Abstract
金屬硫蛋白 (metallothionein, MT) 為一低分子量且富含半胱氨酸 (cysteine)之蛋白質.因其能與金屬結合,故被認為與生物體內金屬離子的 代謝及解毒有關.MT普遍存於各類生物體內;而在哺乳動物中,MT可概分為 MT-I及MT-II兩種異構形(isoform).二者可受金屬離子,glucoc- corticoid hormones,interleukin-1(IL-1),interferon(INF)或紫外線 (UV)等的誘導而合成.我們實驗室已從CHO Cdr細胞中選殖到MT-II基因及 其啟動子(promoter)的部份.經核酸序列的分析比對後得知, MT-II promoter上含有可能的metal responsive elements(MREs)以及basal- level enhancer(BLE) sequences.在本實驗中,我們將此 promoter接於一 報導基因(reportergene)之前以分析其特性.結果發現,在我們處理的條 件下,鎘和鋅能夠誘導MT-IIpromoter之活性,然而銅和dexamethasone 似 乎不能.此外,經promoter活性之比較得知,在CHO K1及Cdr細胞中,MT-II promoter constructs之basal level及受金屬刺激後的表現量均比MT-I者 為高;但其誘導倍數相近.最後,我們檢視了UV誘導MT基因表現的情形得知, UV能誘使CHO Cdr細胞MT mRNA之合成.進一步分析發現,不含AP-1 binding sequence而僅含MRE之MT-II promoter仍可經UV照射而活化.因 此,我們認為MRE在UV誘導MT基因表現中扮演著相當重要的角色. Metallothionein (MT) is a low-molecular-weight, cysteine-rich and metal-binding protein which is found to present ubiquitously in all organisms examined. In our laboratory, we have cloned MT gen- es with their promoters from Chinese hamster ovary cadmium resis- tance (CHO Cdr) cells. Sequence analysis of CHO MT-II promoter r- eveals the transcription initiation site, putative metal respons- ive elements (MREs) and basal-level enhancer (BLE) sequences. In this study, we characterized the promoter activity of CHO MT-II gene by inserting the putative promoter in front of a reporter g- ene and investigated the level of transcriptional activation aft- er induction. It was found that cadmium and zinc stimulated the activities of the promoter at the condition we applied, but copp- er and dexamethasone did not. Moreover, we also investigated the differential expression of MT-I and MT-II genes in CHO K1 and Cdr cells by comparing their promoter activities. The basal level ex- pression of MT-II gene ie much higher than that of MT-I gene whi- le the fold of induction after metal treatments were similar. Fi- nally, we examined the effect of UV irradiation on the expression of MT gene and obserbed that MT mRNA was induced in CHO Cdr cells four hours after UV irradiation. We then further investigated the UV effect by various MT promoter-reporter gene constructs and su- rprisingly found that the promoter can be activated without the presence of AP-1 binding sequence. It is thus suggested that MREs may play the major role in responding the acute challenge of UV irradiation and stimulate the synthesis of MT mRNA.