Fragile X-linked mental retardation is a relatively frequent syndrome associated with a fragile site at band Xq27.3. The objective of this proposal is the cloning of DNA sequences derived from the fragile X site using two independent approaches. Somatic cell hybrids, from fusions between patient's cells and CHO cells deficient HGPRT and G6PD activities, will used to test whether physical breakage occurs at the fragile X site. Since the HGPRT and G6PD loci flank the fragile X site, screening for G6PD activity should allow for the isolation of hybrids with either deletions or translocations of region distal to the fragile site. We have data showing the occurrence of such hybrids and have been recently successful in isolating human HGPRT+ G6PD hybrids, in which we hope to demonstrate deletions at or near the fragile X site. Cosmid libraries from translocation hybrids will be constructed and the junctional sequence of the translocation identified by screening with human and rodent repetitive sequences. The human portion of the junctional sequence should be derived from DNA located at or very near the fragile X site. Alternatively, DNA from the region of the fragile X site will be isolated by chromosome microdissection and microcloning. This should result in a small number of clones with inserts from the area of the fragile X site. Cloned DNA from both approaches will be screened for those closest to the fragile X site by gene mapping and linkage analyses. Those inserts closest to the fragile site will be used as probes to identify large DNA fragments on pulsed field gradient gels, and cosmid libraries from such fragments will be constructed. Cosmids whose inserts may contain the fragile X site will be used to probe for differences between normal and affected nucleic acids. Cloned sequences will also be tested by co-transfection for the ability to induce fragile sites. The proposed experiments should result in cloned sequences at and very near the fragile X site which could then be used for further investigation into the mechanism of chromosome fragility as well as for clinical diagnosis.
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