FIG. 4.

Newly synthesized ss RNA can serve as template for further rounds of RNA synthesis. (A) Labeled ss RNA was chased into ds RNA and RNA species longer than monomeric replicon RNA. RNA products were pulse-labeled for 60 min in standard reactions (mixtures contained 10 μCi of [α-³³P] UTP). After the labeling step, a 300-fold excess of unlabeled UTP was added to the reaction mixtures, and the labeled products were chased for 0, 5, 10, 20, 40, and 60 min (lanes 1, 2, 3, 4, 5, and 6, respectively). The positions of ss and ds RNA are indicated with arrows at the left. (B) Nuclease S1 treatment of the extended RNA products. The labeled products were chased for 0, 20 or 60 min in the presence of excess cold UTP. The reaction mixtures were then separated on a 1% agarose gel after no treatment (lanes 1, 3, and 5) or treatment with nuclease S1 (lanes 2, 4, and 6). The small arrows indicate the positions of the extended products with and without nuclease S1 treatment (as indicated, nuclease treatment resulted in smaller products).