Inhibiton by sulfanilamide of sporulation in Saccharomyces cerevisiae.

The antimetabolite sulfanilamide inhibits sporulation in Saccharomyces cerevisiae strain AP1. Cells exposed to sulfanilamide at various times during the sporulation process become progressively insensitive to the drug, although accumulation of sulfanilamide by the cells increases with time. Vegetative growth of AP1 is practically unaffected by sulfanilamide; pregrowth of the cells in the presence of the drug does not prevent sporulation. Thus, inhibition is confined to the meiotic phase of the cell cycle. Sensitivity to sulfanilamide is independent of pH. Increasing the time cells are exposed to sulfanilamide results in a progressive reduction of ascus formation; however, the inhibition is reversible since sporulation can occur in cells exposed to the drug for greater than 24 h. The drug arrests the cells at a point before commitment to sporulation, since yeast cells exposed to sulfanilamide for 12 h do not complete the sporulation process when returnedto vegetative medium, but resume mitotic growth instead. Meiotic nuclear division is largely prevented by sulfanilamide, and synthesis of RNA and protein is severely retarded. DNA synthesis is inhibited up to 50%; glycogen synthesis is approximately 90% inhibited. Other yeast strains showed varying sensitivity to sulfanilamide; homothallic strains were generally less affected.     

Synthesis of beta-glucanases during sporulation in Saccharomyces cerevisiae: formation of a new, sporulation-specific 1,3-beta-glucanase.

A biphasic synthesis of 1,3-beta-glucanase occurred when cells of Saccharomyces cerevisiae AP-1 (a/alpha) were incubated in sporulation medium. The capacity to degrade laminarin increased very slowly during the first 7 h but at a much faster rate thereafter. Changes occurring during the first period were not sporulation specific since the moderate increase in activity against laminarin was insensitive to glutamine and hydroxyurea and also took place in the nonsporulating strain S. cerevisiae AP-1 (alpha/alpha). However, the changes taking place after 7 h must be included in the group of sporulation-specific events since they were inhibited by glucose, glutamine, and hydroxyurea and did not occur in the nonsporulating diploid. Consequently, only when the cells had been incubated for at least 7 h in sporulation medium did full induction of activity against laminarin take place upon shift to a medium which favored vegetative growth. Changes in the relative proportions of the vegetative glucanases, namely, endo- and exo-1,3-beta-glucanase, and the formation of a new sporulation-specific 1,3-beta-glucanase account for the observed events and are the consequence of the expression of the sporulation program.     

Changes in regulation of ribosomal protein synthesis during vegetative growth and sporulation of Saccharomyces cerevisiae.

When diploid Saccharomyces cerevisiae cells logarithmically growing in acetate medium were placed in sporulation medium, the relative rates of synthesis of 40 or more individual ribosomal proteins (r-proteins) were coordinately depressed to approximately 20% of those of growing cells. These new depressed rates remained constant for at least 10 h into sporulation. If yeast nitrogen base was added 4 yh after the beginning of sporulation to shift the cells back to vegetative growth, the original relative rates of r-protein synthesis were rapidly reestablished. this upshift in the rates occurred even in diploids homozygous for the regulatory mutation rna2 at the restrictive temperature for this mutation (34 degrees C). However, once these mutant cells began to bud and grow at 34 degrees C, the phenotype of rna2 was expressed and the syntheses of r-proteins were again coordinately depressed. At least one protein whose rate of synthesis was not depressed by rna2 in vegetative cells did have a decreased rate of synthesis during sporulation. Another r-protein whose synthesis was depressed by rna2 maintained a high rate of synthesis at the beginning of sporulation. These data suggest that the mechanism responsible for coordinate control of r-protein synthesis during sporulation does not require the gene product of RNA2 and thus defines a separate mechanism by which r-proteins are coordinately controlled in S. cerevisiae.     

Relationship of Glycolytic Intermediates, Glycolytic Enzymes, and Ammonia to Glycogen Metabolism During Sporulation in the Yeast Saccharomyces cerevisiae

To identify the factors which control glycogen synthesis in Saccharomyces cerevisiae, we have studied the regulation of glycogen metabolism during sporulation, since in vivo glycogen has been reported to undergo significant changes in concentration during this process. We examined the concentration of a number of key glycolytic intermediates and enzymes in strains that sporulate at different rates and those that are deficient in sporulation. There were no significant changes found in the adenylate energy charge or cyclic AMP levels throughout sporulation. Although significant alterations occurred in the levels of glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate, phosphoenolpyruvate, and ATP during sporulation, only the fourfold increase in fructose-1,6-bisphosphate appeared to correlate with glycogen synthesis in all of the strains examined. Only limited changes occurred in the level of a number of glycolytic and gluconeogenic enzymes which were examined during this process. Intracellular glucose content underwent a dramatic 30- to 40-fold increase in sporulating cells. Comparison of strains with different rates of sporulation demonstrated that this increase in glucose content coincides with the time of glycogen degradation in each strain. Both the increase in glucose content and the degradation of accumulated glycogen were not observed in nonsporulating alpha/alpha strains, or in cells incubated in NH(4) (+) supplemented sporulation medium. Although glucose appears to be the direct product of glycogen degradation, a 10-fold increase in a nonspecific alkaline phosphatase occurs at this time, which may be degrading phosphorylated sugars to glucose. All of the strains examined released extracellular glucose while suspended in acetate sporulation medium. It is concluded that most of the changes in the glycolytic pathway that occur during sporulation, with the exception of glycogen degradation and the concomitant increase in intracellular glucose pools, are a response to the transfer to sporulation medium and are independent of sporulation-specific processes. Inhibition of sporulation with ammonium ions resulted in a different pattern of change in all of the glycolytic intermediates examined, including a twofold increase in cyclic AMP levels. Ammonia did not interfere with glycogen synthesis, but prevented sporulation-specific glycogen degradation. The levels of the glycolytic enzymes examined were not affected by ammonia.     

Comparative studies on sporulation-promotive actions on cyclic AMP, theophylline and caffeine in Saccharomyces cerevisiae.

Cyclic AMP, theophylline and caffeine promoted sporulation when added to a presporulation medium containing glucose. Caffeine promoted sporulation even when added to a presporulation medium containing acetate as the carbon source, but cyclic AMP and theophylline did not. Caffeine did not increase the intracellular cyclic AMP level, while theophylline did significantly when added to a presporulation medium containing glucose. Caffeine inhibited the vegatative DNA synthesis with little effect on RNA and protein synthesis, resulting in the increase in cell volume, dry weight, and RNA and protein contents, but cyclic AMP and theophylline did not show such effects.     

Developmental regulation of a sporulation-specific enzyme activity in Saccharomyces cerevisiae.

An alpha-glucosidase activity (SAG) occurs in a/alpha Saccharomyces cerevisiae cells beginning at about 8 to 10 h after the initiation of sporulation. This enzyme is responsible for the rapid degradation of intracellular glycogen which follows the completion of meiosis in these cells. SAG differs from similar activities present in vegetative cells and appears to be a sporulation-specific enzyme. Cells arrested at various stages in sporulation (DNA replication, recombination, meiosis I, and meiosis II) were examined for SAG activity; the results show that SAG appearance depends on DNA synthesis and some recombination events but not on the meiotic divisions.     

Effect of yeast extract,peptone, and certain nitrogen compounds on sporulation ofSaccharomyces cerevisiae

Summary Aeration of cells for 24 hrs. previous to placing them in 0.1% sodium acetate solution diminished sporulation, but this decrease was overcome by the addition of 0.1% yeast extract to the acetate solution. Cells starved by growth on Czapek solution agar +0.03% peptone formed very few ascospores in acetate solution. The addition of yeast extract or peptone in low content to the acetate solution increased the yields. However, the cells did not form as many ascospores as well-nourished cells in acetate solution.A comparison was made of the sporulation of cells from basal presporulation medium containing, separately, 18 nitrogen sources. In general, nitrogen sources that supported growth gave cells that sporulated well. Tyrosine and tryptophan were exceptions.Cells multiplied in basal medium with the nitrogen source deleted formed no asci in 0.1% acetate solution. When nitrogen sources were added to the acetate solution, many stimulated sporulation. Yields of asci in these sporulation cultures were, however, lower than the yield obtained from well-nourished cells in 0.1% acetate solution.Based on a thesis submitted byJ. H. Tremaine in May, 1953, to McMaster University in partial fulfilment of the requirements for the degree of Master of Science.     

Double-strand breaks can initiate meiotic recombination in S. cerevisiae

We investigated the effects of double-strand breaks on meiotic recombination in yeast. A double-strand break was introduced at the MATa locus by sporulation of a MAT alpha inc/MATa diploid under inducing conditions for the HO-encoded endonuclease; 14% of the resulting tetrads had undergone 4 alpha:0a conversion. Conversion at MAT was associated with co-conversion of a closely linked marker and an increased recombination frequency for flanking markers. We also studied the sporulation products of a diploid heterozygous at the HIS4 locus for an insertion of a 100 bp fragment of MATa containing the HO endonuclease cut site. Under inducing conditions, a significant number of tetrads were formed that had undergone gene conversions in favor of the HIS4+ allele. Although double-strand breaks can initiate meiotic recombination in yeast, the data suggest that they do not normally do so.     

Induction of multispored asci in two-spored strains of Saccharomyces cerevisiae by amitrole.

Although growth of two yeast strains characterized by consistent production of two diploid spores per ascus was inhibited in complex presporulation media containing amitrole, a fraction of the cells produced were able to form asci with more than two spores after transfer to acetate sporulation medium. Cells grown in a defined presporulation medium containing amitrole did not acquire this ability. The increase in spore numbers per ascus is attributed either to the induction by amitrole in growth medium of cells with more than one nucleus or to the restoration of normal meioses in the multispored asci.     

Differential amino acid requirements for sporulation in Bacillus subtilis

The amino acid requirements for sporulation were studied by use of auxotrophic mutants of Bacillus subtilis 168. Cells were grown to T(0) in medium containing the test amino acid and were then transferred to a minimal medium lacking that amino acid. Omission of leucine caused no reduction in sporulation. Omission of methionine, lysine, and phenylalanine appeared to cause reduced levels of sporulation, and sporulation was completely inhibited when isoleucine, tryptophan, and threonine were omitted. The amino acids in this third class showed a sequence of requirements, with tryptophan required earlier than isoleucine, which in turn was required earlier in the sporulation process than threonine. Isoleucine omission did not affect the early sporulation functions of extracellular protease formation or septum formation, but prevented the increased levels of protein synthesis and oxygen consumption that normally accompany early sporulation stages. Isoleucine did not appear to be metabolized to other compounds in significant amounts during sporulation. The role of isoleucine in the sporulation process remains unclear.     

Evidence for cooperation between cells during sporulation of the yeast Saccharomyces cerevisiae.

Diploid Saccharomyces cerevisiae cells heterozygous for the mating type locus (MATa/MAT alpha) undergo meiosis and sporulation when starved for nitrogen in the presence of a poor carbon source such as potassium acetate. Diploid yeast adenine auxotrophs sporulated well at high cell density (10(7) cells per ml) under these conditions but failed to differentiate at low cell density (10(5) cells per ml). The conditional sporulation-deficient phenotype of adenine auxotrophs could be complemented by wild-type yeast cells, by medium from cultures that sporulate at high cell density, or by exogenously added adenine (or hypoxanthine with some mutants). Adenine and hypoxanthine in addition to guanine, adenosine, and numerous nucleotides were secreted into the medium, each in its unique temporal pattern, by sporulating auxotrophic and prototrophic yeast strains. The major source of these compounds was degradation of RNA. The data indicated that differentiating yeast cells cooperate during sporulation in maintaining sufficiently high concentrations of extracellular purines which are absolutely required for sporulation of adenine auxotrophs. Yeast prototrophs, which also sporulated less efficiently at low cell density (10(3) cells per ml), reutilized secreted purines in preference to de novo-made purine nucleotides whose synthesis was in fact inhibited during sporulation at high cell density. Adenine enhanced sporulation of yeast prototrophs at low cell density. The behavior of adenine auxotrophs bearing additional mutations in purine salvage pathway genes (ade apt1, ade aah1 apt1, ade hpt1) supports a model in which secretion of degradation products, uptake, and reutilization of these products is a signal between cells synchronizing the sporulation process.     

Comparative studies on sporulation-promotive actions of cyclic AMP,theophylline and caffeine in Saccharomyces cerevisiae

Cyclic AMP, theophylline and caffeine promoted sporulation when added to a presporulation medium containing glucose. Caffeine promoted sporulation even when added to a presporulation medium containing acetate as the carbon source, but cyclic AMP and theophylline did not. Caffeine did not increase the intracellular cyclic AMP level, while theophylling did significantly when added to a presporulation medium containing glucose Caffeine inhibited the vegetative DNA synthesis with little effect on RNA and protein synthesis, resulting in the increase in cell volume, dry weight, and RNA and protein contents, but cyclic AMP and theophylline did not show such effects.     

Requirement for peptidoglycan synthesis during sporulation of Bacillus subtilis.

Cultures of Bacillus subtilis were treated during sporulation with antibiotics (bacitracin and vancomycin) that affect peptidoglycan synthesis. The cells were resistant to the effects of the antibiotics only when the drugs were added about 2 h after the beginning of sporulation. This was about 1 h later than the escape time of a temperature-sensitive sporulation mutant that is unable to complete prespore septation. Similar experiments were done with a mutant temperature sensitive for peptidoglycan synthesis. This showed an escape curve similar to that shown by the antibiotics. When sporulating cells were treated with antibiotics, they produced alkaline phosphatase earlier than normal. Enzyme production was unaffected by inhibition of deoxyribonucleic acid synthesis but was inhibited by chloramphenicol. Sporulation mutants that are unable to make alkaline phosphatase under normal conditions were able to make it in the presence of bacitracin. The alkaline phosphatase made under these conditions was under "sporulation-type" control since its synthesis was repressible by casein hydrolysate and unaffected by inorganic phosphate. When cells were treated with bacitracin in the growth medium as well as in the sporulation medium, alkaline phosphatase synthesis was at the same level as in an untreated control. A number of other antibiotics and surfactants were tested for the ability to cause premature production of the phosphatase of those tested, only taurodeoxycholate whowed this behavior. Moreover, incubation of cells with taurodeoxycholate in the growth medium as well as in the sporulation medium prevented premature enzyme production.     

Acetate Utilization and Macromolecular Synthesis During Sporulation of Yeast

Acetate utilization and macromolecule synthesis during sporulation (meiosis) of Saccharomyces cerevisiae were studied. When diploid cells are transferred from glucose nutrient medium to acetate sporulation medium at early stationary phase, respiration of the exogenously supplied acetate proceeds without any apparent lag. At the completion of ascospore development, 62% of the acetate carbon consumed has been respired, 22% remains in the soluble pool, and 16% is incorporated into lipids, protein, nucleic acids, and other cell components. Measurements of the rate of protein synthesis during sporulation reveal two periods of maximal synthetic activity: an early phase coincidental with increases in deoxyribonucleic acid, ribonucleic acid, and protein cellular content and a later phase during ascospore formation. Experiments in which protein synthesis was inhibited at intervals during sporulation indicate that protein synthesis is required both for the initiation and completion of ascus development.     

Role of acetate metabolism in sporulation of Saccharomyces carlsbergensis.

Several aspects of the role of acetate metabolism in the sporulation of Saccaromyces carlsbergensis were investigated. Experiments in which the development of the respiratory system was either stimulated by growth on sugars to which the cells have to adapt, or inhibited by chloramphenicol suggested a correlation between respiratory development and sporulation. In cells in which the respiratory system has been repressed during growth, mitobhondrial protein synthesis and derepression are prerequisites for sporulation. When derepression is complete, sporulation no longer depends on mitochondrial protein synthesis. Incorporation experiments with acetate showed that this compound is an important source of intermediates for biosynthetic processes that occur during sporulation. Its incorporation into macromolecular fractions is tightly coupled to sporutlation.