INTRON MOBILITY IN THE PHAGE T4/E COLI SYSTEM

噬菌体T4的I组内含子在RNA水平上自我剪接。 在过去的资助期内，我们发现运输署和阳光 噬菌体T4的内含子能够进行基于DNA的"迁移"反应 也 根据它们的双重性质，内含子是 二分元素 它们包含折叠成 特征性的RNA结构，核酶核心，其中是 循环一个开放的阅读框架（ORF），其产品是所需的 迁移率 这种流动过程，也称为"归巢"，涉及 将内含子通过一个插入位点转移到无内含子等位基因中。 非互惠基因转换事件。 我们已经证明， 内含子ORF编码位点特异性DNA内切酶，其切割 受体等位基因启动基因转换。 重组 假设通过双链断裂修复（DSBR）发生 通路 移动的噬菌体T4内含子提供了机会， 研究基因转换过程在一个明确的和可操作的 遗传背景，DNA中具有良好特征的突变体 重组和修复功能。 本申请涉及使用噬菌体/细菌模型 系统来剖析内含子的移动过程。 这将涉及 详细定义了重组途径。 我们亦会 利用基因转换系统来促进基因操作 在大肠杆菌中，并测试酵母重组的作用 DSBR中的功能。 此外，我们希望探讨 内含子通过DSBR途径转座到新位点 或通过RNA介导的途径。 最后，我们将进一步 描述我们最近发现的自我剪接内含子 在紫色细菌（E.大肠杆菌），并研究 真细菌界中相似内含子的分布。 因此 我们希望能加深我们对内含子的基本理解， 流动性过程，利用该过程的实际目的， 并解决内含子移动性的进化假设， 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.