Tropomyosin is localized in the nuclear matrix and chromosome scaffold of physarum polycephalum

The nuclei and chromosomes were isolated from plasmodia of Physarum polycephalum.The nuclear matrix and chromosome scaffold were obtained after the DNA and most of the proteins were extracted with DNase I and 2 M NaCl.SD-PAGE analyses revealed that the nuclear matrix and chromosome scaffold contained a 37 kD polypeptide which is equivalent to tropomyosin in molecular weight.Immunofluorescence observations upon slide preparations labeled with anti-tropomyosin antibody showed that the nuclear matrix and chromosome scaffold emanated bright fluorescence,suggesting the presence of the antigen in them.Immunodotting results confirmed the presence of tropomyosin in the nuclear matrix and chromosome scaffold.Immunoelectron microscopic observations further demonstrated that tropomyosin was dispersively distributed in the interphase nuclei and metaphase chromosomes.     

Functional analysis of two putative chromosomal replication origins from Pseudomonas aeruginosa

Two autonomously replicating elements previously isolated from Pseudomonas aeruginosa were characterized in vitro for pre-priming complex formation using combinations of replication proteins from P. aeruginosa and Escherichia coli. The results of these studies showed that the P. aeruginosa DnaA and DnaB proteins could form a pre-priming complex on plasmid templates containing either of the two autonomously replicating elements of P. aeruginosa, pYJ50 (containing oriCI), and pYJ52 (containing oriCII), or the E. coli chromosomal origin (plasmid pYJ2). The E. coli DnaA, DnaB, and DnaC proteins were also able to form a pre-priming complex on pYJ2, pYJ50, and pYJ52. Neither pYJ50 nor pYJ52 could be established in E. coli, suggesting a block in steps subsequent to the formation of the pre-priming complex. Similarly, pYJ2 could not be established in P. aeruginosa. Since pYJ50 and pYJ52 could be established in P. aeruginosa and both putative origins form a pre-priming complex in vitro, attempts were made to delete each of these two putative origins. The results indicate that the oriCI sequence is essential for cell viability under typical laboratory growth conditions but that oriCII is not.     

Identification of the nuclear matrix and chromosome scaffold in dinoflagellate Crypthecodinium cohnii~1

Dinoflagellate is one of the primitive eukaryotes,whosenucleus may represent one of the transition stages fromprokaryotic nucleoid to typical eukaryotic nucleus.Usingselective extraction together with embeddment-free sectionand whole mount electron microscopy,a delicate nuclearmatrix filament network was shown,for the first time,indinoflagellate Crypthecodinium cohnii nucleus.Chromosomeresidues are connected with nuclear matrix filaments to forma complete network spreading over the nucleus.Moreover,we demonstrated that the dinoflagellate chromosome retainsa protein scaffold after the depletion of DNA and solubleproteins.This scaffold preserves the characteristic mor-phology of the chromosome.Two dimensional elec-trophoreses indicated that the nuclear matrix and chromo- some scaffold are mainly composed of acidic proteins.Ourresults demonstrated that a framework similar to the nuclearmatrix and chromosome scaffold in mammalian cells appearsin this primitive eukaryote,suggesting that these structuresmay have been originated from the early stages of eukaryoteevolution.     

The relationship between chromosomal origins of replication and the nuclear matrix during the cell cycle

A cytological investigation into the dynamic behaviour of the origins of replication with respect to the nuclear matrix has been carried out on Xenopus laevis cultured cells. In order to preferentially label origins or 'non-origin' regions along DNA fibres, 5-fluoro-2'-deoxyuridine (FUdR)-treated cells were pulsed with [3H]deoxyadenosine in early or late S phase. Samples were then allowed to proceed through the cell cycle for increasing times. The DNA loops were induced in situ to completely uncoil around the nuclear matrix. The autoradiographic analysis shows that, under the experimental conditions used, 'non-origin' regions behave as expected from previous studies, i.e., they associate with the nuclear matrix only when they become part of a replication fork, whereas active origins of replication remain associated with the matrix throughout the cell cycle.     

Organization of DNA replication in Physarum polycephalum. Attachment of origins of replicons and replication forks to the nuclear matrix.

We have investigated the attachment of the DNA to the nuclear matrix during the division cycle of the plasmodial slime mold Physarum polycephalum. The DNA of plasmodia was pulse labelled at different times during the S phase and the label distribution was studied by graded DNase digestion of the matrix-DNA complexes prepared from nuclei isolated by extraction with 2 M NaCl. Pulse labelled DNA was preferentially recovered from the matrix bound residual DNA at any time of the S phase. Label incorporated at the onset of the S phase remained preferentially associated with the matrix during the G2 phase and the subsequent S phase. The occurrence of the pulse label in the matrix associated DNA regions was transiently elevated at the onset of the subsequent S phase. Label incorporated at the end of the S phase was located at DNA regions which, in the G2 phase, were preferentially released from the matrix by DNase treatment. From the results and previously reported data on the distribution of attachment sites it can be concluded that origins of replicons or DNA sites very close to them are attached to the matrix during the entire nuclear cycle. The data further indicate that initiations of DNA replication occur at the same origins in successive S phases. Replicating DNA is bound to the matrix, in addition, by the replication fork or a region close to it. This binding is loosened after completion of the replication.     

Perichromosomal layer proteins associate with chromosome scaffold and nuclear matrix throughout the cell cycle

According to the radial loop model of chromosome organization, a major role in the formation and maintenance of chromosomes is played by the residual structures (the nuclear matrix in interphase nuclei and the chromosome scaffold in metaphase chromosomes). However, in vivo microscopy has recently revealed that the components of these “static” structures are highly mobile and continuously exchanged between specific target sites and the nucleoplasm or cytoplasm. This contradiction between predicted stability and observed dynamics led us to reexamine the principles underlying the association of proteins with residual structures. In the present paper, we have analyzed the association of two perichromosomal layer proteins, pKi-67 and B23, with the residual structures. The results show that these two proteins are associated with residual structures throughout the cell cycle; only those structures change that contain proteins precipitated by 2 M NaCl (nucleoli, perichromosomal layer, prenucleolar bodies, cytoplasm of mitotic cells). Both pKi-67 and B23 remain associated with the nuclear matrix even when they are translocated to nucleoplasmic foci due to inhibitor action or hypotonic treatment. However, in most cases it remains possible to extract a structurally visible protein fraction with 2 M NaCl (protein distributed in nucleoplasm). One may suppose that the protein fraction associated with residual structures includes molecules interacting with their binding sites at the moment of permeabilization, while the free proteins are extracted (i.e., during the interaction with binding sites, these proteins form salt-resistant complexes; however, on diffusion the same proteins are extractable by the high-salt solution). The residual structures may be considered as a “snapshot” of all proteins transiently (or statically) bound to their target sites at the moment of permeabilization. The article is published in the original.     

The function of the nuclear matrix attachment region of silkworm rDNA as an autonomously replicating sequence in plasmid and chromosomal replication origin in yeast

Nuclear matrix attachment regions (MARs) play a crucial role in chromatin architecture, gene expression, and DNA replication. Although it is well known that yeast autonomously replicating sequences (ARSs) bind nuclear matrix and MARs also function as ARS elements in yeast, whether a heterologous MAR or ARS element acts as a replication origin in the chromosome has not been elucidated. We previously identified a MAR (rMAR) located in the nontranscribed spacer (NTS) of silkworm Attacus ricini rDNA. We report here that this rMAR contains 10 copies of ARS consensus sequence (ACS) and several DNA unwinding regions. The rMAR employs ARS activity in yeast and a rARS element locates in the 3(') region of the rMAR. Furthermore, we have also revealed that either the rMAR or the rARS element functions as a replication origin in the chromosome. Our results provide the first direct evidence to demonstrate that heterologous rMAR and rARS display chromosomal origin activity, suggesting that the chromosome structure and replication origin of rDNA reserve some common features during evolution.     

Protein composition of the chromosomal scaffold and interphase nuclear matrix

Residual protein structures were prepared from isolated chromosomes and interphase nuclei of in vitro cultured bovine liver cells and the protein compositions were analysed. Chromosomes with minimal cytoplasmic contamination were obtained by a simple procedure using a pH 8 isolation medium containing Triton X-100 and polyamines, and residual protein-DNA complexes were prepared by extraction with 2 M NaCl. Residual protein structures were also obtained by digesting isolated chromosomes with staphylococcal nuclease. Protein compositions of both structures as obtained by SDS-polyacrylamide gel electrophoresis were essentially the same. Residual protein structures were prepared from isolated nuclei by the same procedures. The major nuclear matrix proteins, i.e., the lamins A, B, and C, were not found in the chromosomes and chromosome scaffolds. On the other hand, the residual chromosome structures contained two major polypeptides of 37 and 83 kilodalton relative molecular weights that were absent from the nuclear matrix preparations. A few polypeptides with the same or very similar electrophoretic mobilities were found in the residual structures of both the nuclei and the chromosomes.     

Permanent attachment of replication origins to the nuclear matrix in BHK-cells.

The position of replication origins and replication forks relative to the nuclear matrix was analysed by autoradiography. Analysis of 2M NaCl-extracted extracted BHK-nuclei, prepared on coverslips, showed that after brief pulses grains were exclusively found over the central core of the residual nuclei, which corresponds to positions in the nuclear matrix. In asynchronous cells these grains were found to migrate into the DNA-halo surrounding the matrix during a subsequent chase. When the pulse had been administered to synchronous cells at the onset of S-phase, it was observed, however, that in the majority of the structures no such migration had occurred. From this, and from the fact that label incorporated later in S-phase could be chased into the halo, we conclude that, contrary to DNA in replication forks, DNA containing replication origins is permanently attached to the nuclear matrix.     

Cloning and characterization of the chromosomal replication origin region of Amycolatopsis mediterranei U32

The chromosomal replication origins (oriC) of gram positive, acid-fast actinomycetes have been investigated in streptomycetes and mycobacteria. A 1339 bp DNA fragment of the putative oriC region from the rifamycin SV producer Amycolatopsis mediterranei U32 was cloned by PCR amplification employing primers designed based on the conserved flanking genes of dnaA and dnaN. The 884 bp sequence of the intergenic region between dnaA and dnaN genes consists of 19 DnaA-boxes and two 13-mer AT-rich sequences, which is similar to the oriC structure of Streptomyces lividans. A mini-chromosome constructed by cloning the putative U32 oriC DNA fragment into an Escherichia coli plasmid was able to replicate autonomously, but was unstable, in A. mediterranei U32 with an estimated copy number of two per cell. Although efficient replication of the mini-chromosome in U32 requires the complete set of DnaA-boxes and AT-rich regions, only one of the AT-rich sequences together with part of the DnaA-boxes is sufficient, suggesting the presence of combinatorial alternatives for a functional oriC region of A. mediterranei U32. Phylogenetic analysis based on definite oriC sequences among eubacteria reflects well the relationship between these species.     

Regulation of replication at the R/G chromosomal band boundary and pericentromeric heterochromatin of mammalian cells

Mammalian chromosomes consist of multiple replicons; however, in contrast to yeast, the details of this replication process (origin firing, fork progression and termination) relative to specific chromosomal domains remain unclear. Using direct visualization of DNA fibers, here we show that the rate of replication fork movement typically decreases in the early-mid S phase when the replication fork proceeds through the R/G chromosomal band boundary and pericentromeric heterochromatin. To support this, fluorescence in situ hybridization (FISH)-based replication profiles at the human 1q31.1 (R-band)-32.1 (G-band) regions revealed that replication timing switched around at the putative R/G chromosomal band boundary predicted by marked changes in GC content at the sequence level. Thus, the slowdown of replication fork movement is thought to be the general property of the band boundaries separating the functionally different chromosomal domains. By simultaneous visualization of replication fork movement and pericentromeric heterochromatin sequences on DNA fibers, we observed that this region is duplicated by many replication forks, some of which proceed unidirectionally, that originate from clustered replication origins. We showed that histone hyperacetylation is tightly associated with changes in the replication timing of pericentromeric heterochromatin induced by 5-aza-2'-deoxycytidine treatment. These results suggest that, similar to the yeast system, histone modification is involved in controlling the timing of origin firing in mammals.     

The insulator binding protein CTCF associates with the nuclear matrix

Nuclear DNA is organized into chromatin loop domains. At the base of these loops, matrix-associated regions (MARs) of the DNA interact with nuclear matrix proteins. MARs act as structural boundaries within chromatin, and MAR binding proteins may recruit multiprotein complexes that remodel chromatin. The potential tumor suppressor protein CTCF binds to vertebrate insulators and is required for insulator activity. We demonstrate that CTCF is associated with the nuclear matrix and can be cross-linked to DNA by cisplatin, an agent that preferentially cross-links nuclear matrix proteins to DNA in situ. These results suggest that CTCF anchors chromatin to the nuclear matrix, suggesting that there is a functional connection between insulators and the nuclear matrix. We also show that the chromatin-modifying enzymes HDAC1 and HDAC2, which are intrinsic nuclear matrix components and thought to function as corepressors of CTCF, are incapable of associating with CTCF. Hence, the insulator activity of CTCF apparently involves an HDAC-independent association with the nuclear matrix. We propose that CTCF may demarcate nuclear matrix-dependent points of transition in chromatin, thereby forming topologically independent chromatin loops that may support gene silencing.     

The sequence-directed bent DNA detected in the replication origin of Chlamydomonas reinhardtii chloroplast DNA is important for the replication function

Summary We demonstrated that the 1055 by restriction fragment containing OriA, a chloroplast DNA replication origin of Chlamydomonas reinhardtii, has electrophoretic anomalies characteristic of bent DNA. A tandem dimer of the region was constructed. Quantitative measurement of the relative gel mobility of a set of permuted fragments was used to extrapolate the approximate position of the bent DNA segment. By analyzing the gel mobility of short, sequenced fragments of the bent DNA region, the putative bending locus was identified. Two A₄ tracts and two A5 tracts were located in the bending locus. Oligonucleotide-directed mutagenesis was then used to disrupt the A tract or the spacing between A tracts and the effect of site-specific mutation on electrophoretic mobility was analyzed. To assess the functional role of the bent DNA region, subclones containing the bending locus, mutated bending locus, and regions flanking the bending locus were constructed. Each subclone was used as template in an in vitro DNA replication system which preferentially initiated DNA replication at OriA. A 224 by subclone with the bending locus positioned in the middle displayed the highest replication function and was sufficient to initiate DNA replication in vitro. Site-specific mutations or alterations of the A tracts resulted in decreased DNA bending and decreased DNA replication activity.     

Cycle-specific replication of chromosomal and F-plasmid origins

There have been various proposals for the pattern of F-plasmid replication during the division cycle. Here we show that the recent studies of Gordon et al. (Cell 90, 1113–1121, 1997) on the duplication and segregation of green fluorescent protein (GFP) labeled replication origins of the Escherichia coli chromosome and the F plasmid during the division cycle support the proposal that the F plasmid replicates with a cell-cycle-specific (artiocyclic) pattern.     

Neuronal differentiation modulates the dystrophin Dp71d binding to the nuclear matrix

The function of dystrophin Dp71 in neuronal cells remains unknown. To approach this issue, we have selected the PC12 neuronal cell line. These cells express both a Dp71f cytoplasmic variant and a Dp71d nuclear isoform. In this study, we demonstrated by electron and confocal microscopy analyses of in situ nuclear matrices and Western blotting evaluation of cell extracts that Dp71d associates with the nuclear matrix. Interestingly, this binding is modulated during NGF-induced neuronal differentiation of PC12 cells with a twofold increment in the differentiated cells, compared to control cells. Also, distribution of Dp71d along the periphery of the nuclear matrix observed in the undifferentiated cells is replaced by intense fluorescent foci localized in the center of the nucleoskeletal structure. In summary, we revealed that Dp71d is a dynamic component of nuclear matrix that might participate in the nuclear modeling occurring during neuronal differentiation.     

Bromodeoxyuridine DNA fiber technology in plants: replication origins and DNA synthesis in tobacco BY-2 cells under prolonged treatment with aphidicolin

Summary. Newly synthesized DNA can be observed on chromosomes or extended chromatin fibers after incorporation and immunodetection of bromodeoxyuridine. This technique, frequently used in animal cells, was adapted for use in BY-2 cells. For the first time, the origins of replication in plant cells could be visualized and monitored on DNA fibers without the use of radioactive traces. The replicon size for BY-2 cells was estimated to be 12.9 µm; and the fork rate, 1.17 µm/h. These values are comparable to those reported for tomato and mustard cells. Furthermore, the data confirm our previous observation that DNA synthesis is not totally blocked by aphidicolin. Bromodeoxyuridine incorporation into DNA was obvious from 24 h onwards after treatment with aphidicolin.Correspondence and reprints: Department of Biology, Universitaire Instelling Antwerpen, Universiteitsplein 1, 2610 Wilrijk, Belgium.     

Random distribution of mammalian replication origins in matrix and total nuclear DNA

Nuclear matrices from mouse and rat tumour cells were isolated and characterized by their microscopic appearance, protein profiles and DNA content. They presented well-defined structures containing 15-20% of the nuclear protein and 1-3% of the nuclear DNA. Matrix DNAs were immobilized on nitrocellulose filters and hybridized to nick-translation 32P-labelled homologous DNA fragments containing the corresponding replication origins. As control total nuclear DNAs were also immobilized on filters and hybridized to origin-containing DNAs. The origin-containing DNAs hybridized to the same extent to both matrix and total DNAs, which showed that they contained the same proportion of origin sequences. In an alternative series of experiments, plasmids containing either rat or mouse replication origins were immobilized on filters and were hybridized with in vitro 32P-labelled matrix and total nuclear DNAs. Here again both matrix and total nuclear DNAs hybridized to the same extent with the origin-carrying plasmids, which showed that neither rat nor mouse matrix DNAs were enriched in DNA replication origin sequences.     

Automodification of PARP-1 mediates its tight binding to the nuclear matrix

Poly(ADP-ribose) polymerase-1 (PARP-1), a nuclear enzyme that catalyzes the NAD⁺-dependent addition of ADP-ribose polymers on a variety of nuclear proteins, has been shown to be associated with the nuclear matrix. As yet, the properties and conditions of this association are unclear. Here, we show the existence of two PARP-1 pools associated with the nuclear matrix of rat liver and the ability of PARP-1 automodification to facilitate its binding to the nuclear matrix.     

Differential targeting of Tetrahymena ORC to ribosomal DNA and non‐rDNA replication origins

The Tetrahymena thermophila origin recognition complex (ORC) contains an integral RNA subunit, 26T RNA, which confers specificity to the amplified ribosomal DNA (rDNA) origin by base pairing with an essential cis‐acting replication determinant—the type I element. Using a plasmid maintenance assay, we identified a 6.7 kb non‐rDNA fragment containing two closely associated replicators, ARS1‐A (0.8 kb) and ARS1‐B (1.2 kb). Both replicators lack type I elements and hence complementarity to 26T RNA, suggesting that ORC is recruited to these sites by an RNA‐independent mechanism. Consistent with this prediction, although ORC associated exclusively with origin sequences in the 21 kb rDNA minichromosome, the interaction between ORC and the non‐rDNA ARS1 chromosome changed across the cell cycle. In G₂ phase, ORC bound to all tested sequences in a 60 kb interval spanning ARS1‐A/B. Remarkably, ORC and Mcm6 associated with just the ARS1‐A replicator in G₁ phase when pre‐replicative complexes assemble. We propose that ORC is stochastically deposited onto newly replicated non‐rDNA chromosomes and subsequently targeted to preferred initiation sites prior to the next S phase.     

Altered expression of nuclear matrix proteins in etoposide induced apoptosis in HL－60 cells

INTRODUCTIIONThe nuclear matrix is an essential component ofthe nucleus which is important for the nuclear structural integrity and specific genomic functions[1, 2].Several articles have reported that the nuclear matrix, as a higher order framework structures, mightbe disassembled du-ring the apoptotic process[3-5].Accordingly3 nuclear lamins A/C or B have beenfound to decrease in apoptotic thymocytes[6], Tcells[7], and carcinoma cell line[8, 9]. The nucleolar protein B23, an obscure ma…