RNA Sedimenting Research Articles

Isolation and Characterization of 'Dense Particles' from Poliovirus-infected HeLa Cells

Dense poliovirus particles (buoyant density 1.44 g/ml in CsCl) isolated from infected HeLa cells contain the normal four structural polypeptides VP1 to VP4, and 35S poliovirus RNA. In addition, small amounts of VPo and single-stranded RNA sedimenting slower than the poliovirus genome are present. Dense particles have a low specific infectivity, are neutralized by type-specific poliovirus antisera, and are detected during growth as early as normal virus but disappear when virus production stops. They appear to represent a different, more open, conformation of the normal virus capsid.

Synthesis of mammalian mitochondrial rRNA at low temperature

L cells incubated at 19 °C synthesize mitochondrial rRNA but not cytosol rRNA. In addition to 16 and 13S mitochondrial rRNA, mitochondrial RNA sedimenting at higher S values was also synthesized. The processing of mitochondrial rRNA is slower at 19 °C than at 37 °C.

Poly(A)-containing polyribosomal RNA in sea urchin embryos: Changes in proportion during development

Evidence is described which suggests that a large fraction of sea urchin embryo polyribosomal RNA is not polyadenylated, and that the proportion of poly(A)-containing RNA increases during embryological development. Cleavage, early blastula, and mesenchyme blastula stage embryos were treated with cordycepin, and the effects of the drug on polyribosome appearance were determined. Polyribosomes in cleaving embryos and early blastulae were unaffected by the drug. However, there was a significant reduction in the amount of polyribosomes present and in the amount of newly synthesized RNA entering polyribosomes in mesenchyme blastulae after cordycepin treatment. Polyribosomal RNA was isolated from cleavage, early blastula, and mesenchyme blastula stage embryos, and the RNA was characterized by sucrose gradient centrifugation. Cleaving embryo polyribosomal RNA has a prominent 9 S peak and a smaller 20 S peak, and the larger RNA is heterodisperse. There is no poly(A) associated with the 9 S and 20 S material, but there is poly(A) associated with the RNA sedimenting faster than 20 S. Early blastula and mesenchyme blastula polyribosomal RNA have 22 S and 26 S peaks, respectively. This later stage RNA is quite heterodisperse and poly(A) is associated with RNA in all size classes larger than 9 S. Finally, the proportion of polyadenylated light and heavy polyribosome RNA was determined for cleavage, early blastula, and mesenchyme blastula stage embryos. Approximately 20–25% of newly synthesized light polyribosome RNA is polyadenylated and this amount is relatively constant during early development. However, the proportion of newly synthesized RNA in heavy polyribosomes that is polyadenylated increases during development: 25% at cleavage, 33% at early blastula, and 48% at mesenchyme blastula stages.

Messenger RNA species synthesized in vitro by the virion-associated RNA polymerase of vesicular stomatitis virus

The virion-associated RNA-dependent RNA polymerase of vesicular stomatitis virus (VSV) synthesizes in vitro two size classes of RNA products similar to those observed in VSV-infected cells. One RNA product sediments at 31S with an approximate molecular weight of 2.1 × 10 6. The smaller products consist of at least three classes of RNA sedimenting at 17S, 14.5S, and 12S with molecular weights of 0.7 × 10 6, 0.52 × 10 6, and 0.37 × 10 6, respectively. Hybridization experiments show that both the 31S and 12–18S RNA products are complementary to the genome RNA, and that each class is transcribed from different nucleotide sequences. From the molecular weights of the RNA species and the hybridization experiments, it seems that almost the entire VSV genome RNA is transcribed in vitro.

The messenger-like properties of the poly(A) + RNA in mammalian mitochondria

This is the first demonstration that the poly(A)-containing RNA from mammalian mitochondria behaves like messenger RNA in mitochondrial specific protein synthesis. The mitochondrial RNA in HeLa cells is selectively labeled in the presence of camptothecin, the mitochondria isolated, lysed with weak detergents, and the mitochondrial contents sedimented in sucrose gradients. Much of the poly(A)-containing RNA sediments rapidly with polyribosome-like structures. Most important, the association of the poly(A) + RNA with heavy structures is disrupted quantitatively by puromycin, which should be selective for RNA engaged in protein synthesis. The disaggregation by puromycin is largely prevented by the simultaneous addition of chloramphenicol, which demonstrates association with mitochondrial protein synthesis. A minimum lifetime is estimated for mitochondrial RNA of from 1 1 2 –2 hr . This decay rate is observed after the block of new synthesis with either actinomycin or cordycepin. The decay of the poly(A) + RNA is faster than that of mitochondrial poly(A) −. The decay in ethidium bromide is much more rapid and constitutes additional evidence that ethidium has an overall deleterious effect on mitochondrial integrity. The lifetimes observed are long compared to the transcription time. In this sense, the mitochondrial messenger-like RNA more closely resembles eucaryotic rather than procaryotic mRNA.

Physico-chemical characterisation of rinderpest virus.

Rinderpest virus grown in Vero cells was purified by equilibrium sedimentation in potassium tartrate gradients. The characteristics of the virus and its RNA dependend on the passage history of the virus. Undiluted passage virus had a density ofc 1.21 g/ml in potassium tartrate and contained RNA sedimenting atc. 20S and 5S. Virus obtained by dilute passage had a density ofc. 1.24 g/ml and contained 50S in addition to 20S and 5S RNA. Both diluted passage and undiluted passage viruses contained polypeptides with molecular weights of 98, 79, 75, 66, 48, 43 and 37×103. Trace amounts of polypeptides with molecular weights of 29 and 20×103 were also detected. The molecular weights of the major polypeptides are similar to those of the polypeptides present in measles and canine distemper viruses.

Transcription in vivo of a defective parvovirus: Sedimentation and electrophoretic analysis of RNA synthesized by adenovirus-associated virus and its helper adenovirus

We have studied viral RNA synthesized in KB cells coinfected with adenovirus-associated virus type 2 (AAV-2) and adenovirus type 2 (Ad2) as the helper. RNA was analysed in nonaqueous, denaturing solvents either by sucrose density gradient sedimentation in 99% dimethyl sulfoxide or by acrylamide gel electrophoresis in 98% formamide. The analyses revealed two populations of AAV RNA, (i) a single, discrete 20 S species and (ii) a very heterogenous population ranging in size from 4 S to 18 S. In the nucleus both populations of AAV RNA were observed but in the cytoplasm the 20 S RNA was mainly present in the polysome fraction and the heterogenous RNA was mainly in the nonpolysomal fraction. The 20 S RNA therefore appears to be the only functional AAV message species. The heterogenous AAV RNA may arise from incomplete transcription or degradation of the 20 S RNA in the nucleus or cytoplasm. The molecular weight of the 20 S AAV RNA was approximately 0.9 × 10 6–1.0 × 10 6 which accounts for the entire region of the AAV genome that is stably transcribed. This 20 S RNA may be monocistronic and thus AAV DNA may contain only one gene. The 20 S AAV RNA did not appear to be formed by posttranscriptional cleavage of a larger nuclear precursor. In the same experiments the posttranscriptional cleavage of Ad RNA was clearly observed. At least nine species of Ad RNA were present in the cytoplasm late after infection and at least six of these were associated with polysomes. Nonpolysomal, cytoplasmic Ad RNA was heterogenous but contained one discrete, 5.5 S species (VA RNA) which was transported to the cytoplasm more rapidly than either Ad or AAV message RNA or cellular rRNA.

In vitro synthesis of proteins by membrane-bound polyribosomes from vesicular stomatitis virus-infected HeLa cells.

Membrane-bound polysomes from vesicular stomatitis virus (VSV)-infected HeLa cells synthesize predominantly three proteins in an in vitro protein synthesizing system. These three proteins have different molecular weights than the viral structural proteins, i.e., 115,000, 88,000, and 72,000. Addition of preincubated L or HeLa cell S10 or HeLa cell crude initiation factors stimulates amino acid incorporation and, furthermore, alters the pattern of proteins synthesized. Stimulated membrane-bound polysomes synthesize predominantly viral protein G and lesser amounts of N, NS, and M. In vitro synthesized proteins G and N are very similar to virion proteins G and N based on analysis of tryptic methionine-labeled peptides. Most methionine-labeled tryptic peptides of virion G protein contain no carbohydrate moieties, since about 90% of sugar-labeled peptides co-chromatograph with only about 10% of methionine-labeled peptides. Sucrose gradient analysis of the labeled RNA present in VSV-infected membrane-bound polysomes reveals a relative enrichment in a class of viral RNA sedimenting slightly faster than the total population of the 13 to 15S mRNA, as compared to a VSV-infected crude cytoplasmic extract. A number of proteins, other than the viral structural proteins, are synthesized in the cytoplasm of five lines of VSV-infected cells. One of these proteins has the same molecular weight as the major in vitro synthesized protein, P(88). In vitro synthesized protein P(88) does not appear to be a precursor of viral structural proteins G, N, or M based on pulse-chase experiments and tryptic peptide mapping. Nonstimulated membrane-bound polysomes from uninfected HeLa cells synthesize the same size distribution of proteins as nonstimulated VSV-infected membrane-bound polysomes.

In Vitro Transcription of Xenopus Mitochondrial Deoxyribonucleic Acid by Homologous Mitochondrial Ribonucleic Acid Polymerase

Abstract The transcription in vitro of mitochondrial DNA by mitochondrial RNA polymerase from Xenopus laevis has been studied. Nearest neighbor frequencies of the transcribed RNA showed only small deviations from a random pattern. Nascent RNA is bound to the template such that 25 to 35% of the RNA is in a state protected from digestion with ribonuclease. The RNA sediments between 4 and 12 S in sucrose gradients after its release from the template. Mitochondrial RNA polymerase transcribes mtDNA with considerable strand selectivity. With mtDNA-I (closed circular DNA) the heavy (H) strand is preferred about 3:1. With mtDNA-II (nicked circular DNA) the H strand is preferred 20:1. In contrast, denatured mtDNA is transcribed with a 5:1 preference for the light (L) strand. Study of the inhibition of enzyme activity by the rifamycin derivative AF/ABDP suggests that the chain initiation step is more sensitive to the inhibitor than the elongation step. RNA chain initiation in vitro was studied with γ-32P-ribonucleoside triphosphates. Mitochondrial RNA polymerase initiates on mtDNA predominantly with ATP, 2-fold less frequently with GTP, and rarely if at all with the pyrimidine ribonucleoside triphosphates. In pancreatic RNase digests of terminally labeled RNA, at least three but probably many 32P-labeled oligonucleotides were observed, suggesting that in vitro chain initiation occurred at a number of sites on the template.

Studies on the polyribosome-associated RNA in BHK21 cells infected with Semliki Forest virus

We have developed a method that allows us to isolate with high yield RNP particles containing intact RNA from BHK21 cells infected with Semliki Forest virus. Two types of virus-specific RNA sedimenting with about 42 S and 26 S on sucrose gradients are found in the gradient fractions containing the large and intermediate-sized polyribosomes. That the 26 S RNA and at least 50% of the 42 S RNA are presumably part of the polyribosomes has been shown by CsCl density gradient analysis of the fixed ribonucleoprotein particles. We suggest therefore that both molecules are virus specific mRNA. From the quantitative aspects of our results, we conclude that they are the main species of virus specific polyribosome associated RNA in the infected cells. The total virus-specific RNA synthesized during different 2 hr intervals, which is incorporated into polyribosomes, is greater than 20% during the 0 to 2 hr p.i. interval and decreases later. We suggest therefore that especially during the first hours after infection, a major part of the total RNA synthetic capacity of the virus is directed toward mRNA production. Both polyribosome-associated molecules do not hybridize to RNA extracted from the SFV particle.

A precursor to hemoglobin mRNA in nuclei of immature duck red blood cells

A method is described for sedimenting RNA on a preparative scale in the presence of 85% formamide. It is likely that the RNA is fully denatured, since under the same conditions ribosomal RNA develops full hyperchromicity, and formaldehyde-treated and untreated ribosomal and nuclear RNA sediment in an identical manner. RNA from immature duck red blood cells was fractionated on sucrose gradients. The amount of hemoglobin mRNA and poly(A) in each fraction was measured by hybridization with complementary DNA and poly(U), respectively. We observed that hemoglobin mRNA forms aggregates in the presence of phenol, which are dispersed on formamide gradients. Nuclear RNA became more heavily aggregated, and again the aggregates were dispersed by formamide. A possible nuclear precursor of hemoglobin mRNA was identified. The molecular weight of the precursor is 6–7 × 10 5, three times as long as hemoglobin mRNA, and it is attached covalently to a poly(A) sequence.

Expression of the mitochondrial genome in HeLa cells: XIX. Occurrence in mitochondria of polyadenylic acid sequences, “free” and covalently linked to mitochondrial DNA-coded RNA

In the present work we have investigated the distribution, among the components of a mitochondrial Triton X100 lysate from HeLa cells, of poly(A) sequences labeled during a two-hour [8-3H]adenosine pulse in the presence of 0.1 μg actinomycin D ml; these were characterized as to their electrophoretic and sedimentation properties, response to ethidium bromide, physical relationship to mitochondrial RNA molecules and homology to separated mitochondrial DNA strands.Labeled poly(A) segments of a size estimated to correspond to 60 to 80 nucleotides were present in the components of a mitochondrial lysate sedimenting in the mitochondrial polysome and riboaome-subunit region and in the lighter components, and were especially abundant in the heaviest structures (>200 S). The latter structures were characterized as containing mostly labeled RNA components heavier than 18 S ribosomal RNA, but also a considerable amount of newly synthesized and pre-existing mitochondrial ribosomal RNA, with only a relatively small amount of protein synthesizing structures.Substantially all the poly(A) associated with the slowest sedimenting components of the lysate was found to be “free”The labeling of the poly(A) sequences during a two-hour [8-3H]adenosine pulse was found to be partially resistant to ethidium bromide at 1 μg/ml. The ethidium bromide-resistant fraction included most of, and possibly all the “free” poly(A), but also some poly(A) covalently linked to (presumably) unlabeled RNA molecules. These findings strongly suggest that poly(A) synthesis is in mitochondria, as in the nucleus, a post-transcriptional event, and that, in the presence of the drug, addition of newly synthesized poly(A) occurs to pre-existing precursor molecules. RNA-DNA hybridization experiments showed that two-thirds to one-half of the poly(A)-containing mitochondrial BNA molecules labeled during a two-hour [5-3H]uridine or [8-3H]adenosine pulse, and isolated from the mitochondrial polysome or monomer-subunit region of the gradient, are coded for by the heavy mitochondrial DNA strand, and the rest by the light strand. A fairly sharp peak of mitochondrial DNA-coded, poly(A)-containing RNA sedimenting at about 7 S in the native state and about 20% faster than 5 S RNA after formaldehyde treatment, which may represent a discrete species of mitochondrial messenger RNA, has been identified.

Cell-free translation of the globin message within polydisperse high-molecular-weight ribonucleic acid of avian erythrocytes.

Polydisperse high-molecular-weight RNA of nucleated avian erythrocytes includes sequences coding for globin chains. The RNA was extracted from immature erythrocytes of ducks and fractionated under denaturing conditions by sucrose density gradient centrifugation in 99% dimethylsulfoxide. The RNA sedimenting faster than 45 S was able to direct the synthesis of duck globins in the Krebs II ascites cell-free protein-synthesizing system. The newly synthesized globin molecules have been identified by their characteristic electrophoretic properties in polyacrylamide gels containing either urea or sodium dodecyl sulfate, and by immunoprecipitation of the released globin chains by rabbit antibodies against duck hemoglobin. In order to rule out the possibility of a contamination of the high-molecular-weight RNA with duck-globin messenger RNA tailing from the 9-10S region, rabbit-globin messenger RNA was added to duck RNA as an internal control. No rabbit-globin messenger RNA activity could be detected in the RNA fractions sedimenting faster than 45 S. It is concluded that high-molecular-weight RNAs in the nucleated erythroid cell contain sequences of globin messenger RNAs covalently attached to larger polynucleotide chains. These results support the view that polydisperse nuclear RNA is the precursor of the cytoplasmic messenger RNA fraction.

Demonstration of a DNA provirus in the lytic growth of visna virus.

SEVERAL lines of evidence link the RNA viruses responsible for slow diseases in Icelandic sheep with RNA tumour viruses. Visna virus, the prototype of the RNA slow virus group, resembles RNA tumour viruses in its morphology, in maturating by budding from the surfaces of infected cells1 and in having a high molecular weight RNA sedimenting at 60–70S2–4 and a virion-associated RNA-directed DNA polymerase (reverse transcriptase)5–7. However, the RNA slow viruses differ from tumour viruses, in that they do not cause tumours in the natural host or cell transformation in sheep tissue culture cells8. Visna virus in sheep produces an inflammatory demyelinating disease not associated with tumour formation; in tissue culture, infection of ovine cells leads to virus production, syncytium formation and lysis of cells.

Size distributions of high molecular weight RNA synthesized by L1210 cells Effects of DNA-reactive drugs

Formaldehyde treatment was used to obtain size-dependent sedimentation of high molecular weight nucleolar and nucleoplasmic RNA from normal L1210 cells, and from cells treated with drugs thought to affect the size distributions of the RNA synthesized. Reaction with formaldehyde at 70 °C to fully substitute the RNA was accompanied by significant RNA chain scission. Chain scission was minimized by carrying out a limited reaction at 40 °C. Since the reaction at 40 °C under these conditions was incomplete, however, the possible effects of persistant compact conformations and hidden breaks in the RNA were considered. The sedimentation patterns after various reaction times at 70 °C or 40 °C generally fit a simple model for random chain scission at rates of 1.7 · 10 −5 and 4.4 · 10 −7 breaks per nucleotide per min, respectively. Nucleoplasmic RNA after prolonged reaction at 40 °C, however, behaved as if it contained hidden breaks that resist mild formaldehyde treatment but that become exposed after more extensive treatment. Nucleoplasmic RNA synthesized by cells treated with intercalating agents exhibited an increase in sedimentation. This and the finding of hidden breaks may be due to nucleoplasmic RNA processing by cleavage at double-stranded segments, and its inhibition by intercalating agents. Nucleoplasmic RNA synthesized by cells treated with anthramycin or nitrogen mustard, agents that bind irreversibly to DNA, exhibited reduced sedimentation which was demonstrable both after formaldehyde treatment at 40 and 70 °C. The drug effects on sedimentation distributions obtained after mild formaldehyde reaction at 40 °C did not fit simple models of chain termination or DNA inactivation, and were as if a portion of the newly synthesized nucleoplasmic RNA was not susceptible to the drug-induced sedimentation reduction. The effects measured after formaldehyde treatment at 70 °C were equivalent to the introduction of additional random chain breaks due to drug.

A comparison of gel electrophoresis and density gradient centrifugation of heterogeneous nuclear RNA

Rapidly-labelled nRNA, isolated after extensive purification from HeLa cells, has been examined on sucrose density-gradients in conditions which reduce aggregation and intermolecular interactions. The results, which confirm and extend earlier observations, show that the rapid-labelling heterogeneous RNA sediments in a series of broad peaks which lie within or only slightly greater than the rRNA peaks. If this RNA is precipitated from the gradient and subjected to gel electrophoresis, it behaves as if it were of very high molecular weight and gives a virtually identical pattern to nRNA which had not been subjected to sucrose-gradient centrifugation. This provides decisive evidence that degradation has not taken place to account for the lower sedimentation velocity and that this species of RNA is not, therefore, of extremely high molecular weight as currently thought.

Turnover of ribosomal RNA in the submandibular gland of normal and isoproterenol-treated rats

From the decay in specific activity following the administration of 3H-cytidine and [ 3H-methyl]-methionine the half-life of ribosomal RNA in rat submandibular gland was determined. With [ 3H-methyl]methionine a half-life of 12 days of ribosomal RNA was obtained. This may be considered the minimal value since a shorter half-life, 7 days, was measured with 3H-cytidine as precursor. The difference in half-lives with the two methods of labeling is due to the presence of heterogeneous RNA sedimenting with the ribosomal RNA and labeled by 3H-cytidine but not methylated. An attempt was made to measure the half-life of ribosomal RNA in the submandibular gland during an accelerated hypertrophic and hyperplastic growth of the gland produced by the repeated administration of isoproterenol. When correction was made for the increase in RNA and ribosomal RNA that occurs during the enlargement of the gland the specific activity of ribosomal RNA increased instead of declining. During the experimental enlargement of the salivary gland the persistence of a radioactive pool excludes the possibility of measuring the turnover rate of ribosomal RNA accurately with methods presently available.

Characteristics of erythropoietin-induced RNA from foetal mouse liver erythropoietic cell cultures and the effects of 5-Fluorodeoxyuridine

The effect of erythropoietin on the synthesis of different RNA species in cultures of disaggregated mouse embryonic liver cells was investigated. Erythropoietin increased incorporation of uridine into ribosomal precursor RNA, “heterogeneous nuclear RNA” and the RNA sedimenting at greater than 60 S. There was no evidence for the appearance of new species of RNA. 5-Fluorodeoxyuridine (FdUrd) inhibited the stimulation of haemoglobin synthesis but only partially inhibited the stimulation of RNA synthesis caused by erythropoietin. Inhibition of DNA synthesis by FdUrd was not so pronounced in these cultures as might have been expected. Autoradiographs showed that erythropoietin stimulated RNA synthesis in proerythroblasts and basophilic and polychromatic erythroblasts but that the inhibition of this stimulation by FdUrd was confined to basophilic and polychromatic erythroblasts.

Presence of poly (A) in the polyribosome-associated RNA of Sindbis-infected BHK cells.

POLYNUCLEOTIDE sequences rich in adenylic acid (poly (A)) have been found in a variety of eukaryotic messenger, heterogenous nuclear (Hn) and viral RNAs1–5. Poly (A) tracts in HeLa and ascites cell polyribosome-associated RNA sediment at 4-5S in sucrose gradients and contain 85–99% AMP6,7. In Polyacrylamide gels, their mobility suggests a nucleotide content of 150–250 residues. Experiments using actinomycin D and cordycepin indicate that poly (A) in Hn RNA may be added after transcription8. Similar poly (A) sequences have been described in the early and late mRNA produced by vaccinia and adeno viruses9, 10. Vaccinia DNA contains dT sequences which hybridize with poly (A) and may code for the poly (A) in vaccinia mRNA. Adeno DNA does not contain sequences which can hybridize to poly (A) and it was suggested that the poly (A) in adeno mRNA arises, not by transcription of viral DNA, but by addition of cellular poly (A) to viral mRNA in the nucleus of infected cells10. We report here the isolation of poly (A) from viral-specific polyribosomes isolated from BHK 21 cells infected with Sindbis, a group A arbovirus which replicates exclusively in the cytoplasm.

Sedimentation and hybridization analysis of virus-specific RNA in the course of Newcastle disease virus replication cycle.

Self-annealing of 50 S virion RNA of Newcastle disease virus (egg-grown Beaudette strain) reveals no complementary base sequences. Virus-induced 18 S RNA consists exclusively of „minus“ strands,i.e. base sequences complementary to virion RNA. In 22 S RNA a small amount of “plus” strands is revealed both by annealing with an excess of virion RNA and by self-annealing. The sedimentation pattern of virus-specific RNA varies in the course of infection: an increase in the overall rate of synthesis towards the end of the reproduction cycle is due predominantly to the synthesis of smaller (18–35 S) RNA species. The content of “plus” strands in virus-induced RNA as revealed by self-annealing seems to depend mostly on the duration of exposure of cells to actinomycin D and not on the stage of infection.