Isolation and characterization of the human melanin-concentrating hormone gene and a variant gene

Melanin-concentrating hormone (MCH) is a cyclic peptide found expressed almost exclusively in the hypothalamus while MCH-containing fibers project throughout the brain of many vertebrates including man. In fishes, MCH induces melanin concentration within the melanophores and may inhibit ACTH secretion. In mammals, MCH modulates ACTH release in vivo and participates as a neuromediator in the control of complex behaviors such as water and food intake. Salmon, rat and human MCH cDNAs have been cloned and structures of deduced mRNAs and precursors have been elucidated. In this report we determine the nucleotide sequence of two human MCH (hMCH) genes and demonstrate that both genes are expressed in human brain. Cloning from three genomic libraries and sequencing of one class of hMCH genomic DNA reveal high similarity between coding regions and the C-terminal part of the hMCH prohormone. However no sequence identity was found in the N-terminal and 5' end non-coding regions of the gene between them even within 6.5 kilobases (kb) upstream from the truncation point. Using polymerase chain reaction (PCR) analysis we have identified RNA populations that are derived from this gene in human brain. For that reason, this gene is a variant rather than a pseudogene. The authentic hMCH gene could only be cloned by using the PCR technique. With primers specific to 5'-end and 3'-end regions of the MCH mRNA we amplified a 1400 bp fragment as well as other shorter PCR products from human genomic DNA. The longest PCR fragment contains 3 exons encompassing most of the 5' untranslated and all of the coding and 3' untranslated sequences of the hMCH mRNA, that are separated by two introns of 350 and 271 bp, respectively. Interestingly the second intron dissects the hMCH peptide sequence in both the authentic and the variant gene. A strikingly high degree of homology was found between the variant and authentic hMCH genes, including intronic sequences, suggesting that these two genomic sequences diverged very recently during evolution. A strong homology was also noted between the exons and intervening sequences of the human and rat MCH genes. Altogether, our results provide the first strong evidence for the existence of two distinct MCH genes expressing prohormones with different MCH and neuropeptide EI (NEI) sequences in human and along with in vivo and in vitro findings, suggest that these neuropeptides may influence the activity of numerous mammalian neuronal systems.