Shoji Shimada

Direct induction of tetraploids or homozygous diploids in the industrial yeast Saccharomyces cerevisiae by hydrostatic pressure

SummaryHydrostatic pressure and a dye plate method were used to investigate the direct induction of tetraploids or homozygous diploids from the industrial diploid or haploid yeast Saccharomyces cerevisiae. Above 200 MPa, hydrostatic pressure greatly inactivated the strains HF399s1 (α haploid), P-540 (a/α diploid), and P-544 (a/α diploid). At the same time, when pressure-treated cells of these strains were spread on a dye plate, some of the visible colonies were stained red/blue or dark blue (variant colonies); the rest stained violet, similar to colonies originating from diploid cells or haploid cells that were not pressure-treated. In addition, above 100 MPa, the formation of variant colonies increased with increasing pressure, and maximized (1x10-1) at 200 and 250 MPa, respectively. The size of almost all variant cells from P-544, P-540, and HF399s1 was visibly increased compared with that of untreated cells and the measured cellular DNA content of P-540 and HF399s1 was double that of untreated cells. Furthermore, based on random spore analysis and mass-matings, induced variants in the diploid strains were found to be tetraploid with an a/a/α/α genotype at the mating-type locus or, in the haploid strains, homozygous diploid with an α/α genotype. From these results we conclude that pressure treatment in combination with a dye plate is a useful method for strain improvement by direct induction of tetraploids or homozygous diploids from industrial strains whether diploid or haploids.

Changes in microfilaments and microtubules of yeasts induced by pressure stress

Abstract Changes in cytoskeletal elements induced by pressure stress in a budding yeast Saccharomyces cerevisiae , a dimorphic yeast Candida tropicalis and a fission yeast Schizosaccharomyces pombe were investigated by fluorescence microscopy. The cell cycle-specific organization of microfilaments and microtubules in the three yeasts was altered by exposure to hydrostatic pressure of 50–150 MPa for 10 min and their complete disassembly was observed at 150–300 MPa. Similar morphological changes in the cytoskeleton were caused in the three yeasts by acceleration of pressure stress, although their sensitivity differed from that of yeasts; hyphal-form cells of C. tropicalis and S. pombe cells were sensitive to pressure stress.

Morphological effects of pressure stress on yeasts

Abstract To investigate the induction of polyploidy by pressure stress, the ultra- structure and microtubules of Saccharomyces cerevisiae and Schizosaccharomyces pombe were studied by conventional and immunoelectron microscopy. The nuclear membrane was disrupted even at 100 MPa and with increasing pressure mitochondria had electron-dense areas, the cytoplasmic substances changed dramatically and the cellular organelles could hardly be detected. S. pombe cells were more sensitive to low pressure stress than were S. cerevisiae cells. Immunoelectron microscopy confirmed that the microtubules were damaged by pressure stress. The damage to spindle pole bodies, microtubules and the nuclear membrane was thought to be followed by breakdown of the nuclear division apparatus and inhibition of nuclear division.

An Efficient Method for Separating Ascospores from Sporulating Cultures in Saccharomyces cerevisiae by Hydrostatic Pressure

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Response of actin cytoskeleton on Schizosaccharomyces pombe to high pressure-stress

We investigated response to pressure stress on the actin cytoskeleton and actin cytoskeleton-related protein Cdc8 tropomyosin of the cells of a cold-sensitive mutant nda3 -KM311 Schizosaccharomyces pombe by rhodamine-conjugated phalloidin and the specific antibodies of the actin cytoskeleton-related protein Cdc8 tropomyosin. At below 100 MPa actin cytoskeleton and tropomyosin were equally distributed. Tropomyosin was localized at the actin ring and cables, and at 100 MPa it was localized in the long and fine actin cable. Above 150 MPa, however, tropomyosin was dispersed throughout the cytoplasm. We were unable to elucidate the relationship between the actin cytoskeleton and tropomyosin on the degradation pattern of the former. Using a cdc8 temperature-sensitive mutant ( cdc8 –110) grown at a permissive temperature we studied response of ultrastructure of the cell to pressure stress. At 200 MPa septa of cdc8 cells were drastically changed.

Dynamics of Cell Structure by Pressure Stress in the Fission Yeast Schizosaccharomyces pombe

Study of the effect of hydrostatic pressure on yeast cells revealed the impact of ultrastructural changes including microtubules and actin cytoskeletons. We also found that the fission yeast Schizosaccharomyces pombe is more sensitive to pressure stress than the budding yeast Saccharomyces cerevisiae using conventional electron microscopy (CEM), immunoelectron microscopy (immuno-EM), and fluorescence microscopy (FM). To investigate the influence of pressure stress on the cell cycle of S. pombe we used the cells of a cold-sensitive mutant, nda3 KM311, of S. pombe which were arrested highly synchronously at a step similar to mitotic prophase under restrictive temperature at 20°C, for 4 h. We describe here that the morphological changes in actin cytoskeleton were caused by acceleration of pressure stress in nda3 mutant cells related to induction of diploidization in S. pombe. When the nda3 cells were incubated at the restrictive temperature of 20°C, large cells (diploid cells) appeared on a dye plate after pressure stress of 150 MPa. These cells made up over 40% of the colonies on the plate. Nda3 cells were first aerobically grown at 30°C in YPD liquid medium to mid-exponential phase, transferred to restrictive temperature at 20°C for 4 h, and then shifted to a permissive temperature at 36°C for 15 min. The cells grown at 20°C had an abnormal (‘leaf like’) nucleus profile surrounded by normal nuclear membrane. After pressure stress treatment at 100 MPa the nuclear membrane was damaged and the matrix of mitochondria had an electron-dense area. At 150 MPa, other altered features were apparent: the nuclear membrane was broken over a broad area; the vacuoles had fused into large pieces in cells grown at both 20°C and 36°C. The influence of pressure stress on actin cytoskeleton in nda3 cells was revealed by FM. In the cells grown at 20°C, actin patches were concentrated in the central region and actin rings were seen. Even at 100 MPa specific actin distribution was lost. Long and fine actin cables were seen all over the cells: large actin patches remained in the center of the cell and covered the actin rings, then they changed into thick and short cables at 150 MPa; they finally decomposed but the actin ring was visible even with faint fluorescence. Immuno-EM also showed this phenomenon. These results confirmed the process of degradation in actin cytoskeleton of nda3 cells by pressure stress.

Direct induction of homozygous diploidization in the fission yeast Schizosaccharomyces pombe by pressure stress

Hydrostatic pressure stress and a dye plate method were first applied to investigate the direct induction of homozygous diploids from the haploid yeast S. pombe. Above 100 MPa at 25 °C for 10min, pressure stress greatly inactivated the haploid strain JY1(L972h-). At the same time, pressure stressed cells of the former strain at more than 100–200 MPa were spread on a dye plate, some pressure- effected visible colonies were stained violet (variant colonies); the rest were stained pink, similar to colonies originating from haploid cells that were not pressure- stressed. Variant colonies from JY1 began to appear and increased in frequency up to 200 MPa. Around 200 MPa, the maximum measured % cell population of variant colonies presented was approximately 40. Based on the cell size, DNA content, crosses, and random spore analysis for the segregation of mating types or auxotrophic markers, these variant cells originating from color changed colonies after pressure stress were very stable and found to be a homozygous diploid with h-/ h- genotype at the mating- type locus

Ultrastructural effects of pressure stress to Saccharomyces cerevisiae cells revealed by immunoelectron microscopy using frozen thin sectioning

Abstract Effects of hydrostatic pressure on ultrastructure of Saccharomyces cerevisiae were studied by immunoelectron microscopy using frozen thin sections. At 100 MPa bundles of the microtubules (MTs) extended in the nucleus, but spindle pole bodies were not visible. At 150 MPa the deposition of gold particles for anti α-tubulin was recognized in the nucleus, although the filamentous structure of the MTs was not seen. At 200 MPa fewer gold particles were scattered in the nucleus. These results show that elements of the nuclear division apparatus are susceptible to pressure stress. These events were reversible at below 200 MPa.