Patterns of RNA metabolism in a differentiated cell: a rapidly labeled, unstable 60S RNA with messenger properties in duck erythroblasts.

Scherrer, Klaus; Marcaud, Lise; Zajdela, F; London, Irving M.; Gros, François

doi:10.1073/pnas.56.5.1571

Cited by 182 publications

(81 citation statements)

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“…of duck erythroblasts Immature erythroid cells in the circulating blood of anemic ducks or chicken are normal diploid but non-proliferating cells, which synthesize premRNA and mRNA [19] but neither DNA nor ribosomal RNA nor ribosomes; as a net result relatively few proteins are formed. We decided to investigate the pattern of proteins synthesized in duck erythroblasts by radiolabelling proteins in intact isolated cells.…”

Section: Analysis Of In Vivo Synthesized Proteinsmentioning

confidence: 99%

“…Therefore, 2 h incubation were chosen as the op- timum time to qualitatively define the proteins synthesized in duck erythroblasts. Since in the cell, the time required for the complete synthesis of an average M, (50000) protein is about 2 min [27] and the half life of mRNA is generally more than several hours [28,30], proteins labelled after 2 h may be considered as reflecting a steady state situation. In fact, analysis of proteins in cell samples taken after various times of labelling, ranging from 0.25-3 h, showed no major variation in the patterns of labelled proteins (not shown).…”

Section: Analysis Of In Vivo Synthesized Proteinsmentioning

confidence: 99%

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The translation of the messenger for the poly(A)‐binding protein‐associated with translated mRNA is suppressed

et al. 1983

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In vivo protein synthesis in duck erythroblasts was compared to in vitro translation of polyribosomal and free cytoplasmic mRNA. The in vivo study showed the absence of de novo synthesis of the M r 73 000 poly(A)‐binding protein found associated with all polyribosomal mRNA. In vitro translation demonstrated that the mRNA for this protein is absent from the polyribosomal mRNA fraction but constitutes a medium frequency messenger among the repressed free mRNA. This result confirms the existence of a qualitative translational control in terminal differentiating duck erythroblasts leading eventually to the arrest of the protein synthesizing machinery.

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Section: Analysis Of In Vivo Synthesized Proteinsmentioning

confidence: 99%

Section: Analysis Of In Vivo Synthesized Proteinsmentioning

confidence: 99%

The translation of the messenger for the poly(A)‐binding protein‐associated with translated mRNA is suppressed

et al. 1983

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“…La Fig.11 [49,50]. Des travaux anterieurs sur la nature ct la biosynthese des RNA des cellules animales ont Bt6 passes en revue reeemment [51].…”

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Acides ribonucléiques messagers de la glande thyroïde

Cartouzou

Attali

Lissitzky

1968

European Journal of Biochemistry

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( R e p le 2 aoDt 1967/6 dkcembre 1967) I . Nuclear and cytoplasmic RNA from sheep thyroid slices in vitro labeled with [3H]uridine or [32P]phosphate from 5 min up to 9 h, have been obtained by different phenol extraction procedures. Early-labeled RNA was only extracted by phenol containing 0.1 or lo/, (w/v) sodium dodecylsulfate (SDS) at 0" or 60°, whereas stable nuclear or polysomal RNA was easily extracted by phenol at 0".2. Time course of incorporation of label into subcellular particles has shown the early labeling of nuclear RNA and the transfer of radioactivity into the cytoplasm.3. Rapidly-labeled RNA extracted from nuclei by phenol-I O/, (w/v) SDS and analyzed by sedimentation was shown to sediment in three discrete classes 50-30 S, 22 S and 13 S, different from ribosomal RNA. After 9 h o f incubation the 22 S and 13 S labeled RNAs were still present in detectable amounts in the nuclei, indicating a somewhat low turnover rate for these components. 4.The kinetics of incorporation of label into polysomes was studied in thyroid slices incubated from 15 min up to 5 h with [3H]uridine or [32P]phosphate. RNA was dissociated from polysomes by dodecylsulfate treatment and centrifuged on sucrose gradient. As for nuclei, the rapidlylabeled RNAs sediment in discrete size classes, 22 S and 13 S. After a long incubation time (5 h) the label was predominant in 28 S and 18 S ribosomal RNA but 28 S RNA was not completely labeled. Up to 30 min labeling a high molecular weight, early-labeled RNA was present in purified polysomes. Its radioactivity declines after 60 min labeling.5. A part of the rapidly labeled polysomal RNA can be reversibly separated from rRNP by the action of EDTA (1.25 to 5 pmoles EDTA/mg RNP). It sediments in a broad zone (12-30 S) of high specific radioactivity. Increasing Mg++ concentration results in the reassociation of rapidly-labeled RNA to ribosomal sub-particles.6. Buoyant densities of the polysomal RNP particles obtained by EDTA treatment of 32P-labeled polysomes and stabilized by means of formaldehyde fixation, were determined by banding in CsCl gradients. The bulk o f the stable material banded at characteristic densities (d = 1.55 and 1.48), but most of the rapidly-labeled RNA banded at d = 1.38.The specific radioactivity of this material was the same as that of the 12-30 S RNA isolated by sucrose gradient centrifugation from the same EDTA-treated polysomes. Preliminary double labeling experiments and computation of the RNA to protein ratio calculated from the RNA and protein density values indicate this material to be early-labeled nucleoprotein particles with a high protein content.La glande thyroide est un tissu dont la fonction essentielle est la synthhse des hormones thyroidiennes. Morphologiquement la d86renciation du tissu thyroidien se manifeste par une structure folliculaire trhs caractkristique. Chaque follicule est constitu6 [12] le poids moleculaire des sousunites obtenues par dissociation de la thyroglobuline reduite et 8-carboxymethylee serait de 110.000 B 125.000, indiquan...

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“…Several features of the synthesis and structure of hnRNA indicate its role in mRNA metabolism (for review see [l-51). Indirect evidence first led to the postulate that hnRNA contained precursor molecules to mRNA [6,7]. Hybridization-competition experiments later demonstrated base-sequence homology between polyribosomal mRNA and hnRNA from HeLa cells [8].…”

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Size Distribution of Rat Liver Nuclear RNA Containing mRNA Sequences

Sippel

Groner

Hynes

et al. 1977

European Journal of Biochemistry

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Total rat liver poly(A)-containing polysomal mRNA was size-fractionated on polyacrylamide gels in 98 % formamide. Complementary DNA (cDNA) was prepared from the 8 -14-S mRNA fraction and separated into sequences representing abundant and non-abundant mRNAs. The cDNA complementary to the abundant small mRNA of the rat liver cell (approximately 20 species) was hybridized to nuclear RNA of different lengths to determine the size distribution of nuclear RNA molecules which contain these messenger sequences. It was found that :1. All abundant 8 -14 -S poly(A)-containing mRNAs have larger nuclear precursor molecules ; 20% of the different messenger sequences are found in nuclear RNA of several times their cytoplasmic length.2. 70 % of the mass of the examined nuclear messenger sequences is in RNA molecules of a size similar to their polysomal mRNA; 30% are in larger than 18-S RNA and 2% are between 37 S and 44 S.3 . The majority of small messenger-containing RNA molecules in the RNA prepared from isolated nuclei are of true nuclear origin, since their frequency distribution differs significantly from that of the polysomal 8 -14-S mRNA.To understand the regulation of gene expression in eucaryotic cells it is crucial to know more about nuclear processes involved in the biogenesis of mRNA. Several features of the synthesis and structure of hnRNA indicate its role in mRNA metabolism (for review see [l-51). Indirect evidence first led to the postulate that hnRNA contained precursor molecules to mRNA [6,7]. Hybridization-competition experiments later demonstrated base-sequence homology between polyribosomal mRNA and hnRNA from HeLa cells [8]. Furthermore, it was found that viral-specific RNAs in and polyoma-infected [lo] cells are transcribed as giant RNA molecules. However, owing to the enormous sequence complexity, the large size and the high turnover of hnRNA, a detailed describtion of the nuclear pathway of messenger sequences has not yet Abbreviations. Hepes. 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; AMV, avian myeloblastosis virus; cDNA, DNA copy complementary to mRNA ; hnRNA, polydisperse high-molecularweight nuclear RNA; rot value, concentration of RNA (as mol phosphate/l) times time of hybridization (in s); rotllz value, rot value at which half the hybridizable cDNA is hybridized; vgt value, RNA derived from the amount of liver tissue (g), hybridized in IO-pI assay volume, times the hybridization time (h).Enzyme. S 1 nuclease, single-strand-specific endonuclease from Aspergillus oryzae (EC 3.1.4.21).been possible. The use of molecular hybridization with complementary DNA copies of specific mRNA molecules to identify messenger sequences in steadystate nuclear RNA has resulted in an increased amount of new information, but no general picture has emerged from these studies. For example, larger nuclear precursor RNA molecules for globin mRNA have been found, although with conflicting results as to their largest size [ l l -171. For the case of ovalbumin mRNA, no larger nuclear RNA containing the messenger se...

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Patterns of RNA metabolism in a differentiated cell: a rapidly labeled, unstable 60S RNA with messenger properties in duck erythroblasts.

Cited by 182 publications

References 14 publications

The translation of the messenger for the poly(A)‐binding protein‐associated with translated mRNA is suppressed

The translation of the messenger for the poly(A)‐binding protein‐associated with translated mRNA is suppressed

Acides ribonucléiques messagers de la glande thyroïde

Size Distribution of Rat Liver Nuclear RNA Containing mRNA Sequences

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