Election, whereas mutations accumulated in the genome over the entire 250 generations. For example, it is possible that the two lines with individually significant increases in ROS level experienced mutations that affected some feature of ROS metabolism only recently, in which case the increased ROS would have had little time to contribute to the mutational process. Second, since mtDNA mutations are not characterized in these lines, we inhibitor cannot assess the potential contribution of mitochondrial oxidative stress to mutational processes in mtDNA. Third, the (nuclear) mutation rate (which is distinct from the frequency of mutations) does not differ significantly between MA lines; the differences among lines in base substitution frequency is no more extreme than expected if mutations are Poisson distributed among lines with a uniform mutation rate [19]. The fact that mutation rate does not differ between lines suggests that there is no variable process underlying the base substitution process. Fourth, oxidative damage is only one contributor to the base substitution mutation process; baseRelaxed Selection and Oxidative Stressmisincorporation resulting from polymerase errors also contributes. Our marker of oxidative damage, 8-oxodG in total DNA pools (nuclear and mitochondrial), is only one potential cause of transversion mutations and thus may be a less reliable indicator of mutation resulting from oxidative damage than previously thought [13]. Finally, it is important to note that we only considered base substitutions in the nuclear genome, and that there is evidence that the mutagenic effects of oxidative stress primarily result in other types of mutations in somatic tissues, including large deletions and genome rearrangements [63,64]. An additional consideration is that we measured oxidative damage in the soma, whereas we measured heritable mutations that occurred in the germline. Elements of the DNA repair process [1,65,66,67,68,69,70,71] and antioxidant defense systems [18] are known to differ between the soma and the germline; however, evidence is emerging that somatic oxidative stress is associated with and may even contribute to DNA damage and/or mutation in the germline [18]. However, to the extent that the estimates of DM of ROS and 8-oxodG reported here are trustworthy, 23148522 there is every reason to expect that the processes responsible for maintaining the oxidative millieu of the germline will have undergone similar mutational degradation over the 250 generations of relaxed selection. If germline oxidative metabolism has not undergone similar mutational degradation, it could only be for one of two (nonexclusive) reasons: either the mutational target presented by the germline is for some reason much smaller than the target presented by the soma, in which case the inevitable mutational decay would take longer, or the fraction of mutations that can affect germline oxidative metabolism and are strongly deleterious (4Nes,1) is much larger. The not-unreasonablepossibility that mutations affecting germline oxidative metabolism are extremely deleterious has an important implication: it argues against variation in oxidative metabolism having an important role in the process 1676428 of molecular evolution. The study reported here was ultimately motivated by the possibility that oxidative stress is a causal factor underlying Epigenetic Reader Domain condition-dependent mutation. The results provide no direct support for such a causal relationship, at least not with respect to ba.Election, whereas mutations accumulated in the genome over the entire 250 generations. For example, it is possible that the two lines with individually significant increases in ROS level experienced mutations that affected some feature of ROS metabolism only recently, in which case the increased ROS would have had little time to contribute to the mutational process. Second, since mtDNA mutations are not characterized in these lines, we cannot assess the potential contribution of mitochondrial oxidative stress to mutational processes in mtDNA. Third, the (nuclear) mutation rate (which is distinct from the frequency of mutations) does not differ significantly between MA lines; the differences among lines in base substitution frequency is no more extreme than expected if mutations are Poisson distributed among lines with a uniform mutation rate [19]. The fact that mutation rate does not differ between lines suggests that there is no variable process underlying the base substitution process. Fourth, oxidative damage is only one contributor to the base substitution mutation process; baseRelaxed Selection and Oxidative Stressmisincorporation resulting from polymerase errors also contributes. Our marker of oxidative damage, 8-oxodG in total DNA pools (nuclear and mitochondrial), is only one potential cause of transversion mutations and thus may be a less reliable indicator of mutation resulting from oxidative damage than previously thought [13]. Finally, it is important to note that we only considered base substitutions in the nuclear genome, and that there is evidence that the mutagenic effects of oxidative stress primarily result in other types of mutations in somatic tissues, including large deletions and genome rearrangements [63,64]. An additional consideration is that we measured oxidative damage in the soma, whereas we measured heritable mutations that occurred in the germline. Elements of the DNA repair process [1,65,66,67,68,69,70,71] and antioxidant defense systems [18] are known to differ between the soma and the germline; however, evidence is emerging that somatic oxidative stress is associated with and may even contribute to DNA damage and/or mutation in the germline [18]. However, to the extent that the estimates of DM of ROS and 8-oxodG reported here are trustworthy, 23148522 there is every reason to expect that the processes responsible for maintaining the oxidative millieu of the germline will have undergone similar mutational degradation over the 250 generations of relaxed selection. If germline oxidative metabolism has not undergone similar mutational degradation, it could only be for one of two (nonexclusive) reasons: either the mutational target presented by the germline is for some reason much smaller than the target presented by the soma, in which case the inevitable mutational decay would take longer, or the fraction of mutations that can affect germline oxidative metabolism and are strongly deleterious (4Nes,1) is much larger. The not-unreasonablepossibility that mutations affecting germline oxidative metabolism are extremely deleterious has an important implication: it argues against variation in oxidative metabolism having an important role in the process 1676428 of molecular evolution. The study reported here was ultimately motivated by the possibility that oxidative stress is a causal factor underlying condition-dependent mutation. The results provide no direct support for such a causal relationship, at least not with respect to ba.
