The cell structure has been studied using light and electron microscopies for centuries, and it is assumed that the whole structure is clarified by now. Little quantitative and three-dimensional analysis of cell structure, however, has been undertaken. We have coined a new word, 'structome', by combining 'structure' and '-ome', and defined it as the 'quantitative and three-dimensional structural information of a whole cell at the electron microscopic level'. In the present study, we performed structome analysis of Saccharomyces cerevisiae, one of the most widely researched biological materials, by using freeze-substitution and serial ultrathin-sectioning electron microscopy. Our analysis revealed that there were one to three mitochondria, ~220 000 ribosomes in a cell, and 13-28 endoplasmic reticula/Golgi apparatus which do not form networks in the cytoplasm in the G1 phase. Nucleus occupied ~10.5% of the cell volume; cell wall occupied ~17%; vacuole occupied ~5.8%; cytoplasm occupied ~64%; and mitochondria occupied only ~1.7% in the G1 phase. Structome analysis of cells would form a base for the post-genome research.

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The G 2 index of the yeast Cryptococcus neoformans determined by laser scanning cytometer was 2^3 times higher than the budding index during transition to the stationary phase of the culture, indicating that buds emerged in the G 2 phase of the cell cycle. To clarify whether buds also emerge in G 2 during exponential growth of the culture, DNA content for each cell was measured with a fluorescence microscope equipped with a photomultiplier. The DNA content of cells having tiny buds varied rather widely, depending on growth phases and strains used. Typically, buds of C. neoformans emerged soon after initiation of DNA synthesis in the early exponential phase. However, bud emergence was delayed to G 2 during transition to the stationary phase, and in the early stationary phase budding scarcely occurred, although roughly half of the cells completed DNA synthesis. Thus, the timing of budding in C. neoformans was actually shifted to later cell cycle points with progression of the growth phase of the culture. ß

, Vladislav Raclavsky aAims. Limited aeration has been demonstrated to cause slowdown in proliferation and delayed budding, resulting eventually in a unique unbudded G2-arrest in the obligate aerobic pathogenic yeast Cryptococcus neoformans. Also, the ability to adapt to decreased oxygen levels during pathogenesis has been identified as a virulence factor in C. neoformans. The aim of this study was to identify and characterize genes that are necessary for the proliferation slowdown and G2-arrest caused by limited aeration. Methods. Random mutants were prepared and screened for lack of typical slowdown of proliferation under limited aeration. The CNAG_00156.2 gene coding for a zinc-finger transcription factor was identified in mutants showing most distinctive phenotype. Targeted deletion strain and reconstituted strain were prepared to characterize and confirm the gene functions. This gene was also identified in a parallel studies as homologous both to calcineurin responsive (Crz1) and PKC1-dependent (SP1-like) transcription factors. Results. We have confirmed the role of the cryptococcal homologue of CRZ1/SP1-like transcription factor in cell integrity, and newly demonstrated its role in slowdown of proliferation and survival under reduced aeration, in biofilm formation and in susceptibility to fluconazole.Conclusions. Our data demonstrate a tight molecular link between slowdown of proliferation during hypoxic adaptation and maintenance of cell integrity in C. neoformans and present a new role for the CRZ1 family of transcription factors in fungi. The exact positioning of this protein in cryptococcal signalling cascades remains to be clarified.