臺灣博碩士論文加值系統

TDPOZ，為一含腫瘤壞死因子受器結合因子(TD；tumor necrosis factor receptor-associated factor domain)功能區與POZ (poxvirus and zinc finger domain)功能區的新蛋白質家族。TDPOZ蛋白質的功能目前仍未知。不過由TD與POZ功能區於胺基酸序列強烈的保守性顯示出此基因對細胞的功能可能之重要性。
本研究目的在於嘗試製備RTDPOZ重組蛋白提供日後研究分析之用，並對於RTDPOZ的作用結構進行初步的分析。首先針對rat的Rtdpoz基因，構築完整的Rtdpoz序列，並找出編譯讀框，再利用大腸桿菌表現系統表現RTDPOZ蛋白（經電泳分析後發現RTDPOZ蛋白主要存在於不溶性層的包涵體中），並製造抗血清。
另以昆蟲細胞Sf-21為宿主，配合桿狀病毒表現系統表現RTDPOZ，以克服表現蛋白包涵體的形成，日後可供RTDPOZ之活性與功能性研究之用。同時由觀察RTDPOZ普遍分布於細胞膜上和蛋白聚集(aggresome)的現象推測可能與其功能之間有密切的關聯。
另外為了探討RTDPOZ蛋白本身是否會產生同分子雙子，遂以酵母菌雙雜合系統方式，將Rtdpoz基因片段分別選殖至帶有Gal4-AD（activation domain）以及-BD（DNA binding domain）的表現載體中。結果發現於營養限制培養基中並沒有預期的菌落生長；初步推測RTDPOZ並不會形成同分子雙子結構。

TDPOZ, a new protein family member of with contain the tumor necrosis factor receptor-associated factor domain (TD) and the poxvirus and zinc finger domain (POZ). The function of TDPOZ proteins is unknown. However, conservation of the TD and POZ domains strongly suggests that these domains are critical in some cellular functions. This research focused on the rat Rtdpoz gene. The full-length of Rtdpoz DNA sequence was first determine to locate the open reading frame (ORF). The E. coli expression system was then used to express the RTDPOZ protein (existed in inclusion bodies) and to produce anti-serum.
We also used the insect cell Sf-21 and a baculovirus expression vector system to express RTDPOZ to overcome the inclusion body problem. Furthermore, RTDPOZ was widespread at cell membrane and the phenomenon of aggregate may indicate a connection with protein functions.
We also analyzed RTDPOZ’s self-interaction by yeast two-hybrid system by constructing the Rtdpoz sequence in yeast expression vectors which have either an activation domain (AD) or a binding domain (BD). The results indicate that co-transformed yeast would not growth on nutrition-limited medium plate; initial analysis suggests that RTDPOZ does not form homodimer.

中文摘要 -Ⅰ
英文摘要 Ⅱ
目錄 Ⅲ
壹、前言 1
貳、材料和方法 18
一、以大腸桿菌系統表現RTDPOZ 18
(一)、構築含完整的Rtopoz基因序列質體 20
(二)、勝任細胞（competent cell）製備 20
(三)、將重組載體轉型至勝任細胞（DH5-α） 20
(四)、利用cracking buffer打破菌體做初步篩選 21
(五)、擴增Rtdpoz的open reading frame (ORF) 22
(六)、 RTDPOZ蛋白之誘導表現 22
(七)、聚丙烯醯胺膠體電泳：Sodium DodecylSulfate Polyacryl Amide-
Gel-Electrophoresis (SDS-PAGE) 23
(八)、 以FRENCH Press打破大腸桿菌 23
(九)、西方點墨分析(Western blotting) 24
二、RTDPOZ抗血清製備
（一）、抗原的收集 26
（二）、RTDPOZ抗血清測試 26
三、以桿狀病毒系統表現RTDPOZ 29
（一）、昆蟲細胞培養 30
（二）、桿狀病毒轉殖載體構築 30
（三）、共轉染形成重組病毒以及RTDPOZ之表現 31
（四）、免疫螢光染色(Immuno-fluorescence staining) 32
（五）、RTDPOZ之純化與確認 33
（六）、蛋白質定量 34
（七）、純化後產物分析 35
四、RTDPOZ同源雙體分析：酵母菌雙雜合系統(yeast two-hybrid
system)： 37
（一）、酵母菌雙雜合載體構築 38
（二）、酵母菌勝任細胞製備與轉形： 39
（三）、Filter lift assay 41
参、結果 44
肆、討論 53
伍、參考文獻 60
陸、圖表 68
柒、附錄 91

Ansieau, S., Scheffrahn, I., Mosialos, G., Brand, H., Duyster, J., Kaye, K., Harada, J., Dougall, B., Hubinger, G., Kieff, E., et al. (1996). Tumor necrosis factor receptor-associated factor (TRAF)-1, TRAF-2, and TRAF-3 interact in vivo with the CD30 cytoplasmic domain; TRAF-2 mediates CD30-induced nuclear factor kappa B activation. Proc Natl Acad Sci U S A 93, 14053-14058.

Bardwell, V. J., and Treisman, R. (1994). The POZ domain: a conserved protein-protein interaction motif. Genes Dev 8, 1664-1677.

Bartal, M., Mazor, D., Dvilansky, A., and Meyerstein, N. (1993). Iron deficiency anemia: recovery from in vitro oxidative stress. Acta Haematol 90, 94-98.

Bazzoni, F., and Beutler, B. (1996). The tumor necrosis factor ligand and receptor families. N Engl J Med 334, 1717-1725.

Bevilacqua, A., Fiorenza, M. T., and Mangia, F. (2000). A developmentally regulated GAGA box-binding factor and Sp1 are required for transcription of the hsp70.1 gene at the onset of mouse zygotic genome activation. Development 127, 1541-1551.

Bouniol, C., Nguyen, E., and Debey, P. (1995). Endogenous transcription occurs at the 1-cell stage in the mouse embryo. Exp Cell Res 218, 57-62.

Boyer, L. A., Plath, K., Zeitlinger, J., Brambrink, T., Medeiros, L. A., Lee, T. I., Levine, S. S., Wernig, M., Tajonar, A., Ray, M. K., et al. (2006). Polycomb complexes repress developmental regulators in murine embryonic stem cells. Nature 441, 349-353.

Bradley, J. R., and Pober, J. S. (2001). Tumor necrosis factor receptor-associated factors (TRAFs). Oncogene 20, 6482-6491.

Burger, A. M., and Seth, A. K. (2004). The ubiquitin-mediated protein degradation pathway in cancer: therapeutic implications. Eur J Cancer 40, 2217-2229.

Buttice, G., Duterque-Coquillaud, M., Basuyaux, J. P., Carrere, S., Kurkinen, M., and Stehelin, D. (1996). Erg, an Ets-family member, differentially regulates human collagenase1 (MMP1) and stromelysin1 (MMP3) gene expression by physically interacting with the Fos/Jun complex. Oncogene 13, 2297-2306.

Carter, K. C., Bowman, D., Carrington, W., Fogarty, K., McNeil, J. A., Fay, F. S., and Lawrence, J. B. (1993). A three-dimensional view of precursor messenger RNA metabolism within the mammalian nucleus. Science 259, 1330-1335.

Choo, K. B., Chen, H. H., Cheng, W. T., Chang, H. S., and Wang, M. (2001). In silico mining of EST databases for novel pre-implantation embryo-specific zinc finger protein genes. Mol Reprod Dev 59, 249-255.

Collins, T., Stone, J. R., and Williams, A. J. (2001). All in the family: the BTB/POZ, KRAB, and SCAN domains. Mol Cell Biol 21, 3609-3615.

Costanzi, C., and Pehrson, J. R. (1998). Histone macroH2A1 is concentrated in the inactive X chromosome of female mammals. Nature 393, 599-601.

Cote, C., Poirier, J., and Boulet, D. (1990). Role for the mitochondrial inner membrane in the maturation of the precursor to ornithine carbamyl transferase. Biochem Biophys Res Commun 170, 1273-1280.

Dhordain, P., Lin, R. J., Quief, S., Lantoine, D., Kerckaert, J. P., Evans, R. M., and Albagli, O. (1998). The LAZ3(BCL-6) oncoprotein recruits a SMRT/mSIN3A/histone deacetylase containing complex to mediate transcriptional repression. Nucleic Acids Res 26, 4645-4651.

Fields, S., and Song, O. (1989). A novel genetic system to detect protein-protein interactions. Nature 340, 245-246.

Fields, S., and Sternglanz, R. (1994). The two-hybrid system: an assay for protein-protein interactions. Trends Genet 10, 286-292.

Freemont, P. S. (1993). The RING finger. A novel protein sequence motif related to the zinc finger. Ann N Y Acad Sci 684, 174-192.

Garcia-Mata, R., Gao, Y. S., and Sztul, E. (2002). Hassles with taking out the garbage: aggravating aggresomes. Traffic 3, 388-396.

He, J., Tam, A. W., Yarbough, P. O., Reyes, G. R., and Carl, M. (1993). Expression and diagnostic utility of hepatitis E virus putative structural proteins expressed in insect cells. J Clin Microbiol 31, 2167-2173.

Heinzel, T., Lavinsky, R. M., Mullen, T. M., Soderstrom, M., Laherty, C. D., Torchia, J., Yang, W. M., Brard, G., Ngo, S. D., Davie, J. R., et al. (1997). A complex containing N-CoR, mSin3 and histone deacetylase mediates transcriptional repression. Nature 387, 43-48.

Hernandez-Munoz, I., Lund, A. H., van der Stoop, P., Boutsma, E., Muijrers, I., Verhoeven, E., Nusinow, D. A., Panning, B., Marahrens, Y., and van Lohuizen, M. (2005). Stable X chromosome inactivation involves the PRC1 Polycomb complex and requires histone MACROH2A1 and the CULLIN3/SPOP ubiquitin E3 ligase. Proc Natl Acad Sci U S A 102, 7635-7640.

Hideshima, T., Bradner, J. E., Wong, J., Chauhan, D., Richardson, P., Schreiber, S. L., and Anderson, K. C. (2005). Small-molecule inhibition of proteasome and aggresome function induces synergistic antitumor activity in multiple myeloma. Proc Natl Acad Sci U S A 102, 8567-8572.

Hoatlin, M. E., Zhi, Y., Ball, H., Silvey, K., Melnick, A., Stone, S., Arai, S., Hawe, N., Owen, G., Zelent, A., and Licht, J. D. (1999). A novel BTB/POZ transcriptional repressor protein interacts with the Fanconi anemia group C protein and PLZF. Blood 94, 3737-3747.

Hofmann, C., Sandig, V., Jennings, G., Rudolph, M., Schlag, P., and Strauss, M. (1995). Efficient gene transfer into human hepatocytes by baculovirus vectors. Proc Natl Acad Sci U S A 92, 10099-10103.

Hsu, H., Shu, H. B., Pan, M. G., and Goeddel, D. V. (1996). TRADD-TRAF2 and TRADD-FADD interactions define two distinct TNF receptor 1 signal transduction pathways. Cell 84, 299-308.

Huang, C. J., Chen, C. Y., Chen, H. H., Tsai, S. F., and Choo, K. B. (2004). TDPOZ, a family of bipartite animal and plant proteins that contain the TRAF (TD) and POZ/BTB domains. Gene 324, 117-127.

Ishida, T., Mizushima, S., Azuma, S., Kobayashi, N., Tojo, T., Suzuki, K., Aizawa, S., Watanabe, T., Mosialos, G., Kieff, E., et al. (1996a). Identification of TRAF6, a novel tumor necrosis factor receptor-associated factor protein that mediates signaling from an amino-terminal domain of the CD40 cytoplasmic region. J Biol Chem 271, 28745-28748.

Ishida, T. K., Tojo, T., Aoki, T., Kobayashi, N., Ohishi, T., Watanabe, T., Yamamoto, T., and Inoue, J. (1996b). TRAF5, a novel tumor necrosis factor receptor-associated factor family protein, mediates CD40 signaling. Proc Natl Acad Sci U S A 93, 9437-9442.

Kallijarvi, J., Lahtinen, U., Hamalainen, R., Lipsanen-Nyman, M., Palvimo, J. J., and Lehesjoki, A. E. (2005). TRIM37 defective in mulibrey nanism is a novel RING finger ubiquitin E3 ligase. Exp Cell Res 308, 146-155.

Kiriakidou, M., Driscoll, D. A., Lopez-Guisa, J. M., and Strauss, J. F., 3rd (1997). Cloning and expression of primate Daxx cDNAs and mapping of the human gene to chromosome 6p21.3 in the MHC region. DNA Cell Biol 16, 1289-1298.

Klug, A., and Schwabe, J. W. (1995). Protein motifs 5. Zinc fingers. Faseb J 9, 597-604.

La, M., Kim, K., Park, J., Won, J., Lee, J. H., Fu, Y. M., Meadows, G. G., and Joe, C. O. (2004). Daxx-mediated transcriptional repression of MMP1 gene is reversed by SPOP. Biochem Biophys Res Commun 320, 760-765.

Lamond, A. I., and Earnshaw, W. C. (1998). Structure and function in the nucleus. Science 280, 547-553.

Liu, A., Desai, B. M., and Stoffers, D. A. (2004). Identification of PCIF1, a POZ domain protein that inhibits PDX-1 (MODY4) transcriptional activity. Mol Cell Biol 24, 4372-4383.

Lovering, R., Hanson, I. M., Borden, K. L., Martin, S., O'Reilly, N. J., Evan, G. I., Rahman, D., Pappin, D. J., Trowsdale, J., and Freemont, P. S. (1993). Identification and preliminary characterization of a protein motif related to the zinc finger. Proc Natl Acad Sci U S A 90, 2112-2116.

Maeda, S. (1989). Expression of foreign genes in insects using baculovirus vectors. Annu Rev Entomol 34, 351-372.

Maniatis, T. (1997). Catalysis by a multiprotein IkappaB kinase complex. Science 278, 818-819.

Melnick, A., Ahmad, K. F., Arai, S., Polinger, A., Ball, H., Borden, K. L., Carlile, G. W., Prive, G. G., and Licht, J. D. (2000). In-depth mutational analysis of the promyelocytic leukemia zinc finger BTB/POZ domain reveals motifs and residues required for biological and transcriptional functions. Mol Cell Biol 20, 6550-6567.

Mendelsohn, A. R., and Brent, R. (1994). Applications of interaction traps/two-hybrid systems to biotechnology research. Curr Opin Biotechnol 5, 482-486.

Nagai, Y., Kojima, T., Muro, Y., Hachiya, T., Nishizawa, Y., Wakabayashi, T., and Hagiwara, M. (1997). Identification of a novel nuclear speckle-type protein, SPOP. FEBS Lett 418, 23-26.

Nagata, S. (1997). Apoptosis by death factor. Cell 88, 355-365.

Nagy, L., Kao, H. Y., Chakravarti, D., Lin, R. J., Hassig, C. A., Ayer, D. E., Schreiber, S. L., and Evans, R. M. (1997). Nuclear receptor repression mediated by a complex containing SMRT, mSin3A, and histone deacetylase. Cell 89, 373-380.

Natoli, G., Costanzo, A., Moretti, F., Fulco, M., Balsano, C., and Levrero, M. (1997). Tumor necrosis factor (TNF) receptor 1 signaling downstream of TNF receptor-associated factor 2. Nuclear factor kappaB (NFkappaB)-inducing kinase requirement for activation of activating protein 1 and NFkappaB but not of c-Jun N-terminal kinase/stress-activated protein kinase. J Biol Chem 272, 26079-26082.

Nawrocki, S. T., Carew, J. S., Pino, M. S., Highshaw, R. A., Andtbacka, R. H., Dunner, K., Jr., Pal, A., Bornmann, W. G., Chiao, P. J., Huang, P., et al. (2006). Aggresome disruption: a novel strategy to enhance bortezomib-induced apoptosis in pancreatic cancer cells. Cancer Res 66, 3773-3781.

Novokhatskii, A. S., Malakhova, I. V., and Mikheeva, T. G. (1985). [Hybridoma cloning by the end-point dilution method]. Vopr Virusol 30, 602-608.

Pehrson, J. R., Costanzi, C., and Dharia, C. (1997). Developmental and tissue expression patterns of histone macroH2A1 subtypes. J Cell Biochem 65, 107-113.

Pehrson, J. R., and Fried, V. A. (1992). MacroH2A, a core histone containing a large nonhistone region. Science 257, 1398-1400.

Pluta, A. F., Earnshaw, W. C., and Goldberg, I. G. (1998). Interphase-specific association of intrinsic centromere protein CENP-C with HDaxx, a death domain-binding protein implicated in Fas-mediated cell death. J Cell Sci 111 ( Pt 14), 2029-2041.

Rothe, M., Wong, S. C., Henzel, W. J., and Goeddel, D. V. (1994). A novel family of putative signal transducers associated with the cytoplasmic domain of the 75 kDa tumor necrosis factor receptor. Cell 78, 681-692.

Schultz, R. M. (1993). Regulation of zygotic gene activation in the mouse. Bioessays 15, 531-538.

Shu, H. B., Takeuchi, M., and Goeddel, D. V. (1996). The tumor necrosis factor receptor 2 signal transducers TRAF2 and c-IAP1 are components of the tumor necrosis factor receptor 1 signaling complex. Proc Natl Acad Sci U S A 93, 13973-13978.

Smith, G. E., Summers, M. D., and Fraser, M. J. (1983). Production of human beta interferon in insect cells infected with a baculovirus expression vector. Mol Cell Biol 3, 2156-2165.

Stogios, P. J., Downs, G. S., Jauhal, J. J., Nandra, S. K., and Prive, G. G. (2005). Sequence and structural analysis of BTB domain proteins. Genome Biol 6, R82.

Strathdee, C. A., and Buchwald, M. (1992). Molecular and cellular biology of Fanconi anemia. Am J Pediatr Hematol Oncol 14, 177-185.

Suske, G. (1999). The Sp-family of transcription factors. Gene 238, 291-300.

Takahashi, I., Kameoka, Y., and Hashimoto, K. (2002). MacroH2A1.2 binds the nuclear protein Spop. Biochim Biophys Acta 1591, 63-68.

Trojanowska, M. (2000). Ets factors and regulation of the extracellular matrix. Oncogene 19, 6464-6471.

Vallian, S., Chin, K. V., and Chang, K. S. (1998). The promyelocytic leukemia protein interacts with Sp1 and inhibits its transactivation of the epidermal growth factor receptor promoter. Mol Cell Biol 18, 7147-7156.

Vaux, D. L., and Strasser, A. (1996). The molecular biology of apoptosis. Proc Natl Acad Sci U S A 93, 2239-2244.

Waelter, S., Boeddrich, A., Lurz, R., Scherzinger, E., Lueder, G., Lehrach, H., and Wanker, E. E. (2001). Accumulation of mutant huntingtin fragments in aggresome-like inclusion bodies as a result of insufficient protein degradation. Mol Biol Cell 12, 1393-1407.

Wajant, H., Henkler, F., and Scheurich, P. (2001). The TNF-receptor-associated factor family: scaffold molecules for cytokine receptors, kinases and their regulators. Cell Signal 13, 389-400.

Wang, M. Y., Kuo, Y. Y., Lee, M. S., Doong, S. R., Ho, J. Y., and Lee, L. H. (2000). Self-assembly of the infectious bursal disease virus capsid protein, rVP2, expressed in insect cells and purification of immunogenic chimeric rVP2H particles by immobilized metal-ion affinity chromatography. Biotechnol Bioeng 67, 104-111.

Wen, Y., Nguyen, D., Li, Y., and Lai, Z. C. (2000). The N-terminal BTB/POZ domain and C-terminal sequences are essential for Tramtrack69 to specify cell fate in the developing Drosophila eye. Genetics 156, 195-203.

Worrad, D. M., Ram, P. T., and Schultz, R. M. (1994). Regulation of gene expression in the mouse oocyte and early preimplantation embryo: developmental changes in Sp1 and TATA box-binding protein, TBP. Development 120, 2347-2357.

Xing, Y., Johnson, C. V., Moen, P. T., Jr., McNeil, J. A., and Lawrence, J. (1995). Nonrandom gene organization: structural arrangements of specific pre-mRNA transcription and splicing with SC-35 domains. J Cell Biol 131, 1635-1647.

Zapata, J. M., Pawlowski, K., Haas, E., Ware, C. F., Godzik, A., and Reed, J. C. (2001). A diverse family of proteins containing tumor necrosis factor receptor-associated factor domains. J Biol Chem 276, 24242-24252.

Zhang, G., Taneja, K. L., Singer, R. H., and Green, M. R. (1994). Localization of pre-mRNA splicing in mammalian nuclei. Nature 372, 809-812.

Zollman, S., Godt, D., Prive, G. G., Couderc, J. L., and Laski, F. A. (1994). The BTB domain, found primarily in zinc finger proteins, defines an evolutionarily conserved family that includes several developmentally regulated genes in Drosophila. Proc Natl Acad Sci U S A 91, 10717-10721.