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.
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