The overall goal of these proposed studies is to determine the genetic significance and molecular basis for instability of common fragile sites. Common fragile sites are specific and constant regions on chromosomes that appear as gaps or breaks when DNA replication is perturbed, and which display a number of characteristics of unstable and highly recombinogenic DNA in vitro. Only one common fragile site, FRA3B at 3p14.2, has been investigated on the molecular level, and recent data strongly suggests that this instability is also seen in vivo. This fragile site extends over a large region of at least 200 kb. The mechanism of the fragility remains unknown but does not appear to be associated with CCG repeat expansion as are other cloned fragile sites. FRA3B is now known to lie within the FHIT gene. This gene has been shown to be highly unstable and contain deletions in a number of tumors and tumor cell lines, and as a result, FHIT has been proposed to be a tumor suppressor gene. Dr. Glover suggests that FRA3B is mechanistically associated with, or causes, this instability. The fact that FRA3B is associated with a highly mutable gene in cancer leads to the hypothesis that other common fragile sites may also be associated with genes, and that these fragile site regions and associated genes may also show a high rate of instability in tumor cells. The test of this hypothesis, additional studies on the mechanisms responsible for fragility and studies on the evolutionary aspects of common fragile sites are the major aims of this proposal. The P.I. proposes to clone and characterize additional common fragile sites, determine if they are associated with genes and if they are unstable in tumor cells. He will investigate the mouse Fhit gene for similar instability in tumors and presence of a fragile site as one test of the hypothesis that deletions in the FHIT gene are caused by FRA3B and to study the significance of FHIT deletions in cancer. To study the mechanisms of instability, Dr. Glover will analyze and compare sequences form new fragile site regions and will also investigate the influence of p53 and DNA repair/replication pathway mutations on fragile site expression and genome instability at fragile site loci. In addition, the evolution of specific common fragile sites will be traced to determine if they are conserved or if sequences critical for expression have diverged, and to explore if they are involved in chromosome rearrangements during evolution.
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