CHROMOSOME 5 SPECIFIC STSS AND NON-CHIMERIC YAG LIBRARIES

该项目的短期目标是快速生成一个

The short term goal of this project is the rapid generation of a "framework" (0.5-1 Mb) sequenced-tagged-site (STS) map of human chromosome 5, "anchored" with non-chimeric yeast artificial chromosome (YAC) clones. To date, 50 chromosome 5 STSs have been developed from M13 clones generated from flow-sorted DNA, and regionally mapped, in collaboration with Dr. John Wasmuth, UC-Irvine. Additional markers will be generated until approximately 300 STSs are mapped. In parallel, the construction of chromosome 5 specific non-chimeric YAC libraries will be pursued, to eliminate drawbacks associated with first generation total genomic YAC libraries, such as the high (>50%) frequency of chimeric DNA. In pilot studies, chromosome specific YAC libraries for human chromosomes 16 and 21 have been constructed. By 1) maximizing the percentage of fragments with two ligatable ends, 2) performing yeast transformations with less than saturating amounts of DNA, and 3) adding an excess of carrier DNA, YAC libraries with less than a few percent chimeric clones were obtained (0/115 analyzed). A framework STS map at the proposed resolution, with accompanying non- chimeric YAC clones, will cover approximately 60% of chromosome 5, and will form the basis for further contig assembly. At Los Alamos, we will concentrate on the short arm of chromosome 5 (50 Mb). Special emphasis will be placed in the region of chromosome 5 involved in the Cri du chat syndrome, one of the most common terminal deletion syndromes in humans. Given our relevant experience with physical mapping of chromosome 16, the construction of a 2 Mb contig map of the short arm of chromosome 5, with 0.1 Mb STS markers, (the 5-year goal of the hUman Genome Project), can be achieved during the requested funding period.