Figure 1.
The locations of TaqI sites in Drosophila mtDNA.
Schematic depiction of the Drosophila melanogaster mitochondrial genome. Numbers (1-33) indicate the different TaqI sites, color coded according to their utility for RMC analysis as indicated. Boxed numbers indicate the TaqI sites used for RMC analysis in our work. tRNA genes are represented by horizontal lines adjacent to other mitochondrial genes.
Figure 2.
Mitochondrial DNA mutation frequency increases with age and is greater in thoraces than in heads.
(A) The mtDNA mutation frequency is greater in young thoraces than in young heads (**p<0.01). (B) The mtDNA mutation frequency is greater in old heads than in young heads (***p<0.001) (C) and is greater in old thoraces than in young thoraces (*p<0.05). (D) The mtDNA mutation frequency is greater in old thoraces than in old heads (**p<0.001). Horizontal bars represent the median mutation frequency, and error bars indicate the interquartile range. Significance was tested using Mann-Whitney unpaired U tests.
Figure 3.
GC∶TA transversions account for only a small percentage of mtDNA mutations.
The percentage and total number (in parentheses) of each type of mutation identified at TaqI sites in young and old animals. Results depict data obtained separately from heads and thoraces and also combined. The wild-type TaqI sequence (TCGA) is shown for reference. * = Two mutations identified within a single cloned TaqI site.
Figure 4.
Oxidative stress response pathway mutants do not have an increased mtDNA mutation frequency.
(A) Lifespan of w1118 and Sod2 mutant flies. The median lifespan (indicated by dashed lines) was 60 days for w1118 flies and 41 days for Sod2 mutants. (B) The mutation frequency using pooled data from all three TaqI sites in old Sod2 mutants (39- to 41-day-old), young Ogg1ins/Ogg1ins; Sod2 double mutants (1- to 3- day-old), old Ogg1ins/Ogg1ins; Sod2 double mutants (26- to 28- day-old), and age-matched w1118 controls. The 26- to 28- day-old control estimate was obtained from linear regression of the age-dependent w1118 mutation frequency. Horizontal bars represent the median mutation frequency, and error bars indicate the interquartile range. There was no significant difference in mutation frequency between mutants and age-matched controls (Mann-Whitney unpaired U tests).
Figure 5.
An Ogg1 mutation diminishes Ogg1 activity and interacts genetically with Sod2.
(A) Amino acid sequence alignment of human Ogg1 with Drosophila Ogg1 and RpS3. Dark shading denotes amino acid identities; light shading denotes amino acid similarities. The putative N-terminal mitochondrial targeting sequence of Drosophila Ogg1 is underlined. (B) The Ogg1 gene comprises four exons, designated by rectangles, and the coding sequence is indicated with dark gray shading. The Ogg1f08013 piggyBac insertion (designated by a triangle) is located 78 base pairs upstream of the Ogg1 translation start site. (C) 8-oxo-dG repair activity was assayed by monitoring cleavage of a 32P-labeled synthetic probe containing 8-oxo-dG, using protein extracts from flies of the indicated genotypes (Df = the Df(1)BSC627 deletion that removes Ogg1; ins = the Ogg1f08013 piggyBac insertion). Results of the assay are shown following 0, 30, and 60 minutes of incubation. Neg = No protein; Pos = Mouse brain protein extract. (D) Lifespan of w1118, Ogg1ins/Ogg1ins, Sod2, and Ogg1ins/Ogg1ins; Sod2 double mutant flies. The median lifespan (indicated by dashed lines) was 60 days for w1118, 49 days for Ogg1ins mutants, 41 days for Sod2 mutants, and 31 days for Ogg1ins; Sod2 double mutants. All lifespan curves are statistically different from one another using log rank tests (p<0.001). (E) w1118, Ogg1ins/Ogg1ins, Sod2, and Ogg1ins/Ogg1ins; Sod2 double mutant flies were subjected to a mechanical stress sensitivity test at 31 days. Values represent mean time (seconds) of recovery from mechanical stress, and error bars represent standard error of the mean. Significance was determined using one-way ANOVA followed by Tukey's post hoc tests (**p<0.01; ***p<0.001).