MECHANISM OF EUKARYOTIC GENE REGULATION

真核基因调控机制

• 批准号: 3281388
• 负责人: KEITH A BOSTIAN
• 金额: $ 16.21万
• 依托单位: BROWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-04-01 至 1987-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3281388
• 关键词: Saccharomyces; antibody formation; autoradiography; endonuclease; enzyme induction /repression; eukaryote; fungal genetics; gel electrophoresis; gel filtration chromatography; gene complementation; gene dosage; gene expression; gene interaction; gene mutation; genetic manipulation; genetic mapping; genetic regulation; genetic transcription; immunoprecipitation; molecular cloning; nucleic acid sequence; phase contrast microscopy; radiotracer; reagent /indicator; regulatory gene; structural genes

We are attempting to elucidate basic molecular control mechanisms underlying regulation of eucaryotic gene expression. Our approach is a coordinate one utilizing both biochemical and genetic techniques to study the phosphorus metabolizing system of Saccharomyces cerevisiae. The phosphatases are a major constituent of degradation and salvage pathways in yeast, and a major determinant of vacuolar and cellular homeostatis. They provide an opportunity to investigate a system of regulatory gene control which may have broad application to general housekeeping systems in other eucaryotes. They may also help to elucidate mechanisms by which cells arrest in G1 upon starvation. A distinctive property of this system is the action of two classes of regulatory genes. A group of one positive (PHO4) and two negative (PHO80 and PHO85) genes which function in a concerted fashion, mediated by the PHO81 gene, in the regulation of a dispersed cellular family of phosphatases. A second class of positive regulatory genes are specific for individual members of this family. We are interested in understanding funcional aspects of regulatory gene action within this system. We are proposing to determine by direct molecular procedures, the nature of the positive-negative interplay between the PHO4, PHO80 and PHO85 proteins. Specifically, we aim to isolate the PHO4 and PHO80 genes and characterize them by recombinant procedures and DNA sequence analysis. We will determine the constitutivity of their expression and the phenotypes of over-producing and null mutants generated by in vitro modification, using gene disruption procedures. We will prepare antibody probes from synthetic peptides and hybrid proteins to identify the in vivo proteins encoded by these genes, and will determine their cellular location, their constitutivity and their ability to bind specific sequences flanking their target structural genes.

我们试图阐明基本的分子控制机制 真核基因表达的潜在调控。 我们的方法是 协调一个利用生物化学和遗传技术来研究 酿酒酵母的磷代谢系统。 的 磷酸酶是降解和补救途径的主要组成部分， 酵母，以及液泡和细胞内稳态的主要决定因素。 他们 提供了一个研究调控基因控制系统的机会 其可广泛应用于其它的一般内务系统 真核生物 它们还可能有助于阐明细胞 在饥饿状态下，G1被逮捕。 该系统的一个显著特性是两类 调节基因 一组1个阳性（PHO4）和2个阴性（PHO80 和PHO85）基因，其以协同的方式起作用， PHO81基因，在调节一个分散的细胞家族， 磷酸酶 第二类正调控基因特异于 这个家庭的每个成员。 我们有兴趣了解 在这个系统中调控基因作用的功能方面。 我们 建议通过直接的分子程序来确定， PHO4、PHO80和PHO85蛋白之间的正负相互作用。 具体来说，我们的目标是分离PHO4和PHO80基因，并表征 通过重组程序和DNA序列分析， 我们将 确定其表达的组成性和表型， 通过体外修饰产生的过量生产和无效突变体，使用 基因破坏程序。 我们将从合成的抗体探针中制备抗体探针， 肽和杂合蛋白，以鉴定由 这些基因，并将决定他们的细胞位置， 它们的组成性和结合它们侧翼的特定序列的能力， 靶向结构基因。