Characterization of the Editing Apparatus in Physarum Mitochondria

绒泡菌线粒体编辑装置的表征

• 批准号: 9630672
• 负责人: Jonatha Gott
• 金额: $ 27.4万
• 依托单位: Case Western Reserve University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-09-01 至 2000-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9630672&HistoricalAwards=false
• 关键词: Characterization; Editing; Apparatus; Physarum; Mitochondria

9630672 Gott The slime mold Physarum polycephalum is the only organism known to carry out both of the two major forms of RNA editing that have been described, substitution of one base for another and insertion of non-encoded nucleotides. Editing in Physarum mitochondria involves a wide variety of changes, including the addition of each of the four nucleotides (as single or dinucleotide insertions), as well as a number of specific cytidine to uridine changes. These unique features, along with the biochemical and genetic tractability of the organism, make Physarum an excellent model system in which to study editing. The ultimate goal of this work is a mechanistic understanding of this unusual mode of gene expression in Physarum and other organisms. The specific objectives of this research are to characterize the number and types of editing mechanisms that function in Physarum mitochondria to localize the source of the editing information, and to identify the components of the editing machinery. In preliminary studies, conditions have been found that allow only a subset of the insertional editing events normally observed in isolated mitochondria. This observation will be pursued systematically by manipulating labeling conditions and characterizing the RNAs synthesized in this cell-free system. Thus, it should be possible to determine, for example, whether the same apparatus that inserts single nucleotides into RNA is also responsible for dinucleotide addition. The ability to synthesize unedited RNA will also be utilized in pulse-chase studies designed to determine both the extent to which editing and transcription are coupled in Physarum mitochondria, and whether specific RNA sequences are required for editing. Identification of the components of the Physarum editing apparatus is the principal goal of the genetic experiments. Previously it was determined that RNA editing is both accurate and efficient at all stages of the Physarum life cycle, including the haploid amoebal stage that will b e used in genetic studies. Both preexisting temperature sensitive strains and additional mutants generated in the course of this work will be assayed for their ability to edit RNA under non-permissive conditions. Physarum strains defective in any type of RNA editing will be utilized in further genetic and biochemical studies, including those designed to isolate the gene(s) involved in editing and to characterize editing intermediates that may accumulate. ***

9630672粘液霉菌多头绒泡菌是已知的唯一一种同时执行已描述的两种主要核糖核酸编辑形式的生物，即用一个碱基替换另一个碱基和插入非编码核苷酸。绒泡菌线粒体的编辑涉及各种各样的变化，包括添加四个核苷酸中的每一个(作为单核苷酸或二核苷酸插入)，以及一些特定的胞苷到尿苷的变化。这些独特的特征，再加上生物体的生物化学和遗传适应性，使绒泡菌成为研究编辑的极佳模型系统。这项工作的最终目标是从机制上理解这种不寻常的基因在绒泡菌和其他生物体中的表达模式。这项研究的具体目标是确定绒泡菌线粒体中编辑机制的数量和类型，以定位编辑信息的来源，并确定编辑机制的组成部分。在初步研究中，已经发现只允许在分离的线粒体中通常观察到的插入编辑事件的子集的条件。这一观察将通过操纵标记条件和表征在这个无细胞系统中合成的RNA来系统地进行。因此，应该可以确定，例如，将单核苷酸插入RNA的同一装置是否也负责二核苷酸加成。合成未经编辑的RNA的能力也将被用于脉冲追逐研究，旨在确定绒泡菌线粒体中编辑和转录的耦合程度，以及编辑是否需要特定的RNA序列。鉴定绒毛霉菌编辑装置的组件是遗传学实验的主要目标。此前，人们已经确定，在绒泡菌生命周期的所有阶段，包括将用于遗传学研究的单倍体变形虫阶段，RNA编辑都是准确和有效的。将对先前存在的温度敏感菌株和在这项工作过程中产生的其他突变株进行测试，以确定它们在不允许的条件下编辑RNA的能力。在任何类型的核糖核酸编辑中有缺陷的绒泡菌菌株将被用于进一步的遗传和生化研究，包括那些旨在分离参与编辑的基因(S)和表征可能积累的编辑中间产物的那些研究。***