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

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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.