Gene, 42 (1986) 345-349

Elsevier

345

GENE 1584

Use of the lysis gene of bacteriophage @X174 for the construction of a positive selection vector

(Recombinant DNA; cloning vehicle; lac promoter; lac repressor plasmid; inducible tetracycline resistance)

Bernhard Henrich* and Roland Plapp

Fachbereich Biologic. Abteilung Mkrobiologie, Universitiit Kaiserslautern, Postfach 3049, D-6750 Kaiserslautern, (F.R. G.) Tel. (0631)205-2347

(Received December 15th. 1985)

(Revision received February 12th. 1986)

(Accepted February 13th, 1986)

SUMMARY

DNA fragments generated by a variety of restriction enzymes can easily be cloned in the small (3.2-kb) positive-selection vector pUH84, which contains the modified lysis gene of bacteriophage $X174 under transcriptional control of the lac promoter. Plasmid pUH84 does not yield transformants after introduction into Escherichia coli unless the lysis gene is inactivated by insertion of foreign DNA into one of the unique PstI, SalI, AccI, ZZincII, BarnHI, or EcoRI sites. This highIy efficient positive selection of recombinants requires neither the use of a distinct host strain nor a special induction of the lysis function.

Transcription of fragments cloned into pUH84 may be effectively regulated by the lac promoter provided the host cells are cotransformed with the newly constructed plasmid pUH7 which carries the IQ allele of the lac repressor gene.

INTRODUCTION

A variety of cloning vectors have been developed which allow a one-step identification of colonies harbouring recombinant plasmids either by use of indicator media (Rather et al., 1981), or by direct positive selection (Bums and Beacham, 1984, and references therein; Vemet et al., 1985).

*To whom correspondence and reprint requests should be

addressed.

Abbreviations: Ap, ampicillin; bp, base pair(s); Cm, chlor-

amphenicol; IPTG, isopropyl-/?-D-thiogalactoside; kb, 1000 bp;

lac, lactose; PolIk, Klenow (large) fragment of E. coli DNA

polymerase I; R, resistance; Tc, tetracycline; [ 1, designates plas-

mid-carrier state.

One of the most effective mechanisms by which integration of DNA fragments into positive selection vectors leads to survival of clones carrying DNA inserts is insertional inactivation of lethally acting genes. Thus, we decided to exploit gene E of bac- teriophage $X174, whose product (gpE) acts as a potent inducer of cell lysis in E. coli (Hutchison and Sinsheimer, 1966; Henrich and Plapp, 1984), for the development of a new cloning vehicle.

As reported previously, cloning of $X174 gene E

in a multicopy plasmid was achieved by subjecting it to the efficient transcriptional control of the lac

promoter. Expression of the isolated gene showed that gpE is necessary and sufficient to trigger lysis of E. coli (Henrich et al., 1982). Maintenance of the

03J8-1119/86/$03.50 0 1986 Elsevier Science Publishers B.V. (Biomedical Division)

346

cloned lysis gene requires the presence of a re- pressor-overproducing lad allele in the host cells. Transformants, carrying the lad + wild-type allele, are not viable, since the intracellular concentration of luc repressor in this case is not sufficient to prevent the expression of the cloned gene E which is present in multiple copies per cell (Henrich et al., 1983). Taking advantage of these observations, a versatile plasmid vector, pUH84, has been developed by introduction of six unique restriction sites into the gene E coding sequence without destroying the functional integrity of the lysis gene product. Insertion of DNA into one of these sites leads to inactivation of gene E and to the survival of the corresponding transformants.

EXPERIMENTAL AND DISCUSSION

(a) Materials and media

Plasmids pUR2 (Ruther, 1980), pUR222 (Ruther et al., 1981), and pMC7 (Calos, 1978) were obtained from U. Ruther, Heidelberg. Plasmid pBR322 (Bolivar et al., 1977), and 1 DNA were from Boehringer-Mannheim. Genotypes of three E. coli

K-12 strains: F-Z-AM 1 SrecA, CAIQ’ and F’IQ1dM15 have been described (Henrich et al., 1983). Cells were usually grown in LB medium, containing 10 g tryptone, 5 g yeast extract and 5 g NaCl per litre. Antibiotic concentrations used were 200 pg Ap/ml, 7 pg Tc/ml, and 25 pg Cm/ml.

(b) Recombinant DNA techniques

Small-scale preparations of plasmid DNA and isolation of restriction fragments from poly- acrylamide gels were performed by the methods of Maniatis et al. (1982). Restriction endonucleases, T4 DNA ligase, and PolIk (Boehringer-Mannheim; New England Biolabs) were used as recommended by the suppliers. Gel electrophoresis of DNA and transformations of E. colt’ were carried out as de- scribed previously (Henrich et al., 1982).

(c) Construction and lysis inducing properties of the

positive-selection vector pUHS4

The construction of pUH84 is outlined in Fig. 1A. The orientation and the sequence of the 54-bp poly-

linker fragment inserted after the 30th codon of the $X174 lysis gene are shown in Fig. 1B. In this orien- tation the fragment which codes for 18 amino acids neither introduces a nonsense codon nor does it disturb the reading frame of the distal gene E coding sequence. However, after induction of the lysis gene with IPTG in liquid media, CAIQ’[pUH84] trans- formants lysed with a clearly reduced rate and to a smaller extent than CAIQ1 cells harbouring plasmid pUH72 (Fig. 1) which does not contain the poly- linker region.

In spite of this affection of gpE action, the lytic capacity of the modified $X174 gene E is still suf- ficient to completely prevent growth of pUH84 containing lucl + transformants on solid media. This was demonstrated by transformation of several E. coli K-12 strains, carrying the wild-type allele of the luc repressor gene with pUH84. Irrespective of the kind of rich or minimal media used, no viable transformants were obtained with either of the strains tested. Thus, in contrast to several other positive selection vectors, killing of the host cells by pUH84 needs not be induced by treatments such as temperature shift (Honigman et al., 198 1; Cheng and Modrich, 1983) or addition of mitomycin C (Ozaki et al., 1980).

On the other hand pUH84 can be introduced with high efficiency into E. coli strains such as CAIQ1 or F’IQ1dM15 which carry repressor overproducing alleles of the lad gene. These strains yielded more than lo6 ApR clones per pg of pUH84 and are there- fore suitable for propagation, maintenance and pre- paration of the vector.

(d) Cloning in pUHS4

The juxtaposition of six unique restriction sites in the polylinker region of the newly constructed plas- mid pUH84 (Fig. IB) allows cloning and reisolation of DNA fragments generated by a variety of restric- tion enzymes (Ruther et al., 1981).

A direct correlation between the yield of transfor- mants and the concentration of DNA fragments ligated with the vector (see Vernet et al., 1985) was verified by transformation of strain F-Z-dM15recA with ligation mixtures containing increasing amounts of EcoRI-digested bacteriophage J. DNA and a con- stant concentration of EcoRI-linearized pUH84. Analysis of 20 ApR clones revealed that all of them

347

T

ST

R

gH.ST

t

PST1 ACCl y;, I BAHH1 ECOR,

t

Fig. 1. (A) Construction of the positive-selection vector pUH84.

A 436-bpEcoRI-BsfNI fragment ofpUHl2 (Henrich et al., 1982)

containing the $X174 lysis gene was isolated and, after filling-in

the ends with PolIk, inserted into the likewise filled-in EcoRI site

of pUH2, thereby destroying this site. Plasmid pUH2 which

contains the lac promoter-operator region, is identical to pUR2

(Ruther, 1980) with the exception that the unique HincII, and

PstI sites were eliminated by mutagenesis with nitrosoguanidine

(Adelberg et al., 1965) and that the unique AccI site was

destroyed by AccI digestion, PolIk reaction and religation. The

recombinant plasmid pUH72 was detected after transformation

of the lacl Q’ host CAIQ’ by screening for plasmid size and Tag1

restriction analysis. Induction of CAIQ’ [pUH72] transformants

by IPTG resulted in lysis of the bacteria which verified

.the proper orientation and the functional integrity of the lysis

gene. The unique HincII site of pUH72 was used for the insertion

of the polylinker sequence of plasmid pUR222 (Rilther et al.,

1981). To be able to recover this short sequence after its inte-

gration in pUH72 by simple screening for plasmid size, prior to

its isolation from pUR222, the polylinker was “labelled” by

insertion of a 194-bp HaeIII fragment of $X174 RF DNA into

the unique EcoRI site of pUR222 which had been filled-in before

by PolIk. The “labelled” polylinker sequence was excised as a

248-bp AluI-HaeIII fragment and ligated with HincII-linearized

pUH72 giving rise to the hybrid plasmid pUH72-L. To restore

a functional lysis gene, the 194-bp fragment of +X174 RF DNA

was subsequently removed from pUH72-L by EcoRI digestion

contained recombinant plasmids. In three of these clones, the 21-kb EcoRI fragment of 1 DNA was detected, indicating that even large fragments can easily be cloned in pUH84.

(e) Controllable expression of Tc resistance

The genetic structure of the vector pUH84 (Fig. 1) implied that foreign genes could be subjected to transcriptional control by the luc promoter, provided that the cloned DNA fragments contained no inter- nal promoter or terminator sites which interfered with lac regulation. To check this anticipation, the 2038-bp HindIII-PvuII fragment of pBR322 (Sut- cliffe, 1979), containing the TcR gene but not the Tc promoter, was isolated and, after filling-in the Hind111 ends with PolIk, was inserted into the HincII site of pUH84. About 50% of the transfor- mants of strain F-Z-dM15recA obtained on Ap plates were found to be resistant to Tc and contained a plasmid, pUH84-Tc, carrying the pBR322 frag- ment in the orientation which allowed transcription of the TcR gene from the lac promoter.

In lad + strains bearing the hybrid plasmid pUH84-Tc, Tc-resistance is expressed constitution- ally because in such strains the lac repressor is titrated by the lac operator seq&nces present on multiple copies of the plasmid. A convenient possi- bility to nevertheless make use of the efficient control mechanism of the lac operon to achieve regulated transcription of genes cloned in pUH84 is the intro-

and religation. After transformation of strain CAIQ’, the vector

pUH84 was identified among the isolated plasmids by size and

restriction analysis. Plasmid sizes and positions of restriction

sites are drawn to scale. Thin lines represent plasmid portions

originating from pBR322. @X174 sequences are drawn as open

bars, parts of the lac operon are marked by thick lines. Arrows

indicate directions of transcription. A, AccI; B, BarnHI; H,

HincII; N,BstNI; P, &I; R,EcoRI; S, SalI; T, TaqI. Restriction

sites which were eliminated are striked out. The letters p o Z’

refer to the lac promoter, operator, and the N-terminal part of

the 1acZ gene, respectively. E and J are $X174 genes. The scale

of the pUH84 map is in kb. (B)The polylinker sequence of

plasmid pUH84. Based on the strategy of pUH84 construction,

the sequence was deduced from the known sequences ofthe 1acZ

gene (Kalnins et al., 1983), the pUR222 polylinker (Riither et al.,

1981) and of +X174 DNA (Sanger et al., 1978). The upward

arrows point to the ends of the 54-bp polylinker sequence.

Numbers refer to codons of the original 4X174 gene E. The

direction of transcription is given by the horizontal arrow.

348

Fig. 2. Construction of the IUCIQ plasmid pUH7. A 1724-bp

partial Hind11 fragment of the pMB9 derivative pMC7 (Calos,

1978) including the lacIQ gene together with the preceding

promoter, was inserted into the filled-in Hind111 site of the Pl5A

derived vector pACYCl84 (Chang and Cohen, 1978), thereby

inactivating its TcR gene. The resulting construct, pUH7, confers

resistance to Cm and can be propagated concurrently with

ColEl-derived replicons in the same cell. The subcloned pMC7

fragment is marked by a thick line. D, HindII; F, HindIII; R,

EcoRI.

duction of a second multicopy plasmid which is compatible with pUH84 and carries the Ia@ allele. Thereby the concentration of lac repressor in lad +

cells can be increased more than 200-fold (Miiller- Hill et al., 1968). Such a plasmid, pUH7, was con- structed (Fig. 2) and introduced into strain FZ_dMlSrecA[pUH84-Tc]. The double transfor- mants were sensitive to Tc (Fig. 3), indicating that the presence of the pUH7-encoded luclQ allele actu- ally prevented the expression of the TcR gene of pUH84-Tc. As expected, the expression of the TcR phenotype could be induced by addition of IPTG (Fig. 3).

(f ) Conclusions

The main advantages of the new vector pUH84 can be summarized as follows: (1) small plasmid size, hence acceptance of inserts larger than 20 kb; (2) six juxtaposed cloning sites; (3) high efficiency of

1.0 E Q

05

0.1 D f , , , , , , , , , t,ime,[hl ,

0 1 2 3

Fig. 3. Controlled expression of the TcR gene. Strain

F-Z-AMlSrecA[pUH84-Tc, pUH7] was grown in LB medium

supplemented with Ap and Cm at 37’ C and A,,, was measured.

o,atanA,, = 0 1 IPTG was added to give a final concentration

of 1.5 x 10e3M; q , uninduced control. One hour after induction

Tc was added to the induced culture (0) and to the uninduced

control (m).

positive selection for inserted fragments; (4) no requirement for special host strains; (5) no necessity for induction of the lysis gene in ordinary E. coli

strains; (6) controllable expression of cloned genes in the presence of the lucIQ plasmid pUH7.

In addition, plasmid pUH84 appears to be equally suitable for the rapid chemical sequencing of DNA inserts as the vector pUR222 from which the polylinker region was taken (RUther et al., 1981).

In view of these features, pUH84 may prove useful as a universally applicable plasmid vector for both cloning and sequencing of DNA.

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