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Talbot, AT,Smith, TJ,Cairns, MT
2009
April
Fish & Shellfish Immunology
Characterisation of the differentially regulated trout protein 1 (DRTP1) gene in rainbow trout (Oncorhynchus mykiss)
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Quantitative real-time PCR (qPCR) Promoter Genome walking Transfection Luciferase Ly-6/uPAR NF-KAPPA-B SUPPRESSION SUBTRACTIVE HYBRIDIZATION NICOTINIC ACETYLCHOLINE-RECEPTORS SALMON SALMO-SALAR PLASMINOGEN-ACTIVATOR BINDING-SITES TRANSCRIPTION SUPERFAMILY EXPRESSION IDENTIFICATION
26
589
598
Increased levels of differentially regulated trout protein 1 (DRTP1) mRNA transcripts have been reported in fish after activation of the acute phase response. While the function of the DRTP1 protein still remains to be elucidated, this study focused on the genomic organisation of the gene, the quantification of the DRTP1 transcript in various tissues, and the isolation and analysis of the 5' regulatory region of the DRTP1 gene in rainbow trout (Oncorhynchus mykiss). Analysis of the DRTP1 genomic and cDNA sequences showed the gene to consist of four exons separated by three introns. Tissue localisation of the DRTP1 gene was performed by Northern analysis and validated by quantitative real-time PCR (qPCR). Six tissues (liver, intestine, spleen, brain, pituitary, and hypothalamus) were analysed. The tissues with the most abundant transcripts were the liver and the pituitary, with lesser amounts detected in the intestine, hypothalamus, brain and spleen. Genome walking allowed the isolation of a 934 bp sequence of the 5' regulatory region of the gene which was cloned, sequenced and in which potential transcription factor binding sites were identified. Promoter fragments of decreasing size were generated and transiently transfected into the human hepatoma cell line (HepG2). Inducibility of the promoter was determined by stimulation of the HepG2 cells containing the constructs with dexamethasone, polyinosinic:polycytidylic acid (poly I:C) and lipopolysaccharide (LPS) and tumor necrosis factor-alpha (TNF alpha). One construct, containing two potential C-EBP/beta sites and two NF-kappa B sites, exhibited the highest promoter induction (6.34 fold +/- SEM 0.5) when stimulated with human TNF alpha. A slightly shorter fragment containing one C-EBP/beta site and one NF-kappa B site did not show any significant inducibility when treated with TNF alpha. The loss of the C-EBP/beta and NF-kappa B in the shorter construct suggests that these sites, either individually or in combination. are critical for the induction of the DRTP1 promoter by TNF alpha. (C) 2008 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.fsi.2008.09.013
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