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: 10428492
• 负责人: Konstantin Khrapko
• 金额: $ 31.46万
• 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 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10428492
• 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量（ATP） 使用由核基因组和线粒体基因组编码的蛋白质（线粒体DNA， 线粒体DNA）组装线粒体呼吸所需的机器。巧合的是，mtDNA是一种 活性氧物质（ROS）的大分子靶标，它们是在 ATP生产。与核DNA不同，核DNA具有高水平的校对、错误检测和纠正能力， 协调功能，以防止有害的DNA突变传递给后代，mtDNA突变不是 受到同等程度的检测和纠正。事实上，过去的研究表明，线粒体DNA维持着 随着年龄的增长，细胞中的高突变负荷逐渐增加。这种模式从根本上提出了一个 当人们认为线粒体从一代到下一代是单亲的时候， 通过女性生殖细胞（卵子）：有害的mtDNA突变如何在母体生殖细胞中积累 随着时间的推移，细胞被阻止传递给下一代，再下一代，等等？ 已经提出了两个不同但可能相互关联的过程来解释这种现象--线粒体DNA 瓶颈和种系纯化选择。然而，在这两方面的知识仍然存在巨大差距。为 例如，已经提出了几种不同的机制来解释mtDNA瓶颈的作用--没有一种是 这已经被证明，它在生殖系发育中的地位是有争议的。同样，也不知道在哪里 生殖系纯化选择相对于瓶颈发生，或者甚至取决于瓶颈。我们 最近开发了一种创新技术，将PACBIO第三代测序与独特的 分子标识符，它可以对整个单个mtDNA分子进行高保真突变分析。与 有了这一点，我们现在处于独特的地位，可以绘制高质量mtDNA的跨代传递， 分子已经完成。为此，我们提出了以下具体目标（SA）。SA 1：定义结构 和线粒体DNA瓶颈的动力学。我们将建立详细的mtDNA分子系统发育树， 个体生殖细胞，并将这些树与建议的瓶颈模型模拟的树进行比较。SA2： 确定生殖系纯化选择的时机和机制。我们将比较 从系统发育树分支的突变，以确定何时发生纯化选择，以及它如何与 瓶颈。SA 3：确定单个mtDNA分子是否携带无/低与高突变 在早期（植入前）胚胎发生期间优先分配负荷。我们将评估 在2-，4-和8-细胞期的植入前胚胎的单个卵裂球中的mtDNA谱系，以及 如囊胚期的内细胞团（胚胎）和滋养外胚层（胚外 植入前胚胎，以确定是否存在mtDNA突变差异分配的证据。

项目成果

A mitochondria-specific mutational signature of aging: increased rate of A > G substitutions on the heavy strand.

• DOI: 10.1093/nar/gkac779
• 发表时间: 2022-10-14
• 期刊: NUCLEIC ACIDS RESEARCH
• 影响因子: 14.9
• 作者: Mikhailova, Alina G.;Mikhailova, Alina A.;Ushakova, Kristina;Tretiakov, Evgeny O.;Iliushchenko, Dmitrii;Shamansky, Victor;Lobanova, Valeria;Kozenkov, Ivan;Efimenko, Bogdan;Yurchenko, Andrey A.;Kozenkova, Elena;Zdobnov, Evgeny M.;Makeev, Vsevolod;Yurov, Valerian;Tanaka, Masashi;Gostimskaya, Irina;Fleischmann, Zoe;Annis, Sofia;Franco, Melissa;Wasko, Kevin;Denisov, Stepan;Kunz, Wolfram S.;Knorre, Dmitry;Mazunin, Ilya;Nikolaev, Sergey;Fellay, Jacques;Reymond, Alexandre;Khrapko, Konstantin;Gunbin, Konstantin;Popadin, Konstantin
• 通讯作者: Popadin, Konstantin

A nanoscale, multi-parametric flow cytometry-based platform to study mitochondrial heterogeneity and mitochondrial DNA dynamics

• DOI: 10.1038/s42003-019-0513-4
• 发表时间: 2019-07-11
• 期刊: COMMUNICATIONS BIOLOGY
• 影响因子: 5.9
• 作者: MacDonald, Julie A.;Bothun, Alisha M.;Woods, Dori C.
• 通讯作者: Woods, Dori C.

Interpreting Sequence-Levenshtein distance for determining error type and frequency between two embedded sequences of equal length.

解释序列编辑距离以确定两个相等长度的嵌入序列之间的错误类型和频率。

• 发表时间: 2023
• 期刊: ArXiv
• 作者: Logan,Robert;Wehe,AmyWangsness;Woods,DoriC;Tilly,Jon;Khrapko,Konstantin
• 通讯作者: Khrapko,Konstantin

Reanalysis of mtDNA mutations of human primordial germ cells (PGCs) reveals NUMT contamination and suggests that selection in PGCs may be positive

对人类原始生殖细胞 (PGC) mtDNA 突变的重新分析揭示了 NUMT 污染，并表明 PGC 中的选择可能是阳性的

• DOI: 10.1016/j.mito.2023.10.005
• 发表时间: 2024
• 期刊: Mitochondrion
• 影响因子: 4.4
• 作者: Fleischmann, Zoë;Cote-L'Heureux, Auden;Franco, Melissa;Oreshkov, Sergey;Annis, Sofia;Khrapko, Mark;Aidlen, Dylan;Popadin, Konstantin;Woods, Dori C.;Tilly, Jonathan L.
• 通讯作者: Tilly, Jonathan L.