ISOLATION AND CHARACTERIZATION OF HUMAN GENES AFFECTING CHROMOSOME METABOLISM

功能基因组学研究人类基因编码蛋白质的结构和功能。我们在酵母中开发了基于遗传学的方法，以了解可能在DNA和染色体代谢以及基因组稳定性中发挥作用的人类基因的功能。人细胞周期蛋白A1和A2基因改变酵母复制突变体的表型。我们还确定了一个遗传背景，其中表达的hRad 51 p导致合成致死性，提供了hRad 51 p的分子表征及其与克隆到酵母中的其他人蛋白质的相互作用（例如，BRCA 1和p53）。参与DNA复制和修复的人Fen 1瓣状核酸内切酶能够补充酵母rad 27突变体的诱变剂敏感性、遗传不稳定性和与修复和复制突变体的合成致死相互作用。我们确定了一个生物化学活性形式的芬1显性遗传毒性效应。当人类和酵母PCNA结合结构域交换时，Fen 1在酵母中的遗传影响更强。我们的研究定义了一个系统来研究人类FEN 1中潜在的有害突变。在与Kunkel实验室的合作中，基于酵母中共过表达的人类蛋白质的强增变效应，开发了用于人类Msh 2/Msh 6蛋白质的功能分析的酵母模型。人p53的功能分析已被追求使用已建立和新开发的酵母测定。已经评估了从肿瘤样品和细胞系中直接克隆到酵母中的p53基因的反式激活潜力。我们利用酵母中人p53的可变表达和DNA结合和四聚化结构域的随机PCR诱变来筛选具有新表型的p53等位基因。鉴定了在适度表达水平下杀死酵母的p53突变体。这些p53毒素突变体之一阻止了人类细胞的生长。第二次筛选旨在分离与野生型相比具有增加的反式激活潜力的p53变体。这两种类型的p53突变等位基因将是有用的研究，旨在解剖复杂的p53下游途径和开发癌症治疗。- 酵母，人类基因，表型破坏，DNA复制，复制，DNA修复

Summary of Work: Functional genomics elucidates structure and function of proteins encoded by human genes. We developed genetic-based approaches in yeast to understand functions of human genes that may have a role in DNA and chromosome metabolism and genome stability. The human cyclins A1 and A2 genes alter phenotype in a yeast replication mutant. We also determined a genetic background in which expressed hRad51p results in synthetic lethality, providing for molecular characterization of hRad51p and its interactions with other human proteins cloned into yeast (eg. BRCA1 and p53). The human Fen1 flap endonuclease involved in DNA replication & repair was able to complement a yeast rad27 mutant for mutagen sensitivity, genetic instability and synthetic lethal interactions with repair and replication mutants. We identified a biochemically inactive form of Fen1 with dominant genotoxic effects. The genetic impacts of Fen1 in yeast is stronger when the human and yeast PCNA binding domains are exchanged. Our study defines a system to study potentially deleterious mutations in human FEN1. In collaborations with the Kunkel lab, a yeast model for functional analysis of human Msh2/Msh6 protein was developed, based on the strong mutator effect of the co-overexpressed human proteins in yeast. Functional analysis of human p53 has been pursued using established and newly developed yeast assays. The transactivation potential of p53 genes cloned directly in yeast from tumor samples and cell lines has been assessed. We exploited variable expression of human p53 in yeast and random PCR mutagenesis of the DNA binding and tetramerization domains to develop screens for p53 alleles with novel phenotypes. p53 mutants were identified that kill yeast at modest expression levels. One of these p53 toxin mutants prevented growth of human cells. A second screen was aimed at isolating p53 variants with an increased transactivation potential compared to the wild type. Both categories of p53 mutant alleles will be useful in studies aimed at dissecting the complex p53 downstream pathways and in developing cancer therapies. - Yeast, Human Gene, Phenotype Disruption, DNA Recombination, Replication, DNA Repair