mtDNA phylogeny of the germ line: mechanism, structure and function of the mtDNA bottleneck

种系线粒体DNA系统发育：线粒体DNA瓶颈的机制、结构和功能

• 批准号: 9982687
• 负责人: Konstantin Khrapko
• 金额: $ 32.1万
• 依托单位: NORTHEASTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-25 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9982687
• 关键词: Admixture; Age; Biological; Cell physiology; Cells; DNA; DNA Sequence Alteration; Data; Detection; Development; Embryo; Embryonic Development; Energy-Generating Resources; Ensure; Exhibits; Female; Fertilization; Genealogy; Generations; Genetic Materials; Genome; Genotype; Germ Cells; Germ Lines; Hand; Human; Impairment; Individual; Inner Cell Mass; Knowledge; Left; Maps; Mitochondria; Mitochondrial DNA; Modeling; Molecular; Mus; Mutation; Mutation Analysis; Natural Selections; Nuclear; Oocytes; Oxidative Phosphorylation; Phylogenetic Analysis; Phylogeny; Population; Positioning Attribute; Process; Production; Proteins; Reactive Oxygen Species; Respiration; Structure; Structure of primordial sex cell; Surveillance Methods; Third Generation Sequencing; Time; Trees; Work; blastocyst; egg; embryo stage 2; innovation; innovative technologies; mitochondrial DNA mutation; mitochondrial genome; negative affect; new technology; next generation; offspring; preimplantation; prevent; repaired; simulation; transmission process

The principal function of mitochondria is the generation of cellular energy (ATP) by oxidative phosphorylation using proteins encoded for by both the nuclear genome and the mitochondrial genome (mitochondrial DNA, mtDNA) to assemble the machinery needed for mitochondrial respiration. Paradoxically, mtDNA is a macromolecular target of reactive oxygen species (ROS), which are mutagenic by-products generated during ATP production. Unlike nuclear DNA, which has a high-level of proofreading, error detection and correction that coordinately function to prevent passage of harmful DNA mutations to offspring, mtDNA mutations are not subject to the same degree of detection and correction. Indeed, past studies have shown that mtDNA sustains a high mutational burden that progressively increases in cells with age. This paradigm raises a fundamentally critical question when one considers that mitochondrial passage from one generation to the next is uniparental through the female germline (egg): how are detrimental mtDNA mutations that accumulate in maternal germ cells over time prevented from being passed to the next generation, the generation after that, and so on? Two distinct, but likely interrelated, processes have been offered to explain this phenomenon – the mtDNA bottleneck and germline-purifying selection. However, large gaps in knowledge still exist regarding both. For example, several different mechanisms have been proposed for how the mtDNA bottleneck works – none of which have been proven, and its position in germline development is debated. Likewise, it is not known where germline-purifying selection takes place relative to the bottleneck or even if it depends on the bottleneck. We recently developed an innovative technology combining PACBIO third-generation sequencing with unique molecular identifiers, which enables high-fidelity mutational analysis of entire individual mtDNA molecules. With this in hand, we are now uniquely positioned to map how transgenerational passage of high-quality mtDNA molecules is accomplished. To do this, we propose the following Specific Aims (SAs). SA1: Define the structure and dynamics of the mtDNA bottleneck. We will build detailed phylogenetic trees of mtDNA molecules within individual germline cells, and compare these trees to those simulated from proposed bottleneck models. SA2: Determine the timing and mechanisms of germline-purifying selection. We will compare ‘synonymity’ of mutations from phylogenetic tree branches to identify when purifying selection occurs, and how it interfaces with the bottleneck. SA3: Determine if individual mtDNA molecules carrying no/low versus high mutational burdens are preferentially allocated during early (preimplantation) embryogenesis. We will evaluate mtDNA genealogies in individual blastomeres of preimplantation embryos at the 2-, 4- and 8-cell stages, as well as in the inner cell mass (embryo proper) and trophectoderm (extraembryonic) of blastocyst-stage preimplantation embryos, to determine if there exists evidence of differential allocation of mtDNA mutations.

线粒体的主要功能是通过氧化磷酸化产生细胞能（ATP） 使用核基因组和线粒体基因组（线粒体DNA， mtDNA）要组装线粒体呼吸所需的机械。矛盾的是，mtDNA是一个 活性氧（ROS）的大分子靶标，它们是诱变的副产品 ATP生产。与核DNA不同，核DNA具有高级校对，错误检测和校正 协同起作用以防止有害的DNA突变通过后代，MtDNA突变不是 受到相同程度的检测和校正。实际上，过去的研究表明mtDNA维持 高突变燃烧随着年龄的增长而逐渐增加的细胞。这个范式从根本上提出了 当人们认为从一代到第二代的线粒体通道是单元的关键问题 通过雌性种系（鸡蛋）：如何积聚在物物生殖中的有害mtDNA突变 随着时间的流逝，细胞阻止了传递到下一代，之后的一代等等？ 已经提供了两个不同但可能相互关联的过程来解释这一现象 - mtdna 瓶颈和种系纯化选择。但是，关于两者的了解仍然存在很大的差距。为了 例如，已经提出了MTDNA瓶颈的几种不同机制 - 没有 已被证明及其在种系开发中的地位进行了辩论。同样，这也不知道在哪里 种系纯化的选择是相对于瓶颈进行的，即使取决于瓶颈。我们 最近开发了一种创新技术，将PACBIO第三代测序与独特的技术结合在一起 分子标识符，可以对整个单个mtDNA分子进行高保真突变分析。和 这在手上，我们现在是独特的位置，可以映射高质量mtDNA的变革性传递 分子已完成。为此，我们提出以下特定目标（SAS）。 SA1：定义结构 和mtDNA瓶颈的动力学。我们将在内部建造姆特纳分子的详细系统发育树 单个种系细胞，并将这些树与从建议的瓶颈模型模拟的树木进行比较。 SA2： 确定种系纯化选择的时间和机制。我们将比较 系统发育树枝的突变，以识别何时发生净化选择，以及它如何与 瓶颈。 SA3：确定是否携带NO/低突变的单个mtDNA分子 优选在早期（植入前）胚胎发生期间分配了伯元。我们将评估 在2、4和8细胞阶段的植入前胚胎单个胚胎中的mtDNA家谱以及 如内部细胞质量（适当的胚胎）和芽孢杆菌的滋养剂（胚胎） 植入前胚胎，以确定是否存在MTDNA突变差分分配的证据。