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:3.1.26.9 (ribonuclease)
6,589 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Reverse transcription-PCR (RT-PCR) has traditionally required time-consuming RNA extraction and purification. This report demonstrates that one can completely avoid the RNA extraction step in RT-PCR by basing the comparison of samples on cell number rather than micrograms of total RNA. A new method for lysing cells while preserving RNA is described. RT-PCR is carried out (i) by rapidly freezing cells in the presence of ribonuclease inhibitor (RNase inhibitor) plus dithiothreitol and (ii) by using extracts of 250 or fewer cells directly in the RT-PCR assay. Aldolase mRNA, extracted by freeze-thawing cells in the presence of RNase inhibitor, was found to be stable at 42 degrees C for over three hours. Since the RT step can be completed within 1 h, there is minimal degradation of mRNA. This simple procedure avoids the use of harsh reagents, which may inhibit enzymes involved in RT-PCR, and produces results virtually identical to methods that employ guanidinium thiocyanate and phenol for RNA extraction. Optimized conditions for each parameter of the procedure are described that permit amplification of mRNA from as few as four cells.
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PMID:RT-PCR without RNA isolation. 896 38

Yeast HSF is constitutively trimeric and DNA bound. Heat shock is thought to activate HSF by inducing a conformational change. We have developed an assay in which we can follow a conformational change of HSF that correlates with activity and thus appears to be the active conformation. This conformational change requires two HSF trimers bound cooperatively to DNA. The conformational change can be induced in whole cell extracts, and is thus amenable to biochemical analysis. We have purified a factor that triggers the conformational change. The factor is sensitive to dialysis, insensitive to NEM, and is not extractable by phenol. It is small, and apparently not a peptide. Mass spectroscopy identifies a novel guanine nucleotide that tracks with activity on columns. This novel nucleotide, purchased from Sigma, induces the conformational change (although this does not prove the identity of the activating factor unambiguously, because Sigma's preparation is contaminated with other compounds). What is the source of this nucleotide in cells? Activity can be generated by treating extracts with ribonuclease; this implicates RNA degradation as a source of HSF-activating activity. The heat shock response is primarily responsible for monitoring the levels of protein chaperones; how can RNA degradation be involved? Synthetic lethal interactions link HSF activity to ribosome biogenesis, suggesting a possible model. Ribosomal proteins are produced in large quantities, and in excess of rRNA; unassembled r-proteins are rapidly degraded (t1/2 approximately 3 min). Unassembled r-proteins aggregate readily. It is likely that unassembled r-proteins represent a major target of chaperones in vivo, and for proteasome-dependent degradation. Interference with rRNA processing (e.g., by heat shock) requires hsp70s to handle the aggregation-prone r-proteins, and proteasome proteins to help degrade the unassembled r-proteins before they aggregate. A nucleotide signal could be generated from the degradation products of the rRNA itself.
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PMID:A role for RNA metabolism in inducing the heat shock response. 1044 Feb 29

Ribonucleic acid extracted with phenol from Type 1 poliovirus, Coxsackie A-9, Coxsackie B-1, and ECHO 8 viruses infected non-primate cells and animals insusceptible to whole virus as such. Viral RNA was proved infectious for insusceptible cells in test systems of established cell lines, primary monolayer cultures, Maitland type cultures, and living animals inoculated intracerebrally. Cells of rabbit, swine, mouse, guinea pig, chicken, and hamster were infected. Each virus produced was identical with the virus donating RNA, in (a) neutralization by homotypic antiserum, (b) resistance to ribonuclease treatment, and (c) failure to be adsorbed or replicated by nonprimate cells, even of the strain producing the virus from RNA. Produced virus was adsorbed and replicated by susceptible primate cells as usual. Virus in RNA-infected cell cultures was produced in a single cycle unaccompanied by overt cytopathic effect on non-primate cells or disease of intracerebrally inoculated animals. By drastic elution of infective poliovirus associated with rabbit cells exposed to massive inocula of intact virus, intact poliovirus was shown to infect insusceptible non-primate cells to produce progeny indistinguishable from the parent virus population. Under these conditions, infection was accomplished by about 10 virus plaque-forming units per billion inoculated.
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PMID:The mammalian cell-virus relationship. IV. Infection of naturally insusceptible cells with enterovirus ribonucleic acid. 1366 69

A relatively sensitive and adequately reproducible assay of infectious enteroviral RNA was obtained by exposing calf serum-grown HeLa cells to RNA suspended in 2 M magnesium sulfate solution. Highly purified enteroviral preparations yielded RNA exhibiting more than 0.1 per cent of the infectivity of whole original virus and infectivity regressed linearly with dilution. Radioisotope experiments with P(32)-labeled RNA and spectrophotometric studies demonstrated that Gierer-Schramm phenol extraction permits almost quantitative recovery of high molecular weight RNA from poliovirus. Intact chromatography-purified type 2 poliovirus in the analytical ultracentrifuge showed a sharp boundary and a sedimentation coefficient of S(20, w) = 147 +/- 5S. Phenol-extracted poliovirus RNA exhibited a heterodisperse sedimentation pattern with a large proportion of homogeneous, rapidly sedimenting material having a coefficient of S(20, w) = 37 +/- 2S. Although the bulk of extracted poliovirus RNA as measured by radiophosphorus labeling was not taken up by cells, the infectious fraction of RNA was adsorbed rapidly by HeLa-cell or L strain mouse fibroblast monolayers, indicating a possible dissimilarity between the bulk of extracted virus RNA and a relatively small fraction responsible for infectivity. Enhancement of poliovirus RNA infectivity for HeLa cells by high ionic-strength magnesium sulfate solution appeared due partly to an effect of hypertonicity on cells, and partly to an effect of high-concentration divalent cation on RNA itself, but not to enhancement of adsorption. Poliovirus RNA adsorbed by HeLa cells apparently was rapidly received within the cells since it became quickly insusceptible to ribonuclease. Heterologous nucleic acids and degradation products to the level of oligoribonucleotides inhibited infectivity of poliovirus RNA for HeLa cells. This inhibitory activity appeared due to intermolecular complexing, since exposure of cells to heterologous RNA immediately before or after exposure to virus RNA failed to reduce infectivity. Ultraviolet absorption spectra demonstrated that the RNA within intact poliovirus is more hypochromic (and thus more extensively hydrogen-bonded) than is the same RNA isolated by phenol extraction, and suspended in 0.02 M phosphate buffer.
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PMID:Enteroviral ribonucleic acid. I. Recovery from virus and assimilation by cells. 1371 81

Homma, M. (The Wistar Institute, Philadelphia, Pa.), and A. F. Graham. Intracellular fate of Mengo virus ribonucleic acid. J. Bacteriol. 89:64-73. 1965.-P(32)-labeled, purified preparations of Mengo virus adsorbed rapidly and irreversibly to L cells maintained in suspension cultures. At intervals after adsorption of labeled virus, the total ribonucleic acid (RNA) of infected cells was extracted by a phenol technique. Infectivity titrations on this RNA showed that it retained its full biological activity during the early part of the eclipse period. Sucrose gradient sedimentation analyses showed also that this RNA lost none of its structural integrity throughout the eclipse period. No evidence was found for a double-stranded structure involving parental RNA. When cells infected with P(32)-labeled virus were broken open during the first 7 hr after infection, no more than 20% of the parental RNA could be digested with ribonuclease. Electron microscopy indicated that only one particle in five of the purified viral populations was infectious. It is suggested that only one of five adsorbed virus particles was uncoated and that its RNA remained in the cell in an undegraded form during the eclipse period. The other adsorbed particles were not uncoated and took no part in the process of infection. The maximal transfer of parental RNA to progeny virus was 4.5%.
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PMID:INTRACELLULAR FATE OF MENGO VIRUS RIBONUCLEIC ACID. 1425 83

Reilly, Bernard E. (Western Reserve University, Cleveland, Ohio), and John Spizizen. Bacteriophage deoxyribonucleate infection of competent Bacillus subtilis. J. Bacteriol. 89:782-790. 1964.-Phenol extracts of the Bacillus subtilis bacteriophages phi1, phi25, and phi29 contained infectious deoxyribonucleic acid. The infectivity was destroyed by catalytic amounts of deoxyribonuclease but not by specific phage antiserum, ribonuclease, or trypsin. An infectivity of >10(6) infectious centers formed per mug of deoxyribonucleic acid (DNA) added was obtained. The stability of the infectious centers permitted an examination of a single cycle of phage replication in cells unable to adsorb the mature virus. A typical cycle was observed, although the latent period was increased and the burst size slightly reduced after DNA infection. The development of competence for bacterial transformation was strongly correlated with susceptibility to viral DNA infection. Both appeared and disappeared at the same phase of growth in the cell population. More than 4% of the viable cells in the competent population were infected by viral DNA. The kinetics of the transition of phi29 DNA infection to deoxyribonuclease insensitivity, and the relationship of infectivity to DNA dilution, were similar to the results obtained for bacterial transformation of a single marker. The doseresponse curve of phi1 and phi25 DNA was characteristic of that obtained in multiple transformation of unlinked genetic markers. Because of the low efficiency of infection, about 10(-4) per phage equivalent of DNA added, it was not possible to prove that DNA alone was sufficient to initiate infection.
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PMID:BACTERIOPHAGE DEOXYRIBONUCLEATE INFECTION OF COMPETENT BACILLUS SUBTILIS. 1427 61

Towards a goal of detecting scaled-up DNA adducts as altered deoxynucleotides by mass spectrometry, we have set up a practical and general method for isolating DNA-derived deoxyribonucleoside-5'-monophosphates devoid of ribonucleotides starting with a 1 g sample of mammalian tissue. The method is practical because costs have been minimized, and it is general because it can be applied to a more difficult sample such as mouse skin or non-fresh calf liver. The procedure, consisting of a series of steps that were largely gleaned and tuned from prior literature, proceeds as follows: (1) homogenize the tissue in sodium dodecyl sulfate; (2) digest with ribonuclease A, ribonuclease TI, alpha-amylase and proteinase K; (3) partition between water and phenol; (4) precipitate the DNA with ethanol followed by redissolving and dialysis; and (5) digest with nuclease P1 and phosphodiesterase I followed by ultrafiltration and boric acid gel chromatography. The yellow to brown color of DNA from difficult tissues only persisted up to the ultrafiltration step. Apparently this DNA was contaminated with iron-containing proteins. Residual ribonucleotides were not observable (<0.1%) by HPLC in the final sample. Without boric acid gel chromatography, residual contamination by ribonucleotides was about 1% even when the DNA was purified before digestion by phenol partitioning followed by use of a Genomic Tip kit from Qiagen.
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PMID:Phenolic extraction of DNA from mammalian tissues and conversion to deoxyribonucleoside-5'-monophosphates devoid of ribonucleotides. 1554 80

The guanidinium acid-phenol method of RNA extraction is relatively fast (4 h) and is useful for the processing of large numbers of samples, without the need for ultracentrifugation. This protocol produces total RNA that includes ribosomal, transfer, and messenger RNA. This high-quality RNA is suitable for Northern blot analysis, dot-blot hybridization, poly (A) RNA selection, in vitro translation, cDNA library construction, reverse transcriptase-polymerase chain reaction, ribonuclease protection assay, and primer extension experiments.
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PMID:Isolation of RNA from tumor samples: single-step guanidinium acid-phenol method. 1649 93

Blobel, Hans (University of Wisconsin, Madison). Isolation and characterization of deoxyribonucleic acid from a strain of Staphylococcus aureus. J. Bacteriol. 82:425-429. 1961.-Highly polymerized deoxyribonucleic acid was extracted from washed cells of Staphylococcus aureus with a mixture of equal parts of phenol and 2 m NaCl at pH 7.4. The aqueous phase was treated twice with phenol and the deoxyribonucleic acid precipitated with an equal volume of 2-ethoxyethanol. Residual ribonucleic acid was removed by treatment with ribonuclease and subsequent dialysis. Deoxyribonucleic acid was reprecipitated with 2-ethoxyethanol. The final product contained less than 1% protein. The deoxyribonucleic acid obtained from S. aureus strain S(44) had a (adenosine + thymine)/(guanine + cytosine) base ratio of 1.98. Intrinsic viscosity in 0.15 m NaCl + 0.015 m sodium citrate was approximately 76 dl/g. The sedimentation coefficient, S(20)w, was close to 25 S.
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PMID:ISOLATION AND CHARACTERIZATION OF DEOXYRIBONUCLEIC ACID FROM A STRAIN OF STAPHYLOCOCCUS AUREUS. 1656 22

Cotranslational disassembly of tobacco mosaic virus (TMV) particles in the rabbit reticulocyte lysate has recently been demonstrated (T.M.A. Wilson, Virology, in press). A similar phenomenon in wheat germ extracts is now reported. However, in contrast to the reticulocyte lysate, wheat germ cell-free extracts respond approximately three times more efficiently to pH 8 treated TMV particles than to conventional TMV RNA templates prepared by phenol extraction. Dose-response curves indicate that this behaviour is reproducible over a wide range of exogenous RNA or virus concentrations (equivalent to 6 to 400 microg/ml RNA). Enhanced synthesis of the large (126K) virus-specific polypeptide is observed in translations programmed with virus particles. These results probably reflect the protective influence of the capsid protein during protein synthesis in a cell-free extract containing ribonuclease activity. This environment may be similar to that which confronts the virus within the cells of infected host plants.
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PMID:Cotranslational disassembly increases the efficiency of expression of TMV RNA in wheat germ cell-free extracts. 1863 27


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