Defining heteroplasmy at the single mitochondrion level

描述(由申请者提供)：我们的长期目标是了解 线粒体DNA(MtDNA)突变的出现、传播和分布 在细胞中数以千计的线粒体DNA分子中，一种称为 异质性。虽然这些突变与年龄相关的疾病有关 以及衰老过程，预测疾病的异质性程度 症状或与年龄相关的表型几乎是不可能出现的 因为异质性的动力学还没有被很好地理解。中环 这一应用的假设是单个线粒体，包含 在任何地方，从2到10个线粒体DNA拷贝，都可能是异质性的，这种情况导致 线粒体复制时线粒体DNA分子的分离，以及 可能被线粒体间基因组物质的动态交换所修饰 在给定的单元格内。如果这一假设是正确的，一种异质性 线粒体将同时具有突变型和野生型mtDNA，所有的多肽 由线粒体基因组编码，以及正常的线粒体膜 潜力。我们计划开发第一个生物分析技术 研究个体线粒体的异质性。我们将继续 毛细管电泳仪的使用与改进 激光诱导荧光检测(CE-LI-F)技术研究 然后可以收集单个线粒体并进行聚合酶链式反应 他们的DNA扩增。此外，线粒体的多肽图谱 含有突变的线粒体DNA，将通过原位基质辅助检测 激光解吸飞行时间质谱仪将提供更多 异质性的综合表征。作为测试模型，我们将使用 NS-1细胞系，含有7522和4977缺失的两个杂交体细胞系，以及 6、24和28岁Fisher 344大鼠的股直肌和股底肌 月份。NS-1模型将主要用于开发技术和方法。 Cybrid模型具有一定程度的异质性(50%)和宿主 缺失ND5、ND4、ND3、ND4L、COIII、A6、 A8、tRNAL、tRNAS、tRNAH、tRNAR和tRNAG。除了这些基因之外， 携带7522碱基对缺失的环状病毒进一步省略了 CytB、ND6、COIL(2)、tRNAR和tRNAK基因。这些赛伯德模型将被使用 目的：研究细胞系异质性的进展。两种肌肉模型 将被用来研究沿红色粗糙纤维的异质性进展 已鉴定为(COX-，SDH++)表型。我们的个人 线粒体测定将是监测(1)的基础 胞质形成和繁殖后的异质性研究进展 克隆，以及(2)骨骼肌纤维的异质性程度。这个 来自Cybrid和肌肉组织模型的数据将被用来改进 现有的预测异质性克隆扩展的数学模型。 先天性心脏病患者线粒体单基因突变的检测 赛博德和肌肉模型将使我们更接近于揭开 异质性及其在疾病和衰老中的意义。

DESCRIPTION (provided by applicant): Our long-term goal is to understand how mitochondrial DNA (mtDNA) mutations appear, propagate, and are distributed among the thousands of mtDNA molecules in a cell, a condition called heteroplasmy. While these mutations are associated with age-related diseases and the aging process, predicting the degree of heteroplasmy at which disease symptoms or age-related phenotypes will appear is practically impossible because the dynamics of heteroplasmy are not well understood. The central hypothesis of this application is that individual mitochondria, containing anywhere from 2 to 10 mtDNA copies, can be heteroplasmic, a condition resulting from the segregation of mtDNA molecules upon mitochondrial replication, and likely modified by the dynamic exchange of genomic material among mitochondria within a given cell. If this hypothesis is correct, a heteroplasmic mitochondrion will have both mutated and wild-type mtDNA, all the peptides encoded by the mitochondrial genome, and a normal mitochondrial membrane potential. We propose to develop the first bioanalytical technologies capable of investigating heteroplasmy in individual mitochondria. We will continue to use and improve upon an instrument based on capillary electrophoresis with laser-induced fluorescence detection (CE-LI F) to determine the properties of individual mitochondria that can then be collected and subjected to PCR amplification of their DNA. In addition, peptide profiles from mitochondria containing mutated mtDNA that will be determined by in situ matrix-assisted laser-desorption time-of-flight mass spectrometry will provide a more comprehensive characterization of heteroplasmy. As testing models, we will use NS-1 cells lines, two cybrid cell lines harboring 7522 and 4977 deletions, and rectus femoris and soleous muscles from Fisher 344 Rats, aged 6, 24, and 28 months. The NS-1 model will mainly be used to develop technologies and methods. The cybrid models have a defined degree of heteroplasmy (> 50 percent) and host deletions that omit the expression of the genes ND5, ND4, ND3, ND4L, COIII, A6, A8, and tRNAL, tRNAS, tRNAH, tRNAR, and tRNAG. In addition to these genes the cybrid hosting the 7522 base pair deletion further omits expression of the cytb, ND6, COIl (2), tRNAR, and tRNAK genes. These cybrid models will be used to study the progression of heteroplasmy in cell lines. The two muscle models will be used to study the progression of heteroplasmy along red ragged fibers that have been identified by (COX-, SDH++) phenotype. Our individual mitochondrial determinations will be the basis for monitoring (1) the progression of heteroplasmy after the formation and propagation of a cybrid clone, and (2) the degree of heteroplasmy along skeletal muscle fibers. The data resulting from the cybrid and muscle tissue models will be used to refine existing mathematical models that predict the clonal expansion of heteroplasmy. The determination of mtDNA mutations at the single mitochondrion level in the cybrid and muscle models will bring us closer to uncovering the intricacies of heteroplasmy and its implications in disease and aging.