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:3.1.27.4 (ribonuclease)
6,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Methods are described that allow DNA to be prepared from widely different yeasts (Candida utilis, Saccharomyces cerevisiae, and Schizosaccharomyces pombe). The methods are reliably reproducible, and the DNA obtained is of appropriate quality for the construction of gene libraries (upper limit of size range consistently 50-150 kbp). In method A, yeast cells are converted into spheroplasts by treatment with a highly purified mixture of enzymes from Trichoderma harzianum, the spheroplasts are lysed in a lauroylsarcosinate/EDTA buffer, and the lysate is incubated with proteinase K and then directly centrifuged through a cesium trifluoroacetate gradient. DNA is recovered from the appropriate fractions by ethanol precipitation, and the redissolved precipitate is incubated with ribonuclease. For the rest of the isolation, two protocols are given, one avoiding and one including phenol/chloroform extraction. In this way, DNA up to about 150 kbp in size can be obtained. In method B, spheroplasts are not made. Yeast cells are broken by grinding under liquid nitrogen and are then worked up in a manner similar to method A, protocol 2. Subsequent steps depend on the purpose for which the DNA is required. Traditional methods of sucrose or salt density gradient centrifugation or agarose gel electrophoresis are applicable for size selection. A sodium iodide/silica matrix technique allows fast and effective DNA recovery from agarose gels.
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PMID:Isolation of DNA from yeasts. 272 83

An intracellular effect of nickel(II) which may be involved in its carcinogenic action is the alteration of normal DNA-protein binding. This effect of ionic nickel was studied in Chinese hamster ovary cells using several chromatin isolation methods in combination with SDS-polyacrylamide gel electrophoresis. DNA from cells incubated with (35S)-methionine or (35S)-cysteine to radiolabel protein was prepared by three methods: (solation of nuclei or nucleoids followed by chloroform-isoamyl alcohol (24:1 v/v) extraction and in some cases an additional extraction in the absence or presence of 2M NaCl, 40 mM EDTA or SDS; by isopycnic centrifugation through Cs2SO4 gradients containing 0.8% sarkosyl, 2.2 MCs2SO4, 1 mM NaCl and 10 mM EDTA; or by chromatin disaggregation and denaturation using 9 M urea, 2% 2-mercaptoethanol, 4% Nonidet P-40 +/- 2 M NaCl. DNA from nickel-treated cells consistently had more (35S)-methionine radioactivity associated with it than did DNA from untreated cells. This radioactivity was resistant to ribonuclease but sensitive to protease. Differential extraction using denaturing agents and high ionic strength followed by SDS-polyacrylamide gel electrophoresis revealed that most of the tightly bound proteins were nonhistone chromosomal proteins, and possibly histone 1. The enhancement of DNA-protein binding from nickel-treated cells was disrupted by SDS, suggesting that nickel ions do not function as classical bifunctional crosslinking agents. Since regulation of DNA replication and gene expression is dependent upon DNA-protein interactions, the effect of nickel in altering the extent of DNA-protein binding may interfere with this regulation and may contribute to the carcinogenic activity of nickel compounds.
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PMID:Effects of nickel(II) on nuclear protein binding to DNA in intact mammalian cells. 362 Nov 37

A protein kinase which is intimately associated with equine herpesvirus (equine abortion virus) was found by using adenosine triphosphate-gamma-(32)P as a phosphate donor and virus protein as an acceptor. Consistent demonstration of the activity requires prior removal of phosphohydrolase. The kinase activity requires Mg(2+), is not stimulated by cyclic adenosine monophosphate, but is enhanced by added protamine or arginine-rich histone. The labeled product is resistant to ribonuclease, deoxyribonuclease, and chloroform-methanol but is sensitive to Pronase. Other tests suggest that serine and threonine residues are the acceptor sites. In the in vitro reaction, the incorporation represents an average of approximately 4,500 phosphate residues per virion, and all 17 virus protein bands resolved by polyacrylamide gel electrophoresis appear to be labeled.
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PMID:Protein kinase activity in equine herpesvirus. 433 15

Large plaque (4LP) and small plaque (4SP) variants were derived from a parent bovine virus strain by serial plaque passage. Both 4LP and 4SP were resistant to chloroform and stabilized at 50 degrees C for one hour by 1.0 M magnesium chloride. Both 4LP and 4SP had buoyant densities in cesium chloride of 1.36 gm/ml. Antigenically, 4LP and 4SP were reciprocally cross neutralizable. The nucleic acid of 4LP was shown to be ribonucleic acid (RNA) by resistance of its infectivity to deoxynuclease (DNase) but not ribonuclease (RNase) and by increased incorporation of [(3)H]-uridine into cytoplasmic RNA in cells of virus infected cultures. In growth characteristics, both 4LP and 4SP had maximum adsorption times of 75 to 90 minutes but 4LP had more rapid replication and release rates and yielded nearly twice as many infectious units per cell as 4SP. The differences in growth properties correlated directly with the differential in plaque diameter which was 40-50%.
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PMID:Physical, chemical and biological characteristics of two bovine enterovirus plaque variants. 434 Mar 47

Phage X was isolated from sewage as plating on Escherichia coli or Salmonella typhimurium strains harbouring the incompatibility group X plasmid R6K. It also plated on a strain of Serratia marcescens carrying this plasmid. It failed to form plaques on Proteus mirabilis, P. morganii or Providencia alcalifaciens harbouring R6K, but did multiply on them. No phage increase occurred with homologous R- strains. Phage X also plated or registered an increase in titre on E. coli or S. typhimurium strains carrying various plasmids of incompatibility groups M, N, P-1, U or W as well as the unassigned plasmid R775. It adsorbed to pili determined by a group P-10 plasmid in a Pseudomonas aeruginosa strain but did not multiply on this organism. The phage was filamentous and curly, resistant to ribonuclease and diethyl ether and sensitive to chloroform. It adsorbed to the tips of pili.
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PMID:Phage X: a plasmid-dependent, broad host range, filamentous bacterial virus. 612 39

Sixteen strains of Capnocytophaga were isolated from the pocket of a localized juvenile periodontitis patient. These strains were divided into four groups on the basis of morphological and physiological traits. Strains from group I and group III were identified as C. ochracea and group II as C. sputigena. An antigen common to genus Capnocytophaga was purified utilizing immunoabsorbent chromatography from lysates obtained by sodium dodecyl sulfate treatment of C. ochracea strain S1. An antigen specific to C. ochracea was prepared by sequential gel filtration and preparative isoelectric focusing. The genus common and species specific antigens isolated were immunologically unique and pure when tested by immunoelectrophoresis and immunodiffusion against rabbit antisera prepared to Capnocytophaga and other gram-negative rods. The genus common antigen was susceptible to trypsin and pronase digestion, was soluble in chloroform-methanol, but was unaltered by ribonuclease and deoxyribonuclease treatments and periodate oxidation. Antigenicity of the species specific antigen was destroyed by periodate oxidation. The genus common antigen appeared to be lipid-associated protein, while the species specific antigen consisted mainly of carbohydrate. These specific immunological reagents would be valuable in diagnosing and monitoring diseases.
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PMID:Isolation and partial characterization of a genus common antigen and species specific antigen of Capnocytophaga. 618 10

An extracellular bactericidal substance was isolated from the supernatant of Streptococcus mutans Rm-10 culture fluid and partially purified with 60% ammonium sulfate precipitation, differential centrifugation, and gel filtration on Sephadex G-200. There was a good correlation of the sensitivity profiles of indicator strains whether assayed on solid medium or with purified material from cell-free culture fluid, indicating that the same inhibitory substance is produced on solid medium and in broth. Vapor from organic solvents such as chloroform, acetone, ethanol, and ether as well as heat treatment at 100 degrees C for 30 min had little effect on the bactericidal factor. It was sensitive to trypsin and pronase and resistant to deoxyribonuclease, ribonuclease, lysozyme, and phospholipase C. The inhibitor was not infective, and electron microscopic studies failed to reveal phage or phage-like particles in concentrated solutions of the bactericidal material. The results indicate that the extracellular bactericidal substance is indeed a bacteriocin. Activity in broth cultures reached a maximum only after exponential growth had ceased. It was active against other streptococcal strains as well as strains of Actinomyces naeslundii, A. viscosus, Bacillus subtilis and Staphylococcus aureus, but not against strains of Fusobacterium nucleatum and Escherichia coli.
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PMID:Isolation, partial purification and preliminary characterization of a bacteriocin from Streptococcus mutans Rm-10. 641 23

We reported (Scates et al. Carcinogenesis 1994, 15, 2945-2948) that incubating a range of bile acids with DNA in vitro, with or without exogenous metabolic activation, gave no evidence of DNA adduct formation as judged by the nuclease P1 method of 32P-postlabelling. In contrast Hamada et al. (Carcinogenesis 1994, 15, 1911-1915), also using postlabelling, claimed that chenodeoxycholic acid, lithocholic acid, glycolithocholic acid and taurolithocholic acid bound covalently to DNA in vitro. To investigate this discordance we incubated solutions of salmon sperm DNA for 1 h at 37 degrees C with 1 mg/ml of cholic acid, chenodeoxycholic acid, lithocholic acid, glycolithocholic acid or taurolithocholic acid. Each incubate was extracted extensively with diethyl ether after which a sample of DNA was taken and 32P-postlabelled using the nuclease P1 method. The DNA in the remaining incubate was precipitated from high salt solution with ethanol. Aliquots of this DNA were postlabelled. The remainder of the DNA was purified with proteinase-K, ribonuclease, phenol-chloroform, precipitated and postlabelled. Parallel incubates were made with the same bile acids, under the same conditions but in the absence of DNA and were then extracted, precipitated and postlabelled as described above. When DNA was present in the incubate but was not precipitated, chenodeoxycholic acid, lithocholic acid, glycolithocholic acid and taurolithocholic acid, but not cholic acid, produced spots similar to those reported by Hamada et al. No such spots were seen when DNA was postlabelled after precipitation, or after precipitation and purification. These same bile acids produced spots when postlabelled in the absence of DNA, but spots were absent when these incubates were precipitated and purified before postlabelling. We conclude that the spots obtained when bile acids are incubated with DNA which is not precipitated from high salt before it is postlabelled are technical artefacts, and cannot be regarded as evidence that bile acids bind covalently to DNA to form adducts. We also confirm reports (Vulimiri et al. Carcinogenesis 1994, 15, 2061-2064) that bile acids alone can produce spots when incubated with T4 polynucleotide kinase and [gamma-32P]ATP.
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PMID:Appearance of artefacts when using 32P-postlabelling to investigate DNA adduct formation by bile acids in vitro: lack of evidence for covalent binding. 761 81

The Milli-Q PF Plus water polishing system is equipped with high-purity ion and organic removal media and a capillary fiber ultrafiltration device. The system produces ultrapure water practically free of ribonuclease contamination. The necessity for diethyl pyrocarbonate (DEPC) treated solutions in RNA molecular biological procedures was tested by preparing RNA from a variety of tissues and tissue cultured cells using either DEPC-treated, autoclaved solutions or pure Milli-Q PF water dispensed directly from the system. Tissue sources included rabbit brain, heart, lung, liver, kidney, and bladder as well as cultured human corpus cavernosum smooth muscle cells (HCCSMC). RNA was prepared by solubilization in guanidinium isothiocyanate, phenol/chloroform extraction, and isopropanol precipitation followed by Northern blot analysis. Hybridization with fibronectin (approximately 7.6kb) and glyceraldehyde-3-phosphate dehydrogenase (1.2kb) revealed that water from a Milli-Q PF water system performed as well as DEPC-treated, autoclaved solutions. RNA stability at 37 degrees C was examined for various times using rabbit lung RNA in either DEPC-treated water or Milli-Q PF water. Intact RNA was detected after 6 hours in total RNA and by Northern blots hybridized with fibronectin. There was no significant difference in RNA degradation between DEPC-treated water or Milli-Q PF water. We conclude that Milli-Q PF water is an acceptable substitute to DEPC-treated water for the preparation of RNA and Northern blot analysis.
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PMID:Comparison of Milli-Q PF plus water with DEPC-treated water in the preparation and analysis of RNA. 864 47

The Milli-Q PF Plus water-polishing system is equipped with high-purity ion and organic removal media and a capillary fiber ultrafiltration device. The system produces ultrapure water practically free of ribonuclease contamination. The necessity for diethyl pyrocarbonate (DEPC)-treated solutions in RNA molecular biological procedures was tested by preparing RNA from a variety of tissues and tissue-cultured cells using either DEPC-treated, autoclaved solutions or pure Milli-Q PF water dispensed directly from the system. Tissue sources included rabbit brain, heart, lung, liver kidney and bladder as well as cultured human corpus cavernosum smooth muscle cells. RNA was prepared by guanidinium isothiocyanate solubilization, phenol/chloroform extraction and isopropanol precipitation followed by Northern blot analysis. Hybridization with fibronectin (ca. 7.6 kb) and glyceraldehyde-3-phosphate dehydrogenase (1.2 kb) revealed that water from a Milli-Q PF water system performed as well as DEPC-treated, autoclaved solutions. RNA stability at 37 degrees C was examined for various times using rabbit lung RNA in either DEPC-treated water for Milli-Q PF water. Intact RNA was detected after 6 hours in total RNA and by Northern blots hybridized with fibronectin. There was no significant difference in RNA degradation between DEPC-treated water and Milli-Q PF water. We conclude that Milli-Q PF water is an acceptable substitute for DEPC-treated water for the preparation of RNA and Northern blot analysis.
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PMID:Comparison of Milli-Q PF Plus water to DEPC-treated water in the preparation and analysis of RNA. 877 61


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