The DNA between Rz and cosR in bacteriophage lambda is nonessential

363

Gene, 33 (1985) 363-365 Elsevier GENE

1213

The DNA between

Rz and cosRin bacteriophage lambda is nonessential

DNA; cloning vectors; plasmids; promoter; high-level expression; deletions)

(Recombinant

V. James Hernandeza, Thomas D. Edlindb, Ryland F. Young’, and Garret M. Ihler’ * a Department of Biology, Universityof Texas at Dallas, P.O. Box 830688, Richardson, TX 75083-0688 Tel. (214) 690-2500: b Department of Microbiology and Immunology, Medical College of Pennsylvania, Philadelphia. PA 19129. Tel. (215) 842-6908; ’ Department of Medical Biochemistry and Genetics, Texas A&M University, College Station, TX 77843-l 112 (U.S.A.) Tel. (409) 845-2726 (Received

October

22nd,

(Accepted

October

26th, 1984)

1984)

SUMMARY

the right end of phage A DNA, between gene Rz and the cos site, are 2050 bp of apparently non-coding DNA. We have cloned a 2 DNA fragment containing this DNA into a plasmid and constructed a deletion, WI, extending from a site within the Rz gene to a site about 560 bp from cos. This deletion could be recombined into viable ;1 phage at a frequency equal to that observed for the undeleted sequence. Recombinant phage I carrying the wl deletion were demonstrated to have the same burst size and kinetics of phage production as undeleted A. The 01 deletion can be used to extend the capacity of I cloning vectors and to provide a region for the insertion of heterologous DNA which should exhibit controllable high level expression from the ,I late promoter, pk. Near

INTRODUCTION

The bacteriophage i, genome has three regions completely devoid of essential genes defined by point mutations. Two of these regions, 6 and ninR, contain open reading frames (Sanger et al., 1982; Daniels et al., 1983). Polypeptides have been identified which are encoded in the b region (Hendrix, 1971; Reeve * To whom

correspondence

and

reprint

requests

should

be

addressed. Abbreviations:

b, central

region of I genome

1983); bp, base pairs; cos, I cohesive plaque-forming of phage

(see Daniels

units; o, region between

gene Rz and cosR site

I genome.

0378-I 119/85/$03.30

0 1985 Elsevier

et al.,

end site; kb, 1000 bp; pfu,

Science

Publishers

and Shaw, 1979). Although all proteins have not been directly identified for the ninR region, there is evidence for a functional gene, ren (Toothman and Herskowitz, 1980; Kroger and Hobom, 1982). The genes in these two regions might be essential in some other host, as is the case for ren, which is required for growth in 2 lysogens, or in some physiological conditions, as is the case for the lysis gene Rz in medium containing high Mg2+ (Young et al., 1979). The third region, a 2050-bp sequence located between Rz and the cos site at the right end of the 2 genome (Daniels et al., 1983), does not contain open reading frames likely to encode for polypeptides (Sanger et al., 1982), although this region is located in the late operon and is thus heavily transcribed.

364

Several

transposon

insertions

produce no obvious However, tutions

phenotype

deletions,

(Fiandt

to the right of Rz (Young et al., 1979).

without

accompanying

substi-

et al., 1971), of the Rz-cm

(b) Effect on viability of 12 The following deletion

region

results

that the wl

does not affect i viability.

pVJH1 and pVJHlol

have never been reported.

demonstrate

The plasmids

were transformed

into a sup’

lysogen of Mam32 ~I857 Sam7. The lysogens were thermally induced and the A progeny was scored on suppressor

(supF) and nonsuppressor

The frequency

EXPERIMENTAL

tially the same in the two lysates indicates

(a) Deletions

(sup”) hosts.

of M + S + recombinants

that

two independent

was essen-

(Table I). This

double

crossover

events were not required to exclude the 01 deletion. To obtain deletions

in this region, a 9035-bp frag-

ment of A DNA extending from EcoRI site at bp 44972 to the BamHI site at bp 5505, encompassing the S-cos-B region, was inserted into the plasmid pQ, generating the plasmid pVJH1 (Fig. 1). Cleavage with BcfI and religation resulted in the 01 deletion, which begins at bp 46 366 and extends to bp 47 942, which is located 560 bp to the left of cos (Fig. 1; Daniels et al., 1983).

Also, the plaque

sizes of recombinants

from each

induction were indistinguishable (not shown). DNA was prepared from lysates derived from purified plaques and analyzed by agarose gel electrophoresis of restriction enzyme fragments. Fig. 2 shows that the wl deletion was in fact present in the DNA from M’S’ obtained from the induced lysogen carrying pVJHlwl. A single cycle infection revealed no difference in average burst size or kinetics of bacteriophage accumulation between il and hl (Fig. 3). As expected, the /Iwl bacteriophage, which is deleted for a distal portion of the Rz gene, shows the Rz - phenotype, which is a partial defect in lysis in medium containing 20 mM Mg*+ (not shown). (c) Application for cloning vector The 01 deletion thus seems to confer no defect on 1, vegetative growth except the expected conditional Rz

phenotype

TABLE

Fig. 1. Map of pVJH1 and pVJHlwl. l&357

DNA (with ligated

EcoRI-BamHI V., Young, lac promoter

and 1 gene Q, promoterp,. was constructed

of the two Bcfl sites. During taneous

ends) was cloned pQ (Edlind,

R., and Ihler, G., unpublished

pk. The WI deletion

gene Q.

EcoRI + BarnHI-cleaved

cohesive

sites of the plasmid

deletion

occurred

the construction eliminating

containing

induced,

plasmid-containing

lyso-

cI857 Sam”

Resident

pfu/ml

pfu/ml

% M’S’

plasmid

on supF host

on sup0

recombinants

3.3 x lo*

10

pVJH1

5.4 x IO’

5.7 x 10s

1.1

pVJHlw2

1.1 x 10s

5.0 x 105

0.5

il W3101 (Mam32 a

, qut site, and terminator by cleavage

by thermally

gens of Uam

into the

T., Hernandez,

results),

I

Phage produced

000

and therefore defines a nonessential

and ligation

of pVJH1 a spon-

the lac promoter

and

plasmid

~1857 Sam7) carrying

the indicated

was grown in Luria broth with shaking

resident

at 30°C to a cell

density of 5 x lOs/ml, shifted to 42°C for 15 min, and incubated with shaking

at 37°C for 2 h. The culture was frozen at -80°C

and thawed

at 37°C prior to titering on E. co/i Ymel (supF) and

MHI 160 (sup’). centage

% M + S + recombinants

represents

ratio of titers on supF and sup0 (nonsuppressing)

the perhosts.

365

A

region. Use of this deletion would increase the maximal insert size of 1 cloning vectors by 1.6 kb. In addition, this result suggests that it may be feasible to construct 13.expression vectors in which heterologous DNA is inserted into the o region. Since o is transcribed from the very strong J& promoter, very high I-mediated expression might be expected. Furthermore, inappropriate termination of transcription would be minimized by the powerful antitermination and antipolarity properties conferred on late RNA transcription by Q protein and the qut site (Forbes and Herskowitz, 1982; Somasekhar and Szybalski, 1983).

B

15721-

9035

-7459 550546693775+3758_

3530 - 1953

1868-

IIZO-

Fig. 2. Restriction

analysis

of (A) I and (B) lo1

purified phages were used to prepare 1984), and samples phoresed bromide.

DNA. Plaque-

DNA (Benson

were cleaved with EcoRI + BumHI,

in a 0.8%

agarose

gel, and

The 9035 bp band resulted

stained

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D.L.,

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Schroeder,

J.L.,

site of the 3S30-bp (1 right end) and SS05-bp (1 left end) I DNA

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Blattner,

F.R.:

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R.W., Roberts,

The 74S9-bp band resulted

similarly

from annealing

of the 1953-bp (right end with 1576-bp wl deletion) fragments.

The sizes given on margins

and 5505-bp

are in bp.

A.R., Hong,

R.A. (Eds.),

procedure

Szybalski,

W.,

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’

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,

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TIME Fig. 3. One-step LI85701

growth

(triangles). with phages

for phages

0.5% maltose

at a multiplicity

The cells were diluted to approx. 37°C with vigorous

shaking.

cated times and plated

I

60

75

INFECTION (circles)

exponentially

were concentrated of infection

Aliquots

were removed

and

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IO6 cells/ml and incubated

with a lawn of MHl160.

and

at

G. and Szybalski,

W.: Mapping

(qul) required

for antitermination

bacteriophage

lambda.

Toothman,

LI8S7

MHl 160(sup”) cells growing

in Luria broth containing infected

45

AFTER curve

I

,

I.: Rex-dependent

phages, II. Determinants

Virology 102 (1980) 147-160. Young, R., Way, J., Yin, J. and mutagenesis

of phage lambda:

at the indiby A. Campbell.

of sensitivity

Syvanen,

exclusion

of

to exclusion.

M.: Transposition

a new gene affecting

J. Mol. Biol. 132 (1979) 307-322. Communicated

site and

Gene 26 (1983) 291-294.

P. and Herskowitz,

lambdoid

ofQ-utilization

of late transcription

cell lysis.