PROJECT SUMMARY/ABSTRACT All organisms must adapt to new and changing environments. The evolution of new molecular functions by gene duplication and divergence commonly drives this adaptation, allowing organisms to colonize new niches or consume novel compounds. The inefficient and physiologically irrelevant side activities of enzymes, referred to as “promiscuous” activities, can serve as the source material for evolving new functions by gene duplication and divergence. If a promiscuous activity becomes important for fitness due to an environmental change, gene duplication/amplification can rapidly increase the dosage of the now critical promiscuous activity. However, gene duplication/amplification events usually duplicate many genes surrounding the gene under selection for higher dosage. These duplicated genome segments can contain hundreds of genes. Thus, the co-amplified neighboring genes can potentially cause collateral consequences for the organism depending on their function. While the expression of amplified genes typically scales with copy number, the extent to which regulatory mechanisms modulate the expression of recently amplified genes is largely unknown. I hypothesize that the expression and functions of co-amplified neighboring genes influence the evolution of new enzymes by perturbing physiology and impacting fitness after segmental amplification. My sponsor’s lab has developed a model system to study gene duplication/amplification. In this system, an ΔargC Escherichia coli mutant is unable to produce arginine. A point mutation in the gene proA (proA*) increases the promiscuous ArgC activity of the mutant enzyme ProA*, weakly restoring arginine synthesis. Amplification of proA* improves fitness because the inefficient ArgC activity of ProA* is the growth-limiting “weak-link” in metabolism. Previous work from my sponsor’s lab has shown that proA* rapidly amplifies at its native locus (up to 50 copies) within a few hundred generations and that these segmental amplifications typically include dozens to hundreds of other neighboring genes. To address the immediate consequences of segmental amplification, I will modify the ΔargC proA* E. coli model system by deleting the proBA* operon from its native locus and relocating it to five ectopic sites next to genes predicted to perturb physiology if overexpressed and evolve these strains for ≤ 300 generations under conditions selecting for proA* amplification. In Aim 1, I will determine the degree to which mRNA and protein levels expressed from recently amplified genes scale with gene copy number. In Aim 2, I will characterize how the functions of genes within an amplified segment affect global gene expression, physiology, and fitness after amplification but before compensatory mutations can alleviate these effects. Our work will elucidate the extent to which homeostatic mechanisms can regulate the expression of amplified genes. The results will improve our understanding of how the functions ...