Abstract
Apoptosis or programmed cell death is essential in the process of controlling lymphocyte growth and selection. We identified RNA polymerase B transcription factor 3 (BTF3), which is associated with anti-IgM antibody-mediated apoptosis, using a subclone of the human Burkitt lymphoma cell line BL60. To identify the transcription factor BTF3, which is expressed only in minor amounts, we used preparative high-resolution two-dimensional gel electrophoresis (2DE) employing carrier ampholytes for isoelectric focusing. Comparison of the 2DE protein patterns from apoptotic and nonapoptotic cells showed BTF3 as a predominantly altered protein spot. The characterization of the differentially expressed transcription factor and 13 marker proteins described in this study were performed by internal Edman microsequencing and/or by peptide mass fingerprinting using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The proteome analysis was significantly improved by performing the newly developed preparative high-resolution two-dimensional gels employing high protein concentrations.
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REFERENCES
Aebersold, R., and Leavitt, J. (1990). Electrophoresis 11, 517–527.
Bjellqvist, B., Sanchez, J. C., Pasquali, C., Ravier, F., Paquet, N., Frutiger, S., Hughes, G. J., and Hochstrasser, D. (1993). Electrophoresis 14, 1375–1378.
Boise, L. H., and Thompson, C. B. (1996). Science 274, 67–68.
Brockstedt, E., Rickers, A., Kostka, S., Laubersheimer, A., Dörken, B., Wittmann-Liebold, B., Bommert, K., and Otto, A. (1998). J. Biol. Chem. 273, 28057–28064.
Deng, J. M., and Behringer, R. R. (1995). Transgenic Res. 4, 264–269.
Eckerskorn, C., Jungblut, P., Mewes, W., Klose, J., and Lottspeich, F. (1988). Electrophoresis 9, 830–838.
Emoto, Y., Manome, Y., Meinhardt, G., Kisaki, H., Kharbanda, S., Robertson, M., Ghayur, T., Wong, W. W., Kamen, R., Weichselbaum, R., and Kufe, D. (1995). EMBO J. 14, 6148–6156.
Gevaert, K., Rider, M., Puype, M., Van Damme, J., De Boeck, S., and Vandekerckhove, J. (1995). In Methods in Structure Analysis (Atassi, Z. M., and Apella, E., eds.), Plenum Press, New York, pp. 15–20.
Görg, A., Postel, W., Domscheit, A., and Günther, S. (1988). Electrophoresis 9, 531–546.
Henzel, W. J., Billeci, T. M., Stults, J. T., Wong, S. C., Grimley, C., and Watanabe, C. (1993). Proc. Natl. Acad. Sci. USA 90, 5011–5015.
Jacobson, M. D., Weil, M., and Raff, M. C. (1997). Cell 88, 347–354.
Kerr, J. F., Wyllie, A. H., and Currie, A. R. (1972). Br. J. Cancer 26, 239–257.
Klose, J. (1975). Humangenetik 26, 231–243.
Klose, J., and Kobalz, U. (1995). Electrophoresis 16, 1034–1059.
Laemmli, U. K. (1970). Nature 227, 680–685.
Lazebnik, Y. A., Kaufmann, S. H., Desnoyers, S., Poirier, G. G., and Earnshaw, W. C. (1994). Nature 371, 346–347.
Martin, S. J., Green, D. R., and Cotter, T. G. (1994). Trends Biochem. Sci. 19, 26–30.
Mashima, T., Naito, M., Noguchi, K., Miller, D. K., Nicholson, D. W., and Tsuruo, T. (1997). Oncogene 14, 1007–1012.
Meltzer, N. M., Tous, G. I., Gruber, S., and Stein, S. (1987). Anal. Biochem. 160, 356–361.
O'Farrell, P. H. (1975). J. Biol. Chem. 250, 4007–4021.
Otto, A., Thiede, B., Müller, E. C., Scheler, C., Wittmann Liebold, B., and Jungblut, P. (1996). Electrophoresis. 17, 1643–1650.
Pasternack, M. S., Bleier, K. J., and McInerney, T. N. (1991). J. Biol. Chem. 266, 14703–14708.
Rao, L., Perez, D., and White, E. (1996). J. Cell Biol. 135, 1441–1455.
Rickers, A., Brockstedt, E., Mapara, M. Y., Otto, A., Dörken, B., and Bommert, K. (1998). Eur. J. Immunol. 28, 296–304.
Song, Q. I. (1996). EMBO J. 15, 3238–3246.
Steller, H. (1995). Science 267, 1445–1449.
Thiede, B., Otto, A., Zimny-Arndt, U., Müller, E. C., and Jungblut, P. (1996). Electrophoresis 17, 588–599.
Waterhouse, N., et al. (1996). J. Biol. Chem. 271, 29335–29341.
Wittmann-Liebold, B., and Jungblut, P. (1994). In Concepts in Protein Engineering and Design (Wrede, P., and Schneider, G., eds.), de Gruyter, Berlin, pp. 47–107.
Yokote, Y., Arai, K. M., and Akahane, K. (1986). Anal. Biochem. 152, 245–249.
Zheng, X. M., Moncollin, V., Egly, J. M., and Chambon, P. (1987). Cell 50, 361–368.
Zheng, X. M., Black, D., Chambon, P., and Egly, J. M. (1990). Nature 344, 556–559.
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Brockstedt, E., Otto, A., Rickers, A. et al. Preparative High-Resolution Two-Dimensional Electrophoresis Enables the Identification of RNA Polymerase B Transcription Factor 3 as an Apoptosis-Associated Protein in the Human BL60–2 Burkitt Lymphoma Cell Line. J Protein Chem 18, 225–231 (1999). https://doi.org/10.1023/A:1020636308270
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DOI: https://doi.org/10.1023/A:1020636308270