An improved method for high-quality RNA isolation from needles of adult maritime pine trees

When conventional RNA isolation methods optimized for pine seedlings are applied to needles of adult pine trees, poor-quality RNA results. Here we describe a modified procedure to isolate high-quality RNA from needles of 30-year-old maritime pines, exhibiting high levels of phenolics, polysaccharides, and RNases. Major changes are the inclusion of proteinase K in the extraction medium followed by incubation at 42°C. Integrity and purity were evaluated by using denaturing gel electrophoresis and spectrophotometry (A₂₆₀/A₂₃₀ and A₂₆₀/A₂₈₀). The total RNA could be successfully used for poly(A)⁺-RNA isolation and cDNA library construction.     

Interaction of chandipura virus N and P proteins: identification of two mutually exclusive domains of N involved in interaction with P

The nucleocapsid protein (N) and the phosphoprotein (P) of nonsegmented negative-strand (NNS) RNA viruses interact with each other to accomplish two crucial events necessary for the viral replication cycle. First, the P protein binds to the aggregation prone nascent N molecules maintaining them in a soluble monomeric (N(0)) form (N(0)-P complex). It is this form that is competent for specific encapsidation of the viral genome. Second, the P protein binds to oligomeric N in the nucleoprotein complex (N-RNA-P complex), and thereby facilitates the recruitment of the viral polymerase (L) onto its template. All previous attempts to study these complexes relied on co-expression of the two proteins in diverse systems. In this study, we have characterised these different modes of N-P interaction in detail and for the first time have been able to reconstitute these complexes individually in vitro in the chandipura virus (CHPV), a human pathogenic NNS RNA virus. Using a battery of truncated mutants of the N protein, we have been able to identify two mutually exclusive domains of N involved in differential interaction with the P protein. An unique N-terminal binding site, comprising of amino acids (aa) 1-180 form the N(0)-P interacting region, whereas, C-terminal residues spanning aa 320-390 is instrumental in N-RNA-P interactions. Significantly, the ex-vivo data also supports these observations. Based on these results, we suggest that the P protein acts as N-specific chaperone and thereby partially masking the N-N self-association region, which leads to the specific recognition of viral genome RNA by N(0).     

Cell-free synthesis of epoxide hydrase by liver poly (A+)-RNA isolated from phenobarbital treated rats

Total liver RNA has been isolated from normal and 8 day phenobarbital treated rats by guanidine thiocyanate β-mercaptoethanol extraction and fractionated by oligo (dT)-cellulose chromatography to yield poly (A⁺)-RNA. Poly (A⁺)-RNA from normal and phenobarbital treated rats have similar translational activity in the rabbit reticulocyte cell-free system. However major alterations occurred in the polypeptide products directed by these two classes of RNA. The translation products directed by 8 day phenobarbital poly(A⁺)-RNA were immunoprecipitated with rabbit IgG prepared against purified rat liver epoxide hydrase. The immunoprecipitate was subjected to SDS-polyacrylamide gel electrophoresis and the radioactive products detected by fluorography. Analysis of the fluorogram revealed that the major immunoprecipitable product co-electrophoresed with purified epoxide hydrase. These data suggest that the primary translation product of epoxide hydrase messenger RNA has the same molecular weight as the mature form of the enzyme.     

Structure of the vesicular stomatitis virus N⁰-P complex

Replication of non-segmented negative-strand RNA viruses requires the continuous supply of the nucleoprotein (N) in the form of a complex with the phosphoprotein (P). Here, we present the structural characterization of a soluble, heterodimeric complex between a variant of vesicular stomatitis virus N lacking its 21 N-terminal residues (N_Δ21) and a peptide of 60 amino acids (P₆₀) encompassing the molecular recognition element (MoRE) of P that binds RNA-free N (N⁰). The complex crystallized in a decameric circular form, which was solved at 3.0 Å resolution, reveals how the MoRE folds upon binding to N and competes with RNA binding and N polymerization. Small-angle X-ray scattering experiment and NMR spectroscopy on the soluble complex confirms the binding of the MoRE and indicates that its flanking regions remain flexible in the complex. The structure of this complex also suggests a mechanism for the initiation of viral RNA synthesis.     

Preformed and newly synthesized messenger RNA in germinating wheat embryos

In 6 h germinated wheat (Triticum aestivum L. cv. Cama) embryos, more than half of the messenger RNAs are actively involved in translation. Neither preformed nor newly synthesized poly A⁺-RNA is translated preferentially. Germination in the presence of cordycepin showed that the half-life of the templates is about 2 h and that the newly synthesized messengers are essential to support protein synthesis in the embryo from the first hours of germination. Most of the messenger RNAs in 6 h germinated embryos are newly synthesized. The polypeptides coded for by either the endogenous messenger ribonucleoproteins or purified poly A⁺-RNA from both dry and germinated embryos are qualitatively identical; minor quantitative differences can however be observed.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - EDTA ethylenediaminetetraacetic acid - SDS sodium dodecyl sulfate - TCA trichloroacetic acid - mRNP messenger ribonucleoprotein - poly A⁺-RNA polyadenylic acid containing RNA - PB polysome buffer - GM germination medium     

Macronutrient content in leaves of bean plants (Phaseolus vulgaris,L.) grown with differents rates of N/S as fertilizers

Summary The effect of increasing rates of nitrogen (N) and sulphur (S) as fertilizers on the yield, leaf area and N, P, S, Ca, Mg, NO₃ ⁻ and SO₄ ⁼ content in leaves of bean (Phaseolus vulgaris, L.) were studied in a hydroponic culture experiment under greenhouse conditions. Bean plants responded significantly to all treatments with differents N/S ratios. When plants grew with high N/S ratios, the leaf content of N, Ca and NO₃ ⁻ increased while the content of K, P and SO₄ ⁼ decreased. However, optimal yield and leaf area were not obtained. Optimal leaf and fruit dry matter was obtained at N/S ratio value of 1.41. When lower N/S rates were used, optimal leaf and fruit dry matter was only observed when the leaf N/S ratio was between 15 and 16. At high sulphate levels in the nutrient solution there is no interaction with nitrate which is easily observed, resulting in an increase in yield. An interaction between nitrate and sulphate in the nutrient solution was found at a N/S ratio of 0.81 which produced in leaves a synergic effect between P-K, an antagonistic effect between N-P and N-K and a lower yield. This research was supported by Fundacion ‘Ramon Areces’.     

Nucleic acid and protein synthesis and loss of vigour in germinating wheat embryos

A study has been made of the RNA and protein synthesising systems of wheat embryos isolated from seed lots having high viability but differing in vigour. The rate of RNA and protein synthesis in wheat embryos during the early hours of germination is related to the vigour of the seed lot. The imposition of a stress factor, in the nature of a sub-optimal germination temperature, during germination of isolated wheat embryos magnifies the differences in rates of protein and RNA synthesis between high and low vigour seed. Using cell-free protein synthesising systems it has been demonstrated that an important difference between high and low vigour embryos lies in the relative levels of messenger RNA in the embryo. High vigour embryos contain relatively higher levels of poly A⁺-RNA (i.e. potential mRNA species) than lower vigour embryos and furthermore the level of poly A⁺-RNA in high vigour embryos increases during early germination whilst in lower vigour embryos the level decreases. The difference in poly A⁺-RNA levels accounts, at least partially, for the differences in rates of protein synthesis observed between embryos from high and low vigour wheat seed during early germination at both optimal and sub-optimal germination temperatures.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - poly A⁺-RNA polyadenylated RNA - GM germination medium - PMS post-mitochondrial supernatant fraction     

Identification and characterization of the packaging proteins of core 40S hnRNP particles

The proteins involved in protein-RNA and protein-protein interactions to form the core structure of nuclear 40S hnRNP particles in HeLa cells have been identified and characterized. Through complete analysis of nuclear extracts on sucrose density gradients and controlled salt dissociation of particle proteins, six lower molecular weight polypeptides are identified as the protein constituents of the 40S ribonucleoprotein complex which appears in the electron microscope as 210 A spherical particles. 40S hnRNP particles isolated from Chinese hamster lung fibroblasts show a strikingly similar protein composition to the human cells. The proteins are specifically associated with rapidly labeled nonribosomal nuclear RNA. Particle proteins from HeLa cells migrate in polyacrylamide gels as three groups of closely spaced doublets (groups A, B and C) and are present in a simple fixed stoichiometry. The group C proteins (C₁ and C₂ of 42,000 and 44,000 daltons) interact directly with RNA to form a smaller high salt-resistant RNP complex. The group A proteins (A₁ and A₂ of 32,000 and 34,000 daltons) are major nuclear proteins and constitute 60% total particle protein mass. These two proteins are basic with isoelectric points near 9.2 and 8.4, respectively, and are characterized by an unusual amino acid composition, including high glycine (25%) and the unusual modified basic residue identified as N^G,N^G-dimethylarginine. The major particle proteins (A₁ and A₂) interact electrostatically with nucleic acids and apparently function structurally in the packaging and stabilization of hnRNA in a manner analogous to the histones in chromatin υ bodies. The similarity in protein composition of core RNP particles from different cell types (especially in the basic proteins, A₁, A₂ and B₁) is consistent with a conserved particle structure and function in eucaryotes.     

Mechanism of RNA synthesis initiation by the vesicular stomatitis virus polymerase

The minimal RNA synthesis machinery of non-segmented negative-strand RNA viruses comprises a genomic RNA encased within a nucleocapsid protein (N-RNA), and associated with the RNA-dependent RNA polymerase (RdRP). The RdRP is contained within a viral large (L) protein, which associates with N-RNA through a phosphoprotein (P). Here, we define that vesicular stomatitis virus L initiates synthesis via a de-novo mechanism that does not require N or P, but depends on a high concentration of the first two nucleotides and specific template requirements. Purified L copies a template devoid of N, and P stimulates L initiation and processivity. Full processivity of the polymerase requires the template-associated N protein. This work provides new mechanistic insights into the workings of a minimal RNA synthesis machine shared by a broad group of important human, animal and plant pathogens, and defines a mechanism by which specific inhibitors of RNA synthesis function.     

Reduced Model Captures Mg-RNA Interaction Free Energy of Riboswitches

The stability of RNA tertiary structures depends heavily on Mg²⁺. The Mg²⁺-RNA interaction free energy that stabilizes an RNA structure can be computed experimentally through fluorescence-based assays that measure Γ₂₊, the number of excess Mg²⁺ associated with an RNA molecule. Previous explicit-solvent simulations predict that the majority of excess Mg²⁺ ions interact closely and strongly with the RNA, unlike monovalent ions such as K⁺, suggesting that an explicit treatment of Mg²⁺ is important for capturing RNA dynamics. Here we present a reduced model that accurately reproduces the thermodynamics of Mg²⁺-RNA interactions. This model is able to characterize long-timescale RNA dynamics coupled to Mg²⁺ through the explicit representation of Mg²⁺ ions. KCl is described by Debye-Hückel screening and a Manning condensation parameter, which represents condensed K⁺ and models its competition with condensed Mg²⁺. The model contains one fitted parameter, the number of condensed K⁺ ions in the absence of Mg²⁺. Values of Γ₂₊ computed from molecular dynamics simulations using the model show excellent agreement with both experimental data on the adenine riboswitch and previous explicit-solvent simulations of the SAM-I riboswitch. This agreement confirms the thermodynamic accuracy of the model via the direct relation of Γ₂₊ to the Mg²⁺-RNA interaction free energy, and provides further support for the predictions from explicit-solvent calculations. This reduced model will be useful for future studies of the interplay between Mg²⁺ and RNA dynamics.     

Reduced Model Captures Mg2+-RNA Interaction Free Energy of Riboswitches

The stability of RNA tertiary structures depends heavily on Mg²⁺. The Mg²⁺-RNA interaction free energy that stabilizes an RNA structure can be computed experimentally through fluorescence-based assays that measure Γ₂₊, the number of excess Mg²⁺ associated with an RNA molecule. Previous explicit-solvent simulations predict that the majority of excess Mg²⁺ ions interact closely and strongly with the RNA, unlike monovalent ions such as K⁺, suggesting that an explicit treatment of Mg²⁺ is important for capturing RNA dynamics. Here we present a reduced model that accurately reproduces the thermodynamics of Mg²⁺-RNA interactions. This model is able to characterize long-timescale RNA dynamics coupled to Mg²⁺ through the explicit representation of Mg²⁺ ions. KCl is described by Debye-Hückel screening and a Manning condensation parameter, which represents condensed K⁺ and models its competition with condensed Mg²⁺. The model contains one fitted parameter, the number of condensed K⁺ ions in the absence of Mg²⁺. Values of Γ₂₊ computed from molecular dynamics simulations using the model show excellent agreement with both experimental data on the adenine riboswitch and previous explicit-solvent simulations of the SAM-I riboswitch. This agreement confirms the thermodynamic accuracy of the model via the direct relation of Γ₂₊ to the Mg²⁺-RNA interaction free energy, and provides further support for the predictions from explicit-solvent calculations. This reduced model will be useful for future studies of the interplay between Mg²⁺ and RNA dynamics.     

Phosphorus metabolism in coral reef communities: exchange between the water column and bottom biotopes

Exchange of phosphate between components of the reef bottom and the water column were studied on reefs around Heron Island (Great Barrier Reef), both in aquaria and in in situ enclosures, using radioactive phosphorus (³²P) as a tracer. Living corals, dead corals, coral rubble overgrown with periphyton, and soft sediments of coral sand were used in experiments. In all of these components of bottom reef biotopes, two opposite flows of inorganic phosphate were recorded and measured, i.e. the rate of PO₄-P uptake from water (A_c), and its release (A_e). At ambient PO₄-P concentrations in water of 0.1– 0.3 µmoll^–1, both flows varied in living corals and coral rubble between 10 and 70 µg P kg^–1 h^–1, 3–10 mg P m^–2 day^–1, and in coral sand between 10 and 30 µg P kg^–1 h^–1, or 2–7 mg P m^–2 day^–1. Under the latter concentration range (which is typical for coral reef areas), the reciprocal PO₄-P flows almost balanced each other, so that net uptake (A_t) was very low. Often it approached zero or was positive, showing that a net PO₄-P release had taken place. The uptake flow (A_c) in living coral was much more dependent on the PO₄-P content in overlying water than was the release flow (A_e). The influence of conditions of illumination upon the values of A_c and A_e was comparatively low. The data obtained are used to discuss problems of phosphorus balance and dynamics in coral reef ecosystems.