GENOME REORGANIZATION IN HYPOTRICHOUS CILIATED PROTOZOA

观察到了发育性或程序性的DNA重排事件 在各种生物体中。在某些情况下，DNA重排被用作 一种构建蛋白质编码区的方法，如在发育过程中 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。一些致病因素 微生物也利用DNA重排来改变它们的表达 作为逃避免疫系统的一种手段的表面蛋白质模式。这个 目前对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 纤毛虫原生动物Eplotes Crassus。在它的生命周期中，这种有机体 将其染色体微核的副本转化为大核 只包含基因大小的短的线性DNA分子。这一过程 大核发育包括染色体碎裂、DNA 消除和DNA扩增。此外，还有大量的DNA 与DNA相关的断裂和连接或剪接事件 淘汰赛。 初步研究将检查DNA断裂和连接的性质 以便更好地了解它们的分子 机械装置。这将包括确定切除过程是否 精确，鉴定消除的DNA，并确定其性质 参与切除过程的DNA断裂事件。其他研究包括 旨在检测与切除的DNA序列相互作用的蛋白质和 确定与这些蛋白质一起的特定DNA序列 互动。这将使用凝胶流动性的组合来实现 移位分析、体内DNA足迹和体外DNA足迹。AS 识别DNA中涉及的蛋白质的另一种方法 重排，一组针对核的单抗 在DNA切除期间表达的蛋白质将是 已生成。这些研究将提供关于顺位代理的新信息。 指导特定DNA的DNA序列和反式作用因子 破坏和加入活动。此外，这些研究将为 为未来旨在分离和表征的研究奠定基础 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.