Harris S E, Rosen J M, Means A R, O'Malley B W
Biochemistry. 1975 May 20;14(10):2072-81. doi: 10.1021/bi00681a006.
DNA complementary to purified ovalbumin messenger RNA (cDNA ov) was synthesized in vitro using RNA-directed DNA olymerase from avian myeloblastosis virus. This cDNAov was then employed in hybridization assays to determine the effect of estrogen on the number of ovalbumin mRNA (MRNAov) molecules per tubular gland cell of the chick oviduct. The changes in mRNAov were measured in immature chicks during primary stimulation, after hormone withdrawal and again following secondary stimulation of the chick oviduct with estrogen. The number of mRNAov per tubular gland cell was also determined for egg-laving hen. Daily estrogen administration to the immature chick resulted in growth of the oviduct, differentiation of epithelial cells to tubular glands, and a corresponding increase in the concentration of mRNAov in the tubular gland cell from essentially zero before estrogen administration to 48,000 molecules per cell after 18 days of estrogen treatment. Upon withdrawal of estrogen from the chick, the mRNAov concentration decreased to a level of 0-10 molecules/tubular gland cell after 12 days. Readministration of a single dose of estrogen to these chicks resulted in a dramatic and rapid increase in the concentration of mRNAov. Within 30 min, the mRNAov concentration approximately doubled and by 29 hr the tubular gland cell concentration had reached 17,000 molecules. The initial transcription rate for the ovalbumin gene was 12 mRNAov molecules/min. With these data, we have calculated that the half-life of the ovalbumin messinger RNA should be on the order of 40-60 hr and that the steady-state concentration of mRNAov per tubular gland cell was 50,000 molecules. Similarly, each messenger RNA molecule was translated approximately 50,000 times during its lifetime in order to effect the necessary quantity of ovalbumin required for egg production. These data substantiate the hypothisis that estrogen exerts its primary action at the level of transcription to effect the synthesis of nascene mRNA molecules which in turn code for synthesis of hormone-induced proteins.
利用来自禽成髓细胞瘤病毒的RNA定向DNA聚合酶，在体外合成了与纯化的卵清蛋白信使RNA（cDNA ov）互补的DNA。然后将该cDNA ov用于杂交试验，以确定雌激素对雏鸡输卵管每个管状腺细胞中卵清蛋白mRNA（mRNA ov）分子数量的影响。在雏鸡输卵管受到初次刺激、激素撤除后以及再次用雌激素进行二次刺激的过程中，对未成熟雏鸡体内mRNA ov的变化进行了测量。还测定了产蛋母鸡每个管状腺细胞中mRNA ov的数量。每天给未成熟雏鸡施用雌激素，会导致输卵管生长、上皮细胞分化为管状腺，并且管状腺细胞中mRNA ov的浓度相应增加，从施用雌激素前的基本零水平增加到雌激素处理18天后的每个细胞48,000个分子。当雏鸡停止使用雌激素后，12天后mRNA ov浓度降至每个管状腺细胞0 - 10个分子的水平。对这些雏鸡再次施用单剂量的雌激素，会导致mRNA ov浓度急剧快速增加。在30分钟内，mRNA ov浓度大约翻倍，到29小时时，管状腺细胞浓度已达到17,000个分子。卵清蛋白基因的初始转录速率为每分钟12个mRNA ov分子。根据这些数据，我们计算出卵清蛋白信使RNA的半衰期应该在40 - 60小时左右，并且每个管状腺细胞中mRNA ov的稳态浓度为50,000个分子。同样，每个信使RNA分子在其生命周期内大约被翻译50,000次，以产生产蛋所需的足够数量的卵清蛋白。这些数据证实了这样的假说，即雌激素在转录水平发挥其主要作用，以影响新生mRNA分子的合成，而这些mRNA分子进而编码激素诱导蛋白的合成。
