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研究生:張敏麗
研究生(外文):Millie Chang
論文名稱:分離細胞溶質內蛋白質褶合機構失效的酵母菌突變種
論文名稱(外文):Isolation of yeast mutants affecting protein folding in the cytosol
指導教授:鄭明媛
指導教授(外文):Cheng, Ming-Yuan
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:遺傳學研究所
學門:生命科學學門
學類:生物訊息學類
論文種類:學術論文
論文出版年:1994
畢業學年度:82
語文別:中文
中文關鍵詞:遺傳蛋白質酵母菌
外文關鍵詞:HEREDITY
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  • 被引用被引用:1
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原核生物及粒腺體的褶合機制已經較徹底研究。根據粒腺體中HSp70、HSp60之研究,推測不同的分子監護子─HSp70、HSp60對新生多月太在轉譯╱轉位甚至褶合過程中可能扮演連續作用的角色。於真核生物的細胞溶質中,是否有相似的機制,則尚未有深入研究。現有的證據僅知HSp70會與轉譯中的核醣體和新生多月太結合;及細胞溶質的TRiC與α-,β-微管素 (α-,β-tubulin),肌動蛋白(actin)褶合╱組合過程有關。至於TRiC如何參與褶合反應;HSp70族是否與TRiC合作,目前還不清楚。因此真核生物的細胞溶質蛋白質褶合機構尚末完全確認。
在此,我們以遺傳篩選及生化分析,希望分離出細胞溶質蛋白質褶合機構失效的酵母菌突變種。先前篩選37℃無法存活的突變種,同時以β-半乳糖甘酉每低活性作為細胞溶質褶合機構失效的指標;另以正常活性的粒腺體OTCaSe刪除轉譯、轉錄過程失效的突變種。共得到52個突變種,其中6個經四分體分析得知其37℃死亡和β-半乳糖甘酉每失效不呈同步分配。
此次篩選條件除了37℃死亡,β-半乳糖甘酉每失效外,另增加利用嘌呤重新合成步驟中,AIR羧化酉每(ade2基因產物)上游的酵素群為標誌,進行紅白菌落測試。此次篩選實驗一共獲得14個突變種,其主要特性為:37℃時,無法存活;β-半乳糖甘酉每失效及無腺嘌呤呈白色菌落。其中10個皆屬於單一溫度敏感致死突變。同時其溫度敏感致死性和白色菌落呈同步分配。而且至少產生兩個互補群:CAS1,CAS2(Cytosolic assembly species)。其溫度敏感致死性和β-半乳糖甘酉每失效分配呈高度再現性,因此很可能為細胞溶質蛋白質褶合機構失效的酵母菌突變種。
The folding machinery in prokaryotes and in mitochondria has been well-studied. According to the study ofHsp70 and Hsp60 in mitochondria, it has been suggested that Hsp70 and Hsp60 act sequentially during translation/translocation and folding of newly- synthesized polypeptides. It is unclear whether a similar pathway ofchaperoned protein folding exist in the eukaryotic cytosol. Current evidence only indicates that Hsp70 associated with nascent polypeptides and translating ribosomes. In addition, cytosolic protein complex (TRiC) may involve in the folding of α-, β- tubulin and actin. How TRiC involved in the general folding reaction in the cytosol and whether it cooperates with Hsp70 family in folding reaction remain to be elucidated.
Thus, we decided to use genetic and biochemical approaches in order to isolate the yeast mutants defective in the protein folding in the cytosol. From previous screening, 52 mutants were isolated. Their major characters included temperature-sensitive lethal, no activity of β-galactosidase and normal activity of transformed human omithine transcarbarnoylase (OTCase) at non-permissive temperature. The cytosolic β-galactosidase was used as an indicator of the protein folding machinery in the cytosol. The mitochondrial OTCase was a reporter of the abilities of transcription and translation. The six mutants were analyzed. Their loss of β-galactosidase activity and temperature-sensitive lethal did not cosegregate with each other.
In the revised screening scheme, the red/white colony test resulting from activities of the upstream enzymes of AIR carboxylase(ade2 gene product) in de novo biosynthesis of purine is used as an additional criterion. From this screening, 14 mutants whose major characters included temperature-sensitive lethal, low β-galactosidase activities and no adenine, white colonies at 37℃ were isolated. The cosegregation of temperature sensitive lethal and white colony were found in the 10 mutants of above-mentioned mutants. They belong to at least two complementation groups:
CAS1 and CAS2 (cytosolic assembly species). CAS1 and CAS2 are considered as potential mutants defective in protein folding machinery in the cytosol.
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