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Determining the composition of protein complexes is an essential step toward understanding the cell as an integrated system. Using coaffinity purification coupled to mass spectrometry analysis, we examined protein associations involving nearly 5,000 individual, FLAG-HA epitope-tagged Drosophila proteins. Stringent analysis of these data, based on a statistical framework designed to define individual protein-protein interactions, led to the generation of a Drosophila protein interaction map (DPiM) encompassing 556 protein complexes. The high quality of the DPiM and its usefulness as a paradigm for metazoan proteomes are apparent from the recovery of many known complexes, significant enrichment for shared functional attributes, and validation in human cells. The DPiM defines potential novel members for several important protein complexes and assigns functional links to 586 protein-coding genes lacking previous experimental annotation. The DPiM represents, to our knowledge, the largest metazoan protein complex map and provides a valuable resource for analysis of protein complex evolution.
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Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Mapeo de Interacción de Proteínas , Animales , Proteínas de Drosophila/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteómica , Proteínas SNARE/metabolismoRESUMEN
Identifying functional enhancer elements in metazoan systems is a major challenge. Large-scale validation of enhancers predicted by ENCODE reveal false-positive rates of at least 70%. We used the pregrastrula-patterning network of Drosophila melanogaster to demonstrate that loss in accuracy in held-out data results from heterogeneity of functional signatures in enhancer elements. We show that at least two classes of enhancers are active during early Drosophila embryogenesis and that by focusing on a single, relatively homogeneous class of elements, greater than 98% prediction accuracy can be achieved in a balanced, completely held-out test set. The class of well-predicted elements is composed predominantly of enhancers driving multistage segmentation patterns, which we designate segmentation driving enhancers (SDE). Prediction is driven by the DNA occupancy of early developmental transcription factors, with almost no additional power derived from histone modifications. We further show that improved accuracy is not a property of a particular prediction method: after conditioning on the SDE set, naïve Bayes and logistic regression perform as well as more sophisticated tools. Applying this method to a genome-wide scan, we predict 1,640 SDEs that cover 1.6% of the genome. An analysis of 32 SDEs using whole-mount embryonic imaging of stably integrated reporter constructs chosen throughout our prediction rank-list showed >90% drove expression patterns. We achieved 86.7% precision on a genome-wide scan, with an estimated recall of at least 98%, indicating high accuracy and completeness in annotating this class of functional elements.
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Proteínas de Drosophila , Embrión no Mamífero/embriología , Desarrollo Embrionario/fisiología , Elementos de Facilitación Genéticos/fisiología , Análisis de Secuencia de ADN , Factores de Transcripción , Animales , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Estudio de Asociación del Genoma Completo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Animal transcriptomes are dynamic, with each cell type, tissue and organ system expressing an ensemble of transcript isoforms that give rise to substantial diversity. Here we have identified new genes, transcripts and proteins using poly(A)+ RNA sequencing from Drosophila melanogaster in cultured cell lines, dissected organ systems and under environmental perturbations. We found that a small set of mostly neural-specific genes has the potential to encode thousands of transcripts each through extensive alternative promoter usage and RNA splicing. The magnitudes of splicing changes are larger between tissues than between developmental stages, and most sex-specific splicing is gonad-specific. Gonads express hundreds of previously unknown coding and long non-coding RNAs (lncRNAs), some of which are antisense to protein-coding genes and produce short regulatory RNAs. Furthermore, previously identified pervasive intergenic transcription occurs primarily within newly identified introns. The fly transcriptome is substantially more complex than previously recognized, with this complexity arising from combinatorial usage of promoters, splice sites and polyadenylation sites.
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Drosophila melanogaster/genética , Perfilación de la Expresión Génica , Transcriptoma/genética , Empalme Alternativo/genética , Animales , Drosophila melanogaster/anatomía & histología , Drosophila melanogaster/citología , Femenino , Masculino , Anotación de Secuencia Molecular , Tejido Nervioso/metabolismo , Especificidad de Órganos , Poli A/genética , Poliadenilación , Regiones Promotoras Genéticas/genética , ARN Largo no Codificante/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Caracteres Sexuales , Estrés Fisiológico/genéticaRESUMEN
Drosophila melanogaster plays an important role in molecular, genetic, and genomic studies of heredity, development, metabolism, behavior, and human disease. The initial reference genome sequence reported more than a decade ago had a profound impact on progress in Drosophila research, and improving the accuracy and completeness of this sequence continues to be important to further progress. We previously described improvement of the 117-Mb sequence in the euchromatic portion of the genome and 21 Mb in the heterochromatic portion, using a whole-genome shotgun assembly, BAC physical mapping, and clone-based finishing. Here, we report an improved reference sequence of the single-copy and middle-repetitive regions of the genome, produced using cytogenetic mapping to mitotic and polytene chromosomes, clone-based finishing and BAC fingerprint verification, ordering of scaffolds by alignment to cDNA sequences, incorporation of other map and sequence data, and validation by whole-genome optical restriction mapping. These data substantially improve the accuracy and completeness of the reference sequence and the order and orientation of sequence scaffolds into chromosome arm assemblies. Representation of the Y chromosome and other heterochromatic regions is particularly improved. The new 143.9-Mb reference sequence, designated Release 6, effectively exhausts clone-based technologies for mapping and sequencing. Highly repeat-rich regions, including large satellite blocks and functional elements such as the ribosomal RNA genes and the centromeres, are largely inaccessible to current sequencing and assembly methods and remain poorly represented. Further significant improvements will require sequencing technologies that do not depend on molecular cloning and that produce very long reads.
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Drosophila melanogaster/genética , Genoma , Animales , Mapeo Cromosómico , Cromosomas Artificiales Bacterianos , Biología Computacional , Mapeo Contig , Secuenciación de Nucleótidos de Alto Rendimiento , Hibridación Fluorescente in Situ , Datos de Secuencia Molecular , Cromosomas Politénicos , Mapeo RestrictivoRESUMEN
Available online April 7, 2018. This article has been withdrawn at the request of the author(s) and/or editor. The Publisher apologizes for any inconvenience this may cause. The full Elsevier Policy on Article Withdrawal can be found at https://www.elsevier.com/about/our-business/policies/article-withdrawal.
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Drosophila melanogaster is one of the most well studied genetic model organisms; nonetheless, its genome still contains unannotated coding and non-coding genes, transcripts, exons and RNA editing sites. Full discovery and annotation are pre-requisites for understanding how the regulation of transcription, splicing and RNA editing directs the development of this complex organism. Here we used RNA-Seq, tiling microarrays and cDNA sequencing to explore the transcriptome in 30 distinct developmental stages. We identified 111,195 new elements, including thousands of genes, coding and non-coding transcripts, exons, splicing and editing events, and inferred protein isoforms that previously eluded discovery using established experimental, prediction and conservation-based approaches. These data substantially expand the number of known transcribed elements in the Drosophila genome and provide a high-resolution view of transcriptome dynamics throughout development.
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Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/genética , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica/genética , Transcripción Genética/genética , Empalme Alternativo/genética , Animales , Secuencia de Bases , Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Exones/genética , Femenino , Genes de Insecto/genética , Genoma de los Insectos/genética , Masculino , MicroARNs/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Isoformas de Proteínas/genética , Edición de ARN/genética , ARN Mensajero/análisis , ARN Mensajero/genética , ARN Pequeño no Traducido/análisis , ARN Pequeño no Traducido/genética , Análisis de Secuencia , Caracteres SexualesRESUMEN
modENCODE was a 5year NHGRI funded project (2007-2012) to map the function of every base in the genomes of worms and flies characterizing positions of modified histones and other chromatin marks, origins of DNA replication, RNA transcripts and the transcription factor binding sites that control gene expression. Here we describe the Drosophila modENCODE datasets and how best to access and use them for genome wide and individual gene studies.
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Replicación del ADN/genética , Bases de Datos Genéticas , Biología Evolutiva/métodos , Animales , Cromatina/genética , Minería de Datos , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Genoma de los InsectosRESUMEN
Core promoters are critical regions for gene regulation in higher eukaryotes. However, the boundaries of promoter regions, the relative rates of initiation at the transcription start sites (TSSs) distributed within them, and the functional significance of promoter architecture remain poorly understood. We produced a high-resolution map of promoters active in the Drosophila melanogaster embryo by integrating data from three independent and complementary methods: 21 million cap analysis of gene expression (CAGE) tags, 1.2 million RNA ligase mediated rapid amplification of cDNA ends (RLM-RACE) reads, and 50,000 cap-trapped expressed sequence tags (ESTs). We defined 12,454 promoters of 8037 genes. Our analysis indicates that, due to non-promoter-associated RNA background signal, previous studies have likely overestimated the number of promoter-associated CAGE clusters by fivefold. We show that TSS distributions form a complex continuum of shapes, and that promoters active in the embryo and adult have highly similar shapes in 95% of cases. This suggests that these distributions are generally determined by static elements such as local DNA sequence and are not modulated by dynamic signals such as histone modifications. Transcription factor binding motifs are differentially enriched as a function of promoter shape, and peaked promoter shape is correlated with both temporal and spatial regulation of gene expression. Our results contribute to the emerging view that core promoters are functionally diverse and control patterning of gene expression in Drosophila and mammals.
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Biología Computacional , Drosophila melanogaster/genética , Genoma de los Insectos/genética , Regiones Promotoras Genéticas , Regiones no Traducidas 3'/genética , Animales , Mapeo Cromosómico , Drosophila melanogaster/embriología , Etiquetas de Secuencia Expresada , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/genética , Estudio de Asociación del Genoma Completo , Sitio de Iniciación de la TranscripciónRESUMEN
Non-invasive methods of detecting radiation exposure show promise to improve upon current approaches to biological dosimetry in ease, speed, and accuracy. Here we developed a pipeline that employs Fourier transform infrared (FTIR) spectroscopy in the mid-infrared spectrum to identify a signature of low dose ionizing radiation exposure in mouse ear pinnae over time. Mice exposed to 0.1 to 2 Gy total body irradiation were repeatedly measured by FTIR at the stratum corneum of the ear pinnae. We found significant discriminative power for all doses and time-points out to 90 days after exposure. Classification accuracy was maximized when testing 14 days after exposure (specificity > 0.9 with a sensitivity threshold of 0.9) and dropped by roughly 30% sensitivity at 90 days. Infrared frequencies point towards biological changes in DNA conformation, lipid oxidation and accumulation and shifts in protein secondary structure. Since only hundreds of samples were used to learn the highly discriminative signature, developing human-relevant diagnostic capabilities is likely feasible and this non-invasive procedure points toward rapid, non-invasive, and reagent-free biodosimetry applications at population scales.
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Exposición a la Radiación , Radiometría , Humanos , Ratones , Animales , Espectroscopía Infrarroja por Transformada de Fourier , Análisis de Fourier , Radiometría/métodos , Proteínas , Radiación Ionizante , Exposición a la Radiación/análisis , Dosis de RadiaciónRESUMEN
Synthetic biology is creating genetically engineered organisms at an increasing rate for many potentially valuable applications, but this potential comes with the risk of misuse or accidental release. To begin to address this issue, we have developed a system called GUARDIAN that can automatically detect signatures of engineering in DNA sequencing data, and we have conducted a blinded test of this system using a curated Test and Evaluation (T&E) data set. GUARDIAN uses an ensemble approach based on the guiding principle that no single approach is likely to be able to detect engineering with perfect accuracy. Critically, ensembling enables GUARDIAN to detect sequence inserts in 13 target organisms with a high degree of specificity that requires no subject matter expert (SME) review.
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ADN , Análisis de Secuencia de ADN , ADN/genéticaRESUMEN
Short polypeptides encoded by small open reading frames (smORFs) are ubiquitously found in eukaryotic genomes and are important regulators of physiology, development, and mitochondrial processes. Here, we focus on a subset of 298 smORFs that are evolutionarily conserved between Drosophila melanogaster and humans. Many of these smORFs are conserved broadly in the bilaterian lineage, and â¼182 are conserved in plants. We observe remarkably heterogeneous spatial and temporal expression patterns of smORF transcripts-indicating wide-spread tissue-specific and stage-specific mitochondrial architectures. In addition, an analysis of annotated functional domains reveals a predicted enrichment of smORF polypeptides localizing to mitochondria. We conduct an embryonic ribosome profiling experiment and find support for translation of 137 of these smORFs during embryogenesis. We further embark on functional characterization using CRISPR knockout/activation, RNAi knockdown, and cDNA overexpression, revealing diverse phenotypes. This study underscores the importance of identifying smORF function in disease and phenotypic diversity.
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Drosophila melanogaster , Péptidos , Animales , Humanos , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Péptidos/metabolismo , Genoma , Sistemas de Lectura Abierta/genéticaRESUMEN
Microbacterium sp. BDGP8 is a species of facultative anaerobic gram-positive bacterium of the family Microbacteriaceae. The complete genome consists of a single circular chromosome of 3,293,567 bp with a G + C content of 69.84% and two plasmids of 49,365 bp and 32,884 bp.
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Development of effective therapies against SARS-CoV-2 infections relies on mechanistic knowledge of virus-host interface. Abundant physical interactions between viral and host proteins have been identified, but few have been functionally characterized. Harnessing the power of fly genetics, we develop a comprehensive Drosophila COVID-19 resource (DCR) consisting of publicly available strains for conditional tissue-specific expression of all SARS-CoV-2 encoded proteins, UAS-human cDNA transgenic lines encoding established host-viral interacting factors, and GAL4 insertion lines disrupting fly homologs of SARS-CoV-2 human interacting proteins. We demonstrate the utility of the DCR to functionally assess SARS-CoV-2 genes and candidate human binding partners. We show that NSP8 engages in strong genetic interactions with several human candidates, most prominently with the ATE1 arginyltransferase to induce actin arginylation and cytoskeletal disorganization, and that two ATE1 inhibitors can reverse NSP8 phenotypes. The DCR enables parallel global-scale functional analysis of SARS-CoV-2 components in a prime genetic model system.
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COVID-19 , SARS-CoV-2 , Humanos , Animales , SARS-CoV-2/genética , Drosophila , Actinas , Animales Modificados GenéticamenteRESUMEN
Generating reference maps of interactome networks illuminates genetic studies by providing a protein-centric approach to finding new components of existing pathways, complexes, and processes. We apply state-of-the-art methods to identify binary protein-protein interactions (PPIs) for Drosophila melanogaster. Four all-by-all yeast two-hybrid (Y2H) screens of > 10,000 Drosophila proteins result in the 'FlyBi' dataset of 8723 PPIs among 2939 proteins. Testing subsets of data from FlyBi and previous PPI studies using an orthogonal assay allows for normalization of data quality; subsequent integration of FlyBi and previous data results in an expanded binary Drosophila reference interaction network, DroRI, comprising 17,232 interactions among 6511 proteins. We use FlyBi data to generate an autophagy network, then validate in vivo using autophagy-related assays. The deformed wings (dwg) gene encodes a protein that is both a regulator and a target of autophagy. Altogether, these resources provide a foundation for building new hypotheses regarding protein networks and function.
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Proteínas de Drosophila , Mapas de Interacción de Proteínas , Animales , Mapas de Interacción de Proteínas/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Drosophila/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Mapeo de Interacción de Proteínas/métodos , Técnicas del Sistema de Dos HíbridosRESUMEN
We constructed Drosophila melanogaster bacterial artificial chromosome libraries with 21-kilobase and 83-kilobase inserts in the P[acman] system. We mapped clones representing 12-fold coverage and encompassing more than 95% of annotated genes onto the reference genome. These clones can be integrated into predetermined attP sites in the genome using UC31 integrase to rescue mutations. They can be modified through recombineering, for example, to incorporate protein tags and assess expression patterns.
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Animales Modificados Genéticamente/genética , Mapeo Cromosómico/métodos , Cromosomas Artificiales Bacterianos/genética , Clonación Molecular/métodos , Drosophila melanogaster/genética , Biblioteca de Genes , Animales , Secuencia de Bases , Datos de Secuencia MolecularRESUMEN
PURPOSE: The study's purpose was to answer the following clinical question: in patients with mandibular angle fractures requiring open reduction and internal fixation, do those who have fixation screws inserted using a transbuccal approach compared with those with fixation screws inserted using a transoral approach have fewer complications after treatment? The investigators hypothesized that the transoral approach was associated with a higher risk of complications. MATERIALS AND METHODS: A multicenter retrospective cohort study was performed in patients who had open reduction and internal fixation of mandibular angle fractures from 2008 to 2010 within Western Australia. Patients were divided into transbuccal and transoral groups and then further subdivided into groups with and without fixation failures (primary outcome variable) and statistically compared. Binary logistic regression was used to control for possible confounders, which included patient gender, age, a wisdom tooth within the fracture not extracted, dental caries, partial dentition, bilateral/unilateral fractures, and smoking. RESULTS: In total 597 patients were in the study. Sixteen percent of patients in the transoral group had complications after treatment versus 10% in the transbuccal group. For the transoral technique, the odds of having fixation failure was 1.71 times greater than with the transbuccal technique (95% confidence interval, 1.02 to 2.93; P = .04). Incidences of all complication variables (hardware loosening/fracturing, wound dehiscence, secondary infection, surgery redo, nonunion/malunion of fracture, and removal of plate) were lower in the transbuccal group apart from plate fracture. CONCLUSION: The transbuccal technique was associated with fewer complications after treatment compared with the transoral technique.
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Fijación Interna de Fracturas/métodos , Técnicas de Fijación de Maxilares/instrumentación , Fracturas Mandibulares/cirugía , Procedimientos Quirúrgicos Orales/métodos , Complicaciones Posoperatorias/prevención & control , Adolescente , Adulto , Anciano , Placas Óseas , Tornillos Óseos , Distribución de Chi-Cuadrado , Estudios de Cohortes , Factores de Confusión Epidemiológicos , Falla de Equipo , Femenino , Humanos , Modelos Logísticos , Masculino , Persona de Mediana Edad , Oportunidad Relativa , Tempo Operativo , Estudios Retrospectivos , Resultado del Tratamiento , Adulto JovenRESUMEN
SARS-CoV-2 spread rapidly, causing millions of deaths across the globe. As a result, demand for medical supplies and personal protective equipment (PPE) surged and supplies dwindled. Separate entirely, hospital-acquired infections have become commonplace and challenging to treat. To explore the potential of novel sterilization techniques, this study evaluated the disinfection efficacy of Fathhome's ozone-based, dry-sanitizing device by dose and time response. Inactivation of human pathogens was tested on non-porous (plastic) surfaces. 95.42-100% inactivation was observed across all types of vegetative microorganisms and 27.36% inactivation of bacterial endospores tested, with no residual ozone detectable after completion. These results strongly support the hypothesis that Fathhome's commercial implementation of gas-based disinfection is suitable for rapid decontamination of a wide variety of pathogens on PPE and other industrially relevant materials.
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We demonstrate the feasibility of generating thousands of transgenic Drosophila melanogaster lines in which the expression of an exogenous gene is reproducibly directed to distinct small subsets of cells in the adult brain. We expect the expression patterns produced by the collection of 5,000 lines that we are currently generating to encompass all neurons in the brain in a variety of intersecting patterns. Overlapping 3-kb DNA fragments from the flanking noncoding and intronic regions of genes thought to have patterned expression in the adult brain were inserted into a defined genomic location by site-specific recombination. These fragments were then assayed for their ability to function as transcriptional enhancers in conjunction with a synthetic core promoter designed to work with a wide variety of enhancer types. An analysis of 44 fragments from four genes found that >80% drive expression patterns in the brain; the observed patterns were, on average, comprised of <100 cells. Our results suggest that the D. melanogaster genome contains >50,000 enhancers and that multiple enhancers drive distinct subsets of expression of a gene in each tissue and developmental stage. We expect that these lines will be valuable tools for neuroanatomy as well as for the elucidation of neuronal circuits and information flow in the fly brain.
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Drosophila melanogaster , Neuronas/metabolismo , Neurociencias/métodos , Recombinación Genética/genética , Animales , Animales Modificados Genéticamente , Investigación Biomédica/métodos , Encéfalo/citología , Elementos de Facilitación Genéticos/genética , Regulación del Desarrollo de la Expresión Génica , Genes de InsectoRESUMEN
INTRODUCTION: Guidelines recommend that the proximal seminal vesicles (PrSV) should be included in the clinical target volume for locally advanced prostate cancer patients undergoing radiotherapy. Verification and margins for the prostate may not necessarily account for PrSV displacement. The purpose was to determine the inter-fraction displacement of the PrSV relative to the prostate during radiotherapy. METHODS: Fiducials were inserted into the prostate, and right and left PrSV (RSV and LSV) in 30 prostate cancer patients. Correctional shifts for the prostate, right and left PrSV and pelvic bones were determined from each patient's 39 daily orthogonal portal images relative to reference digitally reconstructed radiographs. RESULTS: There was a significant displacement of the RSV relative to the prostate in all directions: on average 0.38 mm (95% confidence interval (CI) 0.26 to 0.50) to the left, 0.80-0.81 mm (CI 0.68 to 0.93) superiorly and 1.51 mm (CI 1.36 to 1.65) posteriorly. The LSV was significantly displaced superiorly to the prostate 1.09-1.13 mm (CI 0.97 to 1.25) and posteriorly 1.81 mm (CI 1.67 to 1.96), but not laterally (mean 0.06, CI -0.06 to 0.18). The calculated PTV margins (left-right, superior-inferior, posterior-anterior) were 4.9, 5.3-5.6 and 4.8 mm for the prostate, 5.2, 7.1-8.0 and 9.7 mm for the RSV, and 7.2, 7.5-7.6 and 8.6 mm for the LSV. CONCLUSION: There is a significant displacement of the PrSV relative to the prostate during radiotherapy. Greater margins are recommended for the PrSV compared to the prostate.
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Huesos Pélvicos , Neoplasias de la Próstata , Radioterapia Guiada por Imagen , Humanos , Masculino , Neoplasias de la Próstata/diagnóstico por imagen , Neoplasias de la Próstata/radioterapia , Dosificación Radioterapéutica , Planificación de la Radioterapia Asistida por Computador , Vesículas Seminales/diagnóstico por imagenRESUMEN
The gut microbiome produces vitamins, nutrients, and neurotransmitters, and helps to modulate the host immune system-and also plays a major role in the metabolism of many exogenous compounds, including drugs and chemical toxicants. However, the extent to which specific microbial species or communities modulate hazard upon exposure to chemicals remains largely opaque. Focusing on the effects of collateral dietary exposure to the widely used herbicide atrazine, we applied integrated omics and phenotypic screening to assess the role of the gut microbiome in modulating host resilience in Drosophila melanogaster. Transcriptional and metabolic responses to these compounds are sex-specific and depend strongly on the presence of the commensal microbiome. Sequencing the genomes of all abundant microbes in the fly gut revealed an enzymatic pathway responsible for atrazine detoxification unique to Acetobacter tropicalis. We find that Acetobacter tropicalis alone, in gnotobiotic animals, is sufficient to rescue increased atrazine toxicity to wild-type, conventionally reared levels. This work points toward the derivation of biotic strategies to improve host resilience to environmental chemical exposures, and illustrates the power of integrative omics to identify pathways responsible for adverse health outcomes.