MINISATELLITE SEQUENCES AND HOMOLOGOUS RECOMBINATION

对哺乳动物同源重组机制的认识 细胞是重要的，因为它对我们知识的普遍贡献 关于遗传过程及其在实现 基因打靶。基因打靶将在分析 通过突变分析克隆基因的功能及其长期意义 为基因治疗程序的发展做出贡献。一直以来都是 理解可能存在促进同源的特定序列 重组，以及近年来的间接证据表明 在高等真核生物中普遍存在的高变数小卫星基因座可能起作用 这样的角色一直在积累。我们最近展示了一种 合成序列SAT，与天然小卫星同源，是一种 人类细胞内质粒间重组的热点。的目标 这项建议是为了研究重组刺激的机制 通过SAT和天然小卫星，分析它们在其他细胞中的作用 重组事件和利用这些序列来改良基因 目标方法论。它旨在将SAT序列引入到 建立基因打靶系统以确定是否以及如何 小卫星可能会影响这一过程。可能参与其中的 将对姐妹染色单体交换过程中的小卫星进行检查 通过使用适当的探针来研究新的 在小卫星座位上形成长度等位基因来确定这是否 由不相等的重组事件发生，以及这些事件是否优先 发生在这些基因座上。理解心力衰竭发病机制的几种途径 将进行微小卫星的复合刺激。这些 包括对一种蛋白质的纯化和鉴定 具体结合到SAT序列，对 重组产物及其对距离和距离的影响分析 小卫星和复合事件之间的异质性以及 底物中存在的小卫星重复数。

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.