Fractionation of the complementary strands of coliphage λ DNA based on the asymmetric distribution of the poly I,G-binding sites

VIROLOGY

32, 633-643 (1967)

Fractionation

of the Complementary

Based on the Asymmetric ZDEXKA MeAdZe

Distribution

HRADECNA

Laboratory,

Strands

Uniuemil~/

of the poly l,G-binding WACLAW

.4ND

of Il’isconsin,

Accepted April

of Coliphage

a DNA Sites’

SZYBALSKI

Jladison,

Wisconsin,

53706

20, 1967

Poly I,G interacts preferentially with one of the two complementary DNAstrands of X and X-related phages 21,434, and @30, thus permit)ting preparative separation of the “dense” fraction, consisting of a complex between the intact strands C and poly I,G, from the less dense (“light”) fraction containing the intact. strands W, which bind 3-4 times less poly 1,G. The isolated and self-annealed fractions are over 99% pure as far as their hybridization properties with complementary RNA are concerned. The interaction of poly 1,G with intact and fragmented DNA of X and its deletion mutants could be interpreted as indicating the asymmetric distribution of poly I,Gbinding deoxycytidine-(dC)-rich clusters between the strands of X DNA; on the left arm (55% G + C) t,he dC-clusters seem to be rest,ricted to the C strand, whereas both complementary strands of the right arm (46% G + C) of h DNA contain these dC clusters. Thus, the two arms of X DNA differ not only in their average base composition (Hershey, 1966), but also in the mode of dist)ribution of the dC-rich clusters, the latter possibly related to the initiation, termination and orientation of the DNA-to-RNA transcription from the complementary DNA strands. INTRODUCTION

(Opara-Hubinski et al., 1964; Kubinski et al., 1966; Sheldrick and Szybalski, 1966)

Preparative separation of the complementary DNA strands is of paramount importance for the detailed study of the

mechanism and orientat’ion of t,he DNAto-RNA transcription process by DNARNA hybridization techniques. No natural bias in buoyant density in a neutral CsCl gradient, similar to that reported for several BuciZZus phages (Cordes et al., 1961; Marmur and Cordes, 1963; Sheldrick and Szybalski, 1966), was detectable for the strands of denatured X DNA. A small density bias observed in an alkaline CsCl gradient (4 mg/ cm3; Hogness et al., 1966) enabled only a marginal and relatively inefficient preparative separation of the complementary strands of coliphage X DXA. On the other hand, it was observed in this laboratory 1 Aided in part by grants from the National Science Foundation (B-14976)) t,he National Cancer Institute (CA-O7175), and the Alexander and Margaret Stewart Trust Fund.

that poly G or poly I ,G have preferential affinity for one of the two strands of many DNA types, including that of phage X. This affinity of single-stranded DNA or guaninerich polyribonucleotides was interpreted as being due to t’he presence of deoxycytidine (dC)-rich sequences (clusters) in DNA (Szybalski et al., 1966). Thus, CsCl density gradient centrifugation of a mixture of denat,ured intact x DNA and poly 1,G results in formahion of two discrete DKA bands, each containing only one of the two complementary strand types. This technique provides an efficient met,hod for preparative fractionation of the complementary DNA strands, and in addit’ion, permits determining the distribution of the poly I,G-binding sites and relating them to the sites of mRNA transcription on the individual DNA strands (Szybalski et al., 1966; Taylor et al., 1967a, b).

633

03-l

III~AI~ECNA

hND

SZYBALSKI

MATERIALS AND METHODS Wisconsin and Dr. G. Kayajanian of the Universit,p of Rochester. Half-molecules of l’urifed concentrated stocks (10’3/ml) of x and X-related coliphages xc1 (xc,~), h DSA were prepared in Dr. A. D. Hershey’s 21, Xizl, Xi434 (Kaiser and Jacob, 1957), laborat’ory in Cold Spring Harbor with the Xcbz, Xbzbs (Kellenberger et al., 1961), and help of Dr. A. Skallca (Hershey, 1966). Analytical and preparat’ive CsCl density@O (Matsushiro et al., 1964) were prepared as described by Thomas and Abelson (1966), gradient ccntrifugat,ion was carried out as described previously using optical or purified using two cycles of high-low speed centrifugation and band sedimentation in a pre- technical grades of CsCl (Szybalski, 1967). formed CsCl gradient. Defective phage Poly I ,G (control No. 158; Miles Laboratories, Elkhart, Indiana) was prepared as an Xdgc,k-,, was obtained by mitomycin inducaqueous st,ock solut,ion (1 mg/ml) and t)ion (1 pg/ml) of the doubly lysogenic stored frozen at -30°C. The pH of all stock strain B304 (X, Xdg,,-,I) (Adler and Templeton, 1963), and separation from the Xf solutions used was adjusted t,o a value of S-8.5. Sarkosyl n’L97 was a gift of the phage in a CsCl gradient (IS hours) by Dr. Geigr Indust’rial Chemicals Corporation, I\‘. Taylor of this laboratory. Phage Xdg(,-,, was prepared in a similar manner by Drs. Ardsiey, New York. A stock solution of Tl 31. Tabaczynski and A. Guha, using t)he RSase (1 mg/ml) (Sankyo Co., Tokyo) was heat,-pret#reated for 10 minut’es at 90°C to singly lysogenic st’rain W3805(X3l+h) (Iiayinact’ivale possible t,races of DNases. ajanian and Campbell, 19G6), and inducing it, with mitomycin and superinfect’ing with RESULTS ANI) 1)18CUHSION Xcbp helper phage. E’igure 1 contains a Release of Phage DNA awl Preparative schematic representation of the genetic and Separation of the Compleinedaq Xt~aurls molecular structure of phage X and several of its deIet)ion and suMitut,ion mutants. Several met,hods for gent)lc release, deThe bact,erial and phage strains were kindl\ proteirlizst,ion, denaturntion, and poly I, Gcontributed h\y Dw. W. F. Dove, G. H. effected strand scpamtion of X DNA n-crc Echols, and J. Adler of the University of evalunt,cd to assure that no singlo-strand W Ab2

83%

Ab2bs

75%

A

hdgCL-J)

SC ABCDEFGHMLKIJ

=III%

C

100%

111%

ABCDEFGHMLKIJ

c,,,No,OP

ABCDEFGHMLKIJ

ABCDEFGHM

b2

k t o _________________--

hd%A-J)

60%

- ----

-

_ -- - - - - - - - - - - - _ 0 ----___--v-m---.. -

QR

0~

5’A CR

c,,,Nc,

OP

QR

c,,,Nc,

OP

QR

flllkl

OP

QR

&~ FIG. 1. Genetic and molecnlar st,ructure of phage h or its point mutants, and its deletioll-sltbstituticJn mutants Xbz (or Xcbz), hb,b: , Xdgca.J, , and Xdgcr,.J, , which were included in this study. The relative molecular sizes are based on the buoyant densities of the phages determined by analytical CsCl densit,y gradient centrifugation [Xcr , 1.508 g/cm3; Xbsbs, 1.483; XdgcA.J, , 1.476; Xdg(L.J) , 1.519; as related to the density of Xcbz equal to 1.491 according to Kellenberger et al. (1961); the buoyant densit,ies of phages X bio, Xdbioc, 01, Xi434, XP, 21, and 480 are 1.510, 1.499, 1.505, 1.501, 1.480, and 1.493 g/cm3, respectively]. The percent figures indicate the lengt,h of the DNA molecules, based 011phage density determinat,ions, and are related to t.he lengt,h of ADNA (loo’%‘,). The letters k, t, e indicate the kinnse, t,ransferase, and epimerase genes of the galactose operon. DNA acquired from h’scherichia coli host is indicat,ed by broken lines. The dotted lines delineate t,he foreign immunity region in phage hbobs The genetic maps are hased on the data of Campbell (1961), Kajayanian and Campbell (1966), Kellenberger et al. (1961), and Adler and Templeton (1963).

SEPARATION

63.5

OF X DNA STRANDS

breaks would be produced by these breatmerits. It was found that t,he simplest and safest, procedure is to combine the DXA release, denaturation, and interaction with poly I ,G in a single step, by exposing a mixture of phage, detergent’, and poly 1,G to a brief thermal treatment, in a solvent of

DROP

NUMBER

101~ ionic strength. Separat’ion of the x DNA into two fractions, each representing OIIC

of t,he complementary

Dn’A

strands,

~:-as accomplished by CsCl density gradient cent’rifugation, a procedure t,hat results also in the removal of proteins which gather at the meniscus of the CsCl gradient. The detailed procedure for the preparative fr:&onation of X DSA strands Ivas as follows. A freshly prepared stock suspension of X phnge, dialyzed during the preceding night against 1V dl EDTA at 2-4”C, was combined in a t,hin-\vall screlvcap tube xvith a stock solution of poly I ,G and 10e3 d;l EDTA to obtain 0.S ml of solution containing a t,ot,al of 150 pg X DiYA and 150 kg of polo- I ,G. To assure more nearly quantitative release of DKA (on t,he average a twofold increase in yield), 2 ~1 of a 30% solution of Sarl
tion of 1 111 KaOH

(usually

3 ~1). This

solut)ion Tvas immersed for 2 minutes in a 95°C bath, immediately chilled in ice and supplement’ed wit’h 0.S ml lop3 X EDTA

and 6.4 ml of a saturated solution of CsCl; the resulting densit’y mas 1.72 g/cm3. Three polyallomer tubes were filled with 2.5ml p&ions of t’his solution, overlayered and b&need wit’h paraffin oil, and centrifuged for SO-70 hours at 30,000 rpm, 5°C in the

SW 391, rotor of the Spinco Model L or I,2 centrifuge.

Four-drop

fractions

lect’ed as described by Szybalski The remaining

solution

were

col-

(1960).

(0.5 ml) was supple-

mented with 0.5 pg of marker D?\TA (usually from Cytophaya joh~zso~~ii, density 1.6945 g/cm3) Spinco 44,770

and banded for 20 hours in bhe Model E analytical centrifuge at rpm, 25°C (Kel-F centerpiece: 2’,

12 mm, or 4”, 3 mm). The results of the analytical and preparative runs are shown in Fig. 2. The two major bands, called C and W, correspond to the two complementary DNA strands. The

BUOYANT

DENSITY


1

FIG. 2. Poly I, G-effected fract,ionation

(separation) of the complementary strands of X phage I>NA in preparative and analytical C&l gradients, according to the procedure described in the text. Upper trace represents the absorbance (260 rnp) of the -l-drop (30 ~1) fractions (total volume 2.5 ml) measured in a 20-J microctlvette (2.mm light path) axing the Perkin & Elmer Rlodel 202 recording spectrophotomet,er. Lower t,race represents the microdensitometric tracing of the photograph of the same undihlted material banded in the analytical ultracerrtrifLlge (-2”, &mm cell) with added density-marker DNA (Qtophaga johnsonii; 1.69&j g/cm”; dashed line). Peak C COW tains the DNA strands C, which preferentially bind poly 1,G; the complementary strands W band under peak W. Symbols dN and NN indicate the positions (densities) of the denatured and native Xcb? l)?JA, respectively.

‘(dense” fraction contains the strands which are characterized by predominance of poly I, G-binding dC-rich clusters (Szybalski et al., 1966) and which will be called C &rands; the complemerkary strands, which will be called W strands, are contained in the “light” (less dense) fraction. A small proportJion of X DKA4 returns to t,he bihelical form during t’he described procedure and forms a band at t’he density of natJive x DNA (Fig. 2, symbol NN). The separately pooled fractions C and W contain the individual strands of X DNA associat,ed with poly 1,G. If required, poly I, G is removed by any of the two following procedures. The fract’ions are dialyzed against SSC (0.15 M SaCI, 0.02 M Na.?. citrate, pH S), and poly 1,G is digested by

636

HRADECNA

AND SZYBALSKI

10 minutes’ incubat)ion at 65°C with 10 units of Tl RNase per milliliter, followed by gentle phenol deproteinization and Sephadex G-200 fractionat’ion. An nlternahive procedure for the removal of poly I ,G consist’s of heating (9O”C, 2 minutes) the pooled W or C fract’ions with an excess of poly C (10 pg/ml); poly C has a preferential affinit’y for poly I,G, and so abolishes its associabion wit,h single-stranded DXA (Kubinski et al., 1966). Jlorcover, poly C has been found not to interact8 wit,h denatured X DiYA in the pH range of 6.5%.0. DSh is separated from t,he poly I ,G-poly C conplexes and from the excess of poly C by preparat’ive CsCl density gradient ccntrifugation or by sediment,ation through a sucrose gradient cont’aining 1 Jl NaCl. Integrity of the X DNA Xtrands The present method of simult’aneous release and denaturation of X DNA does not result in significant strand breakage as assessed by several analytical tests. (I) In neutral 1 11 KaCl, the denatured x DKA sediments as a single band at, SZO,,.= 97 =t 2; t,his value is in good agreement, with t’he sedimentation coefficient for t)he unbroken x DNA strands, as reported by Studier (1965). A similar sedimentation pattern was observed for the separat,ed fractions C and W, wit#h less than 15% of a more slowly sedimenting material being found. (a) Independent evidence for the absence of breaks in the denatured DNA is provided by its banding pattern in CsCl density gradients. A single narrow band is produced by the unbroken denatured DNA, as prepared under presently employed conditions, and banded in the presence of 1 Soformaldehyde (to suppress the cohesion bet’ween the complementary regions), whereas strand breakage results in a bimodal banding pattern of DNA caused by differences in base composition between the left and right arms (Fig. 5). (3) As will be discussed in t’he following sections, the breakage of the C strands, anywhere within the central one-third of the molecule, followed by CsCl gradient cent’rifugation in the presence of poly I,G, results

in appearance of an additional dense band (peak D in trace DEN. DNA + POLY IG; Fig. 3), which corresponds to the left arm of strand C (left, peak in trace B, lcig. 4). Thus, the presence of only two symmetrical bands, as those shown in E’ig. 2, attests to the integrity of &and C’. (4) The integrit,y of W strands was tested by alkaline CsCl density gradient centrifugation (5 ~1 of 1 dl n’nOH/O.;i ml CsCl + lo-” Jl EDTA; 1.76 g/c&; 2’, 12 mm Kel-E’ cell). In such a test, the unbroken W strands were observed to form a single symmetrical band, whereas shearing of strands W results in formation of two bands. It should be point,ed out, however, t,hat’ during centrifugation at pH values above 12.3 (over 10 ~1 of 1 JI NaOH per cell) spontaneous st,rand breakage was observed. (5) The electron microscopy of t#hc W and C’ strands, spread out, on the surface of formamide-cont8aining buffer, provided direct evidence t,hat, over 90 ‘;5 of the singlest,randed DXA molecules have t(he lengt,h of t’he entire X st’rands (Barbara C. W&moreland, personal comm.). Purity of the C and W Strands. There are several lines of evidence that the two fractions correspond to individual DNA st,rands. (1) Fraction C (trace C, Fig. 3) is free of any complementary W strands, since during 4 hours of annealing at 65”C, no renatured DNA is produced, which would have banded at the den&y near to 1.710 g/cm3 (trace RErVAT. C, Fig. 3). Fraction IV (trace W, Fig. 3) is contaminated with the C st,rands, since, upon annealing, lo-20 % of it’ becomes convert,ed to a completely or part’ially renatured material (tract RENAT. W, Fig. 3). A second CsCl densit,y-gradient fract,ionation of such a self-annealed TV fract,ion permits isolation of pure W strands. This second fractionation, however, is not necessary when individual st#rands are prepared for DNA-RNA hybridization cxperimen@ since the self-annealing routine, as described above, converts any contaminating opposit,c strands into double hclices; these do not hybridize with complementary RNA, and thus do not interfere in the hybridization procedure (Taylor et al.,

SEPARATION

OF x DNA

RENATUR. w+c

RENATUR. TOTAL DEN. DNA

I.744 BUOYANT

DENSITY

dN NN / 1.710 1.725 lB94 (G/CM31

FIG. 3. Renaturation analysis of the preparatively separated strands of XCI phage DNA. Trace DEN.DNA + POLY IG represents the banding pat tern of phenol-extracted DNA (2 gg) denatured in the presence of poly I, G (20 rg), and subjected to analytical CsCl density gradient centrifugation under conditions outlined in the text and in the footnotes to Table 1. Banding of the I-pg samples of the preparatively isolated fract,ions W and C, untreated or self-annealed, is represented by traces W, C, RENATUR. W, and RENATUR. C. Trace RENATUR. W + C represents banding pattern of the annealed mixture of W and C fractions, approximately 1 rg of each. Trace RENATUR. TOTAL DEN. DNA represents X DNA, denatured and annealed under identical conditions, but never exposed t.o the poly I,G-effected fractionation procedure. Poly I, G was not removed from the W and C fract,ions used in these experiments. Annealing or self-annealing (symbol RENATUR.)

STRBNDS

637

1967b). Annealing of the eyuimolar mixture of t#he C and W strands results in formation of the renatured DNA (t’races RENATUR. W + C and RENATUR. TOTAL DEN. DNA, Fig. 3.). The particular DNA employed in the experiment illustrated in Fig. 3 was phenol ext,racted from Xc1 phage (a gift, from Dr. A. M. Skalka) and contained a small proport’ion of single-strand breaks. The use of such matserial permits illustrating some banding featuros which are not observed for X D?\‘A free of any single-strand breaks. These undesirable features include the formation of an additional band D (trace DEN. DNA + POLY IG, Fig. 3.), and unequal distribution of DNA between the C and TV bands, as already discussed in the previous section. Also t’he long t,ail on the right side of t,he main peak produced by t,he anrlealed W fraction (trace RENATUR. WV, Fig. 3.) and the peak P represent’ed in t,race RENATUR. W + C (Fig. 3) should be absent in the mat’erial free of singlestrand breaks. (2) The fact t’hat fract,ion C contains less than 0.5 5%of t#hesingle W strands, and thus is more than 99.5% pure, is further confirmed by the lack of hybridization between the C strands and mRnA specific for strand W. Strand W-specific mRNA is produced upon mitomycin induction (Taylor et al., 1967a,b) of the Tll Iysogenic strain, which carries a defective Ml1 prophage with a polar mutat,ion in gene x (Eisen et al., 1966). Over two hundred times more of such mRXA hybridizes with W strands than wit,h C strands (Taylor et al., 1967 b). The self-annealed X DNA fraction W also was carried out for 4 hours, at 65O, and at the concentration of 50 pg DNA per milliliter of 6-7 31 CsCl. The presence of peak D (upper trace), which represents left-arm fragments of strand C (see Figs. 4 and 5, and the text), indicates t)hat the particular phenol-extracted DNA sample contained some single-strand breaks. No such peak is observed for X DNA free of single-strand breaks (see Fig. 2). Symbols dN and NN indicat,e the positions (densit,ies) of the denatured and native Xcr Dh’A’s, respectively. The reasons for the appearance of peak P, which indicates imperfect renaturation between some of the U’ and C strands, are discussed in the text.

63X

FIRADECI\;A

,4NL,

shows practically no capacit(y (200 times less than fraction C) for hybridization wit,h strand C-specific mRNA, which was prepared by prehybridizat’ion of t(he ‘%te” A-mRNA with pure C strands fixed on a nitrocellulose filter, followed by RSase treatment, iodoacetate inactivation of the residual RNase, elution of the strand Cspecific mRNA and it)s final purificat’ion, including DNase treatment, as described by Taylor et al. (196713). (3) When banded in the alka,line CsCl gradient, (5 ~1 of 1 dl XaOH/0.5 ml CsCl + 1O-3 M EDTA; 1.76 g/cm3; 2’, 12-mm KelF cell), the separated fraction W forms a single band, which is 0.0035 g/cm3 denser than fraction C centrifuged under the same conditions, with Cytophaga johusonii DNA serving as a density marker. Thus the “light” strand I and “heavy” strand II isolated by alkaline CsCl gradient centrifugat’ion (Hogness et al., 1966), correspond to t,he C and W strands, respect’ively. Patterns of Poly I ,G Intemction and Fragmented x DNA

with Intact

Although the main purpose of this paper is to present and document’ a pract,ical method for preparative separation of t,he complementary X DNA strands, an at’tempt was also made to evaluate the usefulness of the present technique for the analysis of the distribution of the poly I ,G-binding sites, most probably t’he dC-rich clust.ers (Szybalski et al., 1966), on the individual DNA strands. It was reasoned that the exOent of the poly I,G-effected shift (density incrcment) for the C and W bands should represent a measure of t’he number of dCrich clust,ers per st’andard length of DKL4 molecule. For illustrat,ive purposes let us assume that under the standard experimental conditions (10 pg of poly I ,G/0.5 ml/cell) a density increment of 1 mg/cm3 above the density of poly IG-free denat’ured X DNA, corresponds to one dC-rich c!.lster per one entire single st’rand of X DSA (16 X 10” daltons). It follows that strand C should contain 17 dC-rich clusters since it,s density increases by 17 mg/cm3 in t,he presence of poly I,G. St’rnnd W, which under the same condilions exhibits a densihy

SZYBALSKI

tll--cl.723

I

I .75 pY7D I 1 I I 1 BUOYANT DENSITY (G/CM3

DEN.

I 1

I

I

FIG. -1. The C&l gradient banding pattcrll of the complementary strands of XCI l)NA, (A) elltire molecules (3, whole), (B) “short” left arms (>$ S. left) without the A + T - rich bz region (Figs. 1 and 5), (C) entire right arms (so!id line; 15 h right), and right arms broken into two or more fragments(dotted line;
increment of 5 mg/cm3, should cont,ain five dC-rich clusters. This working hypothesis is illust’rated in Fig. 5, and is based on the numerical data presented in Fig. 4 (trace A) and Table 1 (line 1). The banding patterns for the various fragments of the molecule permit drawing further conclusions concerning the distribut’ion of the dC-rich clusters. There seem t,o be no dC-rich clu&ers on the left half of strand W, since the buoyant density of this fraction is not affect’ed by poly I ,G (trace B, Fig. 4). On the ot’her hand, the shift of 25 mg/cm3 observed for the left, arm of strand C should

639

SEPARATION OF X DNA STRANDS

“LIGHT” W

55% LEFT ARM

42%

ABCDEFGHMLKIJ

b2

*--- mRNA

46% G+C RIGHTARM

5’G

C “DENSE”

a

=III'%~Y=II

Or

.QfJ'

.

5,A

P---p

-------------

mRNA

mRNA

FIG. 5. Tentat,ive

model for the distribution of the dC-rich clusters (-w) on the complementary strands of coliphage A DNA. Symbol C (“DENSE”) indicates the DNA strand which is denser in the poly I,G-containing CsCl gradient than complementary strand 1Y (“LIGHT”). Strand C is less dense in an alkaline CsCl gradient than strand W. The 5’G and 5’A terminals were determined by Wu and Kaiser (1967). The arrows (mRNA) indicate the orientation, the region, and the strand of preference for the DNA-to-RNA transcription as per t,he working hypothesis outlined by Szybalski et al. (1966) compatible with the results of Szybalski el al. (1967), Taylor el al. (1967a, b), and data quoted by Hogness et al. (1966). The genetic map of X is based on the data of Campbell (1961), Kayajanian and Campbell (1966), Eisen et al. (1966), and Kaiser and Jacob (1957).

correspond t’o 25 dC-rich clust’ers per 16 comparison of the banding pattern of Xc1 X lo6 daltons, i.e., to approximately 11-12 and Xcbz DNA’s (Fig. l), the lat’ter having the central bz region (17 % DNA, 42 % G + dC-rich clusters in the Lishort” left half (7 X lo6 daltons) of strand C (Fig. 5). The C) deleted and nevertheless showing a lower poly I,G-effect’ed densit’y shift for its W banding profile of the right arms (46 % G + C) of X DNA indicat’es that each strand of st’rand than for the Xc, st,rand W (Table 1, the right arm cont’ains four t’o five dC-rich lines 1 and 6). Further insight’ into the distribution of clusters, since only one broad band is formed at a density 9 mg/cm3 higher t,han the dC-rich clusters could be derived from the banding pattern of DNA from the that of the denatured right’ arm of DNA (trace C, Fig. 4). Further fragm&ation of defective transducing phage, Xdg,,-,j , which the right arm of X DXA into t’wo quartercarries a part’ of the E. coli genome, including sized fragments results in dissociat#ion of t,he galact’ose operon, as a replacement for t’he single band into two peaks (trace C, t)he 1,-J fragment of the left arm (Fig. 1). dotted line, Fig. 4). One interpretation of In this phage an additional dC-rich cluster these data is presented in Fig. 5: the dC might be present on the W st’rand, since it’s clusters are located on the outer right poly I, G-effect,ed densit,y increase is similar quarter of strand C and on the inner right, or even somewhat higher than t’hat, of quarter of strand W. The discrimination strand W of Xc, phage, although DNA of between t’his and another model, in which Xdg,,-,, appears to be 11 % longer than dC clusters are located on the outer right that of xc1 (Fig. 1). As could be predict#ed, quarter of st’rand W and on the inner right partial or extensive deletion of the left arm quart,er of strand C must’ await further of X DKA result’s in a progressively lowered experiment,s with t,he physically separated density shift for st#rand C of XdgcLeJ, or outer or inner right-quarter molecules, or Xdg(,-,, DNA (Table 1, lines 2 and 3; Fig. with DSA of the appropriate X deletion 4, t’race n). It, should be possible to account8 mutants. Since IIO renat(ured DNA was for both the similarit’y and the difference produced upon annealing of the DSA iso- between the banding patterns of Xdg~,.,,, lated from the dense band (t,rtlce C, dotted and the right arms of X DKL4 (Fig. -1, traces line, Fig. 4) representing t,hese fragments of C and D) by comparing the molecular the right arm which react with poly I,G, struct,ures of these DKA’s (Fig. 1). it is unlikely that the dC-rich clusters are Patternsof I’oly I ,G Interaction with, DNI1 of present on the opposing st’rands of any X-Related Phayes part*icular right’ arm segment’. One of the five dC-rich clusters on st,rand An attempt was also made to determine W is postulated to be located in t,he b, region the DNA banding patt’erns for other tem(Fig. 5). This assignment is based on perate E. coli phages, related to X phage

tra-

Source of phage DNXa

SS”

tIK

AC1

1.7093

1.7245

kkv-.r,

1.7090

1.7249

kb.r J Xc1 right arm entire fragmented XCI “short” left arm

1.7083 1.7OG

1.7230 1.721

tion of poly I,@

C-dN

W-dN

cw

11.1 16.7 18.2 10.9 13.8 11

3.0 5.2 6.3 3.8 5.4 7

8.1 11.0 ll.!) 7.1 8.4 1

(0) 0 2.0 4.G 4.8 2.7 4.3 3.2 6.9 2.5 4.5 2.5 0 .o 2.8 5.9

(1:) 25 8.2 12.1 13.4 10.5 13.8 9.8 13.3 13.1 17.1 8.3 13.1 8.0 9.3

----

I

XCbp

1.715 1.7110

1.729 1.7253

xbzb;

1.7108

1.7208

XP

1.710

1.7241

21

1 .7100~~

1.7250

Xi”34

1.7098

1.7210

1.7121

1.7310; 1.7287;

1.725W 1.7261

1 10 20 1 10 10 10 10 10 1 10 20 1 10 10 1 10 1 10 1 10

9s (16) 25 10.2 16.7 18.2 13.2 18.1 13 0 20.2 15.6 21.9 10.8 19.1 8.5 12.9

11The phages are described in Materials and Methods. Subscripts (L-J) or (A-J) indicate t,he extent of gene delet,ion on the left arm, replaced by the genes of the galactose operon of the host (Fig. 1). h Btmyant density of nat,ive 1)NA (symbol NN) released by 2 minutes’ heat,ing (70°C) of the phage suspension (= 1 rg DNA) in 0.1 ml of 0.2 IV NaCl + 10-X M El)TA in t,he presence of O.lo/, Sarkosyl NL97. Cent,rifugation was performed at, 44,770 rpm (25”C, 20 horn-s) in a 2”, 12.mm cell, after addition of 0.4 ml saturated &Cl, 1 pg of Cgtophaga johnsonii l>NS as a density marker, and adjtlstment of t)he density to approx. 1.71 g/cm3. ” Buoyant density of denat,ured I)NA (symbol dN) released and denatrlred by 2 minutes’ heat.ing (90°C) of the phage srrspension (= 1 rg DNA) in 0.1 ml of 10d3 ,W EI)TA (pH 8.5) in the presence of with 0.4 ml satllrated C&l, and centrifllged 0.2yo Sarkosyl. Samples were chilled (0°C) , srrpplemented as described in footnote b. d I)NA (2 rg per cell) released and denattIred as described in footnote c in the presence of the itldicated amounts of poly I, G (rg/cell/0.5 ml). C-dN, density increment for the denser C band (density of t.he C fraction minus density value dN for the denatured DNA); W-dN, density increment for the less dense W band; C-W, density ditference between t,he C and W fractions (bands). p No perceptible hand separat,ion because of an overlap between peaks (bands). ’ Native 1)NA fragmented by multiple ejection from a spring-loaded syringe (needle gauge 27). Since each band obtained dnring centrifugation in the poly I,G-CsCl gradient most probably contains a mixt.nre of fragments of strands C and W, the figures indicating the density shifts are in parentheses. q This figure dithers from t,hat reported (probably erroneollsly) by Baldwin el al. (1966). h I>enattlred l>NA separates into two bands; the results of two independent experiments indicate some inherent, variability in the natural density bias. For 480 DNA, the density increments were compitted by subtracting the density values for the naturally heavier or lighter fraction of denatured DNA from the densities of band C and W, respectively.

SEPARATION OF X DNA STRANDS (Kaiser and Jacob, 1957; Baldwin et al., 1966). As evident from Table 1, t,he denat#ured DNA of phage 21 shows a somewhat, wider separation between bands C and W (line 9) than Xc, DNA, whereas XP DXA, which contains t#he immunity region of phage 21, exhibits a banding pattern (line 8) iruermediat’e between those of X and 21 DNA’s Similarly, Xb,b, DKA, which has t,he immunity of phage 21 and the bZ region deleted (Fig. l), exhibit’s a banding pattern (line 7) intermediate between Xcbz and 21 DNA. The DSA of Xi434 phage, which differs from X only in t’he cl-r region (Kaiser and Jacob, 1957; Eisen et al., 1966), exhibits a poly I ,G-effect,ed banding pattern (line IO) quite similar to t’hose of X and Xi”’ DKA. The banding pattern of the denat’ured DKA of $80 phage differs from all other “lambdoid” DNA’s, since it forms two bands even in t,he absence of my guaninerich polynucleotides (Table 1, line 11). 1’01~ I, G enhances the separation between t,he C and W bands, although somewhat less effectively t,han for other X and h-like DNA’s t’ested. E,ffect of Other Polyrrucleotides

The effect, of several other guanine-rich polynucleotjides on the banding pattern of denatured X DKA was evaluated. Poly G samples, obt’ained through the courtesy of Dr. RI. Grunberg-Manago (Institut de Biologie Physico-Chimique, Paris), effected a separation between bands C and W similar to that observed wit’h poly I ,G (Miles I,aboratjories, control No. 97 or l;SS), alt’hough band C seemed t,o spread more broadly with poly G than with poly 1,G. A poly G sample purchased from Biopolymers, Inc. (Pinebrook, Sew Jersey) and poly I, G batches supplied after December, 1966, by Miles Laboratories (control 30. 252) or by Schwartz BioResearch, Inc. (I& Ko. 6701; I : G = 1: 2.4), and reported to be prepared by a new method yielding mat~erial of high molecular weight, effect’ed an increase in the buoyant, densit,y of denatured X DKA, but with excessive band spreading and t,herefore hardly any separation between bands C and W. A similarly

641

unfavorable strand separation patstern was observed with poly A, G (1: 1; Miles I,aboratories; cont’rol Ko. 247). Limited alkaline predigestion (Habich et al., 1966) of the Grich polymers improves the strand separation pattern. Poly dG, which was separated from the complementary poly dC strands by the preparative alkaline C&O4 density-gradient cenkifugation (Szybalski, 1967) of a commercial preparation of poly dG .dC (Biopolymers, Inc.), did not interact with &her strand of denat’ured X DNA. ,4 very effect,ive interaction was observed between denatured X DSA and poly U,G (1: 1; Miles Laboratories, control Xo. Z-19). At the same concentrations, poly U,G effected larger density increment’s and wider separation bet,ween t#he C and W bands t,han poly 1,G. For instance, in an experiment similar to that presented in line 6, Table 1, the presence of 1 pg poly U,G/ 0.5 ml cell resuhed in density increments of 29 mg/cn? for krand C (C-dN value), 10 mg/cn? for strand W (W-dN value), and a 19 mg/cm3 separation between bands (C-W value). Using t’his polymer (a-10 pg/O.:i ml cell), it was possible t’o obtain up to 10 mgjcm3 separation bet*ween t,he C and W bands for the phage Xdg,,-,, DKA. By appropriat’e DKA-DSA hybridization tests with the preparatively separated C and W fract~ions, it, was shown by Mr. S. ,IIurata of this laboratory that the dense (+ pal>- U,G) fraction of denatured Xdg,,.,, DKA corresponds to strand C of X DIVA, and t,he less dense fraction to &and W. l’oly U,G was used by Habich et al. (1966) for a CsZS04 density-gradient, separation of non-self-renaturing DXA fractions of Bacillus megateriurn DSA. The density of denatured X DSA increases in the presence of poly U, with all the DNA forming only one band; t,he presence of an excess of poly A abolishes t)he effect’ of poly U (Iiubinski et al., 1966). Similarly, poly C eliminates the effect, of poly G or poly I, G on t’he banding pattern of denat,urcd x DKA. The density incremerks produced by poly U, G are complet,ely abolished by poly C but, are not at’ all affected by poly A. Thus, the irneractions between denatured X D?\TA and poly U,G are basically different from those

(i-l:!

Hl~ADECN.1

AND

CONCLIJSIONS

The complementary st(rands of the X or Xrelated coliphage DNA’s can he effect)ivel:l!, separat’ed by release and gentle heat denat#uration of the DNA in the presence of poly G, poly I ,G, or poly U ,G. The two bands, formed during subsequent C&l density-gradient centrifugat’ion, are set> apart by a density differential of up t’o 20 mg/cm3, and correspond to the two cornplementary DNA strands. These fract8ions were used in a variety of studies--for example, in hybridization experiments bet)ween X DNA and various X-specific mRNA’s where it was found that mRXA produced immediat’ely after infection preferentially hybridized with one (W) of the strands, whereas t’he subsequently produced mRr\‘,4’s hybridized preferentially with t’he ot’hcr (e) DNA strand (Szybalski et al., 1967; Taylor et al., 1967a, b). A comparison of t’he banding patterns for various X DlSA fragments has led t,o a t,ent8at8iveproposal for a model of t#he X DNA, in which t,he poly I,G-binding dCrich clusters are present throughout t,he left arm and on a part of the right arm of the “dense” strand C, whereas only part of the right, arm of the W strand contains such dC clust#ers (Fig. 5). This pattern differs from t#hat observed for unrelat’ed coliphages. For instance, the dC-rich clusters seem to be associat,ed with only one of the DNA strands of T7 phage DNA, since the density of the other st’rand is not affected in the presence of poly I ,G; there is only a very weak interaction between denatured T-even phage DI\-A and poly I, G; whereas a large density increase and separation of denatured DSA into two bands is observed in the presence of poly U (Kubinski et al., 1966; Szybalski et al., 1966). The relationship between the dist#ribution of dC-rich clusters, their possible function in t,he initiation and t,erminat,ion of mRNA synthesis and the orientation of t,he DNA-to-RNA tjranscription (Szybalski et al., 1966, 1967; Taylor et al., 1967b), is discussed elsewhere. ACKNOWLEl>GMENTS The artthors the Uttiversity

are indebted to t.heir colleagttes at of Wiscottsin, especially to l>rs.

SZYBALSKI W. F. Ijove, (;. II. Echols, J. Adler, atttl I). 1’ral.t , :rt~d alsct to their immediate collaborators, Drs. K. Taylor, 11. Tabaczynski. aud A. Grtha for battcrial strains, phage preparations, attd itmtmrcrable discttssions and consult ations; blrs. Barbara C. Westmoreland of Dr. H. Ris’s laborat.ory (I)ept. of Zoology) has kindly permiLted 11s to quote the results of electron-microscopic analysis of our preparat,ion of U’ and C strands. We also thank I)r. (:. Kayajattian of the University of Rochester for the strain of XdgL.1 . The technical aspects of the analyt,ical centzrifngations were skillfully handled by Mr. I). M. Zuhse; to him also we express our thanks. We are deeply grateful to Drs. W. F. Dove, I). Pratt,, and E. II. Szybalski for critical review of the manuscript and for editorial help. The senior author (Z. H.) was on a leave of absence from the Biophysical Institute, Brno, Czechoslovakia. REFERESCES J., and TEMPLEXIN, B. (1963). The atnomlt of galactose genetic material in Xdg bacteriophage with different densities. J. Vol. Hiol. 7, 71&720. B.+LD\IIN, It. L., BM~ILYND, P., FRITSCH, A., GOLDTH~‘MTH, I). A., and JACOH, F. (1966). Cohesive sites on the deoxyribonucleic acids from several temperate coliphages. J. Mol. Riol. Ii, 313-357. CAMI’HELL, A. (1961). Sensitive mutants of bacteriophage X. Virology 14, 22-32. CORDES, S., EPSTEIN, H. T., and X\RMUR, J. (1961). Some properties of t,he deoxyribonucleic acid of phage alpha. Safure 191, 1097-1098. AULER,

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SEPARATION

OF h DNA STRAXDS

genetic properties of selected gal- and gal+ transducing hdg. Virology 30, 482-492. KELLENBERGER,G.,ZICHICHI, M.L.,and WEIGLE, J. (1961). A mutat,ion affecting the DNA content of bacteriophage lambda and its lysogenizing properties. J. Mol. Biol. 3, 399308. KUIIINSKI, H., OPAHL-KUHINSK.\, Z.,and HZYH.ILSBI, W. (1966). Patterns of interaction between polyribonucleotides and individual DNA strands derived from several vertebrates, bacteria and bacteriophages. J. 111ol. Biol. 20, 313-329. MSRMUR, J., and CORDES, S., (1963). Studies on the complementary strands of bacteriophage DNA. In “Informational Molecules” (II. J. Vogel, V. Bryson, and J. 0. Lampen, Eds.) pp. 79-87. Academic Press, New York. MATSUSHIRO, A., &TO, K., and KID.\, S. (1961). Characteristics of the transducing elements of bacteriophage 480. Virology 23, 299-306. OP.4R11-KUBINSK.4, ~.,KUBINSKI, H.,and SZYBBLSKI, w. (1964). Interaction between denatured DNA, polyribonucleotides, and ribosomal RNA: Attempts at preparative separation of the complementary DNA strands. Proc. Natl. Acad. Sci. U.S. 52, 923-930. SHELDRICK. P., and SZYB,~LSKI, W. (1966). Interaction of polyribonucleotides with the complementary st,rands of DNA from certain Bucillus phages. Federation Proc. 25, 707. STUDIER, F. W. (1965). Sedimentation studies of the size and shape of DNA. J. :\lol. Biol. 11, 373-390. SZYB.~LSKI, W. (1960). Sampling of virrls particles and macromolecules sedimented in an equilibrium density gradient. Ezperientia 16, 164.

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SZYB~LSKI, W. (1967). Use of cesium sulfate in equilibrium densit,y gradient cent.rifugation. L In ““Jucleic Acids” (L. Grossmau aud K. Moldave, eds.), “Methods in Enzymology,” Vol. 12. Academic Press, New York (in press). SZ~MLSKI, W., KUBIR-SKI, II., and SHELDRICK, P. (1966). Pyrimidine clust,ers on the trauscribing strand of DNA and their possible role in the initiation of RNA synthesis. Cold Spring Harbor, Symp. Quant. Biol. 31, 123-127. SZYFL~LSKI, W., HRIDECN:~, Z., and TAYLOR, K. (1967). A switch iu the direction of mRNA transcription as related to the distribrltiou of cytosine-rich clusters in coliphage X DNA. dbstr. 7th Intern. Congr. Biochem. Tokyo (in press). T.IYLOH, K., HRADECN.\, Z., and SZYIL~LSKI, W. (1967a). An early switch for transcription of the individual DNA strands in coliphage X. Pederation Proc. 26, 449. T.\YLOR, K., HH.\DECN.\, Z., and SZDALSKI, W. (1967b). Asymmetric distribution of the transcribing regions on the complementary strands of the coliphage x I>NA. Proc. ,$ratZ. Acud. Sci. C.S. 57, 1618-1625. TH~MM, C. A., Jr., and ABELSON, J. (1966). The isolat,ion and charact,erizatiou of I)NA from bacteriophage. Zn “Procedures in Nucleic Acid Research” (G. L. Cantoni and D. R. I)avies, eds.), pp. 553-561. Harper & Row, New York. WI;, R., and KAISER, A. 1). (1967). Mapping the 5’.terminal ullcleotides of the DNA of bacteriophage X and related phages. Proc. X&Z. ilcad. Sci. U.S. 57, 170-177.