Ovalbumin Messenger RNA of Chick Oviduct: Partial Characterization, Estrogen Dependence, and Translation In Vitro

Abstract

A rapidly-labeled RNA fraction can be isolated from hen oviduct polysomes that has characteristics of the messenger RNA (mRNA) for the cell-specific protein, ovalbumin. This RNA, which sediments in the 8-17S region of sucrose gradients, possesses properties suggestive of the presence of a polyadenylic acid sequence and can be translated with fidelity in a cell-free protein synthesizing system derived from rabbit reticulocytes. The identity of the protein product as ovalbumin is confirmed by three methods, and translation of ovalbumin mRNA is shown to be dependent both on amount of exogenous mRNA and incubation time. Both rate and extent of ovalbumin synthesis is enhanced by the addition of a protein extract from ribosomes that contains peptide chain initiation factors. Finally, the presence of this specific mRNA is shown to be estrogen-dependent: it is induced by estrogen administration to immature chicks, disappears upon cessation of estrogen treatment, and can be reinduced by a single injection of estrogen to chicks that have been pretreated with estrogen and then withdrawn from the hormone.The cell-specific synthesis of ovalbumin serves as a characteristic biochemical marker for the estrogen-dependent differentiation of the chick oviduct (1-3). Studies to date have shown that estrogen induces changes in the pattern of synthesis of nuclear RNA before appearance of ovalbumin in the tissue (3-5). These observations are consistent with a hormone-regulated expression of the ovalbumin gene. However, the only direct assessment of messenger RNA (mRNA) is the ability of an RNA fraction to support the de novo synthesis of a specific protein in an in vitro translation system. Recently, rabbit reticulocyte lysate has been used successfully for this purpose by several laboratories, including our own (6-8). We now report data demonstrating that an 8-17S fraction of RNA isolated from oviduct polysomes contains the mRNA for ovalbumin that can be translated with fidelity in the reticulocyte system. Synthesis of ovalbumin in vitro shows a linear dependence on the amount of exogenous mRNA and is stimulated by addition of translation factors required for initiation of protein synthesis. Finally, the presence of the ovalbumin mRNA will be demonstrated to be directly dependent upon the degree of estrogen stimulation of the oviduct. MATERIALS AND METHODSAnimals. Immature chicks used in this study were Rhode Island Reds and were 7-days old at the beginning of the experiments. Young laying hens (5-6 months) were either Rhode Island Red or White Leghorns. For hormone experiments, immature chicks received daily subcutaneous injections of diethylstilbestrol (5 mg) in sesame oil. Precise injection protocols are described in the text.Isolation of Polysomes. Polyribosomes were isolated from chick oviduct as described. Fractionation of isolated polysomes on sucrose gradient has also been described (9, 10). For experiments with rapidly labeled polysomal RNA, oviduct was incubated in Eagle's minimal medium (1 ml/g) that contained [3H]...

Synthesis of [ 3 H]DNA Complementary to Ovalbumin Messenger RNA: Evidence for Limited Copies of the Ovalbumin Gene in Chick Oviduct

“…Total RNA was extracted from mature hen oviduct, and the ovalbumin mRNA fraction was partially purified by adsorption of poly-(adenylic acid)-rich RNA to Millipore filters (11,12). The RNA was eluted from the Millipore filters (11) and then fractionated on 0.3-1.0 M sucrose gradients containing 0.1 M NaCl, 1 mM EDTA, and 0.01 M sodium acetate (pH 5.0).…”

The Complete Amino‐Acid Sequence of Hen Ovalbumin

“…The synthesis of ovalbumin by oviduct tissue ha5 proved to be a suitable system for studying factors affecting the rate of protein synthesis [20], including the influence of steroid hormones [22]. Interest in this system has led to the isolation [22,23] and sequence determination of ovalbumin mRNA [24].…”

“…Interest in this system has led to the isolation [22,23] and sequence determination of ovalbumin mRNA [24]. The isolation of the mRNA has in turn led to the description of 'split genes' in hen DNA [25].…”

Translation of ovalbumin mRNA in Xenopus laevis oocytes. Characterization of the system and effects of estrogen on injected mRNA populations.

“…The induction of these proteins is associated with an accumulation of their respective mRNAs (3)(4)(5). In the case of ovalbumin, detailed kinetic studies led to the hypothesis that the estrogenic induction of ovalbumin mRNA (mRNAov)' was Dr. Chan is an Established Investigator of the American Heart Association.…”