Söhl G, Gillen C, Bosse F, Gleichmann M, Müller H W, Willecke K
Abt. Molekulargenetik, Universität Bonn, Germany.
Eur J Cell Biol. 1996 Mar;69(3):267-75.
Four connexin32 (Cx32) cDNA clones isolated from a rat sciatic nerve cDNA library differ in the nucleotide sequence of their 5' untranslated region (UTR) from the corresponding Cx32 cDNA clones previously characterized from liver. The new Cx32 5'UTR sequence detected in the sciatic nerve cDNA clones is identical to one previously found in the 6.5 kb intron of the murine Cx32 gene. Using primer extension and S1 nuclease protection analysis, we determined the transcriptional starting point of this new alternative Cx32 transcript expressed in the sciatic nerve. This starting point is located 444 bp (409 bp) upstream of exon2 in a region previously described as an intron of the Cx32 gene in the rat (and mouse) genome, respectively. The alternative exon1B comprises 99 bp in rat, but 97 bp in the mouse genome, and is spliced to the same exon2 acceptor site also used for splicing of exon1 in liver. Both transcripts are likely to code for the same Cx32 protein whose reading frame is located in exon2. The putative promoter region, upstream of the alternative exon1B, contains a TATAAA motif and has been sequenced and noticed before by Miller et al. (Biosci. Rep. 8, 455-464, (1988)). The alternative exon1B transcript is highly expressed in the sciatic nerve, (i.e. Schwann cells) and very low in liver (i.e. hepatocytes). Its expression is regulated after sciatic nerve injury. The time course of expression was similar to previously established myelin genes and, therefore, we suggest that the expression of the alternative exon1B Cx32 transcript is related to the process of myelination. Very recently, we have characterized another alternative Cx32 exon1A which is transcribed in mouse embryonic stem cells but not in the sciatic nerve (Dahl et al., submitted for publication, 1995). Thus, the murine Cx32 gene is likely to be regulated by three alternative promoters that appear to be activated in a cell type-specific manner.
从大鼠坐骨神经cDNA文库中分离出的四个连接蛋白32（Cx32）cDNA克隆，其5'非翻译区（UTR）的核苷酸序列与先前从肝脏中鉴定出的相应Cx32 cDNA克隆不同。在坐骨神经cDNA克隆中检测到的新的Cx32 5'UTR序列与先前在小鼠Cx32基因的6.5 kb内含子中发现的序列相同。使用引物延伸和S1核酸酶保护分析，我们确定了在坐骨神经中表达的这种新的可变Cx32转录本的转录起始点。该起始点分别位于大鼠（和小鼠）基因组中先前被描述为Cx32基因内含子的区域中外显子2上游444 bp（409 bp）处。可变外显子1B在大鼠中包含99 bp，但在小鼠基因组中包含97 bp，并剪接到与肝脏中外显子1剪接相同的外显子2受体位点。两种转录本可能编码相同的Cx32蛋白，其阅读框位于外显子2中。可变外显子1B上游的推定启动子区域包含一个TATAAA基序，并且之前已被Miller等人测序并注意到（Biosci.Rep.8,455 - 464,(1988)）。可变外显子1B转录本在坐骨神经（即雪旺细胞）中高度表达，而在肝脏（即肝细胞）中极低表达。其表达在坐骨神经损伤后受到调节。表达的时间进程与先前确定的髓鞘基因相似，因此，我们认为可变外显子1B Cx32转录本的表达与髓鞘形成过程有关。最近，我们鉴定了另一种可变Cx32外显子1A，它在小鼠胚胎干细胞中转录，但不在坐骨神经中转录（Dahl等人，已提交发表，1995）。因此，小鼠Cx32基因可能受三个可变启动子调控，这些启动子似乎以细胞类型特异性方式被激活。
