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 is unknown. We therefore set out to characterize the 5'-untranslated end of both the Cx40 gene and mRNA from different tissues and ages and to identify the Cx40 promoter region.

METHODS

The PCR method 5'-RACE was used to amplify the 5'-end of rat Cx40 mRNAs. Genomic rat Cx40 clones were isolated from a lambda EMBL3 library. The promoter sequence was isolated by long distance PCR. The transcription start site was identified by primer extension and RNase protection assays.

RESULTS

Comparison of Cx40 genomic DNA and mRNA sequences revealed that the Cx40 gene contains a small untranslated exon, exon I, which is separated from the coding sequences by an intron of at least 5.5 Kb. The untranslated 5'-end of Cx40 mRNA sequences from adult rat lung, neonatal and adult rat heart and the rat aortic smooth muscle cell line A7r5 were identical. While the same transcription start site was found for the Cx40 mRNAs from different tissues and ages, and amount of Cx40 mRNA differed between tissues as follows: A7r5 cells > neonatal lung > adult lung > or = neonatal atrium > neonatal ventricle; Cx40 mRNA from adult atrium and ventricle was not readily detected by primer extension and RNase protection analyses. The genomic sequence upstream of the transcription start site contains multiple consensus binding sites for transcription factors putatively responsible for spatio-temporal control of Cx40 gene expression.

CONCLUSIONS

Similar to other connexin genes, the Cx40 gene contains two exons. The same exon I sequence is present in all tissues and developmental stages examined and the relative amounts of Cx40 mRNA in these compare well with published data. Together our data suggest that tissue-specific and developmentally regulated expression of the Cx40 gene is controlled within the same promoter region by mechanisms that have yet to be detailed.