Visvikis A, Pawlak A, Accaoui M J, Ichino K, Leh H, Guellaen G, Wellman M
Centre du Médicament, EA 3117, Faculté de Pharmacie UHP Nancy 1, 30 rue Lionnois, 54000 Nancy, France.
Eur J Biochem. 2001 Jan;268(2):317-25. doi: 10.1046/j.1432-1033.2001.01881.x.
In humans, five distinct mRNAs code for gamma-glutamyltransferase (GGT). Their coding regions are identical and their 5' untranslated regions exhibit both common and type-specific sequences. To elucidate the mecanisms that generate these different mRNAs, we cloned and determined the structure of the 5' region of the human GGT gene. The common regions of the 5' UTR are encoded by five exons, localized within a 2.4-kb region of the genomic DNA. Three of them are separated only by intron-donor or intron-acceptor sites at their boundaries. Alternative splicing of these exons may determine the unique pattern of the different GGT mRNA 5' UTRs in a tissue-specific manner. In addition, we have isolated a genomic fragment containing the most distal 5' sequences of the major GGT mRNA in HepG2 cells. Primer extension analysis revealed one major transcription initiation site while 5' RACE indicated that one more distal initiation site could be present. In the putative promoter sequence neither classical TATA or CAAT boxes were found. However, sites for AP1, AP2, CREB, GRE and SP1 transcription factors were identified. Chimeric plasmids, containing this genomic region fused to the luciferase gene, were transiently expressed in three cell lines of different origin: HeLa cells, ovarian carcinoma A2780 cells and V79 lung fibroblasts. The significant promoter activities obtained indicate a transcription start within this region. However, differences in the level of expression were found between the different cell lines used. These data suggest that the human GGT gene employs regulatory sequences and alternative splicing, and gene expression may therefore be regulated in tissue specific and cell-type-specific manners.
在人类中，五种不同的mRNA编码γ-谷氨酰转移酶（GGT）。它们的编码区相同，其5'非翻译区呈现出共同序列和类型特异性序列。为了阐明产生这些不同mRNA的机制，我们克隆并确定了人类GGT基因5'区域的结构。5'UTR的共同区域由五个外显子编码，定位在基因组DNA的2.4kb区域内。其中三个外显子仅在其边界处由内含子供体或内含子受体位点隔开。这些外显子的可变剪接可能以组织特异性方式决定不同GGT mRNA 5'UTR的独特模式。此外，我们分离出了一个包含HepG2细胞中主要GGT mRNA最远端5'序列的基因组片段。引物延伸分析揭示了一个主要的转录起始位点，而5'RACE表明可能存在一个更远端的起始位点。在推定的启动子序列中未发现经典的TATA或CAAT框。然而，鉴定出了AP1、AP2、CREB、GRE和SP1转录因子的位点。含有与荧光素酶基因融合的该基因组区域的嵌合质粒在三种不同来源的细胞系中瞬时表达：HeLa细胞、卵巢癌A2780细胞和V79肺成纤维细胞。获得的显著启动子活性表明该区域内有转录起始。然而，在所使用的不同细胞系之间发现了表达水平的差异。这些数据表明人类GGT基因采用调控序列和可变剪接，因此基因表达可能以组织特异性和细胞类型特异性方式受到调控。
