Cancers of the digestive tract are a leading cause of cancer related deaths in the United States. Since the majority of these tumors are of epithelial origin characterization of the cellular processes which regulate epithelial renewal in these organs is essential for understanding their malignant transformation. Genetic analyses are particularly suited to the dissection of complex regulatory pathways and for this reason we have begun a molecular analysis of intestinal epithelial renewal using the zebrafish. The zebrafish is the only vertebrate in which large scale forward genetics is feasible and through chemical mutagenesis we have discovered a group of novel mutations which perturb epithelial development in the digestive organs of this organism. From this group of mutants we have defined two mutations, slimjim and meltdown that appear to regulate events in the zebrafish intestinal epithelium that are crucial for orderly renewal of cells in the intestine of mammals. Since only a small number of mammalian genes are known to contribute to the process of epithelial renewal we have designed a strategy for the molecular characterization of the genes responsible for slimjim and meltdown. Both mutations were discovered through chemical mutagenesis so their molecular characterization is dependent upon positional cloning, a complex process but one for which the zebrafish is well suited. In this proposal we outline a strategy for the identification of the slimjim and meltdown genes using positional cloning.
Our first aim i s to identify molecular markers tightly linked to each mutant locus using the Amplified Fragment Length Polymorphism technique (AFLP). Next we will use these markers to generate a physical map of this region of the genome by identifying overlapping contigs from a bacterial artificial chromosome (BAC) library screened with the genetic markers. Using these BAC clones we will further narrow the size of the mutant loci and then identify transcripts from this region using cDNA selection, BAC screening of cDNA libraries, and sequencing of BAC clones. From these candidates the responsible gene will be identified through transcript analysis, sequencing and mutant rescue using BAC clones. An additional aim of this work is to generate new alleles of each mutation using gamma-irradiation, an efficient mutagen in zebrafish. The gamma-alleles
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