CHARACTERIZATION OF MITOCHONDRIAL DNA DELETION MUTATIONS IN RAGGED RED REGIONS OF INDIVIDUAL, AGED RHESUS MONKEY MUSCLE FIBERS





Damian Lee, Zhengjin Cao, Nolan Gokey, Allen Herbst, and Judd M. Aiken.

Department of Animal Health and Biomedical Sciences,
University of Wisconsin-Madison,
1656 Linden Drive,
Madison, WI 53706-1581




Mitochondrial DNA (mtDNA) deletion mutations accumulate in skeletal muscle with age. This accumulation is focal and associated with electron transfer system (ETS) abnormal regions. In this study, we characterized mtDNA deletion mutations in ETS abnormal muscle fibers from rhesus monkey (Macaca mulatta). Vastus lateralis biopsies from aged rhesus monkeys were examined by exhaustive serial cryosectioning and subsequent histochemical staining for cytochrome c oxidase (COX) and succinate dehydrogenase (SDH) activities through 2,000 microns. Individual muscle fibers containing ETS abnormal regions were identified and the ETS abnormal regions dissected by laser capture microdissection. Long extension PCR amplification products of mtDNA recovered from the laser-captured lysates were subcloned and the deletion breakpoints characterized. Twenty-seven ETS abnormal muscle fibers were analyzed and mtDNA deletion mutations were present in all twenty-seven. The size of the deletions ranged from 4.2 kb to 9.9 kb with 25 of the 27 deletions occurring in the major arc region. Unlike the rat in which direct repeat and common deletion sequences were not identified at the deletion breakpoints, twenty-three deletions were flanked by direct repeat sequences and at least two common deletions were identified: a 9.9kb deletion between 5835-15712, and a 4.2kb deletion between 10173-14412. These data taken together with previous investigations on aged rat muscle fibers demonstrate that large mtDNA deletions are associated with the ragged red phenotype in aged rat and rhesus monkey muscle. The accumulation of common deletion mutations and presence of direct repeat sequences at the breakpoints in monkey muscle fibers suggests that the mechanism of mtDNA deletion mutation formation and/or biological response to a deletion event is different in monkey and rat.




Key words: Mitochondrial DNA deletions, electron transfer system, Muscle, Rhesus monkey







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