scholarly journals Fractionation of chick oviduct chromatin. Nuclease-resistant deoxyribonucleic acid

1975 ◽  
Vol 151 (3) ◽  
pp. 497-503 ◽  
Author(s):  
J F Krall ◽  
S H Socher ◽  
N T Van ◽  
B W O'Malley
Keyword(s):  
Dna Sequences ◽  
Thermal Denaturation ◽  
Tissue Specificity ◽  
Deoxyribonucleic Acid ◽  
Micrococcal Nuclease ◽  
Chromosomal Protein ◽  
Nuclease Digestion ◽  
Nuclease Resistance ◽  
Limited Degree ◽  
Chromatin Proteins

Chromatin isolated from several chick tissues was treated with micrococcal nuclease. A limited degree of tissue specificity of chromatin DNA resistance to nuclease digestion was observed. No difference in the extent of nuclease resistance of chromatin DNA was detected during oestrogen-induced oviduct differentiation. This suggested that the amount of non-histone chromosomal protein does not play an important role in the sensitivity of chromatin DNA to nuclease digestion. Studies of nuclease resistance of chromatin DNA after dissociation and reconstitution of chromatin proteins and ethanol extraction of chromatin indicate that the histones protect the DNA from nuclease attack. Slow thermal denaturation of nuclease-resistant DNA suggests that the protected DNA sequences may be (A+T)-rich, and the (G+C)-rich satellites present in total chick DNA are sensitive to nuclease.

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 Single-strand nuclease action on heat-denatured spermiogenic chromatin.

1976 ◽  
Vol 24 (8) ◽  
pp. 901-907 ◽  
Author(s):  
J D Hunter ◽  
A J Bodner ◽  
F T Hatch ◽  
R L Balhorn ◽  
J A Mazrimas ◽  
...  
Keyword(s):  
Thermal Denaturation ◽  
Deoxyribonucleic Acid ◽  
Elevated Temperatures ◽  
Chromatin Condensation ◽  
Heat Denaturation ◽  
S1 Nuclease ◽  
Nuclease Digestion ◽  
Aldehyde Groups ◽  
Phosphate Groups

The aim of this study was to compare the sensitivity of chromatin from representative cellular stages of spermiogenesis to a single-strandeded nuclease after heat denaturation. Thermal denaturation of chromatin was assayed in situ in fixed round, elongating and elongated spermatids and in testicular sperm from mice. Production of single-stranded deoxyribonucleic acid (DNA) at elevated temperatures was monitored by digesting chromatin with endonuclease specific for single-stranded DNA (S1 nuclease), staining the residual DNA with gallocyanin-chrome alum (GAC) and measuring the stain content by absorption cytophotometry. Changes in GCA staining were minimal over the temperature range of 22-90 degrees C in each cell type not exposed to nuclease. Staining of undigested cells decreased progressively with advancing cell maturity. Nuclease had no effect on the GCA content of round spermatids below 60 degrees C, but above this temperature there was a progressive decrease in GCA-stainable chromatin. Both round and elongating spermatid stages showed a significantly greater sensitivity to nuclease digestion than did more mature stages; sperm showed no effects of nuclease action below 80 degrees C. Progressive chromatin condensation and a concomitant decrease in the number of available DNA phosphate groups during spermiogenic cell maturation may be responsible for the observed decline in sensitivity to nuclease and decreased GCA staining. Thermal denaturation of round spermatids labeled with 3H-thymidine produced no change in autoradiographic mean nuclear grain counts, indicating no loss of thymidine-labeled DNA from the slides during denaturation. When round spermatids and sperm were hydrolyzed with hot tricholoroacetic acid before staining, both nuclear GCA content and autoradiograph grain count were partially reduced, indicating incomplete DNA removal. Almost complete loss of Feulgen-stainable material occurred in these cells and may be due to depurination and elimination of Feulgren-reactant aldehyde groups.

scholarly journals Chromatinization ofEscherichia coliwith archaeal histones

2019 ◽  
Author(s):  
Maria Rojec ◽  
Antoine Hocher ◽  
Matthias Merkenschlager ◽  
Tobias Warnecke
Keyword(s):  
Nucleosome Occupancy ◽  
Micrococcal Nuclease ◽  
Ancestral State ◽  
E Coli ◽  
Genome Wide ◽  
Micrococcal Nuclease Digestion ◽  
Nuclease Digestion ◽  
Methanothermus Fervidus ◽  
Chromatin Proteins

ABSTRACTNucleosomes restrict DNA accessibility throughout eukaryotic genomes, with repercussions for replication, transcription, and other DNA-templated processes. How this globally restrictive organization emerged from a presumably more open ancestral state remains poorly understood. Here, to better understand the challenges associated with establishing globally restrictive chromatin, we express histones in a naïve bacterial system that has not evolved to deal with nucleosomal structures:Escherichia coli. We find that histone proteins from the archaeonMethanothermus fervidusassemble on theE. colichromosomein vivoand protect DNA from micrococcal nuclease digestion, allowing us to map binding footprints genome-wide. We provide evidence that nucleosome occupancy along theE. coligenome tracks intrinsic sequence preferences but is disturbed by ongoing transcription and replication. Notably, we show that higher nucleosome occupancy at promoters and across gene bodies is associated with lower transcript levels, consistent with local repressive effects. Surprisingly, however, this sudden enforced chromatinization has only mild repercussions for growth, suggesting that histones can become established as ubiquitous chromatin proteins without interfering critically with key DNA-templated processes. Our results have implications for the evolvability of transcriptional ground states and highlight chromatinization by archaeal histones as a potential avenue for controlling genome accessibility in synthetic prokaryotic systems.

scholarly journals Chromatinization of Escherichia coli with archaeal histones

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Maria Rojec ◽  
Antoine Hocher ◽  
Kathryn M Stevens ◽  
Matthias Merkenschlager ◽  
Tobias Warnecke
Keyword(s):  
Escherichia Coli ◽  
Nucleosome Occupancy ◽  
Micrococcal Nuclease ◽  
E Coli ◽  
Genome Wide ◽  
Micrococcal Nuclease Digestion ◽  
Nuclease Digestion ◽  
Methanothermus Fervidus ◽  
Chromatin Proteins

Nucleosomes restrict DNA accessibility throughout eukaryotic genomes, with repercussions for replication, transcription, and other DNA-templated processes. How this globally restrictive organization emerged during evolution remains poorly understood. Here, to better understand the challenges associated with establishing globally restrictive chromatin, we express histones in a naive system that has not evolved to deal with nucleosomal structures: Escherichia coli. We find that histone proteins from the archaeon Methanothermus fervidus assemble on the E. coli chromosome in vivo and protect DNA from micrococcal nuclease digestion, allowing us to map binding footprints genome-wide. We show that higher nucleosome occupancy at promoters is associated with lower transcript levels, consistent with local repressive effects. Surprisingly, however, this sudden enforced chromatinization has only mild repercussions for growth unless cells experience topological stress. Our results suggest that histones can become established as ubiquitous chromatin proteins without interfering critically with key DNA-templated processes.

scholarly journals Differential compartmentalization of plasmid DNA microinjected into Xenopus laevis embryos relates to replication efficiency.

1991 ◽  
Vol 11 (1) ◽  
pp. 299-308 ◽  
Author(s):  
N J Marini ◽  
R M Benbow
Keyword(s):  
Xenopus Laevis ◽  
Plasmid Dna ◽  
Supramolecular Assembly ◽  
Micrococcal Nuclease ◽  
Dna Molecules ◽  
End Joining ◽  
Stage Of Development ◽  
Nuclease Digestion ◽  
Nuclease Resistance ◽  
Xenopus Laevis Embryos

Circular plasmid DNA molecules and linear concatemers formed from the same plasmid exhibit strikingly different fates following microinjection into Xenopus laevis embryos. In this report, we prove quantitatively that only a minority of small, circular DNA molecules were replicated (mean = 14%) from fertilization through the blastula stage of development. At all concentrations tested, very few molecules (approximately 1%) underwent more than one round of DNA synthesis within these multiple cell cycles. In addition, unlike endogenous chromatin, the majority of circular templates became resistant to cleavage by micrococcal nuclease. The extent of nuclease resistance was similar for both replicated and unreplicated templates. Sequestration of circular molecules within a membranous compartment (pseudonucleus), rather than the formation of nucleosomes with abnormal size or spacing, apparently conferred the nuclease resistance. In contrast, most linearly concatenated DNA molecules (derived from end-to-end joining of microinjected monomeric plasmid DNA) underwent at least two rounds of DNA replication during this same period. Linear concatemers also exhibited micrococcal nuclease digestion patterns similar to those seen for endogenous chromatin yet, as judged by their failure to persist in later stages of embryogenesis, were likely to be replicated and maintained extrachromosomally. We propose, therefore, that template size and conformation determine the efficiency of replication of microinjected plasmid DNA by directing DNA to a particular compartment within the cell following injection. Template-dependent compartmentalization may result from differential localization within endogenous nuclei versus extranuclear compartments or from supramolecular assembly processes that depend on template configuration (e.g., association with nuclear matrix or nuclear envelope).

Reconstituted histone-DNA complexes

1978 ◽  
Vol 283 (997) ◽  
pp. 259-268 ◽  
Keyword(s):  
Dna Sequences ◽  
Micrococcal Nuclease ◽  
Nucleotide Pair ◽  
Nucleosome Core ◽  
Nucleosome Core Particles ◽  
Calf Thymus ◽  
Nuclease Digestion ◽  
Preferential Binding ◽  
One Step ◽  
Pair Fragment

Complexes of λdv 21 plasmid DNA with calf thymus histones are reconstituted in the presence and absence of urea, and, in the absence of urea, at 4 and 37 °C. It has been concluded previously on the basis of restriction nuclease digestion of complexes reconstituted in the presence of urea that the preferential binding of individual histones to certain DNA sequences is abolished, by specific histone-histone interactions, when a mixture of the four small histones is used (Steinmetz, Streeck & Zachau 1975 Nature, Lond. 258, 447). This conclusion holds also for the other conditions tested here. An exception is the pair of arginine-rich histones (H3-H4) which appears to bind specifically when reconstituted in the absence of urea by a one step dialysis procedure and non-specifically when the salt is removed by a stepwise dialysis procedure. The subunits which are formed when the four small histones are present simultaneously are very similar to nucleosome core particles according to the fragment patterns obtained with micrococcal nuclease. Protection of 155, 285, 420, 560 etc. nucleotide pairs is observed. When histone H1 is present in addition to the four small histones a 175 nucleotide pair fragment is protected, but no 200 nucleotide pair fragment or multiples thereof were formed under our conditions.

Differential compartmentalization of plasmid DNA microinjected into Xenopus laevis embryos relates to replication efficiency

1991 ◽  
Vol 11 (1) ◽  
pp. 299-308
Author(s):  
N J Marini ◽  
R M Benbow
Keyword(s):  
Xenopus Laevis ◽  
Plasmid Dna ◽  
Supramolecular Assembly ◽  
Micrococcal Nuclease ◽  
Dna Molecules ◽  
End Joining ◽  
Stage Of Development ◽  
Nuclease Digestion ◽  
Nuclease Resistance ◽  
Xenopus Laevis Embryos

Circular plasmid DNA molecules and linear concatemers formed from the same plasmid exhibit strikingly different fates following microinjection into Xenopus laevis embryos. In this report, we prove quantitatively that only a minority of small, circular DNA molecules were replicated (mean = 14%) from fertilization through the blastula stage of development. At all concentrations tested, very few molecules (approximately 1%) underwent more than one round of DNA synthesis within these multiple cell cycles. In addition, unlike endogenous chromatin, the majority of circular templates became resistant to cleavage by micrococcal nuclease. The extent of nuclease resistance was similar for both replicated and unreplicated templates. Sequestration of circular molecules within a membranous compartment (pseudonucleus), rather than the formation of nucleosomes with abnormal size or spacing, apparently conferred the nuclease resistance. In contrast, most linearly concatenated DNA molecules (derived from end-to-end joining of microinjected monomeric plasmid DNA) underwent at least two rounds of DNA replication during this same period. Linear concatemers also exhibited micrococcal nuclease digestion patterns similar to those seen for endogenous chromatin yet, as judged by their failure to persist in later stages of embryogenesis, were likely to be replicated and maintained extrachromosomally. We propose, therefore, that template size and conformation determine the efficiency of replication of microinjected plasmid DNA by directing DNA to a particular compartment within the cell following injection. Template-dependent compartmentalization may result from differential localization within endogenous nuclei versus extranuclear compartments or from supramolecular assembly processes that depend on template configuration (e.g., association with nuclear matrix or nuclear envelope).

scholarly journals Specific regions of beta-globin RNA are resistant to nuclease digestion in RNA-protein complexes in chicken reticulocyte nuclei.

1985 ◽  
Vol 5 (6) ◽  
pp. 1220-1228 ◽  
Author(s):  
J R Patton ◽  
D A Ross ◽  
C B Chae
Keyword(s):  
Protein Complexes ◽  
Mapping Method ◽  
Micrococcal Nuclease ◽  
Beta Globin ◽  
Nuclear Rna ◽  
Nuclease Digestion ◽  
Nuclease Resistance ◽  
Rna Mapping ◽  
Rna Protein Complexes ◽  
Endogenous Nuclease

The interaction between beta-globin RNA and proteins in chicken reticulocyte nuclei was studied by determining the sequence of nuclease-resistant beta-globin RNA. Two types of nuclease-resistant RNAs were isolated for this study: endogenous nuclease-resistant RNA from 50S heterogeneous nuclear RNA-protein complexes and micrococcal nuclease-resistant nuclear RNA from whole nuclei. The nuclease-resistant regions were identified with the use of a RNA mapping method we recently developed (J.R. Patton and C.-B. Chae, J. Biol. Chem. 258:3991-3995, 1983). We found that beta-globin RNA is assembled into heterogeneous nuclear RNA-protein complexes in a specific manner. There are several regions of nuclease resistance in the first and third exons interrupted at regular intervals by sensitive regions. The second exon has only one nuclease-resistant region. The resistant regions range in size from 20 to 50 nucleotides. This organization may reflect a specific mode of assembly for heterogeneous nuclear RNA-protein complexes.

scholarly journals Specific regions of beta-globin RNA are resistant to nuclease digestion in RNA-protein complexes in chicken reticulocyte nuclei

1985 ◽  
Vol 5 (6) ◽  
pp. 1220-1228
Author(s):  
J R Patton ◽  
D A Ross ◽  
C B Chae
Keyword(s):  
Protein Complexes ◽  
Mapping Method ◽  
Micrococcal Nuclease ◽  
Beta Globin ◽  
Nuclear Rna ◽  
Nuclease Digestion ◽  
Nuclease Resistance ◽  
Rna Mapping ◽  
Rna Protein Complexes ◽  
Endogenous Nuclease

The interaction between beta-globin RNA and proteins in chicken reticulocyte nuclei was studied by determining the sequence of nuclease-resistant beta-globin RNA. Two types of nuclease-resistant RNAs were isolated for this study: endogenous nuclease-resistant RNA from 50S heterogeneous nuclear RNA-protein complexes and micrococcal nuclease-resistant nuclear RNA from whole nuclei. The nuclease-resistant regions were identified with the use of a RNA mapping method we recently developed (J.R. Patton and C.-B. Chae, J. Biol. Chem. 258:3991-3995, 1983). We found that beta-globin RNA is assembled into heterogeneous nuclear RNA-protein complexes in a specific manner. There are several regions of nuclease resistance in the first and third exons interrupted at regular intervals by sensitive regions. The second exon has only one nuclease-resistant region. The resistant regions range in size from 20 to 50 nucleotides. This organization may reflect a specific mode of assembly for heterogeneous nuclear RNA-protein complexes.

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