[Production of directed mutations in T7 phage by transfecting Escherichia coli using locally alkylated T7 DNA].

Modified polyribonucleotides carrying a number of alkylating groups were used for alkylation of complementary sites of DNA to induce gene-directed mutations. Polyfunctional alkylating mustard, N,N,N'-tri-(beta-chloroethyl)-N'-(p-formylphenyl)-propylene diamine-1,3, moieties were attached to T7 phage early transcripts. The transcripts modified to 4-5% were hybridized with T7 DNA to form R-loops, and RNA molecules were covalently bound to the complementary DNA sites as a result of the activation of RNA carried alkylating groups. T7 DNA molecules alkylated by modified RNAs transcribed from 0.3 and 1.1 T7 phage genes were used for transfection of Escherichia coli. In the progeny of phages generated by the transfection of 24 negative colonies, 3 contained double amber mutants in amounts of 2-10%. Complementation tests permitted to assign all mutations to the same complementation group. Recombination analysis established that one of the mutations concerns gene 0.3. The results of complementation studies indicate that the second mutation, with high probability, concerns gene 1.1.