Search tips
Search criteria

Results 1-25 (1049756)

Clipboard (0)

Related Articles

1.  Identification of Genes Periodically Expressed in the Human Cell Cycle and Their Expression in TumorsD⃞ 
Molecular Biology of the Cell  2002;13(6):1977-2000.
The genome-wide program of gene expression during the cell division cycle in a human cancer cell line (HeLa) was characterized using cDNA microarrays. Transcripts of >850 genes showed periodic variation during the cell cycle. Hierarchical clustering of the expression patterns revealed coexpressed groups of previously well-characterized genes involved in essential cell cycle processes such as DNA replication, chromosome segregation, and cell adhesion along with genes of uncharacterized function. Most of the genes whose expression had previously been reported to correlate with the proliferative state of tumors were found herein also to be periodically expressed during the HeLa cell cycle. However, some of the genes periodically expressed in the HeLa cell cycle do not have a consistent correlation with tumor proliferation. Cell cycle-regulated transcripts of genes involved in fundamental processes such as DNA replication and chromosome segregation seem to be more highly expressed in proliferative tumors simply because they contain more cycling cells. The data in this report provide a comprehensive catalog of cell cycle regulated genes that can serve as a starting point for functional discovery. The full dataset is available at
PMCID: PMC117619  PMID: 12058064
2.  Targeted interactomics reveals a complex core cell cycle machinery in Arabidopsis thaliana 
A protein interactome focused towards cell proliferation was mapped comprising 857 interactions among 393 proteins, leading to many new insights in plant cell cycle regulation.A comprehensive view on heterodimeric cyclin-dependent kinase (CDK)/cyclin complexes in plants is obtained, in relation with their regulators.Over 100 new candidate cell cycle proteins were predicted.
The basic underlying mechanisms that govern the cell cycle are conserved among all eukaryotes. Peculiar for plants, however, is that their genome contains a collection of cell cycle regulatory genes that is intriguingly large (Vandepoele et al, 2002; Menges et al, 2005) compared to other eukaryotes. Arabidopsis thaliana (Arabidopsis) encodes 71 genes in five regulatory classes versus only 15 in yeast and 23 in human.
Despite the discovery of numerous cell cycle genes, little is known about the protein complex machinery that steers plant cell division. Therefore, we applied tandem affinity purification (TAP) approach coupled with mass spectrometry (MS) on Arabidopsis cell suspension cultures to isolate and analyze protein complexes involved in the cell cycle. This approach allowed us to successfully map a first draft of the basic cell cycle complex machinery of Arabidopsis, providing many new insights into plant cell division.
To map the interactome, we relied on a streamlined platform comprising generic Gateway-based vectors with high cloning flexibility, the fast generation of transgenic suspension cultures, TAP adapted for plant cells, and matrix-assisted laser desorption ionization (MALDI) tandem-MS for the identification of purified proteins (Van Leene et al, 2007, 2008Van Leene et al, 2007, 2008). Complexes for 102 cell cycle proteins were analyzed using this approach, leading to a non-redundant data set of 857 interactions among 393 proteins (Figure 1A). Two subspaces were identified in this data set, domain I1, containing interactions confirmed in at least two independent experimental repeats or in the reciprocal purification experiment, and domain I2 consisting of uniquely observed interactions.
Several observations underlined the quality of both domains. All tested reverse purifications found the original interaction, and 150 known or predicted interactions were confirmed, meaning that also a huge stack of new interactions was revealed. An in-depth computational analysis revealed enrichment for many cell cycle-related features among the proteins of the network (Figure 1B), and many protein pairs were coregulated at the transcriptional level (Figure 1C). Through integration of known cell cycle-related features, more than 100 new candidate cell cycle proteins were predicted (Figure 1D). Besides common qualities of both interactome domains, their real significance appeared through mutual differences exposing two subspaces in the cell cycle interactome: a central regulatory network of stable complexes that are repeatedly isolated and represent core regulatory units, and a peripheral network comprising transient interactions identified less frequently, which are involved in other aspects of the process, such as crosstalk between core complexes or connections with other pathways. To evaluate the biological relevance of the cell cycle interactome in plants, we validated interactions from both domains by a transient split-luciferase assay in Arabidopsis plants (Marion et al, 2008), further sustaining the hypothesis-generating power of the data set to understand plant growth.
With respect to insights into the cell cycle physiology, the interactome was subdivided according to the functional classes of the baits and core protein complexes were extracted, covering cyclin-dependent kinase (CDK)/cyclin core complexes together with their positive and negative regulation networks, DNA replication complexes, the anaphase-promoting complex, and spindle checkpoint complexes. The data imply that mitotic A- and B-type cyclins exclusively form heterodimeric complexes with the plant-specific B-type CDKs and not with CDKA;1, whereas D-type cyclins seem to associate with CDKA;1. Besides the extraction of complexes previously shown in other organisms, our data also suggested many new functional links; for example, the link coupling cell division with the regulation of transcript splicing. The association of negative regulators of CDK/cyclin complexes with transcription factors suggests that their role in reallocation is not solely targeted to CDK/cyclin complexes. New members of the Siamese-related inhibitory proteins were identified, and for the first time potential inhibitors of plant-specific mitotic B-type CDKs have been found in plants. New evidence that the E2F–DP–RBR network is not only active at G1-to-S, but also at the G2-to-M transition is provided and many complexes involved in DNA replication or repair were isolated. For the first time, a plant APC has been isolated biochemically, identifying three potential new plant-specific APC interactors, and finally, complexes involved in the spindle checkpoint were isolated mapping many new but specific interactions.
Finally, to get a general view on the complex machinery, modules of interacting cyclins and core cell cycle regulators were ranked along the cell cycle phases according to the transcript expression peak of the cyclins, showing an assorted set of CDK–cyclin complexes with high regulatory differentiation (Figure 4). Even within the same subfamily (e.g. cyclin A3, B1, B2, D3, and D4), cyclins differ not only in their functional time frame but also in the type and number of CDKs, inhibitors, and scaffolding proteins they bind, further indicating their functional diversification. According to our interaction data, at least 92 different variants of CDK–cyclin complexes are found in Arabidopsis.
In conclusion, these results reflect how several rounds of gene duplication (Sterck et al, 2007) led to the evolution of a large set of cyclin paralogs and a myriad of regulators, resulting in a significant jump in the complexity of the cell cycle machinery that could accommodate unique plant-specific features such as an indeterminate mode of postembryonic development. Through their extensive regulation and connection with a myriad of up- and downstream pathways, the core cell cycle complexes might offer the plant a flexible toolkit to fine-tune cell proliferation in response to an ever-changing environment.
Cell proliferation is the main driving force for plant growth. Although genome sequence analysis revealed a high number of cell cycle genes in plants, little is known about the molecular complexes steering cell division. In a targeted proteomics approach, we mapped the core complex machinery at the heart of the Arabidopsis thaliana cell cycle control. Besides a central regulatory network of core complexes, we distinguished a peripheral network that links the core machinery to up- and downstream pathways. Over 100 new candidate cell cycle proteins were predicted and an in-depth biological interpretation demonstrated the hypothesis-generating power of the interaction data. The data set provided a comprehensive view on heterodimeric cyclin-dependent kinase (CDK)–cyclin complexes in plants. For the first time, inhibitory proteins of plant-specific B-type CDKs were discovered and the anaphase-promoting complex was characterized and extended. Important conclusions were that mitotic A- and B-type cyclins form complexes with the plant-specific B-type CDKs and not with CDKA;1, and that D-type cyclins and S-phase-specific A-type cyclins seem to be associated exclusively with CDKA;1. Furthermore, we could show that plants have evolved a combinatorial toolkit consisting of at least 92 different CDK–cyclin complex variants, which strongly underscores the functional diversification among the large family of cyclins and reflects the pivotal role of cell cycle regulation in the developmental plasticity of plants.
PMCID: PMC2950081  PMID: 20706207
Arabidopsis thaliana; cell cycle; interactome; protein complex; protein interactions
3.  A model of yeast cell-cycle regulation based on multisite phosphorylation 
Multisite phosphorylation of CDK target proteins provides the requisite nonlinearity for cell cycle modeling using elementary reaction mechanisms.Stochastic simulations, based on Gillespie's algorithm and using realistic numbers of protein and mRNA molecules, compare favorably with single-cell measurements in budding yeast.The role of transcription–translation coupling is critical in the robust operation of protein regulatory networks in yeast cells.
Progression through the eukaryotic cell cycle is governed by the activation and inactivation of a family of cyclin-dependent kinases (CDKs) and auxiliary proteins that regulate CDK activities (Morgan, 2007). The many components of this protein regulatory network are interconnected by positive and negative feedback loops that create bistable switches and transient pulses (Tyson and Novak, 2008). The network must ensure that cell-cycle events proceed in the correct order, that cell division is balanced with respect to cell growth, and that any problems encountered (in replicating the genome or partitioning chromosomes to daughter cells) are corrected before the cell proceeds to the next phase of the cycle. The network must operate robustly in the context of unavoidable molecular fluctuations in a yeast-sized cell. With a volume of only 5×10−14 l, a yeast cell contains one copy of the gene for each component of the network, a handful of mRNA transcripts of each gene, and a few hundreds to thousands of protein molecules carrying out each gene's function. How large are the molecular fluctuations implied by these numbers, and what effects do they have on the functioning of the cell-cycle control system?
To answer these questions, we have built a new model (Figure 1) of the CDK regulatory network in budding yeast, based on the fact that the targets of CDK activity are typically phosphorylated on multiple sites. The activity of each target protein depends on how many sites are phosphorylated. The target proteins feedback on CDK activity by controlling cyclin synthesis (SBF's role) and degradation (Cdh1's role) and by releasing a CDK-counteracting phosphatase (Cdc14). Every reaction in Figure 1 can be described by a mass-action rate law, with an accompanying rate constant that must be estimated from experimental data. As the transcription and translation of mRNA molecules have major effects on fluctuating numbers of protein molecules (Pedraza and Paulsson, 2008), we have included mRNA transcripts for each protein in the model.
To create a deterministic model, the rate laws are combined, according to standard principles of chemical kinetics, into a set of 60 differential equations that govern the temporal dynamics of the control system. In the stochastic version of the model, the rate law for each reaction determines the probability per unit time that a particular reaction occurs, and we use Gillespie's stochastic simulation algorithm (Gillespie, 1976) to compute possible temporal sequences of reaction events. Accurate stochastic simulations require knowledge of the expected numbers of mRNA and protein molecules in a single yeast cell. Fortunately, these numbers are available from several sources (Ghaemmaghami et al, 2003; Zenklusen et al, 2008). Although the experimental estimates are not always in good agreement with each other, they are sufficiently reliable to populate a stochastic model with realistic numbers of molecules.
By simulating thousands of cells (as in Figure 5), we can build up representative samples for computing the mean and s.d. of any measurable cell-cycle property (e.g. interdivision time, size at division, duration of G1 phase). The excellent fit of simulated statistics to observations of cell-cycle variability is documented in the main text and Supplementary Information.
Of particular interest to us are observations of Di Talia et al (2007) of the timing of a crucial G1 event (export of Whi5 protein from the nucleus) in a population of budding yeast cells growing at a specific growth rate α=ln2/(mass-doubling time). Whi5 export is a consequence of Whi5 phosphorylation, and it occurs simultaneously with the release (activation) of SBF (see Figure 1). Using fluorescently labeled Whi5, Di Talia et al could easily measure (in individual yeast cells) the time, T1, from cell birth to the abrupt loss of Whi5 from the nucleus. Correlating T1 to the size of the cell at birth, Vbirth, they found that, for a sample of daughter cells, αT1 versus ln(Vbirth) could be fit with two straight lines of slope −0.7 and −0.3. Our simulation of this experiment (Figure 7 of the main text) compares favorably with Figure 3d and e in Di Talia et al (2007).
The major sources of noise in our model (and in protein regulatory networks in yeast cells, in general) are related to gene transcription and the small number of unique mRNA transcripts. As each mRNA molecule may instruct the synthesis of dozens of protein molecules, the coefficient of variation of molecular fluctuations at the protein level (CVP) may be dominated by fluctuations at the mRNA level, as expressed in the formula (Pedraza and Paulsson, 2008) where NM, NP denote the number of mRNA and protein molecules, respectively, and ρ=τM/τP is the ratio of half-lives of mRNA and protein molecules. For a yeast cell, typical values of NM and NP are 8 and 800, respectively (Ghaemmaghami et al, 2003; Zenklusen et al, 2008). If ρ=1, then CVP≈25%. Such large fluctuations in protein levels are inconsistent with the observed variability of size and age at division in yeast cells, as shown in the simplified cell-cycle model of Kar et al (2009) and as we have confirmed with our more realistic model. The size of these fluctuations can be reduced to a more acceptable level by assuming a shorter half-life for mRNA (say, ρ=0.1).
There must be some mechanisms whereby yeast cells lessen the protein fluctuations implied by transcription–translation coupling. Following Pedraza and Paulsson (2008), we suggest that mRNA gestation and senescence may resolve this problem. Equation (3) is based on a simple, one-stage, birth–death model of mRNA turnover. In Supplementary Appendix 1, we show that a model of mRNA processing, with 10 stages each of mRNA gestation and senescence, gives reasonable fluctuations at the protein level (CVP≈5%), even if the effective half-life of mRNA is 10 min. A one-stage model with τM=1 min gives comparable fluctuations (CVP≈5%). In the main text, we use a simple birth–death model of mRNA turnover with an ‘effective' half-life of 1 min, in order to limit the computational complexity of the full cell-cycle model.
In order for the cell's genome to be passed intact from one generation to the next, the events of the cell cycle (DNA replication, mitosis, cell division) must be executed in the correct order, despite the considerable molecular noise inherent in any protein-based regulatory system residing in the small confines of a eukaryotic cell. To assess the effects of molecular fluctuations on cell-cycle progression in budding yeast cells, we have constructed a new model of the regulation of Cln- and Clb-dependent kinases, based on multisite phosphorylation of their target proteins and on positive and negative feedback loops involving the kinases themselves. To account for the significant role of noise in the transcription and translation steps of gene expression, the model includes mRNAs as well as proteins. The model equations are simulated deterministically and stochastically to reveal the bistable switching behavior on which proper cell-cycle progression depends and to show that this behavior is robust to the level of molecular noise expected in yeast-sized cells (∼50 fL volume). The model gives a quantitatively accurate account of the variability observed in the G1-S transition in budding yeast, which is governed by an underlying sizer+timer control system.
PMCID: PMC2947364  PMID: 20739927
bistability; cell-cycle variability; size control; stochastic model; transcription–translation coupling
4.  Negative regulation of G1 and G2 by S-phase cyclins of Saccharomyces cerevisiae. 
Molecular and Cellular Biology  1995;15(9):5030-5042.
Cell cycle progression in the budding yeast Saccharomyces cerevisiae is controlled by the Cdc28 protein kinase, which is sequentially activated by different sets of cyclins. Previous genetic analysis has revealed that two B-type cyclins, Clb5 and Clb6, have a positive role in DNA replication. In the present study, we show, in addition, that these cyclins negatively regulate G1- and G2-specific functions. The consequences of this negative regulation were most apparent in clb6 mutants, which had a shorter pre-Start G1 phase as well as a shorter G2 phase than congenic wild-type cells. As a consequence, clb6 mutants grew and proliferated more rapidly than wild-type cells. It was more difficult to assess the role of Clb5 in G1 and G2 by genetic analysis because of the extreme prolongation of S phase in clb5 mutants. Nevertheless, both Clb5 and Clb6 were shown to be responsible for down-regulation of the protein kinase activities associated with Cln2, a G1 cyclin, and Clb2, a mitotic cyclin, in vivo. These observations are consistent with the observed cell cycle phase accelerations associated with the clb6 mutant and are suggestive of similar functions for Clb5. Genetic evidence suggested that the inhibition of mitotic cyclin-dependent kinase activities was dependent on and possibly mediated through the CDC6 gene product. Thus, Clb5 and Clb6 may stabilize S phase by promoting DNA replication while inhibiting other cell cycle activities.
PMCID: PMC230750  PMID: 7651421
5.  The Mitotic Cyclins Clb2p and Clb4p Affect Morphogenesis in Candida albicans 
Molecular Biology of the Cell  2005;16(7):3387-3400.
The ability of Candida albicans to switch cellular morphologies is crucial for its ability to cause infection. Because the cell cycle machinery participates in Saccharomyces cerevisiae filamentous growth, we characterized in detail the two C. albicans B-type cyclins, CLB2 and CLB4, to better understand the molecular mechanisms that underlie the C. albicans morphogenic switch. Both Clb2p and Clb4p levels are cell cycle regulated, peaking at G2/M and declining before mitotic exit. On hyphal induction, the accumulation of the G1 cyclin Cln1p was prolonged, whereas the accumulation of both Clb proteins was delayed when compared with yeast form cells, indicating that CLB2 and CLB4 are differentially regulated in the two morphologies and that the dynamics of cyclin appearance differs between yeast and hyphal forms of growth. Clb2p-depleted cells were inviable and arrested with hyper-elongated projections containing two nuclei, suggesting that Clb2p is not required for entry into mitosis. Unlike Clb2p-depleted cells, Clb4p-depleted cells were viable and formed constitutive pseudohyphae. Clb proteins lacking destruction box domains blocked cell cycle progression resulting in the formation of long projections, indicating that both Clb2p and Clb4p must be degraded before mitotic exit. In addition, overexpression of either B-type cyclin reduced the extent of filamentous growth. Taken together, these data indicate that Clb2p and Clb4p regulate C. albicans morphogenesis by negatively regulating polarized growth.
PMCID: PMC1165420  PMID: 15888543
6.  Cell cycle-dependent transcription of CLN2 is conferred by multiple distinct cis-acting regulatory elements. 
Molecular and Cellular Biology  1994;14(7):4788-4801.
The budding yeast Saccharomyces cerevisiae CLN1, CLN2, and CLN3 genes encode functionally redundant G1 cyclins required for cell cycle initiation. CLN1 and CLN2 mRNAs accumulate periodically throughout the cell cycle, peaking in late G1. We show that cell cycle-dependent fluctuation in CLN2 mRNA is regulated at the level of transcriptional initiation. Mutational analysis of the CLN2 promoter revealed that the major cell cycle-dependent upstream activating sequence (UAS) resides within a 100-bp fragment. This UAS contains three putative SWI4-dependent cell cycle boxes (SCBs) and two putative MluI cell cycle boxes (MCBs). Mutational inactivation of these elements substantially decreased CLN2 promoter activity but failed to eliminate periodic transcription. Similarly, inactivation of SWI4 decreased CLN2 transcription without affecting its periodicity. We have identified a second UAS in the CLN2 upstream region that can promote cell cycle-dependent transcription with kinetics similar to that of the intact CLN2 promoter. Unlike the major CLN2 UAS, this newly identified UAS promotes transcription in cells arrested in G1 by inactivation of cdc28. This novel UAS is both necessary and sufficient for regulated transcription driven by a CLN2 promoter lacking functional SCBs and MCBs. Although this UAS itself contains no SCBs or MCBs, its activity is dependent upon SWI4 function. The characteristics of this novel UAS suggest that it might have a role in initiating CLN2 expression early in G1 to activate the positive feedback loop that drives maximal Cln accumulation.
PMCID: PMC358852  PMID: 8007978
7.  Daughter-Specific Transcription Factors Regulate Cell Size Control in Budding Yeast 
PLoS Biology  2009;7(10):e1000221.
The asymmetric localization of cell fate determinants results in asymmetric cell cycle control in budding yeast.
In budding yeast, asymmetric cell division yields a larger mother and a smaller daughter cell, which transcribe different genes due to the daughter-specific transcription factors Ace2 and Ash1. Cell size control at the Start checkpoint has long been considered to be a main regulator of the length of the G1 phase of the cell cycle, resulting in longer G1 in the smaller daughter cells. Our recent data confirmed this concept using quantitative time-lapse microscopy. However, it has been proposed that daughter-specific, Ace2-dependent repression of expression of the G1 cyclin CLN3 had a dominant role in delaying daughters in G1. We wanted to reconcile these two divergent perspectives on the origin of long daughter G1 times. We quantified size control using single-cell time-lapse imaging of fluorescently labeled budding yeast, in the presence or absence of the daughter-specific transcriptional regulators Ace2 and Ash1. Ace2 and Ash1 are not required for efficient size control, but they shift the domain of efficient size control to larger cell size, thus increasing cell size requirement for Start in daughters. Microarray and chromatin immunoprecipitation experiments show that Ace2 and Ash1 are direct transcriptional regulators of the G1 cyclin gene CLN3. Quantification of cell size control in cells expressing titrated levels of Cln3 from ectopic promoters, and from cells with mutated Ace2 and Ash1 sites in the CLN3 promoter, showed that regulation of CLN3 expression by Ace2 and Ash1 can account for the differential regulation of Start in response to cell size in mothers and daughters. We show how daughter-specific transcriptional programs can interact with intrinsic cell size control to differentially regulate Start in mother and daughter cells. This work demonstrates mechanistically how asymmetric localization of cell fate determinants results in cell-type-specific regulation of the cell cycle.
Author Summary
Asymmetric cell division is a universal mechanism for generating differentiated cells. The progeny of such divisions can often display differential cell cycle regulation. This study addresses how differential regulation of gene expression in the progeny of a single division can alter cell cycle control. In budding yeast, asymmetric cell division yields a bigger ‘mother’ cell and a smaller ‘daughter’ cell. Regulation of gene expression is also asymmetric because two transcription factors, Ace2 and Ash1, are specifically localized to the daughter. Cell size has long been proposed as important for the regulation of the cell cycle in yeast. Our work shows that Ace2 and Ash1 regulate size control in daughter cells: daughters ‘interpret’ their size as smaller, making size control more stringent and delaying cell cycle commitment relative to mother cells of the same size. This asymmetric interpretation of cell size is associated with differential regulation of the G1 cyclin CLN3 by Ace2 and Ash1, at least in part via direct binding of these factors to the CLN3 promoter. CLN3 is the most upstream regulator of Start, the initiation point of the yeast cell cycle, and differential regulation of CLN3 accounts for most or all asymmetric regulation of Start in budding yeast mother and daughter cells.
PMCID: PMC2756959  PMID: 19841732
8.  TPR proteins required for anaphase progression mediate ubiquitination of mitotic B-type cyclins in yeast. 
Molecular Biology of the Cell  1996;7(5):791-801.
The abundance of B-type cyclin-CDK complexes is determined by regulated synthesis and degradation of cyclin subunits. Cyclin proteolysis is required for the final exit from mitosis and for the initiation of a new cell cycle. In extracts from frog or clam eggs, degradation is accompanied by ubiquitination of cyclin. Three genes, CDC16, CDC23, and CSE1 have recently been shown to be required specifically for cyclin B proteolysis in yeast. To test whether these genes are required for cyclin ubiquitination, we prepared extracts from G1-arrested yeast cells capable of conjugating ubiquitin to the B-type cyclin Clb2. The ubiquitination activity was cell cycle regulated, required Clb2's destruction box, and was low if not absent in cdc16, cdc23, cdc27, and cse1 mutants. Furthermore all these mutants were also defective in ubiquitination of another mitotic B-type cyclin, Clb3. The Cdc16, Cdc23, and Cdc27 proteins all contain several copies of the tetratricopeptide repeat and are subunits of a complex that is required for the onset of anaphase. The finding that gene products that are required for ubiquitination of Clb2 and Clb3 are also required for cyclin proteolysis in vivo provides the best evidence so far that cyclin B is degraded via the ubiquitin pathway in living cells. Xenopus homologues of Cdc16 and Cdc27 have meanwhile been shown to be associated with a 20S particle that appears to function as a cell cycle-regulated ubiquitin-protein ligase.
PMCID: PMC275930  PMID: 8744951
9.  Dynamics of Cdk1 Substrate Specificity during the Cell Cycle 
Molecular Cell  2011;42(5-4):610-623.
Cdk specificity is determined by the intrinsic selectivity of the active site and by substrate docking sites on the cyclin subunit. There is a long-standing debate about the relative importance of these factors in the timing of Cdk1 substrate phosphorylation. We analyzed major budding yeast cyclins (the G1/S-cyclin Cln2, S-cyclin Clb5, G2/M-cyclin Clb3, and M-cyclin Clb2) and found that the activity of Cdk1 toward the consensus motif increased gradually in the sequence Cln2-Clb5-Clb3-Clb2, in parallel with cell cycle progression. Further, we identified a docking element that compensates for the weak intrinsic specificity of Cln2 toward G1-specific targets. In addition, Cln2-Cdk1 showed distinct consensus site specificity, suggesting that cyclins do not merely activate Cdk1 but also modulate its active-site specificity. Finally, we identified several Cln2-, Clb3-, and Clb2-specific Cdk1 targets. We propose that robust timing and ordering of cell cycle events depend on gradual changes in the substrate specificity of Cdk1.
► Cyclins gradually raise the active-site specificity of Cdk1 during the cell cycle ► A novel substrate docking motif is specific for G1-cyclin-Cdk1 complexes ► G1 cyclin-Cdk1 shows distinct features of substrate site consensus specificity ► Several G1, G2, and M phase-specific Cdk1 targets exist in the cell
PMCID: PMC3115021  PMID: 21658602
10.  Inactivation of the cyclin-dependent kinase Cdc28 abrogates cell cycle arrest induced by DNA damage and disassembly of mitotic spindles in Saccharomyces cerevisiae. 
Molecular and Cellular Biology  1997;17(5):2723-2734.
Eukaryotic cells may halt cell cycle progression following exposure to certain exogenous agents that damage cellular structures such as DNA or microtubules. This phenomenon has been attributed to functions of cellular control mechanisms termed checkpoints. Studies with the fission yeast Schizosaccharomyces pombe and mammalian cells have led to the conclusion that cell cycle arrest in response to inhibition of DNA replication or DNA damage is a result of down-regulation of the cyclin-dependent kinases (CDKs). Based on these studies, it has been proposed that inhibition of the CDK activity may constitute a general mechanism for checkpoint controls. Observations made with the budding yeast Saccharomyces cerevisiae, however, appear to disagree with this model. It has been shown that high levels of mitotic CDK activity are present in the budding yeast cells arrested in G2/mitosis as the result of DNA damage or replication inhibition. In this report, we show that a novel mutant allele of the CDC28 gene, encoding the budding yeast CDK, allowed cell cycle passage through mitosis and nuclear division in the presence of DNA damage and the microtubule toxin nocodazole at a restrictive temperature. Unlike the checkpoint-defective mutations in CDKs of fission yeast and mammalian cells, the cdc28 mutation that we identified was recessive and resulted in a loss of the CDK activity, including the Clb2-, Clb5-, and Clb6-associated, but not the Clb3-associated, CDK activities. Examination of several known alleles of cdc28 revealed that they were also, albeit partially, defective in cell cycle arrest in response to UV-generated DNA damage. These findings suggest that Cdc28 kinase in budding yeast may be required for cell cycle arrest resulting from DNA damage and disassembly of mitotic spindles.
PMCID: PMC232123  PMID: 9111343
11.  Cln3-Associated Kinase Activity in Saccharomyces cerevisiae Is Regulated by the Mating Factor Pathway 
Molecular and Cellular Biology  1998;18(1):433-441.
The Saccharomyces cerevisiae cell cycle is arrested in G1 phase by the mating factor pathway. Genetic evidence has suggested that the G1 cyclins Cln1, Cln2, and Cln3 are targets of this pathway whose inhibition results in G1 arrest. Inhibition of Cln1- and Cln2-associated kinase activity by the mating factor pathway acting through Far1 has been described. Here we report that Cln3-associated kinase activity is inhibited by mating factor treatment, with dose response and timing consistent with involvement in cell cycle arrest. No regulation of Cln3-associated kinase was observed in a fus3 kss1 strain deficient in mating factor pathway mitogen-activated protein (MAP) kinases. Inhibition occurs mainly at the level of specific activity of Cln3-Cdc28 complexes. Inhibition of the C-terminally truncated Cln3-1-associated kinase is not observed; such truncations were previously identified genetically as causing resistance to mating factor-induced cell cycle arrest. Regulation of Cln3-associated kinase specific activity by mating factor treatment requires Far1. Overexpression of Far1 restores inhibition of C-terminally truncated Cln3-1-associated kinase activity. G2/M-arrested cells are unable to regulate Cln3-associated kinase, possibly because of cell cycle regulation of Far1 abundance. Inhibition of Cln3-associated kinase activity by the mating factor pathway may allow this pathway to block the earliest step in normal cell cycle initiation, since Cln3 functions as the most upstream G1-acting cyclin, activating transcription of the G1 cyclins CLN1 and CLN2 as well as of the S-phase cyclins CLB5 and CLB6.
PMCID: PMC121512  PMID: 9418890
12.  Caenorhabditis elegans Cyclin D/CDK4 and Cyclin E/CDK2 Induce Distinct Cell Cycle Re-Entry Programs in Differentiated Muscle Cells 
PLoS Genetics  2011;7(11):e1002362.
Cell proliferation and differentiation are regulated in a highly coordinated and inverse manner during development and tissue homeostasis. Terminal differentiation usually coincides with cell cycle exit and is thought to engage stable transcriptional repression of cell cycle genes. Here, we examine the robustness of the post-mitotic state, using Caenorhabditis elegans muscle cells as a model. We found that expression of a G1 Cyclin and CDK initiates cell cycle re-entry in muscle cells without interfering with the differentiated state. Cyclin D/CDK4 (CYD-1/CDK-4) expression was sufficient to induce DNA synthesis in muscle cells, in contrast to Cyclin E/CDK2 (CYE-1/CDK-2), which triggered mitotic events. Tissue-specific gene-expression profiling and single molecule FISH experiments revealed that Cyclin D and E kinases activate an extensive and overlapping set of cell cycle genes in muscle, yet failed to induce some key activators of G1/S progression. Surprisingly, CYD-1/CDK-4 also induced an additional set of genes primarily associated with growth and metabolism, which were not activated by CYE-1/CDK-2. Moreover, CYD-1/CDK-4 expression also down-regulated a large number of genes enriched for catabolic functions. These results highlight distinct functions for the two G1 Cyclin/CDK complexes and reveal a previously unknown activity of Cyclin D/CDK-4 in regulating metabolic gene expression. Furthermore, our data demonstrate that many cell cycle genes can still be transcriptionally induced in post-mitotic muscle cells, while maintenance of the post-mitotic state might depend on stable repression of a limited number of critical cell cycle regulators.
Author Summary
During development, cells face the important decision whether to continue to proliferate, or to exit the cell-division cycle and fully differentiate. Improved insight into the molecular mechanisms that arrest the cell cycle during terminal differentiation is important for our understanding of normal development, as well as for cancer research and regenerative medicine. To investigate the arrested state of terminally differentiated cells, we examined muscle cells in the model organism C. elegans, which is known for its reproducible cell-division pattern. We found that expression of a single cell cycle kinase with its regulatory partner (Cyclin) induced many cell division genes in muscle. While Cyclin D and E kinases often act similarly, only Cyclin D with CDK-4 triggered DNA replication in muscle, and this combination induced a much broader transcriptional response than Cyclin E/CDK-2. Despite activation of a substantial cell cycle program, Cyclin/CDK expression did not induce complete muscle cell division and failed to induce some key cell cycle regulators. Our results highlight distinct activities of Cyclin D and Cyclin E kinases, and they indicate that cell-cycle gene expression remains remarkably flexible in differentiated cells. We propose that the post-mitotic state of differentiated cells is maintained by tight control of a few regulatory genes.
PMCID: PMC3213155  PMID: 22102824
13.  Candida albicans Cyclin Clb4 Carries S-Phase Cyclin Activity▿† 
Eukaryotic Cell  2010;9(9):1311-1319.
Cyclin-dependent kinases (CDKs) are key regulators of eukaryotic cell cycle progression. The cyclin subunit activates the CDK and also imparts to the complex, at least in some cases, substrate specificity. Saccharomyces cerevisiae, an organism in which the roles of individual cyclins are best studied, contains nine cyclins (three G1 cyclins and six B-type cyclins) capable of activating the main cell cycle CDK, Cdc28. Analysis of the genome of the pathogenic yeast Candida albicans revealed only two sequences corresponding to B-type cyclins, C. albicans Clb2 (CaClb2) and CaClb4. Notably, no homolog of the S. cerevisiae S-phase-specific cyclins, Clb5/Clb6, could be detected. Here, we performed an in vitro analysis of the activity of CaClb2 and CaClb4 and of three G1 cyclins, as well as an analysis of the phenotype of S. cerevisiae cells expressing CaClb2 or CaClb4 instead of Clb5. Remarkably, replacement of CLB5 by CaCLB4 caused rapid diploidization of S. cerevisiae. In addition, both in vivo and in vitro analyses indicate that, in spite of the higher sequence similarity of CaClb2 to Clb5/Clb6, CaClb4 is the functional homolog of Clb5/Clb6. The activity of a CaClb2/CaClb4 cyclin hybrid suggests that the cyclin box domain of CaClb4 carries the functional specificity of the protein. These results have implications for our understanding of the evolution of specificity of the cell cycle cyclins.
PMCID: PMC2937343  PMID: 20639412
14.  Morphogenesis in the yeast cell cycle: regulation by Cdc28 and cyclins 
The Journal of Cell Biology  1993;120(6):1305-1320.
Analysis of cell cycle regulation in the budding yeast Saccharomyces cerevisiae has shown that a central regulatory protein kinase, Cdc28, undergoes changes in activity through the cell cycle by associating with distinct groups of cyclins that accumulate at different times. The various cyclin/Cdc28 complexes control different aspects of cell cycle progression, including the commitment step known as START and mitosis. We found that altering the activity of Cdc28 had profound effects on morphogenesis during the yeast cell cycle. Our results suggest that activation of Cdc28 by G1 cyclins (Cln1, Cln2, or Cln3) in unbudded G1 cells triggers polarization of the cortical actin cytoskeleton to a specialized pre-bud site at one end of the cell, while activation of Cdc28 by mitotic cyclins (Clb1 or Clb2) in budded G2 cells causes depolarization of the cortical actin cytoskeleton and secretory apparatus. Inactivation of Cdc28 following cyclin destruction in mitosis triggers redistribution of cortical actin structures to the neck region for cytokinesis. In the case of pre-bud site assembly following START, we found that the actin rearrangement could be triggered by Cln/Cdc28 activation in the absence of de novo protein synthesis, suggesting that the kinase may directly phosphorylate substrates (such as actin-binding proteins) that regulate actin distribution in cells.
PMCID: PMC2119756  PMID: 8449978
15.  Mutations in RAD27 define a potential link between G1 cyclins and DNA replication. 
Molecular and Cellular Biology  1995;15(8):4291-4302.
The yeast Saccharomyces cerevisiae has three G1 cyclin (CLN) genes with overlapping functions. To analyze the functions of the various CLN genes, we examined mutations that result in lethality in conjunction with loss of cln1 and cln2. We have isolated alleles of RAD27/ERC11/YKL510, the yeast homolog of the gene encoding flap endonuclease 1, FEN-1.cln1 cln2 rad27/erc11 cells arrest in S phase; this cell cycle arrest is suppressed by the expression of CLN1 or CLN2 but not by that of CLN3 or the hyperactive CLN3-2. rad27/erc11 mutants are also defective in DNA damage repair, as determined by their increased sensitivity to a DNA-damaging agent, increased mitotic recombination rates, and increased spontaneous mutation rates. Unlike the block in cell cycle progression, these phenotypes are not suppressed by CLN1 or CLN2. CLN1 and CLN2 may activate an RAD27/ERC11-independent pathway specific for DNA synthesis that CLN3 is incapable of activating. Alternatively, CLN1 and CLN2 may be capable of overriding a checkpoint response which otherwise causes cln1 cln2 rad27/erc11 cells to arrest. These results imply that CLN1 and CLN2 have a role in the regulation of DNA replication. Consistent with this, GAL-CLN1 expression in checkpoint-deficient, mec1-1 mutant cells results in both cell death and increased chromosome loss among survivors, suggesting that CLN1 overexpression either activates defective DNA replication or leads to insensitivity to DNA damage.
PMCID: PMC230668  PMID: 7623823
16.  Bck2 Acts through the MADS Box Protein Mcm1 to Activate Cell-Cycle-Regulated Genes in Budding Yeast 
PLoS Genetics  2013;9(5):e1003507.
The Bck2 protein is a potent genetic regulator of cell-cycle-dependent gene expression in budding yeast. To date, most experiments have focused on assessing a potential role for Bck2 in activation of the G1/S-specific transcription factors SBF (Swi4, Swi6) and MBF (Mbp1, Swi6), yet the mechanism of gene activation by Bck2 has remained obscure. We performed a yeast two-hybrid screen using a truncated version of Bck2 and discovered six novel Bck2-binding partners including Mcm1, an essential protein that binds to and activates M/G1 promoters through Early Cell cycle Box (ECB) elements as well as to G2/M promoters. At M/G1 promoters Mcm1 is inhibited by association with two repressors, Yox1 or Yhp1, and gene activation ensues once repression is relieved by an unknown activating signal. Here, we show that Bck2 interacts physically with Mcm1 to activate genes during G1 phase. We used chromatin immunoprecipitation (ChIP) experiments to show that Bck2 localizes to the promoters of M/G1-specific genes, in a manner dependent on functional ECB elements, as well as to the promoters of G1/S and G2/M genes. The Bck2-Mcm1 interaction requires valine 69 on Mcm1, a residue known to be required for interaction with Yox1. Overexpression of BCK2 decreases Yox1 localization to the early G1-specific CLN3 promoter and rescues the lethality caused by overexpression of YOX1. Our data suggest that Yox1 and Bck2 may compete for access to the Mcm1-ECB scaffold to ensure appropriate activation of the initial suite of genes required for cell cycle commitment.
Author Summary
Cell-cycle-dependent gene expression is a universal feature of cell cycles, with clear transcriptional programs in yeast, bacteria, and metazoans. At the M/G1 transition, many of the up-regulated genes encode key regulators of DNA replication (CDC6) and cyclins that initiate the events of cell cycle commitment (PCL9, CLN3). The promoters of genes activated at M/G1 contain a cis-regulatory sequence called the early cell cycle box (ECB), which is bound by the MADS-box transcription factor Mcm1, as well as the repressor Yox1 or Yhp1. The ECB cluster of genes defines a crucial cell cycle window during which a cell may change its fate; yet how the regulators that appear to act at ECBs are linked to cell cycle position is unclear, and coregulators, which experience tells us must exist, were unknown. Here, we describe our discovery that Bck2, a potent cell-cycle-regulator whose function has remained obscure, functions as a cofactor for Mcm1, to induce ECB–dependent gene expression. We also show that Bck2 has a role in promoting expression of late G1 and M/G2 genes. Our genetic and biochemical experiments reveal a new pathway for regulating gene expression associated with early cell cycle commitment, a process that is highly conserved.
PMCID: PMC3649975  PMID: 23675312
17.  FAR1 is required for posttranscriptional regulation of CLN2 gene expression in response to mating pheromone. 
Molecular and Cellular Biology  1993;13(2):1013-1022.
Yeast cells arrest during the G1 interval of the cell cycle in response to peptide mating pheromones. The FAR1 gene is required for cell cycle arrest but not for a number of other aspects of the pheromone response. Genetic evidence suggests that FAR1 is required specifically for inactivation of the G1 cyclin CLN2. From these observations, the FAR1 gene has been proposed to encode an element of the interface between the mating pheromone signal transduction pathway and the cell cycle regulatory apparatus. We show here that FAR1 is necessary for the decrease in CLN1 and CLN2 transcript accumulation observed in response to mating pheromone but is unnecessary for regulation of the same transcripts during vegetative growth. However, the defect in regulation of CLN1 expression is dependent upon CLN2. We show that pheromone regulates the abundance of Cln2 at a posttranscriptional level and that FAR1 is required for that regulation. From these observations, we suggest that FAR1 function is limited to posttranscriptional regulation of CLN2 expression by mating pheromone. The failure of mating pheromone to repress CLN2 transcript levels in far1 mutants can be explained by the stimulatory effect of the persistent Cln2 protein on CLN2 transcription via the CLN/CDC28-dependent feedback pathway.
PMCID: PMC358986  PMID: 8423774
18.  The transcription factor Swi5 regulates expression of the cyclin kinase inhibitor p40SIC1. 
Molecular and Cellular Biology  1996;16(10):5701-5707.
DNA replication in budding yeast cells depends on the activation of the Cdc28 kinase (Cdk1 of Saccharomyces cerevisiae) associated with B-type cyclins Clb1 to Clb6. Activation of the kinase depends on proteolysis of the Cdk inhibitor p40SIC1 in late G1, which is mediated by the ubiquitin-conjugating enzyme Cdc34 and two other proteins, Cdc4 and Cdc53. Inactivation of any one of these three proteins prevents p40SIC1 degradation and causes cells to arrest in G1 with active Cln kinases but no Clb-associated Cdc28 kinase activity. Deletion of SIC1 allows these mutants to replicate. p40SIC1 disappears at the G1/S transition and reappears only after nuclear division. Cell cycle-regulated proteolysis seems largely responsible for this pattern, but transcriptional control could also contribute; SIC1 RNA accumulates to high levels as cells exit M phase. To identify additional factors necessary for the inhibition of the Cdk1/Cdc28 kinase in G1, we isolated mutants that can replicate DNA in the absence of Cdc4 function. Mutations in three loci (SIC1, SWI5, and RIC3) were identified. We have shown that high SIC1 transcript levels at late M phase depend on Swi5. Swi5 accumulates in the cytoplasm during S, G2, and M phases of the cell cycle but enters the nuclei at late anaphase. Our data suggest that cell cycle-regulated nuclear accumulation of Swi5 is responsible for the burst of SIC1 transcription at the end of anaphase. This transcriptional control may be important for inactivation of the Clb/Cdk1 kinase in G2/M transition and during the subsequent G1 period.
PMCID: PMC231570  PMID: 8816483
19.  PUL21a-Cyclin A2 Interaction is Required to Protect Human Cytomegalovirus-Infected Cells from the Deleterious Consequences of Mitotic Entry 
PLoS Pathogens  2014;10(11):e1004514.
Entry into mitosis is accompanied by dramatic changes in cellular architecture, metabolism and gene expression. Many viruses have evolved cell cycle arrest strategies to prevent mitotic entry, presumably to ensure sustained, uninterrupted viral replication. Here we show for human cytomegalovirus (HCMV) what happens if the viral cell cycle arrest mechanism is disabled and cells engaged in viral replication enter into unscheduled mitosis. We made use of an HCMV mutant that, due to a defective Cyclin A2 binding motif in its UL21a gene product (pUL21a), has lost its ability to down-regulate Cyclin A2 and, therefore, to arrest cells at the G1/S transition. Cyclin A2 up-regulation in infected cells not only triggered the onset of cellular DNA synthesis, but also promoted the accumulation and nuclear translocation of Cyclin B1-CDK1, premature chromatin condensation and mitotic entry. The infected cells were able to enter metaphase as shown by nuclear lamina disassembly and, often irregular, metaphase spindle formation. However, anaphase onset was blocked by the still intact anaphase promoting complex/cyclosome (APC/C) inhibitory function of pUL21a. Remarkably, the essential viral IE2, but not the related chromosome-associated IE1 protein, disappeared upon mitotic entry, suggesting an inherent instability of IE2 under mitotic conditions. Viral DNA synthesis was impaired in mitosis, as demonstrated by the abnormal morphology and strongly reduced BrdU incorporation rates of viral replication compartments. The prolonged metaphase arrest in infected cells coincided with precocious sister chromatid separation and progressive fragmentation of the chromosomal material. We conclude that the Cyclin A2-binding function of pUL21a contributes to the maintenance of a cell cycle state conducive for the completion of the HCMV replication cycle. Unscheduled mitotic entry during the course of the HCMV replication has fatal consequences, leading to abortive infection and cell death.
Author Summary
Cyclin A2 is a key regulator of the cell division cycle. Interactors of Cyclin A2 typically contain short sequence elements (RXL/Cy motifs) that bind with high affinity to a hydrophobic patch in the Cyclin A2 protein. Two types of RXL/Cy-containing factors are known: i) cyclin-dependent kinase (CDK) substrates, which are processed by the CDK subunit that complexes to Cyclin A2, and ii) CDK inhibitors, which stably associate to Cyclin A2-CDK due to the lack of CDK phosphorylation sites. Human cytomegalovirus (HCMV) has evolved a novel type of RXL/Cy-containing protein. Its UL21a gene product, a small and highly unstable protein, binds to Cyclin A2 via an RXL/Cy motif in its N-terminus, leading to efficient degradation of Cyclin A2 by the proteasome. Here, we show that this mechanism is not only essential for viral inhibition of cellular DNA synthesis, but also to prevent entry of infected cells into mitosis. Unscheduled mitotic entry is followed by aberrant spindle formation, metaphase arrest, precocious separation of sister chromatids, chromosomal fragmentation and cell death. Viral DNA replication and expression of the essential viral IE2 protein are abrogated in mitosis. Thus, pUL21a-Cyclin A2 interaction protects HCMV from a collapse of viral and cellular functions in mitosis.
PMCID: PMC4231158  PMID: 25393019
20.  Xbp1 Directs Global Repression of Budding Yeast Transcription during the Transition to Quiescence and Is Important for the Longevity and Reversibility of the Quiescent State 
PLoS Genetics  2013;9(10):e1003854.
Pure populations of quiescent yeast can be obtained from stationary phase cultures that have ceased proliferation after exhausting glucose and other carbon sources from their environment. They are uniformly arrested in the G1 phase of the cell cycle, and display very high thermo-tolerance and longevity. We find that G1 arrest is initiated before all the glucose has been scavenged from the media. Maintaining G1 arrest requires transcriptional repression of the G1 cyclin, CLN3, by Xbp1. Xbp1 is induced as glucose is depleted and it is among the most abundant transcripts in quiescent cells. Xbp1 binds and represses CLN3 transcription and in the absence of Xbp1, or with extra copies of CLN3, cells undergo ectopic divisions and produce very small cells. The Rad53-mediated replication stress checkpoint reinforces the arrest and becomes essential when Cln3 is overproduced. The XBP1 transcript also undergoes metabolic oscillations under glucose limitation and we identified many additional transcripts that oscillate out of phase with XBP1 and have Xbp1 binding sites in their promoters. Further global analysis revealed that Xbp1 represses 15% of all yeast genes as they enter the quiescent state and over 500 of these transcripts contain Xbp1 binding sites in their promoters. Xbp1-repressed transcripts are highly enriched for genes involved in the regulation of cell growth, cell division and metabolism. Failure to repress some or all of these targets leads xbp1 cells to enter a permanent arrest or senescence with a shortened lifespan.
Author Summary
Complex organisms depend on populations of non-dividing quiescent cells for their controlled growth, development and tissue renewal. These quiescent cells are maintained in a resting state, and divide only when stimulated to do so. Unscheduled exit or failure to enter this quiescent state results in uncontrolled proliferation and cancer. Yeast cells also enter a stable, protected and reversible quiescent state. As with higher cells, they exit the cell cycle from G1, reduce growth, conserve and recycle cellular contents. These similarities, and the fact that the mechanisms that start and stop the cell cycle are fundamentally conserved lead us to think that understanding how yeast enter, maintain and reverse quiescence could give important leads into the same processes in complex organisms. We show that yeast cells maintain G1 arrest by expressing a transcription factor that represses conserved activators (cyclins) and hundreds of other genes that are important for cell division and cell growth. Failure to repress some or all of these targets leads to extra cell divisions, prevents reversible arrest and shortens life span. Many Xbp1 targets are conserved cell cycle regulators and may also be actively repressed in the quiescent cells of more complex organisms.
PMCID: PMC3814307  PMID: 24204289
21.  Mechanisms Controlling Subcellular Localization of the G1 Cyclins Cln2p and Cln3p in Budding Yeast 
Molecular and Cellular Biology  2001;21(18):6292-6311.
Different G1 cyclins confer functional specificity to the cyclin-dependent kinase (Cdk) Cdc28p in budding yeast. The Cln3p G1 cyclin is localized primarily to the nucleus, while Cln2p is localized primarily to the cytoplasm. Both binding to Cdc28p and Cdc28p-dependent phosphorylation in the C-terminal region of Cln2p are independently required for efficient nuclear depletion of Cln2p, suggesting that this process may be physiologically regulated. The accumulation of hypophosphorylated Cln2 in the nucleus is an energy-dependent process, but may not involve the RAN GTPase. Phosphorylation of Cln2p is inefficient in small newborn cells obtained by elutriation, and this lowered phosphorylation correlates with reduced Cln2p nuclear depletion in newborn cells. Thus, Cln2p may have a brief period of nuclear residence early in the cell cycle. In contrast, the nuclear localization pattern of Cln3p is not influenced by Cdk activity. Cln3p localization requires a bipartite nuclear localization signal (NLS) located at the C terminus of the protein. This sequence is required for nuclear localization of Cln3p and is sufficient to confer nuclear localization to green fluorescent protein in a RAN-dependent manner. Mislocalized Cln3p, lacking the NLS, is much less active in genetic assays specific for Cln3p, but more active in assays normally specific for Cln2p, consistent with the idea that Cln3p localization explains a significant part of Clnp functional specificity.
PMCID: PMC87357  PMID: 11509671
22.  A family of cyclin-like proteins that interact with the Pho85 cyclin-dependent kinase. 
Molecular and Cellular Biology  1997;17(3):1212-1223.
In budding yeast, entry into the mitotic cell cycle, or Start, requires the Cdc28 cyclin-dependent kinase (Cdk) and one of its three associated G1 cyclins, Cln1, Cln2, or Cln3. In addition, two other G1 cyclins, Pcl1 and Pcl2, associate with a second Cdk, Pho85, to contribute to Start. Although Pho85 is not essential for viability, Pcl1,2-Pho85 kinase complexes become essential for Start in the absence of Cln1,2-Cdc28 kinases. In addition, Pho85 interacts with a third cyclin, Pho80, to regulate acid phosphatase gene expression. Other cellular roles for Pho85 cyclin-Cdk complexes are suggested by the multiple phenotypes associated with deletion of PHO85, in addition to Start defects and deregulated acid phosphatase gene expression. Strains with pho80, pcl1, and pcl2 deletions show only a subset of the pho85 mutant phenotypes, suggesting the existence of additional Pho85 cyclins (Pcls). We used two-hybrid screening and database searching to identify seven additional cyclin-related genes that may interact with Pho85. We found that all of the new genes encode proteins that interacted with Pho85 in an affinity chromatography assay. One of these genes, CLG1, was previously suggested to encode a cyclin, based on the protein's sequence homology to Pcl1 and Pcl2. We have named the other genes PCL5, PCL6, PCL7, PCL8, PCL9, and PCL10. On the basis of sequence similarities, the PCLs can be divided into two subfamilies: the Pcl1,2-like subfamily and the Pho80-like subfamily. We found that deletion of members of the Pcl1,2 class of genes resulted in pronounced morphological abnormalities. In addition, we found that expression of one member of the Pcl1,2 subfamily, PCL9, is cell cycle regulated and is decreased in cells arrested in G1 by pheromone treatment. Our studies suggest that Pho85 associates with multiple cyclins and that subsets of cyclins may direct Pho85 to perform distinct roles in cell growth and division.
PMCID: PMC231846  PMID: 9032248
23.  Cell Size at S Phase Initiation: An Emergent Property of the G1/S Network 
PLoS Computational Biology  2007;3(4):e64.
The eukaryotic cell cycle is the repeated sequence of events that enable the division of a cell into two daughter cells. It is divided into four phases: G1, S, G2, and M. Passage through the cell cycle is strictly regulated by a molecular interaction network, which involves the periodic synthesis and destruction of cyclins that bind and activate cyclin-dependent kinases that are present in nonlimiting amounts. Cyclin-dependent kinase inhibitors contribute to cell cycle control. Budding yeast is an established model organism for cell cycle studies, and several mathematical models have been proposed for its cell cycle. An area of major relevance in cell cycle control is the G1 to S transition. In any given growth condition, it is characterized by the requirement of a specific, critical cell size, PS, to enter S phase. The molecular basis of this control is still under discussion. The authors report a mathematical model of the G1 to S network that newly takes into account nucleo/cytoplasmic localization, the role of the cyclin-dependent kinase Sic1 in facilitating nuclear import of its cognate Cdk1-Clb5, Whi5 control, and carbon source regulation of Sic1 and Sic1-containing complexes. The model was implemented by a set of ordinary differential equations that describe the temporal change of the concentration of the involved proteins and protein complexes. The model was tested by simulation in several genetic and nutritional setups and was found to be neatly consistent with experimental data. To estimate PS, the authors developed a hybrid model including a probabilistic component for firing of DNA replication origins. Sensitivity analysis of PS provides a novel relevant conclusion: PS is an emergent property of the G1 to S network that strongly depends on growth rate.
Author Summary
A major property of living cells is their ability to maintain mass homeostasis throughout cell divisions. It has been proposed that in order to achieve such homeostasis, some critical event(s) in the cell cycle will take place only when the cell has grown beyond a critical cell size. In the budding yeast Saccharomyces cerevisiae, a widely used model for the study of the eukaryotic cell cycle, a large body of evidence indicates that cells have to reach a critical size before they start to replicate their DNA and to form bud, which will give rise to the daughter cell. This critical cell size is modulated by growth rate, hence by nutritional conditions and the multiplicity of genetic material (i.e., ploidy). The authors present a mathematical model of the regulatory molecular network acting at the G1 to S transition. The major novel features of this model compared with previous models of this process are (1) the accounting for cell growth (i.e., the increase in cell volume); (2) the explicit consideration of the fact that cells have a nucleus and a cytoplasm, and that key cell cycle regulatory molecules must move between these different compartments and can only react or regulate each other if they are in the same compartment; and (3) the requirement of sequential overcoming of two molecular thresholds given by a cyclin-dependent kinase/cyclin and a cyclin-dependent kinase inhibitor. The model was tested by simulating the processes during G1 to S transition for different growth conditions or for different mutants and by comparing the results with experimental data. A parameter sensitivity analysis (i.e., testing the model predictions when parameters are varied), newly indicates that the critical cell size is an emergent property of the G1 to S network. The model leads to a unified interpretation of seemingly disparate experimental observations and makes predictions to be experimentally verified.
PMCID: PMC1851985  PMID: 17432928
24.  The More the Merrier: Comparative Analysis of Microarray Studies on Cell Cycle-Regulated Genes in Fission Yeast 
Yeast (Chichester, England)  2006;23(4):261-277.
The last two years saw the publication of three genome-wide gene expression studies of the fission yeast cell cycle. While these microarray papers largely agree on the main patterns of cell cycle-regulated transcription and its control, there are discrepancies with regard to the identity and numbers of periodically expressed genes. We present benchmark and reproducibility analyses showing that the main discrepancies do not reflect differences in the data themselves, microarray or synchronization methods seem to lead only to minor biases, but rather in the interpretation of the data. Our reanalysis of the three data sets reveals that combining all independent information leads to an improved identification of periodically expressed genes. These evaluations suggest that the available microarray data do not allow reliable identification of more than about 500 cell cycle-regulated genes. The temporal expression pattern of the top-500 periodically expressed genes is generally consistent across experiments, and the three studies together with our integrated analysis provide a coherent and rich source of information on cell cycle-regulated gene expression in S. pombe. The reanalyzed data sets and other supplementary information are available from an accompanying website: We hope that this paper will resolve the apparent discrepancies between the previous studies and be useful both for wet-lab biologists and for theoretical scientists who wish to take advantage of the data for follow-up work.
PMCID: PMC1828074  PMID: 16544289
S. pombe; cell cycle; transcription; microarray; cell division; periodic gene expression; S. cerevisiae; computational biology
25.  The Yeast Ser/Thr Phosphatases Sit4 and Ppz1 Play Opposite Roles in Regulation of the Cell Cycle 
Molecular and Cellular Biology  1999;19(3):2408-2415.
Yeast cells overexpressing the Ser/Thr protein phosphatase Ppz1 display a slow-growth phenotype. These cells recover slowly from α-factor or nutrient depletion-induced G1 arrest, showing a considerable delay in bud emergence as well as in the expression of the G1 cyclins Cln2 and Clb5. Therefore, an excess of the Ppz1 phosphatase interferes with the normal transition from G1 to S phase. The growth defect is rescued by overexpression of the HAL3/SIS2 gene, encoding a negative regulator of Ppz1. High-copy-number expression of HAL3/SIS2 has been reported to improve cell growth and to increase expression of G1 cyclins in sit4 phosphatase mutants. We show here that the described effects of HAL3/SIS2 on sit4 mutants are fully mediated by the Ppz1 phosphatase. The growth defect caused by overexpression of PPZ1 is intensified in strains with low G1 cyclin levels (such as bck2Δ or cln3Δ mutants), whereas mutation of PPZ1 rescues the synthetic lethal phenotype of sit4 cln3 mutants. These results reveal a role for Ppz1 as a regulatory component of the yeast cell cycle, reinforce the notion that Hal3/Sis2 serves as a negative modulator of the biological functions of Ppz1, and indicate that the Sit4 and Ppz1 Ser/Thr phosphatases play opposite roles in control of the G1/S transition.
PMCID: PMC84033  PMID: 10022927

Results 1-25 (1049756)