9630672 Gott The slime mold Physarum polycephalum is the only organism known to carry out both of the two major forms of RNA editing that have been described, substitution of one base for another and insertion of non-encoded nucleotides. Editing in Physarum mitochondria involves a wide variety of changes, including the addition of each of the four nucleotides (as single or dinucleotide insertions), as well as a number of specific cytidine to uridine changes. These unique features, along with the biochemical and genetic tractability of the organism, make Physarum an excellent model system in which to study editing. The ultimate goal of this work is a mechanistic understanding of this unusual mode of gene expression in Physarum and other organisms. The specific objectives of this research are to characterize the number and types of editing mechanisms that function in Physarum mitochondria to localize the source of the editing information, and to identify the components of the editing machinery. In preliminary studies, conditions have been found that allow only a subset of the insertional editing events normally observed in isolated mitochondria. This observation will be pursued systematically by manipulating labeling conditions and characterizing the RNAs synthesized in this cell-free system. Thus, it should be possible to determine, for example, whether the same apparatus that inserts single nucleotides into RNA is also responsible for dinucleotide addition. The ability to synthesize unedited RNA will also be utilized in pulse-chase studies designed to determine both the extent to which editing and transcription are coupled in Physarum mitochondria, and whether specific RNA sequences are required for editing. Identification of the components of the Physarum editing apparatus is the principal goal of the genetic experiments. Previously it was determined that RNA editing is both accurate and efficient at all stages of the Physarum life cycle, including the haploid amoebal stage that will b e used in genetic studies. Both preexisting temperature sensitive strains and additional mutants generated in the course of this work will be assayed for their ability to edit RNA under non-permissive conditions. Physarum strains defective in any type of RNA editing will be utilized in further genetic and biochemical studies, including those designed to isolate the gene(s) involved in editing and to characterize editing intermediates that may accumulate. ***
