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: 5.01). 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.

0 Followers
 · 
174 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Faithful chromosome segregation during meiosis is indispensable to prevent birth defects and infertility. Canonical genetic manipulations have not been very useful for studying meiosis II, since mutations of genes involved in cell cycle regulation or chromosome segregation may affect meiosis I, making interpretations of any defects observed in meiosis II complicated. Here we present a powerful strategy to dissect meiosis I and meiosis II, using chemical inhibitors in genetically tractable model organism fission yeast (Schizosaccharomyces pombe). As various chemical probes are not active in fission yeast, mainly due to an effective multidrug resistance (MDR) response, we have recently developed a drug-hypersensitive MDR-sup strain by suppression of the key genes responsible for MDR response. We further developed the MDR-supML (marker-less) strain by deleting 7 MDR genes without commonly used antibiotic markers. The new strain makes fluorescent tagging and gene deletion much simpler, which enables effective protein visualization in varied genetic backgrounds. Using the MDR-supML strain with chemical inhibitors and live cell fluorescence microscopy, we established cell cycle arrest at meiosis I and meiosis II and examined Aurora-dependent spindle assembly checkpoint (SAC) regulation during meiosis. We found that Aurora B/Ark1 kinase activity is required for recruitment of Bub1, an essential SAC kinase, to unattached kinetochore in prometaphase I and prometaphase II as in mitosis. Thus, Aurora's role in SAC activation is likely conserved in mitosis, meiosis I, and meiosis II. Together, our MDR-supML strain will be useful to dissect complex molecular mechanisms in mitosis and 2 successive meiotic divisions.
    Cell cycle (Georgetown, Tex.) 03/2014; 13(8). DOI:10.4161/cc.28294 · 5.01 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: In Schizosaccharomyces pombe, Eso1p is a protein fusion. Two-third of its N-Terminus is conserved to budding yeast Rad30, which functions in error-free replication of UV-damaged DNA. A third of the C-terminus is highly conserved to budding yeast Eco1, a lysine acetyltransferase, which is essential for the establishment of cohesion. Both Rad30p and Eco1p need to be finely tuned in budding yeast. Given the distinct function existed in Rad30p and Eco1p, it is enigmatic how the Eso1p, the protein fusion regulated in S.pombe, works. We have identified 2 forms of the Eso1 protein by Western blot, and detected the Eco1-homology fragment by M/S analysis following TAP purification of Eso1 protein. The result raises the possibility that Eso1 might be processed in vivo to release the Eco1-homology fragment, which allows the independent regulation of Rad30-homology and Eco1-homology fragments.
    Cell Biology International 05/2014; 38(5). DOI:10.1002/cbin.10230 · 1.64 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Analogue-sensitive (as) mutants of kinases are widely used to selectively inhibit a single kinase with few off-target effects. The analogue-sensitive mutant cdc2-as of fission yeast (Schizosaccharomyces pombe) is a powerful tool to study the cell cycle, but the strain displays meiotic defects, and is sensitive to high and low temperature even in the absence of ATP-analogue inhibitors. This has limited the use of the strain for use in these settings. Here, we used in vivo selection for intragenic suppressor mutations of cdc2-as that restore full function in the absence of ATP-analogues. The cdc2-asM17 underwent meiosis and produced viable spores to a similar degree to the wild-type strain. The suppressor mutation also rescued the sensitivity of the cdc2-as strain to high and low temperature, genotoxins and an anti-microtubule drug. We have used cdc2-asM17 to show that Cdc2 activity is required to maintain the activity of the spindle assembly checkpoint. Furthermore, we also demonstrate that maintenance of the Shugoshin Sgo1 at meiotic centromeres does not require Cdc2 activity, whereas localization of the kinase aurora does. The modified cdc2-asM17 allele can be thus used to analyse many aspects of cell-cycle-related events in fission yeast.
    Open Biology 07/2014; 4(7). DOI:10.1098/rsob.140063 · 4.56 Impact Factor

Full-text (2 Sources)

Download
35 Downloads
Available from
May 23, 2014