GENETICS OF MYXOPHAGES

噬菌体遗传学

• 批准号: 2332010
• 负责人: PHILIP A YOUDERIAN
• 金额: $ 16.64万
• 依托单位: UNIVERSITY OF IDAHO
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-02-01 至 1999-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2332010
• 关键词: DNA methylation; Myxococcus; bacterial virus; gene mutation; genetic recombination; lysogeny; microorganism culture; nucleic acid sequence; oligonucleotides; plasmids; transposon /insertion element; virus DNA; virus infection mechanism; virus integration

We will take a molecular genetic approach to study the genes involved in the lysogenic development of temperate phage Mx8 of the complex prokaryote, Myxococcus xanthus. Using a combination of classical phage genetics and recombinant D&A analysis, we will identify and characterize the Mx8 genes involved in site-specific recombination and superinfection immunity. Like phages lambda and P22, Mx8 can integrate its genome as prophage at a preferred attachment site (attB) on the M. xanthus genome. Unlike lambda and P22, Mx8 encodes multiple proteins required for integration. The mechanism underlying Mx8 superinfection immunity is also novel and complex, and involves a hierarchy of at least three genes, one of which may encode a DNA methylase with novel specificity. We will sequence an 8.2 kbp cloned region of Mx8 which encodes the functions required for both integration and immunity. To correlate genes with functions, we will sequence mutations that affect the decision between lytic and lysogenic development. We will also make site-directed mutations that inactivate potential open reading frames and sites involved in integration and immunity, and determine the phenotypes of these mutations on plasmids and phages. Temperature.sensitive mutations in the primary repressor gene of Mx8 will be isolated, and a fragment with a ts repress or gene and strong promoter will be used to construct an inducible, high-level expression system for M. xanthus. Two results will emerge from the successful completion of this work. First, we will begin to understand the biology of the myxophages, about which little is known. The study of a variety of phages has revealed novel mechanisms that regulate gene expression; we have shown that Mx8 offers new surprises. Second, a basic understanding of Mx8 biology will allow us to develop a more powerful genetics to facilitate mapping, cloning, and sequencing studies in M. xanthus. M. xanthus is a major source of natural antibiotics, as well as a model system for the study of cell-cell interactions critical for both gliding motility and multiple, complex developmental cycles.

我们将采用一种分子遗传学方法来研究涉及的基因 该复合物的温带噬菌体Mx8的溶裂发展 原核生物，粘粒球菌。 结合古典噬菌体 遗传学和重组D＆A分析，我们将确定和表征 参与位点特异性重组和超级感染的MX8基因 免疫。 像噬菌体Lambda和P22一样，MX8可以将其基因组整合为 Xanthus基因组上的首选附件（ATTB）的预言。 与Lambda和P22不同，MX8编码需要多种蛋白质 一体化。 MX8超级感染免疫的基础机制是 也是新颖而复杂的，涉及至少三个基因的层次结构， 其中之一可以用新颖的特异性编码DNA甲基酶。 我们将 序列一个8.2 kbp克隆的Mx8，该区域编码功能 整合和免疫力所必需的。 将基因与 功能，我们将测序影响影响决策之间决策的突变 裂解和溶菌发生的发展。 我们还将定向站点 使潜在开放式阅读框和站点失活的突变 参与整合和免疫力，并确定 这些突变在质粒和噬菌体上。 温度敏感突变 将在MX8的主要阻遏物基因中分离出来，并片段 使用TS抑制作用或基因和强启动子将用于构建 叶thus的诱导型高级表达系统。 这项工作的成功完成将产生两个结果。 首先，我们将开始理解有关粘噬细胞的生物学 什么鲜为人知。 各种噬菌体的研究表明 调节基因表达的新型机制；我们已经证明了MX8 提供新的惊喜。第二，对MX8生物学的基本了解将 允许我们开发出更强大的遗传学来促进映射， 克隆和Xanthus的测序研究。 M. Xanthus是主要的 天然抗生素的来源以及研究模型系统 细胞 - 细胞相互作用对于滑行运动至关重要 多个复杂的发育周期。