Isolation and characterization of WHI3, a size-control gene of Saccharomyces cerevisiae

WHI3 is a gene affecting size control and cell cycle in the yeast Saccharomyces cerevisiae. The whi3 mutant has small cells, while extra doses of WHI3 produce large cells, and a large excess of WHI3 produces a lethal arrest in G1 phase. WHI3 seems to be a dose-dependent inhibitor of Start. Whi3 and its partially redundant homolog Whi4 have an RNA-binding domain, and mutagenesis experiments indicate that this RNA-binding domain is essential for Whi3 function. CLN3-1 whi3 cells are extremely small, nearly sterile, and largely nonresponsive to mating factor. Fertility is restored by deletion of CLN2, suggesting that whi3 cells may have abnormally high levels of CLN2 function.     

Metabolic changes during phosphate deprivation in Englena in air and in oxygen

1. Euglena cells were grown in culture media containing either 20mm-phosphate or 20mum-phosphate, with ethanol or glucose as the sole source of carbon, and gassed with either air+carbon dioxide (95:5) or oxygen+carbon dioxide (95:5) at atmospheric pressure. 2. After growth in low-phosphate medium with ethanol as substrate, the cells developed signs of oxygen toxicity, as indicated by a decreased rate of respiration, a decreased net synthesis of paramylum and a failure to resume growth on replenishment of phosphate. 3. After growth in low-phosphate medium with glucose as substrate, the signs of oxygen toxicity were less apparent. 4. During phosphate deprivation the carotenoid content of Euglena increased more than threefold. This increase was largely prevented by exposure of the cells to oxygen+carbon dioxide (95:5) during growth. Oxygenation appears to interfere with ring closure of the common carotenoid precursor. 5. Mitochondria obtained from Euglena exposed to oxygen during phosphate deprivation, i.e. when signs of oxygen toxicity were evident, had greatly decreased activities of succinate dehydrogenase, succinate-cytochrome c oxidoreductase and NADH-cytochrome c oxidoreductase, compared with mitochondria obtained from Euglena exposed to oxygen in medium containing 20mm-phosphate.     

Molecular cloning of WHI2, a gene involved in the regulation of cell proliferation in Saccharomyces cerevisiae

The WHI2 gene of Saccharomyces cerevisiae plays a key role in coordinating cell proliferation and nutrient availability. A 2.6 kb yeast DNA sequence has been cloned which fully suppresses the whi2 mutation. Integration of this sequence to demonstrate that the structural gene itself had been cloned proved difficult. Integration occurred only rarely and only at the LEU2 locus which was also present on the integrating plasmid. To circumvent these difficulties an adjacent sequence, present on the original isolate from the gene library, was subcloned onto the integrating vector YIp5, after which directed integration proved straightforward. The integrated sequence was closely linked to WHI2, confirming that the structural gene had been cloned. A chromosomal restriction map of the WHI2 region is presented; no gross changes were observed in the region as cells entered stationary phase.     

Effects of glucose, tetrapyrroles and protein kinase C activators on cell proliferation in cultures of Saccharomyces cerevisiae

Abstract Saccharomyces cerevisiae was inoculated into a yeast nitrogen base with either glycerol or glucose as carbon source. Cell proliferation was followed by colony counts on agar medium. Cells in the glycerol-supplemented medium divided less than once in 10 days. When glucose, 6-deoxy-glucose or protoporphyrin IX was added, the cells had doubling times of about 24 h and increased in number to about 0.5 × 10⁶ cells ml⁻¹ Addition of either of the protein kinase C activators oleoyl-acetylglycerol or phorbol-12-myristate-13-acetate did not activate cell proliferation in the glycerol medium. However, when (i) glucose was combined with either protoporphyrin IX or chlorophyllin, or (ii) either protoporphyrin IX or chlorophyllin was combined with either of the protein kinase C activators, the cells had doubling times of about 12 h. Hence, (i) glucose can act as both a carbon source and a signalling molecule for proliferation, and (ii) two systems are involved in activating cell proliferation in S. cerevisiae : one operating through a protein kinase C system and another through a guanylate cyclase system.     

Studies on the regulation of X-prolyl dipeptidyl aminopeptidase activity

The specific activity of X-prolyl-dipeptidyl aminopeptidase in Saccharomyces cerevisiae grown on glucose-containing medium remains constant during exponential growth and increases less than twofold when cells reach the stationary phase. In cells harvested from exponential growth on glucose-containing medium the specific activity of the enzyme is found to be 20-30% lower than the specific activity observed in media without glucose, containing acetate or ethanol as the carbon source. X-Prolyl-dipeptidyl aminopeptidase is not inactivated after the addition of glucose to stationary phase cells. Growth of the yeast on poor nitrogen sources or under nitrogen-starvation results in a three- to fourfold increase in the level of the enzyme.     

Expression of the cry11A gene of Bacillus thuringiensis ssp. israelensis in Saccharomyces cerevisiae

The complete cry11A region gene of Bacillus thuringiensis ssp. israelensis was fused in frame to the 3' end of the GST gene under the control of the Saccharomyces cerevisiae HXK1 promoter. The fusion protein GST-cry11A was expressed in S. cerevisiae strain AMW13C+. The fusion gene GST-cry11A was expressed when yeast cells were grown on galactose and a nonfermentable medium containing ethanol as carbon and energy source. When the cells were grown in glucose, mannose, fructose, or glycerol as carbon sources, the GST-cry11A gene was repressed. Thus, a regulated expression in accordance with the regulatory activity of the HXK1 gene promoter has been detected. The GST-cry11A fusion protein was detected in the transformed yeasts as a soluble protein. The fusion protein was purified by affinity chromatography using glutathione-Sepharose beads. Cell-free extracts from transformed yeasts grown in ethanol-containing culture media showed insecticidal activity against third-instar Aedes aegypti larvae. This insecticidal activity was increased about 4-fold when the purified fusion protein was assayed.     

Control of cell morphology of the yeast Saccharomyces cerevisiae by nutrient limitation in continuous culture

Saccharomyces cerevisiae IFO 0203, a polyploid yeast used in ethanol production in Japan, grows as ovoid cells in unstirred batch culture and on fully nutritive agar plates (2% w/v glucose; 0.67% w/v Difco yeast nitrogen base).
Extensively branched pseudohyphae formed on 0.01% w/v ammonium sulphate plates within a few days. In continuous culture with high oxygen supply and limiting glucose, cells were elongated but growth was vigorous and the daughter cells separated well after budding.
Limitation of growth by either nitrogen source or oxygen during continuous culture resulted in formation of truncated, occasionally branched, pseudohyphae up to five cells in length.     

Coordinate control of syntheses of ribosomal ribonucleic acid and ribosomal proteins during nutritional shift-up in Saccharomyces cerevisiae.

We investigated the regulation of ribosome synthesis in Saccharomyces cerevisiae growing at different rates and in response to a growth stimulus. The ribosome content and the rates of synthesis of ribosomal ribonucleic acid and of ribosomal proteins were compared in cultures growing in minimal medium with either glucose or ethanol as a carbon source. The results demonstrated that ribosome content is proportional to growth rate. Moreover, these steady-state concentrations are regulated at the level of synthesis of ribosomal precursor ribonucleic acid and of ribosomal proteins. When cultures growing on ethanol were enriched with glucose, the rate of ribosomal ribonucleic acid synthesis, measured by pulsing cells with [methyl-3H]methionine, increased by 40% within 5 min, doubled within 15 min, and reached a steady state characteristic of the new growth medium by 30 min. Labeling with [3H]leucine reveal a coordinate increase in the rate of synthesis of 30 or more ribosomal proteins as compared with that of total cellular proteins. Their synthesis was stimulated approximately 2.5-fold within 15 min and nearly 4-fold within 60 min. The data suggest that S. cerevisiae responds to a growth stimulus by preferential stimulation of the synthesis of ribosomal ribonucleic acid and ribosomal proteins.     

Polyphosphate synthesis in yeast

Polyphosphate synthesis was studied in phosphate-starved cells of Saccharomyces cerevisiae and Kluyveromyces marxianus. Incubation of these yeasts for a short time with phosphate and either glucose or ethanol resulted in the formation of polyphosphate with a short chain length. With increasing incubation times, polyphosphates with longer chain lengths were formed. Polyphosphates were synthesized faster during incubation with glucose than with ethanol. Antimycin did not affect the glucose-induced polyphosphate synthesis in either yeast. Using ethanol as an energy source, antimycin A treatment blocked both polyphosphate synthesis and accumulation of orthophosphate in the yeast S. cerevisiae. However, in K. marxianus, polyphosphate synthesis and orthophosphate accumulation proceeded normally in antimycin-treated cells, suggesting that endogenous reserves were used as energy source. This was confirmed in experiments, conducted in the absence of an exogenous energy source.     

The cdc30 mutation in Saccharomyces cerevisiae results in a temperature-sensitive isoenzyme of phosphoglucose isomerase

The cdc30 mutation in the yeast Saccharomyces cerevisiae causes cell cycle arrest late in nuclear division when cells are shifted from the permissive temperature of 25 degrees C to the restrictive temperature of 36.5 degrees C. Cell cycle arrest at 36.5 degrees C is dependent upon the carbon source used: a shift-up in glucose containing media results in cell cycle blockade, whereas a shift-up in ethanol, fructose, glycerol, glycerol plus ethanol, or mannose does not. Metabolite analyses showed accumulation of glucose 6-phosphate in a cdc30-bearing strain after a temperature shift-up in glucose-containing medium. Thermal denaturation studies and kinetic measurements indicate the existence of two isoenzymes of phosphoglucose isomerase (EC 5.3.1.9); one of which is apparently altered in the temperature-sensitive cell cycle mutant. We propose that the gene products of both the CDC30 and PG11 genes are required for cell cycle progression in glucose media and that the PGI1 gene product has a regulatory function over the CDC30 gene product.     

Does Giardia intestinalis need glucose as an energy source?

Giardia intestinalis was grown in Diamond's TYI-S-33 medium containing either 50 mM-glucose or no added glucose to assess its dependence on glucose availability as an energy source. The parameters monitored included cell growth, glucose utilization and the accumulation of end products in the medium. In the medium containing no added glucose, G. intestinalis trophozoites achieved a cell density of about half that of the control, and produced the same end products, alanine, ethanol and acetate. Decreased amounts of both ethanol and alanine were observed (10 and 33% of controls, respectively after 4 days) while there was no change in acetate production. These observations indicate that G. intestinalis can utilize carbon sources other than glucose, and is not absolutely dependent upon glucose as an energy source.     

Regulation of phosphoenolpyruvate carboxykinase and pyruvate kinase in Saccharomyces cerevisiae grown in the presence of glycolytic and gluconeogenic carbon sources and the role of mitochondrial function on gluconeogenesis

Phosphoenolpyruvate carboxykinase (PEPCKase) and pyruvate kinase (PKase) were measured in Saccharomyces cerevisiae grown in the presence of glycolytic and gluconeogenic carbon sources. The PEPCKase activity was highest in ethanol-grown cells. However, high PEPCKase activity was also observed in cells grown in 1% glucose, especially as compared with the activity of sucrose-, maltose-, or galactose-grown cells. Activity was first detected after 12 h when glucose was exhausted from the growth medium. The PKase activity was very high in glucose-grown cells; considerable activity was also present in ethanol- and pyruvate-grown cells. The absolute requirement of respiration for gluconeogenesis was demonstrated by the absence or significantly low levels of PEPCKase and fructose-1,6-bisphosphatase activities observed in respiratory deficient mutants, as well as in wild-type S. cerevisiae cells grown in the presence of glucose and antimycin A or chloramphenicol. Obligate glycolytic and gluconeogenic enzymes were present simultaneously only in stationary phase cells, but not in exponential phase cells; hence futile cycling could not occur in log phase cells regardless of the presence of carbon source in the growth medium.     

Carbon starvation can induce energy deprivation and loss of fermentative capacity in Saccharomyces cerevisiae

Seven different strains of Saccharomyces cerevisiae were tested for the ability to maintain their fermentative capacity during 24 h of carbon or nitrogen starvation. Starvation was imposed by transferring cells, exponentially growing in anaerobic batch cultures, to a defined growth medium lacking either a carbon or a nitrogen source. After 24 h of starvation, fermentative capacity was determined by addition of glucose and measurement of the resulting ethanol production rate. The results showed that 24 h of nitrogen starvation reduced the fermentative capacity by 70 to 95%, depending on the strain. Carbon starvation, on the other hand, provoked an almost complete loss of fermentative capacity in all of the strains tested. The absence of ethanol production following carbon starvation occurred even though the cells possessed a substantial glucose transport capacity. In fact, similar uptake capacities were recorded irrespective of whether the cells had been subjected to carbon or nitrogen starvation. Instead, the loss of fermentative capacity observed in carbon-starved cells was almost surely a result of energy deprivation. Carbon starvation drastically reduced the ATP content of the cells to values well below 0.1 micro mol/g, while nitrogen-starved cells still contained approximately 6 micro mol/g after 24 h of treatment. Addition of a small amount of glucose (0.1 g/liter at a cell density of 1.0 g/liter) at the initiation of starvation or use of stationary-phase instead of log-phase cells enabled the cells to preserve their fermentative capacity also during carbon starvation. The prerequisites for successful adaptation to starvation conditions are probably gradual nutrient depletion and access to energy during the adaptation period.     

Regulation of gluconeogenic enzymes during the cell cycle of Saccharomyces cerevisiae growing in a chemostat

Oscillation of the activities of gluconeogenic enzymes (malate dehydrogenase, phosphoenolpyruvate carboxykinase and fructose-1,6-bisphosphatase) was observed during the cell cycle of chemostat cultures of Saccharomyces cerevisiae. Since ethanol is released by the cells at the beginning of the division cycle, its effect on enzyme expression was determined. Pulsing ethanol to a synchronously dividing yeast culture led to a prolongation of the metabolically active phase as indicated by the course of oxygen uptake and carbon dioxide production rates (concomitant ethanol and glucose assimilation). Enzyme activities also remained elevated as long as ethanol was available to the cells. After a substrate shift from glucose to ethanol during cell division, ethanol was used without a lag phase and enzyme induction increased from the level reached at the point of the substrate change. The data confirmed that the small amount of ethanol produced when the cells begin active reproduction acts as an inducer of gluconeogenic enzymes.     

带有木糖还原酶基因和木糖醇脱氢酶基因的重组酿酒酵母的构建

采用双载体系统,将携带有瑞氏木霉木糖醇脱氢酶基因的表达质粒pAJ401－Xdh1转化已带有树干毕赤氏酵母木糖还原酶基因的重组酿酒酵母H475,构建了同时带有毕赤氏酵母木糖还原酶基因和瑞氏木霉木糖醇脱氢酶基因的重组酿酒酵母HX1。研究了重组酿酒酵母HX1对木糖的转化利用情况。     

Adenylate energy charge in Saccharomyces cerevisiae during starvation.

Bakers' yeast cells, Saccharomyces cerevisiae, if grown aerobically on ethanol or if grown aerobically on glucose and allowed to pass into stationary phase, with utilization of accumulated ethanol, maintain a normal value (0.8 to 0.9) of the adenylate energy charge during prolonged starvation. In contrast, cells grown anaerobically on glucose and cells in the early stages of aerobic growth on glucose exhibit a rapid decrease of energy charge if transferred to medium lacking on energy source. These results suggest that functional mitochondria or enzymes of balance of adenine nucleotides during starvation. Yeast cells remain viable at energy charge values below 0.1, in marked contrast to results previously obtained with Escherichia coli. In other respects, the engery charge responses of yeast to starvation and refeeding are generally similar to those previously reported for E. coli.