Characterization of the types of mutational events that spontaneously occur in a plasmid system

The immunity region from a cI857 derivative of bacteriophage λ has been cloned into the EcoRI site of pBR322 to produce a plasmid that can be used to analyze spontaneous mutagenesis. Cells containing this plasmid are temperature-sensitive for growth unless mutations have occurred that somehow prevent the expression of the kil gene in the λ fragment at non-permissive temperature. 678 such temperature-resistant mutants from 10 independent subcultures each of 2 different recA^- E. coli strains have been collected, and the nature of the plasmid mutations obtained has been analyzed. All of the subcultures contained mutants that allowed growth at the restrictive without showing a detectable change in plasmid size. 75% of the total mutants fell in this class. More than half of these mutations involved the λ leftward promoter, pL, and such mutants were found in all 20 subcultures. The remaining 25% of the mutations involved a change in plasmid size and mutations of this class were found in 18 of 20 subcultures. 12% of the total mutants (found in 16 of 20 subcultures) had an insertion of IS1 in the region between pL and the λkil gene. 6% of the total mutants had undergone an IS1-mediated deletion, while 1% were mixed colonies in which multiple IS1-mediated events had occurred. About 1% of the total mutants had undergone complex IS1-mediated DNA rearrangement(s) that have not yet been characterized. In total, 11 of 20 subcultures yielded isolates where IS1-mediated rearrangements had occurred. The remaining 4% of the mutations included insertions of IS5, IS30, and an IS1 family member that appears to be IS1T as well as IS1T-mediated deletions and deletions that do not appear to have been mediated by any insertion sequence. A mutant with both an IS1 insetion and an alteration involving pL has also been isolated.