Signal Mediated Defense Mechanisms of Yeast

酿酒酵母是研究细胞生物学中许多基本过程的一个强有力的模型系统。一个重要的尚未解决的细胞生物学问题是细胞如何感知和保护自己免受其他细胞的化学攻击。为了直接研究这一问题，本研究将开发酵母-小鼠巨噬细胞共培养系统，揭示和分析酵母对巨噬细胞释放的特定有害化学物质（如一氧化氮和超氧阴离子）的信号通路和基因组反应。在先前的研究结果中，已经证明渗透胁迫感知MAP激酶通路（称为HOG通路）也具有感知和响应氧化剂的功能。本研究的目标是：1)两种不同方法的结合将分别用于鉴定由巨噬细胞产生的超氧化物、一氧化氮和非氧化性攻击化学物质激活的酵母信号通路。首先，将使用DNA微阵列杂交技术对mRNA表达进行全基因组分析，以确定巨噬细胞产生一氧化氮而不产生超氧化物或产生超氧化物而不产生一氧化氮等特异性诱导的不同组酵母基因。其次，将分析缺失防御信号通路基因的酵母菌株，并将其与野生型菌株进行比较，分别分析巨噬细胞产生的超氧化物、一氧化氮和非氧化性攻击化学物质对基因诱导和存活的影响。2)将通过三种互补的方法确定HOG途径中参与氧化反应的转录因子靶点。通过对TRX2- lacz报告基因的突变分析，确定HOG途径依赖的氧化诱导硫氧还蛋白基因TRX2表达所需的启动子序列。HOG途径依赖，氧化诱导的一组酵母基因将通过DNA微阵列杂交鉴定。最后，我们将分析HOG通路转录因子缺失的酵母菌株，并将其与野生型菌株进行比较，以了解氧化诱导的TRX2-lacZ表达变化和基因表达的全基因组变化。

The yeast Saccharomyces cerevisiae has been a powerful model system for the study of many fundamental processes in cell biology. One important unsolved cell biology problem is how cells sense and defend themselves against chemical attack by other cells. To directly study this problem, this research will develop a yeast-mouse macrophage co-culture system to uncover and analyze the signaling pathways and genomic responses of yeast to specific noxious chemicals, e.g., nitric oxide and superoxide anion, releasedby macrophages. In previous results, it was demonstrated that the osmostress sensing MAP kinase pathway called the HOG pathway also functions to sense and respond to oxidants.The goals of this research are: 1.) a combination of two different approaches will be used to identify the yeast signaling pathways activated by macrophage-produced superoxide, nitric oxide, and non-oxidant attacking chemicals, respectively. First, genome-wide analysis of mRNA expression using hybridization to DNA microarrays will be used to define the different sets of yeast genes that are specifically induced by macrophages producing nitric oxide and not superoxide or producing superoxide and not nitric oxide, etc. Second, yeast strains with deletions of putative defense signaling pathway genes will be analyzed and compared to a wild type strain for changes in gene induction and survival in response to macrophage-produced superoxide, nitric oxide, and non-oxidant attacking chemicals, respectively. 2.) the transcription factor target of the HOG pathway involved in the oxidant response will be identified by three complementary approaches. The promoter sequences necessary for HOG pathway-dependent, oxidant-induced expression of the thioredoxin gene TRX2 will be identified by mutational analysis of a TRX2-lacZ reporter gene. The HOG pathway dependent, oxidant-induced set of yeast genes will be identified using hybridization to DNA microarrays. Finally, yeast strains with deletions of putative HOG pathway transcription factors will be analyzed and compared to a wild type strain for changes in oxidant-induced TRX2-lacZ expression and genome-wide changes in gene expression.