Ing that, at least in some cases, the genomes of individuals in poor physiological condition tend to mutate more readily than do genomes of individuals in good condition [25,26,27]. One cause of poor condition is a pre-existing load of deleterious mutations. If it can be established that (1) conditions that reduce Title Loaded From File fitness lead to an increase in oxidative stress and (2) an increase in oxidative stress leads to an increase in the rate and/or a change in the spectrum of heritable mutations, then these hypotheses will be tied together and independently strengthened. We found that nematodes from MA lines exhibited higher levels of steady-state oxidative stress in the soma than did nematodes from the ancestral control. Conversely, the correlation between the measures of oxidative stress and the frequencies of base substitution or G-to-T transversions in the nuclear genome was small and not significantly different from zero.Materials and Methods (i) Experimental LinesWe studied five C. elegans MA lines and their common ancestor (MA generation 0, or “G0”) that were generated as part of a large MA experiment [28]. These five particular lines were chosen because whole-genome sequence data are available [19,29]; the nuclear base substitution rates for these MA lines indicated more G:C-T:A transversions than Title Loaded From File observed in nature, a pattern that could be interpreted as evidence of elevated oxidative stress in the MA lines [19], particularly since C. elegans may have limited DNA repair capabilities compared to other metazoans [30,31]. The MA lines are derived from a single, highly inbred N2 strain hermaphrodite; the lines independently experienced 250 generations of serial transfer (a bottleneck; 250 MA generations) of a single individual [32]. Under these conditions, the effective population size, Ne<1 and selection is minimally efficient. Since mutations with selective effect, s,1/4Ne are effectively neutral [20,21], all but the most highly deleterious mutations (s.0.25) are expected to accumulate at the neutral rate. Details of the MA protocol and the mutational declines in fitness in the MA lines (at G200) relative to the ``unmutated'' ancestor (G0) are reported in [28].groups. We performed confocal image analysis on live young adult nematodes using our previously described methods [34,35,36]. Briefly, nematodes were incubated for 24 hours at 20uC in the presence or absence of 10 mM MitoSOX Red (in water; Molecular Probes Inc.), a mitochondria-targeted dye that fluoresces when in contact with (total) mitochondrial oxidants, reflecting both ROS generation and ROS scavenging [37]. Total oxidant production was measured in the pharyngeal bulb, a tissue that is particularly suited for assessment of oxidative stress because it has high metabolic activity and dense populations of mitochondria [38], the primary source of endogenous ROS. It is important to note that the ROS data described mitochondrial oxidative stress while the mutation data were derived from the nuclear genome. Although mitochondrial ROS can damage cytoplasmic and nuclear components [39], the relationship between mitochondrial function and nuclear genetic damage is not straightforward, owing to variation in the stability, longevity and diffusion properties of different ROS [40] and because low levels of ROS may alter DNA repair 1676428 activity [41,42,43]. For each line, fluorescent z-stack images of the pharyngeal bulbs of 15-20 treatment (+MitoSOX) and 5 control (-MitoSOX) nematodes that had.Ing that, at least in some cases, the genomes of individuals in poor physiological condition tend to mutate more readily than do genomes of individuals in good condition [25,26,27]. One cause of poor condition is a pre-existing load of deleterious mutations. If it can be established that (1) conditions that reduce fitness lead to an increase in oxidative stress and (2) an increase in oxidative stress leads to an increase in the rate and/or a change in the spectrum of heritable mutations, then these hypotheses will be tied together and independently strengthened. We found that nematodes from MA lines exhibited higher levels of steady-state oxidative stress in the soma than did nematodes from the ancestral control. Conversely, the correlation between the measures of oxidative stress and the frequencies of base substitution or G-to-T transversions in the nuclear genome was small and not significantly different from zero.Materials and Methods (i) Experimental LinesWe studied five C. elegans MA lines and their common ancestor (MA generation 0, or “G0”) that were generated as part of a large MA experiment [28]. These five particular lines were chosen because whole-genome sequence data are available [19,29]; the nuclear base substitution rates for these MA lines indicated more G:C-T:A transversions than observed in nature, a pattern that could be interpreted as evidence of elevated oxidative stress in the MA lines [19], particularly since C. elegans may have limited DNA repair capabilities compared to other metazoans [30,31]. The MA lines are derived from a single, highly inbred N2 strain hermaphrodite; the lines independently experienced 250 generations of serial transfer (a bottleneck; 250 MA generations) of a single individual [32]. Under these conditions, the effective population size, Ne<1 and selection is minimally efficient. Since mutations with selective effect, s,1/4Ne are effectively neutral [20,21], all but the most highly deleterious mutations (s.0.25) are expected to accumulate at the neutral rate. Details of the MA protocol and the mutational declines in fitness in the MA lines (at G200) relative to the ``unmutated'' ancestor (G0) are reported in [28].groups. We performed confocal image analysis on live young adult nematodes using our previously described methods [34,35,36]. Briefly, nematodes were incubated for 24 hours at 20uC in the presence or absence of 10 mM MitoSOX Red (in water; Molecular Probes Inc.), a mitochondria-targeted dye that fluoresces when in contact with (total) mitochondrial oxidants, reflecting both ROS generation and ROS scavenging [37]. Total oxidant production was measured in the pharyngeal bulb, a tissue that is particularly suited for assessment of oxidative stress because it has high metabolic activity and dense populations of mitochondria [38], the primary source of endogenous ROS. It is important to note that the ROS data described mitochondrial oxidative stress while the mutation data were derived from the nuclear genome. Although mitochondrial ROS can damage cytoplasmic and nuclear components [39], the relationship between mitochondrial function and nuclear genetic damage is not straightforward, owing to variation in the stability, longevity and diffusion properties of different ROS [40] and because low levels of ROS may alter DNA repair 1676428 activity [41,42,43]. For each line, fluorescent z-stack images of the pharyngeal bulbs of 15-20 treatment (+MitoSOX) and 5 control (-MitoSOX) nematodes that had.
