The research here proposed has as its objective the improvement of methods of site-specific mutagenesis which are based on a common strategy--the generation of a short, single-stranded gap at a defined site on a circular DNA molecule, followed by the use of such a gap as a target for efficient in vitro mutagenic reactions. This research will involve refinement of the enzymatic reactions used to introduce nicks at unique sites and to convert such site-specific nicks into short gaps. In addition, a new mutagenic reaction, which induces mutations by stimulating DNA polymerase to misincorporate nucleotides during repair (misrepair) of single-stranded gaps, will be extensively evaluated. Initially, this reaction will be applied to several unique restriction sites on the plasmid pBR322 in order to optimize the efficiency of mutagenesis and to analyze the pattern of induced base substitution mutations. Once these properties have been defined, site-specific mutagenesis with this method will be used in the analysis of two cloned genes. First, mutations causing amino acid substitutions in the signal peptide of the TEM beta-lactamase will be constructed, and their effects on the transport of this enzyme across the bacterial cell membrane will be studied. Secondly, a long-term mutational analysis of the actin gene of Saccharomyces cerevisiae will be conducted. This project will involve the development of methods for efficiently replacing the chromosomal actin gene with mutant alleles constructed in vitro. In addition to providing improved methods for site-specific mutagenesis which should be applicable to any cloned segment of DNA, this research can be expected to yield new information on the biochemistry of protein secretion in prokaryotes. Furthermore, once a collection of conditional lethal mutant alleles of the yeast actin gene have been obtained, many basic questions dealing with the molecular biology of actin--its roles in maintaining cell structure and normal physiology in eukaryotic cells--should be answerable.
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