New cosmid vectors developed for eukaryotic DNA cloning

223

Gene, 21 (1984) 223-232 Elsevier GENE

946

New cosmid vectors developed for eukaryotic DNA cloning + (Recombinant DNA; G418 selection; DNA mediated gene transfer; shuttle vector; cosmid rescue)

Ged Brady, Hans M. Jantzen,’ Hans U. Bernard, Robert Brown, Gtinter Schiitz and Tamotsu HashimotoGotoh * Institute for Cell and Tumor Biology, German Cancer Research Center, Im Neuenheimer Feld. 280. D-6900 Heidelberg (F.R. G.) Tel. 6221-48-4411 (Received

August

(Accepted

September

23rd,

1983)

27th, 1983)

SUMMARY

A series of ColE’l and pSClO1 cosmid vectors have been constructed suitable for cloning large stretches of DNA. All contain a single BamHI site allowing cloning of Suu3A, MboI, BglII, BcZI, and BamHI-generated fragments. These vectors have the following characteristics: (i) they are relatively small (1.7-3.4 kb); (ii) the BumHI cloning site is flanked by restriction enzyme sites enabling direct cloning of unfractionated insert DNA without generating multiple insert or vector ligation products [Ish-Horowitz and Burke, Nucl. Acids Res. 9 (1981) 2989-29981; (iii) two vectors (pHSG272 and pHSG274) contain a hybrid Tn5 KmR/G4 1gR gene which is selectable in both prokaryotic and eukaryotic cells, making them suitable for transferring DNA into eukaryotic cells, and (iv) the different prokaryotic selectable markers available in the other vectors described facilitate cosmid rescue of the transferred DNA sequences from the eukaryotic cell: CmR, ApR, KmR, (pHSG429), CmR, (pHSG439), colicin El immunity (pHSG250), (v) the cosmid pHSG272 was used successfully to construct a shuttle vector based on the BPVI replicon [Matthias et al., EMBO J. 2 (1983) 1487-14921.

INTRODUCTION

A major advance in the field of recombinant DNA has been the development of plasmids and phage

+ Dedicated

to the late Ahmad

the pioneering

work

bacteriophage discussions

Mu system, and in the memory

at his present

Research

who has carried

out

by the use of of the stimulating

with him.

* To whom correspondence rected

I. Bukhari,

in the gene transposition

and Development

ed, Minami-dai,

and reprint

address:

requests

Department

Laboratories,

l-3-2, Kawagoe,

Saitama

should be di-

of Basic Research, Hoechst

Japan Limit-

(Japan)

Tel. 492-43-

vectors, designed for specific types of genetic manipulation. Cosmids are one set of plasmid vectors containing a DNA region including the cohesive end (cos) site required for cleavage and packaging of DNA into ;1 phage heads, the presence of which allows cloning of large DNA fragments via the packaging reaction (Collins and Hohn, 1979). Recombinant cosmids of the approximate size 35-52 kb can be packaged into phage particles in vitro and transfected efficiently into Escherichia coli thus avoiding the problem of poor transformation efficiency associated with large plasmids or preferen-

1234. Abbreviations: type I; Cm,

Ap, ampicillin; chloramphenicol;

0378-l 119/84/$03.00

0

BPVI, bovine FTL,

1984 Elsevier

freeze

Science

papilloma thaw

lysate;

Publishers

kilobase

pairs;

virus,

maximal

allowance

Km, kanamycin;

kb,

tomycin;

Tc, tetracycline.

concentration;

LS, low salt medium; SE, sonic extract;

MAC,

Sm, strep-

“4 __

tial transformation Murray,

by smaller plasmids

1977; Hohn,

the

(Nunberg

possible

size of eukaryotic

large

MATERIALS

genes E. coli HB 10 1 and SK1 592 were used for bacterial

et al., 1980; Wozney et al., 1981), cosmids representing

et al., 1979; Grosveld

an

entire

transformation.

genomic

genome

et al., 198 1). Improvements

have been made in cosmid cloning:

reduction

1982) reduction

and multiple

and Burke, 198 l), selection

(Grosveld

et al., 1982). There are no reports

cosmids.

(1.7-3.4

BHB2688

and

mixes for A. Plasmids

or

ED8767

(N.

for in vitro packaged

BHB2690

(Hohn,

1979)

of the in vitro packaging

used in this study are listed in

Table I.

(Ish-

in eukaryotes (b) Media and antibiotics

of one

vector incorporating all three improvements. Here we describe the construction and characterisation of a series of small cosmids

HBlOl

were used for preparation

of the

insert formation

Horowitz

Either

Murray) were used as recipients

(Royal

cosmid size (Miwa and Matsubara, of polycosmids

AND METHODS

(a) Bacterial strains and plasmids

genomes

size of individual

provide a suitable way of cloning eukaryotic segments

and

1979; Chia et al., 1982).

Owing to the overall and

(Hohn

LS medium is described elsewhere (HashimotoGotoh and Inselberg, 1979b), Km solution (5 mg/ml) is in 35 7; ethanol, Ap and Cm solutions (50 mg/ml)

kb) all of which

can be treated so as to avoid both polycosmids and multiple insert formation. These vectors are designed to allow cosmid rescue by use of their different

are in 707, ethanol, and G418 is in 1 M N-2hydroxyethylpiperazine-N’-2’-ethanesulphonic acid buffer pH 7.3. Final concentration of each drug in the medium is 50 pg/ml (Km, Ap and Cm) or 800 pg/ml (G418), unless otherwise stated. Colicin

selection markers (Lund et al.. 1982). One of these vectors, pHSG272, containing a dually selectable marker (KmR in E. coli and G418R in eukaryotic cells) was successfully used to construct a BPVIbased shuttle vector (Matthias et al., 1983). TABLE

1

Previously

described

plasmids Origin

Plasmid

used in this study of

Relevant

properties

”

Reference

replicon pAG60

pBR322

ApR, G41ER

Colbtre-Garapin

pHSG124

pSClOl~ColEl

TcR.ApR.

Hashimoto-Gotoh

and lnselburg

pHSG306

ColEl

Tn5 (KmR), imr~ +

Hashimoto-Gotoh

and Timmis

pHSG402

ColEl

imm +

Hashimoto-Gotoh

and Inselburg

pHSG415s

pSClO1

KmR. CmR. ApR

Hashimoto-Gotoh

et al.

pHSG422

psc101

KmR, CmR, ApR, cos

Hashimoto-Gotoh

et al. (1981)

pN01523

pBR322

Ap’,

Dean (1981)

pUC8

pBR322

ApR, polylinker

Vieira and Messing

nAN7

pBR322

supF, polylinker

Seed (1983)

nvx

pBR322

supF, polylinker

Seed(1983)

a imm+

confers

immunity

range of eukaryotic

to colicin E 1;

cells. SW+ indicates

irvrn +

str +

cos+. contains the n-$80 cosmid packaging the presence

of the wild-type

et al. (1981)

( 198 1) (1979a and 1979b)

(198I )

(1982)

site. G418R, confers

gene for ribosomal

(197Yb)

S7 protein.

resistance

to G418 in a wide

225

El protein was prepared and used as described elsewhere (Hashimoto-Gotoh and Timmis, 198 1). (c) Enzymes All restriction enzymes were from either BRL or New England Biolabs, calf intestinal alkaline phosphatase from Boehringer, Mannheim; E. coli alkaline phosphatase from P.L. Laboratories; T4 DNA polymerase from BRL; and T4 DNA ligase from New England Biolabs.

(f) Prokaryotic and eukaryotic transformation Highly competent E. co/i HBlOl or SK1592 cells were prepared by CaCl, treatment as described by Dagert and Ehrlich (1979). DNA transfection of eukaryotic cells was carried out essentially as described in Matthias et al. (1983). Mouse NIH3T3, LTK- and Rat 2 cells were used as recipients.

RESULTS

(d) Preparation of insert DNA

(a) Cosmid construction

High-M, total cellular DNA was extracted as described by Colbere-Garapin et al. (198 1). The size of the DNA was estimated by running it on a 0.2% agarose gel alongside 1 Sam7 DNA which had been ligated via the cos ends to give dimers. DNA, of which > 80% migrated above the dimer, was partially digested with Suu3A in 10 mM Tris . HCI, pH 7.4,2 mM MgCl, (Pirrotta, V., personal communication) at 37 ’ C for 30 min followed by heat inactivation for 10 min at 70°C. The extent of digestion was assayed by electrophoresis on a 0.2% agarose gel and the DNA fragments were dephosphorylated as described for the vector preparation (see RESULTS, section b). After inactivation of the phosphatase by heating at 70’ C for 10-30 min in the presence of 10 mM EGTA, the insert DNA was ethanol-precipitated, washed with 70 y0 ethanol and resuspended in 10 mM Tris * HCl pH 8, 1 mM EDTA to a concentration of about 200 pg/ml.

(1) pHSG429,

(e) Ligation, in vitro packaging and transduction Prepared vector (0.5 pg) and insert DNA (1 pg) were ligated in 10~1 overnight at 16°C. In vitro packaging mixes were prepared as described in Scalenghe et al. (198 1). Up to 3 ~1 of ligated vector and insert were packaged by mixing with 5 ~1 of SE and 12 ~1 of FTL and incubating 60 min at room temperature followed by dilution in LS media containing 0.2% maltose and 10 mM MgCl,. Transduction was carried out essentially as described in Grosveld et al. (1981) using either HBlOl or ED8767 cells as recipients.

a pSClOl-derived

Af,

CmR, KmR

cosmid

Plasmid pHSG4 18s is identical to the temperaturesensitive plasmid vector pHSG415s (“s” stands for temperature-sensitive; pHSG415r, a temperatureresistant derivative of pHSG415s, is also available; Hashimoto-Gotoh et al., 198 1; Hashimoto-Gotoh, T., unpublished data) except that the Cm gene is located on the HaeII fragment flanked by the Hue11 Ap and Hue11 rep fragments. pHSG418s was overdigested with PstI, ligated and transformed into SK1592 cells. Most of the transformants were KmR, CmR, but ApS reflecting loss of the PstI site and consequent inactivation of the ApR gene. One of these transformants pHSG425, was analysed by restriction digestion and apart from the absence of a PstI site was identical to the parental pHSG418s. The Bst EII ApR cos+ fragment of pHSG422 was ligated into the single Bst EII site of pHSG425 and transformed into SK1592 (ret + ) to yield CmR, KmR and ApR multiple resistant colonies. A number of these transformants were examined and the smallest taken and designated pHSG429. The Hue11 restriction pattern of pHSG429 differs from that of pHSG422 in the following: the HaeII KmR fragment is smaller and the 1.2-kb Hue11 fragment consisting of $80 DNA segment is missing (Fig. 1). However, pHSG429 still contains the n-$80 cos site since it shows equivalent packaging efficiency in vitro to that of pHSG422. (2) pHSG250 and pHSG262, two ColEI -derived cosmids having imm + and KmR markers respectively pHSG402 is a mini ColEl-plasmid (imm+)

derived from pHSG124 (ColEl::Tn3-pHSG1

com-

PKiG418S

P&f deletion

I ?lVX

polylinker

I

into

,

EcoUIstteandt-emovafofBamHI

inww

deletion

fragment

2.8 kb I

4

4

pHSG272

Fig,

I. Construction

HaeII sites. WI’(0) transcription

case of the pBR322 indicate

outline of(a) cosmids

constructs,

genes confering

resistances

pHSG250,

the origin of replication,

and approximate

(4

pHSG274

pHSG262,

pHSG439.

~-0s (m) the A-+80 cohesive

extent of the genes shown. Restriction pAG60

Km resistance

and pBRG418 in prokaryote

(see also Fig. 2). The drawings

and(b)

pHSG272

end site, arrows

sites destroyed

and pHSG274.

within the plasmids

during cloning procedures

No. 4, the Hue11 sites, oA and resistance and G418 resistance

are not necessarily

to scale.

in eukaryote,

Open triangles indicate

are in brackets.

genes are not shown.

respectively.

represent

the direction

/crrnG confers

of

In the

Km and G418

both Km and G41X

221

posite plasmid) by BumHI digestion and ligation (Hashimoto-Gotoh and Inselburg, 1979a,b). It contains nine WaeII restriction sites and unique EcoRI and BarnHI sites. pHSG402 was partially digested with HueII, ligated and introduced into SK1.592. Transformants (imm+) were selected and pHSG23 1 containing three HaeII sites and single EcoRI and BamHI sites, was chosen for further construction. The zvx poly~ker EcoRI fragment (EcoRI - CZaI- Hind111 - XbaI - BgZII - &I - BamHI EcoRI) was ligated into the EcoRI site of pHSG23 1 and subsequently removed by BamHI digestion to give pHSG232. pHSG232 was digested completely with BarnHI followed by gap filing with T4 poIymerase, and partially digested with Hue11 and ligated to the ~~eII(p~i~~~~II(cornplete) KmR cas .+ fragment of pHSG429. The resulting plasmid, pHSG255, contains three Be11 sites and single EcoRI, BamHI (reconstituted) and HincII sites. The region between the EcoRI site and the cos site was sequenced and the distance between the sites deter-

mined accurately (Hashimoto, S., personal communication; Fig. 2). The imm’ KmS plasmid pHSG250 was produced by removing the Hoe11 KmR fragment of pHSG255. pHSG262 (imm KmR) was made by HueIII(partial) and EcoRI(complete) digestions of pHSG25.5 followed by gap filling with T4 polymerase and ligation (Fig. 1). The EcoRI site was reconstituted in pHSG262. (3) pHSG439, a pSClO1 -derived CmR cosmid pHSG415s DNA was mutagenized by NH,OH, as described by Hashimoto-Gotoh and Sekignchi (1977) and used to transform SK1592. One transformant showing a fourfold increase in Ap resistance (MAC of 200pg/ml) was selected and named pHSG415h (,,t stands for higher copy number). pHSG415h was digested with HaeII, ligated and introduced into SK1592 cells. ApR, CmR and KmS transformants were selected and one was named pHSG413h. The WueII-BarnHI ApR fragment of pHSG413h was replaced by the HaeII-BarnHI cos +

EcoRI HinclI PamHI, Hind

EcoRI

RsaI

AvaI

ClaI

HaeU

EcoRI

pHSG2J2

ori

HaeII

3.4kb

f

SmaI Fig. 2. Scale drawings pHSG274 promoter

represent

ofcosmids the location

(Ptk promoter,

the structural

pHSG250.

pHSG262,

and the direction

see RESULTS,

gene of Tn.5 Km resistance

pHSG272,

pHSG274

of the pBR322

section a, 4). kanG represents and the polyadenylation

and pHSG439;

Pl promoter

internal

arrows

shown in pHSG272

and the Herpes simplev virus thymidine

the hybrid gene consisting

ofthe Pl promoter,

site of Herpes simplex virus thymidine

kinase

kinase

and gene

the Ptk promoter,

gene. ori and cos are

as in Fig. 1.

fragment of pHSG262. The resulting plasmid, pHSG439, had an in vitro packaging efficiency equivalent to pHSG429. (4)pHSG272

that

of the

andpHSG274,

cosmids

pHSG422

and

two ColEl -derived, dually

selectable cosmids

The plasmid pAG60 contains the KmR gene of transposon Tn5 flanked by the promoter (Ptk) and polyadenylation (pAtk) site of the Herpes simplex virus thymidine kinase (HSVtk) gene. This hybrid gene (Ptk-kan/G418-pAtk) efficiently confers resistance in all mammalian cells tested to the toxic antibiotic G418 but does very poorly resist Km in E. coli HBlOl (Jimenez and Davies, 1980; Southern

and Berg, 1982; Colbere-Garapin et al., 1981). To increase the KmR level to a workable value, a partial PvuII fragment of pAG60 containing this hybrid gene was ligated to Hind111 linkers and cloned into the Hind111 site of pBR322 next to the Pl promoter (Stiiber and Bujard, 1981; see Fig. 1). Bacterial clones showing increased KmR levels were picked and analysed, all contained the Tn5 KmR gene in the same orientation with the Pl promoter in tandem with the tk promoter. One of these clones (pBRG4 18 No. 4) had only one Hind111 site, this being 3’ of the Tn5 gene. The ClaI(tilled)-BamHI fragment of this clone containing the Pl promoter plus Ptkkan/G418-pAtk hybrid gene (kanG for short), and the BamHI(complete)-HaeII(partia1) ori fragment

229

and the HincII-Hue11 cos fragment of pHSG262 were isolated and these three fragments were ligated to yield pHSG272 (see Fig. lb). Furthermore, the pBRG418 No. 4 BamHI(filled)-ClaI kanG fragment was cloned between the XhoI(lilled) and ClaI sites of pHSG255. An RsaI fragment containing ColEl sequences downstream of the replication origin, the N’-terminal region of the Tn903 Km gene, and the pBR322 sequences upstream of the PI promoter, was removed by partial RsaI digestion followed by ligation. The C/-terminal region was removed by CZaI (complete-tilled) and HaeII (partial nibbled-filled) digestion. Then the BarnHI-HincII from derived pHSG255 oligonucleotide (G 1GATCCTGCTTTTTGTT r GAC; Hashimoto, S., personal communication) was replaced by the pUC8 BarnHI-HincII oligonucleotide of (G lGATCCGTC t GAC) to have a single SaII site. The resulting plasmid was named pHSG274 (Fig. lb). (b) Preparation of vectors for cosmid cloning Cosmids pHSG250, pHSG262, pHSG272, pHSG274, and pHSG439 were prepared so as to allow cloning into the BamHI site following the strategy described by Ish-Horowitz and Burke (198 1). For example, equal amounts (50-500 pg) of pHSG262 DNA were digested separately with EcoRI and HincII, checked for completion of digestion by gel electrophoresis, treated with calf intestinal or bacterial alkaline phosphatase, followed by phenol-chloroform (1: 1, v/v) extraction and ethanol precipitation. The extent of dephosphorylation was checked by ligation of an aliquot and gel electrophoresis, no bands additional to the linear vector were seen. Both digests were then pooled and cut with BamHI and checked for ligation via the BumHI ends. The same procedure can be used for pHSG250 and a similar protocol can be used for pHSG439 (using the HaeII, bacterial phosphatase at 60°C H&c11 and BamHI sites), pHSG272 (using the NruI, HincII, and BamHI sites) and pHSG274 (using the EcoRI, SalI, and BamHI sites). In the case of pHSG250, pHSG262, pHSG274 and pHSG439, those fragments produced not containing a cos site and therefore not contributing to in vitro packaging are small enough to be removed by either gel filtration or size-selective isopropanol precipi-

tation. Removal of these fragments can be monitored by ligation and gel analysis which will show an increase in the amount of dimer formed when the small fragments are removed. Vector DNAs prepared in this way gave 104 -5 x 104 recombinant cosmid colonies per pg of dephosphorylated insert DNA after ligation and in vitro packaging. (c) Selection for pHSG272 and pHSG274 in E. coli and animal cells MAC for Km was determined for pHSG262, pHSG272, pHSG306 (ColEl::TnS) and pAG60 plasmids in ED8767 cells, as described previously (Hashimoto-Gotoh and Sekiguchi, 1977). Km concentrations of 2.5, 5, 10,20,40,80 pgg/mlin LS agar were used, and the MAC was taken as the highest concentration allowing confluent growth of the strain examined. In our hands the MAC of pAG60 was 2.5 pg Km/ml, whereas pHSG272 and pHSG274 which contain the Tn5 gene of pAG60 plus an additional prokaryotic promoter exhibited a MAC of 40 pg/ml equal to that of the original Tn5 gene contained in pHSG306. pHSG262, unlike pAG60 and pHSG272, derives its KmR gene from Tn903 and has a Km MAC of > 1 mg/ml (not shown). Mouse LTK -, NIH3T3 and Rat 2 cells were used to assay transformation, stable maintenance and expression of G418 resistance of pAG60, pHSG272, and pHSG274. Cells were transfected separately with l-2 pg of pAG60, pHSG272 and pHSG274 plus carrier DNA (see MATERIALSAND METHODS, section f). After a 14-day incubation in G418containing medium, G418-resistant colonies were counted. Reproducibly pHSG272 and pHSG274 gave an equivalent or higher number of G418R colonies per pg DNA as pAG60 (Table II; Matthias et al., 1983).

DISCUSSION Cosmid cloning provides the most practical means to isolate an entire, large (>25 kb) functional eukaryotic gene, since the ,J in vitro packaging reaction will encapsulate ligated vector plus insert DNA of an average 40-45 kb in length. To increase

TABLE Eukaryotic

11 transformation

efficiency

of pHSG272,

pHSG274

and pAG60

.1 _

Eukaryotic

cell line

Plasmid

Rat 2

NIH 3T3

LTK

LTK

a Rat 2, NIH3T3 G418-containing h High-M,

and LTK-

.4mount

G418 transformation

efficiency

pAG60

40

110

pHSG272

40

150

pAG60

40

110

pHSG’72

40

93

pAG60

20

270

pHSG272

20

270

pAG60

40

pHSG274

40

220 660

cells were transformed

( > 30 kb) human placental

media after appropriate

as described

and counted.

or calf thymus

’ At the same time as the cells are transferred differential

DNA h

of viable cells/pg of plasmid

media colonies were stained

plated into non-selective

of carrier

(ng)

in MATERIALS

The two sets of LTK

AND METHODS, results presented

f. After two weeks in

are from independent

experiments.

DNA.

into selective media after transformation, dilution and the resulting

cell death and growth after the transformation

section

per 10’

’

an aliquot was taken from each transformation,

colonies were counted.

The viability count takes into account

step. In our hands the viability varied 20-90”,,

the average size of packaged target DNA it is important that the size of the cosmid vector, including sequences for autonomous replication, a selectable marker, and the 1 cos region be kept small. Recently, Miwa and Matsubara (1982) have identified the minimum size of the 1 cos region required for optimal in vivo and in vitro packaging, and have cloned this 250-bp cos region into pBR322 creating a small functioning cosmid ppBest322 (note that this cos region is 1-2 cos and differs from ours, that is ~-@O cos). In this report we have described even smaller cosmids based on ColEl and pSClO1 replicons. Because of this ability to encompass large segments of DNA recombinant cosmids are particularly useful for studying the expression of cloned eukaryotic genes after introduction into a suitable eukaryotic cell line. Lund et al. (1982) have used a TcR cosmid to “rescue” a eukaryotic gene via its linkage to an integrated prokaryotic selectable marker. For these types of experiment the following features are advantageous. (1) A eukaryotic selectable marker, ensuring identification of those cells taking up DNA: (2) a range of vectors containing different prokaryotic selectable markers, allowing rescue of the initial integrated marker. In this report we have described a series of cosmid vectors which satisfy the above conditions. pHSG250, pHSG262, pHSG272 and

from experiment

to experiment.

pHSG274 are ColEl-based vectors and relatively small, 1.8 kb, 2.8 kb, 3.4 kb and 3.4 kb, respectively. pHSG250 is imm + KmS, pHSG262 is imm KmR, pHSG272 (imm ) and pHSG274 (imm +) are KmR in E. coli and G41gR in eukaryotic cells. For cosmid cloning they can be prepared in such a way as to avoid polycosmids and multiple insert formation and the need to size-fractionate the insert DNA (see RESULTS, section b, and Ish-Horowitz and Burke, 1981). Also the small fragments, containing no cos site, generated

when preparing

“cosmid

arms” from

pHSG250, pHSG262, pHSG274 and pHSG439 (see RESULTS, section b, and Fig. 2) which presumably only act as competitors for insert fragments during vector/insert ligation, are small enough to be removed either by size-selective precipitation (0.1 vol. 2 M Na . acetate pH 9.0, 0.6 ~01s. isopropanol, at room temperature for 30 min) or column chromatography, thus increasing the proportions of usable substrate molecules for in vitro packaging. Furthermore, eukaryotic genes have now been isolated by transformation of an animal cell line with chromosomal DNA physically linked to a bacterial plasmid of marker (tag), identification of the correct eukaryotic clones and recovery of the desired gene via linkage to the bacterial sequences (Perucho et al., 1980; Lowy et al., 1980). Because they contain a

231

single gene selectable in animal and bacterial cells, the use of pHSG272 or pHSG274 as such a tag in the form of a ligation mixture or recombinant cosmids would allow selection of transformed eukaryotic cells via G418 resistance and also ensure that a rescuable KmR gene selectable in E. coli is present. Km rescue has an advantage compared to Ap rescue since no secondary colonies appear even after prolonged incubation. In connection with eukaryotic transformation by bacterial vectors it is worth noting that none of the ColEl-derived cosmids described here contain the so-called poison sequences known in pBR322-derived vectors and thought to interfere with replication in mammalian cells (Lusky and Botchan, 1981; see also Matthias et al., 1983). pHSG272 has also been ligated to the entire bovine papilloma virus genome creating a vector which is capable of dually selectable stable autonomous replication in animal and bacterial cells (Matthias et al., 1983). Apart from the n-$80 cos region, the pSClO1 derived cosmid pHSG439 has no sequence homology known to the ColEl cosmids pHSG250, pHSG262, pHSG272 and pHSG274 and also no homology to pBR322. A pHSG439 derivative lacking the EcoRI site in Cm gene (without inactivating the gene), the HaeII cos fragment and all of the IS1 sequences flanking the Cm gene has been constructed, ligated to the rcAN7 polylinker (EcoRISmaI-BarnHI-SalI-PstI-BgZII-XbaI-HindIII) and designated as pHSG591 2.7 kb). To allow positive selection (SmR) for recombinants, the SstI (nibbled-tilled)-BamHI fragment of pN01523 containing the ribosomal S7 gene was inserted between the SmaI and BamHI sites of this plasmid lacking PstI and BgZII sites and designated pHSG660. Plasmid pHSG660 has the following single sites; EcoRI-HpaI . Sph I * Pst I . SmaI-BumHI-SalI-XbaI-Hind111 (any insertion at or replacement of the underlined region representing str+ gene can be selected by Cm and Sm double selection in stp cells, data not shown and see also Dean, 1981). pHSG591 and pHSG660 are currently being used to develop a cosmid recombination screening method with a combination of pHSG262 or pHSG274, similar to that developed for 1 vectors by Seed (1983). (All final constructions described in this article have been sent to and are available from the Plasmid Reference Center at Stanford University.)

ACKNOWLEDGEMENTS

We thank P. Kourilsky, H. Lehrach, M. Nomura and N. Murray for sending plasmids and bacterial strains used in this study, S. Hashimoto for sequencing data of the 2 cos region, P.D. Matthias for critical comments on the manuscript, Ms. M. Cole and Y. Kurosu for their patience and typing, and K.N. Timmis in whose laboratory (Max-Planck-Institut fur Molekulare Genetik) the initial part of this work was done.

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