Reconstruction of complexes of histone and superhelical nuclear DNA

1981 ◽  
Vol 50 (1) ◽  
pp. 209-224
Author(s):  
J.M. Levin ◽  
P.R. Cook
Keyword(s):  
Nuclear Dna ◽  
Micrococcal Nuclease ◽  
Base Pairs ◽  
The Core ◽  
Ionic Detergent ◽  
Nuclear Rna ◽  
Core Histones ◽  
Non Destructive ◽  
Rna And Dna ◽  
General Method

When HeLa cells are lysed in solutions containing a non-ionic detergent and 2 M-NaCl, structures are released that retain many of the morphological features of nuclei. These nucleoids contain all the nuclear RNA and DNA but few of the proteins characteristic of chromatin. Their DNA is supercoiled and so intact. Using a simple and rapid procedure we have reconstructed nucleohistone complexes from nucleoids and the ‘core’ histones without breaking the DNA. We have probed the integrity and structure of the reconstructed complexes using a non-destructive fluorometric approach, which provides a general method for detecting agents that bind to DNA and alter its supercoiling. The superhelical status of the DNA in the reconstructed complexes is indistinguishable from that found in control nucleoids containing core histones. Experiments with micrococcal nuclease confirm that the DNA in the reconstructed complexes is organized into nucleosome-like structures. These, however, are spaced 145 base-pairs apart and not 200 base-pairs apart as is found in native chromatin.

Conformational changes induced by salt in complexes of histones and superhelical nuclear DNA

1981 ◽  
Vol 50 (1) ◽  
pp. 199-208
Author(s):  
J.M. Levin ◽  
P.R. Cook
Keyword(s):  
Free Energy ◽  
Rna Polymerase ◽  
Conformational Changes ◽  
Salt Concentration ◽  
Nuclear Dna ◽  
The Core ◽  
Ionic Detergent ◽  
Core Histones ◽  
Intercalating Dye ◽  
Negative Supercoiling

When HeLa cells are lysed in solutions containing a non-ionic detergent and 0.75 M-NaCl, structures are released that retain many of the morphological features of nuclei. These nucleoids contain all the nuclear DNA, RNA and the ‘core’ histones, but few other proteins characteristic of chromatin. Their DNA is intact. The core histones dissociate on raising the salt concentration. We have probed the structure of nucleoid-histone complexes using the intercalating dye, ethidium, or the RNA polymerase of Escherichia coli. Both have a higher affinity for superhelical DNA than they do for relaxed DNA. The binding of ethidium is measured fluorometrically, and using this probe we find that the DNA of nucleoids containing all the core histones behaves as if it were supercoiled slightly positively. As the salt concentration is increased, free energy characteristic of negative supercoiling appears between 0.92 M and 0.95 M-NaCl. This transition, which is reversible in the presence of the arginine-rich histones, occurs without dissociation of these histones from the DNA and so must reflect a conformational change in the complex. In contrast to the results with ethidium, we find that RNA polymerase can detect the presence of some negative free energy of supercoiling in nucleoids containing the core histones. The transformations of the free energy that can assist the binding of ethidium and RNA polymerase are discussed.

scholarly journals Heparin increases chromatin accessibility by binding the trypsin-sensitive basic residues in histones

1992 ◽  
Vol 288 (3) ◽  
pp. 953-958 ◽  
Author(s):  
B Villeponteau
Keyword(s):  
Chromatin Structure ◽  
Gradient Analysis ◽  
Chromatin Accessibility ◽  
Micrococcal Nuclease ◽  
The Core ◽  
Core Histones ◽  
Arginine Residues ◽  
Rna And Dna Synthesis ◽  
Rna And Dna ◽  
Regulatory Significance

Recent evidence indicates that chromatin accessibility to transcription factors is of regulatory significance. The polyanion heparin is known to increase chromatin accessibility to DNAase I and to stimulate both RNA and DNA synthesis. In the present study, chromatin structure and its modification by polyanions were examined by using trypsin and micrococcal nuclease as probes. Both heparin and poly(glutamic acid) were found to be equivalent to trypsin digestion of histones in their ability to increase nuclease accessibility in chromatin. However, no increase in nuclease accessibility was observed when trypsin-digested chromatin was further treated with heparin, indicating that polyanions and trypsin are not additive in their effects on chromatin accessibility. Moreover, sucrose-gradient analysis demonstrated that heparin binds tightly to intact nucleosomes but not to trypsin-digested nucleosomes. These data suggest that polyanions interact predominantly with the trypsin-sensitive lysine and arginine residues in histone H1 and the N-terminal segments of the core histones. The possible relevance of these results to the chromatin structure of actively transcribed regions is discussed.

Identification of nonhistone chromatin proteins in chromatin subunits (or mononucleosomes) devoid of histone H1

1979 ◽  
Vol 57 (6) ◽  
pp. 666-672 ◽  
Author(s):  
P. K. Chan ◽  
C. C. Liew
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sucrose Density Gradient ◽  
Polyacrylamide Gel ◽  
Micrococcal Nuclease ◽  
Dna Fragments ◽  
Base Pairs ◽  
The Core ◽  
Liver Chromatin ◽  
Chromatin Proteins

Rat liver chromatin was digested by micrococcal nuclease. Chromatin subunits (or mononucleosomes) were isolated by sucrose density gradient and subsequently fractionated by 6% polyacrylamide gel electrophoresis into two major components. One component (MN1) of the mononucleosomes had a higher mobility, contained histones H2A, H2B, H3, H4, and shorter DNA fragments (140 base pairs) while the other (MN2) contained all five histones and longer DNA fragments (180 base pairs). Both submononucleosomes (MN1 and MN2) were found to contain nonhistone chromatin proteins (NHCP). By electrophoresis in 15% sodium dodecyl sulfate – polyacrylamide gel, 9 and 11 major fractions of NHCP were identified in the submononucleosomes MN1 and MN2, respectively. It was also observed that treatment of mononucleosomes with 0.6 M NaCl removes most of these NHCP and histone H1 except for two major NHCP which remain in the core particles.

Adenovirus chromatin structure at different stages of infection

1981 ◽  
Vol 1 (12) ◽  
pp. 1094-1105
Author(s):  
E Daniell ◽  
D E Groff ◽  
M J Fedor
Keyword(s):  
Dna Synthesis ◽  
Nuclear Dna ◽  
Protein Complexes ◽  
Fragment Size ◽  
Human Adenovirus ◽  
Micrococcal Nuclease ◽  
Base Pairs ◽  
Viral Dna ◽  
Infected Cells ◽  
Cellular Dna

We investigated the structure of adenovirus deoxyribonucleic acid (DNA)-protein complexes in nuclei of infected cells by using micrococcal nuclease. Parental (infecting) DNA was digested into multimers which had a unit fragment size that was indistinguishable from the size of the nucleosomal repeat of cellular chromatin. This pattern was maintained in parenteral DNA throughout infection. Similar repeating units were detected in hamster cells that were nonpermissive for human adenovirus and in cells pretreated with n-butyrate. Late in infection, the pattern of digestion of viral DNA was determined by two different experimental approaches. Nuclear DNA was electrophoresed, blotted, and hybridized with labeled viral sequences; in this procedure all virus-specific DNA was detected. This technique revealed a diffuse protected band of viral DNA that was smaller than 160 base pairs, but no discrete multimers. All regions of the genome were represented in the protected DNA. To examine the nuclease protection of newly replicated viral DNA, infected cells were labeled with [3H]thymidine after blocking of cellular DNA synthesis but not viral DNA synthesis. With this procedure we identified a repeating unit which was distinctly different from the cellular nucleosomal repeat. We found broad bands with midpoints at 200, 400, and 600 base pairs, as well as the limit digest material revealed by blotting. High-resolution acrylamide gel electrophoresis revealed that the viral species comprised a series of closely spaced bands ranging in size from less than 30 to 250 base pairs.

Neutron diffraction from crystals of nucleosome core particles

1980 ◽  
Vol 290 (1043) ◽  
pp. 635-638 ◽  
Keyword(s):  
Neutron Diffraction ◽  
Molecular Mass ◽  
Micrococcal Nuclease ◽  
Core Particle ◽  
Base Pairs ◽  
Histone Protein ◽  
Nucleosome Core ◽  
The Core ◽  
Nucleosome Core Particles ◽  
Protein Molecules

The nucleosome is the basic repeating unit of chromatin (see review by Kornberg (1977)). It is a complex of histone protein molecules with a length of DNA, which digestion studies with the enzyme micrococcal nuclease have shown to be often about 200 base pairs in length but with quite wide variations between different cell species. With sufficient digestion, however, a ‘core’ particle is produced, which for all species so far investigated contains close to 145 base pairs of DNA associated with an octamer of pairs of the histones H3, H4, H2A and H2B. The molecular mass of the core particle is about 200000, roughly equally divided between DNA and protein.

scholarly journals Localization of testis-variant histones in rat testis chromatin

1982 ◽  
Vol 205 (1) ◽  
pp. 15-21 ◽  
Author(s):  
M R S Rao ◽  
B J Rao ◽  
J Ganguly
Keyword(s):  
Sedimentation Coefficient ◽  
Soluble Proteins ◽  
Micrococcal Nuclease ◽  
Base Pairs ◽  
Rat Testis ◽  
Nucleosome Core ◽  
The Core ◽  
Nucleosome Core Particles ◽  
Acid Solubility ◽  
Core Particles

Nucleosome core particles and oligonucleosomes were isolated by digesting rat testis nuclei with micrococcal nuclease to 20% acid-solubility, followed by fractionation of the digest on a Bio-Gel A-5m column. The core particles thus isolated were characterized on the basis of their DNA length of 151 +/- 5 base-pairs and sedimentation coefficient of 11.4S. Analysis of the acid-soluble proteins of the core particles indicated that histones TH2B and X2 are constituents of the core particles, in addition to the somatic histones H2A, H2B, H3 and H4. The acid-soluble proteins of the oligonucleosomes comprised all the histones, including both the somatic (H1, H2A, H2B, H3, H4 and X2) and the testis-specific ones (TH1 and TH2B). It was also observed that histones TH1 and H1 are absent from the core particles and were readily extracted from the chromatin by 0.6 M-NaCl, which indicated that both of them are bound to the linker DNA.

scholarly journals Adenovirus chromatin structure at different stages of infection.

1981 ◽  
Vol 1 (12) ◽  
pp. 1094-1105 ◽  
Author(s):  
E Daniell ◽  
D E Groff ◽  
M J Fedor
Keyword(s):  
Dna Synthesis ◽  
Nuclear Dna ◽  
Protein Complexes ◽  
Fragment Size ◽  
Human Adenovirus ◽  
Micrococcal Nuclease ◽  
Base Pairs ◽  
Viral Dna ◽  
Infected Cells ◽  
Cellular Dna

We investigated the structure of adenovirus deoxyribonucleic acid (DNA)-protein complexes in nuclei of infected cells by using micrococcal nuclease. Parental (infecting) DNA was digested into multimers which had a unit fragment size that was indistinguishable from the size of the nucleosomal repeat of cellular chromatin. This pattern was maintained in parenteral DNA throughout infection. Similar repeating units were detected in hamster cells that were nonpermissive for human adenovirus and in cells pretreated with n-butyrate. Late in infection, the pattern of digestion of viral DNA was determined by two different experimental approaches. Nuclear DNA was electrophoresed, blotted, and hybridized with labeled viral sequences; in this procedure all virus-specific DNA was detected. This technique revealed a diffuse protected band of viral DNA that was smaller than 160 base pairs, but no discrete multimers. All regions of the genome were represented in the protected DNA. To examine the nuclease protection of newly replicated viral DNA, infected cells were labeled with [3H]thymidine after blocking of cellular DNA synthesis but not viral DNA synthesis. With this procedure we identified a repeating unit which was distinctly different from the cellular nucleosomal repeat. We found broad bands with midpoints at 200, 400, and 600 base pairs, as well as the limit digest material revealed by blotting. High-resolution acrylamide gel electrophoresis revealed that the viral species comprised a series of closely spaced bands ranging in size from less than 30 to 250 base pairs.

scholarly journals Localization of specific rDNA spacer sequences to the mouse L-cell nucleolar matrix.

1985 ◽  
Vol 5 (6) ◽  
pp. 1287-1294 ◽  
Author(s):  
R I Bolla ◽  
D C Braaten ◽  
Y Shiomi ◽  
M B Hebert ◽  
D Schlessinger
Keyword(s):  
Nuclear Dna ◽  
Micrococcal Nuclease ◽  
Rna Sequences ◽  
S1 Nuclease ◽  
The Core ◽  
Rdna Sequences ◽  
L Cell ◽  
Cytoplasmic Rna ◽  
Rrna Species ◽  
Tight Association

Mouse L-cell nucleoli were isolated from sonicated nuclei by centrifugation and extensively treated with pancreatic DNase or micrococcal nuclease to obtain "core nucleoli." Core nucleoli still contained the precursors to rRNA and about 1% of the total nuclear DNA, which remained tightly bound even after the removal of some chromatin proteins with 2 M NaCl. The core nucleolar DNA electrophoresed in a series of discrete bands, 20 to about 200 base pairs in length. Hybridization tests with specific DNA probes showed that the DNA was devoid of sequences complementary to mouse satellite, mouse Alu-like, and 5S RNA sequences. It also lacked sequences coding for cytoplasmic rRNA species, since it did not hybridize to the 18S to 28S portion of rDNA in Northern blot analyses and none of it was protected by hybridization to a 100-fold excess of total cytoplasmic RNA in S1 nuclease assays. However, the core nucleolar DNA did hybridize to nontranscribed and external transcribed spacer rDNA sequences. We infer that specific portions of rDNA are protected from DNase action by a tight association with nucleolar structural proteins.

Localization of specific rDNA spacer sequences to the mouse L-cell nucleolar matrix

1985 ◽  
Vol 5 (6) ◽  
pp. 1287-1294 ◽  
Author(s):  
R I Bolla ◽  
D C Braaten ◽  
Y Shiomi ◽  
M B Hebert ◽  
D Schlessinger
Keyword(s):  
Nuclear Dna ◽  
Micrococcal Nuclease ◽  
Rna Sequences ◽  
S1 Nuclease ◽  
The Core ◽  
Rdna Sequences ◽  
L Cell ◽  
Cytoplasmic Rna ◽  
Rrna Species ◽  
Tight Association

Mouse L-cell nucleoli were isolated from sonicated nuclei by centrifugation and extensively treated with pancreatic DNase or micrococcal nuclease to obtain "core nucleoli." Core nucleoli still contained the precursors to rRNA and about 1% of the total nuclear DNA, which remained tightly bound even after the removal of some chromatin proteins with 2 M NaCl. The core nucleolar DNA electrophoresed in a series of discrete bands, 20 to about 200 base pairs in length. Hybridization tests with specific DNA probes showed that the DNA was devoid of sequences complementary to mouse satellite, mouse Alu-like, and 5S RNA sequences. It also lacked sequences coding for cytoplasmic rRNA species, since it did not hybridize to the 18S to 28S portion of rDNA in Northern blot analyses and none of it was protected by hybridization to a 100-fold excess of total cytoplasmic RNA in S1 nuclease assays. However, the core nucleolar DNA did hybridize to nontranscribed and external transcribed spacer rDNA sequences. We infer that specific portions of rDNA are protected from DNase action by a tight association with nucleolar structural proteins.

scholarly journals LENGTH MUTATIONS IN HUMAN MITOCHONDRIAL DNA

Genetics ◽  
1983 ◽  
Vol 104 (4) ◽  
pp. 699-711
Author(s):  
R L Cann ◽  
A C Wilson
Keyword(s):  
Nuclear Dna ◽  
Average Rate ◽  
Rapid Evolution ◽  
Human Species ◽  
Base Pairs ◽  
Human Mitochondrial Dna ◽  
Noncoding Sequences ◽  
Mitochondrial Dnas ◽  
D Loop ◽  
Length Mutations

ABSTRACT By high-resolution, restriction mapping of mitochondrial DNAs purified from 112 human individuals, we have identified 14 length variants caused by small additions and deletions (from about 6 to 14 base pairs in length). Three of the 14 length differences are due to mutations at two locations within the D loop, whereas the remaining 11 occur at seven sites that are probably within other noncoding sequences and at junctions between coding sequences. In five of the nine regions of length polymorphism, there is a sequence of five cytosines in a row, this sequence being comparatively rare in coding DNA. Phylogenetic analysis indicates that, in most of the polymorphic regions, a given length mutation has arisen several times independently in different human lineages. The average rate at which length mutations have been arising and surviving in the human species is estimated to be many times higher for noncoding mtDNA than for noncoding nuclear DNA. The mystery of why vertebrate mtDNA is more prone than nuclear DNA to evolve by point mutation is now compounded by the discovery of a similar bias toward rapid evolution by length mutation.

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