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:3.1.27.1 (RNase)
16,360 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Nuclear matrix was prepared by sequential treatment of oviduct nuclei with Triton X-100, DNase I, and 2 M NaCl. Published procedures were modified such that as many steps as possible were performed at -20 degrees C to minimize endogenous ribonuclease activity. Examination of electron micrographs confirmed the isolation of intact nuclear matrix structures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the proteins in these structures showed an absence of histones and an enrichment of certain nonhistone proteins. RNA was isolated from the nuclear matrix preparations and subjected to denaturing gel electrophoresis. Gels were analyzed by ethidium bromide staining and by hybridization of Northern blots to cloned DNA probes for ovalbumin, ovomucoid, 5.8S ribosomal RNA, and U1 RNA. All of the precursors to ovalbumin and ovomucoid mRNAs (including various splicing intermediates) and all of the precursors to ribosomal RNA were associated exclusively with the nuclear matrix fraction. By contrast, mature ovalbumin and ovomucoid mRNAs were distributed between matrix and nonmatrix fractions. These observations were further supported by quantitative hybridization analysis of the RNA in nuclear and matrix fractions. It was found that less than 50% of the mature message of intact nuclei was recovered in the matrix, while most significantly, over 95% of the mRNA precursors remained associated with the matrix. Finally, mature ribosomal RNAs and virtually all of the small nuclear RNAs (including U1 RNA) were also distributed between matrix and nonmatrix fractions. Our results suggest that all precursor RNAs (be they precursors to mRNA or rRNA) are exclusively associated with the nuclear matrix and support the notion that the nuclear matrix may be the structural site for RNA processing within the nuclei of eucaryotic cells.
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PMID:Ribonucleic acid precursors are associated with the chick oviduct nuclear matrix. 618 7

Intact and fast-sedimenting nucleoids of Bacillus licheniformis were isolated under low-salt conditions and without addition of detergents, polyamines or Mg2+. These nucleoids were partially unfolded by treatment with RNase and completely unfolded by treatments that disrupt protein-DNA interactions, like incubation with proteinase K, 0.1% sodium dodecyl sulphate and high ionic strength. Ethidium bromide intercalation studies on RNase-treated, proteinase-K-treated and non-treated nucleoids in combination with sedimentation analysis of DNase-I-treated nucleoids revealed that DNA is organized in independent, negatively supertwisted domains. In contrast to the DNA organization in bacterial nucleoids, isolated under high-salt conditions and in the presence of detergents (Stonington & Pettijohn, 1971; Worcel & Burgi, 1972), the domains of supertwisted DNA in the low-salt-isolated nucleoids studied here are restrained by protein-DNA interactions. A major role for nascent RNA in restraining supertwisted DNA was not observed. The superhelix density of B. licheniformis nucleoids calculated from the change of the sedimentation coefficient upon ethidium bromide intercalation, was of the same order of magnitude as that of other bacterial nucleoids and eukaryotic chromosomes, isolated under high-salt conditions: namely, -0.150 (corrected to standard conditions: 0.2 M-NaCl, 37 degrees C; Bauer, 1978). Electron microscopy of spread nucleoids showed relaxed DNA and regions of condensed DNA. Spreading in the presence of 100 micrograms ethidium bromide per ml revealed only condensed structures, indicating that nucleoids are intact. From spreadings of proteinase-K-treated nucleoids we infer that supertwisted DNA and the protein-DNA interactions, responsible for restraining the superhelical DNA conformation, are localized in the regions of condensed DNA.
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PMID:Folding of prokaryotic DNA. Isolation and characterization of nucleoids from Bacillus licheniformis. 618 37

To investigate the binding of a nitrofuran to tissue macromolecules in vivo, the urinary bladder carcinogen N-[4-(5-nitro-2-furyl)- 2-[35S]thiazolyl]formamide [[35S]FANFT; 94 mCi/mmol] was given p.o. to conventional [n = 4; 115 +/- 8 g (S.D.)] and germfree [n = 4; 105 + 5 g] female CD rats [1.23 mCi/rat]. After 18 hr, organs were removed, and macromolecules were then isolated from individual livers and kidneys and from pooled urinary bladders. A hydroxylapatite isolation procedure was followed (Beland et al., J. Chromatogr., 174: 177-186, 1979), and the nucleic acids obtained were further purified by digestion with appropriate nucleases and/or centrifugation (105,000 X g). The results are as follows and are given in pairs (conventional/germ-free) expressed as pmol FANFT bound per mg macromolecule. Protein binding levels were: liver, 165 +/- 40/307 +/- 31; kidney, 72 +/- 19/88 +/- 24; bladder, 272/322. RNA levels were: liver, 217 +/- 184/413 +/- 196; kidney, 219 +/- 60/617 +/- 196; bladder, 448/1373. DNA levels were: liver, 9.8 +/- 7.5/17.0 +/- 7.5; kidney, 0.69 +/- 0.38/4.5 +/- 1.0. The quantity of bladder DNA was insufficient for accurate measurement. Diethylaminoethyl cellulose chromatography of liver RNA from a germfree rat, either before or after RNase digestion, showed that the majority of the radioactivity was associated with a polynucleotide fraction that appeared to be RNase resistant and accounted for only a small portion of the total RNA but that also permitted the intercalation of ethidium bromide. The deformylated FANFT metabolite, 2-amino-4-(5-nitro-2-fury)thiazole, reacted with transfer RNA upon reduction with sodium dithionite in vitro to give adduct(s) that also appeared to be RNase resistant. Thus, these results show that the urinary bladder carcinogen FANFT or its metabolites react in vivo with protein and nucleic acid of both target and nontarget organs and that binding levels are elevated in germfree rats.
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PMID:Enhanced macromolecular binding of N-[4-(5-nitro-2-furyl)-2-thiazolyl]- formamide in germfree versus conventional rats. 619 May 54

We have extensively purified from Krebs II ascites cells, although not until homogeneity, a ribonuclease which preferentially cleaves natural or synthetic double-stranded RNA substrates (RNase D); this specificity is also supported by its sensitivity to inhibition by 10(-5) M ethidium bromide. It does not degrade RNA-DNA hybrids and is, therefore, clearly distinct from previously characterized RNases H (Cathala, G., Rech, J., Huet, J., and Jeanteur, Ph. (1979) J. Biol. Chem. 254, 7354-7361). It shows no requirement for a divalent cation and is inhibited by all kinds of nucleic acids regardless of their secondary structure. It acts exclusively as an endonuclease, as shown by the analysis of degradation products, and yields 5'-phosphate termini. This enzyme is able to introduce discrete nicks into purified HeLa 45 S preribosomal RNA as well as into HeLa heterogenous nuclear RNA packaged within naturally occurring nuclear ribonucleoprotein particles. It is, therefore, an interesting candidate for an RNA-processing enzyme.
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PMID:Isolation and characterization of a ribonuclease activity specific for double-stranded RNA (RNase D) from Krebs II ascites cells. 624 30

Concerted structural alterations of viral proteins in the capsid of poliovirions are induced after adsorption to specific receptors on the host cells. Similar changes occur in vitro during exposure to low and high pH, elevated temperature or to denaturing agents. These structural alterations can be monitored conveniently by recording changes in the intrinsic fluorescence of the poliovirus-capsid and by following the fluorescence intensity after addition of ethidium bromide to virus particles. Application of these fluorescence techniques reveals that the uncoating of the virions in vitro occurs in two distinct steps: 1. entry of ions, e.g. ethidium bromide, 2. development of sensitivity of the virion RNA to RNase and release of the RNA. We confirm different structural stabilities of the virions at several pH values to elevated temperatures and a stabilizing effect of arildone on poliovirions.
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PMID:Fluorescence spectrophotometric study of structural alterations in the capsid of poliovirus. 631 93

A very simple, inexpensive procedure for preparing pure plasmid DNA from bacteria is described. In this method, lysozyme-induced spheroplasts are made in presence of 833 micrograms/ml of ethidium bromide which are then lysed by a mixture of Brij 58 and sodium deoxycholate, and the lysate is centrifuged at 48,000 g for 25 min whereby about 99.9% of total chromosomal DNA is pelleted. From the supernatant containing plasmid DNA, the proteins are removed by phenol extraction and the major part of RNA by CaCl2 precipitation, and finally the small amount of residual RNA is removed by RNase treatment. The average yield of pBR322 DNA from 1 liter of amplified culture by this procedure is 2 to 2.5 mg and the preparation is highly pure, containing only about 0.005% of total yield as chromosomal DNA contaminant. Moreover, the substrate activity and the transforming ability of the plasmid DNA prepared by this method remain unaffected.
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PMID:A simple procedure for large-scale preparation of pure plasmid DNA free from chromosomal DNA from bacteria. 635 82

The acid-extractable leaf proteins of potato spindle tuber viroid (PSTV) infected tomato plants were analysed electrophoretically on polyacrylamide gels. The most prominent alteration found during disease development was the appearance of a "pathogenesis-related" protein with an apparent molecular weight of 14,000 (called P14) which is drastically increased in concentration. Its induction, however, is not viroid-specific because it is also accumulating after viral and fungal infections. The degree of P14 accumulation could be directly correlated with the severity of the disease symptoms and its concentration was found to be highest in leaves of the tomato cultivar "Rutgers" four weeks after infection. P14 was isolated from such leaf material by acid-extraction of the leaf proteins, which were concentrated from the clarified homogenates by ultrafiltration through hollow fiber systems or by precipitation at 60 per cent ammonium sulphate saturation. P14 was finally purified by ion exchange chromatography on sulfopropyl (SP-C25) Sephadex and on DEAE cellulose. A protein with properties similar to those of P14 could also be isolated from healthy tomato leaves, where its concentration is about forty to fifty times lower than PSTV-infected tissue. P14 can be stained with Coomassie Brilliant Blue, silver and ethidium bromide, it is sensitive to digestion with pronase and not altered when treated with RNase and DNase. P14 is a basic protein with an estimated isoelectric point of 10.7 and its unusual behaviour during ultrafiltration indicates that it represents an elongated rather than a globular molecule in solution. P14 seems to be different from any of the so-called "pathogenesis-related" proteins described so far in Gynura aurantiaca, "Etrog" citron, potato and tomato after viroid-infection and in tobacco, cucumber and bean leaves after virus- or fungus-induced hypersensitive reactions.
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PMID:Purification and partial characterization of the major "pathogenesis-related" tomato leaf protein P14 from potato spindle tuber viroid (PSTV)-infected tomato leaves. 647 30

A double-stranded RNA specific nuclease (ds RNase) has been purified from the pearl millet Pennisetum typhoides. The purification involved S-30 preparation from the germinating embryos, DEAE-cellulose and DNA-cellulose chromatography. The partially pure enzyme preferentially solubilized the synthetic double-stranded polynucleotide [3H]poly(rA) . poly(rU); the degradation of [3H]poly(rC) was fourteen fold lower under the same assay conditions. Furthermore, the ds RNase activity was inhibited to an extent of 58% by ethidium bromide, which is known to intercalate with double-stranded RNAs. Active sulfhydryl groups were found to be necessary for the ds RNase activity since the enzyme action was inhibited by N-ethylmaleimide. Ethidium bromide and N-ethyl-maleimide did not significantly inhibit the ss RNase activity. In contrast, diethyl pyrocarbonate inhibited ss RNase activity completely and ds RNase by 58%. Heating the enzyme for 20 min at 50 degrees C resulted in drastic loss of both enzyme activities. The ds RNase showed maximum activity in the pH range of 6.5 to 7.5. The enzyme acts in vitro on E. coli 30S precursor ribosomal RNA and the cleavage products migrated in the region of mature 23S and 16S rRNAs.
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PMID:Double-stranded RNA specific nuclease from germinating embryos of Pennisetum typhoides. 652 87

Calf aortic smooth muscle cell cultures produce both type III and type I collagen. Polyadenylated mRNA species purified from these cells direct the synthesis of prepro-alpha 1(III), prepro-alpha 1(I), and prepro-alpha 2(I) in a rabbit reticulocyte cell-free system. These polypeptides were identified by specific immunoprecipitation, cyanogen bromide peptide mapping, and bacterial collagenase digestion. Lower molecular weight collagenase susceptible polypeptides were also produced in translation reactions incubated under conditions optimized for incorporation of radiolabeled amino acids. Their presence did not appear to result from ribonuclease or protease involvement or from premature termination. Increasing the Mg2+ concentration in the translation system significantly reduced the production of these lower molecular weight species. Pulse-chase experiments indicate that the time required for completion of full length preprocollagen at the high Mg2+ concentration is greatly decreased compared to the low concentration. Additional experiments suggest that the incomplete collagen polypeptides result from pausing of ribosome movement during elongation. The relative synthesis of type III and type I chains was examined as a function of mRNA concentration in the cell-free system. At levels of RNA above saturation, the relative production of type III decreased with respect to type I. These data suggest that the ability of the alpha 1(III) mRNA to initiate translation is less efficient than the mRNAs of alpha 1(I) and alpha 2(I).
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PMID:Cell-free translation of calf type III collagen. Effect of magnesium on ribosome movement during elongation. 661 53

A double-stranded RNA-specific nuclease (ds RNase) has been isolated and partially purified from human placenta by DEAE-cellulose and DNA-cellulose column chromatography. Denatured DNA-cellulose retained most of the single-stranded RNA-specific nuclease (ss RNase) activity, whereas the ds RNase came out in the void volume. N-ethylmaleimide at a concentration of 5 mM, selectively inhibited ds RNase activity by 60% under the conditions in which the ss RNase activity was inhibited to an extent of 7%. The ds RNase was specifically inhibited by Penicillium chrysogenum viral ds RNA and by ethidium bromide. The partially purified ds RNase showed requirements for Mg+ whereas Mn2+ and NH4+ ions were inhibitory. The DEAE-enzyme cleaved 32P-labelled 45S ribosomal precursor RNAs from Yoshida ascites sarcoma cells into species that had similar electrophoretic mobilities as the mature rRNAs.
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PMID:Partial purification and characterization of a double-stranded RNA-specific nuclease from human placenta. 663 20


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