Abstract
The nuclear matrix is concealed by a much larger mass of chromatin, which can be removed selectively by digesting nuclei with DNase I followed by elution of chromatin with 0.25 M ammonium sulfate. This mild procedure removes chromatin almost completely and preserves nuclear matrix morphology. The complete nuclear matrix consists of a nuclear lamina with an interior matrix composed of thick, polymorphic fibers and large masses that resemble remnant nucleoli. Further extraction of the nuclear matrices of HeLa or MCF-7 cells with 2 M sodium chloride uncovered a network of core filaments. A few dark masses remained enmeshed in the filament network and may be remnants of the nuclear matrix thick fibers and nucleoli. The highly branched core filaments had diameters of 9 and 13 nm measured relative to the intermediate filaments. They may serve as the core structure around which the matrix is constructed. The core filaments retained 70% of nuclear RNA. This RNA consisted both of ribosomal RNA precursors and of very high molecular weight hnRNA with a modal size of 20 kb. Treatment with RNase A removed the core filaments. When 2 M sodium chloride was used directly to remove chromatin after DNase I digestion without a preceding 0.25 M ammonium sulfate extraction, the core filaments were not revealed. Instead, the nuclear interior was filled with amorphous masses that may cover the filaments. This reflected a requirement for a stepwise increase in ionic strength because gradual addition of sodium chloride to a final concentration of 2 M without an 0.25 M ammonium sulfate extraction uncovered core filaments.
Full Text
The Full Text of this article is available as a PDF (4.5 MB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Barrack E. R., Coffey D. S. The specific binding of estrogens and androgens to the nuclear matrix of sex hormone responsive tissues. J Biol Chem. 1980 Aug 10;255(15):7265–7275. [PubMed] [Google Scholar]
- Barrack E. R. The nuclear matrix of the prostate contains acceptor sites for androgen receptors. Endocrinology. 1983 Jul;113(1):430–432. doi: 10.1210/endo-113-1-430. [DOI] [PubMed] [Google Scholar]
- Berezney R., Coffey D. S. Identification of a nuclear protein matrix. Biochem Biophys Res Commun. 1974 Oct 23;60(4):1410–1417. doi: 10.1016/0006-291x(74)90355-6. [DOI] [PubMed] [Google Scholar]
- Berezney R., Coffey D. S. Nuclear matrix. Isolation and characterization of a framework structure from rat liver nuclei. J Cell Biol. 1977 Jun;73(3):616–637. doi: 10.1083/jcb.73.3.616. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Berezney R., Coffey D. S. Nuclear protein matrix: association with newly synthesized DNA. Science. 1975 Jul 25;189(4199):291–293. doi: 10.1126/science.1145202. [DOI] [PubMed] [Google Scholar]
- Capco D. G., Krochmalnic G., Penman S. A new method of preparing embeddment-free sections for transmission electron microscopy: applications to the cytoskeletal framework and other three-dimensional networks. J Cell Biol. 1984 May;98(5):1878–1885. doi: 10.1083/jcb.98.5.1878. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Capco D. G., Penman S. Mitotic architecture of the cell: the filament networks of the nucleus and cytoplasm. J Cell Biol. 1983 Mar;96(3):896–906. doi: 10.1083/jcb.96.3.896. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Capco D. G., Wan K. M., Penman S. The nuclear matrix: three-dimensional architecture and protein composition. Cell. 1982 Jul;29(3):847–858. doi: 10.1016/0092-8674(82)90446-9. [DOI] [PubMed] [Google Scholar]
- Ciejek E. M., Tsai M. J., O'Malley B. W. Actively transcribed genes are associated with the nuclear matrix. Nature. 1983 Dec 8;306(5943):607–609. doi: 10.1038/306607a0. [DOI] [PubMed] [Google Scholar]
- Fedoroff N., Wellauer P. K., Wall R. Intermolecular duplexes in heterogeneous nuclear RNA from HeLa cells. Cell. 1977 Apr;10(4):597–610. doi: 10.1016/0092-8674(77)90092-7. [DOI] [PubMed] [Google Scholar]
- Fey E. G., Krochmalnic G., Penman S. The nonchromatin substructures of the nucleus: the ribonucleoprotein (RNP)-containing and RNP-depleted matrices analyzed by sequential fractionation and resinless section electron microscopy. J Cell Biol. 1986 May;102(5):1654–1665. doi: 10.1083/jcb.102.5.1654. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fey E. G., Penman S. Nuclear matrix proteins reflect cell type of origin in cultured human cells. Proc Natl Acad Sci U S A. 1988 Jan;85(1):121–125. doi: 10.1073/pnas.85.1.121. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Harpold M. M., Wilson M. C., Darnell J. E., Jr Chinese hamster polyadenylated messenger ribonucleic acid: relationship to non-polyadenylated sequences and relative conservation during messenger ribonucleic acid processing. Mol Cell Biol. 1981 Feb;1(2):188–198. doi: 10.1128/mcb.1.2.188. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hentzen P. C., Rho J. H., Bekhor I. Nuclear matrix DNA from chicken erythrocytes contains beta-globin gene sequences. Proc Natl Acad Sci U S A. 1984 Jan;81(2):304–307. doi: 10.1073/pnas.81.2.304. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jackson D. A., Cook P. R. Transcription occurs at a nucleoskeleton. EMBO J. 1985 Apr;4(4):919–925. doi: 10.1002/j.1460-2075.1985.tb03719.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jackson D. A., Cook P. R. Visualization of a filamentous nucleoskeleton with a 23 nm axial repeat. EMBO J. 1988 Dec 1;7(12):3667–3677. doi: 10.1002/j.1460-2075.1988.tb03248.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lehrach H., Diamond D., Wozney J. M., Boedtker H. RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination. Biochemistry. 1977 Oct 18;16(21):4743–4751. doi: 10.1021/bi00640a033. [DOI] [PubMed] [Google Scholar]
- Lothstein L., Arenstorf H. P., Chung S. Y., Walker B. W., Wooley J. C., LeStourgeon W. M. General organization of protein in HeLa 40S nuclear ribonucleoprotein particles. J Cell Biol. 1985 May;100(5):1570–1581. doi: 10.1083/jcb.100.5.1570. [DOI] [PMC free article] [PubMed] [Google Scholar]
- McCready S. J., Godwin J., Mason D. W., Brazell I. A., Cook P. R. DNA is replicated at the nuclear cage. J Cell Sci. 1980 Dec;46:365–386. doi: 10.1242/jcs.46.1.365. [DOI] [PubMed] [Google Scholar]
- Mirkovitch J., Mirault M. E., Laemmli U. K. Organization of the higher-order chromatin loop: specific DNA attachment sites on nuclear scaffold. Cell. 1984 Nov;39(1):223–232. doi: 10.1016/0092-8674(84)90208-3. [DOI] [PubMed] [Google Scholar]
- Nickerson J. A., Krochmalnic G., Wan K. M., Penman S. Chromatin architecture and nuclear RNA. Proc Natl Acad Sci U S A. 1989 Jan;86(1):177–181. doi: 10.1073/pnas.86.1.177. [DOI] [PMC free article] [PubMed] [Google Scholar]
- O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
- Pardoll D. M., Vogelstein B., Coffey D. S. A fixed site of DNA replication in eucaryotic cells. Cell. 1980 Feb;19(2):527–536. doi: 10.1016/0092-8674(80)90527-9. [DOI] [PubMed] [Google Scholar]
- Penman S., Vesco C., Penman M. Localization and kinetics of formation of nuclear heterodisperse RNA, cytoplasmic heterodisperse RNA and polyribosome-associated messenger RNA in HeLa cells. J Mol Biol. 1968 May 28;34(1):49–60. doi: 10.1016/0022-2836(68)90234-9. [DOI] [PubMed] [Google Scholar]
- Penman S., Vesco C., Weinberg R., Zylber E. The RNA metabolism of nucleoli and mitochondria in mammalian cells. Cold Spring Harb Symp Quant Biol. 1969;34:535–546. doi: 10.1101/sqb.1969.034.01.061. [DOI] [PubMed] [Google Scholar]
- Perry R. P. THE CELLULAR SITES OF SYNTHESIS OF RIBOSOMAL AND 4S RNA. Proc Natl Acad Sci U S A. 1962 Dec;48(12):2179–2186. doi: 10.1073/pnas.48.12.2179. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pollard T. D., Cooper J. A. Actin and actin-binding proteins. A critical evaluation of mechanisms and functions. Annu Rev Biochem. 1986;55:987–1035. doi: 10.1146/annurev.bi.55.070186.005011. [DOI] [PubMed] [Google Scholar]
- Rennie P. S., Bruchovsky N., Cheng H. Isolation of 3 S androgen receptors from salt-resistant fractions and nuclear matrices of prostatic nuclei after mild trypsin digestion. J Biol Chem. 1983 Jun 25;258(12):7623–7630. [PubMed] [Google Scholar]
- Robinson S. I., Nelkin B. D., Vogelstein B. The ovalbumin gene is associated with the nuclear matrix of chicken oviduct cells. Cell. 1982 Jan;28(1):99–106. doi: 10.1016/0092-8674(82)90379-8. [DOI] [PubMed] [Google Scholar]
- Ross D. A., Yen R. W., Chae C. B. Association of globin ribonucleic acid and its precursors with the chicken erythroblast nuclear matrix. Biochemistry. 1982 Feb 16;21(4):764–771. doi: 10.1021/bi00533a029. [DOI] [PubMed] [Google Scholar]
- Salditt-Georgieff M., Harpold M. M., Wilson M. C., Darnell J. E., Jr Large heterogeneous nuclear ribonucleic acid has three times as many 5' caps as polyadenylic acid segments, and most caps do not enter polyribosomes. Mol Cell Biol. 1981 Feb;1(2):179–187. doi: 10.1128/mcb.1.2.179. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Scheller R. H., Costantini F. D., Kozlowski M. R., Britten R. J., Davidson E. H. Specific representation of cloned repetitive DNA sequences in sea urchin RNAs. Cell. 1978 Sep;15(1):189–203. doi: 10.1016/0092-8674(78)90094-6. [DOI] [PubMed] [Google Scholar]
- Simmen R. C., Means A. R., Clark J. H. Estrogen modulation of nuclear matrix-associated steroid hormone binding. Endocrinology. 1984 Sep;115(3):1197–1202. doi: 10.1210/endo-115-3-1197. [DOI] [PubMed] [Google Scholar]
- Small D., Nelkin B., Vogelstein B. The association of transcribed genes with the nuclear matrix of Drosophila cells during heat shock. Nucleic Acids Res. 1985 Apr 11;13(7):2413–2431. doi: 10.1093/nar/13.7.2413. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Thorburn A., Moore R., Knowland J. Attachment of transcriptionally active DNA sequences to the nucleoskeleton under isotonic conditions. Nucleic Acids Res. 1988 Jul 25;16(14B):7183–7183. doi: 10.1093/nar/16.14.7183. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wagner B., Krochmalnic G., Penman S. Resinless section electron microscopy of HeLa cell mitotic architecture. Proc Natl Acad Sci U S A. 1986 Dec;83(23):8996–9000. doi: 10.1073/pnas.83.23.8996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zeitlin S., Parent A., Silverstein S., Efstratiadis A. Pre-mRNA splicing and the nuclear matrix. Mol Cell Biol. 1987 Jan;7(1):111–120. doi: 10.1128/mcb.7.1.111. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zeitlin S., Wilson R. C., Efstratiadis A. Autonomous splicing and complementation of in vivo-assembled spliceosomes. J Cell Biol. 1989 Mar;108(3):765–777. doi: 10.1083/jcb.108.3.765. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zhai Z. H., Nickerson J. A., Krochmalnic G., Penman S. Alterations in nuclear matrix structure after adenovirus infection. J Virol. 1987 Apr;61(4):1007–1018. doi: 10.1128/jvi.61.4.1007-1018.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]