臺灣博碩士論文加值系統

中文摘要 i
英文摘要(Abstract) ii
目錄 iii
附表與附圖目錄 vi
壹、 緒論 1
一、 Y-box binding protein 1 (YB-1) 1
(一) YB-1之結構介紹 1
(二) YB-1調控細胞中之生理功能 2
(1) YB-1參與基因轉錄(Transcription) 2
(2) YB-1參與DNA修復(DNA repair) 3
(3) YB-1參與蛋白質轉譯(Translation) 3
(4) YB-1之轉譯後修飾(Post-translational modification) 4
(三) YB-1作為致癌基因在癌症中的影響 5
二、 Small Ubiquitin-like Modifier (SUMO) 6
(一) SUMO之家族成員 6
(二) SUMO之循環修飾反應 7
三、 錯配位修補(Mismatch repair, MMR) 8
(一) 參與錯配位修補之相關蛋白質 8
(二) 錯配位修補機制與生理功能 9
(三) 錯配位修補在癌症中之重要性 10
貳、 研究目的與策略 12
參、 實驗材料與方法 14
一、 實驗材料 14
1、 菌種(Bacteria strain) 14
2、 質體(Plasmid) 14
3、 細胞株(Cell line) 21
4、 培養液與培養基(Bacterial broth and cell culture medium) 21
5、 溶液(Buffer and solution) 22
6、 抗體(Antibody) 25
7、 化學藥品(Chemical and reagent) 25
8、 引子(Primer) 25
二、 實驗方法 26
1、 質體轉型(Transformation) 26
2、 質體製備(Plasmid preparation) 26
(1) 小量製備(Mini-preparation) 26
(2) 中量製備(Midi-preparation) 26
3、 質體核酸點定位點突變(Site-directed mutagenesis) 27
4、 細胞培養(Cell culture) 29
5、 細胞轉染(Transfection)-磷酸鈣共沉澱法(calcium-phosphate
coprecipitation) 29
6、 SDS-聚丙烯醯胺凝膠製備和電泳(SDS polyacrylamide gel preparation
and electrophoresis) 29
7、 西方墨點法(Western blot) 30
8、 GST融合蛋白質之純化(Purification of GST fusion protein) 31
9、 In vivo SUMO分析(In vivo SUMO assay) 32
10、 In vitro SUMO分析(In vitro SUMO assay) 32
11、 YB-1及其突變蛋白表現過量細胞株之建立(Construct overexpressing
cell line of YB-1 and it mutants) 33
12、 免疫螢光染色分析(Immunofluorescence analysis) 33
13、 Luciferase冷光酵素檢測法(Luciferase assay) 34
14、 共同免疫沉澱檢測法(Co-immunoprecipitation assay) 34
15、 DNA-蛋白質結合試驗(DNA pull-down assay) 35
肆、 實驗結果 36
一、 YB-1胺基酸位置Lys26/301/304是YB-1 SUMOylation修飾重要的位置 36
二、 YB-1可以在細胞外被SUMOylation修飾，並且受到SUMO-1、
SUMO-2以及SUMO-3不同程度的修飾作用 37
三、 YB-1之SUMOylation對於細胞中的分布位置與穩定性影響之探討 38
四、 YB-1之SUMOylation對於MDR-1基因轉錄活性影響之探討 39
五、 YB-1之Lys26/301/304 SUMOylation對於YB-1與PCNA親和力之活性
相當重要 39
六、 YB-1之Lys26 SUMOylation對於MutSα/PCNA結合活性以及MutSα辨識並結合至錯配位點之活性相當重要 40
伍、 討論 42
一、 YB-1之Lys26是主要YB-1 SUMOylation位置，而Lys301/304則是次要
位置 42
二、 YB-1被SUMO修飾的位置不具有ΨKxE/D特徵 43
三、 YB-1的SUMOylation不影響YB-1在細胞中分布的位置、蛋白質穩定
性以及轉錄因子轉活化活性 43
四、 SUMO似乎可以作為YB-1與PCNA相互作用之媒介物 44
五、 YB-1之Lys26 SUMOylation對於MutSα/PCNA複合物形成以及Mutsα
辨識並結合至錯配位點之活性相當重要 44
陸、 參考文獻 46
柒、 附表與附圖 52

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