GENOMIC ORGANIZATION OF THE RAT CONNEXIN40 GENE - IDENTICAL TRANSCRIPTION START SITES IN HEART AND LUNG

Objectives: The gap junction protein connexin(Cx)40 is developmentally and tissue-specifically expressed. How Cx40 expression is regulated i s unknown. We therefore set out to characterize the 5'-untranslated en d of both the Cx40 gene and mRNA from different tissues and ages and t o identify the Cx40 promoter region. Methods: The PCR method 5'-RACE w as used to amplify the 5'-end of rat Cx40 mRNAs. Genomic rat Cx40 clon es were isolated from a lambda EMBL3 library. The promoter sequence wa s isolated by long distance PCR. The transcription start site was iden tified by primer extension and RNase protection assays. Results: Compa rison of Cx40 genomic DNA and mRNA sequences revealed that the Cx40 ge ne contains a small untranslated exon, exon 1, which is separated from the coding sequences by an intron of at least 5.5 Kb. The untranslate d 5'-end of Cx40 mRNA sequences from adult rat lung, neonatal and adul t rat heart and the rat aortic smooth muscle cell line A7r5 were ident ical. While the same transcription start site was found for the Cx40 m RNAs from different tissues and ages, the amount of Cx40 mRNA differed between tissues as follows: A7r5 cells > neonatal lung > adult lung g reater than or equal to neonatal atrium > neonatal ventricle; Cx40 mRN A from adult atrium and ventricle was not readily detected by primer e xtension and RNase protection analyses. The genomic sequence upstream of the transcription start site contains multiple consensus binding si tes for transcription factors putatively responsible for spatio-tempor al control of Cx40 gene expression. Conclusions: Similar to other conn exin genes, the Cx40 gene contains two exons. The same exon 1 sequence is present in all tissues and developmental stages examined and the r elative amounts of Cx40 mRNA in these compare well with published data . Together our data suggest that tissue-specific and developmentally r egulated expression of the Cx40 gene is controlled within the same pro moter region by mechanisms that have yet to be detailed. (C) 1998 Else vier Science B.V. All rights reserved.