Heteroplasmy at the single mitochondrion level

单线粒体水平的异质性

• 批准号: 7214732
• 负责人: EDGAR A ARRIAGA
• 金额: $ 10.81万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7214732
• 关键词: Affect; Age; Aging; Aging-Related Process; Appearance; Automation; Award; Biology; Capillary Electrophoresis; Cell Line; Cell fusion; Cells; Collaborations; Communities; Compatible; Condition; DNA; DNA-Directed DNA Polymerase; Detection; Development; Disease; Educational process of instructing; Exhibits; Fiber; Fluorescence; Genomics; Goals; Independent Scientist Award; Individual; Knowledge; Laboratories; Lasers; Leadership; Length; Link; Measurement; Measures; Membrane Potentials; Mitochondria; Mitochondrial DNA; Mitochondrial Proteins; Modeling; Monitor; Muscle; Muscle Fibers; Mutate; Mutation; Organelles; Peptides; Phenotype; Polymerase Chain Reaction; Process; Proteomics; Range; Rattus; Research; Robotics; Role; Sampling; Scientist; Services; Skeletal Muscle; Solid; Symptoms; Technology; Testing; Time; Tissues; Training; age related; aged; base; drug metabolism; instrument; mathematical model; mitochondrial DNA mutation; mitochondrial genome; mitochondrial membrane; new technology; novel strategies; programs; prototype; segregation

DESCRIPTION (provided by applicant): The immediate goal of the applicant is to devote 75% of his time during the five-year support period of this K02 award to the exploration of novel approaches to subcellular biology. The time off from teaching and service provided by the award would allow him to focus more intensely on the development of new strategies based on individual organelle analysis that would then be used to better understand disease and aging. These strategies are being developed as part of two ongoing R01 projects in his laboratory that investigate (i) the role of mitochondrial DNA (mtDNA) mutations in aging and age-related diseases, and (ii) subcellular drug metabolism. This award would also allow the candidate to devote more time to (i) integrating robotics, cybrid technology, and proteomics expertise into his laboratory; (ii) training scientists, (iii) establishing a solid network of collaborations, and (iv) providing cohesive leadership to a multi-disciplinary research team. In the research plan of this application, the candidate proposes to study the distributions of mtDNA mutations based on individual mitochondrion measurements. While the accumulation of these mutations has been implicated in the aging process and age-related diseases, the link between mutation levels and age-related phenotypes or disease symptoms is not known. The hypothesis of this application is that individual mitochondria contain both wild-type and mutated DNA, a condition known as heteroplasmy, which determines how mutations are distributed and propagated. Two models will be used to test this hypothesis: cybrid cell lines harboring large mtDNA deletions, and skeletal muscle tissue from aged Fisher 344 rats that is expected to have accumulated similar deletions with age. The three goals of the study are: (i) establish the existence of heteroplasmy within individual mitochondria, (ii) monitor changes in heteroplasmy following cybrid fusion, and (iii) measure heteroplasmy along skeletal muscle fibers. Since no technology exists to directly test this hypothesis, this application will require the further development of the applicant's strategies initially on based capillary electrophoresis with laser-induced fluorescence detection for characterizing mtDNA and peptide expression in individual organelles. Upon the completion of this K02 award, the applicant is expected to have provided the scientific community with new technologies and to be directing a widely recognized research program.

描述（由申请人提供）：申请人的直接目标是将他在K02奖的五年支持期间将其时间的75％用于探索新型亚细胞生物学方法。该奖项提供的教学和服务休息时间将使他更加专注于基于单个细胞器分析的新策略的制定，然后将其用于更好地了解疾病和衰老。这些策略是作为他实验室中两个正在进行的R01项目的一部分开发的，该项目研究了（i）线粒体DNA（mtDNA）突变在衰老和年龄相关疾病中的作用，以及（ii）亚细胞药物代谢。该奖项还将允许候选人更多时间（i）将机器人技术，cybrid技术和蛋白质组学专业知识整合到他的实验室中； （ii）培训科学家，（iii）建立坚实的合作网络，（iv）为多学科研究团队提供凝聚力的领导。在本应用的研究计划中，候选人建议根据单个线粒体测量研究mtDNA突变的分布。虽然这些突变的积累与衰老过程和与年龄有关的疾病涉及，但尚不清楚突变水平与与年龄相关的表型或疾病症状之间的联系。该应用的假设是单个线粒体均包含野生型和突变的DNA，即一种称为异质的疾病，它决定了突变的分布和传播。将使用两种模型来检验这一假设：具有较大mtDNA缺失的Cybrid细胞系，以及来自老年Fisher 344大鼠的骨骼肌组织，预计随着年龄的增长会积聚类似的缺失。该研究的三个目标是：（i）在单个线粒体中建立异质的存在，（ii）监测cybrid融合后的杂质变化，以及（iii）测量沿骨骼肌纤维的异质性。由于没有直接检验该假设的技术，因此该应用将需要最初在基于毛细管电泳的基于激光诱导的荧光检测的基于毛细管电泳上的策略，以表征单个细胞器中mtDNA和肽表达。该K02颁奖典礼完成后，预计申请人将为科学界提供新技术，并指导广受认可的研究计划。