RESUMEN
Chinese hamster ovary (CHO) DHFR- cells were converted into the DHFR+ phenotype when they were transfected with a mammalian expression vector carrying human dihydrofolate reductase-encoding cDNAs (DHFR) containing a Ser31 or a Ser34 mutation. Furthermore, transfection of these mutants into wild-type CHO cells resulted in resistance to high levels of methotrexate (MTX), indicating that these human variants can act as dominant selectable markers. Southern blot analysis and polymerase chain reaction amplifications confirmed that the transfected plasmids were integrated into the CHO DNA. Gene copy number analysis revealed that both the Ser3 1 and the Ser3.4 mutants amplifiable when grown in increasing concentrations of MTX. Retrovirus-mediated gene transfer of the Ser31 mutant into mouse marrow progenitor cells also resulted in MTX-resistant CFU-GM (colony-forming unit-granulocyte macrophage) cells.
Asunto(s)
Médula Ósea/metabolismo , ADN Complementario/genética , Metotrexato/metabolismo , Serina/genética , Células Madre/metabolismo , Tetrahidrofolato Deshidrogenasa/genética , Animales , Arginina/genética , Secuencia de Bases , Northern Blotting , Southern Blotting , Células CHO , Ensayo de Unidades Formadoras de Colonias , Cricetinae , Resistencia a Medicamentos/genética , Amplificación de Genes , Técnicas de Transferencia de Gen , Marcadores Genéticos , Humanos , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida/genética , Fenotipo , Plásmidos/genética , Retroviridae/genética , Transfección/genéticaAsunto(s)
Regulación Enzimológica de la Expresión Génica , Biosíntesis de Proteínas , Tetrahidrofolato Deshidrogenasa/biosíntesis , Animales , Secuencia de Bases , Neoplasias de la Mama , Células CHO , Línea Celular , Sistema Libre de Células , Cricetinae , Cartilla de ADN , ADN Complementario/metabolismo , ADN de Neoplasias/aislamiento & purificación , ADN de Neoplasias/metabolismo , Femenino , Antagonistas del Ácido Fólico , Humanos , Metotrexato/farmacología , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismo , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Reticulocitos/metabolismo , Tetrahidrofolato Deshidrogenasa/metabolismo , Transcripción Genética , Transfección , Células Tumorales CultivadasRESUMEN
The 31P nuclear magnetic resonance anisotropies of dispersions of diacylphosphatidic acid and diacylphosphatidylserine were slightly increased in the presence of cytochrome c: no increase was observed with cardiolipin. However, the 31P spin-lattice relaxation times (T1) for all of these lipids were reduced markedly by the protein. As similar effects were observed with ferri-cytochrome c and with the reduced protein, which is diamagnetic, we suggest that the changes in T1 reflect a reduction in the spectral density of fast motions for the lipid headgroups attendant on binding of protein, rather than paramagnetic relaxation of the phosphorus nuclear spin.