RESUMO
Approximately 250 million people worldwide are chronically infected with the hepatitis B virus (HBV) and are at increased risk of developing cirrhosis and hepatocellular carcinoma. The HBV genome persists as covalently closed circular DNA (cccDNA), which serves as the template for all HBV mRNA transcripts. Current nucleos(t)ide analogs used to treat HBV do not directly target the HBV cccDNA genome and thus cannot eradicate HBV infection. Here, we report the discovery of a unique G-quadruplex structure in the pre-core promoter region of the HBV genome that is conserved among nearly all genotypes. This region is central to critical steps in the viral life cycle, including the generation of pregenomic RNA, synthesis of core and polymerase proteins, and genome encapsidation; thus, an increased understanding of the HBV pre-core region may lead to the identification of novel anti-HBV cccDNA targets. We utilized biophysical methods (circular dichroism and small-angle X-ray scattering) to characterize the HBV G-quadruplex and the effect of three distinct G to A mutants. We also used microscale thermophoresis to quantify the binding affinity of G-quadruplex and its mutants with a known quadruplex-binding protein (DHX36). To investigate the physiological relevance of HBV G-quadruplex, we employed assays using DHX36 to pull-down cccDNA and compared HBV infection in HepG2 cells transfected with wild-type and mutant HBV plasmids by monitoring the levels of genomic DNA, pregenomic RNA, and antigens. Further evaluation of this critical host-protein interaction site in the HBV cccDNA genome may facilitate the development of novel anti-HBV therapeutics against the resilient cccDNA template.
Assuntos
DNA Circular/química , DNA Circular/genética , Quadruplex G , Vírus da Hepatite B/genética , Regiões Promotoras Genéticas/genética , Células Hep G2 , Humanos , MutaçãoRESUMO
Fungi of the genus Colletotrichum are economically important and are used as models in plant-pathogen interaction studies. In this study, the complete mitochondrial genomes of two Colletotrichum lindemuthianum isolates were sequenced and compared with the mitochondrial genomes of seven species of Colletotrichum. The mitochondrial genome of C. lindemuthianum is a typical circular molecule 37,446 bp (isolate 89 A2 2-3) and 37,440 bp (isolate 83.501) in length. The difference of six nucleotides between the two genomes is the result of a deletion in the ribosomal protein S3 (rps3) gene in the 83.501 isolate. In addition, substitution of adenine for guanine within the rps3 gene in the mitochondrial genome of the 83.501 isolate was observed. Compared to the previously sequenced C. lindemuthianum mitochondrial genome, an exon no annotated in the cytochrome c oxidase I (cox1) gene and a non-conserved open reading frame (ncORF) were observed. The size of the mitochondrial genomes of the seven species of Colletotrichum was highly variable, being attributed mainly to the ncORF, ranging from one to 10 and also from introns ranging from one to 11 and which encode a total of up to nine homing endonucleases. This paper reports for the first time by means of transcriptome that then ncORFs are transcribed in Colletotrichum spp. Phylogeny data revealed that core mitochondrial genes could be used as an alternative in phylogenetic relationship studies in Colletotrichum spp. This work contributes to the genetic and biological knowledge of Colletotrichum spp., which is of great economic and scientific importance.
Assuntos
Colletotrichum/genética , Genoma Mitocondrial , Colletotrichum/isolamento & purificação , DNA Circular/química , DNA Circular/genética , DNA Mitocondrial/química , DNA Mitocondrial/genética , Éxons , Genes Mitocondriais , Tamanho do Genoma , Fases de Leitura Aberta , Phaseolus/microbiologia , Doenças das Plantas/microbiologia , Mutação Puntual , Análise de Sequência de DNA , Deleção de SequênciaRESUMO
Given their distribution, importance, and richness, Myrtaceae species comprise a model system for studying the evolution of tropical plant diversity. In addition, chloroplast (cp) genome sequencing is an efficient tool for phylogenetic relationship studies. Feijoa [Acca sellowiana (O. Berg) Burret; CN: pineapple-guava] is a Myrtaceae species that occurs naturally in southern Brazil and northern Uruguay. Feijoa is known for its exquisite perfume and flavorful fruits, pharmacological properties, ornamental value and increasing economic relevance. In the present work, we reported the complete cp genome of feijoa. The feijoa cp genome is a circular molecule of 159,370 bp with a quadripartite structure containing two single copy regions, a Large Single Copy region (LSC 88,028 bp) and a Small Single Copy region (SSC 18,598 bp) separated by Inverted Repeat regions (IRs 26,372 bp). The genome structure, gene order, GC content and codon usage are similar to those of typical angiosperm cp genomes. When compared to other cp genome sequences of Myrtaceae, feijoa showed closest relationship with pitanga (Eugenia uniflora L.). Furthermore, a comparison of pitanga synonymous (Ks) and nonsynonymous (Ka) substitution rates revealed extremely low values. Maximum Likelihood and Bayesian Inference analyses produced phylogenomic trees identical in topology. These trees supported monophyly of three Myrtoideae clades.
Assuntos
DNA Circular/genética , Feijoa/genética , Genoma de Cloroplastos/genética , Myrtaceae/genética , Composição de Bases/genética , Teorema de Bayes , Brasil , Códon/genética , DNA Circular/química , Feijoa/classificação , Ordem dos Genes , Genes de Cloroplastos/genética , Cadeias de Markov , Método de Monte Carlo , Mutação , Myrtaceae/classificação , Filogenia , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNARESUMO
In the present study, the complete mitochondrial (mt) genome of Cyclemys dentata was determined using PCR reactions. The structural organization and gene order of C. dentata were equivalent to those of most other vertebrates. The mt genome was 16,489 bp in length, has rich A+T content, consisting of 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes, and a control region (D-loop). All protein-coding genes started with ATG, many genes have complete stop codons, except ND2, COX3, ND3, and cyt-b genes had incomplete stop codons of T. The light-strand replication origin (OL) of C. dentata might fold into a stable stem-loop secondary structure, and its loop had 2 nt less than that of the Cyclemys atripons OL sequence. The D-Loop of C. dentata contained a central domain (CD), 2 extended termination associated sequences (ETAS1, ETAS2) and 3 conserved sequence blocks (CSB1, CSB2, CSB3). The average length of 20 turtles' mt genomes was 16,692.5 bp, including 34.1% A, 27.0% T, 26.0% C and 12.9% G. The C. dentata mitochondrial genome could provide useful data for further studies on phylogenetics and conservation genetics of this species. The phylogenetic relationships of the family Geoemydidae were analyzed by maximum-likelihood (ML) and neighbor-joining (NJ) based on concatenated sequences of 13 protein-coding genes from 20 turtle species. The ML and NJ trees had homologous topologies. The results support the existing classification of the genera of Geoemydidae, that C. dentata was a sister species of C. atripons, Pyxidea nested in Cuora, and Chinemys was synonymous with Mauremys.
Assuntos
DNA Mitocondrial/genética , Genoma Mitocondrial/genética , Filogenia , Tartarugas/genética , Animais , Sequência de Bases , DNA Circular/química , DNA Circular/genética , DNA Mitocondrial/química , DNA Mitocondrial/classificação , Ordem dos Genes , Genes Mitocondriais/genética , Proteínas Mitocondriais/genética , Dados de Sequência Molecular , Análise de Sequência de DNA , Tartarugas/classificaçãoRESUMO
Spodoptera frugiperda (Lepidoptera: Noctuidae) is a major pest in maize crops in Colombia, and affects several regions in America. A granulovirus isolated from S. frugiperda (SfGV VG008) has potential as an enhancer of insecticidal activity of previously described nucleopolyhedrovirus from the same insect species (SfMNPV). The SfGV VG008 genome was sequenced and analyzed showing circular double stranded DNA of 140,913 bp encoding 146 putative ORFs that include 37 Baculoviridae core genes, 88 shared with betabaculoviruses, two shared only with betabaculoviruses from Noctuide insects, two shared with alphabaculoviruses, three copies of own genes (paralogs) and the other 14 corresponding to unique genes without representation in the other baculovirus species. Particularly, the genome encodes for important virulence factors such as 4 chitinases and 2 enhancins. The sequence analysis revealed the existence of eight homologous regions (hrs) and also suggests processes of gene acquisition by horizontal transfer including the SfGV VG008 ORFs 046/047 (paralogs), 059, 089 and 099. The bioinformatics evidence indicates that the genome donors of mentioned genes could be alpha- and/or betabaculovirus species. The previous reported ability of SfGV VG008 to naturally co-infect the same host with other virus show a possible mechanism to capture genes and thus improve its fitness.
Assuntos
Baculoviridae/genética , DNA Viral/química , DNA Viral/genética , Genoma Viral , Spodoptera/virologia , Animais , Baculoviridae/isolamento & purificação , Colômbia , DNA/química , DNA/genética , DNA Circular/química , DNA Circular/genética , Transferência Genética Horizontal , Anotação de Sequência Molecular , Dados de Sequência Molecular , Fases de Leitura Aberta , Recombinação Genética , Análise de Sequência de DNA , Homologia de Sequência , Fatores de Virulência/genéticaRESUMO
Circomics (circular DNA genomics), the combination of rolling circle amplification (RCA), restriction fragment length polymorphism (RFLP) analysis and pyro-sequencing, has been used recently to identify geminiviruses with high efficiency and low costs. Circular DNAs associated with Cuban geminiviruses were characterised by RCA/RFLP analysis and 454 sequencing of two batches of DNA amplified from selected plant samples as well as individual cloning and Sanger sequencing of DNA components and compared to other geminiviral DNAs by phylogenetic analysis. Cuban geminiviruses that were closely related to each other challenged the circomics approach. Ten geminiviral components and one alpha-satellite DNA were determined and compared to three geminiviral components obtained by conventional cloning. New strains of Sida yellow mottle virus (SiYMoV), tomato yellow distortion leaf virus (ToYDLV), Sida golden mosaic Florida virus (SiGMFV) and Sida golden mosaic Liguanea virus (SiGMLV) are described with host plant species being classified by molecular PCR-based bar coding. A new virus species is named Peristrophe mosaic virus. The first alpha-satellite found in Middle America establishes the New World branch of these elements which are related to nanoviruses and were previously thought to be restricted to the Old World. In conclusion, circomics is efficient for complex infections and closely related viruses to detected unexpected viral DNAs, but may need some scrutinisation by direct sequencing and cloning of individual components for certain cases.
Assuntos
DNA Circular/isolamento & purificação , DNA Satélite/isolamento & purificação , Geminiviridae/isolamento & purificação , Doenças das Plantas/virologia , Análise por Conglomerados , Cuba , DNA Circular/química , DNA Circular/genética , DNA Satélite/química , DNA Satélite/classificação , DNA Satélite/genética , Geminiviridae/química , Geminiviridae/classificação , Geminiviridae/genética , Dados de Sequência Molecular , Técnicas de Amplificação de Ácido Nucleico , Filogenia , Plantas/virologia , Polimorfismo de Fragmento de Restrição , Análise de Sequência de DNA , Homologia de Sequência do Ácido NucleicoRESUMO
Previous studies on copper(II) complexes with oxindole-Schiff base ligands have shown their potential antitumor activity towards different cells, inducing apoptosis through a preferential attack to DNA and/or mitochondria. Herein, we better characterize the interactions between some of these copper(II) complexes and DNA. Investigations on its binding ability to DNA were carried out by fluorescence measurements in competitive experiments with ethidium bromide, using plasmidial or calf-thymus DNA. These results indicated an efficient binding process similar to that observed with copper(II)-phenanthroline species, [Cu(o-phen)(2)](2+), with binding constants in the range 3 to 9×10(2) M(-1). DNA cleavage experiments in the presence and absence of distamycin, a recognized binder of DNA, indicated that this binding probably occurs at major or minor groove, leading to double-strand DNA cleavage, and being modulated by the imine ligand. Corroborating these data, discrete changes in EPR spectra of the studied complexes were observed in the presence of DNA, while more remarkable changes were observed in the presence of nucleotides (AMP, GMP, CMP or UMP). Additional evidence for preferential coordination of the copper centers to the bases guanine or cytosine was obtained from titrations of these complexes with each nucleotide, monitored by absorption spectral changes. Therefore, the obtained data point out to their action as groove binders to DNA bases, rather than as intercalators or covalent cross-linkers. Further investigations by SDS PAGE using (32)P-ATP or (32)P-oligonucleotides attested that no hydrolysis of phosphate linkage in DNA or RNA occurs, in the presence of such complexes, confirming their main oxidative mechanism of action.
Assuntos
Complexos de Coordenação/química , Cobre , DNA Circular/química , DNA/química , Indóis/química , Algoritmos , Ligação Competitiva , Dicroísmo Circular , Clivagem do DNA , Distamicinas/química , Espectroscopia de Ressonância de Spin Eletrônica , Etídio/química , Substâncias Intercalantes/química , Oxindóis , Bases de Schiff/química , Espectrometria de FluorescênciaRESUMO
Three platinum-chloroquine complexes, trans-Pt(CQDP)(2)(I)(2) [1], trans-Pt(CQDP)(2)(Cl)(2) [2] and trans-Pt(CQ)(2)(Cl)(2) [3], were prepared and their most probable structure was established through a combination of spectroscopic analysis and density functional theory (DFT) calculations. Their interaction with DNA was studied and their activity against 6 tumor cell lines was evaluated. Compounds 1 and 2 interact with DNA primarily through electrostatic contacts and hydrogen bonding, with a minor contribution of a covalent interaction, while compound 3 binds to DNA predominantly in a covalent fashion, with weaker secondary electrostatic interactions and possibly hydrogen bonding, this complex also exerted greater cytotoxic activity against the tumor cell lines.
Assuntos
Antineoplásicos/síntese química , Quelantes/química , Cloroquina/química , Complexos de Coordenação/síntese química , Platina , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Complexos de Coordenação/farmacologia , DNA , Clivagem do DNA , DNA Circular/química , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Concentração Inibidora 50 , Camundongos , Modelos Moleculares , Conformação MolecularRESUMO
In the search for new therapeutic tools against diseases produced by kinetoplastid parasites five vanadyl complexes, [V(IV)O(L-2H)(phen)], including 1,10-phenanthroline (phen) and tridentate salicylaldehyde semicarbazone derivatives as ligands have been synthesized and characterized in the solid state and in solution by using different techniques. EPR suggested a distorted octahedral geometry with the tridentate semicarbazone occupying three equatorial positions and phen coordinated in an equatorial/axial mode. The compounds were evaluated in vitro on epimastigotes of Trypanosoma cruzi, causative agent of Chagas disease, Leishmania panamensis and Leishmania chagasi and on tumor cells. The complexes showed higher in vitro anti-trypanosomal activities than the reference drug Nifurtimox (IC(50) values in the range 1.6-3.8 µM) and increased activities in respect to the free semicarbazone ligands. In vitro activity on promastigote and amastigote forms of Leishmania showed interesting results. The compounds [VO(L1-2H)(phen)] and [VO(L3-2H)(phen)], where L1 = 2-hydroxybenzaldehyde semicarbazone and L3 = 2-hydroxy-3-methoxybenzaldehyde semicarbazone, resulted active (IC(50) 2.74 and 2.75 µM, respectively, on promastigotes of L. panamensis; IC(50) 19.52 and 20.75 µM, respectively, on intracellular amastigotes of L. panamensis) and showed low toxicity on THP-1 mammalian cells (IC(50) 188.55 and 88.13 µM, respectively). In addition, the complexes showed cytotoxicity on human promyelocytic leukemia HL-60 cells with IC(50) values of the same order of magnitude as cisplatin. The interaction of the complexes with DNA was demonstrated by different techniques, suggesting that this biomolecule could be a potential target either in the parasites or in tumor cells.
Assuntos
Antineoplásicos/síntese química , Complexos de Coordenação/síntese química , Tripanossomicidas/síntese química , Vanádio , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Complexos de Coordenação/farmacologia , DNA/química , DNA Circular/química , Ensaios de Seleção de Medicamentos Antitumorais , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Concentração Inibidora 50 , Leishmania/efeitos dos fármacos , Relação Estrutura-Atividade , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacosRESUMO
The effect of changes in the bulk dielectric constant on the DNA torsional properties was evaluated from plasmid circularization reactions. In these reactions, pUC18 previously linearized by EcoRI digestion was recircularized with T4 DNA ligase. The bulk dielectric constant of the reaction medium was decreased by the addition of different concentrations of neutral solutes: ethylene glycol, glycerol, sorbitol, and sucrose, or increased by the addition of glycine. The topoisomers generated by the ligase reaction were resolved by agarose-gel electrophoresis. The DNA twist energy parameter (kappa), which is an apparent torsional constant, was determined by linearization of the Gaussian topoisomers' distribution. It was observed that the twist energy parameter for the given solutes is almost linearly dependent on the bulk dielectric constant. In the reaction buffer, the twist energy parameter was determined to be 1100 +/- 100. By decreasing the dielectric constant to 74 with the addition of sorbitol, the value of the parameter reaches kappa = 900 +/- 100, whereas the addition of ethylene glycol leads to kappa = 400 +/- 50. Upon addition of glycine, which resulted in a dielectric constant equal to 91, the value of the twist energy parameter increased to kappa = 1750 +/- 100.