Purification and Properties of Ovalbumin Messenger RNA

Haines, Michael E.; Carey, N. H.; Palmiter, Richard D.

doi:10.1111/j.1432-1033.1974.tb03442.x

Cited by 96 publications

(42 citation statements)

References 52 publications

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“…The apparent molecular weight of the estrogen-induced RNA obtained by gel electrophoresis under non-denaturating conditions (2.0 x lo6) is considerably higher than that determined by sedimentation on dodecylsulphate-sucrose gradients (1.5 x lo6). This feature was already observed for other poly(A)-containing RNA's such as myosin-mRNA [23] and ovalbumin mRNA [24]. Since all these separations are very dependent on the secondary structure of the RNA, the molecular weights determined by reference to rRNA are misleading.…”

Section: Discussionmentioning

confidence: 92%

Size, Complexity and Abundance of a Specific Poly(A)‐Containing RNA of Liver from Male Xenopus Induced to Vitellogenin Synthesis by Estrogen

Wahli

Wyler

Weber

et al. 1976

European Journal of Biochemistry

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Estrogen treatment of Xenopus males leads to the appearance of a new species of poly(A)-containing RNA in the liver, at a stage when large amounts of the estrogen-induced yolk precursor protein, vitellogenin, is produced. This estrogen-induced RNA sediments at 28 S and migrates on gels in aqueous solution with an apparent molecular weight of 2.0 x lo6. Contour length measurements under denaturing conditions in the electron microscope reveal a molecular weight of 2.34 x lo6 compared to the mouse 28-S rRNA.Labeling experiments show that the estrogen-induced RNA has a higher stability than the average liver poly(A)-containing RNA and represents 10 -20 of the poly(A)-containing RNA in the cytoplasm after 24 h of labeling.Hybridization of complementary DNA, synthesized on the isolated estrogen-induced RNA, with its template suggests a complexity corresponding to a single species of poly(A)-containing RNA of such a high molecular weight. Hybridization of the complementary DNA with cytoplasmic poly(A)-containing RNA from estrogen-treated Xenopus males and control toads show that the estrogen-induced RNA constitutes 12 -15 % of all cytoplasmic poly(A)-containing RNA, and is at least 2000-fold less abundant in untreated males.Size, complexity and abundance of the estrogen-induced RNA are characteristics expected for a mRNA coding for vitellogenin.

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Section: Discussionmentioning

confidence: 92%

Size, Complexity and Abundance of a Specific Poly(A)‐Containing RNA of Liver from Male Xenopus Induced to Vitellogenin Synthesis by Estrogen

Wahli

Wyler

Weber

et al. 1976

European Journal of Biochemistry

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“…Fractions (0.5-1 ml) were collected from the bottom of the tube. Palmiter (12) demonstrated that the 650 heat treatment method disaggregated OVmRNA without reducing its ability to direct OV synthesis in a reticulocyte lysate. Other workers (13,14) have also found similar heat treatments to effectively disaggregate RNA without causing thermal degradation.…”

Section: Methodsmentioning

confidence: 99%

Ovalbumin Messenger RNA: Evidence That the Initial Product of Transcription Is the Same Size as Polysomal Ovalbumin Messenger

McKnight

Schimke

1974

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

102

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The messenger RNA for ovalbumin, the major secretory protein of the chick oviduct, appears not to be made as a high-molecular-weight precursor when artifacts due to aggregation are eliminated. No ovalbumin messenger RNA sequences that will hybridize to complementary DNA made against ovalbumin mRNA are found in concentrated samples of hen oviduct RNA larger than 28 S. The sensitivity of the hybridization assay is sufficient to detect less than one molecule of ovalbumin mRNA precursor per tubular gland cell. Newly synthesized ovalbumin messenger RNA isolated from immature chicks stimulated briefly by estrogen is the same size as that found in hen polyribosomes. We conclude that ovalbumin messenger RNA does not undergo any significant change in molecular weight from its initial transcription to its incorporation into polyribosomes.The synthesis of messenger RNA in animal cells and its transport from the nucleus to the cytoplasm has received much attention in recent years, but our understanding of this process is incomplete and confused. A model for mRNA synthesis has been proposed by Darnell and coworkers (1) which involves the initial synthesis of a large precursor RNA with subsequent post-transcriptional addition of poly(A) sequences to the 3' terminus. This large precursor is degraded except for the 3' portion containing the mRNA, which is then transported to the cytoplasm and incorporated into polysomes.A large amount of evidence supporting this hypothesis has been reported. Both heterogeneous nuclear RNA (HnRNA) and mRNA contain a 3'-terminal poly(A) region of approximately the same length (2). Jelinek et al. (3) have reported that much of the poly(A) synthesized in the nucleus is conserved and transported to the cytoplasm. However, Perry and coworkers (4) have recently suggested that most of the nuclear poly(A) remains in the nucleus and is degraded there. Further evidence for the precursor-product relationship between HnRNA and mRNA has been obtained for specific mRNAs by either translation of the HnRNA in vitro (5,6) or by its hybridization to complementary DNA (7). Such results are not completely convincing because the possibility of aggregation artifacts in sucrose gradients has not been rigorously excluded. Proof that a large precursor exists for a specific mRNA should involve separation of the precursor from polysomal mRNA and a careful demonstration of its covalent integrity by a variety of denaturing techniques. This has not Abbreviations: OV, ovalbumin; HnRNA, heterogeneous nuclear RNA; NaDodSO4, sodium dodecyl sulfate; Me2SO, been reported for any proposed precursor, and in cases where dimethylsulfoxide (Me2SO) gradients have been used, most of the large mRNA sequences dissaggregate to polysomal mRNA sizes (7). In this study we report evidence suggesting that the mRNA coding for chick oviduct ovalbumin (OV) is synthesized, transported to the cytoplasm, and incorporated into polysomes without a measurable change in its molecular weight. MATERIALS AND METHODSChicks, Injections, and RNA Labelin...

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“…Immediately after the last extraction with chloroform the aqueous phase, containing the RNA, was heated to 600 for 3 min to denature the RNA. This material was then applied directly to linear gradients of 5-20% sucrose in 0.01 M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes), pH 7.5, containing EDTA (0.001 M), as described by Palmiter (15). The gradients were centrifuged at 40 at 67,000 X g., for 16 hr in a SW 27 rotor in a Beckman L-5 65 centrifuge, after which they were fractionated as described above.…”

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confidence: 99%

Induction of vitellogenin synthesis by estrogen in avian liver: relationship between level of vitellogenin mRNA and vitellogenin synthesis.

Mullinix¹,

Wetekam²,

Deeley³

et al. 1976

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

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We have investigated the estrogen-mediated induction of vitellogenin synthesis in rooster liver. We compared the concentrations of vitellogenin messenger RNA (mRNA) in the liver with the concentrations of vitellogenin in the sera of roosters that had received various treatments with estrogen. We found no vitellogenin mRNA in the livers of the unstimulated roosters. An initial injection of estrogen was attended by de novo synthesis of vitellogenin mRNA in the liver and accumulation of vitellogenin in the serum. When vitellogenin was no longer present in the serum or liver (the "post-estrogen-serum-negative" state), the liver was found to contain appreciable amounts of vitellogenin mRNA. This mRNA was of the same size as that found in the liver of the rooster actively synthesizing vitellogenin in response to estrogen. Whereas vitellogenin mRNA was in large polysomes in the livers of the roosters actively synthesizing vitellogenin, the vitellogenin mRNA in the liver of the post-estrogen-serum-negative rooster was not associated with polysomes. The possible relevance of these findings to the fact that the rooster responds differently to a primary stimulation with estrogen than to subsequent stimulations is discussed.

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Purification and Properties of Ovalbumin Messenger RNA

Cited by 96 publications

References 52 publications

Size, Complexity and Abundance of a Specific Poly(A)‐Containing RNA of Liver from Male Xenopus Induced to Vitellogenin Synthesis by Estrogen

Size, Complexity and Abundance of a Specific Poly(A)‐Containing RNA of Liver from Male Xenopus Induced to Vitellogenin Synthesis by Estrogen

Ovalbumin Messenger RNA: Evidence That the Initial Product of Transcription Is the Same Size as Polysomal Ovalbumin Messenger

Induction of vitellogenin synthesis by estrogen in avian liver: relationship between level of vitellogenin mRNA and vitellogenin synthesis.

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