Bud scar-rich preparation, which consisted mostly of crater-like bodies with some fragment of cell wall, was easily obtained from the isolated cell walls of baker's yeast by enzymic treatment under appropriate condition. In spite of low glucosamine content, which is due to the action of chitinase in the enzyme preparation, X-ray diffraction analysis showed that the structure of chitin was well preserved in this preparation.On the other hand, the preparation obtained by chemical treatment of intact yeast cells indicated high glucosamine content.Differing from the results of Bacon et al., the present preparation obtained after 2 cycles of chemical treatment still retained the original cellular shape with remnant cytoplasmic materials. After 5 cycles of the treatment, bud scar-rich fraction nearly equal to that obtained by enzymic treatmet was obtained.By the treatment of the bud scar-rich preparation with chitinase, the characteristic annular structure disappeared with concomitant decrease of glucosamine content, and at the same time, stainability with fluorescent dye, Brightener, of the annular structure disappeared.It was concluded that chitin is the intrinsic entity which constitutes the annular structure of the bud scar region and can be stained with Brightener.The appearance of stainable structures and buds together with the increase in cellular content of glucosamine was observed during culture of age 0 cells. The emergence of buds was preceded by the appearance of the stainable structures ; the increase of glucosamine content proceeded in accordance with the appearance of the annular structure. The morphological basis of the budding was discussed with special reference to the role of the annular structure.Budding yeast cell leaves a bud scar on the surface of mother cell after the daughter cell separated.Attentions were first directed on the conspicuous morphological feature of the bud scar.Many descriptions were made on its morphological structure either on the isolated cell walls or on the ultrathin sections of intact cells ; these are thoroughly described in several reviews (1-4). The number of bud scars of a cell, which corresponds to how many times the cell underwent budding in the past, can be counted by various means. The bud scars can be easily stained with some fluorescent dyes, so that the counting of stained bud scars under fluorescent microscope is the simplest 23

A simple method for the isolation of single ascospores of the fission yeast Schizosaccharomyces pombe was examined. Single spores in the 7-day-old sporulating culture of a homothallic strain were separated from remaining vegetative cells by isopycnic centrifugation in the linear gradient from 10 to 60% of Urografin solution at 700 X g for 20 min. Protein content of isolated spores was very low as compared with that of vegetative cells. The isolated spores germinated through the following steps when cultured in a liquid medium at 25--35 degrees C; loss of refractility (darkening) under a phase-contrast microscope, spherical growth (swelling), emergence of germ tubes, elongation of germ tubes, cell plate formation, and cell separation. The absorbance at 650 nm of the spore suspension initially decreased, accompanied by darkening of spores, and then increased with spherical growth. The germination rate of isolated spores reached almost 100%.

Commercial baker's yeast cells were fractionated into small and large sized cells by repeated centrifugation, and characterization of the small cells was carried out by comparison with the original cells. The small cell preparation consists of more than 90% of small sized age 0 cells, whose length of major axis ranged from 2.5 to 4.5 ,ce. There was no significant difference in DNA content and nuclear appearance between the small and original cell preparations. Respiratory activities were also essentially the same, but a marked difference was observed not only in RNA content but in MAR-column chromatograms. The amount of rRNA corresponding to 28S rRNA was greatly decreased in small cells and that of unidentified peak III was increased. The large cells whose length of major axis ranged from 4.5 to 6.5 showed excellent synchrony when placed in a synthetic medium. The situation of small cells in the cell division cycle was discussed.The cell population of a budding yeast is heterogeneous as to its divisional age as a consequence of its mode of reproduction.Some observations were reported on physiological characters of the age 0 cell which has not undergone the first budding yet. For example, initiation of budding from the age 0 virgin cell is always somewhat delayed from the second budding of its mother cell in agar-mounted slide culture (1) ; ascospore formation in the sporulation culture was hardly observed in the age 0 cell (2), etc. On the other hand, commercial product of baker's yeast in Japan always contains about 40% of small sized cells bearing no bud scars which are easily verified by the vital staining with fluorescent Brightener (1). From this point, it can be used as an experimental material suitable for characterization of the age 0 cell, if effective method of fractionation is established.The present paper deals with the method of obtaining small sized age 0 cells from a baker's yeast and some of its physiological characters.15

No abstract

It has been known that age 0 cells of Saccharonayces cerevisiae, lacking any bud scar, hardly form any spore, while the cells of age 1 or more sporulate abundantly.Based on this finding we attempted to elucidate at what stage of cell age the age 0 cells acquire sporulation activity. Aliquots of the synchronous culture started from small age 0 cells were transferred to sporulation media at time intervals and sporulation in each culture was examined.Results showed that age 0 cells were endowed with the sporulating ability just before the first bud initiation.The sporulation ability decreased after the first budding and increased again at the stage of the second budding. The fluorescent staining of the resultant asci revealed that most asci derived from cultures just before the initiation of the first budding carried neither bud nor bud scar. It was also observed that the number of spores per ascus changed with the cellular age. Cells in synchronous culture of large cells, most of which bear more than one bud scar showed similar changes in the sporulating ability during their cell cycle. Addition of tomato juice to the culture media preceding sporulation stimulated the sporulating ability.