Figure 5.—

Tracking genotypic changes over time. (A) PCR amplification of bursts 34–47 of 600-molecule lineage 5A, which survived at least to burst 50. There is no development of a smear above the 187-bp band, which are the amplicons from wild-type-length RNAs (152 nt). This lineage did not suffer a mutational load imposed by 3′ polyadenylations, while it did evolve both the immunity and the insurance mutations (see C). M denotes size marker (1-kb ladder). (B) PCR amplification of 20 cloned molecules from burst 13 of a 100-molecule lineage that went extinct in burst 18. Size differences are a consequence of 3′ polyadenylations. (C) RFLP analysis of bursts 13–41 of 600-molecule lineage 5A, which survived to burst 50 (shown also in A). The TaqαI digest distinguishes between the wild-type (wt) B16-19 genotype and the immunity (M) mutant; the XmnI digest distinguishes between the B16-19 genotype and the insurance (S) mutant. In this lineage, the immunity mutation arises in about burst 15 and is rapidly fixed in the population by burst 21. The insurance mutation arises later, around burst 20, and slowly accumulates to fixation around burst 43 (not shown on gel).