Mechanism of mtDNA deletion in short lived mutant of Neurospora.

分离到对烷化剂甲基甲烷磺酸盐(MMS)高度敏感的MUS-10突变株。由于MUS-10基因对诱变剂敏感，推测其属于DNA修复途径。该突变体还有其他独特的特征：连续接种几次后不能生长，培养2-3周后停止生长。此外，与野生型菌株的正常(管状)形态相比，这些表型伴随着线粒体DNA的缺失和线粒体特征的碎片化。利用MUS-10对MMS的敏感性互补，克隆了MUS-10的致病基因。该基因编码含有F-box结构域的多肽，缺失的F-box结构域表现出与MUS-10突变株相同的表型。由于F-盒蛋白是已知的SCF(Skp-cullin-F-box)复合体的对应物，它们在MUS-10中降解后通过泛素化降解某些目的蛋白，因此从MUS-10突变体中分离到了哪个…MERE对烷基化试剂甲基甲烷磺酸(MMS)表现出高度的敏感性。由于MUS-10基因对诱变剂敏感，推测其属于DNA修复途径。该突变体还有其他独特的特征：连续接种几次后不能生长，培养2-3周后停止生长。此外，与野生型菌株的正常(管状)形态相比，这些表型伴随着线粒体DNA的缺失和线粒体特征的碎片化。利用MUS-10对MMS的敏感性互补，克隆了MUS-10的致病基因。该基因编码含有F-box结构域的多肽，缺失的F-box结构域表现出与MUS-10突变株相同的表型。由于F-box蛋白是与SCF(Skp-cullin-F-box)Comlex类似的蛋白，在蛋白酶降解后通过泛素化作用降解某些靶蛋白。为了揭示MUS-10基因的功能，我们重点研究了线粒体的特征。我们检测了MUS-10突变体是否1)线粒体融合受到抑制，或2)线粒体分裂受到刺激。线粒体分裂所必需的mus-10和fis-1双突变与野生型菌株的线粒体特征十分相似。该双突变体还抑制了对诱变剂的敏感性和较短的寿命。这些结果表明，MUS-10蛋白通过维持线粒体的特征来防止诱变剂的敏感性和寿命的缩短。MUS-10蛋白被认为具有降解某些目的蛋白的功能。因此，MUS-10蛋白应该与该蛋白结合。考虑到MUS-10蛋白与线粒体功能的维持相关，提出了一个在线粒体融合过程中具有功能的候选基因FZO-1。用免疫沉淀法证明MUS-10蛋白与FZO-1结合。接下来，我们试图获得FZO-1基因敲除菌株，但没有成功。FZO-1基因被认为是必需基因，线粒体的融合对维持脉孢子虫的寿命很重要。此外，我们预测FZO-1蛋白在MUS-10突变体中的结构性表达可能比MUS-10单一突变体表现出更糟糕的表型，因为MUS-10的靶标FZO-1可能通过逃避降解在该菌株中积累。然而，没有观察到任何邪恶的表型。以上结果表明，线粒体特征的维持存在复杂的机制。较少

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 … More 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