Dr. Michael Lynch requests funds for a 5-year project to investigate the nature and consequences of deleterious mutations. The project is comprised of an empirical component and a theoretical component. Empirical studies are proposed to determine the nature and rate of accumulation of mutations in Daphnia and Escherichia coli. Theoretical analyses of the effects of deleterious mutations on extinction rates will be conducted through computer simulation. The accumulation of spontaneous mutations will be monitored in lines derived from natural populations of Daphnia and maintained as clones. Inbred D. pulex and D. pulicaria lines will be generated by inducing sexual reproduction and self-fertilization through 15 generations, and four clonal replicates will be generated from each line. In order to permit the accumulation of mutations without selective elimination, each culture will be maintained by transferring a single randomly chosen offspring to produce the next generation. Fitness, defined as survival and reproduction through six clutches, will be assayed at 10-generation intervals. The rate of change in fitness and the variance in fitness among lines will be estimated. The nature and effects of the mutations that accumulate will be characterized. Aspects to be analyzed include environmental sensitivity, dominance, epistasis, and magnitude of deleterious effects. Estimates obtained from Dr. Lynch's """"""""four-equation"""""""" method using clones derived directly from natural populations will be compared to comparable estimates obtained from the mutation-accumulation experiments. A second empirical study will continue and expand on-going mutation- accumulation experiments using E. coli. All lines will be passed through a single-cell bottleneck each generation, with representative cells frozen every 25 cycles. Various growth characteristics of each line relative to representatives of the line in the initial cycle will be assayed. Aspects of the accumulated mutations that will be estimated include the distribution of effects on growth parameters, sensitivity to environmental conditions, and epistasis. The experiment will be repeated using growth-inhibiting and growth-enhancing environments in order to explore the effects of stress on the rate of generation and nature of mutations. Another extension involves monitoring strains carrying known deficiencies in repair pathways. Numerical simulations will be conducted to characterize the effects of deleterious mutations on extinction rates under various breeding systems including random mating, obligate asexuality, and obligate self- fertilization. Mutational effects will be allowed to vary according to a specified distribution, which may include beneficial as well as deleterious mutations. The effects of the nature of epistasis (positively or negatively synergistic) on the rate of accumulation will be addressed. Mutations affecting fertility as well as viability will be studied. The effects on extinction rates of variation in population size and reproductive rate due to environmental factors as well as mutations will be explored.
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