GENOME REORGANIZATION IN HYPOTRICHOUS CILIATED PROTOZOA

已经观察到程序化的基因组重排事件 在各种原核和真核生物中。 在许多 例如，DNA重排用于控制基因表达， 就像脊椎动物免疫系统的发展一样。 在 此外，一些病原微生物利用DNA 重组作为调节其表达的手段， 表面蛋白，以逃避免疫反应。 广泛 发育基因组重排事件，涉及 消除整个染色体或部分染色体， 也被观察到，但其功能尚不清楚。 拟议的研究将检查基因组重组 下毛类纤毛虫的大核发育事件 原生动物Oxytricha nova和Euplotes crassus。 一生中 在一个循环中，这些生物体将它们的染色体的一个拷贝 微核变成一个大核含有线性，基因大小 DNA分子。 产生大核的过程 涉及多种类型的重排事件，包括 染色体断裂，DNA消除，核酸 剪接和DNA序列添加。 的广泛性 重新排列过程，以及在 实验室，使其成为研究DNA的有利模型系统 真核生物中的重排。 最初的研究将利用特征重组克隆 大核DNA分子和它们的微核 前兆分析的时间和顺序的各种 大核发育过程中的重排事件。 这些 研究将在E. crassus，可以用来 产生大量的细胞， 大核发育，从而提供了一个来源， DNA 进一步的研究将确定染色体位点， 赋予重排过程特异性。 的方法 将克隆的DNA分子显微注射到 经历大核发育的细胞，然后是 再分离和分析DNA进行检测处理。 核 提取物也将从正在发育的细胞中制备 并用于开发体外系统， 催化重排事件或蛋白质， 染色体重排位点 这些研究将提供新的 有关DNA序列和DNA中涉及的蛋白质的信息 重排事件，并扩展我们对分子 基因组重组的机制。

Programmed genomic rearrangement events have been observed in a variety of prokaryotic and eukaryotic organisms. In many instances, DNA rearrangement is used to control gene expression, as in the development of the vertebrate immune system. In addition, some pathogenic microorganisms utilize DNA rearrangement as a means of regulating the expression of their surface proteins so as to evade the immune response. Extensive developmental genomic rearrangement events, involving the elimination of whole chromosomes or parts of chromosomes, have also been observed, but their function is unknown. The proposed studies will examine the genome reorganization events of macronuclear development in the hypotrichous ciliated protozoa Oxytricha nova and Euplotes crassus. During their life cycle, these organisms transform a copy of their chromosomal micronucleus into a macronucleus containing linear, gene-sized DNA molecules. The process of generating a macronucleus involves a number of types of rearrangement events including chromosome fragmentation, DNA elimination, nucleic acid splicing, and DNA sequence addition. The extensive nature of the rearrangement process, and the ability to control it in the laboratory, make this a favorable model system for studying DNA rearrangement in eukaryotes. Initial studies will make use of characterized recombinant clones of macronuclear DNA molecules and their micronuclear precursors to analyze the timing and order of the various rearrangement events during macronuclear development. These studies will be performed on E. crassus, which can be used to generate large numbers of cells synchronously proceeding through macronuclear development, thus providing a source of staged DNAs. Additional studies will define chromosomal sites which impart specificity to the rearrangement process. The method to be used involves microinjection of cloned DNA molecules into cells undergoing macronuclear development,followed by reisolation and analysis of the DNA to detect processing. Nuclear extracts will also be prepared from cells undergoing development and used to develop in vitro systems for either detecting enzymes catalyzing rearrangement events or proteins which interact with chromosomal rearrangement sites. These studies will provide new information on DNA sequences and proteins involved in DNA rearrangement events and extend our knowledge of the molecular mechanisms of genome reorganization.