Abstract
在本篇研究中,我們針對CHO K1及Cdr兩株細胞中MT基因表現量的差異做進一步的分析和探討.首先由北方雜合分析法(Northern Blot Analysis)得知CHO Cdr的MT基因中會有basal level的表現,並可受金屬誘導而大量表現;而CHO K1細胞則否.分析這兩株細胞中MT啟動子序列,發現此兩株細胞MT啟動子序列相同.CHO K1細胞經由5'azacytidine ( 5'aza-C )處理後,MT基因可受誘導而表現,這似乎意味著CHO K1細胞中MT啟動子序列被甲基化 .此外,5'aza-C處理過的CHO K1細胞對鎘的抗性較未處理5'aza-C的CHO K1細胞來得強.從逆轉錄酵素-聚合酵素連鎖反應( RT-PCR )分析結果得知, 5'aza-C處理過的CHO K1細胞其MT-I及MT-II基因均可表現.從逆轉錄酵素-聚合酵素連鎖反應和Msp I / Hpa II截切分析得知當CHO K1細胞處理過鋅後,MT-I基因啟動子會有去甲基化的現象.從Msp I / Hpa II截切分析及南方雜合分析法( Southern Blot Analysis )得知CHO K1細胞中MT啟動子序列被甲基化;而CHO Cdr細胞中MT啟動子則有部份被甲基化,有些部份則未有甲基化現象.藉由sodium bisulfite修飾法,我們更可進一步找出CHO K1細胞中MT-I啟動子序列中5-methylcytosine所在位置的分佈情形.In this study, metallothionein ( MT ) gene expressions inCinese hamster ovary ( CHO ) K1 and CHO cadmium resistant (Cdr) cells were investigated. Northern analysis indicated thatMT genes were highly expressed in Cdr cells after metalinductions while expression was not detectable in CHO K1 cells.Sequence analysis of the MT promoter regions from those celllines revealed that the sequences for both MT-I and MT-IIpromoters are the same despite that the MT genes have beenamplified 60 to 100 folds in Cdr cells. MT genes in CHO K1cells became inducible when treated with 5' azacytidine ( 5'aza-C ), implicating the involvement of methylation in MTgenes. In addition, the 5' aza-C treated CHO K1 cells were moreresistant to cadmium toxicity than the untreated cells. Reversetranscriptase-polymerase cahin reaction demonstrated that MT-Iand MT-II genes were both activated in CHO K1 cells after 5'aza-C treatment. From Reverse transcriptase-polymerase chainreaction and Msp I / Hpa II digestion analysis also revealedthat the MT-I promoter was demethylated in CHO K1 after zinctreatment. From Msp I / Hpa II digestion analysis also revealedthat the MT promoters of CHO K1 were methylated and those ofCHO Cdr were partially methylated. The distribution ofmehylation sites in the MT-I promoter region of CHO K1 cellswere identified by the sodium bisulfite method. The mehylationfrequencies are highest for MREa and MREb, but lowest for MREd.