Rapid discrimination between Candida glabrata, Candida nivariensis, and Candida bracarensis by use of a singleplex PCR.

We report here a PCR-based assay using a single primer pair targeting the RPL31 gene that allows discrimination between Candida glabrata, Candida bracarensis, and Candida nivariensis according to the size of the generated amplicon.

Molecular typing of wine yeast strains Saccharomyces bayanus var. uvarum using microsatellite markers.

The Saccharomyces bayanus var. uvarum yeasts are associated with spontaneous fermentation of must. Some strains were shown to be enological yeasts of interest in different winemaking processes. The molecular typing of S. bayanus var. uvarum at the strain level has become significant for wine microbiologists. Four microsatellite loci were defined from the exploration of genomic DNA sequence of S. bayanus var. uvarum. The 40 strains studied were homozygote for the locus considered. The discriminating capacity of the microsatellite method was found to be equal to that of karyotypes analysis. Links between 37 indigenous strains with the same geographic origin could be established through the analysis of microsatellite patterns. The analysis of microsatellite polymorphism is a reliable method for wine S. bayanus var. uvarum strains and their hybrids with Saccharomyces cerevisiae identification in taxonomic, ecological studies and winemaking applications.

Protein-protein interaction between the RVS161 and RVS167 gene products of Saccharomyces cerevisiae.

As previous studies indicated that the RVS161 and RVS167 gene products of Saccharomyces cerevisiae seem to be involved in the same cellular function, we considered the possibility of a complex between the proteins encoded by these two genes. Using the two hybrid system, we have shown that Rvs167p interacts with Rvs161p, through its N-terminal domain which contains predicted coiled-coil structures. Moreover, if a tagged Rvs 167p protein was immuno adsorbed under non-denaturing conditions, it brought along the Rvs161p protein. These results confirmed the hypothesis of an in vivo complex between the two proteins.

Characterization of a new gene family developing pleiotropic phenotypes upon mutation in Saccharomyces cerevisiae.

The aim of this paper is to gather and complete data about four members of a new gene family. Mutation in SUR4 gene was originally selected as a suppressor of defects caused by mutations in RVS161 or RVS167 genes. Cloning and sequencing of the SUR4 gene were performed. The deduced protein contains six putative transmembrane domains. Sequence comparison revealed that two yeast genes, FEN1 and JO343, shared significant similarities with SUR4. Mutants for SUR4 and FEN1 have the same pleiotropic phenotype, including bud localization defects, resistance to an immunosuppressor and resistance to ergosterol biosynthesis inhibitors. The double inactivation of SUR4 and FEN1 genes is lethal. These data and other aspects implicating SUR4 in glucose metabolism, suggest an involvement of these genes in the dynamics of cortical actin cytoskeleton in response to nutrient availability. Moreover, the existence of a fourth homologous gene in C. elegans extends the family to pluricellular organisms.

Temporal Action of Mutations Inhibiting the Accomplishment of Quiescence or Disrupting Development in the Fungus PODOSPORA ANSERINA.

Two Podospora mutants carrying mutations modE and modF were persumed to be quiescent defective, because, when grown under glucose limitation, they differed from the wild-type strain in an excess of dry weight production and a reduction of cell survival. New insight on the action of modE and modF mutations was provided by the study of double mutants resulting from the association of modE or modF mutations with unrelated developmental mutations.-ModE and modF were first coupled to three allelic mutations ( modC) that inhibit production of all hyphal cell derivatives (late ramifications, aerial hyphae and protoperithecia). Suppression in the double mutants of the excess of proliferation associated with modE and modF and restoration of normal cell survival indicated that modE and modF result in an uncontrolled production of hyphal cell derivatives in which deregulation is presumed to be responsible for the reduction of cell survival following glucose exhaustion.-ModE and modF were associated with mutations of two genes (modD and modG) which abolish production of hyphal cell derivatives (like modC mutations) but also inhibit the renewal of growth of cells situated in the center of colonies. Investigations of eight of these double mutants showed that modE and modF mutations suppress the inhibitory action of modD and modG on production of hyphal cell derivatives and on growth renewal.-Taken together these results lead to the suggestion that the accomplishment of a quiescent state for cell survival under glucose starvation is the final stage in the differentiation of hyphal cells and prerequisite for the production of derivatives of hyphal cells and for a control of their development.

Genomic exploration of the hemiascomycetous yeasts: 5. Saccharomyces bayanus var. uvarum.

Saccharomyces bayanus var. uvarum investigated here is the species closest to Saccharomyces cerevisiae. Random sequence tags (RSTs) allowed us to identify homologues to 2789 open reading frames (ORFs) in S. cerevisiae, ORFs duplicated in S. uvarum but not in S. cerevisiae, centromeres, tRNAs, homologues of Ty1/2 and Ty4 retrotransposons, and a complete rDNA repeat. Only 13 RSTs seem to be homologous to sequences in other organisms but not in S. cerevisiae. As the synteny between the two species is very high, cases in which synteny is lost suggest special mechanisms of genome evolution. The corresponding RSTs revealed that S. uvarum can exist without any S. cerevisiae DNA introgression. Accession numbers are from AL397139 to AL402278 in the EMBL databank.

Genomic exploration of the hemiascomycetous yeasts: 3. Methods and strategies used for sequence analysis and annotation.

The primary analysis of the sequences for our Hemiascomycete random sequence tag (RST) project was performed using a combination of classical methods for sequence comparison and contig assembly, and of specifically written scripts and computer visualization routines. Comparisons were performed first against DNA and protein sequences from Saccharomyces cerevisiae, then against protein sequences from other completely sequenced organisms and, finally, against protein sequences from all other organisms. Blast alignments were individually inspected to help recognize genes within our random genomic sequences despite the fact that only parts of them were available. For each yeast species, validated alignments were used to infer the proper genetic code, to determine codon usage preferences and to calculate their degree of sequence divergence with S. cerevisiae. The quality of each genomic library was monitored from contig analysis of the DNA sequences. Annotated sequences were submitted to the EMBL database, and the general annotation tables produced served as a basis for our comparative description of the evolution, redundancy and function of the Hemiascomycete genomes described in other articles of this issue.

Genomic exploration of the hemiascomycetous yeasts: 1. A set of yeast species for molecular evolution studies.

The identification of molecular evolutionary mechanisms in eukaryotes is approached by a comparative genomics study of a homogeneous group of species classified as Hemiascomycetes. This group includes Saccharomyces cerevisiae, the first eukaryotic genome entirely sequenced, back in 1996. A random sequencing analysis has been performed on 13 different species sharing a small genome size and a low frequency of introns. Detailed information is provided in the 20 following papers. Additional tables available on websites describe the ca. 20000 newly identified genes. This wealth of data, so far unique among eukaryotes, allowed us to examine the conservation of chromosome maps, to identify the 'yeast-specific' genes, and to review the distribution of gene families into functional classes. This project conducted by a network of seven French laboratories has been designated 'Génolevures'.

Genomic exploration of the hemiascomycetous yeasts: 4. The genome of Saccharomyces cerevisiae revisited.

Since its completion more than 4 years ago, the sequence of Saccharomyces cerevisiae has been extensively used and studied. The original sequence has received a few corrections, and the identification of genes has been completed, thanks in particular to transcriptome analyses and to specialized studies on introns, tRNA genes, transposons or multigene families. In order to undertake the extensive comparative sequence analysis of this program, we have entirely revisited the S. cerevisiae sequence using the same criteria for all 16 chromosomes and taking into account publicly available annotations for genes and elements that cannot be predicted. Comparison with the other yeast species of this program indicates the existence of 50 novel genes in segments previously considered as 'intergenic' and suggests extensions for 26 of the previously annotated genes.

Genomic exploration of the hemiascomycetous yeasts: 18. Comparative analysis of chromosome maps and synteny with Saccharomyces cerevisiae.

We have analyzed the evolution of chromosome maps of Hemiascomycetes by comparing gene order and orientation of the 13 yeast species partially sequenced in this program with the genome map of Saccharomyces cerevisiae. From the analysis of nearly 8000 situations in which two distinct genes having homologs in S. cerevisiae could be identified on the sequenced inserts of another yeast species, we have quantified the loss of synteny, the frequency of single gene deletion and the occurrence of gene inversion. Traces of ancestral duplications in the genome of S. cerevisiae could be identified from the comparison with the other species that do not entirely coincide with those identified from the comparison of S. cerevisiae with itself. From such duplications and from the correlation observed between gene inversion and loss of synteny, a model is proposed for the molecular evolution of Hemiascomycetes. This model, which can possibly be extended to other eukaryotes, is based on the reiteration of events of duplication of chromosome segments, creating transient merodiploids that are subsequently resolved by single gene deletion events.

Genomic exploration of the hemiascomycetous yeasts: 19. Ascomycetes-specific genes.

Comparisons of the 6213 predicted Saccharomyces cerevisiae open reading frame (ORF) products with sequences from organisms of other biological phyla differentiate genes commonly conserved in evolution from 'maverick' genes which have no homologue in phyla other than the Ascomycetes. We show that a majority of the 'maverick' genes have homologues among other yeast species and thus define a set of 1892 genes that, from sequence comparisons, appear 'Ascomycetes-specific'. We estimate, retrospectively, that the S. cerevisiae genome contains 5651 actual protein-coding genes, 50 of which were identified for the first time in this work, and that the present public databases contain 612 predicted ORFs that are not real genes. Interestingly, the sequences of the 'Ascomycetes-specific' genes tend to diverge more rapidly in evolution than that of other genes. Half of the 'Ascomycetes-specific' genes are functionally characterized in S. cerevisiae, and a few functional categories are over-represented in them.

Genomic exploration of the hemiascomycetous yeasts: 20. Evolution of gene redundancy compared to Saccharomyces cerevisiae.

We have evaluated the degree of gene redundancy in the nuclear genomes of 13 hemiascomycetous yeast species. Saccharomyces cerevisiae singletons and gene families appear generally conserved in these species as singletons and families of similar size, respectively. Variations of the number of homologues with respect to that expected affect from 7 to less than 24% of each genome. Since S. cerevisiae homologues represent the majority of the genes identified in the genomes studied, the overall degree of gene redundancy seems conserved across all species. This is best explained by a dynamic equilibrium resulting from numerous events of gene duplication and deletion rather than by a massive duplication event occurring in some lineages and not in others.

Genomic exploration of the hemiascomycetous yeasts: 21. Comparative functional classification of genes.

We explored the biological diversity of hemiascomycetous yeasts using a set of 22000 newly identified genes in 13 species through BLASTX searches. Genes without clear homologue in Saccharomyces cerevisiae appeared to be conserved in several species, suggesting that they were recently lost by S. cerevisiae. They often identified well-known species-specific traits. Cases of gene acquisition through horizontal transfer appeared to occur very rarely if at all. All identified genes were ascribed to functional classes. Functional classes were differently represented among species. Species classification by functional clustering roughly paralleled rDNA phylogeny. Unequal distribution of rapidly evolving, ascomycete-specific, genes among species and functions was shown to contribute strongly to this clustering. A few cases of gene family amplification were documented, but no general correlation could be observed between functional differentiation of yeast species and variations of gene family sizes. Yeast biological diversity seems thus to result from limited species-specific gene losses or duplications, and for a large part from rapid evolution of genes and regulatory factors dedicated to specific functions.

The yeast ATP synthase subunit 4: structure and function.

The structure of ATP synthase subunit 4 was determined by using the oligonucleotide probe procedure. This subunit is the fourth polypeptide of the complex when classifying subunits in order of decreasing molecular mass. Its relative molecular mass is 25 kDa. The ATP4 gene was isolated and sequenced. The nucleotide sequence predicts that subunit 4 is probably derived from a precursor protein 244 amino acids long. Mature subunit 4 contains 209 amino acid residues and the predicted molecular mass is 23250 kDa. Subunit 4 shows homology with the b-subunit of Escherichia coli ATP synthase and the b-subunit of beef heart mitochondrial ATP synthase. By using homologous transformation, a mutant lacking wild subunit 4 was constructed. This mutant is devoid of oxidative phosphorylation and F1 is loosely bound to the membrane. Our data are in favor of a structural relationship between subunit 4 and the mitochondrially-translated subunit 6 during biogenesis of F0.

Podospora mutant defective in glucose-dependent growth control.

A mutation (modE), previously described as a membrane mutation, results in several modifications of the female developmental cycle: a high density of protoperithecia, the unscheduled development of protoperithecia into sterile perithecia on the homokaryons of each mating type, and the independence of ascospore outgrowth from the substances normally required for germination. Cultured in liquid medium, the modE strain showed two additional specific features: a higher growth yield than that of wild-type cultures (plus 10% of dry weight) and an extreme reduction of cell life span. Both mutant traits were specific to glucose limitation. Despite the large difference existing in the sensitivity of cells to glucose starvation, the glycogen and trehalose reserves of mutant and wild-type cells were nearly identical. Considered together, these results suggest that the primary effect of the mutation lies in the disruption of a glucose-dependent regulation controlling the transition of the metabolic pattern of cells from growth to quiescence.

Podospora anserina mutant defective in protoperithecium formation, ascospore germination, and cell regeneration.

A mutant (modx) was selected on the basis of the suppression of self-lysis due to a recessive mutation (modB). modx, a dominant mutation, reduced hyphal branching from nonapical cells, abolished protoperithecium formation, and induced the death of stationary cells only when these were isolated to obtain further development. Mutant ascospores, formed in the fruiting bodies which occasionally occur under specific conditions (32 degrees C on starved medium), showed a delay in the germination process (up to 3 months instead of about 5 h for wild-type ascospores) when submitted to incubation under standard conditions (26 degrees C on germination medium) and failed to germinate at 18 degrees C. Revertants from modx strains, selected on the basis of the suppression of the nonrenewal of growth from stationary cells, were wild type for all the other three defects. Indirect arguments suggested that the modx mutant strain might be defective in the control of a specific class of stable messenger ribonucleic acids which would be essential for the physiology of ascospores and stationary cells.

ATP4, the structural gene for yeast F0F1 ATPase subunit 4.

A plasmid containing the gene coding for the Saccharomyces cerevisiae F0F1 ATPase subunit 4 was isolated from a yeast genomic DNA library using the oligonucleotide probe procedure. The gene and the surrounding regions were cloned into M13 tg 130 and M13 tg 131 phage vectors. A 732-base-pair open reading frame encoding a 244-amino-acid polypeptide is described. The nucleotide sequence predicts that subunit 4 is probably derived from a precursor protein with a hydrophilic and basic 35-amino-acid leader sequence. Mature subunit 4 contains 209 amino acid residues and the predicted molecular mass is 23250 Da. This subunit presents amphiphilic behaviour with two distinct domains. A high alpha-helix content of 77% was predicted from the sequence. Subunit 4 shows homology with the b subunit of Escherichia coli ATP synthase.

Expression of the avian gag-myc oncogene in Saccharomyces cerevisiae.

The gag-myc oncogenic sequence of the avian retrovirus MC29 was first inserted in a multicopy expression vector allowing its expression in Saccharomyces cerevisiae. The oncogene transcripts were detected in yeast by Northern blot hybridization and gag-myc proteins were revealed by immunoprecipitation. On solid medium, the average size of gag-myc transformant colonies was smaller than control. In liquid cultures, the gag-myc strains had a doubling time of 4.7 h compared with 3.1 h in the controls. In one of the recipient strains, and after an initial transient period of 5 days, the gag-myc transformants became physiologically indistinguishable from control. In another recipient strain, the slow-growth phenotype is permanent. Plasmid instability is increased in gag-myc transformants. When a single copy of the gag-myc gene was inserted in a yeast chromosome, no phenotype was observed, showing that slow growth is the consequence of plasmid loss.

Evidence for a branched pathway in the polarized cell division of Saccharomyces cerevisiae.

Cells of Saccharomyces cerevisiae can choose a bud site in one of two different spatial patterns (axial or bipolar) determined by their mating type. Genes important for bud-site selection have been identified and a linear model describing the hierarchy of these genes was proposed. We have uncovered a new class of genes which is required only for the bipolar pattern. The phenotype of the corresponding mutants coupled with epistasis experiments with some budding mutants already described suggest the existence of specific genes for the bipolar pathway.

Heterologous insertion of transforming DNA and generation of new deletions associated with transformation in Aspergillus nidulans.

The analysis of four transformants for the proline catabolism (prn) gene cluster of Aspergillus nidulans is reported. Using a combination of traditional genetic methodology and Southern hybridisation we have shown that in two cases multiple copies of the transforming plasmid have been integrated into linkage groups other than VII, which contains the prn cluster. In the other two cases integration of the plasmid has probably occurred homologously. The phenotype of these transformants is broadly consistent with increased copy number resulting in increased expression. Genetic manipulation of these transformants using the sexual or parasexual cycles has shown that recombination events during and possibly also subsequent to integration of the transforming DNA can generate new mutational lesions, in particular, deletions.