Asunto(s)
Vectores Genéticos , Papillomaviridae/genética , Animales , Bovinos , Genotipo , Transcripción Genética , Transformación GenéticaRESUMEN
The promoters from Drosophila and human 70,000-dalton heat shock protein (hsp70) genes were linked to human tissue plasminogen activator (tPA) cDNA. Mouse C127 cells were transformed with bovine papilloma virus (BPV) vectors carrying the hybrid hsp70/tPA genes. Stable BPV-transformed cell lines were selected and analyzed for tPA expression before and after heat shock. In most cell lines, there was a low level of tPA production even in the absence of heat shock or other obvious stress. After heat shock (42 degrees C, 2 hr), there was up to a 40-fold increase in tPA production. Production of tPA protein occurred within the first 5 h after the heat shock and was due to a burst of hsp70/tPA transcription during the heat shock. The hsp70/tPA transcripts appeared to have a short half-life. Thus, stable mouse cell lines carrying hsp70/tPA hybrid genes can be induced by a short heat shock to transcribe high levels of hsp70/tPA mRNAs and, subsequently, to produce elevated levels of tPA protein.
Asunto(s)
Proteínas de Choque Térmico/genética , Regiones Promotoras Genéticas , Activador de Tejido Plasminógeno/genética , Animales , Papillomavirus Bovino 1 , Línea Celular , Transformación Celular Viral , ADN Recombinante , Regulación de la Expresión Génica , Calor , Ratones , Plásmidos , Proteínas/genética , ARN Mensajero/genética , Transcripción GenéticaRESUMEN
We have adapted the fibrin overlay assay for plasminogen activators (Jones et al., 1975) into a gene transfer expression assay which has the advantage of being very sensitive and nondestructive. In this assay plasminogen activators convert plasminogen to plasmin, which then degrades fibrin, resulting in clearings in a fibrin overlay. Furthermore, the assay can be used as a signal indicating the efficiency of gene transfer or the loss of introduced genetic elements in unstable cell lines.
Asunto(s)
Genes , Activadores Plasminogénicos/genética , Transfección , Acetiltransferasas/genética , Virus del Sarcoma Aviar/genética , Secuencia de Bases , Cloranfenicol O-Acetiltransferasa , ADN/metabolismo , Fibrina/metabolismo , Humanos , Plásmidos , Activadores Plasminogénicos/metabolismo , Regiones Promotoras Genéticas , Virus 40 de los Simios/genéticaRESUMEN
HeLa cells were transfected with recombinant DNAs containing the embryonic histone gene repeat of P.miliaris (h22) inserted in either orientation into a pBR-SV40 vector. After 2 to 3 days cytoplasmic RNA was analyzed for authentic sea urchin histone gene transcripts. The correct 5' termini of all five histone genes were detected, three (H2B, H2A and H3) at relatively high levels. In contrast, termination was largely aberrant with the correct 3' terminus of only the H2B mRNA found in significant amounts. The levels of histone gene transcription were dependent on the presence, but not the orientation, of SV40 DNA in the recombinant plasmid. The efficiency of initiation of transcription of the individual sea urchin histone genes in HeLa cells was very similar to that previously observed in Xenopus oocytes. The termination pattern, however, was quite different in that oocytes, all but the H3 gene terminate efficiently. The idiosyncrasies in termination efficiencies for these two heterologous transcription systems may reflect the presence of termination factors which are relatively species or even tissue specific and only some of which recognize the sea urchin "terminators" correctly.
Asunto(s)
ADN Recombinante/metabolismo , Genes , Histonas/genética , Erizos de Mar/genética , Transcripción Genética , Animales , Enzimas de Restricción del ADN , Células HeLa/metabolismo , Humanos , Hibridación de Ácido Nucleico , Plásmidos , Secuencias Repetitivas de Ácidos NucleicosAsunto(s)
Regulación de la Expresión Génica , Genes , Histonas/genética , Operón , Animales , Secuencia de Bases , Cilióforos/genética , ADN Recombinante , ADN Ribosómico , Drosophila/genética , Embrión no Mamífero/metabolismo , Humanos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , Transcripción Genética , Vertebrados/genética , Levaduras/genéticaAsunto(s)
Dexametasona/farmacología , Estradiol/farmacología , Lipoproteínas/biosíntesis , Hígado/metabolismo , Biosíntesis de Proteínas/efectos de los fármacos , Albúmina Sérica/biosíntesis , Vitelogeninas/biosíntesis , Animales , Células Cultivadas , Femenino , Técnica del Anticuerpo Fluorescente , Hígado/citología , Hígado/efectos de los fármacos , Masculino , XenopusAsunto(s)
ARN Polimerasas Dirigidas por ADN/sangre , Eritroblastos/enzimología , Eritrocitos/enzimología , Eritropoyesis , Animales , Núcleo Celular/efectos de los fármacos , Núcleo Celular/enzimología , Femenino , ARN/biosíntesis , ARN Polimerasa II/metabolismo , Tensoactivos/farmacología , Transcripción Genética , XenopusRESUMEN
A method of isolating circular plasmid DNA from cleared lysates of E. coli is described. Purification is achieved by virtue of the rapid re-annealing kinetics or supercoiled DNA. After a brief denaturation step, double stranded plasmid DNA is separated from denatured chromosomal DNA and RNA in a two-phase partition system using dextran and polyethylene glycol. The method is much more rapid than the conventional dye-centrifugation technique and plasmid DNA of comparable purity and yield is obtained.