Studies on factors that affect the rate of frame shift mutations in microsatellite sequences will be expanded. These factors include parameters of the microsatellite sequences themselves and mutations or altered expression of genes involved in the development of cancer. The P.I. is able to determine mutation rates and the nature of mutations in many different types of microsatellite sequences in a variety of cell types, using a plasmid vector through which these sequences are introduced into the host-cell genome. The microsatellite sequence is introduced into the vector, such that it disrupts the normal reading frame of a downstream neomycin-resistance (neo) coding sequence; clones are selected in which the reading frame of the neo gene has been restored as the result of insertions or deletions of integral numbers of repeat units in the microsatellite. A plasmid containing a (CA) 17-repeat has been introduced into human fibroblasts that express telomerase. They propose to determine whether mutation rates in these cells are comparable to those in normal diploid fibroblasts. If so, the P.I. will use these cells for many of the proposed studies. The investigators will ask whether a dominant-negative mutant mismatch-repair gene (hPMS2) results in elevation of mutation rates in the fibroblasts; these rates will be compared with those in a tumor cell line with a mutation in the same gene. They will also ask whether there are increases in mutation rates in CAK cells that have proceeded to anchorage-independence and/or outright malignancy. The P.I. proposes to continue work on the effects of the sequences of different repeat tracts on mutation rates. Besides, continuing work on the consequences of the presence of interruptions in pure-sequence tracts and on comparisons of the rates of deletion vs. insertion, they will analyze mutations of additional tetranucleotide repeats, complex microsatellites and small minisatellite sequences. They will undertake several studies designed to determine the effects of sequence context on microsatellite instability. These will include a set of experiments with a similar plasmid vector in the yeast Saccharomyces cerevisiae. Although the method that has been used for selection of frame shift mutations is sensitive and effective, the experiments each take several months to complete. In order to improve the efficiency with which the fluctuation tests can be carried, they will try using a gene fusion vector with green fluorescent protein as the reporter for mutations. With this marker, mutants can be selected by fluorescence-activated-cell sorting for many of the proposed studies.
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