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PubMed1999

The beta4 integrin interactor p27(BBP/eIF6) is an essential nuclear matrix protein involved in 60S ribosomal subunit assembly.

p27(BBP/eIF6) is an evolutionarily conserved protein that was originally identified as p27(BBP), an interactor of the cytoplasmic domain of integrin beta4 and, independently, as the putative translation initiation factor eIF6. To establish the in vivo function of p27(BBP/eIF6), its topographical distribution was investigated in mammalian cells and the effects of disrupting the corresponding gene was studied in the budding yeast, Saccharomyces cerevisiae. In epithelial cells containing beta4 integrin, p27(BBP/eIF6) is present in the cytoplasm and enriched at hemidesmosomes with a pattern similar to that of beta4 integrin. Surprisingly, in the absence and in the presence of the beta4 integrin subunit, p27(BBP/eIF6) is in the nucleolus and associated with the nuclear matrix. Deletion of the IIH S. cerevisiae gene, encoding the yeast p27(BBP/eIF6) homologue, is lethal, and depletion of the corresponding gene product is associated with a dramatic decrease of the level of free ribosomal 60S subunit. Furthermore, human p27(BBP/eIF6) can rescue the lethal effect of the iihDelta yeast mutation. The data obtained in vivo suggest an evolutionarily conserved function of p27(BBP/eIF6) in ribosome biogenesis or assembly rather than in translation. A further function related to the beta4 integrin subunit may have evolved specifically in higher eukaryotic cells.

Amino Acid Sequence↗
PubMed1989

Heat shock-induced appearance of RNA polymerase II in karyoskeletal protein-enriched (nuclear "matrix") fractions correlates with transcriptional shutdown in Drosophila melanogaster.

Heat shock in vivo or brief incubation at moderately elevated temperatures (15 min at 37 degrees C) in vitro is required for the structural stability of proteinaceous karyoskeletal elements purified from Drosophila melanogaster (McConnell, M., Whalen, A. M., Smith, D. E., and Fisher, P. A. (1987) J. Cell Biol. 105, 1087-1098). We have found that the appearance of the two largest subunits of RNA polymerase II in karyoskeletal preparations is promoted by conditions identical to those which promote in vitro stability of karyoskeletal elements overall. Increased levels of polymerase II in karyoskeletal protein-enriched fractions correlate with decreased levels of nucleotide incorporation in nuclear transcription run-on assays. These results are not easily reconciled with the proposal that putative karyoskeletal elements such as the internal nuclear "matrix" are sites of active transcription in vivo.

Animals↗
PubMed1985

DNA regions associated with the nuclear matrix of Ehrlich ascites cells expose single-stranded sites after deproteinization.

Ehrlich ascites cells were pulse-labeled with [3H]thymidine and subjected to prolonged labeling with [14C]thymidine. The isolated nuclei were digested with the restriction endonuclease BspRI and then processed to yield a 'matrix fraction' and a 'non-matrix fraction'. The DNA fragments purified from these fractions and from whole digested nuclei were examined for nitrocellulose-binding sites before and after digestion with single-strand-specific (S1) nuclease. Both, pulse-labeled and long-time-labeled fragments, isolated from the matrix fraction, exhibited a significantly increased content of nitrocellulose-binding sites. The major portion of these sites were rendered non-binding by digestion with single-strand-specific nuclease and consisted most probably of structures exposing relatively small stretches of non-base-paired DNA. The nature of the minor portion of binding sites which was insensitive to single-strand-specific nuclease is not clear. Both types of binding sites are possible candidates for mediating the attachment of DNA to the nuclear matrix.

Animals↗
PubMed2001

A new spectrin, beta IV, has a major truncated isoform that associates with promyelocytic leukemia protein nuclear bodies and the nuclear matrix.

We isolated cDNAs that encode a 77-kDa peptide similar to repeats 10-16 of beta-spectrins. Its gene localizes to human chromosome 19q13.13-q13.2 and mouse chromosome 7, at 7.5 centimorgans. A 289-kDa isoform, similar to full-length beta-spectrins, was partially assembled from sequences in the human genomic DNA data base and completely cloned and sequenced. RNA transcripts are seen predominantly in the brain, and Western analysis shows a major peptide that migrates as a 72-kDa band. This new gene, spectrin betaIV, thus encodes a full-length minor isoform (SpbetaIVSigma1) and a truncated major isoform (SpbetaIVSigma5). Immunostaining of cells shows a micropunctate pattern in the cytoplasm and nucleus. In mesenchymal stem cells, the staining concentrates at nuclear dots that stain positively for the promyelocytic leukemia protein (PML). Expression of SpbetaIVSigma5 fused to green fluorescence protein in cells produces nuclear dots that include all PML bodies, which double in number in transfected cells. Deletion analysis shows that partial repeats 10 and 16 of SpbetaIVSigma5 are necessary for nuclear dot formation. Immunostaining of whole-mount nuclear matrices reveals diffuse positivity with accentuation at PML bodies. Spectrin betaIV is the first beta-spectrin associated with a subnuclear structure and may be part of a nuclear scaffold to which gene regulatory machinery binds.

Amino Acid Sequence↗
PubMed1986

Varicella-zoster virus p32/p36 complex is present in both the viral capsid and the nuclear matrix of the infected cell.

Varicella-zoster virus (VZV) directs the synthesis of numerous glycosylated and nonglycosylated infected-cell-specific proteins, many of which are later incorporated into the virion as structural components. In this study, we characterized a nonglycosylated polypeptide complex with the aid of a VZV-specific murine monoclonal antibody clone, 251D9. As detected by indirect immunofluorescence, the antibody bound mainly to antigens located within the nuclei of infected cells and did not attach to an uninfected cell substrate. The polypeptide specificity of the monoclonal antibody was determined by immunoblot analysis of electrophoretically separated infected cell extracts to react with a 32,000-molecular-weight VZV-specific protein (p32); in addition, the antibody also bound to a 36,000-molecular-weight polypeptide. The synthesis of these antigens was unaffected by inhibitors of glycosylation. Nonionic or ionic detergents were only marginally effective in solubilization of the p32-p36 complex, and relatively small amounts were eluted from nuclei by high salt concentrations (2 M NaCl). The same proteins remained associated with the nuclear matrix of VZV-infected cells. We also demonstrated that the protein complex was a major component of purified VZV nucleocapsids; p32 was especially prominent in both full and empty capsids. Immunoblot analysis of the nucleocapsid preparation revealed two additional species (p34 and p38) in the p32-p36 complex. Phosphorylation was a distinctive feature of some of the constituents. In summary, these results indicate that the p32-p36 complex represents a family of structural proteins closely associated with the assembly of VZV nucleocapsids and the encapsidation of viral DNA.

Antibodies, Monoclonal↗
PubMed1987

Glycosylation of simian virus 40 T antigen and localization of glycosylated T antigen in the nuclear matrix.

Evidence has been obtained for the glycosylation of simian virus 40 (SV40) T antigen in SV40-infected TC7 cells. Both [3H]mannose and [3H]glucosamine are incorporated into T antigen in cells grown and labeled in medium containing fructose instead of glucose. In addition, T antigen is visualized by a carbohydrate stain specific for mannose and/or glucose residues. Finally, lectin binding studies suggest that T antigen contains galactose and/or galactosamine, since T antigen is specifically eluted from soybean lectin by 0.2 M galactose. When gel-purified, [3H]glucosamine-labeled T antigen is subjected to tryptic peptide mapping, label is found in only one peptide, thought to correspond to the methionine-containing peptide extending from Asn-653 to Arg-691, near the carboxy-terminal end of T antigen. Insensitivity to tunicamycin and the localization of the glycosylation site in the carboxy-terminus of T antigen, and not at Asn-153, suggest that T antigen is not N-glycosylated. Cell fractionation studies show that [3H]glucosamine-labeled T antigen is preferentially associated with the nuclear matrix of SV40-infected TC7 cells.

Antigens, Viral, Tumor↗
PubMed1989

DNA fragments which specifically bind to isolated nuclear matrix in vitro interact with matrix-associated DNA topoisomerase II.

A matrix-associated region (MAR)-containing fragment has been selected from the library of cloned chicken nuclear matrix-associated DNA fragments. Factors, which determine the specific binding of DNA fragments have been studied. Using topoisomerase II-specific inhibitor VM 26 we established that nuclear matrix-associated topoisomerase II interacted with the MAR-containing DNA fragment producing specific cleavage sites on DNA of the fragment.

Animals↗
PubMedDate not supplied

Fine structural observations on the human sperm nuclear matrix.

The nucleus of the mammalian sperm is formed after a series of morphological and biochemical changes during spermatogenesis. The human sperm nucleus, after sequential extraction with detergents, nuclease and ammonium sulfate, consists of a fibroskeletal structure which maintains the original nuclear shape. The chromatin-depleted skeleton is formed by thick and thin fibers as well as electron-dense patches of different sizes. These highly branched matrix fibers had average diameters of 35 and 12 nm. Polarization of the fibroskeletal structure is apparent and can be used as a good model to study the function of nuclear matrix in nuclear compartmentation in germ cells.

Chromatin↗
PubMed1992

A differential location of phosphoinositide kinases, diacylglycerol kinase, and phospholipase C in the nuclear matrix.

Several enzymes involved in the phosphoinositide metabolism have been shown to be present in nuclei of rat liver and Friend cells. In this paper we demonstrate that nuclear matrices of mouse NIH 3T3-fibroblasts and rat liver cells, isolated by nuclease treatment and high salt extraction, contain phosphatidylinositol 4-kinase (PdtIns 4-kinase), phosphatidylinositol 4-phosphate 5-kinase (PtdIns(4)P 5-kinase), diacylglycerol kinase, and phospholipase C. By a selective extraction the nucleus can be dissected in the peripheral matrix (lamina-pore complex) and the internal matrix as shown by using marker antibodies. Surprisingly, PtdIns 4-kinase was found exclusively in the peripheral nuclear matrix, whereas PtdIns(4)P 5-kinase was found to be associated to internal matrix structures. Diacylglycerol kinase and phospholipase C activities were also preferentially detected in the internal matrix. These data demonstrate a differential localization of the phosphoinositide kinases in the nucleus and suggest that the phosphoinositide metabolism may play a specific role in the nucleus.

1-Phosphatidylinositol 4-Kinase↗
PubMed1987

Association of SV40 DNA sequences with nuclear matrix in SV40 transformed hamster fibroblasts.

Nuclear matrices were isolated by the high-salt, non-ionic detergent method from SV40-transformed hamster fibroblasts (TSV5 cell line), and from hamster tumours derived from these cells. DNA isolated from matrices and total nuclei was hybridized with nick-translated SV40 DNA. The enrichment of matrix DNA with SV40 DNA sequences was observed in all five experiments with matrix DNA of TSV5 cells but only in five out of nine matrix DNA isolated from tumour cells.

Animals↗
PubMed2005

Repair of DNA interstrand crosslinks may take place at the nuclear matrix.

Host cell reactivation assay using Trioxsalen-crosslinked plasmid pEGFP-N1 showed that human cells were able to repair Trioxsalen interstrand crosslinks (ICL). To study the mechanism of this repair pathway, cells were transfected with the plasmids pEGFP-1, which did not contain the promoter of the egfp gene, and with pEGFP-G-, which did not contain the egfp gene. Neither of these plasmids alone was able to express the green fluorescent protein. After cotransfection with the two plasmids, 1%-2% of the cells developed fluorescent signal, which showed that recombination events had taken place in these cells to create DNA constructs containing the promoter and the gene properly aligned. When one or both of the plasmids were crosslinked with Trioxsalen, the recombination rate increased several fold. To identify the nuclear compartment where recombination takes place, cells were transfected with crosslinked pEGFP-N1 and the amount of plasmid DNA in the different nuclear fractions was determined. The results showed that Trioxsalen crosslinking increased the percentage of matrix attached plasmid DNA in a dose-dependent way. Immunoblotting experiments showed that after transfection with Trioxsalen crosslinked plasmids the homologous recombination protein Rad51 also associated with the nuclear matrix fraction. These studies provide a model system for investigating the precise molecular mechanisms that appear to couple repair of DNA ICL with nuclear matrix attachment.

Animals↗
PubMed2000

Fatal connections: when DNA ends meet on the nuclear matrix.

A damaged nucleus has long been regarded simply as a "bag of broken chromosomes," with the DNA free ends moving around and forming connections with randomly encountered partners. Recent evidence shows this picture to be fundamentally wrong. Chromosomes occupy specific nuclear domains within which only limited movement is possible. In a human diploid nucleus, 6.6 x 10(9) base pairs (bp) of DNA are compartmentalized into chromosomes in a way that allows stringent control of replication, differential gene expression, recombination and repair. Most of the chromatin is further organized into looped domains by the dynamic binding of tethered bases to a network of intranuclear proteins, the so-called nuclear scaffold or matrix. Thus, DNA movement is severely curtailed, which limits the number of sites where interchanges can occur. This intricate organizational arrangement may render the genome vulnerable to processes that interfere with DNA repair. Both lower and higher eukaryotic cells perform homologous recombination (HR) and illegitimate recombination (IR) as part of their survival strategies. The repair processes comprising IR must be understood in the context of DNA structural organization, which is fundamentally different in prokaryotic and eukaryotic genomes. In this paper we first review important cellular processes including recombination, DNA repair, and apoptosis, and describe the central elements involved. Then we review the different DNA targets of recombination, and present recent evidence implicating the nuclear matrix in processes which can induce either repair, translocation, deletion, or apoptosis. J. Cell. Biochem. Suppl. 35:3-22, 2000.

Animals↗
PubMed2000

Changes in the expression of cytokeratins and nuclear matrix proteins are correlated with the level of differentiation in human prostate cancer.

The nuclear matrix-intermediate filament complex (NM-IF) is a protein scaffold which spans the whole cell, and several lines of evidence suggest that this structural frame represents also a functional unit, which could be involved in the epigenetic control of cancer development. Here we report the characterization by high resolution two-dimensional gel electrophoresis and Western blot analysis of the NM-IF complex isolated from prostate cancer (PCa); tumor-associated proteins were identified by comparing the electrophoretic patterns with those of normal human prostate (NHP). Extensive changes in the expression of both the NM and IF proteins occur; they are, however, related in a different way to tumor progression. Poorly differentiated PCa (Gleason score 8-9) shows a strong down regulation of several constitutive cytokeratins (CKs 8, 18, and 19); their expression significantly (P < 0.05) decreases with respect to both NHP and benign prostatic hyperplasia (BPH) and, more interestingly, also with respect to moderately (Gleason score 6-7) and well (Gleason score 4-5) differentiated tumors. Moreover, we have identified a tumor-associated species which is present in all of the tumors examined, systematically absent in NHP and occurs only in a few samples of BPH; this polypeptide, of M(r) 48,000 and pI 6.0, represent a proteolytic fragment of CK8. At variance with these continuing alterations in the expression, the NM proteins undergo stepwise changes correlating with the level of differentiation. The development of less differentiated tumors is characterized by the appearance of several new proteins and by the decrease in the expression of others. Six proteins were found to be expressed with a frequency equal to one in poorly differentiated tumor, namely in all the samples of tumor examined, while in moderately and well differentiated tumors the frequency is less than one, and decreases with increasing the level of differentiation. When tumors of increasing Gleason score are compared with NHP a dramatic increase in the complexity of the protein patterns is observed, indicating that tumor dedifferentiation results in a considerable increase in the phenotypic diversity. These results suggest that tumor progression can be characterized using an appropriate subset of tumor-associated NM proteins.

Adenocarcinoma↗
PubMed1989

Estradiol receptor-nuclear interactions in aging mouse uteri: the role of DNA and nuclear matrix.

We have studied the interactions of estradiol receptor (ER) with nuclear DNA or matrix (NM) in the uteri from young, middle-aged and old mice. Recombination studies using heat-activated ER and nuclear subfractions from various age groups suggested a 30% reduction for DNA binding (P less than 0.05) but not for NM with aging. Cross-incubation studies of heat-activated ER with nuclear subfractions from mice of all three age subgroups showed that this reduced DNA binding ability followed the onset of anestrous and did not result from proteolytic degradation of ER complexes.

Aging↗
PubMed1985

The interrelation between DNA synthesis rates and DNA polymerases bound to the nuclear matrix in synchronized HeLa cells.

Quantitative rates of DNA synthesis can be determined by DNA:propidium fluorescence measurements of synchronized cells progressing through S-phase. We have previously reported that HeLa cells have discontinuous rates with values of about 2.9, 1.6, and 4.4 pg of DNA/h for early, middle, and late S-phase, respectively. In attempts to understand why two peaks of DNA synthesis rates are observed, we have examined the nuclear DNA polymerases alpha and beta over the S-phase. Nuclear matrices isolated from HeLa cells contained 2% of the alpha polymerase and 12% of the beta polymerase that was present in cell lysates, and about 2% of the original DNA. The amounts of endogenous DNA synthesis in isolated nuclear matrices were comparable to the amounts observed when exogenous DNA was added. DNase treatment abolished the endogenous DNA synthesis but not the exogenous DNA synthesis, suggesting that polymerase alpha binding does not depend on matrix-bound DNA. As synchronized cells progressed through the S-phase, there appeared two peaks of enzymatic activity of alpha polymerase bound to the nuclear matrix which correlated with in vitro DNA synthesis in these nuclear matrices and with the two peaks of quantitative DNA synthesis rates. Two peaks of alpha polymerase activity were also observed with isolated nuclei, but not with cell lysates or cytosol. Our results suggest that, over the S-phase, the differential binding of polymerase alpha to the nuclear matrix determines the differential rates of DNA synthesis.

Cell Nucleus↗
PubMed1996

Properties of nuclear matrix proteins that bind the 5' flanking region of the haptoglobin gene are changed during the acute-phase response.

It was previously shown that during the acute-phase response-induced elevated transcription of the rat haptoglobin gene, protein p55 and the lamins mediate the increased binding of restriction fragment II (-541/-146) via a 38 bp adenine tract lying 147 bp upstream from the haptoglobin gene cis element (-165/-56), to scaffold type II-like nuclear matrices. Here we show that the fragment II binding pattern to > or = 40 kDa proteins of nuclear matrices analogous to type I scaffolds is more complex. Fragment II bound conspicuously to a 40 and less so to p55, a 60 kDa protein and the lamins of control matrices. During increased gene transcription, it bound prominently to p55, the lamins, and less so to 45 and 52 kDa proteins. This was accompanied by the tenacious binding of the DNA to isolated nuclear matrices in vitro and post-translational modifications of certain matrix proteins. The lamins and p55 demonstrated a greater N-acetylglucosamine/sialic acid content and p55 an increased in vitro phosphorylation by a nuclear matrix-associated cyclic-nucleotide-independent kinase. The acute-phase response also caused an increased partitioning of p55 with the nuclear matrix. It was concluded that, as a result of a molecular remodelling of the nuclear matrix at the point of contact with chromatin, the nature of its association with region II DNA changed during elevated haptoglobin gene expression.

Acute-Phase Reaction↗
PubMed1985

Nuclear RNA-associated proteins and their relationship to the nuclear matrix and related structures in HeLa cells.

The ultrastructure and the polypeptide composition of residual nuclear substructures including nuclear matrices, nuclear ghosts, and residual envelopes were investigated by means of electron microscopy and two-dimensional gel electrophoresis. Nuclear matrices were prepared by digesting isolated nuclei with DNase I alone, followed by high-salt extraction in 2 M NaCl. Nuclear ghosts were obtained by high-salt extraction of nuclei previously digested with DNase and RNase in MgCl2-containing buffers. To prepare residual envelopes, nuclei were first digested with RNase in the presence of EDTA, then digested with Mg2+ -activated DNase I, and extracted in 2 M NaCl. The results of this comparative study support the conclusion that the intranuclear matrix is made of two distinct RNA-containing elements. One of these elements appears on ultrathin sections as a thin fibrillar network. It disappears from RNase-digested nuclei, together with numerous basic proteins, whatever the conditions of digestion. Although this element is present in extranucleolar territories, it is a major component of residual nucleoli. The second element appears as coarse-beaded fibers absent from the nucleolar areas. Its preservation in residual nuclear substructures depends on the presence of Mg2+ ions during RNase digestion of nuclei. It is enriched in two minor basic proteins of relative mass 49 000 and 70 000. The involvement of this fibrogranular element in heterogeneous nuclear RNA attachment to the nuclear matrix is discussed.

Cell Fractionation↗
PubMed1985

In situ localization of DNA topoisomerase II, a major polypeptide component of the Drosophila nuclear matrix fraction.

DNA topoisomerase II has been immunochemically identified on protein blots as a major polypeptide component of the Drosophila nuclear matrix-pore complex-lamina fraction. Indirect immunofluorescence analyses of larval cryosections have confirmed the nuclear localization of topoisomerase II in situ. Although apparently excluded from the nucleolus, the topoisomerase protein is otherwise distributed throughout the interior of interphase nuclei. Similar immunocytochemical studies performed with permeabilized whole giant cells from third-instar larval salivary glands have shown topoisomerase II to be largely restricted to the polytene chromosomes. Upon nuclear disassembly during mitosis, the topoisomerase polypeptide appears to redistribute diffusely throughout the cell. Faint immunofluorescent staining of mitotic chromosomes is also observed.

Animals↗