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At least 361 records · Page 20Linked to original sources
PubMed1978

Simian virus 40 (SV40)-specific proteins associated with the nuclear matrix isolated from adenovirus type 2-SV40 hybrid virus-infected HeLa cells carry SV40 U-antigen determinants.

The distribution of simian virus 40 (SV40)-specific proteins in nuclear subfractions of pulse-chase-labeled HeLa cells infected with nondefective adenovirus type 2 (Ad2)-SV40 hybrid viruses was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The SV40-specific proteins of Ad2+ND1, Ad2+ND2, and Ad2+ND5 specifically associate with the nuclear matrix and are virtually absent from the high-salt nuclear extract. In Ad2+ND4-infected HeLa cells, the SV40-specific proteins with molecular weights of 64,000 (64K) and lower also specifically associate with the nuclear matrix. The SV40-specific 72K, 74K, and 95K proteins were found both in the nuclear matrix and in the high-salt nuclear extract. Analyses of the nuclear matrices isolated from hybrid virus-infected cells by immunofluorescence microscopy showed that SV40 U-antigen-positive sera from SV40 tumor-bearing hamsters react with SV40-specific proteins integrated into nuclear matrices of HeLa cells infected by Ad2+ND1, Ad2+ND2, and Ad2+ND4, but not with nuclear matrices of HeLa cells infected by Ad2+ND5. This suggests that SV40-specific proteins of Ad2+ND1, Ad2+ND2, and Ad2+ND4 integrated into the nuclear matrix carry SV40 U-antigen determinants. The apparent discrepancy in the subcellular localization of SV40-specific proteins in hybrid virus-infected cells when analyzed by biochemical cell fractionation procedures and when analyzed by immunofluorescence staining is discussed.

Adenoviruses, Human↗
PubMed1984

Control of late simian virus 40 transcription by the attenuation mechanism and transcriptionally active ternary complexes are associated with the nuclear matrix.

Isolated nuclei derived from simian virus 40 (SV40)-infected cells and incubated with [alpha-32P]UTP can elongate the in vivo preinitiated SV40 late RNA, synthesizing a viral RNA species 94 nucleotides long (attenuator RNA) as well as longer RNA molecules. In contrast to newly synthesized SV40 RNA, the attenuator RNA is not associated with the nuclear matrix. Pretreating the cells with 5,6-dichloro-1-beta-ribofuranosylbenzimidazole before the incubation of isolated nuclei in vitro, enhances the accumulation of the attenuator RNA, but again it is removed from nuclei by DNase and high salt. In contrast, pretreating the cells with proflavine, an intercalating drug that interferes with RNA secondary structure, prevents the accumulation of the attenuator RNA and increases the amount of the long RNA molecules. These RNA molecules become associated with the nuclear matrix. Isolated nuclear matrices from SV40-infected cells are highly enriched in transcriptionally active ternary complexes. Thus, isolated nuclear matrices that contain from 2 to 6% of SV40 DNA are capable of synthesizing at least 35% of the viral RNA synthesized in isolated nuclei after 2 to 15 minutes incubation with [alpha-32P]UTP. The RNA synthesized in vitro on purified nuclear matrices and isolated nuclei is derived from the same regions of the viral genome, suggesting that there is an association between transcribed DNA sequences and the nuclear matrix. The results suggest a major role for the nuclear matrix in controlling SV40 gene expression.

Base Sequence↗
PubMed1997

Subcellular partitioning of transcription factors during osteoblast differentiation: developmental association of the AML/CBF alpha/PEBP2 alpha-related transcription factor-NMP-2 with the nuclear matrix.

The subnuclear location of transcription factors may functionally contribute to the regulation of gene expression. Several classes of gene regulators associate with the nuclear matrix in a cell type, cell growth, or cell cycle related-manner. To understand control of nuclear matrix-transcription factor interactions during tissue development, we systematically analyzed the subnuclear partitioning of a panel of transcription factors (including NMP-1/YY-1, NMP-2/AML, AP-1, and SP-1) during osteoblast differentiation using biochemical fractionation and gel shift analyses. We show that nuclear matrix association of the tissue-specific AML transcription factor NMP-2, but not the ubiquitous transcription factor YY1, is developmentally upregulated during osteoblast differentiation. Moreover, we show that there are multiple AML isoforms in mature osteoblasts, consistent with the multiplicity of AML factors that are derived from different genes and alternatively spliced cDNAs. These AML isoforms include proteins derived from the AML-3 gene and partition between distinct subcellular compartments. We conclude that the selective partitioning of the YY1 and AML transcription factors with the nuclear matrix involves a discriminatory mechanism that targets different classes and specific isoforms of gene regulatory factors to the nuclear matrix at distinct developmental stages. Our results are consistent with a role for the nuclear matrix in regulating the expression of bone-tissue specific genes during development of the mature osteocytic phenotype.

Animals↗
PubMed2002

The interaction of p53 with the nuclear matrix is mediated by F-actin and modulated by DNA damage.

The tumour suppressor protein p53 is localized in the cell nucleus where it serves to initiate cellular responses to a variety of stresses, particularly DNA damage and has the capacity to transactivate stress response genes. An emerging body of evidence indicates that its action is also exerted through direct protein-protein interactions. An approach to understanding p53 function has been to analyse its positioning in relation to nuclear structures and we have shown that p53 can associate with the nuclear matrix. A potential nuclear matrix component for this association is actin. Here we show that p53 interacts with nuclear F-actin and we map the domains involved in this interaction. Using fluorescence resonance energy transfer, we demonstrate that the partition of p53 between F-actin bound and unbound forms is not constant, but is modulated by the presence of DNA damage, which increases binding. Our results indicate that the dynamic interaction of p53 with the nuclear matrix has to be considered for a full understanding of the mechanisms of the p53-mediated cellular response to DNA damage.

3T3 Cells↗
PubMed1993

DNA precursors are channelled to nuclear matrix DNA replication sites.

Studies of replicative DNA synthesis using DNA precursors have shown that the DNA that was replicated most recently is that associated with the nuclear matrix. Consequently, precursors arising via the salvage and the de novo metabolic pathways are first incorporated into a small percentage of the total nuclear DNA that is termed nuclear matrix-associated DNA. These results have been substantiated in cell culture, as well as in intact mammalian systems. Furthermore, when DNA precursors were injected intravenously into regenerating rat liver, a significant lag in the incorporation of orotic acid-derived nucleotides (de novo pathway precursors) into nuclear DNA was observed, when compared with deoxythymidine-derived nucleotides (salvage pathway precursors). This lag in incorporation kinetics was also evident at the nuclear matrix level, although, once incorporated into nuclear matrix-associated DNA, the distribution patterns of both precursors into extra-matrix nuclear DNA fractions were identical. To determine the basis for this kinetic lag, we compared the incorporation kinetics of orotic acid and of deoxythymidine into dTTP and into nuclear matrix-associated DNA, respectively. Orotic acid-derived nucleotides entered the cytosolic dTTP pool before being incorporated into nuclear matrix-associated DNA, that is, traversing the classical metabolic route of DNA precursors. Conversely, deoxythymidine-derived nucleotides by-passed the soluble dTTP cellular pool and engaged directly in DNA synthesis at the nuclear matrix. Not only is this the first evidence for nucleotide channelling in an intact mammalian system, but it also forms direct evidence that salvage pathway DNA precursors are channelled to nuclear matrix-associated sites of DNA replication.

Animals↗
PubMed1993

Evidence for incorporation of N-acetylglucosamine in endogenous nuclear acceptors during the nuclear matrix preparation.

1. The presence of glycoproteins within the nucleus of cell is now well established and the question arises on the nature of the nuclear glycosylation and the site of their glycosylation. 2. In order to study endogenous nuclear proteins acceptors, we have isolated a subnuclear fraction: nuclear matrix characterized by DNA, RNA, phospholipids and proteins content. Nuclear matrix acceptors were obtained from nuclei incubated with UDP-N-acetyl [14C]glucosamine. 3. In this report we describe the presence of three major glycoproteins labeled with N-acetyl [14C]glucosamine in the nuclear matrix fraction. We obtained gP32, gP67 and gP70 with pI values around 6.2, 6.5 and 8.2.

Acetylglucosamine↗
PubMed2000

Genomic organization, chromosomal localization and regulation of expression of the neuronal nuclear matrix protein NRP/B in human brain tumors.

The nuclear matrix and its role in cell physiology are largely unknown, and the discovery of any matrix constituent whose expression is tissue- and/or cell-specific offers a new avenue of exploration. Studies of the novel neuronal nuclear matrix protein, NRP/B, reveal that it is an early and highly specific marker of neuronal induction and development in vertebrates, since its expression is restricted mainly to the developing and mature nervous system. These studies also show that NRP/B is involved in neuronal differentiation. To further examine the structure-function of NRP/B, we have cloned and characterized the murine Nrp/b gene. The murine gene consists of four exons interrupted by three introns that span 7.6kb of DNA. The complete open reading frame is localized in exon 3, suggesting that NRP/B is highly conserved during evolution. Chromosomal analysis shows that NRP/B is localized to chromosome 13 in mouse and chromosome 5q12-13 in human. Since our previous studies demonstrated that NRP/B is expressed in primary hippocampal neurons but not in primary astrocytes, we have characterized NRP/B mRNA and protein expression in various brain cell lines and in human brain tumors. Abundant expression of NRP/B mRNA and protein was observed in human neuroblastoma cell lines (IMR32, SKN-MC, SKN-SH), in glioblastoma cell lines (A172, T98G, U87-MG, U118-MG, U138-MG, and U373-MG), in neuroglioma (H4) and astrocytoma cell lines (CCF-STTG1 and SW1088). Confocal analysis of NRP/B in U87-MG glioblastoma cells indicated nuclear localization of NRP/B. NRP/B expression was also observed in human primary brain tumors including glioblastoma multiformae and astrocytomas (total of five cases). These results suggest that NRP/B expression is upregulated in human brain tumors including glioblastomas and astrocytomas, while under normal conditions NRP/B expression is restricted to neurons. This study implicates a role for NRP/B in brain tumor development.

Animals↗
PubMed1990

Preferential association of a transcriptionally active gene with the nuclear matrix of rat fibroblasts transformed by a simian-virus-40-pBR322 recombinant plasmid.

To study the relationship between the structural organization and function of the eukaryotic genome, DNA associated with nuclear matrix was analysed by using a transformed rat fibroblast cell line. The nuclear matrices were prepared from the isolated nuclei of pSV1-FR, a rat fibroblast cell line transformed by a pBR322-based recombinant plasmid containing an early gene region, which codes for large T-antigen, of simian virus 40. This transformed cell contained a single copy of the plasmid sequence integrated into the chromosomal DNA of the host cell. The early gene of this plasmid was constitutively expressed, as demonstrated by positive immunofluorescence staining of the cell for large T-antigen and by RNA-blot analysis for its specific mRNA. DNAs were extracted from whole isolated nuclei and nuclear-matrix preparations of the cells, and the relative amounts of the sequence similar to that of the plasmid were compared between these DNA preparations. By employing dot hybridization and Southern-blot analyses we found that the plasmid sequence was more enriched in the DNA extracted from the nuclear matrices than in the DNA extracted from the whole nuclei. When an albumin gene sequence that was not transcribed in this cell line was compared similarly as a control, we found no significant enrichment of this sequence in the DNA associated with the nuclear matrix. Our results strongly support the concept that a transcriptionally active gene is preferentially associated with the nuclear matrix.

Animals↗
PubMed1983

Analysis of the distribution of DNA repair patches in the DNA-nuclear matrix complex from human cells.

The distribution of ultraviolet-induced repair patches along DNA loops attached to the nuclear matrix, was investigated by digestion with DNA-degrading enzymes and neutral sucrose gradient centrifugation. When DNA was gradually removed by DNAase 1, pulse label incorporated by ultraviolet-irradiated cells during 10 min in the presence of hydroxyurea or hydroxyurea/arabinosylcytosine showed similar degradation kinetics as prelabelled DNA. No preferential association of pulse label with the nuclear matrix was observed, neither within 30 min nor 13 h after irradiation. When the pulse label was incorporated by replicative synthesis under the same conditions, a preferential association of newly-synthesized DNA with the nuclear matrix was observed. Single-strand specific digestion with nuclease S1 of nuclear lysates from ultraviolet-irradiated cells, pulse labelled in the presence of hydroxyurea/arabinosylcytosine, caused a release of about 70% of the prelabelled DNA and 90% of the pulse-labelled DNA from the rapidly sedimenting material in sucrose gradients. The results suggest no specific involvement of the nuclear matrix in repair synthesis, a random distribution of repair patches along the DNA loops, and simultaneously multiple incision events per DNA loop.

Cell Line↗
PubMed1986

The retention of core ribonucleoproteins in the nuclear matrix isolated from nuclei treated with the phenantroline-copper complex.

The nuclear matrix isolated from rat liver nuclei whose protein sulfhydryl groups were oxidised with the o-phenantroline-copper (OP-Cu) complex was enriched with a set of 32-44 kd polypeptides identified as core proteins of ribonucleoprotein particles (RNP). The most conspicuous protein in the nuclear matrix was a 36 kd protein present as a disulfide-linked homodimer. The propensity of protein 36 to be oxidised and form intermolecular associations suggests that it may contribute to the interaction of RNP particles with the nuclear matrix and thus to their spatial distribution in the nucleus.

Animals↗
PubMed1997

Nuclear matrix proteins in well and poorly differentiated human breast cancer cell lines.

The nuclear matrix, besides providing the structural support of the nucleus, is involved in various cellular functions of the nucleus. Nuclear matrix proteins (NMPs), which are both tissue- and cell type-specific, are altered with transformation and state of differentiation. Furthermore, NMPs have been identified as informative markers of disease states. Here, the NMP profiles from human breast cancer cell lines and breast tumours were analyzed using two-dimension gel electrophoresis. We identified NMPs that are associated with well and poorly differentiated human breast cancer cells in vitro and in vivo. Five NMPs (NMBC 1-5) were found to be exclusive for well-differentiated human breast cancer cells, while one NMP (NMBC-6) was found to be present only in poorly differentiated human breast cancer cells. The identification of these proteins suggests the potential use of nuclear matrix proteins as prognostic indicators.

Aged↗
PubMed1994

Glycoproteins of the nuclear matrix from chicken liver cells.

The nuclear matrix from chicken liver cells contains a small amount of glycoproteins recognized by Concanavalin A. These proteins are present not only in the peripheral matrix, but also in the internal one. In this latter localization many glycoprotein species appear, by cross-linking experiments, to be placed in the proximity of DNA. The effect of a partial enzymatic deglycosylation of matrix preparations suggests that these proteins contribute to the stabilization of the native matrix structure.

Animals↗
PubMed1986

[Heat-shock proteins in the nuclear matrix of Chinese hamster fibroblasts].

Heating of Chinese hamster fibroblasts (46 degrees C, 10 min) results in sharp inhibition of protein biosynthesis in the homogenate, nuclei and, in a lesser degree, in the nuclear matrix. The ratio of specific radioactivity of nuclear matrix 35S-proteins to the homogenate radioactivity taken for 100% increases after the heat shock 2,5-fold. Thus, protein biosynthesis in the nuclear matrix is more stable to the damaging action of heat shock than that in the homogenate and nuclei. The nuclear matrix was shown to contain heat shock proteins with Mr of 82-84, 70 and 26 kD, the 70 kD polypeptide being predominant. This polypeptide revealed in 4 hours is intensively accumulated at the 6th hour and remains unchanged by the 17-24th hours after cell heating. The 70 kD heat shock polypeptide can be separated by two-dimensional electrophoresis into two subfractions differing in terms of pI from other proteins revealed within this time interval in the unheated cells.

Animals↗
PubMed1998

The influences of two plant nuclear matrix attachment regions (MARs) on gene expression in transgenic plants.

Nuclear matrix attachment regions (MARs) are thought to influence gene expression by anchoring active chromatin to the proteinaceous nuclear matrix. In this study, two plant DNA fragments with strong MAR activity were selected and tested for their effects on expression of a linked reporter gene in transgenic tobacco. One MAR was isolated from the 5' flanking region of a pea vicilin gene previously reported to be expressed in a copy number-dependent manner in transgenic tobacco. A second MAR was isolated from the genome of Arabidopsis thaliana by preselection for autonomously replicating sequence (ARS) activity in yeast. Flanking copies of the A. thaliana MAR stimulated median reporter gene expression in transgenic plants by five to ten fold. Neither MAR significantly reduced the variation in transgene expression between individual transformants, or conferred copy number-dependence in gene expression.

Arabidopsis↗
PubMed1992

A normally masked nuclear matrix antigen that appears at mitosis on cytoskeleton filaments adjoining chromosomes, centrioles, and midbodies.

mAbs were generated against HeLa nuclear matrix proteins and one, HIB2, which selectively stained mitotic cells, was selected for further study. Western blot analysis showed H1B2 antibody detected a protein of 240 kD in the nuclear matrix fractions. The H1B2 antigen was completely masked in immunofluorescently stained interphase cells. However, removing chromatin with DNase I digestion and 0.25 M ammonium sulfate extraction exposed the protein epitope. The resulting fluorescence pattern was bright, highly punctate, and entirely nuclear. Further extraction of the nuclear matrix with 2 M NaCl uncovers an underlying, anastomosing network of 9-13 nm core filaments. Most of the H1B2 antigen was retained in the fibrogranular masses enmeshed in the core filament network and not in the filaments themselves. The H1B2 antigen showed remarkable behavior at mitosis. As cells approached prophase the antigen became unmasked to immunofluorescent staining without the removal of chromatin. First appearing as a bright spot, the antibody staining spread through the nucleus finally concentrating in the region around the condensed chromosomes. The antibody also brightly stained the spindle poles and, more weakly, in a punctate pattern in the cytoskeleton around the spindle. As the chromosomes separated at anaphase, H1B2 remained with the separating daughter sets of chromosomes. The H1B2 antigen returned to the reforming nucleus at telophase, but left a bright staining region in the midbody. Immunoelectron microscopy of resinless sections showed that, in the mitotic cell, the H1B2 antibody did not stain chromosomes and centrioles themselves, but decorated a fibrogranular network surrounding and connected to the chromosomes and a fibrogranular structure surrounding the centriole.

Antigens, Nuclear↗
PubMed1998

Matrin CYP, an SR-rich cyclophilin that associates with the nuclear matrix and splicing factors.

We report the identification and cloning of a nuclear matrix protein termed matrin cyclophilin or matrin CYP. The derived sequence of matrin cyp encodes a protein of 752 amino acids with a predicted mass of 88 kDa. A 172-residue stretch at the amino terminus shows high identity with the ubiquitous family of cyclophilins. Clustered throughout the carboxyl half of the protein are a series of serine-arginine (SR) repeats that are a characteristic feature of many RNA splicing factors. Antibodies raised against matrin CYP recognize a 106-kDa antigen that is detected in isolated nuclei and quantitatively subfractionates in the nuclear matrix. Laser scanning confocal microscopy localizes most of the anti-matrin CYP-specific antigen within the nucleus in a pattern of large bright speckles that co-localize with splicing factors and diffuse nucleoplasmic staining. A strikingly similar pattern of staining is observed in cells extracted for in situ nuclear matrices. A fusion protein containing the cyclophilin domain of matrin CYP exhibits cyclosporin A (CsA)-sensitive, peptidylprolyl cis-trans-isomerase activity that is characteristic of native cyclophilins. Although total rat liver nuclei contains predominantly CsA-resistant PPIase activity, the corresponding activity in the nuclear matrix is largely CsA-sensitive.

Amino Acid Sequence↗
PubMed1984

Association of simian virus 40 T antigen with the nuclear matrix of infected and transformed monkey cells.

The subnuclear distribution of simian virus 40 large T antigen within nuclei of transformed Cos and C6 monkey cells was examined. Cos cells express wild-type T antigen but lack viral sequences required for DNA replication, whereas C6 cells contain a functional viral origin but express a replication-defective mutant T antigen which is unable to bind specifically to viral DNA. Discrete subpopulations of T antigen were isolated from the soluble nucleoplasm, chromatin, and nuclear matrix of both cell lines. Although only a small quantity (2 to 12%) of the total nuclear T antigen from Cos cells was associated with the nuclear matrix, a high proportion (25 to 50%) of C6 T antigen was bound to this structure. Results obtained from lytically infected monkey cells showed that early in infection, before viral replication was initiated, a higher proportion (22%) of T antigen was found associated with the nuclear matrix compared with amounts found associated with this structure later in infection (5 to 8%). These results suggest that an increased association of T antigen with this structure is not correlated with viral replication. T antigen isolated from the C6 nuclear matrix was more highly phosphorylated than was soluble C6 T antigen and was capable of binding to the host p53 protein. C6 DNA contains three mutations: two corresponding to N-terminal changes at amino acid positions 30 and 51 and a third located internally at amino acid position 153. By analysis of the subnuclear distribution of T antigen from rat cells transformed by C6 submutant T antigens, it was determined that one or both of the mutations at the NH2 terminus are responsible for the increased quantity of C6 T antigen associated with the nuclear matrix. These results suggest that neither a functional viral DNA replication origin nor the origin binding property of T antigen is required for association of this protein with the nuclear matrix.

Animals↗
PubMed2006

Characterization of a novel Dp71 dystrophin-associated protein complex (DAPC) present in the nucleus of HeLa cells: members of the nuclear DAPC associate with the nuclear matrix.

Dystrophin is an essential component in the assembly and maintenance of the dystrophin-associated protein complex (DAPC), which includes members of the dystroglycan, syntrophin, sarcoglycan and dystrobrevin protein families. Distinctive complexes have been described in the cell membrane of different tissues and cultured cells. In this work, we report the identification and characterization of a novel DAPC present in the nuclei of HeLa cells, which contains dystrophin Dp71 as a key component. Using confocal microscopy and cell fractionation analyses, we found the presence of Dp71, beta-sarcoglycan, beta-dystroglycan, alpha- and beta-syntrophin, alpha1- and beta-dystrobrevin and nNOS in the nuclei of HeLa cells. Furthermore, we demonstrated by co-immunoprecipitation experiments that most of these proteins form a complex in the nuclear compartment. Next, we analyze the possible association of the nuclear DAPC with the nuclear matrix. We found the presence of Dp71, beta-dystroglycan, nNOS, beta-sarcoglycan, alpha/beta syntrophin, alpha1-dystrobrevin and beta-dystrobrevin in the nuclear matrix protein fractions and in situ nuclear matrix preparations from HeLa cells. Moreover, we found that Dp71, beta-dystroglycan and beta-dystrobrevin co-immunoprecipitated with the nuclear matrix proteins lamin B1 and actin. The association of members of the nuclear DAPC with the nuclear matrix indicates that they may work as scaffolding proteins involved in nuclear architecture.

Actins↗