Evidence for two conformational forms of monhistone protein BA which differ in their affinity for DNA

Nonhistone protein BA_free was purified from the 0.075 M NaCl/0.025 M $\text{EDTA}\text{pH}$ 8 extract of whole rat liver nuclei while protein BA_bound was isolated from the 0.05 M Na₂HPO₄/8 M urea/1% β-mercaptoethanol/pH 7.6 extract of dehistonized rat liver chromatin. Chromatin associated protein BA_bound was able to bind 60% of the [³H] DNA in a nitrocellulose filter binding assay while nucleoplasmic protein BA_free showed essentially no DNA binding activity. Circular dichroism analysis of the two forms of protein BA revealed substantial differences in their conformations. Protein BA_free was found to have an α-helix content of 41% while protein BA_bound displayed a spectrum more typical of unordered or β-turn structures.     

Reconstitution of endogenous DNA synthesis in isolated nuclei and template activity of chromatin with respect to mode of inhibition by aphidicolin

We succeeded in reconstituting the endogenous nuclear DNA synthesis of the sea urchin. Endogenous DNA synthesis of isolated nuclei was reconstituted by mixing the salt-treated nuclei (chromatin exhibiting essentially no endogenous DNA synthesis) and the salt extract containing DNA polymerase-alpha. DNA synthesis in this reconstitution system showed a level of activity and a mode of inhibition by aphidicolin similar to those of the original isolated nuclei (noncompetitive with respect to dCTP). On the other hand, the inhibitory mode was competitive with respect to dCTP in DNA synthesis in the reconstituted system obtained from the chromatin and purified DNA polymerase-alpha, indicating that some other factor(s) in addition to DNA polymerase-alpha is necessary for the reconstitution with reference to the inhibitory mode of aphidicolin. We also studied the template activity of the chromatin. When chromatin was used as a template, inhibition by aphidicolin of DNA polymerase-alpha was noncompetitive and uncompetitive with respect to the template at high and low concentrations, respectively. Treatment of chromatin with 5 M urea gave urea-treated chromatin (nonhistone protein-deprived chromatin) and the extract (mainly nonhistone protein fraction). Inhibition by aphidicolin of DNA polymerase-alpha was uncompetitive with respect to the urea-treated chromatin. However, when chromatin reconstituted from the urea-treated chromatin and the extract was used as a template, the inhibitory mode by aphidicolin was similar to that with original chromatin, indicating that the nonhistone protein fraction contained factor(s) which modified the inhibitory mode of aphidicolin. Thus, the inhibitory mode of aphidicolin is a useful parameter for monitoring the resolution and reconstitution of endogenous DNA synthesis of isolated nuclei.     

Nuclear reassemblyin vitro is independent of nucleosome/chromatin assembly

It was show11 that nuclear reassembly was induced by small pieces of DNA fragments in cell-free extracts ofXenopus. In an attempt to learn the relationship between the nuclear reassembly and nucleosome/chromatin assembly, limited amounts of CM-Cellulose are used to eliminate the capacity of the egg extract S-150 to assemble chromatin. while the forming of nucleosomes is checked with DNA supercoiling by plasmid DNA pBR322 incubated in the extract, and further analysed by micrococcal nuclease digestion. This depleted extract is then used to induce nuclear reassembly around demembraned sperms with membrane vesicles. It is found that CM-Cellulose depletes histones H2A and H2B efficiently and blocks the assembly of nucleosomes, the demembraned sperms are yet reconstituted into nuclei in the treated S-150, although the chromatin in reassembled nuclei does not produce protected DNA fragments when digested with micrococcal nuclease. It suggests that in the cell-free system ofXenopus, DNA can be formed into nuclei without assembly of nucleosomes or chromatin.     

Solubilization of chromatin by an endogenous enzymic Ca2+, Mg2+-dependent factor. Activity of residual chromatin]

An endogenous Ca2+, Mg2+-dependent factor of enzymic nature (apparently an endonuclease) digests a part of chromatin in the rat liver nuclei producing DNA fragments of an uniform size. After 60 min of incubation at 15 degrees C and pH 7.50 in the presence of 5 mM MgCl2 and 2 mM CaCl2 87-93% of the total chromatin becomes soluble. The insoluble chromatin however contains 70-85% of the in vivo newly synthesized RNA. In regenerating liver the proportion of the insoluble residual chromatin increases while the radioactivity of the newly synthesized DNA in this fraction is highest. Residual chromatin can be solubilized by ultrasonic treatment only. The Ca2+, Mg2+-dependent dissolving factor is not present either in brain or in PMN leucocyte nuclei.     

Stepwise unfolding of chromatin by urea. A flow linear dichroism and photoaffinity labeling study

The unfolding of chromatin by urea (0-7 M) was studied by means of flow linear dichroism, photoaffinity labeling and nuclease digestion. The linear dichroism results indicate that the unfolding of the DNA is accomplished through two distinct transitions at 1-2 M urea and 6-8 M urea, respectively. The photoaffinity labeling studies indicate that an opening of the nucleosome histone core occurs above 2 M urea, accompanied by general loosening of the structure. Based on the results a model for the unfolding of chromatin fibers by urea is proposed, which includes a stretching of the linker DNA (0-2 M urea) followed by a "loosening" of the nucleosome core, possibly to a one-loop DNA conformation (2-6 M urea), and finally resulting in an almost total stretching of the DNA (greater than 6 M urea).     

Denaturing Urea Polyacrylamide Gel Electrophoresis (Urea PAGE)

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their separation in a polyacrylamide gel matrix based on the molecular weight. Fragments between 2 to 500 bases, with length differences as small as a single nucleotide, can be separated using this method¹. The migration of the sample is dependent on the chosen acrylamide concentration. A higher percentage of polyacrylamide resolves lower molecular weight fragments. The combination of urea and temperatures of 45-55 °C during the gel run allows for the separation of unstructured DNA or RNA molecules.In general this method is required to analyze or purify single stranded DNA or RNA fragments, such as synthesized or labeled oligonucleotides or products from enzymatic cleavage reactions.In this video article we show how to prepare and run the denaturing urea polyacrylamide gels. Technical tips are included, in addition to the original protocol ^1,2.     

Nuclear reassembly in vitro is independent of nucleosome/chromatin assembly

It was show11 that nuclear reassembly was induced by small pieces of DNA fragments in cell-free extracts ofXenopus. In an attempt to learn the relationship between the nuclear reassembly and nucleosome/chromatin assembly, limited amounts of CM-Cellulose are used to eliminate the capacity of the egg extract S-150 to assemble chromatin. while the forming of nucleosomes is checked with DNA supercoiling by plasmid DNA pBR322 incubated in the extract, and further analysed by micrococcal nuclease digestion. This depleted extract is then used to induce nuclear reassembly around demembraned sperms with membrane vesicles. It is found that CM-Cellulose depletes histones H2A and H2B efficiently and blocks the assembly of nucleosomes, the demembraned sperms are yet reconstituted into nuclei in the treated S-150, although the chromatin in reassembled nuclei does not produce protected DNA fragments when digested with micrococcal nuclease. It suggests that in the cell-free system ofXenopus, DNA can be formed into nuclei without assembly of nucleosomes or chromatin. Projrrt supported by the National Natural Science Foundation of China (Grant No. 39730240)     

Whole-genome chromosome distribution during nuclear fragmentation of giant trophoblast cells of Microtus rossiaemeridionalis studied with the use of gonosomal chromatin arrangement

Gonosomal chromatin bodies (GCBs), i.e. blocks of condensed chromatin consisting of heterochromatized region of the sex chromosomes of the field vole M. rossiaemeridionalis, were used as a natural interphase chromosome marker in order to clarify the regularities of GCB rearrangement during nuclear fragmentation of secondary giant trophoblast cells (SGTCs) at the end of their differentiation. Cytophotometrical measurements of DNA content in the nuclei, nuclear fragments and simultaneously in the GCBs were made in the secondary giant SGTCs of field vole M. rossiaemeridionalis. In most cases 1 to 2 GCBs get into the nuclear fragments at different ploidy levels. In the nuclear fragments, GCB DNA content decreased mostly proportionally to DNA content in the whole fragments corresponding to 2c, 4c and 8c. The data obtained demonstrate a regular whole-genome chromosome distribution into nuclear fragments. A possible mechanism of nuclear fragmentation that largely ensures a balanced genome in nuclear fragments is discussed.     

Sperm nuclear chromatin transformations in somatic cell-free extracts

HeLa cell extracts induced decondensation of lysolecithin permeabilized Xenopus, pig, and human sperm chromatin; decondensation began almost immediately on incubation in the extract and was completed within 10–20 min. The average enlargements of human and pig sperm nuclei were 15-fold and 3-fold, respectively. The structural organization of pig and human sperm chromatin was significantly differnt. Decondensation was differentially inhibited by Mg⁺⁺ and polyamines; inhibition was least for Xenopus and most for pig sperm nuclei. The nuclear membrane was disintegrated on chromatin dispersion, whereas the nuclei which failed to decondense exhibited distinct nuclear envelopes. The decondensing factors were stable at 65°C for 15 min. The dispersed chromatin was remodelled to somatic nucleosomal structures within 60 min. The remodelled chromatin could be recondensed to chromosome-like structures, when incubated further in extracts from mitosis arrested HeLa cells. © 1994 Wiley-Liss, Inc.     

Nuclear changes induced by the nematodeXiphinema diversicaudatum in root-tips of strawberry

Summary Feeding by the nematodeX. diversicaudatum caused a progressive increase in the DNA content and size of strawberry nuclei. After four days feeding, nuclei had DNA values intermediate between 8C and 16C and had increased in size from a mean of 17 m² for control root tips to 49 m². Multinucleate cells were present after two and four days feeding. There were no ultrastructural differences in the composition of nuclei from control and parasitized root tips, but strawberry nuclei consisted mainly of dispersed chromatin whereas ryegrass nuclei contained a large proportion of condensed chromatin.     

Whole-genome chromosome distribution during nuclear fragmentation of giant trophoblast cells of Microtus rossiaemeridionalis studied with the use of gonosomal chromatin arrangement

Gonosomal chromatin bodies (GCBs), i.e. blocks of condensed chromatin consisting of heterochromatized region of the sex chromosomes of the field vole M. rossiaemeridionalis, were used as a natural interphase chromosome marker in order to clarify the regularities of GCB rearrangement during nuclear fragmentation of secondary giant trophoblast cells (SGTCs) at the end of their differentiation. Cytophotometrical measurements of DNA content in the nuclei, nuclear fragments and simultaneously in the GCBs were made in the secondary giant SGTCs of field vole M. rossiaemeridionalis. In most cases 1 to 2 GCBs get into the nuclear fragments at different ploidy levels. In the nuclear fragments, GCB DNA content decreased mostly proportionally to DNA content in the whole fragments corresponding to 2c, 4c and 8c. The data obtained demonstrate a regular whole-genome chromosome distribution into nuclear fragments. A possible mechanism of nuclear fragmentation that largely ensures a balanced genome in nuclear fragments is discussed.     

Large scale co-isolation of vimentin and nuclear lamins from ehrlich ascites tumor cells cultured in vitro

Ehrlich ascites tumor (EAT) cells propagated in mass suspension culture were used as a starting material for the simultaneous isolation and purification of large quantities of the intermediate filament protein vimentin and the nuclear lamins A/C and B. Triton cytoskeletons, obtained by repeated washing of cells with a low ionic strength buffer containing Triton X-100 and 4 mM Mg2+, were extracted with 6 M urea at low salt concentration and in the presence of EDTA. Separation of solubilized proteins from unfolded chromatin (DNA) was accomplished by recondensation of the chromatin (DNA) in the presence of Mg2+ before centrifugation. To achieve separation of vimentin from nuclear lamins, the urea extract was subjected to DEAE-Sepharose CL-6B chromatography. Single-stranded DNA-cellulose chromatography was employed for the final purification of vimentin and for the separation of lamin B from lamins A/C. Further purification of lamin B was carried out by CM-Sepharose CL-6B chromatography and of lamins A/C by chromatography on hydroxylapatite. All chromatographies were performed in the presence of 6 M urea. 500 g of pelleted EAT cells yielded approximately 700 mg of vimentin, 225 mg of lamins A/C and 21 mg of lamin B. 2D-polyacrylamide gel electrophoresis revealed great microheterogeneity of lamins A/C, which to a high extent was due to phosphorylation, whereas lamin B was much less heterogeneous. In the absence of urea and at low salt concentration, lamins A/C required pH 5 to stay in solution whereas lamin B required pH 7.5. Increasing the salt concentration to 150 or 250 mM NaCl resulted in the formation of paracrystals from a urea-free mixture of lamins A/C and B. Although the lamins could not be assembled into intermediate filaments under a variety of ionic conditions, the preparations obtained will be useful for further biochemical characterization of these nuclear proteins.     

Pulsed-field gel electrophoresis analysis of higher-order chromatin structures of Zea mays. Highly methylated DNA in the 50 kb chromatin structure

We have investigated the presence of higher-order chromatin structures in different maize tissues. Taking advantage of the pulsed-field gel electrophoresis technique to analyse large DNA fragments from intact nuclei and cells, we have determined the size distribution of the high-molecular-weight DNA fragments obtained from chromatin degradation by endogenous nucleases in isolated nuclei. Chromatin digestion leads to the appearance of stable DNA fragments of about 50 kb in all the tissues examined, suggesting the folding of DNA in higher-order chromatin domain structures. It has been reported that such chromatin domains are formed by loops of the 30 nm fibres anchored to the nuclear matrix by a complex set of proteins, including DNA topoisomerase II. Treatment of maize protoplasts with the calcium ionophore A23187 and the antitumour drug VM-26, which specifically inhibit the religation of the cleaved DNA in the topoisomerase II reaction, also produces the 50 kb structure. Analysis of the DNA contained in the 50 kb chromatin structure shows a higher degree of methylation than in bulk maize chromosomal DNA. The role of methylated DNA in the chromatin folding is discussed.     

Nuclear reassembly in vitro is independent of nucleosome/chromatin assembly

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Isolation and characterization of stable nuclear matrix preparations and associated DNA from avian erythroblasts

A novel procedure for isolation of nuclear matrices from chicken erythroblast cells was elaborated. The influence of variations in the isolation procedure on structural integrity and morphology of nuclear matrices as well as on properties of the nuclear matrix-associated DNA fractions was investigated. The incubation of isolated nuclei in the presence of Cu2+ ions provided significant stabilization of the nuclear matrix. Copper treatment of nuclei did not affect the properties of the nuclear skeleton-associated DNA fraction. In both copper-stabilized as well as unstabilized nuclei, nuclear matrix-attached DNA was digested to the same extent with nucleolytic enzymes, and could be totally removed from nuclear matrices by 2 M NaCl-2 M urea treatment. The fine morphology of the nuclear matrix did not change after extraction of nuclear skeleton-associated DNA fragments. In the presence or absence of copper ions, matrix DNA was found to be qualitatively different compared with total DNA, in particular with respect to the representation of specific repetitive sequences of the chicken beta globin gene domain.     

Nascent DNA in nucleosome like structures from chromatin

We have used chromatin sensitivity to cleavage by micrococcal nuclease as a probe for differences between chromatin containing nascent DNA and that containing bulk DNA. Micrococcal nuclease digested the nascent DNA in chromatin of swimming blastulae of sea urchins more rapidly to acid-soluble nucleotides than the DNA of bulk chromatin. A part of the nascent DNA occurred in micrococcal nuclease-resistant structures which were either different from, or temporary modifications of, the bulk nucleosomes. This was inferred from the size differences between bulk and nascent DNA fragments in 10% polyacrylamide gels after micrococcal nuclease digestion of nuclei from a mixture of ¹⁴C-thymidine long- and ³H-thymidine pulse-labeled embryos. Bulk monomer and dimer DNA fragments contained about 170 and 410 base pairs (bp), respectively, when 18% of the bulk DNA had been rendered acid-soluble. At this level of digestion, “nascent monomer DNA” fragments of about 150 bp as well as 305 bp “large nascent DNA fragments” were observed. Increasing levels of digestion indicated that the large nascent DNA fragment was derived from a chromatin structure which was more resistant to micrococcal nuclease cleavage than bulk dimer chromatin subunits. Peaks of ³H-thymidine-labeled DNA fragments from embryos which had been pulse-labeled and then chased or labeled for several minutes overlapped those of ¹⁴C-thymidine long-labeled monomer, dimer and trimer fragments. This indicated that the chromatin organization at or near the replication fork which had temporarily changed during replication had returned to the organization of its nonreplicating state.     

Variation in the appearance of SV40 tumor antigen in transformed cells

Five types of variation in the appearance of SV40 T antigen have been observed by immunofluorescence in wild-type and tsD^*101 transformed BALB and Swiss/3T3 cells: (1) clones having more than 95% uniformly stained nuclei; (2) clones having variable stained nuclei; (3) clones having T-negative nuclei at the periphery at 39 °C, but not at 33 °C; (4) D101 cells having T-negative nuclei at 39 °C, but not at 33 °C, during growth and after saturation; and (5) wild type cells having T-negative nuclei after confluence at both 33 and 39 °C. This fifth type of variation may involve an immuno-specificity change in the T antigen molecule. Any models proposed for the regulation of SV40 T antigen in transformed cells will have to explain these variations.     

DNA synthesis associated with a DNA-nuclear-membrane complex from rat liver

Summary Ultrastructural analysis of M-band from nuclei of rat liver showed small amounts of chromatin, fragments of inner nuclear membrane, some amorphous nuclear material, and nucleopores. The outer nuclear membrane with its associated ribosomes was removed by Sarkosyl during the preparation of the M-band sample. Morphological features of nucleopores and the inner nuclear membrane were confirmed by freeze-fracture technique. The gross chemical composition of the M-band was similar to that of nuclear membrane fractions prepared by other techniques. The M-band contained the greatest proportion of newly-labeled DNA and also supported DNA synthesis in vitro. Electron-microscopic autoradiography of the M-band showed localization of silver grains of thymidine-³H presumably over newly synthesized DNA. The DNA synthesis could not be attributed to spurious attachment of DNA polymerase to M-band during its isolation. It was partially removed from the M-band by treatment with 0.5 M KC1, phospholipase A or C; and completely, by the action of pancreatic DNase. DNA synthesis was greater in M-band fractions isolated from nuclei of 24-hour regenerating liver.Supported in part by a grant (CA 10298) from the National Cancer Institute     

Dissociation studies of different forms of the estradiol-receptor complex from calf uterus: Higher stability of the complex bound to chromatin

Intact and pure nuclei were isolated by zonal centrifugation from calf uteri preincubated with [³H] estradiol at 37°C. Alternatively, labelling of nuclei was performed by cell-free incubation of filtrate homogenates with radio-active homone at 25°C. The kinetics of dissociation of the estradiol—receptor complex was studied by the tritiated estradiol—non-radioactiv estradiol-exchange method at 22°C using: (a) intact nuclei and isolated chromatin, (b) a 0.5M KCl nuclear extract (5 S) and different cytosolic preparations (4 S, 8 S or heavy aggregates), or (c) cytosolic extracts bound to an inert support like hydroxylapatite or precipitated by nuclear basic proteins. In the three groups the dissociation follows the well established two-stage first-order kinetics patterns, but whereas in Group a the operational half-life of the complex was 5 h, all the preparations of Group b, including the nuclear extract, yielded a half-life of only 10 min. Intermediate values were obtained for the preparations of Group c. Its concluded that the binding to the chromatin endows the estradiol— receptor complex with a higher stability. This might account for the characteristic retention of estradiol in uterine cells in in vivo conditions.     

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)