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

Nguyen-Huu, M C; Sippel, A. A.; Hynes, Nancy E.; Groner, Bernd; Schütz, Günther

doi:10.1073/pnas.75.2.686

Cited by 42 publications

(20 citation statements)

References 34 publications

(26 reference statements)

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“…of HzO, adjusted to 0.5 "/, sodium dodecyl sulfate, and digested with proteinase K (10 mg/ml) at 37°C for 1 h. The nucleic acids were extracted by the hot phenol method [13], and passed through a column of SephadexG-75 in 10 mM Tris/HCl, pH 7.5, containing 1 mM EDTA. The mercurated RNA, synthesized in vitro, was separated from endogenous nuclear RNA by affinity chromatography on a sulfhydryl-Sepharose column (Servachrom A-SH), in the presence of formamide, as described by Nguyen-Huu et al [14].…”

Section: Isolation and Sizing Of The Rna Synthesized In Vitromentioning

confidence: 99%

RNA Synthesis in Rabbit Endometrial Nuclei

Müller

Beato

1980

European Journal of Biochemistry

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Endometrical nuclei, prepared from rabbits subjected to different hormonal treatments, were used for the cell-free synthesis of RNA. Optimal conditions for the incorporation of [3H]UMP into RNA are described, leading to the synthesis of relatively undegraded RNA molecules. Under these conditions there is virtually no initiation of new RNA chains in vitro, and RNA chain elongation is inhibited up to 6004 by low concentrations of a-amanitin and up to 90% by actinomycin D. The synthesis of RNA is slightly inhibited in the presence of Hg-CTP and monothioglycerol, but newly synthesized mercurated RNA can be efficiently separated from endogenous RNA upon chromatography on sulfhydryl-Sepharose under stringent conditions. The RNA synthesized in vitro by endometrial nuclei from pseudopregnant rabbits contains RNA sequences transcribed from the uteroglobin gene, as demonstrated by hybridization to an excess of purified preuteroglobin cDNA. In endometrial cells from pseudopregnant animals the number of RNA polymerase I1 molecules transcribing the uteroglobin gene is 12-fold higher than in control animals, demonstrating that at least part of the hormonally induced accumulation of preuteroglobin mRNA is due to an increased rate of transcription of the uteroglobin gene.In rabbit endometrium, ovarian hormones induce the secretion of large quantities of the steroid-binding protein uteroglobin (for review see [l]). It has been previously shown that uteroglobin induction is accompanied by an increased rate of synthesis of the protein, and by an accumulation of the mRNA for pre-uteroglobin in endometrial polysomes [2 -51. In principle, two mechanisms could account for the accumulation of specific mRNA: an increased rate of synthesis or a decreased rate of degradation. To study the hormonal regulation of the rate of synthesis of pre-uteroglobin mRNA we have developed a nuclear system from rabbit endometrium, in which transcription reflects the situation in vivo.In this article we describe the properties of this system, that efficiently elongates RNA chains initiated in vivo, using mercurated nucleotides as substrate. Mercury-containing RNA can be separated from nonmercurated endogenous RNA upon chromatography on sulfhydryl-Sepharose, and employed for hybridization experiments with purified pre-uteroglobin cDNA. The results demonstrate that this system can be used

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Section: Isolation and Sizing Of The Rna Synthesized In Vitromentioning

confidence: 99%

RNA Synthesis in Rabbit Endometrial Nuclei

Müller

Beato

1980

European Journal of Biochemistry

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“…Injections of estrogen into chickens that had been prestimulated with estrogen and then withdrawn from all hormone (secondary stimulation) induced the accumulation of ovalbumin mRNA (mRNAOv) (4)(5)(6)(7). Recently, it has been shown that the structural sequences of the ovalbumin gene (8)(9)(10) and globin gene are interrupted by multiple regions of nonstructural intervening sequences.…”

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confidence: 99%

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

Swaneck

Nordstrom

Kreuzaler

et al. 1979

Proc. Natl. Acad. Sci. U.S.A.

126

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The transcription of structural and intervening se uences of the chicken ovalbumin gene was studied in nuclei isoated from the oviduct, liver, and spleen of chickens in different states of estrogen stimulation. The concentration of transcripts of structural and intervening DNA sequences was determined by hybridizing the newly synthesized [3HJRNA 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[3H]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.Previous studies on the regulation of gene expression in eukaryotes have shown that steroid hormones induce synthesis of specific proteins in vivo by increasing the concentration of their mRNAs (1-3). Injections of estrogen into chickens that had been prestimulated with estrogen and then withdrawn from all hormone (secondary stimulation) induced the accumulation of ovalbumin mRNA (mRNAOv) (4-7). Recently, it has been shown that the structural sequences of the ovalbumin gene (8-10) and globin gene are interrupted by multiple regions of nonstructural intervening sequences. In addition, the 15S precursor to globin mRNA was shown to contain both structural and intervening sequences of the globin gene (11). Similarly, transcripts of both types of ovalbumin sequences accumulated in vivo in oviduct nuclei after stimulation with estrogen (12).In addition, we have demonstrated the existence of high-mo-The publication costs of this article were def...

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“…Since the cytoplasm is absent from the nuclei in the in vitro system one can control the nuclear environment precisely and in particular for the screening of putative cytoplasmic regulatory molecules. In addition, nuclei incubated in vitro have been shown to add poly(A) tails (7,14,15), initiate RNA synthesis (2,3,6,9,13,14), produce discrete RNA sequences (2,8,10,(11)(12)(13), and process precursor mRNA (15).…”

Section: Introductionmentioning

confidence: 99%

“…We have previously shown that the chicken liver nuclei were able to reinitiate RNA synthesis in vitro (2). RNA synthesis in isolated purified nuclei has been described for a large number of different cell types (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15), but in all cases the endogenous DNA was used as the sole source of template. Since the cytoplasm is absent from the nuclei in the in vitro system one can control the nuclear environment precisely and in particular for the screening of putative cytoplasmic regulatory molecules.…”

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confidence: 99%

Specific modulation of the transcription of cloned avian vitellogenin II gene by estradiol-receptor complex in vitro.

Jost¹,

Geiser²,

Seldran³

1985

Proc. Natl. Acad. Sci. U.S.A.

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Avian vitellogenin-cauliflower mosaic virus hybrid gene is effectively transcribed in vitro in the homologous embryonic liver nuclei system. The transcription of the hybrid gene is modulated by the addition of an estradiol-receptor preparation that has been shown to bind selectively to an upstream region of cloned vitellogenin gene. Stimulation of the transcription of cloned vitellogenin hybrid gene by estradiol receptor is a-amanitin sensitive, hormone dependent, and promoter specific. Simian virus 40 and Escherichia coli promoters are not stimulated by the estradiol-receptor complex. Recently, we have shown that a DNA sequence upstream of the chicken vitellogenin II gene binds preferentially estradiol-receptor complex (1). However, these results did not show whether binding of the receptor complex to this region had any relevant biological function-e.g., the turning on or the modulation of vitellogenin gene expression. As a possible experimental approach to this problem we thought of using an in vitro homologous transcription system. We have previously shown that the chicken liver nuclei were able to reinitiate RNA synthesis in vitro (2). RNA synthesis in isolated purified nuclei has been described for a large number of different cell types (3-15), but in all cases the endogenous DNA was used as the sole source of template. Since the cytoplasm is absent from the nuclei in the in vitro system one can control the nuclear environment precisely and in particular for the screening of putative cytoplasmic regulatory molecules. In addition, nuclei incubated in vitro have been shown to add poly(A) tails (7,14,15), initiate RNA synthesis (2,3,6,9,13,14), produce discrete RNA sequences (2,8,10,(11)(12)(13), and process precursor mRNA (15).We now describe experiments in which we show that the nuclei system transcribes also cloned genes added to the incubation mixture. A promoter-specific modulation of the transcription of cloned genes is also obtained by the addition of estradiol-receptor complex. MATERIALS AND METHODSThe in Vitro Transcription System. The preparation and storage of liver nuclei from 18-day embryos and the in vitro transcription mixture were prepared as outlined by Panyim et al. (2) with the following modifications: endogenous ribonucleases were inhibited by the addition of 10-50 units of human placental ribonuclease inhibitor (RNasin) per 150 1.l of incubation mixture. When nuclear estradiol-receptor preparation was added to the reaction mixture, care was taken to keep the KCI concentration between 0.09 and 0.1 M. One to 3 ug of cloned DNA template was added per ml of incubation mixture. Upon incubation at 250C for 40-60 min, RNA was immediately purified from the incubation mixture as described below. For each time point and estradiol-receptor concentration, assays were run in duplicate and parallel controls were incubated in ice. Thomas (16). Alternatively, the reaction mixture was mixed with 2-3 vol of 8 M guanidine HCl/10 mM sodium acetate, pH 5/5% mercaptoethanol and RNA was centrifuged thr...

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

Cited by 42 publications

References 34 publications

RNA Synthesis in Rabbit Endometrial Nuclei

RNA Synthesis in Rabbit Endometrial Nuclei

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

Specific modulation of the transcription of cloned avian vitellogenin II gene by estradiol-receptor complex in vitro.

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