Abstract
Inbred mouse strains display significant differences in their levels of brain alpha7 nicotinic acetylcholine receptor (alpha7 nAChR) expression, as measured by binding of the alpha7-selective antagonist alpha-bungarotoxin. Variations in alpha-bungarotoxin binding have been shown to correlate with an animal's sensitivity to nicotine-induced seizures and sensory gating. In two inbred mouse strains, C3H/2Ibg (C3H) and DBA/2Ibg (DBA/2), the inter-strain binding differences are linked to a restriction length polymorphism in the alpha7 nAChR gene, Chrna7. Despite this finding, the molecular mechanism(s) through which genetic variability in Chrna7 may contribute to alpha7 nAChR expression differences remains unknown. However, studies of the human alpha7 nAChR gene (CHRNA7) previously have demonstrated that CHRNA7 promoter polymorphisms are associated with differences in promoter activity as well as differences in sensory processing. In the present study, a 947-base pair region of the Chrna7 promoter was cloned from both the C3H and DBA/2 inbred mouse strains in an attempt to identify polymorphisms that may underlie alpha7 nAChR differential expression. Sequence analysis of these fragments identified 14 single nucleotide polymorphisms (SNPs). A combination of two of these SNPs affects promoter activity in an in vitro luciferase reporter assay. These results suggest a mechanism through which the Chrna7 promoter genotype may influence interstrain variations in alpha7 nAChR expression.
Highlights
The expression of ␣7 nAChRs,3 as measured by 125I-labeled ␣-bungarotoxin (␣-BTX), varies significantly across inbred mouse strains [1, 2], and studies have demonstrated that interstrain differences in ␣7 nAChR expression are genetically regulated
Promoter polymorphisms in the gene coding for the human ␣7 nAChR subunit are associated with an auditory gating deficit that is common among schizophrenics [10]
In order to establish whether polymorphisms in the Chrna7 promoter exist between C3H and DBA/2 mice and, if so, whether they affect the function of the Chrna7 promoter, we have cloned a 947-bp region of the putative Chrna7 promoter from each strain
Summary
C3H and DBA/2 Chrna promoter fragments were amplified from the genomic DNA samples using HF2 DNA polymerase (BD Biosciences) and introduced into the vector pCRII TOPO (Invitrogen). Promoter fragments with the strain consensus sequences were subcloned in pGL3 Basic (Promega, Madison, WI) for reporter gene analysis. PC12 cells were grown in high glucose Dulbecco’s modified Eagle’s medium supplemented with 10% horse serum, 5% FBS, and 1% penicillin/streptomycin. In order to differentiate PC12 cells into a neuronal phenotype, the cells were treated with nerve growth factor (100 ng/ml) in Dulbecco’s modified Eagle’s medium supplemented with 4% horse serum, 2% FBS, and 1% penicillin/streptomycin for 1 week as previously reported [12]. To determine a region upstream of the Chrna ATG translation site that contributed to core promoter activity in GH4C1 cells, a series of 5Ј cutdowns were derived from the Ϫ947
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