Characterization and analysis of multiple large-scale rearrangements of mitochondrial DNA in rat liver, heart, and brain

Large-scale rearrangements of mitochondrial DNA have been reported in numerous animal species including humans. These rearrangements are believed to contribute to the etiology of a variety of human neuromuscular diseases and endocrine disorders such as Kearns-Sayre Syndrome, Pearson's Syndrome and diabetes mellitus. Additionally, the accumulation of deletions in mitochondrial DNA has been shown to correlate with the normal ageing process. There is yet much to learn about mtDNA rearrangements before their contribution to disease states can be fully elucidated. Consequently, it is the goal of our laboratory to establish the rat as an animal model to study how mtDNA rearrangements occur, accumulate, and propagate under normal conditions. To that end, the liver, heart, and brain mtDNA from rats ranging in age from 2-day old neonates to 33 month old senescent animals were analyzed for large-scale rearrangements (deletions/insertions) using nested PCR and electron microscopy. The prevalence of both deletions and insertions in the genome was analyzed by measuring the genome length of hundreds of mtDNA molecules obtained from pups and senescent animals. In both the pup and senescent samples mtDNA molecules which deviated from the wild-type mitochondrial genome length were detected. As the magnitude of the deviation increased, the percentage of effected molecules decreased. This indicates that there are more molecules effected by small-scale rearrangements than potentially more detrimental large-scale rearrangements. Through the use of nested PCR, rearrangement junctions were found throughout the genome including the D-Loop, 16S rRNA gene and numerous tRNA genes. Seventy different rearrangements have been sequenced. These were found in all ages and tissue types analyzed. One of the rearrangements was found in several tissue types and in multiple animals. Several other rearrangements had end points within a few bases of this commonly found rearrangement, implicating this region as a hot spot. The majority of the rearrangements lacked direct repeats at their end points. When present, the short direct repeats were up to 6-bp in length with 3- and 4-bp repeats predominating. The majority of the rearrangement end points were located within potential stem-loop structures of the mitochondrial genome. The results from this study indicate that mtDNA rearrangements occur throughout the lifespan in numerous tissues. Hot spots indicate that certain regions of the genome are more susceptible to the rearrangement process. The lack of direct repeats at the end points of all of the rearrangements suggests that multiple mechanisms may exist for the formation of mtDNA rearrangements. One of these mechanisms may involve stem-loop structures.