HEAT SHOCK FACTOR--STRESS AND THE CELL CYCLE

Thiele实验室以前的研究已经确定了羧基- 酵母热休克因子（HSF）末端反式激活（CTA）结构域 作为转录激活的关键区域， 酵母金属硫蛋白基因CUP 1对热氧化反应的研究 应力 这个区域对于热休克激活的 hsp70基因家族最近，我已经确定，失去这个域名 也导致细胞周期进程阻滞，阻滞在G2期 相转移到37 ℃。 这是第一次演示一个域- HSF在细胞周期进程中的特异性作用， 热休克蛋白HSP70家族的基本控制。 基于它作为一种有效的基因调节剂的已知作用，我预测HSF 控制细胞周期进程所需基因的表达， 在热应力条件下通过CTA进行分区。 为了进一步探讨HSF和潜在的靶基因在细胞中的作用， 在压力下分裂，我采取了遗传方法， 温度敏感的多拷贝和基因外抑制基因 缺乏HSF CTA的菌株（HSF（1 - 583））表现出的生长表型。 我已经鉴定了迄今为止未表征的基因YCR 030C作为一种新的基因。 HSF（1 - 583）温度致死性的多拷贝抑制基因。 我建议 表征该基因并确定其缓解G2的机制 在这种背景下逮捕，最终目的是确定其 与HSF和细胞周期控制的关系。 的表型效应 将通过基因破坏研究YCR 030C的失活。 将进行YCR 030 C的基因表达和定位研究 以确定基因产物的时间和空间协调 与细胞周期的进展。YCR 030C在转录调控中的作用 将研究HSF的激活作用，并研究这两种蛋白质 将通过体外生物化学方法测定 实验此外，我将进行基因筛查， HSF（1 - 583）ts表型的基因外抑制因子，目的是 分离可能将HSF与细胞周期进程联系起来的其他基因 在压力下。 本提案中所述的工作与一个关键的 应激反应转录因子在细胞周期中作用 进展

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.