A genetic analysis of the role of calcineurin and calmodulin in Ca++-dependent improvement of NaCl tolerance of Saccharomyces cerevisiae

Mutants of Saccharomyces cerevisiae lacking activity of the Ca²⁺/calmodulin-dependent protein phosphatase calcineurin, show sensitivity to high concentrations of sodium that is partly reversed by the external supply of Ca²⁺. On long-time exposure to NaCl stress the mutants display an increased intracellular Na⁺/K⁺ ratio which is partially corrected by the addition of Ca²⁺, improving the sodium efflux of not only calcineurin-defective cells but also wild-type cells. We also demonstrate that the NaCl sensitivity of cmd mutants, expressing modified forms of calmodulin that do not bind Ca²⁺, is strongly reversed by the addition of Ca²⁺. This effect is highly dependent on calcineurin, since the NaCl tolerance of a cmd1-3 strain, carrying an additional mutation in calcineurin, is only slightly assisted by Ca²⁺. A striking characteristic of the loss of function of calcineurin is a several-fold increased content of intracellular Ca²⁺, localized mainly in subcellular compartment(s). If the compartmentalized Ca²⁺ pool is brought back to normal levels by an additional inactivating mutation of the vacuolar Ca²⁺-transporting ATPase, such double mutants do not significantly improve their tolerance to NaCl. Received: 15 August / 22 August 1996     

Characterization of the Saccharomyces cerevisiaeFMS1 gene related to Candida albicans Corticosteroid-binding protein 1

 In order to investigate ergosterol metabolism in S. cerevisiae we studied the CM8 mutant strain defective in the regulation of this pathway. A genomic multicopy library was screened to reverse the CM8 phenotype. This allowed us to characterize a new gene, FMS1, which relieves mutant phenotype by extragenic functional complementation. FMS1 may encode a 508 amino-acid protein. The predicted protein shares 35% identity with Cbp1p, a Candida albicans corticosteroid binding-protein. Fms1p also shows a weaker homology with monoamine oxidases. The construction of a FMS1 null-allele yeast strain demonstrated that this gene is not essential for yeast in normal usual laboratory culture conditions. The existence of a gene related to CBP1 of C. albicans in S. cerevisiae strongly suggests a possible function of steroid-binding proteins in yeast general physiology rather than in a process related to pathogenicity. Received: 10 December 1995/22 March 1996     

Involvement of the RE V3 gene in the methylated base-excision repair system. Co-operation of two DNA polymerases, δ and Rev3p, in the repair of MMS-induced lesions in the DNA of Saccharomyces cerevisiae

The ability of four yeast DNA polymerase mutant strains to carry out the repair of DNA treated with MMS was studied. Mutation in DNA polymerase Rev3, as well as the already known mutation in the catalytic subunit of DNA polymerase δ, were both found to lead to the accumulation of single-strand breaks, which indicates defective repair. A double-mutant strain carrying mutations in DNA polymerase δ and a deletion in the REV3 gene had a complete repair defect, both at permissive (23°C) and restrictive (38°C) temperatures, which was not observed in other pairwise combinations of tested polymerase mutants. Other polymerases are not involved in the repair of exogenous DNA methylation damage, since neither mutation in the DNA polymerase ɛ, nor deletion in the DNA polymerase IV (β₇₀) gene, caused defective repair. The data obtained suggest that DNA polymerases δ and Rev3p are both necessary to perform repair synthesis in the base-excision repair of methylation damage. The results are discussed in the light of current concepts on the role of DNA polymerase Rev3 in mutagenesis. Received: 18 November / 10 December 1996     

Arabinoxylan degradation by fungi: characterization of the arabinoxylan-arabinofuranohydrolase encoding genes from Aspergillus niger and Aspergillus tubingensis

 The genes encoding the enzyme arabinoxylan arabinofuranohydrolase, which releases L-arabinose from arabinoxylan, have been cloned from the closely related fungi Aspergillus niger and Aspergillus tubingensis and were shown to be functional in A. niger. Integration of multiple copies in the genome resulted in over-expression of the enzymes. The arabinofuranohydrolases encoded comprise 332 amino acids and have 94% amino acid identity. Their primary structure is not related to those of other α-L-arabinofuranosidases, except for a low similarity with XYLC, a bacterial α-L-arabinofuranosidase from Pseudomonas fluorescens which acts on oat spelt xylan. The axhA expression pattern in A. niger differed from that of abfB, since it was strongly induced by birchwood xylan and much less by L-arabitol or L-arabinose. Furthermore, Northern analysis revealed that axhA expression was de repressed in creA ^d mutants and carbon catabolite repressed by D-glucose. Received: 9 August / 29 August 1996     

Isolation of early meiotic recombination genes analogous to S. cerevisiae REC104 from the yeasts S. paradoxus and S. pastorianus

The REC104 gene of Saccharomyces cerevisiae is required to initiate recombination in meiosis. Mutations in REC104 eliminate meiotic recombination and lead to the production of inviable spores. To determine if analogous genes exist in other yeasts, clones that hybridized to a REC104 probe were isolated from the yeasts S.␣paradoxus and S.␣pastorianus. When transformed into a rec104 strain, the REC104 analogs from these two yeasts restored spore viability and meiotic recombination to the same level as a REC104 gene cloned from S.␣cerevisiae. Compared to S.␣cerevisiae, the S.␣paradoxus gene codes for 79% identical amino acids and has 86% nucleic-acid identity in the promoter region and 84% in the coding region. The S.␣pastorianus gene codes for 63% identical amino acids and has 59% and 71% identity in the promoter and the coding regions, respectively. Received: 19 August / 19 September 1996     

Two types of α-factor receptor determinants for pheromone specificity in the mating-incompatible yeasts S. cerevisiae and S. kluyveri

The specificity of mating-pheromone systems helps prevent mating between different species of yeast. The mechanism of specificity of G protein-coupled receptors in other organisms may be similar. To determine the structural basis of species discrimination between α-factor variants, small extracellular segments of the Saccharomyces cerevisiaeα-factor receptor were substituted with residues from the Saccharomyces kluyveriα-factor receptor. Three substitutions altered specificity towards S. cerevisiae and S. kluyveriα-factor pheromone peptides. A substitution at residues 47–49 affected specificity for pheromone binding but not for pheromone activation of response. Substitution of residues 267–269 affected pheromone specificity for activation of cellular responses, but not for pheromone binding (under the assay conditions used). Substitution of residues 104–123 modestly affected both types of specificity. These results suggest two distinct types of receptor/ligand specificity (perhaps corresponding to ligand binding to G protein-coupled or uncoupled conformational states of the receptor) that evolved independently as S. cerevisiae and S. kluyveri diverged as species. Received: 27 September / 12 November 1996     

Group-I introns in the cytochrome c oxidase genes of Dictyostelium discoideum : two related ORFs in one loop of a group-I intron, a cox1/2 hybrid gene and an unusually large cox3 gene

 The DNA sequences of cytochrome oxidase (subunits 1, 2 and 3) genes of the cellular slime mold Dictyostelium discoideum mitochondria were determined. The genes for subunits 1 and 2 have a single continuous ORF (COX1/2) which contains four group-I introns. The insertion sites of the two group-I introns (DdOX1/2.2 and DdOX1/2.3) coincide with those of fungal and algal group-I introns, as well as a liverwort group-I intron, in the cytochrome oxidase subunit 1. Interestingly, intron DdOX1/2.2 has two free-standing ORFs in a loop (L8) which have similar amino-acid sequences and are homologous to ai4 DNA endonuclease (I-Sce II) and bi4 RNA maturase found in group-I introns of Saccharomyces cerevisiae mitochondrial DNA. Two group-I introns (DdOX1/2.3 and DdOX1/2.4) also have a free-standing ORF in loop 1 and loop 2, respectively. These results show that these group-I introns and the intronic ORFs have evolved from the same ancestral origin, but that these ORFs have been propagated independently. Received: 1 May / 16 September 1996     

Transient expression of the β-glucuronidase gene after biolistic transformation of the anaerobic fungus Neocallimastix frontalis

 The rumen anaerobic fungus Neocallimastix frontalis was biolistically transformed using plasmids containing the bacterial β-glucuronidase gene (GUS) fused to the promoter sequences of the enolase gene from N. frontalis. Multiple copies of the plasmids were precipitated onto tungsten particles and delivered into zoosporangia and a mycelial mat by a helium-driven biolistic device. Transformants were detected by histochemical assay for β-glucuronidase. It was found that the enolase promoter sequences tested were responsible for the transient expression of the β-glucuronidase gene. This is the first study presenting results on the transformation of an anaerobic fungus. Received: 9 July / 18 September 1996     

Characterization of the Saccharomyces cerevisiae RTA1 gene involved in 7-aminocholesterol resistance

 7-aminocholesterol has been described as being a strong inhibitor of yeast and of Gram⁺-bacteria proliferation. In order to determine the precise molecular target of the toxicity of this compound, we searched for yeast resistance linked to gene over-expression. We named the new yeast gene that was isolated RTA1 (EMBL X84736). This gene led to strong resistance to the inhibitor. Gene sequencing revealed that RTA1 is adjacent to the NAB1 gene which is orientated in an opposite direction and localized on chromosome VII. The RTA1 gene, which encodes a putative protein with seven potential membrane-spanning segments, was shown to be a non-essential gene in yeast. Received: 25 December 1995/22 March 1996     

The homologue of the Saccharomyces cerevisiae RIM9 gene is required for ambient pH signalling in Candida albicans

In fungi, ambient pH sensing relies on the conserved Rim101 signalling pathway. All components of the pathway have been shown to be functionally conserved in the pathogenic yeast Candida albicans except for Rim9p which, in other fungi, has been suggested to be involved in this process. Here we report that, in C. albicans, the RIM9 homologue is required for Rim-dependent signalling. We also show that overexpressing Vps32p, an endosomal component required for transduction of the pH signal, does not bypass defects in upstream components such as Rim9p, Rim21p and Rim8p.     

Expression and secretion of the Candida wickerhamii extracellular β-glucosidase gene,bglB, in Saccharomyces cerevisiae

The yeast Candida wickerhamii exports a cell-associated β-glucosidase that is active against cellobiose and all soluble cellodextrins. Because of its unique ability to tolerate end-product inhibition by glucose, the bglB gene that encodes this enzyme was previously cloned and sequenced in this laboratory. Using several different promoters and constructs, bglB was expressed in the hosts Escherichia coli, Pichia pastoris, and Saccharomyces cerevisiae. Expression was initially performed in E. coli using either the lacZ or tac promoter. This resulted in intracellular expression of the BglB protein with the protein being rapidly fragmented. Secretion and glycosylation of active β-glucosidase was achieved with several different S. cerevisiae constructs utilizing either the adh1 or the gal1 promoter on 2-μ replicating plasmids. When either the invertase (Suc2) or the BglB secretion signal was used, BglB protein remained associated with the cell wall and appeared to be hyperglycosylated. Expression in P. pastoris was also examined to determine if higher activity and expression could be achieved in a yeast host that usually does not hyperglycosylate. Using the alcohol oxidase promoter in conjunction with either the pho1 or the α-factor secretion signal, the recombinant enzyme was successfully secreted and glycosylated in P. pastoris. However, levels of protein expression from the chromosomally integrated vector were insufficient to detect activity. Received: 28 March / 19 July 1996     

Molecular analysis of UFE1, a Saccharomyces cerevisiae gene essential for spore formation and vegetative growth

 The UFE1 gene of Saccharomyces cerevisiae was cloned, sequenced and characterized. The coding region of UFE1 is separated from the TMP1 gene on chromosome XV by 624 bp. Gene-disruption experiments demonstrated that UFE1 is essential for both the germination of ascospores and for vegetative growth. Translation of the UFE1 coding region generates a protein with significant similarity to cytokeratin and to the coiled-coil region of SED5, USO1 and restin, suggesting that it is involved in the secretory pathway and may also be related to intermediate filament-associated proteins. Received: 6 May/18 June 1996     

New insights into the pyrimidine salvage pathway of Saccharomyces cerevisiae: requirement of six genes for cytidine metabolism

Cytidine metabolism in the yeast Saccharomyces cerevisiae was analyzed by genetic and biochemical approaches. Disruption of a unique ORF (Genbank accession No. U 20865) bearing homology with eucaryotic or bacterial cytidine deaminases abolished cytidine deaminase activity and resulted in 5-fluorocytidine resistance. The gene encoding cytidine deaminase will be referred to as CDD1 (Genbank accession number AF080089). The ability to isolate mutants resistant to 5-fluorocytidine which mapped to five other loci demonstrated the existence of a complex cytidine metabolic network. Deciphering this network revealed several original features: (1) cytidine entry is mediated by the purine-cytosine transporter (Fcy2p), (2) cytidine is cleaved into cytosine by the uridine nucleosidase (Urh1p), (3) cytidine is phosphorylated into CMP by the uridine kinase (Urk1p), (4) a block in cytosine deaminase (Fcy1p), but not in cytidine deaminase (Cdd1p), constitutes a limiting step in cytidine utilisation as a UMP precursor. Received: 21 November 1998/14 April 1999     

Importance of the Sgs1 helicase activity in DNA repair of Saccharomyces cerevisiae

 The Saccharomyces cerevisiae Sgs1 protein, together with Schizosaccharomyces pombe Rqh1 and the human Bloom and Werner proteins, is a DNA helicase of the Escherichia coli RecQ family. Mutation of SGS1 causes premature aging in yeast cells, including the accumulation of extrachromosomal rDNA circles. We have recently shown that Sgs1p interacts with the DNA repair Rad16p protein and is epistatic to Rad16p for UVC, 4-NQO and H₂O₂ lesions. Therefore we tested sgs1 strains containing mutations in the helicase and C-terminal domains. We demonstrate here that the helicase activity of the Sgs1 is important for most elements of the sgs1 mutation phenotype, including sensitivity to UVC, 4-NQO, H₂O₂, MMS and hydroxyurea. Received: 8 August / 18 October 1999     

Over-expression of the NUD1-coded endo-exonuclease in Saccharomyces cerevisiae enhances DNA recombination and repair

  The NUD1(=NUC2) gene of Saccharomyces cerevisiae has been subcloned and over-expressed in multi-copy plasmids. Enhanced expression of this nuclear endo-exonuclease gene was confirmed by Northern hybridization (>10-fold increase), and increased enzymatic activity (2.4-fold increase) was demonstrated by direct immunological assay using antibody raised against the purified Neurospora crassa endo-exonuclease. We found that increased expression of NUD1 was associated with an increase in cell surivival after irradiation treatment with gamma rays, and an increase in radiation-induced mitotic recombination frequencies between duplicated gene sequences. The results presented here are consistent with previous biochemical data and confirm a role for the NUD1 gene product in recombination/repair processes. Received: 10 November 1995 / 16 January 1996     

Expression of the Candida albicans Gene ALS1 in Saccharomyces cerevisiae Induces Adherence to Endothelial and Epithelial Cells

To identify genes encoding adhesins that mediate the binding of Candida albicans to endothelial cells, a genomic library from this organism was constructed and used to transform Saccharomyces cerevisiae. These transformed organisms were screened for adherence to endothelial cells, and a highly adherent clone was identified. The adherence of this clone to endothelial cells was over 100-fold greater than that of control S. cerevisiae transformed with the empty plasmid. This clone also exhibited enhanced adherence to epithelial cells. The C. albicans gene contained within this clone was found to be ALS1. These results indicate that ALS1 may encode a candidal adhesin.