GENOME REORGANIZATION IN HYPOTRICHOUS CILIATED PROTOZOA

已经观察到发育性或程序性DNA重排事件 在各种各样的生物体中。 在一些情况下，DNA重排被用作 一种构建蛋白质编码区的方法，例如在开发 脊椎免疫系统，因此可以被视为一种手段， 发育调节基因表达。 多种病原 微生物也利用DNA重排来改变它们的表达 表面蛋白的模式作为逃避免疫系统的手段。 的 涉及DNA重排的分子机制知之甚少 真核生物 为了更多地了解DNA重排事件，我们建议研究 广泛的基因组重组过程发生在下毛类 纤毛虫原生动物粗游仆虫。 在其生命周期中，这种生物 将其染色体微核的拷贝转化为大核， 只含有短的线性的基因大小的DNA分子。 的过程 大核发育包括染色体断裂、DNA 消除和DNA扩增。 此外，还有大量的DNA 断裂和连接，或剪接，与DNA相关的事件 淘汰 最初的研究将检查DNA断裂和连接的性质 更详细地了解事件，以便更好地了解其分子 机制等 这将包括确定切除过程是否 精确，表征消除的DNA，并确定 切除过程中涉及的DNA断裂事件。 其他研究 旨在检测与切除的DNA序列相互作用的蛋白质， 确定特定的DNA序列， 互动. 这将通过结合凝胶迁移率 偏移测定法、体内DNA足迹法和体外DNA足迹法。 作为 另一种鉴定DNA中蛋白质的方法 重排，一组单克隆抗体针对核 在DNA切除期间表达的蛋白质将被 生成的. 这些研究将提供新的信息，顺式作用 DNA序列和指导特定DNA的反式作用因子 断裂和连接事件。 此外，这些研究将奠定 为今后的研究奠定基础， 调节重排过程的蛋白质。

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.