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ras oncogene activation of a VL30 transcriptional element is linked to transformation
R D Owen1, D M Bortner, M C Ostrowski
1Department of Microbiology and Immunology, Duke University Medical Center, Durham, North Carolina 27710.
Abstract:
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.
Insights
Mutant ras genes enhance VL30 transcriptional activity. This ras-dependent transcription is linked to cell transformation, suggesting distinct signaling pathways for ras and phorbol ester activation.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- The ras oncogene plays a critical role in cell signaling and transformation.
- Transcriptional elements like VL30 can be regulated by cellular and viral factors.
- Understanding oncogene-induced transcriptional changes is key to deciphering cancer mechanisms.
Purpose of the Study:
- To investigate the link between ras-dependent transcriptional activation and cellular transformation.
- To identify the specific DNA elements and nuclear factors involved in ras-mediated VL30 transcription.
- To compare the signaling pathways of ras and phorbol ester (TPA) in transcriptional regulation.
Main Methods:
- Transient transfection assays to measure transcriptional activity.
- Coexpression of mutant ras genes in murine cells.
- Deletion analysis of the VL30 long-terminal-repeat (LTR) U3 region.
- Gel retention assays and exonuclease III footprinting to identify protein-DNA interactions.
Main Results:
- Mutant ras genes induced a 20-fold increase in murine VL30 transcriptional element activity.
- A minimal 53-base-pair segment within the VL30 U3 region was essential for ras-mediated transcriptional activation.
- Two distinct nuclear factors were found to bind to this minimal cis-responsive element.
- Deletion of a specific binding site (TGACTCT) abolished ras responsiveness but not TPA stimulation.
Conclusions:
- Ras-dependent transcriptional alterations are directly linked to ras-mediated cell transformation.
- Distinct nuclear factors mediate the transcriptional response to ras oncogenes.
- The signaling pathways activated by ras and TPA for transcriptional activation are separate and distinct.