A large-scale screen in S. pombe identifies seven novel genes required for critical meiotic events

Abstract

Meiosis is a specialized form of cell division by which sexually reproducing diploid organisms generate haploid gametes. During a long prophase, telomeres cluster into the bouquet configuration to aid chromosome pairing, and DNA replication is followed by high levels of recombination between homologous chromosomes (homologs). This recombination is important for the reductional segregation of homologs at the first meiotic division; without further replication, a second meiotic division yields haploid nuclei. In the fission yeast Schizosaccharomyces pombe, we have deleted 175 meiotically upregulated genes and found seven genes not previously reported to be critical for meiotic events. Three mutants (rec24, rec25, and rec27) had strongly reduced meiosis-specific DNA double-strand breakage and recombination. One mutant (tht2) was deficient in karyogamy, and two (bqt1 and bqt2) were deficient in telomere clustering, explaining their defects in recombination and segregation. The moa1 mutant was delayed in premeiotic S phase progression and nuclear divisions. Further analysis of these mutants will help elucidate the complex machinery governing the special behavior of meiotic chromosomes.

Figure 1

Aberrant Nuclei Behavior in mug Mutants (A) Aberrant nuclear segregation in representative “class a” mutants (Table S1). Wild-type and mutant h⁹⁰ strains were incubated for 2-3 days on malt extract agar medium to induce mating and sporulation, fixed, stained with DAPI and observed under the fluorescence microscope. rec24, rec25, rec27, bqt1, and bqt2 mutants are shown as examples. The phenotype of these “class a” mutants is a mix of the different nuclear segregation defects shown in this figure; details of the frequency of abnormal nuclear segregation in each mutant are in Table 1. (B) Twin horsetail nuclei in the tht2 mutant. Cells were stained with DAPI during the horsetail stage (at 24 h on MEA medium) and examined by microscopy. Approximately half of the cells had twin (side-by-side) horsetails at this time point. (C) Co-localization of Bqt2 and the spindle pole body-component Sad1. Cells expressing Bqt2-CFP and Sad1-GFP were examined during the horsetail stage by microscopy. In all of 20 cells examined the foci co-localized. (D) Bqt2 is required for telomere clustering. Wild-type and bqt2 mutant cells expressing Taz1-GFP were examined during the horsetail stage by microscopy. Taz1-GFP appeared as a single focus in 19/19 wild-type cells and in 0/16 bqt2 cells.

Figure 2

DSB Formation and Meiosis Progression in mug Mutants (A) Meiotic DSB formation in newly isolated rec, tht2, and bqt mutants. DNA was isolated from synchronously induced meiotic cultures (pat1-114 rad50S ade6-3049) and assayed for DSBs on the 501 kb NotI fragment J as described by Young et al. [23]. mbs1 is one of six sites of prominent meiosis-specific DSBs on this fragment. Additional data are in Figure S2. (B) Kinetics of meiotic progression in mug mutants. Diploid pat1-114 leu1-32 strains of the indicated genotypes (the rec12 mutant was leu1⁺) were induced to enter synchronous meiosis as described [32]. The percentage of uninucleated cells that had not yet undergone MI (blue), binucleated cells that had undergone MI (red) and tri- or tetranucleated cells that had undergone MII (green) is shown for times points around MI; complete time courses are in Figure S3. Note that, as in the rec12 mutant included as control, MI entry occurred earlier in the rec24, rec25, and rec27 mutants. Conversely, MI entry was delayed in the moa1 mutant. At least 500 cells were scored for each time point. Two independent experiments were done with similar results. Under each histogram, flow cytometry of the DNA content during the same synchronous meiosis is shown. The bulk of pre-meiotic DNA replication occurred between 1.5 and 2 h (indicated by a black bar) in all strains except in moa1, which showed a delayed premeiotic S-phase (between 2 and 2.5 h).