Passadore M, Feriotto G, Bianchi N, Aguiari G, Mischiati C, Piva R, Gambari R
Department of Biochemistry and Molecular Biology, Ferrara University, Italy.
J Biochem Biophys Methods. 1994 Dec;29(3-4):307-19. doi: 10.1016/0165-022x(94)90041-8.
Sequence-selectivity of DNA-binding drugs was recently reported in a number of studies employing footprinting and gel retardation approaches. In this paper we performed polymerase-chain reaction (PCR) experiments to study the in vitro effects of distamycin, daunomycin, chromomycin and mithramycin. As model systems we employed the human estrogen receptor (ER) gene and the Harvey-ras (Ha-ras) oncogene, in order to obtain PCR products significantly differing for the A + T/G + C frequency ratio. Distamycin, daunomycin, chromomycin and mithramycin are indeed known to differentially bind to different DNA regions depending upon the DNA sequences recognized. The main conclusion of our experiments is that distamycin, daunomycin, chromomycin and mithramycin inhibit polymerase-chain reaction in a sequence-dependent manner. Distamycin inhibits indeed PCR mediated amplification of AT-rich regions of the human estrogen receptor gene, displaying no inhibitory effects on PCR-mediated amplification of GC-rich sequences of Ha-ras oncogene. By contrast daunomycin, chromomycin and mithramycin were found to inhibit PCR-mediated amplification of the Ha-ras GC-rich oncogene sequences. We propose that polymerase-chain reaction technique could be applied to study the in vivo interactions of DNA-binding drugs to specific genes in intact cells.
最近，在一些采用足迹法和凝胶阻滞法的研究中报道了DNA结合药物的序列选择性。在本文中，我们进行了聚合酶链反应（PCR）实验，以研究放线菌素、柔红霉素、色霉素和光神霉素的体外作用。作为模型系统，我们采用了人类雌激素受体（ER）基因和哈维 - 鼠肉瘤病毒（Ha-ras）癌基因，以便获得A + T/G + C频率比有显著差异的PCR产物。放线菌素、柔红霉素、色霉素和光神霉素确实已知会根据所识别的DNA序列与不同的DNA区域进行不同的结合。我们实验的主要结论是，放线菌素、柔红霉素、色霉素和光神霉素以序列依赖的方式抑制聚合酶链反应。放线菌素确实抑制了人类雌激素受体基因富含AT区域的PCR介导扩增，对Ha-ras癌基因富含GC序列的PCR介导扩增没有抑制作用。相比之下，发现柔红霉素、色霉素和光神霉素抑制Ha-ras富含GC的癌基因序列的PCR介导扩增。我们建议聚合酶链反应技术可用于研究DNA结合药物在完整细胞中与特定基因的体内相互作用。
