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Chapter 8,253

CROC-1 encodes a protein which mediates transcriptional activation of the human FOS promoter

Rothofsky, M.L.; Lin, S.L.

Gene 195(2): 141-149

1997


ISSN/ISBN: 0378-1119
PMID: 9305758
DOI: 10.1016/s0378-1119(97)00097-8
Accession: 008252971

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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