The major aim of this research is to isolate, study and map DNA fragments from human chromosome 4. The experiments proposed will fall into two areas which will be investigated simultaneously. First, the method of using yeast artificial chromosomes (YACs) to clone large segments of human DNA will be studied as part of the isolation of chromosome 4. The representation of human sequences in the cloned YAC pool, paying special attention to repeated sequences (VNTRs, LINES, SINES and alphoid repeats) will be examined. In addition, the long term stability and resistance to rearrangement in both wild type yeast and in mutants with alterations in recombination and DNA repair genes will be determined. Human fragments reported to stimulate (""""""""hotspots"""""""") or depress """"""""coldspots"""""""") recombination will also be studied for their effects on recombination in yeast. Second, studies of a novel method for ordering cloned DNA sequences called linkage dis- equilibrium mapping will be expanded. After establishing a 1% genetic linkage map (by traditional methods) for chromosome 4, disequilibrium mapping will be applied to order fragments located less than lcM apart. This technique will serve as a powerful alternative to physical mapping procedures (e.g., pulse field gel electrophoresis (PFGE)) in exactly those regions that are most difficult to physically resolve--coldspots where 1% recombination represents 107 or more bp. Disequilibrium mapping depends upon recombination distances and is as effective in a region where 1% recombination equals 107 bp as in a region where 1% equals 105 bp. The project will then generate several forms of complete maps of chromosome four and a complete library of chromosome four in yeast. Studies of recombination both directly using the maps of chromosome four and indirectly by studying the behavior of particular chromosome four sequences in a yeast recombination system will be made. This project will provide an essential step in establishing,the use of disequilibrium as a tool to confirm or extend physical maps. Finally, it will provide detailed insights into the mechanisms of recombination in yeast and man - one of the basic mechanisms of generating population diversity.
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