Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/20623
DC FieldValueLanguage
dc.contributor.advisor顏宏真zh_TW
dc.contributor.advisorHongyong Fuen_US
dc.contributor.advisor符宏勇zh_TW
dc.contributor.author謝宏政zh_TW
dc.contributor.authorHsieh, Hungchengen_US
dc.date2000zh_TW
dc.date.accessioned2014-06-06T07:14:05Z-
dc.date.available2014-06-06T07:14:05Z-
dc.identifier.urihttp://hdl.handle.net/11455/20623-
dc.description.abstract26S蛋白解體(proteasome)於真核細胞中之ubiquitin/proteasome pathway中扮演著調控蛋白質分解的重要角色。26S蛋白解體是由20S主體(core particle; CP)及19S調控體(regulatory particle; RP)兩個亞體(subcomplex)所組成。20SCP為主要分解去折疊之蛋白質的部分,而19S RP一般認為是扮演辨識宇元化之蛋白受質、蛋白質去折疊(unfolding)及轉送(translocation)去折疊之蛋白質進入20S CP的角色。為了進一步瞭解19S RP的功能與機制,本實驗以酵母菌雙混雜法(two-hybrid)來分析26S蛋白解體次單元之交互作用的關係,初步推測其三級結構之分佈情形,希望對日後了解26S蛋白解體分解蛋白質的機制有所幫助。 本實驗以酵母菌26S蛋白解體為模式,分離其18個RP次單元基因及7個α次單元基因,分別構築於酵母菌雙混雜載體,於酵母菌系YRG2內分析18個RP次單元之間的交互作用(共有18 ×18個測試組合)及分析7個α次單元與18個RP次單元之間的交互作用(共測試了3 ×7 ×18個可能組合)。RPN10/MCB1(Multiubiquitin Chain Binding Protein)為19S RP中一個次單元,是唯一可在生體外(in vitro)與宇元鏈(ubiquitin chain)結合的次單元。但RPN10減基因突變對酵母菌及Physcomitrella patens(moss)並不會造成致死現象,所以RPN10可能參與其它生理功能。為瞭解RPN10於生體內真正的生理功能及其分子作用機制,同時也利用酵母菌雙混雜法,以P. paterns之RPN10(PpRPN10)篩選由P. patens所製備的雙混雜cDNA基因庫,以篩選及分離與RPN10具有交互作用之基因。 本實驗所測試的19S RP次單元間之測試組合中,成功獲得5個組合具交互作用,其中3組已有文獻記載;另2組則目前並無文獻記載,為新發現之組合。另外本實驗20S CP 的7個α次單元與19S RP 18個次單元的測試組合,在HIS3報導基因的表現上並無正反應。當以P. paterns之RPN10來篩選P. paterns之cDNA基因庫可成功地獲得一NTI1(RPN Ten Interaction Factor)因子基因,此基因蛋白產物經過序列比對,與阿拉伯芥ribosomal protein s29-like的相似度(similarity)為80%、相同(identity)為74%;與人類之ribosomal protein s29的相似度為74%、相同為64%;與酵母菌之ribosomal protein s29的相似度為68%、相同為62%。 19S RP次單元間交互作用之組合是在in vivo下成功獲得的,可以進一步確立其19S RP次單元間在三度空間上的關係;而20S CP 7個α次單元與19S RP 18個次單元的測試組合中並無結果。此結果似乎可以說明當7個α次單元組合成複合體(complex)時,20S CPα次單元與19S RP次單元才有交互作用產生,單獨存在的α次單元無法與19S RP次單元進行交互作用。zh_TW
dc.description.abstractThe 26S proteasome plays a central role in degrading cellular proteins conjugated by ubiquitins. The 26S proteasome complex is composed of sub-complexes: the 20S core particle (CP), contains proteolytic active sites in its lumen and the 19S regulatory particle (RP). The exact functions and mechanisms of the 19S RP are unknown. As a first step towards discovering the precise functions of the 19S RP, we have proposed here to investigate its three dimensional structure, i.e. how the individual subunits are organized, by molecular genetic approaches. To serve this purpose, the two-hybrid system was employed here using the yeast 26S proteasome as a model. We isolated genes encoding all 18 RP and 7 CPα—subunits from yeast and constructed separately the isolated RP and CP subunit genes into the yeast two-hybrid vectors. We examined interaction (18 x 18 combinations) among RP subunits using the established chimeric two-hybrid vectors in the yeast YRG2 strain. Among the 18 x 18 combinations of 19S RP subunits interaction tested, eight of them were found to be positive. Four out of these combinations have been reported elsewhere and other four are novel combinations. We also examined the interactions (3 x 7 x 18 combinations) of the seven CP α—subunits and the eighteen RP subunits. However, no interaction was observed. The lack of one-by-one interaction between CP and RP suggests that more than one subunit of CP and RP is involved in the assembly of the complex 26S proteasome. The RPN10/MCB1 (Multiubiquitin Chain Binding Protein) is an important subunit of 19S RP and is also the only subunit that has affinity for multiubiquitin chains in vitro. The fact that P. patens and yeast Δrpn10 strains were viable indicates that in addition to ubiquitin recognition, RPN10 might have other functions besides ubiquitin/proteasome pathway. The RPN10 of P. patens was used to screen the P. patens cDNA library by employing the two-hybrid system. A factor, NTI1 (RPN Ten Interaction), was obtained. The deduced amino sequence of NTI1 showed 80% homology to the arabidopsis s29 ribosomal protein, 74% homology to the human s29 ribosomal protein, and 68% homology to the yeast s29 ribosomal protein.en_US
dc.description.tableofcontents目 錄 中文摘要…………………………………………………………………….1 英文摘要…………………………………………………………………….2 前言………………………………………………………………………….3 一、ubiquitin/proteasome pathway……………………………………..3 二、ubiquitin/proteasome pathway參與植物代謝生理功能………….6 (一)、光敏素的分解………………………………………………….6 (二)、逆境下蛋白質的turnover……………………………………...8 三、20S core particle (CP)結構與功能………………………………..10 四、19S regulatory particle (RP)結構與功能……………………………12 五、RPN10次單元功能………………………………………………….16 六、實驗主題……………………………………………………………..19 (一)、19S RP次單元之交互作用……………………………………..19 (二)、RPN10的結構與功能分析………………………………………21 1.與19S次單元於間之交互作用…………………………………21 2.篩選其他與RPN10交互作用之因子…………………………...22 材料與方法…………………………………………………………………….23 一、實驗材料……………………………………………………………….23 1.材料………………………………………………………………23 2.酵母菌培養基……………………………………………………23 二、實驗方法……………………………………………………………….24 1.酵母菌染色體組DNA抽取方法……………………………….24 2.分離酵母菌19S RP及20SCP次單元基因…………………….25 3.選殖於中間載體…………………………………………………26 4.大腸桿菌之轉化…………………………………………………27 5.Digestion…………………………………………………………27 6.DNA序列分析…………………………………………………..28 7.酵母菌雙混雜載體……………………………………………..28 8.選殖於雙混雜載體………………………………………………28 9.酵母菌之轉化……………………………………………………29 10. 報導基因的活性分析…………………………………………..30 11. 分離PpRPN10CΔ1…………………………………………….31 12.以pBD::PpRPN10CΔ1篩選由P. patens所建立的雙混雜cDNA基因庫……………………………………………………………32 13.由酵母菌分離雙混雜質體………………………………………32 14.序列分析…………………………………………………………33 結果…………………………………………………………………………….34 一、26S proteasome(蛋白解體)次單元之交互作用分析………….….34 1.整體策略………………………………………………………...34 2.19S RP之18個次單元間的交互作用關係…………………….36 3.20S CP α次單元與19S RP次單元之交互作用……………...37 二、PpRPN10與ribosomal protein s29具交互作用……………………..39 1.以PpRPN10CΔ1 clone為篩選由P. patens所建立的雙混雜cDNA基因庫的材料,並成功獲得13個clone……………40 2.NTI1為ribosomal protein s29………………………………41 討論…………………………………………………………………………….43 一、26S proteasome(蛋白解體)次單元之交互作用…………………...43 二、與RPN10次單元交互作用之因子…………………………………..46 三、辨識交互作用方法…………………………………………………...49 四、酵母菌雙混雜載體…………………………………………………...52 參考文獻……………………………………………………………………….78 圖表及附錄 表一、用來分離酵母菌RP次單元基因之DNA 引子序列………………….54 表二、用來分離酵母菌20S主體α次單元基因之DNA 引子序列……………55 表三、不同之目標基因與其接合構築之載體種類………..…….……………56 表四、利用酵母菌雙混雜法測試19S RP次單元交互作用之Lac Z報導基因活性表現。………………………………………………………….57 表五、利用酵母菌雙混雜法測試20S CPα次單元與19S RP次單元交互作用之Lac Z報導基因活性表現…………………………………….58 圖一、酵母菌雙混雜系統使用之酵母菌雙混雜載體………………………...59 圖二、18個RP次單元基因與Gal4 AD或Gal4 BD序列之融合建構…….60 圖三、7個主體(CP)a次單元基因與Gal4 AD或Gal4 BD序列之融合建構…61 圖四、RP次單元基因之交互作用的測試組合……………………………….62 圖五之一、CP a次單元與RP次單元基因之交互作用的所有可能組合……63 圖五之二、CP a次單元與RP次單元基因之交互作用的所有可能組合……64 圖六、藉由HIS3及Lac Z報導基因的表現分析蛋白質之交互作用………65 圖七、19S次單元與pBD-Gal4 Cam及pAD-Gal4-2.1酵母菌雙混雜載體之羧機端構築對照試驗…………………………………………………66 圖八、7個α次單元分別與pBD-Gal4 Cam及pADCT酵母菌雙混雜載體構築之對照試驗……………………………………………………………67 圖九、RP次單元融合於AD之羧基端與RP次單元融合於BD之羧基端的測試組合………………………………………………………………68 圖十、19S RP次單元交互作用之Lac Z報導基因活性表現……………….69 圖十一、α次單元融合於BD之羧基端與RP次單元融合於AD之羧基端的測試組合………………………………………………………………70 圖十二、α次單元融合於BD之羧基端與RP次單元融合於AD之氨基端的測試組合………………………………………………………………71 圖十三、α次單元融合於AD之氨基端與RP次單元融合於BD之羧基端的測試組合………………………………………………………………72 圖十四、pBD::PpRPN10cΔ1(pBD::Ppmcb1cΔ1)篩選由P. patens所建立的雙混雜cDNA基因庫結果………………………………………….73 圖十五、PpRPN10CΔ1(Ppmcb1CΔ1)分別與NTI1及NTI9交互作用的β-gal比活性表現……………………………………………………..74 圖十六、NTI1之核酸序列及轉譯之氨基酸序列……………………………..75 圖十七、NTI1轉譯氨基酸序列與已知ribosomal protein s29轉譯氨基酸序列比對……………………………………………………………………76 圖十八、19S RP次單元間之交互作用模式圖……………………………….77 附錄一、建構允許目標基因與AD或BD序列形成氨基端融合(N-terminal fusion)之改良式酵母菌雙混雜載體………………………………….91zh_TW
dc.language.isoen_USzh_TW
dc.publisher植物學系zh_TW
dc.subjectproteasomeen_US
dc.subject蛋白解體zh_TW
dc.subjectubiquitinen_US
dc.subjecttwo-hybriden_US
dc.subjectsubuniten_US
dc.subjectinteractionen_US
dc.subjectproteinen_US
dc.subject宇元zh_TW
dc.subject酵母菌雙混雜法zh_TW
dc.subject次單元zh_TW
dc.subject交互作用zh_TW
dc.subject蛋白質zh_TW
dc.title26S蛋白解體次單元之交互作用zh_TW
dc.titleSubunit Interaction of the 26S Proteasomeen_US
dc.typeThesis and Dissertationzh_TW
item.cerifentitytypePublications-
item.openairecristypehttp://purl.org/coar/resource_type/c_18cf-
item.languageiso639-1en_US-
item.fulltextno fulltext-
item.grantfulltextnone-
item.openairetypeThesis and Dissertation-
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