臺灣博碩士論文加值系統

阿米巴式趨化運動在許多生理及病理過程中，例如胚胎發育、發炎反應、及癌症轉移，扮演樞紐的角色。由於實驗操控容易、而且其趨化機轉十分類似高等動物細胞，粘菌這個簡單真核生物提供了一個研究趨化運動的絕佳模型系統。
本論文研究利用一以限制脢幫助質體插入基因體的致突變技術建立了一個粘菌突變株庫，並且發展出一個增集及篩尋趨化運動突變株的有效流程。
我們由對一個趨化及發育均異常的粘菌突變株中分析鍾找到了一個極可能和蛋白質醣化有關的新基因large（lrgA）。此lrgA基因產物預測大小約37 kD，其蛋白質C端具有轉醣脢催化區域的相似蛋白序列、且這個區域和人類腦膜瘤中常見到有突變刪除的LARGE基因之產物有49%這麼高的相似性。在lrgA-的突變株中，細胞無法有效地進行趨化運動，且只能發育形成非常小的子實體。北方墨點分析顯示，lrgA基因表現會隨發育時間而有增加的情形。這些結果暗示 LrgA蛋白在趨化運動及發育的調控上扮演著重要角色。
在粘菌中也發現其它兩個LrgA的相關蛋白，LrgB及LrgC。這群在不同物種中發現的同源蛋白顯然界定了一個新的轉醣脢基因家族。透過更進一步對各LARGE基因家族成員的研究，當可擴展我們對醣化作用在趨化運動及多細胞生物發育調控中所扮演角色的暸解。

Amoeboid chemotaxis plays a pivotal role in many physiological or pathological processes, including embryogenesis, inflammation, and tumor metastasis. The simple eukaryote Dictyostelium provides an excellent model system for chemotaxis studies due to its experimental accessibility and remarkable resemblance to higher eukaryotic cells in chemotactic machinery.
In this thesis research, the restriction enzyme-mediated integration (REMI) technique was employed to generate an insertional mutant library of Dictyostelium. A scheme to enrich and screen for chemotaxis mutants from this mutant library was established and proven efficient.
We have identified, from a chemotaxis and developmental mutant, a novel gene, large (lrgA), that is likely involved in protein glycosylation in Dictyostelium discoideum. The lrgA gene encodes for a predicted 37 kD protein with a C-terminal glycosyltransferase catalytic domain and 49% homology with the huamn Large gene product, whose deletion is frequently associated with meningioma. In lrgA- mutant, cells displayed a tiny fruiting body phenotype and were unable to perform chemotaxis efficiently. Northern analysis revealed that the expression of the lrgA gene is regulated during development. These results implicate that lrgA may play an important role in controlling cell chemotaxis and development.
Other LrgA-related proteins, LrgB and LrgC, were also discovered in Dictyostelium by sequence homology. The group of homologous proteins found across diverse species clearly defines a new glycosyltransferase gene family. Further characterization of the LARGE family member genes may expand our understanding of the roles of glycosylation in regulating chemotaxis and multicellular development.

目錄 …………………………i
附表附圖目錄 …………………………ii
縮寫表 …………………………iv
中文摘要 …………………………1
英文摘要 …………………………2
緒論 …………………………3
實驗材料 …………………………10
實驗方法 …………………………14
實驗結果 …………………………24
實驗討論 …………………………31
參考文獻 …………………………34
附表附圖 …………………………42
附錄 …………………………72

1. Downey GP. (1994). “Mechanisms of leukocyte motility and chemotaxis.” Curr Opin Immunol 6: 113-124.
2. Pierce GF, Mustoe TA, Altrock BW, Deuel TF, and Thomason A. (1991). “Role of platelet-derived growth factor in wound healing.” J Cell Biochem 45: 319-326.
3. Forsberg N K, Behar TN, Afrakhte M, Barker JL, and McKay RD. (1998). “Platelet-derived growth factor induces chemotaxis of neuroepithelial stem cells.” J Neurosci Res 53: 521-530.
4. Muller A, Homey B., Soto H, Ge N, Catron D, Buchanan ME, McClanahan T, Murphy E, Yuan W, Wagner SN, Barrera JL, Mohar A, Verastegui E, and Zlotnik A. (2001). “Involvement of chemokine receptors in breast cancer metastasis.” Nature 410: 50-56.
5. Parent CA, and Devreotes PN. (1996). “Molecular genetics of signal transduction in Dictyostelium.” Ann Rev Biochem 65: 411-440.
6. Howard PK, Ahern KG, and Firtel RA. (1988). “Establishment of a transient expression system for Dictyostelium discoideum.” Nucleic Acids Res 16: 2613-2623.
7. Manstein DJ, Titus MA, De Lozanne A, and Spudich JA. (1989). “Gene replacement in Dictyostelium: generation of myosin null mutants.” EMBO J 8: 923-932.
8. Williams KL, and Newell PC. (1976). “A genetic study of aggregation in the cellular slime mould Dictyostelium discoideum using complementation analysis.” Genetics 82: 287-307.
9. Pi M, Sato T, Takemoto K, Yasukawa H, Williams J, Maeda M, Takeuchi I, Ochiai H, and Tanaka Y. (1998). “The Dictyostelium developmental cDNA project: generation and analysis of expressed sequence tags from the first-finger stage of development.” DNA Res 5: 335-340.
10. Kay RR, and Williams JG. (1999). “The Dictyostelium genome project: an invitation to species hopping.” Trends Genet 15: 294-297.
11. Devreotes PN, and Zigmond SH. (1988). “Chemotaxis in eukaryotic cells: a focus on leukocytes and Dictyostelium.” Ann Rev Cell Biol 4: 649-686.
12. Caterina MJ, and Devreotes PN. (1991). “Molecular insights into eukaryotic chemotaxis.” FASEB J 5: 3078-85.
13. Chen MY, Insall RH, Devreotes PN. (1996). “Signaling through chemoattractant receptors in Dictyostelium.” Trends Genet 12: 52-57.
14. van Es S, and Devreotes PN. (1999). “Molecular basis of localized responses during chemotaxis in amoebae and leukocytes.” Cell Mol Life Sci 55: 1341-1351.
15. Aubry L, and Firtel R. (1999). “Integration of signaling networks that regulate Dictyostelium differentiation.” Annu Rev Cell Dev Biol 15: 469-517.
16. Miline JL, Kim JY, and Devreotes PN (1997). Chemoattractant receptor signaling: G protein-dependent and -independent pathways. Adv Sec Mess Phosph Res. 1997: 83-104.
17. Xiao Z, Zhang N, Murphy DB, and Devreotes PN. (1997). “Dynamic distribution of chemoattractant receptors in living cells during chemotaxis and persistent stimulation.” J Cell Biol 139: 365-374.
18. Buczynski G, Grove B, Nomura A, Kleve M, Bush J, Firtel RA, and Cardelli J. (1997). “Inactivation of two Dictyostelium discoideum genes, DdPIK1 and DdPIK2, encoding proteins related to mammalian phosphatidylinositide 3-kinases, results in defects in endocytosis, lysosome to postlysosome transport, and actin cytoskeleton organization.” J Cell Biol 136: 1271-1286.
19. Zhou K, Pandol S, Bokoch G, and Traynor-Kaplan AE. (1998). “Disruption of Dictyostelium PI3K genes reduces [32P] phosphatidylinositol 3,4 bisphosphate and [32P] phosphatidylinositol trisphosphate levels, alters F-actin distribution and impairs pinocytosis.” J Cell Sci 111: 283-294.
20. Funamoto S, Milan K, Meili R, and Firtel RA. (2001). “Role of phosphatidylinositol 3' kinase and a downstream pleckstrin homology domain-containing protein in controlling chemotaxis in Dictyostelium.” J Cell Biol 153: 795-810.
21. Meili R, Ellsworth C, Lee S, Reddy TB, Ma H, and Firtel RA. (1999). “Chemoattractant- mediated transient activation and membrane localization of Akt/PKB is required for efficient chemotaxis to cAMP in Dictyostelium.” EMBO J 15: 2092-2105.
22. Parent CA, Blacklock BJ, Froehlich WM, Murphy DB, and Devreotes PN. (1998). “G protein signaling events are activated at the leading edge of chemotactic cells.” Cell 95: 81-91.
23. Insall RH, Borleis J, and Devreotes PN. (1996). “The Aimless Ras GEF is required for processing of chemotactic signals through G-protein-coupled receptors in Dictyostelium.” Curr Biol 6: 719-729.
24. Zigmond SH, Joyce M, Borleis J, Bokoch GM, and Devreotes PN. (1997). “Regulation of actin polymerization in cell-free systems by GTPgammaS and Cdc42.” J Cell Biol 138: 363-374.
25. Hall A. (1998). “Rho GTPases and the actin cytoskeleton.” Science 279: 509-514.
26. Chung CY, Lee S, Briscoe C, Ellsworth C, and Firtel RA. (2000). “Role of Rac in controlling the actin cytoskeleton and chemotaxis in motile cells.” Proc Natl Acad Sci USA 97: 5225-30.
27. Nobes CD, and Hall A. (1995). “Rho, Rac, and Cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia.” Cell 81: 53-62.
28. Noegel AA, and Schleicher M. (2000). “The actin cytoskeleton of Dictyostelium: a story told by mutants.” J of Cell Sci 113: 759-766.
29. Faix J, Clougherty C, Konzok A, Mintert U, Murphy J, Albrecht R, Muhlbauer B, and Kuhlmann J. (1998). “The IQGAP-related protein DGAP1 interacts with Rac and is involved in the modulation of the F-actin cytoskeleton and control of cell motility.” J Cell Sci 111: 3059-3971.
30. Clow PA, and McNally JG. (1999). “In vivo observations of myosin II dynamics support a role in rear retraction.” Mol Biol Cel 10: 1309-1923.
31. van Haastert PJ, and Kuwayama H. (1997). “cGMP as second messenger during Dictyostelium chemotaxis.” FEBS Lett 410: 25-28.
32. Silveira LA, Smith JL, Tan JL, and Spudich JA. (1998). “MLCK-A, an unconven-tional myosin light chain kinase from Dictyostelium, is activated by a cGMP-dependent pathway.” Proc Natl Acad Sci USA 95: 13000-13005.
33. Dembinsky A, Rubin H, and Ravid S. (1996). “Chemoattractant-mediated increases in cGMP induce changes in Dictyostelium myosin II heavy chain-specific protein kinase C activities.” J Cell Biol 134: 911-921.
34. Ma H, Gamper M, Parent C, and Firtel RA. (1997). “The Dictyostelium MAP kinase kinase DdMEK1 regulates chemotaxis and is essential for chemoattractant-mediated activation of guanylyl cyclase.” EMBO J 16: 4317-4332.
35. Machesky LM, Mullins RD, Higgs HN, Kaiser DA, Blanchoin L, May RC, Hall ME, and Pollard TD. (1999). “Scar, a WASp-related protein, activates nucleation of actin filaments by the Arp2/3 complex.” Proc Natl Acad Sci USA 96: 3739-3744.
36. Machesky LM, and Insall RH. (1998). “Scar1 and the related Wiskott-Aldrich syndrome protein, WASP, regulate the actin cytoskeleton through the Arp2/3 complex.” Curr Biol 8: 1247-1256.
37. Castellano F, Montcourrier P, Guillemot JC, Gouin E, Machesky L, Cossart P, and Chavrier P. (1999). “Inducible recruitment of Cdc42 or WASP to a cell-surface receptor triggers actin polymerization and filopodium formation.” Curr Biol 9: 351-360.
38. Segall JE, Fisher PR, and Gerisch G. (1987). “Selection of chemotaxis mutants of Dictyostelium discoideum.” J Cell Biol 104: 151-161.
39. Kuwayama H, Ishida S, and Van Haastert PJ. (1993). “ Non-chemotactic Dictyostelium discoideum mutants with altered cGMP signal transduction.” J Cell Biol 123: 1453-1462.
40. Kuspa A, and Loomis WF (1992). “Tagging developmental genes in Dictyostelium by restriction enzyme-mediated integration of plasmid DNA.” Proc Natl Acad Sci U S A 89: 8803-8807.
41. Adachi H, Hasebe T, Yoshinaga K, Ohta T, and Sutoh K. (1994). “Isolation of Dictyostelium discoideum cytokinesis mutants by restriction enzyme-mediated integration of the blasticidin S resistance marker.” Biochem Biophys Res Commun. 205: 1808-1814.
42. Boyden S. (1962). “The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leucocytes.” J Exp Med 115: 453-466.
43. Sussman R, and Sussman M. (1967). “Cultivation of Dictyostelium discoideum in axenic medium.” Biochem Biophys Res Commun 29: 53-55.
44. Howard PK, Ahern KG, and Firtel RA. (1988). “Establishment of a transient expression system for Dictyostelium discoideum.” Nucleic Acids Res 16: 2613-2623.
45. Devreotes PN, Fontana D, Klein P, Sherring J, and Theibert A. (1987). “Transmembrane signaling in Dictyostelium.” Methods Cell Biol 28: 299-331.
46. Sun TJ, Van Haastert PJ, and Devreotes PN. (1990). “Surface cAMP receptors mediate multiple responses during development in Dictyostelium: evidenced by antisense mutagenesis.” J Cell Biol 110: 1549-1554.
47. Roisin BC, Jang W, Caprette DR, and Gomer RH. (2000). “A precise group size in Dictyostelium is generated by a cell-counting factor modulating cell-cell adhesion.” Mol Cell 6: 953-959.
48. Segall JE, Kuspa A, Shaulsky G, Ecke M, Maeda M, Gaskins C, Firtel RA, and Loomis WF. (1995). “A MAP kinase necessary for receptor-mediated activation of adenylyl cyclase in Dictyostelium.” J Cell Biol 128: 405-413.
49. Mato JM, Krens FA, van Haastert PJ, and Konijn TM. (1977). “3':5'-cyclic AMP-dependent 3':5'-cyclic GMP accumulation in Dictyostelium discoideum.” Proc Natl Acad Sci U S A 74: 2348-2351.
50. Loomis WF (1988). “Cell-cell adhesion in Dictyostelium discoideum.” Dev Genet 9: 549-559.
51. Brar SK, and Siu CH. (1993). “Characterization of the cell adhesion molecule gp24 in Dictyostelium discoideum. Mediation of cell-cell adhesion via a Ca(2+)-dependent mechanism.” J Biol Chem 268: 24902-24909.
52. Guo HB, Zhang Y, and Chen HL. (2001). “Relationship between metastasis-associated phenotypes and N-glycan structure of surface glycoproteins in human hepatocarcinoma cells.” J Cancer Res Clin 127: 231-236.
53. Peyrard M, Seroussi E, Sandberg-Nordqvist AC, Xie YG, Han FY, Fransson I, Collins J, Dunham I, Kost-Alimova M, Imreh S, and Dumanski JP. (1999). “The human LARGE gene from 22q12.3-q13.1 is a new, distinct member of the glycosyltransferase gene family.” Proc Natl Acad Sci U S A 96: 598-603.
54. Grewal PK, Holzfeind PJ, Bittner RE, and Hewitt JE. (2001). “Mutant glycosyltransferase and altered glycosylation of alpha-dystroglycan in the myodystrophy mouse.” Nat Genet 28: 151-154.
55. Kumagai A, Hadwiger JA, Pupillo M, and Firtel RA. (1991). “Molecular genetic analysis of two G alpha protein subunits in Dictyostelium.” J Biol Chem 266: 1220-1228.
56. Johnson RL, Van Haastert PJ, Kimmel AR, Saxe CL 3rd, Jastorff B, Devreotes PN. (1992). “The cyclic nucleotide specificity of three cAMP receptors in Dictyostelium.” J Biol Chem 267: 4600-4607.
57. Kim JY, Borleis JA, Devreotes PN. (1998). “Switching of chemoattractant receptors programs development and morphogenesis in Dictyostelium: receptor subtypes activate common responses at different agonist concentrations.” Dev Biol 197(117-128).
58. Louis JM, Ginsburg GT, Kimmel AR. (1994). “The cAMP receptor CAR4 regulates axial patterning and cellular differentiation during late development of Dictyostelium.” Genes Dev 8: 2086-2096.
59. Hadwiger JA, Lee S, and Firtel RA. (1994). “The G alpha subunit G alpha 4 couples to pterin receptors and identifies a signaling pathway that is essential for multicellular development in Dictyostelium.” Proc Natl Acad Sci U S A 91: 10566-10570.
60. Peracino, B., Borleis, J., Jin, T., Westphal, M., Schwartz, J.-M., Wu, L.,Bracco, E., Gerisch, G., Devreotes, P. and Bozzaro, S. (1998). “G protein b subunit-null mutants are impaired in phagocytosis and chemotaxis due to inappropriate regulation of the actin cytoskeleton.” J. Cell Biol 141: 1529-1537.
61. Chung CY, Potikyan G, and Firtel RA. (2001). “Control of cell polarity and chemotaxis by Akt/PKB and PI3 kinase through the regulation of PAKa.” Mol Cell 7: 937-947.
62. Chung CY, and Firtel RA. (1999). “PAKa, a putative PAK family member, is required for cytokinesis and the regulation of the cytoskeleton in Dictyostelium discoideum cells during chemotaxis.” J Cell Biol 147: 559-576.
63. Moniakis J, Funamoto S, Fukuzawa M, Meisenhelder J, Araki T, Abe T, Meili R, Hunter T, Williams J, and Firtel RA. (2001). “An SH2-domain-containing kinase negatively regulates the phosphatidylinositol-3 kinase pathway.” Genes Dev 15: 687-698.
64. Lee S, Parent CA, Insall R, and Firtel RA. (1999). “A novel Ras-interacting protein required for chemotaxis and cyclic adenosine monophosphate signal relay in Dictyostelium.” Mol Biol Cell 10: 2829-45.
65. Tuxworth RI, Cheetham JL, Machesky LM, Spiegelmann GB, Weeks G, and Insall RH. (1997). “Dictyostelium RasG is required for normal motility and cytokinesis, but not growth.” J Cell Biol 138: 605-614.
66. Wang Y, Liu J, and Segall JE. (1998). “MAP kinase function in amoeboid chemotaxis.” J Cell Sci 111: 373-83.
67. Kuwayama H, Snippe H, Derks M, Roelofs J, and Van Haastert PJ. (2001). “Identification and characterization of DdPDE3, a cGMP-selective phosphodiesterase from Dictyostelium.” Biochem J 353: 635-644.
68. Coukell MB, and Cameron AM (1986). “Characterization of revertants of stmF mutants of Dictyostelium discoideum: evidence that stmF is the structural gene of the cGMP-specific phosphodiesterase.” Dev Genet 6: 163-177.
69. De Lozanne A, and Spudich, J. A. (1987). “Disruption of the Dictyostelium myosin heavy chain gene by homologous recombination.” Science 236: 1086-1091.
70. Chen TL, Kowalczyk PA, Ho G, and Chisholm RL. (1995). “Targeted disruption of the Dictyostelium myosin essential light chain gene produces cells defective in cytokinesis and morphogenesis.” J Cell Sci 108: 3207-3218.
71. Kolman MF, Futey LM, and Egelhoff TT. (1996). “Dictyostelium myosin heavy chain kinase A regulates myosin localization during growth and development.” J Cell Biol (132): 101-109.
72. Chien S, Chung CY, Sukumaran S, Osborne N, Lee S, Ellsworth C, McNally JG, and Firtel RA. (2000). “The Dictyostelium LIM domain-containing protein LIM2 is essential for proper chemotaxis and morphogenesis.” Mol Biol Cell 11: 1275-1291.
73. Bear JE, Rawls JF, Saxe CL (1998). “Scar, a WASP-related protein, isolated as a suppressor of receptor defects in late Dictyostelium development.” J. Cell Biol 142: 1325-1335.
74. de Hostos EL, Rehfuess C, Bradtke B, Waddell DR, Albrecht R, Murphy J, and Gerisch G. (1993). “Dictyostelium mutants lacking the cytoskeletal protein coronin are defective in cytokinesis and cell motility.” J. Cell Biol 120: 163-173.
75. Rivero F, Koppel B, Peracino B, Bozzaro S, Siegert F, Weijer CJ, Schleicher M, Albrecht R, and Noegel AA. (1996). “The role of the cortical cytoskeleton: F-actin crosslinking proteins protect against osmotic stress, ensure cell size, cell shape and motility, and contribute to phagocytosis and development.” J Cell Sci 109: 2679-2691.
76. Alexander S, Sydow LM, Wessels D, and Soll DR. (1992). “Discoidin proteins of Dictyostelium are necessary for normal cytoskeletal organization and cellular morphology during aggregation.” Differentiation 51: 149-161.
77. Shutt DC, Wessels D, Wagenknecht K, Chandrasekhar A, Hitt AL, Luna EJ, and Soll DR. (1995). “Ponticulin plays a role in the positional stabilization of pseudopods.” J Cell Biol 131: 1495-1506.
78. Haugwitz M, Noegel AA, Karakesisoglou J, and Schleicher M. (1994). “Dictyostelium amoebae that lack G-actin-sequestering profilins show defects in F-actin content, cytokinesis, and development.” Cell 79: 303-314.
79. Gerisch G, Albrecht R, De Hostos E, Wallraff E, Heizer C, Kreitmeier M, and Muller-Taubenberger A. (1993). “Actin-associated proteins in motility and chemotaxis of Dictyostelium cells.” Symp Soc Exp Biol 47: 297-315.
80. Andre E, Brink M, Gerisch G, Isenberg G, Noegel A, Schleicher M, Segall JE, and Wallraff E. (1989). “A Dictyostelium mutant deficient in severin, an F-actin fragmenting protein, shows normal motility and chemotaxis.” J Cell Biol 108: 985-995.
81. Rivero F, K. B., Peracino B, Bozzaro S, Siegert F, Weijer CJ, Schleicher M, Albrecht R, and Noegel AA. (1996). “The role of the cortical cytoskeleton: F-actin crosslinking proteins protect against osmotic stress, ensure cell size, cell shape and motility, and contribute to phagocytosis and development.” J Cell Sci 109: 2679-2691.
82. Wessels D, R. J., Johnson O, Voss E, Burns R, Daniels K, Garrard E, O'Halloran TJ, and Soll DR. (2000). “Clathrin plays a novel role in the regulation of cell polarity, pseudopod formation, uropod stability and motility in Dictyostelium.” J Cell Sci 113: 21-36.
83. Wessels DJ, Z. H., Reynolds J, Daniels K, Heid P, Lu S, Kuspa A, Shaulsky G, Loomis WF, and Soll DR. (2000). “The internal phosphodiesterase RegA is essential for the suppression of lateral pseudopods during Dictyostelium chemotaxis.” Mol Biol Cell 11: 2803-20.
84. Mohanty S, Lee S, Yadava N, Dealy MJ, Johnson RS, and Firtel RA. (2001). “Regulated protein degradation controls PKA function and cell-type differentiation in Dictyostelium.” Genes Dev 15: 1435-1448.
85. van Es S, Wessels D, Soll DR, Borleis J, and Devreotes PN. (2001). “Tortoise, a novel mitochondrial protein, is required for directional responses of Dictyostelium in chemotactic gradients.” J Cell Biol 152: 621-632.
86. Chen MY, Long Y, and Devreotes PN. (1997). “A novel cytosolic regulator, Pianissimo, is required for chemoattractant receptor and G protein-mediated activation of the 12 transmembrane domain adenylyl cyclase in Dictyostelium.” Genes Dev 11: 3218-31.
87. Mohanty S, Jermyn KA, Early A, Kawata T, Aubry L, Ceccarelli A, Schaap P, Williams JG, and Firtel RA. (1999). “Evidence that the Dictyostelium Dd-STATa protein is a repressor that regulates commitment to stalk cell differentiation and is also required for efficient chemotaxis.” Development 126: 3391-405.
88. Lindsey DF, Amerik A, Deery WJ, Bishop JD, Hochstrasser M, and Gomer RH. (1998). “A deubiquitinating enzyme that disassembles free polyubiquitin chains is required for development but not growth in Dictyostelium.” J Biol Chem 273: 29178-29187.
89. Escalante R, Wessels D, Soll DR, and Loomis WF. (1997). “Chemotaxis to cAMP and slug migration in Dictyostelium both depend on migA, a BTB protein.” Mol Biol Cell 8: 1763-1775.