RESUMEN
We evaluated cell survival, DNA double strand breaks (dsbs), and DNA synthesis following camptothecin (CPT) alone or concurrent CPT and X-radiation treatments in exponential-phase cultures of a radioresistant human melanoma cell line. Cell survival was measured by a clonogenic assay. DNA dsbs were measured by pulsed-field gel electrophoresis. DNA synthesis was measured by incorporation of (3)H-thymidine. We found that (i) concurrent CPT and X-radiation interacted additively, unlike previous results with plateau-phase cultures of these cells, which showed synergistic interaction; (ii) there were strong negative correlations (correlation coefficients of at least 0.82) between clonogenic surviving fractions and DNA dsbs following CPT alone or concurrent CPT and radiation treatments; and (iii) concurrent CPT and radiation (10 Gy) treatment did not completely inhibit DNA synthesis, even though addition of radiation to CPT did further decrease DNA synthesis (relative to CPT alone) at CPT concentrations below 20 microM. Our results suggest that during concurrent CPT and radiation treatment residual DNA dsb levels were good indicators of cell killing and that the absence of complete inhibition of DNA synthesis could at least in part explain the additive interaction between CPT and radiation.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Camptotecina/farmacología , Daño del ADN/efectos de los fármacos , Daño del ADN/efectos de la radiación , ADN/efectos de los fármacos , ADN/efectos de la radiación , Inhibidores Enzimáticos/farmacología , Rayos X , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Relación Dosis-Respuesta a Droga , Relación Dosis-Respuesta en la Radiación , Humanos , Timidina/metabolismo , Células Tumorales CultivadasRESUMEN
A-1 frameshift event is required for expression of the pol gene when ribosomes translate the mRNA of human immunodeficiency virus type-1 (HIV-1). In this study, we inserted the frameshift region of HIV-1 (a slippery heptanucleotide motif followed by a stem-loop) in a reporter gene coding for firefly luciferase. The ability of the corresponding mRNA, generated by in vitro transcription, to be translated in an Escherichia coli cell-free extract is the first demonstration that the HIV-1 frameshift can be reproduced in a bacterial cell-free extract, providing a powerful approach for analysis of the frameshift mechanism. The responses of the frameshift signal to chloramphenicol, an inhibitor of peptide bond formation, and spectinomycin, an inhibitor of translocation, suggest that the frameshift complies with the same rules found in eukaryotic translation systems. Furthermore, when translation was performed in the presence of streptomycin and neamine, two error-inducing antibiotics, or with hyperaccurate ribosomes mutated in S12, the frameshift efficiency was increased or decreased, respectively, but only in the presence of the stem-loop, suggesting that the stem-loop can influence the frameshift through a functional interaction with the ribosomes.
Asunto(s)
Sistema de Lectura Ribosómico , VIH-1/genética , Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Viral/genética , Animales , Antibacterianos/farmacología , Secuencia de Bases , Sistema Libre de Células , Cloranfenicol O-Acetiltransferasa/genética , Clonación Molecular , Escarabajos , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Genes Reporteros , Genes gag , Genes pol , Humanos , Luciferasas/genética , Modelos Genéticos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación de Ácido Nucleico , Biosíntesis de Proteínas , ARN Mensajero/química , ARN Viral/química , Proteínas Recombinantes/biosíntesis , Eliminación de SecuenciaRESUMEN
The 3' major domain of Escherichia coli 16S rRNA, which occupies the head of the small ribosomal subunit, is involved in several functions of the ribosome. We have used a site-specific crosslinking procedure to gain further insights into the higher-order structure of this domain. Circularly permuted RNAs were used to introduce an azidophenacyl group at specific positions within the 3' major domain. Crosslinks were generated in a high-ionic strength buffer that has been used for ribosome reconstitution studies and so enables the RNA to adopt a structure recognized by ribosomal proteins. The crosslinking sites were identified by primer extension and confirmed by assessing the mobility of the crosslinked RNA lariats in denaturing polyacrylamide gels. Eight crosslinks were characterized. Among them, one crosslink demonstrates that helix 28 is proximal to the top of helix 34, and two others show that the 1337 region, located in an internal loop at the junction of helices 29, 30, 41, and 42, is proximal to the center of helix 30 and to a segment connecting helix 28 to helix 29. These relationships of vicinity have previously been observed in native 30S subunits, which suggests that the free domain adopts a conformation similar to that within the 30S subunit. Furthermore, crosslinks were obtained in helix 34, which suggest that the upper and lower portions of this helix are in close proximity.
Asunto(s)
Reactivos de Enlaces Cruzados , Conformación de Ácido Nucleico , Etiquetas de Fotoafinidad , ARN Ribosómico 16S/química , Azidas/metabolismo , Secuencia de Bases , Centrifugación por Gradiente de Densidad , Electroforesis en Gel de Poliacrilamida , Escherichia coli/química , Guanina/metabolismo , Reacción en Cadena de la Polimerasa , ARN/biosíntesis , ARN/metabolismo , ARN Bacteriano/química , ARN Bacteriano/metabolismo , ARN Circular , ARN Ribosómico 16S/metabolismo , Proteínas Ribosómicas/biosíntesis , Proteínas Ribosómicas/metabolismo , Azufre/metabolismoRESUMEN
Human immunodeficiency virus type 1 (HIV-1) variants resistant to protease inhibitors have been shown to contain a mutation in the p1/p6 Gag precursor cleavage site. At the messenger RNA level, this mutation generates a U UUU UUU sequence that is reminiscent of the U UUU UUA sequence required for ribosomal frameshifting and Gag-Pol synthesis. To test whether the p1/p6 cleavage site mutation was generating a novel frameshift site, HIV sequences were inserted in translation vectors containing a chloramphenicol acetyltransferase (CAT) reporter gene requiring -1 frameshifting for expression. All sequences containing the original HIV frameshift site supported the synthesis of CAT but expression was increased 3- to 11-fold in the presence of the mutant p1/p6 sequence. When the original frameshift site was abolished by mutation, expression remained unchanged when using constructs containing the mutant p1/p6 sequence, whereas it was decreased 2- to 4.5-fold when using wild-type p1/p6 constructs. Similarly, when introduced into HIV molecular clones, the p1/p6 mutant sequence supported Gag-Pol synthesis and protease activity in the absence of the original frameshift site, indicating that this sequence could also promote ribosomal frameshifting in virus-expressing cells.
Asunto(s)
Mutación del Sistema de Lectura , Proteínas de Fusión gag-pol/genética , Inhibidores de la Proteasa del VIH/farmacología , VIH-1/efectos de los fármacos , Secuencia de Aminoácidos , Secuencia de Bases , Farmacorresistencia Microbiana , VIH-1/genética , Humanos , Datos de Secuencia MolecularRESUMEN
Mutations at positions 13 (U-->A) and/or 914 (A-->U) of Escherichia coli 16S rRNA severely affect cell growth and protein synthesis, when expressed in vivo in a vector encoding an rrn operon under control of an inducible promoter. In vitro assays using extension inhibition indicate that the mutations interfere with the formation of the 30S translational initiation complex, which can account for their effect on cell growth. The two mutations destabilize an adjacent pseudoknot helix in which bases 17-19 pair to bases 916-918. This was shown by the increased binding of an oligodeoxyribonucleotide probe complementary to one strand of the pseudoknot helix, and by the increased reactivity to kethoxal of base G917 within this helix. These observations suggest that this pseudoknot helix participates in the formation of the 30S translational initiation complex.
Asunto(s)
Escherichia coli/metabolismo , Conformación de Ácido Nucleico , Iniciación de la Cadena Peptídica Traduccional , Mutación Puntual , ARN Ribosómico 16S/química , ARN Ribosómico 16S/metabolismo , Ribosomas/metabolismo , Adenina , Aldehídos/farmacología , Proteínas Bacterianas/biosíntesis , Secuencia de Bases , Butanonas , Cloranfenicol O-Acetiltransferasa/biosíntesis , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Cinética , Modelos Estructurales , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , ARN Mensajero/biosíntesis , ARN Mensajero/metabolismo , ARN Ribosómico 16S/genética , Ribosomas/ultraestructura , Espectinomicina/farmacología , UraciloRESUMEN
Ribosomal protein S7 binds to a small RNA fragment of about 100 nucleotides within the lower half of the 3' major domain of E. coli 16 S rRNA. This fragment (D3M) comprises two large internal loops, A and B, connected by helix 29, a six-base-pair helix containing a G.U pair. Two hairpins with non-canonical base-pairs, 42' and 43, protrude from loops A and B, respectively. We used site-directed mutagenesis and molecular probing to further define which parts of D3M are important for S7 binding. Changing the stem of hairpin 42' into a Watson-Crick helix did not affect S7 binding, indicating that the non-canonical pairs of 42' do not provide recognition features for S7. However, deletion of this hairpin decreased S7 binding affinity by about threefold and altered the conformation of loop A. Deletion of the upper part of hairpin 43 (the loop and the adjacent four base-pairs) did not affect S7 binding, whereas the lower part of this hairpin (three base-pairs) was found to be required for proper S7 binding. Moreover, replacing the U.G pair with a C.G pair in this lower part decreased S7 binding affinity by twofold, suggesting that the U.G pair is a recognition signal for S7. S7 binding was also affected by mutations in helix 29. Insertion of one nucleotide 5' to the G or 3' to the U of the G.U pair decreased S7 binding affinity by about threefold and twofold, respectively, whereas replacement of the G.U pair by a G.C pair enhanced the affinity about twofold, and lengthening the helix by inserting a C.G pair upstream from the G.U pair had no effect. Taken together, these results are consistent with a bipartite binding site for S7 on 16 S rRNA, involving two regions of interaction: one centered around helix 29 and extending on the adjacent part of loop A, and the other one centered around the lower part of hairpin 43 and probably extending on the adjoining part of loop B.
Asunto(s)
Escherichia coli/genética , ARN Bacteriano/genética , ARN Ribosómico 16S/genética , Proteínas Ribosómicas/metabolismo , Secuencia de Bases , Sitios de Unión/genética , Análisis Mutacional de ADN , Escherichia coli/metabolismo , Sondas Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Unión Proteica , ARN/química , ARN/metabolismo , ARN Bacteriano/química , ARN Bacteriano/metabolismo , ARN Ribosómico 16S/química , ARN Ribosómico 16S/metabolismo , Relación Estructura-ActividadRESUMEN
Using a conditional expression system with the temperature-inducible lambda PL promoter, we previously showed that the single mutations 13U-->A and 914A-->U, and the double mutation 13U-->A and 914A-->U in Escherichia coli 16S ribosomal RNA impair the binding of streptomycin (Pinard et al., The FASEB Journal, 1993, 7, 173-176). In this study, we found that the two single mutations and the double mutation increase translational fidelity, reducing in vivo readthrough of nonsense codons and frameshifting, and decreasing in vitro misincorporation in a poly(U)-directed system. Using oligodeoxyribonucleotide probes which hybridize to the 530 loop and to the 1400 region of 16S rRNA, two regions involved in the control of tRNA binding to the A site, we observed that the mutations in rRNA increase the binding of the probe to the 530 loop but not to the 1400 region. We suggest that the mutations at positions 13 and 914 of 16S rRNA induce a conformational rearrangement in the 530 loop, which contributes to the increased accuracy of the ribosome.
Asunto(s)
Escherichia coli/genética , Biosíntesis de Proteínas , ARN Bacteriano/genética , ARN Ribosómico 16S/genética , Secuencia de Bases , División Celular/genética , Datos de Secuencia Molecular , Mutación , Conformación de Ácido Nucleico , Sondas de Oligonucleótidos/metabolismoRESUMEN
Single mutations at the end of the 5' proximal helix and in the 915 region (13U-->A or C; 914A-->U or G), and double mutations (13U-->A and 914A-->U; 13U-->C and 914A-->G) were constructed into Escherichia coli 16S ribosomal RNA. The mutations were introduced into an expression plasmid containing the rrnB operon under the transcriptional control of the temperature-inducible lambda PL promoter. None of the mutant 16S rRNAs affected cell growth when expressed. Ribosomes extracted after induction of expression of the mutant 16S rRNAs were assayed for their capacity to bind the error-inducing drug streptomycin and for translational misreading in the presence of streptomycin. All mutations impaired the binding of streptomycin, and consequently its capacity to stimulate misreading. Our results demonstrate the involvement of the 5' proximal helix of 16S rRNA in the binding of streptomycin and confirm the participation of the 915 region. They do not support a previous suggestion [Leclerc, D. and Brakier-Gingras, L. (1991) FEBS Lett., Vol. 279, pp. 171-174] that base pairing between nucleotides 13 and 914 stabilizes the binding of streptomycin.
Asunto(s)
Sulfato de Dihidroestreptomicina/metabolismo , Escherichia coli/metabolismo , Biosíntesis de Proteínas/efectos de los fármacos , ARN Ribosómico 16S/metabolismo , Ribosomas/metabolismo , Secuencia de Bases , Análisis Mutacional de ADN , Sulfato de Dihidroestreptomicina/farmacología , Escherichia coli/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación de Ácido Nucleico , ARN Ribosómico 16S/genética , Relación Estructura-ActividadRESUMEN
After reviewing epidemiological literature on the relationships between depression in parents and onset of a psychiatric disturbance in children as well as investigations of the interactions between a depressed mother and her infant, the authors discuss the psychoanalytical concepts of these interactions and the findings of their research. This epidemiological research is based on the assumption that depression in the mother during pregnancy and the first few months of the post-partum constitutes a risk factor for the onset of an early psychosis in the child. This research is a comparative study between a group of mothers with early onset psychotic children and a group of mothers with non patient children, the age and sex of the children in both groups being equally distributed. The methodology includes two instruments aimed at a retrospective assessment of mothers' depression: the SADS-LA questionnaire and a standardized scoring system for semi-structured interviews investigating the mother's feelings during pregnancy and early development of her baby. Results show that there is a statistically significant relationship between a major depressive condition in the mother during pregnancy and/or during the first year of the infant's life and the onset of an autism in the child. It might be that major depressive conditions starting before delivery could by themselves account for the risk. Depression in the mother therefore constitutes a risk factor for early psychosis, the relative risk being about four. This study also emphasizes particular features of the mother's depressive conditions: difficulties to accept the real child, difficulties in perceiving the infant's psychic evolution, decrease of interactive skills. The statistically significant relationship in no way points to a linear causal relationship, which hypothesis seems to be negated by the statistical findings of this research.
Asunto(s)
Trastornos Generalizados del Desarrollo Infantil/epidemiología , Trastorno Depresivo/complicaciones , Madres/psicología , Trastornos Puerperales/complicaciones , Trastornos Generalizados del Desarrollo Infantil/etiología , Trastornos Generalizados del Desarrollo Infantil/psicología , Preescolar , Trastorno Depresivo/diagnóstico , Trastorno Depresivo/psicología , Humanos , Entrevista Psicológica , Relaciones Madre-Hijo , Trastornos Puerperales/diagnóstico , Trastornos Puerperales/psicología , Factores de RiesgoRESUMEN
The personality traits defined by the Minnesota Multiphasic Personality Inventory (MMPI) and sleep data were analyzed in 45 young subjects with poor quality of sleep. The subjects were divided into three groups: Group 1 had no T score greater than or equal to 70, Group 2 had one or more single T scores greater than or equal to 70, and Group 3 had T scores greater than or equal to 70 in one or more specific groups of scales. The first 2 nights of sleep were polygraphically recorded. Subjects in Group 1 were considered to be normal, those in Group 2 were characterized by depression and anxiety, and those in Group 3 had psychopathic personality traits and somatic disorders. Differences in sleep data were noted among groups. The severity of the sleep disorders was related to the degree of the psychological problems.