Title page for 952204014


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Student Number 952204014
Author Yi-Yi Kuo(郭亦亦)
Author's Email Address No Public.
Statistics This thesis had been viewed 1478 times. Download 529 times.
Department Life Science
Year 2007
Semester 2
Degree Master
Type of Document Master's Thesis
Language zh-TW.Big5 Chinese
Title Translation initiation of HTS1 in yeast
Date of Defense 2008-06-25
Page Count 78
Keyword
  • Histidyl-tRNA synthetase
  • translation initiation
  • Abstract In yeast, there are two sets of aminoacyl-tRNA synthetases, one localized in the cytoplasm and the other in the mitochondria. Most of the mitochondrial tRNA synthetases are encoded by nuclear genes distinct from those encoding their cytoplasmic counterparts. However, some mitochondrial tRNA synthetases are encoded by the same genes that code for their cytoplasmic homologues. For example, the cytoplasmic and mitochondrial histidyl-tRNA synthetases of Saccharomyces cerevisiae (ScHisRS) are encoded by the same nuclear gene, HTS1, through alternative initiation of translation from two in-frame AUG codons. The gene specifies two messages, the longer one with two 5’-end in-frame AUGs and the short one with only the second AUG. In this study, we showed that the HisRS genes of Schizosaccharomyces pombe、Candida albicans and Aspergillus fumigatus have only one gene that also contain two in-frame AUG initiation codons. The mitochodrial and cytosolic forms are translated from the first and second AUG initiation codons, respectively. Besides, we found that ScHisRS had a lysine-rich N-terminal polypeptide extension of 34 residues, which was absent from its E. coli counterpart. Attachment of this appended domain to E. coli glutaminyl-tRNA synthetase did not enhance the enzyme’s tRNA-binding and aminoacylation activity toward yeast tRNAs. Deletion of the appended domain of ScHisRS had little effect on the enzyme’s complementation activity in vivo. These results suggest that the appended domain is not essential for the cytoplasmic and mitochondrial functions of ScHisRS in vivo.
    Table of Content 目錄
    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
    1.1.1 aaRSs的生化功能1
    1.1.2 aaRSs 的分類2
    1.2 原核與真核細胞在蛋白質合成轉譯方式的差異3
    1.2.1 原核與真核細胞內蛋白質的合成3
    1.2.2少數真核細胞aaRS只有一個細胞核基因4
    1.3 Histidyl-tRNA synthetase(HisRS)的簡介5
    1.3.1 HisRS的生化功能5
    1.3.2 HTS1基因的表現6
    1.4酵母菌aaRS的附加區段7
    1.5研究目的8
    第二章 材料與方法9
    2.1使用之菌株、載體及培養基9
    2.2大腸桿菌勝任細胞的製備與轉型作用10
    2.2.1大腸桿菌勝任細胞的製備10
    2.2.2大腸桿菌勝任細胞的轉型作用(Transformation)11
    2.3酵母菌勝任細胞的製備與轉型作用11
    2.3.1酵母菌勝任細胞的製備11
    2.3.2酵母菌勝任細胞的轉型作用(Transformation)12
    2.4質體之選殖12
    2.5點突變(Site-directed Mutagenesis)13
    2.6 hts1剔除株的製備14
    2.7功能性互補試驗(Complementation):測試細胞質功能16
    2.8功能性互補試驗(Complementation):測試粒腺體功能17
    2.9蛋白質製備18
    2.10 SDS-PAGE之蛋白質分子量分析19
    2.11西方點墨法(western blotting)20
    2.12酵母菌融合蛋白質的表現與純化21
    第三章 結果24
    3.1酵母菌HisRS蛋白質序列的比對24
    3.2不同物種間的互補性實驗25
    3.3 ScHTS1基因的表現26
    3.4利用互補性實驗測試SpHTS1與CaHTS1轉譯起始密碼27
    3.5鑑定AfHTS1轉譯起始密碼30
    3.6 ScHisRS的附加區段是否具鍵結tRNA的能力32
    3.7 ScHisRS附加區段對其細胞質與粒腺體酵素的重要性33
    第四章 討論35
    4.1酵母菌HTS1基因的表現35
    4.2 ScHisRS附加區段的特性37
    第五章 參考文獻39
    附錄59

    圖目錄
    圖一、選擇性轉錄及轉譯機制43
    圖二、酵母菌HisRS蛋白質序列的比對44
    圖三、不同物種間的互補性實驗45
    圖四、利用互補試驗分析野生型及突變種ScHisRS的生長狀態46
    圖五、利用互補實驗以低複製載體測試ScHisRS的生長狀態47
    圖六、SpHTS1的基因序列48
    圖七、利用互補試驗分析野生型及突變種SpHisRS的生長狀態49
    圖八、CaHTS1的基因序列50
    圖九、利用互補試驗分析野生型及突變種CaHisRS的生長狀態51
    圖十、AfHTS1的基因序列52
    圖十一、利用互補性實驗鑑定AfHTS1轉譯起始的密碼53
    圖十二、酵母菌及大腸桿菌 HisRS 的比較54
    圖十三、利用E. coli GlnRS的融合蛋白進行互補性試驗55
    圖十四、利用E. coli GlnRS的融合蛋白進行互補性試驗56
    圖十五、刪除ScHisRS附加區段對細胞質和粒腺體功能的影響57
    圖十六、在高複製載體下利用互補實驗測試刪除ScHisRS附加區段對其功能的影響58
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    Advisor
  • Chien-Chia Wang(王健家)
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    Date of Submission 2008-07-02

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