RESUMEN
The human HoxB5 (Hox-2.1) gene product is a sequence-specific DNA binding protein. Cooperative interactions stabilize in vitro DNA binding of the HoxB5 protein to tandem binding sites by at least 100-fold relative to binding to a single site. The HoxB5 homeodomain is sufficient for sequence-specific DNA binding but not for cooperative DNA binding. Here we report that the additional protein sequence required for cooperativity is a small domain adjacent to the homeodomain on the amino-terminal side. We further show that cooperative DNA binding is under redox regulation. The HoxB5 protein binds to DNA in vitro both when oxidized or reduced but binds cooperatively only when oxidized. Mutational analysis has revealed that the cysteine residue in the turn between homeodomain helices 2 and 3 is necessary for cooperative binding and redox regulation. The enhanced DNA binding of oxidized HoxB5 protein is the opposite of the redox regulation reported for other mammalian transcription factors such as Fos, Jun, USF, NF-kappa B, c-Myb, and v-Rel, in which oxidation of cysteine residues inhibits DNA binding. Thus, specific oxidation of nuclear proteins is a potential regulatory mechanism that can act to either decrease or increase their DNA binding activity.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Genes Homeobox , Proteínas de Homeodominio , Regulación Alostérica , Secuencia de Aminoácidos , Clonación Molecular , Cisteína/química , Proteínas de Unión al ADN/química , Humanos , Datos de Secuencia Molecular , Oxidación-Reducción , Proteínas Recombinantes/metabolismoRESUMEN
Heparin-binding epidermal growth factor (HB-EGF) gene transcription is rapidly activated in NIH 3T3 cells transformed by oncogenic Ras and Raf and mediates the autocrine activation of the c-Jun N-terminal kinases (JNKs) observed in these cells. A 1.7-kb fragment of the promoter of the murine HB-EGF gene linked to a luciferase reporter was strongly induced following activation of deltaRaf-1:ER, a conditionally active form of oncogenic human Raf-1. Promoter activation by deltaRaf-1:ER required a composite AP-1/Ets transcription factor binding site located between bp -974 and -988 upstream of the translation initiation site. In vivo genomic footprinting indicated that the basal level of occupancy of this composite AP-1/Ets element increased following deltaRaf-1:ER activation. Cotransfection of Ets-2 and p44 mitogen-activated protein (MAP) kinase expression vectors strongly potentiated HB-EGF promoter activation in response to deltaRaf-1:ER. Potentiated activation required both p44 MAP kinase catalytic activity and threonine 72 in the Pointed domain of Ets-2. Biochemical assays demonstrated the ability of the p42 and p44 MAP kinases to phosphorylate Ets-2 on threonine 72. Importantly, in intact cells, the kinetics of phosphorylation of Ets-2 on this residue closely mirror the activation of the p42 and p44 MAP kinases and the observed onset of HB-EGF gene transcription following deltaRaf-1:ER activation. These data firmly establish Ets-2 as a direct target of the Raf-MEK-MAP kinase signaling pathway and strongly implicate Ets-2 in the regulation of HB-EGF gene expression.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas de Unión al ADN , Factor de Crecimiento Epidérmico/metabolismo , Heparina/metabolismo , Proteínas Serina-Treonina Quinasas/farmacología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/farmacología , Proteínas Represoras/metabolismo , Transactivadores/metabolismo , Transactivadores/farmacología , Factores de Transcripción , Células 3T3 , Animales , Secuencia de Bases , Huella de ADN , Activación Enzimática , Regulación de la Expresión Génica/efectos de los fármacos , Factor de Crecimiento Similar a EGF de Unión a Heparina , Humanos , Péptidos y Proteínas de Señalización Intercelular , Ratones , Datos de Secuencia Molecular , Fosforilación , Mutación Puntual , Regiones Promotoras Genéticas , Proteína Proto-Oncogénica c-ets-2 , Proteínas Proto-Oncogénicas c-raf , Factor de Transcripción AP-1/metabolismoRESUMEN
The Ras oncogene products regulate the expression of genes in transformed cells, and members of the Ets family of transcription factors have been implicated in this process. To determine which Ets factors are the targets of Ras signaling pathways, the abilities of several Ets factors to activate Ras-responsive enhancer (RRE) reporters in the presence of oncogenic Ras were examined. In transient transfection assay, reporters containing RREs composed of Ets-AP-1 binding sites could be activated 30-fold in NIH 3T3 fibroblasts and 80-fold in the macrophage-like line RAW264 by the combination of Ets1 or Ets2 and Ras but not by several other Ets factors that were tested in the assay. Ets2 and Ras also superactivated an RRE composed of Ets-Ets binding sites, but the Ets-responsive promoter of the c-fms gene was not superactivated. Mutation of a threonine residue to alanine in the conserved amino-terminal regions of Ets1 and Ets2 (threonine 38 and threonine 72, respectively) abrogated the ability of each of these proteins to superactivate reporter gene expression. Phosphoamino acid analysis of radiolabeled Ets2 revealed that Ras induced normally absent threonine-specific phosphorylation of the protein. The Ras-dependent increase in threonine phosphorylation was not observed in Ets2 proteins that had the conserved threonine 72 residue mutated to alanine or serine. These data indicate that Ets1 and Ets2 are specific nuclear targets of Ras signaling events and that phosphorylation of a conserved threonine residue is a necessary molecular component of Ras-mediated activation of these transcription factors.
Asunto(s)
Proteínas de Unión al ADN , Proteínas Proto-Oncogénicas/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Proteínas Represoras , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Activación Transcripcional , Proteínas ras/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Secuencia Conservada , Elementos de Facilitación Genéticos , Datos de Secuencia Molecular , Mutación , Fosforilación , Proteína Proto-Oncogénica c-ets-1 , Proteína Proto-Oncogénica c-ets-2 , Proteínas Proto-Oncogénicas c-ets , Transducción de Señal , Treonina/genéticaRESUMEN
Heterozygous female mice carrying a targeted mutation of the Ets2 transcription factor gene were mated with a mouse strain that develops mammary tumors due to the expression of the polyoma virus middle T oncogene. Tumors from females with only one wild-type Ets2 gene were approximately one-half the size of tumors from controls. The smaller size of the tumors was correlated with a more differentiated state of early hyperplastic growths and not to differential growth of the frank tumors or to decreased middle T gene expression. Ets2 may regulate the progression of these aggressive mammary tumors.
Asunto(s)
Alelos , Proteínas de Unión al ADN , Neoplasias Mamarias Experimentales/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Represoras , Transactivadores/genética , Factores de Transcripción , Animales , Femenino , Masculino , Neoplasias Mamarias Experimentales/patología , Neoplasias Mamarias Experimentales/prevención & control , Metaloproteinasa 3 de la Matriz/genética , Ratones , Ratones Transgénicos , Oncogenes , Proteína Proto-Oncogénica c-ets-2RESUMEN
The Ets family of transcription factors are important downstream targets in cellular transformation, as altering Ets activity has been found to reverse the transformed phenotype of Ras transformed mouse fibroblasts and of several human tumor cell lines. To determine whether Ets factors are important targets in the largely uncharacterized aberrant signaling in prostate cancer, we have altered Ets activity in the prostate tumor cell line PPC-1, by stable expression of either full-length Ets2, or a dominant inhibitor of Ets activity, the Ets2 DNA binding domain (Ets2DBD). Analysis of multiple independent clonal cell lines revealed that expression of either Ets2 or the Ets2DBD inhibited the anchorage-independent growth of PPC-1 cells up to 20-fold. In contrast to our previous findings with Ras-transformed NIH3T3 cells, PPC-1 cell lines expressing either Ets2 or the EtsDBD exhibited slower attached cell growth, increased Ets-dependent gene expression, and up to a 10-fold increase in apoptotic cell death. The p21cip gene was identified as a potential target of altered Ets signaling. Interestingly, the two distinct Ets2 constructs had strikingly different effects on in vitro invasiveness. Expression of the Ets2DBD almost completely blocked PPC-1 cell invasion through Matrigel, whereas over-expression of full-length Ets2 did not inhibit invasion. Overall, these results demonstrate that the balance of Ets factor activity can regulate multiple aspects of the transformed phenotype of PPC-1 prostate tumor cells, including anchorage-independent growth, survival, and invasiveness.
Asunto(s)
Proteínas de Ciclo Celular , Transformación Celular Neoplásica , Proteínas de Unión al ADN , Neoplasias de la Próstata/patología , Proteínas Proto-Oncogénicas/fisiología , Proteínas Represoras , Transactivadores/fisiología , Factores de Transcripción , Proteínas Supresoras de Tumor , Células 3T3 , Animales , Apoptosis/fisiología , Sitios de Unión , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Adhesión Celular , División Celular/fisiología , Movimiento Celular/fisiología , Supervivencia Celular/fisiología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Ciclinas/biosíntesis , Regulación Neoplásica de la Expresión Génica , Genes Reporteros , Humanos , Masculino , Ratones , Proteínas Asociadas a Microtúbulos/biosíntesis , Invasividad Neoplásica , Fragmentos de Péptidos/biosíntesis , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/fisiología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Estructura Terciaria de Proteína , Proteína Proto-Oncogénica c-ets-2 , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas/genética , Transactivadores/biosíntesis , Transactivadores/genética , Células Tumorales CultivadasRESUMEN
Oncogenic Ras activates the transcription of a variety of viral and cellular genes through promoter elements consisting of two closely linked binding sites for transcription factors from several distinct families. To better understand what constitutes a promoter oncogene response element (ORE), various transcription factor binding site configurations were inserted into a reporter gene, and transactivation by oncogenic Ras was measured by cotransfection assays in NIH3T3 cells. We show that a single copy of two closely linked binding sites for either AP-1, Ets, NF-kappa B, or single closely linked Ets and AP-1 binding sites, are sufficient to confer at least 10-fold transactivation by similar amounts of oncogenic Ras. Single binding sites for these factors, or several other pairings of binding sites, are not sufficient to confer Ras responsiveness. The effect of altered ORE binding site spacing and orientation was systematically analysed, and limited flexibility was observed. The novel observation that two adjacent c-Ets-2 binding sites are sufficient to act as an ORE, indicates that Ets family proteins are a target of the Ras pathway distinct from AP-1. This ORE also mediates equivalent transactivation by c-Ets-2, and mutant OREs show a parallel decrease in Ras and c-Ets-2 responsiveness. Together, these data help to define transcriptional targets of the Ras signal transduction pathway.
Asunto(s)
Proteínas de Unión al ADN , Genes ras , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras , Transactivadores , Factores de Transcripción , Activación Transcripcional , Células 3T3 , Animales , Secuencia de Bases , Sitios de Unión , Ratones , Datos de Secuencia Molecular , FN-kappa B/metabolismo , Oligodesoxirribonucleótidos , Proteína Proto-Oncogénica c-ets-2 , Proteínas Proto-Oncogénicas c-jun/metabolismo , Transducción de SeñalRESUMEN
Hemophilias A and B are X chromosome-linked bleeding disorders, which are mainly treated by repeated infusions of factor (F)VIII or FIX, respectively. In the present review, we specify the limitations in expression of recombinant (r)FVIII and summarize the bioengineering strategies that are currently being explored for constructing novel rFVIII molecules characterized by high efficiency expression and improved functional properties. We present the strategy to prolong FVIII lifetime by disrupting FVIII interaction with its clearance receptors and demonstrate how construction of human-porcine FVIII hybrid molecules can reduce their reactivity towards inhibitory antibodies. While the progress in improving rFIX is impeded by low recovery rates, the authors are optimistic that the efforts of basic science may ultimately lead to higher efficiency of replacement therapy of both hemophilias A and B.
Asunto(s)
Factor IX/uso terapéutico , Factor VIII/uso terapéutico , Proteínas Recombinantes/uso terapéutico , Factor IX/farmacocinética , Factor VIII/genética , Factor VIII/farmacocinética , Hemofilia A/tratamiento farmacológico , Humanos , Ingeniería de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacocinética , Relación Estructura-ActividadRESUMEN
Because no single assay provides a complete analysis of the transformed phenotype, transactivation assays complement the cell growth and tumorigenicity analyses of oncogene function. Transactivation of ORE-containing genes is such a common feature of a diverse variety of viral and cellular oncogenes that it can be considered one aspect of the oncogene-induced phenotype. After the initial identification of oncogenes that activate transcription, studies of the mechanisms of activation and the identification of the downstream target genes should lead to a better understanding of the events leading to cellular transformation. The fact that cell type specificity of transactivation and transformation can be similar means that the transactivation assay may be a useful tool in dissecting cell type-specific transformation. The transactivation assay of oncogene function also has the advantage that it is easy to perform and significantly more rapid than assays based on altered cell growth. This is of particular advantage when one wishes to examine the function of a large number of oncogene mutants generated in vitro. Overall, transactivation assays provide another tool for examining transforming potential and a starting point for the analysis of the downstream targets of oncogenes.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gi-Go , Proteínas de Unión al GTP/biosíntesis , Proteínas de Unión al GTP/metabolismo , Regulación de la Expresión Génica , Oncogenes , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas/biosíntesis , Transcripción Genética , Activación Transcripcional , Células 3T3 , Animales , División Celular , Transformación Celular Neoplásica , Subunidad alfa de la Proteína de Unión al GTP Gi2 , Proteínas de Unión al GTP/análisis , Sustancias Macromoleculares , Ratones , Fenotipo , Proto-Oncogenes , Proteínas Recombinantes/biosíntesis , Secuencias Reguladoras de Ácidos Nucleicos , Transfección/métodosRESUMEN
In addition to the well-known genomic effects of steroid molecules via intracellular steroid receptors, certain steroids rapidly alter neuronal excitability through binding sites on neurotransmitter-gated ion channels. Several of these steroids accumulate in the brain after local synthesis or after metabolization of adrenal steroids. The 3 alpha-hydroxy ring A-reduced pregnane steroids allopregnanolone and tetrahydrodeoxycorticosterone have been thought not to interact with intracellular receptors but enhance gamma-aminobutyric acid (GABA)-medicated chloride currents. When administered systematically in the rat, these neurosteroids display anxiolytic and hypnotic activities that suggest pronounced systemic effects as well as neuropsychopharmacological potential for modulation of sleep and anxiety. We demonstrated that these neurosteroids can regulate gene expression via the progesterone receptor. The induction of DNA-binding and transcriptional activation of the progesterone receptor requires intracellular oxidation of the neurosteroids into progesterone receptor-active 5 alpha-pregnane steroids. Thus, in physiological concentrations these neurosteroids regulate neuronal function through their concurrent influence on transmitter-gated ion channels and gene expression. These findings extend the concept of a "cross-talk" between membrane and nuclear hormone effects and provide a new role for the therapeutic application of these steroids in neurology and psychiatry.
Asunto(s)
Ansiolíticos/farmacología , Canales de Cloruro/efectos de los fármacos , Hipnóticos y Sedantes/farmacología , Neurotransmisores/fisiología , Receptores de GABA/efectos de los fármacos , Esteroides/fisiología , Animales , Ansiolíticos/uso terapéutico , Biotransformación , Pollos , Canales de Cloruro/fisiología , Chlorocebus aethiops , Regulación de la Expresión Génica/efectos de los fármacos , Hipnóticos y Sedantes/uso terapéutico , Activación del Canal Iónico/efectos de los fármacos , Activación del Canal Iónico/fisiología , Ratones , Modelos Neurológicos , Proteínas del Tejido Nervioso/efectos de los fármacos , Proteínas del Tejido Nervioso/fisiología , Oxidación-Reducción , Ratas , Receptores de GABA/fisiología , Receptores de Progesterona/efectos de los fármacos , Receptores de Progesterona/fisiología , Esteroides/farmacología , Esteroides/uso terapéutico , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiología , Transfección , Células Tumorales CultivadasRESUMEN
The sensitivity of the recombinant alpha 6 beta 2 gamma 2 GABAA receptor expressed in HEK 293 cells to neuroactive steroids was studied. The steroids 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha-OH-DHP), pregnenolone sulfate and 3 alpha-OH-DHP sulfate have different modulatory effects on [3H]muscimol or [3H]Ro15-4513 binding to the alpha 6 beta 2 gamma 2 than to the alpha 1 beta 2 gamma 2 receptor. Binding of both radioactive ligands to the alpha 6 beta 2 gamma 2 receptor was maximally potentiated with each steroid used (10 nM) and decreased with further increases in steroid concentration. Using whole-cell recording, the GABA response of clusters of transfected HEK 293 cells was strongly potentiated by 3 or 10 nM 3 alpha-OH-DHP. In contrast, this response was reduced by 100 nM 3 alpha-OH-DHP. This latter effect appears to be related to the acceleration of the GABA response desensitization, observed in isolated cells. 3 alpha-OH-DHP (10 or 100 nM) was able to activate a response in the absence of GABA. It is proposed that the interaction of neuroactive steroids with the alpha 6 beta 2 gamma 2 receptor involves at least two distinct binding sites. One of them might be located close to the GABA binding site.
Asunto(s)
Receptores de GABA-A/efectos de los fármacos , Proteínas Recombinantes/farmacología , Esteroides/farmacología , Azidas/farmacología , Benzodiazepinas/farmacología , Unión Competitiva , Línea Celular , Células Cultivadas , Electrofisiología , Muscimol/farmacología , Técnicas de Placa-Clamp , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
Neuroactive steroids rapidly alter neuronal excitability through their action via the cell surface. The 3 alpha-hydroxy ring A-reduced pregnane steroids enhance gamma-aminobutyric acid (GABA)-mediated Cl- currents while pregnenolone sulfate and dehydroepiandrosterone sulfate may exert functional antagonistic properties. Based on our previous findings that the 3 alpha-hydroxy ring A-reduced pregnane steroids allotetrahydroprogesterone and allotetrahydrodeoxycorticosterone may regulate gene expression via the progesterone receptor after intracellular oxidation, we have characterized the effects of a series of natural and synthetic neuroactive steroids at the genomic level using a cotransfection system with various steroid receptor expression vectors and a reporter gene in a human neuroblastoma cell line. Pregnanolone and pregnenolone were able to activate both the chicken and the human progesterone receptor while the synthetic 3 alpha-hydroxylated derivative alphaxalone and dehydroepiandrosterone were active via the chicken progesterone receptor but devoid of transcriptional activity via the human progesterone receptor. Moreover, the antiglucocorticoid activity of dehydroepiandrosterone reported at the systemic level could not be reconstituted in the cellular cotransfection system. None of the neuroactive steroids bound directly to steroid receptors. Thus, their genomic activity appears to be mediated via intracellular metabolization. This study provides evidence for differential genomic effects of neuroactive steroids in a structure-specific and species-specific way that may have impact on the development of these steroids for therapeutic application.
Asunto(s)
Neuronas/efectos de los fármacos , Receptores de Progesterona/efectos de los fármacos , Esteroides/farmacología , Animales , Pollos , Deshidroepiandrosterona/farmacología , Humanos , Receptores de Progesterona/análisis , Receptores de Progesterona/metabolismo , Esteroides/metabolismo , Relación Estructura-Actividad , Transcripción Genética/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
The cAMP responsive element binding protein (CREB) and the glucocorticoid receptor (GR) have been reported to bind to a 60 bp promoter fragment of the alpha 1-GABAA receptor gene containing a classic cAMP-responsive element (CRE). We inserted this fragment into a hormone responsive element-deleted mouse mammary tumor virus promoter controlling the expression of luciferase. Activation of GR showed no significant change in luciferase expression, but hormone induction by forskolin revealed a reduction in neuronal cell lines. Furthermore, we demonstrate that cellular factors from neuronal cells can bind to the CRE-containing promoter fragment, although competition by unlabeled CRE and GRE oligo-nucleotides is not present. Mutation of the CRE site and deletion of neighboring DNA sequences indicate that the promoter is probably associated with a complex of different regulatory factors.
Asunto(s)
Proteína Receptora de AMP Cíclico/química , Regiones Promotoras Genéticas , Receptores de GABA/química , Animales , Secuencia de Bases , Células Cultivadas , Chlorocebus aethiops , Clonación Molecular , Proteína Receptora de AMP Cíclico/fisiología , Expresión Génica , Luciferasas/biosíntesis , Virus del Tumor Mamario del Ratón/genética , Ratones , Datos de Secuencia Molecular , Receptores de GABA/fisiología , Transfección , beta-Galactosidasa/químicaAsunto(s)
Proteínas de Unión al ADN/genética , Proteínas Fúngicas/genética , Genes Reporteros , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Saccharomyces cerevisiae , Factores de Transcripción/genética , Activación Transcripcional , Proteínas ras/metabolismo , Células 3T3 , Animales , Luciferasas/genética , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/genética , Transducción de Señal , Transfección , Proteínas ras/genéticaAsunto(s)
Genes ras , Técnicas Genéticas , Activación Transcripcional , Células 3T3 , Animales , Cloranfenicol O-Acetiltransferasa/análisis , Cloranfenicol O-Acetiltransferasa/genética , Genes Reporteros , Factores de Intercambio de Guanina Nucleótido , Luciferasas/análisis , Luciferasas/genética , Ratones , Plásmidos , Proteínas/análisis , Transfección , Factores de Intercambio de Guanina Nucleótido rasRESUMEN
BACKGROUND: Hemophilia A is currently treated by infusions of the coagulation factor (F) VIII, of which production and purification remain a challenging task. Current purification procedures using immunoaffinity chromatography are cumbersome, expensive, and suffer from the instability of the applied antibody ligands, which elute along with the product and contaminate it. Recently, FVIII was purified using octapeptide ligands, but their use is limited due to the low resistance to proteases. OBJECTIVE: Our goal was to develop and evaluate a novel ligand for FVIII purification, overcoming the drawbacks of current procedures. METHODS: Peptide ligands were screened for binding of (125)I-plasma-derived-FVIII (pdFVIII) in a microbead assay. A selected ligand-coated Toyopearl resin was then used for pdFVIII purification from cell-conditioned Delbucco's modified Eagle's medium (DMEM) containing fetal bovine serum. The proteolytic stability of ligand was measured by incubating with human serum and proteinase K, and its cytotoxicity towards human OV-MZ-6 cells was assayed. RESULTS: A high-affinity octapeptidic FVIII ligand was modified into the small, highly stable and non-toxic peptidomimetic ligand L4 by rational and combinatorial design without affecting its affinity for FVIII. Using ligand L4-coated Toyopearl resin, pdFVIII was isolated from cell-conditioned medium with high purity and 89% column retention after elution with a mild buffer containing 0.6 m NaCl at pH 6.8. CONCLUSIONS: Ligand L4 offers a valuable alternative to antibody-based procedures for laboratory and industrial production. Its synthesis by established solid-phase procedures is straightforward and considerably cheaper than the biotechnological production of antibodies, and safety concerns associated with the use of biological material are overcome.
Asunto(s)
Factor VIII/aislamiento & purificación , Biotecnología/métodos , Pruebas de Coagulación Sanguínea , Química Clínica/métodos , Medios de Cultivo Condicionados/farmacología , Endopeptidasa K/química , Factor VIII/química , Humanos , Concentración de Iones de Hidrógeno , Ligandos , Modelos Químicos , Péptido Hidrolasas/química , Péptidos/química , Unión ProteicaRESUMEN
The effect of injections of hormones in utero on fetal rat kidney and liver extramitochondrial phosphoenolpyruvate carboxykinase activity has been studied. Glucagon and thyroxine induced the liber enzyme but none of the hormones tested affected the renal enzyme. In the postnatal rat, the hepatic phosphoenolpyruvate barboxykinase activity is increased after triamcinolone or thyroxine injection but only triamcinolone injection increases the activity of the kidney enzyme. It is suggested that the rise in renal phosphoenolpyruvate carboxykinase activity at about 10 days of age is due to the increase in blood corticosterone content occurring at the same age.
Asunto(s)
Bucladesina/farmacología , Cortisona/farmacología , Glucagón/farmacología , Hormona del Crecimiento/farmacología , Fosfoenolpiruvato Carboxiquinasa (GTP)/metabolismo , Tiroxina/farmacología , Triamcinolona/farmacología , Envejecimiento , Animales , Femenino , Riñón/efectos de los fármacos , Riñón/enzimología , Riñón/crecimiento & desarrollo , Hígado/efectos de los fármacos , Hígado/enzimología , Hígado/crecimiento & desarrollo , Masculino , Especificidad de Órganos , Embarazo , Puromicina/farmacología , RatasRESUMEN
Transcription termination at the ilvB attenuator of Escherichia coli K-12 has been quantitated by measuring the in vivo rate of synthesis and degradation of mRNA segments proximal and distal to the attenuator. This analysis demonstrates that a 4-fold deattenuation results in vivo when the growth of cells is limited by the availability of valine or leucine. The result suggests that attenuation is the major mechanism by which this operon is regulated by these end-product amino acids. On the basis of possible secondary structures of ilvB leader RNA, we predicted that attenuation of this operon should also be affected by growth of cells in limiting amounts of alanine or threonine. We report here that the ilvB operon is deattenuated when cells are starved for either of these amino acids. A rationale for the regulation of this operon by these four amino acids, which represent both the substrates and the products of the ilvB gene product, and by catabolite repression is presented.
Asunto(s)
Proteínas Bacterianas/genética , Genes Bacterianos , Genes , Operón , Secuencia de Bases , Cinética , Hibridación de Ácido Nucleico , Plásmidos , ARN Bacteriano/genéticaRESUMEN
The ilvB gene of Escherichia coli K12 has been cloned into a multicopy plasmid. The regulation of the cloned gene by valine or leucine limitation and by catabolite repression is the same as for the chromosome encoded gene. The nucleotide sequence of a regulatory region preceding the ilvB structural gene has been determined. This DNA sequence includes a promoter, a region which codes for a putative 32 amino acid polypeptide containing multiple valine and leucine codons, and a transcription termination site. In vitro transcription of this region produces a 184 nucleotide terminated leader transcript. Mutually exclusive secondary structures of the leader transcript are predicted. On the basis of these data, a model for multivalent attenuation of the ilvB operon is presented. Data are presented which suggests that at least part of the postulated CRP-cyclic AMP binding site of the ilvB operon precedes the transcription start site by more than 71 base pairs.
Asunto(s)
Clonación Molecular , Escherichia coli/genética , Genes Bacterianos , Genes , Aminoácidos/metabolismo , Secuencia de Bases , Codón/genética , Genes Reguladores , Conformación de Ácido Nucleico , Plásmidos , Transcripción GenéticaRESUMEN
The human homeobox-containing gene Hox 2.1 (hHox 2.1) and its murine cognate mHox 2.1 are part of evolutionarily conserved gene clusters, encode an identical Antennapedia-type homeodomain, and are expressed in a similar pattern in the developing embryo. We have isolated cDNA clones of hHox 2.1 and found that the human/murine Hox 2.1 gene structure is strikingly conserved, with only 3 out of 269 amino acid differences in the entire predicted protein sequence. We show that purified hHox 2.1 protein is a sequence-specific DNA binding protein capable of binding to a variety of DNA sequences, including multiple sites in the promoter of the hHox 2.1 gene. In a footprint titration assay, the apparent affinity of the hHox 2.1 protein for a consensus binding site (LP) increases when the site is present in tandem copies. Quantitative footprint challenge experiments revealed that the in vitro half-life of the protein-DNA complex is less than 30 s for a single LP binding site (kd greater than 1.4 min-1), but 126 min for proteins bound to two tandem LP binding sites (kd = 5.5 x 10(-3) min-1). A domain distinct from the homeodomain is necessary for cooperative DNA binding, because a 61-amino-acid peptide containing only the Hox 2.1 homeodomain can specifically bind to LP sites, but exhibits no cooperativity. A different full-length human homeodomain protein, hHox 1.3, was also found to show cooperative DNA binding quantitatively similar to hHox 2.1. Therefore, cooperative DNA binding to adjacent sites may be a crucial component in the overall affinity of mammalian Antennapedia-type homeodomain proteins for their DNA target sites.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Proteínas de Homeodominio , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , ADN/genética , Proteínas de Unión al ADN/genética , Genes Homeobox , Humanos , Ratones , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Especificidad de la EspecieRESUMEN
Three acetohydroxy acid synthase isozymes, AHAS I (ilvBN), AHAS II (ilvGM) and AHAS III (ilvIH) catalyze the first step of the parallel isoleucine-valine biosynthetic pathway in Escherichia coli. Previous DNA sequence and protein purification data have shown that AHAS II and AHAS III are composed of large and small subunits encoded in the ilvGMEDA and ilvIH operons, respectively. Recent protein purification and characterization data have demonstrated that the AHAS I isozyme is also composed of large and small subunits (L. Eoyang, L. and P. M. Silverman [1984] J. Bacteriol. 157:184-189). Now the complete DNA sequence of the operon encoding the AHAS I isozyme has been determined. These data show that both AHAS I subunits (Mr 60,400 and Mr 11,100) are encoded in this operon. The coordinant regulation of both genes of the ilvBN operon has also been demonstrated. Comparisons of the DNA sequences of the genes encoding all three AHAS isozymes have been performed. Conserved homologies were observed between both the large and small subunits of all three isozymes. The closest homology was seen between the AHAS I and AHAS II isozymes. On the basis of these comparisons a rationale for the evolution of the AHAS isozymes in E. coli has been proposed.