Article

Generation of a set of conditional analog-sensitive alleles of essential protein kinases in the fission yeast Schizosaccharomyces pombe

Max F Perutz Laboratories, University of Vienna, Vienna, Austria.
Cell cycle (Georgetown, Tex.) (Impact Factor: 4.57). 10/2011; 10(20):3527-32. DOI: 10.4161/cc.10.20.17792
Source: PubMed

ABSTRACT

The genome of the fission yeast Schizosaccharomyces pombe encodes for 17 protein kinases that are essential for viability. Studies of the essential kinases often require the use of mutant strains carrying conditional alleles. To inactivate these kinases conditionally, we applied a recently developed chemical genetic strategy. The mutation of a single residue in the ATP-binding pocket confers sensitivity to small-molecule inhibitors, allowing for specific inactivation of the modified kinase. Using this approach, we constructed conditional analog-sensitive alleles of 13 essential protein kinases in the fission yeast S. pombe.

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    • "To create a new sid2-as mutant strain, we employed site-directed mutagenesis (QuikChangeII, Site Directed Mutagenesis Kit) and mutated the methionine M285 gatekeeper residue to a glycine (Gly) as described by Cipak et al. (2011). However, instead of using a hygromycin-resistant sid2-as construct in a sid2D/ sid2 + heterozygous strain (Cipak et al. 2011), we PCR-amplified the sid2-as mutant allele, and the resulting DNA fragment was transformed in the sid2-150 strain. Growing colonies were selected at 35° (including revertants, suppressors, and carrying-sid2-as allele colonies). "
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    • "mutants at the restrictive temperature (data not shown), despite the fact that orb5.as8 cells show sensitivity to 30 mM 3BrB-PP1 (Snell and Nurse, 1994). This is consistent with the observation in Cipak et al. and suggests that either the level of inhibition exerted by the inhibitors is insufficient or that the orb phenotype observed in the temperature-sensitive mutant might not be the direct consequence of the withdrawal of the Orb5 kinase activity (Cipak et al., 2011). To test this latter possibility we shifted orb5.as8 cells to 36˚C as we added the inhibitor to assess whether the Orb phenotype would emerge as a combined consequence of Orb5 inhibition and heat shock, however, no Orb phenotype was seen. "
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