RESUMEN
alpha-Amanitin acts in vitro as a selective inhibitor of the nucleoplasmic form B RNA polymerases. Treatment of Chinese hamster ovary (CHO) cells with this drug leads principally to a severe fragmentation of the nucleoli. While the ultrastructural lesions induced by alpha-amanitin in CHO cells and in rat or mouse liver are quite similar, the results diverge concerning the effect on RNA synthesis. It has been shown that in rat or mouse liver alpha-amanitin blocks both extranucleolar and nucleolar RNA synthesis. Our autoradiographic and biochemical evidence indicates that in CHO cells high molecular weight extranucleolar RNA synthesis (HnRNA) is blocked by the alpha-amanitin treatment, whereas nucleolar RNA (preribosomal RNA) synthesis remains unaffected even several hours after the inhibition of extranucleolar RNA synthesis. Furthermore, the processing of this RNA as well as its transport to the cytoplasm seem only slightly affected by the treatment. Finally, under these conditions, the synthesis of the low molecular RNA species (4-5S) still occurs, though less actively. The results are interpreted as evidence for a selective impairment of HnRNA synthesis by alpha-amanitin in CHO cells.
Asunto(s)
Micotoxinas/farmacología , Oligopéptidos/farmacología , Ovario/metabolismo , ARN/biosíntesis , Animales , Autorradiografía , Basidiomycota , Radioisótopos de Carbono , Fraccionamiento Celular , Nucléolo Celular/metabolismo , Núcleo Celular/metabolismo , Células Cultivadas , Cricetinae , Citoplasma/metabolismo , Femenino , Fibroblastos/ultraestructura , Microscopía Electrónica , Peso Molecular , Ovario/citología , Péptidos Cíclicos/farmacología , Ribosomas/metabolismo , Factores de Tiempo , Tritio , Uridina/metabolismoRESUMEN
STAT transcription factors are induced by a number of growth factors and cytokines. Within minutes of induction, the STAT proteins are phosphorylated on tyrosine and serine residues and translocated to the nucleus, where they bind to their DNA targets. The leukemia inhibitory factor (LIF) mediates pleiotropic and sometimes opposite effects both in vivo and in cultured cells. It is known, for example, to prevent differentiation of embryonic stem (ES) cells in vitro. To get insights into LIF-regulated signaling in ES cells, we have analyzed protein-binding and transcriptional properties of STAT recognition sites in ES cells cultivated in the presence and in the absence of LIF. We have detected a specific LIF-regulated DNA-binding activity implicating the STAT3 protein. We show that STAT3 phosphorylation is essential for this LIF-dependent DNA-binding activity. The possibility that ERK2 or a closely related protein kinase, whose activity is modulated in a LIF-dependent manner, contributes to this phosphorylation is discussed. Finally, we show that the multimerized STAT3-binding DNA element confers LIF responsiveness to a minimal thymidine kinase promoter. This, together with our observation that overexpression of STAT3 dominant-negative mutants abrogates this LIF responsiveness, clearly indicates that STAT3 is involved in LIF-regulated transcriptional events in ES cells. Finally, stable expression of such a dominant negative mutant of STAT3 induces morphological differentiation of ES cells despite continuous LIF supply. Our results suggest that STAT3 is a critical target of the LIF signaling pathway, which maintains pluripotent cell proliferation.
Asunto(s)
Inhibidores de Crecimiento/farmacología , Interleucina-6 , Linfocinas/farmacología , Células Madre/citología , Transcripción Genética/efectos de los fármacos , Proteínas de Fase Aguda/metabolismo , Animales , Especificidad de Anticuerpos , Sitios de Unión/fisiología , Blastocisto/citología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , División Celular/efectos de los fármacos , División Celular/genética , Línea Celular , Proteínas de Unión al ADN/inmunología , Proteínas de Unión al ADN/metabolismo , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Inhibidores de Crecimiento/química , Inhibidores de Crecimiento/genética , Factor Inhibidor de Leucemia , Linfocinas/química , Linfocinas/genética , Ratones , Proteína Quinasa 1 Activada por Mitógenos , Mutagénesis/fisiología , Fosfoproteínas/inmunología , Fosforilación , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Recombinantes de Fusión/genética , Factor de Transcripción STAT3 , Células Madre/química , Células Madre/enzimología , Transactivadores/inmunología , Transactivadores/metabolismo , Tirosina/metabolismoRESUMEN
In vitro genetic techniques were used to study the sequence requirements for the initiation of specific transcription. Deletion mutants were constructed around the putative promoter of the adenovirus-2 major late and chicken conalbumin genes. Specific transcription in vitro by RNA polymerase B together with a HeLa cell cytoplasmic extract was used as the test for promoter function. With this approach sequences which are essential for the initiation of specific transcription in vitro, were shown to be located between 12 and 32 base pairs upstream from the 5' end of these genes.
Asunto(s)
Fenómenos Fisiológicos Celulares , ARN Polimerasas Dirigidas por ADN/metabolismo , Células Eucariotas/fisiología , Operón , ARN Polimerasa II/metabolismo , Transcripción Genética , Secuencia de Bases , Sitios de Unión , ADN/genética , Enzimas de Restricción del ADN , ADN Recombinante , ARN Mensajero/genéticaRESUMEN
We used DNase I footprinting assays on nuclei isolated from adenovirus-infected cells to examine the nucleoprotein configuration of a 250-base-pair segment which encompasses the adenovirus type 5 major late (ML) and IVa2 promoters. At 12 and 20 h postinfection (p.i.), fine DNase I digestion mapping of wild-type adenovirus-infected cells revealed specific sequences protected from digestion which corresponded to promoter elements required for expression of the ML gene in vivo. At 12 h p.i., a G+C-rich region which lies upstream of the IVa2 cap site and is important for maximal IVa2 activity was also found masked to nuclease activity. At 20 h p.i., however, this element became more sensitive to nuclease attack, while the ML promoter elements stayed protected. No major changes in DNA-protein interactions were detected in the region spanning the ML and IVa2 cap sites upon promoter activation, suggesting that the binding properties of the cognate factors for this region are not modified during the process.
Asunto(s)
Adenovirus Humanos/genética , Transformación Celular Viral , Proteínas de Unión al ADN/genética , Regiones Promotoras Genéticas , Factores de Transcripción/genética , Proteínas Virales/genética , Secuencia de Bases , Núcleo Celular/metabolismo , Genes , Genes Virales , Células HeLa/metabolismo , Humanos , Datos de Secuencia MolecularRESUMEN
Specific protein binding on the EIa-inducible adenovirus EIIa early (EIIaE) promoter was analyzed by the sensitive electrophoretic band-shift assay and by protection against DNase I digestion. Three factors were identified, and precise mapping of the cognate-binding sites revealed their correspondence to promoter elements essential for constitutive EIIaE transcription. One binds to the major upstream element located between -82 and -64 (with respect to the major EIIaE cap site), another appears to interact with sequences on either side of this region, and the last one binds to an element located further upstream. Comparison of the binding activities of the factors present in extracts from cells infected with wild-type adenovirus (adenovirus type 5) or with the EIa deletion mutant dl312 did not reveal striking differences. Not only were the general binding patterns indistinguishable, but the concentration of each of the identified factors as well as their affinity for the cognate-binding sites were unchanged. Our results suggest that the EIa-mediated activation of the EIIaE transcription complexes involves appropriate interactions between transcription factors, rather than their increased binding to DNA.
Asunto(s)
ADN Viral/metabolismo , Proteínas de Unión al ADN/fisiología , Proteínas Oncogénicas Virales/genética , Regiones Promotoras Genéticas , Transcripción Genética , Proteínas Precoces de Adenovirus , Secuencia de Bases , Sitios de Unión , Análisis Mutacional de ADN , Regulación de la Expresión Génica , Células HeLa , Técnicas In Vitro , Datos de Secuencia Molecular , Nucleoproteínas/metabolismo , Unión ProteicaRESUMEN
Genomic DNase I footprinting was used to compare specific protein binding to the adenovirus type 5 early, EIa-inducible, EIIa promoter. Identical protection patterns of the promoter region were observed whether EIIa transcription was undetectable or fully induced. These results suggest that EIa-mediated transcriptional induction does not increase binding of limiting transcription factors to the promoter but rather that transactivation results from the proper interactions between factors already bound to their cognate sequences.
Asunto(s)
Adenoviridae/genética , Genes Virales , Proteínas Oncogénicas Virales/farmacología , Regiones Promotoras Genéticas , Proteínas Precoces de Adenovirus , Antígenos Virales de Tumores , ADN Viral/genética , ADN Viral/metabolismo , Desoxirribonucleasa I , Células HeLa , Humanos , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
We recently isolated three cDNA clones encoding closely related proteins (ATFa1, ATFa2, and ATFa3) that belong to the activating transcription factor-cyclic AMP-responsive element family of cellular transcription factors. Using cotransfection experiments, we showed that these proteins mediate the transcriptional activation induced by the adenovirus E1a 13S mRNA gene product and that the zinc-binding domains present in both E1a conserved region 3 and the most N-terminal portion of the ATFa proteins play crucial roles in this activity. Reciprocal coimmunoprecipitation experiments demonstrated direct interactions between these proteins. Neither the conserved region 3 domain of E1a nor the N-terminal metal-binding element of ATFa is essential for these interactions. The simultaneous alteration of both the N-terminal and the C-terminal domains of ATFa abolished E1a binding, while either mutation alone failed to impair these interactions.
Asunto(s)
Proteínas E1A de Adenovirus/fisiología , Adenovirus Humanos/genética , Proteínas Sanguíneas/fisiología , Regulación de la Expresión Génica , Factores de Transcripción/fisiología , Activación Transcripcional , Factores de Transcripción Activadores , Secuencia de Aminoácidos , Secuencia de Bases , ADN/genética , Humanos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Relación Estructura-Actividad , Dedos de ZincRESUMEN
The murine embryonal carcinoma (EC) F9 cells express an adenovirus EIa-like activity which is abolished after differentiation of these cells. We have examined the ability of the adenovirus EIa gene products to transactivate the viral early EIIa (EIIaE) promoter in this cell system. Surprisingly, as shown both by infection and transfection experiments, the EIIaE promoter was refractory to viral EIa-mediated activation in the undifferentiated F9 EC cells, EIa-responsiveness being recovered only after differentiation of these cells. This EC-cell-specific restriction did not correspond to a lack of EIa gene expression, nor to a deficiency of any of the major transcription factors involved in EIIaE basal promoter activity. In these differentiated F9 cells, EIIaE promoter activation correlates with a modification of E2F resulting in its ability to cooperatively interact with the promoter. Our observation that such a modification neither occurs in uninfected F9 EC cells (Jansen-Durr et al., 1989, EMBO J., 8, 3365), nor at early times after infection of these cells suggests that a viral product is required together with the EIa-like activity to induce this modification of E2F. The implication of an EIV gene product in this effect is discussed.
Asunto(s)
Neoplasias de Células Germinales y Embrionarias/patología , Proteínas Oncogénicas Virales/fisiología , Regiones Promotoras Genéticas/fisiología , Proteínas Precoces de Adenovirus , Animales , Secuencia de Bases , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Fibroblastos/metabolismo , Fibroblastos/patología , Fibroblastos/fisiología , Expresión Génica , Células HeLa , Humanos , Ratones , Datos de Secuencia Molecular , Neoplasias de Células Germinales y Embrionarias/metabolismo , Neoplasias de Células Germinales y Embrionarias/fisiopatología , Proteínas Oncogénicas Virales/genética , Proteínas Oncogénicas Virales/metabolismo , Factores de Transcripción/fisiología , Transcripción GenéticaRESUMEN
The reversible interaction of the retinoblastoma susceptibility gene product (Rb) with the cellular transcription factor E2F has recently been demonstrated. Activation of the adenovirus E2a promoter by the products of the viral E1a gene correlates with the ability of both early E1a proteins to sequester Rb, thereby releasing E2F from inactive complexes with this protein. The E2a promoter is also efficiently stimulated by a product (17.5 kDa) of the viral E4 gene. The specific interaction of this E4 protein with E2F results in the formation of complexes that bind cooperatively to the two neighboring E2F binding sites in the E2a promoter. We have previously shown that in undifferentiated F9 cells (F9EC) the E2a promoter is refractory to E2F-mediated activation by E1a, but not by E4. Using both band-shift and transfection experiments, we now demonstrate (i) that in F9EC cells the E4 product, in combination with E2F, recruits Rb into a stable multiprotein complex and (ii) that in these undifferentiated cells, as opposed to their differentiated counterpart, Rb is actively involved in the transcriptional stimulation of the E2a promoter by E4. Our results suggest that, depending on the cell state, Rb may behave either as a transcriptional activator (F9EC cells) or as a transcriptional inhibitor (differentiated F9 cells).
Asunto(s)
Proteínas Portadoras , Proteínas de Ciclo Celular , Proteínas de Unión al ADN , Proteína de Retinoblastoma/fisiología , Transactivadores/fisiología , Proteínas E2 de Adenovirus/genética , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos , Secuencia de Bases , Línea Celular , Factores de Transcripción E2F , Ratones , Datos de Secuencia Molecular , Fosforilación , Regiones Promotoras Genéticas , Proteína 1 de Unión a Retinoblastoma , Factor de Transcripción DP1 , Factores de Transcripción/metabolismoRESUMEN
The ATFa proteins, which are members of the CREB/ATF family of transcription factors, have previously been shown to interact with the adenovirus E1a oncoprotein and to mediate its transcriptional activity; they heterodimerize with Jun, Fos or related transcription factors, possibly altering their DNA-binding specificity; they also stably bind JNK2, a stress-induced protein kinase. Here we report the identification and characterization of a novel protein isolated in a yeast two-hybrid screen using the N-terminal half of ATFa as a bait. This 1306-residue protein (mAM, for mouse ATFa-associated Modulator) is rather acidic (pHi 4.5) and contains high proportions of Ser/Thr (21%) and Pro (11%) residues. It colocalizes and interacts with ATFa in mammalian cells, contains a bipartite nuclear localization signal and possesses an ATPase activity. Transfection experiments show that mAM is able to downregulate transcriptional activity, in an ATPase-independent manner. Our results indicate that mAM interacts with several components of the basal transcription machinery (TFIIE and TFIIH), including RNAPII itself. Together, these findings suggest that mAM may be involved in the fine-tuning of ATFa-regulated gene expression, by interfering with the assembly or stability of specific preinitiation transcription complexes.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción TFII , Factores de Transcripción/metabolismo , Adenosina Trifosfatasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células COS , Núcleo Celular/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , ADN Complementario , Desarrollo Embrionario y Fetal , Humanos , Ratones , Datos de Secuencia Molecular , ARN Polimerasa II/metabolismo , Proteínas Represoras/genética , Factor de Transcripción TFIIH , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
Bloom's syndrome (BS) is a rare human genetic disorder characterized by mutations within the BLM gene whose primary effects are excessive chromosome breakage and increased rates of sister chromatid interchange in somatic cells. We report the characterization of a murine protein (mBLM), highly related to the product of the human BLM gene. This protein exhibits an ATP-dependent DNA-helicase activity that unwinds DNA in a 3'-5' direction. Single amino acid substitutions found in BS cells, abolish both ATPase and helicase activities of this protein, indicating that defects in these BLM functions may be primarily responsible for BS establishment. These results provide the first evidence suggesting that the enzymatic activities of the BLM product are implicated in the upholding of genomic integrity.
Asunto(s)
Adenosina Trifosfatasas/genética , Sustitución de Aminoácidos , Síndrome de Bloom/genética , Cromosomas Humanos Par 15/genética , ADN Helicasas/genética , Ratones/genética , Mutación Puntual , Factores de Transcripción Activadores , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/deficiencia , Adenosina Trifosfatasas/fisiología , Secuencia de Aminoácidos , Animales , Proteínas Sanguíneas/metabolismo , Síndrome de Bloom/enzimología , Células COS , ADN Helicasas/química , ADN Helicasas/deficiencia , ADN Helicasas/fisiología , ADN Complementario/genética , Humanos , Datos de Secuencia Molecular , RecQ Helicasas , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad , Factores de Transcripción/metabolismo , Transfección , Síndrome de Werner/enzimología , Síndrome de Werner/genéticaRESUMEN
The E2F/DP family of transcription factors play an important role in the control of cell cycle progression. By direct regulatory interactions with the retinoblastoma family of proteins, they integrate extracellular growth promoting signals impinging on the cyclin and cyclin dependent kinase complex during the G1 phase, with cell cycle progression. This is accomplished by direct transcriptional activation of genes required for nucleotide biosynthesis and DNA replication in the S phase. In addition, these transcription factors also play a role in the control of genes involved in regulating G1 and S phase progression including, autoregulatory control, as in the case of E2F1 itself. In this report, we describe the characterisation of the genomic locus encoding DP1, a member of this family. The DP1 gene has a TATA-less promoter and transcription initiates at multiple sites. Using transient transfection assays we have delineated sequences in the upstream region which have promoter or enhancer activity. The DP1 gene was localised to mouse chromosome 8 by metaphase chromosome analysis. We describe a dynamic pattern of DP1 expression using in situ hybridisation on cryostat sections of mouse embryos at various stages of development and a variable level of expression by Northern blot analysis of RNA from various adult tissues.
Asunto(s)
Proteínas Portadoras , Proteínas de Ciclo Celular/genética , Proteínas de Unión al ADN , Factores de Transcripción/genética , Animales , Secuencia de Bases , Proteínas de Ciclo Celular/metabolismo , Mapeo Cromosómico/métodos , Factores de Transcripción E2F , Factor de Transcripción E2F1 , Hibridación Fluorescente in Situ , Ratones/embriología , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Proteína 1 de Unión a Retinoblastoma , Factor de Transcripción DP1 , Factores de Transcripción/metabolismo , Transcripción Genética , TransfecciónRESUMEN
Three related clones encoding proteins (ATFa1, 2 and 3) with specific ATF/CRE DNA-binding activities have been isolated from HeLa cell cDNA libraries. All three isoforms have weak effects on the basal activity of the adenovirus E2a promoter. We present evidence suggesting that a C-terminal element of the ATFa molecules negatively interferes with the intrinsic activation function of these proteins. We also show that coexpression of ATFa with c-Jun, Jun-B or Jun-D stimulates ATFa-dependent reporter activity, while coexpression of c-Fos has no effect. Deletion analyses indicate that the metal-binding region of ATFa is dispensible for this effect, but that the domain comprising the leucine-zipper region of ATFa is required. Reciprocal co-immunoprecipitation experiments and electrophoretic band-shift assays with in vitro synthesized proteins reveal direct interactions between ATFa and Jun or Fos. The ATFa/c-Jun heterodimers, but not the ATFa/c-Fos complexes, bind efficiently to ATF, CRE or AP1 sites. The detection of ATFa-Jun complexes in crude extracts from HeLa cells transfected with ATFa and c-Jun expression vectors suggests that such ATFa/c-Jun heterodimers also form in vivo. Altogether these results indicate that the ATFa proteins may contribute to the modulation of the activity of the Jun/Fos complexes by altering their DNA-binding and transcriptional properties.
Asunto(s)
Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/genética , Proteínas Proto-Oncogénicas c-jun/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética/genética , Factores de Transcripción Activadores , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , ADN/genética , Genes Reporteros/genética , Genes fos/genética , Genes jun/genética , Vectores Genéticos , Células HeLa , Humanos , Isomerismo , Leucina Zippers , Datos de Secuencia Molecular , TransfecciónRESUMEN
The human ATFa proteins belong to the CREB/ATF family of transcription factors. We have previously shown that the ATFa proteins may contribute to the modulation of the transcriptional activity of the Jun/Fos complexes (Chatton et al. (1994). Oncogene, 9, 375-385). We now show that a protein kinase activity is strongly associated with ATFa in vivo, as revealed by coimmunoprecipitation of ATFa/kinase complexes from whole cell extracts, with antibodies against ATFa. Two independent regions were found to be implicated in kinase binding: a major interaction site is located within the N-terminal 82 residues comprising an important metal-chelating element; a weaker binding site corresponds to the basic sequence element preceding the C-terminal leucine-zipper of ATFa. Induction experiments suggest that each of these ATFa domains may interact with different kinases. The major activity is associated with the ATFa N-terminal domain. Based on its response to various inducers, on both in vitro and in vivo binding assays, and on its immunological properties, this activity most likely corresponds to the 54/55 kDa JNK2 protein. Taken together, these observations suggest that the ATFa proteins, among other CREB/ATF proteins, may be important effectors of cell signalling pathways.
Asunto(s)
Proteínas Sanguíneas/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos , Quinasas de Proteína Quinasa Activadas por Mitógenos , Proteínas Quinasas/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción Activadores , Animales , Sitios de Unión , Línea Celular , Humanos , MAP Quinasa Quinasa 4 , FosforilaciónRESUMEN
The ATFa proteins, which are members of the CREB/ATF family of transcription factors, display quite versatile properties. We have previously shown that they interact with the adenovirus E1a oncoprotein, mediating part of its transcriptional activity and heterodimerize with the Jun, Fos or related transcription factors, thereby modulating their DNA-binding specificity. In the present study, we report the sequence requirement of the N-terminal activation domain of ATFa and demonstrate the importance of specific threonine residues (Thr51 and Thr53) in addition to that of the metal-binding domain, in transcriptional activation processes. We also show that the N-terminal domain of ATFa which stably binds the Jun N-terminal kinase-2 (JNK2) (Bocco et al., 1996), is not a substrate for this kinase in vivo but, instead, serves as a JNK2-docking site for ATFa-associated partners like JunD, allowing them to be phosphorylated by the bound kinase.
Asunto(s)
Proteínas de Unión al ADN , Proteínas Quinasas Activadas por Mitógenos , Fragmentos de Péptidos/fisiología , Proteínas Quinasas/fisiología , Factores de Transcripción/fisiología , Activación Transcripcional/fisiología , Factor de Transcripción Activador 1 , Animales , Células COS , Línea Celular , Proteína Quinasa 9 Activada por Mitógenos , Fosforilación , Proteínas Proto-Oncogénicas c-jun/metabolismoRESUMEN
It has been reported (Iborra et al. (1979) J. Biol. Chem. 254, 10920-10924) that the third and the fifth largest subunit of yeast RNA polymerase I exhibit ribonuclease H activity. The authors suggested that the third largest subunit is identical with the chromatin-associated ribonuclease H49, the putative yeast equivalent of bovine ribonuclease H IIb. Although the third largest subunit of calf thymus RNA polymerase I and ribonuclease H IIb display nearly identical molecular masses under denaturing conditions, serological analysis reveals that, in contrast to their counterparts in yeast, these mammalian proteins are distinct entities. Interestingly, sera from some patients with mixed connective tissue disease which contain antibodies directed against RNA polymerase I, also react with ribonuclease H IIb epitopes. This observation suggests that a protein displaying ribonuclease H IIb antigenicity could be associated with RNA polymerase I. Additional indications supporting this conclusion are discussed.
Asunto(s)
Endorribonucleasas/metabolismo , ARN Polimerasa I/metabolismo , Timo/enzimología , Animales , Anticuerpos/inmunología , Bovinos , Endorribonucleasas/clasificación , Epítopos/inmunología , Células HeLa , Humanos , Hibridación de Ácido Nucleico , ARN Polimerasa I/inmunología , Ribonucleasa HRESUMEN
Mouse embryonic stem cells remain pluripotent when maintained in the presence of leukemia inhibitory factor (LIF). Upon LIF withdrawal, most cells differentiate into various lineages, while some die by apoptosis within 3 days. We have analyzed the activation pattern of the mitogen-activated protein kinase (MAPK) families and characterized the expression profile of selected genes modulated during differentiation or apoptosis. We show that p38 MAPKs are activated first, during the apoptotic crisis, while extracellular-regulated kinases and c-Jun N-terminal kinases are induced after the apoptotic crisis in differentiated cells. However, by using both p38 kinase inhibitors (PD169316 and SB203580) and a p38alpha(-/-) cell line, we demonstrate that p38alpha activation is rather a consequence than a cause of apoptosis. We thus reveal novel properties of PD169316, which induces cell survival without impairing cell differentiation, and identify PD169316-sensitive targets like the fibroblast growth factor-5, Brachyury and bcl-2 genes. Finally, we demonstrate that overexpression of the PD169316 - regulated bcl-2 gene prevents LIF withdrawal - induced cell death.
Asunto(s)
Apoptosis , Diferenciación Celular , Proteínas Quinasas Activadas por Mitógenos/efectos de los fármacos , Células Madre/citología , Células Madre/efectos de los fármacos , Animales , Western Blotting , Caspasas/efectos de los fármacos , Caspasas/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Células Clonales , Medios de Cultivo , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Citometría de Flujo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas Fluorescentes Verdes , Imidazoles/farmacología , Interleucina-6 , Factor Inhibidor de Leucemia , Proteínas Luminiscentes/metabolismo , Ratones , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/deficiencia , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Modelos Biológicos , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Piridinas/farmacología , Proteínas Recombinantes de Fusión/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Madre/metabolismoRESUMEN
The cDNA of the second largest subunit of RNA polymerase II (or B) from HeLa cells has been cloned and sequenced. A predicted amino acid sequence of 1174 residues (calculated molecular mass of 133,896 Da) was derived from the longest open reading frame and compared to the sequences of homologous subunits of polymerases of eukaryotic, archaeal and bacterial origin. After optimal alignment, about 16% of the residues were found to be conserved throughout evolution, from human to Escherichia coli. About 2/3 of the overall length of the conserved domains delineated by these residues are clustered within the C-terminal half of the human polypeptide, whereas the remaining is spread over its N-terminal half. The putative functional significance of these conserved domains is discussed.
Asunto(s)
ARN Polimerasa II/química , Secuencia de Aminoácidos , Evolución Biológica , Clonación Molecular , Células HeLa , Humanos , Datos de Secuencia Molecular , Homología de Secuencia de Ácido NucleicoRESUMEN
BACKGROUND: The sequences encoding the yeast RNA polymerase II (RPB) subunits are single copy genes. RESULTS: While those characterized so far for the human (h) RPB are also unique, we show that hRPB subunit 11 (hRPB11) is encoded by a multigene family, mapping on chromosome 7 at loci p12, q11.23 and q22. We focused on two members of this family, hRPB11a and hRPB11b: the first encodes subunit hRPB11a, which represents the major RPB11 component of the mammalian RPB complex; the second generates polypeptides hRPB11balpha and hRPB11bbeta through differential splicing of its transcript and shares homologies with components of the hPMS2L multigene family related to genes involved in mismatch-repair functions (MMR). Both hRPB11a and b genes are transcribed in all human tissues tested. Using an inter-species complementation assay, we show that only hRPB11balpha is functional in yeast. In marked contrast, we found that the unique murine homolog of RPB11 gene maps on chromosome 5 (band G), and encodes a single polypeptide which is identical to subunit hRPB11a. CONCLUSIONS: The type hRPB11b gene appears to result from recent genomic recombination events in the evolution of primates, involving sequence elements related to the MMR apparatus.
RESUMEN
By transient expression analysis in HeLa cells of adenovirus-2 E2a early (E2aE) promoter mutants and hybrid E2aE-beta-globin gene constructs, we demonstrate the existence of three nucleotide (nt) sequence elements involved in the E1a-responsiveness of the E2aE transcription unit: element I, localized within a segment (nt -13 to +62) surrounding the major E2aE cap site (nt + 1); element II (between nt -71 and -29), and element III (between nt -146 and -86). Each element is unable by itself to confer E1a responsiveness. Only combinations of sequences including elements I and II (spanning nt -71 and +62) or II and III (spanning nt -146 and -29) ensure maximal inducibility, for which element II appears of central importance.