人MGC5306基因原核、真核表达载体的构建及亚细胞结构的定位
|
摘要
MGC5306基因作为一个新发现的基因,目前国内外对其研究的相关报道还非常少。有实验证据表明MGC5306参与卵巢癌的发生。MGC5306的功能及其作用机理现在还不完全清楚。我们利用基因重组技术构建了MGC5306的原核表达体系,并成功表达了MGC5306(98-204AA)。我们构建了pDsRed-MGC5306,并将其转染至Hela细胞,观察其亚细胞结构的分布及细胞核内的定位。将DsRed-MGC5306和GFP-SUMO1共转染至Hela细胞,通过激光共聚焦发现两者共定位于细胞核内。
As a newly discovered gene, domestic and foreign research reports of MGC5306 are also less now. There is experimental evidence that the MGC5306 involved in the occurrence of ovarian cancer. MGC5306 functions and its mechanism is not entirely clear. We use gene recombination technology to build the prokaryotic MGC5306 expression system, and we have successfully expressed MGC5306(98-204AA).We build eukaryotic expression DsRed- MGC5306, and transfected it into Hela cell to observed sub-cellular distribution and nuclear localization of protein. Eukaryotic expression vectors pDsRed-C1-MGC5306 and pEGFP-N1- SUMO1 were transfected into Hela cell, we found MGC5306 and SUMO1 co-localize in the nucleus by CLSM(Confoca Laser Scanning Microscopel).
As a newly discovered gene, domestic and foreign research reports of MGC5306 are also less now. There is experimental evidence that the MGC5306 involved in the occurrence of ovarian cancer. MGC5306 functions and its mechanism is not entirely clear. We use gene recombination technology to build the prokaryotic MGC5306 expression system, and we have successfully expressed MGC5306(98-204AA).We build eukaryotic expression DsRed- MGC5306, and transfected it into Hela cell to observed sub-cellular distribution and nuclear localization of protein. Eukaryotic expression vectors pDsRed-C1-MGC5306 and pEGFP-N1- SUMO1 were transfected into Hela cell, we found MGC5306 and SUMO1 co-localize in the nucleus by CLSM(Confoca Laser Scanning Microscopel).
引文
[1] Sancar A, Lindsey-Boltz LA, Unsal-Kacmaz K, Linn S. Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints. Annu Rev Biochem2004;73:39–85.
[2] Gu H, Marth JD, Orban PC, et al. Deletion of a DNA polymerase beta gene segment in T cells using cell type-specific gene targeting. Science (Wash. DC) 1994;265:103–6.
[3] Plug AW, Clairmont CA, Sapi E, Ashley T, Sweasy, JB. Evidence for a role for DNA polymerase beta in mammalian meiosis Proc Natl Acad Sci USA 1997;94:1327–31.
[4] Sugo N, Aratani Y, Nagashima Y, Kubota Y, Koyama H. Neonatal lethality with abnormal neurogenesis in mice deficient in DNA polymerase beta. EMBO J 2000; 19:1397– 404.
[5] Ochs K, Sobol RW, Wilson SH, Kaina B. Cells deficient in DNA polymerase beta are hypersensitive to alkylating agent-induced apoptosis and chromosomal breakage. Cancer Res 1999;59:1544–51.
[6] Sweasy JB, Loeb LA. Detection and characterization of mammalian DNA polymerase beta mutants by functional complementation in Escherichia coli. Proc Natl Acad Sci USA 1993;90:4626–30.
[7] Feig DI, Loeb LA. Mechanisms of mutation by oxidative DNA damage: reduced fidelity of mammalian DNA polymerase. Biochemistry 1993;32:4466–73.
[8] Dianov GL, Thybo T, Dianova II, Lipinski LJ, Bohr VA. Single nucleotide patch base excision repair is the major pathway for removal of thymine glycol from DNA in human cell extracts. J Biol Chem 2000;275:11809–13.
[9] Liming Wang, Nandan Bhattacharyya.et al. A Novel Nuclear Protein, MGC5306 Interacts with DNA Polymeraseβand Has a Potential Role in Cellular Phenotype. CANCER RESEARCH, 2004, 64:7673~7677
[10] Julia J Gorski, Shalini Pathak, Kostya Panov, Taciana Kasciukovic, Tanya Panova, Jackie Russell and Joost CBM Zomerdijk. The EMBO Journal (2007) 26, 1560–1568
[11] Hahn S (1998) The role of TAFs in RNA polymerase II transcription. Cell 95: 579–582
[12] Grummt I (1999) Regulation of mammalian ribosomal gene transcription by RNA polymerase I. Prog Nucleic Acid Res Mol Biol 62:109–154
[13] Grummt I (2003) Life on a planet of its own: regulation of RNA polymerase I transcription in the nucleolus. Genes Dev 17:1691–1702
[14] Reeder RH (1999) Regulation of RNA polymerase I transcription in yeast and vertebrates. Prog Nucleic Acid Res Mol Biol 62:293–327
[15] Nomura M (2001) Ribosomal RNA genes, RNA polymerases,nucleolar structures, and synthesis of rRNA in the yeast Saccharomyces cerevisiae. Cold Spring Harb Symp Quant Biol 66: 555–565
[16] Moss T (2004) At the crossroads of growth control; making ribosomal RNA. Curr Opin Genet Dev 14: 210–217
[17] Moss T, Stefanovsky VY (2002) At the center of eukaryotic life. Cell 109: 545–548
[18] Zomerdijk JCBM, Beckmann H, Comai L, Tjian R (1994) Assembly of transcriptionally active RNA polymerase I initiation factor SL1 from recombinant subunits. Science 266: 2015–2018
[19] MELUH P B, KOSHLAND D, et al. Evidence that the MIF2 gene of Saccharomyces cerevisiae encodes a centromere protein with homology to the mammalian centromere protein CENP-C [J]. MolBiol Cell, 1995, 6: 793~807.
[20] BOHREN KM, NADKARN IV, SONG J H, et al. A M55V polymorphism in a novel SUMO gene ( SUMO-4 ) differentially activates heat shock transcription factors and is associated with susceptibility to type I diabetes mellitus [ J ] Bio Chem, 2004, 279(26) : 27233~27238
[21] Jacob S et al. Nature Rev Mol Cell Biol, 2003, 4:690 ~699
[22] PICHLER A, MELCHIOR F, et al. Ubiquitin-related modifer SUMO-1 and nucleo-cytoplasmic transport [J]. Traffic, 2002, 3:381~387
[23]叶晓峰,吴乔.类泛素蛋白---SUMO[J].细胞生物学杂志,2004,26(1):10~14 Ye X F, Wu Q. Chinese Journal of Cell Biology, 2004. 26(1):10~14
[24] Saitoh H, Hinchey J. Functional heterogeneity of small ubiquitin-related protein modifiers SUMO-1 versus SUMO-2/3[J]. J Biol Chem, 2000, 275: 6252~6258
[25] R. S. Hilgarth, L. A. Murphy, H. S. Skaggs, et al. Regulation and function of SUMO modification. J Bio Chem, 2004, 279(52): 53899~53902.
[26] DOHMEN R J. SUMO protein modification [J] 1 SUMO protein modification [J] Biochim Biophys Acta, 2004, 1695 (1 - 3):113~131
[27] Treuter E, Gustafsson J A. Wrestling rules in transrepression: as easy as SUMO-1, -2, -3 [J]. Mol Cell, 2007, 25(2):178~180
[28] Grace Gill, et al. SUMO and ubiquitin in the nucleus: different functions, similar mechanisms? Gene and Development, 2004, 18:2046-2059
[29]陈泉,施蕴渝.小泛素相关修饰物SUMO研究进展[J].生命科学, 2004, 16(1):1~6 Chen Q, Shi Y Y. Chinese Bulletin of Life Sciences, 2004. 16(1):1~6
[30] M. B. Kroetz. SUMO: a ubiquitin-like protein modifier. Yale J Biol Med, 2005, 78(4):197~201.
[31] Alexis Verger, Jose Perdomo, Merlin Crossley. Modification with SUMO A role in transcriptional regulation[J]. European Molecular Biology Organization, 2003, 4(2):137~142.
[32] Gill G. Post-translational modification by the small ubiquitin-related modifier SUMO has big effects on transcription factor activity [J] Curr Opin Genet Dev, 2003, 13(2):108~113
[33] Rodriguez MS. J Biol Chem, 2001, 276:12654~12959
[34] Hay R T. SUMO: a history of modification [J]. Mol Cell, 2005, 18 (1):1~12
[35] Cheng J, Bawa T, Leey P, et al. Role of desumoylation in the development of prostate cancer [J ] . Neoplasia, 2006, 8(8): 667~676
[36] Desterro JM, et al. Mol Cell, 1998, 2: 233 ~239
[37] Sternsdorf T,Jensen K,Freemont P S. SUMO. Curr Biol, 2003, 13:R258~R259
[38] Saitoh H,Hinchey J. Functional beterogeneity of small ubiquitin-related protein modifiers SUMO-1 versus SUMO-2/3. J Biol Chem, 2000, 275:6252~6258
[39] Dellaire G, Farrall R, Bickmore W A. The nuclear protein database (NPD): sub-nuclear localization and functional annotation of the nuclear proteome [J]. Nucl Acids Res, 2003, 31: 328~33
[40] Rodriguez MS, Desterro JM, Lain S, et al. SUMO-1 modification activates the transcriptional response of p53.EMBO J, 1999, 18(22):6455~6461
[41] Muller S, Ledl A, Schmidt D. SUMO: a regulator of gene expression and genome integrity. Oncogene.2004, 23 (11): 1998~2008
[42] Melchior F. SUMO—nonclassical ubiquitin. Annu Rev Cell Dev Biol. 2000, 16:591~626
[43] Mao Y, Sun M, Desai SD, et al. SUMO-1 conjugation to topoisomerase I: A possible repair response to topoisomerase - mediated DNA damage. Proc Natl Acad Sci USA,2000,97(8):4046~4051。
[44] Shen Z, Pardington - Purtymun PE, Comeaux JC, et al. Associatios of UBE2I with RAD52, UBL1, p53 and RAD51 proteins in a yeast two– hybrid system. Genomics,1996, 37(2):183~186。
[45] Gottifredi V, Prives C. P53 and PML: new partners in tumor suppression. Trends Cell Biol,2001, 11(5):184~187
[46] Tojo M, Matsuzaki K, Minami T. et al. The aryl hydrocarbon receptor nuclear transporter is modulated by the SUMO-1 conjugation system.J BioChem, 2002,277 (48):46576~46585
[47] Jeffrey G, Marblestone, Tauseef R. Butt. et al. Comparison of SUMO fusion technology with traditional gene fusion systems: Enhanced expression and solubility with SUMO. Protein Science, 2005, 10(12):182~189
[48] Zhang X, Studier FW. Mechanism of inhibition of bacteriophage T7 RNA polymerase and T7 lysozyme [J]. J Mol Biol, 1997, 269(1):10~27.
[49] Lyakhov D L, He B, Zhang X. Pausing and termination by bacteriophage T7 RNA polymerase [J]. J Mol Biol.1998, 280(2):201~213.
[50] Zhang X, Studier F W. Multiple roles of T7 RNA polymerase and T7 lysozyme during bacteriophage T7 infection [J]. J Mol Biol. 2004, 340(4): 707~730.
[51] Sheen J, H wang S, et al. Green-fluorescent protein as a new vital marker in plant cell [J]. Plant, 1995, 8(5):777~784
[52] Eric J Richards, Singhakowinta A, Brennan MJ. Studes on prolactin in human serum urine and milk. Hormone Res, 1975, 6:310~320
[2] Gu H, Marth JD, Orban PC, et al. Deletion of a DNA polymerase beta gene segment in T cells using cell type-specific gene targeting. Science (Wash. DC) 1994;265:103–6.
[3] Plug AW, Clairmont CA, Sapi E, Ashley T, Sweasy, JB. Evidence for a role for DNA polymerase beta in mammalian meiosis Proc Natl Acad Sci USA 1997;94:1327–31.
[4] Sugo N, Aratani Y, Nagashima Y, Kubota Y, Koyama H. Neonatal lethality with abnormal neurogenesis in mice deficient in DNA polymerase beta. EMBO J 2000; 19:1397– 404.
[5] Ochs K, Sobol RW, Wilson SH, Kaina B. Cells deficient in DNA polymerase beta are hypersensitive to alkylating agent-induced apoptosis and chromosomal breakage. Cancer Res 1999;59:1544–51.
[6] Sweasy JB, Loeb LA. Detection and characterization of mammalian DNA polymerase beta mutants by functional complementation in Escherichia coli. Proc Natl Acad Sci USA 1993;90:4626–30.
[7] Feig DI, Loeb LA. Mechanisms of mutation by oxidative DNA damage: reduced fidelity of mammalian DNA polymerase. Biochemistry 1993;32:4466–73.
[8] Dianov GL, Thybo T, Dianova II, Lipinski LJ, Bohr VA. Single nucleotide patch base excision repair is the major pathway for removal of thymine glycol from DNA in human cell extracts. J Biol Chem 2000;275:11809–13.
[9] Liming Wang, Nandan Bhattacharyya.et al. A Novel Nuclear Protein, MGC5306 Interacts with DNA Polymeraseβand Has a Potential Role in Cellular Phenotype. CANCER RESEARCH, 2004, 64:7673~7677
[10] Julia J Gorski, Shalini Pathak, Kostya Panov, Taciana Kasciukovic, Tanya Panova, Jackie Russell and Joost CBM Zomerdijk. The EMBO Journal (2007) 26, 1560–1568
[11] Hahn S (1998) The role of TAFs in RNA polymerase II transcription. Cell 95: 579–582
[12] Grummt I (1999) Regulation of mammalian ribosomal gene transcription by RNA polymerase I. Prog Nucleic Acid Res Mol Biol 62:109–154
[13] Grummt I (2003) Life on a planet of its own: regulation of RNA polymerase I transcription in the nucleolus. Genes Dev 17:1691–1702
[14] Reeder RH (1999) Regulation of RNA polymerase I transcription in yeast and vertebrates. Prog Nucleic Acid Res Mol Biol 62:293–327
[15] Nomura M (2001) Ribosomal RNA genes, RNA polymerases,nucleolar structures, and synthesis of rRNA in the yeast Saccharomyces cerevisiae. Cold Spring Harb Symp Quant Biol 66: 555–565
[16] Moss T (2004) At the crossroads of growth control; making ribosomal RNA. Curr Opin Genet Dev 14: 210–217
[17] Moss T, Stefanovsky VY (2002) At the center of eukaryotic life. Cell 109: 545–548
[18] Zomerdijk JCBM, Beckmann H, Comai L, Tjian R (1994) Assembly of transcriptionally active RNA polymerase I initiation factor SL1 from recombinant subunits. Science 266: 2015–2018
[19] MELUH P B, KOSHLAND D, et al. Evidence that the MIF2 gene of Saccharomyces cerevisiae encodes a centromere protein with homology to the mammalian centromere protein CENP-C [J]. MolBiol Cell, 1995, 6: 793~807.
[20] BOHREN KM, NADKARN IV, SONG J H, et al. A M55V polymorphism in a novel SUMO gene ( SUMO-4 ) differentially activates heat shock transcription factors and is associated with susceptibility to type I diabetes mellitus [ J ] Bio Chem, 2004, 279(26) : 27233~27238
[21] Jacob S et al. Nature Rev Mol Cell Biol, 2003, 4:690 ~699
[22] PICHLER A, MELCHIOR F, et al. Ubiquitin-related modifer SUMO-1 and nucleo-cytoplasmic transport [J]. Traffic, 2002, 3:381~387
[23]叶晓峰,吴乔.类泛素蛋白---SUMO[J].细胞生物学杂志,2004,26(1):10~14 Ye X F, Wu Q. Chinese Journal of Cell Biology, 2004. 26(1):10~14
[24] Saitoh H, Hinchey J. Functional heterogeneity of small ubiquitin-related protein modifiers SUMO-1 versus SUMO-2/3[J]. J Biol Chem, 2000, 275: 6252~6258
[25] R. S. Hilgarth, L. A. Murphy, H. S. Skaggs, et al. Regulation and function of SUMO modification. J Bio Chem, 2004, 279(52): 53899~53902.
[26] DOHMEN R J. SUMO protein modification [J] 1 SUMO protein modification [J] Biochim Biophys Acta, 2004, 1695 (1 - 3):113~131
[27] Treuter E, Gustafsson J A. Wrestling rules in transrepression: as easy as SUMO-1, -2, -3 [J]. Mol Cell, 2007, 25(2):178~180
[28] Grace Gill, et al. SUMO and ubiquitin in the nucleus: different functions, similar mechanisms? Gene and Development, 2004, 18:2046-2059
[29]陈泉,施蕴渝.小泛素相关修饰物SUMO研究进展[J].生命科学, 2004, 16(1):1~6 Chen Q, Shi Y Y. Chinese Bulletin of Life Sciences, 2004. 16(1):1~6
[30] M. B. Kroetz. SUMO: a ubiquitin-like protein modifier. Yale J Biol Med, 2005, 78(4):197~201.
[31] Alexis Verger, Jose Perdomo, Merlin Crossley. Modification with SUMO A role in transcriptional regulation[J]. European Molecular Biology Organization, 2003, 4(2):137~142.
[32] Gill G. Post-translational modification by the small ubiquitin-related modifier SUMO has big effects on transcription factor activity [J] Curr Opin Genet Dev, 2003, 13(2):108~113
[33] Rodriguez MS. J Biol Chem, 2001, 276:12654~12959
[34] Hay R T. SUMO: a history of modification [J]. Mol Cell, 2005, 18 (1):1~12
[35] Cheng J, Bawa T, Leey P, et al. Role of desumoylation in the development of prostate cancer [J ] . Neoplasia, 2006, 8(8): 667~676
[36] Desterro JM, et al. Mol Cell, 1998, 2: 233 ~239
[37] Sternsdorf T,Jensen K,Freemont P S. SUMO. Curr Biol, 2003, 13:R258~R259
[38] Saitoh H,Hinchey J. Functional beterogeneity of small ubiquitin-related protein modifiers SUMO-1 versus SUMO-2/3. J Biol Chem, 2000, 275:6252~6258
[39] Dellaire G, Farrall R, Bickmore W A. The nuclear protein database (NPD): sub-nuclear localization and functional annotation of the nuclear proteome [J]. Nucl Acids Res, 2003, 31: 328~33
[40] Rodriguez MS, Desterro JM, Lain S, et al. SUMO-1 modification activates the transcriptional response of p53.EMBO J, 1999, 18(22):6455~6461
[41] Muller S, Ledl A, Schmidt D. SUMO: a regulator of gene expression and genome integrity. Oncogene.2004, 23 (11): 1998~2008
[42] Melchior F. SUMO—nonclassical ubiquitin. Annu Rev Cell Dev Biol. 2000, 16:591~626
[43] Mao Y, Sun M, Desai SD, et al. SUMO-1 conjugation to topoisomerase I: A possible repair response to topoisomerase - mediated DNA damage. Proc Natl Acad Sci USA,2000,97(8):4046~4051。
[44] Shen Z, Pardington - Purtymun PE, Comeaux JC, et al. Associatios of UBE2I with RAD52, UBL1, p53 and RAD51 proteins in a yeast two– hybrid system. Genomics,1996, 37(2):183~186。
[45] Gottifredi V, Prives C. P53 and PML: new partners in tumor suppression. Trends Cell Biol,2001, 11(5):184~187
[46] Tojo M, Matsuzaki K, Minami T. et al. The aryl hydrocarbon receptor nuclear transporter is modulated by the SUMO-1 conjugation system.J BioChem, 2002,277 (48):46576~46585
[47] Jeffrey G, Marblestone, Tauseef R. Butt. et al. Comparison of SUMO fusion technology with traditional gene fusion systems: Enhanced expression and solubility with SUMO. Protein Science, 2005, 10(12):182~189
[48] Zhang X, Studier FW. Mechanism of inhibition of bacteriophage T7 RNA polymerase and T7 lysozyme [J]. J Mol Biol, 1997, 269(1):10~27.
[49] Lyakhov D L, He B, Zhang X. Pausing and termination by bacteriophage T7 RNA polymerase [J]. J Mol Biol.1998, 280(2):201~213.
[50] Zhang X, Studier F W. Multiple roles of T7 RNA polymerase and T7 lysozyme during bacteriophage T7 infection [J]. J Mol Biol. 2004, 340(4): 707~730.
[51] Sheen J, H wang S, et al. Green-fluorescent protein as a new vital marker in plant cell [J]. Plant, 1995, 8(5):777~784
[52] Eric J Richards, Singhakowinta A, Brennan MJ. Studes on prolactin in human serum urine and milk. Hormone Res, 1975, 6:310~320