For a full description of our transposon-tagging strategy, please see:
Ross-Macdonald et al. (1999). Nature 402, 413-418.
We have developed a series of novel multipurpose minitransposons (Ross-Macdonald et al., 1997) capable of generating reporter fusions, null mutations, epitope-tagged alleles and hypomorphic mutants all from a single mutagenesis. The Tn3-derived minitransposon mTn-3xHA/lacZ has been used most extensively in our studies. It features the following:
The promoter-less lacZ gene allows production of b-galactosidase in yeast strains containing this mTn inserted within a transcribed and translated region of the genome, typically corresponding to an in-frame fusion of lacZ to yeast protein coding sequence. Productive lacZ fusions can be used to generate expression data by measurement of b-gal activity. Also, mTn insertion creates a truncation of the mutagenized gene, thereby generating disruption alleles for subsequent phenotypic analysis.
Cre-mediated site-specific recombination between the lox sites results in excision of the central body of the transposon. The remaining mTn insertion element encodes 93 amino acids containing three copies of an epitope from the influenza virus hemagglutinin protein (the HA epitope). As this insertion element encodes no translational stops, a yeast strain bearing an in-frame mTn insertion may be used to derive a corresponding strain containing an in-frame HA-tag (useful for immunolocalization of mTn-tagged proteins -- also as a means of generating conditional alleles and hypomorphic mutants).
We have applied this principle to the large-scale analysis of gene function in yeast.
Our multipurpose minitransposons are used as follows: By shuttle mutagenesis, mTn insertions are generated in a plasmid library of yeast genomic DNA in E. coli. Individual tet-resistant colonies are selected, and plasmid DNA is prepared from these transformants in 96-well format.
Plasmid DNA from each transformant is digested with Not I to release the yeast genomic DNA; mutagenized yeast DNA is then used to individually transform a diploid strain of yeast (in 96-well format). By homologous recombination, the mTn-mutagenized fragment will integrate at its native chromosomal locus. Subsequently, Ura+ transformants are selected and assayed for b-gal activity. Yeast strains producing b -gal are stored in a permanent collection. Plasmid DNA samples prepared from corresponding bacterial transformants are sequenced in order to determine the precise chromosomal site of mTn insertion within each strain. This plasmid DNA is also used to transform a haploid yeast strain (in 96-well format) for large-scale analysis of disruption phenotypes.
Yeast strains containing in-frame fusions to lacZ are treated to induce expression of the Cre recombinase; Cre is present on plasmid pGAL-cre, where it has been placed under transcriptional control of the GAL promoter. Following galactose induction, Ura- colonies, presumably having undergone the loxexcision event, are selected and maintained in a collection used for immunolocalization of epitope-tagged proteins with anti-HA antibodies.
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