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博碩士論文 etd-0825108-154115 詳細資訊
Title page for etd-0825108-154115
論文名稱
Title
以DAPA及二維膠體電泳分析結合在TSG101基因啟動子上之Sp1相關轉錄調控因子
Analysis of Sp1 associated transcription regulatory factors bound on TSG101 promoter by DAPA and two dimensional gel electrophoresis
系所名稱
Department
畢業學年期
Year, semester
語文別
Language
學位類別
Degree
頁數
Number of pages
48
研究生
Author
指導教授
Advisor
召集委員
Convenor
口試委員
Advisory Committee
口試日期
Date of Exam
2008-07-26
繳交日期
Date of Submission
2008-08-25
關鍵字
Keywords
二維膠體電泳、轉錄調控因子、基因啟動子
Sp1, TSG101, two dimensional gel electrophoresis
統計
Statistics
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中文摘要
TSG101是一個腫瘤易感染基因,於細胞中有多種功能,包括細胞內膜傳輸、核內受體轉錄活性及細胞的生長與分化等作用之調控,細胞內TSG101蛋白需維持在恆定量,抑制或活化小鼠NIH3T3細胞中的tsg101基因都會使細胞轉型,並產生轉移性腫瘤,然而 TSG101基因調控區的調節機制還未研究明瞭。本實驗室先前的研究中,已證明TSG101具有管家基因之特色,其上游基因調控區,不具有TATA box,但有Sp1結合位,顯示Sp1可能為其基因轉錄調控因子,本研究中以染色質免疫沉澱法證明了甲狀腺癌細胞內,TSG101上游-1~-460調控區確實有Sp1結合,進一步利用帶有Sp1結合序列之寡核苷酸(DAPA probe)與甲狀腺癌細胞核內蛋白進行DNA 親和性沉降分析法(DAPA),以純化其專一性結合蛋白,再以Sp1抗體進行西方墨點法分析,結果證實野生型DAPA probe 上確有Sp1 蛋白結合。此外,並利用DAPA probe 進行Sp1 相關轉錄調控因子複合體之純化,純化之蛋白經二維電泳分析後,再進行基質輔助雷射脫附游離飛行時間(MALDI-TOF)質譜分析,以鑑定DAPA 純化蛋白之身份,結果發現以此方法無法確定出有Sp1 蛋白結合,但鑑定出如參與pre-mRNA 剪接作用的U5small nuclear RNP 和U2af65,還有ATP-dependent DNA helicase、GlutathioneS-transferase Mu 2 及Actin 等意義不明之蛋白。
Abstract
TSG101 is a tumor susceptibility gene exhibits multiple biological functions, including the regulation of vesicular trafficking, transcription, cellular growth and differentiation. The intracellular steady-state level of TSG101 was shown to under stringent control in a narrow range. Either deprivation or overexpression of mouse tsg101 in NIH3T3 cells leads to neoplastic transformation and subsequent tumorigenic potential of the transformed cells. However, the detail mechanism for regulation of TSG101 gene promoter activity is not clear. Our results indicated TSG101 is a housekeeping gene and contains a TATA-less and Sp1 binding site promoter. Here, we demonstrate in vivo binding of Sp1 transcription factor on TSG101 promoter region by chromatin immunoprecipitation(ChIP). In addition, Sp1-associated transcription regulators were purified using DNA affinity precipitation assay (DAPA) method and subjected to two-dimensional gel electrophoresis and the subsequent MALDI-TOF analysis. Our results verify the biding of Sp1 transcription on the DAPA probe containing wildtype but not the mutant Sp1 biding sequence by subsequent western blotting. Our MALDI-TOF analysis of protein spots from two-dimensional gel did not reveal the binding of Sp1 protein, instead the identified a number of cellular proteins, such as U5 small nuclear RNP、ATP-dependent DNA helicase 2 and actin of unknown significance.
目次 Table of Contents
中文摘要………………………………………………. 1
英文摘要………………………………………………. 2
背景介紹………………………………………………. 3
實驗目的………………………………………………..8
材料方法………………………………………………..9
結果……………………………………………………22
討論……………………………………………………26
參考文獻………………………………………………31
圖表……………………………………………………37
附表……………………………………………………46
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