Using Yeast Transposon-Insertion Libraries for Phenotypic Screening and Protein Localization.

Abstract

This protocol details how to use a transposon-insertion library for phenotypic screening and protein localization. The insertion library was generated by mutagenesis of a plasmid-based yeast genomic DNA library by using a multipurpose transposon; the transposon produces gene disruptions, and, by Cre-mediated recombination at lox sites incorporated within the transposon, alleles with an in-frame insertion can be truncated to a residual transposon encoding multiple copies of the hemagglutinin epitope. Insertions are generated in yeast by shuttle mutagenesis. Yeast genomic DNA containing a transposon insertion is released from the library, and the mutagenized DNA sequences are introduced into a desired strain of yeast, where the insertion alleles replace native loci by homologous recombination. The insertion mutants can be screened for phenotypes, and the site of transposon insertion can subsequently be identified in selected mutants by inverse polymerase chain reaction (PCR). In-frame insertions within genes of interest can be truncated to an epitope-tagged allele by Cre-lox recombination, and the subcellular localization of the encoded protein product can be identified by standard methods of indirect immunofluorescence. In summary, the transposon-insertion libraries represent an informative resource for large-scale mutagenesis, presenting a straightforward alternative to labor-intensive targeted approaches for the construction of deletion alleles and fluorescent protein fusions.