Quantitation of Ovalbumin mRNA in Hen and Chick Oviduct by Hybridization to Complementary DNA

Cox, Ronald F.; Haines, Michael E.; Emtage, J.S.

doi:10.1111/j.1432-1033.1974.tb03827.x

Cited by 95 publications

(32 citation statements)

References 36 publications

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“…During stimulation with estradiol the amount of ovalbumin mRNA dramatically increases from a few copies to tens of thousands of copies per cell (2,4,(5)(6)(7). Similar results have been found for the induction of ovomucoid and lysozyme mRNA (7).…”

supporting

confidence: 74%

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

Nguyen-Huu¹,

Sippel²,

Hynes³

et al. 1978

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

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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 sulfhydrylagarose and hybridized to radioactive ovalbumin cDNA. (ii) (3HJUTP 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 a-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 vivo RNA synthesis is retained in isolated nuclei.The synthesis of the egg white proteins in the tubular gland cell of the chicken oviduct is primarily related to the cellular accumulation of the corresponding mRNAs (1-7). During stimulation with estradiol the amount of ovalbumin mRNA dramatically increases from a few copies to tens of thousands of copies per cell (2, 4, 5-7). Similar results have been found for the induction of ovomucoid and lysozyme mRNA (7). The steroid-mediated induction of specific mRNA molecules in the chicken oviduct may result from altered rates of synthesis and/or degradation. We therefore studied the synthesis rate of the egg white protein mRNAs. Because it was not possible to measure the rate of specific mRNA synthesis in the animals, we chose isolated oviduct nuclei as the cell-free system that might best reflect the transcriptional activity of the intact cell (8). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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supporting

confidence: 74%

“…The synthesis of the egg white proteins in the tubular gland cell of the chicken oviduct is primarily related to the cellular accumulation of the corresponding mRNAs (1)(2)(3)(4)(5)(6)(7). During stimulation with estradiol the amount of ovalbumin mRNA dramatically increases from a few copies to tens of thousands of copies per cell (2,4,(5)(6)(7).…”

mentioning

confidence: 99%

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

Nguyen-Huu¹,

Sippel²,

Hynes³

et al. 1978

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

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“…Our approach involved the translation of nuclear RNA into protein with the wheat-germ cell-free system. Although it is now clear that mRNA originates in the eukaryotic nucleus (Molloy & Puckett, 1976) there are very few reports in the literature of the successful translation of nuclear RNA into protein in vitro (Ruiz-Carrillo et al, 1973;Knochel & Teidemann, 1975), and some workers have found that nuclear RNA inhibits protein synthesis in vitro (Cox et al, 1974). We found that nuclear RNA stimulated protein synthesis in the wheat-germ system at concentrations up to 5,g/50pu1 assay, and ascertained that the observed messenger activity was not due to contamination of the nuclear RNA with cytoplasmic RNA.…”

Section: Discussionmentioning

confidence: 99%

“…Preparation of chick intestinal nuclear RNA Nuclei were prepared from chick ileal mucosa by the method described by Cox et al (1974), in order to minimize contamination with ribosomes on the outer nuclear membrane (Knowler et al, 1973), and RNA was isolated from the pellet by a hot phenol/ sodium dodecyl sulphate extraction (Ruiz-Carrillo et al, 1973 It was important for this study to demonstrate that the calcium-binding-protein mRNA designated 'nuclear' did originate from the nucleus and not from contaminating cytoplasmic RNA. In an attempt to obtain a maximum estimate of the extent to which nuclear RNA could be contaminated by cytoplasmic RNA, mucosa from vitamin D-deficient birds (with no calcium-binding-protein mRNA) was homogenized in the post-nuclear supernatant obtained from vitamin D-dosed birds (containing calciumbinding-protein mRNA), and the contamination of nuclei from the deficient birds with trapped cytoplasmic calcium-binding-protein mRNA from the dosed birds was determined by extracting and translating the nuclear RNA in the wheat-germ cell-free system.…”

Section: Nmentioning

confidence: 99%

Stimulation of intestinal calcium-binding-protein mRNA synthesis in the nucleus of vitamin D-deficient chicks by 1,25-dihydroxycholecalciferol

Spencer

Charman

Lawson

1978

Biochemical Journal

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Stimulation of intestinal calcium transport by the hormone 1,25-dihydroxycholecalciferol appears to involve RNA transcriptions and the synthesis of new proteins. Although one of these proteins has been identified as calcium-binding protein, no RNA molecules specifically induced by the hormone in the nucleus have been identified. Nuclear RNA from intestine of vitamin D-deficient chicks before and at various time intervals after treatment with the hormone or cholecalciferol was tested for its ability to code for calcium-binding protein in a cell-free system. Calcium-binding-protein mRNA could only just be detected in the intestinal nuclei 2h after dosing with these steroids which is the same time that it was first observed in the polyribosomes. Thus 1,25-dihydroxycholecalciferol induces the production of new calcium-binding protein by stimulating the formation and rapid release from the nucleus ofnew mRNA molecules for this protein. Polyribosomal translation of the mRNA continued only as long as it was being synthesized, and the maximum rate of synthesis following a pulse dose of 125 ng of the hormone was the same as that observed after prolonged stimulation with cholecalciferol. The possibility that other 1,25-dihydroxycholecalciferol-dependent events may be occurring in the nucleus in the lag period between accumulation of the hormone in the intestine and the appearance of active calcium-binding-protein mRNA, and that these may ultimately control the synthesis of that mRNA, is discussed.

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“…At the end of a 60-min incubation at 25°C, RNA was extracted by the phenol/chloroform method (11) in the presence of wheat germ high molecular weight RNA as carrier (150-fold excess over endogenous nuclear RNA). After 10 min of heat denaturation at 75°C, the Hg-RNA was isolated by SH-agarose column chromatography as described (11,12) 24 hr at 38,000 rpm in a Spinco SW 41 rotor, and an aliquot of each gradient fraction was assayed for radioactivity. X, Position of the 3H-labeled viral marker DNAs centrifuged on a parallel gradient.…”

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confidence: 99%

Glucocorticoid modulation of casein gene transcription in mouse mammary gland.

Ganguly¹,

Mehta²,

Ganguly³

et al. 1979

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

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The influence of cortisol and prolactin on casein gene expression in the mammary gland of lactating BALB/c mice was measured by using a specific cDNA probe to 15S casein mRNA (cDNAcsn Since the establishment that cortisol and prolactin are the two principal hormones required for lactogenesis (2, 3) (differentiation) as well as production of casein (4) in murine mammary gland, numerous studies have attempted to ascertain the role(s) of the adrenal steroid and the pituitary polypeptide hormones in regulation of the milk protein, casein (5-8), and its mRNA (mRNA,,n) (9,10 MATERIALS AND METHODS Animals and Treatments. Two groups of 5-day postpartum BALB/c mice, each mouse nursing 5-7 pups, were bilaterally adrenalectomized under pentabarbitol anesthesia. Five days later mice in one group were killed. The second group of 5-day adrenalectomized mice were given a single subcutaneous injection of 250 ,g of hydrocortisone-21-acetate in 0.1 ml of 0.9% saline. Batches of these animals were killed at different times after the injection. In order to obtain lactating animals with reduced serum prolactin level, unoperated mice in another group were given daily injections of 100 Mug of the prolactin inhibitor (15), 2-bromo-a-ergocryptene (CB-154), for 3 day$ starting on the 8th day of lactation. These animals were killed on the 10th day of lactation, 6 hr after the last injection of the ergot alkaloid. Ten-day lactating mice were used as nontreated controls. All the animals were allowed to nurse their pups throughout the experimental period. Immediately after the animals were killed mammary glands were removed, frozen in liquid nitrogen, and stored at -80°C.In Vitro RNA Synthesis in Isolated Nuclei and SH-Agarose Chromatography. Isolation of the nuclei and in vitro RNA synthesis in the presence of a mercury-labeled nucleotide (Hg-CTP) were as described (11). Essentially, the same number of nuclei (200 ,ig of DNA per ml of reaction mixture) was used in each 1-ml assay mixture. At the end of a 60-min incubation at 25°C, RNA was extracted by the phenol/chloroform method (11) in the presence of wheat germ high molecular weight RNA as carrier (150-fold excess over endogenous nuclear RNA). After 10 min of heat denaturation at 75°C, the Hg-RNA was isolated by SH-agarose column chromatography as described (11,12) and precipitated with ethanol in the presence of Escherichia coli tRNA. For quantitation, Hg-RNA synthesized in vitro was labeled with [a-32P]UTP (New England Nuclear) of low specific activity (50 cpm/pmol).Casein mRNA Purification, Synthesis of cDNAk.n, and Molecular Hybridization. Phenol/chloroform-extracted (16) mammary gland RNA from 8-to 11-day lactating mice was heat denatured and subjected to two successive oligo(dT)-cellulose chromatographic purifications according to standard Abbreviations: Hg-CTP, 5-mercuricytidine triphosphate; Hg-RNA, RNA containing Hg-CTP; mRNA,,,, 15S casein mRNA; cDNAcn, complementary DNA to mRNA.,n; CB-154, 2-bromo-a-ergocryptene; Rot, moles of ribonucleotide per liter x time (sec); Ro...

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Quantitation of Ovalbumin mRNA in Hen and Chick Oviduct by Hybridization to Complementary DNA

Cited by 95 publications

References 36 publications

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

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

Stimulation of intestinal calcium-binding-protein mRNA synthesis in the nucleus of vitamin D-deficient chicks by 1,25-dihydroxycholecalciferol

Glucocorticoid modulation of casein gene transcription in mouse mammary gland.

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