Fauquet M, Boni C
CNRS URA 1414, Laboratoire de Développement et Evolution du Système Nerveux, Ecole Normale Supérieure, Paris, France.
J Neurochem. 1993 Jan;60(1):274-81. doi: 10.1111/j.1471-4159.1993.tb05848.x.
A 1.5-kb genomic DNA fragment situated upstream from the quail tyrosine hydroxylase (TH) gene transcription site was isolated. This upstream region starts 15 bp from the translation initiation site. It contains two canonical TATA boxes, at positions -37 and -297, three putative glucocorticoid-responsive elements, at positions -1487, -1329, and -1268, and one putative cyclic AMP (cAMP)-responsive element (CRE) at position -53, as well as a putative negative regulatory element consensus sequence at position -735. The consensus POU-Oct site is partly conserved. Comparison of the 5' flanking sequences of quail and mammalian (bovine, human, and rat) TH genes revealed a strong sequence conservation within the 230 nucleotides upstream of the TATA box, with a distinct conservation of the CRE region. Constructs in which the bacterial chloramphenicol acetyltransferase (CAT) reporter gene was linked to promoter stretches of increasing lengths were transfected into three cell lines, two of them originating from quail and rat neural crest and the third derived from mouse fibroblasts. Reporter gene expression was specifically high in the quail and rat neural crest-derived cells compared to the fibroblast cell line. The physiological activity of this putative quail CRE was analyzed further in transfected neural crest cells of quail origin. Both cAMP analogues and agents that enhance intracellular cAMP increased CAT activity. The physiological relevance of this finding is sustained by the presence, in quail nuclear extracts, of a protein(s) that binds CRE consensus sequences.
分离出鹌鹑酪氨酸羟化酶(TH)基因转录位点上游的一个1.5kb基因组DNA片段。该上游区域从翻译起始位点起15bp处开始。它包含两个典型的TATA框,分别位于-37和-297位;三个假定的糖皮质激素反应元件,分别位于-1487、-1329和-1268位;一个假定的环磷酸腺苷(cAMP)反应元件(CRE)位于-53位,以及一个位于-735位的假定负调控元件共有序列。共有POU-Oct位点部分保守。鹌鹑和哺乳动物(牛、人、大鼠)TH基因5'侧翼序列的比较显示,TATA框上游230个核苷酸内有很强的序列保守性,CRE区域有明显的保守性。将细菌氯霉素乙酰转移酶(CAT)报告基因与长度不断增加的启动子片段相连的构建体转染到三种细胞系中,其中两种源自鹌鹑和大鼠神经嵴,第三种源自小鼠成纤维细胞。与成纤维细胞系相比,报告基因在鹌鹑和大鼠神经嵴来源的细胞中表达特别高。在源自鹌鹑的转染神经嵴细胞中进一步分析了这个假定的鹌鹑CRE的生理活性。cAMP类似物和增强细胞内cAMP的试剂都增加了CAT活性。鹌鹑核提取物中存在与CRE共有序列结合的一种或多种蛋白质,这一发现支持了这一结果的生理相关性。