RESUMO
Cancer is a serious health problem with a high mortality rate worldwide. Given the relevance of mitochondria in numerous physiological and pathological mechanisms, such as adenosine triphosphate (ATP) synthesis, apoptosis, metabolism, cancer progression and drug resistance, mitochondrial genome (mtDNA) analysis has become of great interest in the study of human diseases, including cancer. To date, a high number of variants and mutations have been identified in different types of tumors, which coexist with normal alleles, a phenomenon named heteroplasmy. This mechanism is considered an intermediate state between the fixation or elimination of the acquired mutations. It is suggested that mutations, which confer adaptive advantages to tumor growth and invasion, are enriched in malignant cells. Notably, many recent studies have reported a heteroplasmy-shifting phenomenon as a potential shaper in tumor progression and treatment response, and we suggest that each cancer type also has a unique mitochondrial heteroplasmy-shifting profile. So far, a plethora of data evidencing correlations among heteroplasmy and cancer-related phenotypes are available, but still, not authentic demonstrations, and whether the heteroplasmy or the variation in mtDNA copy number (mtCNV) in cancer are cause or consequence remained unknown. Further studies are needed to support these findings and decipher their clinical implications and impact in the field of drug discovery aimed at treating human cancer.
Assuntos
Heteroplasmia/genética , Mitocôndrias/genética , Neoplasias/sangue , Neoplasias/genética , Alelos , Biomarcadores/sangue , Enzimas de Restrição do DNA/uso terapêutico , Progressão da Doença , Epigênese Genética , Terapia Genética/métodos , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Neoplasias/metabolismo , Neoplasias/patologia , Microambiente Tumoral/genéticaRESUMO
The morphological identification of mites entails great challenges. Characteristics such as dorsal setae and aedeagus are widely used, but they show variations between populations, and the technique is time consuming and demands specialized taxonomic expertise that is difficult to access. A successful alternative has been to exploit a region of the mitochondrial cytochrome oxidase I (COI) gene to classify specimens to the species level. We analyzed the COI sequences of four mite species associated with cassava and classified them definitively by detailed morphological examinations. We then developed an identification kit based on the restriction fragment length polymorphism-polymerase chain reaction of subunit I of the COI gene focused on the three restriction enzymes AseI, MboII, and ApoI. This set of enzymes permitted the simple, accurate identification of Mononychellus caribbeanae, M. tanajoa, M. mcgregori, and Tetranychus urticae, rapidly and with few resources. This kit could be a vital tool for the surveillance and monitoring of mite pests in cassava crop protection programs in Africa, Asia, and Latin America.
Assuntos
Manihot/parasitologia , Reação em Cadeia da Polimerase/métodos , Tetranychidae/genética , Animais , Proteção de Cultivos/métodos , Enzimas de Restrição do DNA/genética , DNA Mitocondrial/genética , Complexo IV da Cadeia de Transporte de Elétrons/química , Complexo IV da Cadeia de Transporte de Elétrons/genética , Filogenia , Polimorfismo de Fragmento de Restrição , Alinhamento de Sequência , Especificidade da Espécie , Tetranychidae/anatomia & histologia , Tetranychidae/enzimologia , Fatores de TempoRESUMO
OBJECTIVES: To investigate the molecular epidemiology and antimicrobial susceptibility of Clostridioides difficile isolates from patients with C. difficile infection (CDI) from two Phase 3 clinical trials of surotomycin. METHODS: In both trials [Protocol MK-4261-005 (NCT01597505) conducted across Europe, North America and Israel; and Protocol MK-4261-006 (NCT01598311) conducted across North America, Asia-Pacific and South America], patients with CDI were randomized (1:1) to receive oral surotomycin (250 mg twice daily) or oral vancomycin (125 mg four times per day) for 10 days. Stool samples were collected at baseline and C. difficile isolates were characterized by restriction endonuclease analysis (REA) and PCR ribotyping. Susceptibility testing was performed by agar dilution, according to CLSI recommendations. RESULTS: In total, 1147 patients were included in the microbiological modified ITT population. Of 992 recovered isolates, 922 (92.9%) were typed. There was a high association between REA groups and their corresponding predominant PCR ribotype (RT) for BI, DH, G and CF strains. REA group A showed more diverse PCR RTs. Overall, the most common strain was BI/RT027 (20.3%) followed by Y/RT014/020 (15.0%) and DH/RT106 (7.2%). The BI/RT027 strain was particularly prevalent in Europe (29.9%) and Canada (23.6%), with lower prevalence in the USA (16.8%) and Australia/New Zealand (3.4%). Resistance was most prevalent in the BI/RT027 strain, particularly to metronidazole, vancomycin and moxifloxacin. CONCLUSIONS: A wide variation in C. difficile strains, both within and across different geographical regions, was documented by both REA and ribotyping, which showed overall good correlation.
Assuntos
Anti-Infecciosos , Clostridioides difficile , Infecções por Clostridium , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Ásia , Canadá , Clostridioides , Clostridioides difficile/genética , Infecções por Clostridium/tratamento farmacológico , Infecções por Clostridium/epidemiologia , Enzimas de Restrição do DNA , Europa (Continente) , Humanos , Israel , Lipopeptídeos , Testes de Sensibilidade Microbiana , América do Norte , Peptídeos Cíclicos , Reação em Cadeia da Polimerase , Proibitinas , Ribotipagem , América do SulRESUMO
Several studies suggest the relation of DNA methylation to diseases in humans and important phenotypes in plants drawing attention to this epigenetic mark as an important source of variability. In the last decades, several methodologies were developed to assess the methylation state of a genome. However, there is still a lack of affordable and precise methods for genome wide analysis in large sample size studies. Methyl sensitive double digestion MS-DArT sequencing method emerges as a promising alternative for methylation profiling. We developed a computational pipeline for the identification of DNA methylation using MS-DArT-seq data and carried out a pilot study using the Eucalyptus grandis tree sequenced for the species reference genome. Using a statistic framework as in differential expression analysis, 72,515 genomic sites were investigated and 5,846 methylated sites identified, several tissue specific, distributed along the species 11 chromosomes. We highlight a bias towards identification of DNA methylation in genic regions and the identification of 2,783 genes and 842 transposons containing methylated sites. Comparison with WGBS, DNA sequencing after treatment with bisulfite, data demonstrated a precision rate higher than 95% for our approach. The availability of a reference genome is useful for determining the genomic context of methylated sites but not imperative, making this approach suitable for any species. Our approach provides a cost effective, broad and reliable examination of DNA methylation profile on MspI/HpaII restriction sites, is fully reproducible and the source code is available on GitHub (https://github.com/wendelljpereira/ms-dart-seq).
Assuntos
Análise Custo-Benefício , Metilação de DNA/genética , Eucalyptus/genética , Técnicas de Genotipagem/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Folhas de Planta/genética , Análise de Sequência de DNA/métodos , Árvores/genética , Cromossomos de Plantas/genética , Enzimas de Restrição do DNA/genética , Elementos de DNA Transponíveis/genética , Genes de Plantas/genética , Técnicas de Genotipagem/economia , Sequenciamento de Nucleotídeos em Larga Escala/economia , Projetos Piloto , Reprodutibilidade dos Testes , Mapeamento por Restrição , Análise de Sequência de DNA/economia , Sulfitos/farmacologiaRESUMO
Recombinant protein expression is widely used to produce large quantities of protein for diverse uses including functional characterization of selected sequences and vaccination trials. In the postgenomic era, high-throughput techniques that allow us to manipulate several sequences are needed. Cloning by in vivo recombination is a technique that consists in the insertion of a linear DNA into a linearized plasmid DNA by in vivo recombination using a recA+ E. coli strain. This methodology provides high-throughput cloning with high efficiency without the need for restriction enzyme digestion. In this chapter, we describe two protocols for DNA cloning: one using in vivo recombination and the other by using restriction enzymes. We also describe the application of different conditions to produce functional proteins that needs the incorporation of the amino acid selenocysteine (Sec), like thioredoxin-glutathione reductase enzyme.
Assuntos
Códon/genética , Escherichia coli/genética , Proteínas Recombinantes/genética , Clonagem Molecular/métodos , DNA/genética , Enzimas de Restrição do DNA/genética , Plasmídeos/genética , Selenocisteína/genéticaRESUMO
The advent of new DNA sequencing technologies leads to a dramatic increase in the number of available genome sequences and therefore of target genes with potential for functional analysis. The insertion of these sequences into proper expression vectors requires a simple an efficient cloning method. In addition, when expressing a target protein, quite often it is necessary to evaluate different DNA constructs to achieve a soluble and homogeneous expression of the target with satisfactory yields. The development of new molecular methods made possible the cloning of a huge number of DNA sequences in a high-throughput manner, necessary for meeting the increasing demands for soluble protein expression and characterization. In this chapter several molecular methods suitable for high-throughput cloning are reviewed.
Assuntos
Reação em Cadeia da Polimerase/métodos , Clonagem Molecular , Enzimas de Restrição do DNA/genéticaRESUMO
Colletotrichum lindemuthianum, the causal agent of anthracnose, is responsible for significant damage in the common bean (Phaseolus vulgaris L.). Unraveling the genetic mechanisms involved in the plant/pathogen interaction is a powerful approach for devising efficient methods to control this disease. In the present study, we employed the Restriction Enzyme-Mediated Integration (REMI) methodology to identify the gene slnCl1, encoding a histidine kinase protein, as involved in pathogenicity. The mutant strain, MutCl1, generated by REMI, showed an insertion in the slnCl1 gene, deficiency of the production and melanization of appressoria, as well as the absence of pathogenicity on bean leaves when compared with the wild-type strain. The slnCl1 gene encodes a histidine kinase class IV called SlnCl1 showing identity of 97% and 83% with histidine kinases from Colletotrichum orbiculare and Colletotrichum gloesporioides, respectively. RNA interference was used for silencing the histidine kinase gene and confirm slnCl1 as a pathogenicity factor. Furthermore, we identified four major genes involved in the RNA interference-mediated gene silencing in Colletotrichum spp. and demonstrated the functionality of this process in C. lindemuthianum. Silencing of the EGFP reporter gene and slnCl1 were demonstrated using qPCR. This work reports for the first time the isolation and characterization of a HK in C. lindemuthianum and the occurrence of gene silencing mediated by RNA interference in this organism, demonstrating its potential use in the functional characterization of pathogenicity genes.
Assuntos
Colletotrichum/enzimologia , Colletotrichum/patogenicidade , Histidina Quinase/genética , Phaseolus/crescimento & desenvolvimento , Doenças das Plantas/microbiologia , Folhas de Planta/crescimento & desenvolvimento , Sequência de Aminoácidos , Colletotrichum/genética , Enzimas de Restrição do DNA/metabolismo , Histidina Quinase/metabolismo , Mutagênese Insercional , Phaseolus/microbiologia , Doenças das Plantas/terapia , Folhas de Planta/microbiologia , Interferência de RNA , RNA Interferente Pequeno/genéticaRESUMO
Background: The whole-genome sequences of nine Rhizobium species were evaluated using different in silico molecular techniques such as AFLP-PCR, restriction digest, and AMPylating enzymes. The entire genome sequences were aligned with progressiveMauve and visualized by reconstructing phylogenetic tree using NTSYS pc 2.11X. The "insilico.ehu.es" was used to carry out in silico AFLP-PCR and in silico restriction digest of the selected genomes. Post-translational modification (PTM) and AMPylating enzyme diversity between the proteome of Rhizobium species were determined by novPTMenzy. Results: Slight variations were observed in the phylogeny based on AFLP-PCR and PFGE and the tree based on whole genome. Results clearly demonstrated the presence of PTMs, i.e., AMPylation with the GS-ATasE (GlnE), Hydroxylation, Sulfation with their domain, and Deamidation with their specific domains (AMPylating enzymes) GS-ATasE (GlnE), Fic, and Doc (Phosphorylation); Asparagine_hydroxylase and Collagen_prolyl_lysyl_hydroxylase; Sulfotransferase; and CNF (Cytotoxic Necrotizing Factors), respectively. The results pertaining to PTMs are discussed with regard to functional diversities reported in these species. Conclusions: The phylogenetic tree based on AFLP-PCR was slightly different from restriction endonuclease- and PFGE-based trees. Different PTMs were observed in the Rhizobium species, and the most prevailing type of PTM was AMPylation with the domain GS-ATasE (GlnE). Another type of PTM was also observed, i.e., Hydroxylation and Sulfation, with the domains Asparagine_hydroxylase and Collagen_prolyl_lysyl_hydroxylase and Sulfotransferase, respectively. The deamidation type of PTM was present only in Rhizobium sp. NGR234. How to cite: Qureshi MA, Pervez MT, Babar ME, et al. Genomic comparisons of Rhizobium species using in silico AFLP-PCR, endonuclease restrictions and ampylating enzymes.
Assuntos
Rhizobium/genética , Filogenia , Rhizobium/enzimologia , Rhizobium/fisiologia , Simbiose , Simulação por Computador , Enzimas de Restrição do DNA , Reação em Cadeia da Polimerase/métodos , Análise de Sequência , Proteoma , Genômica , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados , Fabaceae/microbiologiaRESUMO
Exposure to ultraviolet (UV) radiation blocks DNA replication and arrests cellular division in Escherichia coli. Restoration of chromosome replication involves nucleoid reorganization, which involves the participation of the recombination-catalyzing proteins RecA, RecO, RecR and RecN. In this work, we evaluated the influence of recN, uvrA and recJ gene mutations on post-irradiation nucleoid reorganization. We used isogenic E. coli strains that are defective for these genes to study post-irradiation kinetics of the nucleoid shape fractions using fluorescence microscopy. The results showed that in the wild-type strain, post-irradiation nucleoid reorganization occurs, which restores the nucleoid shape fractions in the cells to those observed prior to irradiation. First, the nucleoid condenses into the central area of the irradiated cell. Second, the nucleoid disperses along the cell. Third, the cell enters the chromosome replicative phase and cytokinesis. Escherichia coli cells with a recN mutation did not exhibit increased nucleoid condensation, but chromosome replication and cytokinesis occurred. In the uvrA and recJ strains, the condensation step was delayed compared to the wild-type strain, and chromosome replication and cytokinesis did not occur. The results are discussed with an emphasis on the functions of RecN, UvrA and RecJ in nucleoid reorganization in UV-irradiated E. coli cells.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Cromossomos Bacterianos/metabolismo , Enzimas de Restrição do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/efeitos da radiação , Exodesoxirribonucleases/metabolismo , Mutação , Raios Ultravioleta , Adenosina Trifosfatases/genética , Proteínas de Bactérias/genética , Enzimas de Restrição do DNA/genética , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/genética , Escherichia coli/enzimologia , Proteínas de Escherichia coli/genética , Exodesoxirribonucleases/genéticaRESUMO
The most widely used technique for the production of DNA aptamers/oligonucleotides is chemical synthesis. Despite its effectiveness, this technique cannot be performed "in house", making the user fully dependent on a supplier. In this work, we present a simplified method by which it is possible to enzymatically produce DNA aptamers "in house". This new method uses the rolling circle replication followed by a unique cleavage step using the SchI endonuclease. Potentially, any oligonucleotide can be produced by the enzymatic method proposed in this study. To illustrate, we present the production of three variations of the 31-TBA aptamer, a single stranded DNA which has anticoagulant action.
Assuntos
Aptâmeros de Nucleotídeos/biossíntese , DNA de Cadeia Simples/biossíntese , Técnicas de Amplificação de Ácido Nucleico , Oligodesoxirribonucleotídeos/biossíntese , Anticoagulantes/síntese química , Anticoagulantes/metabolismo , Aptâmeros de Nucleotídeos/genética , Sequência de Bases , Enzimas de Restrição do DNA/metabolismo , DNA de Cadeia Simples/genética , Quadruplex G , Humanos , Oligodesoxirribonucleotídeos/genéticaRESUMO
Molecular markers can increase both the efficiency and speed of breeding programs. Functional markers that detect the functional mutations causing phenotypic changes offer a precise method for genetic identification. In this study, we used newly derived cleaved amplified polymorphic sequence markers to detect the functional mutations of tms5, which is a male sterile gene that is widely used in rice production in China. In addition, restriction cutting sites were designed to specifically digest amplicons of tms5 but not wild type (TMS5), in order to avoid the risk of false positive results. By optimizing the condition of the polymerase chain reaction amplifications and restriction enzyme digestions, the newly designed markers could accurately distinguish between tms5 and TMS5. These markers can be applied in marker-assisted selection for breeding novel thermo-sensitive genic male sterile (TGMS) lines, as well as to rapidly identify the TGMS hybrid seed purity.
Assuntos
Quimera/genética , Genes de Plantas , Marcadores Genéticos , Oryza/genética , Melhoramento Vegetal , Infertilidade das Plantas/genética , Mapeamento Cromossômico , Cruzamentos Genéticos , Primers do DNA/síntese química , Enzimas de Restrição do DNA/genética , Técnicas de Amplificação de Ácido Nucleico , Sementes/genéticaRESUMO
The bacteriophage ΦAb-Sp7 was isolated from the cells of the Azospirillum brasilense Sp7. The morphology, size of the gram-negative colonies, and range of lytic activity against other strains and species of the genus Azospirillum was tested. The isolated phage DNA was examined using electrophoretic and restriction analysis, and the size of the genome were established. The electron microscopy. resuIts show that the phage (capsid) has a strand-like form. The electron microscopy study of the bacteriophage ΦAb-Sp7 adsorption on the A. brasilense Sp7 bacterial surface was performed.
Assuntos
Azospirillum brasilense/virologia , Bacteriófagos/genética , DNA Viral/química , Genoma Viral , Adsorção , Azospirillum brasilense/química , Azospirillum brasilense/ultraestrutura , Bacteriófagos/crescimento & desenvolvimento , Bacteriófagos/ultraestrutura , Capsídeo/ultraestrutura , Enzimas de Restrição do DNA/química , Tamanho do Genoma , Lisogenia , Mapeamento por RestriçãoRESUMO
Thirty-six isolates of psittacid herpesvirus (PsHV), obtained from 12 different species of psittacids in Brazil, were genotypically characterized by restriction fragment length polymorphism (RFLP) analysis and PCR amplification. RFLP analysis with the PstI enzyme revealed four distinct restriction patterns (A1, X, W and Y), of which only A1 (corresponding to PsHV-1) had previously been described. To study PCR amplification patterns, six pairs of primers were used. Using this method, six variants were identified, of which, variants 10, 8, and 9 (in this order) were most prevalent, followed by variants 1, 4, and 5. It was not possible to correlate the PCR and RFLP patterns. Twenty-nine of the 36 isolates were shown to contain a 419 bp fragment of the UL16 gene, displaying high similarity to the PsHV-1 sequences available in GenBank. Comparison of the results with the literature data suggests that the 36 Brazilian isolates from this study belong to genotype 1 and serotype 1. (AU)
Assuntos
Animais , Genótipo , Papagaios , Infecções por Herpesviridae , Amplificação de Genes , Enzimas de Restrição do DNARESUMO
Microsatellite markers (also known as SSRs, Simple Sequence Repeats) are widely used in plant science and are among the most informative molecular markers for population genetic investigations, but the development of such markers presents substantial challenges. In this report, we discuss how next generation sequencing can replace the cloning, Sanger sequencing, identification of polymorphic loci, and testing cross-amplification that were previously required to develop microsatellites. We report the development of a large set of microsatellite markers for five species of the Neotropical cactus genus Pilosocereus using a restriction-site-associated DNA sequencing (RAD-seq) on a Roche 454 platform. We identified an average of 165 microsatellites per individual, with the absolute numbers across individuals proportional to the sequence reads obtained per individual. Frequency distribution of the repeat units was similar in the five species, with shorter motifs such as di- and trinucleotide being the most abundant repeats. In addition, we provide 72 microsatellites that could be potentially amplified in the sampled species and 22 polymorphic microsatellites validated in two populations of the species Pilosocereus machrisii. Although low coverage sequencing among individuals was observed for most of the loci, which we suggest to be more related to the nature of the microsatellite markers and the possible bias inserted by the restriction enzymes than to the genome size, our work demonstrates that an NGS approach is an efficient method to isolate multispecies microsatellites even in non-model organisms.
Assuntos
Cactaceae/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Repetições de Microssatélites/genética , Biologia Computacional , Análise Custo-Benefício , Primers do DNA , Enzimas de Restrição do DNA/genética , DNA de Plantas/genética , Marcadores Genéticos/genética , Variação Genética , Genótipo , Desequilíbrio de Ligação , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Polimorfismo Genético , Análise de Sequência de DNA , Especificidade da EspécieRESUMO
Microsatellite markers are widely and evenly distributed, and are highly polymorphic. Rapid and convenient detection through automated analysis means that microsatellite markers are widely used in the construction of plant and animal genetic maps, in quantitative trait loci localization, marker-assisted selection, identification of genetic relationships, and genetic diversity and phylogenetic tree construction. However, few microsatellite markers remain to be isolated. We used streptavidin magnetic beads to affinity-capture and construct a (CA)n microsatellite DNA-enriched library from sika deer. We selected sequences containing more than six repeats to design primers. Clear bands were selected, which were amplified using non-specific primers following PCR amplification to screen polymorphisms in a group of 65 unrelated sika deer. The positive clone rate reached 82.9% by constructing the enriched library, and we then selected positive clones for sequencing. There were 395 sequences with CA repeats, and the CA repeat number was 4-105. We selected sequences containing more than six repeats to design primers, of which 297 pairs were designed. We next selected clear bands and used non-specific primers to amplify following PCR amplification. In total, 245 pairs of primers were screened. We then selected 50 pairs of primers to randomly screen for polymorphisms. We detected 47 polymorphic and 3 monomorphic loci in 65 unrelated sika deer. These newly isolated and characterized microsatellite loci can be used to construct genetic maps and for lineage testing in deer. In addition, they can be used for comparative genomics between Cervidae species.
Assuntos
DNA/genética , DNA/isolamento & purificação , Cervos/genética , Genoma , Repetições de Microssatélites/genética , Animais , Enzimas de Restrição do DNA/metabolismo , Feminino , Biblioteca Gênica , Masculino , Reação em Cadeia da Polimerase , Mapeamento por RestriçãoRESUMO
The aim of this study was to determine whether vitamin D receptor (VDR) genetic polymorphisms are associated with the susceptibility to pulmonary tuberculosis (PTB). MEDLINE and Embase databases and manual literature searches were used. A meta-analysis was conducted on the associations between the VDR FokI, TaqI, BsmI, and ApaI polymorphisms and PTB susceptibility. A total of 16 studies comprising 3231 patients and 3670 controls met the study inclusion criteria, consisting of 14 studies on the VDR FokI polymorphism, 13 on the VDR TaqI polymorphism, 8 on the VDR BsmI polymorphism, and 5 on the VDR ApaI polymorphism. Meta-analysis of the VDR FokI polymorphism showed no association between PTB and the f allele of the VDR FokI polymorphism (long variant) in all subjects (OR = 1.070, 95%CI = 0.979-1.169, P = 0.134). In contrast, after stratification by ethnicity, meta-analysis indicated that the VDR FokI F allele (short variant) was associated with PTB risk in an East Asian population (OR = 1.507, 95%CI = 1.192-1.906, P = 0.001). Meta-analysis revealed no association between PTB susceptibility and the VDR TaqI t allele in all study subjects (OR = 0.986, 95%CI = 0.839-1.159, P = 0.866) or in individual ethnic populations. Furthermore, a risk of PTB was not associated with the BsmI and ApaI polymorphisms. This meta-analysis suggested that the VDR FokI polymorphism is associated with a susceptibility to PTB in East Asians.
Assuntos
Estudos de Associação Genética , Predisposição Genética para Doença , Receptores de Calcitriol/genética , Tuberculose Pulmonar/genética , Alelos , Povo Asiático/genética , Enzimas de Restrição do DNA/genética , Genótipo , Humanos , Polimorfismo Genético , Fatores de Risco , Tuberculose Pulmonar/patologiaRESUMO
Leptospires are usually classified by methods based on DNA-DNA hybridization and the conventional cross-agglutination absorption test, which uses polyclonal antibodies against lipopolysaccharides. In this study, the amplification of the rpoB gene, which encodes the beta-subunit of RNA polymerase, was used as an alternative tool to identify Leptospira. DNA extracts from sixty-eight serovars were obtained, and the hypervariable region located between 1990 and 2500-bp in the rpoB gene was amplified by polymerase chain reaction (PCR). The 600-bp amplicons of the rpoB gene were digested with the restriction endonucleases TaqI, Tru1I, Sau3AI and MslI, and the restriction fragments were separated by 6% polyacrylamide gel electrophoresis. Thirty-five fragment patters were obtained from the combined data of restriction fragment length polymorphism (PCR-RFLP) analysis and used to infer the phylogenetic relationships among the Leptospira species and serovars. The species assignments obtained were in full agreement with the established taxonomic classifications. Twenty-two serovars were effectively identified based on differences in their molecular profiles. However, the other 46 serovars remained clustered in groups that included more than one serovar of different species. This study demonstrates the value of RFLP analysis of PCR-amplified rpoB as an initial method for identifying Leptospira species and serovars.
Assuntos
RNA Polimerases Dirigidas por DNA/genética , Leptospira/classificação , Leptospira/genética , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Análise por Conglomerados , Enzimas de Restrição do DNA/metabolismo , Eletroforese em Gel de Poliacrilamida , Genótipo , Leptospira/isolamento & purificação , Dados de Sequência Molecular , Filogenia , Análise de Sequência de DNA , SorogrupoRESUMO
Leptospires are usually classified by methods based on DNA-DNA hybridization and the conventional cross-agglutination absorption test, which uses polyclonal antibodies against lipopolysaccharides. In this study, the amplification of the rpoB gene, which encodes the beta-subunit of RNA polymerase, was used as an alternative tool to identify Leptospira. DNA extracts from sixty-eight serovars were obtained, and the hypervariable region located between 1990 and 2500-bp in the rpoB gene was amplified by polymerase chain reaction (PCR). The 600-bp amplicons of the rpoB gene were digested with the restriction endonucleases TaqI, Tru1I, Sau3AI and MslI, and the restriction fragments were separated by 6% polyacrylamide gel electrophoresis. Thirty-five fragment patters were obtained from the combined data of restriction fragment length polymorphism (PCR-RFLP) analysis and used to infer the phylogenetic relationships among the Leptospira species and serovars. The species assignments obtained were in full agreement with the established taxonomic classifications. Twenty-two serovars were effectively identified based on differences in their molecular profiles. However, the other 46 serovars remained clustered in groups that included more than one serovar of different species. This study demonstrates the value of RFLP analysis of PCR-amplified rpoB as an initial method for identifying Leptospira species and serovars.
Assuntos
RNA Polimerases Dirigidas por DNA/genética , Leptospira/classificação , Leptospira/genética , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Análise por Conglomerados , Enzimas de Restrição do DNA/metabolismo , Eletroforese em Gel de Poliacrilamida , Genótipo , Leptospira/isolamento & purificação , Dados de Sequência Molecular , Filogenia , Análise de Sequência de DNA , SorogrupoRESUMO
Leptospires are usually classified by methods based on DNA-DNA hybridization and the conventional cross-agglutination absorption test, which uses polyclonal antibodies against lipopolysaccharides. In this study, the amplification of the rpoB gene, which encodes the beta-subunit of RNA polymerase, was used as an alternative tool to identify Leptospira. DNA extracts from sixty-eight serovars were obtained, and the hypervariable region located between 1990 and 2500-bp in the rpoB gene was amplified by polymerase chain reaction (PCR). The 600-bp amplicons of the rpoB gene were digested with the restriction endonucleases TaqI, Tru1I, Sau3AI and MslI, and the restriction fragments were separated by 6% polyacrylamide gel electrophoresis. Thirty-five fragment patters were obtained from the combined data of restriction fragment length polymorphism (PCR-RFLP) analysis and used to infer the phylogenetic relationships among the Leptospira species and serovars. The species assignments obtained were in full agreement with the established taxonomic classifications. Twenty-two serovars were effectively identified based on differences in their molecular profiles. However, the other 46 serovars remained clustered in groups that included more than one serovar of different species. This study demonstrates the value of RFLP analysis of PCR-amplified rpoB as an initial method for identifying Leptospira species and serovars.(AU)
Assuntos
RNA Polimerases Dirigidas por DNA/genética , Leptospira/classificação , Leptospira/genética , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Análise por Conglomerados , Enzimas de Restrição do DNA/metabolismo , Eletroforese em Gel de Poliacrilamida , Genótipo , Leptospira/isolamento & purificação , Dados de Sequência Molecular , Filogenia , Análise de Sequência de DNA , SorogrupoRESUMO
Endangered angelsharks and a protected dolphin species are illegally traded in Brazil. In this study, we determined prospective molecular markers for detecting these species in the trade of angelshark carcasses and 'dolphin' eyeball amulets. We compiled publicly available as well as new and unpublished cytochrome b (cyt b) DNA sequences for species involved in these trades. These sequences were digested in silico using restriction enzymes. We then described prospective polymerase chain reaction (PCR)-restriction fragment length polymorphism markers for distinguishing between protected species and the species whose trade was legally allowed in these two trade groups. The prospective marker for identifying angelshark carcasses consists of cyt b PCR and digestion by BstXI, BsgI, BspMI, BsrDI, and HaeII restriction enzymes. The prospective marker for identifying eyeball amulets consists of cyt b PCR and digestion by ApoI, BtsI, HindII, BsaAI, BplI, and SspI restriction enzymes. This is the first study to deposit in GenBank cyt b sequences for the angelshark species Squatina argentina, Squatina guggenheim, and Squatina occulta. Moreover, the S. argentina haplotype is the first DNA sequence for this species deposited in GenBank.