RESUMO
The preparation of a novel phosphoramidite monomer based on thyminyl acetic acid coupled to the secondary nitrogen of 2-(2-amino-ethylamino)ethanol is described. This monomer can be used to attach a deoxynucleotide to the carboxy terminus of a PNA oligomer by solid-phase synthesis. The resulting PNA primer is recognized as a substrate by various DNA polymerases.
Assuntos
Primers do DNA/síntese química , Primers do DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Ácidos Nucleicos Peptídicos/síntese química , Ácidos Nucleicos Peptídicos/metabolismo , Sequência de Bases , Primers do DNA/química , Indicadores e Reagentes , Dados de Sequência Molecular , Ácidos Nucleicos Peptídicos/química , Especificidade por SubstratoRESUMO
Examination of several commercially available thermostable DNA polymerases identifies 9 degrees N DNA polymerase as single enzyme that could incorporate two components of an expanded genetic alphabet, 2,4-diaminopyrimidine and xanthosine as deoxynucleoside triphosphate opposite their cognate base in a DNA template.
Assuntos
Pareamento de Bases , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Desoxirribonucleotídeos/síntese química , Sequência de Bases , Primers do DNA , Desoxirribonucleotídeos/metabolismo , Estabilidade Enzimática , Ligação de Hidrogênio , Pirimidinas , Pyrococcus/enzimologia , Ribonucleosídeos , Especificidade por Substrato , Moldes Genéticos , Termodinâmica , Thermus/enzimologia , XantinasRESUMO
A research program has applied the tools of synthetic organic chemistry to systematically modify the structure of DNA and RNA oligonucleotides to learn more about the chemical principles underlying their ability to store and transmit genetic information. Oligonucleotides (as opposed to nucleosides) have long been overlooked by synthetic organic chemists as targets for structural modification. Synthetic chemistry has now yielded oligonucleotides with 12 replicatable letters, modified backbones, and new insight into why Nature chose the oligonucleotide structures that she did.
Assuntos
DNA/química , Biologia Molecular/tendências , Ácidos Nucleicos/química , Oligonucleotídeos/síntese química , Catálise , Códon , Estrutura Molecular , Ácidos Nucleicos/síntese química , Oligonucleotídeos/química , Fosfatos/química , Sulfonas/químicaRESUMO
HIV-1 reverse transcriptase (RT) incorporates 2'-deoxyisoguanosine triphosphate (d-isoGTP) opposite thymidine (T) in a DNA template and opposite uracil (U) in an RNA template about 10 times more efficiently than the eukaryotic DNA polymerase alpha, both in the absence and presence of dATP.
Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , Guanosina/metabolismo , Transcriptase Reversa do HIV/metabolismo , Adenosina , Sequência de Bases , DNA , Guanosina/química , Especificidade por Substrato , Moldes GenéticosRESUMO
Mammalian DNA polymerases alpha and epsilon, the Klenow fragment of Escherichia coli DNA polymerase I and HIV-1 reverse transcriptase (RT) were examined for their ability to incorporate components of an expanded genetic alphabet in different forms. Experiments were performed with templates containing 2'-deoxyxanthosine (dX) or 2'-deoxy-7-deazaxanthosine (c7dX), both able to adopt a hydrogen bonding acceptor-donor-acceptor pattern on a purine nucleus (puADA). Thus these heterocycles are able to form a non-standard nucleobase pair with 2,4-diaminopyrimidine (pyDAD) that fits the Watson-Crick geometry, but is joined by a non-standard hydrogen bonding pattern. HIV-1 RT incorporated d(pyDAD)TP opposite dX with a high efficiency that was largely independent of pH. Specific incorporation opposite c7dX was significantly lower and also independent of pH. Mammalian DNA polymerases alpha and epsilon from calf thymus and the Klenow fragment from E. coli DNA polymerase I failed to incorporate d(pyDAD)TP opposite c7dX.
Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , Pirimidinas/metabolismo , Ribonucleosídeos/metabolismo , Animais , Sequência de Bases , Bovinos , Escherichia coli/enzimologia , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/química , Especificidade por Substrato , Moldes Genéticos , XantinasRESUMO
The V3 variable region of the large subunit rRNA was examined for nucleotide sequence signatures as potential taxonomic tools. Data are presented on 117 species, representing 23 genera of basidiomycetous yeasts. The results of nucleotide sequence alignments indicate that strains within species have identical base sequences and that species may differ from one another by one to more than 100 base positions. Phylogenetic analyses of the alignments indicates relationships among species, including the prediction of synonymous species and the clustering of species belonging to the Ustilaginales and Tremellales. These results suggest that species-specific nucleotide sequences can be used for the development of techniques for population analyses of a variety of marine and other microeukaryotes.
Assuntos
RNA Fúngico/química , RNA Ribossômico/química , Microbiologia da Água , Leveduras/classificação , Leveduras/genética , Animais , Sequência de Bases , Humanos , Dados de Sequência Molecular , Filogenia , RNA Fúngico/isolamento & purificação , RNA Ribossômico/isolamento & purificação , Água do Mar , Alinhamento de Sequência , Homologia de Sequência do Ácido NucleicoRESUMO
Chemotactic stimulation of neutrophils can result in the production and release of toxic oxygen metabolites which, in turn, may affect neutrophil function. We have investigated the effects of catalase, superoxide dismutase, hydroxyl radical scavengers and a iron chelator on chemotactic responses of rat peritoneal neutrophils in vitro. Neutrophils were quantitatively assessed in micropore filters for chemotactic migration to several different chemotactic peptides including C5a, present in inulin-activated rat serum, the non-oxidizable tripeptide N-formyl-norleucyl-leucyl-phenylalanine, and the oxidizable peptide N-formyl-methionyl-leucyl-phenylalanine. The presence of catalase in the suspension of neutrophils caused a dose-dependent enhancement of chemotactic responses to each of the three chemotactic stimuli, whereas addition of superoxide dismutase had little effect. Hydroxyl radical scavengers (mannitol, dimethyl sulfoxide) or the iron chelator, deferoxamine, also afforded significant, dose-dependent enhancement of neutrophyl chemotaxis, while the iron-saturated chelator was devoid of chemotaxis-enhancing properties. Furthermore, stimulation of rat neutrophils with phorbol myristate acetate resulted in the appearance of products of lipid peroxidation (conjugated Schiff bases), the generation of which could be prevented by the hydroxyl radical scavenger, dimethyl thiourea. These data suggest that catalase, hydroxyl radical scavengers, and iron chelators protect chemotactically stimulated neutrophils from autotoxicity caused by neutrophil-derived hydrogen peroxide and its iron-catalyzed conversion product, hydroxyl radical.
Assuntos
Quimiotaxia de Leucócito/efeitos dos fármacos , Hidróxidos/farmacologia , Neutrófilos/fisiologia , Animais , Catalase/farmacologia , Complemento C5/fisiologia , Complemento C5a , Desferroxamina/farmacologia , Dimetil Sulfóxido/farmacologia , Peróxido de Hidrogênio/metabolismo , Hidróxidos/metabolismo , Radical Hidroxila , Ferro/metabolismo , Ferro/farmacologia , Peróxidos Lipídicos/metabolismo , Manitol/farmacologia , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/efeitos dos fármacos , Oligopeptídeos/farmacologia , Oxirredução , Ratos , Superóxido Dismutase/farmacologia , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
The authors have previously shown that thermal injury to the skin of rats results in the development of acute lung injury that is susceptible to systemic treatment of animals with catalase and dependent on the presence of neutrophils. The current studies have been expanded for exploration of the nature of the neutrophil-derived oxygen products responsible for the lung injury and have also focused on evidence of the appearance of products of lipid peroxidation (conjugated dienes). With respect to the former, treatment of rats with iron chelators (deferoxamine mesylate, 2,3-dihydroxybenzoic acid), with scavengers of hydroxyl radical (dimethyl sulfoxide, dimethyl thiourea, sodium benzoate), or with vitamin E affords a significant degree of protection from acute lung injury as assessed by changes in lung vascular permeability and by morphologic parameters. These data suggest that lung vascular injury after thermal trauma of the skin is related to the generation by neutrophils of the hydroxyl radical. Conjugated dienes have been demonstrated to appear sequentially both in the burned skin (at 1/4 hour) and in the lungs (at 2 hours), as well as in the plasma (with peaks at 1/2 and at 3 hours) after thermal injury. The appearance of the conjugated dienes in plasma at the two intervals of time is greatly diminished if animals are pretreated with the iron chelator deferoxamine, with catalase, or with scavengers of hydroxyl radical. Furthermore, the appearance of conjugated dienes in plasma at 30 minutes and 3 hours is significantly diminished if animals are depleted of neutrophils, complement-depleted, or the burned skin is excised immediately after thermal injury. These data indicate a linkage between thermal trauma of skin, secondary injury of lung, and appearance in plasma and tissues of products of lipid peroxidation.