MITOCHONDRIAL BIGENESIS AND REVERSE TRASCRIPTASES

线粒体双发生和反向转录酶

• 批准号: 3293839
• 负责人: ALAN M. LAMBOWITZ
• 金额: $ 27.12万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-09-01 至 1995-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3293839
• 关键词: DNA replication; Neurospora; RNA biosynthesis; RNA directed DNA polymerase; autoradiography; cell nucleus; enzyme mechanism; eukaryote; fungal genetics; genetic manipulation; genetic mapping; genetic transcription; immunochemistry; laboratory rabbit; mitochondria; mitochondrial DNA; nucleic acid sequence; nucleoproteins; open reading frames; radionuclides; ribosomal RNA; transposon /insertion element

The proposed research involves continued biochemical-genetic analysis of no retroid elements that propagate as DNA plasmids in Neurospora mitochondria. During the current grant period, we obtained evidence that two such elements, the Mauriceville and Varkud mitochondrial (mt) plasmids, use a novel mechanism of reverse transcription in which (-) strand DNA synthesis is initiated directly at the 3' end of the plasmid transcript, via recognition of a 3' terminal tRNA-like structure. These plasmids also have characteristics of group I introns and may be evolutionarily related to these introns, as well as RNA viruses and retroviruses (Kuiper and Lambowitz, Cell 55, 693-704, 1989). In collaborative experiments, we have also studied a second group I element, Varkud small plasmid (VSP), that appears to utilize the Varkud plasmid RT and may replicate as a satellite of the Varkud plasmid, and the Labelle mt plasmid, an unrelated element that encodes an RT-like protein that may have DNA polymerase activity. In the course of studying the Mauriceville and Varkud plasmids, we developed genetic, biochemical and immunochemical tools that can now be used for detailed analysis of these elements and their reverse transcription/repli- cation mechanisms. Since the mitochondrial elements are unique, we anticipate that these studies will provide novel information about mechanisms and evolution of reverse transcription and DNA synthesis, as well as the evolution of retroid elements, RNA viruses, and introns. Specific aims are: (1) To continue biochemical analysis of the Mauriceville plasmid RT. (2) To characterize other enzymatic activities required for replication and integration of the plasmids. (3) To investigate transcription and processing of plasmid RNA and the mechanism of synthesis of hybrid RNA species. (4) To investigate the mechanism by which the plasmid integrates into mtDNA. (5) To develop methods for transformation of modified plasmids into mitochondria. (6) To investigate the mechanism of reverse transcription of the putative satellite element, VSP. (7) To carry out further biochemical analysis of the RT-like protein encoded by the Labelle mt plasmid.

拟议的研究涉及持续的生化遗传分析 在脉孢菌线粒体中作为 DNA 质粒繁殖的逆转录元件。 在当前的资助期内，我们获得了两个这样的证据： 元素，Mauriceville 和 Varkud 线粒体 (mt) 质粒，使用 逆转录的新机制，其中 (-) 链 DNA 合成 直接在质粒转录本的 3' 端启动，通过 3' 末端 tRNA 样结构的识别。这些质粒还具有 I 组内含子的特征，可能在进化上与 这些内含子，以及 RNA 病毒和逆转录病毒（Kuiper 和 兰博维茨，细胞 55, 693-704, 1989）。在合作实验中，我们有 还研究了第二组 I 元件，Varkud 小质粒 (VSP)， 似乎利用 Varkud 质粒 RT 并且可以作为卫星复制 Varkud 质粒和 Labelle mt 质粒（一个不相关的元件） 编码可能具有 DNA 聚合酶活性的 RT 样蛋白。在 在研究 Mauriceville 和 Varkud 质粒的过程中，我们开发了 遗传、生化和免疫化学工具现在可用于 对这些元件及其逆转录/复制的详细分析 阳离子机制。由于线粒体元件是独特的，我们 预计这些研究将提供关于 逆转录和 DNA 合成的机制和进化，如 以及逆转录元件、RNA 病毒和内含子的进化。 具体目标是： (1) 继续对 Mauriceville 进行生化分析 质粒RT。 (2) 表征所需的其他酶活性 质粒的复制和整合。 (3) 调查 质粒RNA的转录、加工及其合成机制 杂合RNA种类。 (4) 探讨其作用机制 质粒整合到线粒体DNA中。 (5) 制定转化方法 将质粒修饰成线粒体。 (6) 机理研究 推定卫星元件 VSP 的逆转录。 (7) 携带 对 RT 样蛋白编码的进一步生化分析 标签mt质粒。