MITOCHONDRIAL BIGENESIS AND REVERSE TRANSCRIPTASES

这项拟议中的研究涉及对NO的持续生化遗传分析。 在链孢菌线粒体中作为DNA质粒繁殖的逆转录因子。 在目前的赠款期间，我们获得了两个这样的证据， Mauriceville和Varkud线粒体（mt）质粒使用一个 一种新的逆转录机制，其中（-）链DNA合成 直接起始于质粒转录物的3 '末端，通过 识别3 '末端tRNA样结构。这些质粒还具有 I组内含子的特征，并且可能在进化上与 这些内含子，以及RNA病毒和逆转录病毒（Kuiper和 Lambowitz，Cell 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并可作为卫星复制 Varkud质粒和Labelle mt质粒，一个无关元件 它编码一种可能具有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 基因、生物化学和免疫化学工具， 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成分是独特的，我们 预计这些研究将提供新的信息， 逆转录和DNA合成的机制和进化， 以及逆转录酶元件、RNA病毒和内含子的进化。 具体目标是：（1）继续进行Mauriceville的生化分析 （2）为了表征质粒RT所需的其他酶活性， 质粒的复制和整合。(3)探讨 质粒RNA的转录、加工及合成机制 杂交RNA物种。(4)为了研究 质粒整合到mtDNA中。(5)制定方法， 修饰质粒进入线粒体。(6)探讨其作用机制 反转录的推定卫星元件，VSP。(7)携带 进一步的生物化学分析的RT样蛋白编码的 标记mt质粒。

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.