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1.
Figure 5

Figure 5. Genes in the G2/M category can be categorized based upon nuclear DNA staining in large-budded cells.. From: Mechanisms of Cell Cycle Control Revealed by a Systematic and Quantitative Overexpression Screen in S. cerevisiae.

(A) Category I (20 genes): an undivided nucleus in one cell body. (B) Category II (13 genes): undivided nuclei in bud neck. (C) Category III (17 genes): two divided nuclei separated to two cell bodies.

Wei Niu, et al. PLoS Genet. 2008 July;4(7):e1000120.
2.
Figure 3

Figure 3. Independent support for cell cycle delays from histograms of the percentages of cells with no bud, small bud or large bud.. From: Mechanisms of Cell Cycle Control Revealed by a Systematic and Quantitative Overexpression Screen in S. cerevisiae.

(A) shows bud size measurements of strains in the G1 category. Strains are sorted by their percentages of cells without buds. All strains show higher proportions of cells than control strains; 13 strains are more than two standard deviations higher (indicated by red line) than the empty vector control strains (plotted+/−1 s.d.). (B) shows bud size measurements of strains in the G2/M category. Strains are sorted by percentages of cells with large buds. 85 strains showed higher percentages of large-budded cells than empty vector control strains; 47 of these were more than two standard deviations above control strains (indicated by red line). In all plots, ORF gene names are indicated in x-axis, percentages of cells on the y-axis.

Wei Niu, et al. PLoS Genet. 2008 July;4(7):e1000120.
3.
Figure 4

Figure 4. Representative cell images from ORF strains showing G1 or G2/M cell cycle delays.. From: Mechanisms of Cell Cycle Control Revealed by a Systematic and Quantitative Overexpression Screen in S. cerevisiae.

Cell nuclei were stained with Sytox green, and cells visualized through FITC and DIC filters; overlaid images are shown. (A) Empty vector control strain. Overexpression of (B) TRM5 or (C) ARC1 causes G1 cell cycle delays, marked by an accumulation of unbudded cells. (D–F) shows strains with G2/M delays illustrating the three classes of large-budded cell nuclear morphology. (D) Class I (pre-M): overexpression of TUB2 causes elevation in large-budded mononucleate cells. (E) Class II (early-M): overexpression of SPC97 accumulates large budded cells with undivided nuclei at the bud necks. (F) Class III (late-M): increased proportions of large-budded cells that had completed nuclear DNA segregation are apparent upon IME2 overexpression.

Wei Niu, et al. PLoS Genet. 2008 July;4(7):e1000120.
4.
Figure 9

Figure 9. Overexpression phenotypes are generally distinct from loss-of-function phenotypes.. From: Mechanisms of Cell Cycle Control Revealed by a Systematic and Quantitative Overexpression Screen in S. cerevisiae.

Of 108 genes causing cell cycle defects when overexpressed, quantitative cell morphology information for 77 of the corresponding deletion mutants was available in the Saccharomyces cerevisiae Morphology Database (SCMD), with 16 genes in the G1 category and 61 in the G2/M category. Considering only those ORF strains whose bud size index differs from control strains with p<0.05, 12 genes caused significantly higher percentages of cells without buds than control strains when overexpressed. Of these 12 genes, only 1 gene led to significantly high proportions of unbudded cells when deleted. For G2/M genes, 47 genes caused significantly elevated percentages of cells with large buds upon overexpression; only 3 of them also lead to significantly high populations of large-budded cells when deleted. Thus, the large majority of overexpression phenotypes are not mirrored by the corresponding deletion strains, raising the likelihood for overexpression phenotypes to have arisen through gain-of-function mechanisms.

Wei Niu, et al. PLoS Genet. 2008 July;4(7):e1000120.
5.
Figure 6

Figure 6. Overexpression of YPR015C results in mitotic instability and activates the DNA damage checkpoint.. From: Mechanisms of Cell Cycle Control Revealed by a Systematic and Quantitative Overexpression Screen in S. cerevisiae.

(A) Flow cytometry histograms of empty vector control strain and PGAL1-YPR015C strain show the G2/M delay phenotype upon overexpression YPR015C. (B) Summary of the results from analysis of nuclear DNA staining. PGAL1-YPR015C showed a higher percentage of large-budded cells with undivided nuclei at the bud neck than the empty vector control strain. (C) An assay of mitotic instability using a reporter plasmid (pRS412::ADE2 [cir+]) shows that YPR015C overexpression increases mitotic instability, indicated by an increase in red colonies (signifying loss of the centromere-containing plasmid) relative to white colonies (correctly carrying the plasmid). Quantitation of this trend (D) reveals the PGAL1-YPR015C strain to have about twice the rate of centromere loss than that of the empty vector control strain. (E) Flow cytometry indicates that deletion of RAD9 suppressed the G2/M delay caused by overexpression of YPR015C, while deletion of MAD2 did not suppress the G2/M delays caused by overexpression of YPR015C, indicating that the G2/M delay requires RAD9, and thus the DNA damage checkpoint.

Wei Niu, et al. PLoS Genet. 2008 July;4(7):e1000120.
6.
Figure 7

Figure 7. Overexpression of RFA1 causes chromosomal segregation and spindle defects.. From: Mechanisms of Cell Cycle Control Revealed by a Systematic and Quantitative Overexpression Screen in S. cerevisiae.

(A) The G2/M delay phenotype upon overexpression of RFA1 is apparent in flow cytometry histograms of the empty vector control strain and the PGAL1-RFA1 strain. (B) Quantitation of cell microscopy results following nuclear DNA staining indicates that PGAL1-SPC97 and PGAL1-RFA1 strains exhibit considerably higher percentages of large-budded cells with undivided nuclei than the empty vector control strain. (C) Log-phase cultures of the wild type control strain and cells carrying PGAL1-SPC97 or PGAL1-RFA1 were fixed in formaldehyde and stained to visualize DNA (by DAPI) and microtubules (by immunofluorescence). The cells carrying the empty vector correctly showed a long anaphase spindle, with nuclei successfully segregated into two cell bodies. Overexpression of either SPC97 or RFA1 resulted in a failure of chromosome segregation; the spindle morphology of PGAL1-RFA1 cells is distinct from that of PGAL1-SPC97 cells, with shorter mitotic spindles poorly aligned with the division axis.

Wei Niu, et al. PLoS Genet. 2008 July;4(7):e1000120.
7.
Figure 1

Figure 1. Overview of the cell cycle screen.. From: Mechanisms of Cell Cycle Control Revealed by a Systematic and Quantitative Overexpression Screen in S. cerevisiae.

(A) Flowchart summarizing the large-scale screen. 5,556 yeast ORF overexpression strains and 140 replicates of the empty vector (BG1766) control strain (Y258) were induced in 96-well plates with SC-URA, 2% galactose medium, and analyzed via high-throughput flow cytometry. All flow cytometry histograms were analyzed by ModFit LT software to calculate the proportions of cells with one copy (1C) or two copies (2C) of their chromosomal DNA. Cell cycle defects were diagnosed from skews in the proportions of 1C to 2C cells. The ORF strains that showed cell cycle defects in the initial large-scale screen were validated twice manually by flow cytometry. (B) Flow cytometry histograms of control strains and representative ORF strains are shown. The x-axis indicates fluorescence intensity, corresponding to DNA content per cell; the numbers of cells with each given intensity are plotted along the y-axis. (C) Each DNA histogram was fitted with two Gaussian distributions, shown in red, and the percentages of cells in G1 and G2/M phases were calculated as the areas under the 1C and 2C peaks, respectively.

Wei Niu, et al. PLoS Genet. 2008 July;4(7):e1000120.
8.
Figure 8

Figure 8. Overexpression of SKO1 activates the pheromone response pathway.. From: Mechanisms of Cell Cycle Control Revealed by a Systematic and Quantitative Overexpression Screen in S. cerevisiae.

(A) shows flow cytometry analysis of DNA content for the empty vector control strain, PGAL1-SKO1 strain, and the sko1Δ strain with its corresponding control strain. Overproduction of SKO1 causes a strong arrest at the G1 phase (78% of PGAL1-SKO1 cells accumulated at the G1 phase vs. 58% of control cells at the G1 phase). In contrast, there was no obvious G1 arrest in the sko1Δ strain. (B) While the sko1Δ strain exhibits a typical yeast cell morphology, cells from the PGAL1-SKO1 strain resemble yeast cells presented with mating pheromone (shmoos). Overexpression of SKO1 in cells expressing a green-fluorescent protein-tagged version of the mating projection marker Fus1 induces Fus1-GFP localization to the tip of the projection (shown as an overlay of the GFP channel on the DIC image), consistent with SKO1 overexpression inducing shmooing. (C) SKO1 induces shmooing when overexpressed in the deletion strains fus1Δ, fus3Δ, sst2Δ, and dig2Δ, as well as in the corresponding parental strain (BY4741), but not when overexpressed in the deletion strains ste2Δ, ste4Δ, ste5Δ, ste20Δ, ste11Δ, far1Δ, and kar4Δ, indicating that the latter genes are required for SKO1-induced shmoo formation.

Wei Niu, et al. PLoS Genet. 2008 July;4(7):e1000120.
9.
Figure 2

Figure 2. Summary of assay results.. From: Mechanisms of Cell Cycle Control Revealed by a Systematic and Quantitative Overexpression Screen in S. cerevisiae.

(A) The Log2 (1C/2C) ratios of the 5,334 yeast ORF strains with event numbers >5,000 (filled circles; each represents a bin of width 0.06) were approximately normally distributed and fit by a Gaussian distribution (solid line; R2∼0.97). Each strain was assigned a Z-score based upon its Log2 (1C/2C) ratio in order to identify the ORF strains with significantly different proportions of cells in the G1 and G2/M cell cycle phases. (B) A comparison of the resulting distribution of Z-scores for the ORF strains (filled black circles; each represents a bin of width 0.2) relative to Z-scores calculated for the replicate empty vector control strains (filled red triangles) shows that the control strains have a considerably narrower distribution than the ORF strains, with no control strain |Z| scoring higher than 1.96. (C) The numbers of ORF strains showing significantly divergent rations of 1C to 2C cells in the initial screen as a function of different confidence levels. The P-value in the paper is highlighted in bold red text; there were 198 ORF strains identified at this confidence level (p<0.05). Of the 198 genes, 108 were validated at least twice manually; 90 were eliminated for poor reproducibility. (D) The functional classification of the 108 genes reproducibly inducing cell cycle delays upon overexpression.

Wei Niu, et al. PLoS Genet. 2008 July;4(7):e1000120.

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