The effect of CLN3 gene dosage on cell size control. For the model predictions, standard parameters from Chen et al. (2000) were used. Changes in CLN3 gene dosage were simulated by changing the DN3 parameter. bck2 deletion was simulated by reducing the BCK parameter to 0. The modal cell volume from the model was estimated as the midpoint between the size at birth and at cell division, relative to a wild-type value of 1. For the experimental observations, cultures of the appropriate genotype (see MATERIALS AND METHODS) were grown to log phase in YEPD, and the peak modal cell size was determined relative to a wild-type control, set to a value of 1. The total CLN3 gene dosage was taken to be the calculated number of CLN3 genes in the transgene array for strains that were cln3:: URA3 at the endogenous CLN3 locus. CLN3 gene dosage was incremented by one for strains with wild-type CLN3 at this position.

Method of quantitation: sample data for Clb2-PrA. To estimate the number of copies of a PrA fusion per yeast cell, threefold serial dilutions of the GST-PrA standard and of the cell extract from the tagged strain were made in wild-type (untagged) carrier cell extract as described in MATERIALS AND METHODS. SDS-PAGE followed by Western blot analysis was performed with these samples. In addition to the PrA fusion protein band, all blots generated a band at ∼115 kDa (asterisk), derived from the carrier wild-type cell extract. Signal intensities were plotted against dilution factors and points were chosen in the linear parts of the curve (straight line in the figure) for the calculations described in MATERIALS AND METHODS. The points in the graph correspond to the densitometric quantitation of the bands in the autoradiograph. In this example, the middle lane on the GST- PrA gel was determined to contain 1.4 × 10⁹ molecules of GST-PrA based on quantitation of the standard and the dilution factor, and it gave a signal intensity of 2.2, in the linear range of detection. The middle lane of the Clb2-PrA gel derived from 4 × 10⁶ yeast cells and gave a signal intensity of 3.5, also in the linear range of detection. The calculated Clb2-PrA/cell from this example is then (3.5/2.2) × 1.4 × 10⁹ molecules/4 × 10⁶ cells = 557 molecules/cell. Because the strain analyzed was a heterozygous diploid, this number is doubled to give 1114 molecules/cell. Numerous independent determinations of this type (each representing a different protein extraction and quantitation) were averaged to yield the final numbers in Table 1.

Characterization of the elutriation protocol. Log phase cultures of diploid strains heterozygous for various PrA-tagged genes were elutriated as described in MATERIALS AND METHODS. The entire culture was collected. (A) Schematic of the procedure, and sample immunoblotting data. Top: cartoon of an asynchronous population. Middle: the effect of separation on the elutriator, indicated by percentage of unbudded cells. Bottom: sample blot for detection of Clb2-PrA, Clb5-PrA, and Sic1-PrA (all expressed from heterozygous PrA-tagged genes in the diploid strain analyzed). Nop1: loading control detected with anti- Nop1 antibody. (B) The proportion of the total mass of the culture (indicated by OD₆₆₀) as a function of the modal cell volume of the fractions determined by Coulter Channelyzer calibrated with standard 68 fl latex beads. Closed symbols: wild-type diploid strains; open symbols: cdh1 diploid strains. Each symbol represents a different elutriation. The cdh1 strains reproducibly yielded significant mass at slightly smaller cell volume than the wild type. In the first few fractions derived from the cdh1 mutant cultures, two peaks were reproducibly observed in the Coulter Channelyzer electronic cell volume analysis. We do not know the meaning of this observation. For the data reported in Figures 6, B and C, and 7 A and C, we use the larger peak for the x-axis values for these samples. (C) The percentage of unbudded cells in the fractions. Closed symbols: wild-type diploid strains; open symbols: cdh1 diploid strains. Each symbol represents a different elutriation. Arrows above: approximate positions of 50% completion of DNA replication in wild- type (black) and cdh1 (gray) strains, based on FACS analysis of the fractions. Arrowheads above: beginning of nuclear division, indicated by initial accumulation of a significant population of binucleate cells, determined by microscopic examination of propidium iodide-stained cells; in earlier fractions, essentially no binucleate cells are observed. (D) PrA detection from diploids dou-bly heterozygous for CLB2- PrA, CLB5-PrA, and SIC1- PrA (top) or for CLB2-PrA and CLB3-PrA (bottom). Both CDH1 (left) and cdh1 (right) diploids were analyzed. Pgk1 was detected using anti-Pgk1 antibody for a loading control. U, extract from untagged culture. Note that densities of exposure are not comparable between experiments.

Effects of constitutive undegradable Clb2. (A) Model predictions. Loss of transcriptional control of GAL-CLB2 or GAL-CLB2db compared with wild-type CLB2 was modeled by setting ksb2′ = 0.05, ksb2" = 0. Loss of proteolytic control of GAL- CLB2db was modeled by setting kdb2" to 0.01 and kdb2p to 0, eliminating degradation dependent on Cdh1 and Cdc20, respectively. (B) Haploid strains expressing the indicated PrA fusions were transformed with integrating plasmids containing GAL-CLB2-HA or GAL-CLB2db-HA (Jacobson et al., 2000). The untransformed parent and two transformants with each DNA were grown to log phase in YEP- raffinose (noninducing), and then GAL-CLB2- HA or GAL-CLB2db-HA was induced by addition of galactose to 3%. After 3.5 h proteins were extracted for PrA detection using rabbit IgG and for Clb2 and Clb2-HA detection using anti-Clb2 antibody.

Hysteresis in the cell cycle. A strain of genotype cln1 cln2 cln3 GAL-CLN3 cdc14-1 was grown to log phase in YEPGal (galactose medium, GAL-CLN3 on) at 23°C and blocked due to CLN deficiency by incubation in YEPRaff (raffinose medium, GAL- CLN3 off) at 23°C for 6 h. Galactose was then added to 3% final concentration to induce GAL- CLN3. At the indicated times after galactose addition, aliquots of the culture were removed. Some of the aliquot was immediately extracted for immunoblotting with anti-Clb2 antibody. To the rest of the aliquot glucose was added to 2% final concentration to repress GAL-CLN3, and the aliquot was incubated at 37°C for 2.5 h. The aliquot was then extracted for immunoblotting. The percentages of unbudded cells at the time of aliquot removal and after the 2.5-h incubation in glucose at 37°C were determined microscopically. Top: Clb2 immunoblot for cells in continuous galactose (G), after the raffinose block (R), and after the indicated amounts of time after galactose addition (R+G; all at 23°C). Bottom: Clb2 immunoblot for cells incubated in R+G for the indicated amount of time and then shifted to glucose at 37°C for 2.5 h. *An unidentified background band used for standardizing protein loading. The percentage of unbudded cells at the time of protein harvest is indicated below the gel lanes. The top and bottom immunoblots were from the same membrane and film exposure.

Interaction between G1 cyclins and mitotic regulators Sic1 and Cdh1. (A) Model predictions for various genotypes. Parameters: M, cell mass; C, Clb2 levels; S, Sic1 levels. Standard parameters from (Chen et al., 2000) were used throughout, except for the following changes to model the mutants: cln1,2-: ksn2" = 0; CONST CLN2: ksn2′ = 0.05, ksn2" = 0; cdh1-: kdb2" = 0.01; CONST SIC1: ksc1′ = 0.1, ksc1" = 0.1. The model predicts lethality (cell cycle arrest and failure of cell division) in the cln1,2 cdh1, the CONST CLN2 cdh1 (with or without CONST SIC1), and the cln1,2−CONST SIC1 (with or without CDH1) situations. The CONST CLN2 cdh1 CONST SIC1 simulation (not shown) is similar to the CONST CLN2 cdh1 simulation. The cln1,2−CONST SIC1 cdh1 simulation differs from the cln1,2−CONST SIC1 simulation, because Clb2 levels ultimately rise because of cdh1 deletion, but in both cases inviability is predicted. (B) Genetic test of the predictions in A. Strains (BF264-15D background) of the indicated genotypes were constructed by tetrad analysis, using GAL- SIC1/URA3 and GAL-CLN2/TRP1 cassettes and using a cdh1::LEU2 deletion (Schwab et al., 1997) backcrossed five times into the BF264- 15D background. All strains were cln1 cln2 CLN3. cln1 cln2 CLN3 corresponds to absence of Cln2 in the model, and cdh1:: LEU2 corresponds to absence of Cdh1 (called Hct1 in Chen et al., 2000) in the model. The presence of the GAL-SIC1 and GAL-CLN2 cassettes corresponds to CONST SIC1 and CONST CLN2, respectively. Tenfold serial dilutions of cultures of strains of each genotype, grown to stationary phase in YEPD, were spotted on YEPD or YEPGal plates and grown at 30°C for 2 and 3 d, respectively. Another set of strains of these genotypes behaved identically. CLN1,2 controls (wild type, cdh1, and GAL-SIC1) are not shown here but all are viable under these conditions (our unpublished data). The model predicts lethality due to cln1 cln2 cdh1 (YEPD plates, all cdh1- strains, 2nd, 4th, 6th, 8th rows), but this is not observed. It also predicts lethality due to cln1 cln2 cdh1 GAL-CLN2 (YEPGal plates, 4th row), but this is not observed. The model correctly predicts lethality due to constitutive Sic1 expression in the absence of cln1,2 and its rescue by constitutive CLN2, in the presence or absence of cdh1 (YEPGal plates, 3rd, 4th, 7th, and 8th rows).

Selective disadvantage due to clb gene disruption. The selective disadvantage due to the indicated clb gene disruption was determined in a single cycle competitive growth experiment from stationary phase, through a dilution of ∼10⁶-fold, back to stationary phase. The experiment was in liquid YEPD medium, over a 30-h period at 30°C, and the culture initially contained an approximately equal mixture of wild-type and mutant cells. The selective disadvantage parameter will reflect differences in doubling time on the assumption that entry into and exit from stationary phase are identical for all strains, but this has not been tested. In all cases, the clb gene disruption was compared with a control strain with identical auxotrophic markers (see MATERIALS AND METHODS).

Abundance of Clb2, Clb3, Clb5, and Sic1 through the cell cycle, with and without Cdh1: model and experiment. (A) Quantitation of data from Figure 6D, top; diploids heterozygous for CLB2-PrA, CLB5-PrA, and SIC1-PrA. Top: wild type; bottom: cdh1. Levels of the PrA fusions were determined densitometrically, and corrected for loading by quantitation of anti-Pgk1 signal. The scale was normalized to 1 for peak Clb2 levels in wild-type cells; this is probably ∼35 nM (see text). This scale was transferred to the cdh1 case by comparing a sample with peak Clb2-PrA from a wild-type diploid to a similar sample from a cdh1 diploid and standardizing the PrA signal to the Pgk1 signal, resulting in the determination that the peak in the cdh1 mutant was ∼2 times that in the wild type. This correction is somewhat approximate, compared with the scale within an elutriation, which is internally controlled because of coexpression of single chromosomal identically tagged genes. (B) Model predictions for the levels of the same proteins. Wild type: standard parameters (Chen et al., 2000). cdh1: the parameter kdb2" was reduced from 2 to 0.01. Mass is total cell mass in the simulation. Arrows indicate cell division. (C) Quantitation of data from Figure 6D, bottom; diploids doubly heterozygous for CLB2-PrA and CLB3-PrA. Top: wild type; bottom: cdh1 mutant. Analysis as in A.

Effects of unregulatable Cdh1. (A) Model predictions. Loss of negative regulation of Cdh1 was simulated by setting kit1 to zero; this simulates prevention of Cdh1 phosphorylation by all cyclin-Cdc28 complexes. (B) Diploid strains were constructed that were heterozygous for the indicated PrA fusions. For each set of fusions, two diploids (+) were also heterozygous for a double integration of the GALL-HA-HCT1-m11 construct (Zachariae et al., 1998), and one diploid lacked the GALL-HA- HCT1-m11 construct (−), as indicated. GALL-HCT1- m11 encodes a mutant Cdh1 (also called Hct1), lacking all Cdk phosphorylation sites and therefore immune to negative regulation (Zachariae et al., 1998). Strains were grown to log phase in YEP-raffinose (noninducing), and then GALL-HA-HCT1-m11 was induced by addition of galactose to 3%. After 2.5 h proteins were extracted for PrA detection using rabbit IgG. The band in the approximate position of Sic1-PrA in the strain expressing Clb2-PrA and Clb3-PrA is a Clb3-PrA breakdown product. Quantitation of the Cln2-PrA signal (quantitation of an anonymous cross-reacting band from elsewhere in the gel provided a loading control) indicated a three- to fourfold increase in Cln2-PrA and about a 40% decrease in Clb5-PrA, due to induction of GALL-HA-HCT1-m11.