請用此 Handle URI 來引用此文件：
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/19052完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 孫錦虹(Chin-Hung Sun) | |
| dc.contributor.author | Shi-Jun Huang | en |
| dc.contributor.author | 黃實君 | zh_TW |
| dc.date.accessioned | 2021-06-08T01:43:29Z | - |
| dc.date.copyright | 2016-08-26 | |
| dc.date.issued | 2016 | |
| dc.date.submitted | 2016-08-17 | |
| dc.identifier.citation | 1. Lane S, Lloyd D. Current trends in research into the waterborne parasite Giardia. Crit Rev Microbiol 2002; 28(2): 123-47.
2. O'Handley RM, Buret AG, McAllister TA, Jelinski M, Olson ME. Giardiasis in dairy calves: effects of fenbendazole treatment on intestinal structure and function. Int J Parasitol 2001; 31(1): 73-9. 3. Svard SG, Hagblom P, Palm JE. Giardia lamblia -- a model organism for eukaryotic cell differentiation. FEMS Microbiol Lett 2003; 218(1): 3-7. 4. Adam RD. Biology of Giardia lamblia. Clin Microbiol Rev 2001; 14(3): 447-75. 5. Bernander R, Palm JE, Svärd SG. Genome ploidy in different stages of the Giardia lamblia life cycle. Cell Microbiol 2001; 3(1): 55-62. 6. Rendtorff RC. The experimental transmission of human intestinal protozoan parasites. II. Giardia lamblia cysts given in capsules. Am J Hyg 1954; 59(2): 209-20. 7. Farthing MJ. The molecular pathogenesis of giardiasis. J Pediatr Gastroenterol Nutr 1997; 24(1): 79-88. 8. Buret AG. Mechanisms of epithelial dysfunction in giardiasis. Gut 2007; 56(3): 316-7. 9. Ankarklev J, Jerlström-Hultqvist J, Ringqvist E, Troell K, Svärd SG. Behind the smile: cell biology and disease mechanisms of Giardia species. Nat Rev Microbiol 2010; 8(6): 413-22. 10. Tejman-Yarden N, Eckmann L. New approaches to the treatment of giardiasis. Curr Opin Infect Dis 2011; 24(5): 451-6. 11. Gottig N, Elias EV, Quiroga R, Nores MJ, Solari AJ, Touz MC et al. Active and passive mechanisms drive secretory granule biogenesis during differentiation of the intestinal parasite Giardia lamblia. The Journal of biological chemistry 2006; 281(26): 18156-66. 12. Lauwaet T, Davids BJ, Reiner DS, Gillin FD. Encystation of Giardia lamblia: a model for other parasites. Curr Opin Microbiol 2007; 10(6): 554-9. 13. Bittencourt-Silvestre J, Lemgruber L, de Souza W. Encystation process of Giardia lamblia: morphological and regulatory aspects. Arch Microbiol 2010; 192(4): 259-65. 14. Paget TA, Macechko PT, Jarroll EL. Metabolic changes in Giardia intestinalis during differentiation. J Parasitol 1998; 84(2): 222-6. 15. Faghiri Z, Santiago RB, Wu Z, Widmer G. High-throughput screening in suboptimal growth conditions identifies agonists of Giardia lamblia proliferation. Parasitology 2011; 138(2): 194-200. 16. Gillin FD, Boucher SE, Rossi SS, Reiner DS. Giardia lamblia: the roles of bile, lactic acid, and pH in the completion of the life cycle in vitro. Exp Parasitol 1989; 69(2): 164-74. 17. Poxleitner MK, Carpenter ML, Mancuso JJ, Wang C-JR, Dawson SC, Cande WZ. Evidence for karyogamy and exchange of genetic material in the binucleate intestinal parasite Giardia intestinalis. Science 2008; 319(5869): 1530-3. 18. Carpenter ML, Assaf ZJ, Gourguechon S, Cande WZ. Nuclear inheritance and genetic exchange without meiosis in the binucleate parasite Giardia intestinalis. Journal of cell science 2012; 125(Pt 10): 2523-32. 19. Ramesh MA, Malik SB, Logsdon JM, Jr. A phylogenomic inventory of meiotic genes; evidence for sex in Giardia and an early eukaryotic origin of meiosis. Curr Biol 2005; 15(2): 185-91. 20. Malik SB, Pightling AW, Stefaniak LM, Schurko AM, Logsdon JM, Jr. An expanded inventory of conserved meiotic genes provides evidence for sex in Trichomonas vaginalis. PLoS One 2008; 3(8): e2879. 21. Sagolla MS, Dawson SC, Mancuso JJ, Cande WZ. Three-dimensional analysis of mitosis and cytokinesis in the binucleate parasite Giardia intestinalis. Journal of cell science 2006; 119(Pt 23): 4889-900. 22. Jiráková K, Kulda J, Nohýnková E. How nuclei of Giardia pass through cell differentiation: semi-open mitosis followed by nuclear interconnection. Protist 2012; 163(3): 465-79. 23. Cooper MA, Adam RD, Worobey M, Sterling CR. Population genetics provides evidence for recombination in Giardia. Curr Biol 2007; 17(22): 1984-8. 24. Poxleitner MK, Carpenter ML, Mancuso JJ, Wang CJ, Dawson SC, Cande WZ. Evidence for karyogamy and exchange of genetic material in the binucleate intestinal parasite Giardia intestinalis. Science 2008; 319(5869): 1530-3. 25. Shinohara A, Ogawa H, Ogawa T. Rad51 protein involved in repair and recombination in S. cerevisiae is a RecA-like protein. Cell 1992; 69(3): 457-70. 26. Lin Z, Kong H, Nei M, Ma H. Origins and evolution of the recA/RAD51 gene family: evidence for ancient gene duplication and endosymbiotic gene transfer. Proc Natl Acad Sci U S A 2006; 103(27): 10328-33. 27. Lovett ST. Sequence of the RAD55 gene of Saccharomyces cerevisiae: similarity of RAD55 to prokaryotic RecA and other RecA-like proteins. Gene 1994; 142(1): 103-6. 28. Sonoda E, Takata M, Yamashita YM, Morrison C, Takeda S. Homologous DNA recombination in vertebrate cells. Proc Natl Acad Sci U S A 2001; 98(15): 8388-94. 29. Shin DS, Pellegrini L, Daniels DS, Yelent B, Craig L, Bates D et al. Full-length archaeal Rad51 structure and mutants: mechanisms for RAD51 assembly and control by BRCA2. EMBO J 2003; 22(17): 4566-76. 30. Kinebuchi T, Kagawa W, Kurumizaka H, Yokoyama S. Role of the N-terminal domain of the human DMC1 protein in octamer formation and DNA binding. The Journal of biological chemistry 2005; 280(31): 28382-7. 31. Proudfoot C, McCulloch R. Trypanosoma brucei DMC1 does not act in DNA recombination, repair or antigenic variation in bloodstream stage cells. Mol Biochem Parasitol 2006; 145(2): 245-53. 32. Wang TF, Chen LT, Wang AH. Right or left turn? RecA family protein filaments promote homologous recombination through clockwise axial rotation. Bioessays 2008; 30(1): 48-56. 33. Reymer A, Frykholm K, Morimatsu K, Takahashi M, Norden B. Structure of human Rad51 protein filament from molecular modeling and site-specific linear dichroism spectroscopy. Proc Natl Acad Sci U S A 2009; 106(32): 13248-53. 34. Dobson R, Stockdale C, Lapsley C, Wilkes J, McCulloch R. Interactions among Trypanosoma brucei RAD51 paralogues in DNA repair and antigenic variation. Mol Microbiol 2011; 81(2): 434-56. 35. Du L, Luo Y. Structure of a filament of stacked octamers of human DMC1 recombinase. Acta Crystallogr Sect F Struct Biol Cryst Commun 2013; 69(Pt 4): 382-6. 36. Sarai N, Kagawa W, Kinebuchi T, Kagawa A, Tanaka K, Miyagawa K et al. Stimulation of Dmc1-mediated DNA strand exchange by the human Rad54B protein. Nucleic acids research 2006; 34(16): 4429-37. 37. <Posttranslational Modifications of Rad51 Protein and Its Direct Partners.pdf>. 38. Davies AA, Masson JY, McIlwraith MJ, Stasiak AZ, Stasiak A, Venkitaraman AR et al. Role of BRCA2 in control of the RAD51 recombination and DNA repair protein. Mol Cell 2001; 7(2): 273-82. 39. Miller KA, Hinz JM, Yamada NA, Thompson LH, Albala JS. Nuclear localization of Rad51B is independent of Rad51C and BRCA2. Mutagenesis 2005; 20(1): 57-63. 40. Tombline G, Fishel R. Biochemical characterization of the human RAD51 protein. I. ATP hydrolysis. The Journal of biological chemistry 2002; 277(17): 14417-25. 41. Bishop DK, Park D, Xu L, Kleckner N. DMC1: a meiosis-specific yeast homolog of E. coli recA required for recombination, synaptonemal complex formation, and cell cycle progression. Cell 1992; 69(3): 439-56. 42. Kawabata M, Kawabata T, Nishibori M. Role of recA/RAD51 family proteins in mammals. Acta Med Okayama 2005; 59(1): 1-9. 43. Shinohara A, Shinohara M. Roles of RecA homologues Rad51 and Dmc1 during meiotic recombination. Cytogenet Genome Res 2004; 107(3-4): 201-7. 44. Kurzbauer MT, Uanschou C, Chen D, Schlogelhofer P. The recombinases DMC1 and RAD51 are functionally and spatially separated during meiosis in Arabidopsis. Plant Cell 2012; 24(5): 2058-70. 45. Masson JY, West SC. The Rad51 and Dmc1 recombinases: a non-identical twin relationship. Trends Biochem Sci 2001; 26(2): 131-6. 46. Bishop DK. RecA homologs Dmc1 and Rad51 interact to form multiple nuclear complexes prior to meiotic chromosome synapsis. Cell 1994; 79(6): 1081-92. 47. Dresser ME, Ewing DJ, Conrad MN, Dominguez AM, Barstead R, Jiang H et al. DMC1 functions in a Saccharomyces cerevisiae meiotic pathway that is largely independent of the RAD51 pathway. Genetics 1997; 147(2): 533-44. 48. Stassen NY, Logsdon JM, Vora GJ, Offenberg HH, Palmer JD, Zolan ME. Isolation and characterization of rad51 orthologs from Coprinus cinereus and Lycopersicon esculentum, and phylogenetic analysis of eukaryotic recA homologs. Curr Genet 1997; 31(2): 144-57. 49. Sauvageau S, Ploquin M, Masson JY. Exploring the multiple facets of the meiotic recombinase Dmc1. Bioessays 2004; 26(11): 1151-5. 50. Sheridan SD, Yu X, Roth R, Heuser JE, Sehorn MG, Sung P et al. A comparative analysis of Dmc1 and Rad51 nucleoprotein filaments. Nucleic acids research 2008; 36(12): 4057-66. 51. Masson JY, Davies AA, Hajibagheri N, Van Dyck E, Benson FE, Stasiak AZ et al. The meiosis-specific recombinase hDmc1 forms ring structures and interacts with hRad51. EMBO J 1999; 18(22): 6552-60. 52. Chi P, Kwon Y, Moses DN, Seong C, Sehorn MG, Singh AK et al. Functional interactions of meiotic recombination factors Rdh54 and Dmc1. DNA Repair (Amst) 2009; 8(2): 279-84. 53. Habu T, Taki T, West A, Nishimune Y, Morita T. The mouse and human homologs of DMC1, the yeast meiosis-specific homologous recombination gene, have a common unique form of exon-skipped transcript in meiosis. Nucleic acids research 1996; 24(3): 470-7. 54. Liu Y, Gaines WA, Callender T, Busygina V, Oke A, Sung P et al. Down-regulation of Rad51 activity during meiosis in yeast prevents competition with Dmc1 for repair of double-strand breaks. PLoS Genet 2014; 10(1): e1004005. 55. Lim DS, Hasty P. A mutation in mouse rad51 results in an early embryonic lethal that is suppressed by a mutation in p53. Mol Cell Biol 1996; 16(12): 7133-43. 56. Chang HY, Liao CY, Su GC, Lin SW, Wang HW, Chi P. Functional Relationship of ATP Hydrolysis, Presynaptic Filament Stability, and Homologous DNA Pairing Activity of the Human Meiotic Recombinase DMC1. The Journal of biological chemistry 2015. 57. Bugreev DV, Mazin AV. Ca2+ activates human homologous recombination protein Rad51 by modulating its ATPase activity. Proc Natl Acad Sci U S A 2004; 101(27): 9988-93. 58. Chi P, Van Komen S, Sehorn MG, Sigurdsson S, Sung P. Roles of ATP binding and ATP hydrolysis in human Rad51 recombinase function. DNA Repair (Amst) 2006; 5(3): 381-91. 59. Li X, Zhang XP, Solinger JA, Kiianitsa K, Yu X, Egelman EH et al. Rad51 and Rad54 ATPase activities are both required to modulate Rad51-dsDNA filament dynamics. Nucleic acids research 2007; 35(12): 4124-40. 60. Tombline G, Heinen CD, Shim KS, Fishel R. Biochemical characterization of the human RAD51 protein. III. Modulation of DNA binding by adenosine nucleotides. The Journal of biological chemistry 2002; 277(17): 14434-42. 61. Tombline G, Shim KS, Fishel R. Biochemical characterization of the human RAD51 protein. II. Adenosine nucleotide binding and competition. The Journal of biological chemistry 2002; 277(17): 14426-33. 62. Logsdon JM. Evolutionary genetics: sex happens in Giardia. Curr Biol 2008; 18(2): R66-8. 63. Malik S-B, Pightling AW, Stefaniak LM, Schurko AM, Logsdon JM. An expanded inventory of conserved meiotic genes provides evidence for sex in Trichomonas vaginalis. PLoS One 2008; 3(8): e2879. 64. Keister DB. Axenic culture of Giardia lamblia in TYI-S-33 medium supplemented with bile. Trans R Soc Trop Med Hyg 1983; 77(4): 487-8. 65. Chuang S-F, Su L-H, Cho C-C, Pan Y-J, Sun C-H. Functional redundancy of two Pax-like proteins in transcriptional activation of cyst wall protein genes in Giardia lamblia. PLoS One 2012; 7(2): e30614. 66. Symington LS. Role of RAD52 epistasis group genes in homologous recombination and double-strand break repair. Microbiol Mol Biol Rev 2002; 66(4): 630-70, table of contents. 67. Sharma D, Say AF, Ledford LL, Hughes AJ, Sehorn HA, Dwyer DS et al. Role of the conserved lysine within the Walker A motif of human DMC1. DNA Repair (Amst) 2013; 12(1): 53-62. 68. Aihara H, Ito Y, Kurumizaka H, Yokoyama S, Shibata T. The N-terminal domain of the human Rad51 protein binds DNA: structure and a DNA binding surface as revealed by NMR. J Mol Biol 1999; 290(2): 495-504. 69. Egelman EH, Stasiak A. Structure of helical RecA-DNA complexes. Complexes formed in the presence of ATP-gamma-S or ATP. J Mol Biol 1986; 191(4): 677-97. 70. Rivetti C, Guthold M, Bustamante C. Scanning force microscopy of DNA deposited onto mica: equilibration versus kinetic trapping studied by statistical polymer chain analysis. J Mol Biol 1996; 264(5): 919-32. 71. Passy SI, Yu X, Li Z, Radding CM, Masson JY, West SC et al. Human Dmc1 protein binds DNA as an octameric ring. Proc Natl Acad Sci U S A 1999; 96(19): 10684-8. 72. Krejci L, Van Komen S, Li Y, Villemain J, Reddy MS, Klein H et al. DNA helicase Srs2 disrupts the Rad51 presynaptic filament. Nature 2003; 423(6937): 305-9. 73. Veaute X, Jeusset J, Soustelle C, Kowalczykowski SC, Le Cam E, Fabre F. The Srs2 helicase prevents recombination by disrupting Rad51 nucleoprotein filaments. Nature 2003; 423(6937): 309-12. 74. Sehorn MG, Sigurdsson S, Bussen W, Unger VM, Sung P. Human meiotic recombinase Dmc1 promotes ATP-dependent homologous DNA strand exchange. Nature 2004; 429(6990): 433-7. 75. Galkin VE, Esashi F, Yu X, Yang S, West SC, Egelman EH. BRCA2 BRC motifs bind RAD51-DNA filaments. Proc Natl Acad Sci U S A 2005; 102(24): 8537-42. 76. Neale MJ, Keeney S. Clarifying the mechanics of DNA strand exchange in meiotic recombination. Nature 2006; 442(7099): 153-158. 77. Sung P, Klein H. Mechanism of homologous recombination: mediators and helicases take on regulatory functions. Nat Rev Mol Cell Biol 2006; 7(10): 739-750. 78. Matsuo Y, Sakane I, Takizawa Y, Takahashi M, Kurumizaka H. Roles of the human Rad51 L1 and L2 loops in DNA binding. FEBS J 2006; 273(14): 3148-59. 79. Amunugama R, He Y, Willcox S, Forties RA, Shim K-S, Bundschuh R et al. RAD51 protein ATP cap regulates nucleoprotein filament stability. The Journal of biological chemistry 2012; 287(12): 8724-36. 80. Kinebuchi T, Kagawa W, Enomoto R, Tanaka K, Miyagawa K, Shibata T et al. Structural basis for octameric ring formation and DNA interaction of the human homologous-pairing protein Dmc1. Mol Cell 2004; 14(3): 363-74. 81. Lee M-H, Chang Y-C, Hong EL, Grubb J, Chang C-S, Bishop DK et al. Calcium ion promotes yeast Dmc1 activity via formation of long and fine helical filaments with single-stranded DNA. The Journal of biological chemistry 2005; 280(49): 40980-4. 82. Fornander LH, Frykholm K, Reymer A, Renodon-Corniere A, Takahashi M, Norden B. Ca2+ improves organization of single-stranded DNA bases in human Rad51 filament, explaining stimulatory effect on gene recombination. Nucleic acids research 2012; 40(11): 4904-13. 83. Bugreev DV, Golub EI, Stasiak AZ, Stasiak A, Mazin AV. Activation of human meiosis-specific recombinase Dmc1 by Ca2+. The Journal of biological chemistry 2005; 280(29): 26886-95. 84. Lee MH, Chang YC, Hong EL, Grubb J, Chang CS, Bishop DK et al. Calcium ion promotes yeast Dmc1 activity via formation of long and fine helical filaments with single-stranded DNA. The Journal of biological chemistry 2005; 280(49): 40980-4. 85. Cho C-C, Su L-H, Huang Y-C, Pan Y-J, Sun C-H. Regulation of a Myb transcription factor by cyclin-dependent kinase 2 in Giardia lamblia. The Journal of biological chemistry 2012; 287(6): 3733-50. 86. Carranza PG, Lujan HD. New insights regarding the biology of Giardia lamblia. Microbes Infect 2010; 12(1): 71-80. 87. Huang Y-C, Su L-H, Lee GA, Chiu P-W, Cho C-C, Wu J-Y et al. Regulation of cyst wall protein promoters by Myb2 in Giardia lamblia. The Journal of biological chemistry 2008; 283(45): 31021-9. 88. Pan YJ, Cho CC, Kao YY, Sun CH. A novel WRKY-like protein involved in transcriptional activation of cyst wall protein genes in Giardia lamblia. The Journal of biological chemistry 2009; 284(27): 17975-88. 89. Bekker-Jensen S, Mailand N. Assembly and function of DNA double-strand break repair foci in mammalian cells. DNA Repair (Amst) 2010; 9(12): 1219-28. 90. Malewicz M, Perlmann T. Function of transcription factors at DNA lesions in DNA repair. Experimental Cell Research 2014; 329(1): 94-100. 91. Rehrauer WM, Kowalczykowski SC. The DNA binding site(s) of the Escherichia coli RecA protein. The Journal of biological chemistry 1996; 271(20): 11996-2002. 92. VanLoock MS, Yu X, Yang S, Lai AL, Low C, Campbell MJ et al. ATP-mediated conformational changes in the RecA filament. Structure 2003; 11(2): 187-96. 93. Jain SK, Cox MM, Inman RB. On the role of ATP hydrolysis in RecA protein-mediated DNA strand exchange. III. Unidirectional branch migration and extensive hybrid DNA formation. The Journal of biological chemistry 1994; 269(32): 20653-61. 94. Kim PM, Allen C, Wagener BM, Shen Z, Nickoloff JA. Overexpression of human RAD51 and RAD52 reduces double-strand break-induced homologous recombination in mammalian cells. Nucleic acids research 2001; 29(21): 4352-4360. 95. Klein HL. The Consequences of Rad51 Overexpression for Normal and Tumor Cells. DNA repair 2008; 7(5): 686-693. 96. Schild D, Wiese C. Overexpression of RAD51 suppresses recombination defects: a possible mechanism to reverse genomic instability. Nucleic acids research 2010; 38(4): 1061-1070. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/19052 | - |
| dc.description.abstract | 梨形鞭毛蟲(Giardia lamblia)是一種全球性廣泛存在之致病性腸內原蟲寄生蟲，感染途徑為飲用受汙染之水源。其滋養體寄生於小腸，而後隨著腸內環境的改變進行囊體化作用(encystation)形成具有囊壁之感染型 ── 囊體。囊壁是梨形鞭毛蟲在抵禦惡劣環境下生存所需之特殊構造，其主要由蛋白質與多醣構成。梨形鞭毛蟲的生活史有兩種型態，一為擁有兩個核並具有鞭毛運動性之滋養體(trophozoite)以及四個核的成熟囊體(cyst)。在囊體化的過程中，囊體蛋白質(cyst wall protein, cwp)的表現量會增加，而滋養體的雙核在此刻也有機會彼此交換訊息(gene exchange)，進一步的研究指出，有減數分裂基因同源物(meiotic gene homologs)像Dmc1a會參與在雙核之間的同源重組(homologous recombination)過程。
已知在梨形鞭毛蟲的基因庫分析數據中有兩個Dmc1之同源物：Dmc1a及Dmc1b (orf 13104及13346)。推測Dmc1a或Dmc1b或許在梨形鞭毛蟲的囊體化過程扮演不可或缺的角色之一，為了釐清與證實，我們先利用即時定量PCR與西方墨點法分別偵測到Dmc1a的RNA與蛋白質表現量在囊體化時都是上升，而Dmc1b則是在滋養體時表現量上升。而免疫螢光染色發現Dmc1a位於細胞核，而與CWP1共染色結果也確定Dmc1a可促進CWP1基因表現。 我們建立過量表現Dmc1a的細胞株pPDmc1a，發現大量表現Dmc1a時促使CWP1表現與促進生成囊體。我們也建立三個Dmc1a的突變株，針對Dmc1a的ATP酶活性位與N端結構位進行突變，也證實突變位置對Dmc1a促進梨形鞭毛蟲的CWP1蛋白質表現與囊體形成扮演重要的功能。為了與Dmc1a比較，我們同時建立了過量表現的Dmc1b細胞株，結果顯示比起Dmc1a，Dmc1b的過量表現對於促進CWP1蛋白質表現與囊體的形成僅微幅上升。我們推測Dmc1b在滋養體時其可能有未知的調控。另外，有鑑於Dmc1a與DNA的鍵結活性，我們利用染色質免疫沉澱(ChIP)分析發現Dmc1a會與cwp1、cwp3、myb2和wrky的啟動子鍵結，又從免疫沉澱(IP)證實Dmc1a會與囊體化轉錄因子MYB2 、WRKY有交互作用。在梨形鞭毛蟲根據目前實驗結果可推得，Dmc1a參與了囊體化相關基因的調控以促進囊體化進行；比起Dmc1b更顯著的參與囊體化的調控。因此，我們也推測Dmc1a除了參與DNA修復與梨形鞭毛蟲基因多樣性(genome diversity)的增加以外，也與促進囊體化有關。 | zh_TW |
| dc.description.abstract | Giardia lamblia is one of the most common protozoan parasites causing waterborne intestinal infections in humans. G. lamblia have two stages in the life cycle: a flagellated trophozoite with 2 nuclei and an inert cyst with 4 nuclei. During encystation, differentiation from a trophozoite into a cyst, the cyst wall protein (CWPs) is highly synthesized in a concerted manner. Research indicates that the 2 nuclei have chance to exchange the information during the life cycle and the meiotic gene homologs, such as Dmc1a, are involved in mediating homologous recombination between the 2 nuclei. Two genes encoding homologs of Dmc1, Dmc1a and Dmc1b (orf 13104 and 13346), are present in G. lamblia genome. We speculated that the Dmc1a and Dmc1b might play a role in G. lamblia encystation. Using quantitative RT-PCR and Western blots, we found an up-regulation of Dmc1a gene during the encystation stage, but we also found the Dmc1b gene is up-regulated during the trophozoite stage. Immunofluorescence assay also revealed that Dmc1a was localized to the nuclei. We performed the Dmc1a overexpression assay in a stable transfection system and found an induction of the expression of cwp1 gene and cyst formation. Also, we created three Dmc1a mutants, in which mutations located in the ATPase active site and N-terminal domain. We confirmed the ATPase active site and N-terminal domain of Dmc1a are important for cyst formation and CWP1 protein production of G. lamblia. For comparison, we established a Dmc1b overexpression cell line and the result shows a slightly increase but not as significant as Dmc1a. We suggest Dmc1b might have an unknown regulation during trophozoite stage. Furthermore, we found Dmc1a has DNA binding activity. Using chromatin immunoprecipitation assay, we found Dmc1a can bind to the promoters of cwp1, cwp3, myb2 and wrky genes. We also found Dmc1a can interact with encystation transcription factors, MYB2 and WRKY, using immunoprecipitation assay. Taken together, Dmc1a plays much more important role in induction of the cwp genes, which is key to Giardia differentiation into cysts, compared with Dmc1b. Dmc1a might be involved in not only homologous recombination for increasing the diversity of genome of G. lamblia, but also play as transcription factor in induction of Giardia encystation. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T01:43:29Z (GMT). No. of bitstreams: 1 ntu-105-R03445202-1.pdf: 5579643 bytes, checksum: ec0e0e1fb954ceb3f15067d2138cec89 (MD5) Previous issue date: 2016 | en |
| dc.description.tableofcontents | 目錄(CONTENTS)
口試委員會審定書 # 致謝 i 中文摘要 ii ABSTRACT iii 目錄(CONTENTS) iv 圖目錄(LIST OF FIGURES) ix 表目錄(LIST OF TABLES) xi 第一章 前言(Introduction) 1 1.1 梨形鞭毛蟲簡介(Introduction of Giardia lamblia) 1 1.2 梨形鞭毛蟲囊體化(Encystation of Giardia lamblia) 1 1.3 梨形鞭毛蟲中同源的減數分裂特異性基因(homologs of meiosis-specific genes in Giardia lamblia) 2 1.4 recA/Rad51基因家族(recA/Rad51 gene family) 3 1.5 Dmc1 (disrupted meiotic cDNA) 4 1.6 研究動機(Research Motivation) 6 第二章 材料與方法(Materials and Methods) 8 2.1 梨形鞭毛蟲滋養體時期以及囊體化時期之培養(Giardia lamblia culture) 8 2.2 轉殖質體之建構(Plasmid construction) 8 2.2.1 5’Δ5N-pac 8 2.2.2 pPDmc1a 8 2.2.3 pPDmc1a-m1 8 2.2.4 pPDmc1a-m2 9 2.2.5 pPDmc1a-m3 10 2.2.6 pPDmclb 10 2.2.7 13104 5’3’ UM 11 2.3 質體的轉型與萃取(Transformation and extraction) 12 2.3.1 質體的轉型 12 2.3.2 質體的萃取 12 2.4 梨形鞭毛蟲的轉染與選殖(Transfection and Selection) 12 2.5 重組蛋白質之表現與純化(Expression and Purification of Recombinant Dmc1a) 13 2.5.1 蛋白表現量測試 13 2.5.2 重組Dmc1a與重組Dmc1a突變型蛋白質之質體建構 13 2.5.3 Dmc1a與突變株重組蛋白質之表現與純化 14 2.5.4 重組Dmcb1b蛋白質之質體建構 15 2.5.5 Dmc1b重組蛋白質之表現與純化 15 2.6 分子篩管柱層析法(Gel filtration) 16 2.6.1 鎳離子親和性管柱層析法 (Ni-NAT purify) 16 2.6.2 蛋白質濃縮 (Protein concentration) 17 2.6.3 分子篩管柱層析法 (Size exclusion chromatography) 17 2.7 免疫螢光染色(Immunofluorescence assay；IFA) 17 2.8 共同免疫螢光染色(co-IFA) 18 2.9 西方墨點法與Coomassie blue染色(Weatern blot and Coomassie blue stain) 18 2.10 反轉錄聚合酶鍊式反應(RT-PCR) 19 2.11 即時定量反轉錄聚合酶鍊式反應(quantitative RT-PCR；qRT-PCR) 21 2.12 囊體計數(Cyst count) 22 2.13 染色質免疫沉澱 (Chromatia Immunoprecipitation；ChIP) 23 2.14 免疫共沉澱法(Co-immunoprecipitation Assay；Co-IP) 25 2.15 DNA損傷實驗(MMS treatment) 26 2.16 電泳位移實驗(Electrophoretic mobility shift assay, EMSA) 26 2.17 ATP再生分析(ATP regenerating assay) 26 2.18 電顯分析(Electron Microscopy assay) 27 2.19 Dmc1a基因剔除實驗 (Dmc1a gene KO by Cas9 System) 28 第三章 實驗結果(Result) 29 3.1 梨形鞭毛蟲ORF13104胺基酸序列經分析命名為Dmc1a 29 3.1.1 Dmc1a序列分析( Pfam & Protein BLAST) 29 3.1.2 Dmc1a所表現之蛋白質三級結構預測(Swiss-model & PDB) 29 3.1.3 梨形鞭毛蟲蟲體內，是Rad51還是Dmc1? 30 3.1.4 Dmc1a與其他物種Rad51/Dmc1胺基酸序列比對分析 30 3.2 鑑定Dmc1a滋養體時期與囊體化時期之mRNA表現量 31 3.3 建立過量表現Dmc1a蛋白質之梨形鞭毛蟲細胞株 32 3.3.1 梨形鞭毛蟲Dmc1a表現 32 3.3.2 免疫螢光分析Dmc1a-HA與CWP1的分佈 32 3.4 建立過量表現Dmc1a突變蛋白質之梨形鞭毛蟲細胞株 32 3.4.1 建立過量表現Dmc1a-m1蛋白質之梨形鞭毛蟲細胞株 33 3.4.2 建立過量表現Dmc1a-m2蛋白質之梨形鞭毛蟲細胞株 33 3.4.3 建立過量表現Dmc1a-m3蛋白質之梨形鞭毛蟲細胞株 33 3.5 Dmc1a影響囊體的形成 34 3.6 大量表現Dmc1a促使CWP1蛋白質表現量上升 34 3.7 建立過量表現Dmc1b蛋白質之梨形鞭毛蟲細胞株 34 3.7.1 梨形鞭毛蟲Dmc1b表現 34 3.7.2 大量表現Dmc1b對CWP1蛋白質表現量的影響 35 3.7.3 免疫螢光分析Dmc1b-HA與CWP1的分佈 35 3.7.4 Dmc1b影響囊體的形成 35 3.8 利用RT-PCR與qRT-PCR來偵測Dmc1a對基因表現的調控 36 3.8.1 Dmc1a蛋白質會誘導囊體化相關基因的mRNA表現量 36 3.8.2 Dmc1a蛋白質突變株會降低其誘導囊體化相關基因的表現 37 3.9 利用RT-PCR與qRT-PCR來偵測Dmc1b對基因表現的調控 38 3.9.1 滋養體時期，轉染pPDmc1b對囊體化相關基因影響 38 3.9.2 囊體化時期，轉染pPDmc1b對囊體化相關基因影響 39 3.10 Dmc1a蛋白質以八聚體(Octamer)存在梨形鞭毛蟲體內 39 3.11 鑑定Dmc1a蛋白質與DNA結合能力 40 3.12 鑑定Dmc1a之DNA-dependent ATPase水解能力 41 3.13 鑑定Dmc1a與突變株蛋白質ATP水解能力 41 3.14 Dmc1a蛋白質與cwp1、cwp2、cwp3、myb2、wrky基因啟動子結合 42 3.15 Dmc1a與囊體化轉錄因子，MYB2、WRKY交互作用 43 3.16 Dmc1b與囊體化轉錄因子，MYB2交互作用 43 3.17 鑑定Dmc1a與Dmc1b間是否有交叉交互作用 44 3.18 電顯分析(Electron Microscopy assay) 44 第四章 討論(Discussion) 46 4.1 梨形鞭毛蟲Dmc1a蛋白質功能區域分析 46 4.2 大量表現Dmc1a可能參與並促進囊壁蛋白(cwp)基因的轉錄 47 4.3 分析Dmc1a蛋白質能與cwp1、cwp3基因啟動子結合 47 4.4 Dmc1a突變株對囊體化的影響 48 4.5 Dmc1a突變設計 49 4.6 Dmc1a蛋白質對OD280吸光數值低下之問題 49 4.7 ATP結合與ATP水解能力對Dmc1a蛋白質與DNA結合能力的影響 50 4.8 電顯下Dmc1a蛋白質與DNA結合情況 50 4.9 Dmc1b與Dmc1a在梨形鞭毛蟲扮演的角色 50 4.10 DNA損傷下，Dmc1a與Dmc1b表現量與對細胞的影響 51 4.11 Dmc1a KO之細胞株，製備困難 52 附圖(Figures) 53 附表(Table) 99 REFERENCE 100 | |
| dc.language.iso | zh-TW | |
| dc.subject | 梨形鞭毛蟲 | zh_TW |
| dc.subject | 同源重組蛋白 | zh_TW |
| dc.subject | 減數分裂 | zh_TW |
| dc.subject | Giardia lamblia | en |
| dc.subject | meiosis | en |
| dc.subject | Rad51 | en |
| dc.subject | Dmc1 | en |
| dc.title | Dmc1a對調控囊體蛋白基因及對梨形鞭毛蟲分化的影響 | zh_TW |
| dc.title | Dmc1a Impacts the Regulation of Cyst Wall Protein Genes and Differentiation in Giardia lamblia. | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 104-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 李財坤(Tsai-Kun Li),詹迺立(Nei-Li Chan) | |
| dc.subject.keyword | 梨形鞭毛蟲,同源重組蛋白,減數分裂, | zh_TW |
| dc.subject.keyword | Giardia lamblia,meiosis,Rad51,Dmc1, | en |
| dc.relation.page | 109 | |
| dc.identifier.doi | 10.6342/NTU201600413 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2016-08-17 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 微生物學研究所 | zh_TW |
| 顯示於系所單位： | 微生物學科所 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-105-1.pdf 未授權公開取用 | 5.45 MB | Adobe PDF |
系統中的文件，除了特別指名其著作權條款之外，均受到著作權保護，並且保留所有的權利。