m6A RNA methylation of major satellite repeat transcripts facilitates chromatin association and RNA:DNA hybrid formation in mouse heterochromatin

Heterochromatin has essential functions in maintaining chromosome structure, in protecting genome integrity and in stabilizing gene expression programs. Heterochromatin is often nucleated by underlying DNA repeat sequences, such as major satellite repeats (MSR) and long interspersed nuclear elements (LINE). In order to establish heterochromatin, MSR and LINE elements need to be transcriptionally competent and generate non-coding repeat RNA that remain chromatin associated. We explored whether these heterochromatic RNA, similar to DNA and histones, may be methylated, particularly for 5-methylcytosine (5mC) or methyl-6-adenosine (m6A). Our analysis in mouse ES cells identifies only background level of 5mC but significant enrichment for m6A on heterochromatic RNA. Moreover, MSR transcripts are a novel target for m6A RNA modification, and their m6A RNA enrichment is decreased in ES cells that are mutant for Mettl3 or Mettl14, which encode components of a central RNA methyltransferase complex. Importantly, MSR transcripts that are partially deficient in m6A RNA methylation display impaired chromatin association and have a reduced potential to form RNA:DNA hybrids. We propose that m6A modification of MSR RNA will enhance the functions of MSR repeat transcripts to stabilize mouse heterochromatin

Efectividad del nematodo Heterorhabditis bacteriophora (Nematoda: Heterorhabditidae) para el control de larvas de Phyllophaga spp. (Coleoptera: Scarabaeidae)

20 p.Perrera, A. 2009. Efectividad del nematodo Heterorhabditis bacteriophora (Nematoda: Heterorabiditidae) para el control de larvas de Phyllophaga spp. (Coleoptera: Sacarabaeidae). Proyecto especial de graduación para el programa de Ingeniero Agrónomo en Ciencia y Producción Agropecuaria, Zamorano, Honduras. 20 p. Los productores han tenido que luchar por mucho tiempo contra plagas del suelo especialmente, la gallina ciega (Phyllophaga spp.) (Coleoptera: Scarabaeidae), plaga que ataca las raíces de los cultivos. Para la lucha contra esta plaga surgen los controladores biológicos como los nematodos entomopatógenos (Rhabditida: Heterorhabditidae) que se han utilizando con éxito para su control. El objetivo de este estudio fue evaluar la efectividad del nematodo Heterorhabditis bacteriophora para el control de larvas de Phyllophaga spp. El estudio, se dividió en dos fases: En la primera se evaluaron dos dosis de H. bacteriophora (2 × 108 y 4 × 108 JI’s/ha y el insecticida Carbofuran (4 L/ha). Se colocaron 10 larvas de gallina ciega por recipiente plástico de 5500 cm3 de volumen con tres repeticiones por tratamiento en casa malla. Se utilizó un Diseño Completamente al Azar (DCA). La segunda fase se realizó en El Porvenir, La Esperanza, Honduras, y se evaluó una dosis de H. bacteriophora (2 × 108 JI’s/ha) y el insecticida Clorpirifos (5 L/ha), en un cultivo de lechuga. Las aplicaciones se hicieron a través del sistema de riego por goteo. Se utilizó un diseño de Bloques Completamente al Azar (BCA). El porcentaje de mortalidad de Phyllophaga spp. en casa malla fue de 97% para la dosis de 4 × 108 JI’s/ha, 67% para 2 × 108 JI’s/ha y 60% para Carbofuran. Todos los tratamientos presentaron mayor mortalidad que el testigo 3%. El porcentaje de mortalidad obtenido en campo con la dosis 2 × 108 JI’s/ha fue de 81% y 86% con Clorpirifos (Lorsban). El análisis de las muestras de agua muestra una eficiencia de recuperación del nematodo del 60% en las aplicaciones de los juveniles infectivos aplicados a través del sistema de riego. No se encontró diferencia en las cantidades de juveniles infectivos recuperadas a 2, 10 y 18 m en los tratamientos.1. Índice de cuadros y figuras 2. Introducción 3. Materiales y métodos 4. Resultados y discusión 5. Conclusiones 6. Recomendaciones 7. Bibliografí

Methylated RNA Immunoprecipitation Assay to Study m5C Modification in Arabidopsis

Secondary base modifications on RNA, such as m5C, affect the structure and function of the modified RNA molecules. Methylated RNA Immunoprecipitation and sequencing (MeRIP-seq) is a method that aims to enrich for methylated RNA and ultimately identify modified transcripts. Briefly, sonicated RNA is incubated with an antibody for 5-methylated cytosines and precipitated with the assistance of protein G beads. The enriched fragments are then sequenced and the potential methylation sites are mapped based on the distribution of the reads and peak detection. MeRIP can be applied to any organism, as it does not require any prior sequence or modifying enzyme knowledge. In addition, besides fragmentation, RNA is not subjected to any other chemical or temperature treatment. However, MeRIP-seq does not provide single-nucleotide prediction of the methylation site as other methods do, although the methylated area can be narrowed down to a few nucleotides. The use of different modification-specific antibodies allows MeRIP to be adjusted for the different base modifications present on RNA, expanding the possible applications of this method

BrewerIX enables allelic expression analysis of imprinted and X-linked genes from bulk and single-cell transcriptomes

Genomic imprinting and X chromosome inactivation (XCI) are two prototypical epigenetic mechanisms whereby a set of genes is expressed mono-allelically in order to fine-tune their expression levels. Defects in genomic imprinting have been observed in several neurodevelopmental disorders, in a wide range of tumours and in induced pluripotent stem cells (iPSCs). Single Nucleotide Variants (SNVs) are readily detectable by RNA-sequencing allowing the determination of whether imprinted or X-linked genes are aberrantly expressed from both alleles, although standardised analysis methods are still missing. We have developed a tool, named BrewerIX, that provides comprehensive information about the allelic expression of a large, manually-curated set of imprinted and X-linked genes. BrewerIX does not require programming skills, runs on a standard personal computer, and can analyze both bulk and single-cell transcriptomes of human and mouse cells directly from raw sequencing data. BrewerIX confirmed previous observations regarding the bi-allelic expression of some imprinted genes in naive pluripotent cells and extended them to preimplantation embryos. BrewerIX also identified misregulated imprinted genes in breast cancer cells and in human organoids and identified genes escaping XCI in human somatic cells. We believe BrewerIX will be useful for the study of genomic imprinting and XCI during development and reprogramming, and for detecting aberrations in cancer, iPSCs and organoids. Due to its ease of use to non-computational biologists, its implementation could become standard practice during sample assessment, thus raising the robustness and reproducibility of future studies

MERS coronaviruses from camels in Africa exhibit region-dependent genetic diversity [résumé]

Middle East respiratory syndrome coronavirus (MERS-CoV) causes a zoonotic respiratory disease of global public health concern, and dromedary camels are the only proven source of this zoonotic infection. Although MERS-CoV infection is ubiquitous in dromedaries across Africa and the Arabian Peninsula, the continuous appearance of zoonotic MERS cases in humans is confined to the Arabian Peninsula. MERS-CoV from Africa has hitherto been poorly studied. Here, we report the genetic and phenotypic characterization of MERS-CoV from dromedaries in African countries. Phylogenetically, viruses from dromedaries in Africa formed a monophyletic clade, which we have provisionally designated as virus clade C. Molecular dating analyses of MERSCoV, including clade C viruses, suggests that the ancestral MERSCoV in dromedaries could have spread to the two continents within a short timeframe. Camel MERS-CoVs fromwest and north African countries form a subclade (C1) that shares genetic signatures of a major deletion in the accessory gene ORF4b. Compared with human and camel MERS-CoV from Saudi Arabia, virus isolates from Burkina Faso (BF785) and Nigeria (Nig1657) had lower virus replication competence in Calu-3 cells and in ex vivo cultures of human bronchus and lung, and BF785 replicated to lower titer in lungs of human DPP4-transduced mice. However, it is still inconclusive whether ORF4b deletions may lead to the reduced replication competence of BF785 and Nig1657. Genetic and phenotypic differences in West African viruses may be relevant to the zoonotic potential of MERS-CoV

A transcription factor-based mechanism for mouse heterochromatin formation

Heterochromatin is important for genome integrity and stabilization of gene-expression programs. We have identified the transcription factors Pax3 and Pax9 as redundant regulators of mouse heterochromatin, as they repress RNA output from major satellite repeats by associating with DNA within pericentric heterochromatin. Simultaneous depletion of Pax3 and Pax9 resulted in dramatic derepression of major satellite transcripts, persistent impairment of heterochromatic marks and defects in chromosome segregation. Genome-wide analyses of methylated histone H3 at Lys9 showed enrichment at intergenic major satellite repeats only when these sequences retained intact binding sites for Pax and other transcription factors. Additionally, bioinformatic interrogation of all histone methyltransferase Suv39h-dependent heterochromatic repeat regions in the mouse genome revealed a high concordance with the presence of transcription factor binding sites. These data define a general model in which reiterated arrangement of transcription factor binding sites within repeat sequences is an intrinsic mechanism of the formation of heterochromatin

Basic concepts and architectural details of the DELPHI trigger system

Delphi (DEtector with Lepton, Photon and Hadron Identification) is one of the four experiment of the LEP (Large Electron Positron) collider at CERN. The detector is laid out to provide a nearly 4 pi coverage for charged particle tracking, electromagnetic, hadronic calorimetry and extended particle identification. The trigger system consists of four levels. The first two are synchronous with the BCO (Beam Cross Over) and rely on hardwired control units, while the last two are performed asynchronously with respect to the BCO and are driven by the Delphi host computers. The aim of this paper is to give a comprehensive global view of the trigger system architecture, presenting in detail the first two levels, their various hardware components and the latest modifications introduced in order to improve their performance and make more user friendly the whole software user interface