XCEN-XQ21.3 IN OVERLAPPING YEAST ARTIFICIAL CHROMOSOMES

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A first step towards the goal of the Human Genome Initiative is to generate clones that contain overlapping inserts covering a region of interest. This proposal is aimed at generating a complete, overlapping set of human DNA cloned in yeast artificial chromosomes (YACs) covering Xcen to Xq21.3 by starting with a set of markers that are ordered physically and genetically and filling in between the markers with contiguous, overlapping YAC clones (contigs). The specific aims are: 1. Construction of a YAC library with 300 kb inserts from the human X chromosome. 2. Isolation of three types of cloned DNA from Xcen-Xq21.3 to serve as the starting points for building YAC contigs: (a) Genomic DNAs containing exons; (b) HpaII tiny fragment linking clones containing NotI restriction endonuclease sites; and (c) polymorphic genomic sequences consisting of variable number poly(TG)n. These markers will join 17 markers already available in this region whose order relative to each other is indisputably known. 3. Acquisition of sequence information within each marker clone to allow it to serve as a "sequence tagged site" or STS. 4. Mapping of these Xcen-q21.3 STS markers. Physical mapping will be accomplished by (a) a panel of breakpoints in cell lines with X;autosome translocations and interstitial deletions; (b) long-range restriction maps generated by pulsed field gel electrophoresis. 5. Confirmation of physical order of polymorphic STS markers using multipoint linkage analysis of previously documented phase-known recombinant X chromosomes. 6. Isolation of contiguous YAC clones anchored at each STS marker. Probes prepared from the ends of these anchoring YACs will be used to isolate additional YACs to build sets of contigs that extend out from each STS marker until overlap is found between neighboring contigs. YAC contigs spanning Xcen-q21.3 will provide the raw material for complete sequencing of the region. The strategy of anchoring contigs at exons and HTF islands targets DNA segments that are likely to contain genes and therefore to have biological and medical importance.