Characterization of RNA Editing in Physarum Polycephalum

多头绒泡菌 RNA 编辑的表征

• 批准号: 9304465
• 负责人: Jonatha Gott
• 金额: $ 29.3万
• 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: 1993
• 资助国家: 美国
• 起止时间: 1993-08-01 至 1997-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9304465&HistoricalAwards=false
• 关键词: Characterization; RNA; Editing; Physarum; Polycephalum

9304465 Gott RNA editing, the process by which genetic information is altered at the RNA leave, is a fundamental aspect of gene expression and regulation in a variety of systems. Some editing phenomena occur globally within an organelle, while others are tissue specific or developmentally regulated. Despite the almost unlimited potential for the control of gene expression provided by this process, little is known about the mechanisms by which RNA editing is accomplished. In addition, the specific nature of the changes involved makes editing a potential target for therapeutic agents. Thus an understanding, at the molecular level, of the mechanisms of editing may have practical applications as well providing insights into another level of the control of gene expression. Two distinct forms of RNA editing have been described: base substitution and base insertion/deletion. Preliminary results suggest that both types of editing coexist in the slime mold Physarum polycephalum. This unique feature, along with biochemical and developmental tractability of the organism, make Physarum an excellent model system in which to study editing mechanisms. The goals of this research are to identify the intermediates and products of RNA editing in Physarum and to characterize the occurrence and extent of editing relative to the stages of the life cycle. Based on observed differences between mitochondrial DNA and RNA sequences from Physarum, two genes have been identified whose expression is likely to depend upon RNA editing. The proteins encoded by the "edited" versions of these mRNAs are highly homologous to subunits 1 and 2 of other mitochondrial cytochrome oxidases, suggesting that th is process is required for the generation of functional mRNAs. These genes and their RNA and protein products will be characterized further using a combination of techniques, including RNA finger printing, thin layer chromatography, primer extension, nuclease protection, Northern and Southern hybridization, PCR amplification, gel electrophoresis, and in vivo and in vitro isotopic labeling experiments. Although the biochemical experiments described above are essential to an understanding of the editing process, the identification and characterization of the editing apparatus will be facilitated by the development of a genetic framework in which to study editing. A necessary prelude to such experiments is an accurate description of the nature and extent of editing events during the complex life cycle of Physarum. This information will also be useful in the design of future biochemical fractionation experiments and may provide clues regarding potential roles of editing in gene regulation and development in Physarum. %%% This research will help us understand an unusual step in the processing of genetic information. ***

9304465 Gott RNA编辑，即在RNA叶片上改变遗传信息的过程，是各种系统中基因表达和调控的一个基本方面。有些编辑现象发生在细胞器内的整体范围内，而另一些则是特定于组织或受发育调节的。尽管这一过程提供了控制基因表达的几乎无限的潜力，但人们对完成RNA编辑的机制知之甚少。此外，所涉及的变化的特殊性使编辑成为治疗剂的潜在目标。因此，在分子水平上对编辑机制的理解可能会有实际应用，也可以提供对基因表达控制的另一个水平的洞察。已经描述了两种不同形式的RNA编辑：碱基替换和碱基插入/缺失。初步结果表明，这两种类型的编辑在粘菌多头绒泡菌中共存。这种独特的特性，再加上生物体的生物化学和发育适应性，使绒泡菌成为研究编辑机制的极佳模型系统。本研究的目的是确定绒泡菌中RNA编辑的中间体和产物，并根据生命周期的不同阶段描述编辑的发生和程度。根据观察到的绒泡菌线粒体DNA和RNA序列的差异，已经鉴定出两个基因，它们的表达可能依赖于RNA编辑。这些mRNAs的“编辑”版本所编码的蛋白质与其他线粒体细胞色素氧化酶的亚基1和2高度同源，这表明这一过程是产生功能性mRNAs所必需的。这些基因及其RNA和蛋白质产物将使用一系列技术进一步鉴定，包括RNA指纹图谱、薄层层析、引物延伸、核酸酶保护、Northern和Southern杂交、PCR扩增、凝胶电泳以及体内和体外同位素标记实验。尽管上述生化实验对于理解编辑过程是必不可少的，但通过开发研究编辑的遗传框架，将有助于编辑设备的识别和表征。这种实验的一个必要的前奏是准确描述在绒泡菌复杂的生命周期中编辑事件的性质和程度。这些信息也将对未来生化分离实验的设计有用，并可能提供关于编辑在绒泡菌基因调控和发育中的潜在作用的线索。这项研究将帮助我们理解基因信息处理中的一个不寻常的步骤。***