MOLECULAR STUDIES OF HUMAN GENETIC RECOMBINATION

基因信息的重新排列， DNA重组发生在所有生命形式中。 遗传 重组允许基因定位，并已被牵连， 免疫球蛋白基因重排，DNA修复，基因 扩增，癌症的初步发展，以及 防止非整倍体及其导致的出生缺陷。 尽管重组在高等真核生物中很重要， 很难研究，对它的研究知之甚少。 生物化学 在这个提议中，重组的中心步骤， 同源DNA之间的链转移将被研究。 我 先前报道了哺乳动物的第一个特征， 链转移蛋白（重组酶）。 我现在提议 研究这种蛋白质促进的链转移反应。 分析将集中在a）核蛋白网络的形成 通过重组酶及其同源性搜索的性质，B） 平行关节的形成和c）方向性 节理形成和分支迁移。 特殊DNA的作用 也将确定链转移反应的顺序 通过使用新的底物和寡核苷酸。 在上述研究的同时，一种新的测量方法 在培养的人类细胞中进行重组。 这 系统利用串联互补突变抗生素- 逆转录病毒载体中含有的抗性基因。 它将用于 测量染色体外重组，染色体内重组 染色体间重组（姐妹染色单体 交换）和基因转换， 来源于染色体不稳定患者的成纤维细胞系 综合征 通过这些方法可以检测到突变体。 实验 这种突变体的鉴定将有助于 分析哺乳动物细胞中的重组， 提供了一个独特的机会，研究分子病理学， 这些疾病。

The rearrangement of genetic information through the recombination of DNA occurs in all forms of life. Genetic recombination allows gene mapping and has been implicated in immunoglobulin gene rearrangement, DNA repair, gene amplification, the initial development of cancer, and the prevention of aneuploidy with its resultant birth defects. Despite the importance of recombination in higher eukaryotes, it has been difficult to study and little is known about its biochemistry. In this proposal a central step in recombination, strand transfer between homologous DNAs, will be studied. I previously reported the first characterization of a mammalian strand transfer protein (recombinase). I now propose a detained study of the strand transfer reaction promoted by this protein. Analyses will focus on a) the formation of nucleoprotein networks by the recombinase and the nature of its search for homology, b) the formation of paranemic joints and c) the directionality of joint formation and branch migration. The effect of special DNA sequences on the strand-transfer reaction will also be determined by the use of novel substrates and oligonucleotides. In parallel with the above studies, a new method for measuring recombination in human cells in culture with be developed. This system utilizes tandem, complementary mutant antibiotic- resistance genes contained in retroviral vectors. It will be used to measure extrachromosomal recombination, intrachromosomal recombination, interchromosomal recombination (sister chromatid exchange) and gene conversion in normal fibroblast lines and fibroblast lines derived from patients with chromosome instability syndromes. Recombination mutants may be detected by these experiments. The identification of such mutants would aid the analysis of recombination in mammalian cells and may also provide a unique opportunity to study the molecular pathology of these diseases.