Initiation of meiosis in cell cycle initiation mutants of Saccharomyces cerevisiae

Control of the initiation of meiosis in yeast was examined in diploids homozygous for one of four different temperature-sensitive mutations that affect “start” of the mitotic cell cycle. Two of the mutations, cdc28 and tra3, bring about deficiencies in the initiation of meiosis, while cdc25 and cdc35 do not prevent initiation of normal meiosis at both permissive and restrictive temperatures. Moreover, diploids homozygous for the latter two mutations are capable of initiating meiosis in rich growth media upon transfer to the high, non-permissive temperature. This unique feature contrasts with the behavior of other yeast strains which require a starvation sporulation medium for initiation of meiosis. It is suggested that the initiation of meiosis includes functions that are shared with “start” of the mitotic cell cycle, as well as functions related to the choice between the two processes. Meiosis in vegetative media at the restrictive temperature (in cdc25 or cdc35 homozygotes) may be important for the study of chemical and physiological phenomena resulting from the meiotic process and not from adaptation to the sporulation medium.     

Regulation of the Saccharomyces cerevisiae Srs2 helicase during the mitotic cell cycle, meiosis and after irradiation

The expression of theSRS2 gene, which encodes a DNA helicase involved in DNA repair inSaccharomyces cerevisiae, was studied using anSRS2-lacZ fusion integrated at the chromosomalSRS2 locus. It is shown here that this gene is expressed at a low level and is tightly regulated. It is cell-cycle regulated, with induction probably being coordinated with that of the DNA-synthesis genes, which are transcribed at the G1-S boundary. It is also induced by DNA-damaging agents, but only during the G2 phase of the cell cycle; this distinguishes it from a number of other repair genes, which are inducible throughout the cycle. During meiosis, the expression ofSRS2 rises at a time nearly coincident with commitment to recombination. Sincesrs2 null mutants are radiation sensitive essentially when treated in G1, the mitotic regulation pattern described here leads us to postulate that either secondary regulatory events limit Srs2 activity to G1 cells or Srs2 functions in a repair mechanism associated with replication.     

Gene conversion at different points in the mitotic cycle of Saccharomyces cerevisiae

Summary In the Saccharomyces cerevisiae mitotic cycle, the timing of radiation-induced gene conversion has been studied using thermosensitive cell division cycle mutants. The cells were found to perform conversion at different G1 or post-replication steps. A lower yield in induction is found during the G2 phase and is explained by the competition for recombinational repair between sister chromatids and homologous chromosomes. The results are discussed in relation to repair.     

Nuclear pore formation and the cell cycle in Saccharomyces cerevisiae

The total number of nuclear pore complexes/nucleus was determined at intervals through the first cycle of synchronous growth in the yeast, Saccharomyces cerevisiae, using electron micrographs of freeze-fracture replicas of nuclei. Nuclear pore number increased in early G0 phase, attaining a plateau by late G0 which was maintained throughout the S phase. This was followed by a second increase at the time of nuclear division. The significance of these changes in relation to other events of the cell cycle is discussed.     

Calcium uptake during the cell cycle of Saccharomyces cerevisiae

Synchronous culture of the budding yeast Saccharomyces cerevisiae was obtained by sucrose density gradient selection with 90–100% of yeast synchronized by using the cells in the bottom. In these adult cells bud emergence is coincident with an increase in calcium uptake at 100 min of the culture, followed by a return to basal values which are maintained until the end of the first cell cycle of study. The phenothiazine derivatives, trifluoperazine and chlorpromazine inhibit bud emergence and trifluoperazine also increases calcium uptake.     

Mitochondrial DNA synthesis during the cell cycle of Saccharomyces cerevisiae

Logarithmically growing cultures of Saccharomyces cerevisiae were separated into fractions of increasing average age by zonal centrifugation. Nuclear and mitochondrial DNA labeled for both long and short periods were examined at different times in the cell cycle following isolation of the DNA on CsCl gradients. The data thus obtained were used to determine the relative times of replication. In S. cerevisiae the synthesis of nuclear and mitochondrial DNA was found to occur at the same time in the cell cycle. Some data indicate that mitochondrial DNA synthesis was completed before nuclear DNA synthesis.     

Rates of protein synthesis through the cell cycle of Saccharomyces cerevisiae

Exponentially growing cells of Saccharomyces cerevisiae were fractionated by centrifugation in isotonic, self-generated gradients of Percoll. Rapidly growing cells, μ = 0.5 × h⁻¹, with nearly equal length of the daughter and the parental cell cycle were fractionated according to a cell cycle-related density variation. In these cells the net rate of protein synthesis varies nearly 2-fold during the cell cycle. Subsequent separations according to cell size revealed that the highest rate is observed during G2 period. Slow-growing cells, μ = 0.2 × h⁻¹, were fractionated on shallow Percoll gradients in a bimodal fashion, primarily as a dense daughter fraction and a composite light fraction. Thereby a marked high rate of protein synthesis in large unbudded daughter cells was revealed. Separations according to cell size revealed a cell cycle-related separation of budded cells, and the highest rate is observed, as before, in the G2 period. Irrespective of the growth rate a non-exponential increase of cell protein is thereby observed through the cell cycle of budding yeast. Septation and cell separation coincide with a low degree of ribosome exploitation.     

Timing of mitochondrial DNA synthesis during meiosis in Saccharomyces cerevisiae

Mitochondrial DNA (mtDNA) synthesis was examined during meiosis in Saccharomyces cerevisiae using an aneuploid strain disomic (n + 1) for chromosome III. The aneuploid has the advantage over true diploid strains in that it completes early meiotic events, including premeiotic chromosome replication, but does not form mature ascospores. Thus, differential extraction problems, resulting from the simultaneous presence of both unsporulated cells and spores in the population, are eliminated. The kinetic of mtDNA synthesis was monitored by determining the actual mtDNA content of cells following analytical CsCl centrifugation of cell extracts. MtDNA synthesis started soon after the cells were placed in sporulation medium and continued at an approximately constant rate until 24 h, resulting in slightly more than a doubling of the mitochondrial DNA content per cell. [¹⁴C]uracil was incorporated into mtDNA during the entire developmental period. Extensive preferential labeling of mtDNA occurred between 24 and 50 h, when no net DNA synthesis was observed.     

Hierarchical coordination of periodic genes in the cell cycle of Saccharomyces cerevisiae

Background  

Gene networks are a representation of molecular interactions among genes or products thereof and, hence, are forming causal networks. Despite intense studies during the last years most investigations focus so far on inferential methods to reconstruct gene networks from experimental data or on their structural properties, e.g., degree distributions. Their structural analysis to gain functional insights into organizational principles of, e.g., pathways remains so far under appreciated.     

Variations in ozone-sensitivity in relation to cell cycle and ploidy in Saccharomyces cerevisiae

Summary Cyclic variations in resistance to ozone-inactivation were observed during the cell cycle of a haploid strain of Saccharomyces cerevisiae. The lag and G₁ phases showed the highest sensitivity to ozone whereas there was a progressive increase in resistance from the S to G₂ phases as the proportion of dividing cells increased. A related series of S. cerevisiae varying in ploidy from haploid to tetraploid was exposed to ozone. The results demonstrated clearly an enhancement of ozone resistance directly related to the ploidy of the cells.Taken together these data suggest that resistance to ozone is dependent upon the action of an efficient repair mechanism. Similarities and differences between the biological action of ozone and ionizing radiations in S. cerevisiae are discussed.     

Calcium and Saccharomyces cerevisiae

The claim that Ca may be a dispensable element for yeast Saccharomyces cerevisiae has been reexamined. The cells of S. cerevisiae could grow in media which contained no added Ca and were deprived of contaminating Ca²⁺ by filtration through a Chelex 100 column. Also, the cells were able to grow in the presence of fairly high concentrations of EGTA. The apparent intracellular concentrations of Ca, assessed from the content of radioactive ⁴⁵Ca in cells preloaded with ⁴⁵CaCl₂, could vary within the range of approx. 2 nM to 2.8 mM, without adversively affecting growth or morphology of the cells. An extremely low affinity for Ca²⁺ of the system taking up Ca into the cells was corroborated. However, even the Chelex 100-treated media were found in contain 1–5 μM Ca when maintained in glass culture vessels. Also, the ability of the cells to take up Ca from a medium containing surplus of EGTA or EDTA was demonstrated. su14CEDTA, alone or in the presence of Ca, could also be transported into the cells. It has been inferred that Ca must be as essential for yeast as it is for other eucaryotic organisms. The omnipresence of contaminating Ca and peculiarities of the Ca transporting system, combined with an intricate intracellular compartmentation of Ca, would account for the impossibility to prove the importance of Ca for yeast by direct growth studies.     

Estimation of the length of cell cycle phases from asynchronous cultures of Saccharomyces cerevisiae

A method is described which allows estimation of the lengths of the cell cycle phases of Saccharomyces cerevisiae from asynchronous culture data. It is based upon a DNA labelling and nuclear staining procedure which is related to known features of yeast physiology. Estimates obtained indicate that the lengths of the periods of DNA synthesis (S phase) and mitosis (M phase) are relatively independent of the carbon source on which the culture was grown, whilst the lengths of the G1 and G2 phases show considerable variation.     

The isolation of nonsense mutations in cell division cycle genes of the yeast Saccharomyces cerevisiae

Summary Nonsense mutations have been isolated in cell division cycle genes in a strain of Saccharomyces cerevisiae that carries a temperature-sensitive amber suppressor. These mutants may be valuable in identifying the products of genes involved in the cycle and in determining the pattern of their synthesis during the cell cycle.     

Histones from Saccharomyces cerevisiae

Total histones and histone fractions isolated from Saccharomyces cerevisiae chromatin were analysed by polyacrylamide gel electrophoresis. The presence of the four histone fractions H2a, H2b, H3 and H4 was demonstrated. In addition, yeast chromatin contained a protein similar to histone H1 from mammals in molecular weight, charge and association properties with Triton X-100. However, it had a much lower lysine to arginine ratio, equal to about 3, as compared with H1 histones from higher eukaryotes. The order of electrophoretic mobilities of yeast histone fractions in acidic urea-polyacrylamide gels was similar to that observed for histones from plant sources, i.e. H4>H3>H2a>H2b>H1. Previously undetected protein (protein X) was extracted from yeast chromatin with 5 % HClO₄. The properties of this protein are under investigation.     

Synchronous cell growth occurs upon synchronizing the two regulatory steps of the Saccharomyces cerevisiae cell cycle

There are two known asynchronous steps in the budding yeast Saccharomyces cerevisiae cell cycle, where an asynchronous step is one which is completed in different lengths of time by different cells in an isogenic population. It is shown here that elimination of the asynchrony due to cell size by preincubation of cells with the mating pheromone alpha-factor, and decreasing the asynchrony in the cdc28 'start' step by lowering the pH, yields highly synchronous cell growth measured as the time period between the emergence of buds. In one experiment, cell budding for 92% of cells occurred within a 12-min period for at least two generations. Under identical conditions, cell number increase is not as synchronous as bud emergence indicating that there is a third asynchronous step, which is concluded to be at cell separation. These results are consistent with there being two--and only two--asynchronous steps in the cell cycle, measured from bud emergence to bud emergence. Surprisingly, these two steps are also the two major regulatory steps of the cell cycle. It is concluded that asynchrony may be a general feature of cell cycle regulatory steps. The asynchrony in the completion of the cdc28 'start' step which occurs in the first cell cycle after alpha-factor washout is shown here to be almost or entirely eliminated for the second passage through this step after alpha-factor washout. The 'true' time between the onset of budding and the point where 50% of cells have budded (called t50BE) is 17 and less than or equal to 2 min for the first and second budding, respectively, after alpha-factor washout. The cell cycle models requiring a transition probability, or asynchrony, at 'start' for every cell cycle are therefore incorrect.     

Prezygotic reproductive isolation between Saccharomyces cerevisiae and Saccharomyces paradoxus

Background  

Matings between different Saccharomyces sensu stricto yeast species produce sexually sterile hybrids, so individuals should avoid mating with other species. Any mechanism that reduces the frequency of interspecific matings will confer a selective advantage. Here we test the ability of two closely-related Saccharomyces sensu stricto species to select their own species as mates and avoid hybridisation.     

CDC7 protein kinase activity is required for mitosis and meiosis in Saccharomyces cerevisiae

Summary The product of the CDC7 gene of Saccharomyces cerevisiae has multiple cellular functions, being needed for the initiation of DNA synthesis during mitosis as well as for synaptonemal complex formation and commitment to recombination during meiosis. The CDC7 protein has protein kinase activity and contains the conserved residues characteristic of the protein kinase catalytic domain. To determine which of the cellular functions of CDC7 require this protein kinase activity, we have mutated some of the conserved residues within the CDC7 catalytic domain and have examined the ability of the mutant proteins to support mitosis and meiosis. The results indicate that the protein kinase activity of the CDC7 gene product is essential for its function in both mitosis and meiosis and that this activity is potentially regulated by phosphorylation of the CDC7 protein.     

Petite-negative mutants of Saccharomyces cerevisiae

Summary A series of yeast mutants has been isolated with the inability to grow on fermentable carbon sources whilst growing normally on ethanol media. One of the mutants, namely MC16/206 lacks pyruvate decarboxylase activity and does not grow on glucose at 37°C but grows on both ethanol and glucose at 27°C. In this strain rho ^- petites are non-viable.