Cloning, Mapping and Sequencing Rodent Subtelomeric DNA

描述（由申请人提供）： 啮齿动物亚端粒DNA的克隆、定位和测序 本项目的目标是：（1）从小鼠和大鼠基因组中克隆端粒末端大片段DNA，（2）合作获得从小鼠和大鼠常染亚端粒区延伸到端粒末端（TTAGGG）的完整DNA序列，和（3）验证这些克隆预测的长距离序列组织，通过在同源天然染色体DNA上进行物理作图实验，进行基于作图和测序的实验。 哺乳动物端粒的DNA序列组织包括邻近末端端粒重复序列的大量高度相似的重复和低拷贝DNA。这种不寻常的序列组织导致了显着的复杂性方面的映射和测序亚端粒DNA和连接哺乳动物染色体的物理端点与全球克隆为基础的物理地图和草案序列。我们建议使用专门的酵母人工染色体（半YAC）系统，允许在酵母中的线性质粒的大端粒末端DNA片段的繁殖在啮齿动物基因组中解决这个问题。来自单个端粒的半YAC将与全局基于克隆的物理图谱和草图序列相连接，然后半YAC所包含的每个亚端粒区域的特定重复组织和DNA序列将被破译，而不受来自基因组其他地方的重复子的干扰。将使用位点特异性切割和脉冲场凝胶电泳方法对天然染色体DNA进行物理作图实验，以验证基于克隆的作图和测序实验所预测的长程序列组织，并检测特定亚端粒区域中潜在的大多态性。这种基本的方法来完成端粒映射和测序已成功地为大多数人类端粒，我们建议在这里将其应用到小鼠和大鼠的端粒。

DESCRIPTION (provided by applicant): Cloning, mapping, and sequencing rodent subtelomeric DNA The goals of this project are to (1) clone large telomere-terminal DNA fragments from the mouse and rat genomes, (2) to collaboratively acquire finished DNA sequence extending from the euchromatic mouse and rat subtelomere regions to the terminal (TTAGGG) in tracts for individual telomeres, and (3) to validate the long range sequence organization predicted by these clone-based mapping and sequencing experiments by carrying out physical mapping experiments on cognate native chromosomal DNA. The DNA sequence organization of mammalian telomeres includes large stretches of highly similar duplicated and low-copy DNA adjacent to terminal telomere repeat sequences. This unusual sequence organization has led to significant complications with respect to mapping and sequencing subtelomeric DNA and connecting the physical endpoints of mammalian chromosomes with global clone-based physical maps and draft sequences. We propose to address this problem in rodent genomes using a specialized yeast artificial chromosome (half-YAC) system that permits propagation of large telomere-terminal DNA fragments as linear plasmids in yeast. Half-YACs from individual telomeres will be connected with global clone-based physical maps and draft sequences, then the particular repeat organization and DNA sequence of each subtelomeric region encompassed by the half-YACs will be deciphered without interference from duplicons derived from elsewhere in the genome. Physical mapping experiments on native chromosomal DNA using site-specific cleavage and pulsed-field gel electrophoresis methods will be carried out to validate the long-range sequence organization predicted by the clone-based mapping and sequencing experiments, and to detect potentially large polymorphism in specific subtelomere regions. This basic approach to completing telomere mapping and sequencing has worked successfully for most human telomeres; we propose here to apply it to mouse and rat telomeres.