Cis‐ and trans‐acting elements responsible for the cell‐specific expression of the human alpha 1‐antitrypsin gene.
Cis‐ and trans‐acting elements responsible for the cell‐specific expression of the human alpha 1‐antitrypsin gene.
复制标题
顺式和反式作用元件负责人α1-抗胰蛋白酶基因的细胞特异性表达。
作者：
V. Simone;Gennaro Cilibertol;E. Hardon;Giacomo Paonessa;Franco Palla;Lennart Lundberg;Riccardo Cortese
人类α - 1 -抗胰蛋白酶（α - 1 - AT）基因的5 '侧翼区域包含肝脏特异性表达的顺式作用信号，当与报告基因融合时，能够在肝细胞中特异性地驱动该基因的表达。在这里，我们报告了α 1‐AT调控区域的功能解剖结果。通过转染HepG2（肝细胞）和HeLa（非肝细胞）人细胞系，比较了细菌氯霉素转乙酰化酶（CAT）基因的表达，该基因融合到一组因进行性缺失或碱基对替换而缩短的α 1‐AT 5 '侧翼区域。在核苷酸- 137和- 37（来自转录起始位点）之间发现了一个最小的组织特异性元件。该DNA片段在肝癌细胞系中激活异源SV40启动子，但在HeLa细胞中不激活。该元件包含至少两个区域，称为A（‐125/‐100）和B（‐84/‐70）结构域，这两个区域都是转录所必需的。在“最小元素”的上游至少有两个其他监管域；其中最活跃的位于离帽位- 261和- 210之间。这些上游元件在肝癌细胞系和HeLa细胞中激活异源SV40早期启动子。通过对大鼠肝核提取物的分离，鉴定出两种蛋白，α 1TF‐A和α 1TF‐B，它们分别与A和B结构域特异性结合。转录失活的A和B结构域突变体不能结合这些蛋白质。
The 5′ flanking region of the human alpha 1‐antitrypsin (alpha 1‐AT) gene contains cis‐acting signals for liver‐specific expression and, when fused to a reporter gene, is able to drive the expression of this gene specifically in liver cells. Here we report the results of a functional dissection of the alpha 1‐AT regulatory region. The expression of the bacterial chloramphenicol‐transacetylase (CAT) gene, fused to a set of alpha 1‐AT 5′ flanking regions shortened by progressive deletions or mutated by base pair substitutions, has been compared by transfection in HepG2 (hepatocyte) and HeLa (non‐hepatocyte) human cell lines. A minimal tissue‐specific element has been identified between the nucleotides −137 and −37 (from the transcriptional start site). This DNA segment activates the heterologous SV40 promoter in hepatoma cell lines but not in HeLa cells. This element contains at least two regions referred to as the A (‐125/‐100) and B (‐84/‐70) domains, both essential for transcription. There are at least two other regulatory domains located upstream of the ‘minimal element’; the most active of these is located between positions −261 and −210 from the cap site. These upstream elements activate the heterologous SV40 early promoter both in hepatoma cell lines and in HeLa cells. Upon fractionation of rat liver nuclear extracts two proteins have been identified, alpha 1TF‐A and alpha 1TF‐B, which bind specifically to the A and B domains respectively. Transcriptionally inactive A and B domain mutants are not able to bind these proteins.