A combined ultrastructural approach to the study of nuclear matrix thermal stabilization

Summary Using mouse erythroleukaemia cells and different ultrastructural techniques, the morphology was investigated of the nuclear matrix obtained after incubation at 37° C of isolated nuclei. If purified nuclei were heated for 45 min at 37° C, the final matrix exhibited well-recognizable nucleolar remnants, an inner network and a peripheral lamina. Without such incubation only the peripheral lamina was seen surrounding homogeneous, finely granular material. Similar results were obtained with both araldite-embedded and freeze-fractured nuclear matrices, although in the latter case the loose granular material was not evident. Observations of araldite-embedded, heat-treated nuclei revealed clumping of heterochromatin in small, very electron-dense masses with large interchromatin spaces. These ultrastructural aspects were even more striking in freeze-fractured nuclei. Cytochemical matrix analysis by osmium-ammine staining for nucleic acids and DNase-gold labelling for DNA localization demonstrated that also matrix residual nucleic acids, mostly RNA, are stabilized by heat exposure of isolated nuclei. The results demonstrate that the morphology of heat-stabilized nuclear matrix is not artefactually affected during the preparation for conventional electron microscopy and suggest a possible involvement of nucleic acids in the heat-induced stabilization of the nuclear matrix.     

Heat-induced stabilization of the nuclear matrix: A morphological and biochemical analysis in murine erythroleukemia cells

Using mouse erythroleukemia cells we performed a comprehensive morphological and biochemical study of the nuclear matrix obtained after exposure of isolated nuclei to 37 degrees C or from cells heat shocked in vivo at 43 or 45 degrees C. At the ultrastructural level it was possible to see that in the absence of a 37 degrees C incubation of purified nuclei, the final matrix lacked well-defined nucleolar remnants but a peripheral lamina was clearly visible, as well as a sparse fibrogranular network which was located at the periphery of the structures. On the contrary, after a 37 degrees C nuclear incubation, very electron-dense nucleolar remnants were observed along with an abundant meshwork dispersed throughout the interior of the structures. When intact cells were heat shocked in vivo, electron-dense residual nucleoli were present only when isolated nuclei had been exposed to 37 degrees C in vitro, whereas without such an incubation, they were not as easily distinguishable and appeared less electron-dense. In the latter case the inner network was more evenly distributed. After purified nuclei were incubated at 37 degrees C for 45 min, the high salt and DNase I resistant fraction retained about 18% of the nuclear protein whereas if the heating was omitted protein recovery dropped to 6%. An increase in the recovery of intact structures in the matrix fraction was the main reason for the higher protein recovery. Heating nuclei in vitro further increased the amount of nuclear protein present in the matrix fraction even if intact cells had been heat shocked in vivo. No major qualitative differences were seen when the polypeptide pattern of the various types of nuclear matrices was analyzed on one-dimensional polyacrylamide gels and this finding was further supported by Western blot analysis with a monoclonal antibody to lamins A and C. These results show that heating mainly stabilizes the nucleolar remnants of the matrix and to a lesser extent the inner network, but the morphology of the final structures is different depending on whether the stabilization is performed in vivo or in vitro.     

Heat shock-induced changes in the structural stability of proteinaceous karyoskeletal elements in vitro and morphological effects in situ

Karyoskeletal protein fractions prepared from Drosophila melanogaster embryos contain morphologically identifiable remnants of nuclear pore complexes and peripheral lamina as well as what appears to be an internal nuclear "matrix" (Fisher, P. A., M. Berrios, and G. Blobel, 1982, J. Cell Biol., 92: 674-686). Structural stability of these proteinaceous assemblies is dependent on thermal incubation in vitro (37 degrees C, 15 min) before subfractionation of nuclei. In the absence of such incubation, greater than 90% of the total karyoskeletal protein including major polypeptide components of internal "matrix," pore complexes, and the peripheral lamina, is solubilized by 1 M NaCl. In vivo heat shock induces karyoskeletal stabilization resembling that resulting from thermal incubation in vitro. Immunocytochemical studies have been used to establish the effects of heat shock on the organization and distribution of major karyoskeletal marker proteins in situ. Taken together, these results are consistent with the notion that in vivo, regulation of karyoskeletal plasticity (and perhaps form) may be a functionally significant component of the Drosophila heat shock response. They also have broad practical implications for studies pertaining to the structure and function of karyoskeletal protein (nuclear "matrix") fractions isolated from higher eukaryotic cells.     

Morphometric analysis and topological organization of nuclear matrix in freeze-fractured electron microscopy

The ultrastructural organization of nuclear matrix, purified from intact or membrane-denuded rat liver nuclei, has been analysed by means of freeze-fracturing technique. This method avoids dehydration and embedding which, in conventional thin sectioning, partly distort or mask the matrix ultrastructure. The various matrix components, and mainly the peripheral lamina and the inner network revealed complex arrangements undetectable with conventional techniques. Morphometric analyses performed with a Texture Analysis System (TAS) Leitz, allowed to obtain precise information on the matrix constituents, based on the histograms of their size distribution. These textural characteristics have been utilized in order to identify, by means of a particular computer programme, the putative matrix localization within intact freeze-fractured nuclei.     

Further considerations on the thermal stabilization of the nuclear matrix in mouse erythroleukemia cells.

The morphology and the polypeptide composition of the nuclear matrix obtained from 37 degrees C incubated nuclei has been studied in mouse erythroleukemia cells. From a structural point of view, in the absence of heat treatment, the matrix lacked identifiable nucleolar remnants and the internal fibrogranular meshwork whereas a peripheral lamina was seen. On the contrary, the matrix obtained from heat exposed nuclei displayed very electrondense nucleolar remnants and an abundant inner network. These results were obtained irrespective of the type of extracting agent (2M NaCl or 0.2 M (NH4)2SO4) used to remove histones and other soluble proteins. The heat stabilization of the matrix could not be prevented by sulfhydryl blocking chemicals such as iodoacetamide and n-ethylmaleimide, thus suggesting that heat does not stabilize the matrix by inducing the formation of disulfide bonds. Only limited differences in the polypeptide pattern of matrix isolated under different conditions were seen using one-dimensional pore gradient polyacrylamide gels stained with both Coomassie Brilliant Blue and silver despite the fact that the matrix fraction from heat treated nuclei retained about three fold more protein in comparison with controls. The same results were obtained also by means of two-dimensional non-equilibrium gel electrophoresis.     

The effect of in vitro heat exposure on the recovery of nuclear matrix-bound DNA polymerase alpha activity during the different phases of the cell cycle in synchronized HeLa S3 cells.

HeLa S3 cells were synchronized by a double thymidine block or aphidicolin treatment and the levels of nuclear matrix-bound DNA polymerase alpha activity were then measured using activated calf thymus DNA as template. The nuclear matrix was obtained by 2 M NaCl extraction and DNase I digestion of isolated nuclei incubated at 37 degrees C for 45 min prior to subfractionation. In all phases of the cell cycle 25-30% of nuclear DNA polymerase alpha activity remained matrix-bound, even when cells were in the G1 phase. No dynamic association of DNA polymerase alpha activity with the matrix was seen, at variance with previous results obtained in regenerating rat liver. The variations measured in matrix-bound activity closely followed those detected in isolated nuclei throughout the cell cycle. If nuclei were not heat-stabilized very low levels of DNA polymerase alpha activity were measured in the matrix (1-2% of total nuclear activity). Heat incubation of nuclei failed to produce any enrichment in matrix-associated newly replicated DNA, whereas the sulfhydryl cross-linking chemical sodium tetrathionate did. Therefore the results obtained after the heat stabilization procedure do not completely fit with the model that envisions the nuclear matrix as the active site where eucaryotic DNA replication takes place.     

Phospholipid interactions in rat liver nuclear matrix

Rat liver nuclear matrix has been isolated by salt extraction and nuclease digestion of nuclei. Under the electron microscope, the matrix appears as a spongelike network joined by thinner fibrils. Biochemical analysis shows a high protein content and low amounts of nucleic acid and phospholipid. Treatment of the matrix with phospholipase C results in a release of most of the nucleic acid, and a disappearance of the fibrils, however the appearance of the matrix is largely unaffected. It seems likely that phospholipids are responsible for the hydrophobic interactions between nucleic acids and matrix fibrils. From $\text{in vitro}$ labelling studies the released DNA is more recently synthesised than the bulk material, however the matrix bound RNA appears to label less rapidly than total nuclear RNA.     

The protein composition of Friend cell nuclear matrix stabilized by various treatments. Different recovery of nucleolar proteins B23 and C23 and nuclear lamins

Summary— Using two-dimensional polyacrylamide gels stained with Coomassie blue we have studied the protein composition of the nuclear matrix obtained from mouse erythroleukemic nuclei kept at O°C throughout the isolation procedure to prepare the high ionic strength resistant fraction (control matrix) or stabilized in vitro or in vivo by different procedures prior to subfractionation (ie 37°C incubation of isolated nuclei; sodium tetrathionate exposure of purified nuclei; heat shock of intact cells). When the matrix obtained from 37°C incubated nuclei was compared with the control matrix, striking differences in the polypeptide pattern were seen if the protein was obtained in both cases from an equivalent number of nuclei. On the other hand, if the same amount of protein for both the samples was applied to the gels the differences were less evident. Sodium tetrathionate stabilization of isolated nuclei and heat shock of intact cells produced a matrix protein pattern that was very similar and differed from that of the in vitro heat-exposed matrix. Using specific polyclonal antisera, we demonstrate that nucleolar proteins B23/numatrin and C23/nucleolin were very abundant in the matrix obtained from chemically-treated nuclei or in vivo heat-stabilized nuclei but were recovered in very small amounts (B23) or completely absent (C23) in the matrix prepared from nuclei heated to 37°C in vitro. Differences were seen also in the recovery of nuclear lamins, and especially lamin B, that was poorly represented in the sodium tetrathionate-stabilized matrix. The results demonstrate that in mouse erythroleukemia cells the increased recovery of nuclear matrix protein that is seen after in vitro heating of isolated nuclei is predominantly due to an additional recovery of the same types of polypeptides that are detected also in the absence of such a treatment. The data also indicate that in vivo heat shock of intact cells produces a nuclear matrix protein pattern that is more similar to the pattern seen after stabilization of purified nuclei with sodium tetrathionate and differs significantly from that obtained by exposing nuclei to 37°C in vitro, unlike to that what previous reports have indicated.     

Nucleosome rearrangement in vitro. 1. Two phases of salt-induced nucleosome migration in nuclei

We have investigated the salt- and temperature-induced rearrangement of nucleosomes in both intact and H1-depleted nuclei from human cells. In agreement with previous reports on the rearrangement of nucleosomes in isolated chromatin or chromatin fragments, we observed a decrease in the average nucleosome repeat length following incubation of nuclei at 37 degrees C in elevated salt concentrations. However, this decrease occurred in two distinct phases. First, incubation of H1-depleted nuclei at 37 degrees C for as little as 10 min in low-salt, isotonic buffer (containing 0.025 M KCl) resulted in a shift in the limiting repeat value from approximately 190 to 168 base pairs (bp). A similar shift was observed for intact nuclei incubated at 37 degrees C for 1 h in buffer containing near-physiological salt concentrations (i.e., 0.175 M KCl). This limiting repeat value was maintained in both intact and H1-depleted nuclei up to a salt concentration of 0.45 M KCl in the incubation buffer. Second, at salt concentrations of 0.625 M KCl, a limiting repeat of approximately 146 bp was obtained, and the nuclei had clearly lysed. During the first shift in repeat length, little additional exchange of nuclear proteins occurred compared to nuclei kept on ice in a low-salt buffer. This was the case even though the conditions used to monitor exchange were optimized by using a high DNA to chromatin ratio. On the other hand, a significant increase in the exchange of nuclear proteins, and formation of nucleosomes on the naked DNA, was observed during the shift in repeat length to 146 bp.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein disulfide crosslinking stabilizes a polyoma large T antigen-host protein complex on the nuclear matrix

We have investigated the effects of intermolecular disulfide crosslinking and temperature-dependent insolubilization of nuclear proteins in vitro on the association of the polyoma large T antigen with the nuclear matrix in polyomavirus-infected mouse 3T6 cells. Nuclear matrices, prepared from polyomavirus-infected 3T6 cells by sequential extraction of isolated nuclei with 1% Triton X-100 (Triton wash), DNase I, and 2 M NaCl (high salt extract) at 4 degrees C, represented 18% of total nuclear protein. Incubation of nuclei with 1 mM sodium tetrathionate (NaTT) to induce disulfide crosslinks or at 37 degrees C to induce temperature-dependent insolubilization prior to extraction, transferred an additional 9-18% of the nuclear protein from the high salt extract to the nuclear matrix. This additional protein represented primarily an increased recovery of the same nuclear protein subset present in nuclear matrices prepared from untreated nuclei. Major constituents of chromatin including histones, hnRNP core proteins, and 98% of nuclear DNA were removed in the high salt extract following either incubation. Polyoma large T antigen was quantified in subcellular fractions by immunoblotting with rat anti-T ascites. Approximately 60-70% of the T antigen was retained in nuclei isolated in isotonic sucrose buffer at pH 7.2. Most (greater than 95%) of the T antigen retained in untreated nuclei was extracted by DNase-high salt treatment. Incubation at 37 degrees C or with NaTT transferred most (greater than 95%) of the T antigen to the nuclear matrix. T antigen solubilized from NaTT-treated matrices with 1% SDS sedimented on sucrose gradients as a large (50-S) complex. These complexes, isolated by immunoprecipitation with anti-T sera, were dissociated by reduction with 2-mercaptoethanol, and SDS-PAGE analysis revealed that T antigen was crosslinked in stoichiometric amounts to several host proteins: 150, 129, 72, and 70 kDa. These host proteins were not present in anti-T immunoprecipitates of solubilized nuclear matrices prepared from iodoacetamide-treated cells. Our results suggest that the majority of polyomavirus large T antigen in infected cells is localized to a specific subnuclear domain which is distinct from the bulk chromatin and is closely associated with the nuclear matrix.     

6-Iodoacetamidofluorescein labelling to assess the state of sulphhydril groups after thermal stabilization of isolated nuclei

Summary Isolated nuclei and nuclear matrices, prepared from mouse erythroleukaemia cells, were reacted with the sulphhydryl-specific dye 6-iodoacetamidofluorescein. To determine whether in vitro formation of disulphide bonds might play a role in the nuclear matrix stabilization triggered by exposure of isolated nuclei to the physiological temperature of 37°C, a variety of techniques were employed to assess the state of cysteinyl residues after such an incubation. Both flow cytometry and confocal microscopy quantitative analysis did not reveal major differences in the fluorescence intensity of nuclei incubated at 37°C in comparison with those maintained at 0°C. Confocal scanning laser microscopy revealed that 6-iodoacetamidofluorescein labelled a fibrogranular network in isolated nuclei. The fluorescent pattern of the network was not affected by a 37°C exposure of nuclei. However, such a network was not detectable in isolated nuclear matrices, thus suggesting a possible protein re-arrangement during matrix preparation. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of fluorescent-labelled nuclear proteins showed no difference between heat-exposed and control samples. We conclude that oxidation of cysteinyl residues is not a major factor leading to the stabilization of nuclei incubated at 37°C.     

Considerations in the isolation of rat liver nuclear matrix,nuclear envelope,and pore complex lamina

A number of recent studies have demonstrated a salt-, nuclease, and detergent-resistant subnuclear structure termed the nuclear protein matrix which consists of a fibrogranular intranuclear network, residual components of the nucleolus, and a peripheral lamina. Other workers, however, have shown that somewhat similar methods result in the isolation of the peripheral lamina devoid of the intranuclear components. In this report we demonstrate that seemingly slight changes in the isolation procedure cause major changes in the morphology of the residual structures obtained. When freshly purified rat liver nuclei were digested with DNase I and RNase A and then extracted with buffers of low magnesium ion concentration (LS buffer) and high ionic strength (HS buffer), the resulting structures isolated prior to or after Triton X-100 extraction lacked the extensive intranuclear network and the easily identifiable residual nucleoli present in the nuclear protein matrix. Systematic modification of this extraction procedure revealed that morphologically identifiable residual nucleoli were present when digestion with RNase A followed extraction with HS buffer but were absent when the order of these steps was reversed. The removal of the nucleolus by RNase A and HS buffer correlated with the removal of nuclear RNA by the same treatments. These coordinate events could not be prevented by treatment with protease inhibitors but were prevented by treatment of the RNase A with diethylpyrocarbonate, an RNase inhibitor. The extensive intranuclear network seen in the nuclear protein matrix was sparse or absent when residual structures were prepared from DNase- and RNase-treated nuclei under conditions which minimized the oxidation of protein sulfhydryl groups. In contrast, an extensive non-chromatin intranuclear network was seen if the formation of intermolecular protein disulfide bonds was promoted by extraction of nuclei with cationic detergents, by overnight incubation, or by treatment with oxidizing agents like sodium tetrathionate prior to nuclease digestion and subsequent extraction. By varying the order of extraction steps and the extent of disulfide cross-linking, it is possible to isolate from a single batch of nuclei residual structures with a wide range of morphologies and compositions.     

On the association of DNA primase activity with the nuclear matrix in HeLa S3 cells

We have reinvestigated the association of DNA primase activity with the nuclear matrix prepared from exponentially growing HeLa S3 cells. We have found that 25–30 per cent of the nuclear primase activity resists extraction with 2 M NaCl and digestion with Dnase I. Unlike previous investigations, done with the same cell line, the results showed that nuclear matrix-bound DNA primase activity represented less than 10 per cent of the total cell activity. Association of high levels of primase activity with the nuclear matrix was strictly dependent on a 37°C incubation of isolated nuclei prior to subfractionation. Evidence was obtained that the method used for preparing nuclei can have a dramatic effect on the amount of primase activity which is recovered both in the postnuclear supernatant and in isolated nuclei, thus seriously affecting the interpretation of the results about the quantity of DNA primase activity bound to the nuclear matrix.     

The effect of sodium tetrathionate stabilization on the distribution of three nuclear matrix proteins in human K562 erythroleukemia cells

Using both conventional fluorescence and confocal laser scanning microscopy we have investigated wheter or not stabilization of isolated human erythroleukemic nuclei with sodium tetrathionate can maintain in the nuclear matrix the same spatial distribution of three polypeptides (M_r 160 kDa and 125 kDa, previously shown to be components of the internal nuclear matrix plus the 180-kDa nucleolar isoform of DNA topoisomerase II) as seen in permeabilized cells. The incubation of isolated nuclei in the presence of 2 mM sodium tetrathionate was performed at 0° C or 37° C. The matrix fraction retained 20–40% of nuclear protein, depending on the temperature at which the chemical stabilization was executed. Western blot analysis revealed that the proteins studied were completely retained in the high-salt resistant matrix. Indirect immunofluorescence experiments showed that the distribution of the three antigens in the final matrix closely resembled that detected in permeabilized cells, particularly when the stabilization was performed at 37° C. This conclusion was also strengthened by analysis of cells, isolated nuclei and the nuclear matrix by means of confocal laser scanning microscopy. We conclude that sodium tetrathionate stabilization of isolated nuclei does not alter the spatial distribution of some nuclear matrix proteins.     

Spatial distribution of DNA loop attachment and replicational sites in the nuclear matrix

Biochemical fractionation was combined with high resolution electron microscopic autoradiography to study the localization in rat liver nuclear matrix of attached DNA fragments, in vivo replicated DNA, and in vitro synthesized DNA. In particular, we determined the distribution of these DNA components with the peripheral nuclear lamina versus more internally localized structural elements of isolated nuclear matrix. Autoradiography demonstrated that the bulk of in vivo newly replicated DNA associated with the nuclear matrix (71%) was found within internal matrix regions. A similar interior localization was observed in isolated nuclei and in situ in whole liver tissue. Likewise, isolated nuclear lamina contained only a small amount (12%) of the total matrix-bound, newly replicated DNA. The structural localization of matrix-bound DNA fragments was examined following long-term in vivo labeling of the DNA. The radioactive DNA fragments were found predominantly within interior regions of the matrix structure (77%), and isolated nuclear lamina contained less than 15% of the total nuclear matrix-associated DNA. Most of the endogenous DNA template sites for the replicative enzyme DNA polymerase alpha (approximately 70%) were also sequestered within interior regions of the matrix. In contrast, a majority of the endogenous DNA template sites for DNA polymerase beta (a presumptive repair enzyme) were closely associated with the peripheral nuclear lamina. A similar spatial distribution for both polymerase activities was measured in isolated nuclei before matrix fractionation. Furthermore, isolated nuclear lamina contained only a small proportion of total matrix-bound DNA polymerase alpha endogenous and exogenous template activities (3-12%), but a considerable amount of the corresponding beta polymerase activities (47-52%). Our results support the hypothesis that DNA loops are both anchored and replicated at nuclear matrix-bound sites that are predominantly but not exclusively associated with interior components of the matrix structure. Our results also suggest that the sites of nuclear DNA polymerase beta-driven DNA synthesis are uniquely sequestered within the characteristic peripheral heterochromatin shell and associated nuclear envelope structure, where they may potentially participate in DNA repair and/or replicative functions.     

NUP82 is an essential yeast nucleoporin required for poly(A)+ RNA export

We have isolated and characterized the gene encoding a novel essential nucleoporin of 82 kD, termed NUP82. Indirect immunofluorescence of cells containing an epitope tagged copy of the NUP82 localized it to the nuclear pore complex (NPC). Primary structure analysis indicates that the COOH-terminal 195 amino acids contain a putative coiled-coil domain. Deletion of the COOH-terminal 87 amino acids of this domain causes slower cell growth; deletion of the COOH-terminal 108 amino acids results in slower growth at 30 degrees C and lethality at 37 degrees C. Cells in which the last 108 amino acids of NUP82 have been deleted, when shifted to 37 degrees C, do not display any gross morphological defects in their nuclear pore complexes or nuclear envelopes. They do, however, accumulate poly(A)+ RNA in their nuclei at 37 degrees C. We propose that NUP82 acts as a linker to tether nucleoporins directly involved in nuclear transport to pore scaffolding via its coiled-coil domain.     

Nuclear lamina—like filaments and nuclear matrix in allium cepa as revealed by scanning electron microscopy

In this study,freeze-fractured specimens of allium cepa root tip meristems were examined under the scanning electron microscope(SEM),This technique permitted the visualization of the outer membrane of the nuclear envelope with nuclear pore complexes and polyribosomes.Some of the cell nuclei prepared with this procedure had fissures of various widths on their nuclear envelopes through which the nuclear lamina-like filaments(LLF) undernearth the nucleoplasmic side of the envelopes were clearly visible.The diameters of these filaments veried between 25 and 125nm.Many of the LLFs showed granular thickenings at places,and were attached to the inner surface of nuclear envelope in some regions .Similar LLFs were also seen at the peripheries of the freeze-fractured faces of nuclei.Meanwhile,the spatial relation between the nuclear matrix filaments(NMF) and other nuclear structures(nucleoli,chromation and peripheral lamina-like filaments) was revealed in these fractured preparations.In addition,the methods and techniques in studying the nuclear lamina morphology and the roles played by NMFs in activities of various nuclear sturctures were discessed in brief.     

Synthesis of lipids in isolated nuclei from rat thymus and liver cells

Synthesis of lipids was studied in isolated nuclei from rat thymus and liver cells. On incubation of the isolated nuclei with [2-¹⁴C]acetate and [1-¹⁴C]glycerol, the label was intensively incorporated into phospholipids and with a significantly lower intensity into fatty acids and cholesterol. Only trace amounts of radioactivity were detected in the lipids of chromatin prepared from isolated thymus nuclei after their incubation, and this suggested that lipids were mainly synthesized on the nuclear membrane. On the preincubation of thymus tissue homogenate with [2-¹⁴C]acetate and the subsequent isolation of the nuclei and chromatin, the radioactivity of chromatin lipids was comparable to the radioactivity of nuclear lipids. The findings suggested that in the isolated nuclei the newly synthesized lipids were not transported into chromatin from the nuclear membrane. The specific radioactivities of individual phospholipids and fatty acids were different in the isolated nuclei and in nuclei obtained from preincubated homogenate. Mechanisms of lipid synthesis in isolated nuclei and causes of the different radioactivities of lipids in the isolated nuclei and in the nuclei obtained from the preincubated homogenate are discussed.     

Isolation and fractionation of the nuclear matrix from cultured cells

A new method for the isolation of tissue culture cell nuclei is presented which involves incubation of the nuclei in the presence of Cu2+- or Zn2+-ions. This method eliminates the danger of nuclear aggregation and permits nuclear matrix isolation and subsequent fractionation. Stabilization of the inner matrix by Cu2-ions permits analysis of the role of nucleic acids in the maintenance of the matrix structure. It is shown that solubilization of more than 95% of matrix-bound DNA and more than 90% of matrix-bound RNA did not cause any significant changes in the nuclear matrix structure.