Parallel phenotypic analysis of sporulation and postgermination growth in Saccharomyces cerevisiae

Abstract

We have quantitatively monitored the sporulation and germination efficiencies of approximately 4,200 yeast deletion strains in parallel by using a molecular bar coding strategy. In a single study, we doubled the number of genes functionally implicated in sporulation to approximately 400, identifying both positive and negative regulators. Our set of 261 sporulation-deficient genes illustrates the importance of autophagy, carbon utilization, and transcriptional machinery during sporulation. These general cellular factors are more likely to exhibit fitness defects when deleted and less likely to be transcriptionally regulated than sporulation-specific genes. Our postgermination screening assay identified recombinationchromosome segregation genes, aneuploid strains, and possible germination-specific factors. Finally, our results facilitate a genome-wide comparison of expression pattern and mutant phenotype for a developmental process and suggest that 16% of genes differentially expressed during sporulation confer altered efficiency of spore production or defective postgermination growth when disrupted.

Fig 1.

A parallel genetic approach to identify sporulation mutants. (A) Two-color overlay of scanned images from oligonucleotide arrays hybridized with fluorescently labeled molecular tags from a presporulation culture (red channel) and a pure spore suspension (green channel). Red (VAC8) and green (NOT3) probes indicate enrichment in the presporulation sample and the spore sample, respectively. Yellow probes correspond to deletion strains equally represented in the presporulation and spore cultures. (B) The ratio of presporulation signal to spore signal for 4,162 deletion strains is shown. The majority of genes have no effect on sporulation efficiency (PreSpo/Spore ratio of 1). The red and green bars correspond to sporulation-deficient and enhanced sporulation gene sets, respectively.

Fig 2.

Sporulation-specific genes are transcriptionally regulated and not required for optimal fitness in YPD. (A) The 261 sporulation-deficient strains were separated into two classes based on YPD fitness profiling: slow growers (with growth rates less than 0.95 of the pool average) and normal growers. We then hierarchically clustered the genes from each set based on sporulation expression data (7) obtained from strains SK1 and W303. The expression time courses are reflected by the triangles above the cluster: the large end of the triangle represents the end of the corresponding time course. Rows represent individual genes and columns reflect single time points in each experiment. (B) The average YPD fitness profile of the slow growth and normal growth class as a function of original tag signal. (C) Average mRNA expression level of the slow growth and normal growth class during SK1 sporulation. (D) Same as C for the W303 background.

Fig 3.

Identification of negative regulators of sporulation. Strains were added to sporulation media and the percentage of mature asci (2, 3, or 4 spore) was calculated at 1-day intervals. The curves represent the average of three independent experiments.

Fig 4.

A screen to identify strains defective in postgermination growth. (A) Homozygous diploid YPD growth (solid lines) and postgermination growth (dotted lines) of deletion strains [hop1Δ/hop1Δ (open circles), mam1Δ/mam1Δ (open squares), arp1Δ/arp1Δ (filled squares), wsc2Δ/wsc2Δ (open triangles), yhl044wΔ/yhl044wΔ (filled circles), and yjl192cΔ/yjl192cΔ (crosses)] with a postgermination growth defect. The curves are plotted as a function of the initial TAG hybridization signal over time. By 15 generations of postgermination growth, all six strains are absent from the pool. (B) A pure spore suspension (≈2 × 10⁷ cells) derived from each diploid deletion strain was germinated in rich media (YPD). We monitored growth by assaying the culture turbidity at 3-hour intervals. Each curve represents the average of three independent experiments. The strain heterozygous for ALG7, an essential gene, is used as a control.

Fig 5.

Analysis of postgermination defective strains. For the genes shown, tetrads were dissected from the original homozygous diploid deletion strain (white bars) and backcrossed strain (gray bars). Each bar represents the average of three independent tetrad dissections (10 tetrads each) per strain. Plates were scored for the presence of visible colonies after 2 days of growth.