Cloning and sequencing analysis of three amylase cDNAs in the shrimp Penaeus vannamei (Crustacea decapoda): evolutionary aspects.
In Penaeus vannamei, alpha-amylase is the most important glucosidase and is present as at least two major isoenzymes which have been purified. In order to obtain information on their structure, a hepatopancreas cDNA library constructed in phage lambda-Zap II (Strategene) was screened using a synthetic oligonucleotide based on the amino acid sequence of a V8 staphylococcal protease peptide of P. vannamei alpha-amylase. Three clones were selected: AMY SK 37 (EMBL sequence accession number: X 77318) is the most complete of the analyzed clones and was completely sequenced. It contains the complete cDNA sequence coding for one of the major isoenzymes of shrimp amylase. The deduced amino acid sequence shows the existence of a 511-residue-long pre-enzyme containing a highly hydrophobic signal peptide of 16 amino acids. Northern hybridization of total RNA with the amylase cDNA confirms the size of the messenger at around 1,600 bases. AMY SK 28, which contains the complete mature sequence of amylase, belonged to the same family characterized by a common 3' terminus and presented four amino acid changes. Some other variants of this family were also partially sequenced. AMY SK 20 was found to encode a minor variant of the protein with a different 3' terminus and 57 amino acid changes. Phylogenetic analysis established with the conserved amino acid regions of the (beta/alpha) eight-barrel domain and with the total sequence of P. vannamei showed close evolutionary relationships with mammals (59-63% identity) and with insect alpha-amylase (52-62% identity). The use of conserved sequences increased the level of similarity but it did not alter the ordering of the groupings. Location of the secondary structure elements confirmed the high level of sequence similarity of shrimp alpha-amylase with pig alpha-amylase.
Van Wormhoudt
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Laboratoire de Biologie Marine du Collège de France, Concarneau, France.
Sellos
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D000595:Amino Acid Sequence; D000681:Amylases / Q000737:chemistry / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D019143:Evolution, Molecular; D007313:Insecta / Q000235:genetics; D007527:Isoenzymes / Q000235:genetics*; D008322:Mammals / Q000235:genetics; D008969:Molecular Sequence Data; D033561:Penaeidae / Q000235:genetics*; D010802:Phylogeny; D016415:Sequence Alignment; D017385:Sequence Homology; D013045:Species Specificity
D018076:DNA, Complementary; D007527:Isoenzymes; D000681:Amylases