Gaunitz F, Gaunitz C, Papke M, Gebhardt R
Physiologisch-chemisches Institut, Universität Tübingen, Germany.
Biol Chem. 1997 Jan;378(1):11-8. doi: 10.1515/bchm.1997.378.1.11.
In order to identify regulatory elements involved in the hepatocyte specific expression of the enzyme glutamine synthetase [GS (E.C. 6.3.1.2)] we analyzed the first intron of the rat GS gene. A sequence analysis detected clusters of potential transcription factor binding sites in regions that are hypersensitive for DNase I, including sites for Sp1, HNF3 and elements related to binding of members from the C/EBP family. By use of DNA fragments with putative regulatory elements, reporter genes have been constructed that were transfected into isolated hepatocytes in primary culture and into HepG2 hepatoblastoma cells. By these experiments we cold show that sequences from the first intron are able to enhance transcription specifically in hepatocytes but not in cells from the hepatoblastoma cell line. The existence of enhancer effects in the first intron of the GS gene and their restriction to hepatocytes demonstrates that aside from regulatory regions upstream of the transcription start point, there are also downstream regions involved in the specific expression of the gene. We conclude that intronic elements are involved in the pretranslational regulation of the expression of the GS as part of a complex interplay between different regions of the gene.
为了鉴定参与谷氨酰胺合成酶[GS（E.C. 6.3.1.2）]肝细胞特异性表达的调控元件，我们分析了大鼠GS基因的第一个内含子。序列分析在对DNase I高度敏感的区域检测到潜在转录因子结合位点簇，包括Sp1、HNF3的位点以及与C/EBP家族成员结合相关的元件。通过使用带有推定调控元件的DNA片段，构建了报告基因，并将其转染到原代培养的分离肝细胞和HepG2肝癌细胞中。通过这些实验我们能够表明，来自第一个内含子的序列能够特异性增强肝细胞中的转录，但不能增强肝癌细胞系细胞中的转录。GS基因第一个内含子中增强子效应的存在及其对肝细胞的限制表明，除了转录起始点上游的调控区域外，还有下游区域参与该基因的特异性表达。我们得出结论，作为基因不同区域之间复杂相互作用的一部分，内含子元件参与了GS表达的翻译前调控。
