A small RNA response at DNA ends in Drosophila

Small RNAs have been implicated in numerous cellular processes, including effects on chromatin structure and the repression of transposons. We describe the generation of a small RNA response at DNA ends in Drosophila that is analogous to the recently reported double-strand break (DSB)-induced RNAs or Dicer- and Drosha-dependent small RNAs in Arabidopsis and vertebrates. Active transcription in the vicinity of the break amplifies this small RNA response, demonstrating that the normal messenger RNA contributes to the endogenous small interfering RNAs precursor. The double-stranded RNA precursor forms with an antisense transcript that initiates at the DNA break. Breaks are thus sites of transcription initiation, a novel aspect of the cellular DSB response. This response is specific to a double-strand break since nicked DNA structures do not trigger small RNA production. The small RNAs are generated independently of the exact end structure (blunt, 3′- or 5′-overhang), can repress homologous sequences in trans and may therefore—in addition to putative roles in repair—exert a quality control function by clearing potentially truncated messages from genes in the vicinity of the break

Numerical Modeling of Heat Transfer and Thermal Stress at the Czochralski Growth of Neodymium Scandate Single Crystals

The Czochralski growth of NdScO3 single crystals along the [110]-direction is numerically analyzed with the focus on the influence of the optical thickness on the shape of the crystal–melt interface and on the generation of thermal stresses. Due to lack of data, the optical thickness (i.e., the absorption coefficient) is varied over the entire interval between optically thin and thick. While the thermal calculation in the entire furnace is treated as axisymmetric, the stress calculation of the crystal is done three-dimensionally in order to meet the spatial anisotropy of thermal expansion and elastic coefficients. The numerically obtained values of the deflection of the crystal/melt interface meet the experimental ones for absorption coefficients in the range between 40 and 200 m−1. The maximum values of the von Mises stress appear for the case of absorption coefficient between 20 and 40 m−1. Applying absorption coefficients in the range between 3 and 100 m−1 leads to local peaks of high temperature in the shoulder region and the tail region near the end of the cylindrical part

Einige Probleme der wachsenden Rolle der FDJ

Ausgehend von einer Definition der wesentlichen gesellschaftlichen Funktionen der FDJ (ideologische Erziehung, allgemeine Vorbildfunktion) werden Fragen zu einer verbesserungsbedürftigen Jugendarbeit auf organisatorischer, inhaltlicher und ideologischer Ebene gestellt. Es wird deutlich gemacht, daß die Wirksamkeit des Jugendverbandes der DDR auf die junge Generation zu wünschen übrig läßt. (ICB

A Comprehensive Toolbox for Genome Editing in Cultured Drosophila melanogaster Cells

Custom genome editing has become an essential element of molecular biology. In particular, the generation of fusion constructs with epitope tags or fluorescent proteins at the genomic locus facilitates the analysis of protein expression, localization, and interaction partners at physiologic levels. Following up on our initial publication, we now describe a considerably simplified, more efficient, and readily scalable experimental workflow for PCR-based genome editing in cultured Drosophila melanogaster cells. Our analysis at the act5C locus suggests that PCR-based homology arms of 60 bp are sufficient to reach targeting efficiencies of up to 80% after selection;extension to 80 bp (PCR) or 500 bp (targeting vector) did not further improve the yield. We have expanded our targeting system to N-terminal epitope tags;this also allows the generation of cell populations with heterologous expression control of the tagged locus via the copper-inducible mtnDE promoter. We present detailed, quantitative data on editing efficiencies for several genomic loci that may serve as positive controls or benchmarks in other laboratories. While our first PCR-based editing approach offered only blasticidin-resistance for selection, we now introduce puromycin-resistance as a second, independent selection marker;it is thus possible to edit two loci (e.g., for coimmunoprecipitation) without marker removal. Finally, we describe a modified FLP recombinase expression plasmid that improves the efficiency of marker cassette FLP-out. In summary, our technique and reagents enable a flexible, robust, and cloning-free genome editing approach that can be parallelized for scale-up

Homology directed repair is unaffected by the absence of siRNAs in Drosophila melanogaster

Small interfering RNAs (siRNAs) defend the organism against harmful transcripts from exogenous (e.g. viral) or endogenous (e.g. transposons) sources. Recent publications describe the production of siRNAs induced by DNA double-strand breaks (DSB) in Neurospora crassa, Arabidopsis thaliana, Drosophila melanogaster and human cells, which suggests a conserved function. A current hypothesis is that break-induced small RNAs ensure efficient homologous recombination (HR). However, biogenesis of siRNAs is often intertwined with other small RNA species, such as microRNAs (miRNAs), which complicates interpretation of experimental results. In Drosophila, siRNAs are produced by Dcr-2 while miRNAs are processed by Dcr-1. Thus, it is possible to probe siRNA function without miRNA deregulation. We therefore examined DNA double-strand break repair after perturbation of siRNA biogenesis in cultured Drosophila cells as well as mutant flies. Our assays comprised reporters for the single-strand annealing pathway, homologous recombination and sensitivity to the DSB-inducing drug camptothecin. We could not detect any repair defects caused by the lack of siRNAs derived from the broken DNA locus. Since production of these siRNAs depends on local transcription, they may thus participate in RNA metabolism-an established function of siRNAs-rather than DNA repair

Yield trend of winter wheat with varying N fertilization

Dauerversuche können helfen, langfristige Entwicklungen zu identifizieren und zu quantifizieren. In einem Stickstoff-(N)-Steigerungsversuch, der seit 1974 auf dem Versuchsgut Hohenschulen im Östlichen Hügelland Schleswig-Holsteins durchgeführt wird, wurde Winterweizen zu den 3 Terminen Vegetationsbeginn, Schossbeginn EC 30 und Ährenschieben EC 50/51 mit jeweils 0, 40, 80 und 120 kg N ha–1 in allen Kombinationen gedüngt (4*4*4 = 64 N-Varianten, 0–360 kg N ha–1), um ex post das jahresspezifische N-Optimum abschätzen zu können. Die Sorte Diplomat wurde als Standardsorte durchgehend von Versuchsbeginn bis 2002 angebaut, während als zweite, jeweils neuere Sorte Kanzler (1983–1991), Orestis (1992–1995), Ritmo (1996–2004) und Tommi (ab 2005) geprüft wurden. Aus den jahres- und sortenspezifischen Ertragsfunktionen (basierend auf einem quadratischen Ansatz mit der N-Gesamtmenge) wurden der Ertrag in der ungedüngten Variante, die optimale N-Düngung und der entsprechende optimale Ertrag abgeleitet. Daraufhin wurde durch lineare Regression geprüft, ob Ertrags­trends vorlagen. Der Ertrag in der ungedüngten Kontrolle veränderte sich im Zeitablauf nicht signifikant. Dem­gegenüber stieg der Ertrag bei optimaler N-Düngung bei allen Sorten um 0,63 dt ha–1 a–1, allerdings auf unterschiedlichem absoluten Niveau, signifikant während der Versuchsdauer an, wobei sich jedoch die Höhe der optimalen N-Düngung nicht veränderte. Mit steigenden Erträgen sank die Rohproteinkonzentration. Ein statistisch absicherbarer Zusammenhang zwischen der jahresspezifisch optimalen N-Düngermenge und dem entsprechenden Ertragsniveau konnte nicht beobachtet werden. Als mögliche Ursachen für die steigenden Erträge bei opti­maler N-Düngung werden Veränderungen der Produk­tionstechnik, der Jahrestemperatur oder der CO2-Konzentration der Atmosphäre diskutiert.    Long-term field experiments allow identifying and quantifying trends. A field trial was set up in 1974 at the Hohenschulen Experimental Farm in Schleswig-Holstein (Northern Germany) to test different nitrogen (N) treatments in wheat. N fertilization varied in timing and total amount. 0, 40, 80 or 120 kg N ha–1 were applied each at the beginning of spring growth, at stem elongation (GS 30) and at ear emergence (GS 50/51) in any possible combination resulting in 64 (4*4*4) N treatments ranging from 0 to 320 kg N ha–1. The cultivar Diplomat was grown from the trial set up until 2002, whereas Kanzler (1983–1991), Orestis (1992–1995), Ritmo (1996–2004) und Tommi (since 2005) were parallelly tested as newer genotypes. Yield in the unfertilized control, optimal N amount and the respective grain yield were estimated from the year and genotype specific N response curves (quadratic polynomial function based on the total N amount). Linear regression was used to test for trends in these coefficients. Grain yield without N fertilization showed no trend. However, yield of the optimal fertilized treatment increased by 0.063 t ha–1 a–1. Yield trend was similar in all varieties, but at different levels indicating genetic improvements. In contrast, the optimal N amount was not affected. Grain protein concentration correlated negatively with the yield level, whereas only a poor relationship between optimal N amount and the respective yield occurred. As causes for the trend in the optimal yield changes in crop management, average mean temperature or CO2 concentration in the atmosphere are discussed.   &nbsp

Contribution of Particulate and Mineral-Associated Organic Matter to Potential Denitrification of Agricultural Soils

Water-extractable organic carbon (WEOC) is considered as the most important carbon (C) source for denitrifying organisms, but the contribution of individual organic matter (OM) fractions (i.e., particulate (POM) and mineral-associated (MOM)) to its release and, thus, to denitrification remains unresolved. Here we tested short-time effects of POM and MOM on potential denitrification and estimated the contribution of POM- and MOM-derived WEOC to denitrification and CO2 production of three agricultural topsoils. Suspensions of bulk soils with and without addition of soil-derived POM or MOM were incubated for 24 h under anoxic conditions. Acetylene inhibition was used to determine the potential denitrification and respective product ratio at constant nitrate supply. Normalized to added OC, effects of POM on CO2 production, total denitrification, and its product ratios were much stronger than those of MOM. While the addition of OM generally increased the (N2O + N2)-N/CO2-C ratio, the N2O/(N2O + N2) ratio changed differently depending on the soil. Gas emissions and the respective shares of initial WEOC were then used to estimate the contribution of POM and MOM-derived WEOC to total CO2, N2O, and N2O + N2 production. Water-extractable OC derived from POM accounted for 53–85% of total denitrification and WEOC released from MOM accounted for 15–47%. Total gas emissions from bulk soils were partly over- or underestimated, mainly due to nonproportional responses of denitrification to the addition of individual OM fractions. Our findings show that MOM plays a role in providing organic substrates during denitrification but is generally less dominant than POM. We conclude that the denitrification potential of soils is not predictable based on the C distribution over POM and MOM alone. Instead, the source strength of POM and MOM for WEOC plus the WEOC’s quality turned out as the most decisive determinants of potential denitrification

Potential denitrification stimulated by water-soluble organic carbon from plant residues during initial decomposition

Denitrification usually takes place under anoxic conditions and over short periods of time, and depends on readily available nitrate and carbon sources. Variations in CO2 and N2O emissions associated with plant residues have mainly been explained by differences in their decomposability. A factor rarely considered so far is water-extractable organic matter (WEOM) released to the soil during residue decomposition. Here, we examined the potential effect of plant residues on denitrification with special emphasis on WEOM. A range of fresh and leached plant residues was characterized by elemental analyses, 13C-NMR spectroscopy, and extraction with ultrapure water. The obtained solutions were analyzed for the concentrations of organic carbon (OC) and organic nitrogen (ON), and by UV-VIS spectroscopy. To test the potential denitrification induced by plant residues or three different OM solutions, these carbon sources were added to soil suspensions and incubated for 24 h at 20 °C in the dark under anoxic conditions; KNO3 was added to ensure unlimited nitrate supply. Evolving N2O and CO2 were analyzed by gas chromatography, and acetylene inhibition was used to determine denitrification and its product ratio. The production of all gases, as well as the molar (N2O + N2)–N/CO2–C ratio, was directly related to the water-extractable OC (WEOC) content of the plant residues, and the WEOC increased with carboxylic/carbonyl C and decreasing OC/ON ratio of the plant residues. Incubation of OM solutions revealed that the molar (N2O + N2)–N/CO2–C ratio and share of N2O are influenced by the WEOM's chemical composition. In conclusion, our results emphasize the potential of WEOM in largely undecomposed plant residues to support short-term denitrification activity in a typical ˈhot spot–hot momentˈ situation

Differences in labile soil organic matter explain potential denitrification and denitrifying communities in a long-term fertilization experiment

Content and quality of organic matter (OM) may strongly affect the denitrification potential of soils. In particular, the impact of soil OM fractions of differing bioavailability (soluble, particulate, and mineral-associated OM) on denitrification remains unresolved. We determined the potential N2O and N2 as well as CO2 production for samples of a Haplic Chernozem from six treatment plots (control, mineral N and NP, farmyard manure - FYM, and FYM + mineral N or NP) of the Static Fertilization Experiment Bad Lauchstädt (Germany) as related to OM properties and denitrifier gene abundances. Soil OM was analyzed for bulk chemical composition (13C-CPMAS NMR spectroscopy) as well as water-extractable, particulate, and mineral-associated fractions. Soils receiving FYM had more total OM and larger portions of labile fractions such as particulate and water-extractable OM. Incubations were run under anoxic conditions without nitrate limitation for seven days at 25 °C in the dark to determine the denitrification potential (N2O and N2) using the acetylene inhibition technique. Abundances of nirS, nirK, and nosZ (I + II) genes were analyzed before and after incubation. The denitrification potential, defined as the combined amount of N released as N2O + N2 over the experimental period, was larger for plots receiving FYM (25.9–27.2 mg N kg−1) than pure mineral fertilization (17.1–19.2 mg N kg−1) or no fertilization (12.6 mg N kg−1). The CO2 and N2O production were well related and up to three-fold larger for FYM-receiving soils than under pure mineral fertilization. The N2 production differed significantly only between all manured and non-manured soils. Nitrogenous gas emissions related most closely to water-extractable organic carbon (WEOC), which again related well to free particulate OM. The larger contribution of N2 production in soils without FYM application, and thus, with less readily decomposable OM, coincided with decreasing abundances of nirS genes (NO2− reductase) and increasing abundances of genes indicating complete denitrifying organisms (nosZ I) during anoxic conditions. Limited OM sources, thus, favored a microbial community more efficient in resource use. This study suggests that WEOC, representing readily bioavailable OM, is a straightforward indicator of the denitrification potential of soils