Fragile sites are heritable chromosomal loci that form cytogenetically evident gaps and/or breaks after specific biochemical induction. These loci occur throughout the human genome and occur at widely variable population frequencies. The molecular nature of fragile sites and their biological consequences are poorly understood. The fragile site mapping to Xq27.3, referred to as the fragile X site, is the only fragile site associated with a distinct phenotype, being found among patients and carriers of Martin-Bell syndrome (or fragile X syndrome). This X-linked condition is responsible for the most frequent form of inherited mental retardation, occurring at frequencies of >1 per 2000 newborns. It is unusual among mammalian X-linked loci because the gene is penetrant in ~30% of the carrier females and there is a relatively high frequency (~20%) of nonpenetrant carrier males. The fragile site itself is either the basis of this syndrome or is tightly linked to the gene(s) responsible. This proposal requests continuation support for a project to identify and clone the DNA responsible for the fragile X site in order to determine the relationship between the fragile X site and the associated syndrome. In addition, improved DNA-based diagnostic techniques will be developed to allow accurate carrier detection and prenatal diagnosis. To identify DNA of the fragile X site, a somatic cell hybrid system was developed that can monitor chromosome breakage within or near the fragile X site. A large series of independent human-hamster hybrids have been isolated that contain either human Xpter->q27.3->qter with the breakpoints at or very near the fragile X site. Since this breakage was found to be much more frequent in a fragile X chromosome relative to a normal chromosome, it is likely that the majority of the breakpoints are clustered in the vicinity of the sequence responsible for the fragile X site. Using somatic cell hybrid mapping panels, an increasing large number of probes have been found, by several laboratories, to map in the vicinity of the fragile X site. These probes will be used on Southern analysis of pulsed field gel resolved DNA of the hybrids with fragile X-associated chromosome breaks. Those probes that identify the breakpoints will be used to develop a physical map of this region, to initiate chromosome jumps and walks toward the breakpoints, and to identify corresponding YAC clones. The breakpoint junctions of these hybrids will be sequenced as will the corresponding regions of normal and fragile X syndrome genomes. The region surrounding these breakpoints will be searched for highly polymorphic loci which will be used for linkage analysis in both fragile X syndrome and reference families. Sequences that represent candidates for the fragile X site, either by breakpoint clustering, genetic mapping or unusual sequence, will be tested for fragility and/or instability in biological assays utilizing both mammalian and yeast cells. These experiments should illuminate the molecular nature of the fragile X site and provide accurate diagnostic probes for the fragile X syndrome as well as provide the framework for elucidating the molecular basis of this frequent and puzzling genetic disease.