Mutations affecting replication and copy number control in plasmid mini-F both reside in the gene for the 29-kDa protein

Abstract

We have isolated and characterized cop, cop ts , and rep am mutants of plasmid mini-F after in vitro mutagenesis with hydroxylamine. cop mutants exhibit a copy number of about 10 per cell. The cop ts mutants are cold-sensitive and have, at 25 °C, a copy number of about 30–40 copies per cell, which drops to 4 copies at 42 °C. The cop and rep am mutations affect the 29-kDa E protein. The Cop ts phenotype results from the simultaneous occurrence of two mutations, a cop mutation in the E protein and a temperature-dependent mutation (termed ecp) enhancing the Cop phenotype at low temperature. The latter new type of mutation is located within the DNA region 44.1–44.85F. Complementation experiments with plasmid cointegrates show that the wild-type gene is dominant over the cop allele. The nucleotide sequences of the cop and the rep am mutations have been determined.