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    Title: 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
    Other Titles: 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
    Authors: 黃教仁;Huang, Jiao-ren
    Contributors: 淡江大學生命科學研究所碩士班
    陳銘凱;Chern, Ming-kai
    Keywords: 啤酒酵母菌;SSL1;UBC;MMS;孢子分離器;甲基轉移酶;IEF;Saccharomyces cerevisiae;SSL1;UBC;MMS;tetrad dissection;methyltransferase;IEF
    Date: 2008
    Issue Date: 2010-01-11 02:29:59 (UTC+8)
    Abstract: 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.
    Appears in Collections:[生命科學研究所] 學位論文

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