Rusconi S, Severne Y, Georgiev O, Galli I, Wieland S
Institut für Molekularbiologie II, University of Zürich, Switzerland.
Gene. 1990 May 14;89(2):211-21. doi: 10.1016/0378-1119(90)90008-f.
A novel assay to study transcriptional regulation in vivo designated trans-activation-dependent replication (TDR) assay is based on the modulation of a simian virus 40 (SV40)-derived replication system. A mixture of four plasmids (pPARA + pCIS + pTRANS + pREF) is co-transfected into vertebrate cells. After appropriate incubation, the replication of the pPARA plasmid (containing an SV40 origin of replication) is measured with a simple enzymatic test. We demonstrate that the level of replication is dependent on the differential trans-activation of the reporter pCIS (in which SV40 T-antigen is brought under control of the desired promoter) by the specific regulator protein encoded by the pTRANS plasmid. Three advantages make this assay a convenient tool for the systematic analysis of trans-activation in vivo: (1) remarkable sensitivity (higher than conventional assays); (2) rapid sample processing combined with a built-in standard (pREF-plasmid); (3) avoidance of expensive reagents such as freshly radiolabelled probes. We present the application of the TDR assay to the analysis of deletion mutants of the glucocorticoid receptor (GR) and other, GR-based chimeric trans-activators. The results demonstrate that the properties of protein domains are not always additive in a particular chimaera. Further application possibilities of the TDR assay are also discussed.
一种用于研究体内转录调控的新型检测方法——反式激活依赖复制（TDR）检测法，是基于对猿猴病毒40（SV40）衍生的复制系统的调控。将四种质粒（pPARA + pCIS + pTRANS + pREF）的混合物共转染到脊椎动物细胞中。经过适当孵育后，通过简单的酶促检测来测量pPARA质粒（含有SV40复制起点）的复制情况。我们证明，复制水平取决于由pTRANS质粒编码的特定调节蛋白对报告质粒pCIS（其中SV40 T抗原受所需启动子控制）的差异反式激活。该检测方法具有三个优点，使其成为体内反式激活系统分析的便捷工具：（1）灵敏度高（高于传统检测方法）；（2）样品处理快速，并带有内置标准（pREF质粒）；（3）避免使用昂贵的试剂，如新鲜的放射性标记探针。我们展示了TDR检测法在糖皮质激素受体（GR）缺失突变体及其他基于GR的嵌合反式激活因子分析中的应用。结果表明，在特定嵌合体中，蛋白质结构域的特性并非总是具有加和性。还讨论了TDR检测法的其他应用可能性。
