Mouse connexin 45: Genomic cloning and exon usage

Connexin 45 is a gap junction protein that is prominent in early embryos an d is widely expressed in many mature cell types, To elucidate its gene stru cture, expression, and regulation, we isolated mouse Cx45 genomic clones, A lignment of the genomic DNA and cDNA sequences revealed the presence of thr ee exons and two introns, The first two exons contained only 5' untranslate d sequences, while exon 3 contained the remaining 5' UTR, the entire coding region, and the 3' UTR, An RT-PCR with exon-specific primers was utilized to examine exon usage in F9 mouse embryonal carcinoma cells and adult mouse tissues. In all samples, PCR products amplified using exon 2/exon 3 or exo n 3/exon 3 primer pairs were much more abundant than products produced usin g exon 1/exon 2 or exon 1/exon 3 primer pairs, suggesting that Cx45 mRNAs c ontaining exon 1 were relatively rare compared with mRNAs containing the ot her exons. Rapid amplification of cDNA ends (5'-RACE) was performed using a ntisense primers from within exon 3 and template RNA prepared from F9 cells or from adult mouse kidney. We obtained multiple RACE products from both t emplates, including products that contained all three exons and were splice d identically to the cDNA. However, clones were also isolated (from kidney) that began within the region previously identified as intron 1 and continu ed upstream with a sequence identical to the cDNA, including splicing to ex on 3, These results show that mouse Cx45 has a gene structure that differs from that of previously studied connexins and allows the production of hete rogenous Cx45 mRNAs with differing 5' UTRs. These differences might contrib ute to regulation of Cx45 protein levels by modulating mRNA stability or tr anslational efficiency.