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.
Wells
V R
VR
Department of Cell Biology and Molecular Genetics, University of Maryland College Park, College Park, MD 20742, USA.
Plotch
S J
SJ
DeStefano
J J
JJ
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