RESUMO
The plastidic 2-C-methylerythritol 4-phosphate (MEP) pathway supplies the precursors of a large variety of essential plant isoprenoids, but its regulation is still not well understood. Using metabolic control analysis (MCA), we examined the first enzyme of this pathway, 1-deoxyxylulose 5-phosphate synthase (DXS), in multiple grey poplar (Populus × canescens) lines modified in their DXS activity. Single leaves were dynamically labeled with 13CO2 in an illuminated, climate-controlled gas exchange cuvette coupled to a proton transfer reaction mass spectrometer, and the carbon flux through the MEP pathway was calculated. Carbon was rapidly assimilated into MEP pathway intermediates and labeled both the isoprene released and the IDP+DMADP pool by up to 90%. DXS activity was increased by 25% in lines overexpressing the DXS gene and reduced by 50% in RNA interference lines, while the carbon flux in the MEP pathway was 25-35% greater in overexpressing lines and unchanged in RNA interference lines. Isoprene emission was also not altered in these different genetic backgrounds. By correlating absolute flux to DXS activity under different conditions of light and temperature, the flux control coefficient was found to be low. Among isoprenoid end products, isoprene itself was unchanged in DXS transgenic lines, but the levels of the chlorophylls and most carotenoids measured were 20-30% less in RNA interference lines than in overexpression lines. Our data thus demonstrate that DXS in the isoprene-emitting grey poplar plays only a minor part in controlling flux through the MEP pathway.
Assuntos
Eritritol , Eritritol/análogos & derivados , Populus , Fosfatos Açúcares , Transferases , Populus/genética , Populus/metabolismo , Populus/enzimologia , Eritritol/metabolismo , Fosfatos Açúcares/metabolismo , Transferases/metabolismo , Transferases/genética , Hemiterpenos/metabolismo , Butadienos/metabolismo , Folhas de Planta/metabolismo , Folhas de Planta/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Regulação da Expressão Gênica de Plantas , Pentanos/metabolismo , Plantas Geneticamente ModificadasRESUMO
Bone formation and homeostasis are controlled by environmental factors and endocrine regulatory cues that initiate intracellular signaling pathways capable of modulating gene expression in the nucleus. Bone-related gene expression is controlled by nucleosome-based chromatin architecture that limits the accessibility of lineage-specific gene regulatory DNA sequences and sequence-specific transcription factors. From a developmental perspective, bone-specific gene expression must be suppressed during the early stages of embryogenesis to prevent the premature mineralization of skeletal elements during fetal growth in utero. Hence, bone formation is initially inhibited by gene suppressive epigenetic regulators, while other epigenetic regulators actively support osteoblast differentiation. Prominent epigenetic regulators that stimulate or attenuate osteogenesis include lysine methyl transferases (e.g., EZH2, SMYD2, SUV420H2), lysine deacetylases (e.g., HDAC1, HDAC3, HDAC4, HDAC7, SIRT1, SIRT3), arginine methyl transferases (e.g., PRMT1, PRMT4/CARM1, PRMT5), dioxygenases (e.g., TET2), bromodomain proteins (e.g., BRD2, BRD4) and chromodomain proteins (e.g., CBX1, CBX2, CBX5). This narrative review provides a broad overview of the covalent modifications of DNA and histone proteins that involve hundreds of enzymes that add, read, or delete these epigenetic modifications that are relevant for self-renewal and differentiation of mesenchymal stem cells, skeletal stem cells and osteoblasts during osteogenesis.
Assuntos
Osteogênese , Fatores de Transcrição , Osteogênese/genética , Fatores de Transcrição/metabolismo , Lisina/metabolismo , Proteínas Nucleares/genética , Diferenciação Celular/genética , Epigênese Genética , Osteoblastos/metabolismo , Transferases/genética , Transferases/metabolismoRESUMO
Bifidobacterium breve, one of the main bifidobacterial species colonizing the human gastrointestinal tract in early life, has received extensive attention for its purported beneficial effects on human health. However, exploration of the mode of action of such beneficial effects exerted by B. breve is cumbersome due to the lack of effective genetic tools, which limits its synthetic biology application. The widespread presence of CRISPR-Cas systems in the B. breve genome makes endogenous CRISPR-based gene editing toolkits a promising tool. This study revealed that Type I-C CRISPR-Cas systems in B. breve can be divided into two groups based on the amino acid sequences encoded by cas gene clusters. Deletion of the gene coding uracil phosphoribosyl-transferase (upp) was achieved in five B. breve strains from both groups using this system. In addition, translational termination of uracil phosphoribosyl-transferase was successfully achieved in B. breve FJSWX38M7 by single-base substitution of the upp gene and insertion of three stop codons. The gene encoding linoleic acid isomerase (bbi) in B. breve, being a characteristic trait, was deleted after plasmid curing, which rendered it unable to convert linoleic acid into conjugated linoleic acid, demonstrating the feasibility of successive editing. This study expands the toolkit for gene manipulation in B. breve and provides a new approach toward functional genome editing and analysis of B. breve strains.IMPORTANCEThe lack of effective genetic tools for Bifidobacterium breve is an obstacle to studying the molecular mechanisms of its health-promoting effects, hindering the development of next-generation probiotics. Here, we introduce a gene editing method based on the endogenous CRISPR-Cas system, which can achieve gene deletion, single-base substitution, gene insertion, and successive gene editing in B. breve. This study will facilitate discovery of functional genes and elucidation of molecular mechanisms of B. breve pertaining to health-associated benefits.
Assuntos
Bifidobacterium breve , Sistemas CRISPR-Cas , Humanos , Edição de Genes/métodos , Bifidobacterium breve/genética , Ácido Linoleico , Transferases/genética , UracilaRESUMO
Bacterial cell surface glycoconjugates are critical for cell survival and for interactions between bacteria and their hosts. Consequently, the pathways responsible for their biosynthesis have untapped potential as therapeutic targets. The localization of many glycoconjugate biosynthesis enzymes to the membrane represents a significant challenge for expressing, purifying, and characterizing these enzymes. Here, we leverage cutting-edge detergent-free methods to stabilize, purify, and structurally characterize WbaP, a phosphoglycosyl transferase (PGT) from the Salmonella enterica (LT2) O-antigen biosynthesis. From a functional perspective, these studies establish WbaP as a homodimer, reveal the structural elements responsible for dimerization, shed light on the regulatory role of a domain of unknown function embedded within WbaP, and identify conserved structural motifs between PGTs and functionally unrelated UDP-sugar dehydratases. From a technological perspective, the strategy developed here is generalizable and provides a toolkit for studying other classes of small membrane proteins embedded in liponanoparticles beyond PGTs.
Assuntos
Salmonella enterica , Transferases , Transferases/genética , Transferases/química , Antígenos O , Metabolismo dos Carboidratos , Membrana Celular , Salmonella enterica/genéticaRESUMO
PURPOSE: Bagasse, the residue left after extracting juice from sugarcane stalks, is rich in lignocellulosic biomass. The lignin present in this plant biomass is the key factor that hinders the efficient extraction of ethanol from the bagasse. In the current study, γ-irradiated sugarcane mutants were evaluated for variation in lignin content and its corresponding caffeic acid-O-methyl transferase (COMT) gene. MATERIALS AND METHODS: The acetyl bromide method was used to estimate lignin content in sugarcane mutants. PCR-based cloning of the COMT gene was performed in low lignin mutants as well as control plants in E. coli (strain DH5α) to understand the mechanism of variation at the molecular level. The Sanger sequencing for cloned gene was performed to check variation in gene sequence. RESULTS: In comparison to the control (21.5%), the mutant plants' lignin content ranged from 13 to 28%. The Sanger sequencing revealed approximately the same length of the gene from mutants as well as a control plant. In comparison to the reference gene, the mutated gene showed SNPs and indels in different regions, which may have an impact on lignin content. CONCLUSIONS: Therefore, γ-irradiated mutagenesis is an acceptable approach to develop novel mutants of sugarcane with low lignin content to enhance bioethanol production from waste material using bioprocess technology.
Assuntos
Ácidos Cafeicos , Lignina , Saccharum , Transferases/genética , Saccharum/genética , Escherichia coli , MutaçãoRESUMO
Diagnostic classification of soft tissue tumors is based on histology, immunohistochemistry, genetic findings, and radiologic and clinical correlations. Recently, a sarcoma DNA methylation classifier was developed, covering 62 soft tissue and bone tumor entities. The classifier is based on large-scale analysis of methylation sites across the genome. It includes DNA copy number analysis and determines O 6 methylguanine DNA methyl-transferase methylation status. In this study, we evaluated 619 well-studied soft tissue and bone tumors with the sarcoma classifier. Problem cases and typical examples of different entities were included. The classifier had high sensitivity and specificity for fusion sarcomas: Ewing, synovial, CIC -rearranged, and BCOR -rearranged. It also performed well for leiomyosarcoma, malignant peripheral nerve sheath tumors (MPNST), and malignant vascular tumors. There was low sensitivity for diagnoses of desmoid fibromatosis, neurofibroma, and schwannoma. Low specificity of matches was observed for angiomatoid fibrous histiocytoma, inflammatory myofibroblastic tumor, Langerhans histiocytosis, schwannoma, undifferentiated sarcoma, and well-differentiated/dedifferentiated liposarcoma. Diagnosis of lipomatous tumors was greatly assisted by the detection of MDM2 amplification and RB1 loss in the copy plot. The classifier helped to establish diagnoses for KIT-negative gastrointestinal stromal tumors, MPNSTs with unusual immunophenotypes, and undifferentiated melanomas. O 6 methylguanine DNA methyl-transferase methylation was infrequent and most common in melanomas (35%), MPNSTs (11%), and undifferentiated sarcomas (11%). The Sarcoma Methylation Classifier will likely evolve with the addition of new entities and refinement of the present methylation classes. The classifier may also help to define new entities and give new insight into the interrelationships of sarcomas.
Assuntos
Lipossarcoma , Melanoma , Neurilemoma , Neurofibrossarcoma , Patologia Cirúrgica , Sarcoma , Neoplasias de Tecidos Moles , Humanos , Metilação de DNA , Melanoma/genética , Sarcoma/diagnóstico , Sarcoma/genética , Lipossarcoma/genética , Neoplasias de Tecidos Moles/diagnóstico , Neoplasias de Tecidos Moles/genética , Neurilemoma/genética , Neurofibrossarcoma/genética , Transferases/genética , DNA , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/análiseRESUMO
BACKGROUND: Cardiac hypertrophy is an intermediate stage in the development of heart failure. The structural and functional processes occurring in cardiac hypertrophy include extensive gene reprogramming, which is dependent on epigenetic regulation and chromatin remodeling. However, the chromatin remodelers and their regulatory functions involved in the pathogenesis of cardiac hypertrophy are not well characterized. METHODS: Protein interaction was determined by immunoprecipitation assay in primary cardiomyocytes and mouse cardiac samples subjected or not to transverse aortic constriction for 1 week. Chromatin immunoprecipitation and DNA sequencing (ChIP-seq) experiments were performed on the chromatin of adult mouse cardiomyocytes. RESULTS: We report that the calcium-activated protein phosphatase CaN (calcineurin), its endogenous inhibitory protein carabin, the STK24 (STE20-like protein kinase 3), and the histone monomethyltransferase, MLL3 (mixed lineage leukemia 3) form altogether a macromolecular complex at the chromatin of cardiomyocytes. Under basal conditions, carabin prevents CaN activation while the serine/threonine kinase STK24 maintains MLL3 inactive via phosphorylation. After 1 week of transverse aortic constriction, both carabin and STK24 are released from the CaN-MLL3 complex leading to the activation of CaN, dephosphorylation of MLL3, and in turn, histone H3 lysine 4 monomethylation. Selective cardiac MLL3 knockdown mitigates hypertrophy, and chromatin immunoprecipitation and DNA sequencing analysis demonstrates that MLL3 is de novo recruited at the transcriptional start site of genes implicated in cardiomyopathy in stress conditions. We also show that CaN and MLL3 colocalize at chromatin and that CaN activates MLL3 histone methyl transferase activity at distal intergenic regions under hypertrophic conditions. CONCLUSIONS: Our study reveals an unsuspected epigenetic mechanism of CaN that directly regulates MLL3 histone methyl transferase activity to promote cardiac remodeling.
Assuntos
Calcineurina , Histonas , Animais , Camundongos , Calcineurina/metabolismo , Cardiomegalia/metabolismo , Cromatina/metabolismo , Epigênese Genética , Histonas/metabolismo , Miócitos Cardíacos/metabolismo , Transferases/genética , Transferases/metabolismo , Remodelação VentricularRESUMO
Many genes involved in triterpenoid saponins in plants control isoprenoid flux and constitute the precursor pool, which is channeled into various downstream pathways leading to the synthesis of triterpenoid saponins in C. asiatica. Full-length 1-Deoxy-D-Xylulose-5-Phosphate-Synthase (CaDXS) gene was isolated for the study from the previously annotated Centella asiatica leaves transcriptomic data. The CaDXS gene sequence was submitted to the NCBI databases with GenBank accession number MZ997832. The full-length CaDXS gene contained a 2244 base pair open reading frame that encoded a 747 amino acid polypeptide. The predicted molecular weight (MW) and theoretical pI of DXS are 76.28 kDa and 6.86, respectively. Multiple amino acid sequence alignment of amino acids and phylogenetic studies suggest that CaDXS shares high similarities with DXS from other plants DXS belonging to different families. A phylogenetic tree was constructed using Molecular Evolutionary Genetic Analysis (MEGA) version 10.1.6. Structural analysis provided fundamental information about the three-dimensional features and physicochemical parameters of the CaDXS protein. Quantitative expression analysis showed that CaDXS transcripts were maximally expressed in leaf, followed by petiole, roots, and node tissues. CaDXS was cloned into the expression vector pET28a, expressed heterologously in DH5α bacteria, confirmed by sequencing, and subsequently characterized by protein expression and functional complementation. The study focused on understanding the protein structure, biological significance, regulatory mechanism, functional analysis, and gene characterization of the centellosides biosynthetic pathway gene DXS for the first time in the plant. It would provide new information about the metabolic pathway and its relative contribution to isoprenoid biosynthesis.
Assuntos
Centella , Saponinas , Triterpenos , Humanos , Filogenia , Centella/genética , Centella/metabolismo , Transferases/genética , Terpenos/metabolismo , Clonagem Molecular , Regulação da Expressão Gênica de PlantasRESUMO
Several related progeroid disorders are caused by defective post-translational processing of prelamin A, the precursor of the nuclear scaffold protein lamin A, encoded by LMNA. Prelamin A undergoes farnesylation and additional modifications at its C-terminus. Subsequently, the farnesylated C-terminal segment is cleaved off by the zinc metalloprotease ZMPSTE24. The premature aging disorder Hutchinson Gilford progeria syndrome (HGPS) and a related progeroid disease, mandibuloacral dysplasia (MAD-B), are caused by mutations in LMNA and ZMPSTE24, respectively, that result in failure to process the lamin A precursor and accumulate permanently farnesylated forms of prelamin A. The farnesyl transferase inhibitor (FTI) lonafarnib is known to correct the aberrant nuclear morphology of HGPS patient cells and improves lifespan in children with HGPS. Importantly, and in contrast to a previous report, we show here that FTI treatment also improves the aberrant nuclear phenotypes in MAD-B patient cells with mutations in ZMPSTE24 (P248L or L425P). As expected, lonafarnib does not correct nuclear defects for cells with lamin A processing-proficient mutations. We also examine prelamin A processing in fibroblasts from two individuals with a prevalent laminopathy mutation LMNA-R644C. Despite the proximity of residue R644 to the prelamin A cleavage site, neither R644C patient cell line shows a prelamin A processing defect, and both have normal nuclear morphology. This work clarifies the prelamin A processing status and role of FTIs in a variety of laminopathy patient cells and supports the FDA-approved indication for the FTI Zokinvy for patients with processing-deficient progeroid laminopathies, but not for patients with processing-proficient laminopathies.
Assuntos
Lipodistrofia , Progéria , Criança , Humanos , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Progéria/tratamento farmacológico , Progéria/genética , Progéria/metabolismo , Inibidores Enzimáticos/farmacologia , Mutação , Lipodistrofia/metabolismo , Fibroblastos/metabolismo , Transferases/genética , Transferases/metabolismo , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Proteínas de Membrana/metabolismoRESUMO
IMPORTANCE: Plasmid-mediated mobile colistin-resistance genes have been recognized as a global threat because they jeopardize the efficacy of colistin in therapeutic practice. Here, we described the genetic features of two mcr-9.1-carrying Gram-negative bacteria with a colistin-resistant phenotype derived from vegetables in Japan. The colistin-resistant mcr-9.1, which has never been detected in vegetables, was located on a large plasmid in Enterobacter cloacae CST17-2 and Raoultella ornithinolytica CST129-1, suggesting a high chance of horizontal gene transfer. To the best of our knowledge, this is the first report of mcr-9 in R. ornithinolytica. This study indicates that fresh vegetables might be a potential source for the transmission of mcr-9 genes encoding resistance to frontline (colistin) and clinically relevant antimicrobials. The study also provides additional consideration for colistin use and the relevance of routine surveillance in epidemiological perspective to curb the continuous spread of mcr alleles.
Assuntos
Colistina , Enterobacter cloacae , Colistina/farmacologia , Enterobacter cloacae/genética , Antibacterianos/farmacologia , Verduras/microbiologia , Japão , Farmacorresistência Bacteriana/genética , Plasmídeos/genética , Transferases/genética , Testes de Sensibilidade MicrobianaRESUMO
Eukaryotic dehydrodolichyl diphosphate synthases (DHDDSs), cis-prenyltransferases (cis-PTs) synthesizing precursors of dolichols to mediate glycoprotein biosynthesis require partners, for eample Nus1 in yeast and NgBR in animals, which are cis-PTs homologues without activity but to boost the DHDDSs activity. Unlike animals, plants have multiple cis-PT homologues to pair or stand alone to produce various chain-length products with less known physiological roles. We chose Cinnamomum kanehirae, a tree that contains two DHDDS-like and three NgBR-like proteins from genome analysis, and found that one DHDDS-like protein acted as a homodimeric cis-PT to make a medium-chain C55 product, while the other formed heterodimeric complexes with either one of two NgBR homologues to produce longer-chain products. Both complexes were functional to complement the growth defect of the yeast rer2 deficient strain at a higher temperature. From the roles for the polyprenol and dolichol biosynthesis and sequence motifs, their homologues in various species were compared to reveal their possible evolutionary paths.
Assuntos
Alquil e Aril Transferases , Animais , Alquil e Aril Transferases/genética , Biossíntese de Proteínas , Saccharomyces cerevisiae/metabolismo , Transferases/genética , Transferases/metabolismo , Glicoproteínas/química , Glicoproteínas/metabolismoRESUMO
Small peptide aldehydes (SPAs) with protease inhibitory activity are naturally occurring compounds shown to be synthesized by non-ribosomal peptide synthetases (NRPS). SPAs are widely used in biotechnology and have been utilized as therapeutic agents. They are also physiologically relevant and have been postulated to regulate the development of their producing microorganisms. Previously, we identified an NRPS-like biosynthetic gene cluster (BGC) in Streptomyces lividans 66 that lacked a condensation (C) domain but included a tRNA-utilizing enzyme (tRUE) belonging to the leucyl/phenylalanyl (L/F) transferase family. This system was predicted to direct the synthesis of a novel SPA, which we named livipeptin. Using evolutionary genome mining approaches, here, we confirm the presence of L/F transferase tRUEs within the genomes of diverse Streptomyces and related organisms, including fusions with the anticipated C-minus NRPS-like protein. We then demonstrate genetic functional cooperation between the identified L/F-transferase divergent tRUE homolog with the C-minus NRPS, leading to the synthesis of a metabolic fraction with protease inhibitory activity. Semisynthetic assays in the presence of RNAse revealed that the productive interaction between the tRUE and the C-minus NRPS enzymes is indeed tRNA dependent. We expect our findings to boost the discovery of SPAs, as well as the development of protease-mediated biotechnologies, by exploiting the uncovered genetic basis for synthesizing putative acetyl-leu/phe-arginine protease inhibitors. Furthermore, these results will facilitate the purification and structural elucidation of livipeptin, which has proven difficult to chemically characterize. SIGNIFICANCE: The discovery of natural products biosynthetic genes marks a significant advancement in our understanding of these metabolites, for example of their evolution, activity, and biosynthesis, but also opens biotechnological opportunities and knowledge to advance genome mining approaches. We made this possible by uncovering a new biosynthetic pathway in Streptomyces lividans 66 shown to direct the synthesis of a strong protease inhibitor, termed livipeptin, following unprecedented biosynthetic rules and genes. Thus, by shedding light on the genetic mechanisms predicted to govern the production of acetyl-leu/phe-arginine protease inhibitors, including the elusive livipeptin, this study enables novel protease-mediated biotechnologies as well as approaches for discovering protease inhibitors from genome data.
Assuntos
Anti-Infecciosos , Streptomyces lividans , Streptomyces lividans/genética , Streptomyces lividans/metabolismo , Inibidores de Proteases , Peptídeo Sintases/metabolismo , Peptídeos/genética , Peptídeo Hidrolases/genética , RNA de Transferência/genética , Transferases/genética , Arginina , Família MultigênicaRESUMO
BACKGROUND/AIMS: Our previous work identified the dimethyladenosine transferase 1 homolog as a novel prognostic factor for detecting human gastric carcinoma with high sensitivity and specificity. The high expression of dimethyladenosine transferase 1 is closely associated with the occurrence and progression of gastric carcinoma. However, the underlying mechanism of dimethyladenosine transferase 1 for the occurrence and development of gastric carcinoma is not well elucidated yet. MATERIALS AND METHODS: In our present study, the biological role of dimethyladenosine transferase 1 on cell proliferation, apoptosis, and cell cycle progression in human gastric carcinoma cells was investigated through in vitro and in vivo assays by the overexpression and knockdown of dimethyladenosine transferase 1 2-way authentication method. RESULTS: We found that the overexpression of dimethyladenosine transferase 1 significantly promotes cell proliferation (P < .001) and inhibition of cell apoptosis (P < .01) in SGC-7901 cells. However, the in vivo experiment results of the knockdown dimethyladenosine transferase 1 using small interfering RNAs in the MKN-45 are just the opposite. Reverse-transcriptase polymerase chain reaction and western blotting analysis revealed that overexpressed dimethyladenosine transferase 1 in SGC-7901 cells significantly activated the AKT pathway compared to control cells. In contrast, we found that apoptosis genes such as Caspase-3 and Caspase-9 were downregulated in those cells. The xenograft nude mice model exhibited increased tumor growth (P < .01) and weight loss (P < .01), with the overexpression of dimethyladenosine transferase 1 homolog in the SGC-7901 cells. These results have been further confirmed through backward verification in dimethyladenosine transferase 1 knockdown cells. CONCLUSIONS: Taken together, our results indicated that the dimethyladenosine transferase 1 plays a crucial role in stimulating cancer cell proliferation and contributes to apoptosis resistance in human gastric carcinoma. Meanwhile, it provides a potential therapeutic target for gastric carcinoma treatment and is worthy of further studies.
Assuntos
Carcinoma , Neoplasias Gástricas , Animais , Camundongos , Humanos , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transferases/genética , Transferases/metabolismo , Camundongos Nus , Linhagem Celular Tumoral , Apoptose/genética , Carcinoma/patologia , Neoplasias Gástricas/patologia , Proliferação de Células/genética , Regulação Neoplásica da Expressão GênicaRESUMO
Quantitative real-time PCR (qPCR) is a sensitive and commonly used technique for gene expression profiling and provides insight into biological systems. Successful qPCR requires the use of appropriate reference genes for the normalization of data. In the present study, we aimed to identify and assess the best-suited reference genes in near-isogenic resistant (R) and susceptible (S) tomato lines infected with begomovirus Tomato curly stunt virus (ToCSV). Ten candidate reference genes namely, Actin7 (ACT), ß-6 Tubulin (TUB), Ubiquitin 3 (UBI), Clathrin adaptor complexes medium subunit (CAC), Phytoene desaturase (PDS), Expressed protein (EXP), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Adenine phosphoribosyl transferase-like protein (APT1), TAP42-interacting protein (TIP41) and Elongation factor 1-alpha (EF1α) were selected and evaluated for their expression stability in resistant and susceptible tomato leaves using the analytical tools geNorm, NormFinder, BestKeeper, and RefFinder. After ranking the reference genes from most to least stable, the results suggested that a combination of ACT, EXP, and EF1α in the S lines and a combination of TIP41, APT1, and ACT in the R line is appropriate for qPCR normalization. Furthermore, to validate the identified reference genes, iron superoxide dismutase (SOD), heat shock protein 70 (HSP70) and Glutathione-S-transferase (GST) were selected as targets for normalization. The relative expression of the target genes varied when normalized against the most stable reference genes in comparison to the least stable genes. These results highlight the importance of careful selection of reference genes for accurate normalization in qPCR studies.
Assuntos
Geminiviridae , Solanum lycopersicum , Reação em Cadeia da Polimerase em Tempo Real/métodos , Solanum lycopersicum/genética , Geminiviridae/genética , Genes de Plantas , Perfilação da Expressão Gênica/métodos , Transferases/genética , Padrões de ReferênciaRESUMO
The polysaccharide (PS) capsule is essential for immune evasion and virulence of Streptococcus pneumoniae. Existing pneumococcal vaccines are designed to elicit anticapsule antibodies; however, the effectiveness of these vaccines is being challenged by the emergence of new capsule types or variants. Herein, we characterize a newly discovered capsule type, 33E, that appears to have repeatedly emerged from vaccine type 33F via an inactivation mutation in the capsule glycosyltransferase gene, wciE. Structural analysis demonstrated that 33E and 33F share an identical repeat unit backbone [â5)-ß-D-Galf2Ac-(1â3)-ß-D-Galp-(1â3)-α-D-Galp-(1â3)-ß-D-Galf-(1â3)-ß-D-Glcp-(1â], except that a galactose (α-D-Galp) branch is present in 33F but not in 33E. Though the two capsule types were indistinguishable using conventional typing methods, the monoclonal antibody Hyp33FM1 selectively bound 33F but not 33E pneumococci. Further, we confirmed that wciE encodes a glycosyltransferase that catalyzes the addition of the branching α-D-Galp and that its inactivation in 33F strains results in the expression of the 33E capsule type. Though 33F and 33E share a structural and antigenic similarity, our pilot study suggested that immunization with a 23-valent pneumococcal PS vaccine containing 33F PS did not significantly elicit cross-opsonic antibodies to 33E. New conjugate vaccines that target capsule type 33F may not necessarily protect against 33E. Therefore, studies of new conjugate vaccines require knowledge of the newly identified capsule type 33E and reliable pneumococcal typing methods capable of distinguishing it from 33F.
Assuntos
Cápsulas Bacterianas , Genes Bacterianos , Infecções Pneumocócicas , Streptococcus pneumoniae , Transferases , Anticorpos Antibacterianos/imunologia , Projetos Piloto , Infecções Pneumocócicas/microbiologia , Infecções Pneumocócicas/prevenção & controle , Vacinas Pneumocócicas/classificação , Vacinas Pneumocócicas/imunologia , Polissacarídeos/química , Sorogrupo , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/imunologia , Vacinas Conjugadas/classificação , Vacinas Conjugadas/imunologia , Cápsulas Bacterianas/química , Cápsulas Bacterianas/genética , Genes Bacterianos/genética , Genes Bacterianos/imunologia , Inativação Gênica , Transferases/genética , Transferases/metabolismoRESUMO
Mobilized colistin resistance genes (mcr) may confer resistance to the last-resort antimicrobial colistin and can often be transmitted horizontally. mcr encode phosphoethanolamine transferases (PET), which are closely related to chromosomally encoded, intrinsic lipid modification PET (i-PET; e.g., EptA, EptB, CptA). To gain insight into the evolution of mcr within the context of i-PET, we identified 69,814 MCR-like proteins present across 256 bacterial genera (obtained by querying known MCR family representatives against the National Center for Biotechnology Information [NCBI] non-redundant protein database via protein BLAST). We subsequently identified 125 putative novel mcr-like genes, which were located on the same contig as (i) ≥1 plasmid replicon and (ii) ≥1 additional antimicrobial resistance gene (obtained by querying the PlasmidFinder database and NCBI's National Database of Antibiotic Resistant Organisms, respectively, via nucleotide BLAST). At 80% amino acid identity, these putative novel MCR-like proteins formed 13 clusters, five of which represented putative novel MCR families. Sequence similarity and a maximum likelihood phylogeny of mcr, putative novel mcr-like, and ipet genes indicated that sequence similarity was insufficient to discriminate mcr from ipet genes. A mixed-effect model of evolution (MEME) indicated that site- and branch-specific positive selection played a role in the evolution of alleles within the mcr-2 and mcr-9 families. MEME suggested that positive selection played a role in the diversification of several residues in structurally important regions, including (i) a bridging region that connects the membrane-bound and catalytic periplasmic domains, and (ii) a periplasmic loop juxtaposing the substrate entry tunnel. Moreover, eptA and mcr were localized within different genomic contexts. Canonical eptA genes were typically chromosomally encoded in an operon with a two-component regulatory system or adjacent to a TetR-type regulator. Conversely, mcr were represented by single-gene operons or adjacent to pap2 and dgkA, which encode a PAP2 family lipid A phosphatase and diacylglycerol kinase, respectively. Our data suggest that eptA can give rise to "colistin resistance genes" through various mechanisms, including mobilization, selection, and diversification of genomic context and regulatory pathways. These mechanisms likely altered gene expression levels and enzyme activity, allowing bona fide eptA to evolve to function in colistin resistance.
Assuntos
Colistina , Proteínas de Escherichia coli , Humanos , Colistina/farmacologia , Farmacorresistência Bacteriana/genética , Antibacterianos/farmacologia , Plasmídeos/genética , Transferases/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Testes de Sensibilidade MicrobianaRESUMO
BACKGROUND: Glutathione S-transferases (GSTs) are large and multifunctional proteases that play an important role in detoxification, protection against biotic and abiotic stresses, and secondary metabolite transportation which is essential for plant growth and development. However, there is limited research on the identification and function of NtGSTs. RESULTS: This study uses K326 and other six tobacco varieties (Hongda, HG, GDH11, Va116, VG, and GDH88) as materials to conduct comprehensive genome-wide identification and functional characterization of the GST gene in tobacco. A total of 59 NtGSTs were identified and classified into seven subfamilies via the whole-genome sequence analysis, with the Tau type serving as the major subfamily. The NtGSTs in the same branch of the evolutionary tree had similar exon/intron structure and motif constitution. There were more than 42 collinear blocks between tobacco and pepper, tomato, and potato, indicating high homology conservation between them. Twelve segmental duplicated gene pairs and one tandem duplication may have had a substantial impact on the evolution and expansion of the tobacco GST gene family. The RT-qPCR results showed that the expression patterns of NtGSTs varied significantly among tissues, varieties, and multiple abiotic stresses, suggesting that NtGST genes may widely respond to various abiotic stresses and hormones in tobacco, including NtGSTF4, NtGSTL1, NtGSTZ1, and NtGSTU40. CONCLUSIONS: This study provides a comprehensive analysis of the NtGST gene family, including structures and functions. Many NtGSTs play a critical regulatory role in tobacco growth and development, and responses to abiotic stresses. These findings offer novel and valuable insights for understanding the biological function of NtGSTs and the reference materials for cultivating highly resistant varieties and enhancing the yield and quality of crops.
Assuntos
Nicotiana , Estresse Fisiológico , Nicotiana/metabolismo , Estresse Fisiológico/genética , Genoma de Planta , Família Multigênica , Transferases/genética , Glutationa/genética , Glutationa/metabolismo , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regulação da Expressão Gênica de PlantasRESUMO
INTRODUCTION: Genes encoding catechol-O-methyl-transferase (COMT) and adenosine A2A receptor (ADORA2A) have been shown to influence cognitive performances and responses to caffeine intake during prolonged wakefulness. The rs4680 single-nucleotide polymorphism (SNP) of COMT differentiates on memory score and circulating levels of the neurotrophic factor IGF-1. This study aimed to determine the kinetics of IGF-1, testosterone, and cortisol concentrations during prolonged wakefulness under caffeine or placebo intake in 37 healthy participants, and to analyze whether the responses are dependent on COMT rs4680 or ADORA2A rs5751876 SNPs. METHODS: In caffeine (2.5 mg/kg, twice over 24 h) or placebo-controlled condition, blood sampling was performed at 1 h (08:00, baseline), 11 h, 13 h, 25 h (08:00 next day), 35 h, and 37 h of prolonged wakefulness, and at 08:00 after one night of recovery sleep, to assess hormonal concentrations. Genotyping was performed on blood cells. RESULTS: Results indicated a significant increase in IGF-1 levels after 25, 35, and 37 h of prolonged wakefulness in the placebo condition, in subjects carrying the homozygous COMT A/A genotype only (expressed in absolute values [±SEM]: 118 ± 8, 121 ± 10, and 121 ± 10 vs. 105 ± 7 ng/mL for A/A, 127 ± 11, 128 ± 12, and 129 ± 13 vs. 120 ± 11 ng/mL for G/G, and 106 ± 9, 110 ± 10, and 106 ± 10 vs. 101 ± 8 ng/mL for G/A, after 25, 35, and 37 h of wakefulness versus 1 h; p < 0.05, condition X time X SNP). Acute caffeine intake exerted a COMT genotype-dependent reducing effect on IGF-1 kinetic response (104 ± 26, 107 ± 27, and 106 ± 26 vs. 100 ± 25 ng/mL for A/A genotype, at 25, 35, and 37 h of wakefulness vs. 1 h; p < 0.05 condition X time X SNP), plus on resting levels after overnight recovery (102 ± 5 vs. 113 ± 6 ng/mL) (p < 0.05, condition X SNP). Testosterone and cortisol concentrations decreased during wakefulness, and caffeine alleviated the testosterone reduction, unrelated to the COMT polymorphism. No significant main effect of the ADORA2A SNP was shown regardless of hormonal responses. CONCLUSION: Our results indicated that the COMT polymorphism interaction is important in determining the IGF-1 neurotrophic response to sleep deprivation with caffeine intake (NCT03859882).
Assuntos
Cafeína , Privação do Sono , Humanos , Privação do Sono/genética , Cafeína/farmacologia , Estudos Cross-Over , Peptídeos Semelhantes à Insulina , Transferases/genética , Fator de Crescimento Insulin-Like I/genética , Hidrocortisona , Polimorfismo de Nucleotídeo Único , Catecóis , Testosterona , Catecol O-Metiltransferase/genéticaRESUMO
Lettuce produces natural rubber (NR) with an average Mw of > 1 million Da in laticifers, similar to NR from rubber trees. As lettuce is an annual, self-pollinating, and easily transformable plant, it is an excellent model for molecular genetic studies of NR biosynthesis. CRISPR/Cas9 mutagenesis was optimized using lettuce hairy roots, and NR-deficient lettuce was generated via bi-allelic mutations in cis-prenyltransferase (CPT). This is the first null mutant of NR deficiency in plants. In the CPT mutant, orthologous CPT counterparts from guayule (Parthenium argentatum) and goldenrod (Solidago canadensis) were expressed under a laticifer-specific promoter to examine how the average Mw of NR is affected. No developmental defects were observed in the NR-deficient mutants. The lettuce mutants expressing guayule and goldenrod CPT produced 1.8 and 14.5 times longer NR, respectively, than the plants of their origin. This suggests that, although goldenrod cannot synthesize a sufficiently lengthy NR, goldenrod CPT has the catalytic competence to produce high-quality NR in the cellular context of lettuce laticifers. Thus, CPT alone does not determine the length of NR. Other factors, such as substrate concentration, additional proteins, and/or the nature of protein complexes including CPT-binding proteins, influence CPT activity in determining NR length.
Assuntos
Borracha , Solidago , Borracha/química , Borracha/metabolismo , Lactuca/genética , Transferases/genética , Transferases/metabolismoRESUMO
To maintain the integrity of the genome, there is a set of enzymatic systems, one of which is base excision repair (BER), which includes sequential action of DNA glycosylases, apurinic/apyrimidinic endonucleases, DNA polymerases, and DNA ligases. Normally, BER works efficiently, but the enzymes themselves (whose primary function is the recognition and removal of damaged bases) are subject to amino acid substitutions owing to natural single-nucleotide polymorphisms (SNPs). One of the enzymes in BER is DNA polymerase ß (Polß), whose function is to fill gaps in DNA with complementary dNMPs. It is known that many SNPs can cause an amino acid substitution in this enzyme and a significant decrease in the enzymatic activity. In this study, the activity of four natural variants of Polß, containing substitution E154A, G189D, M236T, or R254I in the transferase domain, was analyzed using molecular dynamics simulations and pre-steady-state kinetic analyses. It was shown that all tested substitutions lead to a significant reduction in the ability to form a complex with DNA and with incoming dNTP. The G189D substitution also diminished Polß catalytic activity. Thus, a decrease in the activity of studied mutant forms may be associated with an increased risk of damage to the genome.