Asunto(s)
Drosophila/enzimología , Exorribonucleasas/metabolismo , Animales , Secuencia de Bases , Cromatografía en Gel , Clonación Molecular , Cartilla de ADN , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Exorribonucleasas/genética , Exorribonucleasas/aislamiento & purificación , Ácidos Nucleicos/metabolismo , Unión ProteicaRESUMEN
We have identified and characterized a Drosophila orthologue of SKI2, which, in Saccharomyces cerevisiae, is one of the key components in the cytoplasmic 3'-5' decay of mRNA. The Drosophila orthologue (twister, tst), is expressed as two transcripts which differ in the lengths of their 3'-UTRs, with the smaller transcript being particularly abundant in 0-2 h embryos and the larger transcript reaching its highest levels in 6-8 h embryos. TST protein is expressed in two forms which are differentially expressed in adult tissues and throughout development. Differential expression of TST may modulate activity of the mRNA turnover pathway and could have a major impact on the expression of target RNAs.
Asunto(s)
ADN Helicasas , Proteínas de Drosophila , Drosophila/genética , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Insectos/genética , Proteínas de Saccharomyces cerevisiae , Animales , Drosophila/embriología , Drosophila/crecimiento & desarrollo , Drosophila/metabolismo , Etiquetas de Secuencia Expresada , Femenino , Proteínas Fúngicas/genética , Genes de Insecto , Proteínas de Insectos/biosíntesis , Proteínas de Insectos/química , Masculino , Ovario/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Testículo/metabolismoRESUMEN
CTCF is a widely expressed and highly conserved multi-Zn-finger (ZF) nuclear factor. Binding to various CTCF target sites (CTSs) is mediated by combinatorial contributions of different ZFs. Different CTSs mediate distinct CTCF functions in transcriptional regulation, including promoter repression or activation and hormone-responsive gene silencing. In addition, the necessary and sufficient core sequences of diverse enhancer-blocking (insulator) elements, including CpG methylation-sensitive ones, have recently been pinpointed to CTSs. To determine whether a posttranslational modification may modulate CTCF functions, we studied CTCF phosphorylation. We demonstrated that most of the modifications that occur at the carboxy terminus in vivo can be reproduced in vitro with casein kinase II (CKII). Major modification sites map to four serines within the S(604)KKEDS(609)S(610)DS(612)E motif that is highly conserved in vertebrates. Specific mutations of these serines abrogate phosphorylation of CTCF in vivo and CKII-induced phosphorylation in vitro. In addition, we showed that completely preventing phosphorylation by substituting all serines within this site resulted in markedly enhanced repression of the CTS-bearing vertebrate c-myc promoters, but did not alter CTCF nuclear localization or in vitro DNA-binding characteristics assayed with c-myc CTSs. Moreover, these substitutions manifested a profound effect on negative cell growth regulation by wild-type CTCF. CKII may thus be responsible for attenuation of CTCF activity, either acting on its own or by providing the signal for phosphorylation by other kinases and for CTCF-interacting protein partners.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas Represoras , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Sitios de Unión , Factor de Unión a CCCTC , Quinasa de la Caseína II , División Celular/genética , Línea Celular , Pollos , Proteínas de Unión al ADN/genética , Genes myc , Humanos , Datos de Secuencia Molecular , Mutación , Fosforilación , Regiones Promotoras Genéticas , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Factores de Transcripción/genéticaRESUMEN
CTCF is a unique, highly conserved, and ubiquitously expressed 11 zinc finger (ZF) transcriptional factor with multiple DNA site specificities. It is able to bind to varying target sequences to perform different regulatory roles, including promoter activation or repression, creating hormone-responsive gene silencing elements, and functional block of enhancer-promoter interactions. Because different sets of ZFs are utilized to recognize different CTCF target DNA sites, each of the diverse DNA.CTCF complexes might engage different essential protein partners to define distinct functional readouts. To identify such proteins, we developed an affinity chromatography method based on matrix-immobilized purified recombinant CTCF. This approach resulted in isolation of several CTCF protein partners. One of these was identified as the multifunctional Y-box DNA/RNA-binding factor, YB-1, known to be involved in transcription, replication, and RNA processing. We examined CTCF/YB-1 interaction by reciprocal immunoprecipitation experiments with anti-CTCF and anti-YB-1 antibodies, and found that CTCF and YB-1 form complexes in vivo. We show that the bacterially expressed ZF domain of CTCF is fully sufficient to retain YB-1 in vitro. To assess possible functional significance of CTCF/YB-1 binding, we employed the very first identified by us, negatively regulated, target for CTCF (c-myc oncogene promoter) as a model in co-transfection assays with both CTCF and YB-1 expression vectors. Although expression of YB-1 alone had no effect, co-expression with CTCF resulted in a marked enhancement of CTCF-driven c-myc transcriptional repression. Thus our findings demonstrate, for the first time, the biological relevance of the CTCF/YB-1 interaction.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT , Proteínas de Unión al ADN , Proteínas Represoras , Factores de Transcripción , Secuencia de Bases , Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Factor de Unión a CCCTC , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Células HeLa , Humanos , Datos de Secuencia Molecular , Factores de Transcripción NFI , Proteínas Nucleares , Unión Proteica , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Proteína 1 de Unión a la Caja Y , Dedos de ZincRESUMEN
Immobilization of native proteins, retaining their activity, on the solid support is often crucial for a variety of biochemical assays involving protein-protein interactions. In this study we describe a technique which allows binding of both complex (protein kinase CK2) and simple (calf intestine alkaline phosphatase, CIP) enzymes to the solid support without denaturization of the proteins. This method is based on the covalent cross-linking of the enzymes to the bifunctional resin, containing the secondary amino and thiol groups, in a coupling reaction with the imidoester dimethyl pimelimidate hydrochloride. Both enzymes in their bound form were active in the specific biochemical assays. We also found that the CK2 and CIP resins did not change their activity for at least 3 months, and the quality of these resins were not affected by high salts or reducing agents. Thus, this method can be recommended for general use to generate active enzymes coupled to the solid support.
Asunto(s)
Enzimas Inmovilizadas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Quinasa de la Caseína II , Bovinos , Fosforilación , Especificidad por SustratoRESUMEN
Ebola virus (EV), an extremely infectious pathogen, causes severe hemorrhagic fever in humans and nonhuman primates. The disease pattern includes damage of parenchymal cells of vital organs in association with hemostatic and immune disorders. Vaccination with the inactivated virions does not provide an effective immune protection against the disease. The inadequate immune response may be directly caused by the virus, and, hence, it may presumably be crucial in the pathogenic process and prophylactic treatment of Ebola infection. The suggested immunosuppressive properties of EV were examined in this study. We have demonstrated that the whole heat-inactivated virions can dose-dependently suppress human lymphocyte mitogen-stimulated proliferation in vitro. In further analyses, we identified the viral protein responsible for the suppressive effect, and we showed that it was provided by a protein corresponding to a 125-kDa envelope glycoprotein (GP-125). The protein alone inhibited lymphocyte proliferation, whereas the other viral proteins were without significant effect on blastogenesis. To determine the immunosuppressive properties of different portions of GP-125, deletion mutants of GP were designed based on predicted localisation of antigen sites. They were expressed as recombinant proteins and studied in proliferation assays. We identified a 40-amino acid sequence at the N-terminus of GP-125 that exerted a suppressive effect on blastogenesis.
Asunto(s)
Ebolavirus/inmunología , Inmunosupresores/farmacología , Activación de Linfocitos/efectos de los fármacos , Linfocitos T/inmunología , Proteínas del Envoltorio Viral/farmacología , Ebolavirus/patogenicidad , Humanos , Mutación , Eliminación de Secuencia , Proteínas del Envoltorio Viral/genéticaRESUMEN
CTCF is a transcriptional repressor of the c-myc gene. Although CTCF has been characterized in some detail, there is very little information about the regulation of CTCF activity. Therefore we investigated CTCF expression and phosphorylation during induced differentiation of human myeloid leukemia cells. We found that: (i) both CTCF mRNA and protein are down-regulated during terminal differentiation in most cell lines tested; (ii) CTCF down-regulation is retarded and less pronounced than that of c-myc; (iii) CTCF protein is differentially phosphorylated and the phosphorylation profiles depend on the differentiation pathway. We concluded that CTCF expression and activity is controlled at transcriptional and post-transcriptional levels.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Eritrocitos/citología , Regulación de la Expresión Génica , Leucocitos/citología , Megacariocitos/citología , Proteínas Proto-Oncogénicas c-myc/genética , Factores de Transcripción/metabolismo , Northern Blotting , Western Blotting , Linfoma de Burkitt/patología , Factor de Unión a CCCTC , Diferenciación Celular/efectos de los fármacos , Citarabina/farmacología , Proteínas de Unión al ADN/genética , Dimetilsulfóxido/farmacología , Regulación hacia Abajo/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Focalización Isoeléctrica , Leucemia Mieloide/patología , Fosforilación/efectos de los fármacos , Procesamiento Postranscripcional del ARN/efectos de los fármacos , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Estaurosporina/farmacología , Acetato de Tetradecanoilforbol/farmacología , Factores de Transcripción/genética , Tretinoina/farmacología , Células Tumorales CultivadasRESUMEN
The effect of Ebola virus antigen on the growth of hemopoietic precursors was studied. Incubation of mononuclear cells with the viral antigen led to a dose-dependent decrease of erythroid colony formation but did not alter the growth of the granulocyto-macrophagal precursors. Hence, Ebola virus antigen is capable of directly affecting the hemopoietic activity of precursors in man by inhibiting the growth of erythroid colonies.
Asunto(s)
Antígenos Virales/inmunología , Ebolavirus/inmunología , Células Madre Hematopoyéticas/citología , Vacunas Virales/inmunología , División Celular , Ensayo de Unidades Formadoras de Colonias , Relación Dosis-Respuesta Inmunológica , Células Madre Hematopoyéticas/virología , HumanosRESUMEN
Data on the immunopathogenesis of Ebola fever in laboratory animals are presented and the efficacy of some methods of vaccine prophylaxis discussed. Antiviral immunity induced in guinea pigs by injection of inactivated viral agents did not protect them from infection, whereas injections of a nonlethal strain of the virus in ascending doses led to the formation of immunity preventing the development of disease upon inoculation with a lethal strain in high doses. The role of some viral peptides in the development of immune response is shown and variants of recombinant constructions for the prevention of Ebola fever are offered.
Asunto(s)
Fiebre Hemorrágica Ebola/prevención & control , Vacunas de Productos Inactivados/biosíntesis , Vacunas Virales/biosíntesis , Animales , Anticuerpos Antivirales/biosíntesis , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Relación Dosis-Respuesta Inmunológica , Epítopos/inmunología , Cobayas , Fiebre Hemorrágica Ebola/inmunología , Dosificación Letal Mediana , Papio , Conejos , Pase Seriado , Vacunas de Productos Inactivados/administración & dosificación , Vacunas de Productos Inactivados/inmunología , Vacunas Virales/administración & dosificación , Vacunas Virales/inmunologíaRESUMEN
Incubation of murine spleen cells with antisense oligonucleotide complementary to initiation site of this gene highly increased RNA synthesis relative to the normal T- and B-lymphocytes from spleen. In macrophages, inhibition of gene env expression stimulated phagocytosis and IL-1 production. Under these conditions, the level of expression of proviral envelope transmembrane p15E protein, which in infectious type C retroviruses is known to be immunosuppressive, decreased in spleen cells. Antisense oligonucleotide stimulatory effect on murine spleen cell RNA synthesis is presumably related to the reduced production of endogenous p15E.
Asunto(s)
Linfocitos B/inmunología , Genes env , Macrófagos/inmunología , Virus Inductores de Focos en Células del Visón/genética , Linfocitos T/inmunología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células Cultivadas , Interleucina-1/biosíntesis , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Virus Inductores de Focos en Células del Visón/inmunología , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Fagocitosis/genética , ARN Viral/biosíntesis , Bazo/citología , Bazo/inmunologíaRESUMEN
A biological function of endogenously expressed MuLV p15E-related proteins for lymphocyte and hematopoietic precursor activity in mice was examined. A high level of endogenous p15E-related peptide expression in spleen cells of mice with hemolytic anemia rendered by phenylhydrazine (PHZ) treatment was observed, detected by hyperimmune rabbit antisera against amino acid sequence which compose the immunosuppressive domain (ISD) of exogenous viral transmembrane (TM) p15E protein. The conditioned medium of these cultured cells (PHZ/CM) was inhibitory for lymphocyte blastogenesis and granulocyte-macrophage (GM) precursor activity, but stimulatory for the erythroid colony growth. When added to PHZ/CM, anti-ISD/p15E antibodies were capable to abrogate these effects. These antibodies bound 14K and 48K structural peptides contented in PHZ/CM as presumably smaller components of env gene products. Given together, the results indicate that erythroid immature cells produce proteins appearing in cell culture medium which exert p15E-related properties. These peptides are suggested to exert a down regulation for both lymphocyte and GM precursor activities, and the colony-promoting effect towards erythroid compartment cells.
Asunto(s)
Células Madre Hematopoyéticas/metabolismo , Linfocitos/metabolismo , Retroviridae , Proteínas del Envoltorio Viral/metabolismo , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Fenilhidrazinas/farmacologíaRESUMEN
We report in vivo growth of human promonocytic cells infected with HIV-1 presented in new mouse model. Cloned U937 cells chronically infected with HIV-1 were grafted in (CBA*C57B1/6)F1 mice deprived of immunity by thymectomia and total body irradiation with subsequent marrow reconstitution. Nine weeks after cell inoculation, HIV-1-positive cells were found only in mice that received an additional single dose of cyclophosphamide (100 mg/kg bw) prior to transplantation, whereas, in mice without further immune deprivation, the complete elimination of cells bearing viral antigen occurred already on the seventh day after transplantation. The approach described may be suitable for in vivo development of antiviral drugs against latent infection in macrophage-like cells which represent a serious problem in therapy of AIDS in humans.
Asunto(s)
Modelos Animales de Enfermedad , Infecciones por VIH/inmunología , Infecciones por VIH/patología , VIH-1 , Animales , División Celular , Línea Celular , Enfermedad Crónica , Femenino , Humanos , Huésped Inmunocomprometido , Linfoma de Células B Grandes Difuso/patología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBARESUMEN
A possible biologic activity of endogenously expressed env sequence of retroviral mink cell focus-forming virus (MCF) genome for hematopoietic colony formation was studied in mice. Antisense 20-mer complementary to MCF env sequence was used to detect the result of blockage of this gene translation on the potency of marrow cells to form colonies of erythroid (BFU-E), myeloid granulocyte-macrophage (CFU-GM), and stem cell (day 11 CFU-S) hematopoietic compartments. A large relative decrease in BFU-E number was found in bone marrow cell cultures preincubated with antisense oligonucleotide during 4 h, whereas CFU-GM colonies remained unaffected. A marked reduction of CFU-S colony formation was also registered under antisense oligomer influence. Following a decreased proliferation of erythroid progenitors, we suggest the mechanism by which antisense oligonucleotide could cause the loss of colony formation. Taken together, these data allow to propose that the expression of this gene is naturally significant for hematopoietic progenitor activity exerting some property of env gene products to regulate the growth of erythroid and multilineage hematopoietic precursors.
Asunto(s)
Genes env , Células Madre Hematopoyéticas/efectos de los fármacos , Virus Inductores de Focos en Células del Visón/genética , Oligonucleótidos Antisentido/farmacología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , División Celular/efectos de los fármacos , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Células Madre Hematopoyéticas/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Oligonucleótidos Antisentido/genéticaRESUMEN
The retroviral transmembrane p15E peptide is known to suppress a wide variety of immune cell functions, suggesting a role for immunosuppression associated with retroviral infection. The 10-amino acid sequence from the highly conserved portion of p15E (CKS-10) is capable of reproducing this inhibitory activity. In this study we set out to determine the influence of this decapeptide on murine spleen cell mitogen-induced proliferation and hematopoietic granulocyte-macrophage and erythroid precursor colony formation in vitro. A dose- and time-dependent suppression of spleen cell blastogenic response was produced by the CKS-10 peptide. When bone marrow cells were incubated with decapeptide, the significant decrease of CFU-GM colony number was also dose-dependent. In contrast, the same doses of CKS-10 peptide which induced a most significant inhibition of CFU-GM colony formation caused a marked increase of BFU-E colonies. A most pronounced effect of the peptide on bone marrow hematopoietic progenitor activity was produced by prolonged exposure to the peptide. Given the results of this study, it seems likely that, in addition to the cytopathic effect of retroviruses on the lymphocytes, viral peptide-mediated hematopoiesis disorders may also play an important role in the pathogenesis of immunodeficiency associated with retroviral infections.