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

项目总结/摘要 所有生物都必须适应新的和不断变化的环境。通过基因进化新的分子功能 复制和分化通常驱动这种适应，使生物体能够殖民新的生态位， 消耗新的化合物。酶的低效和生理上不相关的副作用， 作为“混杂”活动，可以作为通过基因复制进化新功能的源材料， 发散如果由于环境的变化，一种混杂的活动对健康变得重要， 复制/扩增可以迅速增加现在关键的混杂活性的剂量。然而，基因 复制/扩增事件通常复制选择中的基因周围的许多基因，以获得更高的生物学活性。 剂量.这些重复的基因组片段可以包含数百个基因。因此，共同放大的相邻 基因根据其功能可能对生物体造成附带后果。而 扩增基因的表达通常与拷贝数成比例，即调控机制 调节最近扩增的基因的表达在很大程度上是未知的。我假设这个表达和 共同扩增的邻近基因的功能通过干扰生理学来影响新酶的进化 以及节段性扩增后影响适应性。我赞助人的实验室开发了一个模型系统来研究 基因复制/扩增。在该系统中，ΔargC大肠杆菌突变体不能产生精氨酸。 基因proA（proA*）中的点突变增加了突变酶ProA* 的混杂ArgC活性， 弱恢复精氨酸合成。proA* 的扩增提高了适应性，因为低效的ArgC活性 ProA* 是新陈代谢中限制生长的“弱环节”。我的赞助人实验室之前的工作表明， proA* 在其天然基因座上在几百代内迅速扩增（高达50个拷贝），并且这些 片段扩增通常包括几十到几百个其它邻近基因。解决 片段扩增的直接后果，我将修改ΔargC proA* E。大肠杆菌模型系统， 将proBA* 操纵子从其天然基因座删除，并将其重新定位到预测为 如果过表达，则干扰生理学，并在选择条件下使这些菌株进化≤ 300代 用于proA* 扩增。在目标1中，我将确定在何种程度上表达的mRNA和蛋白质水平， 最近扩增的基因与基因拷贝数成比例。在目标2中，我将描述基因的功能 在扩增片段内影响扩增后但扩增前的整体基因表达、生理学和适应性 补偿突变可以减轻这些影响。我们的工作将阐明体内平衡 机制可以调节扩增基因的表达。结果将提高我们对如何 共同扩增基因的功能可以引起系统范围的后果，并立即影响健康。 节段性放大的后果。

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.