PHAGE T4 HEAD ASSEMBLY AND INITIATION OF INFECTION

描述：讨论了两个主要主题。第一个问题是表达 以及包装外源蛋白质或T4中的蛋白质融合。一个系统， 利用与T4 SoC基因的融合，使蛋白质能够在 T4衣壳致密。蛋白质可以装饰已经形成的衣壳或 基因可以被整合到T4克隆载体中进行表达和 与衣壳组装成有活力的噬菌体或多头体。蛋白质可以是 组装成密集的阵列，适合于结构研究，或在较低的 复印号。对大肠杆菌有毒性的蛋白质可以被合成并 在本系统中显示。另一种噬菌体包埋系统 外来蛋白质涉及与IPIII的融合，IPIII是一种内部蛋白质 被隔离在脑袋里。IPIII与绿色荧光蛋白的融合 (GFP)将被用来测量头部、拴系和 使用荧光光谱学，在头部内未与IPIII捆绑。绿色荧光蛋白 融合可以以展开的非荧光形式并入头部， 允许注射后的复性和激活参数 学习。GFP融合将被纳入T4衍生物中，以吸附 对特定的病原体，允许通过荧光识别它们 显微镜。最后，T4的三个内部蛋白实际上是成员 属于一个大的基因家族，存在着许多不同的基因组合 每一个轨迹。这些基因都有一个短的靶向/处理序列 N-末端，允许所有的同时打包。不同 类T4噬菌体的分离株在内部呈现广泛的多态 蛋白质基因座。调查人员之前曾描述过其中的一种， T4的IPI，对于克服在一种 临床衍生宿主。他将回到对这一制度的研究中，定义 T4IPI克服的抗性基因及其特征 其他致病性大肠杆菌中的变异基因可能是 对大量IPI样变异体在 T4的亲属。 该提案的第二个主要主题涉及T4中的DNA包装， 特别注意终止期、衣壳入口及其可能的情况。 互动。终止酶小亚基(Gp16)控制包装 由连体DNA上的PAC位点突触而起。调查员 将通过足迹来研究这个建筑群，并确定预测的 位点决定了DNA在成熟噬菌体中的末端。其他因素在其中的作用 将确定gp16与PAC位点的组装，并进行超微结构 将继续对蛋白质及其与DNA的复合体进行分析。 与门脉复合体和DNA的相互作用将用荧光研究 用门静脉蛋白-GFP融合和5-BrdU标记的DNA进行测量， 通过激光捕获和原子力显微镜。最后，合并后的 研究人员在形态形成和包装方面的经验将是 用于完善T4及其巨型版本的使用，以交付 用于170-1,000 kb大小范围的克隆DNA的试剂，使用T4 连接酶表达菌环化T4注射的线状DNA。

DESCRIPTION: Two major themes are addressed. The first concerns expression and packaging of foreign proteins or protein fusions in T4. One system, using fusions to the T4 soc gene, allow proteins to be displayed at high density on T4 capsids. Proteins can decorate already preformed capsids or genes can be incorporated into T4 cloning vectors for expression and assembly with the capsid into viable phage or polyheads. Proteins can be assembled into dense arrays, suitable for structural studies, or at lower copy number. Proteins that are toxic to E. coli can be synthesized and displayed in this system. A second system for phage encapsidation of foreign proteins involves fusions to IPIII, an internal protein that is sequestered inside the head. Fusions of IPIII to Green Fluorescent Protein (GFP) will be used to measure protein mobility inside the head, tethered and untethered to IPIII inside the head, using fluorescent spectroscopy. GFP fusions can be incorporated into heads in an unfolded non-fluorescent form, allowing parameters for refolding and activation after injection to be studied. GFP fusions will be incorporated into T4 derivatives that adsorb to specific pathogens, allowing their identification by fluorescent microscopy. Finally, the three internal proteins of T4 are actually members of a large gene family, with many combinations of different genes present at each locus. These genes all have a short targeting/processing sequence at the N-terminus, allowing all to be packaged simultaneously. Different isolates of T4-like phages exhibit extensive polymorphism at the internal protein loci. The investigator has previously characterized one of these, IPI of T4, as essential for overcoming a resistance mechanism found in a clinical derived host. He will return to the study of this system, defining the resistance gene that is overcome by T4 IpI and going on to characterize the variant genes in other pathogenic E. coli that are presumably responsible for the existence of the large number of IPI-like variants in relatives of T4. The second major theme of the proposal concerns DNA packaging in T4, with particular attention to terminase, the capsid portal, and their possible interaction. The small subunit of terminase (gp16) controls packaging initiation by synapsis of pac sites on concatemeric DNA. The investigator will study this complex by footprinting and determine whether the predicted site determines DNA ends in mature phage. The role of other factors in assembly of gp16 with pac sites will be determined, and ultrastructural analysis of the protein and its complex with DNA will be pursued. Interaction with the portal complex and DNA will be studied by fluorescence measurements using portal protein-GFP fusions and DNA labeled with 5-BrdU, and by laser trapping and atomic force microscopy. Finally, the combined experience of the investigator in morphogenesis and packaging will be utilized to perfect the use of T4, and giant versions thereof, into delivery agents for cloned DNA in the 170-1,000 kb size range, making use of T4 ligase expressing bacteria to circularize the linear DNA injected by T4.