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.     

Growth and the cell cycle of the yeast Saccharomyces cerevisiae. II. Relief of cell-cycle constraints allows accelerated cell divisions

For cells of the yeast Saccharomyces cerevisiae, conditions which limit S phase or nuclear division allow steady-state division kinetics without significant effects on growth. Such cells become unusually large. When large proliferating cells were released from any one of several conditions which slowed progress through the DNA-division sequence, they underwent a period of accelerated division with a cell cycle devoid of a G1 interval, as evidenced by low proportions of unbudded cells and shifted execution points for the 'start' cell cycle step. We interpret these results to mean that when released from conditions slowing the DNA-division sequence these large cells continue for several cell doublings to accumulate mass fast enough to eliminate the need for a G1 interval. The results support the conclusion that the yeast G1 interval is the for most part only an interval of growth.     

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.     

Acquisition of meiotic competence in mouse oocytes: absolute amounts of p34(cdc2), cyclin B1, cdc25C, and wee1 in meiotically incompetent and competent oocytes

M-Phase promoting factor (MPF) is a complex of p34(cdc2) and cyclin B. Results of previous studies in which relative mass amounts of these cell cycle regulators were determined suggested that the accumulation of p34(cdc2), rather than cyclin B, could be a limiting factor in the acquisition of meiotic competence in mouse oocytes. Nevertheless, in the absence of measurements of the absolute amount of these components of MPF, it is possible that the molar amount of p34(cdc2) is in excess to that of cyclin B, i.e., the accumulation of p34(cdc2) is not a limiting factor. We report measurements of the absolute mass of p34(cdc2) and cyclin B1, as well as the two proximal regulators of MPF, namely cdc25C and wee1, in meiotically incompetent and competent mouse oocytes. We find that the numbers of molecules of p34(cdc2), cyclin B1, cdc25C, and wee1 in meiotically incompetent oocytes are 1.4 x 10(6), 11.3 x 10(6), 24.6 x 10(6), 15. 6 x 10(6), respectively, and in meiotically competent oocytes the numbers are 14.3 x 10(6), 95.5 x 10(6), 80.0 x 10(6), 40.1 x 10(6), respectively. Thus, the concentration of cyclin B1 is always in excess to that of p34(cdc2), and this is consistent with the hypothesis that the accumulation of p34(cdc2) plays a role in the acquisition of meiotic competence. Last, the concentration of cdc25C is greater than that of wee1 and the concentration of each is greater than that of p34(cdc2) in both meiotically incompetent and competent oocytes.     

Involvement of a type 1 protein phosphatase encoded by bws1+ in fission yeast mitotic control

Fission yeast cdc25+ and wee1+ interact genetically with cdc2+ in the regulation of cell division, respectively as a mitotic activator and inhibitor. cdc25+ is normally essential for mitosis, but this requirement is alleviated in a loss-of-function wee1 mutant background. A plasmid-borne sequence, other than wee1+, that causes a cdc25ts wee1- double mutant to revert to a temperature-sensitive cdc phenotype has been isolated. The gene carried by this plasmid is called bws1+ (for bypass of wee suppression). bws1+ also bypasses the ability of alleles of cdc2 that confer a wee phenotype (cdc2w) to suppress loss-of-function cdc25 mutants. The nucleotide sequence of bws1+ shows that the predicted protein shares 81% amino acid identity with the catalytic subunit of mammalian type 1 protein phosphatase. Thus a genetic screen that might have yielded a protein kinase (wee1+) uncovered a phosphatase that also appears to be involved in the pathway of mitotic control.     

Transformation in Tetrahymena pyriformis: description of an inducible phenotype.

Transformation of Tetrahymena pyriformis to a rapid-swimming (presumably dispersal) form can be induced by washing cells and suspending them in distilled H2O, Dryl's solution or 10 mM Tris. Transformation is possible with high efficiency in mass cultures of axenically grown cells within approximately 5 h at 30 C. The radically different phenotype produced during transformation is characterized by a more elongate body form, increased numbers of somatic basal bodies and cilia, a long caudal cilium and oral membranelles positioned beneath the cell surface. DNA quantities characteristic of G1, S, and G2 cells are found in these transformed ciliates, suggesting that achievement of a particular stage in the DNA-division cycle is not a prerequisite for transformation. Preliminary observations on cells belonging to syngens 2-12 indicate that they also have a capacity to form a caudal cilium, but that the amicronucleate strain GL-C does not. Possible relevance of the transformed phenotype for taxonomy of Tetrahymena is discussed.     

Tome-1, a trigger of mitotic entry,is degraded during G1 via the APC

Entry into mitosis requires the activation of cdk1/cyclin B, while mitotic exit is achieved when the same kinase activity decreases, as cyclin B is degraded. Cyclin B proteolysis is mediated by the anaphase promoting complex, or APC, an E3 ligase that is active at anaphase in mitosis through G1. We have identified a G1 substrate of the APC that we have termed Tome-1, for trigger of mitotic entry. Tome-1 is a cytosolic protein required for proper activation of cdk1/cyclin B and mitotic entry. Tome-1 associates with Skp-1 and is required for degradation of the cdk1 inhibitory tyrosine kinase wee1; Tome-1 therefore appears to be acting as part of an SCF-type E3 for wee1. Degradation of Tome-1 during G1 allows for wee 1 accumulation during interphase, thereby providing a critical link between the APC and SCF pathways in regulation of cdk1/cyclin B activity and thus mitotic entry and exit.     

Midcycle doubling of uptake rates of adenine and serine in Saccharomyces cerevisiae.

Rates of uptake of serine and of adenine were measured as a function of cell size, and therefore age, in asynchronous, exponential phase cultures of diploid Saccharomyces cerevisiae strain Y55. In both cases, uptake rates were constant during the initial third of the cell cycle and doubled during the S period in the middle part of the cycle to a constant value during the final third. Cell size and age at mid-step doubling were indistinguishable for serine and adenine uptake, and occurred during the period of DNA synthesis. The results extend an earlier hypothesis of constancy of cell growth rates (mass accumulation rates) and rates of uptake of all or almost all compounds into cells in exponential phase growth to one of piecewise constancy, with an abrupt doubling of growth and uptake rates during DNA synthesis.     

Mutation of fission yeast cell cycle control genes abolishes dependence of mitosis on DNA replication

Entry into mitosis in fission yeast is controlled by the p34cdc2 protein kinase, which is activated by cdc25+ and inhibited by wee1+. In "wee" mutants one or the other of these controls is circumvented resulting in advancement of mitosis. We report that dependence of mitosis on DNA synthesis is lost in wee mutants in which cdc25+ control is circumvented either by mutations in cdc2+ or by overproduction of cdc25+. In contrast, dependence is maintained when the wee1+ control is bypassed. We propose that cdc25+ activity requires completion of earlier cell-cycle events such as DNA synthesis, and thus links p34cdc2 kinase activation to completion of these earlier events. Constitutive expression of cdc25+ homologs could explain why mitosis is not dependent on DNA replication in some early embryos.     

Cell cycle analysis of Taxus suspension cultures at the single cell level as an indicator of culture heterogeneity

Single cell growth and division was measured via flow cytometry in order to characterize the metabolic variability of Taxus cuspidata suspension cultures, which produce the valuable secondary metabolite Taxol. Good agreement was observed between the cell cycle distribution and biomass accumulation over the batch culture period. Specific growth rates of 0.13 days(-1) by fresh weight and 0.15 days(-1) by dry weight were measured. Elicitation with methyl jasmonate (MJ) significantly decreased both cell cycle progression and biomass accumulation, as the specific growth rate decreased to 0.027 days(-1) by fresh and dry weight. Despite the decrease in biomass accumulation for MJ elicited cultures, sucrose utilization was not significantly different from control cultures. MJ elicitation also increased the accumulation of paclitaxel and other taxanes. The accumulation of upstream taxanes (baccatin III and 10-deactylbaccatin III) increased during exponential growth, reached a maximum around day 12, and then declined throughout the stationary phase. The paclitaxel concentration increased during both exponential growth and stationary phase, reaching a maximum around days 20-25. Throughout the culture period, greater than 70% of the cells were in G(0)/G(1) phase of the cell cycle. Studies using bromodeoxyuridine (BrdU) incorporation showed that approximately 65% of the Taxus cells are noncycling, even during exponential growth. Although the role of these cells is currently unknown, the presence of a large, noncycling subpopulation can have a significant impact on the utilization of plant cell culture technology for the large-scale production of paclitaxel. These results demonstrate that there is a high degree of metabolic heterogeneity in Taxus cuspidata suspension cultures. Understanding this heterogeneity is important for the optimization of plant cell cultures, particularly the reduction of production variability.     

Evidence for an intraclonal random shift between two types of cell cycle times in an embryonal carcinoma cell line

In a recent paper we reported the discovery of an intraclonal bimodal-like cell cycle time variation within the multipotent embryonal carcinoma (EC) PCC3 N/1 line growing in the exponential phase in the undifferentiated state. The variability was found to be localized in the G1 period. Furthermore, an inverse relation between cell size and cell generation time was found in the cell system analysed. It was suggested that the bimodal-like intraclonal time variability previously reported was attributable to an intraclonal shift between two types of cell-growth-rate cycles and that the cell-growth cycle has a supramitotic character, being dissociated from the DNA-division cycle. The growth rate heterogeneity in the cell population was found to need three cell cycles to reach full dispersion in time. This was assumed to be due to a decreased inheritance from sister cell pairs to second cousin cell pairs. Thus, the interesting feature is that in one and the same multipotent cell line there was evidence for an intraclonal instability with a random shift between two types of cell cycle differing in the duration of their G1 period.     

Transformation in Tetrahymena pyriformis: Description of an Inducible Phenotype*†

SYNOPSIS. Transformation of Tetrahymena pyriformis to a rapid-swimming (presumably dispersal) form can be induced by washing cells and suspending them in distilled H₂O, Dryl's solution or 10 mm Tris. Transformation is possible with high efficiency in mass cultures of axenically grown cells within ～ 5 h at 30 C. The radically different phenotype produced during transformation is characterized by a more elongate body form, increased numbers of somatic basal bodies and cilia, a long caudal cilium and oral membranelles positioned beneath the cell surface. DNA quantities characteristic of G1, S, and G2 cells are found in these transformed ciliates, suggesting that achievement of a particular stage in the DNA-division cycle is not a prerequisite for transformation. Preliminary observations on cells belonging to syngens 2–12 indicate that they also have a capacity to form a caudal cilium, but that the amicronucleate strain GL-C does not. Possible relevance of the transformed phenotype for taxonomy of Tetrahymena is discussed.     

Mcs4, a Two-Component System Response Regulator Homologue, Regulates the Schizosaccharomyces Pombe Cell Cycle Control

The Schizosaccharomyces pombe cdc2-3w wee1-50 double mutant displays a temperature-sensitive lethal phenotype termed mitotic catastrophe. Six mitotic catastrophe suppressor (mcs1-6) genes were identified in a genetic screen designed to identify regulators of cdc2. Mutations in mcs1-6 suppress the cdc2-3w wee1-50 temperature-sensitive growth defect. Here, the cloning of mcs4 is described. The mcs4 gene product displays significant sequence homology to members of the two-component system response regulator protein family. Strains carrying the mcs4 and cdc25 mutations display a synthetic osmotic lethal phenotype along with an inability to grow on minimal synthetic medium. These phenotypes are suppressed by a mutation in wee1. In addition, the wis1 gene, encoding a stress-activated mitogen-activated protein kinase kinase, was identified as a dosage suppressor in this screen. These findings link the two-component signal transduction system to stress response and cell cycle control in S. pombe.     

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.     

Modulation of cell-cycle regulatory signaling network by 2-methoxyestradiol in prostate cancer cells is mediated through multiple signal transduction pathways

2-Methoxyestradiol (2-ME(2)), a promising anticancer drug, induces growth arrest and apoptosis in various androgen-dependent (LNCaP) and -independent (DU145 and PC-3) prostate cancer cell lines. Moreover, flow cytometric analysis indicated a novel dual impact of 2-ME(2) on the cell division cycle of prostate cancer cells. Chronic exposure of high doses of 2-ME(2) enhance the accumulation of cells in S and G2/M phases, while cell numbers in the G1 phase were reduced significantly by this treatment. Because cyclin B1 overexpression, induction of cdc2 phosphorylation, and its regulatory proteins wee1 and phospho-cdc25C (interphase and mitotic forms) by 2-ME(2) treatment correlated with the induction of apoptosis, growth arrest at the G2/M phase, and accumulation of the S phase, we reasoned that cyclin B1 and cdc2 phosphorylation and its upstream regulatory molecular networks may be associated with the ultimate impacts of 2-ME(2). Because phosphorylation of cdc2 and upregulation of wee1 by 2-ME(2) can be abolished by both extracellular receptor kinase (ERK) inhibitor (U0126) and c-Jun N-terminal kinase (JNK) inhibitor (SP600125), our studies indicate that the 2-ME(2)-induced upregulation of wee1 and subsequent cdc2 phosphorylation are mediated through mitogen-activated protein kinase (MAPK)-ERK-JNK signaling pathways.     

Amber mutation affecting the length of Escherichia coli cells.

An amber mutation in a newly found gene (wee) of Escherichia coli has been isolated from strain OV-2, which harbors a temperature-sensitive suppressor. At 42 degrees C cells of the mutant, OV-25, increased in mass and deoxyribonucleic acid content and divided at normal rates, compared with the wild type under the same growth conditions. Total cell length increased under the restrictive conditions, although at a slightly lower rate. Values of mean cell length and cell volume, contrary to what would be expected from the increment in the rate of increase in particles, mass, and deoxyribonucleic acid, became at 42 degrees C smaller than those found in the wild type. A parallel increase in protein content per length and cell density and a loss of viability were found to occur after four generations at the restrictive temperature. The behavior of strain OV-25 in the absence of the wee gene product could be interpreted in terms of either a faulty regulation of the elongation processes or their abnormal coordination with the cell cycle. The genetic location of the wee gene has been found to be at 83.5 min on the E. coli genetic map.     

Leucine uptake and protein synthesis are exponential during the division cycle of Escherichia coli B/r.

The rate of leucine uptake, which is a measure of protein synthesis, was measured during the division cycle of Escherichia coli B/r by the membrane elution technique. The rate of leucine uptake was exponential, indicating that protein synthesis is exponential, and not linear, during the division cycle. These results, coupled with the results of other work on the exponential rate of RNA synthesis during the division cycle, indicate that the accumulation of mass in E. coli and other gram-negative organisms is exponential during the division cycle.     

Changes in regulatory phosphorylation of Cdc25C Ser287 and Wee1 Ser549 during normal cell cycle progression and checkpoint arrests

Entry into mitosis is catalyzed by cdc2 kinase. Previous work identified the cdc2-activating phosphatase cdc25C and the cdc2-inhibitory kinase wee1 as targets of the incomplete replication-induced kinase Chk1. Further work led to the model that checkpoint kinases block mitotic entry by inhibiting cdc25C through phosphorylation on Ser287 and activating wee1 through phosphorylation on Ser549. However, almost all conclusions underlying this idea were drawn from work using recombinant proteins. Here, we report that in the early Xenopus egg cell cycles, phosphorylation of endogenous cdc25C Ser287 is normally high during interphase and shows no obvious increase after checkpoint activation. By contrast, endogenous wee1 Ser549 phosphorylation is low during interphase and increases after activation of either the DNA damage or replication checkpoints; this is accompanied by a slight increase in wee1 kinase activity. Blocking mitotic entry by adding the catalytic subunit of PKA also results in increased wee1 Ser549 phosphorylation and maintenance of cdc25C Ser287 phosphorylation. These results argue that in response to checkpoint activation, endogenous wee1 is indeed a critical responder that functions by repressing the cdc2-cdc25C positive feedback loop. Surprisingly, endogenous wee1 Ser549 phosphorylation is highest during mitosis just after the peak of cdc2 activity. Treatments that block inactivation of cdc2 result in further increases in wee1 Ser549 phosphorylation, suggesting a previously unsuspected role for wee1 in mitosis.     

Negative regulation of mitosis by wee1+, a gene encoding a protein kinase homolog

Fission yeast wee1- mutants initiate mitosis at half the cell size of wild type. The wee1+ activity is required to prevent lethal premature mitosis in cells that overproduce the mitotic inducer cdc25+. This lethal phenotype was used to clone wee1+ by complementation. When wee1+ expression is increased, mitosis is delayed until cells grow to a larger size. Thus wee1+ functions as a dose-dependent inhibitor of mitosis, the first such element to be specifically identified and cloned. The carboxy-terminal region of the predicted 112 kd wee1+ protein contains protein kinase consensus sequences, suggesting that negative regulation of mitosis involves protein phosphorylation. Genetic evidence indicates that wee1+ and cdc25+ compete in a control system regulating the cdc2+ protein kinase, which is required for mitotic initiation.