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Show THE UNIVERSITY OF UTAH UNDERGRADUATE RESEARCH ABSTRACTS Assaying Loss of Heterozygosity in Zebrafish Tyson J. Edwards (David J. Grunwald) Department of Human Genetics University of Utah The conversion of a normal cell into a cancer cell may be facilitated by a genetic change that has been termed loss of heterozygosity (LOH). In LOH the second wild-type copy of a specific gene is mutated, destroyed, or otherwise lost. Alfred Knudson first proposed the model for this genetic event in the early 1970s with the revolutionary "two-hit" hypothesis. He suggested that two hits, or mutations, affecting each copy of a critical gene present on homologous chromosomes would be sufficient to cause the retinal cancer. In many cases, the first hit occurs in the germ line such that all cells in the organism become heterozygous for that particular gene. A second hit can then occur in the soma, thereby rendering the cell without a functional copy of the critical gene. If this occurs in a tumor-suppressor gene then a likely effect may be uncontrolled proliferation and tumorogenesis. Many years later Cavenee proposed possible explanations for this loss of heterozygosity (LOH), specifically citing mitotic recombination and mitotic nondisjunction . As some evidence already suggests, we hypothesize that any factor, genetic or environmental, that increases the frequency of LOH will also increase the frequency of cancer formation. In order to measure LOH events we developed a genetic screen involving the mosaic eye assay in zebrafish. Using the golden gene (gol) that encodes for pigment in the eye, we were able to establish a baseline frequency for LOH in our fish population and to develop a dose-sensitive positive control using gamma ray induction. In order to obtain more-sensitive measurements of LOH, a transgenic line of zebrafish with fluorescence genes linked to the keratin8 promoter is being generated. Using this assay we will then 1) test several FDA approved drugs for their affect on the rate of LOH; 2) test other mutant fish to determine whether their mutation affects LOH; 3) analyze the types of genetic changes that occur in cells displaying LOH; and 4) determine the frequency of tumor formation in fish that display LOH. Tyson J. Edwards David J. Grunwald 24 |
