MOLECULAR-STRUCTURE AND TRANSCRIPTIONAL FUNCTION OF THE RAT VASCULAR AT(1A) ANGIOTENSIN RECEPTOR GENE
MOLECULAR-STRUCTURE AND TRANSCRIPTIONAL FUNCTION OF THE RAT VASCULAR AT(1A) ANGIOTENSIN RECEPTOR GENE
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DOI：
10.1161/01.res.73.4.612
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发表时间：
1993-10-01
影响因子：
20.1
通讯作者：
MURPHY, TJ
中科院分区：
文献类型：
--
作者：
TAKEUCHI, K;ALEXANDER, RW;MURPHY, TJ
大鼠血管紧张素受体（AT1a受体）由两个具有相同受体编码序列的mRNA转录本编码，但其5‘和3’非翻译序列不同。我们筛选雄性Sprague-Dawley大鼠基因组文库克隆血管AT1a受体基因。分离出两组重叠克隆，编码AT1a受体基因周围超过90kb的基因组序列。从基因的5'侧翼部分鉴定出四个重叠的克隆。它们包含启动子区域和两个外显子，大小分别为141 bp和89 bp，编码交替剪接的5'非翻译mRNA序列。另外六个克隆彼此重叠，但不重叠从基因的5'侧翼区域的一组克隆。它们包含一个1977 bp的外显子，编码900 bp的5‘和3’未翻译序列，以及一个1077 bp的开放阅读框，与血管平滑肌细胞AT1a受体cdna中发现的相同。引物延伸和RNase保护研究表明，该基因的转录起始位点在AT1a受体cdna中发现的最多5'序列上游9 bp处开始。我们对克隆基因的定位研究表明，转录起始位点在受体编码外显子上游不少于67 kb。迄今为止，克隆基因在第二个内含子内包含一个未克隆的缺口。启动子报告基因检测是通过转染血管平滑肌细胞，将缺失的3.2 kb启动子区域融合到荧光素酶cDNA报告质粒中进行的。在启动子最5'处的2kb处观察到相对较强的基础转录活性，并且随着在早期启动子区域1kb内的缺失而显著减弱，这表明在基因的上游区域有较强的启动子元件。删除包含转录起始位点和假定的TATA盒的53-bp早期启动子区域完全消除了上游元件驱动荧光素酶cDNA转录的能力。这些结果表明我们已经分离出了AT1a受体基因及其功能启动子。
Rat vascular angiotensin receptors (AT1a receptors) are encoded by two mRNA transcripts sharing an identical receptor coding sequence but differing in their 5' and 3' untranslated sequences. We screened male Sprague-Dawley rat genomic libraries to clone the vascular AT1a receptor gene. Two sets of overlapping clones were isolated that encode over 90 kb of genomic sequence around the AT1a receptor gene. Four overlapping clones were identified from the 5' flanking portion of the gene. These contain the promoter region,and two exons, 141 bp and 89 bp in size, respectively, encoding the alternately spliced 5' untranslated mRNA sequence. Six additional clones overlap each other but do not overlap the set of clones from the 5' flanking region of the gene. These contain a single 1977-bp exon that encodes 900 bp of the 5' and 3' untranslated sequences in addition to a 1077-bp open reading frame identical to that found in vascular smooth muscle cell AT1a receptor cDNAs. Primer extension and RNase protection studies indicate that the transcription start site for this gene begins 9 bp upstream from the most 5' sequence found within the AT1a receptor cDNAs. Our mapping studies of the cloned gene, which so far includes an uncloned gap within the second intron, indicate that the transcription start site is no less than 67 kb upstream from the receptor coding exon. Promoter-reporter assays were performed by transfection of vascular smooth muscle cells with deletions of a 3.2-kb promoter region fused to a luciferase cDNA reporter plasmid. Relatively strong basal transcriptional activity is observed from the 5'-most 2 kb of the promoter and diminishes markedly with deletions within 1 kb of the early promoter region, suggesting strong promoter elements in the more upstream regions of the gene. Deletion of a 53-bp early promoter region containing the transcription start site and a putative TATA box completely abolishes the ability of upstream elements to drive transcription of the luciferase cDNA. These results indicate that we have isolated the AT1a receptor gene and its functional promoter.