Delimiting a rice wide-compatibility gene <Emphasis Type="Italic">S</Emphasis><Stack><Subscript>5</Subscript><Superscript><Emphasis Type="Italic">n</Emphasis></Superscript></Stack> to a 50 kb region

Wide-compatibility (WC) is one of the most important traits in rice, which can overcome the fertility barrier in the indica/japonica hybrids, and hence to make it possible to utilize the higher yield potential of inter-subspecific hybrids. The S ₅ ⁿ gene located on chromosome 6 has been previously reported to be responsible for the wide-compatibility in rice. Here we report the precise location of the S ₅ ⁿ gene. In the first-pass mapping, the S ₅ ⁿ gene was restricted within a 200 kb region by using a population of 242 isogenic lines in combination with high-density markers developed in the S ₅ region. In the fine mapping, the S ₅ region was further saturated with newly developed markers and more isogenic lines (549 in total) were investigated. Eventually, the S ₅ ⁿ gene was mapped within a 50 kb region delimited by the left marker J13 and the right marker J17. One BAC clone screened from the BAC library of the WC rice variety 02428 covered the whole S ₅ region. Sequence analysis of the 50 kb region revealed two candidate genes, coding an aspartyl protease and a hypothetical protein. This result would greatly accelerate both cloning and marker-assisted selection of this important S ₅ ⁿ gene. Qing Ji and Jufei Lu have contributed equally to this paper.     

Estimation of denitrification potential in a karst aquifer using the <Superscript>15</Superscript>N and <Superscript>18</Superscript>O isotopes of NO<Stack><Subscript>3</Subscript><Superscript>−</Superscript></Stack>

A confined aquifer in the Malm Karst of the Franconian Alb, South Germany was investigated in order to understand the role of the vadose zone in denitrifiaction processes. The concentrations of chemical tracers Sr²⁺ and Cl^– and concentrations of stable isotope ¹⁸O were measured in spring water and precipitation during storm events. Based on these measurements a conceptual model for runoff was constructed. The results indicate that pre-event water, already stored in the system at the beginning of the event, flows downslope on vertical and lateral preferential flow paths. Chemical tracers used in a mixing model for hydrograph separation have shown that the pre-event water contribution is up to 30%. Applying this information to a conceptual runoff generation model, the values of ¹⁵N and ¹⁸O in nitrate could be calculated. Field observations showed the occurence of significant microbial denitrification processes above the soil/bedrock interface before nitrate percolates through to the deeper horizon of the vadose zone. The source of nitrate could be determined and denitrification processes were calculated. Assuming that the nitrate reduction follows a Rayleigh process one could approximate a nitrate input concentration of about 170 mg/l and a residual nitrate concentration of only about 15%. The results of the chemical and isotopic tracers postulate fertilizers as nitrate source with some influence of atmospheric nitrate. The combined application of hydrograph separation and determination of isotope values in ¹⁵N and ¹⁸O of nitrate lead to an improved understanding of microbial processes (nitrification, denitrification) in dynamic systems.     

Sequence specific <Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N resonance assignments of a cataract-related variant G57W of human γS-crystallin

γS-crystallin is a major structural component of the human eye lens, which maintains its stability over the lifetime of an organism with negligible turnover. The G57W mutant of human γS-crystallin (abbreviated hereafter as γS-G57W) is associated with dominant congenital cataracts. In order to provide a structural basis for the ability of γS-G57W causing cataract, we have cloned, overexpressed, isolated and purified the protein. The 2D [¹⁵N–¹H]-HSQC spectrum recorded with uniformly ¹³C/¹⁵N-labelled γS-G57W was highly dispersed indicating the protein to adopt an ordered conformation. In this paper, we report almost complete sequence-specific ¹H, ¹³C and ¹⁵N resonance assignments of γS-G57W using a suite of heteronuclear 3D NMR experiments.     

Simultaneous α/β spin-state selection for <Superscript>13</Superscript>C and <Superscript>15</Superscript>N from a time-shared HSQC-IPAP experiment

Two novel HSQC-IPAP approaches are proposed to achieve α/β spin-state editing simultaneously for ¹³C and ¹⁵N in a single NMR experiment. The pulse schemes are based on a time-shared (TS) 2D ¹H,¹³C/¹H,¹⁵N-HSQC correlation experiment that combines concatenated echo elements for simultaneous J(CH) and J(NH) coupling constants evolution, TS evolution of ¹³C and ¹⁵N chemical shifts in the indirect dimension and heteronuclear α/β-spin-state selection by means of the IPAP principle. Heteronuclear α/β-editing for all CH _n (n = 1–3) and NH _n (1–2) multiplicities can be achieved in the detected F2 dimension of a single TS-HSQC-F2-IPAP experiment. On the other hand, an alternative TS-HSQC-F1-IPAP experiment is also proposed to achieve α/β-editing in the indirect F1 dimension. Experimental and simulated data is provided to evaluate these principles in terms of sensitivity and performance simultaneously on backbone and side-chain CH, CH₂, CH₃, NH, and NH₂ spin systems in uniformly ¹³C/¹⁵N-labeled proteins and in small natural-abundance peptides.     

Projected [<Superscript>1</Superscript>H,<Superscript>15</Superscript>N]-HMQC-[<Superscript>1</Superscript>H,<Superscript>1</Superscript>H]-NOESY for large molecular systems: application to a 121 kDa protein-DNA complex

We present a projected [¹H,¹⁵N]-HMQC-[¹H,¹H]-NOESY experiment for observation of NOE interactions between amide protons with degenerate ¹⁵N chemical shifts in large molecular systems. The projection is achieved by simultaneous evolution of the multiple quantum coherence of the nitrogen spin and the attached proton spin. In this way NOE signals can be separated from direct-correlation peaks also in spectra with low resolution by fully exploiting both ¹H and ¹⁵N frequency differences, such that sensitivity can be increased by using short maximum evolution times. The sensitivity of the experiment is not dependent on the projection angle for projections up to 45° and no additional pulses or delays are required as compared to the conventional 2D [¹H,¹⁵N]-HMQC-NOESY. The experiment provides two distinct 2D spectra corresponding to the positive and negative angle projections, respectively. With a linear combination of 1D cross-sections from the two projections the unavoidable sensitivity loss in projection spectra can be compensated for each particular NOE interaction. We demonstrate the application of the novel projection experiment for the observation of an NOE interaction between two sequential glycines with degenerate ¹⁵N chemical shifts in a 121.3 kDa complex of the linker H1 histone protein with a 152 bp linear DNA.     

Chronic Atmospheric NO<Stack><Subscript>3</Subscript><Superscript>−</Superscript></Stack> Deposition Does Not Induce NO<Stack><Subscript>3</Subscript><Superscript>−</Superscript></Stack> Use by <Emphasis Type="Italic">Acer saccharum</Emphasis> Marsh.

The ability of an ecosystem to retain anthropogenic nitrogen (N) deposition is dependent upon plant and soil sinks for N, the strengths of which may be altered by chronic atmospheric N deposition. Sugar maple (Acer saccharum Marsh.), the dominant overstory tree in northern hardwood forests of the Lake States region, has a limited capacity to take up and assimilate NO₃⁻. However, it is uncertain whether long-term exposure to NO₃⁻ deposition might induce NO₃⁻ uptake by this ecologically important overstory tree. Here, we investigate whether 10 years of experimental NO₃⁻ deposition (30 kg N ha⁻¹ y⁻¹) could induce NO₃⁻ uptake and assimilation in overstory sugar maple (approximately 90 years old), which would enable this species to function as a direct sink for atmospheric NO₃⁻ deposition. Kinetic parameters for NH₄⁺ and NO₃⁻ uptake in fine roots, as well as leaf and root NO₃⁻ reductase activity, were measured under conditions of ambient and experimental NO₃⁻ deposition in four sugar maple-dominated stands spanning the geographic distribution of northern hardwood forests in the Upper Lake States. Chronic NO₃⁻ deposition did not alter the V _max or K _m for NO₃⁻ and NH₄⁺ uptake nor did it influence NO₃⁻ reductase activity in leaves and fine roots. Moreover, the mean V _max for NH₄⁺ uptake (5.15 μmol ¹⁵N g⁻¹ h⁻¹) was eight times greater than the V _max for NO₃⁻ uptake (0.63 μmol ¹⁵N g⁻¹ h⁻¹), indicating a much greater physiological capacity for NH₄⁺ uptake in this species. Additionally, NO₃⁻ reductase activity was lower than most values for woody plants previously reported in the literature, further indicating a low physiological potential for NO₃⁻ assimilation in sugar maple. Our results demonstrate that chronic NO₃⁻ deposition has not induced the physiological capacity for NO₃⁻ uptake and assimilation by sugar maple, making this dominant species an unlikely direct sink for anthropogenic NO₃⁻ deposition.     

Flow of Deposited Inorganic N in Two Gleysol-dominated Mountain Catchments Traced with <Superscript>15</Superscript>NO<Subscript>3</Subscript><Superscript>−</Superscript> and <Superscript>15</Superscript>NH<Subscript>4</Subscript><Superscript>+</Superscript>

In two mountain ecosystems at the Alptal research site in central Switzerland, pulses of ¹⁵NO₃ and ¹⁵NH₄ were separately applied to trace deposited inorganic N. One forested and one litter meadow catchment, each approximately 1600 m², were delimited by trenches in the Gleysols. K¹⁵NO₃ was applied weekly or fortnightly over one year with a backpack sprayer, thus labelling the atmospheric nitrate deposition. After the sampling and a one-year break, ¹⁵NH₄Cl was applied as a second one-year pulse, followed by a second sampling campaign. Trees (needles, branches and bole wood), ground vegetation, litter layer and soil (LF, A and B horizon) were sampled at the end of each labelling period. Extractable inorganic N, microbial N, and immobilised soil N were analysed in the LF and A horizons. During the whole labelling period, the runoff water was sampled as well. Most of the added tracer remained in both ecosystems. More NO₃⁻ than NH₄⁺ tracer was retained, especially in the forest. The highest recovery was in the soil, mainly in the organic horizon, and in the ground vegetation, especially in the mosses. Event-based runoff analyses showed an immediate response of ¹⁵NO₃⁻ in runoff, with sharp ¹⁵N peaks corresponding to discharge peaks. NO₃⁻ leaching showed a clear seasonal pattern, being highest in spring during snowmelt. The high capacity of N retention in these ecosystems leads to the assumption that deposited N accumulates in the soil organic matter, causing a progressive decline of its C:N ratio.     

Negative molecular ion of deuterium D<Stack><Subscript>2</Subscript><Superscript>−</Superscript></Stack>

D₂⁻ ions produced in collisions of D⁻ ions with relative energies of 2.5–9.2 eV were detected for the first time. It is shown that the effective cross section for this reaction is no less than 1.5 × 10⁻¹⁴ cm². Along with the theoretically predicted short-lived state of negative molecular deuterium ions, a state existing for more than 1 μs was observed.     

<Emphasis Type="Italic">Glycine max</Emphasis> non-nodulation locus <Emphasis Type="Italic">rj1</Emphasis>: a recombinogenic region encompassing a SNP in a lysine motif receptor-like kinase (<Emphasis Type="Italic">GmNFR1α</Emphasis>)

The rj1 mutation of soybean is a simple recessive allele in a single line that arose as a spontaneous mutation in a population; it exhibits non-nodulation with virtually all Bradyrhizobium and Sinorhizobium strains. Here, we described fine genetic and physical mapping of the rj1 locus on soybean chromosome 2. The initial mapping of the rj1 locus using public markers indicated that A343.p2, a sequence-based marker that contains sequence similar to a part of the LjNFR1 gene regulating nodule formation as a member of lysin motif-type receptor-like kinase (LYK) family, maps very close to or cosegregates with the rj1 locus. The sequence of A343.p2 is 100% identical to parts of two BAC clone sequences (GM_WBb0002O19 and GM_WBb098N11) that contain three members of the LYK family. We analyzed the sequence contig (262 kbp) of the two BAC clones by resequencing and subsequent fine genetic and physical mapping. The results indicated that rj1 is located in a gene-rich region with a recombination rate of 120 kbp/cM: several fold higher than the genome average. Among the LYK genes, NFR1α is most likely the gene encoded at the Rj1 locus. The non-nodulating rj1 allele was created by a single base-pair deletion that results in a premature stop codon. Taken together, the fine genetic and physical mapping of the Rj1-residing chromosomal region, combined with the unexpected observation of a putative recombination hotspot, allowed us to demonstrate that the Rj1 locus most likely encodes the NFR1α gene.     

Optimal choice of word length when comparing two Markov sequences using a <Emphasis Type="Italic">χ</Emphasis><Superscript>2</Superscript>-statistic

Background

Alignment-free sequence comparison using counts of word patterns (grams, k-tuples) has become an active research topic due to the large amount of sequence data from the new sequencing technologies. Genome sequences are frequently modelled by Markov chains and the likelihood ratio test or the corresponding approximate χ ²-statistic has been suggested to compare two sequences. However, it is not known how to best choose the word length k in such studies.

Results

We develop an optimal strategy to choose k by maximizing the statistical power of detecting differences between two sequences. Let the orders of the Markov chains for the two sequences be r ₁ and r ₂, respectively. We show through both simulations and theoretical studies that the optimal k= max(r ₁,r ₂)+1 for both long sequences and next generation sequencing (NGS) read data. The orders of the Markov chains may be unknown and several methods have been developed to estimate the orders of Markov chains based on both long sequences and NGS reads. We study the power loss of the statistics when the estimated orders are used. It is shown that the power loss is minimal for some of the estimators of the orders of Markov chains.

Conclusion

Our studies provide guidelines on choosing the optimal word length for the comparison of Markov sequences.

     

Determination of the lifetime of D<Stack><Subscript>2</Subscript><Superscript>−</Superscript></Stack> and HD<Superscript>−</Superscript> ions

A method for determining the lifetime of unstable ions is described. The method is based on measuring the decrease in the ion beam current onto a fixed detector with increasing path length of the ion beam from the ion source to the detector. The measurements performed for D^? ₂ and HD^? molecular ions have shown that their lifetimes are 3.5 ± 0.1 and 4.4 ± 0.1 μs, respectively.     

CACA-TOCSY with alternate <Superscript>13</Superscript>C–<Superscript>12</Superscript>C labeling: a <Superscript>13</Superscript>C<Superscript>α</Superscript> direct detection experiment for mainchain resonance assignment,dihedral angle information,and amino acid type identification

We present a ¹³C direct detection CACA-TOCSY experiment for samples with alternate ¹³C–¹²C labeling. It provides inter-residue correlations between ¹³C^α resonances of residue i and adjacent C^αs at positions i − 1 and i + 1. Furthermore, longer mixing times yield correlations to C^α nuclei separated by more than one residue. The experiment also provides C^α-to-sidechain correlations, some amino acid type identifications and estimates for ψ dihedral angles. The power of the experiment derives from the alternate ¹³C–¹²C labeling with [1,3-¹³C] glycerol or [2-¹³C] glycerol, which allows utilizing the small scalar ³J_CC couplings that are masked by strong ¹J_CC couplings in uniformly ¹³C labeled samples.     

Decline in Ecosystem <Emphasis Type="Italic">δ</Emphasis><Superscript>13</Superscript>C and Mid-Successional Nitrogen Loss in a Two-Century Postglacial Chronosequence

Uncertainty about controls on long-term carbon (C) and nitrogen (N) balance, turnover, and isotopic composition currently limits our ability to predict ecosystem response to disturbance and landscape change. We used a two-century, postglacial chronosequence in Glacier Bay, Alaska, to explore the influence of C and N dynamics on soil and leaf stable isotopes. C dynamics were closely linked to soil hydrology, with increasing soil water retention during ecosystem development resulting in a linear decrease in foliar and soil δ¹³C, independent of shifts in vegetation cover and despite constant precipitation across sites. N dynamics responded to interactions among soil development, vegetation type, microbial activity, and topography. Contrary to the predictions of nutrient retention theory, potential nitrification and denitrification were high, relative to inorganic N stocks, from the beginning of the chronosequence, and gaseous and hydrological N losses were highest at mid-successional sites, 140–165 years since deglaciation. Though leaching of dissolved N is considered the predominant pathway of N loss at high latitudes, we found that gaseous N loss was more tightly correlated with δ¹⁵N enrichment. These results suggest that δ¹³C in leaves and soil can depend as much on soil development and associated water availability as on climate and that N availability and export depend on interactions between physical and biological state factors.     

Sources of variation in consumer-diet δ<Superscript>15</Superscript>N enrichment: a meta-analysis

Measurements of ¹⁵N of consumers are usually higher than those of their diet. This general pattern is widely used to make inferences about trophic relationships in ecological studies, although the underlying mechanisms causing the pattern are poorly understood. However, there can be substantial variation in consumer-diet ¹⁵N enrichment within this general pattern. We conducted an extensive literature review, which yielded 134 estimates from controlled studies of consumer-diet ¹⁵N enrichment, to test the significance of several potential sources of variation by means of meta-analyses. We found patterns related to processes of nitrogen assimilation and excretion. There was a significant effect of the main biochemical form of nitrogenous waste: ammonotelic organisms show lower ¹⁵N enrichment than ureotelic or uricotelic organisms. There were no significant differences between animals feeding on plant food, animal food, or manufactured mixtures, but detritivores yielded significantly lower estimates of enrichment. ¹⁵N enrichment was found to increase significantly with the C:N ratio of the diet, suggesting that a nitrogen-poor diet can have an effect similar to that already documented for fasting organisms. There were also differences among taxonomic classes: molluscs and crustaceans generally yielded lower ¹⁵N enrichment. The lower ¹⁵N enrichment might be related to the fact that molluscs and crustaceans excrete mainly ammonia, or to the fact that many were detritivores. Organisms inhabiting marine environments yielded significantly lower estimates of ¹⁵N enrichment than organisms inhabiting terrestrial or freshwater environments, a pattern that was influenced by the number of marine, ammonotelic, crustaceans and molluscs. Overall, our analyses point to several important sources of variation in ¹⁵N enrichment and suggest that the most important of them are the main biochemical form of nitrogen excretion and nutritional status. The variance of estimates of ¹⁵N enrichment, as well as the fact that enrichment may be different in certain groups of organisms should be taken into account in statistical approaches for studying diet and trophic relationships.     

FiatFlux – a software for metabolic flux analysis from <Superscript>13</Superscript>C-glucose experiments

Background  

Quantitative knowledge of intracellular fluxes is important for a comprehensive characterization of metabolic networks and their functional operation. In contrast to direct assessment of metabolite concentrations, in vivo metabolite fluxes must be inferred indirectly from measurable quantities in ¹³C experiments. The required experience, the complicated network models, large and heterogeneous data sets, and the time-consuming set-up of highly controlled experimental conditions largely restricted metabolic flux analysis to few expert groups. A conceptual simplification of flux analysis is the analytical determination of metabolic flux ratios exclusively from MS data, which can then be used in a second step to estimate absolute in vivo fluxes.