【作者】 彭雪瑞；

【导师】 严庆丰；

【作者基本信息】 浙江大学 ， 细胞生物学， 2008， 硕士

【摘要】 核修饰基因所编码的蛋白质本身不诱导任何病变,但是可以抑制或增强线粒体DNA（mtDNA）突变的致病性,也可能是定位于线粒体的一种组织特异的功能性多态,对mtDNA引起的致病表达有重要的调控作用。因线粒体功能缺陷导致的线粒体疾病,涉及线粒体基因组突变、核基因突变、及环境因素等复杂机制,深入阐明三者的相互作用关系具有重要的理论意义,进而指导线粒体疾病的诊断、预警、和治疗等,具有重要的潜在应用价值。核修饰基因MTO2编码5-甲基-氨甲基-2-硫甲基转移酶。本实验室曾率先克隆了该基因,并对其功能进行了初步研究。表明MTO2是核基因编码的、进化上高度保守的线粒体功能蛋白。当酵母mtDNA存在C1409G（与人类mtDNA编码的12SrRNA的A1555G同源突变）,即巴龙霉素抗性突变(P₄₅₄^R)时,核修饰基因MTO2突变菌株显示呼吸缺陷表型。在上述研究的基础上,并论文主要探讨了核修饰基因MTO2与氨基糖苷类抗生素（主要是新霉素）的相互作用,并获得以下创新成果。1、首次发现敲除MTO2基因显著抑制酵母mtDNA P₄₅₄^R突变引起的氨基糖苷类抗生素敏感性。具体结果如下:（1）在含有新霉素的葡萄糖培养基（GYP）上,mtDNA为野生型、MTO2敲除与否,相应的酵母菌株的生长没有显著差异;mtDNA完全缺失、MTO2敲除与否,相应的酵母菌株的生长也没有显著差异;然而,当mtDNA存在P₄₅₄^R突变时,MTO2野生型菌株几乎完全不能生长,而MTO2敲除菌株的生长只受到部分抑制,表现二者对新霉素的敏感性存在显著差异。（2）在不含新霉素的GYP培养基上,所有菌株的生长没有显著差异。2、从全基因组水平对上述MTO2新功能进行了初步机理研究。基因芯片结果显示:当mtDNA存在P₄₅₄^R突变时,不经新霉素处理,敲除MTO2基因显著促进rRNA代谢相关基因表达,降低葡萄糖酵解相关基因的表达水平;新霉素处理后,MTO2基因敲除与否,rRNA代谢相关基因表达水平没有显著差异,却极大促进了糖酵解途径相关基因的表达。表明敲除MTO2基因抑制了P₄₅₄^R突变相关的氨基糖苷类抗生素的敏感性。其分子机制并不是改变了P₄₅₄^R突变引起的rRNA结构,而是通过促进糖酵解途径产生更多的ATP,从而弥补因P₄₅₄^R突变和氨基糖苷类抗生素造成的线粒体功能缺陷。更多还原

【Abstract】 Nuclear modifier genes do not induce any pathology per se,but contribute to the pathogenic effect of the mitochondrial mutations.The nuclear modifier could be a common functional polymorphism in a tissue-specific protein,possibly with mitochondrial location.MtDNA mutations were necessary for mitochondrial diseases that caused by mitochondrial dysfunction,but not sufficient to induce the pathology.Nuclear modifier genes,environment factors,also contributes to the pathogenic effect of the mitochondrial mutations.In-depth clarify the interaction between this three factors, has an important social significance for guiding mitochondrial disease diagnosis、early warning and treatment.Nuclear modifier gene MTO2 encoding the mitochondrial 5-methylaminomethyl-2-thiouridylate-methyltransferase. Our laboratory first identified and characterized this gene.MTO2 is a nuclear genes encoding protein,which was highly evolutionarily conserved,mto2 null mutants expressed a respiratory-deficient phenotype when coexisting with the C1409G mutation of mitochondrial 15 S rRNA(paromomycin resistance mutation p₄₅₄^R)at the very conservative site for human deafness-associated 12S rRNA A1555G mutations.In this study,we focus on the interaction between nuclear modifier gene MTO2 and aminoglycosides（mainly neomycin）,and have received the following innovations.1,We here report first that the knockout of MTO2 significantly inhibited mtDNA p₄₅₄^R mutant`s sensitivity to aminoglycoside antibiotics.Here are the results:（1）In neomycin GYP medium,when mitochondria genes were wild type,whether MTO2 knockout or not,the growth of yeast strains has no significant difference;when mitochondria genes were complete deletion,whether MTO2 knockout or not,the growth of yeast strains also has no significant difference;Interesting,when mtDNA P₄₅₄^R,wild-type MTO2 strains were hardly survival in neomycin GYP medium,but the growth of MTO2 knockout strains were only partly inhibited,there was a significant difference performance of neomycin sensitivity between them.（2）In the non-neomycin GYP medium,the growth of all strains there is no significant difference.2,from the genome-wide level,we doing some research on the MTO2.The gene chip results showed:under mtDNA p₄₅₄^R mutation,without a treatment with neomycin,the knockout of MTO2,significantly promote the expression of genes involved in rRNA metabolism,but lower expression of genes involved in glucose fermentation.After the treatment with neomycin,there was significantly increased expression of genes involved in glucose fermentation,but rRNA metabolism genes has no significant difference expression.Knockout of MTO2 significantly inhibited p₄₅₄^R associated aminoglycoside antibiotics sensitivity.The molecular mechanism of which was not changed rRNA structure caused by p₄₅₄^R mutation,but enhanced yeast capability of using glucose fermentation to produce more ATP,partly restored mitochondrial defects caused by p₄₅₄^R mutation and the aminoglycoside antibiotics.更多还原