Molecular analysis of the amylase gene and its expression during development in the winter flounder, Pleuronectes americanus

Determination of the onset of amylase production in marine fish larvae is d ifficult due to their small size and the possible presence of exogenous amy lases from prey organisms in the diet or from the gut flora. In order to de velop a sensitive PCR-based assay for the detection of fish-specific amylas e in larvae, a complete cDNA and partial genomic sequence, the first report ed from a teleost fish, were determined from winter flounder. The complete cDNA for alpha amylase is 1539 bp and the deduced polypeptide sequence is 512 amino acids, including a putative 15 amino acid signal pept ide. The molecular weight of the mature protein is 55,769 Da and the predic ted isoelectric point is 6.76. Southern hybridisation analysis showed that the winter flounder amylase cDNA could be used to detect homologs in other species, particularly flatfish, and that there are likely two copies of the gene in the winter flounder genome. The winter flounder genomic sequence c orresponding to amino acids 194-404 (including three introns) was amplified by the polymerase chain reaction (PCR) and the sequence used to design pri mers for PCR-based assays for amylase gene expression in larval and adult f ish. The levels of expression of the amylase gene from larvae sampled at 5, 13, 20, 27 and 41 days post-hatch (dph) were determined using the housekeeping gene, actin, as a control. Amylase transcripts were first detected at 5 dph , peaked at 20 dph and then decreased during metamorphosis. The amylase gen e is highly expressed in adult winter flounder. This sensitive assay will b e useful for investigating amylase gene expression under different feeding conditions and help in the development of optimal diets. (C) 2000 Elsevier Science B.V. All rights reserved.