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研究生: 陳念慈
Nien-tzu Chen
論文名稱: 探討酵母菌細胞質 glutamyl-tRNA synthetase 的粒腺體標的訊號
Identification of the mitochondrial targeting signal of yeast glutamyl-tRNA synthetase
指導教授: 王健家
Chien-chia Wang
口試委員:
學位類別: 碩士
Master
系所名稱: 生醫理工學院 - 生命科學系
Department of Life Science
畢業學年度: 98
語文別: 中文
論文頁數: 69
中文關鍵詞: 粒腺體標的訊號
外文關鍵詞: GluRS, MTS
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  • 酵母菌Saccharomyces cerevisiae中cytoplasmic glutamyl-tRNA synthetase (cGluRS) 是由細胞核基因GUS1解碼而來。在細胞質中cGluRS會將Glu接到相對應的tRNAGlu形成Glu-tRNAGlu,但是最近的研究報告指出cGluRS也會被送到粒腺體中參與Gln-tRNAGln的形成。在粒腺體中cGluRS首先會將Glu接到tRNAGln形成Glu-tNRAGln,再藉由Glu-tNRAGln amidotransferase的轉胺作用形成正確的Gln-tRNAGln。先前,我們實驗室指出,在細胞質中與cGluRS結合的分子Arc1p有粒腺體標的訊號,因此cGluRS有可能是藉由Arc1p而進入粒腺體的,經由點突變的實驗我們發現cGluRS和Arc1p各自攜帶粒腺體標的訊號,不需靠彼此交互作用而進入粒腺體。cGluRS的粒腺體標的訊號位於其胺基端1-29胺基酸。相反地,在酵母菌Schizosaccharomyces pombe及Candida albicans的cGluRS中並沒有發現類似的粒腺體標的訊號。而在Pichia guilliermodii、Candida albicans、Schizosaccharomyces pombe這三株酵母菌的Arc1p也沒有粒腺體標的訊號。


    In Saccharomyces cerevisiae, the cytoplasmic glutamyl-tRNA synthetase (cGluRS) is encoded by GUS1. In cytoplasm, cGluRS attaches Glu to the cognate tRNAGlu to form Glu-tRNAGlu. In addition to the cytoplasmic activity, cGluRS can enter the mitochodria to synthesize Glu-tNRAGln (a misacylated product), which is then converted to correct Gln-tRNAGln by Glu-tNRAGln amidotransferase. A pervious study in our lab indicated that Arc1p, which binds cGluRS in cytoplasm, also has a mitochondrial targeting signal. Our results showed that cGluRS and Arc1p each carry a mitochondrial targeting signal and thus can enter the mitochondria by itself. Next, we used the cytoplasmic form of valyl-tRNA synthetase as the reporter gene to map the mitochondrial targeting signal of cGluRS. As it turned out, this signal was embedded in the N-terminal amino acids 1-29 of cGluRS. In contrast, no mitochondrial targeting signal was found in Schizosaccharomyces pombe or Candida albicans GluRS. Moreover, Arc1p from Pichia guilliermodii, Candida albicans, and Schizosaccharomyces pombe does not possess a mitochondrial targeting signal.

    中文摘要i 英文摘要ii 誌 謝iii 目 錄iv 圖 目 錄vii 縮寫檢索表viii 第一章 緒論1 1.1 Aminoacyl-tRNA synthetases (aaRSs)的簡介1 1.2 原核與真核細胞在轉譯方式上的差異2 1.3 Glutaminyl-tRNA synthetase (GluRS)的簡介4 1.4 非專一性的tRNA 結合蛋白5 1.4.1 Arc1p 5 1.4.2 Ad(ScGluRS)5 1.5 粒腺體標的訊號的特性6 1.6 研究目的6 第二章 材料與方法8 2.1 菌株、載體及培養基8 2.2 大腸桿菌勝任細胞的製備與轉型作用 9 2.2.1 大腸桿菌勝任細胞的製備9 2.2.2 大腸桿菌勝任細胞的轉型作用 (transformation)10 2.3 酵母菌勝任細胞的製備與轉型作用10 2.3.1 酵母菌勝任細胞的製備10 2.3.2 酵母菌勝任細胞的轉型作用11 2.4 質體之建構 11 2.5 點突變 (Site-directed Mutagenesis)12 2.6 功能性互補試驗 (Complementation)―測試細胞質功能13 2.7 功能性互補試驗 (Complementation)―測試粒腺體功能14 2.8 蛋白質製備15 2.9 SDS-PAGE 之蛋白質分子量分析16 2.10 西方點墨法 (Western Blotting) 16 2.11酵母菌雙雜交系統 (Yeast Two-hybrid Assay)17 2.12 In Vitro Pull Down Assay 18 2.13酵母菌融合蛋白質的表現與純化18 第三章 結果 22 3.1 測定Arc1p的粒腺體標的訊號22 3.2利用點突變方法證實Arc1p及ScGluRS的胺基端粒腺體標的訊號之活性23 3.3 利用two-hybrid及pull-down的方法證實Arc1p及ScGluRS胺基端的交互作用24 3.4利用回報基因找出ScGluRS的粒腺體標的訊號25 3.5 鑑定不同物種的GluRS是否具有粒腺體標的訊號25 3.6 鑑定不同物種的Arc1p是否具有粒腺體標的訊號26 第四章 討論 28 4.1 ScGluRS 對於粒腺體功能必要性之探討28 4.2 ScGluRS的粒腺體標的訊號之預測30 第五章 參考文獻 32

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