This proposal describes genetic and biochemical studies designed to understand how eukaryotic organisms respond to hazardous chemicals found at Superfund sites and aimed at development of a sensitive yeast growth assay that signals the presence of environmental toxins. These studies will utilizes the fission yeast Schizosaccharomyces pombe. Fission yeast has served as an outstanding model system in studies of the cellular responses to genotoxic and cytotoxic agents. Recent studies of fission yeast have uncovered the stress-activated protein kinase (SAPK) Spc1 and transcription factors Pap1 and Atf1 that resemble stress response factors in higher eukaryotes. These proteins regulate the transcriptional induction of stress response genes. DNA microarray technologies, combined with the genome sequence of S. pombe, provide the opportunity to develop a comprehensive picture of the transcriptional response to stress in fission yeast.
The Specific Aims are: (1) Decipher pathways that control the transcriptional response to oxidative stress. Transcriptional regulation of a small group of oxidative stress genes will be analyzed in response to various agents and in different genetic backgrounds. Regulation of the transcription factors Atf1 and Pap1 by stress-activated protein kinases (SAPKs) will be elucidated. (2) Identify all oxidative stress genes in fission yeast. Microarrays containing DNA sequences from all fission yeast genes will be available in the near future. Expression profiling will be performed in response to oxidative stress agents and in different genetic backgrounds. (3) Establish the physiological importance of oxidative stress-response mechanisms. Genes transcriptionally induced by oxidative stress will be disrupted. Physiological consequences of single and multiple mutants that will be evaluated. (4) Develop yeast strains that are biosensors for environmental toxins will be developed and tested with a range of toxins.
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