The fidelity of poliovirus RNA-dependent RNA polymerase (3D(pol)) was deter
mined using a system based on the fidelity of synthesis of the alpha -lac g
ene which codes for a subunit of beta -galactosidase. Synthesis products ar
e screened for mutations by an alpha -complementation assay, in which the p
rotein product from alpha -lac is used in trans to complement beta -galacto
sidase activity in bacteria that do not express alpha -Lac. Several polymer
ases 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 f
or the N-terminal region of alpha -Lac. The product of this reaction was us
ed 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 bacteri
a were screened for beta -galactosidase activity by blue-white phenotype an
alysis with white or faint blue colonies scored as errors made during synth
esis on alpha -lac. Results showed a mutation rate for 3D(pol) correspondin
g to approximate to 4.5 x 10(-4) errors per base (one error in approximate
to 2200 bases). Analysis of mutations showed that base substitutions occurr
ed with greater frequency than deletions and insertions. (C) 2001 Elsevier
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