Yeast Saccharomyces cerevisiae Cells

Water as the unique natural medium for native cells contribntes a major portion of their weight. Water (with dissolved inorganic and organic compounds) can penetrate into or from cells throngh membrane channels or by transport protein molecules (e.g., aquaporins) (Finkelstein 1987, Murata et al. 2000, Vorisek 2000, Fujiyoshi et al. 2002). The length of transmembrane channels 

Nuclear Magnetic Resonance Studies of Interfacial Phenomena 

FIGURE 7.1 (continued) Size matters, (c) Overview of water metabolism in cells. Transport of water across 

FIGURE 7.2 300 MHz water-suppressed H NMR spectra of a freshly excised liver sample obtained from 

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It was tempting to base the study of membrane transport in eukaryotic cells on similar simple principles. For this purpose, as well as for molecular biology as a whole, the yeast Saccharomyces cerevisiae appeared to be the best suited organism. From early times on, this yeast has occupied a privileged place for mankind. Due to... 

Fitch, I., Dahmann, C., Surana, U., Amon, A., Nasmyth, K., Goetsch, L., Byers, B., and Futcher, B. (1992). Characterization of four B-type cyclin genes of the budding yeast Saccharomyces cerevisiae. Mol. Biol. Cell 3 805-818. 

Fosburg, S. L., and Nurse, P. (1991). Cell cycle regulation in the yeasts Saccharomyces cerevisiae and Schizosaccaromycespombe. Annu, Rev. Cell Biol. 7 227-256. 

Spellman, P. T., Sherlock, G., Zhang, M. Q., Iyer, V. R., Anders, K., Eisen, M. B., Brown, P. O., Bot-stein, D. and Futcher, B. (1998), Comprehensive identification of cell cycle-regulated genes of the yeast Saccharomyces cerevisiae by microarray hybridization , Mol Biol. Cell, 9, 3273-3297. 

There are numerous protocols for polysomal gradients preparations that differ mainly at the step for harvesting the cells, and the gradient composition and separation times. The protocol presented later was optimized for isolation of polysomal mRNA from the yeast Saccharomyces cerevisiae, yet many steps will be similar to other eukaryotes and the procedure can easily be modified for other organisms. We will use this protocol as a template on which we will indicate and highlight points that are critical for the microarray analysis. Generally, the RNA isolated by this protocol can be used for analysis by DNA microarray, Northern blot, or RT-PCR. 

GFP has also been proposed as a successor to the Ames and SOS chromotest. Billinton et al. [8] obtained a reporter system, employed as genotoxicity biosensor, that uses eukaryotic cells (the baker yeast Saccharomyces cerevisiae) instead of bacteria. The strain produces green fluorescent protein, codon optimized for yeast, when DNA damage has occurred. It was demonstrated that the reporter does not falsely respond to chemicals that delay mitosis, and responds appropriately to the genetic regulation of DNA repair. 

Weeks et al. 1979) or DNA damage (Nakamura et al. 1987) following hexachloroethane treatment. Similar results were reported for eukaryotic cells. Hexachloroethane did not cause gene mutation in cells harvested from the stationary growth phase (Bronzetti et al. 1989) or DNA damage in yeast (Saccharomyces cerevisiae) (Simmon and Kauhanen 1978), chromosomal aberrations in fungi (Aspergillus nidulans) (Crebelli et al. 

Our approach recently developed utilizes DNA repair- or recombination-deficient mutants of the yeast Saccharomyces cerevisiae [8]. An important feature of many tumor cells is that they have defects in their ability to repair damage to DNA as compared with normal cells, suggesting that agents with selective toxicity towards repair-deficient cells might be potential anticancer agents. 

Histone kinases responsible for N-phosphorylation have been isolated from regenerating rat liver [109] and Walker-256 carcinosarcoma cells [110]. One kinase with a pH optimum of 9.5 phosphorylated His-18 and His-75 of H4, while the other with a pH optimum of 6.5 phosphorylated lysine of HI. The enzyme from regenerating rat liver phosphorylated H4 at 1-phosphoryl histidine, while the carcinosarcoma enzyme phosphorylated H4 His at the position 3 [111]. Both kinases were cAMP independent [110]. Matthews and colleagues purified a 32-kDa histidine H4 kinase from yeast, Saccharomyces cerevisiae [112,113]. The enzyme phosphorylated His-75 (1-phosphoryl histidine) in H4. His-18 of H4 and other histidines in other core histones were not phosphorylated by this kinase [112]. Protein phosphatases 1, 2A, and 2C could dephosphorylate His-75 of H4 [114]. Applying a gel kinase approach to detect mammalian H4 histidine kinases, Besant and Attwood detected four activities in the 34-41 kDa range with extracts from porcine thymus [115]. 

Davis, J.L., Kunisawa, R., and Thorner, J. (1992) A presumptive helicase (MOTl gene product) affects gene expression and is required for viability in the yeast Saccharomyces cerevisiae. Mol. Cell. Biol. 12, 1879-1892. 

DePace AH, Santoso A, Hillner P, Weissman JS (1998) A critical role for amino-terminal glu-tamine/asparagine repeats in the formation and propagation of a yeast prion. Cell 93 1241-1252 Didichenko SA, Ter-Avanesyan MD, Smirnov VN (1991) Ribosome-bound EF-1 alpha-like protein of yeast Saccharomyces cerevisiae. Eur J Biochem 198 705-711 Dong H, Kurland CG (1995) Ribosome mutants with altered accuracy translate with reduced processivity. J Mol Biol 248 551-561... 

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