Strong purifying selection in transmission of mammalian mitochondrial DNA

Abstract

There is an intense debate concerning whether selection or demographics has been most important in shaping the sequence variation observed in modern human mitochondrial DNA (mtDNA). Purifying selection is thought to be important in shaping mtDNA sequence evolution, but the strength of this selection has been debated, mainly due to the threshold effect of pathogenic mtDNA mutations and an observed excess of new mtDNA mutations in human population data. We experimentally addressed this issue by studying the maternal transmission of random mtDNA mutations in mtDNA mutator mice expressing a proofreading-deficient mitochondrial DNA polymerase. We report a rapid and strong elimination of nonsynonymous changes in protein-coding genes; the hallmark of purifying selection. There are striking similarities between the mutational patterns in our experimental mouse system and human mtDNA polymorphisms. These data show strong purifying selection against mutations within mtDNA protein-coding genes. To our knowledge, our study presents the first direct experimental observations of the fate of random mtDNA mutations in the mammalian germ line and demonstrates the importance of purifying selection in shaping mitochondrial sequence diversity.

Figure 1. The Breeding Scheme for the Mutagenesis of Mouse mtDNA

The PolgA^mut/PolgA^mut mtDNA mutator females were derived from mothers with wild-type C57Bl/6 mtDNA. The mtDNA mutators, and all subsequent generations, were crossed to wild-type C57Bl/6 males. Heterozygous (+/PolgA^mut) N1 female progeny were used to establish the 13 mtDNA mutator lines. Only females that were homozygous for the wild-type PolgA allele (+/+) were bred from generation N2 and onwards.

Figure 2. Mutation Distribution by Codon Position Reveals Purifying Selection on Protein-Coding Genes in mtDNA Mutator Lines

Plot of observed mtDNA mutations grouped by positional category. (A) Observed mutations per base pair for each codon position from mtDNA mutator lines compared to (B), mtDNA sequences of 21 mouse strains obtained from GenBank, and (C), human mtDNA sequences obtained from the mtDB database. The reduction in observed first and second codon position mutations signifies selection against amino acid–changing mutations. Due to the larger number of human sequences available, the y-axis is larger for the human dataset.

Figure 3. Distribution of Mutations by Gene for mtDNA Mutator Lines and Human mtDNA Sequence Data

Plot of observed minus expected ratio of 4-fold degenerate sites versus all other protein-coding sites. (A) For mtDNA mutator mouse lines, the ratio of observed to expected sites is plotted for third codon position mutations at 4-fold degenerate sites (filled bars), and for all other protein-coding gene mutations observed (open bars). The line represents the observed expected values normalized to 1.0. (B) The same plot for observed human variants found on the mtDB database. (C) Plot of observed minus expected for non–4-fold degenerate sites for mouse (filled bar) and human (open bars). Genes are grouped by mitochondrial respiratory chain complexes. Expected values are derived from an assumption of equal distribution of the observed mutations in the dataset, and observed values are derived from a count of the detected mutations for that gene (see Material and Methods).

Figure 4. Relaxed Selection on rRNA and tRNA Genes in mtDNA Mutator Lines

Plot of observed mtDNA mutations grouped by positional category. (A) Observed mutations in tRNA, rRNA, and control region sequences compared with (B) mouse strain sequences and (C) human mtDNA sequences. Due to the larger number of human sequences available, the y-axis is larger for the human dataset.