REGULATION OF THE GAL GENES IN YEAST

我们工作的长期目标是了解， 在真核生物中，调节蛋白与特定的DNA序列结合， 开启和关闭基因 我们的实验集中在酵母上 与所谓的半乳糖上游结合的激活剂GAL 4 序列（UASG），并打开侧翼基因的转录， 跨越数百个碱基对的距离。 该活化剂还 在组织培养的小鼠细胞中启动基因表达， 了解GAL 4的作用机制可以为我们提供 对基因调控的分子基础有了非常全面的了解。 我们将继续我们的活化表面的表征 酵母转录激活因子：我们将分离突变体 影响GAL 4的活化区域，设计合成的 激活区域，并尝试分离新的激活类别 刺激不被GAL 4激活的基因的区域。 我们将研究 酵母激活剂在哺乳动物和果蝇细胞中的作用。 我们将研究GAL 4的DNA结合特异性的基础： 我们将分离出特异性改变或放松的突变体， 各种定义的突变操作符，并测试是否“半胱氨酸 GAL 4和PPR 1的指负责序列识别 通过研究这两种分子的混合体。 我们将定义 GAL 4的部分，其介导GAL 4与 体内多个部位。 我们将尝试复制合作 GAL 4与体外重复位点的结合。 我们会研究 各种GAL 4衍生的激活剂对构建体的活性， 改变GAL 4结合位点与转录因子的距离， 启动站点。 我们将确定GAL 4的基因外抑制因子， 缺乏激活功能的突变体;这些抑制因子 可能定义转录因子（例如RNA聚合酶？） 与GAL 4的激活功能相互作用。 我们将 表征葡萄糖生长阻断的机制 GAL 4的刺激活性。 我们将研究阴性对照， 通过将GAL 1启动子置于HMR的控制下， 沉默子，并分析其对GAL 4结合和活性的影响。 vivo.

The longterm objective of our work is to understand how, in eukaryotes, regulatory proteins bind to specific DNA sequences and turn genes on and off. Our experiments focus on the yeast activator GAL4 that binds to the so called galactose upstream sequence (UASG) and turns on transcription of flanking genes at distance spanning many hundreds of base pairs. This activator also turns on gene expression in mouse cells in tissue culture, and understanding the mechanism of action of GAL4 may provide us with very general insights into the molecular basis of gene regulation. We will continue our characterization of the activating surfaces of yeast transcriptional activators: we will isolate mutants affecting the activating region(s) of GAL4, design synthetic activating regions and attempt to isolate new classes of activating regions that stimulate genes not activated by GAL4. We will study the effects of yeast activatorS in mammalian and Drosophila cells. we will study the basis of the DNA binding specificity of GAL4: we will isolate mutants with altered or relaxed specificity for various defined mutant operators and test whether the "cysteine fIngers of GAL4 and PPRl are responsible for sequence recognition by studying hybrids of these two molecules. We will define the part(s) of GAL4 that mediate(s) cooperative binding of GAL4 to multiple sites in vivo. We will attempt to reproduce cooperative binding of GAL4 to reiterated sites in vitro. We will study the activities of various GAL4-derived activators on constructs that vary the distance of the GAL4 binding site from the transcriptional start site. We will identify extragenic suppressors of GAL4 mutants deficient in the activation function; these suppressors might define the transcription factor(s) (e.g. RNA polymerase?) that interact with the activation function of GAL4. We will characterize the mechanism(s) by which growth in glucose blocks GAL4's stimulatory activity. We will study negative control at a distance by placing the GAL1 promoter under control of the HMR silencer and analyzing the effect on GAL4 binding and activity in vivo.