Barley stripe mosaic virus replicative form RNA: Preparation and characterization

Abstract

The replicative form (RF) of barley stripe mosaic virus RNA was isolated after in vivo 32P-labeling followed by LiCl separation of the phenol-extracted nucleic acids and density gradient centrifugation. The RF was resistant to 0.1μg ribonuclease/ml in SSC (0.15 M sodium chloride, 0.015 M sodium citrate) susceptible to the enzyme at 1 μg/ml in 0.01 × SSC, resistant to deoxyribonuclease, and susceptible to alkaline degradation. The RF sedimented at 12.7 S, prior to and after formaldehyde treatment, and had an estimated molecular weight of 1.73–1.88 × 10 6 daltons. Single-stranded viral RNA (21.3 S) had an estimated molecular weight of 8.85 × 10 5 daltons, based on sedimentation after formaldehyde treatment. In addition to the major 21.3 S zone, a slower sedimenting RNA (19.5 S) was detected. A shoulder RF species, apparently corresponding to the 19.5 S single-stranded RNA, sedimented at 11.2 S. The buoyant density of the RF in Cs 2SO 4 was 1.595 g/cc, and that of single-stranded RNA was 1.630 g/cc. Maximum 32P incorporation into RF occurred prior to the period of maximum concentration of single-stranded viral RNA, but 32P-labeled single-stranded viral RNA was detected before RF could be isolated.