Site-Specific DNA Deletion in Tetrahymena thermophila

Cox98038381基因组重排伴随着许多生物体的发育过程，但在纤毛原生动物的大核发育过程中，基因组重组最为广泛。在这一过程中，旧的大核被破坏，新的从微核发展，伴随着微核染色体的断裂和片段的扩增。一个相当大的部分的微核基因组也抛弃了数千个位点特异性缺失的形式（内部消除序列，或IES元素）。在四膜虫嗜热细胞中的两个发育编程的DNA缺失反应的体外系统的发展将继续。将研究缺失途径的机制解释。重点是IES的两个要素。来自几个实验室的证据表明，这种切除是一种类似转位的机制。IES元件一端的双链切割之后将通过一步酯交换机制进行链转移，以在一条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链中的DNA分支。已经实现了用于该反应的链转移步骤的体外系统。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和探索体外反应机制。一个完全重建的体外系统将可能打开的方式，不仅删除反应的生化分析，而且在不寻常的DNA代谢的纤毛原生动物的许多其他过程。拟议的研究的最终目标是一个完整的分子描述的删除途径，来自在体外系统重建形式纯化的酶和底物的研究。虽然所描述的工作是集中在机制，它也应该有助于更好地理解的IES元素的进化起源，以及它们的功能在macronuclear发展。它还将有助于更完整地了解一般的重组机制，这是相关的和重要的，因为重组过程是正在或将要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.