Although a number of gene and genome rearrangement processes have been defined in eukaryotic organisms, the mechanisms that mediate these events are poorly understood. We plan to study the extensive genome reorganization process that occurs in hypotrichous ciliated protozoa, such as Oxytricha nova and Stylonychia mytilus, in order to extend our knowledge of eukaryotic rearrangement processes as well as to define the types of DNA sequences and enzymes that mediate such events. In these organisms a copy of the chromosomal micronuclear genome is transformed into a macronucleus containing small gene-size DNA molecules. This genome rearrangement process occurs at a defined time in the organism's life cycle and involves steps of DNA sequence elimination, sequence addition, and sequence rearrangement. We plan to study this genome rearrangement process by constructing recombinant DNA clones of macronuclear DNA molecules and the regions of the micronuclear chromosome from which they are derived. These materials will be characterized in order to define DNA sequences that are retained and eliminated during development. We will also clone and characterize intermediate DNA forms generated during the reorganization process in order to determine the sequence of events or steps involved in producing gene-size macronuclear DNA molecules from micronuclear chromosomes. Finally, we will develop two systems that will allow us to study the particular DNA sequences and enzymes mediating genome reorganization. In the first, we will directly microinject cloned micronuclear DNA sequences into developing macronuclei and follow their fate in order to precisely define sequences involved in rearrangement. Our second approach will be to develop in vitro assays for enzymes involved in rearrangement to ultimately purify and characterize their activities.
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