GENETICS OF MYXOPHAGES

我们将采用分子遗传学的方法来研究与 温带噬菌体Mx8复合体的溶原性发育 原核生物，黄色粘球菌。使用经典噬菌体的组合 遗传学和重组D&A分析，我们将鉴定和表征 参与定点重组和重叠感染的Mx8基因 豁免权。与噬菌体lambda和P22一样，Mx8可以将其基因组整合为 在黄花莲基因组上的首选附着位置(AttB)的前驱噬菌体。 与lambda和P22不同，Mx8编码多种蛋白质， 整合。Mx8重叠感染免疫的机制是 同样新颖和复杂，涉及至少三个基因的层次结构， 其中一个可能编码具有新的特异性的DNA甲基酶。我们会 对Mx8的一个8.2kbp的克隆区进行测序，该区编码这些功能 对于整合和免疫都是必需的。使基因与…相关联 函数，我们将在影响序列突变的决定之间 裂解和溶源发育。我们还将使站点定向 使潜在的开放阅读框架和位点失活的突变 参与整合和免疫，并确定表型 质粒和噬菌体上的这些突变。温度.敏感突变 在Mx8的初级阻遏基因中将分离出一段 带有ts抑制或基因和强启动子的人将被用来构建 一种可诱导的、高水平的黄花草表达系统。 圆满完成这项工作将产生两个结果。 首先，我们将开始了解粘噬菌体的生物学，关于 我们对此知之甚少。对多种噬菌体的研究揭示了 调节基因表达的新机制；我们已经证明Mx8 提供了新的惊喜。其次，对Mx8生物学的基本理解将 允许我们开发更强大的遗传学来促进地图绘制， 黄花莲的克隆和测序研究。黄花假单胞菌是一个 天然抗生素的来源，以及研究的模型系统 细胞与细胞之间的相互作用对滑动运动和 多个复杂的发育周期。

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.