A Novel Mitochondrial DNA (mtDNA) - Theses - Flinders University

Chapter IV Molecular Analysis of the Effect(s) of the 3229.A Mutation onmtDNA Transcription and Translation ............................................................ 994.1 Introduction ............................................................................................. 1004.1.1 Analysis of mtDNA transcription ................................................... 1004.1.2 Analysis of mt-mRNA translation................................................... 1054.2 Results....................................................................................................... 1064.2.1 Analysis of mtDNA expression by real-time RT-PCR (qPCR).... 1064.2.2 Analysis of mRNA levels by Northern blotting ............................. 1124.2.3 Immunoblot analysis of COX subunit levels.................................. 1154.2.4 BN-PAGE analysis of mitochondrial holocomplexes .................... 1154.3 Discussion................................................................................................. 118Chapter V Analysis of the Pathological Effect(s) of the 3229.A Mutation... 1215.1 Introduction ............................................................................................. 1225.1.1 Measurement of respiratory chain activity.................................... 1225.1.2 Measurement of oxidative stress ..................................................... 1245.1.3 Analysis of apoptotic effects of the 3229.A mutation .................... 1275.2 Results....................................................................................................... 1285.2.1 Respiratory chain activity in the proband ..................................... 1285.2.2 Measurement of oxidative stress in proband cells......................... 1345.2.3 Analysis of apoptogenic effects of the 3229.A mutation................ 1345.3 Discussion................................................................................................. 139viii