Brack C, Mattaj I W, Gautschi J, Cammisuli S
Exp Cell Res. 1984 Apr;151(2):314-21. doi: 10.1016/0014-4827(84)90382-3.
In order to investigate the molecular mechanism of biological action of the drug cyclosporin A (CsA), we have studied its effect on eukaryotic RNA polymerase activities in two systems: (a) in vitro transcription; (b) injection into frog oocytes. The electron microscopic transcription R-loop method [1] has been used to assay for specific transcription of an immunoglobulin gene clone in HeLa cell lysates. In this system, CsA inhibits efficiently the transcription of immunoglobulin genes. Dilution experiments show that RNA polymerase II activity is inhibited by nanomolar concentrations (i.e. amounts comparable to the ones needed for the inhibition of lymphocyte stimulation), whereas polymerase III is inhibited at micromolar concentrations. In vitro transcription with E. coli RNA polymerase is not inhibited. Parallel experiments with the mushroom toxin alpha-amanitin have shown that the eukaryotic RNA polymerase II displays similar sensitivity towards the two cyclic peptides CsA and alpha-amanitin in vitro. When CsA is injected into frog oocytes, together with a cloned Xenopus laevis U1 small nuclear RNA gene, transcription of this gene by endogenous RNA polymerase II is inhibited by 60%. RNA polymerase III and RNA polymerase I transcription is not inhibited in the oocyte. Various possibilities for the selective action of CsA on stimulated lymphocytes are discussed.
为了研究环孢菌素A（CsA）药物生物作用的分子机制，我们在两个系统中研究了其对真核RNA聚合酶活性的影响：（a）体外转录；（b）注射到蛙卵母细胞中。电子显微镜转录R环法[1]已用于检测HeLa细胞裂解物中免疫球蛋白基因克隆的特异性转录。在该系统中，CsA有效抑制免疫球蛋白基因的转录。稀释实验表明，RNA聚合酶II的活性受到纳摩尔浓度（即与抑制淋巴细胞刺激所需的浓度相当）的抑制，而聚合酶III则受到微摩尔浓度的抑制。用大肠杆菌RNA聚合酶进行的体外转录不受抑制。与蘑菇毒素α-鹅膏菌素的平行实验表明，真核RNA聚合酶II在体外对两种环肽CsA和α-鹅膏菌素表现出相似的敏感性。当将CsA与克隆的非洲爪蟾U1小核RNA基因一起注射到蛙卵母细胞中时，内源性RNA聚合酶II对该基因的转录受到60%的抑制。卵母细胞中的RNA聚合酶III和RNA聚合酶I转录不受抑制。讨论了CsA对受刺激淋巴细胞选择性作用的各种可能性。
