RESUMO
The aim of the study was to demonstrate that the bZIP-type transcription factor AtfA regulates different types of stress responses in Aspergillus nidulans similarly to Atf1, the orthologous 'all-purpose' transcription factor of Schizosaccharomyces pombe. Heterologous expression of atfA in a S. pombe Deltaatf1 mutant restored the osmotic stress tolerance of fission yeast in surface cultures to the same level as recorded in complementation studies with the atf1 gene, and a partial complementation of the osmotic and oxidative-stress-sensitive phenotypes was also achieved in submerged cultures. AtfA is therefore a true functional ortholog of fission yeast's Atf1. As demonstrated by RT-PCR experiments, elements of both oxidative (e.g. catalase B) and osmotic (e.g. glycerol-3-phosphate dehydrogenase B) stress defense systems were transcriptionally regulated by AtfA in a stress-type-specific manner. Deletion of atfA resulted in oxidative-stress-sensitive phenotypes while the high-osmolarity stress sensitivity of the fungus was not affected significantly. In A. nidulans, the glutathione/glutathione disulfide redox status of the cells as well as apoptotic cell death and autolysis seemed to be controlled by regulatory elements other than AtfA. In conclusion, the orchestrations of stress responses in the aspergilli and in fission yeast share several common features, but further studies are needed to answer the important question of whether a fission yeast-like core environmental stress response also operates in the euascomycete genus Aspergillus.
Assuntos
Fatores Ativadores da Transcrição/metabolismo , Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores Ativadores da Transcrição/genética , Aspergillus nidulans/enzimologia , Sequência de Bases , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genótipo , Glutationa/metabolismo , Dissulfeto de Glutationa/metabolismo , Cinética , Oxirredução , Estresse Oxidativo/genética , Fenótipo , Proteínas/genética , Proteínas/metabolismo , RNA Fúngico/genética , RNA Mensageiro/genética , DNA Polimerase Dirigida por RNA/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismoRESUMO
Exponentially growing human erythroleukemia K562 cells were permeabilized and the dose dependent decrease of DNA synthesis rate was measured after ultraviolet (UV B, 290 nm) irradiation. Cells were able to overcome 2 and 5 J/m2 UV doses, partial recovery was observed at 15 J/m2, while at high (25 J/m2) UV dose replicative DNA synthesis remained suppressed. K562 cells were subjected to synchronization prior to and after UV irradiation (24 J/m2) and 18 fractions were collected by centrifugal elutriation. Cell cycle analysis by flow cytometry did not show early apoptotic cells after UV irradiation. The gradual increase in DNA content typical for non-irradiated cells was contrasted by an early S phase block between 2.2 and 2.4 C-values after UV irradiation. Cell cycle dependent chromatin changes after ultraviolet irradiation were seen as a fine fibrillary network covering the mainly fibrous chromatin structures and incompletely folded primitive chromosomes. Based on observations after UV irradiation and on earlier results with cadmium treatment and gamma irradiation, we confirm that typical chromatin changes characteristic to genotoxic agents can be recognized and classified.