Isolation and Characterization of Highly Purified Globin Messenger RNA from Anaemic-Rabbit Spleen

(2,3,13) were resolved by common procedures (zones 1, 2, and 3, possibly corresponding to protein classes A, B, and C, respectively). The resolution of chorion mRNAs seemed considerably lower than that attained with histone mRNAs, which lack poly(A) (25). We reasoned that the presence of poly(A) sequences, which are known to be polydisperse in size (13), might broaden the size distribution for individual mRNA species to a degree sufficient for overlap between species to occur. This might create a single diffuse zone out of what would otherwise be a series of distinct mRNA bands. Here we report that specific removal of the poly(A) by calf thymus RNase H indeed leads to sharpening of the electrophoretic band for a model mRNA, globin mRNA. In the case of chorion messages, deadenylation leads to the formation of distinct electrophoretic bands, nearly as numerous as the protein components that can be resolved by NaDodSO4/ polyacrylamide gel electrophoresis.MATERIALS AND METHODS Animals. Chorionating follicles were dissected from commercially obtained Antheraea polyphemus and Antheraea Abbreviations: PEI, polyethyleneimine; NT, nucleotides; NaDod-S04, sodium dodecyl sulfate./ pernyt as described (1, 2).Radioactive Labeling and Purification of Chorion mRNAs. Chorion mRNAs were purified and labeled in 6-to 8-hr organ culture as described (3, 4, 5). The mRNA was recovered from Mg+2-precipitated polysomes and was bound to oligo(dT)-cellulose at least once. 125I-Labeled chorion or rabbit globin mRNAs were prepared as described (5) Recovery of Deadenylylated mRNA and of Low Molecular Weight Products of RNase H Digestion. After addition of 1, 0.2, and 0.2 volume of 0.05 M KCI, 0.2 M EDTA, and glycerol, respectively, reaction mixtures were passed through a freshly made Sephadex G-150 plus nitrocellulose column assembly (7). The elution buffer was 0.1 M KC1. Low molecular weight digestion products and the deadenylated RNA were recovered after passage through the nitrocellulose part of the column assembly (back and front peaks, respectively).RNA Electrophoresis. As indicated, 6%, 8.5%, or 12% polyacrylamide slab gels containing 7 M urea were used (3, 4). Labeled RNA was detected by autoradiography. Using the autoradiogram as a guide, bands were sometimes excised and the RNA was eluted (4,8).Nucleotide Base Compositions. RNA of oligonucleotide samples was lyophilized to dryness and hydrolyzed in 20 ul of 0.3 M KOH at 950 for 1-2 hr, under mineral oil. The products were spotted on Whatman 540 paper and analyzed by electrophoresis (9).Polynucleotide Phosphorylase Digestion of RNA. Samples of RNA (<1 Mg) dissolved in 10 or 20 Ml of water were digested with Micrococcus luteus polynucleotide phosphorylase (EC 2.7.7.8; Worthington), by mixing with an equal volume of 2X buffer (0.10 M Tris-HCI, pH 7.5, 0.03 M MgCl2, 0.03 M potassium phosphate) and with 0.5 volume of enzyme solution (5 mg/ml in 1X of the above buffer).Analysis of Polynucleotide Phosphorylase Digestion Products. Nucleotide diphosphates were fractionated on 20 X 20 cm po...

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“…3). The overall decrease in size upon digestion is consistent with recent estimates of the average poly(A) size in this message (23).…”

Section: Discussionsupporting

confidence: 90%

Electrophoretic patterns of deadenylylated chorion and globin mRNAs.

Vournakis¹,

Efstratiadis²,

Kafatos³

1975

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

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“…We employed the method described by Bishop et al (1974) since this is a method which is well characterized stoichiometrically. We have confirmed that a one to one ribonuclease-resistant hybrid between poly(A) and radioactive poly(U) was formed (Figure 1), contrary to what some authors have reported (Nokin et al, 1975), under similar experimental conditions.…”

Section: Resultssupporting

confidence: 78%

Length of the polyadenylated sequence in cytoplasmic ribonucleic acid isolated from fetal calf myoblasts differentiating in vitro

Goto¹,

Buckingham²,

Gros³

1981

Biochemistry

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Poly(adenylic acid) [poly(A)] containing cytoplasmic ribonucleic acid (RNA) in differentiating fetal calf myoblasts cultivated in vitro was examined by hybridization with radioactive poly(uridylic acid). The size distribution of the poly(A)-containing RNA after sucrose-gradient centrifugation was similar in cells before and after differentiation. There was no apparent correlation between the length of the poly(A) segment and the change in stability of messenger RNA which occurs on differentiation, nor with the polysomal or nonpolysomal localization of the RNA in the cytoplasm. The average length of the poly(A) segments in cytoplasmic RNA in the steady state was found to be dependent on the size of the RNA: the longer the RNA, the longer the average length of the poly(A) sequence. In contrast, in pulse-labeled RNa, the length of poly(A) is similar in all size classes of RNa.

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“…lose-retained RNA was further bound to a poly(U) -Sepharose column. Three fractions were obtained: (i) unbound; (ii) bound and eluted in low salt at 20 C; (iii) bound and eluted in low salt at 45 C, according to Nokin et al (28). After chromatography of the active mRNA preparation on poly(U)-Sepharose, the three fractions obtained were assayed in the wheat germ system and 35S-labeled products were analyzed as before by polyacrylamide gel electrophoresis.…”

Section: Fpv Structural Proteinsmentioning

confidence: 99%

Cell-free translation of influenza virus mRNA

Content¹

1976

J Virol

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Cytoplasmic poly(A)-rich RNA extracted from fowl plague virus-infected cells was found to program efficiently the translation of two major peptides in the wheat germ cell-free system. These peptides have the same electrophoretic mobility, on polyacrylamide gels, as the two major virion proteins M and NP. I35S]methionine tryptic peptide analysis by one-dimensional paper ionophoresis and finger printing by two-dimensional thin-layer ionophoresis and chromatography show a high degree of similarity between the two in vitro products and the authentic viral proteins M and NP. Although virion RNA is devoid of any poly(A) sequence, it is confirmed here that the viral complementary cytoplasmic RNA contains poly(A) stretches of varying lengths. Intact purified virion was found to promote the synthesis of very low amounts of the same NP and M proteins in this cell-free system. Quantitative aspects of the data would indicate that this is due to minute amounts of complementary viral RNA associated with the virion or with the virion RNA itself. In conclusion, it is shown directly by cell-free translation of authentic viral products that the influenza virion is "negative stranded" (Baltimore, 1971), at least for its two major structural proteins.

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Isolation and Characterization of Highly Purified Globin Messenger RNA from Anaemic-Rabbit Spleen

Cited by 15 publications

References 26 publications

Electrophoretic patterns of deadenylylated chorion and globin mRNAs.

Electrophoretic patterns of deadenylylated chorion and globin mRNAs.

Length of the polyadenylated sequence in cytoplasmic ribonucleic acid isolated from fetal calf myoblasts differentiating in vitro

Cell-free translation of influenza virus mRNA

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