A fluctuation test was carried out using the reversion of an amber mutation as selectable phenotype (1).  In each individual culture, the number of initial pfu was high but the virus underwent a single cell infection cycle.  For each of three amber mutants, P0 = 646 / 740, P0 = 679 / 778, and P0 = 510 / 602.  The corresponding burst sizes were B = 167, B = 28, and B = 51, and the final numbers of pfu were 9.0 ´ 107, 5.5 ´ 107, and 2.6 ´ 109, respectively.  Hence, N1 - N0 = 9.0 ´ 107 / 740 - 9.0 ´ 107 / 740 / 167 = 1.2 ´ 105 for the first mutant, and analogously, N1 - N0 = 6.8 ´ 104 and N1 - N0 = 4.2 ´ 106 for the second and third mutants.  Using the null-class method, m = 1.1 ´ 10-6 s/r, m = 2.0 ´ 10-6 s/r, and m = 3.9 ´ 10-8 s/r, respectively, with geometric mean ms = 4.5 ´ 10-7 s/r.  If all amber revertants were to the wild-type, ms/n/r = 3 ´ 4.5 ´ 10-7 = 1.4 ´ 10-6.  However, there are eight possible single-nucleotide revertants and, if all were viable, the mutation rate would be ms/n/r = 3 ´ 4.5 ´ 10-7 / 8 = 1.7 ´ 10-7.  For this phage, the estimated lethal fraction is P = 0.2 (2) and hence the expected number of viable revertants is 8 ´ 0.8 = 6.4, which gives ms/n/r = 2.1 ´ 10-7.

 

 

    1.    Denhardt, D. T. and R. B. Silver. 1966. An analysis of the clone size distribution of phi-X-174 mutants and recombinants. Virology 30:10-19.

    2.    Domingo-Calap, P., J. M. Cuevas, and R. Sanjuán. 2009. The fitness effects of random mutations in single-stranded DNA and RNA bacteriophages. PLoS Genet. 5:e1000742.