PHAGE T4 HEAD ASSEMBLY AND INITIATION OF INFECTION

噬菌体 T4 头部组装和感染起始

• 批准号: 6372951
• 负责人: LINDSAY W BLACK
• 金额: $ 33.69万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 1977
• 资助国家: 美国
• 起止时间: 1977-07-01 至 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6372951
• 关键词: DNA binding protein; DNA footprinting; DNA replication; Escherichia coli; atomic force microscopy; bacteriophage T4; capsid; fluorescence spectrometry; green fluorescent proteins; host organism interaction; laser spectrometry; protein biosynthesis; virus DNA; virus assembly; virus genetics; virus infection mechanism; virus protein

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.

描述：两个主要主题的解决。 第一个问题涉及表达 以及在T4中包装外源蛋白或蛋白融合物。 一个系统， 使用与T4 β基因的融合，使蛋白质以高水平展示， T4衣壳上的密度。 蛋白质可以修饰已经形成的衣壳， 基因可以被整合到T4克隆载体中用于表达， 与衣壳组装成活的噬菌体或多聚头。 蛋白可 组装成密集阵列，适合结构研究，或在较低的 副本编号。 对E.大肠杆菌可以合成， 显示在这个系统中。 用于噬菌体噬菌体酸化的第二系统 外源蛋白包括与IPIII的融合，IPIII是一种内部蛋白， 隐藏在大脑里 IPIII与绿色荧光蛋白的融合 (GFP)将用于测量头部内的蛋白质流动性， 不受IPIII的束缚，使用荧光光谱法。 GFP 融合体可以以未折叠的非荧光形式掺入头部， 允许注射后的再折叠和激活参数 研究了 GFP融合物将被掺入T4衍生物中， 特定的病原体，使他们的识别荧光 显微镜 最后，T4的三种内部蛋白质实际上是 一个大的基因家族，有许多不同基因的组合， 每个轨迹 这些基因都有一个短的靶向/加工序列， N-末端，允许所有同时包装。 不同 T4-样大肠杆菌的分离株在内部表现出广泛的多态性， 蛋白位点 研究人员以前曾描述过其中一种， T4的IPI对于克服在一种免疫缺陷病毒中发现的耐药机制至关重要。 临床衍生宿主 他将回到这个系统的研究，定义 被T4 IpI克服的抗性基因， 其它致病性大肠杆菌的变异基因。大肠杆菌， 负责存在大量的IPI样变异体， T4的亲戚 该提案的第二个主要主题涉及T4中的DNA包装， 特别注意终止酶，衣壳门户，及其可能的 互动 末端酶的小亚基（gp 16）控制包装 多联体DNA上堆积位点突触启动。 研究者 将通过足迹研究这个综合体，并确定预测的 位点决定成熟噬菌体中的DNA末端。 其他因素在其中的作用 将确定gp 16与PAC位点的组装，并进行超微结构分析。 将对蛋白质及其与DNA的复合物进行分析。 将通过荧光研究门静脉复合物和DNA的相互作用 使用门静脉蛋白-GFP融合物和用5-BrdU标记的DNA进行测量， 以及激光捕获和原子力显微镜。 最后，结合 研究者在形态发生和包装方面的经验将 用于完善T4及其巨型版本的使用， 用于克隆170- 1，000 kb大小范围的DNA的试剂，利用T4 连接酶表达细菌环化T4注射的线性DNA。