Site-Specific DNA Deletion in Tetrahymena thermophila

基因组重排伴随着许多生物的发育过程，但在纤毛虫原生动物的大核发育过程中，基因组重组的范围最为广泛。在这个过程中，旧的宏核被破坏，新的微核从微核发育而来，伴随着微核染色体的断裂和片段的扩增。微核基因组的很大一部分也以数千个特定位点缺失的形式被抛弃（内部消除序列，或IES元件）。在嗜热四膜虫中两个发育程序DNA缺失反应的体外系统的开发将继续进行。一个机制解释的删除途径将被调查。重点是IES的两个要素。来自几个实验室的证据表明，这种切除的机制类似于移位。在IES元件一端的双链切割之后，通过一步反式酯化机制进行链转移，从而在一条DNA链上产生缺失连接。需要额外的步骤来处理另一条DNA链上留下的DNA分支。该反应的链转移步骤的体外系统已经实现。在寻找互补酶活性和探索体外反应机制的同时，将继续对所观察到的反应负责的酶进行纯化。一个完全重建的体外系统可能为生化分析开辟道路，不仅是对缺失反应，而且对纤毛原生动物不寻常的DNA代谢过程的许多其他过程。提出的研究的最终目标是一个完整的缺失途径的分子描述，从纯化酶和底物的体外系统重构的研究中得到。虽然所描述的工作集中在机制上，但它也应该有助于更好地理解IES元素的进化起源以及它们在宏观核发展中的功能。它还将有助于更全面地了解重组机制，这是相关的和重要的，因为重组过程是基因治疗正在或将实现的分子手段。

Cox98038381Genome rearrangements accompany the developmental process in many organisms, but nowhere is genome reorganization more extensive than during macronuclear development in ciliated protozoans. During that process, the old macronuclei are destroyed and new ones are developed from the micronuclei, accompanied by the fragmentation of the micronuclear chromosomes and the amplification of the fragments. A substantial fraction of the micronuclear genome is also jettisoned in the form of thousands of site-specific deletions (internal eliminated sequences, or IES elements).The development of an in vitro system for two of the developmentally programmed DNA deletion reactions in Tetrahymena thermophila will be continued. A mechanistic explanation for the deletion pathway will be investigated. The focus is on two of the IES elements. Evidence from several laboratories have suggested a transposition-like mechanism for this excision. A double-strand cleavage at one end of the IES element would be followed by strand transfer via a one-step trans-esterification mechanism to generate the deletion junction on one DNA strand. Additional steps are required to process the DNA branch left in the other DNA strand. An in vitro system for the strand transfer step of this reaction has been achieved. The purification of the enzymes responsible for the observed reaction will be continued while searching for complementary enzymatic activities and exploring in vitro reaction mechanisms. A completely reconstituted in vitro system would likely open the way for biochemical analysis not only of the deletion reactions, but also of many other processes in the unusual DNA metabolism of ciliated protozoans. The ultimate goal of the proposed research is a complete molecular description of the deletion pathway, derived from studies of an in vitro system reconstituted form purified enzymes and substrates. Although the described work is focused on mechanism, it should also contribute to a better understanding of the evolutionary origin of the IES elements as well as their function during macronuclear development. It will also contribute to a more complete appreciation of recombination mechanisms in general which is relevant and important because recombination processes are the molecular means by which gene therapy is being or will be achieved.