IMR Press / FBL / Volume 11 / Issue 2 / DOI: 10.2741/1905

Frontiers in Bioscience-Landmark (FBL) is published by IMR Press from Volume 26 Issue 5 (2021). Previous articles were published by another publisher on a subscription basis, and they are hosted by IMR Press on imrpress.com as a courtesy and upon agreement with Frontiers in Bioscience.

Open Access Article
The promoter competition assay (PCA): a new approach to identify motifs involved in the transcriptional activity of reporter genes
Show Less
1 Department of Pathology, University of Manitoba, Winnipeg R3E 0W3, Manitoba, Canada
2 Department of Biochemistry and Medical Genetics, University of Manitoba, 770 Bannatyne Avenue, Winnipeg R3E 0W3, Manitoba, Canada
Front. Biosci. (Landmark Ed) 2006, 11(2), 1577–1584; https://doi.org/10.2741/1905
Published: 1 May 2006
Abstract

Identifying particular motifs responsible for promoter activity is a crucial step toward the development of new gene-based preventive and therapeutic strategies. However, to date, experimental methods to study promoter activity remain limited. We present in this report a promoter competition assay designed to identify, within a given promoter region, motifs critical for its activity. This assay consists in co-transfecting the promoter to be analyzed and double-stranded oligonucleotides which will compete for the binding of transcription factors. Using the recently characterized SBEM promoter as model, we first delineated the feasibility of the method and optimized the experimental conditions. We then identified, within an 87-bp region responsible for a strong expression of the reporter gene, an octamer-binding site essential for its transcriptional regulation. The importance of this motif has been confirmed by site-directed mutagenesis. The promoter competition assay appears to be a fast and efficient approach to identify, within a given promoter sequence, sites critical for its activity.

Keywords
Promoter Competition Assay
Transcription Factor Binding Site
Small Breast Epithelial Mucin
Luciferase Reporter Gene
Share
Back to top