Growth and the DNA-division sequence in the yeast Saccharomyces cerevisiae

Cells of the yeast S. cerevisiae can be cultured under conditions in which the DNA-division sequence, and not cellular growth, is the rate-limiting feature for cell proliferation. Relief of these limiting conditions, which has been shown to allow accelerated cell division, did not result in increased rates of cell mass accumulation during the time of rapid cell division. Moreover, under conditions of constant DNA-division sequence constraint, populations of smaller cells produced by slowing growth with cycloheximide gave rise to large cells when cycloheximide was removed. These observations suggest that in proliferating cells of S. cerevisiae the DNA-division sequences does not affect cellular growth.     

Novel cell cycle regulation in the yeast Schizosaccharomyces pombe: The DNA-division sequence modulates mass accumulation

For wee1 mutant cells of Schizosaccharomyces pombe the DNA-division sequence of the cell cycle can be differentially slowed by the presence of low concentrations of the S-phase inhibitor hydroxyurea, or by semipermissive temperatures for certain wee1 cdc double mutants. Under these conditions the rate of proliferation is decreased, yet still exponential. Relief of these constraints slowing the DNA-division sequence resulted in prompt increases in the exponential rates of mass accumulation, to rates greater than those normally found. These observations suggest that mass accumulation by this yeast is always modulated by performance of the DNA-division sequence.     

Unequal division in Saccharomyces cerevisiae and its implications for the control of cell division

The budding yeast, Saccharomyces cerevisiae, was grown exponentially at different rates in the presence of growth rate-limiting concentrations of a protein synthesis inhibitor, cycloheximide. The volumes of the parent cell and the bud were determined as were the intervals of the cell cycle devoted to the unbudded and budded periods. We found that S. cerevisiae cells divide unequally. The daughter cell (the cell produced at division by the bud of the previous cycle) is smaller and has a longer subsequent cell cycle than the parent cell which produced it. During the budded period most of the volume increase occurs in the bud and very little in the parent cell, while during the unbudded period both the daughter and the parent cell increase significantly in volume. The length of the budded interval of the cell cycle varies little as a function of population doubling time; the unbudded interval of the parent cell varies moderately; and the unbudded interval for the daughter cell varies greatly (in the latter case an increase of 100 min in population doubling time results in an increase of 124 min in the daughter cell's unbudded interval). All of the increase in the unbudded period occurs in that interval of G1 that precedes the point of cell cycle arrest by the S. cerevisiae alpha-mating factor. These results are qualitatively consistent with and support the model for the coordination of growth and division (Johnston, G. C., J. R. Pringle, and L. H. Hartwell. 1977. Exp. Cell. Res. 105:79-98.) This model states that growth and not the events of the DNA division cycle are rate limiting for cellular proliferation and that the attainment of a critical cell size is a necessary prerequisite for the "start" event in the DNA-division cycle, the event that requires the cdc 28 gene product, is inhibited by mating factor and results in duplication of the spindle pole body.     

The G1 interval in the mammalian cell cycle: dual control by mass accumulation and stage-specific activities

The temporal determinants of the G1 cell cycle interval were investigated using nine mammalian cell lines. In each case, cells were allowed to proliferate for many cell cycles under conditions that slowed progress through S phase without an equivalent impairment of overall mass accumulation. This disproportionate inhibition of progress through the cell cycle caused newly produced cells to be more massive than usual. Under these growth conditions, the determinants of the length of the G1 interval became evident. For two cell lines, HeLa S3 and NIH 3T3, a protracted S phase, and the resultant increase in mass, resulted in a dramatically shortened G1 interval. Thus, for these cell lines, a major portion of G1 time exists to accommodate mass accumulation needed to initiate the subsequent S phase. Nevertheless, under conditions that protracted S phase and shortened the G1 interval, cells still exhibited a measurable G1 time, reflecting the stage-specific activities within G1. One activity that may be responsible for this obligatory G1 time is the synthesis of a labile protein. For other cells studied here, protraction of S phase also caused proliferating cells to become more massive, but in these cases there was no diminution of the G1 time. For these cells, the entire G1 interval must accommodate G1-specific activities necessary to initiate a new cell cycle. A unifying view of the G1 interval recognizes the two distinct influences that determine the time spent in G1: the need to accumulate sufficient mass to initiate a new DNA-division sequence; and the stage-specific events necessary for the subsequent S phase. The length of the G1 interval is dictated by the longer of these two time-consuming activities.     

Yeast-phase cell cycle of the polymorphic fungus Wangiella dermatitidis.

The yeast-phase cell cycle of Wangiella dermatitidis was studied using flow microfluorimetry and the deoxyribonucleic acid (DNA) synthesis inhibitor hydroxyurea (HU). Exposure of exponential-phase yeastlike cells to 0.1 M HU for 3 to 6 h resulted in the arrest of the cells in DNA synthesis and produced a nearly homogeneous population of unbudded cells. Treatment of the yeast-phase cells with HU for 9 h or longer resulted in the accumulation of the cells predominantly as budded forms having either a single nucleus in the mother cell or a single nucleus arrested in the isthmus between the mother cell and the daughter bud. Exposure of unbudded stationary-phase cells to 0.1 M HU resulted in the accumulation of the cells in the same phenotypes. Analysis by flow microfluorimetry and cell counts of HU-inhibited mithramycin-stained cells indicated that the eventual progress of HU-inhibited cells from unbudded to the two budded forms was due to the limited continuation of the growth sequence of the cell cycle even in the absence of DNA synthesis, nuclear division, and in some cases nuclear migration. On the basis of these observations and the results of flow microfluorimetric analysis of exponential-phase cells, a map of the yeast-phase cell cycle was constructed. The cycle appears to consist of two independent sequences of events, a budding growth sequence and a DNA division sequence. The nuclear division cycle of yeast-phase cells growing exponentially with a 4.5-h generation time is composed of a G1 interval of 148 min, as S phase of 16 min, and a G2 plus M interval of 107 min.     

A model of cell cycle control: effects of thymidine on synchronous cell cultures.

Further evidence is presented in support of a model for growth control in which commitment for cell division is determined by an event in the preceding cell cycle. A study was made of conditions affecting synchronous growth following treatment of murine mastocytoma cells with excess thymidine at different phases of the cell cycle. Cells were synchronized by a physical procedure involving velocity sedimentation in a zonal rotor. Pulse treatment of such cultures with thymidine at times corresponding to the S, G2, and M periods had no effect on further growth. However, addition at G1, although having no immediate effect, arrested cell growth in the next cell cycle. This temporal effect may account for the decay of synchrony observed during double thymidine blockade or thymidine-FUdR blockade. When the time interval between two such blocks was 7 hr or less, P815Y cells were arrested after one synchronous division. At this critical time a majority of cells were at, or near, G1. It is suggested that thymidine exerts a hitherto unrecognized effect at the G1 interval.     

Cell cycle phases in the unequal mother/daughter cell cycles of Saccharomyces cerevisiae.

During cell division in the yeast Saccharomyces cerevisiae mother cells produce buds (daughter cells) which are smaller and have longer cell cycles. We performed experiments to compare the lengths of cell cycle phases in mothers and daughters. As anticipated from earlier indirect observations, the longer cell cycle time of daughter cells is accounted for by a longer G1 interval. The S-phase and the G2-phase are of the same duration in mother and daughter cells. An analysis of five isogenic strains shows that cell cycle phase lengths are independent of cell ploidy and mating type.     

Partial elimination of G1 and G2 periods in higher plant cells by increasing the S period

Meristematic cells from Allium cepa L roots can attain a steady-state of growth at both 15 and 25 °C in the presence of drugs, hydroxyurea and 5-amino-uracil, which reduce the rate of DNA synthesis. These drugs, at used concentrations, significantly lengthen the S period without altering the cell growth rate, as indicated by the maintenance of the generation time. It has been observed that steady-state populations respond to a gradual increase in S by a reduction of G2 until a minimum value; with larger lengthening of S, both G1 and G2 are reduced. Natural synchronous populations have been used to study cell cycle parameters during transition from the physiological steady-state to the new one created by the presence of the drug. G2 (but not G1) is reduced during transition even in the presence of maximum drug concentrations that do not alter the cell growth rate. Both the S period and the division time are lengthened during transition. These observations support the concept that certain fractions of G1 and G2 are expendable, because they have no role in the DNA-division sequence of cell cycle events. We conclude that cell size regulates the length of these fractions by means of a negative correlation.     

Controls over the timing of DNA replication during the cell cycle of fission yeast

The controls acting over the timing of DNA replication (S) during the cell cycle have been investigated in the fission yeast Schizosaccharomyces pombe. The cell size at which DNA replication takes place has been determined in a number of experimental situations such as growth of nitrogen-starved cells, spore germination and synchronous culture of wee mutant and wild-type strains. It is shown that in wee mutant strains and in wild type grown under conditions in which the cells are small, DNA replication takes place in cells of the same size. This suggests that there is a minimum cell size beneath which the cell cannot initiate DNA replication and it is this control which determines the timing of S during the cell cycle of the wee mutant. Fast growing wild-type cells are too large for this size control to be expressed. In these cells the timing of S may be controlled by the completion of the previous nuclear division coupled with a requirement for a minimum period in G1. Thus in S. pombe there are two different controls over the timing of S, either of which can be operative depending upon the size of the cell at cell division. It is suggested that these two controls may form a useful conceptual framework for considering the timing control over S in mammalian cells.     

Specification of sites for polarized growth in Saccharomyces cerevisiae and the influence of external factors on site selection.

Many eucaryotic cell types exhibit polarized cell growth and polarized cell division at nonrandom sites. The sites of polarized growth were investigated in G1 arrested haploid Saccharomyces cerevisiae cells. When yeast cells are arrested during G1 either by treatment with alpha-factor or by shifting temperature-sensitive cdc28-1 cells to the restrictive temperature, the cells form a projection. Staining with Calcofluor reveals that in both cases the projection usually forms at axial sites (i.e., next to the previous bud scar); these are the same sites where bud formation is expected to occur. These results indicate that sites of polarized growth are specified before the end of G1. Sites of polarized growth can be influenced by external conditions. Cells grown to stationary phase and diluted into fresh medium preferentially select sites for polarized growth opposite the previous bud scar (i.e., distal sites). Incubation of cells in a mating mixture results in projection formation at nonaxial sites: presumably cells form projections toward their mating partner. These observations have important implications in understanding three aspects of cell polarity in yeast: 1) how yeast cell shape is influenced by growth conditions 2) how sites of polarized growth are chosen, and 3) the pathway by which polarity is affected and redirected during the mating process.     

Regulation of cell size in the yeast Saccharomyces cerevisiae.

For cells of the yeast Saccharomyces cerevisiae, the size at initiation of budding is proportional to growth rate for rates from 0.33 to 0.23 h-1. At growth rates lower than 0.23 h-1, cells displayed a minimum cell size at bud initiation independent of growth rate. Regardless of growth rate, cells displayed an increase in volume each time budding was initiated. When abnormally small cells, produced by starvation for nitrogen, were placed in fresh medium containing nitrogen but with different carbon sources, they did not initiate budding until they had grown to the critical size characteristic of that medium. Moreover, when cells were shifted from a medium supporting a low growth rate and small size at bud initiation to a medium supporting a higher growth rate and larger size at bud initiation, there was a transient accumulation of cells within G1. These results suggest that yeast cells are able to initiate cell division at different cell sizes and that regulation of cell size occurs within G1.     

Ras-mediated cell cycle arrest is altered by nuclear oncogenes to induce Schwann cell transformation.

The cellular responses to ras and nuclear oncogenes were investigated in purified populations of rat Schwann cells. v-Ha-ras and SV40 large T cooperate to transform Schwann cells, inducing growth in soft agar and allowing proliferation in the absence of added mitogens. Expression of large T alone reduces their growth factor requirements but is insufficient to induce full transformation. In contrast, expression of v-Ha-ras leads to proliferation arrest in Schwann cells expressing a temperature-sensitive mutant of large T at the restrictive temperature. Cells arrest in either the G1 or G2/M phases of the cell cycle, and can re-enter cell division at the permissive temperature even after prolonged periods at the restrictive conditions. Oncogenic ras proteins also inhibit DNA synthesis when microinjected into Schwann cells. Adenovirus E1a and c-myc oncogenes behave similarly to SV40 large T. They cooperate with Ha-ras oncogenes to transform Schwann cells, and prevent ras-induced growth arrest. Thus nuclear oncogenes fundamentally alter the response of Schwann cells to a ras oncogene from cell cycle arrest to transformation.     

Control of cell division in Paramecium tetraurelia. Effects of abrupt changes in nutrient level on accumulation of macronuclear DNA and cell mass

In the cell cycle of Paramecium there are three points of interaction between cell growth-related processes and the processes of macronuclear DNA replication and cell division: initiation of DNA synthesis, regulation of the rates of growth and DNA accumulation, and initiation of cell division. This study examines the regulation of the latter two processes by analysis of the response of each to abrupt changes in nutrient level brought about either by transferring dividing cells from a steady-state chemostat culture to medium with unlimited food, or by transferring well-fed dividing cells to exhausted medium. The rates of DNA accumulation and cell growth respond quickly to changes in nutrient level. The amounts of these cell components accumulated during the cell cycle following a shift in nutrient level are typical of those occurring during equilibrium growth under post-shift conditions. Commitment to division occurs at a fixed interval prior to fission that is similar in well-fed and nutrient-limited cells. Initiation of cell division in Paramecium is associated with accumulation of a threshold DNA increment, whose level is largely independent of nutritive conditions. The amount of DNA accumulated during the cell cycle varies with nutritional conditions because the rates of growth and DNA accumulation are affected by nutrient level; slowly growing cells accumulated relatively little DNA during the fixed interval between commitment to cell division and fission.     

Indirect suppression of the wee1 mutant phenotype in Schizosaccharomyces pombe

For S. pombe cells mutations in the wee1 regulatory gene have been shown previously to allow cells to be smaller than normal at cell division, to endow the cell with a significantly long G1 cell cycle interval, and to alter the timing in the cell cycle of certain mutationally-defined cell cycle steps in G2. We show here that situations which lengthen S phase in proliferating wee1 mutant cells 'suppress' to varying degrees these wee1-mediated cell cycle alterations. Conditions chosen to protract S phase were use of cdc22.M45 mutant cells at semipermissive temperatures, and the presence of sub-arresting concentrations of the S phase inhibitors hydroxyurea or deoxyadenosine. Proliferation in the presence of each of these inhibitors was shown directly to result in protracted S phase. Residual cell division measurements were used to measure the cell cycle timing of G1 and G2 cell-cycle steps. The indirect suppression of the wee1 phenotype shown here can be understood in terms of the proposed role of the wee1+ gene product in coordinating cell division with cellular growth.     

Calmodulin in Zajdela hepatoma cell growth

Calmodulin levels were measured in Zajdela hepatoma cells growing both in vivo and in culture, with respect to the distribution of the cells into G1 and S+G2 phases of the cell cycle and growth conditions. These levels, expressed on a per-microgram of protein basis, were significantly elevated at the G1-S boundary and maintained throughout the remainder of the cell cycle. This elevation of calmodulin took place independently of the culture conditions. Taken together with previous observations, these data suggest that a threshold concentration for calmodulin is required for progression through the cell cycle, DNA synthesis and cell division.     

Systematic identification of cell size regulators in budding yeast

Cell size is determined by a complex interplay between growth and division, involving multiple cellular pathways. To identify systematically processes affecting size control in G1 in budding yeast, we imaged and analyzed the cell cycle of millions of individual cells representing 591 mutants implicated in size control. Quantitative metric distinguished mutants affecting the mechanism of size control from the majority of mutants that have a perturbed size due to indirect effects modulating cell growth. Overall, we identified 17 negative and dozens positive size control regulators, with the negative regulators forming a small network centered on elements of mitotic exit network. Some elements of the translation machinery affected size control with a notable distinction between the deletions of parts of small and large ribosomal subunit: parts of small ribosomal subunit tended to regulate size control, while parts of the large subunit affected cell growth. Analysis of small cells revealed additional size control mechanism that functions in G2/M, complementing the primary size control in G1. Our study provides new insights about size control mechanisms in budding yeast.     

A model for restriction point control of the mammalian cell cycle

Inhibition of protein synthesis by cycloheximide blocks subsequent division of a mammalian cell, but only if the cell is exposed to the drug before the "restriction point" (i.e. within the first several hours after birth). If exposed to cycloheximide after the restriction point, a cell proceeds with DNA synthesis, mitosis and cell division and halts in the next cell cycle. If cycloheximide is later removed from the culture medium, treated cells will return to the division cycle, showing a complex pattern of division times post-treatment, as first measured by Zetterberg and colleagues. We simulate these physiological responses of mammalian cells to transient inhibition of growth, using a set of nonlinear differential equations based on a realistic model of the molecular events underlying progression through the cell cycle. The model relies on our earlier work on the regulation of cyclin-dependent protein kinases during the cell division cycle of yeast. The yeast model is supplemented with equations describing the effects of retinoblastoma protein on cell growth and the synthesis of cyclins A and E, and with a primitive representation of the signaling pathway that controls synthesis of cyclin D.     

cAMP-mediated increase in the critical cell size required for the G1 to S transition in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, cyclic AMP is required for cellular growth. In this study we show that cAMP also specifically inhibits the G1-S transition of the S. cerevisiae cell cycle by increasing the critical cell size required at start, the major yeast cell cycle control step. In fact: (a) addition of cAMP delays the time of entering into the S budded phase of small G1 cells, while it is ineffective on large fast-growing cells. (b) If cell growth is strongly depressed, cAMP permanently inhibits cell cycle commitment of cells arrested at the alpha-factor-sensitive step. The cell fraction inhibited by cAMP is inversely correlated with the average cell size of treated populations. (c) The critical protein content (Ps) and the critical cell volume (VB) required for budding in unperturbed exponentially growing yeast populations are largely increased by cAMP. On these bases, we propose a new cAMP role at start.