Genomic studies of chimeric mitochondria in cybrids from Solanaceae

描述(申请人提供)：“茄科线虫嵌合线粒体的基因组研究”这项研究将主要在阿根廷国立库约大学与马尔马·维吉尼亚·桑切斯·普尔塔博士合作进行，作为R01 GM070612-4的延伸。研究线粒体基因组的进化、动力学、外源DNA的摄取以及与核基因组的相互作用对于深入理解真核细胞是必不可少的。线粒体基因组提供了一个极好的系统来增加我们对基因组重排、嵌合基因、核质不亲和性和水平基因转移(HGT)的了解。植物线粒体通过HGT获得基因的倾向是独一无二的。HGT在真核生物中最普遍的例子涉及cox1内含子，它编码一个假定的归巢内切酶，通过水平转移增加内含子的固定频率。我们建议研究两个不同的线粒体基因组之间的相互作用，这两个基因组重组在一个杂交类植物中，通过原生质体融合实验获得。在囊体形成过程中，两种亲本类型的线粒体及其基因组(只有一种含有cox1内含子)会融合，形成一个杂交的线粒体基因组。我们将从含有cox1内含子的物种和缺乏cox1内含子的物种之间的体细胞杂交中建立大约20个循环系，以解决以下两个目标：首先，我们将检验cox1内含子编码植物中功能定位内切酶的假设，评估内含子定殖率，并测量伴随内含子插入的外显子共转换区的长度。其次，我们将对多达20个线虫品系的整个线粒体基因组进行测序和分析。这将为重现植物线粒体中HGT的自然过程提供一个实验模型，并将在不同水平上提供丰富的线粒体基因组重组过程和模式的图景。维持一个完整的、功能正常的线粒体基因组是生存的关键。鉴于植物和动物之间有许多分子上的相似之处，该项目将对动物基因治疗和植物基因工程的发展都有价值。与公共卫生相关：基因组重排、核苷酸替换和外来DNA的引入塑造了真核生物的线粒体基因组，并经常对人类健康造成严重后果，几种重要的线粒体遗传病就是明证。此外，本研究的关键实验生物(烟草)被广泛用作疫苗、抗生素和一些治疗性蛋白质的生物制药生产的表达系统。因此，了解线粒体DNA进化的分子机制对人类健康和疾病具有相当重要的意义。

DESCRIPTION (provided by applicant): "Genomic studies of chimeric mitochondria in cybrids from Solanaceae" This research will be conducted primarily in Argentina at Universidad Nacional de Cuyo in collaboration with Dr. Marma Virginia Sanchez Puerta, as an extension of R01 GM070612-4. The study of mitochondrial genome evolution, dynamics, uptake of foreign DNA, and interactions with the nuclear genome is essential to a deep understanding of the eukaryotic cell. Mitochondrial genomes provide an excellent system to increase our knowledge of genome rearrangements, chimeric genes, nuclear- cytoplasmic incompatibilities, and horizontal gene transfer (HGT). Plant mitochondria are unique in their propensity to acquire genes by HGT. The most pervasive example of HGT in eukaryotes involves the cox1 intron, which encodes a putative homing endonuclease that increases the frequency of the intron's fixation via horizontal transfer. We propose to study aspects of the interactions between two distinct mitochondrial genomes that recombine in a cybrid plant obtained by protoplast fusion experiments. During cybrid formation, the two mitochondrial parental types and their genomes (only one containing the cox1 intron) will fuse, resulting in a hybrid mitochondrial genome. We will establish some 20 cybrid lines derived from somatic crosses between cox1 intron-containing and -lacking species in order to address the following two aims: First, we will test the hypothesis that the cox1 intron encodes a functional homing endonuclease in plants, assess rates of intron colonization, and measure lengths of exonic coconversion tracts that accompany intron insertion. Second, we will sequence and analyze the entire mitochondrial genomes of up to 20 cybrid lines. This will serve as an experimental model for recapitulating the natural process of HGT in plant mitochondria and will provide a rich picture of the process and pattern of mitochondrial genome recombination at various levels. Maintenance of an intact, functioning mitochondrial genome is critical for survival. Given the many molecular parallels between plants and animals, this project will be valuable for the development of both animal gene therapy and plant genetic engineering. PUBLIC HEALTH RELEVANCE: Genome rearrangements, nucleotide substitutions, and the introduction of foreign DNA shape the mitochondrial genomes of eukaryotes and often have severe consequences for human health, as evidenced by several important mitochondrially-inherited diseases. In addition, the key experimental organism of this study (tobacco) is widely used as an expression system for biopharmaceutical production of vaccines, antibiotics, and a number of therapeutic proteins. Understanding the molecular mechanisms of mitochondrial DNA evolution is therefore of considerable importance to human health and disease.