RESUMEN
A zebrafish sodium channel cDNA encoding a 1949-amino acid polypeptide, Na(v)1.6, was isolated. Two transcripts were detected in zebrafish adult brain but not in cardiac or skeletal muscle. The RNase protection analysis confirmed the neural specificity of zebrafish Na(v)1.6 24 hours postfertilization (hpf) Na(v)1.6 was expressed in the trigeminal ganglion, anterior and posterior lateral line ganglia, rhombomeres, and Rohon-Beard neurons. This preferential localization suggests that Na(v)1.6 plays an important role in tactile sensitivity. The abundance of zebrafish Na(v) 1.6 mRNA in the central and peripheral nervous systems increased markedly between 48 and 72 hpf, during the maturation of the nervous system.
Asunto(s)
Neuronas Aferentes/metabolismo , Canales de Sodio/química , Canales de Sodio/metabolismo , Proteínas de Pez Cebra , Pez Cebra/metabolismo , Secuencia de Aminoácidos , Animales , Encéfalo/metabolismo , Clonación Molecular , Embrión no Mamífero/metabolismo , Ganglios Espinales/química , Ganglios Espinales/citología , Ganglios Espinales/metabolismo , Inmunohistoquímica , Hibridación in Situ , Datos de Secuencia Molecular , Neuronas Aferentes/química , Filogenia , Conformación Proteica , Células Ganglionares de la Retina/química , Células Ganglionares de la Retina/metabolismo , Canales de Sodio/genética , Colículos Superiores/química , Colículos Superiores/metabolismo , Tacto , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/crecimiento & desarrolloRESUMEN
The expression pattern of zebrafish hoxa1a mRNA during embryonic development was studied. Herein, we show that hoxa1a mRNA is expressed in the ventral region of both the midbrain and anterior hindbrain during the developmental period from the pharyngula to the protruding-mouth stages via whole-mount in situ hybridization. Furthermore, double-labeling with anti-zHu antibody confirms that the zebrafish hoxa1a gene is expressed in neuronal cells. The observed temporal and spatial distributions of zebrafish hoxa1a mRNA differ greatly from the expression patterns of zebrafish hoxb1a and hoxb1b paralagous genes. In addition, in embryos injected with mouse ihh mRNA, hoxa1a-expressing cells increase in number with a dorsalized expression pattern in the midbrain.
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Proteínas de Homeodominio/biosíntesis , Mesencéfalo/embriología , Rombencéfalo/embriología , Factores de Transcripción/biosíntesis , Pez Cebra/embriología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Hibridación in Situ , Ratones , Datos de Secuencia Molecular , Neuronas/metabolismo , Homología de Secuencia de Aminoácido , Factores de Tiempo , Distribución TisularRESUMEN
Oxygen free radicals have been proposed to be involved in the process of aging. Superoxide dismutase (SOD) and catalase are important for antioxidative defense. In this study, profiles of SOD, catalase, and their mRNA levels were investigated in the frontal, parietal, temporal and occipital lobes, subcortex and cerebellum of male Wistar rats at ages 1-21 months. The total SOD and Mn SOD activities increased with age and exhibited higher levels at 6 and 12 months but decreased thereafter. Activity of catalase showed a similar trend and notably peaked at 12 months. The mRNA levels of Cu/Zn SOD, Mn SOD, and catalase remained constant in all areas tested (frontal, parietal, temporal and occipital lobes, and subcortex) except the cerebellum. Post-transcriptional regulation was involved in modulating the enzymes' activities during aging. Furthermore, the rate of mitochondrial generation of the superoxide anion (O(2)(-).) increased gradually with aging. Taken together, the results suggest that the increase of oxidative potential and the loss of proper antioxidant defense in the rats appear to be highly involved in the aging process of the brain.
Asunto(s)
Envejecimiento/metabolismo , Encéfalo/enzimología , Catalasa/metabolismo , Superóxido Dismutasa/metabolismo , Envejecimiento/genética , Animales , Encéfalo/metabolismo , Catalasa/genética , Expresión Génica , Masculino , Mitocondrias/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa/genética , Superóxidos/metabolismo , Distribución TisularRESUMEN
The stimulus-transcriptional coupling during ischemia/hypoxia was examined for ATP-stimulated expression of immediate early genes (IEGs; c-fos, zif268, c-myc and nur77) in a rat brain-derived type 2 astrocyte cell line, RBA-2. Incubation of cells with 1 mM of extracellular ATP stimulated time-dependent expression of c-fos and zif268. ATP induced the largest increases in zif268 mRNA and a lesser one in c-fos mRNA. ATP also induced a slight increase in nur77 mRNA but was ineffective in inducing c-myc expression in these cells. Brief exposure of cells to potassium cyanide to simulate chemical hypoxia induced 9-fold and 7-fold transient increases in c-fos and zif268 expression, respectively, but did not affect c-myc or nur77 expression. When cyanide and ATP were added together, the expression of c-fos and zif268 expression was inhibited, and the effect was mimicked by simulating chemical hypoxia with sodium azide. To elucidate the mechanism involved, the effect of cyanide on ATP-stimulated increases in intracellular Ca(2+) concentrations, [Ca(2+)](i), and phospholipase D (PLD) activities were measured. Cyanide induced an increase in [Ca(2&plus);](i) and further enhanced the ATP-stimulated increases in [Ca(2+)](i) and PLD activities. Nevertheless, metabolic inhibitor, iodoacetate, blocked the ATP-induced c-fos and partially inhibited zif268 expression, and deprivation of cells with glucose also inhibited the ATP-induced c-fos expression. Taken together, these results demonstrate that both extracellular ATP and chemical hypoxia induce c-fos and zif268 expression in RBA-2 type 2 astrocytes. The chemical hypoxia inhibited ATP-stimulated c-fos and zif268 expression is not due to alterations in Ca(2+) and PLD signaling, and is at least partially related to metabolic disturbance in these cells.
Asunto(s)
Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Hipoxia de la Célula/genética , Proteínas de Unión al ADN/genética , Genes Inmediatos-Precoces , Genes fos , Proteínas Inmediatas-Precoces , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Transcripción/genética , Adenosina Trifosfato/farmacología , Animales , Astrocitos/citología , Encéfalo/citología , Encéfalo/metabolismo , Calcio/metabolismo , Hipoxia de la Célula/efectos de los fármacos , Línea Celular , Proteína 1 de la Respuesta de Crecimiento Precoz , Expresión Génica/efectos de los fármacos , Genes Inmediatos-Precoces/efectos de los fármacos , Genes fos/efectos de los fármacos , Genes myc/efectos de los fármacos , Yodoacetatos/toxicidad , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares , Fosfolipasa D/metabolismo , Cianuro de Potasio/toxicidad , Ratas , Receptores Citoplasmáticos y Nucleares , Receptores de Esteroides , Azida Sódica/toxicidadRESUMEN
It has been proposed that persistent oxidative stress accounts for the increased levels of DNA damage in cancer tissues. We have examined the profile of anti-oxidant enzymes in a transplanted hepatic tumor model by injecting N1S1 rat hepatoma cells into the liver of Sprague-Dawley rats. The transplanted N1S1 tumors displayed characteristics resembling human hepatocellular carcinoma. The immunoreactivities of catalase (CAT), manganese-superoxide dismutase (Mn SOD), copper/zinc-SOD (Cu/Zn SOD), and glutathione peroxidase (GPx) were found to decrease significantly. The enzyme activity in tumors decreased 26.2-, 4.2-, 4.5-, and 5.4-fold for CAT, Mn SOD, Cu/Zn SOD, and GPx, respectively, relative to those in normal liver tissue from the same animals. In contrast, the mRNA levels of CAT and GPx in tumors decreased only 5- and 2-fold, respectively, and the mRNA levels of Cu/Zn SOD and Mn SOD showed either no change or an increase as compared to those of normal liver tissue. The contents of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) and thiobarbituric acid-reactive substances (TBARS) were comparable to those of normal controls. Furthermore, mitochondrial production of superoxide in tumors was 4 times lower than that in normal tissues. In conclusion, the data indicate that the reduced activities of anti-oxidant enzymes in the N1S1 tumor did not cause significant oxidative stress.
Asunto(s)
Antioxidantes/metabolismo , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas Experimentales/metabolismo , Estrés Oxidativo , Animales , Carcinoma Hepatocelular/patología , Catalasa/metabolismo , Glutatión Peroxidasa/metabolismo , Humanos , Neoplasias Hepáticas Experimentales/patología , Trasplante de Hígado , Ratas , Ratas Sprague-Dawley , Superóxido Dismutasa/metabolismoRESUMEN
We induced heat stress in urethane-anesthetized rats (the animals were exposed to an ambient temperature at 42 degrees C), and monitored their colon temperature, mean arterial pressure and local cerebral blood flow. Rats 0, 20, 40 or 80 min after heat stress were sacrificed for determination of c-fos mRNA and protein expression in the paraventricular nucleus (PVN), supraoptic nucleus (SON) and preoptic nucleus (PON). The heatstroke, which appears as profound decreases in both mean arterial pressure and local cerebral blood flow and increases in colon temperature, is produced 80 min after heat stress. We show the c-fos mRNA and protein is strongly induced in all these nuclei of rat hypothalamus after the onset of heatstroke. We conclude that c-fos expression in the hypothalamus during rat heatstroke is associated with hyperthermia, arterial hypotension and cerebral ischemia.
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Golpe de Calor/metabolismo , Hipotálamo/metabolismo , Proteínas Proto-Oncogénicas c-fos/biosíntesis , Animales , Isquemia Encefálica/metabolismo , Isquemia Encefálica/fisiopatología , Golpe de Calor/fisiopatología , Hipotálamo/fisiología , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Phospho-DARPP-32 (where DARPP-32 is dopamine- and cAMP-regulated phosphoprotein, Mr 32,000), its homolog, phospho-inhibitor-1, and inhibitor-2 are potent inhibitors (IC50 approximately 1 nM) of the catalytic subunit of protein phosphatase-1 (PP1). Our previous studies have indicated that a region encompassing residues 6-11 (RKKIQF) and phospho-Thr-34, of phospho-DARPP-32, interacts with PP1. However, little is known about specific regions of inhibitor-2 that interact with PP1. We have now characterized in detail the interaction of phospho-DARPP-32 and inhibitor-2 with PP1. Mutagenesis studies indicate that within DARPP-32 Phe-11 and Ile-9 play critical roles, with Lys-7 playing a lesser role in inhibition of PP1. Pro-33 and Pro-35 are also important, as is the number of amino acids between residues 7 and 11 and phospho-Thr-34. For inhibitor-2, deletion of amino acids 1-8 (I2-(9-204)) or 100-204 (I2-(1-99)) had little effect on the ability of the mutant proteins to inhibit PP1. Further deletion of residues 9-13 (I2-(14-204)) resulted in a large decrease in inhibitory potency (IC50 approximately 800 nM), whereas further COOH-terminal deletion (I2-(1-84)) caused a moderate decrease in inhibitory potency (IC50 approximately 10 nM). Within residues 9-13 (PIKGI), mutagenesis indicated that Ile-10, Lys-11, and Ile-13 play critical roles. The peptide I2-(6-20) antagonized the inhibition of PP-1 by inhibitor-2 but had no effect on inhibition by phospho-DARPP-32. In contrast, the peptide D32-(6-38) antagonized the inhibition of PP1 by phospho-DARPP-32, inhibitor-2, and I2-(1-120) but not I2-(85-204). These results indicate that distinct amino acid motifs contained within the NH2 termini of phospho-DARPP-32 (KKIQF, where italics indicate important residues) and inhibitor-2 (IKGI) are critical for inhibition of PP1. Moreover, residues 14-84 of inhibitor-2 and residues 6-38 of phospho-DARPP-32 share elements that are important for interaction with PP1.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Proteínas Musculares/farmacología , Proteínas del Tejido Nervioso/farmacología , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosfoproteínas , Proteínas/farmacología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Dominio Catalítico , Fosfoproteína 32 Regulada por Dopamina y AMPc , Inhibidores Enzimáticos/química , Humanos , Datos de Secuencia Molecular , Peso Molecular , Proteínas Musculares/química , Proteínas Musculares/genética , Mutagénesis Sitio-Dirigida , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Fosforilación , Proteína Fosfatasa 1 , Proteínas/química , Proteínas/genética , Conejos , Serina/metabolismo , Treonina/metabolismoRESUMEN
Rats 0, 16, or 48 h after heat shock (42 degrees C core temperature for 15 min) or chemical stress (5 mg/kg sodium arsenite, i.p.) were exposed to a high ambient temperature (43 degrees C) to induce heatstroke onset. The moment in which the mean arterial pressure and cerebral blood flow began to decrease from their peak values was taken as the onset of heatstroke. Prior heat shock or chemical stress conferred significant protection against heatstroke-induced arterial hypotension, cerebral ischemia, cerebral neuronal damage and death, and correlated with expression of HSP72 in brain, heart, liver and kidney at 16 h. However, at 48 h, when HSP72 expression returned to basal values, the above responses that occurred after the onset of heatstroke of two groups (0 h group VS 48 h group) were indistinguishable. The data suggest that HSP72 presence increases survival in rat heatstroke by attenuating arterial hypotension, cerebral ischemia and neuronal damage.
Asunto(s)
Isquemia Encefálica/metabolismo , Golpe de Calor/metabolismo , Proteínas de Choque Térmico/biosíntesis , Neuronas/citología , Animales , Arsenitos , Presión Sanguínea , Química Encefálica/fisiología , Isquemia Encefálica/inducido químicamente , Isquemia Encefálica/mortalidad , Muerte Celular/fisiología , Supervivencia Celular/fisiología , Circulación Cerebrovascular/fisiología , Inhibidores Enzimáticos , Proteínas del Choque Térmico HSP72 , Interleucina-1/fisiología , Riñón/química , Flujometría por Láser-Doppler , Hígado/química , Masculino , Miocardio/química , Neuronas/metabolismo , Ratas , Ratas Wistar , Compuestos de Sodio , Análisis de SupervivenciaRESUMEN
Experiments were carried out to determine the effects of altering the serotonin (5-HT) levels in the hypothalamus on thermoregulatory function in unanesthetized restrained rats. Local perfusion of the hypothalamus with dialysis solution containing 5-hydroxytryptophan (a 5-HT precursor), fluoxetine (a 5-HT reuptake inhibitor), or high potassium significantly increased both colonic temperature (Tco) and the extracellular concentrations of 5-HT in the hypothalamus. Reciprocally, both extracellular concentration of 5-HT in the hypothalamus and Tco were decreased with a dialysis solution containing tetrodotoxin (which blocks the voltage-dependent sodium channel), zero calcium concentration, or systemic administration of 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT,5-HT1A agonist). Intrahypothalamic administration of 8-OH-DPAT and (2,5-dimethoxy-4-iodophenyl)-2-aminopropane (a 5-HT2 agonist) produced hypothermic and hyperthermic effects, respectively. The results indicate that elevating the 5-HT levels in the hypothalamus activates postsynaptic 5-HT2 receptors and results in hyperthermic effects, whereas stimulation of presynaptic 5-HT1A receptors in the hypothalamus reduces the endogenous 5-HT release and results in hypothermic effects.
Asunto(s)
Regulación de la Temperatura Corporal/fisiología , Hipotálamo/fisiología , Serotonina/fisiología , Animales , Espacio Extracelular/metabolismo , Hipotálamo/ultraestructura , Masculino , Microdiálisis , Ratas , Ratas Sprague-Dawley , TetrodotoxinaRESUMEN
Motor activity blocks the extrasynaptic expression of many genes in skeletal muscle, including those encoding ion channels, receptors, and adhesion molecules. Denervation reinduces transcription throughout the multinucleated myofiber, restoring the developmental pattern of expression, especially of the genes coding for the acetylcholine receptor. A screen for trans-acting factors binding to the enhancer region of the alpha-subunit gene of the acetylcholine receptor identified CTF4, a ubiquitously expressed and alternatively spliced chicken homologue of the human E protein transcription factor HTF4/HEB. Expression of the CTF4 locus closely parallels that of myogenin and acetylcholine receptor during development and maturation of skeletal muscle, but transcription is not similarly regulated by neuronal cues. Alternative splicing within the region encoding the transactivation domain generates two CTF4 isoforms with different tissue distributions, but similar binding affinities for the acetylcholine receptor alpha-subunit enhancer and similar transcriptional potential when complexed to myogenin. Direct injection of a myogenin, but not a MyoD, antisense expression vector into denervated skeletal muscle caused a significant decrease in the transcriptional activation of a depolarization-sensitive reporter gene. Similarly, injection of a CTF4, but less so of an E12, antisense expression vector impaired the denervation response, further implicating the involvement of a myogenin/CTF4 heterodimer in the expression of AChR genes in vivo.
Asunto(s)
Proteínas de Unión al ADN/genética , Receptores Colinérgicos/genética , Factores de Transcripción/genética , Células 3T3 , Secuencia de Aminoácidos , Animales , Proteínas Aviares , Secuencia de Bases , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Encéfalo/embriología , Encéfalo/metabolismo , Pollos , ADN Complementario , Proteínas de Unión al ADN/metabolismo , Desnervación , Dimerización , Regulación del Desarrollo de la Expresión Génica , Secuencias Hélice-Asa-Hélice , Humanos , Masculino , Ratones , Datos de Secuencia Molecular , Músculo Esquelético/embriología , Músculo Esquelético/inervación , Músculo Esquelético/metabolismo , Miogenina/metabolismo , Receptores Colinérgicos/metabolismo , Alineación de Secuencia , Factores de Transcripción/metabolismoRESUMEN
Beta-actin, cyclophilin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are all constantly expressed proteins that regulate cellular structures and endogenous cytoarchitectural functions. In this study, we used an in vivo N1S1 rat hepatoma model to examine changes in the expression levels of these housekeeping genes in normal and tumor liver samples. The beta-actin, cyclophilin and GAPDH genes were all up-regulated in tumor groups as compared to the controls. Our results suggest that up-regulation of beta-actin, cyclophilin and GAPDH genes may be essential for oncogenesis in hepatoma.
Asunto(s)
Actinas/metabolismo , Regulación de la Expresión Génica/genética , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Neoplasias Hepáticas Experimentales/metabolismo , Isomerasa de Peptidilprolil/metabolismo , Regulación hacia Arriba , Actinas/genética , Animales , Northern Blotting , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Neoplasias Hepáticas Experimentales/genética , Masculino , Trasplante de Neoplasias , Isomerasa de Peptidilprolil/genética , ARN Neoplásico/aislamiento & purificación , ARN Neoplásico/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Neural-specific expression of a sodium channel mini-gene has been shown to be mediated by a 28 bp silencer element, RE1, located in the 5' flanking region of the gene. This element is active exclusively in cell lines that do not express the endogenous brain type II sodium channel gene, including fibroblast, skeletal muscle, and certain neuronal cell lines. All of these non-type II expressing cells contain RE1-binding complexes. On the basis of mutational analysis and in vivo "repressor trap" experiments, we propose that cell-specific RE1-binding proteins are responsible, at least in part, for restricting expression of the type II sodium channel gene to specific neurons in the vertebrate nervous system.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Genes Reguladores/genética , Neuronas/fisiología , Canales de Sodio/genética , Neoplasias de las Glándulas Suprarrenales/patología , Neoplasias de las Glándulas Suprarrenales/ultraestructura , Animales , Secuencia de Bases , Células Cultivadas , ADN/genética , ADN de Neoplasias/genética , Desoxirribonucleasas , Fibroblastos/citología , Fibroblastos/ultraestructura , Genes Reguladores/fisiología , Datos de Secuencia Molecular , Músculos/citología , Músculos/ultraestructura , Mutación/genética , Neuronas/citología , Neuronas/ultraestructura , Feocromocitoma/patología , Feocromocitoma/ultraestructura , Ratas , Secuencias Reguladoras de Ácidos Nucleicos , Canales de Sodio/fisiología , Transfección , Células Tumorales Cultivadas/patología , Células Tumorales Cultivadas/ultraestructuraRESUMEN
1. The skeletal muscle acetylcholine receptor comprises several subunits whose coordinated expression during myogenesis is probably controlled by cis elements in the individual subunit genes. We have previously analyzed promoter regions of the alpha and delta genes (Wang et al., 1988, 1990); to gain further insight into receptor regulation, we have now studied the promoter of the chick muscle gamma-subunit gene. 2. This analysis was faciliated by the close upstream proximity of the coding region of the delta-subunit gene and the consequent brevity (740 bp) of the untranslated linker connecting the two genes (Nef et al., 1984). Nuclease protection and primer extension analysis revealed that transcription of the gamma-subunit gene starts at position 56 upstream of the translational initiation site. 3. Nested deletions of the promoter region were employed to identify functionally important elements. A 360-bp sequence (-324 to +36) was found to activate transcription, in a position- and orientation-independent manner, during myotube formation. This sequence comprises 5 M-CAT (Nikovits et al., 1986) similarities and contains, at positions -52/-47 and -33/-28, two CANNTG (Lassar et al., 1989) motifs. 4. Binding experiments were performed by means of gel retardation, gel shift competition, and footprint analysis. The CANNTG motifs were found to bind MyoD and myogenin fusion proteins and to interact with proteins in nuclear extracts from cultured myotubes. 5. Point mutations in the CANNTG motifs revealed that these elements are crucial for full promoter activity in myotubes and essential in fibroblasts cotransfected with a myogenin expression vector. 6. We conclude that the activity of the gamma-subunit gene is determined largely by E boxes, which in vivo are likely to be activated by MyoD family proteins; in addition, other transactivators such as the M-CAT binding protein presumably play a role. Both CANNTG elements and M-CAT motifs are also present in the alpha- and delta-subunit enhancer and may therefore account for the coordinate expression of the three subunits during muscle differentiation.
Asunto(s)
Proteínas Musculares/genética , Músculos/metabolismo , Receptores Colinérgicos/genética , Secuencias Reguladoras de Ácidos Nucleicos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células Cultivadas , Pollos/genética , Secuencia de Consenso , Elementos de Facilitación Genéticos , Fibroblastos/metabolismo , Genes Sintéticos , Glutatión Transferasa/metabolismo , Datos de Secuencia Molecular , Proteínas Musculares/metabolismo , Proteínas Musculares/farmacología , Mutagénesis , Proteína MioD , Miogenina , Especificidad de Órganos , Regiones Promotoras Genéticas/efectos de los fármacos , Unión Proteica , Receptores Colinérgicos/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Transactivadores/metabolismo , Transactivadores/farmacologíaAsunto(s)
Proteínas de Unión al ADN/genética , Factores de Transcripción/genética , Células 3T3 , Secuencia de Aminoácidos , Animales , Proteínas Aviares , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Embrión de Pollo , Proteínas de Unión al ADN/química , Elementos de Facilitación Genéticos/genética , Ratones , Datos de Secuencia Molecular , Receptores Colinérgicos/genética , Homología de Secuencia de Ácido Nucleico , Factores de Transcripción TCF , Proteína 1 Similar al Factor de Transcripción 7 , Factores de Transcripción/química , TransfecciónRESUMEN
The effect of cycloheximide (CHX) on denervation-induced acetylcholine receptor (AChR) expression was investigated in chickens one day after nerve section, using probe excess solution hybridization to quantitate AChR alpha-subunit gene transcript levels and run-on analysis to measure subunit gene activity. The increase in alpha-subunit transcripts that normally follows denervation was prevented when drug treatment was commenced 2 h before or after denervation but was not blocked when CHX administration was begun 6 h after the operation. Drug-induced reduction of transcript levels results from decreased activity of genes coding for the alpha-, delta-, and gamma-subunits; in contrast, the transcription rates of several non-receptor genes are not affected by CHX. The results suggest that the de novo synthesis of a transcriptional activator is required as a mediating event in the signalling pathway linking the plasma membrane and AChR gene expression.
Asunto(s)
Desnervación , Regulación de la Expresión Génica , Biosíntesis de Proteínas , Receptores Colinérgicos/genética , Nervio Ciático/fisiología , Animales , Núcleo Celular/metabolismo , Pollos , Cicloheximida/farmacología , Sondas de ADN , Dactinomicina/farmacología , Sustancias Macromoleculares , ARN Mensajero/análisis , ARN Mensajero/genéticaRESUMEN
The acetylcholine receptor is a multimeric membrane protein whose expression is activated during muscle differentiation and upon denervation of adult muscle. To gain insight into the coordinate expression of receptor subunits during myogenesis we have analyzed the chick muscle receptor delta-subunit gene upstream region. The delta-subunit gene lacks canonical promoter elements (CCAAT and TATA boxes). Nuclease protection and primer extension analysis revealed that transcription starts at six major and several minor sites between -110 and -30 upstream of the translational initiation site; two sites, at positions -77 and -66, give rise to approximately 50% of all transcripts. Using nested deletions of the proximal 960 bp of the 5' flanking region of this gene we have identified a 62 bp sequence (-207 to -146) that activates transcription in a position independent manner. This enhancer-like element is activated during myotube formation; it contains two distinct functional moieties, each resembling the same 16 bp portion of the stage and tissue specific alpha-subunit gene enhancer which we have characterized previously [Wang et al. (1988) Neuron, 1, 527-534]. This common element, which also comprises several previously proposed skeletal muscle specific motifs [Buskin, J. N. and Hauschka, S. D. (1989) Mol. Cell Biol., 9, 2627-2640; Mar, J. H. and Ordahl, C. P. (1988) Proc. Natl. Acad. Sci. USA, 85, 6404-6408], may account for the coordinate expression of the two subunits. The cell specificity of the delta-subunit gene 5' flanking region is partly due to the enhancer, partly to an inhibitory element upstream of -207.
Asunto(s)
Elementos de Facilitación Genéticos , Genes , Músculos/fisiología , Receptores Colinérgicos/genética , Animales , Composición de Base , Secuencia de Bases , Diferenciación Celular , Línea Celular , Pollos , Deleción Cromosómica , Sustancias Macromoleculares , Datos de Secuencia Molecular , Plásmidos , Regiones Promotoras Genéticas , Señales de Clasificación de Proteína/genética , Mapeo Restrictivo , Transcripción Genética , TransfecciónRESUMEN
Transcriptional activity of acetylcholine receptor subunit genes was investigated in innervated and denervated chick skeletal muscle. The sciatic nerve of 3-d-old White Leghorn chicks was sectioned unilaterally; after various intervals, nuclei were isolated from operated and sham-operated animals, and run-on assays performed. Nuclei were incubated with 32P-UTP, and total RNA was extracted and hybridized onto filters containing an excess of subunit-specific DNA. Specific transcripts were detected by autoradiography and quantitated densitometrically. A sharp increase in transcriptional activity was observed to begin approximately 1/2 d after the operation and peak 1 d later when transcriptional rates reached approximately seven-, six-, and fivefold control levels for the alpha-, delta-, and gamma-subunit genes, respectively. The specificity of the effect was ascertained by normalization to total RNA synthesis and by the demonstration that several nonreceptor genes respond differently to denervation. These results suggest that a denervation signal reaches the genome to induce receptor expression. In addition, since the increase in mRNA levels significantly exceeds what can be accounted for by increased gene activity, posttranscriptional effects are suggested.