bluuebunny/crossref_metadata_2025 huggingface dataset:
Physiology of mutants with reduced expression of plasma membrane H+‐ATPase
Authors: Vallejo Carmen G. , Serrano Ramon
Published: May 2005

Two mutations containing insertions and deletions in the promoter in the plasma membrane H+‐ATPase gene (PMA1) of Saccharomyces cerevisiae have been introduced into the genome by homologous recombination, replacing the wild‐type gene. The resulting strains have 15 and 23% of the wild‐type ATPase content. Decreased levels of ATPase correlate with decreased rates of proton efflux and decreased uptake rates of amino acids, methylamine, hygromycin B and tetraphenylphosphonium. This supports a central role of the enzyme in yeast bioenergetics. However, the final accumulation gradient of tetraphenylphosphonium is not affected by the mutations and that of methylamine and 2‐aminoisobutyric acid is only decreased in the most extreme mutant. Apparently, kinetic constraints seem to prevent the equilibration of yeast active transports with the electrochemical proton gradient. As expected from their transport defects, the ATPase‐deficient mutants are more resistant to hygromycin B and more sensitive to acidification than wild‐type yeast. Mutant cells are very elongated, suggesting a structural role of the ATPase in the yeast surface.
Occurrence of peroxisomal membrane proteins in methylotrophic yeasts grown under different conditions
Authors: Sulter G. J. , Looyenga L. , Veenhuis M. , Harder W.
Published: May 2005

We have the substructure and polypeptide composition of the peroxisomal membranes in two methylotrophic yeasts in relation to different growth conditions. The results obtained that no significant ultrastructural differences existed between the membranes of variously grown cells.

The presence of specific peroxisomal membrane proteins (PMPs) was studied biochemically. On sodium dodecyl sulphate‐polyacrylamide gels of purified microbody membranes isolated from methanol‐grown Hansenula polymorpha, prominent proteins bands were observed at 22, 31, 35, 42, 49 and 51 kD. These proteins were also present when the cells were grown in media containing ethanol and/or ethylamine. Apart from these, several other PMPs were specifically induced under these conditions, namely 24, 29, 37 and 62 kD proteins. The polypeptide composition of peroxisomal membranes from H. polymorpha was compared with that of another methylotroph. Candida biodinii. In the latter organism a specific PMP with a molecular weight of 23 kD was induced during growth on D‐alanine instead of ammonium sulphate as the nitrogen source.
Immunocytochemical demonstration of the peroxisomal ATPase of yeasts
Authors: Douma A. C. , Veenhuis M. , Waterham H. R. , Harder W.
Published: May 2005

The presence of an ATpase on yeast peroxisomal membranes was studied by immunological methods. Western blot analysis of purified peroxisomal membranes from several yeasts revealed distinct cross‐reaction with specific antibodies against the F1‐part or the β‐subunit of the mitochondrial ATpase of Saccharomyces cerevisiae. This was not due to mitochondrial contamination as was demonstrated by analytical sucrose gradient centrifugation.

Protein A‐gold labelling carried out on Lowicryl‐embedded methanol‐grown Hansenula polymorpha using these antibodies did not result in significant staining. However, when organelles isolated from this yeast were successively incubated with antibodies and protein A‐gold prior to embedding, specific labelling was observed on both the peroxisomal membrane and the membrane of damaged mitochondria but not on intact mitochondira. Specific labelling of the peroxisomal membrane was confirmed by freeze‐fracture immunocytochemistry. In addition to the peroxisomal membrane, the mitochondrial membrane was also labelled in these experiments. Freeze‐fracture immunocytochemistry was also successful for the localization of peroxisomal matrix proteins, e.g. alcohol oxidase and dihydroxyacetone synthase and of mitochondrial membrane proteins, e.g. cytochrome c oxidase.
Comparative study on cytochrome P‐450 of yeasts using specific antibodies to cytochromes P‐450alk and P‐45014DM
Authors: Kaergel E. , Aoyama Y. , Schunck W.‐H. , Mueller H.‐G. , Yoshida Y.
Published: May 2005

The occurrence of cytochrome P‐45014DM(lanosterol 14α‐demethylase) and cytochrome P‐450alk (long‐chain alkane terminal hydroxylase) in various yeast strain was determined with immunological procedures. Cytochrome P‐45014DM, which is a constitutive or housekeeping enzyme playing an essential role in ergosterol biogenesis, was found in all yeast strains so far tested. Cytochromes P‐45014DM from different species of yeast were immunologically different, although they may have a few common antigenic sites. In contrast, cytochrome P‐450alk was detected only in the alkane‐assimilating yeasts.
A K2 neutral Saccharomyces cerevisiae strain contains a variant K2 M genome
Authors: Wingfield Brenda D. , Southgate Valerie J. , Pretorius Isak S. , Van Vuuren Hendrik J. J.
Published: May 2005

K2 neutral strain Saccharomyces cerevisiae USM12 was identified and characterized. This strain carried an M double‐stranded RNA (dsRNA) genome encoding for resistance to K2 toxin. The M dsRNA was larger than the K2 killer yeast M dsRNA and homoduplex analysis of denatured and reannealed K2 neutral M dsRNA revealed an inverted duplication. Heteroduplex analysis showed that two thirds of the K2 M genome had homology with K2 neutral M genome. Hybridization showed that the USM12 M dsRNA has significant homology with the K2M dsRNA. Protein profiles of extracellular proteins from USMA12 and cured strain indicated that USM12 did not secrete any toxin. This is the first time that a K2 neutral yeast strain has been characterized.
Gaba transport in Saccharomyces cerevisiae
Authors: McKelvey Joyce , Rai Rajendra , Cooper Terrance G.
Published: May 2005

Gamma‐aminobutyrate (GABA) accumulation in growing cultures of Saccharomyces cerevisiae was shown to occur by means of an active transport system that is inhibited by proton ionophores, azide, fluoride and arsenate ions. Transport occurred maximally at pH 5·0 and exhibited apparent Km values of 12 μM and 0·1 mM. Accumulated GABA did not efflux upon treatment with proton ionophores and exchanged with extracellular material only very slowly. However, release was complete upon treatment with nystatin. These observations raise the possibility that a major portion of intracellular GABA is sequestered in the vacuole. The response of GABA uptake to growth on various nitrogen sources suggested that uptake may be subject to several types of regulation.
An essential gene in Saccharomyces cerevisiae shares an upstream regulatory element with PRP4
Authors: Petersen‐Bjørn Sara , Harrington T. R. , Friesen James D.
Published: May 2005

ORF2 is an essential gene immediately upstream of PRP4 (formeryl RNA4), a gene involved in nuclear mRNA processing in Saccharomyces cerevisiae. The two genes are arranged head‐to‐head. An 8 base‐pair conserved sequences element is found upstream of both genes, as well as upstream of certain other genes that are known to be involved in pre‐mRNA processing. Through deletion analysis we have found that both of the conserved sequence elements are important for transcription of both genes. We have cloned ORF2 and have isolated temperature‐sensitive orf2 mutants. The phenotype of these mutants does not suggest a role for ORF2 in mRNA processing. The deduced amino acid sequence of ORF2 indicates significant similarity to DPR1, a gene encoding a protein that is involved in the carboxy‐terminal processing of G‐protein.
Yeast Saccharomyces cerevisiae selectable markers in pUC18 polylinkers
Authors: Jones Jeffery S. , Prakash Louise
Published: May 2005

A set of plasmids was constructed that contain the yeast selectable markers HIS3, LEU2, TRP1 or URA3 embedded in the multiple cloning site of pUC18.
Characterization of the cytochrome c gene from the starch‐fermenting yeast Schwanniomyces occidentalis and its expression in bakers's yeast
Authors: Amegadzie Bernardy Y. , Zitomer Richard S. , Hollenberg Cornelis P.
Published: May 2005

A cytochrome c protein gene, CYC10, of the dextran‐ and starch‐fermenting yeast, Schwanniomyces occidentalis was cloned and characterized. The DNA sequence was determined, and the predicted amino acid sequence of the protein‐coding region shares close homologies to the cytochrome c genes. A. S. occidentalis strain with a disruption of the gene revealed that CYC10 was the only functional cytochrome c protein‐encoding gene in S. occidentalis, unlike the two cytochrome c protein genes (CYC1 and CYC7) in Saccharomyces cerevisiaear The CYC10 gene was oxygen‐induced but not subject to catabolite repression.

The expression of the CYC10 gene was studied in the heterologous yeast. S. cerevisiae. The oxygen induction of the gene was found to be identical to that of the CYC1 gene, indecating these two genes share similar or closely related cis‐ and trans‐ acting oxygen regulatory elements. However, the CYC10 gene was glucose repressed in S. cerevisiae strains; a phenomenon which was not observed in the native S. accidentalis cells. Search in the 5′ unstranslated region of the CYC10 gene revealed some homologies at −425 to −405 to UAS1 of the S. cerevisiae CYC1 gene. A deletion of a segment of upstream region including this sequence abolished expression in S. cerevisiae.

Finally the phylogenetic relationships of different yeasts and fungi were determined based upon the amino acid sequences of the cytochrome c proteins. These relationships do not completely agree with classical divisions.
The omnipotent suppressor SUP45 affects nucleic acid metabolism and mitochondrial structure
Authors: Pocklington Michael J. , Johnston Lee , Jenkins John R. , Orr Elisha
Published: May 2005

Yeast (Saccharomyces cerevisiae) strains sensitive to a variety drugs were used to select for novobiocin‐resistant mutants that were simultaneously temperature‐sensitive. The mutants remained as sensitive as the parent strains to a wide range of drugs other than novobiocin, and did not exhibit any suppression of suppressible auxotrophic markers. At the non‐permissive temperature, the mutant cells arrested mainly as unbudded cells, and were instantly defective in DNA and RNA synthesis, but not protein synthesis. The cloned wild‐type gene was identified as SUP45, which has been previously implicated in the translation process. Our results suggest that SUP45 may have a function in addition to, or different from, the one that has been assigned to it previously.
Effect of leader primary structure on the translational efficiency of phosphoglycerate kinase mRNA in yeast
Authors: Van Den Heuvel Joop J. , Planta Rudi J. , Raué Hendrik A.
Published: May 2005

In order to determine the effect of nucleotide composition of the 5′‐untranslated (leader) region on the translational efficiency of mRNA in yeast, we replaced a large part of the leader region of the phosphoglycerate kinase (PGK) gene by various deoxyoligonucleotides of defined sequence. All mutations left the context of the transcription initiations site and AUG start codon intact. The mutant genes were introduced into yeast cells on a multicopy vector and the ratio of the steady‐state levels of PGKmRNA and protein were determined.

We found the translational efficiency to be unaffected by the presence of either an 18 nucleotides (nt) long poly A or poly C tract or by sequences consisting of mixtures of A and C residues in any proportion. In contrast, a polyU tract, as well as mixtures of U and C residues, reduced translational efficiency by a factor of two to three, presumably by long‐rang base ‐pairing between the leader and sequences elsewhere in the coding or 3′‐non‐coding regions of the messenger. In agreement with this hypothesis, a five‐fold reduction in translational efficiency was found for an mRNA carrying a polyC tract in the leader as well as a polyG tract in the trailer, neither of which had any effect on translational efficiency by itself. Therefore, we conclude that the leader and trailer regions (including the polyA tail) of PGK mRNA are sufficiently close to base‐pair when containing complementary sequences. The resulting secondary structure evidently constitutes a barrier for incoming 40S subunits on their way to the AUG start codon.

The presence of an 18 nt long polyG tract in the leader completely abolished translation of the PGK mRNA in accordance with earlier observations. However, we found the leaders containing up to 40% G residues interspersed with either A or U, still allow highly efficient translation. This value is about four times as high as the average G content of leader sequence in naturally occurring yeast mRNAS.

Finally, neither deletion of about 40% of the trailer sequence of PGK mRNA, not replacement of this sequence by homopolymer tracts had any effect on translational efficiency.
Evaluation of phylogenetic relationships among fission yeast by nDNA/nDNA reassociation and conventional taxonomic criteria
Authors: Martini Ann Vaughan
Published: May 2005

The genus Schizosaccharomyces comprises a small, somewhat heterogeneous group of yeast species which have in common a unique mode of vegetative reproduction by cross‐wall formation without constriction as well as a certain degree of osmophilia, most strains having been isolated from habitats of high sugar concentration. This study evaluated inter‐ and intraspecific relationships utilizing nDNA/nDNA optical reassociation and by the analysis of physiological profiles of several strains of each species. Results demonstrate that the genus should be divided into three species: Schiz. japonicus, Schiz. octosporus and Schiz. pombe.
Increased endocytosis in the Saccharomyces cerevisiae fragile mutant VY1160
Authors: Waltschewa L. , Kotyk A. , Venkov P.
Published: May 2005

The VY1160 mutant is characterized by cell lysis in hypotonic solutions and generally increased permeability to substances for which Saccharomyces cerevisiae cells are not permeable. Two mutations, srb1 and ts1, have been identified in VY1160 mutant, and previous studies (Kozhina et al., 1979) have shown srb1 to be responsible for cell lysis. We now present evidence that the ts1 mutation leads to increased endocytosis in VY1160 cells. The internalization of lucifer yellow carbohydrazide in VY1160 cells is time‐, temperature‐ and energy‐dependent an consistent with a fluid‐phase mechanism of endocytosis. The rate of steady‐state accumulation of hte dye at 37°C is 145 ng/μg DNA per h for VY1160 mutant and 23 ng/μg DNA per h for S288C parental strain. Studies with isogenic strains having either the srb1 or the ts1 mutation, or SRB1 TS1 wild‐type alleles have shown that only ts1 strains possess increased endocytosis. Quantitation of endocytosis in cells grown at 24°C and shifted at 38°C shows that ts1 strains, but not srb1 and wild‐type strains, increase ten‐fold the internalization of lucifer yellow 2 h after the shift at 38°C. The analysis of ts1 × wild‐type crosses provides evidence that the temperature‐sensitive phenotype segregates together with the enhanced endocytosis. It is concluded that the increased endocytosis might the generally increased permeability of VY1160 mutant cells.
Killer system of Kluyveromyces lactis: The open reading frame 10 of the pGK12 plasmid encodes a putative DNA binding protein
Authors: Tommasino Massimo
Published: May 2005

ORF 10 of the K2 plasmid from Kluyveromyces lactis encodes a small basic protein (22·3% lysine). The function of its product has been investigated. Western blot analysis, using an antibody against MS2 RNA polymeras/ORF 10 fusion protein, reveals a protein band with an apparent molecular weight of 14 kDa. The protein can bind a DNA‐Sepharose column, and is eluted by 350 mM‐salt. Immunoprecipitation experiments show that the ORF 10 protein coprecipitates with the linear genomic DNAs of the two killer plasmids (K1 and K2). From Western/Southern blot data, it is possible to conclude that the interaction between protein and DNA occurs directly, rather than via other proteins(s). ORF 10 is easily detected by Western blot and its transcript is one of the most abundant of the K2 plasmid, suggesting that this protein may have a structural rather than a regulatory function. This possibility is also suggested by the observed sequeence homology between the ORF 10 protein and the family of histone‐like proteins.
CDC15, an essential cell cycle gene in Saccharomyces cerevisiae, encodes a protein kinased domain
Authors: Schweitzer Bert , Philippsen Peter
Published: May 2005

The cell division cycle gene CDC 15 is essential for the late nucler division in the yeast Saccharomyces cerevisiae. The amino acid sequence of the 974 amino acids/110 kDa CDC 15 gene product, as deduced from the nucletide sequence, includes an aminoterminal protein kinase domain which contains a primary sequence mosaic showing patterns specific for protein serine/theonine kinases besides those for protein tyrosine kinases. Many protein kinases non‐essential for growth are known. CDC 15 represents an essential protein kinase like CDC 7 and CDC 28. A carboxyterminal deletion of 32 amino acids renders the protein inactive.
Optimization of Bacillus α‐amylase production by Saccharomyces cerevisiae
Authors: Ruohonen L. , Penttilä M. , Keränen S.
Published: May 2005

Production of Bacillus amyloliquefaciens α‐amylase by Saccharomyces cerevisiae using the multicopy plasmid pAAH5 and ways of improving the yields of secreted enzyme were studied. In standard non‐buffered medium, α‐amylase was rapidly inactivated but stabilization of the pH at 6 led to stable accumulation of α‐amylase in the culture medium. Removal of 1100 bp of the upstream sequence of the ADH1 promoter present on pAAH5 resulted in delayed but increased α‐amylase production: 29‐fold in selective medium, two‐fold in non‐selective medium. With the original ADH1 promoter, accumulation of α‐amylase in the medium started to level off before the cultures reached stationary phase and was very low when exponentially growing cells were transferred from glucose to ethanol. This coincided with the appearance of a mRNA larger than the α‐amylase messenger. With the shortened promoter, the normal‐size α‐amylase mRNA was detected under all growth conditions and α‐amylase was efficiently secreted into the medium also late in stationary phase and after transfer to ethanol. Highest total yields of α‐amylase were obtained with the short promoter in non‐selective glucose‐containing medium; this may be explained by the greater final cell density obtained. However, the production of α‐amylase per cell mass was higher in ethanol‐containing selective medium. Seventy to eighty per cent of the α‐amylase activity was secreted into the medium independent of the total amount produced.
Induction of a heat‐shock‐type response in Saccharomyces cerevisiae following glucose limitation
Authors: Bataillé Nelly , Régnacq Matthieu , Boucherie Hélian
Published: May 2005

The protein pattern of yeast cells which have arrested proliferation in response to glucose exhaustion is drastically different from that of exponentially growing cells (Boucherie, 1985). In this study, we used two‐dimensional gel electrophoresis to characterize the protein events responsible for these alterations. We found that the induction of heat‐shock proteins is one of the major events responsible for these changes. This induction accounts for the synthesis of 18 of the 35 novel polypeptides observed in glucose‐limited cells. It was shown to occur in combination with two other protein events: the derepression of carbon catabolite repressed proteins, which accounts for the synthesis of the other novel polypeptides, and an arrest of the synthesis of almost all the proteins present in exponentially growing cells.

The time course of each of these events was determined by carrying out a detailed analysis of the pattern of proteins synthesized at vaious stages of a culture exhausting its glucose supply, and by the measurement of the rate of synthesis of individual polypeptides. The results showed in particular that the synthesis of most of the heat‐shock proteins synthesized in glucose‐limited cells was induced closely before glucose exhaustion, and that this synthesis was transient, climaxing by the time glucose was exhausted. Under the culture condition investigated, the entry into stationary phase associated with glucose limitation began several hours before glucose exhaustion. It was thus concluded that the observed induction of heat‐shock proteins is directly related to the nutritional limitation and is independent from the arrest of cell proliferation.
The YDp plasmids: A uniform set of vectors bearing versatile gene disruption cassettes for Saccharomyces cerevisiae
Authors: Berben Gilbert , Dumont Jacques , Gilliquet Véronique , Bolle Paul‐André , Hilger François
Published: May 2005

The YDp plasmids (Yeast Disruption plasmids) are pUC9 vectors bearing a set of yeast gene disruption cassettes, all uniform in structure and differing only in the selectable marker used (HIS3, LEU2, LYS2, TRP1 or URA3). The markers, surrounded by translational termination codons, are embedded in the slightly modified sequence of the pUC9 multiple cloning sites.
The determinants of heat‐shock element‐directed lacZ expression in Saccharomyces cerevisiae
Authors: Kirk Niall , Piper Peter W.
Published: May 2005

Heat‐shock induction of heat‐shock protein genes is due to a specific promoter element (the heat‐shock element, HSE). This study used lacZ under HSE control (HSE‐lacZ) to characterize HSE activity in Saccharomyces cerevisiae cells of different physiological states and differing genetic backgrounds.

In batch fermentations HSE‐lacZ induction by heat shock was maximal in exponential growth, and showed marked decline with the approach to stationary phase. Expression in the absence of heat shock was unaffected by growth phase, indicating that the growth‐dependent expression of many yeast heat‐shock genes uses promoter elements in addition to the HSE. Heat‐induced expression was strongly influenced by the temperature at which cultures were grown. While basal, uninduced expression was constant during growth at different temperatures to 30°C, induction by transfer to 39°C was reduced by increases in growth temperature as low as 18–24°C. Maximal HSE‐lacZ induction (30‐ to 50‐fold) was in cultures grown at low temperatures (18–24°C), then heat shocked at 39°C. Ethanol was a poor inducer. Mutations having little effect on HSE‐lacZ expression included a respiratory petite; ubi4 (which inactivates the polyubiquitin gene); also ubc4 and ubc5 (which each inactivate one of the ubiquitin ligases involved in degradation of aberrant protein). pep4‐3 increased both basal and induced β‐galactosidase about two‐fold, probably because of slower turnover of this enzyme in pep4‐3 strains.
The control of trehalose biosynthesis in Saccharomyces cerevisiae: Evidence for a catabolite inactivation and repression of trehalose‐6‐phosphate synthase and trehalose‐6‐phosphate phosphatase
Authors: François Jean , Neves Maria‐José , Hers Henri‐Géry
Published: May 2005

During diauxic growth of yeast in glucose‐rich medium, the accumulation of trehalose started well after complete exhaustion of glucose from the medium. The accumulation of the disaccharide was concomitant with a resumption of cell growth on the ethanol accumulated in the medium, but not with a degrdation of glycogen which occurred as soon as glucose had been consumed. In contrast, in a mutant deficient in phosphoenlpyruvate carboxykinase, the synthesis of trehalose coincided exactly with the degradation of glycogen. Upon inoculation of stationary phase wild‐type cells into a glucose medium, the activites of trehalose‐6‐phosphate (Tre6P) synthase and Tre6P phosphatase dropped in parallel to reach only 15% of their initial values after 3 h, and only recovered their original values as cell re‐entered staionary phase. In the presence of cycloheximide, the decrease in Tre6P synthase and Tre6P phosphatase activities was restricted to 50–60%, the remaining decrease being inhibited by the drug. Furthermore, the reappearance of the enzyme activities following transfer of cells to an acetate medium was blocked by cycloheximide. It was also shown that loss of activity of these two enzymes required a combination of metabolizable sugars together with a nitrogen source. Low activities of Tre6P synthase and Tre6P phosphatase were measured in mutants with increased adenylate cyclase activity (RAS2ala18val19 mutants). Moreover, derepression of these enzymes at the approach of stationary phase was prevented in a pde2 mutant when it was cultivated in the presence of exogenous cyclic nucleotide. The mechanism of this effect is not clear, but may involve a transcriptional regulation by cAMP of the genes encoding these proteins.
The use of proline as a nitrogen source causes hypersensitivity to, and allows more economical use of 5FOA in Saccharomyces cerevisiae
Authors: McCusker John H. , Davis Ronald W.
Published: May 2005

The use of proline as a nitrogen soucre causes hypersensitivity to 5‐fluoro‐orotic acid (5FOA) and allows up to 40‐fold less of this drug to be used to select for the loss of URA3 function in Saccharomyces cerevisiae. 5FOA hypersensitivity is presumably due to the absence of nitrogen catabolite repression when proline is substituted for (NH4)2SO4 as a nitrogen source. There are two constraints to the use of the proline–5FOA combination: (1) S288c genetic background strains are hypersensitive to 5FOA when grown in proline as a nitrogen source but at least one other genetic background is resistant to low levels of 5FOA under these conditions. (2) The addition of some nutritional supplements confers phenotypic resistance to the 5FOA–proline combination.
A family of low and high copy replicative, integrative and single‐stranded S. cerevisiae/E. coli shuttle vectors
Authors: Bonneaud Nathalie , Ozier‐Kalogeropoulos Odile , Li Guoya , Labouesse Michel , Minvielle‐Sebastia Lionel , Lacroute Francois
Published: May 2005

We describe a set of replicative, integrative and single‐stranded shuttle vectors constructed from the pUC19 plasmid that we use routinely in our experiments. They bear a yeast selectable marker: URA3, TRP1 or LEU2. Replicative vectors carrying different yeast replication origins have been constructed in order to have plasmids based on the same construction with a high or low copy number per cell and with different mitotic stabilities. All the vectors are small in size, provide a high yield in Escherichia coli and efficiently transform Saccharomyces cerevisiae. These plasmids have many of the unique sites of the pUC19 multicloning region and many of them allow for the screening of plasmids with an insert by alpha‐complementation. The nucleotide sequence of each of them is completely known.
The sequence of 8·8 kb of yeast chromosome III cloned in lambda PM3270 contains an unusual long ORF (YCR601)
Authors: Rodriguez Francesco , Martegani Enzo , Mauri Isabella , Alberghina Lilia
Published: May 2005

We have determined the nucleotide sequence of a segment of chromosome III contained in the right part of the lambda PM3270 clone, for a total of 8824 bp. This sequence contains an unusual long open reading frame, YCR601, of 6501 bp that encodes for a protein of 2167 amino acids that show no homology with other known proteins. YCR601 was disrupted by internal deletion and insertion of LEU2 gene and is a non‐essential gene, however it is transcribed during vegetative growth yielding a polyadenylated mRNA of approximately 7 kb.
Yeast mutant affected for viability upon nutrient starvation: Characterization and cloning of the RVS161 gene
Authors: Crouzet Marc , Urdaci Maria , Dulau Laurent , Aigle Michel
Published: May 2005

In yeast, nutrient starvation leads to entry into stationary phase. Mutants that do not respond properly to starvation conditons have been isolated in Saccharomyces cerevisiae. Among them the rvs161 mutant (RVS for Reduced Viability upon Starvation) is sensitive to carbon, nitrogen and sulphur starvation. When these nutrients are depleted in the medium, mutant cells show cellular viability loss with morphological changes. The mutation rvs161‐1 is very pleiotropic, and besides the defects in stationary phase entry, the mutant strain presents other alterations: sensitivity to high salt concentrations, hypersensitivity to amino acid analogs, no growth on lactate or acetate medium. The addition of salts or amino acid analogs leads to the same morphological defects observed in starved cells, suggesting that the gene could be implicated mainly in the control of cellular viability. The gene RVS161 was cloned; it codes for a 30,252 daltons protein. No homology was detected with the proteins contained in the databases. Moreover, Southern analysis revealed the presence of other sequences homologous to the RVS161 gene in the yeast genome.
Four major transcriptional responses in the methionine/threonine biosynthetic pathway of Saccharomyces cerevisiae
Authors: Mountain Harry A. , Byström Anders S. , Larsen Jörgen Tang , Korch Christopher
Published: May 2005

Genes encoding enzymes in the threonin/methionine biosynthetic pathwa were cloned and used to investigate their transcriptional response to signals known to affect gene expression on the basis of enzyme specific‐activities. Four major responses were evident: strong repression by methionine of MET3, MET5 and MET14, as previously described for MET3, MET2 and MET25; weak repression by methionine of MET6; weak stimulation by methionine but no response to threonine was seen for THR1, HOM2 and HOM3; no response to any of the signals tested, for HOM6 and MES1. In a BOR3 mutant, THR1, HOM2 and HOM3 mRNA levels were increased slightly. The stimulation of transcription by methionine for HOM2, HOM3 and THR1 is mediated by the GCN4 gene product and hence these genes are under the general amino acid control. In addition to the strong repression by methionine, MET5 is also regulated by the general control.
Electroporation‐stimulated recombination in yeast
Authors: Higgins David R. , Strathern Jeffrey N.
Published: May 2005

Saccharomyces cerevisiae cells treated by high voltage and made transformation‐competent (electroporation) are also made hyper‐recombinational as determined by an assay that measures interchromosomal mitotic recombination between chromosome III homologs, each containing mutant heteroallelic copies of the trp1 and his3 genes. There is a 10‐fold stimulation of Trp+ and 21‐fold stimulation of His+ prototrophs. Although this stimulation coincides with conditions for maximal transformation competence it is independent of the presence of transforming plasmid DNA. Electroporation does not increase the reversion frequency of these mutations, nor is there a stimulation in Ty transposition. Among the electroporation‐stimulated Trp+ and His+ recombinants there is no dramatic difference in the pattern of events: that is to say that, while there is an increase in the number of recombinants, the distribution of gene conversion and cross–over events among the stimulated recombinants is not significantly altered compared to spontaneously arising Trp+ and His+ recombinants. This electroporation‐stimulated recombination is abolished in an isogenic rad52 mutant strain consistent with the increase in Trp+ and His+ prototrophs being the result of a stimulation of a RAD52‐dependent recombination pathway.
The cysteine transport system of Saccharomyces cerevisiae
Authors: Ono Bun‐Ichiro , Naito Kazuhide
Published: May 2005

Although Saccharomyces cerevisiae strains had different cysteine uptake activities, they revealed monophasic uptake kinetics and had the same KT(83·3 μM). The optimal pH of cysteine uptake was between 4·5 and 5·0, but the activity was quickly lost if cells were kept in buffer. When the activity was measured in the growth medium, it increased in the presence of EDTA and greatly decreased in the presence of mercuric chloride. Thioglycol as well as metabolic inhibitors such as dinitropherol and azide were inhibitory. Homocysteine and methionine were competitive and non‐competitive inhibitors, respectively. Cysteamine and cysteic acid were not inhibitory. From these observations, we conclude that the system mediating uptake of cysteine is specific (we thus name it the cysteine transport system) and that the cysteine transport system recognizes not only the SH‐group but also amino‐ and carboxyl‐groups.

In wild‐type strains the cysteine transport system was derepressed only when the cells were incubated without any sulfur source. On the other hand, in cysteine‐dependent mutants, cysteine uptake activity increased with increase of exogenous supply of cysteine, glutathione or methionine. From this result, we suspect that the cellular cysteine level is the limiting factor for biosynthesis of the cysteine transport system in cysteine‐dependent strains.
Participation of proteinase yscA in the in vitro formation of the smaller subunit of glycogen phosphorylase in extracts of Saccharomyces cerevisiae
Authors: Ziegler Karl‐Josef , Schwartz Arnold C.
Published: May 2005

Analyses by sodium dodecyl sulphate–polyacrylamide gradient‐gel electrophoresis and Western blotting of the proteins from boiled cell samples from all growth phases of yeast cultures, and from all stages of extract preparation indicate that the smaller subunit (s‐monomer), which is found in purified glycogen phosphorylase (EC 2.4.1.1) from baker's yeast, is not present in the living cell. It is observed in extracts of Saccharomyces carlsbergensis and S. cerevisiae after incubation at ambient temperatures or even after storage in the frozen state at −25°C. Its formation is sensitive towards pepstatin A, and it is absent from extracts of several mutants of S. cerevisiae that do not contain active proteinase yscA (EC 3.4.33.6). When purified proteinase yscA‐deficient strains are grown with a reduced amount of complex nitrogen compounds, the slightly smaller sc‐monomer is formed in their extracts. This event must be attributed to a different proteinase, since it is sensitive towards p‐hydroxymercuriphenylsulphonate, but not towards pepstatin A. The N‐terminal amino acid of the sc‐monomer was found to be blocked, as in the case of the native 1‐monomer, but not the s‐monomer.
On the level of plasmid‐bearing cells in transformed cultures of Saccharomyces cerevisiae
Authors: Guerrini Anna Maria , Boglione Clara , Ascenzioni Fiorentina , Donini Pierluigi
Published: May 2005

LC1, a YIP5‐derived plasmid containing a human DNA fragment with ARS activity in yeast, has been used to study the replication of ARS plasmids in Saccharomyces cerevisiae. ARS plasmids carried in yeast hosts are normally mitotically unstable. In transformed cultures the fraction of cells that contain plasmid, measured by plating on selective media, is lower than would be expected from measured rates of plasmid loss. In the case of S. cerevisiae carrying either the plasmid LC1 or YRP17, the assay yields values of the order of 10–20% or 30–50% respectively. We have found that by doing a double nutritional upshift that involves conditioned medium and casamino acids, a population of cells can be defined that carry plasmid but are unable to grow on media that select for the plasmid marker. Thus the total fraction of cells that can be shown to contain plasmid increases to greater than 70%. To distinguish between the inability of plasmid to replicate in these cells and lack of expression of the selectable gene, cultures grown from single cells were analysed for the presence of plasmid DNA. In a substantial fraction of the population, plasmid DNA could be detected only by polymerase chain reaction and not by standard blotting and hydribization. These results suggest that plasmid is unable to replicate in these cells. Growth kinetics experiments with transformed cultures are consistent with the notion that only a small fraction of the cells contains plasmid capable of replication upon dilution into selective medium. Possible explanations for the phenomena observed are discussed.
Cloning and disruption of the LEU2 gene of Kluyveromyces marxianus CBS 6556
Authors: Bergkamp Ronald J. M. , Geerse Ruud H. , Verbakel John M. A. , Musters Wouter , Planta Rudi J.
Published: May 2005

The LEU2 gene, coding for β‐isopropylmalate dehydrogenase, of the yeast Kluyveromyces marxianus was isolated and sequenced. An open reading frame, coding for a protein with a molecular weight of 38kDa was found. Comparison of the deduced amino acid sequence of the LEU2 gene with the corresponding enzymes of three other yeasts and two thermophilic bacteria, revealed extensive sequence similarities. The cloned gene could complement a leuB mutation of Escherichia coli and a leu2 mutation of Saccharomyces cerevisiae. Using orthogonal field alternation gel electrophoresis, the genomic copy of the gene was found to be located at chromosome VI or VII. Analysis of the 5′‐untranslated region indicated the presence of a putative binding site for the LEU3 protein, which is involved in the leucine‐specific regulation of transcription. We show that the cloned gene can be used for the construction of a non‐reverting K. marxianus leu2 mutant.
The nucleotide sequence of a third cyclophilin‐homologous gene from Saccharomyces cerevisiae
Authors: Franco L. , Jiménez A. , Demolder J. , Molemans F. , Fiers W. , Contreras R.
Published: May 2005

The nucleotide sequence of a 1558 bp DNA fragment from the right arm of chromosome III of Saccharomyces cerevisiae contains an open reading frame of 954 nucleotides with coding potential for a protein with high similarity to the ubiquitous cyclophilins which are both peptidyl‐prolyl cis‐trans isomerases and cyclosporin A‐binding proteins. It should, therefore, represent the third gene (SCC3) of this kind from S. cerevisiae. SCC3 is present in a single copy in the genome of S. cerevisiae and results in a constitutively expressed 1·2 kb transcript during cell growth. Its putative protein product (Scc3) contains two hydrophobic cores, one at the amino terminal, 20 amino acids long, which could serve as a signal peptide, and the other one at the carboxyl end with a structure similar to a transmembrane helix. These findings suggest that Scc3 could be a secretory or, more likely, a transmembrane protein. The only cyclophilin with similar structure to that of Scc3 is ninaA from Drosophila melanogaster, a transmembrane protein which seems to be implicated in the correct folding and/or intercalation of rhodopsin in the endoplasmic reticulum of the fly photoreceptors (Stamnes, M. A. et al., Cell 65, 219–227, 1991). In addition, the amino and the carboxy regions of Scc3 and ninaA share a significant level of homology, which suggests that they have a similar function, albeit for different target proteins.
Flow cytometric analysis of Saccharomyces cerevisiae autolytic mutants and protoplasts
Authors: de la Fuente J. M. , Nombela C. , Sanchez M. , Alvarez A.
Published: May 2005

Simple methods, based on the technique of flow cytometry, have been developed for the phenotypic characterization of yeast autolytic mutants and for the analysis of the formation and regeneration of the yeast protoplasts. The expression of lytic mutations determined uptake of the fluorescent dye propidium iodide, which could be carefully monitored by flow cytometry. Mixed populations of lysed and viable cells were precisely quantified and sorted, and the technique was also applied to demonstrate protection from lysis of mutant cells with cell wall defects, in the presence of osmotic stabilizers. Protoplast formation and regeneration was monitored by analysing relative cell size; this was facilitated by the preparation of homogeneous protoplast preparations. The technique of flow cytometry proved superior to other conventional methods for these types of study.
Effects of phosphoglycerate kinase overproduction in Saccheromyces cerevisiae on the physiology and plasmid stability
Authors: van der Aar Paul C. , Röling Wilfred F. M. , Stouthamer Adriaan H. , van Verseveld Henk W. , Raué Hendrik A. , van den Heuvel Joop J.
Published: May 2005

In this report the effects of phosphoglycerate kinase (PGK) overproduction on the physiology and plasmid stability in baker's yeast Saccharomyciae cerevisiae containing the PGK1 gene on an episomal plasmid are described. This examination reveals that there is a preferred intracellular level for this enzyme, amounting to 10–15% of the total soluble protein. Strains containing the plasmid and the host strain were grown in non‐selective batch cultures and continuous culture, under different growth conditons. Plasmid‐containing yeast strains stabilize the copy number of the episomal plasmid at a level at which the PGK concentration is about 12%. This stabilization is due to an equilibrium between normal plasmid loss and selective pressure because of advantages resulting from the increased amount of PGK under glucose‐limited conditions. During respiro‐fermentative growth, PGK‐overproducing cells showed an increased respiration rate and decreased fermentative activity, compared to the host strain.

The PGK1 gene can be applied as a direct positive selection marker to obtain a high episomal plasmid stability during growth on glucose. The results are consistent with previously reported data on the physiology and gene stability of PGK‐overproducing yeast cells that contain multiple copies of the PGK1 gene integrated into the genome.
The complete sequence of a 10·8kb fragment to the right of the chromosome III centromere of Saccharomyces cerevisiae
Authors: Biteau Nicolas , Fremaux Christophe , Hebrard Sylvie , Menara Annie , Aigle Michael , Crouzet Marc
Published: May 2005

The complete nucleotide sequence of the D10H fragment (10850 bp) was determined. The D10H fragment is located on the right arm of chromosome III near the centromere and contains the SUF2 gene. Six open reading frames (ORFs) larger than 300 bp were found. One of them is the CIT2 gene encoding the cytoplasmic citrate synthase. The others are new putative genes and show no significant similarly with any known gene. In addition two tRNA genes (Asn and Pro) and a solo delta element were identified. Two ORFs were disrupted; no peculier phenotype was observed.
Thioredoxin genes in Saccharomyces cerevisiae: Map positions of TRX1 and TRX2
Authors: Muller Eric G. D.
Published: May 2005

The two genes encoding thioredoxims in Saccharomyces cerevisiae, TRX1 and TRX2, map to chromosome XII and VII, respectively. From the DNA sequence of the intragenic region TRX1 is 500 bp downstream of PDC1. Tetrad analysis places TRX21·1 cM from ADE3, while a physical map of this region positions TRX2 4·5 kb downstreams of ADE3. The mapping of TRX1 adjacent to PDC1 clarifies previous results (Muller, E. G. D. J. Biol. Chem. 266, 9194–9202, 1991) that suggested a third thioredoxims gene.
Characterization of mutants of the yeast Yarrowia lipolytica defective in acetyl‐coenzyme a synthetase
Authors: Kujau Marian , Weber Herbert , Barth Gerold
Published: May 2005

The expression of the glyoxylate cycle enzymes is required for growth of the yeast Yarrowia lipolytica on acetate or fatty acids as sole carbon source. Acetyl‐coenzyme A, which is produced by acetyl‐coenzyme A synthetase (ACS) from acetate, is needed for induction of this expression. Acetate‐non‐utilizing mutants of this yeast were investigated in order to identify mutants which express no or strongly reduced activity of this enzyme. Mutations in gene ICL2 exhibited the strongest effects on the activity. In icl2 mutants, lack of ACS activity resulted in a non‐induced glyoxylate cycle on acetate; however, induction on fatty acids was not affected. Gene ICL2 was identified as the sstructural gene encoding the monomer of ACS. It is shown that a high level of ACS activity is necessary for full expression of the glyoxylate cycle enzymes. Mutations in gene ICL1, which encodes isocitrate lyase, resulted in overproduction of ACS without any growth on acetate. A new gene (GPR1 = glyoxylate pathway rergulation) was detected in which trans‐dominant mutations inhibit expression of ACS and the glyoxylate cycle on acetate as carbon source.
Genetic and molecular analysis of DNA43 and DNA52: Two new cell‐cycle genes in Saccharomyces cerevisiae
Authors: Solomon Natalie A. , Wright Matthew B. , Chang Soo , Buckley Ann M. , Dumas Lawrence B. , Gaber Richard F.
Published: May 2005

Two Saccharomyces cerevisiae genes previously unknown to be required for DNA synthesis have ben identified by screening a collection of temperature‐sensitive mutants. The effects of mutations in DNA43 and DNA52 on the rate of S phase DNA synthesis were detected by monitoring DNA synthesis in synchronous populations that were obtained by isopycnic density centrifugation. dna43‐1 and dna52‐1 cells undergo cell‐cycle arrest at the restrictive temperature (37°C), exhibiting a large‐budded terminal phenotype; the nuclei of arrested cells are located at the neck of the bud have failed to undergo DNA replication. These phenotypes suggest that DNA43 and DNA52 are required for entry into or completion of S phase. DNA43 and DNA52 were cloned by their abilities to suppress the temperature‐sensitive lethal phenotypes of dna43‐1and dna52‐1 cells, respectively. DNA sequence analysis suggested that DNA43 and DNA52 encode proteins of 59.6 and 80.6 kDa, respectively. Both DNA43 and DNA52 are essential for viability and genetic mapping experiments indicate that they represent previously unidentified genes: DNA43 is located on chromosome IX, 32 cM distal from his5 and DNA52 is located on chromosome IV, 0.9 cM from cdc34.
Sequence of the novel essential gene YJU2 and two flanking reading frames located within a 3.2 kb EcoRI fragment from chromosome X of Saccharomyces cerevisiae
Authors: Forrová Helena , Kolarov Jordan , Ghislain Michel , Goffeau André
Published: May 2005

We have isolated and sequenced a novel gene from Saccharomyces cerevisiae coding for an essential protein of unknown function. The gene called YJU2 was borne on a chromosome X fragments shown by hybridization to intact S. cerevisiae chromosomal DNA fractionated by orthogonal pulsed field electrophoresis. Northern hybridization analysis indicated that the 0.8‐kb transcript of YJU2 is expressed in exponential‐phase cells grown in rich medium (data not shown). Figure 1 shows the nucleotide and deduced amino‐acid sequences of the 834‐bp coding region as well as the nucleotide sequences of the 5′ upstream region and of the 3′ downstream region, together with the flanking neighbouring open reading frames (ORFs), YJU1 and YJU3.