Cloning, Mapping and Sequencing Rodent Subtelomeric DNA

点击翻译按钮获取中文摘要

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.