MECHANISM OF EUKARYOTIC GENE REGULATION

我们正试图阐明基本的分子调控机制 真核基因表达的潜在调控。我们的方法是 协调利用生化和遗传技术进行研究 酿酒酵母磷代谢系统的研究这个 磷酸酶是降解和挽救途径的主要组成部分。 酵母菌，空泡和细胞内稳态的主要决定因素。他们 为研究调控基因控制系统提供了机会 可广泛应用于其他地方的通用家政系统 真核生物。它们也可能有助于阐明细胞 饥饿后在G1中被逮捕。 这一系统的一个独特性质是两类 调控基因。一组一个阳性(PHO4)和两个阴性(PHO80) 和PHO85)基因以协调方式发挥作用，由 PHO81基因，在一个分散的细胞家族的调节中 磷酸酶。第二类正性调控基因是针对 这个家庭中的个别成员。我们有兴趣了解 这个系统中调控基因作用的功能方面。我们是 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。 具体地说，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。我们会 确定其表达的构成和表型 通过体外修饰产生的过量和无效突变，使用 基因破坏程序。我们将用合成的抗体制备抗体探针 多肽和杂交蛋白用于鉴定编码的体内蛋白 这些基因，并将决定它们的细胞位置，它们的 构造性及其结合其侧翼特定序列的能力 以结构基因为目标。

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.