The role of a template sugar-phosphate backbone in the ribosomal decoding mechanism. Comparative study of poly(U) and poly(dT) template activity

To study the role of a template sugar-phosphate backbone in the ribosomal decoding process, poly(U), poly(dT) and poly(dU)-directed cell-free amino acid incorporation was investigated under the influence of neomycin and high concentrations of Mg2+. The specificity of a factor-dependent translation system of Escherichia coli was shown to change according to the principle: "either ribo- or deoxyribopolynucleotide messenger". Poly(dT) is shown to be effectively translated in the absence of elongation factors, both at low (2 degrees C) and high (37 degrees C) temperature. Neomycin inhibits factor-free poly(dT) translation. Little or no poly(U) translation is observed in this system. A chromatographic analysis of the oligophenylalanine residues synthesized seems to show that translocation is the main step responsible for ribosome specificity to the ribo- or deoxyribopolynucleotide template in both factor-dependent and factor-free translation systems.     

Cell-free translation in reversed micelles.

Cell-free translation in reversed micelles (RM) of surfactants in organic solvents is demonstrated using as an example the synthesis of human interleukin-2 by the wheat germ translation system solubilized in Brij 96 (oleyl-poly(10)oxyethylene ether) RM in cyclohexane. The translation system components and the product were recovered from the RM system by acetone precipitation. The recovery and translation reaction yields depended on the degree of surfactant hydration. The translation yields in Brij 96 RM were close to that observed in regular aqueous solution. The Brij 96 RM system is regarded as a promising media for the cell-free synthesis of hydrophobic proteins. Meanwhile, no translation reaction was observed in Aerosol OT (sodium bis(2-ethylhexyl) sulfosuccinate) RM in octane, which presumably is due to the ability of Aerosol OT to bind Mg2+ ions necessary for the functioning of the translation apparatus.     

Chickpea genotypes differ in their sensitivity to Zn deficiency

Zinc (Zn) deficiency is common in most of the chickpea growing areas of the world and growing Zn-efficient genotypes on Zn-deficient soil is a benign approach of universal interest. Response of 13 chickpea genotypes (10 desi and 3 kabuli) to Zn nutrition was studied in a pot experiment under glasshouse conditions. Plants were grown in a Zn-deficient siliceous sand for 6 weeks and fertilized with 0 (Zn–) and 2.5 mg Zn per kg soil (Zn+). When grown with no added Zn, Zn deficiency symptoms (chlorosis of younger leaves and stipules followed by necrosis of leaf margins) appeared 3–4 weeks after planting and were more apparent in cultivars Tyson, Amethyst and Dooen than Kaniva and T-1587. Zn deficiency reduced shoot growth, but it was less affected in breeding lines T-1587 and CTS 11308 than cultivars Tyson, Dooen, Amethyst and Barwon. Among all genotypes, Tyson produced the lowest root dry weight in Zn– treatment. Zinc efficiency based on shoot dry weight showed marked differences among genotypes; breeding lines CTS-60543, CTS-11308 and T-1587 were 2-fold more Zn-efficient than cultivars Tyson and Dooen. A higher Zn accumulation per plant and higher Zn uptake per g. of root dry weight were recorded in T-1587 and CTS-11308 when compared with Tyson. Root:shoot ratio was increased and proportionally more Zn was transported to the shoot when the soil was deficient. Cultivars that were very sensitive to Zn deficiency tended to have their root:shoot ratio increased by Zn deficiency more than less sensitive cultivars. The insensitive lines T-1587 and CTS-11308 transported more than 70% of the total absorbed Zn to the shoot. It is concluded that chickpea genotypes vary in their sensitivity to Zn deficiency. Advanced breeding lines T-1587 and CTS-11308 are relatively more Zn-efficient compared with Australian chickpea cultivar Tyson. Zn efficiency in chickpea genotypes is probably related to an efficient Zn absorption coupled with a better root to shoot transport.     

Glycolipid-anchored acetylcholinesterases from rabbit lymphocytes and erythrocytes differ in their sensitivity to phosphatidylinositol-specific phospholipase C.

The type of membrane association of acetylcholinesterase (AChE, EC 3.1.1.7) was studied in rabbit lymphocytes and erythrocytes. In both cases, the unique AChE molecular form was an amphiphilic dimer (referred to as G2a) anchored in the membrane by a glycosylphosphatidylinositol. In lymphocytes, G2a AChE was directly converted into its hydrophilic G2h counterpart by a treatment with Bacillus thuringiensis phosphatidylinositol-phospholipase C (PI-PLC, EC 3.1.4.10). In erythrocytes, AChE was resistant to PI-PLC but was rendered sensitive by a prior deacylation with alkaline hydroxylamine. This observation suggests that, as previously reported for human erythrocyte AChE, an acylation of the inositol ring in the glycolipid anchor of rabbit erythrocyte AChE (that does not occur in lymphocytes) prevents the cleavage.     

Cell-free systems for capsid assembly of primate lentiviruses from three different lineages

We recently demonstrated that capsids from three main primate lentiviral lineages appear to form via a pathway of assembly intermediates in primate cells. Retroviral capsid assembly intermediates were initially identified and characterized using a cell-free system for assembly of immature HIV-1 capsids. Because cell-free capsid assembly systems are useful tools, we are interested in developing such systems for other primate lentiviruses besides HIV-1. Here we extend previous cell-free studies by showing that Gag proteins of HIV-2, from a second primate lentiviral lineage, progress from early intermediates to late intermediates and completed capsids over time. Additionally, we demonstrate that Gag proteins of SIVagm, from a third primate lentiviral lineage, associate with the cellular factor HP68 and complete assembly in this system. Therefore, cell-free systems reproduce assembly of Gag from three main primate lentiviral lineages, and can be used to compare mechanistic features of capsid assembly of genetically divergent primate lentiviruses.     

Cell-free translation systems prepared from starfish oocytes faithfully reflect in vivo activity; mRNA and initiation factors stimulate supernatants from immature oocytes.

Meiotic maturation stimulates a change in the translation of stored mRNAs: mRNAs encoding proteins needed for growth of oocytes are translated before meiotic maturation, whereas those encoding proteins required for cleavage are translated after meiotic maturation. Studies of translational regulation during meiotic maturation have been limited by the lack of translationally active cell-free supernatants. Starfish oocytes are ideal for preparing cell-free translation systems because experimental application of the hormone 1-methyladenine induces their maturation, synchronizing meiosis. We have prepared such systems from both immature and mature oocytes of starfish. Changes in protein synthesis rates and the specificity of proteins synthesized in these cell-free translation supernatants mimic those seen in vivo. Supernatants both from immature and mature oocytes have a high capacity to initiate new translation because 90% of the proteins made are newly initiated from mRNAs. Cell-free supernatants from mature oocytes have a much higher rate of initiation of translation than those from immature oocytes and use the 43S preinitiation complexes more efficiently in initiation of translation. Similarly, we have shown that mRNAs and initiation factors are rate limiting in cell-free translation systems prepared from immature oocytes. In addition, cell-free translation systems prepared from immature oocytes are only slightly, if at all, inhibitory to cell-free translation systems from mature oocytes. Thus, soluble inhibitors, if they exist, are rapidly converted by cell-free supernatants from mature oocytes. The similarities between translation in our starfish cell-free translation systems and in intact oocytes suggests that the cell-free translation systems will be useful tools for further studies of maturation events and translational control during meiosis.     

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

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

Cell-free translation of virion RNA from nondefective and transformation-defective Rous sarcoma viruses.

Nondefective and transformation-defective virion subunit RNAs from two strains of Rous sarcoma virus (RSV) were translated in cell-free systems derived from Krebs IIA ascites cells, wheat germ, and L-cells. In each case the predominant viral-specific product was a polypeptide of molecular weight 76,000 that is related to the internal viral group-specific antigens, as judged by immunoprecipitation with monospecific antisera and tryptic peptide fingerprinting. No difference could be detected between the translation products of 35S RNA from nondefective and transformation-defective RSV virions, nor of 35S RNA from different strains of RSV. The 76,000-molecular-weight polypeptide synthesized in response to 35S RNA in vitro was labeled with formyl-methionine from initiator tRNA. Models for viral protein synthesis are discussed in the light of these results, and arguments positioning the group-specific antigen gene at the 5' end of the 35S RNA are presented.     

Fungal polygalacturonases exhibit different substrate degradation patterns and differ in their susceptibilities to polygalacturonase-inhibiting proteins.

Polygalacturonic acid (PGA) was hydrolyzed by polygalacturonases (PGs) purified from six fungi. The oligogalacturonide products were analyzed by HPAEC-PAD (high performance anion exchange chromatography-pulsed amperimetric detection) to assess their relative amounts and degrees of polymerization. The abilities of the fungal PGs to reduce the viscosity of a solution of PGA were also determined. The potential abilities of four polygalacturonase-inhibiting proteins (PGIPs) from three plant species to inhibit or to modify the hydrolytic activity of the fungal PGs were determined by colorimetric and HPAEC-PAD analyses, respectively. Normalized activities of the different PGs acting upon the same substrate resulted in one of two distinct oligogalacturonide profiles. Viscometric analysis of the effect of PGs on the same substrate also supports two distinct patterns of cleavage. A wide range of susceptibility of the various PGs to inhibition by PGIPs was observed. The four PGs that were inhibited by all PGIPs tested exhibited an endo/exo mode of substrate cleavage, while the three PGs that were resistant to inhibition by one or more of the PGIPs proceed by a classic endo pattern of cleavage.     

Cell-free translation of exogenous mRNA in extracts from dry pea primary axes

Extracts from the primary axes of dry pea (Pisum sativum L.) seeds are able to perform an initiation-dependent translation of exogenous mRNA. SDS polyacrylamide gel electrophoresis of the products synthesized under direction of alfalfa mosaic virus RNA (AMV-RNA) and tobacco mosaic virus RNA (TMV-RNA) shows that the fidelity of translation in this pea system is at least as high as in a wheat embryo cell-free protein synthesizing system. The endogenous messengers are also efficiently translated in extracts from the primary axes of pea seeds. The direct translation of these messengers in a homologous cell-free system may be of interest for a study of the products coded for by the long-lived messengers present in this plant.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-ethanesulfonic acid - SDS sodium dodecyl sulphate - mRNP messenger ribonucleoprotein - AMV-RNA alfalfa mosaic virus RNA - TMV-RNA tobacco mosaic virus RNA - ATA aurin tricarboxylic acid - TCA trichloroacetic acid - S.A. specific activity     

Cell-free RNA replication systems based on a human cell extracts-derived in vitro translation system with the encephalomyocarditisvirus RNA

To study the relationship between translation and replication of encephalomyocarditisvirus (EMCV) RNA, we established a cell-free RNA replication system by employing a human cell extracts-based in vitro translation system. In this system, a cis-EMCV RNA replicon encoding the Renilla luciferase (R-luc) or GFP and the viral regulatory proteins efficiently replicated with simultaneous translation of the encoded protein. To examine how translation of the replicon RNA, but not the translated products, affected replication, a trans-EMCV RNA replicon encoding R-luc and the RNA replication elements was next constructed. The trans-replicon RNA replicated only in the presence of the regulatory proteins pre-expressed in trans. Incubation with cycloheximide, puromycin or a dominant-negative eukaryotic translation initiation factor 4A following expression of the regulatory proteins almost completely inhibited not only translation of the trans-replicon RNA but also replication of the RNA, suggesting that EMCV RNA translation promotes replication of the RNA. In conclusion, the cell-free RNA replication systems should become useful tools for the study of the viral RNA replication.     

Pathogen fitness components and genotypes differ in their sensitivity to nutrient and temperature variation in a wild plant-pathogen association

Understanding processes maintaining variation in pathogen life-history stages affecting infectivity and reproduction is a key challenge in evolutionary ecology. Models of host-parasite coevolution are based on the assumption that genetic variation for host-parasite interactions is a significant cause of variation in infection, and that variation in environmental conditions does not overwhelm the genetic basis. However, surprisingly little is known about the stability of genotype-genotype interactions under variable environmental conditions. Here, using a naturally occurring plant-pathogen interaction, I tested whether the two distinct aspects of the infection process - infectivity and transmission potential - vary over realistic nutrient and temperature gradients. I show that the initial pathogen infectivity and host resistance responses are robust over the environmental gradients. However, for compatible responses there were striking differences in how different pathogen life-history stages and host and pathogen genotypes responded to environmental variation. For some pathogen genotypes even slight changes in temperature arrested spore production, rendering the developing infection ineffectual. The response of pathogen genotypes to environmental gradients varied in magnitude and even direction, so that their rankings changed across the abiotic gradients. Hence, the variable environment of spatially structured host-parasite interactions may strongly influence the maintenance of polymorphism in pathogen life-history stages governing transmission, whereas evolutionary trajectories of infectivity may be unaffected by the surrounding environment.     

Cell-free translation in lysates from Spodoptera frugiperda (Lepidoptera: Noctuidae) cells

1. Conditions for in vitro translation of mRNA in cell-free extracts from cultured Spodoptera frugiperda cells were defined. 2. Incorporation of [35S]methionine into acid-precipitable material increased for approximately 1 hr, and was sensitive to the protein synthesis inhibitors pactamycin and cycloheximide. 3. Micrococcal nuclease-treated lysate, primed with purified rabbit globin mRNA, synthesized a major protein with the size of full length globin, indicating that the lysate supported correct initiation and elongation of polypeptides.     

Chemical reactions in double-stranded nucleic acids. VI. N-hydroxybenzotriazole esters of oligonucleotides--reagents for synthesis of DNA-duplexes with modified sugar-phosphate backbone

With the aid of a new type of phosphorylating agents, N-hydroxybenzotriazole phosphodiesters of oligonucleotides, template-directed synthesis of DNA duplexes was carried out with ribonucleotide, aliphatic diamine or amino acid residues at a predetermined position of the sugar-phosphate backbone. Introduction of a ribonucleotide into an oligonucleotide strand gives rise to a mixture of 2'-5' and 3'-5' isomers, whose ratio depends on the condensation conditions. Residues of amino acids (lysine, serine, tyrosine) or aliphatic diamines were substituted for one or two mononucleotides in the duplex. Phosphoamide bonds with the participation of an aliphatic diamine or lysine are formed most efficiently in the presence of N-methylimidazole and if the reacting groups are in the close proximity to each other. Phosphodiester bond synthesis with the participation of hydroxy groups of serine or tyrosine proceeds less effectively. Oligonucleotide N-hydroxybenzotriazole esters exceeds previously used phosphoimidazolides in efficiency of the chemical ligation. An amino acid residue incorporated into the duplex may be used in construction of new compounds by joining the carboxyl with various groups.