RESUMO
Currently, a number of structurally and functionally different temperature-sensitive elements like as RNA thermometers which control a variety of biological processes of bacteria, including virulence, are known. Well-known RNA thermometers correspond to one long step-loop structure or few hairpins which can be matched or mismatched. Based on the computer and thermodynamical analysis of 25 isolates of Salmonella enterica with complete genome, algorithm and the criteria of search for putative RNA thermometers were developed. It will permit to perform the search of potential riboswitchers in genome of socially significant pathogens in the future. For S. enterica, in addition to well-known 4U RNA thermometer, four step-loop structures that may be new RNA thermometers were identified and two of them are localized in 5'-UTR of virulence regulators gltB and yaeQ. They correspond to necessary and sufficient conditions of RNA thermometer formation as far as these highly conservative structures are found in genome of all 25 isolates of S. enterica. Matched hairpins forming cruciform structure in supercoiled pUC8 plasmid were visualized by atomic force microscopy.
Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Genes de Troca , Genoma Bacteriano , Salmonella enterica/genética , Salmonella enterica/patogenicidade , Regiões 5' não Traduzidas , Adaptação Fisiológica , Sequências Repetidas Invertidas , Microscopia de Força Atômica , Conformação de Ácido Nucleico , Plasmídeos/química , Plasmídeos/ultraestrutura , Temperatura , VirulênciaRESUMO
Two types of techniques for detection of single nucleotide polymorphism in 315 codon of katG gene of MTB are developed. Isoniazid resistance of MTB is associated with point mutations in the mentioned codon. Two primer sets with additional competitive blocking primer containing 3'-terminal phosphate group (for elimination of unspecific amplification) allow detecting the most frequent point mutations AGC --> ACC and AGC --> AGA in 315 codon of katG gene. PCR with primer set of two primers one of which contains five LNA-monomers allows to determine an occurrence of any type from six known mutations in 315 codon of katG gene, i.e. to differentiate wild type and isoniazid-resistant MTB. Purity and structure of 17 bp long primers with LNA-modified nucleotides were characterized by time-of-flight MALDI-mass spectrometry. Duplex of 17 bp length formed by two complementary oligonucleotides with LNA-monomers was studied using melting.
Assuntos
Proteínas de Bactérias/genética , Técnicas de Tipagem Bacteriana , Catalase/genética , Primers do DNA/genética , Sondas de DNA/genética , Mycobacterium tuberculosis/genética , Reação em Cadeia da Polimerase/métodos , Tuberculose Resistente a Múltiplos Medicamentos , Antituberculosos/administração & dosagem , Códon , Primers do DNA/síntese química , Sondas de DNA/síntese química , DNA Bacteriano/análise , DNA Bacteriano/genética , Humanos , Isoniazida/administração & dosagem , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/isolamento & purificação , Mutação Puntual , Polimorfismo Genético , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tuberculose Resistente a Múltiplos Medicamentos/diagnóstico , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/microbiologiaRESUMO
Perfect interstranded triplexes that can potentially arise in the proviral DNA of wide-spread bovine retroviruses like as bovine leukemia virus (BLV) and bovine immunodeficiency virus (BIV) have been determined. In the BLV and BIV genomes 2 and 5 fragments respectively were found to form triple helixes under acidic conditions. One of those fragments that is localized on the BLV gag gene can exist as cruciform structure too. Experimentally the existence of triplexes is confirmed by atomic force microscopic visualization of supercoiled pGEMEX DNA for which genome 6 fragments are found with mirror symmetry that is necessary for intramolecular triplex formation. The diagrams of triplexes (one of the elements of signaling genome function) localization on the genome of bovine retroviruses are obtained.
Assuntos
DNA Viral/química , DNA/química , Vírus da Imunodeficiência Bovina/química , Vírus da Leucemia Bovina/química , Purinas/química , Pirimidinas/química , Animais , Sequência de Bases , Bovinos , DNA/genética , DNA Viral/genética , Genoma Viral , Vírus da Imunodeficiência Bovina/genética , Vírus da Leucemia Bovina/genética , Microscopia de Força Atômica , Dados de Sequência Molecular , Conformação de Ácido NucleicoRESUMO
The primer set for detection of bovine immunodeficiency virus (BIV) proviral DNA, retrovirus of unknown pathology, by standard polymerase chain reaction was developed. Amplicon of the expected size was detected in DNA from peripheral blood mononuclear cells of seropositive experimentally BIV infected sheep and cattle. Primers targeted the short fragment of BIV pol gene. Sequences of BIV proviral DNA extracted from BIV infected cell culture as well as from experimentally infected cows were taken for targeted pol BI BPX gene locus. Problems of BIV detection from the clinical material of the experimentally and naturally infected animals are discussed.
Assuntos
Primers do DNA/análise , DNA Viral/análise , Vírus da Imunodeficiência Bovina/isolamento & purificação , Infecções por Lentivirus/virologia , Reação em Cadeia da Polimerase/veterinária , Provírus/isolamento & purificação , Animais , Bovinos , Doenças dos Bovinos/virologia , Modelos Animais de Doenças , Vírus da Imunodeficiência Bovina/genética , Infecções por Lentivirus/veterinária , Linfócitos/virologia , Reação em Cadeia da Polimerase/métodos , Provírus/genética , Ovinos , Doenças dos Ovinos/virologiaRESUMO
Methodical approaches for studying of living cells in aqueous solutions by atomic force microscopy (AFM) are demonstrated. Images of intact cyanobacteria Synechocystis PCC 6803 in TES buffer were captured in tapping mode using aminomodified mica as AFM substrate. Modification of freshly cleaved mica has been done in 3-aminopropyltri-ethoxysilane vapours. The average size of cyanobacteria was determined from AFM images. The linear size of Synechocystis PCC 6803 in TES buffer was equal to 70 x 90 nm and their height was about 20 nm. Possible causes of insufficiently high resolution of the cyanobacteria AFM images in aqueous solutions and possible ways for gaining molecular resolution in studies of structural, functional and micromechanical properties of living cells are discussed.
Assuntos
Cianobactérias/ultraestrutura , Silicatos de Alumínio , Soluções Tampão , Microscopia de Força Atômica , SoluçõesRESUMO
The potential of atomic force microscopy (AFM) for the investigation of peculiarities of microorganisms genome structure is demonstrated. AFM images of phage lambda DNA linear molecules and supercoiled mica in buffer solution was imaged in air. New experimental method of DNA stretching based on using amino-modified mica with a decreased surface density of active amino-groups is proposed. Stretched molecules of phage lambda DNA were imaged by AFM.
