| Co-Investigator(Kenkyū-buntansha) |
IIDA Hidetoshi National Institute for Basic Biology, Research Associate, 助手 (70124435)
OHYA Yoshikazu Univ. of Tokyo, Dept. of Biology, Research Associate, 理学部, 助手 (20183767)
NAKANO Akihiko Univ. of Tokyo, Dept. of Biology, Assistant Prof., 理学部, 講師 (90142140)
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| Budget Amount *help |
¥4,100,000 (Direct Cost: ¥4,100,000)
Fiscal Year 1989: ¥4,100,000 (Direct Cost: ¥4,100,000)
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| Research Abstract |
Roles of calcium ion in regulation of bell proliferation, the mating pheromone response and morphogenesis of Saccharomyces cerevisiae have been investigated using genetic, molecular biological and physiological approaches. The research is particularly focused on the following four projects. 1) We have established an experimental system suitable for study of cell cycle regulation by Ca^<2+>. Using this system, we have shown that Ca^<2+> is essential for G_1 and G_2/M events during the cell cycle and regulates cAMP level. This system will be useful to isolate conditional lethal mutants which can grow only when sufficient concentrations of Ca^<2+> are present in media. 2) Techniques for measuring the cytosolic free Ca^<2+> concentration ([Ca^<2+>]i) in individual yeast cells have been established. We have employed fura-2 as a calcium specific fluorescent probe in conjunction with digital lmage processing. Images of fura-2 fluorescence under a Nikon Microphot-FX microscope are acquired by
… More
a SIT camera and relayed into a TV monitor and a Hamamatsu ARGUS-100 image processor. In S.cerevisiae, the mating process of haploid cells is controlled by the mating pheromones, a__- and alpha-factors, that induce several responses in cells of opposite mating type and are essential for mating. Using this system, we have shown that addition of alpha-factor to cells of a__- mating type raises [Ca^<2+>]i to 500-800 nM from a basal level of 100 nM and that this rise is essential for maintaining viability of yeast cells late in the mating pheromone response pathway. 3) To establish another measurement system suitable for detecting a rapid and transient rise in [Ca^<2+>]i in response to extracellular stimuli, we have employed a luminescent protein, aequorin, as a calcium specific probe. We have constructed plasmeds in which the aequorin cDNA is joined downstream of either the GAL1 promoter or the GAP promoter. We could successfully regenerate aequorin inside intact yeast cells and detect luminescent activity of it when Ca^<2+> was introduced into the cells. 4) The coding region of a yeast calmodulin gene was fused to the GAL1 promoter and a conditional-lethal mutant of S.cerevisiae, in which the expression of calmodulin was regulated by galactose, was constructed. The mutant grew normally in galactose medium, but in glucose medium, it ceased growing. The growth arrest was found to be associated with a decrease in intracellular calmodulin levels. Analysis of the terminal phenotype showed that when the cell stopped growing, it had a bud, a nucleus after S-phase and a short mitotic spindle. We concluded that the defect is mainly in nuclear division. Less
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