Composition and structure of nucleolar skeleton (nucleolar matrix)-Actin and fibrillarin are two main protein components of nucleolar skeleton

Purified nucleoli of HeLa cells were treated sequentially with nonionic detergent, nucleic acid enzyme, low salt and high salt. The residual nucleolar structure termed nucleolar skeleton (nucleolar matrix) was shown as a fine network under electron microscope with DGD embedding-unembedding technique. Such structures of BHK-21 cell and mouse liver cell are similar to that of HeLa cell. The protein composition of the nucleolar skeleton of HeLa cells was analyzed. The protein composition of such nucleolar residual shows obvious difference from the compositions of nuclear matrix and chromosome scaffold. The major protein composition of the nucleolar skeleton of HeLa cells contains 6–7 polypeptides. Their molecular weights are about 48, 43, 36 and 33 ku. Further studies show that actin and fibrillarin are two major protein components of nucleolar skeleton of HeLa cells.     

Composition and structure of nucleolar skeleton (nucleolar matrix)——Actin and fibrillarin are two main protein components of nucleolar skeleton

Purified nucleoli of HeLa cells were treated sequentially with nonionic detergent, nucleic acid enzyme, low salt and high salt. The residual nucleolar structure termed nucleolar skeleton (nucleolar matrix) was shown as a fine network under electron microscope with DGD embedding-unembedding technique. Such structures of BHK-21 cell and mouse liver cell are similar to that of HeLa cell. The protein composition of the nucleolar skeleton of HeLa cells was analyzed. The protein composition of such nucleolar residual shows obvious difference from the compositions of nuclear matrix and chromosome scaffold. The major protein composition of the nucleolar skeleton of HeLa cells contains 6-7 polypeptides. Their molecular weights are about 48, 43, 36 and 33 ku. Further studies show that actin and fib-rillarin are two major protein components of nucleolar skeleton of HeLa cells.     

Composition and structure of nucleolar skeleton (nucleolar matrix)

Purified nucleoli of HeLa cells were treated sequentially with nonionic detergent, nucleic acid enzyme, low salt and high salt. The residual nucleolar structure termed nucleolar skeleton (nucleolar matrix) was shown as a fine network under electron microscope with DGD embedding-unembedding technique. Such structures of BHK-21 cell and mouse liver cell are similar to that of HeLa cell. The protein composition of the nucleolar skeleton of HeLa cells was analyzed. The protein composition of such nucleolar residual shows obvious difference from the compositions of nuclear matrix and chromosome scaffold. The major protein composition of the nucleolar skeleton of HeLa cells contains 6–7 polypeptides. Their molecular weights are about 48, 43, 36 and 33 ku. Further studies show that actin and fibrillarin are two major protein components of nucleolar skeleton of HeLa cells.     

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.     

On the history of nuclear matrix manifestation

The nonchromatin proteinous residue of the cell nucleus was revealed in our laboratory as early as in 1948 and then identified by light and electron microscopy as residual nucleoli,intranuclear network and nuclear envelope before 1960,This structure termed afterwards as “nuclear residue“,“nuclear skeleton“,“nuclear cage“,“nuclear carcass“etc.,was much later(in 1974) isolated,studied and entitled as “nuclear matrix“ by Berezney and Coffey,to whom the discovery of this residual structure is often wronly ascribed.The real history of nuclear matrix manifestation is reported in this paper.     

Analysis of the internal nuclear matrix. Oligomers of a 38 kD nucleolar polypeptide stabilized by disulfide bonds

When rat liver nuclei are treated with the sulfhydryl cross-linking reagent sodium tetrathionate (NaTT) prior to nuclease treatment and extraction with 1.6 M NaCl, residual nucleoli and an extensive non-chromatin intranuclear network remain associated with the nuclear envelope. Subsequent treatment of this structure with 1 M NaCl containing 20 mM dithiothreitol (DTT) solubilizes the intranuclear material, while the nuclear envelope remains structurally intact. We have isolated and partially characterized a major polypeptide of the disulfide-stabilized internal nuclear matrix. The polypeptide, which has an apparent molecular mass 38 kD and isoelectric point 5.3, has been localized to the nucleolus of rat liver nuclei by indirect immunofluorescence using a specific polyclonal chicken antiserum. Based on its molecular mass, isoelectric point, intracellular localization and amino acid composition, the 38 kD polypeptide appears to be analogous to the nucleolar phosphoprotein B23 described by Prestayko et al. (Biochemistry 13 (1974) 1945) [20]. Immunologically related polypeptides have likewise been localized to the nucleoli of both hamster and human tissue culture cell lines as well as the cellular slime mold Physarum polycephalum. By immunoblotting, a single 38 kD polypeptide is recognized by the antiserum in rat, mouse, hamster and human cell lines. The antiserum has been utilized to investigate the oligomeric structure of the 38 kD polypeptide and the nature of its association with the rat liver nuclear matrix. By introducing varying numbers of disulfide bonds, we have found that the 38 kD polypeptide becomes incorporated into the internal nuclear matrix in a two-step process. Soluble disulfide-bonded homodimers of the polypeptide are first formed and then are rendered salt-insoluble by more extensive disulfide cross-linking.     

Distribution of nuclear matrix proteins in interphase CHO cells and rearrangements during the cell cycle. An ultrastructural study

The nuclear matrix contains a group of residual non-histone proteins which remain structurally organized after extensive extraction of isolated nuclei with a high salt buffer, nucleases and a non-ionic detergent. Electron microscopic examination shows that the nuclear matrix is composed of a pore-complex lamina, an intranuclear network and residual nucleoli. In CHO cells biochemical analyses performed by one-dimensional SDS-PAGE show three major nuclear matrix polypeptides with molecular weights between 60 and 70 kDa. Polyclonal antibodies produced against these polypeptides were used to determine their nuclear distribution. Using immunoblotting, these proteins were found in whole nuclei, nuclear matrix, and in the intranuclear network but not in the pore-complex lamina. In order to determine the relationship between these structural proteins and the organization of the nucleus, the proteins were localized in situ. Ultrastructural detection was carried out by immunogold staining of thin sections of Lowicryl K4M-embedded cells. In interphase nuclei all condensed chromatin clumps were labelled. The nucleolus and the interchromatin granules were never immunogold-stained. During mitosis, the label was found to be associated with the chromosomes. This study shows that unlike the lamins, these 60-70 kDa nuclear matrix proteins are associated with the condensed chromatin throughout the cell cycle.     

Nuclear matrix of HeLa S3 cells. Polypeptide composition during adenovirus infection and in phases of the cell cycle

A subnuclear fraction has been isolated from HeLa S3 nuclei after treatment with high salt buffer, deoxyribonuclease, and dithiothreitol. This fraction retains the approximate size and shape of nuclei and resembles the nuclear matrix recently isolated from rat liver nuclei. Ultrastructural and biochemical analyses indicate that this structure consists of nonmembranous elements as well as some membranous elements. Its chemical composition is 87% protein, 12% phospholipid, 1% DNA, and 0.1% RNA by weight. The protein constituents are resolved in SDS- polyacrylamide slab gels into 30-35 distinguishable bands in the apparent molecular weight range of 14,000 - 200,000 with major peptides at 14,000 - 18,000 and 45,000 - 75,000. Analysis of newly synthesized polypeptides by cylindrical gel electrophoresis reveals another cluster in the 90,000-130,000 molecular weight range. Infection with adenovirus results in an altered polypeptide profile. Additional polypeptides with apparent molecular weights of 21,000, 23,000, and 92,000 become major components by 22 h after infection. Concomitantly, some peptides in the 45,000-75,000 mol wt range become less prominent. In synchronized cells the relative staining capacity of the six bands in the 45,000-75,000 mol wt range changes during the cell cycle. Synthesis of at least some matrix polypeptides occures in all phases of the cell cycle, although there is decreased synthesis in late S/G2. In the absence of protein synthesis after cell division, at least some polypeptides in the 45,000- 75,000 mol wt range survive nuclear dispersal and subsequent reformation during mitosis. The possible significance of this subnuclear structure with regard to structure-function relationships within the nucleus during virus replication and during the life cycle of the cell is discussed.     

The major polypeptides of the nuclear pore complex

Nuclear envelopes of maturing oocytes of various amphibia contain an unusually high number of pore complexes in very close packing. Consequently, nuclear envelopes, which can be manually isolated in great purity, provide a remarkable enrichment of nuclear pore complex material, relative to membranous and other interporous structures. When the polypeptides of nuclear envelopes isolated from oocytes of Xenopus laevis and Triturus alpestris are examined by gel electrophoresis, visualized either by staining with Coomassie blue or by radiofluorography after in vitro reaction with [³H]dansyl chloride, a characteristic pattern is obtained (10 major and 15 minor bands). This polypeptide pattern is radically different from that of the nuclear contents isolated from the same cell. Extraction of the nuclear envelope with high salt concentrations and moderately active detergents such as Triton X-100 results in the removal of membrane material but leaves most of the non-membranous structure of the pore complexes. The dry weight of the pore complex (about 0.2 femtograms) remains essentially unchanged during such extractions as measured by quantitative electron microscopy. The extracted preparations which are highly enriched in nuclear pore complex material contain only two major polypeptide components with apparent molecular weights of 150 000 and 73 000. Components of such an electrophoretic mobility are not present as major bands, if at all, in nuclear contents extracted in the same way. It is concluded that these two polypeptides are the major constituent protein(s) of the oocyte nuclear pore complex and are specific for this structure. When nuclear envelopes are isolated from rat liver and extracted with high salt buffers and Triton X-100 similar bands are predominant, but two additional major components of molecular weights of 78 000 and 66 000 are also recognized. When the rat liver nuclear membranes are further subfractionated material enriched in the 66 000 molecular weight component can be separated from the membrane material, indicating that this is relatively loosely associated material, probably a part of the nuclear matrix. The results suggest that the nuclear pore complex is not only a characteristic ubiquitous structure but also contains similar, if not identical, skeletal proteins that are remarkably resistant to drastic changes of ionic strength as well as to treatments with detergents and thiol reagents.     

Heat shock (45 degrees C) results in an increase of nuclear matrix protein mass in HeLa cells

The nuclear matrix from HeLa cells heated at 45 degrees C was isolated to determine the effect of thermal shock on its composition and structure. The matrix from unheated cells contained about 10 per cent of total cell protein and was observed to be spherical particle with a diameter ranging from 3 to 5 microns with the major constituent polypeptides having molecular weights of 45, 47, 55, 57, 59 and 65 kilodaltons. The nuclear-matrix protein mass increased linearly with increasing exposure time at 45 degrees C with no observable change in its size or shape. The additional proteins were observed in general to have molecular weights greater than 45 kilodaltons, with marked increases in polypeptides of 28.5, 38.5, 60, 66, 75, 81, 88, 100 and 115 kilodaltons. An exponential relationship was observed between heat-induced cytotoxicity and the nuclear matrix protein mass increase. A 15 per cent increase in matrix protein mass was sustained prior to the onset of cytotoxicity, while a 35 per cent increase in matrix protein content was associated with a 63 per cent probability of cell killing. The results indicate that redistribution of cell protein or alterations in the mass or structure of the nuclear matrix may be involved in heat-induced cytotoxicity.     

Immunocytochemical Detection of Structural and Regulatory Proteins in Rat Adrenal Nuclear Matrix

The nuclear matrix is a specific cell structure consisting of a residual nucleoskeleton that extends from the nucleoli to the nuclear envelope. The nuclear matrix of steroido-genic cells was isolated previously from a purified nuclear fraction. We present here an in situ extraction method, modified Lutz's method, for rat glandular adrenal cell nuclear matrix. This residual organelle was characterized and studied using immunocytochemical methods. The adrenal glands were removed, the cells prepared in suspension and deposited by cytospin onto Poly-L-lysine glass slides. The nuclear matrix was extracted with Nonidet P-40, DNase I and high and low ionic strength buffers. Structural proteins, nuclear lamins, coilin and fibrillarin were detected immunocytochemically. The adrenal fasciculata cells were easily identified by this method because of their large nuclei and abundant lipid droplets in the cytoplasm. After immunocytochemical detection by antibodies against lamins A and C, a marked brown layer at the periphery of the nucleus was observed. The intensity of the staining was lower using the antibody against nuclear lamin B. Immunocytochemical detection of the protein coilin revealed punctuated stained areas, 2-6 per nucleus, that probably correspond to the coiled bodies. The protein fibrillarin was detected at the nucleolus and coiled bodies. Our technique is simple, reveals well preserved adrenal nuclear matrices, and may be a useful method for immunocytochemical analysis and in situ hybridization.     

Reversibly contractile nuclear matrix. Its isolation, structure, and composition

From Tetrahymena macronuclei we have isolated a reversibly contractile nucleo-skeleton, i.e., an "expanded" nuclear matrix which reversibly contracts when the total concentration of the bivalent cations, Ca and Mg (3:2), is decreased to 5 mM or increased to 125 mM. During contraction the average diameter of the expanded matrix becomes reduced by about 24%; this corresponds to a volume contraction of about 55%. The reversible contraction of the nuclear matrix does not depend on ATP and cannot be inhibited by salygran. The expanded matrix is obtained by removing carefully from the macronuclei 89.7% of the phospholipid, 99.6% of the DNA, 98.5% of the RNA, and 74.8% of the protein by treatment with Triton X-100 and digestion with DNase and RNase followed by an extraction with 2 M NaCl. Electron microscopy reveals, within the expanded matrix, residual equivalents to the structures characteristic for macronuclei: (a) a residual nuclear envelope with nuclear pore complexes; (b) residual nucleoli at the periphery; (c) a fibrillar internal network. The expanded matrix is essentially composed of proteins (96.2%) and traces of DNA (0.8%), RNA (0.5%), phospholipid (1.6%), and carbohydrates (0.9%). The last, which have been determined by gas chromatography, contain glucose, mannose, and an unidentified sugar in the ratio 1:5.4:5.7. The ratio of acidic to basic amino acids of the expanded matrix is 1.55. Sodium dodecyl sulfate (SDS) gel electrophoresis reveals a predominant protein with a mol wt of 18,000 which is apparently involved in the reversible contractile process. The mechanism of this reversible contraction of the expanded matrix remains to be elucidated, but it differs both from actin-myosin contraction systems and from the contractile spasmoneme system in vorticellids.     

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.     

Adrenal nuclear matrix isolation. A morphologic and biochemical study

Summary— The nuclear matrix of adrenal cells was isolated by using the methods proposed by Commerford et al and Kaufmann et al for the liver nuclear matrix isolation. Both methods permitted, to the best of our knowledge for the first time, to prepare the nuclear matrix of a steroidogenic cell and therefore to study some regulatory mechanisms governing steroidogenesis. Commerford et al's method retains nuclear envelope and so produces a higher contamination; Kaufmann et al's method presents a higher purity since the nuclear envelope was removed by Triton X-100. No RNase digestion has been employed for the isolation of the residual nuclear matrices. Both methods however, permit the isolation of fractions with a good morphology, retaining a reticular nucleolus, interchromatinic granules, and a fibrogranular scaffold extending from the nucleolus to the nuclear lamina. The major peptides detected by 1-D SDS-PAGE were 123, 56, 46 and 41 kDa; with both methods protein profiles were similar. Identification of proteins by immunodetection reveals lamins A and C, 80 and 65 kDa respectively; no labeling was found for actin (45 kDa) and vimentin (57 kDa). In short, adrenal nuclear matrix was isolated, Kaufmann et al's method being the method of choice.     

Nuclear skeleton structures in some normal and tumor cells

Nuclear protein fractions, described earlier, were identified as constituents of the nuclear sap (the globulin fraction), that of the nucleoli and ribonucleoprotein network (the acidic protein), and of the nuclear envelope (the residual protein). The latter two fractions compose the protein skeleton of the cell nucleus.An essential difference between electrophoretic profiles of nuclear skeleton structures in experimental tumors and those of normal tissues was revealed. Tumor preparations contained more high molecular weight polypeptides and, in earlier stages of growth, low molecular weight components as well. Fractionation of the nuclear matrix proteins showed that the bulk of them are soluble in diluted alkali. The alkali-insoluble fraction retains the shape of the nucleus and appears in the electron microscope as a spongy nuclear skeleton. A finely dispersed fraction sedimenting from the alkaline suspension is enriched with the pore complexes. The fractions obtained differ in protein composition and probably contain protein components which are similar in molecular weights but non-identical.Abbreviations KD kilodaltons - NM nuclear matrix - PAAG polyacrylamide gel - PC pore complex - RP residual protein     

Nuclear proteins : III. The fibrillar nature of the nuclear matrix

The nuclear matrix of mouse liver nuclei was examined after extraction of the chromatin with high salt, deoxyribonuclease and Triton X-100. The residual nuclear matrix is composed of a nuclear pore-lamina complex, fibrillar nucleoli, and intranuclear matrix. Whole mount electron microscopy shows that a portion of the nuclear matrix is composed of 20–30 Å protein fibers which we call matrixin. The fibers may associate to form larger 100–300 Å fibers. When mouse testicular cells were used, intact synaptonemal complexes and the sex vesicle were intimately associated with the matrix and we suggest these structures may be composed of matrixin. SDS gel electrophoresis of the matrix shows three major polypeptides of 65 000, 67 000 and 68 000 D. Several observations suggest DNA is attached to the matrix at many sites throughout the nucleus. The matrix may play a role in the arrangement of chromatin into the chromomeres of meiotic and mitotic chromosomes.     

Distribution and induction of cytochrome P-450 in rat liver nuclear envelope

Induction of cytochrome P-450s by 3-methylcholanthrene (MC) and phenobarbital (PB) and distribution of P-450s in the rat liver nuclear envelope were investigated by biochemical analyses and ferritin immunoelectron microscopy using specific antibodies against the major molecular species of MC- and PB-induced cytochrome P-450. It was found, in agreement with Kasper (J. Biol. Chem., 1971, 246: 577-581), that the total amount of cytochrome P-450s determined by biochemical analysis was markedly increased by MC, but not by PB, treatment. Immunoelectron microscopic analysis, however, showed marked and slight increases in ferritin labeling by MC and PB treatment, respectively. The latter finding was interpreted as resulting from the induction of a particular molecular species of PB-induced cytochrome P-450s. Ferritin immunoelectron microscopic analysis of intact isolated nuclei, naked nuclei from which the outer membrane of the nuclear envelope was partially detached (mechanically), and isolated nuclear envelopes have shown that the ferritin particles are found exclusively on the cytoplasmic face of the outer nuclear envelopes. Neither the nucleoplasmic face of the inner membrane of the nuclear envelope nor the cisternal face of both membranes of the nuclear envelope showed any labeling with ferritin. This indicates that cytochrome P-450 is located only on the outer membrane of the nuclear envelope and does not diffuse laterally into the domain of the inner membrane of the nuclear envelope across the nuclear pores. Our results suggest that a marked heterogeneity exists in the enzyme distribution between the outer and inner membrane of the nuclear envelope and that microsomal marker enzymes such as cytochrome P-450 exist exclusively in the outer membrane. In addition, it appears that cytochrome P-450 is probably not a transmembrane protein but an intrinsic protein located on the cytoplasmic face of the outer membrane of the nuclear envelope.     

Evidence for the existence of a nucleolar skeleton attached to the pore complex-lamina in human fibroblasts

This work deals with the types of nuclear skeletal structures obtained from human fibroblast nuclei isolated by different procedures. It is confirmed that, in somatic vertebrate cells, the pore complex-lamina is always observed, whereas the presence of internal nucleolar and extranucleolar residual structures depends upon the method of nuclear isolation used. Furthermore, the results reported here argue for the existence of a nucleolar skeleton different from the nucleolar matrix often observed in different cell types by other investigators. The conditions of nuclear isolation which allow us to visualize this nucleolar skeleton without any other internal residual structures are described. The attachment of the nucleolar skeleton to the lamina suggested by the present data is considered in relation to the in situ position of nucleoli near the nuclear envelope.     

A subset of non-histone nuclear proteins reversibly stabilized by the sulfhydryl cross-linking reagent tetrathionate : Polypeptides of the internal nuclear matrix

When rat liver nuclei are isolated in the presence of the irreversible sulfhydryl-blocking reagent iodoacetamide, digested with DNase I and RNase A, and extracted with 1.6 M NaCl, nuclear envelope (NE) spheres depleted of intranuclear material, as analysed by thin-section electron microscopy, are obtained. Two-dimensional isoelectric focusing (IEF)/SDS-PAGE and non-equilibrium pH gradient electrophoresis (NEPHGE)/SDS-PAGE reveal that the predominant polypeptides are lamins A, B and C. Nuclei isolated in the absence of sulfhydryl blocking reagents yield salt- and nuclease-resistant structures which contain sparse but demonstrable intranuclear material. A number of non-histone polypeptides are seen in addition to the lamins. Nuclei treated with the sulfhydryl cross-linking reagent sodium tetrathionate (NaTT) yield, after exposure to nucleases and 1.6 M NaCl, nuclear matrix-like structures containing an extensive intranuclear network and components of the nucleolus in addition to the NE. Increased amounts of the non-lamin, non-histone polypeptides are recovered with these structures. Subsequent treatment of these NaTT-cross-linked structures with reducing agents in 1.0 M NaCl selectively solubilizes the intranuclear components but leaves the nuclear envelope apparently intact. The lamins remain sedimentable and are virtually absent from the soluble (intranuclear) material. Instead, the major solubilized polypeptides are (a) 68 and 63 kD polypeptides which migrate in the vicinity of lamins B and C, respectively, but are distinguishable from the lamins by immunoblotting and by uni-dimensional peptide mapping; (b) a series of basic 60-70 kD polypeptides (pI greater than 8.0) which are not recognized by anti-lamin antisera; (c) an acidic (pI 5.3) 38 kD polypeptide; and (d) a number of high molecular mass (greater than 100 kD) polypeptides. These observations not only suggest a convenient method for fractionating matrix structures from rat liver nuclei into biochemically and morphologically discrete components, but also identify a subset of major non-lamin, non-histone nuclear polypeptides (comprising approx. 20% of the total nuclear protein) whose intermolecular interactions can be reversibly stabilized apparently by intermolecular disulfide bond formation by NaTT.