Mutagenesis is a fundamental biological process whose elucidation is necessary for understanding a variety of topics involving genetics, evolution, carcinogenesis and aging. In order to explain spontaneous and induced molecular mechanisms of mutagenesis, mutation specificity must first be discerned. We propose to examine mutational specificity in Escherichia coli using two complementary approaches. First we will determine the reversion spectra of trpA base-pair substitution and frameshift mutations. The amino acid changes in the trytophan synthetase A protein resulting from these trpA mutants and their Trp+ revertants are known, allowing the deduction of nucleotide changes associated with the revision events. The trpA reversion system is ideal for the detection of rare or poorly induced mutational events because of the ease of selection of Trp revertants for the various trpA mutant alleles. However only a small number of sites (8-10) within the trpA gene are available to detect reversion events. This precludes the opportunity to characterize sites of mutational preference (hotspots). Furthermore complex mutational events, i.e., large insertions, rearrangement and deletions, can't be detected with the trpA system. Our second approach, recently developed, involves the rapid repeated cloning of mutant lacI repressor genes (lacI genes). This procedure allows the efficient recovery of lacI- genes from F lac onto a single-stranded M13 phage vector. Recovery of the lacI mutation in the phage from the F plasmid is based on the conversion of this lacI+Z phage to lac-Z+ by recombination with F' lacI-Z+. This double change produces -complementing ability in the phage vector. The recombinant phage can be readily selected for (by its - complementing ability) and the lacI- mutation characterized by direct DNA sequence determination. With this procedure hundreds of independent lacI- mutations can be analyzed. Any mutant site within the lacI gene that produces a lacI- phenotype is amenable to analysis with this system. Thus hotspots and complete mutational spectra can be deduced by DNA sequencing. We intend to utilizing both of the above approaches to study spontaneous and induced mutations.
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