scholarly journals Synthesis and turnover of mitochondrial ribonucleic acid in HeLa cells: the mature ribosomal and messenger ribonucleic acid species are metabolically unstable.

1981 ◽  
Vol 1 (6) ◽  
pp. 497-511 ◽  
Author(s):  
R Gelfand ◽  
G Attardi
Keyword(s):  
Ribosomal Rna ◽  
Hela Cells ◽  
Ribonucleic Acid ◽  
Half Life ◽  
Messenger Rna ◽  
Specific Activity ◽  
Mitochondrial Rna ◽  
Messenger Rnas ◽  
Precursor Pool ◽  
Messenger Ribonucleic Acid

The synthesis rates and half-lives of the individual mitochondrial ribosomal ribonucleic acid (RNA) and polyadenylic acid-containing RNA species in HeLa cells have been determined by analyzing their kinetics of labeling with [5-3H]-uridine and the changes in specific activity of the mitochondrial nucleotide precursor pools. In one experiment, a novel method for determining the nucleotide precursor pool specific activities, using nascent RNA chains, has been utilized. All mitochondrial RNA species analyzed were found to be metabolically unstable, with half-lives of 2.5 to 3.5 h for the two ribosomal RNA components and between 25 and 90 min for the various putative messenger RNAs. A cordycepin "chase" experiment yielded half-life values for the messenger RNA species which were, in general, larger by a factor of 1.5 to 2.5 than those estimated in the labeling kinetics experiments. On the basis of previous observations, a model is proposed whereby the rate of mitochondrial RNA decay is under feedback control by some mechanism linked to RNA synthesis or processing. A short half-life was determined for five large polyadenylated RNAs, which are probably precursors of mature species. A rate of synthesis of one to two molecules per minute per cell was estimated for the various H-strand-coded messenger RNA species, and a rate of synthesis 50 to 100 times higher was estimated for the ribosomal RNA species. These data indicate that the major portion of the H-strand in each mitochondrial deoxyribonucleic acid molecule is transcribed very infrequently, possibly as rarely as once or twice per cell generation. Furthermore, these results are consistent with a previously proposed model of H-strand transcription in the form of a single polycistronic molecule.

scholarly journals Synthesis and turnover of mitochondrial ribonucleic acid in HeLa cells: the mature ribosomal and messenger ribonucleic acid species are metabolically unstable

1981 ◽  
Vol 1 (6) ◽  
pp. 497-511
Author(s):  
R Gelfand ◽  
G Attardi
Keyword(s):  
Ribosomal Rna ◽  
Hela Cells ◽  
Ribonucleic Acid ◽  
Half Life ◽  
Messenger Rna ◽  
Specific Activity ◽  
Mitochondrial Rna ◽  
Messenger Rnas ◽  
Precursor Pool ◽  
Messenger Ribonucleic Acid

The synthesis rates and half-lives of the individual mitochondrial ribosomal ribonucleic acid (RNA) and polyadenylic acid-containing RNA species in HeLa cells have been determined by analyzing their kinetics of labeling with [5-3H]-uridine and the changes in specific activity of the mitochondrial nucleotide precursor pools. In one experiment, a novel method for determining the nucleotide precursor pool specific activities, using nascent RNA chains, has been utilized. All mitochondrial RNA species analyzed were found to be metabolically unstable, with half-lives of 2.5 to 3.5 h for the two ribosomal RNA components and between 25 and 90 min for the various putative messenger RNAs. A cordycepin "chase" experiment yielded half-life values for the messenger RNA species which were, in general, larger by a factor of 1.5 to 2.5 than those estimated in the labeling kinetics experiments. On the basis of previous observations, a model is proposed whereby the rate of mitochondrial RNA decay is under feedback control by some mechanism linked to RNA synthesis or processing. A short half-life was determined for five large polyadenylated RNAs, which are probably precursors of mature species. A rate of synthesis of one to two molecules per minute per cell was estimated for the various H-strand-coded messenger RNA species, and a rate of synthesis 50 to 100 times higher was estimated for the ribosomal RNA species. These data indicate that the major portion of the H-strand in each mitochondrial deoxyribonucleic acid molecule is transcribed very infrequently, possibly as rarely as once or twice per cell generation. Furthermore, these results are consistent with a previously proposed model of H-strand transcription in the form of a single polycistronic molecule.

scholarly journals The control of ribonucleic acid synthesis in bacteria. Steady-state content of messenger ribonucleic acid in Escherichia coli M.R.E. 600

1970 ◽  
Vol 120 (2) ◽  
pp. 279-288 ◽  
Author(s):  
W. J. H. Gray ◽  
J. E. M. Midgley
Keyword(s):  
Escherichia Coli ◽  
Ribosomal Rna ◽  
Ribonucleic Acid ◽  
Messenger Rna ◽  
Acid Synthesis ◽  
Growth Conditions ◽  
Transfer Rna ◽  
Average Lifetime ◽  
Messenger Ribonucleic Acid ◽  
Wide Range

1. The technique of DNA–RNA hybridization was used to follow changes in the amount and average lifetime of unstable messenger RNA in Escherichia coli M.R.E. 600 over a wide range of different growth conditions. The method of analysis was based on the kinetics of incorporation of exogenous labelled nucleic acid bases into the RNA of steadily growing cultures, as described by Bolton & McCarthy (1962). 2. The ratio of the average lifetime of messenger RNA to the mean generation time of E. coli cultures was constant over the temperature range 25–45°C in a given medium, but the constant varied with the nature of the growth medium. For cultures growing in sodium lactate–salts or glucose–salts media the ratio was 0.046±0.005 and in enriched broth it was 0.087±0.009. Measurements of the amounts of transfer RNA, ribosomal RNA and messenger RNA were also made. The results confirmed earlier reports that the ratio of the amount of messenger RNA to the amount of ribosomes in the cells is virtually constant. On the other hand, the ratio of the amount of transfer RNA to the amount of ribosomal RNA decreased with increasing growth rate at a given temperature. 3. In cultures at temperatures higher than necessary for optimum rates of growth the average lifetime of messenger RNA lengthened in harmony with the increased time required for cell division. It seems that suboptimum growth rates at higher temperatures cannot be explained simply as a combination of increased rates of synthesis and breakdown of messenger RNA with a grossly decreased efficiency of translation. The absolute rate of messenger RNA synthesis was lowered, and its amount in the cells was typical of all other cultures grown at lower temperatures in the same medium. 4. The rate of entry of exogenous labelled uracil into unstable messenger RNA and stable ribosomal RNA was constant in all media at all temperatures in the approximate ratio 1:2. In media supporting a lower rate of growth, e.g. lactate–salts or glucose–salts media, the messenger RNA fraction constituted 2.2±0.3% of the total cellular RNA. In enriched broth 3.6±0.3% of the total RNA was messenger.

scholarly journals A method for measuring the content of a specific messenger ribonucleic acid in Escherichia coli

1971 ◽  
Vol 121 (1) ◽  
pp. 105-108 ◽  
Author(s):  
J. O. Bishop ◽  
M. I. Irving
Keyword(s):  
Escherichia Coli ◽  
Ribosomal Rna ◽  
Ribonucleic Acid ◽  
Messenger Rna ◽  
Model System ◽  
Total Rna ◽  
Messenger Ribonucleic Acid

A method is described for measuring the porportion of a specific messenger RNA in the total RNA extracted from pulse-labelled cells. A model system consisting of total ribosomal RNA and Escherichia coli DNA is used to validate the method and to define the conditions under which it can be used.

The appearance and quantitation of cytoplasmic ribonucleic acid in the early chick embryo

Development ◽  
1972 ◽  
Vol 28 (2) ◽  
pp. 367-384
Author(s):  
C. C. Wylie
Keyword(s):  
Chick Embryo ◽  
Ribosomal Rna ◽  
Rna Synthesis ◽  
Specific Activity ◽  
Cell Number ◽  
Present Knowledge ◽  
Precursor Pool ◽  
Cytoplasmic Rna ◽  
Labelling Experiment ◽  
Neurula Stage

This paper seeks to extend our knowledge about RNA synthesis in early embryogenesis to the domestic fowl, Gallus domesticus. Using this species for research, apart from increasing our knowledge of higher vertebrate embryology, has certain advantages such as rapid uptake of isotopic precursors and ease of microdissection in culture. The following results are presented: (1) The cell number in the whole chick embryos is shown to be increasing logarithmically between the time of laying and the early neurula stage; with a doubling time of 7·4 h. (2) The onset of ribosomal RNA synthesis has been shown to be during mid-cleavage of the chick embryo, while development is taking place in the oviduct and uterus of the mother. (3) In a cumulative labelling experiment, embryos were labelled at the unincubated-egg stage, allowed to develop to various morphological stages up to neurulation, and their cytoplasmic RNA prepared and analysed by gel electrophoresis. (4) The specific activity of the precursor pool for RNA synthesis was measured at several stages, using the same labelling conditions, and the results were used to quantitate the RNA synthesis from the incorporated radioactivity. (5) Using these techniques, it was found that newly synthesized cytoplasmic RNA accumulates steadily in the whole chick embryo, reaching a level of 104 μg by the early neurula stage. On a per cell basis, however, the amount of newly synthesized cytoplasmic RNA seems to decrease slightly. These findings are discussed in the light of present knowledge about embryos of other vertebrates and certain invertebrates.

scholarly journals Rate of synthesis and half-life of globin messenger ribonucleic acid. Rate of synthesis of globin messenger ribonucleic acid calculated from data of cell haemoglobin content

1974 ◽  
Vol 138 (3) ◽  
pp. 499-510 ◽  
Author(s):  
John A. Hunt
Keyword(s):  
Ribonucleic Acid ◽  
Half Life ◽  
Chain Elongation ◽  
Erythroid Cell ◽  
Synthesis Rate ◽  
Mrna Synthesis ◽  
Base Pairs ◽  
Messenger Ribonucleic Acid ◽  
Nucleotide Base ◽  
Mrna Half Life

By the use of the favoured models defining mRNA synthesis and half-life from the preceding paper (Hunt, 1974) and the known content of globin in a reticulocyte it is possible to estimate the absolute rate of mRNA and globin synthesis and the mRNA and globin content in each type of erythroid cell. The best model requires an mRNA-synthetic rate of 3000 molecules per h/cell. This rate compares favourably with the estimated chain-extension rate of 43 nucleotides/s in Escherichia coli (Manor et al., 1969) provided that the four α- and β-chain cistrons per cell are transcribed by polymerases spaced 50 nucleotide base pairs apart. Similar calculations can be made for erythropoiesis in the chick embryo, where cell times and relative globin content at each mitosis have been measured (Campbell et al., 1971), but where no reliable estimates of mRNA half-life have been made. In this case it was estimated that a constant rate of mRNA synthesis at 10000 molecules per h/cell through six cell divisions is necessary if the mRNA half-life is 15h; after the sixth mitosis the mRNA synthesis would stop and its half-life would increase to approx. 20h. If an mRNA half-life of 4.5h is used, the synthesis rate through the six mitoses would be 21000 molecules per h/cell, ceasing at the sixth mitosis, when the half-life would need to increase to 25h. The chain-elongation rate for the four α- and β-globin cistrons per cell would be 1–2 times higher than in E. coli and would either require a greater rate, polymerases spaced between 25 and 50 nucleotide base pairs apart on the DNA, or limited gene replication. These possibilities are discussed in the light of the low values found for globin cistron multiplicity in ducks and mice.

scholarly journals Deoxyribonucleic acid–ribonucleic acid hybridization. Annealing and quantitative recovery of intact ribosomal ribonucleic acid molecules from hybrids

1969 ◽  
Vol 115 (2) ◽  
pp. 287-294 ◽  
Author(s):  
Michael Fry ◽  
Michael Artman
Keyword(s):  
Ribosomal Rna ◽  
Ribonucleic Acid ◽  
Messenger Rna ◽  
Deoxyribonucleic Acid ◽  
Cellulose Nitrate ◽  
Absolute Size ◽  
Rna Molecules ◽  
Membrane Filters ◽  
Ribosomal Ribonucleic Acid

A simple and efficient method for hybridization and subsequent recovery of non-fragmented ribosomal RNA from the hybrid is described. The procedure involves annealing of immobilized denatured DNA bound on cellulose nitrate membrane filters to complementary RNA in 50% (v/v) formamide–0·33m-potassium chloride–10mm-tris–hydrochloric acid buffer, pH7·4, at 33° for 3hr. Under these conditions no detectable changes in the sedimentation coefficients of the input RNA were detected. The RNA can subsequently be recovered quantitatively from the hybrid in intact form by incubating the filters in formamide or in 85% (v/v) dimethyl sulphoxide. The applicability of the method for the evaluation of the absolute size of ribosomal RNA cistrons in Escherichia coli DNA and for the determination of the size of messenger RNA molecules is discussed.

Location of oligo(uridylic acid) sequences within messenger ribonucleic acid molecules of HeLa cells

Biochemistry ◽  
1985 ◽  
Vol 24 (14) ◽  
pp. 3678-3686 ◽  
Author(s):  
Joseph W. Kulkosky ◽  
William M. Wood ◽  
Mary Edmonds
Keyword(s):  
Hela Cells ◽  
Ribonucleic Acid ◽  
Messenger Ribonucleic Acid

scholarly journals Characterization of rapidly labelled ribonucleic acid in Escherichia coli by deoxyribonucleic acid–ribonucleic acid hybridization

1968 ◽  
Vol 110 (2) ◽  
pp. 251-263 ◽  
Author(s):  
G. H. Pigott ◽  
J. E. M. Midgley
Keyword(s):  
Escherichia Coli ◽  
Ribosomal Rna ◽  
Ribonucleic Acid ◽  
Messenger Rna ◽  
Rna Binding ◽  
Hybridization Technique ◽  
E Coli ◽  
K 12 ◽  
Dna 2

1. Rapidly labelled RNA from Escherichia coli K 12 was characterized by hybridization to denatured E. coli DNA on cellulose nitrate membrane filters. The experiments were designed to show that, if sufficient denatured DNA is offered in a single challenge, practically all the rapidly labelled RNA will hybridize. With the technique employed, 75–80% hybridization efficiency could be obtained as a maximum. Even if an excess of DNA sites were offered, this value could not be improved upon in any single challenge of rapidly labelled RNA with denatured E. coli DNA. 2. It was confirmed that the hybridization technique can separate the rapidly labelled RNA into two fractions. One of these (30% of the total) was efficiently hybridized with the low DNA/RNA ratio (10:1, w/w) used in tests. The other fraction (70% of the total) was hybridized to DNA at low efficiencies with the DNA/RNA ratio 10:1, and was hybridized progressively more effectively as the amount of denatured DNA was increased. A practical maximum of 80% hybridization of all the rapidly labelled RNA was first achieved at a DNA/RNA ratio 210:1 (±10:1). This fraction was fully representative of the rapidly labelled RNA with regard to kind and relative amount of materials hybridized. 3. In competition experiments, where additions were made of unlabelled RNA prepared from E. coli DNA, DNA-dependent RNA polymerase (EC 2.7.7.6) and nucleoside 5′-triphosphates, the rapidly labelled RNA fraction hybridized at a low (10:1) DNA/RNA ratio was shown to be competitive with a product from genes other than those responsible for ribosomal RNA synthesis and thus was presumably messenger RNA. At higher DNA/rapidly labelled RNA ratios (200:1), competition with added unlabelled E. coli ribosomal RNA (without messenger RNA contaminants) lowered the hybridization of the rapidly labelled RNA from its 80% maximum to 23%. This proportion of rapidly labelled RNA was not competitive with E. coli ribosomal RNA even when the latter was in large excess. The ribosomal RNA would also not compete with the 23% rapidly labelled RNA bound to DNA at low DNA/RNA ratios. It was thus demonstrated that the major part of E. coli rapidly labelled RNA (70%) is ribosomal RNA, presumably a precursor to the RNA in mature ribosomes. 4. These studies have shown that, when earlier workers used low DNA/RNA ratios (about 10:1) in the assay of messenger RNA in bacterial rapidly labelled RNA, a reasonable estimate of this fraction was achieved. Criticisms that individual messenger RNA species may be synthesized from single DNA sites in E. coli at rates that lead to low efficiencies of messenger RNA binding at low DNA/RNA ratios are refuted. In accordance with earlier results, estimations of the messenger RNA content of E. coli in both rapidly labelled and randomly labelled RNA show that this fraction is 1·8–1·9% of the total RNA. This shows that, if any messenger RNA of relatively long life exists in E. coli, it does not contribute a measurable weight to that of rapidly labelled messenger RNA.

scholarly journals Direct association of messenger RNA labeled in the presence of fluoroorotate with membranes of the endoplasmic reticulum in rat liver.

1976 ◽  
Vol 70 (1) ◽  
pp. 47-58 ◽  
Author(s):  
J Cardelli ◽  
B Long ◽  
H C Pitot
Keyword(s):  
Endoplasmic Reticulum ◽  
Ribosomal Rna ◽  
Messenger Rna ◽  
Translational Regulation ◽  
Orotic Acid ◽  
Complete Removal ◽  
High Ionic Strength ◽  
Messenger Rnas ◽  
Direct Association ◽  
Pancreatic Rnase

Liver rough endoplasmic reticulum (RER) membranes were isolated from rats given [3H]orotic acid for 48 h (ribosomal RNA [rRNA] label) or for 3 h along with 5-fluoroorotate; this latter procedure permits the labeling of cytoplasmic messenger RNAs (mRNAs) in the absence of rRNA labeling. More than 50% of the labeled mRNA remained attached to membranes of the RER after complete removal of ribosomes with a buffer of high ionic strength in the presence of puromycin. Under similar conditions, membranes retained 40% of their polyadenylate as determined by a [3H]-polyuridylate hybridization assay. Treatment of mRNA-labeled endoplasmic reticulum membranes with pancreatic RNase indicates that the polyadenylate and possibly nonpolyadenylate-pyrimidine portions of the messenger are involved in the binding of mRNA to the membranes. The implication of these results in furthering our understanding of the mechanisms of the translational regulation of genetic expression is discussed.

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