Structure of the m1 muscarinic acetylcholine receptor gene and its promoter

The mi receptor is one of five muscarinic receptors that mediate the metabotropic actions of acetylcholine in the nervous system where it is expressed predominantly in the telencephalon and autonomic ganglia, RNase protection, primer extension, and 5'-rapid amplification of cDNA ends analysis of a rat cosmid clone containing the entire mi gene demonstrated that the rat mi. gene consists of a single 657-base pairs (bp) noncoding exon separated by a 13.5-kilobase (kb) intron from a 2.54-kb coding exon that contains the entire open reading frame. The splice acceptor for the coding exon starting at -71 bp relative to the adenine of the initiating methionine. This genomic structure is similar to that of the m4 gene (Wood, I. C., Roopra, A., Harrington, C. A., and Buckley, N. J. (1995) J. Biol, Chem. 270, 30933-30940 and Wood, I, C., Roopra, A., and Buckley, N. J. (1996) J. Biol. Chem. 271, 14221-14225). Like the m4 gene, the mi promoter lacks TATA and CAAT consensus motifs, and the first exon and 5'-flanking region are not gc-rich, The 5'-flanking region also contains the consensus regulatory elements Sp-1, NZF-1, AP-I, AP-2, E-box, NF kappa B, and Oct-1, Unike the m4 promoter, there is no evidence of a RE1/NRSE silencer element in the mi promoter. Deletional analysis and transient transfection assays demonstrates that reporter constructs containing 0.9 kb of 5'-flanking sequence and the first exon are sufficient to drive cell-specific expression of reporter gene in IMR32 neuroblastoma cells while remaining silent in 3T3 fibrobasts.