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研究生:盧志嘉
研究生(外文):Chih-Chia Lu
論文名稱:Sp1轉錄因子的氨基端區域對基因調控的重要性
論文名稱(外文):Significance of the amino terminus of Sp1 in gene regulation
指導教授:黃金鼎黃金鼎引用關係
指導教授(外文):Jin-Ding Huang
學位類別:碩士
校院名稱:國立成功大學
系所名稱:藥理學研究所
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:2004
畢業學年度:92
語文別:中文
論文頁數:82
中文關鍵詞:基因調控氨基端
外文關鍵詞:gene regulationamino terminus
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  Sp1為遍佈於體內且調控許多哺乳類動物基因的轉錄因子。先前研究使用缺少胺基端82個胺基酸的不完全Sp1 cDNA (696個胺基酸) 。已有文獻報導Sp1及C/EBP�猁漕韟P性作用參與大鼠CYP2D5基因的轉錄活化(MCB 14: 1383, 1994)。我們發現全長(778個胺基酸)的Sp1對CYP2D5啟動子的活化作用比Sp1 (696)較低。因此,我們將探討Sp1的胺基端對於基因調控所扮演的角色。
  利用缺少內生性Sp1活性的果蠅細胞(SL2細胞),我們知道Sp1 (696)對CYP2D5啟動子的活化作用較Sp1 (778)高;大鼠Mrp3啟動子亦有相似的結果。Sp1的胺基端會明顯干擾在CYP2D5啟動子Sp1與C/EBP的協同性作用;在Mrp3啟動子則仍會適度地影響Sp1與C/EBP�啋漕韟P性作用。Sp1胺基端區會阻礙其與C/EBP��,或CBP/p300對CYP2D5啟動子協同性活化作用。我們更進一步建構分別缺少胺基端28;46;64個胺基酸的一系列Sp1 truncated constructs(分別命名為Sp1-N1;Sp1-N2;Sp1-N3)。我們發現這些不同長度的constructs對CYP2D5啟動子轉錄活性與Sp1(696)相差不多。利用SUMOplot™分析軟體,該胺基端前方28個胺基酸含有一個高可能性的SUMO標的區。利用對該標的區進行單點突變分析,發現可增加CYP2D5啟動子約2-3倍作用,但是Mrp3啟動子卻只有些微上升;Sp1變異子與C/EBP�圇蛝�於Sp1 (778)與C/EBP�埳餬rp3啟動子活性可增加約2~3倍。我們在利用西方點墨法及DAPA assay之下,發現Sp1變異子的蛋白質表現量較高而DNA 結合力不變及變異子在translocate至核內的能力並未改變的情形。綜合而言,該結果推測胺基端的sumoylation作用會調控Sp1的基因轉錄活性。
  Sp1 is a ubiquitous transcription factor that regulates a variety of mammalian genes. Previous studies utilized a truncated Sp1 cDNA lacking the first 82 amino acids of the Sp1 coding sequence (696 amino acids). It has been reported that transcriptional activation of the rat CYP2D5 gene involves synergism between Sp1 and C/EBPb (MCB 14:1383, 1994). We found that the full-length (778 amino acids) Sp1 was less active than Sp1 (696) in transactivating CYP2D5 promoter. We therefore examined the role of this amino terminus of Sp1 in gene regulation.
  Using Drosophila cells that lack endogenous Sp1 activity, we showed that Sp1 (696) was more potent than Sp1 (778) in transactivation of CYP2D5 promoter. Similar result was observed in rat Mrp3 promoter. However, the amino terminus of Sp1 significantly interfered synergistic effect between Sp1 and C/EBPb in CYP2D5 promoter, but moderately affect the synergistic effect between Sp1 and C/EBP�� in Mrp3 promoter. The amino terminus of Sp1 prevent Sp1, C/EBPb, or CBP/p300 synergistic transactivation of CYP2D5 promoter. We further construct truncated Sp1 cDNA lacking the first 28; 46; 64 amino acids of the Sp1 coding sequence (Sp1-N1; Sp1-N2; Sp1-N3 respectively). We observed that these deletion constructs were almost equal to Sp1 (696) in transactivation of CYP2D5 promoter in Drosophila SL2 cells. By using SUMOplot�� prediction software, it contains one SUMO targeted motifs with high probability within the first 28 a.a. Mutational analyses of this site enhance the activity of the CYP2D5 promoter about two to three folds, but the activity of Mrp3 promoter was no obvious increase. Coexpression of Sp1 mutants with C/EBP�� enhance the activity of Mrp3 promoter about two to three folds than Sp1 (778) with C/EBP��. We found that Sp1 mutants with higher protein expression and equal DNA binding in Western blotting and DAPA assay, and the ability of of these mutants translocation into nuclear is not altered. Taken together, it suggests that sumoylation at the amino terminus of Sp1 regulated its transcriptional activity.
目錄…………………………………………………………………… I
表目錄………………………………………………………………… II
圖目錄………………………………………………………………… III
中文摘要……………………………………………………………… 1
英文摘要……………………………………………………………… 3
縮寫檢索表…………………………………………………………… 5

第一章 緒論………………………………………………………… 6
第二章 實驗材料……………………………………………………… 14
第三章 實驗方法……………………………………………………… 19
第四章 實驗結果……………………………………………………… 32
第一節 探討Sp1轉錄因子的氨基端對基因調控的影響
第二節 進一步研究Sp1胺基端負向調控反應區
第三節 探討Sp1變異子與野生型蛋白質表現量及DNA 結合能力
第五章 總結與討論…………………………………………………… 40
第六章 參考文獻……………………………………………………… 47

附表…………………………………………………………………… 57
附圖…………………………………………………………………… 59
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