A system to characterize mutations arising from in vitro nucleotide misincorporation, which avoids the effects of in vivo mismatch repair on recovery of mutants, was constructed and evaluated. The lacI gene of Escherichia coli was inserted into phage M13 and the M13-lacI recombinant was introduced into a strain of E. coli lacking a resident lacI gene. In this system the function of the M13-bearing lacI gene can be detected by plaque color. Mutants in the 5′-region of the lacI gene (encoding operator-binding domain) are seen as blue plaques when the host strain is grown in the presence of chromogenic substrate, X-gal, in the absence of inducer. The use of uracil-containing single stranded DNA from M13-lacI as template for DNA synthesis avoids the contribution of mismatch repair (in transfection recipients) on the recovery of mutants. To demonstrate the usefulness of the M13-lacI system we produced nucleotide misincorporations by in vitro DNA synthesis in the N-terminal region of the lacI template in the presence of only 3 deoxynucleoside triphosphates (dNTPs). Such mutagenic reactions were conducted in the absence of dATP with 4 different primers and in the absence of dGTP with 2 primers. The type of mutants produced by these reactions were identified through sequencing of DNA from progeny phage after screening for i-(blue plaque) phenotype. Mutations recovered in this system consisted of single and multiple base substitutions in the region of the template near the 3′-terminus of the primer. Nearly all of the mutants induced by '-A' conditions were T → C base substitutions, and those induced by '-G' conditions were C → T transitions. In general, the results were consistent with the spectrum of spontaneous mutants produced in strains deficient in mismatch repair, although some differences were noted. Several new base substitutions within the lacI gene (producing i-phenotype and unobserved by others) were isolated by the procedures described in this paper. © 1991.
|Original language||American English|
|Number of pages||179|
|Journal||Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis|
|State||Published - 1 Jan 1991|