Investigators' Abstract) High-resolution physical maps of human chromosomes depend on knowing the order of a series of genetic markers and the distance between adjacent markers. A limitation of some current approaches to top-down physical mapping is their dependence on the availability of relatively rare, naturally occurring chromosome translocations and deletions. To generate an unlimited number of well-ordered breakpoints for physical mapping, the investigators have developed a somatic cell hybrid approach in which an individual human chromosome in a rodent/human hybrid is simultaneously subjected to both positive and negative selection pressure. Hybrids surviving this counter-selection (called """"""""Pushmi-pullyu"""""""" hybrids) have segregated the portion of the chromosome carrying the gene under negative selection, while retaining the portion of the chromosome carrying the gene under positive selection. A series of such hybrids represents a collection of evenly distributed breakpoints along the chromosome for use in physical mapping. A unique order of markers can be deduced from the linear deletion map generated from hybridization patterns of a number of DNA probes against the Pushmi-pullyu hybrid panel. The investigators propose to use this scheme to generate a high-resolution, one megabase physical map of the human X chromosome, using as selectable markers the HPRT gene located in Xq26 and the A1S9T temperature-sensitivity complementing gene located in Xp11. The specific goals of the proposal are to: 1) isolate a series of 200-250 Pushmi-pullyu hybrids of the X chromosome; 2) characterize the hybrids by fluorescence in situ hybridization analysis and by Southern blotting with a series of reference markers to assign individual breakpoints to one of a series of primary map intervals on the X; 3) saturate the Pushmi-pullyu hybrid mapping panel with 250-500 new X-linked DNA probes, including a series of yeast artificial chromosome probes from the X in order to derive a best-fit linear map of all markers; 4) use X-linked YAC clones in pulsed-field gel analysis to localize precisely fragments detected by the YACs; and 5) assess the general applicability of Pushmi-pullyu hybrid mapping by expanding the approach to include the selectable MIC2 gene in Xp22.3. The use of these three, well-spaced genetic markers on the X will lead to the development of a one-megabase physical map for virtually the entire length of the human X chromosome.
