Yeast

Uemura, Hiroshi; Watanabe‐Yoshida, Miyuki; Ishii, Nobuya; Shinzato, Tomoko; Haw, Robin; Aoki, Yuko

doi: 10.1002/yea.1048pmid: 14745778

To study the function of RAP1, a Candida albicans gene (CaRAP1) that shows sequence similarity to RAP1 of Saccharomyces cerevisiae was isolated by colony hybridization. DNA sequencing predicted an open reading frame of 429 amino acids with an overall identity of 24% to the ScRap1p. The DNA binding domain (DBD) was highly conserved, and EMSA using a GST–CaRap1p fusion protein confirmed its binding ability to the RPG‐box of S. cerevisiae ENO1. In contrast, the N‐terminus was less conserved and a moderate homology was observed in the BRCT domain. Interestingly, CaRap1p did not contain the C‐terminal activation/repression region of ScRap1p. Its DDBJ/EMBL/GenBank Accession No. is AB036055. Copyright © 2003 John Wiley & Sons, Ltd.

Babour, A.; Beckerich, J.‐M.; Gaillardin, C.

doi: 10.1002/yea.1051pmid: 14745779

The UDP‐Glc:glycoprotein glucosyltransferase (UGT) is a soluble protein of the endoplasmic reticulum (ER) that plays a determining part in the mechanism by which unfolded, partially folded or misfolded glycoproteins are retained into the ER. We have identified an UGT in the yeast Yarrowia lipolytica. This protein, of a predicted molecular weight of 165.7 kDa, is encoded by a 5054 bp coding sequence containing a 643 bp intron at position 682–1323. The N‐terminal part of the protein displays a signal sequence whereas its C‐terminal part carries an ER retrieval signal HDEL. An interruption of the gene that removes the 1075 last nucleotides of its sequence did not lead to any evident phenotype except for a slight increased sensitivity to tunicamycin. YlUGT1 mRNA levels respond to tunicamycin treatment by an induction factor of 2–4, which indicates that the gene product participates in the quality control mechanism in this yeast. Finally, an immunofluorescence study of the protein localization, shows that the protein distribution is different from that of previously studied ER resident proteins. This could indicate that UGT distribution in the secretory pathway is not confined to the ER. Copyright © 2003 John Wiley & Sons, Ltd.

Xue, Yongtao; Haas, Stefan A.; Brino, Laurent; Gusnanto, Arief; Reimers, Mark; Talibi, Driss; Vingron, Martin; Ekwall, Karl; Wright, Anthony P. H.

doi: 10.1002/yea.1053pmid: 14745780

Completion of the fission yeast genome sequence has opened up possibilities for post‐genomic approaches. We have constructed a DNA microarray for genome‐wide gene expression analysis in fission yeast. The microarray contains DNA fragments, PCR‐amplified from a genomic DNA template, that represent > 99% of the 5000 or so annotated fission yeast genes, as well as a number of control sequences. The GenomePRIDE software used attempts to design similarly sized DNA fragments corresponding to gene regions within single exons, near the 3′‐end of genes that lack homology to other fission yeast genes. To validate the design and utility of the array, we studied expression changes after a 2 h temperature shift from 25 °C to 36 °C, conditions widely used when studying temperature‐sensitive mutants. Obligingly, the vast majority of genes do not change more than two‐fold, supporting the widely held view that temperature‐shift experiments specifically reveal phenotypes associated with temperature‐sensitive mutants. However, we did identify a small group of genes that showed a reproducible change in expression. Importantly, most of these corresponded to previously characterized heat‐shock genes, whose expression has been reported to change after more extreme temperature shifts than those used here. We conclude that the DNA microarray represents a useful resource for fission yeast researchers as well as the broader yeast community, since it will facilitate comparison with the distantly related budding yeast, Saccharomyces cerevisiae. To maximize the utility of this resource, the array and its component parts are fully described in On‐line Supplementary Information and are also available commercially. Copyright © 2003 John Wiley & Sons, Ltd.

Staneva, Dessislava; Uccelletti, Daniela; Farina, Francesca; Venkov, Pencho; Palleschi, Claudio

doi: 10.1002/yea.1055pmid: 14745781

Phosphomannomutase (PMM) is a key enzyme, which catalyses one of the first steps in the glycosylation pathway, the conversion of D‐mannose‐6‐phosphate to D‐mannose‐1‐phosphate. The latter is the substrate for the synthesis of GDP‐mannose, which serves as the mannosyl donor for the glycosylation reactions in eukaryotic cells. In the yeast Saccharomyces cerevisiae PMM is encoded by the gene SEC53 (ScSEC53) and the deficiency of PMM activity leads to severe defects in both protein glycosylation and secretion. We report here on the isolation of the Kluyveromyces lactis SEC53 (KlSEC53) gene from a genomic library by virtue of its ability to complement a Saccharomyces cerevisiae sec53 mutation. The sequenced DNA fragment contained an open reading frame of 765 bp, coding for a predicted polypeptide, KlSec53p, of 254 amino acids. The KlSec53p displays a high degree of homology with phosphomannomutases from other yeast species, protozoans, plants and humans. Our results have demonstrated that KlSEC53 is the functional homologue of the ScSEC53 gene. Like ScSEC53, the KlSEC53 gene is essential for K. lactis cell viability. Phenotypic analysis of a K. lactis strain overexpressing the KlSEC53 gene revealed defects expected for impaired cell wall integrity. The sequence of the KlSEC53 has been deposited in the EMBL database under Accession No. AJ428418. Copyright © 2003 John Wiley & Sons, Ltd.

Ito‐Harashima, Sayoko; McCusker, John H.

doi: 10.1002/yea.1057pmid: 14745782

Novel MX cassettes are described that contain the open reading frames (ORFs) of Saccharomyces cerevisiae or Candida albicans LYS5. The LYS5MX and CaLYS5MX cassettes, the targeting efficiencies of which are equivalent to those of other MX cassettes, are positively selected for Lys+ in a lys5 background. Unlike most of the other MX cassettes, the LYS5MX cassettes are also negatively selectable (α‐aminoadipate‐resistant), which will allow the use of the LYS5MX cassettes in plasmid shuffling and will also greatly facilitate marker recycling. Copyright © 2003 John Wiley & Sons, Ltd.

Schlösser, Thomas; Gätgens, Cornelia; Weber, Ulrike; Stahmann, K.‐Peter

doi: 10.1002/yea.1058pmid: 14745783

Alanine : glyoxylate aminotransferase is one of three different enzymes used for glycine synthesis in Saccharomyces cerevisiae. The open reading frame YFL030w (named AGX1 in the following), encoding this enzyme, was identified by comparing enzyme specific activities in knockout strains. While 100% activity was detectable in the parental strain, 2% was found in a YFL030w::kanMX4 strain. The ORF found at that locus was suspected to encode alanine : glyoxylate aminotransferase because its predicted amino acid sequence showed 23% identity to the human homologue. Since the YFL030w::kanMX4 strain showed no glycine auxtrophic phenotype, AGX1 was replaced by KanMX4 in a Δ GLY1Δ SHM1Δ SHM2 background. These background mutations, which cause inactivation of threonine aldolase, mitochondrial and cytosolic serine hydroxymethyltransferase, respectively, lead to a conditional glycine auxotrophy. This means that growth is not possible on glucose but on ethanol as the sole carbon source. Additional disruption of AGX1 revealed a complete glycine auxotrophy. Complementation was observed by transformation with a plasmid‐encoded AGX1. Copyright © 2003 John Wiley & Sons, Ltd.

Versele, Matthias; Thevelein, Johan M.; Van Dijck, Patrick

doi: 10.1002/yea.1065pmid: 14745784

The initiation of fermentation in the yeast Saccharomyces cerevisiae is associated with a rapid drop in general stress resistance. Previously we identified a mutant which is deficient in fermentation‐induced loss of stress resistance (fil1), as a partially inactivating mutant in adenylate cyclase. We have now investigated possible causes of its high stress resistance. Deletion of the TPS1 gene, encoding the first enzyme in the biosynthesis of trehalose, or the heat shock protein gene HSP104 only resulted in a minor effect on heat stress resistance compared with deletion of these genes in a wild‐type background. A strain with a deletion of both genes still showed a higher stress resistance in the fil1 background compared to the corresponding wild‐type background. Deletion of the transcription factor genes MSN2 and MSN4, which are required for the expression of STRE‐regulated genes, resulted in a dramatic drop in heat resistance in the wild‐type background but had much less effect in the fil1 mutant. The fil1 msn2Δmsn4Δ strain remained more heat‐resistant than a wild‐type strain. A strain in which all four genes, TPS1, HSP104, MSN2 and MSN4, are deleted was very sensitive to heat stress and also to oxidative and salt stress. Presence of the fil1 mutation in such a strain, however, still clearly enhanced heat, oxidative and salt stress resistance. These results indicate that, in addition to trehalose, Hsp104 and the Msn2/4‐controlled genes, other factors exist in S. cerevisiae that can, significantly and independently of the known factors, enhance general stress resistance. The mutants described in this work provide a tool to identify these novel components. Copyright © 2003 John Wiley & Sons, Ltd.

doi: 10.1002/yea.1083pmid: 14768639

In order to keep subscribers up‐to‐date with the latest developments in their field, this current awareness service is provided by John Wiley & Sons and contains newly‐published material on yeasts. Each bibliography is divided into 10 sections. 1 Books, Reviews & Symposia; 2 General; 3 Biochemistry; 4 Biotechnology; 5 Cell Biology; 6 Gene Expression; 7 Genetics; 8 Physiology; 9 Medical Mycology; 10 Recombinant DNA Technology. Within each section, articles are listed in alphabetical order with respect to author. If, in the preceding period, no publications are located relevant to any one of these headings, that section will be omitted. (9 weeks journals ‐ search completed 5th. Nov. 2003)