Nocillado Josephine N, Elizur Abigail, Avitan Ayelet, Carrick Frank, Levavi-Sivan Berta
Department of Primary Industries and Fisheries, Bribie Island Aquaculture Research Centre, 144 North Street, Woorim, Qld 4507, Australia.
Mol Cell Endocrinol. 2007 Jan 15;263(1-2):65-78. doi: 10.1016/j.mce.2006.08.013. Epub 2006 Oct 31.
In a study towards elucidating the role of aromatases during puberty in female grey mullet, the cDNAs of the brain (muCyp19b) and ovarian (muCyp19a) aromatase were isolated by RT-PCR and their relative expression levels were determined by quantitative real-time RT-PCR. The muCyp19a ORF of 1515bp encoded 505 predicted amino acid residues, while that of muCyp19b was 1485 bp and encoded 495 predicted amino acid residues. The expression level of muCyp19b significantly increased in the brain as puberty advanced; however, its expression level in the pituitary increased only slightly with pubertal development. In the ovary, the muCyp19a expression level markedly increased as puberty progressed. The promoter regions of the two genes were also isolated and their functionality evaluated in vitro using luciferase as the reporter gene. The muCyp19a promoter sequence (650 bp) contained a consensus TATA box and putative transcription factor binding sites, including two half EREs, an SF-1, an AhR/Arnt, a PR and two GATA-3 s. The muCyp19b promoter sequence (2500 bp) showed consensus TATA and CCAAT boxes and putative transcription binding sites, namely: a PR, an ERE, a half ERE, a SP-1, two GATA-binding factor, one half GATA-1, two C/EBPs, a GRE, a NFkappaB, three STATs, a PPAR/RXR, an Ahr/Arnt and a CRE. Basal activity of serially deleted promoter constructs transiently transfected into COS-7, alphaT3 and TE671 cells demonstrated the enhancing and silencing roles of the putative transcription factor binding sites. Quinpirole, a dopamine agonist, significantly reduced the promoter activity of muCyp19b in TE671. The results suggest tissue-specific regulation of the muCyp19 genes and a putative alternative promoter for muCyp19b.
在一项旨在阐明芳香化酶在雌性鲻鱼青春期作用的研究中，通过逆转录聚合酶链反应（RT-PCR）分离出大脑（muCyp19b）和卵巢（muCyp19a）芳香化酶的互补脱氧核糖核酸（cDNA），并通过定量实时逆转录聚合酶链反应确定它们的相对表达水平。muCyp19a的开放阅读框（ORF）为1515碱基对（bp），编码505个预测的氨基酸残基，而muCyp19b的开放阅读框为1485 bp，编码495个预测的氨基酸残基。随着青春期的推进，大脑中muCyp19b的表达水平显著增加；然而，随着青春期发育，其在垂体中的表达水平仅略有增加。在卵巢中，随着青春期的进展，muCyp19a的表达水平显著增加。还分离出这两个基因的启动子区域，并使用荧光素酶作为报告基因在体外评估其功能。muCyp19a启动子序列（650 bp）包含一个共有TATA框和假定的转录因子结合位点，包括两个半雌激素反应元件（ERE）、一个类固醇生成因子1（SF-1）、一个芳烃受体/芳烃受体核转运蛋白（AhR/Arnt）、一个孕激素受体（PR）和两个GATA结合蛋白3（GATA-3）。muCyp19b启动子序列（2500 bp）显示出共有TATA框和CCAAT框以及假定的转录结合位点，即：一个PR、一个ERE、一个半ERE、一个特异性蛋白1（SP-1）、两个GATA结合因子、一个半GATA-1、两个CCAAT/增强子结合蛋白（C/EBP）、一个糖皮质激素反应元件（GRE）、一个核因子κB（NFκB）、三个信号转导和转录激活因子（STAT）、一个过氧化物酶体增殖物激活受体/维甲酸X受体（PPAR/RXR）、一个Ahr/Arnt和一个环磷腺苷反应元件（CRE）。将连续缺失的启动子构建体瞬时转染到COS-7、alphaT3和TE671细胞中的基础活性证明了假定的转录因子结合位点的增强和沉默作用。多巴胺激动剂喹吡罗显著降低了TE671中muCyp19b的启动子活性。结果表明muCyp19基因存在组织特异性调控，并且muCyp19b存在一个假定的替代启动子。
