Mutations in the bacteriophage λ pL/oL region that spontaneously occur in plasmid pRPZ126

In a previous study (Chen and Porter, 1988), we isolated spontaneous mutations in a test plasmid that had occurred under non-selective conditions and assigned them to 1 of 6 different categories or groups. The test plasmid, pRPZ126, is a pBR322 derivative containing the bacteriophage λ immunity region with the cI₈₅₇ allele so that plasmid-containing cells shifted to 42°C survive only if the expression of th λkil gene is prevented by mutation. 75% of the total spontaneous mutations obtained fall into two of these groups where there is no readily detectable change in plasmid size. The two groups differ in that the plasmids from the group 4 mutations are missing a specific HincII site while the plasmids from the group 5 mutations had no detectable plasmid change whatsoever. In this study, we randomly selected ten group 4 mutants and ten group 5 mutants and sequenced the λ pL/oL region of the mutant plasmid. Of the ten group 4 mutants (HincII site missing), five involved a 24- or 44-basepair detection in the pL/oL region of the plasmid. The other five group 4 mutants and four of the ten group 5 mutants were A-T to G-C transitions in the pL/oL region. The remaining six group 5 mutants did not demonstrate any sequence change in the pL/oL region of the plasmids. 8 out of 9 of these transition mutations occurred next to the 3′ end of 3 different 5′-PyGGNPuNTG-3′ sequences in the λ operator region, and this same sequence is found adjacent to the A-T to G-C transition hotspot in the lac operator region (Schaaper et al., 1986). The 9th mutation, where the A-T to G-C transition occurred one basepair away from the λ operator, was adjacent to a very similar sequence.