Encapsidation of Sendai virus genome RNAs by purified NP protein during in vitro replication.

The ability of the Sendai virus major nucleocapsid protein, NP, to support the in vitro synthesis and encapsidation of viral genome RNA during Sendai virus RNA replication was studied. NP protein was purified from viral nucleocapsids isolated from Sendai virus-infected BHK cells and shown to be a soluble monomer under the reaction conditions used for RNA synthesis. The purified NP protein alone was necessary and sufficient for in vitro genome RNA synthesis and encapsidation from preinitiated intracellular Sendai virus defective interfering particle (DI-H) nucleocapsid templates. The amount of DI-H RNA replication increased linearly with the addition of increasing amounts of NP protein. With purified detergent-disrupted DI-H virions as the template, however, there was no genome RNA synthesis in either the absence or presence of the NP protein. Furthermore, addition of the soluble protein fraction of uninfected cells alone or in the presence of purified NP protein also did not support DI-H genome RNA synthesis from purified DI-H. Another viral component in addition to the NP protein appears to be required for the initiation of encapsidation, since the soluble protein fraction of infected but not uninfected cells did support DI-H genome replication from purified DI-H.     

On the mechanism of erythropoietin-induced differentiation. XII. A cytoplasmic protein mediating induced nuclear RNA synthesis

Erythropoietin, the primary inducer of red blood cell differentiation, has no effect on RNA synthesis by isolated bone marrow nuclei. A cytoplasmic fraction from marrow cells exposed to erythropoietin does, however, stimulate RNA synthesis by such nuclei. This marrow cell cytoplasmic factor (MCF) also stimulates RNA synthesis by liver and kidney nuclei, whereas erythropoietin has no effect on intact kidney or lung cells. MCF appears rapidly in cells after addition of erythropoietin, and its formation does not require protein synthesis. MCF is inactivated by trypsin, but not by ribonuclease. The data suggest that erythropoietin acts on the responsive cells to generate a cytoplasmic protein that mediates the effect of the hormone on nuclear RNA synthesis.     

Mutagenic SOS repair of site-specific psoralen damage in plasmid pBR322

The mutagenic repair of psoralen damage was examined by transforming Escherichia coli with psoralen-treated pBR322. Plasmid DNA randomly reacted with psoralen was repaired only when the E. coli was uvrA⁺ and recA⁺, and only when the cells were pre-irradiated with far-ultraviolet light. The recA dependence and requirement for pre-irradiation are characteristics of SOS repair.Psoralens were placed specifically near the BamHI site, in the tetracycline-resistance gene of pBR322, using a sulfhydryl-containing psoralen derivative. Repair of this damage also required pre-irradiation of the host cells. This repair was accompanied by a 4% frequency of mutagenesis to a tetraeycline-sensitive phenotype. Sequence analysis of these mutant plasmids revealed that 75% had mutations within the targeted region, while 25% had no sequence changes within 100 bases of the BamHI site. In up to five independent isolates only one kind of mutation was observed at each site, suggesting that mutagenic SOS repair is influenced by DNA structure at the site of the psoralen. Most mutations were transitions, primarily G-C to A-T changes. Some transitions occurred at sites where psoralen crosslinks could not have formed, and these may have arisen from the repair of psoralen monoadducts.     

Cell-free synthesis of tryptophanase from Escherichia coli. Use of ribonucleic acid isolated from induced cells and a comparison of the product from a system employing ribosomes with that from one employing ribosomes and exogenous ribonucleic acid

1. Polyribosomes and RNA were isolated from cultures in which tryptophanase (EC 4.2.1.-) was induced. The polyribosomes were incubated under conditions of protein synthesis, in the presence of a radioactive amino acid and a post-ribosomal supernatant fraction obtained from repressed cells. The RNA preparations were incubated under conditions of protein synthesis in the presence of a radioactive amino acid and a supernatant fraction containing ribosomes from repressed cells. 2. The system was characterized and the synthesis of a radioactive protein with the same chromatographic properties as tryptophanase was demonstrated. This synthesis was shown to be time-dependent and required the presence of RNA from induced cultures, ribosomes and an energy supply; it was inhibited by chloramphenicol. 3. The maximum activity for the synthesis of this protein was found to be associated with 23S rRNA isolated from sucrose gradients. 4. The N-terminal amino acid of tryptophanase was labelled in the protein synthesized in this system but not in the protein synthesized by polyribosomes (without added RNA). Conversely, the C-terminal amino acid of tryptophanase was labelled in the polyribosome system but not in the RNA-containing system. 5. Tryptic digests of protein labelled in vitro were compared with those of tryptophanase. No labelled tryptic peptides were identified other than tryptophanase tryptic peptides. An analysis of the results implied that in the polyribosome system almost the complete tryptophanase subunit chain was labelled but that in the RNA-containing system these chains were incompletely synthesized. 6. Sucrose-gradient analysis of protein synthesized in the RNA-containing system suggested that it cannot be converted into structures with the same sedimentation properties as native tryptophanase. 7. The significance of these results for the assay of tryptophanase mRNA and for an understanding of the control of the translation of this mRNA in vivo is discussed.     

Hormonal stimulation of 3H-orotic acid incorporation into RNA by serum-free cultured hepatocytes

Insulin and dexamethasone greatly stimulate the incorporation of 3H-orotic acid into RNA. Such a stimulation is associated to an increase in the uptake of the labelled precursor into the acid soluble fraction as well as in the the specific radioactivity of the nucleoside plus nucleotide pool suggesting that hormone supplementation does not affect RNA synthesis by cultured cells. The lack of effect of insulin and dexamethasone on the level of total RNA polymerase activity in nuclei isolated from cultured hepatocytes is in line with this assumption. The hormone stimulated uptake of orotic acid is dependent on protein synthesis since it is completely abolished by cycloheximide.     

RNA biosynthesis in mitochondria under conditions of prolonged inhibition of protein biosynthesis in rat liver cytoplasm]

When cytoplasmic protein synthesis is inhibited by cycloheximide (CHI) in vivo synthesis of water-soluble mitochondrial proteins and of mitochondrial RNA is decreased. These changes measured in isolated rat liver mitochondria are similar to those observed in vivo and correlate with the changes the synthesis of water-soluble proteins in mitochondria. When the cytoplasmic fraction (30,000 g-supernatant) had been added to the mitochondria showing decreased RNA synthesis, the RNA synthesis increased to the control level (the incubation conditions were favourable for the protein transport from microsomes to mitochondria). RNA synthesis in mitochondria was not stimulated by cytoplasmic fractions from the CHI-pretreated rats. After prolonged dialysis these fraction stimulated RNA synthesis even to a greater extent than cytoplasmic fractions from the untreated animals. Mitochondrial RNA polymerase activity (measured in mitochondrial extracts supplemented with exogenous DNA) was higher in extracts of mitochondria from livers of normal rats than in extracts of mitochondria from livers of animals injected with CHI.     

Free messenger ribonucleoprotein complexes of chicken primary muscle cells following modification of protein synthesis by heat-shock treatment

When primary cultures of chicken myoblasts were subjected to incubation at a temperature higher than their normal growing temperature of 36-37 degrees C, the pattern of protein synthesis was altered. This condition of heat shock induced a vigorous production of a number of proteins collectively known as 'heat-shock proteins'. The synthesis of heat-shock proteins was achieved without a significant decrease in the production of a broad spectrum of proteins by muscle cells. The synthesis of three major heat-shock polypeptides with Mr values of 81 000, 65 000 and 25 000 was observed in both mononucleated dividing myoblast cells and terminally differentiated myotubes. Two-dimensional electrophoretic separation of the heat-induced polypeptides synthesized by myogenetic cultures further established that same set of polypeptides with Mr of 65 000 (pI 6.0 and 5.5), 81 000 (pI 6.2) and 25 000 (pI 5.6 and 5.3) were produced in myoblasts and myotubes. The effect of the changes in pattern of protein synthesis on the mRNA and protein moieties of non-polysomal cytoplasmic mRNA-protein complexes (free mRNP) was examined. Free mRNP complexes sedimenting at 20-35 S were isolated from the post-ribosomal supernatant of both normal and heat-shocked myotube cultures by centrifugation in a sucrose gradient. A 10-20S RNA fraction isolated from these complexes stimulated protein synthesis in a cell-free system. The RNA fraction obtained from heat-shocked cells appeared to direct the synthesis of all three major heat-shock proteins. In contrast, synthesis of these polypeptides was not detected when RNA from free mRNP complexes of normal cells was used for translation. The free mRNP complexes of both normal and heat-shocked cells showed a buoyant density of 1.195 g/cm3 in metrizamide gradients. A large number of polypeptides of Mr = 35 000-105 000 were present in the highly purified free mRNP complexes isolated from the metrizamide gradient. Similar sets of polypeptides were found in these complexes from both normal and heat-shocked myotube culture. However, the relative proportion of a 65 000-Mr polypeptide was dramatically increased in the free mRNP complexes of heat-shocked cells. Two-dimensional gel electrophoretic analysis revealed that this polypeptide and the 65 000-Mr heat-shock polypeptide exhibit similar electrophoretic migration properties. These observations suggest that, following heat-shock treatment of chicken myotube cultures, the changes in the pattern of protein synthesis is accompanied by alteration of the mRNA and protein composition of free mRNP complexes.     

Control of ribosomal protein synthesis during wheat embryo imbibition

Dry wheat embryos contain large quantities of ribosomes, synthesized and assembled during embryogenesis. When messenger RNA isolated from dry embryos is translated, in vitro, a significant proportion of the total translation products (approx. 10%) is identifiable as ribosomal proteins, by electrophoresis in two distinct two-dimensional polyacrylamide gel electrophoretic systems. When germinating embryos are labelled with [³⁵S]methionine, during the first 24 h of imbibition, the appearance of newly synthesized ribosomal proteins in the cytosolic fraction is barely detectable. However, this low level (< 1% of total cytosolic protein synthesis) of observed ribosomal protein synthesis is not correlated with a correspondingly low level of ribosomal protein mRNA. Ribosomal proteins constitute at least 10% of the products of translation, in vitro, of mRNA isolated from germinating wheat embryos. Ribosomal proteins are also conspicuous products of translation when polyribosomes isolated from imbibing embryos are used to direct protein synthesis in a cell-free ‘run-off’ system, and newly synthesized ribosomal proteins can be detected in the nuclei isolated from germinating embryos. It is proposed that their absence from the cytosolic fraction is a consequence of post-translational regulatory events.     

RNA synthesis in isolated HeLa cell nuclei.

RNA synthesis has been studied in isolated nuclei of HeLa cells. The incubation medium has been optimized for RNA synthesis and the requirements for the presence of specific components previously used by other investigators has been examined. Nuclei isolated by centrifugation through 2 M sucrose synthesize RNA linearly for at least 1 h only at low temperature (25 degrees C). Low molecular weight RNA is found in the supernatant fraction after incubation; this RNA accounts for about 10% of the RNA synthesized. The RNA which remains within nuclei is of high molecular weight and processing of this RNA into molecules of the size of cytoplasmic mRNA does not seem to occur in isolated nuclei. We have studied the effect of an inhibitor of protein-nucleic acid interaction - aurintricarboxylic acid - on RNA synthesis by isolated nuclei. At concentrations below 0.1 mM, this drug does not inhibit RNA synthesis effectively, whereas at concentrations above 0.1 mM it inhibits RNA synthesis by about 80%. In view of the proposed mechanism of action of aurintricarboxylic acid, we suggest that completion of nucleotide chains initiated before nuclei isolation accounts for 20% of the RNA synthesized in our system by isolated nuclei, whereas nucleotide chains initiated during the in vitro incubation account for 80% of the RNA synthesized.     

RNA synthesis by villus and crypt cell nuclei of rat intestinal mucosa

Rat intestinal mucosa was separated by eversion and vibration to provide a sequence of fractions from predominantly villus cells to predominantly crypt cells. The proportions of these cell types in each fraction were computed from the concentrations of alkaline phosphatase (villus cells) and thymidine kinase (crypt cells) in each population. The isolated mucosal fractions varied from about 90% villus cells to 90% crypt cells. Following injection of the rats with [³H]thymidine, the nuclei were isolated from each mucosal cell fraction and the amount of radioactivity incorporated into DNA was measured as an index of crypt cell abundance. The isolated nuclei were also incubated with ribonucleoside triphosphates and the amount of RNA synthesized was measured. Nuclei labeled with [³H]thymidine were found only in fractions rich in crypt cells, whereas capacity for RNA synthesis remained very active in mucosal fractions consisting predominantly of villus cells. It is concluded that non-dividing villus cells continue to make RNA.     

Macromolecular synthesis in E. coli, isolated mitochondria and L5178Y cells in the presence of hydroxyurea or formamidoxime

The effects of formamidoxime and hydroxyurea over a 10⁵ concentration range were studied on macromolecular synthesis in E. coli, L5178Y mouse leukemic cells, isolated rat liver mitochondria and isolated rat cerebral cortex mitochondria. In E. coli 2 mg per ml of formamidoxime and hydroxyurea inhibited, respectively, RNA synthesis by 20% and 17%, DNA synthesis by 91% and 96%, protein synthesis by 54% and 60% and lipopolysaccharide synthesis by 65% and 48%. In L5178Y mouse leukemic cells 2 mg/ml of formamidoxime and hydroxyurea inhibited, respectively, RNA synthesis by 41% and 24%, DNA synthesis by 90% and 97%, protein synthesis by 59% and 44% and glycoprotein synthesis by 83% and 50%. In isolated rat liver mitochondria 2 mg/ml of formamidoxime and hydroxyurea inhibited, respectively, RNA synthesis by 43% and 52%, DNA synthesis by 42% and 56% and protein synthesis by 18% and 30%. Glycoprotein synthesis was not affected. In isolated rat cerebral cortex mitochondria 2 mg/ml of formamidoxime and hydroxyurea inhibited, respectively, RNA synthesis by 50% and 44%, DNA synthesis by 59% and 66% and protein synthesis by 48% and 40%. Glycoprotein synthesis again was not affected. Lower concentrations of the drugs produced less inhibition of macromolecular synthesis in each of the systems.     

Role of the Cytoplasmic Membrane in the Synthesis of Ribonucleic Acid by Disrupted Spheroplasts of Pseudomonas schuylkilliensis

Disrupted spheroplast preparations of Pseudomonas schuylkilliensis strain P contained fragments of cytoplasmic membrane and approximately 82% of the total cellular phospholipid. The protoplast-bursting factor (PB-factor), partially purified from pig pancreas, and a heat-treated pancreatic lipase fraction both inhibited ribonucleic acid (RNA) synthesis by disrupted spheroplasts but did not inhibit or only slightly inhibited RNA synthesis by intact cells or intact spheroplasts. The PB-factor preparation and the heat-treated pancreatic lipase fraction catalyzed partial (15 to 50%) deacylation of diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine in disrupted spheroplasts but not in intact spheroplasts. Phospholipase A activity was demonstrated in the PB-factor preparation by use of isolated phospholipids as substrates. Treatment of disrupted spheroplasts with the PB-factor preparation caused a 70% inhibition in oxidative phosphorylation and RNA synthesis, but had little effect on electron transport. Addition of adenosine-5'-triphosphate or adenosine-5'-diphosphate and a mixture of ribonucleosides after treatment with the PB-factor preparation partially restored oxidative phosphorylation but did not relieve the inhibition in RNA synthesis. The most reasonable explanation for the latter observation appears to be that the concentrations of newly synthesized nucleotides retained by the preparations with partially deacylated membrane phospholipids were insufficient to permit the synthesis of RNA.     

Separation and characterization of low-molecular-weight nuclear RNA species that inhibit cell-free protein synthesis

Various RNA fractions were isolated from nuclei of 12-day lactating rat mammary glands and examined for their ability to inhibit cell-free protein synthesis. Although total nuclear RNA was generally inactive, material contained in the poly(A)+ nuclear RNA fraction and the low-molecular-weight RNA derived from total nuclear RNA by sucrose gradient centrifugation, inhibited the translation of several mRNAs but not poly(U) or poly(A). Separation of the small nuclear RNAs by preparative polyacrylamide-urea gel electrophoresis allowed the identification of at least three active inhibitor RNA species. These differed both with respect to their ability to inhibit protein synthesis, and in their mechanism of action. While two of the RNA species inhibited elongation the other inhibited initiation of protein synthesis.     

Glucocorticoid-mediated selective reduction of functioning collagen messenger ribonucleic acid

Polysomes were isolated from both lung and dermis of neonatal rats and the poly(A) RNA therein was isolated by oligo(dT)-cellulose chromatography. The RNA fractions were then translated in the nuclease-treated reticulocyte lysate in the presence of radioactive proline. Optimal collagen and noncollagen protein synthesis directed by lung poly(A) RNA occurred at 0.7 mm magnesium and 100 mm potassium. The RNA fraction directed the synthesis of both proα₁ and proα₂ chains as determined by polyacrylamide gel electrophoresis. The poly(A) RNA isolated from both lung and dermis polysomes of rats treated with triamcinolone diacetate synthesized significantly less collagen peptides as determined by collagenase digestion as did the RNA isolated from polysomes of nontreated animals. Noncollagen protein synthesis was decreased to a lesser extent than collagen synthesis. Glucocorticoid treatment did not affect the ability of either polysomes in wheat germ extract or polysomal poly(A) RNA in nuclease-treated reticulocyte lysate to synthesize prolyl hydroxylase as determined by immunoprecipitation. These data indicate the glucorticoid-mediated inhibition of collagen polypeptide synthesis does not result from nonspecific effect on total cellular protein synthesis of normal fibroblasts, but a selective reduction of poly some-associated messenger RNA. Furthermore these data provide a molecular basis for selective inhibitory effect of synthetic anti-inflammatory steroids on collagen synthesis.