A retroviral vector containing the lacZ a-complementation gene region as a neutral mutational target was used to score null mutations appearing during a single infection cycle (2). Out of 16867 clones, 37 carried null mutations in the lacZ a-complementation gene region based on the white/blue assay of transformed Escherichia coli colonies. Sequencing showed that 11 were nucleotide substitutions (including two nonsense mutations), 24 were indels (5 frameshifts), and two were 15-base G®A hypermutations. The coding region the lacZ region is 258 bases long (280 bases including the promoter region), but the mutational target is smaller because many mutations will not lead to the null phenotype. In a previous study, it was determined that T_s = 219 (1). Hence, for substitutions not caused by host-mediated hypermutation, m_s/n/c = 3 ´ 11 / 16867 / 219 = 8.9 ´ 10^-6. Alternatively, we used the method based on scoring stop codons. Since there are 20 possible nonsense substitutions in the lacZ a-complementation sequence and all should lead to the null phenotype, the mutation rate is m_s/n/c = 3 ´ 2 / 16867 / 20 = 1.8 ´ 10^-5. We used the latter value. Considering that all G®A hypermutations should lead to loss of function and including the promoter in the mutational target (T_s = 280), the G®A mutation rate due to host-mediated hypermutation is 2 ´ 15 / 16867 / 280 = 6.3 ´ 10^-6. Hence, the total mutation rate to substitutions is m_s/n/c = 6.3 ´ 10^-6 + 1.8 ´ 10^-5 = 2.4 ´ 10^-5. For indels, it was determined that T_i = 150 for frameshifts T_i = 280 for the other indels. Hence m_i/n/c = 5 / 16867 / 150 + 19 / 16867 / 280 = 6.0 ´ 10^-6. The indel ratio is d = 0.25.

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. Pathak, V. K. and H. M. Temin. 1990. Broad spectrum of in vivo forward mutations, hypermutations, and mutational hotspots in a retroviral shuttle vector after a single replication cycle: substitutions, frameshifts, and hypermutations. Proc. Natl. Acad. Sci. USA 87:6019-6023.