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Transposon mutagenesis allows for the discovery and characterization of genes by creating mutations that can be easily mapped and sequenced. Moreover, this method allows for a relatively unbiased approach to isolating genes of interest. Recently, a system of transposon based mutagenesis for Schizosaccharomyces pombe became available. This mutagenesis relies on Hermes, a DNA transposon from the house fly that readily integrates into the chromosomes of S. pombe. The Hermes system is distinct from the retrotransposons of S. pombe because it efficiently integrates into open reading frames. To mutagenize S. pombe, cells are transformed with a plasmid that contains a drug resistance marker flanked by the terminal inverted repeats of Hermes. The Hermes transposase expressed from a second plasmid excises the resistance marker with the inverted repeats and inserts this DNA into chromosomal sites. After S. pombe with these two plasmids grow 25 generations, approximately 2% of the cells contain insertions. Of the cells with insertions, 68% contain single integration events. The protocols listed here provide the detailed information necessary to mutagenize a strain of interest, screen for specific phenotypes, and sequence the positions of insertion.
Transposon mutagenesis is an important tool used in many model organisms to screen for genes involved in a variety of processes (1–6). Unfortunately, in the yeasts the endogenous transposons possess unique targeting mechanisms that direct integration to specific sites in the host chromosomes (7–9). As a result, these transposons do not actively target open reading frames (ORFs) and can not function as efficient mutagens. To overcome this problem, we have tested an exogenous DNA transposon called Hermes from the house fly (10). In the yeast Schizosaccharomyces pombe, Hermes has high transposition activity and in 54% (14 of 26) of insertions, open reading frames are disrupted (10). Although the proportion of inserts that disrupt ORFs may vary from experiment to experiment, we have been able to generate insertion mutations in specific genes with frequencies expected for unbiased integration (10). The Hermes system can be used in virtually any strain background, making it more versatile than screening a set of deletions. The ease of creating a mutant library and identifying the insertion sites via inverse PCR also make it desirable. That some of these mutations result in hypomorphic alleles of essential genes is another valuable feature that distinguishes insertional mutagenesis from deletion sets.
The Hermes transposon when mobilized from a plasmid and grown in S. pombe for 25 generations produces insertions in approximately 2% of the cells (10). About 70% of the strains contain a single integration of Hermes. When 106,000 colonies with integration events are screened for disruptions of genes required for adenine biosynthesis, five disruptions of ade6 and two of ade7 can be isolated (10).
The mutagenesis system available with Hermes requires that two plasmids be introduced into the strain of S. pombe chosen for mutagenesis (10). One plasmid contains the transposase gene driven by the Rep81X nmt1 promoter. This plasmid also contains LEU2 to select for its presence in S. pombe (Fig. 1, pHL2578). While the Rep41X version of the nmt1 promoter allows for higher levels of expression and greater numbers of cells with insertions, the Rep81X version tends to avoid fluctuations in the proportion of the cells that have insertions (Evertts and Levin, unpublished). The second plasmid introduced into S. pombe contains the donor sequence, which consists of kanMX6 flanked by the inverted terminal repeats, Hermes right and Hermes left. The donor plasmid is marked with URA3, to allow for selection in S. pombe (Fig. 1, pHL2577). Transposition occurs when the transposase (Tpase) cleaves the Hermes right and left sequences in the donor plasmid and inserts this fragment containing kanMX6 into chromosomal sequences. Cells with copies of kanMX6 inserted into S. pombe chromosomes are selected on medium containing G418. The direct selection for integration is made possible because we also include 5-fluoroorotic acid (5-FOA) in the medium. 5-FOA selects against cells containing the donor plasmid with the original copy of kanMX6. Using this system of two plasmids, extensive libraries of transposon insertions can be generated, the mutant strains can be screened for phenotypes, and positions of the insertion events can be sequenced, all with a relatively simple set of procedures (Fig. 2).
The high level of Hermes integration in exogenous hosts provides a unique opportunity to apply the highly valued technique of transposon-mediated mutagenesis to study the genes of S. pombe. The reagents necessary for these experiments are readily available from our laboratory.
This research was supported by the Intramural Research Program of the NIH from the Eunice Kennedy Shriver National Institute of Child Health and Human Development.
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