XCEN-XQ21.3 IN OVERLAPPING YEAST ARTIFICIAL CHROMOSOMES

重叠酵母人工染色体中的 XCEN-XQ21.3

• 批准号: 3333274
• 负责人: Jennifer M. Puck
• 金额: $ 21.47万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-04-01 至 1994-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3333274
• 关键词: DNA; biotechnology; chromosomes; endonuclease; fungal genetics; gel electrophoresis; genetic library; genetic manipulation; genetic mapping; genetic markers; genetic polymorphism; human genetic material tag; hybrid cells; linkage mapping; molecular cloning; nucleic acid hybridization; nucleic acid structure; polymerase chain reaction; sex chromosomes; yeasts

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.

实现人类基因组计划目标的第一步是 包含覆盖感兴趣区域的重叠插入物的克隆。 该提案旨在生成一组完整的、重叠的人类 在酵母人工染色体（YAC）中克隆的DNA覆盖Xcen至Xq21.3 通过从一组物理上有序的标记开始， 在遗传上， YAC克隆（重叠群）。 具体目标是：1. 300 kb YAC文库的构建 从人类X染色体上插入的基因。 2. 从Xcen-Xq 21.3中分离三种类型的克隆DNA，以用作 构建YAC重叠群的起始点：（a）基因组DNA，其含有 外显子;（B）含有NotI限制性酶切的HpaII微小片段连接克隆 核酸内切酶位点;和（c）多态基因组序列，由以下组成 可变数聚（TG） 这些标记将加入17个标记已经 在这个区域中，它们之间的顺序是无可争议的。 知道的 3. 获得每个标记克隆内的序列信息，以允许 它被用作“序列标记位点”或STS。 4. 这些Xcen-q21.3 STS标记的定位。 物理映射将是 通过（a）具有X的细胞系中的一组断裂点;常染色体 易位和间质缺失;（B）长距离限制性酶切图谱 通过脉冲场凝胶电泳产生。 5. 多态性STS标记的物理顺序的确认， 多点连锁分析先前记录的阶段已知 重组X染色体。 6. 锚定在每个STS标记处的连续YAC克隆的分离。 探针 从这些锚定YAC的末端制备的材料将用于隔离 额外的YAC以构建从每个STS延伸出的重叠群组 标记，直到在相邻重叠群之间发现重叠。 跨越Xcen-q21.3的YAC重叠群将为完整的Xcen-q21. 区域的排序。 将重叠群锚定在外显子和 HTF岛靶向可能含有基因的DNA片段， 因此具有生物学和医学上的重要性。