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

Improvements in rating models for the German corporate sector

Group-specific estimations can significantly improve the predictive power of accountingbased rating models. This is shown using a binary logistic regression model applied to the Deutsche Bundesbank's USTAN dataset, which contains 300,000 financial statements provided by German companies for the years 1994 to 2002, i. e. throughout a complete business-cycle. The robustness and the representability of this result is verified through out-of-sample tests and through comparisons with a benchmark model which applies the variables of Moody's RiskCalcTM for Germany. --Credit Risk,Credit Rating,Probability of Default,Logistic Regression

Transposon Defense by Endo-siRNAs, piRNAs and Somatic pilRNAs in Drosophila: Contributions of Loqs-PD and R2D2

Transposable elements are a serious threat for genome integrity and their control via small RNA mediated silencing pathways is an ancient strategy. The fruit fly Drosophila melanogaster has two silencing activities that target transposons: endogenous siRNAs (esiRNAs or endo-siRNAs) and Piwi-interacting small RNAs (piRNAs). The biogenesis of endo-siRNAs involves the Dicer-2 co-factors Loqs-PD, which acts predominantly during processing of dsRNA by Dcr-2, and R2D2, which primarily helps to direct siRNAs into the RNA interference effector Ago2. Nonetheless, loss of either protein is not sufficient to produce a phenotype comparable with a dcr-2 mutation. We provide further deep sequencing evidence supporting the notion that R2D2 and Loqs-PD have partially overlapping function. Certain transposons display a preference for either dsRBD-protein during production or loading;this appeared to correlate neither with overall abundance, classification of the transposon or a specific site of genomic origin. The endo-siRNA biogenesis pathway in germline operates according to the same principles as the existing model for the soma, and its impairment does not significantly affect piRNAs. Expanding the analysis, we confirmed the occurrence of somatic piRNA-like RNAs (pilRNAs) that show a ping-pong signature. We detected expression of the Piwi-family protein mRNAs only barely above background, indicating that the somatic pilRNAs may arise from a small sub-population of somatic cells that express a functional piRNA pathway

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

Vergleichende Untersuchung möglicher pro- oder antioxidativer Eigenschaften verschiedener Pflanzen-Phenole in vitro

Es ist bekannt, daß Nahrungsmittel auf Grund ihres Gehaltes an spezifischen Wirkstoffen das physische und psychische Wohlbefinden positiv beeinflussen können. Zu den wichtigsten potentiell nützlichen Wirkstoffen zählen die sekundären Pflanzenstoffe. Unter diesem Begriff werden über 100.000 Substanzen zusammengefaßt, einige Tausend davon sind Bestandteil unserer Nahrung. Der größte Teil davon sind phenolische Verbindungen. Ausgehend von den bereits nachgewiesenen Radikalfängereigenschaften der Vitamine A, C und E sowie der Harnsäure sollte in der vorliegenden Arbeit untersucht werden, ob Pflanzen-Phenole in Abhängigkeit von der eingesetzten Methode zum Nachweis von Scavengereigenschaften unterschiedliche antioxidative/prooxidative Eigenschaften aufweisen. Der Schwerpunkt der vorliegenden Arbeit umfaßte folgende Modellreaktionen an Rattenlebermikrosomen: a) die stimulierte Lipidperoxidation (LPO), b) die Wasserstoffperoxidbildung (H2O2-Bildung), c) die Luminol-verstärkte Chemilumineszenz (Lm-CL) und d) die Lucigenin-verstärkte Chemilumineszenz (Lc-CL). Insgesamt konnten für alle untersuchten Pflanzenphenole, in Abhängigkeit von der Konzentration, in allen Modellversuchen Scavengereffekte nachgewiesen werden. Sowohl für die Wirkungsstärke als auch die Wirkungsqualität ließen sich in den Modellreaktionen für die Pflanzenphenole mehr oder weniger stark ausgeprägte Unterschiede nachweisen

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

Reversible perturbations of gene regulation after genome editing in Drosophila cells

The prokaryotic phage defense CRISPR/cas-system has developed into a versatile toolbox for genome engineering and genetic studies in many organisms. While many efforts were spent on analyzing the consequences of off-target effects, only few studies addressed side-effects that occur due to the targeted manipulation of the genome. Here, we show that the CRISPR/cas9-mediated integration of an epitope tag in combination with a selection cassette can trigger an siRNA-mediated, epigenetic genome surveillance pathway in Drosophila melanogaster cells. After homology-directed insertion of the sequence coding for the epitope tag and the selection marker, a moderate level of siRNAs covering the inserted sequence and extending into the targeted locus was detected. This response affected protein levels less than two-fold and it persisted even after single cell cloning. However, removal of the selection cassette abolished the siRNA generation, demonstrating that this response is reversible. Consistently, marker-free genome engineering did not trigger the same surveillance mechanism. These two observations indicate that the selection cassette we employed induces an aberrant transcriptional arrangement and ultimately sets off the siRNA production. There have been prior concerns about undesirable effects induced by selection markers, but fortunately we were able to show that at least one of the epigenetic changes reverts as the marker gene is excised. Although the effects observed were rather weak (less than twofold de-repression upon ago2 or dcr-2 knock-down), we recommend that when selection markers are used during genome editing, a strategy for their subsequent removal should always be included

The System of Company Pension Scheme in Germany

Die betriebliche Altersversorgung wird neben der gesetzlichen Rentenversicherung und der privaten Altersvorsorge häufi g als zweite von drei Säulen der Alterssicherung bezeichnet. Die Arten dieser Altersversorgung variieren stark und werden unterschiedlich gefördert und reguliert. Sie sind dabei wenig transparent. Die Regulierung der betrieblichen Altersversorgung sollte entsprechend angepasst werden.Due to differentiated regulation and tax treatment, occupational pension schemes in Germany are fairly complex. Consequently, administrative costs tend to be higher than necessary, and subsidisation is fiscally expensive. A lack of transparency not only complicates individuals' pension planning but also raises questions about risks to financial stability

Normal microRNA Maturation and Germ-Line Stem Cell Maintenance Requires Loquacious, a Double-Stranded RNA-Binding Domain Protein

microRNAs (miRNAs) are single-stranded, 21- to 23-nucleotide cellular RNAs that control the expression of cognate target genes. Primary miRNA (pri-miRNA) transcripts are transformed to mature miRNA by the successive actions of two RNase III endonucleases. Drosha converts pri-miRNA transcripts to precursor miRNA (pre-miRNA); Dicer, in turn, converts pre-miRNA to mature miRNA. Here, we show that normal processing of Drosophila pre-miRNAs by Dicer-1 requires the double-stranded RNA-binding domain (dsRBD) protein Loquacious (Loqs), a homolog of human TRBP, a protein first identified as binding the HIV trans-activator RNA (TAR). Efficient miRNA-directed silencing of a reporter transgene, complete repression of white by a dsRNA trigger, and silencing of the endogenous Stellate locus by Suppressor of Stellate, all require Loqs. In loqs (f00791) mutant ovaries, germ-line stem cells are not appropriately maintained. Loqs associates with Dcr-1, the Drosophila RNase III enzyme that processes pre-miRNA into mature miRNA. Thus, every known Drosophila RNase-III endonuclease is paired with a dsRBD protein that facilitates its function in small RNA biogenesis

Rudimentary G-Quadruplex-Based Telomere Capping In Saccharomyces Cerevisiae

Telomere capping conceals chromosome ends from exonucleases and checkpoints, but the full range of capping mechanisms is not well defined. Telomeres have the potential to form G-quadruplex (G4) DNA, although evidence for telomere G4 DNA function in vivo is limited. In budding yeast, capping requires the Cdc13 protein and is lost at nonpermissive temperatures in cdc13-1 mutants. Here, we use several independent G4 DNA-stabilizing treatments to suppress cdc13-1 capping defects. These include overexpression of three different G4 DNA binding proteins, loss of the G4 DNA unwinding helicase Sgs1, or treatment with small molecule G4 DNA ligands. In vitro, we show that protein-bound G4 DNA at a 3\u27 overhang inhibits 5\u27-\u3e 3\u27 resection of a paired strand by exonuclease I. These findings demonstrate that, at least in the absence of full natural capping, G4 DNA can play a positive role at telomeres in vivo