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GENETIC ANALYSIS OF MAMMALIAN MITOCHONDRIAL INHERITANCE

哺乳动物线粒体遗传的基因分析

基本信息

批准号：
2177052
负责人：
PHILIP J LAIPIS
金额：
$ 19.85万
依托单位：
UNIVERSITY OF FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
1984
资助国家：
美国
起止时间：
1984-12-01 至 1995-06-30
项目状态：
已结题

项目摘要

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）从母亲到后代的限制，这是线粒体遗传多样性的起源机制目前尚不清楚。线粒体DNA序列的频繁出现一个物种内的多态性表明，女性的线粒体补充她的后代不会发生，遵循一条更复杂的路径，母体线粒体转移到新胚胎中。通过这个瓶颈会让新的线粒体快速出现，在人群中的基因型为了测试该模型，异质性小鼠（其含有两种不同的 mtDNA基因组）通过单细胞胚胎融合构建水平对异质性雌性小鼠后代的分析表明，线粒体DNA物种被传递给后代;他们似乎是在不同的后代中以不同的水平传递。描述了实验，这将确定是否偏析率不同核基因背景的两种mtDNA中，相同，以及是否整体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种类分离的速率将确定来源于这些动物的细胞系中的细胞数。最后描述了一种检测父系mtDNA存在的敏感试验。这些实验将有助于确定线粒体DNA变异的种类和数量在哺乳动物身上发生的。该提案的第二个主要目标是开发线粒体DNA的方法转型各种来源的线粒体将被转移到从各种类型的体细胞转化成胚胎。一旦这样完成后，将尝试将mtDNA导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转化细胞系或分离的线粒体与外源性mtDNA。这些实验将使构建携带体外突变mtDNA的动物，并允许 mtDNA功能在肌肉和神经中的作用肌病这些动物和技术，使他们的构建也有助于理解细胞质运动在早期胚胎发育，细胞谱系的运动，线粒体在早期发育过程中发生的变化。