HEAT SHOCK FACTOR--STRESS AND THE CELL CYCLE

热休克因子——压力和细胞周期

• 批准号: 6018363
• 负责人: KEVIN ANTHONY MORANO
• 金额: $ 3.67万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-06-29 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6018363
• 关键词: cell cycle; gene expression; gene mutation; genetic regulation; genetic regulatory element; genetic screening; genetic techniques; genetic transcription; in situ hybridization; metallothionein; oxidative stress; phenotype; phosphorylation; protein localization; stress proteins; suppressor mutations; temperature; transcription factor

Previous studies in the Thiele laboratory have identified the carboxyl- terminal transactivation (CTA) domain of yeast heat shock factor (HSF) as a critical region required for transcriptional activation of the yeast metallothionein gene CUP1 in response to heat and oxidative stress. This domain is dispensible for heat shock activation of the hsp70 gene family. Recently, I have determined that loss of this domain also results in a block in cell cycle progression, with arrest at G2 phase upon shift to 37 C. This is the first demonstration of a domain- specific role for HSF in cell cycle progression and is distinct from basal control of the essential family of hsp70 heat shock proteins. Based on its known role as a potent gene regulator, I predict that HSF controls the expression of genes required for cell cycle progression and division under conditions of thermal stress through the CTA. To further explore the roles HSF and potential target genes play in cell division during stress, I have taken a genetic approach and isolated both multicopy and extragenic suppressors of the temperature sensitive growth phenotype exhibited by strains lacking the HSF CTA (HSF(1-583)). I have identified the heretofore uncharacterized gene YCR030C as a multicopy suppressor of HSF(1-583) temperature lethality. I propose to characterize this gene and determine its mechanism for alleviating G2 arrest in this background with the ultimate goal of determining its relationship to HSF and cell cycle control. The phenotypic effects of inactivation of YCR030C will be investigated through gene disruption. Gene expression and localization studies of YCR030C will be conducted to determine the temporal and spatial coordination of the gene product with cell cycle progression. The role of YCR030C in transcriptional activation by HSF will be studied, and the ability of these two proteins to physically interact will be assayed by in vitro biochemical experiments. Furthermore, I will carry out a genetic screen for extragenic suppressors of the HSF(1-583) ts phenotype with the goal of isolating other genes which may link HSF with cell cycle progression during stress. The work described in this proposal links a critical stress-responsive transcription factor to a role in cell cycle progression.

Thiele 实验室之前的研究已经确定了羧基- 酵母热休克因子 (HSF) 的末端反式激活 (CTA) 结构域 作为转录激活所需的关键区域 酵母金属硫蛋白基因 CUP1 对热和氧化的反应 压力。 该域对于热激激活是可有可无的 hsp70 基因家族。最近，我确定该域名丢失 也会导致细胞周期进程受阻，在 G2 停滞 转变到 37 C 时的相位。这是域的首次演示 HSF 在细胞周期进展中的特定作用，与 hsp70 热休克蛋白基本家族的基础控制。 基于其作为有效基因调节因子的已知作用，我预测 HSF 控制细胞周期进展所需基因的表达， 通过 CTA 在热应力条件下进行划分。 进一步探讨HSF和潜在靶基因在细胞中的作用 在压力期间分裂，我采取了遗传方法并分离 温度敏感的多拷贝和外源抑制因子 缺乏 HSF CTA (HSF(1-583)) 的菌株表现出的生长表型。 我已将迄今为止未表征的基因 YCR030C 确定为 HSF(1-583) 温度致死性的多拷贝抑制子。 我建议 表征该基因并确定其缓解 G2 的机制 在此背景下逮捕的最终目的是确定其 HSF 和细胞周期控制的关系。 表型效应 将通过基因破坏来研究 YCR030C 的失活。 将进行YCR030C的基因表达和定位研究 确定基因产物的时间和空间协调 随着细胞周期的进展。 YCR030C 在转录中的作用 将研究 HSF 的激活，以及这两种蛋白质的能力 物理相互作用将通过体外生化进行测定 实验。此外，我还将进行基因筛查 HSF(1-583) ts 表型的基因外抑制因子，其目标是 分离可能将 HSF 与细胞周期进展联系起来的其他基因 在压力期间。 本提案中描述的工作链接了一个关键的 应激反应转录因子在细胞周期中的作用 进展。