CLONING AND SEQUENCE-ANALYSIS OF HYPOTHALAMUS CDNA-ENCODING TILAPIA MELANIN-CONCENTRATING HORMONE

Melanin-concentrating hormone (MCH) is a neuroendocrine peptide involv ed in the regulation of skin pigmentation in teleosts. We isolated and sequenced a 543 bp hypothalamic cDNA encoding the MCH-preprohormone o f tilapia (Oreochromis mossambicus). Initially, polymerase chain react ion (PCR) experiments were performed on hypothalamic RNA with a synthe tic oligonucleotide primer corresponding to a conserved region of salm on and mammalian MCH peptide and an oligo dT primer. A 0.2 kb PCR frag ment was obtained and found to have low but significant nucleotide seq uence similarity with the 3' ends of known MCH-mRNAs. Subsequently, th e PCR fragment was used to screen lambdaZAP cDNA libraries constructed from tilapia hypothalamic poly(A+) RNA. The cloned tilapia MCH prepro hormone cDNA encodes a 133-amino acid protein of which 17 amino acids belong to the signal peptide. The MCH peptide sequence is located at t he carboxy terminus of the preprohormone structure and is preceded by a pair of arginine residues which can serve as a proteolytic cleavage site. 23 to 25 amino acids further upstream in the prohormone structur e three consecutive basic residues are present. Cleavage at this site would yield a 22-amino acid MCH gene-related peptide (Mgrp), which is much larger than (12- to 13-amino acid) salmon and mammalian Mgrp. A c omparative structural analysis between tilapia preproMCH and its salmo n and mammalian counterparts revealed that the MCH peptide sequence is very well conserved (100% identity with salmon and 75% identity with both rat and human MCH). In contrast, the remaining parts of the prepr oMCH structures have diverged considerably. Northern blot analysis rev ealed the presence of tilapia preproMCH mRNA in the hypothalamus and n ot in other brain regions nor in several peripheral tissues.