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Molecular and General Genetics MGG

, Volume 178, Issue 3, pp 525–533 | Cite as

Establishment of Escherichia coli cells with an integrated high copy number plasmid

  • Kazuo Yamaguchi
  • Jun-ichi Tomizawa
Article

Summary

The dnaA46 cells can grow at high temperature when a high copy number plasmid pKY31, a derivative of pBR322 carrying a segment of the E. coli chromosome, integrates into the bacterial chromosome. In contrast, the dnaA46 polA- cells with the integrated plasmid can not grow at high temperature. Therefore, integration of the plasmid can suppress the dnaA mutation and this suppression requires DNA polymerase I which has been known to be required for plasmid replication. Full reversion of polA or lysogenization of λpolA+ is lethal for the dnaA46polA- bacteria that carry the plasmid only in integrated state. Partial reversion of polA allows these cells to grow at both low and high temperatures. Introduction of the plasmid pBR322 into cytoplasm of these bacteria suppresses the lethal effect caused by full reversion of polA or lysogenization of λpolA+. This lethal effect expresses independent of the presence or absence of the dnaA mutation. In partial revertants of polA which have only integrated plasmid, the number of copies of a region near the replication origin of integrated plasmid increases. The number is reduced by the presence of extrachromosomal pBR322. It is suggested that the lethal effect of normal levels of DNA polymerase I in strains that carry only the integrated plasmid is due to excessive initiation of replication of the bacterial chromosome from the plasmid origin and high potential of initiation can be absorbed in many copies of cytoplasmic plasmid, probably, in their replication origins.

Keywords

Normal Level High Potential Integrate State Lethal Effect Replication Origin 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Abbreviations

Ampr

ampicillin resistant (resistance)

Tets

tetracycline sensitive

Tetr

tetracycline resistant

MMSr

methyl methane sulfonate resistant (resistance)

ts

temperature sensitive

Kb

kilobase pairs

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References

  1. Bazaral, M., Helinski, D.R.: Replication of a bacterial plasmid and an episome in Escherichia coli. Biochem. 9, 399–406 (1970)Google Scholar
  2. Bird, R.E., Chandler, M., Caro, L.: Suppression of an E. coli dnaA mutation by the integrated R factor R100.1: Change of replication origin in synchronized cultures. J. Bacteriol. 126, 1215–1223 (1976)Google Scholar
  3. Bird, R.E., Louarn, J.M., Martuscelli, J., Caro, L.: Origin and sequence of chromosome replication in Escherichia coli. J. Mol. Biot. 70, 549–566 (1972)Google Scholar
  4. Chandler, M., Silver, L., Caro, L.: Suppression of an E. coli dnaA mutation by the integrated R factor R100.1: Origin of chromosome replication during exponential growth. J. Bacteriol. 131, 421–430 (1977)Google Scholar
  5. 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. Natl. Acad. Sci. U.S.A. 62, 1159–1166 (1969)Google Scholar
  6. Cohen, S.N., Chang, A.C.Y., Hsu, L.: Nonchromosomal antibiotic resistance in bacteria: Genetic transformation of Escherichia coli by R-factor DNA. Proc. Natl. Acad. Sci. U.S.A. 69, 2110–2114 (1972)Google Scholar
  7. Denhardt, D.T.: A membrane filter technique for the detection of complementary DNA. Biochem. Biophys. Res. Commun. 23, 641–646 (1966)Google Scholar
  8. Frey, J., Chandler, M., Caro, L.: The effects of an Escherichia coli dnaAts mutation on the replication of the plasmids ColE1, pSC101, R100.1 and TRF-TC. Mol. Gen. Genet. 174, 117–126 (1979)Google Scholar
  9. Friedman, E.I., Yarmolinsky, M.B.: Prevention of the lethality of induced λ prophage by an isogenic λ plasmid. Virol. 50, 472–481 (1972)Google Scholar
  10. Goebel, W.: Integrative suppression of temperature-sensitive mutants with a lesion in the initiation of DNA replication of autonomous plasmids in the suppressed state. Eur. J. Biochem. 43, 125–130 (1974)Google Scholar
  11. Hasunuma, K., Sekiguchi, M.: Replication of plasmid pSC101 in Escherichia coli K12: requirement for dnaA function. Mol. Gen. Genet. 154, 225–230 (1977)Google Scholar
  12. Hirota, Y., Ryter, A., Jacob, F.: Thermosensitive mutants of E. coli affected in the processes of DNA synthesis and cellular division. Cold Spring Harbor Symp. Quant. Biol. 33, 677–693 (1968)Google Scholar
  13. Hirota, Y., Yasuda, S., Yamada, M., Nishimura, A., Sugimoto, K., Sugisaki, H., Oka, A., Takanami, M.: Structural and functional properties of the Escherichia coli origin of DNA replication. Cold Spring Harbor Symp. Quant. Biol. 43, 129–138 (1978)Google Scholar
  14. Inselburg, J.: Replication of Colicin E1 plasmid DNA in minicells from a unique replication initiation site. Proc. Natl. Acad. Sci. U.S.A. 71, 2256–2259 (1974)Google Scholar
  15. Jacob, F., Brenner, b., Cuzin, F.: On the regulation of DNA replication in bacteria. Cold Spring Harbor Symp. Quant. Biol. 28, 329–348 (1963)Google Scholar
  16. Kelley, W.S., Chalmer, K., Murray, N.E.: Isolation and characterization of a λpolA transducing phage. Proc. Natl. Acad. Sci. U.S.A. 74, 5632–5636 (1977)Google Scholar
  17. Kingsbury, D.T., Helinski, D.R.: DNA polymerase as a requirement for the maintenance of the bacterial plasmid colicinogenic factor E1. Biochem. Biophys. Res. Commun. 41, 1538–1544 (1970)Google Scholar
  18. Kuempel, P.L., Duerr, S.A., Seeley, N.R.: Termination region of the chromosome in E. coli inhibits replication forks. Proc. Natl. Acad. Sci. U.S.A. 74, 3927–3931 (1977)Google Scholar
  19. Lovett, M.A., Katz, L., Helinsky, D.R.: Unidirectional replication of plasmid ColE1 DNA and relation of the origin-terminus to the nicked site in the ColE1 relaxation complex. Nature 251, 337–340 (1974)Google Scholar
  20. Lucia, P., de, Cairns, J.: Isolation of an E. coli strain with a mutation affecting DNA polymerase. Nature 224, 1164–1166 (1969)Google Scholar
  21. Marsh, R.S., Worcel, A.: A DNA fragment containing the origin of replication of the Escherichia coli chromosome. Proc. Natl. Acad. Sci. U.S.A. 74, 2720–2724 (1977)Google Scholar
  22. Messer, W., Meijer, M., Bergamans, H.E.N., Hansen, F.G., Meyenburg, K., von, Beck, E., Schaller, H.: Origin of replication, oriC, of the Escherichia coli K12 chromosome: Nucleotide sequence. Cold Spring Harbor Symp. Quant. Biol. 43, 139–146 (1978)Google Scholar
  23. Miller, J.H.: Experiments in molecular genetics. New York: Cold Spring Harbor Laboratory 1972Google Scholar
  24. Monk, M., Kinross, J.: Conditional lethality of recA and recB derivatives of a strain of E. coli K12 with a temperature-sensitive DNA polymerase I. J. Bacteriol. 109, 971–978 (1972)Google Scholar
  25. Nishimura, Y., Caro, L., Berg, C.M., Hirota, Y.: Chromosome replication in E. coli. IV. Control of chromosome replication and cell division by an integrated episome. J. Mol. Biol. 55, 441–456 (1971)Google Scholar
  26. Pritchard, R.H.: Control of DNA replication in bacteria. In: DNA synthesis-present and future (Molineux, I. and Kohiyama, M., eds.) pp. 1–26. New York: Plenum 1977Google Scholar
  27. Radloff, R., Bauer, W., Vinograd, J.: A dye-buoyant-density method for the detection and isolation of closed circular duplex DNA: The closed circular DNA in HeLa cells. Proc. Natl. Acad. Sci. U.S.A. 57, 1514–1521 (1967)Google Scholar
  28. Sakakibara, Y., Tomizawa, J.: Replication of colicin E1 plasmid in cell extracts. Proc. Natl. Acad. Sci. U.S.A. 71, 802–806 (1974)Google Scholar
  29. Sotomura, M., Yoshikawa, M.: Reinitiation of chromosome replication in the presence of chloramphenicol under an integratively suppressed state by R6K. J. Bacteriol. 122, 623–628 (1975)Google Scholar
  30. Sutcliffe, J.G.: pBR322 restriction map derived from the DNA sequence: Accurate DNA size markers up to 4361 nucleotides pairs long. Nucleic Acids Res. 5, 2721–2728 (1978a)Google Scholar
  31. Sutcliffe, J.G.: Complete nucleotide sequence of the Escherichia coli plasmid pBR322. Cold Spring Harbor Symp. Quant. Biol. 43, 77–90 (1978b)Google Scholar
  32. Tomizawa, J., Sakakibara, Y., Kakefuda, T.: Replication of Colicin E1 plasmid DNA in cell extracts Origin and direction of replication. Proc. Natl. Acad. Sci. U.S.A. 71, 2260–2264 (1974)Google Scholar
  33. Yasuda, S., Hirota, Y.: Cloning and mapping of the replication origin of Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 74, 5458–5462 (1977)Google Scholar

Copyright information

© Springer-Verlag 1980

Authors and Affiliations

  • Kazuo Yamaguchi
    • 1
  • Jun-ichi Tomizawa
    • 1
  1. 1.Laboratory of Molecular Biology, National Institute of Arthritis, Metabolism and Digestive DiseasesNational Institutes of HealthBethesdaUSA

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