Genome Reorganization in Hypotrichous Ciliated Protozoa

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Developmental, or programmed, DNA rearrangement occurs in a variety of organisms. The molecular mechanisms involved are, in some cases, well understood but little information exists for the large-scale rearrangement processes that occur in numerous eukaryotes. The hypotrichous ciliated protozoan, Euplotes crassus, undergoes extensive genome reorganization during sexual reproduction. A copy of the germinal micronucleus eliminates about 100,000 interstitial segments of DNA or internal eliminated sequences (IES) with concomitant rejoining of flanking sequences to form the transcriptionally active macronucleus. The eliminated IESs consists of two large, related families of transposons, as well as short unique segments of DNA. Chromosome fragmentation generates the gene size macronuclear DNA molecules. Simple repeats are added to the ends of the gene size pieces as telomeres. Models have been developed to explain the mechanisms involved in this extensive rearrangement and the models will be tested. Further characterization of the molecular mechanisms of the two major rearrangement processes will be done. Highly sensitive ligation-mediated PC procedures will be used to search for in vivo rearrangement intermediates that have undergone chromosome fragmentation, but not telomere addition. A similar series of PCR analysis will be performed to identify intermediates in the IES excision process. In vitro chromosome fragmentation activity in extracts derived from appropriately staged developing macronuclei will be sought. Finally, additional in vitro studies with partially purified telomerase preparations will be performed to test the current model of de novo telomere synthesis.E. crassus is one of the few cases where there is strong evidence for the involvement of transposable elements in developmental DNA rearrangement. Further insight into this process will allow this mechanism to be validated as well as providing information relevant to the co-evolution of transposons and their host genomes. Ultimately these studies may also lead to tools that are of practical use in generating artificial chromosomes both in vivo and in vitro, as well as suggesting strategies for mitigating the harmful effects of chromosome breaks.