Mutational and evolutionary impact of mitochondrial dysfunction

描述(由申请人提供)： 拟议研究项目的主要目标是提供关于线粒体功能障碍引起的活性氧物种(ROS)突变和进化后果的知识。ROS是人类各种疾病和衰老过程中的核心角色，也在对抗寄生虫的先天免疫中发挥着重要的保护作用。主要目标将通过模式线虫秀丽线虫及其姊妹种布氏线虫实现。第一个具体目标是确定ROS对全基因组生殖系突变过程的影响。这一目标将通过首先从秀丽线虫的遗传突变体(与野生型相比经历更高或更低水平的ROS)和从因自然发生的线粒体基因组缺失而经历不同水平的ROS变异的线虫菌株中创建突变-积累(MA)系来实现。将使用Solexa高通量DNA测序技术对产生的MA系的核基因组进行全面的突变分析。这一目标的实现将证实或否认广泛存在的、但未经证实的假设，即线粒体来源的ROS导致核基因组中突变率上升。第二个具体目标是表征ROS的体细胞突变效应随年龄的变化。一个简单而强大的体细胞突变报告转基因系统将被用来检测用于第一个目的的相同线虫遗传背景中的马赛克体细胞突变过程。第二个目标的实现将提供有关ROS在体细胞突变积累中的作用的基本知识，以及对种系和体细胞突变过程耦合程度的新见解。第三个具体目标是确定自然发生的布里奇萨线虫线粒体基因组缺失的进化原因。这一目标将通过检验两个相反的假说来实现：第一个非适应性假说认为，缺失是由于与小种群进化相关的弱自然选择而被动进化的，第二个适应性假说是由于缺失导致的ROS在保护线虫免受细胞内寄生虫感染方面发挥作用。第一个假设将通过实验室比较种群大小研究进行验证，其中包括不同线粒体基因组缺失水平的布里奇萨线虫；第二个假设将使用涉及杀线虫的感染和竞争分析进行验证。1，为新发现的一种寄生于自然界中的桥小球虫。缺失水平和感染状态之间的相关性也将在布氏梭菌自然分离株中进行测试。这三个目标的实现将提供关于ROS对核基因组突变过程的影响的前所未有的知识，并为理解针对ROS高度负面影响的选择和ROS诱导的寄生虫抗性的正选择之间的相互作用提供了一种新的范式的潜力。 公共卫生相关性： 突变是所有人类基因变异的最终来源，几乎是所有可遗传的遗传病和癌症的根源。特别是，线粒体基因组突变和相关的活性氧水平升高与衰老、阿尔茨海默病、帕金森病和许多线粒体肌病有关。了解突变的基本特性对于了解它在各种遗传疾病、癌症和衰老过程中的作用至关重要。

DESCRIPTION (provided by applicant): The main objective of the proposed research project is to provide knowledge on the mutational and evolutionary consequences of reactive oxygen species (ROS) that result from mitochondrial dysfunction. ROS are central players in a variety of human disorders and the aging process, but also play important protective roles in innate immunity against parasites. The main objective will be achieved using the model nematode Caenorhabditis elegans and its sister species C. briggsae. The first specific aim is to determine the impact of ROS on genome-wide germline mutation processes. This aim will be accomplished by first creating mutation-accumulation (MA) lines from C. elegans genetic mutants that experience higher or lower levels of ROS as compared to wild-type, and from C. briggsae strains that experience varying levels of ROS due variation in naturally-occurring mitochondrial genome deletions. The nuclear genomes of the resultant MA lines will be comprehensively analyzed for mutation using Solexa high-throughput DNA sequencing technology. The achievement of this aim will confirm or deny the widely held, but unproven hypothesis that ROS of mitochondrial origin cause elevated mutation rates in the nuclear genome. The second specific aim is to characterize the somatic mutation effects of ROS as a function of age. A simple yet powerful somatic mutation reporter transgene system will be employed to examine the mosaic somatic mutation process in the same C. elegans genetic backgrounds used for the first aim. Achievement of the second aim will provide essential knowledge on the role of ROS in somatic mutation accumulation, and novel insights into the extent to which germline and somatic mutation processes are coupled. The third specific aim is to identify the evolutionary causes of naturally- occurring mitochondrial genome deletions in C. briggsae. This aim will be accomplished by testing two opposing hypotheses: a first non-adaptive hypothesis that the deletions passively evolved due to weak natural selection associated with evolution in small populations, and a second adaptive hypothesis that ROS resulting from the deletions play a functional role in protecting nematodes against intracellular parasite infections. The first hypothesis will be tested using laboratory comparative population size studies involving C. briggsae of varying mitochondrial genome deletion levels; the second hypothesis will be tested using infection and competition assays involving Nematocida sp. 1, a newly discovered microsporidia parasite that infects C. briggsae in nature. Correlations between deletion levels and infection status will also be tested for in C. briggsae natural isolates. Achievement of the three aims will provide unprecedented knowledge on the effects of ROS on nuclear genome mutation processes, and the potential of a new paradigm for understanding the interplay between selection against the highly negative effects of ROS, and positive selection for ROS-induced resistance to parasites. PUBLIC HEALTH RELEVANCE: Mutations are the ultimate source of all human genetic variation, and virtually all heritable genetic diseases and cancers. In particular, mitochondrial genome mutations and associated elevations in reactive oxygen species levels are implicated in aging, Alzheimer's disease, Parkinson's disease and numerous mitochondrial myopathies. Understanding the fundamental properties of mutation is of central importance to understanding its role in diverse genetic disorders, cancers and the aging process.