Publicação
The yeast DEL assay with dominant genetic markers
| Resumo: | The yeast DEL assay is a genotoxicity test that works by measuring the frequency of reversion of two disrupted his3 alleles sharing approximately 400 bp of homology by intrachromosomal recombination. This event leads to recovery of histidine prototrophy and deletion (DEL) of the intervening LEU2 marker and leucine auxotrophy. This assay has provided results displaying high correlation for well-known carcinogens with standardized and validated in vitro genotoxicity assays such as the Ames test. In this work, a novel version of the yeast DEL assay containing dominant genetic markers (dDEL) using antibiotic resistance genes KanR and Hph was developed to expand its application to wild-type yeast strains. The dDEL system enables quantitative measurement of genotoxicity using industrially relevant yeast strains lacking auxotrophic markers. For the construction of the dDEL assay, a shuttle plasmid was assembled from seven linear DNA fragments as a genetic support of the dDEL system, part of which was subsequently integrated in the HIS3 locus of the desired strains. The genetic constructions were performed through the yeast homologous recombination machinery. The yeast dDEL system was established in the laboratory strains CEN.PK 112-3A and RS112 (substituting the original DEL cassette), and in the industrial ethanol production strain PE-2. The functionality of the dDEL assay was assessed with hydrogen peroxide in a comparison with the original DEL assay. The DEL and dDEL systems in laboratory strains performed in a similar manner, and, interestingly, dDEL presented the highest relative increase in the recombination frequency. The dDEL assay in the PE-2 strain was used to evaluate the genotoxicity of furfural, which is a main fermentative inhibitor present in lignocellulosic hydrolysates. In this work, furfural was positively detected as genotoxic by the RS112 strain, although it displayed no clear genotoxic damage in the PE-2 strain, perhaps reflecting differences in the DNA repair machinery. This result supports the notion that performing genotoxicity resistance and other tests in the desired relevant biotechnological strains is vital in order to select stronger and more robust industrial yeast strains for a specific purpose. |
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| Autores principais: | Silva, Paulo César Fernandes da |
| Assunto: | Ciências Naturais::Ciências Biológicas |
| Ano: | 2017 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | The yeast DEL assay is a genotoxicity test that works by measuring the frequency of reversion of two disrupted his3 alleles sharing approximately 400 bp of homology by intrachromosomal recombination. This event leads to recovery of histidine prototrophy and deletion (DEL) of the intervening LEU2 marker and leucine auxotrophy. This assay has provided results displaying high correlation for well-known carcinogens with standardized and validated in vitro genotoxicity assays such as the Ames test. In this work, a novel version of the yeast DEL assay containing dominant genetic markers (dDEL) using antibiotic resistance genes KanR and Hph was developed to expand its application to wild-type yeast strains. The dDEL system enables quantitative measurement of genotoxicity using industrially relevant yeast strains lacking auxotrophic markers. For the construction of the dDEL assay, a shuttle plasmid was assembled from seven linear DNA fragments as a genetic support of the dDEL system, part of which was subsequently integrated in the HIS3 locus of the desired strains. The genetic constructions were performed through the yeast homologous recombination machinery. The yeast dDEL system was established in the laboratory strains CEN.PK 112-3A and RS112 (substituting the original DEL cassette), and in the industrial ethanol production strain PE-2. The functionality of the dDEL assay was assessed with hydrogen peroxide in a comparison with the original DEL assay. The DEL and dDEL systems in laboratory strains performed in a similar manner, and, interestingly, dDEL presented the highest relative increase in the recombination frequency. The dDEL assay in the PE-2 strain was used to evaluate the genotoxicity of furfural, which is a main fermentative inhibitor present in lignocellulosic hydrolysates. In this work, furfural was positively detected as genotoxic by the RS112 strain, although it displayed no clear genotoxic damage in the PE-2 strain, perhaps reflecting differences in the DNA repair machinery. This result supports the notion that performing genotoxicity resistance and other tests in the desired relevant biotechnological strains is vital in order to select stronger and more robust industrial yeast strains for a specific purpose. |
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