Examination of SBF and MBF targets at 2 hours.

<p><i>A</i>, Distributions of log₂ (2 h/0 h) ratios for SBF and MBF targets in WT and <i>ime2Δ</i> cells. <i>B</i>, Direct comparison of WT and <i>ime2Δ</i> log₂ (2 h/0 h) ratios for each SBF and MBF target.</p

Model of early Ime2 function.

<p>Pathways are shown leading from Ime1 induction early in meiosis to DNA replication. Factors relevant to the discussion in the text are included. Ime1 activates early gene expression through de-repression at the URS1 element <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031575#pone.0031575-Mallory1" target="_blank">[45]</a>, which is found in many meiosis-specific promoters including that of <i>IME2 </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031575#pone.0031575-Mitchell2" target="_blank">[43]</a>. Ime2 in turn upregulates gene expression <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031575#pone.0031575-Mitchell2" target="_blank">[43]</a>, and negatively affects Ime1 expression transcriptionally and post- transcriptionally <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031575#pone.0031575-Mitchell1" target="_blank">[30]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031575#pone.0031575-GuttmannRaviv1" target="_blank">[44]</a>. We hypothesize that Ime2 activation of the meiotic (mei) SBF/MBF transcriptional cascade leads to activation of a protein kinase with CDK specificity that enables Sic1 destruction, Clb5,-6/Cdk1 activation, and initiation of DNA replication.</p

Yeast strains used in this study.

1<p>Strains are congenic with SK1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031575#pone.0031575-Kane1" target="_blank">[42]</a> and are listed in the order that they appear in the text.</p

Analysis of consensus motif gene sets.

<p><i>A</i>, Gene expression data from our time course were analyzed by T-profiler for average expression of gene groups defined by consensus promoter motifs. Results for selected gene groups characterized by the indicated sequences are shown (R = A or G; W = A or T). Comparisons were made between expression levels at 2, 4, and 6 h <i>v.</i> expression levels at 0 h. Asterisks indicate statistically significant values (E<0.05). <i>B</i>, Distributions of log₂ (2 h/0 h) ratios for the CRCGAAA (left) and ACGCGT (right) gene sets are shown for WT and <i>ime2Δ</i> cells.</p

Epistasis analysis of <i>IME2</i> and <i>WHI5</i>.

<p>WT and mutant cells were induced to enter meiosis synchronously and DNA content was analyzed by flow cytometry at the indicated time points. This experiment was conducted in conjunction with the microarray experiment; therefore, sets of WT and <i>ime2Δ</i> histograms shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031575#pone-0031575-g002" target="_blank">Fig. 2</a> are reproduced here. Note that we have observed variability in the degree of DNA replication observed in <i>ime2Δ</i> cells at 24 h (<i>e.g.</i> compare with <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031575#pone-0031575-g001" target="_blank">Fig. 1</a>). Strains used were DSY1089, YGB221, YGB752, and YGB753.</p

T-profiler analysis of consensus motifs for WT <i>v. ime2Δ</i> at 2 hours.

<p>T-profiler analysis of consensus motifs for WT <i>v. ime2Δ</i> at 2 hours.</p

Expression of SBF and MBF targets in early meiosis.

<p>Gene-normalized hierarchical clustering analysis of MBF and SBF targets. For clustering procedure, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031575#s4" target="_blank"><i>Materials and Methods</i></a>.</p

Sic1 steady-state levels.

<p>WT and indicated mutant cells were induced to enter meiosis in a synchronous fashion and followed through time (h = hours). DNA content was analyzed by flow cytometry to detect pre-meiotic DNA replication (2C to 4C transition). Sic1^13myc and tubulin were detected by western blotting. For each time point, Sic1^13myc level was quantified by determining the relative band intensities of Sic1^13myc (red) and tubulin (green) and normalizing the resulting Sic1^13myc/tubulin ratio to the corresponding 0 hour ratio. Results are shown in graphical form (a.u., arbitrary units). Prior to immunodetection, membranes were stained with Ponceau S for total protein content assessment; regions that include Sic1^13myc and tubulin are shown. Strains used were YGB803, YGB787, and YGB804.</p