New cassettes for single-step drug-resistance and prototrophic marker switching in fission yeast

Construction of multiply mutated strains for genetic interaction analysis and of strains carrying different epitope tags at multiple open reading frames for testing protein localization, abundance and protein-protein interactions is hampered by the availability of a sufficient number of different selectable markers. Moreover, strains with single gene deletions or tags often already exist in strain collections; for historical reasons these will mostly carry the ura4(+) gene or the G418-resistance kanMX as marker. Because it is rather cumbersome to produce multiply deleted or tagged strains using the same marker or to completely reconstruct a particular strain with a different marker, single-step exchange protocols of markers are a time-saving alternative. In recent years dominant drug resistance markers (DDRMs) against clonNAT, Hygromycin B, and Bleomycin have been adapted and successfully used in Schizosaccharomyces pombe. The corresponding DDRM cassettes - natMX, hphMX, and bleMX - all carry the TEF-promotor and -terminator sequences from Ashbya gossypii as kanMX, this provides flanking homologies to enable single-step marker swapping by homologous gene targeting. To expand this very useful toolset for single-step marker exchange I constructed MX-cassettes containing the nutritional markers arg3(+) , his3(+) , leu1(+) , and ura4(+) . Furthermore, a set of constructs was created to enable single-step exchange of ura4(+) to kanMX6, natMX4, and hphMX4. The functionality of the cassettes is demonstrated by successful single-step marker swapping at several loci. These constructs allow a straight-forward and rapid re-marking of existing ura4(+) - and MX-deleted and -tagged strains.