A study of virus-induced RNA polymerase from TMV-infected tobacco leaves

Abstract

The properties of a virus-induced RNA polymerase have been studied in cell-free extracts of TMV-infected tobacco leaves. UTP-³H in the presence of actinomycin D and unlabeled ATP, GTP, and CTP was incorporated into acid-insoluble material following incubation with a cytoplasmic fraction from infected leaves. UTP- ³H incorporation was completely inhibited by actinomycin D in similar preparations from healthy leaves. Viral RNA polymerase activity was strongly surpressed when the three unlabeled nucleoside triphosphates were left out of the incubation mixture, suggesting that the incorporation of UTP- ³H was due to actual RNA synthesis and not simply to incorporation onto existing RNA molecules. Differential and density gradient centrifugation of cell-free extracts of infected leaves demonstrated that the viral RNA polymerase was in a cytoplasmic fraction that sedimented at 12,000 g and was not associated with nuclei and chloroplasts. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Viral RNA polymerase activity was detected in preparations from infected leaves as early as 16 hours after inoculation; the activity increased to a peak 36-48 hours after inoculation and then rapidly decreased. A corresponding rise and fall in total RNA polymerase activity (determined by omitting actinomycin D from the assay mixture) was observed. Optimum production of viral RNA polymerase was obtained using upper leaves and assaying 36 hours after direct inoculation. attached leaves were shown to be better for the production of viral RNA polymerase than detached leaves.