Mullinix K P, Meyers M B, Christmann J L, Deeley R G, Gordon J I, Goldberger R F
J Biol Chem. 1979 Oct 10;254(19):9860-6.
We have developed a system for the in vitro transcription of specific genes in rooster liver chromatin by endogenous RNA polymerase II that maintains the specificity of transcription in vivo. Radioactive transcripts synthesized in vitro were identified and quantitated by hybridization to a vast excess of cloned cDNA. The cDNA preparations employed corresponded to vitellogenin mRNA, the synthesis of which is responsive to estrogen stimulation in vivo, and chicken serum albumin mRNA, the synthesis of which is not significantly affected by estrogen stimulation in vivo. Comparing the pattern of transcription of the albumin and vitellogenin genes in chromatin from the liver of the normal rooster with the pattern in chromatin from the liver of the estrogen-stimulated rooster, we found that prior estrogen treatment of the rooster is attended by a slight decrease in the differential rate of transcription of the albumin gene and approximately a 10-fold increase in the differential rate of transcription of the vitellogenin gene. Because this pattern of transcription reflects the estrogen-induced changes in transcription observed in vivo, chromatin preparations from the livers of normal and estrogen-stimulated roosters can be used to investigate regulation of specific gene transcription at the molecular level in vitro.
我们开发了一种系统,可通过内源RNA聚合酶II在公鸡肝脏染色质中对特定基因进行体外转录,该系统能维持体内转录的特异性。体外合成的放射性转录本通过与大量克隆的cDNA杂交进行鉴定和定量。所使用的cDNA制剂分别对应于卵黄生成素mRNA(其合成在体内对雌激素刺激有反应)和鸡血清白蛋白mRNA(其合成在体内不受雌激素刺激的显著影响)。比较正常公鸡肝脏染色质中白蛋白基因和卵黄生成素基因的转录模式与雌激素刺激的公鸡肝脏染色质中的转录模式,我们发现,对公鸡进行雌激素预处理后,白蛋白基因的差异转录率略有下降,而卵黄生成素基因的差异转录率则增加了约10倍。由于这种转录模式反映了体内观察到的雌激素诱导的转录变化,因此来自正常和雌激素刺激的公鸡肝脏的染色质制剂可用于在体外分子水平研究特定基因转录的调控。