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研究生:黃瓊嬅
研究生(外文):Chiung-Hua Huang
論文名稱:Sp1變異型及c-Jun在上皮生長因子誘導人類十二位脂氧酵素基因表現之交互作用
論文名稱(外文):Interaction between Sp1 Variants and c-Jun in Epidermal Growth Factor-induced Gene Expression of Human 12(S)-Lipoxygenase
指導教授:黃金鼎黃金鼎引用關係周辰熹周辰熹引用關係
指導教授(外文):Jing-Ding HuangChen-Hsi Chou
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:114
中文關鍵詞:十二位脂氧酵素基因上皮生長因子
外文關鍵詞:Sp1c-JunEpidermal growth factor and 12(S)-lipoxygenase g
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在調控真核基因轉錄的分子機制中DNA和蛋白質及蛋白質和蛋白質之間的交互作用是扮演著重要的角色,在這過程中基因的啟動區需和一些轉錄因子共同參與RNA-polymerase II的轉錄反應。其中轉錄因子Sp1會和一些細胞性及病毒性因子產生交互作用。雖然Sp1是普遍存在,但在特定的基因及細胞經由訊息傳遞過程中和其他因子結合而產生多樣性的交互作用。在人類上皮癌A431細胞中,EGF能誘導c-Jun/Sp1交互作用調控十二脂氧酵素基因的轉錄活性,而這過程中Sp1扮演著“攜帶者”的角色來幫助c-Jun至基因啟動區。在A431細胞中,EGF能誘導c-Jun蛋白質表現及增加十二脂氧酵素基因的轉錄活性。在細胞中大量表現Sp1能協同EGF所誘導的c-Jun而活化十二脂氧酵素基因的轉錄活性,因此在這Ras-Raf-ERK及Ras-Rac-JNK的訊息傳導過程中可能有將c-Jun或Sp1蛋白質磷酸化。為了進一步釐清Sp1和c-Jun或十二脂氧酵素基因作用的重要胺基酸區域,利用點突變製備各種Sp1變異型質體。經由大量表現Sp1變異型的報告基因分析及凝膠電泳位移實驗發現,無論有無處理EGF,變異型R590E及P718I都能使十二脂氧酵素基因啟動區的轉錄活性比野生型增加2-5倍。但相反的變異型H701G及T419E則會降低十二脂氧酵素基因啟動區的轉錄活性。結果證實在Sp1/c-Jun interaction調控十二脂氧酵素基因啟動區轉錄活性的過程中,Sp1蛋白質的B或zinc finger區域會因為改變transactivation或DNA binding的能力而影響十二脂氧酵素基因啟動區轉錄活性。除此之外,變異型R590E及P718I因為改變了Sp1蛋白質的C及D區域結構而增加啟動區的轉錄活性。綜合而言,Sp1及c-Jun能產生協同性作用而共同調控十二脂氧酵素基因啟動區轉錄活性,而且Sp1蛋白質上的B、C、D及zinc finger區域在這調控機制中扮演著重要的角色。
The molecular mechanisms involved in the transcription of eukaryotic genes are controlled by the ordered interplay of DNA-protein and protein-protein interaction. The factors responsible for basal RNA polymerase II transcription reaction are the core promoter elements and the general transcription factors. One transcription factor, Sp1, interacts with several cellular factors as well as viral gene products. The interaction indicates that multiple protein interactions is a mechanism by which Sp1 can mediate signal transduction in a gene- and cell type-dependent manner despite its ubiquitous expression. The interaction between c-Jun and Sp1 mediated EGF-induced gene expression of the 12(S)-lipoxygenase gene, and that Sp1 might function as a carrier bringing c-Jun to the promoter region. EGF increased abundance of c-Jun protein and transcription of the 12(S)-lipoxygenase gene in human epidermoid A431 cells. Overexpression Sp1 was synergistic with the effect of EGF-inducible c-Jun suggesting that the possibility of modification in Sp1 or c-Jun by the Ras-Raf-ERK and Ras-Rac-JNK signaling cascade. To further characterize the amino acid of Sp1 within the region interacted with c-Jun or 12(S)-lipoxygenase gene, site-directed mutagenesis was carried out to create Sp1 mutations. Overexpression studies and electrophoresis mobility shift assays shows that Sp1 variants, R590E and P718I, increased 12(S)-lipoxygenase promoter activity 2-5 fold during presence or absence of EGF, whereas H701G and T419E markedly attenuated in response. These data demonstrated that Sp1/c-Jun interaction modulates 12(S)-lipoxygenase promoter activity through either B domain or zinc finger domain of Sp1 that are responsible for the transactivation activity and the DNA binding affinity, respectively. Furthermore, elevated promoter activity on transfection of R590E and P718I may be due to structural change in C and D domains. Taken together, our results suggest a synergistic effect between Sp1 and c-Jun to cooperatively activate 12(S)-lipoxygenase promoter activity and provide an insight into that B, C, D and zinc finger domains of Sp1 appeared to be significant modules of the Sp1/c-Jun interaction to modulate 12(S)-lipoxygenase transcription activity.
中文摘要…………………………………………………………………… I
英文摘要…………………………………………………………………… III
縮寫檢索表………………………………………………………………… V
目錄………………………………………………………………………… VI
表目錄……………………………………………………………………… VII
圖目錄……………………………………………………………………… VIII
第一章 緒論……………………………………………………………… 1
第二章 實驗材料………………………………………………………… 15
第三章 實驗方法………………………………………………………… 26
第四章 實驗結果
   第一節 探討EGF對Sp1及c-Jun之蛋白質表現之影響………… 57
   第二節 C-Jun overexpression對12(S)-lipoxygenase基因
       啟動區轉錄活性之影響………………………………… 58
   第三節 探討野生型Sp1是否調12(S)-lipoxygenase基因
       啟動區的轉錄活性……………………………………… 59
   第四節 探討變異型Sp1對於人類子宮頸上皮A431細胞中
       Sp1蛋白質表現之影響…………………………………… 61
   第五節 探討變異型Sp1對於12(S)-lipoxygenase基因
       啟動區轉錄活性之影響………………………………… 62
第五章 總結與討論………………………………………………………… 64
第六章 參考文獻…………………………………………………………… 73
附表……………………………………………………………………………85
附圖……………………………………………………………………………93
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