The non-histone proteins of the rat liver nucleus and their distribution amongst chromatin fractions as produced by nuclease digestion.

The search for proteins involved in maintaining higher order chromatin structures has led to a systematic examination of the non-histone proteins (NHP) of rat liver nuclei in the context of nuclease digestion studies. 40-45% of the 3H-tryptophan labelled NHP originally present could be removed by extensive washing in a "physiological" buffer, incubation at 37 degrees C with or without nuclease and a further wash step. Nuclei at this stage had a remarkably constant NHP content (ca. 0.73 micrograms/micrograms DNA), independent of the degree of digestion with micrococcal nuclease or HaeIII. The solubilized chromatin produced by limited digestion with either nuclease contained 0.3-0.5 microgram NHP/microgram DNA, this value falling to ca. 0.16 after more extensive cleavage. Insoluble chromatin fractions were between 2-fold (very limited digestion) and 16-fold (extensive digestion) richer in NHP than the corresponding soluble fractions. Gel electrophoresis revealed about 12 NHP bands in soluble fractions, the most prominent of M.Wt. 41.400, while the insoluble material had at least 50 components. These properties were independent of whether lysis of nuclei occurred in 0.2 or 50 mM ionic strength. The large disparity in NHP content between complementary soluble and insoluble chromatin fractions is considered in terms of chromatin organization in vivo and the possible role of NHP migration.     

Conformational changes in rat liver chromatin after liver regeneration.

N-Pyrenemaleimide, a fluorescent probe that specifically labels histone H3 of rat liver chromatin in situ, was used to monitor the accessibility of histone H3 in chromatin isolated from rat liver at different times during degeneration. At times of maximum DNA synthesis (18--24 h after hepatectomy), the accessibility of the probe was found to be markedly (40--50%) increased. This increase is abolished, however, by treatment of the chromatin fibres with high salt (2 M-NaCl) or detergent. Tryptophan fluorescence was also enhanced at points of maximum DNA synthesis, suggesting that some non-histone tryptophan-containing protein was being synthesized. The polarization of the labelled histone H3 is not markedly altered, suggesting that fibre aggregation or dissociation does not occur. Mononucleosomes extracted from sham-operated and hepatectomized animals did not exhibit any difference in binding to the probe. Also, analysis of the chromatin protein by electrophoresis on detergent- and acid/urea/ Triton-X-100-containing polyacrylamide gels showed no detectable difference in histone H3 : 1, H3 : 2 or H3 : 3 subclasses.     

Isolation of chromatin fragments rich in non-histone protein after endonuclease digestion of rat liver nuclei

Detergent-washed rat liver nuclei, prepared in the presence of a protease inhibitor, were incubated for up to 60 min at 37 °C. The action of endonucleases produced chromatin fragments which could be removed from the nuclei by extraction with 8 M urea 50 mM phosphate, pH 7.6, 15% of the total nuclear DNA being extracted. No DNA could be detected in this extract after incubation of nuclei at 4 °C. The chromatin fragments were sedimented by centrifugation at 90000 g or by chromatography on Sepharose 4B. The DNA fragment sizes were similar to those found in nucleosome particles but the protein/DNA ratio was approx. 5.3:1. The nuclei were prelabelled with [³H]tryptophan and 60% of the label present in the 8 M urea extract was found to sediment with the chromatin fraction. SDS polyacrylamide gel electrophoresis of the latter showed the presence, in addition to histones, of at least 25 polypeptide species of tightly bound non-histone proteins with molecular weights in excess of 30000.     

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.     

Distribution of tightly bound non-histone proteins in chromatin fractions obtained by DNAase II digestion

Digestion of pig liver chromatin with DNAse II afforded three different fractions which were characterized in terms of their DNA, RNA and tightly bound non-histone protein content, their DNA fragment size and their template activity. Two of these fractions are soluble after digestion with DNAase II and have been separated on the basis of their different solubility in MgCl2. A third fraction is not solubilized even after extensive digestion, although the size of its DNA is comparable to that of the enzyme solubilized fractions. The three fractions show qualitatively and quantitatively different distribution of tightly bound non-histone proteins, with specific protein components in each fraction; furthermore the non-solubilized fraction is greatly enriched in proteins tightly bound to DNA. From all the data obtained it can be suggested that the tightly bound proteins of the insoluble fraction may play, directly or indirectly, a role in maintaining an organized chromatin structure.     

Fractionation of chromatin of liver cell nuclei after mild micrococcal nuclease digestion

Mild micrococcal nuclease treatment of rat and mouse nuclei and fractionation were based on the method of Tata and Baker. Three chromatin fractions, S, P1, P2, were separated, and for each of these fractions the sensitivity to the DNase 1 action was determined. The relative content in these fractions of non-transcribed DNA sequences was established by hydridization with a mouse satellite DNA, and the relative content of transcribed DNA sequences--by hydridization with DNA synthesised on the total poly (A) mRNA. None of the fractions displayed the properties characteristic of active chromatin.     

Isolation of Golgi fractions from colchicine-treated rat liver. II. Electrophoretic characterization.

1. Intact Golgi fractions, three from colchicine- or ethanol-treated rat livers and two from a control, were analyzed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. All the fractions showed very similar electrophoretic profiles with 33 protein bands, some of which, especially albumin, had rather higher density in the secretory vesicle fraction than those in the cisternal fraction. 2. Using albumin as the content marker, the Golgi fractions were subfractionated into membranes and contents by freezing-thawing and sonication followed by centrifugation. Distribution of galactosyltransferase among these membrane preparations showed that this enzyme was more enriched in the Golgi cisternal membranes than in the secretory vesicle membranes. 3. All the membrane preparations from the Golgi complex showed very similar patterns on electrophoresis, which were distinctly different from those of microsomal membranes and of plasma membrane. Furthermore, all the Golgi content subfractions had similar protein components, most of which were also found in serum. The microsomal contents, however, showed a considerably different pattern from those of the Golgi contents. 4. From these results it could be concluded that the secretory vesicles are indeed a member of the Golgi complex despite their different appearance and morphology.     

The distribution of nuclear proteins and transcriptionally-active sequences in rat liver chromatin fractions

Chromatin fractions from rat liver nuclei digested by nucleases were separated by differential solubility into several fractions. Material solubilized during digestion (predominantly monomer nucleosomes and polynucleosomes) had the highest HMG14 + 17/DNA ratios but were not enriched in active gene sequences (albumin and c-Ha-ras1 genes). Material soluble in a low ionic strength buffer containing 0.2 mM MgCl2 (monomer nucleosomes and polynucleosomes) contained in addition to the histones, HMG14 and 17 plus a 41K non-histone protein. This fraction was depleted in active gene sequences and enriched in inactive sequences. The insoluble material was highly enriched in active sequences and had the lowest HMG14 + 17/DNA ratio. This fraction could be further fractionated into a histone-containing 2 M NaCl-soluble fraction and a 2 M NaCl-insoluble matrix-bound fraction, both of which were enriched in active sequences. The results show that the HMG proteins do not partition with active sequences during fractionation of chromatin. The 41K protein may be associated with inactive chromatin fraction.     

Arachidonic acid hydroperoxide stimulates lipid peroxidation in rat liver nuclei and chromatin fractions

Arachidonic acid, the most abundant polyunsaturated fatty acid in rat liver nuclei phospholipids is a major target of free radical attack, which induces lipid peroxidation. The non-enzymatic lipid peroxidation process in intact rat liver nuclei and in several chromatin fractions indicated that the most sensitive fatty acid for peroxidation is arachidonic acid C20:4 n-6. In this study, the effect of different amounts of arachidonic acid hydroperoxide on the lipid peroxidation of rat liver nuclei and chromatin fractions was studied; rat liver nuclei and chromatin fractions deprived of exogenous added hydroperoxide were utilized as control. The addition of arachidonic acid hydroperoxide to liver nuclei produces a marked increase in light emission that was hydroperoxide concentration dependent. The maximal peak of chemiluminescence displayed by the different chromatin fractions analyzed was observed between 20 and 80 min of incubation. The highest value of light emission was displayed by the high-density chromatin fractions, the 27.5 K fraction showed intermediate values of light emission, whereas the lowest density fraction produced very low chemiluminescence. A high correlation between arachidonic acid hydroperoxide concentration and chemiluminescence in the different chromatin fractions was observed. AC is Members of Carrera del Investigador Científico, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina.     

Stimulation of DNA synthesis in isolated nuclei of rat liver cells by neutral Mn-dependent DNase of chromatin

It was demonstrated that neutral Mn-dependent DNAase from rat liver chromatin stimulates the incorporation of labeled precursors of DNA into high molecular weight fractions of isolated nuclear DNA. The effects of DNA-polymerase inhibitors and the properties of DNA synthesis products suggest that neutral Mn-dependent DNAase can induce replicative synthesis of DNA in the nuclei of normal and regenerating rat liver.     

DNase I site mapping and micrococcal nuclease digestion of pachytene chromatin reveal novel structural features

A comparison of the DNase I digestion products of the 32P-5'-end-labeled pachytene nucleosome core particles (containing histones H2A, TH2A, X2, H2B, TH2B, H3, and H4) and liver nucleosome core particles (containing somatic histones H2A, H2B, H3, and H4) revealed that the cleavage sites that are 30, 40, and 110 nucleotides away from the 5'-end are significantly more accessible in the pachytene core particles than in the liver core particles. These cleavage sites correspond to the region wherein H2B interacts with the nucleosome core DNA. These results, therefore, suggest that the histone-DNA interaction at these sites in the pachytene core particles is weaker, possibly because of the presence of the histone variant TH2B interacting at similar topological positions in the nucleosome core as that of its somatic counterpart H2B. Such a loosened structure may also be maintained even in the native pachytene chromatin since micrococcal nuclease digestion of pachytene nuclei resulted in a higher ratio of subnucleosomes (SN4 + SN7) to mononucleosomes than that observed in liver chromatin.     

Release of ribonucleoprotein during digestion of rat testis chromatin with deoxyribonuclease II (3.1.4.6)

The composition of rat testis chromatin proteins in fractions produced by limited DNase II digestion followed by differential precipitation with MgCl2 has been studied. Over 50% of the acid-soluble proteins in the soluble chromatin fraction appeared to be quite similar to proteins which are associated with ribonucleoprotein (RNP) particles in HeLa cells. Although the ratios of the testis RNP protein components differed from those of HeLa RNP particles, the three major polypeptides were most similar to the HeLa components designated A2, B2, and C1. The soluble chromatin fraction was also enriched in the high mobility group proteins HMG1 and HMG2.