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研究生:張育銘
研究生(外文):Chang Yu Ming
論文名稱:p53反應片段主結合位中AT間距之容許度
論文名稱(外文):The tolerance of AT gap in the core sequence of p53 response element
指導教授:陳正繹呂美華呂美華引用關係趙壯飛
指導教授(外文):Chen Jang YiLu Mei HuaChao Chung Faye
學位類別:碩士
校院名稱:國防醫學院
系所名稱:生物及解剖學研究所
學門:生命科學學門
學類:生物訊息學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:39
中文關鍵詞:AT間距鹼基數
外文關鍵詞:p53core sequenceconsensus sequence
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p53蛋白為調控細胞週期中的一個重要控制者。當DNA受損時,p53便會透過多個檢查點(check point)的活化,中止正在進行中的細胞週期。其中p53可以分別藉由p21 和 14-3-3σ來調控 G1-S期和 G2-M期。整個p53結合序列為兩組重複之10個鹼基組成。在只有10個鹼基時稱為half-site,5個鹼基稱為quarter-site。而15個鹼基就相當於1.5倍的half-site,稱為1.5 half-site。雖然p53可結合於half-site,但是卻無法活化。另外在1.5 half-site,p53雖然有活性表現,但是卻沒有whole-site的活性表現來的高。p21和14-3-3σ啟動子(promoter)上都有兩個p53 response elements。除了14-3-3σ site 1,p53在p21 site 1、p21 site 2和 14-3-3σsite 2上均有活性表現。在14-3-3σ site 1的側結合區(flanking sequence)有幾個鹼基和p53 consensus sequence不相符,但是在14-3-3σ site 2只有一個鹼基不符。p21 site 1、p21 site 2 和14-3-3σ site 1的主結合區(core sequence)上的AT gap 都只有兩個鹼基,而14-3-3σ site 2卻有四個。為了探討在p53能表現的狀態下AT間距鹼基數有多少容許度? 本論文構築數個不同AT間距鹼基數的p53 response element報導載體,並進行報導基因分析(reporter assay),以探討p53反應片段主結合位中AT間距之容許度。我們發現在5’端或3’端的AT間距鹼基數可以為2或4。但若兩端的AT間距鹼基數都為4時,則無活性表現。根據此結果,我們可能可以找出和p53有關的新調控模式和基因。
p53 is a key controller of the cell cycle. After DNA damage, the p53 can be activated to block cell cycle progression through multiple check points. The p53 downstream genes, p21 and 14-3-3σ, can be induced by activated p53 to arrest the G1 to S and G2 to M respectively. The whole- site of p53 binding sequence is the ten bases direct repeat sequence. Only ten bases and five bases of p53 binding sequence are as the half-site and quarter-site of p53 binding site respectively. Ten plus five bases of p53 binding sequence is equal to the 1.5 folds of p53 half-sites. Although one half-site of p53 binding site has p53 binding affinity, it can’t be activated by p53. On the other hand, the 1.5 folds of p53 half-sites can be activated by p53, but this activity is much lower than whole-site of p53 binding site. Both p21 and 14-3-3σ have two p53 response elements in their promoter. The p21 site one, p21 site two, and 14-3-3σ site two can be activated by p53 but 14-3-3σ site one can’t. There are several mismatches in the flanking sequence of the p53 binding site in 14-3-3σ site one, but only one base mismatch in the flanking sequence of the p53 binding site in 14-3-3 σ site two. Only two bases of AT gap are in the core sequence of p53 binding site in the p21 site one, p21 site two, and 14-3-3σ site one, but there are four bases in the 14-3-3σ site two. In order to understand how many bases of AT gaps in p53 core sequence could be activated by p53. In this research, several vectors of oligo-nucleotides with different AT gap of p53 response elements were constructed and used for reporter assay to study the tolerance of AT gap in the core sequence of p53 response element. We found that one of AT gap must keep in two bases, and the other could be as two or four bases. According to the results, we might find out the new rule of p53 regulation pattern and the novel p53 target gene.
目錄
目錄 I
圖表目錄 Ⅲ
中文摘要 Ⅳ
英文摘要 Ⅵ

本文
壹、緒論 1
貳、材料與方法 6
一、實驗材料 6
1.腫瘤細胞株 6
2.實驗藥品與試劑 6
3.重要儀器 8
二、實驗方法 10
1.腫瘤細胞繼代培養 10
2.構築p53 response element的報導載體 10
3.以site-directed mutagenesis製備p53 response element的報導載體 10
4.轉形作用 11
5.質體DNA之抽取 11
6.質體轉染細胞 12
7.蟲螢光酶分析 13
叁、結果………………………………………………………………………..14
1.p53 response element 報導載體的構築 14
2.以site-directed mutagenesis製備p53 response element的報導載體 14
3.14-3-3σ site 2活性確認 15
4. p53在14-3-3σ site 2的3’端中不同AT間距鹼基數的片段之活性表現 16
5. p53在p53 response element為2-2g、2-4g、4-2g、4-4g的活性表現
17
6. p63在p53 response element為2-2g、2-4g、4-2g、4-4g的活性表現
18
7. p53在14-3-3σ site 2的5’端中不同AT間距鹼基數的片段之活性表現 .................................................................................................................18
肆、討論 19
伍、實驗圖表 24
陸、參考文獻 36
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