STRUCTURE OF THE M1 MUSCARINIC ACETYLCHOLINE-RECEPTOR GENE AND ITS PROMOTER

The mi receptor is one of five muscarinic receptors that mediate the m etabotropic actions of acetylcholine in the nervous system where it is expressed predominantly in the telencephalon and autonomic ganglia, R Nase protection, primer extension, and 5'-rapid amplification of cDNA ends analysis of a rat cosmid clone containing the entire mi gene demo nstrated 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 adeni ne of the initiating methionine. This genomic structure is similar to that of the m4 gene (Wood, I. C., Roopra, A., Harrington, C. A., and B uckley, 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 moti fs, 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 promot er. Deletional analysis and transient transfection assays demonstrates that reporter constructs containing 0.9 kb of 5'-flanking sequence an d the first exon are sufficient to drive cell-specific expression of r eporter gene in IMR32 neuroblastoma cells while remaining silent in 3T 3 fibrobasts.