Abstract
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.
Highlights
IntroductionMeiosis is a highly conserved process that produces haploid sex cells (gametes) as an integral part of sexual reproduction
Meiosis is a highly conserved process that produces haploid sex cells as an integral part of sexual reproductionThis article is part of a Special Issue on Recent advances in meiosis from DNA replication to chromosome segregation Bedited by Valérie Borde and Francesca Cole, co-edited by Paula Cohen and Scott Keeney^.Electronic supplementary material The online version of this article contains supplementary material, which is available to authorized users.(Hunter 2015)
To avoid ectopic recombination events between the Peis1 and Ppil2 constructs and the upstream regions of endogenous eis1 and pil2, we decided to follow a similar strategy as Keeney and co-workers (Thacker et al 2011), and investigated whether Peis1 and Ppil2 from Schizosaccharomyces species other than Sz. pombe can be used as spore-autonomous promoters in Sz. pombe
Summary
Meiosis is a highly conserved process that produces haploid sex cells (gametes) as an integral part of sexual reproduction. Chromosomes are deliberately broken to initiate homologous (meiotic) recombination that physically connects the equivalent maternal and paternal (homologous) chromosomes; this is absolutely essential for correct chromosome segregation (Petronczki et al 2003; Lam and Keeney 2015). If these connections (chiasmata) are achieved accurately, healthy gametes containing a single chromosome complement will result from the two meiotic cell divisions. Failure to perform meiosis correctly has been shown to cause infertility, miscarriages, and hereditary disorders in mammals (Hassold and Hunt 2001); meiosis is fundamental to sexual reproduction
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