RESUMEN
OBJECTIVE: This study investigates the prevalence of pathogenic variants in the mechanistic target of rapamycin (mTOR) pathway in surgical specimens of malformations of cortical development (MCDs) and cases with negative histology. The study also aims to evaluate the predictive value of genotype-histotype findings on the surgical outcome. METHODS: The study included patients with drug-resistant focal epilepsy who underwent epilepsy surgery. Cases were selected based on histopathological diagnosis, focusing on MCDs and negative findings. We included brain tissues both as formalin-fixed, paraffin-embedded (FFPE) or fresh frozen (FF) samples. Single-molecule molecular inversion probes (smMIPs) analysis was conducted, targeting the MTOR gene in FFPE samples and 10 genes within the mTOR pathway in FF samples. Correlations between genotype-histotype and surgical outcome were examined. RESULTS: We included 78 patients for whom we obtained 28 FFPE samples and 50 FF tissues. Seventeen pathogenic variants (22 %) were identified and validated, with 13 being somatic within the MTOR gene and 4 germlines (2 DEPDC5, 1 TSC1, 1 TSC2). Pathogenic variants in mTOR pathway genes were exclusively found in FCDII and TSC cases, with a significant association between FCD type IIb and MTOR genotype (P = 0.003). Patients carrying mutations had a slightly better surgical outcome than the overall cohort, however it results not significant. The FCDII diagnosed cases more frequently had normal neuropsychological test, a higher incidence of auras, fewer multiple seizure types, lower occurrence of seizures with awareness impairment, less ictal automatisms, fewer Stereo-EEG investigations, and a longer period long-life of seizure freedom before surgery. SIGNIFICANCE: This study confirms that somatic MTOR variants represent the primary genetic alteration detected in brain specimens from FCDII/TSC cases, while germline DEPDC5, TSC1/TSC2 variants are relatively rare. Systematic screening for these mutations in surgically treated patients' brain specimens can aid histopathological diagnoses and serve as a biomarker for positive surgical outcomes. Certain clinical features associated with pathogenic variants in mTOR pathway genes may suggest a genetic etiology in FCDII patients.
Asunto(s)
Epilepsia Refractaria , Epilepsias Parciales , Epilepsia , Malformaciones del Desarrollo Cortical de Grupo I , Malformaciones del Desarrollo Cortical , Adulto , Humanos , Epilepsia Refractaria/genética , Epilepsia Refractaria/cirugía , Serina-Treonina Quinasas TOR , Epilepsias Parciales/genética , Epilepsias Parciales/diagnóstico , Convulsiones , Células Germinativas/patología , Malformaciones del Desarrollo Cortical/patologíaRESUMEN
Over the past 10 years, it has emerged that pervasive transcription in mammalian genomes has a tremendous impact on several biological functions. Most of transcribed RNAs are lncRNAs and repetitive elements. In this review, we will detail the discovery of a new functional class of natural and synthetic antisense lncRNAs that stimulate translation of sense mRNAs. These molecules have been named SINEUPs since their function requires the activity of an embedded inverted SINEB2 sequence to UP-regulate translation. Natural SINEUPs suggest that embedded Transposable Elements may represent functional domains in long non-coding RNAs. Synthetic SINEUPs may be designed by targeting the antisense sequence to the mRNA of choice representing the first scalable tool to increase protein synthesis of potentially any gene of interest. We will discuss potential applications of SINEUP technology in the field of molecular biology experiments, in protein manufacturing as well as in therapy of haploinsufficiencies.
Asunto(s)
Biosíntesis de Proteínas , ARN sin Sentido/genética , ARN Largo no Codificante/genética , ARN Mensajero/genética , Animales , Sitios de Unión/genética , Humanos , Modelos Genéticos , ARN sin Sentido/metabolismo , ARN Largo no Codificante/metabolismo , ARN Mensajero/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos/genéticaRESUMEN
TRAF and TNF receptor-associated protein (TTRAP) is a multifunctional protein that can act in the nucleus as a 5'-tyrosyl DNA phosphodiesterase and in the cytoplasm as a regulator of cell signaling. In this paper we show that in response to proteasome inhibition TTRAP accumulates in nucleolar cavities in a promyelocytic leukemia protein-dependent manner. In the nucleolus, TTRAP contributes to control levels of ribosomal RNA precursor and processing intermediates, and this phenotype is independent from its 5'-tyrosyl DNA phosphodiesterase activity. Our findings suggest a previously unidentified function for TTRAP and nucleolar cavities in ribosome biogenesis under stress.
Asunto(s)
Nucléolo Celular/metabolismo , Proteínas Nucleares/metabolismo , Inhibidores de Proteasoma , Procesamiento Postranscripcional del ARN/fisiología , ARN Ribosómico/metabolismo , Ribosomas/metabolismo , Transducción de Señal/fisiología , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Línea Celular Tumoral , Nucléolo Celular/genética , Proteínas de Unión al ADN , Células HEK293 , Humanos , Proteínas Nucleares/genética , Hidrolasas Diéster Fosfóricas , Proteína de la Leucemia Promielocítica , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , ARN Ribosómico/genética , Ribosomas/genética , Estrés Fisiológico/fisiología , Factores de Transcripción/genética , Proteínas Supresoras de Tumor/genéticaRESUMEN
Mutations in PARK7 DJ-1 have been associated with autosomal-recessive early-onset Parkinson's disease (PD). This gene encodes for an atypical peroxiredoxin-like peroxidase that may act as a regulator of transcription and a redox-dependent chaperone. Although large gene deletions have been associated with a loss-of-function phenotype, the pathogenic mechanism of several missense mutations is less clear. By performing a yeast two-hybrid screening from a human fetal brain library, we identified TRAF and TNF receptor-associated protein (TTRAP), an ubiquitin-binding domain-containing protein, as a novel DJ-1 interactor, which was able to bind the PD-associated mutations M26I and L166P more strongly than wild type. TTRAP protected neuroblastoma cells from apoptosis induced by proteasome impairment. In these conditions, endogenous TTRAP relocalized to a detergent-insoluble fraction and formed cytoplasmic aggresome-like structures. Interestingly, both DJ-1 mutants blocked the TTRAP protective activity unmasking a c-jun N-terminal kinase (JNK)- and p38-MAPK (mitogen-activated protein kinase)-mediated apoptosis. These results suggest an active role of DJ-1 missense mutants in the control of cell death and position TTRAP as a new player in the arena of neurodegeneration.
Asunto(s)
Apoptosis/fisiología , Cuerpos de Inclusión/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Mutación Missense , Proteínas Nucleares/metabolismo , Proteínas Oncogénicas/genética , Enfermedad de Parkinson , Factores de Transcripción/metabolismo , Antineoplásicos/metabolismo , Neoplasias Encefálicas , Línea Celular , Proteínas de Unión al ADN , Dopamina/metabolismo , Activación Enzimática , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Leupeptinas/metabolismo , Neuroblastoma , Proteínas Nucleares/genética , Proteínas Oncogénicas/metabolismo , Estrés Oxidativo , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Hidrolasas Diéster Fosfóricas , Unión Proteica , Proteína Desglicasa DJ-1 , Sustancia Negra/citología , Sustancia Negra/metabolismo , Factores de Transcripción/genética , Técnicas del Sistema de Dos Híbridos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoAsunto(s)
Encéfalo/citología , Encéfalo/fisiología , Regulación de la Expresión Génica/fisiología , Transcripción Genética/fisiología , Animales , Genómica/métodos , Humanos , Mamíferos/genética , Mamíferos/fisiología , Neuronas/citología , Neuronas/fisiología , Biosíntesis de Proteínas/fisiología , ARN/genéticaRESUMEN
Only a small proportion of the mouse genome is transcribed into mature messenger RNA transcripts. There is an international collaborative effort to identify all full-length mRNA transcripts from the mouse, and to ensure that each is represented in a physical collection of clones. Here we report the manual annotation of 60,770 full-length mouse complementary DNA sequences. These are clustered into 33,409 'transcriptional units', contributing 90.1% of a newly established mouse transcriptome database. Of these transcriptional units, 4,258 are new protein-coding and 11,665 are new non-coding messages, indicating that non-coding RNA is a major component of the transcriptome. 41% of all transcriptional units showed evidence of alternative splicing. In protein-coding transcripts, 79% of splice variations altered the protein product. Whole-transcriptome analyses resulted in the identification of 2,431 sense-antisense pairs. The present work, completely supported by physical clones, provides the most comprehensive survey of a mammalian transcriptome so far, and is a valuable resource for functional genomics.
Asunto(s)
ADN Complementario/genética , Genómica , Ratones/genética , Transcripción Genética/genética , Empalme Alternativo/genética , Secuencias de Aminoácidos , Animales , Cromosomas de los Mamíferos/genética , Clonación Molecular , Bases de Datos Genéticas , Etiquetas de Secuencia Expresada , Genes/genética , Genómica/métodos , Humanos , Proteínas de la Membrana/genética , Mapeo Físico de Cromosoma , Estructura Terciaria de Proteína , Proteoma/química , Proteoma/genética , ARN sin Sentido/genética , ARN Mensajero/análisis , ARN Mensajero/genética , ARN no Traducido/análisis , ARN no Traducido/genética , Sitio de Iniciación de la TranscripciónRESUMEN
Extrasynaptic release of dopamine is well documented, but its relation to the physiological activity of the neuron is unclear. Here we show that in absence of presynaptic active zones, solitary cell bodies of retinal dopaminergic neurons release by exocytosis packets of approximately 40,000 molecules of dopamine at irregular intervals and low frequency. The release is triggered by the action potentials that the neurons generate in a rhythmic fashion upon removal of all synaptic influences and therefore depends upon the electrical events at the neuronal surface. Furthermore, it is stimulated by kainate and abolished by GABA and quinpirole, an agonist at the D(2) dopamine receptor. Since the somatic receptors for these ligands are extrasynaptic, we suggest that the composition of the extracellular fluid directly modulates extrasynaptic release.
Asunto(s)
Potenciales de Acción/fisiología , Membrana Celular/metabolismo , Dopamina/metabolismo , Exocitosis/fisiología , Espacio Extracelular/metabolismo , Proteínas de Transporte de Membrana , Neuronas/metabolismo , Neuropéptidos , Retina/metabolismo , Potenciales de Acción/efectos de los fármacos , Animales , Relojes Biológicos/efectos de los fármacos , Relojes Biológicos/fisiología , Comunicación Celular/efectos de los fármacos , Comunicación Celular/fisiología , Membrana Celular/efectos de los fármacos , Membrana Celular/ultraestructura , Tamaño de la Célula/fisiología , Células Cultivadas , Exocitosis/efectos de los fármacos , Espacio Extracelular/efectos de los fármacos , Inmunohistoquímica , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica , Neuronas/efectos de los fármacos , Neuronas/ultraestructura , Neurosecreción/efectos de los fármacos , Neurosecreción/fisiología , Tamaño de la Partícula , Periodicidad , Retina/efectos de los fármacos , Retina/ultraestructura , Transmisión Sináptica/fisiología , Proteínas de Transporte Vesicular de Aminas BiógenasRESUMEN
When the vertebrate retina is stimulated by light, a class of amacrine or interplexiform cells release dopamine, a modulator responsible for neural adaptation to light. In the intact retina, dopamine release can be pharmacologically manipulated with agonists and antagonists at GABA(A) receptors, and dopaminergic (DA) cells receive input from GABAergic amacrines. Because there are only 450 DA cells in each mouse retina and they cannot be distinguished in the living state from other cells on the basis of their morphology, we used transgenic technology to label DA cells with human placental alkaline phosphatase, an enzyme that resides on the outer surface of the cell membrane. We could therefore identify DA cells in vitro after dissociation of the retina and investigate their activity with whole cell voltage clamp. We describe here the pharmacological properties of the GABA(A) receptors of solitary DA cells. GABA application induces a large inward current carried by chloride ions. The receptors are of the GABA(A) type because the GABA-evoked current is blocked by bicuculline. Their affinity for GABA is very high with an EC(50) value of 7.4 microM. Co-application of benzodiazepine receptor ligands causes a strong increase in the peak current induced by GABA (maximal enhancement: CL-218872 220%; flunitrazepam 214%; zolpidem 348%) proving that DA cells express a type I benzodiazepine-receptor (BZ1). GABA-evoked currents are inhibited by Zn(2+) with an IC(50) of 58.9 +/- 8.9 microM. Furthermore, these receptors are strongly potentiated by the modulator alphaxalone with an EC(50) of 340 +/- 4 nM. The allosteric modulator loreclezole increases GABA receptor currents by 43% (1 microM) and by 107% (10 microM). Using outside-out patches, we measured in single-channel recordings a main conductance (29 pS) and two subconductance (20 and 9 pS) states. We have previously shown by single-cell RT-PCR and immunocytochemistry that DA cells express seven different GABA(A) receptor subunits (alpha1, alpha3, alpha4, beta1, beta3, gamma1, gamma2(S), and gamma2(L)) and by immunocytochemistry that all subunits are expressed in the intact retina. We show here that at least alpha1, beta3 and gamma2 subunits are assembled into functional receptors.
Asunto(s)
Dopamina/metabolismo , Neuronas/metabolismo , Picrotoxina/análogos & derivados , Receptores de GABA-A/metabolismo , Retina/metabolismo , Fosfatasa Alcalina/genética , Animales , Benzodiazepinas/metabolismo , Bicuculina/farmacología , Sitios de Unión , Relación Dosis-Respuesta a Droga , Antagonistas del GABA/farmacología , Antagonistas de Receptores de GABA-A , Humanos , Ratones , Ratones Transgénicos/genética , Concentración Osmolar , Técnicas de Placa-Clamp , Picrotoxina/farmacología , Pregnanodionas/farmacología , Isoformas de Proteínas/metabolismo , Receptores de GABA-A/efectos de los fármacos , Receptores de GABA-A/fisiología , Retina/citología , Sesterterpenos , Triazoles/farmacología , Zinc/farmacología , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
Amperometry with carbon-fiber microelectrodes provides a unique way to measure very small chemical concentration changes at the surface of biological cells. In this work, an investigation of dopamine release from individual neurons isolated from the mouse retina is described. The mice were genetically modified so that, in cells that expressed the protein responsible for catecholamine synthesis, tyrosine hydroxylase, the marker protein, placental alkaline phosphatase, was also expressed. This modification allowed for identification of the dopamine-containing cells among the many present in the freshly dissociated retina. Release of dopamine was evoked by chemical secretagogues delivered from micropipets that were calibrated with respect to response time and concentration delivered. Amperometric measurements were recorded with low-noise patch clamp amplifiers, and the primary noise source was found to be the electrode capacitance. Dopamine release occurred in the form of transient concentration spikes, consistent with release from small intracellular vesicles. With optimized filtering of the data, the quantity secreted during each release event could be determined. The average quantity determined at one cell was 52 zmol. However, the spikes were quite variable in size and the amount released per event ranged from 8 to 170 zmol. These measurements allow an estimation of the concentration of released transmitter in a synapse.
Asunto(s)
Técnicas Biosensibles , Dopamina/análisis , Neuronas/metabolismo , Animales , Calibración , Dopamina/metabolismo , Exocitosis , Técnicas In Vitro , Ratones , Microelectrodos , Retina/citologíaRESUMEN
Transgenic technology, single-cell RT-PCR, and immunocytochemistry were combined to investigate the composition of the GABA(A) receptors of dopaminergic (interplexiform) amacrine (DA) cells. A mouse line was used in which these neurons were labeled with human placental alkaline phosphatase and could therefore be identified in vitro after dissociation of the retina. We performed single-cell RT-PCR on the isolated cells and showed that (1) DA cells contained the messages for alpha1, alpha3, alpha4, beta1, beta3, gamma1, gamma2(S), and gamma2(L) subunits; (2) this transcript repertory did not change on dissociation of the retina and throughout the time required for cell harvesting; and (3) all DA cells contained the entire transcript repertory. Immunocytochemistry with subunit-specific antibodies showed that all subunits were expressed and appeared homogeneously distributed throughout the cell membrane at a low concentration. In addition, with the exception of alpha4, the subunits formed clusters at the surface of the dendrites and on the inner pole of the cell body. Because of their size, shape, and topographic coincidence with GABAergic endings, the clusters were interpreted as postsynaptic active zones containing GABA(A) receptors. The composition of the synaptic receptors was not uniform: clusters distributed throughout the dendritic tree contained alpha3, beta3, and, less frequently, beta1 subunits, whereas clusters containing the alpha1 subunit were confined to large dendrites. Therefore, DA cells possess at least two types of GABA(A) receptors localized in different synapses. Furthermore, they exhibit multiple extrasynaptic GABA(A) receptors.
Asunto(s)
Neuronas/fisiología , Receptores de GABA-A/análisis , Receptores de GABA-A/genética , Retina/fisiología , Transcripción Genética , Fosfatasa Alcalina/análisis , Fosfatasa Alcalina/genética , Animales , Dopamina/análisis , Humanos , Inmunohistoquímica , Sustancias Macromoleculares , Ratones , Ratones Transgénicos , Neuronas/citología , Receptores de GABA-A/química , Mapeo Restrictivo , Retina/citología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sinapsis/fisiología , Sinapsis/ultraestructura , Tirosina 3-Monooxigenasa/análisis , Tirosina 3-Monooxigenasa/genéticaRESUMEN
The serum deprivation response gene (SDPR, alias sdr) has been previously isolated for its high mRNA expression in serum-starved cells compared to contact-inhibited NIH3T3 cells; such regulation is not observed in single-oncogene transformed NIH3T3 cells after serum starvation. More recently Sdpr has been identified as a substrate of protein kinase C (PKC): this interaction determines the compartimentalization of PKC to caveolae, a plasma membrane invagination of which Sdpr is a major component. Lack of Sdpr-PKC interaction in transformed cells has been proposed to be involved in the alteration of PKC subcellular localization and substrate specificity. Here we report the cloning of the human SDPR homologue (HGMW-approved symbol SDPR) and its mapping to 2q32-q33 in the human genome. In analogy with the murine system, SDPR mRNA expression is increased when human fibroblasts are serum starved, it becomes down-regulated during synchronous cell-cycle reentry, but it is not induced in cells arrested by contact inhibition. Analysis of SDPR expression in human tissues reveals a near ubiquitous expression, with highest levels found in heart and lung. We show that human SDPR encodes PS-p68, a previously characterized phosphatidylserine-binding protein purified from human platelets. Accordingly, recombinant Sdpr is able to specifically bind phosphatidylserine in the absence of Ca2+. SDPR is homologous to two genes in the databank, one of which, srbc, is similarly regulated during growth arrest and encodes a phosphatidylserine-binding protein that is a substrate of PKC.
Asunto(s)
Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Cromosomas Humanos Par 2 , Fosfatidilserinas/metabolismo , Secuencia de Aminoácidos , Northern Blotting , Ciclo Celular/fisiología , Clonación Molecular , Fibroblastos/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Hibridación Fluorescente in Situ , Hígado/metabolismo , Modelos Genéticos , Datos de Secuencia Molecular , Proteínas de Unión a Fosfato , Proteínas Recombinantes , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Factores de Tiempo , Distribución Tisular , TransfecciónRESUMEN
Dopaminergic interplexiform amacrine cells were labeled in transgenic mice with human placental alkaline phosphatase and could therefore be identified after dissociation of the retina and used for whole-cell current and voltage clamp. In absence of synaptic inputs, dopaminergic amacrines spontaneously fired action potentials in a rhythmic pattern. This activity was remarkably robust in the face of inhibition of various voltage-dependent ion channels. It was minimally affected by external cesium or cobalt, suggesting no involvement of either the hyperpolarization-activated cation current Ih or voltage-dependent calcium channels. Inhibiting calcium-activated potassium channels by charybdotoxin or tetraethylammonium slowed the repolarizing phase of the action potentials and eliminated a slow afterhyperpolarization but had a scarce effect on the frequency of spontaneous firing. Voltage-clamp experiments showed that the interspike depolarization leading to threshold results from tetrodotoxin-sensitive sodium channels active at the interspike voltages of -60 to -40 mV. Because dopamine acts on distant targets in the retina, the pacemaker activity of dopaminergic amacrines may be necessary to ensure a tonic release of the modulator from their dendritic tree. Pacemaking is a property that this type of retinal amacrine cell shares with the dopaminergic mesencephalic neurons, but the ionic mechanisms responsible for the spontaneous firing are apparently different.
Asunto(s)
Canales de Calcio/fisiología , Dopamina/fisiología , Retina/citología , Animales , Potenciales de la Membrana/fisiología , Ratones , Ratones Transgénicos , Técnicas de Placa-Clamp , Canales de Potasio/efectos de los fármacos , Canales de Sodio/efectos de los fármacos , Tetrodotoxina/farmacologíaRESUMEN
Dopaminergic, interplexiform amacrines (DA cells) were labeled in transgenic mice with human placental alkaline phosphatase, an enzyme that resides on the outer surface of the cell membrane. It was therefore possible to investigate their activity in vitro after dissociation of the retina with whole-cell current and voltage clamp, as well as their connections in the intact retina with the electron microscope. DA cells generate action potentials even in the absence of synaptic inputs. This activity is abolished by the amacrine cell transmitters GABA and glycine, which induce an inward current carried by chloride ions, and is stimulated by kainate, an agonist at the receptor for the bipolar cell transmitter glutamate, which opens nonselective cation channels. Since DA cells are postsynaptic to amacrine and bipolar cells, we suggest that the spontaneous discharge of DA cells is inhibited in the dark by GABAergic amacrines that receive their input from off-bipolars. Upon illumination, the GABA-inhibition is removed, DA cells generate action potentials, and their firing is modulated by the excitation received from on-bipolars.
Asunto(s)
Dopamina/metabolismo , Ratones Transgénicos/fisiología , Retina/enzimología , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Fosfatasa Alcalina/análisis , Animales , Bicuculina/farmacología , Canales de Calcio/efectos de los fármacos , Canales de Calcio/fisiología , Separación Celular , ADN Complementario , Agonistas de Aminoácidos Excitadores/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Femenino , Antagonistas del GABA/farmacología , Proteínas Ligadas a GPI , Glicina/farmacología , Glicinérgicos/farmacología , Humanos , Activación del Canal Iónico/fisiología , Isoenzimas/análisis , Ácido Kaínico/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica , Vías Nerviosas/fisiología , Neuronas/citología , Neuronas/enzimología , Neuronas/ultraestructura , Técnicas de Placa-Clamp , Canales de Potasio/efectos de los fármacos , Canales de Potasio/fisiología , Regiones Promotoras Genéticas/fisiología , Ratas , Retina/química , Retina/citología , Estricnina/farmacología , Sinapsis/efectos de los fármacos , Sinapsis/fisiología , Tirosina 3-Monooxigenasa/genéticaRESUMEN
The product of the c-myc proto-oncogene is an important regulator of cell proliferation and apoptosis in murine fibroblasts. Addition of the tumor promoter, phorbol myristate acetate (PMA), prevents apoptotic cell death induced by low serum concentrations in NIH3T3 cells that constitutively express and are transformed by v-myc. The protective effect of PMA allowed us to analyse the ability of normal c-Myc and Myc deletion mutants to induce serum starved, untransformed NIH3T3 cells to enter S phase. By microinjecting these quiescent cells with wild type and mutant human c-myc plasmids, we showed that full length c-myc is able to induce S phase entry in presence of PMA, but that c-Myc mutants that delete amino acids delta 7/91, delta 41/53, delta 56/103, delta 106/143, delta 265/317 and delta 414/433 are totally inactive. c-Myc did not shorten the period before entry into S phase, since Myc overexpressing cells entered S phase with the same kinetics as control cells when both were stimulated with 20% fetal calf serum (FCS). However, c-Myc overexpression did increase the percentage of cells entering S phase when these cells were stimulated with 2% fetal calf serum. Interestingly, this ability to enhance stimulation by a suboptimal concentration of FCS was retained to a significant degree by Myc mutants that delete amino acids delta 41/53, delta 56/103 or delta 265/317. Finally, Myc mutants that delete delta 106/143 or delta 414/433 exerted a dominant negative effect on S phase entry both in quiescent cells stimulated with 2% FCS and in unsynchronized, cycling cells.
Asunto(s)
Genes myc , Fase S , Células 3T3 , Animales , Apoptosis , Células Cultivadas , Ratones , Microinyecciones , Mutación , Proto-Oncogenes Mas , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
This report describes the construction of a new family of lambda phage and plasmid cloning vectors. lambda GDST3/T7 allows cDNA insertion up to 14 kb; it is derived from lambda NM1151 by the insertion of a multiple cloning site containing eight unique restriction sites. The two asymmetrical SfiI sites are flanked by the T3 and T7 promoters for direct sequencing and in vitro transcription/translation. The same multiple cloning site is also present in both orientations in the eukaryotic expression plasmids, pGDSV3 and pGDSV7. By exploiting the superior discrimination of the signal-to-noise ratio of the lambda vectors for primary screening (by either nucleic acids or antibody probes), relevant cDNAs can thus be efficiently transferred through SfiI sites into the plasmids pGDSV3/7 for functional secondary screening by expression in mammalian cells.
Asunto(s)
Bacteriófago lambda/genética , Clonación Molecular , Vectores Genéticos , Plásmidos , Animales , Secuencia de Bases , Datos de Secuencia MolecularRESUMEN
The relationship between quiescence induced by serum deprivation and that induced by high cell density (contact inhibition) is still unclear. Here, we describe a gene, named sdr (serum deprivation response), whose mRNA level is highly induced in serum starved but not in density dependent growth arrested NIH3T3 cells. sdr induction seems to be coupled to growth arrest since it does not become apparent when transformed NIH3T3 cells are cultured in low serum. The expression of sdr is down-regulated within 6 h after the addition of serum or epidermal growth factor to serum starved cells. Although a transient reinduction is noticed at later times after fetal calf serum stimulation, this is not the case for epidermal growth factor, for which the sdr mRNA level remains down-regulated. The data presented here point to the possibility that the density dependent growth arrest state cannot be simply explained by local growth factor depletion, as occurs in overcrowded cultures, but it could be the consequence of a more complex pathway mediated by cellular interactions.
Asunto(s)
Fenómenos Fisiológicos Sanguíneos , Proteínas Portadoras/genética , Inhibición de Contacto/fisiología , Medio de Cultivo Libre de Suero , Regulación de la Expresión Génica/fisiología , Células 3T3 , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Recuento de Células , División Celular/fisiología , Línea Celular Transformada , Clonación Molecular , Ratones , Datos de Secuencia Molecular , Proteínas de Unión a Fosfato , Fase de Descanso del Ciclo Celular/fisiología , Fase S/fisiologíaRESUMEN
We have initiated the screening of the delta F508 cystic fibrosis (CF) mutation in our country basing our study on the polymerase chain reaction technique. In a sample of 10 western and a northeastern Mexican families that have at least one CF affected child, we found a delta F508 frequency of 59.1% among CF chromosomes. This frequency is similar to those reported for Spain. Furthermore, we found a correlation between the delta F508 allele and pancreatic insufficiency. This paper points out the advantage of the polymerase chain reaction technology for the analysis of Mendelian disorders.
Asunto(s)
Alelos , Fibrosis Quística/genética , Fibrosis Quística/etnología , Frecuencia de los Genes/genética , Tamización de Portadores Genéticos , Genotipo , Homocigoto , Humanos , México/etnología , Mutación/genética , Reacción en Cadena de la PolimerasaRESUMEN
A simple and fast protocol is described for the purification of genomic DNA from 0.3 ml of whole human blood. The recovery of DNA is quantitative and reproducible; the quality is such that it can be used for all relevant molecular biology techniques.
Asunto(s)
ADN/aislamiento & purificación , Secuencia de Bases , Southern Blotting , ADN/sangre , Electroforesis en Gel de Campo Pulsado , Técnicas Genéticas , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la PolimerasaAsunto(s)
Ciclo Celular , División Celular , Animales , Diferenciación Celular , Humanos , MamíferosRESUMEN
A total of 120 human samples of blood, saliva and semen stains, hair roots, bone and skin fragments, obtained from 30 males and 16 females were analyzed in Southern blots with probe cY97. Only the male samples gave a specific band of 5.7 kb. In dot blot, under high stringency conditions, male DNA gave signals equivalent to a quantity of female DNA eight times higher. Probe cY97 did not react with 9 different vertebrate species but gave a signal for monkey DNA when used at low stringency. The advantage of using a probe specific for the centromeric region for sex determination and species exclusion is discussed.