Stimulation Of Amino Acid Incorporation Research Articles

Polymorphism in fowl serum albumin—V. Protein synthesis in cell-free systems from regenerating liver of the fowl ( Gallus domesticus)

1. 1. Removal of the left hepatic lobe from 2–6 week old chicks results in liver regeneration. 2. 2. Polyribosomes from regenerating livers contain fewer 80 S monomers than those from normal or sham-operated livers, but were as active in cell-free protein synthesis. 3. 3. High speed supernatant from regenerating liver stimulated amino acid incorporation more than preparations from sham-operated controls over wide ranges of time and supernatant, Mg 2+ and GTP concentrations. Supernatant activity was maximal (160% of control) 12–18 hr after partial hepatectomy. 4. 4. Neither the activity of amino acid activating enzymes nor ribonuclease differed sufficiently between regenerating and control liver preparations to explain the protein biosynthesis differences. 5. 5. The data were consistent with the hypothesis of a temporary increase in elongation factor activity following partial hepatectomy.

Template properties of bacteriophage T4 vegetative DNA. II. Effect of maturation and DNA-arrest mutations

The DNA in gently lysates of T4-infected Escherichia coli cells sediments in sucrose gradients as two major components; the slower sedimenting component is designated as the S-5 fraction and the faster sedimenting component as the pad fraction. The distribution of these fractions in lysates of cells infected with T4 maturation-defective and DNA-arrest mutants was determined, and their template activities were compared in a DNA-dependent amino acid-incorporating system. The S-5 DNA template was found to be completely absent in E. coli B cells infected with a T4 maturation-defective mutant (gene 55). On the other hand, DNA sedimenting as the S-5 component is greatly increased, while that sedimenting as the pad component is virtually absent in nonpermissive cells infected with a DNA-arrest mutant (gene 46). The S-5 fractions prepared from cells infected with a DNA ligase mutant (gene 30) and a gene 30 gene 46 double mutant are reduced in their ability to stimulate amino acid incorporation compared to similar preparations from cells infected with wild type T4 or a gene 46 mutant. Moreover, the template activity of partially purified replicative DNA prepared from cells infected with phage-carrying mutations either on gene 30 or in both genes 46 and 56 (dCTPase) is lower than that of DNA obtained from cells infected with wild type phage. The polypeptide products of reaction mixtures programmed with several of the mutant DNAs were found to be qualitatively different from polypeptides synthesized in response to either mature DNA or replicative DNA prepared from cells infected with wild type phage. These data suggest that the expression of phage DNA may be significantly influenced by physical changes in the DNA arising from abnormal replication.

Polyanion-induced release of polyribosomes from HeLa cell nuclei

Intact detergent-washed HeLa nuclei contain a population of polyribisomes that were released by exposure to polyanions such as RNA or poly(U). The released material appeared by electron microscopic examination to be particles averaging about 200 to 300 angstroms in diameter. Sedimentation velocity analysis of the released particles indicated that the particles had S20,w values of 75 and 110. The particles stimulated amino acid incorporation in an ascites S-30 or S-100 extract at 2.5 mM Mg2+. Studies with a variety of antibiotics indicated that these polyribosomes were capable of elongating but not initiating protein synthesis. Although these polyribosomes may be of cytoplasmic origin, they appear unique in that agents thought to disperse chromatin are required for their release from the nucleus.

Template properties of bacteriophage T4 vegetative DNA. I. Isolation and characterization of two template fractions from gently lysed T4-infected bacteria

The synthesis and template properties of T4 vegetative DNA were studied. The DNA-containing material in lysates of cells taken 20 min past T4 infection sediments in sucrose gradients as two major components. Both fractions function as templates for amino acid incorporation in a DNA-dependent in vitro system (coupled transcription-translation). The slower sedimenting activity is not present in uninfected cells and appears in wild type T4-infected cells only after 12 min at 30 degrees, shortly after DNA synthesis starts. It is dependent for its activity on an added S-30 fraction from either uninfected or T4-infected cells and is completely inhibited by deoxyribonuclease or rifampin. On a weight basis the slower sedimenting template is about 30 to 70% as active as mature T4 DNA when supplemented with S-30 extracts from uninfected cells. The spectrum of proteins synthesized in response to the slower sedimenting template is different from that produced in response to mature T4 DNA. In contrast to mature DNA, this template is capable of directing the synthesis of material that precipitates with antiserum directed against whole T4 particles. Thus, it appears capable of directing the synthesis of mRNA for phage structural proteins, i.e. late proteins. The faster sedimenting component is about 8-fold less active for stimulating amino acid incorporation than mature DNA. Significant amounts of RNA polymerase are associated with this DNA in active transcription complexes, yet polyacrylamide gel electrophoresis of the proteins synthesized in response to this fraction show a pattern that resembles the early proteins made from mature T4 DNA in extracts from uninfected cells.

The early synthesis and possible function of a 0.5 × 10 6[formula omitted]r RNA after transfer of dark-grown [formula omitted] plants to light

A 0.5 × 10 6 M r RNA found in plastids of the aquatic angiosperm Spirodela , is synthesized at a much higher rate than any other rapidly labeling RNA species about 3–3 1 2 h after dark-grown plants are transferred to light. The pulse labeling kinetics of the 0.5 × 10 6 M r RNA after transfer to light, argue against its involvement in the biogenesis of plant rRNAs. Although poly(A) RNA is found in Spirodela , poly(A) sequences are not detected in the 0.5 × 10 6 M r RNA; yet a sucrose gradient fraction which includes RNA of this M r stimulates amino acid incorporation by an E. coli cell free extract more than other RNA fractions. The possible involvement of the 0.5 × 10 6 M r RNA as a chloroplast messenger is discussed.

Effect of plumbous ion on messenger RNA

The effects of Pb2+, a potent catalyst for the depolymerization of RNA have been studied on brome mosaic virus (BMV) RNA, rabbit globin m-RNA and polyuridylic acid. After exposure of these natural and synthetic messengers to a sufficiently high concentration of Pb2+, they all lost their ability to stimulate amino acid incorporation in cell-free protein-synthesizing systems. There were differences in the susceptibilities of the messengers; gloing the m-RNA for 40 min revealed that there was a threshold Pb2+ concentration below which no loss of m-RNA activity was observed. The threshold concentration was considerably greater than the Pb2+ concentration at which protein synthesis is inhibited in reticulocytes and overt symptoms of plumbism are observed. However, when m-RNA were incubated for an extended period (24 h), even with sub-threshold concentrations of Pb2+, there was destruction of messenger function and globin m-RNA was more susceptible than BMV-RNA. Also the susceptibility of m-RNA to Pb2+ is temperature-dependent, which would indicate that m-RNA, like t-RNA, exists as a population of molecules in different conformational states that are not readily interconvertible.

Absence of messenger ribonucleic acid specific for flagellin in non-flagellate mutants of Salmonella

The flagellin synthesizing activity of RNAs prepared from the fla mutants of Salmonella was examined using the Escherichia coli cell-free system for protein synthesis. The fla complementation groups tested covered the following: fla-A, -B, -C, -D, -E and -F in Salmonella typhimurium and fla-L, -N, -P, -Q and -R in S. abortus-equi. None of the RNAs were found to stimulate in vitro synthesis of flagellin, although stimulation of amino acid incorporation into general protein was high enough. Complementation at a translational level was not observed in any of the following combinations of the RNAs prepared from the fla mutants: A-F, B-C, B-E, C-D, E-F, F-L and N-P. A mutation in one of the fla genes tested resulted in failure to produce flagellin mRNA in both flagellar phrases. The products of fla + were assumed to participate in the transcription of the structural gene for flagellin.

Enhanced differential synthesis of proteins in a mammalian cell-free system by addition of polyamines.

Addition of the polyamines spermidine, spermine, or putrescine to a fractionated mammalian cell-free protein-synthesis system programmed by a variety of mRNAs results in a 3- to 5-fold stimulation of amino acid incorporation over that found in the absence of added polyamine. The mRNAs used as template were adenovirus mRNA, globin 9s mRNA, and RNA from the bacteriophages R17, Qbeta, and MS2. The relative amounts of 10 adenovirus polypeptides synthesized in vitro are altered by the addition of polyamines to the translation system to reflect more closely the relative amounts of these polypeptides synthesized in vivo. This qualititive improvement in translation products on addition of polyamines allow the analysis of a number of products which are at best only marginally synthesized in the absence of added polyamines. The low level of synthesis due to endogenous mRNA is stimulated by spermidine and spermine but a lesser extent by putrescine.

Studies on the differential response to insulin on the stimulation of amino acid incorporation into protein in isolated hepatocytes containing different levels of glycogen

Effect of insulin on amino acid incorporation into protein by isolated rat liver hepatocytes was studied. A two to three-fold increase in the incorporation of U- 14C-Leucine and U- 14C-Phenylalanine into protein by insulin (100 μUnits) was observed in isolated hepatocytes containing high glycogen. This effect was abolished by the addition of glucagon (3 × 10 −6M). No stimulation in amino acid incorporation by insulin was observed when isolated hepatocytes contained low or no glycogen. Electron micrographs of incubated cells show that in the presence of insulin more normal parallel strands of polyribosomes are maintained as compared to control cell preparation.

The cell-free translation of Rauscher leukemia virus RNA into high molecular weight polypeptides

Rauscher leukemia virus (RLV) 65-S RNA, 35-S mengovirus RNA and reticulocyte A-rich RNA each stimulated cell-free protein synthesis in a JLS-V5 cell-derived S-30 system. rRNA, however, was not stimulatory in this system. Of the stimulated protein products only those synthesized in response to added RLV RNA were immune-precipitable with anti-RLV rabbit serum. Furthermore, cell-free incubations with pactamycin at a concentration which specifically inhibits initiation and not elongation prevented the stimulation of amino acid incorporation in response to added RLV RNA. Analysis of the polypeptides synthesized by the cell-free system in response to reticulocyte A-rich RNA, showed them to be globin-like and, therefore, also mRNA specific. The RLV RNA-directed product included at least two classes of polypeptides (mol. wts of 140 000–185 000 and 50 000–75 000) both of which were larger than the group specific polypeptides of mature virions. None of the internal structural polypeptides of mature virions were synthesized in response to RLV RNA. The large molecular weight, viral-specific polypeptides are candidate precursor polyproteins which may represent the translational products of a polycistronic mRNA with a single initiation site.

Effect of sclerin on amino acid incorporation into mitochondria isolated from rat liver.

Though sclerin (SCL) stimulated amino acid incorporation into the protein fraction of post mitochondrial supernatant of rat liver homogenate, it had no effect on the incorporation into the isolated mitochondria at pH 7.2, despite of its stimulating effect on mitochondrial oxidative phosphorylation. SCL stimulated amino acid incorporation into the mitochondria at pH 6.1, and to some extent maintained the activity on that in mitochondria during aging in hypotonic Tris-HCl buffer (pH 7.2). Since SCL prevented leakage of amino acids from the mitochondria into these buffers, it was suggested that SCL may protect a structure of mitochondrial membrane which appeared to have a significance on transport of amino acids. In liver slices, SCL stimulated amino acid incorporation only into the extra-mitochondrial fraction for the first 3 min, but gradually turned to stimulate incorporation into mitochondria within 30 min.

Protein synthesis in chloroplasts: V. Translation of messenger RNA for the large subunit of fraction I protein in a heterologous cell-free system

The addition of spinach chloroplast total RNA to cell-free extracts from Escherichia coli stimulates amino acid incorporation into protein. The products were characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and were qualitatively and quantitatively similar to those synthesized in intact isolated chloroplasts. There are two major discrete products of both systems with molecular weights of 52,000 and 35,000. The [ 35S]methionine-containing chymotryptic peptides of the 52,000 M r polypeptide synthesized in the E. coli cell-free system have been compared with those of fraction I protein large subunit labelled with [ 35S]methionine in vivo. From the close similarity in chromatographic properties of the peptides of the two polypeptides, we conclude that the 52,000 M r product of chloroplast RNA-directed protein synthesis in E. coli extracts is the large subunit of fraction I protein.

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.

Translation in vitro of total nuclear RNA from HeLa cell nuclei infected with adenovirus 2.

Total heterogeneous nuclear RNA from HeLa cells infected with adenovirus for 18-20 hr stimulates amino acid incorporation into protein in a cell-free system from Ehrlich ascites tumors. This stimulation of protein synthesis by the nuclear RNA requires intact nuclei. The role of nuclei in this system is unknown, but evidence is presented that the nuclei are involved in the conversion of high-molecular-weight RNA to low-molecular-weight species. Some of the newly synthesized polypeptides appear to resemble the virion polypeptides.

Purification and Properties of Chain Initiation Factor 3 from T4-infected and Uninfected Escherichia coli MRE 600: STIMULATION OF TRANSLATION OF SYNTHETIC AND NATURAL MESSENGERS

Abstract The requirement for chain initiation factor 3 in the translation of synthetic polynucleotides lacking an ApUpG codon was examined. Highly purified IF-3 stimulates amino acid incorporation 6- to 10-fold in the presence of 4 µg of poly(U). Based on this observation, a simple and specific assay was developed for the purification of IF-3 from the 1.0 m NH4Cl wash of Escherichia coli MRE 600 ribosomes. At all steps of purification, IF-3 activity detected by the poly(U) assay coincided with that determined by R17 RNA-dependent lysine incorporation. The purified factor was required for maximal rates of amino acid incorporation with poly(U), poly(A), and other synthetic polynucleotides. Samples of purified IF-3 isolated from uninfected and T4-infected E. coli MRE 600 migrated as one major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with a molecular weight of 22,600. Both standard IF-3 and T4-IF-3 were found to promote the translation of R17 RNA and late T4 RNA with equal efficiency.

Further studies on eukaryote DNA stimulation of amino acid incorporation in E. coli extracts

DNA from adenovirus-2 and mouse myeloma tumors stimulate RNA synthesis and amino acid incorporation into protein in a cell-free extract from Escherichia coli. The RNA synthesis is dependent on exogenous DNA, and the RNA can be hybridized to respective template DNA. A major part of this RNA is also found attached to E. coli polysomes suggesting that RNA with messenger-like activity has been synthesized. However, the in vitro-synthesized polypeptides using adenovirus DNA or myeloma DNA do not correspond in size or antigenic activity to either the virion proteins or immunoglobulins, respectively.

Tobacco mosaic virus RNA directs the synthesis of a coat protein peptide in a cell-free system from wheat

Tobacco mosaic virus (TMV) RNA stimulates amino acid incorporation into protein in cell-free extracts from wheat germ, rye embryo and Escherichia coli. The properties of the wheat germ system are examined and the nature of the viral RNA-induced products studied with the aid of a virus mutant carrying a threonine → methionine replacement in its coat protein. A peptide containing this methionine residue is present in tryptic digests of mutant RNA-directed cell-free products, and is absent from digests of wild type RNA-directed products. The undigested cell-free product contains a very large number of polypeptides with molecular weights from 10,000 to 140,000, but little or no synthesis of correct sized coat protein is observed.

Nuclear binding of oestradiol-17beta and induction of protein synthesis in the rat uterus during postnatal development.

1. The uterine response to a single injection of oestradiol-17beta during postnatal development of the rat was studied with respect to (i) nuclear binding of oestradiol-17beta; (ii) induction of the synthesis of a specific cytoplasmic protein (;induced protein' of Gorski); (iii) rate of incorporation of (3)H-labelled amino acids into total protein and into nuclear acid-soluble and acid-insoluble protein; and (iv) rate of [(3)H]thymidine incorporation into DNA. 2. Specific nuclear binding of oestradiol-17beta could be demonstrated even at birth. Administration of oestradiol-17beta in vivo caused a significant increase in the number of nuclear binding sites in rats aged 10 days or older. 3. A rapid method is described for the detection of the ;induced protein', based on cellulose acetate electrophoresis. Induction of this protein could be demonstrated at the age of 10, 15 and 20 days, but not in 5-day-old rats. 4. In 20-day-old rats the rate of (3)H-labelled amino acid incorporation into protein increased by 3h after oestradiol administration. Incorporation into the different protein fractions reached peak values asynchronously: at 3-4h for acid-insoluble nuclear protein, at 6h for total protein and at about 12h for acid-soluble protein. 5. Treatment with oestradiol failed to stimulate amino acid incorporation into protein in 5- or 10-day-old rats; at the age of 15 to 30 days the hormone caused a significant increase in incorporation into total protein and into both types of nuclear protein. 6. Since the capacity for nuclear binding of oestradiol and for synthesis of the induced protein is demonstrable in the rat uterus before it acquires the ability to respond to the hormone with enhanced general protein synthesis and DNA synthesis, it appears that nuclear binding and the synthesis of the induced protein may be necessary but not sufficient conditions for the trophic action of oestradiol.

Effect of eukaryote DNA on amino acid incorporation in extracts of E. coli

Rat liver mitochondria DNA and nuclear DNA from several sources stimulate amino acid incorporation in a cell-free extract of E. coli. In all cases, the endogenous E. coli polymerase synthesized RNA that specifically hybridizes with the added DNA. Although the newly synthesized RNA may be protecting endogenous E. coli mRNA from degradation, evidence is presented that some of the polypeptides synthesized are products of the added DNA.

Thyroxine stimulation of amino acid incorporation into mitochondrial protein: differences between in vivo and in vitro effects.

ABSTRACT The in vivo and in vitro stimulation of rat hepatic mitochondrial protein synthesis by thyroxine (T4) was compared. In confirmation of Buchanan & Tapley (1966). T4 added to isolated mitochondria rapidly stimulated [14C] leucine incorporation into mitochondrial protein. The in vitro stimulation was reversed after T4 was removed by incubating the mitochondria with bovine serum albumin (BSA). The decrease in T4 stimulation of protein synthesis appeared proportional to the T4 removed by BSA. Thus, it appears probable that exchangeable T4 controls the in vitro system. In contrast, the increase in mitochondrial protein synthesis which was observed 3 to 4 days after pretreatment of hypothyroid rats with labelled and non-radioactive T4 was not reversed by BSA treatment. Moreover, mitochondrial radioactivity could not be extracted with albumin. The in vivo phenomenon does not, therefore, appear to be related to exchangeable hormone in the mitochondria. Furthermore, the estimated quantity of T4 associated with mitochondria after in vivo stimulation was at least two orders of magnitude less than that required to produce comparable stimulation of mitochondrial protein synthesis in vitro. These findings strongly suggest that in vitro and in vivo stimulation of amino acid incorporation by T4 may be mediated by different biochemical mechanisms.