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，已转移了自己的大部分遗传 进化过程中核基因组的材料。然而，所有线粒体基因组中仍然存在少数基因， 尽管它们易于破坏代谢副产品和突变。 mtDNA的后果 突变很重要：它们与一系列严重疾病有关，突变积累 据信，一生中会导致神经退行性疾病和衰老过程本身。这加剧了 尽管可能对线粒体基因组存在严重后果，但仍存在线粒体基因组的问题 所有真核生物的适应性，以及限制或支持线粒体基因的细胞过程 核表达？这些问题可以通过合成的“同种异体”表达来回答这些问题 来自核保护环境的基因。最近的研究表明缺乏成功 有了这种策略，不仅需要在同倍蛋白中进行适应，还需要在几个细胞中进行适应 过程。该项目的目的是使用组合系统地研究酵母中的同种异体表达 高通量和机械生化方法。酵母非常适合研究这个问题 因为它是可以操纵mtDNA的少数生物之一，并且可以适合基因组和 合成生物学技术。尚未实验的4种酵母基因的同种异体表达 到目前为止转移的每一个都与疾病有关，将通过多种版本测试 利用成本效益的下一代寡核苷酸合成技术。应用遗传的力量 屏幕，微弱成功的同种异体菌株将用于发现改善同种异体的遗传抑制剂 通过基因组筛选和LAB进化的表达，揭示了参与核基因的途径 转移和线粒体生物发生。这些发现将用于生产“超主机”酵母菌菌株 其背景强烈偏爱同种异体表达。发现阻止同种异体的障碍 表达和测试竞争假设是为什么保留mtDNA基因的蛋白质定位， 将跟踪贩运，降解和线粒体运输的易感性。这些融合的压力将 在转录组，生物能和机械水平上进行表征。最后，同种异体表达 基因将逐步合并，以最小的线粒体基因组产生菌株。这项工作将是 由主要基因组学，线粒体生物能学和进化的领先组进行。它将揭示 进化过程中线粒体基因的核转移的障碍，线粒体基因产物如何 表达和处理，并对线粒体生物发生的关键因素产生系统的理解。 该项目还将为研究mtDNA在衰老和神经退行性中的作用开放新途径 疾病。