Figure 1.
A schematic model illustrating the transcriptional cascade that governs meiosis in S. cerevisiae.
Figure 2.
Normalized level of transcription of representatives of meiosis-specific genes.
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 ACT1 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.
Figure 3.
Increase in IME1 copy number results in an increase in the level of expression of IME1 and all meiosis-specific genes.
Isogenic diploids carrying 1 (Y1639, diamonds), 2 (Y1631, squares), 3 (Y1663, triangles) and 5 (Y1662, circles) copies of IME1 were shifted to meiotic conditions (SPM). Samples were taken at the indicated times to isolate RNA and estimate transcript levels by qPCR. A. The RNA level of the indicated gene relative to RNA levels of ACT1. B. The relative RNA level is drawn in a log scale. The experiment was repeated 3 times and a representative result is shown.
Figure 4.
Meiosis is insensitive to the level of IME1 mRNA.
Isogenic diploids carrying different copy numbers of IME1 were shifted to meiotic inducing conditions. A. The correlation between IME1 copy number and its maximal level of expression. The maximal level of IME1 RNA (from Fig. 3A) in comparison to the copy number of the IME1 gene was drawn. The fitted trend line (dotted) and its formula are given. 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.
Figure 5.
The level of IME1 affects the time cells initiate premeiotic DNA replication, recombination and nuclear division.
Isogenic diploids carrying different copy numbers of IME1 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 ADE2 locus (B), and to stain with DAPI to count the percentage of cells with more than 2 nuclei (C).
Figure 6.
Premature transcription of NDT80 results in a premature entry into nuclear division, and consequently a defective meiosis.
Isogenic NDT80/NDT80 (Y1631, squares) and ndt80ΔC'::IME2p-NDT80-TRP/ndt80ΔC'::IME2p-NDT80-TRP1 (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.
Figure 7.
The ime2-3SA mutation resulted in premature entry into premeiotic DNA replication.
Isogenic IME2/IME2 (Y1631, squares) and ime2-3SA/ime2-3SA (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. A. FACS and budding index. The percentage of budded cells is given at the left side of each FACS. B. DNA replication. The percentage of ime2-3SA/ime2-3SA 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 C. Commitment to intragenic recombination at the ADE2 locus. D. The percentage of wild type (square) and mutant cells with more than 2 nuclei (grey lines). The relative level of NDT80 RNA in comparison to RNA levels of ACT1 is shown (black line). The experiment was repeated 3 times and a representative result is shown.
Figure 8.
Ectopic overexpresion of Cln3 increases the transcription of IME1, but delays initiation of premeiotic DNA replication and NDT80 transcription.
A wt strain (Y1631) carrying pIME2-CLN3 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 IME1 and NDT80 by qPCR (B).
Figure 9.
A schematic model illustrating the double negative feedback loop between Ime2 and Cdk1.
Black arrows and lines illustrate graded type of regulation; grey lines and arrows represent a threshold mode of regulation.
Table 1.
List of strains.