CONTROL OF VIRUS INDUCED LYSIS

这项研究工作的长期目标是阐明 噬菌体裂解宿主的分子机制。 噬菌体裂解是一种涉及相互作用的分子的基本现象 宿主细胞膜和细菌细胞壁结构。 此外，宿主裂解的过程必须由宿主仔细安排。 噬菌体以优化病毒粒子的产生。因此， 从噬菌体裂解的研究中了解到的可能对 了解细胞膜的结构和动力学，以及 一般的病毒增殖，以及提供深入了解 结构，功能和细菌包膜的代谢。噬菌体 系统提供了强大的遗传工具，我们建议在研究中使用 在λ和相关噬菌体以及噬菌体phiX174的裂解系统中， 代表宿主裂解的两种基本策略。我们会进行 对编码原型“holin”的λ S基因进行遗传分析， 其功能是允许可溶性内溶素，或溶菌酶， 穿过细胞膜我们的方法使用积极和消极的 使用质粒和噬菌体载体选择holin功能。的突变 将进行来自噬菌体21的功能同源物的研究以确定 21个holin的哪些特征与lambda holin相同。基因内 抑制和互补将用于建立点对点 Holin序列内的相互作用图。生物钟突变体 将分离并表征裂解的时间。体外孔 将使用平面脂质膜或小的 单层囊泡。phiX174 E裂解蛋白之间的相互作用 和宿主蛋白SlyD，其是人FKBP的同源物， 免疫亲和素，将使用遗传和分子手段进行研究。

The long term objective of this research effort is to elucidate the molecular mechanisms by which bacteriophage accomplish lysis of the host. Phage lysis is a fundamental phenomenon involving molecules which interact with the host cell membrane and with bacterial cell wall structures. Moreover, the process of host lysis must be carefully scheduled by the bacteriophage in order to optimize the production of virions. Thus lessons learned from the study of phage lysis are likely to be important to the understanding of cell membrane structure and dynamics and the regulation of viral proliferation in general, as well as providing insights into the structure, function, and metabolism of the bacterial envelope. Phage systems provide powerful genetic tools, which we propose to use in a study of the lysis systems of lambda and related phage and of the phage phiX174, representing the two fundamental strategies of host lysis. We will conduct a genetic analysis of the lambda S gene, encoding the prototype "holin", which functions to allow passage of the soluble endolysin, or lysozyme, across the cell membrane. Our approach uses positive and negative selections for holin function using plasmid and phage vectors. A mutational study of a functional homolog from phage 21 will be conducted to determine what features of the 21 holin are common with the lambda holin. Intragenic suppression and complementation will be used to establish a point-to-point interaction-map within the holin sequence. Clock mutants defective in the timing of lysis will be isolated and characterized. An in vitro hole forming assay will be developed using planar lipid membranes or small unilamellar vesicles. The interaction between the phiX174 E lysis protein and the host protein SlyD, which is a homolog of the human FKBP immunophilin, will be investigated using genetic and molecular means.