INSTABILITY OF MICROSATELLITES IN MAMMALIAN CELLS
Principal Investigator: ROSANN FARBER
Abstract: DESCRIPTION (Adapted from investigator's abstract): 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.
Funding Period: 1995-07-13 - 2007-06-30
more information: NIH RePORT
- Microsatellite instability testing in colorectal carcinoma: choice of markers affects sensitivity of detection of mismatch repair-deficient tumorsStephanie B Hatch
Curriculum in Genetics and Molecular Biology, Department of Genetics, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
Clin Cancer Res 11:2180-7. 2005..We investigated the relationship between instability of these markers and MMR protein expression in a cohort of sporadic colorectal cancer patients...
- Variation in efficiency of DNA mismatch repair at different sites in the yeast genomeJoshua D Hawk
Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC 27599, USA
Proc Natl Acad Sci U S A 102:8639-43. 2005..The simplest interpretation of this result is that the efficiency of DNA mismatch repair varies in different regions of the genome, perhaps reflecting some aspect of chromosome structure...
- Ninety-six haploid yeast strains with individual disruptions of open reading frames between YOR097C and YOR192C, constructed for the Saccharomyces genome deletion project, have an additional mutation in the mismatch repair gene MSH3Kevin R Lehner
Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina 27710, USA
Genetics 177:1951-3. 2007..We found that one set of 96 strains (containing deletions of ORFs located between YOR097C and YOR192C) in the collection, which was derived from the haploid BY4741, has an additional mutation in the MSH3 mismatch repair gene...
- Sequence-dependent effect of interruptions on microsatellite mutation rate in mismatch repair-deficient human cellsJayne C Boyer
Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, CB 7525, Chapel Hill, NC 27599, United States
Mutat Res 640:89-96. 2008..We conclude that interruptions decrease microsatellite mutation rate and influence the spectrum of frameshift mutations. The sequence of the interrupting base(s) determines the magnitude of the effect on mutation rate...
- MLH1 mediates PARP-dependent cell death in response to the methylating agent N-methyl-N-nitrosoureaJ R McDaid
Stem Cells and Epigenetics Research Group, Centre for Molecular Biosciences, School of Biomedical Sciences, University of Ulster, Coleraine BT52 1SA, Northern Ireland, UK
Br J Cancer 101:441-51. 2009..We wished to investigate the possible role of MLH1 in signalling cell death through PARP...