Xiphophorus Gene Map Saturation--Genes Regulating Tumor Development

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DESCRIPTION: (Applicant's Description) The gene map of Xiphophorus fishes (Teleostei: Poeciliiidae) is one of the largest among vertebrates, with more than 100 genes among 350 markers assigned to independently assorting multipoint linkage groups residing on probably all 24 acrocentric chromosome pairs. This detailed linkage map has been used to identify a specific sex chromosome-linked oncogene and an autosomal CDNK2 tumor suppressor gene associated with susceptibility to spontaneous or UV-induced melanoma formation in interspecific backcross hybrids. Goals of this proposal include: 1) expansion of the gene map to more than 200 loci by mapping cellular oncogenes, potential tumor suppressor genes, cell cycle control genes, and members of other multigene families; 2) to saturate the linkage map with more than 500 anonymous DNA markers, primarily utilizing arbitrarily primed polymerase chain reaction (AP-PCR) techniques to generate polymorphic amplified DNA sequences; and 3) to increase the number of sequence tagged products in the Xiphophorus genome to facilitate positional cloning of genes of interest and to relate recombination distance to physical distance in the genome. A number of genetic crosses, each consisting of more than 100 backcross hybrids, will be utilized to assess linkage relationships of all informative genetic markers, including isozyme, pigment pattern, DNA restriction fragment length (RFL), specifically primed PCR amplification, and AP- PCR polymorphisms. Initially, most crosses will segregate approximately 100 markers which will establish most of the multipoint linkage groups. Linkage of markers in multiple crosses coupled with expansion of numbers of informative polymorphisms will finally coalesce multipoint linkage groups into 24 chromosomal linkage groups. By the end of the project period, placement of approximately 750 markers will result in an average of a marker every 2-5 centimorgans, and will provide many opportunities to correlate physical distances of markers in one or contiguous YAC segments with recombination frequencies. The most important contribution of a densely marked genetic map will be the potential to detect through linkage analyses new tumor regulatory genes in Xiphophorus models. Addition of a large number of characterized genes may provide candidates for such regulatory, or alternatively, positional cloning may identify previously uncharacterized regulatory genes whose homologues control tumorigenesis in man.