Development Of A PiggyBac Transposon System For Genetic Mutation In Budding Yeast

The piggyBac(PB)transposon is a TTAA-specific transposon isolated from the cabbage looper moth,Trichoplusia ni.The PB transposon system works in various eukaryotic cells,and is used as a tool for insertional mutagenesis.Chemical mutagenesis and irradiation have been generally used to obtain mutant cells.However,identification of the gene responsible for a mutant phenotype requires plasmid complementation or positional cloning,which takes time and is sometimes difficult.Compared with classical mutagenesis,PB transposon insertional mutagenesis is advantageous for identifying the gene responsible for a mutation,because common sequences in the insertional module can be used to determine the insertion site.It has been reported that the PB transposon system can be used as a mutagenesis tool in many organisms including mammal cells,plants,insects,and fission yeast.However,it has not yet been adapted for genetic mutation in budding yeast.In this study,we established screening methods using the PB transposon in budding yeast(Saccharomyces cerevisiae).We constructed a yeast strain with a 3?PB-TEFp-HIS3-5?PB element,which was inserted in the ADE2 gene coding sequence.The PBase was expressed under a galactose-inducible promoter.After the PBase was expressed in galactose medium,the PB element was precisely excised from and re-inserted into the genome in yeast.The PB element keeps one copy during transposition,and only one insertion site was found in each mutant cell.The DNA region containing the inserted PB elements can be easily identified.These results indicated the PB system we constructed worked well in budding yeast,and genetic screening in budding yeast can be done through PB insertion.To check the usefulness of the PB transposon system for in vivo mutagenesis in yeast,we adapted it to screen for factors involved in localization of the Golgi-localized ?-1,6-mannosyltransferase(Och1p).Och1 p is mainly localized in the cis-Golgi,but is constantly recycled within the Golgi.Once Och1 p moves to the trans-Golgi,it is recycled back to the cisGolgi by anterograde retrieval transport.It has been reported that several factors are involved in Och1 p retention in the cis-Golgi,but how Och1 p localization is regulated is not well understood.We constructed short Och1-Suc2 fusion protein.Because it was mainly localized in the intracellular organelle,expression of this fusion protein in suc2? yeast strain made the cell show slow growth in sucrose plate.If this fusion protein was secreted or localized on the cell surface by mutation,the mutant cells would grow faster on sucrose plate.With this growth defect and PB screening system,one mutant strain named M6 grew faster than parental strain.The PB element was integrated into the promoter region of the SCY1 gene.The amount of SCY1 gene expression level in M6 mutant strain was about 70% of parental strain.Overexpression of SCY1 caused short-Och1 protein go to vacuole,resulting the decrease in the amount of short-Och1 protein in the Golgi.Also the glycosylation and secretion of invertase were impaired in SCY1-overexpressing cells.In the screen,the SCY1 gene was identified as a candidate gene required for correct localization of Och1 p.This observation suggests our PB-based screening is useful for isolating genes required for phenotypes of interest in budding yeast.