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


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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Available from: Eva Miadokova, Oct 10, 2015
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    ABSTRACT: In the fission yeast Schizosaccharomyces pombe, meiosis is inhibited by the protein kinase Pat1, which phosphorylates and inactivates Mei2, an RNA binding protein essential for the initiation of meiosis. When diploid cells are deprived of nutrients, they initiate a cascade of events leading to the inactivation of Pat1 and entry into meiosis. Strains carrying the temperature-sensitive pat1-114 allele are forced to enter into meiosis when shifted to the non-permissive temperature, independently of the ploidity of the cell. This system has been extensively used, since it is possible to achieve a highly synchronous meiosis, which is a must for any molecular or microscopic approach that aims to decipher the mechanisms governing meiosis. Here, we have designed a new system to obtain a similarly synchronous meiosis, but independently of temperature shifts. Thus, by introducing a mutation in the ATP pocket of Pat1, we have generated a protein kinase that, in the presence of small specific inhibitors, can be inactivated. This results in forced entry into meiosis without the need of a temperature shift, minimizing the introduction of heat shock or any other stress responses along the meiotic waves of transcription.
    Cell cycle (Georgetown, Tex.) 04/2012; 11(8):1621-5. DOI:10.4161/cc.20051 · 4.57 Impact Factor
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    ABSTRACT: To study meiosis, synchronous cultures are often indispensable, especially for physical analyses of DNA and proteins. A temperature-sensitive allele of the Pat1 protein kinase (pat1-114) has been widely used to induce synchronous meiosis in the fission yeast Schizosaccharomyces pombe, but pat1-114-induced meiosis differs from wild-type meiosis, and some of these abnormalities might be due to higher temperature needed to inactivate the Pat1 kinase. Here, we report an ATP analog-sensitive allele of Pat1 [Pat1(L95A), designated pat1-as2] that can be used to generate synchronous meiotic cultures at physiological temperature. In pat1-as2 meiosis, chromosomes segregate with higher fidelity, and spore viability is higher than in pat1-114 meiosis, although recombination is lower by a factor of 2–3 in these mutants than in starvation-induced pat1+ meiosis. Addition of the mat-Pc gene improved chromosome segregation and spore viability to nearly the level of starvation-induced meiosis. We conclude that pat1-as2 mat-Pc cells offer synchronous meiosis with most tested properties similar to those of wild-type meiosis.
    Cell cycle (Georgetown, Tex.) 04/2012; 11(8):1626-33. DOI:10.4161/cc.20052 · 4.57 Impact Factor
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    ABSTRACT: Comment on: Cipak L, et al. Cell Cycle 2012; 11:1625-32 and Guerra-Moreno A, et al. Cell Cycle 2012; 11:1620-4.
    Cell cycle (Georgetown, Tex.) 05/2012; 11(9):1755-6. DOI:10.4161/cc.20314 · 4.57 Impact Factor
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