蛋白質的修飾和轉錄調控，一直以來都是細胞分子生物學研究的重要課題，蛋白質的作用，作為細胞傳遞訊息的一種方式，尤其在轉譯後的修飾，參與著蛋白質調控的任務，這些修飾作用包含：甲基化(methylation)、去甲基化(demethylation)、乙醯化(acetylation)、去乙醯化(deacetylation)、磷酸化(phosphorylation)、泛素化(ubiquitination)、糖基化(glycosylation)，目前已知部分蛋白質甲基轉移酶(methyltransferase)和去甲基轉移酶(demethyltransferase)扮演細胞調控的重要角色，但是其功能和詳細作用，所知依然有限。
     根據兩篇不同的文獻資料，我們分別以啤酒酵母菌(Saccharomyces cerevisiae)基因YHR129c、YDR096w作為研究目標，再利用重組蛋白技術，分別表現重組蛋白質在大腸桿菌(Escherichia coli)中，並且設計前後帶有His-tag，純化出重組蛋白，配合去除所表現基因的酵母菌為受質，來研究其甲基化和去甲基化作用。

Protein modification and transcriptional regulation are two areas in cellular and molecular biology. Protein expression is the way of conveying the message of DNA. Modification of the protein includes methylation, demethylation, phosphorylation, acetylation, deacetylation and ubiquitination.Methyltransferase and demethyltransferase play an important role in cell regulation.
     According to different references, we selected Saccharomyces cerevisiae gene YHR129c and YDR096w as research targets. Using recombinant protein technology, expression of recombinant protein was made in Escherichia coli. We designed the fusion protein with His-tag, and-purified the recombinant protein. Using △YHR129c and △YDR096w yeast the source of substrate and S-adenosyl-L-methionine as co-substrate, I study methylation and demethylation of the respective recombinant proteins.

謝誌.....................................................I
中文摘要................................................III
英文摘要..................................................V
縮寫檢索表..............................................VII
目錄....................................................IX
第一章 緒論...............................................1
一、蛋白質甲基化作用和去甲基化作用.............................1
二、研究動機...............................................2
第二章 實驗材料............................................3
一、菌種..................................................3
二、培養基................................................4
三、緩衝液................................................4
四、膠片的配製.............................................5
五、實驗藥品...............................................6
六、實驗儀器...............................................7
第三章 實驗方法............................................9
一、分別建構啤酒酵母菌基因ARP1、GIS1於大腸桿菌(cloning).........9
1.抽取酵母菌(S. cerevisiae) genomic DNA....................9
2.設計PCR引子(primer).....................................9
3.聚合酶鏈鎖反應( polymerase chain reaction ; PCR).........10
4.DNA瓊脂膠體電泳(gel electrophoresis).....................10
5.純化聚合酶鏈鎖反應之產物(purification).....................10
6.產物補A(在純化產物兩端加上adenosine).......................11
7.TA Cloning.............................................12
8.轉形作用(transformation)................................13
9.藍白篩選(blue and white screening)......................13
10.菌種保存...............................................14
11.質體萃取(plasmid)......................................14
12.限制酶酵素確認切位、片段大小(digestion)....................15
13.DNA定序、確認片段(sequencing)...........................15
二、ARP1基因次選殖到pET28c和pET43.1b，GIS1基因次選殖到pET43.1b表現載體(subcloning)..........................................15
1.限制酵素剪切(digestion)..................................15
2.切位接合(ligation)......................................16
3.確認基因接入表現質體(checking)............................16
三、ARP1p、GIS1p蛋白質表現與純化.............................17
1.使用 E. coli 菌種表現ARP1p、GIS1p重組蛋白質................17
2.超音波破菌(sonication)...................................18
3.His-tag 融合純化法純化重組蛋白.............................19
四、測定ARP1p重組蛋白之甲基活性、GIS1p重組蛋白去甲基活性..........20
1.ARP1p受質製備(substrate).................................20
2.ARP1p甲基酶反應活性測定(methylation).......................21
3.GIS1p受質製備(substrate).................................21
4.GIS1p去甲基酶反應活性測定(demethylation)...................22
5.SDS-PAGE 測定甲基酶和去甲基酶活性...........................23
第四章 實驗結果與討論........................................24
一、ARP1蛋白質表現結果與放射線壓片結果..........................24
二、GIS1蛋白質表現結果與放射線壓片結果..........................25
第五章 結論................................................27
一、ARP1p.................................................27
二、GIS1p.................................................28
第六章 參考文獻.............................................29
表格與圖片.................................................32
表一.引子設計...............................................32
表二.ARP1之PCR條件.........................................32
表三.GIS1之PCR條件.........................................33
表四.ARP1限制酶使用材料......................................33
表五.GIS1限制切位使用材料....................................35
表六.ARP1序列定序比對和切位比對...............................36
表七.GIS1序列定序比對和切位比對...............................37
表八.甲基化反應放射性標定.....................................39
表九.去甲基化反應放射性標定...................................39
圖一.ARP1 PCR產物電泳圖.....................................40
圖二.GIS1 PCR產物電泳圖.....................................41
圖三.pGEM-T vector-ARP1，Nhe I / -EcoR I電泳圖..............42
圖四.pGEM-T vector-ARP1，BamHⅠ/-HindⅢ電泳圖.................43
圖五.pGEM-T vector-GIS1，SacⅡ/-NotⅠ電泳圖....................44
圖六.pET28c-ARP1，Nhe I / -EcoR I電泳圖.....................45
圖七.pET43.1b-ARP1，BamHⅠ/-HindⅢ電泳圖......................46
圖八.pET43.1b-GIS1，SacⅡ/-NotⅠ電泳圖.........................47
圖九.pET28c-ARP1-BL21小量誘導測試............................48
圖十.pET28c-ARP1-Rosata小量誘導測試..........................49
圖十一.pET43.1b-ARP1p-BL21小量誘導測試.......................50
圖十二.ARP1p His-tag管柱純化................................51
圖十三.pET43.1b-GIS1-BL21小量誘導測試........................52
圖十四.GIS1p His-tag管柱純化................................53
圖十五.pET43.1b載體誘導蛋白質................................54
圖十六.ARP1p甲基化活性測定SDS PAGE...........................55
圖十七.ARP1p甲基化活性測定SDS PAGE放射線感應底片................56
圖十八.GIS1p去甲基化活性測定SDS PAGE..........................57
圖十九.GIS1p去甲基化活性測定SDS PAGE放射線感應底片..............58
附錄一pGEMR-T Easy Vector..................................59
附錄二.pET28c載體..........................................60
附錄三.pET43.1b載體........................................61
附錄四.重組蛋白質流程圖.......................................62

【1】	Malakhova, O. A., M. Yan, et al. (2003). "Protein ISGylation modulates the JAK-STAT signaling pathway." Genes Dev 17(4): 455-60.
【2】	Martin, J. L. and F. M. McMillan (2002). "SAM (dependent) I AM: the S-adenosylmethionine-dependent methyltransferase fold." Curr OpinStruct Biol 12(6): 783-93.
【3】	Lee, D. Y., C. Teyssier, et al. (2005). "Role of protein methylation in regulation of transcription." Endocr Rev 26(2):147-70.
【4】	van Leeuwen, F., P. R. Gafken, et al. (2002). "Dot1p modulates silencing in yeast by methylation of the nucleosome core." Cell 109(6): 745-56.
【5】	Friesen, W. J., S. Paushkin, et al. (2001). "The methylosome, a 20S complex containing JBP1 and pICln, produces dimethylarginine-modified Sm proteins." Mol Cell Biol 21(24): 8289-300.
【6】	Forneris,F.,C. Binda, et al. (2007). "Structural basis of LSD1-CoREST selectivity in histone H3 recognition. "J Biol Chem 282(28):20070-4.
【7】	Hou, H. and H. Yu (2010). "Structural insights into histone lysine demethylation."Curr Opin Struct Biol(6):739-48.
【8】	Kwon, D. W. and S. H. Ahn (2011). "Role of yeast JmjC-domain containing histone demethylases in actively transcribed regions." Biochem Biophys Res Commun
【9】	Whetstine, J. R., A. Nottke, et al. (2006) "Reversal of histone lysine trimethylation by the JMJD2 family of histone demethylases." Cell 125(3):467-81.
【10】	Klose, R. J., K. E. Gardner, et al. (2007) "Demethylation of histone H3K36 and H3K9 by Rph1: a vestige of an H3K9 methylation system in Saccharomyces cerevisiae."Mol Cell Biol 27(11):3951-61.
【11】	Woodruff, J. B., D. G. Drubin, et al. (2009) "Dynein-Driven Mitotic Spindle Positioning Restricted to Anaphase by She1p Inhibition of Dynactin Recruitment." Mol Biol Cell 20(13):3003-11.
【12】	Banerjee, H. N., and M. Verma (2009) "Epigenetic mechanisms in cancer. "Biomark Med 3(4):397-410.
【13】	Sharma, S. K., Y. Wu, et al. (2010) "(Bis)urea and (Bis)thiourea Inhibitors of Lysine-Specific Demethylase 1 as Epigenetic Modulators." J Med Chem 53(14):5197-212.
【14】	Deligezer, U., F. Yaman, et al. (2010) "Post-treatment circulating plasma BMP6 mRNA and H3K27 methylation levels discriminate metastatic prostate cancer from localized disease." Clin Chim Acta 411(19-20):1452-6.
【15】	Duns, G., E. van den Berg, et al. (2010) "Histone methyltransferase gene SETD2 is a novel tumor suppressor gene in clear cell renal cell carcinoma."Cancer Res 70(11):4287-91.
【16】	Yoshimatsu, M., G. Toyokawa, et al. (2011) "Dysregulation of PRMT1 and PRMT6, Type I arginine methyltransferases, is involved in various types of human cancers. " Int J Cancer 128(3):562-73.
【17】	Lambrot, R., and S. Kimmins (2011) "Histone methylation is a critical regulator of the abnormal expression of POU5F1 and RASSF1A in testis cancer cell lines."Int J Androl .34(2):110-23.
【18】	Katz, J. E., M. Dlakic, et al. (2003). "Automated identification of putative methyltransferases from genomic open reading frames." Mol Cell Proteomics 2(8): 525-40.
【19】	Zhang, N., J. Wu, et al. (2009) "Gis1 is required for transcriptional reprogramming of carbon metabolism and the stress response during transition into stationary phase in yeast. " Microbiology 155(Pt 5):1690-8.
【20】	Tu, S., E. M. Bulloch, (2007) "Identification of histone demethylases in Saccharomyces cerevisiae." J Biol Chem 282(19):14262-71.