The induction of gene expression in eukaryotic cells in response to small effector molecules or various environmental stimuli is a complex process involving many stages. A signal is received by the cell at its surface and in turn transmitted to the cell nucleus where a specific transcriptional response is elicited. In an attempt to understand various stages in this signaling process, focusing primarily at the stages within the cell nucleus, we have chosen to study the molecular mechanism of the transcriptionsl regulation of the phosphate-repressible acid phosphatase gene of the yeast Saccharomyces cerevisiae which is transcriptionally regulated in response to phosphate level in the growth medium. This proposal utilizes both in vivo and in vitro approaches to investigate the nucleoprotein structure of the active and inactive gene and the factors responsible for inducing the transcriptional response. Specific experiments are proposed to investigate the protein:nucleic acid interactions involved in gene structure and expression and how the interactions are mediated by the presence of low levels of phosphate: (1) Fine structure analysis of the nucleoprotein interactions using a high copy extrachromosomal plasmid system containing the acid phosphatase gene. (2) Analysis of the role of topoisomerase activity and DNA replication in the establishment and maintenance of transcriptionally active and inactive states. (3) Analysis of the structure and function of the upstream regulatory sequences in regards to nucleosome phasing and the interaction with promoter proximal sequences. (4) Development and characterization of an in vitro protein:DNA binding assay to isolate factors involved in acid phosphatase regulation and transcription. Results obtained from these experiments will provide valuable information concerning the mechanisms responsible for induction of a transcriptional response to a change in environmental conditions and general information concerning the role of nucleosomal structure in gene expression.
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