Comparative genomics of protoploid Saccharomycetaceae.

Our knowledge of yeast genomes remains largely dominated by the extensive studies on Saccharomyces cerevisiae and the consequences of its ancestral duplication, leaving the evolution of the entire class of hemiascomycetes only partly explored. We concentrate here on five species of Saccharomycetaceae, a large subdivision of hemiascomycetes, that we call "protoploid" because they diverged from the S. cerevisiae lineage prior to its genome duplication. We determined the complete genome sequences of three of these species: Kluyveromyces (Lachancea) thermotolerans and Saccharomyces (Lachancea) kluyveri (two members of the newly described Lachancea clade), and Zygosaccharomyces rouxii. We included in our comparisons the previously available sequences of Kluyveromyces lactis and Ashbya (Eremothecium) gossypii. Despite their broad evolutionary range and significant individual variations in each lineage, the five protoploid Saccharomycetaceae share a core repertoire of approximately 3300 protein families and a high degree of conserved synteny. Synteny blocks were used to define gene orthology and to infer ancestors. Far from representing minimal genomes without redundancy, the five protoploid yeasts contain numerous copies of paralogous genes, either dispersed or in tandem arrays, that, altogether, constitute a third of each genome. Ancient, conserved paralogs as well as novel, lineage-specific paralogs were identified.

Ploidy influences cellular responses to gross chromosomal rearrangements in Saccharomyces cerevisiae.

BACKGROUND: Gross chromosomal rearrangements (GCRs) such as aneuploidy are key factors in genome evolution as well as being common features of human cancer. Their role in tumour initiation and progression has not yet been completely elucidated and the effects of additional chromosomes in cancer cells are still unknown. Most previous studies in which Saccharomyces cerevisiae has been used as a model for cancer cells have been carried out in the haploid context. To obtain new insights on the role of ploidy, the cellular effects of GCRs were compared between the haploid and diploid contexts. RESULTS: A total number of 21 haploid and diploid S. cerevisiae strains carrying various types of GCRs (aneuploidies, nonreciprocal translocations, segmental duplications and deletions) were studied with a view to determining the effects of ploidy on the cellular responses. Differences in colony and cell morphology as well as in the growth rates were observed between mutant and parental strains. These results suggest that cells are impaired physiologically in both contexts. We also investigated the variation in genomic expression in all the mutants. We observed that gene expression was significantly altered. The data obtained here clearly show that genes involved in energy metabolism, especially in the tricarboxylic acid cycle, are up-regulated in all these mutants. However, the genes involved in the composition of the ribosome or in RNA processing are down-regulated in diploids but up-regulated in haploids. Over-expression of genes involved in the regulation of the proteasome was found to occur only in haploid mutants. CONCLUSION: The present comparisons between the cellular responses of strains carrying GCRs in different ploidy contexts bring to light two main findings. First, GCRs induce a general stress response in all studied mutants, regardless of their ploidy. Secondly, the ploidy context plays a crucial role in maintaining the stoichiometric balance of the proteins: the translation rates decrease in diploid strains, whereas the excess protein synthesized is degraded in haploids by proteasome activity.

The Ty1 LTR-retrotransposon population in Saccharomyces cerevisiae genome: dynamics and sequence variations during mobility.

Transposable element (TE) evolution in genomes has mostly been deduced from comparative genome analyses. TEs often account for a large proportion of the eukaryotic nuclear genome (up to 50%, depending on the species). Among the many existing genomic copies, only a small fraction may contribute to the mobility of a TE family. We have identified here, using a genetic screening procedure to trap Ty1 long terminal repeat-retrotransposon insertions in Saccharomyces cerevisiae, which among the populations of resident Ty1 copies are responsible for Ty1 mobility. Although the newly inserted Ty1 copies resulting from a single round of transposition were found to originate from a limited subset of Ty1 resident copies, they showed a high degree of diversity at the nucleotide level, mainly due to the reverse transcription-mediated recombination. In this process, highly expressed and strikingly nonautonomous mutant Ty1 were found to be the most frequently used resident copies, which suggests that nonautonomous elements play a key role in the dynamics of the Ty1 family.

Complete mitochondrial genome sequence of the yeast Pichia farinosa and comparative analysis of closely related species.

Yeasts of the Pichia genus have been isolated from different natural environments. Phylogenies based on multigene sequence analysis have shown that the genus is polyphyletic. Some species of this genus are member of the CTG group. In order to have a better insight into the relationship among species assigned to the yeast genera Pichia into the CTG group, we first sequenced the mitochondrial genome of the osmotolerant yeast Pichia farinosa. We then compared this genome with mitochondrial genomes of yeasts of the CTG group. The P. farinosa mitochondrial DNA is a circular-mapping genome of 32,065 bp, which contains 43 genes transcribed from both strands. It contains a complete set of tRNAs, the small and the large rRNAs, as well as 14 protein-coding genes. Yeasts of the CTG group contain the same core of mitochondrial genes. Phylogenetic analysis based on mitochondrial sequences clearly shows that the CTG group is divided into two distinct clades: the first one contains diploid Candida species, whereas the second mainly contains haploid Pichia species. Moreover, this analysis provides clear evidence that Pichia farinosa and Pichia sorbitophila, which were known to be unique species, are two distinct species.

Genome evolution in yeasts.

Identifying the mechanisms of eukaryotic genome evolution by comparative genomics is often complicated by the multiplicity of events that have taken place throughout the history of individual lineages, leaving only distorted and superimposed traces in the genome of each living organism. The hemiascomycete yeasts, with their compact genomes, similar lifestyle and distinct sexual and physiological properties, provide a unique opportunity to explore such mechanisms. We present here the complete, assembled genome sequences of four yeast species, selected to represent a broad evolutionary range within a single eukaryotic phylum, that after analysis proved to be molecularly as diverse as the entire phylum of chordates. A total of approximately 24,200 novel genes were identified, the translation products of which were classified together with Saccharomyces cerevisiae proteins into about 4,700 families, forming the basis for interspecific comparisons. Analysis of chromosome maps and genome redundancies reveal that the different yeast lineages have evolved through a marked interplay between several distinct molecular mechanisms, including tandem gene repeat formation, segmental duplication, a massive genome duplication and extensive gene loss.

La Société de Biologie de Strasbourg : 100 ans au service de la science et de la société.

Founded in 1919, the Society of Biology of Strasbourg (SBS) is a learned society whose purpose is the dissemination and promotion of scientific knowledge in biology. Subsidiary of the Society of Biology, the SBS celebrated its Centenary on Wednesday, the 16th of October 2019 on the Strasbourg University campus and at the Strasbourg City Hall. This day allowed retracing the various milestones of the SBS, through its main strengths, its difficulties and its permanent goal to meet scientific and societal challenges. The common thread of this day was the transmission of knowledge related to the past, the present, but also the future. At the start of the 21st century, the SBS must continue to reinvent itself to pursue its objective of transmitting scientific knowledge in biology and beyond. Scientific talks performed by senior scientists and former SBS thesis prizes awardees, a round table, and informal discussions reflected the history and the dynamism of the SBS association. All SBS Centennial participants have set the first milestone for the SBS Bicentennial.

TITLE: La Société de Biologie de Strasbourg : 100 ans au service de la science et de la société. ABSTRACT: Filiale de la Société de Biologie, la Société de Biologie de Strasbourg (SBS) est une société savante qui a pour objet la diffusion et la promotion du savoir scientifique en biologie et en médecine. Fondée en 1919, La SBS a célébré son Centenaire le mercredi 16 octobre 2019. Cette journée a permis de retracer les différents jalons de la SBS, à travers ses lignes de forces, ses difficultés et sa volonté permanente de mettre en exergue les défis scientifiques et sociétaux auxquels participent les recherches strasbourgeoises. Le fil rouge de cette journée a été la transmission d'un savoir en lien avec le passé, le présent, mais également le futur. En ce début du 21e siècle, la SBS se doit de continuer de se réinventer pour poursuivre son objectif de transmission des connaissances scientifiques en biologie et au-delà. L'ensemble des participants du Centenaire de la SBS a ainsi posé la première pierre du Bicentenaire de la SBS.

Influence of genetic background on the occurrence of chromosomal rearrangements in Saccharomyces cerevisiae.

BACKGROUND: Chromosomal rearrangements such as duplications and deletions are key factors in evolutionary processes because they promote genomic plasticity. Although the genetic variations in the Saccharomyces cerevisiae species have been well documented, there is little known to date about the impact of the genetic background on the appearance of rearrangements. RESULTS: Using the same genetic screening, the type of rearrangements and the mutation rates observed in the S288c S. cerevisiae strain were compared to previous findings obtained in the FL100 background. Transposon-associated rearrangements, a major chromosomal rearrangement event selected in FL100, were not detected in S288c. The mechanisms involved in the occurrence of deletions and duplications in the S288c strain were also tackled, using strains deleted for genes implicated in homologous recombination (HR) or non-homologous end joining (NHEJ). Our results indicate that an Yku80p-independent NHEJ pathway is involved in the occurrence of these rearrangements in the S288c background. CONCLUSION: The comparison of two different S. cerevisiae strains, FL100 and S288c, allowed us to conclude that intra-species genomic variations have an important impact on the occurrence of chromosomal rearrangement and that this variability can partly be explained by differences in Ty1 retrotransposon activity.

An evolutionary scenario for one of the largest yeast gene families.

The DUP gene family of Saccharomyces cerevisiae comprises 23 members that can be divided into two subfamilies--DUP240 and DUP380. The location of the DUP loci suggests that at least three mechanisms were responsible for their genomic dispersion: nonreciprocal translocation at chromosomal ends, tandem duplication and Ty-associated duplication. The data we present here suggest that these nonessential genes encode proteins that facilitate membrane trafficking processes. Dup240 proteins have three conserved domains (C1, C2 and C3) and two predicted transmembrane segments (H1 and H2). A direct repetition of the C1-H1-H2-C2 module is observed in Dup380p sequences. In this article, we propose an evolutionary model to account for the emergence of the two gene subfamilies.

The complete mitochondrial genome of the yeast Pichia sorbitophila.

Here, we report the complete nucleotide sequence of the 39 107-bp mitochondrial genome of the yeast Pichia sorbitophila. This genome is closely related to those of Candida parapsilosis and Debaryomyces hansenii, as judged from sequence similarities and synteny conservation. It encodes three subunits of cytochrome oxidase (COX1, COX2 and COX3), three subunits of ATP synthase (ATP6, ATP8 and ATP9), the seven subunits of NADH dehydrogenase (NAD1-6 and NAD4L), the apocytochrome b (COB), the large and small rRNAs and a complete set of tRNAs. Although the mitochondrial genome of P. sorbitophila contains the same core of mitochondrial genes observed in the ascomycetous yeasts, those coding for the RNAse P and the ribosomal protein VAR1p are missing. Moreover, the mtDNA of P. sorbitophila contains several introns in its genes and has the particularity of possessing an intron, which is not linked to any upstream exon.

Spontaneous duplications in diploid Saccharomyces cerevisiae cells.

The duplication of DNA sequences is a powerful determinant of genomic plasticity and is known to be one of the key factors responsible for evolution. Recent genomic sequence data demonstrate the abundance of duplicated genes in all surveyed organisms. Over the past years, experimental systems were adequately designed to explore the molecular mechanisms involved in their formation in haploid Saccharomyces cerevisiae strains. To obtain a more global and accurate view of the events leading to DNA sequence duplications, we have selected and characterized duplication occurrences in diploid S. cerevisiae cells. The molecular analysis showed that two other predominant ways lead to duplication in this context: formation of extra chimeric chromosomes and non-reciprocal translocation events. Moreover, we demonstrated that these two types of rearrangements are RAD52 independent and therefore that homologous recombination plays no part in their formation. Finally, our results show the multiplicity of mechanisms involved in duplication events and provide the first experimental evidence that these mechanisms might be ploidy dependent.

Duplication processes in Saccharomyces cerevisiae haploid strains.

Duplication is thought to be one of the main processes providing a substrate on which the effects of evolution are visible. The mechanisms underlying this chromosomal rearrangement were investigated here in the yeast Saccharomyces cerevisiae. Spontaneous revertants containing a duplication event were selected and analyzed. In addition to the single gene duplication described in a previous study, we demonstrated here that direct tandem duplicated regions ranging from 5 to 90 kb in size can also occur spontaneously. To further investigate the mechanisms in the duplication events, we examined whether homologous recombination contributes to these processes. The results obtained show that the mechanisms involved in segmental duplication are RAD52-independent, contrary to those involved in single gene duplication. Moreover, this study shows that the duplication of a given gene can occur in S.cerevisiae haploid strains via at least two ways: single gene or segmental duplication.

Expansion and contraction of the DUP240 multigene family in Saccharomyces cerevisiae populations.

The influence of duplicated sequences on chromosomal stability is poorly understood. To characterize chromosomal rearrangements involving duplicated sequences, we compared the organization of tandem repeats of the DUP240 gene family in 15 Saccharomyces cerevisiae strains of various origins. The DUP240 gene family consists of 10 members of unknown function in the reference strain S288C. Five DUP240 paralogs on chromosome I and two on chromosome VII are arranged as tandem repeats that are highly polymorphic in copy number and sequence. We characterized DNA sequences that are likely involved in homologous or nonhomologous recombination events and are responsible for intra- and interchromosomal rearrangements that cause the creation and disappearance of DUP240 paralogs. The tandemly repeated DUP240 genes seem to be privileged sites of gene birth and death.

Integrated allosteric regulation in the S. cerevisiae carbamylphosphate synthetase - aspartate transcarbamylase multifunctional protein.

BACKGROUND: The S. cerevisiae carbamylphosphate synthetase - aspartate transcarbamylase multifunctional protein catalyses the first two reactions of the pyrimidine pathway. In this organism, these two reactions are feedback inhibited by the end product UTP. In the present work, the mechanisms of these integrated inhibitions were studied. RESULTS: The results obtained show that the inhibition is competitive in the case of carbamylphosphate synthetase and non-competitive in the case of aspartate transcarbamylase. They also identify the substrate whose binding is altered by this nucleotide and the step of the carbamylphosphate synthetase reaction which is inhibited. Furthermore, the structure of the domains catalyzing these two reactions were modelled in order to localize the mutations which, specifically, alter the aspartate transcarbamylase sensitivity to the feedback inhibitor UTP. Taken together, the results make it possible to propose a model for the integrated regulation of the two activities of the complex. UTP binds to a regulatory site located in the vicinity of the carbamylphosphate synthetase catalytic subsite which catalyzes the third step of this enzyme reaction. Through a local conformational change, this binding decreases, competitively, the affinity of this site for the substrate ATP. At the same time, through a long distance signal transmission process it allosterically decreases the affinity of the aspartate transcarbamylase catalytic site for the substrate aspartate. CONCLUSION: This investigation provides informations about the mechanisms of allosteric inhibition of the two activities of the CPSase-ATCase complex. Although many allosteric monofunctional enzymes were studied, this is the first report on integrated allosteric regulation in a multifunctional protein. The positions of the point mutations which specifically abolish the sensitivity of aspartate transcarbamylase to UTP define an interface between the carbamylphosphate synthetase and aspartate transcarbamylase domains, through which the allosteric signal for the regulation of aspartate transcarbamylase must be propagated.

Spontaneous deletions and reciprocal translocations in Saccharomyces cerevisiae: influence of ploidy.

Studying spontaneous chromosomal rearrangements throws light on the rules underlying the genome reshaping events occurring in eukaryotic cells, which are part of the evolutionary process. In Saccharomyces cerevisiae, translocation and deletion processes have been frequently described in haploids, but little is known so far about these processes at the diploid level. Here we investigated the nature and the frequency of the chromosomal rearrangements occurring at this ploidy level. Using a positive selection screen based on a particular mutated allele of the URA2 gene, spontaneous diploid revertants were selected and analysed. Surprisingly, the diploid state was found to be correlated with a decrease in chromosome rearrangement frequency, along with an increase in the complexity of the rearrangements occurring in the target gene. The presence of short DNA tandem repeat sequences seems to be a key requirement for deletion and reciprocal translocation processes to occur in diploids. After discussing the differences between the haploid and diploid levels, some mechanisms possibly involved in chromosome shortening and arm exchange are suggested.

Paleogenomics or the search for remnant duplicated copies of the yeast DUP240 gene family in intergenic areas.

Duplication, resulting in gene redundancy, is well known to be a driving force of evolutionary change. Gene families are therefore useful targets for approaching genome evolution. To address the gene death process, we examined the fate of the 10-member-large S288C DUP240 family in 15 Saccharomyces cerevisiae strains. Using an original three-step method of analysis reported here, both slightly and highly degenerate DUP240 copies, called pseudo-open reading frames (ORFs) and relics, respectively, were detected in strain S288C. It was concluded that two previously annotated ORFs correspond, in fact, to pseudo-ORFs and three additional relics were identified in intergenic areas. Comparative intraspecies analysis of these degenerate DUP240 loci revealed that the two pseudo-ORFs are present in a nondegenerate state in some other strains. This suggests that within a given gene family different loci are the target of the gene erasure process, which is therefore strain dependent. Besides, the variable positions observed indicate that the relic sequence may diverge faster than the flanking regions. All in all, this study shows that short conserved protein motifs provide a useful tool for detecting and accurately mapping degenerate gene remnants. The present results also highlight the strong contribution of comparative genomics for gene relic detection because the possibility of finding short conserved protein motifs in intergenic regions (IRs) largely depends on the choice of the most closely related paralog or ortholog. By mapping new genetic components in previously annotated IRs, our study constitutes a further refinement step in the crucial stage of genome annotation and provides a strategy for retracing ancient chromosomal reshaping events and, hence, for deciphering genome history.

Difference in substrate specificity divides the yeast alkali-metal-cation/H(+) antiporters into two subfamilies.

Yeast plasma membrane Na(+)/H(+) antiporters (TC 2.A.36) share a high degree of similarity at the protein level. Expression of four antiporters (Saccharomyces cerevisiae Nha1p, Candida albicans Cnh1p, Zygosaccharomyces rouxii ZrSod2-22p and Schizosaccharomyces pombe sod2p) in a SACCH: cerevisiae mutant strain lacking both Na(+)-ATPase and Na(+)/H(+) antiporter genes made it possible to study the transport properties and contribution to cell salt tolerance of all antiporters under the same conditions. The ZrSod2-22p of the osmotolerant yeast Z. rouxii has the highest transport capacity for lithium and sodium but, like the SCHIZ: pombe sod2p, it does not recognize K(+) and Rb(+) as substrates. The SACCH: cerevisiae Nha1p and C. albicans Cnh1p have a broad substrate specificity for at least four alkali metal cations (Na(+), Li(+), K(+), Rb(+)), but their contribution to overall cell tolerance to high external concentration of toxic Na(+) and Li(+) cations seems to be lower compared to the antiporters of SCHIZ: pombe and especially Z. rouxii.

Two-dimensional protein map of an "ale"-brewing yeast strain: proteome dynamics during fermentation.

The first protein map of an ale-fermenting yeast is presented in this paper: 205 spots corresponding to 133 different proteins were identified. Comparison of the proteome of this ale strain with a lager brewing yeast and the Saccharomyces cerevisiae strain S288c confirmed that this ale strain is much closer to S288c than the lager strain at the proteome level. The dynamics of the ale-brewing yeast proteome during production-scale fermentation was analysed at the beginning and end of the first and the third usage of the yeast (called generation in the brewing industry). During the first generation, most changes were related to the switch from aerobic propagation to anaerobic fermentation. Fewer changes were observed during the third generation but certain stress-response proteins such as Hsp26p, Ssa4p and Pnc1p exhibited constitutive expression in subsequent generations. The ale brewing yeast strain appears to be quite well adapted to fermentation conditions and stresses.

Recovery of a function involving gene duplication by retroposition in Saccharomyces cerevisiae.

The duplication of DNA sequences contributes to genomic plasticity and is known to be one of the key factors responsible for evolution. The mechanisms underlying these rare events, which have been frequently mentioned by authors performing genomic analysis, have not yet been completely elucidated. These mechanisms were approached here in the yeast Saccharomyces cerevisiae, using a positive selection screen based on a particular mutated allele of the URA2 gene. Spontaneous revertants containing a duplication of the terminal part of the URA2 gene were selected and analyzed. Some important features of the duplicated regions, such as their chromosome location, size, and insertion sites, were characterized. The events selected correspond to a single inter- or intrachromosomal gene duplication process. The duplicated ATCase sequence is generally punctuated by a poly(A) tract and is always located in Ty1 sequences. In addition, the activation of a Ty1 transcription process increased the frequency of the duplication events. All in all, these data suggest that the duplication mechanism involves the reverse transcription of mRNA and the subsequent integration of the cDNA into a Ty1 area. The Ty1 elements and the retrotransposon-encoded function are key factors contributing to chromosomal reshaping. The genomic rearrangements described constitute experimental evidence for the recovery of a function involving duplication by retroposition.

The Nha1 antiporter of Saccharomyces cerevisiae mediates sodium and potassium efflux.

The NHA1 gene of Saccharomyces cerevisiae, transcribed into a 3.5 kb mRNA, encodes a protein mediating Na+ and K+ efflux through the plasma membrane that is required for alkali cation tolerance at acidic pH. Deletion of the gene in a wild-type strain resulted in higher sensitivity to both K+ and Na+ at acidic pH. Measurements of cation loss in strains carrying deleted or overexpressed alleles of NHA1 demonstrated its role in K+ and Na+ efflux. In addition, high K+ and Na+ efflux observed upon alkalinization of the cytoplasm implies a role of Nha1p in the regulation of intracellular pH. Moreover, the overexpression of ENA1 and NHA1 genes in an ena1-4 delta-nha1 delta strain showed that the Nha1 alkalication antiporter is responsible for growth on high concentrations of KCl and NaCl at acidic pH, and Ena alkali-cation ATPases are necessary at higher pH values. Both systems have a complementary action to maintain the intracellular steady-state concentration of K+ and Na+.

Cation/H+ antiporters mediate potassium and sodium fluxes in Pichia sorbitophila. Cloning of the PsNHA1 and PsNHA2 genes and expression in Saccharomyces cerevisiae.

Pichia sorbitophila grows rapidly in the presence of very high NaCl concentrations. Under these conditions, even when the K(+) concentration is low, P. sorbitophila cells can maintain low Na(+) and high K(+) contents. This remarkable capacity of P. sorbitophila fails when the external pH is not acidic. This indicates that Na(+) efflux is mediated by an electroneutral Na(+)/H(+) antiporter. We have cloned and sequenced two genes designated as PsNHA1 and PsNHA2, which probably encode two antiporters of this type. The genes present high similarity with the corresponding genes from other yeasts. The heterologous expression of PsNHA1 or PsNHA2 in a Saccharomyces cerevisiae mutant lacking the Na(+) efflux systems and sensitive to high concentrations of Na(+) and K(+) rescued the tolerance and the ability to extrude both cations. The Accession Nos of the sequenced DNA fragments are: PsNHA1, AJ496431; PsNHA2, AJ496432. (TC 2.A.36)