Biochemical and Biophysical Research Communications
Fission yeast dam1-A8 mutant is resistant to and rescued by an anti-microtubule agent
Section snippets
Materials and methods
Schizosaccharomyces pombe strains, media and genetic methods. The strains used in this study are listed in Table 1. The growth and the maintenance of the strains were carried out according to standard methods [11].
Generation of temperature sensitive mutants. The screening approach we formulated involved PCR-based random mutagenesis of a template DNA fragment, which contains the dam1+ gene tagged with GFP and a selectable drug resistance marker kanr (kanamycin). Out of ∼400 kanamycin-resistant
Isolation of a temperature sensitive dam1 mutant in a klp5 null background
It has previously been shown that Dam1-complex deletion mutants are synthetic lethal when combined with deletion mutants of the fission yeast kinesin-8 members Klp5 or Klp6 [8]. In order to garner further understanding of the essential overlapping function of the Dam1 complex and kinesin-8, we screened for a temperature sensitive dam1 mutant in a Δklp5 background. We identified dam1-A8 (Fig. 1A), which contains two C-G point mutations, resulting in a change of amino acids H126R and E149G in the
Discussion
Fission yeast Dam1 complex, albeit not essential, has been implicated in the establishment of chromosome bi-orientation and plays an essential overlapping role with kinesin-8 members Klp5 and Klp6 [7], [8], [9], [10]. In this study, we have generated a temperature sensitive dam1 mutant in a Δklp5 mutant background and characterised the phenotypes of this double mutant to uncover the functional interplay between Dam1 and Klp5/6. The dam1-A8 mutant contained two point mutations in the C-terminal
Acknowledgments
We thank Keith Gull, Yasushi Hiraoka, Nirada Koonrugsa, Osami Niwa, Amy Unsworth, and Ayumu Yamamoto for providing materials used in this study. We thank Rafael Carazo-Salas for his technical support and training for the Deltavision microscope. We are grateful to Frank Uhlmann for discussion. Cancer Research UK (T.T.) supported this work.
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2013, Current BiologyCitation Excerpt :However, the temperature-sensitive double mutant dam1-A8:klp5Δ is viable (but very sick) at room temperature and lethal at the nonpermissive 37°C [33]. Our numerous attempts to construct the triple deletion-inactivation mutant cut7.24ts:dam1-A8:klp5Δ proved unsuccessful, most likely because dam1-A8:klp5Δ itself is very sick, even at permissive temperature [33]. Nevertheless, the dam1-A8:klp5Δ double mutant exhibits longer metaphase spindles compared to the individual mutants dam1Δ and klp5Δ or to the wild-type cells (Figures S2B and S2C), consistent with the tug-of-war analogy.
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