CHROMOSOME 5 SPECIFIC STSS AND NON-CHIMERIC YAG LIBRARIES

该项目的短期目标是快速生成 人类“框架”(0.5-1Mb)序列标记位点(STS)图 5号染色体，与非嵌合酵母人工染色体“锚定” (YAC)克隆。到目前为止，已经有50个5号染色体STS从 从流动排序的DNA生成的M13克隆，并按区域映射，在 与加州大学欧文分校约翰·瓦斯穆斯博士合作。其他标记将 直到绘制出大约300个STS为止。同时， 5号染色体特异性非嵌合YAC文库的构建 追求，以消除与第一代总 基因组YAC文库，例如嵌合体的高频率(&gt；50%) DNA在初步研究中，人类染色体特异的YAC文库 16号和21号染色体已经构建完成。通过1)最大化 具有两个可连接末端的片段的百分比，2)执行酵母 DNA含量低于饱和量的转化，以及3)添加 过量的载体DNA，YAC文库只有不到几个百分点 获得嵌合克隆(分析0/115)。 建议分辨率下的框架STS地图，附带非 嵌合YAC克隆，将覆盖大约60%的5号染色体，以及 将形成进一步重叠群组装的基础。在洛斯阿拉莫斯，我们将 集中在5号染色体的短臂(50Mb)。特别强调 将被放置在Cri Du Chat所涉及的5号染色体区域 综合征，人类最常见的末端缺失综合征之一。 鉴于我们对16号染色体物理图谱的相关经验， 5号染色体短臂2Mb重叠群图的构建 拥有0.1Mb的STS标记(人类基因组计划的5年目标)， 可以在所要求的供资期间实现这一目标。

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.