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This Article Full Text Full Text (PDF) All Versions of this Article: 297/4/R1075    most recent 00267.2009v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Esbaugh, A. J. Articles by Walsh, P. J. PubMed PubMed Citation Articles by Esbaugh, A. J. Articles by Walsh, P. J.

Articles

Identification of two glucocorticoid response elements in the promoter region of the ubiquitous isoform of glutamine synthetase in gulf toadfish, Opsanus beta

Department of Biology and Centre for Advanced Research in Environmental Genomics, University of Ottawa, Ottawa, Ontario, Canada

Submitted May 13, 2009 ; accepted in final form July 29, 2009

Unlike most teleosts, gulf toadfish have the capacity to switch from ammoniotely to ureotely as the predominate means of nitrogen excretion during periods of stress. The switch to ureotely is a result of increased glutamine synthetase (GS) mRNA expression/enzyme activity in the liver and muscle, which is initiated by cortisol. Cortisol typically affects gene expression through the action of cortisol-activated transcription factors, such as glucocorticoid receptors, which bind to glucocorticoid response elements (GRE) in the upstream regulatory region of genes. The purpose of the present study was to identify the GRE responsible for increased GS gene expression during crowding/confinement in gulf toadfish using an in vivo luciferase reporter assay. Upstream promoter regions for both the ubiquitous and gill GS isoforms were amplified by PCR. Additionally, an intron was amplified from the ubiquitous GS isoform that suggested the possibility of two discreet transcripts for the mitochondrial and cytoplasmic proteins. When tested via in vivo reporter assays, both the cytoplasmic and mitochondrial ubiquitous GS promoters showed increased luciferase activity during crowding vs. noncrowded controls; the gill GS promoter showed no effects in response to crowding. In silico analysis of the mitochondrial and cytoplasmic ubiquitous GS promoter constructs showed an overlapping section of 565 bp containing two potential GREs. Mutation of either site alone had no effect on luciferase activity vs. wild-type controls. However, when both sites were mutated a significant decrease in luciferase activity was observed. We conclude that two functional GREs combine to confer cortisol-inducible GS expression in the liver of gulf toadfish.

ammonia; urea; nitrogen metabolism; cortisol; ureotely; ornithine-urea cycle