自人類及多種生物的基因體被解碼後，生命科學界立即邁向後基因體時代，而基因功能及蛋白質體學成為研究重點。學者們常有機會因為由某個生物體的genome之序列中找到的功能未知基因而需要面對必須找出該基因所轉譯出的蛋白質之生物學功能的挑戰。找出新基因產物之功能的方法就如同是去了解生命系統的組成。
啤酒酵母菌（Saccharomyces cerevisiae）中的 YHR209W 基因又被稱為 CRG1，被認為是參與調節抵抗斑蝥素（cantharidin）的相關作用的基因。由於 YHR209W 也具有甲基轉移酶之特徵序列，而 YHR209W 基因所轉譯出的蛋白質經由蛋白質序列比對（Blast-P）發現與許多甲基轉移酶較為相似且具有形成與S-腺苷甲硫胺酸（S-adenosyl-L-methionine，AdoMet；簡稱為SAM）結合之三級結構部位的序列，因此 YHR209Wp 被推測為可能具有甲基轉移酶之活性。 在本研究中為了探討 YHR209W 是否具有甲基轉移酶之活性以及探討其可能的受質，我們將啤酒酵母菌中的 YHR209W 基因構築到大腸桿菌（Escherichia coli; E. coli ) 之中使其表現出重組蛋白 YHR209Wp ，再以已剔除 YHR209W 基因的啤酒酵母菌 lysate為受質，利用具有放射線性質的 S-腺苷甲硫胺酸作為輔基質來測定重組蛋白 YHR209Wp 之活性。

Since the genome of human and other creatures has been decoded, the life science gets into the post-genomic era, and the gene functions and proteomics become the main research areas. Researchers often have chances to face the challenge of the protein bio-function translated by a function unknown gene found in the genome of an organism. Methods to find out the functions of new gene’s products are as to realize the components of the life system.
YHR209W, also called CRG1, has been known to be involved in mediating cantharidin resistance of Saccharomysec cereviseae. YHR209W has been inferred as a methyltransferase by sequence comparison, and the Protein translated by YHR209W is called Yhr209wp. Because Yhr209wp is similar with many methyltransferases in Blast-P, and has S-adenosyl-L-methionine binding domain sequence, YHR209W is a putative S-adenosylmethionine-dependent methyltransferase. In order to confirm if YHR209W has methyltransferase activity and possible substrate, in this research, we construct the yeast YHR209W gene into E. coli (Escherichia coli) in different host cell strains and conditions to express the recombinant protein Yhr209wp. The activity is determined by a reaction containing Yhr209wp, protein substrates ΔYHR209W yeast genome and cosubstrate isotope labeled S-adenosyl-L-methionine.

謝    誌------------------------------------------------------Ⅰ
中文摘要----------------------------------------------------Ⅱ
英文摘要----------------------------------------------------Ⅲ
目    錄-----------------------------------------------------Ⅳ
表 目 錄----------------------------------------------------Ⅶ
圖 目 錄----------------------------------------------------Ⅷ
縮寫檢索表--------------------------------------------------Ⅸ

壹、緒論----------------------------------------------------1
第一節  生物體中的甲基化作用-------------------------------------1
第二節  研究動機與目的------------------------------------------2
貳、研究材料與方法--------------------------------------------4 
第一節  酵母菌CRG1（YHR209W）基因的----------------------------4
1.	設計複製放大目標基因 YHR209w 之引子--------------------------4
2.	聚合酶連鎖反應模版製備--------------------------------------4
3.	利用聚合酶鏈鎖反應大量複製YHR209w基因------------------------5
4.	瓊脂凝膠電泳分析鑑定PCR產物---------------------------------7
5.	切膠純化-------------------------------------------------8
6.	PCR產物片段3'端補A----------------------------------------9 
7.	TA- cloning---------------------------------------------10
1)	DNA ligation--------------------------------------------10
2)	E. coli competent Cell之製備---------------------------------11
3)	大腸桿菌之質體轉型----------------------------------------12
4)	少量質體DNA的製備---------------------------------------14
5)	利用限制酶EcoRI 之digestion來確認plasmid DNA------------------15
第二節 重組基因之次選殖----------------------------------------16
1.	建構選殖基因之限制酶切位-----------------------------------16
2.	選殖基因YHR209W與表現載體pET28c之接合----------------------18
3.	轉型至DH5α---------------------------------------------18
4.	利用限制酶篩選正確之plasmid DNA----------------------------19
第三節	蛋白質表現與純化重組蛋白--------------------------------20
1.	轉型至表現宿主BL21（DE3）、Rosetta（DE3）以及tRNA380---------21
2.	確認轉型後之質體DNA--------------------------------------22
3.	目標蛋白質表現-------------------------------------------23
1)	初步誘導------------------------------------------------23
2)	初步破菌分析蛋白質----------------------------------------24
3)	SDS-PAGE 分析蛋白質--------------------------------------25
4)	生長曲線及大量誘導蛋白表現---------------------------------27
4.	分析粗抽蛋白質-------------------------------------------29
5.	純化蛋白質----------------------------------------------31
第四節  蛋白質甲基化活性測定------------------------------------33
1.	SDS-PAGE----------------------------------------------33
2.	IEF-PAGE等電聚焦電泳-------------------------------------35
參、實驗結果與討論-------------------------------------------37
肆、結論---------------------------------------------------46
參考文獻---------------------------------------------------48
附錄------------------------------------------------------52


表 格 目 錄
表一、PCR實驗之反應溶液---------------------------------------6
表二、PCR實驗之反應溫度與時間設定-------------------------------7
表三、配製補A之反應溶液成分------------------------------------9
表四、接合反應之溶液------------------------------------------10
表五、限制酶反應條件------------------------------------------16
表六、EcoR I限制酶反應條件-------------------------------------17
表七、NdeⅠ限制酶反應條件--------------------------------------17
表八、接合反應溶液-------------------------------------------18
表九、EcoR I限制酶反應條件Ⅱ------------------------------------22
表十、NdeⅠ限制酶反應條件Ⅱ-------------------------------------22
表十一、配製12.5%分離凝膠-------------------------------------25
表十二、配製3.7%焦集凝膠--------------------------------------26
表十三、YHR209Wp反應溶液配方---------------------------------34
表十四、BL21（DE3）、Rosetta（DE3）以及tRNA380以30℃誘導各時間點的
        細胞生長（OD600）數值------------------------------------38


圖 目 錄
圖1、Blast-P比對YHR209Wp之結果-------------------------------6
圖2、BL21在30℃的生長曲線------------------------------------37
圖3、tRNA380在30℃的生長曲線---------------------------------37
圖4、Rosetta在30℃的生長曲線----------------------------------38
圖5、30℃ 24小時誘導及未誘導上清物SDS-PAGE比較------------------40
圖6、30℃ 24小時誘導及未誘導沉澱物SDS-PAGE比較------------------40
圖7、YHR209W在Rosetta中加了sorbitol及betaine在18℃誘導48小時破菌後經
     由His-Tag管柱純化後的結果-----------------------------------41
圖8、YHR209Wp反應後的SDS-PAGE------------------------------42
圖9、YHR209Wp反應後的SDS-PAGE底片圖--------------------------43
圖10、YHR209Wp反應後的IEF-PAGE電泳圖-------------------------44
圖11、YHR209Wp反應後的IEF-PAGE電泳圖底片圖---------------------45

1.	Niewmierzycka A and Clarke S: S-Adenosylmethionine-dependent methylation in Saccharomyces cerevisiae. Identification of a novel protein arginine methyltransferase. J Biol Chem 274: 814-24, 1999.
2.	Finkelstein JD and Martin JJ: Homocysteine. Int J Biochem Cell Biol 32: 385-9, 2000.
3.	Dillon SC, Zhang X, Trievel RC and Cheng X: The SET-domain protein superfamily: protein lysine methyltransferases. Genome Biol 6: 227, 2005.
4.	Bogarapu S, Bishop JR, Krueger CD and Pavuluri MN: Complementary medicines in pediatric bipolar disorder. Minerva Pediatr 60: 103-14, 2008.
5.	Kagan BL, Sultzer DL, Rosenlicht N and Gerner RH: Oral S-adenosylmethionine in depression: a randomized, double-blind, placebo-controlled trial. Am J Psychiatry 147: 591-5, 1990.
6.	Rosenbaum JF, Fava M, Falk WE, Pollack MH, Cohen LS, Cohen BM and Zubenko GS: The antidepressant potential of oral S-adenosyl-l-methionine. Acta Psychiatr Scand 81: 432-6, 1990.
7.	Samson DJ, Grant MD, Ratko TA, Bonnell CJ, Ziegler KM and Aronson N: Treatment of primary and secondary osteoarthritis of the knee. Evid Rep Technol Assess (Full Rep): 1-157, 2007.
8.	Camara M, Williams P and Hardman A: Controlling infection by tuning in and turning down the volume of bacterial small-talk. Lancet Infect Dis 2: 667-76, 2002.
9.	Schauder S and Bassler BL: The languages of bacteria. Genes Dev 15: 1468-80, 2001.
10.	Whitehead NA, Byers JT, Commander P, Corbett MJ, Coulthurst SJ, Everson L, Harris AK, Pemberton CL, Simpson NJ, Slater H, Smith DS, Welch M, Williamson N and Salmond GP: The regulation of virulence in phytopathogenic Erwinia species: quorum sensing, antibiotics and ecological considerations. Antonie Van Leeuwenhoek 81: 223-31, 2002.
11.	Hoon S, Smith AM, Wallace IM, Suresh S, Miranda M, Fung E, Proctor M, Shokat KM, Zhang C, Davis RW, Giaever G, St Onge RP and Nislow C: An integrated platform of genomic assays reveals small-molecule bioactivities. Nat Chem Biol 4: 498-506, 2008.
12.	Studier FW and Moffatt BA: Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol 189: 113-30, 1986.
13.	Porath J, Carlsson J, Olsson I and Belfrage G: Metal chelate affinity chromatography, a new approach to protein fractionation. Nature 258: 598-9, 1975.
14.	Lin LL and Hsu WH: Lactose-induced expression of Bacillus sp. TS-23 amylase gene in Escherichia coli regulated by a T7 promoter. Lett Appl Microbiol 24: 365-8, 1997.
15.	Monteiro RA, Souza EM, Yates MG, Pedrosa FO and Chubatsu LS: Use of lactose to induce expression of soluble NifA protein domains of Herbaspirillum seropedicae in Escherichia coli. Can J Microbiol 46: 1087-90, 2000.
16.	Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA and Arnheim N: Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230: 1350-4, 1985.
17.	Petrossian TC and Clarke SG: Multiple Motif Scanning to identify methyltransferases from the yeast proteome. Mol Cell Proteomics 8: 1516-26, 2009.
18.	Boorsma A, de Nobel H, ter Riet B, Bargmann B, Brul S, Hellingwerf KJ and Klis FM: Characterization of the transcriptional response to cell wall stress in Saccharomyces cerevisiae. Yeast 21: 413-27, 2004.
19.	Garcia R, Bermejo C, Grau C, Perez R, Rodriguez-Pena JM, Francois J, Nombela C and Arroyo J: The global transcriptional response to transient cell wall damage in Saccharomyces cerevisiae and its regulation by the cell integrity signaling pathway. J Biol Chem 279: 15183-95, 2004.
20.	Garcia R, Rodriguez-Pena JM, Bermejo C, Nombela C and Arroyo J: The high osmotic response and cell wall integrity pathways cooperate to regulate transcriptional responses to zymolyase-induced cell wall stress in Saccharomyces cerevisiae. J Biol Chem 284: 10901-11, 2009.
21.	Harris K, Lamson RE, Nelson B, Hughes TR, Marton MJ, Roberts CJ, Boone C and Pryciak PM: Role of scaffolds in MAP kinase pathway specificity revealed by custom design of pathway-dedicated signaling proteins. Curr Biol 11: 1815-24, 2001.
22.	Iwahashi H, Odani M, Ishidou E and Kitagawa E: Adaptation of Saccharomyces cerevisiae to high hydrostatic pressure causing growth inhibition. FEBS Lett 579: 2847-52, 2005.
23.	Wu WS and Li WH: Identifying gene regulatory modules of heat shock response in yeast. BMC Genomics 9: 439, 2008.
24.	Schneiter R and Daum G: Extraction of yeast lipids. Methods Mol Biol 313: 41-5, 2006.
25.	Athenstaedt K, Zweytick D, Jandrositz A, Kohlwein SD and Daum G: Identification and characterization of major lipid particle proteins of the yeast Saccharomyces cerevisiae. J Bacteriol 181: 6441-8, 1999.
26.	Bligh EG and Dyer WJ: A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37: 911-7, 1959.
27.	Folch J, Lees M and Sloane Stanley GH: A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem 226: 497-509, 1957.