GENETIC ANALYSIS OF MAMMALIAN MITOCHONDRIAL INHERITANCE

哺乳动物线粒体遗传的基因分析

• 批准号: 3283408
• 负责人: PHILIP J LAIPIS
• 金额: $ 18.61万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-12-01 至 1994-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3283408
• 关键词: animal population genetics; biological polymorphism; cell fusion; cell transformation; cytogenetics; early embryonic stage; extrachromosomal DNA; genetic markers; genotype; growth /development; heterozygote; laboratory mouse; liposomes; mitochondria; mitochondrial DNA; newborn animals; nucleic acid sequence; single cell analysis; tissue /cell culture; transfection

The details of the mechanism which transfers mammalian mitochondrial DNA (mtDNA) from mother to offspring and the constraints which this mechanism would place on the origin of mitochondrial genetic diversity are currently unknown. The frequent occurrence of mtDNA sequence polymorphisms within a species suggests that simple transfer of a female's mitochondrial complement to her offspring does not occur and that a more complex path is followed which transfers only a small subset of the maternal mitochondria to the new embryo. Passage through this bottleneck would allow for the rapid appearance of new mitochondrial genotypes in the population. To test this model, heteroplasmic mice (which contain two different mtDNA genomes) were constructed by embryo fusion at the single cell level. Analysis of offspring of heteroplasmic female mice indicate both mtDNA species are transmitted to offspring; they appear to be transmitted at different levels in different progeny. Experiments are described which will determine if the segregation rates of the two mtDNA species in different nuclear gene backgrounds are identical, and whether both the level of overall mtDNA heteroplasmy and possible tissue differences in the levels of heteroplasmy occur. In addition, the rates at which the two different mtDNA species segregate in cell lines derived from these animals will be determined. Finally, a sensitive test for the presence of paternal mtDNA is described. These experiments will help define the kind and amounts of mtDNA variation that can occur in mammals. A second major goal of this proposal is to develop methods for mtDNA transformation. Mitochondria from various sources will be transferred into embryos, from somatic cells of various types. Once this is accomplished, mtDNA introduction into the organelle will be attempted using chemical treatment, liposome fusion, electroporation, or use of a particle accelerator to transform either cell lines or isolated mitochondria with exogenous mtDNA. These experiments will allow the construction of animals carrying in vitro mutated mtDNAs, and permit an examination of the role of mtDNA function in muscle and nerve myopathies. These animals and the techniques that allow their construction should also help in understanding cytoplasmic movement during early embryo development, movement of cell lineages, and the changes in mitochondria which occur during early development.

哺乳动物线粒体DNA转移机制的细节 （mtDNA）从母亲到后代以及这的限制 机制将放在线粒体遗传多样性的起源上 目前未知。 mtDNA序列频繁出现 物种内的多态性表明，简单的转移 女性的线粒体对其后代的补充不会发生，并且 遵循更复杂的路径，仅传输一小部分 将母体线粒体传递给新胚胎。 通过这个 瓶颈将允许新线粒体的快速出现 人群中的基因型。 为了测试这个模型，异质小鼠（含有两种不同的 mtDNA基因组）是通过单细胞胚胎融合构建的 等级。 对异质雌性小鼠后代的分析表明 mtDNA 物种遗传给后代；他们似乎是 在不同的后代中以不同的水平传播。 描述了实验，该实验将确定分离率是否 不同核基因背景下的两个 mtDNA 物种的 相同，以及总体 mtDNA 异质性水平和 异质性水平可能存在组织差异。 在 此外，两种不同 mtDNA 物种分离的速率 将确定源自这些动物的细胞系中的。 最后，一个 描述了父系 mtDNA 存在的敏感测试。 这些 实验将有助于确定 mtDNA 变异的种类和数量 这可能发生在哺乳动物身上。 该提案的第二个主要目标是开发 mtDNA 方法 转变。 各种来源的线粒体将被转移 从各种类型的体细胞进入胚胎。 一旦这是 完成后，将尝试将 mtDNA 引入细胞器 使用化学处理、脂质体融合、电穿孔或使用 粒子加速器可转化细胞系或分离的细胞 线粒体与外源mtDNA。 这些实验将允许 构建携带体外突变 mtDNA 的动物，并允许 检查 mtDNA 功能在肌肉和神经中的作用 肌病。 这些动物和允许它们的技术 构建还应该有助于理解细胞质运动 在早期胚胎发育过程中，细胞谱系的运动以及 早期发育过程中发生的线粒体变化。