Arrangement of mitochondrial nucleoids during life cycle of Saccharomyces cerevisiae.

Double staining of mitochondria and mitochondrial nucleoids in the living yeast during the life cycle.

“…They have also revealed that there is a single and highly branched mitochondrion per cell during sporulation and each ascospore also contains a single, branched mitochondrion that closely encircles the nucleus (18). In spite of the difference of strains and culture conditions used, these electron-microscopic studies well coincided with our earlier observations (8,11,14) .…”

“…Saccharomyces cerevisiae, diploid strain G2-2, obtained by crossing a haploid strain 24428 (mating type a) with a haploid strain 3626 (mating type a) was aerobically cultured under reciprocal shaking at 30°C in a modified Burkholder's medium (BT medium) (15), except for the omission of amino acids, purine and pyrimidine bases and supplementation with tomato juice (3.6%). Fractionation of large cells at stationary-phase and sporulation cultures were the same as we described previously (8,14).…”

“…The method 1 was used to stain cells at vegetative growth, germination and zygote formation. Appropriate volume (0.1-1.5 ml) of the culture (approximately 1 X 10' cells/ml) was placed in an Eppendorf tube and cells were washed twice with NS buffer (20 mM Tris-HCI, pH 7.6, 0.25 M sucrose, 1 mM EDTA, 1 mM MgC12, 0.1 mM ZnSO4i 0.1 mM CaC12, 0.8 mM phenylmethylsulphonylfluoride, 0.05% 2-mercaptoethanol) (14) by repeated centrifugation (1,700 X g, 30 sec). Cells were suspended in 100,ul of NS buffer and, then, DiOC6 (3) (Eastman Kodak Co., New York, U.S.A.) and DAPI (Sigma Chemical Co., St. Louis, U.S.A.) were added to final concentrations of 1.l,ug/ml and 0.8,ag/ml respectively, from the stock solutions (8.6 a g/ml DiOC6 (3) and 60 ,u g/ml DAPI, each dissolved in water).…”

“…We have investigated the morphological changes of mt-nucleoids in growing cells (14) and sporulating cells (8,11), as well as the examination of mitochondrial morphology (8,14). In those studies, we have shown that yeast mitochondria and mt-nucleoids change their morphology drastically depending on the stage of life cycle or culture conditions.…”

“…In yeast cells, dimethyl aminostyrylmethylpyridiniumiodine (DASPMI) (3,6,8,14), Janus green B and rhodamine 123 (20) have been used to stain mitochondria in living cells, and 4',6-diamidino-2-phenylindole (DAPI) to stain mitochondrial nucleoids (mt-nucleoids) in both living and fixed cells (3,8,9,11,14,22). DAPI staining has been performed in combination with indirect immunofluorescence microscopy (10).…”