Filament formation in Saccharomyces cerevisiae--a review.

Many yeasts can produce filamentous elongated cells identifiable as hyphae, pseudohyphae or invasive filaments. Filament formation has been understood as a foraging response that occurs in nutrient-poor conditions. However, fusel alcohols were observed to induce filament formation in rich nutrient conditions in every yeast species examined. Fusel alcohols, e.g., 3-methyl-1-butanol (3Me-BuOH; 'isoamyl alcohol'), 2-methyl-1-propanol (isobutyl alcohol), (-)-2-methyl-1-butanol ('active amyl alcohol'), 2-phenylethanol and 3-(2-hydroxyethyl)indole (tryptophol) (the end products of leucine, valine, isoleucine, phenylalanine and tryptophan catabolism, respectively) are the end products of amino acid catabolism that accumulate when nutrients become limiting. Thus, yeast responds to its own metabolic by-products. Considerable effort was made to define the cell biological and biochemical changes that take place during 3Me-BuOH-induced filamentation. In Saccharomyces cerevisiae filaments contain significantly greater mitochondrial mass and increased chitin content in comparison with yeast-form cells. The global transcriptional response of S. cerevisiae during the early stages of 3Me-BuOH-induced filament formation has been described. Four ORFs displayed very significant (more than 10-fold) increases in their RNA species, and 12 ORFs displayed increases in transcription of more than 5-fold. The transcription of five genes (all of which encode transporters) decreased by similar amounts. Where examined, the activity of the proteins encoded reflected the transcriptional pattern of their respective mRNAs. To understand this regulation, studies were performed to see whether deletion or overexpression of key genes affects the ability to filament and invade solid YEPD medium. This has led to identification of those proteins that are essential for filament formation, repressors and those which are simply not required. It also leads to the conclusion that 3Me-BuOH-induced filament formation is not a foraging response but a response to reduced growth rate.

Sequence of the Escherichia coli K-12 edd and eda genes of the Entner-Doudoroff pathway.

A 3.5-kb DNA fragment from the Clarke and Carbon Escherichia coli genomic clone, pLC37-44, was sequenced on both strands. Part of the zwf gene, encoding glucose-6-phosphate dehydrogenase, and all of the edd and eda genes, encoding 6-phosphogluconate dehydratase and 2-keto-3-deoxy-6-phosphogluconate aldolase, respectively, of the Entner-Doudoroff pathway, were identified. These data are compared with those of Egan et al. [J. Bacteriol. 174 (1992) 4638-4646] and important differences were noted.

A study of branched-chain amino acid aminotransferase and isolation of mutations affecting the catabolism of branched-chain amino acids in Saccharomyces cerevisiae.

The specific activity of branched-chain amino acid aminotransferase was highest when S. cerevisiae was grown in minimal medium containing a branched-chain amino acid as nitrogen source. Growth in complex media with glycerol or ethanol gave moderately high levels, whereas with glucose and fructose the specific activity was very low. Mutagenesis defined three genes (BAA1 to BAA3) required for branched-chain amino acid catabolism. The baa1 mutation reduced the specific activity of the aminotransferase, the stationary phase density in YEPD and caused gross morphological disturbance. Branched-chain amino acid aminotransferase is essential for sporulation.

Irreversible formation of pseudohyphae by haploid Saccharomyces cerevisiae.

Culturing haploid strains of Saccharomyces cerevisiae in liquid minimal medium with 2% ethanol and 2% leucine resulted in the formation of long anucleate pseudohyphae. This occurred only with the combination of ethanol as carbon source and leucine as nitrogen source and was independent of mating type. The transition to a pseudohyphal form observed under these conditions appears to be irreversible. These findings further extend our view of the developmental alternatives in this important model eukaryote.

A rapid and convenient screening technique for developmental pathway mutants of Saccharomyces cerevisiae.

A brief exposure to iodine vapour was used to screen for mutants of the yeast Saccharomyces cerevisiae affected in development. Besides obtaining a large number of asporogenous mutants, two novel mutations were identified that permitted germination of spores to occur in conditions (sporulation medium) in which the wild-type would not germinate. These two mutations were named gdr1 and gdr2 for germination derepressed. Both alter nutritional control of germination, but not the kinetics of germination in glucose-containing medium.

Exercise on-transient gas exchange kinetics are slowed as a function of age.

The purpose was to characterize gas exchange kinetics following the on-transient of exercise in men aged 30-80 yr. Forty-six men completed square wave exercise tests from loadless cycling to subventilatory threshold (V(E)T) work rates with gas exchange measured breath-by-breath. Signal averaged data were fit with a monoexponential equation to derive time constants (tau) for gas exchange and ventilation (tau VO2, tau VCO2, tau VE) and heart rate (tau HR). There was a significant slowing of ventilation and gas exchange kinetics across age with linear regression yielding an increase of 0.67 s.yr-1 for tau VO2 (39 s in young to 61 s in old), 0.57 s.yr-1 for tau VCO2, and 0.65 s.yr-1 for tau VE, whereas tau HR (44 to 41 s) was not changed significantly. The slowed VO2 kinetics with age may reflect limitations in muscle blood flow or in control of the rate of oxidative metabolism. The less marked slowing of tau VCO2 compared with tau VO2 across age may reflect reduced CO2 storage capacity with loss of muscle tissue. The tau VE change across age was similar to that for tau VCO2 (tau VE/tau VCO2 unchanged). The present study demonstrated marked age-related slowing of gas exchange dynamics at exercise onset.

Yeasts: providing questions and answers for modern biology.

Yeasts are to be found in virtually every conceivable niche on this planet and are amazingly varied in their shapes ('morphologies'), life cycles, metabolic capabilities, potentials for use in industrial processes, abilities to spoil food and drink or to act as dangerous human pathogens. This review describes four very different species of yeast to illustrate some of the diversity which exists and, in the case of one of them, Saccharomyces cerevisiae (the familiar baker's or brewer's yeast), the extent of both our knowledge and ignorance.

Interviewer's role-playing and responses to sensory deprivation: a clinical demonstration.

10 Ss with a history of intransigent hypochondriacal personality disorder were subjected to 2-1/2 hr. of sensory deprivation preceded and followed by planned interviewing procedures. Each interview was designed to prestructure the interpersonal meaning of the experience of sensory deprivation and selectively reinforce social roles antithetical to S's characteristic, maladaptive interpersonal behavior. As predicted, Ss showed a significant (p smaller than .01) shift from passively hostile to an actively warm social role. The changes in social role were also reflected in a significant (p smaller than .01) reduction in number of medical clinic visits. These effects were still operative 30 days following the procedure, whereas an equated baseline group of 10 Ss showed no significant change in behavior over the same period of time.

Are yeasts free-living unicellular eukaryotes?

Yeasts are defined as unicellular fungi, yet many recent observations suggest their whole lifestyle is anything but unicellular. This review surveys the evidence that yeasts are really social organisms with cell-to-cell communication.

The Arabidopsis CDC25 induces a short cell length when overexpressed in fission yeast: evidence for cell cycle function.

The putative mitotic inducer gene, Arath;CDC25 cloned in Arabidopsis thaliana, was screened for cell cycle function by overexpressing it in Schizosaccharomyces pombe (fission yeast). The expression pattern of Arath;CDC25 was also examined in different tissues of A. thaliana. Fission yeast was transformed with plasmids pREP1 and pREP81 with the Arath;CDC25 gene under the control of the thiamine-repressible nmt promoter. Using reverse transcription-polymerase chain reaction (RT-PCR), the expression of Arath;CDC25 was examined in seedlings, flower buds, mature leaves and stems of A. thaliana; actin (ACT2) was used as a control. In three independent transformants of fission yeast, cultured in the absence of thiamine (T), pREP1::Arath;CDC25 induced a highly significant reduction in mitotic cell length compared with wild type, pREP::Arath;CDC25 +T, and empty vector (pREP1 +/- T). The extent of cell shortening was greater using the stronger pREP1 compared with the weaker pREP81. However, Arath;CDC25 was expressed at low levels in all tissues examined. The data indicate that Arath;CDC25 can function as a mitotic accelerator in fission yeast. However, unlike other plant cell cycle genes, expression of Arath;CDC25 was not enhanced in rapidly dividing compared with non-proliferative Arabidopsis tissues.

Biochemical and genetic studies on the function of, and relationship between, the PGI1- and CDC30-encoded phosphoglucose isomerases in Saccharomyces cerevisiae.

Isoelectric focusing was used to compare the complement of phosphoglucose isomerase isoenzymes in a wild-type strain of Saccharomyces cerevisiae and in a strain with a deletion in the PGI1 structural gene. Deletion of the PGI1 gene did not result in the absence of the high-Km isoenzyme I but the low-Km isoenzyme II was absent. Hence, the isoenzymes must be the products of two genes. If PGI1 were the sole structural gene its deletion would result in the disappearance of both isoenzymes. After a temperature shift-up a cdc30-bearing strain had cell cycle arrested and contained only 8% of the polysaccharide in the wild-type. Phosphoglucose isomerase is required for the synthesis of fructose 6-phosphate (F6-P), a precursor of the cell wall components chitin and mannoprotein ('mannan'), which are a polysaccharide and contain polysaccharide, respectively. Since the cdc30 mutation confers a temperature-sensitive phosphoglucose isomerase, the likely explanation for cell cycle arrest caused by this mutation is that the defective phosphoglucose isomerase results in a reduction of F6-P and hence an inability to synthesize the mannan and chitin needed for cytokinesis and cell separation. Revertants of a pgi1-1 bearing strain were selected for their ability to grow on glucose at 25 degrees C and this yielded a number of different phenotypes. Amongst the isolates was a strain which had undergone an intragenic reversion at the pgi1 locus, designated pgi1-1,100. This mutation permits growth and cell division at 25 degrees C but results in cell cycle arrest at 36 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

The cdc 22 mutation by Schizosaccharomyces pombe is a temperature-sensitive defect in nucleoside diphosphokinase.

A number of temperature-sensitive cdc- mutants of Schizosaccharomyces pombe that are affected in DNA replication, were screened for the absence of deoxynucleoside triphosphate(s) when blocked at their restrictive temperature. The preliminary screening simply involved analysis of perchloric acid-soluble cell extracts by two-dimensional thin-layer chromatography on poly(ethyleneimine)-impregnated cellulose. One mutant strain, cdc 22-M45, was found which apparently lacked dTTP. Pulse-labelling of intracellular nucleotides revealed that not only did dTTP become depleted, but that dTDP accumulated when this mutant was blocked by a temperature shift-up, indicating a defective nucleoside diphosphokinase. Nucleoside diphosphokinase from cdc 22-M45 was less active than that from wild-type strain 972 when assayed at high temperatures. The nucleoside diphosphokinase of the mutant also has an altered Km for dTDP at both permissive (25 degrees C), and at the restrictive (36.8 degrees C) temperatures. At the restrictive temperature the Km for dTDP of the mutant enzyme is more than 11-times greater than that of the wild type. Characterisation of the biochemical basis of the defect in this cdc- mutant has shown that in S. pombe, despite its having an apparently complex system of genetic control over progression through S-phase, one factor at least is merely availability of a nucleoside triphosphate precursor to DNA synthesis.

Nucleoside diphosphokinase and cell cycle control in the fission yeast Schizosaccharomyces pombe.

Centrifugal elutriation was used to prepare synchronous cultures of Schizosaccharomyces pombe. Nucleoside diphosphokinase activity was measured throughout the cell cycle. In the wild-type strain (972) nucleoside diphosphokinase activity doubled in a stepwise fashion. The midpoint of the rise in enzyme activity was at 0.65 of a cycle, 0.29 of a cycle before the next S phase. Synchronous cultures of the mutant wee 1-6 were also prepared. In this strain S phase is delayed, occurring about 0.3 cycle later than in the wild-type. In wee 1-6 the midpoint of the stepwise doubling in nucleoside diphosphokinase activity occurred at 0.084; showing that the rise in enzyme activity is also delayed. Addition of cycloheximide to an exponentially growing culture caused an immediate inhibition of protein synthesis, yet nucleoside diphosphokinase activity continued to increase exponentially for a further 300 min. This indicates that the stepwise doubling of nucleoside diphosphokinase activity during the cell cycle is not achieved by a simple control on protein synthesis. Two temperature-sensitive cdc- mutants were also used: cdc2-33, a mutant whose single genetic lesion results in the twin defects of a loss of mitotic control and a loss of commitment to the cell cycle; and cdc 10-129, which has a defect in DNA replication. In both mutants a temperature shift-up of an asynchronously growing culture from the permissive (25 degrees C) to the restrictive temperature (36.5 degrees C) results in a rapid inhibition of DNA replication. In both mutants nucleoside diphosphokinase continues to increase exponentially. Therefore, although nucleoside diphosphokinase is required for DNA replication, apparently DNA replication is not required for an increase in nucleoside diphosphokinase activity.

The metabolism of sporulation in yeast.

An holistic view of the metabolism of sporulation in Saccharomyces cerevisiae is emerging. Furthermore, there may be a general biochemical signal for the initiation of differentiation that is conserved in both prokaryotes and eukaryotes.