Groenewegen W A, van Veen T A, van der Velden H M, Jongsma H J
Utrecht University, Department of Medical Physiology and Sports Medicine, The Netherlands.
Cardiovasc Res. 1998 May;38(2):463-71. doi: 10.1016/s0008-6363(97)00325-8.
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.
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.
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.
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.
缝隙连接蛋白连接蛋白（Cx）40在发育过程中及组织中呈特异性表达。Cx40的表达是如何调控的尚不清楚。因此，我们着手对来自不同组织和年龄的Cx40基因及mRNA的5'-非翻译区进行表征，并确定Cx40启动子区域。
采用PCR方法5'-RACE扩增大鼠Cx40 mRNA的5'-末端。从λEMBL3文库中分离出基因组大鼠Cx40克隆。通过长距离PCR分离启动子序列。通过引物延伸和RNase保护试验确定转录起始位点。
Cx40基因组DNA与mRNA序列的比较显示，Cx40基因包含一个小的非翻译外显子，即外显子I，它与编码序列被至少5.5 Kb的内含子隔开。成年大鼠肺、新生和成年大鼠心脏以及大鼠主动脉平滑肌细胞系A7r5的Cx40 mRNA序列的5'-非翻译末端是相同的。虽然在不同组织和年龄的Cx40 mRNA中发现了相同的转录起始位点，但不同组织中Cx40 mRNA的量如下：A7r5细胞>新生肺>成年肺>或=新生心房>新生心室；通过引物延伸和RNase保护分析不易检测到成年心房和心室中的Cx40 mRNA。转录起始位点上游的基因组序列包含多个转录因子的共有结合位点，这些转录因子可能负责Cx40基因表达的时空控制。
与其他连接蛋白基因相似，Cx40基因包含两个外显子。在所检测的所有组织和发育阶段中都存在相同的外显子I序列，并且这些组织中Cx40 mRNA的相对量与已发表的数据比较吻合。我们的数据共同表明，Cx40基因的组织特异性和发育调控表达是由尚未详细阐明的机制在同一启动子区域内控制的。
