Increase in <i>IME1</i> copy number results in an increase in the level of expression of <i>IME1</i> and all meiosis-specific genes.

<p>Isogenic diploids carrying 1 (Y1639, diamonds), 2 (Y1631, squares), 3 (Y1663, triangles) and 5 (Y1662, circles) copies of <i>IME1</i> were shifted to meiotic conditions (SPM). Samples were taken at the indicated times to isolate RNA and estimate transcript levels by qPCR. <b>A.</b> The RNA level of the indicated gene relative to RNA levels of <i>ACT1</i>. <b>B.</b> The relative RNA level is drawn in a log scale. The experiment was repeated 3 times and a representative result is shown.</p

Ectopic overexpresion of Cln3 increases the transcription of <i>IME1</i>, but delays initiation of premeiotic DNA replication and <i>NDT80</i> transcription.

<p>A wt strain (Y1631) carrying <i>pIME2-CLN3</i> on a 2μ plasmid (YEp3212, triangles) or the vector plasmid (squares) were shifted to meiotic conditions. At the indicated hours samples were taken to process for FACS analysis and calculate the percentage of cells with 4C DNA content (A), and to isolate RNA and determine the relative transcripts levels of <i>IME1</i> and <i>NDT80</i> by qPCR (B).</p

Meiosis is insensitive to the level of <i>IME1</i> mRNA.

<p>Isogenic diploids carrying different copy numbers of <i>IME1</i> were shifted to meiotic inducing conditions. <b>A.</b> The correlation between <i>IME1</i> copy number and its maximal level of expression. The maximal level of <i>IME1</i> RNA (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011005#pone-0011005-g003" target="_blank">Fig. 3A</a>) in comparison to the copy number of the <i>IME1</i> gene was drawn. The fitted trend line (dotted) and its formula are given. <b>B.</b> Efficiency of meiosis. The percentage of asci (line) and the number of cells that can form colonies (viability, column) at 48 hours in SPM. The results are the averages of three independent transformants and a count of ≥200 cells. Error bars represent standard deviations.</p

A schematic model illustrating the transcriptional cascade that governs meiosis in <i>S. cerevisiae</i>.

<p>A schematic model illustrating the transcriptional cascade that governs meiosis in <i>S. cerevisiae</i>.</p

The <i>ime2-3SA</i> mutation resulted in premature entry into premeiotic DNA replication.

<p>Isogenic <i>IME2</i>/<i>IME2</i> (Y1631, squares) and <i>ime2-3SA/ime2-3SA</i> (Y1740, triangles or circles) cells were shifted to meiotic conditions (SPM). At the indicated hours samples were taken to process for FACS analysis, to count the number of unbudded cells, to plate on minus adenine and YEPD plates, to isolate RNA, and to stain with DAPI. <b>A.</b> FACS and budding index. The percentage of budded cells is given at the left side of each FACS. <b>B.</b> DNA replication. The percentage of <i>ime2-3SA/ime2-3SA</i> cells with 2C DNA content (open circles, grey line), in S phase (triangle, dashed line), or with 4C DNA content (triangle, line) was calculated. The percentage of wt cells with 4C DNA content (square, line) was calculated <b>C.</b> Commitment to intragenic recombination at the <i>ADE2</i> locus. <b>D.</b> The percentage of wild type (square) and mutant cells with more than 2 nuclei (grey lines). The relative level of <i>NDT80</i> RNA in comparison to RNA levels of <i>ACT1</i> is shown (black line). The experiment was repeated 3 times and a representative result is shown.</p

Normalized level of transcription of representatives of meiosis-specific genes.

<p>Wild type diploid cells (Y1631) were shifted to meiotic conditions (sporulation medium, SPM), and RNA was isolated at the indicated hours. Transcripts levels were measured by qPCR. The relative level of RNA in comparison to RNA levels of <i>ACT1</i> was normalized for the maximal level of expression of each gene, and set to 1. The experiment was repeated 3 times and a representative result is shown.</p

The level of <i>IME1</i> affects the time cells initiate premeiotic DNA replication, recombination and nuclear division.

<p>Isogenic diploids carrying different copy numbers of <i>IME1</i> were shifted to meiotic conditions. Samples were taken at the indicated times for FACS analysis and to calculate the percentage of cells with 4C DNA content (A), to plate on minus adenine medium and YEPD to measure the level of intragenic recombination at the <i>ADE2</i> locus (B), and to stain with DAPI to count the percentage of cells with more than 2 nuclei (C).</p

List of strains.

<p>List of strains.</p

Premature transcription of <i>NDT80</i> results in a premature entry into nuclear division, and consequently a defective meiosis.

<p>Isogenic <i>NDT80</i>/<i>NDT80</i> (Y1631, squares) and <i>ndt80</i>Δ<i>C'::IME2p-NDT80-TRP/ndt80</i>Δ<i>C'::IME2p-NDT80-TRP1</i> (Y1764, triangles) cells were shifted to meiotic conditions (SPM), and at the indicated hours samples were taken to extract RNA (A), to process for FACS analysis and calculate the percentage of cells with 4C DNA content (B), and to stain with DAPI to count the percentage of cells with 2 (open squares and triangles, dashed lines) and 4 nuclei (filled squares and triangles) (C). Arrows mark initiation time for DNA replication and nuclear division.</p

The role of signal transduction pathways and putative TFS on the activity of UASru.

<p>Rate (log2 of gene expression vs. background control expression level) was determined using the R-SGA assay <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078920#pone.0078920-Kahana1" target="_blank">[20]</a>. P-values were calculated on the basis of a normal distribution. NT – Not tested.</p