s a l lowed to s tan d 8 o r more hou rs in the re f r ige ra to r in o rde r to e f fec ts te r i li za t ion . Th e a lcoho l p rec ip i t a ted m ate r ia l is cen t r i fuged , the supe rna tan td i sca rded , and th e tube con ta in ing the p rec ip i t a te i s a l lowed to d ra in fo r a f ew minu tesin the inver ted pos i t ion to remove excess a lcoho l . Th e mo uth o f the tube i s thencare fu l ly f l amed and a d ry , s t e r i le co t ton p lug i s inse r ted . The p rec ip i t a te i s r ed i s -so lved in the o r ig ina l vo lume of sa line. S te r i l iza t ion o f ac t ive mate r ia l b y th i stechn ique has invar iab ly p rov ed e ffec t ive . Th is p rocedure avo ids the loss o f ac t ivesubs tance which may occur when the so lu t ion i s passed th rough a Berkefe ld f i l t e r o ri s hea ted a t the h igh t empera tu res requ i red fo r s t e r i l i za t ion .

To the charcoa l -adsorbed b ro th desc r ibed above i s added 10 pe r cen t o f the s te r i l easc i t i c o r l~ leura l f lu id which has p rev ious ly been hea ted a t 60C. fo r 30 minu tes , ino r d e r t o d e s tr o y t h e e n z y m e k n o w n t o i n a c t iv a t e t h e t r a n s f o r m i n g pr i n ci p le . T h eenr iched medium i s d i s t r ibu ted under asep t ic cond i t ions in 2 .0 cc . amounts in s te r i l etubes measu r ing 15 100 ram. The s te r i li zed ex t rac t is d i lu ted se r ia l ly in sa lineneu t ra l i zed t o pH 7 .2 -7 .6 by add i t ion o f 0.1 r~ Na OH , Or i t m ay be s imi la r ly d i lu tedin u /4 0 phosp ha te buf fe r , pH 7 .4. 0 .2 cc. o f each d i lu tion i s adde d to a t l eas t 3 o r 4t u b e s o f t h e s e r u m m e d i u m . T h e t u b e s a r e t h e n s e ed e d w i t h a 5 t o 8 h o u r b l o o dbro th cu l tu re o f R36A. 0 .0~ cc . o f a 10 4 d i lu t ion o f th i s cu l tu re i s added to eachtube , and the cu l tu res a re incuba ted a t 37C. fo r 18 to 24 hours .



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14 2 TILa.-NS~'OI~ATION O ~ PN'EU~OCOCCAL TYPEST h e a n t i - R p r o p e r t i e s o f t h e s e r u m i n t h e m e d i u m c a u s e t h e R c e ll s t o

a g g l u t i n a t e d u r i n g g r o w t h , a n d c l u m p s o f t h e a g g l u t i n a t e d c e ll s s e t t le t o t h eb o t t o m o f t h e t u b e le a v i n g a c l ea r s u p e r n a t a n t . W h e n t r a n s f o r m a t i o n o c c u rs ,t h e e n c a p s u l a t e d S c e l ls , n o t b e i n g a f f e c t e d b y t h e s e a n t i b o d i e s , g r o w d i f f u s e lyt h r o u g h o u t t h e m e d i u m . O n t h e o t h e r h a n d , i n th e a b s e n ce o f t r a n s f o r m a t i o nt h e s u p e r n a t a n t r e m a i n s c l e a r , a n d o n l y s e d i m e n t e d g r o w t h o f R o r g a n i s m so c c u r s . T h i s d i f f er e n c e i n t h e c h a r a c t e r o f g r o w t h m a k e s i t p o s s ib l e b y i n s p e c-t i o n a l o n e t o d i s t i n g u i s h t e n t a t i v e l y b e t w e e n p o s i t i v e a n d n e g a t i v e r e s u l t s .A s r o u t i n e a l l th e c u l t u r e s a r e p l a t e d o n b l o o d a g a r f o r c o n f i r m a t i o n a n d f u r t h e rb a c t e r io l o g i c a l i d e n t if i c a ti o n . S i n ce t h e e x t r a c t s u s e d i n t h e p r e s e n t s t u d yw e r e d e r i v e d f r o m P n e u m o c o c c u s T y p e I I I , t h e d i f f e r e n t ia t i o n b e t w e e n t h ec o l o n i e s o f t h e o r i g i n a l K o r g a n i s m a n d t h o s e o f t h e t r a n s f o r m e d S c e l l s i se spec i a l l y s t r i k i ng , t he l a t t e r be i ng l a rge , g l is t en i ng , mu co i d co l on i es t yp i ca l o fP n e u m o c o c c u s T y p e I I I . F i g s. 1 a n d 2 i l lu s t r a t e th e s e d if f e re n c e s i n c o l o n yf o r m .A t y p i c a l p r o t o c o l o f a t i t r a t i o n o f t h e t r a n s f o r m i n g a c t i v i t y o f a h i g h l yp u r i f ie d p r e p a r a t i o n i s g i v e n i n T a b l e I V .

Preparative MethodsSource MateriaL--In the present investigat ion a s tock labora tory s t ra in of Pneu -mococcus Ty pe I I I (A66) has been used as source mater ia l for obtaining the a ct iveprinciple. Ma ss cultures of these organisms are g row n in 50 to 75 l i ter lots of plainbeef hear t infusion broth. M ter 16 to 18 hours ' incubat ion a t 37C. the bacter ia lceils are collected in a steam-driv en steri lizable Sharpies centrifuge. Th e centrifugeis equipped with cooling coils immersed in ice wa ter so th at the cu lture fluid is t h o r -oughly chi lled before f lowing into the machine. This proced ure re tards autolysisdur ing the course of cent ri fugation. Th e sedimented bacter ia are remov ed f rom thecollecting cylinder and resuspended in ap prox ima tely !50 cc. of chil led ~aline (0,85per cent NaC1 ) , and care i s taken th at a l l c lumps are thorough ly emulsi fied. Theglass vessel containing the thick, creamy suspension of cells is immersed in a waterba t h , and t he t empera t u re of the suspension rapidly raised to 65C. Du ring theheat ing process the m ater ia l is Constant ly s t i r red, an d the tem perature m aintainedat 6 5C. for 30 minutes . Heat ing a t thi s tem peratu re inact ivates the int racel lularenzym e known to dest roy the t ransforming pr inciple.Extraction of Heat-Killed Cdls.--Although var ious procedures have been used,only that which has been found most sa t i s factory wi ll be descr ibed here . Th e heat -kil led cells are washed with saline 3 t imes. Th e chief value of the washing processis to remove a large excess of capsular poly ~cch ar ide together wi th m uch of the pro-te in, ribonucleic acid, and somat ic "C" polysacchar ide. Qu ant i ta t iye t i tra t ions oft ransforming act ivi ty have shown that not mo re than 10 to 15 per cent of the act ivematerial is lost in the washing, a loss which is small in comparison to the amount of

iner t substances which are rem oved by thi s procedure.M ter the final washing, th e cells are ex tracted in 150 cc. of saline contain ing sodiumdesoxycholate in f inal concent rat ion of 0.5 per cent by shaking the mixture me-



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OSWALD T. AVERY, COL IN M. MACLEOD, AND MACLYN McCARTY 143chan ica l ly 30 to 60 minu tes . Th e cel ls a re separa ted by cen t r i fuga t ion , and the ex-t rac t ion p rocess i s r epea ted 2 o r 3 times . Th e desoxycho la te ex t rac t s p repared inth i s mann er a re c lea r and co lo rless . These ex t rac t s a re combined and p rec ip i t a tedb y t h e a d d i t i o n o f 3 t o 4 v o lu m e s o f a b s o l u t e e t h y l a l co h o l. T h e s o d i u m d e s o x y c h o -la te be ing so lub le in a lcoho l r emains in the sup erna ta n t an d i s thus remov ed a t th i ss tep . The p rec ip i t a te fo rms a f ib rous mass which f loa ts to the su r face o f the a lcoho land can be removed d i rec t ly by li f t ing i t ou t wi th a spa tu la . The excess a lcoho l i sd ra ined f ro m the p rec ip i t a te which i s then red i sso lved in abou t 50 ce. o f sa line . Th eso lu t ion ob ta ined i s usua l ly v i scous , opa lescen t , and somew hat c loudy .Deproteinization and Removal of Capsular Polysaccharide.--The so lu t ion i s thendepro te in ized by the ch lo ro form me thod desc r ibed by Sevag (12) . Th e p rocedure isrepea ted 2 o r 3 times un t i l the so lu t ion becomes dea r . Af te r th i s p re l iminary t r ea t -me nt the m ate r ia l i s r ep rec ip i t a ted in 3 to 4 vo lumes o f a lcoho l . Th e p rec ip i t a teob ta ined i s d i s so lved in a l a rge r vo lume of sa l ine (150 cc . ) to which i s added 3 to 5nag. o f a pur i f i ed p repara t ion o f the bac te r ia l enzym e capab le o f hydro lyz ing theT y p e I I I c a p s u l a r p o l y s a c ch a r i d e (1 3 ). T h e m i x t u r e i s i n c u b a t e d a t 3 7 C ., a n d t h edes t ruc t ion o f the capsu la r po lysacchar ide i s de te rmined by se ro log ica l t e s t s wi thT y p e I I I a n t i b o d y s o l u t i o n p r e p a r e d b y d i s s o c i a t i o n o f i m m u n e p r e c i p i t a t e a c c o r d -i n g to t h e m e t h o d d e s c ri b e d b y L i n a n d W u ( 14 ). T h e a d v a n t a g e s o f u s i n g t h ean t i bod y so lu t ion fo r th i s purpose a re tha t i t does no t r eac t wi th o the r se ro log ica l lyac t ive subs tances in the ex t rac t and tha t i t se lec t ive ly de tec t s the p resence o f the cap-su la r po lysacchar ide in d i lu t ions as h igh as 1 : 6 ,000 ,000. Th e enzym at ic b re akdow nof the po lysacchar ide i s usua l ly comple te wi th in 4 to 6 hours , a s ev idenced by the lossof se ro log ica l r eac t iv i ty . Th e d iges t is then p rec ip i t a ted in 3 to 4 v o lumes o f e thy la lcoho l , and th e p rec ip i t a te i s red i s so lved in 50 cc . o f sa line. Depro te in iza t ion by thec h l o ro f o r m p ro c e s s i s a g a i n u s e d t o r e m o v e t h e a d d e d e n z y m e p r o t e i n a n d r e m a i n i n gt races o f pneum ococca l p ro te in . Th e p rocedure i s r epea ted un t i l no fu r th e r f i lm ofpro te in -ch lo ro form ge l i s v is ib le a t th e in te r face .Alcohol Fractionation.--FoUow ingdepro te 'miza t ion and enzymat ic d iges t ion o f thecapsu la r po lysacchar ide , the mate r ia l i s r epea ted ly f rac t iona ted in e thy l a lcoho l a sfo llows. Abso lu te e thy l a lcoho l i s added d ropwise to the so lu t ion wi th cons tan ts t i r ring . At a c r i t i ca l concen t ra t ion va ry ing f rom 0 .8 to 1 .0 vo lum e of a lcoho l theac t ive m ate r ia l separa te s ou t in the fo rm of f ib rous s t rands tha t w ind themse lvesa roun d the s t i rr ing rod . Th is p rec ip i t a te i s r emov ed on the rod and washed in a 50per cen t mix tu re o f a lcoho l and sa l ine . A l thou gh the bu lk o f ac t ive mate r ia l i s r e -m o v e d b y f r a c t i o n a t io n a t t h e c r i t i ca l c o n c e n t r a ti o n , a s m a l l b u t a p p r e c i a bl e a m o u n tremains in so lu tion . How ever , upon inc reas ing the concen t ra t ion o f a lcoho l to 3vo lumes , the res idua l f rac t ion i s th row n down to ge the r wi th ine r t m a te r ia l in the fo rmof a f loccu len t p rec ip i t a te . Th is f loccu len t p rec ip i t a te i s t ake n up in a smal l vo lum eof sal ine (5 to 10 cc.) and the solut ion aga in f rac t iona ted by the add i t ion o f 0 .8 to 1 .0v o l u m e o f a l c o h oL Addi t iona l f ib rous mate r ia l i s ob ta ined w hich is combined wi ththa t r ecovered f rom the o r ig ina l so lu t ion . Alcoho l ic f rac t iona t ion i s r epea ted 4 to 5t imes . Th e y ie ld o f f ib rous mate r ia l ob ta in ed by th i s me thod va r ies f rom 10 to 25rag . pe r 75 li t e r s o f cu l tu re and represen t s the m ajor por t ion o f ac t ive mate r ia l p resen tin the o r ig ina l c rude ex t rac t .Effect of Temperature.--As a rou t ine p rocedure a l l s teps in pur i f i ca t ion were ca r r i ed



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144 TRANS]~OR.M.ATION O F PNE U3K OCO CCA L T YPE S

out at room tempe rature unless specifically stated otherwise. Because of the the o-retical advantage of working at low temperature in the preparation of biologicallyactive material, th e purification of one lot (preparation 44) was carried ou t in the cold.In this instance all the above procedures with the exception of desoxycholate ex-extraction an d enzyme treatm ent were conducted in a cold room maintained at 0-4C.This prepara tion proved to have significantly higher activ ity than did m aterial simi-larly prepared at room temperature.Desoxycholate extraction of the heat-killed cells at low temp erature is less efficientand yie lds smaUer amou nts of the act ive fract ion. I t has been demonstra ted t h a thigher tempera tures facilitate extraction of the active principle, although act ivit y isbest preserved a t low temperatures.Analysis of Purified Transform ing M aterial

General Properties.--Saline so lu t ions con ta in ing 0 . 5 to 1 .0 rag . pe r co . o f thepur i f i ed subs tance a re co lor le ss an d c lea r in d i f fuse l igh t . How ever , in s t rongt r a n s m i t t e d l i g h t t h e s o l u t io n i s n o t e n t i r e l y c l ea r a n d w h e n s t i r re d e x h i b i t s as i lky sheen . S o lu t ions a t these concen t ra t ions a re h igh ly v i s cous .

P ur i f i ed m a te r ia l d i s so lved in phys io log ica l s a l t so lu t ion and s to re d a t 2 -4C.r e t a i n s i t s a c t i v i t y i n u n d i m i n i s h e d t i t e r f o r a t l e a s t 3 m o n t h s . H o w e v e r , w h e ndis so lved in d i st i l l ed wa te r , i t rap id ly dec reases in ac t iv i ty and becom es com -ple te ly ine r t wi th in a few days . S a l ine so lu t ions s to red in the f rozen s ta te in aCO2 ice box ( - -70C . ) re ta in fu l l po te ncy fo r s eve ra l m o nths . S im i la r ly ,m a t e r i a l p r e c i p i t a t e d f r o m s a l in e s o l u t i o n b y a lc o h o l a n d s t o r e d u n d e r t h es u p e r n a t a n t r e m a i n s a c t i v e o v e r a l o n g p e r i o d o f t i m e . P a r t i a l l y p u r i fi e dm a t e r i a l c a n b e p r e s e r v e d b y d r y i n g f r o m t h e f r o z e n s t a t e i n t h e l y o p h i l ea p p a r a t u s . H o w e v e r , w h e n t h e s a m e p r o c e d u r e i s u s e d fo r th e p r e s e r v a t i o no f t h e h i g h l y p u r i f ie d s u b s t a n c e, i t i s f o u n d t h a t t h e m a t e r i a l u n d e r g o e s c h a n g e sresu l t ing in dec rease in so lub i l i ty and loss o f ac t iv i ty .

T h e a c t i v i t y o f t h e t r a n s f o r m i n g p r i n c i p l e i n c r u d e e x t r a c t s w i t h s t a n d sh e a t i n g f o r 30 t o 6 0 m i n u t e s a t 6 5 C . H i g h l y p u r i f ie d p r e p a r a ti o n s o f a c ti v em ate r ia l a re l e s s s t ab le , and som e los s o f ac t iv i ty occurs a t th i s t em pera tu re .A q u a n t i t a t i v e s t u d y o f t h e e f f e c t o f h e a t i n g p u r i fi e d m a t e r i a l a t h i g h e r t e m -p e r a t u r e s h a s n o t a s y e t b e e n m a d e . A l l o w a y (6 ) , u s i n g c r u d e e x t r a c t s p re -p a r e d f r o m T y p e I I I p n e u m o c o c c a l ce il s, f o u n d t h a t o c c a s io n a l l y a c t i v i t y c o u l ds t il l b e d e m o n s t r a t e d a f t e r 1 0 m i n u t e s ' e x p o s u r e i n t h e w a t e r b a t h t o t e m -pe ra tu res a s h igh a s 90C.

T h e p r o c e d u re s m e n t i o n e d a b o v e w e r e c a r r ie d o u t w i t h s o l u t io n s a d j u s t e dt o n e u t r a l r e a c t io n , s in c e i t h a s b e e n s h o w n t h a t h y d r o g e n i o n c o n c e n t r a t i o n sin the ac id range re su l t in p rogres s ive los s o f ac t iv i ty . Ina c t iva t ion occursr a p i d l y a t p H 5 a n d b e l o w .Qualitative Chemical Te sts .--Th e p u r i fi e d m a t e r i a l i n c o n c e n t r a t e d s o l u t i o ng i v es n e g a t i v e b i u r e t a n d M i l l o n t e s t s. T h e s e t e s t s h a v e b e e n d o n e d i r e c t l yo n d r y m a t e r i a l w i t h n e g a t iv e r es u l ts . T h e D i s c h e d i p h e n y l a m i n e re a c t i o n



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OSWALD T . AVERY, COLIN M. MAC LEOD, AND MACLYN McCARTY 14 5f or desoxyr ibonuc le ic ac id i s s t r ong ly pos i t ive . The or c ino l t e s t ( B ia l) f o rr i bo n u c l ei c a c i d is w e a k l y p o s i ti v e . H o w e v e r , i t h a s b e e n f o u n d t h a t i n s i m i l a rconce n t r a t ions pur e p r epa r a t ions o f desoxyr ibonuc le ic ac id o f an im a l o r ig inp r e p a r e d b y d i f fe r e n t m e t h o d s g i v e a B l a l r e a c t i o n o f c o r r es p o n d i n g i n t e n s i t y .

Al though no spec i f i c t e s t s f o r the p r esence o f l ip id in the pur i f i ed m a te r ia lh a v e b e e n m a d e , i t h a s b e e n f o u n d t h a t c r u d e m a t e r i a l c a n b e r e p e a t e d l y ex -t r a c t e d w i t h a l c o h ol a n d e t h e r a t - - 12 C . w i t h o u t lo s s o f a c t i v i t y . I n a d d i t i o n ,a s wi l l be no te d in the p r ep a r a t ive p r ocedu r es , r epea ted a lcohol p r ec ip i t a t iona n d t r e a t m e n t w i t h c h l o ro f o r m r e su l t i n n o d e c r e a se i n b i o l o g ic a l a c t i v i t y .

Elementary Chemica l Analys i s . L - Four p u r i fi e d p r e p a r a t i o n s w e r e a n a l y z e df o r c o n t e n t o f n i t ro g e n , p h o s p h o r u s, c a rb o n , a n d h y d r o g e n . T h e r e s u l ts a r epr esen ted in Tab le I . The n i t r ogen- pho sphor us r a t ios va r y f r om 1. 58 to 1 .75wi th a n ave r age va lu e o f 1 .67 which is in close agr eem ent wi th t ha t ca lcu la ted

TABLE IElementary CieraicalA nalysis o f P urified Preparations of tie Transforming Substanc, .Preparation No.

3738B4244

Carbon

34.2735.50

Hydrogenp~ G~t3.893.76

Nitrogenpet ~~14.2115.9315.3613.40

Phosphorus~er c ~8.579.099.048.45

N / P r a ti o

1.661.751.691.58Theory for sodiumdesoxyribonucleate . . . . 34 .20 3.21 15.32 9.05 ! . 69

on the bas i s o f the theor e t i ca l s t r uc tur e o f sod ium desoxyr ibonuc lea te ( t e t r a -n u c l e o t id e ) . T h e a n a l y t i c a l f ig u r es b y t h e m s e l v e s d o n o t e s t a b l is h t h a t t h esubs tance i so la ted i s a pur e chem ica l en t i t y . How ever , on the bas i s o f then i t r o g e n -p h o s p h o r u s r a t i o , i t w o u l d a p p e a r t h a t l i t tl e p r o t e i n o r o t h e r s u b -s tances con ta in ing n i t r ogen or phosphor us a r e p r esen t a s im pur i t i e s s ince i ft h e y w e r e t h i s r a t i o w o u l d b e c o n s i d e r a b l y al t e re d .

E n z y m a t i c A n a l y s i s . - - V a r i o u s c r u d e a n d c r y s t a l l i n e e n z y m e s2 h a v e b e e nt e s t e d f o r t h e i r c a p a c i t y t o d e s t r o y t h e b i o lo g i ca l a c t i v i t y o f p o t e n t b a c t e r i a le x t r a c ts . E x t r a c t s b u f f e r ed a t t h e o p t i m a l p H , t o w h i c h w er e a d d e d c r y s t a ll i n et r y p s i n a n d c h y m o t r y p s i n O r c o m b i n a t i o n s o f b o t h , s u f fe r e d n o l o s s in a c t i v i t yf o ll o w i n g t r e a t m e n t w i t h t h e s e e n z y m e s . P e p s i n c o u l d n o t b e t e s t e d b e c a u se

t The elem entary chemical analyses were mad e by D r. A. Elek o f The RockefellerIns t i tute .The authors are indebted to Dr . John H. Nor throp and Dr . M. Kunitz of TheRockefeller Institute for Medical Research, Princeton, N. J. , for the samples ofcrystalline tryp sin, chym otrypsin , and ribonuclease used in th is work.



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1 4 5 TRANS~'ORMATION O ~ PNEUMOCOCCAL TYPES

ex t r ac t s a r e r ap id ly inac t iva ted a t the low pH r equ i r ed fo r i t s use . P r o longedt r e a t m e n t w i t h c r y s t a l l i n e r i b o n u c l e a s e u n d e r o p t i m a l c o n d i t i o n s c a u s e d n od e m o n s t r a b l e d ec r e as e in t r a n s f o rm i n g a c t i v i t y . T h e f a c t t h a t t r y p s i n ,chym ot r yps in , and r ibonuc lease had no e f f ec t on the t r ans f or m ing pr inc ip le i sf u r the r ev idence th a t th i s subs tance i s no t r ibonuc le ic ac id o r a p r o te in suscep-t i b le t o t h e a c t i o n o f t r y p t i c e n z y m e s .

I n a d d i t i o n t o t h e c r y s t a l l i n e e n z y m e s , s e r a a n d p r e p a r a t i o n s o f e n z y m e so b t a i n e d f r o m t h e o r g a n s o f v a r i o u s a n i m a l s w e r e t e s t e d t o d e t e r m i n e t h e i re f f ec t on t r ans f or m in g ac t iv i ty . Cer ta in o f these wer e f ound to be capab le o fc o m p l e t e l y d e s t r o y i n g b i o lo g i ca l a c t i v i t y . T h e v a r i o u s e n z y m e p r e p a r a t i o n st e s t e d i n c l u d e d h i g h l y a c t i v e p h o s p h a t a s es o b t a i n e d f r o m r a b b i t b o n e b y t h em e t h o d o f M a r t l a n d a n d R o b i s o n ( 15 ) a n d f r o m s w in e k i d n e y as d e s c ri b e d b y

TABLE I IThe Inacti~ogion o f Transform ing Princip le by Crude Enzy me Preparations

Crude enzym e preparations

D o g i n t e s t in a l m u c o s a .Rabbi t bone phospha tase . . . . . . . . . . . . . . . .Swine kidney " . . . . . . . . . . . . . . . . .P n e u m o c o c c u s a u t o l y s a t es . . . . . . . . . . . . . . . .N o r m a l d o g a n d r a b b it s e ru m . . . . . . . . . . . . .

Ph o sp h a ta se

++++

Enzymatic a c t i v i tyn e p o l y m e r -Tr i b u ty r i n a se fo re s te r a se desox.yribo-nucleate

+ +++ ++ +

In a c t i v a t i o nof transformingprlnc~p~e+

++

H . a n d E . A l b er s (1 6) . I n a d d i t i o n , a p r e p a r a t io n m a d e f r o m t h e i n t e st i n a lm u c o s a o f d o g s b y L e v e n e a n d D i l l o n ( 1 7) a n d c o n t a i n i n g a p o l y n u c l e o t id a s ef o r t h y m u s n u c le i c a c i d w a s u s e d . P n e u m o c o c c a l a u t o l y s a t e s a n d a c o m m e r -c ia l p r epa r a t ion of panc r ea t in wer e a l so t e s ted . The a lka l ine phosp ha tasea c t i v i t y o f t h e s e p r e p a r a t i o n s w a s d e t e r m i n e d b y t h e i r a c t i o n o n ~ - g l y c e r o -p h o s p h a t e a n d p h e n y l p h o s p h a t e , a n d t h e e s te r a se a c t i v i t y b y t h e i r c a p a c i t yto sp l i t t r ibu tyr in . S ince the h igh ly pur i f i ed t r ans f or m ing m a te r ia l i so la tedf r o m p n e u m o c o c c a l e x t r a c t s w a s f o u n d t o c o n t a i n d e s o x y r i bo n u c l ei c a c i d ,t h e s e s a m e e n z y m e s w e r e t e s t e d f o r d e p o l y m e r a s e a c t i v i t y o n k n o w n s a m p l esof desoxyr ibonuc le ic ac id i so la ted by M ir sky s f r o m f is h sp e r m a n d m a m m a l i a nt i ss u e s. T h e r e s u lt s ar e s u m m a r i z e d i n T a b l e I I i n w h i c h th e p h o s p h a t a s e ,e s te r a se , a n d n u c l e o d e p o ly m e r a s e a c t i v i t y o f t h e s e e n z y m e s i s c o m p a r e d w i t hthe i r capac i ty to des t r oy the t r ans f or m in g pr inc ip le. Ana lys i s o f these r e su l t sshows tha t i r r e spec t ive o f the p r esence o f phospha tase o r e s te r a se on ly those

3 The authors express their thanks to Dr . A . E. Mirsky of the Hospita l of TheRockefeller Ins titu te for these preparations of desoxyribonucleic acid.



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OSWALD T. AVERY, COLIN ~r. MACLEOD~ AND MACLYN MCCARTY 147

p r e p a r a t i o n s s h o w n t o c o n t a i n a n e n z y m e c a p a b l e o f d e p o l y m e r i z i n g a u t h e n t i cs a m p l e s o f d e s o x y r i b o n u c l e i c a c i d w e r e f o u n d t o i n a c t i v a t e t h e t r a n s f o r m i n gpr i nc i p l e .

G r e e n s t e i n a n d J ' e n r e t t e ( 1 8 ) h a v e s h o w n t h a t t i ss u e e x t r a c t s , a s w e l l a s t h em i l k a n d s e r u m o f s e v e r a l m a m m a l i a n s p e c ie s , c o n t a i n a n e n z y m e s y s t e m w h i c hc a u s e s d e p o l y m e r i z a t i o n o f d e s o x y r i b o n u c l e i c a c i d . T o t h i s e n z y m e s y s t e mG r e e n s t e i n h a s l a t e r g i v e n t h e n a m e d e s o x y r i b o n u c l e o d e p o l y m e r a s e ( 1 9 ).T h e s e i n v e s t i g a t o r s d e t e r m i n e d d e p o l y m e r a s e a c t i v i t y b y f o l l o w i n g t h e r e d u c -t i o n i n v i s c o s i ty o f s o l u t io n s o f s o d i u m d e s o x y r i b o n u c l e a t e . T h e n u c l e a t ea n d e n z y m e w e r e m i x e d i n t h e v i s c os i m e te r a n d v i s c o s i t y m e a s u r e m e n t s m a d ea t i n t e r v a l s d u r i n g i n c u b a t i o n a t 3 0 C . I n t h e p r e s e n t s t u d y th i s m e t h o d w a su s e d i n t h e m e a s u r e m e n t o f d e p o l y m e r a s e a c t i v i t y e x c e p t t h a t i n c u b a t io n w a sc a r r i e d o u t a t 3 7 C . a n d , i n a d d i t i o n t o t h e r e d u c t i o n o f v is c o s i t y , t h e a c t i o no f t h e e n z y m e w a s f u r t h e r t e s t e d b y t h e p r o g r e s s iv e d e c r e a s e in a ci d p r e c i p -i t a b i li t y o f t h e n u c l e a t e d u r i n g e n z y m a t i c b r e a k d o w n .T h e e f fe c t o f fr e sh n o r m a l d o g a n d r a b b i t s e r u m o n t h e a c t i v i t y o f t h et r a n s f o r m i n g s u b s t a n c e i s s h o w n i n t h e f o l l o w i n g e x p e r i m e n t .

Sera obtained f rom a normal dog and normal rabbi t were di luted wi th an equalvolume of physiological sal ine . Th e di luted serum was divided into three equ alpor t ions . One par t was heated a t 65C . for 30 minutes , anoth er a t 50C. for 30minutes , and the thi rd was used unheated as cont rol . A par t ia l ly pur if ied prepara-t ion of t ransforming m ater ia l which had previously been dr ied in the lyophi le appara-tus was dissolved in saline in a con centratio n of 3.7 rag. per cc. 1.0 cc. of this solutionwas mixed with 0.5 cc. of the various samples of heated and unheated diluted sera,and the m ixtures a t p H 7.4 were incubated a t 37C. for 2 hours . Af ter the serum hadbeen al lowed to act o n the t ransforming mater ia l for this per iod, a l l tubes were heatedat 65C. for 30 minutes to s top enzy mat ic action. Seria l di lut ions were then m ade insal ine and tes ted in t r ipl icate for t ransforming act ivi ty according to the proceduredescr ibed un der Metho d of t i t ra t ion. Th e resul t s given in Table I I I i l lus tra te thedi fferent ia l h eat inact ivat ion of the enzy mes in dog an d rab bi t serum w hich dest roythe transforming principle.

F r o m t h e d a t a p r e s e n t e d i n T a b l e I I I i t i s e v i d e n t t h a t b o t h d o g a n d r a b b i ts e r u m i n t h e u n h e a t e d s t a t e a r e c a p a b l e o f c o m p l e t e l y d e s t r o y i n g t r a n s f o r m i n ga c t i v i t y . O n t h e o t h e r h a n d , w h e n s a m p l e s o f d o g s e r u m w h i ch h a v e b e e nh e a t e d e i t h e r a t 6 0 C . o r a t 6 5 C . f o r 3 0 m i n u t e s a r e u s e d , t h e r e i s n o l o s s o ft r a n s f o r m i n g a c t i v i t y . T h u s , i n t h is s p e ci e s t h e s e r u m e n z y m e r e s p o n s ib l ef o r d e s t r u c t i o n o f t h e t r a n s f o r m i n g p r i n c i p l e is c o m p l e t e l y i n a c t i v a t e d a t6 0 C . I n c o n t r a s t t o t h e s e r e s u lt s , e x p o s u r e to 6 5 C . f o r 3 0 m i n u t e s w a sr e q u i r e d f o r c o m p l e t e d e s t r u c t i o n o f t h e c o r r e s p o n d i n g e n z y m e i n r a b b i t s e r u m .

T h e s a m e s a m p l es o f d o g a n d r a b b i t s e r u m u s e d i n t h e p r e c e d i n g e x p e r i m e n tw e r e a ls o t e s t e d f o r t h e i r d e p o l y m e r a s e a c t i v i t y o n a p r e p a r a t i o n o f s o d i u md e s o x y r i b o n u c l e a t e i s o la t e d b y M i r s k y f r o m s h a d s p e rm .



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14 8 T R ANSFOR MAT I ON 0 ~ ' P N ~ ' M O C O C C A L T Y P E SA h i g h l y v i s c o u s s o l u t i on o f t h e n u c l e a t e i n d is t i ll e d a t e r i n a c o n c e n t r a t i o n o f 1

r a g . p e r c c . w a s u s e d . 1 . 0 c c . a m o u n t s o f h e a t e d a n d u n h e a t e d s e r a d i l u t e d i n s a l i nea s s h o w n i n t h e p r e c e d i ng p r o t oc o l w e r e m i x e d i n O s t w a l d v i s co s i me t e rs w i t h 4 . 0 cc .

T A B L E H IDifferential Heat Inactivation of Enzym es in Dog and Rabbit Serum Wkic,k Destroy th eTransforming Substanze

D o g s e r u m

R a b b i t s e r u m

H e a t treatmentof serum

U n h e a t e d

6 0 C . f o r 3 0m i n .

6 5 C . f o r 3 0m ~ .

U n h e a t e d

6 0 C . f o r 3 0m i n .

Dilution*T r i p l i c a t e t e s t s

1 2 3~I ~ Colony ~ Colony ~ ~ Colony

U n d i l u t e d - - ; R o n l y - - R o n l y - - R o n l y1 : 5 - - I R " - - R " - - R "1 : 2 5 - - R " - - R " - - R "

U n d i l ut e d + S l l I + S i l l + S i l l1 : 5 + S l l I + S I I I + S I I I1 : 25 + S i l l + S i l l + S i l l

U n d i l u t e d + S I I I + S i l l + S i l l1 : 5 + S I I I + S i l l + S H I1 : 2 5 + S i l l + S i l l + S i l l

U n d i l u t e d - - R o n l y1 : 5 - - R "1 : 2 5 - - I R "

- - R o n l ym l ~ t ~

C o n t r o ls e l q l m )

6 5 C . f o r 3 0m i n .

( n o N o n e

- - R o n l y

U n d i l u t e d - - R o n l y - - R o n l y - - R o n l y1 :5 - - R " - - R " - - R "1 : 2 5 - - R " - - R " - - R "I

U n d i l u t e d + ~ S i l l + S I I I + S i l l1:5 + ~ SII I "b S il l + SII I1 : 2 5 + S I I I + S l l I + S i l l

U n d i l u t e d + ' S i l l + S l l I [ + S I I I1 : 5 + S i l l + S I I I I + S I I I1 : 2 5 + S I I I + S I I I - b S i l l

* D i l u t i o n o f t h e d i g e s t m i x t u r e o f s e r u m a n d t r a n s f o r m i n g s u b s t a n c e .o f t h e a q u e o u s s o l u t i o n of t h e n u c l e a t e . D e t e r m i n a t i o n s o f v i s c o s i t y w e r e m a d e~ m m e d i a t e l y a n d a t i n t e r v a l s o v e r a p e r i o d o f 2 4 h o u r s d u r i n g i n c u b a t i o n a t 3 7 C .

T h e r e s u l t s of t h i s e x p e r i m e n t a r e g ra p h i c a l l y p r e s e n t e d i n C h a r t 1 . I n t h ec a s e of u n h e a t e d s e r u m o f b o t h d o g a n d r a b b i t , t h e v i s c o s i t y fe l l t o t h a t o fw a t e r i n 5 t o 7 h o u r s . D o g s e r u m h e a t e d a t 6 0 C , fo r 3 0 m i n u t e s b r o u g h t a b o u t



13/23

OSWALD T. AVERY, COLIN M. MACI,EOD, AND MACLYN McCARTY 14 9no signi ficant reduc t ion in v isco si ty af ter 22 hours . On the other band, heat ingrabb i t se rum a t 50C . mere ly r educed t he r a t e o f depo lymerase ac t i on , andaf t e r 24 hour s t he v i scos i t y was b rough t t o t he same l eve l as wi th t he unhea t edserum. Hea t i ng a t 65oc . , however , comple t e ly des toyed t he r abb i t se rumdepo lymerase .Thus , i n t he case o f dog and r abb i t se ra t here i s a s t r l]dng para ll e li sm be tweenthe t empera tu re o f i nac t i va t i on o f t he depo lymerase and t ha t o f t he enzymewhich des t roys t he ac t i v i t y o f t he t r ans fo rming p r inc ip le . The f ac t t ha t t h i sd i ff e rence i n t empe ra tu re o f i nac t iva t i on i s no t m ere ly a genera l p rope r ty o f a l lenzymes i n t he sera i s ev iden t f rom exper imen t s on t he hea t i nac t i va t i on o f

])iffe~ential Hea~ I n a ~ a t A o n

~ [ \ l l l e a t e d 6 5 ~ " S O'

3L_ - .~ ~ I-I ~ed ~ fo~ 30. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .[ ~ , - o . . i . ie a t ~ L 6 0 o ~ , 3 0 ,

11- i i i ,HI,#. 5 10 15 Z0'l'imoCHART I

t r i bu ty r i n es t e rase i n t he same. samples o f se rum. In t he l a t t e r i n s tance , t heresu l t s a r e t he r ever se o f those observed wi th depo lymerase s ince t he es t e raseo f r abb i t se rum i s a lmos t comple t e ly i nac t i va t ed a t 60"C . wh i le t ha t i n dogserum i s on ly s l igh t l y a f f ec ted by exposu re t o t h i s t empera tu re .

Of a number o f subs t ances t es t ed fo r t he i r capac i t y t o i nh ib i t t he ac t i onof t he enzym e known to des t roy t he t r ans fo rming p r i nc ipl e , on ly sod ium f l uor idehas been found to hav e a s igni ficant inh ibi tory effect . Regard less of wh etherth i s enzym e i s der i ved f rom pneum ococca l cel ls , dog i n t es ti na l m ucosa , pan -creat in , or norm al sera i ts act iv i ty i s inhibi ted by f luor ide. Simi lar ly i t hasbeen found t h a t f luo ride i n t he sam e concen t r a t ion a l so i nh ib it s t he enzym at i cdepolymerizat ion of desoxyribonucleic acid .The f ac t t ha t t r ans fo rming ac t i v i t y i s des t royed on ly by t hose p repara t ionscon t a in ing depo lymerase fo r desoxyribonucle i c ac id and t he fu r t her f ac t t ha t



14/23

150 T R A N S ] ~ O R 3 ~ A T I O N 0 ~ ' P N E U 3 ~ O C O C C A L T Y P E S

i n b o t h i n s t an c e s t h e e n z y m e s c o n c e r n ed a r e i n a c t i v a t e d a t t h e s a m e t e m p e r a -tu r e and inh ib i ted by f luor ide p r ov ide add i t iona l ev idence f o r the be l ie f tha tthe ac t ive p r inc ip le i s a nuc le ic ac id o f the desoxyr ibose type .Serological Analysis . - - In the cour se o f chem ica l i so la t ion of the ac t ivem ate r ia l i t was f ound tha t a s c r ude ex t r ac t s wer e pur i f i ed , the i r s e r o log ica la c t i v i t y i n T y p e I I I a n t i s e r u m p r o g r e s si v e l y d e c r ea s e d w i t h o u t c o r re s p o n d in gloss in b io log ica l ac t iv i ty . S o lu t ions o f the h igh ly pur i f i ed subs tance i t s e l fg a v e o n l y f a i n t t r a c e r e a c t io n s i n p r e c i p i ti n t e s t s w i t h h i g h t i t e r T y p e I I Ia n t i p n e u m o c o c c u s ra b b i t s e ru m . 4 I t i s w e l l k n o w n t h a t p n e u m o c o c c a l p ro t e i ncan be d e tec ted b y se r o log ica l m e th ods in d i lu t ions a s h igh a s 1 : 50 ,000 and thecapsu la r a s we l l a s the som at ic p o lysaccha r ide in d i lu t ions o f a t l eas t 1 : 5 ,000, 000 .I n v iew of these f ac t s , the los s o f s e r o log ica l r eac t iv i ty ind ica te s th a t these ce l lc o n s t i t u e n t s h a v e b e e n a l m o s t c o m p l e t e l y r e m o v e d f r o m t h e f i n al p r e p a ra t i o n s .The f ac t tha t the t r ans f or m ing subs tance in pur i f i ed s ta te exh ib i t s l i t t l e o r nose r o log ica l r eac t iv i ty i s in s t r ik ing con t r a s t to i t s b io log ica l spec i f i c i ty ini n d u c i n g p n e u m o c o c c a l t r a n s f o r m a t i o n .Phys icochemica l S t ud i es ) - -A p u r i f i e d a n d a c t i v e p r e p a r a t i o n o f t h e t r a n s -f o r m i n g s u b s t a n c e ( p r e p a r a t i o n 4 4 ) w a s e x a m i n e d i n t h e a n a l y t i c a l u l t r a -c e n t ri g u e . T h e m a t e r i a l g a v e a s i n g le a n d u n u s u a l l y s h a r p b o u n d a r yi n d i c a t in g t h a t t h e s u b s t a n c e w a s h o m o g e n e o u s a n d t h a t t h e m o l ec u l es w e r eu n i f o r m in s iz e a n d v e r y a s y m m e t r i c . B i o l og i c al a c t i v i t y w a s f o u n d t o b es e d i m e n t e d a t t h e s a m e r a t e a s t h e o p t i c a l l y o b s e r v e d b o u n d a r y , s h o w i n gt h a t a c t i v i t y c o u l d n o t b e d u e t o t h e p r e s e n c e o f a n e n t i t y m u c h d i f f e r e n ti n si ze . T h e m o l e c u l a r w e i g h t c a n n o t b e a c c u r a t e l y d e te r m i n e d u n t i l m e a s -u r e m e n t s o f t h e d i f fu s i o n c o n s t a n t a n d p a r t i a l s p e c if ic v o l u m e h a v e b e e nm a d e . H o w e v e r , T e n n e n t a n d V i l b r a n d t ( 20 ) h a v e d e t e r m i n e d t h e d i f fu s i onc o n s t a n t o f s e v e r al p r e p a r a t io n s o f t h y m u s n u c l e ic a c i d t h e s e d i m e n t a t i o n r a t eof which i s in c lose agr eem ent wi th the va lues obse r ved in the p r esen t s tudy .A s s u m i n g t h a t t h e a s y m m e t r y o f t h e m o l e c u le s i s t h e s a m e i n b o t h i n s ta n c e s ,i t i s e s t i m a t e d t h a t t h e m o l e c u l a r w e i g h t o f t h e p n e u m o c o c c a l p r e p a r a t i o n i s o fthe order of 500,000.

E x a m i n a t i o n o f t h e s a m e a c t iv e p r e p a r a t i o n w a s c a r r i ed o u t b y e l e c t ro p h o -r esi s in the T ise l ius app a r a tu s and r evea led on ly a s ing le e lec t r ophor e t i c com po-n e n t o f r e la t i v e l y h i g h m o b i l i t y c o m p a ra b l e t o t h a t o f a n u c le i c a c i d . T r a n s -f o r m i n g a c t i v i t y w a s a s s o c i a t ed w i t h t h e f a s t m o v i n g c o m p o n e n t g i v i n g th e

4 The Type I I I antipneumococcus rabbit serum employed in this s tud y was fur -nished through the co urtesy of Dr. Jules T. Freu nd, Burea u of Laboratories, D epart-ment of Health, City of New York.5 Studies on sedimentation in the ultracentrifuge were carried o ut by D r. A .Ro then ; the electrophoretic analyses were mad e by Dr. T . Shedlovsky, and the ult ra-violet absorption curves by Dr. G. I . Lav in. The au thors gratefully acknowledgetheir indebtedness to these members of the staff of The Rockefeller Institute.



15/23

OSWAL D T . AVE R Y, C OL IN M. MAC L E OD, AND M AC L Y N MC C AR T Y 15 1o p t i c a l l y v i s ib l e b o u n d a r y . T h u s i n b o t h t h e e l e c t r ic a l a n d c e n t r i f u g a l f ie l ds ,t h e b e h a v i o r o f t h e p u r i f i e d s u b s t a n c e i s c o n s i s t e n t w i t h t h e c o n c e p t t h a t b i o -l o g ic a l a c t i v i t y i s a p r o p e r t y o f t h e h i g h l y p o l y m e r i z e d n u c l e i c a c i d .

U l t r a v i o le t a b s o r p t io n c u r v e s s ho w e d m a x i m a i n t h e r e g i o n o f 2 6 0 0 / ~ a n dm i n i m a i n t h e r e g i o n o f 2 3 5 0 / ~ . T h e s e f in d i n g s a r e c h a r a c t e r i s t i c o f n u c l e icac i ds .Q.uanti ta ti~e Determination of Biological A ct{d ty.-- In i t s h i gh l y pur i f i ed s t a t et h e m a t e r i a l a s i s o la t e d h a s b e e n f o u n d t o b e c a p a b l e o f i n d u c i n g t r a n s f o r m a t i o ni n a m o u n t s r a n g i n g f r o m 0 . 0 2 t o 0 .0 0 3 / z g . P r e p a r a t i o n 4 4 , t h e p u r i fi c a t i o n o fw h i c h w a s c a r ri e d o u t a t l o w t e m p e r a t u r e a n d w h i c h h a d a n i t r o g e n - p h o s p h o r u s

T A B L E I VTitration of Transforming Activity of Preparation 44

Transformln$ principle Quadruplicate t e s t sPreparation 44 * 1 2

D i l u t i o n ? k m o t m ta d d e d/Jg.

10 -~ 1 . 0I0 -~'s 0 .310 a O. 110 3"5 O. 0310 ~ 0 .01104. 5 0 . 0 0 310 -~ O. 001C o n t r o l N o n e

D i f f u ~growth _ _

+

' Colony

s I I IS I I IS I I IS I I IS I I IR o n l y

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Diffusegrowth Colony D i f f u s ef o r m g r o w t h

S I I IS I I IS I I IS I I ISIII -bS I I IR o n l y

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3 4Colony D i ffuse Colonyf o r m g r o w t h f o r m

S I I I ~ S I I ISIII SILlS I I I ~ S I I IS I I I SIIISIII ~ SIH

R o n l y S I I IR " R on l yR " R "

* S o l u t i on f r om w h i c h d i l u t i on s w e r e m ad e c on ta i n e d 0 . S r ag. p e r c c . o f p u r i f i e d m ate r i a l .0 . 2 c c . o f e ac h d i l u t i on ad d e d to q u ad r u p l i c a te tu b e s c on ta i n i n g 2 . 0 c c . o f s tan d ar d s e r u mb r oth . 0 . 05 c c . o f a 10 ' ~ d i l u t i on o f a b l ood b r o th c u l tu r e o f R 36 A i s ad d e d to e ac h tu b e .

r a t i o o f 1 .5 8 , e x h i b i t e d h i g h t r a n s f o r m i n g a c t i v i t y . T i t r a t i o n o f t h e a c t i v i t yo f t h i s p r e p a r a t i o n i s g i v e n i n T a b l e I V .

A solut ion containing 0.5 rag. per cc . was ser ia lly di luted as shown in the pro toco l0.2 cc . of each of these di lut ions was added to qu adrupl icate tubes containing 2.0 cc .of s tandard serum broth. Al l tubes were then inoculated wi th 0.05 ec. of a 10 4di lut ion of s 5 to 8 hour blood broth cul ture of R36A. Transforming act ivi ty wasdetermined by the procedure descr ibed under Method of t i t ra t ion.T h e d a t a p r e s e n t e d i n T a b l e I V s h o w t h a t o n t h e b a s i s o f d r y w e i g h t 0 .0 0 3

/~ g. o f t h e a c t i v e m a t e r i a l b r o u g h t a b o u t t r a n s f o r m a t i o n . S i n c e t h e r e a c t i o ns y s t e m c o n t a i n i n g t h e 0 . 0 0 3 /~ g . h a s a v o l u m e o f 2 .2 5 c c . , t h i s r e p r e s e n t s a f i n a lc o n c e n t r a t i o n o f t h e p u r i f i e d s u b s t a n c e o f 1 p a r t i n 6 0 0 ,0 0 0 ,0 0 0 .



16/23

15 2 T R A N S F O R M A T I O N O 1 P N E U M O C O C C A I , T Y P E S

D I S C U S S I O N

The p resen t s t ud y dea l s wi th t he r esu lt s o f an a t t em pt t o de t e rmine t he chem-ical nature of the substance inducing speci f ic t ransformat ion of pneumococcalt ypes . A desoxyr ibonucl e ic ac id f rac t i on has been i so la t ed f rom T ype I I Ipneumoeocci wh ich i s capab l e o f t r ans fo rming unencapsu l a t ed R var i an t sder ived f rom Pneumococcus Type I I i n to fu l l y encapsu l a t ed Type I I I ce l l s .Thom pson and D ubos (21 ) have i so l a ted f rom pneumococei a nuc l e i c ac id o f t her ibose type . So far as the wri ters are aware, howev er , a nucleic acid of thedesoxyr ibose t ype has no t here to fo re been r ecovered f rom pneumococci no r hasspeci f ic t ransformat ion been exper imental ly induced i r b v i t r o by a chemica l l ydef ined subs t ance .

Al though t he observa t i ons a re l imi t ed t o a s i ng l e example , t hey acqu i r ebroader s igni f icance f rom the work of ear l ier invest igators who demonst ratedthe i n t e rconver t i b i l i t y o f var i ous pneumococca l t ypes and showed t ha t t hespec i fi c it y o f t he changes i nduced i s in each i n s t ance de t e rm ined b y t he par -t i cu l a r t ype o f encapsu l a t ed ce ll s u sed t o evoke t he r eac ti on . F rom the po in tof v iew of the phenom enon in general , therefore, i t i s of special in terest tha t inthe ex ample s tudied, h ighly pur i f ied and protein-free mater ial consist ing largely ,i f not exclusively , of desoxyribonucleic acid i s capa ble of s t imulat ing unenc ap-su l a t ed R var i an t s o f Pneum ococcus Ty pe I I t o p roduce a capsu l ar po lysac-char ide ident ical in type sp eci f ici ty wi th th at of the cel ls f rom which theinducing subs t ance was i so la t ed . Equa l l y s tr i k ing i s t he f ac t t ha t t he su b -s t ance evok ing t he r eac t i on and t he capsu l ar subs t ance p roduced i n r esponseto i t a re chem ical ly d is t inct , eac h belonging to a w hol ly d if ferent class of chem-ical compounds.

The i nducing subs t ance , on t he bas i s o f i t s chemica l and phys i ca l p roper ti es ,appear s t o be a h igh ly po lymer i zed and v i scous fo rm o f sod ium desoxyr ibo -nucl ea t e . On t he o ther hand , the Type I I I capsu l ar subs t ance , t he syn thes i sof which i s evoke d by th is t ransform ing a gent , consist s chief ly of a non-ni t rog-enous polysacchar ide const i tu ted of g lucose-glucuronic acid uni t s l inked inglycosidic union (22) . The presence of the newly formed capsule containingthis type-speci f ic polysacch ar ide confers on the t ran sform ed cell s al l the d is-t ingnishing character i s t ics of Pneu moc occus Ty pe HI . Thu s, i t i s evid entt ha t t he i nducing subs t ance and t he subs t ance p roduced i n t u rn a re chem ica ll ydist inct and biological ly specif ic in thei r act ion and tha t bo th are requisi te inde t ermin ing t he t ype spec i fi c it y o f t he ce ll o f wh ich t he y fo rm a par t .

The exper imen ta l da t a p resen t ed i n t h is paper s t rong ly sugges t tha t nuc l e icacids, at least thos e of the desoxyribose type, possess d i f ferent specif ici ties asevidenc ed by the select ive act ion of the t ransforming pr inciple. Indeed , thepossibi l i ty of the existence of speci fic d i f ferences in b iological behavior of nucleicac ids has p rev ious ly been sugges t ed (23 , 24 ) bu t has never been exper imen ta l l ydem ons t r a t ed ow ing in par t a t l eas t t o t he l ack o f su i tab l e b io log i ca l methods .



17/23

O S W A L D T. A V E R Y , C O L IN M. M A c LE O D , A N D M A C L Y N M c C A R T Y 153

The techniques used in the study of transformation appear to afford a sensitivemeans of testing the validit y 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 t hat the transformingact ivi ty of the mater ial described is actually an inherent property of the nucleicacid, one must still account on a chemical basis for the biological specificity ofits action. At first glance, immunological methods would appear to offer theideal means of determining the differential specificity of this group of biologicallyimportant substances. Although the constituent units and general patternof the nucleic acid molecule have been defined, there is as yet relatively littleknown of the possible effect that subtle differences in molecular configurationmay exert on the biological specificity of these substances. However, sincenucleic acids free or combined with histones or protamines are not known tofunction antigenically, one would not anticipate t hat such differences would berevealed by immunological techniques. Consequently, it is perhaps not sur-prising that h ighly purified and protein-free prepara tions of desoxyribonucleicacid, although extremely active in inducing transformation, showed only fainttrace reactions in precipitin tests with potent Type III antipneumococcusrabbit sera.

From these limited observations it would be unwise to draw any conclusionconcerning the immunological significance of the nucleic acids until furtherknowledge on this phase of the problem is available. Recent observations byLackrnan and his collaborators (25) have shown that nucleic acids of both theyeast and thymus type derived from hemolytiC streptococci and from animaland plant sources precipitate with certain antipneumococcal sera. The reac-tions varied with different lots of immune serum and occurred more frequentlyin 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 theresults. Unless special immunochemical methods can be devised similar tothose so successfully used in demonstrat ing the serological specificity of simplenon-antigenic substances, it appears t hat the techniques employed in the s tudyof transformation are the only ones available at present for testing possibledifferences in the biological behavior of nucleic acids.

Admittedly there are many phases of the problem of transformation thatrequire further study and many questions th at remain unanswered largelybecause of technical di~cult ies. For example, it would be of interest to knowthe relation between rate of reaction and concentration of the transformingsubstance; the proportion of cells transformed to those that remain unaffectedin the reaction system. However, from a bacteriological point of view, nu-merical estimations based on colony counts might prove more misleading thanenlightening because of the aggregation and sedimentation of the R ceils ag-



18/23

15 4 T R A N S F O R M A T I O N O F P N ~ O C O C C A L TYPESg lu t i na t ed by t he an t i se rum in t he med ium. At t em pt s t o i nduce tr ans fo rma-t ion in suspensions of rest ing cei ls held under con di t ions inhibit ing gro wth andmul t ip l icat ion have thus far proved unsuccessful , and i t seems probable thatt r ans fo rmat ion occu r s on ly du r ing ac t i ve r ep roduct i on o f t he ce ll s. Im por t a n tin th is connect ion i s the fact tha t th e R cei l s, as wel l as those that hav e unde r-gone t r ans fo rmat ion , p resumab ly a l so a l l o ther var i an t s and t ypes o f pneu -mococci , contain an in t racel lu lar enzyme which i s released dur ing autolysisand i n t he f r ee s ta t e i s capab l e o f r ap id ly and com ple t e ly des t roy ing t he ac t i v i t yo f t he t r ans fo rming agen t . I t w ou ld appear , t herefo re , t ha t du r ing t he l oga-r i thmic phase o f g rowth when ce ll d iv is i on is mo s t ac t i ve an d au to lys i s l eas tapparen t , t he cu l t u ra l cond i t ions a re op t imal fo r t he main t enance o f t he ba l ancebetween m ax imal r eac t i v i t y o f the R ce ll and min imal des t ruc t i on o f t he t r ans -forming agent throug h th e release of auto lyt ic ferments .

In t he p resen t s t a t e o f knowledge any i n t e rp re t a t i on o f t he mechan i sm in -vo lved i n tr ans fo rmat ion m us t o f necess i t y be pu re ly t heo re t i ca l. The b io -chemica l even t s under ly ing t he phenomenon sugges t t ha t t he t r ans fo rmingprinciple in teracts w i th the R cell g iv ing ri se to a coord inated ser ies of enzym at icreac t i ons t ha t cu lmina t e i n t he syn thes i s o f t he Type I I I capsu l ar an t i gen .The exper imen ta l fi nd ings have c l ear ly demons t r a t ed t ha t t he i nduced a l t e r a -t i ons a re no t r andom changes bu t a r e p red i c t ab l e , a lways co r respond ing i ntype spec i fi c it y t o t h a t o f t he e ncapsu l a t ed ce l ls f rom which t he t r ans fo rmingsubs t ance was i so la t ed . Once tr ans fo rmat ion has occu r red , the newly acqu i r edcharacter i s t ics are thereaf ter t ransmi t ted in ser ies through innumerable t rans-f e r s in a r t if i c ia l med i a w i thou t an y fu r t her add i t i on o f t he t r ans fo rming agen t .Moreover , f rom the t r ans fo rmed ce l l s t hemselves , a subs t ance o f i den t i ca lac t i v i t y can aga in be r ecovered i n amou n t s f a r i n excess o f t ha t o r i g ina l ly ad dedto induce the change. I t i s eviden t , therefore, that not only i s the capsu larmater i a l r ep roduced i n success ive genera t ions bu t t h a t t he p r im ary f ac to r ,which con t rols the occurrence an d spe ci f ici ty of capsular dev elopm ent , i s al soredup l i ca t ed in t he daugh t er ce ll s. The i nduced changes a re no t t empo rarymod i f i ca t i ons bu t a r e permanen t a l t e r a t i ons wh ich per s i s t p rov ided t he cu l -t u ra l cond i ti ons a re f avo rab l e fo r t he main t enance o f capsu l e fo rmat ion . Thet r ans fo rmed ce l l s can be r ead i l y d i s t i ngu i shed f rom the paren t R fo rms no ta lone by sero log ica l r eac t i ons b u t by t he p resence o f a new ly fo rmed and v i s ib l ecapsu l e wh ich i s the immuno log i cal un i t o f t yp e spec i f ic i ty and t he a ccesso ryst ructure essent ial in determining the infect ive capaci ty of the microorganismin t he an imal body .I t i s par t icula r ly s igni ficant in the case of pneum ococc i tha t the exper i -men ta l l y i nduced a l t e r a t ions a re def i n i te l y co r re la t ed wi th t he deve lopme n t o f anew m orpho log i ca l s truc tu re and t he co nsequen t acqu i s i ti on o f new an t i gen i cand i nvas ive p roper t ies . Equa l l y if no t m ore si gn if i can t is the f ac t t ha t t hesechanges are predictable, type-speci f ic, and her i table.



19/23

OSWALD T. AVERY, COLIN M. MACLEOD, AND MACLY'N McCARTY 15S

V a r i o u s h y p o t h e s e s h a v e b e e n a d v a n c e d i n e x p l a n a t i o n o f t h e n a t u r e o f t h echanges induced . I n h i s o r ig ina l desc r ip t ion of the pheno m en on Gr i f f ith ( 1)s u g g e s te d t h a t t h e d e a d b a c t e r i a i n t h e i n o c u l u m m i g h t f u r n i s h s o m e s p ec i fi cp r o t e i n t h a t s e rv e s a s a " p a b u l u m " a n d e n a b l es t h e R f o r m t o m a n u f a c t u r e ac a p s u l a r c a r b o h y d r a t e .

M o r e r e c e n t l y t h e p h e n o m e n o n h a s b e e n i n t e r p r e t e d f r o m a g e n e t i c p o i n t o fv iew ( 26 , 27). The indu c ing subs tance has been l ikened to a gene , and thecapsu la r an t ige n which i s p r odu ced in r e sponse to i t has been r ega r ded a s a genep r o d u c t . I n d i s c u ss i n g t h e p h e n o m e n o n o f t r a n s f o r m a t i o n D o b z h a n s k y (2 7)h a s s t a te d t h a t "If t h i s t r a n s f o r m a t i o n i s de s c ri b e d a s a g e n e t i c m u t a t i o n - - a n di t i s d i ff i cu l t to avo id so desc r ib ing i t - - w e a r e dea l ing wi th a u th en t ic cases o fi n d u c t i o n o f s p e ci fi c m u t a t i o n s b y s p ec if ic t r e a t m e n t s . . . . "

A n o t h e r i n t e r p r e t a t i o n o f t h e p h e n o m e n o n h a s b e e n s u g g e s t e d b y S t a n l e y( 28 ) w h o h a s d r a w n t h e a n a l o g y b e t w e e n t h e a c t i v i t y o f t h e t r a n s f o r m i n g a g e n ta n d t h a t o f a v i r u s. O n t h e o t h e r h a n d , M u r p h y ( 29 ) h a s co m p a r e d t h e c a u s a -t i v e a g e n t s o f f o w l t u m o r s w i t h t h e t r a n s f o rm i n g p r i nc i p le o f P n e u m o c o c c u s .H e h a s s u g g e s te d t h a t b o t h t h e s e g r o u p s o f a g e n t s b e t e r m e d " t r a n sm i s s i b l em u t a g e n s " in o r d er to d i f fe r e n t ia t e t h e m f r o m t h e v i ru s g r o u p . W h a t e v e rm ay pr ov e to be th e cor r ec t in te r pr e ta t ion , these d i f fe r ences in v iewpoin t ind i -ca te the im pl ica t ions o f the phen om e non of t r ans f or m a t io n in r e la t ion to s im i la rpr ob lem s in the f i elds o f gene t ic s , v i r o logy , and cance r r e sea rch .

I t i s , o f course , pos sib le tha t the b io log ica l ac t i v i ty o f the sub s tance desc r ibedi s n o t a n i n h e r e n t p r o p e r t y o f t h e n u c le i c a c i d b u t i s d u e to m i n u t e a m o u n t so f s o m e o t h e r s u b s t a n c e a d s o r b e d t o i t o r s o i n t i m a t e l y a s s o c i a te d w i t h i t a s t oescape de tec t ion . I f , however , the b io log ica l ly ac t ive subs tance i so la ted inh igh ly pur i fi ed f o r m as the sod ium sa l t o f desoxyr ibonuc le ic ac id ac tu a l ly p r ovesto be th e t r ans f o r m ing pr inc ip le , a s the ava i lab le ev idence s t r ong ly sugges t s,t h e n n u c l ei c a ci d s o f th i s t y p e m u s t b e r e g a r d e d n o t m e r e l y a s s t r u c t u r a l l yi m p o r t a n t b u t a s f u n c t i o n a l l y a c t i v e i n d e t e r m i n i n g t h e b i o c h em i c a l a c t i v it i e san d spec if i c cha r ac te r i s t i c s o f pneum ococca l ce il s. Assum ing tha t the sod iumdesoxyr ibonu c lea te an d the ac t ive p r inc ip le a r e one and the s am e subs tance ,then the t r an s f or m a t ion desc r ibed r epr esen t s a change tha t i s chem ica l ly in -duced and spec i f ica l ly d i r ec ted by a known chem ica l com p ound . I f the r e su l t so f t h e p r e s e n t s t u d y o n t h e c h e m i c a l n a t u r e o f t h e t r a n s fo r m i n g p r in c i p le a r econf i r m ed , then nuc le ic ac ids m us t be r ega r ded a s pos ses s ing b io log ica lspec i f i c ity the ch em ica l bas i s o f which i s a s ye t u nde te r m ined .

SUMMARYI . F r o m T y p e I I I pneumococcia b io log ica l ly ac t ive f r ac t io n has b een i so la ted

in h igh ly puTi fied f o r m which in exceed ing ly m in u te am ou nts i s capab le un dera p p r o p r i a t e c u l t u r a l c o n d i t i o n s o f in d u c i n g t h e t r a n s f o r m a t i o n o f u n e n c a p s u -l a t e d R v a r i a n t s o f P n e u m o c o c c u s T y p e I I i n t o f u l l y e n c a p s u l a t e d c e ll s o f t h e



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156 TRANSFORMATION O F PNEUMOC~CAL TYPESs a m e s p e c if i c t y p e a s t h a t o f t h e h e a t - k i l l e d m i c r o o r g a n i s m s f r o m w h i c h t h ei n d u c i n g m a t e r i a l w a s r e c o v e r e d .2 . M e t h o d s f o r t h e i s o l a t io n a n d p u r i f i c a t io n o f th e a c t i v e t r a n s f o r m i n g m a -t e r i a l a r e d e s c r i b e d .

3 . T h e d a t a o b t a i n e d b y c h e m i c a l, e n z y m a t i c , a n d s e r o l o g i c a l a n a l y s e st o g e t h e r w i t h t h e r e s u l t s o f p r e l i m i n a r y s t u d i e s b y e l e c t r o p h o r e s is , u l t r a c e n -t r i f u g a t i o n , a n d u l t r a v i o l e t s p e c t r o s c o p y i n d i c a t e t h a t , w i t h i n t h e l im i t s o f t h em e t h o d s , t h e a c t i v e f r a c t io n c o n t a in s n o d e m o n s t r a b l e p r o t e i n , u n b o u n d l i p i d,o r s e r o l o g i c a ll y r e a c t iv e p o l y s a c c h a r i d e a n d c o n s i s t s p r in c i p a l l y , i f n o t s o l e ly , o fa h i g h l y p o l y m e r i z e d , v i s c o u s f o r m o f d e s o x y r i b o n u c l e i c a c i d .

4 . E v i d e n c e i s p r e s e n t e d t h a t t h e c h e m i c a l l y i n d u c e d a l t e r a t i o n s i n c e l lu l a rs t r u c t u r e a n d f u n c t i o n a r e p r e d i c t a b l e , t y p e - s p e c i f i c , a n d t r a n s m i s s i b l e i ns e ri es . T h e v a r i o u s h y p o t h e s e s t h a t h a v e b e e n a d v a n c e d c o n c e rn i n g t h en a t u r e o f t h e s e c h a n g e s a r e r e v ie w e d .

CONCLUSIONT h e e v i d e n c e p r e s e n t e d s u p p o r t s t h e b e l i e f t h a t a n u c l e ic a c i d o f t h e d e s o x y -

r i b os e t y p e i s t h e f u n d a m e n t a l u n i t o f t h e t r a n s f o r m i n g p r i n ci p le o f P n e u m o -c o c c u s T y p e I I I .

B I B L I O G R A P H Y1. Griifith, F., 3". Hy g., Cam br idge , En g. , 1928, 27 , 113 .2 . Neufe ld , F . , and Levin tha l , W., Z. Immunitgtsforsch. , 1928, 5, 324.3 . B a u r h e n n , W . , Centr. Bakt., 1. Abt., Orig., 1932, 126, 68.4 . D a w s o n , M . H . , J . E x p . M e d . , 1930, 51, 123.5 . D a w s o n , M . H . , a n d S i a, R . H . P . , Y . E x p . M e d . , 1931, 54 , 681 .6. Alloway, J . L. , Y. E x p . M e d ., 1932, 55, 91; 1933, ST, 265.7 . B e r r y , G . P . , a n d D e d r i c k , H . M . , Y . B a a . , 1936, 31, 50.8 . Ber ry , G. P . , Arch. Path ., 1937, 24 , 533 .9 . H u r s t , E . W . , Bri t . Y . Exp . Path . , 1937, 18 , 23 . Ho ffs tad t , R . E . , and Pi lcher ,K. S. , Y. Infect . Dis. , 1 9 4 1 , 6 8 , 6 7. G a r d n e r , R . E . , a n d H y d e , R . R . , Y .Infect . Dis. , 1942, 71 , 47 . Hou l ihan , R. B. , Proc . Soc . Exp . Bio l . and Med. ,1 9 4 2 , 5 1 , 2 5 9 .10 . M acLeo d, C. M. , and M ir ick , G. S . , Y. B aa . , 1942, 44 , 277 .1 1 . D a w s o n , M . H . , Y . Exp. Mecl . , 1928, 47 , 577; 1930, 51 , 99 .12. Sevag, M. G. , Biochem. Z. , 1 9 3 4 , 2 7 3 , 4 1 9 . S e v ag , M . G . , L a c k m a n , D . B . ,and Smolens , J . , Y. Biol . Chem. , 1938, 124, 4 25.1 3 . D u b o s , R . J . , a n d A v e r y , O . T . , J . E x p . M e d . , 1 9 3 1 , 5 4 , 5 1. D u b o s , R . J . ,a n d B a u e r , J . H . , J . E x p . M c d . , 1935, 62 , 271 .14 . Liu , S . , and Wu, H. , Chinese J. Physiol . , 1938, 13 , 449 .1 5 . M a r t l a n d , M . , a n d R o b i s o n , R . , Biochem. J . , 1929, 23, 237.

16. Albers, H. , and Albers, E. , Z. physiol . Chem. , 1935, 232 , 189 .17 . Leven e , P . A. , and D i llon , R. T . , J . B io l . Chem. , 1933, 96 , 461 .18 . Greens te in , J . P . , and Je nre t te , W . Y. , Y. Nat . Cancer Ins t . , 1 9 4 0 , 1 , 8 4 5 .



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O S W A L D T . A V E R Y , C O L I N ~f. M A C L F O D ~ A N D M A C L Y N M c C A R T Y 15719. Greenstein, J . P. , J. 2Vat. Cancer Inst . , 1943, 4, 55.20 . Tennent~ H. G. , and V i lbrandt , C. F. , J . A m. Chem. Soc., 1943, 65, 424.2 1 . T h o m p s o n , R . H . S . , a n d D u b o s , R. J., J. Biol . Chem., 1938, 125, 65.2 2 . R e e v e s , R . E . , a n d G o e b d , W . F. , J . B iol . Chem. , 1941, 139, 511.23 . Schul tz , J . , in Genes and chromosomes . S t ruc tu re and organiza t ion , ColdSpr ing Ha rbo r sym pos ia on quant i ta t iv e b io logy , Cold Spr ing H arbo r , LongIslan d Biological Association, 1941, 9, 55.2 4 . M i r s k y , A . E . , i n A d v a n c e s i n e n z y m o l o g y a n d r e l a te d s u b j e c t s o f b i o c h e m i s t r y ,( F . F . N o r d a n d C . H . W e r k m a n , e d i t o r s ) , N e w Y o r k , I n t e r sc i e n c e P u b l is h e r s ,Inc. , 1943, 3, 1.25 . L ackm an, D. , Mu dd, S . , Sevag , M . G. , Smolens , ] . , and W iener, M. , J . Immunol . ,1941, 40 , 1 .26 . Go r tner , R . A. , Out l ines of b iochem is t ry , N ew Y ork , W i ley , 2nd ed it ion , 1938 ,547.27 . Dobzhansky , T . , Genet ics and the or ig in of the spec ies , New York , Columbia

Un ivers i ty Press , 1941 , 47 .28 . S tan ley , W. M. , in Doer r , R . , and Hal iauer , C . , Handbuch der Vi rusforschung,Vienna, Ju lius Springer, 1938, 1, 491.2 9 . M u r p h y , J . B . , T r . A ssn . A m. P hysn . , 1931, 46 , 182; Bul l . Johns Hopkins Hosp. ,1935, 56, 1.



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158 TRANSF ORMATI ON O~" PNEITMOCOCC AL TYPES

EXPLANATION OF PLATEThe 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 thetransforming substance. X 3.5.FIO. 2. Colonies on blood agar of the same cells after induction of transformationduring growth in the same medium with the addition of active transforming prin-ciple isolated from Type n I pneumococci. The smooth, glistening, mucoid coloniesshown are characteristic of Pneumococcus Type In and readily distinguishable fromthe small, rough colonies of the parent R strain illustrated in Fig. 1. X3.5.


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