The Kluyver effect for trehalose in Saccharomyces cerevisiae

Saccharomyces cerevisiae cells are able to grow using trehalose as a sole source of carbon and energy. However, the biomass yield obtained with trehalose was higher, and the specific growth rate lower, than that obtained with glucose or maltose. The respiratory inhibitor antimycin A prevented cell growth on trehalose, and no ethanol or glycerol was formed during batch growth on this carbon source. Thus, S. cerevisiae exhibits the KLUYVER effect for trehalose: this disaccharide is assimilated and respired, but, in contrast to glucose or maltose, it cannot be fermented. The high-affinity trehalose-H+ symporter encoded by the AGT1 gene is required for growth on trehalose. Analysis of the differences in the metabolism of maltose and trehalose (both disaccharides of glucose transported by active transport systems) indicated that the absence of trehalose fermentation is a consequence of low sugar influx into the cells during growth on this carbon source.     

The effect of aging on protein synthesis in the yeast Saccharomyces cerevisiae.

The protein synthetic rate in the yeast S. cerevisiae, measured by the incorporation of radioactive amino acids per unit amount of proteins, decreased linearly with age reaching 50% of the rate of 2nd generation cells (young cells) in 20th generation cells (old cells), whereas the RNA content of the old cells was increased three times. Using a cell-free system for poly(U)-directed poly-phenylalanine synthesis, the activity of run-off ribosomes from old cells was shown to be about 40% less than the activity of ribosomes from young cells and the polysome level in old cells was much decreased compared to that in young cells. However, as protein content was increased twice in 20 generations, the cell is considered to maintain a constant level of protein synthesis during the process of aging compensating the decrease in the activity of ribosomes. Thus, it is likely that the decrease in the synthesis of certain proteins whose requirement was raised by the increase in cell volume, which is twice the increase in protein content, causes prolongation of the unbudded phase in old cells.     

Chromosomal proteins in Saccharomyces cerevisiae

Summary Proteins were isolated from purified yeast chromatin and subjected to two-dimensional electrophoresis. The cellular and the chromosomal content of the major nonhistone proteins was measured. Two polypeptides of molecular weights 55,000 and 53,000, identified as and tubulin, and a polypeptide of molecular weight 63,000, associated with the nuclear DNA to a very high degree, account for nearly 50% of the nonhistone proteins present in chromatin. Only one tenth of the RNA polymerase subunit with the molecular weight of 23,000 was associated with nuclear DNA following chromatin purification in metrizamide gradients.     

Mitochondrial mutagenesis in Saccharomyces cerevisiae

Summary Four types of mit ^– mutations induced with manganese are found in the following relative proportions: oxi3 ^– > cob-box ^– > oxi2 ^– oxi1 ^–1. The frequences of loss of their respective mit ⁺ alleles in manganese-induced rho ^–] primary and secondary clones follow the same order. The possible interdependence between these two sets of data is discussed.     

UV-inducible proteins in Saccharomyces cerevisiae

Summary Two UV-inducible proteins have been detected in the yeast, Saccharomyces cerevisiae. The proteins have molecular weights of 78,000 Daltons and 23,000 Daltons. This induction is specific for UV-irradiation as exposure to X-rays, mitomycin C and heat shock does not result in the synthesis of the proteins. The larger (78 kD) protein is induced in various rad strains and in a ° cir° strain. Attempts are being made to isolate the genes coding for these inducible proteins.     

Pathogenicity of Saccharomyces cerevisiae in complement factor five-deficient mice.

We have previously determined the relative virulence of isolates of Saccharomyces cerevisiae on the basis of differences in proliferation and resistance to clearance in CD-1 mice. These infections were not fatal. To further characterize S. cerevisiae pathogenesis, we studied a virulent clinical isolate, YJM128, and an avirulent nonclinical isolate, Y55, in C5-deficient mice. DBA/2N mice were infected intravenously with YJM128 or Y55, and temporal burdens of yeast cells in various organs were determined. After infection with 10(7) CFU, Y55 increased by 13-fold and YJM128 increased by 20-fold in the brain from day 0 to 3. In addition, YJM128 increased by 4-fold in the kidneys, whereas Y55 decreased by 16-fold. Both isolates declined in number in other organs. In all studies, 90% of mice infected with 10(7) CFU of YJM128 died between days 2 and 7, whereas no mice infected with equivalent numbers of Y55 died. No mice died after infection with 10(6) CFU of Y55 or YJM128. The importance of C5 was confirmed by studies using B10.D2/oSnJ (C5-) mice and their congenic C5+ counterparts. Again, the C5- mice were most susceptible to infection with S. cerevisiae, with 63% infected with YJM128 dying by day 7; no C5+ mice died. No Y55-infected mice died, and mean burdens in the brain at day 14 were sevenfold lower in C5+ mice than in C5- mice. Seven of 10 other S. cerevisiae isolates were also more virulent in DBA/2N than CD-1 mice, causing > or = 40% mortality. These data indicate that C5 is a critical factor in host resistance against S. cerevisiae infections and further confirm the pathogenic potential of some isolates of S. cerevisiae.     

Expression of human papillomavirus proteins in yeast Saccharomyces cerevisiae.

The L1 and L2 proteins of human papillomavirus (HPV) types 1, 6, and 16 and the E6 and E7 proteins of HPV 16 were expressed in Saccharomyces cerevisiae. The yeast expressed proteins were readily detected by immune blotting and were generally intact. The HPV 1 L1 and L2 proteins expressed in yeast were indistinguishable from the major and minor capsid proteins purified from HPV 1 virions as judged by gel electrophoresis and immunoblotting. The HPV 6 and HPV 16 L2 proteins and HPV 16 E7 proteins were secreted from yeast by fusion to the yeast pre-pro-alpha-factor leader sequence. Following secretion of the HPV 16 E7 protein a rapid method of purification was developed. The yeast expressed proteins were used as antigen targets to study the human immune response in Western blot assay, ELISA, and immune precipitation. One human serum reacted with intact, but not denatured HPV 16 L2 proteins, suggesting that the yeast expressed proteins will be useful to detect antibodies reactive with conformational epitopes.     

Radioprotective effect of some pyrazolone derivatives

Analgin, antipyrine, and aminopyrine, if administered to mice in large doses 3 h before irradiation (800 R), increased the survival rate and prolonged the life of the dying animals. In combination with cystamine, these compounds increased the changes of survival of the mice after the period of acute intestinal death following irradiation in a dose of 1050 R. In therapeutic doses the pyrazolone derivatives had a less marked radioprotective effect.     

Non-selective transformation of Saccharomyces cerevisiae

Summary Wild or industrial yeast strains cannot be transformed by most selective vectors due to a lack of auxotrophic mutations. To enable identification of transformants of such yeast species, we have developed a 2-µm DNA vector with an indicator gene that can be used without any additional marker. The Escherichia coli gene for -lactamase (bla) was placed under the control of the yeast promoter for the structural gene encoding ADHI. This increased the amount of -lactamase produced in Saccharomyces cerevisiae 100-fold giving an enzyme activity in transformant colonies which is high enough to be detected directly on indicator plates. Non-selectively, the transformation frequency is even higher than under selective conditions indicating that selection does not assist the establishment of new plasmids. Transformants isolated non-selectively were found to retain the endogenous 2-µm DNA. Under control of appropriate promoters, the bacterial bla gene may also provide a convenient marker for other eukaryotic transformation systems.     

Instability of Saccharomyces cerevisiae heterokaryons

Summary We have constructed heterokaryons of Saccharomyces cerevisiae by crossing kar1 — mutants incapable of nuclear fusion. Approximately 1% of the total zygotes from kar1 — crosses can form heterokaryotic clones. These are very small as compared to diploid colonies, and are composed mainly of a mixture of both types of heteroplasmons (cells which contain the cytoplasmic components of both parents, but the nuclear genotype of only one of them). This fact indicates that heterokaryons are unstable.This instability is already observed by pedigree analysis in the first zygotic divisions. We suggest that missegregation is the main factor in heterokaryon instability and results from an unequal nuclear transmission, which occurs when one of the mother nuclei divides and, although viable, does not migrate to the daughter bud. However, the proportion of inviable zygotes and buds found in the pedigree analysis, as well as the recovery of only one type of heteroplasmon, indicates the complete loss of one of the parental nuclei. Consequently nuclear inactivation is suggested as the second reason for heterokaryon instability.     

Stationary-phase mitophagy in respiring Saccharomyces cerevisiae

The clearance of malfunctioning mitochondria is an important housekeeping function in respiring eukaryotic cells and plays a role in physiological homeostasis as well as in the progression of late-onset diseases. This clearance is thought to occur by a specific form of autophagic degradation called mitophagy. Although the mechanism of nonspecific macroautophagy is relatively well established, the selective autophagic degradation of mitochondria has only recently begun to receive significant attention. An important step toward elucidating the mechanism by which defective mitochondria are selected and degraded is the establishment of conditions under which mitophagy is induced. This review covers our current understanding of mitophagy in the model organism Saccharomyces cerevisiae and its modes of activation, with a focus on stationary-phase mitophagy-a form of mitophagy that holds promise as a potential quality control mechanism.     

Polypeptide chain termination in Saccharomyces cerevisiae

Summary The study of translational termination in yeast has been approached largely through the identification of a range of mutations which either increase or decrease the efficiency of stop-codon recognition. Subsequent cloning of the genes encoding these factors has identified a number of proteins important for maintaining the fidelity of termination, including at least three ribosomal proteins (S5, S13, S28). Other non-ribosomal proteins have been identified by mutations which produce gross termination-accuracy defects, namely the SUP35 and SUP45 gene products which have closely-related higher eukaryote homologues (GST1-h and SUP45-h respectively) and which can complement the corresponding defective yeast proteins, implying that the yeast ribosome may be a good model for the termination apparatus existing in higher translation systems.While the yeast mitochondrial release factor has been cloned (Pel et al. 1992), the corresponding cytosolic RF has not yet been identified. It seems likely, however, that the identification of the gene encoding eRF could be achieved using a multicopy antisuppressor screen such as that employed to clone the E. coli prfA gene (Weiss et al. 1984). Identification of the yeast eRF and an investigation of its interaction with other components of the yeast translational machinery will no doubt further the definition of the translational termination process.While a large number of mutations have been isolated in which the efficiency of termination-codon recognition is impaired, it seems probable that a proportion of mutations within this class will comprise those where the accuracy of A site codon-anticodon interaction is compromised: such defects would also have an effect on termination-codon suppression, allowing mis- or non-cognate tRNAs to bind stop-condons, causing nonsense suppression. The remainder of mutatoons affecting termination fidelity should represent mutations in genes coding for components of the termination apparatus, including the eRF: these mutations reduce the efficiency of termination, allowing nonsense suppression by low-efficiency natural suppressor tRNAs. Elucidation of the mechanism of termination in yeast will require discrimination between these two classes of mutations, thus allowing definition of termination-specific gene products.     

Time-dependent mitotic recombination in Saccharomyces cerevisiae

The time-dependent appearance of prototrophic recombinants between heterologously located artificial repeats has been studied in Saccharomyces cerevisiae. While initial prototrophic colony numbers from independent cultures were highly variable, additional recombinants were found to arise daily at roughly constant rates irrespective of culture. These late-appearing recombinants could be accounted for neither by detectable growth on the selective media nor by delayed appearance of recombinants present at the time of selective plating. Significantly, at no time did the distributions of recombinants fully match those expected according to the Luria-Delbruck model and, in fact, after the first day, the distributions much more closely approximated a Poisson distribution. Prototrophic recombinants accumulated not only on the relevant selective medium, but also on media unrelated to the acquired prototrophy.     

Biosynthesis of hydroxymethylpyrimidine pyrophosphate in Saccharomyces cerevisiae

Two redundant genes, THI20 and THI21, of Saccharomyces cerevisiae encode a 2-methyl-4-amino-5-hydroxymethylpyrimidine monophosphate (HMP-P) kinase required for thiamin biosynthesis. Using functional complementation analysis with an Escherichia coli mutant strain and a defined biochemical system containing partially purified proteins for the reconstitution of thiamin monophosphate synthesis, we demonstrate that both Thi20p and Thi21p proteins also have HMP kinase activity. Although each isoform independently can synthesize HMP pyrophosphate (HMP-PP) from HMP, there is a marked difference in efficiency between the two proteins. The thi20 deletion strain grows at the same rate as the parental strain in minimal medium without thiamin, but its ability to synthesize HMP-PP from HMP is significantly decreased. We discuss the possibility that HMP is not involved in the pathway of de novo thiamin synthesis in S. cerevisiae.     

Regulation of isoleucine-valine biosynthesis in Saccharomyces cerevisiae

Summary The threonine deaminase gene (ILV1) of Saccharomyces cerevisiae has been designated multifunctional since Bollon (1974) indicated its involvement both in the catalysis of the first step in isoleucine biosynthesis and in the regulation of the isoleucine-valine pathway. Its role in regulation is characterized by a decrease in the activity of the five isoleucine-valine enzymes when cells are grown in the presence of the three branched-chain amino acids, isoleucine, valine and leucine (multivalent repression). We have demonstrated that the regulation of AHA reductoisomerase (encoded by ILV5) and branched-chain amino acid transaminase is unaffected by the deletion of ILV1, subsequently revealing that the two enzymes can be regulated in the absence of threonine deaminase. Both threonine deaminase activity and ILV1 mRNA levels increase in mutants (gcd2 and gcd3) having constitutively derepressed levels of enzymes under the general control of amino acid biosynthesis, as well as in response to starvation for tryptophan and branched-chain amino acid imbalance. Thus, the ILV1 gene is under general amino acid control, as is the case for both the ILV5 and the transaminase gene. Multivalent repression of reductoisomerase and transaminase can be observed in mutants defective in general control (gcn and gcd), whereas this is not the case for threonine deaminase. Our analysis suggests that repression effected by general control is not complete in minimal medium. Amino acid dependent regulation of threonine deaminase is only through general control, while the branched-chain amino acid repression of AHA reducto isomerase and the transaminase is caused both by general control and an amino acid-specific regulation.     

Nature of abortive transformation in Saccharomyces cerevisiae

Disruption mutagenesis by homologous recombination in Saccharomyces cerevisiae is carried out by transforming-DNA fragments containing the target gene disrupted by a selectable marker. A large number of transient (abortive) transformants are often formed that may hinder the isolation of integrants containing the gene disruption. We show that abortive transformants result from re-circularization of the linear transforming-DNA in vivo. Their number was greatly reduced when the cut DNA could not readily re-ligate, either by digestions that gave non-compatible or blunt ends, or by de-phosphorylation. In addition, true integrants could be readily distinguished from abortive transformants through replica plating onto selective media. Enhanced disruption-mutagenesis was also observed when non-compatible ends were generated in an ARS-containing insertion vector.     

Cytoplasmic splicing of tRNA in Saccharomyces cerevisiae

The splicing of nuclear encoded RNAs, including tRNAs, has been widely believed to occur in the nucleus. However, we recently found that one of the tRNA splicing enzymes, splicing endonuclease, is localized to the outer surface of mitochondria in Saccharomyces cerevisiae. These results suggested the unexpected possibility of tRNA splicing in the cytoplasm. To investigate this possibility, we examined whether cytoplasmic pre-tRNAs are bona fide intermediates for tRNA maturation in vivo. We isolated a new reversible allele of temperature-sensitive (ts) sen2 (HA-sen2-42), which encodes a mutant form of one of the catalytic subunits of yeast splicing endonuclease. The HA-sen2-42 cells accumulated large amounts of pre-tRNAs in the cytoplasm at a restrictive temperature, but the pre-tRNAs were diminished when the cells were transferred to a permissive temperature. Using pulse-chase/hybrid-precipitation techniques, we showed that the pre-tRNAs were not degraded but rather converted into mature tRNAs during incubation at the permissive temperature. These and other results indicate that, in S. cerevisiae, pre-tRNAs in the cytoplasm are genuine substrates for splicing, and that the splicing is indeed carried out in the cytoplasm.     

Sensors of extracellular nutrients in Saccharomyces cerevisiae

It has been known for a long time that yeast are capable of making rapid metabolic adjustments in response to changing extracellular nutrient conditions. Until recently it was thought that yeast, in contrast to mammalian cells, primarily monitored nutrient availability through the activity of intracellular sensors. Recent advances in our understanding of nutrient sensing indicate that yeast cells possess several nutrient-sensing systems localized in the plasma membrane that transduce information regarding the presence of extracellular amino acids, ammonium. and glucose. Strikingly, the transmembrane components of several of these sensors, Ssylp, Mep2p, Snf3p. and Rgt2p, are unique members of nutrient-transport protein families. Perhaps with the exception of Mep2p, the ability of these transporter homologues to transduce nutrient-(ligand)-induced signals across the plasma membrane appears to be independent of nutrient uptake; and thus these sensor components may function analogously to traditional ligand-dependent receptors. Additionally, the G protein-coupled receptor Gpr1p has been shown to exhibit properties consistent with it being a sensor. These recent advances indicate that yeast cells obtain information regarding their growth environments using sensing systems that are more similar to those present in mammalian cells than previously thought. The fact that yeast plasma membrane nutrient sensors have only recently been discovered reveals how little is understood regarding the molecular signals that enable eukaryotic cells to adapt to changing environments.