An Improved System for Selection of Forward Mutations in an Escherichia coli supF Gene Carried by Plasmids

Abstract

An improved system to examine forward mutations that occurred in the supF gene of Escherichai coli carried on a multicopy plasmid is described. The system was validated by measuring spontaneous mutations of supF plasmids propagated in wild-type, recA and mutM- mutY- E. coli strains, the mutation frequencies of which were 1.3 × 10-7, 6.3 × 10-7 and 1.5 × 10-6, respectively. Sequence analysis of the supF mutant plasmids revealed that G:C→T:A and G:C→C:G transversions dominated. This improved system allows rapid scoring and sequencing forward mutations in the supF gene, thus permitting its use as a genetic target for repair and mutagenesis studies in bacteria and mammalian cells.