The capacity of polyadenylated RNA from myogenic cells treated with actinomycin D to direct protein synthesis in a cell-free system

Cytoplasmic polyadenylated RNA of myogenic cells was shown to decay with biphasic kinetics, suggesting the existence of two main populations of mRNA with respect to stability. In the present study, the stability of mRNA extracted from actinomycin-D-treated cultures of a myogenic cell line was tested by its capacity to direct protein synthesis in the wheat germ cell-free system. The products were analyzed by dodecylsulphate/polyacrylamide gel electrophoresis. All major radioactive bands found in gels used for analyzing the products of the cell-free system directed by polyadenylated RNA extracted from untreated cultures were also found in similar gels containing products of RNA extracted after many hours of application of actinomycin D. The capacity to code for specific protein bands decays with a half-life ranging between 11 and 40 h. No fast-decaying translatable mRNA could be detected by this method. Instead, it was found that during the first 4--6 h following application of actinomycin D, the capacity of RNA to stimulate incorporation of amino acids into total acid-insoluble material increased by 20--30%. The synthesis of specific products increased by up to 100%. The possibility that the fast-decaying polyadenylated RNA or part of it is nontranslatable RNA is discussed.     

Ribonucleic acid synthesis in streptomyces antibioticus: Stable ribonucleic acid species synthesized by young and old cells

In studies of RNA synthesis by intact cells and cell-free extracts of $\text{Streptomyces antibioticus}$, it has been found that 48 hr cells (producing actinomycin) and cell-free extracts are less efficient than 12 hr cells (not producing actinomycin) and extracts in the synthesis of RNA. Analysis of the products of “in vivo” and “in vitro” RNA synthesis by sucrose gradient centrifugation reveals that both 12 and 48 hr cultures and cell-free extracts synthesize ribosomal RNA as well as RNA species of higher and lower molecular weights. However, 50–60% of the ³H-uridine labelled RNA synthesized by intact cells sediments as rRNA as compared with only 5–10% of the cell-free product. The addition of 2 × 10^?5 M actinomycin D to incubation mixtures for cell-free RNA synthesis does not significantly alter the relative amounts of the various RNA species synthesized by 12 or 48 hr extracts.     

Synthesis of tropomyosin in myogenic cultures and in RNA-directed cell-free systems: qualitative changes in the polypeptides

The synthesis of polypeptides with the properties of alpha and beta tropomyosin was investigated in differentiating cultures of a myogenic cell line and in a wheat germ cell-free system directed by purified RNA extracted at different stages of differentiation. The polypeptides co-migrate with tropomyosin in isoelectric focusing and SDS two-dimensional gel electrophoresis and SDS-urea/SDS two-dimensional gels. Like authentic tropomyosin, these polypeptides change their mobility greatly in the presence of urea and do not become labeled with proline. The beta tropomyosin synthesized in the intact cells and in the cell-free system can be separated by isoelectric focusing into at least two components. One component (designated beta1) is present in a small amount at all developmental stages examined, and a more basic component (beta2) is specific for differentiated cultures. The synthesis of beta2 in the intact cells and the capacity of purified RNA to direct its synthesis in a cell-free system become detectable and increase greatly during the period of fusion of the mononucleated cells into multinucleated fibers. The results suggest that the beta1 and beta2 tropomyosins are coded for by different genes.     

Protein biosynthesis in a homologous, cell-free system in the presence of chick embryo RNA isolated from free and membrane-bound polyribosomes.

Protein synthesis has been studied in a cell-free system from chick embryo, in the presence of homologous RNA isolated from free and endoplasmic reticulum-bound polyribosomes. The two RNA fractions showed equal activities in total protein synthesis. However, while the RNA from bound polyribosomes mainly supported synthesis of high molecular weight, TCA-insoluble polypeptides, the RNA from free polyribosomes was more active in the synthesis of low molecular weight, TCA-soluble polypeptides. Optimal conditions for translation of the two RNA's under study were different when studied in a cell-free system with reduced content of endogenous matrix. Collagen synthesized in the system was identified by collagenase digestion. Collagen synthesis was demonstrated only in the presence of RNA from endoplasmic reticulum-bound polyribosomes, and represented 16-19% of total protein synthesis.     

Translation of chicken interferon messenger in a cell-free protein synthesis system and in a cell culture

A highly effective cell-free system for protein synthesis was obtained from rabbit reticulocytes and for the first time used for synthesis of biologically active chicken interferon. The optimal conditions for translation of its mRNA were developed. The translation efficacy in the cell-free system was 10-50 times higher than that in the culture of heterologous cells. The higher the purity level of RNA, the higher the translation level. With respect to poly (A+) RNA sedimenting in the sucrose gradient 9S the efficacy reached 2560 units per 1 microgram of RNA. By the content of poly (A), sequences and rate of the sedimentation, mRNA of the chicken interferon was similar to that of the human fibroblast cell interferon. The possible translation of mRNA of the chicken interferon at low concentrations of exogenic potassium ions in the cell-free system is explained by production of interferon in infected cells where the concentration of the intracellular potassium significantly decreases which is indicative of the mRNA interferon similarity with virus templates. It was found that only albino New Zealand rabbits, but also chinchilla may be used for preparation of the cell-free protein synthesizing system. Various exogenic templates in the mRNA-dependent cell-free system prepared from reticulocyte nonfractionated lysate by treatment with micrococcal nuclease stimulated the protein synthesis by 7-15 times.     

Comparative Studies with Three Antibiotics Binding to Deoxyribonucleic Acid

The effects of the three antibiotics U-12,241, nogalamycin, and U-20,661 on (i) deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) synthesis in KB cell cultures and cell-free systems of bacterial and mammalian origin and on (ii) oxidative phosphorylation in rat liver mitochondria were compared. Nogalamycin and U-12,241 inhibited RNA synthesis more strongly than DNA synthesis in all test systems. Antibiotic U-20,661 inhibited DNA and RNA synthesis equally in whole mammalian cells and their corresponding cell-free systems. The RNA polymerase from Escherichia coli, however, was at least 100 times more sensitive to U-20,661 than was the DNA polymerase. U-12,241 caused significant uncoupling of oxidative phosphorylation in mitochondria.     

Regulation of synthesis of a brain-specific protein in monolayer cultures of clonal rat glial cells

C6 cells were grown in monolayer culture under conditions permitting continued exponential cell division after attainment of a density at which extensive intercellular contacts were formed. An increase in the relative synthesis of S100 protein coincided with the time of formation of extensive intercellular contacts and preceded the onset of the stationary phase of growth by three generations. These observations suggested that the induction of S100 protein synthesis was mediated by cell contact and not by an arrest of cellular growth. The mechanism of this induction was first studied in a homologous non-initiating cell-free protein-synthesizing system from C6 cells, using fixed amounts of free amino acids or fully charged rat liver aminoacyl-tRNA as a source of precursors for protein synthesis. Real synthesis of total soluble proteins decreased as the cells progressed from logarithmic to stationary growth while synthesis of S100 protein increased during this period. The capacity of poly(A)+ RNA from logarithmic and stationary cultures to direct the synthesis of S100 protein was estimated in a cell-free protein-synthesizing system derived from wheat embryos. Increased synthesis of S100 protein in stationary cultures was directly correlated with an increase in translatable S100 protein mRNA.     

Poly(A)-containing RNA from Petroselinum hortense: Isolation,properties and messenger function in vitro

Cell suspension cultures from parsley (Petroselinum hortense Hoffm.) were labelled in vivo with [2-3H] adenosine. The RNA isolated from the ribosomal pellet was fractionated on an oligo(dT)-cellulose column. Approximately 1.5% of the RNA, representing about 15% of the total radioactivity, was retained at high salt concentrations and eluted at low ionic strength. As determined by two independent methods, this fraction contained poly(A) segments with an average length of about 80 nucleotides. It was active as template in a cell-free system from wheat germ, directing the synthesis of peptides ranging in molecular weight from about 4000-40000 daltons.     

Cell-free synthesis of a large translation product of prolactin messenger RNA.

RNA prepared from rat anterior pituitaries or from prolactin-secreting pituitary tumors has been shown to direct the synthesis of a large form of prolactin in a cell-free system derived from wheat germ. Immunoprecipitation of cell-free reactions demonstrated the synthesis of a product which was recognized by a specific antiprolactin antisera. Analysis of the immunoprecipitate on sodium dodecyl sulfate containing polyacrylamide gels suggested that the cell-free product has a molecular weight of approximately 28,000 compared to 22,500 for prolactin. RNA prepared by completely different techniques from rat pituitary and a pituitary tumor resulted in identical large translation products. Translation of tumor RNA in a cell-free system from Krebs ascites cells also resulted in a similar large product. The identity of the cell-free product as prolactin was confirmed by comparing peptides derived from the cell-free product and prolactin. The results of these studies suggest that prolactin messenger RNA directs the cell-free synthesis of a product which contains the amino acid sequence of prolactin but which has an addition at one or both ends of the molecule.     

Encephalomyocarditis Virus Infection of Mouse Plasmacytoma Cells I. Inhibition of Cellular Protein Synthesis

Mouse plasmacytoma ascites tumor cells (MOPC 460) were efficiently infected with encephalomyocarditis virus. Inhibition of host protein synthesis was evident after 2 h and complete by 4 h postinfection. The mechanism by which virus infection results in inhibition of host cell protein synthesis was studied in vitro. Cell-free protein-synthesizing systems, prepared from uninfected and infected cells, were found to be equally active with respect to their abilities to translate cellular and viral mRNAs. The plasmacytoma cell-free system was also shown to be insensitive to the addition of double-stranded viral RNA. Host cellular mRNA was isolated from uninfected and infected cells. No difference in the amount or size distribution of the mRNA was detected. However, the mRNA from infected cells was translated only 46 to 49% as actively as that from uninfected cells. mRNA isolated from cells in which initiation of protein synthesis was inhibited with pactamycin was similarly inactivated. Simultaneous addition of viral RNA and cellular mRNA to the plasmacytoma cell-free system resulted in a complete suppression of the translation of the cellular message, whereas viral RNA was translated normally.     

Inhibition of eukaryotic cell-free protein synthesis by thionins from wheat endosperm

Thionins are polypeptide toxins of about 5000 molecular weight, present in the endosperms of many Gramineae, which modify membrane permeability and inhibit macromolecular synthesis in cultured mammalian cells. Evidence is presented that they inhibit in vitro protein synthesis at micromolar concentrations in cell-free systems derived from wheat germ or from rabbit reticulocytes. Inhibition seems to occur by direct binding of mRNA by the toxin, as judged by the ability of thionins to mediate retention of RNA in nitrocellulose filters and by the dependence of inhibitory concentrations on the amount of exogenous RNA added to the wheat-germ translation system. Commercial preparations of wheat-germ have been found to include some endosperm contamination (up to 15%), which may result in at least partially inhibitory concentrations of the toxin in the cell-free extracts.     

Murine mammary gland RNA directed synthesis of casein in a heterologous cell-free protein synthesis system.

A cell-free protein synthesis system derived from Ehrlich ascites tumor cell ribosomes (S30) plus rabbit reticulocyte tRNA was developed and the activity of the system was dependent on rabbit reticulocyte ribosomal salt (0.5 M KC1) wash factors, The exogenous mRNAs from BALB/c mouse liver and the mammary gland were translated with a high efficiency in this heterologous cell-free system. Furthermore, the RNA from the lactating mammary gland faithfully directed the synthesis of casein. The presence of mouse casein in the reaction product was identified by radioimmunoprecipitation with mouse casein antiserum, co-electrophoresis of the reaction product and mouse casein the urea-polyacrylamide gel and by electrophoresis in sodium dodecyl sulfate (SDS) polyacrylamide gel. The major portion of the lactating mammary gland RNA directed synthesis of the milk protein in the cell-free system appeared to be analogous to alphas casein,     

Inhibition of cell-free protein synthesis by low-molecular-weight nuclear polyadenylate-containing ribonucleic acid species isolated from the lactating guinea pig.

1. Poly(A)-containing RNA was isolated from the nuclei of mammary gland, liver and brain of lactating guinea pigs. 2. Total nuclear poly(A)-containing RNA from mammary gland inhibited mRNA-directed protein synthesis by a wheat-germ cell-free system. It also inhibited the endogenous activity of the wheat-germ and other cell-free systems. It did not inhibit a wheat-germ cell-free system directed by poly(U). 3. Total nuclear poly(A)-containing RNA from liver and brain did not inhibit the mRNA-directed wheat-germ system. 4. Fractionation of the nuclear poly(A)-containing RNA revealed inhibitory activity in the less than 10 S fraction from mammary gland as well as that from liver and brain. 5. The mechanism of protein-synthesis inhibition appeared to be at the level of elongation. 6. The inhibitory activity could be reversed in a wheat-germ system by increasing the amount of S-30 supernatant. 7. The mechanism of inhibition of protein synthesis is discussed in relation to other RNA species known to inhibit such systems.     

Nitrogen source regulates glutamine synthetase mRNA levels in Neurospora crassa.

Neurospora crassa glutamine synthetase mRNA was measured by its capacity to direct the synthesis of the specific protein in a cell-free system derived from rabbit reticulocytes. N. crassa cultures grown on glutamate as the sole nitrogen source had higher mRNA activities than did those grown on glutamine. The differences were about 10-fold when polysomal RNA was used for translation and about 5-fold when either total cellular RNA or polyadenylic acid-enriched cellular RNA was used. These data indicate that in exponentially growing N. crassa, the nitrogen source regulates glutamine synthetase by adjusting specific mRNA levels.     

Antigenic proteins of Eimeria maxima gametocytes: cell-free translation and detection with recovered chicken serum

RNA was extracted from isolated Eimeria maxima gametocytes and translated in a rabbit reticulocyte cell-free protein synthesis system. The major cell-free translation products from E. maxima gametocyte RNA ranged from 225 to 50 kDa, distinct and different from uninfected chicken intestine cell-free translation products. Rabbit antiserum to E. maxima gametocytes as well as recovered chicken sera specifically precipitated some of the major gametocyte cell-free products. A time course of infected intestine RNA indicated that these cell-free synthesized gametocyte antigens appear at 130 to 138 hr postinfection.     

Subcellular distribution of total poly (A) -containing RNA and ferritin-mRNA in the cytoplasm of rat liver.

Poly(A)-containing RNA was isolated from rat liver microsomes and from the post-microsomal supernatant fraction. Approximately 15% of total rat liver poly(A)-containing RNA was found to be present in the post-microsomal supernatant. The relative capacity for apoferritin synthesis of each poly(A)-containing RNA preparation was measured in a cell-free system derived from wheat germ. The post-microsomal supernatant fraction was found to be highly enriched with ferritin mRNA and accounted for 40–50% of the total ferritin-mRNA present in the cytoplasm of rat liver.     

RNA metabolism in cultures of corneal stromal cells from patients with keratoconus

Total cellular RNA was extracted from cultured keratoconus and normal human corneal stromal cells. The translational activity of these RNAs was examined in a cell-free translation system derived from reticulocyte lysate. Results indicated that keratoconus cells can be separated into two groups, as has been shown previously. Group I keratoconus cells contained the same amount of total RNA as normal cells. RNA activity and the rate of mRNA synthesis in this group of keratoconus cells were also normal. By these criteria it seems that the protein synthesizing system is functioning properly, and group I keratoconus cells should have a normal rate of protein synthesis. These results correlate well with previous findings. Group II keratoconus cells, in contrast, contained more RNA than normal cells. The translational efficiency of RNA was so markedly reduced that the elevation in RNA content did not compensate for the decrease in translational efficiency. It is likely that the reduced protein and collagen synthesis in this group of cells is related to the reduction in the RNA activity. An inhibitory component was present in the keratoconus RNA which affected synthesis of all proteins and suppressed translation of normal RNA.