CLONING, SEQUENCE-ANALYSIS, TISSUE-SPECIFIC EXPRESSION, AND PROHORMONE ISOLATION OF EEL ATRIAL-NATRIURETIC-PEPTIDE

A complementary DNA (cDNA) encoding eel atrial natriuretic peptide (AN P) precursor was specifically amplified from eel atrial mRNAs by rapid -amplification polymerase chain reaction. The sequence analysis of the cDNA using multiple clones revealed that the preproANP consists of 14 0 amino acid residues carrying a signal sequence at its N-terminus and a mature ANP at its C-terminus. An additional glycine residue was att ached to the C-terminus of previously isolated eel ANP. The glycine re sidue may be used for amidation of the C-terminus or removed after pro cessing. The cleavage site of a signal peptide with 22 amino acid resi dues was confirmed by isolation of proANP protein from eel atria. The proANP sequence deduced from the cDNA was also confirmed for 71% of th e isolated protein. Sequence comparison with other natriuretic peptide s revealed that eel ANP is more similar to mammalian ANP than to B-typ e natriuretic peptide (BNP) at both amino acid and nucleotide sequence levels. The eel ANP gene was a single copy gene as shown by Southern blot analysis. Northern blot analysis showed that eel ANP mRNA is appr oximately 0.8 kb in size and exclusively detected in the atrium. Thus, eel ANP is a true atrial hormone judging from both the sequence and t he site of production. However, reverse transcription-polymerase chain reaction detected ANP message in the brain, gill, cardiac ventricle, red body of swim bladder (rete mirabilis), intestine, head kidney (inc luding interrenal and chromaffin tissues) and kidney. Most of these ti ssues are involved in ion and/or gas exchange in fishes.