Determination of the mutation rate of poliovirus RNA-dependent RNA polymerase.
The fidelity of poliovirus RNA-dependent RNA polymerase (3D(pol)) was determined using a system based on the fidelity of synthesis of the alpha-lac gene which codes for a subunit of beta-galactosidase. Synthesis products are screened for mutations by an alpha-complementation assay, in which the protein product from alpha-lac is used in trans to complement beta-galactosidase activity in bacteria that do not express alpha-Lac. Several polymerases have been analyzed by this approach allowing comparisons to be drawn. The assay included RNA synthesis by 3D(pol) on an RNA template that coded for the N-terminal region of alpha-Lac. The product of this reaction was used as a template for a second round of 3D(pol) synthesis and the resulting RNA was reverse transcribed to DNA by MMLV-RT. The DNA was amplified by PCR and inserted into a vector used to transform Escherichia coli. The bacteria were screened for beta-galactosidase activity by blue-white phenotype analysis with white or faint blue colonies scored as errors made during synthesis on alpha-lac. Results showed a mutation rate for 3D(pol) corresponding to approximately 4.5x10(-4) errors per base (one error in approximately 2200 bases). Analysis of mutations showed that base substitutions occurred with greater frequency than deletions and insertions.
Department of Cell Biology and Molecular Genetics, University of Maryland College Park, College Park, MD 20742, USA.
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Wells
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Plotch
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DeStefano
D001483:Base Sequence; D004252:DNA Mutational Analysis; D004274:DNA, Recombinant; D004926:Escherichia coli; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009154:Mutation; D017955:Poliovirus / Q000201:enzymology* / Q000235:genetics; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics* / Q000378:metabolism; D013379:Substrate Specificity; D013698:Templates, Genetic; D001616:beta-Galactosidase / Q000235:genetics
D004274:DNA, Recombinant; C090443:polymerase 3Dpol, poliovirus; D012324:RNA-Dependent RNA Polymerase; D001616:beta-Galactosidase