We find that ΔdinB cells expressing the DinB(F13V) variant have a modest increase in both MMS and NFZ-induced mutant frequency when compared to either ΔdinB or to those expressing DinB(D103N) (Fig. 7).
This plot gives a slope of −1 when mutant frequencies show a Luria Delbrück distribution (Luria 1951).
Using flow cytometry, the main subsets of mutant mice displayed similar relative frequencies when compared to age-matched wild-type controls in both neonatal and adult mice (data not shown and Figure S2A).
For large populations, our method is found to provide a much better approximation to the mutant-frequency distribution when selection is strong, while all three methods perform comparably when selection is weak.
For example, without AZT when epistasis is highest, the relative mutant frequencies are around 10−8 to 10−9 and epistasis affect those frequencies up to 3-fold (Figure 4 and Table S3).
In contrast, the insertion-deletion mutant frequencies of the two forms were similar (data not shown) when examined using a 6-nucleotide gap-filling substrate containing a template TTTT run in the +1 reading frame.
According to the mutant frequencies the isolates were grouped in strong mutators (20 times the mutant frequency of PAO1 (≥2×10−7), weak mutators (>1×10−8<2×10−7) or nonmutators [46], [47].
A mutant frequency of 100% was observed when 0.05 pmole of the first flanking primer was used in the first PCR, but the efficiency decreased drastically (36%) when a normal amount of primer (1.0 pmole) was used.
Combined across age, a significant difference was observed when the mean mutant frequency of hippocampus or hypothalamus were compared to the mean mutant frequency of cortex (p = 0.0305; p<0.00001), remainder (p = 0.0148; p<0.0001) or when the mutant frequency of hypothalamus was compared to total brain (p<0.0001) (Table S1).
This result is unsurprising since random departures from the mean mutant frequency will be approximately normal only when the population size is large and selection is strong enough to prevent stochastic loss.
