Region- and strand-specific mutagensis of a recombinant plasmid

Techniques were developed to mutagenize a single DNA strand in a specific region of the tetracycline-resistance (tetr) gene of the plasmid pKB280 that also carries the lambda repressor gene. Separate annealings of complementary single strands gave two isomeric, circular plasmids containing a 275-nucleotide, single-stranded region (gap) in the tetr gene. One of the isomeric, gapped plasmids was mutagenized specifically with sodium bisulfite such that an estimated 98% of the molecules had suffered at least one C to U conversion in the gap. The mutagenized gap was filled in with DNA polymerase. These molecules transformed Escherichia coli strain MM294 to lambda-immunity with the same frequency as unmutagenized, gap-filled pKB280. Of the lambda-immune transformants, 32% were Tcr and 68% were Tcs. Restriction analysis of plasmids from some Tcs transformants showed losses of restriction sites within the gap and at the gap termini, but none outside the gap. No deletions were detected.