MINISATELLITE SEQUENCES AND HOMOLOGOUS RECOMBINATION

小卫星序列和同源重组

• 批准号: 3304490
• 负责人: PETER D MOORE
• 金额: $ 17.2万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-01-01 至 1995-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3304490
• 关键词: alleles; binding proteins; cell cycle; eukaryote; gene mutation; gene therapy; genetic crossing over; genetic manipulation; genetic recombination; genotype; heterozygote; homozygote; hypoxanthine phosphoribosyltransferase; molecular cloning; nucleic acid sequence; plasmids; point mutation; protein purification; recombinant DNA; tissue /cell culture

An understanding of the mechanisms of homologous recombination in mammalian cells is of importance both for its general contribution to our knowledge of genetic processes and for its practical application in accomplishing gene targeting. Gene targeting will play a major role in analyzing the function of cloned genes by mutational analysis and will in the long term contribute to the development of gene therapy procedures. It has long been understood that specific sequences may exist that promote homologous recombination, and in recent years circumstantial evidence that the hypervariable minisatellite loci, ubiquitous in higher eukaryotes, may play such a role has been accumulating. We have recently demonstrated that a synthetic sequence SAT, showing homology with natural minisatellites is a hot spot for recombination between plasmids in human cells. The goals of this proposal are to investigate the mechanism of recombination stimulation by SAT and natural minisatellites, to analyse their role in other cellular recombination events and to exploit these sequences to improve gene targeting methodologies. It is intended to introduce SAT sequences into an established gene targeting system to determine whether and how minisatellites may affect this process. The possible involvement of minisatellites in the process of sister chromatid exchange will be examined by using appropriate probes to investigate the recognized process of new length allele formation in minisatellite loci to determine whether this occurs by unequal recombination events and whether these preferentially occur at these loci. Several approaches to understanding the mechanism of recombination stimulation by minisatellites will be undertaken. These include the purification and characterization of a protein shown to specifically bind to the SAT sequence, an extensive analysis of the products of recombination and analysis of the affects of distance and heterology between the minisatellites and the recombination events and of the number of minisatellite repeats present in the substrates.

哺乳动物同源重组机制的探讨