Identification of a previously unrecognized promoter that drives expression of the UXP transcription unit in the human adenovirus type 5 genome.
Identification of a previously unrecognized promoter that drives expression of the UXP transcription unit in the human adenovirus type 5 genome.
复制标题
鉴定了一个先前未被识别的启动子，该启动子驱动人类 5 型腺病毒基因组中 UXP 转录单元的表达。
DOI：
10.1128/jvi.01338-10
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发表时间：
2010
影响因子：
5.4
通讯作者：
Wold,WilliamSM
中科院分区：
文献类型：
--
作者：
Ying,Baoling;Tollefson,AnnE;Wold,WilliamSM
我们之前鉴定了一种名为 U 外显子蛋白 (UXP) 的腺病毒 (Ad) 蛋白，由左链 (l 链) 转录单元编码。在这里，我们识别并描述了 UXP 启动子。引物延伸和 RNase 保护测定将转录起始位点定位在 UXP 基因起始密码子上游 32 个核苷酸处。产生了一系列在两个假定的反向CCAAT (I-CCAAT)盒和两个E2F位点处具有突变的病毒突变体。通过定量逆转录 PCR 测量，对于缺乏近端 I-CCAAT 盒的突变体，UXP mRNA 水平显着降低至 Ad 5 型 (Ad5) mRNA 水平的 30%。通过免疫印迹和免疫荧光也观察到 UXP 降低。对于缺乏远端 I-CCAAT 盒或两个假定的 E2F 位点的突变体，UXP mRNA 和蛋白质水平与 Ad5 相似。在感染后期，突变型和野生型 Ad5 感染细胞中的 Ad DNA 水平相似，强烈表明缺乏近端 I-CCAAT 盒的突变体中 UXP mRNA 和蛋白质的减少是由于启动子活性降低所致。电泳迁移率变动分析 (EMSA) 表明细胞因子与 UXP 启动子的近端 I-CCAAT 盒特异性结合。体外荧光素酶报告基因测定表明，基础启动子活性位于转录起始位点的 bp -158 和 +30 之间。与 A549 细胞相比，293 细胞中未观察到 E1A 介导的启动子反式激活。因此，我们推测存在一个先前未识别的 Ad5 启动子驱动 UXP 转录单元的表达。该启动子嵌入纤维基因内，并且包含一个对 UXP mRNA 转录至关重要的近端 I-CCAAT 盒。
We previously identified an adenovirus (Ad) protein named U exon protein (UXP) encoded by a leftward-strand (l-strand) transcription unit. Here we identify and characterize the UXP promoter. Primer extension and RNase protection assays mapped the transcription initiation site at 32 nucleotides upstream of the UXP gene initiation codon. A series of viral mutants with mutations at two putative inverted CCAAT (I-CCAAT) boxes and two E2F sites were generated. With mutants lacking the proximal I-CCAAT box, the UXP mRNA level decreased significantly to 30% of the Ad type 5 (Ad5) mRNA level as measured by quantitative reverse transcription-PCR. Decreased UXP was also observed by immunoblotting and immunofluorescence. UXP mRNA and protein levels were similar to those of Ad5 for mutants lacking the distal I-CCAAT box or both putative E2F sites. Ad DNA levels were similar in mutant- and wild-type Ad5-infected cells during the late stage of infection, strongly suggesting that the decreased UXP mRNA and protein from mutants lacking the proximal I-CCAAT box was due to decreased promoter activity. Electrophoretic mobility shift assays (EMSA) indicated that a cellular factor binds specifically to the proximal I-CCAAT box of the UXP promoter. Anin vitroluciferase reporter assay demonstrated that basal promoter activity lies between bp −158 and +30 of the transcription initiation site. No E1A-mediated promoter transactivation was observed in 293 cells compared with A549 cells. Thus, we propose that there is a previously unidentified Ad5 promoter that drives expression of the UXP transcription unit. This promoter is embedded within the gene for fiber, and it contains a proximal I-CCAAT box critical for UXP mRNA transcription.