Protective role and regulation of Rad9 from the fission yeastSchizosaccharomyces pombe

Abstract

To assess novel cellular roles and regulation of Rad9 in the fission yeast Schizosaccharomyces pombe, the full-length rad9 gene was cloned into the shuttle vector pRS316, generating pYFRad9. The rad9 mRNA level was significantly increased in the S. pombe cells harboring the plasmid pYFRad9, suggesting that the cloned rad9 gene is functioning. The S. pombe cells harboring pYFRad9 showed higher survival in the minimal media containing nitric oxide (NO)-generating sodium nitroprusside (SNP, 20 muM) and no nitrogen than the vector control cells. SNP and nitrogen starvation notably enhanced the synthesis of beta-galactosidase from the rad9-lacZ fusion gene in the Pap1-positive cells but not in the Pap1-negative cells. The rad9 mRNA level, detected by semi-quantitative reverse transcriptase (RT)-PCR, was elevated in the Pap1-positive cells but not in the Pap1-negative cells by SNP and nitrogen starvation. It was also increased only in the Pap1-positive cells by diethylmaleate, which activates Pap1. Collectively, the results imply that Rad9 plays a protective role against nitrosative and nutritional stress and is positively regulated by NO and nitrogen starvation in a Pap1-dependent manner.