Publication Date:
2015-08-26
Description:
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.
Print ISSN:
0749-503X
Electronic ISSN:
1097-0061
Topics:
Biology
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