Somatic mutations in mitochondrial DNA (mtDNA) have been implicated in the loss of function that accompanies mammalian aging. However, there is no way at present to easily measure rare mtDNA point mutations (frequency < 10-5) in a background of wild-type molecules. A method will be developed for quantifying point mutations, small deletions, and small insertions in mtDNA. The technique -- called Translational Arrest Assay (TAAssay) -- will detect mutations which introduce stop codons into the target sequence or change its reading frame. A suitable restriction fragment from mtDNA samples will be fused to the amino-terminus of the lacZ gene encoding beta- galactosidase, such that a functional mtDNA/lacZ fusion protein will be produced in transformed E. coli carrying the recombinant plasmid. Mutations which introduce stop codons or shift the reading frame will interrupt translation, resulting in lac colonies. By comparing the frequency of lac colonies resulting from TAAssay of mtDNA from young versus old rats, an estimate of the rate of mtDNA somatic mutation will be obtained. The rate of mutation at neutral sites will be compared with that at replacement sites, and the rate of mtDNA mutation will be compared in different tissues (dividing versus nondividing). Overall, these experiments will provide a measure of mtDNA sequence fidelity in the aging mammal, thereby critically testing the """"""""extranuclear somatic mutation"""""""" theory of aging.
