TEMMA Kyosuke Kitasato University, Toxicology, Prof., 獣医畜産学部, 教授 (50050654)
SAIDA Kaname National Institute of Advanced Industrial Science and Technology (AIST), Institute for Biological Resources and Functions, Senior Research Scientist, 分子細胞, 主任研究官 (00357055)
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Fiscal Year 2001: ¥2,000,000 (Direct Cost: ¥2,000,000)
Endothelia-2(ET2), which was originally identified in human, is a bioactive peptide of 21 amino acids with strong vasoconstrictive and pressor effects. Here we report the cDNA cloning and characterization of bovine preproendothelin-2(PPET2), the precursor form of ET2. The bovine cDNA encodes 177 amino acids of the PPET2 polypeptide, in which a 21-amino acid mature ET2 peptide and a 16-amino acid ET2-like peptide as well as a 23-amino acid putative signal peptide were found. The bovine ET2-like peptide sequence was missing a dibasic amino acid pair at the C-terminal, in contrast to human, mouse and rat, for which the ET2-like sequence flanked dibasic pairs at both the N-and C-terminals. Gene expression analysis by RT-PCR showed that the transcript is expressed in various organs including heart, lung, liver, kidney, gastrointestinal tract, uterus and ovary, but not in spleen. Within the gastrointestinal tract, gene expression was detected in rumen, a ruminant-specific digestive organ, as well as stomach, duodenum and colon. Based on the full-length bovine PPET2 cDNA sequence obtained, we designed gene-specific primers and TagMan probe, and then established a rapid and precise quantitative method for the gene expression using a real-time PCR system.