ISOLATION AND CHARACTERIZATION OF HUMAN GENES AFFECTING CHROMOSOME METABOLISM

工作总结：人类基因组计划正在从早期的 高通量大规模测序的阶段， 基因组学，即阐明生物化学结构和功能， 人类转录本编码的蛋白质。 迄今为止， 基因组学主要依赖于低通量方法， 反向遗传学、互补分析和通过PCR的基因分离 利用简并寡核苷酸。 除此之外，大- 许多不同基因组的大规模测序导致了 比较基因组学，其中基因功能是在电脑上推导。 作为 作为这些方法的替代，我们开发了一种新的方法，称为 表型破坏，这使我们能够快速功能性地识别 可能在DNA和染色体代谢中起作用的人类基因， 基因组稳定性 表型破坏方法依赖于 评估过表达的人cDNA在人类中的表型影响， 遗传致敏的微生物突变体，因为它们与特定的 基因终点 我们提出，一个既定的遗传相互作用 过表达的人cDNA和特定微生物突变体之间的关系可以 帮助我们了解人类蛋白质在正常细胞中的功能 环境。 特别是，我们已经筛选并分离了两种- 特征和未知的人类基因，专门防止 酵母聚合酶d的生长以及诱导大肠杆菌 紧急呼救。虽然两种表型破坏测定都促进了 快速分离基因组稳定性相关因子， 系统还提供了额外的分子 分离基因的特征。在相关的工作中，我们已经表明， 人的RAD 51增强了辐射敏感性， 检查点突变体DNA聚合酶突变体中的缺陷。 这将形成 研究与其他人为因素相互作用的基础 包括hBRCA 1和hp 53。 总之，我们的方法提供了一个 功能基因组分析的宝贵工具。

Summary of Work: The Human Genome Project is progressing from the early stages of high throughput large scale sequencing to one of functional genomics, i.e. elucidation of both biochemical structure and function of proteins encoded by identified human transcripts. To date, functional genomics has primarily depended on low throughput approaches such as reverse genetics, complementation analysis and gene isolation via PCR utilizing degenerate oligos. In addition to these approaches, large- scale sequencing of many diverse genomes has led to the emergence of comparative genomics whereby gene function is deduced in silico. As an alternative to these approaches, we developed a new approach, termed phenotype disruption, which allows us to quickly functionally identify human genes that may have a role in DNA and chromosome metabolism and genome stability. The phenotype disruption approach relies upon assessing the phenotypic impact that over-expressed human cDNAs have in genetically sensitized microbial mutants as they relate to specific genetic endpoints. We proposed that an established genetic interaction between an over-expressed human cDNA and a specific microbial mutant can lend insight to the human protein function in their normal human cell milieu. Specifically, we have screened for and isolated both well- characterized and unknown human genes that specifically prevent the growth of a yeast polymerase d as well as genes that induce the E.coli SOS response. While both of the phenotype disruption assays facilitate the rapid isolation of factors involved in genome stability, these systems also provide the opportunity for additional molecular characterization of the isolated genes. In related work we have shown that human RAD51 elicits increased radiation sensitivity and a growth defect in a checkpoint mutant the DNA polymerase mutant. This will form the basis for investigation of interactions with other human factors including hBRCA1 and hp53. Altogether, our approach has provided a valuable tool for functional genomic analysis.