Mitochondrial genetics revisited.

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.

My route to the intimacy of genomes.

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.

Yeast evolutionary genomics.

Over the past few years, genome sequences have become available from an increasing range of yeast species, which has led to notable advances in our understanding of evolutionary mechanisms in eukaryotes. Yeasts offer us a unique opportunity to examine how molecular and reproductive mechanisms combine to affect genome architectures and drive evolutionary changes over a broad range of species. This Review summarizes recent progress in understanding the molecular mechanisms--such as gene duplication, mutation and acquisition of novel genetic material--that underlie yeast evolutionary genomics. I also discuss how results from yeasts can be extended to other eukaryotes.

Genome-wide replication landscape of Candida glabrata.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Basic principles of yeast genomics, a personal recollection.

The genomes of many yeast species or strain isolates have now been sequenced with an accelerating momentum that quickly relegates initial data to history, albeit that they are 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 data, heterogenous in quality and completion but informative about the origin and evolution of this heterogeneous collection of unicellular modern fungi. However, 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 with general applications were originally developed with this yeast; and surprises emerged from the results. Here, I have tried to recollect the gradual building up 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.

Purification of G1 daughter cells from different Saccharomycetes species through an optimized centrifugal elutriation procedure.

Centrifugal elutriation discriminates cells according to their sedimentation coefficients, generating homogeneous samples well suited for genomic comparative approaches. It can, for instance, isolate G1 daughter cells from a Saccharomyces cerevisiae unsynchronized population, alleviating ageing and cell-cycle biases when conducting genome-wide/single-cell studies. The present report describes a straightforward and robust procedure to determine whether a cell population of virtually any yeast species can be efficiently elutriated, while offering solutions to optimize success. This approach was used to characterize elutriation parameters and S-phase progression of four yeast species (S. cerevisiae, Candida glabrata, Lachancea kluyveri and Pichia sorbitophila) and could theoretically be applied to any culture of single, individual cells.

Detection and characterization of megasatellites in orthologous and nonorthologous genes of 21 fungal genomes.

Megasatellites are large DNA tandem repeats, originally described in Candida glabrata, in protein-coding genes. Most of the genes in which megasatellites are found are of unknown function. In this work, we extended the search for megasatellites to 20 additional completely sequenced fungal genomes and extracted 216 megasatellites in 203 out of 142,121 genes, corresponding to the most exhaustive description of such genetic elements available today. We show that half of the megasatellites detected encode threonine-rich peptides predicted to be intrinsically disordered, suggesting that they may interact with several partners or serve as flexible linkers. Megasatellite motifs were clustered into several families. Their distribution in fungal genes shows that different motifs are found in orthologous genes and similar motifs are found in unrelated genes, suggesting that megasatellite formation or spreading does not necessarily track the evolution of their host genes. Altogether, these results suggest that megasatellites are created and lost during evolution of fungal genomes, probably sharing similar functions, although their primary sequences are not necessarily conserved.

Genome-wide analysis of heteroduplex DNA in mismatch repair-deficient yeast cells reveals novel properties of meiotic recombination pathways.

Meiotic DNA double-strand breaks (DSBs) initiate crossover (CO) recombination, which is necessary for accurate chromosome segregation, but DSBs may also repair as non-crossovers (NCOs). Multiple recombination pathways with specific intermediates are expected to lead to COs and NCOs. We revisited the mechanisms of meiotic DSB repair and the regulation of CO formation, by conducting a genome-wide analysis of strand-transfer intermediates associated with recombination events. We performed this analysis in a SK1 × S288C Saccharomyces cerevisiae hybrid lacking the mismatch repair (MMR) protein Msh2, to allow efficient detection of heteroduplex DNAs (hDNAs). First, we observed that the anti-recombinogenic activity of MMR is responsible for a 20% drop in CO number, suggesting that in MMR-proficient cells some DSBs are repaired using the sister chromatid as a template when polymorphisms are present. Second, we observed that a large fraction of NCOs were associated with trans-hDNA tracts constrained to a single chromatid. This unexpected finding is compatible with dissolution of double Holliday junctions (dHJs) during repair, and it suggests the existence of a novel control point for CO formation at the level of the dHJ intermediate, in addition to the previously described control point before the dHJ formation step. Finally, we observed that COs are associated with complex hDNA patterns, confirming that the canonical double-strand break repair model is not sufficient to explain the formation of most COs. We propose that multiple factors contribute to the complexity of recombination intermediates. These factors include repair of nicks and double-stranded gaps, template switches between non-sister and sister chromatids, and HJ branch migration. Finally, the good correlation between the strand transfer properties observed in the absence of and in the presence of Msh2 suggests that the intermediates detected in the absence of Msh2 reflect normal intermediates.

Comparative genomics of emerging pathogens in the Candida glabrata clade.

BACKGROUND: Candida glabrata follows C. albicans as the second or third most prevalent cause of candidemia worldwide. These two pathogenic yeasts are distantly related, C. glabrata being part of the Nakaseomyces, a group more closely related to Saccharomyces cerevisiae. Although C. glabrata was thought to be the only pathogenic Nakaseomyces, two new pathogens have recently been described within this group: C. nivariensis and C. bracarensis. To gain insight into the genomic changes underlying the emergence of virulence, we sequenced the genomes of these two, and three other non-pathogenic Nakaseomyces, and compared them to other sequenced yeasts. RESULTS: Our results indicate that the two new pathogens are more closely related to the non-pathogenic N. delphensis than to C. glabrata. We uncover duplications and accelerated evolution that specifically affected genes in the lineage preceding the group containing N. delphensis and the three pathogens, which may provide clues to the higher propensity of this group to infect humans. Finally, the number of Epa-like adhesins is specifically enriched in the pathogens, particularly in C. glabrata. CONCLUSIONS: Remarkably, some features thought to be the result of adaptation of C. glabrata to a pathogenic lifestyle, are present throughout the Nakaseomyces, indicating these are rather ancient adaptations to other environments. Phylogeny suggests that human pathogenesis evolved several times, independently within the clade. The expansion of the EPA gene family in pathogens establishes an evolutionary link between adhesion and virulence phenotypes. Our analyses thus shed light onto the relationships between virulence and the recent genomic changes that occurred within the Nakaseomyces. SEQUENCE ACCESSION NUMBERS: Nakaseomyces delphensis: CAPT01000001 to CAPT01000179Candida bracarensis: CAPU01000001 to CAPU01000251Candida nivariensis: CAPV01000001 to CAPV01000123Candida castellii: CAPW01000001 to CAPW01000101Nakaseomyces bacillisporus: CAPX01000001 to CAPX01000186.

Chromosome arm length and nuclear constraints determine the dynamic relationship of yeast subtelomeres.

Physical interactions between distinct chromosomal genomic loci are important for genomic functions including recombination and gene expression, but the mechanisms by which these interactions occur remain obscure. Using telomeric association as a model system, we analyzed here the in vivo organization of chromosome ends of haploid yeast cells during interphase. We separately labeled most of the 32 subtelomeres and analyzed their positions both in nuclear space and relative to three representative reference subtelomeres by high-throughput 3D microscopy and image processing. We show that subtelomeres are positioned nonrandomly at the nuclear periphery, depending on the genomic size of their chromosome arm, centromere attachment to the microtubule organizing center (spindle pole body, SPB), and the volume of the nucleolus. The distance of subtelomeres to the SPB increases consistently with chromosome arm length up to approximately 300 kb; for larger arms the influence of chromosome arm length is weaker, but the effect of the nucleolar volume is stronger. Distances between pairs of subtelomeres also exhibit arm-length dependence and suggest, together with dynamic tracking experiments, that potential associations between subtelomeres are unexpectedly infrequent and transient. Our results suggest that interactions between subtelomeres are nonspecific and instead governed by physical constraints, including chromosome structure, attachment to the SPB, and nuclear crowding.

Unusual composition of a yeast chromosome arm is associated with its delayed replication.

The 11.3-Mb genome of the yeast Lachancea (Saccharomyces) kluyveri displays an intriguing compositional heterogeneity: a region of approximately 1 Mb, covering almost the whole left arm of chromosome C (C-left), has an average GC content of 52.9%, which is significantly higher than the 40.4% global GC content of the rest of the genome. This region contains the MAT locus, which remains normal in composition. The excess of GC base pairs affects both coding and noncoding sequences, and thus is not due to selective pressure acting on protein sequences. It leads to a strong codon usage bias and alters the amino acid composition of the 457 proteins encoded on C-left that do not show obvious bias for functional categories, or the presence of paralogs or orthologs of essential genes of Saccharomyces cerevisiae. They share significant synteny conservation with other species of the Saccharomycetaceae, and phylogenetic analysis indicates that C-left originates from a Lachancea species. In contrast, there is a complete absence of transposable elements in C-left, whereas 18 elements per megabase are distributed across the rest of the genome. Comparative hybridization of synchronized cells using high-density genome arrays reveals that C-left is replicated later during S phase than the rest of the genome. Two possible primary causes of this major compositional heterogeneity are discussed: an ancient hybridization of two related species with very distinct GC composition, or an intrinsic mechanism, possibly associated with the loss of the silent cassettes from C-left that progressively increased the GC content and generated the delayed replication of this chromosomal arm.

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.

Dynamic evolution of megasatellites in yeasts.

Megasatellites are a new family of long tandem repeats, recently discovered in the yeast Candida glabrata. Compared to shorter tandem repeats, such as minisatellites, megasatellite motifs range in size from 135 to more than 300 bp, and allow calculation of evolutionary distances between individual motifs. Using divergence based on nucleotide substitutions among similar motifs, we determined the smallest distance between two motifs, allowing their subsequent clustering. Motifs belonging to the same cluster are recurrently found in different megasatellites located on different chromosomes, showing transfer of genetic information between megasatellites. In comparison, evolution of the few similar tandem repeats in Saccharomyces cerevisiae FLO genes mainly involves subtelomeric homologous recombination. We estimated selective constraints acting on megasatellite motifs and their host genes, and found that motifs are under strong purifying selection. Surprisingly, motifs inserted within pseudogenes are also under purifying selection, whereas the pseudogenes themselves evolve neutrally. We propose that megasatellite motifs propagate by a combination of three different molecular mechanisms: (i) gene duplication, (ii) ectopic homologous recombination and (iii) transfer of motifs from one megasatellite to another one. These mechanisms actively cooperate to create new megasatellites, that may play an important role in the adaptation of Candida glabrata to its human host.

Functional variability in adhesion and flocculation of yeast megasatellite genes.

Megasatellites are large tandem repeats found in all fungal genomes but especially abundant in the opportunistic pathogen Candida glabrata. They are encoded in genes involved in cell-cell interactions, either between yeasts or between yeast and human cells. In the present work, we have been using an iterative genetic system to delete several Candida glabrata megasatellite-containing genes and found that 2 of them were positively involved in adhesion to epithelial cells, whereas 3 genes negatively controlled adhesion. Two of the latter, CAGL0B05061g or CAGL0A04851g, were also negative regulators of yeast-to-yeast adhesion, making them central players in controlling Candida glabrata adherence properties. Using a series of synthetic Saccharomyces cerevisiae strains in which the FLO1 megasatellite was replaced by other tandem repeats of similar length but different sequences, we showed that the capacity of a strain to flocculate in liquid culture was unrelated to its capacity to adhere to epithelial cells or to invade agar. Finally, to understand how megasatellites were initially created and subsequently expanded, an experimental evolution system was set up, in which modified yeast strains containing different megasatellite seeds were grown in bioreactors for more than 200 generations and selected for their ability to sediment at the bottom of the culture tube. Several flocculation-positive mutants were isolated. Functionally relevant mutations included general transcription factors as well as a 230-kbp segmental duplication.

Telomere tethering at the nuclear periphery is essential for efficient DNA double strand break repair in subtelomeric region.

In the yeast Saccharomyces cerevisiae that lacks lamins, the nuclear pore complex (NPC) has been proposed to serve a role in chromatin organization. Here, using fluorescence microscopy in living cells, we show that nuclear pore proteins of the Nup84 core complex, Nup84p, Nup145Cp, Nup120p, and Nup133p, serve to anchor telomere XI-L at the nuclear periphery. The integrity of this complex is shown to be required for repression of a URA3 gene inserted in the subtelomeric region of this chromosome end. Furthermore, altering the integrity of this complex decreases the efficiency of repair of a DNA double-strand break (DSB) only when it is generated in the subtelomeric region, even though the repair machinery is functional. These effects are specific to the Nup84 complex. Our observations thus confirm and extend the role played by the NPC, through the Nup84 complex, in the functional organization of chromatin. They also indicate that anchoring of telomeres is essential for efficient repair of DSBs occurring therein and is important for preserving genome integrity.

Megasatellites: a new class of large tandem repeats discovered in the pathogenic yeast Candida glabrata.

Megasatellites are DNA tandem arrays made of large motifs; they were discovered in the yeast Candida glabrata. They are widespread in this species (40 copies) but are not found in any other hemiascomycete so far, raising the intriguing question of their origin. They are found mainly in genes encoding cell wall products, suggesting that megasatellites were selected for a function linked to cell-cell adhesion or to pathogenicity. Their putative role in promoting genome rearrangements by interfering with DNA replication will also be discussed.

Population polymorphism of nuclear mitochondrial DNA insertions reveals widespread diploidy associated with loss of heterozygosity in Debaryomyces hansenii.

Debaryomyces hansenii, a yeast that participates in the elaboration of foodstuff, displays important genetic diversity. Our recent phylogenetic classification of this species led to the subdivision of the species into three distinct clades. D. hansenii harbors the highest number of nuclear mitochondrial DNA (NUMT) insertions known so far for hemiascomycetous yeasts. Here we assessed the intraspecific variability of the NUMTs in this species by testing their presence/absence first in 28 strains, with 21 loci previously detected in the completely sequenced strain CBS 767(T), and second in a larger panel of 77 strains, with 8 most informative loci. We were able for the first time to structure populations in D. hansenii, although we observed little NUMT insertion variability within the clades. We determined the chronology of the NUMT insertions, which turned out to correlate with the previously defined taxonomy and provided additional evidence that colonization of nuclear genomes by mitochondrial DNA is a dynamic process in yeast. In combination with flow cytometry experiments, the NUMT analysis revealed the existence of both haploid and diploid strains, the latter being heterozygous and resulting from at least four crosses among strains from the various clades. As in the diploid pathogen Candida albicans, to which D. hansenii is phylogenetically related, we observed a differential loss of heterozygosity in the diploid strains, which can explain some of the large genetic diversity found in D. hansenii over the years.

Segmental duplications arise from Pol32-dependent repair of broken forks through two alternative replication-based mechanisms.

The propensity of segmental duplications (SDs) to promote genomic instability is of increasing interest since their involvement in numerous human genomic diseases and cancers was revealed. However, the mechanism(s) responsible for their appearance remain mostly speculative. Here, we show that in budding yeast, replication accidents, which are most likely transformed into broken forks, play a causal role in the formation of SDs. The Pol32 subunit of the major replicative polymerase Poldelta is required for all SD formation, demonstrating that SDs result from untimely DNA synthesis rather than from unequal crossing-over. Although Pol32 is known to be required for classical (Rad52-dependant) break-induced replication, only half of the SDs can be attributed to this mechanism. The remaining SDs are generated through a Rad52-independent mechanism of template switching between microsatellites or microhomologous sequences. This new mechanism, named microhomology/microsatellite-induced replication (MMIR), differs from all known DNA double-strand break repair pathways, as MMIR-mediated duplications still occur in the combined absence of homologous recombination, microhomology-mediated, and nonhomologous end joining machineries. The interplay between these two replication-based pathways explains important features of higher eukaryotic genomes, such as the strong, but not strict, association between SDs and transposable elements, as well as the frequent formation of oncogenic fusion genes generating protein innovations at SD junctions.

On the origin of the genetic code: a 27-codon hypothetical precursor of an intricate 64-codon intermediate shaped the modern code.

The modern genetic code reveals numerous traces of specific relationships between the early codons which, together with its internal asymmetries, suggest a sequential appearance of the nucleobases in primitive RNA molecules. Keeping the hypothesis of triplet pairings between primitive RNA molecules at the origin of the code, this work systematically examines complete codon-anticodon interaction matrices assuming distinct pairing options at each position of the triplet duplexes. Application of these principles suggests that a 27-codon precursor having a reasonable coding capacity for short peptide synthesis could have started with primitive RNA molecules able to form two distinct pairs with different free energies between a single purine and two pyrimidines (such as G with C and U). Conservation of the same pairing options at positions 1 and 2 of codons at the arrival of a second purine with distinct pairing preferences (such as A) generated a 64-codon intermediate code made of interrelated pairs or groups of codons (designated here as intricacy). The numerous traces of this hypothetical scheme that are visible in the standard and variant forms of the modern code demonstrate without ambiguity that the ancestral codon-anticodon duplexes required high energetic pairings at their central position (Watson-Crick) but tolerated less energetic pairings at the first codon position (G • U type). Combined with the sequential appearance of the nucleobases, the predicted codon intricacy allows a stepwise reconstruction of the evolution of the coding repertoire, by simple a posteriori comparison to the modern code. This reconstruction reveals a remarkable internal coherence in terms of amino acids and tRNA synthetases recruitment. The code started with a group of amino acids (Ala, Gly, Pro, Ser and Thr) that are now all activated by class II tRNA synthetases before reaching an intermediate period during which up to 14 distinct amino acids could be encoded by a full set of intricated codons. The perfect coincidence between the last 6 amino acids predicted in this reconstruction and the speculated action of the arrival of free atmospheric oxygen on proteins is spectacular, and suggests that the code has only reached its present form after the great oxidation event.

Le code génétique moderne révèle de nombreuses traces de relations spécifiques entre les premiers codons qui, avec ses asymétries internes, suggèrent une apparition séquentielle des nucléobases dans les molécules d'ARN primitives. Gardant l'hypothèse d'appariements de triplets entre molécules d'ARN primitives à l'origine du code, ce travail examine systématiquement des matrices d'interaction codon­anticodon complètes en supposant des options d'appariement distinctes à chaque position des duplex des triplets. L'application de ces principes suggère qu'un précurseur de 27 codons ayant une capacité de codage raisonnable pour la synthèse de peptides courts pourrait avoir commencé avec des molécules d'ARN primitives capables de former deux paires distinctes avec des énergies libres différentes entre une seule purine et deux pyrimidines (comme G avec C et U). La conservation des mêmes options d'appariement aux positions 1 et 2 des codons à l'arrivée d'une seconde purine avec des préférences d'appariement distinctes (comme A) a généré un code intermédiaire de 64 codons constitué de paires ou de groupes de codons interconnectés (appelé ici intriqués). Les nombreuses traces de ce schéma hypothétique qui sont visibles dans les formes standard et variante du code moderne démontrent sans ambiguïté que les duplex ancestraux codon­anticodon exigeaient des appariements très énergétiques à leur position centrale (Watson­Crick) mais toléraient des appariements moins énergétiques à la première position des codons (type G • U). Combinée à l'apparition séquentielle des bases azotées (nucléobases), l'intrication prédite des codons permet une reconstruction progressive de l'évolution du répertoire de codage, par simple comparaison a posteriori avec le code moderne. Cette reconstruction révèle une cohérence interne remarquable en termes de recrutement des acides aminés et des ARNt synthétases. Le code a commencé avec un groupe d'acides aminés (Ala, Gly, Pro, Ser et Thr) qui sont maintenant tous activés par des ARNt synthétases de classe II avant d'atteindre une période intermédiaire pendant laquelle jusqu'à 14 acides aminés distincts pouvaient être codés par un ensemble complet de codons intriqués. La coïncidence parfaite entre les 6 derniers acides aminés prédits dans cette reconstruction et l'action spéculée de l'arrivée de l'oxygène atmosphérique libre sur les protéines est spectaculaire, et suggère que le code n'a atteint sa forme actuelle qu'après le grand événement d'oxydation.

The Formation of Neochromosomes during Experimental Evolution in the Yeast Saccharomyces cerevisiae.

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.