Unknown,Transcriptomics,Genomics,Proteomics

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Expression analysis of the singlet oxygen resistant 1 (sor1) mutant

ABSTRACT: In a screen for singlet oxygen resistant mutants, a mutant called sor1 was isolated with increased resistance to different oxidative and electrophilic stress conditions. This mutant was found to have a constitutive high expression of three known singlet oxygen response genes, a glutathione peroxidase GPX5 and two glutathione-S-transferase GSTS1 and GSTS2. The sor1 mutation was mapped to the gene of a 393-amino acid protein for which blast searches revealed only poor homology to other proteins but predicted a putative basic leucine zipper DNA binding domain indicating that it might function as transcription factor to activate gene expression during oxidative stress.Global expression analysis of sor1 compared to the untreated wild-type enabled to identify additional genes differentially regulated in the sor1 mutant including several other genes involved in oxidative stress response and detoxification of endogenous xenobiotics. The sor1 mutant and the corresponding wild-type strain 4A+ were grown mixotrophically in a Tris-acetate phosphate in three independent biological replicates. Then the cells of each replicate were harvested by centrifugation and total RNA was isolated using the RNeasy Mini Kit (Qiagen). DNA microarrays were performed using the ‘One-Color Microarray-Based Gene Expression Analysis’ system and a custom made 4 × 44 K ‘Chlamydomonas Whole Genome DNA Microarrays’ (Agilent Technologies) containing 15143 specific probes designed based on the Chlamydomonas version 4.0 transcript models provided by the DOE Joint Genome Institute (JGI), with an average of three replicates for each probe

ORGANISM(S): Chlamydomonas reinhardtii

SUBMITTER: Beat Fischer

PROVIDER: E-GEOD-30645 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Acclimation to singlet oxygen was shown to induce various oxidative stress response genes of which some were also strongly overexpressed in the singlet oxygen resistant mutant sor1. Because sor1 was also more tolerant to other oxidative and electrophilic stress conditions, and because many of the sor1 overexpressed genes are known to be involved in the detoxification of reactive electrophile species, the response of the C. reinhardtii wild-type strain to various oxidative and electrophilic stress conditions was determined. Therefore, cultures were exposed to the reactive oxygen species-producing photosensitizer neutral red, the organic hydroperoxide tert-butylhydroperoxide, the photosynthetic herbicide 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) and the lipophilic electrophile 2(E)-Hexenal for two hours and the global genetic response was analyzed. Cluster analysis revealed the most similar expression pattern between DBMIB and 2(E)-Hexenal and to a lower degree between NR and tBOOH. Still, there were many common induced genes including several of the oxidative stress response and detoxification genes overexpressed in the sor1 mutant. The 4A+ wild-type strain was grown mixotrophically in a Tris-acetate phosphate to a density of 2x10^6 cells/ml. Then cultures were split into 20 ml subcultures, and exposed to either of the four chemicals DBMIB (2 µM), 2(E)-Hexenal (0.3 mM), neutral red (NR, 3 µM), tert-butylhydroperoxide (tBOOH, 100 µM) or no chemical (control) for 2 hours, in three independent biological replicates. The cells of each replicate were harvested by centrifugation and total RNA was isolated using the RNeasy Mini Kit (Qiagen). DNA microarrays were performed using the ‘One-Color Microarray-Based Gene Expression Analysis’ system and a custom made 4 × 44 K ‘Chlamydomonas Whole Genome DNA Microarrays’ (Agilent Technologies) containing 15143 specific probes designed based on the Chlamydomonas version 4.0 transcript models provided by the DOE Joint Genome Institute (JGI), with an average of three replicates for each probe

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Expression analysis of the singlet oxygen resistant 1 (sor1) mutant

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