Cells were synchronized in G1 and released in the presence of galactose to induce expression of SIC1Δ3P and CLN2^4t3s from the GAL1 promoter. Cells expressing CLN2^4t3s, grey; cells not expressing CLN2^4t3s, black. (A) Bud formation was measured over time and averaged among three experiments at each time point; percent cells with one bud, solid line, percent cells with two or more buds, broken line. Representative images are shown (B); bar 5μm. Protein levels of Cln2^4t3s-HA₃ or Cln2-HA₃ were measured (D) in cells expressing CLN2^4t3s (bottom panel) or control cells (top panel) and normalized to the anti-PSTAIR loading control. (C) Normalized protein levels are expressed as log₂-fold change relative to the mean protein level for each experiment.

Transcript dynamics were measured in a synchronous population of cdc28-4 cells at the restrictive temperature, and periodic transcripts were identified by comparison to transcript dynamics in wild-type cells grown under the same conditions. Genes that are periodically expressed in wild-type cells and cdc28-4 cells are shown in (A) cdc28-4, (B) wild-type cells. Genes are aligned to a cell-cycle time line and expressed as log₂-fold change relative to mean expression in the interval shown. Each row represents transcript levels for a single gene in (A, B). The grey bar designates cell cycle phases through which cells do not progress. (C-H) Line graphs showing the expression of single genes expressed as log₂-fold change relative to the mean in wild-type cells (black), and cdc28-4 cells (grey). (I) Venn diagram showing overlap of periodically expressed genes in wild-type cells (blue), Δclb1-6 cells (red) and cdc28-4 cells (green). (J) Network graph of TFs remaining periodic in cdc28-4 cells. All nodes were placed on a cell-cycle time-line based on time of peak expression; activators, green; repressors, red. Edges were drawn based on evidence for TF-promoter interactions in the literature. Also see Figure S1, Tables S1 and S2.

Cells with altered TF expression and control cells were synchronized in G1 by mating pheromone and released into the cycle in the presence (3J) or absence (3A-3I) of B-cyclin-CDK activity. Oscillations in bud formation were monitored. Representative budding curves for cells lacking B-cyclin-CDK activities are shown for GAL1p-SWI6 cells (A), Δmbp1 cells (B), Δyox1 cells (C), Δyhp1 cells (D), Δyox1; Δyhp1 cells (E), GAL1p-HCM1 cells (F) and Δsfg1 cells (G). Control cells, black; perturbed cells, grey; cells with 1 bud, solid line; cells with 2 buds, broken line. (H) A representative CLOCCS fit for cells lacking B-cyclin-CDK activity; cells with 0 buds, circles; cells with 1 bud, squares; cells with 2 buds, triangles. The width of the colored band reflects the degree of posterior uncertainty in the fit data. Learned period lengths for three experiments were averaged and are shown as % change from control for cells lacking B-cyclin activities (I) and cycling cells (J). Bars indicate standard deviation and asterisks (*) indicate periods significantly different than control cells (paired t-test; p<.05). Also see Figure S3, Table S3.

(A and B) Examples of CDK regulation of transcription through positive (B) and both positive and negative feedback (A); kinase activities, blue; transcriptional activators, green; transcriptional repressors, red. (C) Box-and-whisker plots of PTRs for genes periodic in both Δclb1-6 and cdc28-4 cells calculated as the percent of wild-type control PTR (grown at 30° C for and 37° C for cdc28-4 cells). Whiskers extend to 1.5 times the interquartile range; asterisk (*) indicates significantly different PTRs between Δclb1-6 or cdc28-4 cells with respect to wild-type cells. (D) Model of amplitude loss; solid line, wild-type expression; broken line, expression in the absence of CDK activities. (E) Expression of HCM1, black, and NDD1, grey, normalized to maximum expression in wild-type cells; wild-type cells, solid line; Δclb1-6 cells, broken line. (F) Average period length in wild-type cells (including similar periods observed at both 30° and 37° C), B-cyclin mutant cells, and cdc28-4 cells. Values for wild-type cells were determined by CLOCCS (Orlando et al., 2007). For Δclb1-6 cells and cdc28-4 cells, average period length is determined through MCMC mixture modeling. Error bars represent standard deviation between the replicates, percent increase over appropriate wild-type periods are indicated for Δclb1-6 and cdc28-4 cells. Also see Table S4.

Δclb1-5; GAL1pr-CLB1 cells were synchronized in G1 and released in presence of dextrose. Cells were monitored for bud emergence (A; solid line, one bud, broken line 2 buds), expression of CLN2 (grey) and CLB6 (black) by northern blot (B and C) and DNA content (D). (B) mRNA levels are plotted as the level of cyclin mRNA divided by the level of ACT1 at that time point. In all panels grey arrows signify the first (120 min) and second (300 min) peak of CLB6 expression. (D) DNA content is compared to Δclb1-6; GAL1pr-CLB1 cells. Accumulation of cells with less than 1C DNA content reflects the lysis of Δclb1-6 cells at late time points. Also see Figure S4.

Box indicates elements required for robust oscillations.