Recent evidence from human studies strongly suggest that the aging- related syndrome Parkinson's disease as well as the aging process itself may be the result of a loss of cellular energy caused by the accumulation of mtDNA molecules harboring substantial deletion mutations. The missing genes in the deleted molecules are all subunits of the mitochondrial energy-generating systems and their absence is particularly crucial in brain function. The underlying mechanism that leads to deletion of large segments (up to 8 kb) mtDNA is probably slipped mispairing between direct repeat sequences in displaced single strand segments of mtDNA during replication. Mammalian mitochondria contain a nuclear-encoded single strand binding protein (called """"""""P16"""""""") that inhibits renaturation and thus probably protects the replicative intermediates against accidental slipped mispairing under normal circumstances. The goals of this proposal are (1.) to establish the widespread occurrence of deletion mutation events in mtDNA; (2.) to measure the extent of accumulation of partially deleted mtDNA as a function of aging, and (3.) to study the link between P16 and the mechanism of the deletion process. Since our past studies of P16 (including amino acid sequence data), were carried out in rat tissue, the aging model will be developed in rat as well. Several tissues (brain, heart, and liver) from fetal, newborn, adult and senescent rats will be screened by Southern blot analysis and polymerase chain reaction (PCR) amplification for the occurrence of mtDNA with deletion mutations. The deletion mutations will be sequenced by the dideoxy termination method. Based on previous amino acid sequence data, PCR primers were synthesized and used to amplify the NH2-terminal cDNA sequence of P16 from a lambda phage library. The complete cDNA sequence will be obtained from either anchored PCR experiments or from complete cDNA clones identified by screening with the confirmed PCR-probe directed to the known NH2-terminal end of P16. The deduced complete amino acid sequence will be computer analyzed for prediction of structural motifs and functional correlations. The importance of P16 in the generation of deletion mutations in mtDNA will be studied in a suitable rat cell culture system using antisense oligodeoxynucleotides to P16 mRNA in order to specifically inhibit P16 biosynthesis. The long term effects of insufficient P16 will then be examined by screening for enhanced production and accumulation of mtDNAs having substantial deletion mutations.
| Van Tuyle, G C; Gudikote, J P; Hurt, V R et al. (1996) Multiple, large deletions in rat mitochondrial DNA: evidence for a major hot spot. Mutat Res 349:95-107 |
