GENETIC REGULATION OF YEAST CELL TYPE

这项建议考察了差异基因的分子基础。 酵母在单细胞真核生物中的表达 酿酒酵母。酵母细胞表现为三种细胞之一 特殊的细胞类型--α、α和α--每一种 产生一组独特的蛋白质，形成独特的细胞 类型。编码这些细胞的结构基因的表达- 特定类型的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 为了了解调控蛋白的机制 由MATAlpha编码的Alphal激活α-的表达 特定的基因。我们关注的是一种特定于阿尔法的 STE3基因，其转录受两个输入的影响 除了对激活器字母的要求之外。在……里面 特别是，另外五个基因(STE4、STE5、STE7、 STE11和STE12)是有效转录所必需的 对分泌的a因子多肽信息素的部分反应 由一个细胞增加STE3的转录。我们已将一个 STE3的部分(称为ITS UAS)是必要和充分的 传递特定于阿尔法的表达。我们已经证明了Alphal 蛋白质，与一种尚未确定特征的蛋白质结合在一起， 绑定到STE3 UAS元素。我们将使用凝胶迁移率变换 应用DNA酶足迹分析和DNA酶足迹分析研究 这种蛋白质-DNA复合体。具体地说，我们将确定 无人机内的核苷酸是必不可少的，以及 STE4/12基因或对a因子的反应影响 很复杂。我们将确定Alphal是否明确 与UAS DNA接触，并鉴定另一种蛋白质(S) 这是建筑群的一部分。我们的观点是，另一种蛋白质 一种通用的转录因子，表达许多 基因，并且它只能与STE3无人机相互作用 阿尔法尔的帮助。 这项提议的第二个目标是通过 哪种a因子信息素改变了阿尔法细胞的生理， 实例以导致细胞分裂周期停滞。我们已经展示了 STE3编码a因子的细胞表面受体。我们会 利用STE3基因的体外诱变来鉴定 STE3蛋白的一部分，参与产生 细胞内的第二信使。表型分离 STE3突变的抑制者将识别起作用的基因 在对a因子的正常反应中。

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.