Here we describe a novel expression system for mammalian cells which is based on transcription of hybrid genes containing T7 phage promoters by a T7 phage RNA polymerase targeted to the nucleus of the host cells. The RNA polymerase gene of T7 phage has been modified by substituting a sequence encoding the nuclear location signal of SV40 large T antigen for the N-terminal part of the polymerase gene. Expression of the modified gene is driven by the mouse metallothionein promoter in transfected mouse Ltk- cells resulting in high concentration of the polymerase in the nucleus. Nuclear T7 RNA polymerase directs efficient transcription of the cat gene under control of a T7 promoter. T7 constructs are expressed at a level at least 6 fold higher than the prototype pRSVcat. The unique properties of this heterologeous expression system are discussed.
The bovine papillomavirus type 1 (BPV-1) genome replicates as a plasmid within the nuclei of BPV-1-transformed murine C127 cells at a constant multiple copy number, and spontaneous amplification of the viral DNA is rarely observed. We report here that a mutant BPV-1 plasmid within a contact-inhibited C127 cell line replicated as a stable multicopy plasmid in exponentially growing cells but amplified to a high level in confluent cell culture. In situ hybridization analysis revealed that most of the mutant viral DNA amplification occurred in a minor subpopulation of cells within the culture. These consisted of giant nondividing cells with greatly enlarged nuclei, a cell form which was specifically induced in stationary-phase cultures. These observations indicated that expression of a viral DNA replication factor was cell growth stage specific. Consistent with this hypothesis, considerable amplification of wild-type BPV-1 DNA associated with characteristic giant cell formation was observed in typical wild-type virus-transformed C127 cultures following a period of growth arrest achieved by serum deprivation. Further observations indicated that induction of the giant-cell phenotype was dependent on BPV-1 gene expression and implicated a viral El replication factor in this process. Moreover, heterogeneity in virus genome copy numbers within the giant-cell population suggested a complex regulation of induction of DNA synthesis in these cells. It appears that this process represents a mechanism employed by the virus to ensure maximal viral DNA synthesis within a growth-arrested cell. Fundamental questions concerning the integration of the virus-cell control circuitry in proliferating and resting cells are discussed.
The bovine papillomavirus type 1 (BPV-1) genome replicates as a latent plasmid in mouse C127 cells transformed in vitro by the virus. However, we have recently shown that BPV-1 DNA amplification can be induced in a subpopulation of cells under culture conditions which suppress cell proliferation, a finding which led us to hypothesize that expression of a viral replication factor was regulated by cell growth stage. In this report, we describe the detection in these cells of abundant BPV-1 nuclear E2 antigen by immunofluorescence analysis. Expression of E2 antigen in fibropapilloma tissue was similarly localized to nonproliferating epidermal cells of the lower spinous layers-the natural site of induction of vegetative viral DNA replication. Immunoprecipitation analysis showed that the previously characterized 48-kilodalton (transactivator) and 31-kilodalton (repressor) E2 proteins were both induced in growth-arrested cell cultures. In parallel with E2 antigen synthesis under conditions of serum-deprivation in vitro, we observed a significant increase in levels of BPV-1 early region mRNAs. Furthermore, we present evidence for preferential induction of the P2443 promoter, in addition to specific induction of the P7940 promoter in response to serum deprivation. These observations indicate a central role for E2 transcription factors in the induction of viral DNA amplification in division-arrested cells in vitro and in vivo and suggest that this process is associated with a qualitative switch in the expression of viral early region genes.
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