RESUMEN
In vitro selection with either DNA or RNA libraries was performed against the TAR RNA element of HIV-1. The role of the selection conditions on the outcome of the selection was evaluated by varying the magnesium concentration and the temperature. The selection stringency was demonstrated to determine i) the affinity of the best identified aptamers for the TAR target, and ii) the type of interaction between the two partners. Selections performed with a DNA library under low (4 degrees C, 10 mM magnesium) and high stringency (23 degrees C, 3 mM magnesium) led to the emergence of "kissing aptamers"; but even if the motif interacting directly with the TAR loop were identical in the two kinds of aptamers, the consensus was extended from eight to thirteen nucleotides when the Mg(2+) concentration was decreased from 10 to 3 mM. Similar kissing aptamers were selected at 23 degrees C and 37 degrees C starting with two different RNA libraries under identical ionic conditions. In addition, selection performed at 37 degrees C yielded a significant proportion of antisense sequences. Only antisense RNAs complementary to the TAR loop competitively inhibited the association of a Tat peptide with TAR.
Asunto(s)
ADN Viral/química , VIH-1/genética , Magnesio/química , ARN Viral/química , Temperatura , Secuencia de Bases , ADN Viral/genética , Ensayo de Cambio de Movilidad Electroforética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Oligonucleótidos , ARN Viral/genéticaRESUMEN
We used in vitro selection to identify RNA aptamers able to selectively bind to the TAR RNA motif of HIV-1, an unperfect RNA hairpin involved in the transcription of the retroviral genome. We selected aptameric RNA hairpins giving rise to kissing complexes with TAR. The N3'-->P5' phosphoramidate variant of the aptamer bind to TAR with a Kd in the low nanomolar range. However, only the RNA-RNA loop-loop complex is recognized by the Rop protein of E. coli which is specific for kissing complexes.
Asunto(s)
Duplicado del Terminal Largo de VIH/genética , VIH-1/genética , ARN/genética , ARN/metabolismo , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Humanos , Conformación de Ácido Nucleico , Oligonucleótidos/síntesis química , Oligonucleótidos/genética , Oligonucleótidos/farmacología , Proteínas de Unión al ARN/biosíntesis , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Elementos de Respuesta/genética , Especificidad por Sustrato , Resonancia por Plasmón de Superficie , Activación TranscripcionalRESUMEN
Regardless of their targets and modes of action, subinhibitory concentrations of antibiotics can have an impact on cell physiology and trigger a large variety of cellular responses in different bacterial species. Subinhibitory concentrations of ß-lactam antibiotics cause reactive oxygen species production and induce PolIV-dependent mutagenesis in Escherichia coli. Here we show that subinhibitory concentrations of ß-lactam antibiotics induce the RpoS regulon. RpoS-regulon induction is required for PolIV-dependent mutagenesis because it diminishes the control of DNA-replication fidelity by depleting MutS in E. coli, Vibrio cholerae and Pseudomonas aeruginosa. We also show that in E. coli, the reduction in mismatch-repair activity is mediated by SdsR, the RpoS-controlled small RNA. In summary, we show that mutagenesis induced by subinhibitory concentrations of antibiotics is a genetically controlled process. Because this mutagenesis can generate mutations conferring antibiotic resistance, it should be taken into consideration for the development of more efficient antimicrobial therapeutic strategies.
Asunto(s)
Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Proteínas Bacterianas/fisiología , Replicación del ADN/efectos de los fármacos , Mutagénesis , Factor sigma/fisiología , beta-Lactamas/farmacología , Bacterias/genética , Replicación del ADN/fisiología , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Vibrio cholerae/efectos de los fármacos , Vibrio cholerae/genética , Vibrio cholerae/metabolismoRESUMEN
Chronic infection by Helicobacter pylori is a major risk factor for gastric adenocarcinoma and mucosa-associated lymphoid tissue lymphoma. H. pylori possesses a set of virulence factors, including the CagA effector, which interferes with intracellular signalling pathways and mediates phenotypic alterations, strongly evoking neoplasic transformation. MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression involved in development, cell proliferation and immune responses. miRNAs are frequently altered in cancers, revealing their functions as oncogenes or tumour suppressors. However, the role, if any, that miRNAs play in the host cell responses to H. pylori remains unknown. This review considers the possible involvement of some miRNAs, including miR-146, miR-155, miR-21, miR-27a, miR-106-93-25 and miR-221-222 clusters and the miR-200 family in H. pylori-induced infection and gastric cancers. Further exploration of miRNA-mediated gene silencing, taking into account the relationship between host targets and bacterial effectors, will most certainly bring new insights into the control of gene expression in human gastric cells chronically infected by H. pylori.