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研究生:蔡炎富
研究生(外文):Yen-Fu Tsai
論文名稱:Sp1變異型及C/EBPdelta在大鼠Mrp3基因表現之交互作用
論文名稱(外文):Interaction of Sp1 variants and C/EBP delta in Mrp3 gene expression
指導教授:周辰熹周辰熹引用關係黃金鼎黃金鼎引用關係
指導教授(外文):Chen-Hsi ChouJin-Ding Huang
學位類別:碩士
校院名稱:國立成功大學
系所名稱:臨床藥學研究所
學門:醫藥衛生學門
學類:藥學學類
論文種類:學術論文
論文出版年:2003
畢業學年度:91
語文別:中文
論文頁數:109
中文關鍵詞:多重抗藥性相關轉錄器轉錄因子
外文關鍵詞:Sp1Mrp3C/EBP deltatranscription factor
相關次數:
  • 被引用被引用:1
  • 點閱點閱:162
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  • 下載下載:7
  • 收藏至我的研究室書目清單書目收藏:0
中文摘要
啟動子專一因子Sp1對於許多細胞或病毒的基因是具有轉錄活性的。之前在我們實驗室所發表的文獻中指出,大鼠Mrp3 promoter上在-157~-19的第三和第四個Sp1 binding site對大鼠Mrp3 promoter的活性扮演著重要的角色。且在最近,本實驗室未發表的實驗結果,亦發現在大鼠Mrp3 promoter的第三個Sp1 binding site附近有C/EBP protein binding site,也進一步證明C/EBP δ會鍵結在此C/EBP binding site,並與Sp1產生協同作用,影響大鼠Mrp3 prmoter的表現。然而在Sp1調控大鼠Mrp3啟動子的機轉至今尚未明確,為了能了解Sp1蛋白是如何藉由本身的四級結構而影響大鼠Mrp3 promoter的表現,並探討哪一個胺基酸具有舉足輕重的重要性。故選用不含內生性Sp1的 Drosophila Schneider''s Line 2 (SL2)作為細胞株,大鼠Mrp3 promoter作為所欲觀察的基因表現模式,將Sp1做不同的胺基酸點突變,觀察大鼠Mrp3 promoter的表現量不同,以確知這些變異型是否能直接或間接的影響基因調控。
由實驗結果顯示,Sp1變異型中的H701G能明顯調降大鼠Mrp3 啟動子的表現達50%,幾乎是完全抑制Sp1的活性,而T419E、T476E、S713D、P718I相較於C'-his Sp1在大鼠 Mrp3 啟動子的表現具有升高的趨勢,顯示突變於glutamine-rich region的B domain和zinc finger、D domain能影響Sp1對大鼠Mrp3啟動子的調控。而在Sp1變異型於C/EBP δ的加入,在大鼠Mrp3 啟動子的協同作用上, E65K、S353A、W457S、A499Y、R590E則有下降的趨勢,其突變區域分布於A至C domain。而本實驗的另一個主題,證明E65K、T419E、W457S、H701G在無表皮成長因子的處理下,12-(S)-lipoxygenase啟動子的調控具有明顯抑制,而A499Y、R590E、P718I則為增加調控,此項結果雖和本實驗有所差異,可能和不同細胞的轉錄機制相關,但同時亦佐證了這些變異型於C/EBP δ和c-Jun間的交互作用是不同的。
The promoter specificity factor Sp1 is required for efficient transcriptional activation of many cellular and viral genes. Our previous studies indicated that there are five Sp1 binding site in the rat Mrp3 promoter region, and the third and the fourth Sp1 binding sites in —157~+19 region of rat Mrp3 promoter play important role in the gene transcription. Our recent data revealed that there is a C/EBP protein binding site in front of the third Sp1 binding site, and Sp1 has synergistic effect with C/EBPδin rat Mrp3 transcription. However, the mechanism is unclear. In this study, we studied how Sp1 mutants affect the interaction in the rat Mrp3 promoter. We designed a series of Sp1 variants, and transfected them into Drosophila Schneider cell line 2 (SL2 cell), which is lack of Sp1 protein. Then, We observed the difference of rat Mrp3 promoter activity to detect if these Sp1 variants could influence gene regulation.
In overexpression studies, we found that H701G of Sp1 variants could significantly diminish rat Mrp3 promoter activity by 50%, almost completely inhibited Sp1 activity. T419E, T476E, S713D and P718I increased promoter activity . They locate at glutamine-rich regions of B domain and D domain of Sp1. E65K, S353A, W457S, A499Y and R590E located at A, B and C domain of Sp1 protein could down-regulate the synergestic effect of Sp1 and C/EBPδ. In the other topic associated with Sp1 variants and c-Jun in epidermal growth factor-induced gene expression of human 12(S)-lipoxygenase, the result evidences that E65K, T419E, W457S and H701G of Sp1 variants with EGF treatment could decrease 12(S)-lipoxygenase promoter activity, and A499Y, R590E and P718I could increase the activity. Although the results of these two studies are not the same, it may be due to the different transcription mechanism of two cell lines. The studies also suggest that C/EBPδ and c-jun may interact with Sp1 differently.
中文摘要…………………………………………………….1
英文摘要……………………………………………………2
縮寫檢索表…………………………………………………4
第一章 緒論………………………………………………5
第二章 實驗材料………………………………………… 14
第三章 實驗方法………………………………………… 25
第四章 實驗結果………………………………………… 53
第一節探討Sp1變異型是否影響調控Mrp3啟動子的表現
第二節探討Sp1變異型是否影響和C/EBPδ產生的協同作用
第五章 總結與討論………………………………………57
文獻參考…………………………………………………63
附表…………………………………………………………79
附圖………………………………………………………… 86
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