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PubMed1984

Monoclonal antibodies to the nuclear matrix of chick embryonal erythrocytes.

Monoclonal antibodies were prepared from mice that had been immunized with the nuclear matrix from chick embryonal erythrocytes. Seven stable clones were obtained by an ELISA that used nuclear lysate as the solid phase. Six clones of them reacted with the nuclear matrix, and one reacted with nuclear components other than the matrix. Immunoblotting showed that one clone recognized the 72K polypeptide, two clones recognized the 69K polypeptide and three clones recognized both the 69K and 44K polypeptides. Indirect immunofluorescence that used antibodies to the nuclear matrix showed homogeneous nuclear fluorescence in cultured chick embryonal fibroblasts, and intense fluorescence was present in the peripheral part of the nucleus in thin-sectioned chick embryos. Only weak nuclear fluorescence was seen in fibroblasts from humans and rats when two of the antibodies which recognized only 69K polypeptide were used. The rest of the antibodies to the nuclear matrix produced no nuclear fluorescence in human and rat fibroblasts. The metaphase-rich population of chick embryonal fibroblasts were stained diffusely over the entire cytoplasm, but not the chromosomes, when antibodies to the nuclear matrix were used. These results indicate that monoclonal antibodies we prepared are directed to the major proteins of the nuclear matrix that correspond to the lamin A and B defined in rat liver.

Animals↗
PubMed1998

Thinking about a nuclear matrix.

The possible existence in eukaryotic cells of an internal, non-chromatin nuclear structural framework that facilitates gene readout as a set of spatially concerted reactions has become a popular but controversial theater of investigation. This article endeavors to present a circumspect review of the nuclear matrix concept as we presently know it, framed around two contrasting hypotheses: (1) that an internal nuclear framework actively enhances gene expression (in much the same way the cytoskeleton mediates cell locomotion, mitosis and intracellular vesicular traffic) versus (2) that the interphase chromosomes have fixed, inherited positions and that the DNA replication, transcripton and RNA processing machinery diffusionally arrives at sites of gene readout, with some aspects of nuclear structure thus being more a result than a cause of gene expression. On balance, the available information suggests that interactions among various gene expression machines may contribute to isolated nuclear matrix preparations. Some components of isolated nuclear matrix preparations may also reflect induced or reconfigured protein-protein associations. The protein characterization and ultrastructural analysis of the isolated nuclear matrix has advanced significantly in recent years, although controversies remain. Important new clues are now coming in from promising contemporary lines of research that report on nuclear structure in living cells.

Cell Nucleus↗
PubMed1994

Association of chromosomal telomere DNA with nuclear matrix in HeLa cell.

Using Electron Spectroscopic Imaging (ESI), we visualized the in situ binding of nucleic acids to nuclear matrix and 3H-thymidine incorporation which indicates that a small partial DNA bound to nuclear matrix tightly. Furthermore we found that chromosomal telomere DNA could bind to nuclear matrix specifically by the dot and Southern hybridization. The result of the Southwestern blot suggests that telomere DNA has high affinity to lamin B, vimentin and some nuclear matrix proteins. Therefore, the nuclear matrix and lamina of HeLa cell are possibly associated with spatial organization and action of chromosome.

DNA↗
PubMed1994

Association of nuclear matrix antigens with exon-containing splicing complexes.

mAbs raised against the human nuclear matrix (anti-NM)1 mAbs have been used to investigate the role of nuclear matrix antigens in pre-mRNA processing. The three anti-NM mAbs used in this study recognize antigens that are highly localized to nuclear matrix speckles. Surprisingly, all three of these mAbs preferentially immunoprecipitate splicing complexes containing exon sequences. The anti-NM mAbs efficiently immunoprecipitate the exon product complex but not complexes containing the lariat product after the second step of splicing. Two of the anti-NM mAbs completely inhibit pre-mRNA splicing in vitro. However, none of the anti-NM mAbs appear to recognize factors stably associated with splicing snRNPs. The three anti-NM mAbs predominantly react with distinct high molecular weight antigens, which belong to a class of nuclear proteins that selectively precipitate with Ser-Arg protein-splicing factors in the presence of high Mg2+ concentrations. Immunological, biochemical, and cell biological data indicate that two of the NM antigens are related to the defined set of Ser-Arg proteins. The results suggest the existence of an extended Ser-Arg family as a component of the nuclear matrix.

Amino Acid Sequence↗
PubMed2005

Natural ageing in the rat liver correlates with progressive stabilisation of DNA-nuclear matrix interactions and withdrawal of genes from the nuclear substructure.

In the interphase nucleus, the DNA of higher eukaryotes is organised in supercoiled loops anchored to a nuclear matrix (NM). Replication, transcription and splicing seem to occur at macromolecular complexes organised upon the NM. Thus, the topological relationship between genes located in the loops and the NM appears to be very important for nuclear physiology. Here, we report that natural ageing in the rat liver correlates with a progressive strengthening of the NM framework and the stabilisation of the DNA loop-NM interactions, as well as with a progressive increase in the relative distance of genes to the NM. Both phenomena correlate with the gradual loss of proliferating potential and progression towards terminal differentiation in the hepatocytes, suggesting that wholesale modifications in the topological relationships within the cell nucleus are markers of tissue ageing and senescence, at least in the mammalian liver. We discuss the possible functional implications of such structural modifications that may underlie both terminal hepatocyte differentiation and their eventual replicative senescence.

Aging↗
PubMed1999

Identification of nuclear matrix and associated proteins that bind the haptoglobin gene cis-element.

To identify the major nuclear matrix proteins that bind to the rat haptoglobin gene cis-element, we isolated a soluble nuclear matrix protein fraction and analysed it by gel retardation. Two major DNA-binding proteins exhibiting different types of protein-DNA interactions were detected: a DNA sequence-specific 32-kDa isoform of transcription factor C/EBP beta, and a nuclear matrix protein p55 that bound to the DNA nonspecifically. During increased transcription of the haptoglobin gene in the course of the acute-phase reaction, the DNA-binding affinities and concentrations of these proteins in the soluble nuclear matrix fraction were increased. These data lend further evidence that the nuclear matrix is an active support structure that localizes gene regulatory proteins and participates in transcriptional regulation.

Acute-Phase Reaction↗
PubMed1996

Nuclear lamina and nuclear matrix organization in sperm pronuclei assembled in Xenopus egg extract.

Nuclear lamina and matrices were prepared from sperm pronuclei assembled in Xenopus egg extracts using a fractionation and extraction procedure. Indirect immunofluorescence revealed that while chromatin was efficiently removed from nuclei during the extraction procedure, the distribution of lamins was unaffected. Consistent with this data, the amount of lamin B3, determined by immunoblotting, was not affected through the extraction procedure. Nuclear matrices were visualised in DGD sections by TEM. Within these sections filaments were observed both at the boundary of the nucleus (the lamina) and within the body of the nucleus (internal nuclear matrix filaments). To improve resolution, nuclear matrices were also prepared as whole mounts and viewed using field emission in lens scanning electron microscopy (FEISEM). This technique revealed two distinct networks of filaments. Filaments lying at the surface of nuclear matrices interconnected nuclear pores. These filaments were readily labelled with monoclonal anti-lamin B3 antibodies. Filaments lying within the body of the nuclear matrix were highly branched but were not readily labelled with antilamin B3 antibodies. Nuclear matrices were also prepared from sperm pronuclei assembled in lamin B3 depleted extracts. Using FEISEM, filaments were also detected in these preparations. However, these filaments were poorly organised and often appeared to aggregate. To confirm these results nuclear matrices were also observed as whole mounts using TEM. Nuclear matrices prepared from control nuclei contained a dense array of interconnected filaments. Many (but not all) of these filaments were labelled with anti-lamin B3 antibodies. In contrast, nuclear matrices prepared from "lamin depleted nuclei' contained poorly organised or aggregated filaments which were not specifically labelled with anti-lamin B3 antibodies.

Animals↗
PubMed2001

Association of the telomere-telomere-binding protein complex of hypotrichous ciliates with the nuclear matrix and dissociation during replication.

Telomeric interactions with the nuclear matrix have been described in a variety of eukaryotic cells and seem to be essential for specific nuclear localization. Macronuclear DNA of hypotrichous ciliates occurs in small gene-sized DNA molecules, each being terminated by telomeres. Each macronucleus contains over 10(8 )individual DNA molecules. Owing to the high number of telomeres present in this nucleus it provides an excellent model to study telomere behaviour throughout the cell cycle. In this study we provide experimental evidence that the telomere-telomere-binding protein (TEBP) complex specifically interacts with components of the nuclear matrix in vivo. In the course of replication the specific interaction of the TEBP with components of the nuclear matrix is resolved and an attachment of the telomeres to the matrix no longer occurs.

Animals↗
PubMed1997

[Nuclear matrix protein pattern in human hepatocellular carcinoma].

OBJECTIVE: To compare nuclear matrix proteins of normal liver with those of hepatocellular carcinoma (HCC) to see whether there are HCC-specific proteins. METHODS: Using high resolution two-dimensional polyacrylamide gel electrophoresis, the nuclear matrix proteins of 3 normal livers and 8 HCC were compared. RESULTS: There was a high degree of similarity between nuclear proteins of normal liver and those of HCC. Four HCC-specific nuclear matrix proteins were identified. Among them, one protein(Mr: 62,000; pI: 5.3) appeared in all tumor samples. The other three proteins appeared in most of the 8 HCC cases. These proteins were not detected in 3 normal livers. CONCLUSION: There are HCC-specific nuclear matrix proteins which may be related to the etiology and pathogenesis of HCC.

Antigens, Nuclear↗
PubMed1998

NRP/B, a novel nuclear matrix protein, associates with p110(RB) and is involved in neuronal differentiation.

The nuclear matrix is defined as the insoluble framework of the nucleus and has been implicated in the regulation of gene expression, the cell cycle, and nuclear structural integrity via linkage to intermediate filaments of the cytoskeleton. We have discovered a novel nuclear matrix protein, NRP/B (nuclear restricted protein/brain), which contains two major structural elements: a BTB domain-like structure in the predicted NH2 terminus, and a "kelch motif" in the predicted COOH-terminal domain. NRP/B mRNA (5.5 kb) is predominantly expressed in human fetal and adult brain with minor expression in kidney and pancreas. During mouse embryogenesis, NRP/B mRNA expression is upregulated in the nervous system. The NRP/B protein is expressed in rat primary hippocampal neurons, but not in primary astrocytes. NRP/B expression was upregulated during the differentiation of murine Neuro 2A and human SH-SY5Y neuroblastoma cells. Overexpression of NRP/B in these cells augmented neuronal process formation. Treatment with antisense NRP/B oligodeoxynucleotides inhibited the neurite development of rat primary hippocampal neurons as well as the neuronal process formation during neuronal differentiation of PC-12 cells. Since the hypophosphorylated form of retinoblastoma protein (p110(RB)) is found to be associated with the nuclear matrix and overexpression of p110(RB) induces neuronal differentiation, we investigated whether NRP/B is associated with p110(RB). Both in vivo and in vitro experiments demonstrate that NRP/B can be phosphorylated and can bind to the functionally active hypophosphorylated form of the p110(RB) during neuronal differentiation of SH-SY5Y neuroblastoma cells induced by retinoic acid. Our studies indicate that NRP/B is a novel nuclear matrix protein, specifically expressed in primary neurons, that interacts with p110(RB) and participates in the regulation of neuronal process formation.

Adult↗
PubMed1984

Identification of nuclear matrix proteins tightly bound to soluble simian virus 40 chromosomes.

Nuclear matrix proteins (defined as the nuclear proteins which were highly enriched in an insoluble fraction following extraction of lipids, loosely bound proteins, and nucleic acids) from mock- and SV40-infected cells were identified by two-dimensional polyacrylamide gel electrophoresis, consisting of nonequilibrium pH gradients in the first dimension and sodium dodecyl sulfate gel electrophoresis in the second dimension. The proteins identified in the mock-infected nuclear matrix included M1 (molecular weight, 71K), M2 (69K) M3 (58K), M4 (50K), M5 (49K), M6 (36K), M7 (36K), and M8 (31K), while the nuclear matrix from SV40-infected cells included, in addition to all these proteins, VP-1 (45K), VP-1' (44K), VP-3 (25K), V1 (36K), V2 (35K), and V3 (35K). Except for M7 all of these proteins sedimented with SV40 chromosomes isolated and partially purified by glycerol gradient sedimentation at low ionic strength, and only M6 and M8 were removed from the SV40 chromosomes during more extensive purification of the SV40 chromosomes by subsequent sedimentation at high ionic strength (0.5 M NaCl). When the structures of the SV40 chromosomes were destroyed by digestion with DNAase I, these tightly bound proteins no longer sedimented to the position of SV40 chromosomes. Further subfractionation of SV40 chromosomes indicated that the proteins M1 to M4 were preferentially associated with the nonreplicating SV40 chromosomes, whereas M5 was associated with encapsidating SV40 chromosomes and virions.

Animals↗
PubMed1985

A constitutively transcribed actin gene is associated with the nuclear matrix in a Drosophila cell line.

The relationship between transcriptional activity and gene association with the nuclear matrix has been investigated in Drosophila melanogaster. The nuclear matrix of Schneider cell line 2 of Drosophila was isolated and observed to conform to expected dimensions in phase contrast and scanning electron microscopic preparations. This structure contains proteins that appear similar to the intact nucleus. High salt extracted nuclei digested with DNase I released 98% of the DNA, whereas digestion with Eco RI released a maximum of 80%. These and other nuclease digestions indicate that satellite DNA as well as some unique sequence DNA are bound to the nuclear matrix. A constitutively transcribed actin gene was enriched in the nuclear matrix bound DNA. Two other nontranscribed genes, a muscle-specific actin gene and the myosin heavy chain gene, showed no enrichment in nuclear matrix DNA.

Actins↗
PubMed1995

The nuclear matrix: a structural milieu for genomic function.

While significant progress has been made in elucidating molecular properties of specific genes and their regulation, our understanding of how the whole genome is coordinated has lagged behind. To understand how the genome functions as a coordinated whole, we must understand how the nucleus is put together and functions as a whole. An important step in that direction occurred with the isolation and characterization of the nuclear matrix. Aside from the plethora of functional properties associated with these isolated nuclear structures, they have enabled the first direct examination and molecular cloning of specific nuclear matrix proteins. The isolated nuclear matrix can be used for providing an in vitro model for understanding nuclear matrix organization in whole cells. Recent development of high-resolution and three-dimensional approaches for visualizing domains of genomic organization and function in situ has provided corroborative evidence for the nuclear matrix as the site of organization for replication, transcription, and post-transcriptional processing. As more is learned about these in situ functional sites, appropriate experiments could be designed to test molecular mechanisms with the in vitro nuclear matrix systems. This is illustrated in this chapter by the studies of nuclear matrix-associated DNA replication which have evolved from biochemical studies of in vitro nuclear matrix systems toward three-dimensional computer image analysis of replication sites for individual genes.

Animals↗
PubMed1990

Progressive changes in the protein composition of the nuclear matrix during rat osteoblast differentiation.

Primary cultures of fetal rat calvarial osteoblasts undergo a developmental sequence with respect to the temporal expression of genes encoding osteoblast phenotypic markers. Based on previous suggestions that gene-nuclear matrix associations are involved in regulating cell- and tissue-specific gene expression, we investigated the protein composition of the nuclear matrix during this developmental sequence by using high-resolution two-dimensional gel electrophoresis. The nuclear matrix was isolated at times during a 4-week culture period that represent the three principal osteoblast phenotypic stages: proliferation, extracellular matrix (ECM) maturation, and mineralization. The most dramatic changes in the nuclear matrix protein patterns occurred during transitions from the proliferation to the ECM maturation stage and from ECM maturation to the mineralization period, with only minor variations in the profiles within each period. These stage-specific changes, corresponding to the major transition points in gene expression, indicate that the nuclear matrix proteins reflect the progressive differentiation of the bone cell phenotype. Subcultivation of primary cells delays mineralization, and a corresponding delay was observed for the nuclear matrix protein patterns. Thus, the sequential changes in protein composition of the nuclear matrix that occur during osteoblast differentiation represent distinct stage-specific markers for maturation of the osteoblast to an osteocytic cell in a bone-like mineralized ECM. These changes are consistent with a functional involvement of the nuclear matrix in mediating modifications of developmental gene expression.

Animals↗
PubMedDate not supplied

[Inhibition by cycloheximide of the incorporation of radioactive amino acids into nuclear matrix proteins of Zajdela ascites hepatoma].

Proteins of nuclear matrix readily incorporated labelled amino acids after incubation of ascites Zajdela hepatoma cells with 14C-hydrolyzate of chlorella proteins. Cycloheximide at the concentrations, inhibiting the cytoplasmic protein synthesizing system, significantly decreased the labelled amino acid incorporation both into nuclear proteins and into proteins of nuclear matrix but the antibiotic effect was less distinct in the matrix. The incorporation into proteins with molecular mass below 26 KD and above 150 KD was especially distinctly inhibited as shown in experiments on separation of the matrix proteins by means of SDS-polyacrylamide gel electrophoresis. Besides the inhibition of the incorporation of amino acids, a decrease in content was found in studies of the fraction of matrix low molecular proteins. The high rate of metabolism of nuclear matrix low molecular proteins is discussed.

Amino Acids↗
PubMed1982

Chinese hamster nuclear proteins. An electrophoretic analysis of interphase, metaphase and nuclear matrix preparations.

A comparison, by two-dimensional gel electrophoresis, of total interphase nuclear, metaphase chromosomal and nuclear matrix proteins from Chinese hamster V-79 cells was undertaken to examine the distribution of these proteins during mitosis. We have found a number of differences among these populations, although the two-dimensional gel patterns are generally similar. The most striking observation is that a loose cluster of six interphase nuclear polypeptides, with isoelectric points in urea between 5.7 and 6.7 and molecular masses ranging from 53 to 75 kDa, is greatly enriched in chromosome preparations. Each of these species is prominent also in the nuclear matrix. Preliminary evidence suggests that one of these polypeptides is the intermediate filament protein, vimentin. In addition, two major polypeptides of interphase nuclear preparations, a basic 94-kDa species and an approximately 65-kDa species, are absent from chromosomes. The latter polypeptide is the nuclear pore-lamina complex lamin B. Actin is present in all of these fractions, but tubulin has not been observed. hnRNP particle polypeptides are major components of the nuclear matrix, but are markedly reduced in metaphase chromosomes. The intermediate and basic 65-75-kDa nuclear matrix polypeptides we have previously demonstrated to be major components of rat liver nuclear matrix, are reduced in Chinese hamster matrix preparations and at least one of these species, a minor, basic, 68-kDa polypeptide, is missing entirely from metaphase chromosomes. These results are discussed in relation to nuclear and chromosome structure and the possibility of contamination of nuclear protein preparations from cultured cell lines with intermediate filaments.

Animals↗
PubMed1985

An association between replicating adenovirus DNA and the nuclear matrix of infected HeLa cells.

An association between newly synthesized human adenovirus type 5 DNA and the nuclear matrix of infected HeLa cells is described. Adenovirus-infected cells were pulsed labeled with [3H]thymidine late in infection and the nuclear matrix was prepared. After a 1-min pulse more than 95% of the labeled viral DNA was matrix associated and, when compared with total cell DNA, was resistant to DNase I digestion. When the pulse is longer or is followed by a chase period, the viral DNA remains nuclear matrix associated and less nuclease sensitive than bulk cellular DNA. The resistance to nuclease digestion may result from the close association of viral DNA with the nuclear matrix or could be due to a number of viral-specific proteins which are nuclear matrix associated. It is concluded that viral DNA synthesis occurs in association with the nuclear matrix and the newly synthesized DNA remains matrix associated until it is incorporated into a mature virus particle.

Adenoviruses, Human↗
PubMed1998

Identification and characterization of the ubiquitously occurring nuclear matrix protein NMP 238.

By systematic comparison of two-dimensional electrophoretic patterns of nuclear matrix proteins an ubiquitously occurring (common) nuclear matrix protein, termed NMP 238, was detected. Localization of the protein in isolated nuclear matrices and in nuclear and cytoplasmic regions of cells was determined by confocal immunofluorescence microscopy. N-terminal protein sequencing, mass spectrometry, and sequencing of a human EST cDNA clone showed identity of the protein with a nuclear protein, termed TIP49, of as yet uncertain function. Expression of the corresponding gene in diverse human and rat cells was confirmed by Northern blotting. The protein displays two nuclear localization signals. Sequence homologies indicate evolutionary related proteins in nematodes, yeast, and archaebacteria. Similarities to the AAA family of proteins and to a subgroup of chaperones suggest that the nuclear matrix protein may play a role in the assembly and ATP-dependent anchorage of proteins.

Amino Acid Sequence↗