跳到主要內容

臺灣博碩士論文加值系統

(34.204.169.230) 您好!臺灣時間:2024/02/22 00:02
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

我願授權國圖
: 
twitterline
研究生:潘承澤
研究生(外文):Pan Cheng-Tza
論文名稱:人類bcl-x基因啟動子活性分析之研究
論文名稱(外文):Study on the Functional Analysis of Human bcl-x Gene Promoter
指導教授:張自忠周慰遠鄭石通鄭石通引用關係姜淑媛,應靜雯
學位類別:碩士
校院名稱:國防醫學院
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:1998
畢業學年度:86
語文別:中文
論文頁數:70
中文關鍵詞:bcl-x基因啟動子糖皮質固醇巨分子生合成抑制劑
外文關鍵詞:bcl-x genepromoterdexamethasone(Dex)56-Dichloro-1-b-D-ribofuranosylbenzimidazole(DRB)
相關次數:
  • 被引用被引用:0
  • 點閱點閱:273
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
為了探究人類bcl-x基因表現之調控以及受糖皮質固醇(Dex)誘導之影響,本實驗成功地構建人類bcl-x基因5'端未轉錄區一系列的刪除片段之質體,並且應用於細胞轉染實驗。為提高轉染反應之效率,我們應也發展修正傳統之轉染反應方法。結果顯示改良的HBSS-磷酸鈣沈澱法(HBSS-CaPO4)對TMK-1、MCF-7與HeLa細胞的轉染效率顯著提高。利用此HBSS-CaPO4方法,我們將一系列刪除片段的構築質體進行轉染於TMK-1及HEK-293兩種不同來源的細胞。由實驗結果顯示Dex對TMK-1細胞中bcl-x啟動子序列有不十分明顯之誘導作用,僅能使相對的luciferase活性提高至1.7倍。同樣地,Dex在HEK-293細胞中也無明顯誘導現象發生。雖是如此,但我們的實驗也顯示了啟動子在此兩種細胞中的活性表現非常類似。當啟動子序列從5'端刪除-552至-402 bp時,相對的螢光活性在TMK-1及HEK-293細胞中均有急遽下降的趨勢。由此結果顯示此段DNA序列150 bp可能具有正向調節單位,對轉錄活性影響甚大。為了更深入瞭解-552至-402 bp間DNA的活化部位,我們將此段DNA序列構建出另一系列bcl-x基因啟動區的報告質體。由轉染反應結果顯示DNA序列從5'端刪除-494至-465 bp時,相對的螢光活性在兩細胞中皆明顯降低了2.7倍與3.5倍。顯然地,此反應序列具有活化轉錄的能力。以凝膠位移法與競爭性實驗分析兩細胞核萃取液,結果證實了細胞中存有某些轉錄蛋白因子會與-494至-434 bp之間的DNA活化序列結合且具有專一性。此外,為了瞭解DRB誘發TMK-1 c-myc基因表現之作用,本實驗也構建了人類c-myc基因啟動區一系列刪除片段的質體,分別轉染TMK-1與HEK-293兩種不同的細胞株,結果DRB可刺激-1457至-763 bp以及- 763至-254 bp間之DNA序列,增加螢光活性1.4至2.1倍。此外,當DNA序列刪除-763至-254 bp以及-254至+24 bp時,則相對的螢光活性在此兩細胞中大幅降低了3.7倍與10倍。顯示此兩段啟動區序列對c-myc基因轉錄活性之表現相當重要。
In order to study the regulation of bcl-x gene expression, the bcl-x gene promoter was isolated and subcloned into the luciferase reporter. A series of promoter deletions were made and the effect of these deletions and dexamethasone on promoter activities were analyzed by transient transfection. To increase the transfection efficiency, we modified the conventional method of calciumphosphate-DNA coprecipitation. This Hank's balanced salt solution (HBSS)-modified method showed marked improvement in transfection efficiency in cultured cell lines including TMK-1, HeLa, and MCF-7. By using this modified method, we carried out the bcl-x promoter analysis in TMK-1 and HEK-293 cells. Our results indicated that dexamethasone conferred only little (1.7 fold) of activation of bcl-x gene promoter and the promoter constructs exhibited very similar activity patterns in both of TMK-1 and HEK293 cells. A marked reduction in promoter activity was observed in constructs with deletions between -552 to - 402, suggest the presence of potential regulatory elements in this region. Detailed analysis of this region revealed that the region between -494 and -465 is important for full promoter activity. Competitive gel mobility shift assay using nuclear extracts from TMK-1 or HEK293 cells demonstrate the presence of a protein factor which binds with high affinity and specificity to this region. Similar experimental approaches were conducted for studying the extraordinary induction of c-myc gene expression in response to the transcription inhibitor, DRB in TMK- 1 cells. The regions correspond to P0 (-1457 to -763) and P1 (-763 to -254) promoters conferred 1.4 and 2.1 fold of DRB-induced activation, respectively, indicated that these c-myc promoter constructs may reflect the in vivo observations correctly. The regions which are responsible for major promoter activities were identified between -763 to -254 and -254 to +24, which correspond to the P1 and P2 promoters of c-myc gene. Taken together, these results revealed that P1 promoter may be responsible for the DRB-mediated c-myc transcription.
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top