Defining heteroplasmy at the single mitochondrion level

描述（由申请人提供）：我们的长期目标是了解如何 线粒体DNA（mtDNA）突变出现、传播和分布 在细胞中成千上万的mtDNA分子中， 异质性虽然这些突变与年龄相关的疾病有关， 和衰老过程，预测疾病的异质性程度， 症状或年龄相关的表型将出现几乎是不可能的 因为异质性的动力学还没有被很好地理解。中央 本申请的假设是，含有 任何地方从2到10个mtDNA拷贝，可以是异质性的，这种情况导致 线粒体复制时线粒体DNA分子的分离， 很可能是通过线粒体之间基因组物质的动态交换而改变的 在一个给定的细胞。如果这个假设是正确的， 突变的线粒体DNA和野生型的线粒体DNA， 由线粒体基因组编码，和正常的线粒体膜 潜力我们建议开发第一种生物分析技术， 研究个体线粒体中的异质性。我们将继续 使用和改进基于毛细管电泳的仪器， 激光诱导荧光检测（CE-LI F），以确定 然后可以收集单个线粒体并进行PCR 扩增他们的DNA。此外，线粒体的肽谱 含有突变的mtDNA，将通过原位基质辅助 激光解吸飞行时间质谱法将提供更多的 异质性的综合表征。作为测试模型，我们将使用 NS-1细胞系，两种含有7522和4977缺失的胞质杂种细胞系，和 6、24和28岁Fisher 344大鼠的股直肌和比目鱼肌 个月NS-1模型将主要用于开发技术和方法。 胞质杂种模型具有确定的异质性程度（> 50%）和宿主 缺失缺失基因ND 5、ND 4、ND 3、ND 4L、COIII、A6的表达， A8和tRNAL、tRNAS、tRNAH、tRNAR和tRNAG。除了这些基因， 含有7522个碱基对缺失的胞质杂交体进一步省略了 cytb、ND 6、COII（2）、tRNAR和tRNAK基因。这些赛伯人模型将被用于 来研究细胞系中异质性的发展。两个肌肉模型 将被用来研究异质性的进展沿着红色参差不齐的纤维 已通过（考克斯-，SDH++）表型鉴定。我们个人 线粒体测定将是监测的基础（1） 胞质杂种形成和繁殖后异质性的发展 克隆;（2）沿沿着骨骼肌纤维的异质性程度。的 从胞质杂种和肌肉组织模型得到的数据将用于改进 现有的数学模型预测异质性的克隆扩张。 中国汉族人群mtDNA单碱基突变的检测 赛伯人和肌肉模型将使我们更接近于揭示 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.