The mitochondrial (MT) genome of the sea anemone Metridium senile is a 17.5 kb circular molecule that contains the 13 protein and two ribosomal RNA genes characteristic of other metazoan mtDNAs. However, Metridium mtDNA is unique among metazoan mtDNAs in that it contains genes for only two tRNAs and a single group I intron is found in each of two of the protein genes, ND5 and COI. The investigator proposes to isolate, fractionate by 2D gel electrophoresis, charge and sequence tRNAs from mitochondria and from the cytoplasm of Metridium. The tRNA sequence information gained will be used to determine whether most of the tRNAs in mitochondria are nuclear encoded and to isolate genes for both mt-tRNAs and cytoplasmic tRNAs. The precise sites of insertion of group I introns in the Metridium ND5 and COI genes will be determined. Experiments will be carried out to elucidate steps in the processing of primary transcripts of the COI and ND5 gene complexes, and conditions will be sought under which the COI and ND5 group I introns will self-splice in vitro. The protein encoded in the group I intron of the Metridium COI gene will be synthesized in E. coli cells and assayed for its ability to cleave the COI gene from which the introns have been removed, and to catalyze splicing-out of the COI gene group I intron in vitro. The mtDNAs of other cnidarians (sea anemones, corals, jellyfish and Hydra) with different degrees of relatedness to Metridium, and ctenophores (comb jellies) will be examined for the presence of group I introns in their ND5 and COI genes or in other genes. Introns that are located will be sequenced and compared to gain information on the evolution of group I introns in these phyla. MtDNA, chloroplast DNA, and nuclear DNA of the dinoflagellate (Zooxanthellae) endosymbiont of the sea anemone Anthopleura elegantissima will be isolated and an attempt will be made to detect in these different DNAs group I introns related to any group I introns that occur in the mtDNA of the host anemone. Further experiments are proposed to determine whether multiple transcription initiation sites might occur at various intergenic locations around the Metridium mtDNA molecule. Secondary structure models of the unusually large Metridium mt-rRNA genes will be constructed. Finally, the origin of replication will be mapped, using electron microscopy. Data from the proposed work should advance the investigators overall understanding of the structure, functioning and evolution of animal mt-genomes.
