ECB elements are important for cell cycle regulation of CLN3. (A and B) Cycling of CLN3 mRNA in wild type (BY2125) and cln3ecb6 mutant (BY2690), respectively. Cells were grown in YEPGal and synchronized with α-factor. Cells were released into fresh medium at time point 0 and samples were taken every 10 min, as indicated by the numbers above the lanes. The expression of CLN3 and ACT1 was analyzed by Northern blot hybridizations. The blots were hybridized at the same time to the same probes, and exposure times were identical. (C) CLN3 transcript levels in the wild-type strain (BY2125) (closed squares) and cln3ecb5 (BY2278) (open squares) and cln3ecb6 (BY2690) (open circles) mutant strains grown in parallel were measured throughout the cell cycle, and the results of Northern blotting were quantitated with a PhosphorImager.

ECB elements confer rapid, synchronous progression through G₁ in daughter-cell fractions isolated by elutriation. Late-exponential-phase cultures (ca. 5 × 10⁷ cells/ml) grown in YEPGal were fractionated by elutriation. Fractions containing <5% budded cells were diluted into fresh YEPGal at densities between 2.4 × 10⁶ and 2 × 10⁷ cells/ml, grown at 30°C, and sampled at 15-min intervals for determinations of size and budding indexes. The median cell volumes (in femtoliters) were determined by sizing on a Coulter model Z2 analyzer), as described in Materials and Methods. (A) Budding profiles of cells lacking the ECB-mediated burst of CLN3 expression. Open symbols, wild-type [BY2679(CLN3 SWI)] (starting fractions had median volumes of from 13.0 to 15.8 fl); closed circles, cln3ecb mutant (BY2681) (starting fractions had median volumes of from 22.7 to 25.9 fl). (B) Budding profiles of cells lacking the ECB-mediated burst of SWI4 expression. Circles, swi4ecb (BY2682) (starting fractions had median volumes of from 14.8 to 17.2 fl); open squares, wild type (profile shown for comparison). (C) Budding profiles of cells lacking ECB activation of both CLN3 and SWI4. Circles, cln3ecb swi4ecb (BY2680) (starting fractions had median volumes of from 15.0 and 28.4 fl); open squares, wild type (profile shown for comparison). (D) Phase-contrast photomicrographs of fractions of wild-type (initial median volume = 16.9 fl) and cln3ecb swi4ecb (initial median volume = 18.8 fl) elutriated cells after outgrowth to ca. 50% budded cells. The images shown are at the same magnification.

Coupling between the G₁-to-S phase transition and initiation of budding is maintained in cln3ecb swi4ecb mutants. Unbudded daughter-cell fractions from elutriation were sampled at 15-min intervals during outgrowth for budding (squares) and initiation of DNA synthesis (circles). The percentage of cells in each sample that had exited G₁ was determined by subtracting the percentage of cells in G₁ (determined by analysis of the FACS profiles with MultiCycle software) from 100. Open symbols, wild-type (BY2679); closed symbols, cln3ecb swi4ecb (BY2680).

ECB elements are required for transcriptional regulation of CLN3 and SWI4 and their target genes (CLN1, CLN2, and HO) through the cell cycle. Unbudded daughter-cell fractions from elutriation were grown in YEPGal, and samples were taken at 15-min intervals for isolation of total RNA, as well as for size determination, FACS analysis, and budding index. Data shown are from a single Northern blot iteratively hybridized with probes for the indicated transcripts; multiple blotting experiments (data not shown) were carried out to confirm the validity of the cycling pattern shown. Initial cell volumes of these preparative elutriation fractions were 18.4 fl (wild-type strain [BY2679]) (open squares) and 23.4 fl (cln3ecb swi4ecb strain [BY2680]) (filled squares). The graph at the upper left shows budding kinetics, and the other five graphs show quantitated hybridization signals for the transcripts indicated (data normalized to the ACT1 signal and plotted with the lowest point in the wild-type samples set at 1.0). Below, hybridization patterns for each transcript scanned by a PhosphorImager are shown.

CLN3 promoter region showing the positions of the ECB elements (solid boxes) and the glucose response elements (GREs) (open boxes). Below and to the left the sequence alignment of the six potential ECB elements of the CLN3 promoter is shown. Bases identical to the consensus sequence are shaded. To the right the mutations (in italics and lowercase) that have been introduced to prevent Mcm1 binding and to inactivate the ECB elements are shown. The boldface type indicates key residues for Mcm1 binding. uORF, untranslated ORF.

Loss of ECB-mediated transcription of key cell cycle regulators leads to increased cell volume. Exponential-phase cultures of strains BY2679, BY2680, BY2681, and BY2682 grown in YEPGlu (A) or YEPGal (B) and of strains BY2125, BY2270, and BY2278 grown in YEPGlu (C) or YEPGal (D) were analyzed for cell size profile as described in Materials and Methods. The relevant genotypes are indicated.

Wild-type (BY2679) and cln3ecb swi4ecb (BY2680) daughter cells exhibit very different budding kinetics in spite of their identical initial size distributions. Elutriated fractions of wild-type and cln3ecb swi4ecb daughter cells with identical size distributions (A) and starting median volumes of 15.1 and 15.0 fl, respectively, were sampled during outgrowth and monitored for budding and median cell size (B).

ECB mutants are more sensitive to α-factor and recover from α-factor arrest more slowly than wild-type cells. The upper portion of the figure shows 3,000, 1,000 and 300 cells of the genotypes indicated (strains BY2125, BY2680, BY2681, and BY2684) that were spotted on YEPglu plates containing 0 or 0.6 μM α-factor and allowed to grow for 2 days at 30°C. The lower portion of the figure shows FACS profiles of wild-type (BY2125) and cln3ecb swi4ecb (BY2680) cells that were grown in YEPGal. Growth was arrested by treatment with 5 μg of α-factor/ml for 135 min, and then the cells were released into the same medium and sampled at 15-min intervals.

Ectopic expression of CLN2 suppresses the increased cell size and heterogeneity of cln3ecb swi4ecb cells. A DNA construct with CLN2 under the control of the MET3 promoter or a control vector was integrated at leu2 in the cln3ecb swi4ecb strain and the comparable wild-type strain. For analysis, cultures were grown in synthetic minimal glucose medium lacking methionine to derepress the promoter, and size profiles were determined on a Coulter model Z2 analyzer.