Genome-Wide Screening of Genes Required for XXXX Biosynthesis.

@entity1 @entity4495 @entity10611 @entity1009
UNASSIGNED: @entity4495 ( @entity10611 ) is synthesized and transferred to proteins in the endoplasmic reticulum (ER). @entity10611 -anchored proteins are then transported from the ER to the plasma membrane through the Golgi apparatus. To date, at least 17 steps have been identified to be required for the @entity10611 biosynthetic pathway. Here, we aimed to establish a comprehensive screening method to identify genes involved in @entity10611 biosynthesis using @entity1 haploid screens. @entity1 haploid cells were mutagenized by the integration of gene trap vectors into the genome. Mutagenized cells were then treated with a bacterial pore-forming toxin, aerolysin, which binds to @entity10611 -anchored proteins for targeting to the cell membrane. Cells that showed low surface expression of @entity1009 , a @entity10611 -anchored protein, were further enriched for. Gene trap insertion sites in the non-selected population and in the enriched population were determined by deep sequencing. This screening enriched 23 gene regions among the 26 known @entity10611 biosynthetic genes, which when mutated are expected to decrease the surface expression of @entity10611 -anchored proteins. Our results indicate that the forward genetic approach using haploid cells is a useful and powerful technique to identify factors involved in phenotypes of interest.
