Distribucion de Metilcytosina en La Piramide de Sec de Ac Nucleicos

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    B I O C H I M I C A E T B I O P H Y S I C A A C TA 953

    DISTRIBUTION OF 5-METHYLCYTOSINEIN PYR IM ID IN E SEQUENCES OF DEOXYRIB ONUCLEI C ACIDS

    J . D O S K O ~ I L AND F. ~ O R M

    Institute o/Organic Chemistry and Biochemistry, Czechoslovak Academy o/Sciences,Praha Czechoslovakia)

    R e c e i v e d O c t o b e r 6 t h , 1 9 6 1 )

    S U M M A R Y

    Several samples of DNA from different mammalian organs and from wheat germ weredegraded with Burton reagent and the location of derivatives of 5-methylcytosine inseparate fractions of the hydrolysate was investigated. In mammal ian DNA s 5-meth-ylcytosine was found to occur almost exclusively in the frac tion of solit ary pyrimi-dine nucleotides and in the terminal groups of polypyrimidine series carry ing mono-esterified phosphorus on C-3 of deoxyribose. The ratio of 5-methylcytosine to cytosinewas nearly identica l in these positions. In the DNA from wheat germ the highest de-gree of replacement occurred in similar positions, but a fair ly high amount of 5-methyl-cytosine was found in the sequence MpT. The relat ion of these findings to earlierevidence obtained by enzymic degradat ion of DNA is discussed. It is concluded th atthe replacement of cytosine by 5-methylcytosine is determined by the nature of the

    nucleotide attached to C-3 of deoxycytidine. The replacement occurs with highestprobability in the sequence CpG; somewhat less probable is the replacement in thesequence CpT, whereas in the sequences CpM, CpC and CpA the substitution takesplace only rarely, even in preparations with high overall content of 5-methylcytosine.

    I N T R O D U C T I O N

    DNA from several plant and animal sources contains small quantities of 5-methylcy-tosineL 2. According to the Watson-Crick double-helix model of DNA, 5-methylcyto-sine should be freely exchangeable with cytosine. However, when determining the

    content of 5-methylcytosine in fractions 3 and degradation products 4, 5 of DNA, signif-icant deviations from random distribution and free exchangeability were found.SINSHEIMER 4 demonstrated tha t in the hydrolysate of calf-thymus DNA, large quan-tities of the dinucleotide pMpG occur, whereas other dinucleotides are comple tely freeof methylcytosine. SI~APII~O A N D C H A R G A F F 5investigating the composition of theacid hydrolysa te of rye-germ DNA, found 5-methylcytosine to be accumulated inthe fraction of solitary nucleotides and in dipyrimidine fragments, thus showing somesimilarity to the relative distribution of thymidylic acid.

    A b b r e v i a t i o n s : G , A , C , M . T, d e o x y g u a n o s i n e , d e o x y a d e n o s i n e , d e o x y c y t i d i n e , d e o x y - 5 -m e t h y l c y t i d i n e a n d t h y m i d i n e , r e s p e c t iv e l y ; X , a n y o f t h e s e n u c l e os i d es ; P u , p u r i n e d e o x y r i b o n u -c l e o s i d e s ;Py, p y r i m i d i n e d e o x y r i b o n u c l eo s i d e s ; p r e p r e s e n t s o r t h o p h o s p h a t e e s t e ri f ie d w i t h t h e s en u c l e o s i d e s ; f o r a b b r e v i a t i o n s o f n u c l e o t i d e s s e e r e f. 6 .

    Bioehim. Biophys. Acta, 5 5 1 9 6 2 ) 9 5 3 - 9 5 9

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    95 4 j. DOSKOCIL, F. SORM

    I n t h e p r e s e n t s t u d y t h e l o c a t i o n o f 5 - m e t h y l c y t i d y l i c a c i d i n p o l y p y r i m i d i n es e q u e n c e s w a s i n v e s ti g a t e d . P r e p a r a t i o n s o f D N A f r o m c a l f t h y m u s , r a t s p le e n , m o u s el e u k em i c l iv e r a n d w h e a t g e r m w e r e h y d r o l y s e d w i t h f o r m i c a c id a n d d i p h e n y l a m i n e6.U s i n g t h is m e t h o d o f h y d ro l y s i s th e p u r i n e d e o x y r i b o n u c l eo t i d e s a r e d e g r a d e d t op u r i n e b a s e s a n d i n o rg a n i c o r t h o p h o s p h a t e i s f o r m e d f r o m t i le p h o s p h o d i e s t e r g r o u p sj o i n in g t w o p u r i n e n u c l eo s i de s ; t h e p y r i m i d i n e n u c le o s id e s , h o w e v e r, a r e n o t d e g r a d e da n d r e m a i n a s n u c l e o t id e s o f t h e g e n e r a l s t r u c t u r e P Y . P. + v T h e f r a c t i o n s o f n - n u -c le o si de ( n + I ) - p h o s p h a t e s w e r e d e g r a d e d s u c c e si v el y w i t h p r o s t a t e p h o s p h o m o n o -e s t e r a s e a n d w i t h p h o s p h o d i e s t e r a s e f r o m c a l f s p le e n . W i t h t h i s t y p e o f d e g r a d a t i o n ,d e o x y c y t i d y l i c ac i d o c c u r in g i n t h e s e q u en c e C p P y a p p e a r s i n t h e f o r m o f d e o x y c y -t i d i n e 3 ' -p h o s p h a t e , w h e r e a s t h a t f o r m i n g p a r t o f t h e s e q u e n c e C p P u i n t h e o r i g i n a lc h a i n o f D N A is re c o v e r e d a s d e o x y c y t i d i n e . T h e e x t e n t o f re p l a c e m e n t o f cy t o s i n ew i t h 5 - m e t h y l c y t o s i n e i n t h e s e p o s it i o n s w a s t h e n d e t e r m i n e d b y a n a l y si s o f t h e d e o x y -r i b o n u c l e o s i d e 3 '- p h o s p h a t e a n d d e o x y r i b o n u c l e o s i d e f r a c t io n s .

    EXPERIMENTAL

    D N A f r o m r a t s p l e e n a n d m o u s e l e u k e m i c li v e r ( s t r a in B l a c k C 57 , t r a n s p l a n t a b l ep o s t - i r r a d i a t i o n b l a s t i c l e u k e m i a , 2 0 - 3 0 % b l a s t ic i n f i lt r a t io n ) w a s p r e p a r e d a c c o r d i n gto KAY, SIMMON S AND D OUNC E7. D N A f r o m c a l f t h y m u s w a s i s ol a te d b y t h e m e t h o dof SCHWANDER AND SIGNER 8. The method of LIPSHITZAND CHARGAFF 9 wa s used , top r e p a r e D N A f r o m w h e a t g er m e x c e pt t h a t R N A w a s r e m o v e d b y t r e a t m e n t w i t hr i b o n u c l e a s e i n s t e a d o f t h e a b s o r p t i o n o n c h a r c o a l . T h e a n a l y s e s o f t h e p r e p a r a t i o n sa r e g i v e n i n Ta b l e I .

    TABLE ICOMPOSITION O F THE SAMPLI~S O F D N

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    Wh ea t ge rm 9.0 1.5 22.6 27. 3 15.8 6. 7 27.6

    T h e p h o s p h o m o n o e s t e r a s e w a s p r e p a r e d f r o m h u m a n h y p e r t r o p h i c p r o s t a t i ct i s sue a ccord ing to DAVlDSON AND FISHMAN o . Th e ac t iv i ty was de t e rm ine d w i ths o d i u m g l y c e r o p h o s p h a t e , u s in g a o .2 5 % ( w / v ) s o l u ti o n in o .o 5 M a c e t a t e b u ff e r, p H5 .5 . o. o5 m l o f t h e s o lu t i o n o f t h e e n z y m e ( a b s o r b a n c y a t 2 8 o m e q u a l t o o. 31 5 ) w a sa d d e d / m l o f th e b u f f e r e d s o lu t io n o f s o d iu m g l y c e r o p h o s p h a t e a n d t h e m i x t u r e w a si n c u b a t e d a t3 7 i m l o f t h e s o l ut io n o f t h e e n z y m e p r o d u c e d 3 9 o o g P i /h u n d e r t h e s ec o n d i t i o n s .

    T h e s p l e n i c p h o s p h o d i e s t e r a s e w a s i s o l a t e d f r o m c a l f s p l e e n b y t h e m e t h o d o fHILMOEn . A m i x t u r e o f o l ig o n u cl e ot id e s f r o m t h e h y d r o l y s i s of c a l f- t h y m u s D N A w i t hs p le n ic D N A a s e I I w a s u s e d a s s u b s t r a t e f o r t e s t i n g t h e p h o s p h o d i e s t e r a s e a c t i v i t y.T h e h y d r o l y s a t e of D N A w i t h D N A a s e I I w a s c k r o m a t o g r a p h e d o n a c o lu m n o f

    D o w e x - I X 2 . T h e f r a c t i o n e lu t e d b y 1 . o- 1 .2 M a m m o n i u m f o r m a t e ( p H 4 .6 5 ) w a se v a p o r a t e d t o d r y n e s s a n d t h e f o r m a t e w a s r e m o v e d b y v a c u u m s u b l im a t i o n a t

    B i o c h i m . B i o p h y s . A c t a ,55 1962) 953-9 59

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    DISTRIBUTION OF 5-METHYLCYTOSINE IN D N A 955

    4 o . T h e r e s i d u e , c o n s i s t i n g m o s t l y o f t e t r a n u c l e o t i d e s , w a s d i s s o l v e d i n a n a p p r o p r i -a t e a m o u n t o f 0 .0 5 M a c e t a t e b u f f e r, p H 6 .5 , t o g i v e a s o l u t io n h a v i n g A 2 eo e q u a l t o5 . 0 - 5 . 5 . 0 . 0 2 5 m l o f e n z y m e w a s a d d e d / m l o f t h i s so l u ti o n a n d t h e m i x t u r e w a s i n -c u b a t e d a t 3 7 . T h e p r o d u c t i o n o f m o n o n u c l e o t i d e s w a s f o ll o w e d b y c i r c u l a r p a p e rc h r o m a t o g r a p h y i n e t h a n o l - I M a m m o n i u m a c e t a t eTM, p H 7 -5 ( 7 5: 3 0 ) . W i t h u s u a lp r e p a r a t i o n s o f t h e e n z y m e ( a b s o r b a n c y a t 2 80 m a b o u t I O ) t h e s p l i tt i n g to m o n o n u -c l e ot i de s w a s v i r t u a l l y c o m p l e t e i n 3 o m i n o f in c u b a t i o n . T h e p r e p a r a t i o n c o n t a i n e ds o m e p h o s p h o m o n o e s t e r a s e a c t i v i t y, b u t i f c a r e w a s t a k e n t o i n t e r r u p t t h e i n c u b a t i o nj u s t a s t h e c l e a v a g e o f p h o s p h o d i e s t e r b o n d s w a s c o m p l e t e , e q u a l a m o u n t s O f n u c l eo -t i d e s a n d n u c l e o s id e s w e r e a l w a y s o b t a i n e d i n a n a l y s e s o f d i n u c l eo s i d e m o n o p h o s -p h a t e s .

    T h e h y d r o l y s i s o f D N A a n d t h e r e m o v a ] o f f o r m i c a c i d w e r e c a r r i e d o u t a s d e-sc r ib ed by BURTON AND PETERSEN6. 4 0 0 m g o f m a m m a l i a n D N A ( e x c ep t in t h e c a s eo f D N A f l: om m o u s e l e u k e m i c li v e r, w h e r e o n l y 1 8 o m g w e r e a v a i la b l e ) a n d 2 00 m g o f

    w h e a t - g e r m D N A w e r e u s e d fo r o n e ex p e r i m e n t . T h e h y d r o l y s a t e w a s c h r o m a t o g r a p h -e d on a c o lu m n o f D o w e x - I X 2 (2 0 I c m ) w i t h a m m o n i u m f o r m a t e b u ff e r, p H4 . 65 4 -0 .0 .5 . A s t o c k s o lu t i o n o f f o r m a t e b u f f e r, 4 M i n a m m o n i u m f o r m a t e a n d a p p r o x .I M i n f o r m i c a c id , w a s a p p r o p r i a t e l y d i l u t e d w i t h w a t e l t o o b t a i n t h e i n d i c a t e dc o n c e n t r a t i o n o f f o r m a t e . E x a c t c o n t r o l o f p H o f th e b u f f e r w a s e s se n t ia l , s in c e e v e ns l ig h t d e v i a t i o n s f r o m t h e c o r r e c t v a l u e c a u s e d i n c o m p l e t e s e p a r a t i o n o f p C p C p f r o mp T p o r f r o m p T p C p a n d p C p T p . Tw o l i n e a r c o n c e n t r a t i o n g r a d i e n t s w e r e u s e d s u c -c e s i v e l y t o e l u t e t h e n - n u c l e o s i d e ( n + I ) - p h o s p h a t e s : 2 5 0 m l o. i M t o 2 5 o m l 0 . 6 M a n d2 5 0 m l o . 6 M t o 2 5 0 m l 1 .2 M f o r m a t e . A b o u t 9 % o f t h e t o t a l u l t r a v i o l e t - a b s o r b i n gm a t e r i a l w a s e lu t e d . T h e e l u t i o n c u r v e w a s r e g i s t e re d b y m e a n s o f a U v i c o r d , t y p e

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    Fig. I. Elution curves of the hydrolysate of DNA with Burton reagent. The column of Dowex-IX2 z 20 cm) was charged wit h a hydr olys ate of 4o0 mg DNA an d elut ed with t wo succesivelinear gradie nts of ammon ium f orm ate buffer , pH 4.65 25o ml o.i M to 25o ml 0.6 M and 25 mlo. 6M to 25oml 1.2 M formate). The volume of the fractions collected was 7 ml. A, ca lf-thymu sDNA; B, whea t-g erm DNA. F ract ion I, pCp, pMp, 2, pTp; 3, pCpCp, pCpMp; 3a, pMpNIp, pCpMp,pMpCp; 3 b, pCpCp; 4, pCpTp, pTpCp, pTpMp; 4 a, pTpMp, pMpTp; 4 b, pTpCp, pCpTp; 5, pTpTp,

    pCpC]?Cp; 6, trin ucleot ides TCC; 7, trin ucleot ides TTC; 8, mostl y tetr anucle otid es.

    Biochim. Biophys. Acta 55 1962) 953-959

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    956 3. DOSKO~IL, F. ~ORM

    4 7O l A ( L K B , S w e d e n ). T h e f r a c t io n s f o r m i n g s e p a r a t e p e a k s w e r e p o o l e d a n d e v a p o -r a t e d t o d r y n e s s i n v a c u o . A m m o n i u m f o r m a t e w a s r e m o v e d b y s u b l i m a t i o nat 4 0i n v a c u o . T h e o l i g o n u c le o t i d e s w e r e t h e n d i s s o l v e d i n 2 m l o f 0 .0 5 M a c e t a t e b u f -f e r, p H 5 .5 . o . I m l o f p r o s t a t i c p h o s p h o m o n o e s t e r a s e w a s a d d e d a n d t h e s o l u t io n i n -c u b a t e d f o r 8 h a t 3 7 . T h e p H o f t h e m i x t u r e w a s b r o u g h t t o 6 .5 b y a d d i t i o n o f a p -p r o x . 0 . 02 m l o f 0 .5 M a m m o n i a a n d t h e p h o s p h o m o n o e s t e r a s e w a s i n a c t i v a t e d b yh e a t i n g t h e s o l u ti o n o n a w a t e r b a t h a t 8 o f o r 15 m i n . A f t e r c o o li n g 0.0 5 m l o f p h o s -p h o d i e s t e r a s e w a s a d d e d a n d t h e s o l u t io n w a s i n c u b a t e d a t 3 7 u s u a l l y f o r 2 h . S i n c et h e p r e p a r a t i o n o f p h o s p h o d i e s t e r a s e w a s n o t e n t i r e l y f r e e o f p h o s p h o m o n o e s t e r a s ea c t i v i t y, t h e i n c u b a t i o n h a d t o b e i n t e r r u p t e d a s s o o n a s t h e s u b s t r a t e w a s c o m p l e t e l yd e c o m p o s e d t o m o n o n u c l e o t i d e s a n d n u c l eo s id e s. F o r t h i s p u r p o s e s a m p l e s w e r ew i t h d r a w n f r o m t h e r e a c t i o n m i x t u r e i n 3 o- m i n i n t e r v a l s a n d a n a l y s e d b y c i r c u l a rp a p e r c h r o m a t o g r a p h y o n a d is k o f W h a t m a n N o . 4 p a p e r w i t h e t h a n o l - I M a m m o n i u ma c e t a t e , p H 7 .5 (7 5 : 3 0 ). A s s o o n a s n o r e s i d u a l s p o t o f o li g o n u c l e o t id e s c o u l d b e d i s -

    c e r n e d o n t il e c h r o m a t o g r a m , t h e r e a c t i o n w a s i n t e r r u p t e d a n d t h e s a m p l e s w er e:r a p i d l y c o n c e n t r a t e d t o s m a l l v o l u m e o n a h o r iz o n t a l r o t a r y v a c u u m e v a p o r a t o r .T h e m i x t u r e w a s th e n a p p l i e d o n a s h e e t o f W h a t m a n N o . 3 p a p e r i n a l in e I O c m o fl e n g th a n d s u b j e c te d to d e s c en d in g c h r o m a t o g r a p h y w i t h e t h a n o l - a m m o n i u m a c e t a t e ,p H 7 .5 , fo r a p p r o x . 1 4 h . T h e z o n e s of n u c l e o t i d e s a n d n u c l e o s id e s w e r e e l u t e d w i t hw a t e r ; t h e e l u a t e s w e r e e v a p o r a t e d t o d r y n e s s a n d h y d r o l y s e d w i t h c o n c e n t r a t e dfo rm ic ac id acco rd ing to VISCHER AND CHARGAFF 3 us ing sea led cap i l l a r y tu be sa p p r o x . I m m o f i n n er d i a m e t e r . T h e h y d r o l y s a t e s w e r e e v a p o r a t e d t o d r y n e s si nv a c u o d i ss o lv e d i n o . i N H C I a n d c h r o m a t o g r a p h e d o n W h a t m a n N o . 3 p a p e r i n as y s t e m1 i s o p r o p a n o l - H C l - w a t e r ( 17 o : 4 1 : 3 9 ) . U s u a l l y s a t i s f a c t o r y s e p a r a t i o n o f 5-m e t h y l c y t o s i n e a n d c y t os i n e w a s a c h i e v e d b y t h e fi r s t c h r o m a t o g r a p h y , s o t h a t r e -c h r o m a t o g r a p h y 1 w a s n o t n e c e s s a r y, I f , h o w e v e r , t h e e l u t ed 5 - m e t h y l c y t o s i n e w a sn o t s p e c t r o s c o p i c a l l y p u r e , r e c h r o m a t o g r a p h y i n t h e s a m e s o l v e n t w a s u s e d f o r f u r -t h e r p u r i f i c a t i o n . I f n o d i s t in c t z o n e o f 5 - m e t h y l c y t o s i n e c o u l d b e d e t e c t e d o n t h ec h r o m a t o g r a m , t h e z o n e o f c y t o s in e w a s a ls o r e c h r o m a t o g r a p h e d t o b e s u r e t h a t n om e t h y l c y t o s i n e r e m a i n e d i n t h e c y t o s i n e z o n e .

    T h e z o n e s o f p y r i m i d i n e s w e r e c u t o u t o f t il e c h r o m a t o g r a m a n d e l u t e d w i t h 4 m lo . I N H C I . U l t r a v i o l e t s p e c t r a w e r e t h e n r e c o r d e d i n t h e r a n g e 2 5 0 -3 0 0 m i n 5 - mi n t e r v a ls . T h e a m o u n t s o f p y r i m i d i n e s w e r e c a l c u l at e d u s i n g t h e f o ll o w in g v a l u e sof m i l l im ola r ex t inc t ion coeff ic i en t s14 : lO . 5 a t 275 m fo r cy tos in e , 9 .8 a t 283 m fo r5 - m e t h y l c y t o s i n e a n d 7 .9 5 a t 2 65 m f o r t h y m i n e .

    To c h e c k t h e r e l i a b il i t y o f t h e a n a l y t i c a l m e t h o d a l i q u o t s o f f r a c t i o n s o f t h e h y -d r o l y s a t e w e r e a n a l y s e d f o r 5 - m e t h y l c y t o s i n e c o n t e n t w i t h o u t p r e v i o u s e n z y m i c h y -d r o ly s i s. T h e r e s u l ts o b t a i n e d w e r e t h e n c o m p a r e d w i t h t h e c o n t e n t o f 5 - m e t h y l c y t o -s in e c a lc u l a t e d f r o m t h e d e t e r m i n a t i o n s a f t e r e n z y m i c d e g r a d a t io n . I t m a y b e s e e nf r o m Ta b l e I I t h a t t h e a g r e e m e n t is s a t i s f a c to r y. S i m i l a r ly, g o o d a g r e e m e n t w a so b t a i n e d w h e n c a l c u la t i n g th e c o n t e n t o f 5 - m e t h y l c y t o s i n e i n w h o le D N A f r o m t h ea n a l y s e s o f s e p a r a t e f r a c t io n s a n d c o m p a r i n g i t w i t h t h e d a t a o b t a i n e d b y d i r e c t a n a -l y si s, i n d ic a t i n g t h a t t h e h y d r o l y s i s m e t h o d u s i n g f o rm i c a c i d to d e t e r m i n e n i t r o g e -n o u s c o n s t i t u e n t s i s t r u s t w o r t h y.

    W h e n a n a l y s i n g th e m i x e d d i p y r i m i d i n e s a d d i t i o n a l i n f o r m a t i o n w a s o b t a i n e dg i v i n g t h e r a t i o o f b o t h s e q u e n t i a l is o m e r s , p C p T p a n d p T p C p , w i t h o u t t h e i r a c t u a ls e p a r a t i o n . I t h a s b e e n s h o w n b y B U RTO N A ND P ET E RS EN6,15, th a t th i s r a t io i s s ig -

    Biochim. Biophys. Acta 55 1962) 953 -959

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    D I S T R IB U T I O N O F 5 M E T H Y L C Y T O S I N E I N D N A 9 5 7

    nificantly different from unit y in many preparations of DNA from different organ-isms. The ratios given in Table II are derived from the ratio of cytosine (plus 5-methylcytosine) to thymine in the nucleoside fraction, which is equal to the recipro-cal of this ra tio in the 3'-phosphate fraction of the enzymic hydrolysate. This equal-ity provides a test for the correctness of the degradative and analytical proceduresemployed.

    R E S U LT S

    The experiments summarized in Table II show that in all three preparations ofmammalian DNA 5-methylcytosine occurs almost exclusively in the fraction pCp andin the terminal nucleotide of polypyrimidine units bearing monoesterified phospho-rus on the C-3' atom of deoxyribose. This fact indicates that methylcytosinereplaces cytosine only in the sequence PupCpPu (sohtary deoxycytidyhc acid) orPypCpPu (3'-phosphate terminus of po]ypyrimidine sequences), but not in the se-quence PupCpPy. E . g . in the isomeric mixed dipyrimidine nucleotides partial re-placement of cytosine with 5-methylcytosine occurs in the less frequent pTpCp iso-mer, but not in the more frequent pCpTp isomer; the preponderance of the lattersequential isomer observed by BURTON AND PETERSEN 6 in calf -thymus DNA seemsto be quite common in prepara tions of DNA from mammal ian tissues, since almostequal ratios of both sequential isomers were observed with calf-thymus, rat-spleenand mouse-leukemic-hver DNA's.

    It can therefore be readily understood that the solitary cytosine is replacedwith 5-methylcytosine about twice as frequently as the total cytosine in the dipyri-midine sequences and that even less 5-methylcytosine is contained in higher poly-

    pyrimidine sequences TM. A similar type of distribution was suspected by SHAPIROAND CHARGAFF to exist in rye-germ DNA, accounting for the observed tendency of5-methylcytosine to be accumulated in the fraction of solitary pyrimidine nucleo-tides and in lower polypyrimidine sequences.

    The extent of actual replacement of exchangeable cytosine with 5-methylcyto-sine is near ly the same in all types of sequences, name ly PupMpPu, CpMpPu, TpMpPuas well as in terminal deoxycytidylic acid occuring at the 3'-phosphate end of tri-pyrimidine sequences. This fact indicates that, in mammalian preparations studied,the replacement of cytosine with 5-methylcytosine is determined exclusively by thenature of the nucleotide attached to 3'-carbon atom of deoxycytidylic acid, whereasthe nucle, otide on the 5'-carbon atom is irrelevant in this respect.

    In the DNA from wheat germ the distribut ion of 5-methylcytosine is somewhatdifferent. The greater part of 5-methylcytosine occurs in the sequence MpPu, but adefinite, though smaller, amount of it is found in the sequence MpPy as well. Espe-cially the sequence MpT is favored, being only slightly less frequent tha n TpM. I tcannot be decided by the present method, whether the II Mp occuring in the hy-drolysate of the dicytidyhc acid fraction comes from the sequence pMpCp or pMpMp;the isolation of appreciable amount of pMpMp from acid hydrolysates of rye-germDNA by SHAPIRO AND CHARGAFF indicates that a pa rt of the Mp from the dicy-tidyhc acid fraction was originally bound in the form of dideoxy-5-methylcytidylicacid.

    Another significant difference from mammalian preparations is apparent inwheat-germ DNA. The degree of replacement of deoxycyt idyhc acid occurring in the

    Biochim. Biophys . dc ta55 (I962) 953-959

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    6/7

    9 8 j. DOSKOCIL, F. SORM

    s e q u e n c e s C p P u i s c o n s i d e r a b l y h i g h e r i n d i p y r i m i d i n e f r a g m e n t s t h a n i n h i g h e r p o l y -

    p y r i m i d i n e s e q u e n c e s .

    TA B L E I I

    D I S T R I U T I O N OF 5-METHYLCYTOSINE IN THE HYDROLYSATES OF D N A WITH FORMIC ACID ANDDIPHENYLAMINE

    T h e f r a c t i o n s of t h e h y d r o l y s a t e w e r e d e g r a d e d w i t h p h o s p h o m o n o e s t e r a s e a n d s p l en i c p h o s p h o -d ies te rase .

    5-Melkylcytosine as a percentage o] the s um cytosine + 5 methylcytosine

    Composition oI maio r In the who le/ractio ncomponent in the ]raction In nucIeoside In nucleosides

    3 phosphates Fo un d Calculated

    C a l l - th y m u s D N A

    p e p - - x i 11 . o o . 8 -

    pCpCp

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