Studies on conformational changes of mitochondrial nucleoids and the effect on gene separation in cells of the yeast, Saccharomyces cerevisiae

Research Project

Project/Area Number	61540488
Research Category	Grant-in-Aid for General Scientific Research (C)
Allocation Type	Single-year Grants
Research Field	植物生理学
Research Institution	Yamaguchi University
Principal Investigator	SANDO Nobundo Facul. of Sci., Yamaguchi Univ., Professor, 理学部, 教授 (80007062)
Co-Investigator(Kenkyū-buntansha)	MIYAKAWA Isamu Facul. of Sci., Yamaguchi Univ., Assistant, 理学部, 助手 (50136165)
Project Period (FY)	1986 – 1987
Project Status	Completed (Fiscal Year 1987)
Budget Amount *help	¥1,600,000 (Direct Cost: ¥1,600,000) Fiscal Year 1987: ¥500,000 (Direct Cost: ¥500,000) Fiscal Year 1986: ¥1,100,000 (Direct Cost: ¥1,100,000)
Keywords	Saccharomyces cerevisiae / Mitochondrial DNA / Mitochondrial nucleoid / Protoplast fusion / Mating-type interconversion / ミトコンドリア遺伝子の分配 / 酵母 / ミトコンドリア核様体の状態変化 / ミトコンドリア遺伝子の分配・組換え / 突然変異の誘起 / 細胞融合 / 制限酵素処理
Research Abstract	Mitochondria(mt) of Saccharomyces cerevisiae contain their inherent DNA(mtDNA), with which proteins constitute a superstructure, "mt-nucleoid", that change the conformation from the dispersd state to the integrated and vice versa under various growth conditions. In this study, we tried to elucidate mechanisms of these morphological changes regulated by mitochondrial(mt) or nuclear(nc) genes and control of nc-gene expression by a mt-gene. 1. Induction of mitochondrial-character mutants with different mutagens Haploid yeasts were treated with ethidium bromide(EB), laser, MnCl_2 and ultraviolet light(UV), then only EB and UV unduced respiratory-deficient mt- and nc-mutants, respectively. 2. Analysis of characters of the mutants (1)Mutants for conformational changes of mt-nucleids Mt-nucleoids in EB-induced rhoー mutant cells aggregated during growth phases. Some ncmutants produced normal cells at 25゜C and respiratory-deficient ones carrying aggregated nucleoids or no mtDNA at 36゜C. The other mutants consisted of cells containing the aggregated nucleoids or no nucleoid, irrespective of temperature. (2)Effect of mt-gene on mating-type interconversion and sporulating ability During spore germination and growth of homothallic strain FE-1, respiratory-deficient cells were formed at frequency of 10^<-3> and also were found to be mainly <alpha>-type haploids. On the other hand, protoplasts of <alpha>-type rho゜ YGU-20 regenerated respiration-positive a/<alpha> cells after fused with anuclear "miniprotoplasts" of YGU-4 carrying normal mitochondria. 3. Isolation, analyses and cloning of mtDNA and the application methods to protoplasts Techniques for isolating mtDNA from mt-nuclioids or disrupted whole cells and for obtaining the electrophoresis profiles of the restriction fragments have been examined. However, no fragment of mtDNA was cloned yet. Works on devising methods for chemical or electric fusion of protoplasts are in progress.