Two screening assays for sporulation efficiency.

<p>(A) Schematic of the fluorescence-based assay used to identify sporulation inhibitors. Meiotic landmark events indicated as Ent (entry), DS (pre-meiotic DNA replication), Rec (recombination), MI and MII (first and second meiotic division), and Spo (spore formation). The normal final product is depicted as an ascus with four spores (green). Compounds that inhibit sporulation or that are cytotoxic in sporulating yeast cells will suppress the expression of the sporulation-specific gene <i>CDA2</i> and therefore also <i>CDA2</i> promotor-driven <i>GFP</i> expression. (B) Real-time measurement of fluorescence intensities in cells harboring the <i>GFP</i>-reporter, which were sporulated in the presence of varying concentrations of ammonium sulfate (AS) as indicated. Fluorescence of the sporulation culture was measured every 15 min over the course of 20 hours (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042853#s4" target="_blank">Methods</a>). (C) Schematic of the post-recombination growth assay. The two defective alleles (<i>his4x</i> and <i>his4B</i>) can give rise to a functional <i>HIS4</i> allele upon meiotic recombination. The histidine-auxotrophic diploid mother cells can therefore produce histidine-prototrophic spores (indicated in blue). If this event is suppressed by a compound, either because it directly inhibits meiotic recombination or because it is cytotoxic to sporulating yeast cells, no histidine-prototrophs are formed. (D) Proof-of-concept of the <i>his4x</i>/<i>his4B</i> assay. Cells were sporulated for 5 hours in the presence of varying concentrations of ammonium sulfate (AS) as indicated. Aliquots of cells were then transferred to agar plates that lack histidine (-his) or leucine (-leu) and incubated for two days at 30°C, prior to measuring colony density (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042853#s4" target="_blank">Methods</a>) of each spot on the -his agar. Agar plates lacking leucine served as a loading control. The barplot in the upper panel shows mean and standard deviation data from 4 independent experiments.