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German Larriba - One of the best experts on this subject based on the ideXlab platform.
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partner choice in spontaneous mitotic recombination in wild type and homologous recombination mutants of candida albicans
G3: Genes Genomes Genetics, 2019Co-Authors: Alberto Bellido, Toni Ciudad, Belen Hermosa, Encarnacion Andaluz, Anja Forche, German LarribaAbstract:Candida albicans, the most common fungal pathogen, is a diploid with a genome that is rich in repeats and has high levels of heterozygosity. To study the role of different recombination pathways on direct-repeat recombination, we replaced either Allele of the RAD52 gene (Chr6) with the URA-blaster cassette (hisG-URA3-hisG), measured rates of URA3 loss as resistance to 5-fluoroorotic acid (5FOAR) and used CHEF Southern hybridization and SNP-RFLP analysis to identify recombination mechanisms and their frequency in wildtype and recombination mutants. FOAR rates varied little across different strain backgrounds. In contrast, the type and frequency of mechanisms underlying direct repeat recombination varied greatly. For example, wildtype, rad59 and lig4 strains all displayed a bias for URA3 loss via pop-out/deletion versus inter-homolog recombination and this bias was reduced in rad51 mutants. In addition, in rad51-derived 5FOAR strains direct repeat recombination was associated with ectopic translocation (5%), chromosome loss/truncation (14%) and inter-homolog recombination (6%). In the absence of RAD52, URA3 loss was mostly due to chromosome loss and truncation (80-90%), and the bias of retained Allele frequency points to the presence of a recessive Lethal Allele on Chr6B. However, a few single-strand annealing (SSA)-like events were identified and these were independent of either Rad59 or Lig4. Finally, the specific sizes of Chr6 truncations suggest that the inserted URA-blaster could represent a fragile site.
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Partner Choice in Spontaneous Mitotic Recombination in Wild Type and Homologous Recombination Mutants of Candida albicans
'Genetics Society of America', 2019Co-Authors: Alberto Bellido, Toni Ciudad, Belen Hermosa, Encarnacion Andaluz, Anja Forche, German LarribaAbstract:Candida albicans, the most common fungal pathogen, is a diploid with a genome that is rich in repeats and has high levels of heterozygosity. To study the role of different recombination pathways on direct-repeat recombination, we replaced either Allele of the RAD52 gene (Chr6) with the URA-blaster cassette (hisG-URA3-hisG), measured rates of URA3 loss as resistance to 5-fluoroorotic acid (5FOAR) and used CHEF Southern hybridization and SNP-RFLP analysis to identify recombination mechanisms and their frequency in wildtype and recombination mutants. FOAR rates varied little across different strain backgrounds. In contrast, the type and frequency of mechanisms underlying direct repeat recombination varied greatly. For example, wildtype, rad59 and lig4 strains all displayed a bias for URA3 loss via pop-out/deletion vs. inter-homolog recombination and this bias was reduced in rad51 mutants. In addition, in rad51-derived 5FOAR strains direct repeat recombination was associated with ectopic translocation (5%), chromosome loss/truncation (14%) and inter-homolog recombination (6%). In the absence of RAD52, URA3 loss was mostly due to chromosome loss and truncation (80–90%), and the bias of retained Allele frequency points to the presence of a recessive Lethal Allele on Chr6B. However, a few single-strand annealing (SSA)-like events were identified and these were independent of either Rad59 or Lig4. Finally, the specific sizes of Chr6 truncations suggest that the inserted URA-blaster could represent a fragile site
Alberto Bellido - One of the best experts on this subject based on the ideXlab platform.
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partner choice in spontaneous mitotic recombination in wild type and homologous recombination mutants of candida albicans
G3: Genes Genomes Genetics, 2019Co-Authors: Alberto Bellido, Toni Ciudad, Belen Hermosa, Encarnacion Andaluz, Anja Forche, German LarribaAbstract:Candida albicans, the most common fungal pathogen, is a diploid with a genome that is rich in repeats and has high levels of heterozygosity. To study the role of different recombination pathways on direct-repeat recombination, we replaced either Allele of the RAD52 gene (Chr6) with the URA-blaster cassette (hisG-URA3-hisG), measured rates of URA3 loss as resistance to 5-fluoroorotic acid (5FOAR) and used CHEF Southern hybridization and SNP-RFLP analysis to identify recombination mechanisms and their frequency in wildtype and recombination mutants. FOAR rates varied little across different strain backgrounds. In contrast, the type and frequency of mechanisms underlying direct repeat recombination varied greatly. For example, wildtype, rad59 and lig4 strains all displayed a bias for URA3 loss via pop-out/deletion versus inter-homolog recombination and this bias was reduced in rad51 mutants. In addition, in rad51-derived 5FOAR strains direct repeat recombination was associated with ectopic translocation (5%), chromosome loss/truncation (14%) and inter-homolog recombination (6%). In the absence of RAD52, URA3 loss was mostly due to chromosome loss and truncation (80-90%), and the bias of retained Allele frequency points to the presence of a recessive Lethal Allele on Chr6B. However, a few single-strand annealing (SSA)-like events were identified and these were independent of either Rad59 or Lig4. Finally, the specific sizes of Chr6 truncations suggest that the inserted URA-blaster could represent a fragile site.
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Partner Choice in Spontaneous Mitotic Recombination in Wild Type and Homologous Recombination Mutants of Candida albicans
'Genetics Society of America', 2019Co-Authors: Alberto Bellido, Toni Ciudad, Belen Hermosa, Encarnacion Andaluz, Anja Forche, German LarribaAbstract:Candida albicans, the most common fungal pathogen, is a diploid with a genome that is rich in repeats and has high levels of heterozygosity. To study the role of different recombination pathways on direct-repeat recombination, we replaced either Allele of the RAD52 gene (Chr6) with the URA-blaster cassette (hisG-URA3-hisG), measured rates of URA3 loss as resistance to 5-fluoroorotic acid (5FOAR) and used CHEF Southern hybridization and SNP-RFLP analysis to identify recombination mechanisms and their frequency in wildtype and recombination mutants. FOAR rates varied little across different strain backgrounds. In contrast, the type and frequency of mechanisms underlying direct repeat recombination varied greatly. For example, wildtype, rad59 and lig4 strains all displayed a bias for URA3 loss via pop-out/deletion vs. inter-homolog recombination and this bias was reduced in rad51 mutants. In addition, in rad51-derived 5FOAR strains direct repeat recombination was associated with ectopic translocation (5%), chromosome loss/truncation (14%) and inter-homolog recombination (6%). In the absence of RAD52, URA3 loss was mostly due to chromosome loss and truncation (80–90%), and the bias of retained Allele frequency points to the presence of a recessive Lethal Allele on Chr6B. However, a few single-strand annealing (SSA)-like events were identified and these were independent of either Rad59 or Lig4. Finally, the specific sizes of Chr6 truncations suggest that the inserted URA-blaster could represent a fragile site
Legrand Mélanie - One of the best experts on this subject based on the ideXlab platform.
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Identification of recessive Lethal Alleles in the diploid genome of a candida albicans laboratory strain unveils a potential role of repetitive sequences in buffering their deleterious impact.
'American Society for Microbiology', 2019Co-Authors: Marton Timea, Feri Adeline, Commere Pierre-henri, Maufrais Corinne, D'enfert Christophe, Legrand MélanieAbstract:International audienceThe heterozygous diploid genome of Candida albicans is highly plastic, with frequent loss of heterozygosity (LOH) events. In the SC5314 laboratory strain, while LOH events are ubiquitous, a chromosome homozygosis bias is observed for certain chromosomes, whereby only one of the two homologs can occur in the homozygous state. This suggests the occurrence of recessive Lethal Allele(s) (RLA) preventing large-scale LOH events on these chromosomes from being stably maintained. To verify the presence of an RLA on chromosome 7 (Chr7), we utilized a system that allows (i) DNA double-strand break (DSB) induction on Chr7 by the I-SceI endonuclease and (ii) detection of the resulting long-range homozygosis. I-SceI successfully induced a DNA DSB on both Chr7 homologs, generally repaired by gene conversion. Notably, cells homozygous for the right arm of Chr7B were not recovered, confirming the presence of RLA(s) in this region. Genome data mining for RLA candidates identified a premature nonsense-generating single nucleotide polymorphism (SNP) within the HapB Allele of C7_03400c whose Saccharomyces cerevisiae ortholog encodes the essential Mtr4 RNA helicase. Complementation with a wild-type copy of MTR4 rescued cells homozygous for the right arm of Chr7B, demonstrating that the mtr4K880* RLA is responsible for the Chr7 homozygosis bias in strain SC5314. Furthermore, we observed that the major repeat sequences (MRS) on Chr7 acted as hot spots for interhomolog recombination. Such recombination events provide C. albicans with increased opportunities to survive DNA DSBs whose repair can lead to homozygosis of recessive Lethal or deleterious Alleles. This might explain the maintenance of MRS in this species.IMPORTANCE Candida albicans is a major fungal pathogen, whose mode of reproduction is mainly clonal. Its genome is highly tolerant to rearrangements, in particular loss of heterozygosity events, known to unmask recessive Lethal and deleterious Alleles in heterozygous diploid organisms such as C. albicans By combining a site-specific DSB-inducing system and mining genome sequencing data of 182 C. albicans isolates, we were able to ascribe the chromosome 7 homozygosis bias of the C. albicans laboratory strain SC5314 to an heterozygous SNP introducing a premature STOP codon in the MTR4 gene. We have also proposed genome-wide candidates for new recessive Lethal Alleles. We additionally observed that the major repeat sequences (MRS) on chromosome 7 acted as hot spots for interhomolog recombination. Maintaining MRS in C. albicans could favor haplotype exchange, of vital importance to LOH events, leading to homozygosis of recessive Lethal or deleterious Alleles that inevitably accumulate upon clonality
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Analysis of repair mechanisms following an induced double-strand break uncovers recessive deleterious Alleles in the candida albicans diploid genome
2016Co-Authors: Feri Adeline, Loll-krippleber Raphaël, Commere Pierre-henri, Maufrais Corinne, Sertour Natacha, Schwartz Katja, Sherlock Gavin, Bougnoux Marie-elisabeth, Legrand MélanieAbstract:The diploid genome of the yeast Candida albicans is highly plastic, exhibiting frequent loss-of-heterozygosity (LOH) events. To provide a deeper understanding of the mechanisms leading to LOH, we investigated the repair of a unique DNA double-strand break (DSB) in the laboratory C. albicans SC5314 strain using the I-SceI meganuclease. Upon I-SceI induction, we detected a strong increase in the frequency of LOH events at an I-SceI target locus positioned on chromosome 4 (Chr4), including events spreading from this locus to the proximal telomere. Characterization of the repair events by single nucleotide polymorphism (SNP) typing and whole-genome sequencing revealed a predominance of gene conversions, but we also observed mitotic crossover or break-induced replication events, as well as combinations of independent events. Importantly, progeny that had undergone homozygosis of part or all of Chr4 haplotype B (Chr4B) were inviable. Mining of genome sequencing data for 155 C. albicans isolates allowed the identification of a recessive Lethal Allele in the GPI16 gene on Chr4B unique to C. albicans strain SC5314 which is responsible for this inviability. Additional recessive Lethal or deleterious Alleles were identified in the genomes of strain SC5314 and two clinical isolates. Our results demonstrate that recessive Lethal Alleles in the genomes of C. albicans isolates prevent the occurrence of specific extended LOH events. While these and other recessive Lethal and deleterious Alleles are likely to accumulate in C. albicans due to clonal reproduction, their occurrence may in turn promote the maintenance of corresponding nondeleterious Alleles and, consequently, heterozygosity in the C. albicans species. IMPORTANCE Recessive Lethal Alleles impose significant constraints on the biology of diploid organisms. Using a combination of an I-SceI meganuclease-mediated DNA DSB, a fluorescence-activated cell sorter (FACS)-optimized reporter of LOH, and a compendium of 155 genome sequences, we were able to unmask and identify recessive Lethal and deleterious Alleles in isolates of Candida albicans, a diploid yeast and the major fungal pathogen of humans. Accumulation of recessive deleterious mutations upon clonal reproduction of C. albicans could contribute to the maintenance of heterozygosity despite the high frequency of LOH events in this species
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Analysis of Repair Mechanisms following an Induced Double-Strand Break Uncovers Recessive Deleterious Alleles in the Candida albicans Diploid Genome
'American Society for Microbiology', 2016Co-Authors: Feri Adeline, Loll-krippleber Raphaël, Commere Pierre-henri, Maufrais Corinne, Sertour Natacha, Schwartz Katja, Sherlock Gavin, Bougnoux Marie-elisabeth, D’enfert Christophe, Legrand MélanieAbstract:International audienceThe diploid genome of the yeast Candida albicans is highly plastic, exhibiting frequent loss-of-heterozygosity (LOH) events. To provide a deeper understanding of the mechanisms leading to LOH, we investigated the repair of a unique DNA double-strand break (DSB) in the laboratory C. albicans SC5314 strain using the I-SceI meganuclease. Upon I-SceI induction , we detected a strong increase in the frequency of LOH events at an I-SceI target locus positioned on chromosome 4 (Chr4), including events spreading from this locus to the proximal telomere. Characterization of the repair events by single nucleotide polymorphism (SNP) typing and whole-genome sequencing revealed a predominance of gene conversions, but we also observed mitotic crossover or break-induced replication events, as well as combinations of independent events. Importantly, progeny that had undergone homozygosis of part or all of Chr4 haplotype B (Chr4B) were inviable. Mining of genome sequencing data for 155 C. albicans isolates allowed the identification of a recessive Lethal Allele in the GPI16 gene on Chr4B unique to C. albicans strain SC5314 which is responsible for this inviability. Additional recessive Lethal or deleterious Alleles were identified in the genomes of strain SC5314 and two clinical isolates. Our results demonstrate that recessive Lethal Alleles in the genomes of C. albicans isolates prevent the occurrence of specific extended LOH events. While these and other recessive Lethal and deleterious Alleles are likely to accumulate in C. albicans due to clonal reproduction, their occurrence may in turn promote the maintenance of corresponding nondeleterious Alleles and, consequently, heterozygosity in the C. albicans species. IMPORTANCE Recessive Lethal Alleles impose significant constraints on the biology of diploid organisms. Using a combination of an I-SceI meganuclease-mediated DNA DSB, a fluorescence-activated cell sorter (FACS)-optimized reporter of LOH, and a compendium of 155 genome sequences, we were able to unmask and identify recessive Lethal and deleterious Alleles in isolates of Candida albicans, a diploid yeast and the major fungal pathogen of humans. Accumulation of recessive deleterious mutations upon clonal reproduction of C. albicans could contribute to the maintenance of heterozygosity despite the high frequency of LOH events in this species. Citation Feri A, Loll-Krippleber R, Commere P, Maufrais C, Sertour N, Schwartz K, Sherlock G, Bougnoux M, d'Enfert C, Legrand M. 2016. Analysis of repair mechanisms following an induced double-strand break uncovers recessive deleterious Alleles in the Candida albicans diploid genome. mBio 7(5):e01109-16
Julie M Jones - One of the best experts on this subject based on the ideXlab platform.
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mutation of vps54 causes motor neuron disease and defective spermiogenesis in the wobbler mouse
Nature Genetics, 2005Co-Authors: Thomas Schmittjohn, Carsten Drepper, Anke Mussmann, Phillip Hahn, Melanie Kuhlmann, Cora S Thiel, Martin Hafner, Andreas Lengeling, Peter Heimann, Julie M JonesAbstract:Vacuolar-vesicular protein sorting (Vps) factors are involved in vesicular trafficking in eukaryotic cells. We identified the missense mutation L967Q in Vps54 in the wobbler mouse, an animal model of amyotrophic lateral sclerosis, and also characterized a Lethal Allele, Vps54(beta-geo). Motoneuron survival and spermiogenesis are severely compromised in the wobbler mouse, indicating that Vps54 has an essential role in these processes.
John M Belote - One of the best experts on this subject based on the ideXlab platform.
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identification of an essential gene l 3 73ai with a dominant temperature sensitive Lethal Allele encoding a drosophila proteasome subunit
Proceedings of the National Academy of Sciences of the United States of America, 1993Co-Authors: Kenneth J Saville, John M BeloteAbstract:Abstract Proteasomes are multicatalytic proteinase complexes that function as a major nonlysosomal proteolytic system in all eukaryotes. These particles are made up of 13-15 nonidentical subunits, and they exhibit multiple endopeptidase activities that promote the intracellular turnover of abnormal polypeptides and short-lived regulatory proteins. Although the biochemical characterization of proteasomes has been quite extensive, and although a number of the genes encoding proteasome subunits have been cloned from various organisms, there is still much to be learned about their function in vivo and what role(s) they might play during development. Here, we report the identification of the l(3)73Ai1 Allele of Drosophila melanogaster as a dominant temperature-sensitive Lethal mutation in a gene encoding a component of the proteasome, thus opening the way for future genetic and developmental studies on this important proteolytic system in a higher eukaryote.