GENOME REORGANIZATION IN HYPOTRICHOUS CILIATED PROTOZOA

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Developmental, or programmed, DNA rearrangement events have been observed in a variety of organisms. In some instances, DNA rearrangement is used as a means of constructing protein coding regions, such as in the development of the vertebrae immune system, and can thus be viewed as a means of developmentally regulating gene expression. a number of pathogenic microorganisms also employ DNA rearrangement to alter their expression pattern of surface proteins as a means of evading the immune system. The molecular mechanisms involved in DNA rearrangement are poorly understood in eukaryotic organisms. To learn more about DNA rearrangement events, we propose to study the extensive genome reorganization process that occurs in the hypotrichous ciliated protozoan Euplotes crassus. During its life cycle, this organism transforms a copy of its chromosomal micronucleus into a macronucleus that contains only short, linear, gene-sized DNA molecules. The process of macronuclear development involves steps of chromosome fragmentation, DNA elimination, and DNA amplification. In addition, there are numerous DNA breakage and joining, or splicing, events that are associated with DNA elimination. Initial studies will examine the nature of the DNA breakage and joining events in greater detail so as to better understand their molecular mechanisms. This will include determining if the excision process is precise, characterizing the eliminated DNA, and determining the nature of the DNA breakage event involved in the excision process. Other studies are aimed at detecting proteins that interact with excised DNA sequences and determining the particular DNA sequences with which these proteins interact. This will be accomplished using a combination of gel mobility shift assays, in vivo DNA footprinting, and in vitro DNA footprinting. As an additional approach to identifying proteins involved in DNA rearrangement, a panel of monoclonal antibodies directed against nuclear proteins that are expressed during periods of DNA excision will be generated. These studies will provide new information on the cis-acting DNA sequences and trans-acting factors that direct the specific DNA breakage and joining events. In addition, these studies will lay the groundwork for future studies aimed at the isolation and characterization of the proteins that mediate the rearrangement process.