Bernard Dujon

Novel, large-scale structural mutations were previously discovered during the cultivation of engineered Saccharomyces cerevisiae strains in which essential tRNA synthetase genes were replaced by their orthologs from the distantly related yeast Yarrowia lipolytica. Among those were internal segmental amplifications forming giant chromosomes as well as complex segmental rearrangements associated with massive amplifications at an unselected short locus. The formation of such novel structures, whose stability is high enough to propagate over multiple generations, involved short repeated sequences dispersed in the genome (as expected), but also novel junctions between unrelated sequences likely triggered by accidental template switching within replication forks. Using the same evolutionary protocol, we now describe yet another type of major structural mutation in the yeast genome, the formation of neochromosomes, with functional centromeres and telomeres, made of extra copies of very long chromosomal segments ligated together in novel arrangements. The novel junctions occurred between short repeated sequences dispersed in the genome. They first resulted in the formation of an instable neochromosome present in a single copy in the diploid cells, followed by its replacement by a shorter, partially palindromic neochromosome present in two copies, whose stability eventually increased the chromosome number of the diploid strains harboring it.

Mitochondrial genetics started decades ago with the discovery of yeast mutants that ignored the Mendelian rules of inheritance. Today, the many known DNA sequences of this second eukaryotic genome illustrate its eccentricity in terms of informational content and functional organisation, suggesting a yet incomplete understanding of its evolution. The hereditary transmission of mitochondrial alleles relies on complex mixes of molecular and cellular mechanisms in which recombination and limited sampling, two sources of rapid genetic changes, play central roles. It is also under the influence of invasive genetic elements whose inconstant distribution in mitochondrial genomes suggests rapid turnovers in evolving populations. This susceptibility to changes contrasts with the development of specific functional interactions between the mitochondrial and nuclear genetic compartments, a trend that is prone to limit the genetic exchanges between distinct lineages. It is perhaps this opposition and the discordant inheritance between mitochondrial and nuclear genomes that best explain the maintenance of a second genome and a second independent protein synthesising machinery in eukaryotic cells.

Being invited by a prestigious journal to write the retrospective of one's life is first a great honor, and then a chore when starting to do it. These feelings did not spare me. But trying to recall my past to the best of my memory, I learned how lucky I was to have been born to a generation that witnessed so many scientific discoveries. There is little in common between the genetic courses I taught recently and those that I received more than 50 years ago. Thinking that a tiny bit of this fantastic evolution might come from my accidental encountering with yeasts is a stunning experience. I wish the same for the new generation.

Considerable progress in our understanding of yeast genomes and their evolution has been made over the last decade with the sequencing, analysis, and comparisons of numerous species, strains, or isolates of diverse origins. The role played by yeasts in natural environments as well as in artificial manufactures, combined with the importance of some species as model experimental systems sustained this effort. At the same time, their enormous evolutionary diversity (there are yeast species in every subphylum of Dikarya) sparked curiosity but necessitated further efforts to obtain appropriate reference genomes. Today, yeast genomes have been very informative about basic mechanisms of evolution, speciation, hybridization, domestication, as well as about the molecular machineries underlying them. They are also irreplaceable to investigate in detail the complex relationship between genotypes and phenotypes with both theoretical and practical implications. This review examines these questions at two distinct levels offered by the broad evolutionary range of yeasts: inside the best-studied Saccharomyces species complex, and across the entire and diversified subphylum of Saccharomycotina. While obviously revealing evolutionary histories at different scales, data converge to a remarkably coherent picture in which one can estimate the relative importance of intrinsic genome dynamics, including gene birth and loss, vs. horizontal genetic accidents in the making of populations. The facility with which novel yeast genomes can now be studied, combined with the already numerous available reference genomes, offer privileged perspectives to further examine these fundamental biological questions using yeasts both as eukaryotic models and as fungi of practical importance.

Gene amplification has been observed in different organisms in response to environmental constraints, such as limited nutrients or exposure to a variety of toxic compounds, conferring them with specific phenotypic adaptations via increased expression levels. However, the presence of multiple gene copies in natural genomes has generally not been found in the absence of specific functional selection. Here, we show that the massive amplification of a chromosomal locus (up to 880 copies per cell) occurs in the absence of any direct selection, and is associated with low-order amplifications of flanking segments in complex chromosomal alterations. These results were obtained from mutants with restored phenotypes that spontaneously appeared from genetically engineered strains of the yeast Saccharomyces cerevisiae suffering from severe fitness reduction. Grossly extended chromosomes (macrotene) were formed, with complex structural alterations but sufficient stability to propagate unchanged over successive generations. Their detailed molecular analysis, including complete genome sequencing, identification of sequence breakpoints, and comparisons between mutants, revealed novel mechanisms causing their formation, whose combined action underlies the astonishing dynamics of eukaryotic chromosomes and their consequences.

The opportunistic pathogen Candida glabrata is a member of the Saccharomycetaceae yeasts. Like its close relative Saccharomyces cerevisiae, it underwent a whole-genome duplication followed by an extensive loss of genes. Its genome contains a large number of very long tandem repeats, called megasatellites. In order to determine the whole replication program of the C. glabrata genome and its general chromosomal organization, we used deep-sequencing and chromosome conformation capture experiments. We identified 253 replication fork origins, genome wide. Centromeres, HML and HMR loci, and most histone genes are replicated early, whereas natural chromosomal breakpoints are located in late-replicating regions. In addition, 275 autonomously replicating sequences (ARS) were identified during ARS-capture experiments, and their relative fitness was determined during growth competition. Analysis of ARSs allowed us to identify a 17-bp consensus, similar to the S. cerevisiae ARS consensus sequence but slightly more constrained. Megasatellites are not in close proximity to replication origins or termini. Using chromosome conformation capture, we also show that early origins tend to cluster whereas non-subtelomeric megasatellites do not cluster in the yeast nucleus. Despite a shorter cell cycle, the C. glabrata replication program shares unexpected striking similarities to S. cerevisiae, in spite of their large evolutionary distance and the presence of highly repetitive large tandem repeats in C. glabrata. No correlation could be found between the replication program and megasatellites, suggesting that their formation and propagation might not be directly caused by replication fork initiation or termination.

The genomes of many yeast species or strain isolates have now been sequenced in an accelerating momentum that quickly relegates initial data to history, albeit less than two decades-old. Today, novel yeast genomes are entirely sequenced for a variety of reasons, often only to identify a few expected genes of specific interest, thus providing a wealth of information of unequal quality and completion about the origin and evolution of this heterogeneous collection of unicellular modern fungi. But how many scientists fully appreciate the important conceptual and technological roles played by yeasts in the extraordinary development of today's genomics ? Novel notions of general significance emerged from the very first eukaryote sequenced, Saccharomyces cerevisiae , and were successively refined and extended over time. Tools of general applications were originally developed with this yeast. And surprises sprung from results. Here, I have tried to recollect the gradual building of knowledge as yeast genomics developed, and then briefly summarize our present views about the basic nature of yeast genomes, based on the most recent data.

Copy number variation of chromosomal segments is now recognized as a major source of genetic polymorphism within natural populations of eukaryotes, as well as a possible cause of genetic diseases in humans, including cancer, but its molecular bases remain incompletely understood. In the baker’s yeast Saccharomyces cerevisiae, a variety of low-order amplifications (segmental duplications) were observed after adaptation to limiting environmental conditions or recovery from gene dosage imbalance, and interpreted in terms of replication-based mechanisms associated or not with homologous recombination. Here we show the emergence of novel high-order amplification structures, with corresponding overexpression of embedded genes, during evolution under favourable growth conditions of severely unfit yeast cells bearing genetically disabled genomes. Such events form massively extended chromosomes, which we propose to call macrotene, whose characteristics suggest the products of intrachromosomal rolling-circle type of replication structures, probably initiated by increased accidental template switches under important cellular stress conditions.

The industrially important yeast Blastobotrys (Arxula) adeninivorans is an asexual hemiascomycete phylogenetically very distant from Saccharomyces cerevisiae. Its unusual metabolic flexibility allows it to use a wide range of carbon and nitrogen sources, while being thermotolerant, xerotolerant and osmotolerant.