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1.
Nat Commun ; 11(1): 4079, 2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32796936

RESUMO

DNA methylation is an epigenetic modification that specifies the basic state of pluripotent stem cells and regulates the developmental transition from stem cells to various cell types. In flowering plants, the shoot apical meristem (SAM) contains a pluripotent stem cell population which generates the aerial part of plants including the germ cells. Under appropriate conditions, the SAM undergoes a developmental transition from a leaf-forming vegetative SAM to an inflorescence- and flower-forming reproductive SAM. While SAM characteristics are largely altered in this transition, the complete picture of DNA methylation remains elusive. Here, by analyzing whole-genome DNA methylation of isolated rice SAMs in the vegetative and reproductive stages, we show that methylation at CHH sites is kept high, particularly at transposable elements (TEs), in the vegetative SAM relative to the differentiated leaf, and increases in the reproductive SAM via the RNA-dependent DNA methylation pathway. We also show that half of the TEs that were highly methylated in gametes had already undergone CHH hypermethylation in the SAM. Our results indicate that changes in DNA methylation begin in the SAM long before germ cell differentiation to protect the genome from harmful TEs.


Assuntos
Metilação de DNA , Meristema/crescimento & desenvolvimento , Meristema/genética , Oryza/genética , Brotos de Planta/crescimento & desenvolvimento , Brotos de Planta/genética , Elementos de DNA Transponíveis , Biologia do Desenvolvimento , Epigenômica , Flores , Regulação da Expressão Gênica de Plantas , Inflorescência , Folhas de Planta/metabolismo , Proteínas de Plantas/genética
2.
Nat Commun ; 11(1): 4058, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792480

RESUMO

Tomatoes come in a multitude of shapes and flavors despite a narrow genetic pool. Here, we leverage whole-genome resequencing data available for 602 cultivated and wild accessions to determine the contribution of transposable elements (TEs) to tomato diversity. We identify 6,906 TE insertions polymorphisms (TIPs), which result from the mobilization of 337 distinct TE families. Most TIPs are low frequency variants and TIPs are disproportionately located within or adjacent to genes involved in environmental responses. In addition, genic TE insertions tend to have strong transcriptional effects and they can notably lead to the generation of multiple transcript isoforms. Using genome-wide association studies (GWAS), we identify at least 40 TIPs robustly associated with extreme variation in major agronomic traits or secondary metabolites and in most cases, no SNP tags the TE insertion allele. Collectively, these findings highlight the unique role of TE mobilization in tomato diversification, with important implications for breeding.


Assuntos
Elementos de DNA Transponíveis/genética , Lycopersicon esculentum/genética , Evolução Molecular , Genoma de Planta/genética , Estudo de Associação Genômica Ampla , Polimorfismo Genético/genética
3.
J Med Microbiol ; 69(8): 1089-1094, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32692646

RESUMO

Introduction. The bla CTX-M-3 gene has rarely been reported in Morganella morganii strains and its genetic environment has not yet been investigated.Aim. To identify the bla CTX-M-3 gene in M. morganii isolated from swine and characterize its genetic environment.Methodology. A M. morganii isolate (named MM1L5) from a deceased swine was identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and subjected to antimicrobial susceptibility testing. The bla genes were detected and then the genetic location and environment of bla CTX-M-3 were investigated by Southern blot and PCR mapping, respectively. The M. morganii bla CTX-M-3 gene was cloned and expressed in Escherichia coli.Results. Isolate MM1L5 harboured the bla CTX-M-3 and bla TEM-1 genes. The bla CTX-M-3 gene, located on the chromosome, was co-carried with an IS26 and bla TEM-1 gene by a novel 6361 bp IS26-flanked composite transposon, designated Tn6741. This transposon consisted of a novel bla CTX-M-3-containing module, IS26-ΔISEcp1-bla CTX-M-3-Δorf477-IS26 (named Tn6710), and a bla TEM-1-containing module, IS26-Δorf477-bla TEM-1-tnpR-IS26, differing from previous reports. Phylogenetic analysis showed a significant variation based on the sequence of Tn6741, as compared to those of other related transposons. Interestingly, although the cloned bla CTX-M-3 gene could confer resistance to ceftiofur, cefquinome, ceftriaxone and cefotaxime, one amino acid substitution (Ile-142-Thr) resulted in a significant reduction of resistance to these antimicrobials.Conclusion. This is the first time that bla CTX-M-3 has been identified on a chromosome from a M. morganii isolate. Furthermore, the bla CTX-M-3 gene was located with an IS26 element and bla TEM-1 gene on a novel IS26-flanked composite transposon, Tn6741, suggesting that Tn6741 might act as a reservoir for the bla CTX-M-3 and bla TEM-1 genes and may become an important vehicle for their dissemination among M. morganii.


Assuntos
Elementos de DNA Transponíveis/genética , Morganella morganii/genética , beta-Lactamases/genética , Animais , Anti-Infecciosos/farmacologia , Sequência de Bases , Clonagem Molecular , Morganella morganii/classificação , Morganella morganii/efeitos dos fármacos , Filogenia , Reação em Cadeia da Polimerase , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Suínos
4.
Nat Protoc ; 15(8): 2705-2727, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32681154

RESUMO

Invasive fungal infections caused by Candida species are life threatening with high mortality, posing a severe public health threat. New technologies for rapid, genome-wide identification of virulence genes and therapeutic targets are urgently needed. Our recent engineering of a piggyBac (PB) transposon-mediated mutagenesis system in haploid Candida albicans provides a powerful discovery tool, which we anticipate should be adaptable to other haploid Candida species. In this protocol, we use haploid C. albicans as an example to present an improved version of the mutagenesis system and provide a detailed description of the protocol for constructing high-quality mutant libraries. We also describe a method for quantitative PB insertion site sequencing, PBISeq. The PBISeq library preparation procedure exploits tagmentation to quickly and efficiently construct sequencing libraries. Finally, we present a pipeline to analyze PB insertion sites in a de novo assembled genome of our engineered haploid C. albicans strain. The entire protocol takes ~7 d from transposition induction to having a final library ready for sequencing. This protocol is highly efficient and less labor intensive than alternative approaches and significantly accelerates genetic studies of Candida.


Assuntos
Candida/genética , Elementos de DNA Transponíveis/genética , Genoma Fúngico/genética , Haploidia , Mutagênese Insercional/métodos
5.
PLoS Genet ; 16(7): e1008931, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32644999

RESUMO

Shigella species are specialised lineages of Escherichia coli that have converged to become human-adapted and cause dysentery by invading human gut epithelial cells. Most studies of Shigella evolution have been restricted to comparisons of single representatives of each species; and population genomic studies of individual Shigella species have focused on genomic variation caused by single nucleotide variants and ignored the contribution of insertion sequences (IS) which are highly prevalent in Shigella genomes. Here, we investigate the distribution and evolutionary dynamics of IS within populations of Shigella dysenteriae Sd1, Shigella sonnei and Shigella flexneri. We find that five IS (IS1, IS2, IS4, IS600 and IS911) have undergone expansion in all Shigella species, creating substantial strain-to-strain variation within each population and contributing to convergent patterns of functional gene loss within and between species. We find that IS expansion and genome degradation are most advanced in S. dysenteriae and least advanced in S. sonnei; and using genome-scale models of metabolism we show that Shigella species display convergent loss of core E. coli metabolic capabilities, with S. sonnei and S. flexneri following a similar trajectory of metabolic streamlining to that of S. dysenteriae. This study highlights the importance of IS to the evolution of Shigella and provides a framework for the investigation of IS dynamics and metabolic reduction in other bacterial species.


Assuntos
Elementos de DNA Transponíveis/genética , Disenteria/genética , Evolução Molecular , Shigella dysenteriae/genética , DNA Bacteriano/genética , Disenteria/microbiologia , Escherichia coli/genética , Escherichia coli/patogenicidade , Genoma Bacteriano/genética , Humanos , Shigella dysenteriae/patogenicidade
6.
PLoS Genet ; 16(7): e1008872, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32673310

RESUMO

Transposable elements (TEs) are genomic parasites that selfishly replicate at the expense of host fitness. Fifty years of evolutionary studies of TEs have concentrated on the deleterious genetic effects of TEs, such as their effects on disrupting genes and regulatory sequences. However, a flurry of recent work suggests that there is another important source of TEs' harmful effects-epigenetic silencing. Host genomes typically silence TEs by the deposition of repressive epigenetic marks. While this silencing reduces the selfish replication of TEs and should benefit hosts, a picture is emerging that the epigenetic silencing of TEs triggers inadvertent spreading of repressive marks to otherwise expressed neighboring genes, ultimately jeopardizing host fitness. In this Review, we provide a long-overdue overview of the recent genome-wide evidence for the presence and prevalence of TEs' epigenetic effects, highlighting both the similarities and differences across mammals, insects, and plants. We lay out the current understanding of the functional and fitness consequences of TEs' epigenetic effects, and propose possible influences of such effects on the evolution of both hosts and TEs themselves. These unique evolutionary consequences indicate that TEs' epigenetic effect is not only a crucial component of TE biology but could also be a significant contributor to genome function and evolution.


Assuntos
Elementos de DNA Transponíveis/genética , Epigênese Genética , Evolução Molecular , Inativação Gênica , Animais , Repressão Epigenética/genética , Regulação da Expressão Gênica/genética , Insetos/genética , Mamíferos/genética , Plantas/genética
7.
Nat Commun ; 11(1): 3739, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32719317

RESUMO

The PIWI protein MIWI2 and its associated PIWI-interacting RNAs (piRNAs) instruct DNA methylation of young active transposable elements (TEs) in the male germline. piRNAs are proposed to recruit MIWI2 to the transcriptionally active TE loci by base pairing to nascent transcripts, however the downstream mechanisms and effector proteins utilized by MIWI2 in directing de novo TE methylation remain incompletely understood. Here, we show that MIWI2 associates with TEX15 in foetal gonocytes. TEX15 is predominantly a nuclear protein that is not required for piRNA biogenesis but is essential for piRNA-directed TE de novo methylation and silencing. In summary, TEX15 is an essential executor of mammalian piRNA-directed DNA methylation.


Assuntos
Proteínas Argonauta/metabolismo , Proteínas de Ciclo Celular/metabolismo , Metilação de DNA/genética , Elementos de DNA Transponíveis/genética , Inativação Gênica , Animais , Proteínas Argonauta/genética , Feminino , Feto/citologia , Genoma , Células Germinativas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , Testículo/metabolismo
8.
Nat Commun ; 11(1): 3446, 2020 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-32651359

RESUMO

The piggyBac DNA transposon is used widely in genome engineering applications. Unlike other transposons, its excision site can be precisely repaired without leaving footprints and it integrates specifically at TTAA tetranucleotides. We present cryo-EM structures of piggyBac transpososomes: a synaptic complex with hairpin DNA intermediates and a strand transfer complex capturing the integration step. The results show that the excised TTAA hairpin intermediate and the TTAA target adopt essentially identical conformations, providing a mechanistic link connecting the two unique properties of piggyBac. The transposase forms an asymmetric dimer in which the two central domains synapse the ends while two C-terminal domains form a separate dimer that contacts only one transposon end. In the strand transfer structure, target DNA is severely bent and the TTAA target is unpaired. In-cell data suggest that asymmetry promotes synaptic complex formation, and modifying ends with additional transposase binding sites stimulates activity.


Assuntos
Elementos de DNA Transponíveis/genética , Transposases/metabolismo , Microscopia Crioeletrônica , Humanos , Espectroscopia de Ressonância Magnética , Ligação Proteica , Estrutura Secundária de Proteína , Transposases/genética
9.
Nat Commun ; 11(1): 3506, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32665538

RESUMO

Acute myeloid leukemia (AML) is characterised by a series of genetic and epigenetic alterations that result in deregulation of transcriptional networks. One understudied source of transcriptional regulators are transposable elements (TEs), whose aberrant usage could contribute to oncogenic transcriptional circuits. However, the regulatory influence of TEs and their links to AML pathogenesis remain unexplored. Here we identify six endogenous retrovirus (ERV) families with AML-associated enhancer chromatin signatures that are enriched in binding of key regulators of hematopoiesis and AML pathogenesis. Using both locus-specific genetic editing and simultaneous epigenetic silencing of multiple ERVs, we demonstrate that ERV deregulation directly alters the expression of adjacent genes in AML. Strikingly, deletion or epigenetic silencing of an ERV-derived enhancer suppresses cell growth by inducing apoptosis in leukemia cell lines. This work reveals that ERVs are a previously unappreciated source of AML enhancers that may be exploited by cancer cells to help drive tumour heterogeneity and evolution.


Assuntos
Cromatina/metabolismo , Leucemia Mieloide Aguda/genética , Animais , Linhagem Celular , Cromatina/genética , Elementos de DNA Transponíveis/genética , Retrovirus Endógenos/genética , Epigênese Genética/genética , Redes Reguladoras de Genes/genética , Redes Reguladoras de Genes/fisiologia , Genoma Humano/genética , Humanos , Sequências Repetitivas Dispersas/genética
10.
PLoS Comput Biol ; 16(7): e1007980, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32678849

RESUMO

Bacteria need to survive in a wide range of environments. Currently, there is an incomplete understanding of the genetic basis for mechanisms underpinning survival in stressful conditions, such as the presence of anti-microbials. Transposon directed insertion-site sequencing (TraDIS) is a powerful tool to identify genes and networks which are involved in survival and fitness under a given condition by simultaneously assaying the fitness of millions of mutants, thereby relating genotype to phenotype and contributing to an understanding of bacterial cell biology. A recent refinement of this approach allows the roles of essential genes in conditional stress survival to be inferred by altering their expression. These advancements combined with the rapidly falling costs of sequencing now allows comparisons between multiple experiments to identify commonalities in stress responses to different conditions. This capacity however poses a new challenge for analysis of multiple data sets in conjunction. To address this analysis need, we have developed 'AlbaTraDIS'; a software application for rapid large-scale comparative analysis of TraDIS experiments that predicts the impact of transposon insertions on nearby genes. AlbaTraDIS can identify genes which are up or down regulated, or inactivated, between multiple conditions, producing a filtered list of genes for further experimental validation as well as several accompanying data visualisations. We demonstrate the utility of our new approach by applying it to identify genes used by Escherichia coli to survive in a wide range of different concentrations of the biocide Triclosan. AlbaTraDIS identified all well characterised Triclosan resistance genes, including the primary target, fabI. A number of new loci were also implicated in Triclosan resistance and the predicted phenotypes for a selection of these were validated experimentally with results being consistent with predictions. AlbaTraDIS provides a simple and rapid method to analyse multiple transposon mutagenesis data sets allowing this technology to be used at large scale. To our knowledge this is the only tool currently available that can perform these tasks. AlbaTraDIS is written in Python 3 and is available under the open source licence GNU GPL 3 from https://github.com/quadram-institute-bioscience/albatradis.


Assuntos
Biologia Computacional , Elementos de DNA Transponíveis , Escherichia coli/genética , Mutagênese Insercional , Software , Algoritmos , Anti-Infecciosos/farmacologia , Farmacorresistência Bacteriana , Biblioteca Gênica , Genes Essenciais , Genoma Bacteriano , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Mutação , Fenótipo , Biossíntese de Proteínas , Triclosan/farmacologia
11.
Science ; 368(6495)2020 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-32499410

RESUMO

Strecker et al (Research Articles, 5 July 2019, p. 48) described a system for exploiting a Tn7-type transposon-encoded CRISPR-Cas system to make RNA-guided, programmable insertions. Although this system has great promise, we note that the well-established biochemistry of Tn7 suggests that the particular system used may insert not only the transposon but also the entire donor plasmid.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Transposases , Sistemas CRISPR-Cas , Elementos de DNA Transponíveis , RNA
12.
Science ; 368(6495)2020 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-32499411

RESUMO

Rice et al suggest that the CRISPR-associated transposase ShCAST system could lead to additional insertion products beyond simple integration of the donor. We clarify the outcomes of ShCAST-mediated insertions in Escherichia coli, which consist of both simple insertions and integration of the donor plasmid. This latter outcome can be avoided by use of a 5' nicked DNA donor.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Transposases , Elementos de DNA Transponíveis , Plasmídeos , RNA
13.
PLoS Genet ; 16(6): e1008861, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32525870

RESUMO

In metazoan germlines, the piRNA pathway acts as a genomic immune system, employing small RNA-mediated silencing to defend host DNA from the harmful effects of transposable elements (TEs). Expression of genomic TEs is proposed to initiate self regulation by increasing the production of repressive piRNAs, thereby "adapting" piRNA-mediated control to the most active TE families. Surprisingly, however, piRNA pathway proteins, which execute piRNA biogenesis and enforce silencing of targeted sequences, evolve rapidly and adaptively in animals. If TE silencing is ensured through piRNA biogenesis, what necessitates changes in piRNA pathway proteins? Here we used interspecific complementation to test for functional differences between Drosophila melanogaster and D. simulans alleles of three adaptively evolving piRNA pathway proteins: Armitage, Aubergine and Spindle-E. In contrast to piRNA-mediated transcriptional regulators examined in previous studies, these three proteins have cytoplasmic functions in piRNA maturation and post-transcriptional silencing. Across all three proteins we observed interspecific divergence in the regulation of only a handful of TE families, which were more robustly silenced by the heterospecific piRNA pathway protein. This unexpected result suggests that unlike transcriptional regulators, positive selection has not acted on cytoplasmic piRNA effector proteins to enhance their function in TE repression. Rather, TEs may evolve to "escape" silencing by host proteins. We further discovered that D. simulans alleles of aub and armi exhibit enhanced off-target effects on host transcripts in a D. melanogaster background, as well as modest reductions in the efficiency of piRNA biogenesis, suggesting that promiscuous binding of D. simulans Aub and Armi proteins to host transcripts reduces their participation in piRNA production. Avoidance of genomic auto-immunity may therefore be a critical target of selection. Our observations suggest that piRNA effector proteins are subject to an evolutionary trade-off between defending the host genome from the harmful effect of TEs while also minimizing collateral damage to host genes.


Assuntos
Autoimunidade/genética , Elementos de DNA Transponíveis/imunologia , Drosophila simulans/genética , Evolução Molecular , Genoma de Inseto/imunologia , RNA Interferente Pequeno/biossíntese , Alelos , Animais , Animais Geneticamente Modificados , Citoplasma/genética , Citoplasma/metabolismo , Elementos de DNA Transponíveis/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/imunologia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/imunologia , Drosophila melanogaster/metabolismo , Drosophila simulans/metabolismo , Feminino , Regulação da Expressão Gênica/imunologia , Genoma de Inseto/genética , Masculino , Mutação , Interferência de RNA/imunologia
14.
PLoS Genet ; 16(6): e1008864, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32584820

RESUMO

Cytosine methylation is an ancient epigenetic modification yet its function and extent within genomes is highly variable across eukaryotes. In mammals, methylation controls transposable elements and regulates the promoters of genes. In insects, DNA methylation is generally restricted to a small subset of transcribed genes, with both intergenic regions and transposable elements (TEs) depleted of methylation. The evolutionary origin and the function of these methylation patterns are poorly understood. Here we characterise the evolution of DNA methylation across the arthropod phylum. While the common ancestor of the arthropods had low levels of TE methylation and did not methylate promoters, both of these functions have evolved independently in centipedes and mealybugs. In contrast, methylation of the exons of a subset of transcribed genes is ancestral and widely conserved across the phylum, but has been lost in specific lineages. A similar set of genes is methylated in all species that retained exon-enriched methylation. We show that these genes have characteristic patterns of expression correlating to broad transcription initiation sites and well-positioned nucleosomes, providing new insights into potential mechanisms driving methylation patterns over hundreds of millions of years.


Assuntos
Artrópodes/genética , Metilação de DNA , Epigênese Genética , Evolução Molecular , Animais , Ilhas de CpG/genética , Elementos de DNA Transponíveis/genética , Éxons/genética , Filogenia , Regiões Promotoras Genéticas/genética
15.
Nat Commun ; 11(1): 3256, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32591500

RESUMO

BRCA1 mutation carriers have a higher risk of developing triple-negative breast cancer (TNBC), which is a refractory disease due to its non-responsiveness to current clinical targeted therapies. Using the Sleeping Beauty transposon system in Brca1-deficient mice, we identified 169 putative cancer drivers, among which Notch1 is a top candidate for accelerating TNBC by promoting the epithelial-mesenchymal transition (EMT) and regulating the cell cycle. Activation of NOTCH1 suppresses mitotic catastrophe caused by BRCA1 deficiency by restoring S/G2 and G2/M cell cycle checkpoints, which may through activation of ATR-CHK1 signalling pathway. Consistently, analysis of human breast cancer tissue demonstrates NOTCH1 is highly expressed in TNBCs, and the activated form of NOTCH1 correlates positively with increased phosphorylation of ATR. Additionally, we demonstrate that inhibition of the NOTCH1-ATR-CHK1 cascade together with cisplatin synergistically kills TNBC by targeting the cell cycle checkpoint, DNA damage and EMT, providing a potent clinical option for this fatal disease.


Assuntos
Proteína BRCA1/deficiência , Carcinogênese/patologia , Receptor Notch1/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Alelos , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/metabolismo , Morte Celular , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem/metabolismo , Elementos de DNA Transponíveis/genética , Progressão da Doença , Transição Epitelial-Mesenquimal , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos Knockout , Mitose , Mutação/genética , Transdução de Sinais , Neoplasias de Mama Triplo Negativas/genética
16.
Nat Commun ; 11(1): 3224, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32591528

RESUMO

In plants, epigenetic regulation is critical for silencing transposons and maintaining proper gene expression. However, its impact on the genome-wide transcription initiation landscape remains elusive. By conducting a genome-wide analysis of transcription start sites (TSSs) using cap analysis of gene expression (CAGE) sequencing, we show that thousands of TSSs are exclusively activated in various epigenetic mutants of Arabidopsis thaliana and referred to as cryptic TSSs. Many have not been identified in previous studies, of which up to 65% are contributed by transposons. They possess similar genetic features to regular TSSs and their activation is strongly associated with the ectopic recruitment of RNAPII machinery. The activation of cryptic TSSs significantly alters transcription of nearby TSSs, including those of genes important for development and stress responses. Our study, therefore, sheds light on the role of epigenetic regulation in maintaining proper gene functions in plants by suppressing transcription from cryptic TSSs.


Assuntos
Arabidopsis/genética , Epigênese Genética , Regulação da Expressão Gênica de Plantas , Transcrição Genética , Sequência de Bases , Sequência Consenso/genética , Metilação de DNA/genética , DNA Polimerase beta/metabolismo , Elementos de DNA Transponíveis/genética , Genes de Plantas , Mutação/genética , RNA Polimerase II/metabolismo , Sítio de Iniciação de Transcrição , Transcriptoma/genética
17.
PLoS Genet ; 16(6): e1008894, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32598340

RESUMO

Meiotic crossovers (COs) are important for reshuffling genetic information between homologous chromosomes and they are essential for their correct segregation. COs are unevenly distributed along chromosomes and the underlying mechanisms controlling CO localization are not well understood. We previously showed that meiotic COs are mis-localized in the absence of AXR1, an enzyme involved in the neddylation/rubylation protein modification pathway in Arabidopsis thaliana. Here, we report that in axr1-/-, male meiocytes show a strong defect in chromosome pairing whereas the formation of the telomere bouquet is not affected. COs are also redistributed towards subtelomeric chromosomal ends where they frequently form clusters, in contrast to large central regions depleted in recombination. The CO suppressed regions correlate with DNA hypermethylation of transposable elements (TEs) in the CHH context in axr1-/- meiocytes. Through examining somatic methylomes, we found axr1-/- affects DNA methylation in a plant, causing hypermethylation in all sequence contexts (CG, CHG and CHH) in TEs. Impairment of the main pathways involved in DNA methylation is epistatic over axr1-/- for DNA methylation in somatic cells but does not restore regular chromosome segregation during meiosis. Collectively, our findings reveal that the neddylation pathway not only regulates hormonal perception and CO distribution but is also, directly or indirectly, a major limiting pathway of TE DNA methylation in somatic cells.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Cromossomos de Plantas/genética , Metilação de DNA , Meiose/genética , Proteínas de Arabidopsis/genética , Pareamento Cromossômico , Segregação de Cromossomos , Troca Genética , Quebras de DNA de Cadeia Dupla , Elementos de DNA Transponíveis/genética , Técnicas de Inativação de Genes , Plantas Geneticamente Modificadas
18.
PLoS Genet ; 16(6): e1008822, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32497040

RESUMO

Insecticide resistance in malaria vectors threatens to reverse recent gains in malaria control. Deciphering patterns of gene flow and resistance evolution in malaria vectors is crucial to improving control strategies and preventing malaria resurgence. A genome-wide survey of Anopheles funestus genetic diversity Africa-wide revealed evidences of a major division between southern Africa and elsewhere, associated with different population histories. Three genomic regions exhibited strong signatures of selective sweeps, each spanning major resistance loci (CYP6P9a/b, GSTe2 and CYP9K1). However, a sharp regional contrast was observed between populations correlating with gene flow barriers. Signatures of complex molecular evolution of resistance were detected with evidence of copy number variation, transposon insertion and a gene conversion between CYP6P9a/b paralog genes. Temporal analyses of samples before and after bed net scale up suggest that these genomic changes are driven by this control intervention. Multiple independent selective sweeps at the same locus in different parts of Africa suggests that local evolution of resistance in malaria vectors may be a greater threat than trans-regional spread of resistance haplotypes.


Assuntos
Anopheles/genética , Evolução Molecular , Genoma de Inseto/genética , Resistência a Inseticidas/genética , Malária/prevenção & controle , Mosquitos Vetores/genética , África , Alelos , Animais , Anopheles/parasitologia , Família 6 do Citocromo P450/genética , Variações do Número de Cópias de DNA , Elementos de DNA Transponíveis/genética , Fluxo Gênico , Loci Gênicos , Haplótipos , Humanos , Proteínas de Insetos/genética , Malária/parasitologia , Malária/transmissão , Metagenômica , Controle de Mosquitos/métodos , Polimorfismo Genético , Piretrinas , Sequenciamento Completo do Genoma
19.
J Med Microbiol ; 69(7): 979-985, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32579099

RESUMO

Introduction. Childhood tuberculosis meningitis is a severe form of tuberculosis with high morbidity and mortality. The diagnosis is frequently missed and delayed due to lack of sensitive tests like acid-fast bacilli (AFB) smear and delayed results by culture.Aims. To compare the role of IS6110 and protein antigen b PCR in cerebrospinal fluid (CSF) for rapid diagnosis of tuberculous meningitis (TBM) in children.Methodology. Forty-five cases of TBM and 20 controls were enrolled in this prospective study.Results. The mean ages of cases and controls were 4.2±0.5 years and 4.5±0.7 years, respectively. In the TBM group, two-thirds of the children were <4 years of age, and 62 % were males. Sensitivities of AFB smear examination, Löwenstein-Jensen (LJ) medium and bactenecin (BACTEC) culture in cases were 4.4, 0 and 2.2%, respectively. The protein antigen b PCR was most sensitive as it was positive in 35 (77.8 %) of TBM patients; IS6110 PCR was positive in 27 (60 %) patients. Both PCR-based tests had higher positivity than conventional tests and BACTEC culture. No significant difference was seen between the PCR tests. Excellent agreement was observed between both PCR-based tests as they were concordant for 26 positive samples and 35 negative samples.Conclusion. Protein b PCR is a sensitive and rapid method for the diagnosis of TBM (sensitivity 77.8 %). Both PCRs were more sensitive than smear, LJ and BACTEC. The specificity of both PCR was 100 %.


Assuntos
Mycobacterium tuberculosis/genética , Reação em Cadeia da Polimerase/métodos , Tuberculose Meníngea/diagnóstico , Antígenos de Bactérias/genética , Líquido Cefalorraquidiano , Pré-Escolar , Elementos de DNA Transponíveis/genética , DNA Bacteriano , Feminino , Humanos , Masculino , Estudos Prospectivos , Sensibilidade e Especificidade , Tuberculose Meníngea/líquido cefalorraquidiano
20.
PLoS Negl Trop Dis ; 14(6): e0008051, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32569298

RESUMO

BACKGROUND: In Japan, Buruli ulcer cases are often advanced, requiring surgical treatment. However, extensive debridement is often difficult because of cosmetic and functional sequelae. Moreover, the lesions are complicated and composed of edematous erythema, necrotic ulcer, and erythematous skin lesions caused by a paradoxical reaction, which also make it difficult to perform adequate debridement. METHODOLOGY/PRINCIPAL FINDINGS: We performed quantitative polymerase chain reaction (PCR) analysis for IS2404 using 29 samples taken from mapping biopsy. We evaluated the relationship among mycobacterial burden, histopathological findings, and clinical outcomes using 83 tissue samples taken from mapping biopsy and debrided Buruli ulcer. On quantitative PCR, the Cp values of IS2404 amplification were substantially different in each site. The major histological findings could be divided into massive subcutaneous necrosis with scant inflammatory cell infiltration and dense inflammatory cell infiltration. Of the 84 sites, 34 were subjected to repeated histological evaluations. In these sites, histological necrosis did not disappear over time despite standard antibiotic treatment. In contrast, the ulcers were cured and no recurrences were observed without resecting the 11 biopsied sites that lacked histological necrosis. Although quantitative PCR revealed that a lower Cp value of IS2404 was associated with histological massive necrosis, sites that showed lower Cp values clinically did not always need debridement. CONCLUSION/SIGNIFICANCE: Our descriptive study revealed that the histological findings and amounts of mycobacterial DNA differed according to the sites despite being found in one lesion. Our results showed that the need for surgical debridement in each site was correlated with histological necrosis without inflammatory cell infiltration, as the inflammation is supposed to represent an active host immune response rather than mycobacterial burden. We suggest that the debridement of lesions with histological necrosis in mapping biopsy may be useful for Japanese cases with unsuccessful standard antibiotic treatment to achieve sufficient clinical improvement.


Assuntos
Carga Bacteriana , Úlcera de Buruli/microbiologia , Úlcera de Buruli/patologia , Histocitoquímica , Mycobacterium ulcerans/isolamento & purificação , Adulto , Biópsia , Elementos de DNA Transponíveis , DNA Bacteriano/análise , DNA Bacteriano/genética , Humanos , Japão , Masculino , Mycobacterium ulcerans/genética , Reação em Cadeia da Polimerase em Tempo Real , Adulto Jovem
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