Screen in Saccharomyces cerevisiae for transposon insertion sites able to rescue phenotype of MTM1 deletion mutant using mTn-lacZ/LEU2 transposon library

Abstract:

[Objective] MTM1 gene is essential for superoxide dismutase 2 activity and normal mitochondrial functions. MTM1 deletion results in decreased superoxide dismutase 2 activity, impaired mitochondrial functions and growth defect on nonfermentable carbon source. To promote understanding of MTM1 gene, we started a genetic screen for transposon insertions which are able to rescue the growth defect resulting from MTM1 deletion. [Methods] Routine screening strategy didn’t work because of the irreversible damage caused by MTM1 deletion. So we adopted the following screening strategy: we transformed a plasmid overexpressing MTM1 into wild type before deleting MTM1 in chromosome and got the resulting strain, designated YES2MTM1. Then we transformed mTn-lacZ/LEU2 transposon library into the YES2MTM1 strain. Transformants lost the plasmid overexpressing MTM1 after 5-Fluoroorotic acid treatment. We picked up the yeast strains able to grow on nonfermentable carbon source with MTM1 deletion and some transposon insertion and identified the insertion sites. [Results] We found transposon insertions in two genes were able to rescue the growth defect resulting from MTM1 deletion on nonfermentable carbon source. [Conclusion] Our study will provide reference for thorough understanding of MTM1 gene function.