Construction Of Saccharomyces Cerevisiae Mutants Deleted ERG 6 Gene And Application In Genotoxic Chemical Detection

Both natural and man-made genotoxic chemicals can damage DNA, resulting in cell death or cancers due to accumulated genetic mutations (Friedberg, E.C. et al.1995). Sensitive, simple methods are therefore required for screening and monitoring a wide range of environmental pollutants and synthetic compounds for potential genotoxic activity.A genotoxic testing system based on yeast transcriptional response to DNA damage(Walmsley et al.1997, Afanassiev et al. 2000, Jia, X. et al. 2002) has shown some advantages: this testing system has not only traits of eukaryotes, but also traits of prokaryotic bacteria for easy culture and gene operation, which can detect various kinds of genotoxic chemicals and appears to be more sensitive than other similar tests in microorganisms. Due to the permeability of yeast cells and its simpler metabolic activation system, this system has been compromised for practical applications. In order to apply this testing system for environmental detection, further enhancement of detection sensitivity and spectrum is desired. In this desertation, we have attempted genetic manipulation in the following areas:1. Enhance permeability of yeast cells:In this study, a foreign gene targeting cassette was constructed from plasmid pBlue-Kan and Saccharomyces cerevisia by PCR. It consisted of a G418 resistance gene with long flanking homology regions of ERG 6 gene, and transferred into the AD1-8. It integrated into Saccharomyces cerevisia genome due to homologous recombination. Thus ERG 6 gene was disrupted and an mutant was obtained. The results show that inactivation of ERG6 gene encoding C-24 sterol methyltransferase has profound effects on the cell wall structure and permeability, and markedly enhances the system sensitivity to genotoxic chemicals.2. Select gene promoters that are sensitively and specifically induced by DNA damage:In this study, we select RNR2 promoter as a promoter of DNA damage inducible, and introduce the LexA yeast two-hybrid system into the detection of genetic toxicity system at the same time.The results show that the deletion of ERG6 could enhance the induction of b-gal activity by causing cell wall defects and thus increasing cellular permeability.In summary, we had enhanced sensitivity of the yeast genotoxic testing system through altering different biological pathways, which provide more research groundwork for this as well as other similar testing systems into practical applications. This study also provides insights into the mechanisms of cellular response to DNA damage in eukaryotes.