1996 Fiscal Year Final Research Report Summary

MUTAGEN-INDCED ALTERATION OF MITOCHONDRIAL GENOME in vivo.

Research Project

Project/Area Number	07670737
Research Category	Grant-in-Aid for Scientific Research (C)
Allocation Type	Single-year Grants
Section	一般
Research Field	Neurology
Research Institution	OKINAKA MEMORIAL INSTTUTE FOR MEDICAL RESEARCH
Principal Investigator	NAKASE Hirofumi , 財団法人・冲中記念成人病研究所, 研究員 (80155738)
Project Period (FY)	1995 – 1996
Keywords	MITOCHONDRIA / MUTAGEN / PCR-SSCP / METHOTOREXATE / ETHIDIUM BROMIDE / HISTOCHEMISTRY
Research Abstract	We studied alteration of mitochondrial genome induced with minimal dose of two mutagens, methothexate or ethidium bromide in vivo. These two mutagens have srong mutagenic action to especiaaly mitochondrial genomes and toxicity for mitochodrial respiratory function. Each reagent was injected for 4 weeks into 8-weeks old female B10 mice, which were well-studied for muscle pathology. Histochemical study of quadriceps muscles of the treated mice showed almost mormal histological features, except for a minimal alteration of myofibrillar networks in a few muscle fibers of an EtBr-terated mouse. Using PCR primers that can amplify tRNA-Lys region of mice mitochondrial genomes, we determined a good condition to obtain a single band even in the silver-stained PHAST gel. PCR-SSCP analysis of mitochondrial genome obtained from mice skeletal muscles showed that the number of bands on the SSCP gel were inceased and varied, compared with ones from the untreated skeletal muscle. This implicated these mutagens actually induced mutations of mitochondrial genomes. Deacreased number of mitochondrial DNA copies has been reported after treatment of this kind of mitochondrial mutagens. It has been reported that "petit" mutation was induced by ethidium bromide in yeast. Therefore, this study provide a good evidence that these mutagens may act with mitochondrial genomes directly or indirectly and alter nucleotide sequences of mitochondrial genomes. The next step of this research should be an experiment to see if the mutagen-induced mtation of mitochondrial genome chould be induced in oocytes or germ-line cells when these mutagens are injected into pregnant mice. If mutated mitochondrial genome could be inheritated to the next generation, animal model of mitochondrial cytopathy chould be available.