Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Small oligonucleotides from DNA and RNA have been separated according to their base composition by high-performance anion-exchange liquid chromatography on Partisil-10 SAX using triethylammonium acetate buffer as the eluent. Fifteen of the 16 possible deoxydinucleoside monophosphates and all 16 dinucleoside monophosphates have been separated. All pairs of sequence isomers were all resolved. The 15 commercially available deoxydinucleotides were resolved into 13 fractions. A good resolution of deoxytrinucleoside diphosphates isolated from an alkaline phosphatase-Mg2+-activated DNase I digest of calf thymus DNA was achieved by this technique. A large number of sequence isomers could be fully separated. The base sequence of the eluted individual constituents has been determined by their hydrolysis with snake venom and spleen phosphodiesterase followed by high-performance liquid chromatographic analysis of the nucleotides released. The eight trinucleoside diphosphates isolated from an alkaline phosphatase-pancreatic RNase digest of yeast RNA have also been separated according to base composition. Their sequence was determined as above. The described technique is fast and gave very good separation. Most of the sequence isomers could be separated. Moreover, the eluent triethylammonium acetate can easily be removed from column effluents by freeze-drying in order to facilitate subsequent sequence analysis of the eluted compounds. The observed elution orders of the sequence isomers obey certain rules which are discussed in detail.
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PMID:Separation of small DNA and RNA oligonucleotides by high-performance anion-exchange liquid chromatography. 9 13

When rat liver nuclei prepared with polyamines as stabilising cations are digested with DNAase II, release of both inactive chromatin and Mg-soluble, active chromatin is greatly reduced, in comparison to digestion of liver nuclei prepared with Mg2+ as stabilising cation. Chromatin release from polyamine stabilised nuclei is also inhibited relative to Mg-stabilised nuclei following digestion with micrococcal nuclease under two very different cation conditions. Nuclei prepared with polyamines and monovalent ions as stabilising cations exhibit properties intermediate between these two extremes with both nucleases. These effects are due to residual binding of polyamines to chromatin, which is thus maintained in a condensed state, inaccessible to nucleases. Since polyamine binding is not easily reversed, concentrations of polyamines and other cations must be rigidly controlled in experiments on chromatin structure if artefacts are to be avoided. The significance of these findings to the nature and properties of active chromatin within the intact nucleus is considered.
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PMID:Cations and the accessibility of chromatin to nucleases. 46 Dec 8

Initial results of an approach to the isolation of functionally active chromatin are described. Slight digestion of mouse myeloma nuclei at 0 degrees C with micrococcal nuclease, followed by dialysis against near-physiological saline solution containing 1 mM Mg2+, caused release of up to 17% of the nuclear DNA as soluble nucleoproteins. This soluble (S) fraction was relatively depleted in H1 histones and methylated DNA (5-methylcytosine) but highly enriched in RNA, single-stranded DNA, and nonhistone chromosomal proteins, particularly two species of the high mobility group identified as HMG 1 and HMG 2. The S fraction released most rapidly (6--8% of the total DNA) consisted mainly of mono- and small oligonucleosomes. The mononucleosomes appeared normal in terms of sedimentation behavior, DNA length, and content of histones H2A, H2B, H3, and H4, but lacked H1, and instead were associated with approximately stoichiometric amounts of HMG 1 and HMG 2. Studies using isolated, fluorescence-labeled, total mouse HMG proteins indicated that added HMG 1 and HMG 2 do not bind strongly to S-fraction nucleoproteins but that two smaller HMG species (probably HMG 14 and HMG 17) do bind preferentially to S-fraction mono- and dinucleosomes. These results argue against artifactual redistribution of HMG 1 and HMG 2 during this fractionation but suggest caution in interpreting the distribution of smaller HMG proteins after digestion of chromatin. The potential relationship of this soluble fraction to transcriptionally active chromatin is discussed.
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PMID:Chromatin fractionation procedure that yields nucleosomes containing near-stoichiometric amounts of high mobility group nonhistone chromosomal proteins. 47 83

It has been demonstrated by digestion studies with micrococcal nuclease that reconstitution of complexes from DNA and a mixture of the four small calf thymus histones H2A, H2B, H3, and H4 leads to subunits closely spaced in a 137 +/- 7-nucleotide-pair register. Subunits isolated from the reconstituted complex contain nearly equimolar amounts of the four histones and sediment at 11.6S. On DNase I digestion both the reconstituted complex and the separated subunits gave rise to series of single-stranded DNA fragments with a 10-nucleotide periodicity. This indicates that the reconstitution leads to subunits very similar to nucleosome cores. Nucleosome cores closely spaced in a 140-nucleotide-pair register were also obtained upon removal of histone H1 from chromatin by dissociation with 0.63 M NaCl and subsequent ultracentrifugation. In reconstitution experiments with all five histones (including histone H1) our procedure did not lead to tandemly arranged nucleosomes containing about 200 nucleotide pairs of DNA. In the presence of EDTA, DNase II cleaved calf thymus nuclei and chromatin at about 200-nucleotide-pair intervals whereas in the presence of Mg2+ cleavage at intervals of approximately half this size was observed. The change in the nature of the cleavage pattern, however, was no longer found after removal of histone H1 from chromatin. This indicates that H1 influences the accessibility of DNase II cleavage sites in chromatin. This finding is discussed with respect to the influence of histone H1 on chromatin superstructure.
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PMID:Closely spaced nucleosome cores in reconstituted histone.DNA complexes and histone-H1-depleted chromatin. 63 Nov 38

Autodigestion of chromosomal DNA does not take place during the brain nuclei incubation in the presence of Ca2+ and Mg2+. The kinetic of chromatin digestion in brain and liver nuclei by staphylococcal nuclease and the formation of DNP-fragments suggest that subnucleosomes are generated in both cases by digesting of monomer specific sites. This monomer contains 185--200 DNA base pairs and the most starting DNA going throughout it. However the quantity of nuclease-resistant DNA in brain chromatin is more and the rate of subnucleosome formation is less than in liver chromatin. Redigestion of isolated monomers of brain chromatin results in the appearance of subnucleosomes similar to those which are formed under limited digestion of nuclear chromatin. The incubation of brain nuclei in the presence of Ca, Mg-dependent endonuclease prepared from liver nuclei results in the appearance of fragment. DNA-spectra of these fragments are similar to those prepared under digestion of liver chromatin in situ. These data suggest definite resemblance of subunit organization in brain and liver chromatin.
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PMID:[Analysis of brain chromatin subunit organization]. 64 83

Bacteriophage T4-infected Escherichia coli rendered permeable to nucleotides by sucrose plasmolysis exhibited two apparently separate pathways or channels to T4 DNA with respect to the utilization of exogenously supplied substrates. By one pathway, individual labeled ribonucleotides, thymidine (tdR), and 5-hydroxymethyl-dCMP could be incorporated into phage DNA. Incorporation of each of these labeled compounds was not dependent upon the addition of the other deoxyribonucleotide precursors, suggesting that a functioning de novo pathway to deoxyribonucleotides was being monitored. The second pathway or reaction required all four deoxyribonucleoside triphosphates or the deoxyribonucleoside monophosphates together with ATP. However, in this reaction, dTTP was not replaced by TdR. The two pathways were also distinguished on the basis of their apparent Mg2+ requirements and responses to N-ethylmaleimide, micrococcal nuclease, and to hydroxyurea, which is a specific inhibitor of ribonucleoside diphosphate reductase. Separate products were synthesized by the two channels, as shown by density-gradient experiments and velocity sedimentation analysis. Each of the pathways required the products of the T4 DNA synthesis genes. Furthermore, DNA synthesis by each pathway appeared to be coupled to the functioning of several of the phage-induced enzymes involved in deoxyribonucleotide biosynthesis. Both systems represent replicative phage DNA synthesis as determined by CsCl density-gradient analysis. Autoradiographic and other studies provided evidence that both pathways occur in the same cell. Further studies were carried out on the direct role of dCMP hydroxymethylase in T4 DNA replication. Temperature-shift experiments in plasmolyzed cells using a temperature-sensitive mutant furnished strong evidence that this gene product is necessary in DNA replication and is not functioning by allowing preinitiation of DNA before plasmolysis.
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PMID:Replicative bacteriophage DNA synthesis in plasmolyzed T4-infected cells: evidence for two independent pathways to DNA. 78 11

DNA isolated from (a) liver chromatin digested in situ with endogenous Ca2+, Mg2+-dependent endonuclease, (b) prostate chromatin digested in situ with micrococcal nuclease or pancreatic DNAase I, and (c) isolated liver chromatin digested with micrococcal nuclease or pancreatic DNAase I has been analyzed electrophoretically on polyacrylamide gels. The electrophoretic patterns of DNA prepared from chromatin digested in situ with either endogenous endonuclease (liver nuclei) or micrococcal nuclease (prostate nuclei) are virtually identical. Each pattern consists of a series of discrete bands representing multiples of the smallest fragment of DNA 200 +/- 20 base pairs in length. The smallest DNA fragment (monomer) accumulates during prolonged digestion of chromatin in situ until it accounts for nearly all of the DNA on the gel; approx. 20% of the DNA of chromatin is rendered acid soluble during this period. Digestion of liver chromatin in situ in the presence of micrococcal nuclease results initially in the reduction of the size of the monomer from 200 to 170 base pairs of DNA and subsequently results in its conversion to as many as eight smaller fragments. The electrophoretic pattern obtained with DNA prepared from micrococcal nuclease digests of isolated liver chromatin is similar, but not identical, to that obtained with liver chromatin in situ. These preparations are more heterogeneous and contain DNA fragments smaller than 200 base pairs in length. These results suggest that not all of the chromatin isolated from liver nuclei retains its native structure. In contrast to endogenous endonuclease and micrococcal nuclease digests of chromatin, pancreatic DNAase I digests of isolated chromatin and of chromatin in situ consist of an extremely heterogeneous population of DNA fragments which migrates as a continuum on gels. A similar electrophoretic pattern is obtained with purified DNA digested by micrococcal nuclease. The presence of spermine (0.15 mM) and spermidine (0.5 mM) in preparative and incubation buffers decreases the rate of digestion of chromatin by endogenous endonuclease in situ approx. 10-fold, without affecting the size of the resulting DNA fragments. The rates of production of the smallest DNA fragments, monomer, dimer, and trimer, are nearly identical when high molecular weight DNA is present in excess, indicating that all of the chromatin multimers are equally susceptible to endogenous endonuclease. These observations points out the effects of various experimental conditions on the digestion of chromatin by nucleases.
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PMID:Structure of eukaryotic chromatin. Evaluation of periodicity using endogenous and exogenous nucleases. 124 19

The virion host shutoff (vhs) gene of herpes simplex virus encodes a virion polypeptide that induces degradation of host mRNAs at early times and rapid turnover of viral mRNAs throughout infection. To better investigate the vhs function, an in vitro mRNA degradation system was developed, consisting of cytoplasmic extracts from HeLa cells infected with wild-type herpes simplex virus type 1 or a mutant encoding a defective vhs polypeptide. Host and viral mRNAs were degraded rapidly in extracts from cells productively infected with wild-type herpes simplex virus type 1 but not in extracts from mock-infected cells or cells infected with the mutant vhs1. In contrast, 28S rRNA was stable in all three kinds of extract. Accelerated turnover of host mRNAs was also observed in extracts from cells infected with wild-type virus in the presence of dactinomycin, indicating that the activity was induced by a structural component of the infecting virions. The in vitro vhs activity was inactivated by heat or proteinase K digestion but was insensitive to brief treatment of the extracts with micrococcal nuclease. It was not inhibited by placental RNase inhibitor, it exhibited a strong dependence upon added Mg2+, it was active at concentrations of K+ up to 200 mM, and it did not require the components of an energy-generating system. In summary, the in vitro mRNA degradation system appears to accurately reproduce the vhs-mediated decay of host and viral mRNAs and should be useful for studies of the mechanism of vhs action.
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PMID:In vitro mRNA degradation system to study the virion host shutoff function of herpes simplex virus. 184 79

We report on a dynamic aspect of nucleosome positioning, in the absence of transcription-related events, on sea urchin 5 S rDNA. On tandem repeats of nucleosome length DNA of this strongly positioning sequence, histone octamers assemble in one dominant position surrounded by minor positions, ten base-pairs apart and therefore with identical rotational setting of the DNA coil. The existence of this cluster of positions, determined using micrococcal nuclease is confirmed by the results from DNase I footprinting and restriction enzyme analysis. The results from these techniques and from two-dimensional nucleoprotein polyacrylamide gel analysis indicate that the cluster of octamer positions is in dynamic equilibrium, in low ionic conditions, suggesting that the minor positions reflect fluctuations around the major nucleosome site. Histone octamer mobility appears to be temperature dependent and is reversibly inhibited by Mg2+.
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PMID:Mobility of positioned nucleosomes on 5 S rDNA. 206 9

Flow cytometric assessment of DNA digestion with micrococcal nuclease has been performed on isolated HeLa nuclei by determining the relative reduction in stainability with the DNA-specific fluorochrome, propidium iodide. At the nuclease concentrations used, DNA histograms of digested nuclei showed the typical bimodal pattern, when the enzymatic reaction was performed in a medium maintaining chromatin in its native (i.e. condensed) or partially decondensed from. In contrast, when nuclei were digested in a buffer lacking both the mono- and divalent cations K+ and Mg2+, an extensive decrease in fluorescence intensity, with loss of the histogram shape, was observed. In nuclei with native chromatin, DNA stainability decreased as a function of time and enzyme concentration, to reach a lower limit of about 46%, as compared with undigested control samples. Removal of the histone H1 induced a significant increase (approximately by a factor of 2) in the extent of digestion, although only in nuclei with partially decondensed chromatin. These results suggest that the sensitivity of DNA to digestion with micrococcal nuclease can be quantitatively monitored with flow cytometry when appropriate reaction conditions are chosen.
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PMID:Flow cytometric evaluation of DNA digestion with micrococcal nuclease on isolated HeLa nuclei. 224 61


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