D'Souza U M, Wang W, Gao D Q, Kanda S, Lee G, Junn E, Hwang C K, Jose P A, Mouradian M M
Genetic Pharmacology Unit, Experimental Therapeutics Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Neurochem. 2001 Mar;76(6):1736-44. doi: 10.1046/j.1471-4159.2001.00155.x.
The D(3) dopamine receptor has a restricted regional distribution in brain and is regulated by dopaminergic agents. Additionally, the D(3) gene is implicated in the pathogenesis of several neuropsychiatric disorders or in their response to pharmacological agents. Elucidating its transcription control mechanisms is therefore of interest in order to explain these biological features of the D(3) gene. In this study, the 5' flanking region of the rat D(3) gene was characterized by isolating the 5' end of its cDNA as well as 4.6 kb of genomic sequence. Analysis of this region revealed the presence of two new exons 196-bp and 120-bp long, separated by an 855-bp intron, located several kilobases upstream of the previously published coding exons. Thus, current evidence indicates that the rat D(3) gene is organized into eight exons. Transcription initiation site was determined by primer extension analysis and repeated rounds of 5' RACE and was found to localize at a pyrimidine-rich consensus 'initiator' sequence, similar to the rat D(2) gene. The D(3) promoter lacks TATA or CAAT boxes but unlike that of other dopamine receptor genes has only 52% GC content. Functional analysis of D(3) promoter deletion mutants fused to a reporter gene in TE671 cells, which endogenously express this gene, revealed strong transcriptional activity localized within 36 nucleotides upstream of transcription start site, and a potent silencer between bases --37 and --537. The D(3) promoter is inactive in C6 and COS7 cells. We conclude that the D(3) gene, similar to the closely related D(2) gene, is transcribed from a tissue specific promoter which is under intense negative control.
D3多巴胺受体在脑中具有局限的区域分布，并受多巴胺能药物调节。此外，D3基因与几种神经精神疾病的发病机制或它们对药物的反应有关。因此，阐明其转录控制机制对于解释D3基因的这些生物学特性具有重要意义。在本研究中，通过分离大鼠D3基因cDNA的5′末端以及4.6 kb的基因组序列，对其5′侧翼区域进行了表征。对该区域的分析揭示了两个新的外显子，长度分别为196 bp和120 bp，被一个855 bp的内含子隔开，位于先前发表的编码外显子上游数千碱基处。因此，目前的证据表明大鼠D3基因由八个外显子组成。通过引物延伸分析和多轮5′RACE确定了转录起始位点，发现其定位于富含嘧啶的共有“起始子”序列处，类似于大鼠D2基因。D3启动子缺乏TATA或CAAT框，但与其他多巴胺受体基因不同的是，其GC含量仅为52%。在TE671细胞（内源性表达该基因）中，将D3启动子缺失突变体与报告基因融合进行功能分析，结果显示强转录活性定位于转录起始位点上游36个核苷酸内，并且在碱基-37和-537之间存在一个有效的沉默子。D3启动子在C6和COS7细胞中无活性。我们得出结论，与密切相关的D2基因类似，D3基因从一个受到强烈负调控的组织特异性启动子转录。
