ROLE OF FRAGILE SITES IN CHROMOSOME BREAKAGE AND CANCER

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The overall aims of this proposal are to clone and characterize DNA sequences at the 3p14 fragile site (FRA3B). This fragile site will be studied because of its contribution to chromosome structure and due to its possible mechanistic involvement in chromosomal deletions seen in nearly all small cell lung cancers and many renal cell tumors. We have previously shown that this fragile site is a hot spot for chromosomal recombination as measured by sister chromatid exchange analysis and the induction of deletions and translocations at this site. As part of these experiments, we have utilized somatic cell hybrids containing human chromosome 3 to construct derivatives of this chromosome with deletions and translocations at FRA3B. The translocations have occurred with hamster chromosomes. We will use our translocation and deletion chromosomes as physical markers in complementary reverse genetics approaches to clone the fragile sites. We will attempt to identify the FRA3B fragile site translocation breakpoints with pulsed-field gel electrophoresis (PFGE) using currently available probes. We will also generate additional probes for this approach by preparing linking libraries from irradiation-reduced hybrids containing small segments of chromosome 3, including the fragile site, and physically map these clones with conventional hybrid panels that minimize the region around the fragile sites and by in situ hybridization. Once a probe is identified within close physical distance by PFGE analysis, protocols of chromosome jumping, cosmid walking and screening a general YAC library will be utilized to move close to, and ultimately clone, the fragile site sequences themselves. The irradiation-induced hybrids will be constructed in such a manner as to facilitate a complementary direct cloning strategy. Based on the observed high rate of chromosomal recombination at these sites, we will test the hypothesis that a selectable marker (the neor gene) can be inserted into the fragile sites and rescued together with flanking DNA sequences. Detection of the insertion event will be accomplished in part by co-segregation of the neor gene with DNA sequences close to FRA3B. Fragile site clones will be characterized at the DNA and RNA levels and tested in a series of biological experiments including testing for variation in normal individuals, transfection experiments, and in a direct test of the hypothesis that FRA3B plays a mechanistic role in chromosome breakage and deletions of chromosome 3 in human tumors including small cell carcinoma of the lung.