Expression of transcripts for human pro-melanin concentrating hormone (pMCH) were studied in the hypothalamus, the primary location for pMCH producing cells in the mammalian CNS. Human hypothalamic tissue was extracted for total RNA and the cDNA generated with reverse transcriptase (RT). PCR amplification with primers spanning exons 2 and 3 of the pMCH human-variant genes (pMCHL), yielded an unspliced product, confirming prior work [T.B. Campbell, C.K. McDonald, M. Hagen, The effect of structure in a long target RNA on ribozyme cleavage efficiency, Nucleic Acids Res. 25 (1997) 4985-4993]. In addition, this product was shown to be exclusively antisense, and to be derived from the 5p (pMCHL1), not the 5q (pMCHL2) locus. Thus, there is no evidence that the MCH peptide-precursor molecule is produced in the brain by the human-variant pMCHL loci. In contrast, corresponding RT-PCR for pMCH RNA generated by the locus on 12q, demonstrated the presence of both sense and antisense spliced RNA. Partial sequencing of the spliced product confirmed that production of at least the two C-terminal peptides would occur from the 12q pMCH locus. The significance of the findings for pMCH and pMCHL1 are discussed relative to what is known about the function of endogenous antisense RNA.
Copyright 1998 Published by Elsevier Science B.V.