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1.
Genesis ; 56(8): e23237, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30005136

RESUMO

While developmental studies of Drosophila neural stem cell lineages have identified transcription factors (TFs) important to cell identity decisions, currently only an incomplete understanding exists of the cis-regulatory elements that control the dynamic expression of these TFs. Our previous studies have identified multiple enhancers that regulate the POU-domain TF paralogs nubbin and pdm-2 genes. Evolutionary comparative analysis of these enhancers reveals that they each contain multiple conserved sequence blocks (CSBs) that span TF DNA-binding sites for known regulators of neuroblast (NB) gene expression in addition to novel sequences. This study functionally analyzes the conserved DNA sequence elements within a NB enhancer located within the nubbin gene and highlights a high level of complexity underlying enhancer structure. Mutational analysis has revealed CSBs that are important for enhancer activation and silencing in the developing CNS. We have also observed that adjusting the number and relative positions of the TF binding sites within these CSBs alters enhancer function.


Assuntos
Proteínas de Drosophila/genética , Drosophila/genética , Proteínas de Homeodomínio/genética , Fatores do Domínio POU/genética , Animais , Sequência de Bases , Sítios de Ligação , Sistema Nervoso Central/embriologia , Sequência Conservada/genética , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Genes Reporter , Células-Tronco Neurais/metabolismo , Filogenia , Fatores de Transcrição/genética , Transcrição Gênica
2.
Genesis ; 56(3): e23094, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29417712

RESUMO

Evolutionary analysis of cis-regulatory DNA reveals that enhancers consist of clusters of conserved sequence blocks (CSBs) that are made up of both unique and repeated sequence elements. This study seeks to address the basis for spatial and temporal regulation of neuroblas. A search for temporally restricted CNS NB enhancers identified one within the transcription factor grainyhead (grh) gene locus. The intronic enhancer, grh-15, contains two separable semi-autonomous activities, one that drives expression predominantly within the developing brain NBs and another in ventral cord NBs. To gain insight into the function of the CSBs constituting the brain-specific enhancer, we have systematically deleted each CSB and compared the activity of the altered enhancer to that of the full brain-specific enhancer. While our results indicate that information regulating enhancer activity is highly redundant, we have found that individual CSBs convey expression in subsets of larval lineages that are generated from either Type I or Type II NBs. These studies also highlight how evolutionary sequence conservation can be used as a guide the functional analysis of cis-regulatory DNA.


Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Drosophila/genética , Elementos Facilitadores Genéticos , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição/genética , Animais , Animais Geneticamente Modificados , Sequência de Bases , Sequência Conservada , Imunofluorescência , Deleção de Genes , Expressão Gênica , Regulação da Expressão Gênica , Genes Reporter , Genoma de Inseto , Genômica/métodos , Imuno-Histoquímica , Fenótipo , Análise de Sequência de DNA
3.
Development ; 141(2): 253-8, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24306106

RESUMO

Drosophila type II neuroblasts (NBs), like mammalian neural stem cells, deposit neurons through intermediate neural progenitors (INPs) that can each produce a series of neurons. Both type II NBs and INPs exhibit age-dependent expression of various transcription factors, potentially specifying an array of diverse neurons by combinatorial temporal patterning. Not knowing which mature neurons are made by specific INPs, however, conceals the actual variety of neuron types and limits further molecular studies. Here we mapped neurons derived from specific type II NB lineages and found that sibling INPs produced a morphologically similar but temporally regulated series of distinct neuron types. This suggests a common fate diversification program operating within each INP that is modulated by NB age to generate slightly different sets of diverse neurons based on the INP birth order. Analogous mechanisms might underlie the expansion of neuron diversity via INPs in mammalian brain.


Assuntos
Drosophila/citologia , Drosophila/crescimento & desenvolvimento , Células-Tronco Neurais/citologia , Animais , Animais Geneticamente Modificados , Encéfalo/citologia , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Linhagem da Célula , Drosophila/genética , Modelos Neurológicos , Células-Tronco Neurais/classificação , Células-Tronco Neurais/metabolismo , Neurogênese
4.
BMC Genomics ; 16: 700, 2015 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-26377945

RESUMO

BACKGROUND: One of the major challenges in developmental biology is to understand the regulatory events that generate neuronal diversity. During Drosophila embryonic neural lineage development, cellular temporal identity is established in part by a transcription factor (TF) regulatory network that mediates a cascade of cellular identity decisions. Two of the regulators essential to this network are the POU-domain TFs Nubbin and Pdm-2, encoded by adjacent genes collectively known as pdm. The focus of this study is the discovery and characterization of cis-regulatory DNA that governs their expression. RESULTS: Phylogenetic footprinting analysis of a 125 kb genomic region that spans the pdm locus identified 116 conserved sequence clusters. To determine which of these regions function as cis-regulatory enhancers that regulate the dynamics of pdm gene expression, we tested each for in vivo enhancer activity during embryonic development and postembryonic neurogenesis. Our screen revealed 77 unique enhancers positioned throughout the noncoding region of the pdm locus. Many of these activated neural-specific gene expression during different developmental stages and many drove expression in overlapping patterns. Sequence comparisons of functionally related enhancers that activate overlapping expression patterns revealed that they share conserved elements that can be predictive of enhancer behavior. To facilitate data accessibility, the results of our analysis are catalogued in cisPatterns, an online database of the structure and function of these and other Drosophila enhancers. CONCLUSIONS: These studies reveal a diversity of modular enhancers that most likely regulate pdm gene expression during embryonic and adult development, highlighting a high level of temporal and spatial expression specificity. In addition, we discovered clusters of functionally related enhancers throughout the pdm locus. A subset of these enhancers share conserved elements including sequences that correspond to known TF DNA binding sites. Although comparative analysis of the nubbin and pdm-2 encoding sequences indicate that these two genes most likely arose from a duplication event, we found only partial evidence of sequence duplication between their enhancers, suggesting that after the putative duplication their cis-regulatory DNA diverged at a higher rate than their coding sequences.


Assuntos
Proteínas de Drosophila/genética , Drosophila/genética , Elementos Facilitadores Genéticos , Loci Gênicos , Proteínas de Homeodomínio/genética , Neurogênese/genética , Fatores do Domínio POU/genética , Animais , Sequência de Bases , Encéfalo/metabolismo , Análise por Conglomerados , Sequência Conservada , Proteínas de Drosophila/química , Regulação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Ordem dos Genes , Genes Reporter , Proteínas de Homeodomínio/química , Dados de Sequência Molecular , Especificidade de Órgãos/genética , Fatores do Domínio POU/química , Alinhamento de Sequência , Análise de Sequência de DNA , Transgenes
5.
Dev Cell ; 59(9): 1210-1230.e9, 2024 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-38569548

RESUMO

The Drosophila larval ventral nerve cord (VNC) shares many similarities with the spinal cord of vertebrates and has emerged as a major model for understanding the development and function of motor systems. Here, we use high-quality scRNA-seq, validated by anatomical identification, to create a comprehensive census of larval VNC cell types. We show that the neural lineages that comprise the adult VNC are already defined, but quiescent, at the larval stage. Using fluorescence-activated cell sorting (FACS)-enriched populations, we separate all motor neuron bundles and link individual neuron clusters to morphologically characterized known subtypes. We discovered a glutamate receptor subunit required for basal neurotransmission and homeostasis at the larval neuromuscular junction. We describe larval glia and endorse the general view that glia perform consistent activities throughout development. This census represents an extensive resource and a powerful platform for future discoveries of cellular and molecular mechanisms in repair, regeneration, plasticity, homeostasis, and behavioral coordination.


Assuntos
Drosophila melanogaster , Larva , Neurônios Motores , Animais , Larva/genética , Larva/metabolismo , Neurônios Motores/metabolismo , Neurônios Motores/citologia , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Neuroglia/metabolismo , Neuroglia/citologia , Junção Neuromuscular/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , RNA-Seq/métodos , Análise da Expressão Gênica de Célula Única
6.
Dev Dyn ; 241(1): 169-89, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22174086

RESUMO

BACKGROUND: Phylogenetic footprinting has revealed that cis-regulatory enhancers consist of conserved DNA sequence clusters (CSCs). Currently, there is no systematic approach for enhancer discovery and analysis that takes full-advantage of the sequence information within enhancer CSCs. RESULTS: We have generated a Drosophila genome-wide database of conserved DNA consisting of >100,000 CSCs derived from EvoPrints spanning over 90% of the genome. cis-Decoder database search and alignment algorithms enable the discovery of functionally related enhancers. The program first identifies conserved repeat elements within an input enhancer and then searches the database for CSCs that score highly against the input CSC. Scoring is based on shared repeats as well as uniquely shared matches, and includes measures of the balance of shared elements, a diagnostic that has proven to be useful in predicting cis-regulatory function. To demonstrate the utility of these tools, a temporally-restricted CNS neuroblast enhancer was used to identify other functionally related enhancers and analyze their structural organization. CONCLUSIONS: cis-Decoder reveals that co-regulating enhancers consist of combinations of overlapping shared sequence elements, providing insights into the mode of integration of multiple regulating transcription factors. The database and accompanying algorithms should prove useful in the discovery and analysis of enhancers involved in any developmental process.


Assuntos
Bases de Dados Genéticas , Drosophila melanogaster/genética , Elementos Facilitadores Genéticos , Genoma de Inseto , Algoritmos , Animais , Sequência de Bases , Biologia Computacional/métodos , Drosophila melanogaster/anatomia & histologia , Drosophila melanogaster/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Dados de Sequência Molecular , Filogenia , Transgenes
7.
Bioinformatics ; 27(23): 3319-20, 2011 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-21994220

RESUMO

SUMMARY: Images containing spatial expression patterns illuminate the roles of different genes during embryogenesis. In order to generate initial clues to regulatory interactions, biologists frequently need to know the set of genes expressed at the same time at specific locations in a developing embryo, as well as related research publications. However, text-based mining of image annotations and research articles cannot produce all relevant results, because the primary data are images that exist as graphical objects. We have developed a unique knowledge base (FlyExpress) to facilitate visual mining of images from Drosophila melanogaster embryogenesis. By clicking on specific locations in pictures of fly embryos from different stages of development and different visual projections, users can produce a list of genes and publications instantly. In FlyExpress, each queryable embryo picture is a heat-map that captures the expression patterns of more than 4500 genes and more than 2600 published articles. In addition, one can view spatial patterns for particular genes over time as well as find other genes with similar expression patterns at a given developmental stage. Therefore, FlyExpress is a unique tool for mining spatiotemporal expression patterns in a format readily accessible to the scientific community. AVAILABILITY: http://www.flyexpress.net CONTACT: s.kumar@asu.edu.


Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Animais , Recursos Audiovisuais , Mineração de Dados , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Desenvolvimento Embrionário , Perfilação da Expressão Gênica
8.
Curr Opin Cell Biol ; 17(6): 672-5, 2005 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16243502

RESUMO

One of the major goals of neurobiology is to describe, in molecular terms, how a neural progenitor cell can generate an ordered series of uniquely fated neurons and glia. It has become clear that many, or all, neural-subtype identities can be linked to sequentially changing regulatory programs within neural precursors. Recent studies shed light on regulatory inputs and timing mechanisms that generate temporally defined cell identities, and new contributions are beginning to establish a link between the temporal network and cell function.


Assuntos
Linhagem da Célula/fisiologia , Sistema Nervoso Central/embriologia , Drosophila/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Neurônios/fisiologia , Células-Tronco/fisiologia , Animais , Sistema Nervoso Central/citologia , Drosophila/citologia
9.
Risk Anal ; 32(10): 1741-9, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22591382

RESUMO

Although coastal oil spills tend to be highly publicized, crude oil spills in the United States affect inland areas relatively often. Spills to inland areas often affect sensitive environments and can have greater impacts to health and welfare than spills to coastal areas. For these reasons, the authors investigated inland crude oil spill threats, vulnerabilities, and emergency response in the midwestern U.S. states of Illinois, Indiana, Michigan, Minnesota, Ohio, and Wisconsin. These states work with the Region 5 Offices of the U.S. Environmental Protection Agency. Region 5's geospatial data in the Inland Sensitivity Atlas were turned into metrics indicating inland crude oil spill threats and vulnerabilities among the Region's sub-watersheds. These threats and vulnerabilities were weighted using data from the National Response Center and the Department of Energy's Environmental Restoration Priority System. The locations of the Region's emergency responders were geocoded in GIS. The GIS calculated the emergency response times to the Region's sub-watersheds. The resulting scatter plots are connected to the sub-watersheds in the map so stakeholders can (1) see the outlying sub-watersheds of concern and (2) better understand how reducing threats and better response time can reduce the risk of inland crude oil spills.

10.
Curr Protoc ; 1(2): e37, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33600085

RESUMO

Single-cell RNA sequencing provides a new approach to an old problem: how to study cellular diversity in complex biological systems. This powerful tool has been instrumental in profiling different cell types and investigating, at the single-cell level, cell states, functions, and responses. However, mining these data requires new analytical and statistical methods for high-dimensional analyses that must be customized and adapted to specific goals. Here we present a custom multistage analysis pipeline which integrates modules contained in different R packages to ensure flexible, high-quality RNA-seq data analysis. We describe this workflow step by step, providing the codes, explaining the rationale for each function, and discussing the results and the limitations. We apply this pipeline to analyze different datasets of Drosophila larval ventral cords, identifying and describing rare cell types, such as astrocytes and neuroendocrine cells. This multistage analysis pipeline can be easily implemented by both novice and experienced scientists interested in neuronal and/or cellular diversity beyond the Drosophila model system. © 2021 US Government.


Assuntos
Análise de Célula Única , Software , Animais , Drosophila/genética , Perfilação da Expressão Gênica , Larva/genética
11.
Curr Protoc ; 1(2): e38, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33620770

RESUMO

Drosophila provides a powerful genetic system and an excellent model to study the development and function of the nervous system. The fly's small brain and complex behavior has been instrumental in mapping neuronal circuits and elucidating the neural basis of behavior. The fast pace of fly development and the wealth of genetic tools has enabled systematic studies on cell differentiation and fate specification, and has uncovered strategies for axon guidance and targeting. The accessibility of neuronal structures and the ability to edit and manipulate gene expression in selective cells and/or synaptic compartments has revealed mechanisms for synapse assembly and neuronal connectivity. Recent advances in single-cell RNA sequencing (scRNA-seq) have further enhanced our appreciation and understanding of neuronal diversity in a fly brain. However, due to the small size of the fly brain and its constituent cells, scRNA-seq methodologies require a few adaptations. Here, we describe a set of protocols optimized for scRNA-seq analysis of the Drosophila larval ventral nerve cord, starting from tissue dissection and cell dissociation to cDNA library preparation, sequencing, and data analysis. We apply this workflow to three separate samples and detail the technical challenges associated with successful application of scRNA-seq to studies on neuronal diversity. An accompanying article (Vicidomini, Nguyen, Choudhury, Brody, & Serpe, 2021) presents a custom multistage analysis pipeline that integrates modules contained in different R packages to ensure high-flexibility, high-quality RNA-seq data analysis. These protocols are developed for Drosophila larval ventral nerve cord, but could easily be adapted to other tissues and model organisms. © 2021 U.S. Government. Basic Protocol 1: Dissection of larval ventral nerve cords and preparation of single-cell suspensions Basic Protocol 2: Preparation and sequencing of single-cell transcriptome libraries Basic Protocol 3: Alignment of raw sequencing data to indexed genome and generation of count matrices.


Assuntos
Drosophila , Análise de Célula Única , Animais , Drosophila/genética , Larva/genética , Análise de Sequência de RNA , Software
12.
G3 (Bethesda) ; 10(9): 3015-3024, 2020 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-32601058

RESUMO

This study has taken advantage of the availability of the assembled genomic sequence of flies, mosquitos, ants and bees to explore the presence of ultraconserved sequence elements in these phylogenetic groups. We compared non-coding sequences found within and flanking Drosophila developmental genes to homologous sequences in Ceratitis capitata and Musca domestica Many of the conserved sequence blocks (CSBs) that constitute Drosophila cis-regulatory DNA, recognized by EvoPrinter alignment protocols, are also conserved in Ceratitis and Musca Also conserved is the position but not necessarily the orientation of many of these ultraconserved CSBs (uCSBs) with respect to flanking genes. Using the mosquito EvoPrint algorithm, we have also identified uCSBs shared among distantly related mosquito species. Side by side comparison of bee and ant EvoPrints of selected developmental genes identify uCSBs shared between these two Hymenoptera, as well as less conserved CSBs in either one or the other taxon but not in both. Analysis of uCSBs in these dipterans and Hymenoptera will lead to a greater understanding of their evolutionary origin and function of their conserved non-coding sequences and aid in discovery of core elements of enhancers.This study applies the phylogenetic footprinting program EvoPrinter to detection of ultraconserved non-coding sequence elements in Diptera, including flies and mosquitos, and Hymenoptera, including ants and bees. EvoPrinter outputs an interspecies comparison as a single sequence in terms of the input reference sequence. Ultraconserved sequences flanking known developmental genes were detected in Ceratitis and Musca when compared with Drosophila species, in Aedes and Culex when compared with Anopheles, and between ants and bees. Our methods are useful in detecting and understanding the core evolutionarily hardened sequences required for gene regulation.


Assuntos
Dípteros , Himenópteros , Animais , Abelhas , Sequência Conservada , DNA , Dípteros/genética , Drosophila/genética , Himenópteros/genética , Filogenia
13.
Gene Expr Patterns ; 9(2): 65-72, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19056518

RESUMO

We have identified clusters of conserved sequences constituting discrete modular enhancers within the Drosophilanerfin-1 locus. nerfin-1 encodes a Zn-finger transcription factor that directs pioneer interneuron axon guidance. nerfin-1 mRNA is detected in many early delaminating neuroblasts, ganglion mother cells and transiently in nascent neurons. The comparative genomics analysis program EvoPrinter revealed conserved sequence blocks both upstream and downstream of the transcribed region. By using the aligning regions of different drosophilids as the reference DNA, EvoPrinter detects sequence length flexibility between clusters of conserved sequences and thus facilitates differentiation between closely associated modular enhancers. Expression analysis of enhancer-reporter transgenes identified enhancers that drive expression in different regions of the developing embryonic and adult nervous system, including subsets of embryonic CNS neuroblasts, GMCs, neurons and PNS neurons. In summary, EvoPrinter facilitates the discovery and analysis of enhancers that control crucial aspects of nerfin-1 expression.


Assuntos
Sistema Nervoso Central/embriologia , Sequência Conservada , Proteínas de Drosophila/genética , Drosophila/genética , Elementos Facilitadores Genéticos/genética , Família Multigênica/fisiologia , Fatores de Transcrição/genética , Região 5'-Flanqueadora/genética , Animais , Animais Geneticamente Modificados , Sequência de Bases , Sistema Nervoso Central/metabolismo , Hibridização Genômica Comparativa , Biologia Computacional , Drosophila/embriologia , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Embrião não Mamífero , Elementos Facilitadores Genéticos/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Variação Genética/fisiologia , Dados de Sequência Molecular , Software , Fatores de Transcrição/metabolismo
14.
Curr Protoc Neurosci ; 89(1): e82, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31532915

RESUMO

The goal of neurogenetics is an understanding of the genetic basis of brain structure and function. Neurogenetics deals with multiple areas of investigation, including the genetic basis of neural induction, patterning, cell fate specification, neuron maturation, axonal and dendritic organization, synapse function, neural information processing, and learning and behavior. This appendix provides links to databases and other Web sites used by neurobiologists for discovery of information about genes and cellular networks involved in neural development and neuron function. Special care has been taken to curate sites involving model organisms, since great strides have been made using Drosophila and C. elegans for understanding neural development and function. Published 2019. U.S. Government.


Assuntos
Axônios/fisiologia , Gerenciamento de Dados , Neurônios/fisiologia , Neurociências , Animais , Encéfalo/fisiologia , Caenorhabditis elegans , Dendritos/fisiologia , Drosophila , Humanos , Sinapses/fisiologia
15.
BMC Genomics ; 9: 371, 2008 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-18673565

RESUMO

BACKGROUND: The presence of highly conserved sequences within cis-regulatory regions can serve as a valuable starting point for elucidating the basis of enhancer function. This study focuses on regulation of gene expression during the early events of Drosophila neural development. We describe the use of EvoPrinter and cis-Decoder, a suite of interrelated phylogenetic footprinting and alignment programs, to characterize highly conserved sequences that are shared among co-regulating enhancers. RESULTS: Analysis of in vivo characterized enhancers that drive neural precursor gene expression has revealed that they contain clusters of highly conserved sequence blocks (CSBs) made up of shorter shared sequence elements which are present in different combinations and orientations within the different co-regulating enhancers; these elements contain either known consensus transcription factor binding sites or consist of novel sequences that have not been functionally characterized. The CSBs of co-regulated enhancers share a large number of sequence elements, suggesting that a diverse repertoire of transcription factors may interact in a highly combinatorial fashion to coordinately regulate gene expression. We have used information gained from our comparative analysis to discover an enhancer that directs expression of the nervy gene in neural precursor cells of the CNS and PNS. CONCLUSION: The combined use EvoPrinter and cis-Decoder has yielded important insights into the combinatorial appearance of fundamental sequence elements required for neural enhancer function. Each of the 30 enhancers examined conformed to a pattern of highly conserved blocks of sequences containing shared constituent elements. These data establish a basis for further analysis and understanding of neural enhancer function.


Assuntos
Sequência Conservada , Drosophila/genética , Elementos Facilitadores Genéticos , Neurônios , Software , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Sequência de Bases , Biologia Computacional , Proteínas de Drosophila/genética , Regulação da Expressão Gênica no Desenvolvimento , Biblioteca Gênica , Genes de Insetos , Alinhamento de Sequência , Células-Tronco , Fatores de Transcrição/genética
16.
BMC Genomics ; 9: 106, 2008 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-18307801

RESUMO

BACKGROUND: Multi-genome comparative analysis has yielded important insights into the molecular details of gene regulation. We have developed EvoPrinter, a web-accessed genomics tool that provides a single uninterrupted view of conserved sequences as they appear in a species of interest. An EvoPrint reveals with near base-pair resolution those sequences that are essential for gene function. RESULTS: We describe here EvoPrinterHD, a 2nd-generation comparative genomics tool that automatically generates from a single input sequence an enhanced view of sequence conservation between evolutionarily distant species. Currently available for 5 nematode, 3 mosquito, 12 Drosophila, 20 vertebrate, 17 Staphylococcus and 20 enteric bacteria genomes, EvoPrinterHD employs a modified BLAT algorithm [enhanced-BLAT (eBLAT)], which detects up to 75% more conserved bases than identified by the BLAT alignments used in the earlier EvoPrinter program. The new program also identifies conserved sequences within rearranged DNA, highlights repetitive DNA, and detects sequencing gaps. EvoPrinterHD currently holds over 112 billion bp of indexed genomes in memory and has the flexibility of selecting a subset of genomes for analysis. An EvoDifferences profile is also generated to portray conserved sequences that are uniquely lost in any one of the orthologs. Finally, EvoPrinterHD incorporates options that allow for (1) re-initiation of the analysis using a different genome's aligning region as the reference DNA to detect species-specific changes in less-conserved regions, (2) rapid extraction and curation of conserved sequences, and (3) for bacteria, identifies unique or uniquely shared sequences present in subsets of genomes. CONCLUSION: EvoPrinterHD is a fast, high-resolution comparative genomics tool that automatically generates an uninterrupted species-centric view of sequence conservation and enables the discovery of conserved sequences within rearranged DNA. When combined with cis-Decoder, a program that discovers sequence elements shared among tissue specific enhancers, EvoPrinterHD facilitates the analysis of conserved sequences that are essential for coordinate gene regulation.


Assuntos
Biologia Computacional/métodos , Sequência Conservada/genética , DNA/análise , DNA/genética , Genômica/métodos , Software , Algoritmos , Animais , Duplicação Gênica , Rearranjo Gênico/genética , Genoma Bacteriano/genética , Humanos , Sequências Repetitivas de Ácido Nucleico/genética , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Especificidade da Espécie , Fatores de Tempo
17.
PLoS Negl Trop Dis ; 11(6): e0005673, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28622346

RESUMO

BACKGROUND: Flavivirus and Filovirus infections are serious epidemic threats to human populations. Multi-genome comparative analysis of these evolving pathogens affords a view of their essential, conserved sequence elements as well as progressive evolutionary changes. While phylogenetic analysis has yielded important insights, the growing number of available genomic sequences makes comparisons between hundreds of viral strains challenging. We report here a new approach for the comparative analysis of these hemorrhagic fever viruses that can superimpose an unlimited number of one-on-one alignments to identify important features within genomes of interest. METHODOLOGY/PRINCIPAL FINDING: We have adapted EvoPrinter alignment algorithms for the rapid comparative analysis of Flavivirus or Filovirus sequences including Zika and Ebola strains. The user can input a full genome or partial viral sequence and then view either individual comparisons or generate color-coded readouts that superimpose hundreds of one-on-one alignments to identify unique or shared identity SNPs that reveal ancestral relationships between strains. The user can also opt to select a database genome in order to access a library of pre-aligned genomes of either 1,094 Flaviviruses or 460 Filoviruses for rapid comparative analysis with all database entries or a select subset. Using EvoPrinter search and alignment programs, we show the following: 1) superimposing alignment data from many related strains identifies lineage identity SNPs, which enable the assessment of sublineage complexity within viral outbreaks; 2) whole-genome SNP profile screens uncover novel Dengue2 and Zika recombinant strains and their parental lineages; 3) differential SNP profiling identifies host cell A-to-I hyper-editing within Ebola and Marburg viruses, and 4) hundreds of superimposed one-on-one Ebola genome alignments highlight ultra-conserved regulatory sequences, invariant amino acid codons and evolutionarily variable protein-encoding domains within a single genome. CONCLUSIONS/SIGNIFICANCE: EvoPrinter allows for the assessment of lineage complexity within Flavivirus or Filovirus outbreaks, identification of recombinant strains, highlights sequences that have undergone host cell A-to-I editing, and identifies unique input and database SNPs within highly conserved sequences. EvoPrinter's ability to superimpose alignment data from hundreds of strains onto a single genome has allowed us to identify unique Zika virus sublineages that are currently spreading in South, Central and North America, the Caribbean, and in China. This new set of integrated alignment programs should serve as a useful addition to existing tools for the comparative analysis of these viruses.


Assuntos
Biologia Computacional/métodos , Filoviridae/classificação , Filoviridae/genética , Flavivirus/classificação , Flavivirus/genética , Alinhamento de Sequência/métodos , Animais , Evolução Molecular , Humanos , Epidemiologia Molecular/métodos
18.
Mech Dev ; 113(1): 41-59, 2002 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11900973

RESUMO

During Drosophila neuroblast lineage development, temporally ordered transitions in neuroblast gene expression have been shown to accompany the changing repertoire of functionally diverse cells generated by neuroblasts. To broaden our understanding of the biological significance of these ordered transitions in neuroblast gene expression and the events that regulate them, additional genes have been sought that participate in the timing and execution of these temporally controlled events. To identify dynamically expressed neural precursor genes, we have performed a differential cDNA hybridization screen on a stage specific embryonic head cDNA library, followed by whole-mount embryo in situ hybridizations. Described here are the embryonic expression profiles of 57 developmentally regulated neural precursor genes. Information about 2389 additional genes identified in this screen, including 1614 uncharacterized genes, is available on-line at 'BrainGenes: a search for Drosophila neural precursor genes' (http://sdb.bio.purdue.edu/fly/brain/ahome.htm).


Assuntos
Neurônios/citologia , Animais , Clonagem Molecular , Citosol/metabolismo , DNA Complementar/metabolismo , Drosophila , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Biblioteca Gênica , Hibridização In Situ , Neurônios/metabolismo , Hibridização de Ácido Nucleico , Ligação Proteica , RNA/metabolismo , RNA Mensageiro/metabolismo , Fatores de Tempo , Transcrição Gênica
19.
Nat Neurosci ; 17(4): 631-7, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24561995

RESUMO

The Drosophila cerebrum originates from about 100 neuroblasts per hemisphere, with each neuroblast producing a characteristic set of neurons. Neurons from a neuroblast are often so diverse that many neuron types remain unexplored. We developed new genetic tools that target neuroblasts and their diverse descendants, increasing our ability to study fly brain structure and development. Common enhancer-based drivers label neurons on the basis of terminal identities rather than origins, which provides limited labeling in the heterogeneous neuronal lineages. We successfully converted conventional drivers that are temporarily expressed in neuroblasts, into drivers expressed in all subsequent neuroblast progeny. One technique involves immortalizing GAL4 expression in neuroblasts and their descendants. Another depends on loss of the GAL4 repressor, GAL80, from neuroblasts during early neurogenesis. Furthermore, we expanded the diversity of MARCM-based reagents and established another site-specific mitotic recombination system. Our transgenic tools can be combined to map individual neurons in specific lineages of various genotypes.


Assuntos
Linhagem da Célula , Cérebro/citologia , Proteínas de Drosophila , Drosophila/citologia , Técnicas Genéticas , Células-Tronco Neurais/citologia , Animais , Linhagem da Célula/fisiologia , Cérebro/fisiologia , Drosophila/fisiologia , Proteínas de Drosophila/biossíntese , Proteínas de Drosophila/genética , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/fisiologia , Receptores Notch/biossíntese , Receptores Notch/genética , Recombinação Genética , Transgenes
20.
PLoS One ; 8(4): e60137, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23613719

RESUMO

Analysis of cis-regulatory enhancers has revealed that they consist of clustered blocks of highly conserved sequences. Although most characterized enhancers reside near their target genes, a growing number of studies have shown that enhancers located over 50 kb from their minimal promoter(s) are required for appropriate gene expression and many of these 'long-range' enhancers are found in genomic regions that are devoid of identified exons. To gain insight into the complexity of Drosophila cis-regulatory sequences within exon-poor regions, we have undertaken an evolutionary analysis of 39 of these regions located throughout the genome. This survey revealed that within these genomic expanses, clusters of conserved sequence blocks (CSBs) are positioned once every 1.1 kb, on average, and that a typical cluster contains multiple (5 to 30 or more) CSBs that have been maintained for at least 190 My of evolutionary divergence. As an initial step toward assessing the cis-regulatory activity of conserved clusters within gene-free genomic expanses, we have tested the in-vivo enhancer activity of 19 consecutive CSB clusters located in the middle of a 115 kb gene-poor region on the 3(rd) chromosome. Our studies revealed that each cluster functions independently as a specific spatial/temporal enhancer. In total, the enhancers possess a diversity of regulatory functions, including dynamically activating expression in defined patterns within subsets of cells in discrete regions of the embryo, larvae and/or adult. We also observed that many of the enhancers are multifunctional-that is, they activate expression during multiple developmental stages. By extending these results to the rest of the Drosophila genome, which contains over 70,000 non-coding CSB clusters, we suggest that most function as enhancers.


Assuntos
Drosophila/genética , Elementos Facilitadores Genéticos/genética , Genoma de Inseto/genética , Animais , Sequência de Bases , Cromossomos de Insetos/genética , Sequência Conservada/genética , Genes de Insetos/genética , Genes Reporter/genética , Genômica , Dados de Sequência Molecular , Família Multigênica/genética , Especificidade da Espécie
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