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研究生:蔡七女
研究生(外文):Tsai, Chi-Neu
論文名稱:EBV第一核蛋白啟動子於人類B細胞及上皮細胞中細胞因子之調控與研究
論文名稱(外文):Studies of the factor(s) that control differential usage of EBV nuclear antigen I promoter in B iymphocytes and epithelial cells
指導教授:張玉生張玉生引用關係
指導教授(外文):Chang, Yu-Sun
學位類別:博士
校院名稱:國立陽明大學
系所名稱:微生物及免疫學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:1997
畢業學年度:85
語文別:中文
論文頁數:128
中文關鍵詞:微生物學免疫學EBV第一核蛋白上皮細胞
外文關鍵詞:MICROBIOLOGYIMMUNOLOGY
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EB病毒可感染人類B淋巴球細胞及表皮細胞,是引起傳染性單核球增多症的致病原,並與巴克氏淋巴瘤及鼻咽癌有關。在這些腫瘤細胞中除了具有EB病毒遺傳體,還能偵測到潛伏性基因產物EBNA-1及LMP-1之表現。因此一般相信在這些腫瘤細胞中EBV是以潛伏期的狀態存在的,而在這些潛伏基因產物中,EBNA-1同時能在有EBV感染的鼻咽癌及巴克氏淋巴瘤中偵測到的。
在先前的研究中指出,在鼻咽癌及巴克氏淋巴瘤中,EBNA-1的訊息RNA是由F啟動子所引導,這個啟動子有別於活體外建立之淋巴芽細胞株所得之結果,因在活體外建立之淋巴芽細胞株中,訊息RNA是由BamH1C/W啟動子所產生,此結果顯示腫瘤細胞中與活體外建立之細胞株之細胞調控截然不同,對於F啟動子而言,其調節區域有二:一是EBNA-1蛋白本身的附著點(EBNA-1 binding site III),可自動調控F啟動子;另一則是細胞因子附著區域,位於F啟動子下游+24-+270之間。為了了解EBNA-1基因表現的調控機轉,首先利用包含長短不一的調節區域片段的luciferase及CAT報告基因送入人類上皮細胞株及第一類巴氏淋巴瘤細胞中,以報告基因的活性分析來確定最小的啟動子區域,本實驗中發現真正具有啟動子能力的片段並非以前認為的F啟動子,而在其下游之BamH1 Q區域 ,才是具有下列啟動子的特色:如具最高的luciferase及CAT活性,活性具有方向性,且這個啟動子受到EBNA-1蛋白本身的附著點的自動調控。為了再進一步確定這個啟動子的功用,以RNA水解酉每保護法分析顯示在潛伏期感染的第一型巴克氏淋巴瘤細胞確實有訊息由此啟動子產生,此外利用RT-PCR在鼻咽癌及第一型淋巴瘤細胞中皆可偵測到Qp之轉錄產物,以上之結果證實在BamH1 Q區域確實有啟動子的功用,我們稱它為BAMH1Q promoter,簡稱為Qp。
Qp除了受到EBNA-1蛋白附著點之調節外,還受到細胞因子之影響。利用報告基因及體外足跡反應等作分析,發現一重要之區域(Qp的-70--39)可正向調節Qp之活性,而以電泳帶阻滯分析中亦證實細胞因子之存在,因此利用此段區域構築具有此段區域五次重覆之酵母菌株YB-His,及YB-Blue,並加以篩選鼻咽癌檢體及轉移之鼻咽癌細胞建立的兩個表現基因庫。從YB-His中共得到四十個cDNA clones,再將此四十個clones送入YB-Blue中分析β-galactosidase之活性,發現只有三個clones會顯示藍色反應。其中兩個clones含有相同的一段胺基酸序列(Cys2/His2 Zinc finger),另一則是DPC4(為一腫瘤抑制基因)。分析其β-galactosidase之單位顯示含Zinc finger蛋白可導至20-30倍活化。至於DPC4則有7-10倍之活化,然而其真實之生物特性仍待進一步證實。
Epstein-Barr virus (EBV) is a ubiquitious human herpesvirus that infects both B lymphocytes and epithelial cells in vitro. This virus can cause self-limiting infectious mononucleosis (IM) and is closely associated with Burkitt''s lymphoma (BL) and nasopharyngeal carcinoma (NPC). Within these maligant cells, EBV is latent and only very few latent genes, including EBNA-1 and LMP-1 genes are expressed. Among these latent gene products, EBNA-1 is the only protein expressed in both EBV-infected NPC and BL cells. Previous studies have reported that the mRNA of EBNA-1 in these two maligancies is initiated from the promoter located in BamH1 F fragment (Fp) in the viral genome, instead of the conventional BamH1 C/W promoter preferentially used in B lymphoblastoid cell lines. Two regulatory elements, an EBNA-1 binding site for autoregulation of EBNA-1 and a positive regulatory region located in the downstream BamH1 Q region between Fp and EBNA-1 binding site are identified. These data strongly suggest that cellular factors may modulate the selection of Fp. In order to understand the regulation of EBNA-I gene in the type I BL and NPC cells, a series of luciferase gene and chloramphenicol acetyl transferase gene containing plasmids that contained various portions of the Fp region were constructed. Plasmid DNA was then introduced into cells and the promoter activity was determined by the reporter gene activity. By this method, a 186 bp fragment within the BamHI Q region was identified. This promoter activity was orientation-dependent and was down-regulated by EBNA-1 in both type I BL cells and a human epithelial cell line, C33A. Furthermore, RNase protection assay showed that the transcripts were indeed initiated from nt 62416 within the BamH1 Q region of EBV. In addition, transcipts initiated from Qp were present in type I BL cell lines and in NPC biopies as detected by the RT-PCR method. The above data suggested that BamH1 Q region contained a novel promoter (Qp) for EBNA-1 expression in the type I BL and NPC cells which was autoregulated by the EBNA1 antigen .
To search for the cellular factor(s) that might be involved in the positive regulation of the Qp, a region (-70 to -39 of the Qp) was identified by using DNase1 footprinting and gel mobility shift assay (EMSA). Reporter gene analysis also showed that this region was essential for Qp activity in C33A cells. This 32-bp DNA fragment was then used to construct two yeast cell clones, YB-His and YB- Blue. Both cell clones contained five copies of 32-bp region inserted upstream from the His3 and LacZ genes, individually. Two cDNA libraries, derived from pooled NPC biopies and from a metastasized NPC tumor were transformed into the yeast cell clone YB-His, respectively. Fourty cDNA clones from these two libraries were able to grow on the His- plates and were then transformed into yeast cell clone YB-Blue. The β-galactosidase activity was assayed and only three out of forty clones turned blue whithin 2 hours. Two of them contained two cys2/his2 zinc finger motifs. This cDNA was 602 bp in length and this region was identical to several important DNA binding proteins, such as HIV enhancer binding protein 2 (EP-2), MHC class II binding protein... etc. The third one was 100% homologous to the pancreatic tumor suppressor gene, DPC4. To confirm their function in the yeast one-hybrid system, these three cDNA clones were transformed into the yeast YB-Blue cell clones. Data showed that the introduction of the zinc finger clones into yeast YB-Blue cell clone resulted in a 20-30 fold increase of the β-galactosidase activity. Introduction of DPC4 gene into YB-Blue resulted in a 7-10 fold increase. However, the biological significance of these factors needs to be proven in the future.
封面
目錄
中文摘要
Abstract
一、緒論
二、材料與方法
三、結果
四、討論
五、參考文獻
圖一至三
表一
圖四至圖二十二
表二,三
圖二十三至二十七
表四 A-D
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