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In eukaryotic cells, the production of cellular energy requires close interplay between nuclear and mitochondrial genomes. The mitochondrial genome is essential in that it encodes several genes involved in oxidative phosphorylation. Each cell contains several mitochondrial genome copies and mitochondrial DNA recombination is a widespread process occurring in plants, fungi, protists, and invertebrates. Saccharomyces cerevisiae has proved to be an excellent model to dissect mitochondrial biology. Several studies have focused on DNA recombination in this organelle, yet mostly relied on reporter genes or artificial systems. However, no complete mitochondrial recombination map has been released for any eukaryote so far. In the present work, we sequenced pools of diploids originating from a cross between two different S. cerevisiae strains to detect recombination events. This strategy allowed us to generate the first genome-wide map of recombination for yeast mitochondrial DNA. We demonstrated that recombination events are enriched in specific hotspots preferentially localized in non-protein-coding regions. Additionally, comparison of the recombination profiles of two different crosses showed that the genetic background affects hotspot localization and recombination rates. Finally, to gain insights into the mechanisms involved in mitochondrial recombination, we assessed the impact of individual depletion of four genes previously associated with this process. Deletion of NTG1 and MGT1 did not substantially influence the recombination landscape, alluding to the potential presence of additional regulatory factors. Our findings also revealed the loss of large mitochondrial DNA regions in the absence of MHR1, suggesting a pivotal role for Mhr1 in mitochondrial genome maintenance during mating. This study provides a comprehensive overview of mitochondrial DNA recombination in yeast and thus paves the way for future mechanistic studies of mitochondrial recombination and genome maintenance.

Dissecting the molecular basis of quantitative traits is a significant challenge and is essential for understanding complex diseases. Even in model organisms, precisely determining causative genes and their interactions has remained elusive, due in part to difficulty in narrowing intervals to single genes and in detecting epistasis or linked quantitative trait loci. These difficulties are exacerbated by limitations in experimental design, such as low numbers of analyzed individuals or of polymorphisms between parental genomes. We address these challenges by applying three independent high-throughput approaches for QTL mapping to map the genetic variants underlying 11 phenotypes in two genetically distant Saccharomyces cerevisiae strains, namely (1) individual analysis of >700 meiotic segregants, (2) bulk segregant analysis, and (3) reciprocal hemizygosity scanning, a new genome-wide method that we developed. We reveal differences in the performance of each approach and, by combining them, identify eight polymorphic genes that affect eight different phenotypes: colony shape, flocculation, growth on two nonfermentable carbon sources, and resistance to two drugs, salt, and high temperature. Our results demonstrate the power of individual segregant analysis to dissect QTL and address the underestimated contribution of interactions between variants. We also reveal confounding factors like mutations and aneuploidy in pooled approaches, providing valuable lessons for future designs of complex trait mapping studies.

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.

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.

Mitochondrial diseases are systemic, prevalent and often fatal; yet treatments remain scarce. Identifying molecular intervention points that can be therapeutically targeted remains a major challenge, which we confronted via a screening assay we developed. Using yeast models of mitochondrial ATP synthase disorders, we screened a drug repurposing library, and applied genomic and biochemical techniques to identify pathways of interest. Here we demonstrate that modulating the sorting of nuclear-encoded proteins into mitochondria, mediated by the TIM23 complex, proves therapeutic in both yeast and patient-derived cells exhibiting ATP synthase deficiency. Targeting TIM23-dependent protein sorting improves an array of phenotypes associated with ATP synthase disorders, including biogenesis and activity of the oxidative phosphorylation machinery. Our study establishes mitochondrial protein sorting as an intervention point for ATP synthase disorders, and because of the central role of this pathway in mitochondrial biogenesis, it holds broad value for the treatment of mitochondrial diseases., Effective treatment options for mitochondrial diseases are scarce. Here, Aiyar et al. identify the TIM23 mitochondrial protein sorting machinery as a potential intervention point for mitochondrial ATP synthase disorders.

Background The throughput of next-generation sequencing machines has increased dramatically over the last few years; yet the cost and time for library preparation have not changed proportionally, thus representing the main bottleneck for sequencing large numbers of samples. Here we present an economical, high-throughput library preparation method for the Illumina platform, comprising a 96-well based method for DNA isolation for yeast cells, a low-cost DNA shearing alternative, and adapter ligation using heat inactivation of enzymes instead of bead cleanups. Results Up to 384 whole-genome libraries can be prepared from yeast cells in one week using this method, for less than 15 euros per sample. We demonstrate the robustness of this protocol by sequencing over 1000 yeast genomes at ~30x coverage. The sequence information from 768 yeast segregants derived from two divergent S. cerevisiae strains was used to generate a meiotic recombination map at unprecedented resolution. Comparisons to other datasets indicate a high conservation of recombination at a chromosome-wide scale, but differences at the local scale. Additionally, we detected a high degree of aneuploidy (3.6%) by examining the sequencing coverage in these segregants. Differences in allele frequency allowed us to attribute instances of aneuploidy to gains of chromosomes during meiosis or mitosis, both of which showed a strong tendency to missegregate specific chromosomes. Conclusions Here we present a high throughput workflow to sequence genomes of large number of yeast strains at a low price. We have used this workflow to obtain recombination and aneuploidy data from hundreds of segregants, which can serve as a foundation for future studies of linkage, recombination, and chromosomal aberrations in yeast and higher eukaryotes.

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.

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.

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.