Dynamic DNA helicase-DNA polymerase interactions assure processive replication fork movement

A single copy of bacteriophage T7 DNA polymerase and DNA helicase advance the replication fork with a processivity greater than 17,000 nucleotides. Nonetheless, the polymerase transiently dissociates from the DNA without leaving the replisome. Ensemble and single-molecule techniques demonstrate that this dynamic processivity is made possible by two modes of DNA polymerase-helicase interaction. During DNA synthesis the polymerase and the helicase interact at a high-affinity site. In this polymerizing mode, the polymerase dissociates from the DNA approximately every 5000 bases. The polymerase, however, remains bound to the helicase via an electrostatic binding mode that involves the acidic C-terminal tail of the helicase and a basic region in the polymerase to which the processivity factor also binds. The polymerase transfers via the electrostatic interaction around the hexameric helicase in search of the primer-template.     

Dynamics of Vertical Leaf Nitrogen Distribution in a Vegetative Wheat Canopy. Impact on Canopy Photosynthesis

The development of vertical canopy gradients of leaf N has beenregarded as an adaptation to the light gradient that helps tomaximize canopy photosynthesis. In this study we report thedynamics of vertical leaf N distribution during vegetative growthof wheat in response to changes in N availability and sowingdensity. The question of to what extent the observed verticalleaf N distribution maximized canopy photosynthesis was addressedwith a leaf layer model of canopy photosynthesis that integratesN-dependent leaf photosynthesis according to the canopy lightand leaf N distribution. Plants were grown hydroponically attwo amounts of N, supplied in proportion to calculated growthrates. Photosynthesis at light saturation correlated with leafN. The vertical leaf N distribution was associated with thegradient of absorbed light. The leaf N profile changed duringcrop development and was responsive to N availability. At highN supply, the leaf N profiles were constant during crop development.At low N supply, the leaf N profiles fluctuated between moreuniform and steep distributions. These changes were associatedwith reduced leaf area expansion and increasing N remobilizationfrom lower leaf layers. The distribution of leaf N with respectto the gradient of absorbed irradiance was close to the theoreticaloptimum maximizing canopy photosynthesis. Sensitivity analysisof the photosynthesis model suggested that plants maintain anoptimal vertical leaf N distribution by balancing the capacityfor photosynthesis at high and low light. Copyright 2000 Annalsof Botany Company Canopy photosynthesis, leaf nitrogen distribution, nitrogen, Triticum aestivum L, wheat     

Effects of tree species composition on within‐forest distribution of understorey species

Question: Do tree species, with different litter qualities, affect the within‐forest distribution of forest understorey species on intermediate to base‐rich soils? Since habitat loss and fragmentation have caused ancient forest species to decline, those species are the main focus of this study. Location: Three ancient forests, along a soil gradient from acidification‐sensitive to base‐rich, were studied: Limbrichterbosch and Savelsbos in The Netherlands and Holtkrat in Denmark. Methods: Canopy and soil surveys along transects generated data for Redundancy Analysis on tree – humus relationships. We analysed the distribution of forest plant species with Canonical Correspondence Analysis. The explanatory factors were soil characteristics (pH, organic matter, loam content and thickness of the humus layers), external crown projection, ground water and canopy data. We further analysed the relationship between forest species and humus characteristics with Spearman correlations. Results: Tree species have a significant impact on humus characteristics through the nature of their litter. Humus characteristics significantly explain the distribution of forest understorey species. The pH of the first 25 cm mineral soil and the thickness of the F‐ (fermentation) layer are the primary factors affecting the distribution of ancient forest species. Conclusion: This study indicates that the species composition of the forest canopy affects the distribution of forest understorey species. Ancient forest species are more abundant and frequent underneath trees with base‐rich litter. On acidification‐sensitive soils these relationships were stronger than on more base‐rich, loamy soils.     

A simulation model of root and shoot growth at different levels of nitrogen availability

Conclusions The model calculations correspond in a qualitative way rather well with experimental data and the model appears to be quite stable. This means that a) the division into three plant parts (growing and mature shoot parts and roots) and b) the mechanisms simulated (chemical conversions and transport processes) can form a good basis for describing plant growth.     

Coupling dTTP hydrolysis with DNA unwinding by the DNA helicase of bacteriophage T7

The DNA helicase encoded by gene 4 of bacteriophage T7 assembles on single-stranded DNA as a hexamer of six identical subunits with the DNA passing through the center of the toroid. The helicase couples the hydrolysis of dTTP to unidirectional translocation on single-stranded DNA and the unwinding of duplex DNA. Phe(523), positioned in a β-hairpin loop at the subunit interface, plays a key role in coupling the hydrolysis of dTTP to DNA unwinding. Replacement of Phe(523) with alanine or valine abolishes the ability of the helicase to unwind DNA or allow T7 polymerase to mediate strand-displacement synthesis on duplex DNA. In vivo complementation studies reveal a requirement for a hydrophobic residue with long side chains at this position. In a crystal structure of T7 helicase, when a nucleotide is bound at a subunit interface, Phe(523) is buried within the interface. However, in the unbound state, it is more exposed on the outer surface of the helicase. This structural difference suggests that the β-hairpin bearing the Phe(523) may undergo a conformational change during nucleotide hydrolysis. We postulate that upon hydrolysis of dTTP, Phe(523) moves from within the subunit interface to a more exposed position where it contacts the displaced complementary strand and facilitates unwinding.     

Gastrointestinal symptoms in low-dose aspirin users: a comparison between plain and buffered aspirin

Background

Aspirin is associated with gastrointestinal side effects such as gastric ulcers, gastric bleeding and dyspepsia. High-dose effervescent calcium carbasalate (ECC), a buffered formulation of aspirin, is associated with reduced gastric toxicity compared with plain aspirin in healthy volunteers, but at lower cardiovascular doses no beneficial effects were observed.

Aim

To compare the prevalence of self-reported gastrointestinal symptoms between low-dose plain aspirin and ECC.

Methods

A total of 51,869 questionnaires were sent to a representative sample of the Dutch adult general population in December 2008. Questions about demographics, gastrointestinal symptoms in general and specific symptoms, comorbidity, and medication use including bioequivalent doses of ECC (100 mg) and plain aspirin (80 mg) were stated. We investigated the prevalence of self-reported gastrointestinal symptoms on ECC compared with plain aspirin using univariate and multivariate logistic regression analyses.

Results

A total of 16,715 questionnaires (32 %) were returned and eligible for analysis. Of these, 911 (5 %) respondents reported the use of plain aspirin, 633 (4 %) ECC and 15,171 reported using neither form of aspirin (91 %). The prevalence of self-reported gastrointestinal symptoms in general was higher in respondents using ECC (27.5 %) compared with plain aspirin (26.3 %), but did not differ significantly with either univariate (OR 1.06, 95 %CI 0.84–1.33), or multivariate analysis (aOR 1.08, 95 %CI 0.83–1.41). Also, none of the specific types of symptoms differed between the two aspirin formulations.

Conclusions

In this large cohort representative of the general Dutch population, low-dose ECC is not associated with a reduction in self-reported gastrointestinal symptoms compared with plain aspirin.     

Single-Molecule Imaging at High Fluorophore Concentrations by Local Activation of Dye

Single-molecule fluorescence microscopy is a powerful tool for observing biomolecular interactions with high spatial and temporal resolution. Detecting fluorescent signals from individual labeled proteins above high levels of background fluorescence remains challenging, however. For this reason, the concentrations of labeled proteins in in vitro assays are often kept low compared to their in vivo concentrations. Here, we present a new fluorescence imaging technique by which single fluorescent molecules can be observed in real time at high, physiologically relevant concentrations. The technique requires a protein and its macromolecular substrate to be labeled each with a different fluorophore. Making use of short-distance energy-transfer mechanisms, only the fluorescence from those proteins that bind to their substrate is activated. This approach is demonstrated by labeling a DNA substrate with an intercalating stain, exciting the stain, and using energy transfer from the stain to activate the fluorescence of only those labeled DNA-binding proteins bound to the DNA. Such an experimental design allowed us to observe the sequence-independent interaction of Cy5-labeled interferon-inducible protein 16 with DNA and the sliding via one-dimensional diffusion of Cy5-labeled adenovirus protease on DNA in the presence of a background of hundreds of nanomolar Cy5 fluorophore.     

Gaia as a complex adaptive system

We define the Gaia system of life and its environment on Earth, review the status of the Gaia theory, introduce potentially relevant concepts from complexity theory, then try to apply them to Gaia. We consider whether Gaia is a complex adaptive system (CAS) in terms of its behaviour and suggest that the system is self-organizing but does not reside in a critical state. Gaia has supported abundant life for most of the last 3.8 Gyr. Large perturbations have occasionally suppressed life but the system has always recovered without losing the capacity for large-scale free energy capture and recycling of essential elements. To illustrate how complexity theory can help us understand the emergence of planetary-scale order, we present a simple cellular automata (CA) model of the imaginary planet Daisyworld. This exhibits emergent self-regulation as a consequence of feedback coupling between life and its environment. Local spatial interaction, which was absent from the original model, can destabilize the system by generating bifurcation regimes. Variation and natural selection tend to remove this instability. With mutation in the model system, it exhibits self-organizing adaptive behaviour in its response to forcing. We close by suggesting how artificial life ('Alife') techniques may enable more comprehensive feasibility tests of Gaia.     

A comparison of soil core sampling and minirhizotrons to quantify root development of field-grown potatoes

Root growth of potato (Solanum tuberosum L.) is sensitive to soil conditions. A reduced root system size can result in reduced uptake of water and/or nutrients, leading to impaired crop growth. To understand the mechanisms by which soil conditions affect crop growth, study of temporal and spatial development of roots is required.In field experiments, effects of soil temperature, soil compaction and potato cyst nematodes (Globodera pallida) on root growth of potato cultivars were studied using two methods: core sampling and vertically oriented minirhizotrons.Minirhizotrons showed relatively more roots in deeper soil layers than core sampling, probably because of preferential root growth along the tube. Spatial distribution of roots should therefore be analysed by core sampling.To eliminate differences in spatial distribution, total root systems as measured by both methods were compared. Nematodes, cultivars and time did not affect the relationship between both methods. Soil compaction, however, affected it because of a strong response of root length in bulk soil and small differences in root number against the minirhizotron, suggesting that soil coring has to be used to study effects of different bulk densities.With both methods, sequential measurements of roots give the net effect of root growth and decay. Data on root turnover can only be obtained with minirhizotrons by comparing video recordings of different dates. Other information obtained with minirhizotrons is the average orientation of roots. Moreover, the minirhizotron method has the advantage of demanding less labour.