MITOCHONDRIAL BIGENESIS AND REVERSE TRASCRIPTASES

拟议的研究包括对NO的持续生化遗传分析。 在脉孢子菌线粒体中以DNA质粒的形式繁殖的逆转录因子。 在目前的授权期内，我们获得了两个这样的证据 Mauriceville和Varkud线粒体(Mt)质粒使用 反转录合成(-)链DNA的新机制 直接在转录本的3‘端启动，通过 识别3‘末端的tRNA样结构。这些质粒也有 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，手机55,693-704,1989)。在合作实验中，我们有 还研究了第二个I族元素，Varkud小质粒(VSP)，它 似乎利用了瓦尔库德质粒RT，并可能作为卫星复制 Varkud质粒，和Labelle Mt质粒，一个不相关的元件 它编码一种类似RT的蛋白，可能具有DNA聚合酶活性。在……里面 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，现在可以用于 对这些元件及其逆转录/复制的详细分析 阳离子机制。由于线粒体元素是独一无二的，我们 预计这些研究将提供有关 反转录和DNA合成的机制和进展 以及逆转录病毒、RNA病毒和内含子的进化。 具体目标是：(1)继续对毛利切维尔进行生化分析 质粒RT。(2)鉴定所需的其他酶活性 质粒的复制和整合。(三)调查研究 质粒RNA的转录、加工及其合成机制 杂交核糖核酸物种。(4)调查有关机制。 质粒整合到线粒体DNA中。(5)发展转型的方法 将修饰后的质粒化成线粒体。(6)探讨心力衰竭的发病机制。 推测的卫星元件VSP的逆转录。(七)携带 对该基因编码的类RT蛋白进行了进一步的生化分析 LaBelle Mt.

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.