Gene/Protein Disease Symptom Drug Enzyme Compound
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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)

A small proportion (0.1-0.5%) of the total DNA content of native Chinese hamster metaphase chromosomes is protected from nucleolytic degradation following the removal of histones by extraction with either 0.2 N HCl or 2 M NaCl, and remains attached to the nonhistone protein core. Acid extraction followed by DNase I digestion leads to small fragments of 10-30 bases. Salt extraction followed by micrococcal nuclease digestion gives approx. 140 b.p. fragments which are undistinguishable in size from nucleosome core DNA fragments. Furthermore, DNase I treatment of salt extracted chromosomes gives DNA fragments containing single strands which are multiples of 10 bases in length, again characteristic of the nucleosome structure. Reassociation kinetics using the 32P-labelled 140 b.p. fragments as probes suggests they are enriched for rapidly reassociating sequences.
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PMID:A partial characterization of DNA fragments protected from nuclease degradation in histone depleted metaphase chromosomes of the Chinese hamster. 49 44

RNA synthesis in the nuclei of liver from newly hatched (4-d-old) chicks is enhanced by intake of food. The enhanced synthesis was ascribed not to an increase in the activity of solubilized DNA-dependent RNA polymerase but to an increase in the initiation of RNA synthesis. Enhanced RNA synthesis in fed chicks was accompanied by greater susceptibility of nuclei to digestion by micrococcal nuclease. Salt extraction abolished the difference in nuclease sensitivity between the fed and fasted groups. Reconstitution with either 0.35 M NaCl extracts or high mobility group (HMG) nonhistone proteins restored digestion susceptibility, but changing the source of extracted proteins did not equalize the extent of digestion in nuclei from livers of fed and fasted chicks. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of HMG proteins revealed the presence of HMGs 1 and 2 as well as a 38,000-dalton protein. The nuclear HMG protein content in fed chicks was greater than that of fasted chicks (121 +/- 17 micrograms/mg DNA vs. 31 +/- 12 micrograms/mg DNA). The electron microscopic examination of hepatocyte nuclei revealed the enlargment of nucleoli and scarcity of aggregated heterochromatin structures in the fed chicks as compared with the fasted chicks. These morphological features are compatible with the high transcriptional activity in liver of fed chicks.
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PMID:Enhancement of RNA synthesis in chick liver by food intake: possible role of high mobility group nonhistone proteins. 241 21

In order to characterize the nuclear thyroid hormone receptors in human tissue, an improved method for isolation of nuclei from cultured human fibroblasts was developed. This method provided nuclei with a protein/DNA ratio of 2.8 and recovery of 42%. The purity of nuclei was verified by phase contrast and electron microscopy, which showed normal appearance of chromatin structure. Nuclear binding assay was performed by incubation of whole cells at 37 degrees C or isolated nuclei at 22 degrees C with L-triiodothyronine (T3). In both cases, an affinity constant (Ka) of 2.0-3.0 X 10(10) M-1 and an average binding capacity of 41 femtomoles of T3/100 micrograms DNA (3,100 binding sites/nucleus) were obtained. During incubation of the nuclei, 13% to 16% of receptors that had an identical Ka was released into the medium. Salt extraction recovered 85% to 90% of the receptors, which had a Ka of 4.5 X 10(10) M-1 and the capacity of 0.13 pmol of T3/mg protein. The Ka fo. L-thyroxine (T4) was seven to 18 times lower than that for T3, but the capacity was the same in isolated nuclei, receptors released during incubation of nuclei, and in salt-extracted receptors. Of the iodothyronines examined, affinity for triiodothyroacetic acid was the highest, followed by L-T3, D-T3, L-T4. Isokinetic glycerol gradient analysis revealed that salt-extracted receptors had a sedimentation coefficient of 3.4 S, whereas micrococcal nuclease digested receptors showed two major (6.0 to 6.5 and 12.5 S), and two minor (17 and 19 S) peaks. These results were virtually identical to those obtained with rat liver nuclei analyzed in parallel studies.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Nuclear thyroid hormone receptors in cultured human fibroblasts: improved method of isolation, partial characterization, and interaction with chromatin. 301 26

When 125I-labeled H6 was incubated with trout testis nuclei under conditions of pH and ionic strength approximating those in vivo, the radioactivity bound nearly quantitatively to the chromatin. Under comparable conditions, most non-nuclear proteins do not bind. Binding was neither tissue- nor species-specific, and HMG-17, a mammalian homolog of H6, behaved similarly to H6. The labeled and the endogenous H6 molecules were equivalent by several criteria: 1) Both were released nearly quantitatively upon treatment of the chromatin with DNase I, whereas neither was released by digestion with micrococcal nuclease, suggesting that the labeled molecules, like those of endogenous H6, were bound primarily to the core particles in transcriptionally competent portions of the genome. 2) Salt extraction curves were similar for both the labeled and unlabeled proteins, although about 15% of the labeled molecules were bound to the chromatin more loosely than those of the endogenous H6. Taken together, these results suggest that chromatin contains specific, well defined sites to which H6 binds. Upon increasing the concentration of H6 in the incubation mixture, progressively greater numbers of low affinity, presumably nonspecific binding sites become occupied. This observation has important implications for studies in which nucleases are employed to probe chromatin structure, since they suggest that H6 molecules released from specific, high affinity sites by the action of the nuclease might rebind more loosely to other regions of the chromatin.
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PMID:Binding of the high mobility group protein, H6, to trout testis chromatin. 644 56

In mealybugs, chromatin condensation is related to both genomic imprinting and sex determination. The paternal chromosomal complement is condensed and genetically inactive in sons but not in daughters. During a study of chromatin organization in Planococcus lilacinus, digestion with micrococcal nuclease showed that 3% to 5% of the male genome is resistant to the enzyme. This Nuclease Resistant Chromatin (NRC) apparently has a nucleosomal organization. Southern hybridization of genomic DNA suggests that NRC sequences are present in both sexes and occur throughout the genome. Cloned NRC DNA is A+T-rich with stretches of adenines similar to those present in mouse alpha-satellite sequences. NRC DNA also contains sequence motifs that are typically associated with the nuclear matrix. Salt-fractionation experiments showed that NRC sequences are matrix associated. These observations are discussed in relation to the unusual cytological features of mealybug chromosomes, including the possible existence of multiple centres of inactivation.
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PMID:A male-specific nuclease-resistant chromatin fraction in the mealybug Planococcus lilacinus. 857 51

We have previously reported the existence of pollen mother cell nuclear protein (PMCP) which appears during microsporogenesis in lily (Lilium spesiosum). It is very similar to mammalian testis specific H1 histone, H1t. In this paper, we describe the PMCP distribution in lily nucleosomes. Isolated nuclei were treated with micrococcal nuclease, and template active and inactive chromatin fractions were prepared. The nucleosome repeat length of pollen mother cells was determined to be 210 base pairs. The majority of the PMCP was found in the template inactive chromatin fraction, similar to other histones. PMCP was contained in the nucleosome monomer, but not in the core particle. However, PMCP was mainly found in the nucleosome dimer when slightly digested. Salt extraction from isolated nuclei indicated that PMCP and H1 histone share similar binding affinities to DNA. Judging from our results, it seems probable that PMCP links two core particles more strongly than H1 histone does. Since it is known that meiotic chromatin includes nick transferase and nuclease activity, one possible role of PMCP is the protection of its own chromatin. Other possible functions of PMCP are also discussed.
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PMID:Binding form of pollen mother cell protein in the nucleosomes of lily. 1666 14

We applied genome-wide profiling to successive salt-extracted fractions of micrococcal nuclease-treated Drosophila chromatin. Chromatin fractions extracted with 80 mM or 150 mM NaCl after digestion contain predominantly mononucleosomes and represent classical "active" chromatin. Profiles of these low-salt soluble fractions display phased nucleosomes over transcriptionally active genes that are locally depleted of histone H3.3 and correspond closely to profiles of histone H2Av (H2A.Z) and RNA polymerase II. This correspondence suggests that transcription can result in loss of H3.3+H2Av nucleosomes and generate low-salt soluble nucleosomes. Nearly quantitative recovery of chromatin is obtained with 600 mM NaCl; however, the remaining insoluble chromatin is enriched in actively transcribed regions. Salt-insoluble chromatin likely represents oligonucleosomes that are attached to large protein complexes. Both low-salt extracted and insoluble chromatin are rich in sequences that correspond to epigenetic regulatory elements genome-wide. The presence of active chromatin at both extremes of salt solubility suggests that these salt fractions capture bound and unbound intermediates in active processes, thus providing a simple, powerful strategy for mapping epigenome dynamics.
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PMID:Genome-wide profiling of salt fractions maps physical properties of chromatin. 1908 6

INTRODUCTIONIn the salt gradient dialysis method, purified core histones are incubated with a DNA template in a buffer containing a high concentration of NaCl. As the salt is slowly dialyzed away, nucleosomes spontaneously assemble on the DNA, and their translational positioning along the DNA is directed by the DNA sequence. In the absence of nucleosome-positioning elements (e.g., 5S rDNA genes), the nucleosomes can adopt a closely packed nonphysiological structure with little space between the nucleosomes. Removal of remaining free histones, as well as templates with closely packed nucleosomes, can be achieved by fractionation over sucrose gradients. The chromatin assembled in these reactions can then be analyzed using micrococcal nuclease digestion. Salt dialysis reconstitutions are easy to perform, but they are time-consuming because of multiple changes of the dialysis buffer. The reconstitution method presented here takes a total of 1.5-2 d to complete.
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PMID:Salt gradient dialysis reconstitution of nucleosomes. 2135 58

Salt fractionation of nucleosomes, a classical method for defining "active" chromatin based on nucleosome solubility, has recently been adapted for genome-scale profiling. This method has several advantages for profiling chromatin dynamics, including general applicability to cell lines and tissues, quantitative recovery of chromatin, base-pair resolution of nucleosomes, and overall simplicity both in concept and execution. This chapter provides detailed protocols for nuclear isolation, chromatin fragmentation by micrococcal nuclease digestion, successive solubilization of chromatin fractions by addition of increasing concentrations of salt, and genome-wide analyses through microarray hybridization and next-generation sequencing.
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PMID:Salt fractionation of nucleosomes for genome-wide profiling. 2218 8