Nucleotide Sequence Change in A Cole1 Copy Number Mutant

Abstract

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).