Stimulation Of Amino Acid Incorporation Research Articles

Synthesis of lens protein in vitro. 3. Ribonucleic acid with template activity isolated from calf lens tissue.

RNA was isolated from calf lens polysomes and crude nuclear preparations. Template activity, base composition, sedimentation behaviour, and electrophoretic properties were compared with corresponding RNA fractions from rat liver. 8–9‐fold stimulation of amino acid incorporation was obtained in the cell‐free test system derived from Escherichia coli. Template activity was widely distributed throughout sucrose density gradients. However, the major activity consistently was found at the bottom of the tubes. All fractions from the gradient revealed a heterogeneous pattern when subjected to polyacrylamide gel electrophoresis. The bottom fraction constistently showed a diffuse pattern instead of discrete bands. Only RNA fractions extracted at 65° and at pH 8.3 showed a G‐C content which was significantly different from ribosomal RNA.

The apparent template activity of RNA from developing wings of the cecropia silkmoth

1. 1. RNA from wing epidermis of the cecropia silkmoth stimulated amino acid incorporation in ribosomal systems prepared from Escherichia coli. Most of the stimulatory activity was found in a small fraction of the total RNA which was rapidly labeled and extracted with phenol and sodium dodecyl sulfate at 45°. 2. 2. A comparison of the effects of cecropia RNA with those of f2 viral RNA in the E. coli ribosomal system showed differences which suggest that part, at least, of the stimulation obtained with the cecropia RNA may be due to a “non-template” mechanism. While it is still possible that template activity may be responsible for much of the stimulation, the results indicate that assay with bacterial ribosomes cannot be assumed to give a quantitative measure of animal messenger RNA. 3. 3. RNA from different stages of early wing development was assayed. There appear to be two phases of accumulation of stimulatory RNA: during the first two days of development and between days 7 and 9, with decreasing activity at days 3–5. Neither period of activity coincided with one in which total RNA was rising most rapidly but they do correlate with other biochemical and morphological changes. Following integumental injury, which enhances RNA synthesis, there is no appreciable change in the level of stimulatory RNA. The stimulatory RNA appears therefore to have developmental significance and, taken together with its other properties, this strongly suggests that it may be messenger RNA.

A plausible mechanism of simultaneous read-out of genetic information from both strands of DNA

A molecular mechanism of protein synthesis at the site where DNA exists is considered. A model is presented which describes RNA and protein syntheses on chromosomes on the basis of data, mainly resulting from stimulation of amino acid incorporation by addition of DNA in vitro, from the formation of a ribosome-DNA complex and from the known feature of in vivo transcription catalyzed by a DNA-dependent RNA polymerase. This model is concerned with the simultaneous read-out of the genetic information from both strands of DNA molecule. Once the transcription process by the DNA dependent RNA polymerase is initiated on one strand of the DNA molecule, the translation process takes place simultaneously on the other DNA strand, which is kept single and free from RNA synthesis, but is attached by the ribosomes and forms a ribosome-DNA complex. During this translation process, the genetic information encoded in one of the DNA strands is directly transferred to proteins without any mediation through mRNA (messenger RNA) in the ribosome-DNA complex. The model presented here predicts that both strands of DNA duplex molecule could yield proteins with the same amino acid composition and sequence and the same polarity of peptide linkages. Unless the newly synthesized mRNA is degraded during transportation from chromosomes to the cytoplasm or translational inhibition takes place in the cytoplasm or nucleus, the identical protein molecules could be separately formed on chromosomes and in the cytoplasm. This prediction may be of significance in the nuclear-cytoplasmic interaction within a cell.

Stimulation of globin synthesis by RNA purified from reticulocyte supernatant fraction

Rabbit reticulocytes were preincubated in sodium fluoride to dissociate polyribosomes to 80S ribosomes, and RNA was phenol-extracted from the supernatant fraction of these cells after removing ribosomes. This purified RNA was fractionated by sucrose density gradient centrifugation, and found to contain a fraction corresponding to 6S to 14S which induced formation of polyribosomes active in protein synthesis and specifically stimulated rabbit α- and β-globin chain formation at least 2.5-fold in a cell-free system containing ribosomes prepared from fluoride-treated rabbit reticulocytes. No stimulation of protein synthesis was observed with addition of the RNA fraction to a cell-free system containing ribosomes prepared from reticulocytes not preincubated with sodium fluoride. Addition of this rabbit fluoride RNA to a mouse cell-free system stimulated amino acid incorporation into protein, but no formation of rabbit globin chains was detectable.

Studies on messenger RNA from peanut plants: In vitro polyribosome formation and protein synthesis

Preparations from cotyledons of unimbibed peanut seed show a low level of amino acid incorporation into protein. Similar preparations from imbibed seed exhibit increased activity. RNA extracted from germinating peanut cotyledons and then eluted after ribosomal RNA on the methylated albumin-kieselguhr (MAK) column had the highest template activity. This fraction stimulates amino acid incorporation into protein in an in vitro system. Isolated messenger RNA binds to monoribosomes obtained from dry seed and the polyribosomes thus produced are active in protein synthesis. It is suggested from these results that native mRNA from peanut cotyledons forms a stable complex with monoribosomes in the presence of Mg 2+. The reaction does not require an ATP energy source or 105 000 × g supernatant enzymes.

Acides ribonucléiques messagers de la glande thyroïde de mouton

The messenger character of nuclear and polysomal rapidly‐labeled thyroid RNA has been established using stimulation of amino acid incorporation into trichloracetic‐insoluble material in the Escherichia coli system, mononucleotide composition of the early‐labeled material and tenacity of adsorption on columns of methylated serumalbumin adsorbed on Kieselguhr (MAK columns).A relation between the difficulty to extract nuclear or cytoplasmic thyroid RNA by phenol at 0°, their rapidity of labeling and their capacity to stimulate protein synthesis in vitro has been demonstrated.80% of the nRNA labeled for 15 min was tenaciously bound to MAK columns. After elution with a buffer containing sodium dodecylsulphate at 35–70°, it showed a DNA‐like base composition and sedimented in 4 discrete size classes: 30 S, 22 S, 13 S. The remaining 20% has been identified as preribosomal RNA. After a 9 h‐period of labeling the total nRNA fraction still contained labeled preribosomal RNA, rRNA and DNA‐like RNA.Most of the early‐labeled polysomal RNA has been identified as mRNA by tenacity of adsorption on MAK columns and base composition. Even after a 1 h‐pulse the labeled rRNA accounted only for 30–40% of the total RNA. Absence of preribosomal RNA in polysomes as shown by MAK column chromatography was considered as a good index of the absence of contamination of polysomes by nuclear material. Polysomes pulse‐labeled with [32P]orthophosphate and [14C]protein hydrolysate were dissociated by ethylenediaminetetraacetic acid and fixed with formaldehyde. The buoyant densities of the nucleoprotein particles were determined by banding in CsCI gradients. Under these conditions, the bulk of the material in ribosomes banded at characteristic densities (d= 1.57 and 1.52) but most of RNA with high specific radioactivity banded at a density approximately 1.38. This material contained [32P]RNA and [14C]protein, and was identified as an association of mRNA and protein by base composition of its RNA: (G + C)/(A + U) = 0.83.Purification of pulse‐labeled nuclear mRNA was carried out by the hot‐phenol fractionation procedure of Georgiev et al. [9,10]. Specific radioactivity, specific stimulating activity of protein synthesis in vitro and base composition of the five fractions obtained were determined. The RNAs extracted from nuclei between 55–65° and 65–85° were considerably enriched in mRNA. Sucrose‐density gradient sedimentation showed substantial polydispersity of the stable RNA of all the fractions. However, the main component of the RNA extracted by phenol between 65° and 85° culminated in a metabolically homogeneous 16 S peak. A definite correlation between pulse‐labeled nRNA isolated by the hot‐phenol procedure and pulse‐labeled polysomal RNA was noticed.As compared with 18 S and 28 S rRNA, the fractions of stable polysomal thyroid RNA which corresponded in sedimentation to the rapidly‐labeled RNA (30–40 S, 22 S, 13 S and 7 S) definitely stimulated polypeptide synthesis in vitro.The size of the monocistronic mRNA which codes for the peptide chains of thyroglobulin is discussed. If a molecular weight of 73,000 for the two different chains is assumed, their amino acid sequence might be encoded in the 13 S polysomal RNA. The functional significance of the 30–40 S and 22 S mRNA and the possible importance of polysomal messenger nucleoprotein particles are also discussed.

Quelques Propriétés des RNA à Marquage Rapide Extraits de Cellules Foliaires

32P incorporation was followed in Chinese cabbage leaves (Brassica chinensis var. Pe tsai) isolated from plants. After pulses of different lengths, the total RNA of leaves was extracted and run on a sucrose gradient. It has been possible to isolate different rapidly labelled RNA's with sedimentation coefficients increasing from 5 to 35 s while the pulse length increased from 7 to 25 minutes. These heterogeneous RNA's were divided into four major classes: 4 to 10 s; 15 s; 23 s; 35 s. The following points were established. o (1) Base composition of rapidly labelled RNA's is very different from those of ribosomal and soluble RNA's. The base composition varies also with the size of the molecule. (2) The participation of rapidly labelled RNA's in polysome building and their ability to stimulate amino acid incorporation into proteins within a cell-free system are in agreement with messenger function for these RNA's. (3) The meaning of the increase in size of rapidly labelled RNA with the length of 32P pulse is discussed. (4) 18 hours incubation with high concentration of actinomycin D inhibits the synthesis both of ribosomal and soluble RNA's to the extent of 85 and 75%, respectively. After the same treatment, the decrease of 32P incorporation into rapidly labelled RNA is about 60%. At the same time, protein synthesis is not affected by actinomycin D treatment. This result implies a very high stability of leaf mRNA. (5) Preliminary studies on the effect of light on rapidly labelled RNA synthesis illustrates the importance of this factor.

Some properties of the stimulatory effect of thyroid hormones on amino acid transport by embryonic chick bone.

Some characteristics of the stimulation by thyroid hormones of the intracellular accumulation of methionine and cycloleucine by embryonic chick bones are described. The stimulation by triiodothyronine exceeded that of Lthyroxine. 3,3′-Diiodothyronine was as effective as l-thyroxine, which was more active than 3,5-diiodothyronine or d-thyroxine. Diiodotyrosine, thyronine and inorganic iodide were inactive at comparable concentrations. Stimulation of the accumulation of methionine appeared to be immediate. The effect was not accompanied by a stimulation of amino acid incorporation and was not inhibited by puromycin or by actinomycin D prior to the inhibition by these antibiotics of transport per se. Chelation of metals by ethylenediamine tetraacetate did not produce similar effects. Stimulation of the accumulation of free methionine by costal and xiphoid cartilage of mature thyroidectomized rats was also observed. (Endocrinology 81: 1372, 1967)

Messenger ribonucleic acid of cerebral nuclei

1. RNA was isolated from crude nuclear preparations and from ribosomes derived from rat brain and liver. Nuclear RNA was obtained by lysis of the nuclei with sodium dodecyl sulphate, followed by denaturation and removal of DNA and protein with hot phenol. 2. Base composition analyses indicated that the cerebral nuclear RNA preparation contained a higher proportion of non-ribosomal RNA than the analogous hepatic preparation. 3. Sucrose-density-gradient analyses revealed a heterogeneous profile for each nuclear RNA preparation, with two major peaks possessing the sedimentation properties of ribosomal RNA (18s and 28s). 4. Template activities of both preparations were widely distributed through the sucrose density gradients. 5. The cerebral nuclear RNA preparation was more active than the hepatic nuclear RNA preparation in promoting amino acid incorporation in cell-free systems from Escherichia coli and rat brain. 6. Cerebral nuclear RNA stimulated amino acid incorporation in a cerebral ribosomal system even in the presence of an excess of purified E. coli transfer RNA. 7. It is concluded that a significant proportion of cerebral nuclear RNA has the characteristics of messenger RNA.

The metabolism of benzene in vitro

Benzene- 14C metabolism was studied in rabbit and rat liver preparations. Phenol and its conjugates, phenyl sulfate and phenyl glucuronide, were the major metabolites. At low benzene concentrations most of the phenol was conjugated. At higher benzene concentrations considerably more free phenol remained. Pretreatment of animals with phenobarbital or benzene resulted in a stimulation of benzene metabolism in vitro. The stimulation of benzene metabolism by benzene was accompanied by a stimulation of amino acid incorporation into microsomal protein, suggesting that the stimulation of benzene metabolism resulted from induction of more benzene-metabolizing enzymes.

A direct effect of growth hormone on the incorporation of precursors into proteins and nucleic acids of perfused rat liver.

1. The livers of rats were perfused in situ. When the amino acid concentration in the perfusing medium was that present in rat plasma, the addition of growth hormone to the medium stimulated the incorporation of labelled amino acids into liver protein only marginally and not to a statistically significant extent. When, however, the amino acid concentration was raised to three times that present in rat plasma, growth hormone significantly and substantially stimulated amino acid incorporation into protein within 30min. of perfusion of normal rat liver. 2. A significant effect of growth hormone on labelling of normal rat-liver protein was seen with concentrations not much greater than those reported to be present in rat plasma. 3. The labelling of nucleic acids of normal and hypophysectomized rat liver by [(3)H]orotic acid was enhanced by addition of growth hormone to the perfusing medium when normal concentrations of amino acids were used. 4. At elevated concentrations of amino acids, growth hormone stimulated labelling of nucleic acids of hypophysectomized rat liver at 30 and 60min. of perfusion. Under these conditions, nucleic acids of normal rats were labelled to about the same extent in control and hormone-treated livers at 30min. and, because of a fall in the radioactivity of the control livers, there was more labelled nucleic acids in growth-hormone-treated livers at 60min. than in the control livers. 5. Growth hormone, unlike insulin, had no inhibitory effect on the release of glucose by the perfused liver. 6. It is concluded that growth hormone can stimulate the incorporation of precursor into proteins and nucleic acids of liver directly and without the mediation of other organs or of insulin.

Effect of Threonine on Tyrosine Metabolism of Rats Fed a Low Protein Diet

The alleviation of tyrosine toxicity obtained by the addition of threonine to a 3% tyrosine low protein diet was studied to determine the mode of action of threonine. Rats fed a 3% L-tyrosine and 1.25% L-threonine low protein diet had a lower plasma tyrosine concentration as compared with rats fed a 3% L-tyrosine low protein diet. Injection of these rats with L-tyrosine-U-14C indicated that the effect of threonine was not to increase the catabolism of tyrosine to 14CO2 but to increase the excretion of radioactivity in the urine and the incorporation of radioactive tyrosine into plasma and liver proteins. In rats receiving an excess of tyrosine or threonine or both amino acids, the incorporation of DL-alanine-1-14C into tissue proteins was also increased over the control rats indicating a general stimulation of amino acid incorporation into proteins.

The stimulation of amino acid incorporation into the protein of unfertilized sea urchin eggs by gamma-irradiation.

AbstractControl and γ‐irradiated sea urchin (Arbacia punctulata) eggs were incubated with H3‐leucine. No incorporation of the amino acid into the proteins of most unirradiated eggs was detectable by autoradiography. All irradiated eggs incorporated the amino acid and the amount of autoradiographic labelling varied greatly from cell to cell.

Occurrence, isolation, and characterization of polyribosomes in yeast.

This report details the procedural requirements for preparing cell-free extracts of yeast rich in polyribosomes. This enabled us to demonstrate the occurrence of polyribosomes in yeast, to show their role in protein synthesis, and to devise methods for their resolution and isolation. When certain precautions are met (the use of log phase cells, rapidly halting cell growth, gentle methods of disruption, sedimentation through exponential density gradients, etc.), individual polyribosome size classes ranging up to the heptosome can be fractionated and separated from their nearest neighbors. Larger size classes are resolved partially among themselves, free of smaller polyribosomes. This was confirmed by extensive electron micrographic studies of material from the various fractions obtained upon density gradient centrifugation of yeast extracts. Modifications of the gradients and procedure should allow fractionation and isolation of the larger polyribosomes, including those containing polycistronic messages. Yeast polyribosomes are disaggregated to single ribosomes by longer term grinding, cell disruption by the French pressure cell, the Hughes press, or by incubation with dilute RNAse. Yeast polyribosomes are active in the incorporation of amino acids into polypeptide; the single ribosomes exhibit only slight activity. The latter activity is probably due to the presence of a small fraction of monosomes still containing mRNA. Poly-U stimulates amino acid incorporation only in the single ribosomes.

Effects of Thyroxine on Initiation and Completion of Protein Chains of Hemoglobin in Vitro

Abstract Thyroxine added to a rabbit reticulocyte lysate system in vitro stimulates amino acid incorporation into the lysate protein. In agreement with previous findings in other cell-free preparations, the thyroxine effect is dependent on the addition of mitochondria and an oxidizable substrate to the reaction system. The products of the thyroxine-stimulated amino acid-incorporation reaction include both the α and β chains of hemoglobin. By analysis of both the amino-terminal and the interior valine positions labeled with l-valine-14C during polypeptide chain formation, it was found that the thyroxine stimulation of β chain synthesis involves both chain initiation and completion, i.e. complete synthesis de novo, although the effect on completion is considerably greater than that on initiation. In the case of α chain synthesis, thyroxine stimulated the completion of chains previously begun in vivo without any apparent effect on the initiation of new chains during the incubation. The data suggest that the primary site of the thyroxine stimulation of protein biosynthesis is at a late stage in polypeptide chain elongation, chain termination, or chain release, and that chain initiation may be secondarily increased when the rates of the earlier reactions are favorable.

Characteristics of in vitro amino acid incorporation by the ribosomal fraction from tobacco leaves

Amino acid incorporation by the ribosomal fraction from tobacco leaves was studied. The incorporation was dependent on ATP and GTP. An energy-generating system [phosphoenol pyruvate (PEP) and PEP-kinase] was able to meet part of the energy requirements. The incorporation was very sensitive to ribonuclease and was dependent on the presence of soluble RNA and enzymes from the soluble fraction. These latter dependencies were particularly clear when the ribosomes were purified by centrifugation through sucrose layers. The ribosomal fraction could be separated into two fractions, one of 70 S and 80 S particles and one containing polysomes and some 80 S particles. Both fractions had the same specific incorporating activity. Addition of polyuridylic acid to the ribosomes stimulated the incorporation of phenylalanine 3- to 4-fold. Tobacco Mosaic Virus (TMV)-RNA had no measurable effect on the amino acid incorporation. The stimulation of amino acid incorporation by TMV-RNA in the ribosomal system of Escherichia coli was not inhibited by the addition of tobacco leaf ribosomes. Therefore the failure of TMV-RNA to stimulate the ribosomal system from leaves may be ascribed to faulty interaction between TMV-RNA and the ribosomes.

Comparison of amino acid incorporation into nuclear protein of rat liver and thymus in cell free systems.

1. Amino acid incorporation into proteins of rat liver and thymus nuclei were compared using cell free systems. 2. Both thymus and liver nuclei incorporated amino acids into their proteins without addition of high-energy phosphates. Amino acid incorporation into thymus nuclear proteins was not modified by ATP, CTP, GTP, and UTP added to the incubation medium, whereas amino acid incorporation into liver nuclear proteins was stimulated by ATP and GTP. In the presence of phosphoenolpyruvate and pyruvate kinase [EC 2.7.1.40] in the incubation medium, oligomycin, an inhibitor of oxidative phosphorylation, inhibited amino acid incorporation into thymus nuclear proteins.and this inhibition was not restored by ATP added. Under the same conditions oligomycin did, however, not inhibit amino acid incorporation into liver nuclear proteins. 3. Potassium and sodium ions inhibited amino acid incorporation into rat thymus nuclear proteins under the conditions of this study, but these ions stimulated amino acid incorporation into liver nuclear proteins. 4. RNase [EC 2.7.7.16] inhibited amino acid incorporation into liver nuclear proteins, but it did not affect amino acid incorporation into thymus nuclear proteins. DNase [EC 3.1.4.5] did not affect amino acid incorporation into both liver and thymus nuclear proteins.

Effects of purine nucleoside triphosphates on amino acid incorporation in a cell-free system from 8-azaguanine-treated Bacillus cereus

1. 1. The cell-free amino acid-incorporating systems from normal and 8-azaguanine-treated Bacillus cereus were examined to determine the effect of supplementation with increasing amounts of GTP and ATP on incorporation of l-[ 14C]phenylalanine into protein. 2. 2. The addition of GTP to the 30-S fraction from normal cells stimulated amino acid incorporation to a greater extent than when added to the 30-S fraction from 8-azaguanine-treated cells. The higher endogenous GTP activity in the 30-S fraction from 8-azaguanine-treated cells was not due to 8-azaguanosine triphosphate, since this compound failed to stimulate amino acid incorporation in the 30-S fraction from normal or 8-azaguanine-treated cells. 3. 3. Amino acid incorporation in the 30-S fraction from normal cells was stimulated when supplemented with ATP. In contrast, the 30-S fraction from 8-azaguanine-treated cells maintained a high level of amino acid incorporation in the absence or presence of added ATP. The concentration of ATP, measured by the luciferase assay, was found to be higher in the 30-S fraction from drug-treated cells than from normal cells.

Stimulation of amino acid incorporation in vitro by DNA: Different effects of aminoglycoside antibiotics in calf-thymus nuclear and bacterial ribosome systems

Stimulation of amino acid incorporation in vitro by DNA: Different effects of aminoglycoside antibiotics in calf-thymus nuclear and bacterial ribosome systems

Thyroxine stimulation of amino acid incorporation into protein of skeletal muscle in vitro.

Thyroxine stimulation of amino acid incorporation into protein of skeletal muscle in vitro.