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PURPOSE: To present the proof of concept and the experimental validation of tomographic dosimetry (tomodosimetry), where a tomographic acquisition of the incident deposited dose is performed using long scintillating fibers. METHOD: 2D tomodosimetry: 50 long scintillating fibers were aligned on a 20cm diameter disk inside a 30cm diameter masonite phantom. 3D tomodosimetry: 128 long scintillating fibers of various orientation were simulated on the surface of two cylindrical regions of radius 7.5 and 3.75cm inside a 20cm diameter, 20cm long cylindrical phantom. In both case, the dose projections were acquire each 5 degrees over a 180 degrees (2D) or 360 degrees (3D) rotation of the device, and the dose in each scintillating fiber plane was reconstructed using a total variation minimization reconstruction iterative algorithm at a resolution of 1×1mm2 . The 3D dose was obtained by interpolating between in each cylindrical plane in the 3D prototype. RESULTS: 3%/3mm gamma tests conducted in the isocentre plane for both configurations achieved a success rate of more than 99% of the dose pixels in the region over 50% of the maximum dose. Absolute dose differences in the high dose low gradient region of each scintillating fiber plane were on average below 1% for the 2D configuration and below 1.3% for the 3D configuration. CONCLUSIONS: This work illustrates the potential and capacity of scintillating fiber based 2D and 3D tomodosimeters. The presented methodology allows for millimeter resolution dosimetry in a whole 2D plane or 3D volumes in real-time using only a limited number of detectors.
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PURPOSE: To ensure the quality assurance of small field, dynamic radiotherapy, we present and validate a radiation tracking system based on long scintillating fibers that allows for the real-time measurement of the position and energetic fluence of a small incident radiation field. METHOD: We aligned 60 parallel scintillating fibers on a thin grooved acrylic slab with a 100-cm source-to-fibers distance. Both ends of each scintillating fiber were coupled to clear optical fibers to enable light collection by a single CCD camera using an f/0.95, 50 mm focal length lens. Using a small, static photon radiation field of 2×2 cm2 of a Varian Clinac iX, we changed the interaction position on the prototype using the linac treatment couch. The interaction position parallel and perpendicular to the scintillating fiber array were deduced using the optical attenuation of the scintillating fibers. The energetic fluence of the incident field was calculated from the fibers light fluxes, corrected for the position dependent optical attenuation and scintillation efficiency. RESULTS: Considering a treatment couch positioning error of ±0.5 mm, the system was able to measure the field position with a mean error of 0.1 mm perpendicular and 0.8 mm parallel to the scintillating fiber array. The maximum error measured using this setup was of 0.13 mm perpendicular and 3.2 mm parallel to the scintillating fiber array. The energetic fluence was determined with a mean error of 0.5% and a maximum error of 2.2%. CONCLUSIONS: This work demonstrates the capacity of a long scintillating fibers array to detect in real-time both the position and the energetic fluence of an incident small radiation field. Such methodology would allow for the real-time tracking of small field in both photon and particle radiation therapy.
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PURPOSE: To demonstrate that the independence of the calibration coefficients of plastic scintillation detector (PSD) for both photon and electron beams in the megavoltage energy range. METHOD AND MATERIALS: The PSD consists in a small 1 mm diameter and 2 mm long plastic scintillating fiber made of a polystyrene core (BCF-12, Saint-Gobain, inc.). The scintillator was coupled to a 2 meters long non-scintillating plastic optical fiber and a color CCD camera (Apogee instruments inc.) was used as photodetector. The calibration coefficients of the PSD where extracted for 6 MV, 23 MV photon beams and 9,12,15 and 18 MeV electron beams using a Farmer ionization chamber (Exradin). Complete removal of the Cerenkov radiation produced in the optical fiber was performed with a chromatic discrimination technique using the blue and green channel of the CCD camera. All measurements were performed according to the recommendations of the AAPM TG-51 protocol for clinical dosimetry. RESULTS: The PSD exhibits a maximum deviation of less than 1.7 % (about the mean) of its calibration coefficients over the measured energy range for both irradiation modalities. CONCLUSION: The energy independence of the calibration coefficients for PSD was demonstrated experimentally for the first time for both photons and electrons. PSDs have the potential to simplify and improve accuracy of dose measurements in clinical situations where photons and electrons are both present in the beam such as electron contamination in photon beams or bremsstrahlung contaminated electron beams.
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In the case of non-small cell lung cancer, doses typically prescribed (60-66 Gy) are not sufficient to ensure a satisfactory tumor control probability. Dose escalation needs to be realized, but dose to organs at risk (OARs) must be kept under widely accepted clinical thresholds. Also, lung functionality is not homogeneously distributed over all the volume: single-photon emission computed tomography (SPECT) allows spatial characterization of perfusion, open the way to the design of treatments plans that could preferentially avoid highly-functional lung. In this study, three cases of lung cancer were retrospectively used to assess the capacity of an anatomy-based aperture inverse planning system to realize dose escalation while limiting dose to perfused lung. Plans were generated for four-beam non-coplanar configurations, mixing 6 and 23 MV photon beams. All dose calculations were performed using Pinnacle3 superposition/convolution algorithm. An increasing dose was prescribed to a subvolume of the initial planning target volume. Levels of escalation achieved for the three cases studied were 81 Gy, 111 Gy and 66 Gy to the subvolume. Escalation was limited in two cases by the dose to the esophagus and in the other case by the presence of overdosages near beam entry ports. Calculation of dose-volume parameters for OARs shows that they respect clinical thresholds. Plans generated by the system are less complex than plans generated in beamlet-based IMRT, because of the use of few, large segments. The approach used in this study allows important dose escalation, potentially improving treatment outcome.
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PURPOSE: To perform full 3D heterogeneous dose calculations for total body irradiation (TBI) cases and compare different treatment planning softwares (TPS). METHOD: A retrospective study was performed on 7 patients. Dose distributions obtained with Pinnacle3 v.7.9u (Philips Medical Systems) were compared with the ones calculated using our actual TBI planning system Theraplan Plus (TPP) by MDS Nordion/Nucletron. Two different Pinnacle3 models were studied: standard beam commissioning (std_Pinnacle3 ) and TBI commissioning (TBI_Pinnacle3 ). For the later case, commissioning was adapted for the special TBI conditions (extended SSD of 190cm, large field, acrylic beam spoiler, and out of field dose (OFD)). RESULTS: Significant differences are found between the TPP, std_Pinnacle3 and TBI_Pinnacle3 dose distributions. For the relative mid-line doses, differences up to 12% were observed. Systematic overestimations of 5% were found in patients extremities between TPP and TBI_Pinnacle3 . Average dose underestimation of 3% was observed between std_Pinnacle3 and TBI_Pinnacle3 . Differences in patient extremities are attributed to the OFD contribution which is not correctly computed in TPP and std_Pinnacle3 . Dose comparison outside the patient's mid-line showed greater differences (up to 20%) between models. Accurate 3D heterogeneous dose calculations with TBI_Pinnacle3 model show major differences (homogeneous versus heterogeneous) in high and low density regions. Dose overestimation of 5% was observed in bony regions and dose underestimation of 5% to 10% was observed in lung regions. CONCLUSION: Those results are of major interest since they show a strong dependence of the dose calculation outcome on both TPS and commissioning used, potentially leading to significant dose misevaluation.
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Ribosomal protein S7 from Escherichia coli binds to the lower half of the 3' major domain of 16S rRNA and initiates its folding. It also binds to its own mRNA, the str mRNA, and represses its translation. Using filter binding assays, we show in this study that the same mutations that interfere with S7 binding to 16S rRNA also weaken its affinity for its mRNA. This suggests that the same protein regions are responsible for mRNA and rRNA binding affinities, and that S7 recognizes identical sequence elements within the two RNA targets, although they have dissimilar secondary structures. Overexpression of S7 is known to inhibit bacterial growth. This phenotypic growth defect was relieved in cells overexpressing S7 mutants that bind poorly the str mRNA, confirming that growth impairment is controlled by the binding of S7 to its mRNA. Interestingly, a mutant with a short deletion at the C-terminus of S7 was more detrimental to cell growth than wild-type S7. This suggests that the C-terminal portion of S7 plays an important role in ribosome function, which is perturbed by the deletion.
Assuntos
Escherichia coli/química , RNA Mensageiro/química , RNA Ribossômico 16S/química , Proteínas Ribossômicas/química , Sequência de Bases , Sítios de Ligação , Ligação Competitiva , Divisão Celular/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Genótipo , Dados de Sequência Molecular , Estrutura Molecular , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Ribossômico 16S/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismoRESUMO
Bacterial ribosomal protein S7 initiates the folding of the 3' major domain of 16S ribosomal RNA by binding to its lower half. The X-ray structure of protein S7 from thermophilic bacteria was recently solved and found to be a modular structure, consisting of an alpha-helical domain with a beta-ribbon extension. To gain further insights into its interaction with rRNA, we cloned the S7 gene from Escherichia coli K12 into a pET expression vector and introduced 4 deletions and 12 amino acid substitutions in the protein sequence. The binding of each mutant to the lower half of the 3' major domain of 16S rRNA was assessed by filtration on nitrocellulose membranes. Deletion of the N-terminal 17 residues or deletion of the B hairpins (residues 72-89) severely decreased S7 affinity for the rRNA. Truncation of the C-terminal portion (residues 138-178), which includes part of the terminal alpha-helix, significantly affected S7 binding, whereas a shorter truncation (residues 148-178) only marginally influenced its binding. Severe effects were also observed with several strategic point mutations located throughout the protein, including Q8A and F17G in the N-terminal region, and K35Q, G54S, K113Q, and M115G in loops connecting the alpha-helices. Our results are consistent with the occurrence of several sites of contact between S7 and the 16S rRNA, in line with its role in the folding of the 3' major domain.
Assuntos
Escherichia coli/genética , RNA Ribossômico 16S/química , RNA Ribossômico 16S/metabolismo , Proteínas Ribossômicas/química , Proteínas Ribossômicas/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Sequência de Bases , Sítios de Ligação , Dicroísmo Circular , Escherichia coli/metabolismo , Genes Bacterianos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , Mutação Puntual , Conformação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Deleção de SequênciaAssuntos
Censos , Demografia , Fertilidade , Censos/história , Características da Família , Feminino , História do Século XX , Humanos , Paridade , Gravidez , QuebequeRESUMO
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.
Assuntos
Mudança da Fase de Leitura do Gene Ribossômico , HIV-1/genética , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Viral/genética , Animais , Antibacterianos/farmacologia , Sequência de Bases , Sistema Livre de Células , Cloranfenicol O-Acetiltransferase/genética , Clonagem Molecular , Besouros , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Genes Reporter , Genes gag , Genes pol , Humanos , Luciferases/genética , Modelos Genéticos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , Biossíntese de Proteínas , RNA Mensageiro/química , RNA Viral/química , Proteínas Recombinantes/biossíntese , Deleção de SequênciaRESUMO
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.
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Reagentes de Ligações Cruzadas , Conformação de Ácido Nucleico , Marcadores de Fotoafinidade , RNA Ribossômico 16S/química , Azidas/metabolismo , Sequência de Bases , Centrifugação com Gradiente de Concentração , Eletroforese em Gel de Poliacrilamida , Escherichia coli/química , Guanina/metabolismo , Reação em Cadeia da Polimerase , RNA/biossíntese , RNA/metabolismo , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA Circular , RNA Ribossômico 16S/metabolismo , Proteínas Ribossômicas/biossíntese , Proteínas Ribossômicas/metabolismo , Enxofre/metabolismoRESUMO
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.
Assuntos
Mutação da Fase de Leitura , Proteínas de Fusão gag-pol/genética , Inibidores da Protease de HIV/farmacologia , HIV-1/efeitos dos fármacos , Sequência de Aminoácidos , Sequência de Bases , Resistência Microbiana a Medicamentos , HIV-1/genética , Humanos , Dados de Sequência MolecularRESUMO
Streptomycin, an error-inducing aminoglycoside antibiotic, binds to a single site on the small ribosomal subunit of bacteria, but this site has not yet been defined precisely. Here, we demonstrate that streptomycin binds to E. coli 16 S rRNA in the absence of ribosomal proteins, and protects a set of bases in the decoding region against dimethyl sulfate attack. The binding studies were performed in a high ionic strength buffer containing 20 mM Mg2+. The pattern of protection in the decoding region was similar to that observed when streptomycin binds to the 30 S subunit. However, streptomycin also protects the 915 region of 16 S rRNA within the 30 S subunit, whereas it did not protect the 915 region of the naked 16 S rRNA. The interaction of streptomycin with 16 S rRNA was further defined by using two fragments that correspond to the 3' minor domain of 16 S rRNA and to the decoding analog, a portion of this domain encompassing the decoding center. In the presence of streptomycin, the pattern of protection against dimethyl sulfate attack for the two fragments was similar to that seen with the full-length 16 S rRNA. This indicates that the 3' minor domain as well as the decoding analog contain the recognition signals for the binding of streptomycin. However, streptomycin could not bind to the decoding analog in the absence of Mg2+. This contrasts with neomycin, another error-inducing aminoglycoside antibiotic, that binds to the decoding analog in the absence of Mg2+, but not at 20 mM Mg2+. Our results suggest that both neomycin and streptomycin interact with the decoding center, but recognize alternative conformations of this region.
Assuntos
Antibacterianos/metabolismo , RNA Bacteriano/metabolismo , RNA Ribossômico 16S/metabolismo , Estreptomicina/metabolismo , Antibacterianos/farmacologia , Sequência de Bases , Sítios de Ligação , Relação Dose-Resposta a Droga , Escherichia coli/metabolismo , Magnésio/farmacologia , Dados de Sequência Molecular , Mutagênicos/farmacologia , Neomicina/metabolismo , Neomicina/farmacologia , Conformação de Ácido Nucleico , RNA Bacteriano/química , RNA Bacteriano/efeitos dos fármacos , RNA Ribossômico 16S/química , RNA Ribossômico 16S/efeitos dos fármacos , Estreptomicina/farmacologia , Ésteres do Ácido Sulfúrico/farmacologiaRESUMO
An increased density of alpha1-adrenergic receptors (AR) has been linked to the development of necrotic lesions in the heart of hamsters with hereditary cardiomyopathy. To determine whether this increase results from an upregulation of the receptor mRNA(s), Northern blot analyses were carried out in the heart of 60-day-old control and cardiomyopathic hamsters using selective DNA probes for the three subtypes of alpha1-ARs (alpha1A, alpha1B and alpha1D). Transcripts for the three alpha1-ARs were detected in both control and cardiomyopathic hamsters. A two-fold increase in the alpha1A- and alpha1B-AR mRNA levels was observed in the cardiomyopathic hearts when compared to controls. In contrast, no change in the alpha1D-AR mRNA level could be detected. The enhancement in alpha1A- and alpha1B-AR mRNA levels was paralleled by a 20% increase in the total number of alpha1-ARs, as assessed by [3H]prazosin radioligand binding assays. Competition binding assays using subtype selective ligands indicated that the increased density of both alpha1A and alpha1B receptors contributes to the total alpha1-AR upregulation. Taken together, these data suggest that the early development of hereditary cardiomyopathy in hamsters is accompanied by a specific overexpression of the alpha1A- and alpha1B-ARs. A discrete increase of the alpha1-AR density could contribute to eliciting coronary microspasms, therefore participating in the development of focal necrotic lesions that are characteristic of the hamster cardiomyopathic model.
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Cardiomiopatias/metabolismo , Miocárdio/metabolismo , RNA Mensageiro/metabolismo , Receptores Adrenérgicos alfa 1/genética , Animais , Ligação Competitiva , Northern Blotting , Cricetinae , Código Genético , Modelos Logísticos , Masculino , Mesocricetus , Ensaio Radioligante , Valores de Referência , Análise de Regressão , Regulação para CimaRESUMO
Mutations at position 13 or 914 of Escherichia coli 16S ribosomal RNA exert pleiotropic effects on protein synthesis. They interfere with the binding of streptomycin, a translational miscoding drug, to the ribosomes. They increase translational fidelity, and this effect can be related to a perturbation of the higher order structure of the 530 stem-loop, a key region for tRNA selection. In contrast, the structure of the decoding center is not perturbed. The mutations also affect translational initiation, slowing down the formation of the 30S initiation complex. This effect can be related to a destabilization of the pseudoknot helix (17-19/916-918), at the convergence of the three major domains of 16S ribosomal RNA.
Assuntos
Escherichia coli/genética , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Sequência de Bases , Escherichia coli/metabolismo , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , Fenótipo , RNA Bacteriano/metabolismo , RNA Ribossômico 16S/metabolismo , Estreptomicina/metabolismoRESUMO
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.
Assuntos
Escherichia coli/metabolismo , Conformação de Ácido Nucleico , Iniciação Traducional da Cadeia Peptídica , Mutação Puntual , RNA Ribossômico 16S/química , RNA Ribossômico 16S/metabolismo , Ribossomos/metabolismo , Adenina , Aldeídos/farmacologia , Proteínas de Bactérias/biossíntese , Sequência de Bases , Butanonas , Cloranfenicol O-Acetiltransferase/biossíntese , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Cinética , Modelos Estruturais , Dados de Sequência Molecular , Regiões Promotoras Genéticas , RNA Mensageiro/biossíntese , RNA Mensageiro/metabolismo , RNA Ribossômico 16S/genética , Ribossomos/ultraestrutura , Espectinomicina/farmacologia , UracilaRESUMO
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.
Assuntos
Escherichia coli/genética , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Proteínas Ribossômicas/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Análise Mutacional de DNA , Escherichia coli/metabolismo , Sondas Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Ligação Proteica , RNA/química , RNA/metabolismo , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA Ribossômico 16S/química , RNA Ribossômico 16S/metabolismo , Relação Estrutura-AtividadeRESUMO
The cDNA for hamster preproenkephalin A (ENK) was cloned from an adrenal gland cDNA library constructed in the lambda ZapII vector. A nearly full-length cDNA was obtained and its 5' end region was completed using the technique of rapid amplification of cDNA ends (RACE). The coding and 3' untranslated regions of the hamster ENK cDNA share a high sequence identity with the rat, human, and bovine cDNAs, whereas the sequence identity is lower for the 5' untranslated region. Southern blot analysis of genomic DNA digests showed that a single copy of the ENK gene is present in the hamster haploid genome. Northern blot analysis of poly(A)+RNA from various hamster tissues indicated the following rank order for ENK messenger RNA abundance: adrenal glands > right atrium > brain > left atrium > right ventricle > ventricular septum > left ventricle, whereas primer extension analysis showed a single, identical transcriptional initiation site for the ENK mRNA in all these tissues. The sequence of the 5' untranslated region of the heart ENK cDNA was found to be identical to that from adrenal glands. This rules out the possibility that structural divergences in the 5' untranslated region of the heart ENK mRNA could decrease its translation efficiency and contribute to the very low level of enkephalin-containing peptides in the heart, compared to the adrenal glands.
Assuntos
DNA Complementar/genética , Encefalinas/genética , Precursores de Proteínas/genética , Glândulas Suprarrenais/química , Animais , Clonagem Molecular , Cricetinae , Encefalinas/química , Dosagem de Genes , Biblioteca Gênica , Humanos , Masculino , Mesocricetus , Dados de Sequência Molecular , Miocárdio/química , Especificidade de Órgãos , Precursores de Proteínas/química , RNA Mensageiro/análise , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
The beta-adrenergic receptor (beta AR)/adenylyl cyclase signalling pathway was examined in cardiac membranes from cardiomyopathic Syrian hamsters. Three stages were examined during the progression of this hereditary cardiomyopathy (30 days old, prenecrotic phase; 60 days old, necrotic phase; and 120 days old, compensatory phase). Isoproterenol-stimulated adenylyl cyclase activity was decreased by 32 +/- 16% in 30-day-old cardiomyopathic hamsters, compared with age-matched controls. This was not accompanied by any change in the fluoride- or forskolin-stimulated activities, suggesting that the decrease reflects a perturbation of the receptor-mediated stimulation. Neither the density nor the subcellular distribution of the beta AR, as assessed by [125I]iodocyanopindolol binding assays, was affected in these animals. However, the agonist binding properties of the beta AR were significantly affected. Indeed, the effect of guanyl nucleotides on isoproterenol binding was decreased in 30-day-old cardiomyopathic hamsters. Given that guanyl nucleotide sensitivity is correlated with the ability of the beta AR to productively interact with Gs protein, these results suggest that the decreased beta-adrenergic-stimulated adenylyl cyclase activity results from a functional uncoupling of the beta AR with no change in receptor density. The desensitization of the beta-adrenergic-stimulated adenylyl cyclase was transient, since no change in isoproterenol-stimulated adenylyl cyclase was detected in 60- and 120-day-old hamsters, compared with age-matched controls. Similarly, the receptor number and distribution were not affected at those ages.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Cardiomiopatias/fisiopatologia , Receptores Adrenérgicos beta/fisiologia , Adenilil Ciclases/análise , Envelhecimento/fisiologia , Animais , Ligação Competitiva , Cardiomiopatias/genética , Células Cultivadas , Cricetinae , Masculino , Mesocricetus , Ensaio Radioligante , Receptores Adrenérgicos beta/metabolismoRESUMO
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.