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研究生:曾品維
研究生(外文):Pin-Wei Tseng
論文名稱:大腸桿菌Thioredoxin活性區域突變株對噬菌體感染之影響
論文名稱(外文):Effects of active site Mutations of Escherichia coli Thioredoxin on Bacteriophage Infection
指導教授:林苕吟
指導教授(外文):Tiao-Yin Lin
學位類別:碩士
校院名稱:國立交通大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:法文
論文頁數:106
中文關鍵詞:噬菌體
外文關鍵詞:Thioredoxinphage T7
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  • 被引用被引用:2
  • 點閱點閱:121
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中文摘要
Thioredoxin是一個小型且普遍存在於細菌至所有動植物的細胞的熱穩定性蛋白質。在E.coli中參與生理上的氧化還原反應,促進去氧核糖核酸的形成,也可幫助T7、f1、M13的生長。在噬菌體T7中,thioredoxin為T7 DNA polymerase的次單元;在線形噬菌體f1、M13參與噬菌體組裝的過程。本實驗中,我們在thioredoxin W31、G33、P34、C35、K36、M37位置建構了19株E.coli thioredoxin突變株,分析它們對噬菌體T3/T7、f1、M13、T7生長的影響。
由本實驗的各E.coli thioredoxin突變株的感染平盤效率(E.O.P值)得知P34D、P34E、P34K、C35A在37℃及42℃時嚴重抑制f1、M13、T3/T7的生長。另外K36R在42℃時,會促進f1、M13的生長。由T3/T7、f1、M13的實驗數據比較得知W31、P34、C35、K36應該是thioredoxin蛋白與f1 gene I蛋白交互作用時重要的接觸點。
i
Effects of active site Mutations of Escherichia coli Thioredoxin on Bacteriophage Infection
Student:Pin-Wei Tseng Advisor: Dr. Tiao-Yin Lin
Institute of Biological Science and Technology National Chiao Tung University
Abstract
Thioredoxin is a small heat stable protein that exists generally in bacteria to all animal and plant cells. In E.coli, it participates in physiologically oxidation-reduction reaction to form deoxyribonucleic acid as well as supports f1、M13 and T3/T7 growth. In phage T7, thioredoxin is a T7 DNA polymerase subunit. For phage f1 and M13, it participates in the assembly of filamentous phage.
In this study, we used site-directed mutagenesis to construct thioredoxin mutants to analyze the effect on f1, M13 and T3/T7 growth. Thioredoxin mutants P34D、P34E、P34K and C35A would suppress seriously f1, M13 and T3/T7 growth at 37℃ and 42℃. Thioredoxin mutant K36R could help f1 and M13 growth at 42℃. By comparing experimental data of f1, M13 and T3/T7, we found residues 31,34,35,36 of thioredoxin could be the contact positions that thioredoxin interacts with f1 geneI protein.
ii
一、緒論----------------------------------------------------------------------------1
1.1 Thioredoxin簡介--------------------------------1
1.2大腸桿菌Thioredoxin----------------------------1
1.3噬菌體T7與T3/T7的簡介------------------------ 2
1.4Thioredoxin與噬菌體T7的關係-------------------3
1.5線形噬菌體f1與M13的簡介----------------------3
1.6線形噬菌體f1與M13與thioredoxin的關係--------4
1.7實驗目的---------------------------------5
二、實驗材料-------------------------------------------------6
2.1菌株-----------------------------------------------------------------6
2.2質體----------------------------------------------------------------7
2.3引導子-------------------------------------------------------------8
2.4噬菌體-------------------------------------------------------------9
2.5培養基---------------------------------------------------9
2.6溶液及緩衝液------------------------------------------11
2.7分析材料-----------------------------------------------------------------12
三、實驗方法-----------------------------------------------13
3.1定點突變---------------------------------------------------------13
3.2 Thioredoxin蛋白表現-----------------------------------------18
3.3噬菌體感染作用------------------------------------------------18
四、實驗結果----------------------------------------------23
4.1定點突變-------------------------------------23
4.2 Thioredoxin基因表現-------------------26
4.3噬菌體感染作用--------------------------31
五、討論----------------------------------47
六、結論-------------------------------------------------------------53
七、未來展望與方向---------------------------------------------54
八、參考文獻-------------------------------------------------63
八、參考文獻:
1. Holmegren, A. (1985) Thioredoxin. Ann. Rev. Biochem. Annual Reviews Ins. 54:237-271
2. Holmegren, A. (1979) Reduction of disulfides by thioredoxin:Exceptional reactivity of insulin and suggested functions of thioredoxin in mechanism of hormone action. J.Biol. Chem. 254:9113-9119
3. Holmegren, A., Krause, G. (1991) Substitution of the conserved tryptophan 31 in thioredoxin by site-directed mutagenesis and structure-function analysis. J.Biol. Chem. 266:4056-4066
4. Laurent, T. C., Moore, E.C., & Richard, P.(1964) Enzymatic synthesis of deoxyribonucleotides. IV. Isolation and characterization of thioredoxin, the hydrogen donor from Escherichia coli B. J. Biol. Chem. 239:3436-3444
5. Gonzalez Porque P., Baldesten, A.,& Reichard, P.(1970) Purification of a thioredoxin system from yeast. J .Biol. Chem. 245:2371-2374
6. Russel, M., Model,P.,& Holmegren, A. (1990) Thioredoxin or gluyaredoxin in Escherichia coli is essential for sulfate reduction but not for deoxyribonucletide sythesis
7. Clarke, F. M., Orozco, C., & Perkins, A. V. (1991) Identification of molecules involved in the ‘early pregnancy factor’ phenomenon. J. Reprod. Fert. 93,525-539
8. Wolosiuk, R. A., Buchanan, B. B. (1997) Thioredoxin and glutathione regulate photosynthesis in chloroplasts. Nature 266:565-567
9. Holmgren, A., Ohlsson, I., & Grankvist, M.-L. (1978) Thioredoxin from Escherichia coli radioimmunological and enzymatic determinations in wild type cells and mutants defective in phage T7 DNA replication. J . Biol. Chem. 253:430-436
10. Holmgren, A. (1976) Hydrogen donor system for Escherichia coli ribonucleoside-diphosphate reductase dependent upon glutathione. Proc. Natl. Acad. Sci. U.S.A. 73:2275-2279
11. The Bacteriophage (Volume 1): 259-289 Edited by Richard Calendar
12. Chamberlin, M. (1974). Isolation and characterization of prototrophic mutants of Escherichia coli unable to support the intracellular growth of T7. J. Virol.14: 509-16
63
13. Modrich, P., and C. C. Richardson. (1975). Bacteriophage T7 deoxyribonucleic acid replication in virto: Bacteriophage T7 DNA polymerase: an enzyme composed of phage and host-specified subunits. J . Biol. Chem. 250:5515-5522
14. Modrich, P., and C. C. Richardson. (1975). Bacteriophage T7 deoxyribonucleic acid replication in virto: a protein of Escherichia coli required for bacteriophage T7 DNA polymerase activity. J . Biol. Chem. 250:5508-5514
15. Marvin, D.A., Hohn, B. (1969) Filamentous bacterial viruses. Bacteriol. Rev. 33:172-209
16. Hill, D. F.,and Petersen G.B. (1982) Nucleotide sequence of bacteriophage f1 DNA. J. Virol. 44(1): 32-46
17. Russel, M. (1991) Filamentous phage assembly. Molecular Microbiology 5(7): 1607-1613
18. Russel, M., Model, P. (1984) characterization of the cloned fip gene and its product. J. Bacterial. 157:526-532
19. Lim, C.J., Haller, B., Fuchs, J.A. (1985) Thioredoxin is the bacterial protein encoded by fip that is required for filamentous bacteriophage f1 assembly. J. Bacterial. 161:799-802
20. Russel, M., Model, P. (1986)The role of thioredoxin in filamentous phage assembly. J. Biol. Chem. 261:14997-15005
21. Guy-Caffey, J.K., Rapoza,M.P., Jolley, K. A.,and Webste, R.E.(1992) Membrane localization and topology of a viral assembly protein. J. Bacterial. 174:2460-2465
22. Spence, J.L., Mooney, P.O., Molineux I. J. (1983) Physiological properties of a T7-T3 recombinant bacteriophage that productively infects strains of Escherichia coli that harbor the F plasmid. J. Virol. 46:895-900
23. Bedford E, Tabor S, Richardson CC. (1997) The thioredoxin binding domain of bacteriophage T7 DNA polymerase confers processivity on Escherichia coli DNA polymerase I. Proc Natl Acad Sci U S A. 94:479-84
24. Himawan, J. S., and Richardson, C. C. (1996) Amino Acid Residues Critical for the Interaction between Bacteriophage T7 DNA Polymerase and Escherichia coli Thioredoxin. J. Biol. Chem. 271: 19999-20008
25. Alder, S., Modrich, P. (1983) T7-induced DNA polymerase. Requirement for
thioredoxin sulfhydryl groups. J. Biol. Chem.250:6956-6962
26. Studier, F.W. (1991) Use of bacteriophage T7 lysozyme to improve an inducible T7 expression system. J. Mol. Biol. 219:37-44
64
27. Lunn, C.A., Kathju, S., Wallace, B.J., Kushner, S.R., and Pigiet, V. (1984) Aplification and purificationof plasmid-encoded thioredoxin from Escherichia coli K12. J. Biol. Chem.256: 10469-10474
28. Russel, M., Model, P. (1984) Replacement of the fip gene of Escherichia coli by an inactive gene cloned on a plasmid. J.Bacterial.159: 1034-1039
29. Kato M, Frick DN, Lee J, Tabor S, Richardson CC, Ellenberger T. (2001) A complex of the bacteriophage T7 primase-helicase and DNA polymerase directs primer utilization. J Biol Chem.276: 21809-20
30. Lee J, Chastain PD 2nd, Griffith JD, Richardson CC. (2002) Lagging strand synthesis in coordinated DNA synthesis by bacteriophage t7 replication proteins. J Mol Biol. 316: 19-34
31. Hollis T, Stattel JM, Walther DS, Richardson CC, Ellenberger T. (2001) Structure of the gene 2.5 protein, a single-stranded DNA binding protein encoded by bacteriophage T7. Proc Natl Acad Sci U S A. 98: 9557-62
32. Kim YT, Richardson CC. (1994) Acidic carboxyl-terminal domain of gene 2.5 protein of bacteriophage T7 is essential for protein-protein interactions. : J Biol Chem 269: 5270-8
33. Kong D, Richardson CC. (1998) Role of the acidic carboxyl-terminal domain of the single-stranded DNA-binding protein of bacteriophage T7 in specific protein-protein interactions. J Biol Chem 273: 6556-64
34. Huber, H. E., Russel, M., Model, P., Richardson, C.C. (1986) Interaction of mutant thioredoxin of Escherichia coli with the gene 5 protein of phage T7. The redox capacity of thioredoxin is not required for stimulation of DNA polymerase activity. J. Biol. Chem. 261:15006-15012
35. 林燕慈 (2000) 大腸桿菌Thioredoxin C32、P34與G92點突變對噬菌體感染之分析 交通大學生物科技研究所碩士論文
36. Tabor, S., Huber, H. E. & Richardson, C. C. (1987) Escherichia coli thioredoxin confers processivity on the DNA polymerase activity of the gene 5 protein of bacteriophage T7. J. Biol. Chem. 262: 16212-16223
37. Yang, X. -M. & Richardson, C. C. (1996) Structural and Functional Organization of the DNA Polymerase of Bacteriophage T7. J. Biol. Chem.271: 24207-24212
38. Russel M. (1994) Phage assembly: a paradigm for bacterial virulence factor export. Science, Jul, 265:5172,612-4
65
39. Russel M; Linderoth NA; Sali A. (1997) Filamentous phage assembly: variation on a protein export theme. Gene. 192:1,23-32
40. Kumar JK, Kremsdorf R, Tabor S, Richardson CC. (2001) A Mutation in the gene-encoding bacteriophage T7 DNA polymerase that renders the phage temperature-sensitive. J Biol Chem 276(49):46151-9
41. Kumar JK, Tabor S, Richardson CC. (2001) Role of the C-terminal residue of the DNA polymerase of bacteriophage T7. J Biol Chem 276(37):34905-12
42. 廖鈞芃(1999) 大腸桿菌Thioredoxin表現量不同與質體f1複製原點對噬菌體感染之影響 交通大學生物科技研究所碩士論文
43. 陳藤賢(1999) 大腸桿菌Thioredoxin G33與G74點突變對噬菌體感染之分析 交通大學生物科技研究所碩士論文
1. Holmegren, A. (1985) Thioredoxin. Ann. Rev. Biochem. Annual Reviews Ins. 54:237-271
2. Holmegren, A. (1979) Reduction of disulfides by thioredoxin:Exceptional reactivity of insulin and suggested functions of thioredoxin in mechanism of hormone action. J.Biol. Chem. 254:9113-9119
3. Holmegren, A., Krause, G. (1991) Substitution of the conserved tryptophan 31 in thioredoxin by site-directed mutagenesis and structure-function analysis. J.Biol. Chem. 266:4056-4066
4. Laurent, T. C., Moore, E.C., & Richard, P.(1964) Enzymatic synthesis of deoxyribonucleotides. IV. Isolation and characterization of thioredoxin, the hydrogen donor from Escherichia coli B. J. Biol. Chem. 239:3436-3444
5. Gonzalez Porque P., Baldesten, A.,& Reichard, P.(1970) Purification of a thioredoxin system from yeast. J .Biol. Chem. 245:2371-2374
6. Russel, M., Model,P.,& Holmegren, A. (1990) Thioredoxin or gluyaredoxin in Escherichia coli is essential for sulfate reduction but not for deoxyribonucletide sythesis
7. Clarke, F. M., Orozco, C., & Perkins, A. V. (1991) Identification of molecules involved in the ‘early pregnancy factor’ phenomenon. J. Reprod. Fert. 93,525-539
8. Wolosiuk, R. A., Buchanan, B. B. (1997) Thioredoxin and glutathione regulate photosynthesis in chloroplasts. Nature 266:565-567
9. Holmgren, A., Ohlsson, I., & Grankvist, M.-L. (1978) Thioredoxin from Escherichia coli radioimmunological and enzymatic determinations in wild type cells and mutants defective in phage T7 DNA replication. J . Biol. Chem. 253:430-436
10. Holmgren, A. (1976) Hydrogen donor system for Escherichia coli ribonucleoside-diphosphate reductase dependent upon glutathione. Proc. Natl. Acad. Sci. U.S.A. 73:2275-2279
11. The Bacteriophage (Volume 1): 259-289 Edited by Richard Calendar
12. Chamberlin, M. (1974). Isolation and characterization of prototrophic mutants of Escherichia coli unable to support the intracellular growth of T7. J. Virol.14: 509-16
13. Modrich, P., and C. C. Richardson. (1975). Bacteriophage T7 deoxyribonucleic acid replication in virto: Bacteriophage T7 DNA polymerase: an enzyme composed of phage and host-specified subunits. J . Biol. Chem. 250:5515-5522
14. Modrich, P., and C. C. Richardson. (1975). Bacteriophage T7 deoxyribonucleic acid replication in virto: a protein of Escherichia coli required for bacteriophage T7 DNA polymerase activity. J . Biol. Chem. 250:5508-5514
15. Marvin, D.A., Hohn, B. (1969) Filamentous bacterial viruses. Bacteriol. Rev. 33:172-209
16. Hill, D. F.,and Petersen G.B. (1982) Nucleotide sequence of bacteriophage f1 DNA. J. Virol. 44(1): 32-46
17. Russel, M. (1991) Filamentous phage assembly. Molecular Microbiology 5(7): 1607-1613
18. Russel, M., Model, P. (1984) characterization of the cloned fip gene and its product. J. Bacterial. 157:526-532
19. Lim, C.J., Haller, B., Fuchs, J.A. (1985) Thioredoxin is the bacterial protein encoded by fip that is required for filamentous bacteriophage f1 assembly. J. Bacterial. 161:799-802
20. Russel, M., Model, P. (1986)The role of thioredoxin in filamentous phage assembly. J. Biol. Chem. 261:14997-15005
21. Guy-Caffey, J.K., Rapoza,M.P., Jolley, K. A.,and Webste, R.E.(1992) Membrane localization and topology of a viral assembly protein. J. Bacterial. 174:2460-2465
22. Spence, J.L., Mooney, P.O., Molineux I. J. (1983) Physiological properties of a T7-T3 recombinant bacteriophage that productively infects strains of Escherichia coli that harbor the F plasmid. J. Virol. 46:895-900
23. Bedford E, Tabor S, Richardson CC. (1997) The thioredoxin binding domain of bacteriophage T7 DNA polymerase confers processivity on Escherichia coli DNA polymerase I. Proc Natl Acad Sci U S A. 94:479-84
24. Himawan, J. S., and Richardson, C. C. (1996) Amino Acid Residues Critical for the Interaction between Bacteriophage T7 DNA Polymerase and Escherichia coli Thioredoxin. J. Biol. Chem. 271: 19999-20008
25. Alder, S., Modrich, P. (1983) T7-induced DNA polymerase. Requirement for thioredoxin sulfhydryl groups. J. Biol. Chem.250:6956-6962
26. Studier, F.W. (1991) Use of bacteriophage T7 lysozyme to improve an inducible T7 expression system. J. Mol. Biol. 219:37-44
27. Lunn, C.A., Kathju, S., Wallace, B.J., Kushner, S.R., and Pigiet, V. (1984) Aplification and purificationof plasmid-encoded thioredoxin from Escherichia coli K12. J. Biol. Chem.256: 10469-10474
28. Russel, M., Model, P. (1984) Replacement of the fip gene of Escherichia coli by an inactive gene cloned on a plasmid. J.Bacterial.159: 1034-1039
29. Kato M, Frick DN, Lee J, Tabor S, Richardson CC, Ellenberger T. (2001) A complex of the bacteriophage T7 primase-helicase and DNA polymerase directs primer utilization. J Biol Chem.276: 21809-20
30. Lee J, Chastain PD 2nd, Griffith JD, Richardson CC. (2002) Lagging strand synthesis in coordinated DNA synthesis by bacteriophage t7 replication proteins. J Mol Biol. 316: 19-34
31. Hollis T, Stattel JM, Walther DS, Richardson CC, Ellenberger T. (2001) Structure of the gene 2.5 protein, a single-stranded DNA binding protein encoded by bacteriophage T7. Proc Natl Acad Sci U S A. 98: 9557-62
32. Kim YT, Richardson CC. (1994) Acidic carboxyl-terminal domain of gene 2.5 protein of bacteriophage T7 is essential for protein-protein interactions. : J Biol Chem 269: 5270-8
33. Kong D, Richardson CC. (1998) Role of the acidic carboxyl-terminal domain of the single-stranded DNA-binding protein of bacteriophage T7 in specific protein-protein interactions. J Biol Chem 273: 6556-64
34. Huber, H. E., Russel, M., Model, P., Richardson, C.C. (1986) Interaction of mutant thioredoxin of Escherichia coli with the gene 5 protein of phage T7. The redox capacity of thioredoxin is not required for stimulation of DNA polymerase activity. J. Biol. Chem. 261:15006-15012
林燕慈 (2000) 大腸桿菌Thioredoxin C32、P34與G92點突變對噬菌體感染之分析 交通大學生物科技研究所碩士論文
36. Tabor, S., Huber, H. E. & Richardson, C. C. (1987) Escherichia coli thioredoxin confers processivity on the DNA polymerase activity of the gene 5 protein of bacteriophage T7. J. Biol. Chem. 262: 16212-16223
37. Yang, X. -M. & Richardson, C. C. (1996) Structural and Functional Organization of the DNA Polymerase of Bacteriophage T7. J. Biol. Chem.271: 24207-24212
38. Russel M. (1994) Phage assembly: a paradigm for bacterial virulence factor export. Science, Jul, 265:5172,612-4
39. Russel M; Linderoth NA; Sali A. (1997) Filamentous phage assembly: variation on a protein export theme. Gene. 192:1,23-32
40. Kumar JK, Kremsdorf R, Tabor S, Richardson CC. (2001) A Mutation in the gene-encoding bacteriophage T7 DNA polymerase that renders the phage temperature-sensitive. J Biol Chem 276(49):46151-9
41. Kumar JK, Tabor S, Richardson CC. (2001) Role of the C-terminal residue of the DNA polymerase of bacteriophage T7. J Biol Chem 276(37):34905-12
42. 廖鈞芃(1999) 大腸桿菌Thioredoxin表現量不同與質體f1複製原點對噬菌體感染之影響 交通大學生物科技研究所碩士論文
43. 陳藤賢(1999) 大腸桿菌Thioredoxin G33與G74點突變對噬菌體感染之分析 交通大學生物科技研究所碩士論文
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