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.
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