CHARACTERIZATION OF THE S POMBE CAMP SIGNAL PATHWAY

S Pombe Camp 信号通路的特征

• 批准号: 3468475
• 负责人: CHARLES S. HOFFMAN
• 金额: $ 9.83万
• 依托单位: BOSTON COLLEGE
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-07-01 至 1996-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3468475
• 关键词: Saccharomyces; adenylate cyclase; alleles; biological signal transduction; cyclic AMP; enzyme mechanism; fungal genetics; gene complementation; gene expression; gene induction /repression; gene mutation; genetic manipulation; genetic regulation; genetic transcription; glucose; molecular cloning; molecular genetics; northern blottings; nucleic acid probes; nucleic acid sequence; polymerase chain reaction; radioimmunoassay

Transcriptional regulation in response to environmental signals involves important mechanisms employed by cells to control the processes of cell growth, differentiation and development. In unicellular organisms, changes in the carbon source available can trigger dramatic alterations in transcription of genes involved in carbon source utilization and gluconeogenesis. For example, glucose causes the repression of transcription of genes required for the utilization of other carbon sources. This phenomenon has been termed glucose repression or catabolite repression. My work focuses on glucose repression of fbp1 (encoding fructose-1,6bisphosphatase) transcription in the yeast Schizosaccharomyces pombe. S. pombe is a genetically pliable organism that resembles higher eukaryotic cells in many respects. While this organism has been widely and very successfuly exploited in the area of cell cycle control, few studies have been carried out in the area of transcriptional regulation. I have identified aset of genes, designated git genes (git=glucose insensitive transcription), required for glucose repression of fbp1 transcription, including the adenylate cyclase gene (git2). The trasncriptional defect in strains carrying a mutation in git2 or any six additional git genes is suppressed by exogenous cAMP. This suppression, along with other genetic and biochemical observations, suggests that these genes function to stimulate adenylate cyclase in response to glucose. I therefore propose to measure cAMP levels in wild type cells upon exposure to glucose, and to analyze further git2 mutants that appear to be defective in response to glucose. I also propose to carry out molecular analyses of these git genes and of nft genes (nft mutants are defective in depression of fbp1 transcription; nft= nonderepressible fbp1 transcription). I will clone the wild type copies of these genes, determine their DNA sequences, construct null alleles, and examine the null phenotypes with regard to cAMP levels and fbp1 transcription. I will also characterize spontaneous git and nft mutants by cAMP assays, genetic analyses and fbp1 transcriptional assays to identify the pathway for activation of adenylate cyclase in S. pombe.

响应环境信号的转录调控涉及 细胞用于控制细胞过程的重要机制 生长、分化和发展。 在单细胞生物中， 可用碳源的变化可能引发巨大的变化 参与碳源利用的基因转录 糖异生。 例如，葡萄糖会抑制 利用其他碳所需的基因转录 来源。 这种现象被称为葡萄糖抑制或 分解代谢物抑制。 我的工作重点是 fbp1 的葡萄糖抑制 （编码果糖-1,6双磷酸酶）在酵母中的转录 粟酒裂殖酵母。粟酒裂殖酵母是一种遗传柔韧的生物体 在许多方面类似于高等真核细胞。 虽然这 有机体已在以下领域得到广泛且非常成功的利用 细胞周期调控方面的研究还很少 转录调控。 我已经确定了一组基因，指定为 git 基因（git=葡萄糖不敏感转录），葡萄糖所需 抑制 fbp1 转录，包括腺苷酸环化酶基因 （git2）。 携带突变的菌株的转录缺陷 git2 或任何六个其他 git 基因被外源 cAMP 抑制。 这种抑制，连同其他遗传和生化观察， 表明这些基因的功能是刺激腺苷酸环化酶 对葡萄糖的反应。 因此，我建议测量野生环境中的 cAMP 水平 对暴露于葡萄糖的细胞进行分型，并进一步分析 git2 突变体 对葡萄糖的反应似乎有缺陷。我也提议 对这些 git 基因和 nft 基因（nft 突变体在 fbp1 转录抑制方面存在缺陷； nft= 不可抑制的 fbp1 转录）。 我将克隆野生型副本 这些基因，确定它们的 DNA 序列，构建无效等位基因， 并检查 cAMP 水平和 fbp1 的无效表型 转录。 我还将描述自发的 git 和 nft 突变体 通过 cAMP 测定、遗传分析和 fbp1 转录测定 鉴定粟酒裂殖酵母中腺苷酸环化酶的激活途径。