Honscha W, Oesch F, Friedberg T
Institute of Toxicology, University of Mainz, Germany.
Arch Biochem Biophys. 1991 Jun;287(2):380-5. doi: 10.1016/0003-9861(91)90493-3.
mRNA was isolated from several rat tissues and subjected to either the nuclease S1 or the RNAseA protection assay with probes covering the 5' end, the middle part, and the 3' end of the microsomal epoxide hydrolase (mEHb) cDNA. Whereas probes directed against the latter two regions yielded a single protected fragment, a probe which covered base pairs -148 to +453 (+1 defines the start of protein biosynthesis) yielded two protected fragments. The degree of protection of the two fragments was strongly dependent on the tissue from which the mRNA had been isolated. Thus at least two mEHb mRNAs which differ at their 5' ends are differentially expressed in various tissues. In addition the mRNAs corresponding to the two protected fragments were clearly differentially inducible by Aroclor 1254 treatment of the animals. Primer extension analysis with hepatic RNA from untreated animals yielded three primer-extended products corresponding to three mRNAs which differ at their 5' ends. As already seen in the nuclease S1 protection assay, one of the mRNAs was induced by Aroclor 1254 treatment. The expression of the two other mRNAs was either repressed or stable. Thus besides the mRNA already characterized for mEHb, there are at least two other mEHb mRNAs. This result was confirmed by the isolation of a mEHb cDNA which is completely distinct in its sequence in a region just preceding the initiation codon for protein biosynthesis. From that point on, the sequence of our cDNA becomes identical to the published mEHb cDNA. This point corresponds exactly to the start of exon 2 as determined from the genomic sequence. Thus the region where both mEHb cDNAs differ is encoded by two different exons 1, which are joined to exon 2 by alternative splicing. The tissue-specific expression and the different inducibility of the various mEHb mRNAs might indicate that their expression is governed by different promoters.
从几只大鼠的多种组织中分离出信使核糖核酸（mRNA），并使用覆盖微粒体环氧化物水解酶（mEHb）互补脱氧核糖核酸（cDNA）5'端、中间部分和3'端的探针，对其进行核酸酶S1或核糖核酸酶A保护分析。针对后两个区域的探针产生了一个单一的受保护片段，而覆盖碱基对-148至+453（+1定义蛋白质生物合成的起始点）的探针产生了两个受保护片段。这两个片段的保护程度强烈依赖于分离mRNA的组织。因此，至少有两种在5'端不同的mEHb mRNA在各种组织中差异表达。此外，对应于两个受保护片段的mRNA在经阿罗氯丹1254处理的动物中明显具有不同的诱导性。用未处理动物的肝脏RNA进行引物延伸分析，产生了三种引物延伸产物，对应于三种在5'端不同的mRNA。正如在核酸酶S1保护分析中已经看到的那样，其中一种mRNA在阿罗氯丹1254处理后被诱导。另外两种mRNA的表达要么被抑制，要么保持稳定。因此，除了已经鉴定的mEHb mRNA之外，至少还有另外两种mEHb mRNA。通过分离一个mEHb cDNA证实了这一结果，该cDNA在蛋白质生物合成起始密码子之前的一个区域的序列完全不同。从这一点开始，我们的cDNA序列与已发表的mEHb cDNA相同。这一点与根据基因组序列确定的外显子2的起始点完全对应。因此，两个mEHb cDNA不同的区域由两个不同的外显子1编码，它们通过可变剪接与外显子2相连。各种mEHb mRNA的组织特异性表达和不同的诱导性可能表明它们的表达受不同启动子的调控。
