The localization of pea enation mosaic virus-induced RNA-dependent RNA polymerase in infected peas

Powell, Charles A.; Zoeten, G. A. de; Gaard, G.

doi:10.1016/0042-6822(77)90085-x

Cited by 26 publications

(11 citation statements)

References 12 publications

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“…The discontinuous glycerol gradient procedure yielded pea nuclei that were virtually free of other host organelles and bacteria as judged by light and electron microscopy (6). The procedure appears to be no better or worse than the procedure of Hamilton et al (14), the chief advantage of the glycerol gradient procedure being that no detergent is used so that the nuclear membranes are more likely to be in a "native" state.…”

Section: Resultsmentioning

confidence: 84%

“…However, bacterial nucleic acid synthesis did not contribute significantly to the data. No bacteria were detected in sections of the pelleted nuclear fraction; no leaf bacteria grew on media containing 10 ,ug of AMD per ml; and chloroplast fractions, which did contain some bacteria, showed little PEMV RNA synthesis (6).…”

Section: Discussionmentioning

confidence: 99%

“…The substrate requirements of the RNA-dependent RNA-polymerase in nuclei from peas systemically infected with PEMV have been previously investigated (6). It was, therefore, of interest to determine the substrate requirements of the polymerase from healthy nuclei primed with PEMV RNA.…”

Section: Resultsmentioning

confidence: 99%

“…The assay for RNA-dependent RNA polymerase was performed as reported by Zabel et al (11). The details of this assay, including some minor modifications, are described by Powell et al (6). The Hybridization.…”

Section: Methodsmentioning

confidence: 99%

“…The nonsenescing leaves were harvested when the plants were about 15 cm high. Fifty grams of these leaves was homogenized and fractionated on discontinuous glycerol gradients as described (6). The glycerol gradient pellets contained predominantly nuclei and starch with a few chloroplast granar-lamellae.…”

Section: Methodsmentioning

confidence: 99%

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Replication of pea enation mosaic virus RNA in isolated pea nuclei

Powell¹,

Zoeten²

1977

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

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The only plant virus that has been unequivocally shown to replicate in association with the nucleus is pea enation mosaic virus (PEMV) (5, 6). Thus, PEMV was a suitable choice for studying organelle-associated virus replication at the molecular level. To facilitate this study, a procedure was developed by which nuclei were isolated from healthy peas and treated with various viral RNAs or viruses. This system was subsequently used to study the specificity of RNA replication in nuclei and the early events of PEMV RNA replication. MATERIALS AND METHODSVirus and Virus RNA. PEMV and southern bean mosaic virus (SBMV) were purified as described by German and de Zoeten (7) and Ball and Brakke (8), respectively. Radish mosaic virus (RdMV) was purified by Steere's (9) chloroform/butanol method. Tobacco mosaic virus (TMV) was obtained in purified form from R. W. Fulton. The RNAs from these four viruses were prepared by a sodium dodecyl sulfate disruption buffer method (10), followed by sucrose density gradient centrifugation (5). The RNAs were tested for homogeneity on 2.5% polyacrylamide gels and for infectivity on appropriate indicator hosts.Purification of Nuclei. Peas (Pisum sativum L. cv. "Per- fected Wales") were grown in a controlled environment (21°, day; 16°, night; 12 hr of light). The nonsenescing leaves were harvested when the plants were about 15 cm high. Fifty grams of these leaves was homogenized and fractionated on discontinuous glycerol gradients as described (6). The glycerol gradient pellets contained predominantly nuclei and starch with a few chloroplast granar-lamellae. The pellets were resuspended in a total volume of 2. were also prepared for electron microscopy (6) and evaluated ultrastructurally. Treatment of Nuclei. The purified nuclei were primed within 1 hr after resuspension in polymerase assay buffer. A sample (0.1 ml) of the nuclei suspension was mixed with appropriate amounts of viral RNA or intact virus, 10-fold concentrated polymerase assay buffer, bentonite, and H20 so that the final concentrations of the ingredients were 33 ,ug of RNA per standard polymerase assay buffer and 100 jg of bentonite per ml in a total volume of 0.3 ml. When intact virus was the primer, a virus concentration was used that would yield 33 ,g of RNA per ml on the basis of 100% uncoating (controls contained no virus or virus RNA). The primed nuclei were then shaken at 300 for 16 hr unless stated otherwise.Polymerase Assay. The assay for RNA-dependent RNA polymerase was performed as reported by Zabel et al. (11 Hybridization. The total nucleic acid was isolated, by phenol extraction followed by ethanol precipitation (7), from 0.3-ml nuclei samples that had been incubated with PEMV RNA or buffer for 16 hr. The concentration of total nucleic acid was determined spectrophotometrically (5), and the percentages of DNA and RNA were determined by the diphenylamine reaction (12) and the orcinol reaction (13), respectively. PEMV (+) strand RNA was prepared as previously described and labeled with 125I (5). Hybridizat...

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Section: Resultsmentioning

confidence: 84%

Section: Discussionmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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