This application proposes an investigation of the processes by which genes duplicate and amplify further during growth under selection. We've applied this process to explain the phenomenon of """"""""adaptive mutation"""""""" and have shown that selective stress is not actually mutagenic (as claimed) but appears so because selection favors growth of cells with additional copies of the rate-limiting gene formed by duplication and amplification. Selection enhances the frequency of mutants by increasing the number of mutation targets and can do so with no change in the mutation rate. Our model is general for all genetic systems and leads to an extremely powerful process for genetic adaptation-nested serial clonal expansions. This process is highly relevant to the origins of cancer and adaptation of pathogens to hosts-2 situations in which populations of single cells adapt genetically during the course of a disease. Gene amplifications are proving important to development of cancer and resistance to cancer chemotherapies. We suggest that the approach taken here is important because it investigates the interface between recombination mechanism and population biology. We will characterize 2 bacterial systems (in addition to Cairns') that have been used (incorrectly, we believe) to support the idea of stress-induced mutagenesis. Our hope is to reveal new mechanisms of genetic adaptation and to resolve the controversy surrounding """"""""adaptive mutation"""""""". Thus far, we've learned that amplifications are remodeled during growth under selection to shorten their repeated unit and increase their copy number. We've discovered a new form of amplification (inversion-duplication) and will test a model for how these rearrangments arise and remodel under selection. A new recombination-independent assay shows that RecA is not essential for duplication formation; this assay should help characterize the functional requirements of duplication. We've found that the reversibiltiy of duplications causes their frequency in a population to approach a steady state level. The segregation of duplications provides an assay for internal recombination that does supply DNA ends (as does sexual exchange) but relies on spontaneous production of initiating structures. Therefore, this assay allows study of spontaneous events that initiate recombination. We've developed an assay for duplication segregation rate that avoids growth rate problems and shows that palindromic sequence stimulates duplication formation. ? ? ?
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