XCEN-XQ21.3 IN OVERLAPPING YEAST ARTIFICIAL CHROMOSOMES

迈向人类基因组计划目标的第一步是产生 包含覆盖感兴趣区域的重叠插入的克隆。 这项提议旨在生成一套完整的、重叠的人类 覆盖Xcen至Xq21.3的酵母人工染色体中的DNA克隆 通过从一组标记开始，这些标记是物理排序的，并且 在遗传上并用连续、重叠的标记填充在标记之间 YAC克隆(重叠群)。 具体目标是：1.构建300kb的YAC文库 来自人类X染色体的插入片段。 2.从Xcen-Xq21.3中分离出三种类型的克隆DNA 构建YAC重叠群的起点：(A)包含以下内容的基因组DNA 外显子；(B)连接含有Noti限制的克隆的HpaII小片段 以及(C)由以下组成的多态基因组序列 可变数字聚(TG)n。这些标记将加入17个标记 在这个地区可用，其相对顺序是无可争辩的 为人所知。 3.获取每个标记克隆内的序列信息以允许 它将被用作“序列标记位点”或STS。 4.Xcen-q21.3 STS标记的定位。物理映射将是 通过(A)带有X的细胞系中的一组断点；常染色体 移位和间隙缺失；(B)远程限制图 由脉冲场凝胶电泳法产生。 5.利用以下方法确定多态STS标记的物理顺序 已有文献记载的相位已知的多点连锁分析 重组X染色体。 6.分离锚定在每个STS标记上的连续YAC克隆。探头 从这些锚定的YAC末端制备的YAC将用于分离 用于构建从每个STS向外延伸的重叠群集合的附加YAC 标记，直到找到相邻重叠群之间的重叠。 跨越Xcen-q21.3的YAC重叠群将为完整的 对该区域进行测序。外显子和外显子锚定重叠群的策略 HTF岛的目标DNA片段可能包含基因和 因此具有生物学和医学上的重要性。

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.