Size, number, and distribution of poly G binding sites on the separated DNA strands of coliphage T7

BIOCHIMICAET BIOPHYSICAACTA

371

BBA 95965

SIZE, NUMBER, AND DISTRIBUTION OF POLY G BINDING SITES ON THE SEPARATED DNA STRANDS OF COLIPHAGE TT*

WILLIAM C. SUMMERS** AND WACLAW SZYBALSKI

McArdle Laboratory, Univerisity o/ Wisconson, Madison, Wisc. (U.S.A.) (Received February Igth, 1968) (Revised manuscript received May 6th, 1968)

SUMMARY"

Poly G binds to only one of the two complementary strands of coliphage T 7 DNA, as detected by measuring the buoyant density increments in the CsC1gradient and by trapping of the ESH]polyG.denatured DNA complexes on nitrocellulose membranes. The poly G binding sites on DNA most probably consist of deoxycytidylate-rich clusters, 15-4 ° nucleotides long, as judged from the "melting" temperatures of the complexes and the sedimentation rates of the E3HJpolyG fragments recovered from the ribonuclease Tl-treated complexes. Since the ribonuclease Tl-resistant fraction of the poly G represents 3.3 % of the complex, one could estimate that there are approx. 30-75 poly G binding sites per one T 7 DNA molecule. These sites appear to be evenly distributed along the T 7 DNA, as shown by the patterns of interaction between poly G and fragmented, denatured DNA. As reported earlier by SUMMERS AND SZYBALSKI 3, only the poly G binding ("heavy") DNA strand is transcribed into RNA during all phases of T 7 phage development.

INTRODUCTIO N

It was suggested by SZYBALSKI, KUBINSKIAND SHELDRICK1 that pyrimidine-rich clusters, which were found in all DNA's tested, are related to the initiation and termination regions for RNA transcription. These pyrimidine-rich clusters were inferred from the existence of sites in single-stranded (denatured) DNA which bind certain complementary polyribonucleotides (e.g., poly G) *. We have investigated the nature of this binding reaction for one specific DNA, in order to determine (a) the average size of the binding sites, (b) the number of sites on a DNA molecule, and (c) the distribution of sites between and along the DNA strands. T 7 phage DNA was selected for these experiments since only one of the two strands binds poly G~,3. The experimental results suggest that approx. 30-75 poly G binding sites, each encompassing 15-4o nucleotides, are rather uniformly distributed Abbreviations: SSC, o.15 M ~laCl+o.oI 5 M trisodium citrate (pH 7.6); o.z × SSC, 2 × SSC etc. indicate concentration multiples of SSC; tRNA, transfer RNA. * A preliminary account of this work was presented at the American Chemical Society Meeting, September 14, 1967, Chicago, Ill. (U.S.A.). *~ Present address: Radiobiology Laboratory, Yale University School of Medicine, 333 Cedar Street, New Haven, Conn. o65IO, U.S.A.

Biochim. Biophys. Acta, 166 (1968) 371-378

372

w.c.

SUMMERS, W. SZYBALSKI

along the T 7 DNA molecule. It might be significant that there appears to be a correlation between these data and the number, implicated size and distribution of RNA polymerase binding sites on T 7 DNA as reported by RICHARDSON4, JONES AND BERG5, and SLATER AND HALL s.

MATERIALS AND METHODS

Preparation and purification of coliphage T 7, release of its DNA by heating to 7 °0 in the presence of detergent, and CsC1 density-gradient fractionation of the complementary H and L DNA strands in the presence of guanine-rich ribopolymers (e.g., poly G) was described b y SUMMERS AND SZYBALSKIa. [aH]poly G (2 • IOs counts/rain per #mole) was a gift from Dr. M. GRUNBERG-MANAGO. The binding of [3Hlpoly G to denatured DN'A was measured by two alternative methods. One was based on direct trapping of the [aHJpoly G. denatured DNA complex dissolved in 6 x SSC on nitrocellusose filters, and the other on the isolation of this complex b y preparative CsC1 density gradient centrifugation as described b y SUMMERS AND SZYBALSKI a. In the latter method o.I-ml fractions were collected after 48 h of equilibrium centrifugation, their absorbance (260 m/z) was determined, and then the radioactivity was assayed in a liquid scintillator in two ways (Fig. I). One 2o-/,1 aliquot was counted directly, whereas another was added to 5 ml of 6 x SSC and slowly filtered through a nitrocellulose filter (Schleicher and Schuell Co., B6, 25 ram), which was then counted. Both methods gave practically identical specific activity measurements for the complex, when appropriate background and efficiency corrections were made.

RESULTS AND DISCUSSION

The results presented in Fig. I permit the quantitative determination of the amount of poly G bound to one of the T 7 DNA strands. It is also possible to show that the buoyant density increase is a linear function of [3Hlpoly G binding (Fig. 2). This justifies previous assumptions ~,8 that the amount of polynucleotide binding is proportional to the density increase of the complex. Furthermore, from this data one can roughly extrapolate the buoyant density of E3H]poly G in the CsC1 gradient (i.e., when the per cent E3HJpoly G in the complex reaches lOO%) to be about 1.95 g/cm a. The stability of this poly G. denatured DNA complex was examined under a variety of conditions. Subfractions (5 #1, each containing o.15 #g DNA) from the "heavy" (H) peak were diluted at 20 ° into 2 ml of solutions of different ionic strengths, then made to 5 ml of 6 x SSC by adding appropriate volumes of io x SSC and subsequently filtered through nitrocellulose to measure the amount of EaH]poly G retained in the complex. At 2o ° the complex was unstable in o.195 M Na + (SSC) but was completely stable in 0.585 N[ Na + (3 × SSC). It was reported by KUBINSKI, OPARA-KUBINSKA AND SZYBALSKI2 and confirmed by our experiments that addition of poly C or formaldehyde prevents complex formation and can even dissociate the complex once formed. However, denatured DNA subsequently mixed with the isolated complex (2/zg denatured DNA/2.8 #g complex/o.5 ml CsC1 solution of density Biochim. Biophys. Acta, 166 (1968) 371-378

NATURE OF d C - R I C H CLUSTERS IN

T7

DNA

373

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Fig. 1. F r a c t i o n a t i o n of t h e c o m p l e m e n t a r y s t r a n d s of coliphage T 7 D N A in t h e p r e p a r a t i v e CsC1 d e n s i t y g r a d i e n t in t h e p r e s e n c e of [SH]poly G. H e a t - d e n a t u r e d T 7 D N A (80 Fg) w a s c o m b i n e d w i t h [SH]poly G (4° / ~ g ) in 7.7 M CsC1 (2. 5 ml) a n d c e n t r i f u g e d for 48 h a t 3 ° ooo r e v . / m i n , io ° in t h e S W - 3 9 r o t o r of t h e Spinco L-2 u l t r a c e n t r i f u g e . F r a c t i o n s (ioo #1) were collected f r o m t h e b o t t o m of t h e t u b e *x a n d t h e i r a b s o r b a n c e (26o m # , I e r a ) d e t e r m i n e d . T h e a b s o r b a n c e c u r v e ( Q - O ) w a s c o r r e c t e d for t h e s k e w e d b a c k g r o u n d c a u s e d b y t h e a b s o r b a n c e of u n b o u n d [3H~p o l y G, as d e t e r m i n e d f r o m a parallel a n a l y t i c a l CsC1 d e n s i t y g r a d i e n t run. T h e r a d i o a c t i v i t y of t h e f r a c t i o n s ( A... A) w a s a s s a y e d as described in MATERIALS AND METHODS. T h e b u o y a n t d e n s i t i e s of t h e b a n d s were d e t e r m i n e d in a parallel a n a l y t i c a l r u n , u s i n g 0.2 m l of t h e s a m e CsC1 solution in a c e n t r i f u g e cell w i t h a 3 m m light p a t h a n d a bihelical reference D N A (Cytophaga johnsonii D N A , 1.694 g/cmS). T h e s y m b o l s H a n d L i n d i c a t e t h e " h e a v y " a n d " l i g h t " s t r a n d fractions, respectively. P o l y G s e e m s to b i n d to o n l y one (H) of t h e two D N A s t r a n d s . Fig. 2. T h e b u o y a n t d e n s i t y i n c r e m e n t (density increase over t h a t of d e n a t u r e d T 7 D N A , 1.723 g/cm 3) as a f u n c t i o n of t h e a m o u n t of b o u n d ? H ] p o l y G (expressed as wt. % of t h e poly G . d e n a t u r e d D N A c o m p l e x ) . T h e a m o u n t b o u n d w a s c a l c u l a t e d b y u s i n g t h e specific a c t i v i t y of t h e [SH]poly G a n d t h e r a d i o a c t i v e c o u n t of t h e f r a c t i o n s f r o m t h e CsC1 g r a d i e n t (as in Fig. I) to estim a t e t h e q u a n t i t y of [3H]poly G in t h e fraction, a n d u s i n g t h e a b s o r b a n c e (26o m/u) of t h e fraction (c/. Fig. i) to e s t i m a t e t h e t o t a l a m o u n t of nucleic acids (i.e., p o l y G . d e n a t u r e d D N A complex) in t h e fraction. T h e v a r i o u s s y m b o l s i n d i c a t e t h r e e i n d e p e n d e n t e x p e r i m e n t s .

1.7642 g/cmz) was not able to compete for poly G molecules already bound to the denatured DNA, since the added DNA banded at the density of denatured T 7 DNA (1.723 g/cm3) and the density of the complex remained unchanged (1.7636 g/cm3). Schematically these results may be summarized: poly G. denatured DNA+poly C ~ poly G. poly C+denatured DNA poly G. denatured DNA+2 HCHO -~ poly G-HCHO+denatured DNA-HCHO poly G. denatured DNA m +denatured DNA(21 t - ~ 1/2 poly G. denatured DNA m + 1/2 poly G. denatured DNA(2I These observations suggest that the complex is hydrogen-bonded, most probably by Watson-Crick base pairing, as symbolized by colons. An estimate of the size of the sites was obtained from thermal stability data (Fig. 3). NIYOGI AND THOMAS9 measured hybrid formation between enzymatically synthesized T7 RNA fragments of uniform length and denatured T 7 DNA at various temperatures in 5 × SSC (0.98 M Na+), and suggested that a perfect homology of at least 11-14 bases is required for hybrid stability in 5 × SSC between 3°0 and 60 °. The present study was done in 3 x SSC (0.585 M Na+), in which hybrids would be expected to be only slightly less stable 1°. Moreover, there probably exist regions of imperfect homology within the poly G. denatured DNA complex (i.e., bases other Biochim. Biophys. Acta, 166 (1968) 371-378

374

W. C. SUMMERS, W. SZYBALSKY

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Fig. 3. T h e r m a l s t a b i l i t y of t h e [ S H ] p o l y G . d e n a t u r e d D N A c o m p l e x ( F r a c t i o n io, Fig. I ) in 3 × SSC (0.585 M Na+). T h e s a m p l e , c o n t a i n i n g 1. 5 ~ug of t h e c o m p l e x in 2.2 m l 3 × SSC, w a s h e a t e d (3 ° p e r min), a n d o.2-ml a l i q u o t s were w i t h d r a w n a t v a r i o u s t e m p e r a t u r e s a n d d i l u t e d into 5 m l of 6 x SSC a t 2o °. T h e s e " q u e n c h e d " s a m p l e s were slowly filtered t h r o u g h nitrocellulose filters, w h i c h b i n d t h e d e n a t u r e d D N A a n d t h e c o m p l e x , a n d w h i c h were t h e n a s s a y e d for radioa c t i v i t y in a t o l u e n e ~ l i p h e n y l o x a z o l e liquid scintillator. A p p r o x . 8o % of t h e [SH]poly G a p p e a r s to dissociate f r o m t h e d e n a t u r e d D N A in t h e 3 o - 8 o ° t e m p a r a t u r e range, w i t h t h e m i d p o i n t , T m = 52°. N o corrections were m a d e for non-specific t r a p p i n g of u n b o u n d [3H]poly G b y t h e filters, w h i c h u s u a l l y a m o u n t e d to a b o u t 16 % in t h i s a n d a n a l o g o u s e x p e r i m e n t s .

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Fig. 4. Zone s e d i m e n t a t i o n 14 of [3H]poly G f r a g m e n t s r e s i s t a n t to ribonuclease T 1 b y v i r t u e of b e i n g c o m p l e x e d w i t h d e n a t u r e d T 7 D N A . C o m p l e x of d e n a t u r e d T 7 DIKA a n d [SH]poly G w a s isolated f r o m a p r e p a r a t i v e CsC1 d e n s i t y g r a d i e n t (c]. Fig. I, F r a c t i o n s 7-8) a n d t h e n t r e a t e d w i t h ioo u n i t s of r i b o n u c l e a s e T 1 p e r m l of 3 × SSC for 3 h a t 22 °. R i b o n u c l e a s e T 1 w a s t h e n i n a c t i v a t e d w i t h i o d o a c e t a t e 18. O n e a l i q u o t w a s s e d i m e n t e d directly ( O - - - O ), while a n o t h e r a l i q u o t w a s first dissociated b y h e a t i n g a t 95 ° for 3 rain w i t h i % f o r m a l d e h y d e ([N-V]).These s a m p l e s were t h e n l a y e r e d over CsC1 (I.3O g ] c m 3) a n d s e d i m e n t e d a t 35 ooo r e v . / m i n for 25 h in a SW-39 r o t o r in a Spinco L-2 u l t r a c e n t r i f u g e . Control s a m p l e s c o n t a i n e d E. coli t R N A , [3H]poly G, a n d GMP. T h e m i d p o i n t s of t h e p o s i t i o n s of t h e s e r e f e r e n c e s are indicated. F r a c t i o n s were collected a n d c o u n t e d in A N P O scintillator s0 for a t i m e sufficient to r e d u c e t h e s t a t i s t i c a l c o u n t i n g error to less t h a n 3 %. Fig. 5- A s y m p t o t i c l i m i t of r i b o n u c l e a s e T x d i g e s t i o n of a c o m p l e x of d e n a t u r e d T 7 D N A a n d [SH]poly G ( F r a c t i o n 13; Fig. i). T h e p e r c e n t of t h e u n d i g e s t e d c o n t r o l s a m p l e is p l o t t e d vs. I/t of d i g e s t i o n (min -1 • ioo) for long d i g e s t i o n times. A limit of 34, w h i c h is e s t i m a t e d f r o m t h e diag r a m , w a s f u r t h e r corrected b y s u b t r a c t i o n of 16 % of t h e digested [SH]poly G (i.e., 16 % × 66 % = lO.6 %) to a c c o u n t for non-specific t r a p p i n g of d i g e s t e d [3H]poly G o n t h e nitrocellulose filters. T h i s a m o u n t (i6 % of t h e total bound [SH]poly G) of non-specific t r a p p i n g w a s d e t e r m i n e d f r o m s e p a r a t e c o n t r o l e x p e r i m e n t s ; a similar f r a c t i o n of t h e [SH]poly G w a s non-specifically t r a p p e d b y t h e B6 filters d u r i n g filtration of t h e r m a l l y dissociated (above 9o °) [SH]poly G . d e n a t u r e d D N A c o m p l e x e s (see Fig. 3). To d e t e r m i n e t h e f r a c t i o n of t h e c o m p l e x t h a t w a s r e s i s t a n t to r i b o n u c l e a s e T 1 t h e corrected l i m i t of d i g e s t i o n (23. 4 %) w a s m u l t i p l i e d b y t h e f r a c t i o n of t h e u n d i g e s t e d c o m p l e x t h a t w a s [SH]poly G (14 % a s d e t e r m i n e d for F r a c t i o n I3, Fig. I, u s i n g t h e relation in Fig. 2). I n t h i s way, it w a s e s t i m a t e d t h a t a b o u t 3.3 % of t h e c o m p l e x r e p r e s e n t s t h a t p o l y G f r a c t i o n w h i c h is r e s i s t a n t to d i g e s t i o n b y r i b o n u c l e a s e T 1.

Biochim. Biophys. Acta, 166 (1968) 371-378

NATURE OF dC-RICH CLUSTERS IN T 7 D N A

375

than cytosine scattered along the binding site). These two considerations, together with the results of N'IYOGIAND THOMAS9, suggest that a reasonable lower limit on the size of the binding region appears to be about 15 nucleotides. The observed melting (Fig. 3) was a result of poly G. denatured DNA dissociation rather than poly G. poly G disaggregation since the latter complex is shown to have a very high (> 95 °) "melting" temperature l°,n. Even guanylic oligonucleotides containing only 5-12 nucleotides exhibited thermal transition at temperatures as high as 75° in o.oi M sodium phosphate buffer at pH 7 (ref. 12). Sedimentation analyses of the poly G used in the present experiments, which were performed in 7 M urea in an attempt to diminish aggregation, yielded an average S2o,~value of 3, which as discussed below very roughly indicates a polyribonucleotide with about 44 bases. This determines a trivial upper limit on the size of the binding sites for each molecule of poly G. Another approach permitting an estimation of the sizes of the binding sites was based on measuring the length of the poly G fragments which are rendered resistant to ribonuclease T 1 (EC 2.7.7.26; Worthington Chemical Corp.) by virtue of forming a complex with the denatured DNA. The preparatively isolated complex was treated with ribonuclease T1 under conditions assuring complete digestion of all poly G not hydrogen-bonded to denatured DNA (see Figs. 4 and 5). Ribonuclease T1 was then inactivated with o.15 IV[iodoacetateTM, the complex dissociated (3 rain, 95 °, 1% formaldehyde to prevent aggregation of poly G fragments), and the [~Hlpoly G fragments subjected to sedimentation analysis in a self-generating CsC1 gradient14 (1.3 g/cms CsC1, 25 h, 35 ooo rev./min, Spinco SW39 rotor), employing 4-S tRNA as a reference. These fragments, which sedimented with an average s20,w=2.1 S, formed a broad and asymmetric band skewed toward low molecular weights (Fig. 4). Assuming that the 4-S tRNA contains 76 nucleotides and that the sedimentation constant is proportional to the 0.5 power of the molecular weight, the average ribonuclease T1protected E3Hlpoly G fragment was 20 nucleotides long. This result suggests that the poly G binding sites in T 7 DNA are shorter than the [3H]poly G used in these experiments, since on the average only half of each poly G molecule is protected by binding to denatured DNA. Thus, the deoxycytidylate-richclusters in DNA appear to contain an average of about 20 nucleotides, which estimate is in fair agreement with the sizes estimated from the thermal stability of the complexes. An estimate of the total number of bases in all the binding sites was made by treating the isolated complex with ribonuclease T1 to cleave those guanine residues not protected by hydrogen bonding to denatured DNA and then measuring the remaining bound [3H]poly G by trapping the complex on nitrocellulose filters. The complex was diluted in 6 ml of 3 × SSC (Fraction 13, Fig. I) and treated with 50 units per ml of ribonuclease T1 at 24% Aliquots were removed at o (ribonuclease T1free control), 7, 17, 48, 90 and 15o rain, diluted to 5 ml in 6 × SSC and filtered through prewashed B6 filters, which were then exhaustively washed with 6 × SSC, dried, and assayed for radioactivity. The asymptotic limit of ribonuclease T1 digestion was obtained from a linear plot (Fig. 5) of per cent of control (undigested) counts vs. I/t (t = time of reaction). This limit is considered to represent primarily that portion of the E3H]poly G which was protected from ribonuclease T1, most probably by its intimate association with the DNA strands (bihelical configuration?), but also some contaminating ESH]polyG fragments non-specifically trapped on the filter. The latter count, which usually amounted to 15-2o% of the total detached FSH]poly G (as Biochim. Biophys. Acta, 166 (1968) 371-378

376

w.c.

SUMMERS, W. SZYBALSKY

determined in separate control experiments in which the amount of ribonuclease Ttdigested [SH]poly G trapped on the B6 filters was measured), was subtracted from the total count retained by the B6 filters after ribonuclease T 1 digestion. The data obtained in the above experiment and corrected in this manner showed that ribonuclease Tl-resistant [aH]poly G accounts for about 3.3% of the complex. This corresponds to about 115o nucleotides. Division of this number by the approximate size of each site (i.e., 15-4 ° nucleotides) gives an estimate of about 30-75 poly G binding sites per T 7 DNA molecule (mol.wt. = 25 •Ios; ref. 15). This estimate might be on the high side, since there is no assurance that ribonuclease T 1 removed all the [~HIpoly G which was not in perfect hybrid configuration with the complementary deoxycytidine sequences in the DNA. To determine the distribution of the poly G binding sites along the length of the T 7 genome, bihelical T 7 DNA was sonicated prior to denaturation and complex formation. Although the DNA was fragmented into eighths (Table I), the amount of TABLE I BINDING OF POLY C~ TO INTACT AND FRAGMENTED DENATURED T 7 DNA Molecular w e i g h t e s t i m a t e d f r o m b o u n d a r y s e d i m e n t a t i o n v e l o c i t y u n d e r c o n d i t i o n s p r e v i o u s l y r e p o r t e d (ref. 20). ~dN is t h e i n c r e a s e in b u o y a n t d e n s i t y (CsCI) a b o v e t h a t of d e n a t u r e d T 7 D N A , c a u s e d b y poly G binding. H : L is t h e ratio of t h e a m o u n t of D N A w h i c h b i n d s p o l y G (H fraction) to t h a t w h i c h does n o t (L fraction), as j u d g e d b y t h e a r e a s u n d e r t h e c u r v e s f r o m a n a l y t i c a l CsC1 d e n s i t y g r a d i e n t p y c n o g r a m s , w h i c h were m e a s u r e d b y resolution into G a u s s i a n c o m p o n e n t s w i t h a D u P o n t 31o C u r v e Resolver. DNA

Mol. wt. × zo -s

OdN(mg/cm ~)

H :L

T7 D N A , i n t a c t , d e n a t u r e d T7 D N A , sonicated, d e n a t u r e d

12. 5 1. 5

14.2 13.9

49 : 51 48 : 5z

poly G bound per unit length of denatured DN'A was unchanged, as indicated b y the constancy of the density increment. In addition, the ratio of the amounts of DNA in the two fractions (H and L) remained I : I. This indicates that no significant dissociation (cleavage) of DNA with binding sites from DNA without binding sites occurred. It may be concluded that the binding regions are rather evenly distributed along the whole of the T 7 DNA molecule. This contrasts with coliphage 2 DNA, where it was shown that the poly G binding clusters are unevenly distributed s.

CONCLUSIONS

The observations reported here show that there are approximately 30-75 sites, evenly distributed along the H strand of the T 7 DNA molecule, which are capable of binding poly G. These regions are 15-4o nucleotides long and most probably are rich in cytosine residues. However, it would be very difficult to determine the exact size of these deoxycytidylate clusters until they are isolated and the exact base sequence determined, since the presence of bases other than cytosine within such clusters might strongly affect the ribonuclease T 1 sensitively and thermal dissociation of the poly Gdenatured DNA complexes. The correlation of poly G binding with mRNA transcripBiochim. Biophys. Acta, 166 (1968) 3 7 1 - 3 7 8

NATURE OF dC-RICH CLUSTERS IN

T7

DNA

377

tion has been previously reported in coliphages T 3 (ref. 3),T5 (ref. 16), T 7 (ref. 3), 2 (refs. I, 8, 13 I7), Bacillus subtilis phage SP82 (ref. 18) and in B. megaterium TM. The physical data presented here on the poly G binding, deoxycytidylate-rich clusters (i.e., their size, number and distribution) show a striking correlation with the biochemical4,5 and electron microscopical6 studies, which indicated that T 7 DNA contained 35-75 binding sites for RNA polymerase. Perhaps this relationship is more than fortuitous. It should not be overlooked, however, that several additional conditions might have to be met to permit a successful initiation in vivo of transcription at every RNA polymerase binding site. Thus, the number of binding sites might certainly be higher than the actual number of operons. Whatever the role of the poly G binding sites, they represent novel structural singularities in the bihelical DNA, since the stacking forces between the purines, all accumulated on one strand, confer upon DNA new structural properties 1. Thus, such sites could act as recognition points for various regulatory and synthesizing entities. Moreover, highly specific binding sites for as many as 64 various proteins or other macromolecules could be provided by 64 permutations of nucleotide triplets when their unique specificity depends on their position within or next to the deoxycytidylate clusters. With all the various sequences composed of only four nucleotides which are obligatorily coupled to two kinds of structural singularities (e.g., deoxycytidylate and deoxythymidylate clusters) over 500 specific recognition sites in bihelical DNA can be provided for initiating, terminating and binding functions for various entities, including repressors, activators, polymerases, specific nucleases (e.g., those opening the circular DNA of coliphage 2), or ribosomes 1,2,22. These and other biological and molecular implications, posed by the presence of the structural singularities in all naturally occurring DNA's (refs. I, 2, 3, 8, I8), were and will be discussed elsewhere (refs. I, 2, 22).

ACKNOWLEDGEMENTS

We are greatly indebted to Dr. M. GRUNBERG-MANAGOof the Institut de Biologie Physico-Chimique, Paris for the sample of [3H~poly G. We are also grateful to Mr. D. M. ZUI~SE for handling the analytical ultracentrifugation and to Dr. E. H. SZYBALSKIfor editorial help. This work was supported by grants from the National Cancer Institute (Cao7175 ), the National Science Foundation (B-I4976), and the Alexander and Margaret Stewart Trust Fund. The stipend of the senior author (W.C.S.) was provided by Training Grant ToI Ca 5002 from the National Institutes of Health, U.S. Public Health Service. REFERENCES I W. SZYBALSKI, H. KUBINSKI AND P. SHELDRICK, Cold Spring Harbor Symp. Quant. Biol., 31 (1966) 123 . 2 H. KUBINSKI, Z. OPARA-KUBINSK& AND W. SZYBALSKI, J. Mol. Biol., 20 (1966) 313 . 3 W. C. SUMMERS AND W. SZYBALSKI, Virology, 34 (1968) 94 J. P- RICHARDSON, J. Mol. Biol., 21 (1966) 83. 5 0 . W. JONES AND P. B~RG, J. Mol. Biol., 22 (1966) 199. 6 H. S. SLATER AND C. E. HALL, J. Mol. Biol., 21 (1966) 113.

Biochim. Biophys. Acta, 166 (I968) 371-378

378 7 8 9 IO II 12 13 14 15 16 17 18 19 20 2I

22 23

W. C. SUMMERS, W. SZYBALSKY

Z. OPARA-KUBINSKA,H. KUBINSKI AND W . SZYBALSKI,Proc. Natl. Acad. Sci. U.S., 52 (1964) 923. Z. HRADECNA AND W. SZYBALSKI, Virology, 32 (1967) 633. S. K. NIYOGI AND C. A. THOMAS, Jr., Biochem. Biophys. Res. Commun. 26 (1967) 51. W . SZYBALSKI,in A. H. R o s E , Thermobiology, A c a d e m i c Press, L o n d o n , 1967, p. 73. A. M. MICHELSON, J. MASSOULI~ AND W. GUSCHLBAUER,Progress in Nucleic Acid Research and Molecular Biology, Vol. 6, A c a d e m i c Press, N e w York, 1967, p. 83. H. HAYASHI AND F. EGAMI, J. Biochem. T o k y o , 53 (1963) 176. K. TAYLOR, Z. HRADECNA AND W . SZYBALSKI, Proc. Natl. Acad. Sci. U.S., 57 (1967) 1618. J. VINOGRAD, R. BRUNER, R. KENT a n d J. WEIGLE, Proc. Natl. Acad. Sci. U.S., 49 (1963) 9o2. S. B. LEIGHTON AND I. RUBINSTEIN, Biophys. Soc. Abstr., (1968) T A I 4. Y. T. LANNI AND W. SZYBALSKI,in p r e p a r a t i o n . S. N. COHEN AND J. HURWlTZ, Proc. Natl. Acad. Sci. U.S., 57 (1967) 1759P. SHELDRICK AND W. SZYBALSKI, J. Mol. Biol., 29 (1967) 217. A. HABICH, C. WEISSMANN, M. LIBONATI AND R. C. WARNER, J. Mol. Biol., 21 (1966) 255. W . C. SUMMERS AND W . SZYBALSKI, J. Mol. Biol., 26 (1967) lO 7. W. SZYBALSKI,Experientia, i6 (196o) x64. W . F. D o v E , Ann. Rev. Genetics, 2 (1968) in press. B. WEISS AND C. C. RICHARDSON, J. Mol. Biol., 23 (1967) 4o5 .

NOTE ADDED IN PROOF

(Received July 3° , 1968 )

In analogy with the presently accepted nomenclature for the complementary strands of coliphage ~l DNA we suggest that the poly G binding strand H of phage T 7 should be named r (transcribed to the right) and the other less dense strand L should be named l (non-transcribed or potentially transcribed to the left), and the T 7 DNA molecule be oriented as indicated in Fig. 6, with the 5'-pTpC terminus on the left and the 5'-pApG terminus on the right 23. L STRAND l 5' pTpCp

pCpT 3'

DNA STRAND r 3' ApGp . . . . . . . . . . . . . . . . . . . . . . H

pGpAp 5'

mRNA

Fig. 6. S c h e m a t i c r e p r e s e n t a t i o n of c o l i p h a g e T 7 D N A ( h e a v y lines) a n d of m e s s e n g e r R N A (open arrow), w h i c h is t r a n s c r i b e d f r o m t h e r (H) s t r a n d . T h e k n o b s o n t h e r - s t r a n d s y m b o l i z e t h e p o l y G-binding, c y t o s i n e - r i c h clusters. T h e 5 ' - t e r m i n a l d i n a c l e o t i d e s o n t h e l a n d r s t r a n d s were d e t e r m i n e d b y W E i s s AND RICHARDSONza.

Biochim. Biophys. Acta, 166 (1968) 371-378