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。

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.

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

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