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研究生:林弘富
研究生(外文):Lin Hong-Fu
論文名稱:核仁磷酸蛋白對E2F1之轉錄調控
論文名稱(外文):The role of nucleophosmin/B23 in transcriptional regulation of E2F1
指導教授:翁一鳴
指導教授(外文):BYM Yung
學位類別:碩士
校院名稱:長庚大學
系所名稱:基礎醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:英文
論文頁數:42
中文關鍵詞:核仁磷酸蛋白轉錄調控
外文關鍵詞:nucleophosminB23E2F1
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核仁磷酸蛋白(B23)是細胞核中重要的磷酸蛋白,在中心體的複製、細胞凋亡、細胞分化及細胞週期的調控扮演重要的角色。根據實驗室以前的研究發現,B23會對E2F1行轉錄調控,又E2F1對於細胞週期與細胞凋亡扮演重要角色。因此B23如何對E2F1進行轉錄調控就成了一個重要的問題。為了解答這個問題,使用過度表達B23的系統去測量E2F1的基因表現分析,並藉由建立B23 磷酸化與乙醯化的突變clone,來解答B23是利用那一種後轉譯修飾來調控E2F1。藉由實驗發現,B23似乎不會藉由乙醯化的修飾調控E2F1.而在磷酸化的後轉譯修飾方面,我們發現B23-Threonine 199去磷酸化時,E2F1的基因表現比wild type B23還要高。這顯示出B23可能藉由Thr199這個鄰酸化位置去調控E2F1,我們也利用免疫染色法去看每個後轉譯修飾B23的位置,來確定是否因為位置的不同而造成E2F1的基因表現不同。實驗結果顯示,所有的後轉譯修飾跟wild type B23一樣都位於核仁。
B23 is a major nucleolar protein in nucleus, and it plays role in centrosome duplication, cell apopotosis, cell differentiation and cell cycle progression. According to the previous data from our lab, it has shown that B23 will regulate E2F1 promoter. E2F1 is a important in cell apoptosis and cell cycle. Thus, it is a important to answer how B23 regulate E2F1 promoter. In order to answer the question , we use overexpression B23 system to measure E2F1 promoter activity. And by constructing phosphorylation and acetylation mutant B23 answer how B23 regulate E2F1. We found that B23 seem do not regulate E2F1 by acetylation. Per contra , we found E2F1 promoter activity is higher, when we transfect FlagB23/T199A. It seem that phosphor-Thr199 B23 involved in regulating E2F1 promoter. In order to find whether the localization of post-modification B23 will affect E2F1 promoter activity. We use immunostain to visualize all the mutant form of B23. We found the localization of all mutant B23 are the same with the wild type B23.
Chapter I Introduction………………………………………………….1
B23………………………………………………………………….1
E2F1………………………………………………………………...4
Objectives of this study……………………………………..............6
Chapter II Material and Method………………………………………..7
Cell line and Cell culture……………………………………………7
Antibodies...…………………………………………………………7
Plasmid……………………………………………………………...7
Preparation of protein samples……………………………………...8
Determination of protein concentration…………………………………..8
SDS-PAGE………………………………………………………….9
Western blot analysis……………………………………………….9
Cell transfection and luciferase activity assays……………………10
Imunoprecipitation…………………………………………………11
Chromatin immunoprecipitation…………………………………11
Transformation of plasmid into competent cells…………………..12
Mini – and large – preparation of plasmid DNA…………………..13
Confocal microscopy………………………………………………13
Chapter III Result……………………………………………………..15
Exogenous B23 over-expression can up regulate E2F1 promoter activity……………………………………………………………..15
Using different Vector also can upregulate E2F1 promoter activity.15
FLAG-tagged non-phosphorylatable mutant NPM/T199A stimulate higher E2F1 activity than wild type FlagB23……………………...15
FLAG-tagged acetylation mutant NPM was not involved in regulating E2F1 promoter activity…………………………………16
The localization of all phosphorylatable and acetylatable mutant site was the same with wild type FlagB23……………………………..16
Chapter IV Discussion………………………………………………...18
Chpater V Figure……………………………………………………..21
Chapter IV References………………………………………………...32
Table.1……………………………………………………37
Table.2……………………………………………………37
Table.3……………………………………………………38
Table.4……………………………………………………39
Appendix.1……………………………………………….40
Appendix.2……………………………………………….41
Appendix.3……………………………………………….42
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