Production of heat shock protein is independent of cell cycle blockage in the yeast Saccharomyces cerevisiae.

In response to certain environmental stresses, cells display a response characterized by the production of heat shock proteins. In this study we showed that blockage of cells of the yeast Saccharomyces cerevisiae at specific points in the mitotic cell cycle was not in itself a stress that induced the production of heat shock proteins. Nevertheless, cell cycle blockage did not preclude a normal heat shock response in arrested cells subjected to elevated temperatures.     

Control of the yeast cell cycle by protein synthesis

The increased synthesis of ribosomal RNA (rRNA) is correlated with enhanced cell proliferation, and it has been suggested that rRNA metabolism may have a regulatory role in the progression of the cell cycle. Alternatively, it might be the ensuing more active protein synthesis that drives the cell cycle progression. We have found that treatment with low doses of cycloheximide dissociates rRNA and protein synthesis. In fact, after the addition of cycloheximide the protein synthesis rate is strongly inhibited, whereas the rate of rRNA synthesis is unaffected for some time. The progression of the cell cycle, monitored as analysis of DNA distribution by flow cytometry and as bud emergence, is quickly and largely inhibited, thus indicating that a sustained rRNA metabolism is not sufficient to allow continuous cycle progression. The effects of cycloheximide on the daughter and mother duplication times, on the mean cell volume, and on the volume at budding were also analyzed. The results suggest that protein synthesis, rather than rRNA synthesis, may have a key role in the control of cell cycle progression in Saccharomyces cerevisiae.     

Synthesis of ribosomal proteins during the cell cycle of the yeast Saccharomyces cerevisiae.

Centrifugal elutriation was used to separate yeast cells by their cell cycle position. The rate of synthesis of ribosomal proteins showed a constant exponential increase through the cell cycle.     

Nuclear envelope transport capacity and the cell cycle in yeast (Saccharomyces cerevisiae).

Changes in the nuclear envelope transport capacity, as measured by the number of nuclear pore complexes/unit nuclear volume/cell, were followed during the Saccharomyces cerevisiae cell cycle using data obtained by freeze-fracture electron microscopy. Pore number per unit nuclear volume decreased sharply in early G0, remained steady from mid-GO through S to G2, and showed a further slight decrease at M and G1. These periods of decline apparently resulted from nuclear enlargement without sufficient formation of new nuclear pore complexes to maintain the pore number to nuclear volume ratio. However, marked nuclear pore formation did accompany both increases in nuclear volume. The significance of these changes in relation to other events in the cell cycle is discussed. The validity of using nuclear pore number/unit nuclear volume and other pore number data as indices of nuclear envelope transport capacity and cell activity is critically examined.     

Telomeric protein distributions and remodeling through the cell cycle in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, telomeric DNA is protected by a nonnucleosomal protein complex, tethered by the protein Rap1. Rif and Sir proteins, which interact with Rap1p, are thought to have further interactions with conventional nucleosomic chromatin to create a repressive structure that protects the chromosome end. We showed by microarray analysis that Rif1p association with the chromosome ends extends to subtelomeric regions many kilobases internal to the terminal telomeric repeats and correlates strongly with the previously determined genomic footprints of Rap1p and the Sir2-4 proteins in these regions. Although the end-protection function of telomeres is essential for genomic stability, telomeric DNA must also be copied by the conventional DNA replication machinery and replenished by telomerase, suggesting that transient remodeling of the telomeric chromatin might result in distinct protein complexes at different stages of the cell cycle. Using chromatin immunoprecipitation, we monitored the association of Rap1p, Rif1p, Rif2p, and the protein component of telomerase, Est2p, with telomeric DNA through the cell cycle. We provide evidence for dynamic remodeling of these components at telomeres.     

Phospholipid accumulation during the cell cycle in synchronous cultures of the yeast, Saccharomyces cerevisiae

Phospholipid concentrations have been examined throughout successive cell cycles in synchronously growing cultures of the yeast, Saccharomyces cerevisiae. Total phospholipid phosphorus, as well as lecithin and phosphatidylethanolamine levels, exhibited stepwise increases during the cell cycle with step increments beginning just prior to new rounds of bud formation. Phosphatidylinositol and phosphatidylserine levels, on the other hand, showed what have been interpreted to be peak concentrations near the time of bud formation. Cardiolipin content varied considerably and was dependent upon the carbon source of the growth medium. Glucose-grown cells exhibited peak concentrations of cardiolipin near the time of bud formation, with marked decreases after this time. In contrast, galactose-grown synchronous cells exhibited stepwise increments in cardiolipin content, with step increases occurring near the time of new rounds of bud formation. Step or peak increases in cardiolipin, as well as all other phospholipids, were found to coincide with the time of stepwise increases in cytochrome c oxidase activity in these cells. No correlations were observed between the elaboration of mitochondrial membranes during the synchronous cell cycle and the observed patterns of phospholipid increase.     

Immunological evidence for the involvement of cell wall proteins in phosphate uptake in the yeast Saccharomyces cerevisiae

Immunological cross-reactivity between cell wall proteins obtained from two yeast genera (Candida tropicalis and Saccharomyces cerevisiae) is reported. Specific retention of two cell wall proteins from Saccharomyces cerevisiae by an immunoabsorbent column coupled with antibodies against phosphate binding protein 2 (PiBP2) from Candida tropicalis allowed to generate antibodies against the proteins from S. cerevisiae. These antibodies were effective in inhibiting phosphate uptake by S. cerevisiae cells. The proteins from S. cerevisiae displayed a phosphate binding activity which was inhibited in the presence of the forementioned antibodies. These results and the observation that the amount of these proteins in the shock fluid was dependent of the growth conditions (i.e., in the presence or in the absence of phosphate) support the idea that these proteins are involved in the high affinity phosphate transport system.Abbreviations Pi inorganic phosphate - PiBP2 phosphate binding protein 2 obtained from Candida tropicalis - Tris Tris(hydroxymethyl)-aminoethane - MES [2-(N-Morpholino)] ethanesulfonic acid - EDTA ethylene diamine tetraacetic acid, disoldium salt - PMSF phenylmethyl sulfonyl fluoride - SDS sodium dodecyl sulfate - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Estimating the per-base-pair mutation rate in the yeast Saccharomyces cerevisiae

Although mutation rates are a key determinant of the rate of evolution they are difficult to measure precisely and global mutations rates (mutations per genome per generation) are often extrapolated from the per-base-pair mutation rate assuming that mutation rate is uniform across the genome. Using budding yeast, we describe an improved method for the accurate calculation of mutation rates based on the fluctuation assay. Our analysis suggests that the per-base-pair mutation rates at two genes differ significantly (3.80x10(-10) at URA3 and 6.44x10(-10) at CAN1) and we propose a definition for the effective target size of genes (the probability that a mutation inactivates the gene) that acknowledges that the mutation rate is nonuniform across the genome.     

Spindle dynamics and cell cycle regulation of dynein in the budding yeast, Saccharomyces cerevisiae

We have used time-lapse digital- and video-enhanced differential interference contrast (DE-DIC, VE-DIC) microscopy to study the role of dynein in spindle and nuclear dynamics in the yeast Saccharomyces cerevisiae. The real-time analysis reveals six stages in the spindle cycle. Anaphase B onset appears marked by a rapid phase of spindle elongation, simultaneous with nuclear migration into the daughter cell. The onset and kinetics of rapid spindle elongation are identical in wild type and dynein mutants. In the absence of dynein the nucleus does not migrate as close to the neck as in wild-type cells and initial spindle elongation is confined primarily to the mother cell. Rapid oscillations of the elongating spindle between the mother and bud are observed in wild-type cells, followed by a slower growth phase until the spindle reaches its maximal length. This stage is protracted in the dynein mutants and devoid of oscillatory motion. Thus dynein is required for rapid penetration of the nucleus into the bud and anaphase B spindle dynamics. Genetic analysis reveals that in the absence of a functional central spindle (ndcl), dynein is essential for chromosome movement into the bud. Immunofluorescent localization of dynein-beta- galactosidase fusion proteins reveals that dynein is associated with spindle pole bodies and the cell cortex: with spindle pole body localization dependent on intact microtubules. A kinetic analysis of nuclear movement also revealed that cytokinesis is delayed until nuclear translocation is completed, indicative of a surveillance pathway monitoring nuclear transit into the bud.     

Relationship between cytoplasmic and mitochondrial apparatus of protein synthesis in yeast Saccharomyces cerevisiae

A conditional respiratory deficiency in yeast Saccharomyces cerevisiae is expressed as a result of a nuclear mutation in sup1 and sup2 genes (II and IV chromosomes, respectively), coding for a component of cytoplasmic ribosomes (Ter-Avanesyan et al. 1982). One such strain is studied here in detail. The strain is temperature-dependent and expresses a respiratory deficient phenotype at 20 degrees C but not at 30 degrees C. Moreover, the strain is simultaneously chloramphenicol-dependent and is able to grow on media containing glycerol or ethanol as a sole carbon source only in the presence of the drug. Chloramphenicol has a differential effect on protein synthesis in mitochondria of the parent strain and the mutant. Since chloramphenicol is a ribosome-targeting antibiotic we suggest that the differential effect of the drug on parent and mutant mitochondrial protein synthesis is due to the altered properties of mito-ribosomes of the mutant compared to those of the parent strain. Mitochondria of the mutant synthesize all the mitochondrially encoded polypeptides, however, in significantly lowered amounts. A suggestion is put forward for the existence of a common component (a ribosomal protein) for mito and cyto-ribosomes.     

Synthesis and modification of proteins during the cell cycle of the yeast Saccharomyces cerevisiae.

We have used a novel technique to study the synthesis, modification and degradation of proteins during the cell cycle in Saccharomyces cerevisiae. Logarithmically growing cells were pulse-labeled twice, with the pulses separated in time by more than one generation. Subsequently, the cells were fractionated as to their position in the cell cycle by centrifugal elutriation, and for different proteins the ratio of radioactive material from the two pulses was then determined. Periodic degradation, synthesis, or modification would produce periodic variations in the ratio of counts. Two-dimensional gel electrophoresis was used to examine 110 different proteins at different times of the cell cycle. All but two proteins had a constant ratio of counts through the cell cycle. This indicates that the rate of synthesis of individual proteins increases exponentially during the cell cycle and that periodic degradation or modification of proteins is not a general feature of the cell cycle in S. cerevisiae.     

Functions of microtubules in the Saccharomyces cerevisiae cell cycle

We used the inhibitor nocodazole in conjunction with immunofluorescence and electron microscopy to investigate microtubule function in the yeast cell cycle. Under appropriate conditions, this drug produced a rapid and essentially complete disassembly of cytoplasmic and intranuclear microtubules, accompanied by a rapid and essentially complete block of cellular and nuclear division. These effects were similar to, but more profound than, the effects of the related drug methyl benzimidazole carbamate (MBC). In the nocodazole-treated cells, the selection of nonrandom budding sites, the formation of chitin rings and rings of 10-nm filaments at those sites, bud emergence, differential bud enlargement, and apical bud growth appeared to proceed normally, and the intracellular distribution of actin was not detectably perturbed. Thus, the cytoplasmic microtubules are apparently not essential for the establishment of cell polarity and the localization of cell-surface growth. In contrast, nocodazole profoundly affected the behavior of the nucleus. Although spindle-pole bodies (SPBs) could duplicate in the absence of microtubules, SPB separation was blocked. Moreover, complete spindles present at the beginning of drug treatment appeared to collapse, drawing the opposed SPBs and associated nuclear envelope close together. Nuclei did not migrate to the mother-bud necks in nocodazole-treated cells, although nuclei that had reached the necks before drug treatment remained there. Moreover, the double SPBs in arrested cells were often not oriented toward the budding sites, in contrast to the situation in normal cells. Thus, microtubules (cytoplasmic, intranuclear, or both) appear to be necessary for the migration and proper orientation of the nucleus, as well as for SPB separation, spindle function, and nuclear division.