INTRODUCTION

Top Abstract Introduction Materials & Methods Results Discussion References

A budding yeast divides by an asymmetrical process. The degree of asymmetry at division depends on the growth rate, with slow-growing cells dividing more asymmetrically (14, 16, 19-21, 31, 37). In an asynchronously growing S. cerevisiae population, individual cells differ in their positions within the cell division cycle, their genealogical ages (i.e., daughters, parents of first generation, parents of second generation, etc.), and their sizes, although cells of the same size do not necessarily have the same age or cell cycle position (8, 9, 14, 16, 17, 20-22, 28, 31, 34, 35, 38). All of these differences determine the cell size distribution (i.e., the cell protein content distribution) of the growing population. It has been shown that the protein content distribution of a given population in balanced exponential growth is stable and characteristic of each growth condition (1, 3, 4, 24, 27, 29, 35). This homeostasis depends on the mechanism that coordinates cell growth to DNA division cycle and prevents cells from becoming too small or too large (14, 28). Changes in the environment modify the protein content distribution of a given population in steady state, adjusting it, after a transitory period, to the protein distribution characteristic of the new growth condition (1, 3, 4, 29); this behavior indicates that the homeostasis mechanism is operating under constraints different from those active in steady-state environments. These experimental conditions could be very informative in investigating the effects of cell size on the control of cell cycle progression. In order to adequately describe a transitory state it would be useful to determine the relationship between the cell size of the newborn daughter cells (called Po) and the fraction of budded cells. In this study, we describe a set of experiments used to analyze the Po value and the duration of the budded phase during transitory states of growth. Studies on the dynamics of the growth of individual yeast cells and their relationships with factors affecting cell cycle regulation are generally done by cell synchronization procedures (38) or, with a relatively small number of cells, by time-lapse studies (14, 37, 38). Time-lapse studies and synchronous cell population analyses usually require elaborate experimental procedures and tend to perturb the physiological state of the cells. We used a recently developed flow cytometric approach that allows, in asynchronously growing S. cerevisiae populations, the tagging of both the cell age and the cell protein content of individual cells (24-27). This approach permitted characterization of the properties of growth at the single cell level for yeast populations exponentially growing at different specific growth rates.

The findings obtained during transient states of growth (i.e., in a nutritional shift-up, in a nutritional shift-down, and following the addition of cyclic AMP [cAMP]) indicate that the whole population needs several hours to reach the new balanced growth condition. We found that both the nutritional shift-up and the addition of cAMP delay the entrance into S phase and the exit from the cycle, although according to different schedules.

	MATERIALS AND METHODS

Top Abstract Introduction Materials & Methods Results Discussion References

Strains and growth conditions. S. cerevisiae S288C (29) and OL214 (7) were used in this study. Cells were grown in flasks by being shaken at 30°C in Difco yeast nitrogen base (YNB, 0.67% [wt/vol]) medium. The carbon sources were 2% (wt/vol) glucose or 2% (vol/vol) ethanol. cAMP was added to a final concentration of 3 mM as previously described (7).

Staining conditions. Cells growing under conditions of balanced exponential growth (balanced growth was typically observed in a cell concentration range of between 1 × 10⁶ and 6 × 10⁶ cells ml¹) were collected by centrifugation (5 min, 5,000 rpm) and sonicated in order to allow the cell wall staining of each individual cell. Cells were stained by resuspension in precooled fresh YNB-based medium containing 120 µg of conjugated concanavalinA-fluorescein isothiocyanate (ConA-FITC) ml¹ (ca. 3.6 mol of FITC per mol of lectin) at a cell concentration of 2 × 10⁸ cells ml¹. The operations were carried out at 4°C. After 7 min of staining, the cells were recovered by rapid centrifugation and resuspended in a prewarmed fresh medium. We previously showed that the staining procedure does not perturb the normal growth behavior of the population (25). At different times after resuspension and new growth, yeast cells were collected by centrifugation, washed and fixed in 70% (vol/vol) ethanol for 20 min at 4°C. The fixed cells were centrifuged and washed once with phosphate buffer (pH 7.4), and the cell proteins were stained by resuspending the cells in 0.5 M sodium bicarbonate containing 50 µg of tetramethylrhodamine isothiocyanate (TRITC) ml¹. After 30 min, cells were recovered by centrifugation, washed three times, and resuspended in phosphate buffer.

Cell number, fraction of budded cells, budded phase, and Td-d determinations. Cells were counted after sonication with a Coulter Counter ZBI. The specific growth rate was obtained by fitting the cell number against time. The fraction of total budded cells (FBC) was calculated after microscopic examination (at least 500 cells were scored). The duration of the budded phase during the balanced growth period or the time of budding (Tb), comprising the S, G₂, M, and G₁* cell cycle phases, was determined from the following equation (19):

(1)

where Td is the duplication period for the whole cell population. Table 1 shows the budded-phase durations for the daughter cells; however, it is generally accepted that, at any given specific growth rate, the budded period is appreciably constant for both daughter and parent cells (3, 19, 25, 35). The duplication times of the daughter subpopulations (Td-d) were calculated with a mathematical model that takes into account four different subpopulations of parent and daughter cells (1, 3, 23, 24, 26, 27) or by using the following equation (19):

(2)

where FDB represents the fraction of daughter cells that are budded.

Flow cytometric analysis. FITC and TRITC fluorescence signal intensities were determined with a FACStarplus (Becton Dickinson) equipped with an argon-ion laser (excitation wavelength, 488 nm; laser power, 200 mW) (24, 26, 27). The sample flow rate during analysis did not exceed 500 to 600 cells per s. Typically, 50,000 cells were analyzed per sample. Only raw data have been used to prepare figures.

	RESULTS

Top Abstract Introduction Materials & Methods Results Discussion References

Analysis of the dynamics of growth during a nutritional shift-up. Figure 1 shows the growth curve, the FBC, and the cell protein content distributions of an S288C yeast population that was grown on ethanol-YNB, harvested during the exponential phase of growth (specific growth rate, 0.156 h¹; duplication time, 4.42 h), washed, and then resuspended in a glucose-YNB fresh medium at time t = 0. The parameters obtained during the nutritional shift-up indicate a very strong modulation of the FBC (Fig. 1A), the specific growth rate (Fig. 1A), and the distributions of the cellular protein content (Fig. 1B). It is important to note the oscillatory change of the FBC value and the increase of the protein content distribution of the growing population (Fig. 1B). The FBC represents a sensitive determination of the relative rates of the entrance into the S phase-budded phase and of the exit from the mitosis-cell division phase, while the increased size of the cells suggests that the mechanism which coordinates the cell growth with the DNA division cycle has undergone a resetting.

View larger version (29K): [in this window] [in a new window]   FIG. 1.   Effects of a nutritional shift-up on the FBC value, cell concentration, and distribution of the cellular protein content. (A) S288C yeast cells were grown on ethanol-YNB medium to balanced exponential phase (0.156 h1), harvested, washed, and inoculated in fresh glucose-YNB medium at t = 0. The specific growth rate and the FBC of the control yeast population exponentially growing on glucose-YNB were 0.352 h1 and 0.58, respectively. The specific growth rate of the yeast population upon reaching the balanced growth conditions was 0.327 h1. Symbols: , number of cells per milliliter for the control yeast population growing on ethanol-YNB; , number of cells per milliliter for the control yeast population growing on glucose-YNB; , number of cells per milliliter for the yeast population growing during the nutritional shift-up from ethanol to glucose; , FBC for the yeast population growing during the nutritional shift-up from ethanol to glucose. (B) At different times after resuspension, samples of the culture were withdrawn, and the cell proteins were stained with TRITC and analyzed at t = 0 h (A), t = 1 h (B), t = 2 h (C), t = 3 h (D), t = 4 h (E), and t = 6 h (F). GLU, distribution of the cellular protein content of the control yeast population growing on glucose-YNB.

1

1

Identification of newborn daughter cells and determination of their size during the transitory state. We used the previously described procedure to analyze individual cells growing during the nutritional shift-up depicted in Fig. 1. Figure 2A shows the time course of the double-staining pattern. At time t = 0 (i.e., the time of resuspension) the cells belong to all of the different cell cycle positions and are completely stained with both of the fluorescent tags, ConA-FITC (see insert at the top) and TRITC; each individual cell of the yeast population is represented by a single dot. Since the synthesis of new cell wall material in the budded cells is restricted to the bud (5, 10, 24-27, 32), the first effect of the new cell growth is the production of newborn daughter cells with a gradually decreasing amount of surface stain (i.e., partially stained daughter cells). The analysis of the cytograms clearly indicates a growing tail of newborn partially stained cells and the accumulation of cells with the same degree of staining (i.e., unstained cells). The partially stained cells are newborn daughter cells that originated from cells stained during the budded phase (S-G₂-M-G₁*) of the cell cycle (24-27) (Fig. 2A). Completely unstained cells represent the newborn daughter cells that originated from cells stained while in the unbudded phase (G₁) (24-27). The time of appearance (i.e., after 3.5 h) of this last subpopulation of new daughter cells is a function of the duration of the S-G₂-M-G₁* phase (24-27). Therefore, this analysis allows a direct determination of the length of the budded phase during the transitory state (Table 1) (i.e., being 2.48 h for the budded phase of the yeast population growing on ethanol and 1.39 h for the budded phase during the balanced growth condition on glucose). Examples of selection of newborn daughter cells that originated during the transient state of growth are shown in the Fig. 2A: the gate R1 on the cytogram t = 0.66, the gate R2 on the cytogram t = 1, the gate R3 on the cytogram t = 2, the gate R4 on the cytogram t = 3, and the gate R5 on the cytogram t = 4 indicate the diverse regions where newborn daughter cells were found. In this way, it is possible to identify the newborn daughter cells (i.e., on the abscissas) and to determine their size (i.e., on the ordinates). Figure 2B shows the protein content value of diverse cohorts of newborn daughter cells that originated during the shift-up. The figure also shows the cell protein content of newborn daughter cells that originated during the balanced cell growth period on ethanol and glucose (i.e., the controls). As previously indicated, the analysis of such data also suggests that the yeast population needs about 4 h to reach the new balanced growth condition. After that point, the cell size of the newborn daughter cells appears to be constant and very close to that of the control population growing on glucose.

View larger version (47K): [in this window] [in a new window]   FIG. 2.   Dynamic of the double tagging over time for cells grown on ethanol-YNB, stained with ConA-FITC, and resuspended in glucose-YNB medium. (A) At different times after resuspension, samples of the culture were withdrawn and cell proteins were stained with TRITC. The cell wall tag (abscissa: ConA-FITC, channel number) and cell size tag (ordinate: TRITC, channel number) signals were acquired with a linear scale. During the acquisition of data at time t = 0, only the cells with lower ConA-FITC signals (i.e., the smaller cells) were acquired (St., completely stained), and the settings were not changed during the experiment. This approach provides evidence of even the smallest differences in the ConA-FITC fluorescence signals between individual cells (26, 27). The evolution of partially stained (P.St.) and then unstained (Un.) cells over time is clearly visible. The diagram at the top of panel A shows the cell cycle phases of a growing yeast cell. (B) Cell protein content value of cohorts of newborn daughter Po cells born at different times after resuspension. S288C yeast cells were grown in ethanol-YNB medium till the exponential phase; they were then stained with ConA-FITC, resuspended in glucose-YNB medium, and processed as described in Fig. 2A. Examples of selection of the regions where newborn daughter cells were located after birth are indicated by gates R1 (t = 0.66), R2 (t = 1.0), R3 (t = 2), R4 (t = 3) and R5 (t = 4) in Fig. 2A. To obtain the data after t = 4, cells from parallel and independent experiments were collected, completely stained with ConA-FITC, resuspended in the same medium, and processed as already indicated. Newborn daughter cells born on ethanol or glucose (controls, open symbols) were selected by applying the same procedure to yeast cells exponentially growing on ethanol or glucose. Data are expressed as the average channel number of the relative protein content distributions (TRITC signals).

Determination of the specific growth rate of individual daughter cells during the transitory state. We previously showed that, since the stained cells retain the surface label over the subsequent growth period (i.e., the dye on the single cells is only diluted by the new growth) (25), it is possible to follow over time the growth dynamics of a cohort of selected daughter cells born at the same time (24-27). In Fig. 2A, an example of the selection of such a cohort of daughter cells over time (gate R1 in the cytograms t = 0.66 through t = 6) is reported. The dynamics of the cell protein content values over time are shown in Fig. 3. Different cohorts of daughter cells, born at different times during the same experiment (i.e., 0.666, 1, and 2 h after resuspension), have been analyzed. Experimental data acquired from birth till t = 5 clearly indicate an exponential increase of the cell size over time, reflecting an exponential growth of the individual cells (Fig. 3, open symbols). The coefficient of correlation for all cases is higher than 0.99. The exponential fitting of the data yields the CSIR, i.e., the specific growth rate (27), of the different cohorts of daughter cells. The CSIR values for the three different cohorts of daughter cells are the same (CSIR = 0.324 ± 0.05 hr¹ from birth till t = 5) and are identical to the specific growth rate of the overall population during the newly reached balanced growth condition as determined by the increase of the cell number concentration (0.327 h¹). Suddenly, 5 h after resuspension, an exponential rate law cannot be utilized any more (Fig. 3, closed symbols). However, it is important to note that the time at which such deviation from the exponential increase occurs is the same for the three different cohorts of cells. At the time of deviation, the cell sizes of the cells belonging to the different cohorts are also the same. We have previously shown for yeast populations exponentially growing with different duplication times that this deviation depends on, and slightly precedes in time, the cellular division of the daughter cells (i.e., 31 min for a yeast population growing with a specific growth rate of 0.25 h¹ and 15 min and 5 to 7 min for specific growth rates of 0.224 and 0.211 h¹, respectively) (27). Figure 3 also reports the cell size increase, from birth to division, of daughter cells born during the balanced cell growth on glucose. It is interesting that the cell size of the dividing daughter cells during balanced growth on glucose (i.e., 441.76) also represents the cell size value reached from the selected daughter cells at 40 min (t = 5.66) after the deviation observed at t = 5. The CISR values as well as the coefficients of correlation for the three cohorts of cells selected do not change following inclusion of these last data, and therefore it is reasonable to assume that the selected daughter cells divide at t = 5.66. 

View larger version (21K): [in this window] [in a new window]   FIG. 3.   Average cell protein content (TRITC signals) of selected cohorts of partially stained daughter cells as a function of time after birth during the transitory state of growth. S288C yeast cells were grown in ethanol-YNB medium until the exponential phase; they were then stained with ConA-FITC, resuspended in glucose-YNB medium, and processed as described for Fig. 2A. (Since, from birth to division, the surface label does not change, the gate R1 in Fig. 2A selects the same cohort of partially stained daughter cells over time.) The figure shows the average cell protein content (TRITC signal) of selected cohorts of daughter cells born at different times (i.e., 0.666 h [ and ], 1 h [ and ], and 2 h [ and ]; see arrows) after resuspension. Data represented with the open symbols clearly fit an exponential increase of the cell size over time (in each case the coefficient of correlation is higher than 0.99), while the data represented by the closed symbols show that an exponential rate law cannot be utilized. Starting from t = 0 are also reported data for the cell size increase for daughter cells exponentially growing on ethanol or during the balanced-growth phase on glucose (i.e., the controls). These values have been calculated by taking into consideration the estimated specific growth rate of the daughter populations (0.156 and 0.327 h1, respectively), the estimated cell size values of the daughter cells at birth, and the calculated generation times of the daughter subpopulations (the generation times of the daughter populations were calculated, by using a previously developed mathematical model, to be 6.56 and 2.74 h for the yeast populations growing on ethanol and glucose, respectively). The same data were obtained by using equation 2 as described in the text). The cell size value of the dividing daughter cells during exponential growth on glucose (i.e., the control) has been used to extrapolate (dashed arrow) the correct cell sizes of the different cohorts of daughter cells at division (data in the circle). Data are expressed as the average channel number of the relative protein content distributions (TRITC signals).

Determination of generation time, duration of the G₁ phase, and duration of the budded phase of diverse cohorts of daughter cells born before, during, and after the transitory state. By using the experimental procedure previously described in Fig. 2 and 3, the generation times for the daughter cells selected at different times after resuspension were determined (Table 1). These generation times were found to be 5.0 h for daughter cells born at t = 0.66 and grown till t = 5.66; 4.66 h for daughter cells born at t = 1.0 and grown till t = 5.66, and 3.66 h for daughter cells born at t = 2.0 and grown till t = 5.66. It is interesting that 5, 4.66, and 3.66 h are periods of time much longer than the duplication time of the overall population during the steady state of growth on glucose (i.e., 2.11 h = ln2/0.327).

Cell size of the newborn daughter cells as related to their G₁-phase length. The nutritional shift-up produced newborn daughter cells of increasing size (Fig. 2B), growing with the same specific growth rate (Fig. 3) and having different duplication times and different G₁ phase lengths (Fig. 3 and Table 1). More importantly, these new daughter cells share the same budded-phase period and begin to bud and then divide at the same Ps and Pd values (Table 1). This observation suggests that the different cohorts of daughter cells that originated at different times after resuspension are able to actively control their commitment to Start phase and to cell division. A similar process of resetting of the individual cell cycles for daughter cells is also operative during balanced cell growth. In fact, newborn daughter cells which originated from parent cells of different genealogical ages have different sizes at birth but begin to bud and then divide showing similar protein contents (3, 26, 27, 35). A simple explanation is that all daughter cells are endowed at birth of the same amount of an inhibitor of the entrance into S phase that would be inactivated by a growth-dependent protein produced during G₁ phase (2). Figure 4A shows the relationship of the cell size of the newborn daughter cells produced over time during the nutritional shift-up (see also Fig. 3) compared to their G₁ durations (see also Table 1); Fig. 4B shows the relationship between the cell size of a single cohort of daughter cells during cell cycle progression and the remaining G₁ phase duration. The linear correlations found (in both cases the coefficients are higher than 0.99) suggest a relation between the cell size of the daughter cells and the duration of their G₁ phase. The daughter cells under examination in Fig. 4A were produced from cells that crossed Start before the nutritional shift-up. Since all of the cells increased in size at the same specific rate (i.e., the amount of protein synthesized per unit of cell mass per unit of time; see Fig. 3 and text) and grow through the same cell cycle phases, the synthesis rates (i.e., the amount of protein synthesized per unit of cell mass) of the constitutively synthesized proteins per cell may be assumed to be the same. However, if a protein(s) is compartmentalized in the nucleus and it is assumed that the dimension of the nucleus is constant and independent of the cell size, that protein will be inherited at increasing concentrations from the newborn daughter cells produced over time. The increased concentration(s) could determine a proportionally faster commitment to Start phase (Fig. 4). The growth-dependent factor would neutralize the above-mentioned inhibitor and induce a sharp and reproducible commitment to S phase at a given threshold (2). A tentative model that involves the Cln3 protein (and its constant accumulation rate through the cell cycle together with its compartmentalization in the nucleus) as the growth-dependent factor has recently been proposed (12).

View larger version (15K): [in this window] [in a new window]   FIG. 4.   Cell size of newborn daughter cells produced during the shift-up relative to their G1 durations. (A) The cell sizes of the newborn daughter cells were determined as described in Fig. 2; their G1 durations were determined as described in Table 1. (B) Cell sizes during cell cycle progression for the cohort of daughter cells originated at t = 0.666 (Fig. 3) relative to the remaining G1 phase duration. Data are expressed as the average channel number of the relative protein content distributions (TRITC signals).

Analysis of the dynamics of growth after the addition of cAMP. The same flow cytometric approach has been used to gain insight into the effects of the addition of cyclic AMP (cAMP) to yeast cells exponentially growing on glucose. In our laboratory it has been previously shown that the addition of cAMP to yeast cells growing on glucose determines an oscillatory behavior in the fraction of budded cells and an increase of the cell size (7). For such investigations, a strain permeable to cAMP is required (strain OL214). Figures 5A and B show the growth curve, FBC, and distributions of the cellular protein contents of a yeast population grown on glucose, harvested in the exponential phase of growth (Td = 1.86 h, with a budded-phase duration of 1.2 to 1.3 h), washed, and then resuspended in a glucose-YNB fresh culture medium containing 3 mM cAMP at time t = 0. The behavior of the FBC value and of the distribution of the cellular protein content is similar to that observed during a nutritional shift-up (see Fig. 1). However, several differences should be noted. Although following a nutritional shift-up the FBC value remains constant for about 0.666 h (Fig. 1), after the addition of cAMP it decreases immediately. A second main difference relies on the fact that after the addition of cAMP, the budded-phase period for cells stained at Start at the time of resuspension does not change compared to the value observed before the addition of cAMP (1.2 to 1.3 h [data not shown]). It is important to note that by 1.2 to 1.3 h after the addition of cAMP, the increase in the number of cells per milliliter is almost blocked (Fig. 5A), indicating that at this time also the rate of exit from the cycle is strongly reduced. On the other hand, confirming data previously obtained by determining the protein and RNA synthesis rates in the whole population (7), we did not detect any difference in the CSIR for cohorts of daughter cells selected before and after the addition of cAMP (data not shown). As a consequence, the size of the newborn daughter cells increases (Fig. 5C).

View larger version (21K): [in this window] [in a new window]   FIG. 5.   Effect of the addition of cAMP on the FBC value, number of cells per milliliter, distribution of the cellular protein content, and Po cell values. To OL214 yeast cells grown on glucose-YNB medium to balanced exponential phase (duplication time, 1.86 h), cAMP was added at time t = 0 (final concentration, 3 mM). (A) Symbols: , number of cells per milliliter for the control yeast population growing on glucose; , number of cells per milliliter for the yeast population after addition of cAMP; , FBC for the yeast population after the addition of cAMP. (B) At different times after resuspension, samples of the culture were withdrawn, and the cell proteins were stained with TRITC and analyzed at t = 0 h (A), t = 1.5 h (B), t = 1.83 h (C), t = 3 h (D), t = 3.5 h (E), t = 4.5 h (F), and t = 6 h (G). (C) Behavior of the average cell size of the population (), the average cell size of the newborn daughter cells, Po (), and the FBC in the population () for the yeast population growing after the addition of cAMP. Po and the average cell size of the population are expressed as the average channel number of the relative protein content distributions (TRITC signals).

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

Control of the cell cycle progression by nutrients has a key role in the regulation of cell proliferation in all organisms. Mechanisms are operative to coordinate nutrient availability to cell cycle transitions. Therefore, controlled manipulations of the cellular environment represent a useful methodological approach to gaining better understanding of the dynamics and the controls determining the cell cycle progression of a growing population.

In budding yeast, the main control of the cell division cycle takes place late in the G₁ phase at an area called Start (14, 28). At Start, three essential processes begin: budding, DNA replication, and spindle-pole body duplication. The cyclin-dependent kinase Cdc28, which is associated with the Cln3 cyclin, seems to be the most upstream activator of Start and acts as a potent inducer of two distinct transcriptional complexes, SBF and MBF, respectively (11, 12, 18, 30, 33).

The analysis of the findings obtained during the nutritional shift-up shown in Fig. 1 to 4 indicates that during this transition two delays are induced. The cells that at the moment of the nutritional shift-up existed before Start delay their entrance into S phase; cells cycling after Start delay their exit from the cycle. The fast decrease in the FBC (from 47.5 to 22.5%) that was observed during the first 2 h shows that the rate of entrance into S phase is much more reduced than the rate of exit from the cycle. When deprived of essential nutrients, yeast cells are arrested in G₁ phase. It has recently been shown that nitrogen-deprived G₁-phase-arrested cells are able to grow, reaching a volume much larger than the average size required for budding of nonstarved cells and thus suggesting a mechanism that causes the accumulation of cells in G₁ phase even when the critical mass for budding seems to be largely overcome (13). Such as response appears to be related to a faster degradation of Cln3 cyclin as a consequence of the changed nutritional conditions (13). In shift-up there is a prolonged stay in the G₁ phase, although the cell population is actively growing; a diminished availability of Cln3 in shifting cells could be the cause of the observed delay.

The second delay involves the cells shifted and stained after Start at the time of resuspension. The increased duration of the budded-phase period (from 2.48 to 3.5 h [Table 1]) indicates a delay in mitosis and cellular division. The main effect of such as delay is the production of newborn daughter cells of increasing size (Fig. 2B). These daughter cells grow with a specific rate characteristic of the new medium and are all committed to Start for the same Ps value characteristic for the new medium. The combined effects of the two delays allow the cell population that preexisted the shift-up to quickly adjust to the new growth condition.

The experimental approach described here has also been applied to gain insight into the effects of a nutritional shift-down. Cells were grown on fructose-yeast extract-peptone, harvested in the exponential phase of growth, stained with ConA-FITC, and then resuspended in a raffinose-YNB fresh culture medium. The Po cell value changes over time following a pattern similar to but opposite that shown for a nutritional shift-up. On the other hand, the qualitative behavior of the FBC value is similar to that observed during a nutritional shift-up, except that it shifts from higher (71% at time t = 0) to lower values (58% upon reaching the balanced growth condition) (data not shown).

Since the addition of glucose stimulates the level of cAMP in budding yeast cells, it has been proposed that at least part of the effects of glucose on growth and cell cycle progression could be mediated by cAMP, whose connection to the early events of Start is well known (6, 7). The analysis of the transition following the addition of cAMP to permeable yeast cells indicates both similarities and differences. While the shift-up both delays the entrance into S phase and the exit from the cycle for existing cells, the addition of cAMP first delays entrance into S phase and only after 1.2 to 1.3 h delays the exit from the cycle. The nutrients and cAMP modulations of the cell cycle, both at Start and at exit from the cycle, could offer useful experimental approaches for investigating the physiologically relevant events in cell cycle progression.