The major research effort in my lab involves the use of a cell culture system for detection of frameshift mutations in short tandem repeat sequences (microsatellites). We are using this system to quantitate the rates of mutation of these tandem repeat sequences in colorectal cancer cell lines with microsatellite instability, which is a phenotype consistently observed in patients with Hereditary Nonpolyposis Colorectal Cancer (HNPCC). These patients have defects in DNA mismatch repair leading to the high rates of mutation in mirosatellites. We have constructed a plasmid vector that contains a fusion of a (CA)17 sequence to the 5' end of the neomycin-resistance gene, such that the gene is out of frame and, therefore, nonfunctional. This plasmid is introduced into cells by transfection, and frameshift revertants are isolated. Revertants generally have insertions or deletions of integral numbers of CA-repeat units that restore the reading frame of the neo gene. We are using this approach to gain a general understanding of the involvement of this type of instability in cancer. We have found that two neoplastically transformed cell lines with normal mismatch repair have microsatellite mutation rates that are substantially higher than those of normal fibroblasts. We are asking whether this elevation in mutation rate results from disruption of the function of genes other than those involved in mismatch repair. In addition, we are studying parameters of the repeated sequence that affect the instability (tract length, repeat-unit length, and sequence composition). For example, we have determined that the rates of insertion mutations in CA-repeats are higher than the rates of deletion; this finding may have significant implications regarding the evolution of these sequences.