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At least 1,351 records · Page 75Linked to original sources
PubMed2003

Active poly(ADPribose) metabolism in DNAase- and salt-resistant rat testis chromatin with high transcriptional activity/competence.

A chromatin fraction, named pP fraction, was prepared from rat testis nuclei, which had been digested with nuclease in order to separate soluble and insoluble chromatin. This fraction resembled nuclear matrix as it was highly resistant to DNAase digestion, had a high content of proteins compared to the low DNA percentage, and a noticeable transcriptional activity. Moreover, poly(ADPribosyl)ation system (i.e., poly(ADPR)polymerase, poly(ADPribose), and acceptor proteins) was still present at high levels. In order to study whether it might be identified as the protein support surrounding chromatin loops, this pP fraction was further analyzed after 3 M NaCl extraction. The 3 M NaCl extract and the highly insoluble pellet, named Nuclear Matrix Pellet, were characterized as it regards DNA, newly synthesized RNA and proteins. Furthermore, poly(ADPribose) metabolism was analyzed by measuring both poly(ADPribose) polymerase and poly(ADPribose) glycohydrolase activities, poly(ADPribose) distribution and by identifying protein acceptors. The final pellet had features of nuclear matrix containing less than 10% DNA and high percentage of proteins; 28% of newly synthesized RNA was still associated with this fraction. Long and branched polyADPribose were found in the nuclear matrix-like pellet, although ADPribose acceptors (mainly H1 and core histones) appeared to be modified mostly with short ADPribose oligomers. Longest and branched polymers were retained on the top of protein gel, likely bound to automodified poly(ADPribose) polymerase.

Animals↗
PubMedDate not supplied

[Localization of matrix-associated elements in the 5'-region of the rat estrogen sulfotransferase gene].

Potential matrix-associated elements in the 5'-region of the rat estrogen sulfotransferase gene (Ste1) were searched for and characterized. The DNA fragments corresponding to the regions -800/+1048 and +1049/+2038 relative to the main point of transcription initiation were found to be bound to the nuclear matrix in vitro. A permanent association of the 5'-region of the Ste1 gene with the nuclear matrix in rat hepatocytes was found, the most probable site of attachment being the region -352/-152. No differences were found in the attachment of the 5'-region of the Ste1 gene to the nuclear matrix in the liver of males where the gene is actively transcribed and in that of females where it is inactive.

5' Flanking Region↗
PubMed1997

Quantitative immunofluorescence and immunoelectron microscopy of the topoisomerase II alpha associated with nuclear matrices from wild-type and drug-resistant chinese hamster ovary cell lines.

Topo II alpha is considered an important constituent of the nuclear matrix, serving as a fastener of DNA loops to the underlying filamentous scaffolding network. To further define a mechanism of drug resistance to topo II poisons, we studied the quantity of topo II alpha associated with the nuclear matrix in drug-resistant SMR16 and parental cells in the presence and absence of VP-16. Nuclear matrices were prepared from nuclei isolated in EDTA buffer, followed by nuclease digestion with DNase II in the absence of RNase treatment and extraction with 2 M NaCl. Whole-mount spreading of residual structures permits, by means of isoform-specific antibody and colloidal-gold secondary antibodies, an estimate of the amount of topo II alpha in individual nuclear matrices. There are significant variations in topo II alpha amounts between individual nuclear matrices due to the cell cycle distribution. The parental cell line contained eight to ten times more nuclear matrix-associated topo II alpha than the resistant cell line matrices. Nuclear matrix-associated topo II alpha from wild-type and resistant cell lines correlated well with the immunofluorescent staining of the enzyme in nuclei of intact cells. The amount of DNA associated with residual nuclear structures was five times greater in the resistant cell line. This quantity of DNA was not proportional to the quantity of topo II alpha in the same matrix; in fact they were inversely related. In situ whole-mount nuclear matrix preparations were obtained from cells grown on grids and confirmed the results from labeling of isolated residual structures.

Animals↗
PubMed1992

Electrical-ionic control of gene expression.

1. Changes in turgor, in cell volume, in membrane potential, in intracellular ionic activities and, more recently, in spontaneous electrical activity have been reported to be causally linked to the expression of specific genes. 2. As a result, it has become clear that changes in membrane properties and/or in the intracellular "ionic environment" can play an important role in generating cell type specific physiological responses which indirectly--or maybe directly--affect gene expression. 3. Possible targets of the ionic "environment" are: the selective transport across biological membranes; the activity of certain (regulatory) enzymes; the conformation of some (regulatory) proteins; of chromatin; of the cytoskeleton; of the nuclear matrix; the association of the cytoskeleton with plasmamembrane proteins or RNA; the association chromatin-nuclear matrix; protein-DNA and protein-protein interactions etc. All these sites may be instrumental to "fine or coarse" tuning of gene expression. 4. The exact mechanisms by which changes in intracellular ionic environment are transduced, directly or indirectly, into alterations of the activity of trans-acting factors have not yet been fully uncovered. Changes in the degree of phosphorylation of regulatory proteins and/or of trans-acting factors may provoke fine tuning effects on cell type specific gene expression activity. 5. The intranuclear ionic environment is difficult to measure in an exact way. It can be influenced in a number of ways. The location of a gene, as determined by the position of the nucleus in the cytoplasm and by the association of chromatin to the nuclear matrix may be especially important in cells which can generate some type of intracellular gradient or in excitable cells. 6. In some somatic cell types--germinal vesicles may behave differently--the intranuclear inorganic ionic "environment" has been reported to be distinct from the cytoplasmic one. This challenges the widespread assumption that the nuclear envelope is always freely permeable to small molecules and inorganic ions. 7. It can be expected that the fast progress in the cloning of "electrically" controlled genes, in the identification of trans-acting factors, in their mode of interaction with genes and in the precise localization of genes within the nucleus may soon lead to substantial progress in this domain.

Animals↗
PubMed1989

The limits of the DNase I-sensitive domain of the human apolipoprotein B gene coincide with the locations of chromosomal anchorage loops and define the 5' and 3' boundaries of the gene.

In eukaryotic cells, chromatin is organized as domains or loops that are generated by periodic attachment of the chromatin fiber to protein components of a nuclear matrix, or scaffold. These chromosomal loops may have a function in gene regulation. The length of the chromatin domain encompassing the human apolipoprotein B gene was studied by determining the locations of nuclear matrix attachment sites as well as the boundaries of the DNase I-sensitive domain in cells that express the gene (such as HepG2 and CaCo-2 cells) and in those that do not (HeLa cells). Three nuclear matrix attachment regions (MARs) of the human apolipoprotein B gene have been localized: a 3' -proximal MAR, between nucleotides +43,186 and +43,850; a 5' -proximal MAR, between nucleotides -2,765 and -1,801; and a 5' -distal MAR, between nucleotides -5,262 and -4,048. Both the 3' -proximal and the 5' -distal MARS were present in cells that express the gene (HepG2 and CaCo-2 cells) as well as in cells that do not (HeLa cells), whereas the 5' -proximal MAR was detected only in HepG2 cells. These MARs were located at the bases of chromosomal loops in histone-extracted nuclei in all three cell lines. Various classes of A/T-rich sequences resembling the recognition site for topoisomerase II were present within the MAR-containing fragments. The boundaries of the DNase I-sensitive domain coincide with the positions of the 3' -proximal and 5' -distal matrix attachment sites. These results suggest the existence of a 47.5-kilobase domain that represents a topologically sequestered functional unit containing the coding region and all known cis-acting regulatory elements of the human apolipoprotein B gene.

Apolipoproteins B↗
PubMed1992

Binding of matrix attachment regions to lamin B1.

Eukaryotic chromatin is organized into topologically constrained loops that are attached to the nuclear matrix. The regions of DNA that interact with the matrix are called matrix attachment regions (MARs). We studied the spatial distribution of MAR-binding sites in the nuclear matrix from rat liver cells, following a combined biochemical and ultrastructural approach. We found that MAR-binding sites are distributed equally over the internal fibrogranular network and the peripheral nuclear lamina. Internal and peripheral binding sites have similar binding characteristics: both sets of binding sites show specific and saturable binding of MARs from different organisms. By means of a DNA-binding protein blot assay and in vitro binding studies, we identified lamin B1 as a MAR-binding protein, which provides evidence for a specific interaction of DNA with the nuclear lamina.

Animals↗
PubMed2004

Expression of C/EBP delta in rat liver during development and the acute-phase response.

Using Western analysis, C/EBP delta was established in the nuclear extract and nuclear matrix throughout rat liver development and in the adult. During the acute-phase response (APR), C/EBP delta increased in the nuclear extract but remained unchanged in the nuclear matrix of fetal and postnatal rats, whereas it increased in both the nuclear extract and nuclear matrix of the adult. The solubility partitioning of gene regulatory proteins in the nucleus is important for their functioning (Uskoković et al. 2002). The obtained different solubility partitioning profiles of C/EBP delta suggest that its activity is regulated by different mechanisms during development and in the adult.

Acute-Phase Proteins↗
PubMed1992

Matrix attachment sites in the murine alpha-globin gene.

DNA sequences with a high affinity for nuclear matrix proteins have been identified and localized in the mouse alpha-globin gene. These matrix association regions (MARs) are adjacent, covering the first intron and part of the 5'-coding sequence. The binding sites are in close proximity to DNase I hypersensitive sites and other important signal sequences. The proteins of the nuclear lamina do not bind the alpha-globin gene MARs in the in vitro binding assay. The finding of MARs in the mouse alpha-globin gene creates an apparent paradox, since works from other authors and our results presented here indicate that this gene is not bound to the nuclear matrix in vivo. This contradiction is difficult to explain at present but different possibilities are accounted for in the text.

Animals↗
PubMed1994

Discrete localization of different DNA topoisomerases in HeLa and K562 cell nuclei and subnuclear fractions.

Monoclonal antibodies raised against DNA topoisomerase I and against topoisomerase II alpha and beta isoforms, which have been previously demonstrated to be highly specific and capable of detecting cell cycle-related variations of the topoisomerase II isoforms (Negri et al., 1992, Exp. Cell Res. 200, 452-459), have been utilized for a fine subcellular localization. Immunocytochemistry by confocal and electron microscopy have been used for a topological and quantitative evaluation of the fine distribution of the different topoisomerases in HeLa and K562 cells. Topoisomerase I and topoisomerase II alpha are present both in the nucleoplasm and in the nucleolus, though at different relative ratios, while topoisomerase II beta is exclusively present at the nucleolar level. This is further confirmed by immunoblotting and immunocytochemical quantitative evaluations performed on purified nuclear matrix fractions obtained from K562 cells. In fact, the amount of topoisomerase I and topoisomerase II alpha present in the whole cell nuclei is partly lost in isolated nuclei but, while topoisomerase I is further significantly reduced in nuclear matrix preparations, the topoisomerase II alpha content is only slightly decreased. On the other hand, the great majority of topoisomerase II beta is retained in the nuclear matrix and can be detected exclusively in association with the nucleolar remnant. These results are consistent with specific functional roles hypothesized for the different topoisomerase types.

Antibodies, Monoclonal↗
PubMed2000

Radioprotective thiolamines WR-1065 and WR-33278 selectively denature nonhistone nuclear proteins.

Differential scanning calorimetry was used to study the interactions of nuclei isolated from Chinese hamster V79 cells with the radioprotector WR-1065, other thiol compounds, and polyamines. Differential scanning calorimetry monitors denaturation of macromolecules and resolves the major nuclear components (e.g. constrained and relaxed DNA, nucleosome core, and nuclear matrix) of intact nuclei on the basis of thermal stability. WR-1065 treatment (0.5-10 mM) of isolated nuclei led to the irreversible denaturation of nuclear proteins, a fraction of which are nuclear matrix proteins. Denaturation of 50% of the total nonhistone nuclear protein content of isolated nuclei occurred after exposure to 4.7 mM WR-1065 for 20 min at 23 degrees C. In addition, a 22% increase in the insoluble protein content of nuclei isolated from V79 cells that had been treated with 4 mM WR-1065 for 30 min at 37 degrees C was observed, indicating that WR-1065-induced protein denaturation occurs not only in isolated nuclei but also in the nuclei of intact cells. From the extent of the increase in insoluble protein in the nucleus, protein denaturation by WR-1065 is expected to contribute to drug toxicity at concentrations greater than approximately 4 mM. WR-33278, the disulfide form of WR-1065, was approximately twice as effective as the free thiol at denaturing nuclear proteins. The proposed mechanism for nucleoprotein denaturation is through direct interactions with protein cysteine groups with the formation of destabilizing protein-WR-1065 disulfides. In comparison to its effect on nuclear proteins in isolated nuclei, WR-1065 had only a very small effect on non-nuclear proteins of whole cells, isolated nuclear matrix, or the thiol-rich Ca(2+)ATPase of sarcoplasmic reticulum, indicating that WR-1065 can effectively denature protein only inside an intact nucleus, probably due to the increased concentration of the positively charged drug in the vicinity of DNA.

Animals↗
PubMed2000

Structural and functional aspects of nuclear lipids in normal and tumor cells.

This review summarizes available data on the structural and functional role of neutral lipids and phospholipids in normal and tumor eukaryotic cells. The role of acidic phospholipids (cardiolipin, phosphatidylinositol, and phosphatidylglycerol) in regulation of activities of DNA- and RNA-polymerases, DNA-topoisomerases I and II, DNA-methylases, and replication initiation proteins (dnaA and T-antigen) is discussed. The role of sphingolipids is emphasized considering, on one hand, the involvement of sphingosines in signal transduction, chromatin association-dissociation, and regulation of DNA and RNA synthesis and protein kinase C and, on the other hand, participation of ceramides and dihydroceramides in apoptosis. The possible role of sphingomyelin, sphingosine, cardiolipin, and diglycerides in the contacts of DNA loops with nuclear matrix is analyzed. Lipid hormones indirectly influence supercoiled DNA conformation; the effect of hormones on metabolism of phospholipids and neutral lipids in chromatin and nuclear matrix is reviewed. Characteristics of lipid composition in chromatin and nuclear matrix of the tumor cells are discussed.

Cell Nucleus↗
PubMed1997

Structural protein 4.1 in the nucleus of human cells: dynamic rearrangements during cell division.

Structural protein 4.1, first identified as a crucial 80-kD protein in the mature red cell membrane skeleton, is now known to be a diverse family of protein isoforms generated by complex alternative mRNA splicing, variable usage of translation initiation sites, and posttranslational modification. Protein 4.1 epitopes are detected at multiple intracellular sites in nucleated mammalian cells. We report here investigations of protein 4.1 in the nucleus. Reconstructions of optical sections of human diploid fibroblast nuclei using antibodies specific for 80-kD red cell 4.1 and for 4.1 peptides showed 4.1 immunofluorescent signals were intranuclear and distributed throughout the volume of the nucleus. After sequential extractions of cells in situ, 4.1 epitopes were detected in nuclear matrix both by immunofluorescence light microscopy and resinless section immunoelectron microscopy. Western blot analysis of fibroblast nuclear matrix protein fractions, isolated under identical extraction conditions as those for microscopy, revealed several polypeptide bands reactive to multiple 4.1 antibodies against different domains. Epitope-tagged protein 4.1 was detected in fibroblast nuclei after transient transfections using a construct encoding red cell 80-kD 4.1 fused to an epitope tag. Endogenous protein 4.1 epitopes were detected throughout the cell cycle but underwent dynamic spatial rearrangements during cell division. Protein 4.1 was observed in nucleoplasm and centrosomes at interphase, in the mitotic spindle during mitosis, in perichromatin during telophase, as well as in the midbody during cytokinesis. These results suggest that multiple protein 4.1 isoforms may contribute significantly to nuclear architecture and ultimately to nuclear function.

3T3 Cells↗
PubMed2001

Delaying S-phase progression rescues cells from heat-induced S-phase hypertoxicity.

The mechanism by which a cell protects itself from the lethal effects of heat shock and other stress-inducing agents is the subject of much research. We have investigated the relationship between heat-induced damage to DNA replication machinery and the lethal effects of heat shock, in S-phase cells, which are more sensitive to heat shock than either G1 or G2. We found that maintaining cells in aphidicolin, which prevents the passage of cells through S-phase, can rescue S-phase HeLa cells from the lethal effects of heat shock. When S-phase, HeLa cells were held for 5-6 h in 3 microM aphidicolin the measured clonogenic survival was similar to that for exponentially growing cells. It is known, that heat shock induces denaturation or unfolding of proteins, rendering them less soluble and more likely to co-isolate with the nuclear matrix. Here, we show that enhanced binding of proteins involved in DNA replication (PCNA, RPA, and cyclin A), with the nuclear matrix, correlates with lethality of S-phase cells following heat shock under four different experimental conditions. Specifically, the amounts of RPA, PCNA, and cyclin A associated with the nuclear matrix when cells resumed progression through S-phase correlated with cell killing. Heat-induced enhanced binding of nuclear proteins involved with other aspects of DNA metabolism, (Mrell, PDI), do not show this correlation. These results support the hypothesis that heat-induced changes in the binding of proteins associated with DNA replication factories are the potentially lethal lesions, which become fixed to lethal lesions by S-phase progression but are repairable if S-phase progression is delayed.

Aphidicolin↗
PubMed1996

Nonhistone protein reorganization in normal and hepatoma cells.

Previously we described (Dong et al., 1990) a nuclear protein (mol. wt. 112 kD) which is expressed abundantly in hepatoma cells and also in hepatocyte cells committed to carcinogenesis. In this report, we further characterize its chemical properties and cellular localization in normal and hepatoma cells. 112 kD hepatoma-associate nonhistone protein is not a cytokeratin-related protein as described by Fukuda et al. (1991). Protein purification experiments revealed that 112 kD protein is a dimer of 56 kD polypeptide present in normal rat liver nuclei. Intranuclear distribution pattern indicated that 112 kD nonhistone protein localizes exclusively in hepatoma nuclear matrix. The data from this study suggest that dimerization of 56 kD nonhistone protein is involved in nuclear matrix reorganization during neoplastic transformation.

Animals↗
PubMed1991

Visualizing DNA replication sites in the cell nucleus.

Studies of DNA replication associated with the nuclear matrix have led to a radically new view of replication at the macroscopic level. It is proposed that individual replicons and their associated replicational assemblies (replisomes) are clustered together during active replication by attachment to the nuclear matrix at special sites termed 'clustersomes'. Direct visualization of replication sites in permeabilized cells by fluorescence microscopy following biotin-11-dUTP incorporation provides support for this model. Discrete replication granules are observed with sizes and numbers consistent with each granule being a site of replicon cluster synthesis. Distinct patterns of these sites are seen in different periods of S-phase. Both the individual granules and their early and late S-phase dependent patterns are strikingly maintained following extraction of the cells for in situ nuclear matrix structures. Similar results were obtained when probing in vivo sites of replication following incorporation of 5-bromodeoxyuridine. The three-dimensional organization of these replicational granules (clustersomes) is studied using confocal light microscopy and an appropriate multidimensional image analysis system.

Animals↗
PubMed2003

Functional cooperativity between osteoblast transcription factors: evidence for the importance of subnuclear macromolecular complexes?

The accompanying article by Drs. Lian and Stein describes current thinking on how genes are organized in the nucleus and suggests that subnuclear localization is critical for the control of gene expression in bone. In particular, it is proposed that a major function of the osteoblast transcription factor, Runx2, is to tether genes that are active in osteoblasts to the nuclear matrix and serve as an organizing center for other nuclear factors which together form osteoblast-specific transcriptional units. Although it is still not established that the nuclear matrix localization function of Runx2 is essential for all its biological activities, there is no question that this factor plays a central role in mediating the response of osteoblasts to a variety of signals, as would be expected if Runx2 were involved in organizing the transcriptional apparatus. As will be discussed, Runx2 is required for the response of osteoblasts to other lineage-specific transcription factors and signals initiated by extracellular matrix-integrin binding, growth factors, hormones, and morphogens. We hypothesize that Runx2 transduces this wide range of responses by cycling between active, phosphorylated and a less active, dephosphorylated states which can selectively interact with other nuclear factors to form macromolecular complexes active in transcription. The possible relationship between these complexes and the subnuclear localization of the osteoblast transcriptional apparatus will also be discussed.

Animals↗
PubMed2002

Organization of the macronuclear gene-sized pieces of stichotrichous ciliates into a higher order structure via telomere-matrix interactions.

Macronuclear DNA of stichotrichous ciliates occurs in small 'gene-sized' molecules with sizes of about 0.5 to 40 kb. Each of these molecules is terminated by telomeric sequences of defined length. A single macronucleus contains up to 10(8) DNA molecules; due to the high concentration of telomeric sequences in this nucleus it is an attractive model to study telomere behaviour. We recently provided evidence that macronuclear telomeres are attached to the nuclear matrix and that this interaction is mediated by the telomere binding protein (TeBP). Using various experimental approaches, we now demonstrate that telomeres as well as both subunits of the telomere binding protein are associated with the nuclear matrix. However, there is no direct binding of telomeric DNA to the matrix but telomere matrix interaction is exclusively mediated by the TeBP. In addition, we show that telomeric sequences adopt in vivo the antiparallel G-quartet structure when bound to the nuclear matrix. These data not only allow us to propose a model for macronuclear architecture but may also be relevant for further analysis of telomere-matrix interactions in higher eukaryotes.

Animals↗
PubMedDate not supplied

[Spatial organization of replicons in the eukaryotic nucleus: attachment of replication initiation regions to the nuclear skeleton].

The DNA fraction enriched in replication origins was isolated from chicken erythroblastosis cells with the aid of nascent DNA strand extrusion method. In renaturation experiments, the sequence specificity of this DNA fraction (oriDNA) was compared to the sequence specificity of the nuclear matrix DNA. The oriDNA was shown to comprise a specific subset of unique genes all of which being represented also in the nuclear matrix DNA. The same unique DNA sequences were found to be depleted from the oriDNA and from the nuclear matrix DNA. A conclusion has been drawn that replication origins are located at the basements of DNA loops. The putative positions of replication origins within the domain of chicken alpha globin genes were mapped by hybridization experiments and were found to coincide with the positions of stable (not depending on the transcriptional state of globin genes) sites of DNA attachment to the nuclear skeleton.

Animals↗