MOUSE CONNEXIN40 - GENE STRUCTURE AND PROMOTER ANALYSIS

A family of related connexin genes encodes the subunit gap junction pr oteins that form intercellular channels ill different tissues. Connexi n40 (Cx40) is one of these proteins, and it exhibits limited expressio n only in a few cells of the cardiovascular system. To begin to analyz e Cx40 expression, we isolated a 3.3-kb rat Cx40 cDNA by hybridization screening of a bacteriophage library prepared from BWEM cells and iso lated corresponding mouse genomic clones from a bacterial artificial c hromosome library. Restriction mapping, sequencing and comparison to t he rat cDNA showed that the mouse Cx40 gene contained a short first ex on, an 11.4-kb intron, and a second exon containing the complete codin g region and 3'-UTR. Exon I contained only 1 base that differed betwee n rat and mouse. Primer extension experiments yielded a single band an d confirmed the position of the transcriptional start site, We obtaine d 1,2 kb of sequence 5' of the transcriptional start site and 400 bp 5 ' of exon I, Exon I was closely preceded by a consensus TATA box, The flanking sequences contained a number of potential transcription facto r binding sites (including AP-1, AP-2; SP1, TRE, and p53). To identify transcriptional regulatory elements in the Cx40 promoter region, a se ries of DNA deletion fragments flanking exon I was prepared, subcloned adjacent to a luciferase reporter gene, and used for transient transf ections of BWEM, SHM, and N2A cells, The resulting luciferase activity determinations suggested that an area of 300 bp 5' of the transcripti on start site acted as a basal promoter for Cx40 and that there was a strong negative regulatory element in the region from +100 to +297. (C ) 1997 Academic Press.