QUANTITATIVE LONGITUDINAL ANALYSIS OF MITOCHONDRIAL DNA DELETION MUTATIONS IN ELECTRON TRANSPORT SYSTEM ABNORMAL REGIONS





M. Bassiouni, A. Herbst, J.M. Aiken

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



Mitochondrial DNA (mtDNA) deletion mutations accumulate in skeletal muscle with age and are associated with electron transport system (ETS) abnormalities. These segmental abnormalities are identified within muscle fibers by the absence of cytochrome c oxidase and the over-expression of succinate dehydrogenase activities. Mitochondrial myopathy studies have determined the threshold of deletion mutation load leading to an ETS abnormal phenotype, but none have quantified deletion mutations and the associated phenotype with age. We hypothesize that mitochondrial DNA deletion mutations result in electron transport system abnormalities and that the highest mtDNA deletion load is found in the ETS abnormal region of ragged red fibers.

Rectus femoris skeletal muscle from 36-month old rats was examined by exhaustive serial cryo-sectioning and subsequent histochemical staining for cytochrome c oxidase (COX), and succinate dehydrogenase (SDH) enzymatic activities through 2,000 microns. An individual muscle fiber containing an ETS abnormal region (COX- and SDH++) was identified and dissected by laser capture microdissection. Long extension PCR of mtDNA was used to amplify a unique deletion product from the laser-captured cell lysate. The deletion mutation was cloned and sequenced. Quantitation of the mtDNA deletion mutation, wildtype genome and total mtDNA levels were determined using quantitative real-time PCR on laser captured fiber sections. This study will delineate the relationship between mtDNA deletion mutations and ETS phenotypes.




Key words: mitochondrial DNA deletion, ETS abnormal region, laser capture microdissection, quantitative real-time PCR







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