Takahashi Akiyoshi, Nakata Osamu, Kosugi Takayoshi, Kako Koichiro, Sakai Takafumi, Ito Michihiko, Takamatsu Nobuhiko, Shiba Tadayoshi, Sower Stacia A, Kawauchi Hiroshi
School of Fisheries Sciences, Kitasato University, Ofunato, Iwate, Japan.
Gen Comp Endocrinol. 2005 Nov;144(2):182-7. doi: 10.1016/j.ygcen.2005.05.012. Epub 2005 Jul 21.
Adrenocorticotropic hormone (ACTH) and melanophore-stimulating hormone (MSH) are produced in the pars distalis and pars intermedia, respectively, throughout vertebrates. These hormones together with beta-endorphin are encoded on a single gene proopiomelanocortin (POMC) in gnathostomes, but in the sea lamprey, an agnathan, ACTH and MSH are encoded on two separate genes, proopiocortin (POC) and proopiomelanotropin (POM), respectively. Moreover, the nucleotide sequences of 5'-flanking regions of the POC and POM genes are significantly different from each other. To investigate the potential promoter activities of the POC and POM genes, we constructed promoter reporter plasmids by fusing the 5' flanking sequences (nucleotides -1151 to +31 and -2510 to +51, respectively) to a firefly luciferase gene. Transient transfection studies in AtT-20/D16v cells, which derived from a mouse pituitary tumor cell line, revealed that the 5'-flanking sequence of the POC gene did not exhibit promoter activity, whereas that of the POM gene showed the activity at high levels nearly equivalent to SV40 promoter. Analysis of a series of the 5'-deleted reporter for the POM gene in the AtT-20/D16v cells demonstrated that the 422 bp 5'-flanking sequence was sufficient for promoter activity, while the sequence from -853 to -574 may contain negatively acting regulatory elements. Because the POC and POM genes are supposed to have differentiated from a common ancestor, during evolution, the POC gene may lack essential element(s) for expression in the AtT-20/D16v cells.
促肾上腺皮质激素（ACTH）和促黑素细胞激素（MSH）在整个脊椎动物中分别由远侧部和中间部分泌。在有颌类动物中，这些激素与β-内啡肽一起由单一基因阿黑皮素原（POMC）编码，但在无颌类动物海七鳃鳗中，ACTH和MSH分别由两个独立的基因促皮质素原（POC）和促黑素原（POM）编码。此外，POC和POM基因5'-侧翼区域的核苷酸序列彼此显著不同。为了研究POC和POM基因的潜在启动子活性，我们通过将5'侧翼序列（分别为核苷酸-1151至+31和-2510至+51）与萤火虫荧光素酶基因融合，构建了启动子报告质粒。在源自小鼠垂体肿瘤细胞系的AtT-20/D16v细胞中的瞬时转染研究表明，POC基因的5'-侧翼序列没有表现出启动子活性，而POM基因的5'-侧翼序列表现出与SV40启动子几乎相当的高水平活性。对AtT-20/D16v细胞中POM基因的一系列5'-缺失报告基因的分析表明，422 bp的5'-侧翼序列足以产生启动子活性，而-853至-574的序列可能含有负性作用的调控元件。由于POC和POM基因被认为是从一个共同祖先进化而来的，在进化过程中，POC基因可能缺乏在AtT-20/D16v细胞中表达所需的必需元件。
