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1.
BMC Bioinformatics ; 20(1): 421, 2019 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-31409274

RESUMO

BACKGROUND: Ultra-fast pseudo-alignment approaches are the tool of choice in transcript-level RNA sequencing (RNA-seq) analyses. Unfortunately, these methods couple the tasks of pseudo-alignment and transcript quantification. This coupling precludes the direct usage of pseudo-alignment to other expression analyses, including alternative splicing or differential gene expression analysis, without including a non-essential transcript quantification step. RESULTS: In this paper, we introduce a transcriptome segmentation approach to decouple these two tasks. We propose an efficient algorithm to generate maximal disjoint segments given a transcriptome reference library on which ultra-fast pseudo-alignment can be used to produce per-sample segment counts. We show how to apply these maximally unambiguous count statistics in two specific expression analyses - alternative splicing and gene differential expression - without the need of a transcript quantification step. Our experiments based on simulated and experimental data showed that the use of segment counts, like other methods that rely on local coverage statistics, provides an advantage over approaches that rely on transcript quantification in detecting and correctly estimating local splicing in the case of incomplete transcript annotations. CONCLUSIONS: The transcriptome segmentation approach implemented in Yanagi exploits the computational and space efficiency of pseudo-alignment approaches. It significantly expands their applicability and interpretability in a variety of RNA-seq analyses by providing the means to model and capture local coverage variation in these analyses.


Assuntos
Algoritmos , Transcriptoma , Processamento Alternativo , Animais , Área Sob a Curva , Drosophila/genética , Humanos , RNA/química , RNA/metabolismo , Curva ROC , Análise de Sequência de RNA
2.
BMC Evol Biol ; 19(1): 99, 2019 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-31068148

RESUMO

BACKGROUND: RNA interference (RNAi) related pathways provide defense against viruses and transposable elements, and have been implicated in the suppression of meiotic drive elements. Genes in these pathways often exhibit high levels of adaptive substitution, and over longer timescales show gene duplication and loss-most likely as a consequence of their role in mediating conflict with these parasites. This is particularly striking for Argonaute 2 (Ago2), which is ancestrally the key effector of antiviral RNAi in insects, but has repeatedly formed new testis-specific duplicates in the recent history of the obscura species-group of Drosophila. RESULTS: Here we take advantage of publicly available genomic and transcriptomic data to identify six further RNAi-pathway genes that have duplicated in this clade of Drosophila, and examine their evolutionary history. As seen for Ago2, we observe high levels of adaptive amino-acid substitution and changes in sex-biased expression in many of the paralogs. However, our phylogenetic analysis suggests that co-duplications of the RNAi machinery were not synchronous, and our expression analysis fails to identify consistent male-specific expression. CONCLUSIONS: These results confirm that RNAi genes, including genes of the antiviral and piRNA pathways, have undergone multiple independent duplications and that their history has been particularly labile within the obscura group. However, they also suggest that the selective pressures driving these changes have not been consistent, implying that more than one selective agent may be responsible.


Assuntos
Adaptação Fisiológica/genética , Drosophila/genética , Duplicação Gênica , Genes de Insetos , Interferência de RNA , Substituição de Aminoácidos/genética , Animais , Teorema de Bayes , Proteínas CLOCK/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Evolução Molecular , Regulação da Expressão Gênica , Masculino , Filogenia
3.
Nat Commun ; 10(1): 2113, 2019 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-31068592

RESUMO

Gene editing by CRISPR/Cas9 is commonly used to generate germline mutations or perform in vitro screens, but applicability for in vivo screening has so far been limited. Recently, it was shown that in Drosophila, Cas9 expression could be limited to a desired group of cells, allowing tissue-specific mutagenesis. Here, we thoroughly characterize tissue-specific (ts)CRISPR within the complex neuronal system of the Drosophila mushroom body. We report the generation of a library of gRNA-expressing plasmids and fly lines using optimized tools, which provides a valuable resource to the fly community. We demonstrate the application of our library in a large-scale in vivo screen, which reveals insights into developmental neuronal remodeling.


Assuntos
Animais Geneticamente Modificados/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Drosophila/genética , Edição de Genes/métodos , Animais , Sistemas CRISPR-Cas/genética , Feminino , Masculino , Corpos Pedunculados/metabolismo , Mutagênese , Sistema Nervoso/crescimento & desenvolvimento , Plasticidade Neuronal/genética , Neurônios/fisiologia , Plasmídeos/genética , RNA Guia/genética
4.
Yi Chuan ; 41(5): 422-429, 2019 May 20.
Artigo em Chinês | MEDLINE | ID: mdl-31106778

RESUMO

The type2 CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR- associated protein 9) is an efficient RNA-guided genome-editing technique. Guided by sgRNA, the Cas9 endonuclease generates site-specific double-stranded breaks (DSB) at specific site, which is amenable to repair by homology-directed repair (HDR) to generate a designed knock-out or knock-in transgene. In combination with CRISPR/Cas9 and Cre/loxP or FLP/FRT system, efficient gene targeting can be achieved, and meanwhile screening markers introduced can be readily removed except a 34-base pair residual fragment. Thus, difficulties remain in accurate editing of the genome without introducing any extraneous sequences. In human induced pluripotent stem cells (iPSCs), a two-step strategy has been developed using CRISPR/Cas9 and the piggyBac system to establish a seamless genomic editing, in which CRISPR/Cas9 is initially used to introduce mutations along with screening markers by HDR, then the markers are precisely excised by piggyBac transposase. Using this strategy, we have successfully transformed the tyrosine to cysteine at position 21 within the 18th exon of the CG4894 gene in the Drosophila genome without introducing any extraneous sequence. Hence, this strategy provides more options for precise and seamless editing of the Drosophila genome.


Assuntos
Sistemas CRISPR-Cas , Drosophila/genética , Edição de Genes , Genoma de Inseto , Transposases/genética , Animais
5.
Dev Growth Differ ; 61(4): 265-275, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31037730

RESUMO

The CRISPR-Cas9 technology has been a powerful means to manipulate the genome in a wide range of organisms. A series of GFP knocked-in (GFPKI ) Drosophila strains have been generated through CRISPR-Cas9-induced double strand breaks coupled with homology-directed repairs in the presence of donor plasmids. They visualized specific cell types or intracellular structures in both fixed and live specimen. We provide a rapid and efficient strategy to identify KI lines. This method requires neither co-integration of a selection marker nor prior establishment of sgRNA-expressing transgenic lines. The injection of the mixture of a sgRNA/Cas9 expression plasmid and a donor plasmid into cleavage stage embryos efficiently generated multiple independent KI lines. A PCR-based selection allows to identify KI fly lines at the F1 generation (approximately 4 weeks after injection). These GFPKI strains have been deposited in the Kyoto Drosophila stock center, and made freely available to researchers at non-profit organizations. Thus, they will be useful resources for Drosophila research.


Assuntos
Sistemas CRISPR-Cas/genética , Drosophila/genética , Edição de Genes/métodos , Técnicas de Introdução de Genes/métodos , Proteínas de Fluorescência Verde/genética , Animais , Fatores de Tempo
6.
Mol Cells ; 42(4): 301-312, 2019 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-31091556

RESUMO

Post-transcriptional regulation underlies the circadian control of gene expression and animal behaviors. However, the role of mRNA surveillance via the nonsense-mediated mRNA decay (NMD) pathway in circadian rhythms remains elusive. Here, we report that Drosophila NMD pathway acts in a subset of circadian pacemaker neurons to maintain robust 24 h rhythms of free-running locomotor activity. RNA interference-mediated depletion of key NMD factors in timeless-expressing clock cells decreased the amplitude of circadian locomotor behaviors. Transgenic manipulation of the NMD pathway in clock neurons expressing a neuropeptide PIGMENT-DISPERSING FACTOR (PDF) was sufficient to dampen or lengthen free-running locomotor rhythms. Confocal imaging of a transgenic NMD reporter revealed that arrhythmic Clock mutants exhibited stronger NMD activity in PDF-expressing neurons than wild-type. We further found that hypomorphic mutations in Suppressor with morphogenetic effect on genitalia 5 (Smg5 ) or Smg6 impaired circadian behaviors. These NMD mutants normally developed PDF-expressing clock neurons and displayed daily oscillations in the transcript levels of core clock genes. By contrast, the loss of Smg5 or Smg6 function affected the relative transcript levels of cAMP response element-binding protein B (CrebB ) in an isoform-specific manner. Moreover, the overexpression of a transcriptional repressor form of CrebB rescued free-running locomotor rhythms in Smg5-depleted flies. These data demonstrate that CrebB is a rate-limiting substrate of the genetic NMD pathway important for the behavioral output of circadian clocks in Drosophila.


Assuntos
Relógios Circadianos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/genética , Mutação , Degradação do RNAm Mediada por Códon sem Sentido , Transativadores/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas CLOCK/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Endorribonucleases/genética , Endorribonucleases/metabolismo , Neurônios/metabolismo , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Processamento Pós-Transcricional do RNA , Transdução de Sinais
7.
Int J Mol Sci ; 20(9)2019 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-31060255

RESUMO

GSK3 (glycogen synthase kinase 3) is a conserved protein kinase governing numerous regulatory pathways. In Drosophila melanogaster, GSK3 is encoded by shaggy (sgg), which forms 17 annotated transcripts corresponding to 10 protein isoforms. Our goal was to demonstrate how differential sgg transcription affects lifespan, which GSK3 isoforms are important for the nervous system, and which changes in the nervous system accompany accelerated aging. Overexpression of three sgg transcripts affected the lifespan in a stage- and tissue-specific way: sgg-RA and sgg-RO affected the lifespan only when overexpressed in muscles and in embryos, respectively; the essential sgg-RB transcript affected lifespan when overexpressed in all tissues tested. In the nervous system, only sgg-RB overexpression affected lifespan, causing accelerated aging in a neuron-specific way, with the strongest effects in dopaminergic neurons and the weakest effects in GABAergic neurons. Pan-neuronal sgg-RB overexpression violated the properties of the nervous system, including the integrity of neuron bodies; the number, distribution, and structure of mitochondria; cytoskeletal characteristics; and synaptic activity. Such changes observed in young individuals indicated premature aging of their nervous system, which paralleled a decline in survival. Our findings demonstrated the key role of GSK3 in ensuring the link between the pathology of neurons and lifespan.


Assuntos
Proteínas de Drosophila/genética , Drosophila/genética , Regulação da Expressão Gênica , Quinase 3 da Glicogênio Sintase/genética , Estágios do Ciclo de Vida/genética , Longevidade/genética , Animais , Drosophila/crescimento & desenvolvimento , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Feminino , Quinase 3 da Glicogênio Sintase/metabolismo , Masculino , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Neurônios/metabolismo , Neurônios/ultraestrutura , Especificidade de Órgãos/genética , Fenótipo
8.
Genetics ; 212(1): 13-24, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31053615

RESUMO

Transcription by RNA polymerase II initiates at the core promoter, which is sometimes referred to as the "gateway to transcription." Here, we describe the properties of the RNA polymerase II core promoter in Drosophila The core promoter is at a strategic position in the expression of genes, as it is the site of convergence of the signals that lead to transcriptional activation. Importantly, core promoters are diverse in terms of their structure and function. They are composed of various combinations of sequence motifs such as the TATA box, initiator (Inr), and downstream core promoter element (DPE). Different types of core promoters are transcribed via distinct mechanisms. Moreover, some transcriptional enhancers exhibit specificity for particular types of core promoters. These findings indicate that the core promoter is a central component of the transcriptional apparatus that regulates gene expression.


Assuntos
Drosophila/genética , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Transcrição Genética , Animais , Drosophila/enzimologia , Proteínas de Drosophila/metabolismo , Ativação Transcricional
9.
Nat Commun ; 10(1): 1640, 2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30967548

RESUMO

Gene-drive systems developed in several organisms result in super-Mendelian inheritance of transgenic insertions. Here, we generalize this "active genetic" approach to preferentially transmit allelic variants (allelic-drive) resulting from only a single or a few nucleotide alterations. We test two configurations for allelic-drive: one, copy-cutting, in which a non-preferred allele is selectively targeted for Cas9/guide RNA (gRNA) cleavage, and a more general approach, copy-grafting, that permits selective inheritance of a desired allele located in close proximity to the gRNA cut site. We also characterize a phenomenon we refer to as lethal-mosaicism that dominantly eliminates NHEJ-induced mutations and favors inheritance of functional cleavage-resistant alleles. These two efficient allelic-drive methods, enhanced by lethal mosaicism and a trans-generational drive process we refer to as "shadow-drive", have broad practical applications in improving health and agriculture and greatly extend the active genetics toolbox.


Assuntos
Alelos , Reparo do DNA por Junção de Extremidades/genética , Drosophila/genética , Tecnologia de Impulso Genético/métodos , Agricultura/métodos , Animais , Animais Geneticamente Modificados/genética , Sistemas CRISPR-Cas/genética , Análise Mutacional de DNA , Feminino , Edição de Genes/métodos , Padrões de Herança/genética , Masculino , Mosaicismo , RNA Guia/genética
10.
PLoS Comput Biol ; 15(4): e1006949, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30986215

RESUMO

Understanding genomic structural variation such as inversions and translocations is a key challenge in evolutionary genetics. We develop a novel statistical approach to comparative genetic mapping to detect large-scale structural mutations from low-level sequencing data. The procedure, called Genome Order Optimization by Genetic Algorithm (GOOGA), couples a Hidden Markov Model with a Genetic Algorithm to analyze data from genetic mapping populations. We demonstrate the method using both simulated data (calibrated from experiments on Drosophila melanogaster) and real data from five distinct crosses within the flowering plant genus Mimulus. Application of GOOGA to the Mimulus data corrects numerous errors (misplaced sequences) in the M. guttatus reference genome and confirms or detects eight large inversions polymorphic within the species complex. Finally, we show how this method can be applied in genomic scans to improve the accuracy and resolution of Quantitative Trait Locus (QTL) mapping.


Assuntos
Mapeamento Cromossômico/métodos , Biologia Computacional/métodos , Variação Genética/genética , Algoritmos , Animais , Evolução Biológica , Drosophila/genética , Genética Populacional/métodos , Genoma/fisiologia , Genômica , Hibridização Genética/genética , Cadeias de Markov , Mimulus/genética , Fenótipo , Locos de Características Quantitativas/genética
11.
Mol Cell ; 74(1): 3-4, 2019 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-30951650

RESUMO

Cardozo Gizzi et al. (2019) develop a new sequential imaging methodology (Hi-M) for observing chromosome structure in the Drosophila blastoderm and find that topological domains in single nuclei change in response to transcriptional activation.


Assuntos
Proteínas de Drosophila/genética , Drosophila/genética , Animais , Cromossomos , Genoma , Microscopia
12.
Med Sci (Paris) ; 35(3): 245-251, 2019 Mar.
Artigo em Francês | MEDLINE | ID: mdl-30931909

RESUMO

Alfred H. Sturtevant was the first to raise the question: why does the mutation rate not become reduced to zero? Indeed, most new mutations with a phenotypic effect are deleterious. Therefore, individuals who produce less mutants produce more viable and fertile offspring. Consequently, natural selection should increase the frequency of antimutator genotypes and progressively reduce the mutation rate to zero. However, no species has ever been found with a mutation rate equal to zero. Recent analyses suggest that setting the mutation rate above zero depends mainly on the effective size of the genome and the effective population size. The mutation rate is a trade-off between natural selection that operates to improve replication fidelity and the random genetic drift that sets the ultimate lower limit. This trade off illustrates the limitation of the power of natural selection in a world where natural populations have a finite size.


Assuntos
Evolução Molecular , Taxa de Mutação , Animais , Drosophila/genética , Genes Letais/fisiologia , Deriva Genética , Humanos , Relação entre Gerações , Modelos Genéticos , Mutação/fisiologia , Seleção Genética/genética
13.
Mol Biol Evol ; 36(6): 1316-1332, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30847475

RESUMO

There are numerous sources of variation in the rate of synonymous substitutions inside genes, such as direct selection on the nucleotide sequence, or mutation rate variation. Yet scans for positive selection rely on codon models which incorporate an assumption of effectively neutral synonymous substitution rate, constant between sites of each gene. Here we perform a large-scale comparison of approaches which incorporate codon substitution rate variation and propose our own simple yet effective modification of existing models. We find strong effects of substitution rate variation on positive selection inference. More than 70% of the genes detected by the classical branch-site model are presumably false positives caused by the incorrect assumption of uniform synonymous substitution rate. We propose a new model which is strongly favored by the data while remaining computationally tractable. With the new model we can capture signatures of nucleotide level selection acting on translation initiation and on splicing sites within the coding region. Finally, we show that rate variation is highest in the highly recombining regions, and we propose that recombination and mutation rate variation, such as high CpG mutation rate, are the two main sources of nucleotide rate variation. Although we detect fewer genes under positive selection in Drosophila than without rate variation, the genes which we detect contain a stronger signal of adaptation of dynein, which could be associated with Wolbachia infection. We provide software to perform positive selection analysis using the new model.


Assuntos
Códon , Modelos Genéticos , Taxa de Mutação , Seleção Genética , Mutação Silenciosa , Animais , Simulação por Computador , Drosophila/genética , Recombinação Genética , Vertebrados/genética
14.
BMC Genomics ; 20(1): 223, 2019 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-30885123

RESUMO

BACKGROUND: Drosophila subobscura has long been a central model in evolutionary genetics. Presently, its use is hindered by the lack of a reference genome. To bridge this gap, here we used PacBio long-read technology, together with the available wealth of genetic marker information, to assemble and annotate a high-quality nuclear and complete mitochondrial genome for the species. With the obtained assembly, we performed the first synteny analysis of genome structure evolution in the subobscura subgroup. RESULTS: We generated a highly-contiguous ~ 129 Mb-long nuclear genome, consisting of six pseudochromosomes corresponding to the six chromosomes of a female haploid set, and a complete 15,764 bp-long mitogenome, and provide an account of their numbers and distributions of codifying and repetitive content. All 12 identified paracentric inversion differences in the subobscura subgroup would have originated by chromosomal breakage and repair, with some associated duplications, but no evidence of direct gene disruptions by the breakpoints. Between lineages, inversion fixation rates were 10 times higher in continental D. subobscura than in the two small oceanic-island endemics D. guanche and D. madeirensis. Within D. subobscura, we found contrasting ratios of chromosomal divergence to polymorphism between the A sex chromosome and the autosomes. CONCLUSIONS: We present the first high-quality, long-read sequencing of a D. subobscura genome. Our findings generally support genome structure evolution in this species being driven indirectly, through the inversions' recombination-suppression effects in maintaining sets of adaptive alleles together in the face of gene flow. The resources developed will serve to further establish the subobscura subgroup as model for comparative genomics and evolutionary indicator of global change.


Assuntos
Inversão Cromossômica , Cromossomos de Insetos , Drosophila/genética , Evolução Molecular , Genoma de Inseto , Recombinação Genética , Sintenia , Animais , Drosophila/classificação , Feminino , Fluxo Gênico , Marcadores Genéticos , Proteínas de Insetos , Masculino , Filogenia , Polimorfismo Genético
15.
Mech Dev ; 156: 20-31, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30904594

RESUMO

In a screen for human kinases that regulate Xenopus laevis embryogenesis, we identified Nagk and other components of the UDP-GlcNAc glycosylation salvage pathway as regulators of anteroposterior patterning and Wnt signaling. We find that the salvage pathway does not affect other major embryonic signaling pathways (Fgf, TGFß, Notch, or Shh), thereby demonstrating specificity for Wnt signaling. We show that the role of the salvage pathway in Wnt signaling is evolutionarily conserved in zebrafish and Drosophila. Finally, we show that GlcNAc is essential for the growth of intestinal enteroids, which are highly dependent on Wnt signaling for growth and maintenance. We propose that the Wnt pathway is sensitive to alterations in the glycosylation state of a cell and acts as a nutritional sensor in order to couple growth/proliferation with its metabolic status. We also propose that the clinical manifestations observed in congenital disorders of glycosylation (CDG) in humans may be due, in part, to their effects on Wnt signaling during development.


Assuntos
Desenvolvimento Embrionário/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Via de Sinalização Wnt/genética , Xenopus laevis/crescimento & desenvolvimento , Animais , Padronização Corporal/genética , Drosophila/genética , Drosophila/crescimento & desenvolvimento , Evolução Molecular , Regulação da Expressão Gênica no Desenvolvimento , Glicosilação , Humanos , Xenopus laevis/genética , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento
16.
BMC Bioinformatics ; 20(1): 122, 2019 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-30866794

RESUMO

BACKGROUND: Balancer chromosomes are tools used by fruit fly geneticists to prevent meiotic recombination. Recently, CRISPR/Cas9 genome editing has been shown capable of generating inversions similar to the chromosomal rearrangements present in balancer chromosomes. Extending the benefits of balancer chromosomes to other multicellular organisms could significantly accelerate biomedical and plant genetics research. RESULTS: Here, we present GRIBCG (Guide RNA Identifier for Balancer Chromosome Generation), a tool for the rational design of balancer chromosomes. GRIBCG identifies single guide RNAs (sgRNAs) for use with Streptococcus pyogenes Cas9 (SpCas9). These sgRNAs would efficiently cut a chromosome multiple times while minimizing off-target cutting in the rest of the genome. We describe the performance of this tool on six model organisms and compare our results to two routinely used fruit fly balancer chromosomes. CONCLUSION: GRIBCG is the first of its kind tool for the design of balancer chromosomes using CRISPR/Cas9. GRIBCG can accelerate genetics research by providing a fast, systematic and simple to use framework to induce chromosomal rearrangements.


Assuntos
Cromossomos de Insetos/genética , RNA Guia/isolamento & purificação , Software , Animais , Proteína 9 Associada à CRISPR/genética , Inversão Cromossômica , Drosophila/genética , Edição de Genes , RNA Guia/genética , Streptococcus pyogenes
17.
Genetics ; 212(1): 343-360, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30842209

RESUMO

Cis-regulatory sequences known as enhancers play a key role in regulating gene expression. Evolutionary changes in these DNA sequences contribute to phenotypic evolution. The Drosophila yellow gene, which is required for pigmentation, has emerged as a model system for understanding how cis-regulatory sequences evolve, providing some of the most detailed insights available into how activities of orthologous enhancers have diverged between species. Here, we examine the evolution of yellow cis-regulatory sequences on a broader scale, by comparing the distribution and function of yellow enhancer activities throughout the 5' intergenic and intronic sequences of Drosophila melanogaster, D. pseudoobscura, and D. willistoni We find that cis-regulatory sequences driving expression in a particular tissue are not as modular as previously described, but rather have many redundant and cryptic enhancer activities distributed throughout the regions surveyed. Interestingly, cryptic enhancer activities of sequences from one species often drove patterns of expression observed in other species, suggesting that the frequent evolutionary changes in yellow expression observed among Drosophila species may be facilitated by gaining and losing repression of preexisting cis-regulatory sequences.


Assuntos
Proteínas de Drosophila/genética , Drosophila/genética , Elementos Facilitadores Genéticos , Evolução Molecular , Animais , Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Pigmentação/genética , Especificidade da Espécie
18.
Genetics ; 212(1): 53-63, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30862621

RESUMO

The Q-system is a binary expression system that works well across species. Here, we report the development and demonstrate the applications of a split-QF system that drives strong expression in Drosophila, is repressible by QS, and is inducible by a small nontoxic molecule (quinic acid). The split-QF system is fully compatible with existing split-GAL4 and split-LexA lines, thus greatly expanding the range of possible advanced intersectional experiments and anatomical, physiological, and behavioral assays in Drosophila, and in other organisms.


Assuntos
Drosophila/genética , Expressão Gênica , Transgenes , Animais , Animais Geneticamente Modificados , Proteínas de Bactérias/genética , Proteínas de Drosophila/genética , Feminino , Técnicas Genéticas , Masculino , Ácido Quínico , Serina Endopeptidases/genética , Fatores de Transcrição/genética
19.
Genetics ; 212(1): 187-211, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30867197

RESUMO

Centrosomes play a critical role in mitotic spindle assembly through their role in microtubule nucleation and bipolar spindle assembly. Loss of centrosomes can impair the ability of some cells to properly conduct mitotic division, leading to chromosomal instability, cell stress, and aneuploidy. Multiple aspects of the cellular response to mitotic error associated with centrosome loss appear to involve activation of JNK signaling. To further characterize the transcriptional effects of centrosome loss, we compared gene expression profiles of wild-type and acentrosomal cells from Drosophila wing imaginal discs. We found elevation of expression of JNK target genes, which we verified at the protein level. Consistent with this, the upregulated gene set showed significant enrichment for the AP-1 consensus DNA-binding sequence. We also found significant elevation in expression of genes regulating redox balance. Based on those findings, we examined oxidative stress after centrosome loss, revealing that acentrosomal wing cells have significant increases in reactive oxygen species (ROS). We then performed a candidate genetic screen and found that one of the genes upregulated in acentrosomal cells, glucose-6-phosphate dehydrogenase, plays an important role in buffering acentrosomal cells against increased ROS and helps protect those cells from cell death. Our data and other recent studies have revealed a complex network of signaling pathways, transcriptional programs, and cellular processes that epithelial cells use to respond to stressors, like mitotic errors, to help limit cell damage and maintain normal tissue development.


Assuntos
Apoptose , Centrossomo , Drosophila/metabolismo , Sistema de Sinalização das MAP Quinases , Estresse Oxidativo , Animais , Drosophila/genética , Drosophila/fisiologia , Células Epiteliais/metabolismo , Perfilação da Expressão Gênica , Mitose , Análise de Sequência de RNA , Fuso Acromático , Ativação Transcricional
20.
Malar J ; 18(1): 62, 2019 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-30845961

RESUMO

BACKGROUND: UDP-glycosyltransferase (UGT) is an important biotransformation superfamily of enzymes. They catalyze the transfer of glycosyl residues from activated nucleotide sugars to acceptor hydrophobic molecules, and function in several physiological processes, including detoxification, olfaction, cuticle formation, pigmentation. The diversity, classification, scaffold location, characteristics, phylogenetics, and evolution of the superfamily of genes at whole genome level, and their association and mutations associated with pyrethroid resistance are still little known. METHODS: The present study identified UGT genes in Anopheles sinensis genome, classified UGT genes in An. sinensis, Anopheles gambiae, Aedes aegypti and Drosophila melanogaster genomes, and analysed the scaffold location, characteristics, phylogenetics, and evolution of An. sinensis UGT genes using bioinformatics methods. The present study also identified the UGTs associated with pyrethroid resistance using three field pyrethroid-resistant populations with RNA-seq and RT-qPCR, and the mutations associated with pyrethroid resistance with genome re-sequencing in An. sinensis. RESULTS: There are 30 putative UGTs in An. sinensis genome, which are classified into 12 families (UGT301, UGT302, UGT306, UGT308, UGT309, UGT310, UGT313, UGT314, UGT315, UGT36, UGT49, UGT50) and further into 23 sub-families. The UGT308 is significantly expanded in gene number compared with other families. A total of 119 UGTs from An. sinensis, An. gambiae, Aedes aegypti and Drosophila melanogaster genomes are classified into 19 families, of which seven are specific for three mosquito species and seven are specific for Drosophila melanogaster. The UGT308 and UGT302 are proposed to main families involved in pyrethroid resistance. The AsUGT308D3 is proposed to be the essential UGT gene for the participation in biotransformation in pyrethroid detoxification process, which is possibly regulated by eight SNPs in its 3' flanking region. The UGT302A3 is also associated with pyrethroid resistance, and four amino acid mutations in its coding sequences might enhance its catalytic activity and further result in higher insecticide resistance. CONCLUSIONS: This study provides the diversity, phylogenetics and evolution of UGT genes, and potential UGT members and mutations involved in pyrethroid resistance in An. sinensis, and lays an important basis for the better understanding and further research on UGT function in defense against insecticide stress.


Assuntos
Anopheles/efeitos dos fármacos , Anopheles/enzimologia , Glicosiltransferases/genética , Resistência a Inseticidas , Inseticidas/farmacologia , Proteínas Mutantes/genética , Piretrinas/farmacologia , Aedes/enzimologia , Aedes/genética , Animais , Anopheles/genética , Biologia Computacional , Drosophila/enzimologia , Drosophila/genética , Feminino , Perfilação da Expressão Gênica , Glicosiltransferases/metabolismo , Proteínas Mutantes/metabolismo , Mutação , Filogenia , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de RNA
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