| Abstrakt: |
The cloning of signal transducing molecules capable of activating the human FOS proto-oncogene promoter was achieved by co-transfecting a modified human FOS promoter-driven polyomavirus large T antigen gene (P(f)LAG-8) with a human brain cDNA library, incorporated into a replication-competent mammalian retroviral expression vector whose replication occurs in the presence of T antigen. In murine cells, transcriptional activation of the P(f)LAG-8 promoter by a biologically active, cDNA-encoded signalling molecule resulted in plasmid replication. Replicated plasmids, following selective cleavage of unreplicated plasmids by Dpn1, were recovered by transformation into competent bacteria. Successive P(f)LAG-8/cDNA library co-transfections, using library plasmids resulting from prior transfections, ultimately resulted in the identification of individual plasmids capable of transcriptionally activating the FOS promoter. DNA sequencing revealed the first plasmid, denoted CROC-1, to contain a 1.8-kb cDNA encoding a 16.5-kDa nuclear protein possessing a bipartite structure comprised an amino-terminal acidic domain and a carboxy-terminal basic domain, and displaying partial homology to the HMG domain of the TAF(II)250 transcription cofactor. Co-transfection of CROC-1 with various FOS/CAT reporter genes revealed that the human FOS promoter region spanning -56 to - 105, encompassing two identical 8-bp DR enhancer sequences, was necessary for CROC-1-mediated transcriptional activation. Results suggest that CROC-1 participates in intracellular signalling pathways involved in induction of the human FOS promoter. |
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