A single plaque of a recombinant virus carrying the lacZ a-complementation sequence (258 bases) as neutral mutational target (a = 1) was used to inoculate a large E. coli culture and incubated overnight (3). Viral DNA was extracted and transfected to score null mutations in the lacZ sequence (based on the blue/white colony assay). After discarding 11 false positives, f = 117 / 199655, with 67 plaques containing single-nucleotide substitutions and 50 containing indels (11 frameshifts and 39 deletions or rearrangements). In a previous study (1), it was determined that T_s = 219. Thus, c ´ m_s/n/c = 3 ´ 67 / 199655 / 219 = 4.6 ´ 10^-6. For indels, assuming T_i = 150 for frameshifts and T_i = 280 for other indels (see SNV (i) estimate), c ´ m_i/n/c = 11 / 150 / 199655 + 39 / 280 / 199655 = 1.1 ´ 10^-6. At the very least, c = 3 (two cycles during the formation of the plaque and another one during the infection of the liquid culture). According to Drake (2), the initial and final viral population sizes were 1 and ca. 1.0 ´ 10¹⁵. Our own data suggest that under relatively optimal conditions, the exponential growth rate of the virus is ca 4.0 h^-1 and the duration of the cell infection cycle is ca. 1 h. According to this and assuming exponential growth, an increase in population size by a factor of 1.0 ´ 10¹⁵ would require approximately c = 8.6. Taking the average of the 3 and 8.6, c = 5.8. This gives m_s/n/c = 4.6 ´ 10^-6 / 5.8 = 7.9 ´ 10^-7, m_i/n/c = 1.1 ´ 10^-6 / 5.8 = 1.9 ´ 10^-7, and d = 0.19. The main source of error in this estimate is the undetermined c-value. This could lead to a maximal underestimation of 1.9-fold and a maximal overestimation which, although not determined, probably does not exceed 1.5-fold.

1. Bebenek, K., J. Abbotts, J. D. Roberts, S. H. Wilson, and T. A. Kunkel. 1989. Specificity and mechanism of error-prone replication by human immunodeficiency virus-1 reverse transcriptase. J Biol. Chem. 264:16948-16956.

2. Drake, J. W. 1991. A constant rate of spontaneous mutation in DNA-based microbes. Proc. Natl. Acad. Sci. USA 88:7160-7164.

3. Kunkel, T. A. 1985. The mutational specificity of DNA polymerase-beta during in vitro DNA synthesis. Production of frameshift, base substitution, and deletion mutations. J Biol. Chem. 260:5787-5796.