On the desoxypentose nucleic acids from several microorganisms

402

BIOCHIMICA ET BIOPHYSICA ACTA

ON T H E D E S O X Y P E N T O S E

VOL. 9

(1952)

N U C L E I C ACIDS FROM

SEVERAL MICROORGANISMS* by STEPHEN

Z A M E N H O F , G E O R G E B R A W E R M A N * * , AND ER'vVIN C t t A R G A F F

Department o[ Biochemistry, College o/ Physicians and Surgeons, Columbia University, New York, N . Y . (U.S.A,)

The present study continues the attempts of this laboratory to gain an insight into the diffelences in composition, and therefore, presumably, in nucleotide sequence, distinguishing the desoxypentose nucleic acids (DNA's) derived from different species. As has been pointed out on a previous occasion 2, the investigation of microbial DNA's is of particular interest in this connection, not only because of the range of cellular morphology and physiology, much wider than in higher organisms, that may thus be covered, but also because of the role currently assigned to certain microbial DNA's in the phenomena of bacterial transformation 3. One of the DNA preparations examined here, namely that of Hemophilus influenzae, was, in fact, endowed with transforming activity 4. EXPERIMENTAL Sources R e p r e s e n t a t i v e s of three different bacterial families served for the isolation of DNA. T h e y were : (i) Serratia marcescens, m u t a n t VII-I-2 (BUNTING), obtained from strain H y 5 b y ultraviolet irradiation. (2) Bacillus Schatz, a facultatively autotrophic, Gram-positive h y d r o g e n organism, kindly given us b y Drs C. ]3. VAN NII~L AND A. SCHATZ,H o p k i n s Marine Station, Pacific Grove, California. (3) Hemophilus influenzae, t y p e c.

Preparations DATA o[ Serratia marcescens. The o r g a n i s m s were cultivated in R o u x bottles a t r o o m t e m p e r a t u r e for 48 h o u r s on Difco N u t r i e n t Agar. The suspension of the cells in physiol, saline exhibited desoxyribonuclease activity; it contained a p p r o x i m a t e l y 7 ° units 6 per g of wet cells. W h e n the organisms were g r o u n d in a m o r t a r w i t h P y r e x p o w d e r and t h e n extracted w i t h physiol, saline (1.6 ml per g of cells), the e x t r a c t contained a b o u t 25 u n i t s p e r g of bacteria. The assays were carried o u t in the a r r a n g e m e n t described previously 7. This relatively s t r o n g depolymerase, which could h a v e precluded t h e isolation of highly polymerized DNA, was retarded, b u t n o t inhibited, b y o.i M sodium citrate. I t s inhibition was, however, a l m o s t complete in 3-5 M s o d i u m chloride solution. F o r the isolation of DNA, the 48 h o u r s old organisms were r e m o v e d from each bottle b y w a s h i n g w i t h 30 ml of o.i M s o d i u m citrate buffer (pH 7-3), recovered b y centrifugation at 18,ooo × g for 3 ° m i n u t e s and w a s h e d three t i m e s b y suspension in 5 v o l u m e s of 3.5 M a q u e o u s s o d i u m chloride. The cells deposited by centrifugation, were frozen i m m e d i a t e l y and g r o u n d for 3 ° m i n u t e s in a chilled m o r t a r w i t h one p a r t (by weight) of w a s h e d P y r e x p o w d e r (diameter 3/~) and o.18 p a r t of solid sodium chloride. After e x t r a c t i o n w i t h one v o l u m e of 3.5 h i a q u e o u s sodium chloride and centrifugation at 18,ooo × g * This w o r k has been s u p p o r t e d b y research g r a n t s from the N a t i o n a l I n s t i t u t e s of Health, United States Public H e a l t h Service, and from the Rockefeller F o u n d a t i o n . Some of the results h a v e formed the subject of a brief note 1. ** U.S. Public H e a l t h Research Fellow.

Re/erences p. 4o5.

VOL. 9

(1952)

DESOXYPENTOSE NUCLEIC ACIDS FROM MICROORGANISMS

403

for 3 ° m i n u t e s , t h e s u p e r n a t a n t w a s injected into t h r e e v o l u m e s of ice-cold 9o % e t h a n o l . T h e prec i p i t a t e d t h r e a d s were w a s h e d w i t h 9o a n d s t o r e d in 95 % e t h a n o l . T h e e x t r a c t i o n residue w a s ree x t r a c t e d IO to 15 t i m e s w i t h d e c r e a s i n g q u a n t i t i e s of salt solution, a n d fibers were collected from each e x t r a c t . T h e c o m b i n e d t h r e a d s were dissolved in io % a q u e o u s s o d i u m chloride ( a b o u t o. 5 m g D N A p e r ml) a n d freed of p r o t e i n a n d purified, as described before s. T h e final yield of D N A was o.o8 % of t h e w e t cell weight. I t c o n t a i n e d 8.99 % P, less t h a n 2 % p e n t o s e nucleic acid, a n d a s s a y e d for 95 % of D N A , w h e n c o m p a r e d w i t h a calf t h y m u s D N A s t a n d a r d 2. D N A o[ Bacillus Schatz. T h e o r g a n i s m s were g r o w n in R o u x b o t t l e s a t 3 o° for 48 h o u r s on Difco Y e a s t Beef Agar. T h e yield was a b o u t I g of p a c k e d cells (wet weight) p e r bottle. W i t h a few exceptions, t h e p r o c e d u r e for t h e isolation of D N A followed t h a t described in t h e p r e c e d i n g p a r a g r a p h . O w i n g to t h e a b s e n c e of d e t e c t a b l e d e s o x y r i b o n u c l e a s e a c t i v i t y o.I M a q u e o u s s o d i u m c i t r a t e (pH 7.1) could be u s e d for t h e w a s h i n g of t h e cells a n d l O % a q u e o u s s o d i u m chloride for t h e ext r a c t i o n of t h e c r u s h e d o r g a n i s m s . T h e p r e c i p i t a t i o n of p e n t o s e nucleic acid as a n insoluble c a l c i u m salt s failed to give s a t i s f a c t o r y r e s u l t s in t h i s case. F o r t h i s reason, recourse w a s h a d to t h e r e m o v a l of c o n t a m i n a t i n g p e n t o s e nucleic acid b y its c o n v e r s i o n to dialyzable p e n t o s e n u c l e o t i d e s * . A solution of t h e c r u d e D N A fibers, collected a f t e r t h e d e p r o t e i n i z a t i o n , in a q u e o u s s o d i u m h y d r o x i d e of p H 13.5 (1.6 m g D N A p e r ml) w a s dialyzed w i t h r o c k i n g a t 3 ° ° for 18 h o u r s a g a i n s t t h e 25e-fold v o l u m e of t h e s a m e solvent, for 24 h o u r s a g a i n s t r u n n i n g t a p water, a n d for t h e s a m e period a g a i n s t ice-cold distilled water. T h e D N A , recovered b y lyophilization of t h e dialyzed solution, a m o u n t e d to o.i % of t h e cells (wet weight). I t c o n t a i n e d 8.83 % P, less t h a n 2 % p e n t o s e nucleic acid, a n d a s s a y e d for 9 4 % of D N A . D N A o[ Hemophilus influenzae, type c. T h e isolation of t h i s p r e p a r a t i o n h a s been discussed in a s e p a r a t e p a p e r 1°.

Procedures T h e a n a l y t i c a l m e t h o d s for t h e e s t i m a t i o n of p u r i n e s a n d p y r i m i d i n e s a n d t h e d e t e r m i n a t i o n of t o t a l D N A a n d p e n t o s e nucleic acid a n d of p h o s p h o r u s h a v e , w i t h one exception, b e e n outlined in detail in a r e c e n t p u b l i c a t i o n 11. A n a d d i t i o n a l procedure, w h i c h p r o v e d useful in t h e p l e s e n t a n d in o t h e r work, is b a s e d on t h e u n e x p e c t e d finding t h a t t h e p u r i n e s a d e n i n e a n d g u a n i n e m a y be s e p a r a t e d f r o m t h e p y r i m i d i n e s b y w h a t c a n be considered as real a d s o r p t i o n c h r o m a t o g r a p h y on filter paper, w i t h w a t e r as t h e sole s o l v e n t . T h e D N A h y d r o l y s a t e w a s p r e p a r e d in t h e u s u a l m a n n e r w i t h cone. formic acid 11 a n d applied to t h e t o p of a strip of filter p a p e r (Schleicher a n d Schuell, No. 597), w i t h t h e longer edge (at l e a s t 60 cm) paralleling t h e w a t e r - m a r k s of t h e p a p e r . T h e lower p o r t i o n of t h e strip w a s w o u n d i n t o a roll held t o g e t h e r b y p a p e r clips, so t h a t t h e p a p e r fitted into t h e c h r o m a t o g r a p h y a s s e m b l y n o r m a l l y e m p l o y e d 13. A f t e r n e u t r a l i z a t i o n of t h e d i s p e n s e d h y d r o l y s a t e w i t h g a s e o u s a m m o n i a , c h r o m a t o g r a p h y w a s carried o u t w i t h w a t e r or w i t h o.oi M p h o s p h a t e buffer of p H 7.1 as t h e solvent. A sufficient s e p a r a t i o n w a s a c h i e v e d w i t h i n a b o u t 165 m i n u t e s a t r o o m t e m p e r a t u r e . W i t h a m i x t u r e of adenine, g u a n i n e , cytosine, a n d t h y m i n e , t h e p y r i m i d i n e zone w a s f o u n d to t r a v e l a b o u t twice as f a s t as t h e p u r i n e zone. Following t h e location of t h e s e p a r a t e d a r e a s b y m e a n s of a s u i t a b l e u l t r a v i o l e t l a m p , t h e zone c o n t a i n i n g t h e p u r i n e s w a s c u t off, t h e r e m a i n i n g strip w a s unrolled a n d t h e p y r i m i d i n e s s e p a r a t e d f r o m e a c h o t h e r b y c h r o m a t o g r a p h y in t h e u s u a l f a s h i o n 18.

Composition T a b l e I p r e s e n t s t h e a n a l y t i c a l r e s u l t s on t h e c o n t e n t s of i n d i v i d u a l p u r i n e s a n d p y r i m i d i n e s in t h e D N A p r e p a r a t i o n s e x a m i n e d here. T h e figures are a v e r a g e s b a s e d on b e t w e e n 3 a n d io indep e n d e n t h y d r o l y s i s e x p e r i m e n t s a n d on a large n u m b e r of i n d i v i d u a l d e t e r m i n a t i o n s . T h e m a n n e r of p r e s e n t a t i o n of t h e d a t a follows t h a t a d o p t e d p r e v i o u s l y 11,12,14. DISCUSSION The quality

of the DNA

preparations

was fairly satisfactory,

with

the exception

o f t h e D N A o f t h e h y d r o g e n o r g a n i s m B a c i l l u s Schatz, w h i c h w a s n o t a n a l y z e d a s t h e highly polymerized product. Work in this laboratory, as yet unpublished, has, however, yielded no indications that the depolymerization of a DNA by alkali to non-dialyzable * T h e r e c e n t l y p u b l i s h e d p r o c e d u r e for r i d d i n g D N A of p e n t o s e nucleic acid 9 w a s n o t y e t a v a i l a b l e a t t h e t i m e t h i s w o r k w a s carried ont. ** T h i s m e t h o d h a s been f o u n d of v a l u e for t h e c h r o m a t o g r a p h i c d e t e c t i o n of t r a c e c o m p o n e n t s a n d has, for i n s t a n c e , been utilized r e c e n t l y for t h e e s t i m a t i o n of 5 - m e t h y l c y t o s i n e in t h e D N A of Paracentrotus lividus 12. I t also is useful, w h e n t o t a l D N A h y d r o l y s a t e s are to be t e s t e d for t h e presence of uracil, w h i c h otherwise is o v e r s h a d o w e d b y a d e n i n e .

Re[erences p. 4o5.

S. ZAMENHOF, G. BRAWERI~IAN, E. CHARGAFF

404

VOL. 9 (1952)

TABLE I DNA'S

OF

SEVERAL

MICROORGANISMS;

MOLAR

PROPORTIONS

AND

RELATIONSHIPS

Source of preparation Serratia marcescens

Moles per mole t s Adenine Guanine Cytosine Thymine P accounted for, % P in hydrolysate Molar ratio Adenine to guanine Thymine to cytosine Adenine to thymine Guanine to cytosine Purines to pyrimidines Amino groups to enolic hydroxyls

o.18o 0.237 o.278 o.i75 87,o

o,76 o,63 1,o3 0-85 o.92 1.69

Bacillus Sehatz

o.173 o.253 o.281 o.162 86.9

0.68 0.58 1.o7 0.9o 0.96 1.7o

Hemophilus Influenzae, Type c

0.296 o.169 o.182 0.280 92. 7

1.75 1.54 1.o6 0.93 i.oi 1.44

fragments is attended by appreciable changes in the proportions of the constituents. The figures reported here m a y be considered as representative of the composition of the DNA's under examination, the more so, since contamination with pentose nucleic acid had been reduced to insignificant levels. The three DNA specimens are good examples of two of the main types of DNA encountered so fa11,15: the infrequent "GC type" (represented by the DNA's of Serralia and the hydrogen organism) and the much more prevalent "AT type" (here r e p r ~ e n t e d by the Hemophilus DNA). Quite apart from differences in the proportion: of individual nitrogenous constituents, which appear to be characteristic of the species furnishing the paltieular DNA TM, and from regularities governing the composition of all DNA's analyzed is, the majority seems to belong to a class in which adenine and thymine exceed guanine and cytosine b y 35 to 9o%. In the case of microbial DNA's, this "AT t y p e " is represented b y the Hemophilus DNA described here, by the DNA of yeast s and that of Pneumococcus, Type 11117. An intermediate type, containing all bases in nearly equimolar quantities, has been found in several strains of E. coli14,15,TM. The "GC type", containing larger amounts of guanine and cytosine than of adenine and thymine, is exemplified by the DNA of different strains of tubercle bacilliZ, TM and by two of the DNA prepalations discussed in the present communication. I t is worthy of mention that until now only microorganisms have yielded representatives of all three types. I t is, however, clear t h a t the correlation between the composition and the biological functions of a DNA and the t a x o n o m y and phylogeny of the species from which it is derived will not be possible without supplementary methods of investigation, whether they aim at distinction b y physical means (compare TM) or b y analysis of the nucleotide sequence. We should like to express our appreciation to Miss BERTA GANDELMAN AND Mr. LEONARD KERIN for help in some of the experiments.

References p. 405.

VOL. 9 ( 1 9 5 2 )

DESOXYPENTOSE NUCLEIC ACIDS FROM MICROORGANISMS

405

SUMMARY T h e isolation of t h e d e s o x y p e n t o s e nucleic acids of t h r e e m i c r o o r g a n i s m s , viz. Serratia marcescens, a f a c u l t a t i v e l y a u t o t r o p h i c h y d r o g e n o r g a n i s m Bacillus Schatz, a n d Hemophilus influenzae, t y p e c, is described. T h e c o m p o s i t i o n of t h e s e s u b s t a n c e s w i t h r e s p e c t to t h e c o n t e n t s of adenine, g u a n i n e , cytosine, a n d t h y m i n e w a s d e t e r m i n e d ; t h i s led to t h e i r classification in r e g a r d to t h e D N A t y p e s to w h i c h t h e y belong. A p r o c e d u r e for t h e s e p a r a t i o n of t h e p u r i n e s a d e n i n e a n d g u a n i n e f r o m t h e p y r i n i i d i n e s c y t o s i n e a n d t h y m i n e b y a d s o r p t i o n c h r o m a t o g r a p h y on filter p a p e r w i t h w a t e r as t h e s o l v e n t h a s been developed. RI~SUMI~ L e s a u t e u r s d ~ c r i v e n t le m o d e d ' o b t e n t i o n des acides d 6 s o x y p e n t o s e n u c l 6 i q u e s de trois microo r g a n i s m e s , Serratia marcescens, Bacillus Schatz (organisme f a c u l t a t i v e m e n t a u t o t r o p h e ) et Haemophilus influenzae, t y p e c. L a d 6 t e r m i n a t i o n de la t e n e u r de ces s u b s t a n c e s en ad6nine, g u a n i n e , c y t o s i n e et t h y m i n e p e r m e t leur g r o u p e m e n t p a r r a p p o r t a u t y p e d ' a c i d e nucldique a u q u e l elles a p p a r t i e n n e n t . Le proc6d6 de s 6 p a r a t i o n des p u r i n e s (adenine et guanine) des p y r i m i d i n e s (cytosine et t h y m i n e ) en solution a q u e u s e p a r a d s o r p t i o n c h r o m a t o g r a p h i q u e s u r p a p i e r filtre est d6crit. ZUSAMMENFASSUNG Die ]3ereitung d e r D e s o x y p e n t o s e n u k l e i n s g u r e n a u s drei M i k r o b e n s t i i m m e n , n g m l i c h Serratia marceseens, Bacillus Schatz (einem f a k u l t a t i v a u t o t r o p h e n W a s s e r s t o f f o r g a n i s m u s ) u n d Haemophilus influenzae, T y p u s c, wird geschildert. Die q u a n t i t a t i v e B e s t i m m u n g des G e h a l t e s dieser V e r b i n d u n g e n a n Adenin, G u a n i n , C y t o s i n u n d T h y m i n e r m 6 g l i c h t e ihre G r u p p i e r u n g in B e z u g a u f die Nukleins~iuret y p e n , d e n e n sie a n g e h 6 r t e n . Die T r e n n u n g der P u r i n e A d e n i n u n d G u a n i n y o n d e n P y r i m i d i n e n C y t o s i n u n d T h y m i n in rein wiisseriger L 6 s u n g m i t t e l s A d s o r p t i o n s c h r o m a t o g r a p h i e a u f F i l t r i e r p a p i e r wird beschrieben. REFERENCES 1 E. CHARGAFF, S. ZAMENHOF, G. BRAWERMAN, AND L. KERIN, J. Am. Chem. Soe., 72 (195o) 3825. 2 g . VISCHER, S. ZAMENHOF, AND E. CHARGAFF, J. Biol. Chem., 177 (1949) 429. 3 0 . T. AVERY, C. M. MACLEOD, AND M. MCCARTY, J. Exp. Med., 79 (1944) 137. 4 S . E. ALEXANDER AND G. LEIDY, Proc. Soc. Exptl Biol. Med., 73 (195o) 485 • s M. I. BUNTING, E. L. LABRUM, AND J. HEMMERLEY, Am. Naturalist, 85 (1951) 331. 6 M. MCCARTY, J. Gen. Physiol., 29 (1946) I23. ? S. ZAMENHOF AND E. CHARGAFF, J. Biol. Chem., 18o (1949) 727. 8 E. CHARGAFF AND S. ZAMENHOF, J. Biol. Chem., 173 (1948) 327. 9 S. ZAMENHOF AND E. CHARGAFF, Nature, 168 (I95 I) 6o 4. 10 S. ZAMENHOF, G. LEIDY, H. E. ALEXANDER, P. L. FITZGERALD, AND E. CHARGAFF, Arch. Biochem. Biophys., in t h e press. 11 E. CHARGAFF, R. LIPSHITZ, C. GREEN, AND M. E. HODES, J. Biol. Chem., 192 (1951) 223. 12 E. CHARGAFF, R. LIPSHITZ, AND C. GREEN, J. Biol. Chem., 195 (1952) 155. 13 E. VISCHER AND E" CHARGAFF, J. Biol. Chem., 176 (1948) 703 . 14 B. GANDELMAN, S. ZAMENHOF, AND E. CHARGAFF, Biochim. Biophys. Acta, 9 (1952) 39915 E. CHARGAFF, Federation Proc,, IO (1951) 654. 16 E. CHARGAFF,Experientia, 6 (195 o) 2 o i ; J. Cell. and Comp. Physiol., 38, suppl, i, (1951) 41. IT M. M. DALY, V. G. ALLFREY, AND A. E. MIRSKY, J. Gen. Physiol., 33 (195o) 497. 18 j . D. SMITH AND G. R. WYATT, Bioehem. J., 49 (1951) 144. 19 S. ZAMENHOF AND E. CHARGAFF, 9/. Biol. Chem., 186 (195o) 207. Received

July 4th, 1952