Abstract
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.
Original language | English |
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Pages (from-to) | 703-710 |
Number of pages | 8 |
Journal | Yeast |
Volume | 32 |
Issue number | 12 |
Early online date | 17 Sept 2015 |
DOIs | |
Publication status | Published - Dec 2015 |
Bibliographical note
Copyright © 2015 John Wiley & Sons, Ltd.Funded by College of Life Science and Medicine, University of Aberdeen, UK
This work was funded by a start-up
grant from the College of Life Science and Medicine, University
of Aberdeen, UK. I am grateful to J. Bähler, E. Hartsuiker, F. Klein,
J. Kohli, K. Nasmyth, M. C. Whitby, the Leibniz Institute – German
Collection of Microorganisms and Cell Cultures (DMSZ) and the
National BioResource Project Japan (NBRP) for providing
materials used in this study. I thank Alistair J. P. Brown and
Takashi Kubota for critically reading this manuscript.
Keywords
- Schizosaccharomyces pombe
- selectable marker
- marker switch
- plasmid
- PCR
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Plasmid sequences - New cassettes for single-step drug-resistance and prototrophic marker switching in fission yeast
Lorenz, A. (Creator), Figshare, 12 Aug 2015
DOI: 10.6084/m9.figshare.1468419.v1
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