Jost J P, Moncharmont B, Jiricny J, Saluz H, Hertner T
Proc Natl Acad Sci U S A. 1986 Jan;83(1):43-7. doi: 10.1073/pnas.83.1.43.
The vitellogenin II gene is specifically reactivated in vitro (secondary stimulation, memory effect) in purified liver nuclei that had ceased to express the gene in vivo a month after the roosters had received a single injection of estradiol (primary stimulation). The in vitro reactivation depends on the addition to the nuclei of nuclear and cytoplasmic extracts from estradiol-stimulated livers, polyamines (0.1-1.0 mM), and calmodulin (0.1 mM). Under identical incubation conditions the vitellogenin gene could not be reactivated in oviduct, embryonic, and immature chicken liver nuclei. Two other genes, those for ovalbumin and lysozyme, which are regulated by estradiol in the oviduct, could not be activated in the liver nuclei. The correct initiation of vitellogenin gene transcription in the liver nuclei was tested by primer extension studies. Addition of the antiestrogen tamoxifen (0.1 microM) to the system decreased vitellogenin mRNA synthesis by about 45% without affecting total RNA synthesis. Addition of quercetin (0.1 mM) and trans-flupenthixol (0.2 mM), inhibitors of nuclear protein kinase II and calmodulin-dependent kinase, respectively, inhibited the synthesis of vitellogenin mRNA by about 55% without affecting total RNA synthesis. The inhibitory effects of the antiestrogen and the kinase inhibitors were not additive, suggesting that both classes of inhibitor act on the same target or related targets. Depleting the estradiol receptors from the cell and nuclear extracts by means of estradiol-receptor antibodies covalently bound to Matrex beads reduced the stimulation of the vitellogenin gene by 40%. We conclude that in addition to the estradiol receptor and phosphorylation of nuclear protein(s) there are additional factors responsible for the in vitro secondary activation of the avian vitellogenin II gene.
在公鸡单次注射雌二醇（初次刺激）一个月后，体内已停止表达卵黄生成素II基因的纯化肝细胞核，在体外（二次刺激，记忆效应）可特异性地重新激活该基因。体外重新激活取决于向细胞核中添加来自经雌二醇刺激的肝脏的核提取物和细胞质提取物、多胺（0.1 - 1.0 mM）以及钙调蛋白（0.1 mM）。在相同的孵育条件下，卵黄生成素基因在输卵管、胚胎和未成熟鸡肝细胞核中无法重新激活。另外两个受雌二醇调控的基因，即卵清蛋白和溶菌酶的基因，在肝细胞核中也无法被激活。通过引物延伸研究检测了肝细胞核中卵黄生成素基因转录的正确起始。向系统中添加抗雌激素他莫昔芬（0.1 microM）可使卵黄生成素mRNA合成减少约45%，而不影响总RNA合成。分别添加核蛋白激酶II抑制剂槲皮素（0.1 mM）和钙调蛋白依赖性激酶抑制剂反式氟奋乃静（0.2 mM）可使卵黄生成素mRNA合成减少约55%，而不影响总RNA合成。抗雌激素和激酶抑制剂的抑制作用并非相加的，这表明这两类抑制剂作用于同一靶点或相关靶点。通过与Matrex珠共价结合的雌二醇受体抗体从细胞和核提取物中耗尽雌二醇受体，可使卵黄生成素基因的刺激减少40%。我们得出结论，除了雌二醇受体和核蛋白的磷酸化外，还有其他因素负责禽类卵黄生成素II基因的体外二次激活。
