J. Mol. Biol. (1975) 92, 261-277

Mapping and Characterization of Promoters in Bacteriophages fd, fl and Ml3 PETER H. SEEBURG AND HEINZ

Max-Plunck-Institut

SCHALLER

fiir Virusforschung, Tiibingen, Germany and

Lehrstuhl fi.ir Mikrobiologie der Universitdt Heidelberg 6900 Heidelberg, Im NeuenheimerFebd 280, Germany (Received 16 July 1974, and in revisedform 31 October1974) The RF? DNAs of bacteriophages fd, f 1 and Ml3 are cleaved by nuclease Hpa II at 13 sites. As compared to fd RF DNA, f 1 and Ml3 RF DNA have one cleavage site in a different position. DNA fragments were ordered by using fragments as primers for the ia vitro synthesis of their neighbours. The resulting physical map was correlated with the genetic map by marker rescue experiments with amber mutant phage DNAs and purified wild-type DNA fragments (Hutchison et al., 1971). RNA polymerase binding sites were detected in six of the 13 Hpa II fragments. Each site promotes the synthesis of RNA, with four species being initiated by GTP and two by ATP. The sites display different affinities for the enzyme and are distributed throughout the genome. A site that is occupied preferentially by the enzyme is located at the distal end of gene II near a unique site on the genome where all phage RNA species seem to be terminated. It promotes the synthesis of RNA from a region of the genome that is expressed very actively ilz uivo. In view of these findings, a mechanism for constitutive transcriptional control is proposed.

1. Introduction Interaction of RNA polymerase with promoter regions on a DNA template is a necessary event for any specific RNA chain initiation leading to correct gene expression, and possibly also to initiation of DNA replication. The filamentous Escherichia coli phagesfd, fl and Ml3 (for review seeMarvin & Hohn, 1969) provide a model system for studying the functional and structural aspectsof this interaction. In this system the frequency of initiation at the phage promoters seemsnot to be influenced by interaction with regulatory proteins other than host RNA polymerase itself. Furthermore, the small size of the phage genome (approximately 6060 bases) facilitates a structural analysis of regulatory regions. Experiments with fd RFT DNA and E. coli RNA polymerase in vitro have so far led to the isolation and characterization of several unique binding sites for the enzyme (Okamoto et d., 1972; Heyden et al., 1972). These sites specify the initiation of several RNA species(Heyden, 1974) that have already been characterized with respect to size and initiation sequence(Okamoto et al., 1969; Takanami et al., 1970). Control of transcription would be better understood if positions and interactions t Abbreviations

used: RF DNA, replicative form DNA. 261

262

P. H. SEEBURG

AND

H. SCHALLER

of the promoters on the phage genome would be determined. To this end we have dissected the genome into unique fragments, using two bacterial restriction endonucleases. The fragments were analysed binding sites and were arranged on a physical

for their

content

of RNA

polymerase

and genetic map of the phage DNAs.

2. Materials and Methods (a) Chemicals and enzymes Ribonucleoside and deoxyribonucleoside triphosphates were from Boehringer Mannheim GmbH, Msnnheim. [u-~~P]TTP was from New England Nuclear Company, Chicago. Carrier-free 32P0,3was purchased from Buchler, Braunschweig. Acrylamide, N,N’methylene bis acrylamide and N,N,N’,N’-tetramethylethylenediamine were from Serva Feinbiochemioa, Heidelberg, Polyethyleneglycol (mol. wt 6000) was from Roth, Karlsruhe. Dithiothreitol was from Calbiochem. Nitrocellulose filters (pore size 0.15 pm) were from Sartorius, Gdttingen. Calf thymus DNA type V was purchased from Sigma, St Louis. E. coli DNA polymerase I (fraction 7) was prepared according to Jovin et al. (1969). E. ooli RNA polymerase was prepared by Ch. Niisslein (Niisslein & Heyden, 1972). The enzyme was electrophoretically at least 95% pure and contained stoichiometrical amounts of u-factor. Endonuclease Hpa II was prepared and enzyme activity was monitored according to Sharp et al. (1973), using fd RF DNA as a substrate: 1 ~1 of the final preparation cleaved 1 pg fd RF DNA (O-25 pmol RF DNA rings) to completion in 1 h at 37°C; 1 ~1 of this preparation released less than 0.2% acid-soluble material from 0.25 pg single-stranded fd DNA when incubated for 12 h at 37°C. The preparation contained trace amounts of endonuolease Hpa I. It did not unspecifically nick double-stranded DNA as determined by polyacrylamide gel electrophoresis of fd RF DNA fragments in the presence of formamide. Endonuclease Hind II was a gift of H. Tabak: 2 ~1 of this preparation cleaved 0.5 pg DNA in 1 h at 37°C. Pancreatic DNAase (electrophoretically pure DNA&se I) was purchased from Worthington, Freehold, N.J. (b) Bacterial straim, phage strain8 and preparatiolz of phage DNA Haemophilus parain&enzae was obtained from E. Kopecka. E. wli strains C600 and K37 SuI+ were from A. Klein and T. J. Henry, respectively. The following phage strains were used: phage fd amber mutants fd22 (gene I), fd150 (gene II), fdlll (gene III), fd9, fdl0, fd12, fd13 (gene IV), fd122 (gene V or VII). Phage Ml3 amber mutants 5.H27 (gene V), 6-Hl (gene VI) and S-H1 (gene l’111). Phage fl mutants R21 and R86, both amber in gene 17, carrying the respective markers IIs and llso. The fd, Ml3 and f 1 strains were kindly provided by D. Marvin, T. Henry and P. Model, respectively. All amber mutants were grown on E. coli K37 SuI. Reversion rates of these mutants ranged from lo-* to lo-*. Phage DNA and 32P-labelled phage DNA was prepared as described by Marvin & Schaller (1966) with the modification that phage was purified by 2 successive precipitations with 4% and 2% polyethyleneglycol in 0.5 M-N&I (Yamamoto et al., 1970). DNA was obtained from purified phage by extraction with phenol. L&balled synthetic RF DNA was prepared according to Goulian & Kornberg (1967) as modified by Heyden (1970). (c) Fragmentation of double-stranded phage DNA by restriction endonudease A typical reaction mixture (100 ~1) contained 5% glycerol, 0.02 M-Tris.HCl (pH 8.0), O-01 M-MgCI,, 0.005 M-KC& 0.001 M-dithiothreitol (cleavage buffer), 10 pg phage RF DNA and 5 ~1 Hpa II. Occasionally 20 ~1 Hind II were included. When Hind II was used, the concentration of KC1 was 0.05 M. The mixtures were incubated at 37°C for 2 h and the reaction was stopped by the addition of EDTA (final concentration 0.02 Ed).

PROMOTERS

IN

PHAGES

fd,

fl

AND

Ml3

263

(d) Polyacrylamide gel electrophoresis For gel electrophoresis, samples of digested DNA were made 0.2% in sodium dodecyl sulphate, 20% in sucrose and O*l”/o in bromophenol blue. Electrophoresis was carried out in (1) discontinuous polyacrylamide gel slabs (14 cm x 10 cm x 0.2 cm) filled to a height of 5 cm with a 12% aqueous acrylamide solution containing 20% sucrose and topped by 5 cm of an aqueous 4.5% acrylamide solution, in electrophoresis buffer consisting of 0.04 MTris- acetate (pH 8*0), 0.001 M-EDTA. For gel electrophoresis under denaturing conditions, discontinuous (3%, 10%) or continuous (12%) slab gels were poured in the presence of, and the eleotrophoresis buffer contained, 7 M-urea. (2) Polyacrylamide gel columns (10 cm x 0.6 cm) of 45% gels in eleotrophoresis buffer. Gels contained 0.2% iV,N,N’,N’, tetramethylethylenediamine, 0.1% ammonium persulphate. Gels were pre-run for at least 0.5 h at 1 mA per gel column and 5 mA per gel slab. Electrophoresis was at 4 mA per column and 30 mA per slab. Slab gels were used for autoradiographio analysis of 3al?labelled DNA or RNA. Gels were dried as described by Maize1 (1971) and placed on DNA fragments were Kodak X-ray film (Kodirex). Mixtures of 32P and 3H-labelled analysed on gel columns. After electrophoresis these gels were sliced into 1 mm segments. Each slice was incubated for 2 h at 40°C in 0.5 ml of a solution containing 0.01 M-Tris*HCl (pH S-O), 0.01 M-MgCl,, 1 mM-Cd&, 0.1 m-NaCl and 5 pg pancreatic DNAase. With this treatment, recovery of 3H radioactivity was over 80% and of 32P radioactivity over 95%. For preparative separation of RF DNA fragments, gel columns (18 cm x 0.6 cm) were loaded with LIP to 100 pg 32P-labelled RF DNA (about lo4 cts/min per pg) and submitted to electrophoresis for up to 15 h. Gels were sliced, Cerenkov radiation was determined and peak segments were pooled. Judging from the radioactivity profiles, the separation of fd RF DNA fra-gents B,C (cross contamination up to 20%) and D,E (cross contamination up to 30%) was incomplete. DNA was eluted from gel segments for 24 h at 4°C by the addition of 200 ~1 0.01 M-Tris.HCl (pH 8-O), 10e4 M-EDTA per gel slice. Recovery of DNA fragments from gel slices was between 40% and SO%, increasing with decreasing fragment size. Eluates were concentrated by rotary evaporation, desalted and freed from gel remnants by passage over columns of Sephadex G50 (8 cm x O-4 cm) in 0.005 M-Tris*HCl (pH 8*0), 5 x lo-* M-EDTA. Pool fractions were again concentrated by rotary evaporation and either directly used for hybridization or stored at - 20°C. (e) Hybridization of RF DNA fragments to phage DNA viral strands Hybridization mixtures (50 ~1) contained 0.02 M-Tris*HCl (pH 8*0), 2 x 10m4 M-EDTA, 0.1 M-NaCl, 1 pmol purified wild-type fd RF DNA fragment and 3 pmol circular singlestranded wild-type or mutant phage DNA. The mixtures were heated to 100°C for 5 min and incubated for 1 h at 55°C. (f) Fragment-primed DNA synthesis DNA synthesis was routinely carried out in 0.05 M-Tris.HCl (pH 75), O-1 M-NaCl, 0.01 M-MgCl,, 5 x 10e4 M-EDTA. A lo-p1 standard reaction mixture contained in addition: 10 pmol [32P]TTP (lo4 to lo5 cts/min), 1 nmol each unlabelled dATP, dGTP, dCTP, 0.01 pmol primer template DNA hybrid and 0.02 unit DNA poIymerase I. Label incorporation was allowed to proceed at 15°C for 2 to 25 min. Then 1 nmol unlabelled TTP in 1 ~1 was added to the mixture and incubation continued for 5 min at 30°C. Synthesis was stopped by the addition of 1 ~1 O-2 M-EDTA and DNA polymerase was inactivated by heating at 65°C for 5 min. For Hpa II cleavage of the DNA product, the mixture was diluted with 90 ~1 cleavage buffer (see section (c), above) containing 1 pg 3H-labelled fd RF DNA (5000 to 10,000 cts/min), and 1 ~1 Hpa II. The reaction was allowed to proceed for 2 h at 37% and was stopped by the addition of EDTA to a final concentration of 0.02 M. (g) Bioassay E. coli C600 was infected with phage DNA using a variation of the procedure of Mandel & Higa (1970) as modified by Taketo (1972) and by Cohen et al. (1972). One ~1 of a hybridization mixture containing, with respect to the wild-type Hpa fragment, 0.02 pmol

264

P. H.

SEEBURG

AND

H.

SCHALLER

heteroduplex DNA (see section (e), above) was diluted with 50 ~1 0.01 M-Tris*HCl (pH S-O), 0.05 M-CaCl,. Then 0.8 0.D.650 unit of an E. ooli cell suspension in 0.1 ml 0.03 M-CaCI, was added at 0°C. The mixture was inoubated at 0°C for 15 min, subjected to a heat pulse at 40°C for 2 min, chilled and plated directly on nutrient agar together with 0.1 ml of an overnight culture of E. co& Hfr 3300 in 2 ml soft agar. Plates were kept at 37°C for 6 h and were scored for infective centres. In this test system the efficiency of transfection as calculated by the number of infective centres divided by the number of input molecules ranged from lo-’ to 10-s for single-stranded phage DNA and from 10W5 to 1O-6 for RF DNA.

RNA polymerase

(h)

binding

to DNA fragmennts

Reaction mixtures (10 ~1) contained 5% glycerol, 0.02 M-Tris*HCl (pH 8*0), 0.01 MMgCl,, 10Y4 M-EDTA, 10m4 M-dithiothreitol (binding buffer), and either 0.04 M or 0.12 M KCl, O-1 pmol Hpa II-digested 32P-labelled RF DNA (approximately 2 x lo5 &s/mm) and between O-1 and 2 pmol RNA polymerase holoenzyme. Incubation was at 37°C for 10 min. The binding reaction was stopped by the addition of 5 pg unlabelled denatured calf-thymus DNA in 1 ~1 water and incubation was continued for 10 min. The reaction mixtures were diluted by the addition of 90 ~1 binding buffer prewarmed to 37°C and containing 0.04 MKCl. The mixtures were filtered by gentle suction through cellulose nitrate filters (Jones & Berg, 1966) of 6 mm diameter, which had been prewashed with binding buffer. After filtration, filters were washed with O-2 ml binding buffer and retained DNA was eluted by immersing filters in 40 ~1 O-01 nn-Tris*HCl (pH S-O), 0.2% Sarkosyl (Ciba, Geigy), at 0%. Samples of the eluates were electrophoresed as described. (i)

RNA

chain initiation

on

DNA fragments

RNA polymerase was bound to DNA fragments as described in section (h), above. The unlabelled competitor DNA was added together with the 4 ribonuoleoside triphosphates (to 100 PM each), which doubled the volume of the reaction mixtures. To assay for A-starts, ATP was 100 PM and GTP 1 pM. To assay for G-starts, GTP was 100 PM and ATP 1 PM. RNA synthesis was allowed to proceed for 1 min at 37°C and was stopped by the addition of EDTA to 0.02 M. The incubation mixtures were chilled to 0°C and KC1 was added to a final concentration of 0.25 M. Exposure to these conditions was for 30 min. Analysis of complexed DNA is described above.

(j) Sylzthesis and analysis

of RNA from Hpafragments

RNA polymerase was complexed with 0.2 pmol Hpa II-digested unlabelled fd RF DNA in 20 ~1 binding buffer containing 0.12 M-KCl. Molar ratio of enzyme/RF was 1.5 or 10. After 10 min at 37”C, RNA synthesis was started by the addition of 20 ~1 of this buffer containing (each at a concentration of 200 pM) GTP, UTP, CTP and [32P]ATP (500 cts/min per pmol) and 20 pg rifampicin/ml. Incubation was continued for 30 min at 37°C and synthesis was stopped by the addition of EDTA to 0.02 M. Samples (10 ~1) of the reaction mixtures were diluted with 30 ~1 formamide containing 30% sucrose, 0.2% Sarkosyl, 0.1 y0 bromophenol blue, heated at 90°C for 2 min, quickly cooled in ice, and applied to a discontinuous 3%, 10% gel slab containing 7 M-urea. Samples were electrophoresed at room temperature and the gel was dried for radioautography. 32P-labelled Hpa II-cleaved fd RF DNA, heat-denatured in formamide was run in parallel to serve as a size standard for the RNA products.

3. Results (a) Speci$e cleavage of the RF DNA of phages fcl, fl and

Ml3

Before the enzymic cleavage, the single-stranded phage DNAs were converted to their double-stranded form (RF DNA) by in vitro DNA synthesis (Heyden, 1974). Restriction endonucleaseHpa II, which has been reported to cleave Ml3 RF DNA into at Ieast nine fragments (Tabak et al., 1974), was chosen for the production of a

PROMOTERS

IN

PHAGES

fd,

fl

AND

Ml3

265

relatively high number of specific fragments from the phage genomes. Radioactively labelled phage RF DNAs were incubated with Hpa II and the digests were analysed by electrophoresis in polyacrylamide gels. As shown in Plate I, limit digests were each resolved into 12 bands. For each band a comparison of label content to electrophoretic mobility indicated the presence of one fragment except in one case. This band contained the two fragments Hpa-J and Hpa-K of fd RF DNA and Hpa-K and Hpa-L of fl and Ml3 RF DNA. Thus, Hpa II produces 13 fragments from each of the RF DNAs. It should be noted that the cleavage patterns of Ml3 and fl RF DNA are identical. The differences from the cleavage pattern of fd RF DNA originate from one additional and one missing scission: Hpa II cleaves the f 1 analogue of fd fragment Hpa-E, yielding the fl fragments Hpa-G and Hpa-I and cuts within the fd analogue of f 1 fragment Hpa-E, resulting in the appearance of the fd fragments Hpa-F and Hpa-M. TABLE

Chain Hpa

fragment A B C D E F G H I J K L M

1

lengths of fd RP DNA Radioactivity per fragment 25.0&0+3 13.3f0.7 12.7&O-7 9*7-&0.5 9.4f0.5 7Y5&0*6 7*1*0*5 7*0*0*5 2*6+0*3 2*0*0.2 2.OAO.2 1.010.2 0.850.2

(%)

fragments Chain length in base-pairs 1500 800 780 590 570 450 410 400 150 120 120 60 50

Figures represent average values obtained from analysis of 5 Hpa II-fragmented fd RF DNA preparations, uniformly Iabelled with 32P in the viral strand. Fragment size was calculated from the relative percentage radioactivity recovered from the respective gel bands by assuming a total of 6000 base-pairs per RF DNA.

The sizes of the cleavage products by Hpa II were calculated on the basis of the relative amount of radioactivity recovered from the gel bands by assuming a total of 6000 base-pairsper RF DNA. The sizesof the fd RF DNA fragments are listed in Table 1. Restriction endonucleaseHap II from Haemwphilus aphrophilus has been shown to produce a set of fragments from fd RF DNA (Takanami, 1973) identical to that listed in Table 1, suggesting an identical recognition sequence for endonucleasesHpa II and Hap II. This suggestion has been further substantiated by sequence analysis of their DNA cleavage sites (Sugisaki & Takanami, 1973; Garfin & Goodman, 1974). (lo) Construction of a physical map offa, fI and Ml3 RF DNA To determine the original arrangement of the Hpa II fragments, neighbourhood relations among fd RF DNA fragments were studied using fragment-primed DNA synthesis directed by E. coli DNA polymerase I.

266

P.

H.

SEEBURG

AND

H.

SCHALLER

A set of template-primer DNAs was constructed by denaturing and reannealing purified fd RF DNA fragments in the presence of circular full-length fd DNA viral strands. By offering 32P-labelled deoxyribonucleoside triphosphates for short pulse periods followed by a chase of unlabelled deoxyribonucleoside triphosphates, DNA polymerase I was made to synthesize specifically started DNA chains, which were 32P-labelled only in regions close to the 3’ end of the primer. The products of DNA synthesis were cleaved by Hpa II and the resulting digests were analysed in polyacrylamide gels, using [3H]TMP-labelled fd RF DNA fragments as internal marker. To demonstrate the validity of this procedure, radioactivity profiles of DNA primed by fragment Hpa-A and pulse-labelled for different times (5 and 15 min) are shown in Figure 1. In both profiles the primer fragment itself (peak 1) contained 32Pradioactivity indicating that DNA polymerase I had incorporated deoxyribonucleoside monophosphates into the primer fragment. This was also found with other primers and is suggestive of repair synthesis at single-strand breaks in these fragments. Beside peak 1, 32P radioactivity from the product of five minutes of pulse-labelling was detected only in peak 3 (Hpa-D and Hpa-E), whereas after 15 minutes of pulselabelling, peaks 3 and 2 (Hpa-B and Hpa-C), and possibly also 5 (Hpa-G) and 7 (Hpa-I), contained 32P label. The position of 32P label relative to 3H label within the composite peaks 2 and 3 suggests that Hpa-B and Hpa-E are the respective

I

5

IO

15 Slice

20

I’

45

50

number

FIG. 1. Polacrylamide gel electrophoresis of products of fragment-primed DNA radioactivity profiles are from DNA primed by fragment Hpa-A and pulse-labelled (b) 15 min. DNA was cleaved by Hpa II and dectrophoresed on 4.5% polyaorylamide with W-labelled fd RF DNA fragments as internal markers. Numbers indicate activity. --O-O---, 3H radioactivity; -- l -- @ --, 32P radioactivity.

synthesis. szP for (a) 6 and gel columns peaks of radio-

P----y

IL.---F-.-“:+--“--G-----j---Ii/ /

PLATE I. Radioautogram of 32P-labelled fd, fl, and Ml3 RF DNA II. DNA fragments were resolved on a discontinuous polyacrylamide concentration. Arrow indicates origin. The gel was dried and exposed are designated by capital letters in order of fragment size.

i---

fragments produced gel slab of 4.5% to X-ray film. Hpa

by Hpa and 12% fragments

[facing p. 266

PLATE II. Interaction of RNA polymerme with phage RF DNA fragments. (a) fd RF DNA fra@nents that form stable complexes wxth RNA polymerase at 0.04 &r-KU1 (a, b) and 0.12 iv-KC1 f&RF DNA fragments and polymer&se, stable to competition by unlabelled denatured onlf thymns I>NR, wmn tra.pped eluted and electrophoresed on dmoontinuous gel slabs with complete Hpa digest marker (M). Molar ratios of enzyme/RF (h) fd RF l)NA fmgmcnts containing RNA polymarnso initiation niton. After complex format,ion at, a molar rat,io of 0.04 M-KC1 (f, g, i, j) and 0.12 ~-Kc1 (11) the 4 ribonucleoside triphosphates (g and h), ATP, G’I’P, c”I’P and UTP, 100 and IJTP (100 ,LM onch); j, ATP (1 @x), GTP, (:TP rind TJTP (100 @VI onch) wove a. ddr?d together with unlabelled, denatured side triphosphates were omitted frmn f. incubation was for 1 min at 37°C. RNA synthesis was sluppad by Lhe addition at 0°C (O-25 M-KCI) for 30 min. Analysis of complexed DNA was as described under (a). (c) fl DNA fragmenls Lhal lorm stable complexes with RNA polyrrlerase at 0.04 M-KC1 (11) and 0.12 RI-KC1 (1, m, n). DNA was as described under (a). Molar ratios of enzyme/RF DNA were k, 5; 1, 2; xx, 5; n, 10.

Isolation

and

analysis

of complnx~d

(c, d, e). Complexes of ~“P-l~b~&d cm nitrocellnlone filters. DNA was DNA were a, 2; b, b; c, 2; d, 5; e, 10. RNA polymerase/RF DNA of 12 in @I eaoh; i, GTP (1 pm), ATP, CTP calf thymus DNA. Ribonucleoof EDTA. The mixtures were kept

0.0

o-o o-o 9.8 0.0

0.9 0.0 0-c: 0.0 0.9 8.0

0.0 0.0 9-P 0.0 0.0

Z 3-Z’dH

9.0 0.0 6.0

93

91 R-“dH

9.1

8.1

P.0

0.0

0.0 0.2 O-0 0+x 0.0 0.0

L-0

9.0

o-0

0.2 P.0 o-0 6573 0.0 o-0

L*l:

0.0

0.91 o-0 0% 0.0 o-0 0.0

0.0

0.0

0.0

0.0 0.0

0.0

Z I-BdH

8.9 0.0 0.0 0.0 6.0 o-o

9-Z 0.8 o-0 O-E o-0 0.0

0.0 9.0 0.0 vo 0.0 8.Z

0.0 0.0 0.0 0.0 0.0 9-z

0.2 0.0 z-z 0.0 8-Z 0.0

6.P 0.0 O-1 0.0 P-E 0.0

0.0 Z-0 o-0 0.0 0.0 0.9

0.0 0.0 0.0 0.0 VP o-o

0.6 0.0 9.2 0.0 0.9 O-O

6.Z

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0.0

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0.0

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9.1

0.0 0.0

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o-0 6.0

0.0

9

9.0 E-O

0.0

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o-=+x

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268

P.

H.

SEEBURG

AND

H.

SCHALLER

fragments labelled by Hpa-A-primed DNA synthesis. Thus Hpa fragments B,E,G and I were generated by Hpa-A-primed synthesis and, therefore, encompass a eontiguous part of the fd genome. Such sets of contiguous fragments were also obtained by priming with other fd RF DNA fragments. In order to establish the correct arrangement of the fragments within each set, the respective gel profiles were quantitated by calculating a relative value for each fragment based on the 32P radioactivity content and fragment size (see Table 2). For each pulse period these values gradually declined with increasing distance of the fragments from their respective primer, reflecting non-synchrony of DNA synthesis. Arranging Hpa-A-primed fragments by this decline yielded the order of Hpa-A + Hpa-E -+ Hpa-B -+ Hpa-(G,I) in the 5’ + 3’ direction of the complementary strand. A- +E

-+B(C) +I,G E +B B +I -+G,J(K) I -+G +C(B) -c-J(K) -G+C C - -+J(K),D

J(K)

+F D - +F

aH +J(K),H F -+J(K) J(K)

+H +H

+D H -+A,E -

FIU. 2. Overlapping groups of fd RF DNA fragments. The order of the fragments within each group is based on the decrease of the relative percentage of 3zP label cakulated for these fragments (see Table 2). Primer fragments are underlined. Arrows indicate the direction of DNA synthesis (6’ -t 3’ of the complementary strand). Fragment symbols in parentheses or separated by a comma denote alternative fragments or alternative fragm.ent order. The fragment groups are arranged in overlapping order. The resulting arrangement of all fragments is shown at the bottom. The order of the symbols J and K is arbitrary.

In the same manner, the fragments within other sets were ordered, and the sets were aligned as shown in Figure 2. Ambiguities in some of the secondary and tertiary overlaps were observed, which could be attributed both to impurities in the primers (see Materials & Methods) and errors due to gel analysis. In these cases the primary overlaps alone were used to obtain the correct arrangement. Knowledge of nearest neighbour was sufficient throughout to order all the fragments. The small fragments Hpa-L and Hpa-M, which were not analysed in the experiment described in Table 2, have since been located between Hpa-F and Hpa-K by using similar procedures (data not shown). The arrangement of fd RF DNA fragments served as a guide for ordering the fragments from fl and Ml3 RF DNA, using the unique cleavage site by restriction endonuclease Hind II (Tabak et al., 1974; Takanami, 1973) as a point of reference. On scission with Hind II, fd fragment Hpa-F (450 base-pairs) is split into two hagments of 280 and 170 base pairs, whereas f 1 and Ml3 fragment Hpa-E (500 base-pairs) yields two fragments of 280 and 220 base-pairs. Since Hpa-M (50 base-pairs) is contiguous to Hpa-F in fd RF DNA and its analogue is missing from the Hpa II

PROMOTERS

IN

PHAGES

fd,

fl

AND

Ml3

269

cleavage products of fl and Ml3 RF DNA, the appearance of fl and Ml3 fragment Hpa-E is best explained by a missing Hpa II cleavage site in this region of fl and Ml3 RF DNA:

M I I

$

, I

F + 280 : Hind lI I 220 i 280 E 170

I ’

fd

, I

fl,M

I3

Conversely, Hpa II cuts the fl and Ml3 analogue of fd fragment Hpa-E into two pieces, namely Hpa-P and Hpa-I, the order of which has not been determined. The physical maps of the three phage RF DNAs are shown in Figure 3. A cleavage map identical to that shown in Figure 3 has been obtained for fd RF DNA by Takanami (1973) and for Ml3 RF DNA by Hondel $ Schoenmakers (personal communications) with the use of a different method to order the phage DNA fragments produced by endonuclease Hpa II. (c) Correlation of the physical map with the genetic map Each Hpa II fragment represents a unique part of the phage genome. This genome is known to code for at least eight gene products, whose molecular weights and functions have been characterized in some cases (Henry & Pratt, 1969; Mode1 t Zinder, 1971). To determine the genetic origin of the fragments, we tested their ability to rescuemarkers in each gene. The method used (Hutchison & Edgell, 19’71) involved annealing one strand of the DNA fragments from wild-type fd RF DNA to single-stranded circular DNAs of the phagesfd, fl and M13, bearing amber mutations as genetic markers. These DNA hybrids were used to infect E. coli cells treated with CaC1,(Mandel & Higa, 1970) and production of wild-type phage indicated the presence of the wild-type allele in the fragment. The results of the bioassy are given in Table 3. The yield of wild-type phage varied in a reproducible manner with different hybrids. Each mutant could be rescued by a unique wild-type fd DNA fragment. Reflecting their incomplete separation on gels, fragments Hpa-D and Hpa-E were both found to rescue the same genetic marker in gene I, with Hpa-E displaying a threefold activity over Hpa-D. Hpa-D also specified the wild-type allele to a marker carried by mutant fd 12.2,whereas Hpa-E showed only little activity in its rescue. Mutant fd 122 lies either in gene V or in gene VII (D. Marvin, personal communication). Fragment Hpa-C rescuedmarkers in both geneIII and gene VIII, indicating that these genesoccupy a contiguous region on the genetic map. A distribution of genetic markers along the physical map was obtained that allowed the arrangement of all the known phage genes, with the possible exception of gene VII. If fd 122 is a mutant in gene VII, its rescue by Hpa-D indicates that this gene is located between gene V and gene VIII (seeFig. 3), in keeping with the arrangement of phage fl genesobtained by Lyons & Zinder (1972). Recently a gene order of VIII, VII, V has been inferred from experiments similar to ours, using phage Ml3 (Hondel & Schoenmakers, persona1 communication). Such a gene order is compatible with our results, provided fd 122 is amber in gene V.

130

166

-

0

-

190 0 -

fdl0

90 0 0 0 -

fd9

IV

0

180

-

240 0 -

fd12

0

220

-

340 0 0 0 -

fd13

I

520

220 2

4 600 1 0 -

fd22

VI

460

-

0 270 9 -

6-Hl

III

96

1 2

2 3 2 70 5

fdlll

VIII

12

0 0

0 0 1 15 0

SRI

V

1

3

70

-

1 60

-

6-H27

0

9

85

76 -

-

-

155

0 120 3 140 140 2

-

2 1 0 0

fd150

II

0

110

130 2 140 135 1

-

-

RS6

0

146

136 1 190 160 6

-

-

R21

A portion (0.8 o.D.~~~ unit) of a cell suspension of E. coli C600 cells treated with CaCl, were infected with 0.05 pmol DNA hybrid constructed from wild-type fd RF DNA fragments and circular viral amber mutant DNA. Infection mixtures were plated together with 4. coZi Hfr 3300 as indicator strain and the plates were scored for infective centres. The figures listed are the mean values of results compiled from at least 2 assays. The deviation of scored figures from listed values was in some oases as high as 50%.

Fragmented fd RF DNA

Fragment Hpa-A Hpa-E Hpa-B Hpa-C Hpa-J, K Hpa-D Hpa-F, G, H Hpa-G, H Hpa-F Kin Hpa-PI Hin Hpa-Fa

Gene Mutant

3

Bioassay

TABLE

PROMOTERS Fractional length

0.3 I

0.2 I

IN

0.1

PHAGES

0 I

I

0.9 I

fd,

0.8

fl

AND

0.7 I

I

Ml3

0.6 I

271

0.5 I

0.4 I

0.3

c HindlI

fl,M13RFDNA fragment I

A

fd RF DNA fragment I

I

H LM III

I

HKLMiF II Ill,,

A

!E 1 I I ’ I I

D

I I

D

K I I

C

J I I

C

GJ

GI I

B I

I

I II

I

(F

I)

t

B

A

I,

E 1

A I

I I

FIG. 3. Correlation of physical map with genetic map. The genetic map was aligned with the physical map by genetic marker rescue with wild-type fd RF DNA fragments. IIzo and II,, are mapped markers in gene II and were salvaged by Hin Hpa-F,. The sizes of genes were obtained by converting the sizes of gene products to nuoleotide lengths. Genes VI and VII, the products of which are unknown, are represented by 150 nuoleotides each. As to the location of gene VII see text.

Map

units

are given

as distahoe

from

Hind

II cleavage

site per length

of phage

DNA:

O-1 unit equals 600 nuoleotides.

In order to correlate the genetic and physical map in terms of nucleotides, both maps were drawn to scale in a linear fashion (seeFig. 3) on the basisof the calculated fragment lengths and the published molecular weights of the products of genesI, II,

III, IV, V and VIII; molecular weights 35,000, 40,000, 68,000, 50,000, 10,000, and 5,200, respectively. If all viral DNA codesfor protein and if the total coding capacity is a molecular weight of protein of 220,000, there remains a molecular weight of 12,060, corresponding to approximately 300 nucleotides, for the unknown products of genes VI and VII. Each of these genesis represented in Figure 3 by 150 nucleotides. The lack of information concerning the exact number and size of phage genesand intercistronic regions, markers constituted congruency between the region of gene II (mutant R86) were

together with the paucity of accurately mapped genetic inherent ditliculties in precisely correlating both maps. Good the physical and genetic map could be obtained, however, in and of gene V. The de6ned markers II,, (mutant R21) and II,, rescued by the larger of the two fragments (Hin Hpa-F,)

generated by Hind II scissionof fd fragment Hpa-F. This fragment covers approximately 300 nucleotides, which is also the distance separating the two markers (Model & Zinder, 1974). The small fragment Hpa-J was found to rescue a marker in the small gene V. Although the alignment of the genetic and physical map aspresented lacks accuracy in regions other than of genes V and II, it provides a suitable basis for analysing functions of the genome. (d) Promoters on the genomeof the phagesfd, fl and Ml3 DNA molecules with specific RNA polymerase initiation regions (promoters) are known to form stable promoter complexes with RNA polymerase in the absenceof ribonucleoside triphosphates (Hinkle & Chamberlin, 1970). Making use of this fact, a grossdistribution of such regions on the genome of the phages fd, fl and Ml3 was

272

P. H. SEEBURG

AND

H. SCHALLER

determined by analysisng the Hpa II-produced phage DNA fragments for their ability to stably bind RNA polymerase. DNA complexed with the enzyme was isolated by retention on nitrocellulose filters (seeMaterials and Methods) and, after elution, was resolved by gel electrophoresis to yield the fragment profiles shown in Plate II. It will be noted that the number and type of fragments present in these profiles were dependent on ionic conditions of binding and molar ratios of RNA polymerase/ RF DNA, indicating intrinsically different affinities of the several sites for the enzyme. Up to six fragments from each of the RF DNAs formed stable complexes with polymerase at low ionic strength (O-04 M-KCI). These fragments were for fd RF DNA: Hpa-A, -B, 4, -D, -F, -H and for Ml3 and fl RF DNA: Hpa-A, -B, -C, -D, -E, -H. The same sets of fragments were obtained when complex formation preceded fragmentation of the RF DNAs, implying that fragment ends did not interfere with the specificity of the binding reaction and that the sites were intact after scission. On raising the ionic strength of the binding buffer (to 0.12 M-KC%), only fragments Hpa-A, -B, -D, to someextent Hpa-C, and the fl and Ml3 fragment Hpa-E, could be detected in the corresponding gel profiles. Of these, Hpa-D was preferentially bound by polymerase at low molar ratios of enzyme/RF DNA. These findings are consistent with an estimate of the number of RNA polymerase binding sites as calculated from the fraction of polymerase-protected, nuclease-resistant fd RF DNA. The equivalent of approximately five (Okamoto et al., 1972) and three (Heyden et al., 1972) sites was protected in low salt and high salt, respectively. Low molar ratios of enzyme/RF DNA in the presence of high salt resulted in the protection of a single site (Heyden et al., 1972). This site lies on fragment Hpa-D, as indicated by the preferential binding of polymerase to this fragment under similar conditions. It is of interest to note the striking difference in polymerase binding by the analogous fragments Hpa-F (fd) and Hpa-E (fl, M13) (seePlate II). Whereas Hpa-F (fd) is stably bound by the enzyme at low salt only and with a low efficiency, enzyme complexes with Hpa-E (fl, M13) are obtained even at high salt. This was found after saturation with polymerase and regardless of whether the binding occurred before or after the fragmentation of RF DNA. Our failure to obtain the complexes in equimolar yields suggestsa phage-specific difference in the structure of the nucleotide regions involved in polymerase complex formation. In fact, a diverse nucleotide sequenceis found at the fd-specific Hpa II cleavage site in map position O-03. Two findings suggest a position of the polymerase binding site near this cleavage site. When fl fragment Hpa-E is cleaved with Hind II, only Hin Hpa-E,, the smaller of the two pieces generated is bound by the enzyme (data not shown). Furthermore, the absence of a particular GTP-initiated RNA specieson prior Hind II cleavage of the fd RF DNA template (Takanami & Okamoto, 1973) places the respective promoter to the left of the Hin site. No Hpa fragments were bound to RNA polymerase under highly stringent conditions (OW, 0.25 M-KCl), and preformed complexes dissociated when exposed to such treatment. Dissociation could be prevented by a short incubation of the complexes with the four ribonucleoside triphosphates ATP, GTP, UTP and CTP, which leads to the formation of stable ternary complexes of DNA, enzyme and RNA (Richardson, 1966). Such ternary complexes were only formed with fragments that also formed promoter complexes (see Plate II), indicating that DNA ohains were

PROMOTERS

IN

PHAGES

fd,

fl

AND

Ml3

273

initiated at the complexed regions. In an attempt to estimate the homogeneity of the initiation sequences within each fragment, chain initiations by either ATP or GTP were suppressed by limiting the concentration of the respective purine triphosphate. In this test situation, ternary complexes with fd DNA fragments Hpa-A and Hpa-B were formed when A-starts were favoured, whereas G-starts yielded ternary complexes involving fd DNA fragments Hpa-C, -D, -F and -H (see Plate II). Thus RNA polymerase activity seems to be directed ir, vitro by six sites, of which four promote the synthesis of RNA with GTP at the 5’ end, and two with ATP. The specific transcription of the fragments was used to locate two of the promoters with some precision by measuring the size of the transcription products relative to denatured Hpa fragments (see Materials and Methods). Under conditions of preincubation when polymerase was preferentially looked on fragment Hpa-D (see Plate II), a single RNA specieswas synthesized that migrated just above denatured Hpa-F in a polyacrylamide-urea gel corresponding to a length of about 450 nucleotides for the Hpa-D transcript. When the molar ratio of polymerase to RF was raised, additional bands appeared in the gel. One of these bands had a mobility between Hpa-A and Hpa-B, corresponding to at least 1000 nucleotides. On the basis of size, Hpa-A was the template for this transcript. These data suggestthe presence of initiation sites around map positions 0.33 and O-93, at the distal ends of genesI and 11 (seeFigs 3 and 4).

FIU. 4. Positional pattern of phagepromoters.Phage genes are arranged on the outer circle. The inner circle specifies map distances E-M fractional lengths of the phage DNA. Solid bars indicate regions of uncertainty for promoter location. The direction of transcription and the approximate position of the terminator rare indicated. A and G stand for ATP and GTP initiators.

The accurate location of the promoter within Hpa-D was determined in a second way, owing to the availability of the respective binding site as a unique fd DNA segment (Heyden et al., 1972). This segment served as a primer for the synthesis of DNA by a pulse-chaseprocedure, essentially as described for the Hpa fragments. The product of synthesis was cleaved by either Hpa II or Hind II and the resulting digests were analysed for size distribution of 32P-labelledDNA by gel electrophoresis

274

P.

H.

SEEBURG

AND

H.

SCHALLER

under denaturing conditions. Results indicated that DNA polymerase had traversed the junction of fd fragments Hpa-D and Hpa-F after incorporating 80 nucleotides and had reached the unique cleavage site by Hind II on extension of the primer by approximately 400 nucleotides. Thus, in keeping with the length of the RNA chain synthesized on Hpa-D, the promoter site on this fragment is situated close to its proximal end, at map position 0.93. When fd RF DNA was transcribed in vitro, the RNA species initiated at the Hpa-D promoter grew to a length of about 1000 nucleotides, implying the presence of a terminator site around map position 0.75, within fragment Hpa-C and downstream of gene VIII. An analogous RNA species synthesized in vitro on fl RF DNA has been observed to code in a cell-free system for the products of genes I’ and V.Z11 (P. Model, personal communication).

4. Discussion A physical map of the circular genomes of E. coli phages fd, fl and Ml3 was constructed by dissecting each phage RI? DNA with the restriction endonuoleases Hpa II and Hind II, and ordering the resulting fragments by using them as primers for the synthesis of their respective neighbours. This method is of general applicability to locate DNA and RNA fragments in relation to known markers on the parental DNA molecule. The cleavage map of the phage DNAs was aligned with the genetic map. Ordered sets of phage DNA fragments with defined genetic content are a valuable tool for analysing functions of the phage genome. For example, evidence has been presented indicating that the origin and termination of conversion of Ml3 viral DNA to its duplex form in vitro is confined to a region encompassedby Ml3 fragments Hpa-H and Hpa-L (Tabak et al., 1974). According to our data, this region maps in the vicinity of the junction of genesII and IV (map position O-07). In order to approach an understanding of how RNA polymerase activity is regulated by the phage DNA, the interaction of this enzyme with the specific phage DNA fragments was studied and led to the detection of six RNA polymerase binding sites. It should be pointed out that the method used can lead to an error in the number of promoters determined on the phage genome. First, an overestimation of this number will result from stable binding of polymerase to DNA regions artifactually opened under the test conditions. The specific RNA initiation at the detected sites, however, argues in favour of their natural promoter function. Second, an underestimation of the number of promoters will ensue from the presence of more than one promoter on the same fragment. Likewise, a promoter could be lost by Hpa II cleavage, in analogy to the phages X and T7, where promoters and Hind II cleavage sites have been shown to overlap in some instances (Allet et al., 1974; Maurer et al., 1974). This possibility is disposed of by our finding that polymerase binding before Hpa II cleavage of RF DNA yields the samenumber of fragments bound. Several properties of the detected sites deserve comment. A11sites appear to be parts of promoters, since RNA polymerase is capable of starting RNA chains at the complexed sites. The sites display marked differences in their rates of RNA polymerase complex formation (seePlate II) and decay (Seeburg, 1975), which may be considered as a prerequisite for transcriptional regulation. The site within fragment Hpa-D has the highest affinity for the enzyme, It is of inter& to note that this

PROMOTERS

IN

PHAGES

fd,

fl

AND

Ml3

275

affinity is not influenced by a double-strand soission as close as 80 nucleotides upstream of the respective binding site. This indicates that only a small DNA region, if any, is needed to direct the enzyme to this binding site, which has been shown to contain the start point of RNA chain formation (Heyden, 1974). The gross positional pattern of the six phage promoters determined on the circular genome is shown in Figure 4. The two ATP initiators map towards the distal ends of genes III and I at the approximate map positions of 0.54 and 0.33, respectively. The four GTP initiation sites (approx. map positions 0.03, 0.1, 0.8, O-93) are distributed over one-third of the genome, spanning genes VIII, V, VII and 11, with three closely spaced sites in the region of gene II. This region is of special interest, since the gene II product is important in phage DNA replication (Pratt & Erdahl, 1968) and the origin of replication is thought to lie in this gene, in analogy to gene A of phage #X174 (Baas & Jansz, 1972), and in analogy to other systems where the origin of DNA replication is located close to genes whose products are involved in DNA replication. If DNA replication starts in gene II, either one of two GTP initiators at map position O-1 and 0.03 could be functionally involved in the synthesis of the proposed RNA primer (Brutlag et al., 1971). As mentioned above, the conversion of the single-stranded viral DNA to its duplex form begins and ends around map position 0.07. No information is available concerning the number and size of the in vivo products specified by the determined phage promoters. Analysis of in vitro fd RNA led to the detection of at least four species with unique initiation sequences and lengths, ATPinitiated chains being of nearly full genome length and GTP-initiated chains approximately one-third and less (Okamoto et al., 1969; Takanami et al., 1970). These findings suggested the termination of all RNA chains at only one site on fd RF DNA (Okamoto et al., 1969). To accord with our data on the position and number of the initiation sites and on the length of one of the in vitro transcripts, the single terminator can only be in the region of map position 0.75, i.e. downstream of gene VIII. The presence of a terminator in this region is also indicated by the in vitro synthesis of a short messenger RNA species that codes for gene VIII product only (Model and Konings, personal communications) and seems to be initiated at the promoter on Hpa-C. When transcription on a circular genome starts at several promoters but ends at a single site, one expects to find a stepwise gradient of transcriptional activity along the genome with its minimum and maximum level just downstream and upstream of the terminator. The overall shape of the gradient will be determined by number, position and strength of the promoters, the difference in transcriptional activity between downstream and upstream parts of the genome being enhanced by a higher density of promoters towards the terminator and amplified by a positioning of the strongest promoters next to the terminator. According to this scheme, infrequently transcribed genes are located downstream and frequently transcribed genes upstream of the terminator site. Such a design provides a simple mechanism for differential constitutive gene expression and appears to be well-realized in Uamentous bacteriophage. Genes V and VIII (unwinding protein and major coat protein), which are preferentially expressed in viva (Henry & Pratt, 1969) and in vitro (Konings, 1973; Model & Zinder, 1974), lie right upstream of the terminator. They are controlled by the promoter sites in map positions 093 and 0.8, which are the strongest phage promoters (Seeburg,

276

P.

H.

SEEBURG

AND

H.

SCHALLER

1975). Gene III (a minor coat protein), which is expressed in very low amounts (Henry & Pratt, 1969), is located just downstream of the terminator site where transcriptional activity seems to be at its lowest level. Our failure to detect an initiator for gene III raises the interesting possibility that transcription of a limited region on the phage genome is effected by a leakiness in the termination event. Read-through is also suggested by oversize in, vitro transcripts from fd RF DNA (Okamoto et al., 1969). This indicates that not only the position of the terminator but also its e%ciency may be a factor used in this system to control RNA synthesis. A mechanism for transcriptional regulation as described here can be considered as a general scheme that allows for economy of gene expression without the partioipation of regulatory proteins other than RNA polymerase itself. REFERENCES Allet,

B., Roberts, R. J., Gesteland, R. F. & Solem, R. (1974). Nature (London), 249, 217-221. Baas, P. D. I% Jansz, G. 8. (1972). J. Mol. BioZ. 63, 569-576. Brutlag, D., Schekman, R. & Kornberg, A. (1971). Proc. Nut. Acad. SC%., U.S.A. 68, 2826-2829. Cohen, S. N., Chang, A. C. Y. & Hsu, L. (1972). Proc. Nat. Accd Sci., U.S.A. 69,2110-2114. Gd, D. E. C% Goodman, H. M. (1974). Biochem. Biophys. Res. Commun. 59, 108-116. Goulian, M. & Kornberg, A. (1967). Proc. Nat. Acad. Sk, U.S.A. 58, 1723-1730. Henry, T. J. & Pratt, D. (1969). Proc. Nat. Acad. Sk, U.S.A. 62, 800-807. Heyden, B. (1974). Ph.D. thesis, University of Tubingen, Germany. Heyden, B., Niisslein, Ch. & Schaller, H. (1972). Nature New Biol. 246, 9-12. Hinkle, D. C. & Chamberlin, M. (1970). Cold Spting Harbor Syrnp. Qwznt. Bill. 35, 65-72. Hutchison, C. A., III & Edgel, M. H. (1971). J. F&roZ. 8, 181-189. Jones, 0. W. & Berg, P. (1966). J. Mol. BioZ. 22, 199-209. Jovin, T. M., Eglnnd, P. T. & Bertsch, L. L. (1969). J. BioZ. Chem. 244, 2996-3008. Lyons, L. B. & Zinder, N. D. (1972). Z’@oZogy, 49, 45-60. Konings, R. H. H. (1973). FEBS Letters, 35, 155-160. Maizel, J. V., Jr (1971). In Methods of ‘c’irology (Maramosch, K. & Koprowski, H., eds), vol. 5, pp. 18&247, Academic Press, New York. Mandel, M. & Higa, A. (1970). J. Mol. BioZ. 53, 159-162. Marvin, D. A. & Hohn, B. (1969). Bacterial. Rev. 33, 172-209. Marvin, D. A. & Schaller, H. (1966). J. Mol. BioZ. 15, l-7. Maurer, R., Maniatis, T. & Ptashne, M. (1974). Nature (London), 249, 221-223. Model, P. & Zinder, N. D. (1974). J. Mol. Bid. 83, 231-251. Niisslein, Ch. & Heyden, B. (1972). Biochem. Biophys. Res. Commun. 47, 282-289. Okamoto, T., Suguira, M. & Takanami, M. (1969). J. Mol. BioZ. 45, 101-111. Okamoto, T., Suguira, M. & Takanami, M. (1972). Natwre New BioZ. 237, 108-109. Pratt, D. & Erdahl, W. S. (1968). J. Mol. BioZ. 37, 181-199. Richardson, J. P. (1966). J. Mol. BioZ. 21, 115-127. Seeburg, P. H. (1975). Ph.D. thesis, University of Tiibingen, Germany. Sharp, P. A., Sugden, B. & Sambrook, J. (1973). Biochemistry, 12, 3055-3063. Sugisaki, H. & Takanami, M. (1973). Nature New Biol. 246, 138-140. Tabak, H., Grif%th, J., Geider, K., Schaller, H. & Kornberg, A. (1974). J. BioZ. Chem. 249, 3049-3054. Takanami, M. (1973). FEBS Letters, 24, 318-322. Takanami, M., Okamoto, T. & Sugiura, M. (1970). CoM; Spring Harbor Symp. Quant. Biol. 35, 179-187. Takanami, M. & Okamoto, T. (1973). Bose Institute Symposium, in the press. Taketo, A. (1972). J. Biochem. 72, 973-979. L. & Treiber, G. (1970). Yamamoto, K. R., Alberta B. M., Benzinger, R., Lawhorne, Firology, 40, 734-744.

PROMOTERS

IN

PHAGES

fd,

fl

AND

Ml3

277

Note added in. proof: Further analysis of DNA fragment Hpa-C showed that this fragment is cleaved once by restriction endonuclease Hae III and that RNA polymerase forms stable complexes with both cleavage products, indicating that Hpa-C contains two promoters. The smaller fragment Hae Hpa-Cz (map position 0:‘79 to 043) contains the strong gene VIII promoter. The promoter in Hae Hpa-C1 (map position 0.69 to 0.79) lies probably downstream of the single terminator contained in this fragment and may control gene III expression.