Dataset Information

Genomic instability induced by furfural-treated in S. cerevisiae [sectored colonies]

ABSTRACT: Furfural is a key inhibitor in S. cerevisiae fermentation causing serious economic loss. To disclose of the recombinational DNA lesions induced by furfural, we analyzed 19 JSC25-derived sectored colonies using chromosome IV SNP-specific microarrays. The parent diploid S. cerevisiae strain JSC25 (constructed by crossing two haploid strains isogenic W303-1A and YJM789) was homologous for ade2-1 alleles. One copy of gene SUP4-o, an ochre-suppressing tRNA, was inserted at the end of right arm of chromosome IV. Under normal conditions, the colony color of JSC25 is pink. A crossover event occurred on the right arm of chromosome IV at the first cell cycle would result in a white/red sectored colony on solid plate.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE104880 | GEO | 2017/10/13

SECONDARY ACCESSION(S): PRJNA414089

REPOSITORIES: GEO

ACCESS DATA

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

Project description:Two types of RNA:DNA associations can lead to genome instability: the formation of R-loops during transcription and the incorporation of ribonucleotide monophosphates (rNMPs) into DNA during replication. Both ribonuclease (RNase) H1 and RNase H2 degrade the RNA component of R-loops, whereas only RNase H2 can remove one or a few rNMPs from DNA. We performed high-resolution mapping of mitotic recombination events throughout the yeast genome in diploid strains of Saccharomyces cerevisiae lacking RNase H1 (rnh1Δ), RNase H2 (rnh201Δ), or both RNase H1 and RNase H2 (rnh1Δ rnh201Δ). We found little effect on recombination in the rnh1Δ strain, but elevated recombination in both the rnh201Δ and the double-mutant strains; levels of recombination in the double mutant were about 50% higher than in the rnh201 single-mutant strain. An rnh201Δ mutant that additionally contained a mutation that reduces rNMP incorporation by DNA polymerase ε (pol2-M644L) had a level of instability similar to that observed in the presence of wild-type Polε. This result suggests that the elevated recombination observed in the absence of only RNase H2 is primarily a consequence of R loops rather than misincorporated rNMPs. The details of these experiments are in press in Genetics This diploid S.cerevisiae strain used here is a hybrid between W303-1A and YJM789. The backgrounds are diverged by about 50,000 polymorphisms that are surveyed by custom CGH microarrays containing SNP-specific probes (St. Charles et al., 2012; Genetics 190: 1267-1284; PMID 22267500). The whole genome array surveyed loss of heterozygosity (LOH) events throughout the genome of in wild type, pol2-M644L, rnh1Δ, rnh201Δ, rnh201Δ pol2-M644L, and rnh1Δ rnh201Δ mutant backgrounds in 10, 12, 10, 19, 17, and 22 samples, respectively. The names of these strains are KO_198,KO_234, KO_73, KO_75, KO_244, and KO_5, respectively. Another assay was performed mapping reciprocal crossover events on the right arm of chromosome IV in the rnh201Δ (strains KO_135 and KO188) and the rnh1Δrnh201Δ (strain KO_132) mutant backgrounds. A reporter system with a wild type ADE2 on the end of chromosome IV allowed us to detect crossovers by seeing a red-white sectored colony. Each reciprocal event was mapped by doing a microarray specific for the right arm of chromosome IV (St. Charles and Petes, 2013; PLoS Genet. 1267-1284 PMCID PMC3616911) on the red and white sides of sectors. 21 sectors were analyzed for the rnh201Δ mutant while 14 were analyzed for the rnh1Δ rnh201Δ mutant. We mapped crossovers on the right arm of chromosome IV in the rnh201Δ mutant in four different diploid isolates: KO135_5 (1R/1W, 2R/2W, 4R/4W, 5R/5W, 12R/12W, and 13R/13W), KO_135_6 (1R/1W, 4R/4W, 5R/5W, 7R/7W, 9R/9W, 11R/11W, 13R/13W, and 17R/17W), KO 188_1 (1R/1W, and 4R/4W), and KO188_2 (6R/6W, 7R/7W, 11R/11W, and 12R/12W). We mapped crossovers on the right arm of chromosome IV in the rnh1Δ rnh201Δ mutant in two different diploid isolates: KO_132_31 (2R/2W, 6R/6W, 8R/8W, 15R/15W, 17R/17W, 21R/21W, 22R/22W, and 24R/24W) and KO_132_29 (3R/3W, 7R/7W, 18R/18W, 24R/24W, and 34R/34W).

Project description:Resistance of Saccharomyces cerevisiae to high furfural concentration is based on NADPH-dependent reduction by at least two oxireductases. Biofuels derived from lignocellulosic biomass hold promises for a sustainable fuel economy, but several problems hamper their economical feasibility. One important problem is the presence of toxic compounds in processed lignocellulosic hydrolysates with furfural as a key toxin. While Saccharomyces cerevisiae has some intrinsic ability to reduce furfural to the less toxic furfuryl alcohol, higher resistance is necessary for process conditions. By comparing an evolved, furfural resistant strain and its parent in micro-aerobic, glucose-limited chemostats at increasing furfural challenge, we elucidate key mechanism and the molecular basis of both natural and high-level furfural resistance. At lower furfural concentrations, NADH-dependent oxireductases are the main defence mechanism. At concentrations above 15 mM, however, [1-13C]-flux and global array-based transcript analysis demonstrated that the NADPH-generating flux through pentose-phosphate pathway increases and that NADPH-dependent oxireductases became the major resistance mechanism. The transcript analysis further revealed that iron transmembrane transport is up-regulated in response to furfural. While these responses occur in both strains, high-level resistance in the evolved strain was based on strong induction of ADH7, the uncharacterised ORF YKL071W and 4 further, likely NADPH-dependent oxireductases. By overexpressing the ADH7 gene and the ORF YKL071W, we inverse engineered significantly increased furfural resistance in the parent strain, thereby demonstrating these two enzymes to be key elements of the resistance phenotype. Experiment Overall Design: RNA levels were measured in glucose limited, micro-aerobic chemostat cultures with different concentrations of the growth inhibitor furfural. Two strains were compared: TMB3400-FT30-3 is a strain that has been evolutionary adapted to withstand high furfural concentrations. TMB3400 is its less resistant parent. Number of biological replicates: 2-3.

Dataset Information

Genomic instability induced by furfural-treated in S. cerevisiae [sectored colonies]

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets