UTP-3H in the presence of the factors necessary for RNA synthesis, was incorporated into an acid-insoluble product upon incubation in cell-free homogenates from barley leaves infected with bromegrass mosaic virus. RNA was extracted by treatment of the homogenate with phenol and detergent, precipitated with 2 volumes of ethanol, resuspended in appropriate buffer, and the acid-insoluble radioactivity was determined after Millipore filtration. The incorporation of UTP-3H was suppressed in the absence of the three other unlabeled nucleotides, or in the presence of 0.1 M sodium pyrophosphate. Under suitable conditions of infection and fractionation, the incorporation of UTP as acid-insoluble product in the homogenate from infected leaves was resistant to actinomycin D (20 μg/ml) and to 0.1 M sodium orthophosphate. When assayed under similar conditions, incorporation of UTP-3H in the homogenate from healthy leaves was inhibited 85% by actinomycin, and the labeled product was almost completely hydrolyzed by RNase (10 μg/ml). Part of the labeled acid-insoluble product synthesized in homogenates from infected leaves was resistant to RNase (10 μg/ml) in 0.14 M NaCl, 0.014 M sodium citrate, pH 7.2, and the RNase-resistant material was also resistant to DNase (50 μg/ml). When the RNase-resistant product was heated for 15 min at 100° and rapidly cooled, it was rendered completely acid-soluble upon subsequent treatment with 5 μg/ml of RNase. The labeled product was also made acid-soluble after incubation in 0.2 N NaOH. These results indicate that the product of 3H incorporation by infected homogenates is partially in the form of double-stranded RNA. The RNA's were fractionated by centrifugation in sucrose density gradients. The labeled product extracted from healthy homogenates sedimented slowly, with a peak in the 4-6 S region. The labeled material extracted from infected homogenates was heterogeneous; a major peak was obtained in the 12-14 S region, with sedimentation properties comparable to those of double-stranded structures described for other RNA viruses. © 1968.
