臺灣博碩士論文加值系統

於Esherichia coli具有一組以操作子 (operon) 型式存在的熱休克基因 (heat-shock genes) clpQY，其中ClpQ為peptidase，分子量為19kDa，與廣泛分布於真核生物中之20S proteasome中的β-type subunit具有同源性；而ClpY為chaperone-like ATPase，分子量為49kDa，與E.coli中的另一個ATPase ClpA具有50% 的相同度。ClpQ和ClpY兩者協同作用而成一組ATP-dependent protease。直至目前，對clpQ和clpY的研究多侷限於E. coli中，並以in vitro的報告為主。因此，我們想進一步了解clpQ和clpY基因於細菌中分布的情形，及蛋白的功能是否與E. coli的ClpQ和ClpY相同。故而，我們依據Bergey’s Manual of Systematic Bacteriology的分類找出19個菌株，以南方氏及西方氏雜交分析clpQ和clpY分布的情形，我們發現，於腸內道菌科中普遍存在與E. coli的clpQ和clpY具保守性的同源性基因。我們藉由基因選殖的方式選殖出與E. coli同為腸內道菌科的Salmonella typhimurium及已知具有clpQ及clpY基因的格蘭氏陽性菌Bacillus subtilis的clpQ及clpY基因，並於E. coli中過量表現，試圖由此了解ClpQY這組ATP-dependent protease於格蘭氏陰性菌和陽性菌間功能上的差異。研究結果顯示，B. subtilis的ClpQY蛋白於本系統中不具分解蛋白酶功能，但其ClpQ卻可與E. coli的ClpY協同作用分解SulA及RcsA。從選殖S.typhimurium clpQ及clpY基因的表現結果發現，其ClpQ及ClpY於本系統中可協同作用作用分解SulA及RcsA，並且ClpY蛋白C端的11個胺基酸對ClpY蛋白的功能具有影響性。

The clpQ and clpY genes were identified as an operon in Esherichia coli. The ClpQ is a 19 kDa peptidase and is a homolog of theβ-type subunit of eukaryptic 20S proteasome. ClpY is a 49 kDa chaperone-like ATPase similar to another E.coli ATPase ClpA (50% similarity). ClpQ together with ClpY form a founctional ATP-dependent protease. Most studies of the ClpQY protease were in E. coli and focused on the invitro analysis. In our study, we investigated the scattering of clpQ and clpY genes in different bacteria. We also examined their proteolytic function compared with that of the ClpQY from E. coli. In accordance with the classification of Bergey’s Manual of Systematic Bacteriology, the southern and the western blot analyses were used to identify the homologs of clpQ /ClpQ and clpY/ClpY in different bacteria. We found out that the E. coli ClpQ and ClpY homologs are conserved in Enterobacteriaceae family. To understand the functional difference of the ATP-dependent ClpQY protease between Gram’s negative and positive bacteria, we separately cloned clpQ and clpY genes from Salmonella typhimurium (Enterobacteriaceae bacteria) and Bacillus subtilis (genome sequence was published). Our data revealed that ClpQY of B. subtilis couldn’t complement the E. coli ClpQY activities, but the ClpQ of B. subtilis could interact with the ClpY of E. coli to degrade RcsA or SulA. An overexpression of the S. typhimurium clpQ+clpY+ genes from the multicopy plasmids could complement the E. coli ClpQY activities. We also demonstrated that the last 11 amino acid residues at the C-terminii of the ClpY were functionally essential.

目 錄
壹、前言
頁次
一、簡介 ………………………………………………………… 1
二、ClpQY為依賴ATP蛋白酶之研究 …………………………… 3
三、ClpQY蛋白複合體的結構 ………………………………… 6
四、有關ClpQY protease基質辨識區域的研究 ……………… 8
五、有關ClpQY protease分解之基質的研究 ………………… 8
六、ClpQY蛋白的晶體繞射結果 ……………………………… 11
七、Salmonella typhimurium 中之ClpY同源蛋白的報告 … 12
八、Bacillus subtilis中Clp family蛋白酶的簡介 ……… 13
九、實驗目的及源起 ………………………………………… 13
貳、材料與方法
一、實驗材料 …………………………………………….… 15
二、方法
1.一般性的DNA實驗方法 ……………..……………………… 17
1.1少量萃取細菌染色體DNA …………………………… . 17
1.2少量萃取質體DNA …………………………………………. 20
2.南方式雜交方法 …………………………………………… 21
2.1核酸探針的製備 …………………………………………. 21
2.2南方氏漬膜之製備 ………………………………………… 23
2.3 放射性標定核酸探針 …………………………………… 25
2.4 雜交反應 ………………………………………………… 27
3.選殖基因之表現系統的建立 ……………………………… 29
3.1 選殖基因片段的製備 …………………………………… 29
3.2 載體的製備 ……………………………….……………… 31
3.3 選殖片段的處理 …………………………………………. 33
3.4 接合反應 ………………………………………………… 33
3.5 轉殖及選殖質體的篩選 ………………………………… 34
3.6 將選殖質體轉殖至宿主細胞 …………………………… 34
4.蛋白質膠體電泳分析 ……………………………………… 35
5.西方氏雜交方法 …………………………………………… 39
5.1 蛋白質轉印 ……………………...……………………… 39
5.2 雜交反應 ………………………………………………… 41
5.3 雜交訊號的偵測 ………………………………………… 42
6.ClpQY protease於胞內大量表現的特性分析方法 ……… 43
6.1β-galactosidase 活性分析 …………………………… 43
6.2 MMS的抗性分析 ………………………………………… 45
參、結果
一、細菌中clpQY同源基因的研究結果 ………………… 47
二、clpQY基因的選殖及蛋白的表現 ………………………… 49
三、B. subtilis及S. typhimurium ClpQY蛋白的功能確認 51
四、B. subtilis及S. typhimurium之ClpQ同源蛋白與
E. coli ClpY蛋白協同作用之結果 …………………… 52
五、S. typhimurium完整基因的選殖及其功能確認 ……… 54
六、B. subtilis及S. typhimurium與E. coli clpQY基因上
游序列比對之結果 ……………………………………… 56
肆、討論
一、細菌中clpQY基因之存在情形 ………………………….. 57
二、選殖質體pBS211及pST28蛋白的表現結果之探討 ……. 59
三、選殖S. typhimurium clpQY基因之質體pST28無法
取代E. coli ClpQY蛋白功能的原因探討 ……………… 59
四、選殖B. subtils clpQY基因之質體pBS211無法
取代E. coli ClpQY蛋白功能的原因探討 ……………… 60
五、B. subtilis及S. typhimurium之ClpQ蛋白可與
E. coli ClpY蛋白協同作用的探討 ………………………… 61
六、選殖基因片段上游啟動子的探討 ……………………… 62
伍、結論 ……………………………………………………… 63
表 次
頁次
表一、 本實驗所使用之菌株及其資料 ………………………….… 70
表二、 本實驗所使用之菌株及質體 …………………….………… 71
表三、 本實驗所使用的聚合酶連鎖反應引子 ……………………. 72
表四、 各菌種之 clpY基因對E. coli clpY 基因比對結果 …… 73
表五、 各菌種之clpQ基因對E. coli clpQ 基因比對結果 ………. 74
表六、 利用β—galactosidase報告基因分析B. subtilis ClpQY
分解RcsA之結果 ………………………………………….. 75
表七、 利用β—galactosidase報告基因分析S. tyhimurium LT2
ClpQY分解RcsA之結果 …………………………………. 76
表八、 B. subtilis clpQ+clpY+對MMS抗性的實驗結果 …….… 77
表九、 S. typhimurium LT2 clpQ+clpY+對MMS抗性的實驗結果 . 78
表十、 利用β—galactosidase報告基因分析B. subtilis ClpQ與
E. coli ClpY協同分解RcsA之結果 ………………………. 79
表十一、利用β—galactosidase報告基因分析S. typhimurium
LT2 ClpQ與E. coli ClpY協同分解RcsA之結果 ………… 80
表十二、B. subtilis ClpQ與E. coli ClpY協同作用對
MMS抗性的實驗結果 …………………………..……….…. 81
表十三、S. typhimurium LT2 ClpQ與E. coli ClpY協同作用對
MMS抗性的實驗結果 …………………………………….. 82
表十四、利用β—galactosidase報告基因分析S. typhimurium
LT2 ClpQY分解RcsA之結果 ………………………….…. 83
表十五、S. typhimurium LT2 clpQ+clpY+對MMS抗性的實驗結果 … 84
表十六、南方氏雜交結果 …………………………………………….. 85
圖 次
頁次
圖一、 染色體DNA經過Bgl I截切後以clpY1100為
核酸探針的南方氏雜交結果 ……………………………… 86
圖二、 染色體DNA經過Pvu II截切後以clpY1100為
核酸探針的南方氏雜交結果 ……………………………… 87
圖三、 染色體DNA經過Bgl I截切後以clpQ300為
核酸探針的南方氏雜交結果 ……………………………… 88
圖四、 染色體DNA經過Pvu II截切後以clpQ300為
核酸探針的南方氏雜交結果 ……………………………… 89
圖五、 利用E. coli ClpY的多株抗體之西方氏雜交結果 ………. 90
圖六、 利用E. coli ClpQ的多株抗體之西方氏雜交結果 ………..91
圖七、 選殖質體pBS211之B. subtilis clpQY的DNA
及胺基酸序列 ……………………………………………… 92
圖八、 選殖質體pST28之S. typhimurium LT2 clpQY之
DNA及胺基酸序列 ……………………………………….. 95
圖九、 表現質體 pBS211、pBS212、pST28 and pST29
之建構 …………………………………………………….. 98
圖十、 選殖基因之蛋白表現的SDS-PAGE蛋白質電泳分析 ….. 99
圖十一、選殖基因於不同誘導條件下蛋白質之表現結果 …….…. 100
圖十二、選殖質體pBS211於不同誘導條件下蛋白質之
表現結果 …………………………………………………. 101
圖十三、 B. subtilis clpQ及clpY基因經PCR後之片段的構造 .…102
圖十四、 S. typhimurium LT2 clpQ 及 clpY 基因經PCR後之
片段的構造 ………………………………………………...103
圖十五、選殖質體pSTQ及pBSQ的蛋白質表現 …………………. 104
圖十六、選殖質體pST513之S. typhimurium LT2的
DNA及胺基酸序列 ……………………………………….. 105
圖十七、S. typhimurium LT2之clpQY基因的完整
DNA及胺基酸序列 ………………………………………. 108
圖十八、表現質體pST513的建構 ……………………………….… 111
圖十九、ClpY同源蛋白之C端胺基酸序列 ………………………. 112

莊榮輝, 蘇仲卿. 1987. 蛋白質膠體電泳檢法. 電泳分離技術研討會論文集.9: 69-85
Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Simth, K. Sturhl, eds. 1987. Current Protocol in Molecular Biology. John Wilwy. N. Y.
Bochlter, M., C. Hartmann, H. K. Song, G. P. Bourenkov, H. D. Bartunik, and R. Huber. 2000. The structures of HslU and the ATP-dependent protease HslU-HslV. Nature 403: 800-805.
Bochtler, M., L. Ditzel, M. Groll, and R. Huber. 1997. Crystal structure of heat shock locus V (HslV) from Escherichia coli. Proc. Natl. Acad. Sci. USA. 94: 6070-6074.
Bukau, B. 1993. Regulation of the Escherichia coli heat shock response. Mol. Microbial. 9: 671-680.
Chuang , S. E., V. Burland, G. Plunkett III, D. L. Daniels, and R. R. Blattner. 1993. Sequence analysis of four new heat-shock genes constituting the hslTS/ibpAB and hslVU operons in Escherichia coli. Gene 134:1-6.
Cowing, D.W., J. C. A. Bardwell, E. A. Craing, C. Woolford, R. W. Hendrix, and C. A. Gross. 1985. Concensus sequence for Escherichia coli heat shock gene promoters. Proc. Natl. Acad. Sci. USA 82: 2679-2683.
Dahlmann, B., F. Kopp, L. Kuehn, B. Niedel, G. Pfeifer, R. Hegerl, and W. Baumeister. 1989. The multicatalytic proteinase (proteasome) is ubiquitous from eukaryotes to archaebacteria. FEBS Lett. 251: 125-131.
Dahlmann, B., L. Kuehn, A. Grziwa, P. Zwickl, and W. Baumeister. 1992. Biochemical properties of the proteasome from Thermoplasma acidophilum. Eur. J. Biochem. 208: 789-797.
Gerth, U., E. Kruger, I. Derre, T. Msadek, and M. Hecker. 1998. Stress induction of the Bacillus subtilis clpP gene encoding the proteolytic component of the ClpP protease and involvement of ClpP and ClpX in stress tolerance. Mol. Microbiol. 28: 787-802.
Gottesman, S. and M. R. Maurizi. 1992. Regulation by proteases and their targets. Microbial. Rev. 56: 592-621.
Gottesman, S., W. F. Wu, and D. Missiakas. 1997. ClpY. In Gething, M.-J. and Sambrook, J (eds), Guidebook to Molecular Chaperones and Protein Folding Catalysis. Oxfold University Press, Oxford, United Kingdom.
Gottesman, S., W. P. Clark, V. de Crecy-Lagard, and M.R. Maurizi. 1993. ClpX, an alternative subunit for the ATP-dependent Clp protease of Escherichia coli. J. Biol. Chem. 268: 22618-22626.
Gross, C. A. 1996. Function and regulation of the heat shock proteins. In Neidhardt, F. C (ed.), Escherichia coli and Salmonella : Cellular and Molecular Biology. 2nd edn .American Society for Microbiology, Washington, D. C.
Guzman, L. M., D. Belin, M. J. Carson, and J. Beckwith. 1995. Tight regulation, modulation, and high-level expression by vector containing the arabinose PBAD promoter. J. Bacteriol. 177: 4121-4130.
Kanemori, M., H. Yanaji, and T. Yura. 1999. The ATP-dependent HslVU/ClpQY protease participates in turnover of cell division inhibitor SulA in Escherichia coli. J. Bacteriol. 181: 3674-3680.
Katayama, T., T. Kubota, M. Takata, N. Akimitsu, and K. Sekimizu. 1996. Disruption of the hslU gene, which encodes an ATPase subunit of the eukaryotic 26S proteasome homolog in Escherichia coli, suppresses the temperature-sensitive dnaA46 mutation. Biochim. Biophys. Research Communications 229: 219-224.
Kessel, M., W. F. Wu, S. Gottesman, E. Kocsis, A. C. Steven, and M. R. Maurizi. 1996. Six-fold rotational symmetry of ClpQ, the E.coli homology of 20S proteasome, and its ATP-dependent activator, ClpY. FEBS Lett. 398: 274-278.
Kunst, F., N. Ogasawara, I. Moszer, A. M. Albertini, G. Alloni, V. Azevedo, M. G. Bertero, P. Bessieres, A. Bolotin, S. Borchert, R. Borriss, L. Boursier, A. Brans, M. Braun, S. C. Brignell, S. Bron, S. Brouillet, C. V. Bruschi, B. Caldwell, V. Capuano, N. M. Carter, S. K. Choi, J. J. Codani, I. F. Connerton, A. Danchin, et. al. 1997. The complete genome sequence of the gram-positive bacterium Bacillus subtilis. Nature 390: 249-256.
Kruger, E., E. Witt, S. Ohlmeier, R. Hanschke, and M. Hecker. 2000. The Clp proteases of Bacillus subtilis are directly involved in degradation of misfolded proteins. J.Bacteriol.182: 3259-3265.
Levchenko, I., C. K. Smith, N. P. Walsh, R. T. Sauer, and T. A. Baker. 1997. PDZ-like domains mediate binding specificity in the Clp/Hsp100 family of chaperones and protease regulatory subunits. Cell 91: 939-947.
Medhat M. Khattar. 1997. Overexpression of the hslVU operon suppresses SOS-
mediated inhibition of cell division in Escherichia coli. FEBS Lett. 414: 402-404.
Miller, J. H. 1992. A short course in bacterial genetics. Cold Spring Harbor Laboratory Press, N. Y. 72-74.
Missiakas, D., F. Schwager, J. M. Betton, C. Georgopoulos, and S. Raina. 1996. Identification and Characterization of HslV and HslU (ClpQClpY) proteins involved in overall proteolysis of misfolded proteins in Escherichia coli. EMBO J. 15: 6899-6909.
Missiakas, D., S. Raina, and C. Georgopoulos. 1996. The heat shock system. In Lin, E. C. C. and Lynch, S. A. (eds), Regulation of Gene Expression in Escherichia coli. Landes Company, Austin, TX.
Misuzawa, S. and S. Gottesman. 1983. Protein degradation in Escherichia coli: the lon gene controls the stability of the SulA protein. Proc. Natl. Acad. Sci. USA 80: 358-362.
Msadek, T., V. Dartois, F. Kunst, M. L. Herbaud, F. Denizot, and G. Rapoport. 1998. ClpP of Bacillus subtilis is required for competence development, degradative enzyme synthesis, motility, growth at high temperature and sporulation. Mol. Microbiol. 27: 899-914.
Raina, S. and C. Georgopoulos. 1990. A new Escherichia coli heat shock gene , htrC, whose product is essential for viability only at hight temperatures. J. Bacteriol. 172 : 3417-3426.
Rohrwild, M., O. Coux, H. C. Huang, R. P. Moerschell, S. J. Yoo, J. H. Seol, C. H. Chung, and A. L. Goldberg. 1996. HslV-HslU: a novel ATP-dependent protease complex in Escherichia coli related to the eukaryotic proteasome. Proc. Natl. Acad. Sci. USA 98: 5808-5813.
Rohrwild, M., G. Pfeifer, U. Santarius, S. A. Muller, H. C. Huang, A Engel, W. Baumeister, and A. L. Goldberg. 1997. The ATP-dependent HslVU protease from Escherichia coli is a four-ring structure resembling the proteasome. Natural Structure Biology 4: 133-139.
Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y.
Seemuler, E., A. Lupas, D. Stock, J. Lowe, R. Huber, and W. Baumeister. 1995. Proteasome from Thermoplasma acidophilum : a threonine protease. Science 268: 579-582.
Shin, D. H., S. J. Yoo, Y. K. Shim, J. H. Seol, M. S. Kang, and C. H. Chung. 1996. Mutational analysis of the ATP-binding site in HslU, the ATPase component of HslVU protease in Escherichia coli. FEBS Lett. 398: 151-154.
Smith, C.K., T. A. Baker, and R. T. Sauer. 1999. Lon and Clp family proteases and chaperones share homologous substrate-recognition domains. Proc. Natl. Acad. Sci. USA 96: 6678-6682.
Valdivia H. R. and S. Falkow. 1997. Fluorescence-based isolation of bacterial gene expressed within host cells. Science. 277: 2007-2011.
Wawrzynow, A., D. Wojtkowiak, J. Marszalek, B. Banecki, M. Jonsen, B. Graves, C. Georgopoulos, and M. Zylicz. 1995. The ClpX heat shock protein of Escherichia coli, the ATP-dependent substrate specificity component of the ClpP/ClpX protease, is a novel molecular chaperone. EMBO J. 14: 1867-1877.
Wu, W. F., Y. N Zhou, and S. Gottesman. 1999. Redundant in vivo proteolytic activities of Escherichia coli Lon and the ClpYQ (HslUV) protease. J. Bacteriol. 181: 3681-3687.
Yoo, S. J., J. H. Seol, D. H. Shin, M. Rohrwild, M. S. Kang, K. Tanaka, A. L. Goldberg, and C. H. Chung. 1996. Purification and characterization of the heat shock proteins HslV and HslU that form a new ATP-dependent protease in Escherichia coli . J. Biol. Chem. 271: 14035-14040
Yura, T., H. Nagai, and H. Mori. 1993. Regulation of the heat shock response in bacteria. Annu. Rev. Microbiol. 47 : 321-350.