Graduate Thesis Or Dissertation

 

Isolation and characteristics of the DNA from nuclear polyhedrosis virus Public Deposited

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https://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/41687m171

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  • The genome of the nuclear polyhedrosis virus which infects the larvae of the tussock moth, the lepidopteran, Hemerocampa pseudotsugata, was isolated, identified as deoxyribonucleic acid, and characterized in terms of its physical and biological properties. An isolation procedure for the viral DNA was established which consisted of the following steps: the polyhedra were isolated by isopycnic centrifugation in a sucrose density gradient; the virus bundles were released from the polyhedra by solubilization of the polyhedron protein at pH 11.6 and concentrated by centrifugation; the virus bundles were disrupted by sodium dodecyl sulfate treatment to release the viral DNA genome; the viral DNA from the virus bundle lysate was banded in a cesium chloride density gradient; and the viral DNA fraction was recovered in a relatively pure state and dialyzed against buffer. The isolated viral genome was identified as DNA by its susceptibility to deoxyribonuclease, its buoyant density in cesium chloride, its ultraviolet absorption spectrum, and its melting curve. The viral DNA was found to have a buoyant density in cesium chloride of 1.710 gm/ml, a melting temperature of 92°C, and a guanine + cytosine base composition of 47 molar percent. The viral DNA genome was shown to exist, at times, as a circular molecule by ethidium bromidecesium chloride density gradient analysis. The molecular weight of the molecule was found to be 96 x 10⁶ daltons by velocity sedimentation analysis. The isolated viral DNA genome was shown to cause about 60% lethal polyhedrosis when injected into H. pseudotsugata larvae at a concentration of 25 mμg of DNA per larva. The nuclear polyhedrosis virus of H. pseudotsugata was compared to other insect viruses and to other major groups of animal and bacterial deoxyriboviruses on the basis of the properties of the viral DNA. The nuclear polyhedrosis virus of H. pseudotsugata fits easily into the group of insect polyhedrosis viruses, but the polyhedrosis viruses, as a group, must be considered separately from other deoxyriboviruses. The nuclear polyhedrosis viruses have several features which make them a desirable model with which to study DNA replication, transcription, translation and other virus-host relationships.
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