Fractionation of eukaryotic DNA in a pulsating electrical field. I. Detection and properties of discrete DNA fragments]

The fractionation of eukaryotic DNA by field inversion gel electrophoresis results in the appearance of discrete DNA-fragments. The set of these fragments is similar to that of different eukaryotic representatives and consists of various chromosomal DNAs, unified by size. The physical properties of DNA-fragments suggest that they can form multimeric structures due to the presence of sticky ends flanking discrete fragments. We suppose that the set of discrete DNA-fragments results in a specific cleavage of intact nuclear DNA and can reflect different levels of chromatin structural organization.     

Fractionation, isolation and characterization of nonhistone chromosomal protein from calf thymus.

1) A method is described for the separation and fractionation of nonhistone chromosomal proteins from salt-urea dissociated calf thymus chromatin. After precipitating DNA in the dissociated chromatin solution with LaCl3, the chromosomal proteins in the supernatant were fractionated by SP-Sephadex C-25 column chromatography using a combination of NaCl stepwise and linear gradient elutions. Much care was taken to prevent proteolytic degradation of the chromosomal proteins during the preparation. 2) Among the protein fractions separated by this chromatography, twenty subfractions were found to be homogeneous on SDS-polyacrylamide gel electrophoresis. These purified proteins account for about 18% of the whole chromosomal protein. Eleven subfractions of these purified nonhistone proteins had ratios of acidic to basic amino acids above 1.0 and the nine remaining subfractions had ratios below 1.0, corresponding to nonhistone proteins of basic character. 3) The molecular weights of the purified nonhistone proteins ranged from 7,400 to 19,000.     

Role of nonhistone chromosomal proteins in determining circular dichroism spectra of chromatin.

Complexing of histone proteins, from WI-38 cells with pure DNA from WI-38 cells, causes a marked decrease in the amplitude of the positive ellipticity band and a red shift in circular dichroism spectra in the 250–300 nm region. Total nonhistone chromosomal proteins from WI-38 cells (without histones) cause an analogous effect, but of significantly reduced magnitude. However, the two effects are not additive, because, when DNA is complexed with both histones and nonhistones, the amplitude of the positive ellipticity band has an intermediate value, between the histone-DNA complex and the nonhistone-DNA complex. Removal of certain nonhistone proteins from chromatin of WI-38 cells, by extraction with 0.25–0.35 m NaCl, causes a decrease in the positive circular dichroism band in the 250–300 nm region. Removal of histones and other nonhistone proteins from chromatin by extraction with 0.75 and 1.5 m NaCl causes a strong increase in positive ellipticity. This suggests the existence of modest but definite effects of nonhistone proteins in determining DNA conformation in native chromatin. Taken as a whole, nonhistone chromosomal proteins have a weaker but analogous effect to that of histones, while the nonhistone proteins extractable with 0.25–0.35 m NaCl have an opposite effect.     

Properties of the genome in experimental hepatomas: variations in the composition of chromatin

The chromosomal proteins of two rapidly growing and poorly differentiated Morris hepatomas were compared with those of liver from normal and tumor bearing animals. While the total quantity of histone associated with DNA in all tumor and liver chromatin preparations studied were similar, tumor chromatin contained an increased quantity of nonhistone chromosomal proteins. Variations in specific classes of histones and nonhistone chromosomal proteins associated with the genome of the two tumors, host liver and liver of tumor bearing animals were observed.     

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.     

DNA-binding activity of tightly-bound nonhistone chromosomal proteins in chicken liver chromatin.

We have isolated a nonhistone chromosomal protein fraction from chicken liver chromatin which possesses high affinity and preferential sequence DNA binding. Residually DNA-bound nonhistone chromosomal proteins after 2.0 M NaCl extraction of bulk chromatin are isolated. Bound proteins are released by dissociation of the complexes in 5.0 M urea/3.0 M NaCl. We have investigated the in vitro DNA-binding properties of this class. In contrast to other DNA-binding NHCP whose activities have been studied, direct DNA-binding activity is observed which is not abolished under conditions of high ionic strength (to 3.0 M NaCl). Strong preference in binding fractionated homologous DNA is observed, while binding of heterologous (E. Coli) DNA is negligible. The fractionation of homologous DNA permits the isolation of DNA for which this protein class displays strong binding preference, presumably through a concentration of binding sites. The composite data suggest sequence-specific interaction between this protein class and DNA, which is not abolished by high ionic strength.     

Release of a globin gene enriched chromatin fraction from chicken erythrocyte nuclei following DNase II digestion

Mild digestion of chicken erythrocyte nuclei with deoxyribonuclease II results in the release of a chromatin fraction which is 4- to 13-fold enriched for the globin coding sequences when compared to total chicken DNA. The remaining nuclear pellet is depleted in these sequences. A maximum of 25% of the globin genes have been recovered in the released fraction. The addition of 5 mM sodium butyrate to the digestion buffer is required to obtain reproducible globin gene enrichment. The released fraction contains equimolar amounts of the four core histones and a subset of the nonhistone chromosomal proteins. The globin genes are released as large chromatin fragments which exceed the 1.6 kilobase size of the transcribed portion of the gene.     

Isolation and characterization of chromosomal proteins from the mosquito Anopheles albimanus Weidemann

1. Nuclei were isolated from adult anopheline mosquitoes and fractionated into nucleolar chromatin, nucleoplasmic chromatin and ribonucleoprotein particles by sucrose density gradients. 2. Histones and nonhistone proteins were selectively dissociated from chromatin by treatment with sodium chloride, urea and guanidine HC1. 3. A special class of nonhistone proteins (tight proteins) were extracted from chromatin with Na4P2O7. 4. The electrophoretic properties of the histones, nonhistone proteins and ribonucleoprotein particles were examined by isoelectric focusing and SDS multiphase polyacrylamide gel electrophoresis. 5. By contrast to the histones, the nonhistone proteins displayed considerable heterogeneity. 6. Possible functional implications of the chromosomal proteins are discussed.     

Cell-specific antigens in chicken erythroid nuclei: species specificity.

Antisera raised to dehistonized chicken reticulocyte chromatin were tested for their cell and species specificity. Quantitative microcomplement fixation and immunohistochemical localization revealed the presence in chromatin of erythroid cell-specific nonhistone protein antigen(s). The antigenic specificity was shown to depend on the association of the antigenic protein(s) with deoxyribonucleic acid (DNA). Although the antisera were exceptionally cell specific, they cross-reacted with erythroid cells of other avian species. The extent of cross-reactivity was found to approximate the phylogenetic distances of the tested avian species. Erythroid cells from fish and amphibians were not reactive. Reconstitution experiments of partially purified chicken reticulocyte chromosomal nonhistone protein antigens with DNAs isolated from several vertebrate species showed that the species specificity of the antigenic complexes is determined principally by the species origin of the nonhistone proteins. Our results show that a cell-specific chromosomal nonhistone protein(s) has undergone evolutionary change and the relative immunological differences are consistent with the accepted phylogenetic distances of the species examined.     

Monoclonal antibodies specific for tight-binding human chromatin antigens reveal structural rearrangements within the nucleus during the cell cycle

The class of nonhistone chromosomal proteins that remains bound to DNA in chromatin in the presence of 2.5 M NaCl-5 M urea has proven refractile to biochemical analysis. In order to study its role in chromatin organization, we have produced monoclonal antibodies that are specific for the HeLa DNA-protein complex that remains after extraction of chromatin with high salt and urea. The antibody-producing clones were identified with an ELISA assay. Of the six clones selected, five were stabilized by limiting dilution. All clones are IgG producers. None cross-react significantly with native DNA, core histones, or the high-mobility group nonhistone proteins. All antibodies are specific for nuclear or juxtanuclear antigens. Indirect immunofluorescence shows that three antibodies, which are nonidentical, stain three different nuclear networks. Available evidence indicates that two of these networks are the nuclear matrix. A fourth antibody reveals structures reminiscent of chromocenters. A fifth antibody, AhNA-1, binds to interphase HeLa chromatin and specifically decorates metaphase chromosomes. AhNA-1 similarly recognizes rat chromosomes. Each of these monoclonal antibodies also reveals a changing pattern of nuclear staining as cells progress through the cell cycle. Presumably, this reflects the rearrangement of the cognate antigens.     

Tissue specificity of nonhistone proteins from human chromatin

Summary Nonhistone proteins were isolated from human placental and tonsillar chromatins. Antiserum was prepared against a complex from some nonhistone proteins and DNA (NP-DNA) from placental chromatin. With the help of polyacrylamide gel electrophoresis and immunological methods the tissue specificity of human chromatin nonhistone proteins was established. The described organ immunogenic specificity of the complex of DNA and nonhistone proteins (NP-DNA) from human chromatin is in accordance with data published on similar complexes from different animal organs. Besides, it is shown that shearing of chromatin leads to large chifts in NP-DNA concentrations required for maximum complement fixation in the presence of the prepared antiserum. This may probably be due to a damage of certain chromatin super structures which involve some of the nonhistone proteins and DNA sequences from both the more condensed and less condensed parts of chromatin.     

The effect of 20-hydroxyecdysone on synthesis of chromosomal and cytosol proteins in imaginal discs

Over 600 cytosol and 300 nonhistone chromosomal proteins of mass-isolated imaginal discs of Drosophila melanogaster have been resolved by two-dimensional electrophoresis. More than half of the nonhistone chromosomal proteins fall into families with effectively constant apparent molecular weight but varying isoelectric points. At least six chromosomal proteins differ distinctly in proportions between embryos and imaginal discs. The synthesis of six cytosol proteins is increased, and one decreased with incubation of the discs in vitro with 20-hydroxyecdysone. Two disc acidic chromosomal proteins are specifically synthesized in the presence of 20-hydroxyecdysone. Their isoelectric points and molecular weights are similar to those of the subunits of vertebrate steroid hormone receptor proteins. However, although ecdysteroid receptor activity is associated with purified chromatin, no ecdysteroid-dependent increase in receptor activity is detected during in vitro culture of discs.     

Isolation and characterization of chromatin subfractions from rat liver

Rat-liver chromatin was digested with micrococcal nuclease at low ionic strength in the presence of a low concentration of CaCl2. The nuclease digest was successfully separated into three fractions, P1, P2, and P3, by gel filtration on a column of Sepharose 2B. P1 fraction was shown to be a mixture of long fragments of partially digested chromatin by the sedimentation profile or by electrophoresis of DNA. P2 fraction contained four histones H2A, H2B, H3, and H4 in almost equal amounts, together with nonhistone protein of low molecular weight. The DNA was composed of three or four fragments less than 300 base pairs long. From the Kav value of the P2 fraction, the average size was estimated to be about 240 base pairs. On analytical ultracentrifugation, this fraction exhibited a monophasic boundary and a sedimentation value of 13.7S. P3 fraction contained nonhistone proteins which showed a molecular weight larger than that of H1 histone. The size of DNA was estimated to be less than 50 base pairs from the Kav value. Based on these results, the P2 fraction was concluded to consist of nucleosome monomer enriched in nonhistone proteins. The P3 fraction is presumably the nuclease-sensitive or internucleosome portion, which contains small amounts of nonhistone proteins.     

Proteins of metaphase chromosomes and interphase chromatin.

Metaphase chromosomal and interphase chromatin proteins from cells of two species have been compared by polyacrylamide gel electrophoresis. Consistent, common changes in the quantitative distribution of the nonhistone chromosomal proteins are observed in both species. Proteins of ca. 65,000 and 68,000 MW are enriched in interphase chromatin while proteins of ca. 50,000 and 200,000 are more prominent components of metaphase chromosomes. A group of proteins of 90,000-100,000 are also increased in metaphase chromosomes compared to interphase chromatin. By two dimensional gel analysis, the most abundant proteins from chromosomes of both cell types are similar, suggesting a structural role for these nonhistone proteins (1).     

Large-scale isolation of a native deoxyribonucleohistone complex from baker''s yeast.

A method for large scale isolation of a native deoxyribonucleohistone complex from yeast is described. Crude chromatin, obtained after disrupting yeast cells at low ionic strength, contains a large amount of lipids, partially due to contaminating membranes. Most of them are removed by a Triton X-100 treatment, followed by step-gradient centrifugation. About 90% of the pellet may be solubilized by mild procedures, the composition of the soluble material being: histone/DNA = 1.0;nonhistone proteins/DNA = 0.55; RNA/DNA = 0.18. Histones can be obtained with high purity. Micrococcal nuclease digests DNA to yield a series of oligomeric fragments, with an average repeat length of about 160 base pairs. Circular dichroism spectra show that (theta) 270 is reduced by about 30% when compared to pure DNA and that chromosomal proteins are not denatured. These results indicate that the components of the complex conserve the native state.