ras oncogene activation of a VL30 transcriptional element is linked to transformation.
The activity of a murine VL30 transcriptional element was increased 20-fold in transient assays by coexpression of mutant ras genes. The cis element did not respond to ras in a revertant cell line that was transformation defective. Therefore, ras-dependent alterations in transcription and ras transformation are linked. Deletion analysis of the VL30 long-terminal-repeat U3 region showed that a minimal 53-base-pair segment is required in cis for oncogene activation of transcription. Gel retention assays using a probe that contained the minimal cis element revealed that a unique complex was formed with nuclear proteins prepared from transformed cells. Exonuclease III footprinting and gel retention experiments that used oligonucleotide probes and competitors indicated that two distinct nuclear factors interact with the minimal cis-responsive element. Site-directed deletion of the 5'-proximal binding site (TGACTCT) resulted in a complete loss of ras responsiveness. However, deletion of this site did not affect stimulation by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). These data are consistent with the hypothesis that ras and TPA signal transduction mechanisms for transcriptional activation are distinct.
Department of Microbiology and Immunology, Duke University Medical Center, Durham, North Carolina 27710.
R D
RD
Owen
D M
DM
Bortner
M C
MC
Ostrowski
D000818:Animals; D001483:Base Sequence; D002471:Cell Transformation, Neoplastic; D004252:DNA Mutational Analysis; D005786:Gene Expression Regulation; D011905:Genes, ras; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000502:physiology*; D015689:Oncogene Protein p21(ras) / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid; D012091:Repetitive Sequences, Nucleic Acid; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014157:Transcription Factors / Q000502:physiology*
D009687:Nuclear Proteins; D014157:Transcription Factors; D015689:Oncogene Protein p21(ras); D013755:Tetradecanoylphorbol Acetate