Miller C L, Burmeister M, Thompson R C
Mental Health Research Institute, University of Michigan, Ann Arbor, MI 48109, USA.
Brain Res. 1998 Aug 24;803(1-2):86-94. doi: 10.1016/s0006-8993(98)00626-x.
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.
研究了人类促黑素浓缩激素（pMCH）转录本在大脑下丘脑的表达情况，下丘脑是哺乳动物中枢神经系统中产生pMCH细胞的主要位置。提取人类下丘脑组织的总RNA，并用逆转录酶（RT）生成cDNA。使用跨越pMCH人类变体基因（pMCHL）外显子2和3的引物进行PCR扩增，产生了一个未剪接的产物，证实了之前的研究工作【T.B.坎贝尔、C.K.麦克唐纳、M.哈根，长靶RNA结构对核酶切割效率的影响，《核酸研究》25（1997）4985 - 4993】。此外，该产物显示为完全反义，且来源于5p（pMCHL1），而非5q（pMCHL2）位点。因此，没有证据表明人类变体pMCHL位点在大脑中产生MCH肽前体分子。相比之下，对12号染色体上该位点产生的pMCH RNA进行的相应RT - PCR显示，存在有义链和反义链剪接RNA。对剪接产物的部分测序证实，至少两种C末端肽将由12号染色体上的pMCH位点产生。结合对内源反义RNA功能的已知认识，讨论了这些关于pMCH和pMCHL1研究结果的意义。
