GENOME REORGANIZATION IN HYPOTRICHOUS CILIATED PROTOZOA

尽管许多基因和基因组重排过程已经被发现， 在真核生物中定义，介导这些事件的机制 我们对此知之甚少。 我们计划研究 重组过程发生在下毛纤毛原生动物，如 Oxytricha nova和Stylonychia mytilus，以扩展我们的知识 真核生物的重排过程以及定义DNA的类型 介导这些事件的序列和酶。 在这些生物体中， 染色体微核基因组的一部分转化为大核 含有基因大小的DNA小分子。 这种基因组重排 过程发生在生物体生命周期的一个确定的时间， DNA序列消除、序列添加和测序步骤 重排 我们计划通过构建 大核DNA分子的重组DNA克隆和 它们所来源的微核染色体。 这些材料将 为了定义保留的DNA序列， 在发展过程中被淘汰。 我们还将克隆并描述 重组过程中产生的中间DNA形式， 确定生产过程中所涉及的事件或步骤的顺序 基因大小的大核DNA分子从微核染色体。 最后，我们将开发两个系统，使我们能够研究 特定的DNA序列和酶介导基因组重组。 在 第一，我们将直接显微注射克隆的微核DNA序列， 进入发育中的大核，并跟随它们的命运， 定义重排中涉及的序列。 我们的第二种方法是 开发参与重排的酶的体外测定， 净化和表征其活动。

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.