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)

Mammalian intestinal apolipoprotein B (apoB) messenger RNA (mRNA) undergoes posttranscriptional editing, changing codon 2153 from CAA in apoB100 mRNA to an in-frame translational stop codon (UAA) in apoB48 mRNA. By contrast, chicken intestinal apoB cDNA contains a CAA codon at the corresponding site and apoB mRNA from chicken enterocytes, kidney, and liver is unedited. The cDNA sequence of chicken apoB spanning the edited base is divergent from mammalian apoB cDNA sequence, with 70% homology over the conserved 29-nucleotide sequence (6662-6690) flanking codon 2153. Efficient in vitro editing of both human and rat, but not chicken, synthetic apoB RNA was achieved using rat enterocyte S-100 extracts. By contrast, chicken enterocyte S-100 extracts failed to edit chicken, rat, or human synthetic apoB RNA. Mixing experiments, however, revealed that chicken enterocyte S-100 extracts enhance the in vitro editing activity of rat, pig, and human enterocyte S-100 extracts upon homologous RNAs. The editing enhancement activity of chicken enterocyte S-100 extracts is tissue-specific, heat-sensitive, substrate-saturable, and sensitive to proteinase K, but resistant to micrococcal nuclease. The activity was partially purified by Q-Sepharose chromatography and has an average molecular mass of 49 kDa when analyzed by gel filtration chromatography. We conclude that the evolutionary adaptation of intestinal apoB mRNA editing requires both a requisite RNA motif and tissue-specific factors which mediate the site-specific modification.
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PMID:Evolution of intestinal apolipoprotein B mRNA editing. Chicken apolipoprotein B mRNA is not edited, but chicken enterocytes contain in vitro editing enhancement factor(s). 140 Apr 37

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

Apolipoprotein B (apoB) circulates in human plasma as two isoforms, apoB-100 (512 kDa) and apoB-48 (242 kDa). ApoB-48 is generated by a novel RNA editing mechanism which post-transcriptionally modifies apoB mRNA in the intestine by converting cytidine at nucleotide 6666 to uridine. This converts codon 2153 from glutamine (CAA) to a premature stop codon (UAA). To characterize the activity which edits apoB mRNA, extracts were prepared from enterocytes isolated from baboon small intestine. These extracts efficiently edit synthetic apoB RNA in vitro. Editing was detected by primer extension, and the specificity of the reaction was confirmed by DNA sequencing. Extracts prepared from other baboon tissues did not edit apoB RNA in vitro. The editing activity was partially purified by chromatography of the enterocyte extracts on DEAE-cellulose. The activity is sensitive to proteinase K but resistant to micrococcal nuclease and has an average molecular mass of 125 kDa when analyzed by gel filtration chromatography.
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PMID:Characterization of the apolipoprotein B mRNA editing activity in enterocyte extracts. 225

Z chromatin-chromium (Cr) complex, prepared from mouse liver chromatin and CrCl3, showed a significantly enhanced template activity for in vitro RNA synthesis. Digestion experiments with this complex using micrococcal nuclease and DNase I suggested that Cr(III) preferentially binds to linker regions rather than core regions of chromatin. Further, it was found that Cr(III) binds to DNA and nonhistone proteins (NHP), but hardly to histones. Moreover, the template activity of an NHP-Cr complex, when added to a DNA-histones complex, was inhibited remarkably. The template activity of the chromatin-Cr complex was not significantly altered by proteinase K digestion. Furthermore, experiments using rifampicin and [gamma-32P]guanosine 5'-triphosphate (GTP) demonstrated an increase in the number of initiation sites in the chromatin-Cr complex. These results suggest that, in this in vitro system, Cr(III) preferentially binds to DNA in chromatin and causes an increase in the number of initiation sites, thus enhancing RNA synthesis.
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PMID:Enhancement of in vitro ribonucleic acid synthesis on chromium(III)-bound chromatin. 242 31

A differential Giemsa staining between sister chromatids was obtained by treating chromosomes replicated twice in medium containing 5-bromodeoxyuridine (BrdU) with Hoechst 33258 plus black light at 55 degrees C (HB pretreatment) and deoxyribonuclease (DNase) I, II, or micrococcal nuclease. In this staining pattern the BrdU bifilarly substituted chromatids were darkly and the unifilarly substituted chromatids lightly stained. This staining pattern was obtained only by staining the HB-DNase I-treated chromosomes with Giemsa and methylene blue, not by several other dyes tested. Relatively more DNA labelling was removed from the non-BrdU-substituted than the BrdU-substituted chromosomes, when the HB-pretreated chromosomes were digested with DNase I. But the protein labelling was not removed appreciably in the same treatment. The differential DNase I sensitivity between the non-BrdU-substituted and BrdU-substituted chromosomes disappeared when the HB-pretreated chromosomes were incubated with proteinase K before The DNase I digestion. Moreover, no differential DNase I sensitivity was found between the HB-pretreated isolated DNA containing and not containing BrdU. We propose that during the HB pretreatment, more DNA-protein cross-linkings are induced in BrdU bifilarly substituted than the unifilarly substituted chromatids. This structure protects the chromosomal DNA against the DNase I digestion. Thus, a reverse differential Giemsa staining between sister chromatids is obtained by the HB-DNase I treatment.
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PMID:Reverse differential staining of sister chromatids induced by Hoechst plus black light and endonuclease. 257 33

35 S rRNA is the major intracellular precursor to 18, 5.8, and 25 S rRNAs in Saccharomyces cerevisiae. In this report, we show that the 3' termini of 35 S rRNA as well as 25 S rRNA are generated by post-transcriptional RNA processing rather than transcription termination. Using a partially purified yeast whole cell extract, efficient site-specific cleavage of a synthetic rRNA precursor was demonstrated in vitro. The 3' termini of the processed precursor were established by S1 nuclease protection analysis. RNA molecules containing the mature 3' termini of 35 and 25 S rRNA as well as molecules with a 3' terminus located 12 nucleotides beyond the 3' terminus of 25 S rRNA were the major products of the in vitro processing reaction. Processing activity required Mg2+ but was independent of ribonucleotides. Pretreatment of the yeast whole cell extract with proteinase K abolished processing activity, whereas micrococcal nuclease pretreatment of the extract had no effect on processing activity. These results show that RNA polymerase I-dependent transcription of yeast ribosomal cistrons continues beyond sequences that encode the 3' terminus of 35 S rRNA into the spacer region that separates 35 S rRNA transcription units.
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PMID:In vitro RNA processing generates mature 3' termini of yeast 35 and 25 S ribosomal RNAs. 264 84

Hen oviduct N alpha-acetyltransferase was clarified to have a nucleic acid as an existing constituent by the following three results: (i) an ultraviolet absorption spectrum of the purified N alpha-acetyltransferase free of S-acetyl coenzyme A (Ac-CoA) had an absorption maximum at 260 nm. (ii) A nucleic acid band stained with ethidium bromide was detected on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. (iii) An ethidium bromide band co-migrated with a fluorescent band of the protein treated with N-(7-dimethylamino-4-methylcoumarinyl)maleimide, a reagent specific for thiol groups, on polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate. N alpha-Acetyltransferase lost its activity partially or completely by digestion with bovine pancreatic RNase A, Staphylococcus aureus nuclease, or proteinase K, showing that both the nucleic acid and the protein subunit were necessary for the enzyme activity. The nucleic acid component was identified as an RNA but not a DNA because the RNase T2 digest of the nucleic acid was composed of four 3'-ribomononucleotides and completely separated from 3'- and 5'-deoxyribomononucleotides on TLC. The chain length of the nucleic acid of 260 nucleotides estimated by formamide-polyacrylamide gel electrophoresis was calculated to be about 83,000 of the molecular weight. The contents of RNA (35.0%) and protein (65.0%) in N alpha-acetyltransferase determined on weight basis corresponded reasonably well to the contents of RNA (34.4%) and protein (65.6%) calculated based on the assumption that N alpha-acetyltransferase consisted of one molecule of 7 S RNA (Mr 83,000) and two identical Mr 79,000 protein subunits. The total molecular weight (241,000) of the holoenzyme calculated based on the above result was identical to the molecular weight (240,000) of N alpha-acetyltransferase estimated by Sepharose 6B gel filtration.
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PMID:Hen oviduct N alpha-acetyltransferase is a ribonucleoprotein having 7 S RNA. 275 10

Incubation of extracts of Cp-1-infected Streptococcus pneumoniae with [alpha-32P]dATP produced a labeled treatment with micrococcal nuclease and sensitive to treatment with proteinase K. Incubation of the 32P-labeled protein with 5 M piperidine for 4 h at 50 degrees C released 5'-dAMP, indicating that a covalent complex between the terminal protein and 5'-dAMP was formed in vitro. When the four deoxynucleoside triphosphates were included in the reaction mixture, a labeled complex of slower electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels than the terminal protein-dAMP complex was also found, indicating that the Cp-1 terminal protein-dAMP complex can be elongated and, therefore, that it is an initiation complex. Treatment of the 32P-labeled terminal protein-dAMP complex with 5.8 M HCl at 110 degrees C for 2 h yielded phosphothreonine. These results, together with the resistance of the terminal protein-DNA linkage to hydroxylamine, suggest that the Cp-1 terminal protein is covalently linked to the DNA through a phosphoester bond between L-threonine and 5'-dAMP, namely, a O-5'-deoxyadenylyl-L-threonine bond.
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PMID:Formation of a covalent complex between the terminal protein of pneumococcal bacteriophage Cp-1 and 5'-dAMP. 308 36

Binding of exogenous DNA to the nuclear scaffold was investigated using a plasmid DNA (pBR322, EcoRI site deleted) of various topological forms and nuclear subfractions with different levels of nuclear DNA depletion. When supercoiled DNA was incubated with histone-depleted nuclei (nuclear halo), a dose-dependent binding of the DNA occurred, whereas no binding was observed with relaxed and linear forms of DNA. The bound DNA was released upon linearization with BamHI or digestion of the scaffolding structure with proteinase K. Extensive digestion of the halo with micrococcal nuclease generated additional sites which bind both relaxed and linear DNA. In the presence of a large excess of calf thymus DNA, these sites were effectively blocked and the specificity to supercoiled DNA was restored. The binding of all forms of DNA was abolished by heat-denatured DNA. There was no detectable change in linking number of the scaffold-associated supercoils. Competitive binding was observed between supercoiled DNAs with unrelated sequences, indicating that no specific nucleotide sequence is required for the binding. RNA was found to be a weak competitor. A DNA binding assay performed on electrophoretic blots of solubilized nuclear scaffold revealed a protein component with apparent molecular weight of 120,000 which retained selective binding to supercoils. These results suggest that the nuclear scaffold possesses DNA-binding sites for torsionally strained domains of chromatin and that an integral protein factor is involved in the binding. Implications of the findings are discussed in connection with proposed functions of the nuclear scaffold and topoisomerase II.
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PMID:The nuclear scaffold exhibits DNA-binding sites selective for supercoiled DNA. 336 76

Nucleosomes composed of 195 base pairs of DNA associated with histones H2A, H2B, H3, and H4 purified from chicken erythrocyte nuclei were used to elicit antibodies in rabbits. Specific serological reaction between the antisera and the nucleosomes is demonstrated by immunodiffusion, immunofluorescence, microcomplement fixation, solid-phase radioimmunoassay, immunosedimentation, and polyacrylamide gel electrophoresis of 5'-32P end-labeled nucleosomes. The antisera did not react with DNA extracted from these nucleosomes, core histones, or the cross-linked histone octamer from chicken erythocytes, calf thymus total histones, or chromosomal proteins HMG-1 or HMG-17. Nucleosome antigenicity was not affected by redigestion with micrococcal nuclease. Digestion with DNase I brought about 50% loss of reactivity while digestion with trypsin or proteinase K resulted in total loss of activity. The antisera reacted strongly with trimer, dimer, and monomer nucleosomes as well as with the core particle (145 base pairs of DNA) and subnucleosome (greater than 145 base pairs) obtained from chicken. It reacted less well with nucleosomes obtained from HeLa cells and was almost totally devoid of activity against chromatin particles obtained from rat liver or wheat germ. Experiments employing the technique of transferring proteins from a polyacrylamide gel to diazobenzyloxymethyl paper and visualization of antigens by autoradiography excluded the possibility that the serum contains antibodies against tissue-specific antigens which are found in small amounts but are very immunogenic. It is concluded that most of the anitbodies in the sera are directed against nucleoprotein antigenic determinants composed of the N-terminal portion of the histones and segments of DNA. Antibody binding is dependent on contact between the histone and DNA segments and is independent of the integrity of the entire nucleosome. Thus, certain histone DNA contacts remain intact even though the structure of the nucleosome has been disrupted.
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PMID:Chromatin subunits elicit species-specific antibodies against nucleoprotein antigenic determinants. 615 7


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