RESUMO
Many heterologous genes when cloned into bacterial expression vectors are poorly expressed because of an inefficient ribosome binding site (RBS). We have constructed a plasmid which expresses human gamma-interferon (gamma-IF), where the level of expression is limited by the RBS. Expression was increased by placing the gamma-IF sequence immediately downstream of a small translated sequence. The production of gamma-IF was dependent upon the efficiency of translation of this upstream cistron and could be increased to very high levels. The same upstream cistron would greatly improve the expression of gamma-IF in a plasmid where the RBS was very poor due to inhibitory secondary structure at the 5' end of its mRNA. However, it would not improve the efficiency of a poor RBS containing a weak Shine-Dalgarno sequence. The general utility of the two-cistron expression strategy to diagnose a weak RBS is discussed.
Assuntos
Clonagem Molecular/métodos , Escherichia coli/genética , Expressão Gênica , Genes , Interferon gama/genética , Sequência de Aminoácidos , Sequência de Bases , Humanos , Interferon gama/isolamento & purificação , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Plasmídeos , RNA Mensageiro/genética , Proteínas Recombinantes , Mapeamento por Restrição , Homologia de Sequência do Ácido NucleicoRESUMO
The glutathione peroxidase activity of blood, liver and brain of sexually mature male and female rats has been determined using both hydrogen peroxide and cumene hydroperoxide as substrates. No significant differences were found on comparison of the glutathione peroxidase activity of the brain tissues of male and female animals. Observed mathematically significant differences between the enzyme activity in the blood of male and females were probably of little physiological significance. Both selenium and non-selenium dependent glutathione peroxidase activities were significantly higher in the livers of female rats but this was much more apparent when cumene hydroperoxide was reduced. Castrated male rats had significantly higher hepatic enzyme activities, approaching those of the female animals. The lipoperoxide concentration was significantly higher in the livers of female rats.
Assuntos
Glutationa Peroxidase/metabolismo , Animais , Encéfalo/enzimologia , Castração , Feminino , Glutationa Peroxidase/sangue , Peróxidos Lipídicos/metabolismo , Fígado/enzimologia , Masculino , Ratos , Fatores SexuaisRESUMO
Rats of various ages were subjected to stress by confinement in restraining cages at 2-4 degrees C. Analysis of the plasma of these animals revealed an elevation in corticosteroids of approximately 50% above the control level. The livers of all the groups of cold-restrained animals contained significantly more lipoperoxide (estimated as thiobarbituric-acid-reactive material) than did control hepatic tissue. The plasma of the 12-, 24-, and 32-wk-old groups of rats subjected to stressful treatment also contained significantly higher lipoperoxide levels. There was no significant difference between the lipoperoxide levels of the brain tissue of control or stress-treated rats. The activities of both glutathione peroxidase and glutathione reductase were increased in hepatic, but not brain, tissue of the stressed animals. The perturbation of the activities of these enzymes did not produce any significant change in the ratio of reduced, oxidized glutathione. The livers of the stressed animals had significantly less total glutathione than those of the controls.
