Wheat Germ Translation System Research Articles

Synthesis in Vitro of the coat protein of papaya mosaic virus

Papaya mosaic virus (PMV) RNA directs the synthesis in vitro in either the wheat germ or reticulocyte lysate translation systems of numerous products. Three polypeptides are most prominent: pA (155,000 molecular weight), pB (73,000), and pC (22,000). The last, pC, displays the same molecular weight as coat protein and is precipitated by antibodies raised against PMV. The identities of pA and pB are less clear, although peptide mapping suggests that they and many of the minor products of translation are related to each other, but not to pC. The ability of partially encapsidated PMV ribonucleoprotein particles to direct protein synthesis has been assessed. As the extent of encapsidation increased, the relative synthesis of pA and pB decreased markedly. In contrast, the synthesis of pC persisted until a substantial fraction of the PMV RNA was completely encapsidated. In view of the polarity of assembly of PMV in vitro (M. Abouhaidar and J. B. Bancroft, (1978), Virology 90, 54–59) this finding indicates that the coding sequences for PMV coat protein are localized toward the 3′ end of PMV RNA. A kinetic analysis of coat protein synthesis, coupled with Northern blot analysis of PMV RNA during translation suggest that PMV RNA must be cleaved to activate it for in vitro coat protein synthesis.

Salt‐tolerance in plants. II. In vitro translation of m‐RNAs from salt‐tolerant and salt‐sensitive plants on wheat germ ribosomes. Responses to ions and compatible organic solutes

Abstract Messenger RNA from salt‐sensitive and salt‐tolerant plants Triticum aestivum. Beta vulgaris, Pisum sativum, Chenopodium album and Atriplex nummularia was translated in vitro in a wheatgerm translation system. The optimal monovalent and divalent ion concentrations for translation were independent of the salt tolerance of the plants from which the m‐RNAs were derived. Translation was optimal in 100 120 mol m−3 potassium acetate and 1.5–2.0 mol m−3 Mg2+. Substitution of Na+ for K+, or of Cl− for acetate, was inhibitory. The pattern of polypeptides synthesized from cytoplasmic m‐RNAs of salt‐sensitive and salt‐tolerant plants remained constant in all the conditions examined. The effects of adding the ‘compatible' organic solutes glycine‐betaine and mannitol were examined in the wheat‐germ system primed with RNA from the leaves of Triticum aestivum or Beta vulgaris. The rate of translation, the optimum ionic concentrations and the distribution of polypeptide products were maintained in organic solute concentrations of up to 500 mol m−3. Proline above 300 mol m−3 and surcose above 100 mol m−3 did inhibit translation. The results indicate that translation in plants is unlikely in cytoplasmic K+ concentrations exceeding 180 mol m−3, but would proceed in the presence of up to 500 mol m−3 mannitol or glyinebetaine, or of up to 300 mol m−3 proline.

Isolation and characterization of a translation inhibitor from human term placenta

An inhibitor of protein synthesis has been isolated from free cytoplasmic ribonucleoprotein particles of human term placenta. The inhibitor is resistant to phenol, DNase, proteinase K, and heating at 100 °C, but is sensitive to alkaline hydrolysis. These data suggest that the inhibitor is RNA. Experiments provide evidence that this preparation contains no RNase contaminant and does not induce an RNase in this assay system. Three lines of evidence suggest that the inhibitor acts at the initiation of protein synthesis in the wheat germ translation system. First, a lag occurs before cessation of translation when the inhibitor is added to translating polyribosomes. This lag is identical to that seen upon the addition of aurintricarboxylic acid, a known inhibitor of initiation. Second, sucrose gradient analyses demonstrate that, when the inhibitor is present at the start of translation, 40 S complexes form, but neither 80 S complexes nor polyribosomes are seen. Third, gradient analyses show that, when the inhibitor is added to translating polyribosomes, 40 S complexes accumulate with a progressive loss of polyribosomes. Finally, the extent of inhibition depends upon the amount of wheat germ extract added to the reaction mixture and not the amount of mRNA present. This suggests an interaction between the inhibitor and a component of the wheat germ extract.

Chloroplast ribosomal proteins of Chlamydomonas synthesized in the cytoplasm are made as precursors.

Polyadenylated RNA from Chlamydomonas was translated in a cell-free rabbit reticulocyte system that employed [35S]methionine. Antibodies made to four chloroplast ribosomal proteins synthesized in the cytoplasm and imported into the organelle were used for indirect immunoprecipitation of the labeled translation products, which were subsequently visualized on fluorographs of SDS gels. The cytoplasmically synthesized chloroplast ribosomal proteins were first seen as precursors with apparent molecular weights of 1,000 to 6,000 greater than their respective mature forms. Processing of the ribosomal protein precursors to mature proteins was affected by adding a postribosomal supernatant that had been extracted from cells of Chlamydomonas. In contrast to the chloroplast ribosomal proteins synthesized in the cytoplasm, two such proteins made within the chloroplast were found to be synthesized in mature form in cell-free wheat germ translation systems programmed with nonpolyadenylated RNA.

Evidence that 2-allyl-2-isopropylacetamide, phenobarbital and 3,5-diethoxycarbonyl-1,4-dihydrocollidine induce the same cytochrome P450 mRNA in chick embryo liver.

The induction of cytochrome P450 in chick embryo liver has been studied using three different porphyrinogenic drugs, 2-allyl-2-isopropylacetamide, 3,5-diethoxycarbonyl-1,4-dihydrocollidine and phenobarbital. Pulse-labelling studies have shown that for each drug the cytochrome P450 synthesized either in ovo or in a wheat germ translation system reacted immunologically with antibody raised against the purified 2-allyl-2-isopropylacetamide-induced enzyme (Mr = 50000). To investigate whether this is due to the three drugs inducing the same protein or different proteins with common immunological determinants, nucleic acid hybridization studies have been carried out using a recently characterised 2-allyl-2-isopropylacetamide-induced cytochrome P450 cloned cDNA probe [Brooker, J. D. et al. (1982) Eur. J. Biochem. 129, 325-333]. It has been shown that the mRNA induced by each drug hybridizes with this probe and all are of similar size. The melting profile of the mRNA . cDNA hybrids indicates that the mRNAs induced by the three drugs have at least 98% homology with the cDNA probe. Restriction endonuclease digestions of total chick embryo genomal DNA and a chick cytochrome P450 genomal clone indicates that the cytochrome P450 gene homologous with the cDNA probe is represented in the genome only once. These results strongly suggest that the three drugs cause increased levels of the same cytochrome P450 mRNA, possibly due to enhanced expression of the same gene. Results are also presented which show that other cytochrome-P450-inducing drugs, 3-methylcholanthrene, beta-naphthoflavone or pregnenolone-16 alpha-carbonitrile do not increase the level of the 2-allyl-2-isopropylacetamide-inducible mRNA but rather reduce it to a level which was lower than that of the untreated controls.

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.

56] In vitro processing of plant preproteins

This chapter proposes to develop a cell-free system for the translation, processing and packaging of plant preproteins that demonstrates the synthesis, processing, and deposition of zein polypeptides and α-amylase. The method uses a nuclease-treated rough endoplasmic reticulum (RER)-enriched preparation derived from developing maize endosperm in combination with a wheat germ translation system. The procedure involves material required for membrane preparation, isolation of membranes, preparation of treated membranes, and in vitro protein synthesis and processing. Nonpigmented kernels, such as Illinois High Protein, are preferred to colored kernels. In vivo , zein is synthesized in the maize endosperm on polyribosomes of the RER cisternae that subsequently enlarge to form globular protein bodies. The procedures for the isolation and preparation of treated maize RER membranes can probably be readily adapted to other plant species. Before setting up a synthesis and processing reaction, the membranes are treated with micrococcal nuclease to destroy endogenous zein messages.

Stability of Polysome-Associated mRNA in Potato Tuber Cells during Aging of Tissue Discs

The stability of polysome-associated mRNA in potato tuber discs in the early stage of aging was examined by pulse-chase labeling experiments and the change in the translational capacity of the RNA was studied using a wheat germ translation system. The incorporation of pulse-fed 3H-uridine into polysomal RNA was not arrested immediately after the addition of actinomycin D to the tissue, but increased by 25% during 4 hr of chasing. The radioactivity in the polysomal RNA then decreased by only 30% of the value at the 4th hr during the next 9 hr in the presence of actinomycin D. The remaining radioactivity in the polysomal RNA was stable at least for 18 hr. The proportion of radioactivity in polyadenylated RNA to that in non-polyadenylated RNA did not vary appreciably during the chasing period. Non-polyadenylated RNA of high molecular weight degraded faster than that of low molecular weight, but polyadenylated RNA did not show such size-selective degradation. The translational capacity of the polysomal RNA also decreased by about 23% within 9 hr during the period of inhibited RNA synthesis. In vivo experiments of 14C-leucine incorporation into proteins in the absence of RNA synthesis suggested that stable polysome-associated mRNA was actually functioning in the cells. SDS-polyacrylamide gel electrophoresis of the in vitro translation products indicated that mRNA coding for polypeptides with relatively high molecular weights turned over slightly faster than those for low molecular weight polypeptides.

Translation of mRNAs for subunits of chloroplast coupling factor 1 in spinach

The chloroplast coupling factor 1 consists of five nonidentical subunits, three of which (alpha, beta, and epsilon subunits) have been shown in several laboratories to be synthesized within chloroplasts. The site of synthesis of the remaining two (gamma and delta subunits) was investigated by analyzing products directed by spinach leaf RNAs in wheat germ and reticulocyte translation systems in vitro. It was found that poly(A)(+) RNA directs the synthesis of two distinct polypeptides, one of which is immunochemically related to the gamma subunit but is 4,000 daltons larger. The other shares antigenic sites with the delta subunit but is 8,000 daltons larger. When wheat germ or reticulocyte translation systems were programmed with RNAs from purified chloroplasts, the only products related to CF(1) that we could detect were a putative precursor of beta, 2,000 daltons larger than the mature subunit, and some smaller polypeptides, which appear to be incomplete translation products of beta. From these results it appears likely that the gamma and delta subunits are synthesized in the cytoplasm as larger precursors and that beta is synthesized within the chloroplast as a precursor.

In Vitro Synthesis and Processing of Wheat α-Amylase

Wheat (Triticum aestivum) RNA was used to program synthesis of the alpha-amylase protein by Xenopus laevis oocytes. A 41,500-dalton protein was made which was identified as alpha-amylase by immunoprecipitation with rabbit anti-alpha-amylase antiserum raised against the purified wheat protein and by its co-migration with authentic alpha-amylase on sodium dodecyl sulfate polyacrylamide gels. Synthesis of alpha-amylase was dependent upon injection of RNA extracted from gibberellic acid-induced aleurone layers from wheat. The amount of alpha-amylase produced was proportional to the amount of RNA injected and reached a plateau within 4 hours after injection. When the same RNA was translated in a wheat germ cell-free translation system, a 43,000-dalton protein was produced. Addition of dog pancreas microsomal membranes to the wheat germ translation system resulted in processing of the alpha-amylase protein to a form which co-migrated with authentic alpha-amylase purified from malted wheat and with the protein synthesized in oocytes.

Regulation of calcium-binding protein messenger RNA by 1,25-dihydroxycholecalciferol.

1,25-Dihydroxycholecalciferol modulates the synthesis of the intestinal calcium-binding protein. To determine if this effect is due to an increase in calcium-binding protein mRNA activity, we measured total cytoplasmic protein-specific mRNA activity from chick intestine in an in vitro wheat germ translation system. 1,25-Dihydroxycholecalciferol enhances calcium-binding protein specific mRNA activity. The sterol does not induce a general increase in mRNA synthesis since the concentration of only a few proteins increased. Thus 1,25-dihydroxycholecalciferol regulates only a subset of genes and specifically affects a transcriptional process involving calcium-binding protein synthesis.

Glucocorticoid-dependent induction of mRNA coding for phosphoenolpyruvate carboxykinase (GTP) in rat kidney. Its inhibition by cycloheximide.

The glucocorticoids induce the synthesis of phosphoenolpyruvate carboxykinase (GTP) in rat kidney as a consequence of an increase in the level of the specific enzyme mRNa. The mRNA induction was characterized with respect to its time course after hormone administration and its sensitivity to cycloheximide. The level of rat kidney mRNA directing the synthesis of phosphoenolpyruvate carboxykinase in wheat germ translation system nearly doubled within 2 h of a dexamethasone injection and further increased to four times the initial value at 6 h of treatment and to five times at 10 h. Cycloheximide injected 30 min prior to dexamethasone prevented the mRNA increase. When injected 5 h after dexamethasone, the inhibitor of protein synthesis blocked the rise of phosphoenolpyruvate carboxykinase mRNA occurring normally between 5 h and 10 h after treatment with dexamethasone. Maximal inhibitions of protein synthesis on the one hand and of mRNA induction on the other were achieved at the same dose of cycloheximine, suggesting that the two effect might be related. Dexamethasone caused an increase in the functional level of several as yet unidentified mRNAs in addition to that coding for phosphoenolpyruvate carboxykinase. The main points emerging from this study are: (a) the virtual absence of lag between dexamethasone administration and increase in phosphoenolpyruvate carboxykinase mRNa; (b) the inhibition of phosphoenolpyruvate carboxykinase mRNA induction by cycloheximide, suggesting a possible requirement for ongoing protein synthesis; (c) the existence in the kidney of a glucocorticoid-responsive domain comprising several distinct proteins.

Different species of messenger RNA encode receptor and secretory IgM mu chains differing at their carboxy termini.

Biosynthetic studies in the presence of an inhibitor of glycosylation indicate that individual human lymphoma-derived cell lines can synthesize both membrne receptor and presumptive secretory forms of IgM mu chains. The receptor form has a larger polypeptide chain than the secretory form and possesses a different C-terminus, but similar N-terminus, consistent with the presence of a C-terminal hydrophobic "tail" for integral membrane binding. Messenger RNA isolated from these cells directs the synthesis of both forms of mu chains in a wheat germ translation system, indicating the presence of independent mRNAs for each form. It is proposed that the synthetic pathways for receptor and secretory IgM diverge at the post-transcriptional level, possibly by differential RNA splicing to give mRNA molecules with or without a translatable "tail" segment.

Hormonal control of tyrosine aminotransferase, prolactin, and growth hormone induction in somatic cell hybrids

Hybrids between cells containing glucocorticoid-inducible functions and cells naturally lacking those functions usually show loss of such responses. The data supporting this conclusion is reviewed, emphasizing the control of tyrosine aminotransferase. This dominant negative control in hybrids suggests that non-inducible cells usually prevent induction by negative controls rather than by lacking appropriate receptors or acceptors. New hybrid studies, between prolactin- and growth hormone-producing (rat pituitary carcinoma) GH3 cells and non prolactin- and growth hormone-producing (transformed mouse fibroblast) L cells, will be described. Both cell types possess glucocorticoid (G) and estrogen (E) receptors, but only GH3 cells have thyrotropin-releasing hormone (TRH) receptors. In GH3 cells, prolactin is induced by E and TRH, while growth hormone is induced by G. GH3 × L cell hybrids therefore allow the first examination of the control over a sex steroid- and TRH-induced product as well as a glucoconicoid-inductd product. Fresh hybrids were (prolactin −, E and G receptor +, TRH receptor −, growth hormone −. They lacked translatable prolactin mRNA, measured in a wheat germ translation system. After extensive loss of mouse chromosomes, one hybrid regained prolactin production.

Differences in the distribution of poly(A) size classes in individual messenger RNAs from neuroblastoma cells.

Neuroblastoma polysomal mRNAs have been fractionated by a combination of oligo(dT)-cellulose and Millipore filter techniques into four classes, each containing a different average length of poly(A). These classes are present in mRNAs isolated using different extraction techniques and they probably represent the steady state mRNA distribution in neuroblastoma cells. Quantitation of the biological activity of each fraction in a wheat germ translation system shows that 32% of the mRNAs contain no oligo(A)s longer than 6; 4% have poly(A)s between 6 and 20 nucleotides, 22% have between 20 and 50 nucleotides, and 42% have greater than 50 nucleotides. Two-dimensional gel electrophoresis of the proteins synthesized by the unfractionated and fractionated mRNAs showed there was no mRNA degradation during the fractionation procedures. Analysis of the most abundant peptides synthesized by each RNA fraction shows that most mRNAs are found in each of the four populations. However, the distribution of some individual mRNAs varies widely. The (Yand fi-tubulin mRNAs are examples of those which are more abundant in the population with long poly(A)s. In contrast, a significant proportion of the mRNAs coding for actin and the R1 and RB CAMP-binding proteins are nonadenylated. The histone mRNAs are unique in that they are more abundant in the nonadenylated mRNAs than in the mRNAs containing short stretches of poly(A). These results are consistent with a model wherein the poly(A) termini in some mRNA species are more rapidly cleaved than in others.

Cell-free synthesis of cod preproinsulin.

Poly(A)-containing ribonucleic acid from cod islet greatly stimulated the incorporation of radioactive amino acids into proteins when assayed in a wheat germ translation system. The translation products were examined by specific immunoprecipitation with guinea pig anti cod insulin antibodies and by extraction with acid--ethanol. These measurements revealed at least a fivefold increase in incorporation of labelled amino acid over the nonprogrammed system. Sodium dodecyl sulfate disc gel electrophoresis and gel filtration chromatography showed a product of molecular weight 12 500, a size considerably larger than cod proinsulin (9000). It is concluded that cod proinsulin is synthesized via a larger precursor, preproinsulin.

Translation “in vitro” of globin mRNA from [formula omitted

RNA isolated from Xenopus laevis reticulocytes and characterized as globin mRNA (Meza et al., 1978) was tested for its capacity to stimulate “in vitro” a wheat germ translation system, and the ability to synthesize a polypeptide. The latter was identified as globin by its electrophoretic mobility and immunoprecipitation with antiglobin antibody.

Identification and Properties of the Messenger RNA Activity in Chlamydomonas reinhardi Coding for the Large Subunit of d-ribulose-1,5-bisphosphate Carboxylase

Properties of the mRNA coding for the large subunit of ribulose-1,5-bisphosphate carboxylase from Chlamydomonas reinhardi were determined. Large subunit synthesis, directed by RNA from partially purified whole cell extracts, was detected by specific immunoprecipitation of polypeptide products synthesized in a heterologous translation system derived from Escherichia coli. Large subunit synthesis showed sharp RNA concentration dependence in an E. coli translation system, and at optimal RNA concentrations, immunoprecipitable large subunit synthesis accounted for 2% of the total incorporation. Large subunit messenger activity sedimented at 12 to 14S on nondenaturing sucrose gradients and did not bind to oligo(dT)-cellulose suggesting the mRNA is not polyadenylated. The immunoprecipitable products translated in vitro are not complete polypeptide chains, but are smaller peptides identifiable as large subunit fragments by tryptic fingerprint analysis. No immunoprecipitable product was obtained when similar RNA fractions were tested in a wheat germ translation system.

Cell-free translation of simian virus 40 early messenger RNA coding for viral T-antigen.

Simian virus 40 (SV40) mRNA was isolated by hybridization of cytoplasmic RNA, from SV40-infected BS-C-1 monkey cells early in lytic infection, to SV40 DNA immobilized on Sepharose. The early viral mRNA, when added to a wheat-germ translation system, directed the synthesis of a unique class of products including a 90,000 molecular weight (Mr) polypeptide. It was found that this 90,000 Mr product as well as a prominent 17,000 Mr polypeptide could be specifically immunoprecipitated with hamster antiserum to SV40 T-antigen, but not with hamster control serum. Similar immunoprecipitation of extracts of SV40-infected cells with hamster anti-T serum yielded 90,000 Mr and 17,000 Mr polypeptides; these polypeptides were not found in immunoprecipitates of uninfected cell extracts. SV40 cRNA, prepared by asymmetric transcription of plaque-purified SV40 DNA, directed the cell-free synthesis of several products, including a 70,000 Mr polypeptide that could be specifically immunoprecipitated with anti-T serum. However, no T-antigen-related polypeptide was found in infected cells that corresponded in size to the major immunoprecipitated cRNA product.

Synthesis of very low density lipoproteins in the cockerel. Effects of estrogen.

The effect of estrogen on the synthesis of plasma very low density lipoproteins (VLDL) in the cockerel was studied both in vivo and in vitro. Synthesis was studied by immunoprecipitation techniques with antisera prepared against VLDL and a major VLDL protein. VLDL were isolated from the plasma of white Leghorn hens and estrogen-treated white Leghorn cockerels by ultracentrifugal flotation at d 1.006 g/ml. After delipidation, the lipid-free proteins (apoproteins) were fractionated on Sephadex G-150 and DEAE-cellulose. Both the hen and the estrogen-treated cockerel VLDL were shown to contain an identical apoprotein with a mol wt of approximately 12,000; the apoprotein is designated fraction B. Reduction and S-carboxy-methylation of fraction B resulted in a reduction of the molecular weight by approximately one-half, indicating a dimer-monomer relationship. Antiserum prepared to the hen VLDL dimer protein gave precipitin lines of complete identity to both the hen and cockerel dimer, monomer, VLDL, apoVLDL, low density lipoproteins, and plasma; no precipitin line was formed with either hen or cockerel high density lipoproteins. After a single subcutaneous injection of diethylstilbestrol into the cockerel, plasma VLDL protein, cholesterol, and triglyceride increased, reaching a maximum 24--48 h after hormone administration. Liver slices from similarly treated animals were incubated in vitro in culture medium in the presence of [3H]lysine for 2 h. Immunoprecipitable radioactivity in VLDL increased within 2 h of diethylstilbestrol treatment and reached a maximum at 24 h; VLDL radioactivity returned to base-line levels by 72 h. At the peak of induction, newly synthesized VLDL represented 11% of the total soluble protein synthesized. When actinomycin-D (5 mg/kg) was administered simultaneously with estrogen, the induction of VLDL synthesis was totally inhibited. To determine whether the effect of estrogen on VLDL synthesis was mediated at the level of transcription, partially-purified cockerel liver mRNA was prepared from estrogen-treated animals and the mRNA activity for fraction B was quantitated in a wheat germ translation system. Fraction B mRNA was found to increase from a low base-line value to a maximum 16-24 h after estrogen treatment, returning towards baseline values at 30 h. At the peak of induction, fraction B constituted 12% of the total protein synthesized. The kinetics of induction of fraction B mRNA activity in the cell-free translation system is very similar to that observed in liver slice experiments. This finding suggests that estrogen stimulates VLDL synthesis, at least partially, by enhancing the accumulation of the mRNA for one of their major apoproteins.