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1.
BMC Genomics ; 22(1): 420, 2021 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-34090348

RESUMO

BACKGROUND: Single-cell RNA sequencing (scRNA-seq) has led to remarkable progress in our understanding of tissue heterogeneity in health and disease. Recently, the need for scRNA-seq sample fixation has emerged in many scenarios, such as when samples need long-term transportation, or when experiments need to be temporally synchronized. Methanol fixation is a simple and gentle method that has been routinely applied in scRNA-sEq. Yet, concerns remain that fixation may result in biases which may change the RNA-seq outcome. RESULTS: We adapted an existing methanol fixation protocol and performed scRNA-seq on both live and methanol fixed cells. Analyses of the results show methanol fixation can faithfully preserve biological related signals, while the discrepancy caused by fixation is subtle and relevant to library construction methods. By grouping transcripts based on their lengths and GC content, we find that transcripts with different features are affected by fixation to different degrees in full-length sequencing data, while the effect is alleviated in Drop-seq result. CONCLUSIONS: Our deep analysis reveals the effects of methanol fixation on sample RNA integrity and elucidates the potential consequences of using fixation in various scRNA-seq experiment designs.


Assuntos
Metanol , RNA , Sequência de Bases , RNA/genética , Análise de Sequência de RNA , Análise de Célula Única
2.
Mol Biol (Mosk) ; 55(3): 431-440, 2021.
Artigo em Russo | MEDLINE | ID: mdl-34097678

RESUMO

This paper reports the analysis of the nucleotide sequences of the 5'-untranslated region (5'-UTR) of tick-borne encephalitis virus (TBEV) genomic RNA isolated from 39 individual taiga ticks collected in several regions of Northern Eurasia. The sequences of 5'-UTRs of the Siberian and Far East TBEV genotypes were 89% and 95% identical to the prototype strains (Zausaev and 205), respectively. The detected nucleotide substitutions were typical for these two TBEV genotypes, which made possible unambiguous identification. Both conservative and variable motifs were detected in the 5'-UTR RNA. The B2, C1, and C2 elements of the Y-shaped 5'-UTR structure and the presumable viral RNA-dependent RNA-polymerase binding site were the most variable. The A2, CS A, CS В elements as well as the start codon were conservative. Interestingly, five substitutions in the 5'-UTR C1 variable element of the TBEVs isolated in different geographical regions were strictly conservative, while 11 different substitutions were detected in this element among the laboratory TBEV variants. A little less that a third of all nucleotide substitutions were mapped outside the main elements of the Y-shaped structure. In general, nucleotide substitutions were localized to stem structures, not being found in the hairpin regions of the TBEV 5'-UTR. The results indicated significant variability of the genomic RNA 5'-UTR in the TBEV laboratory strains and field isolates obtained from different geographical regions. It has been suggested that genetic variability of 5'-UTR is characteristic of the TBEV genome 5'-UTR organization and may serve as a structural basis for virus efficient replication in various avian, mammalian, and ixodic tick cells.


Assuntos
Vírus da Encefalite Transmitidos por Carrapatos , Encefalite Transmitida por Carrapatos , Ixodes , Animais , Sequência de Bases , Vírus da Encefalite Transmitidos por Carrapatos/genética , Encefalite Transmitida por Carrapatos/genética , Genoma Viral/genética , Filogenia , RNA Viral/genética
3.
BMC Genomics ; 22(1): 412, 2021 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-34088266

RESUMO

BACKGROUND: The development of RNA sequencing (RNAseq) and the corresponding emergence of public datasets have created new avenues of transcriptional marker search. The long non-coding RNAs (lncRNAs) constitute an emerging class of transcripts with a potential for high tissue specificity and function. Therefore, we tested the biomarker potential of lncRNAs on Mesenchymal Stem Cells (MSCs), a complex type of adult multipotent stem cells of diverse tissue origins, that is frequently used in clinics but which is lacking extensive characterization. RESULTS: We developed a dedicated bioinformatics pipeline for the purpose of building a cell-specific catalogue of unannotated lncRNAs. The pipeline performs ab initio transcript identification, pseudoalignment and uses new methodologies such as a specific k-mer approach for naive quantification of expression in numerous RNAseq data. We next applied it on MSCs, and our pipeline was able to highlight novel lncRNAs with high cell specificity. Furthermore, with original and efficient approaches for functional prediction, we demonstrated that each candidate represents one specific state of MSCs biology. CONCLUSIONS: We showed that our approach can be employed to harness lncRNAs as cell markers. More specifically, our results suggest different candidates as potential actors in MSCs biology and propose promising directions for future experimental investigations.


Assuntos
Células-Tronco Mesenquimais , RNA Longo não Codificante , Sequência de Bases , Biologia Computacional , RNA Longo não Codificante/genética , Análise de Sequência de RNA
4.
Int J Mol Sci ; 22(10)2021 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-34068033

RESUMO

Conjugation, besides transformation and transduction, is one of the main mechanisms of horizontal transmission of genetic information among bacteria. Conjugational transfer, due to its essential role in shaping bacterial genomes and spreading of antibiotics resistance genes, has been widely studied for more than 70 years. However, new and intriguing facts concerning the molecular basis of this process are still being revealed. Most recently, a novel family of conjugative relaxases (Mob proteins) was distinguished. The characteristic feature of these proteins is that they are not related to any of Mobs described so far. Instead of this, they share significant similarity to tyrosine recombinases. In this study MobK-a tyrosine recombinase-like Mob protein, encoded by pIGRK cryptic plasmid from the Klebsiella pneumoniae clinical strain, was characterized. This study revealed that MobK is a site-specific nuclease and its relaxase activity is dependent on both a conserved catalytic tyrosine residue (Y179) that is characteristic of tyrosine recombinases and the presence of Mg2+ divalent cations. The pIGRK minimal origin of transfer sequence (oriT) was also characterized. This is one of the first reports presenting tyrosine recombinase-like conjugative relaxase protein. It also demonstrates that MobK is a convenient model for studying this new protein family.


Assuntos
Proteínas de Bactérias/metabolismo , Conjugação Genética , DNA Bacteriano/genética , Endodesoxirribonucleases/metabolismo , Klebsiella pneumoniae/enzimologia , Plasmídeos/genética , Recombinação Genética , Proteínas de Bactérias/genética , Sequência de Bases , Endodesoxirribonucleases/genética , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/crescimento & desenvolvimento , Plasmídeos/metabolismo
5.
Commun Biol ; 4(1): 698, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34083758

RESUMO

Given the global impact and severity of COVID-19, there is a pressing need for a better understanding of the SARS-CoV-2 genome and mutations. Multi-strain sequence alignments of coronaviruses (CoV) provide important information for interpreting the genome and its variation. We apply a comparative genomics method, ConsHMM, to the multi-strain alignments of CoV to annotate every base of the SARS-CoV-2 genome with conservation states based on sequence alignment patterns among CoV. The learned conservation states show distinct enrichment patterns for genes, protein domains, and other regions of interest. Certain states are strongly enriched or depleted of SARS-CoV-2 mutations, which can be used to predict potentially consequential mutations. We expect the conservation states to be a resource for interpreting the SARS-CoV-2 genome and mutations.


Assuntos
COVID-19/virologia , Genoma Viral , SARS-CoV-2/genética , Animais , Sequência de Bases , Sequência Conservada , Evolução Molecular , Genômica , Humanos , Mutação , Nucleotídeos/genética , Alinhamento de Sequência
6.
Artigo em Inglês | MEDLINE | ID: mdl-34106822

RESUMO

A novel acidophilic actinobacterium, designated strain NEAU-YB345T, was isolated from a pumpkin root collected from Mudanjiang, Heilongjiang Province, northeast PR China. Based on 16S rRNA gene sequence similarity and chemotaxonomic and morphological properties, the isolate was assigned to the genus Streptacidiphilus, with the high 16S rRNA gene sequence similarities to Streptacidiphilus melanogenes JCM 16224T (99.2 %), Streptacidiphilus anmyonensis JCM 16223T (99.1 %) and Streptacidiphilus jiangxiensis JCM 12277T (98.7 %). Its cell wall contained ll-diaminopimelic acid as the major diamino acid. Rhamnose, ribose, glucose and galactose were the detected sugars from the whole-cell hydrolysates. The phospholipid profile consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside and an unidentified phospholipid. The menaquinones were MK-9(H8) and MK-9(H6). Major fatty acids were C16 : 0, iso-C16 : 0, iso-C15 : 0 and anteiso-C15 : 0. Phylogenetic analysis using 16S rRNA gene and whole-genome sequences placed the strain in distinct clades but within the genus Streptacidiphilus. The DNA G+C content was 71.2 mol%. Based on DNA-DNA relatedness and physiological and biochemical data, the isolate could be distinguished from its closest relatives. Therefore, strain NEAU-YB345T represents a novel species of the genus Streptacidiphilus, for which the name Streptacidiphilus fuscans sp. nov. is proposed. The type strain is NEAU-YB345T (=CCTCC AA 2020030T=JCM 33976T).


Assuntos
Actinobacteria/isolamento & purificação , Cucurbita/microbiologia , Raízes de Plantas/microbiologia , Actinobacteria/genética , Actinobacteria/crescimento & desenvolvimento , Actinobacteria/ultraestrutura , Sequência de Bases , DNA Bacteriano/genética , Genoma Bacteriano , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Especificidade da Espécie
7.
Artigo em Inglês | MEDLINE | ID: mdl-34106824

RESUMO

Six bacterial strains isolated from root nodules of soybean plants that had been inoculated with root-zone soil of legumes native to Canada were previously characterized and 1) placed in two novel lineages within the genus Bradyrhizobium and 2) assigned to symbiovar septentrionale. Here we verified the taxonomic status of these strains using genomic and phenotypic analyses. Phylogenetic analyses of five protein encoding partial gene sequences as well as 52 full length ribosome protein subunit gene sequences confirmed placement of the novel strains in two highly supported lineages distinct from named Bradyrhizobium species. The highest average nucleotide identity values of strains representing these two lineages relative to type strains of closest relatives were 90.7 and 92.3% which is well below the threshold value for bacterial species circumscription. The genomes of representative strains 1S1T, 162S2 and 66S1MBT have sizes of 10598256, 10733150 and 9032145 bp with DNA G+C contents of 63.5, 63.4 and 63.8 mol%, respectively. These strains possess between one and three plasmids based on copy number of plasmid replication and segregation (repABC) genes. Novel strains also possess numerous insertion sequences, and, relative to reference strain Bradyrhizobium diazoefficiens USDA110T, exhibit inversion and fragmentation of nodulation (nod) and nitrogen-fixation (nif) gene clusters. Phylogenetic analyses of nodC and nifH gene sequences confirmed placement of novel strains in a distinct lineage corresponding to symbiovar septentrionale. Data for morphological, physiological and symbiotic characteristics complement the sequence-based results. The data presented here support the description of two new species for which the names Bradyrhizobium septentrionale sp. nov. (sv. septentrionale) and Bradyrhizobium quebecense sp. nov. (sv. septentrionale) are proposed, with 1S1T (=LMG 29930T=HAMBI 3676T) and 66S1MBT (=LMG 31547T=HAMBI 3720T) as type strains, respectively.


Assuntos
Bradyrhizobium/genética , Bradyrhizobium/fisiologia , Fabaceae/microbiologia , Rearranjo Gênico , Mutagênese Insercional/genética , Simbiose/genética , Composição de Bases , Sequência de Bases , Teorema de Bayes , Bradyrhizobium/classificação , Canadá , Fenótipo , Filogenia , Nodulação/genética , RNA Ribossômico 16S/genética , Subunidades Ribossômicas/genética , Nódulos Radiculares de Plantas/microbiologia
8.
Artigo em Inglês | MEDLINE | ID: mdl-34106825

RESUMO

An endophytic actinobacterium, designated strain CA1R205T, was isolated from the surface-sterilized root of Coffea arabica L. collected from Ratchaburi province, Thailand. The taxonomic position of this strain was evaluated using a polyphasic approach. The strain produced light yellowish brown to dark brownish black substrate mycelium and greyish white aerial mycelium. The spiral spore chains were produced directly on aerial mycelium. CA1R205T was found to have ll-diaminopimelic acid in the cell peptidoglycan, galactose, glucose, mannose and ribose as whole-cell reducing sugars, MK-10(H4), MK-9(H6), MK-10(H2), MK-9(H4), MK-10(H6) and MK-10(H8) as menaquinones and iso-C15 : 0, anteiso-C15 : 0, iso-C16 : 0 and C16 : 0 as major fatty acids. Diphosphatidylglycerol, phosphatidylethanolamine, hydroxyphosphatidylethanolamine, phosphatidylglycerol and phosphatidylinositol were detected in the cells. These characteristics were consistent the typical chemotaxonomic properties of members the genus Streptomyces. The taxonomic affiliation at the genus level of this strain could be confirmed using its 16S rRNA gene sequence data. CA1R205T showed the highest 16S rRNA gene sequence similarity value to Streptomyces rapamycinicus NRRL B-5491T (98.9 %), followed by Streptomyces iranensis HM 35T (98.8 %). Digital DNA-DNA hybridization and average nucleotide identity-by blast (ANIb) values between CA1R205T and S. rapamycinicus NRRL B-5491T were 27.2 and 81.5 %, respectively. The DNA G+C content of genomic DNA was 70.7 mol%. Due to the differences in physiological, biochemical and genotypic data, CA1R205T could be discriminated from its closest neighbour. Thus, CA1R205T should be recognized as representing a novel species of the genus Streptomyces, for which the name Streptomyces coffeae sp. nov. is proposed. The type strain is CA1R205T (=TBRC 11244T=NBRC 114295T).


Assuntos
Coffea/microbiologia , Endófitos/isolamento & purificação , Raízes de Plantas/microbiologia , Streptomyces/isolamento & purificação , Composição de Bases/genética , Sequência de Bases , DNA Bacteriano/genética , Endófitos/genética , Funções Verossimilhança , Filogenia , RNA Ribossômico 16S/genética , Streptomyces/classificação , Tailândia
9.
Nat Commun ; 12(1): 3297, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34078885

RESUMO

Using the Cap Analysis of Gene Expression (CAGE) technology, the FANTOM5 consortium provided one of the most comprehensive maps of transcription start sites (TSSs) in several species. Strikingly, ~72% of them could not be assigned to a specific gene and initiate at unconventional regions, outside promoters or enhancers. Here, we probe these unassigned TSSs and show that, in all species studied, a significant fraction of CAGE peaks initiate at microsatellites, also called short tandem repeats (STRs). To confirm this transcription, we develop Cap Trap RNA-seq, a technology which combines cap trapping and long read MinION sequencing. We train sequence-based deep learning models able to predict CAGE signal at STRs with high accuracy. These models unveil the importance of STR surrounding sequences not only to distinguish STR classes, but also to predict the level of transcription initiation. Importantly, genetic variants linked to human diseases are preferentially found at STRs with high transcription initiation level, supporting the biological and clinical relevance of transcription initiation at STRs. Together, our results extend the repertoire of non-coding transcription associated with DNA tandem repeats and complexify STR polymorphism.


Assuntos
Repetições de Microssatélites , Redes Neurais de Computação , Doenças Neurodegenerativas/genética , Sítio de Iniciação de Transcrição , Iniciação da Transcrição Genética , Células A549 , Animais , Sequência de Bases , Biologia Computacional/métodos , Aprendizado Profundo , Elementos Facilitadores Genéticos , Genoma Humano , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Camundongos , Doenças Neurodegenerativas/diagnóstico , Doenças Neurodegenerativas/metabolismo , Polimorfismo Genético , Regiões Promotoras Genéticas
10.
Ann Saudi Med ; 41(3): 141-146, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085542

RESUMO

BACKGROUND: Angiotensin-converting enzyme (ACE) insertion/deletion (I/D) polymorphism may play a role in the pathogenesis of coronavirus-19 disease (COVID-19). OBJECTIVES: Investigate the relationship between ACE I/D polymorphism and the clinical severity of COVID-19. DESIGN: Prospective cohort study. SETTING: Tertiary care hospital. PATIENTS AND METHODS: The study included COVID-19 patients with asymptomatic, mild, and severe disease with clinical data and whole blood samples collected from 1 April 2020 to 1 July 2020. ACE I/D genotypes were determined by polymerase chain reaction and agarose gel electrophoresis. MAIN OUTCOME MEASURE: ACE DD, DI and II genotypes frequencies. SAMPLE SIZE: 90 cases, 30 in each disease severity group. RESULTS: Age and the frequency of general comorbidity increased significantly from the asymptomatic disease group to the severe disease group. Advanced age, diabetes mellitus and presence of ischemic heart disease were independent risk factors for severe COVID-19 [OR and 95 % CI: 1.052 (1.021-1.083), 5.204 (1.006-26.892) and 5.922 (1.109-31.633), respectively]. The ACE II genotype was the dominant genotype (50%) in asymptomatic patients, while the DD genotype was the dominant genotype (63.3 %) in severe disease. The ACE II geno-type was protective against severe COVID-19 [OR and 95% CI: .323 (.112-.929)]. All nine patients (8.9%) who died had severe disease. CONCLUSIONS: The clinical severity of COVID-19 infection may be associated with the ACE I/D polymorphism. LIMITATIONS: Small sample size and single center. CONFLICT OF INTEREST: None.


Assuntos
COVID-19/genética , Peptidil Dipeptidase A/genética , Polimorfismo Genético , Índice de Gravidade de Doença , Adulto , Idoso , Sequência de Bases , COVID-19/diagnóstico , Feminino , Seguimentos , Marcadores Genéticos , Genótipo , Técnicas de Genotipagem , Humanos , Masculino , Pessoa de Meia-Idade , Mutagênese Insercional , Estudos Prospectivos , Deleção de Sequência
11.
Nat Commun ; 12(1): 3431, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34103499

RESUMO

The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We demonstrate that despite the large size of the viral RNA genome (~30 kb), infectious full-length cDNA is readily assembled in vitro by a circular polymerase extension reaction (CPER) methodology without the need for technically demanding intermediate steps. Overlapping cDNA fragments are generated from viral RNA and assembled together with a linker fragment containing CMV promoter into a circular full-length viral cDNA in a single reaction. Transfection of the circular cDNA into mammalian cells results in the recovery of infectious SARS-CoV-2 virus that exhibits properties comparable to the parental virus in vitro and in vivo. CPER is also used to generate insect-specific Casuarina virus with ~20 kb genome and the human pathogens Ross River virus (Alphavirus) and Norovirus (Calicivirus), with the latter from a clinical sample. Additionally, reporter and mutant viruses are generated and employed to study virus replication and virus-receptor interactions.


Assuntos
Genética Reversa , SARS-CoV-2/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Chlorocebus aethiops , Culicidae/virologia , Furina/metabolismo , Genoma Viral , Células HEK293 , Humanos , Camundongos , Mutação/genética , Células NIH 3T3 , Reação em Cadeia da Polimerase , Células RAW 264.7 , Receptores Virais/metabolismo , Células Vero , Proteínas Virais/química , Replicação Viral
12.
Nat Commun ; 12(1): 3436, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34103525

RESUMO

Clostridioides difficile infections are an urgent medical problem. The newly discovered C. difficile adenine methyltransferase A (CamA) is specified by all C. difficile genomes sequenced to date (>300), but is rare among other bacteria. CamA is an orphan methyltransferase, unassociated with a restriction endonuclease. CamA-mediated methylation at CAAAAA is required for normal sporulation, biofilm formation, and intestinal colonization by C. difficile. We characterized CamA kinetic parameters, and determined its structure bound to DNA containing the recognition sequence. CamA contains an N-terminal domain for catalyzing methyl transfer, and a C-terminal DNA recognition domain. Major and minor groove DNA contacts in the recognition site involve base-specific hydrogen bonds, van der Waals contacts and the Watson-Crick pairing of a rearranged A:T base pair. These provide sufficient sequence discrimination to ensure high specificity. Finally, the surprisingly weak binding of the methyl donor S-adenosyl-L-methionine (SAM) might provide avenues for inhibiting CamA activity using SAM analogs.


Assuntos
Adenina/metabolismo , Clostridioides/enzimologia , DNA Bacteriano/química , Conformação de Ácido Nucleico , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , Pareamento de Bases , Sequência de Bases , Coenzimas/metabolismo , Modelos Moleculares , Motivos de Nucleotídeos , S-Adenosil-Homocisteína/metabolismo , DNA Metiltransferases Sítio Específica (Adenina-Específica)/química , Especificidade da Espécie , Especificidade por Substrato
13.
Nat Commun ; 12(1): 2675, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33976148

RESUMO

Developing molecules that emulate the properties of naturally occurring ice-binding proteins (IBPs) is a daunting challenge. Rather than relying on the (limited) existing structure-property relationships that have been established for IBPs, here we report the use of phage display for the identification of short peptide mimics of IBPs. To this end, an ice-affinity selection protocol is developed, which enables the selection of a cyclic ice-binding peptide containing just 14 amino acids. Mutational analysis identifies three residues, Asp8, Thr10 and Thr14, which are found to be essential for ice binding. Molecular dynamics simulations reveal that the side chain of Thr10 hydrophobically binds to ice revealing a potential mechanism. To demonstrate the biotechnological potential of this peptide, it is expressed as a fusion ('Ice-Tag') with mCherry and used to purify proteins directly from cell lysate.


Assuntos
Proteínas Anticongelantes/genética , Técnicas de Visualização da Superfície Celular/métodos , Mutação , Peptídeos Cíclicos/genética , Aminoácidos/química , Aminoácidos/genética , Aminoácidos/metabolismo , Proteínas Anticongelantes/química , Proteínas Anticongelantes/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Cristalização , Interações Hidrofóbicas e Hidrofílicas , Gelo , Simulação de Dinâmica Molecular , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos
14.
Nat Commun ; 12(1): 2668, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33976151

RESUMO

Telomeres are bound by dedicated proteins, which protect them from DNA damage and regulate telomere length homeostasis. In the nematode Caenorhabditis elegans, a comprehensive understanding of the proteins interacting with the telomere sequence is lacking. Here, we harnessed a quantitative proteomics approach to identify TEBP-1 and TEBP-2, two paralogs expressed in the germline and embryogenesis that associate to telomeres in vitro and in vivo. tebp-1 and tebp-2 mutants display strikingly distinct phenotypes: tebp-1 mutants have longer telomeres than wild-type animals, while tebp-2 mutants display shorter telomeres and a Mortal Germline. Notably, tebp-1;tebp-2 double mutant animals have synthetic sterility, with germlines showing signs of severe mitotic and meiotic arrest. Furthermore, we show that POT-1 forms a telomeric complex with TEBP-1 and TEBP-2, which bridges TEBP-1/-2 with POT-2/MRT-1. These results provide insights into the composition and organization of a telomeric protein complex in C. elegans.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Telômero/metabolismo , Animais , Animais Geneticamente Modificados , Sequência de Bases , Sítios de Ligação/genética , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/classificação , Proteínas de Caenorhabditis elegans/genética , DNA/genética , Proteínas de Ligação a DNA/genética , Células Germinativas/metabolismo , Microscopia de Fluorescência/métodos , Complexos Multiproteicos/genética , Mutação , Filogenia , Ligação Proteica , Isoformas de Proteínas/classificação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Telômero/genética , Proteínas de Ligação a Telômeros/classificação , Proteínas de Ligação a Telômeros/genética
15.
Nat Commun ; 12(1): 2661, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33976182

RESUMO

Precursor messenger RNA (pre-mRNA) splicing is an essential and tightly regulated process in eukaryotic cells; however, the regulatory mechanisms for the splicing are not well understood. Here, we characterize a RNA binding protein named FgRbp1 in Fusarium graminearum, a fungal pathogen of cereal crops worldwide. Deletion of FgRbp1 leads to reduced splicing efficiency in 47% of the F. graminearum intron-containing gene transcripts that are involved in various cellular processes including vegetative growth, development, and virulence. The human ortholog RBM42 is able to fully rescue the growth defects of ΔFgRbp1. FgRbp1 binds to the motif CAAGR in its target mRNAs, and interacts with the splicing factor FgU2AF23, a highly conserved protein involved in 3' splice site recognition, leading to enhanced recruitment of FgU2AF23 to the target mRNAs. This study demonstrates that FgRbp1 is a splicing regulator and regulates the pre-mRNA splicing in a sequence-dependent manner in F. graminearum.


Assuntos
Proteínas Fúngicas/metabolismo , Fusarium/metabolismo , Precursores de RNA/metabolismo , Splicing de RNA , Proteínas de Ligação a RNA/metabolismo , Fator de Processamento U2AF/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Grão Comestível/microbiologia , Proteínas Fúngicas/genética , Fusarium/genética , Fusarium/patogenicidade , Humanos , Íntrons/genética , Ligação Proteica , Precursores de RNA/genética , Sítios de Splice de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Virulência
16.
BMC Infect Dis ; 21(1): 464, 2021 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-34020601

RESUMO

BACKGROUND: Leishmaniasis is one of the most neglected tropical diseases in the world and remains endemic in some underdeveloped regions, including western China. The phylogeny and classification of Chinese Leishmania has not been completely clarified to date, especially within the Leishmania (L.) donovani complex, although phylogenetic analyses based on a series of gene markers have been performed. More analytic methods and data are still needed. Random amplified polymorphic DNA (RAPD) technology can sensitively identify slight intraspecific differences, and it is a powerful tool to seek species-specific markers. This work attempted to identify Chinese Leishmania isolates from diverse geographic regions at the genomic level. Meanwhile, specific markers of the L. donovani complex were also developed by RAPD. METHODS: RAPD was applied to 14 Chinese Leishmania isolates from diverse geographic regions and 3 WHO reference strains. The polymorphic sites of amplification were transformed into a data matrix, based on which genetic similarity was calculated, and a UPGMA dendrogram was constructed to analyse the genetic diversity of these Leishmania isolates. Meanwhile, the specific amplification loci of the L. donovani complex were TA-cloned, sequenced and converted into sequence characterized amplified region (SCAR) markers, which were validated preliminarily in 17 available Leishmania strains in this study and analysed by bioinformatics. RESULTS: The cluster analyses showed that the three Leishmania sp. isolates SC10H2, SD and GL clustered together and apart from others, the strains of the L. donovani complex clearly divided into two clades, and the three isolates Cy, WenChuan and 801 formed a subclade. Three specific SCAR markers of the L. donovani complex, i.e., 1-AD17, 2-A816 and 3-O13, were successfully obtained and validated on 17 available Leishmania strains in this study. Through bioinformatic analyses, Marker 1-AD17 may have more specificity for PCR detection of VL, and Marker 3-O13 has the potential to encode a protein. CONCLUSIONS: The RAPD results verified that the undescribed Leishmania species causing visceral leishmaniasis (VL) in China was a unique clade distinguished from L. donovani and revealed that there was genetic differentiation among Chinese L. donovani. The identification of L. donovani-specific markers may help to provide a foundation for future research attempting to develop new specific diagnostic markers of VL and identify specific gene functions.


Assuntos
Variação Genética , Leishmania donovani/classificação , Leishmania donovani/genética , Leishmaniose Visceral/epidemiologia , Técnica de Amplificação ao Acaso de DNA Polimórfico/métodos , Animais , Sequência de Bases , China/epidemiologia , Análise por Conglomerados , DNA de Protozoário/genética , DNA de Protozoário/isolamento & purificação , Marcadores Genéticos , Humanos , Leishmania donovani/isolamento & purificação , Leishmaniose Visceral/parasitologia , Filogenia , Reação em Cadeia da Polimerase , Especificidade da Espécie
17.
J Am Chem Soc ; 143(22): 8333-8343, 2021 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-34039006

RESUMO

The 5' untranslated region (UTR) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome is a conserved, functional and structured genomic region consisting of several RNA stem-loop elements. While the secondary structure of such elements has been determined experimentally, their three-dimensional structures are not known yet. Here, we predict structure and dynamics of five RNA stem loops in the 5'-UTR of SARS-CoV-2 by extensive atomistic molecular dynamics simulations, more than 0.5 ms of aggregate simulation time, in combination with enhanced sampling techniques. We compare simulations with available experimental data, describe the resulting conformational ensembles, and identify the presence of specific structural rearrangements in apical and internal loops that may be functionally relevant. Our atomic-detailed structural predictions reveal a rich dynamics in these RNA molecules, could help the experimental characterization of these systems, and provide putative three-dimensional models for structure-based drug design studies.


Assuntos
COVID-19/virologia , RNA Viral/química , SARS-CoV-2/genética , Regiões 5' não Traduzidas , Sequência de Bases , Genoma Viral , Humanos , Simulação de Dinâmica Molecular , Estrutura Molecular , Conformação de Ácido Nucleico , RNA Viral/genética , SARS-CoV-2/química
18.
Curr Protoc ; 1(5): e108, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33945676

RESUMO

The preparation of controlled pore glass (CPG) supports, functionalized with several hexaethylene glycol spacers, to alleviate the problems associated with the porosity of commercial CPG supports is described in this article. The pore size of CPG restricts the diffusion of reagents to the leader nucleoside embedded in porous supports; this inhibits efficient solid-phase syntheses of DNA and RNA sequences and, by default, the purity of those sequences through formation of a shorter than full-length oligonucleotide. Functionalization of a CPG support with five hexaethylene glycol spacers led to a 42% reduction in process-related impurities contaminating oligonucleotide sequences, compared to that obtained using the commercial long-chain alkylamine (LCAA) CPG support. © 2021 Wiley Periodicals LLC. This article has been contributed to by US Government employees and their work is in the public domain in the USA. Basic Protocol 1: Preparation of the hydroxylated CPG support 3 Basic Protocol 2: Automated preparation of the CPG support 6 Basic Protocol 3: Automated preparation of the poly(hexaethylene glycol)-derived CPG 7 Basic Protocol 4: Automated functionalization of the poly(hexaethylene glycol)-derived CPG support 7 with leader deoxyribo- and ribonucleosides to provide the CPG support 9 Basic Protocol 5: Automated syntheses of DNA and RNA sequences on poly(hexaethylene glycol)-derived CPG support 9 and on a commercial long-chain alkylamine (LCAA) CPG support Support Protocol: Release and deprotection of the DNA and RNA sequences linked to the poly(hexaethylene glycol)-derived CPG support 10 and commercial LCAA-CPG support Basic Protocol 6: Comparative RP-HPLC analyses of crude, fully deprotected DNA or RNA sequences released from the poly(hexaethylene glycol)-derived CPG support 10 and from a commercial LCAA-CPG support.


Assuntos
DNA , Técnicas de Síntese em Fase Sólida , Sequência de Bases , Vidro , Oligonucleotídeos
19.
Biomed Res Int ; 2021: 6699910, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33937412

RESUMO

Cartilage injury of the knee joint is very common. Due to the limited self-healing ability of articular cartilage, osteoarthritis is very likely to occur if left untreated. Bone marrow mesenchymal stem cells (BMMSCs) are widely used in the study of cartilage injury due to their low immunity and good amplification ability, but they still have disadvantages, such as heterogeneous undifferentiated cells. MicroRNAs can regulate the chondrogenic differentiation ability of MSCs by inhibiting or promoting mRNA translation and degradation. In this research, we primarily investigated the effect of microRNA-210-3p (miR-210-3p) on chondrogenic and adipogenic differentiation of BMMSCs in vitro. Our results demonstrate that miR-210-3p promoted chondrogenic differentiation and inhibited adipogenic differentiation of rat BMMSCs, which was related to the HIF-3α signalling pathway. Additionally, miR-210-3p promotes mRNA and protein levels of the chondrogenic expression genes COLII and SOX9 and inhibits mRNA and protein levels of the adipogenic expression genes PPARγ and LPL. Thus, miR-210-3p combined with BMMSCs is a candidate for future clinical applications in cartilage regeneration and could represent a promising new therapeutic target for OA.


Assuntos
Adipogenia/genética , Condrogênese/genética , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Masculino , MicroRNAs/genética , Modelos Biológicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Fatores de Transcrição/genética
20.
Biomed Res Int ; 2021: 7509825, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33969125

RESUMO

Juvenile myoclonic epilepsy (JME) is the most prevalent and genetically heterogeneous form of epilepsy and accounts for 10-30% of all the cases worldwide. Ef-hand domain- (c-terminal-) containing protein 1 (EFHC1) encodes for a nonion channel protein and mutations in this gene have been extensively reported in different populations to play a causative role in JME. Linkage between JME and 6p11-12 locus has already been confirmed in Mexican and Dutch families. A case-control study was conducted on Pakistani JME patients for the first time, aimed at finding out EFHC1 mutations that have been reported in different populations. For this purpose, 66 clinically diagnosed JME patients and 108 control subjects were included in the study. Blood samples were collected from all the participants, and DNA was isolated from the lymphocytes by the modified organic method. Total 3 exons of EFHC1, harboring extensively reported mutations, were selected for genotypic analysis. We identified three heterozygous variants, R159W, V460A, P436P, and one insertion in the current study. V460A, an uncommon variant identified herein, has recently been reported in public databases in an unphenotyped American individual. This missense variant was found in 3 Pakistani JME patients from 2 unrelated families. However, in silico analysis showed that V460A may possibly be a neutral variant. While the absence of a majority of previously reported mutations in our population suggests that most of the mutations of EFHC1 are confined to particular ethnicities and are not evenly distributed across the world. However, to imply the causation, the whole gene and larger number of JME patients should be screened in this understudied population.


Assuntos
Proteínas de Ligação ao Cálcio/genética , Epilepsia Mioclônica Juvenil/genética , Adolescente , Sequência de Bases , Análise Mutacional de DNA , Éxons/genética , Feminino , Humanos , Masculino , Mutação/genética , Paquistão
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