RESUMEN
The integrase encoded by the lambdoid phage HK022 (Int-HK022) resembles its coliphage λ counterpart (Int-λ) in the roles of the cognate DNA arm binding sites and in controlling the direction of the reaction. We show here that within mammalian cells, Int-HK022 does not exhibit such a control. Rather, Int-HK022 recombined between all ten possible pairwise att site combinations, including attB × attB that was more effective than the conventional integrative attP × attB reaction. We further show that Int-HK022 depends on the accessory integration host factor (IHF) protein considerably less than Int-λ and exhibits stronger binding affinity to the att core. These differences explain why wild-type Int-HK022 is active in mammalian cells whereas Int-λ is active there only as an IHF-independent mutant.
Asunto(s)
Bacteriófago HK022/enzimología , Bacteriófago HK022/genética , Integrasas/genética , Bacteriófago lambda/enzimología , Bacteriófago lambda/genética , Escherichia coli/virología , Humanos , Recombinación Genética , Proteínas Virales/genética , Integración ViralRESUMEN
It has been previously demonstrated that the wild type integrase (Int) protein of coliphage HK022 can catalyze site-specific recombination in human cells between attachment (att) sites that were placed on extrachromosomal plasmids. In the present report it is shown that Int can catalyze the site-specific recombination reactions in a human cell culture on the chromosomal level. These include integrative (attP x attB) as well as excisive (attL x attR) reactions each in two configurations. In the cis configuration both sites are on the same chromosome, in the trans configuration one site is on a chromosome and the other on an episome. The reactions in cis were observed without any selection force, using the green fluorescent protein (GFP) as a reporter. The reactions in trans could be detected only when a selection force was applied, using the hygromycin-resistant (Hyg(R)) phenotype as a selective marker. All reactions were catalyzed without the need to supply any of the accessory proteins that are required by Int in its Escherichia coli host. The versatility of the att sites may be an advantage in the utilization of Int to integrate plasmid DNA into the genome, followed by a partial exclusion of the integrated plasmid.
Asunto(s)
Bacteriófago HK022/enzimología , Genoma Humano/genética , Integrasas/metabolismo , Recombinación Genética/genética , Southern Blotting , Línea Celular , ADN/genética , ADN/metabolismo , Humanos , Modelos Genéticos , Plásmidos/genéticaRESUMEN
Glutathione synthesis and growth properties were studied in the gamma-glutamyl transpeptidase(GGT)-negative, non-tumorigenic rat liver oval cell line OC/CDE22, and in its GGT-positive, tumorigenic counterpart line M22. gamma-Glutamylcysteine synthetase (GGCS) activities were comparable. Growth rates of M22 cells exceeded those of OC/CDE22 cells at non-limiting and limiting exogenous cysteine concentrations. A monoclonal antibody (Ab 5F10) that inhibits the transpeptidatic but not the hydrolytic activity of GGT did not affect the growth rates of OC/CDE22, and decreased those of M22 to the OC/CDE22 level. In GSH-depleted M22, but not in OC/CDE22 cells, the rate and extent of GSH repletion with exogenous cysteine and glutamine exceeded those obtained with exogenous cysteine and glutamate. With Ab 5F10, repletion with cysteine/glutamine was similar to that obtained with cysteine/glutamate. Repletion with exogenous GSH occurred only in M22 cells, and was abolished by the GGT inhibitor acivicin. Repletion with gamma-glutamylcysteine (GGC) in OC/CDE22 was resistant to acivicin whereas that in M22 was inhibited by acivicin. Repletion with exogenous GSH or cysteinylglycine (CG) required aminopeptidase activity and was lower than that obtained with cysteine. Unless reduced, CG disulfide did not support GSH repletion. The findings are compatible with the notions that (i) GGT-catalyzed transpeptidation was largely responsible for the growth advantage of M22 cells at limiting cysteine concentration, and for their high GSH content via the formation of GGC from a gamma-glutamyl donor (glutamine) and cyst(e)ine, and (ii) aminopeptidase/dipeptidase activity is rate-limiting in GSH repletion when GSH or CG serve as cysteine sources.
Asunto(s)
Glutatión/biosíntesis , Hígado/citología , gamma-Glutamiltransferasa/metabolismo , Aminopeptidasas/antagonistas & inhibidores , Aminopeptidasas/metabolismo , Animales , División Celular/efectos de los fármacos , Línea Celular , Cistina/metabolismo , Inhibidores Enzimáticos/farmacología , Ácido Glutámico/metabolismo , Glutamina/metabolismo , Glutatión/metabolismo , Glutatión/farmacología , Cinética , Hígado/efectos de los fármacos , Hígado/enzimología , Ratas , Células Tumorales Cultivadas , gamma-Glutamiltransferasa/antagonistas & inhibidoresRESUMEN
An assay of gamma-glutamylcysteine synthetase (gamma-GCS) and glutathione synthetase (GS) in crude extracts of cultured cells and tissues is described. It represents a novel combination of known methods, and is based on the formation of glutathione (GSH) from cysteine, glutamate and glycine in the presence of rat kidney GS for the assay of gamma-GCS, or from gamma-glutamylcysteine and glycine for the assay of GS. GSH is then quantified by the Tietze recycling method. Assay mixtures contain the gamma-glutamyl transpeptidase (GGT) inhibitor acivicin in order to prevent the degradation of gamma-glutamylcysteine and of the accumulating GSH, and dithiothreitol in order to prevent the oxidation of cysteine and gamma-glutamylcysteine. gamma-GCS and GS levels determined by this method are comparable to those determined by others. The method is suitable for the rapid determination of gamma-GCS GS in GGT-containing tissues and for the studies of induction of gamma-GCS and GS in tissue cultures.