INTRON MOBILITY IN THE PHAGE T4/E COLI SYSTEM

噬菌体T4的I组内含子在RNA水平上进行自我剪接。 在过去的资助期内，我们发现TD和SUNY 噬菌体T4的内含子能够进行基于DNA的“移动”反应 也是。根据它们的双重属性，内含子是 二部元素。它们包含的序列折叠成一个 特有的RNA结构，核酶核心，从中出来的是 循环开放阅读框(ORF)，其产物需要用于 机动性。这一流动过程，也被称为“归位”，涉及 将内含子转移到无内含子的等位基因 非互惠基因转化事件。我们已经证明了 内含子ORF编码一种位点特异的DNA内切酶，该酶可切割 启动基因转换的受体等位基因。重组 被假设通过双链断裂修复(DSBR)发生 路径。可移动的噬菌体T4内含子提供了 在定义明确和可操作的情况下研究基因转化过程 遗传背景，DNA中具有良好特征的突变 重组和修复功能。 该应用程序针对使用噬菌体/细菌模型 系统来剖析内含子移动过程。这将涉及到 对重组途径进行了详细的定义。我们还将 开发基因转换系统以促进基因操作 在大肠杆菌中，并测试酵母重组的作用 DSBR中的函数。此外，我们希望探讨是否有可能 通过DSBR途径将内含子转座到新的位置 或者通过RNA介导的途径。最后，我们将进一步 描述我们最近发现的自剪接内含子 在紫色细菌(大肠杆菌的近亲)中，并研究 真细菌王国中相似内含子的分布。因此， 我们希望加强我们对内含子的基本理解 流动过程，将该过程用于实际目的， 并提出了关于内含子迁移率和 内含子分布。

The group I introns of phage T4 self-splice at the RNA level. During the past funding period we discovered that the td and sunY introns of phage T4 are capable of DNA-based "mobility" reactions as well. In accordance with their dual properties, the introns are bipartite elements. They comprise sequences that fold into a characteristic RNA structure, the ribozyme core, out of which is looped an open reading frame (ORF), whose product is required for mobility. This mobility process, also termed "homing", involves transfer of the intron into an intronless allele via a non-reciprocal gene conversion event. We have shown that the intron ORF encodes a site-specific DNA endonuclease which cleaves the recipient allele to initiate gene conversion. Recombination is hypothesized to occur via the double-strand-break repair (DSBR) pathway. The mobile phage T4 introns provide the opportunity to study the gene conversion process in a well-defined and manipulable genetic background, with well-characterized mutants in DNA recombination and repair functions. This application is directed toward using the phage/bacterial model system to dissect the intron mobility process. This will involve defining the recombination pathway in detail. We shall also exploit the gene conversion system to facilitate gene manipulation in Escherichia coli, and to test the role of yeast recombination functions in DSBR. Furthermore, we wish to explore the possibility of intron transposition to new sites via either the DSBR pathway or by an RNA-mediated pathway. Finally, we shall further characterize the self-splicing introns which we recently identified in purple bacteria (close relatives of E. coli) and study the distribution of similar introns in the eubacterial kingdom. Thus we hope to enhance our fundamental understanding of the intron mobility process, to utilize the process for practical purposes, and to address evolutionary hypotheses for intron mobility and intron distribution.