UBC Theses and Dissertations
Isolation and partial characterization of a virus-like particle from a eucaryotic alga Cole, Anabel
Large icosahedral virus-like particles (VLP's) have been isolated and partially characterized from a eucaryotic freshwater green alga. The algal host, Uronema gicras, was reported to contain VLP's in an earlier ultrastructural study. Subcultures of this alga were found to release VLP's into the liquid medium in which the alga was grown, from which the VLP's were purified without homogenization of intact cells. The VLP's in situ within infected cells, or when purified and fixed in glutaraldehyde, have a diameter of approximately 400 nm. Sectioned VLP's exhibit an external membrane-like shell enclosing some regions of densely-staining, homogeneous material and other regions of fibrillar material. About 10% of the particles are tailed, the tails measure up to one micron in length and are attached to one vertex of the icosahedron. The development of the VLP's appears to involve the nucleus, and algal cells show extensive disorganization of all cellular membrane systems as the formation of the VLP's progresses. Tailed particles have been observed forming within algal cells. The VLP's are never released in high concentrations, so all biochemical characterizations have been hampered by the limited amount of material. Purified VLP's from U. gigas have a density of approximately 1.32 g/ml in sucrose, a sedimentation coefficient of 6300 S, and are highly light scattering. The polypeptides from the VLP's were analyzed by SDS-polyacrylamide gel electrophoresis. Ten protein bands were detected; the major species had a molecular weight of 45,000 daltons. The nucleic acid contained within the intact VLP was determined to be double-stranded DNA by the following methods: acridine orange staining, diphenylamine and orcinol tests, and specific enzyme digestions of VLP pellets prepared for electron microscopy. Purified DNA from the VLP's was found to have a buoyant density of 1.719 g/ml in cesium chloride, corresponding to a molar fraction of cytosine plus guanine of about 60%. The DNA appeared to be linear and double-stranded using electron microscopical techniques. Length measurements of the DNA were variable, representing DNA molecular weights of 8 x 10⁶ to 72 x 10⁶ daltons, although the accuracy of the technique was confirmed with DNA's of known lengths. Algal germlings seemed to be the most susceptible stage in the life cycle of U. gigas for synthesis and release of VLP's. No particles were observed in thin sections of elongated cells of the mature filament. Scanning electron microscopical views of large germling populations showed that a certain percentage of the cells contained opaque spheres of a diameter similar to the VLP, and by x-ray microanalysis these spheres were found to contain more phosphorus than the surrounding algal cytoplasm. A heat shock of 38 C administered in the dark, during the period of zoospore settling, seemed to greatly increase the yield of VLP's. Every culture of U. gigas examined contained VLP's; a demonstration of the infectivity of this VLP has not been possible without a healthy culture. Structural and biochemical aspects of the VLP from U. gigas are unusual. The particle does not appear to be related to any existing group of viruses.
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