In vitro viral RNA synthesis in a crude membrane fraction from tobacco [Nicotiana tabacum] protoplasts inoculated with potato virus X
1998
Que, X.F. (Tokyo Univ. of Agriculture and Technology, Fuchu (Japan). Faculty of Agriculture) | Takamatsu, N. | Hosokawa, D.
A membrane fraction was isolated by differential centrifugation from PVX-inoculated tobacco protoplasts to investigate in vitro viral RNA synthesis. This fraction was capable of catalyzing the in vitro synthesis of a genomic-length (6.4 kb) and tow subgenomic-length RNAs (2.1 and 0.9 kb). The different sensitivities f the RNAs to SI nuclease at high and low salt concentrations suggested that the synthesized genomic-length RNA was mainly the replicative intermediate RNA (RI) and that the two subgenomic-length RNAs were single-stranded. Most of the synthesized RNAs were shown to be of a positive polarity based on ribonuclease protection assay. Dependent on the exogenous template, the fraction treated with micrococcal nuclease synthesized only the genomic-length RNA abut not the subgenomic RNAs. Treating the membrane fraction with protease abolished synthesis of the subgenomic-length RNAs, but not that of genomic-length RNA. The synthesizing activity for the subgenomic-length RNAs was released into the soluble fraction after treatment with the detergent Brij 58. These results suggested that genomic and subgenomic RNAs are replicated at different sites in the membrane; genomic RNA synthesis occurs in side the membranes, whereas subgenomic RNA synthesis occurs on their surface
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