Are doping tests in sports trustworthy?: Athletes suffer from insufficiently defined criteria for doping tests: Athletes suffer from insufficiently defined criteria for doping tests.

The lack of clearly defined criteria for doping tests carries a great risk of punishing innocent athletes and undermines the fight against doping in international sports.

Cosegregation of asymmetric features during cell division.

Cellular asymmetry plays a major role in the ageing and evolution of multicellular organisms. However, it remains unknown how the cell distinguishes 'old' from 'new' and whether asymmetry is an attribute of highly specialized cells or a feature inherent in all cells. Here, we investigate the segregation of three asymmetric features: old and new DNA, the spindle pole body (SPB, the centrosome analogue) and the old and new cell ends, using a simple unicellular eukaryote, Schizosaccharomyces pombe. To our knowledge, this is the first study exploring three asymmetric features in the same cells. We show that of the three chromosomes of S. pombe, chromosome I containing the new parental strand, preferentially segregated to the cells inheriting the old cell end. Furthermore, the new SPB also preferentially segregated to the cells inheriting the old end. Our results suggest that the ability to distinguish 'old' from 'new' and to segregate DNA asymmetrically are inherent features even in simple unicellular eukaryotes.

eIF2α phosphorylation and the regulation of translation.

We discuss novel insight into the role and consequences of the phosphorylation of the translation initiation factor eIF2α in the context of stress responses and cell-cycle regulation. eIF2α is centrally located to regulate translation and its phosphorylation in response to different environmental challenges is one of the best characterized stress-response pathways. In addition to its role in stress management, eIF2α phosphorylation is also linked to cell-cycle progression and memory consolidation in the nervous system. The best known consequences of eIF2α phosphorylation are downregulation of global translation and stimulation of translation of some mRNAs. However, recent evidence shows that (i) eIF2α phosphorylation is not always required for the downregulation of global translation after exposure to stress and (ii) eIF2α phosphorylation does not necessarily lead to the downregulation of global translation. These results suggest that the textbook view of eIF2α phosphorylation needs to be revised and that there must be additional regulatory mechanisms at play.

Improving scientific practice in sports-associated drug testing.

Antidoping work is heavily based on scientific analyses of biological material, such as urine and blood. Because of the high stakes both for sports and for the athletes involved it is important that analyses are performed and interpreted in agreement with established scientific standards and professional norms. This is not always the case, as we document here. It is our experience that the antidoping movement does not appear willing to consider that errors can occur and should be corrected. The consequences of the lack of transparency and responsibility are carried by unlucky athletes. Scientific, ethical and legal considerations urge the antidoping movement to reform some of their rules and regulations and to include the possibility that the World Anti-Doping Agency position could, in some cases, be incorrect.

Regulation of global translation during the cell cycle.

It is generally accepted that global translation varies during the cell cycle and is low during mitosis. However, addressing this issue is challenging because it involves cell synchronization, which evokes stress responses that, in turn, affect translation rates. Here, we have used two approaches to measure global translation rates in different cell-cycle phases. First, synchrony in different cell-cycle phases was obtained involving the same stress, by using temperature-sensitive mutants. Second, translation and DNA content were measured by flow cytometry in exponentially growing, single cells. We found no major variation in global translation rates through the cell cycle in either fission yeast or mammalian cells. We also measured phosphorylation of eukaryotic initiation factor-2α, an event that is thought to downregulate global translation in mitosis. In contrast with the prevailing view, eIF2α phosphorylation correlated poorly with downregulation of global translation and ectopically induced eIF2α phosphorylation inhibited global translation only at high levels.

A novel role for ATR/Rad3 in G1 phase.

Checkpoint kinases are important in cellular surveillance pathways that help cells to cope with DNA damage and protect their genomes. In cycling cells, DNA replication is one of the most sensitive processes and therefore all organisms carefully regulate replication initiation and progression. The checkpoint kinase ATR plays important roles both in response to DNA damage and replication stress, and ATR inhibitors are currently in clinical trials for cancer treatment. Therefore, it is important to understand the roles of ATR in detail. Here we show that the fission yeast homologue Rad3 and the human ATR regulate events also in G1 phase in an unperturbed cell cycle. Rad3Δ mutants or human cells exposed to ATR inhibitor in G1 enter S phase prematurely, which results in increased DNA damage. Furthermore, ATR inhibition in a single G1 reduces clonogenic survival, demonstrating that long-term effects of ATR inhibition during G1 are deleterious for the cell. Interestingly, ATR inhibition through G1 and S phase reduces survival in an additive manner, strongly arguing that different functions of ATR are targeted in the different cell-cycle phases. We propose that potential effects of ATR inhibitors in G1 should be considered when designing future treatment protocols with such inhibitors.

A checkpoint-independent mechanism delays entry into mitosis after UV irradiation.

When cells are exposed to stress they delay entry into mitosis. The most extensively studied mechanism behind this delay is the DNA-damage-induced G2/M checkpoint. Here, we show the existence of an additional stress-response pathway in Schizosaccharomyces pombe that is independent of the classic ATR/Rad3-dependent checkpoint. This novel mechanism delays entry mitosis independently of the spindle assembly checkpoint and the mitotic kinases Fin1, Ark1 and Plo1. The pathway delays activation of the mitotic cyclin-dependent kinase (CDK) Cdc2 after UV irradiation. Furthermore, we demonstrate that translation of the mitotic cyclin Cdc13 is selectively downregulated after UV irradiation, and we propose that this downregulation of Cdc13 contributes to the delayed activation of Cdc2 and the delayed mitosis.

Analyzing Schizosaccharomyces pombe DNA Content by Flow Cytometry.

Flow cytometry can be used to measure the DNA content of individual cells. The data are usually presented as DNA histograms that can be used to examine the cells' progression through the cell cycle. Under standard growth conditions, fission yeast cells do not complete cytokinesis until after G1 phase; therefore, DNA histograms show one major peak representing cells in G1 (2×1C DNA) and G2 phase (1×2C DNA). By analysis of the duration of the fluorescence signal as well as the intensity of the DNA-related signal, it is possible to discriminate between cells in M/G1, S, and G2 This protocol describes how to prepare cells for flow cytometry and analyze them. We also describe the application of barcoding for more accurate comparison of samples.

Consequences of abnormal CDK activity in S phase.

Cyclin Dependent Kinases (CDKs) are important regulators of DNA replication. In this work we have investigated the consequences of increasing or decreasing the CDK activity in S phase. To this end we identified S-phase regulators of the fission yeast CDK, Cdc2, and used appropriate mutants to modulate Cdc2 activity. In fission yeast Mik1 has been thought to be the main regulator of Cdc2 activity in S phase. However, we find that Wee1 has a major function in S phase and thus we used wee1 mutants to investigate the consequences of increased Cdc2 activity. These wee1 mutants display increased replication stress and, particularly in the absence of the S-phase checkpoint, accumulate DNA damage. Notably, more cells incorporate EdU in a wee1(-) strain as compared to wildtype, suggesting altered regulation of DNA replication. In addition, a higher number of cells contain chromatin-bound Cdc45, an indicator of active replication forks. In addition, we found that Cdc25 is required to activate Cdc2 in S phase and used a cdc25 mutant to explore a situation where Cdc2 activity is reduced. Interestingly, a cdc25 mutant has a higher tolerance for replication stress than wild-type cells, suggesting that reduced CDK activity in S phase confers resistance to at least some forms of replication stress.

Stress-induced inhibition of translation independently of eIF2α phosphorylation.

Exposure of fission yeast cells to ultraviolet (UV) light leads to inhibition of translation and phosphorylation of the eukaryotic initiation factor-2α (eIF2α). This phosphorylation is a common response to stress in all eukaryotes. It leads to inhibition of translation at the initiation stage and is thought to be the main reason why stressed cells dramatically reduce protein synthesis. Phosphorylation of eIF2α has been taken as a readout for downregulation of translation, but the role of eIF2α phosphorylation in the downregulation of general translation has not been much investigated. We show here that UV-induced global inhibition of translation in fission yeast cells is independent of eIF2α phosphorylation and the eIF2α kinase general control nonderepressible-2 protein (Gcn2). Also, in budding yeast and mammalian cells, the UV-induced translational depression is largely independent of GCN2 and eIF2α phosphorylation. Furthermore, exposure of fission yeast cells to oxidative stress generated by hydrogen peroxide induced an inhibition of translation that is also independent of Gcn2 and of eIF2α phosphorylation. Our findings show that stress-induced translational inhibition occurs through an unknown mechanism that is likely to be conserved through evolution.

Cell-cycle analyses using thymidine analogues in fission yeast.

Thymidine analogues are powerful tools when studying DNA synthesis including DNA replication, repair and recombination. However, these analogues have been reported to have severe effects on cell-cycle progression and growth, the very processes being investigated in most of these studies. Here, we have analyzed the effects of 5-ethynyl-2'-deoxyuridine (EdU) and 5-Chloro-2'-deoxyuridine (CldU) using fission yeast cells and optimized the labelling procedure. We find that both analogues affect the cell cycle, but that the effects can be mitigated by using the appropriate analogue, short pulses of labelling and low concentrations. In addition, we report sequential labelling of two consecutive S phases using EdU and 5-bromo-2'-deoxyuridine (BrdU). Furthermore, we show that detection of replicative DNA synthesis is much more sensitive than DNA-measurements by flow cytometry.

Crosstalk between the Tor and Gcn2 pathways in response to different stresses.

Regulating growth and the cell cycle in response to environmental fluctuations is important for all organisms in order to maintain viability. Two major pathways for translational regulation are found in higher eukaryotes: the Tor signaling pathway and those operating through the eIF2α kinases. Studies from several organisms indicate that the two pathways are interlinked, in that Tor complex 1 (TORC1) negatively regulates the Gcn2 kinase. Furthermore, inactivation of TORC1 may be required for activation of Gcn2 in response to stress. Here, we use the model organism Schizosaccharomyces pombe to investigate this crosstalk further. We find that the relationship is more complex than previously thought. First, in response to UV irradiation and oxidative stress, Gcn2 is fully activated in the presence of TORC1 signaling. Second, during amino-acid starvation, activation of Gcn2 is dependent on Tor2 activity, and Gcn2 is required for timely inactivation of the Tor pathway. Our data show that the crosstalk between the two pathways varies with the actual stress applied.

GCN2, an old dog with new tricks.

Gcn2 was first described in budding yeast as a serine/threonine protein kinase involved in the response to amino acid starvation and this is its best characterized role to date. Recent work has revealed new and exciting roles for Gcn2, which affect many aspects of cellular physiology in response to a number of stresses in addition to starvation. Furthermore, the Gcn2 pathway has been implicated in diseases such as cancer and Alzheimer's disease, and therefore elucidating the new roles of Gcn2 seems ever more important.

Hpz1 modulates the G1-S transition in fission yeast.

Here we characterize a novel protein in S. pombe. It has a high degree of homology with the Zn-finger domain of the human Poly(ADP-ribose) polymerase (PARP). Surprisingly, the gene for this protein is, in many fungi, fused with and in the same reading frame as that encoding Rad3, the homologue of the human ATR checkpoint protein. We name the protein Hpz1 (Homologue of PARP-type Zn-finger). Hpz1 does not possess PARP activity, but is important for resistance to ultraviolet light in the G1 phase and to treatment with hydroxyurea, a drug that arrests DNA replication forks in the S phase. However, we find no evidence of a checkpoint function of Hpz1. Furthermore, absence of Hpz1 results in an advancement of S-phase entry after a G1 arrest as well as earlier recovery from a hydroxyurea block. The hpz1 gene is expressed mainly in the G1 phase and Hpz1 is localized to the nucleus. We conclude that Hpz1 regulates the initiation of the S phase and may cooperate with Rad3 in this function.

Induction of a G1-S checkpoint in fission yeast.

Entry into S phase is carefully regulated and, in most organisms, under the control of a G(1)-S checkpoint. We have previously described a G(1)-S checkpoint in fission yeast that delays formation of the prereplicative complex at chromosomal replication origins after exposure to UV light (UVC). This checkpoint absolutely depends on the Gcn2 kinase. Here, we explore the signal for activation of the Gcn2-dependent G(1)-S checkpoint in fission yeast. If some form of DNA damage can activate the checkpoint, deficient DNA repair should affect the length of the checkpoint-induced delay. We find that the cell-cycle delay differs in repair-deficient mutants from that in wild-type cells. However, the duration of the delay depends not only on the repair capacity of the cells, but also on the nature of the repair deficiency. First, the delay is abolished in cells that are deficient in the early steps of repair. Second, the delay is prolonged in repair mutants that fail to complete repair after the incision stage. We conclude that the G(1)-S delay depends on damage to the DNA and that the activating signal derives not from the initial DNA damage, but from a repair intermediate(s). Surprisingly, we find that activation of Gcn2 does not depend on the processing of DNA damage and that activated Gcn2 alone is not sufficient to delay entry into S phase in UVC-irradiated cells. Thus, the G(1)-S delay depends on at least two different inputs.

Cell-cycle analysis of fission yeast cells by flow cytometry.

The cell cycle of the fission yeast, Schizosaccharomyces pombe, does not easily lend itself to analysis by flow cytometry, mainly because cells in G(1) and G(2) phase contain the same amount of DNA. This occurs because fission yeast cells under standard growth conditions do not complete cytokinesis until after G(1) phase. We have devised a flow cytometric method exploiting the fact that cells in G(1) phase contain two nuclei, whereas cells in G(2) are mononuclear. Measurements of the width as well as the total area of the DNA-associated fluorescence signal allows the discrimination between cells in G(1) and in G(2) phase and the cell-cycle progression of fission yeast can be followed in detail by flow cytometry. Furthermore, we show how this method can be used to monitor the timing of cell entry into anaphase. Fission yeast cells tend to form multimers, which represents another problem of flow cytometry-based cell-cycle analysis. Here we present a method employing light-scatter measurements to enable the exclusion of cell doublets, thereby further improving the analysis of fission yeast cells by flow cytometry.

Global transcriptional response after exposure of fission yeast cells to ultraviolet light.

BACKGROUND: In many cell types, including the fission yeast Schizosaccharomyces pombe, a set of checkpoints are induced by perturbations of the cell cycle or by DNA damage. Many of the checkpoint responses include a substantial change of the transcriptional pattern. As part of characterising a novel G1/S checkpoint in fission yeast we have investigated whether a transcriptional response is induced after irradiation with ultraviolet light. RESULTS: Microarray analyses were used to measure the global transcription levels of all open reading frames of fission yeast after 254 nm ultraviolet irradiation, which is known to induce a G1/S checkpoint. We discovered a surprisingly weak transcriptional response, which is quite unlike the marked changes detected after some other types of treatment and in several other checkpoints. Interestingly, the alterations in gene expression after ultraviolet irradiation were not similar to those observed after ionising radiation or oxidative stress. Pathway analysis suggests that there is little systematic transcriptional response to the irradiation by ultraviolet light, but a marked, coordinated transcriptional response was noted on progression of the cells from G1 to S phase. CONCLUSION: There is little response in fission yeast to ultraviolet light at the transcriptional level. Amongst the genes induced or repressed after ultraviolet irradiation we found none that are likely to be involved in the G1/S checkpoint mechanism, suggesting that the checkpoint is not dependent upon transcriptional regulation.

The right half of the Escherichia coli replication origin is not essential for viability, but facilitates multi-forked replication.

Replication initiation is a key event in the cell cycle of all organisms and oriC, the replication origin in Escherichia coli, serves as the prototypical model for this process. The minimal sequence required for oriC function was originally determined entirely from plasmid studies using cloned origin fragments, which have previously been shown to differ dramatically in sequence requirement from the chromosome. Using an in vivo recombineering strategy to exchange wt oriCs for mutated ones regardless of whether they are functional origins or not, we have determined the minimal origin sequence that will support chromosome replication. Nearly the entire right half of oriC could be deleted without loss of origin function, demanding a reassessment of existing models for initiation. Cells carrying the new DnaA box-depleted 163 bp minimal oriC exhibited little or no loss of fitness under slow-growth conditions, but were sensitive to rich medium, suggesting that the dense packing of initiator binding sites that is a hallmark of prokaryotic origins, has likely evolved to support the increased demands of multi-forked replication.

Checkpoint regulation of DNA replication.

We discuss the mechanisms regulating entry into and progression through S phase in eukaryotic cells. Methods to study the G1/S transition are briefly reviewed and an overview of G1/S-checkpoints is given, with particular emphasis on fission yeast. Thereafter we discuss different aspects of the intra-S checkpoint and introduce the main molecular players and mechanisms.