EXPRESSION OF AN INTERRUPTED PROKARYOTIC GENE

本研究的长期目标是利用裂解的TD基因 噬菌体T4作为理解RNA和DNA的分子范例 自剪接、移动内含子的交易。在前5年内 资助期(由NSF拨款DMB8502961支持)我们确定 TD插入序列是I组自剪接内含子。我们用了这个 检测T4中另外两个内含子的知识，并发现其中一个 这些基因以及Td内含子都是可移动的，有效地转移到 它们各自基因的无内含子等位基因。至于它们的真核群 I对应的，内含子的移动取决于编码的内切酶 由内含子本身决定。拟议的研究旨在进一步推动我们的 对基于RNA的(剪接)和 基于DNA的(移动)过程，使用TD基因作为模型系统。对双方都是 最终，我们将继续利用强大的积极和消极 由该噬菌体遗传系统和Td基因提供的选择 很特别。对于剪接的研究，我们将继续使用分子 遗传学分析原核酶的功能。我们会 进行平行研究以调查大肠杆菌可能的作用 Coli作为自剪接过程的附件发挥作用。关于…… DNA的迁移率，我们将检测TD内切酶的功能，它 调节这一过程，以及它与DNA靶基因内含子的相互作用 寻的地点。我们还将问一些更一般的问题，关于 内含子-噬菌体的关系，并以TD基因为模型来探索 原核系统中内含子丢失的可能性。最后，要理顺 在简化的噬菌体基因组中，我们希望解决内含子的存在 假设内含子通过增强 噬菌体重组原性。总之，拟议的实验代表了一种 继续利用简便易行的原核基因进行的工作 阐明第一类内含子的多方面反应的技术， 它们是原核生物和真核生物共有的动态遗传元件 王国。

The long-term objective of this research is to use the split td gene of phage T4 as the molecular paradigm for understanding the RNA and DNA transactions of a self-splicing, mobile intron. During the first 5-year funding period (supported by NSF grant DMB8502961) we established that the td intervening sequence is a group I self-splicing intron. We used this knowledge to detect two other introns in T4 and discovered that one of these as well as the td intron are mobile, transferring efficiently to intronless alleles of their respective genes. As for their eukaryotic group I counterparts, intron mobility is dependent upon an endonuclease encoded by the intron itself. The proposed research is intended to further our fundamental understanding of aspects of both the RNA-based (splicing) and DNA-based (mobility) processes, using the td gene as model system. To both ends we shall continue to exploit the powerful positive and negative selections provided by this phage genetic system and by the td gene in particular. For the splicing studies we shall continue to use molecular genetics to analyze the function of a prokaryotic ribozyme. We shall conduct parallel studies to investigate the possible role of Escherichia coli functions as accessories to the self-splicing process. With respect to DNA mobility, we shall examine the function of the td endonuclease, which mediates the process, and its interaction with its DNA target, the intron homing site. We shall also ask some more general questions about the intron-phage relationship and use the td gene as a model for exploring the possibility of intron loss in a prokaryotic system. Finally, to rationalize the existence of introns , in streamlined phage genomes, we wish to address the hypothesis that introns provide a selective advantage by enhancing phage recombinogenicity. Together, the proposed experiments represent a continuation of ongoing work that exploits facile prokaryotic genetic techniques to shed light on the multifaceted reactions of group I introns, which are dynamic genetic elements in common to the pro- and eukaryotic kingdoms.