Segmental amplification: the collateral effects of co-amplifying genes near a gene under selection for higher dosage

项目摘要/摘要 所有有机体都必须适应新的和不断变化的环境。基因对新分子功能的进化 复制和分化通常推动这种适应，使有机体能够在新的生态位或 食用新奇的化合物。酶的低效和生理上无关的副作用，指 可以作为通过基因复制和进化新功能的原始材料 分歧。如果一项杂乱无章的活动由于环境变化而变得对健康很重要， 复制/扩增可以迅速增加现在至关重要的混杂活动的剂量。然而，基因 复制/扩增事件通常复制基因周围的许多基因，以获得更高的 剂量。这些复制的基因组片段可能包含数百个基因。因此，共同放大的邻居 基因可能会根据它们的功能对生物体造成附带后果。而当 扩增基因的表达通常随拷贝数、调控机制的程度而变化 调控最近扩增的基因的表达在很大程度上是未知的。我假设这个表情和 共扩增相邻基因的功能通过干扰生理来影响新酶的进化 节段性扩增后影响适合度。我的赞助商的实验室已经开发了一个模型系统来研究 基因复制/扩增。在这个系统中，Δargc大肠杆菌突变体不能产生精氨酸。 ProA基因的点突变(ProA*)增加了突变酶ProA*的混杂ARGC活性， 精氨酸合成恢复乏力。ProA*的扩增改善了体能，因为低效的ARGC活性 ProA*是新陈代谢中限制生长的“薄弱环节”。我的赞助人实验室之前的研究表明 ProA*在几百代内在其天然基因座(最多50个拷贝)迅速扩增，这些 片段扩增通常包括数十到数百个其他邻近基因。要解决这个问题 片段扩增的直接后果，我将通过以下方式修改ΔARGC ProA*E.Coli模型系统 将Proba*操纵子从其天然位点删除，并将其重新定位到与预测的 如果过度表达会扰乱生理，并在条件选择下将这些菌株进化到≤300代 用于ProA*扩增。在目标1中，我将确定mRNA和蛋白质水平表达的程度 最近扩增的基因随着基因拷贝数的增加而变化。在目标2中，我将描述基因的功能如何 在扩增后但在扩增之前，扩增片段内会影响全局基因表达、生理和适合度 代偿性突变可以减轻这些影响。我们的工作将阐明体内平衡在多大程度上 机制可以调节扩增基因的表达。结果将提高我们对如何 共扩增基因的功能可以导致系统范围的后果，并在即刻影响健康 节段性放大的后遗症。

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 of co-amplified genes can cause system-wide consequences and impact fitness in the immediate aftermath of segmental amplification.