STRUCTURE OF THE M4 CHOLINERGIC MUSCARINIC RECEPTOR GENE AND ITS PROMOTER

Cholinergic muscarinic receptor genes are members of the G-protein rec eptor gene superfamily, In this study we describe the structure of the gene and promoter of the rat m4 muscarinic receptor gene, A rat cosmi d clone containing the coding region for the m4 gene and 25 kilobases of upstream sequence was isolated, This clone directed expression of t he rat m4 gene when introduced in IMR32 cells, a human neuroblastoma t hat expresses m4, but did not drive expression when introduced into Ch inese hamster ovary cells, a line that does not express the m4 gene, S 1 nuclease, modified 5'-rapid amplification of cDNA ends and polymeras e chain reaction analysis of rat cosmid DNA and cDNA showed that the g ene consists of a 2.6-kilobase coding exon, extending 34 base pairs (b p) upstream from the initiating ATG, separated from a 460-493 bp nonco ding exon by a 4.8-kilobase intron, DNA sequence analysis shows that t he noncoding exon is CC-rich and that the promoter does not contain a TATA or CAAT box and has several consensus sequences for enhancer elem ents including five Sp-1 binding sites, one AP-2 site, one AP-3 bindin g site and two E-boxes within the proximal 600 bp, A reporter construc t consisting of 1440 bp of flanking DNA and 80 bp of the first exon cl oned into a luciferase reporter plasmid, drove cell specific expressio n in transient transfection assays, Removal of 1088 bp of the 5' end o f this construct resulted in expression in non-m4 expressing cell line s suggesting there is a repressor element in this region.