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:3.1.27.5 (RNase)
17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The study of messenger RNA in mammalian cells by Northern analysis requires the extraction of intact RNA in pure form. Although a number of reliable techniques have been developed for the purpose, most are fairly complex, involving steps such as ultracentrifugation and multiple extractions with large volumes of phenol and chloroform. When the number of cell samples to be analyzed is large, these techniques can be unwieldy. I now describe an RNA purification procedure which is simple enough to allow handling of a large number of cultured cell samples. It uses safe and inexpensive reagents and produces a high yield of pure total cell RNA, essentially free of DNA and ribonuclease, suitable for Northern analysis. The procedure also allows extraction of intact RNA from human granulocytes, cells which are rich in ribonuclease and contain very low amounts of RNA.
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PMID:Rapid extraction of high molecular weight RNA from cultured cells and granulocytes for Northern analysis. 245 Mar 33

A rapid procedure for the isolation of total RNA from small amounts of mammalian tissue (35 to 150 mg) is described. Tissues were homogenized in the presence of RNase inhibitors but in the absence of strong detergents. Contaminants were removed by phenol/chloroform extraction and Sephadex column chromatography. Total RNAs were precipitated with ethanol and sodium acetate. The RNAs isolated were intact and suitable for mRNA quantitation via Northern blot or slot-blot analyses. This procedure isolates total RNAs in high yield and purity, without CsCl ultracentrifugation, and is especially useful when mRNAs must be quantitated from many samples.
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PMID:Rapid isolation of total RNA from mammalian tissues. 246 82

A method is described which facilitates the rapid purification of high molecular weight chromosomal DNA from gram positive and gram negative bacteria grown on solid media. A total of 32 reference strains and fresh isolates were examined in this study. The purification procedure involved lysis of cells with SDS in the presence of proteinase K, followed by removal of cellular polysaccharides and proteins with hexadecyltrimethyl ammonium bromide (CTAB) and phenol:chloroform:isoamyl alcohol. Preparations were incubated with RNase and, after removal of the enzyme, DNA was precipitated with ethanol. Several hundred micrograms of DNA could be prepared within 5 h from cells grown on 1-2 agar plates. None of the final preparations contained RNA; protein was detected in 12/32 preparations. The resultant DNA proved suitable for restriction enzyme digestion and biotin-labelling by a random primer technique. DNA probes constructed from these preparations were capable of detecting 100 pg of homologous target DNA fixed to nitrocellulose. Cross reactions between closely related species displayed weaker signal intensities than, and, thus, were easily distinguished from, true positive reactions between homologous species. DNA obtained by this procedure may also be suitable for DNA-DNA homology studies, recombinant DNA experiments and molecular fingerprinting.
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PMID:Rapid method for the purification of DNA from subgingival microorganisms. 262 68

A simplified method for preparing genomic DNA at and human brain has been established. The procedure includes the following steps: (1) mincing and homogenizing in SSC-EDTA, (2) solubilizing in high concentration detergent (2% SDS or Triton X-100), (3) digesting with proteinase K and RNase A, (4) extracting with phenol-chloroform, (5) finally dialyzing the sample against TE. This method could result in a threefold increase in yield with advantages of simplification and time-saving. The DNA obtained by this method is of a size over 30 kd, and is suitable for the molecular biological and molecular genetical study of brain.
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PMID:[A molecular biology technique for the brain--preparation of genomic DNA from the brain]. 263 87

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

Rapid and sensitive methods are needed for the detection of enteric viruses to ensure proper drinking water quality. Gene probes have been shown to be useful for this purpose. Previously, samples to be assayed were treated with a series of phenol-chloroform extractions to release the viral nucleic acid. We have developed a more rapid procedure for liberating or exposing the genome of poliovirus for probing. In this study, a poliovirus model was used to test the ability of heat (65 degrees C for 30 min) for release or exposure of viral nucleic acid. Several different RNase inhibitors were tested for their ability to prevent viral RNA degradation. A comparison of the two methods indicates phenol-chloroform extraction is not necessary before probing. In addition to saving 2-4 h of time, maximum sensitivity levels were consistently obtained using this novel procedure.
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PMID:A novel method for liberating viral nucleic acid for assay of water samples with cDNA probes. 284 56

Two rapid methods were evaluated for their extraction of plasmids from Clostridium perfringens. The first method involved lysis of 1 to 2 ml of C. perfringens culture by treatment with hyaluronidase, lysozyme, and sarcosyl. DNA, extracted with phenol-chloroform, was treated with RNase, boiled, and electrophoresed in a 1.2% agarose gel. The second method involved lysis of 2 ml of culture by lysozyme treatment and extraction with alkaline sodium dodecyl sulfate (SDS). Extracted DNA was treated with RNase, boiled, and electrophoresed in a 0.7% agarose gel. Of 57 strains of C. perfringens analyzed by both extraction procedures, 11 were shown to have plasmids by the alkaline SDS method which were missed by the phenol-chloroform extraction method. These new plasmids were of higher molecular mass and ranged up to 68 megadaltons. Use of the DNase inhibitor diethyl pyrocarbonate did not further improve the yield of plasmid DNA. An additional 159 isolates of C. perfringens screened by the alkaline SDS method revealed plasmids up to 80 megadaltons in mass and an overall plasmid carriage rate of 69%.
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PMID:Rapid extraction of plasmids from Clostridium perfringens. 287 Jun 80

A new gel filtration method was developed for purification of R-type lipopolysaccharides (lipooligosaccharides) from some nonenteric gram-negative bacteria, including Neisseria meningitidis, Haemophilus influenzae, and Bordetella pertussis. These wild-type lipooligosaccharides are poorly extractable by the phenol-chloroform-ether extraction method of C. Galanos, O. Luderitz, and O. Westphal [1969) Eur. J. Biochem. 9, 245-249) and therefore a new procedure was developed for their isolation. The lipooligosaccharides (LOS) were first extracted by hot phenol-water, treated with RNase, then disaggregated in deoxycholic acid, and purified by gel filtration on Sephadex G-75. By comparison the conventional hot phenol-water purification method using repeated ultracentrifugations yielded less LOS. The yield of LOS by gel filtration was 30 to 108% higher and the purity was better.
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PMID:A method for purification of bacterial R-type lipopolysaccharides (lipooligosaccharides). 288 9

A method is described for the isolation of high molecular weight DNA in solution using the principles that have allowed electrophoresis of chromosome-sized DNA in pulse field gradient electrophoresis. Stationary phase yeast cells are converted to spheroplasts by the action of zymolyase in 1 M sorbitol. In the presence of EDTA and sodium lauroyl sarcosinate, proteins are digested with proteinase K. DNA is extracted with phenol and chloroform, and high molecular weight DNA is collected by ethanol precipitation. RNA is removed by RNase digestion of the redissolved pellet, and RNase is removed by chloroform extraction followed by a second ethanol precipitation. The method is rapid and gives a high yield of DNA that is readily digestible by restriction endonucleases.
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PMID:A method for the preparation of high molecular weight yeast DNA. 333 61

Macrophage migration enhancement factor (MEF), a lymphokine produced in the spleen by suppressor-like lymphoid cells, may be an important immunoregulatory molecule of macrophage function. MEF appears to be a potent positive chemokinetic factor and is unusual in that it lacks chemotactic activity. To aid in the development of a purification scheme for MEF we have employed biophysical characterization techniques to define its physical properties. Using the technique of velocity sedimentation in isokinetic sucrose gradients, the S20w for MEF was determined to be 2.25. The Stoke's radius for MEF was determined by Sephadex G-100 gel filtration to be 28.9 A. From these measurements the D20w was calculated to be 7.55 x 10(-7) cm2/sec, the mol. wt was calculated to be 28,000, the frictional ratio (f/f0) was calculated to be 1.45, the axial ratio was calculated to be 1:8, and the dimensions of the molecule were estimated to be 20 x 160 A. Using the technique of isoelectric focusing in liquid density gradients, the isoelectric point for MEF was estimated to be 8.8. We have also determined by enzyme treatment that MEF is resistant to DNase and RNase and susceptible to proteinase K and L-fucosidase. In addition, MEF partitioned to the aqueous phase during methanol-chloroform extraction procedures. MEF was inactivated at pH 12; at 100 degrees C MEF was stable for 10 min but was inactive after 1 hr. Collectively, these data will facilitate the development of a purification scheme for MEF which will ultimately permit the analysis of the molecule and its function.
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PMID:Biophysical characterization of macrophage migration enhancement factor (MEF). 343 51


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