The terminal band of the human X chromosome long arm, Xq28, is estimated to be <10,000 kb. Although small in size, over 20 loci have been mapped to this region, 13 of whom have alleles that lead to genetic diseases of which little or nothing is understood at the molecular level, such as Emery- Dreifuss muscular dystrophy and an X-linked form of manic-depressive illness. A somatic cell genetic approach has been developed which allows for the isolation of Xq28 within a hamster cell line, as the sole human DNA present, permitting rapid saturational cloning of this small region of the human genome. For these reasons, Xq28 is an ideal region of the human genome for in-depth molecular analysis. Somatic cell hybrids, isolated from fusions between hamster cells deficient in both HGPRT and G6PD activities and human cells derived from fragile X syndrome males, will be used to select clones that have lost all of the human X chromosome except for Xq28. this will be accomplished by inducing, in the hybrid cells, chromosome breakage at the fragile X site (Xq27.3) with thymidine stress and identifying those cells which have lost HGPRT (which maps proximal to the fragile site) but retained G6PD (which maps to Xq28). A strategy for isolating such hybrids is based upon selection against HGPRT with 6- thioguanine and subsequent enrichment for G6PD-positive cells by oxidative stress. Multiple independent hybrids will be isolated which appear to contain only human Xq28 and they will be subjected to careful and complete characterization to verify the presence of the unrearranged authentic human chromosomal band. Cosmid libraries of hybrid cells will be prepared and clones containing human inserts identified and isolated to provide a panel of cosmids of sufficient number to saturate Xq28. This cosmid panel will be screened for those that detect new polymorphic loci. Loci which detect relatively frequent plymorphisms will be placed upon the genetic and physical maps of Xq28. A genetic map, utilizing known and newly identified loci, will be constructed to approach 1 cM resolution. These loci will also be placed upon a physical map of this region, using pulsed filed gel electrophoresis, to allow for the correlation of the two maps. Such a composite map, as well as the resource of cosmids which saturate Xq28, should prove valuable for the identification and isolation of candidate genes for the genetic disorders which map to Xq28 as well as provide the substrates necessary for a future contig and sequence analysis of Xq28.
