Characterization of The 5'-Flanking Region of the Stress Response Gene, Osp94

为了研究Osp94基因在这些胁迫下转录调控的分子机制，我们克隆并表征了该基因的5 '侧翼区。对该基因近端4kb的5 '侧翼区进行序列分析，发现一个TATA缺失的富含G/C的启动子区，该启动子区含有一簇Sp1位点。我们还确定了类似于共识热休克元件（HSE）以及共识张力/渗透反应元件（TonE/ORE）的上游序列基序。为了阐明启动子区的功能活性，使用5-侧翼区的截短/缺失构建体进行荧光素酶报告基因测定。经42 ℃热休克处理3 h后，转染HSE报告基因的IMCD3细胞荧光素酶活性明显高于对照组。在高渗应激期间，用含有TonE/ORE样元件（Osp94-TonE）的报告构建体转染的细胞中的荧光素酶活性增强报告基因表达，并且这种反应用点突变体Osp94-TonE废除。电泳凝胶迁移率变动分析显示与Osp94-TonE探针结合的缓慢迁移条带。这些结果表明，Osp94基因的5 '侧翼区含有一个高渗敏感的顺式作用元件Osp94-TonE，以及一个功能性HSE。

To investigate the molecular mechanism of transcriptional regulation of the Osp94 gene under these stresses, we cloned and characterized the 5'-flanking region of the gene. Sequence analysis of the proximal 4 kb 5'-flanking region revealed a TATA-less G/C rich promoter region containing a cluster of Sp1 sites. We also identified upstream sequence motifs similar to the consensus heat shock element (HSE) as well as the consensus tonicity/osmotic response element (TonE/ORE). To elucidate the functional activity of the promoter region, luciferase reporter assays were performed using truncation/deletion constructs of the 5-flanking region. After exposure to heat shock stress at 42 ℃ for 3 hrs, IMCD3 cells transfected with HSE reporter constructs showed a higher luciferase activity than that observed in control vector. Luciferase activities in cells transfected with reporter constructs containing a TonE/ORE-like element (Osp94-TonE) enhanced reporter gene expression during hypertonic stress, and this response was abolished with a point mutant Osp94-TonE. Electrophoretic gel mobility shift assay showed a slowly migrating band binding to the Osp94-TonE probe. These results indicate that the 5'-flanking region of Osp94 gene contains a hypertonicity sensitive cis-acting element, Osp94-TonE, as well as a functional HSE.