This 1973 study characterizes double-stranded RNA (dsRNA) isolated from Vigna leaves infected with cowpea mosaic virus (CPMV). The researchers developed a standardized isolation procedure using DNase treatment, high-salt precipitation, and gel filtration to obtain RNase-resistant RNA from the chloroplast-nucleoli fraction. They characterized the dsRNA through cesium sulfate density gradient centrifugation, finding a buoyant density of 1.595 g/cm³, consistent with CPMV's 40% guanine-cytosine content. Thermal melting analysis yielded a melting temperature of 87-94°C. Electron microscopy of 397 molecules revealed a heterogeneous size distribution with two populations: one at 1.2-1.4 μm corresponding to middle component RNA and another at 2.0-2.4 μm corresponding to bottom component RNA. These sizes matched the expected lengths for middle and bottom component RNAs based on molecular weights. Hybridization experiments demonstrated virus specificity—labeled CPMV RNA hybridized to the dsRNA but labeled TYMV RNA did not, confirming the dsRNA contained CPMV sequences. The findings support the hypothesis that middle and bottom component RNAs each induce formation of their own replicative forms in infected cells.

Key findings

• Double-stranded RNA isolated from CPMV-infected leaves has a buoyant density of 1.595 g/cm³ in cesium sulfate, corresponding to the expected density for CPMV dsRNA with 40% G-C content
• Electron microscopy identified two distinct populations of dsRNA molecules: 1.2-1.4 μm (middle component) and 2.0-2.4 μm (bottom component), consistent with each viral RNA component having its own replicative form
• Melting temperature analysis (Tm = 87-94°C) confirmed the dsRNA structure and correlated appropriately with the established relationship between Tm and G-C content for other virus RNAs
• Hybridization experiments demonstrated that dsRNA specifically incorporates labeled CPMV RNA but not TYMV RNA, confirming virus specificity and supporting the biological relevance of the isolated material
• The successful isolation procedure enabled purification of viral dsRNA using DNase treatment, high-salt precipitation, and equilibrium density gradient centrifugation, establishing a standard method for studying RNA virus replication