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系統識別號 U0002-2602200813291700
中文論文名稱 I. 酵母菌泛素共軛酶對MMS致變劑之專一性探討 II.酵母菌假設性甲基轉移酶YJR129Cp之蛋白質基質之IEF分析
英文論文名稱 I.Exploration of the specificity of ubiquitin-conjugating enzymes UBCs to MMS mutagen in Saccharomyces cerevisiae II.IEF analysis of the protein substrate for putative methyltransferase YJR129Cp of 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 96
學期 1
出版年 97
研究生中文姓名 黃教仁
研究生英文姓名 Jiao-Ren Huang
學號 694290288
學位類別 碩士
語文別 中文
口試日期 2008-01-28
論文頁數 128頁
口試委員 指導教授-陳銘凱
共同指導教授-陳曜鴻
委員-林賜恩
委員-陳銘凱
委員-章為皓
中文關鍵字 啤酒酵母菌  SSL1  UBC  MMS  孢子分離器  甲基轉移酶  IEF 
英文關鍵字 Saccharomyces cerevisiae  SSL1  UBC  MMS  tetrad dissection  methyltransferase  IEF 
學科別分類 學科別醫學與生命科學生物學
中文摘要 在酵母菌中,轉譯出來的蛋白質後常會有一些修飾作用,其中包含了甲基化、泛素化、磷酸化、醣基化等修飾作用。本研究主要在探討泛素化及甲基化的修飾作用,共分兩部分:

第一部分:
酵母菌Saccharomyces cerevisiae核心轉錄因子TFIIH具有轉錄與nucleotide excision repair DNA修復(NER)兩種功能。其SSL1次單元中的RING finger domain具有E3泛素連接酶活性(Ub ligase),且涉及base excision repair (BER) DNA修復路徑。至於此泛素化反應路徑之專一的共軛酶UBC,則尚無所悉。

先以定點突變及質體互換的方式建構出S. cerevisiae SSL1之BER機制缺陷的C403A菌株,並將S. cerevisiae各別UBC基因剔除之單倍體菌株△UBCs,做致變劑MMS反應測試。對於異合子之UBC剔除菌株,則先以孢子分離器(tetrad dissection)分離出單倍體以進行MMS反應測試。以此篩選出與C403A菌株對MMS反應有相同敏感度的UBC剔除菌株。

第二部分:
由於S. cerevisiae的開啟讀碼框YJR129C具有甲基轉移酶(methyltransferase)的特徵序列,且先前也測得蛋白質甲基轉移酶活性。本研究利用His-tag融合純化出所需之YJR129Cp,以△YJR129C蛋白質粗抽液作為受質,並用氚化甲基S-腺苷甲硫氨酸([C3H3]S- adenosyl-L-methionnine)為輔受質,進行甲基化反應,並再進行受質蛋白質分離工作。

分離工作利用垂直式等電點聚焦電泳,將其受質蛋白質依照等電點做分離後,再以SDS –PAGE做第二維分離,並將其萃取與水解後以質譜做鑑定分析。
英文摘要 In Saccharomyces cerevisiae, proteins are subject to post-translation modification, including methylation, ubiquitination, phosphorylation, glycosylation. This research was performed to explore ubiquitination and methylation, respectively:
I:
The core transcription factor TFIIH of Saccharomyces cerevisiae is essential for both transcription and nucleotide excision repair (NER). Core TFIIH purified from yeast possesses an E3 ubiquitin ligase activity, which resides in a RING finger domain of the SSL1 subunit and is involved in the base excision repair (BER) DNA repair pathway. The ubiquitin conjugating enzyme (UBC) that is specifically linked to the SSL1 ubiquitination pathway remained to unknown.
The SSL1C403A mutant strain was first constructed by site-directed mutagenesis and plasmid shuffle. The haploid deletion strains of individual UBC gene △UBCs, were subject to MMS response test. For the heterozygous diploid deletion strains, tetrad dissection was carried out to obtain the haploid deletion strains prior to the MMS test. Thus, we isolated a UBC deletion strain which exhibits similar MMS response with the SSL1C403A mutant strain.
II:
The ORF of YJR129C of S. cerevisiae has a close match with the methyltransferase on database and has proved to be active with protein substrates. Using the purified recombinant YJR129Cp fused with His-tag, the protein extract from △YJR129C strain was methylated with the cosubstrate [C3H3]S-adenosyl-L- methionine prior to separation.
The labeled proteins were first separated by isoelectric focusing (IEF) on a vertical slab gel. The labeled spot was further separated on SDS-PAGE. Finally the labeled spot was extracted from SDS-PAGE and digested for identification by mass spectrometry.

論文目次 目錄
謝誌 I
中文摘要 II
英文摘要 III
目錄 V
圖表目錄 IX
第一部分:酵母菌泛素共軛酶對MMS致變劑之專一性探討

第一章 緒論---------------------------------------------1
第二章 研究材料與方法-----------------------------------7
第一節 SSL1基因之突變、選殖與表現-----------------------7
1-1 SSL1/pRS315質體轉型並且確認片段長度
1-1-1大腸桿菌勝任細胞(competent cell)製備---------------8
1-1-2大腸桿菌之質體轉型(transformation):氯化鈣轉型法---9
1-1-3以Cla I處理並確認片段-------------------------------10
1-2 聚合酶連鎖反應(PCR)方法大量複製ssl1基因
1-2-1 引子設計----------------------------------------12
1-2-2 模板製備----------------------------------------13
1-2-3 聚合酶連鎖反應(polymerase chain reaction, PCR)
1-2-3.a PCR-based mutagenesis之mega-primer製備----------16
1-2-3.b 鑑定PCR產物-------------------------------------17
1-2-3.c 切膠純化----------------------------------------18
1-2-3.d 利用mega-primer與external primer進行反應--------19
1-2-3.e 利用外側引子增幅目標產物------------------------20
1-3 以定點突變方法複製放大ssl1基因
1-3-1 突變合成反應(thermal cycler)-----------------22
1-3-2 酵素Dpn I處理-------------------------------------25
1-3-3 轉型至大腸桿菌勝任細胞DH5α------------------------25
1-3-4 突變產物之質體抽取與確認--------------------------27
1-3-4.a 野生型與突變型引子設計--------------------------27
1-3-4.b 質體抽取----------------------------------------27
1-3-4.c 利用PCR方法初步確認-----------------------------28
1-4 突變質體(ssl1/pRS315)之之酵母菌質體轉型-------29
1-5 質體互換(plasmid shuffle)---------------------31
1-6 質體互換後產物之確認
1-6-1 Plasmid shuffle後之酵母菌質體抽取-----------------33
1-6-2 以電破法轉型至大腸桿菌勝任細胞--------------------34
1-6-3 抽取轉型後質體------------------------------------36
1-6-4 PCR方法確認及定序---------------------------------36
第二節 UBCs的選殖與異核子的分離
2-1 剔除菌株(Deletion strains)--------------------37
2-2 異核子之分離------------------------------------38
2-3 單倍體之確認------------------------------------39
第三節 MMS突變試劑測試---------------------------------40
第三章 結果與討論
第一節 SSL1基因突變、選殖與表現
1-1 SSL1/pRS315質體轉型並且確認片段長度-------------42
1-2 聚合酶連鎖反應(PCR)方法放大SSL1基因-----------43
1-3 以QuikChange定點突變方法複製放大SSL1基因--------46
1-4 質體互換(plasmid shuffle)---------------------47
第二節 UBCs的選殖與異核子的分離-----------------------49
第三節 MMS突變試劑對於UBCs的影響----------------------52
第四章 結論----------------------------------------------57

第二部分:酵母菌假設性甲基轉移酶YJR129Cp之蛋白質基質之鑑定分析
第一章 緒論--------------------------------------------58
第二章 研究材料與方法
第一節 垂直式等電點聚焦電泳(IEF)最佳條件-------------60
第二節 蛋白質甲基化反應與IEF分析-----------------------61
第三節 SDS-PAGE分析 -----------------------------------64
第三章 結果與討論
第一節 垂直式等電點聚焦電泳之最佳條件------------------68
第二節 蛋白質甲基化反應與IEF分析-----------------------71
第三節 SDS-PAGE分析------------------------------------73
第四章 結論-------------------------------------------74
附錄
附錄一、SSL1/pRS315的製備-------------------------------75
附錄二、GST-SSL1p的誘導純化與表現-----------------------78
A.GST-SSL1質體確認與轉型--------------------------------78
B.GST-ssl1的製作與確認----------------------------------80
C.目標蛋白質表現-轉型至表現載體與確認-------------------81
D.目標蛋白質表現-初步誘導-------------------------------83
E.目標蛋白質表現:生長曲線及大量誘導蛋白質表現----------84
F.分析粗抽蛋白質:超音波破菌與SDS-PAGE分析--------------85
G.Glutathione S-transferase fusion protein純化----------88
H.純化後蛋白質分析--------------------------------------89
附錄三、Drop-out powder成分及濃度表---------------------91
附錄四、培養基組成成分----------------------------------92
附錄五、所使用的緩衝液組成------------------------------94
附錄六、使用到的引子及使用目的--------------------------97
附錄七、QuikChange Site-Directed Mutagenesis Kit流程示意圖---------------------------------------------------------98
附錄八、Tetrad Dissection Microscope TDM400-------------99
附錄九、Map of the glutathione S-transferase fusion vectors------------------------------------------------100
附錄十、使用的marker-----------------------------------101
附錄十一、定序比對結果---------------------------------103
附錄十二、測試菌株之相對存活率-------------------------107
附錄十三、縮寫表---------------------------------------112
附錄十四、儀器設備-------------------------------------114
參考文獻-----------------------------------------------117














圖表目錄
表一、酵母菌菌種品系----------------------------------------------------------------7
表二、大腸桿菌菌種品系-------------------------------------------------------------7
表三、限制酶Cla I切位建立之反應組成-----------------------------------------10
表四、製備mega-primer之一產物α之PCR反應組成--------------------------16
表五、製備mega-primer之一產物β之PCR反應組成--------------------------16
表六、one side SOE-PCR初步製備融合產物之反應組成---------------------20
表七、利用外側引子增殖突變融合產物之反應組成---------------------------21
表八、點突變合成反應控制組之反應組成--------------------------------------24
表九、點突變合成反應實驗組之反應組成---------------------------------------24
表十、利用PCR方式確認產物之反應組成--------------------------------------28
表十一、IEF-PAGE組成------------------------------------------------------------62
表十二、3H放射性標定之甲基化反應組成-------------------------------------63
表十三、3H放射性標定之甲基化反應組成(含neuclease)----------------63
表十四、配置12.5 % SDS-PAGE之分離凝膠(separating gel)-------------66
表十五、配置3.7 % SDS-PAGE之焦集凝膠(stacking gel)---------------66
表十六、IEF-PAGE最佳化條件---------------------------------------------------68
表D-1、GST-ssl1p有無利用乳糖誘導之菌數-----------------------------------85




圖a、剔除菌株之建立方法--------------------------------------------------------37
圖b、出芽生殖-----------------------------------------------------------------------38
圖c、酵母菌生活史------------------------------------------------------------------38
圖1-1、SSL1/pRS315之Cla I切位建立圖----------------------------------------42
圖1-2、以Cla I切割SSL1/pRS315質體之瓊脂凝膠電泳圖-------------------42
圖1-3、利用PCR-based mutagenesis方法合成出其中間產物之瓊脂凝膠電泳圖-------------------------------------------------------------------------------------43
圖1-4、利用one-side SOE-PCR合成目標產物之瓊脂凝膠電泳圖----------44
圖1-5、利用外側引子增幅目標產物之瓊脂凝膠電泳圖---------------------44
圖1-6、利用Cla I切割轉型後ssl1質體之瓊脂凝膠電泳圖-----------------46
圖1-7、利用野生型與突變型引子對ssl1質體進行PCR篩選----------------47
圖1-8、利用Cla I切割質體轉移後之質體DNA--------------------------------48
圖1-9、利用野生型與突變型引子對質體轉移後之質體DNA進行PCR篩選----------------------------------------------------------------------------------------49
圖1-10、酵母菌細胞在sporulation情況下,以相位差顯微鏡觀察到的細胞型態-------------------------------------------------------------------------------49
圖1-11、ΔUBC1在進行TDM後再YPD medium生長情況-------------------50
圖1-12、ΔUBC3在進行TDM後再YPD medium生長情況----------------50
圖1-13、ΔUBC4在進行TDM後再YPD medium生長情況----------------51
圖1-14、利用YPD-G篩選ΔUBC4之目標菌株---------------------------------51
圖1-15、ΔUBC4與pTa之交配狀-------------------------------------------------52
圖1-16、ΔUBC4與pTα之交配狀-------------------------------------------------52
圖1-17、異核子之ΔUBCs與MMS反應後,於YPD medium之存活情況
-------------------------------------------------------------------------------------------53
圖1-18、異核子之ΔUBCs與MMS反應後,於SC medium之存活情況
-------------------------------------------------------------------------------------------53
圖1-19、單倍體之ΔUBCs與MMS反應後,於YPD medium之存活情況
-------------------------------------------------------------------------------------------54
圖1-20、單倍體之ΔUBCs與MMS反應後,於SC medium之存活情況
--------------------------------------------------------------------------------------------------54
圖1-21、以YPD medium測試單套剔除核子之結果進行二變相分析------55
圖1-22、以SC medium測試單套剔除核子之結果進行二變相分析---------55
圖1-23、以YPD medium測試單套剔除核子之結果進行集群分析----------56
圖1-24、以SC medium測試單套剔除核子之結果進行集群分析------------56
圖2-1、pI範圍pH 3~10之最佳化測試----------------------------------------69
圖2-2、pI範圍pH 4~6之最佳化測試-------------------------------------------69
圖2-3、模擬3H標定後,進行IEF之底片(pI範圍pH 3~10)------------70
圖2-4、模擬3H標定後,進行IEF之底片(pI範圍pH 4~6)--------------------70
圖2-5、ΔYJR129Cp實驗組與控制組之膠片與底片(pI範圍pH 3~10)----71
圖2-6、ΔYJR129Cp實驗組與控制組之膠片與底片(pI範圍pH 4~6)------72
圖2-7、將不同蛋白質區帶萃取透析,進行SDS-PAGE後壓片,所得到的底片結果-------------------------------------------------------------------------------73
圖A-1、GST-SSL1質體DNA與Cla I反應確認片段----------------------------79
圖B-1、抽取出突變產物GST-ssl1質體DNA並以野生及突變引子進行PCR反應-------------------------------------------------------------------------------------81
圖D-1、GST-ssl1p有無利用乳糖誘導之生長曲線-----------------------------85
圖F-1、比較12及14小時下有無誘導之SDS-PAGE分析圖------------------87
圖F-2、比較16及20小時下有無誘導之SDS-PAGE分析圖------------------88
圖H-1、以GST-ssl1p進純化後之SDS-PAGE分析圖--------------------------89







參考文獻 參考文獻
【1】 Rappsilber J. and Mann M.(2002)What does it mean to identify a protein in proteomics? Trends in Biochemical Sciences; 27 : 74-78
【2】 Yanagida M. (2002) Functional proteomics current achievements. Journal of Chromatography B-Analytical Technologies in The Biomedical and Life Sciences;771 : 89-106
【3】 Graves P. R. and Haystead T. A.(2002)Molecular biologist's guide to proteomics. Journal of Molecular Microbiology and Biotechnology Reviews; 66 : 39-63
【4】 王惠鈞, 吳啓裕. 蛋白質體學之新進展
【5】 Malakhova O. A., Yan M., Malakhov M. P., Yuan Y., Ritchie K. J., Kim K. I., Peterson L. F., Shuai K., and Dong-Er Z.(2003)Protein ISGylation modulates the JAK-STAT signaling pathway. Genes & Development ;17 : 455-460
【6】 David J. Garfinkel , Adam M. Bailis. (2002)Nucleotide Excision Repair, Genome Stability,and Human Disease: New Insight from Model Systems. Journal of Biomedicine and Biotechnology; 2:2 :55–60
【7】 網路資料,取自http://www.myoops.org/twocw/mit/NR/rdonlyres
/Biology/7-340Fall-2004/42231E59-7742-4CD0-A518-726D9147BCA9/0/intro_s9.pdf
【8】 Shang F, Gong X, Palmer HJ, Nowell Jr. TR, Taylor A.(1997)Age-related decline in ubiquitin conjugation in response to oxidative stress in the lens. Experimental Eye Research;64
:21–30
【9】 Bulteau AL, Szweda LI, Friguet B.(2002)Age-dependent declines in proteasome activity in the heart. Archives of Biochemistry and Biophysics ; 397: 298–304.
【10】 Konishi Y, Beach T, Sue LI, Hampel H, Lindholm K, Shen Y. (2003)The temporal localization of frame-shift ubiq-uitin-B and amyloid precursor protein, and comple¬ment proteins in the brain of non-demented control patients with increasing Alzheimer’s disease pathol¬ogy. Neuroscience Letters; 348 : 46–50.
【11】 Sungmin Song and Yong-Keun Jung. (2004)Alzheimer’s disease meets the ubiquitin–proteasome system. TRENDS in Molecular Medicine. 10 : 565-570
【12】 E. M. Taylor and A. R. Lehmann(1998)Review: Conservation of eukaryotic DNA repair mechanisms. International Journal of Radiation Biology; 74(3): 277- 286
【13】 Qi-En Wang, Mette Prætorius-Ibba, Qianzheng Zhu, Mohamed A. El-Mahdy, Gulzar Wani, Qun Zhao, Song Qin, Srinivas Patnaik,and Altaf A. Wani.(2007)Ubiquitylation-independent degradation of Xeroderma pigmentosum group C protein is required for efficient nucleotide excision repair. Nucleic Acids Research. 35(16): 5338–5350
【14】 劉宣良, 蘇永成, 趙建華. 2004諾貝爾化學獎-蛋白質的死亡之吻
【15】 Michael P. Malakhov, Michael R. Mattern, Oxana A. Malakhova, Mark Drinker, Stephen D.Weeks & Tauseef R. Butt(2004) SUMO fusions and SUMO-specific protease for efficient expression and purification of proteins. Journal of Structural and Functional Genomics ; 5 : 75–86,
【16】 網路資料,取自http://www.acris-antibodies.com/focus_review /focus0024-Ubiquitin.php
【17】 Mark Hochstrasser(2000)Evolution and function of ubiquitin-like protein-conjugation systems. Nature Cell Biology; 2 :152-157
【18】 Markus Biel, Veit Wascholowski, and Athanassios Giannis(2004)A Fatal Affair: The Ubiquitinylation of Proteins. Angew. Angewandte Chemie International Edition.; 43 : 6414 –6416
【19】 Simon H. Reed, Thomas G. Gillette(2007)Nucleotide excision repair and the ubiquitin proteasome pathway—Do all roads lead to Rome? DNA Repair; 6 :149–156
【20】 David J. Garfinkel1and Adam M. Bailis(2002)Nucleotide Excision Repair, Genome Stability, and Human Disease: New Insight from Model Systems. Journal of Biomedicine and Biotechnology; 2(2): 55–60
【21】 Keith D.Wilkinson,Karen H.Ventii,Kenneth L. Friedrich & James E.Mullally (2005)The ubiquitin signal: assembly, recognition.and termination . EMBO reports; 6 : 815–820
【22】 Debdyuti Mukhopadhyay and Howard Riezman(2007)Proteasome-Independent Functions of Ubiquitin in Endo-
cytosis and Signaling. Science ; 315 : 201-205
【23】 Rasmus Hartmann-Petersen , Colin A.M. Semple , Chris P. Ponting ,Klavs B. Hendil , Colin Gordon(2003) UBA domain containing proteins in fission yeast. The International Journal of Biochemistry & Cell Biology: 35 : 629–636
【24】 Rasmus Hartmann-Petersen1, Michael Seeger2 and Colin Gordon (2003)Transferring substrates to the 26S proteasome. TRENDS in Biochemical Sciences ; 28(1): 26-31
【25】 Andre´ Catic and Hidde L. Ploegh(2005) Ubiquitin – conserved protein or selfish gene? TRENDS in Biochemical Sciences;30 (11) :600-603
【26】 Borden, K.L., and Freemont, P.S. (1996). The RING finger domain a recent example of a sequence-structure family. Current Opinion in Structural Biology; 6 : 395–401.
【27】 Zhimin Lu, Shuichan Xu,Claudio Joazeiro, Melanie H. Cobb,2 and Tony Hunter (2002) The PHD Domain of MEKK1 Acts as an E3 Ubiquitin Ligase and Mediates Ubiquitination and Degradation of ERK1/2. Molecular Cell; 9 :945–956
【28】 Johnson, E.S., Ma, P.C., Ota, I.M., and Varshavsky, A. (1995). A proteolytic pathway that recognizes ubiquitin as a degrada-
tion signal. Journal of Biological Chemistry; 270 :17442
–17456.
【29】 Koegl, M., Hoppe, T., Schlenker, S., Ulrich, H.D., Mayer, T.U., and Jentsch, S. (1999). A novel ubiquitination factor, E4, is involved in multiubiquitin chain assembly. Cell ; 96 :635– 644.
【30】 Bart M. Kus, Catherine E. Caldon, Ronit Andorn-Broza, and Aled M. Edwards(2004)Functional Interaction of 13 Yeast SCF Complexes with a Set of Yeast E2 Enzymes In Vitro. PROTEINS: Structure, Function, and Bioinformatics; 54:455–467
【31】 Hartmut Scheel and Kay Hofmann(2003) No evidence for PHD fingers as ubiquitin ligases. TRENDS in Cell Biology ;13(6):285-287
【32】 Coscoy, L. and Ganem, D. (2003) PHD domains and E3 ubiquitin ligases: viruses make the connection. Trends Cell Biol;13:7–12
【33】 Lee Bardwell, A. Jane Bradwell, W. John Feaver, Jesper Q. Svejstrupt, Roger D. Korngergf, and Errol C. Friedberg(1994) Yeast RAD3 protein binds directly to both SSL2 and SSL1 Implications for the structure and function of transcription /repair factor b. Proceedings of the National Academy of Scienves of the United States of America;19:3926 -3930
【34】 Zhigng Wang, Stephen Buratowski, Jesper Q. Svejstrup, William J. feaver, Xiaohua Wu, Roger D. Kornberg, Thomas F. Donahue, and Errol C. Friedberg(1995)The Yeast TFB1 and SSL1 Genes, Which Encode Subunits of Transcription Factor IIH, Are Required for Nucleotide Excision Repair and RNA Polymerase II Transcription. Molecular and Cellular Biology ;15(4):2288-2293
【35】 Errol C. Friedberg.(2000)Nucleotide Excision Repair and Cancer Predisposition. American Journal of Pathology;157 (3):693-701
【36】 網路資料,取自http://db.yeastgenome.org/cgi-bin/locus.pl
?locus=ssl1
【37】 P Matsui, J DePaulo, and S Buratowski(1995)An interaction between the Tfb1 and Ssl1 subunits of yeast TFIIH correlates with DNA repair activity. Nucleic Acids Research; 23(5): 767–772
【38】 Yuichiro Takagi, Claudio A. Masuda, Wei-Hau Chang, Hirofumi Komori, Dong Wang, Tony Hunter, Claudio A.P. Joazeiro,and Roger D. Kornberg (2005) Ubiquitin Ligase Activity of TFIIH and the Transcriptional Response to DNA Damage. Molecular Cell;18(2): 237-243
【39】 Oliver Zolk , Carolus Schenke, Antonio Sarikas(2006)The ubiquitin–proteasome system: Focus on the heart. Cardiovascular Research ;70 : 410 – 421
【40】 Baggavalli P. Somesh,Stefan Sigurdsson, Hideaki Saeki, Hediye Erdjument-Bromage, Paul Tempst, and Jesper Q. Svejstrup(2007)Communication between Distant Sites in RNA Polymerase II through Ubiquitylation Factors and the Polymerase CTD.Cell;129 :57-68
【41】 Birnboim H. C. and Doly J. A.(1979) Rapid alkine extraction procedure for screening recombinant plasmid DNA. Nucleic
Acids; 7 : 1513-1523
【42】 Richard J. Reece(2004)PCR Based Mutagenesis. Analysis of genes and genomes;7-4:241-248
【43】 Russell Higuchi, Barbara Krummell and Randall K.Saiki(1988)A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions .Nucleic Acids Research;16 :7359-7366
【44】 Matthias Kammann, Jurgen Laufs, Jeff Schell and Bruno Gronenborn(1989)Rapid insertional mutagenesis of DNA by polymerase chain reaction (PCR). Nucleic Acids Research ;
17:5404
【45】 M. J McPherson and S.G. Moller(2000)Point mutations by SOEing. PCR;5:152-156
【46】 Steffan N. Hoa, Henry D. Hunt a, Robert M. Horton b, Jeffrey K. Pullen” and Larry R. Peasea(1989)Site-directed mutagenesis by overlap extension using the polymerase chain reaction Gene ;77 :51-59
【47】 Bert Vogelstein and David Gillespie(1979)Preparative and Analytical Purification of DNA from Agarose. Proceedings of the National Academy of Sciences of the United States of America ;76(2):615-619
【48】 Kunkel, T. A.(1985)Rapid and Efficient Site-Specific Mutagenesis without Phenotypic Selection. Proceedings of the National Academy of Sciences of the United States of America ;82:488
【49】 Vandeyar MA, Weiner MP, Hutton CJ, Batt CA. A simple and rapid method for the selection of oligodeoxynucleotide- directed mutants. Gene; 65(1):129–133.
【50】 Ruey-Shyang Chen1, Shin-Ling Yang1, Yu-Wen Hua2, Meng-Chun Chi and Long-Liu Lin(2005)Use of DNA methyltransferase/endonuclease enzyme combinations for megabase mapping of chromosomes. Methods in Enzymology;
216 :279-303
【51】 網路資料,取自http://www.qcbio.com/stratagene/stratagene.htm
【52】 Sikorski, JD Boeke (1991)In vitro mutagenesis and plasmid shuffing: from cloned gene to mutant yeast. Methods in Enzymology; 194:302-318
【53】 網路資料,取自http://www.bio.davidson.edu/courses/MolBio/
plasmidshuffle/plasmidshuffle.html
【54】 Robert S. Sikorski and Jef D. Boeke(1991)In vitro Mutagenesis and Plasmid Shuffling: From Cloned Gene to Mutant Yeast. Methods in Enzymology;194(20):302-319
【55】 JD Boeke, F Croute, GR Fink(1984)A positive selection for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast. Molecular & general genetics;197:345
【56】 Chatoo, B. B., F. Sherman, D. A. Azubalis, T. A. Fjellstedt, D.
Mehnert, and M. Ogur.(1979) Selection of Iys2 mutants of the yeast Saccharomyces cerevisiae by the utilization of aminoadipate. Genetics; 93:51-65.
【57】 Giaever, G., et al. (2002) Functional Profiling of the Saccharomyces cerevisiae Genome. Nature ;418, 387-391
【58】 Gu, Z., et al. (2003) Role of duplicate genes in genetic robustness against null mutations; Nature, 42, 63-66
【59】 Steinmetz, L., et al.(2002) Systematic screen for human disease genes in yeast. Nature Genetics. 31, 400-404
【60】 Fraser, H. B., et al. (2002)Evoluationary Rate in the Protein Interaction Network. Science, 296, 750-752
【61】 Baudin et al.(1993)A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae .Nucleic Acids Research; 21: 3329-3330
【62】 Wach et al.(1994)New Heterologous Modules for Classical or PCR-Based Gene Disruptions in Saccharomyces cerevisiae. Yeast;10:1793-1808
【63】 Gangloff S, de Massy B, Arthur L, Rothstein R, Fabre F.(1999)The essential role of yeast topoisomerase III in meiosis depends on recombination. European Molecular Biology Organization;18:1701-1711
【64】 Fred Sherman and James Hicks(1991)Micromanipulation and Dissection of Asci. Method in enzymology ;194:21-37
【65】 高宇(2004)Cancer and Oncogene.分子生物學(下);18:9-12
【66】 Leslie Barbour, Michelle Hanna, and Wei Xiao(2005)Yeast Protocols: mutagenesis. Methods in Molecular Biology; 313(13):121-127
【67】 Sung-Keun Lee, Sung-Lim Yu, Louise Prakash, and Satya Prakash(2002)Yeast RAD26, a Homolog of the Human CSB Gene, Functions Independently of Nucleotide Excision Repair and Base Excision Repair in Promoting Transcription through Damaged Bases. Molecular and Cellular Biology;22(12):4383-4576
【68】 Lee, S.K., Yu, S.L., Prakash, L., and Prakash, S. (2002). Yeast RAD26, a homolog of the human CSB gene, functions indepen- dently of nucleotide excision repair and base excision repair in promoting transcription through damaged bases. Molecular and Cellular Biology;22: 4383–4389.
【69】 Frank Marini III, Amyr Naeem and Jean-Numa Lapeyre(1993)An efficient 1-tube PCR method for internal site-directed mutagenesis of large amplified molecules. Nucleic Acids Research;21(9):2277-2278
【70】 Neuner,A., Jannasch, H.W., Belkin,S. and Stetter,K.O. (1990) Thermococcus litoralis sp. nov.: A new species of extremely thermophilic marine Archaebacteria. Archives of Microbiology;153:205-207
【71】 PA Garrity and BJ Wold(1992)Effects of Different DNA Polymerases in Ligation-Mediated PCR: Enhanced Genomic Sequencing and In vivo Footprinting. Proceedings of the National Academy of Sciences of the United States of America ;89: 1021-1025
【72】 Giaever G, et al. (2002) Functional profiling of the Saccharomyces cerevisiae genome. Nature ;418(6896):387-391
【73】 Clarke S.(1993) Protein Methylation. Current Opinion in
Cell Biology; 5 : 977-983
【74】 Clarke S.(1992)The biological function of protein
methylation. In Fundamentals of Medical Cell Biology;3: 413-436
【75】 Jennifer L. M. and Fiona M. M.(2002)SAM(dependent)I AM : the S-adenosylmethionine-dependent methyltransferase fold. Current Opinion in Structural Biology;12 : 783-793
【76】 Chiang PK, Gordon RK, Tal J, Zeng GC, Doctor BP, Pardhasaradhi K, McCann PP.(1996) S-Adenosylmethionine and methylation. Federation of American Societies for Experimental Biology; 10(4):471-80
【77】 網路資料,取自http://brc.se.fju.edu.tw/protein/purify/
isoelect.htm
【78】 Pharmacia Fine Chemicals AB.(1982)Theory of Isoelectric Focusing. Isoelectric Focusing:principle & methods;2:5-6
【79】 Pharmacia Biotech(1994)IEF in polyacrylamide gels. Acrylamide gel casting handbook;8:47
【80】 網路資料,取自http://www.bbioo.com/bio101/2007/
19917_3.htm
【81】 Jyh-Cheng Hsieh, Fu-Pang Lin, and Ming F. Tam(1988)Electroblotting onto Glass-Fiber Filter from an Analytical lsoelectrofocusing Gel: A Preparative Method for Isolating Proteins for N-Terminal Microsequencing. Analytical Biochemistry ;170: 1-8
【82】 Pharmacia Biotech(1994)Guide to PAGE Techniques. Acrylamide gel casting handbook;4:19-20
【83】 Pier Giorgio Righetti(1983)Theory and fundamental aspects of isoelectric focusing; Isoelectric Focusing:Theory, Methodology and Applications;11(1):17-24
【84】 Pharmacia Fine Chemicals AB.(1982)Theory of Isoelectric Focusing. Isoelectric Focusing:principle & methods;2:16-41
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