Symmetrical in vivo transcription of polyoma DNA and the separation of self-complementary viral and cell RNA

Abstract

Mouse kidney cells infected with polyoma virus were labeled for 20 min with [5,6- 3H]uridine late in infection. The rapidly-labeled RNA was extracted from whole cells and from the Hirt SDS/high salt supernatant fraction. The RNA was self-annealed and became resistant to ribonuclease digestion. The RNase-resistant RNA was isolated by chromatography on Sephadex G-100 and shown to be double-stranded RNA by several different criteria: resistance to RNase and to the combined effects of RNase and DNase, the characteristic buoyant density of double-stranded RNA in Cs 2SO 4, a buoyant density increase upon denaturation to that of single-stranded viral RNA marker, an inability to hybridize with complementary DNA unless denatured, and a high T m value with a sharp transition to RNase sensitivity. Separated strands of the double-stranded RNA hybridized with high efficiency with component I of polyoma DNA. After self-annealing in formamide at low temperature, from 18 to 30% of the total rapidly-labeled viral RNA of infected cells sedimented at 11 S; 11 S corresponds in size to about 30% of the polyoma DNA. The extraction procedure for double-stranded viral RNA also yields double-stranded cellular RNA, but the double-stranded viral RNA can be further purified by using the Hirt supernatant, the RNaseresistance of the viral RNA coupled with its higher T m , its greater sedimentation coefficient. These observations indicate that, late in infection of mouse cells, polyoma DNA is transcribed symmetrically over a considerable portion of its length, yielding self-complementary RNA that is distinguishable from the cellular self-complementary RNA.