RESUMO
Reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR) is the most used, fast, and reproducible method to confirm large-scale gene expression data. The use of stable reference genes for the normalization of RT-qPCR assays is recognized worldwide. No systematic study for selecting appropriate reference genes for usage in RT-qPCR experiments comparing gene expression levels at different Schistosoma mansoni life-cycle stages has been performed. Most studies rely on genes commonly used in other organisms, such as actin, tubulin, and GAPDH. Therefore, the present study focused on identifying reference genes suitable for RT-qPCR assays across six S. mansoni developmental stages. The expression levels of 25 novel candidates that we selected based on the analysis of public RNA-Seq datasets, along with eight commonly used reference genes, were systematically tested by RT-qPCR across six developmental stages of S. mansoni (eggs, miracidia, cercariae, schistosomula, adult males and adult females). The stability of genes was evaluated with geNorm, NormFinder and RefFinder algorithms. The least stable candidate reference genes tested were actin, tubulin and GAPDH. The two most stable reference genes suitable for RT-qPCR normalization were Smp_101310 (Histone H4 transcription factor) and Smp_196510 (Ubiquitin recognition factor in ER-associated degradation protein 1). Performance of these two genes as normalizers was successfully evaluated with females maintained unpaired or paired to males in culture for 8 days, or with worm pairs exposed for 16 days to double-stranded RNAs to silence a protein-coding gene. This study provides reliable reference genes for RT-qPCR analysis using samples from six different S. mansoni life-cycle stages.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Reação em Cadeia da Polimerase em Tempo Real/normas , Schistosoma mansoni/genética , Animais , Feminino , Perfilação da Expressão Gênica , Inativação Gênica , Estágios do Ciclo de Vida/genética , Masculino , Fases de Leitura Aberta/genética , Padrões de Referência , Transcriptoma/genéticaRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of COVID-19, is a readily transmissible and potentially deadly pathogen which is currently re-defining human susceptibility to pandemic viruses in the modern world. The recent emergence of several genetically distinct descendants known as variants of concern (VOCs) is further challenging public health disease management, due to increased rates of virus transmission and potential constraints on vaccine effectiveness. We report the isolation of SARS-CoV-2 VOCs imported into Australia belonging to the B.1.351 lineage, first described in the Republic of South Africa (RSA), and the B.1.1.7 lineage originally reported in the United Kingdom, and directly compare the replication kinetics of these two VOCs in Vero E6 cells. In this analysis, we also investigated a B.1.1.7 VOC (QLD1516/2021) carrying a 7-nucleotide deletion in the open reading frame 7a (ORF7a) gene, likely truncating and rendering the ORF7a protein of this virus defective. We demonstrate that the replication of the B.1.351 VOC (QLD1520/2020) in Vero E6 cells can be detected earlier than the B.1.1.7 VOCs (QLD1516/2021 and QLD1517/2021), before peaking at 48 h post infection (p.i.), with significantly higher levels of virus progeny. Whilst replication of the ORF7a defective isolate QLD1516/2021 was delayed longer than the other viruses, slightly more viral progeny was produced by the mutant compared to the unmutated isolate QLD1517/2021 at 72 h p.i. Collectively, these findings contribute to our understanding of SARS-CoV-2 replication and evolutionary dynamics, which have important implications in the development of future vaccination, antiviral therapies, and epidemiological control strategies for COVID-19.
Assuntos
Fases de Leitura Aberta/genética , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , Proteínas Virais/genética , Replicação Viral , Adulto , Animais , Austrália , COVID-19/prevenção & controle , COVID-19/transmissão , COVID-19/virologia , Chlorocebus aethiops , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Cinética , Pessoa de Meia-Idade , Mutação , Nasofaringe/virologia , Filogenia , SARS-CoV-2/classificação , África do Sul , Reino Unido , Células VeroRESUMO
Small open reading frames (smORFs) and their encoded microproteins play central roles in microbes. However, there is a vast unexplored space of smORFs within human-associated microbes. A recent bioinformatic analysis used evolutionary conservation signals to enhance prediction of small protein families. To facilitate the annotation of specific smORFs, we introduce SmORFinder. This tool combines profile hidden Markov models of each smORF family and deep learning models that better generalize to smORF families not seen in the training set, resulting in predictions enriched for Ribo-seq translation signals. Feature importance analysis reveals that the deep learning models learn to identify Shine-Dalgarno sequences, deprioritize the wobble position in each codon, and group codon synonyms found in the codon table. A core-genome analysis of 26 bacterial species identifies several core smORFs of unknown function. We pre-compute smORF annotations for thousands of RefSeq isolate genomes and Human Microbiome Project metagenomes and provide these data through a public web portal.
Assuntos
Bactérias/genética , Genoma Bacteriano , Anotação de Sequência Molecular , Fases de Leitura Aberta , Proteínas de Bactérias/genética , Biologia Computacional , Aprendizado Profundo , Humanos , Cadeias de Markov , Microbiota , Modelos TeóricosRESUMO
The generation of omic resources is central to develop adequate management strategies for species with economic value. Here, we provide high-coverage RNA-seq datasets of liver tissue (containing between 80,2 and 88,4 million of paired-end reads) from four wildtype teleost species with high commercial value: Trachurus trachurus (TTR; Atlantic horse mackerel), Scomber scombrus (SSC; Atlantic mackerel), Trisopterus luscus (TLU; pout), and Micromesistius poutassou (MPO; blue whiting). A comprehensive assembly pipeline, using de novo single and multi-kmer assembly approaches, produced 64 single high-quality liver transcriptomes - 16 per species. The final assemblies, with N50 values ranging from 2,543-3,700 bp and BUSCO (Benchmarking Universal Single-Copy Orthologs) completeness values between 81.8-86.5% of the Actinopterygii gene set, were subjected to open reading frame (ORF) prediction and functional annotation. Our study provides the first transcriptomic resources for these species and offers valuable tools to evaluate both neutral and selected genetic variation among populations, and to identify candidate genes for environmental adaptation assisting in the investigation of the effects of global changes in fisheries.
Assuntos
Peixes/genética , Fígado/metabolismo , Transcriptoma , Animais , Pesqueiros , Variação Genética , Genética Populacional , Fases de Leitura AbertaRESUMO
MOTIVATION: Nanopore long-read sequencing technology offers promising alternatives to high-throughput short read sequencing, especially in the context of RNA-sequencing. However this technology is currently hindered by high error rates in the output data that affect analyses such as the identification of isoforms, exon boundaries, open reading frames and creation of gene catalogues. Due to the novelty of such data, computational methods are still actively being developed and options for the error correction of Nanopore RNA-sequencing long reads remain limited. RESULTS: In this article, we evaluate the extent to which existing long-read DNA error correction methods are capable of correcting cDNA Nanopore reads. We provide an automatic and extensive benchmark tool that not only reports classical error correction metrics but also the effect of correction on gene families, isoform diversity, bias toward the major isoform and splice site detection. We find that long read error correction tools that were originally developed for DNA are also suitable for the correction of Nanopore RNA-sequencing data, especially in terms of increasing base pair accuracy. Yet investigators should be warned that the correction process perturbs gene family sizes and isoform diversity. This work provides guidelines on which (or whether) error correction tools should be used, depending on the application type. BENCHMARKING SOFTWARE: https://gitlab.com/leoisl/LR_EC_analyser.
Assuntos
Nanoporos , Análise de Sequência de RNA/métodos , Software , Éxons , Fases de Leitura AbertaRESUMO
BACKGROUND: A century ago, pantheras were abundant across Asia. Illegal hunting and trading along with loss of habitat have resulted in the designation of Panthera as a genus of endangered species. In addition to the onslaught from humans, pantheras are also susceptible to outbreaks of several infectious diseases, including babesiosis. The latter is a hemoprotozoan disease whose causative agents are the eukaryotic parasites of the apicomplexan genus Babesia. Babesiosis affects a varied range of animals including humans (Homo sapiens), bovines (e.g. Bos taurus), pantheras (e.g. Panthera tigris, P. leo, P. pardus) and equines. Babesia spp. are transmitted by the tick vector Ixodes scapularis or ticks of domestic animals, namely Rhipicephalus (Boophilus) microplus and R. (B.) decoloratus. At the level of protein translation within these organisms, the conserved aminoacyl tRNA synthetase (aaRS) family offers an opportunity to identify the sequence and structural differences in the host (Panthera) and parasites (Babesia spp.) in order to exploit these for drug targeting Babesia spp. METHODS: Using computational tools we investigated the genomes of Babesia spp. and Panthera tigris so as to annotate their aaRSs. The sequences were analysed and their subcellular localizations were predicted using Target P1.1, SignalP 3.0, TMHMM v.2.0 and Deeploc 1.0 web servers. Structure-based analysis of the aaRSs from P. tigris and its protozoan pathogens Babesia spp. was performed using Phyre2 and chimera. RESULTS: We identified 33 (B. bovis), 34 (B. microti), 33 (B. bigemina) and 33 (P. tigris) aaRSs in these respective organisms. Poor sequence identity (~ 20-50%) between aaRSs from Babesia spp. and P. tigris was observed and this merits future experiments to validate new drug targets against Babesia spp. CONCLUSIONS: Overall this work provides a foundation for experimental investigation of druggable aaRSs from Babesia sp. in an effort to control Babesiosis in Panthera.
Assuntos
Aminoacil-tRNA Sintetases/efeitos dos fármacos , Babesia/enzimologia , Babesiose/tratamento farmacológico , Panthera/parasitologia , Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/genética , Animais , Babesia/classificação , Babesia/genética , Babesiose/transmissão , Domínio Catalítico , Biologia Computacional , Sistemas de Liberação de Medicamentos/veterinária , Espécies em Perigo de Extinção , Inibidores Enzimáticos/metabolismo , Genoma de Protozoário , Isocumarinas/metabolismo , Cadeias de Markov , Anotação de Sequência Molecular , Fases de Leitura Aberta , Panthera/genética , Panthera/metabolismo , Alinhamento de Sequência/veterináriaRESUMO
BACKGROUND: Long Adapter Single-Stranded Oligonucleotide (LASSO) probes were developed as a novel tool for massively parallel cloning of kilobase-long genomic DNA sequences. LASSO dramatically improves the capture length limit of current DNA padlock probe technology from approximately 150 bps to several kbps. High-throughput LASSO capture involves the parallel assembly of thousands of probes. However, malformed probes are indiscernible from properly formed probes using gel electrophoretic techniques. Therefore, we used next-generation sequencing (NGS) to assess the efficiency of LASSO probe assembly and how it relates to the nature of DNA capture and amplification. Additionally, we introduce a simplified single target LASSO protocol using classic molecular biology techniques for qualitative and quantitative assessment of probe specificity. RESULTS: A LASSO probe library targeting 3164 unique E. coli ORFs was assembled using two different probe assembly reaction conditions with a 40-fold difference in DNA concentration. Unique probe sequences are located within the first 50 bps of the 5' and 3' ends, therefore we used paired-end NGS to assess probe library quality. Properly mapped read pairs, representing correctly formed probes, accounted for 10.81 and 0.65% of total reads, corresponding to ~ 80% and ~ 20% coverage of the total probe library for the lower and higher DNA concentration conditions, respectively. Subsequently, we used single-end NGS to correlate probe assembly efficiency and capture quality. Significant enrichment of LASSO targets over non-targets was only observed for captures done using probes assembled with a lower DNA concentration. Additionally, semi-quantitative polyacrylamide gel electrophoresis revealed a ~ 10-fold signal-to-noise ratio of LASSO capture in a simplified system. CONCLUSIONS: These results suggest that LASSO probe coverage for target sequences is more predictive of successful capture than probe assembly depth-enrichment. Concomitantly, these results demonstrate that DNA concentration at a critical step in the probe assembly reaction significantly impacts probe formation. Additionally, we show that a simplified LASSO capture protocol coupled to PAGE (polyacrylamide gel electrophoresis) is highly specific and more amenable to small-scale LASSO approaches, such as screening novel probes and templates.
Assuntos
Clonagem Molecular/métodos , Sondas de DNA/genética , DNA de Cadeia Simples/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Oligonucleotídeos/genética , DNA/análise , DNA/genética , Primers do DNA/genética , Eletroforese em Gel de Poliacrilamida/métodos , Proteínas de Escherichia coli/genética , Amplificação de Genes , Biblioteca Gênica , Fases de Leitura Aberta/genética , Reação em Cadeia da Polimerase/métodos , Reprodutibilidade dos TestesRESUMO
The human T cell lymphotropic virus type 1 (HTLV-1) infects 5 to 10 million individuals and remains without specific treatment. This retrovirus genome is composed of the genes gag, pol, env, and a region known as pX. This region contains four open reading frames (ORFs) that encode specific proteins. The ORF-I produces the protein p12 and its cleavage product, p8. In this study, we analyzed the genetic diversity of 32 ORF-I sequences from patients with different clinical profiles. Seven amino acid changes with frequency over 5% were identified: G29S, P34L, L55F, F61L, S63P, F78L, and S91P. The identification of regions where the posttranslational sites were identified showed a high identity among the sequences and the amino acid changes exclusive of specific clinical profile were found in less than 5% of the samples. We compare the findings with 2.406 sequences available in GenBank. The low overall genetic diversity found suggested that this region could be used in the HTLV-1 vaccine development.
Assuntos
Variação Genética , Infecções por HTLV-I/virologia , Vírus Linfotrópico T Tipo 1 Humano/genética , Fases de Leitura Aberta , Proteínas Virais Reguladoras e Acessórias/genética , Infecções Assintomáticas , Bases de Dados de Ácidos Nucleicos , Endocardite/virologia , Humanos , Leucemia-Linfoma de Células T do Adulto/virologia , Mutação , Paraparesia Espástica Tropical/virologiaRESUMO
As specific pathogens of noctuid pests, including Spodoptera exigua, S. litura, Helicoverpa armigera, and Mythimna separata, ascoviruses are suitable for the development of bioinsecticides. In this study, the infectivity of Heliothis virescens ascovirus 3j (HvAV-3j) on insect and mammalian cells was evaluated. HvAV-3j infection induced drastic morphological changes in Sf9, HzAM1, SeFB, and HaFB cells, including swelling and detachment. Notably, the latter phenomena did not occur in HvAV-3j-inoculated mammalian cells (HEK293, 7402, HePG2, PK15, ST, and TM3). MTT assays indicated that HvAV-3j inhibited the growth of host insect cells from the 6th hpi, but no effects were detected in the HvAV-3j-inoculated mammalian cells. Furthermore, viral DNA replication, gene transcription, and protein expression were investigated, and the results consistently suggested that HvAV-3j viruses were not able to replicate their genomic DNA, transcribe, or express their proteins in the non-target vertebrate cells. The HvAV-3j genes were only transcribed and expressed in the four insect cell lines. These results indicated that HvAV-3j was infectious to cells derived from S. frugiperda, S. exigua, H. armigera, and H. zea but not to cells derived from human, pig, and mouse, suggesting that ascoviruses are safe to non-target vertebrate cells.
Assuntos
Ascoviridae/genética , Ascoviridae/fisiologia , Interações entre Hospedeiro e Microrganismos , Replicação Viral , Animais , Replicação do DNA , DNA Viral/genética , Células HEK293 , Humanos , Larva/virologia , Camundongos , Mariposas/virologia , Fases de Leitura Aberta , Filogenia , Medição de Risco , Células Sf9 , Spodoptera/virologia , SuínosRESUMO
We report complete mitochondrial genome of Northern Indian red muntjac, Muntiacus vaginalis, and its phylogenetic inferences. Mitogenome composition was 16,352 bp in length and its overall base composition in the circular genome was A = 33.2%, T = 29.0%, C = 24.50% and G = 13.30%. It exhibited a typical mitogenome structure, including 22 transfer RNA genes, 13 protein-coding genes, two ribosomal RNA genes and a major non-coding control region (D-loop region). All the genes except ND6 and eight tRNA's were encoded on the heavy strand. Phylogenetic analyses showed that M. vaginalis is closely related to M. muntjak and formed a sister relationship with Elaphodus cephalophus. In view of the unclear distribution range and escalating habitat loss, it is important to identify its population genetic status. The complete mitogenome described in this study can be used in further phylogenetics, identification of extant maternal lineage, evolutionary significance unit and its genetic conservation.
Assuntos
Genoma Mitocondrial , Cervo Muntjac/genética , Filogenia , Animais , Teorema de Bayes , Índia , Cadeias de Markov , Método de Monte Carlo , Nucleotídeos/genética , Fases de Leitura Aberta/genética , RNA Ribossômico/genética , RNA de Transferência/genéticaRESUMO
Purpose: To determine the progression rate and the variability of rod and cone sensitivities in patients with X-linked retinitis pigmentosa (XLRP) caused by mutations in ORF15-RPGR. Methods: ORF15-RPGR-XLRP patients (n = 15) were studied prospectively over 2 years with static perimetry sampling the visual field under dark-adapted and light-adapted conditions on a 12° square grid covering 168° width and 84° height. Natural history of rod and cone sensitivity loss and test-retest variability were estimated. Data were analyzed pointwise as well as averaged across small regions of neighboring loci of approximately 80 mm2 (900 deg2) in size representing the likely extent of localized gene therapy injections. Results: Retinal loci with mild to moderate loss of sensitivity tended to be in the mid- to far-peripheral retina in most patients. When averaged across small regions, dark-adapted rod vision progressed at an average of 2 dB per year with a coefficient of repeatability (CR) of 6.3 dB, and light-adapted cone vision with white stimulus progressed at an average of 0.9 dB per year with a CR of 3.8 dB. For an average patient enrolled in an early-phase clinical trial, significant (α = 0.05) progression would be predicted to occur with 80% power in 4.5 years for rod vision and 6.1 years for cone vision. Localization of regions in the temporal hemifield and grouping of results from multiple patients would permit trial designs of shorter duration. Conclusions: Measurement of rod sensitivity under dark-adapted conditions averaged across a small region showed the greatest potential for detectability of progression in the shortest period.
Assuntos
Proteínas do Olho/genética , Doenças Genéticas Ligadas ao Cromossomo X/genética , Mutação , Fases de Leitura Aberta/genética , Retinose Pigmentar/genética , Transtornos da Visão/fisiopatologia , Campos Visuais/fisiologia , Adolescente , Adulto , Adaptação à Escuridão , Progressão da Doença , Eletrorretinografia , Feminino , Doenças Genéticas Ligadas ao Cromossomo X/fisiopatologia , Humanos , Masculino , Células Fotorreceptoras de Vertebrados/fisiologia , Estudos Prospectivos , Retinose Pigmentar/fisiopatologia , Acuidade Visual/fisiologia , Testes de Campo Visual , Adulto JovemRESUMO
Organisms regulate gene expression through changes in the activity of transcription factors (TFs). In yeast, the response of genes to changes in TF activity is generally assumed to be encoded in the promoter. To directly test this assumption, we chose 42 genes and, for each, replaced the promoter with a synthetic inducible promoter and measured how protein expression changes as a function of TF activity. Most genes exhibited gene-specific TF dose-response curves not due to differences in mRNA stability, translation, or protein stability. Instead, most genes have an intrinsic ability to buffer the effects of promoter activity. This can be encoded in the open reading frame and the 3' end of genes and can be implemented by both autoregulatory feedback and by titration of limiting trans regulators. We show experimentally and computationally that, when misexpression of a gene is deleterious, this buffering insulates cells from fitness defects due to misregulation.
Assuntos
Regulação Fúngica da Expressão Gênica , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/genética , Sequência de Bases , Mecanismo Genético de Compensação de Dose , Retroalimentação Fisiológica , Genes Fúngicos , Homeostase , Modelos Genéticos , Fases de Leitura Aberta/genética , Ploidias , Fatores de Transcrição/metabolismoRESUMO
Despite decades of porcine reproductive and respiratory syndrome (PRRS) research, outbreaks with emerging and re-emerging PRRS virus (PRRSV) strains are not uncommon in North America. The role of area spread, commonly referred but not limited to airborne transmission, in originating such outbreaks is currently unknown. The main objective of this study was to explore the role of area spread on the occurrence of new PRRSV cases by combining information on genetic similarity among recovered PRRSV isolate's open-reading frame (ORF) 5 sequences and publicly available weather data. Three small regions were enrolled in the study for which high farm-level participation rate was achieved, and swine sites within those regions were readily sampled after reporting of an outbreak in a sow farm. Oral fluid PCR testing was used to determine PRRSV status of farms, and wind roses were generated for assessment of prevailing wind directions during 2-14 days preceding the outbreak. Under the conditions of this study, the data did not support the area spread theory as the main cause for these outbreaks. We suggest that for future studies, analysis of animal movement and other links between farms such as personnel, equipment and sharing of service providers should be incorporated for better insights on source of the virus. Furthermore, the development of rapid and easy diagnostic methods for ruling out resident PRRSV is urgently needed.
Assuntos
Surtos de Doenças/veterinária , Transmissão de Doença Infecciosa/veterinária , Fazendas , Síndrome Respiratória e Reprodutiva Suína/epidemiologia , Síndrome Respiratória e Reprodutiva Suína/transmissão , Vírus da Síndrome Respiratória e Reprodutiva Suína/isolamento & purificação , Criação de Animais Domésticos/métodos , Animais , Feminino , Fases de Leitura Aberta , Reação em Cadeia da Polimerase/veterinária , Síndrome Respiratória e Reprodutiva Suína/virologia , Suínos , Estados Unidos/epidemiologiaRESUMO
Massive amounts of metagenomics data are currently being produced, and in all such projects a sizeable fraction of the resulting data shows no or little homology to known sequences. It is likely that this fraction contains novel viruses, but identification is challenging since they frequently lack homology to known viruses. To overcome this problem, we developed a strategy to detect ORFan protein families in shotgun metagenomics data, using similarity-based clustering and a set of filters to extract bona fide protein families. We applied this method to 17 virus-enriched libraries originating from human nasopharyngeal aspirates, serum, feces, and cerebrospinal fluid samples. This resulted in 32 predicted putative novel gene families. Some families showed detectable homology to sequences in metagenomics datasets and protein databases after reannotation. Notably, one predicted family matches an ORF from the highly variable Torque Teno virus (TTV). Furthermore, follow-up from a predicted ORFan resulted in the complete reconstruction of a novel circular genome. Its organisation suggests that it most likely corresponds to a novel bacteriophage in the microviridae family, hence it was named bacteriophage HFM.
Assuntos
Genoma Viral , Metagenoma , Metagenômica , Proteínas Virais/genética , Sequência de Bases , Análise por Conglomerados , Biologia Computacional/métodos , Humanos , Cadeias de Markov , Metagenômica/métodos , Anotação de Sequência Molecular , Fases de Leitura AbertaRESUMO
The physiology of a cell can be viewed as the product of thousands of proteins acting in concert to shape the cellular response. Coordination is achieved in part through networks of protein-protein interactions that assemble functionally related proteins into complexes, organelles, and signal transduction pathways. Understanding the architecture of the human proteome has the potential to inform cellular, structural, and evolutionary mechanisms and is critical to elucidating how genome variation contributes to disease. Here we present BioPlex 2.0 (Biophysical Interactions of ORFeome-derived complexes), which uses robust affinity purification-mass spectrometry methodology to elucidate protein interaction networks and co-complexes nucleated by more than 25% of protein-coding genes from the human genome, and constitutes, to our knowledge, the largest such network so far. With more than 56,000 candidate interactions, BioPlex 2.0 contains more than 29,000 previously unknown co-associations and provides functional insights into hundreds of poorly characterized proteins while enhancing network-based analyses of domain associations, subcellular localization, and co-complex formation. Unsupervised Markov clustering of interacting proteins identified more than 1,300 protein communities representing diverse cellular activities. Genes essential for cell fitness are enriched within 53 communities representing central cellular functions. Moreover, we identified 442 communities associated with more than 2,000 disease annotations, placing numerous candidate disease genes into a cellular framework. BioPlex 2.0 exceeds previous experimentally derived interaction networks in depth and breadth, and will be a valuable resource for exploring the biology of incompletely characterized proteins and for elucidating larger-scale patterns of proteome organization.
Assuntos
Bases de Dados de Proteínas , Doença , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas , Proteoma/metabolismo , Fenômenos Fisiológicos Celulares/genética , Genoma Humano , Humanos , Espaço Intracelular/metabolismo , Cadeias de Markov , Espectrometria de Massas , Anotação de Sequência Molecular , Fases de Leitura Aberta , Proteoma/análise , Proteoma/química , Proteoma/genéticaRESUMO
Controlling plant disease has been a struggle for humankind since the advent of agriculture. Studies of plant immune mechanisms have led to strategies of engineering resistant crops through ectopic transcription of plants' own defence genes, such as the master immune regulatory gene NPR1 (ref. 1). However, enhanced resistance obtained through such strategies is often associated with substantial penalties to fitness, making the resulting products undesirable for agricultural applications. To remedy this problem, we sought more stringent mechanisms of expressing defence proteins. On the basis of our latest finding that translation of key immune regulators, such as TBF1 (ref. 3), is rapidly and transiently induced upon pathogen challenge (see accompanying paper), we developed a 'TBF1-cassette' consisting of not only the immune-inducible promoter but also two pathogen-responsive upstream open reading frames (uORFsTBF1) of the TBF1 gene. Here we demonstrate that inclusion of uORFsTBF1-mediated translational control over the production of snc1-1 (an autoactivated immune receptor) in Arabidopsis thaliana and AtNPR1 in rice enables us to engineer broad-spectrum disease resistance without compromising plant fitness in the laboratory or in the field. This broadly applicable strategy may lead to decreased pesticide use and reduce the selective pressure for resistant pathogens.
Assuntos
Regulação da Expressão Gênica de Plantas , Aptidão Genética/genética , Fases de Leitura Aberta/genética , Doenças das Plantas/genética , Doenças das Plantas/imunologia , Imunidade Vegetal/genética , Biossíntese de Proteínas , Arabidopsis/genética , Arabidopsis/imunologia , Proteínas de Arabidopsis/biossíntese , Proteínas de Arabidopsis/genética , Produtos Agrícolas/genética , Produtos Agrícolas/imunologia , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Fatores de Transcrição de Choque Térmico , Proteínas de Choque Térmico/biossíntese , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/imunologia , Oryza/genética , Oryza/imunologia , Proteínas de Plantas/biossíntese , Proteínas de Plantas/genética , Proteínas de Plantas/imunologia , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Fatores de Transcrição/imunologia , Transcrição GênicaRESUMO
Staphylococcus phages of the Myoviridae family have a wide host range and potential applications in phage therapy. In this report, safety assessments of these phages were conducted based on their complete genome sequences. The complete genomes of Staphylococcus phages of the Myoviridae family were analyzed, and the Open Reading Frame (ORFs) were compared with a pool of virulence and antibiotic resistance genes using the BLAST algorithm. In addition, the lifestyle of the phages (virulent or temperate) was also confirmed using PHACTS. The results showed that all phages were lytic and did not contain resistance or virulence genes based on bioinformatic analyses, excluding the possibility that they could be vectors for the dissemination of these undesirable genes. These findings suggest that the phages are safe at the genome level. The SceD-like transglycosylase, which is a biomarker for vancomycin-intermediate strains, was widely distributed in the phage genomes. Approximately 70% of the ORFs encoded in the phage genomes have unknown functions; therefore, their roles in the antibiotic resistance and virulence of Staphylococcus aureus are still unknown and require consideration before use in phage therapy.
Assuntos
Genoma Viral , Myoviridae/genética , Fagos de Staphylococcus/genética , Sequência de Bases , Análise por Conglomerados , Myoviridae/patogenicidade , Myoviridae/fisiologia , Fases de Leitura Aberta/genética , Filogenia , Fagos de Staphylococcus/patogenicidade , Fagos de Staphylococcus/fisiologia , Virulência/genéticaRESUMO
Ziziphus jujuba is an important woody plant with high economic and medicinal value. Here, we analyzed and characterized the complete chloroplast (cp) genome of Z. jujuba, the first member of the Rhamnaceae family for which the chloroplast genome sequence has been reported. We also built a web browser for navigating the cp genome of Z. jujuba ( http://bio.njfu.edu.cn/gb2/gbrowse/Ziziphus_jujuba_cp/ ). Sequence analysis showed that this cp genome is 161,466 bp long and has a typical quadripartite structure of large (LSC, 89,120 bp) and small (SSC, 19,348 bp) single-copy regions separated by a pair of inverted repeats (IRs, 26,499 bp). The sequence contained 112 unique genes, including 78 protein-coding genes, 30 transfer RNAs, and four ribosomal RNAs. The genome structure, gene order, GC content, and codon usage are similar to other typical angiosperm cp genomes. A total of 38 tandem repeats, two forward repeats, and three palindromic repeats were detected in the Z. jujuba cp genome. Simple sequence repeat (SSR) analysis revealed that most SSRs were AT-rich. The homopolymer regions in the cp genome of Z. jujuba were verified and manually corrected by Sanger sequencing. One-third of mononucleotide repeats were found to be erroneously sequenced by the 454 pyrosequencing, which resulted in sequences of 1-4 bases shorter than that by the Sanger sequencing. Analyzing the cp genome of Z. jujuba revealed that the IR contraction and expansion events resulted in ycf1 and rps19 pseudogenes. A phylogenetic analysis based on 64 protein-coding genes showed that Z. jujuba was closely related to members of the Elaeagnaceae family, which will be helpful for phylogenetic studies of other Rosales species. The complete cp genome sequence of Z. jujuba will facilitate population, phylogenetic, and cp genetic engineering studies of this economic plant.
Assuntos
Genoma de Cloroplastos , Análise de Sequência de DNA , Ziziphus/genética , Composição de Bases , Códon , Biologia Computacional/métodos , Evolução Molecular , Ordem dos Genes , Genes de Plantas , Repetições de Microssatélites , Anotação de Sequência Molecular , Fases de Leitura Aberta , Filogenia , Sequências Repetitivas de Ácido Nucleico , Ziziphus/classificaçãoRESUMO
Structural birth defects are a leading cause of mortality and morbidity in children world-wide, affecting as much as 6% of all live births. Among these conditions, neural tube defects (NTDs), including spina bifida and anencephaly, arise from a combination of complex gene and environment interactions that are as yet poorly understood within human populations. Rapid advances in massively parallel DNA sequencing and bioinformatics allow for analyses of the entire genome beyond the 2% of the genomic sequence covering protein coding regions. Efforts to collect and analyze these large datasets hold promise for illuminating gene network variations and eventually epigenetic events that increase individual risk for failure to close the neural tube. In this review, we discuss current challenges for DNA genome sequence analysis of NTD affected populations, and compare experience in the field with other complex genetic disorders for which large datasets are accumulating. The ultimate goal of this research is to find strategies for optimizing conditions that promote healthy birth outcomes for individual couples. Birth Defects Research 109:120-128, 2017. © 2016 Wiley Periodicals, Inc.