Revisiting the mouse mitochondrial DNA sequence

Abstract

The existence of reliable mtDNA reference sequences for each species is of great relevance in a variety of fields, from phylogenetic and population genetics studies to pathogenetic determination of mtDNA variants in humans or in animal models of mtDNA-linked diseases. We present compelling evidence for the existence of sequencing errors on the current mouse mtDNA reference sequence. This includes the deletion of a full codon in two genes, the substitution of one amino acid on five occasions and also the involvement of tRNA and rRNA genes. The conclusions are supported by: (i) the re-sequencing of the original cell line used by Bibb and Clayton, the LA9 cell line, (ii) the sequencing of a second L-derivative clone (L929), and (iii) the comparison with 12 other mtDNA sequences from live mice, 10 of them maternally related with the mouse from which the L cells were generated. Two of the latest sequences are reported for the first time in this study (Balb/cJ and C57BL/6J). In addition, we found that both the LA9 and L929 mtDNAs also contain private clone polymorphic variants that, at least in the case of L929, promote functional impairment of the oxidative phosphorylation system. Consequently, the mtDNA of the strain used for the mouse genome project (C57BL/6J) is proposed as the new standard for the mouse mtDNA sequence.

Figure 1

RFLP confirmation and degree of heteroplasmy of private mutations observed in LA9 (A and F) and L929 mtDNAs (B and C) and specific polymorphysms detected in C57BL/6J mtDNA (D and E). The number under each panel indicates the nucleotide position responsible for the RFLP. The amplification products and the different restriction enzymes used are described in Material and Methods.

Figure 2

Allele-specific termination of primer extension assays designed to establish the size of three polymorphic mononucleotide tracks observed in the tRNA^Arg gene (A), the Nd6 gene (B) and the L-strand replication origin (C). Note that the extension of the C3H mtDNA sample in (A) cannot be longer than 1 nt due to its particular four Ts + nine As sequence, while the rest of the samples harbor three Ts + n As, with n = eight, nine or 10. Lines without label in (A) and (C) represent un-extended primer. Lines labeled 7/C, 11/A or 12/A in (B) or (C) represent controls for seven Cs or 11 or 12 As in the respective polymorphic track.

Figure 3

Representative chromatograms that illustrate the variety of viable sequences described to date in the highly polymorphic locus at the mouse mtDNA-encoded tRNA^Arg gene. The source of the DNA is indicated in each panel.