Fluorometric quantification of RNA and DNA in solutions containing both nucleic acids

A fluorescence-based method for quantitative determination of RNA and DNA in probes containing both nucleic acids has been developed. The total concentration of nucleic acids is determined using SYBR Green II dye under conditions providing independent binding of the fluorophore with DNA and RNA. The concentration of DNA is specifically measured using the Hoechst 33258 dye and the RNA concentration is calculated from these data. The procedure allows for accurate determination of DNA concentration in the range 10-1000 ng/ml in the presence of 200-fold excess of RNA and determination of RNA concentrations in the range 10-1000 ng/ml in the presence of large excess of DNA. An absence of the treatment of mixed samples with RNase-free DNase I provides rapid, reproducible, and accurate RNA quantification.     

Taxonomic revision of the cestodes of the family Progynotaeniidae (Cyclophyllidea) parasitising stone curlews (Charadriiformes: Burhinidae)

The type series of Progynotaenia evaginata Fuhrmann, 1909 from Burhinus senegalensis in Sudan, P. foetida Meggitt, 1928 from B. oedicnemus in Egypt and Angularia australis Maplestone, 1921 from B. grallarius in Australia are redescribed. As a comparative material, specimens of P. evaginata from B. oedicnemus in Kazakhstan were studied. The type-series of P. evaginata and P. foetida were found to be heterogeneous due to the presence of scoleces and fragments of cestodes of the genus Stenovaria Spasskii & Borgarenko, 1973 (Dilepididae). For P. foetida, a lectotype is designated. P. foetida is recognised as a synonym of P. evaginata (new synonymy). Angularia australis, previously considered a member of the Dilepididae, is transferred to the Progynotaeniidae as a synonym of P. evaginata (new synonymy). The synonymy of P. indica Johri, 1963 with P. evaginata, proposed by Ryzhikov & Tolkacheva (1981), is supported. The host range and the geographical distribution of P. evaginata are restricted to birds of the genus Burhinus from the Eastern Hemisphere.     

Simultaneous increase in cellular content and volumetric concentration of lipids in <Emphasis Type="Italic">Bracteacoccus bullatus</Emphasis> cultivated at reduced nitrogen and phosphorus concentrations

Manipulation of the nutrient concentration is an inexpensive and efficient method for increasing lipid and TAG accumulation in algal cells. However, high volumetric production requires finding a proper balance between the decrease of biomass production and the increase in the total lipid content. We isolated a strain of green microalga Bracteacoccus bullatus and increased its lipid content from 17 to 59% of biomass dry weight by manipulating of nitrogen and phosphorus content in the medium. The 10-fold reduction of the nitrogen and phosphorus concentration in the medium was the most efficient method of the lipid induction compared to nutrient deplete and high nutrient conditions. The oleic (48–64% mass of total fatty acids) and linoleic (14–24% mass of total fatty acids) acids dominated in the fatty acid profile, thus making this strain a suitable candidate for biodiesel production.     

Climate Change Increases Drought Stress of Juniper Trees in the Mountains of Central Asia

Assessments of climate change impacts on forests and their vitality are essential for semi-arid environments such as Central Asia, where the mountain regions belong to the globally important biodiversity hotspots. Alterations in species distribution or drought-induced tree mortality might not only result in a loss of biodiversity but also in a loss of other ecosystem services. Here, we evaluate spatial trends and patterns of the growth-climate relationship in a tree-ring network comprising 33 juniper sites from the northern Pamir-Alay and Tien Shan mountain ranges in eastern Uzbekistan and across Kyrgyzstan for the common period 1935–2011. Junipers growing at lower elevations are sensitive to summer drought, which has increased in intensity during the studied period. At higher elevations, juniper growth, previously favored by warm summer temperatures, has in the recent few decades become negatively affected by increasing summer aridity. Moreover, response shifts are observed during all seasons. Rising temperatures and alterations in precipitation patterns during the past eight decades can account for the observed increase in drought stress of junipers at all altitudes. The implications of our findings are vital for the application of adequate long-term measures of ecosystem conservation, but also for paleo-climatic approaches and coupled climate-vegetation model simulations for Central Asia.     

DNA polymorphism in the beta-Esterase gene cluster of Drosophila melanogaster

We have analyzed nucleotide polymorphism within a 5.3-kb region encompassing the functional Est-6 gene and the psiEst-6 putative pseudogene in 28 strains of Drosophila melanogaster and one of D. simulans. Two divergent sequence types were detected, which are not perfectly associated with Est-6 allozyme variation. The level of variation (pi) is very close in the 5'-flanking region (0.0059) and Est-6 gene (0.0057), but significantly higher in the intergenic region (0.0141) and putative pseudogene (0.0122). The variation in the 3'-flanking region is intermediate (0.0083). These observations may reflect different levels of purifying selection in the different regions. Strong linkage disequilibrium occurs within the region studied, with the largest values revealed in the putative pseudogene and 3'-flanking region. Moreover, recombination is restricted within psiEst-6. Gene conversion is detected both within and (to a lesser extent) between Est-6 and psiEst-6. The data indicate that psiEst-6 exhibits some characteristics that are typical of nonfunctional genes, while other characteristics are typically attributed to functional genes; the same situation has been observed in other pseudogenes (including Drosophila). The results of structural entropy analysis demonstrate higher structural ordering in Est-6 than in psiEst-6, in accordance with expectations if psiEst-6 is indeed a pseudogene. Taking into account that the function of psiEst-6 is not known (but could exist) and following the terminology of J. Brosius and S. J. Gould, we suggest that the term "potogene" may be appropriate for psiEst-6, indicating that it is a potential gene that may have acquired some distinctive but unknown function.     

Membrane order perturbation in the presence of antimicrobial peptides by H solid-state NMR spectroscopy

The ²H solid-state NMR spectra of deuterated fatty acyl chains provide direct access to the order of the hydrophobic membrane interior. From the deuterium order parameter profiles of perdeuterated fatty acyl chains the membrane hydrophobic thickness can be calculated. Here we show data obtained from POPC, POPE and mixed POPE/POPG bilayers, representative of bacterial membranes, in the presence of cholesterol or ergosterol and antimicrobial peptaibols. Whereas sterols have a strong ordering effect also on these membranes, the peptides exhibit neutral or disordering effects. By comparing with data from the literature it becomes obvious that cationic amphipathic peptides that probably reside within the interface of phospholipid membranes tend to strongly disorder the packing of the fatty acyl chains, an effect that has been correlated to antimicrobial and DNA transfection activities. In contrast transmembrane sequences or hydrophobic peptides that probably partition deeply into the membrane tend to have only modest disordering activities. The ²H solid-state NMR approach has also been used to monitor the lateral separation of domains rich in anionic phospholipids in the presence of cationic peptides and has thereby provided important insights into their mechanisms of action.     

Extensive phenotypic diversification coexists with little genetic divergence and a lack of population structure in the White Wagtail subspecies complex (Motacilla alba)

Geographically clustered phenotypes often demonstrate consistent patterns in molecular markers, particularly mitochondrial DNA (mtDNA) traditionally used in phylogeographic studies. However, distinct evolutionary trajectories among traits and markers can lead to their discordance. First, geographic structure in phenotypic traits and nuclear molecular markers can be co‐aligned but inconsistent with mtDNA (mito‐nuclear discordance). Alternatively, phenotypic variation can have little to do with patterns in neither mtDNA nor nuclear markers. Disentangling between these distinct patterns can provide insight into the role of selection, demography and gene flow in population divergence. Here, we examined a previously reported case of strong inconsistency between geographic structure in mtDNA and plumage traits in a widespread polytypic bird species, the White Wagtail (Motacilla alba). We tested whether this pattern is due to mito‐nuclear discordance or discrepancy between morphological evolution and both nuclear and mtDNA markers. We analysed population differentiation and structure across six out of nine commonly recognized subspecies using 17 microsatellite loci and a combination of microsatellites and plumage indices in a comprehensively sampled region of a contact between two subspecies. We did not find support for the mito‐nuclear discordance hypothesis: nuclear markers indicated a subtle signal of genetic clustering only partially consistent with plumage groups, similar to previous findings that relied on mtDNA. We discuss evolutionary factors that could have shaped the intricate patterns of phenotypic diversification in the White wagtail and the role that repeated selection on plumage ‘hotspots’ and hybridization may have played.     

Dendroclimatological potential of three juniper species from the Turkestan range,northwestern Pamir-Alay Mountains,Uzbekistan

Key message

Intensity and magnitude of the growth-climate relationship depends on juniper species and sites. Juniperus seravschanica at low elevations shows highest potential for April–September drought reconstruction in the Turkestan range (Pamir-Alay), Uzbekistan.

Abstract

We present a detailed dendroclimatological study of three juniper species, Juniperus seravschanica Kom., Juniperus semiglobosa Regel and Juniperus turkistanica Kom., sampled at six sites of different elevation (2100–2700 m a.s.l.), exposition (west and south) and steepness (10°–30°) in the Zaamin National Park, Turkestan range, Pamir-Alay mountain system in eastern Uzbekistan. Simple correlation statistics and redundancy analyses were applied to detect species- and site-specific climate responses during the twentieth century, which were additionally investigated in the high-frequency domain by identifying extreme growth years. Our results show that tree-ring formation of J. seravschanica at our low-elevation site is strongly limited by April to September drought conditions, while J. semiglobosa inherits a weak and variable climate response with respect to elevation. J. turkistanica growth at high altitudes is positively associated with warm spring and summer temperatures. Species-specific growth extremes are triggered by incoming air masses from the Atlantic and Arctic, highlighting the connection of synoptic climate regimes across Eurasia. From a dendroclimatic perspective, J. seravschanica exhibits a high potential for reconstructing past drought and pluvials, but under sustained temperature rise also J. semiglobosa will likely increase its sensitivity to drought. Moreover, J. turkistanica at its distribution limit at the tree line is a suitable proxy of summer temperature. Our findings clearly demonstrate that a careful selection of the site, overall topography and elevation as well as the different juniper species are important for successfully reconstructing past climate in Uzbekistan.

     

Use of proteinase K nonspecific digestion for selective and comprehensive identification of interpeptide cross-links: application to prion proteins

Chemical cross-linking combined with mass spectrometry is a rapidly developing technique for structural proteomics. Cross-linked proteins are usually digested with trypsin to generate cross-linked peptides, which are then analyzed by mass spectrometry. The most informative cross-links, the interpeptide cross-links, are often large in size, because they consist of two peptides that are connected by a cross-linker. In addition, trypsin targets the same residues as amino-reactive cross-linkers, and cleavage will not occur at these cross-linker-modified residues. This produces high molecular weight cross-linked peptides, which complicates their mass spectrometric analysis and identification. In this paper, we examine a nonspecific protease, proteinase K, as an alternative to trypsin for cross-linking studies. Initial tests on a model peptide that was digested by proteinase K resulted in a "family" of related cross-linked peptides, all of which contained the same cross-linking sites, thus providing additional verification of the cross-linking results, as was previously noted for other post-translational modification studies. The procedure was next applied to the native (PrP(C)) and oligomeric form of prion protein (PrPβ). Using proteinase K, the affinity-purifiable CID-cleavable and isotopically coded cross-linker cyanurbiotindipropionylsuccinimide and MALDI-MS cross-links were found for all of the possible cross-linking sites. After digestion with proteinase K, we obtained a mass distribution of the cross-linked peptides that is very suitable for MALDI-MS analysis. Using this new method, we were able to detect over 60 interpeptide cross-links in the native PrP(C) and PrPβ prion protein. The set of cross-links for the native form was used as distance constraints in developing a model of the native prion protein structure, which includes the 90-124-amino acid N-terminal portion of the protein. Several cross-links were unique to each form of the prion protein, including a Lys(185)-Lys(220) cross-link, which is unique to the PrPβ and thus may be indicative of the conformational change involved in the formation of prion protein oligomers.