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Single-stranded circular DNA generated from broad host range plasmid R1162 and its derivatives
PLASMID
21,238-241 (1989)
NOTE Single-Stranded Circular DNA Generated from Broad Host Range Plasmid R1162 and its Derivatives MENNO KoK,*,’
ANNIKA
C. ARNBERG,~
AND BERNARD WITHOLT**~
‘Groningen Biotechnology Center, Nijenborgh 16 9747AG Groningen, The Netherlands, and TDepartment of Biochemistry, University of Groningen, Nijenborgh 16 9747AG Groningen, The Netherlands Received June 6, 1988; revised January 23, 1989 Extraction of R1162 plasmid DNA with the alkaline lysis method yields considerable amounts of single-stranded circular plasmid DNA. Destabilization of plasmid DNA is stimulated by the RI 162 mob region in cis. The formation of single-strandedcircular DNA is initiated at a specific site on the plasmid, presumably the origin of transfer (oriT). Q 1989 Academic press,IX.
The 8.7-kb2 IncQ plasmid R1162 is a multicopy number broad host range plasmid (Bagdasarian et al., 1981) and is nearly identical to the independently isolated plasmids R300-B and RSFlOlO (Barth and Grinter, 1974; Grinter and Barth, 1976). These plasmids can be transferred efficiently between Gram-negative bacteria with the aid of a conjugative plasmid (Derbyshire and Willetts, 1987; Meyer et al., 1982). In addition to the transfer functions encoded by the conjugative (helper) plasmid, at least three R1162 encoded mob genes are required (Brash and Meyer, 1986; Derbyshire and Willetts, 1987; Derbyshire et al., 1987) for efficient plasmid mobilization. Transfer of R1162 DNA is preceded by strand-specific nicking at the origin of transfer (oriT) resulting in the formation of a protein-DNA relaxation complex. The relaxation complex can be converted to the open circular configuration by treatment with a protease or with alkali in vitro (Nordheim et al., 1980). During the characterization of R 1162 derivatives, we observed an additional nucleic acid species on agarose gels with the high electrophoretic mobility typical of single* To whom correspondence should be addressed. ’ Abbreviations used: kb, kilobase pairs; n, number of molecules measured, ::, novel joint.
0147-619X/89 $3.00 Copyright 0 1989 by Academic Pres, Inc. All rights of repmduclion in any form resewed.
stranded circular DNA. In this paper we report its identification as such and show that unwinding of plasmid DNA is intiated in the mob region of R1162. Plasmid DNA is often extracted from cells with the alkaline lysis procedure (Bimboim and Daly, 1979). Although this procedure involves denaturation of double-stranded DNA, only minute amounts of single-stranded DNA have been seento be formed (Birnboim, 1983) and the alkaline denaturation of DNA is therefore regarded as reversible. In this communication we show that under some conditions, however, DNA may be denaturated irreversibly during alkaline lysis. When analyzing alkaline lysis preparations of R1162 recombinants, we reproducibly found large amounts of an additional nucleic acid species resembling single-stranded DNA in its electrophoretic mobility. The same results were obtained when derivatives of the closely related plasmid RSF 1010 were analyzed. This nucleic acid speciescould be removed selectively from the DNA preparations by nucleaseS1 or mung bean nuclease, strongly suggestingthat it was single-stranded DNA. Electron microscopic investigation of the plasmid preparations (Fig. 1) confirmed this conclusion. Single-stranded circular DNA molecules were found to be abundantly pres-
238
ent in all plasmid preparations of RI 162 derivatives. In some casesmore than 70% of the circular DNA molecules (II > 100) were single stranded. The relationship between the singlestranded circular and the double-stranded plasmid DNA was establishedby heteroduplex analysis (Fig. IA) (Arnberg et al., 1980; Davis et al., 1971). Purified single-stranded circular RI 162 DNA forms a perfect duplex with linearized pGEc32 1 (R 1162::pBR322) DNA, the noncomplementary loop corresponding to the pBR322 sequences(4.35 +- 0.32 kb; II = 35). This shows that the single-stranded circular DNA species,present in plasmid preparations of R 1162, is identical to one strand of the double-stranded plasmid DNA. Isolation of RI 162 with the cleared lysate method (Davis et al., 1980), which does not involve large pH variations, did not yield detectable amounts of single-stranded DNA. Thus, single-stranded DNA is formed during the extraction procedure and has no significance in vivo. Fusions of the plasmids pBR322, pACYC 177 (Maniatis et al., 1982), or pSa727 (Tait et al., 1983) with RI 162 all yielded large amounts of single-stranded circular DNA when extracted from Escherichia coli with the alkaline lysis procedure, whereas none of the three plasmids produced single-strandedDNA when the RI 162 sequences were present in trans. To establish which sequencesof R 1162 enhanced the formation of single-stranded DNA in cis, we constructed a number of pBR322 recombinants carrying various portions of R1162. All the recombinants that could be mobilized efficiently (Mob+) by the IncP-1 plasmid R75 1 also yielded considerable amounts of single-stranded plasmid DNA. These Mob’ plasmids had the 2.5-kb R1162 mobilization region (oriV, oriT, mobABC) in common. In contrast, the plasmids that lacked (part of) the mob region did not produce detectable amounts of single-stranded DNA. Inhibition of protein synthesisin E. coli with chloramphenicol prevents the formation of a
relaxation complex at oriT while plasmid DNA synthesis continues (Clewell, 1972). With this procedure we amplified the copy number of pGEc321 (pBR322::R1162) 20- to 30-fold prior to extraction of the plasmid DNA. As a result of the chloramphenicol treatment no single-stranded plasmid DNA was observed in alkaline lysis preparations. Thus, newly synthesized proteins are involved in the formation of single-stranded RI 162 DNA. These results suggestthat plasmid unwinding is initiated in the mob region of R1162. The DNA structures shown in Fig. 1 support this view. In the partially denatured dimer (Fig. 1E) exactly half of the molecule is single stranded, whereas the other half has retained its double-stranded conformation. In this case unwinding was apparently initiated as well as terminated at the same (specific) site on the plasmid, as would be expected if singlestranded circular DNA formation involves the relaxation complex. We assume that the structures shown in Figs. 1C and lD, which contain small single-stranded loops, represent intermediate structures in the DNA unwinding process, suggesting that the unwinding of duplex plasmid DNA proceeds a stepwise manner. We found that the R1162 mob region enhances the formation of single-stranded circular DNA in cis. The plasmid DNA structures observed suggestthat DNA unwinding is initiated as well as terminated at a specific site on the plasmid, presumably oriT. In the absence of nicking at oriT no single-stranded circular DNA is expected to be formed. This wasin fact observed,since inhibition of protein synthesis during plasmid replication reduced the amount of single-stranded DNA generated from pBR322::R 1162 hybrids dramatically. Other mobilizable vectors, such as ColEl (Boyd and Sherratt, 1986),pRK20 13 (Figurski and Helinski, 1979), and R75 1 (Meyer and Shapiro, 1980), which can be isolated as a relaxation complex, fail to exhibit the behavior observed for R1162. The formation of singlestranded circular DNA during alkaline ex-
FIG. I. DNA structures generated from R 1162 derivatives. (A) pGEc32 1, an R 1162-pBR322 fusion plasmid, was digested with Sac1 and annealed to purified single-stranded R1162 DNA. The resulting heteroduplex molecules show a single-stranded loop that corresponds to the pBR322 sequences. (B-F) Alkaline lysis preparation of pGEc324, an 8.82-kb pMB8 (Heffron et al., 1977) based vector that carries the 2.5kb R1162 mob region. pGEc324 yields relatively large amounts of plasmid multimers which allowed the identification
240
241
NO TE
traction therefore appears to be a unique feature of the mob regions of IncQ plasmids R 1162, RSFlO 10, and presumably R300-B. ACKNOWLEDGMENTS We thank Drs. J. Davison, J. Frey, and K. N. Timmis for helpful discussions,Drs. J. A. Shapiro and J. Davison for supplying strains and plasmids.Janny de Wit for expert technical assistance,and K. Gilissen for printing the photographs.These investigations were carried out in part with financial support from the Netherlands Foundation for Chemical Research (SON) with financial aid from the Netherlands Organization for the Advancement of Pure Research (NWG) and the Commission of the European Communities Biotechnology Action Program (BAP0047NL).
REFERENCES ARNBERG,A. C., VAN OMMEN,G. J. B., GRIVELL,L. A., VAN BRUGCEN,E. F. J., AND BORST,P. (1980). Some yeast mitochondrial RNAs are circular. Cell 19, 3 I3319. BAGDASARIAN,M., LURZ, R., RUKERT, B., FRANKLIN, F. C. H., BAGDASARIAN,M. M., FREY,J., AND TIMMIS, K. N. (198 1). Specific-purpose plasmid cloning vectors II. Broad host range, high copy number, RSFlOlOderived vectors, and a host-vector system for gene cloning in Pseudomonas. Gene I&237-247. BARTH, P. T., AND GRINTER,N. J. (1974). Comparison of the deoxyribonucleic acid molecular weights and homologies of plasmids conferring linked resistance to streptomycins and sulphonamides. J. Bacterial. 120, 6 18-630. BIRNBOIM, H. C. (1983). A rapid alkaline extraction method for the isolation of plasmid DNA. In “Methods in Enzymology” (R. Wu, L. Grossman, K. Moldave, S. P. Colowick, and N. 0. Kaplan, Eds.), Vol. 100, pp. 243-255. Academic Press,Orlando, FL. BIRNBOIM,H. C., AND DOLY, J. (1979). A rapid alkaline extraction procedure for screeningrecombinant plasmid DNA. NucleicAcids Res. 7, 1513-1523. BOYD, A. C., AND SHERRATT,D. J. (1986). Polar mobil&ion of the E. coli chromosome by the ColE 1 transfer origin. Mol. Gen. Genet. 203,496-504. BRASH,M. A., AND MEYER, R. J. (1986). Genetic organization of plasmid RI 162 DNA involved in plasmid mobilization. J. Bacterial. 167, 703-710. CLEWELL,D. B. (1972). Nature of ColE 1 plasmid replication in E. coli in the presenceof chloramphenicol. J.
Advanced Bacterial Genetics: A Manual for Genetic Engineering. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. DAVIS,R. W., SIMON,M., AND DAVISON,N. (1971). Electron microscope heteroduplex methods for mapping regions of base sequence homology in nucleic acids. In “Methods in Enzymology” (L. Grossman, K. Moldave, S. P. Colowick, and N. 0. Kaplan, Eds.), Vol. 2 1D, pp. 413-428. Academic Press,Orlando, FL. DERBYSHIRE,K. M., HATFWLL, G., AND WILLETTS, N. S. (1987). Mobilization of the non-conjugative plasmid RSFl010: A genetic and DNA sequence analysis of the mobilisation region. Mol. Gen. Genet. 206, 161168. DERBYSHIRE,K. M., AND WILLETTS,N. S. (1987). Mobilization of the non-conjugative plasmid RSFlOlO: A genetic analysisof its origin of transfer. Mol. Gen. Genet. 206, 154-160. RGURSKI,D. H., AND HELINSKI,D. R. (1979).Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc. Natl. Acad. Sci. USA 76, 1648-1652. GRINTER,N. J., AND BARTH, P. T. (1976). Characterization of SmSu plasmids by restriction endonuclease cleavage and incompatibility. J. Bacterial. 128, 394400. HEFFRON,F., BEDINGER,P., CHAMFQUX,J., AND FAL KOW,S. (1977). Deletions affecting the transposition of an antibiotic resistancegene.Proc. Natl. Acad. Sci. USA 74,702-706. MANIATIS,T., FRITSCH,E. F., AND SAMBROOK,J. ( 1982). Molecular Cloning, A Laboratory Manual. Cold Spring Harbor laboratory, Cold Spring Harbor, NY. MEYER, R., HINDS, M., AND BRASCH,M. (1982). Properties of R 1162,a broad-host-range, high-copy-number plasmid. J. Bacterial. 150, 552-562. MEYER, R. J., AND SHAPIRO,J. (1980). Genetic organisation of the broad host range plasmid R751. J. Bacteriol. 143, 1362-1373. NORDHEIM,A., HASHIMOTOGOTOH, T., AND TIMMIS, K. N. (1980). Location of two relaxation nick sites in R6K and single sites in pSClO1 and RSFlOlO close to origins of vegetative replication: Implications for conjugal transfer of plasmid deoxyribonucleic acid. .Z.Bacteriol. 144, 923-932.
TAIT, R. C., CLOSE,T. J., LUNDQUIST,R. C., HAGIYA, M., RODRIQUEZ,R. L., AND KADO, C. I. (1983). Construction and characterization of a versatile broad host range DNA cloning system for Gram-negative bacteria. Bio/Technology
1, 269-275.
Bacterial. 110, 667.
DAVIS, R. W., BOTSTEIN,D., AND ROTH, J. R. (1980).
Communicated
by Kurt Nordstriim
of intermediate structuresin the DNA unwinding process.The most abundant speciesin the DNA preparation, double-stranded circular and (fully) single-strandedcircular pGEc324, are shown in (B) and(F), respectively. In (C) and (D) small (approximately 0.8 kb) single-strandedloops are present on the duplex circular pGEc324 monomer (C) and dimer (D), and in (E) half of the pGEc324 dimer is in the single-stranded conformation (arrows mark the boundaries between single-stranded and double-stranded DNA). Bar represents0.5 rm.
21,238-241 (1989)
NOTE Single-Stranded Circular DNA Generated from Broad Host Range Plasmid R1162 and its Derivatives MENNO KoK,*,’
ANNIKA
C. ARNBERG,~
AND BERNARD WITHOLT**~
‘Groningen Biotechnology Center, Nijenborgh 16 9747AG Groningen, The Netherlands, and TDepartment of Biochemistry, University of Groningen, Nijenborgh 16 9747AG Groningen, The Netherlands Received June 6, 1988; revised January 23, 1989 Extraction of R1162 plasmid DNA with the alkaline lysis method yields considerable amounts of single-stranded circular plasmid DNA. Destabilization of plasmid DNA is stimulated by the RI 162 mob region in cis. The formation of single-strandedcircular DNA is initiated at a specific site on the plasmid, presumably the origin of transfer (oriT). Q 1989 Academic press,IX.
The 8.7-kb2 IncQ plasmid R1162 is a multicopy number broad host range plasmid (Bagdasarian et al., 1981) and is nearly identical to the independently isolated plasmids R300-B and RSFlOlO (Barth and Grinter, 1974; Grinter and Barth, 1976). These plasmids can be transferred efficiently between Gram-negative bacteria with the aid of a conjugative plasmid (Derbyshire and Willetts, 1987; Meyer et al., 1982). In addition to the transfer functions encoded by the conjugative (helper) plasmid, at least three R1162 encoded mob genes are required (Brash and Meyer, 1986; Derbyshire and Willetts, 1987; Derbyshire et al., 1987) for efficient plasmid mobilization. Transfer of R1162 DNA is preceded by strand-specific nicking at the origin of transfer (oriT) resulting in the formation of a protein-DNA relaxation complex. The relaxation complex can be converted to the open circular configuration by treatment with a protease or with alkali in vitro (Nordheim et al., 1980). During the characterization of R 1162 derivatives, we observed an additional nucleic acid species on agarose gels with the high electrophoretic mobility typical of single* To whom correspondence should be addressed. ’ Abbreviations used: kb, kilobase pairs; n, number of molecules measured, ::, novel joint.
0147-619X/89 $3.00 Copyright 0 1989 by Academic Pres, Inc. All rights of repmduclion in any form resewed.
stranded circular DNA. In this paper we report its identification as such and show that unwinding of plasmid DNA is intiated in the mob region of R1162. Plasmid DNA is often extracted from cells with the alkaline lysis procedure (Bimboim and Daly, 1979). Although this procedure involves denaturation of double-stranded DNA, only minute amounts of single-stranded DNA have been seento be formed (Birnboim, 1983) and the alkaline denaturation of DNA is therefore regarded as reversible. In this communication we show that under some conditions, however, DNA may be denaturated irreversibly during alkaline lysis. When analyzing alkaline lysis preparations of R1162 recombinants, we reproducibly found large amounts of an additional nucleic acid species resembling single-stranded DNA in its electrophoretic mobility. The same results were obtained when derivatives of the closely related plasmid RSF 1010 were analyzed. This nucleic acid speciescould be removed selectively from the DNA preparations by nucleaseS1 or mung bean nuclease, strongly suggestingthat it was single-stranded DNA. Electron microscopic investigation of the plasmid preparations (Fig. 1) confirmed this conclusion. Single-stranded circular DNA molecules were found to be abundantly pres-
238
ent in all plasmid preparations of RI 162 derivatives. In some casesmore than 70% of the circular DNA molecules (II > 100) were single stranded. The relationship between the singlestranded circular and the double-stranded plasmid DNA was establishedby heteroduplex analysis (Fig. IA) (Arnberg et al., 1980; Davis et al., 1971). Purified single-stranded circular RI 162 DNA forms a perfect duplex with linearized pGEc32 1 (R 1162::pBR322) DNA, the noncomplementary loop corresponding to the pBR322 sequences(4.35 +- 0.32 kb; II = 35). This shows that the single-stranded circular DNA species,present in plasmid preparations of R 1162, is identical to one strand of the double-stranded plasmid DNA. Isolation of RI 162 with the cleared lysate method (Davis et al., 1980), which does not involve large pH variations, did not yield detectable amounts of single-stranded DNA. Thus, single-stranded DNA is formed during the extraction procedure and has no significance in vivo. Fusions of the plasmids pBR322, pACYC 177 (Maniatis et al., 1982), or pSa727 (Tait et al., 1983) with RI 162 all yielded large amounts of single-stranded circular DNA when extracted from Escherichia coli with the alkaline lysis procedure, whereas none of the three plasmids produced single-strandedDNA when the RI 162 sequences were present in trans. To establish which sequencesof R 1162 enhanced the formation of single-stranded DNA in cis, we constructed a number of pBR322 recombinants carrying various portions of R1162. All the recombinants that could be mobilized efficiently (Mob+) by the IncP-1 plasmid R75 1 also yielded considerable amounts of single-stranded plasmid DNA. These Mob’ plasmids had the 2.5-kb R1162 mobilization region (oriV, oriT, mobABC) in common. In contrast, the plasmids that lacked (part of) the mob region did not produce detectable amounts of single-stranded DNA. Inhibition of protein synthesisin E. coli with chloramphenicol prevents the formation of a
relaxation complex at oriT while plasmid DNA synthesis continues (Clewell, 1972). With this procedure we amplified the copy number of pGEc321 (pBR322::R1162) 20- to 30-fold prior to extraction of the plasmid DNA. As a result of the chloramphenicol treatment no single-stranded plasmid DNA was observed in alkaline lysis preparations. Thus, newly synthesized proteins are involved in the formation of single-stranded RI 162 DNA. These results suggestthat plasmid unwinding is initiated in the mob region of R1162. The DNA structures shown in Fig. 1 support this view. In the partially denatured dimer (Fig. 1E) exactly half of the molecule is single stranded, whereas the other half has retained its double-stranded conformation. In this case unwinding was apparently initiated as well as terminated at the same (specific) site on the plasmid, as would be expected if singlestranded circular DNA formation involves the relaxation complex. We assume that the structures shown in Figs. 1C and lD, which contain small single-stranded loops, represent intermediate structures in the DNA unwinding process, suggesting that the unwinding of duplex plasmid DNA proceeds a stepwise manner. We found that the R1162 mob region enhances the formation of single-stranded circular DNA in cis. The plasmid DNA structures observed suggestthat DNA unwinding is initiated as well as terminated at a specific site on the plasmid, presumably oriT. In the absence of nicking at oriT no single-stranded circular DNA is expected to be formed. This wasin fact observed,since inhibition of protein synthesis during plasmid replication reduced the amount of single-stranded DNA generated from pBR322::R 1162 hybrids dramatically. Other mobilizable vectors, such as ColEl (Boyd and Sherratt, 1986),pRK20 13 (Figurski and Helinski, 1979), and R75 1 (Meyer and Shapiro, 1980), which can be isolated as a relaxation complex, fail to exhibit the behavior observed for R1162. The formation of singlestranded circular DNA during alkaline ex-
FIG. I. DNA structures generated from R 1162 derivatives. (A) pGEc32 1, an R 1162-pBR322 fusion plasmid, was digested with Sac1 and annealed to purified single-stranded R1162 DNA. The resulting heteroduplex molecules show a single-stranded loop that corresponds to the pBR322 sequences. (B-F) Alkaline lysis preparation of pGEc324, an 8.82-kb pMB8 (Heffron et al., 1977) based vector that carries the 2.5kb R1162 mob region. pGEc324 yields relatively large amounts of plasmid multimers which allowed the identification
240
241
NO TE
traction therefore appears to be a unique feature of the mob regions of IncQ plasmids R 1162, RSFlO 10, and presumably R300-B. ACKNOWLEDGMENTS We thank Drs. J. Davison, J. Frey, and K. N. Timmis for helpful discussions,Drs. J. A. Shapiro and J. Davison for supplying strains and plasmids.Janny de Wit for expert technical assistance,and K. Gilissen for printing the photographs.These investigations were carried out in part with financial support from the Netherlands Foundation for Chemical Research (SON) with financial aid from the Netherlands Organization for the Advancement of Pure Research (NWG) and the Commission of the European Communities Biotechnology Action Program (BAP0047NL).
REFERENCES ARNBERG,A. C., VAN OMMEN,G. J. B., GRIVELL,L. A., VAN BRUGCEN,E. F. J., AND BORST,P. (1980). Some yeast mitochondrial RNAs are circular. Cell 19, 3 I3319. BAGDASARIAN,M., LURZ, R., RUKERT, B., FRANKLIN, F. C. H., BAGDASARIAN,M. M., FREY,J., AND TIMMIS, K. N. (198 1). Specific-purpose plasmid cloning vectors II. Broad host range, high copy number, RSFlOlOderived vectors, and a host-vector system for gene cloning in Pseudomonas. Gene I&237-247. BARTH, P. T., AND GRINTER,N. J. (1974). Comparison of the deoxyribonucleic acid molecular weights and homologies of plasmids conferring linked resistance to streptomycins and sulphonamides. J. Bacterial. 120, 6 18-630. BIRNBOIM, H. C. (1983). A rapid alkaline extraction method for the isolation of plasmid DNA. In “Methods in Enzymology” (R. Wu, L. Grossman, K. Moldave, S. P. Colowick, and N. 0. Kaplan, Eds.), Vol. 100, pp. 243-255. Academic Press,Orlando, FL. BIRNBOIM,H. C., AND DOLY, J. (1979). A rapid alkaline extraction procedure for screeningrecombinant plasmid DNA. NucleicAcids Res. 7, 1513-1523. BOYD, A. C., AND SHERRATT,D. J. (1986). Polar mobil&ion of the E. coli chromosome by the ColE 1 transfer origin. Mol. Gen. Genet. 203,496-504. BRASH,M. A., AND MEYER, R. J. (1986). Genetic organization of plasmid RI 162 DNA involved in plasmid mobilization. J. Bacterial. 167, 703-710. CLEWELL,D. B. (1972). Nature of ColE 1 plasmid replication in E. coli in the presenceof chloramphenicol. J.
Advanced Bacterial Genetics: A Manual for Genetic Engineering. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. DAVIS,R. W., SIMON,M., AND DAVISON,N. (1971). Electron microscope heteroduplex methods for mapping regions of base sequence homology in nucleic acids. In “Methods in Enzymology” (L. Grossman, K. Moldave, S. P. Colowick, and N. 0. Kaplan, Eds.), Vol. 2 1D, pp. 413-428. Academic Press,Orlando, FL. DERBYSHIRE,K. M., HATFWLL, G., AND WILLETTS, N. S. (1987). Mobilization of the non-conjugative plasmid RSFl010: A genetic and DNA sequence analysis of the mobilisation region. Mol. Gen. Genet. 206, 161168. DERBYSHIRE,K. M., AND WILLETTS,N. S. (1987). Mobilization of the non-conjugative plasmid RSFlOlO: A genetic analysisof its origin of transfer. Mol. Gen. Genet. 206, 154-160. RGURSKI,D. H., AND HELINSKI,D. R. (1979).Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc. Natl. Acad. Sci. USA 76, 1648-1652. GRINTER,N. J., AND BARTH, P. T. (1976). Characterization of SmSu plasmids by restriction endonuclease cleavage and incompatibility. J. Bacterial. 128, 394400. HEFFRON,F., BEDINGER,P., CHAMFQUX,J., AND FAL KOW,S. (1977). Deletions affecting the transposition of an antibiotic resistancegene.Proc. Natl. Acad. Sci. USA 74,702-706. MANIATIS,T., FRITSCH,E. F., AND SAMBROOK,J. ( 1982). Molecular Cloning, A Laboratory Manual. Cold Spring Harbor laboratory, Cold Spring Harbor, NY. MEYER, R., HINDS, M., AND BRASCH,M. (1982). Properties of R 1162,a broad-host-range, high-copy-number plasmid. J. Bacterial. 150, 552-562. MEYER, R. J., AND SHAPIRO,J. (1980). Genetic organisation of the broad host range plasmid R751. J. Bacteriol. 143, 1362-1373. NORDHEIM,A., HASHIMOTOGOTOH, T., AND TIMMIS, K. N. (1980). Location of two relaxation nick sites in R6K and single sites in pSClO1 and RSFlOlO close to origins of vegetative replication: Implications for conjugal transfer of plasmid deoxyribonucleic acid. .Z.Bacteriol. 144, 923-932.
TAIT, R. C., CLOSE,T. J., LUNDQUIST,R. C., HAGIYA, M., RODRIQUEZ,R. L., AND KADO, C. I. (1983). Construction and characterization of a versatile broad host range DNA cloning system for Gram-negative bacteria. Bio/Technology
1, 269-275.
Bacterial. 110, 667.
DAVIS, R. W., BOTSTEIN,D., AND ROTH, J. R. (1980).
Communicated
by Kurt Nordstriim
of intermediate structuresin the DNA unwinding process.The most abundant speciesin the DNA preparation, double-stranded circular and (fully) single-strandedcircular pGEc324, are shown in (B) and(F), respectively. In (C) and (D) small (approximately 0.8 kb) single-strandedloops are present on the duplex circular pGEc324 monomer (C) and dimer (D), and in (E) half of the pGEc324 dimer is in the single-stranded conformation (arrows mark the boundaries between single-stranded and double-stranded DNA). Bar represents0.5 rm.