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1.
Artigo em Inglês | MEDLINE | ID: mdl-33906706

RESUMO

Three strains (YZ01T, YZ02 and YZ03) of Gram-stain-positive, facultatively anaerobic rods were isolated from the forestomach contents collected from a captive male proboscis monkey (Nasalis larvatus) at Yokohama Zoo in Japan. Phylogenetic analysis of the 16S rRNA gene sequences revealed that these strains belonged to the genus Lactobacillus. Based on the sequence similarity of the 16S rRNA gene, Lactobacillus delbrueckii subsp. indicus JCM 15610T was the closest phylogenetic neighbour to YZ01T. Sequence analyses of two partial concatenated housekeeping genes, the RNA polymerase alpha subunit (rpoA) and phenylalanyl-tRNA synthase alpha subunit (pheS) also indicated that the novel strains belonged to the genus Lactobacillus. The average nucleotide identity and digital DNA-DNA hybridization (dDDH) between L. delbrueckii subsp. indicus and YZ01T were 85.9 and 31.4 %, respectively. The phylogenetic tree based on the whole genomic data of strains YZ01T, YZ02 and YZ03 suggested that these three strains formed a single monophyletic cluster in the genus Lactobacillus, indicating that it belonged to a new species. The DNA G+C content of strain YZ01T was 51.6 mol%. The major fatty acids were C16 : 0 and C18 : 1 ω9c. Therefore, based on phylogenetic, phenotypic and physiological evidence, strains YZ01T, YZ02 and YZ03 represent a novel species of the genus Lactobacillus, for which the name Lactobacillus nasalidis sp. nov. is proposed with the type strain YZ01T (=JCM 33769T=DSM 110539T).


Assuntos
Lactobacillus/classificação , Filogenia , Presbytini/microbiologia , Estômago/microbiologia , Animais , Animais de Zoológico/microbiologia , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , RNA Polimerases Dirigidas por DNA/genética , Ácidos Graxos/química , Genes Bacterianos , Japão , Lactobacillus/isolamento & purificação , Hibridização de Ácido Nucleico , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
2.
BMC Bioinformatics ; 22(1): 210, 2021 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-33888055

RESUMO

BACKGROUND: Mutations in an enzyme target are one of the most common mechanisms whereby antibiotic resistance arises. Identification of the resistance mutations in bacteria is essential for understanding the structural basis of antibiotic resistance and design of new drugs. However, the traditionally used experimental approaches to identify resistance mutations were usually labor-intensive and costly. RESULTS: We present a machine learning (ML)-based classifier for predicting rifampicin (Rif) resistance mutations in bacterial RNA Polymerase subunit ß (RpoB). A total of 186 mutations were gathered from the literature for developing the classifier, using 80% of the data as the training set and the rest as the test set. The features of the mutated RpoB and their binding energies with Rif were calculated through computational methods, and used as the mutation attributes for modeling. Classifiers based on five ML algorithms, i.e. decision tree, k nearest neighbors, naïve Bayes, probabilistic neural network and support vector machine, were first built, and a majority consensus (MC) approach was then used to obtain a new classifier based on the classifications of the five individual ML algorithms. The MC classifier comprehensively improved the predictive performance, with accuracy, F-measure and AUC of 0.78, 0.83 and 0.81for training set whilst 0.84, 0.87 and 0.83 for test set, respectively. CONCLUSION: The MC classifier provides an alternative methodology for rapid identification of resistance mutations in bacteria, which may help with early detection of antibiotic resistance and new drug discovery.


Assuntos
RNA Bacteriano , Rifampina , Bactérias , Teorema de Bayes , Consenso , RNA Polimerases Dirigidas por DNA/genética , Farmacorresistência Bacteriana/genética , Mutação , Rifampina/farmacologia
3.
Int J Mol Sci ; 22(6)2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-33804237

RESUMO

Ataxia in children is a common clinical sign of numerous neurological disorders consisting of impaired coordination of voluntary muscle movement. Its most common form, cerebellar ataxia, describes a heterogeneous array of neurologic conditions with uncountable causes broadly divided as acquired or genetic. Numerous genetic disorders are associated with chronic progressive ataxia, which complicates clinical management, particularly on the diagnostic stage. Advances in omics technologies enable improvements in clinical practice and research, so we proposed a multi-omics approach to aid in the genetic diagnosis and molecular elucidation of an undiagnosed infantile condition of chronic progressive cerebellar ataxia. Using whole-exome sequencing, RNA-seq, and untargeted metabolomics, we identified three clinically relevant mutations (rs141471029, rs191582628 and rs398124292) and an altered metabolic profile in our patient. Two POLR1C diagnostic variants already classified as pathogenic were found, and a diagnosis of hypomyelinating leukodystrophy was achieved. A mutation on the MMACHC gene, known to be associated with methylmalonic aciduria and homocystinuria cblC type, was also found. Additionally, preliminary metabolome analysis revealed alterations in our patient's amino acid, fatty acid and carbohydrate metabolism. Our findings provided a definitive genetic diagnosis reinforcing the association between POLR1C mutations and hypomyelinating leukodystrophy and highlighted the relevance of multi-omics approaches to the disease.


Assuntos
Ataxia Cerebelar/diagnóstico , RNA Polimerases Dirigidas por DNA/genética , Genoma/genética , Oxirredutases/genética , Transcriptoma/genética , Adolescente , Adulto , Ataxia Cerebelar/genética , Ataxia Cerebelar/patologia , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Metaboloma/genética , Mutação/genética , Linhagem , RNA-Seq , Deficiência de Vitamina B 12/genética , Sequenciamento Completo do Exoma/métodos , Adulto Jovem
4.
Int J Mol Sci ; 22(5)2021 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-33673662

RESUMO

Two different molecular mechanisms, sliding and hopping, are employed by DNA-binding proteins for their one-dimensional facilitated diffusion on nonspecific DNA regions until reaching their specific target sequences. While it has been controversial whether RNA polymerases (RNAPs) use one-dimensional diffusion in targeting their promoters for transcription initiation, two recent single-molecule studies discovered that post-terminational RNAPs use one-dimensional diffusion for their reinitiation on the same DNA molecules. Escherichia coli RNAP, after synthesizing and releasing product RNA at intrinsic termination, mostly remains bound on DNA and diffuses in both forward and backward directions for recycling, which facilitates reinitiation on nearby promoters. However, it has remained unsolved which mechanism of one-dimensional diffusion is employed by recycling RNAP between termination and reinitiation. Single-molecule fluorescence measurements in this study reveal that post-terminational RNAPs undergo hopping diffusion during recycling on DNA, as their one-dimensional diffusion coefficients increase with rising salt concentrations. We additionally find that reinitiation can occur on promoters positioned in sense and antisense orientations with comparable efficiencies, so reinitiation efficiency depends primarily on distance rather than direction of recycling diffusion. This additional finding confirms that orientation change or flipping of RNAP with respect to DNA efficiently occurs as expected from hopping diffusion.


Assuntos
DNA Bacteriano/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Iniciação da Transcrição Genética , Terminação da Transcrição Genética , DNA Bacteriano/genética , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Regiões Promotoras Genéticas
5.
Nat Commun ; 12(1): 906, 2021 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-33568644

RESUMO

Promoter-proximal pausing regulates eukaryotic gene expression and serves as checkpoints to assemble elongation/splicing machinery. Little is known how broadly this type of pausing regulates transcription in bacteria. We apply nascent elongating transcript sequencing combined with RNase I footprinting for genome-wide analysis of σ70-dependent transcription pauses in Escherichia coli. Retention of σ70 induces strong backtracked pauses at a 10-20-bp distance from many promoters. The pauses in the 10-15-bp register of the promoter are dictated by the canonical -10 element, 6-7 nt spacer and "YR+1Y" motif centered at the transcription start site. The promoters for the pauses in the 16-20-bp register contain an additional -10-like sequence recognized by σ70. Our in vitro analysis reveals that DNA scrunching is involved in these pauses relieved by Gre cleavage factors. The genes coding for transcription factors are enriched in these pauses, suggesting that σ70 and Gre proteins regulate transcription in response to changing environmental cues.


Assuntos
RNA Polimerases Dirigidas por DNA/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , RNA Bacteriano/genética , Fator sigma/genética , Fatores de Transcrição/metabolismo , Transcrição Genética , Fatores de Elongação da Transcrição/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Regiões Promotoras Genéticas , RNA Bacteriano/metabolismo , Análise de Sequência de RNA , Fator sigma/metabolismo , Fatores de Transcrição/genética , Fatores de Elongação da Transcrição/genética
6.
Viruses ; 13(2)2021 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-33572652

RESUMO

Rabies virus (RABV) causes fatal neurological encephalitis and results in approximately 6000 human death cases worldwide every year. The large (L) protein of RABV, possessing conserved domains, is considered as the target for detection. In this study, three monoclonal antibodies (mAbs), designated as 3F3, 3A6 and L-C, against L protein were generated by using the recombinant truncated L protein (aa 1431-1754) and the epitopes were also identified using a series of overlapping truncated polypeptides for testing the reactivity of mAbs with different RABV strains. The 1479EIFSIP1484, 1659RALSK1663 and 1724VFNSL1728 were identified as the minimal linear epitopes recognized by mAbs 3F3, 3A6 and L-C, respectively. Amino acid alignment showed epitope 1724VFNSL1728 recognized by mAb L-C is completely conserved among RABV strains, indicating that mAb L-C could be used to detect all of the RABV strains. Epitope 1479EIFSIP1484 is highly conserved among RABV strains except for a P1484S substitution in a China I sub-lineage strain of Asian lineage, which eliminated the reactivity of the epitope with mAb 3F3. However, the epitope 1659RALSK1663 was only completely conserved in the Africa-2 and Indian lineages, and a single A1660T substitution, mainly appeared in strains of the China I belonging to Asian lineage and a Cosmopolitan lineage strain, still retained the reactivity of the epitope with mAb 3A6. While both A1660T and K1663R substitutions in a China I lineage strain, single K1663R/Q substitution in some China II strains of Asian lineage and some Arctic-like lineage strains and R1659Q mutation in a strain of Africa-3 lineage eliminated the reactivity of the epitope with mAb 3A6, suggesting mAb 3A6 could be used for differentiation of variable epitopes of some strains in different lineages. Thus, variability and conservation of the three epitopes of L protein showed the reactive difference of mAbs among RABV strains of different lineages. These results may facilitate future studies in development of detection methods for RABV infection, the structure and function of RABV L protein.


Assuntos
Anticorpos Monoclonais/análise , RNA Polimerases Dirigidas por DNA/imunologia , Epitopos/imunologia , Vírus da Raiva/imunologia , Raiva/virologia , Proteínas Virais/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/genética , Mapeamento de Epitopos , Epitopos/química , Epitopos/genética , Humanos , Filogenia , Vírus da Raiva/química , Vírus da Raiva/classificação , Vírus da Raiva/genética , Alinhamento de Sequência , Proteínas Virais/química , Proteínas Virais/genética
7.
Biochem Biophys Res Commun ; 545: 98-104, 2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33548630

RESUMO

A large class of bacterial RNA polymerase (RNAP) from low-G + C-content Gram-positive bacterial strains, such as the major human pathogen Staphylococcus aureus, not only contain five conserved subunits (αI, αII, ß, ß' and ω), but also has a δ subunit. Despite being first identified as unique, Gram-positive specific component of RNAP apoenzyme more than 30 years ago and reported to be essential for transcription, the structural basis and molecular mechanism of δ subunit in the regulation of transcription remain poorly understood. Here, we performed structural analyses, site-directed mutagenesis and biochemical assays to uncover the interactions of S. aureus δ subunit with RNAP core enzyme and DNA towards the understanding of its role in transcription regulation. Microscale thermophoresis (MST) and electrophoretic mobility shift assay (EMSA) of the wild-type and mutated S. aureus δ subunit revealed the N-terminal domain of δ subunit directly binds to the ß' jaw of S. aureus RNAP (SauRNAP), identified the key amino acid residues (F58, D61, D65, R67 and W81) of δ subunit involving in the binding with SauRNAP core enzyme, and uncovered the δ subunit C-terminal domain interferes with the interaction between DNA and SauRNAP core enzyme, by which transcription is regulated. Our results provide an excellent starting point for understanding the unique regulatory role and physiological function of δ subunit on transcription regulation in Gram-positive bacteria.


Assuntos
Proteínas de Bactérias/química , RNA Polimerases Dirigidas por DNA/química , Staphylococcus aureus/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Genes Bacterianos , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas , Homologia de Sequência de Aminoácidos , Staphylococcus aureus/genética , Transcrição Genética
8.
Nat Commun ; 12(1): 1135, 2021 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-33602924

RESUMO

While >300 disease-causing variants have been identified in the mitochondrial DNA (mtDNA) polymerase γ, no mitochondrial phenotypes have been associated with POLRMT, the RNA polymerase responsible for transcription of the mitochondrial genome. Here, we characterise the clinical and molecular nature of POLRMT variants in eight individuals from seven unrelated families. Patients present with global developmental delay, hypotonia, short stature, and speech/intellectual disability in childhood; one subject displayed an indolent progressive external ophthalmoplegia phenotype. Massive parallel sequencing of all subjects identifies recessive and dominant variants in the POLRMT gene. Patient fibroblasts have a defect in mitochondrial mRNA synthesis, but no mtDNA deletions or copy number abnormalities. The in vitro characterisation of the recombinant POLRMT mutants reveals variable, but deleterious effects on mitochondrial transcription. Together, our in vivo and in vitro functional studies of POLRMT variants establish defective mitochondrial transcription as an important disease mechanism.


Assuntos
RNA Polimerases Dirigidas por DNA/genética , Mitocôndrias/genética , Mutação/genética , Doenças do Sistema Nervoso/genética , Transcrição Genética , Adolescente , Adulto , Criança , DNA Mitocondrial/genética , RNA Polimerases Dirigidas por DNA/química , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Lactente , Masculino , Doenças do Sistema Nervoso/patologia , Fosforilação Oxidativa , Linhagem , Domínios Proteicos , Subunidades Proteicas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Adulto Jovem
9.
Nat Commun ; 12(1): 796, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33542236

RESUMO

RNA polymerases (RNAPs) synthesize RNA from NTPs, whereas DNA polymerases synthesize DNA from 2'dNTPs. DNA polymerases select against NTPs by using steric gates to exclude the 2'OH, but RNAPs have to employ alternative selection strategies. In single-subunit RNAPs, a conserved Tyr residue discriminates against 2'dNTPs, whereas selectivity mechanisms of multi-subunit RNAPs remain hitherto unknown. Here, we show that a conserved Arg residue uses a two-pronged strategy to select against 2'dNTPs in multi-subunit RNAPs. The conserved Arg interacts with the 2'OH group to promote NTP binding, but selectively inhibits incorporation of 2'dNTPs by interacting with their 3'OH group to favor the catalytically-inert 2'-endo conformation of the deoxyribose moiety. This deformative action is an elegant example of an active selection against a substrate that is a substructure of the correct substrate. Our findings provide important insights into the evolutionary origins of biopolymers and the design of selective inhibitors of viral RNAPs.


Assuntos
Proteínas de Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Desoxirribonucleotídeos/metabolismo , Desoxirribose/metabolismo , Arginina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/ultraestrutura , Cristalografia por Raios X , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/isolamento & purificação , RNA Polimerases Dirigidas por DNA/ultraestrutura , Escherichia coli/enzimologia , Escherichia coli/genética , Cinética , Simulação de Acoplamento Molecular , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Especificidade por Substrato , Thermus thermophilus/enzimologia , Thermus thermophilus/genética
10.
Arch Virol ; 166(2): 627-632, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33423081

RESUMO

Enterovirus 71 (EV71) is a causative agent of hand, foot and, mouth disease (HFMD) in young children. It is valuable for virologists to develop a fast method to rescue infectious virus from a viral cDNA clone. Here, we report a method for rapid rescue of infectious EV71 by using cells expressing T7 polymerase. The full-length EV71 genome was amplified in one step by long-distance PCR with a T7 promoter at the 5' end, and the T7 polymerase gene was cloned into a lentivirus vector for construction of a stable cell line expressing T7 polymerase. The infectious virus was rapidly and efficiently rescued by single transfection of cells with the infectious cDNA clone. Further experiments showed that the rescued virus had characteristics similar to those of the parental virus. This method circumvented the difficulty in performing in vitro transcription of a long linear DNA to obtain high-quality RNA. The construction of the viral cDNA clone and the fast rescue of the infectious virus will greatly benefit future investigations.


Assuntos
Clonagem Molecular/métodos , DNA Complementar/genética , RNA Polimerases Dirigidas por DNA/genética , Enterovirus Humano A/genética , Transfecção/métodos , Proteínas Virais/genética , Viroses/genética , Linhagem Celular , Genoma Viral/genética , Humanos , Lentivirus/genética , Reação em Cadeia da Polimerase/métodos , Regiões Promotoras Genéticas/genética , RNA Viral/genética
11.
Proc Natl Acad Sci U S A ; 118(1)2021 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-33443179

RESUMO

RNA polymerase (RNAP) encounters various roadblocks during transcription. These obstacles can impede RNAP movement and influence transcription, ultimately necessitating the activity of RNAP-associated factors. One such factor is the bacterial protein Mfd, a highly conserved DNA translocase and evolvability factor that interacts with RNAP. Although Mfd is thought to function primarily in the repair of DNA lesions that stall RNAP, increasing evidence suggests that it may also be important for transcription regulation. However, this is yet to be fully characterized. To shed light on Mfd's in vivo functions, we identified the chromosomal regions where it associates. We analyzed Mfd's impact on RNAP association and transcription regulation genome-wide. We found that Mfd represses RNAP association at many chromosomal regions. We found that these regions show increased RNAP pausing, suggesting that they are hard to transcribe. Interestingly, we noticed that the majority of the regions where Mfd regulates transcription contain highly structured regulatory RNAs. The RNAs identified regulate a myriad of biological processes, ranging from metabolism to transfer RNA regulation to toxin-antitoxin (TA) functions. We found that cells lacking Mfd are highly sensitive to toxin overexpression. Finally, we found that Mfd promotes mutagenesis in at least one toxin gene, suggesting that its function in regulating transcription may promote evolution of certain TA systems and other regions containing strong RNA secondary structures. We conclude that Mfd is an RNAP cofactor that is important, and at times critical, for transcription regulation at hard-to-transcribe regions, especially those that express structured regulatory RNAs.


Assuntos
Proteínas de Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética/fisiologia , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , DNA/metabolismo , Reparo do DNA/genética , Reparo do DNA/fisiologia , DNA Bacteriano/genética , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/metabolismo , RNA/metabolismo , Fatores de Transcrição/genética , Transcrição Genética/genética
12.
J Med Microbiol ; 70(3)2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33507146

RESUMO

Introduction. Resistance to rifampin (RIF) in Mycobacterium tuberculosis infection is associated with mutations in the rpoB gene coding for the ß-subunit of RNA polymerase. The contribution of various rpoB mutations to the development and level of RIF resistance remains elusive.Hypothesis/Gap Statement. Various rpoB mutations may be associated with differential levels of RIF resistance.Aim. This study aimed to investigate the relationship between specific rpoB mutations and the MICs of RIF and rifabutin (RFB) against M. tuberculosis.Methodology. Of the 195 clinical isolates, 105 and 90 isolates were randomly selected from isolates resistant to RIF and sensitive to RIF, respectively. The MICs of 12 agents for M. tuberculosis isolates were determined using commercial Sensititre M. tuberculosis MIC plates and the broth microdilution method. Strains were screened for rpoB mutations by DNA extraction, rpoB gene amplification and DNA sequence analysis.Results. One hundred isolates (95.24 %) were found to have mutations in the RIF-resistance-determining region (RRDR) of the rpoB gene. Three rpoB mutations were identified in 90 RIF-susceptible isolates. Out of 105 isolates, 86 (81.90 %) were cross-resistant to both RIF and RFB. The most frequent mutation occurred at codons 450 and 445. We also found a novel nine-nucleotide (ATCATGCAT) deletion (between positions 1543 and 1551) in the rpoB gene in two strains (1.90 %) with resistance to RIF, but susceptibility to RFB. In addition, the mutation frequency at codon 450 was significantly higher in RIF-resistant/RFB-resistant (RIFR/RFBR) strains than in RIFR/RFBS strains (75.58 % versus 21.05 %, P<0.01), whereas the mutation frequency at codon 435 was significantly lower in RIFR/RFBR strains than in RIFR/RFBS strains (1.16 % versus 26.32 %, P<0.01).Conclusion. Our data support previous findings, which reported that various rpoB mutations are associated with differential levels of RIF resistance. The specific mutations in the rpoB gene in RIFR/RFBR isolates differed from those in the RIFR/RFBS isolates. A novel deletion mutation in the RRDR might be associated with resistance to RIF, but not to RFB. Further clinical studies are required to investigate the efficacy of RFB in the treatment of infections caused by M. tuberculosis strains harbouring these mutations.


Assuntos
Antibióticos Antituberculose/farmacologia , Proteínas de Bactérias/genética , RNA Polimerases Dirigidas por DNA/genética , Farmacorresistência Bacteriana/genética , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Rifampina/farmacologia , China , Humanos , Testes de Sensibilidade Microbiana , Mutação , Mycobacterium tuberculosis/isolamento & purificação , Rifabutina/farmacologia , Tuberculose/microbiologia
13.
Int J Mol Sci ; 22(2)2021 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-33430258

RESUMO

Plants are in continuous conflict with the environmental constraints and their sessile nature demands a fine-tuned, well-designed defense mechanism that can cope with a multitude of biotic and abiotic assaults. Therefore, plants have developed innate immunity, R-gene-mediated resistance, and systemic acquired resistance to ensure their survival. Transcription factors (TFs) are among the most important genetic components for the regulation of gene expression and several other biological processes. They bind to specific sequences in the DNA called transcription factor binding sites (TFBSs) that are present in the regulatory regions of genes. Depending on the environmental conditions, TFs can either enhance or suppress transcriptional processes. In the last couple of decades, nitric oxide (NO) emerged as a crucial molecule for signaling and regulating biological processes. Here, we have overviewed the plant defense system, the role of TFs in mediating the defense response, and that how NO can manipulate transcriptional changes including direct post-translational modifications of TFs. We also propose that NO might regulate gene expression by regulating the recruitment of RNA polymerase during transcription.


Assuntos
Resistência à Doença/genética , Óxido Nítrico/genética , Doenças das Plantas/genética , Fatores de Transcrição/genética , RNA Polimerases Dirigidas por DNA/genética , Regulação da Expressão Gênica de Plantas/genética , Óxido Nítrico/metabolismo , Transcrição Genética/genética
14.
PLoS Negl Trop Dis ; 14(12): e0008888, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33373362

RESUMO

Brucella spp. are facultative intracellular pathogens that can persistently colonize host cells and cause the zoonosis- brucellosis. The WHO recommended a treatment for brucellosis that involves a combination of doxycycline, rifampicin, or streptomycin. The aim of this study was to screen rifampicin-resistance related genes by transcriptomic analysis and gene recombination method at low rifampicin concentrations and to predict the major rifampicin- resistance pathways in Brucella spp. The results showed that the MIC value of rifampicin for B. melitensis bv.3 Ether was 0.5 µg / mL. Meanwhile, B. melitensis had an adaptive response to the resistance of low rifampicin in the early stages of growth, while the SNPs changed in the rpoB gene in the late stages of growth when incubated at 37°C with shaking. The transcriptome results of rifampicin induction showed that the functions of significant differentially expressed genes were focused on metabolic process, catalytic activity and membrane and membrane part. The VirB operon, ß-resistance genes, ABC transporters, quorum-sensing genes, DNA repair- and replication -related genes were associated with rifampicin resistance when no variations of the in rpoB were detected. Among the VirB operons, VirB7-11 may play a central role in rifampicin resistance. This study provided new insights for screening rifampicin resistance-related genes and also provided basic data for the prevention and control of rifampicin-resistant Brucella isolates.


Assuntos
Antibióticos Antituberculose/farmacologia , Brucella melitensis/efeitos dos fármacos , Rifampina/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Membrana Celular , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Farmacorresistência Bacteriana , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Testes de Sensibilidade Microbiana , Rifampina/administração & dosagem
15.
PLoS One ; 15(12): e0244482, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33370358

RESUMO

Harmful algal blooms are commonly thought to be dominated by a single genus, but they are not homogenous communities. Current approaches, both molecular and culture-based, often overlook fine-scale variations in community composition that can influence bloom dynamics. We combined homology-based searches (BLASTX) and phylogenetics to distinguish and quantify Microcystis host and phage members across a summer season during a 2014 Microcystis- dominated bloom that occurred in Lake Tai (Taihu), China. We found 47 different genotypes of the Microcystis-specific DNA-dependent RNA polymerase (rpoB), which included several morphospecies. Microcystis flos-aquae and Microcystis wesenbergii accounted for ~86% of total Microcystis transcripts, while the more commonly studied Microcystis aeruginosa only accounted for ~7%. Microcystis genotypes were classified into three temporal groups according to their expression patterns across the course of the bloom: early, constant and late. All Microcystis morphospecies were present in each group, indicating that expression patterns were likely dictated by competition driven by environmental factors, not phylogeny. We identified three primary Microcystis-infecting phages based on the viral terminase, including a novel Siphoviridae phage that may be capable of lysogeny. Within our dataset, Myoviridae phages consistent with those infecting Microcystis in a lytic manner were positively correlated to the early host genotypes, while the Siphoviridae phages were positively correlated to the late host genotypes, when the Myoviridae phages express putative genetic markers for lysogeny. The expression of genes in the microcystin-encoding mcy cassette was estimated using mcyA, which revealed 24 Microcystis-specific genotypes that were negatively correlated to the early host genotypes. Of all environmental factors measured, pH best described the temporal shift in the Microcystis community genotypic composition, promoting hypotheses regarding carbon concentration mechanisms and oxidative stress. Our work expounds on the complexity of HAB events, using a well-studied dataset to highlight the need for increased resolution of community dynamics.


Assuntos
Proliferação Nociva de Algas , Lagos/microbiologia , Microcystis/genética , Siphoviridae/genética , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , China , DNA Bacteriano/genética , RNA Polimerases Dirigidas por DNA/genética , Conjuntos de Dados como Assunto , Variação Genética , Lisogenia , Microcistinas/genética , Microcystis/virologia , Filogenia , Homologia de Sequência do Ácido Nucleico
16.
Int J Mol Sci ; 22(1)2020 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-33374812

RESUMO

5-Hydroxymethylcytosine (5hmC) is a functionally active epigenetic modification. We analyzed whether changes in DNA 5-hydroxymethylation are an element of age-related epigenetic drift. We tested primary fibroblast cultures originating from individuals aged 22-35 years and 74-94 years. Global quantities of methylation-related DNA modifications were estimated by the dot blot and colorimetric methods. Regions of the genome differentially hydroxymethylated with age (DHMRs) were identified by hMeDIP-seq and the MEDIPS and DiffBind algorithms. Global levels of DNA modifications were not associated with age. We identified numerous DHMRs that were enriched within introns and intergenic regions and most commonly associated with the H3K4me1 histone mark, promoter-flanking regions, and CCCTC-binding factor (CTCF) binding sites. However, only seven DHMRs were identified by both algorithms and all of their settings. Among them, hypo-hydroxymethylated DHMR in the intron of Rab Escort Protein 1 (CHM) coexisted with increased expression in old cells, while increased 5-hydroxymethylation in the bodies of Arginine and Serine Rich Protein 1 (RSRP1) and Mitochondrial Poly(A) Polymerase (MTPAP) did not change their expression. These age-related differences were not associated with changes in the expression of any of the ten-eleven translocation (TET) enzymes or their activity. In conclusion, the distribution of 5hmC in DNA of in vivo aged human fibroblasts underwent age-associated modifications. The identified DHMRs are, likely, marker changes.


Assuntos
5-Metilcitosina/análogos & derivados , Metilação de DNA , Envelhecimento da Pele/genética , 5-Metilcitosina/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Fator de Ligação a CCCTC/genética , Fator de Ligação a CCCTC/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Feminino , Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Histonas/genética , Histonas/metabolismo , Humanos , Masculino , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Regiões Promotoras Genéticas
17.
Nat Commun ; 11(1): 6409, 2020 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-33335104

RESUMO

In eukaryotes, RNA Polymerase (Pol) III is specialized for the transcription of tRNAs and other short, untranslated RNAs. Pol III is a determinant of cellular growth and lifespan across eukaryotes. Upregulation of Pol III transcription is observed in cancer and causative Pol III mutations have been described in neurodevelopmental disorders and hypersensitivity to viral infection. Here, we report a cryo-EM reconstruction at 4.0 Å of human Pol III, allowing mapping and rationalization of reported genetic mutations. Mutations causing neurodevelopmental defects cluster in hotspots affecting Pol III stability and/or biogenesis, whereas mutations affecting viral sensing are located in proximity to DNA binding regions, suggesting an impairment of Pol III cytosolic viral DNA-sensing. Integrating x-ray crystallography and SAXS, we also describe the structure of the higher eukaryote specific RPC5 C-terminal extension. Surprisingly, experiments in living cells highlight a role for this module in the assembly and stability of human Pol III.


Assuntos
RNA Polimerase III/química , Microscopia Crioeletrônica , RNA Polimerases Dirigidas por DNA/genética , Estabilidade Enzimática , Células HeLa , Humanos , Modelos Moleculares , Mutação , Conformação Proteica , Subunidades Proteicas , RNA Polimerase III/genética , RNA Polimerase III/metabolismo , Espalhamento a Baixo Ângulo , Difração de Raios X
18.
PLoS Genet ; 16(12): e1009243, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33320854

RESUMO

Paramutations represent directed and meiotically-heritable changes in gene regulation leading to apparent violations of Mendelian inheritance. Although the mechanism and evolutionary importance of paramutation behaviors remain largely unknown, genetic screens in maize (Zea mays) identify five components affecting 24 nucleotide RNA biogenesis as required to maintain repression of a paramutant purple plant1 (pl1) allele. Currently, the RNA polymerase IV largest subunit represents the only component also specifying proper development. Here we identify a chromodomain helicase DNA-binding 3 (CHD3) protein orthologous to Arabidopsis (Arabidopsis thaliana) PICKLE as another component maintaining both pl1 paramutation and normal somatic development but without affecting overall small RNA biogenesis. In addition, genetic tests show this protein contributes to proper male gametophyte function. The similar mutant phenotypes documented in Arabidopsis and maize implicate some evolutionarily-conserved gene regulation while developmental defects associated with the two paramutation mutants are largely distinct. Our results show that a CHD3 protein responsible for normal plant ontogeny and sperm transmission also helps maintain meiotically-heritable epigenetic regulatory variation for specific alleles. This finding implicates an intersection of RNA polymerase IV function and nucleosome positioning in the paramutation process.


Assuntos
Montagem e Desmontagem da Cromatina/genética , DNA Helicases/metabolismo , Proteínas de Plantas/metabolismo , Pólen/metabolismo , Zea mays/genética , Alelos , Proteínas de Arabidopsis/genética , DNA Helicases/genética , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Epigênese Genética , Regulação da Expressão Gênica de Plantas , Genótipo , Mutação , Fenótipo , Filogenia , Proteínas de Plantas/genética , Pólen/genética , RNA de Plantas/genética , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismo , Zea mays/crescimento & desenvolvimento , Zea mays/metabolismo
19.
Viruses ; 13(1)2020 12 27.
Artigo em Inglês | MEDLINE | ID: mdl-33375489

RESUMO

Severe fever with thrombocytopenia syndrome virus subclone B7 shows strong plaque formation and cytopathic effect induction compared with other subclones and the parental strain YG1. Compared to YG1 and the other subclones, only B7 possesses a single substitution in the L protein at the amino acid position 1891, in which N is changed to K (N1891K). In this study, we evaluate the effects of this mutation on L protein activity via a cell-based minigenome assay. Substitutions of N with basic amino acids (K or R) enhanced polymerase activity, while substitutions with an acidic amino acid (E) decreased this activity. Mutation to other neutral amino acids showed no significant effect on activity. These results suggest that the characteristic of the amino acid at position 1891 of the L protein are critical for its function, especially with respect to the charge status. Our data indicate that this C-terminal domain of the L protein may be crucial to its functions in genome transcription and viral replication.


Assuntos
Aminoácidos , RNA Polimerases Dirigidas por DNA/metabolismo , Phlebovirus/fisiologia , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Aminoácidos/química , Animais , Linhagem Celular , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/genética , Imunofluorescência , Humanos , Modelos Moleculares , Conformação Proteica , Proteínas Virais/química , Proteínas Virais/genética
20.
BMC Infect Dis ; 20(1): 791, 2020 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-33096996

RESUMO

BACKGROUND: Tuberculosis (TB) remains a major public health concern in many low-income countries accounting for approximately two-thirds of deaths in people living with human immunodeficiency virus (HIV) infection. With prompt, accurate and appropriate treatment, almost all TB disease can be cured. The present study was to evaluate the diagnostic performance of an in-house duplex PCR (D-PCR) using IS1610 and rpoB specific primers in sputum samples from TB suspected patients. METHODS: A hospital-based cross-sectional study was conducted at the Limbe and Buea Regional Hospitals of the South West Region of Cameroon from June 2016 to April 2017. Sputum samples, decontaminated with hypertonic saline/sodium hydroxide solution were centrifuged and pellets processed for smear microscopy, culture and DNA extraction. Suspected inhibition was resolved by serial dilution of genomic DNA. Results were compared to culture as gold standard as well as a Composite Reference Standard (CRS). RESULTS: A total of 129 participants aged between 5 to 82 years were enrolled in to the study. The median age of the participants was 37 years (interquartile range, IQR: 27-50 years), with 54.3% being male. Forty-seven samples (36.4%) were positive by direct sputum microscopy, 49 (38%) by microscopy after concentration, 51 (39.5%) by culture and 62 (40.1%) by D-PCR. PCR inhibition was resolved in 85.7% (18/21) of the samples that had inhibition. The overall sensitivity, specificity, positive and negative predictive values, positive and negative likelihood ratios and area under the curve AUC) of the D-PCR was 93.5, 94, 94, 94%, 15.6, 0.005 and 89.0% respectively using CRS as reference. The sensitivities of D-PCR observed among different sample categories were 95.7, 87.5 and 87.5% for smear-and culture-positives, smear-negative/culture-positive, and clinically diagnosed cases respectively. CONCLUSION: IS1610 and rpoB duplex PCR using relatively cheap decontamination and DNA extraction methods in addition to simple serial dilutions to resolve PCR inhibition shows high sensitivity in the diagnosis of paucibacillary tuberculosis.


Assuntos
Proteínas de Bactérias/genética , RNA Polimerases Dirigidas por DNA/genética , Genes Bacterianos , Mycobacterium tuberculosis/genética , Reação em Cadeia da Polimerase/métodos , Tuberculose Pulmonar/diagnóstico , Tuberculose Pulmonar/epidemiologia , Infecções Oportunistas Relacionadas com a AIDS/diagnóstico , Infecções Oportunistas Relacionadas com a AIDS/virologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Camarões/epidemiologia , Criança , Pré-Escolar , Estudos Transversais , Primers do DNA , Feminino , HIV , Humanos , Masculino , Microscopia , Pessoa de Meia-Idade , Mycobacterium tuberculosis/isolamento & purificação , Sensibilidade e Especificidade , Escarro/microbiologia , Tuberculose Pulmonar/microbiologia , Adulto Jovem
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