The long-term objective of this proposal is the preparation of a molecular linkage map of the human X-chromosomes using polymorphism at DNA restriction enzyme sites (RFLP=restriction fragment length polymorphism) as genetic markers. X-linked RFLP's can be most easily identified by hybridizing single-copy, X-specific DNA probes to Southern blots of restriction enzyme digested human DNA. We will prepare such probes by isolating cloned, human X-chromosome DNA from our Charon 28-A9/HRBC2 DNA library. A9/HRBC2 is a reduced mouse-human hybrid cell which has regularly retained only the human X and a small fragment of chromosome 2. Single-copy segments of these purified human X DNA sequences will be identified and subcloned in appropriate plasmid vectors to establish a permanent collection of probes to screen for X-RFLP's. To further characterize these single copy sequences, they will be hydridize to Southern blots of restriction enzyme digested DNA from mouse-human hybrid cells which have retained only defined portions of the human X-chromosome or used in in situ hybridization experiments to human metaphase cells containing identifyable, aberrant X-chromosomes. The information provided by these experiments will alllow us to select DNA recombinant clones which may fall within measurable linkage of X-linked mendelian markers of known subregional location. We intend to use these probes to screen for RFLP's among the progenitors of a series of Sardinian pedigrees segregating for common as well as rare X-linked markers. The informative pedigrees will provide data on the location of X-RFLP's relative to the already mapped X-linked loci and to other X-RFLP's. Approximately, 10 well-spaced X-RFLP's would be sufficient to construct a complete linkage map of the human-X which would be subsequently used to map other X-linked loci (over 100 such loci are known) of unknown position. Particular efforts will be devoted to the screening for common RFLP's in linkage disequilibrium with X-linked lethal recessive mutants in view of the importance that such types of probes may have for the detection of silent heterozygous carriers and for the prenatal diagnosis of the lethal genotypes.
