Specific rearrangements of human chromosome 3 are characteristically associated with certain malignant disorders. Loss of heterozygosity studies have revealed a consistent deletion of 3p2l.1 in spontaneous carcinoma of the kidney. Cytogenetic studies of studies of several hundred small cell and non-small cell lung cancer cell lines reveal three distinct chromosome 3 regions consistently deleted; 3pl3-l4.2, 3p2l and 3p24.2-pter. Chromosome 3pl4.2 is also the location of the most common constitutive fragile site which may be involved in the pathogenesis of some of these rearrangements. The large size of 3pl3-p2l precludes identifying all genes in this region and testing them individually for their role in tumor development. In the present application we propose to build upon the already constructed physical map to clone and characterize several chromosome breakpoints that may represent a short-cut to identifying .genes involved in cancer development. The first is a t(3;6)(p2l.l;pl3) breakpoint associated with hematologic malignancies. We have already isolated a cosmid which spans this breakpoint and contains a gene apparently disrupted by the translocation. We will isolate and characterize this gene and all other genes in a 200 kb region around this breakpoint. The second is the t(3;8)(pl4.2;ql4.13) breakpoint associated with hereditary renal cell carcinoma. We have already isolated markers which flank this breakpoint and propose to walk from the closest proximal marker (less than 500 kb) using overlapping YAC clones. We will then characterize this region and all genes within several hundred kb of the breakpoint. Finally we plan to extend our studies on the analysis of aphidicolin-induced fragile site breaks that occur in the 3pl3-l4.2 region. Using a sensitive PCR-based assay we will screen large numbers of chromosome 3-only somatic cell hybrids in which chromosome breakage is induced with aphidicolin. This will show whether there is a clustering of fragile site breaks. We will then use the closest flanking probes as startpoints for chromosome walking to this region. The entire region will be isolated in overlapping YAC clones and tested to determine if sequences from this region can induce chromosome fragility in an in vivo system. In addition to elucidating the structure of the most common fragile site in the genome this project may also lead to the identification of genes that are consistently deleted in small cell lung and renal cell carcinoma.
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