Abstract
We describe the structural and functional features of the human α3 nicotinic receptor subunit promoter. A 0.35-kb region immediately upstream of the start codon was identified that when transfected in human neuroblastoma cells was able to drive the expression of the luciferase reporter gene with a strength comparable to that of the well-characterized simian virus 40 promoter/enhancer. This region displayed the features of a multistart-site, GC-rich, TATA-less, and CAAT-less promoter, containing many overlapping Sp1 and AP-2 putative binding sites. Further dissections of the 0.35-kb fragment revealed that its 3′ region, specifying the 5′ UT of the mRNA, plays a relevant positive effect in determining the strength of the promoter. This region contains putative cis-acting elements for AP-2, nuclear factor-κB, and the recently described multiple-start site element downstream-1. By mutation analysis, we showed that these sites are functional and when combined increase the promoter activity by 4-fold. The 0.35-kb promoter was found to be under the negative control of upstream sequences that include a modern Alu repeat. The α3 Alu repeat works as a composite region, containing both positive and negative elements that control the activity of the downstream promoter. Finally, we investigated the tissue-specific activity of the human α3 gene 5′ regulatory sequences, showing that they are able to drive the expression of the reporter gene preferentially in neuronal cells.
Footnotes
- Received July 8, 1996.
- Accepted October 25, 1996.
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Send reprint requests to: Dr. Diego Fornasari, CNR Cellular and Molecular Pharmacology Center, Department of Medical Pharmacology, University of Milan, Via Vanvitelli 32, 20129 Milan, Italy. E-mail: diegof{at}farma9.csfic.mi.cnr.it
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This work was supported in part by Fabriques de Tabac Reunies (Neuchotel, Switzerland).
- The American Society for Pharmacology and Experimental Therapeutics
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