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Results for “Nuclear Matrix”

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At least 19 recordsLinked to original sources

PubMed1988

Association of Autographa californica nuclear polyhedrosis virus (AcMNPV) with the nuclear matrix.

Nuclear matrices from uninfected Spodoptera frugiperda cells and those infected with Autographa californica nuclear polyhedrosis virus (AcMNPV) were isolated and their protein constituents were compared. Proteins were characterized according to size and several different antibodies to Drosophila nuclear proteins were employed in an attempt to identify the proteins comprising this nuclear substructure. Three species of lamins were identified as major constituents of the nuclear matrix of Spodoptera cells. Two DNA-binding proteins having molecular weights of 54 and 36 kDa were also identified as components of the nuclear matrix of uninfected cells. Infection resulted in a superimposition of viral proteins upon the nuclear matrix of the host cell. Polyhedrin, the basic viral DNA-binding protein (p6.9), and the major capsid protein of AcMNPV were identified immunologically as components of the nuclear matrix fraction of infected cells. Infection also resulted in the increased association of cellular histones with the nuclear matrix. DNA-binding assays demonstrated histones and p6.9 were the predominant DNA-binding proteins associated with the nuclear matrix of infected cells. Nuclear matrices from uninfected cells and cells infected with AcMNPV for 10 and 24 hr were examined using transmission electron microscopy. Morphologically, the nuclear matrix of the uninfected cell consists of the outer nuclear lamina (including nuclear pore complexes), an internal fibrogranular protein constituent, and a residual nucleolar structure. Numerous viral capsids were observed associated with the nuclear matrix in cells infected with either wild-type AcMNPV or a polyhedrin-deletion mutant by 10 hr p.i. The capsids appeared to be attached in an end-on association with the internal fibrogranular protein network of the nuclear matrix. The matrix-associated capsids were similar in width and length to those packaged within the polyhedra. In addition to the capsids, polyhedra in various stages of maturation were seen at 24 hr following infection of the cells with the wild-type virus. The nuclear matrix of the infected cell appears to play an important role in baculovirus assembly.

Animals↗

PubMed1992

Nuclear distribution of histone deacetylase: a marker enzyme for the internal nuclear matrix.

Nuclear matrins are proteins that localize to the internal nuclear matrix. In a previous study, we reported that histone deacetylase is a component of the internal matrix, suggesting that histone deacetylase is a nuclear matrin. Here, we demonstrate that the majority of the histone deacetylase activity is associated with the internal nuclear matrices of chicken and trout liver. Thus, the association of the histone deacetylase with the internal nuclear matrix is neither tissue- nor species-specific. Using histone deacetylase as a marker enzyme for the partitioning of the internal nuclear matrix during nuclear fractionations, we show that in contrast to the internal nuclear matrices of trout liver, trout hepatocellular carcinoma and chicken liver, the stability of the chicken erythrocyte internal nuclear matrix is temperature-dependent. Our results support a model that has the histone deacetylase mediating transient interactions between the internal nuclear matrix and chromatin regions undergoing dynamic acetylation, for example transcriptionally active chromatin regions.

Acetylation↗

PubMed1991

DNA binding properties of the nuclear matrix and individual nuclear matrix proteins. Evidence for salt-resistant DNA binding sites.

The DNA binding characteristics of the rat nuclear matrix were investigated. A saturable and temperature-dependent, salt-resistant DNA binding to the nuclear matrix was discovered, with 70-80% of total bound DNA resistant to extraction with high concentrations of salt at 37 degrees C, compared to less than 5% at 0 degrees C. The initial binding of DNA to nuclear matrix is sensitive to salt concentration, indicating a transition to a salt-resistant binding state. The nuclear matrix shows a preference for single-stranded DNA, both in saturation and competition assays, with little binding of RNA or double-stranded DNA. Further competition studies show a preference for matrix-attached DNA probably involving predominantly AT-rich sequences, while a specific sequence defined previously as a matrix-attached region (MAR; Cockerill, P. N., and Garrard, W. T. (1986) Cell 46, 273-282) only showed preference for a limited number of the total matrix binding sites. These results and estimates from saturation data of approximately 150,000 single-stranded DNA binding sites per matrix lead us to propose that the nuclear matrix contains different classes of DNA binding sites, each with a separate sequence specificity. Binding of DNA to individual matrix polypeptides separated on sodium dodecyl sulfate-polyacrylamide gels and transferred to nitrocellulose blots was also temperature-dependent, salt-resistant, and showed a preference for binding DNA over RNA and nuclear matrix DNA over total genomic DNA. Subnuclear fractionation experiments further demonstrated that the nuclear matrix is enriched in the subset of higher molecular weight (greater than 50,000) DNA binding proteins of isolated nuclei and correspondingly depleted of the lower molecular weight ones. Of the approximately 12 major proteins separated on nonequilibrium two-dimensional gels, 7 were identified as specific DNA binding proteins including lamins A and C (but not B), and the internal nuclear matrix proteins, matrins D, E, F, G, and 4.

Animals↗

PubMed1991

DNA attachment to the hamster sperm nuclear matrix and nuclear annulus.

Hamster sperm DNA is packaged so tightly that it is the most highly condensed eukaryotic DNA known. Nevertheless, the sperm genome is also organized in a very specific fashion by two nuclear structures, the nuclear matrix and the nuclear annulus. The nuclear matrix organizes sperm DNA into loop domains approximately 46 Kb in length by specific attachment sequences. When the sperm nucleus is induced to decondense, the nuclear matrix almost completely dissipates, leaving only the nuclear annulus, a structure located at the implantation fossa within the nucleus. All the DNA remains anchored to the annulus during decondensation. Rotary shadowing transmission electron microscopy of isolated nuclear annuli retaining only a small portion of this anchored DNA suggests that the DNA is anchored to the annulus in a series of mini-loops, 5-10 Kb in length. These data support our previous suggestions that sperm DNA is organized in a very specific manner.

Animals↗

PubMed1986

Transcribed human ribosomal RNA genes are attached to the nuclear matrix.

Nuclear matrices were prepared from HeLa nuclei in high or low-salt buffers. After digestion with restriction enzymes, the DNA associated with the nuclear matrix was analysed for its content of ribosomal DNA sequences (rDNA). In both salt conditions, the entire rDNA repeat unit was found to be enriched in the matrix DNA. No enrichment, however, was detected in the absence of rRNA transcription either due to treatment with actinomycin D or when cells arrested in the G0 stage were used to prepare matrices. rDNA was found to be linked to chromosomal scaffolding structures when similar experiments were performed on chromosomes. Moreover, a gradient of enrichment along the rDNA repeat unit was observed, in which the promoter-containing portion was the most highly enriched. I conclude from these experiments that the tandem repeats of rDNA are not randomly associated with matrix or scaffold structure but are probably attached at transcription complexes.

Autoradiography↗

PubMed1986

Monoclonal antibodies against nuclear matrix detect nuclear antigens in mammalian, insect and plant cells: an immunofluorescence study.

We have applied monoclonal antibodies generated against nuclear matrix in an immunofluorescence study of a variety of plant and animal species. Antibodies P1 and I1 detected antigens in all species examined, including higher and lower plants. Antibodies PI1 and PI2 stained only animal cells, and showed some tissue and/or species-specific variability in staining pattern. The presence of similar nuclear matrix components in such diverse species suggests that nuclear order may be maintained by similar mechanisms in all eukaryotes.

Animals↗

PubMed1985

Modification of nuclear matrix proteins by ADP-ribosylation. Association of nuclear ADP-ribosyltransferase with the nuclear matrix.

Nuclear matrices were isolated by treatment of isolated HeLa cell nuclei with high DNase I, pancreatic RNase and salt concentrations. ADP-ribosylated nuclear matrix proteins were identified by electrophoresis, blotting and autoradiography. In one experimental approach nuclear matrix proteins were labeled by exposure of permeabilized cells to the labeled precursor [32P]NAD. Alternatively, the cellular proteins were prelabeled with [35S]methionine and the ADP-ribosylated nuclear matrix proteins separated by aminophenyl boronate column chromatography. By both methods bands of modified proteins, though with differing intensities, were detected at 41, 43, 46, 51, 60, 64, 69, 73, 116, 140, 220 and 300 kDa. Approximately 2% of the total nuclear ADP-ribosyltransferase activity, but only 0.07% of the nuclear DNA, was tightly associated with the isolated nuclear matrix. The matrix-associated enzyme catalyzes the incorporation of [32P]ADP-ribose into acid-insoluble products of molecular mass 116 kDa and above, in a 3-aminobenzamide-inhibited, time-dependent reaction. The possible function of ADP-ribosylation of nuclear matrix proteins and of the attachment of ADP-ribosyltransferase to the nuclear matrix in the regulation of matrix-associated biochemical processes is discussed.

Adenosine Diphosphate Ribose↗

PubMed1979

Isolation and characterization of the nuclear matrix in Friend erythroleukemia cells: chromatin and hnRNA interactions with the nuclear matrix.

Nuclear matrices from undifferentiated and differentiated Friend erythroleukemia cells have been obtained by a method which removes DNA in a physiological buffer. These matrices preserved the characteristic topographical distribution of condensed and diffuse "chromatin" regions, as do nuclei in situ or isolated nuclei. Histone H1 was released from the nuclear matrix of undifferentiated cells by 0.3 M KCl; inner core histones were released by 1 M KCl. Nuclear matrix from differentiated cells did not maintain H1, and histone cores were fully released in 0.7 M KCl. KCl removed the core histones as an octameric structure with no evidence of preferential release of any single histone. Electron microscopy of KCl-treated matrix revealed no condensed regions but rather a network of fibrils in the whole DNA-depleted nuclei. When nuclear matrices from both types of cell were exposed to conditions of very low ionic strength, inner core histones and condensed regions remained. These observations support the contention that inner core histones are bound to matrix through natural ionic bonds or saline-labile elements, and that these interactions are implicated in chromatin condensation. hnRNA remained undegraded and tenaciously associated to the matrix fibrils, and was released only by chemical means which, by breaking hydrophobic and hydrogen bonds, produced matrix lysis. Very few nonhistone proteins were released upon complete digestion of DNA from either type of nuclei. The remaining nonhistone proteins represent a large number of species of which the majority may be matrix components. The molecular architecture in both condensed and diffuse regions of interphase nuclei appears to be constructed of two distinct kinds of fibers; the thicker chromatin fibers are interwoven with the thinner matrix fibers. The latter are formed by a heteropolymer of many different proteins.

Animals↗

PubMed1991

Sequence-specific binding of a c-myc nuclear-matrix-associated region shows increased nuclear matrix retention after leukemic cell (HL-60) differentiation.

HL-60 cells, a human promyelocytic leukemia cell line, contain amplified c-myc DNA sequences and mRNA transcripts. These cells can be induced to undergo macrophage differentiation by phorbol esters, which results in suppression of c-myc expression and cessation of cell proliferation. The nuclear matrix (NM), a nuclear skeleton resistant to DNase I digestion and high salt extraction, is proposed to be involved in DNA replication, gene regulation, and the correct distribution of DNA at mitosis. We have previously identified a nuclear-matrix-associated region (MAR) of the c-myc protooncogene to reside in a 1.4-kb region between Cla I and Eco RI restriction sites at the 3'-end of the gene. A 172-bp Dra I/Dra I subfragment of the 1.4-kb region was shown to be a major component of the MAR (myc-MAR), and this subfragment was demonstrated to be recognized by a nuclear protein (p25). In this report we demonstrate that phi X174 DNA, or the synthetic copolymers poly[d(G.C)] and poly[d(A.T)], are not effective suppressors of the binding of the myc-MAR to isolated NM, indicating that the binding sequence(s) are unique. We find that the addition of partially purified protein p25 increases the relative affinity of the myc-MAR for HL-60 NM in an in vitro assay system. NM isolated from HL-60 macrophages induced by phorbol esters retains significantly more myc-MAR DNA fragment in the presence of an excess amount of competitor DNA than does NM from untreated HL-60 cells. These data suggest that a change of the myc-MAR association with the NM occurs after monocytic differentiation of HL-60 cells.

Base Composition↗

PubMed1990

Core filaments of the nuclear matrix.

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.

Cell Line↗

PubMed1986

[Binding of triiodothyronine to the nuclear matrix of the rat liver. The effect of thyroid hormones on the phosphorylation of nuclear matrix proteins].

The interaction of thyroid hormones with rat liver nuclear matrix proteins was studied. It was shown that the nuclear matrix contains the sites which bind triiodothyronine with a high affinity (Ka = 1.07 X 10(9) M-1) and limited capacity (maximal binding capacity--28.5 fmol triiodothyronine/100 micrograms protein). Electrophoretic analysis of triiodothyronine-binding matrix proteins revealed that the molecular mass of the major triiodothyronine-binding fraction is 50 000-52 000 Da. Injections of triiodothyronine to thyroidectomized animals stimulated the phosphorylation of all protein fractions of the nuclear matrix.

Animals↗

PubMed1991

Immunologically-related nucleic acid-binding proteins associated with the nuclear matrix of Physarum polycephalum.

The nuclear matrix of Physarum polycephalum is composed of two abundant polypeptides of 27 and 38 kDa as well as numerous minor polypeptides of various molecular weight. By contrast, the nuclear matrix of vertebrates consists of three major (the lamins) and many minor polypeptides mainly in the 60-70 kDa molecular weight range. In order to better characterize the major nuclear matrix proteins of P. polycephalum and, perhaps, define their relationship with the major nuclear matrix proteins of vertebrates, we have purified the abundant nuclear matrix proteins of P. polycephalum. In Western blot analyses, polyclonal antibodies raised against the purified 27 kDa polypeptide recognised polypeptides of 50 kDa, 45 kDa and several low molecular weight species (14-21 kDa) in the P. polycephalum nuclear matrix. The polyclonal antibodies did not react with the other abundant nuclear matrix protein of 38 kDa from P. polycephalum nor with polypeptides of the mouse nuclear matrix. Two-dimensional gel electrophoresis showed that the major nuclear matrix proteins of P. polycephalum were more basic than the major nuclear matrix proteins of vertebrates, the lamins. Moreover, both the 27 and 38 kDa polypeptides are post-translationally modified by either D-mannosyl or D-glycosyl moieties, and not by phosphorylation as has been demonstrated for the vertebrate lamins. DNA-binding assays further revealed that the immunologically related polypeptides of 50 kDa, 45 kDa, 27 kDa and low molecular weight species of 14-21 kDa preferentially bound single-stranded DNA, but the 38 kDa polypeptide of Physarum matrix did not. Based on these findings, we conclude that the abundant nuclear matrix protein of 27 kDa belongs to a group of immunologically-related nucleic acid-binding proteins, and is immunologically and functionally distinct from the other major nuclear matrix protein of 38 kDa from P. polycephalum and the vertebrate lamins.

Autoradiography↗

PubMed1986

Role of the nuclear matrix in adenovirus maturation.

The nuclear matrix has been implicated in several important cellular processes. In this paper, we investigate the role of the nuclear matrix in adenovirus type 2 assembly. Electron microscopic examination of nuclear matrices isolated from adenovirus infected Hep-2 cells clearly reveals that late in the lytic cycle, adenovirus capsids are intimately associated with the nuclear matrix. SDS-PAGE analysis showed that the viral core polypeptides V, PVII and 11 kDa were enriched in the nuclear matrix fraction. After a 3 h chase period a constant high ratio of PVII to VII prevailed in the nuclear matrix suggesting that mostly young virions and viral cores are bound to this structure. Most of the virus maturation endoproteinase activity co-purified with the nuclear matrix and the data suggest that the enzyme may be released from fragile young virions or assembly intermediates. Together these experiments suggest that the nuclear matrix is the site of adenovirus assembly and that mature virions may be released from the matrix by the viral endoproteinase.

Adenoviruses, Human↗

PubMed1992

[Association of chromosomal telomere DNA with nuclear matrix in HeLa cells].

Nuclear matrix from HeLa cells was gently extracted with a high salt solution and treated with DNase I. DNA that remained associated with the nuclear matrix (N. M. DNA) and DNA fragments released into the supernatant (SN.DNA) were isolated respectively and dot hybridized to human telomere sequence (AGGGTT/TCCCAA)40 probe. As the time of DNase I treatment was extended, the amount of N. M. DNA decreased while the concentration of telomere sequence in N.M. DNA proportionally increased. These preliminary results suggest that the telomere sequence is tightly bound to nuclear matrix in HeLa cells.

Base Sequence↗

PubMed1989

Cytochemical localization of DNA loop attachment sites to the nuclear lamina and to the inner nuclear matrix.

The rat liver nuclear matrix, obtained by endogenous nuclease digestion and extraction with low and high ionic strength media, contains residual DNA fragments that are considered to represent the attachment sites of the chromatin domains to the nucleoskeleton. These sites, protected against nuclease digestion by their binding with the nucleoskeleton proteins, should be either mainly linked to the peripheral lamina or to the inner nuclear matrix. The DNA fragment distribution at the level of the different components of the nuclear matrix has been evaluated in samples embedded in Epon and in hydrophilic resins by means of the DNase-gold technique. The labeling obtained suggests that the chromatin loops are prevailingly associated with the interior of the matrix; in fact about twice of the label is present in the inner matrix with respect to the peripheral lamina area. These results confirm the hypothesis that in interphase the chromatin maintains an organization similar to that of chromosomes, with loops radiating from a central scaffold, instead of being mainly attached to the lamina as otherwise suggested.

Animals↗

PubMed1986

Characterization of nuclear matrix from cultured normal human fibroblasts.

Nuclear matrix was isolated from cultured human fibroblasts by extraction of nuclei with 2 M NaCl. Electron microscopic observation on the isolated nuclear matrix revealed a fine network structure. The matrix fraction contained approximately 15% of total nuclear DNA and the matrix DNA was about 3- to 4-fold enriched in transcriptionally active collagen I (alpha 2) gene sequences, whereas transcriptionally inactive beta-globin gene sequences were not enriched. The nuclear matrix contained two major proteins of 65,000 and 45,000 daltons (pI 5.9 and 5.6, respectively). The DNA-binding activity of these nuclear matrix proteins was examined by Western blotting or by nitrocellulose filter-binding assay using cloned specific gene probes. The results suggest that there is no base sequence specificity in the binding, and that protein species of 60,000 to 200,000 daltons showed DNA-binding activity. These results indicate that association of transcribing genes with the nuclear matrix may reflect the functional state of the genes and may not be determined solely by the base sequence specificity of DNA binding. The nuclear matrix protein of 65,000 daltons was phosphorylated in vivo, and was the main substrate for protein kinase(s) associated with the nuclear matrix.

Cell Nucleus↗

PubMed1990

The nuclear matrix from cells of different origin. Evidence for a common set of matrix proteins.

We compared the protein composition of the nuclear matrix isolated from several murine embryonal carcinoma cells and mature tissues by two-dimensional gel electrophoresis. Two nuclear matrix fractions were investigated: the "peripheral" nuclear matrix (matrix proteins that remain insoluble after reduction), and the "internal" nuclear matrix (matrix proteins released by reduction). The two subfractions have completely different protein compositions. Although numerous differences in nuclear matrix protein composition among different cell types were observed, a limited set of polypeptides common to all mouse cell types was identified. A majority of these common proteins was also present in cells from other mammalian species (i.e. rat and human). For this set of proteins, we coin the term "minimal matrix." As expected, lamin B, known to be expressed throughout differentiation, is part of the common set of peripheral nuclear matrix proteins. Lamins A and C are not because these proteins were absent from undifferentiated embryonal carcinoma cells. Since these common nuclear matrix proteins occur in all mammalian nuclear matrices analyzed so far, it is likely that they have a basic role in nuclear organization and function.

Animals↗

PubMed1991

Electrophoretic analysis of nuclear matrix proteins and the potential clinical applications.

Nuclear matrix proteins form the skeleton of the nucleus and participate in the various cellular functions of the nucleus. These proteins have been demonstrated to be tissue-type specific and can potentially reflect changes in the state of differentiation of the cell. Elucidating nuclear matrix protein changes necessitates the use of high-resolution two-dimensional polyacrylamide gel electrophoresis. Separation of this complex mixture into its component parts resolves protein changes when comparing the normal state to a diseased state of a cell. Evidence has been reviewed which shows the potential use of nuclear matrix proteins and antibodies to nuclear matrix proteins as diagnostic tools for various cancers, autoimmune diseases, adenoviral infection, and other diseases. Consequently, the central functions of the nuclear matrix in the cell allow it to have significant potential as a diagnostic agent.

Electrophoresis, Gel, Two-Dimensional↗