| Budget Amount *help |
¥4,810,000 (Direct Cost: ¥3,700,000、Indirect Cost: ¥1,110,000)
Fiscal Year 2010: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000)
Fiscal Year 2009: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
Fiscal Year 2008: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
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| Research Abstract |
The mus-10 mutant was isolated which showed highly sensitivity to alkylating agent methylmethane sulfonate (MMS). It had been forecasted that mus-10 gene belonged to the some DNA repair pathway, because of it sensitivity to mutagen. This mutant have other unique characteristics; unable to grow after several times sequential inoculation, or stop growing after 2 to 3 weeks culture. Furthermore, these phenotypes are accompanied the deletion of mitochondrial DNA and fragmented mitochondrial feature comparing to the normal (tubular) shape in wild type strain. The responsible gene of mus-10 was cloned by complementation of its MMS sensitivity. This gene encodes the F-box domain containing polypeptide, and deletion of F-box domain showed identical phenotype with mus-10 mutant. Since F-box protein is known as a counterpart of SCF (Skp-Cullin-F-box) comlex, which have a role for the degradation of some target protein via ubiquitination following degrading in The mus-10 mutant was isolated which
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showed highly sensitivity to alkylating agent methylmethane sulfonate (MMS). It had been forecasted that mus-10 gene belonged to the some DNA repair pathway, because of it sensitivity to mutagen. This mutant have other unique characteristics ; unable to grow after several times sequential inoculation, or stop growing after 2 to 3 weeks culture. Furthermore, these phenotypes are accompanied the deletion of mitochondrial DNA and fragmented mitochondrial feature comparing to the normal (tubular) shape in wild type strain. The responsible gene of mus-10 was cloned by complementation of its MMS sensitivity. This gene encodes the F-box domain containing polypeptide, and deletion of F-box domain showed identical phenotype with mus-10 mutant. Since F-box protein is known as a counterpart of SCF (Skp-Cullin-F-box) comlex, which have a role for the degradation of some target protein via ubiquitination following degrading in proteasome.
To uncover the mus-10 gene function, we focused the feature of mitochondria. We examined whether 1) mitochondrial fusion is inhibited, or 2) mitochondrial fission is stimulated in mus-10 mutant. Double mutation of mus-10 and fis-1, which was essential for mitochondrial fission, showed quite resemble feature of mitochondria with wild type strain. And also this double mutant suppressed sensitivity to mutagen and short life span. These results suggested that MUS-10 protein prevent from the mutagen sensitivity and short life span according to maintain a mitochondrial feature. MUS-10 protein was considered to have a function of degradation of some target protein. So MUS-10 protein should be bound to that protein. Considering MUS-10 protein was correlated to maintenance of mitochondria feature, one candidate FZO-1 arose which had functions in the mitochondrial fusion. Using immunoprecipitation mthod, we could show that MUS-10 protein bound to FZO-1. Next, we tried to make fzo-1 knock out strain, but couldn't. The fzo-1 gene thought to be essential gene and fusion of mitochondria was important for maintenance of life span in Neurospora. Further, we forecasted that constitutive expression of FZO-1 protein in mus-10 mutant might show eviler phenotype than the mus-10 single mutant, because FZO-1, target of MUS-10, might be accumulated in that strain by escaping degradation. However, any evil phenotypes were not observed. Above these results, it was suggested that there were complex mechanism to maintain the mitochondrial feature. Less
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