Nucleotide Sequence Change in A Cole1 Copy Number Mutant

  • Barry Polisky
  • Mark Muesing
  • Joseph Tamm


Despite a great deal of knowledge about the enzymology of DNA replication, the elements that regulate initiation of DNA replication are largely unknown. The definition and ultimate analysis of such elements depends initially on genetic identification of mutations affecting their function. In turn, the genetic analysis requires that the mutant be viable under certain conditions. For complex replicons such as the IS. coli chromosome, such mutants have not been described. We have studied the multicopy plasmid ColEl and its derivatives as a model system for the analysis of replication control elements. This plasmid is stably inherited and exists at a characteristic copy number of 10–15 copies per host chromosome. Our approach has been to perturb the control mechanism by isolating plasmid mutants which have altered copy number and then investigating the molecular consequences of the lesion. We have studied a high copy number mutant of the ColEl-derived cloning vehicle pBGP120 (Polisky, Bishop and Gelfand, 1976). The mutant plasmid, pOPl, and its derivatives, such as pOPlA6, comprise about 30% of intracellular DNA, compared to about 5% for the parent, pBGP120 (Gelfand et al., 1978). Previously, we localized the mutation to a 2kb region near the plasmid replication origin and demonstrated that the mutation was recessive, i.e., in cells containing both a copy number mutant and a wild-type plasmid, the copy number of the mutant was lowered to wild-type levels (Shepard, Gelfand and Polisky, 1979). The turn-down of copy number in trans was not observed when an unrelated plasmid co-resided with the mutant, suggesting the existence of a specific, plasmid-encoded, negative regulator of replication (Pritchard, 1978).


Replication Origin Mutant Plasmid Alter Copy Number Deletion Derivative Plasmid Genome 
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  1. Backman, K., Betlach, M., Boyer, H.W., and Yanofsky, S. (1978). Genetic and physical studies on the replication of ColEl-type Plasmids. Cold Spring Harbor Symp. 43, 69–76.CrossRefGoogle Scholar
  2. Chan, P.T., Lebowitz, J., and Bastia, D. (1979). Nucleotide sequence determination of a strong promoter of the colicin El plasmid. Analysis of restriction sites protected by RNA polymerase interactions before and after limited transcription. Nuc. Acids Res. 7, 1247–1262.CrossRefGoogle Scholar
  3. Conrad, S.E., and Campbell, J.L. (1979). Role of plasmid-coded RNA and ribonuclease III in plasmid DNA replication. Cell 18, 61–71.PubMedCrossRefGoogle Scholar
  4. Gelfand, D.H., Shepard, H.M., O’Farrell, P.H., and Polisky, B. (1978). Isolation and characterization of a ColEl-derived plasmid copy number mutant. Proc. Natl. Acad. Sci. USA 75, 5869–5873.PubMedCrossRefGoogle Scholar
  5. Inselburg, J. (1977). Studies of colicin El plasmid functions by analysis of deletions and TnA insertions of the plasmid. J. Bacteriol. 132, 332–340.PubMedGoogle Scholar
  6. Itoh, T., and Tomizawa, J. (1980). Formation of an RNA primer for initiation of replication of ColEl DNA by ribonuclease H. Proc. Natl. Acad. Sci. USA 77, 2450–2454.PubMedCrossRefGoogle Scholar
  7. Levine, A.D., and Rupp, W.D. (1978). Small RNA product from the in vitro transcription of ColEl DNA. In Microbiology—1978. D. Schlessinger, ed. (Washington, D.C., American Society for Microbiology), pp. 163–166.Google Scholar
  8. Maxam, A.M., and Gilbert, W. (1980). Sequencing end-labeled DNA with base-specific chemical cleavages. In Methods in Enzymology, Vol 65, L. Grossman and K. Moldave, eds. (New York: Academic Press), pp. 499–580.Google Scholar
  9. Miller, J.H. (1972). Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.Google Scholar
  10. Morita, M., and Oka, A. (1979). The structure of a transcriptional unit on Colicin El plasmid. Eur. J. Biochem. 97, 435–443.PubMedCrossRefGoogle Scholar
  11. O’Callaghan, C.H., Morris, A., Kirby, S., and Shingler, A.H. (1972). Novel method for detection of β-lactamases using a chromogenic cephalosporin substrate. Antimicrobial Agents and Chemotherapy 1, 283–288.PubMedCrossRefGoogle Scholar
  12. O’Farrell, P.H., Polisky, B., and Gelfand, D.H. (1978). Regulated expression by read-through translation from a plasraid-encoded β-galactosidase. J. Bacteriol. 134, 645–654.PubMedGoogle Scholar
  13. Oka, A., Nomura, N., Morita, M., Sugisaki, H., Sugimoto, K., and Takanami, M. (1979). Nucleotide sequence of small ColEl derivatives: Structure of the regions essential for autonomous replication and colicin El immunity. Mol. Gen. Genet. 172, 151–159.PubMedCrossRefGoogle Scholar
  14. Polisky, B., Bishop, R.J., and Gelfand, D.H. (1976). A plasmid cloning vehicle allowing regulated expression of eukaryotic DNA in bacteria. Proc. Natl. Acad. Sci. USA 73, 3900–3904.PubMedCrossRefGoogle Scholar
  15. Pritchard, R.H. (1978). Control of DNA replication in bacteria. In DNA Synthesis, I. Molineaux and M. Kohiyama, eds. (Plenum Press: New York), pp. 1–26.CrossRefGoogle Scholar
  16. Shepard, H.M., Gelfand, D.H., and Polisky, B. (1979). Analysis of a recessive plasmid copy number mutant; evidence for negative control of ColEl replication. Cell 18, 267–275.PubMedCrossRefGoogle Scholar
  17. Stuitje, A.R., Veitkamp, E., Maat, J., and Heynecker, H.L. (1980). The nucleotide sequence surrounding the replication origin of the Cop 3 mutant of the bacteriocinogenic plasmid CloDF13. Nuc. Acids Res. 8, 1459–1473.CrossRefGoogle Scholar
  18. Tinoco, I., Borer, P., Dengler, B., Levine, B., Uhlenbeck, O., Crothers, D., and Gralla, J. (1973). Improved estimation of secondary structure in ribonucleic acids. Nature New. Biol. 246: 40–41.PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 1981

Authors and Affiliations

  • Barry Polisky
    • 1
  • Mark Muesing
    • 1
  • Joseph Tamm
    • 1
  1. 1.Department of BiologyIndiana University BloomingtonIndianaUSA

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