The baker's yeast, Saccharomyces cerevisiae, plays a primary role in basic genetic research and was the first eukaryotic organism whose genomic sequence was completely determined. The Unité played an important role in this work, published in 1996. Since that date, research activity of the Unité was continued in several directions as summarized below.
Functional analysis of the genome of the yeast Saccharomyces cerevisiaePersons in charge : DUJON Bernard, BOYER Jeanne, FAIRHEAD Cécile, LLORENTE Bertrand, THIERRY Agnès
Following a systematic approach, begun as part of a European program (EUROFAN) and continued as an international collaboration, the Unité now possesses a collection of mutants of all yeast genes. In each mutant, the targetted gene was entirely deleted and replaced by an artificial selection cassette. Four strains are available for each non essential gene (two haploids, MATa et MATa, one diploid homozygous for the mutation and another diploid, heterozygous for the mutation). Obviously, for essential genes (ca. 15 % of total), only the hererozygous diploid strain is available. The complete collection contains 24000 strains. All strains are derived from the sequenced strain and are isogenic except for the deleted genes and for auxotrophic markers facilitating crosses.
As part of the European program EUROFAN 2, completed this year, the Unité has concentrated on the problem of functional redundancy within paralogous gene families originated from ancestral duplications, a common signature to all eukaryotic genomes. The functions of several previously uncharacterized gene families were described. As a general rule, the functional differentiation between members of a same family is primarily due to a modification of the relative importance of the genes keeping a common function. One of the genes becomes predominant (it is that gene which, generally, is found by genetic screening), the other play secondary roles. In other cases, such as the transporters, recruitement of existing structures for novel substrates is the rule. Finally, there exists cases of protein specialisation for intracellular compartments.
The Unité has also started a systematic screening of dominant negative mutants. A genomic library was constructed in a replicative yeast artificial plasmid allowing the expression of translation products fused in frame with an artificial epitope, under the control of a tetracycline responsive promoter. Tetracycline has no other target in yeast. This library is being screened. Over 500 genes have been identified whose overexpression interferes with normal cell growth.
Comparative genomics of yeasts Persons in charge : DUJON Bernard, BLANDIN Gaëlle, KALOGEROPOULOS Odile, LLORENTE B., MALPERTUY Alain, TEKAIA Fredj
The first phase of a comparative genomics project devoted to Hemiascomycetous yeasts was completed at the end of year 2000 by the publication of a special issue of FEBS letters containing 21 original articles, among which 14 were signed by members of the Unité (9 as first and/or last authors), and by the opening of a web site (http://cbi.labri.u-bordeaux.fr/Genolevures). This project was a collaborative effort in which a low coverage partial genomic sequencing was undertaken on 13 distinct species of Hemiascomycetes selected for their physiological properties, their phylogenetic location and their biotechnological or biomedical interest. Sequences were determined by Génoscope and their analysis was done by 6 laboratories including our Unité. Over 20000 genes were identified, offering an unprecedented set of sequences from a phylogenetically homogeneous group of Eukaryotes.
This work permitted a new annotation of the S. cerevisiae genome, with the discovery of 50 previously ignored genes and with the definition of an important set of "ascomycete-specific" genes whose functions need to be studied. This project is now continued as part of a CNRS structure (GDR) in which our Unité is engaged. Finally, with the help of the Institut Pasteur Génopole, we have started the sequencing of a human pathogenic yeast Candida glabrata. About 2500 genes from this species are now identified, thanks to a low coverage sequencing.
Chromosome dynamics and evolution Persons in charge : DUJON Bernard, FAIRHEAD Cécile, FISCHER Gilles, RICCHETTI Miria, RICHARD Guy-Franck
Molecular mechanisms ensuring the stability and the rearrangements of eukaryotic chromosomes have been a long standing interest of the Unité. The mode of repair of double strand breaks, artificially induced by the I-Sce I system that we have previously developed, identifies specific properties within chromosomal subtelomeric regions that are under analysis. Mitotic and meiotic instabilities of long trinucleotide repeats (microsatellites) are also studied in different mutants or wild-type backgrounds. Finally, we have recently started the screening of putative genes whose expression would be induced by chromosomal double strand breaks.
Our results of comparative genomics on Hemiascomycetous yeasts were very useful in this topic. An in-depth analysis of the loss of linkage between neighboring genes and of their relative orientation, has lead us to conclude that the major mechanism of eukaryotic chromosomal evolution is segmental chromosomal duplication, leading to a transient merodiploid state, which is subsequently resolved by the loss of the duplicated genes or their sequence divergence. Our results show the quantitative importance of chromosomal dynamics in evolution and its role in the formation and elimination of gene families. The genomes that one can observe should be considered as the temporary stage of a rapidly evolving equilibrium between chromosome segment duplications and sequence loss.
The comparison between S. cerevisiae and S. bayanus var. uvarum shows that, even between related species (the two species show 20 % amino-acid susbtitutions and share 99 % conserved synteny), chromosomal segmental duplications are more important than translocations to rearrange the genetic maps.
Molecular typing of patient isolates of S. cerevisiae Person in charge : HENNEQUIN Christophe
Natural polymorphism of trinucleotide-containing microsatellites has allowed us to define a sufficient number of loci for the molecular typing of S. cerevisiae strains. This method, developed on laboratory strains, was then validated and applied on strains of biotechnological origins as well as on isolates from hospitalized patients with local or general S. cerevisiae infections. While patients not receiving "Ultralevure" (a specific strain of S. cerevisiae called S. boulardii) show a large variety of origin of the infectious strain, those under "Ultralevure" treatment all show infection by the S. boulardii strain. This result is important to consider by those in charge of defining hospital practices.
The nucleus Persons in charge : FABRE Emmanuelle, RICHARD Guy-Franck
Nucleo-cytoplasmic RNA transport has been studied in the Unité during the last few years. After the detailed charaterization of a nucleoporin that catalyzes its own proteolytic processing prior to assembly, attention is now focussed on intranuclear retention of transcripts containing long trinucleotide repeats. An artificial system allowing the analysis of this phenomenon has been developed and is now used.
A novel high specificity, intron-encoded endonuclase: I-Spom I Persons in charge : DUJON Bernard, HARINGTON Alexis, PELLENZ Stefan
A new intron-encoded endonuclease has been characterized this year by the Unité. Like previous ones, this enzyme is encoded by a mitochondrial group I intron. In this case, the intron is inserted into the mitochondrial cytochrome oxidase gene of the yeast Schizosaccharomyces pombe. The enzyme was partially purified after heterologous expression in E. coli and its enzymatic features and recognition site were determined. Since I-Spom I is a new highly specific endonuclease, like I-Sce I but having a distinct recognition site, a patent has been deposited.
Genomes and bioinformatics Persons in charge : TALLA Emmanuel, TEKAIA Fredj
Systematic comparison of proteins predicted from completely sequenced genomes was continued. Some of these results are the basis for the definition of the "ascomycete-specific" genes previously mentioned. Quantitative analysis of the composition of proteomes was also done from which it appears a significant bias between glutamine and glutamic acid linked to the extreme life conditions of the organisms studied.
Finally, a specific effort was made to redefine hybridization arrays, taking into account the possible cross hybridization between paralogous genes in available arrays.