Project description:Array data detailing the progression of DNA replication in the yeast Lachancea waltii. L. waltii cells were pregrown in heavy isotope medium and synchronized at early S phase. They were then released into normal medium, wherein DNA replication proceeds. Replicated DNA molecules are thus of heavy-light (HL) composition as compared to unreplicated molecules, which are heavy-heavy (HH). 4 time points were taken and the percent of heavy-light DNA was determined at each time point. The heavy-heavy and heavy-light DNA molecules were separated by ultracentrifugation, differentially labeled, and hybridized to a genomic array for L. waltii. The array thus shows the progression of DNA replication.
Project description:Haploid cells of Lachancea kluyveri were arrested in G1 phase using Saccharomices cerevisiae alpha factor. After, release in a new media, cells go synchronously through S-phase. One sample is taken every five minutes. Microarrays are used to monitor the change of DNA copy number from 1 to 2, all along the genome during S-phase. Two-condition experiment, G1 cells vs. S-phase cells at different time points. Biological replicates: 3 biological replicates.
Project description:Haploid cells of Lachancea kluyveri were arrested in G1 phase using Saccharomices cerevisiae alpha factor. After, release in a new media, cells go synchronously through S-phase. One sample is taken every five minutes. Microarrays are used to monitor the change of DNA copy number from 1 to 2, all along the genome during S-phase.
Project description:Dissecting the genetic basis of complex trait remains a real challenge. The budding yeast Saccharomyces cerevisiae has become a model organism for studying quantitative traits, successfully increasing our knowledge in many aspects. However, the exploration of the genotype-phenotype relationship in non-model yeast species could provide a deeper insight into the genetic basis of complex traits. Here, we have studied this relationship in the Lachancea waltii species which diverged from the S. cerevisiae lineage prior to the whole-genome duplication. By performing linkage mapping analyses in this species, we identified 86 quantitative trait loci (QTL) impacting the growth in a large number of conditions. The distribution of these loci across the genome has revealed two major QTL hotspots. A first hotspot corresponds to a general growth QTL, impacting a wide range of conditions. By contrast, the second hotspot highlighted a trade-off with a disadvantageous allele for drug-free conditions which proved to be advantageous in the presence of several drugs. Finally, a comparison of the detected QTL in L. waltii with those which had been previously identified for the same trait in a closely related species, Lachancea kluyveri was performed. This analysis clearly showed the absence of shared QTL across these species. Altogether, our results represent a first step toward the exploration of the genetic architecture of quantitative trait across different yeast species.
Project description:Meiotic recombination is a driving force for genome evolution, deeply characterized in a few model species, notably in the budding yeast Saccharomyces cerevisiae. Interestingly, Zip2, Zip3, Zip4, Spo16, Msh4, and Msh5, members of the so-called ZMM pathway that implements the interfering meiotic crossover pathway in S. cerevisiae, have been lost in Lachancea yeast species after the divergence of Lachancea kluyveri from the rest of the clade. In this context, after investigating meiosis in L. kluyveri, we determined the meiotic recombination landscape of Lachancea waltii. Attempts to generate diploid strains with fully hybrid genomes invariably resulted in strains with frequent whole-chromosome aneuploidy and multiple extended regions of loss of heterozygosity (LOH), which mechanistic origin is so far unclear. Despite the lack of multiple ZMM pro-crossover factors in L. waltii, numbers of crossovers and noncrossovers per meiosis were higher than in L. kluyveri but lower than in S. cerevisiae, for comparable genome sizes. Similar to L. kluyveri but opposite to S. cerevisiae, L. waltii exhibits an elevated frequency of zero-crossover bivalents. Lengths of gene conversion tracts for both crossovers and non-crossovers in L. waltii were comparable to those observed in S. cerevisiae and shorter than in L. kluyveri despite the lack of Mlh2, a factor limiting conversion tract size in S. cerevisiae. L. waltii recombination hotspots were not shared with either S. cerevisiae or L. kluyveri, showing that meiotic recombination hotspots can evolve at a rather limited evolutionary scale within budding yeasts. Finally, L. waltii crossover interference was reduced relative to S. cerevisiae, with interference being detected only in the 25 kb distance range. Detection of positive inference only at short distance scales in the absence of multiple ZMM factors required for interference-sensitive crossovers in other systems likely reflects interference between early recombination precursors such as DSBs.
Project description:AID promotes chromosomal translocations by inducing DNA double-strand breaks (DSBs) at immunoglobulin (Ig) genes and oncogenes in G1. RPA is a ssDNA-binding protein that associates with resected DSBs in the S phase and facilitates the assembly of factors involved in homologous repair (HR) such as Rad51. Notably, RPA deposition also marks sites of AID-mediated damage, but its role in Ig gene recombination remains unclear. Here we demonstrate that RPA associates asymmetrically with resected ssDNA in response to lesions created by AID, RAG, or other nucleases. Small amounts of RPA are deposited at AID targets in G1 in an ATM-dependent manner. In contrast, recruitment in S-G2/M is extensive, ATM-independent, and associated with Rad51 accumulation. RPA in S-G2/M increases in NHEJ-deficient lymphocytes, where there is more extensive DNA-end resection. Thus, most RPA recruitment during CSR represents salvage of un-repaired breaks by homology-based pathways during the S-G2/M phases of the cell cycle. Chip-Seq of RPA from mouse activated B cells (n = 40), mouse thymocytes (n = 6), and MEFs (n = 1). Different genotypes and/or inhibitors were used.