The oncogenic virus Bovine Papilloma Virus (BPV-1) replicates in synchrony with cellular DNA in transformed cells as a supercoiled plasmid, and as an autonomous replicon it provides an ideal system to study the replication of DNA in the cell cycle. We propose a program of experiments whose major goal is to understand the differences between regulated (latent) replication and vegetative replication. These experiments should add to our knowledge of how viral oncogenes usurp normal cell cycle controls. We propose to: 1. Clone and express from various vectors the viral genes which regulate and mediate this replication. 2. Continue our genetic analysis of the trans and cis requirements for latent replication. 3. Define when in the cell cycle the virus replicates and if the order of viral gene expression is critical to its apparent ability to replicate once per cell cycle. 4. Determine if the viral DNA encodes for a """"""""repressor of amplification."""""""" 5. Measure accurately the loss of plasmids as cells divide. Methods are described which will be very sensitive to mitotic instability. 6. Characterize the regulation of BPV-1 promoters both through genetic and in vitro transcription experiments. 7. Characterize the infectious """"""""BPV retroviruses"""""""" that we have made. These viruses will be useful in defining the genetic potential of various cell lines to support plasmid replication. 8. Raise polyclonal antibodies to BPV-1 early proteins.
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