Amount and distribution of virus-specific sequences in giant RNA molecules isolated from polyoma-infected mouse kidney cells

Abstract

A two-step hybridization with polyoma DNA was used to study the composition of giant RNA molecules synthesized in mouse kidney cells late in productive infection by polyoma virus. Giant molecules longer than a complete transcript of the polyoma genome were purified from cells that had been pulse-labeled for 30 min with [3H]uridine and annealed, under mild conditions (50% formamide, 37 degrees C), with polyoma DNA loaded on nitrocellulose filters. Hybridized RNA (6 to 7% of the entire population of 3H-labeled molecules and up to 15% of the molecules containing polyadenylic acid [poly(A)]] was eluted and annealed a second time with polyoma DNA under more stringent conditions. In this second step, 75% of the 3H-labeled RNA formed an RNase-resistant hybrid. Under the same conditions, complementary RNA hybridized with polyoma DNA to a maximal extent of 80%. Since the difference between 75 and 80% is within the experimental error of the hybridization assay, it is inferred that the giant molecules selected by the first hybridization may consist entirely of virus-specific sequences or contain, at the most, a minor fraction of nonviral sequences. To examine the possibility that such nonviral sequences are clustered at the 3'-terminus of these molecules, poly(A)+ giant RNA, which had not been preselected by hybridization with polyoma DNA, was fragmented by a limited alkaline hydrolysis. Fragments linked to the poly(A) segment were separated from the rest of the cleavage products. A one-step hybridization with polyoma DNA revealed that both fractions contain 8 to 10% of virus-specific sequences. These results indicate that the 3'-termini of the poly(A)+ polyoma-specific giant RNA molecules consist of viral rather than nonviral sequences.