scholarly journals Fractionation of nucleosomes by salt elution from micrococcal nuclease-digested nuclei.

1978 ◽  
Vol 79 (1) ◽  
pp. 97-109 ◽  
Author(s):  
M M Sanders
Keyword(s):  
Chromatin Condensation ◽  
Apparent Molecular Weight ◽  
Histone H1 ◽  
Micrococcal Nuclease ◽  
Morphological Properties ◽  
Chromosomal Proteins ◽  
Nuclease Digestion ◽  
Liver Nuclei ◽  
Packing Interactions ◽  
Macromolecular Composition

The solubilization of nucleosomes and histone H1 with increasing concentrations of NaCl has been investigated in rat liver nuclei that had been digested with micrococcal nuclease under conditions that did not substantially alter morphological properties with respect to differences in the extent of chromatin condensation. The pattern of nucleosome and H1 solubilization was gradual and noncoordinate and at least three different types of nucleosome packing interactions could be distinguished from the pattern. A class of nucleosomes containing 13--17% of the DNA and comprising the chromatin structures most available for micrococcal nuclease attack was eluted by 0.2 M NaCl. This fraction was solubilized with an acid-soluble protein of apparent molecular weight of 20,000 daltons and no histone H1. It differed from the nucleosomes released at higher NaCl concentrations in content of nonhistone chromosomal proteins. 40--60% of the nucleosomes were released by 0.3 M NaCl with 30% of the total nuclear histone H1 bound. The remaining nucleosomes and H1 were solublized by 0.4 M or 0.6 M NaCl. H1 was not nucleosome bound at these ionic strengths, and these fractions contained, respectively, 1.5 and 1.8 times more H1 per nucleosome than the population released by 0.3 M NaCl. These fractions contained the DNA least available for micrococcal nuclease attach. The strikingly different macromolecular composition, availability for nuclease digestion, and strength of the packing interactions of the nucleosomes released by 0.2 M NaCl suggest that this population is involved in a special function.

scholarly journals Mechanism of enhanced RNA synthesis in acute-phase rat liver and its relationship to chromatin structure

1984 ◽  
Vol 219 (1) ◽  
pp. 165-171 ◽  
Author(s):  
L Schiaffonati ◽  
L Bardella ◽  
G Cairo ◽  
V Giancotti ◽  
A Bernelli-Zazzera
Keyword(s):  
Acute Phase ◽  
Chromatin Structure ◽  
Rna Synthesis ◽  
Elongation Rate ◽  
Micrococcal Nuclease ◽  
Chromosomal Proteins ◽  
Micrococcal Nuclease Digestion ◽  
Nuclease Digestion ◽  
Liver Nuclei ◽  
Phase Process

Nuclei isolated from the liver of rats undergoing an acute inflammatory reaction induced by turpentine treatment show increased RNA synthesis. This increase is essentially determined by a faster polyribonucleotide-elongation rate while the number of transcribing polymerase molecules is unchanged. The sensitivity of chromatin to micrococcal-nuclease digestion and the composition of chromosomal proteins are not affected by the acute-phase process. Therefore the increased RNA synthesis by liver nuclei from acutely inflamed rats does not seem to correlate with major changes in chromatin structure.

Evidence for Nucleosomal Phasing and a Novel Protein Specifically Binding to Cucumber Satellite DNA

1994 ◽  
Vol 49 (1-2) ◽  
pp. 79-86 ◽  
Author(s):  
Thilo C. Fischer ◽  
Sabine Groner ◽  
Ulrike Zentgraf ◽  
Vera Hemleben
Keyword(s):  
Satellite Dna ◽  
Specific Binding ◽  
Apparent Molecular Weight ◽  
Micrococcal Nuclease ◽  
Type I ◽  
Dna Repeats ◽  
Cucumis Sativus L ◽  
Nuclease Digestion

The nucleosomal organization and the protein-binding capability of highly repeated and methylated satellite DNA of cucumber (Cucumis sativus L.), comprising approx. 30% of the genome, were analyzed. Nucleosomal core DNA from satellite type I was prepared after micrococcal nuclease digestion of chromatin and sequenced. Most of the core sequences obtained could be grouped in two main (A and B) and two minor groups (C and D) indicating a specific and complex phasing of nucleosomes on this satellite DNA. In vitro, gel retardation assays with cloned satellite DNA repeats (types I-IV) demonstrated a specific binding of nuclear proteins. These specific binding effects are also obtained with genomic, in vivo methylated and sequence heterogeneous (1 to 10% diversity) satellite type I DNA. For the first time in plants, a satellite DNA-binding protein with an apparent molecular weight of 14 kDa (SAT 14) was identified.

Hyper(ADP-ribosyl)ation of histone H1

1982 ◽  
Vol 60 (12) ◽  
pp. 1085-1094 ◽  
Author(s):  
R. J. Aubin ◽  
V. T. Dam ◽  
J. Miclette ◽  
Y. Brousseau ◽  
A. Huletsky ◽  
...  
Keyword(s):  
Repeat Unit ◽  
Selective Extraction ◽  
Histone H1 ◽  
Polymerase Activity ◽  
Micrococcal Nuclease ◽  
Micrococcal Nuclease Digestion ◽  
Nuclease Digestion ◽  
Nonionic Detergent ◽  
Minimal Modification ◽  
Triton X

Nucleosomal chains of various repeat unit lengths were generated by a mild micrococcal nuclease digestion of purified pancreatic nuclei. Maximal nucleosome associated poly(ADP-ribose) polymerase activity was recovered in trimeric to tetrameric chromatin fragments, after which the enzyme activity gradually decreased and stabilized towards oligomeric periodicities of 11 to 16 nucleosomes. Electrophoresis of [32P] ADP-ribosylated histones on first-dimension acid–urea or acid–urea–Triton gels and on second-dimension acid – urea – cetyltriammonium bromide gels revealed that, of all histones, only histone H1 could be significantly poly(ADP-ribosyl)ated while only minimal modification could be recovered with histone H10. Furthermore, the extent of ADP-ribosylation present on pancreatic histone H1 is shown to proportionally retard this protein's electrophoretic mobility in all gel systems and to consist of a distinct series of at least 12 modification intermediates which can be evidenced, in nuclei or nucleosomes, and fully recovered along with histone H1 upon its selective extraction with 5% perchloric acid. The generation of these increasingly ADP-ribosylated forms of histone H1 is also demonstrated to be time dependent and the more complex ADP-ribosylated forms of this histone are favored at high NAD+ concentrations. Moreover, the electrophoretic mobilities of all intermediates are unaffected by the presence of the nonionic detergent Triton X-100.

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 Different Proteins Mediate Step-wise Chromosome Architectures in Thermoplasma acidophilum and Pyrobaculum calidifontis

2020 ◽  
Author(s):  
Hugo Maruyama ◽  
Eloise I. Prieto ◽  
Takayuki Nambu ◽  
Chiho Mashimo ◽  
Kosuke Kashiwagi ◽  
...  
Keyword(s):  
Higher Order ◽  
Micrococcal Nuclease ◽  
Thermoplasma Acidophilum ◽  
Chromosomal Proteins ◽  
Pyrobaculum Calidifontis ◽  
Archaeal Species ◽  
Nuclease Digestion ◽  
Chromosomal Architecture ◽  
Order Structures

AbstractArchaeal species encode a variety of distinct lineage-specific chromosomal proteins. We have previously shown that in Thermococcus kodakarensis, histone, Alba, and TrmBL2 play distinct roles in chromosome organization. Although our understanding of individual archaeal chromosomal proteins has been advancing, how archaeal chromosomes are folded into higher-order structures and how they are regulated are largely unknown. Here, we investigated the primary and higher-order structures of archaeal chromosomes from different archaeal lineages. Atomic force microscopy of chromosome spreads out of Thermoplasma acidophilum and Pyrobaculum calidifontis cells revealed 10-nm fibers and 30–40-nm globular structures, suggesting the occurrence of higher-order chromosomal folding. Our results also indicated that chromosome compaction occurs toward the stationary phase. Micrococcal nuclease digestion indicated that fundamental structural units of the chromosome exist in T. acidophilum and T. kodakarensis but not in P. calidifontis or Sulfolobus solfataricus. In vitro reconstitution showed that, in T. acidophilum, the bacterial HU protein homolog HTa formed a 6-nm fiber by wrapping DNA, and that Alba was responsible for the formation of the 10-nm fiber by binding along the DNA without wrapping. Remarkably, Alba could form different higher-order complexes with histone or HTa on DNA in vitro. Mass spectrometry detected HTa in the T. acidophilum chromosome but not in other species. A putative transcriptional regulator of the AsnC/Lrp family (Pcal_1183) was detected on the P. calidifontis chromosome, but not on that of other species studied. Putative membrane-associated proteins were detected in the chromosomes of the three archaeal species studied, including T. acidophilum, P. calidifontis, and T. kodakarensis. Collectively, our data show that Archaea use different combinations of proteins to achieve chromosomal architecture and functional regulation.

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.

Chromatin structural transitions following histone H1 displacement by phosphatidylserine vesicles and low pH treatment. A multiparametric analysis involving flow cytometry, electron microscopy, and nuclease digestion.

1988 ◽  
Vol 36 (1) ◽  
pp. 65-71 ◽  
Author(s):  
L Cocco ◽  
S Papa ◽  
N M Maraldi ◽  
P Santi ◽  
A M Martelli ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Flow Cytometry ◽  
Histone H1 ◽  
Low Ph ◽  
Ion Concentration ◽  
Light Scatter ◽  
Chromatin Decondensation ◽  
Nuclease Digestion ◽  
Chromatin Structural ◽  
Liver Nuclei

We describe several morphological and functional modifications in isolated rat liver nuclei incubated in the presence of phosphatidylserine (PS) multilamellar vesicles (MLV). These effects, which occur through the release of histone H1, induce chromatin decondensation, as shown by electron microscopy and nuclease digestion. Flow cytometry was employed to monitor these changes in chromatin structure in isolated nuclei by means of perpendicular light scatter (PLS) and fluorescence signals. Chromatin decondensation induced by PS or by low pH treatment was accompanied by an increase in perpendicular light scatter and by less efficient binding of ethidium bromide. These flow cytometric findings are peculiar to chromatin decondensation induced by displacement of histone H1. Conversely, chromatin decondensation caused by lowering of the divalent ion concentration, without displacement of histone H1, is characterized only by an increase in perpendicular light scatter.

Effects of Mg2+ on Chromatic Structure in Isolated Chicken Liver Nuclei

1990 ◽  
Vol 48 (3) ◽  
pp. 130-131
Author(s):  
Soichiro Arai ◽  
Yuh H. Nakanishi
Keyword(s):  
Chromatin Structure ◽  
Room Temperature ◽  
Osmium Tetroxide ◽  
Divalent Cations ◽  
Chromatin Condensation ◽  
Chicken Liver ◽  
Electron Microscopic ◽  
Razor Blade ◽  
Microscopic Studies ◽  
Liver Nuclei

Although many electron microscopic studies on extracted chromatin have provided considerable information on chromatin condensation induced by divalent cations, there is only a little literature available on the effects of divalent cations on chromatin structure in intact nuclei. In the present study, the effects of Mg2+ on chromatin structure in isolated chicken liver nuclei were examined over a wide concentration range of Mg2+ by scanning electron microscopy.Nuclei were prepared from chicken liver by the method of Chauveau et al. with some modifications. The nuclei were suspended in 25 mM triethanolamine chloride buffer (pH7.4) with 1 mM EDTA or in the buffer with concentrations of MgCl2 varying from 1 to 50 mM. After incubation for 1 min at 0°C, glutaraldehyde was added to 1.8% and the nuclei were fixed for 1 h at 4°C. The fixed nuclei were mixed with 15% gelatin solution warmed at about 40°C, and kept at room temperature until the mixture set. The gelatin containing the nuclei was fixed with 2% glutaraldehyde for 2-4 h, and cut into small blocks. The gelatin blocks were conductive-stained with 2% tannic acid and 2% osmium tetroxide, dehydrated in a graded series of ethanol, and freeze-cracked with a razor blade in liquid nitrogen.

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