Rapid isolation of undegraded polysomal RNA without phenol

Wiegers, U.; Hilz, Helmuth

doi:10.1016/0014-5793(72)80289-8

Cited by 55 publications

(12 citation statements)

References 15 publications

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“…In B. subtiZis, one group of workers (8) reported a poly(A)-RNA content of 5-11% during growth and 3-6% during sporulation, while others (9) were unable to detect poly(A)-RNA in growing B. subtiZis but found up to 20% during sporulation. A possible explanation for the consistently high levels of polyadenylated RNA observed by us is that our procedure, being very rapid and involving the use of ribonuclease inhibitors heparin and o-phenanthroline (18) as well as proteinase K, which will rapidly degrade other enzymes in the presence of sodium dodecyl sulfate, minimizes RNA degradation (24,25). Indeed, in parallel experiments in which RNA was isolated by the conventional procedure involving deproteinization by phenol extraction at pH 9-(3) rather than by proteinase K treatment, a poly(A)-RNA content of only 0.3% and 1% was found in pulse-labeled RNA from E. coli B and B. subtilis, respectively, even though the total recovery of pulse-labeled RNA was about 50%.…”

Section: Resultsmentioning

confidence: 99%

Detection of high levels of polyadenylate-containing RNA in bacteria by the use of a single-step RNA isolation procedure

1981

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A new one-step procedure for the isolation of bacterial RNA, involving lysis by proteinase K in the presence of sodium dodecyl sulfate, is described.Pulse-labeled RNA isolated by this procedure from Bacillus brevis, Bacillus subtiZis, and Escherichia coZi B has been found to contain a substantial fraction (15-40%) of polyadenylated RNA as determined by adsorption to oligo (dT)-cellulose. This contrasts with RNA isolated by procedures involving phenol extraction, a process which appears to lead to the selective loss of polyadenylated RNA. The presence of polyadenylated RNA in E. coZi was confirmed by an independent method which involved hybridization with [3H]polyuridylic acid. Using the proteinase K method for RNA isolation, it was possible to demonstrate the in vitro synthesis of polyadenylated RNA by toluene-

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Section: Resultsmentioning

confidence: 99%

Detection of high levels of polyadenylate-containing RNA in bacteria by the use of a single-step RNA isolation procedure

1981

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“…The NlS1 strain of Novikoff hepatoma cells was grown in culture in Swimm's S-77 medium supplemented with 4 mM glutamine and 10% (v/v) dialyzed calf serum (13 (15). From this point, the RNA extraction procedure followed the technique described by Singer and Penman (16 Enzymatic Degradation of RNA and Resolution of Nucleosides.…”

Section: Methodsmentioning

confidence: 99%

Identification of Methylated Nucleosides in Messenger RNA from Novikoff Hepatoma Cells

Desrosiers

Friderici

Rottman

1974

Proc. Natl. Acad. Sci. U.S.A.

1,475

1,448

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The poly(A) tract Since post-transcriptional events may represent an important control mechanism in the regulation of genetic expression, we have investigated the possibility of methylation as an additional post-transcriptional modification of mRNA. Earlier work with bacterial and phage mRNA produced strong evidence for the essential absence of methylation in these systems, being no higher than one per 3500 nucleotides (8). Other early studies with mammalian heterogeneous nuclear RNA (HnRNA) indicated that methylation was either nonexistent or very low (9, 10). The discovery of poly-(A) has now made it possible to obtain pure mRNA fractions through affinity chromatography and, therefore, to search for low levels of methylation in mRNA without interference from rRNA contamination. Recently Perry and Kelley reported the existence of methylation in mouse L cell mRNA at about one-sixth the level found in rRNA, and both base and ribose methylations were found (11). This paper reports the existence of methylated nucleosides in the mRNA of Novikoff hepatoma cells and identifies the unique distribution of methylated moieties. A preliminary report of these results has appeared elsewhere (12). METHODSCell Culture and Labeling. The NlS1 strain of Novikoff hepatoma cells was grown in culture in Swimm's S-77 medium supplemented with 4 mM glutamine and 10% (v/v) dialyzed calf serum (13

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“…Before use in the experiments described below, RNA and DNA solutions (lmg/ml) were dialysed exhaustively, in the presence of 5,ug of proteinase K (Boehringer)/ml, against 5mM-triethanolamine/HCl+ 1 MM-MgCl2, pH 7.7, at 40C (Wiegers & Hilz, 1972). At least 90% of the proteinase K activity was removed from the dialysed nucleic acid by passage through a column (l0cmx 1cm) of Sephadex G-25 and discarding the retarded material.…”

Section: P S Agutter J R Harris and L Stevensonmentioning

confidence: 99%

Ribonucleic acid stimulation of mammalian liver nuclear-envelope nucleoside triphosphatase. A possible enzymic marker for the nuclear envelope

Agutter¹,

Harris²,

Stevenson³

1977

Biochemical Journal

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1. The specific activity of rat and pig liver nuclear-envelope nucleoside triphosphatase (EC 3.6.1.3) decreases when the system is depleted of RNA. The activity can be restored by adding high concentrations of yeast RNA to the assay medium. 2. Exogenous RNA also increases the activity of the enzyme in control envelopes (not RNA-depleted). The effect appears to be largely specific for poly(A) and poly(G); it is not stimulated by rRNA or tRNA preparations, ribonuclease-hydrolysed RNA, AMP, or double-or singlestranded DNA. 3. Inhibitors of the enzyme, in concentrations at which half-maximal inhibition of the enzyme is achieved, do not affect the percentage stimulation of the enzyme by yeast RNA. 4. The stimulation is abolished by the inclusion of l5OnM-KCl or -NaCl in the assay medium, but not by increasing the assay pH to 8.5. 5. The results are discussed in the light of the possible role of the nucleoside triphosphatase in vivo in nucleo-cytoplasmic ribonucleoprotein translocation. 6. It is proposed that poly(G)-stimulated Mg2+-activated adenosine triphosphatase activity should be adopted as an enzymic marker for the nuclear envelope.

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Rapid isolation of undegraded polysomal RNA without phenol

Cited by 55 publications

References 15 publications

Detection of high levels of polyadenylate-containing RNA in bacteria by the use of a single-step RNA isolation procedure

Detection of high levels of polyadenylate-containing RNA in bacteria by the use of a single-step RNA isolation procedure

Identification of Methylated Nucleosides in Messenger RNA from Novikoff Hepatoma Cells

Ribonucleic acid stimulation of mammalian liver nuclear-envelope nucleoside triphosphatase. A possible enzymic marker for the nuclear envelope

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