Ovalbumin mRNA Sequences Research Articles

Estrogen receptors as mediators of gene transcription

Three high affinity estrogen binding proteins which bind estradiol with equilibrium dissociation constants ( K d ) of 0.1 nM, 4nM and 30 nM have been quantitated in chick oviduct cytosol. The two highest affinity binding proteins with K d s of 0.1 nM and 4 nM have the characteristics of extrogen receptors. The concentration of these receptors in isolated nuclei is highly correlated with ovalbumin mRNA sequences synthesized in vitro in these same nuclei. The two receptors have been separated using a low capacity estrogen affinity resin and the steroid binding specificities of each was determined. When the higher affinity receptor ( K d 0.1 nM) is highly purified and added to nuclei from estrogen withdrawn oviducts RNA polymerase II activity is increased 2–3-fold, but selective transcription of the ovalbumin gene is only observed when nuclei from short term estrogen withdrawn oviducts (24 hr) are utilized. The administration ofDanazol (17α-pregn-4-en-20-yno-[2,3- disoxazol-17-ol) to estrogen stimulated chicks blocks the formation of the lower affinity receptor ( K d , 4 nM). Upon stimulation with estrogen the nuclei from the Oanazol treated oviducts have the same RNA polymerase activity as control nuclei but ovalbumin mRNA synthesis is inhibited. It would appear that both receptors are required for estrogen mediation of ovalbumin gene transcription.

Isolation of a nuclear ribonucleoprotein fraction from chick oviduct containing ovalbumin messenger RNA sequences

A method was developed for the isolation of a ribonucleoprotein fraction from chick oviduct nuclei that contains 70% of the pulse-labeled RNA. These fractions also contain about 1% of the nuclear DNA and have an average RNA to DNA ratio of about 4:1. The major nuclear RNP proteins of 32,000 M r are present along with many additional proteins including histories. However, polysomal proteins and major oviduct cytoplasmic proteins are absent. Nuclei from fully stimulated chick oviduct contain about 3000 copies of ovalbumin messenger RNA sequences of which about 200 are in the RNP complexes: these complexes have sedimentation coefficients of 30 to 350 S and are resistant to disruption by EDTA. The level of ovalbumin mRNA sequences in these complexes reflects the overall rate of synthesis of this RNA. Withdrawal of estrogen leads to a parallel decline of nuclear estrogen receptors and ovalbumin mRNA sequences in the RNP complexes and a subsequent loss of cytoplasmic ovalbumin mRNA about three hours later. The 300-fold decrease in the level of ovalbumin mRNA sequences in these complexes and the eightfold decrease in stability of cytoplasmic ovalbumin mRNA account for the 2500-fold decrease in the level of cytoplasmic ovalbumin mRNA observed during withdrawal. Upon stimulation with estrogen, the kinetics of reappearance of ovalbumin mRNA sequences in the RNP complexes apparently accounts for the accumulation of cytoplasmic ovalbumin mRNA. Thus the nuclear RNP has some of the properties expected of nascent RNP complexes. The levels of ovalbumin and conalbumin mRNA sequences increase in the nuclear RNP with markedly different kinetics: conalbumin mRNA sequences reach half maximum by 1.5 hours, whereas ovalbumin mRNA sequences in these complexes reach half maximum at about eight hours. In the analysis in the accompanying Appendix, we show that the immediate increase of conalbumin mRNA sequences in the nuclear RNP may be accounted for by interaction of the hormone receptor complex with a single regulatory site, whereas the delayed increase of ovalbumin mRNA sequences in the RNP may be due to a requirement for interaction of the hormone receptor complex with multiple regulatory sites.

Effect of estrogen on gene expression in chicken oviduct: Evidence for transcriptional control of ovalbumin gene

The transcription of structural and intervening sequences of the chicken ovalbumin gene was studied in nuclei isolated from the oviduct, liver, and spleen of chickens in different states of estrogen simulation. The concentration of transcripts of structural and intervening DNA sequences was determined by hybridizing the newly synthesized [(3)H]RNA to filters containing cloned ovalbumin cDNA (pOV230) or fragments of the natural ovalbumin gene (pOV2.4 and pOV1.8). Of the RNA synthesized by oviduct nuclei from chickens chronically stimulated with diethylstilbestrol, 0.23% corresponded to ovalbumin mRNA and 0.17% were transcripts of intervening sequences. No detectable ovalbumin mRNA sequences were synthesized by nuclei from spleen and liver. After 60 hr of hormone withdrawal, synthesis of ovalbumin mRNA by oviduct nuclei could not be detected. After readministration of estrogen, a gradual increase in ovalbumin mRNA synthesis was observed which began at 1 hr and reached a plateau by 8 hr. For the intervening sequences, similar kinetics were observed for the initial 4 hr. Previously we had identified multiple species of putative precursors of ovalbumin mRNA in oviduct nuclei from chickens chronically stimulated with diethylstilbestrol. We demonstrate here that withdrawal of diethylstilbestrol resulted in a depletion of high-molecular-weight ovalbumin RNA and of mature ovalbumin mRNA and that readministration of the estrogen induced the nuclear accumulation of both forms of ovalbumin RNA. These findings indicate that: (i) a method exists to assay synthesis of hormone-inducible specific eukaryotic [(3)H]mRNA in vitro; (ii) the estrogen-mediated preferential expression of the ovalbumin gene is maintained in isolated oviduct nuclei; (iii) after hormone withdrawal, a single injection of diethylstilbestrol induces transcription of ovalbumin structural and intervening sequences, with nuclear accumulation of high-molecular-weight ovalbumin RNA and mature ovalbumin mRNA; and (iv) these results are consistent with regulation of ovalbumin mRNA at the level of ovalbumin gene transcription.

Regulation of translation of ovalbumin messenger RNA by estrogens and progesterone in oviduct of withdrawn chicks.

In the oviduct of chicks withdrawn from previous treatment with estrogens, no ovalbumin synthesis can be detected, although there are a limited number of ovalbumin mRNA sequences. These sequences are predominately associated with membrane-bound ribosomes. However, the size of the polysomes is small compared to those from the laying hen, suggesting that the inability to detect ovalbumin synthesis is the result of inefficient initiation of ovalbumin synthesis. When the rate of peptide chain elongation is reduced by treatment of chicks with cycloheximide, there is an increase in the average size of polysomes and a shift of ovalbumin mRNA sequences from small to large-sized polysomes. Readministration of estrogen to withdrawn chicks results in a time-dependent shift of monosomes to polysomes and a proportional shift of ovalbumin mRNA sequences between the two fractions, indicating that estrogen stimulates the rate of initiation of all mRNA species in the oviduct to essentially the same extent. In contrast, progesterone administration results in a preferential shift of ovalbumin mRNA relative to total RNA, suggesting a preferential effect of progesterone on initiation of protein synthesis with ovalbumin mRNA.

Preferential transcription of the ovalbumin gene in isolated hen oviduct nuclei by RNA polymerase B.

The synthesis of ovalbumin mRNA sequences was studied in isolated nuclei from hen oviduct. Two different methods of analysis were used to distinguish in vitro synthesized from preexisting mRNA sequences: (i) Mercurated ribonucleotides were used for in vitro RNA synthesis, and the newly synthesized RNA was purified by chromatography on sulfhydryl-agarose and hybridized to radioactive ovalbumin cDNA. (ii) [3H]UTP was used to label the in vitro synthesized RNA. Hybridization to unlabeled mercurated cDNA, RNase A digestion, and subsequent purification of the hybrids on SH-agarose allowed the quantitation of newly synthesized ovalbumin mRNA sequences. Approximately 0.1% of the newly synthesized RNA was identified as ovalbumin RNA by both methods. The synthesis of ovalbumin RNA progressed during the incubation of nuclei and was sensitive to actinomycin D and low concentrations of alpha-amanitin. The preferential in vitro transcription of the ovalbumin gene (1000-fold over random transcription of the chicken genome) by RNA polymerase B (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) suggests that the specificity of in vitro RNA synthesis is retained in isolated nuclei.

The ovalbumin split gene: molecular cloning of Eco RI fragments “c” and “d”

The Eco RI fragments "c" and "d" of the ovalbumin gene (1, 2) have been isolated by molecular cloning. Restriction enzyme mapping and electron microscopy have confirmed that the two fragments contain the same ovalbumin mRNA coding sequences. These sequences are split into two regions which have been mapped in fragments "c" and "d". There is no evidence that the ovalbumin mRNA sequences contained in these fragments could be further interrupted. Our results confirm that the presence of Eco RI fragment "d" in some chickens is due to the existence of an allelic variant of the ovalbumin gene which contains an additional Eco RI site within the region corresponding to Eco RI fragment "c". This additional Eco RI site appears to be the main difference between the two alleles. Finally, our results provide a direct demonstration that most of the ovalbumin mRNA sequences are encoded for by Eco RI fragments "a", "b" and "c".

Effect of estrogen on gene expression in the chick oviduct.

Mercurated UTP was used as a substrate for RNA polymerases in the in vitro transcription of chromatin so that newly synthesized RNA could be efficiently separated from endogenous chromatin RNA by means of sulfhydryl-Sepharose affinity chromatography. Utilizing this technique, it was possible to examine the effect of varying enzyme to DNA ratios on the transcription of specific genes from chromatin. For both Escherichia coli RNA polymerase and wheat germ RNA polymerase II, lowering the enzyme to DNA ration resulted in an increase in the percentage of ovalbumin mRNA sequences transcribed from chick oviduct chromatin. Similar results were also obtained for the transcription of the globin gene from chick reticulocyte chromatin. On the other hand, transcription of the globin gene from oviduct chromatin or the ovalbumin gene from reticulocyte chromatin or deproteinized chick DNA was not significantly affected by varying enzyme to DNA ratios. These results indicate that preferential transcription of certain chromatin genes relative to total RNA synthesis can occur and that this process is dependent on the presence of chromosomal proteins. Utilizing a cDNA probe complementary to the anticoding strand of the ovalbumin gene, the degree of asymmetry of the in vitro transcription of this gene was also examined. The percentage of ovalbumin RNA sequences homologous to the anticoding strand was not significantly affected when the enzyme to DNA ratio was lowered 16-fold. Since the percentage of coding ovalbumin mRNA sequences increased more than 6-fold over the same range, the percentage of asymmetric transcription of this gene increased. At the lowest enzyme to DNA ratio tested, the transcription of the ovalbumin gene from oviduct chromatin was almost totally asymmetric and, thus, closely resembled the pattern of gene transcription characteristic of the in vivo state.

A RNA-dependent RNA polymerase activity: implications for chromatin transcription experiments

Mercurated nucleoside triphosphates have been used for transcription of chicken oviduct chromatin with E. coli RNA polymerase. The newly synthesized RNA was purified from preexisting RNA by SH-agarose chromatography and analyzed for the content of specific mRNA sequences. The apparent preferential production of ovalbumin mRNA sequences was not inhibited by actinomycin D, although total RNA synthesis was reduced by more than 90%. Furthermore, when globin mRNA alone, or added to oviduct chromatin, was incubated in the transcription assay, a significant fraction of this mRNA was retained on SH-agarose. The copurification of chromatin associated RNA with in vitro synthesized mercurated RNA was mainly due to a RNA-dependent synthesis of complementary sequences by the bacterial enzyme. Although denaturation of the transcripts prior to SH-agarose chromatography leads to a reduced contamination with endogenous ovalbumin specific RNA, we are unable to show that the messenger-specific RNA sequences purified with the newly mercurated RNA results from a DNA-dependent reaction.

Effects of estrogen on gene expression in chick oviduct. The role of chromatin proteins in regulating transcription of the ovalbumin gene.

Histones, extractable non-histone proteins, and a tightly bound non-histone protein DNA complex were fractionated from chromatins which were isolated from estrogen-stimulated and hormone-withdrawn chick oviducts. Reconstitution of homologous constituents was performed and RNA was transcribed from these reconstituted chromatins using Escherichia coli RNA polymerase. DNA complementary to ovalbumin mRNA was then used as a hybridization probe to estimate the concentration of ovalbumin messenger RNA sequences in these in vitro transcripts. Our results demonstrated that 0.011% of the in vitro RNA transcripts produced from reconstituted estrogen-stimulated chromatin was ovalbumin mRNA sequences as compared to 0.0015% obtained for reconstituted hormone-withdrawn chromatin. Thus, the ratio of ovalbumin mRNA sequences in the in vitro transcripts of reconstituted stimulated and withdrawn chromatins was 8 to 1, identical with the value obtained from native estrogen-stimulated and hormone-withdrawn chromatins. Furthermore, the number of initiation sites for RNA synthesis on reconstituted and native chromatins are indistinguishable. Thus, the specificity of transcription of isolated chromatins appears to be conserved upon dissociation and fractionation of the chromatin proteins followed by reconstitution of these constituents to DNA. The effect of chromatin proteins on gene expression was further examined by reconstituting components from different development stages. Reconstitution of extractable non-histone proteins from estrogen-stimulated chromatins to tightly bound non-histone protein. DNA complexes from hormone-withdrawn chromatins resulted in the synthesis of a substantial amount of ovalbumin mRNA sequences. Conversely, when extractable non-histone proteins from withdrawn chromatins were reconstituted to tightly bound non-histone protein DNA complexes from estrogen-stimulated chromatins, very low levels of mRNAov sequences were detected. In contrast, interchange of the histones and tightly bound non-histone protein DNA complexes from hormone-withdrawn and estrogen-stimulated chromatins during reconstitution did not affect the level of mRNAOV sequences produced. Therefore, the extractable non-histone proteins of chromatin appear to be extremely important in regulating specific gene expression.

Induction of ovalbumin mRNA sequences by estrogen and progesterone in chick oviduct as measured by hybridization to complementary DNA.

A complementary DNA synthesized from ovalbumin mRNA was used in hybridization experiments to study the early effect of estrogen and progesterone on the accumulation of ovalbumin mRNA sequences in the chick oviduct. Chicks treated with estrogen withdrawn from the hormone maintain a steady level of 60 molecules of ovalbumin mRNA per tubular gland cell, at least 80% of which are localized in the cytoplasm. After estrogen administration, there is a 3- to 4-hour lag before a rapid increase in the number of ovalbumin mRNA sequences and a parallel increase in ovalbumin synthesis. Progesterone causes a more rapid increase in both ovalbumin mRNA sequences and ovalbumin synthesis with a lag period of only 90 min. The hybridization results demonstrate that both estrogen and pregesterone affect the amount of ovalbumin mRNA per cell. The 3-hour lag period seen with estrogen appears to be caused by some event after the binding of the estrogen receptor to chromatin but prior to change in the rate of transcription of the ovalbumin gene.

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.

Estrogen induction of ovalbumin mRNA: evidence for transcription control.

Estrogen induces the synthesis and accumulation of the specific messenger RNA for the egg white protein ovalbumin. The messenger RNA has been purified to apparent homogeneity on a preparative scale and utilized to synthesize a radioactive complementary DNA copy. This complementary DNA probe was first used to reveal that although ovalbumin constitutes 60% of the total protein of chick oviduct the gene which codes for the ovalbumin nRNA is represented only once in each haploid genome: The induction of genetranscription and subsequent accumulation of ovalbumin mRNA during estrogen-mediated tissue differentiation was also investigated. Ovalbumin mRNA sequences were quantifiedusing the complementary DNA probe and by an in vitro heterologous translation system. Similar experiments were performed using chicks which were withdrawn from hormone treatment and then given a single injection of estrogen. The data suggest pure transcriptional control for the mechanism by which estrogen regulates the synthesis of the tissuespecific protein ovalbumin. Finally, several in vitro translation systems are comparedwith respect to their usefullness to assess the effects of hormones on mRNA production. It is concluded that the protein synthesis system derived from wheat germ offers thegreatest advantages for initial studies.

Quantitation of ovalbumin mRNA in hen and chick oviduct by hybridization to complementary DNA. Accumulation of specific mRNA in response to estradiol.

DNA complementary to ovalbumin mRNA was synthesized using reverse transcriptase from avian myelobastosis virus and ovalbumin mRNA, purified 60–70‐fold with respect to polysomal RNA, as template. The quantitative distribution of ovalbumin mRNA sequences in hen and chick oviduct was determined by hybridization with ovalbumin complementary DNA (cDNA). In hen, we calculate that there are approximately 36000 ovalbumin mRNA molecules per oviduct tubular gland cell. The polysome fraction contains 93% of ovalbumin mRNA; nuclear and non‐polysomal cytoplasmic RNA fractions contain 5% and 2% respectively. In the nucleus, the majority of sequences exist as the 16‐S “translatable” form when analyzed on formamide gradients. High‐molecular‐weight RNA species (> 30 S) were detected, and contained less than 2% of total nuclear mRNA sequences. The appearance of ovalbumin mRNA sequences in total, nuclear and polysomal RNA fractions from chick oviduct was measured in response to estradiol. Prior to hormone treatment, low levels of sequences were present in all fractions, approximately one mRNA molecule per gland cell being detected in nuclei. Up to 2 h after treatment a small increase in ovalbumin mRNA concentration was observed in nuclear and, to a lesser extent, in polysomal RNA. Between 3 and 6 h ovalbumin mRNA accumulates rapidly in the polysome fraction and after 9 h the rate of appearance of this mRNA is approximately 7 molecules per min per gland cell. Our results indicate that the induction by estradiol of ovalbumin synthesis in oviduct is mediated either by the synthesis of new ovalbumin gene transcripts or the stabilization of ovalbumin gene products.