CDKA and CDKB kinases from Chlamydomonas reinhardtii are able to complement cdc28 temperature-sensitive mutants of Saccharomyces cerevisiae.

Cyclin-dependent kinases (CDK) play a key role in coordinating cell division in all eukaryotes. We investigated the capability of cyclin-dependent kinases CDKA and CDKB from the green alga Chlamydomonas reinhardtii to complement a Saccharomyces cerevisiae cdc28 temperature-sensitive mutant. The full-length coding regions of algal CDKA and CDKB cDNA were amplified by RT-PCR and cloned into the yeast expression vector pYES-DEST52, yielding pYD52-CDKA and pYD52-CDKB. The S. cerevisiae cdc28-1N strain transformed with these constructs exhibited growth at 36 degrees C in inducing (galactose) medium, but not in repressing (glucose) medium. Microscopic observation showed that the complemented cells had the irregular cylindrical shape typical for G2 phase-arrested cells when grown on glucose at 36 degrees C, but appeared as normal budded cells when grown on galactose at 36 degrees C. Sequence analysis and complementation tests proved that both CDKA and CDKB are functional CDC28/cdc2 homologs in C. reinhardtii. The complementation of the mitotic phenotype of the S. cerevisiae cdc28-1N mutant suggests a mitotic role for both of the kinases.

Aged mother cells of Saccharomyces cerevisiae show markers of oxidative stress and apoptosis.

Recently, we and others have shown that genetic and environmental changes that increase the load of yeast cells with reactive oxygen species (ROS) lead to a shortening of the life span of yeast mother cells. Deletions of yeast genes coding for the superoxide dismutases or the catalases, as well as changes in atmospheric oxygen concentration, considerably shortened the life span. The presence of the physiological antioxidant glutathione, on the other hand, increased the life span of yeast cells. Taken together, these results pointed to a role for oxygen in the yeast ageing process. Here, we show by staining with dihydrorhodamine that old yeast mother cells isolated by elutriation, but not young cells, contain ROS that are localized in the mitochondria. A relatively large proportion of the old mother cells shows phenotypic markers of yeast apoptosis, i.e. TUNEL (TdT-mediated dUTP nick end labelling) and annexin V staining. Although it has been shown previously that apoptosis in yeast can be induced by a cdc48 allele, by expressing pro-apoptotic human cDNAs or by stressing the cells with hydrogen peroxide, we are now showing a physiological role for apoptosis in unstressed but aged wild-type yeast mother cells.

The influence of oxygen toxicity on yeast mother cell-specific aging.

The effect of deleting both catalase genes and of increased oxygen as well as paraquat (a pro-oxidant) on the replicative life span of yeast mother cells has been investigated to test the so-called oxygen theory of aging. This is well established in higher organisms, but has not been extensively tested in the unicellular yeast model system. Life span determinations were performed in ambient air or in a controlled atmosphere (55% oxygen) and an isogenic series of strains deleted for one or both yeast catalases was used and compared with wild type. In the absence of cellular catalase, increased oxygen caused a marked decrease in life span that could be completely reversed by adding 1 mM GSH, a physiological antioxidant, to the yeast growth medium. In a second unrelated strain, the effects were similar although even the wild type showed a decrease in life span when oxygen was increased. The effect could again be compensated by addition of extracellular GSH. Our results show that manipulating the detoxification of reactive oxygen species has a profound effect on yeast aging. These findings are discussed in the light of recent results relating to oxygen toxicity in the aging process of higher organisms.

GRC5 and NMD3 function in translational control of gene expression and interact genetically.

The yeast gene, GRC5 (growth control), is a member of the highly conserved QM gene family, the human member of which has been associated with the suppression of Wilms' tumor. GRC5 encodes ribosomal protein L10, which is thought to play a regulatory role in the translational control of gene expression. A revertant screen identified four spontaneous revertants of the mutant grc5-1ts allele. Genetic and phenotypic analysis showed that these represent one gene, NMD3, and that the interaction of NMD3 and GRC5 is gene-specific. NMD3 was previously identified as a component of the nonsense-mediated mRNA decay pathway. The point mutations within NMD3 reported here may define a domain important for the functional interaction of Grc5p and Nmd3p.

Mutants in the Saccharomyces cerevisiae RAS2 gene influence life span, cytoskeleton, and regulation of mitosis.

We investigated the phenotypic consequences in Saccharomyces cerevisiae of a disruption allele (ras2::LEU2) and of a dominant mutant form (RAS2ala18,val19) of RAS2. In addition to the phenotypes described earlier for these mutants, we observed a small increase in the life span for the disruption allele and a drastic decrease of life span for the dominant mutant form, as compared with the isogenic wild type. This was found by analyzing these alleles in two different genetic backgrounds with nearly the same results. Life spans were determined by micromanipulating mother cells and counting generations until no further cell division occurred. A morphological analysis of the terminal phenotypes of very old mother cells was performed showing enlarged or rounded cells and in some cases elongated buds, some of which were difficult to separate from the mother cell. This was observed in wild-type cells, as well as mutant cells. However, the dominant RAS2 mutant (but not the wild-type or ras2::LEU2 mutant cells) after 2 days on complex media displayed phenotypes similar to the terminal phenotype of old mothers. A substantial fraction of the cells were enlarged and generated elongated buds, they lost Calcofluor staining of the bud scars, the cell surface appeared folded, the actin cytoskeleton was aberrant, and the mitotic spindle and the cytoplasmic microtubles were defective in their proper orientation, resulting in aberrant mitoses and empty buds. These phenotypic characteristics of the RAS2ala18,val19 mutation could be causative for the previously observed rapid loss of viability of these cells in stationary phase.

Changes in cell wall composition of deformed ras1- cells of Schizosaccharomyces pombe.

Disruption of the Schizosaccharomyces pombe ras1 gene results in a morphological transformation to large spheres, in contrast to wild-type cells which grow as rods. Chemical analysis of isolated cell walls showed no significant changes in saccharide content but an increase in protein and phosphate contents in ras1- walls relative to parent walls. Polymers tightly bound to the cell wall were solubilized by SDS treatment. Several compounds with molar mass ranging from 22 to 130 kDa and more were resolved by gel filtration and SDS-PAGE. Among low-molar-mass species, a component moving as a band at 31 kDa was conspicuous in ras1- cell walls. It was solubilized by heating in Tris-HCl buffer and shown to have a beta-1,3-glucanase activity against laminarin. The level of the enzyme was by 30% higher in the ras1- cell wall than in the wild-type cell wall. This enzyme may participate in the remodelling of the rigid glucan network and account (at least partially) for the aberrant cell shape. The ras1- cell wall contained a high level of charged polymers, especially phosphoproteins, raising the appealing possibility that ras1- is involved in a putative kinase cascade required to sense and respond to external stimuli destined for the cell wall. Although the present study shows that ras1 loss of function and altered cell wall composition are closely linked defects, it has still to be shown that the ras1 protein is directly involved in alterations found in the mutant cell walls.

Fed-batch cultivation of Saccharomyces cerevisiae with increased content of delta 5,7-sterols.

Cultivation of Saccharomyces cerevisiae with a linear nutrient feed was used to test the validity of a mathematical model describing changes in the physiological state of the culture. Markers of these changes were the concentrations of proteins and delta 5,7-sterols in yeast dry mass. The model was used to optimize the production of these sterols with regard to the magnitude and composition of nutrient feed.

Ergosterol synthesis and population analysis of a fed-batch fermentation in Saccharomyces cerevisiae.

Saccharomyces cerevisiae with an increased content of ergosterol or delta 5,7-sterols, growing on a molasses medium with a feed of ethanol and (NH4)2HPO4, was analyzed as to the age of cell population. The analysis was done by centrifugation in a dextran gradient and by a fluorescence-microscopic technique. In the phase of batch fermentation at a mean specific growth rate of 0.22 h-1 daughter cells contained less than 1% ergosterol while the ergosterol content of mother cells depended on the time of cultivation, a maximum level (4%) being found after two generation times. In the fed-batch phase at a mean growth rate of 0.052 h-1, both daughter and mother cells contained about the same amount of ergosterol (4.7-5.5%). Differences between daughter and mother cells are discussed in view of the relationship between the growth rate and the growth cycle.