RESUMEN
DNA replication origins are located at random with respect to DNA sequence in Xenopus early embryos and on DNA replicated in Xenopus egg extracts. We have recently shown that origins fire throughout the S phase in Xenopus egg extracts. To study the temporal regulation of origin firing, we have analyzed origin activation in sperm nuclei treated with the DNA polymerase inhibitor aphidicolin. Sperm chromatin was incubated in Xenopus egg extracts in the presence of aphidicolin and transferred to a fresh extract, and digoxigenin-dUTP and biotin-dUTP were added at various times after aphidicolin release to selectively label early and late replicating DNA. Molecular combing analysis of single DNA fibers showed that only a fraction of potential origins were able to initiate in the presence of aphidicolin. After release from aphidicolin, the remaining origins fired asynchronously throughout the S phase. Therefore, initiation during the S phase depends on the normal progression of replication forks assembled at earlier activated origins. Caffeine, an inhibitor of the checkpoint kinases ATR and ATM, did not relieve the aphidicolin-induced block to origin firing. We conclude that a caffeine-insensitive intra-S phase checkpoint regulates origin activation when DNA synthesis is inhibited in Xenopus egg extracts.
Asunto(s)
Afidicolina/farmacología , Núcleo Celular/fisiología , Cromatina/fisiología , Replicación del ADN/fisiología , Oocitos/fisiología , Origen de Réplica/fisiología , Espermatozoides/fisiología , Animales , Cafeína/farmacología , Núcleo Celular/efectos de los fármacos , Núcleo Celular/ultraestructura , Replicación del ADN/efectos de los fármacos , Femenino , Cinética , Masculino , Inhibidores de la Síntesis del Ácido Nucleico , Origen de Réplica/efectos de los fármacos , Fase S , Extractos de Tejidos/metabolismo , Xenopus laevisRESUMEN
We exploit an improved mammalian cell-free DNA replication system to analyse quiescence and Cdc6 function. Quiescent 3T3 nuclei cannot initiate replication in S phase cytosol from HeLa or 3T3 cells. Following release from quiescence, nuclei become competent to initiate semiconservative DNA replication in S phase cytosol, but not in G0 phase cytosol. Immunoblots show that quiescent cells lack Cdc6 and that minichromosome maintenance (MCM) proteins are not associated with chromatin. Competence of G1 phase nuclei to replicate in vitro coincides with maximum Cdc6 accumulation and MCM protein binding to chromatin in vivo. Addition of recombinant Cdc6 to permeabilized, but not intact, G1 nuclei causes up to 82% of the nuclei to initiate and accelerates G1 progression, making nuclei competent to replicate prematurely.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Fase S/fisiología , Proteínas de Saccharomyces cerevisiae , Células 3T3 , Animales , Sistema Libre de Células , Cromatina/metabolismo , Citosol/metabolismo , Replicación del ADN , Fase G1 , Células HeLa , Humanos , Ratones , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Permeabilidad , Unión Proteica , Fase de Descanso del Ciclo CelularRESUMEN
The subcellular distribution and posttranslational modification of human chromatin assembly factor 1 (CAF-1) have been investigated after UV irradiation of HeLa cells. In an asynchronous cell population only a subfraction of the two large CAF-1 subunits, p150 and p60, were found to exist in a chromatin-associated fraction. This fraction is most abundant during S phase in nonirradiated cells and is much reduced in G2 cells. After UV irradiation, the chromatin-associated form of CAF-1 dramatically increased in all cells irrespective of their position in the cell cycle. Such chromatin recruitment resembles that seen for PCNA, a DNA replication and repair factor. The chromatin-associated fraction of p60 was predominantly hypophosphorylated in nonirradiated G2 cells. UV irradiation resulted in the rapid recruitment to chromatin of phosphorylated forms of the p60 subunit. Furthermore, the amount of the p60 and p150 subunits of CAF-1 associated with chromatin was a function of the dose of UV irradiation. Consistent with these in vivo observations, we found that the amount of CAF-1 required to stimulate nucleosome assembly during the repair of UV photoproducts in vitro depended upon both the number of lesions and the phosphorylation state of CAF-1. The recruitment of CAF-1 to chromatin in response to UV irradiation of human cells described here supports a physiological role for CAF-1 in linking chromatin assembly to DNA repair.
Asunto(s)
Cromatina/metabolismo , Proteínas Cromosómicas no Histona , Proteínas de Unión al ADN/metabolismo , Rayos Ultravioleta , Animales , Fraccionamiento Celular , Núcleo Celular/metabolismo , Cromatina/efectos de la radiación , Factor 1 de Ensamblaje de la Cromatina , Proteínas de Unión al ADN/fisiología , Proteínas de Unión al ADN/efectos de la radiación , Fase G2 , Células HeLa , Humanos , Octoxinol , Fosforilación , Antígeno Nuclear de Célula en Proliferación/fisiología , Conejos , Factores de TranscripciónRESUMEN
1. Two eukaryotic viral systems, the baculovirus/insect cell and the Semliki Forest virus systems, were tested for heterologous expression of human gonadotropin-releasing hormone receptor (GnRHR) cDNA. 2. An unmodified as well as a c-myc epitope-tagged human GnRH receptor was produced in two insect cell lines (Spodoptera frugiperda, Trichoplusia ni) after infection with the respective recombinant baculoviruses. In both insect cell lines, the receptor was identified by immunoblot analysis as a triplet of bands between 35 and 40 kDa. After deglycosylation of the receptor the molecular mass decreased to 35 kDa. The GnRH receptor was localized in membrane compartments within the infected insect cells. However, only in membranes of infected Trichoplusia ni insect cells could approximately 2000 receptors per cell be detected. 3. Production of the GnRH receptor in BHK cells using the Semliki Forest virus system resulted in approximately 50,000 receptors per cell. A maximal yield of 0.42 pmol/mg membrane protein was obtained 24 hr after electroporation of BHK cells with in vitro synthesized RNA. Binding of the antagonist [125I]Cetrorelix was saturable with a KD of 1.3 nM. The receptor produced in the BHK cells was further characterized by ligand displacement studies. The rank order of agonist and antagonist affinities was Cetrorelix > Triptorelin > Antide > GnRH.
Asunto(s)
Vectores Genéticos/genética , Nucleopoliedrovirus/genética , Receptores LHRH/biosíntesis , Proteínas Recombinantes de Fusión/biosíntesis , Virus de los Bosques Semliki/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Clonación Molecular , Cricetinae , ADN Complementario/genética , Hormona Liberadora de Gonadotropina/análogos & derivados , Hormona Liberadora de Gonadotropina/metabolismo , Humanos , Inmunohistoquímica , Mesocricetus , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Mariposas Nocturnas/citología , Hipófisis/química , Receptores LHRH/genética , Receptores LHRH/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Spodoptera/citologíaRESUMEN
We characterized changes of nucleosome assembly activity, intracellular localization, and reversible phosphorylation of the human chromatin assembly factor CAF-1 during the somatic cell division cycle. HeLa cells were synchronized in the G1, S, G2, and M phases of the cell cycle. All three subunits of human CAF-1 (p150, p60, and p48) are present during the entire cell cycle. In interphase, p150 and p60 are bound to the nucleus, but they predominantly dissociate from chromatin during mitosis. During S phase, p150 and p60 are concentrated at sites of intranuclear DNA replication. Only a fraction of total p48 is associated with p150 and p60, and the majority is present in other high molecular weight complexes. The other nucleosome assembly protein, NAP-1, is predominantly cytosolic throughout the cell cycle. Human CAF-1 efficiently mediates nucleosome assembly during complementary DNA strand synthesis in G1, S, and G2 phase cytosolic extracts. Active CAF-1 can be isolated as a 6.5 S complex from G1, S, and G2 phase nuclei. In contrast, CAF-1 isolated from mitotic cytosol does not support nucleosome assembly during DNA synthesis. In mitosis, the p60 subunit of inactive CAF-1 is hyperphosphorylated, whereas active CAF-1 in interphase contains hypophosphorylated and/or phosphorylated forms of p60.
Asunto(s)
Ciclo Celular/fisiología , Proteínas Cromosómicas no Histona , Proteínas de Unión al ADN/química , Nucleosomas/metabolismo , Proteínas de Ciclo Celular , Cromatina/metabolismo , Factor 1 de Ensamblaje de la Cromatina , Replicación del ADN/fisiología , Citometría de Flujo , Células HeLa , Humanos , Nucleasa Microcócica/metabolismo , Mitosis/fisiología , Proteínas Nucleares/análisis , Proteína 1 de Ensamblaje de Nucleosomas , Fosforilación , Proteínas/metabolismo , Factores de TranscripciónRESUMEN
1. cDNA of the human dopamine transporter (hDAT) was cloned into a cloning vector based on the Semliki Forest virus. Electroporation of in vitro transcribed mRNA from this plasmid into BHK-21 cells resulted in production of the transporter as measured by [3H]dopamine uptake (Km = 2.0 +/- 0.4 microM), which was specifically inhibited in the presence of cocaine. 2. The recombinant transporter protein exhibited an apparent molecular mass of 56 kDa, which was reduced to 50 kDa after tunicamycin treatment of the producing BHK-21 cells. Tunicamycin treatment of the electroporated cells also resulted in a decrease in transport activity with no change in the Km value (2.1 +/- 0.4 microM). 3. The localization of the heterologously produced transporter in the BHK cells either with or without tunicamycin treatment was studied by electron microscopic immunogold staining. The glycosylated transporter was found to be localized at the plasma membrane, whereas in the case of the unglycosylated transporter, transport to the plasma membrane was blocked.
Asunto(s)
Proteínas Portadoras/biosíntesis , Proteínas Portadoras/química , Dopamina/metabolismo , Glicoproteínas de Membrana , Proteínas de Transporte de Membrana , Proteínas del Tejido Nervioso , Animales , Proteínas Portadoras/genética , Línea Celular , Cricetinae , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática , Fibroblastos/química , Fibroblastos/metabolismo , Humanos , Plásmidos/química , Plásmidos/metabolismo , Pruebas de Precipitina , Virus de los Bosques Semliki/genética , TransfecciónRESUMEN
The lipid composition of two different insect cell lines from Spodoptera frugiperda (Sf9) and Trichoplusia ni (Tn) which are established cell lines for infection with recombinant baculovirus was analyzed by high-performance liquid chromatography and gas-liquid chromatography. The major phospholipids found were phosphatidylcholine and phosphatidylethanolamine, the major mono-unsaturated fatty acids were oleic acid and palmitoleic acid, the major saturated fatty acid was stearic acid. The cholesterol to phospholipid ratio was demonstrated to be lower than in mammalian cell lines. Infection with a recombinant baculovirus Autographa californica resulted in increased levels of phosphatidylcholine in the insect cells. The baculovirus/insect cell system has become a popular system for heterologous protein production. Functional changes of membrane proteins produced in these two cell lines might be correlated to a different lipid profile of their cellular membranes.
Asunto(s)
Baculoviridae , Insectos/química , Lípidos/análisis , Spodoptera/química , Animales , Línea Celular/química , Cromatografía de Gases , Cromatografía Líquida de Alta Presión , Fosfolípidos/análisis , Transfección/métodosRESUMEN
The cGMP-gated cation channel is responsible for the last step in the vertebrate phototransduction cascade which couples light activation of rhodopsin to a change in membrane permeability. Two different expression systems, baculovirus-infected Spodoptera frugiperda (Sf9) insect cells and Saccharomyces cerevisiae, were used for the overproduction of the cGMP-gated cation channel subunit 1 of bovine rod cells. Presence of recombinant channel protein was monitored by SDS-PAGE and Western-blot analysis. Through immunogold labeling, the heterologously expressed cation channel was found to be localized predominantly in the inner compartments of the infected insect cells and in the vacuole of recombinant yeast cells.
Asunto(s)
Proteínas del Ojo/biosíntesis , Canales Iónicos/biosíntesis , Células Fotorreceptoras Retinianas Bastones/metabolismo , Animales , Baculoviridae , Secuencia de Bases , Western Blotting , Bovinos , Línea Celular , Clonación Molecular , Canales Catiónicos Regulados por Nucleótidos Cíclicos , Cartilla de ADN , Electroforesis en Gel de Poliacrilamida , Proteínas del Ojo/análisis , Canales Iónicos/análisis , Sustancias Macromoleculares , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Peso Molecular , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/análisis , Proteínas Recombinantes/biosíntesis , Mapeo Restrictivo , Rodopsina/metabolismo , Saccharomyces cerevisiae , Spodoptera , TransfecciónRESUMEN
The cDNA encoding the receptor for gonadoliberin (GnRH or LH-RH) was isolated from a human pituitary cDNA library and heterologously expressed in the murine fibroblast cell line LTK-. By using a dicistronic expression strategy utilizing the internal ribosomal-entry-site sequence of poliovirus, single cell clones with stable and high expression of human gonadoliberin receptors were selected. In radioligand saturation-binding experiments, the gonadoliberin antagonist Cetrorelix showed high-affinity binding to the heterologously expressed human gonadoliberin receptor with a Kd of 0.1 nM. The pharmacological profile using 125I-Cetrorelix as radioligand and the authentic gonadoliberin or agonistic and antagonistic derivatives as competitors, showed a distinct rank order of binding potencies. Superagonistic gonadoliberin derivatives had more than ten-times higher binding affinities in comparison to gonadoliberin with a Kd of 3.47 nM. The gonadoliberin receptor expressed in stably transfected LTK- cells coupled to the inositol phosphate signal-transduction pathway. Gonadoliberin stimulated the synthesis of inositol 1,4,5-trisphosphate in a dose-dependent way with an EC50 of 5 nM. This stimulatory effect of gonadoliberin was completely antagonized by Cetrorelix in equimolar concentrations, demonstrating the high potency of this competitive receptor antagonist. In growth-arrested cells, a transient expression of the c-fos protooncogene was induced by gonadoliberin or [D-Trp6]gonadoliberin, showing that the gonadoliberin receptor couples to a putative mitogenic signal-transduction pathway in this heterologous cell system.