These plasmids have been designed for tagging fission yeast genes with the DHFR degron (Dohman et al 1994). This degron can confer temperature sensitivity on proteins in fission yeast as in S. cerevisiae - the degron is recognized by an E3 enzyme at 36°C but not at 25°C. We have used this approach to make existing ts alleles tighter (Gregan et al 2003; Lindner et al. 2002). In some cases, just tagging the wild type version of the gene with the degron will generate a tight ts allele, although the success we have had here is more variable (see also Rajagopalan et al 2004). We find that deletion of ubr11 removes the ts phenotype associated with the degron, implying that this gene encodes the relevant E3 activity. The strategy of overexpressing the E3 encoding gene under the nmt1 (wt) promoter to enhance the ts phenotype of the degron (Labib et al. 2000), does not appear to work in S. pombe (unpublished results, also see Rajagopalan et al 2004).
Plasmids available:
The degron is expressed from either the thiamine-repressible nmt1 promoter, or the constitutitive mcm4 (cdc21) promoter. The degron is expressed fused to N-terminal ubiquitin; cleavage of the ubiquitin yields a degron-HA epitope-protein fusion (this has N-terminal arginine which is a destabilizing amino acid according to the N-end rule).
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Plasmid
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Code
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Promoter
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Other markers
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pSMUG2-mcm4-degronHA
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453
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mcm4 (cdc21)
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ura4+
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pSMRG2-mcm4-degronHA
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455
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mcm4 (cdc21)
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KanR
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pSMRG2-nmt41-degronHA
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501
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nmt1 (41)
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KanR
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pSMUG2-nmt41-degronHA
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502
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nmt1 (41)
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ura4+
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pSMRG2-nmt1-degronHA
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503
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nmt1 (wt) |
KanR
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pSMUG2-nmt1-degronHA
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504
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nmt1 (wt) |
ura4+
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pSMRG2-nmt81-degronHA
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552
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nmt1 (81)
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KanR
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pSMUG2-nmt81-degronHA
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554
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nmt1 (81)
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ura4+
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Promoter strength nmt1 (wt strongest) > 41 ex REP41X plasmid (middle strength) > 81 ex REP81X plasmid (weakest) promoter. The non-regulatable cdc21/mcm4 promoter expresses at a level comparable to nmt1(41).
To modify your gene of interest:
1. Use long oligos to amplify the segment
NgoMI-(ura4+or KanR)-NgoM1- promoter-ubiquitin-DHFR tsdegron-HA,
so as to fuse DHFR-HA with the N terminus of the reading frame to be modified. Approximate regions to use as pcr primers are shown on the map as long oligo 1 (forward), long oligo 2 (reverse).
2. Or clone the N terminal region of your gene into the region downstream of the DHFR degron to give an in frame fusion: the unique sites XhoI, BglII, SmaI, SalI can be used for this. Cut the resulting plasmid in the N terminal region of your gene (make sure there is a unique site when you design your oligos) to integrate (the resulting strain will express an N terminal truncated version of your gene as well as the degron version).
References:
Dohman, RJ, Wu, P, and Varshavsky, A. (1994) Heat-inducible degron: a method for constructing temperature-sensitive mutants. Science. 263:1273-1276.
Gregan, J., Lindner, K., Brimage, L., Franklin, R., Namdar, M., Hart, E.A., Aves, S.J. and Kearsey, S.E. (2003) Fission yeast Cdc23/Mcm10 functions after pre-replicative complex formation to promote Cdc45 chromatin binding. Molecular Biology of the Cell, 14, 3876-3887.
Labib, K., Tercero, J. and Diffley, J. (2000) Uninterrupted MCM2-7 function required for DNA replication fork progression. Science, 288, 1643-1647.
Rajagopalan, S., Liling, Z., Liu, J. and Balasubramanian, M. (2004) The N-degron approach to create temperature-sensitive mutants in Schizosaccharomyces pombe. Methods, 33, 206-212.
Stephen Kearsey
stephen.kearsey@zoo.ox.ac.uk