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Mutations in a new chromosomal gene of Escherichia coli K-12, pcnB, reduce plasmid copy number of pBR322 and its derivatives

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Summary

We describe mutants of Escherichia coli that decrease the plasmid copy number of pBR322 derivatives. One mutant was partially characterized genetically and its mutation, designated pcnB for plasmid copy number, was mapped to approximately 3 min on the E. coli chromosome. This locus is distinct from other genes whose products are known to affect plasmid replication or stable plasmid maintenance. The pcnB mutant strain should be useful for cloning genes into pBR322 that have aberrant or deleterious effects on the cell when present in high copy number.

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References

  • Bassett CL, Kushner SR (1984) Exonucleases I, III, and V are required for stability of ColE1-related plasmids in Escherichia coli. J Bacteriol 157:661–664

    Google Scholar 

  • Berkower I, Leis J, Hurwitz J (1973) Isolation and characterization of an endonuclease from Escherichia coli specific for ribonucleic acid hybrid structures. J Biol Chem 248:5914–5921

    Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Google Scholar 

  • Burton K (1956) A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem J 62:315–323

    Google Scholar 

  • Cabello F, Timmis K, Cohen SN (1976) Replication control in a composite plasmid constructed by in vitro linkage of two distinct replicons. Nature 259:285–290

    Google Scholar 

  • Clewell DB, Evenchik BG (1973) Effects of rifampicin, streptolydigin and actinomycin D on the replication of ColE1 plasmid DNA in Escherichia coli. J Mol Biol 75:503–513

    Google Scholar 

  • Cress DE, Kline BC (1976) Isolation and characterization of Escherichia coli chromosomal mutants affecting plasmid copy number. J Bacteriol 125:635–642

    Google Scholar 

  • Friedman DI, Plantefaber LC, Olson EJ, Carver D, O'Dea MH, Gellert M (1984) Mutations in the DNA gyrB gene that are temperature sensitive for Lambda site-specific recombination, Mu growth, and plasmid maintenance. J Bacteriol 157:490–497

    Google Scholar 

  • Horiuchi T, Maki H, Sekiguchi M (1984) RNase H-defective mutants of Escherichia coli: A possible discriminatory role of RNase H in initiation of DNA replication. Mol Gen Genet 195:17–22

    Google Scholar 

  • Iida A, Harayama S, Iino T, Hazelbauer GL (1984) Molecular cloning and characterization of genes required for ribose transport and utilization in Escherichia coli K-12. J Bacteriol 158:674–682

    Google Scholar 

  • Itoh T, Tomizawa J (1978) Initiation of replication of plasmid ColE1 DNA by RNA polymerase, ribonuclease H and DNA polymerase I. Cold Spring Harbor Symp Quant Biol 43:409–418

    Google Scholar 

  • Itoh T, Tomizawa J (1980) Formation of an RNA primer for initiation of replication of ColE1 DNA by ribonuclease H. Proc Natl Acad Sci USA 77:2450–2454

    Google Scholar 

  • Kanaya S, Crouch RJ (1984) The rnh gene is essential for growth of Escherichia coli. Proc Natl Acad Sci USA 81:3447–3451

    Google Scholar 

  • Keller W, Crouch R (1972) Degradation of DNA RNA hybrids by ribonuclease H and DNA polymerases of cellular and viral origin. Proc Natl Acad Sci USA 69:3360–3364

    Google Scholar 

  • Kingsbury DT, Helinski DR (1970) DNA polymerase as a requirement for the maintenance of the bacterial plasmid colicinogenic factor E1. Biochem Biophys Res Commun 41:1538–1544

    Google Scholar 

  • Kingsbury DT, Helinski DR (1973) Temperatue-sensitive mutants for the replication of plasmids in Escherichia coli I. Isolation and specificity of host and plasmid mutations. Genetics 74:17–31

    Google Scholar 

  • Kumamoto CA, Beckwith J (1983) Mutations in a new gene, secB, cause defective protein localization in Escherichia coli. J Bacteriol 154:253–260

    Google Scholar 

  • Kumamoto CA, Beckwith J (1985) Evidence for specificity at an early step in protein export in Escherichia coli. J Bacteriol 163:267–274

    Google Scholar 

  • Lopilato JE, Garwin JL, Emr SD, Silhavy TJ, Beckwith JR (1984) D-ribose metabolism Escherichia coli K-12: Genetics, regulation and transport. J Bacteriol 158:665–673

    Google Scholar 

  • Macrina FL, Weatherly GG, Curtiss III R (1974) R6K plasmid replication: Influence of chromosomal genotype in minicellproducing strains of Escherichia coli K-12. J Bacteriol 120:1387–1400

    Google Scholar 

  • Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

    Google Scholar 

  • Mekalanos JJ (1983) Duplication and amplification of toxin genes in Vibrio cholerae. Cell 35:253–263

    Google Scholar 

  • Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

    Google Scholar 

  • Naito S, Kitani T, Ogawa T, Okazaki T, Uchida H (1984) Escherichia coli mutants suppressing replication-defective mutations of the ColE1 plasmid. Proc Natl Acad Sci USA 81:550–554

    Google Scholar 

  • O'Calloghan CH, Morris A, Kirby SM, Shingler AH (1972) Novel method for detection of β-lactamase by using a chromogenic cephalosporin substrate. Antimicrobial Agents Chemother 1:283–288

    Google Scholar 

  • Oliver DB, Beckwith J (1981) Escherichia coli mutant pleiotropically defective in the export of secreted proteins. Cell 25:765–772

    Google Scholar 

  • Silhavy TJ, Berman ML, Enquist LW (1984) Experiments with gene fusions. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

    Google Scholar 

  • Southern E (1979) Gel electrophoresis of restriction fragments. Methods Enzymol 68:152–176

    Google Scholar 

  • Sullivan NF, Donachie WD (1984) Transcriptional organization within an Escherichia coli cell division gene cluster: Direction of transcription of the cell separation gene envA. J Bacteriol 160:724–732

    Google Scholar 

  • Summers DK, Sherratt DJ (1984) Mutimerization of high copy number plasmids causes instability: ColE1 encodes a determinant essential for plasmid monomerization and stability. Cell 36:1097–1103

    Google Scholar 

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Communicated by N.D.F. Grindley

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Lopilato, J., Bortner, S. & Beckwith, J. Mutations in a new chromosomal gene of Escherichia coli K-12, pcnB, reduce plasmid copy number of pBR322 and its derivatives. Molec. Gen. Genet. 205, 285–290 (1986). https://doi.org/10.1007/BF00430440

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  • DOI: https://doi.org/10.1007/BF00430440

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