GENETIC REGULATION OF YEAST CELL TYPE

酵母细胞类型的遗传调控

• 批准号: 3277650
• 负责人: GEORGE F. SPRAGUE
• 金额: $ 14.23万
• 依托单位: UNIVERSITY OF OREGON
• 依托单位国家: 美国
• 财政年份: 1982
• 资助国家: 美国
• 起止时间: 1982-02-01 至 1993-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3277650
• 关键词: DNA binding protein; Saccharomyces; alleles; cell cycle; cell type; chemical binding; chromatography; complementary DNA; fungal genetics; gene expression; gene mutation; genetic manipulation; genetic mapping; genetic regulation; genetic transcription; laboratory rabbit; micromanipulator; microscopy; molecular cloning; molecular genetics; mutagens; nucleic acid hybridization; nucleic acid probes; nucleic acid sequence; pheromone; protein biosynthesis; regulatory gene; structural genes

This proposal examines the molecular basis of differential gene expression in the unicellular eucaryotic organism, the yeast Saccharomyces cerevisiae. Yeast cells exhibit one of three specialized cell types -- a, alpha, and a alpha -- each of which produces a unique set of proteins that create the distinctive cell types. Expression of the structural genes that encode these cell- type-specific proteins is regulated by alleles of a single genetic locus, the mating-type locus (MATa and MATalpha). Our goal is to understand the mechanism by which the regulatory protein alphal, encoded by MATalpha, activates expression of alpha- specific genes. We have focused on a particular alpha-specific gene, STE3, whose transcription is subject to two inputs in addition to the requirement for the activator alphal. In particular, the products of five others genes (STE4, STE5, STE7, STE11, and STE12) are required for efficient transcription and part of the response to the a-factor peptide pheromone secreted by a cells is increased transcription of STE3. We have delimited a segment of STE3 (called its UAS) that is necessary and sufficient to impart alpha-specific expression. We have shown that alphal protein, in conjunction with an as yet uncharacterized protein, binds to the STE3 UAS element. We will use gel mobility shift assays and DNAase footprinting assays to study the formation of this protein-DNA complex. Specifically we will determine which nucleotides within the UAS are essential, and whether the STE4/12 genes or response to a-factor affect the formation of the complex. We will determine whether alphal makes specific contacts with the UAS DNA and also identify the other protein(s) that is part of the complex. Our views is that this other protein is a general transcription factor required for expression of many genes, and that it can interact with the STE3 UAS only with the help of alphal. A second goal of this proposal is to understand the mechanism by which a-factor pheromone alters the physiology of alpha cells, for instance to cause arrest of the cell division cycle. We have shown that STE3 encodes the cell surface receptor for a-factor. We will use in vitro mutagenesis of the STE3 gene in an effort to identify the part of the STE3 protein that is involved in production of the intracellular second-messenger. Isolation of phenotype suppressors of STE3 mutations will identify genes that play a role in the normal response to a-factor.

该建议探讨了差异基因的分子基础 在单细胞真核生物酵母中的表达 酿酒酵母 酵母细胞表现出三种 专门的细胞类型--α，α，和α--每一种都是 产生一组独特的蛋白质， 类型 编码这些细胞的结构基因的表达- 型特异性蛋白质受单个基因的等位基因调控， 基因座，交配型基因座（MATa和MATalpha）。 我们的目标是 来了解调节蛋白 由MAT α编码的α-淀粉酶激活α-淀粉酶的表达， 特定基因 我们专注于一种特定的阿尔法 基因，STE3，其转录受到两个输入， 除了对活化剂的要求之外。 在 特别地，五个其他基因（STE4，STE5，STE7， STE11和STE12）是有效转录所必需的， 对a因子肽信息素的部分反应分泌 增加了STE3的转录。 我们已经划定了一个 STE3段（称为其UAS），这是必要和充分的 以赋予α特异性表达。 我们已经证明， 蛋白质，与尚未表征的蛋白质结合， 与STE3 UAS元件结合。 我们将使用凝胶迁移率变化 分析和DNA酶足迹分析，以研究 这种蛋白质-DNA复合物。 具体来说，我们将确定 UAS内的核苷酸是必不可少的， STE4/12基因或对a因子的反应影响了 复杂. 我们将决定是否将具体的 与UAS DNA接触，并识别其他蛋白质 这是复杂的一部分。 我们的观点是这种蛋白质 一种表达许多转录因子所需的通用转录因子， 基因，并且它只能与STE3 UAS相互作用 帮助阿提哈德。 本建议的第二个目标是通过以下方式了解该机制： 其中a因子信息素改变阿尔法细胞的生理机能， 导致细胞分裂周期停滞的例子。 我们已经表明 STE3编码α因子的细胞表面受体。 我们将 使用STE3基因的体外诱变来鉴定 STE3蛋白的一部分，参与生产的 细胞内第二信使 表型分离 STE3突变的抑制基因将识别出在基因突变中起作用的基因。 对A因子的正常反应。