PCR amplification of cDNAs of fish hemoglobin beta chains using a consensus primer: cDNA-derived amino acid sequences of beta chains from the catfish Parasilurus asotus and the scad Decapterus maruadsi.
Hemoglobin beta chains were isolated from the catfish Parasilurus asotus, the scad Decapterus maruadsi, the filefish Thamnaconus modestus, and the scorpaenoid Sebastiscus marmoratus by reverse-phase chromatography, and the N-terminal sequences were determined. To obtain the complete amino acid sequence, a 20-meric redundant consensus primer based on the N-terminal amino acid sequences of the beta chains was designed. Using this primer and oligo-dT adaptor, we amplified successfully the beta-chain cDNAs of about 600 bp from the four fishes. The amplified products from Parasilurus and Decapterus were subcloned in the SmaI site of pUC18 and cDNA-derived amino acid sequences of 147 residues were determined, of which 69 and 76 residues, respectively, were identified by the chemical amino acid sequencing of internal peptides. Thus this PCR methodology using the consensus primer should be widely applicable for amplifying hemoglobin beta chains from teleosts.
Suzuki
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T
Department of Biology, Faculty of Science, Kochi University, Japan. suzuki@ cc.kochi.u.ac.jp
Nishikawa
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002845:Chromatography; D017931:DNA Primers; D018076:DNA, Complementary / Q000737:chemistry; D005399:Fishes; D005914:Globins / Q000737:chemistry* / Q000302:isolation & purification; D006454:Hemoglobins / Q000737:chemistry* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D018076:DNA, Complementary; D006454:Hemoglobins; D005914:Globins