Gene-specific Suppressor Strains of Escherichia coli Carrying a Functional Gene G from ϕX174 on a Plasmid

Abstract

A majority of carcinogenic agents covalently modify DNA and produce mutations (Miller and Miller 1974). At present, no direct methods are available for determining either which of the many different kinds of modifications caused by such agents lead to mutation or the type of mutation generated by specific premutational modifications. We are exploring a direct approach to this problem in which (1) a well-characterized modification is introduced at a preselected single site in the genome of a suitable organism by a combination chemical and enzymatic synthesis; (2) the biological response to this modified DNA is followed by isolating any mutants that are produced from this modified DNA in vivo; and (3) the nature of the mutation is determined by DNA sequencing. Phage ϕX174 is an attractive choice for our initial studies because extensive information on its chemistry and biology is available (Sinsheimer 1968; Denhardt 1975; Sanger et al. 1977).

To test this approach, we have chosen an essential gene, gene G, which codes for a spike protein. However, propagation of non-conditionally lethal mutations in a virulent phage such as ϕX presents a problem. An attractive solution is to provide a source of the gene product in a host cell by introducing a biochemically constructed plasmid carrying a functional copy of the gene under investigation. Bacterial strains harboring such plasmids should act as gene-specific suppressors; they should be permissive for frameshift and large deletion mutations as well as base substitutions within the chosen gene. In this article we will summarize the results...