Harris S E, Schwartz R J, Tsai M J, O'Malley B W, Roy A K
J Biol Chem. 1976 Jan 25;251(2):524-9.
RNA was transcribed from chromatin isolated from chick oviduct and spleen by using RNA polymerase from Escherichia coli. RNA was also transcribed from whole chick DNA using E. coli RNA polymerase. DNA complementary to ovalbumin messenger RNA (cDNAov) was then used as a hybridization probe to estimate the concentration of ovalbumin messenger RNA sequences (mRNAov) in these in vitro transcripts. Although chromatin from unstimulated chick oviduct was capable of substantial RNA synthesis, no detectable mRNAov sequences could be found in the transcript. Likewise, mRNAov sequences could not be found in RNA synthesized from spleen chromatin using E. coli RNA polymerase. However, chromatin from estrogen-stimulated chick oviducts was capable of supporting synthesis of ovalbumin mRNA. We estimate that approximately 0.01% of the RNA synthesized from estrogen-stimulated chromatin was mRNAov sequences. When RNA synthesized from chick DNA was tested with the cDNAov probe, mRNAov sequences could be detected in a concentration of approximately 10% that found in the RNA transcript from estrogen-stimulated chromatin. This was as expected if the ovalbumin gene is considered to be in the "open or derepressed" region of the estrogen-stimulated oviduct chromatin. Chromatin isolated from chicks withdrawn from hormone for 12 days was only capable of supporting mRNAov synthesis in vitro at a level of 5 to 10% of that observed in chromatin prepared from estrogen-stimulated chicks, thus indicating the requirement for estrogen to maintain the ovalbumin gene in the available or "open" state in the majority of oviduct cells. These data militate against post-transcriptional control as the primary mechanism of steroid hormone regulation of specific mRNA synthesis in the chick oviduct system, and favor primary gene derepression as the most likely mechanism for estrogen induction of ovalbumin synthesis.
使用来自大肠杆菌的RNA聚合酶，从鸡输卵管和脾脏分离的染色质中转录RNA。也使用大肠杆菌RNA聚合酶从完整的鸡DNA中转录RNA。然后将与卵清蛋白信使RNA互补的DNA（cDNAov）用作杂交探针，以估计这些体外转录物中卵清蛋白信使RNA序列（mRNAov）的浓度。尽管来自未受刺激的鸡输卵管的染色质能够进行大量的RNA合成，但在转录物中未发现可检测到的mRNAov序列。同样，使用大肠杆菌RNA聚合酶从脾脏染色质合成的RNA中也未发现mRNAov序列。然而，来自雌激素刺激的鸡输卵管的染色质能够支持卵清蛋白mRNA的合成。我们估计，从雌激素刺激的染色质合成的RNA中约0.01%是mRNAov序列。当用cDNAov探针检测从鸡DNA合成的RNA时，可检测到mRNAov序列，其浓度约为雌激素刺激的染色质RNA转录物中发现浓度的10%。如果认为卵清蛋白基因位于雌激素刺激的输卵管染色质的“开放或去抑制”区域，这是预期的结果。从停止激素处理12天的鸡中分离的染色质，在体外仅能支持mRNAov合成，其水平为从雌激素刺激的鸡制备的染色质中观察到水平的5%至10%，因此表明需要雌激素来维持大多数输卵管细胞中卵清蛋白基因处于可用或“开放”状态。这些数据不利于转录后控制作为鸡输卵管系统中类固醇激素调节特定mRNA合成的主要机制，而支持基因去抑制作为雌激素诱导卵清蛋白合成最可能的机制。
