Genomic analysis of the rhg1 locus: candidate genes that underlie soybean resistance to the cyst nematode

The rhg1 gene or genes lie at a recessive or co-dominant locus, necessary for resistance to all Hg types of the soybean (Glycine max (L.) Merr.) cyst nematode (Heterodera glycines I.). The aim here was to identify nucleotide changes within a candidate gene found at the rhg1 locus that were capable of altering resistance to Hg types 0 (race 3). A 1.5 ± 0.25 cM region of chromosome 18 (linkage group G) was shown to encompass rhg1 using recombination events from four near isogenic line populations and nine DNA markers. The DNA markers anchored two bacterial artificial chromosome (BAC) clones 21d9 and 73p6. A single receptor like kinase (RLK; leucine rich repeat-transmembrane-protein kinase) candidate resistance gene was amplified from both BACs using redundant primers. The DNA sequence showed nine alleles of the RLK at Rhg1 in the soybean germplasm. Markers designed to detect alleles showed perfect association between allele 1 and resistance to soybean cyst nematode Hg types 0 in three segregating populations, fifteen additional selected recombination events and twenty-two Plant Introductions. A quantitative trait nucleotide in the RLK at rhg1 was inferred that alters A47 to V47 in the context of H297 rather than N297. Contiguous DNA sequence of 315 kbp of chromosome 18 (about 2 cM) contained additional gene candidates that may modulate resistance to other Hg-types including a variant laccase, a hydrogen-sodium ion antiport and two proteins of unknown function. A molecular basis for recessive and co-dominant resistance that involves interactions among paralagous disease-resistance genes was inferred that would improve methods for developing new nematode-resistant soybean cultivars.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Gain of allelic gene expression for IGF-II occurs frequently in Barrett's esophagus

The IGF-II gene normally exhibits genomic imprinting, a DNA modification that allows the expression of only one of the two inherited alleles. With loss of imprinting, there is a gain of allelic gene expression (GOAGE) due to IGF-II being expressed by both alleles. GOAGE for IGF-II has been demonstrated in a number of malignancies and in normal epithelia surrounding malignancies, but not in epithelia without associated neoplasia. We hypothesized that nonneoplastic Barrett's epithelium might have GOAGE for IGF-II that could facilitate its progression to neoplasia. Endoscopic biopsies were obtained from metaplastic esophageal, normal gastric, and normal duodenal epithelia from 43 patients with Barrett's esophagus. Genomic DNA were analyzed using PCR followed by ApaI restriction enzyme digestion or allele-specific PCR to identify an ApaI polymorphism of IGF-II. cDNA from patients with the ApaI polymorphism were analyzed for IGF-II GOAGE using exon connection PCR, followed by a secondary nested PCR and ApaI restriction enzyme digestion. We found that 13 (30%) of 43 samples of Barrett's metaplasia contained the ApaI polymorphism and were thus informative for IGF-II, and sufficient material was available for GOAGE analysis in 9 of those 13 cases. GOAGE for IGF-II was demonstrated in five (56%) of those nine cases. All patients with GOAGE in Barrett's metaplasia also demonstrated GOAGE in the gastric and duodenal epithelia. In contrast, patients without GOAGE in Barrett's metaplasia also had no GOAGE in their gastric and duodenal epithelia. We conclude that in patients with Barrett's esophagus, GOAGE for IGF-II is found frequently in the metaplastic esophageal epithelium as well as in normal gastric and duodenal epithelia.     

Life Cycle Assessment of Water Supply Plans in Mediterranean Spain

Life cycle assessment (LCA) was used to compare the current water supply planning in Mediterranean Spain, the so‐called AGUA Programme, with its predecessor, the Ebro river water transfer (ERWT). Whereas the ERWT was based on a single interbasin transfer, the AGUA Programme excludes new transfers and focuses instead on different types of resources, including seawater and brackish water desalination and wastewater reuse, among others. The study includes not only water supply but the whole anthropic cycle of water, from water abstraction to wastewater treatment. In addition to standard LCA impact categories, a specific impact category focusing on freshwater resources is included, which takes into account freshwater scarcity in the affected water catchments. In most impact categories the AGUA Programme obtains similar or even lower impact scores than ERWT. Concerning impacts on freshwater resources, the AGUA Programme obtains an impact score 49% lower than the ERWT. Although the current water planning appears to perform better in many impact categories than its predecessor, this study shows that water supply in Spanish Mediterranean regions is substantially increasing its energy intensity and that Mediterranean basins suffer a very high level of water stress due to increasing demand and limited resources.     

Integrating Chemical Footprinting Data into RNA Secondary Structure Prediction

Chemical and enzymatic footprinting experiments, such as shape (selective 2′-hydroxyl acylation analyzed by primer extension), yield important information about RNA secondary structure. Indeed, since the -hydroxyl is reactive at flexible (loop) regions, but unreactive at base-paired regions, shape yields quantitative data about which RNA nucleotides are base-paired. Recently, low error rates in secondary structure prediction have been reported for three RNAs of moderate size, by including base stacking pseudo-energy terms derived from shape data into the computation of minimum free energy secondary structure. Here, we describe a novel method, RNAsc (RNA soft constraints), which includes pseudo-energy terms for each nucleotide position, rather than only for base stacking positions. We prove that RNAsc is self-consistent, in the sense that the nucleotide-specific probabilities of being unpaired in the low energy Boltzmann ensemble always become more closely correlated with the input shape data after application of RNAsc. From this mathematical perspective, the secondary structure predicted by RNAsc should be ‘correct’, in as much as the shape data is ‘correct’. We benchmark RNAsc against the previously mentioned method for eight RNAs, for which both shape data and native structures are known, to find the same accuracy in 7 out of 8 cases, and an improvement of 25% in one case. Furthermore, we present what appears to be the first direct comparison of shape data and in-line probing data, by comparing yeast asp-tRNA shape data from the literature with data from in-line probing experiments we have recently performed. With respect to several criteria, we find that shape data appear to be more robust than in-line probing data, at least in the case of asp-tRNA.     

15Alpha-hydroxyprogesterone in male sea lampreys, Petromyzon marinus L

There is growing evidence that sea lampreys, Petromyzon marinus L., produce gonadal steroids differing from those of other vertebrates by possessing an additional hydroxyl group at the C15 position. Here we demonstrate that sea lamprey testes produce 15alpha-hydroxyprogesterone (15alpha-P) in vitro when incubated with tritiated progesterone, that 15alpha-P is present in the plasma of sea lampreys, and that plasma concentrations of immunoreactive (ir) 15alpha-P rise dramatically in response to injections of gonadotropin-releasing hormone (GnRH). The identity of the tritiated 15alpha-P produced in vitro was confirmed by co-elution with standard 15alpha-P on high performance liquid chromatography, co-elution with standard and acetylated 15alpha-P on thin layer chromatography, and specific binding to antibodies raised against standard 15alpha-P. The in vitro conversion was used to produce tritiated 15alpha-P label for a radioimmunoassay (RIA), which is able to detect 15alpha-P in amounts as low as 2 pg per tube. The RIA has been used to measure the plasma concentrations of 15alpha-P in males given two serial injections, 24 h apart, of either lamprey GnRH I or GnRH III (50, 100, or 200 microg/kg) or saline control, with plasma being sampled 8 and 24 h after the second injection. Plasma concentrations of ir-15alpha-P rose from < 1 to 36 ng/ml (mean of all treatments) 8 h after injection and declined within 24 h. This is the first time that an RIA has detected such high steroid concentrations in lampreys. This finding is suggestive of a role for 15alpha-P in control of reproduction in the sea lamprey.     

REVCOM: a robust Bayesian method for evolutionary rate estimation

MOTIVATION: Evolutionary conservation estimated from a multiple sequence alignment is a powerful indicator of the functional significance of a residue and helps to predict active sites, ligand binding sites, and protein interaction interfaces. Many algorithms that calculate conservation work well, provided an accurate and balanced alignment is used. However, such a strong dependence on the alignment makes the results highly variable. We attempted to improve the conservation prediction algorithm by making it more robust and less sensitive to (1) local alignment errors, (2) overrepresentation of sequences in some branches and (3) occasional presence of unrelated sequences. RESULTS: A novel method is presented for robust constrained Bayesian estimation of evolutionary rates that avoids overfitting independent rates and satisfies the above requirements. The method is evaluated and compared with an entropy-based conservation measure on a set of 1494 protein interfaces. We demonstrated that approximately 62% of the analyzed protein interfaces are more conserved than the remaining surface at the 5% significance level. A consistent method to incorporate alignment reliability is proposed and demonstrated to reduce arbitrary variation of calculated rates upon inclusion of distantly related or unrelated sequences into the alignment.     

DNA Barcodes of Rosy Tetras and Allied Species (Characiformes: Characidae: Hyphessobrycon) from the Brazilian Amazon Basin

DNA barcoding can be an effective tool for fast and accurate species-level identification based on sequencing of the mitochondrial cytochrome c oxidase subunit (COI) gene. The diversity of this fragment can be used to estimate the richness of the respective species. In this study, we explored the use of DNA barcoding in a group of ornamental freshwater fish of the genus Hyphessobrycon. We sequenced the COI from 10 species of Hyphessobrycon belonging to the “Rosy Tetra Clade” collected from the Amazon and Negro River basins and combined our results with published data. The average conspecific and congeneric Kimura 2-parameter distances were 2.3% and 19.3%, respectively. Six of the 10 species were easily distinguishable by DNA barcoding (H. bentosi, H. copelandi, H. eques, H. epicharis, H. pulchrippinis, and H. sweglesi), whereas the remaining species (H. erythrostigma, H. pyrrhonotus, H. rosaceus and H. socolofi) lacked reciprocal monophyly. Although the COI gene was not fully diagnostic, the discovery of distinct evolutionary units in certain Hyphessobrycon species under the same specific epithet as well as haplotype sharing between different species suggest that DNA barcoding is useful for species identification in this speciose genus.     

Pure MnTBAP selectively scavenges peroxynitrite over superoxide: Comparison of pure and commercial MnTBAP samples to MnTE-2-PyP in two models of oxidative stress injury,an SOD-specific Escherichia coli model and carrageenan-induced pleurisy

MnTBAP is often referred to as an SOD mimic in numerous models of oxidative stress. We have recently reported that pure MnTBAP does not dismute superoxide, but commercial or poorly purified samples are able to perform O₂^·?dismutation with low-to-moderate efficacy via non-innocent Mn-containing impurities. Herein, we show that neither commercial nor pure MnTBAP could substitute for SOD enzyme in a SOD-deficient Escherichia coli model, whereas MnTE-2-PyP-treated SOD-deficient E. coli grew as well as a wild-type strain. This SOD-specific system indicates that MnTBAP does not act as an SOD mimic in vivo. In another model, carrageenan-induced pleurisy in mice, inflammation was evidenced by increased pleural fluid exudate and neutrophil infiltration and activation: these events were blocked by 0.3 mg/kg MnTE-2-PyP and, to a slightly lesser extent, by 10 mg/kg of either MnTBAP. Also, 3-nitrotyrosine formation, an indication of peroxynitrite existence in vivo, was blocked by both compounds; again MnTE-2-PyP was 33-fold more effective. Pleurisy model data indicate that MnTBAP exerts some protective actions in common with MnTE-2-PyP, which are not O₂^·? related and can be fully rationalized if one considers that the common biological role shared by MnTBAP and MnTE-2-PyP is related to their reduction of peroxynitrite and carbonate radical, the latter arising from ONOOCO₂ adduct. The log k_cat (O₂^·?) value for MnTBAP is estimated to be about 3.16, which is ~ 5 and ~ 6 orders of magnitude smaller than the SOD activities of the potent SOD mimic MnTE-2-PyP and Cu,Zn-SOD, respectively. This very low value indicates that MnTBAP is too inefficient at dismuting superoxide to be of any biological impact, which was confirmed in the SOD-deficient E. coli model. The peroxynitrite scavenging ability of MnTBAP, however, is only ~ 2.5 orders of magnitude smaller than that of MnTE-2-PyP and is not significantly affected by the presence of the SOD-active impurities in the commercial MnTBAP sample (log k_red (ONOO^?) = 5.06 for pure and 4.97 for commercial sample). The reduction of carbonate radical is equally fast with MnTBAP and MnTE-2-PyP. The dose of MnTBAP required to yield oxidative stress protection and block nitrotyrosine formation in the pleurisy model is > 1.5 orders of magnitude higher than that of MnTE-2-PyP, which could be related to the lower ability of MnTBAP to scavenge peroxynitrite. The slightly better protection observed with the commercial MnTBAP sample (relative to the pure MnTBAP) could arise from its impurities, which, by scavenging O₂^·?, reduce consequently the overall peroxynitrite and secondary ROS/RNS levels. These observations have profound biological repercussions as they may suggest that the effect of MnTBAP observed in numerous studies may conceivably relate to peroxynitrite scavenging. Moreover, provided that pure MnTBAP is unable to dismute superoxide at any significant extent, but is able to partially scavenge peroxynitrite and carbonate radical, this compound may prove valuable in distinguishing ONOO^?/CO₃^·? from O₂^·? pathways.     

Paramagnetic relaxation enhancements in unfolded proteins: Theory and application to drkN SH3 domain

Site‐directed spin labeling in combination with paramagnetic relaxation enhancement (PRE) measurements is one of the most promising techniques for studying unfolded proteins. Since the pioneering work of Gillespie and Shortle (J Mol Biol 1997;268:158), PRE data from unfolded proteins have been interpreted using the theory that was originally developed for rotational spin relaxation. At the same time, it can be readily recognized that the relative motion of the paramagnetic tag attached to the peptide chain and the reporter spin such as ¹H^N is best described as a translation. With this notion in mind, we developed a number of models for the PRE effect in unfolded proteins: (i) mutual diffusion of the two tethered spheres, (ii) mutual diffusion of the two tethered spheres subject to a harmonic potential, (iii) mutual diffusion of the two tethered spheres subject to a simulated mean‐force potential (Smoluchowski equation); (iv) explicit‐atom molecular dynamics simulation. The new models were used to predict the dependences of the PRE rates on the ¹H^N residue number and static magnetic field strength; the results are appreciably different from the Gillespie–Shortle model. At the same time, the Gillespie–Shortle approach is expected to be generally adequate if the goal is to reconstruct the distance distributions between ¹H^N spins and the paramagnetic center (provided that the characteristic correlation time is known with a reasonable accuracy). The theory has been tested by measuring the PRE rates in three spin‐labeled mutants of the drkN SH3 domain in 2M guanidinium chloride. Two modifications introduced into the measurement scheme—using a reference compound to calibrate the signals from the two samples (oxidized and reduced) and using peak volumes instead of intensities to determine the PRE rates—lead to a substantial improvement in the quality of data. The PRE data from the denatured drkN SH3 are mostly consistent with the model of moderately expanded random‐coil protein, although part of the data point toward a more compact structure (local hydrophobic cluster). At the same time, the radius of gyration reported by Choy et al. (J Mol Biol 2002;316:101) suggests that the protein is highly expanded. This seemingly contradictory evidence can be reconciled if one assumes that denatured drkN SH3 forms a conformational ensemble that is dominated by extended conformations, yet also contains compact (collapsed) species. Such behavior is apparently more complex than predicted by the model of a random‐coil protein in good solvent/poor solvent.