CAREER: The evolutionary genetics of constraint and evolvability in an RNA bacteriophage

该项目的目标是研究基因之间的相互作用如何限制进化。利用只感染细菌的病毒，研究病毒感染和适应新宿主时所采取的步骤。了解进化极限对病毒尤其重要，因为它们可能对人类、牲畜和作物的健康构成威胁。成功地转换到一个新的宿主物种需要病毒基因的多次变化，并且有许多可能的变化集或途径。由于这些病毒对人类没有致病性，城市特许学校的中学生可以在课后活动中参与一些实际的实验工作。本研究将为本科生开设实验进化实验课程，而获资助的学员将在教学技巧、课程发展和研究方法方面得到指导和培训。通过这种方法，50名新不伦瑞克省的中学生和40名罗格斯大学的本科生将被介绍到病毒进化的研究中，而两名初级科学家将为研究和教学相结合的学术生涯做好准备。双链RNA噬菌体phi6感染不同菌株和种类的假单胞菌，将用于研究初始宿主范围突变和先前进化（在一个宿主特化过程中固定的突变）如何影响噬菌体在不同新宿主中的进化途径和出现。复制种群的实验进化将使噬菌体一次暴露在单个或多个宿主中超过100代。测序将确定相同突变被复制谱系或不同基因型固定的频率。最后，基因杂交将创造杂交病毒基因型，这将探索突变之间的相互作用如何影响替代菌株的出现，以及有多少突变通过限制其可进化性来捕获一个谱系。这些实验将证明，为了适应另一种环境，RNA病毒必须经常逆转在一种环境中适应的替代。

The goal of this project is to study how interactions among genes limits evolution. Using viruses that only infect bacteria, the steps that viruses take when infecting and adapting to new hosts will be studied. Understanding evolutionary limits is especially important for viruses as they can be threats to the health of humans, livestock, and crops. Successfully switching to a new host species requires multiple changes in a virus' genes, and there are many possible sets or pathways of change. Because these viruses are not pathogenic to humans, middle-school students at an urban charter school can be involved in some of the actual experimental work during an after-school activity. This research will be furthered by a laboratory course in experimental evolution for undergraduate students, and trainees supported by the grant will be mentored and trained in teaching techniques and curriculum development as well as research methods. Through this approach, 50 New Brunswick middle school students and 40 Rutgers undergraduates will be introduced to research in viral evolution, while two junior scientists will be prepared for an academic career that combines research and teaching. The double-stranded RNA bacteriophage phi6, which infects different strains and species of Pseudomonas bacteria, will be used to study how initial host range mutations and previous evolution (mutations fixed during specialization on one host) affects phage evolutionary pathways and emergence in different novel hosts. Experimental evolution of replicate populations will expose phages to single or multiple hosts for over 100 generations at a time. Sequencing will determine how often the same mutations were fixed by replicate lineages, or by different genotypes. Finally, genetic crosses will create hybrid viral genotypes that will explore how interactions between mutations affect emergence on alternate strains of bacteria, and how many mutations trap a lineage by limiting its evolvability. These experiments will demonstrate how often RNA viruses must reverse substitutions that were adaptive in one environment in order to adapt to another.