O'Malkey B W, Woo S L, Harris S E, Rosen J M, Means A R
J Cell Physiol. 1975 Apr;85(2 Pt 2 Suppl 1):343-56. doi: 10.1002/jcp.1040850403.
Evidence is presented that the induction of specific proteins in the chick oviduct by the steroid hormones estrogen and progesterone, involves a primary effect at the level of gene transcription. The intracellular levels of mRNA's which code for the synthesis of the egg-white proteins, ovalbumin and avidin, have been quantitated in a heterologous protein synthesizing system. It is demonstrated that these levels are directly dependent upon the inducing steroid, estrogen or progesterone, respectively. Ovalbumin mRNA has been purified to apparent homogeneity. This ovalbumin mRNA was then used as a template for the synthesis of a complementary DNA copy catalyzed by the enzyme reverse transcriptase which was isolated from avian myeloblastosis virus. This radioactively labeled complementary DNA was used to demonstrate, by means of DNA excess hybridization, that the ovalbumin gene is represented only once in each haploid genome of the chick cell. Next the complementary DNA copy of the ovalbumin mRNA was used as a genetic probe to determine the precise number of sequences of ovalbumin mRNA present at any one time after the administration of estrogen. It was demonstrated that the unstimulated chick contained no sequences of ovalbumin mRNA. Within a very short period of time after estrogen is administered the ovalbumin sequences begin to appear and reach a steady state level of 140,000 molecules per tubular gland cell. It could also be calculated that each ovalbumin molecule is probably translated some 50,000 times during its life which explains why ovalbumin comprises some 60% of the total protein in the oviduct cell. Following withdrawal of the oviduct from estrogen treatment, ovalbumin mRNA sequences again drop to undetectable levels. However, following a single injection of estrogen to these withdrawn animals, new ovalbumin mRNA sequences could be detected within 30 minutes. These data suggest that estrogen controls the activity of the ovalbumin gene via a pure transcriptional control mechanism. It is also demonstrated that the efficiency of the complementary DNA as a means of quantitating specific mRNA sequences is some 1,000 times more sensitive than the best available in vitro translation system. Finally, the efficacy of four popular translation systems is compared. It is suggested that for initial studies involving hormonal control of mRNA levels, the translation system derived from wheat germ is the simplest and most sensitive.
有证据表明，类固醇激素雌激素和孕酮在鸡输卵管中诱导特定蛋白质的产生，涉及基因转录水平的初级效应。在异源蛋白质合成系统中，对编码蛋清蛋白、卵清蛋白和抗生物素蛋白合成的mRNA的细胞内水平进行了定量。结果表明，这些水平分别直接依赖于诱导类固醇激素雌激素或孕酮。卵清蛋白mRNA已被纯化至表观均一性。然后，将这种卵清蛋白mRNA用作模板，用于合成由从禽成髓细胞瘤病毒中分离的逆转录酶催化的互补DNA拷贝。这种放射性标记的互补DNA通过DNA过量杂交用于证明，卵清蛋白基因在鸡细胞的每个单倍体基因组中仅出现一次。接下来，将卵清蛋白mRNA的互补DNA拷贝用作遗传探针，以确定在给予雌激素后任何时间存在的卵清蛋白mRNA序列的精确数量。结果表明，未受刺激的鸡不含卵清蛋白mRNA序列。在给予雌激素后的很短时间内，卵清蛋白序列开始出现，并达到每个管状腺细胞140,000个分子的稳定水平。还可以计算出，每个卵清蛋白分子在其生命周期中可能被翻译约50,000次，这解释了为什么卵清蛋白占输卵管细胞总蛋白的约60%。在停止对输卵管进行雌激素处理后，卵清蛋白mRNA序列再次降至无法检测的水平。然而，在对这些停止处理的动物单次注射雌激素后，30分钟内即可检测到新的卵清蛋白mRNA序列。这些数据表明，雌激素通过纯转录控制机制控制卵清蛋白基因的活性。还证明，互补DNA作为定量特定mRNA序列的手段，其效率比现有的最佳体外翻译系统敏感约1000倍。最后，比较了四种常用翻译系统的功效。建议对于涉及mRNA水平激素控制的初步研究，从小麦胚芽衍生的翻译系统是最简单和最敏感的。
