Purification of viral RNA by means of bentonite

Fraenkel‐Conrat, H.; Singer, Brett C.; Tsugita, Akira

doi:10.1016/0042-6822(61)90131-3

Cited by 653 publications

(132 citation statements)

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“…TMV, a Japanese common strain OM, was purified by polyethylene glycol precipitation (12) followed by differential centrifugation. TMV protein and RNA were isolated by the acetic acid method (13) and by phenolbentonite extraction (14), respectively. TMV RNA radioactively labeled at its termini was prepared by periodate oxidation and subsequent reduction with tritiated sodium borohydride (8).…”

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Kinetics of biphasic reconstitution of tobacco mosaic virus in vitro.

Fukuda¹,

Ohno²,

Okada³

et al. 1978

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

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The kinetics of the in vitro reconstitution of tobacco mosaic virus from its RNA and protein were studied by measuring the increase in turbidity, the development of ribonuclease-resistant infectivity, the encapsidation of the terminal ends of the RNA, and the growth of rod length. Recently, several laboratories independently reached the conclusion that reconstitution starts at an internal region of the RNA, about 800-1000 nucleotides from the 3' terminus, and that the elongation occurs bidirectionally (7-10). In an early phase of the reaction, the rod elongation toward the 5' end is much favored over that toward the 3' end (9-11). The present study deals with kinetics of the bidirectional elongation of TMV rods during the reconstitution reaction, studied by measuring turbidity, infectivity, RNase resistance of the two ends of RNA, and rod length. The results show that reconstitution takes place in two successive phases that differ not only in the direction, but also in the rate of rod elongation. Rods with a length of 260 nm were identified as the product of the early process of reconstitution.MATERIALS AND METHODS Preparation of TMV, TMV Protein, TMV RNA, and Tritium-Labeled TMV RNA. TMV, a Japanese common strain OM, was purified by polyethylene glycol precipitation (12) followed by differential centrifugation. TMV protein and RNA were isolated by the acetic acid method (13) and by phenolbentonite extraction (14), respectively. TMV RNA radioactively labeled at its termini was prepared by periodate oxidation and subsequent reduction with tritiated sodium borohydride (8). Both the 5'-and the 3'-ribosyl ends of TMV RNA were labeled with about the same efficiency.The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.Reconstitution Reaction. A protein disk preparation was obtained by incubation of the protein (6.6 mg/ml) for 24 hr in 0.1 M phosphate buffer (pH 7.0) at 20°. Reconstitution reactions were carried out in the same buffer at 200 by mixing the protein disk preparation (5.6 mg/ml) with a mixture of TMV RNA (0.13 mg/ml) and tritium-labeled TMV RNA (0.05 mg/ml). At intervals, aliquots were withdrawn to determine the rod lengths, the ribonuclease resistance of tritium-labeled termini, and the infectivity.Turbidimetric Measurement during Reconstitution Reaction. The reconstitution reaction during the initial 90 min of the reaction was followed by the increase in absorbance at 310 nm with a Gilford model 250 spectrophotometer.Rod Length Measurements by Electron Microscopy. An aliquot from the reaction mixture was diluted 1:100 with icecold 0.01% (wt/vol) bovine serum albumin solution, and minute drops of the mixture were placed on carbon-coated electron microscope grids. The grids were immediately transferred to a desiccator and kept under reduced pressure overnight. The specimens were then negatively stained with 1% (wt/vol) phosphotungst...

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confidence: 99%

Kinetics of biphasic reconstitution of tobacco mosaic virus in vitro.

Fukuda¹,

Ohno²,

Okada³

et al. 1978

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

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“…All glassware was treated with 0.1% ethyl oxidiformate (Eastman Kodak Co.) and then autoclaved; all plastic was treated with 2.0% Na dodecyl sulfate. 1 to 2 X 109 cells were suspended in 10 ml of isotonic saline and added by drops to an extraction mixture consisting of 40 ml of water-saturated distilled phenol, 20 ml of buffer R [46 mM NaCl-20 mM sodium acetate-2 mM NaEDTA (pH 5.2) made 2% with Na dodecyl sulfate], and 50 mg of bentonite dry weight, prepared by the method of Fraenkel-Conrat et al (13). After the mixture was shaken for 30 min at 40, the aqueous and phenol phases were separated by centrifugation at 17,300 X g. 10 ml of phenol was added to the aqueous phase, which was kept on ice while the phenol phase and the interphase were again extracted with 20 ml of buffer R by shaking for 15 min at 4°.…”

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confidence: 99%

Effects of 5-Bromo-2′-Deoxyuridine on Production of Globin Messenger RNA in Dimethyl Sulfoxide-Stimulated Friend Leukemia Cells

Preisler¹,

Housman

Scher³

et al. 1973

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

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From these studies, where low doses which did not significantly interfere with cell replication had been used, synthesis of proteins characteristic of the final differentiated state was inhibited. For example, hemoglobin synthesis by chick embryo was inhibited during culture in the presence of BrdU (8, 9).We previously reported that dimethyl sulfoxide [(CH3)2S0 I enhances heme synthesis and erythroid differentiation of Friend leukemia cells in vitro (10) and that inhibition of these effects by BrdU was dependent on its incorporation into DNA (7). Similarly, other investigators have observed inhibition by BrdU of (CH3)2S0-induced stimulation of globin synthesis (11). Since appreciable amounts of globin mRNA were detected in (CH3)2S0-stimulated, but not in control, Friend leukemia cells cultures, it appeared that (CH3)2S0 might act at a transcriptional level (12, 21.)The present studies were designed to determine whether BrdU might also affect transcription of globin mRNA by Friend leukemia cells. We found that a decrease in the amount of globin mRNA accompanied inhibition of (CH3) BrdU-and (CH3)2S0-treated-cells cultured in the presence of both 2% (CH3)2S0 and 3 ,g/ml of BrdU. The cultures in each experiment were seeded simultaneously and maintained in the dark.Preparation of RNA RNA was prepared by a modification of the method of Natta (29). All glassware was treated with 0.1% ethyl oxidiformate (Eastman Kodak Co.) and then autoclaved; all plastic was treated with 2.0% Na dodecyl sulfate. 1 to 2 X 109 cells were suspended in 10 ml of isotonic saline and added by drops to an extraction mixture consisting of 40 ml of water-saturated distilled phenol, 20 ml of buffer R [46 mM NaCl-20 mM sodium acetate-2 mM NaEDTA (pH 5.2) made 2% with Na dodecyl sulfate], and 50 mg of bentonite dry weight, prepared by the method of Fraenkel-Conrat et al. (13). After the mixture was shaken for 30 min at 40, the aqueous and phenol phases were separated by centrifugation at 17,300 X g. 10 ml of phenol was added to the aqueous phase, which was kept on ice while the phenol phase and the interphase were again extracted with 20 ml of buffer R by shaking for 15 min at 4°. The aqueous and phenol phases were again separated by centrifugation. The aqueous phases were combined and extracted with 30 ml of additional phenol by shaking for 20 min at 4°. After centrifugation for 5 min, the phenol phase was discarded. The aqueous phase was centrifuged for 20 min at 30,900 X g and the pellet was discarded. 0.1 Volume of 3 M NaCl and 2 volumes of absolute ethanol were added to the aqueous phase, and the mixture was allowed to stand over-2956 Abbreviations: BrdU, 5-bromo-2'-deoxyuridine; (CH3)2SO, dimethyl sulfoxide; cDNA, complementary DNA. t Present address:

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“…The resulting pellet was washed twice with distilled water and resuspended in 0.1 M-disodium ethylenediaminetetra-acetic acid and stirred overnight. Crude bentonite has a high metal ion content which decreases protein-binding capacity, and EDTA is effective in ion removal (Fraenkel-Conrat et al, 1961;Watts & Mathias, 1967). The EDTA was removed by overnight dialysis against distilled water.…”

Section: Methods Of Isolation and Analysismentioning

confidence: 99%