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Title: Characterization of the seabass pancreatic α-amylase gene and promoter
Authors: Ma, P.
Liu, Y.
Reddy, K.P. 
Chan, W.K. 
Lam, T.J. 
Keywords: α-Amylase
AR42-J cell
Glucocorticoid response element
Issue Date: 15-May-2004
Citation: Ma, P., Liu, Y., Reddy, K.P., Chan, W.K., Lam, T.J. (2004-05-15). Characterization of the seabass pancreatic α-amylase gene and promoter. General and Comparative Endocrinology 137 (1) : 78-88. ScholarBank@NUS Repository.
Abstract: Seabass (Lates calcarifer) pancreatic α-amylase gene was cloned and characterized. The α-amylase cDNA has 1620bp and the deduced polypeptide has 522 amino acids. Southern blot indicated that there are two gene copies in the seabass genome. Sequence analysis showed that except for the loss of an intron in seabass, the coding region and the exon/intron boundaries are highly homologous to those of mammalian amylases. However, the promoter regions are distinctively divergent. To investigate the seabass amylase promoter, a series of deletion mutants was generated and fused to the luciferase reporter gene, followed by studies of their functional activity in rat AR42J cell line. Besides identifying several potential regulatory elements that have been previously identified in the human and mouse pancreatic amylase promoter, we have identified a glucocorticoid response element (GRE). However, while the human and mouse pancreatic amylase promoters are highly homologous between nucleotide -160 and transcription start site where GRE is located, the 5′ promoter deletion mutants revealed that the GRE of the seabass amylase promoter was located far upstream -947 to -776bp of the promoter. Site-directed mutagenesis of the putative GRE and electrophoretic mobility shift assays (EMSA) confirmed that this region was responsible for dexamethasone induction. However, no functional PTF-1 binding site, which is responsible for pancreas-specific transcription in higher vertebrates, was identified in seabass amylase promoter. Instead a Hepatocyte Nuclear Factor 3 binding site was found to modulate the amylase promoter expression. The evolutionary significance of this divergence in promoter regulation between seabass and mammals requires further studies. © 2004 Elsevier Inc. All rights reserved.
Source Title: General and Comparative Endocrinology
ISSN: 00166480
DOI: 10.1016/j.ygcen.2004.02.018
Appears in Collections:Staff Publications

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