The objective of the proposed research is to identify and characterize a gene involved in lung cancer development and progression on the centromeric portion of chromosome 11p15.5. The appliant s previous studies have provided evidence for the existence of two regions with frequent allelic loss on chromosome 11p in lung cancer which suggests the presence of tumor suppressor genes. A region in the centromeric part of chromosome band 11p15.5, which was identified by the polymorphic marker D11S12, had loss of heterozygosity (LOH) in 57% of all 156 lung cancer cases analyzed. This region is approximately 0.5 megabases (Mb) in size and is located between the markers D11S1758 and D11S860. The objective of this research proposal is to clone the tumor suppressor gene/s in this region using a positional cloning approach. The applicant proposes 1) to complement the partial Pl phage and cosmid contig of the target region (D11S1758 to D11S860) with P1 phage and cosmid clones using the """"""""DuPont"""""""" and """"""""PAC"""""""" human genomic Pl libraries and the National Laboratory and ICRF chromosome 11 specific cosmid libraries. He will assemble the contig by """"""""chromosome walking"""""""" using Pl phage and cosmid-specific end-labeled probes generated by PCR with SP6, T3, and T7 primers and with Pl phage-specific primers generated by direct sequencing. Contig gaps will be filled with bacterial artificial chromosome (BAC) clones by screening the BAC human genomic library available from GenomeSystems, Inc.; 2) to refine the presently known region of allele loss to less than 500 Kb with new microsatellite markers. New markers will be generated by hybridization of a poly(CA/TG) probe to contig clones and by direct sequencing. Allele frequencies of these new markers will be assessed in an unrelated population survey. 3) to positionally clone genes within this region with a solution hybridization and magnetic bead capture method and by screening human cDNA libraries with contig subclones. For solution hybridization and magnetic bead capture cosmid and Pl clones from the contig will serve as """"""""selector"""""""" templates. """"""""Driver"""""""" templates will be synthesized from normal lung polyA RNA. Selected sequences will be analyzed for an open reading frame, and they will be compared with published sequences in data bases. Genes identified (""""""""candidate"""""""" genes) will then be screened for evolutionary conservation, expression in normal lung, and mutations.
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