Mitochondrial to nuclear gene transfer via synthetic evolution

通过合成进化从线粒体到核基因转移

• 批准号: 9269097
• 负责人: Lars M Steinmetz
• 金额: $ 32.81万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-05-01 至 2019-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9269097
• 关键词: Address; Aging; Aging-Related Process; Alleles; Biochemical; Bioenergetics; Biogenesis; Budgets; Cell Nucleus; Cell physiology; Cells; Competence; Complement; Complex; DNA; Defect; Disease; Engineering; Environment; Eukaryota; Evolution; Gene Expression; Gene Expression Regulation; Gene Transfer; Genes; Genetic; Genetic Materials; Genetic Screening; Genome; Genomics; Goals; Human; Investigation; Knowledge; Lead; Life; Location; Maintenance; Measures; Medical; Metabolic; Mitochondria; Mitochondrial DNA; Molecular; Molecular Target; Mutation; Neurodegenerative Disorders; Nuclear; Oligonucleotides; Organism; PTGS1 gene; Pathway interactions; Predisposition; Process; Production; Proteins; Reporting; Research Personnel; Respiration; Respiratory physiology; Role; Saccharomyces cerevisiae; System; Techniques; Technology; Testing; Time; Work; Yeasts; combinatorial; cost effective; fitness; functional genomics; gene product; genome sequencing; genome-wide; improved; insight; mitochondrial DNA mutation; mitochondrial genome; mutant; next generation; novel; novel strategies; nuclear transfer; overexpression; prevent; protein expression; respiratory; success; synthetic biology; trafficking; transcriptomics; whole genome

Mitochondria, the centers of cellular energy production, have transferred the majority of their own genetic material to the nuclear genome during evolution. Yet a handful of genes remain in all mitochondrial genomes, despite their susceptibility to damaging metabolic byproducts and mutations. The consequences of mtDNA mutations are significant: they are implicated in a range of severe diseases, and the mutations accumulated during a lifetime are believed to lead to neurodegenerative disorders and the ageing process itself. This raises the question of why the mitochondrial genome still exists, despite the potentially severe consequences on fitness in all eukaryotes, and what are the cellular processes that limit or support mitochondrial gene expression from the nucleus? These questions can be answered by synthetic 'allotopic' expression of these genes from the protected environment of the nucleus. Recent studies have suggested that the lack of success with this strategy is due to the need for adaptations not only in the allotopic protein, but also in several cellular processes. The goal of this project is to systematically study allotopic expression in yeast using a combination of high-throughput and mechanistic biochemical approaches. Yeast is uniquely suited to study this problem because it is one of few organisms where mtDNA can be manipulated, and is amenable to genomic and synthetic biology techniques. Allotopic expression of the 4 yeast genes that have not been experimentally transferred thus far, each of which have been implicated in disease, will be tested in multiple versions by exploiting cost-effective, next-generation oligonucleotide synthesis technology. Applying the power of genetic screens, weakly successful allotopic strains will be used to discover genetic suppressors that improve allotopic expression through genomic screens and in-lab evolution, revealing pathways involved in nuclear gene transfer and mitochondrial biogenesis. These discoveries will be used to produce 'superhost' yeast strains whose backgrounds strongly favour allotopic expression. To discover the roadblocks that prevent allotopic expression and test competing hypotheses for why mtDNA genes have been retained, protein localization, trafficking, susceptibility to degradation, and mitochondrial transport will be tracked. These rewired strains will be characterized at the transcriptomic, bioenergetic, and mechanistic levels. Finally, the allotopically expressed genes will be combined stepwise to generate a strain with a minimal mitochondrial genome. This work will be carried out by leading groups in functional genomics, mitochondrial bioenergetics, and evolution. It will reveal obstacles facing nuclear transfer of mitochondrial genes during evolution, how mitochondrial gene products are expressed and processed, and build a systematic understanding of the key factors in mitochondrial biogenesis. This project will also open new avenues for studying the role of mtDNA in ageing and neurodegenerative disorders.

线粒体是细胞能量产生的中心， 在进化过程中向核基因组提供物质。然而，在所有线粒体基因组中仍然存在少数基因， 尽管它们对破坏性代谢副产物和突变敏感。mtDNA的影响 突变是重要的：它们与一系列严重疾病有关，突变积累 在一生中被认为会导致神经退行性疾病和衰老过程本身。这就提出 为什么线粒体基因组仍然存在的问题，尽管潜在的严重后果， 在所有真核生物中，限制或支持线粒体基因的细胞过程是什么？ 从细胞核中表达出来？这些问题可以通过这些的合成“异位”表达来回答 从受保护的细胞核环境中提取基因。最近的研究表明，缺乏成功 这种策略是由于不仅在异位蛋白中需要适应，而且在几种细胞中也需要适应。 流程.该项目的目标是系统地研究酵母中的异位表达， 高通量和机械生化方法。酵母是研究这个问题的唯一合适的材料 因为它是少数几种可以操纵mtDNA的生物之一，并且适合基因组和 合成生物学技术4个酵母基因的异位表达，这些基因尚未在实验中得到证实。 到目前为止，每一种都与疾病有关，将在多个版本中进行测试， 开发具有成本效益的下一代寡核苷酸合成技术。运用基因的力量 筛选，弱成功的异位菌株将用于发现遗传抑制，提高异位 通过基因组筛选和实验室进化表达，揭示了核基因 转移和线粒体生物发生。这些发现将用于生产“超级宿主”酵母菌株 其背景强烈支持异位表达。去发现阻止异位的障碍 表达和测试竞争的假设，为什么mtDNA基因被保留，蛋白质定位， 将跟踪运输、易降解性和线粒体运输。这些重新连接的菌株 在转录组学、生物能量学和机械学水平上进行表征。最后，异位表达的 基因将逐步组合以产生具有最小线粒体基因组的菌株。这项工作将 由功能基因组学、线粒体生物能量学和进化的领先小组进行。它将揭示 在进化过程中线粒体基因的核转移面临的障碍，线粒体基因产物是如何 表达和加工，并建立线粒体生物发生的关键因素的系统理解。 该项目还将为研究mtDNA在衰老和神经退行性疾病中的作用开辟新的途径。 紊乱