MOLECULAR STUDIES OF HUMAN GENETIC RECOMBINATION

基因信息的重新排列通过 DNA的重组发生在所有形式的生命中。遗传 重组允许基因作图，并已被牵连到 免疫球蛋白基因重排，DNA修复，基因 放大，癌症的最初发展，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，但它 一直很难研究，对它的了解也很少 生物化学。在这项提议中，重组的核心步骤是， 将研究同源DNA之间的链转移。我 此前首次报道了一种哺乳动物的特征 链转移蛋白(重组酶)。我现在提议拘留一名 该蛋白促进链转移反应的研究。 分析将集中在a)核蛋白网络的形成 根据重组酶及其同源搜索的性质，b) 贫血关节的形成和c)的方向性 节理形成和分支迁移。特殊DNA的作用 链转移反应的序列也将被确定 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 重组技术在人类细胞培养中的应用有待发展。这 系统利用串联、互补的突变抗生素- 逆转录病毒载体中包含的抗性基因。它将被用来 测量染色体外重组、染色体内重组 重组，染色体间重组(姐妹染色单体 交换)和基因转化的正常成纤维细胞系 染色体不稳定患者来源的成纤维细胞系 综合症。重组突变体可以通过这些方式检测到 实验。这种突变体的鉴定将有助于 在哺乳动物细胞中的重组分析，也可能 提供了一个独特的研究分子病理学的机会 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.