Aminopeptidase-A. II. Genomic cloning and characterization of the rat promoter.
Aminopeptidase-A. II. Genomic cloning and characterization of the rat promoter.
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氨肽酶-A。
DOI：
10.1152/ajpregu.2000.278.2.r425
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发表时间：
2000
期刊：
影响因子：
--
通讯作者：
Healy,DP
中科院分区：
文献类型：
--
作者：
Jiang,Q;Troyanovskaya,M;Jayaraman,G;Healy,DP
氨肽酶-A（阿帕）具有广泛的组织分布，与循环或局部产生的ANG II或CCK-8代谢中的作用一致。阿帕在前B淋巴细胞中也高度表达，但其在淋巴细胞发育中的作用尚不清楚。为了开始理解阿帕表达的细胞特异性调控的基础，我们试图克隆和表征大鼠基因启动子。用部分大鼠阿帕cDNA筛选大鼠基因组文库，分离出一个12 kb的克隆，发现其含有第一外显子和>3 kb的5′-侧翼序列。对大鼠肾脏mRNA的引物延伸分析表明，该基因的主要转录起始位点位于翻译起始密码子上游312 bp和TATA盒下游22 bp处。含有氯霉素乙酰转移酶报告基因上游的5′-侧翼区部分的构建体表明表达是细胞特异性的，并且用含有少至110 bp的5′-侧翼区序列的构建体可以获得高活性。我们进一步确定了-1063和-348之间的上游调控元件，该元件以细胞特异性方式抑制转录。该元件（称为阿帕或USA的上游抑制子）也抑制异源启动子的转录。这些结果表明，大鼠阿帕的组织和调节是不一致的，它是一个管家基因，并进一步表明，大鼠阿帕基因的转录可能是通过一个新的强大的上游抑制元件的存在下进行调节。
Aminopeptidase-A (APA) has a widespread tissue distribution consistent with a role in the metabolism of circulating or locally produced ANG II or CCK-8. APA is also highly expressed in pre-B lymphocytes, but its role in lymphoid cell development is unknown. To begin to understand the basis for cell-specific regulation of APA expression, we sought to clone and characterize the rat gene promoter. Screening of a rat genomic library with a partial rat APA cDNA resulted in isolation of a 12-kb clone found to contain the first exon and >3 kb of 5′-flanking sequence. Primer extension of rat kidney mRNA indicated that the major transcription start site was 312 bp upstream of the translation start codon and 22 bp downstream from a TATA box. Constructs containing portions of the 5′-flanking region placed upstream of a chloramphenicol acetyltransferase reporter gene indicated that expression was cell specific and that high activity could be obtained with constructs containing as little as 110 bp of 5′-flanking region sequence. We further identified an upstream regulatory element between −1063 and −348 that suppressed transcription in a cell-specific manner. This element (termed upstream suppressor of APA, or USA) also suppressed transcription of a heterologous promoter. These results indicate that the organization and regulation of the rat APA is not consistent with it being a housekeeping gene and further suggest that rat APA gene transcription might be regulated through the presence of a novel strong upstream suppressor element.