Abstract
Cloning and characterization of the promoter region for the MCT-1 oncogene is described. We used luciferase assays to identify cis-acting elements responsible for human MCT-1 promoter function. The MCT-1 promoter is TATA-less with a consensus initiator element located at the transcription start site and facilitated by two Sp1 sites that directs basal transcription. Deletion of a region of the MCT-1 promoter (-133 to -122) resulted in significant decrease in luciferase activity, suggesting that this region contains a positive cis-acting element. Using mobility shift assays with a 26-mer oligonucleotide, which contains this fragment and its flanking regions, we demonstrated the presence of sequence-specific DNA-binding protein in both Jurkat and Hela nuclear extracts that we designated as LMBF (for lymphoid MCT-1 binding factor). This 26-mer oligonucleotide containing the LMBF binding site is required for maximum transcriptional activity of the MCT-1 promoter. Although the 26-mer oligonucleotide contains a sequence with strong homology to a heat-shock factor consensus, competitive electrophoretic mobility shift assay (EMSA) analysis demonstrated that the binding protein is not a known member of heat shock family. Furthermore, this sequence when placed in reverse orientation downstream of the luciferase gene was able to enhance luciferase activity driven by a minimal promoter. These data are consistent with this sequence behaving as an enhancer. Finally, Southwestern blot analysis revealed a 96-kDa protein capable of binding a probe containing the LMBF binding site.
Original language | English (US) |
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Pages (from-to) | 68-79 |
Number of pages | 12 |
Journal | Journal of Cellular Biochemistry |
Volume | 90 |
Issue number | 1 |
DOIs | |
State | Published - Sep 1 2003 |
Keywords
- DNA-binding protein
- EMSA
- Enhancer
- Heat shock factors
- Jurkat
- MCT-1
- Oncogene
- Promoter
- Southwestern
- Transcription factor
ASJC Scopus subject areas
- Biochemistry
- Molecular Biology
- Cell Biology