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Sequential Synthesis of Double-stranded and Single-stranded RNA by Cell-free Extracts of Barley Leaves Infected with Brome Mosaic Virus

Faculté des Sciences agronomiques, 5800 Gembloux, Belgium

A particulate cell-free fraction of barley leaves infected with brome mosaic virus had RNA polymerase activity in the presence of actinomycin D and EDTA. The RNA produced during a short pulse (3 min.) of ³H-labelled UTP was mostly resistant to RNase in high salt concentration, whereas longer labelling periods gave an increasingly RNase-sensitive product. ‘Pulse-chase’ experiments showed that the first product labelled sedimented at about 14 s, was presumably a double-stranded RNA and was the precursor of single-stranded RNA, most of which sedimented more slowly. Production of the single-stranded RNA depended upon the presence of all four ribonucleotides. Little complete radioactive brome mosaic virus RNA could be found, possibly because of the effect of nucleases in the reaction mixture. When heated, the 14 s RNase-resistant RNA gave polydisperse single-stranded RNA, some of which sedimented at a similar rate to the large component of brome mosaic virus RNA. Most of the RNase-resistant RNA did not behave like replicative intermediates that have been described.

Received 13 July 1970; accepted 15 September 1970.