LOCALIZATION OF THE WILMS TUMOR SUSCEPTIBILITY GENE(S)

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Wilm's tumor is one of the most common malignancies of children. Strong evidence indicates that tumor suppressor genes are located at 11p13 and 11p15.5. Deletions in these regions contribute to the genesis of Wilm's tumor. To understand the basic defect in this cancer, the Wilms tumor genes (WTG) at 11p13 and 11p15.5 must be isolated and their products analyzed. To this end, an overlapping library of human chromosome 11 will be made in yeast artificial chromosome (YAC) vectors using DNA from a human:rodent hybrid cell line. Insert DNA will be partially digested and size selected >400kb. The YAC library will be screened with known probes from the 11p13 and 11p15.5 regions. Comprehensive overlapping libraries of the 1500kb of 11p13 and the 20mb of 11p15.5 will be created. The YAC inserts will be compared to existing maps of the regions. YAC chromosome walking will be used to fill in gaps between known probes. Any gaps that are not able to be cloned in YACs will be cloned in cosmid or lambda vectors. The regions will be physically mapped with pulsed field gel electrophoresis capable of resolving 50-5000kb fragments of DNA. Panels of DNA from Wilm's tumors will be used to more precisely map the WTG locus in 11p15.5. Transcribed regions of the 11p13 and 11p15.5 libraries will be isolated by several methods: 1) insertional mutagenesis with a selectable marker; 2) hybridization with cDNA from human kidney; 3) identification of unmethylated CpG islands; and 4) cross-species hybridization. Probes that detect expressed genes will be hybridized to Northern blots of mRNA from multiple Wilm's tumors. Probes that detect variable sizes or decreased levels mRNA from some of the Wilm's tumors will be candidate WTG exon probes. Additionally, the YACs will be tested for tumor suppression function by spheroplast fusion into a Wilm's tumor cell line, G401. If a YAC clone is able to suppress tumorigenicity, deletion analysis and insertional mutagenesis would greatly facilitate localization of a WTG.