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PubMed1984

Differential association with cellular substructures of pseudorabies virus DNA during early and late phases of replication.

Pseudorabies virus DNA synthesis can be divided into two phases, early and late, which can be distinguished from each other on the basis of the structures of the replicating DNA. The two types of replicating virus DNA can also be distinguished from each other on the basis of the cellular substructures with which each is associated. Analysis by electron microscopic autoradiography showed that during the first round of replication, nascent virus DNA was found in the vicinity of the nuclear membrane; during later rounds of replication the nascent virus DNA was located centrally within the nucleus. The degree of association of virus DNA synthesized at early and late phases with the nuclear matrix fractions also differed; a larger proportion of late than of early nascent virus DNA was associated with this fraction. While nascent cellular DNA only was associated in significant amounts with the nuclear matrix fraction, a large part (up to 40%) of all the virus DNA remained associated with this fraction. However, no retention of specific virus proteins in this fraction was observed. Except for two virus proteins, which were preferentially extracted from the nuclear matrix, approximately 20% of all virus proteins remained in the nuclear matrix fraction. The large proportion of virus DNA associated with the nuclear fraction indicated that virus DNA may be intimately associated with some proteins. Indeed, protease-treated, "purified" DNA preparations contained two proteins (15K and 10K) with histone-like properties which were protected by the DNA from deproteinization, probably by virtue of being at the center of the concatemeric tangles of virus DNA. It is possible that these proteins play a role in anchoring virus DNA to the nuclear matrices.

Animals↗
PubMed1999

Characterization of randomly-obtained matrix attachment regions (MARs) from higher plants.

Matrix attachment regions (MARs) can be operationally defined as DNA fragments that bind to the nuclear matrix. We have created a library of randomly obtained MARs from tobacco (Nicotiana tobacum) by cloning DNA fragments that co-isolate with nuclear matrixes prepared by a method involving lithium diiodosalicylate. The interactions of several of the cloned MARs with nuclear matrixes were tested by an in vitro binding assay in which genomic DNA was used as competitor. Based on this assay, the MARs were classified as strong, medium, and weak binders. Examples of each of the binding classes were further studied by in vitro binding using self- and cross-competition. Estimates of dissociation constants for several MARs ranged from 6 to 11 nM and correlated inversely with binding strength. The number of binding sites per matrix for several MARs ranged from 4 x 10(5) to 9 x 10(5) and correlated directly with binding strength. We conclude that binding strength, as we have measured it, is a function of both numbers of binding sites and affinity for the sites. The tobacco MARs were sequenced and analyzed for overall AT content, for distribution of AT-rich regions, and for the abundance of several MAR-related motifs. Previously identified MAR motifs correlate to various degrees with binding strength. Notably, the Drosophila topoisomerase II motif does not correlate with binding strength of the tobacco MARs. A newly identified motif, the "90%AT Box," correlates better with binding strength than any of the previously identified motifs we investigated.

Base Sequence↗
PubMed1994

Nuclear structure and the microdistribution of radiation damage in DNA.

Evidence for the roles of proteins and metal ions in the microheterogeneity of DNA damage is reviewed. Decondensation of chromatin in hypotonic buffers markedly sensitizes the DNA to radiation, while treatment of nuclei with hypertonic buffers strips the DNA of histones and other nuclear proteins and enhances the radiosensitivity of the DNA with respect to double-strand break (dsb) formation. Addition of the radical scavenger DMSO reduces the yield of strand breaks, but dehistonized chromatin remains approximately 2.5 times more sensitive to radiation than does native chromatin at 0.1 M DMSO. DNA-protein crosslink (DPC) formation is relatively unaffected by the removal of the majority of histones from chromatin. Most DPC form at or near the nuclear matrix, and matrix is stabilized and radiosensitized by Cu++. To elucidate the role of Cu++, the induction of dsb and DPC by gamma-radiation has been compared with that by hydroxyl radical from Fe(++)-EDTA, or Cu++ catalysed Fenton reactions. Data comparing the size of DNA fragments produced, the effect of expanding or dehistonizing chromatin, and the effects of radical scavengers suggest that gamma-radiation and Fe(++)-EDTA produce dsb at open chromatin sites, whereas Cu(++)-generated dsb are similar to radiation-induced DPC in their location at the nuclear matrix. Both metal ions appeared to produce damage by site-specific generation of hydroxyl radicals. The nuclear matrix, the proteinaceous skeleton which anchors chromosomal loops and provides sites for DNA replication and transcription, binds metal ions and matrix-attachment DNA regions (MARs) consisting of 300 + bp of AT-rich DNA. The interaction of cloned MARs with isolated nuclear matrices has been found to be hypersensitive to crosslinking upon gamma-irradiation, in comparison with associations formed by similarly sized DNA fragments lacking MAR sequences. Thus, the non-random distribution of radiation damage is partially explained by the protection of DNA afforded by histones and chromatin structure and partially by the hypersensitivity of DNA-nuclear matrix associations.

Binding Sites↗
PubMed1998

A coactivator of pre-mRNA splicing.

The nuclear matrix antigen recognized by the monoclonal antibody (mAb) B1C8 is a novel serine (S) and arginine (R)-rich protein associated with splicing complexes and is named here SRm160 (SR-related matrix protein of 160 kD). SRm160 contains multiple SR repeats, but unlike proteins of the SR family of splicing factors, lacks an RNA recognition motif. SRm160 and a related protein SRm300 (the 300-kD nuclear matrix antigen recognized by mAb B4A11) form a complex that is required for the splicing of specific pre-mRNAs. The SRm160/300 complex associates with splicing complexes and promotes splicing through interactions with SR family proteins. Binding of SRm160/300 to pre-mRNA is normally also dependent on U1 snRNP and is stabilized by U2 snRNP. Thus, SRm160/300 forms multiple interactions with components bound directly to important sites within pre-mRNA. The results suggest that a complex of the nuclear matrix proteins SRm160 and SRm300 functions as a coactivator of pre-mRNA splicing.

Amino Acid Sequence↗
PubMed1991

The nucleus: a black box being opened.

Until recently our knowledge about the structural and functional organization of the cell nucleus was very limited. Recent technical developments in the field of ultrastructural analysis, combined with ongoing research on the properties of the nuclear matrix, give new insight into how the nucleus is structured. Two types of observations shape our ideas about nuclear organization. First, most nuclear functions (replication, transcription, RNA processing, and RNA transport) are highly localized within the nucleus, rather than diffusely distributed. Moreover, they are associated with the nuclear matrix. Second, chromatin is organized in discrete loops, bordered by nuclear matrix attachment sequences (MARs). Each loop may contain one or several genes. The arrangement of chromatin in loops has profound consequences for the regulation of gene expression.

Animals↗
PubMed1991

Proteolysis of nuclear proteins by mu-calpain and m-calpain.

Purified calpains are capable of proteolyzing several high Mr nuclear proteins and solubilizing a histone H1 kinase activity from rat liver nuclei upon exposure to 10(-6) - 10(-5) M Ca2+. Major nuclear substrates displayed apparent molecular masses of 200, 130, 120, and 60 kDa on Coomassie Blue-stained SDS-PAGE gels. The nuclear proteins and the H1 kinase were released from Triton-treated nuclei following incubation with buffer containing 0.5 M NaCl. They therefore appeared to be internal nuclear matrix proteins. The nuclear H1 kinase activity solubilized by incubation with m-calpain was eluted in the void volume of a Bio-Gel A-1.5m column, indicating an apparent mass greater than 1,500 kDa. Treatment of the calpain-solubilized kinase with 0.5 M NaCl dissociated it to a form having an apparent mass of 300 kDa (Stokes radius = 5.6 nm), suggesting that the 300-kDa (Stokes radius = 5.6 nm), nuclei by calpain treatment as a large complex containing other internal matrix proteins. Purified human erythrocyte mu-calpain was capable of proteolyzing the nuclear matrix proteins at 10(-6) M Ca2+. In contrast, human erythrocyte multicatalytic protease complex produced little cleavage of the nuclear proteins. Proteolysis of nuclear proteins by either mu-calpain or m-calpain was inhibited by calpastatin. These experiments suggest a physiologic role for the calpains in the turnover of nuclear proteins.

Animals↗
PubMed1996

Nuclear autoantigen p330d/CENP-F: a marker for cell proliferation in human malignancies.

p330d, also known as CENP-F, is a newly characterized cell cycle specific nuclear autoantigen which is associated both with the centromeres and the nuclear matrix. It is expressed in low amounts in G0/G1-cells and accumulates in the nuclear matrix during S-phase with a maximum expression in G2/M-cells. In the present study we have investigated if p330d/CENP-F could be used as a marker for proliferation in different human malignancies. A flow cytometric method was developed by which p330d/CENP-F expression and DNA-content could be assessed on hematopoietic and solid tumors. Twenty-four different human hematopoietic malignancies, 12 breast cancers, and several cell lines were analyzed and the number of p330d/CENP-F positive cells and the S-phase fraction were determined. The percentage of p330d/CENP-F positive cells correlated with the fraction of S-phase cells in all human malignancies tested. Various cell lines revealed a similar cell cycle specific distribution. The association of p330d/CENP-F with the nuclear matrix facilitated the flow cytometric analysis of this protein due to its resistance to different preparation and fixation procedures. In summary, p330d/CENP-F seems to be a potentially valuable proliferation marker which can be applied to different tumors.

Antigens, Neoplasm↗
PubMed1996

Post-mitotic assembly of the nucleolus. I. The internal matrix network is a recruitment site for processing nucleolar components in prenucleolar bodies.

The aim of this work was to investigate whether the nuclear matrix could provide the nucleation sites for dispersed parental nucleolar components to form post-mitotic prenucleolar bodies (PNBs). For this purpose, nuclear matrices from asynchronous populations of onion cells were fractionated, and the distribution of the insoluble components of the nucleolar processing complexes in the matrices were analysed by fibrillarin immunolabelling. The ultra-structural organization of the nuclear matrix of cells from late telophase to late G1, corresponding to the period of nucleolar reassembly and activation, was also analysed. Our results demonstrate that PNBs are structural components of the telophasic nuclear matrix and that this structure provides recruitment and assembly sites for the components of the nucleolar processing machinery, and suggests that the telophasic matrix network is involved in the early steps of post-mitotic nucleologenesis.

Allium↗
PubMed1999

Textural analysis of nuclear mitotic apparatus antigen (NuMA) spatial distribution in interphase nuclei from human drug-resistant CEM lymphoblasts.

In tumour cell lines, the resistance of cancer cells to a variety of structurally unrelated chemotherapeutic drugs is termed multidrug-resistance or MDR. We reported previously [6] that MDR leukemic cells displayed nuclear texture changes, as assessed by image cytometry. The nature of these changes remained uncertain but they could be associated with alterations of the nuclear matrix which could serve an important role in DNA organization and chromatin structure. Therefore, we have compared the textural features observed in G0/G1 nuclei from human leukemic CEM cells and their MDR variant CEM-VLB, after staining of either DNA by Feulgen method or nuclear matrix by immunodetection of NuMA antigen on DNase treated samples. Chromatin or NuMA distributions within the nucleus were evaluated by image cytometry. Changes in textural parameters indicate that modifications of NuMA distribution observed in MDR cells are parallel to those observed at the whole chromatin level (i.e., a more decondensed and coarse texture with increase of Energy and Long-run sections and decrease of Contrast and Short-run sections). Moreover, Optical Densities measurements indicate that MDR cells seem to contain less NuMA, a datum confirmed by immunoblotting of nuclear proteins. In conclusion, chromatin changes observed by image cytometry in drug-resistant human leukemic CEM cells appear associated with modifications of the nuclear matrix structure.

Antigens, Nuclear↗
PubMed1996

Comparison of DNA-protein interactions in intact nuclei from avian liver and erythrocytes: a cross-linking study.

DNA-protein cross-linkages were formed in intact nuclei of chicken erythrocytes and liver cells by the action of cis-diammine dichloroplatinum (II). Most cross-linked proteins were components of the nuclear matrix, and their heterogeneity reflected the different complexity of liver and erythrocytes matrices, respectively. Some basic proteins, including histones, were also cross-linked, particularly in erythrocyte nuclei. South-Western blotting revealed that a variety of proteins isolated from the cross-linked liver nuclei recognized DNA specifically. In this group of proteins two relatively abundant, acidic, species of 38 and 66 kDa, respectively, might represent novel DNA-binding proteins from the nuclear matrix. In the case of erythrocytes, only the basic proteins showed a DNA-recognition capacity, and among them there were some unidentified species, absent from liver. Lamin B2 was cross-linked but was unable to recognize DNA, and the same was true for other abundant, cross-linked proteins from both types of nuclei. This led to the hypothesis that for some DNA-nuclear matrix interactions the aggregation typical of matrix proteins is essential for the specificity of DNA recognition. Hybridization analysis of the DNA isolated from the cross-linked complexes showed that SARs (scaffold attachment regions) and telomeric sequences were well represented in the cross-linked fragments, that the cross-linked DNA of liver was partially different from that of erythrocytes and that two defined SAR sequences were found to be present only in the cross-linked DNA. These results are in agreement with the present views on DNA-nuclear matrix interactions, which are usually studied on isolated nuclear matrices or purified proteins. Instead, our results provide experimental evidence obtained directly from intact nuclei.

Animals↗
PubMed1997

Cell cycle independent interaction of CDC2 with the centrosome, which is associated with the nuclear matrix-intermediate filament scaffold.

The cell cycle regulating Cdc2 protein kinase helps orchestrate cell cycle dependent changes in cell structure and function. This report shows that Cdc2 is localized to the centrosome region and is tightly bound to the nuclear matrix-intermediate filament scaffold. Antibodies to Cdc2 and to the centrosome-specific protein, pericentrin, label the centrosome in an apparently cell cycle independent manner. Isolated centrosomes also label similarly with both antibodies. Essentially, all cells show Cdc2 labeling of the centrosomes, implying an independence of the stage in the cell cycle, a conclusion supported by studies of synchronized cells. In contrast to the labeling of every cell with the Cdc2 monoclonal antibody, fewer centrosomes were labeled with an antibody to the PSTAIRE domain of Cdc2. Embedment-free, immunogold electron micrographs of extracted cell whole mounts show the centrioles and a pericentriolar network of filaments. Both Cdc2 and pericentrin antibodies decorate the amorphous pericentriolar material, while the Cdc2 antibodies also decorate the centrioles themselves. The constitutive presence of Cdc2 at the centrosome suggests a continuing role in the dynamics of centrosome function throughout the cell cycle.

CDC2 Protein Kinase↗
PubMed2002

[Structure of genomic domains of mammalian and avian globin genes].

The data on the genomic domain structure of both mammalian and avian alpha- and beta-globin genes are reviewed. The specific features of chromatin, DNA binding to the nuclear matrix, and domain-specific transcripts are discussed. In humans, the beta-globin gene domain is located in the GC-depleted isochore and contains multiple nuclear matrix attachment regions. The locus is controlled by six chromatin regions hypersensitive to DNase located far upstream of the first structural gene. Some of these regions display enhancer activity to support normal transcription level in the domain. Other mammalian beta-globin domains are similarly organized. The avian beta-globin genes are specifically arranged and their expression is less dependent from the locus control region. The human alpha-globin gene is located in the GC-rich isochore. The nuclear matrix attachment sites are not identified in this gene. An analog of the locus control region is located 40 kb upstream of the zeta-globin gene. The avian alpha-globin gene domains contain numerous nuclear matrix attachment regions. In these domains, an element located far upstream the genes regulates positive rather than negative transcription. An unidentified housekeeping gene as well as some other transcripts not encoding the structural globin genes is transcribed in the direction opposite to that of the globin genes in both mammalian and avian domains.

Animals↗
PubMed2005

[Preparation and characterization of nuclear matrix/chromosome scaffold in situ].

A method of nuclear matrix and chromosomal scaffold preparation from cultured animal cells was developed. After the high-salt extraction, interphase and mitotic cells were not detached from the coverslips that enabled us to analyse the nuclear matrix and chromosomal scaffold in cells at all mitotic phases. Morphological methods (phase contrast microscopy and electron microscopy of ultrathin sections) did not reveal any structures that could be identified as a chromosomal scaffold. However, after staining with antibodies to XCAP-E and topoisomerase IIalpha some structures were revealed in metaphase cells having both localization and morphology of a chromosomal scaffold. The cell residuals were not stained with antibodies to XCAP-E and topoisomerase IIalpha, if the nuclear matrix and chromosomal scaffold were destabilized by addition of beta-mercaptoethanol.

Animals↗
PubMed2006

Involvement of the N-terminal unique domain of Chk tyrosine kinase in Chk-induced tyrosine phosphorylation in the nucleus.

Chk tyrosine kinase phosphorylates Src-family kinases and suppresses their kinase activity. We recently showed that Chk localizes to the nucleus as well as the cytoplasm and inhibits cell proliferation. In this study, we explored the role of the N-terminal unique domain of Chk in nuclear localization and Chk-induced tyrosine phosphorylation in the nucleus. In situ binding experiments showed that the N-terminal domain of Chk was associated with the nucleus and the nuclear matrix. The presence of the N-terminal domain of Chk led to a fourfold increase in cell population exhibiting Chk-induced tyrosine phosphorylation in the nucleus. Expression of Chk but not kinase-deficient Chk induced tyrosine phosphorylation of a variety of proteins ranging from 23 kDa to approximately 200 kDa, especially in Triton X-100-insoluble fraction that included chromatin and the nuclear matrix. Intriguingly, in situ subnuclear fractionations revealed that Chk induced tyrosine phosphorylation of proteins that were associated with the nuclear matrix. These results suggest that various unidentified substrates of Chk, besides Src-family kinases, may be present in the nucleus. Thus, our findings indicate that the importance of the N-terminal domain to Chk-induced tyrosine phosphorylation in the nucleus, implicating that these nuclear tyrosine-phosphorylated proteins may contribute to inhibition of cell proliferation.

Amino Acid Sequence↗
PubMed1991

Structural topography of simian virus 40 DNA replication.

Applying an in situ cell fractionation procedure, we analyzed structural systems of the cell nucleus for the presence of mature and replicating simian virus 40 (SV40) DNA. Replicating SV40 DNA intermediates were tightly and quantitatively associated with the nuclear matrix, indicating that elongation processes of SV40 DNA replication proceed at this structure. Isolated nuclei as well as nuclear matrices were able to continue SV40 DNA elongation under replication conditions in situ, arguing for a coordinated and functional association of SV40 DNA and large T molecules at nuclear structures. SV40 DNA replication also was terminated at the nuclear matrix. While the bulk of newly synthesized, mature SV40 DNA molecules then remained at this structure, some left the nuclear matrix and accumulated at the chromatin.

Animals↗
PubMed2004

Nuclear scaffold/matrix attached region modules linked to a transcription unit are sufficient for replication and maintenance of a mammalian episome.

The activation of mammalian origins of replication depends so far on ill understood epigenetic events, such as binding of transcription factors, chromatin structure, and nuclear localization. Understanding these mechanisms is not only a scientific challenge but also represents a prerequisite for the rational design of nonviral episomal vectors for mammalian cells. In this paper, we demonstrate that a tetramer of a 155-bp minimal nuclear scaffold/matrix attached region DNA module linked to an upstream transcription unit is sufficient for replication and mitotic stability of a mammalian episome in the absence of selection. Fluorescence in situ hybridization analyses, crosslinking with cis-diammineplatinum(II)-dichloride and chromatin immunoprecipitation demonstrate that this vector associates with the nuclear matrix or scaffold in vivo by means of specific interaction of the nuclear scaffold/matrix attached region with the nuclear matrix protein SAF-A. Results presented in this paper define the minimal requirements of an episomal vector for mammalian cells on the molecular level.

Animals↗
PubMed2000

Rearrangement of chromatin domains during development in Xenopus.

A dynamic change in the organization of different gene domains transcribed by RNA polymerase I, II, or III occurs during the progression from quiescent [pre-midblastula transition (pre-MBT)] to active (post-MBT) embryos during Xenopus development. In the rDNA, c-myc, and somatic 5S gene domains, a transition from random to specific anchorage to the nuclear matrix occurs when chromatin domains become active. The keratin gene domain was also randomly associated to the nuclear matrix before MBT, whereas a defined attachment site was found in keratinocytes. In agreement with this specification, ligation-mediated (LM)-PCR genomic footprinting carried out on the subpopulation of 5S domains specifically attached to the matrix reveals the hallmarks of determined chromatin after the midblastula transition. In contrast, the same analysis performed on the total 5S gene population does not reveal specific chromatin organization, validating the use of nuclear matrix fractionation to unveil active chromatin domains. These data provide a means for the determination of active chromosomal territories in the embryo and emphasize the role of nuclear architecture in regulated gene expression during development.

Animals↗
PubMed2002

Translated Alu sequence determines nuclear localization of a novel catalytic subunit of casein kinase 2.

Casein kinase 2 (CK2) is a tetrameric enzyme constitutively expressed in all eukaryotic tissues. The two known isoforms of the catalytic subunit, CK2alpha and CK2alpha', have been reported to have distinct tissue-dependent subcellular distributions. We recently described a third isoform of the catalytic subunit, designated CK2alpha", which is highly expressed in liver. Immunoblot analysis of HuH-7 human hepatoma cell fractions as well as immunofluorescent microscopy revealed that CK2alpha" was exclusively localized to the nucleus and preferentially associated with the nuclear matrix. CK2alpha and CK2alpha' were found in nuclear, membrane, and cytosolic compartments. Deletion of the carboxy-terminal 32 amino acids from the CK2alpha" sequence resulted in release of the truncated green fluorescent protein fusion protein from the nuclear matrix and redistribution to both the nucleus and the cytoplasm. Demonstration that the carboxy terminus is necessary but not sufficient for nuclear retention indicates that the underlying mechanism of CK2alpha" nuclear localization is dependent on the secondary structure of the holoenzyme directed by the carboxy-terminal sequence.

Active Transport, Cell Nucleus↗