Nitric oxide as a signaling molecule in the fission yeast Schizosaccharomyces pombe

Nitric oxide synthases (NOS) catalyze the synthesis of ubiquitous signaling molecule nitric oxide (NO) which controls numerous biological processes. Using a spectrofluorometric NOS assay, we have measured the rate of total NO production in the crude cell extracts of Schizosaccharomyces pombe. NO production was reduced in the absence of NOS cofactors calmodulin and tetrahydrobiopterin, and a competitive NOS inhibitor NG-nitro-l-arginine methyl ester (l-NAME) was able to cause a statistically significant inhibition on the rate of total NO production. These results, for the first time, provide evidence that an enzyme with a NOS-like activity may be present in the fission yeast. In order to assess the possible regulatory roles of NO as a signaling molecule in this yeast, using the differential display technique, we screened for NO-responsive genes whose expression decreased upon exposure to l-NAME and increased in response to an NO donor, sodium nitroprusside treatment. Differential expression patterns of byr1, pek1, sid1, and wis1 genes were confirmed by quantitative real-time PCR. The physiological experiments performed based on the functions and molecular interactions of these genes have pointed to the possibility that NO production might be required for sporulation in S. pombe. Taken together, these findings suggest that NO may function as a signaling molecule which can induce both transcriptional and physiological changes in the fission yeast. Hence, these data also imply that S. pombe can be used as a model system for investigating the mechanisms underlying NO-related complex signaling pathways

The 3 ' terminal sequence of the inosine monophosphate dehydrogenase gene encodes an active domain in the yeast Schizosaccharomyces pombe

The gua1 gene encoding inosine monophosphate dehydrogenase (IMPDH), which catalyses the first step in de novo biosynthesis of guanosine monophosphate (GMP), was cloned in the yeast Schizosaccharomyces pombe by functional complementation of a gua1ura4-D18 mutant strain from a S. pombe DNA genomic library. Complementation analysis revealed a 1.2 kb fragment which segregation analysis confirmed did not code for a suppressor gene. Only 446 nucleotides of the gua1 gene encoding the IMPDH C-terminal residues were found within this 1.2 kb sequence (GenBank, AJ293460). The comparison of this wild-type fragment with the same fragment from the gua1ura4-D18 mutant revealed that there was a point mutation at position 1261 (guanine -> adenine) from the 5' end, corresponding to the amino acid residue 421 (glycine -> serine) of the enzyme. Dot and Northern analyses showed that the gua1 gene was expressed in transformants as well as in the wild-type and the gua1ura4-D18 mutant, but enzyme activity was only detected in wild-type and transformant cells. It seems likely that a 446 by fragment from the 3' end of the gua1 gene abolished the point mutation in the mutant strain, suggesting that this fragment participates in the sequences encoding the active domain of IMPDH in S. pombe

Investigation of the Relationship Between Oxidative Stress and Glucose Signaling in Schizosaccharomyces pombe

The invertase mutant defective in the glucose signaling pathway of () is resistant to glucose repression. This mutant is able to consume sucrose alongside glucose and grows in glucose-containing media with a generation time close to that of the wild type. Intracellular oxidation, protein carbonyl, and reduced glutathione levels and catalase, superoxide dismutase, and glutathione peroxidase activity were investigated in , to determine the relationship between oxidative stress response and glucose signaling. The expression profiles of some genes involved in regulation of glucose repression ( fructose-1,6-bis-phosphatase; hexokinase) and stress response ( and transcription factors; catalase; Cu,Zn superoxide dismutase) were analyzed using the quantitative real-time PCR technique. Oxidative stress response in seems to be affected by glucose signaling in a manner different from that caused by glucose deprivation

Expression of human cytochrome p450 3A4 gene in Schizosaccharomyces pombe

Heterologous expression systems can be utilized to great advantage in the study of cytochrome P450 enzymes. P450 3A4 is one of the major forms of cytochrome P450 found in liver. It is also involved in the metabolism of numerous widely used drugs and xenobiotics. In the present study human liver cytochrome P450 3A4 gene was transferred into the fission yeast Schizosaccharomyces pombe via two different S. pombe expression vectors carrying thiamine repressible promoter - nmt1 (pREP42) and constitutive promoter - adh1 (pART1). Heterologously expressed cytochrome P450 3A4 was detected in the cells grown in minimal (EMM) or rich medium (YEL) containing 0.5% (w/v) glucose. A typical cytochrome P450 peak for 3A4 was observed at 448 nm in microsomal fraction. The presence of heterologous expression of 3A4 form was also determined by SDS-PAGE and it molecular mass was identified as 52 kDa. The enzyme activity was confirmed by HPLC analysis, using testosterone as substrate

Expression of human cytochrome p450 3A4 gene in Schizosaccharomyces pombe

Heterologous expression systems can be utilized to great advantage in the study of cytochrome P450 enzymes.P450 3A4 is one of the major forms of cytochrome P450 found in liver. It is also involved in the metabolism of numerouswidely used drugs and xenobiotics. In the present study human liver cytochrome P450 3A4 gene was transferred intothe fission yeast Schizosaccharomyces pombe via two different S. pombe expression vectors carrying thiamine repressiblepromoter – nmt1 (pREP42) and constitutive promoter – adh1 (pART1). Heterologously expressed cytochrome P450 3A4 wasdetected in the cells grown in minimal (EMM) or rich medium (YEL) containing 0.5% (w/v) glucose. A typical cytochromeP450 peak for 3A4 was observed at 448 nm in microsomal fraction. The presence of heterologous expression of 3A4 formwas also determined by SDS-PAGE and it molecular mass was identified as 52 kDa. The enzyme activity was confirmed byHPLC analysis, using testosterone as substrate