Viral dsRNA and killer system in Saccharomyces paradoxus
File(s)
Author(s)
Nematbakhsh, Mahsan
Type
Thesis
Abstract
Some yeast strains (killer strains) release killer toxins which kill other strains of the same or different species of yeast but not the strain producing the toxin itself. In Saccharomyces cerevisiae, killer toxins are encoded by either dsRNA viruses or nuclear genes.
This study aims to characterise the genetic basis of the killer toxin synthesised in Saccharomyces paradoxus, a wild non-domesticated relative of S. cerevisiae. One hundred and nine stains of S. paradoxus gathered from Silwood Park, Continental Europe, Far East, and North America were screened for the killer-immune phenotype against killer and sensitive yeast strains. In order to find whether the killer toxin is encoded by dsRNA or a nuclear gene, the killer strains were treated by cyclohexamide that removes the dsRNA which carries the killer toxin gene. The strains were also screened for presence and absence of dsRNA by directly visualising the dsRNA on the agarose gel and also by Next Generation Sequencing (NGS).
Several strains were found to have the killer phenotype (30% of the screened strains). In the majority of killer strains the toxins were encoded by dsRNA except the strains from Canada and one strain from continental Europe, which seems to have other genetic bases. Sixteen full sequences of large dsRNA variants (L-A; with the length of about 4.5 kb) composed of L-A-Q, L-A-D1, L-A-C, L-A-P1.1, L-A-P1.2, L-A-P1.3, L-A-P1.4, L-A-P1.5, L-A-P1.6, L-A-P1.7, L-A-P2.1, L-A-P2.2, L-A-P2.3, L-A-P2.4, L-A-P2.5, and L-A-P2.6, and seven new types of medium size dsRNA (M; with the length between 0.8 and 2kb) composed of MQ, MC, M-P1G1, M-P1G2, M-P1G3-1, M-P1G3-2, M-P1G5, and M-P1SG, have been identified in this study. M28 was the only M dsRNA in S. paradoxus which was nearly identical to the M28 from S. cerevisiae; therefore likely to have been transferred between the two species.
To test whether the killer toxins were encoded from the new types of M dsRNAs, the MQ ORF sequence from S. paradoxus was cloned and expressed into S. cerevisiae and then tested for the killer phenotype. The S. paradoxus MQ protein was successfully expressed in the S. cerevisiae strain and a positive killer response in the killer assay confirmed its function as a killer toxin.
This study aims to characterise the genetic basis of the killer toxin synthesised in Saccharomyces paradoxus, a wild non-domesticated relative of S. cerevisiae. One hundred and nine stains of S. paradoxus gathered from Silwood Park, Continental Europe, Far East, and North America were screened for the killer-immune phenotype against killer and sensitive yeast strains. In order to find whether the killer toxin is encoded by dsRNA or a nuclear gene, the killer strains were treated by cyclohexamide that removes the dsRNA which carries the killer toxin gene. The strains were also screened for presence and absence of dsRNA by directly visualising the dsRNA on the agarose gel and also by Next Generation Sequencing (NGS).
Several strains were found to have the killer phenotype (30% of the screened strains). In the majority of killer strains the toxins were encoded by dsRNA except the strains from Canada and one strain from continental Europe, which seems to have other genetic bases. Sixteen full sequences of large dsRNA variants (L-A; with the length of about 4.5 kb) composed of L-A-Q, L-A-D1, L-A-C, L-A-P1.1, L-A-P1.2, L-A-P1.3, L-A-P1.4, L-A-P1.5, L-A-P1.6, L-A-P1.7, L-A-P2.1, L-A-P2.2, L-A-P2.3, L-A-P2.4, L-A-P2.5, and L-A-P2.6, and seven new types of medium size dsRNA (M; with the length between 0.8 and 2kb) composed of MQ, MC, M-P1G1, M-P1G2, M-P1G3-1, M-P1G3-2, M-P1G5, and M-P1SG, have been identified in this study. M28 was the only M dsRNA in S. paradoxus which was nearly identical to the M28 from S. cerevisiae; therefore likely to have been transferred between the two species.
To test whether the killer toxins were encoded from the new types of M dsRNAs, the MQ ORF sequence from S. paradoxus was cloned and expressed into S. cerevisiae and then tested for the killer phenotype. The S. paradoxus MQ protein was successfully expressed in the S. cerevisiae strain and a positive killer response in the killer assay confirmed its function as a killer toxin.
Version
Open Access
Date Issued
2018-05
Date Awarded
2019-01
Copyright Statement
Creative Commons Attribution NonCommercial Licence
Advisor
Burt, Austin
Koufopanou, Vassiliki
Publisher Department
Life Sciences
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)