Investigations of mitochondrial dna point mutations and mitochondrial oxidants in aging and disease

The extranuc[ear somatic mutation theory of aging proposes that the accumulation of mutations in the mitochondrial genome may be responsible in part for the phenoinenology of aging. Recent studies of mtDNA deletions have suggested that they increase with age in humans and other mammals. There have been few quantitative studies of the accumulation mtDNA point mutations. We have developed a method for the detection of mtDNA point mutations, called the Translational Arrest Assay (TAAssay). A fragment of restriction-endonuclease digested rat mtDNA is cloned into a TAA vector such that a functional, expressed fusion gene of the mtDNA and 3-galactosidase results. Frameshift and nonsense mutations in the continuous reading frame of the mtDNA result in lac- bacterial colonies. The ratio of lac-:lac+ colonies is a measure of the frequency of somatic mutations, and an increase in the ratio represents the accumulation of mutations over time. The free radical theory of aging argues that oxidative damage plays a key role in degenerative senescence. Leakage of superoxide anion from the mitochondrial electron transport chain is of particular interest, since a correlation between metabolic rate and life span has been observed. The eytokine TNF- has been shown to promote mitochondrial generation of oxidants. We have used TNF-treatment of cells lacking mitochondrial DNA (pcells), in combination with their parent cell lines, in order to understand the role of mitochondrially encoded electron transport chain components in mitochondrial oxidant generation.