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An antisense promoter of the murine c-myc gene is localized within intron 2
1Department of Biochemistry, Boston University School of Medicine, Massachusetts 02118.
Abstract:
Previously we have demonstrated the existence of stable transcripts from the noncoding strand of a rearranged c-myc gene in murine plasmacytomas in which the oncogene has translocated to an immunoglobulin constant-region gene element (M. Dean, R. B. Kent, and G. E. Sonenshein, Nature [London] 305:443-446, 1983). The resulting RNAs are chimeric, containing c-myc antisense and immunoglobulin sense sequences. A normal unrearranged murine c-myc gene is transcribed in the antisense orientation throughout much of the gene; however, stable transcripts have not been detected. In this study, using Northern (RNA) blot, S1 nuclease, and primer extension analyses, we have mapped the 5' end of the stable chimeric transcripts to a site 175 bp from the start of exon 3, within intron 2 of the c-myc gene. In vitro transcription assays with constructs containing this site and 400 bp upstream, in the antisense orientation, and nuclear extracts from plasmacytoma cells, as well as a number of cell lines with normal unrearranged c-myc genes, indicated that this promoter was functional. This finding was confirmed in transient transfection assays using the antisense promoter linked to the chloramphenicol acetyltransferase reporter gene. These results suggest that a normal promoter of antisense transcription is used following c-myc gene translocation.
Insights
Stable antisense transcripts from rearranged c-myc genes are chimeric RNAs. A functional promoter for antisense transcription was identified within intron 2 of the c-myc gene, active after gene translocation.
Area of Science:
- Molecular Biology
- Oncogenesis
- Gene Regulation
Background:
- Stable chimeric transcripts containing c-myc antisense sequences were previously observed in murine plasmacytomas with translocated c-myc genes.
- Normal unrearranged c-myc genes exhibit antisense transcription, but stable transcripts are not typically detected.
Purpose of the Study:
- To map the 5' end of stable chimeric transcripts originating from rearranged c-myc genes.
- To identify and characterize the promoter responsible for antisense transcription in the context of c-myc gene rearrangement.
Main Methods:
- Northern (RNA) blot analysis
- S1 nuclease assay
- Primer extension analysis
- In vitro transcription assays
- Transient transfection assays with chloramphenicol acetyltransferase reporter gene
Main Results:
- The 5' end of stable chimeric transcripts was mapped to intron 2 of the c-myc gene, 175 bp upstream of exon 3.
- A functional promoter for antisense transcription was identified in this region using in vitro and transient transfection assays.
- This promoter was found to be active in plasmacytoma cells and other cell lines with normal c-myc genes.
Conclusions:
- A normal promoter within intron 2 of the c-myc gene is utilized for antisense transcription following gene translocation.
- This finding provides insight into the regulatory mechanisms governing antisense transcription of the c-myc oncogene in cancer.