Steroid hormone regulation of specific messenger RNA and protein synthesis in eucaryotic cells.
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 However, following a single injection of estrogen to these withdrawn animals, new ovalbumin mRNA sequences could be detected within 30 minutes.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.
O'Malkey, BW; Woo, SL; Harris, SE; Rosen, JM; Means, AR
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