Molec. gen. Genet. 157, 175-182 (1977) © by Springer-Verlag 1977

Molecular Cloning and in vitro Transcription of Bacillus subtilis Plasmid in Escherichia coli Sueharu Horinouchi, Takeshi Uozumi, Takayuki Hoshino, Akio Ozaki, Sadayo Nakajima, Teruhiko Beppu, and Kei Arima Department of Argricultural Chemistry, The University of Tokyo, Bunkyo-ku, Tokyo, Japan

Summary. A composite plasmid (pAT2010) has been constructed in vitro from RSF2124 and Bacillus subtilis IFO3022 plasmid (pAT1060) by covalent joining of the two DNA molecules by means of Escherichia coli DNA ligase through the cohesive ends generated by restriction endonuclease RI (EcoRI) cleavage. The composite plasmid was selected by transformation of E. coli C600 r- m - with the ligated mixture after enrichment for composite plasmid by preparative agarose gel electrophoresis, and plating of the transformants on a medium containing ampicillin and colicin El. Treatment of the composite plasmid with EcoRI yielded two fragments corresponding to the linear forms of the parental plasmids. The composite plasmids replicated as biologically functionally units in E. coli, and expressed genetic information carried by RSF2124. In the presence of chloramphenicol, the composite plasmids continued to replicate and the copy number gradually increased. Such nature of replication in the presence of chloramphenicol is characteristic to RSF2124 derived from colicin E1 factor, and so it is suggested that the replicator of RSF2124 is functional in the composite plasmid. The composite plasmid was found to synthesize mRNA of B. subtilis plasmid in cell-free extracts of E. coli, by hybridization of the mRNA to the original plasmid DNA of pAT1060.

Introduction Molecular cloning is a powerful tool for purification and amplification of D N A fragments. These techFor offprints contact." Sueharu Horinouchi, Laboratory of Fer-

mentation, Department of Agricultural Chemistry, The University of Tokyo, /-1-1 Yayoi, Bunkyo-ku, Tokyo, Japan

niques consisting of two steps have recently been developed which allow the cloning in E. coli of bacterial or eukaryotic genome segments. The first step is that the fragments to be cloned are joined in vitro to an autonomously replicating vehicle molecule, such as a drug resistance factor (Chang et al., 1974), colicin E1 factor (Hershfield et al., 1974; Tanaka and Weisblum, 1975; Collins et al., 1976) or bacteriophage 2 genome (Thomas et al., 1974; Struhl et al., 1976). Secondaly, the composite DNA molecules are selected after transformation or transfection in E. coli, and then cloned by single colony isolation or plaquing. These techniques have been used for the genetic mapping of the genome of higher organisms such as Drosophila melanogaster (Wensink et al., 1974) or sea urchin (Wu et al., 1976) and the genetic control of plasmid replication (Timmis et al., 1974). EcoRI endonuclease cuts at specific sites in a double-stranded DNA and therefore EcoRI-fragments can be reannealed and ligated for constructing composite DNA molecules. RSF2124 is a ColE1-Ap r cloning vehicle constructed by Magdalene et al. (1975), which have one EcoRI cleavage site. The use of the ColE1 plasmid and several derivatives of this plasmid as a cloning vehicle for certain DNA fragments of genome has become increasingly important. The replication of RSF2124 continues in the cells of late log phase culture in the presence of chloramphenicol (CAP). Elimination of chromosomal replication increases the number of copies of this plasmid and at the same time the number of inserted sequences in a RSF2124 recombinant molecule. Recently it has been reported that Bacillus subtilis (Lovett and Bramcci, 1975; Tanaka et al., 1977) and Bacillus pumilus (Lovett, 1973) have covalently closed circular (CCC) duplex plasmid. Those plasmids but one (Lovett et al., 1976) have not been known as having detectable genetic markers conferred on the host. We have also screened the plasmid DNA of

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B. subtilis and isolated six different types of plasmids, which were classified by molecular weight and restriction endonuclease sensitivity. Of these six types of plasmids, five were the same as those found by Tanaka et al. (1977) and Tanaka (1977, J. Bact. in press), and one was a new plasmid which is present in B. subtilis IAM1075. But we could not find any detectable genetic markers on any of these plasmids (Uozumi et al., manuscript in preparation). Despite of the usefulness of recombinant DNA molecule techniques, the in vitro recombination systems have not been established in B. subtilis which is non-pathogenic and does not inhabit the human body. To establish these techniques in B. subtilis, we have isolated a restrictionless and modificationless mutant (B. subtilis RMI25) of B. subtilis 168 YSll (Uozumi et al., 1977), which is possible to use as a recipient of foreign DNA. Then we have tried to construct composite plasmid consisted of E. coli plasmid and B. subtilis plasmid. It seems possible to use such a composite plasmid as a vehicle for B. subtilis. This report describes the in vitro construction of a composite plasmid between RSF2124 and a B. subtilis plasmid pAT1060 and in vitro transcription of the B. subtilis plasmid in E. coli.

Materials and Methods Bacterial Strains The strain of E. coli used were: C600 r~ m(c (thr, leu, thi; Meselson and Yuan, 1968) obtained from R.W. Davis (1974), C600 (carrying RSF2124; Magdalene et al., 1975) from T. Tanaka, RY13 (carrying the RI plasmid; Yoshimori, 1971) from H.W. Boyer, JC411 (thy, met, his, arg, lac, carrying ColE1) from Y. Sakakibara (1974), N1625 (ligase over-producing strain; Gottesman et al., 1973) from I.R. Lehman, AD1 (Sin", (proA/B, cxm, argF, lac)V,argIV; Kikuchi et al., 1975) from M. Dohi. Bacillus subtilis IFO3022 carrying pAT1060 was a stock culture in our laboratory.

Enzymes EcoRI was prepared from E. coli RY13 by the method of Tanaka and Weisblum (1975). D N A ligase was from E. coli N1625 by preparative disc gel electrophoresis in succession after the purification method of Modrich et al. (1973). HindIII was purchased from Miles Laboratories Inc. BsuG was prepared from B. subtilis IAM1247 by streptomycin sulfate precipitation, DEAE-cellulose column chromatography, and phosphocellulose column chromatography (Hoshino et al., manuscript in preparation).

ethylene glycol precipitation method (Humphreys et al., 1975). For preparation of B. subtilis plasmid, ceils of B. subtilis IFO 3022 carrying the plasmid were grown to late log phase in L broth, harvested, and lysed by the same method as for preparation of RSF2124 and ColE1. Solid cesium chloride (CsCI) and ethidium bromide (final concentration was 500 ~tg/ml) were added, and the final density was adjusted to 1.615 g/ml. The CsCl-ethidium bromide equilibrium density gradient centrifugation was performed at 15 ° C and 39,000 rpm for 48 h in a Hitachi 65T angle rotor. After centrifugation, the lower band (closed circular DNA) was collected with an aid of peristaltic pump under an ultraviolet (UV) illuminator. The solution containing superhelical D N A was extracted with isoamyl alcohol five or six times in order to remove ethidium bromide, and CsC1 was removed by dialysis against 0.5 x SSC (SSC contains 0.15 M NaCI+0.015 M sodium citrate) + 1 m M EDTA, pH 7.5.

Ethidium Bromide-Agarose Gel Electrophoresis Gel electrophoresis was carried out according to Sharp et al. (1973). D N A solutions (20-50 gl) were subjected to electrophoresis in 0.7% (W/V) agarose dissolved in Tris:HC1 buffer (0.04 M Tris:HCl, pH 7.9, 0.005 M sodium acetate, 0.001 M EDTA, pH 7.7) containing 0.5 gg/ml of ethidium bromide. The samples were adjusted to 8% sucrose-0.025% bromophenol blue before loading on the gels. For direct electrophoretic analysis of ethanol precipitated D N A from cleared lysates, 2 ~tl of ethidium bromide (5 mg/ml) was also added to the sample in order to ensure the availability of sufficient ethidium bromide for intercalation into DNA. Electrophoresis was performed for 3 h at 2.5 mA/gel (0.5 x 9 cm) at room temperature. After the run, the gels were blown gently out of the tubes and examined by direct illumination from short wave UV light. The D N A was seen as fluorescent bands and photographed through an orange filter on Kodak Tri-X pan film. For electrophoretic analysis of fragments of D N A digested with restriction endonucleases, 1% agarose slab gel (2 mm thickness x 13 cm width x 12 cm height) was employed, and the electrophoresis was carried out at constant I00 volts for 3 h.

Construction of the Composite Plasmid EcoRI digestion of the two plasmid was carried out as follows. The reaction mixture in a final volume of 400 ~tl containing 0.09 M Tris:HCl, pH 7.4, 0.01 M MgCI2, 1 m M EDTA, plasmid DNAs (8.7 gg of RSF2124 and 14.8 ~tg of pAT1060), and EcoRI (80 lal) was incubated at 37 ° C for 1 h and the reaction was terminated by heating at 65 ° C for 10 min (A mixture). Completeness of digestion was determined by electrophoresis of a 50 ~tl sample of A mixture on agarose gel as described above. The ligase reaction was performed in a final volume of 510 gl. A portion of A mixture (300 gl) was placed on ice and 155 gl of the following buffer was added: 0.07 M Tris:HC1, pH 8.1, 2.5 mM EDTA, 0.02 M MgC12, 0.3 m M NAD, 0.025 M ammonium sulfate, 0.05 M NaC1, 0.02% bovine serum albumin (crystallized and lyophilized), and D N A ligase (50 lal). The reaction mixture was incubated at 12°C for 40 h (B mixture).

Plasmid DNAs

Enrichment for Composite PIasmid by Preparative Agarose Gel Electrophoresis

Cleared lysates ofE. eoli C600 (RSF2124) and JC411 (ColE1) were prepared by Iysozyme-EDTA-SDS procedure (Tanaka and Weisblum, 1975) after amplification by the incubation for 20 h in the presence of chloramphenicol (Clewell, 1972), followed by the poly-

To enrich the composite plasmid, B mixture was subjected to electrophoresis according to Tanaka and Weisblum (1975). A portion (100 gl) of B mixture was layered on agarose gel and electrophoresis was carried out as described above. After the run, the D N A bands

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177

with mobilities slower than linear form of RSF2124 were excised with a razor blade. The gel segments were extruded through a size 19 hypodermic needle into 1 ml of cold Tris:HC1, pH 8.0, 0.3 M NaC1, and 0.01 M EDTA. The suspension was kept on ice for 12 h, and frozen with acetone-dry ice. After storage at - 8 0 ° C for 3 h, it was thawed and gel particles were removed by ultracentrifugation at 30,000 x g for 30 rain at 4 ° C. The supernatant was filtered through a membrane filter (HAWP-02500; Millipore Corp.) to remove fine particles of agarose, extracted three times with isoamyl alcohol to remove ethidium bromide, and dialyzed against 0.1 x S S C + I m M EDTA (pH 7.5) (C mixture).

Table 1. D N A - R N A hybridization data

Transformation and Selection

DNA to be immobilized on filter was denatured by heating to 100° C for 4 min in 0.5 N NaOH in 6 x SSC, following the liuearization with EeoRI-digestion, and rapidly cooled in an ice bath. The DNA solution containing 25 gg D N A was passed through a filter prewashed with 6 × SSC (pH 7.0). The loaded DNA filters were washed with 50 ml of 6 x SSC, dried at room temperature overnight, and finally dried in vacuum at 80°C for 3 h, essentially according to GilIespie and Spiegelman (1965). Hybrids were formed by immersing the filters in vials containing [3H] labeled R N A in 30% formamide-0.1% SDS in 2 x S S C , at 24°C for 30h as described in materials and methods. The filters were removed from the hybridization solution and each side was washed with 50 ml of 2 x SSC. To degrade the nonspecific hybrids, the filters were then immersed in 10 ml of 2 x SSC containing 20 ~tg/ml of heattreated pancreatic RNase and RNase T1. After RNase treatment, the filters were again washed on each side with 50 ml of 2 x SSC, dried, and counted in a scintiilation counter. Transcription in vitro with S100 extracts was carried out as described in materials and methods. Control experiment (S100 reaction without pAT2010 DNA) was performed in the same buffer as described above, in which pAT2010 DNA as template was omitted

Transformation was carried out according to Lederberg and Cohen (1974). To 200 gl of the competent cells of E. coli C600 r m - , 100 gl of cold C mixture was added, and incubated at 0 ° C for 30 rain. The ceI1-DNA mixture was then subjected to a heat pulse at 42°C for 2 min, chilled, and finally diluted in 10 volumes of fresh L broth. The cells were allowed to grow at 37 ° C for 90 min on a s h a k e r and aliquots were plated on nutrient agar plates containing 30 gg/ml of ampicillin and colicin E1 to select E. coli transformants. Crude colicin El was prepared as described by Foulds and Barett (1973). After single colony isolation, all transformants were examined by agarose gel electrophoresis of the cleared lysates, whether they had plasmids of higher molecular weight than that of RSF2124, and were tested as for colicin immunity.

Electron Microscopy The D N A was picked up using Hostaflex-coated grids by the technique of Davis et al. (1971), and rotary shadowed with platinumpalladium. A Hitachi HU-12 electron microscope was used.

In vitro Transcription 1. Preparation of SlO0 Extracts. The strain AD1 was used for the production of $30 extracts according to Zubay et aI. (1970) except that the incubation step was omitted. Supernatant solutions were prepared by centrifugation of $30 extracts for 3 h at 105,000 x g at 4 ° C. After centrifugation the upper two-thirds of the cellular extracts were collected and dialyzed against 0.01 M Tris:HC1, pH 8.2, 0.014 M magnesium acetate, 0.06 M KCI, 0.001 M dithiothreitol. This solution (S100) was rapidly frozen with acetonedry ice and stored in 0.6 ml aliquots at - 8 0 ° C.

2. Transcription of pATiO60 in the Composite Plasmid. Standard reaction mixture for in vitro transcription, which is slightly modified from that of Sens and James (1975), contained per ml: 0.023 M Tris:HCI, pH 7.9, 0.015 M magnesium acetate, 0.1 m M dithiothreitol, 0.15 mM ATP, CTP, GTP, 0.075 mM UTP, 0.1 millicurrie of [5, 6-3H] UTP (specific activity, 45 Ci/mmoI, 1 mCi/ml; purchased from The Radiochemical Centre, Amersham), 60 ~tg of the recombinant D N A (pAT2010), and 350 gl of S100 extracts. The reaction was initiated by the addition of NTP's, following the preincubation without NTP's for 5 min at 37 ° C. After 15 rain, the reaction was terminated by the addition of 2.2 ml of cold solution containing 0.1 M Tris:HCl, pH 7.0, 3 mM magnesium acetate, 0.2 mg/ml t R N A (from yeast) and 25 ~tg/ml of DNase I (RNase-free). [3H] labeled R N A was extracted with an equal volume of distilled phenol saturated with 50 m M sodium acetate (pH 5.2) and 0.01 M MgC12. R N A was precipitated by the addition of 2 volumes of 95% ethanol and standing at - 2 0 ° C overnight. The precipitate was collected by centrifugation at 10,000×g for 30 min at 4 ° C and dissolved in 2 × SSC (pH 7.0).

DNA on filter

pAT2010 RSF2124 pAT1060 none

Hybridization (c.p.m.) of RNA synthesized in S100 with pAT2010

Without pAT2010

1972 + 108 716± 16 1388_+ 88 97+ 5

101 _+5 81+1 74+4 58_+5

3. Hybridization Procedure. The quantity of pAT1060 m R N A synthesized in vitro was determined using the hybridization method of Bonner et al. (1967). The in vitro transcripts were hybridized for 30 h at 24 ° C, in a volume of 1 mI containing 30% formamide in 2 x S S C and 0.l% SDS, to pATI060 D N A immobilized on a nitrocellulose filter (HAWP-02500). The other details were in the legend of Table 1. Radioactivity fixed on the filter was determined in a toluenePPO cocktail in a Aloka LSC-670 scintillation spectrometer.

Results

Construction o f a Composite Plasmid Between R S F 2 1 2 4 and Bacilhts subtilis Plasmid A schematic outline of the composite p l a s m i d is p r e s e n t e d in F i g u r e 1. E. coli p l a s m i d R S F 2 1 2 4 a n d

B. subtilis I F O 3 0 2 2 ethidium

plasmid (pAT1060) purified by bromide-CsC1 centrifugation method were

digested completely with EcoRI to generate cohesive e n d s a n d l i g a t e d w i t h E. coli D N A l i g a s e t h r o u g h the cohesive ends as described in materials and m e t h o d s . B o t h p l a s m i d s h a v e o n l y o n e E c o R I site. They were converted into linear forms by EcoRI ( F i g . 2 a). T o e n r i c h c o m p o s i t e p l a s m i d s a f t e r l i g a t i o n , the ligase mixture was subjected to agarose gel electro-

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B. subtilis plasmid RSF2124

chromosomal

CCC-cqmposite

( pATIO60 )

Q @

Ap r

EcoRI

EcoRI

b C

colicin El imm

d e

f g

Apr colicin El irnm

Fig, 1. Schematic outline of the construction of composite plasmid between RSF2124 (7.3 x 106 daltons) and pAT1060 (5.4 x 106 daltons)

b

a

j R S F 2124 ~pAT 1060

Fig. 3a-g. Agarose gel electrophoresis of ethanol-precipitated cleared lysates obtained from Apr and colicin El imm clones: a cleared lysate from C600 (RSF2124), as a reference; b-g, from the transformants. Five clones shown in h 4 have the composite plasmid between RSF2124 and pAT1060. Most of Ap'-colicin E1 ~mm clones were simple RSF2124 transformants as shown in g. In b-f, DNA bands with lower mobilities than chromosomal DNA bands are seen, and these must be multimers of the composite plasmids reported by Bedbrook and Ausubel (1976)

E1 immunity. Cleared lysates were prepared from the selected clones after amplifying plasmid DNA by the addition of chloramphenicol to detect plasmid DNA (Fig. 3). Of 102 Apr-colicin E1imm clones five had the plasmid DNA with higher molecular weight than that of RSF2124. Although these five clones did not produce colicin El, they had colicin El-immunity but were sensitive to colicin E2 when tested by crossstreaking method on L-broth. Relaxed circular DNA of one of those five clones was examined for its length by electron microscopy using ColE1 double stranded DNA (4.2 x 106 daltons, or 6.34 kilobase pairs) as an internal standard (Fig. 4). Projections of the molecules were traced on paper to determine the length of DNA with a map measure. The plasmid was 3.0 times as long as ColE1 DNA or about 12.8 x 106 daltons, 19.0 kilobase pairs, which corresponded to the sum of lengths of RSF2124 and pAT1060. The plasmid was named pAT2010.

Digestion of the Composite Plasmid with EcoRL HindlII, and BsuG Fig. 2a and h. Agarose gel electrophoresis of a EcoRI-cleaved DNAs of RSF2124 and pAT1060, and of b the ligation mixture prepared as described in materials and methods. After the run, gel segments indicated by arrows were cut off and DNA was extracted from the segments

phoresis. The DNA bands with lower mobilities than that of the linear form of RSF2124 were extracted from agarose gel as described in materials and methods (Fig. 2b). E. coli C600 r m was transformed with this extracted DNA. Transformants were selected by ampicillin (Ap) resistance and colicin

DNA of the composite plasmid, pAT2010, was purified by eithidium bromide-CsC1 centrifugation method and subjected to 1% agarose slab gel electrophoresis with DNA preparations of RSF2124 and pAT1060 as references. As shown in Figure hA, all preparations consisted of both superhelical and relaxed circular forms of DNA. Treatment of pAT2010 DNA with EcoRI yielded two fragments which had the same mobilities as the linear forms of the parental plasmids (RSF2124 and pAT1060) (Fig. 5B). HindIII did not cut RSF2124 DNA, whereas

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179

Fig. 4. Eiectron micrograph of the composite plasmid DNA. ColE1 DNA was used as a standard (pointed by arrow). The composite plasmid DNA is 3.0 times the length of ColE1 DNA or approximately 12.6 × 106 daltons

1

A 2

3

1

8 2

D

C 3

t

2

3

1

2

3

Fig. 5A-D. Agarose gel electrophoresis of restriction endonucleases, EcoR1, HindIII, BsuG-fragments of the composite plasmid and the individual DNAs, RSF2124 and pATI060. BsuG reaction was carried out at 37° C for 1 h in 0.05 M Tris:HC1, 10 mM MgCI2, and 50 mM NaC1 (pH 7.5) in a final volume of 50 ~tl. A native DNAs (supercoils and relaxed circles), B EcoRI-cleaved DNAs, C HindIII-cleaved, D BsuG-cleaved. (1) RSF2124, (2) pAT1060, (3) composite plasmid, pAT2010

pAT1060 was cut into five fragments by HindIII. Under the same condition with HindIII, pAT2010 DNA was cut into five fragments, of which four fragments corresponded to HindIII-pATl060 fragments (Fig. 5C). The largest fragment of pAT2010 corresponded to the sum of RSF2124 and one of the HindlII-pATl060 fragments. Restriction endonuclease R.BsuG obtained from B. subtilis IAM1247 generates cohesive ends in fragmented DNA (Hoshino et al., manuscript in preparation). Digestion of RSF2124 with BsuG

yielded four fragments, while pAT1060 was fragmented with BsuG into two. As shown in Figure 5C, the BsuG treatment of pAT2010 yielded six fragments (not so clearly separated in this particular experiment), four bands of them corresponded to BsuG-fragments of the parental plasmids, while two bands of them should be derived from joint portions of parental plasmids. The digestion patterns of the composite plasmid DNA with the three endonucleases confirm that pAT2010 consists of RSF2124 and pAT1060.

180

(el

S. Horinouchi et al. : Cloning and in vitro Transcription of Bacillus Plasmid in E. coli

Oh

(b)

4h

(C)

12h

(d)

24h

Fig. 6a-d. Replication of the composite plasmid in the presence of chloramphenicol; photographs of UV-illuminated dye-buoyant density gradient of cleared lysate prepared from cells treated for varying lengths of time with chloramphenicol. Upper band represents chromosomal DNA; lower band represents covalently closed circular DNA

Replication of the Composite Plasmid in the Presence of Chloramphenicol

Replication of colicin E1 factor and its derivatives does not require de novo protein synthesis and continues for at least 6 h in the presence of chloramphenicol (CAP) to reach more than 1000 copies per cell. RSF2124 (ColE1-Ap0 was also shown to replicate continuously in the presence of CAP, and amplified to 280 copies per cell (Magdalene et al., 1975). To confirm the replication nature of the composite plasmid in the presence of CAP, a culture (1500 ml) of E. coli (pAT2010) was treated with CAP, and an equal portion (250 ml) of the culture was taken at various time intervals to determine the amount of synthesized plasmid as described in materials and methods. The D N A bands in the gradients were photographed under UV lamp to promote ethidium bromide-mediated fluorescence of the DNA bands (Fig. 6). The higher-density DNA band (lower band) representing covalently closed circular DNA gradually increased compared to the lower-density DNA band (chromosomal DNA). Further examination on the replication nature of the composite plasmid was carried out as described by Clewell (1972). A culture of E. coli (pAT2010) was grown for several generations in glucose minimal medium contaning [14C]-thymidine (1 ~tCi/ml) to label uniformally all cellular DNA. The cells were then centrifuged, washed with the fresh same medium, and suspended in a M9-0.2% glucose-2% casamino acids medium (20 ml) containing [3H]-thymidine (10 ~tCi/ ml) in place of [14C]-thymidine. The culture was incubated further in the presence of CAP at 37 ° C with shaking. Immediately after samples were removed at a given time interval (2, 4, 6, 8, 12, and 24 h), the cells were lysed by the lysozyme-EDTA-SDS procedure. An equal portion of the lysates was precipitated

~2 ~.. ~= i

o

' 2

~ 4

' 6

II 8

12

24

HOURS AFTER MEDIUM SHIFT

Fig. 7. The extent of replication of the composite plasmid in the presence of chloramephenicol; quantitative analysis of the [3H]thymidine labeled product. The ratio of 3H to 14C was calculated and the data were normalized to a value of 1 for the 2 h point

with ethanol and determined for the radioactivity by liquid scintillation counting. The ratio of 3H to 14C is depicted in Figure 7. Under these conditions, [3H]thymidine was incorporated into plasmid DNA, since the replication of chromosomal DNA was ceased by CAP. In the presence of CAP, the composite plasmid continued to replicate and the copy number gradually increased. We have found six types of B. subtilis plasmid, but none of them, including pAT1060, continued to replicate in the presence of CAP. All those data suggest that pAT2010, a composite plasmid of RSF2124 pAT1060, exists as a stable replicon in E. coli, where it can utilize the replication function specified by the RSF2124 portion, which results in the amplificatfon of pAT1060 DNA.

In vitro Transcription of pATiO60 DNA in SIO0 Extracts of E. coli

To determine whether B. subtilis plasmid, pAT1060, inserted into RSF2124, is transcribed in E. coli cell-

S. Horinouchi et al. : Cloning and in vitro Transcription of Bacillus Plasmid in E. coli

free extracts, the composite plasmid DNA was used as template in the $100 extracts system as described by Zubay et al. (1970), since this system has been utilized for the analysis of specific mRNA synthesis (Sens and James, 1975; Bennett etal., 1976). As shown in Table 1, 3H-RNA extracted from the S100 reaction mixture was hybridized to denatured pAT1060 DNA on a nitrocellulose filter as well as the composite plasmid and RSF2124 DNA. The amount of 3H-RNA hybridized to RSF2124 (716 c.p.m.) was less than that to pAT1060 (1388 c.p.m.), although RSF2124 DNA (7.3 x 10 6 daltons) had higher molecular weight than pAT1060 (5.4 × 10 6 daltons). This tendency was always observed in three different experiments. These results suggest that some parts of RSF2124 sequences in the composite plasmid are not transcribed, or that RSF2124 in the composite plasmid may have longer parts of untranscribed sequences than pAT1060. However, the total counts of RNA hybridized to the individual fragments [716 (to RSF2124)+ 1388 (to pAT1060)=2104 c.p.m.] were very close to those hybridized to the composite plasmid (1792 c.p.m.). Therefore, these data show that the inserted DNA or pAT1060 sequences are transcribed almost to the same extent as the sequences of the vector plasmid, RSF2124. Discussion

This paper describes the in vitro construction of a composite plasmid which consists of RSF2124 and B. subtiIis plasmid, pATI060, and the in vitro transcription of the composite plasmid (RSF2124pAT1060) in E. coli cell-free extracts. Composite plasmids formed in the ligase reaction were enriched first by preparative agarose gel electrophoresis before transformation, in order to get a high yield of transformants containing composite plasmids, since pAT1060 had no available selective markers. Of 102 Apr-colicin E1 imm transformants five had the composite plasmid (frequency, 2.5%). This enrichment technique might be simpler than those described by Cohen and Chang (1974) and could be used to isolate in a high yield transformants containing composite plasmids when selective markers do not exist. We have so far constructed composite plasmid, such as RSF2124-pSC101 (Magdalene et al., 1975) and RSF2124-trp (from ~80pt190; Hershfield et al., 1974) using RSF2124 as a cloning vector. RSF2124RSF2124 dimer, however, was not detected, since RSF2124-RSF2124 dimer produced by EcoRI-ligase method seems not to survive in E. coli. We confirmed that pAT2010 was a composite plasmid consisted of RSF2124 and pAT1060, from

181

the data of colicin sensitivity, electron microscopy, and the fragmented DNA patterns with the three endonucleases. The composite plasmid continued to replicate when chromosomal DNA replication was interrupted by chloramphenicol. This result suggests the replicator of RSF2124 in the composite plasmid is functional, since none of the plasmids of B. subtilis, including pAT1060, showed relaxed replication. Although it is not known whether pAT1060 codes for any proteins or not, we detected the transcription of pAT1060 DNA in cell-free extracts of E. coli. Some eukaryotic DNAs cloned in E. coIi were transcribed (Morrow et al., 1974; Kedes et al., 1975; Change et al., 1975) and translated (Struhl et al., 1976; Ratzkin and Carbon, 1977; Mergher et al., 1977; Miller et al., 1977) in E. coli minicells. Among these cloned eukaryotic DNAs, only yeast DNA was expressed functionally in E. coIi, complementing his or leuauxotrophy (Struhl et al., 1976; Ratzkin and Carbon, 1977). Our present data clearly indicate the transcription of pAT1060 in E. coli cell-free extracts, but we do not know whether the RNA complementary to pAT1060 DNA was synthesized in bona fide or whether it is merely nonsence RNA read through from the origin of transcription on RSF2124. In this respect, we are now trying to detect the protein coded for pAT1060. These problems of transcription and translation of pAT1060 will be confirmed in the near future. Acknowledgements. We thank Dr. T. Tanaka for his advice for the preparation of plasmid D N A a n d E c o R I endonuclease. We also acknowledge the expert technical assistance for electron microscopy of plasmid DNA to Dr. H. Hirokawa and S. Mizusawa, and for the preparation of S100 extracts to Dr. M. Dohi.

References Bedbook, J.R., Ausubel, F.M.: Recombination between bacterial plasmids leading to the formation of plasmid multimers. Cell 9, 707 716 (1976) Bennet, G.N., Schweingruber, M.E., Brown, K.D., Squires, C., Yanofsky, C.: Nucleotide sequence of region preceding trp mRNA initiation site and its role in promoter and operator function. Pro. nat. Acad. Sci. (Wash.) 73, 2351 2355 (1976) Bonner, J., Kung, G., Bekhor, I.: A method for the hybridization of nucleic acid molecules at low temperature. Biochemistry 6, 3650 3653 (1967) Chang, A.C.Y., Cohen, S.N. : Genome construction between bacterial species in vitro: replication and expression of Staphylococcus plasmid genes in Escherichia coll. Pro. nat. Acad. Sci. (Wash.) 71, 1030 1034 (1974) Clewell, D.B. : Nature of ColEI plasmid replication in the presence of chloramphenicol. J. Bact. 110, 667-676 (1972) Cohen, S.N., Chang, A.C.Y.: A method for selective cloning of eukaryotic DNA fragments in Escherichia coil by repeated transformation. Molec. gen. Genet. 134, 133 141 (1974) Collins, C.J., Jackson, D.A., Devries, F.A.J.: Biochemical construction of specific chimeric plasmids from ColE1 DNA and unfractionated Escherichia coli DNA. Proc. nat. Acad. Sci. (Wash.) 73, 3838-3842 (1976)

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Communicated by F. Kaudewitz Received August 2, 1977