RESUMO
The glutathione peroxidase homologous gene (Gpxh gene) in Chlamydomonas reinhardtii is up-regulated under oxidative stress conditions. The Gpxh gene showed a remarkably strong and fast induction by the singlet oxygen-generating photosensitizers neutral red, methylene blue and rose Bengal. The Gpxh mRNA levels strongly increased, albeit much more slowly, upon exposure to the organic hydroperoxides tert-butyl hydroperoxide (t-BOOH) and cumene hydroperoxide. In contrast, the Gpxh mRNA levels were only weakly induced by exposure to the superoxide-generating compound paraquat and by hydrogen peroxide. A comparison of the Gpxh mRNA levels with those of the heat shock protein HSP70A and the iron superoxide dismutase gene showed qualitative and quantitative differences for the three genes under oxidative stress conditions tested. The Gpxh gene is specifically induced by singlet-oxygen photosensitizers and the relative induction by other compounds is much weaker for Gpxh than for the other genes investigated. Using Gpxh promoter fusions with the arylsulfatase reporter gene, we have shown that the Gpxh was transcriptionally up-regulated by singlet-oxygen photosensitizers. It is also shown that the Gpxh promoter contains a region between 104 and 179 bp upstream of the transcription start that is responsible for the mRNA up-regulation upon exposure to 1O2 but not t-BOOH. Within this region a regulatory sequence homologous to the mammalian cAMP response element (CRE) and activator protein 1 (AP-1) binding site was identified within a 16 bp palindrome.
Assuntos
Proteínas de Algas/genética , Chlamydomonas reinhardtii/genética , Glutationa Peroxidase/genética , Oxigênio/farmacologia , Animais , Arilsulfatases/efeitos dos fármacos , Arilsulfatases/genética , Arilsulfatases/metabolismo , Relação Dose-Resposta a Droga , Proteínas de Choque Térmico HSP70/genética , Peróxido de Hidrogênio/farmacologia , Luz , Estresse Oxidativo , Oxigênio/metabolismo , Fármacos Fotossensibilizantes/farmacologia , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , RNA Mensageiro/efeitos da radiação , Proteínas Recombinantes de Fusão/efeitos dos fármacos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência , Oxigênio Singlete , Superóxido Dismutase/genética , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/efeitos da radiação , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/efeitos da radiaçãoRESUMO
The Saccharomyces cerevisiae yap1 null strain was transformed with a Chlamydomonas reinhardtii cDNA expression library. A 688-bp cDNA fragment, coding for the 60S ribosomal protein L10a (RPL10a), restored the capacity of the S. cerevisiae yap1 null strain to resist oxidative stress. The rpl10a gene is a single-copy gene in C. reinhardtii and encodes a constitutively produced 1.35-kb mRNA. The deduced 214-residue amino acid sequence was highly related with RPL10a proteins from eukarya (between 46.1 and 63.7% identity) and archaea (between 24.5 and 29.2% identity). Resistant transformants were pink, due to increased carotenoid levels, with the same chemical structure as torularhodin, the main carotenoid of the pink yeast Rhodotorula mucilaginosa. The pink transformants showed high resistance levels against H(2)O(2), paraquat, menadione, and UV light. Partial inhibition of the carotenoid synthesis by diphenylamine reduced the resistance levels, demonstrating the role of excess carotenoid synthesis in the resistance mechanism.
