Magalini A, Ferrari F, Savoldi G, Ingrassia R, Albertini A, Pollio G, Patrone C, Maggi A, Di Lorenzo D
Institute of Chemistry, School of Medicine, University of Brescia, Italy.
DNA Cell Biol. 1995 Aug;14(8):665-71. doi: 10.1089/dna.1995.14.665.
In this work, we have studied the activity of a tetracycline modulatable trans-activator (tTA) generated by fusing the DNA binding domain of the tetracycline repressor to the trans-activation domain of the Herpes simplex virus protein 16 (HSV VP16) (plasmid pUHD15-1Neo). In the three different cell lines studied (HTC, rat hepatoma; T47D, human breast cancer; SK-N-BE, human neuroblastoma), the expression of the luciferase gene under the control of a tetracycline operator sequence (plasmid pUHC13-3) was used as a control of the incorporation and the functionality of the trans-activator. Clones selected from these cells responded in a time and dose-dependent manner to the withdrawal of tetracycline. In all these clones, the tTA trans-activator not only modulates the activity of the luciferase gene, but also modulates the activity of a number of endogenous proteins, including C/EBP beta, the glucocorticoid receptor (GR), and SP1. In the transfected cells, the level of these transcription factors was strongly inhibited in the presence of tetracycline and was highly increased after tetracycline removal. Electrophoresis mobility shift assay (EMSA) and footprint experiments proved that the induced proteins are perfectly efficient in binding the DNA. Their transcriptional activity was also determined. In HTC/A9 cells, the level of the chloramphenicol acetyltransferase (CAT) expression driven by the promoter of the alpha 1-glycoprotein (AGP) gene was strongly enhanced at 72-84 hr following removal of tetracycline from the growth media. The accumulation of the endogenous AGP mRNA also increased at 84 hr. In the T47D/TA11 and SK-N-BE/C2.6 cells, a general activation of protein synthesis was also evidenced.
在本研究中，我们研究了一种四环素可调节反式激活因子（tTA）的活性，该因子通过将四环素阻遏物的DNA结合结构域与单纯疱疹病毒蛋白16（HSV VP16）的反式激活结构域融合而产生（质粒pUHD15-1Neo）。在所研究的三种不同细胞系（HTC，大鼠肝癌细胞；T47D，人乳腺癌细胞；SK-N-BE，人神经母细胞瘤细胞）中，将四环素操纵序列控制下的荧光素酶基因表达（质粒pUHC13-3）用作反式激活因子掺入和功能的对照。从这些细胞中选出的克隆对四环素撤除呈现出时间和剂量依赖性反应。在所有这些克隆中，tTA反式激活因子不仅调节荧光素酶基因的活性，还调节许多内源性蛋白质的活性，包括C/EBPβ、糖皮质激素受体（GR）和SP1。在转染细胞中，这些转录因子的水平在四环素存在时受到强烈抑制，在四环素去除后则大幅增加。电泳迁移率变动分析（EMSA）和足迹实验证明，诱导产生的蛋白质能完美有效地结合DNA。还测定了它们的转录活性。在HTC/A9细胞中，从生长培养基中去除四环素后72 - 84小时，由α1-糖蛋白（AGP）基因启动子驱动的氯霉素乙酰转移酶（CAT）表达水平显著增强。内源性AGP mRNA在84小时时的积累也增加了。在T47D/TA11和SK-N-BE/C2.6细胞中，也证明了蛋白质合成的普遍激活。
