Large Scale Identification and Characterization of Hsf1-mediated Heat Shock Respo

描述（由申请人提供）：感知和响应不断变化的环境条件的能力是所有生物系统的基本方面。酿酒酵母中保守的热休克反应已被证明是一般真核生物应激反应的范例。尽管在基因表达水平和这些表达基因的功能方面进行了大量分析，但对激活保守的热休克转录因子Hsf1蛋白（简称Hsf1）的细胞传感器或潜在的信号转导途径知之甚少。 该提案中概述的研究旨在鉴定和表征作为Hsf1调节剂的蛋白质。进行了初步筛选，并导致在11个潜在的积极Hsf1监管机构的鉴定。本提案的第一部分致力于研究这些基因在Hsf1介导的热休克反应中的功能和保守作用。这将使用经典的遗传和生物化学技术（如缺失分析和蛋白质印迹分析）和系统级生物技术（如竞争性健身测定和微阵列）的组合来完成。该提案的第二部分致力于开发一种通用的定量系统，用于识别转录因子的正调控因子和负调控因子，该系统将用于进一步识别Hsf1调控因子。除了使用下一代测序技术（Illumina基因组分析仪II）进行荧光激活细胞分选（FACS）和条形码DNA测序外，还将使用常见的重组DNA技术来实现这一点。总的来说，这项工作将显着增加我们的知识，热休克反应，真核细胞应激反应信号转导，一般。

DESCRIPTION (provided by applicant): The ability to sense and respond to changing environmental conditions is a fundamental aspect of all biological systems. The conserved heat shock response in Saccharomyces cerevisiae has proven to be a paradigm for general eukaryotic stress responses. Despite much analysis at the level of gene expression and the functions of those expressed genes, very little is known about the cellular sensor or potential signal transduction pathway that activates the conserved heat shock transcription factor, the Hsf1 protein (referred to as Hsf1). The research outlined in this proposal seeks to identify and characterize proteins that act as regulators of Hsf1. A preliminary screen was performed and resulted in identification of 11 potential positive Hsf1 regulators. The first part of this proposal is dedicated to examining each of these genes for their functional and conserved role in the Hsf1-mediated heat shock response. This will be accomplished using a combination of classic genetic and biochemical techniques (such as deletion analysis and Western blot analysis) and systems-level biological techniques (such as competitive fitness assays and microarrays). The second part of this proposal is dedicated to developing a versatile, quantitative system for identification of both positive and negative regulators of transcription factors, which will be used to further identify Hsf1 regulators. This will be accomplished using common recombinant DNA technology in addition to fluorescent activated cell sorting (FACS) and barcode DNA sequencing using next generation sequencing technology (Illumina Genome Analyzer II). Overall, this work will significantly increase our knowledge of the heat shock response, and of eukaryotic stress- responsive signal transduction, in general.