Download Supplementary information

Supplementary information.
meiosis could be induced at various phases of the cell cycle
To investigate whether meiosis can be induced from other phases of the cell cycle apart from
G1, various cell cycle mutants of the fission yeast Schizosaccharomyces pombe were tested for their
ability to undergo meiotic differentiation. Meiotic initiation requires heterozygosity at the mating type
locus and nitrogen starvation, and so diploid h+/h- cells harboring various temperature sensitive mutations
were grown at 25 C and shifted to nitrogen free media at 33 C. Two mutants which have extended G1
periods under these conditions (cdc10-129 and wee1-50) entered meiosis efficiently. In contrast, the two
mutants which have extended G2 periods (cdc25-22 and rum1∆) failed to enter meiosis (Fig. a). These
results suggest that cells cannot normally undergo meiosis from G2. Meiosis is induced by the RNA
binding protein Mei2, which is kept inactive by Pat1 protein kinase mediated phosphorylation. When
diploid h+/h- cells are nutritionally starved, mei3 transcription is induced and the Mei3 protein inhibits the
Pat1 protein kinase leading to Mei2 activation (Fig. b). Therefore we monitored mei3 transcript level as a
marker for activation of the meiotic regulatory network. Mei3 transcript levels increased in the cdc10-129
and wee1-50 diploids, but not in the cdc25-22 and rum1∆ diploids (Fig. a). The restriction of transcription
to G1 cells was specific for mei3, because another gene isp6, which is induced by nitrogen starvation, was
found to be transcribed in all the cell cycle mutants. Thus we conclude that the meiotic programme
normally starts from G1 due to the restriction of Mei2 activation to this phase of the cell cycle (Fig. b).
Next we examined if ectopic activation of the meiotic regulatory network at stages of the cell
cycle other than G1 could induce meiosis. This was tested in rum1∆ cells, which have a very short G1
phase, in three different ways. Firstly, mei3 was ectopically expressed, secondly Pat1 was inactivated
using a temperature sensitive pat-114 mutation, and thirdly mei2-SATA, a constitutively activated form of
Mei2 that cannot be inhibited by Pat1, was ectopically expressed. All three procedures efficiently induced
meiosis and sporulation in rum1∆ cells. We further examined ectopic expression of nmt1-mei2-SATA at
other cell cycle stages, blocked with cdc10 (at G1), cdc25 (at G2), and cdc2 (at G2) ts mutations or HU
(at S). The mutant cells were grown at 25 C for 16 h after removal of thiamine, during which time the
nmt1 promoter was still switched off. Then they were shifted to 36C for 4 h, resulting in cells arresting at
each cell cycle block point, and accumulating mei2-SATA transcripts. Cells were then released to 25C
and monitored for meiosis and sporulation using DAPI. The HU block and release experiment was
performed in wild type cells using the same conditions, adding HU at 36 C and removing it at 25C. In
every case, meiosis was efficiently induced, and cell number remained constant after release, confirming
that all cells entered meiosis from the cell cycle block points.
Recombination during G1-exit and G2-exit meiosis.
G1-exit and G2-exit meioses were induced using
an h+/h+ cdc2-L7/cdc2-L7 pat1-114/pat1-114 leu2+/leu2-120 lys1-131/lys1+ ade6-M26/ade6-469 diploid,
to examine the intragenic recombination between ade6-M26 and ade6-469. The identical diploid
mitotically proliferating at permisive temperature and wild type h+/h- diploid undergoing physiological
meiosis were tested for the recombination as well. The average recombination of each case is 6500
(G1-exit meiosis), 330 (G2-exit meiosis), 70 (mitosis) and 7500 (physiological meiosis) per 106 cells,
respectively. Intergenic recombination between leu2 and lys1 was simultaneously examined during
G2-exit meiosis and gave the 20 times less frequency than that calculated from map distance, which was
determined in normal meiosis.
Manipulation of rec8 expression.
The rec8-GFP fusion gene, including the rec8 promoter region
(-108~-1), was subcloned into pUC118. The 0.6 kb sequence extending downstream from the spo5 stop
codon (Y. W. and M. Y. unpublished results) was inserted behind rec8-GFP to provide a transcriptional
stop element. Subsequently ura4+ and kanMX6 cassettes were amplified by PCR and subcloned into the
flanking regions of the plasmid. The plasmid was then linearised within kanMX6 to allow targeted
integration at the chromosomal rec8 locus, which had previously been replaced with kanMX6. Thus the
[rec8-promoter]-rec8-GFP integrant was constructed. For the [spo5-promoter]-rec8-GFP integrant, we
replaced the rec8 promoter region with the 0.4 kb spo5 promoter sequence (Y. W. and M. Y. unpublished
results). We further changed the Mlu I sequence located near the N-terminus in the rec8 ORF by in vitro
mutagenesis without changing the encoded amino acid sequence because Mlu I boxes in rec genes may
act as regulatory sequence for transcription. Thus the [spo5-promoter]-rec8-GFP integrant was similarly
constructed. Subsequently we made a diploid h+/h+ cdc2-L7/cdc2-L7 pat1-114/pat1-114 leu2+/leu2-120
lys1-131/lys1+ ade6-M210/ade6-M216 carrying either an integrated [rec8-promoter]-rec8-GFP or
[spo5-promoter]-rec8-GFP construct to induce synchronous G1-exit meiosis. The constitutive expressing
construct [adh1-promoter]-rec8-GFP was similarly constructed and integrated at the chromosomal rec8
locus.