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The waveguide losses from a range of surface plasmon and double metal waveguides for Ge/Si1-xGex THz quantum cascade laser gain media are investigated at 4.79 THz (62.6 µm wavelength). Double metal waveguides demonstrate lower losses than surface plasmonic guiding with minimum losses for a 10 µm thick active gain region with silver metal of 21 cm-1 at 300 K reducing to 14.5 cm-1 at 10 K. Losses for silicon foundry compatible metals including Al and Cu are also provided for comparison and to provide a guide for gain requirements to enable lasers to be fabricated in commercial silicon foundries. To allow these losses to be calculated for a range of designs, the complex refractive index of a range of nominally undoped Si1-xGex with x = 0.7, 0.8 and 0.9 and doped Ge heterolayers were extracted from Fourier transform infrared spectroscopy measurements between 0.1 and 10 THz and from 300 K down to 10 K. The results demonstrate losses comparable to similar designs of GaAs/AlGaAs quantum cascade laser plasmon waveguides indicating that a gain threshold of 15.1 cm-1 and 23.8 cm-1 are required to produce a 4.79 THz Ge/SiGe THz laser at 10 K and 300 K, respectively, for 2 mm long double metal waveguide quantum cascade lasers with facet coatings.
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BACKGROUND: Little is known about xeroderma pigmentosum (XP) in Himalayan countries. OBJECTIVE: To describe clinical characteristics of XP in Nepal and investigate its genetic bases. METHODS: This study was carried out on all consecutive patients referred for XP to a Nepalese tertiary referral centre in 2014-2015. Clinical data were collected using a standardized questionnaire. DNA was extracted from salivary samples, and next-generation sequencing (NGS) was conducted using a panel covering all 8 known XP genes (classical XP (XP-A to XP-G) and XP variant) and a skin cancer modifier gene, the melanocortin 1 receptor gene (MC1R). RESULTS: Seventeen patients (median age: 15 years; range: 1-32) were included. Twelve had skin cancers (including a total of 8 squamous cell carcinomas, 60 basal cell carcinomas, ocular carcinomas requiring an orbital exenteration in 3 patients, but no melanoma). Fifteen patients carried the same homozygous non-sense XPC mutation c.1243C>T, p.R415X. A homozygous non-sense XPA mutation (p.W235X) was found in the only patient with a history of early severe sunburn reaction and associated neurological symptoms. Associated genetic alterations included heterozygous missense variants in XPD/ERCC2 gene and the presence of MC1R variant R163Q in 5 and 9 patients, respectively. CONCLUSION: Although not previously reported, XP seems frequent in Nepal. Patients often presented with a very severe phenotype after a long history of excessive sun exposure without knowledge of the disease. Fifteen of 17 had the same p.R415X XPC mutation, which seems very specific of XP in Nepal, suggesting a founder effect. NGS analyses frequently revealed associated genetic alterations which could play a modifier role in the clinical expression of the disease.
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Carcinoma Basocelular/etiología , Carcinoma de Células Escamosas/etiología , Proteínas de Unión al ADN/genética , Neoplasias del Ojo/etiología , Neoplasias Primarias Múltiples/etiología , Neoplasias Cutáneas/etiología , Xerodermia Pigmentosa/genética , Adolescente , Adulto , Niño , Preescolar , Análisis Mutacional de ADN , Femenino , Heterocigoto , Secuenciación de Nucleótidos de Alto Rendimiento , Homocigoto , Humanos , Lactante , Queratosis Actínica/etiología , Masculino , Mutación , Nepal , Fenotipo , Proyectos Piloto , Estudios Prospectivos , Receptor de Melanocortina Tipo 1/genética , Xerodermia Pigmentosa/complicaciones , Proteína de la Xerodermia Pigmentosa del Grupo A/genética , Proteína de la Xerodermia Pigmentosa del Grupo D/genética , Adulto JovenRESUMEN
Solid-state emitters are excellent candidates for developing integrated sources of single photons. Yet, phonons degrade the photon indistinguishability both through pure dephasing of the zero-phonon line and through phonon-assisted emission. Here, we study theoretically and experimentally the indistinguishability of photons emitted by a semiconductor quantum dot in a microcavity as a function of temperature. We show that a large coupling to a high quality factor cavity can simultaneously reduce the effect of both phonon-induced sources of decoherence. It first limits the effect of pure dephasing on the zero-phonon line with indistinguishabilities above 97% up to 18 K. Moreover, it efficiently redirects the phonon sidebands into the zero-phonon line and brings the indistinguishability of the full emission spectrum from 87% (24%) without cavity effect to more than 99% (76%) at 0K (20K). We provide guidelines for optimal cavity designs that further minimize the phonon-induced decoherence.
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We present a cost-effective metabarcoding approach, aMPlex Torrent, which relies on an improved multiplex PCR adapted to highly degraded DNA, combining barcoding and next-generation sequencing to simultaneously analyse many heterogeneous samples. We demonstrate the strength of these improvements by generating a phylochronology through the genotyping of ancient rodent remains from a Moroccan cave whose stratigraphy covers the last 120 000 years. Rodents are important for epidemiology, agronomy and ecological investigations and can act as bioindicators for human- and/or climate-induced environmental changes. Efficient and reliable genotyping of ancient rodent remains has the potential to deliver valuable phylogenetic and paleoecological information. The analysis of multiple ancient skeletal remains of very small size with poor DNA preservation, however, requires a sensitive high-throughput method to generate sufficient data. We show this approach to be particularly adapted at accessing this otherwise difficult taxonomic and genetic resource. As a highly scalable, lower cost and less labour-intensive alternative to targeted sequence capture approaches, we propose the aMPlex Torrent strategy to be a useful tool for the genetic analysis of multiple degraded samples in studies involving ecology, archaeology, conservation and evolutionary biology.
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Código de Barras del ADN Taxonómico , Roedores/clasificación , Animales , Arqueología , Genotipo , Secuenciación de Nucleótidos de Alto Rendimiento , Marruecos , FilogeniaRESUMEN
In a quantum network based on atoms and photons, a single atom should control the photon state and, reciprocally, a single photon should allow the coherent manipulation of the atom. Both operations require controlling the atom environment and developing efficient atom-photon interfaces, for instance by coupling the natural or artificial atom to cavities. So far, much attention has been drown on manipulating the light field with atomic transitions, recently at the few-photon limit. Here we report on the reciprocal operation and demonstrate the coherent manipulation of an artificial atom by few photons. We study a quantum dot-cavity system with a record cooperativity of 13. Incident photons interact with the atom with probability 0.95, which radiates back in the cavity mode with probability 0.96. Inversion of the atomic transition is achieved for 3.8 photons on average, showing that our artificial atom performs as if fully isolated from the solid-state environment.
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The potential for scale-up coupled with minimized system size is likely to be a major determining factor in the realization of applicable quantum information systems. Nanofabrication technology utilizing the III-V semiconductor system provides a path to scalable quantum bit (qubit) integration and a materials platform with combined electronic/photonic functionality. Here, we address the key requirement of qubit-site and emission energy control for scale-up by demonstrating uniform arrays of III-V nanowires, where each nanowire contains a single quantum dot. Optical studies of single nanowire quantum dots reveal narrow linewidth exciton and biexciton emission and clear state-filling at higher powers. Individual nanowire quantum dots are shown to emit nonclassically with clear evidence of photon antibunching. A model is developed to explain unexpectedly large excited state separations as revealed by photoluminescence emission spectra. From measurements of more than 40 nanowire quantum dots, we find emission energies with an ensemble broadening of 15 meV. The combination of deterministic site control and the narrow distribution in ensemble emission energy results in a system readily capable of scaling for multiqubit quantum information applications.
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The cheetah (Acinonyx jubatus) has been described as a species with low levels of genetic variation. This has been suggested to be the consequence of a demographic bottleneck 10 000-12 000 years ago (ya) and also led to the assumption that only small genetic differences exist between the described subspecies. However, analysing mitochondrial DNA and microsatellites in cheetah samples from most of the historic range of the species we found relatively deep phylogeographic breaks between some of the investigated populations, and most of the methods assessed divergence time estimates predating the postulated bottleneck. Mitochondrial DNA monophyly and overall levels of genetic differentiation support the distinctiveness of Northern-East African cheetahs (Acinonyx jubatus soemmeringii). Moreover, combining archaeozoological and contemporary samples, we show that Asiatic cheetahs (Acinonyx jubatus venaticus) are unambiguously separated from African subspecies. Divergence time estimates from mitochondrial and nuclear data place the split between Asiatic and Southern African cheetahs (Acinonyx jubatus jubatus) at 32 000-67 000 ya using an average mammalian microsatellite mutation rate and at 4700-44 000 ya employing human microsatellite mutation rates. Cheetahs are vulnerable to extinction globally and critically endangered in their Asiatic range, where the last 70-110 individuals survive only in Iran. We demonstrate that these extant Iranian cheetahs are an autochthonous monophyletic population and the last representatives of the Asiatic subspecies A. j. venaticus. We advocate that conservation strategies should consider the uncovered independent evolutionary histories of Asiatic and African cheetahs, as well as among some African subspecies. This would facilitate the dual conservation priorities of maintaining locally adapted ecotypes and genetic diversity.
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Acinonyx/genética , Genética de Población , Filogeografía , África , Animales , Asia , Núcleo Celular/genética , Conservación de los Recursos Naturales , ADN Mitocondrial/genética , Evolución Molecular , Variación Genética , Genotipo , Irán , Repeticiones de Microsatélite , Análisis de Secuencia de ADNRESUMEN
Carrier relaxation is a key issue in determining the efficiency of semiconductor optoelectronic device operation. Devices incorporating semiconductor quantum dots have the potential to overcome many of the limitations of quantum-well-based devices because of the predicted long quantum-dot excited-state lifetimes. For example, the population inversion required for terahertz laser operation in quantum-well-based devices (quantum-cascade lasers) is fundamentally limited by efficient scattering between the laser levels, which form a continuum in the plane of the quantum well. In this context, semiconductor quantum dots are a highly attractive alternative for terahertz devices, because of their intrinsic discrete energy levels. Here, we present the first measurements, and theoretical description, of the intersublevel carrier relaxation in quantum dots for transition energies in the few terahertz range. Long intradot relaxation times (1.5 ns) are found for level separations of 14 meV (3.4 THz), decreasing very strongly to approximately 2 ps at 30 meV (7 THz), in very good agreement with our microscopic theory of the carrier relaxation process. Our studies pave the way for quantum-dot terahertz device development, providing the fundamental knowledge of carrier relaxation times required for optimum device design.
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Nonsense-mediated mRNA decay (NMD) is a eukaryotic surveillance process that promotes selective degradation of imperfect messages containing premature translation termination codons (PTCs). In yeast, PTCs trigger both deadenylylation-independent mRNA decapping, thereby allowing their rapid degradation by a 5' to 3' exonuclease, and to a smaller extent accelerated deadenylylation. It is not clear to what extent this decay pathway is conserved in higher eukaryotes. We used a transcriptional pulse strategy relying on a tetracycline-regulated promoter to study the decay of a PTC- containing beta-globin mRNA in human cells. We show that a PTC destabilizes the mRNA and decreases its half-life from >16 h to 3 h. The deadenylylation rate is increased, but not sufficiently to account for the decreased half-life on its own. Using a circularization RT-PCR (cRT-PCR) strategy, we could detect decapped degradation intermediates and measure simultaneously their poly(A) tail length. This allowed us to show that a PTC enhances the rate of mRNA decapping and that decapped products have been deadenylylated to a certain extent. Thus the major feature of the NMD pathway, enhanced decapping, is conserved from yeast to man even though the kinetic details might differ between various mRNAs and/or species.
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Codón sin Sentido/genética , Globinas/genética , Caperuzas de ARN/genética , Caperuzas de ARN/metabolismo , Estabilidad del ARN/genética , Ingeniería Genética , Semivida , Células HeLa , Humanos , Cinética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
We are studying the mechanisms of transcriptional activation by nuclear receptors and we focus our studies on the glucocorticoid regulation of the model tyrosine aminotransferase gene. Rather than using in vitro biochemical approaches, we determine the actual events occurring in the cells. Our experimental approaches include genomic footprinting, chromatin immunoprecipitation, in situ hybridization and transgenic mice. Our results show that the glucocorticoid receptor uses a dynamic multistep mechanism to recruit successively accessory DNA binding proteins that assist in the activation process. Chromatin is first remodelled, DNA is then demethylated, and the synthesis of an accessory factor is induced. Efficient transcription induction is finally achieved upon the formation of a 'stable' multiprotein complex interacting with the regulatory element. We discuss: the relative contribution of histone acetyltransferases and ATP-dependent remodelling machines to the chromatin remodelling event; the nature of the remodelled state; the contribution of regulated DNA demethylation to gene memory during development; the mechanisms of regulated DNA demethylation; the dynamics of protein recruitment at regulatory elements; the control of the frequency of transcription pulses and the control levels of the cell-type specificity of the glucocorticoid response.
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Receptores de Glucocorticoides/fisiología , Tirosina Transaminasa/genética , Animales , Cromatina/genética , Cromatina/metabolismo , Humanos , Modelos Biológicos , Activación Transcripcional , Tirosina Transaminasa/metabolismoRESUMEN
In vertebrates, cytosine methylation is an epigenetic DNA modification that participates in genome stability and gene repression. Methylation patterns are either maintained throughout cell division, or modified by global or local de novo methylation and demethylation. Site-specific demethylation is a rather elusive process that occurs mainly in parallel to gene activation during development. In light of our studies of the glucocorticoid-dependent DNA demethylation of the tyrosine aminotransferase gene, we discuss the potential biochemical mechanisms allowing DNA demethylation and its targeting to specific sequences by transcription factors as well as possible links to DNA replication and chromatin remodelling.
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Metilación de ADN , Modelos Genéticos , Animales , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Islas de CpG/genética , ADN/química , ADN/genética , ADN/metabolismo , Replicación del ADN , Regulación de la Expresión Génica , Receptores de Glucocorticoides/metabolismo , Especificidad por Sustrato , Activación Transcripcional , VertebradosRESUMEN
Glucocorticoid hormones were found to regulate DNA demethylation within a key enhancer of the rat liver-specific tyrosine aminotransferase (Tat) gene. Genomic footprinting analysis shows that the glucocorticoid receptor uses local DNA demethylation as one of several steps to recruit transcription factors in hepatoma cells. Demethylation occurs within 2-3 days following rapid (< 1 h) chromatin remodeling and recruitment of a first transcription factor, HNF-3. Upon demethylation, two additional transcription factors are recruited when chromatin is remodeled. In contrast to chromatin remodeling, the demethylation is stable following hormone withdrawal. As a stronger subsequent glucocorticoid response is observed, demethylation appears to provide memory of the first stimulation. During development, this demethylation occurs before birth, at a stage where the Tat gene is not yet inducible, and it could thus prepare the enhancer for subsequent stimulation by hypoglycemia at birth. In vitro cultures of fetal hepatocytes recapitulate the regulation analyzed in hepatoma cells. There fore, demethylation appears to contribute to the fine-tuning of the enhancer and to the memorization of a regulatory event during development.
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Cromatina/metabolismo , Metilación de ADN , Dexametasona/farmacología , Regulación de la Expresión Génica , Glucocorticoides/farmacología , Hígado/efectos de los fármacos , Receptores de Glucocorticoides/genética , Tirosina Transaminasa/genética , 5-Metilcitosina , Animales , Citosina/análogos & derivados , Citosina/metabolismo , Elementos de Facilitación Genéticos , Silenciador del Gen , Hígado/citología , Hígado/embriología , Hígado/patología , Ratas , Factores de Transcripción/metabolismo , Células Tumorales CultivadasRESUMEN
Cytosine methylation is attracting new attention for regulatory roles in gene expression and there is an increasing interest in detecting, at a single-base resolution, any 5-methylcytosine in genes from complex genomes. Differential base modification by chemicals followed by PCR-based genomic sequencing procedures can provide the resolution, sensitivity, and specificity required for such a goal. The various methods available are not devoid of artifacts but if used carefully and in combination, very reliable information can be obtained. We compare the methods using bisulfite and conventional PCR with those using either hydrazine or potassium permanganate and ligation-mediated PCR and provide a step-by-step description of the corresponding procedures.
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Núcleo Celular/química , Citosina/análogos & derivados , Metilación de ADN , Genoma , Biología Molecular/métodos , 5-Metilcitosina , Núcleo Celular/genética , Citosina/análisisRESUMEN
In the liver, genes are expressed along a portocentral gradient. Based on their adaptive behavior, a gradient versus compartment type, and a dynamic versus stable type of gradient have been recognized. To understand at least in principle the development and maintenance of these gradients in gene expression in relation to the limited number of signal gradients, we propose a simple and testable model. The model uses portocentral gradients of signal molecules as input, while the output depends on two gene-specific variables, viz., the affinity of the gene for its regulatory factors and the degree of cooperativity that determines the response in the signal-transduction pathways. As a preliminary validity test for its performance, the model was tested on control and hormonally induced expression patterns of phosphoenolpyruvate carboxykinase (PCK), carbamoylphosphate synthetase I (CPS), and glutamine synthetase (GS). Affinity was found to determine the overall steepness of the gradient, whereas cooperativity causes these gradients to steepen locally, as is necessary for a compartment-like expression pattern. Interaction between two or more different signal gradients is necessary to ensure a stable expression pattern under different conditions. The diversity in sequence and arrangement of related DNA-response elements of genes appears to account for the gene-specific shape of the portocentral gradients in expression. The feasibility of testing the function of hepatocyte-specific DNA-response units in vivo is demonstrated by integrating such units into a ubiquitously active promoter/enhancer and analyzing the pattern of expression of these constructs in transgenic mice.
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Regulación de la Expresión Génica/fisiología , Hígado/metabolismo , Modelos Biológicos , Animales , Velocidad del Flujo Sanguíneo/fisiología , ADN/metabolismo , Hígado/fisiología , Circulación Hepática , Ratones , ARN Mensajero/metabolismo , Ratas , Transducción de Señal , Factores de Transcripción/fisiologíaRESUMEN
A single far-upstream enhancer is sufficient to confer hepatocyte-specific, glucocorticoid- and cyclic AMP-inducible periportal expression to the carbamoylphosphate synthetase I (CPS) gene. To identify the mechanism of hormone-dependent activation, the composition and function of the enhancer have been analyzed. DNase I protection and gel mobility shift assays revealed the presence of a cyclic AMP response element, a glucocorticoid response element (GRE), and several sites for the liver-enriched transcription factor families HNF3 and C/EBP. The in vivo relevance of the transcription factors interacting with the enhancer in the regulation of CPS expression in the liver was assessed by the analysis of knockout mice. A strong reduction of CPS mRNA levels was observed in glucocorticoid receptor- and C/EBPalpha-deficient mice, whereas the CPS mRNA was normally expressed in C/EBPbeta knockout mice and in HNF3alpha and -gamma double-knockout mice. (The role of HNFbeta could not be assessed, because the corresponding knockout mice die at embryonic day 10). In hepatoma cells, most of the activity of the enhancer is contained within a 103-bp fragment, which depends for its activity on the simultaneous occupation of the GRE, HNF3, and C/EBP sites, thus meeting the requirement of a glucocorticoid response unit. In fibroblast-like CHO cells, on the other hand, the GRE in the CPS enhancer does not cooperate with the C/EBP and HNF3 elements in transactivation of the CPS promoter. In both hepatoma and CHO cells, stimulation of expression by cyclic AMP depends mainly on the integrity of the glucocorticoid pathway, demonstrating cross talk between this pathway and the cyclic AMP (protein kinase A) pathway.
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Carbamoil-Fosfato Sintasa (Amoniaco)/genética , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Proteínas de Unión al ADN/fisiología , Proteínas Nucleares/fisiología , Receptores de Glucocorticoides/fisiología , Factores de Transcripción/fisiología , Activación Transcripcional/fisiología , Animales , Secuencia de Bases , Proteínas Potenciadoras de Unión a CCAAT , Células CHO , Cricetinae , AMP Cíclico/genética , Proteínas de Unión al ADN/análisis , Desoxirribonucleasa I/metabolismo , Elementos de Facilitación Genéticos/genética , Regulación de la Expresión Génica/genética , Factor Nuclear 3-beta del Hepatocito , Hibridación in Situ , Hígado/enzimología , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Proteínas Nucleares/análisis , ARN Mensajero/metabolismo , Células Tumorales CultivadasRESUMEN
Glucocorticoids and their receptor (GR) play a key role in perinatal gene induction. In the liver, the GR is essential for the neonatal induction of a number of genes, including that coding for tyrosine aminotransferase (TAT). To assess the function of the GR in the perinatal period, we have compared the activity of two types of glucocorticoid responsive elements in transgenic mice; one is the Tat gene glucocorticoid-responsive unit (GRU), an assembly of numerous binding sites for transcription factors, including the GR; the other is a simple dimer of high-affinity GR binding sites (GREs). Both elements confer strong glucocorticoid response in the adult liver. However, only the Tat GRUs are able to promote neonatal induction; the GRE dimer is unresponsive. Because this dimer is responsive to glucocorticoid administration in the neonate, the absence of neonatal induction is not due to the inactivity of the GR at this stage. At birth, the neonate has to withstand a brief period of starvation and hypoglycemia, a nutritional and hormonal situation that resembles fasting in the adult. In transgenic mice, the responses at birth and after fasting in the adult are similar: the Tat GRUs but not the dimeric GREs are activated. Our results show that, in rodents, glucocorticoids are not sufficient for neonatal gene induction in the liver and support the conclusion that the hypoglycemia at birth is the main trigger for expression.
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Regulación del Desarrollo de la Expresión Génica , Glucocorticoides/fisiología , Hígado/crecimiento & desarrollo , Animales , Animales Recién Nacidos , ADN/metabolismo , Dimerización , Privación de Alimentos , Hipoglucemia/genética , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Receptores de Glucocorticoides/metabolismo , TATA Box , Activación TranscripcionalRESUMEN
In yeast, the major mRNA degradation pathway is initiated by poly(A) tail shortening that triggers mRNA decapping. The mRNA is then degraded by 5'-to-3' exonucleolysis. In mammalian cells, even though poly(A) tail shortening also precedes mRNA degradation, the degradation pathway has not been elucidated. We have used a reverse transcription-PCR approach that relies on mRNA circularization to measure the poly(A) tail length of four mammalian mRNAs. This approach allows for the simultaneous analysis of the 5' and 3' ends of the same mRNA molecule. For all four mRNAs analyzed, this strategy permitted us to demonstrate the existence of small amounts of decapped mRNA species which have a shorter poly(A) tail than their capped counterparts. Kinetic analysis of one of these mRNAs indicates that the decapped species with a short poly(A) tail are mRNA degradation products. Therefore, our results indicate that decapping is preceded by a shortening of the poly(A) tail in mammalian cells, as it is in yeast, suggesting that this mRNA degradation pathway is conserved throughout eukaryotic evolution.
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Apolipoproteínas/biosíntesis , Hígado/metabolismo , Caperuzas de ARN/metabolismo , ARN Mensajero/metabolismo , Proteína Amiloide A Sérica/biosíntesis , Animales , Secuencia de Bases , Evolución Biológica , Caseínas , Cartilla de ADN , Femenino , Inflamación , Cinética , Mamíferos , Ratones , Ratones Endogámicos CBA , Fosfoenolpiruvato Carboxiquinasa (GTP)/biosíntesis , Plantas Tóxicas , Poli A/metabolismo , Reacción en Cadena de la Polimerasa , Pirofosfatasas/biosíntesis , ARN Mensajero/química , ARN Mensajero/aislamiento & purificación , Albúmina Sérica/biosíntesis , Nicotiana/enzimologíaRESUMEN
Analysis of the interaction of proteins with either DNA or RNA sequences by in vivo footprinting involves two steps: (i) the in situ modification of nucleic acids by the footprinting reagent and (ii) the visualization of the footprints. Ligation-mediated PCR (LM-PCR) procedures provide a level of sensitivity and specificity that is suitable for visualization of footprints of single-copy genes or low-abundance mRNAs in higher eukaryotes. In this article, we discuss several of the technical aspects of these multistep procedures that contribute to the quality of the results, particularly the parameters that affect the specificity and fidelity of the reactions: (i) the design of the primers, which is important to achieve optimal specificity; (ii) the choice of polymerases so that the amplified material represents faithfully the initial nucleic acid population; and (iii) the impact of the plateau effect within the PCR on the interpretation of the data. We then discuss aspects of in vivo nucleic acid manipulation that may affect the quality of the footprinting image, in particular the choice of the footprinting reagent and its condition of use (e.g., on intact or permeabilized cells or prepared nuclei) and the extent of nucleic acid modification. Finally, we provide detailed experimental procedures corresponding to the techniques we have developed or modified: LM-PCR, reverse ligation-mediated PCR, and nuclease treatment of RNAs in vivo.
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Huella de ADN/métodos , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Reacción en Cadena de la Polimerasa/métodos , Proteínas de Unión al ARN/metabolismo , ARN/metabolismo , Secuencia de Bases , Carcinoma Hepatocelular , Línea Celular , Cartilla de ADN , ADN Polimerasa Dirigida por ADN , Desoxirribonucleasa I , Humanos , Neoplasias Hepáticas , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Regiones Promotoras Genéticas , ARN Mensajero/biosíntesis , ARN Mensajero/química , Receptores de Transferrina/biosíntesis , Receptores de Transferrina/química , Sensibilidad y Especificidad , Polimerasa TaqRESUMEN
In order to better understand the influence of RNA transcript context on RNA localization and catalytic RNA efficacy in vivo, we have constructed and characterized several expression cassettes useful for transcribing short RNAs with well defined 5' and 3' appended flanking sequences. These cassettes contain promoter sequences from the human U1 snRNA, U6 snRNA, or tRNA Meti genes, fused to various processing/stabilizing sequences. The levels of expression and the sub-cellular localization of the resulting RNAs were determined and compared with those obtained from Pol II promoters normally linked to mRNA production, which include a cap and polyadenylation signal. The tRNA, Ul, and U6 transcripts were nuclear in localization and expressed at the highest levels, while the standard Pol II promoted transcripts were cytoplasmic and present at lower levels. The ability of these cassettes to confer ribozyme activity in vivo was tested with two assays. First, an SIV-growth hormone reporter gene was transiently transfected into human embryonic kidney cells expressing an anti-SIV ribozyme. Second, cultured T lymphocytes expressing an anti-HIV ribozyme were challenged with HIV. In both cases, we found that the ribozymes were effective only when expressed as capped, polyadenylated RNAs transcribed from Pol II cassettes that generate a cytoplasmically localized ribozyme that facilitates co-localization with its target. We also show that the inability of the other cassettes to support ribozyme-mediated inhibitory activity against their cytoplasmic target is very likely due to the resulting nuclear localization of these ribozymes. These studies demonstrate that the ribozyme expression cassette determines its intracellular localization and, hence, its corresponding functional activity.