bluuebunny/crossref_metadata_2025 huggingface dataset:
Metabolic regulation in the yeast Hansenula polymorpha. Growth of dihydroxyacetone kinase/glycerol kinase‐negative mutants on mixtures of methanol and xylose in continuous cultures
Authors: De Koning W. , Weusthuis R. A. , Harder W. , Dijkhuizen L.
Published: May 2005

The physiological responses of Hansenula polymorpha wild‐type and mutant strains 17B (dihydroxyacetone kinase‐negative) and 17BG51 (dihydroxyacetone kinase‐ and glycerol kinase‐negative) to growth on mixtures of xylose and methanol in chemostats were investigated. Increasing methanol concentrations (0–110 mM) in the feed of the wild‐type culture resulted in increasing cell densities and a gradual switch towards methanol metabolism. At the lower methanol feed concentrations the mutant cultures used methanol and xylose to completion and changes in enzyme patterns comparable to the wild type were observed. This was not reflected in significant changes in cell densities. Instead, formaldehyde assimilation resulted in dihydroxyacetone (DHA) production, which was proportional to the amount of methanol added. At intermediate methanol concentration the cultures showed a strong variation in DHA levels and cell densities. Further increased in the methanol feed concentrations resulted in a drop in DHA accumulation rates, repression of alcohol oxidase synthesis and accumulation of residual methanol. The phenomena were studied in more detail in transition experiments and with gradients of methanol. The results indicate that xylulose‐5‐phosphate (Xu5P) generated in xylose metabolism served as acceptor molecule for formaldehyde assimilation by the peroxisomal enzyme DHA synthase. Accumulation of DHA in the mutant cultures, however, further diminished the availability of carbon for growth. The data suggest that with increasing methanol concentrations Xu5P eventually became growth rate limiting. This resulted in an unstable situation but wash‐out of the culture did not occur to a significant extent. Instead, DHA accumulation ceased and cell densities, and enzymes specifically involved in xylose metabolism increase, indicating that the organism resumed its xylose metabolism. The molecular mechanisms controlling the partitioning of Xu5P over xylose (pentose phosphate pathway) and methanol (peroxisome) metabolism under these conditions remain to be elucidated.
Cloning and sequencing of the ornithine carbamoyltransferase gene from Pachysolen tannophilus
Authors: Skrzypek M. , Borsuk P. , Maleszka R.
Published: May 2005

A fragment of DNA from a yeast Pachysolen tannophilus, bearing the ornithine carbamoyltransferase gene (OCTase, EC 2.1.3.3) has been cloned from a genomic library by functional complementation of the Escherichia coli OCT‐negative mutant. The gene was located within the cloned segment of DNA and its coding sequence identified by DNA sequencing. This has indicated that P. tannophilus OCT gene encodes a 347 amino acid polypeptide, which shows 60% identity to the homologous Saccharomyces cerevisiae protein. The amino acid composition of its N‐terminus indicates that this protein is translocated across the mitochondrial membrane. The gene can be expressed in E. coli as well as in S. cerevisiae. Comparison with other OCTases confirms a high degree of conservation among these proteins.
Bacterial plasmid pBR322 sequences serve as upstream activating sequences in Saccahromyces cerevisiae
Authors: Sidhu Rajinder S. , Bollon Arthur P.
Published: May 2005

The expression of acid phosphatase (Apase) from PHO5 and MFα‐PHO5 hybrid genes is regulated by inorganic phosphate and mating type locus respectively, as well as the PHO4 and MATα1 gene products respectively. When PHO5 and MFα‐PHO5 hybrid genes were cloned in the BamHI site of the pBR322 sequence of the yeast shuttle vectors (YRp7 or YEp9T), in one orientation they were regulated normally but in the other orientation their expression was not regulated but expressed constitutively. The pBR322 sequences present upstream of the inserted genes are responsible for the constitutive expression. By replacing the PHO5 upstream activating sequences (UAS) element with pBR322 fragments, we have identified three pBR322 sequences, rom nucleotides 376 to 650, 2068 to 2116 and 2136 to 2247, which were able to promote expression of APase. A comparison of these three pBR322 fragments revealed 5′ ATCGCGCGAG 3′ and 5′ CGGTGATGNCGG 3′ to be the common sequences likely to act as USAs in Saccharomyces cerevisiae. By using synthetic oligonucleotides, it was found that both sequences are required for maximum expression of APase activity.
Mapping of the trifunctional fatty acid synthetase gene FAS2 on chromosome XVI of Saccharomyces cerevisiae
Authors: Siebenlist Ursula , Nix Johanna , Schweizer Michael , Jäger Daniel , Schweizer Eckhart
Published: May 2005

The trifunctional FAS2 gene encoding subunits α of the Saccharomyces cerevisiae fatty acid synthetase complex was mapped on the left arm of chromosome XVI 24 centinorgans proximal to GAL4 and 39 centimorgans distal and PEP4 relative to the centromere. Mapping was achieved by three‐independent methods: meiotic co‐segragation of FAS2 and ARO7 in recombination‐deficient spo11‐mutants; tetrad analysis of crosses between FAS2, GAL4 and PEP4; and Southern hybridization of purified FAS2 DBA with individual yeast chromosomes separated by pulsed‐field gel electrophoresis.
Mating type locus‐dependent stability of the Kluyveromyces linear pGKL plasmids in Saccharomyces cerevisiae
Authors: Gunge Norio , Murakami Kazuo , Takesako Tosikazu , Moriyama Hideaki
Published: May 2005

The linear killer plasmids, pGKL1 and pGKL2, from Kluyveromyces lactis stably replicated in mitochondria1 DNA‐deficient (ρ0) MATa or MATα haploids of Saccharomyces cerevisiae, but were unstable and frequently lost in ρ° MATa/MATα diploids, suggesting that the replication of pGKL plasmids was under the control of the MAT locus. In MATa/MATα cells of S. cerevisiae, the MATα gene product (α2) is combined with the MATa gene product (a1) and the resultant protein, a1‐α2, acts to repress the expression of haploid specific genes. Experiments showed that the K. lactis linear plasmids were stably maintained in ρ0 mata1/MATα diploids, indicating that the a1‐α2 repressor interfered with the stability of pGKL2. It was revealed by computer analysis that the consensus sequence homologous to the a1‐α2 repressor binding site occurred within the coding regions of pGKL2 genes which were presumed to be essential for the plasmid replication. Since the plasmids were stably maintained in diploids of K. lactis, the mating type control must not be working there.
Assembly of amine oxidase and D‐amino acid oxidase in the cytosol of peroxisome‐deficient mutants of the yeast Hansenula polymorpha during growth of cells on glucose in the presence of primary amines or D‐alanine as the sole nitrogen source
Authors: Sulter G. J. , Van Der Klei I. J. , Harder W. , Veenhuis M.
Published: May 2005

We have studied growth of two peroxisome‐deficient mutant strains of Hansenula polymorpha on glucose in the presence of different organic nitrogen sources (methylamine, ethylamine and D‐alanine), the metabolism of which is mediated by peroxisome‐borne oxidases in wild‐type (WT) cells. Both strains grew well on each of these substrates with growth rates comparable to WT cells. Growth on both methylamine and ethylamine was associated with enhanced levels of catalase and amine oxidase in the cells; in D‐alanine‐grown cells D‐amino acid oxidase activity and increased. In WT cells of H‐polymorpha the activities of these enzymes were confined to the peroxisomal matrix; however, in both peroxisome‐deficient strains their activities were localized in the cytosol. Electron microscopy indicated that, dependent on the stage of growth, the enzymes may form large protein aggregates.

The molecular masses of both amine oxidase and D‐amino acid oxidase in the mutant strains were identical to their respective counterparts in WT cells, indicating that both proteins were correctly assembled and active in the cytosol.
Mapping of the ARS‐like activity and transcription initiation sites in the non‐canonical yeast mitochondrial ori 6 region
Authors: Delouya Daniel , Nobrega Francisco G.
Published: May 2005

The insert‐containing, non‐canonical ori 6 region of yeast mitochrondrial DNA of Saccharomyces cerevisiae was dissected into 15 different segments that were ligated to the integrative yeast vector YIp5. Six recombinant plasmids exhibited replicative ability in yeast and carried consensus sequences similar to the previously described 11 bp motifs active as autonomous replication sequences (ARS). In addition, all active constructions carry one or more of the characteristic GC‐rich domains A, B or C present in the ori 6 region, thus confirming and expanding the study of Blanc (Gene 30 (1984) 47–61) with the canonical ori 5. Also a new transcriptional origin is activated in the ori 6 region, apparently circumventing a disruption by insertion of a GC‐rich sequence that, in this ori, removes the mitochondrial promoter usually present next to the Celement. The ARS‐positive constructions correspond to the retained segments of spontaneous well‐characterized suppressive or neutral petite genomes that contain segments of the ori sequence.
Phylogenetic relationships among species of Saccharomyces, Schizosaccharomyces, Debaryomyces and Schwanniomyces determined from partial ribosomal RNA sequences
Authors: Kurtzman Cletus P. , Robnett Christie J.
Published: May 2005

Species of the genera Saccharomyces, Schizosaccharomyces, Debaryomyces and Schwanniomyces were compared from their extent of divergence in three regions from small (18S) and large (25S) subunit ribosomal RNAs comprising a total of 900 nucleotides. With the exception of the closely related Saccharomyces bayanus and S. pastorianus, which appear to have identical sequences, all other species could be distinguished by nucleotide differences in a variable region of the large subunit, and genus‐specific nucleotides were discernible in all three regions. The taxon D. tamarii differed markedly from other species and is excluded from Debaryomyces. By contrast, Schwanniomyces occidentalis showed few nucleotide differences with Debaryomyces spp. and its transfer to Debaryomyces is proposed. Schizosaccharomyces proved to be somewhat more divergent than Saccharomyces and Debaryomyces, but species differences appear insufficient for dividing the genus. Some of the factors influencing estimates of phylogenetic distances from rRNA sequences are discussed.
Synthesis of superoxide dismutase, catalase and other enzymes and oxygen and superoxide toxicity during changes in oxygen concentration in cultures of brewing yeast
Authors: Clarkson Simon P. , Large Peter J. , Boulton Christopher A. , Bamforth Charles W.
Published: May 2005

The physiological effects on brewing yeast, growing in semi‐defined wort medium, of a sudden transition from aerobiosis to anaerobiosis were studied. Two yeast strains were examined, used for ale and lager fermentations respectively. The reverse transition (from anaerobiosis to aerobiosis) was also examined. Transitions were applied by changing the sparging gas during growth or in stationary phase, and the effects on the specific activities of certain key enzymes and on the viability of the cultures were examined.

Neither type of transition led to significant changes in growth rate, the rate of ethanol production or the specific activities of alcohol dehydrogenase and pyruvate decarboxylase. The most significant change was in the specific activity of CuZn‐superoxide dismutase, which showed a rapid increase in activity on transition from anaerobiosis to aerobiosis, and a decrease in activity on the reverse transition. Catalase activity in the ale yeast generally followed that of CuZn‐superoxide dismutase, whereas in the lager yeast it remained unchanged by the transitions. The transition from anaerobiosis to aerobiosis caused increases in citrate synthase and Mn‐superoxide dismutase, though only after a significant lag period. Aerobic to anaerobic transitions caused a decrease in Mn‐superoxide dismutase activity, while citrate synthase remained unchanged.

Anaerobically grown cells showed a rapid loss in viability on exposure to oxygen (5–7% in the first hour), while aerobically grown cells were unaffected. When anaerobically grown cells were exposed to 0·25 mM‐potassium superoxide, there was an 8% loss of viability within 10 min, whereas aerobic cells were not affected.

It is concluded that the toxic effect of oxygen is due to superoxide (or a species derived from it) and that the CuZn‐superoxide dismutase (but not the Mn‐isoenzyme) plays a role in protecting the cells. The de novo synthesis of the CuZn‐enzyme is not always rapid enough to confer full protection.
Hydrogen peroxide as an electron acceptor for mitochondrial respiration in the yeast Hansenula polymorpha
Authors: Verduyn Cornelis , Van Wijngaarden Connie J. , Alexander Scheffers W. , Van Dijken Johannes P.
Published: May 2005

Chemostat cultures of a catalase‐negative mutant of Hansenula polymorpha CBS 4732 were able to decompose hydrogen peroxide at a high rate. This was apparent from experiments in which yeast was grown under carbon limitation in chemostat culture on mixtures of glucose and H2O2. The enzyme responsible for H2O2 degradation is probably the mitochondrial enzyme cytochrome c peroxidase (CCP), which was present at very high activities. This enzyme was partially purified and shown to be specific for reduced cytochrome c as an electron donor; no reaction was observed with NAD(P)H. Thus, reducing equivalents for H2O2 degradation by CCP must be provided by the respiratory chain.

That H2O2 can act as an electron acceptor for reducing equivalents could be confirmed with experiments in which cells were incubated with ethanol and H2O2 in the absence of oxygen. This resulted in oxidation of ethanol to equimolar amounts of acetate.

Energetic aspects of mitochondrial H2O2 decomposition via CCP and the physiological function of CCP in yeasts are discussed.
Ribosomal DNA spacer probes for yeast identification: Studies in the genus Metschnikowia
Authors: Henriques Marília , Sá‐Nogueira Isabel , Giménez‐Jurado Gabriella , Van Uden N.
Published: May 2005

To test whether DNA probes derived from ribosomal DNA spacer sequences are suitable for rapid and species‐specific yeast identification, a pilot study was undertaken. A 7·7 kb entire ribosomal DNA unit of the type strain of Metschnikowia reukaufii was isolated, cloned and mapped. A 0·65 kb BamHI‐HpaI fragment containing nontranscribed spacer sequences was amplified and selected for testing as a 32P hybridization probe with total DNA from the type strains of M. reukaufii, M. pulcherrima, M. lunata, M. bicuspidata, M. australis, M. zobellii, M. krissii, five other strains identified as M. reukaufii and strains of Schizosaccharomyces pombe, Hansenula canadensis, Saccharomyces cerevisiae and Yarrowia lipolytica. The probe hybridized exclsively with DNA from the type strain and four other strains of M. reukaufii. DNA from one strain labelled M. reukaufii did not hybridize with the probe. Subsequent % G+C comparison and DNA–DNA reassociation with the type strain revealed that the non‐hybridizing strain does not belong to the species M. reukaufii.
Inositol biosynthesis: Candida albicans and Saccharomyces cerevisiae genes share common regulation
Authors: Klig Lisa S. , Antonsson Bruno , Schmid Elisabeth , Friedli Laurence
Published: May 2005

The Candida albicans inositol biosynthetic gene and its regulation have been studied. The gene, CalNO1, was cloned on a multicopy vector by complementation of a Saccharomyces cerevisae mutant strain. Southern blot analysis established that the cloned DNA was C. albicans genomic DNA in origin; neither rearrangements nor pseudogenes were evident. Blot hybridization analysis using RNA isolated from C. albicans revealed that a single RNA species (1·8 kilobases) was homologous to the cloned DNA fragment. The steady‐state levels of these transcripts were shown to be regulated in response to inositol in the growth media. In addition, the steady‐state levels of the RNA encoded by the cloned C. albicans DNA present in S. cerevisiae on a plasmid (YRpCalNO1) were regulated in response to exogenously provided inositol. The cloned C. albicans DNA fragment was shown to restore inositol‐1‐phosphate synthase activity to a S. cerevisiae mutant strain defective in this enzyme. This activity was also shown to be regulated in response to the presence of inositol in the growth media.
III. Yeast sequencing reports. Sequence of the CDC10 region at chromosome III of Saccharomyces cerevisiae
Authors: Steensma H. Yde , Van Der Aart Quirina J. M.
Published: May 2005

A 4·74 kb DNA fragment from the right arm of chromosome III of Saccharomyces cerevisiae, adjacent to the centromere region was sequenced. Four open reading frames with an ATG initiation codon and larger than 200 bp were found in this fragment. The largest open reading frame of 966 bp was identified as the CDC10 gene.
Assignment of most genes encoding major peroxisomal polypeptides to chromosomal band V of the asporogenic yeast Candida tropicalis
Authors: Kamiryo Tatsuyuki , Mito Naruo , Niki Toshiro , Suzuki Takahito
Published: May 2005

The peroxisomes of the asporogenic yeast Candida tropicalis contain about 20 major polypeptides (PXPs). We have isolated a number of genes encoding them; 11 POX genes encoded independent PXPs and three POY genes were likely to encode three other PXPs. To locate these genes on the chromosomes, chromosomes of C. tropicalis were separated by pulsed‐field gel electrophoresis. Eight chromosomal bands were observed over the range of 1·0 Mbp (band I) to 2·8 Mbp (band VIII); the genome size was estimated to be about 20 Mbp. Southern blot analysis showed that ten genes were on band V, three genes were on band IV, and the other gene was on band VI. Three genes gave hybridization signals of nearly equal intensity on two different chromosomal bands: POX6A and POX8B, on bands V and VII; and POX8A, on bands IV and VI. Ribosomal RNA genes also hybridized to two bands, VI and VII. Most genes assigned to only one band hybridized to two restriction fragments produced by either NotI or SfiI endonuclease. The results suggested that C. tropicalis was diploid and that restriction sites were conserved little between homologues. The three POX genes that were found on two chromosomal bands hybridized to not more than two restriction fragments, implying that the allelic genes were present on different chromosomal bands.
Genetic transformation of auxotrophic mutants of the filamentous yeast Trichosporon cutaneum using homologous and heterologous marker genes
Authors: Ochsner Urs A. , Glumoff Virpi , Kälin Markus , Fiechter Armin , Reiser Jakob
Published: May 2005

A transformation system for the filamentous yeast Trichosporon cutaneum based on auxotrophic markers is presented and techniques for the induction, isolation and characterization of mutants are described. A number of auxotrophic mutants were isolated and characterized by using biosynthetic precursors and/or inhibitors. A mutant unable to grow in the presence of ornithine could be complemented successfully in spheroplast transformation experiments using the cloned Aspergillus nidulans ornithine transcarbamoylase gene (argB gene) as selection marker with an efficiency of 5–100 transformants per μg of DNA. In these transformants the heterologous argB gene was present in multiple tandem copies and the transforming DNA was found to remain stable after more than 50 generations in non‐selective media. The same mutant could be complemented by a T. cutaneum cosmid gene library and a complementing cosmid was subsequently isolated from this library by a sib‐selection strategy. This cosmid transformed. T. cutaneum spheroplasts with an efficiency of 50–200 colonies per μg of DNA. Southern blot analyses were consistent with the view that the transforming sequences became stably integrated into the host genome at the homologous site.
Yeast flocculation: Flo1 and NewFlo phenotypes and receptor structure
Authors: Stratford Malcolm , Assinder Stephen
Published: May 2005

Flocculation characteristics of 42 flocculet strains of Saccharomyces cerevisiae were examined. Two entirely distinct ‘lectin‐like’ mechanisms of flocculation were distinguished by sugar, salt, and low pH inhibitions, protease sensitivity, and selective expression of flocculation. One group, termed Flo1 phenotype, was inhibited by mannopyranoses and contained all strains bearing known genes affecting flocculation. The other group, termed NewFlo phenotype, contained the majority of brewery ale strains and was inhibited by manno‐ and glucopyranoses. Detailed sugar‐inhibition work revealed the probable receptor identity of both Flo1 and NewFlo flocculation, as being non‐reducing termini of α‐(1–3)‐linked mannan side branches, two or three mannopyranose residues in length.
The ureidoglycollate hydrolase (DAL3) gene in Saccharomyces cerevisiae
Authors: Yoo Hyang Sook , Cooper Terrance G.
Published: May 2005

The DAL3 gene has been sequenced and found to encode a 195 amino acid protein with a molecular weight of 21 727. The four carboxy‐terminal amino acids of DAL3 product (Cys‐Ile‐Ile‐Ile) are homologous to those (CAAX) previously shown to be the primary structural signal for post‐translational farnesylation of yeast RAS protein and mating factor. This modification is reported to be responsible for membrane localization of proteins containing it. The upstream region of DAL3 contains six copies of a sequence that is homologous to the positively acting DAL UASNTR reported to be required for transcriptional activation of the DAL5 and DAL7 genes. Missing from the DAL3 upstream region were any sequences related to those shown to be required for a DAL7 response to inducer, the UIS element. This correlates with the previous report that DAL3 expression is independent of the allantoin pathway inducer.
Selective inactivation of alcohol oxidase in two peroxisome‐deficient mutants of the yeast Hansenula polymorpha
Authors: Van Der Klei I. J. , Harder W. , Veenhuis M.
Published: May 2005

We have studied selective inactivation of alcohol oxidase (AO) in two peroxisome‐deficient (PER) mutants of the yeast Hansenula polymorpha. In these mutants high activities of cytosolic AO are induced by different growth conditions. At enhanced expression rates AO is arranged in large crystalloids in the cytosol, whereas smaller crystalloids are often observed inside the nucleus. Transfer of cells of the PER mutant 125‐2E, which completely lacks peroxisomes, to glucose‐excess conditions did not lead to degradative inactivation of AO and catalase as observed in wild‐type (WT) cells used as a control. The gradual decrease in enzyme activities in the PER mutant could be accounted for by dilution of existing enzyme into newly formed cells as a result of growth. Morphologically, degradation of the cytosolic crystalloids was also not observed. Similar results were obtained with a second PER mutant (strain 124‐2D), impaired in the import of peroxisomal matrix proteins. This mutant is characterized by the presence of small peroxisomes and large cytosolic AO crystalloids. Upon a shift of cells to glucose‐excess conditions only part of the small peroxisomes present in these cells were degraded by mechanisms similar to those observed in WT cells placed under identical conditions. These results indicate that degradative inactivation of AO in H. polymorpha is strictly dependent on the localization of the enzyme inside peroxisomes and furthermore suggests that the mechanisms triggering this process are not directed against AO protein, but instead, to the membrane surrounding the organelle. Transfer of cells to methanolor ethanol‐containing media both resulted in modification inactivation of AO. Under these conditions also the AO crystalloids remained unaffected by incubation in the new environment.
The major exoglucanase from Candida albicans: A non‐glycosylated secretory monomer related to its counterpart from Saccharomyces cerevisiae
Authors: Luna‐Arias Juan P. , Andaluz Encarnación , Ridruejo Juan C. , Olivero Isabel , Larriba Germán
Published: May 2005

Exoglucanases secreted by two different strains from Candida albicans have been purified to homogeneity. The purified enzyme from each strain behaved as a non‐glycosylated monomer (molecular weight 38 000) that was identical in terms of sodium dodecyl sulphate/polyacrylamide gel electrophoresis comigratin, amino acid analysis and amino terminal sequence. The amino acid composition was similar to that of the major exoglucanase from Saccharomyces cerevisiae. In addition, these two enzymes displayed a 50% homology in the first 35 amino acids of the amino terminus. Antibodies against the deglycosylated exoglucanase (treated with Endo H) from S. cerevisiae were reactive with the exoglucanase from C. albicans and vice versa. Immunoblotting proved to be a semiquantitative method to detect C. albicans antigen in culture fluids. The exoglucanase from C. albicans appears to enter the secretory pathway without undergoing N‐glycosylation.
Regulation of cystathionine γ‐lyase in Saccharomyces cerevisiae
Authors: Ono Bun‐Ichiro , Naito Kazuhide , Shirahige Yoh‐Ichi , Yamamoto Mahoko
Published: May 2005

Regulation of the two enzymes in reverse trans‐sulfuration was investigated in Saccharomyces cerevisiae. In wild‐type strains, cystathionine γ‐lyase, but not cystathionine β‐synthase, was derepressed nearly 15‐fold if cells were starved for both inorganic and organic sulfur compounds. In a met17 strain which is defective of O‐acetylserine and O‐acetylhomoserine sulfhydrylase, the same enzyme was derepressed if organic sulfur compounds were limited; the repressive effect was in the order of glutathione > methionine > cysteine. The repressive effect of methionine was not observed, however, in a cys2 cys4 strain which is deficient of serine O‐acetyltransferase and cystathionine β‐synthase, indicating that methionine itself is not the effector. The weak repressive effect of cysteine was attributed to inefficient uptake of this amino acid.

Our observations indicate that cystathionine γ‐lyase is the target of regulation in reverse trans‐sulfuration and that cysteine is very likely to be the effector of this regulation.
The PHO80/TUP7 locus in Saccharomyces cerevisiae is on the left arm of chromosome XV: Mapping by chromosome engineering
Authors: Kawasaki Hideki , Matsuzaki Hiroaki , Nakajima Ryoichi , Oshima Yasuji
Published: May 2005

The PHO80/TUP7 locus in Saccharomyces cerevisiae is reported to be located on the right arm of chromosome XV close to its centromere. In the present study, the locus has been reassigned to the left arm of the same chromosome by reciprocal recombination between chromosomes V and XV at URA3 (on chromosome V) and PHO80/TUP7 loci by using the site‐specific recombination system of the yeast plasmid pSR1.
The allantoinase (DAL1) gene of Saccharomyces cerevisiae
Authors: Buckholz Richard G. , Cooper Terrance G.
Published: May 2005

The allantoinase (DAL1) gene from Saccharomyces cerevisiae has been cloned, sequenced, and found to encode a 472 amino acid protein with a Mr of 52 028. DAL1 is expressed in an inducer‐independent manner in strain M970 (∑1278b genetic background) and modestly responds to mutation of the da180 locus. Expression was also sensitive to nitrogen catabolite repression (NCR). Correlated with these expression characteristics, the upstream region of DAL1 contained five copies of a sequence that is homolgous to the DAL UASNTR element previously shown to be required for transcriptional activation and NCR sensitivity of the DAL5 and DAL7 genes. Missing from the DAL1 5′ flanking region were any sequences with significant homology to the DAL7 UIS element required for response to inducer. These observations further support the roles of UASNTR and DAL7 UIS in the regulation of allantoin pathway gene expression.
Molecular cloning of CIF1, a yeast gene necessary for growth on glucose
Authors: González M. Isabel , Blázquez Miguel A. , Gancedo Carlos , Stucka Rolf , Feldmann Horst
Published: May 2005

The cif1 mutation of Saccharomyces cerevisia (Navon et al., Biochemistry 18, 4487–4499, 1979) causes inability to grow on glucose and absence of catabolite inactivation. We have cloned the CIF1 gene by complementation of funcion and licated it in a 2·75 kb SphI‐BstEII fragment situated at ca. 18 kb centomere distal of LYS2 and ca. 80 kb centromere proximal of TYRI on chromosome II. Southern analysis demostrated that CIF1 is present in a single copy in the yeast genome. Northern analysis revealed that the corresponding mRNA of 1·8 kb is more abundant in cells grown on galactose than in those grown on glucose. A protein of ca. 54 kDa was predicted from the open reading frame in the sequenced fragment. In strains carrying the cif1 mutation the intracellular concentration of ATP decreased immediately after addition of glucose while the intracellular concentration of cAMP did not increse. cAMP concentration increases in response to galactose or 2,4‐dinitrophenol. Disruption of BCY1 or overexpression of CDC25 in a cif1/, background did not restore growth on glucose, suggesting that the absence of cAMP signal is not primary cause of lack of growth on glucose. Complementation tests showed that cif1 is not allelic to fdp1 although the two genes seem to be functionally related.
Efficient secretion in yeast based on fragments from K1 killer preprotoxin
Authors: Cartwright Charles P. , Zhu Yun‐Song , Tipper Donald J.
Published: May 2005

The α and β components of the secreted K1 killer toxin of Saccharaomyces cerevisiae are derived from residues 45–147 and 234–316, respectively, of the 316 residue prepotoxin (ppTox). The β N‐terminus is produced by Kex2 cleavage after Lys Arg233, when β1a(the mature sequence of β‐lactamase)is fused at this site and the fusion is expressed form the PGK promoter in pDT17, a multicopy plasmid, unexpectedly modest levels of βla secretion resulted. Over‐expression of Kex2 failed to increase βla secretion while a kex2‐null mutation reduced secretion by 98%. βla secretion in a Kex+ strain was not enhanced by inactivation of the a toxin component or by deletion of most of its central hydrophobic segments. However SP‐βla, produced by deletion of ppTox residues 35–176, expressed 10‐fold higher βla activity and the precursor was not secreted with similar efficiency in a kex − 2 null strain. Fusions of βla to ppTox at Ala34 or Ala46 also led to efficient secretion in both KEX2 and kex − 2‐null strains. Since these βla fusions differ only in segments well downstream of the signal peptide and all had similar transcript levels, the efficiency of βla secretion is apparently determined by the efficiency with efficiency with which these fusions are translocated to the Golgi compartment where Kex2 is active. Efficiency is high for the shorter fusions but is 10% or less for the longer fusions; even this fraction is apparently diverted to the vacuole if not cleaved by Kex2. SP‐βla was athe most efficient construct tested; secreted βla reacahed 4% of total cell protein, modestly exceeding levels produced by fusion to the MFα1‐encoded preproα‐factor, suggesting potential for the production of foreign proteins in yeast.
Control of peroxisome proliferation in Saccharomyces cerevisiae by ADR1, SNF1 (CAT1, CCR1) and SNF4 (CAT3)
Authors: Simon Manuel , Binder Maximilian , Adam Gerhard , Hartig Andreas , Ruis Helmut
Published: May 2005

The Saccharomyces cerevisiae ADR1 gene has recently been demonstrated to control transcription of several genes encoding peroxisomal proteins or proteins necessary for peroxisome formation. Therefore, the effect of two other genes (SNF1 (CAT1, CCR1) and SNF4 (CAT3)) known to control derepression of glucose‐repressible genes was studied. Levels of transcripts of genes encoding catalase A, fatty acid β‐oxidation enzymes and of the PAS1 gene are reduced in snf1 and snf4 mutants of ethanol as well as on oleic acid medium. By immunogold labelling with an antibody directed against peroxisomal thiolase, clusters of peroxisomes were detected in wild‐types cells, whereas smaller single peroxisomes were observed in adr1 mutant cells. Results of immunofluorescence experiments are consistent with these observations. No peroxisomes were detected in snf1 and snf4 mutants by immunogold labelling as well as by imunofluorescence.
Sequence of a segment of yeast chromosome XI identifies a new mitochondrial carrier,a new member of the G protein family, and a protein with the PAAKK motif of the H1 histones
Authors: Colleaux Laurence , Richard Guy‐Franck , Thierry Agnès , Dujon Bernard
Published: May 2005

We have entirely sequaenced an 8.3 kb segment localized on the left arm of chromosome XI of Saccharomyces cerevisiae. Five new open reading frames have been uncovered. One of them encodes a new mitochondrial carrier protein which is dispensable for growth on glycerol medium. Another could be a new member of the G protein family. A third possesses the PAAKK motif common to H1 histones.
Genetic analysis of maintenance and expression of L and M double‐stranded RNAs from yeast killer virus K28
Authors: Schmitt Manfred J. , Tipper Donald J.
Published: May 2005

The killer phenotype expressed by Saccharomyces cerevisiae strain 28 differs fron that of the more extensively studied K1 and K2 killers with respect to immunity, mode of toxin action and cell wall primary toxin receptor. We previosly demonstrated that the M28 and L28 dsRNAs found in strain 28 are present in virus‐like particles (VLPs) and that transfection with these VLPs is sufficient to confer the complete K28 phenotype on a dsRNA‐free recipient cell. We also demonstrated that L28, like the L‐A‐H species in K1 killers, has [HOK] activity required for maintenance of M1‐dsRNA, and predicted that M28 would share with M1 dependence on L‐A for replication. We now confirm this prediction by genetic and biochemical analysis of the effects of representative mak, ski and mkt mutations on M28 maintenance, demonstrating that M28 replication resebles M1 in all respects. We also show that L28 is an L‐A‐H species lacking [B] activity, and that M28 excludes both M1 and M2 from the same cytoplasm. Stable coexpression of K28 phenotype from M28 and of K1 phenotype from an M1‐cDNA clone was demonstrated. Exclusion, therefore, acts at the level of dsRNA replication, presumably reflecting competition for the L‐A‐H encoded capsid and cap‐pol fusion protein, rather than reflecting incompatibility of toxin or immunity expression. Finally, we show that expression of active K28 toxin, bu t not of K28 immunity, requires the Kex2 endoprotease.
The complete sequence of a 10.8 kb segment distal of SUF2 on the right arm of chromosome III from Saccharomyces cerevisiae reveals seven open reading frames including the RVS161, ADP1 and PGK genes
Authors: Skala Jacek , Purnelle Bénedicte , Goffeau André
Published: May 2005

We have entirely sequenced a 10,835 bp segment of the right arm from chromosome III contained in the J11D and J11D‐K3B GF clones. The segment contains seven open reading frames longer than 100 amino acids. Three of them, RVS161 (Urdaci et al., 1990; Crouzet et al., 1991), ADP1 (Purnelle et al., 1991) and PGK1, (Hitzeman et al., 1982) have been described previously. YCR10C, encodes a putative membrane protein. YCR8W, (encoding a putative protein kinase) and YCR14c extend inside the D10H (Skala et al., 1991) and 62B5‐2D clones respectively. Four ARS elements previously reported by Palzkill et al., (1986) are located between RVS161 and YCR10C.
Factors affecting homologus overexpression of the Sacchromyces cerevisiae Lanosterol 14α‐demethylase gene
Authors: Weber Jakob M. , Ponti Charly G. , Käppeli Othmar , Reiser Jakob
Published: May 2005

The Saccharomyces cerevisiae Lanosterol 14α‐demethylase (14DM) gene was overxpressed in S. cerevisiae using Promoter sequences of the highly expressed S. cerevisiae glyceraldehyde‐3‐phosphate dehydrogenase TDH3 gene. To investigate factors affecting 14DM overproduction, the levels of 14DM‐specific specific RNAs, apoprotein, and heme protein, repectively, were determined and the 14DM‐specific RNA levels compared with the RNA levels originating from the enodogenous TDH gene(s). The quantitative measurements revealed that the 14DM steady‐state RNA levels reached were some three‐to five‐fold below the theoretically expected values. With a View towards futrher improving expression of the 14DM gene, the specing between the TDH3 promoter and the AUG was adjusted precisely and to rule out possible toxic effects exerted by the 14DM protein, the TDH3 promoter was placed under galactose regulation by introducing an UASG segment. Furthermore, the effects of the gene copy number on 14DM overproduction were investigated. From the analysis of the improved expression constructs five conclusions could be reached: (1) experssion from the native 14DM gene is comparable to the expression driven by the TDH3 promoter‐14DM fusion construct on single copy plasmid vectors; (2) expression from the TDH3 promoter‐14DM construct on single‐copy vectors is nearly as effcient as expression from the corresponding endogenous TDH3 gene; (3) the gene copy number has an effect on the relative expression levels of the TDH3 promoter‐14DM constructs; (4) the steady‐state amounts of protein produced are very nearly proportional to gene dosage; and (5) protein toxicity does not have a major impact on 14DM production. The maximum yield of 14DM was in the order of 7% of the total yeast protein and the maximum production of functional 14DM heme protein appears to be limited by the availability of heme.
Analysis of the MSS51 region on chromosome XII of Saccharomyces cerevisiae
Authors: Simon Michael , Seta Flavio Della , Sor Frederic , Faye Gerard
Published: May 2005

We have localized gene MSS51 on chromosome XII of Saccharomyces cerevisiae between the RDN1 and CDC42 loci. ‘Head to head’ with MSS51 is another gene, QRI5, the function of which is unkown. However, the proximity of these genes, the structure of the intergenic region and the presence of an ABFl binding site right in the middle of this region suggest that the MSS51 and QRI5 expressions are submitted to a common regulatory process.
The complete sequence of a 6146 bp fragment of Saccharomyces cerevisiae chromosome III contains two new open reading frames
Authors: Wilson Cathal , Grisanti Paola , Frontal Laura
Published: May 2005

As part of the EEC project to sequence the entire chromosome III of Saccharomyces cerevisiae we have sequenced a total of 11,040 bp from near the right end of the chromosome. A new protein kinase gene was found at one extremity of the sequenced region (Wilson et al., 1992), while the previouslysequenced actin binding protein gene, ABPI, (Drubin et al., 1990) was found at the other extremity. We present here the sequence of the region between these two genes which has the potential to code for two new open reading frames (ORFs).
II. Yeast sequencing reports. An 11·4 kb DNA segment on the left arm of yeast chromosome II carries the carboxypeptidase Y sorting gene PEP1, as well as ACH1, FUS3 and a putative ars
Authors: Van Dyck Luc , Purnelle Bénédicte , Skala Jacek , Goffeau André
Published: May 2005

We report the nucleotide sequence of an 11.4 kb DNA segment from the left arm of Saccharomyces cerevisiae chromosome II. This sequence contains a typical structure of a functional ARS as well as five open reading frames (ORFs) longer than 300 bp. One is PEP1, a gene encoding a transmembrane protein of 1579 amino acids which transits through the secretory pathway and is involved in vacuolar protein sorting. Two genes were previously sequenced: ACH1 (Lee et al., 1990) and FUS3 (Elion et al., 1990), which encode an acetyl‐CoA hydrolase and a protein kinase involved in the cell division cycle, respectively. The last two ORFs localized on the complementary strand of ACH1 are not likely to be expressed.
Isolation and characterization of peroxisomal protein import (Pim−) mutants of Hansenula polymorpha
Authors: Waterham Hans R. , Titorenko Vladimir I. , Van Der Klei Ida J. , Harder Wim , Veenhuis Marten
Published: May 2005

In the course of our studies on the molecular mechanisms involved in peroxisome biogenesis, we have isolated several mutants of the methylotrophic yeast Hansenula polymorpha impaired in the import of peroximal matrix proteins. These mutants are characterized by the presence of small intact peroxisomes, while the bulk of the peroxisomal matrix protein is not imported and resides in the cytosol (Pim− phenotype). Genetic analysis of back‐crossed mutants revealed five different complementation groups, which were designated PERI–PER5. Mapping studies to determine the linkage relationships indicated that the observed Pim− phenotypes were determined by single recessive nuclear mutations.

The different mutants had comparable phenotypes: (i) they were impaired to utilize methanol as the sole source of carbon and energy but grew well on various other compounds, including nitrogen sources, the metabolism of which is known to be mediated by peroxisome‐borne enzymes in wild‐type cells; (ii) all peroxisomal enzymes tested were induced, assembled and activated as in wild‐type cells although their activities varied between the different representative mutants; (iii) all peroxisomal proteins, whether constitutive or inducible, were found both in the cytosol and in the small peroxisomes. These results suggest that a general, major import mechanism is affected in all mutants.
Effect of sterol alterations on conjugation in Saccharomyces cerevisiae
Authors: Tomeo Michele E. , Fenner Gregeory , Tove Shirley R. , Parks Leo W.
Published: May 2005

Sterol auxotrophic strains of Saccharomyces cerevisiae were grown and allowed to conjugate on media supplemented with various sterols.

The mating efficiency of the auxotrophs is perturbed by the relacement of the normal yeast sterol, ergsterol, with other sterols. After 4 h of mating, cells grown on ergosterol a 30‐fold higher productive mating efficiency than those cells grown in stigmasterol. Aberrant budding by the conjugants was enhanced following incubation on stigmasterol and other non‐ergosterol sterols. Using light and electron microscopy, we demonstrated that there is a reduced ability for stigmasterol‐grown cells to undergo cytoplasmic fusion during conjugation. Many of the mated pairs remained adherent but Prezygotic even after 12 h of incubation. The addition of ergosterol to cells previously grown on stigmasterol rescued the organisms, allowing for zygote formation and normal budding.
The SCH9 protein kinase mRNA contains a long 5′ leader with a small open reading frame
Authors: Di Blasi Francesco , Carra Elena , De Vendittis Emmanuele , Masturzo Pietro , Burderi Emanuele , Lambrinoudaki Irene , Mirisola Mario G. , Seidita Gregorio , Fasano Ottavio
Published: May 2005

The SCH9 yeast gene, that was previously identified as a suppressor of cdc25 and ras1− ras2‐ts temperature‐sensitive mutants, encodes a putative protein kinase that positively regulates the progression of yeast cells through the G1 phase of the cell cycle. We have determined the structure of the SCH9 transcription unit, using primer extension and S1 mapping techniques. The corresponding mRNA included an unusually long 5′ region of more than 600 nucleotides preceding the major open reading frame (ORF). While the latter corresponded to a protein of 824 amino acids, an upstream open reading frame (uORF) within the 5′ leader could potentially encode a 54 amino acid peptide. To investigate the role of the AUGs within the uORF, we engineered chimaeric plasmid vectors in which SCH9 sequences including the promoter, the mRNA leader and the first 514 nucleotides of the major ORF were fused in‐frame with β‐galactosidase‐coding sequences. Upon introduction into yeast cells, the fusion protein was efficiently expressed. However, mutational disruption of the uORF using oligonucleotide‐directed mutagenesis did not affect the level of expression of the fusion protein. This indicates that regulatory mechanisms in Saccharomyces cerevisiae prevent upstream AUGs within the SCH9 mRNA leader sequence from influencing translation from downstream initiation codons.
Cloning and genetic analysis of the gene encoding a new protein kinase in Saccharomyces cerevisiae
Authors: Kambouris Nicholas G. , Burke Daniel J. , Creutz Carl E.
Published: May 2005

We have isolated a single gene from the yeast Saccharomyces cerevisiae encoding a potential 800 amino acid polypeptide of calculated Mr 90 098 Da. This protein consists of an N‐terminal region that shares significant homology with the catalytic domains of several serine‐ and threonine‐specific protein kinases, as well as a large, unique, C‐terminal domain of unknown function. Haploid disruption mutants are viable and do not exhibit any readily observable growth defects under varying conditions of temperature, nutrients or osmotic strength. Due to the apparent structural similarity between this kinase and the protein products of the KIN1 and KIN2 genes, we have chosen to name this new gene KIN3.
Control of Saccharomyces cerevisiae carboxypeptidase S (CPS1) gene expression under nutrient limitation
Authors: Bordallo Javier , Suárez‐Rendueles Paz
Published: May 2005

Expression of the vacuolar carboxypeptidase S (CPS1) gene in Saccharomyces cerevisiae is regulated by the availability of nutrients. Enzyme production is sensitive to nitrogen catabolite repression; i.e. the presence of ammonium ions maintains expression of the gene at a low level. Transfer of ammonium–glucose pre‐grown cells to a medium deprived of nitrogen causes a drastic increase in CPS1 RNA level provided that a readily usable carbon source, such as glucose or fructose, is available to the cells. Derepression of the gene by nitrogen limitation is cycloheximide‐insensitive. Neither glycerol, ethanol, acetate nor galactose support derepression of CPS1 expression under nitrogen starvation conditions. Non‐metabolizable sugar analogs (2‐deoxyglucose, 6‐methyl‐glucose or glucosamine) do not allow derepression of CPS1, showing that the process is energy‐dependent. Production of carboxypeptidase yscS also increases several‐fold when ammonium‐pregrown cells are transferred to media containing glucose and a non‐readily metabolizable nitrogen source such as proline, leucine, valine or leucyl‐glycine. Analysis of CPS1 expression in RAS2+ (high cAMP) and ras2 mutant (low cAMP) strains and in cells grown at low temperature (23°C) and in heat‐shocked cells (38°C) shows that steady‐state levels of CPS1 mRNA are not controlled by a low cAMP level‐signalling pathway.
A new promoter‐probe vector for Saccharomyces cerevisiae using fungal glucoamylase cDNA as the reporter gene
Authors: Scorpione Regina Céli , De Camargo Solange Soares , Astolfi‐Filho Spartaco , Schenberg Ana Clara Guerrini
Published: May 2005

A system is described for the selection of DNA sequences showing promoter activity in the yeast Saccharomyces cerevisiae using a heterologous reporter enzyme which is efficiently secreted by the yeast host. A multicopy shuttle plasmid of the YEp‐type was constructed so as to carry multiple unique cloning sites at the 5′ end of the Aspergillus awamori glucoamylase cDNA. Glucoamylase can only be expressed upon insertion at one of these unique cloning sites of a DNA fragment from any source, provided it is endowed with promoter function in S. cerevisiae. As the glucoamylase signal‐peptide is functional in S. cerevisiae, the enzyme is efficiently secreted by the yeast transformants. This phenotype can be very easily detected on plate assays and accurately quantified by spectrophotometric analysis of the culture supernatant. Since S. cerevisiae naturally lacks amylolytic activity, any wild‐type strain can be used as a host in this system. To evaluate the system, a DNA pool of random fusions was created by ligating sau 3A digested S. cerevisiae genomic DNA to the BglII‐linearized vector. The resulting hybrid plasmids were transformed into S. cerevisiae and several transformants secreting glucoamylase to varying degrees were obtained.