SYNTHESIS AND STORAGE OF MICROTUBULE PROTEINS BY SEA URCHIN EMBRYOS

Studies employing colchicine binding, precipitation with vinblastine sulfate, and acrylamide gel electrophoresis confirm earlier proposals that Arbacia punctulata and Lytechinus pictus eggs and embryos contain a store of microtubule proteins. Treatment of 150,000 g supernatants from sea urchin homogenates with vinblastine sulfate precipitates about 5% of the total soluble protein, and 75% of the colchicine-binding activity. Electrophoretic examination of the precipitate reveals two very prominent bands. These have migration rates identical to those of the A and B microtubule proteins of cilia. These proteins can be made radioactive at the 16 cell stage and at hatching by pulse labeling with tritiated amino acids. By labeling for 1 hr with leucine-³H in early cleavage, then culturing embryos in the presence of unlabeled leucine, removal of newly synthesized microtubule proteins from the soluble pool can be demonstrated. Incorporation of labeled amino acids into microtubule proteins is not affected by culturing embryos continuously in 20 µg/ml of actinomycin D. Microtubule proteins appear, therefore, to be synthesized on "maternal" messenger RNA. This provides the first protein encoded by stored or "masked" mRNA in sea urchin embryos to be identified.     

The rye embryo system as an alternative to the wheat system for protein synthesis in vitro.

Isolated rye embryos are a readily available source for the preparation of very active, cell-free, protein-synthesizing systems. Incorporation levels up to 2000 pmol leucine per 50 mul assay are routinely obtained at saturating TMV (Tobacco mosaic virus) RNA concentrations; at limiting messenger RNA concentrations the incorporation exceeds 1000 leucine molecules per TMV RNA molecule. The characteristics of this cell-free system for the translation of TMV RNA are identical with those of a similarly prepared wheat germ system. The major advantage of the rye embryo system is its high reliability as compared to the unpredictable wheat germ system. Sucrose gradient analysis of the reaction mixture during the incubation shows an extensive polysome formation with TMV RNA and demonstrates efficient polypeptide chain release.     

Further characterization of ribosome binding to thylakoid membranes

Previous work indicated more polysomes bound to pea (Pisum sativum cv Progress No. 9) thylakoids in light than in the dark, in vivo (LE Fish, AT Jagendorf 1982 Plant Physiol 69: 814-825). With isolated intact chloroplasts incubated in darkness, addition of MgATP had no effect but 24 to 74% more RNA was thylakoid-bound at pH 8.3 than at pH 7. Thus, the major effect of light on ribosome-binding in vivo may be due to higher stroma pH. In isolated pea chloroplasts, initiation inhibitors (pactamycin and kanamycin) decreased the extent of RNA binding, and elongation inhibitors (lincomycin and streptomycin) increased it. Thus, cycling of ribosomes is controlled by translation, initiation, and termination. Bound RNA accounted for 19 to 24% of the total chloroplast RNA and the incorporation of [³H]leucine into thylakoids was proportional to the amount of this bound RNA. These data support the concept that stroma ribosomes are recruited into thylakoid polysomes, which are active in synthesizing thylakoid proteins.     

POLYADENYLIC ACID-CONTAINING RNA FROM RAT BRAIN SYNAPTOSOMES

Abstract— Synaptosomal RNA of rat brain was labelled in vivo by intracranial injection of tritiated uridine. The change in the specific activity of this material with time was similar to that of polysomal RNA. The percent of the radioactive synaptosomal RNA which bound to oligo(dT)-cellulose columns decreased with time after intracranial labelling. The percent of the total synaptosomal RNA which bound to oligo(dT)-cellulose was greater than that of polysomes. The length of the polyadenylate (poly(A)) sequence of synaptosomal RNA was approximately one-half that of polysomal RNA, and about the same as that from mitochondria. Investigation of synaptosomal RNA using sucrose gradients and polyacrylamide gel electrophoresis indicated that there were several distinct species present, and that they were similar to those from the mitochondria. The poly(A)-containing RNA isolated from synaptosomes stimulated the incorporation of radioactive leucine into TCA-precipitable material in a cell-free protein synthesis system. Isolation of RNA from subsynaptosomal components indicated that most, if not all, of the synaptosomal messenger activity was localized in the synaptic mitochondria.     

Synthesis of Protein, Ribonucleic Acid, and Ribosomes by Individual Bacterial Cells in Balanced Growth

Relative rates of protein synthesis in individual cells were determined by allowing random populations to incorporate tritiated leucine for very short periods (pulses) and then examining autoradiographs of these cells to assess the amount of incorporation (grains per cell) as a function of cell size. Relative rates of ribonucleic acid (RNA) synthesis were determined in the same way by using tritiated uracil. Unless the uracil pulse was very short (less than 1/20 generation), the RNA labeled during the pulse was predominantly ribosomal. The rate of protein synthesis in individual cells is directly proportional to cell size. The rate of RNA synthesis also increases linearly with size in larger cells, but there appears to be a slight delay in RNA synthesis immediately after cell division. Total cellular content of protein, RNA, and ribosomes is directly proportional to cell size. Thus, we conclude that, in individual cells during the cell cycle (i) the average rate of protein synthesis per ribosome is constant and (ii) the increase in macromolecular mass of the cell is exponential with age.     

Leucine regulates α-amylase and trypsin synthesis in dairy calf pancreatic tissue in vitro via the mammalian target of rapamycin signalling pathway

Starch digestion in the small intestines of the dairy cow is low, to a large extent, due to a shortage of syntheses of α-amylase. One strategy to improve the situation is to enhance the synthesis of α-amylase. The mammalian target of rapamycin (mTOR) signalling pathway, which acts as a central regulator of protein synthesis, can be activated by leucine. Our objectives were to investigate the effects of leucine on the mTOR signalling pathway and to define the associations between these signalling activities and the synthesis of pancreatic enzymes using an in vitro model of cultured Holstein dairy calf pancreatic tissue. The pancreatic tissue was incubated in culture medium containing l-leucine for 3 h, and samples were collected hourly, with the control being included but not containing l-leucine. The leucine supplementation increased α-amylase and trypsin activities and the messenger RNA expression of their coding genes (P <0.05), and it enhanced the mTOR synthesis and the phosphorylation of mTOR, ribosomal protein S6 kinase 1 and eukaryotic initiation factor 4E-binding protein 1 (P <0.05). In addition, rapamycin inhibited the mTOR signal pathway factors during leucine treatment. In sum, the leucine regulates α-amylase and trypsin synthesis in dairy calves through the regulation of the mTOR signal pathways.     

Inhibitors of Ribonucleic Acid Synthesis in Saccharomyces cerevisiae: Decay Rate of Messenger Ribonucleic Acid

Daunomycin and ethidium bromide, two deoxyribonucleic acid-intercalating drugs, inhibit ribonucleic acid (RNA) and protein synthesis in Saccharomyces cerevisiae. Both agents rapidly curtail uptake of radioactive adenine, whereas the kinetics of radioactive leucine uptake after drug addition are consistent with translation of a pool of exponentially decaying messenger RNA. Messenger RNA half-life determinations from these experiments gave identical results over a range of drug concentrations; this value is 21 +/- 4 min at 30 C. In a temperature-sensitive mutant in which RNA synthesis is curtailed at the nonpermissive temperature, a similar half-life for messenger RNA decay is found both in the absence and in the presence of either drug. This indicates that at the concentrations used in this study, neither daunomycin nor ethidium bromide has an appreciable direct effect on translation and do not increase the lability of messenger RNA.     

Autoradiographic studies of the synthesis of RNA and protein as a function of cell volume in Streptococcus faecium

Mid-exponential-phase cultures were either labeled continuously with tritiated leucine and uracil or pulse-labeled with tritiated leucine. The amount of leucine and uracil incorporated into protein or RNA per cell was determined by grain counts of autoradiographs of cells seen in electron micrographs; the volume of each cell was determined by three-dimensional reconstruction. The average number of autoradiographic grains around cells continuously labeled with uracil and leucine increased linearly with cell volume. In contrast, while the average grain count around cells pulse-labeled with leucine increased in a near-linear fashion over most of the volume classes, less than the expected number of grains were seen around cells in large- and small-size classes. The distribution of grains around cells from both the continuously and pulse-labeled populations could be fit at the 5% confidence level with a Poisson distribution modified to take into consideration the volume distribution of each population of cells analyzed. These findings suggested that large changes in the density of RNA and protein do not occur in most cells as they increase in size; however, there may be decreases in the rate of protein synthesis in some large and small cells. The decrease in the rate of protein synthesis appears consistent with the hypothesis that new sites of envelope growth must be introduced into cells that are close to the division event to restore rapid growth.     

Effects of ecdysone analogues on development and metabolic activity of wing disks of the fleshfly, Sarcophaga peregrina,in vitro

Effects of ecdysone analogues on development and metabolic activities of Sarcophaga wing disks were studied in cultures. Development of disks was induced by ecdysterone, ponasterone A, and cyasterone in vitro, whereas rubrosterone was quite inactive in inducing development.As well as morphogenetic effects, a proper concentration (3 × 10^?5 M to 3 × 10^?7 M) was required to induce the incorporation of tritiated uridine, thymidine, and leucine into RNA, DNA, and protein, respectively. Higher concentration of the hormone was more favourable to development of disks and enhancement of RNA synthesis. However, the hormone at concentration higher than 2 × 10^?9 M seemed to be rather toxic to both development and metabolic activity.     

Regulation of uptake of purines, pyrimidines and amino acids by Candida utilis

Uptake of uracil by Candida utilis is increased by addition of leucine to a minimal medium in which organisms are growing. This response requires protein synthesis and has kinetics consistent with the induction of additional uracil transport by the amino acid or a derivative. Consequently, the contribution of exogenous radioactive uracil to the pyrimidine nucleotide pools increases so that RNA made after the amino acid is added is of greater specific radioactivity. Some other amino acids are as effective as leucine in increasing the incorporation of uracil into RNA. Growth with leucine present also increases to different extents the initial rates of uptake of adenine, cytosine, uridine, lysine, histidine, threonine, phenylalanine, aspartic acid and leucine itself. The action of leucine on lysine transport appears to involve induction. These effects are not restricted to leucine; growth with aspartic acid or phenylalanine in the medium gives similar results. Lysine, on the other hand, is without action on the uptake of leucine, aspartic acid, phenylalanine, threonine or uracil but decreases the initial rates of uptake of both histidine and lysine. We suggest that lysine represses its own transport. Similarly, there is a specific decrease in uracil uptake caused by growth with this pyrimidine. Thus in C. utilis there are complex interrelationships in the uptake of nitrogen-containing compounds.     

Non-parallel kinetics and the role of tissue-specific factors in the secretion of chicken ovalbumin and lysozyme from Xenopus oocytes

Ovalbumin and lysozyme made in Xenopus oocytes under the direction of injected chicken oviduct messenger RNA accumulate at different rates in the surrounding culture medium. Pulse-chase experiments confirm that the intrinsic rate of lysozyme secretion from oocytes is 12 times that of ovalbumin. This slower rate of ovalbumin export is maintained following injection of either diluted oviduct RNA or purified ovalbumin messenger, the latter having been obtained by hybridization to cloned ovalbumin complementary DNA. These results suggest that the differential rates of transport observed in oocytes are not the consequence of competition for amphibian or avian factors and show that oviduct-specific proteins are not required for ovalbumin secretion.     

A possible mechanism of inhibition of protein synthesis by dimethylnitrosamine

1. The incorporation of [¹⁴C]leucine into liver proteins of rats was measured in vivo at various times after treatment of the animals with dimethylnitrosamine and was correlated with the state of the liver ribosomal aggregates. Inhibition of incorporation ran parallel with breakdown of the aggregates. 2. Inhibition of leucine incorporation into protein and breakdown of ribosomal aggregates were not preceded by inhibition of incorporation of [¹⁴C]orotate into nuclear RNA of the liver. 3. Evidence was obtained of methylation of nuclear RNA in the livers of rats treated with [¹⁴C]dimethylnitrosamine. 4. Zonal centrifugation analysis of radioactive, nuclear, ribosomal and transfer RNA from livers of rats treated with [¹⁴C]dimethylnitrosamine revealed labelling of all centrifugal fractions to about the same extent. 5. It is suggested that methylation of messenger RNA might occur in the livers of dimethylnitrosamine-treated rats and the possible relation of this to inhibition of hepatic protein synthesis is discussed.     

Isolation of myosin-synthesizing polysomes from cultures of embryonic chicken myoblasts before fusion.

Mononucleated myoblasts and multinucleated myotubes were obtained by culturing embryonic chicken skeletal muscle cells. Comparison of total polysomes isolated from these mononucleated and multinucleated cell cultures by density gradient centrifugation and electron microscopy revealed that mononucleated myoblasts contain polysomes similar to those contained by multinucleated myotubes and large enough to synthesize the 200,000-dalton subunit of myosin. When placed in an in vitro protein-synthesizing assay containing [³H]leucine, total polysomes from both mononucleated and multinucleated myogenic cultures were active in synthesizing polypeptides indistinguishable from myosin heavy chains as detected by measurement of radioactivity in slices through the myosin band on sodium dodecyl sulfate (SDS)-polyacrylamide gels. Fractionation of total polysomes on sucrose density gradients showed that myosin-synthesizing polysomes from mononucleated myoblasts may be slightly smaller than myosin-synthesizing polysomes from myotubes. Multinucleated myotubes contain approximately two times more myosin-synthesizing polysomes per unit of DNA than mononucleated myoblasts, and the proportion of total polysomes constituted by myosin polysomes is only 1.2 times higher in multinucleated myotubes than it is in mononucleated myoblasts. The results of this study suggest that mononucleated myoblasts contain significant amounts of myosin messenger RNA before the burst of myosin synthesis that accompanies muscle differentiation and that a portion of this messenger RNA is associated with ribosomes to form polysomes that will actively translate myosin heavy chains in an in vitro protein-synthesizing assay.     

Messenger ribonucleic acid content of Bacillus amyloliquefaciens throughout its growth cycle compared with Bacillus subtilis 168

The messenger RNA contents of Bacillus amyloliquefaciens and B. subtilis 168, grown in a 1% maltose-0.5% casein hydrolysate complex medium, were determined throughout their growth cycles by a hybridization technique. In both cases there was a level equal to about 3% of the total cellular RNA during the exponential phase. In B. subtilis this level was maintained into the stationary phase. By contrast, in B. amyloliquefaciens the proportion of messenger RNA increased after the end of exponential growth levelling off in the stationary phase at a value twice that observed in exponential growth. The total messenger RNA in each organism was resolved into two components, that involved in the formation of cell proteins and that concerned in extracellular protein production, by determining the relative rates of incorporation of l-[¹⁴C]valine into the two protein fractions. In both cases the cell protein component was the same and remained a relatively constant proportion of the total cellular material throughout the growth cycles. The exoprotein mRNA paralleled exoprotein secretion in each species, remaining at a constant low level in B. subtilis and undergoing a tenfold increase after the end of exponential growth in B. amyloliquefaciens. Applying a serial hybridization procedure to B. amyloliquefaciens, no evidence was obtained for the accumulation of a specific component of the messenger RNA in the exponential or post-exponential phase of growth, which was not detected by hybridization.     

The translation of rabbit hemoglobin messenger RNA in a cell-free extract from chick embryo brain

The translation of rabbit hemoglobin messenger RNA in an unfractionated cytoplasmic extract from chick embryo brain was studied. This translation was not dependent upon reticulocyte-specific factors. An analysis of the product synthesized in vitro with the embryo brain cell-free extract and rabbit hemoglobin messenger RNA by carboxymethyl cellulose chromatography showed that the system was capable of synthesizing both the α and β globin chains. Analysis of the tryptic peptides of the in vitro synthesized α chain by ion-exchange chromatography showed that the embryo brain extract with rabbit hemoglobin messenger RNA was capable of synthesizing the complete α chain of rabbit hemoglobin. The results suggest that no stringent tissue-specific controls exist for the translation of globin messenger RNA and were discussed in this context.     

Isoleucine and Valine Metabolism in Escherichia coli K-12: Detection and Measurement of ilv-Specific Messenger Ribonucleic Acid

Ribonucleic acid-deoxyribonucleic acid (RNA-DNA) hybridization was employed for the determination of messenger RNA transcribed from the ilv gene cluster of Escherichia coli K-12. Strains with derepressed levels of the isoleucine and valine biosynthetic enzymes owing to linked or unlinked genetic lesions were found to exhibit ilv messenger RNA levels from 1.5- to 4-fold higher than did their isogenic parents. When grown under conditions that specifically repressed the synthesis of isoleucine- and valine-forming enzymes, most strains exhibited drastically reduced ilv messenger RNA levels. Hybridization performed with the separated strands of ilv DNA showed that all the ilv genes are transcribed from the same strand, the "l strand" of lambdaphi80CI857St68dilv DNA. Sucrose gradient analyses of RNA extracted from cells starved for isoleucine, valine, or leucine resulted in the detection of at least two distinct types of ilv messenger RNA.