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 该项目的目标是(1)从小鼠和大鼠基因组中克隆大的端粒末端DNA片段，(2)合作获得从小鼠和大鼠等染亚端粒区域延伸到单个端粒末端(TTAGGG)的完整DNA序列，以及(3)通过对同源天然染色体DNA进行物理作图实验来验证这些基于克隆的作图和测序实验所预测的长范围序列组织。 哺乳动物端粒的DNA序列组织包括与末端端粒重复序列相邻的大量高度相似的复制和低拷贝DNA。这种不寻常的序列组织导致了在绘制和测序亚端粒DNA以及将哺乳动物染色体的物理端点与基于克隆的全球物理图谱和草稿序列连接方面的显著复杂性。我们建议在啮齿动物基因组中使用专门的酵母人工染色体(Half-YAC)系统来解决这个问题，该系统允许大的端粒末端DNA片段作为线性质粒在酵母中繁殖。来自单个端粒的Half-YAC将与基于全球克隆的物理图谱和草稿序列相连接，然后将破译Half-YAC所包含的每个亚端粒区域的特定重复组织和DNA序列，而不会受到来自基因组其他地方的复制子的干扰。将利用定点切割和脉冲场凝胶电泳法对天然染色体DNA进行物理作图实验，以验证克隆作图和测序实验所预测的长范围序列组织，并检测特定亚端粒区域潜在的大多态性。这种完成端粒映射和测序的基本方法已经成功地应用于大多数人类端粒；我们建议将其应用于小鼠和大鼠的端粒。

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.