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Residual structure and constituent proteins of the peripheral framework of the cell nucleus in somatic embryos from Daucus carota L.

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Abstract

Nuclei were isolated from somatic embryos of carrot (Daucus carota L.) using a buffer system containing non-ionic detergent. To prepare nuclear matrices, the purified membrane-depleted nuclei were digested with DNase I in combination with RNase A, followed by extraction with 1 M NaCl. The DNA residue in the final insoluble fraction was less than 4% of that in isolated nuclei, and most of the residual nuclei retained their sphericity. Electron microscopy revealed that the nuclear matrix was composed of a distinct peripheral layer, an internal matrix structure and some fibrils; residual nucleoli were observed when exogeneous RNase was not incorporated. The proteins extracted from the nuclei and nuclear subfractions were compared by gel electrophoresis, which showed that the residual fraction contained many minor proteins. To identify proteins showing specific localization at the nuclear periphery, we prepared monoclonal antibodies (MAbs) against an ion-exchange chromatography fraction extracted from carrot nuclear matrices. Immunofluorescence microscopy with one of the MAbs, CML-1, showed exclusive staining of the nuclear periphery. The MAb recognized several spots showing microheterogeneity, with a narrow range of pI and molecular mass upon immunoblotting. A complete set of these spots was shown to be conserved in nuclear matrices. On the other hand, MAb CML-13 appeared to react with the nuclear interior as well as the periphery, recognizing a 96-kDa polypeptide of the nuclear matrix. These proteins were thus demonstrated to lie at the nuclear periphery, and to constitute the nuclear matrices in carrot. The 96-kDa polypeptide is suggested to be similar to the 92-kDa nuclear protein reported by Beven et al. in carrot (Beven et al., 1991, J. Cell Sci. 98, 293–302).

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Abbreviations

DEAE:

diethylaminoethyl

MAb:

monoclonal antibody

NEPHGE:

nonequilibrium pH gradient electrophoresis

References

  1. Aebi, U., Chon, J., Buhle, L., Gerace, L. (1986) The nuclear lamina is a meshwork of intermediate-type filaments. Nature 323, 560–564

  2. Aaronson, R.P., Blobel, G. (1975) Isolation of pore complex in association with a lamina. Proc. Natl. Acad. Sci. USA 72, 1007–1011

  3. Berezney, R. (1980) Fractionation of the nuclear matrix. I. Partial separation into matrix protein fibrils and a residual ribonucleoprotein fraction. J. Cell Biol. 85, 641–650

  4. Berezney, R., Coffey, D.S. (1974) Identification of a nuclear protein matrix. Biochem. Biophys. Res. Commun. 60, 1410–1417

  5. Berezney, R., Coffey, D.S. (1977) Nuclear matrix. Isolation and characterization of a framework structure from rat liver nuclei. J. Cell Biol. 73, 616–637

  6. Beven, A., Guan, Y., Peart, J., Cooper, C., Shaw, P. (1991) Monoclonal antibodies to plant nuclear matrix reveal intermediate filament-related components within the nucleus. J. Cell Sci. 98, 293–302

  7. Dougall, D.K., Wetherell, D.F. (1974) Storage of wild carrot cultures in the frozen state. Cryobiology 11, 410–415

  8. Dwyer, N., Blobel, G. (1976) A modified procedure for the isolation of a pore complex-lamina fraction from rat liver nuclei. J. Cell Biol. 70, 581–591

  9. Fey, E.G., Krochmalnic, G., Penman, S. (1986a) The nonchromatin substructures of the nucleus: the ribonucleoprotein (RNP)-containing and RNA-depleted matrices analyzed by sequential fractionation and resinless section electron microscopy. J. Cell Biol. 102, 1654–1665

  10. Fey, E.G., Ornelles, D.A., Penman S. (1986b) Association of RNA with the cytoskeleton and the nuclear matrix. J. Cell Sci. Suppl. 5, 99–119

  11. Fisher, D.Z., Chaudhary, N., Blobel, G. (1986) cDNA sequencing of nuclear lamins A and C reveals primary and secondary structural homology to intermediate filament proteins. Proc. Natl. Acad. Sci. USA 83, 6450–6454

  12. Franke, W.W. (1987) Nuclear lamins and cytoplasmic intermediate filament proteins: a growing multigene family. Cell 48, 3–4

  13. Frearson, E.M., Power, J.B., Cocking, E.C. (1973) The isolation, culture and regeneration of Petunia leaf protoplasts. Dev. Biol. 33, 130–137

  14. Frederick, S.E., Mangan, M.E., Carey, J.B., Gruber, P.J. (1992) Intermediate filament antigens of 60 and 65 kDa in the nuclear matrix of plants: their detection and localization. Exp. Cell Res. 199, 213–222

  15. Fujimura, T., Komamine, A., Matsumoto, H. (1980) Aspects of DNA, RNA and protein synthesis during somatic embryogenesis in a carrot cell suspension culture. Physiol. Plant. 49, 255–260

  16. Galcheva-Gargova, Z.I., Marinova, E.I., Koleva, S.T. (1988) Isolation of nuclear shells from plant cells. Plant Cell Environ. 11, 819–825

  17. Georgatos, S.D., Maroulakou, I., Blobel, G. (1989) Lamin A, lamin B and lamin B receptor analogues in yeast. J. Cell Biol. 108, 2069–2082

  18. Gerace, L., Blum, A., Blobel, G. (1978) Immunochemical localization of the major polypeptides of the nuclear pore complex-lamina fraction: interphase and mitotic distribution. J. Cell Biol. 79, 546–566

  19. Ghosh, S., Dey, R. (1986) Nuclear matrix network in Allium cepa. Chromosoma 93, 429–434

  20. Hancock, R., Hughes, M.E. (1982) Organization of DNA in the interphase nucleus. Biol. Cell 44, 201–212

  21. Holt, G.D., Snow, C.M., Senior, A., Haltiwanger, R.S., Gerace, L., Hart, G.W. (1987) Nuclear pore complex glycoproteins contain cytoplasmically disposed O-linked N-acetylglucosamine. J. Cell Biol. 104, 1157–1164

  22. Inagaki, M., Gonda, Y., Matsuyama, M., Nishizawa, K., Nishi, Y., Sato, C. (1988) Intermediate filament reconstitution in vitro: The role of phosphorylation on the assembly-disassembly of desmin. J. Biol. Chem. 263, 5970–5978

  23. Inoue, M., Hosaka, M., Matsumoto, T. (1984) Antigenic characterization of a Japanese common strain of tobacco mosaic virus by monoclonal antibodies. Ann. Phytopath. Soc. Japan 50, 19–26

  24. Jordan, E.G. (1984) Nucleolar nomenclature. J. Cell Sci. 67, 217–220

  25. Kaufmann, S.H., Shaper, J.H. (1984) A subset of non-histone nuclear proteins reversibly stabilized by the sulfhydryl crosslinking reagent tetrathionate. Exp. Cell Res. 155, 477–495

  26. Krachmarov, C., Stoilov, L., Zlatanova, J. (1991) Nuclear matrix from transcriptionally active and inactive plant cells. Plant Sci. 76, 35–41

  27. Krohne, G., Benavente, R. (1986) The nuclear lamins: a multigene family of proteins in evolution and differentiation. Exp. Cell Res. 162, 1–10

  28. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–684

  29. Lebkowski, J.S., Laemmli, U.K. (1982) Non-histone proteins and long-range organization of HeLa interphase DNA. J. Mol. Biol. 156, 325–344

  30. LeStourgeon, W.C., Beyer, A.L. (1977) The rapid isolation, highresolution electrophoretic characterization, and purification of nuclear proteins. Methods Cell Biol. 16, 387–406

  31. Masuda, K., Takahashi, S., Nomura, K., Inoue, M (1991) A simple procedure for the isolation of pure nuclei from carrot embryos in synchronized cultures. Plant Cell Rep. 10, 329–333

  32. McKeon, F.D., Kirschner, M.W., Caput, D. (1986) Homologies in both primary and secondary structure between nuclear envelope and intermediate filament proteins. Nature 319, 463–468

  33. Moreno Diaz de la Espina, S., Barthellemy, I., Cerezuela, M.A. (1991) Isolation and ultrastructural characterization of the residual nuclear matrix in a plant cell system. Chromosoma 100, 110–117

  34. Murashige, T., Skoog, F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473–497

  35. O'Farrell, P.Z., Goodman, H.M., O'Farrell, P.H. (1977) High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell 12, 1133–1142

  36. Pruss, R.M., Mirsky, R., Raff, M.C., Thorpe, R., Dowding, A.J., Anderson, B.H. (1981) All classes of intermediate filaments share a common antigenic determinant defined by a monoclonal antibody. Cell 27, 419–428

  37. Scofield, G.N., Beven, A.F., Shaw, P.J., Doonan, J.H. (1992) Identification and localization of a nucleoporin-like protein component of the plant nuclear matrix. Planta 187, 414–420

  38. Snow, C.M., Senior, A., Gerace, L. (1987) Monoclonal antibodies identify a group of nuclear pore complex glycoproteins. J. Cell Biol. 104, 1143–1156

  39. Towbin, J., Staehelin, T., Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. Proc. Natl. Acad. Sci. USA 76, 4350–4354

  40. Traub, P., Scherbarth, A., Willingale-Theune, J., Traub, U. (1988) Large scale co-isolation of vimentin and nuclear lamins from Ehrlich ascites tumor cells cultured in vitro. Prep. Biochem. 18, 381–404

  41. Verheijen, R., van Venrooij, W., Ramaekers, F. (1988) The nuclear matrix: structure and composition. J. Cell Sci. 90, 11–36

  42. Worman, H.J., Henriquez, R., Schnell, D.J., Newman, J.L., Georgatos, S.D., Blobel, G (1989) Identification of a lamin B-like protein in plant cell nuclei. J. Cell Biol. 109, 133a

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Author information

Correspondence to Kiyoshi Masuda.

Additional information

We wish to thank Ms. Akiko Itoh for excellent technical assistance. This work was supported by a Grant-in-Aid (05640738) from the Ministry of Education of Japan.

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Masuda, K., Takahashi, S., Nomura, K. et al. Residual structure and constituent proteins of the peripheral framework of the cell nucleus in somatic embryos from Daucus carota L.. Planta 191, 532–540 (1993). https://doi.org/10.1007/BF00195755

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Key words

  • Daucus (nuclear structure)
  • Nuclear matrix
  • Nuclear protein
  • Nucleus (structure)
  • Somatic embryo