Studies On Point Mutation Of Human Mitochondrial TRNA～(Leu(UUR)) Gene

Human mitochondria contain multiple copies of a 16,569bp closed circular DNA molecule, encoding 13 polypeptides,2 rRNAs and 22 tRNAs that are involved in the generation of bioenergy for cell's functioning. The application of molecular genetics opened possibilities for understanding the pathogenesis of mitochondrial disorders. In 1988, the first pathogenic mtDNA mutation was identified. A missense mutation at nucleotide position (np) 11778 was found in association with Leber's hereditary optic neuropathy(LHON). Soon thereafter, A3243G mutation of the tRNALeu(UUR) gene(MTTLl) was reported in patients \vith mitochondrial myopathy, encephalopathy, lactic acidosis, stroke-like episodes(MELAS) and a heteroplasmic A8344G transition was found in the majority of patients with myoclonic epilepsy, ragged red fibers (MERRF). Now , the roles of mtDNA mutations in human health are increasingly recognized, in the process of both aging and carcinogenesis. Further studies on the etilogy and the pathogenetic mechanism of mtDNA mutation are needed to advance our understanding of human mitochondrial disorders and enable, in rum, thedevelopment of novel therapies and genetic rescue strategies for the treatment of these diseases.One of the most perplexing aspects of mitochondrial genetics is the marked clinical variability associated with mtDNA mutations. This is dramatically demonstrated for the MTTL1 gene where base substitutions atnp3243,3252,3271,and 3291 give MEL AS, but the np 3243 mutation can also result in maternally inherited diabetes and deafness. The MTTL1 gene mutations at np3303 and 3260 result in hypertrophic cardiomyopathy and myopathy, but the former has a pediatric onset while the latter is adult-onset. So, the MTTL1 gene is an ideal target gene for us to search for the mutation formation and the relationship between the mtDNA mutations and the mitochondrial disorders.1 .Oxidative damage and the repair response under oxidative stress in MTTL1 gene of human mitochondrial DNAMitochondrial DNA is considered to suffer much more extensive oxidative damage than does nuclear DNA. Base-excision repair which is well established in mitochondria may be involved in the prevention of nucleotides from oxidative damage. We should need to consider which severe damage or inadequate repair mainly contributes to the mutations in rntDNA. Therefore, this study was designed to investigate oxidative damage to mtDNA and its subsequent repair. Human LO2 liver cells were exposed to 9mmol/L alloxan for 1h and then incubated in fresh culture media for 0,2, 8 and 24h. The frequencies of oxidative base damage in mtDNA were measured by a quantitative Southern technique coupled with digestion by the enzymes endonuclease III and formamidopyrimiding DNA glycosylase. Next, ligation-mediated PCR (LM-PCR) was performed to map damage to specific nucleotides along a ~100bp fragment including MTTL1 gene. The addition of alloxan to cultured human cells increased the rate of oxidative base damage and ,by several fold, the lesion frequency in mtDNA. After removal of this DNA damaging agent from culture, the lesion frequency decreased to levels slightly higher than normal at 8h and returned to normal levels at 24h. The result of LM-PCR showed 20 hot spots of MTTLl gene where nucleotides were receiving a majority of damage. The pattern of oxidative damage is like that of point mutation identified in this gene, suggesting the possibility that oxidative damage mainly contributes to the formation of the point mutation in MTTL1 gene.2.Studies on the pathogenetic mechanism of the A3243G mutation of human mitochondrial MTTL1 gene.To investigate the interactions of the mitochondrial wild and mutated tRNA Leu(UUR) with mitochondrial leucyl-tRNA synthetase (mtLeuRS),we have expressed mtLeuRS in E.coli and used the purified recombinant enzyme for in vitro assays of aminoacylation of wild type and mutated mitochondrial tRNA Leu(UUR).Measurement of the kinetic parameters for aminoacylation of wild and mutated tRNAs expressed in E.coli s...