Reciprocal translocations in Saccharomyces cerevisiae formed by nonhomologous end joining

Reciprocal translocations are common in cancer cells, but their creation is poorly understood. We have developed an assay system in Saccharomyces cerevisiae to study reciprocal translocation formation in the absence of homology. We induce two specific double-strand breaks (DSBs) simultaneously on separate chromosomes with HO endonuclease and analyze the subsequent chromosomal rearrangements among surviving cells. Under these conditions, reciprocal translocations via nonhomologous end joining (NHEJ) occur at frequencies of approximately 2-7 x 10(-5)/cell exposed to the DSBs. Yku80p is a component of the cell's NHEJ machinery. In its absence, reciprocal translocations still occur, but the junctions are associated with deletions and extended overlapping sequences. After induction of a single DSB, translocations and inversions are recovered in wild-type and rad52 strains. In these rearrangements, a nonrandom assortment of sites have fused to the DSB, and their junctions show typical signs of NHEJ. The sites tend to be between open reading frames or within Ty1 LTRs. In some cases the translocation partner is formed by a break at a cryptic HO recognition site. Our results demonstrate that NHEJ-mediated reciprocal translocations can form in S. cerevisiae as a consequence of DSB repair.     

Additions, Losses, and Rearrangements on the Evolutionary Route from a Reconstructed Ancestor to the Modern Saccharomyces cerevisiae Genome

Comparative genomics can be used to infer the history of genomic rearrangements that occurred during the evolution of a species. We used the principle of parsimony, applied to aligned synteny blocks from 11 yeast species, to infer the gene content and gene order that existed in the genome of an extinct ancestral yeast about 100 Mya, immediately before it underwent whole-genome duplication (WGD). The reconstructed ancestral genome contains 4,703 ordered loci on eight chromosomes. The reconstruction is complete except for the subtelomeric regions. We then inferred the series of rearrangement steps that led from this ancestor to the current Saccharomyces cerevisiae genome; relative to the ancestral genome we observe 73 inversions, 66 reciprocal translocations, and five translocations involving telomeres. Some fragile chromosomal sites were reused as evolutionary breakpoints multiple times. We identified 124 genes that have been gained by S. cerevisiae in the time since the WGD, including one that is derived from a hAT family transposon, and 88 ancestral loci at which S. cerevisiae did not retain either of the gene copies that were formed by WGD. Sites of gene gain and evolutionary breakpoints both tend to be associated with tRNA genes and, to a lesser extent, with origins of replication. Many of the gained genes in S. cerevisiae have functions associated with ethanol production, growth in hypoxic environments, or the uptake of alternative nutrient sources.     

Convergent adaptation of Saccharomyces uvarum to sulfite,an antimicrobial preservative widely used in human-driven fermentations

Different species can find convergent solutions to adapt their genome to the same evolutionary constraints, although functional convergence promoted by chromosomal rearrangements in different species has not previously been found. In this work, we discovered that two domesticated yeast species, Saccharomyces cerevisiae, and Saccharomyces uvarum, acquired chromosomal rearrangements to convergently adapt to the presence of sulfite in fermentation environments. We found two new heterologous chromosomal translocations in fermentative strains of S. uvarum at the SSU1 locus, involved in sulfite resistance, an antimicrobial additive widely used in food production. These are convergent events that share similarities with other SSU1 locus chromosomal translocations previously described in domesticated S. cerevisiae strains. In S. uvarum, the newly described VII^XVI and XI^XVI chromosomal translocations generate an overexpression of the SSU1 gene and confer increased sulfite resistance. This study highlights the relevance of chromosomal rearrangements to promote the adaptation of yeast to anthropic environments.     

Pure and mixed genetic lines of Saccharomyces bayanus and Saccharomyces pastorianus and their contribution to the lager brewing strain genome

The yeast species Saccharomyces bayanus and Saccharomyces pastorianus are of industrial importance since they are involved in the production process of common beverages such as wine and lager beer; however, they contain strains whose variability has been neither fully investigated nor exploited in genetic improvement programs. We evaluated this variability by using PCR-restriction fragment length polymorphism analysis of 48 genes and partial sequences of 16. Within these two species, we identified "pure" strains containing a single type of genome and "hybrid" strains that contained portions of the genomes from the "pure" lines, as well as alleles termed "Lager" that represent a third genome commonly associated with lager brewing strains. The two pure lines represent S. uvarum and S. bayanus, the latter a novel group of strains that may be of use in strain improvement programs. Hybrid lines identified include (i) S. cerevisiae/S. bayanus/Lager, (ii) S. bayanus/S. uvarum/Lager, and (iii) S. cerevisiae/S. bayanus/S. uvarum/Lager. The genome of the lager strains may have resulted from chromosomal loss, replacement, or rearrangement within the hybrid genetic lines. This study identifies brewing strains that could be used as novel genetic sources in strain improvement programs and provides data that can be used to generate a model of how naturally occurring and industrial hybrid strains may have evolved.     

After the duplication: gene loss and adaptation in Saccharomyces genomes

The ancient duplication of the Saccharomyces cerevisiae genome and subsequent massive loss of duplicated genes is apparent when it is compared to the genomes of related species that diverged before the duplication event. To learn more about the evolutionary effects of the duplication event, we compared the S. cerevisiae genome to other Saccharomyces genomes. We demonstrate that the whole genome duplication occurred before S. castellii diverged from S. cerevisiae. In addition to more accurately dating the duplication event, this finding allowed us to study the effects of the duplication on two separate lineages. Analyses of the duplication regions of the genomes indicate that most of the duplicated genes (approximately 85%) were lost before the speciation. Only a small amount of paralogous gene loss (4-6%) occurred after speciation. On the other hand, S. castellii appears to have lost several hundred genes that were not retained as duplicated paralogs. These losses could be related to genomic rearrangements that reduced the number of chromosomes from 16 to 9. In addition to S. castellii, other Saccharomyces sensu lato species likely diverged from S. cerevisiae after the duplication. A thorough analysis of these species will likely reveal other important outcomes of the whole genome duplication.     

The genome sequence of the wine yeast VIN7 reveals an allotriploid hybrid genome with Saccharomyces cerevisiae and Saccharomyces kudriavzevii origins

The vast majority of wine fermentations are performed principally by Saccharomyces cerevisiae. However, there are a growing number of instances in which other species of Saccharomyces play a predominant role. Interestingly, the presence of these other yeast species generally occurs via the formation of interspecific hybrids that contain genomic contributions from both S.?cerevisiae and non-S.?cerevisiae species. However, despite the large number of wine strains that are characterized at the genomic level, there remains limited information regarding the detailed genomic structure of hybrids used in winemaking. To address this, we describe the genome sequence of the thiol-releasing commercial wine yeast hybrid VIN7. VIN7 is shown to be an almost complete allotriploid interspecific hybrid that is comprised of a heterozygous diploid complement of S.?cerevisiae chromosomes and a haploid Saccharomyces kudriavzevii genomic contribution. Both parental strains appear to be of European origin, with the S.?cerevisiae parent being closely related to, but distinct from, the commercial wine yeasts QA23 and EC1118. In addition, several instances of chromosomal rearrangement between S.?cerevisiae and S.?kudriavzevii sequences were observed that may mark the early stages of hybrid genome consolidation.     

Sequence diversity, reproductive isolation and species concepts in Saccharomyces

Using the biological species definition, yeasts of the genus Saccharomyces sensu stricto comprise six species and one natural hybrid. Previous work has shown that reproductive isolation between the species is due primarily to sequence divergence acted upon by the mismatch repair system and not due to major gene differences or chromosomal rearrangements. Sequence divergence through mismatch repair has also been shown to cause partial reproductive isolation among populations within a species. We have surveyed sequence variation in populations of Saccharomyces sensu stricto yeasts and measured meiotic sterility in hybrids. This allows us to determine the divergence necessary to produce the reproductive isolation seen among species. Rather than a sharp transition from fertility to sterility, which may have been expected, we find a smooth monotonic relationship between diversity and reproductive isolation, even as far as the well-accepted designations of S. paradoxus and S. cerevisiae as distinct species. Furthermore, we show that one species of Saccharomyces--S. cariocanus--differs from a population of S. paradoxus by four translocations, but not by sequence. There is molecular evidence of recent introgression from S. cerevisiae into the European population of S. paradoxus, supporting the idea that in nature the boundary between these species is fuzzy.     

Non-reciprocal chromosomal bridge-induced translocation (BIT) by targeted DNA integration in yeast

Several experimental in vivo systems exist that generate reciprocal translocations between engineered chromosomal loci of yeast or Drosophila, but not without previous genome modifications. Here we report the successful induction of chromosome translocations in unmodified yeast cells via targeted DNA integration of the KAN^R selectable marker flanked by sequences homologous to two chromosomal loci randomly chosen on the genome. Using this bridge-induced translocation system, 2% of the integrants showed targeted translocations between chromosomes V-VIII and VIII-XV in two wild-type Saccharomyces cerevisiae strains. All the translocation events studied were found to be non-reciprocal and the fate of their chromosomal fragments that were not included in the translocated chromosome was followed. The recovery of discrete-sized fragments suggested multiple pathway repair of their free DNA ends. We propose that centromere-distal chromosome fragments may be processed by a break-induced replication mechanism ensuing in partial trisomy. The experimental feasibility of inducing chromosomal translocations between any two desired genetic loci in a eukaryotic model system will be instrumental in elucidating the molecular mechanism underlying genome rearrangements generated by DNA integration and the gross chromosomal rearrangements characteristic of many types of cancer.Electronic Supplementary Material Supplementary material is available for this article at     

Conservation of ARS elements and chromosomal DNA replication origins on chromosomes III of Saccharomyces cerevisiae and S. carlsbergensis.

DNA replication origins, specified by ARS elements in Saccharomyces cerevisiae, play an essential role in the stable transmission of chromosomes. Little is known about the evolution of ARS elements. We have isolated and characterized ARS elements from a chromosome III recovered from an alloploid Carlsberg brewing yeast that has diverged from its S. cerevisiae homeologue. The positions of seven ARS elements identified in this S. carlsbergensis chromosome are conserved: they are located in intergenic regions flanked by open reading frames homologous to those that flank seven ARS elements of the S. cerevisiae chromosome. The S. carlsbergensis ARS elements were active both in S. cerevisiae and S. monacensis, which has been proposed to be the source of the diverged genome present in brewing yeast. Moreover, their function as chromosomal replication origins correlated strongly with the activity of S. cerevisiae ARS elements, demonstrating the conservation of ARS activity and replication origin function in these two species.     

Genetic aspects of targeted insertion mutagenesis in yeasts

Targeted insertion mutagenesis is a main molecular tool of yeast science initially applied in Saccharomyces cerevisiae. The method was extended to fission yeast Schizosaccharomyces pombe and to "non-conventional" yeast species, which show specific properties of special interest to both basic and applied research. Consequently, the behaviour of such non-Saccharomyces yeasts is reviewed against the background of the knowledge of targeted insertion mutagenesis in S. cerevisiae. Data of homologous integration efficiencies obtained with circular, ends-in or ends-out vectors in several yeasts are compared. We follow details of targeted insertion mutagenesis in order to recognize possible rate-limiting steps. The route of the vector to the target and possible mechanisms of its integration into chromosomal genes are considered. Specific features of some yeast species are discussed. In addition, similar approaches based on homologous recombination that have been established for the mitochondrial genome of S. cerevisiae are described.     

Intergenomic translocations in unisexual salamanders of the genus Ambystoma (Amphibia, Caudata)

Intergenomic interactions that include homoeologous recombinations and intergenomic translocations are commonly observed in plant allopolyploids. Homoeologous recombinations have recently been documented in unisexual salamanders in the genus Ambystoma and revealed exchanged chromosomal segments between A. laterale and A.jeffersonianum genomes in individual unisexuals. We discovered intergenomic translocations in two widespread unisexual triploids A.laterale--2 jeffersonianum (or LJJ) and its tetraploid derivative A.laterale--3 jeffersonianum (or LJJJ) by genomic in situ hybridization (GISH). Two different types of intergenomic translocations were observed in two unisexual populations and one contained novel chromosomes generated by an intergenomic reciprocal translocation. We also observed chromosome deletions in several individuals and these chromosome fragmentations were all derived from the A. jeffersonianum genome. These observed intergenomic reciprocal translocations are believed to be caused by non-homologous pairing during meiosis followed by breakage-rejoining events. Genomes of unisexual Ambystoma undergo complicated structural changes that include various intergenomic exchanges that offer unisexuals genetic and phenotypic complexity to escape their evolutionary demise. Unisexual Ambystoma have persisted as natural nuclear genomic hybrids for about four million years. These unisexuals provide a vertebrate model system to examine the interaction of distinct genomes and to evaluate the corresponding genetic, developmental and evolutionary implications of intergenomic exchanges. Intergenomic translocations and homoeologous recombinations appear to be frequent chromosome reconstruction events among unisexual Ambystoma.     

Incipient mitochondrial evolution in yeasts. I. The physical map and gene order of Saccharomyces douglasii mitochondrial DNA discloses a translocation of a segment of 15,000 base-pairs and the presence of new introns in comparison with Saccharomyces cerevisiae

We have determined the physical and genetic map of the 73,000 base-pair mitochondrial genome of a novel yeast species Saccharomyces douglasii. Most of the protein and RNA-coding genes known to be present in the mitochondrial DNA of Saccharomyces cerevisiae have been identified and located on the S. douglasii mitochondrial genome. The nuclear genomes of the two species are thought to have diverged some 50 to 80 million years ago and their nucleo-mitochondrial hybrids are viable but respiratorily deficient. The mitochondrial genome of S. douglasii displays many interesting features in comparison with that of S. cerevisiae. The three mosaic genes present in both genomes are quite different with regard to their structure. The S. douglasii COXI gene has two new introns and is missing the five introns of the S. cerevisiae gene. The S. douglasii cytochrome b gene has one new intron and lacks two introns of the S. cerevisiae gene. Finally, the L-rRNA gene of S. douglasii, like that of S. cerevisiae, has one intron of which the structure is different. Another salient feature of the S. douglasii mitochondrial genome reported here is that the gene order is different in comparison with S. cerevisiae mitochondrial DNA. In particular, a segment of approximately 15,000 base-pairs including the genes coding for COXIII and S-rRNA has been translocated to a position between the genes coding for varl and L-rRNA.     

Reciprocal chromosome painting shows that genomic rearrangement between rat and mouse proceeds ten times faster than between humans and cats.

Reciprocal chromosome painting between mouse and rat using complete chromosome probe sets of both species permitted us to assign the chromosomal homology between these rodents. The comparative gene mapping data and chromosome painting have a better than 90% correspondence. The reciprocal painting results graphically show that mouse and rat have strikingly different karyotypes. At least 14 translocations have occurred in the 10-20 million years of evolution that separates these two species. The evolutionary rate of chromosome translocations between these two rodents appears to be up to 10 times greater than that found between humans and cats, or between humans and chimpanzees, where over the last 5-6 million years just one translocation has occurred. Outgroup comparison shows that the mouse genome has incorporated at least three times the amount of interchromosomal rearrangements compared to the rat genome. The utility of chromosome painting was also illustrated by the assignment of two new chromosome homologies between rat and mouse unsuspected by gene mapping: between mouse 11 and rat 20 and between mouse 17 and rat 6. We conclude that reciprocal chromosome painting is a powerful method, which can be used with confidence to chart the genome and predict the chromosome location of genes. Reciprocal painting combined with gene mapping data will allow the construction of large-scale comparative chromosome maps between placental mammals and perhaps other animals.     

Comparative genomics among Saccharomyces cerevisiae x Saccharomyces kudriavzevii natural hybrid strains isolated from wine and beer reveals different origins

ABSTRACT: BACKGROUND: Interspecific hybrids between S. cerevisiae x S. kudriavzevii have frequently been detected in wine and beer fermentations. Significant physiological differences among parental and hybrid strains under different stress conditions have been evidenced. In this study, we used comparative genome hybridization analysis to evaluate the genome composition of different S. cerevisiae x S. kudriavzevii natural hybrids isolated from wine and beer fermentations to infer their evolutionary origins and to figure out the potential role of common S. kudriavzevii gene fraction present in these hybrids. RESULTS: Comparative genomic hybridization (CGH) and ploidy analyses carried out in this study confirmed the presence of individual and differential chromosomal composition patterns for most S. cerevisiae x S. kudriavzevii hybrids from beer and wine. All hybrids share a common set of depleted S. cerevisiae genes, which also are depleted or absent in the wine strains studied so far, and the presence a common set of S. kudriavzevii genes, which may be associated with their capability to grow at low temperatures. Finally, a maximum parsimony analysis of chromosomal rearrangement events, occurred in the hybrid genomes, indicated the presence of two main groups of wine hybrids and different divergent lineages of brewing strains. CONCLUSION: Our data suggest that wine and beer S. cerevisiae x S. kudriavzevii hybrids have been originated by different rare-mating events involving a diploid wine S. cerevisiae and a haploid or diploid European S. kudriavzevii strains. Hybrids maintain several S. kudriavzevii genes involved in cold adaptation as well as those related to S. kudriavzevii mitochondrial functions.     

Comparative genetics of yeast Saccharomyces cerevisiae: chromosomal translocations carrying the SUC2 marker

Three different translocations involving chromosome IX have been detected in natural Saccharomyces cerevisiae strains using pulsed-field gel electrophoresis with intact chromosomal DNA and their hybridization with the SUC2 probe. Hybrids of these strains with genetic lines having normal molecular karyotype were shown to have back dislocation of at least marker SUC2 due to crossingover. The significance of the detected translocations is discussed.     

Rapid analysis of Saccharomyces cerevisiae genome rearrangements by multiplex ligation-dependent probe amplification

Aneuploidy and gross chromosomal rearrangements (GCRs) can lead to genetic diseases and the development of cancer. We previously demonstrated that introduction of the repetitive retrotransposon Ty912 onto a nonessential chromosome arm of Saccharomyces cerevisiae led to increased genome instability predominantly due to increased rates of formation of monocentric nonreciprocal translocations. In this study, we adapted Multiplex Ligation-dependent Probe Amplification (MLPA) to analyze a large numbers of these GCRs. Using MLPA, we found that the distribution of translocations induced by the presence of Ty912 in a wild-type strain was nonrandom and that the majority of these translocations were mediated by only six translocation targets on four different chromosomes, even though there were 254 potential Ty-related translocation targets in the S. cerevisiae genome. While the majority of Ty912-mediated translocations resulted from RAD52-dependent recombination, we observed a number of nonreciprocal translocations mediated by RAD52-independent recombination between Ty1 elements. The formation of these RAD52-independent translocations did not require the Rad51 or Rad59 homologous pairing proteins or the Rad1-Rad10 endonuclease complex that processes branched DNAs during recombination. Finally, we found that defects in ASF1-RTT109-dependent acetylation of histone H3 lysine residue 56 (H3K56) resulted in increased accumulation of both GCRs and whole-chromosome duplications, and resulted in aneuploidy that tended to occur simultaneously with GCRs. Overall, we found that MLPA is a versatile technique for the rapid analysis of GCRs and can facilitate the genetic analysis of the pathways that prevent and promote GCRs and aneuploidy.     

Saccharomyces cerevisiae as a model system to define the chromosomal instability phenotype

Translocations, deletions, and chromosome fusions are frequent events seen in cancers with genome instability. Here we analyzed 358 genome rearrangements generated in Saccharomyces cerevisiae selected by the loss of the nonessential terminal segment of chromosome V. The rearrangements appeared to be generated by both nonhomologous end joining and homologous recombination and targeted all chromosomes. Fifteen percent of the rearrangements occurred independently more than once. High levels of specific classes of rearrangements were isolated from strains with specific mutations: translocations to Ty elements were increased in telomerase-defective mutants, potential dicentric translocations and dicentric isochromosomes were associated with cell cycle checkpoint defects, chromosome fusions were frequent in strains with both telomerase and cell cycle checkpoint defects, and translocations to homolog genes were seen in strains with defects allowing homoeologous recombination. An analysis of human cancer-associated rearrangements revealed parallels to the effects that strain genotypes have on classes of rearrangement in S. cerevisiae.     

Rate asymmetry after genome duplication causes substantial long-branch attraction artifacts in the phylogeny of Saccharomyces species

Whole-genome duplication (WGD) produces sets of gene pairs that are all of the same age. We therefore expect that phylogenetic trees that relate these pairs to their orthologs in other species should show a single consistent topology. However, a previous study of gene pairs formed by WGD in the yeast Saccharomyces cerevisiae found conflicting topologies among neighbor-joining (NJ) trees drawn from different loci and suggested that this conflict was the result of "asynchronous functional divergence" of duplicated genes (Langkjaer, R. B., P. F. Cliften, M. Johnston, and J. Piskur. 2003. Yeast genome duplication was followed by asynchronous differentiation of duplicated genes. Nature 421:848-852). Here, we test whether the conflicting topologies might instead be due to asymmetrical rates of evolution leading to long-branch attraction (LBA) artifacts in phylogenetic trees. We constructed trees for 433 pairs of WGD paralogs in S. cerevisiae with their single orthologs in Saccharomyces kluyveri and Candida albicans. We find a strong correlation between the asymmetry of evolutionary rates of a pair of S. cerevisiae paralogs and the topology of the tree inferred for that pair. Saccharomyces cerevisiae gene pairs with approximately equal rates of evolution tend to give phylogenies in which the WGD postdates the speciation between S. cerevisiae and S. kluyveri (B-trees), whereas trees drawn from gene pairs with asymmetrical rates tend to show WGD pre-dating this speciation (A-trees). Gene order data from throughout the genome indicate that the "A-trees" are artifacts, even though more than 50% of gene pairs are inferred to have this topology when the NJ method as implemented in ClustalW (i.e., with Poisson correction of distances) is used to construct the trees. This LBA artifact can be ameliorated, but not eliminated, by using gamma-corrected distances or by using maximum likelihood trees with robustness estimated by the Shimodaira-Hasegawa test. Tests for adaptive evolution indicated that positive selection might be the cause of rate asymmetry in a substantial fraction (19%) of the paralog pairs.     

Meiotic recombination at the ends of chromosomes in Saccharomyces cerevisiae

Meiotic reciprocal recombination (crossing over) was examined in the outermost 60-80 kb of almost all Saccharomyces cerevisiae chromosomes. These sequences included both repetitive gene-poor subtelomeric heterochromatin-like regions and their adjacent unique gene-rich euchromatin-like regions. Subtelomeric sequences underwent very little crossing over, exhibiting approximately two- to threefold fewer crossovers per kilobase of DNA than the genomic average. Surprisingly, the adjacent euchromatic regions underwent crossing over at twice the average genomic rate and contained at least nine new recombination "hot spots." These results prompted an analysis of existing genetic mapping data, which showed that meiotic reciprocal recombination rates were on average greater near chromosome ends exclusive of the subtelomeres. Thus, the distribution of crossovers in S. cerevisiae appears to resemble that found in several higher eukaryotes where the outermost chromosomal regions show increased crossing over.