The long-term of objective of this research is to understand the mechanisms used by nuclear polyhedrosis viruses to control their gene expression. This virus has been chosen as a model for gene expression in eukaryotes; it has the advantage of being relatively simple and yet complex enough to employ a variety of interesting control mechanisms. A better understanding of the control of gene expression in this virus will probably shed light on the control of gene expression that occurs during human development and during the progression of a normal cell to a cancer cell. The present project contains two major specific aims: 1) to identify the viral gene(s) that confer(s) alpha-amanitin resistance on Spodoptera (host) cells. It is possible that these genes play a role in the switch from alpha-amanitin-sensitive transcription of early viral genes by host polymerase to the alpha-amanitin-resistant transcription of the late viral genes.
This specific aim will be approached by transfecting Spodoptera cells with cloned viral DNA fragments and assaying the transfected cells for alpha-amanitin-resistance. (It is already clear that viral infection very rapidly confers alpha-amanitin-resistance.) When the relevant genes are identified, they and their transcripts will be mapped using restriction mapping, S1-mapping and Northern blotting. The genes will also be sequenced. 2) The second specific aim is to compare the structure of a novel alpha-amanitin-resistant RNA polymerase appearing in the late phase of infection with host RNA polymerase II. The enzymes will be purified using monoclonal antibodies directed against them, if possible. If this proves impractical, the enzymes will be purified by conventional means, employing column chromatography, liquid chromatography and ultracentrifugation. The subunit strucures of the enzymes will be determined. If they appear identical, possible charge differences in the enzyme subunits will be investigated. The genes for any virus-coded subunits in the novel enzyme will be mapped.
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