This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Although double-stranded DNA-containing bacteriophages and eukaryotic viruses all have the same core genes for morphogenesis (reviewed in Bamford et al., 2005), the length of bacteriophage genomes varies from 20 Kb to 670 Kb; 1,200 Kb eukaryotic virus genomes have been found (reviewed in Serwer, 2007a). The long-genome bacteriophages are of interest for understanding how and why complexity evolves in biological systems. Statistically, the data indicate a systematic under-sampling of long-genome bacteriophages during isolation from the environment. We recently have found procedures to more comprehensively isolate long-genome bacteriophages (Serwer et al., 2007a). A result was the isolation of bacteriophage 0305?8-36, a virulent double-stranded DNA bacteriophage that, however, clears bacterial cultures only when grown in dilute agarose gels. This bacteriophage has atypical tail fibers that appear to be corkscrew-shaped when a negatively stained specimen is observed by electron microscopy (Serwer et al., 2007a). In situ (in-plaque) fluorescence microscopy analysis of Bacteriophage 0305?8-36 revealed that bacteriophage 0305?8-36 also (1) partitions to one of two liquid phases that form in part because of the presence of the bacteriophage particles and (2) forms aggregates that range in size from 3-20 particles to hundreds of bacteriophage particles (Serwer et al., 2007b). Thus, the data indicate that 0305?8-36 is programmed for complex interactions within the extracellular environment.Our sequencing of the 218.948 Kb 0305?8-36 genome (6.479 Kb terminal repeat) revealed that, while 0305?8-36 had the classical core genes for virus morphogenesis, 0305?8-36 also had many others; 55 total proteins were found in its capsid by SDSPAGE/mass spectrometry. All genes were highly diverged from known genes. The 55 capsid proteins are twice the number for bacteriophage T4, the most complex bacteriophage previously studied Thomas et al., 2007).The long-range objective of the proposal is to determine the reason(s) that double-stranded DNA bacteriophages in the 0305?8-36 class have numerous bacteriophage particle-associated proteins that are not classical bacteriophage/virus proteins.The working hypothesis is that these proteins were selected to optimize complex interactions of bacteriophage particles after release into the environment. Testing this hypothesis will provide a leading edge prototype of the evolution of biological complexity. Understanding both environmental interactions and the genomes of these bacteriophages are also important for (1) constructing models for the evolution of the bacterial hosts, (2) optimizing bacteriophage therapy of infectious disease, (3) analyzing vertical gene descent with minimal interference from horizontal gene transfer, (4) developing improved, large capacity cloning and display vectors, including those useful for both DNA and protein vaccines and (5) setting standards for conducting controlled evolution. We propose to use cryo-electron microscopy to provide a basis in detailed structure for determining the function of the Sextra ? proteins. To understand the function of the Sextra ? capsid proteins, we will perform either mutagenesis or controlled evolution and then compare genotype to phenotype for the modified bacteriophages. We propose to make cryo-electron microscopy-derived structure a major aspect of the phenotype. We will also compare the structure of 0305?8-36 to the structure of other known corkscrew fibered bacteriophages.
| Bucero, Marta Abril; Bajaj, Chandrajit; Mourrain, Bernard (2016) On the construction of general cubature formula by flat extensions. Linear Algebra Appl 502:104-125 |
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| Baker, Mariah R; Fan, Guizhen; Serysheva, Irina I (2015) Single-particle cryo-EM of the ryanodine receptor channel in an aqueous environment. Eur J Transl Myol 25:35-48 |
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