Showing papers on "Yeast published in 1993"

Xylose fermentation by Saccharomyces cerevisiae

Abstract: We have performed a comparative study of xylose utilization in Saccharomyces cerevisiae transformants expressing two key enzymes in xylose metabolism, xylose reductase (XR) and xylitol dehydrogenase (XDH), and in a prototypic xylose-utilizing yeast, Pichia stipitis. In the absence of respiration (see text), baker's yeast cells convert half of the xylose to xylitol and ethanol, whereas P. stipilis cells display rather a homofermentative conversion of xylose to ethanol. Xylitol production by baker's yeast is interpreted as a result of the dual cofactor dependence of the XR and the generation of NADPH by the pentose phosphate pathway. Further limitations of xylose utilization in S. cerevisiae cells are very likely caused by an insufficient capacity of the non-oxidative pentose phosphate pathway, as indicated by accumulation of sedoheptulose-7-phosphate and the absence of fructose-1,6-bisphosphate and pyruvate accumulation. By contrast, uptake at high substrate concentrations probably does not limit xylose conversion in S. cerevisiae XYL1/XYL2 transformants.

Germ-line transmission and expression of a human-derived yeast artificial chromosome

Abstract: Introduction of DNA fragments, hundreds of kilobases in size, into mouse embryonic stem (ES) cells would greatly advance the ability to manipulate the mouse genome. Mice generated from such modified cells would permit investigation of the function and expression of very large or crudely mapped genes. Large DNA molecules cloned into yeast artificial chromosomes (YACs) are stable and genetically manipulable within yeast, suggesting yeast-cell fusion as an ideal method for transferring large DNA segments into mammalian cells. Introduction of YACs into different cell types by this technique has been reported; however, the incorporation of yeast DNA along with the YAC has raised doubts as to whether ES cells, modified in this way, would be able to recolonize the mouse germ line. Here we provide, to our knowledge, the first demonstration of germ-line transmission and expression of a large human DNA fragment, introduced into ES cells by fusion with yeast spheroplasts. Proper development was not impaired by the cointegration of a large portion of the yeast genome with the YAC.

Glycoprotein biosynthesis in yeast

Abstract: Many proteins in the yeast Saccharomyces cerevisiae are modified by the attachment of N-linked saccharides to asparagine, of O-linked mannose glycans to serine or threonine, and of glycosylphosphoinositol membrane anchors. The biosynthetic events leading to these modifications are coupled to the secretory pathway. Early stages of N-linked glycosylation and the formation of glycosylphosphoinositol anchors have been conserved through evolution of eukaryotes. Studies of yeast offer a variety of genetic and molecular biological approaches, which have led to the isolation of different glycosylation mutants and of genes for enzymes involved in glycosylation. Yeast mutants are useful to identify biosynthetic intermediates, to establish whether a given enzyme is essential for viability, and to determine how cellular functions are affected when glycosylation is perturbed. Yeast glycosylation mutants and genes can be used to identify their counterparts in other eukaryotes.

Preparation of yeast RNA

Abstract: This unit provides two protocols for extraction of RNA from yeast that differ primarily in the method for lysing the yeast cells. The first protocol isolates RNA directly from intact yeast cells by extraction with hot acidic phenol. This yields RNA that is relatively free of contaminating DNA, is convenient to perform with multiple samples, and gives little or no sample-to-sample variation. In contrast, an alternate protocol relies upon disruption of cells by vigorous mixing with glass beads and denaturing agents. Although this procedure results in efficient breaking of the cells, the product is associated with residual DNA, and the procedure itself is troublesome when one is working with multiple samples. A second alternate protocol describes the scaling up of the first two procedures to isolate enough total RNA for poly (A)+ RNA preparation.

A yeast protein similar to bacterial two-component regulators

Abstract: Many bacterial signaling pathways involve a two-component design. In these pathways, a sensor kinase, when activated by a signal, phosphorylates its own histidine, which then serves as a phosphoryl donor to an aspartate in a response regulator protein. The Sln1 protein of the yeast Saccharomyces cerevisiae has sequence similarities to both the histidine kinase and the response regulator proteins of bacteria. A missense mutation in SLN1 is lethal in the absence but not in the presence of the N-end rule pathway, a ubiquitin-dependent proteolytic system. The finding of SLN1 demonstrates that a mode of signal transduction similar to the bacterial two-component design operates in eukaryotes as well.

S. cerevisiae 26S protease mutants arrest cell division in G2/metaphase

Abstract: We isolated two mutants from the yeast Saccharomyces cerevisiae, cim3-1 and cim5-1, that arrest cell division in G2/metaphase at 37 degrees C. CIM3 (identical to SUG1; ref. 1) and CIM5 are similar to each other and are members of a family of putative ATPases that have been proposed to be 26S protease subunits. We show here that CIM5 is the functional yeast homologue of the human MSS1 protein and that homologues of CIM3 and CIM5 are present in a highly purified preparation of the Drosophila 26S protease. The short-lived ubiquitin-proline-beta-galactosidase fusion protein is stabilized in cim mutants, but Leu-beta-galactosidase is not. The CLB2 and CLB3 cyclins also accumulate in the cim mutants. Thus the 26S protease is required in vivo for the degradation of ubiquitinated substrates and for anaphase chromosome separation.

Identification of Ras farnesyltransferase inhibitors by microbial screening.

Abstract: A microbial screen using a yeast strain with conditional deficiency in the GPA1 gene was carried out to search for inhibitors of protein farnesyltransferase (PFT). A strain of Streptomyces was found to produce active compounds named UCF1-A, UCF1-B, and UCF1-C. Structural determination of these compounds revealed that UCF1-C is identical to the known antibiotic, manumycin, whereas UCF1-A and UCF1-B are structurally related to manumycin. All three UCF1 compounds suppress the lethality of gpa1 disruption, with UCF1-C exhibiting the strongest activity. UCF1 inhibits yeast as well as rat brain PFT. Fifty percent inhibition of yeast PFT activity is observed with 5 microM UCF1-C. Kinetic analyses of the inhibition suggest that UCF1-C acts as a competitive inhibitor of PFT with respect to farnesyl pyrophosphate, exhibiting a Ki of 1.2 microM, whereas the same compound appears to act as a noncompetitive inhibitor of PFT with respect to the farnesyl acceptor, the Ras protein. UCF1-C shows significant activity to inhibit the growth of Ki-ras-transformed fibrosarcoma, raising the possibility of its use as an antitumor drug.

Cloning and characterization of ECE1, a gene expressed in association with cell elongation of the dimorphic pathogen Candida albicans

Abstract: The gene ECE1 (extent of cell elongation 1) was isolated by differential hybridization screening of a Candida albicans cDNA library by using probes derived from populations of yeast cells or hyphae. Expression of this gene was not detected when C. albicans grew as a budding yeast cell but was observed within 30 min after cells had been induced to form hyphae. In all strains tested, regardless of the induction signal, ECE1 expression correlated with the extent of cell elongation. The genomic version of ECE1 was cloned and sequenced. The deduced 271-amino-acid polypeptide consisted of eight tandem repeats of a degenerate 34-amino-acid sequence which contained no discernible homology with other known sequences. An ECE1 null mutant displayed no morphological alterations, which demonstrated that ECE1 is not essential for cell elongation or hypha formation despite the strict morphological association of its expression.

Yeast and mammalian metallothioneins functionally substitute for yeast copper-zinc superoxide dismutase

Abstract: Copper-zinc superoxide dismutase catalyzes the disproportionation of superoxide anion to hydrogen peroxide and dioxygen and is thought to play an important role in protecting cells from oxygen toxicity. Saccharomyces cerevisiae strains lacking copper-zinc superoxide dismutase, which is encoded by the SOD1 gene, are sensitive to oxidative stress and exhibit a variety of growth defects including hypersensitivity to dioxygen and to superoxide-generating drugs such as paraquat. We have found that in addition to these known phenotypes, SOD1-deletion strains fail to grow on agar containing the respiratory carbon source lactate. We demonstrate here that expression of the yeast or monkey metallothionein proteins in the presence of copper suppresses the lactate growth defect and some other phenotypes associated with SOD1-deletion strains, indicating that copper metallothioneins substitute for copper-zinc superoxide dismutase in vivo to protect cells from oxygen toxicity. Consistent with these results, we show that yeast metallothionein mRNA levels are dramatically elevated under conditions of oxidative stress. Furthermore, in vitro assays demonstrate that yeast metallothionein, purified or from whole-cell extracts, exhibits copper-dependent antioxidant activity. Taken together, these data suggest that both yeast and mammalian metallothioneins may play a direct role in the cellular defense against oxidative stress by functioning as antioxidants.

Identification of yeast strains using the polymerase chain reaction

Abstract: Commonly used techniques for the identification of industrial yeast strains are usually time-consuming and cumbersome. Moreover, some of these methods may give ambiguous results. A novel strategy has been developed for identifying yeast strain employing polymerase chain reaction technology. Using customised oligonucleotides, some regions of the yeast genome between δ elements are amplified to give an ‘amplified’ sequence polymorphisml (Skolnick and Wallace 1988) characteristic of the strains. With this technique it is possible to identify individual strains of Saccharomyces cerevisiae.

Kinetics of growth and sugar consumption in yeasts

Abstract: An overview is presented of the steady- and transient state kinetics of growth and formation of metabolic byproducts in yeasts. Saccharomyces cerevisiae is strongly inclined to perform alcoholic fermentation. Even under fully aerobic conditions, ethanol is produced by this yeast when sugars are present in excess. This so-called 'Crabtree effect' probably results from a multiplicity of factors, including the mode of sugar transport and the regulation of enzyme activities involved in respiration and alcoholic fermentation. The Crabtree effect in S. cerevisiae is not caused by an intrinsic inability to adjust its respiratory activity to high glycolytic fluxes. Under certain cultivation conditions, for example during growth in the presence of weak organic acids, very high respiration rates can be achieved by this yeast. S. cerevisiae is an exceptional yeast since, in contrast to most other species that are able to perform alcoholic fermentation, it can grow under strictly anaerobic conditions. 'Non-Saccharomyces' yeasts require a growth-limiting supply of oxygen (i.e. oxygen-limited growth conditions) to trigger alcoholic fermentation. However, complete absence of oxygen results in cessation of growth and therefore, ultimately, of alcoholic fermentation. Since it is very difficult to reproducibly achieve the right oxygen dosage in large-scale fermentations, non-Saccharomyces yeasts are therefore not suitable for large-scale alcoholic fermentation of sugar-containing waste streams. In these yeasts, alcoholic fermentation is also dependent on the type of sugar. For example, the facultatively fermentative yeast Candida utilis does not ferment maltose, not even under oxygen-limited growth conditions, although this disaccharide supports rapid oxidative growth.

Gene SNQ2 of Saccharomyces cerevisiae, which confers resistance to 4-nitroquinoline-N-oxide and other chemicals, encodes a 169 kDa protein homologous to ATP-dependent permeases.

Abstract: The yeast gene SNQ2 confers hyper-resistance to the mutagens 4-nitroquinoline-N-oxide (4-NQO) and Triaziquone, as well as to the chemicals sulphomethuron methyl and phenanthroline when present in multiple copies in transformants of Saccharomyces cerevisiae. Subcloning and sequencing of a 5.5 kb yeast DNA fragment revealed that SNQ2 has an open reading frame of 4.5 kb. The putative encoded polypeptide of 1501 amino acids has a predicted molecular weight of 169 kDa and has several hydrophobic regions. Northern analysis showed a transcript of 5.5 kb. Haploid cells with a disrupted SNQ2 reading frame are viable. The SNQ2-encoded protein has domains believed to be involved in ATP binding and is likely to be membrane associated. It most probably serves as an ATP-dependent permease.

Expression cloning in yeast of a cDNA encoding a broad specificity amino acid permease from Arabidopsis thaliana.

Abstract: To study amino acid transport in plants at the molecular level, we have isolated an amino acid permease cDNA from Arabidopsis thaliana by complementation of a yeast mutant defective in proline uptake with a cDNA. The predicted polypeptide of 53 kDa is highly hydrophobic with 12 putative membrane-spanning regions and shows no significant homologies to other known transporters. Expression of the cDNA enables the yeast mutant to take up L-[14C]proline. Competition studies argue for a broad but stereospecific substrate recognition by the permease, which resembles neutral or general amino acid transport systems from Chlorella and higher plants. Both pH dependence and inhibition by protonophores are consistent with a proton symport mechanism.

BUD2 encodes a GTPase-activating protein for Bud1/Rsr1 necessary for proper bud-site selection in yeast

Abstract: Cells of the budding yeast Saccharomyces cerevisiae bud at either axial or bipolar sites depending on their cell type. Bud-site selection directs both cell polarity and the cytoskeletal orientation during budding and is determined by at least five genes: BUD1/RSR1, BUD2, BUD3, BUD4 and BUD5. Mutants defective in BUD1, BUD2 or BUD5 choose bud sites randomly. Bud1 protein (Bud1p) has sequence similarity to Ras, a small GTP-binding protein, and Bud5p is similar to Cdc25p (refs 4, 5), a GDP-GTP exchange factor. Here we report that Bud2p is a GTPase-activating protein (GAP) for Bud1p with a sequence similar to the catalytic domain of rasGAPs, and that Bud2p purified from yeast stimulates GTP hydrolysis by Bud1p. Chromosomal deletion of BUD2 causes a random budding pattern but no obvious growth defect. Overexpression of BUD2 also causes a random budding pattern by wild-type cells.

Alteration of a yeast SH3 protein leads to conditional viability with defects in cytoskeletal and budding patterns.

Abstract: Mutations in genes necessary for survival in stationary phase were isolated to understand the ability of wild-type Saccharomyces cerevisiae to remain viable during prolonged periods of nutritional deprivation. Here we report results concerning one of these mutants, rvs167, which shows reduced viability and abnormal cell morphology upon carbon and nitrogen starvation. The mutant exhibits the same response when cells are grown in high salt concentrations and other unfavorable growth conditions. The RVS167 gene product displays significant homology with the Rvs161 protein and contains a SH3 domain at the C-terminal end. Abnormal actin distribution is associated with the mutant phenotype. In addition, while the budding pattern of haploid strains remains axial in standard growth conditions, the budding pattern of diploid mutant strains is random. The gene RVS167 therefore could be implicated in cytoskeletal reorganization in response to environmental stresses and could act in the budding site selection mechanism.

A human mitochondrial transcriptional activator can functionally replace a yeast mitochondrial HMG-box protein both in vivo and in vitro.

Abstract: Human mitochondrial transcription factor A is a 25-kDa protein that binds immediately upstream of the two major mitochondrial promoters, thereby leading to correct and efficient initiation of transcription. Although the nature of yeast mitochondrial promoters is significantly different from that of human promoters, a potential functional homolog of the human transcriptional activator protein has been previously identified in yeast mitochondria. The importance of the yeast protein in yeast mitochondrial DNA function has been shown by inactivation of its nuclear gene (ABF2) in Saccharomyces cerevisiae cells resulting in loss of mitochondrial DNA. We report here that the nuclear gene for human mitochondrial transcription factor A can be stably expressed in yeast cells devoid of the yeast homolog protein. The human protein is imported efficiently into yeast mitochondria, is processed correctly, and rescues the loss-of-mitochondrial DNA phenotype in a yeast abf2 strain, thus functionally substituting for the yeast protein. Both human and yeast proteins affect yeast mitochondrial transcription initiation in vitro, suggesting that the two proteins may have a common role in this fundamental process.

Analysis of yeast diversity during spontaneous and induced alcoholic fermentations

Abstract: The diversity of yeast species and strains was monitored by physiological tests and a simplified method of karyotyping of yeast chromosomes. During the first phase of investigated alcoholic fermentations, the yeast species Metschnikowia pulcherrima and Hanseniaspora uvarum were predominant, irrespective of the origin of the grape must. At the beginning of fermentation H. uvarum was even present in the case of induced fermentations with dried yeast. Middle and end phase of the alcoholic fermentation were clearly dominated by the yeast species Saccharomyces cerevisiae. In the case of spontaneous fermentations, several different strains of S. cerevisiae were present and competed with each other, whereas in induced fermentations only the inoculated strain of S. cerevisiae was observed. A competition of strains of S. cerevisiae also occurred during the fermentation with dried yeast product consisting of two different strains. An effect of H. uvarum on taste and flavour of wines can be postulated according to the frequency of its appearance during the first phase of fermentation. With the method of rapid karyotyping and supplementary physiological tests it was possible to make reliable assertions about the yeast diversity during alcoholic fermentation.

Cloning and improving the expression of Pichia stipitis xylose reductase gene in Saccharomyces cerevisiae.

Abstract: The intactPichia stipitis xylose reductase gene (XR) has been cloned and expressed inSaccharomyces cerevisiae. The possible further improvement of the expression of thePichia gene in the new host was studied. To improve the expression of the XR gene in yeast (Saccharomyces cerevisiae), its 5′-noncoding sequence containing the genetic elements for transcription and translation was systematically replaced by that from the yeast genes. It was found that thePichia genetic signal for transcription of XR is more effective than the yeast TRP5 promoter, but is about half as effective as the yeast strong promoter of the alcohol dehydrogenase gene (ADC1). However, the nucleotide sequence immediately adjacent to the initiation codon of XR, which controls the translation of the gene product, seemed to be five times less effective than the corresponding sequence of the ADC1 gene. By totally replacing its 5′-noncoding sequence with that of the yeast ADC1 gene, the expression of XR in yeast was found to be nearly ten times higher. Furthermore, the clonedPichia XR described in this article contains very little of its 3′-noncoding sequence. In order to study whether the 3′-noncoding sequence is important to its expression inS. cerevisiae, the intact 3′-noncoding sequences of the yeast xylulokinase gene was spliced to the 3′ end of theP ADC1-XR structural gene. This latter modification has resulted in a twofold further increase in the expression of thePichia XR in yeast.

An essential yeast protein, CBF5p, binds in vitro to centromeres and microtubules.

Abstract: Yeast centromere DNA (CEN) affinity column chromatography has been used to purify several putative centromere and kinetochore proteins from yeast chromatin extracts. The single yeast gene (CBF5) specifying one of the major low-affinity centromere-binding proteins (p64'/CBF5p) has been cloned and shown to be essential for viability of Saccharomyces cerevisiae. CBF5 specifies a 55-kDa highly charged protein that contains a repeating KKD/E sequence domain near the C terminus, similar to known microtubule-binding domains in microtubule-associated proteins 1A and 1B, CBF5p, obtained by overexpression in bacterial cells, binds microtubules in vitro, whereas C-terminal deleted proteins lacking the (KKD/E)n domain do not. Dividing yeast cells containing a C-terminal truncated CBF5 gene, producing CBF5p containing only three copies of the KKD/E repeat, delay with replicated genomes at the G2/M phase of the cell cycle, while depletion of CBF5p arrests most cells in G1/S. Overproduction of CBF5p in S. cerevisiae complements a temperature sensitivity mutation in the gene (CBF2) specifying the 110-kDa subunit of the high-affinity CEN DNA-binding factor CBF3, suggesting in vivo interaction of CBF5p and CBF3. A second low-affinity centromere-binding factor has been identified as topoisomerase II.

The putative phosphoinositide-specific phospholipase C gene, PLC1, of the yeast Saccharomyces cerevisiae is important for cell growth.

Abstract: Using the polymerase chain reaction technique, we have isolated a gene that encodes a putative phosphoinositide-specific phospholipase C (PLC) in the yeast Saccharomyces cerevisiae. The nucleotide sequence indicates that the gene encodes a polypeptide of 869 amino acid residues with a calculated molecular mass of 101 kDa. This polypeptide has both the X and Y regions conserved among mammalian PLC-beta, -gamma, and -delta, and the structure is most similar to that of mammalian PLC-delta. This putative yeast PLC gene has been designated PLC1. Disruption of PLC1 results in slow growth or lethality for cells, depending on their genetic background and the medium, indicating that PLC1 is important for cell growth. Expression of rat PLC-delta 1 cDNA suppressed the growth defect of plc1 disruptants, strongly suggesting that PLC1 encodes PLC.

Catabolism of benzene compounds by ascomycetous and basidiomycetous yeasts and yeastlike fungi. A literature review and an experimental approach.

Abstract: A literature review is given on growth of yeasts on benzene compounds and on the catabolic pathways involved. Additionally, a yeast collection was screened for assimilation of phenol and 3-hydroxybenzoic acid. Fifteen ascomycetous and thirteen basidiomycetous yeast species were selected and were tested for growth on 84 benzene compounds. It appeared that 63 of these compounds supported growth of one or more yeast species. The black yeast Exophiala jeanselmei assimilated 54 of these compounds. The catechol branch of the 3-oxoadipate pathway and its hydroxyhydroquinone variant were involved in phenol and resorcinol catabolism of ascomycetes as well as of basidiomycetes. However, these two groups of yeasts showed characteristic differences in hydroxybenzoate catabolism. In the yeastlike fungus E. jeanselmei and in basidiomycetes of the genera Cryptococcus, Leucosporidium and Rhodotorula, the protocatechuate branch of the 3-oxoadipate pathway was induced by growth on 3- and 4-hydroxybenzoic acids. In three Trichosporon species and in all ascomycetous yeasts tested, 4-hydroxybenzoic acid was catabolyzed via protocatechuate and hydroxyhydroquinone. These yeasts were unable to cleave protocatechuate. 3-Hydroxybenzoic and 3-hydroxycinnamic acids were catabolized in ascomycetous yeasts via the gentisate pathway, but in basidiomycetes via protocatechuate. Incomplete oxidation of phenol, some chlorophenols, cresols and xylenols was observed in cultures of Candida parapsilosis growing on hydroquinone. Most compounds transformed by the growing culture were also converted by the phenol monooxygenase present in cell-free extracts of this yeast. They did not support growth. The relationship between the ability of ascomycetous yeasts to assimilate n-alkanes, amines and benzene compounds, and the presence of Coenzyme Q9 is discussed.

Use of yeast in the study of anticancer drugs targeting DNA topoisomerases: expression of a functional recombinant human DNA topoisomerase II alpha in yeast.

Abstract: A plasmid was constructed for the expression of human DNA topoisomerase IIα in yeast from a galactose-inducible promoter of the yeast GAL1 gene. Expression of a recombinant human enzyme, in which the first 28 of the 1531 codons of human DNA topoisomerase IIα were replaced by the first five codons of yeast DNA topoisomerase II, was shown to rescue the lethal phenotype of thermal sensitive yeast DNA topoisomerase II mutants at 35°C. Purification of the human enzyme overexpressed in yeast yielded a single polypeptide with an apparent mass of 170 kDa, and the properties of the purified recombinant enzyme were found to be the same as those reported for human DNA topoisomerase IIα purified from HeLa cells. Studies with the anticancer drug amsacrine indicated that the human enzyme, either inside yeast cells or in its purified form, is a target of the drug; inhibition of the purified enzyme by teniposide (VM-26) and merbarone was also demonstrated. These studies demonstrate that yeast strains expressing human DNA topoisomerase IIα provide a convenient system for studying drugs targeting the enzyme; unlike mammalian systems, potential complications due to the presence of human DNA topoisomerase IIβ can be eliminated in this system. Overexpression of human DNA topoisomerase IIα in yeast also provides a convenient source of the enzyme for in vitro studies.