RESUMEN
Nuclei were isolated from synchronized HeLa cells in the S-phase by a modification of the non-aqueous method described by Kirsch et al. (Science (1970) 168, 1592-1595). The method involved lyophilization of the cells, homogenization in non-aqueous glycerol and centrifugation in a gradient of 0-35% (w/w) 3-chloro-1,2-propanediol in glycerol. Such nucleic incorporated deoxyribonucleotides into DNA when incubated in an aqueous buffer containing Mg2+, ATP, dATP, dGTP, dCTP and dTTP. The product was sensitive to DNAase and banded with bulk DNA in isopycnic centrifugation. Sedimentation of the product in alkaline sucrose gradients after labelling of the nuclei for 2 min revealed labelled material in the 5 S peak and in the 18 S area. The material in the 5 S peak moved into the 12 S area after a 13 min chase.
Asunto(s)
Núcleo Celular/metabolismo , Replicación del ADN , ADN de Neoplasias/biosíntesis , Células HeLa/metabolismo , Fraccionamiento Celular , Núcleo Celular/efectos de los fármacos , Núcleo Celular/ultraestructura , Centrifugación por Gradiente de Densidad , Reductasas del Citocromo/metabolismo , Replicación del ADN/efectos de los fármacos , Desoxirribonucleasas , Ditiotreitol/farmacología , Etilmaleimida/farmacología , Glicerol , Humanos , Magnesio/farmacología , Microscopía Electrónica , Mitosis , NADH NADPH Oxidorreductasas/metabolismo , Timidina/metabolismo , Nucleótidos de Timina/metabolismo , Factores de TiempoAsunto(s)
Enzimas , Escherichia coli/enzimología , Transferasas/metabolismo , Adenosina Trifosfato/metabolismo , Ácido Aspártico , Cadmio , Fenómenos Químicos , Química , Cobalto , Cobre , Nucleótidos de Citosina , Magnesio , Manganeso , Níquel , ZincAsunto(s)
Globinas/biosíntesis , Reticulocitos/metabolismo , Ribosomas/metabolismo , Animales , Isótopos de Carbono , Centrifugación por Gradiente de Densidad , Cromatografía por Intercambio Iónico , Globinas/aislamiento & purificación , Leucina/metabolismo , Ratones , Cloruro de Potasio , ARN Mensajero/farmacología , Conejos , Reticulocitos/citología , Reticulocitos/efectos de los fármacos , Ribosomas/efectos de los fármacos , TritioAsunto(s)
Hígado/metabolismo , ARN/metabolismo , Ribosomas/metabolismo , Animales , Unión Competitiva , Isótopos de Carbono , Núcleo Celular , Centrifugación por Gradiente de Densidad , Precipitación Química , Ácido Edético/farmacología , Etanol , Formaldehído/farmacología , Hígado/citología , Hígado/efectos de los fármacos , Poli U/farmacología , Polirribosomas/efectos de los fármacos , Polirribosomas/metabolismo , Puromicina/farmacología , ARN/aislamiento & purificación , ARN Ribosómico/farmacología , Ratas , Ribosomas/efectos de los fármacos , TritioAsunto(s)
Carcinógenos/farmacología , Hígado/efectos de los fármacos , Hígado/enzimología , Ácidos Nucleicos/metabolismo , Animales , Ácido Aspártico/metabolismo , Dieta , Femenino , Hígado/patología , Neoplasias Hepáticas/enzimología , N-Glicosil Hidrolasas/análisis , Nucleotidasas/análisis , Monoéster Fosfórico Hidrolasas/análisis , Fosfotransferasas/análisis , Lesiones Precancerosas/enzimología , Ratas , Transferasas/análisisRESUMEN
1. An improved 11000g cell-free system for the incorporation of [(14)C]valine into gramicidin S has been obtained. The cell-free extract used was the supernatant obtained by treating Bacillus brevis with ultrasonics for 1min. followed by centrifugation at 11000g. The optimum pH for the incorporation was 8.2-8.4 and the optimum Mg(2+) concentration 0.05m. The presence of ammonium sulphate (0.1m) and K(+) (0.01m) increased the incorporation. 2. Cell-free extracts prepared from cells harvested in the early phase of growth (extinction value 0.1) incorporated negligible amounts of [(14)C]valine into gramicidin S compared with that incorporated by cell-free extracts prepared from cells harvested in the late phase of growth (extinction value 0.5). This was not due to the presence of inhibitors in the cell-free extracts prepared from cells harvested early, since there was no marked decrease in gramicidin S synthesis in a mixture of extracts prepared from cells harvested early and late in the growth phase. 3. The small incorporation of [(14)C]valine into protein, which took place in cell-free extracts from cells harvested in the late growth phase, was not inhibited by puromycin, chloramphenicol and ribonuclease. However, the substantial incorporation that took place in cell-free extracts prepared from cells harvested in the early phase of growth was completely inhibited by puromycin, chloramphenicol and ribonuclease. On mixing cell-free extracts prepared from cells harvested early and late in the growth phase, it appeared that the small incorporation that occurs in extracts from cells harvested in the late phase of growth was not due to cellular inhibitors.