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PubMed1984

Nuclear matrix DNA from chicken erythrocytes contains beta-globin gene sequences.

Nuclear matrices containing residual DNA were isolated from chicken erythrocytes after extraction of purified nuclei with buffered 2 M NaCl. After further purification of this residual DNA, it was found to contain high concentrations of beta-globin gene sequences as assayed by dot hybridization with 32P-labeled nick-translated pHB1001. Electron microscopy of a random sample of this residual DNA fraction shows the DNA to be intimately associated with protein at various intervals. A hypothesis for enrichment of active genes in residual DNA from purified chromatin or in nuclear matrix DNA is also discussed.

Animals↗
PubMed1984

[Inhibition by antibiotics of the incorporation of labelled amino acids into the nuclear matrix proteins of the Zajdela hepatoma].

The incorporation of radioactivity into nuclear matrix proteins during incubation of Zajdela hepatoma cells with labelled amino acids was strongly inhibited by chloramphenicol and cycloheximide and slightly inhibited by actinomycin D and mitomycin C. The antibiotics studied inhibited the incorporation of the radioactive label preferentially into proteins with Mr 150 000-220 000, approximately 55 000 and less than 26 000. During incubation of ascites tumour cells with antibiotics, predominantly with chloramphenicol, a decrease in the content of some protein components was observed as well. As in the low molecular weight protein fraction, the intense inhibition of the radioactive label and a decrease of its content was observed, a conclusion is drawn that this protein fraction is characterized by a high turnover rate.

Amino Acids↗
PubMed1984

Interphase nuclear matrix and metaphase scaffolding structures.

The protein compositions of purified metaphase chromosomes, nuclei and their residual scaffold and matrix structures, are reported. The protein pattern of nuclei on sodium dodecyl sulphate/polyacrylamide gels is considerably more complex and rich in non-histone proteins than that of chromosomes. Nuclei contain about three to four times more non-histone proteins relative to their histones than chromosomes. Besides the protein components of the peripheral lamina, several protein bands are specific or at least highly enriched in nuclei. Conversely, two proteins X0 (33 X 10(3) Mr) and X1 (37 X 10(3) Mr) are highly enriched in the pattern of metaphase chromosomes. We have compared morphologically the previously defined nuclear matrices type I and II. The type I nuclear matrix is composed of the known lamina proteins, which form the peripheral lamina structure, and a complex series of proteins that form the internal network of the matrix as observed by electron microscopy. This internal network is stabilized similarly to the metaphase scaffolding by metalloprotein interaction. Both the scaffolding and the internal network of the matrix dissociate if thiols or certain metal chelators are used in the extraction buffer. Under these conditions the resulting nuclear structure, called matrix type II, appears empty in the electron microscope, with the exception of some residual nucleolar material. This latter material can be extracted from the internal network by exhaustive treatment of the nuclei with RNase before extraction with high salt. Immunoblotting and activity studies show RNA polymerase II to be tightly bound to the type I, but not to the type II matrix, or to the scaffolding structure. No polymerase II enzyme was detected in isolated metaphase chromosomes. Another nuclear enzyme, poly(ADP-ribose) polymerase is not bound to either of the residual nuclear matrices or to the scaffolding structures. The association of RNA polymerase with the internal network of the nuclear matrix is consistent with the idea that transcription occurs in close association with this structure.

Cell Nucleus↗
PubMed2002

Translocation of Cockayne syndrome group A protein to the nuclear matrix: possible relevance to transcription-coupled DNA repair.

Transcription-coupled repair (TCR) efficiently removes a variety of lesions from the transcribed strand of active genes. By allowing rapid resumption of RNA synthesis, the process is of major importance for cellular resistance to transcription-blocking genotoxic damage. Mutations in the Cockayne syndrome group A or B (CSA or CSB) gene result in defective TCR. However, the exact mechanism of TCR in mammalian cells remains to be elucidated. We found that CSA protein is rapidly translocated to the nuclear matrix after UV irradiation. The translocation of CSA was independent of Xeroderma pigmentosum group C, which is specific to the global genome repair subpathway of nucleotide excision repair (NER) and of the core NER factor Xeroderma pigmentosum group A but required the CSB protein. In UV-irradiated cells, CSA protein colocalized with the hyperphosphorylated form of RNA polymerase II, engaged in transcription elongation. The translocation of CSA was also induced by treatment of the cells with cisplatin or hydrogen peroxide, both of which produce damage that is subjected to TCR but not induced by treatment with dimethyl sulfate, which produces damage that is not subjected to TCR. The hydrogen peroxide-induced translocation of CSA was also CSB dependent. These findings establish a link between TCR and the nuclear matrix mediated by CSA.

Active Transport, Cell Nucleus↗
PubMed1984

Tight association of DNA polymerase alpha with granular structures in the nuclear matrix of chick embryo cell: immunocytochemical detection with monoclonal antibody against DNA polymerase alpha.

Immunofluorescent methods using a monoclonal antibody against chick DNA polymerase alpha and a rabbit antibody against chick DNA polymerase beta demonstrated that both DNA polymerases alpha and beta are present mainly in nuclei of cultured chick embryo cells. Fluorescence produced by anti-DNA polymerase alpha was more intense in the small granules than in other parts of the nucleus but, fluorescence produced by anti-DNA polymerase beta was distributed evenly in the nucleus. Cells first were treated with Nonidet P-40, followed by treatment with 50 micrograms/ml pancreatic DNase and 2 M NaCl in order to prepare the nuclear matrix. Fluorescence produced by anti-DNA polymerase alpha was still detectable in the granules after these treatments, but most of the fluorescence produced by anti-DNA polymerase beta disappeared. Our results indicate that a part of DNA polymerase alpha is tightly bound to a special structure present in the nuclear matrix which presumably is the DNA replication machinery.

Animals↗
PubMed1995

CENP-F is a protein of the nuclear matrix that assembles onto kinetochores at late G2 and is rapidly degraded after mitosis.

Centromere protein-F (CENP-F) is mammalian kinetochore protein that was recently identified by an autoimmune serum (Rattner, J. B., A. Rao, M. J. Fritzler, D. W. Valencia, and T. J. Yen. Cell Motil. Cytoskeleton. 26:214-226). We report here the human cDNA sequence of CENP-F, along with its expression and localization patterns at different stages of the HeLa cell cycle. CENP-F is protein of the nuclear matrix that gradually accumulates during the cell cycle until it reaches peak levels in G2 and M phase cells and is rapidly degraded upon completion of mitosis. CENP-F is first detected at the prekinetochore complex during late G2, and is clearly detectable as paired foci that correspond to all the centromeres by prophase. During mitosis, CENP-F is associated with kinetochores from prometaphase until early anaphase and is then detected at the spindle midzone throughout the remainder of anaphase. By telophase, CENP-F is concentrated within the intracellular bridge at either side of the mid-body. The predicted structure of the 367-kD CENP-F protein consists of two 1,600-amino acid-long coil domains that flank a central flexible core. A putative P-loop nucleotide binding site (ADIPTGKT) is located within the globular carboxy terminus. The structural features deduced from our sequence studies and the spatial and temperal distribution of CENP-F revealed in our cytological and biochemical studies suggest that it may play a role in several mitotic events.

Amino Acid Sequence↗
PubMed1994

TRiC-P5, a novel TCP1-related protein, is localized in the cytoplasm and in the nuclear matrix.

We have recently reported the cloning of a novel protein, TRiC-P5, with significant homology with protein 1 of the t-complex (TCP1). In the present study, the cellular localization of TRiC-P5 in Raji cells has been determined using an antiserum raised against a 18.5 kDa fusion protein. Results from cell fractionation and immunoblot studies indicate that TRiC-P5 is mainly localized in the cytoplasm. In addition, a significant part of TRiC-P5 is also found in the nucleus where it is attached to the nuclear matrix, a complex filament network involved in essential cellular functions such as DNA replication, and RNA transcription and maturation. Immunofluorescence experiments using the anti-TRiC-P5 antibodies confirm these results. We also provide evidence that, in the cytoplasm, TRiC-P5 is part of a large protein complex, most probably the TCP1-ring complex (TRiC), a hetero-oligomeric ring complex that plays a role of molecular chaperone in the folding of actin and tubulin.

Chaperonin Containing TCP-1↗
PubMed1994

Nuclear matrix and the regulation of gene expression: tissue specificity.

Tissue specific regulation of gene expression by a single transcription factor or group of transcription factors cannot be explained simply by DNA sequence alone. For example, in the same animal a particular transcription factor is capable of interacting with DNA in the nucleus of many different cell types, resulting in unique gene expressions despite the presence of a similar genome in all cells. Historically, these differences in response to a single type of factor within target tissues in the same animal have been suggested to occur through different alterations in chromatin structure. Recent, data has demonstrated that combinations of hormones and transcription factors working together may cooperatively play a role in the regulation of gene expression [Pearce and Yamamoto (1993): Science 259:1161-1165]. However, the molecular mechanisms of this tissue specific regulation of gene expression still remains largely unexplained. Current evidence suggests that in different cell types the interplay between the specific three-dimensional organization of the genome and the structural components of the nucleus, the nuclear matrix, may accomplish the regulation of specific gene expression.

Animals↗
PubMed1994

The amino-terminal region of the retinoblastoma gene product binds a novel nuclear matrix protein that co-localizes to centers for RNA processing.

The tumor suppressing capacity of the retinoblastoma protein (p110RB) is dependent on interactions made with cellular proteins through its carboxy-terminal domains. How the p110RB amino-terminal region contributes to this activity is unclear, though evidence now indicates it is important for both growth suppression and regulation of the full-length protein. We have used the yeast two-hybrid system to screen for cellular proteins which bind to the first 300 amino acids of p110RB. The only gene isolated from this screen encodes a novel 84-kD nuclear matrix protein that localizes to subnuclear regions associated with RNA processing. This protein, p84, requires a structurally defined domain in the amino terminus of p110RB for binding. Furthermore, both in vivo and in vitro experiments demonstrate that p84 binds preferentially to the functionally active, hypophosphorylated form of p110RB. Thus, the amino terminus of p110RB may function in part to facilitate the binding of growth promoting factors at subnuclear regions actively involved in RNA metabolism.

Amino Acid Sequence↗
PubMed1985

Double stranded RNA and the nuclear matrix--implications for the 2-5A system.

RNA species present on rat liver nuclear matrices were investigated. Nuclear matrices prepared by extensive digestion of isolated nucleii with DNase and RNaseA followed by low and high (2M NaC1) salt washes were labelled in vitro with T4 RNA ligase and [5'-32P]pCp and the labelled RNA analysed by gel electrophoresis. Despite the extensive RNaseA treatment, a prominent RNA species migrating as a heterodisperse band of 220-300 nucleotides (termed MX220-300), was observed--only minor amounts of other RNA molecules were seen. A comparison of RNA isolated from in-vitro labelled nuclear matrices with isolated matrix RNA that was subsequently labelled, indicated that part of MX220-300 was preferentially exposed on the nuclear matrix structure. Analysis of MX220-300 indicated that it was composed of a polyadenylic acid moiety hydrogen bonded to a smaller molecule of polyuridylic acid. No evidence was found for the presence of guanosine or cytosine residues. Control experiments in which labelled polyuridylic acid was added to nucleii prior to the preparation of MX220-300, virtually excluded the possibility that the partial double stranded RNA structure was an artefact of matrix preparation. An analysis of proteins in the nuclear matrix structure that interact with double stranded (ds)RNA showed at least 2 proteins having molecular weights of 62K and 66K daltons that recognized and bound polyadenylic/polyuridylic acid. Competition experiments with unlabelled polyinosinic/polycytidylic acid indicated that these proteins specifically recognized the dsRNA structure. The 62K and 66K dalton matrix proteins that specifically bound dsRNA were observed in nuclear matrices prepared from HeLa, Ehrlich ascites tumor and rat liver cells. It is not known whether these matrix located dsRNA binding proteins have 2-5A synthetase activity. The relevance of the above findings to the 2-5A system will be discussed.

Adenine Nucleotides↗
PubMed1990

Complexes of nuclear matrix DNA with proteins tightly bound to DNA contain a specific small-size RNA of a novel type.

Analysis of DNA-protein structures composed of nuclear matrix attached DNA and the most tightly bound proteins was performed. Although the previously described non-histone proteins (1) were present the buoyant density of the complex was the same as that of pure DNA. RNA inaccessible to RNase in 0.4 M NaCl but digestible in low ionic strength buffer was detected. This RNA is not a nascent one. It turned out to be homogeneous and represent a novel type of small nuclear RNA. Partial sequence of this RNA is presented.

Animals↗
PubMed1983

Steroid receptor-nuclear matrix interactions. The role of DNA.

The interaction of sex steroid hormone receptors with the nuclear matrix (NM) of target and non-target tissue was investigated using a simple in vitro binding assay. Steroid receptors can recognize acceptor sites on the NM of target cells; androgen receptor binds with the highest apparent affinity to rat prostate NM; similarly estrogen receptor binds with the highest apparent affinity to uterine NM. Furthermore, the steroid receptor-NM interaction depends upon the hormonal status of the animal. The binding of androgen receptor to rat prostate NM was drastically reduced upon hormone withdrawal (castration) and fully recovered upon hormonal stimulation. When NM were prepared by an alternate method (DNase I digestion prior to high salt extraction) known to digest "active" chromatin, no preferential receptor binding to target tissue NM was observed. Although the NM fraction contains less than 1% of the total nuclear DNA, the matrix-associated DNA sequences seem to be, at least in part, responsible for specific receptor recognition. DNA extracted from the prostate NM was shown to be a potent competitor for androgen receptor binding as measured by DNA-cellulose competition experiments. Moreover, this DNA recognition also depends upon the hormonal status of the animal. These studies are consistent with the notion that hormonal manipulation induces changes in the NM-associated DNA sequences of steroid hormone target tissue.

Animals↗
PubMed1999

Flanking nuclear matrix attachment regions synergize with the T cell receptor delta enhancer to promote V(D)J recombination.

Previous studies have identified nuclear matrix attachment regions (MARs) that are closely associated with transcriptional enhancers in the IgH, Igkappa, and T cell receptor (TCR) beta loci, but have yielded conflicting information regarding their functional significance. In this report, a combination of in vitro and in situ mapping approaches was used to localize three MARs associated with the human TCR delta gene. Two of these are located within the Jdelta3-Cdelta intron, flanking the core TCR delta enhancer (Edelta) both 5' and 3' in a fashion reminiscent of the Ig heavy chain intronic enhancer-associated MARs. The third is located about 20 kb upstream, tightly linked to Ddelta1 and Ddelta2. We have previously used a transgenic minilocus V(D)J recombination reporter to establish that Edelta functions as a developmental regulator of V(D)J recombination, and that it does so by modulating substrate accessibility to the V(D)J recombinase. We show here that the Edelta-associated MARs function synergistically with the core Edelta to promote V(D)J recombination in this system, as they are required for enhancer-dependent transgene rearrangement in single-copy transgene integrants.

Animals↗
PubMed2004

Comparison of nuclear matrix proteins between gastric cancer and normal gastric tissue.

AIM: To study the alteration of nuclear matrix proteins (NMPs) in gastric cancer. METHODS: The NMPs extracted from 22 cases of gastric cancer and normal gastric tissues were investigated by SDS-PAGE technique and the data were analyzed using Genetools analysis software. RESULTS: Compared with normal gastric tissue, the expression of 30 ku and 28 ku NMPs in gastric cancer decreased significantly (P=0.002, P=0.001, P<0.05). No significant difference was found in the expression of the two NMPs between the various differentiated grades (P=0.947, P=0.356) and clinical stages of gastric cancer (P=0.920, P=0.243, P>0.05). CONCLUSION: The results suggested that the alteration of NMPs in gastric cancer occurred at the early stage of gastric cancer development.

Cell Nucleus↗
PubMedDate not supplied

[Effect of alpha-tocopherol and nuclear tocopherol-binding proteins on DNA-polymerase activity of isolated nuclei and nuclear matrix].

It was shown, that tocopherol addition to the incubation medium increased the DNA-polymerase activity of isolated rat liver nuclei. This phenomenon was not discovered in nuclei of vitamin E-depleted rats. The action of tocopherol changed after the extraction of nuclei with triton X-100. In this situation tocopherol decreased DNA-polymerase activity of nuclei. The differences between rates of DNA synthesis was found after simultaneous addition of tocopherol and fraction of tocopherol-binding protein to the nuclei of normal and vitamin E-depleted rats. In this case DNA-polymerase activity of normal nuclei increased, but did not change in nuclei of vitamin E undernourished rats. The difference between them was about 30%. The differences between DNA-polymerase activity of normal and vitamin E-depleted rats after the addition of tocopherol and fraction of tocopherol-binding protein were also found in nuclear matrix preparation. It was supposed that tocopherol was able to take part in functioning of cell nuclei modulating DNA synthesis as well.

Animals↗
PubMed1991

Resinless section immunogold electron microscopy of karyo-cytoskeletal frameworks of eukaryotic cells cultured in vitro. Absence of a salt-stable nuclear matrix from mouse plasmacytoma MPC-11 cells.

The karyo-cytoskeleton of cells cultured in vitro was investigated employing resinless section immunogold electron microscopy. Cells were entrapped in low-melting agarose, sequentially extracted with various buffers and digested with nucleases to obtain karyo-cytoskeletal frameworks and reacted with specific primary and gold-conjugated secondary antibodies or gold-conjugated protein A to decorate structural elements of these frameworks. Following embedment of the gold-labeled residual cell structures in diethylene glycol distearate and their sectioning, the embedding material was removed with organic solvent and the sections were finally subjected to CO2 critical point drying. When this technique was applied to mouse skin fibroblasts (MSF), it revealed a dense and salt-stable intranuclear network of fibrogranular material. Antibodies directed against vimentin and lamin B detected a cytoplasmic meshwork of intermediate filaments (IFs) and a nuclear lamina, respectively; the latter, however, only after removal of chromatin from nuclei by nuclease digestion of DNA. Intranuclear filaments free of adhering globular material were morphologically very similar to cytoplasmic vimentin filaments. By contrast, mouse plasmacytoma MPC-11 cells lacking detectable amounts of cytoplasmic IF proteins and lamins A and C were devoid of a salt-stable internal nuclear matrix. The same holds true for MPC-11 cells that had been treated with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate to induce vimentin synthesis and establish a cytoplasmically extended IF network. These findings were in accordance with the biochemical behavior of Triton X-100-treated MSF and MPC-11 cells and their appearance in immunofluorescence microscopy upon extraction with high ionic strength buffer. While the chromatin was quantitatively retained in the residual cell structures derived from MSF cells, in those obtained from MPC-11 cells the nuclear lamina was disrupted and the chromatin was released from the nuclei, suggesting that MPC-11 cells lack the salt-stable nuclear scaffold to which chromatin is normally anchored.

Animals↗
PubMed1990

Immunolocalization in three dimensions: immunogold staining of cytoskeletal and nuclear matrix proteins in resinless electron microscopy sections.

We describe two methods for staining resinless thin sections with antibodies and gold-conjugated second antibodies. Immunolocalization of specific proteins is a powerful tool for cell structure studies but current techniques do not develop its full potential. Immunofluorescence provides only low-resolution localization, whereas conventional thin-section electron microscopy images and immunostains only the section surface. Resinless sections of extracted cell structures offer a simple and effective means of immuno-electron microscopy. Without embedding plastic or soluble proteins, the cell cytostructure produces high-contrast, three-dimensional images. Resinless sections of detergent-extracted cells are prepared by embedding in diethylene glycol distearate, sectioning, and removing diethylene glycol distearate before microscopy. In the first method of immunostaining, extracted cells were fixed and stained with antibodies before embedment, sectioning, removal of the embedding resin, and critical point drying. In the postembedment method, the sample was embedded and sectioned, the diethylene glycol distearate was removed, and the sample was rehydrated before antibody staining. With these techniques, specific proteins were localized with high resolution throughout the entire section. Stereoscopic micrographs of resinless sections revealed the precise localization of specific cytoskeleton and nuclear matrix proteins in three dimensions with unprecedented clarity.

Cell Line↗
PubMed1993

The Saccharomyces cerevisiae LOS1 gene involved in pre-tRNA splicing encodes a nuclear protein that behaves as a component of the nuclear matrix.

Mutations of the Saccharomyces cerevisiae LOS1 gene cause the accumulation of end matured intron-containing pre-tRNAs at elevated temperatures. In an effort to decipher the role of the LOS1 protein in pre-tRNA splicing, we have analyzed the LOS1 gene and its protein product. The LOS1 gene is located on the left arm of chromosome XI and the order of genes in this area of the chromosome is .... URA1 ... SAC1 TRP3 UBA1 STE6 LOS1 .... FAS1..... The LOS1 open reading frame encodes a putative protein of 1100 amino acids that shows no significant homology to other genes. The LOS1 open reading frame was tagged with the influenza virus hemagglutinin epitope recognized by the 12CA5 antibody. The 12CA5 antibody recognizes an epitope-tagged protein of the size predicted by the LOS1 open reading frame. Using this antibody for indirect immunofluorescence and cell fractionation studies we show that the LOS1 protein is located in nuclei. Los1p cannot be extracted from nuclei by treatment with nucleases, salts, or Triton X-100. This insolubility suggests that Los1p is a component of the nucleoskeleton. We propose that LOS1 mutations may affect pre-tRNA processing via alteration of the nuclear matrix.

Amino Acid Sequence↗