RESUMEN
Invadopodia are actin-rich cell membrane projections used by invasive cells to penetrate the basement membrane. Control of invadopodia stability is critical for efficient degradation of the extracellular matrix (ECM); however, the underlying molecular mechanisms remain poorly understood. Here, we uncover a new role for podoplanin, a transmembrane glycoprotein closely associated with malignant progression of squamous cell carcinomas (SCCs), in the regulation of invadopodia-mediated matrix degradation. Podoplanin downregulation in SCC cells impairs invadopodia stability, thereby reducing the efficiency of ECM degradation. We report podoplanin as a novel component of invadopodia-associated adhesion rings, where it clusters prior to matrix degradation. Early podoplanin recruitment to invadopodia is dependent on lipid rafts, whereas ezrin/moesin proteins mediate podoplanin ring assembly. Finally, we demonstrate that podoplanin regulates invadopodia maturation by acting upstream of the ROCK-LIMK-Cofilin pathway through the control of RhoC GTPase activity. Thus, podoplanin has a key role in the regulation of invadopodia function in SCC cells, controlling the initial steps of cancer cell invasion.
Asunto(s)
Carcinoma de Células Escamosas/patología , Extensiones de la Superficie Celular/fisiología , Matriz Extracelular/metabolismo , Glicoproteínas de Membrana/fisiología , Carcinoma de Células Escamosas/metabolismo , Línea Celular Tumoral , Humanos , Quinasas Lim/fisiología , Microdominios de Membrana/fisiología , Transducción de Señal , Proteínas de Unión al GTP rho/fisiología , Quinasas Asociadas a rho/fisiología , Proteína rhoC de Unión a GTPRESUMEN
Phospholipid Hydroperoxide Glutathione Peroxidase (PHGPx) is the only known enzyme able to reduce lipid peroxides bound to cell membranes. Moreover it has been involved in apoptosis and can influence intracellular signaling. To investigate the possible relationship between PHGPx and human cancer we have quantified PHGPx expression levels by real-time quantitative PCR and immunohistochemistry in tissue samples of human breast invasive ductal carcinoma from 34 patients compared with their own controls of benign breast tissue. PHGPx expression levels were compared with the clinical and pathological data of these patients. The results showed that PHGPx expression levels are downregulated in poorly differentiated (grade 3) breast invasive ductal carcinoma (P = 0.0043). PHGPx expression levels decreased gradually with tumor grade from grade 1 to grade 3. We also found a downregulation of PHGPx in cases that showed p53 accumulation compared with cases without p53 immunostaining (P = 0.0011). PHGPx was also downregulated in cases without progesterone receptors (PR) immunostaining compared with cases with PR immunostaining (P = 0.0165). Grade 3, p53 immunostaining and absence of PR immunostaining are poor prognostic factors. These results suggest that PHGPx downregulation could be related with a poorer prognosis in breast invasive ductal carcinoma.
Asunto(s)
Neoplasias de la Mama/enzimología , Carcinoma Ductal de Mama/enzimología , Diferenciación Celular , Regulación Enzimológica de la Expresión Génica , Glutatión Peroxidasa/metabolismo , Adulto , Anciano , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/patología , Regulación hacia Abajo , Femenino , Glutatión Peroxidasa/genética , Humanos , Técnicas para Inmunoenzimas , Persona de Mediana Edad , Fosfolípido Hidroperóxido Glutatión Peroxidasa , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Receptores de Estrógenos/metabolismo , Receptores de Progesterona/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
This study aimed at determining the distribution and expression levels of different subtypes of Ca(2+) channels in the bovine adrenal medulla, and whether individual subtypes were more abundant in chromaffin cells exhibiting an adrenergic or a noradrenergic phenotype. In situ hybridization using riboprobes specific for the pore-forming Ca(2+) channel alpha(1D) (L-type channel), alpha(1B) (N-type channel), and alpha(1A) (P/Q-type channel) subunits of bovine chromaffin cells showed a broad distribution of the three transcripts in adrenal medulla tissue. However, a tissue-specific expression pattern of individual subunits was found; whereas alpha(1B) mRNA was homogeneously distributed throughout the medulla, alpha(1D) and alpha(1A) transcripts were present at higher densities in the internal medullary area, far away from the adrenal cortex. These results were corroborated by comparative analysis of the alpha(1B), alpha(1D), and alpha(1A) products amplified by RT-PCR from total RNA extracted from small pieces of tissue dissected out from external or internal medullary areas. Interestingly, immunohistochemical experiments performed in adrenal gland sections, using antidopamine-beta-hydroxylase and anti-phenylethanolamine-N-methyltransferase antibodies, indicated a higher density of noradrenergic over adrenergic chromaffin cells in the internal medullary region. These results provide direct evidence in favor of a heterogeneous distribution of Ca(2+) channel subtypes in the adrenal medulla, in agreement with previous functional data showing that blockade of the high K+ -elicited responses by dihydropyridines was greater in noradrenergic than in adrenergic chromaffin cells. These differences may be relevant for the differential release regulation of each catecholamine under physiological and pathophysiological conditions.
Asunto(s)
Médula Suprarrenal/metabolismo , Canales de Calcio/biosíntesis , Secuencia de Aminoácidos , Animales , Northern Blotting , Bovinos , Células Cromafines/efectos de los fármacos , Células Cromafines/metabolismo , Dopamina beta-Hidroxilasa/biosíntesis , Inmunohistoquímica , Hibridación in Situ , Datos de Secuencia Molecular , ARN Mensajero/biosíntesisRESUMEN
We have used a heterologous system of expression of N-methyl-D-aspartate (NMDA) receptors based on the use of vaccinia virus to analyse the maturation, transport, assembly and differential expression of the NR1 and NR2A subunits of the receptors. We have demonstrated that the NR1 subunit is efficiently transported to the plasma membrane in cells expressing NR1 alone, similarly to cells producing NR1 and NR2A together. In contrast, NR2A requires NR1 expression to be located at the cell surface. The stability of both receptor subunits expressed alone is similar to that obtained in cells producing NR1 and NR2A. In pulse-chase experiments, the NR1 subunit displays a biphasic decay, with a fraction of the protein having a half-life of only 1 h and the remaining presenting a turnover longer than 24 h, similar to values obtained for the NR2A subunit. Our results also show a maturation process affecting the carbohydrate moiety in the NR1 subunit, such that immature NR1 has a much shorter half-life than the mature form or the NR2A subunit. Finally, we show that only a fraction of mature NR1 interacts with NR2A to form multimeric functional complexes.
Asunto(s)
Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/metabolismo , Transporte Biológico Activo , Línea Celular , Membrana Celular/metabolismo , Expresión Génica , Humanos , Procesamiento Proteico-Postraduccional , Subunidades de Proteína , Receptores de N-Metil-D-Aspartato/genética , Fracciones Subcelulares/metabolismo , Transfección , Virus Vaccinia/genéticaRESUMEN
The inhibitor of the Hsp90 chaperone Geldanamycin has been reported to have several cellular effects, such as inhibition of v-src activity or destabilization of Raf-1 among others. We show now that Geldanamycin treatment induces different phenotypes in different cell lines. In PC12 cells, it triggers apoptosis, whereas in the murine neuroblastoma N2A, it induces differentiation with neurite outgrowth. Geldanamycin effects cannot be mimicked by inhibition of the c-src protein tyrosine kinases, and nerve growth factor does not protect PC12 cells from apoptosis. Mitogen-activated protein kinase activities ERK and JNK are activated differently according to cell type: in PC12 cells JNK is activated, and its inhibition abolishes apoptosis, but not ERK; in N2A cells, both ERK and JNK are activated, but with peak activities at different times.
Asunto(s)
Apoptosis , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Quinonas/farmacología , Animales , Antibióticos Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Benzoquinonas , Diferenciación Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Imidazoles/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos , Cinética , Lactamas Macrocíclicas , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Factor de Crecimiento Nervioso/metabolismo , Neuroblastoma/metabolismo , Células PC12 , Fenotipo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-raf/metabolismo , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Piridinas/farmacología , Ratas , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
Using reverse transcription followed by PCR amplification (RT-PCR), we have identified multiple messenger RNAs encoding for the neuronal pore-forming Ca(2+) channel subunits alpha(1A) (P/Q channel), alpha(1B) (N channel), alpha(1D) (neuronal/endocrine L channel), alpha(1E) (R channel), alpha(1G-H) (T channel) and alpha(1S) (skeletal muscle L channel) in bovine chromaffin cells. mRNAs for the auxiliary beta(2), beta(3), beta(4), alpha(2)/delta and gamma(2) subunits were also identified. In agreement with these molecular data, perforated patch-clamp recordings of whole-cell Ca(2+) currents reveal the existence of functional R-type Ca(2+) channels in these cells that were previously undetected with other techniques. Our results provide a molecular frame for a much wider functional diversity of Ca(2+) channels in chromaffin cells than that previously established using pharmacological and electrophysiological approaches.
Asunto(s)
Canales de Calcio/clasificación , Canales de Calcio/genética , Células Cromafines/metabolismo , ARN Mensajero/aislamiento & purificación , Animales , Canales de Calcio/aislamiento & purificación , Canales de Calcio/fisiología , Bovinos , Células Cultivadas , Células Cromafines/fisiología , Técnicas de Placa-Clamp , ARN Mensajero/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The relationship between fluctuating cytokine concentrations in plasma and the outcome of sepsis is complex. We postulated that early measurement of the activation of nuclear factor kappaB (NF-kappaB), a transcriptional regulatory protein involved in proinflammatory cytokine expression, may help to predict the outcome of sepsis. We determined NF-kappaB activation in peripheral blood mononuclear cells of 34 patients with severe sepsis (23 survivors and 11 nonsurvivors) and serial concentrations of inflammatory cytokines (interleukin-6, interleukin-1, and tumor necrosis factor) and various endogenous antagonists in plasma. NF-kappaB activity was significantly higher in nonsurvivors and correlated strongly with the severity of illness (APACHE II score), although neither was related to the cytokine levels. Apart from NF-kappaB activity, the interleukin-1 receptor antagonist was the only cytokine tested whose level in plasma was of value in predicting mortality by logistic regression analysis. These results underscore the prognostic value of early measurement of NF-kappaB activity in patients with severe sepsis.
Asunto(s)
Citocinas/sangre , FN-kappa B/metabolismo , Sepsis/sangre , Adulto , Anciano , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Interleucina-1/sangre , Interleucina-10/sangre , Interleucina-6/sangre , Leucocitos Mononucleares/metabolismo , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Pronóstico , Curva ROC , Sepsis/diagnóstico , Sepsis/mortalidad , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Incubation of bovine adrenal chromaffin cells in high K+ (38 mM) during 24-48 h enhanced 2.5 to five times the expression of SNAP-25 protein and mRNA, respectively. This increase was reduced 86% by furnidipine (an L-type Ca2+ channel blocker) but was unaffected by either omega-conotoxin GVIA (an N-type Ca2+ channel blocker) or -agatoxin IVA (a P/Q-type Ca2+ channel blocker). Combined blockade of N and P/Q channels with omega-conotoxin MVIIC did, however, block by 76% the protein expression. The inhibitory effects of fumidipine were partially reversed when the external Ca2+ concentration was raised from 1.6 to 5 mM. These findings, together with the fact that nicotinic receptor activation or Ca2+ release from internal stores also enhanced SNAP-25 protein expression, suggest that an increment of cytosolic Ca2+ concentration ([Ca2+]), rather than its source or Ca2+ entry pathway, is the critical signal to induce the protein expression. The greater coupling between L-type Ca2+ channels and protein expression might be due to two facts: (a) L channels contributed 50% to the global [Ca2+]i rise induced by 38 mM K+ in indo-1-loaded chromaffin cells and (b) L channels undergo less inactivation than N or P/Q channels on sustained stimulation of these cells.
Asunto(s)
Calcio/metabolismo , Células Cromafines/metabolismo , Proteínas de la Membrana , Proteínas del Tejido Nervioso/metabolismo , Animales , Cafeína/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Bovinos , Células Cultivadas , Células Cromafines/efectos de los fármacos , Células Cromafines/fisiología , Citosol/metabolismo , Dihidropiridinas/farmacología , Yoduro de Dimetilfenilpiperazina/farmacología , Electrofisiología , Espacio Extracelular/metabolismo , Membranas Intracelulares/metabolismo , Proteínas del Tejido Nervioso/genética , Agonistas Nicotínicos/farmacología , Concentración Osmolar , Potasio/farmacología , ARN Mensajero/metabolismo , Proteína 25 Asociada a Sinaptosomas , omega-Conotoxinas/farmacologíaRESUMEN
N-Methyl-D-Aspartate (NMDA) receptors containing NR1 and NR2A subunits have been expressed with high efficiency in Human Embryonic Kidney 293 cells with the aid of a recombinant vaccinia virus. This expression system produced functional receptors that sustained calcium influxes dependent on receptor agonists and inhibited by receptor antagonists. Immunocytochemistry of the recombinant receptors demonstrated that they were properly arranged in membrane structures. The entrance of calcium through the recombinant receptors induced delayed toxicity, demonstrated by approximately a three-fold increase in the number of dead cells obtained 12 h after the antagonist 2-amino-phosphopentanoic acid (DL-AP5) was removed from the culture. This result correlated with more than 88% inhibition in the expression of a reporter gene 24 h after antagonist removal. Calcium toxicity was completely abolished by specific antagonists of the NMDA receptor. Treatment of cell extracts with N-glycosydase showed that both receptor subunits were N-glycosylated. Tunicamycin prevented calcium toxicity; gel electrophoresis studies showed that this protection was likely due to degradation of the NR1 subunit.
Asunto(s)
Aminoácidos Excitadores/farmacología , Riñón/embriología , Receptores de N-Metil-D-Aspartato/genética , Virus Vaccinia/genética , Calcio/farmacocinética , Calcio/toxicidad , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Regulación de la Expresión Génica/efectos de los fármacos , Genes Reporteros/genética , Glicósido Hidrolasas/metabolismo , Glicosilación , Humanos , Inmunohistoquímica , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Receptores de N-Metil-D-Aspartato/metabolismo , Proteínas Recombinantes/genética , Transfección , Tunicamicina/farmacología , Valina/análogos & derivados , Valina/farmacologíaRESUMEN
In strips of pig coronary arteries incubated in oxygenated Krebs-bicarbonate solution at 37 degrees C, dotarizine blocked the phasic contractions evoked by 5-HT (0.5 microM) or K+ depolarization (35 mM K+) with an IC50 of 0.22 and 3.7 microM, respectively. Flunarizine inhibited both types of contractions with IC50 values of 1.7 microM for 5-HT and 2.4 microM for K+ responses. In Xenopus oocytes injected with in vitro transcribed RNA encoding for 5-HT2A or 5-HT2C receptors, 5-HT (100 nM for 20 s) applied every 10 min caused, in both cases, a reproducible inward current through Ca2(+)-activated Cl- channels (ICl). Dotarizine inhibited the 5-HT2A response in a concentration-dependent manner, with an IC50 of 2.2 nM. In contrast, the 5-HT2C response was unaffected by 1 microM dotarizine and blocked around 62% by 10 microM of this drug. The ICl activated either by intracellular injection of inositol 1,4,5-trisphosphate (IP3) in oocytes or by direct photorelease of Ca2+ in DM-nitrophen-injected oocytes was unaffected by 10 microM dotarizine. It is concluded that dotarizine blocks 5-HT2A receptors with a high affinity; the compound is devoid of intracellular effects on any further steps of the transduction pathway (i.e., IP3 receptor). Contrary to flunarizine that blocks equally well the serotonergic and the K+ vascular responses, dotarizine exhibits 17-fold higher affinity for vascular 5-HT receptors. These findings might be relevant to an understanding of the mechanism involved in the use of dotarizine and flunarizine as prophylactic agents in migraine.
Asunto(s)
Compuestos de Bencidrilo/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Vasos Coronarios/efectos de los fármacos , Oocitos/efectos de los fármacos , Piperazinas/farmacología , Receptores de Serotonina/metabolismo , Antagonistas de la Serotonina/farmacología , Animales , Arterias/efectos de los fármacos , Vasos Coronarios/metabolismo , Femenino , Flunarizina/farmacología , Masculino , Microinyecciones , Oocitos/metabolismo , Porcinos , Vasoconstricción/efectos de los fármacos , Vasodilatadores/farmacología , Xenopus laevisRESUMEN
We have examined the effects of oleoylanilide, one of the main candidates in the etiology of the toxic oil syndrome, in the neuroblastoma cell line N2A. Oleoylanilide treatment causes two kinds of phenomena: alteration of the actin cytoskeleton, creating a brush-like protrusion of actin at the periphery of the cells, and reduction of the adhesiveness of these cells to laminin and fibronectin, two of the main components of the extracellular matrix in the central nervous system. These effects could be correlated with symptoms shown in the acute and chronic phases of the disease.
Asunto(s)
Anilidas/toxicidad , Citoesqueleto/efectos de los fármacos , Enfermedades Transmitidas por los Alimentos/etiología , Ácidos Oléicos/toxicidad , Células Tumorales Cultivadas/efectos de los fármacos , Actinas/efectos de los fármacos , Animales , Supervivencia Celular/efectos de los fármacos , Citoesqueleto/patología , Matriz Extracelular/metabolismo , Ácidos Grasos Monoinsaturados , Fibronectinas/metabolismo , Laminina/metabolismo , Ratones , Neuroblastoma/patología , Aceites de Plantas/envenenamiento , Aceite de Brassica napus , Síndrome , Células Tumorales Cultivadas/patologíaRESUMEN
The protein kinase C inhibitor bisindolylmaleimide GF109203X has a dual effect on the behavior of the neuroblastoma cell line Neuro-2A; when the inhibitor is added in conditions that induce differentiation (absence of serum), neurite outgrowth is potentiated in a dose-dependent manner. However, if the inhibitor is added in growth-promoting conditions (presence of serum), programmed cell death (apoptosis) is induced, as assessed by internucleosomal DNA cleavage and specific immunoassays. This effect is also seen with other specific protein kinase C inhibitors. Bcl2 gene overexpression protects Neuro-2A cells from apoptosis, as has been found in other systems. We also show that calpain I, a neutral Ca(2+)-activated proteinase, participates in this apoptotic pathway. Our results point to a key role of protein kinase C in the regulation of growth and differentiation in Neuro-2A cells.
Asunto(s)
Apoptosis/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Indoles/farmacología , Maleimidas/farmacología , Neuroblastoma , Proteína Quinasa C/antagonistas & inhibidores , Animales , Proteínas Sanguíneas/farmacología , Calpaína/farmacología , Diferenciación Celular/fisiología , División Celular/efectos de los fármacos , Expresión Génica/fisiología , Ratones , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-bcl-2 , Células Tumorales Cultivadas/citologíaRESUMEN
We have characterized two polyubiquitin genes from the crustacean Artemia franciscana. One of them, Ubi1, has nine ubiquitin units and an intron of a minimum size of 3.5 kb that ends 7 bp before the initiator ATG. The 5' end of the transcript from this gene has been identified by anchored PCR. The existence of the other gene (Ubi2) was inferred from several cDNA clones that differ from Ubi1 in the C-terminal extension and in the 3' untranslated region as well as in the nucleotide sequence of the coding region. We find two transcripts of ubiquitin genes, of 2.7 and 3.3 kb. Hybridization of RNA blots with an oligonucleotide specific for Ubi2 gene demonstrates that this gene codes for the 3.3 kb transcript. Ubiquitin messenger RNAs are present in the dormant embryos and their steady-state levels are maximum at 8 h after resumption of development, declining thereafter. The Ubi2 gene transcripts are less abundant but its proportion in relation to the other transcript does not vary with development.
Asunto(s)
Artemia/genética , Biopolímeros/genética , Ubiquitinas/genética , Secuencia de Aminoácidos , Animales , Artemia/embriología , Secuencia de Bases , Clonación Molecular , ADN Complementario , ADN Recombinante , Regulación del Desarrollo de la Expresión Génica , Datos de Secuencia Molecular , PoliubiquitinaRESUMEN
The biochemical properties of Artemia ras proteins (p21) have been studied after immunoprecipitation with the monoclonal antibody Y13-259. The ras products bind GTP and GDP, and have GTPase activity. Artemia p21 was unable to hydrolyze Gp4G, although this dinucleotide exhibits high affinity for the protein. Our results demonstrate that the protein(s) recognized by the Y13-259 antibody in this crustacean behave as typical mammalian ras p21s.
Asunto(s)
Artemia/química , Proteínas Proto-Oncogénicas p21(ras)/química , Animales , Unión Competitiva , Fosfatos de Dinucleósidos/metabolismo , GTP Fosfohidrolasas/análisis , Guanosina Trifosfato/metabolismo , Pruebas de Precipitina , Proteínas Proto-Oncogénicas p21(ras)/inmunología , Especificidad por SustratoRESUMEN
5S rRNA genes are linked to the histone genes in the 13 populations of the crustacean Artemia that we have studied. In all cases, two types of repeat units are found. Southern blot analysis of all populations shows that they can be grouped into three classes: a) American bisexuals; b) Eurasian bisexuals, and c) parthenogenetic organisms (all from Eurasia). Restriction analysis of a bisexual population from San Francisco Bay shows that the two repeat units are of 9.0 and 8.5 kb (with minor heterogeneities of restriction sites). In parthenogenetic organisms, the two repeat units are of approximately 12 kb. Sequencing data from the region of the 5S rRNA from the San Francisco Bay population, shows that in both types of units, the single 5S rRNA gene (315 bp in length), is located 430 bp downstream the 3' regulatory sequences of the H2A gene, the last gene in the histone cluster. We have isolated three clones that contain 5S rRNA sequences. Two of them (one from an American bisexual and the other from a parthenogenetic population) contain histone and 5S rRNA genes, both with the same transcriptional polarity. The third clone, lacking histone genes, is likely to be an orphon derived from the parthenogenetic population.
Asunto(s)
Artemia/genética , Variación Genética , Histonas/genética , Polimorfismo Genético , Polimorfismo de Longitud del Fragmento de Restricción , ARN Ribosómico 5S/genética , ARN Ribosómico/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN/genética , Enzimas de Restricción del ADN , Femenino , Ligamiento Genético , Masculino , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Partenogénesis , Secuencias Repetitivas de Ácidos Nucleicos , Mapeo Restrictivo , Especificidad de la EspecieRESUMEN
We report the identification of p21ras and a cDNA coding for it in the crustacean Artemia. The monoclonal antibody Y13-259 immunoprecipitates a polypeptide of 21.5 kDa in 24 hr-old larvae. The homology of p21ras with the Drosophila melanogaster and the mammalian p21s is in the order of 75-80% mRNA (of 1.2 kb in length) is already present in encysted gastrulae, and the levels increase, reaching a maximum before emergence. On the contrary, both the amount of p21ras and its GTP-binding activity are low prior to emergence and rises afterwards. These results suggest that p21ras expression is regulated post-transcriptionally, and that its function(s) is needed for post-hatching events, the more likely being the resumption of cell proliferation.
Asunto(s)
Artemia/crecimiento & desarrollo , Regulación de la Expresión Génica , Proteínas Proto-Oncogénicas/genética , Proto-Oncogenes , Secuencia de Aminoácidos , Animales , Artemia/genética , Secuencia de Bases , ADN/genética , ADN/aislamiento & purificación , Drosophila melanogaster/genética , Técnicas de Inmunoadsorción , Larva/metabolismo , Datos de Secuencia Molecular , Proteínas Proto-Oncogénicas p21(ras) , ARN Mensajero/biosíntesis , Homología de Secuencia de Ácido NucleicoRESUMEN
cDNA clones coding for Artemia mitochondrial 16S ribosomal RNA (rRNA) have been isolated. The clones cover from nucleotide 650 of the RNA molecule to its 3' end. The comparison of Artemia sequence with both vertebrate and invertebrate mitochondrial 16S rRNA sequences has shown the existence of regions of high similarity between them. A model for the secondary structure of the 3' half of Artemia mitochondrial 16S rRNA is proposed. The size of the rRNA molecule has been estimated at 1.35 kb. Despite the similarity of the Artemia gene to insect rRNA in size, sequence and secondary structure, the G + C content of the Artemia gene (42%) is closer to that of mammals than to the insect genes. The number of mitochondria in Artemia has been estimated at 1500 per diploid genome in the cyst and 4000 in the nauplius. In contrast, the amount of mt 16S rRNA is constant at all stages of Artemia development.
Asunto(s)
Artemia/genética , Clonación Molecular , ADN Mitocondrial/genética , ADN Ribosómico/genética , ARN Ribosómico 16S/genética , ARN Ribosómico/genética , Animales , Secuencia de Bases , ADN Mitocondrial/aislamiento & purificación , ADN Ribosómico/aislamiento & purificación , Sistemas de Información , Mitocondrias/metabolismo , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Mapeo Restrictivo , Homología de Secuencia de Ácido Nucleico , Estados UnidosRESUMEN
The proximal part of the Intergenic Spacer, as well as most of the External Transcribed Spacer of the ribosomal RNA type I genes from the crustacean Artemia have been sequenced. We have identified in the Intergenic Spacer five repeats of around 600 bp in length and, possibly, two imperfect or truncated repeats, derived from the principal ones. These sequences are separated by 485 bp from the 17S rRNA coding sequence. We have also identified the start point of transcription by S1 nuclease analysis. This start point is found 248 bp inside the first repeat. The sequence around the start point shows homology with that described for other members of the same phylum, mostly insects. The most conserved regions are from -1 to +25, and the G residue at position -16. At least the three 600-bp repeats upstream from that containing the promoter also contain the start point sequence, and could therefore act as initiation sites for snPIRNA and/or as enhancer sequences for ribosomal RNA gene transcription.
Asunto(s)
Artemia/genética , Mapeo Cromosómico , ARN Mensajero/análisis , Transcripción Genética , Animales , Secuencias Repetitivas de Ácidos Nucleicos , Homología de Secuencia de Ácido NucleicoRESUMEN
Rabbit antibodies against Artemia RNA polymerase II have been raised and utilized to study the immunological relationships between the subunits from RNA polymerases I, II and III from this organism and RNA polymerase II from other eukaryotes. We describe here for the first time the subunit structure of Artemia RNA polymerases I and III. These enzymes have 9 and 13 subunits respectively. The anti-RNA polymerase II antibodies recognize two subunits of 19.4 and 18 kDa common to the three enzymes, and another subunit of 25.6 kDa common to RNA polymerases II and III. The antibodies against Artemia RNA polymerase II also react with the subunits of high molecular weight and with subunits of around 25 and 33 kDa of RNA polymerase II from other eukaryotes (Drosophila melanogaster, Chironomus thummi, triticum (wheat) and Rattus (rat]. This interspecies relatedness is a common feature of eukaryotic RNA polymerases.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/inmunología , ARN Polimerasa III/inmunología , ARN Polimerasa II/inmunología , ARN Polimerasa I/inmunología , Animales , Artemia , Chironomidae , Reacciones Cruzadas , Drosophila melanogaster , Electroforesis en Gel de Poliacrilamida , ARN Polimerasa I/aislamiento & purificación , ARN Polimerasa II/aislamiento & purificación , ARN Polimerasa III/aislamiento & purificación , Conejos , Ratas , TriticumRESUMEN
We have isolated a satellite fraction from the Artemia genome by both restriction endonuclease digestion and equilibrium density centrifugation in CsCl gradients containing ligand dye Hoechst 33258. Satellite DNA was arranged in long stretches (approx. 23 kb) of tandem repeats of a basic unit of 113 bp. The basic unit has been sequenced, showing a G + C content very close to that of total DNA. Different amounts of satellite were present in several populations of Artemia, whereas it was absent from others.