Molecular mechanisms regulating transcription of the gene encoding peroxisomal acyl-CoA oxidase in Saccharomyces cerevisiae.
Item
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Title
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Molecular mechanisms regulating transcription of the gene encoding peroxisomal acyl-CoA oxidase in Saccharomyces cerevisiae.
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Identifier
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AAI9720112
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identifier
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9720112
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Creator
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Luo, Yi.
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Contributor
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Adviser: Gillian M. Small
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Date
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1997
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, Molecular | Biology, Cell | Biology, Microbiology
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Abstract
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Peroxisomes play an important role in the metabolism of fatty acids. In the yeast Saccharomyces cerevisiae, fatty acid {dollar}\beta{dollar}-oxidation occurs in peroxisomes. The first and rate-limiting enzyme of the {dollar}\beta{dollar}-oxidation cycle is fatty acyl-CoA oxidase, which is encoded by a sing1 copy gene, POX1. The transcription of POX1 is regulated by the carbon source provided for the growth of yeast cells. The gene is repressed in glucose, derepressed in glycerol and induced by oleate. The goal of this study is to analyze the molecular mechanisms regulating the expression of POX1.;The regulation of POX1 expression is controlled by cis-acting elements in the 5{dollar}\sp\prime{dollar} regulatory region of the gene. Previous work in our lab led to the identification of an upstream repression sequence (URS1). By further deletion analysis and DNA band shift assays, another repression sequence, URS2, and an activation sequence, UAS1, were identified. The functionality of these elements was confirmed in a heterologous CYC1 promoter. By DNA band shift assays, a protein or protein complex, was found to bind to UAS1 in an oleate dependent manner.;As a first step to characterize the oleate induction pathway, a transcription factor (Oaf1p) was purified by standard protein purification methods and was subjected to peptide sequencing analysis. Using the obtained peptide sequences, Oaf1p was found to be encoded by an open reading frame in yeast chromosome I, and the predicted molecular mass of Oaf1p is approximately 118 kD. By disruption of the OAF1 gene in yeast, OAF1 was identified to be involved in the oleate induction of POX1 and peroxisome proliferation.;The expression of OAF1 is not induced by oleate. Oaf1p is subjected to regulated phosphorylation in glycerol and oleate growth conditions. The activation domain of Oaf1p is located at the carboxyl terminus and the oleate responsive domain is located in a different region of the protein. Oaf1p appears to form a complex with Oaf2p, a transcription factor which is 39% identical to Oaf1p. A model for the regulation of POX1 is proposed.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
