A rapid and inexpensive method for the direct PCR amplification of DNA from plants

? Premise of the study: We present a rapid and inexpensive alternative to DNA isolation for polymerase chain reaction (PCR) amplification from plants. ? Methods and Results: The method involves direct PCR amplification from material macerated in one buffer, followed by dilution and incubation in a second buffer. We describe the procedure and demonstrate its application for nuclear and plastid DNA amplification across a broad range of vascular plants. ? Conclusions: The method is fast, easy to perform, cost-effective, and consequently ideal for large sample numbers. It represents a considerable simplification of present approaches requiring DNA isolation prior to PCR amplification and will be useful in plant systematics and biotechnology, including applications such as DNA barcoding.     

Thin casein films as prepared by spin-coating: influence of film thickness and of pH

Casein films were successfully prepared with the spin-coating technique of aqueous casein solutions on base-treated glass surfaces. The film structure is investigated in real space with optical microscopy and atomic force microscopy and for the first time in reciprocal space with grazing incidence small-angle X-ray scattering (GISAXS). The size of the substructures detected in the film increases with pH from 170 nm (pH 5.1) up to 490 nm (pH 9.4). Dynamic light scattering experiments reveal that the average diameters of casein micelles in solution exhibit the same quantitative increase. This result suggests that the substructures detected in the bulklike films with GISAXS reflect intact casein micelles. However, with thin homogeneous casein films, the micelle size diminishes with decreasing film thickness. This indicates that the moderate pressures introduced by spin-coating force the micelles to rearrange into a more compact structure.     

Degenerate in vitro genetic selection reveals mutations that diminish alfalfa mosaic virus RNA replication without affecting coat protein binding

The alfalfa mosaic virus (AMV) RNAs are infectious only in the presence of the viral coat protein; however, the mechanisms describing coat protein's role during replication are disputed. We reasoned that mechanistic details might be revealed by identifying RNA mutations in the 3'-terminal coat protein binding domain that increased or decreased RNA replication without affecting coat protein binding. Degenerate (doped) in vitro genetic selection, based on a pool of randomized 39-mers, was used to select 30 variant RNAs that bound coat protein with high affinity. AUGC sequences that are conserved among AMV and ilarvirus RNAs were among the invariant nucleotides in the selected RNAs. Five representative clones were analyzed in functional assays, revealing diminished viral RNA expression resulting from apparent defects in replication and/or translation. These data identify a set of mutations, including G-U wobble pairs and nucleotide mismatches in the 5' hairpin, which affect viral RNA functions without significant impact on coat protein binding. Because the mutations associated with diminished function were scattered over the 3'-terminal nucleotides, we considered the possibility that RNA conformational changes rather than disruption of a precise motif might limit activity. Native polyacrylamide gel electrophoresis experiments showed that the 3' RNA conformation was indeed altered by nucleotide substitutions. One interpretation of the data is that coat protein binding to the AUGC sequences determines the orientation of the 3' hairpins relative to one another, while local structural features within these hairpins are also critical determinants of functional activity.     

Subcellular localization of collapsin response mediator proteins to lipid rafts

Collapsin response mediator proteins (CRMPs) are involved in signal transduction after exposure of neural cells to the axon guidance molecule Semaphorin 3A/collapsin. All five known CRMPs are expressed in the developing cerebral cortex and neocortical neurons are responsive to Semaphorin 3A. Here, we examine the expression and subcellular localization of CRMPs in neocortical neurons and in neonatal rat brain. In neocortical neurons CRMP-4 was detected in the perikaryon with a diffuse cytosolic distribution. In neurites and at growth cones punctate staining patterns were observed. Extraction of neuron cultures with methyl-beta-cyclodextrin to deplete cholesterol caused rapid redistribution of the punctate CRMP-4 staining into larger patches and abundant growth cone collapse. Western blotting of brain extracts demonstrated for all CRMPs the existence of soluble, detergent-extractable, and Triton X-100-resistant forms. Furthermore, sucrose density gradient centrifugation after solubilization of brain membranes with Triton X-100 revealed that CRMP-1, -3, -5, and to a lower extent CRMP-4 are associated with a detergent-resistant fraction with low buoyant density, but CRMP-2 was not detectable in this fraction. Thus, we propose that lipid rafts form sites for the compartmentalization of signaling events involving specific CRMPs and that the integrity of these membrane microdomains is essential for the maintenance of growth cones.     

Characterization of a unique enzyme complex composed of S-adenosyl-L-methionine-tRNA-methyltransferase and aminoacyl-tRNA synthetase activities

S-adenosyl-L-methionine-tRNA methyltransferases of a murine leukemia cell line were found to exist in a high molecular weight enzyme complex. Aminoacyl-tRNA synthetase activity always co-chromatographed and co-sedimented with methyltransferase activity in evidence of a unique association of these two groups of enzymes. Molecular weight studies showed a probable molecular weight of 9 X 10(5) daltsons for the intact complex which dissociates to complexes of 6 X 10(5) and 3 X 10(5) daltons. The complexes contain discrete polypeptides of 25,000-90,000 daltons as determined from SDS-gel electrophoresis. High resolution fatty acid analysis showed that only very small amounts of saponifiable lipids were associated with the purified enzyme complex. Similarly very little protein-bound sugars was found within the complex indicating that neither lipids nor sugars were involved in the protein-protein interactions of the complex. Analysis of tRNA methylated in vitro indicated the presence of most methyltransferase activities in the purified complex. Of note was the absence from the complex of the methyltransferase responsible for the production of ribo Tp.     

CO2 environment,microclimate and photosynthetic characteristics of the moss Hylocomium splendens in a subarctic habitat

Summary In order to document the natural CO₂ environment of the moss Hylocomium splendens, and ascertain whether or not the moss was adapted to this, and its interactions with other microenvironmental factors, two studies were carried out. Firstly, the seasonal variations of CO₂ concentration, photosynthetically active radiation (PAR), tissue water content and temperature were measured in the natural microenvironment of H. splendens in a subarctic forest during the summer period (July–September). Secondly, the photosynthetic responses of the species to controlled CO₂ concentrations, PAR, temperature, and hydration were measured in the laboratory. CO₂ concentrations around the upper parts of the plant, when PAR was above the compensation point (30 mol m^–2 s^–1), were mostly between 400 and 450 ppm. They occasionally increased up to 1143 ppm for short periods. PAR flux densities below saturating light levels for photosynthesis (100 mol m^–2 s^–1), occurred during 65% (July), 76% (August) and 96% (September) of the hours of the summer period. The temperature optimum of photosynthesis was 20° C: this temperature coincided with PAR above the compensation point during 5%, 6% and 0% of the time in July, August and September, respectively. Optimal hydration of tissues was infrequent. Hence PAR, temperature and water limit CO₂ uptake for most of the growing season. Our data suggest that the higher than normal ambient CO₂ concentration in the immediate environment of the plant counteracts some of the limitations in PAR supply that it experiences in its habitat. This species already experiences concentrations of atmospheric CO₂ predicted to occur over the next 50 years.     

Quantitative enzymatic hydrolysis of tRNAs : Reversed-phase high-performance liquid chromatography of tRNA nucleosides

A rapid quantitative method for enzymatic hydrolysis of microgram amounts of tRNA has been developed, specifically to take full advantage of our precise, accurate, and selective reversed-phase high-performance liquid chromatographic (HPLC) system for separation and measurement of the major and modified nucleosides in tRNA. After study of several enzyme systems, nuclease P1 and bacterial alkaline phosphatase were selected and the hydrolysis parameters were systematically studied. Optimized hydrolysis conditions give quantitative hydrolysis in 2 h and this short incubation time prevents loss of unstable nucleosides. The chromatographic system can tolerate relatively high levels of protein in the sample allowing high enzyme—substrate ratios and direct injection of hydrolysates. This enzymatic hydrolysis—HPLC method is the best described to date for quantitative determination of the nucleoside composition of tRNAs and has already provided important information for investigation of the role of modification in the function of RNAs.