title: Determination of the poliovirus RNA polymerase error frequency at eight sites in the viral genome.
abstract: The poliovirus RNA polymerase error frequency was measured in vivo at eight sites in the poliovirus genome. The frequency at which specific G residues in poliovirion RNA changed to another base during one round of viral RNA replication was determined. Poliovirion RNA uniformly labeled with 32Pi was hybridized to a synthetic DNA oligonucleotide that was complementary to a sequence in the viral genome that contained a single internal G residue. The nonhybridized viral RNA was digested with RNase T1, and the protected RNA oligonucleotide was purified by gel electrophoresis. The base substitution frequency at the internal G residue was measured by finding the fraction of this RNA oligonucleotide that was resistant to RNase T1 digestion. A mean value of 2.0 x 10(-3) +/- 1.2 x 10(-3) was obtained at two sites. A modification of the above procedure involved the use of 5'-end-labeled RNA oligonucleotides. The mean value of the error frequency determined at eight sites in the viral genome by using this technique was 4.1 x 10(-3) +/- 0.6 x 10(-3). Sequencing two of the RNase T1-resistant RNA oligonucleotides confirmed that the internal G was changed to a C, A, or U residue in most of these oligonucleotides. Thus, our results indicated that the polymerase had a high error frequency in vivo and that there was no significant variation in the values determined at the specific sites examined in this study.
authors.affiliation: Department of Immunology and Medical Microbiology, College of Medicine, University of Florida, Gainesville 32610-0266.
authors.forename: C D
authors.initials: CD
authors.lastname: Ward
authors.forename: J B
authors.initials: JB
authors.lastname: Flanegan
mesh_terms: D001483:Base Sequence; D002874:Chromosome Mapping; D016679:Genome, Viral; D006151:Guanosine / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000378:metabolism; D017955:Poliovirus / Q000235:genetics*; D012367:RNA, Viral / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism*; D006163:Ribonuclease T1 / Q000378:metabolism; D014771:Virion / Q000254:growth & development; D014779:Virus Replication
chemical_list: D009841:Oligonucleotides; D012367:RNA, Viral; D006151:Guanosine; D012324:RNA-Dependent RNA Polymerase; D006163:Ribonuclease T1