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系統識別號 U0002-2602200714563000
中文論文名稱 啤酒酵母菌假設性甲基轉移酶YJR129Cp之體外活性研究
英文論文名稱 In vitro study of the activity of YJR129Cp, a putative methyltransferase from Saccharomyces cerevisiae
校院名稱 淡江大學
系所名稱(中) 生命科學研究所碩士班
系所名稱(英) Graduate Institute of Life Sciences
學年度 95
學期 1
出版年 96
研究生中文姓名 溫淑萍
研究生英文姓名 Shu-Ping Wen
學號 693290081
學位類別 碩士
語文別 中文
口試日期 2007-01-11
論文頁數 80頁
口試委員 指導教授-陳銘凱
委員-王三郎
委員-官宜靜
中文關鍵字 啤酒酵母菌  甲基轉移酶  S-腺苷甲硫氨酸 
英文關鍵字 Saccharomyces cerevisiae  methylltransferase  S-adenosyl-L-methionine 
學科別分類 學科別醫學與生命科學生物學
中文摘要 現今因為生物醫學對模式生物的基因序列研究已具有相當的代表性,使得蛋白質體學的研究價值快速提升,其中又以蛋白質的修飾及轉錄調控兩部分為主要研究指標。蛋白質的甲基化在這兩部份佔了重要的位置,它是一種常見的轉譯後修飾行為,已知有些蛋白質甲基化扮演重要的細胞調控角色,然而大部分的蛋白質甲基化所產生的功能目前還所知有限。近年來,組蛋白的離胺酸甲基化被發現在基因的表現及染色質的重塑上具有決定性的調控作用。雖然如此,但還有許多蛋白質甲基化所需的轉移酶尚未找到。
啤酒酵母菌(Saccharomyces cerevisiae)的開放讀碼框YJR129C具有甲基轉移酶之特徵序列,且在資料庫中與幽門螺旋菌(Helicobacter pylori)的核糖體蛋白L11甲基轉移酶最為相似;而幽門螺旋菌的L11則與啤酒酵母菌核糖體蛋白L12及粒線體核糖體蛋白L19(MRPL19)具有同源關係。為找出離胺酸甲基化所扮演的功能,以及YJR129Cp之可能的甲基轉移酶活性,因此先測定YJR129Cp是否具有甲基化L12或MRPL19p或MRPL11p之活性。本研究利用His-tag融合純化出所需之YJR129Cp、MRPRL19p及MRPL11p,並以L12、MRPL19p、MRPL11p及ΔYJR129C為基質,以S-腺苷甲硫氨酸(S-adenosyl-L-methionine)為輔基質來測定YJR129Cp之活性。本實驗證實YJR129Cp確實具有甲基轉移酶活性,也證實L12、MRPL19p及MRPL11p不是YJR129Cp的基質。
英文摘要 With genome sequencing for the model organisms used in biomedical research, there is a rapidly growing appreciation that proteomics, the study of modification to proteins, and transcriptional regulation will likely dominate the research headlines. Protein methylation plays a central role in both of these fields, and it is a post-translation modification which is frequent occurrence. Although in many cases the roles of protein methylation are poorly understood, some have been known for positive to play regulatory roles. Recently, the lysine methylation of histone has been found to play an important role in regulating gene expression and chromatin remodeling. However, many proteins have been long known to be methylated but still miss their methylltransferase partners.
The ORF YJR129C of Saccharomyces cerevisiae has closest match with the methyltransferase of lysine in ribosomal protein L11 of Helicobacter pylori on database. The ribosomal protein L11 of H. pylori is homologous to yeast ribosomal protein L12 and mitochondrial ribosomal protein MRPL19 of S. cerevisiae. In order to find the role of lysine methylation on L12 and the putative methylltransferase activity of YJR129Cp, we manage to determine whether YJR129Cp is an enzyme that methylates L12 or MRPL19p or MRPL11p. Thus YJR129Cp, MRPL19p and MRPL11p fused with his-tag were purified. The activity is determined by a reaction containing YJR129Cp, protein substrates L12, MRPL19, MRPL11, ΔYJR129C and cosubstrate S-adenosyl-L-methionine.
論文目次 目 錄
謝 誌------------------------------------------------------------------------------ I
中文摘要----------------------------------------------------------------------------- II
英文摘要---------------------------------------------------------------------------- III
縮 寫 表--------------------------------------------------------------------------- IV
目 錄----------------------------------------------------------------------------- V
圖表目錄--------------------------------------------------------------------------- VII
第一章 緒論------------------------------------------------------------------------ 1
第二章 研究材料與方法--------------------------------------------------------- 6
第一節 基因選殖----------------------------------------------------------- 7
第二節 重組基因之次選殖----------------------------------------------- 23
第三節 蛋白質表現與純化重組蛋白---------------------------------- 29
第四節 蛋白質甲基化活性測定---------------------------------------- 40
第三章 結果與討論-------------------------------------------------------------- 44
第一節 基因選殖----------------------------------------------------------- 44
第二節 重組基因之次選殖----------------------------------------------- 46
第三節 蛋白質表現與純化重組蛋白----------------------------------- 50
第四節 蛋白質甲基化活性測定----------------------------------------- 66
第四章 結論----------------------------------------------------------------------- 70
參考文獻---------------------------------------------------------------------------- 71
附 錄---------------------------------------------------------------------------- 76


圖表目錄
表一、酵母菌菌種品系------------------------------------------------------------- 6
表二、大腸桿菌菌種品系---------------------------------------------------------- 6
表三、PCR反應溶液-------------------------------------------------------------- 10
表四、PCR反應條件-------------------------------------------------------------- 10
表五、PCR產物補A反應溶液------------------------------------------------- 13
表六、TA接合反應溶液---------------------------------------------------------- 15
表七、EcoR I限制酶進行確認TA接合結果反應溶液--------------------- 21
表八、EcoR I限制酶建立切位反應溶液-------------------------------------- 24
表九、Nde I限制酶建立切位反應溶液---------------------------------------- 24
表十、次選殖基因接合反應溶液----------------------------------------------- 25
表十一、PCR確認次選殖基因質體-------------------------------------------- 27
表十二、EcoR I與Nde I限制酶確認次選殖基因質體--------------------- 28
表十三、EcoR I與Nde I限制酶確認轉殖至表現菌株基因質體--------- 29
表十四、配製12.5%分離凝膠(Separating gel)----------------------------- 36
表十五、配製3.7%焦集凝膠(Stacking gel)--------------------------------- 36
表十六、測定活性之反應溶液-------------------------------------------------- 41
表十七、測定活性之反應溶液-------------------------------------------------- 42
表十八、蛋白質濃度-------------------------------------------------------------- 65
圖A、基因體與蛋白質體之關係------------------------------------------------- 1
圖B、蛋白質體學之研究範疇---------------------------------------------------- 2
圖1-1、PCR目標基因YJR129C之瓊脂凝膠電泳圖---------------------- 45
圖1-2、PCR目標基因MRPL11之瓊脂凝膠電泳圖----------------------- 45
圖1-3、PCR目標基因MRPL19之瓊脂凝膠電泳圖----------------------- 45
圖1-4、以EcoR I限制酶切割確認抽出的質體DNA片段--------------- 46
圖2-1、利用限制酶EcoR I和Nde I切割pGEM-T-目標基因----------- 47
圖2-2、利用限制酶EcoR I和Nde I切割表現載體pET28c-------------- 48
圖2-3、利用PCR確認pET28c-YJR129C片段----------------------------- 49
圖2-4、利用PCR確認pET28c-MRPL11片段----------------------------- 49
圖2-5、利用PCR確認pET28c-MRPL19片段----------------------------- 49
圖2-6、限制酶EcoR I +Nde I確認pET28c-YJR129C片段-------------- 50
圖2-7、限制酶EcoR I +Nde I確認pET28c-MRPL11片段--------------- 50
圖2-8、限制酶EcoR I +Nde I確認pET28c-MRPL19片段--------------- 50
圖3-1、限制酶EcoR I +Nde I確認轉型到BL21(DE3)後產物------- 51
圖3-2、PCR確認轉型到BL21(DE3)後之pET28c–MRPL19-------- 52
圖3-3、YJR129Cp及MRPL11p初步誘導結果----------------------------- 53
圖3-4、MRPL19p初步誘導結果----------------------------------------------- 54
圖3-5、MRPL19p蛋白質表現之生長曲線----------------------------------- 56
圖3-6、MRPL11p蛋白質表現之生長曲線----------------------------------- 56
圖3-7、MRPL19p在不同時間點誘導之SDS-PAGE電泳圖------------- 57
圖3-8、MRPL11p在不同時間點誘導之SDS-PAGE電泳圖------------- 58
圖3-9、YJR129Cp蛋白質表現之生長曲線---------------------------------- 59
圖3-10、YJR129Cp在不同時間點誘導之SDS-PAGE電泳圖------------- 60
圖3-11、YJR129Cp進行純化後之SDS-PAGE電泳圖---------------------- 62
圖3-12、MRPL19p進行純化後之SDS-PAGE電泳圖---------------------- 63
圖3-13、MRPL11p進行純化後之SDS-PAGE電泳圖---------------------- 64
圖4-1、活性測試之SDS-PAGE電泳圖--------------------------------------- 68
圖4-2、活性測試之底片---------------------------------------------------------- 68
圖4-3、活性測試之SDS-PAGE電泳圖--------------------------------------- 69
圖4-4、活性測試之底片---------------------------------------------------------- 69
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