Characterization of a novel liver-specific enhancer in the human prothrombin gene.
Characterization of a novel liver-specific enhancer in the human prothrombin gene.
复制标题
人类凝血酶原基因中新型肝脏特异性增强子的表征。
DOI：
10.1016/s0021-9258(18)55152-8
复制
发表时间：
1991
期刊：
影响因子：
--
通讯作者：
R. Macgillivray
中科院分区：
文献类型：
--
作者：
B. Chow;V. Ting;F. Tufaro;R. Macgillivray
通过用人凝血酶原cDNA作为杂交探针筛选人肝噬菌体文库来分离人凝血酶原基因的5'-侧翼序列。鉴定出噬菌体在起始甲硫氨酸密码子上游含有 3 KB 对 DNA。引物延伸研究表明主要转录起始位点位于起始密码子上游 23 和 36 个碱基对处。然后使用人生长激素基因作为报告基因，通过瞬时表达系统分析人凝血酶原基因5'侧翼区域的DNA序列的顺式激活转录活性。将嵌合表达载体导入HepG2细胞，并使用放射免疫测定法监测分泌的人生长激素。这些研究表明，3 公斤碱基对片段包含足以在 HepG2 细胞中启动转录的序列。随后的删除研究表明，这个3-kilobase对片段包含两个元件：位于mRNA编码序列上游区域的弱启动子和位于核苷酸-860和-940之间的增强子。增强剂元件在一定距离处并且在任一方向上都是活跃的。此外，增强子是肝细胞特异性的，作用于异源启动子，包括单纯疱疹病毒胸苷激酶启动子和小鼠金属硫蛋白I启动子。增强子的核苷酸序列与DNA序列数据库的比较表明增强子序列是独特的。增强子序列两侧是反向重复序列 5' CCTCCC 3'，并包含肝核因子 1 的推定结合位点。脱氧核糖核酸酶 I 足迹分析和接头扫描诱变表明，增强子包含多个蛋白质结合基序。 3' 边界 CCTCCC 序列的诱变消除了增强子活性。与其他肝脏基因的比较显示，乙型肝炎病毒增强子、α1-抗胰蛋白酶启动子和纤维蛋白原β链启动子中存在CCTCCC序列，表明该基序的功能作用。
The 5'-flanking sequence of the human prothrombin gene was isolated by screening a human liver phage library with a human prothrombin cDNA as a hybridization probe. A phage was identified that contained 3 kilobase pairs of DNA upstream of the initiator methionine codon. Primer extension studies showed that the major transcription initiation sites were located 23 and 36 base pairs upstream of the initiator codon. DNA sequences in the 5'-flanking region of the human prothrombin gene were then analyzed for cis-activating transcriptional activity by a transient expression system using the human growth hormone gene as the reporter gene. The chimeric expression vector was introduced into HepG2 cells, and secreted human growth hormone was monitored by using a radio-immunoassay. These studies showed that the 3-kilo-base pair fragment contained sequences that were sufficient for the initiation of transcription in HepG2 cells. Subsequent deletion studies showed that the 3-kilobase pair fragment contained two elements: a weak promoter in the region immediately upstream of the mRNA coding sequence and an enhancer located between nucleotides -860 and -940. The enhancer element was active at a distance and in either orientation. In addition, the enhancer was liver cell-specific and acted on heterologous promoters including the herpes simplex virus thymidine kinase promoter and the mouse metallothionein I promoter. Comparison of the nucleotide sequence of the enhancer with a DNA sequence data base showed the enhancer sequence to be unique. The enhancer sequence is flanked by an inverted repeat 5' CCTCCC 3' and contains a putative binding site for hepatic nuclear factor 1. Deoxyribonuclease I footprint analysis and linker scanning mutagenesis showed that the enhancer contains multiple protein binding motifs. Mutagenesis of the 3' boundary CCTCCC sequence eliminated the enhancer activity. Comparison with other liver genes showed the presence of the CCTCCC sequence in the hepatitis B virus enhancer, the alpha 1-antitrypsin promoter, and the fibrinogen beta-chain promoter, suggesting a functional role for this motif.