Sharon Wretzel

Rutgers, The State University of New Jersey, New Brunswick, New Jersey, United States

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Publications (4)18.26 Total impact

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    ABSTRACT: In Saccharomyces cerevisiae, OLE1 encodes a delta9 fatty acid desaturase, an enzyme that plays a critical role in maintaining the correct ratio of saturated to monounsaturated fatty acids in the cell membrane. Previous studies have demonstrated that (i) OLE1 expression is repressed by unsaturated fatty acids (UFAs) and induced by low oxygen tension, (ii) a component of this regulation is mediated through the same low oxygen response element (LORE) in the OLE1 promoter, and (iii) Mga2p is involved in LORE-dependent hypoxic induction of OLE1. We now report that LORE-CYC1 basal promoter-lacZ fusion reporter assays demonstrate that UFAs repress the reporter expression under hypoxic conditions in a dose-dependent manner via LORE. Electrophoretic mobility shift assays show that UFAs repress the hypoxia-induced complex formation with LORE. Studies with a construct encoding a truncated form of Mga2p support the hypothesis that both hypoxia and UFA signals affect the processing of Mga2p and the UFA repression of OLE1 hypoxic induction is mediated through Mga2p. Data from Western blot assays provide evidence that under normoxic conditions, Mga2p processing produces approximately equimolar levels of the membrane-bound and processed forms and is unaffected by UFAs. Hypoxic induction of OLE1, however, is associated with increased processing of the protein, resulting in an approximately fivefold increase in the soluble active form that is counteracted by exposure of the cells to unsaturated fatty acids. Data from this study suggest that the Mga2p-LORE interaction plays an important role in OLE1 expression under both normoxic and hypoxic conditions.
    Eukaryotic Cell 07/2002; 1(3):481-90. · 3.59 Impact Factor
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    ABSTRACT: Eukaryotes have the ability to respond to changes in oxygen tension by alterations in gene expression. For example, OLE1 expression in Saccharomyces cerevisiae is upregulated under hypoxic conditions. Previous studies have suggested that the pathway regulating OLE1 expression by unsaturated fatty acids may involve Mga2p and Spt23p, two structurally and functionally related proteins. To define the possible roles of each of these genes on hypoxia-induced OLE1 expression, we examined OLE1 expression under normoxia, hypoxia, and cobalt treatment conditions in Deltamga2 or Deltaspt23 deletion strains. The results of OLE1 promoter-lacZ reporter gene and Northern blot analyses showed that hypoxia- and cobalt-induced OLE1 expression was dramatically decreased in a Deltamga2 strain but not in a Deltaspt23 strain. Further analyses using low-oxygen response element (LORE)-CYC1-lacZ fusion reporter assays and electrophoretic mobility shift assays (EMSAs) demonstrated that MGA2 significantly affects the LORE-dependent hypoxic induction pathway of gene expression. When MGA2 was supplied by a plasmid, the LORE-dependent hypoxia-inducible reporter expression was recovered, as was the hypoxia-inducible complex in EMSAs in the S. cerevisiae Deltamga2 strain. Supershift analysis of EMSAs using crude extracts containing mycMga2p indicated that Mga2p is a component of the LORE-binding complex. Another LORE-dependent, hypoxia-inducible gene, ATF1, was similarly affected in the Deltamga2 strain. These results indicate that MGA2 is required for the LORE-dependent hypoxic gene induction in S. cerevisiae.
    Molecular and Cellular Biology 10/2001; 21(18):6161-9. · 5.37 Impact Factor
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    ABSTRACT: An organism's ability to respond to changes in oxygen tension depends in large part on alterations in gene expression. The oxygen sensing and signaling mechanisms in eukaryotic cells are not fully understood. To further define these processes, we have studied the Delta9 fatty acid desaturase gene OLE1 in Saccharomyces cerevisiae. We have confirmed previous data showing that the expression of OLE1 mRNA is increased in hypoxia and in the presence of certain transition metals. OLE1 expression was also increased in the presence of the iron chelator 1,10-phenanthroline. A 142-base pair (bp) region 3' to the previously identified fatty acid response element was identified as critical for the induction of OLE1 in response to these stimuli using OLE1 promoter-lacZ reporter constructs. Electromobility shift assays confirmed the presence of an inducible band shift in response to hypoxia and cobalt. Mutational analysis defined the nonameric sequence ACTCAACAA as necessary for transactivation. A 20-base pair oligonucleotide containing this nonamer confers up-regulation by hypoxia and inhibition by unsaturated fatty acids when placed upstream of a heterologous promoter in a lacZ reporter construct. Additional yeast genes were identified which respond to hypoxia and cobalt in a manner similar to OLE1. A number of mammalian genes are also up-regulated by hypoxia, cobalt, nickel, and iron chelators. Hence, the identification of a family of yeast genes regulated in a similar manner has implications for understanding oxygen sensing and signaling in eukaryotes.
    Journal of Biological Chemistry 05/2001; 276(17):14374-84. · 4.65 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: An organism's ability to respond to changes in oxygen tension depends in large part on alterations in gene expression. The oxygen sensing and signaling mechanisms in eukaryotic cells are not fully understood. To further define these processes, we have studied the Δ9 fatty acid desaturase gene OLE1 inSaccharomyces cerevisiae. We have confirmed previous data showing that the expression of OLE1 mRNA is increased in hypoxia and in the presence of certain transition metals.OLE1 expression was also increased in the presence of the iron chelator 1,10-phenanthroline. A 142-base pair (bp) region 3′ to the previously identified fatty acid response element was identified as critical for the induction of OLE1 in response to these stimuli using OLE1 promoter-lacZ reporter constructs. Electromobility shift assays confirmed the presence of an inducible band shift in response to hypoxia and cobalt. Mutational analysis defined the nonameric sequence ACTCAACAA as necessary for transactivation. A 20-base pair oligonucleotide containing this nonamer confers up-regulation by hypoxia and inhibition by unsaturated fatty acids when placed upstream of a heterologous promoter in alacZ reporter construct. Additional yeast genes were identified which respond to hypoxia and cobalt in a manner similar toOLE1. A number of mammalian genes are also up-regulated by hypoxia, cobalt, nickel, and iron chelators. Hence, the identification of a family of yeast genes regulated in a similar manner has implications for understanding oxygen sensing and signaling in eukaryotes.
    Journal of Biological Chemistry 04/2001; 276(17):14374-14384. · 4.65 Impact Factor

Publication Stats

123 Citations
18.26 Total Impact Points

Institutions

  • 2001–2002
    • Rutgers, The State University of New Jersey
      • Department of Cell Biology and Neuroscience
      New Brunswick, New Jersey, United States