MINISATELLITE SEQUENCES AND HOMOLOGOUS RECOMBINATION

小卫星序列和同源重组

• 批准号: 3304487
• 负责人: PETER D MOORE
• 金额: $ 14.93万
• 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/3304487
• 关键词: alleles; binding proteins; eukaryote; gene mutation; gene therapy; 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.

对哺乳动物同源重组机制的认识 细胞的重要性不仅在于它对我们知识的贡献 遗传过程及其在实现 基因靶向。基因靶向将在分析 通过突变分析克隆基因的功能， 有助于基因治疗程序的发展。人们早就 应当理解，可以存在启动同源性的特定序列， 近年来，有证据表明， 在高等真核生物中普遍存在的高变小卫星基因座， 这种作用是不断积累的。我们最近证明， 合成序列SAT，显示与天然小卫星的同源性，是一个 人类细胞中质粒重组热点。的目标 本研究旨在探讨重组刺激的机制 卫星和天然小型卫星，以分析其在其他蜂窝通信系统中的作用。 重组事件，并利用这些序列来改善基因表达。 确定目标的方法。它旨在将SAT序列引入到一个 建立了基因打靶系统，以确定是否以及如何 小型卫星可能会影响这一进程。可能参与的 将检查姐妹染色单体交换过程中的小卫星 通过使用适当的探针来研究新的识别过程， 在小卫星基因座中的长度等位基因形成，以确定这是否 发生不平等的重组事件，这些是否优先 发生在这些地方。了解其机制的几种方法 将利用小型卫星进行复合刺激。这些 包括蛋白质的纯化和表征，所述蛋白质 特异性地结合到SAT序列上，一个广泛的分析， 分析了距离、 小卫星和重组事件之间的异质性， 存在于基底中的小卫星重复的数目。