Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 25
Filtrar
1.
Nature ; 512(7515): 393-9, 2014 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-24670639

RESUMO

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.


Assuntos
Drosophila melanogaster/genética , Perfilação da Expressão Gênica , Transcriptoma/genética , Processamento Alternativo/genética , Animais , Drosophila melanogaster/anatomia & histologia , Drosophila melanogaster/citologia , Feminino , Masculino , Anotação de Sequência Molecular , Tecido Nervoso/metabolismo , Especificidade de Órgãos , Poli A/genética , Poliadenilação , Regiões Promotoras Genéticas/genética , RNA Longo não Codificante/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Caracteres Sexuais , Estresse Fisiológico/genética
2.
Nature ; 512(7515): 445-8, 2014 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-25164755

RESUMO

The transcriptome is the readout of the genome. Identifying common features in it across distant species can reveal fundamental principles. To this end, the ENCODE and modENCODE consortia have generated large amounts of matched RNA-sequencing data for human, worm and fly. Uniform processing and comprehensive annotation of these data allow comparison across metazoan phyla, extending beyond earlier within-phylum transcriptome comparisons and revealing ancient, conserved features. Specifically, we discover co-expression modules shared across animals, many of which are enriched in developmental genes. Moreover, we use expression patterns to align the stages in worm and fly development and find a novel pairing between worm embryo and fly pupae, in addition to the embryo-to-embryo and larvae-to-larvae pairings. Furthermore, we find that the extent of non-canonical, non-coding transcription is similar in each organism, per base pair. Finally, we find in all three organisms that the gene-expression levels, both coding and non-coding, can be quantitatively predicted from chromatin features at the promoter using a 'universal model' based on a single set of organism-independent parameters.


Assuntos
Caenorhabditis elegans/genética , Drosophila melanogaster/genética , Perfilação da Expressão Gênica , Transcriptoma/genética , Animais , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/crescimento & desenvolvimento , Cromatina/genética , Análise por Conglomerados , Drosophila melanogaster/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento/genética , Histonas/metabolismo , Humanos , Larva/genética , Larva/crescimento & desenvolvimento , Modelos Genéticos , Anotação de Sequência Molecular , Regiões Promotoras Genéticas/genética , Pupa/genética , Pupa/crescimento & desenvolvimento , RNA não Traduzido/genética , Análise de Sequência de RNA
3.
Genome Res ; 24(7): 1236-50, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24985917

RESUMO

We expanded the knowledge base for Drosophila cell line transcriptomes by deeply sequencing their small RNAs. In total, we analyzed more than 1 billion raw reads from 53 libraries across 25 cell lines. We verify reproducibility of biological replicate data sets, determine common and distinct aspects of miRNA expression across cell lines, and infer the global impact of miRNAs on cell line transcriptomes. We next characterize their commonalities and differences in endo-siRNA populations. Interestingly, most cell lines exhibit enhanced TE-siRNA production relative to tissues, suggesting this as a common aspect of cell immortalization. We also broadly extend annotations of cis-NAT-siRNA loci, identifying ones with common expression across diverse cells and tissues, as well as cell-restricted loci. Finally, we characterize small RNAs in a set of ovary-derived cell lines, including somatic cells (OSS and OSC) and a mixed germline/somatic cell population (fGS/OSS) that exhibits ping-pong piRNA signatures. Collectively, the ovary data reveal new genic piRNA loci, including unusual configurations of piRNA-generating regions. Together with the companion analysis of mRNAs described in a previous study, these small RNA data provide comprehensive information on the transcriptional landscape of diverse Drosophila cell lines. These data should encourage broader usage of fly cell lines, beyond the few that are presently in common usage.


Assuntos
Drosophila/genética , Variação Genética , MicroRNAs/genética , RNA Interferente Pequeno/genética , Animais , Sequência de Bases , Linhagem Celular , Biologia Computacional/métodos , Expressão Gênica , Loci Gênicos , Células Germinativas , Sequenciamento de Nucleotídeos em Larga Escala , MicroRNAs/química , Anotação de Sequência Molecular , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA Interferente Pequeno/química , Alinhamento de Sequência
4.
Genome Res ; 24(7): 1209-23, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24985915

RESUMO

Accurate gene model annotation of reference genomes is critical for making them useful. The modENCODE project has improved the D. melanogaster genome annotation by using deep and diverse high-throughput data. Since transcriptional activity that has been evolutionarily conserved is likely to have an advantageous function, we have performed large-scale interspecific comparisons to increase confidence in predicted annotations. To support comparative genomics, we filled in divergence gaps in the Drosophila phylogeny by generating draft genomes for eight new species. For comparative transcriptome analysis, we generated mRNA expression profiles on 81 samples from multiple tissues and developmental stages of 15 Drosophila species, and we performed cap analysis of gene expression in D. melanogaster and D. pseudoobscura. We also describe conservation of four distinct core promoter structures composed of combinations of elements at three positions. Overall, each type of genomic feature shows a characteristic divergence rate relative to neutral models, highlighting the value of multispecies alignment in annotating a target genome that should prove useful in the annotation of other high priority genomes, especially human and other mammalian genomes that are rich in noncoding sequences. We report that the vast majority of elements in the annotation are evolutionarily conserved, indicating that the annotation will be an important springboard for functional genetic testing by the Drosophila community.


Assuntos
Biologia Computacional/métodos , Drosophila melanogaster/genética , Perfilação da Expressão Gênica , Anotação de Sequência Molecular , Transcriptoma , Animais , Análise por Conglomerados , Drosophila melanogaster/classificação , Evolução Molecular , Éxons , Feminino , Genoma de Inseto , Humanos , Masculino , Motivos de Nucleotídeos , Filogenia , Matrizes de Pontuação de Posição Específica , Regiões Promotoras Genéticas , Edição de RNA , Sítios de Splice de RNA , Splicing de RNA , Reprodutibilidade dos Testes , Sítio de Iniciação de Transcrição
5.
Nature ; 471(7339): 473-9, 2011 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-21179090

RESUMO

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.


Assuntos
Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/genética , Transcrição Gênica/genética , Processamento Alternativo/genética , Animais , Sequência de Bases , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Éxons/genética , Feminino , Genes de Insetos/genética , Genoma de Inseto/genética , Masculino , MicroRNAs/genética , Análise de Sequência com Séries de Oligonucleotídeos , Isoformas de Proteínas/genética , Edição de RNA/genética , RNA Mensageiro/análise , RNA Mensageiro/genética , Pequeno RNA não Traduzido/análise , Pequeno RNA não Traduzido/genética , Análise de Sequência , Caracteres Sexuais
6.
PLoS Genet ; 8(8): e1002883, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22912598

RESUMO

The ecdysone receptor is a heterodimer of two nuclear receptors, the Ecdysone receptor (EcR) and Ultraspiracle (USP). In Drosophila melanogaster, three EcR isoforms share common DNA and ligand-binding domains, but these proteins differ in their most N-terminal regions and, consequently, in the activation domains (AF1s) contained therein. The transcriptional coactivators for these domains, which impart unique transcriptional regulatory properties to the EcR isoforms, are unknown. Activating transcription factor 4 (ATF4) is a basic-leucine zipper transcription factor that plays a central role in the stress response of mammals. Here we show that Cryptocephal (CRC), the Drosophila homolog of ATF4, is an ecdysone receptor coactivator that is specific for isoform B2. CRC interacts with EcR-B2 to promote ecdysone-dependent expression of ecdysis-triggering hormone (ETH), an essential regulator of insect molting behavior. We propose that this interaction explains some of the differences in transcriptional properties that are displayed by the EcR isoforms, and similar interactions may underlie the differential activities of other nuclear receptors with distinct AF1-coactivators.


Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Receptores de Esteroides/genética , Fatores de Transcrição/genética , Ativação Transcricional , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Animais , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Hormônios de Inseto/genética , Hormônios de Inseto/metabolismo , Muda , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerização Proteica , Estrutura Terciária de Proteína , Receptores de Esteroides/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica
7.
Genome Res ; 21(2): 301-14, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21177962

RESUMO

Drosophila melanogaster cell lines are important resources for cell biologists. Here, we catalog the expression of exons, genes, and unannotated transcriptional signals for 25 lines. Unannotated transcription is substantial (typically 19% of euchromatic signal). Conservatively, we identify 1405 novel transcribed regions; 684 of these appear to be new exons of neighboring, often distant, genes. Sixty-four percent of genes are expressed detectably in at least one line, but only 21% are detected in all lines. Each cell line expresses, on average, 5885 genes, including a common set of 3109. Expression levels vary over several orders of magnitude. Major signaling pathways are well represented: most differentiation pathways are "off" and survival/growth pathways "on." Roughly 50% of the genes expressed by each line are not part of the common set, and these show considerable individuality. Thirty-one percent are expressed at a higher level in at least one cell line than in any single developmental stage, suggesting that each line is enriched for genes characteristic of small sets of cells. Most remarkable is that imaginal disc-derived lines can generally be assigned, on the basis of expression, to small territories within developing discs. These mappings reveal unexpected stability of even fine-grained spatial determination. No two cell lines show identical transcription factor expression. We conclude that each line has retained features of an individual founder cell superimposed on a common "cell line" gene expression pattern.


Assuntos
Drosophila melanogaster/genética , Variação Genética , Transcrição Gênica , Animais , Linhagem Celular , Análise por Conglomerados , Éxons , Feminino , Perfilação da Expressão Gênica , Masculino , Dados de Sequência Molecular , Transdução de Sinais/genética , Fatores de Transcrição/genética
8.
Nature ; 426(6962): 78-83, 2003 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-14603321

RESUMO

Steroid hormones fulfil important functions in animal development. In Drosophila, ecdysone triggers moulting and metamorphosis through its effects on gene expression. Ecdysone works by binding to a nuclear receptor, EcR, which heterodimerizes with the retinoid X receptor homologue Ultraspiracle. Both partners are required for binding to ligand or DNA. Like most DNA-binding transcription factors, nuclear receptors activate or repress gene expression by recruiting co-regulators, some of which function as chromatin-modifying complexes. For example, p160 class coactivators associate with histone acetyltransferases and arginine histone methyltransferases. The Trithorax-related gene of Drosophila encodes the SET domain protein TRR. Here we report that TRR is a histone methyltransferases capable of trimethylating lysine 4 of histone H3 (H3-K4). trr acts upstream of hedgehog (hh) in progression of the morphogenetic furrow, and is required for retinal differentiation. Mutations in trr interact in eye development with EcR, and EcR and TRR can be co-immunoprecipitated on ecdysone treatment. TRR, EcR and trimethylated H3-K4 are detected at the ecdysone-inducible promoters of hh and BR-C in cultured cells, and H3-K4 trimethylation at these promoters is decreased in embryos lacking a functional copy of trr. We propose that TRR functions as a coactivator of EcR by altering the chromatin structure at ecdysone-responsive promoters.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/efeitos dos fármacos , Drosophila/embriologia , Ecdisona/farmacologia , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Animais , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Olho/embriologia , Olho/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Proteínas Hedgehog , Histona-Lisina N-Metiltransferase/genética , Masculino , Metilação/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Ligação Proteica , Receptores de Esteroides/metabolismo
10.
Methods Mol Biol ; 388: 317-40, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17951778

RESUMO

Techniques and experimental applications are described for exogenous protein expression in Drosophila cell lines. Ways in which the Drosophila cell lines and the baculovirus expression vector system differ in their applications are emphasized.


Assuntos
Baculoviridae/genética , Drosophila/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Animais , Linhagem Celular , Drosophila/citologia , Drosophila/genética , Expressão Gênica , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Plasmídeos/genética , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes de Fusão/genética , Transfecção/métodos
11.
G3 (Bethesda) ; 6(3): 683-94, 2016 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-26772746

RESUMO

Steroid hormones induce cascades of gene activation and repression with transformative effects on cell fate . Steroid transduction plays a major role in the development and physiology of nearly all metazoan species, and in the progression of the most common forms of cancer. Despite the paramount importance of steroids in developmental and translational biology, a complete map of transcriptional response has not been developed for any hormone . In the case of 20-hydroxyecdysone (ecdysone) in Drosophila melanogaster, these trajectories range from apoptosis to immortalization. We mapped the ecdysone transduction network in a cohort of 41 cell lines, the largest such atlas yet assembled. We found that the early transcriptional response mirrors the distinctiveness of physiological origins: genes respond in restricted patterns, conditional on the expression levels of dozens of transcription factors. Only a small cohort of genes is constitutively modulated independent of initial cell state. Ecdysone-responsive genes tend to organize into directional same-stranded units, with consecutive genes induced from the same strand. Here, we identify half of the ecdysone receptor heterodimer as the primary rate-limiting step in the response, and find that initial receptor isoform levels modulate the activated cohort of target transcription factors. This atlas of steroid response reveals organizing principles of gene regulation by a model type II nuclear receptor and lays the foundation for comprehensive and predictive understanding of the ecdysone transduction network in the fruit fly.


Assuntos
Drosophila/genética , Drosophila/metabolismo , Regulação da Expressão Gênica , Hormônios/metabolismo , Transdução de Sinais , Animais , Linhagem Celular , Análise por Conglomerados , Ecdisona/metabolismo , Ecdisona/farmacologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Hormônios/farmacologia , Isoformas de Proteínas , Receptores de Esteroides/metabolismo , Esteroides/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacos , Transcriptoma
12.
Mol Endocrinol ; 17(4): 716-31, 2003 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-12554759

RESUMO

The ecdysone receptor is a heterodimer of the two nuclear receptors EcR and ultraspiracle (USP). We have identified the regions of Drosophila EcR and USP responsible for transcriptional activation of a semisynthetic Eip71CD promoter in Kc cells. The isoform-specific A/B domains of EcR-B1 and B2, but not those of EcR-A or USP, exhibit strong activation activity [activation function 1 (AF1)], both in isolation and in the context of the intact receptor. AF1 activity in isoform B1 derives from dispersed elements; the B2-specific AF1 consists of a 17-residue amphipathic helix. AF2 function was studied using a two-hybrid assay in Kc cells, based on the observation that potent hormone-dependent activation by the EcR/USP ligand-binding domain heterodimer requires the participation of both partners. Mutagenesis reveals that AF2 function depends on EcR helix 12, but not on the cognate USP region. EcR helix 12 mutants (F645A and W650A) exhibit a dominant negative phenotype. Thus, in the setting tested, the ecdysone receptor can activate transcription using the AF1 regions of EcR-B1 or -B2 and the AF2 region of EcR. USP acts as an allosteric effector for EcR, but does not contribute any intrinsic function.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Receptores de Esteroides/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional , Sequência de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular , Sequência Conservada , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Ecdisona/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Dominantes , Dados de Sequência Molecular , Mutação , Regiões Promotoras Genéticas , Conformação Proteica , Isoformas de Proteínas , Receptores de Esteroides/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fatores de Transcrição/genética , Técnicas do Sistema de Duplo-Híbrido
13.
Genetics ; 201(4): 1307-18, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26450921

RESUMO

We describe an adaptation of φC31 integrase-mediated targeted cassette exchange for use in Drosophila cell lines. Single copies of an attP-bounded docking platform carrying a GFP-expression marker, with or without insulator elements flanking the attP sites, were inserted by P-element transformation into the Kc167 and Sg4 cell lines; each of the resulting docking-site lines carries a single mapped copy of one of the docking platforms. Vectors for targeted substitution contain a cloning cassette flanked by attB sites. Targeted substitution occurs by integrase-mediated substitution between the attP sites (integrated) and the attB sites (vector). We describe procedures for isolating cells carrying the substitutions and for eliminating the products of secondary off-target events. We demonstrate the technology by integrating a cassette containing a Cu(2+)-inducible mCherry marker, and we report the expression properties of those lines. When compared with clonal lines made by traditional transformation methods, which lead to the illegitimate insertion of tandem arrays, targeted insertion lines give more uniform expression, lower basal expression, and higher induction ratios. Targeted substitution, though intricate, affords results that should greatly improve comparative expression assays-a major emphasis of cell-based studies.


Assuntos
Drosophila melanogaster/genética , Marcação de Genes/métodos , Genoma de Inseto , Mutagênese Insercional , Animais , Linhagem Celular , Células Clonais , Engenharia Genética , Marcadores Genéticos , Integrases/metabolismo , Transgenes
14.
Fly (Austin) ; 8(4): 226-31, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25833067

RESUMO

In insects, localized tissue injury often leads to global (organism-wide) delays in development and retarded metamorphosis. In Drosophila, for example, injuries to the larval imaginal discs can retard pupariation and prolong metamorphosis. Injuries induced by treatments such as radiation, mechanical damage and induction of localized cell death can trigger similar delays. In most cases, the duration of the developmental delay appears to be correlated with the extent of damage, but the effect is also sensitive to the developmental stage of the treated animal. The proximate cause of the delays is likely a disruption of the ecdysone signaling pathway, but the intermediate steps leading from tissue injury and/or regeneration to that disruption remain unknown. Here, we review the evidence for injury-induced developmental delays, and for a checkpoint or checkpoints associated with the temporal progression of development and the on-going efforts to define the mechanisms involved.


Assuntos
Pontos de Checagem do Ciclo Celular/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Insetos/metabolismo , Insetos/crescimento & desenvolvimento , Insetos/fisiologia , Animais , Proteínas de Insetos/genética , Larva
15.
Genome Biol ; 15(8): R70, 2014 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-25262759

RESUMO

BACKGROUND: Structural rearrangements of the genome resulting in genic imbalance due to copy number change are often deleterious at the organismal level, but are common in immortalized cell lines and tumors, where they may be an advantage to cells. In order to explore the biological consequences of copy number changes in the Drosophila genome, we resequenced the genomes of 19 tissue-culture cell lines and generated RNA-Seq profiles. RESULTS: Our work revealed dramatic duplications and deletions in all cell lines. We found three lines of evidence indicating that copy number changes were due to selection during tissue culture. First, we found that copy numbers correlated to maintain stoichiometric balance in protein complexes and biochemical pathways, consistent with the gene balance hypothesis. Second, while most copy number changes were cell line-specific, we identified some copy number changes shared by many of the independent cell lines. These included dramatic recurrence of increased copy number of the PDGF/VEGF receptor, which is also over-expressed in many cancer cells, and of bantam, an anti-apoptosis miRNA. Third, even when copy number changes seemed distinct between lines, there was strong evidence that they supported a common phenotypic outcome. For example, we found that proto-oncogenes were over-represented in one cell line (S2-DRSC), whereas tumor suppressor genes were under-represented in another (Kc167). CONCLUSION: Our study illustrates how genome structure changes may contribute to selection of cell lines in vitro. This has implications for other cell-level natural selection progressions, including tumorigenesis.


Assuntos
Linhagem Celular , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Evolução Molecular , Dosagem de Genes , Animais , Sobrevivência Celular , DNA/análise , Proteínas de Drosophila/genética , Feminino , Aptidão Genética , Variação Genética , Masculino , MicroRNAs/genética , Receptores Proteína Tirosina Quinases/genética , Seleção Genética , Análise de Sequência de DNA , Cromossomos Sexuais/genética , Técnicas de Cultura de Tecidos
16.
PLoS One ; 7(11): e49105, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23166607

RESUMO

In humans, chronic inflammation, severe injury, infection and disease can result in changes in steroid hormone titers and delayed onset of puberty; however the pathway by which this occurs remains largely unknown. Similarly, in insects injury to specific tissues can result in a global developmental delay (e.g. prolonged larval/pupal stages) often associated with decreased levels of ecdysone - a steroid hormone that regulates developmental transitions in insects. We use Drosophila melanogaster as a model to examine the pathway by which tissue injury disrupts developmental progression. Imaginal disc damage inflicted early in larval development triggers developmental delays while the effects are minimized in older larvae. We find that the switch in injury response (e.g. delay/no delay) is coincident with the mid-3rd instar transition - a developmental time-point that is characterized by widespread changes in gene expression and marks the initial steps of metamorphosis. Finally, we show that developmental delays induced by tissue damage are associated with decreased expression of genes involved in ecdysteroid synthesis and signaling.


Assuntos
Drosophila melanogaster/crescimento & desenvolvimento , Ecdisteroides/biossíntese , Discos Imaginais/lesões , Transdução de Sinais/genética , Animais , Primers do DNA/genética , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Larva/crescimento & desenvolvimento , Larva/metabolismo , Metamorfose Biológica/fisiologia , Reação em Cadeia da Polimerase em Tempo Real
17.
Cell Rep ; 1(3): 277-89, 2012 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-22685694

RESUMO

We analyzed the usage and consequences of alternative cleavage and polyadenylation (APA) in Drosophila melanogaster by using >1 billion reads of stranded mRNA-seq across a variety of dissected tissues. Beyond demonstrating that a majority of fly transcripts are subject to APA, we observed broad trends for 3' untranslated region (UTR) shortening in the testis and lengthening in the central nervous system (CNS); the latter included hundreds of unannotated extensions ranging up to 18 kb. Extensive northern analyses validated the accumulation of full-length neural extended transcripts, and in situ hybridization indicated their spatial restriction to the CNS. Genes encoding RNA binding proteins (RBPs) and transcription factors were preferentially subject to 3' UTR extensions. Motif analysis indicated enrichment of miRNA and RBP sites in the neural extensions, and their termini were enriched in canonical cis elements that promote cleavage and polyadenylation. Altogether, we reveal broad tissue-specific patterns of APA in Drosophila and transcripts with unprecedented 3' UTR length in the nervous system.


Assuntos
Drosophila melanogaster/genética , Especificidade de Órgãos/genética , Poliadenilação/genética , Regiões 3' não Traduzidas/genética , Animais , Sequência de Bases , Northern Blotting , Sequência Conservada/genética , Proteínas de Ligação a DNA/metabolismo , Drosophila melanogaster/embriologia , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genes de Insetos/genética , Hibridização In Situ , Masculino , Dados de Sequência Molecular , Neurônios/citologia , Neurônios/metabolismo , Motivos de Nucleotídeos/genética , Poli A/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Análise de Sequência de RNA , Testículo/metabolismo , Transcriptoma/genética
18.
Cold Spring Harb Protoc ; 2010(6): pdb.prot5001, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20516171

RESUMO

Most commonly used Drosophila cell lines adhere only loosely to the substrate. For some purposes (e.g., time-lapse photography and cytological examination of living cells), it is desirable to fix the growing cells more firmly to the substrate. As described in this protocol, this can be accomplished by treating the substrate with poly-L-lysine, with no obvious detrimental effect on the growth of the cells.


Assuntos
Técnicas de Cultura de Células/métodos , Drosophila melanogaster/citologia , Polilisina/farmacologia , Animais , Células Cultivadas , Propriedades de Superfície/efeitos dos fármacos
20.
CSH Protoc ; 2007: pdb.top6, 2007 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-21357155

RESUMO

INTRODUCTIONPermanent Drosophila cell lines derived from mixed embryonic tissues including the most commonly used lines, S2 and Kc, have been available for ~30 yr. More recently, lines derived from specific tissues, imaginal discs, and the larval central nervous system have come into use. Although cultured cells were originally used by Drosophilists mainly as convenient sources of DNA or carrier RNA, that situation has changed, as an armamentarium of techniques for using the cells has slowly but steadily evolved. Most investigators use Drosophila cell lines as hosts for transformation experiments. The goal may be to characterize a promoter, to investigate the role of a transcription factor, to overexpress a polypeptide, or to do something more novel. This article provides an organized collection of pointers to published protocols.

SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA