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，我们克隆并鉴定了该基因的5‘侧翼区。近端4kb 5‘侧翼区的序列分析显示，一个富含TATA的G/C启动子区域含有一簇Sp1位点。我们还鉴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。为了阐明启动子区域的功能活性，利用5-侧翼区域的截短/缺失结构进行了荧光素酶报告基因的分析。在42℃热休克应激3h后，报告基因表达载体的荧光素酶活性明显高于对照载体。在高渗应激过程中，含有Tone/Ore-like元件的报告构建体(Osp94-Tone)的荧光素酶活性增强了报告基因的表达，而这种反应被点突变Osp94-Tone取消。凝胶迁移率改变分析显示有一条缓慢迁移的条带与Osp94-Tone探针结合。这些结果表明，Osp94基因的5‘侧翼区含有一个高张敏感的顺式作用元件Osp94-Tone，以及一个功能性的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.