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V. A. Smyth, M. E. Morrissey, D. D. Lorenzo, P. O'Gaora, M. Flanagan, C. Cagney, B. N. Kennedy; A Novel 20 bp Enhancer Region Controls Cone-Specific Expression. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4665.
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The molecular mechanism of differential photoreceptor development and gene expression remains largely undefined. The cone transducin α subunit (TαC) is expressed exclusively in cone photoreceptors in the eye and pineal of vertebrates. TαC specificity makes it an ideal tool to investigate the molecular mechanisms of cone photoreceptor gene expression. This study examines cis-elements responsible for the cell specific expression pattern of TαC in vivo.
To identify cis-elements controlling cone specific expression a serial deletion of the zebrafish TαC promoter (zTαCP) was performed. These 5’ flanking deletions were constructed upstream of enhanced green fluorescent protein (EGFP) and tested in vivo by means of a transient transgenic assay (Luo et al. (2004), J. Biol. Chem. 279, 19286-93). Identified cis-elements were further characterised by Electrophoretic mobility shift assay (EMSA). Chimeric constructs containing identified zTαCP cis-elements were placed upstream of a 800 bp minimal zebrafish UV opsin promoter (zUVOP) to test their ability to control a heterologous promoter.
Serial deletions identified a 55 bp region controlling robust EGFP expression in zebrafish cones. Deletion of this enhancer region from the distal promoter resulted in a significant reduction of EGFP expression in both heterologous and wild type promoter constructs. The putative enhancer sequence was narrowed to a 20 bp region by EMSA and deletion construct analyses. EMSA analysis of sequential three base pair mutations, identified a 9 bp and 6 bp sequence necessary for binding eye nuclear protein.
An evolutionary conserved 20 bp sequence of the zebrafish TαC promoter region acts as an enhancer capable of driving strong cone specific expression. 9 bp and 6 bp sequence within the 20 bp sequence are essential for the binding of eye nuclear protein. Progress in characterising the binding trans-factor(s), and the importance of the nine and six base pair region for the enhancer’s activity, will be presented.
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