The ovalbumin gene: Cloning of the natural gene

The structural ovalbumin DNA sequences are not contiguous and are separated by multiple "intervening regions" in native chicken DNA. EcoRI, a restriction endonuclease that does not cleave the structural ovalbumin DNA sequences, digests the natural ovalbumin gene into three distinct fragments of 2.4, 1.8, and 9.5 kilobase pairs in length by cleaving within these "intervening regions." The 2.4-kilobase pair fragment contains only about 450 nucleotide pairs of coding sequence, with the rest being intervening sequences. This DNA fragment was cloned in bacteria by using the certified EK2 vector lambdagtWES.lambdaB after enrichment from total EcoRI-digested chicken DNA by a combination of RPC-5 column chromatography and preparative agarose gel electrophoresis. Five out of approximately 20,000 recombinant phage plaques were capable of hybridizing with a (32)P-labeled Hha I fragment of a recombinant plasmid pOV230 containing the entire structural ovalbumin gene. DNA amplified in these recombinant phages, lambdagtWES.OV2.4, was shown to contain the same restriction endonuclease cleavage sites as in the 2.4-kilobase pair EcoRI fragment previously determined by restriction mapping of total genomic chicken DNA. The intervening sequences were allowed to hybridize with excess total chicken DNA and oviduct nuclear RNA after nick-translation. They were found to be unique chicken DNA sequences, and appeared to be transcribed in their entireties during gene expression. Like the structural gene sequences, the expression of the intervening sequences is also inducible by steroid hormones.     

Desensitization of the chick oviduct to estrogen: mediation at different levels of gene expression

We have investigated the effects of changing the dosage or kind of estrogen administered to immature chicks on the synthesis of two egg white proteins, ovalbumin and conalbumin, and the accumulation of their mRNAs. The results suggest that the oviduct can become desensitized to estrogen. Ovalbumin accounted for 25% of oviduct protein synthesis in chicks treated with low dose of diethylstilbestrol (DES; 0.5 mg/day) for 14 days. Within several days after the dosage of estrogen had been increased 10-fold, ovalbumin synthesis fell to undetectable levels. The synthesis of conalbumin also became undetectable when the dosage of estrogen was increased. Throughout the above experiment both ovalbumin and conalbumin mRNA activity, as measured by cell-free translation, remained elevated. This implies that expression of the ovalbumin and conalbumin genes may be regulated at the level of translation. In a separate experiment, ovalbumin and conalbumin synthesis also decreased when chicks primed with DES pellets were given an increased dosage of estrogen. Ovalbumin synthesis fluctuated, but overall decreased from 25% of oviduct protein synthesis after priming with DES pellets to 16% of oviduct protein synthesis after 7-12 days of injections of estradiol benzoate (1 mg/day). At the same time, conalbumin synthesis decreased from 10% to 6% of oviduct protein synthesis. In contrast with the previous results, changes in ovalbumin and conalbumin mRNA activity paralleled changes in ovalbumin and conalbumin synthesis. Thus, not only can the oviduct become desensitized to estrogen, desensitization can be mediated at different levels of gene expression.     

Rates of Induction of Specific Translatable Messenger RNAs for Ovalbumin and Avidin by Steroid Hormones

In the chick oviduct, injections of estrogen and progesterone induce synthesis of the specific proteins ovalbumin and avidin, respectively. We have studied the rate of induction of the specific messenger RNA molecules for these proteins after a single injection of estrogen or progesterone. The mRNAs were extracted, partially purified, and quantified in vitro in a heterologous protein-synthesizing system. Single injections of estrogen in chicks previously withdrawn from all steroid hormones for 2 weeks led to rapid increases in ovalbumin mRNA with 3 hr, which coincided with increases in the rate of ovalbumin synthesis. Maximal ovalbumin mRNA activity occurred by 18-20 hr. The half-life of the mRNA was estimated to be 8-10 hr and corresponded to the half-life for cessation of intracellular ovalbumin synthesis after a single injection of an estrogen. Similarly, after an injection of progesterone into chicks first treated with estrogen, appearance of avidin mRNA preceded demonstrable accumulation of this specific protein in oviduct cells. The mRNA for avidin was first apparent at 6 hr, and reached maximal concentrations between 18 and 24 hr after injection. These data confirm that both estrogen and progesterone act on oviduct to induce rapid accumulation of specific mRNAs before and coincident with the appearance of the cell-specific induced proteins. The overall results of these experiments are compatible with the hypothesis that the production of mRNA is a rate-limiting step in the steroid hormone-mediated induction of protein synthesis.     

Rapid Inactivation of Ovalbumin Messenger Ribonucleic Acid after Acute Withdrawal of Estrogen

Synthesis of ovalbumin mRNA is induced and maintained in the avian oviduct by estrogen. When estrogen is rapidly removed from circulation, there is a general involution of the oviduct and an unusually rapid decay of ovalbumin mRNA activity. The kinetics of ovalbumin mRNA decay were not first order; instead, the rate of degradation increased about 10-fold over a 20-hr period after removal of estrogen. These results are in contrast with first-order decay kinetics observed for ovalbumin mRNA in estrogen-stimulated chicks (t(1/2) = about 24 hr) and in cell-free extracts. The degradative response triggered by hormonal withdrawal becomes more rapid between 1 and 4 days of estrogen-stimulated growth. We conclude that in the process of inducing egg-white protein synthesis, estrogen produces a cellular environment in which the egg-white protein mRNAs are relatively stable; removal of estrogen initiates cellular catabolism in a manner that is not understood.     

The ovalbumin gene: structural sequences in native chicken DNA are not contiguous.

The sequence organization of the structural ovalbumin gene and flanking sequences in native chicken DNA was studied by restriction mapping and filter hybridization using a nick-translated probe generated from pOV230, a recombinant plasmid that contains a full-length ovalbumin DNA synthesized from ovalbumin mRNA. The structural sequences of the ovalbumin gene in native chicken DNA were found to be noncontiguous because at least two restriction endonucleases that do not cut the structural sequence do cleave the natural gene into multiple fragments by cleaving within nonstructural sequences interspersed between the structural sequences. The observation that all ovalbumin DNA-containing sequences were contained within a single DNA fragment generated by BamHI digestion of total chicken DNA has allowed us to construct an inclusive restriction map of the natural ovalbumin gene which contains at least two "insert regions." These regions may be further subdivided into alternating structural and insert sequences. Both insert regions were located within the peptide-coding regions of the gene and the sizes of these insert regions were estimated to be approximately 1.0 and 1.5 kilobase pairs, respectively.     

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

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.     

Chicken lysozyme gene contains several intervening sequences.

The organization of the chicken lysozyme gene and its neighboring sequences was examined by a comparison of the restriction map of the lysozyme structural gene with the map of the lysozyme gene in genomic DNA. Chicken DNA was cleaved with restriction endonucleases and the DNA fragments were separated by agarose gel electrophoresis. After transfer of the fragments onto nitrocellulose filters, those fragments that contain lysozyme mRNA sequences were detected by hybridization of the filters to labeled probes generated from pls-1, a recombinant plasmid carrying the lysozyme structural gene. This analysis revealed the presence of at least three intervening sequences, two of which interrupt the protein coding region and one of which is located in the 3' untranslated region. When oviduct DNA and sperm DNA were compared, no difference was observed in the size and number of restriction fragments that contain either lysozyme or ovalbumin structural gene sequences.     

Estrogen-Induced Changes in Translation, and Specific Messenger RNA Levels during Oviduct Differentiation

Estrogen-induced morphologic differentiation of chick oviduct is accompanied by increases in the total endogenous mRNA activity of oviduct polysomes. Concomitant increases are also noted in ribosome translational capacity and activity of peptide chain initiation factors. Once the differentiation process nears completion (about 7 days of estrogen administration), total ribosomebound mRNA activity decreases, but the translational machinery remains very active. In addition, estrogen induces the accumulation of ovalbumin mRNA before ovalbumin is demonstrable in the oviduct. The data suggest that the rate-limiting event in the hormonal induction of cell-specific proteins, such as ovalbumin, is the synthesis and intracellular accumulation of specific mRNA for such proteins.