Characterization of the seabass pancreatic alpha-amylase gene and promoter.
Seabass (Lates calcarifer) pancreatic alpha-amylase gene was cloned and characterized. The alpha-amylase cDNA has 1620 bp 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 -776 bp 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.
Ma
Peisong
P
Department of Biological Sciences, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260, Singapore.
Liu
Yansong
Y
Reddy
Konda P
KP
Chan
Woon Khiong
WK
Lam
T J
TJ
D000818:Animals; D001492:Bass / Q000235:genetics*; D015139:Blotting, Southern; D002478:Cells, Cultured; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D003907:Dexamethasone / Q000494:pharmacology; D024202:Electrophoretic Mobility Shift Assay; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D005938:Glucocorticoids / Q000494:pharmacology; D006801:Humans; D007814:Larva; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013045:Species Specificity; D014158:Transcription, Genetic; D000516:alpha-Amylases / Q000096:biosynthesis / Q000235:genetics*
D018076:DNA, Complementary; D005938:Glucocorticoids; D012333:RNA, Messenger; D003907:Dexamethasone; D000516:alpha-Amylases