The main objective of the proposed research project is to provide knowledge on the mutational and evolutionary consequences of reactive oxygen species (ROS) that result from mitochondrial dysfunction. ROS are central players in a variety of human disorders and the aging process, but also play important protective roles in innate immunity against parasites. The main objective will be achieved using the model nematode Caenorhabditis elegans and its sister species C. briggsae. The first specific aim is to determine the impact of ROS on genome-wide germline mutation processes.
This aim will be accomplished by first creating mutation-accumulation (MA) lines from C. elegans genetic mutants that experience higher or lower levels of ROS as compared to wild-type, and from C. briggsae strains that experience varying levels of ROS due variation in naturally-occurring mitochondrial genome deletions. The nuclear genomes of the resultant MA lines will be comprehensively analyzed for mutation using Solexa high-throughput DNA sequencing technology. The achievement of this aim will confirm or deny the widely held, but unproven hypothesis that ROS of mitochondrial origin cause elevated mutation rates in the nuclear genome. The second specific aim is to characterize the somatic mutation effects of ROS as a function of age. A simple yet powerful somatic mutation reporter transgene system will be employed to examine the mosaic somatic mutation process in the same C. elegans genetic backgrounds used for the first aim. Achievement of the second aim will provide essential knowledge on the role of ROS in somatic mutation accumulation, and novel insights into the extent to which germline and somatic mutation processes are coupled. The third specific aim is to identify the evolutionary causes of naturally- occurring mitochondrial genome deletions in C. briggsae.
This aim will be accomplished by testing two opposing hypotheses: a first non-adaptive hypothesis that the deletions passively evolved due to weak natural selection associated with evolution in small populations, and a second adaptive hypothesis that ROS resulting from the deletions play a functional role in protecting nematodes against intracellular parasite infections. The first hypothesis will be tested using laboratory comparative population size studies involving C. briggsae of varying mitochondrial genome deletion levels;the second hypothesis will be tested using infection and competition assays involving Nematocida sp. 1, a newly discovered microsporidia parasite that infects C. briggsae in nature. Correlations between deletion levels and infection status will also be tested for in C. briggsae natural isolates. Achievement of the three aims will provide unprecedented knowledge on the effects of ROS on nuclear genome mutation processes, and the potential of a new paradigm for understanding the interplay between selection against the highly negative effects of ROS, and positive selection for ROS-induced resistance to parasites.
Mutations are the ultimate source of all human genetic variation, and virtually all heritable genetic diseases and cancers. In particular, mitochondrial genome mutations and associated elevations in reactive oxygen species levels are implicated in aging, Alzheimer's disease, Parkinson's disease and numerous mitochondrial myopathies. Understanding the fundamental properties of mutation is of central importance to understanding its role in diverse genetic disorders, cancers and the aging process.
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