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研究生:詹宗憲
研究生(外文):Chan, Tsunghsien
論文名稱:酵母菌拓樸異構酶II的C末端區功能探究
論文名稱(外文):Functional analysis of the C-terminal domain of yeast topoisomerase II
指導教授:樊琳樊琳引用關係
指導教授(外文):Farh, Lynn
口試委員:楊文仁黃鐘慶
口試委員(外文):Yang, WenjenHuang, Jongchin
口試日期:2013-01-11
學位類別:碩士
校院名稱:國立屏東教育大學
系所名稱:化學生物系碩士班
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:52
中文關鍵詞:拓樸異構酶 IIC末端區膠體電泳位移分析免疫螢光
外文關鍵詞:Topisomerase IIC-terminal domainElectrophoretic mobiolity shift assayImmunofluorescence
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DNA在轉錄及複製完成後,需要將DNA分離到兩個子細胞中卻完全一樣的相同基因組的複製,此關鍵步驟需要DNA 拓樸異構酶扮演關鍵角色。拓樸異構酶主要分成第一型拓樸異構酶I(Type I)與第二型拓樸異構酶II(TypeII)。然而在酵母菌的DNA拓樸異構酶II(topos II)主要是作用在染色體分離時。但其C末端區(胺基酸位置在1202-1429)的結構至今尚未有任何文獻報導。先前已有文獻指出,在C末端區具有核定位訊號,SUMO化等結合位。
本研究將具有yCTD的載體送入Rosetta(DE3)LysS大腸桿菌菌株與BY4741酵母菌菌株以進行活體外及活體內的研究分析。在活體外分析方面,利用Rosetta(DE3)LysS去大量表現帶有his-tag的yCTD,利用膠體電泳位移分析Electrophoretic mobility shift assay(EMSA)來檢視yCTD與ssDNA與dsDNA結合能力。結果顯示yCTD並不會對ssDNA具有結合能力,只對dsDNA具有結合的功能。此外也試著了解dsDNA與yCTD結合時鎂離子(Mg2+)是否會影響與yCTD的結合。結果顯示鎂離子(Mg2+)並不參與yCTD與dsDNA結合的過程。在分析condensin與topoisomerase共同結合在染色體的片段發現具有GC-rich的現象,因此設計一40mer GC-rich的DNA片段與yCTD結合,觀察yCTD是否是藉由GC-rich而結合在DNA上,結果yCTD並非藉由GC-rich 的因素與DNA結合。
在活體內分析方面,利用BY4741去大量表現帶有GST融合蛋白的yCTD。利用免疫螢光觀察yCTD在有絲分裂(mitosis)期間的停留位置。結果顯示在加入nocodazole藥物時,使酵母菌生長期可以形成有絲分裂(mitosis),並停滯在中期(metaphase)但無法進入到後期(anaphase),藉由免疫螢光染色可發現在yCTD會在中期前由核膜進到核內。

DNA topoisomerase plays an important role in cell growth. They are divided into two classes: type I topoisomerase and type II topoisomerase. In yeast , the topoisomerase II major role is on the chromosome separate . NLS、SUMO-
ylation binding site of the C-terminal domain had been identified in recent years. Howerer , there is no research about the C-terminal domain (the amino acid at 1202-1429) struc-
ture until now . In this study , yCTD was cloned to E.coli Rosetta (DE3) LysS strain and yeast BY4741 strain for in vitro and in vivo assay. We used an in vitro yCTD , which was overexpressed in Rosetta (DE3) LysS , assay re-
combinant His-tagged. Then we tested the yCTD combination
with the dsDNA and ssDNA by the Electrophoretic mobility shift assay (EMSA) . The result shows that the yCTD can only bind with dsDNA , but not with the ssDNA . On the other hand , we want to find out whether Mg2+ has effect on the binding of dsDNA and yCTD or not. The result shows that the Mg2+ was not involved in the process of yCTD and DNA combination . During the analysis the condensing and topoisomerase combination on chromosome sequence , we find out that the sequence has GC-rich. To exam whether yCTD recognizes the DNA by the GC-rich , we then design an GC-rich sequence in our assay. Unfortunately , the answer is no. In the in vivo assay , yCTD was tagged with GST fusion protein , over-
expressed in By4741 . Then we observed the location of yCTD in mitosis by immunofluorescence. The result shows that the yeast with nocodazole can arrest in metaphase , but not entry into the anaphase. Furthermore , the asses-
sment of yeast by immunofluorescence shows yCTD can enter the nucleus from the nuclear membrane before the metaphase.

目錄 I
圖表目錄 IV
縮字表 VI

第一章 緒論 1
1-1 DNA拓樸異構酶的功能及種類 1
1-2 第二型拓樸異構酶的結構 2
1-3 原核生物有關C末端區的文獻探討 3
1-4 真核生物有關C末端區的文獻探討 4
1-5 研究動機、目的及實驗大綱 5
  實驗流程圖 6
  實驗流程圖 7

第二章 實驗方法 9
2-1 大腸桿菌融合蛋白質的表現及萃取 9
2-2-1 管柱層析純化(histrap) 11
2-2-2 管柱層析純化(Superdex 75) 12
2-3 濃縮 13
2-4 正十二烷硫酸鈉-聚丙醯胺凝膠電泳(Sodium dodecyl sulfate polyacrylamide gel electrophoresis,SDS-PAGE) 14
2-5 膠體電泳位移分析 electrophoretic mobility shift assay (EMSA) 15
2-6 西方墨點法 (Western blotting) 16
2-7 酵母菌融合蛋白質的表現 18
2-8 酵母菌免疫螢光染色 19
2-9 酵母菌融合蛋白質的萃取 21

第三章 實驗結果 22
3-1 yCTD 的蛋白質表現及純化 22
3-2 yCTD 與DNA 在EMSA中的膠體移動 23
3-3 yCTD 在酵母菌中大量表現的免疫螢光分布 25

第四章 討論及未來展望 26
4-1 yCTD與DNA結合能力 26
4-2 yCTD的免疫螢光分布與其可能性推論 27
4-3 總結 28

第五章 參考文獻 29

圖表 32

附錄 50

圖表目錄
表一、加入nocodazole觀察大量表現yCTD析計算細胞型態之統計 32
圖一、Type IIA拓樸異構酶的催化反應 33
圖二、Type IA拓樸異構酶的催化反應 34
圖三、Type IB拓樸異構酶的催化反應 35
圖四、Type II DNA topoisomerase作用機制 36
圖五、真核Topoisomerase II結構與示意圖 37
圖六、Type II topoisomerase 之序列在不同物種間C末端區的差異性 38
圖七、利用GE histrap 1ml 純化pET28a-yCTD的層析記錄與SDS-PAGE圖 39
圖八、利用Superdex 75分離Histrap純化後目標蛋白的層析紀錄與SDS-page圖 40
圖九、利用Superdex 75分離Histrap純化後濃縮目標蛋白SDS-page圖 41
圖十、pET28a-yCTD 在EMSA中的westernblotting與commassin blue染色 42
圖十一、Scatchard analysisi of EMSA 43
圖十二、pET28a-yCTD 在EMSA中 觀察鎂離子的影響 44
圖十三、pET28a-yCTD 在EMSA中 觀察dsDNA或與ssDNA的結合能力 45
圖十四、pET28a-yCTD 在EMSA中 觀察與GC-rich的結合能力 46
圖十五、pEG(KT)-yCTD在BY4741中大量表現的免疫螢光分析圖 47
圖十六、pEG(KT)-yCTD在BY4741中大量表現的Western blotting 48
圖十七、pEG(KT)-yCTD在BY4741中大量表現加藥後的免疫螢光分析圖 49

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