Site-Specific DNA Deletion in Tetrahymena thermophila

Cox98038381基因组重排伴随着许多生物的发育过程，但没有任何地方的基因组重组比纤毛虫大核发育期间的基因组重组更广泛。在这个过程中，旧的大核被破坏，新的大核从微核发育而来，伴随着微核染色体的碎裂和片段的放大。很大一部分微核基因组也以数千个位点特定缺失(内部消除序列或IES元件)的形式被丢弃。嗜热四膜虫两个发育程序化DNA缺失反应的体外系统的开发将继续下去。对缺失途径的机制解释将被研究。重点放在两个IES元素上。来自几个实验室的证据表明，这种切除是一种类似转位的机制。IES元件一端的双链切割后，通过一步转酯化机制进行链转移，在一条DNA链上产生缺失连接。需要额外的步骤来处理留在另一条DNA链上的DNA分支。建立了该反应链转移步骤的体外体系。负责观察到的反应的酶的纯化将继续进行，同时寻找互补的酶活性并探索体外反应机制。一个完全重组的体外系统不仅可能为缺失反应的生化分析开辟道路，而且可能为纤毛原生动物异常的DNA代谢的许多其他过程开辟道路。这项拟议研究的最终目标是对缺失途径进行完整的分子描述，这源于对由纯化的酶和底物重组的体外系统的研究。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元件的进化起源以及它们在大核发育过程中的功能。它还将有助于更全面地了解重组机制，这是相关和重要的，因为重组过程是正在或将实现基因治疗的分子手段。

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.