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Synthetic Lethality Resulted from AFR1 Over-Expression and MIH1/MPK1 Deletion in Saccharomyces Cerevisiae

Author: FanChun
Tutor: MaPingSheng
School: Tianjin University
Course: Biochemistry and Molecular Biology
Keywords: Saccharomyces cerevisiae synthetic lethal Afr1 Mpk1 Mih1
CLC: Q78
Type: Master's thesis
Year: 2009
Downloads: 24
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Abstract


The MAPK signal transduction pathways in Saccharomyces cerevisiae have important cross talks with each other. AFR1p works in the mating pheromone pathway and the cell wall integrity pathway respectively. It is found that AFR1p also has a role in the morphogenesis checkpoint. This work is mainly about the synthetic lethal phenomenon and synthetic lethal mechanism of Gal1p-AFR1-mpk1Δstrain and Gal1p-AFR1-mih1Δstrain.Plasmid pUC18-RYu-E-Gal1p-Afr1p-Afr1 was constructed and then transformed into W303-A strain. Through homologous recombination, Gal1 promoter was integrated into chromosom locus up stream of the AFR1 gene, generating Gal1p-AFR1 strain. This strain was subsequently crossed with a mpk1Δand a mik1Δstrain respectively and, after sporulation and tetrad dissection, strain Gal1p-AFR1-mpk1Δand Gal1p-AFR1-mih1Δwere obtained.Phenotipic analysis showed that, while galactose-induced AFR1 over-expression could induce hyperpolarization, the presence of mih1Δmutation further enhanced the hyperpolarization phenotype of the Gal1p-AFR1 strain.Gal1p-AFR1-mpk1Δstrain showed no growth on YPGal plates demonstrating that deletion of MPK1 and over-expression of AFR1 is synthetic lethal. This phenomenon was also observed at lower temperature. Gal1p-AFR1-mih1Δstrain did not form colonies on YPGal plate in the presence of 1 M sorbitol, suggesting that over-expression of AFR1 and knockout of MIH1 is synthetic lethal in the presence of 1M sorbitol .We explored the mechanism causing the above mentioned synthetic lethality by measuring zymolyase sensitivity of the double mutant strains. We found both Gal1p-AFR1-mpk1Δstrain and mpk1Δstrain were more sensitieve to zymolyase treatment compared to wild type strain, but the synthetic lethal phenotype and the cell wall integraty were not necessarily linked. Gal1p-AFR1-mih1Δstrain showed no difference with the wild type strain in terms of zymolyase sensitivity. This study also found that using galactose as a carbon source or adding osmotic supporting agent resulted increased resistance to zymolyase.

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