Summary
The plasmids pSC138 and pML31 each contain the EcoRI-generated f5 replicator fragment of the conjugative plasmid F in addition to an EcoRI fragment encoding antibiotic resistance: ampicillin resistance derived from Staphylococcus aureus in pSC138 and kanamycin resistance from Escherichia coli in pML31. We have mapped one HindIII and two BamHI restriction sites in the f5 region of these plasmids and one HindIII site in the antibiotic resistance region of each plasmid. The HindIII site in the Km region of pML31 occurs in the kan gene whereas the HindIII site in the Ap region of pSC138 appears to occur in an area important for the regulation of β-lactamase production.
By means of in vitro recombinant DNA manipulation of plasmids pML31 and pSC138, we have shown that ∼ 1.9x106 daltons of the 6.0x106 dalton f5 fragment can be deleted without disrupting plasmid stability. In addition, we have used these same techniques to isolate a novel F-controlled Ap plasmid cloning vehicle which contains a single restriction site for each of the enzymes EcoRI, HindIII, and BamHI. This cloning vehicle has been linked via either its EcoRI or HindIII site to a ColE1 plasmid replicon to yield stable recombinants.
Similar content being viewed by others
References
Berg, P., Baltimore, D., Brenner, S., Roblin, R.O., Singer, M.F.: Summary statement of the Asilomar conference on recombinant DNA molecules. Proc. nat. Acad. Sci. (Wash.) 72, 1981–1984 (1975)
Cabello, F., Timmis, K., Cohen, S.N.: Replication control in a composite plasmid constructed by in vitro linkage of two distinct replicons. Nature (Lond.) 259, 285–290 (1976)
Clewell, D.B., Helinski, D.R.: Supercoiled circular DNA-protein complex in Escherichia coli: Purification and induced conversion to an open circular DNA form. Proc. nat. Acad. Sci. (Wash.) 62, 1159–1166 (1969)
Clewell, D.B., Helinski, D.R.: Properties of a deoxyribonucleic acid-protein relaxation complex and strand specificity of the relaxation event. Biochemistry 9, 4428–4440 (1970)
Guyer, M.S., Figurski, D., Davidson, N.: Electron microscope study of a plasmid chimera containing the replication region of the Escherichia coli F plasmid. J. Bact. 127, 988–989 (1976)
Helling, R.B., Goodman, H.M., Boyer, H.W.: Analysis of endonuclease R · EcoRI fragments of DNA from lamdoid bacteriophages and other viruses by agarose gel electrophoresis. J. Virol. 14, 1235–1244 (1974)
Imsande, J., Lilleholn, J.L.: Characterization of mutations in the penicillinase operon of Staphylococcus aureus. Molec. gen. Genet. 147, 23–27 (1976)
Kline, B.C., Miller, J.R.: Detection of nonintegrated plasmid deoxyribonucleic acid in the folded chromosome of Escherichia coli: Physicochemical approach to studying the unit of segregation. J. Bact. 121, 165–172 (1975)
Lederberg, E.M., Cohen, S.N.: Transformation of Salmonella typhimurium by plasmid deoxyribonucleic acid. J. Bact. 119, 1072–1074 (1974)
Lennox, E.S.: Transduction of linked genetic characters of the host by bacteriophage P1. Virology 1, 190–206 (1955)
Lovett, M.A., Helinski, D.R.: Method for the isolation of the replication region of a bacterial replicon: Construction of a mini-F′Km plasmid. J. Bact. 127, 982–987 (1976)
Miller, J.H.: Experiments in molecular genetics. New York: Cold Spring Harbor Laboratory 1972
Novick, R.P., Clowes, R.C., Cohen, S.N., Curtis, III, R., Datta, N., Falkow, S.: Uniform nomenclature for bacterial plasmids: A proposal. Bact. Rev. 40, 168–189 (1976)
Richmond, M.H.: Dominance of the inducible state in strains of Staphyloccus aureus containing two distinct penicillinase plasmids. J. Bact. 90, 370–374 (1965)
Sharp, P.A., Sugden, J., Sambrook, J.: Detection of two restriction endonuclease activities in Haemophilus parainfluenzae using analytical agarose-ethidium bromide electrophoresis. Biochemistry 12, 3055–3063 (1973)
Skurray, R.A., Nagaishi, H., Clark, A.J.: Molecular cloning of DNA from F sex factor of Escherichia coli K-12. Proc. nat. Acad. Sci. (Wash.) 73, 64–68 (1976a)
Skurray, R.A., Guyer, M.S., Timmis, K., Cabello, F., Cohen, S.N., Davidson, N., Clark, A.J.: Replication region fragments cloned from Flac + are identical to EcoRI fragment f5 of F. J. Bact. 127, 1571–1575 (1976b)
Tanaka, T., Weisblum, B.: Construction of a colicin E1-R factor composite plasmid in vitro: Means for amplification of deoxyribonucleic acid. J. Bact. 121, 354–362 (1975)
Taylor, A.L., Trotter, C.D.: Linkage map of Escherichia coli strain K-12. Bact. Rev. 36, 504–524 (1972)
Timmis, K., Cabello, F., Cohen, S.N.: Cloning, isolation and characterization of replication regions of complex plasmid genomes. Proc. nat. Acad. Sci. (Wash.) 72, 2242–2246 (1975)
Author information
Authors and Affiliations
Additional information
Communicated by B.A. Bridges
Rights and permissions
About this article
Cite this article
Manis, J.J., Kline, B.C. Restriction endonuclease mapping and mutagenesis of the F sex factor replication region. Molec. Gen. Genet. 152, 175–182 (1977). https://doi.org/10.1007/BF00268815
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00268815