Insertional mutagenesis: Transposon-insertion libraries as mutagens in yeast

Abstract

Publisher Summary Transposon-based insertional libraries are exceptionally useful as a means of screening for desired phenotypes and expression patterns. As compared to mutations generated by classical methods of chemical treatment and ultraviolet irradiation, transposon insertions are easy to identify: the transposon-encoded sequence serves as a marker or tag through which sites of insertion may be rapidly identified by polymerase chain reaction (PCR) amplification or plasmid rescue. Additionally, transposon insertion alleles can be uniquely informative: a single insertion may be sufficient to generate a gene disruption, reporter gene fusion, epitope-tagged allele, and conditional mutation. Transposons, if engineered appropriately, may also serve as gene traps identifying novel coding sequences. The chapter provides comprehensive protocols for the use of insertional libraries as mutagens in yeast. From the protocols presented, a plasmid-based library of transposon-mutagenized yeast DNA may be used to generate and identify yeast mutants of interest; transposon insertion sites within these mutants may be characterized by vectorette PCR as also described in the chapter. Also, by employing insertional libraries carrying a specially designed multipurpose transposon, insertions may be modified in yeast to generate corresponding epitope-tagged alleles for a variety of functional studies.