Choi D S, Handa M, Young H, Gordon A S, Diamond I, Messing R O
Ernest Gallo Clinic and Research Center, Department of Neurology, Graduate Program in Neuroscience, University of California, San Francisco, 5858 Horton Street, Suite 200, Emeryville, California, USA.
Biochem Biophys Res Commun. 2000 Oct 14;277(1):200-8. doi: 10.1006/bbrc.2000.3665.
We have cloned and characterized the genomic structure of the mouse gene for the NBMPR-sensitive equilibrative nucleoside transporter (mENT1), which is located on chromosome 17C. About 12-kb of genomic DNA was sequenced including the promoter region, 12 exons, 11 introns, and the 3'-untranslated region. All exon-intron junction sequences conform to the GT/AG rule. Primer extension analysis demonstrated a transcription initiation site located 252 bp upstream of the translation start site. Analysis of the 2.5-kb 5'-flanking sequence shows putative binding sites for several transcription factors, including GATA-1, IRF-2, Pit-1, myogenin, CREB, Sp-1, Ap-2, MAZ, and GR. We demonstrated that mouse ENT1 mRNA was highly expressed in heart, spleen, lung, liver, and testis. Lower levels of expression were detected in brain and kidney. Functional analysis of the 5'-flanking region showed that the nucleotide sequence from -652 to -111 contains cis-regulatory elements that promote gene expression. We found two Sp-1 binding sites (-296/-303, -307/-313) and two MAZ binding sites (-353/-359, -522/-528) in this region. Luciferase assay results suggest that MAZ and Sp-1 transcription factors are important positive regulators of transcription for the ENT1 gene in NG108-15 cells.
我们已经克隆并鉴定了位于17C染色体上的对NBMPR敏感的平衡核苷转运体（mENT1）的小鼠基因的基因组结构。对约12kb的基因组DNA进行了测序，包括启动子区域、12个外显子、11个内含子和3'非翻译区。所有外显子-内含子连接序列均符合GT/AG规则。引物延伸分析表明转录起始位点位于翻译起始位点上游252bp处。对2.5kb的5'侧翼序列分析显示了几个转录因子的推定结合位点，包括GATA-1、IRF-2、Pit-1、肌细胞生成素、CREB、Sp-1、Ap-2、MAZ和GR。我们证明小鼠ENT1 mRNA在心脏、脾脏、肺、肝脏和睾丸中高表达。在脑和肾中检测到较低水平的表达。对5'侧翼区域的功能分析表明从-652到-111的核苷酸序列包含促进基因表达的顺式调控元件。我们在该区域发现了两个Sp-1结合位点（-296/-303，-307/-313）和两个MAZ结合位点（-353/-359，-522/-528）。荧光素酶测定结果表明MAZ和Sp-1转录因子是NG108-15细胞中ENT1基因转录的重要正调控因子。
