Reovirus replicase-directed synthesis of double-stranded ribonucleic acid.
After the incubation of reovirus replicase reaction mixtures (containing labeled ribonucleoside triphosphates), partially double-stranded ribonucleic acid (pdsRNA) products were isolated by cellulose column chromatography followed by precipitation with 2 m NaCl. The pulse-labeled reaction product contained a significantly large amount of pdsRNA that became complete dsRNA as reaction time increased, indicating that pdsRNA was an intermediate of the replicase reaction. The newly synthesized RNA strand ((3)H-labeled) of the pdsRNA was resistant to ribonuclease digestion, suggesting that single-stranded RNA regions were part of a preexistent unlabeled RNA template. These observations, together with the electrophoretic behavior of the pdsRNA in polyacrylamide gel, are consistent with the hypothesis that dsRNA is synthesized by the elongation of a complementary RNA strand upon a preexistent template of single-stranded RNA (i.e., messenger RNA). The direction of the RNA strand elongation was determined by carrying out the replicase reaction in the presence of (3)H-cytidine triphosphate (or (3)H-uridine triphosphate) and adenine triphosphate-alpha-(32)P followed by a chase with excess unlabeled cytidine triphosphate (or uridine triphosphate). The dsRNA product was digested with T1 ribonuclease and the resulting 3'-terminal fragments were isolated by chromatography on a dihydroxyboryl derivative of cellulose. Examination of the ratio of (3)H to (32)P in these fragments indicated that RNA synthesis proceeded from the 5' to 3' terminus.
D000818:Animals; D002247:Carbon Isotopes; D002474:Cell-Free System; D002499:Centrifugation, Density Gradient; D011232:Chemical Precipitation; D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D007739:L Cells; D051379:Mice; D009691:Nucleic Acid Denaturation; D010759:Phosphorus Isotopes; D012316:RNA Nucleotidyltransferases / Q000378:metabolism*; D012333:RNA, Messenger; D012367:RNA, Viral / Q000032:analysis / Q000096:biosynthesis* / Q000302:isolation & purification; D012087:Reoviridae / Q000201:enzymology / Q000254:growth & development / Q000378:metabolism*; D012260:Ribonucleases; D012265:Ribonucleotides / Q000378:metabolism; D012965:Sodium Chloride; D013698:Templates, Genetic; D014316:Tritium
D002247:Carbon Isotopes; D010759:Phosphorus Isotopes; D012333:RNA, Messenger; D012367:RNA, Viral; D012265:Ribonucleotides; D014316:Tritium; D012965:Sodium Chloride; D012316:RNA Nucleotidyltransferases; D012260:Ribonucleases