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Query: EC:3.1.31.1 (micrococcal nuclease)
 2,818 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Monomeric nucleosomes from micrococcal nuclease digest of rat liver nuclei have been purified on Sepharose columns in the presence of divalent cations and 0.6 M NaCl. The particles contain histones H2A, H2B, H3, H4, and a complement of nonhistone chromosomal proteins. In 0.6 M NaCl, the nucleosome mixture sedimented at 10 S; however, when the NaCl was removed approximately 30% of the particles aggregated and precipitated, and the remaining soluble fraction sedimented at 11 S. The aggregation phenomenon was divalent cation-dependent and reversible. Characterization of the macromolecular components of the subfractions of nucleosomes showed that the subfractions differed in composition of species of histone H3 as well as of several nonhistone chromosomal proteins but not in the size of the DNA fragment present. The aggregation properties of the isolated nucleosomes showed similarities to the divalent cation-dependent differences in the extent of chromatin condensation in the intact eukaryote nucleus.
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PMID:Fractionation of purified nucleosomes on the basis of aggregation properties. 85 56

Calf thymus chromatin and nuclease-produced chromatin fragments have been examined by thermal denaturation measurements. Native chromatin gave a series of distinct melting transitions at 64, 73,79, and 85 degrees C in 0.25 mM EDTA pH8. Treatments such as dialysis, mechanical shearing, or sulfhydryl oxidation of histone H3 carried out on native chromatin significantly altered the derivative melting profiles by blurring the distinct transitions and shifting the highest melting transition to a lower temperature. Derivative melting profiles for electrophoretically purified chromatin fragments, monomer through hexamer, all resembled that obtained from dialyzed chromatin. These results suggest that higher order structures exist in chromatin that are easily disrupted. Since the products of micrococcal nuclease (EC3.1.4.7) digestion of the altered chromatins did not exhibit any major electrophoretic differences from those obtained from nuclei, than most likely the primary arrangements of histones along the DNA are the main determinant for cleavage sites.
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PMID:A thermal denaturation study of chromatin and nuclease-produced chromatin fragments. 89 May 68

Reactivation of chicken erythrocyte nuclei for DNA replication in Xenopus egg extracts involves two phases of chromatin remodelling: a fast decondensation leading to a small volume increase and chromatin dispersion occurring within a few minutes (termed stage I decondensation), followed by a slower membrane-dependent decondensation and enlargement of up to 40-fold from the initial volume (stage II decondensation). Chromatin decondensation as measured by nuclear swelling and micrococcal nuclease digestion required ATP. We observed a characteristic change in the phosphorylation pattern of erythrocyte proteins upon incubation in egg extract. While histones H5, H2A, and H4 became selectively phosphorylated during decondensation, the phosphorylation of histone H3 and of several nonhistone proteins was prevented. Furthermore, histone H5 was selectively released from erythrocyte nuclei in an energy-dependent reaction. These molecular changes already occurred during stage I decondensation and they persisted during stage II decondensation. DNA replication was confined to nuclei of stage II decondensation which incorporated lamin LIII from the egg extract. These results show that initiation of DNA replication in chicken erythrocytes requires in addition to ATP-dependent chromatin remodelling (stage I), further changes in chromatin structure that correlates with lamin LIII incorporation, and stage II decondensation.
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PMID:Reactivation of DNA replication in erythrocyte nuclei by Xenopus egg extract involves energy-dependent chromatin decondensation and changes in histone phosphorylation. 139 77

The relationship between histone methylation and the transcriptionally active chromatin state was investigated. Immature chicken erythrocytes, which were obtained from the peripheral blood of anemic birds, were incubated with L-[methyl-3H]methionine and cycloheximide. Under these conditions histones H3 and H4 are methylated. The erythrocyte nuclei were incubated with micrococcal nuclease, and the chromatin fragments were fractionated according to their solubility in EDTA and 0.15 M NaCl. Chromatin fractions, which were enriched in transcriptionally active genes, were enriched in methylated histones. Moreover, the acetylated species of histones H3 and H4, which are complexed with active genes (Hebbes, T. R., Thorne, A. W., and Crane-Robinson, C. (1988) EMBO J. 7, 1395-1402), were preferentially methylated. The methylation of these histones was not dependent on ongoing transcription. The distribution of histone H3 methyltransferase activity among the various chromatin regions was also studied. This enzyme activity was greatest for the chromatin fragments that were enriched in active/competent genes. However, our results suggest that histone H3 methyltransferase is bound to the nucleosome. The enzyme, which may be localized in the active gene chromatin domains, may ensure that the histones associated with active genes are methylated. Histone methylation, which has a slow turnover rate, may contribute to the maintenance of the transcriptionally active chromatin state.
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PMID:Distribution of methylated histones and histone methyltransferases in chicken erythrocyte chromatin. 280 20

Chicken erythrocyte nuclei previously incubated separately with two novel mercury compounds (N-chloromercuribenzoyl)-biocytin and bis(p-(chloromercuribenzoyl))-[3H]lysine diamide) were digested with micrococcal nuclease and the digest products fractionated according to their solubility in 0.15 M NaCl and molecular size. The identity and quantitation of the chromatin fractions and proteins containing covalently bound mercury were determined by Western blotting, autoradiography, and scintillation counting. The most highly acetylated species of histone H3 in the 0.15 M NaCl-soluble polynucleosome fraction also contained the highest proportion of bound mercury. This fraction contains hyperacetylated core histones, is depleted in linker histones, and enriched in nonhistone proteins. Histone H3 in the 0.15 M NaCl-soluble mononucleosomes, which are unacetylated and lack linker histones, was 45% less labeled than histone H3 in the 0.15 M NaCl-soluble polynucleosome fraction. In the 0.15 M NaCl-insoluble polynucleosomes, which contain unacetylated histones and molar proportions of linker histones, histone H3 was 63% less labeled. Allowing for the differential abundance of these subfractions in the nucleus, the relative H3 reactivities are 50, 7, and 1 for 0.15 M NaCl-soluble polynucleosomes, mononucleosomes, and 0.15 M NaCl-insoluble polynucleosomes, respectively. Thus a gradation of reactivities exists which correlates with increasing hyperacetylation and linker histone depletion. High mobility group proteins 1 and 2, found in subnucleosome particles in the 0.15 M NaCl-soluble fraction, are extensively mercury-labeled. Distribution of histone acetyltransferase activity among salt- and size-resolved micrococcal nuclease produced fractions was almost 5-fold greater in the 0.15 M NaCl-soluble supernatant than in the 0.15 M NaCl-insoluble pellet. Furthermore, the acetyltransferase activity, which is tightly bound to undigested chromatin, is rapidly released by both micrococcal nuclease and DNase I. For short digestion times the enzyme is associated with the salt-soluble polynucleosomes, but at longer times of digestion the enzyme appears to be free from intact nucleosomes. The enzyme may be localized in the globin domain in erythrocytes and maintains that region in a hyperacetylated state which results in an altered linker histone binding reflected in a change in the reactivity of the usually inaccessible H3 cysteine 110.
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PMID:Histone H3 thiol reactivity and acetyltransferases in chicken erythrocyte nuclei. 317 Jun 3

A procedure for the isolation of transcriptionally active nucleosomes was used to monitor changes in chromatin structure during the activation, repression, and superinduction of the protooncogenes c-fos and c-myc. Nuclei were isolated from murine fibroblasts at successive times after stimulation of quiescent cell cultures with serum or platelet-derived growth factor. The nucleosomes released by a brief micrococcal nuclease digestion were fractionated by HgII-affinity chromatography to separate the unfolded nucleosomes of transcriptionally active genes (in which the sulfhydryl groups of histone H3 are accessible for binding to HgII) from the compactly beaded nucleosomes of transcriptionally inert DNA sequences (in which the H3 sulfhydryl groups are not accessible). The DNA sequence contents of the HgII-bound and unbound nucleosome fractions were compared by slot-blot hybridizations to 32P-labeled cloned probes for c-fos and c-myc. The binding of the c-fos and c-myc nucleosomes to the HgII column accurately reflected both the timing and the degree of their expression, as determined by run-off transcription assays with the isolated nuclei. The superinduction of c-fos and c-myc expression by an inhibitor of protein synthesis (cycloheximide) was reflected in the persistence of the unfolded, transcriptionally active state of their component nucleosomes. These results provide direct evidence that rapid and reversible changes in nucleosome topography accompany the program of oncogene expression, and they suggest a way to monitor aberrant gene activity during malignant transformation.
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PMID:Rapid and reversible changes in nucleosome structure accompany the activation, repression, and superinduction of murine fibroblast protooncogenes c-fos and c-myc. 347 51

The unfolding of nucleosome cores in transcriptionally active chromatin uncovers the sulfhydryl groups of histone H3, making them accessible to SH-reagents. This has suggested that nucleosomes from active genes could be retained selectively on organomercurial/agarose columns. When nucleosomes released from rat liver nuclei by limited digestion with micrococcal nuclease were passed through an Hg affinity column, a run-off fraction of compact, beaded nucleosomes was separated from a retained nucleosome fraction. Although both contained monomer-length DNA and a full complement of core histones, histones in the retained fraction were hyperacetylated. Dot blot hybridizations showed the Hg-bound nucleosome fraction to be enriched in DNA sequences transcribed by hepatocytes (serum albumin and transferrin genes), while a brain-specific gene (preproenkephalin) was not retained, but appeared in the nucleosomes of the run-off fraction. The results are discussed in light of other evidence linking hyperacetylation of histones H3 and H4 to conformational changes at the middle of the nucleosome core.
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PMID:Affinity chromatographic purification of nucleosomes containing transcriptionally active DNA sequences. 365 49

The histone lysine methyltransferases catalyze the transfer of methyl groups from S-adenosylmethionine to specific epsilon-N-lysine residues in the N-terminal regions of histones H3 and H4. These enzymes are located exclusively within the nucleus and are firmly bound to chromatin. The chromosomal bound enzymes do not methylate free or nonspecifically associated histones, while histones H3 and H4 within newly synthesized chromatin are methylated. These enzymes can be solubilized by limited digestion (10-16%) of chromosomal DNA from rapidly proliferating rat brain chromatin with micrococcal nuclease. Histone H3 lysine methyltransferase remained associated with a short DNA fragment throughout purification. Dissociation of the enzyme from the DNA fragment with DNAase digestion resulted in complete loss of enzyme activity; however, when this enzyme remained associated with DNA it was quite stable. Activity of the dissociated enzyme could not be restored upon the addition of sheared calf thymus or Escherichia coli DNA. Histone H3 lysine methyltransferase was found to methylate lysine residues in chromosomal bound or soluble histone H3, while H3 associated with mature nucleosomes was not methylated. The histone H4 lysine methyltransferase which was detectable in the crude nuclease digest was extremely labile, losing all activity upon further purification. We isolated a methyltransferase by DEAE-cellulose chromatography, which would transfer methyl groups to arginine residues in soluble histone H4. However, this enzyme would not methylate nucleosomal or chromosomal bound histone H4, nor were methylated arginine nucleosomal or chromosomal bound histone H4, nor were methylated arginine residues detectable upon incubating intact nuclei or chromatin with S-adenosylmethionine.
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PMID:Specificity of the histone lysine methyltransferases from rat brain chromatin. 393 70

Infection of BHK cells with foot-and-mouth disease virus (FMDV) causes a thorough change in the electrophoretic profile of whole nuclear histones. It consists in the disappearance of histone H3 and the appearance of a new polypeptide (Pi) which migrates between histones H2A and H4 on SDS-polyacrylamide gels. Protein Pi is detected at 2 hr postinfection (pi), the time in which viral RNA synthesis begins to increase, and reaches equimolecular amounts with the remaining core histones 1 hr later, when the disappearance of histone H3 is almost complete. Labeling of cells prior to infection demonstrates that Pi is not a novo product but the result of a viral-induced processing of a host precursor synthetized beforehand. Protein Pi comigrates with histone H2A/B in acetic acid/urea polyacrylamide gels and it shares common major peptides with histone H3 under controlled proteolysis with protease V8 or trypsin. The mononucleosomal and nucleosomal DNA pattern analysis after micrococcal nuclease treatment of nuclei from infected and mock-infected cells did not show any significant differences even though after 3 hr (p.i.), protein Pi replaces histone H3 in the nucleosomal structure. It was concluded that FMDV infection is responsible for a specific modification in the nucleus of infected cells which leads, after 3 hr (p.i.), to a complete histone H3 protein Pi transition in the nucleosomes.
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PMID:Histone H3 modification in BHK cells infected with foot-and-mouth disease virus. 633 Sep 87

A series of mono- and dinucleosomal DNAs characterized by an about ten-base periodicity in the size were revealed in the micrococcal nuclease digests of Drosophila chromatin which have 180 +/- 5 base pair (bp) nucleosomal repeat. 20, 30, and 40 bp spacers were found to be predominant in chromatin by trimming DNA in dinucleosomes to the core position. Among several identified mononucleosomes (MN), MN170, MN180 and MN190 were isolated from different sources (the figures indicate the DNA length in bp). The presence of the 10, 20, and 30 bp long spacers was shown in these mononucleosomes by crosslinking experiments. The interaction of histone H3 with the spacer in the Drosophila MN180 particle was also shown by the crosslinking /5/. We conclude from these results that the 10 n bp long intercore DNA (n = 2, 3 and 4) is organized by histone H3, in particular, and together with the core DNA forms a continuous superhelix. Taken together, these data suggest that Drosophila chromatin consists of the regularly aligned and tightly packed MN180, as a repeating unit, containing 10 and 20 bp spacers at the ends of 180 bp DNA. Within the asymmetric and randomly oriented in chromatin MN180, the cores occupy two alternative positions spaced by 10 bp.
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PMID:Alignment of nucleosomes along DNA and organization of spacer DNA in Drosophila chromatin. 681 25


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