Schizosaccharomyces pombe, also known as fission yeast, is an established model for studying chromosome biological processes. Over the years, research employing fission yeast has made important contributions to our knowledge about chromosome segregation during meiosis, as well as meiotic recombination and its regulation. Quantification of meiotic recombination frequency is not a straightforward undertaking, either requiring viable progeny for a genetic plating assay, or relying on laborious Southern blot analysis of recombination intermediates. Neither of these methods lends itself to high-throughput screens to identify novel meiotic factors. Here, we establish visual assays novel to Sz. pombe for characterizing chromosome segregation and meiotic recombination phenotypes. Genes expressing red, yellow, and/or cyan fluorophores from spore-autonomous promoters have been integrated into the fission yeast genomes, either close to the centromere of chromosome 1 to monitor chromosome segregation, or on the arm of chromosome 3 to form a genetic interval at which recombination frequency can be determined. The visual recombination assay allows straightforward and immediate assessment of the genetic outcome of a single meiosis by epi-fluorescence microscopy without requiring tetrad dissection. We also demonstrate that the recombination frequency analysis can be automatized by utilizing imaging flow cytometry to enable high-throughput screens. These assays have several advantages over traditional methods for analyzing meiotic phenotypes.
Bibliographical noteWe are grateful to Scott Keeney, Franz Klein, Jürg Kohli, Josef Loidl, Kim Nasmyth, Ken E. Sawin, Gerald R. Smith, Takashi Toda, Matthew C. Whitby, and the National BioResource Project (NBRP) Japan for providing strains and/or plasmids; to M.N. Asogwa, A. Bebes, and L. Duncan for technical assistance; and to M. De Carvalho for spotting a critical typographical error in an earlier version of the manuscript. Microscopy was performed at the University of Aberdeen Microscopy & Histology facility (Kevin Mackenzie). This work was supported by a Carnegie Trust for the Universities of Scotland Research Incentive Grant (No. 70021), and the University of Aberdeen (College of Life Sciences and Medicine Start-up grant).
Open Access via the Springer Compact Agreement.
- Schizosaccharomyces pombe
- chromosome segregation
- meiotic recombination
- spore autonomous promoters
- imaging flow cytometry
- Meiotic recombination
- Spore-autonomous promoters
- Imaging flow cytometry
- Chromosome segregation
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Andrea Holme (Manager)Iain Fraser Cytometry Centre
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