scholarly journals Nucleosomes from normal and regenerating rat liver

1979 ◽  
Vol 178 (1) ◽  
pp. 173-185 ◽  
Author(s):  
Margery G. Ord ◽  
Lloyd A. Stocken
Keyword(s):  
Rat Liver ◽  
Base Pair ◽  
Specific Radioactivity ◽  
Histone H1 ◽  
S Phase ◽  
Micrococcal Nuclease ◽  
Core Histones ◽  
32P Uptake ◽  
Liver Nuclei ◽  
Core Particles

Micrococcal-nuclease digestion of rat liver nuclei selectively released mononucleosomes associated with ADP-ribosylated [Caplan, Ord & Stocken (1978) Biochem. J.174, 475–483] histone H1. Two classes of mononucleosome were detected, those that leaked out during digestion and those that were subsequently released by 5mm-sodium phosphate buffer (pH6.8)/0.2mm-NaEDTA. The former, from which histone H1 had been dissociated, contained 140-base-pair-length DNA and core histones;the latter contained core particles and mononucleosomes with histone H1 and 200-base-pair-length DNA. When normal liver nuclei were phosphorylated with [γ-32P]ATP, dissociated histone H1, which could be separated from core particles with Sephadex G-200, showed 32P uptake. 32P uptake into histones H2A and poly(ADP-ribosyl)ated H3 was appreciable in core particles, but was less evident in nucleosomes still containing histone H1. When [3H]-thymidine was given to partially hepatectomized rats in S-phase, 5–10min pulses in animals of over 300g body wt. showed the presence of high-specific-radioactivity DNA in released core particles and mononucleosomes compared with DNA retained in the nuclear pellets. Mononucleosomes from rat livers in S-phase with new, [3H]lysine-containing histones, had higher 32P incorporation in histones H1 and their core histones, than for di- or tri-nucleosomes. Thermal-denaturation properties of control and phosphorylated mononucleosomes and core particles were very similar; removal of histone H1 and non-histone chromosomal proteins in 0.5m-NaCl markedly increased the proportion of DNA ‘melting’ below 70°C.

Structure of rat liver nuclei after depletion and reassociation of histone H1

1981 ◽  
Vol 132 (2) ◽  
pp. 399-409 ◽  
Author(s):  
Reiner Klingholz ◽  
Wolf H. Strätling ◽  
Hansjörg Schäfer
Keyword(s):  
Rat Liver ◽  
Histone H1 ◽  
Rat Liver Nuclei ◽  
Liver Nuclei

scholarly journals A chromosomal phosphoprotein is preferentially released by mild micrococcal-nuclease digestion

1984 ◽  
Vol 220 (2) ◽  
pp. 539-545 ◽  
Author(s):  
C C Liew ◽  
M J Halikowski ◽  
M S Zhao
Keyword(s):  
Gradient Centrifugation ◽  
Specific Radioactivity ◽  
Sucrose Density Gradient ◽  
Micrococcal Nuclease ◽  
Chromosomal Protein ◽  
Sucrose Density Gradient Centrifugation ◽  
Micrococcal Nuclease Digestion ◽  
Nuclease Digestion ◽  
32P Incorporation ◽  
Liver Nuclei

[32P]Pi was administered to rats (5mCi/rat) 2h before the isolation of liver nuclei. The isolated nuclei were subjected to mild micrococcal-nuclease digestion for 2.5, 5 and 10 min at 37 degrees C, and the mononucleosomal fraction was subsequently isolated by sucrose-density-gradient centrifugation. The specific radioactivity of 32P-labelled mononucleosomal fractions decreased with increased digestion times. A phosphorylated chromosomal protein, B2 (Mr 68000, pI6.5-8.2), was demonstrated immunologically in the mononucleosomal fraction by using an antibody specific to this electrophoretically purified phosphoprotein. The incorporation of 32P into this phosphoprotein, previously shown to be mainly through covalent linkage, was revealed by antibody precipitation followed by gel electrophoresis. The rate of release of acid-soluble nucleotides by micrococcal-nuclease digestion of liver nuclei from partially hepatectomized rats 16 h after operation was strikingly higher than that for sham-operated controls. After partial hepatectomy, an increase in 32P incorporation into phosphoprotein in the monomer fractions specifically precipitated by this antibody was also found. This suggests that the phosphorylated non-histone chromatin protein B2 is preferentially associated with the transcriptionally active chromatin.

scholarly journals Subnuclear fractionation by mild micrococcal-nuclease treatment of nuclei of different transcriptional activities causes a partition of expressed and non-expressed genes

1980 ◽  
Vol 187 (2) ◽  
pp. 467-467 ◽  
Author(s):  
G J Dimitriadis ◽  
J R Tata
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rat Liver ◽  
Nuclear Dna ◽  
Globin Gene ◽  
Micrococcal Nuclease ◽  
Deoxyribonuclease I ◽  
Liver Nuclei ◽  
Albumin Mrna

Extremely mild treatment with micrococcal nuclease of isolated nuclei yields subnuclear fractions in which the majority of RNA polymerase II transcriptional complexes formed in vivo are segregated [Tata & Baker (1978) J. Mol. Biol. 118, 249-272]. We now describe different approaches followed to established whether or not the nuclei are thus resolved into transcribed and non-transcribed DNA. First, we have compared the sensitivity to deoxyribonuclease I, which is known to digest preferably expressed genes as present in nuclei or chromatin, of three micrococcal-nuclease-derived fractions from nuclei of different transcriptional activities. In transcriptionally active nuclei (rat liver, hen liver and oviduct, and Xenopus liver), the DNA in a polynucleosomal fraction comprising 6-15% of DNA and the majority of template-engaged RNA polymerase II (fraction P2) was 10-50 times as sensitive to deoxyribonuclease I as the DNA in the other two fractions (fractions P1 and S, comprising 78-88% of total nuclear DNA as large polynucleosomal aggregates and 2-6% of DNA mostly as mononucleosomes, respectively). In transcriptionally inactive nuclei obtained from hen erythrocytes, micrococcal nuclease did not separate DNA into fractions exhibiting such differential sensitivities. Second, we have monitored the partition of an expressed gene. Hybridization of complementary DNA to Xenopus albumin mRNA revealed a 5-10-fold enrichment of the albumin (but not the globin) gene in the P2 fraction of nuclei from Xenopus liver in which this gene is fully expressed. Third, a large part of the nascent rapidly labelled RNA synthesized in vivo in rat liver nuclei was recovered in the micrococcal-nuclease-derived fraction that is more susceptible to digestion with deoxyribonuclease I. It is concluded that mild micrococcal-nuclease treatment of nuclei causes their separation into transcribed and non-transcribed DNA as determined by a number of very different criteria.

scholarly journals Initiation of transcription in normal and phosphorylated rat liver nucleosomes

1978 ◽  
Vol 176 (2) ◽  
pp. 615-618 ◽  
Author(s):  
M G Ord ◽  
L A Stocken
Keyword(s):  
Rna Polymerase ◽  
Rat Liver ◽  
Base Pair ◽  
S Phase ◽  
Initiation Of Transcription

Initiation sites were enumerated in rat liver nucleosomes with Echerichia coli RNA polymerase. One site was present in approx. 3.5 mononucleosomes and in a 1200-base-pair length of polynucleosomes. S-phase nuclei or normal nuclei phosphorylated in vitro showed increased numbers of sites; the elongation rates were unchanged.

Influence of Distamycin, Chromomycin, and UV-irradiation on Extraction of Histone H1 from Rat Liver Nuclei by Polyglutamic Acid

2010 ◽  
Vol 75 (11) ◽  
pp. 1331-1341 ◽  
Author(s):  
A. N. Prusov ◽  
T. A. Smirnova ◽  
L. P. Kurochkina ◽  
G. Ya. Kolomijtseva
Keyword(s):  
Rat Liver ◽  
Uv Irradiation ◽  
Histone H1 ◽  
Polyglutamic Acid ◽  
Rat Liver Nuclei ◽  
Liver Nuclei

scholarly journals Nucleosome binding by the polymerase I transactivator upstream binding factor displaces linker histone H1.

1997 ◽  
Vol 17 (10) ◽  
pp. 5833-5842 ◽  
Author(s):  
M Kermekchiev ◽  
J L Workman ◽  
C S Pikaard
Keyword(s):  
Histone H1 ◽  
Linker Histone ◽  
Micrococcal Nuclease ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Nucleosome Core ◽  
Binding Factor ◽  
Core Histones ◽  
Upstream Binding Factor ◽  
Polymerase I

Upstream binding factor (UBF) is a vertebrate RNA polymerase I transcription factor that can bend and wrap DNA. To investigate UBF's likely role as an architectural protein of rRNA genes organized in chromatin, we tested UBF's ability to bind rRNA gene enhancers assembled into nucleosome cores (DNA plus core histones) and nucleosomes (DNA plus core histones plus histone H1). UBF bound with low affinity to nucleosome cores formed with enhancer DNA probes of 162 bp. However, on nucleosome cores which contained approximately 60 bp of additional linker DNA, UBF bound with high affinity similar to its binding to naked DNA, forming a ternary DNA-core histone-UBF complex. UBF could be stripped from ternary complexes with competitor DNA to liberate nucleosome cores, rather than free DNA, suggesting that UBF binding to nucleosome cores does not displace the core histones H2A, H2B, H3, and H4. DNase I, micrococcal nuclease, and exonuclease III footprinting suggests that UBF and histone H1 interact with DNA on both sides flanking the histone octamer. Footprinting shows that UBF outcompetes histone H1 for binding to a nucleosome core and will displace, if not dissociate, H1 from its binding site on a preassembled nucleosome. These data suggest that UBF may act to prevent or reverse the assembly of transcriptionally inactive chromatin structures catalyzed by linker histone binding.

scholarly journals Processing and migration of ribosomal ribonculeic acids in the nucleolus and nucleoplasm of rat liver nuclei

1978 ◽  
Vol 171 (2) ◽  
pp. 375-383 ◽  
Author(s):  
K P Dudov ◽  
M D Dabeva ◽  
A A Hadjiolov ◽  
B N Todorov
Keyword(s):  
Rat Liver ◽  
Specific Radioactivity ◽  
Kinetic Studies ◽  
Simultaneous Occurrence ◽  
Turnover Rates ◽  
Mathematical Methods ◽  
Processing Pathways ◽  
Liver Nuclei ◽  
And Migration

Kinetic studies on the labelling in vivo with [14C]orotate of rat liver nucleolar and nucleoplasmic pre-rRNA (precursor of rRNA) and rRNA, isolated from detergent-purified nuclei, were carried out. The mathematical methods used for the computer analysis of specific-radioactivity curves are described. Evaluation of the experimental data permitted the selection of the most probable models for the processing of pre-rRNA and the nucleo-cytoplasmic transfer of rRNA. It was shown that considerable flexibility exists in the sequence of endonuclease attacks at critical sites of 45 and 41 S pre-rRNA chains, resulting in the simultaneous occurrence of several processing pathways. However, the phosphodiester bonds involved in the formation of mature 28 and 18 S rRNA appear to be protected until the generation of their immediate pre-rRNA. The turnover rates and half-lives of all pre-rRNA and rRNA pools were determined. The turnover rate of 45 S pre-rRNA corresponds to the formation of 1100 ribosomes/min per nucleus. The model for the nucleolus-nucleoplasm-cytoplasm migration of rRNA includes a ‘nucleoplasm’ compartment in which the small ribosomal subparticle is in rapid equilibrium with the respective cytoplasmic pool. At equimolar amounts of nuclear 28 and 18 S rRNA this model explains the faster appearance of labelled small ribosomal subparticles in the cytoplasm simultaneous with a lower labelling of nuclear 18 S rRNA as compared with 28 S rRNA.

The lack of histone H1 in the peripheral chromatin of rat liver nuclei

1982 ◽  
Vol 6 (5) ◽  
pp. 433-441 ◽  
Author(s):  
D FAIS ◽  
A PRUSOV ◽  
V POLYAKOV
Keyword(s):  
Rat Liver ◽  
Histone H1 ◽  
Rat Liver Nuclei ◽  
Liver Nuclei
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