The three species of virus RNA (45s, 26s and 20s) formed during infection of chick embryo cells with Semliki Forest virus were isolated by sequential extraction of the cells with phenol and then with phenol+sodium dodecyl sulphate. They were then separated either by sucrose density gradient centrifugation, or by polyacrylamide gel electrophoresis. The 20s RNA was relatively resistant to the action of ribonuclease, in contrast to the single-stranded 26s and 45s species. The 20s RNA was the first species to be formed in infected cells followed by 26s and then by 45s RNA. An attempt to demonstrate the intermediate role of 20s RNA in virus RNA synthesis by the displacement of radioactive by non-radioactive uridine (‘pulse-chase’ experiment) was unsuccessful, but both 26s and 45s RNA displaced radioactivity from 20s RNA on heating above the thermal transition temperature and slow cooling. Comparison of the sedimentation velocities and electrophoretic mobilities of the 26s and 45s RNA species, together with the sharp thermal transition at 65° of 45s RNA to 26s RNA suggested that 45s RNA consisted of two 26s RNA species, and that the different sedimentation characteristics did not indicate different conformations of the same molecule.


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