Non-phenotypic selection of N-methyl-N'-nitro-N-nitrosoguanidine-directed mutation at a predicted hotspot site

The striking mutational specificity of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) exhibited in the lacI gene in Escherichia coli allows comment on the phenotypic consequences of mutation at specific sequences that are not recovered after MNNG mutagenesis. We predict that the I+ phenotype is maintained when such silent positions are substituted by amino acids whose codons are generated by the MNNG-directed G:C----A:T transition. We chose the mutationally silent Gly200 codon (an MNNG hotspot motif sequence) to test this prediction. Through MNNG mutagenesis we have generated, identified and isolated a G:C----A:T transition at position 627 (5'-G-G-C-3') under non-selective conditions which creates the Gly200----Asp substitution. The I+ phenotype is retained for this altered repressor.