THE JOURNAL OF Blo~oCrcM. CHEMISTRY vel. 257,,No. 15, h u e of August 1% PP. 9205-92103 1982 Printed ut U.S.A.

Precise Location of ’pwo Promoters for the P-Lactamase Gene of pBR322 SI MAPPING OF RIBONUCLEIC ACID ISOLATED FROM ESCHERICHIA COLI OR SYNTHESIZED IN VITRO*

(Received for publication, March 22, 1982)

Jiirgen Brosiusf, Richard L. Catefi, and Aaron P. Perlmutter From the Department of Biochemistry ana Molecular Biology, Harvard Uniuersity, Cambridge, Massachusetts 02138

We identify two promoters for the p-ladamase gene of plasmid pBR322. RNA isolated from bacteria con- taining pBR322 or RNA transcribed in vitro on pBR322 templates was hybridized to 5’ end-labeled single- stranded plasmid probes (Berk, A. J., and Sharp, P. A. (1977) Cell 12,721-732). Electrophoretic analysis of the nuclease S1 digestion products next to Maxam-Gilbert sequencing ladders closely defines the transcriptional initiation points. The natural promoter lies near the coding sequence of the 8-lactamase gene, initiating transcription at -35 bases before the ATG initiation codon, while a second promoter initiates at positions -244 and/or -245 (on the opposite side of the Eco RI site). This promoter overlaps the promoter transcribing in the opposite direction toward the tetracycline gene(s) and starts in the -10 region of that promoter. SI mapping of procaryotic mRNA, transcribed in uiuo, allows both an accurate identification of promoters and the analysis of their transcriptional regulation.

Plasmid pBR322 is one of the most widely employed cloning vehicles (1). Its many uses include the isolation and amplif1- cation of foreign DNA fragments and their expression utilizing the bacterial control elements for the initiation of transcrip- tion and translation (2-5). The complete nucleotide sequence of pBR322 was determined some time ago (6), but knowledge of the precise location of transcriptional control elements has been accumulating slowly. A large number of bacterial and bacteriophage promoter sequences are known, and consensus structures have been proposed for the -10 (7-10, 13) and -35 regions (10-13). Nevertheless, it is often difficult to identify a promoter structure merely by comparing DNA sequences with the consensus structures. This is the case for the promoter located upstream from the 0-lactamase (Ma) gene of pBR322, since there are several sequences which have homologies to the consensus structures. Therefore we have used a 5‘ end analysis of specific RNA’s to identify unambiguously the promoters.

We employed the Berk-Sharp S1 nuclease protection method (14), previously used to locate the 5‘ ends of eucaryotic mRNAs (15), to study the RNA transcripts hom pBR322, RNA transcribed in vitro from pBR322 templates or RNA

* Funds were obtained from Grant GM21514-07 from the National Institutes of Health to Walter Gilbert. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertbement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Recipient of a postdoctoral fellowship from the Deutsche For- schungsgemeinschaft.

of Health. 3 Recipient of postdoctoral fellowship from the National Institutes

isolated from bacteria harboring pBR322 can protect single- stranded DNA probes from S1 nuclease digestion. We identi- fied the 5‘ ends of the mRNAs by electrophoresing these protected fragments next to Maxam-Gilbert sequencing iad- ders (16, 17) of the undigested 5’ end-labeled probe. The 5’ ends of the in uiuo and in vitro messages are identical. The precise identity of the initiating nucleotide was confi ied by sizing ZR vitro transcription products singly end-labeled with [Y-~’P]ATP, [yBP]GTP, or [y-32P)UTP.

Stuber and Bujard (18) identified three promoters in this region by electron microscopic mapping (+80 base pairs). Two of these, P1 and P3, transcribe toward the bla gene. P2, which is roughly coincident with P1 transcribes in the opposite direction toward the tet gene($). By sizing the in vitro message and identifying the fmt two nucleotides, Russell and Bennett (19) previously mapped the P3 promoter. This is the first report of the exact start of the blu promoters in vivo.

EXPERIMENTAL PROCEDURES

Preparation of DNA Templates and Probes and DNA Sequenc- ing-Plasmid pBR322 was digested with restriction enzymes from New England Biolabs or Bethesda Research Laboratories using the supplier’s suggested conditions. Restriction fragments were phenol- extracted, ethanol-precipitated, and separated on 5% acrylamide, 0.16% bisacrylamide gels. Specific fragments were located by UV shadowing, excised, and eluted by diffusion (17). Probes for Sl map- ping were 5’ end-labeled, strand separated on 6% acrylamide, 0.1% bisacrylamide gels, and eluted (17). DNA sequencing was done ac- cording to the procedure of Maxam and Gilbert (16, 17).

In Vitro Transcription of RNA-RNA was synthesized using 100 nM Escherichia coli RNA polymerase (a f l from W. McClure, Carnegie-MeUon University, Pittsburgh, PA) in a 20-pl reaction vol- ume containing 40 m~ Tris-C1, pH 7.9, 10 m~ MgClz, 150 mM KC1, 0.1 m~ dithiothreitol, 0.1 mM EDTA, 0.15 m~ ribonucleotide triphos- phates, and 5 nni double-stranded DNA template (20). Radiospecific activity of [y3*P]ATP, [Y-~~P)GTP, or [Y-~’P]UTP (Amersham and New England Nuclear) was 2 Ci/mmol and of [a-32P]UTP (Amer- sham), it was 0.02 Ci/mmol. After incubation for 30 min at 37 “C, the reaction was quenched by addition of sodium acetate, pH 5.2, to a final concentration of 100 m ~ , Sarkosyl to 0.5%, and EDTA to 10 mM. The RNA was extracted with phenol, chloroform, and finally ethanol- precipitated twice.

Preparation of in Vivo RNA-E, coli strain HB 101 (21) or HB 101-harboring plasmid pBR322 was grown in YT medium (22) without addition of antibiotics to A5m = 0.8. Six ml of cells were collected by centrifugation at 2500 rpm for 5 min at 4 “C. The cells were resus- pended in 210 pl of cold 50 mM Tris-C1, pH 7.9, and transferred to a 1.5-ml Eppendorf tube. Twenty pl of coId aqueous lysozyme solution (5 mg/ml) and 40 pl of cold 0.25 M EDTA, pH 8, were added. The tubes were inverted several times and incubated for 8 min on ice, After addition of 100 pl of a cold Triton solution (0.3% (v/v) Triton X-100,0.18 M EDTA, pH 8, and 0.15 M Tris-C1, pH 8), the tubes were again inverted and incubated for another 8 min on ice. The samples were spun for 12 min in an Eppendorf centrifuge at 4 ”C. The supernatant was extracted two times with 400 pl of phenol and the aqueous phase ethanol-precipitated twice, ethanol-washed, and dried.

9205

9206 SI Mapping of pBR322 P-Lactamase Promoters

The pellet was dissolved in 100 p1 of H20 at about 2 pg of nucleic acids/pl and stored at -80 "C.

S1 Protection-SI mapping was performed by the procedure of Weaver and Weissmann (15). RNA synthesised in uitro, or total RNA isolated from bacteria containing pBR322, was ethanol-precipitated with 10 pg of carrier tRNA, dried, and resuspended in 20 pl of 0.04 M 1,4-piperazinediethanesulfonic acid, pH 6.4, 0.4 M NaCI, and 1 KIM EDTA. One pl of end-labeled probe (about 5 ng) was added, the mixture covered with paraffin oil, and incubated at 65 "C for 90 min.

a "

b x - "

A

C

~~ B "- -

... D

P S I I

FIG. 1. Region of pBR322 which contains promoters P1 and P3 (transcribing toward the 8-lactamase gene) and P2 tran- scribing toward the tet gene(s)). Their respective start sites are given in parentheses. The numbering of Sutcliffe (6) has been used. Locations of the single-stranded DNA fragments used as probes for S1 mapping are shown: a, fast strand of the 666-base pair Suu 3AI fragment (positions 4046-340); b, fast strand of the 378-base pair Eco RI/Bum HI fragment (position 4360-375); c, slow strand of the same fragment. Asterisks indicate the position of the 5' end label. The templates for in uitro transcription are: A, the 753 base pair Pst I/ Eco RI fragment (positions 3607-4360); B, the 378-base pair Eco Ri/ Barn HI fragment (positions 4360-375); C, pBR322 linearized with Pst; D, the 666-base pair Sau 3AI fragment (positions 4046-340).

M 1' 2 3 4 3a 4a

99r 9 34

r40- a73

506/516 631

3 9 6 34 4

2 90,

The aqueous phase was removed with a micropipette and diluted into 200 pl of SI buffer (0.25 M NaCI, 0.03 M sodium acetate, pH 4.6,0.001 M ZnS04, 20 p g / d of denatured salmon sperm DNA) at 0 "C. Two thousand units of SI nuclease (Boehringer Mannheim) were added and the mixture was incubated at 37 "C for 30 min. The samples were ethanol-precipitated and resuspended in 10 pl of 95% formamide, 1 mM EDTA, 0.015% xylene cyanol, and bromphenol blue. After heat denaturation for 90 s at 90 O C , 5 pl were loaded on 6% or 12% polyacrylamide gels containing 7 M urea. The remainder was saved for electrophoresis on sequencing gels.

RESULTS

RNA was synthesised in vitro from double-stranded DNA templates A-C and hybridized to the single-stranded probes a-c shown in Fig. 1. After digestion with S1 nuclease, the protected fragments were resolved on acrylamide gels (Fig. 2; see Table I). RNA synthesized in vitro from template A protected a 140-nucleotide fragment of probe a (Fig. 2, lane 1). Since the 5' end of probe a is located at nucleotide 4046 of pBR322, initiation of transcription from one promoter can be placed around position 4190. The additional 350-nucleotide fragment (Fig. 2, lane 2) produced by hybridizing transcripts from the longer template C to the same probe indicated another promoter, P1, located about 210 nucleotides upstream from P3 transcribing toward the p-lactamase gene. This was confirmed by using probe b labeled at the Eco R1 site (position 4361). Hybridization of this probe with RNA transcribed from templates B or C produced a fragment of about 35 nucleotides after S1 digestion (Fig. 2, lanes 5 and 6, respectively). Thus we conclude that transcription toward the p-lactamase gene initiates in vitro near positions 4190 and 35 of pBR322. These start sites are promoters P3 and PI.

M 9 10 11 1

748 - 506116-

631 -

396 - 344 - 0 298 - - c 221R-

154-

154 -

rs -

2

FIG. 2. S1 nuclease-protected fragments. Fragments were electrophoresed on 6% acrylamide, 0.2% bisacrylamide gels (lanes 1-4 and 9- 12), or 12% acrylamide, 0.4% bisacrylamide gels (lanes 5-8). All gels contain 7 M urea. The experiments in each lane are summarized in Table I. Samples in lanes 3a, 4a, 7u, and 8a are identical with samples in lanes 3, 4, 7, and 8, respectively, except that a different preparation of bacterial RNA was used.

SI Mapping of pBR322 P-Lactamase Promoters 9207

TABLE I Summary of SI nuclease experiments using various sources of RNA and different single-strandedprobes

For identification of templates and probes see Fig. 1. The approximate amounts of in vivo isolated nucleic acids are given. Fragments protected from SI digestion by in vitro end to end transcripts are marked with superscript 1. Prominent fragments which are believed to be generated by degradation products of a given message are marked by the superscript 2. For details see text.

vitro vitro 1, in 2 , in

Template A C Bacterial strain

Amount of RNA used Probe a a Relevant protected frag- 310' 660'

ments (nucleotide 140 350 lengths)

3, in vivo

HB101/ pBR322

4-5 Pg a

350 325' 3152

3252 140 315* 140 120'

4 , in vivo

HBlOl/ pBR322 22.5 pg

a 350 325* 3152 140

1 20'

vitro 5! in 5; 7, in vivo

B C HBlOl/ pBR322

b b 4.5 pg

b 370' 370' 35 35 35

a, in vivo 9, in vivo

HB101/ HB101/ pBR322 pBR322 22.5 pg 4.5 p g

b a 35 350

325' 315$

(140)

IO, in 11, in 12, in vivo uivo vivo

HBlOl HB101/ HBlOl pBR322

a C C

330

4.5 pg 4.5 p g 4.5 pg

The larger fragments appearing in Fig. 2 (the 310-nucleotide fragment in lane 1, the 666-nucleotide fragment in lanes 2, 5, and 6) result from protection of the respective probes by end to end transcripts. These full length transcripts are artifacts frequently found with in vitro transcriptions.

Are these promoters also the in vivo initiation points? We isolated RNA directly from E. coEi-harboring pBR322 for S1 mapping. Fig. 2 shows that this RNA protected fragments of 350 and 140 nucleotides when hybridized to probe a (lanes 3 and 4) and a 35-nucleotide fragment when hybridized to probe b (lanes 7 and 8). Since the in uitro and in vivo protection patterns are identical (lanes 1 and 2 versus 3 and 4 and lanes 5 and 6 versus 7 and 8), the in vitro start sites for P1 and P3 are used in uivo. In lane 3, a doublet at 325/315 and a strong band at 120 nucleotides were also observed (see also Fig. 3). We believe that such bands reflect major breakdown products of the p-lactamase mRNAs synthesized from P1 and P3 in vivo rather than further initiation points for transcription. This is supported by the lesser intensity of these bands in S1 mapping experiments using in vitro synthesized RNA (Fig. 2) since this RNA is not subjected to possible degradation during metabolic turnover or during the isolation procedure. Also we do not see RNA transcripts that initiate in vitro near these positions with either [Y-~~P]ATP, [Y-~'P]GTP, or [Y-~'P]UTP (see Fig. 6).

We used probe c to map the start site of promoter P2 which transcribes in the opposite direction toward the gene(s) for tetracycline resistance on pBR322. Fig. 2, lane 11, shows that the in vivo RNA protected a 330-nucleotide fragment of probe c. Since the probe is end-labeled at position 378, this defrnes the start point for promoter P2 close to position 48.' RNA isolated from E. coli strain HB 101 lacking pBR322 did not protect probes a and c (lanes 10 and 12, respectively) from SI digestion.

In order to assign the exact start sites of transcription for promoters P1 and P3, DNA fragments of probe a or b, pro- tected by in vitro or in vivo synthesized RNA, were electro- phoresed next to sequencing ladders of the undigested probe. In Fig. 3, DNA fragments of probe a protected by in vitro RNA (lanes 1 and 2) or by in viuo RNA (lanes 3 and 3a) were run next to the A f G and T + C sequencing patterns of probe a. Similarly in Fig. 4, lanes 6 and 8 contain the DNA fragments of probe b protected by in vitro and in uivo RNA, respectively, electrophoresed next to the sequencing ladder of probe b. These sequencing gels demonstrate with greater resolution

' J. Lawrie, J. Hedgpeth, and C. Harley (personal communication) have shown that P2 begins in vitro ai base 45.

that the in vitro and in vivo start sites are identical. The arrows in Fig. 5 indicate the nucleotides at the 3' end of the SI-resistant bands observed in Fig. 3 for promoter P3 and in Fig. 4 for promoter P1. The length of the arrows is approxi- mately proportional to the intensity of the bands on the sequencing gels. The sequencing reactions yield DNA mole- cules which are chemically different than S1 digestion prod- ucts. The absence of a 3"phosphate group on the S1-protected fragments slightly decreases their mobility (about half a nu- cleotide) with respect to the fragments derived from the chemical sequencing reactions. A further correction of one base is necessary since the 3' modified base including the sugar has been eliminated in the DNA chemistry.

We resolved the ambiguity of the multiple S1-generated bands present in Figs. 3 and 4 (see also Fig. 5) by using different y-labeled ribonucleotide triphosphates for in vitro transcription. This enabled us to determine the triphosphate residues at the 5' end of the mRNAs transcribed from P1 and P3. In vitro transcription from the P3 promoter of template D (Fig. 1) using [y-32P]GTP generates an RNA transcript of about 150 nucleotides (Fig. 6, lane 2). This is consistent with transcription starting at promoter P3 and terminating at the Sau 3AI site (position 4046) and thus defines the start site of P3 as a G residue. If template A is used with [y-=P]GTP, we observe a 580-nucleotide long transcript (data not shown) initiating at P3 and terminating at the Pst I site (position 3607). The S1 mapping data summarized in Fig. 5 placed the initiation site for P3 near position 4190. Thus we assign the start site at G-4189, since there is no other G residue between nucleotides 4182 and 4201. In this case the two major S1- resistant bands contain one or two additional nucleotides beyond the 5'-G residue of the p-lactamase mRNA. It is likely that the 5'-terminal triphosphate group of the procaryotic mRNA sterically inhibits the SI nuclease from cleaving the probe exactly at the junction of the RNA/DNA hybrid. Weaver and Weissmann (15) have described a similar phe- nomenon; the cap structure of eucaryotic mRNAs leads to SI- protected products which are up to 5 nucleotides larger than expected from the known start point of the message. When transcription is initiated with [Y-~'P]ATP on template A, we also observe a weakly labeled 580-nucleotide fragment (data not shown). This is consistent with a minor initiation site at A residue 4188; an in vitro start site also reported by Russell and Bennett (19).

We used the same strategy to determine the exact nucleo- tide at the 5' end of the RNA initiated at P1. Transcription from template D using [y3'P]UTP produced a transcript of about 350 nucleotides (Fig. 6, lane 3). This is the expected

9208 S1 Mapping of pBR322 P-Lactamase Promoters

A C

G T 1 2 + + 3 3a

6 + + 8 A C

G T

II ” 98

” ”-

L e c c

”

FIG. 3. Ladder of the G + A and T + C sequencing reactions of probe a. Ladder of the C + A and T + C sequencing reactions (16, FIG. 4. Ladder of the G + A and T + C sequencing reactions 17) of probe a coelectrophoresed with fragments of probe c protected of probe b. Ladder of the G + A and T + C sequencing reactions (16, by: I , RNA synthesized from template A, 2, RNA synthesized from 17) of probe b electrophoresed with fragments of probe b protected template C; 3 and 3a, RNA isolated from bacteria containing pBR322. by: 6, in vitro transcribed RNA 8, in vivo isolated RNA. The gel The gel (0.38 X 200 X 400 cm) contained 6% acrylamide, 0.3% (same size as in Fig. 3) contained 20% acrylamide, 1% bisacrylamide, bisacrylamide, and 8.3 M urea and was run at 1400 V until the xylene and 8.3 M urea and was run until the xylene cyanol migrated about cyanol dye reached the bottom of the gel. 22.5 cm.

SI Mapping of pBR322 P-Lactamase Promoters 9209

no 70 60 50 P1 4 0 P2 ~ ~ T ~ ~ C C T G A C T G C C T T A C C A A T T T A A C T C T ~ A ~ T A A A C T A ] C C ~ ~ C A ~ ~ : ~ : A A A ~ ~ C

C A C C C A C T ~ A C C C A A T C ~ T T A A A T T ~ A C A C T A T T T ~ A T ~ ~ ~ ~ C ~ ~ T A A T T ~ ~ C ~

-" -"

4 t t 4 4

30 PO I 4360 4:lblJ !O T T A T C C A T t i A T A A t i L T C T C A A A C A T C A G A A T t i , ~ t i A A T l ~ T T t i A A t i A ~ t i A . .... A A T A C C T A C T A T T C t i A C A t i T T T t i T A C T C T T A A C A A C T T C T t i C T .....

4230 4220 4210 42v0 P3 4190

A - ~ ? A C A T T C A A A T A T ~ - ~ A T C ~ t i C T C A T t i A l C A C A A T A l 4 ~ C C l ~ A T A A A T t i C

T T A T ~ ~ T A A ~ ~ T T T A T A C A T A ~ C C C A G T A T T C C C , ~ C T A T T T A C G

4 1 h O 41 70 4!60

T I L A A .r A A T A T T c A A A A A ti c A A c A c ~m .... 0-lucramasc

A A L T T A T T A T A A C T T T T T C C T T C T C A T A C . . . . .

50 6" 396" 3 4 4 " 2 9 8 - -. 'SP2 P2)

P3) - P1) L "

"

7 5 -

FIG. 6. RNA transcripts synthesized from template D. RNA transcripts synthesized from template D (lunes 1-4) or template D digested with Dde I (lunes 5-8) resolved on a 6% polyacrylamide gel containing 7 M urea. The labeled ribonucleotide triphosphate used is indicated for each lune.

length for an RNA species initiating at P1 and terminating at the Sau 3AI site (position 4046). Furthermore, after shorten- ing template D by digestion with Dde I, we observe a tran- script of about 110 nucleotides (Fig. 6, lane 7). Since there are 2 T residues at positions 36 and 37, we cannot say with certainty which residue is the actual start site of transcription. A direct comparison of the S1 patterns generated by the P1 RNA and the P3 RNA would favor base 36 as the initiation site. However, since the P3 mRNA/probe a hybrid ends with a G/C base pair, while the P mRNA/probe b hybrid ends with an A/U base pair, the latter hybrid might be less stable at the end and thus more susceptible to cleavage by SI. Therefore, we cannot exclude a U start at position 37, or two start sites at positions 36/37. We also observe weak bands (Fig. 6, lanes 1 and 5) when transcription is initiated with [y-

PIATP, suggesting minor in uitro start sites at position(s) 35 and/or 38 of promoter P1. 32

FIG. 5. Nucleotide sequence from the pBR322 containing the two pro- moters P1 and P3 transcribing to- ward the 8-lactamase gene and P2 transcribing toward the tet gene(s). The symbols PI, P2, and P3 are located above the boxed -10 regions of the re- spective promoters. The ATG initiation codon of the p-lactamase gene is also boxed. The -35 regions of promoters P1 and P3 are overlined and initiation sites of transcription are circled. Since we cannot discriminate between T-36 and T-37 as start sites for P1, both are cir- cled. The 3'-nucleotides of the protected fragments are denoted by arrows whose lengths are approximately proportional to the intensities of the bands on gels in Figs. 3 and 4. See text for details.

DISCUSSION

We have located the exact sites for two promoters transcrib- ing toward the /3-lactamase gene of pBR322. P3, the nautral promoter of the bla gene initiates predominantly at G residue 4189, while P1, located upstream from P3, initiates at T-36 and/or T-37. We have demonstrated the existence of these promoters in vivo and in uitro, and shown that P1 transcribes into the bla gene and probably contributes to its expression in pBR322.

Fig. 5 shows the -10 and -35 regions for promoters P1 and P3, and the Pribnow box of P2. Five (underlined) out of seven positions located in the Pribnow box G-A-C-A-A-T-A of P3, and X-&A-A"C-I-Aof P1 are in agreement with the consen- sus sequences for -10 regions (10, 13). Many of the -10 se- quences of known promoters have up to three deviations from the Rosenberg-Court consensus structure (10, 13). The -35 regions of P1 and P3 show more variation from the consensus structure (10,13). The -35 region of P3 shows partial homology with the -35 region of the Ipp promoter (23), while the -35 region of P1 has the octamer C-C-T-G-A-C-T-G (positions 70-77, Fig. 5) in common with the -35 region of the fd V promoter (10,24).

Curiously, P1 and P2 partially overlap on opposite strands, perhaps making simultaneous initiation of transcription from P1 and P2 impossible. A similar arrangement has been de- scribed for the regulatory region of the biotin operon in E. coli (25). Our data also show that these three promoters operate in the absence of antibiotics (see Fig. 2, lanes 3, 4, 9, and 11).

Although P1 promotes transcription of the p-lactamase gene, this is an unnatural role created by the ligation of two different DNA fragments via an Eco RI site to construct vector pBR322 (1). P1 might be the promoter for the gene encoding a 14,000-dalton polypeptide on pSC101, the plasmid from which part of pBR322 was derived and which still contains the original Salmonella sequences adjacent to the Eco RI site (26). This protein is altered in cells containing pSClOl recombinants with DNA insertions at the Eco RI site (27).

It is well established that in E. coli most mRNA species have a short half-life (about 2 min; e.g. Ref. 31). However, from our data it is clear that the chemical half-lives of the 5' portions of messages transcribed from pBR322 promoters P1, P2, and P3 are sufficient for detection by S1 mapping. A

9210 SI Mapping of pBR322 P-Lactamase Promoters

degradation mechanism involving a 5’-exonuclease can be ruled out because there is not a continuous ladder of fragments (see Figs. 3 and 4). The degradation products we observe in vivo (see Fig. 2, lanes 3, 4, 9, and 11) indicate the action of endonucleases during initial steps of mRNA degradation.

We have demonstrated that precise procaryotic transcrip- tional start sites can be identified using the Berk-Sharp S1 nuclease protection method with RNA isolated from bacteria. Moran et al. (28), Dunn et al. (29), and Aiba et al. (30) have used similar approaches to map other procaryotic promoters.

Acknowledgments-We thank Walter Gilbert, in whose laboratory this work was carried out, for support and discussions. Special thanks to F. Fliegen.

REFERENCES 1. Bolivar, F., Rodriguez, R. L., Greene, P. J., Betlach, M. C.,

Heyneker, H. L., Boyer, H. W., Crosa, J. H., and Falkow, S. (1977) Gene 2.95-113

2. Chang, A. C. Y., Nunberg, J. H., Kaufmann, R. J., Ehrlich, H. A., Schimke, R. T., and Cohen, S. N. (1978) Nature (Lond.) 275,

3. Villa-Komaroff, L., Efstratiadis, A., Broome, S., Lomedico, P., Tizard, R., Naber, S. P., Chick, W. L., and Gilbert, W. (1978) Proc. Natl. Acad. Sci. U. S. A. 75,3727-3731

4. Talmadge, K., Stahl, S., and Gilbert, W. (1980) Proc. Natl. Acad. Sci. U. S. A. 77,3369-3373

5. Talmadge, K., and Gilbert, W. (1980) Gene 12,235-241 6. Sutcliffe, J. G. (1978) Cold Spring Harbor Symp. Quant. Biol.

7. Pribnow, D. (1975) Proc. Natl. Acad. Sei. U. S. A. 72, 784-788 8. Pribnow, D. (1975) J. Mol. Biol. 99,419-443 9. Schaller, H., Gray, C., and Herrmann, K. (1975) Proc. Natl. Acad.

10. Rosenberg, M., and Court, D. (1979) Annu. Reu. Genet. 13, 319-

11. Takanami, M., Sugimoto, K., Sugisaki, H., and Okamoto, T.

617-624

43, 77-90

Sci. U. S. A. 72, 737-741

353

(1976) Nature (Lond.) 260,297-302

12. Seeburg, P. H., Nusslein, C., and Schaller, H. (1977) Eur. J.

13. Siebenlist, U., Simpson, R. B., and Gilbert, W. (1980) Cell 20,

14. Berk, A. J., and Sharp, P. A. (1977) Cell 12, 721-732 15. Weaver, R. F., and Weissmann, C. (1979) Nucleic Acids Res. 7,

16. Maxam, A. M., and Gilbert, W. (1977) Proc. Natl. Acad. Sei. U.

17. Maxam, A. M., and Gilbert, W. (1980) Methods Enzymol. 65,

18. Stuber, D., and Bujard, H. (1981) Proc. Natl. Acad. Sei. U. S. A.

19. Russell, D. R., and Bennett, G. N. (1981) Nucleic Acids Res. 9,

20. Burgess, R. R., and Travers, A. A. (1971) Methods Enzymol. 21D,

21. Boyer, H. W., and Roulland-Dussoix, D. (1969) J. Mol. Biol. 41,

22. Miller, J. (1972) Experiments in Molecular Genetics, p. 433, Cold

23. Nakamura, K., and Inouye, M. (1979) Cell 18, 1109-1117 24. Schaller, H., Beck, E., and Takanami, M. (1978) in The Single

Stranded DNA Phages (Denhardt, D. T., Dressler, D., and Ray, D. S., eds) pp. 139-163, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

Biochem. 74, 107-113

269-281

1175-1193

S. A. 74,560-564

499-559

78, 167-171

2517-2533

500-528

459-472

Spring Harbor Laboratory, Cold Spring Harbor, NY

25. Otsuka, A., and Abelson, J. (1978) Nature (Lond.) 276,689-694 26. Cohen, S. N., Chang, A. C. Y., Boyer, H. W., and Helling, R. B.

(1973) Proc. Natl. Acad. Sei. U. S. A. 70.3240-3244 27. Tait, R. C., Rodriguez, R. L., and Boyer, H. W. (1977) Mol. Gen.

Genet. 151,327-331 28. Moran, C. P., Jr., Lang, N., Banner, C. D. B., Haldenwang, W. G.,

and Losick, R. (1981) Cell 25,783-791 29. Dunn, R., McCoy, J., Simsek, M., Majumdar, A., Chang, S. H.,

RajBhandary, U. L., and Khorana, H. G. (1981) Proc. Natl. Acad. Sei. U. S. A. 78,6744-6748

30. Aiba, H., Adhya, S., and de Crombrugghe, B. (1981) J. Biol. Chem. 256,11905-11910

31. Salser, W., Janin, J., and Levinthal, C. (1968) J. Mol. Biol. 31, 237-266