鑑定酵母菌中具高親和力的tRNA結合蛋白

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姓名

王如玉(Ju-Yu Wang) 查詢紙本館藏

畢業系所

生命科學系

論文名稱

鑑定酵母菌中具高親和力的tRNA結合蛋白
(Identification of high affinity tRNA-binding proteins in yeast.)

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摘要(中)

在真核細胞中，非專一性的tRNA結合蛋白質大致上可分為兩大類：第一類參與細胞內生理功能，例如：Arc1p可促進aminoacyl-tRNA synthetase與其相對應tRNA的親和力，進而提高醯胺化作用的效率；另一類蛋白質則是參與tRNA的相關運輸機制，例如： Utp8p可協助tRNA自細胞核運送到細胞質，然而，此方面的研究仍僅冰山一角。為了探討細胞內其他tRNA結合蛋白質的存在，我使用three-hybrid system對酵母菌的基因庫進行篩選，以tRNAGln當作餌（bait），結果得到了19種群組，再選取其中分別代表族群數目最多及反應強弱的六個群組進行序列分析及比對後發現，其中四個並不屬於任何一個已知的酵母菌基因讀碼框，亦即，在自然的狀態下，這些序列並不會解碼成胜肽；其餘二個群組，則可分別轉譯為活化醣解作用蛋白質GCR2以及一個細胞壁蛋白質Yol155c。此外，我更進一步刪除這六個陽性候選質體多餘的序列，再進行three-hybrid測試後，發現他們仍呈現陽性反應。目前正分別純化這些胜肽片段，以便進一步做活體外試驗，測試這些蛋白質或胜肽片段與tRNA的結合能力，以期進一步了解其在細胞內所扮演的角色。

摘要(英)

There are two kinds of non-specific tRNA-binding proteins in the eukaryotic cell. The first kind of non-specific tRNA-binding protein participates in physiological functions of the cell; an example is Arc1p, which improves the binding affinity between aminoacyl-tRNA synthetases and their cognate tRNAs. The other kind of non-specific tRNA-binding protein plays a part in tRNA export; for example, Utp8p is involved in nuclear tRNA export. However, current knowledge of tRNA-binding proteins is still very limited. Therefore, we chose the three-hybrid screening system (using tRNAGln as bait) to gain insight into other tRNA binding proteins in vivo. After screening a yeast genomic DNA library, the positive candidates were classified into nineteen groups based on the size of the library fragment. Six groups, representing the groups with the most clones or groups with the strongest interaction between bait and prey, were chosen for sequencing. After BLAST searching the sequences against the database, two kinds of results were achieved. In four of the clones, an in-frame open reading frame (ORF) corresponding to the sequence of the library fragment could not be found in the yeast database; in other words, these peptides did not exist. The other clones were successfully matched to in-frame ORFs in the yeast database― one was GCR2, a transcriptional activator of glycolytic genes, and the other was Yol155c, a cell wall protein. After appropriate deletion of unnecessary fragments, we retested the clones by the three-hybrid system and found the library-bait interaction to still be positive. We will purify these library peptides and further test for tRNA binding ability in vitro. These results will give us a more in-depth understanding of what roles they play in vivo.

關鍵字(中)

★ three-hybrid 篩選系統
★ tRNA-結合蛋白
★ 高親和力

關鍵字(英)

★ three-hybrid system
★ tRNA-binding protein
★ high affinity

論文目次

目錄……………………………………………………………..……....I
圖表目錄……………………………………...………….…..……..... Ⅲ
縮寫檢索表……………………………………………………….…....IV
英文摘要……………………………………………………….….…....1
中文摘要……………………………………………………………......3
第一章序論……………………………………………………….….4
第二章材料與方法
l 使用之菌株、載體....………………………………………..…....10
l 大腸桿菌勝任細胞的製備與轉型作用……………………...…...11
l 構築質體………………………………………………..…...…….13
l 小量酵母菌勝任細胞的製備與轉型作用……………..…….…...13
l 酵母菌基因庫的轉型作用及篩選……………......……….……...15
l 抽取酵母菌的質體………………………………………………………..17
l 大腸桿菌融合蛋白質的表現與純化…………….……… ...….…19
l SDS-PAGE之蛋白質分子量分析…………..……...………………23
第三章結果
I. tRNA bait的構築…………………….……….……..….…..……26
II. Three-hybrid的篩選…………………………………......….…..26
III. 序列分析結果……………………………………......…….....…...28
Ⅳ. 尋找tRNA結合區域…………………………………..……...…….29
V. 測試已知的tRNA結合蛋白…………………………..…….……..30
VI. 純化tRNA結合區域…………………………………...……..…....30
第四章討論………………………………………………………...…32
第五章參考文獻……………………………………………….….…36
圖表…………………………………………………….…………….…40
附錄……………………………………………………………………..54

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指導教授

王健家(Chien-Chia Wang)

審核日期

2004-7-16

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