ABSTRACT
BACKGROUND: Universal screening for postpartum depression is recommended in many countries. Knowledge of whether the disclosure of depressive symptoms in the postpartum period differs across cultures could improve detection and provide new insights into the pathogenesis. Moreover, it is a necessary step to evaluate the universal use of screening instruments in research and clinical practice. In the current study we sought to assess whether the Edinburgh Postnatal Depression Scale (EPDS), the most widely used screening tool for postpartum depression, measures the same underlying construct across cultural groups in a large international dataset. METHOD: Ordinal regression and measurement invariance were used to explore the association between culture, operationalized as education, ethnicity/race and continent, and endorsement of depressive symptoms using the EPDS on 8209 new mothers from Europe and the USA. RESULTS: Education, but not ethnicity/race, influenced the reporting of postpartum depression [difference between robust comparative fit indexes (∆*CFI) 0.01), but not between European countries (∆*CFI < 0.01). CONCLUSIONS: Investigators and clinicians should be aware of the potential differences in expression of phenotype of postpartum depression that women of different educational backgrounds may manifest. The increasing cultural heterogeneity of societies together with the tendency towards globalization requires a culturally sensitive approach to patients, research and policies, that takes into account, beyond rhetoric, the context of a person's experiences and the context in which the research is conducted.
Subject(s)
Cross-Cultural Comparison , Depression, Postpartum/diagnosis , Depression, Postpartum/ethnology , Psychiatric Status Rating Scales , Self Report , Adolescent , Adult , Female , Humans , Middle Aged , Young AdultABSTRACT
Carbon dioxide off-setting policy in the agricultural sector is focused on manipulating the terrestrial carbon cycle by reafforestation and increasing the retention of carbon within agricultural soils. We quantified the amount of carbon stored in the living and dead biomass and the surface soils of a previously grazed woodland ecosystem. We demonstrate that modification of coarse woody debris management could potentially store 8 to 15 t C ha. This large carbon pool raises the prospect that appropriate management of temperate woodlands to retain coarse woody debris and increase its volume into the future could achieve increased landscape carbon storage.
ABSTRACT
The fruit fly Drosophila melanogaster has emerged in recent years as a tractable system for studying sleep. The sleep-wake dichotomy represents one of the principal transitions in global brain state, and neurohormones and neuromodulators are well known for their ability to change global brain states. Here, we describe studies of two brain systems that regulate sleep in Drosophila, the neurohormonal epidermal growth factor receptor system and the neuromodulatory dopaminergic system, each of which acts through a discrete anatomical locus in the dorsal brain. Both control systems display considerable mechanistic similarity to those in mammals, suggesting possible functional homologies.
Subject(s)
Drosophila melanogaster/physiology , Sleep/physiology , Animals , Arousal/genetics , Arousal/physiology , Brain/physiology , Dopamine/physiology , Drosophila Proteins/genetics , Drosophila Proteins/physiology , Drosophila melanogaster/genetics , ErbB Receptors/genetics , ErbB Receptors/physiology , Genes, Insect , Insect Hormones/physiology , Neurotransmitter Agents/physiology , Signal Transduction , Sleep/geneticsABSTRACT
When DNA replication is inhibited during the synthesis (S) phase of the cell cycle, a signaling pathway (checkpoint) is activated that serves to prevent mitosis from initiating before completion of replication. This replication checkpoint acts by down-regulating the activity of the mitotic inducer cdc2-cyclin B. Here, we report the relation between chromatin structure and induction of the replication checkpoint. Chromatin was competent to initiate a checkpoint response only after the DNA was unwound and DNA polymerase alpha had been loaded. Checkpoint induction did not require new DNA synthesis on the unwound template strand but did require RNA primer synthesis by primase. These findings identify the RNA portion of the primer as an important component of the signal that activates the replication checkpoint.
Subject(s)
CDC2-CDC28 Kinases , Chromatin/metabolism , DNA Primase/metabolism , DNA Replication , RNA/biosynthesis , Saccharomyces cerevisiae Proteins , Animals , Aphidicolin/pharmacology , Carrier Proteins/metabolism , Checkpoint Kinase 1 , Cyclin E/metabolism , Cyclin-Dependent Kinase 2 , Cyclin-Dependent Kinases/metabolism , DNA Helicases/metabolism , DNA Polymerase I/antagonists & inhibitors , DNA Polymerase I/metabolism , DNA Replication/drug effects , DNA, Single-Stranded/metabolism , DNA-Binding Proteins/metabolism , Mitosis , Nuclear Proteins/metabolism , Phosphorylation , Protein Kinases/metabolism , Protein Serine-Threonine Kinases/metabolism , Recombinant Proteins/metabolism , S Phase , Signal Transduction , Xenopus , Xenopus ProteinsABSTRACT
Human Peroxisome Proliferator-Activated Receptor gamma (PPARgamma) was originally cloned from a human bone marrow library. What role does this ligand activated transcription factor play in hematopoiesis and the immune system? We note that: a) PPARgamma has potential to interact/interfere or synergize with retinoid biology, b) fatty acids and a prostaglandin have been identified as ligands, and c) lymphocytes, monocytes and neutrophils use fatty acids as a major source of energy production, d) PPARgamma has been shown to oppose TNFalpha and down regulate cytokine production in monocytes. Therefore, we undertook a review of the literature and an expression survey of PPARgamma in a number of major organs and cells involved in the hematopoietic system, for the purpose of building a database towards understanding the role and function of PPARgamma gene regulation in the developing blood and immune systems. PPARgamma is expressed before mesodermal induction in tissue in and around Speymann's organizer in the xenopus blastocyst, in erythroid precursors of blood islands and in the circulation of the day 10.0 murine embryo, in human 19 week fetal liver, in some but not all murine and human bone marrow erythroid, myeloid, and monocytoid progenitors, bone marrow stromal cells and adipocytes, osteoblasts, endothelial cells, some T, and B lymphocytes, monocytes, macrophages, and other monocytic derivatives. It can be found in the cells of Peyer's patches, lymphoid follicles, spleen, and thymus. It is not clear if it is ever or transiently expressed in megakaryocytes, mast cells, or neutrophils. Based on the above data and a review of the literature, PPARgamma seems to play a role during the elicitation of immune responses. We propose PPARgamma may be involved in changes in energy states required during activation and development of many cell types involved, and has additional immunologically relevant effects in erythroid, myeloid, monocytic, T and B lymphocytic, stromal, and endothelial cell function.
Subject(s)
Bone Marrow/physiology , Receptors, Cytoplasmic and Nuclear/physiology , Transcription Factors/physiology , Amino Acid Sequence , Gene Expression Regulation/physiology , Humans , Transcription, Genetic/physiologyABSTRACT
We report that a plasmid replicating in Xenopus egg extracts becomes negatively supercoiled during replication initiation. Supercoiling requires the initiation factor Cdc45, as well as the single-stranded DNA-binding protein RPA, and therefore likely represents origin unwinding. When unwinding is prevented, Cdc45 binds to chromatin whereas DNA polymerase alpha does not, indicating that Cdc45, RPA, and DNA polymerase alpha bind chromatin sequentially at the G1/S transition. Whereas the extent of origin unwinding is normally limited, it increases dramatically when DNA polymerase alpha is inhibited, indicating that the helicase that unwinds DNA during initiation can become uncoupled from the replication fork. We discuss the implications of these results for the location of replication start sites relative to the prereplication complex.
Subject(s)
CDC2-CDC28 Kinases , Chromatin/metabolism , DNA Replication , DNA-Binding Proteins/metabolism , Replication Origin , Saccharomyces cerevisiae Proteins , Animals , Carrier Proteins/metabolism , Cell-Free System , Cyclin-Dependent Kinase 2 , Cyclin-Dependent Kinases/metabolism , Cytosol/metabolism , DNA Helicases/metabolism , DNA Polymerase I/metabolism , DNA, Single-Stranded/metabolism , DNA, Superhelical/metabolism , Eukaryotic Cells , Models, Genetic , Nuclear Proteins/metabolism , Nucleic Acid Conformation , Plasmids/metabolism , Protein Serine-Threonine Kinases/metabolism , Replication Protein A , Xenopus , Xenopus ProteinsABSTRACT
BACKGROUND: The DNA replication checkpoint ensures that mitosis is not initiated before DNA synthesis is completed. Recent studies using Xenopus extracts have demonstrated that activation of the replication checkpoint and phosphorylation of the Chk1 kinase are dependent on RNA primer synthesis by DNA polymerase alpha, and it has been suggested that the ATR kinase-so-called because it is related to the product of the gene that is mutated in ataxia telangiectasia (ATM) and to Rad3 kinase-may be an upstream component of this response. It has been difficult to test this hypothesis as an ATR-deficient system suitable for biochemical studies has not been available. RESULTS: We have cloned the Xenopus laevis homolog of ATR (XATR) and studied the function of the protein in Xenopus egg extracts. Using a chromatin-binding assay, we found that ATR associates with chromatin after initiation of replication, dissociates from chromatin upon completion of replication, and accumulates in the presence of aphidicolin, an inhibitor of DNA replication. Its association with chromatin was inhibited by treatment with actinomycin D, an inhibitor of RNA primase. There was an early rise in the activity of Cdc2-cyclin B in egg extracts depleted of ATR both in the presence or absence of aphidicolin. In addition, the premature mitosis observed upon depletion of ATR was accompanied by the loss of Chk1 phosphorylation. CONCLUSIONS: ATR is a replication-dependent chromatin-binding protein, and its association with chromatin is dependent on RNA synthesis by DNA polymerase alpha. Depletion of ATR leads to premature mitosis in the presence and absence of aphidicolin, indicating that ATR is required for the DNA replication checkpoint.
Subject(s)
Cell Cycle Proteins/metabolism , Chromatin/metabolism , DNA Replication , Genes, cdc/physiology , Protein Serine-Threonine Kinases , Xenopus Proteins , Amino Acid Sequence , Animals , Aphidicolin/pharmacology , Ataxia Telangiectasia Mutated Proteins , Blotting, Western , Cell Cycle/drug effects , Cell Cycle/physiology , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/genetics , Cell Nucleus/metabolism , Checkpoint Kinase 1 , Cloning, Molecular , DNA Replication/drug effects , Dactinomycin/pharmacology , Enzyme Inhibitors/pharmacology , Humans , Male , Molecular Sequence Data , Oocytes/physiology , Protein Kinases/metabolism , Protein Synthesis Inhibitors/pharmacology , Recombinant Proteins/metabolism , Sequence Analysis, DNA , Spermatozoa/physiology , Xenopus laevisABSTRACT
Protein phosphatase 2A (PP2A) is an abundant, multifunctional serine/threonine-specific phosphatase that stimulates simian virus 40 DNA replication. The question as to whether chromosomal DNA replication also depends on PP2A was addressed by using a cell-free replication system derived from Xenopus laevis eggs. Immunodepletion of PP2A from Xenopus egg extract resulted in strong inhibition of DNA replication. PP2A was required for the initiation of replication but not for the elongation of previously engaged replication forks. Therefore, the initiation of chromosomal DNA replication depends not only on phosphorylation by protein kinases but also on dephosphorylation by PP2A.
Subject(s)
DNA Replication , Phosphoprotein Phosphatases/metabolism , Animals , Phosphoprotein Phosphatases/genetics , Phosphorylation , Protein Phosphatase 2 , Xenopus laevisABSTRACT
The dependence of mitosis on the completion of the period of DNA replication in the cell cycle [synthesis (S) phase] ensures that chromosome segregation occurs only after the genome has been fully duplicated. A key negative regulator of mitosis, the protein kinase Wee1, was degraded in a Cdc34-dependent fashion in Xenopus egg extracts. This proteolysis event was required for a timely entrance into mitosis and was inhibited when DNA replication was blocked. Therefore, the DNA replication checkpoint can prevent mitosis by suppressing the proteolysis of Wee1 during S phase.
Subject(s)
Ligases/metabolism , Mitosis , Nuclear Proteins , Protein-Tyrosine Kinases/metabolism , S Phase , Tumor Suppressor Proteins , Ubiquitin-Protein Ligase Complexes , Xenopus Proteins , Anaphase-Promoting Complex-Cyclosome , Animals , Aphidicolin/pharmacology , Cell Cycle Proteins/metabolism , Cell Nucleus/metabolism , Cyclin-Dependent Kinase Inhibitor p27 , DNA Replication/drug effects , Female , G2 Phase , Leupeptins/pharmacology , Male , Maturation-Promoting Factor/metabolism , Microtubule-Associated Proteins/metabolism , Okadaic Acid/pharmacology , Ovum , Phosphoprotein Phosphatases/metabolism , Protein Serine-Threonine Kinases/metabolism , Proteins , Recombinant Proteins/metabolism , Spermatozoa , Ubiquitin-Protein Ligases , Ubiquitins/analogs & derivatives , Ubiquitins/metabolism , Ubiquitins/pharmacology , Xenopus , cdc25 PhosphatasesABSTRACT
The initiation of DNA replication involves a minimum of four factors: a specific DNA sequence (origin), an initiator protein which binds to the origin, a helicase that unwinds the origin and a protein that binds single-stranded DNA that stabilizes the unwound origin. In eukaryotic cells, the origin recognition complex (ORC) is the initiator protein and replication protein A (RPA; ref. 3) is the single-stranded DNA-binding protein. However, the helicase has not been identified and the nature of origins remains elusive, except in the case of Saccharomyces cerevisiae. A unique feature of eukaryotic DNA replication is that it occurs at a few-hundred discrete foci. It has thus been proposed that a real origin must contain a specific DNA sequence and must be attached to replication foci. Using Xenopus laevis egg extracts, we have identified and purified a 170-kD protein, focus-forming activity 1 (FFA-1), which is required for the formation of replication foci. Here we report that FFA-1 has DNA-helicase activity. Moreover, it is a homologue of the human Werner syndrome gene product WRN, a protein associated with premature ageing in humans.
Subject(s)
DNA Helicases/genetics , DNA Helicases/metabolism , DNA Replication/physiology , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Animals , Cloning, Molecular , DNA/metabolism , DNA Helicases/isolation & purification , DNA-Binding Proteins/metabolism , Exodeoxyribonucleases , Humans , Molecular Sequence Data , RecQ Helicases , Replication Protein A , Sequence Homology, Amino Acid , Werner Syndrome/genetics , Werner Syndrome Helicase , Xenopus laevisABSTRACT
Using Xenopus egg extracts, we have developed a completely soluble system for eukaryotic chromosomal DNA replication. In the absence of a nuclear envelope, a single, complete round of ORC-dependent DNA replication is catalyzed by cytosolic and nuclear extracts added sequentially to demembranated sperm chromatin or prokaryotic plasmid DNA. The absence of rereplication is explained by an activity present in the nucleus that prevents the binding of MCM to chromatin. Our results indicate that the role of the nuclear envelope in DNA replication is to concentrate activators and inhibitors of replication inside the nucleus. In addition, they provide direct evidence that metazoans use the same strategy as yeast to activate DNA replication and to restrict it to a single round per cell cycle.
Subject(s)
Chromosomes/metabolism , Cytoplasm/metabolism , DNA Replication , Nuclear Envelope/metabolism , Oocytes/metabolism , Animals , Cell Cycle Proteins/metabolism , Cell Extracts/pharmacology , Interphase/physiology , Male , Plasmids , Spermatozoa/drug effects , Xenopus , Yeasts/genetics , Yeasts/metabolismABSTRACT
Before initiation of DNA replication, origin recognition complex (ORC) proteins, cdc6, and minichromosome maintenance (MCM) proteins bind to chromatin sequentially and form preinitiation complexes. Using Xenopus laevis egg extracts, we find that after the formation of these complexes and before initiation of DNA replication, cdc6 is rapidly removed from chromatin, possibly degraded by a cdk2-activated, ubiquitin-dependent proteolytic pathway. If this displacement is inhibited, DNA replication fails to initiate. We also find that after assembly of MCM proteins into preinitiation complexes, removal of the ORC from DNA does not block the subsequent initiation of replication. Importantly, under conditions in which both ORC and cdc6 protein are absent from preinitiation complexes, DNA replication is still dependent on cdk2 activity. Therefore, the final steps in the process leading to initiation of DNA replication during S phase of the cell cycle are independent of ORC and cdc6 proteins, but dependent on cdk2 activity.
Subject(s)
CDC2-CDC28 Kinases , Cell Cycle Proteins/metabolism , Cyclin-Dependent Kinases/metabolism , DNA Replication , DNA-Binding Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Saccharomyces cerevisiae Proteins , Xenopus Proteins , Animals , Chromatin/metabolism , Cyclin A/metabolism , Cyclin-Dependent Kinase 2 , Female , Male , Molecular Sequence Data , Oocytes/metabolism , Origin Recognition Complex , Spermatozoa/metabolism , Xenopus laevisABSTRACT
Previous work identified a developmental timer that controls the stability of cyclin A protein in interphase-arrested Xenopus embryos. It was shown that cyclins A1 and A2 abruptly become unstable in hydroxyurea-treated embryos at the time that untreated embryos are beginning gastrulation (early gastrulation transition; EGT). We have demonstrated here that cyclins A1 and A2 are degraded at the equivalent of the EGT by the ICE-like caspases that are responsible for programmed cell death or apoptosis. Analysis of embryos treated with hydroxyurea or cycloheximide showed widespread cellular apoptosis coincident with cyclin A cleavage. Our data further indicate that the apoptotic pathway is present in Xenopus embryos prior to the EGT; however, it is maintained in an inactive state in early cleaving embryos by maternally encoded inhibitors. Characterization of the timing of the activation of apoptosis implicates the initiation of zygotic transcription at the mid-blastula transition (MBT) in the suppression of apoptosis in normal embryos. The decreased biosynthetic capacity of embryos treated with hydroxyurea or cycloheximide most likely interferes with the ability to maintain sufficient levels of apoptotic inhibitors and results in widespread apoptosis. Our results suggest a scenario whereby the apoptotic pathway is suppressed in the early cleaving embryo by maternally contributed inhibitors. Degradation at the EGT of maternal RNAs encoding these inhibitors is compensated for by new zygotic transcription beginning at the MBT. This indicates that the interval between the MBT and the EGT represents a critical developmental period during which the regulation of embryonic cellular processes is transferred from maternal to zygotic control.
Subject(s)
Apoptosis/physiology , Caspases , Cyclin A , Cyclins/metabolism , Gastrula/cytology , Gastrula/metabolism , Interphase/physiology , Animals , Blastocyst/cytology , Caspase 3 , Cleavage Stage, Ovum/cytology , Cycloheximide/pharmacology , Cysteine Endopeptidases/physiology , Cysteine Proteinase Inhibitors/pharmacology , Cytochrome c Group/pharmacology , Hydroxyurea/pharmacology , Mitosis , Protein Synthesis Inhibitors/pharmacology , S Phase/drug effects , Transcription, Genetic , XenopusABSTRACT
Using cell-free extracts made from Xenopus eggs, we show that cdk2-cyclin E and A kinases play an important role in negatively regulating DNA replication. Specifically, we demonstrate that the cdk2 kinase concentration surrounding chromatin in extracts increases 200-fold once the chromatin is assembled into nuclei. Further, we find that if the cdk2-cyclin E or A concentration in egg cytosol is increased 16-fold before the addition of sperm chromatin, the chromatin fails to initiate DNA replication once assembled into nuclei. This demonstrates that cdk2-cyclin E or A can negatively regulate DNA replication. With respect to how this negative regulation occurs, we show that high levels of cdk2-cyclin E do not block the association of the protein complex ORC with sperm chromatin but do prevent association of MCM3, a protein essential for replication. Importantly, we find that MCM3 that is prebound to chromatin does not dissociate when cdk2-cyclin E levels are increased. Taken together our results strongly suggest that during the embryonic cell cycle, the low concentrations of cdk2-cyclin E present in the cytosol after mitosis and before nuclear formation allow proteins essential for potentiating DNA replication to bind to chromatin, and that the high concentration of cdk2-cyclin E within nuclei prevents MCM from reassociating with chromatin after replication. This situation could serve, in part, to limit DNA replication to a single round per cell cycle.
Subject(s)
CDC2-CDC28 Kinases , Cyclin-Dependent Kinases/metabolism , DNA Replication/physiology , Protein Serine-Threonine Kinases/metabolism , S Phase/physiology , Animals , Cell Cycle Proteins/metabolism , Cell Nucleus/physiology , Chromatin/metabolism , Cyclin-Dependent Kinase 2 , Cyclins/metabolism , Cyclins/pharmacology , Cytosol/chemistry , Female , G1 Phase/physiology , G2 Phase/physiology , Male , Oocytes/cytology , Oocytes/enzymology , Protein Binding/physiology , Replication Origin/physiology , Spermatozoa/chemistry , Xenopus , Xenopus ProteinsABSTRACT
Once a specific number of cells have been produced in the early Xenopus laevis embryo, replicon size during the S phase of the cell cycle increases. Here, it is reported that similar increase in replicon size occurred when the concentration of nuclei in replication-competent Xenopus egg extracts exceeded a critical threshold. In this system, the origin recognition complex (ORC) did not become stoichiometrically limiting for initiation, and similar amounts of this complex bound to chromatin regardless of replicon size. These data suggest that in early development, an unidentified factor controls how many preformed ORC-DNA complexes initiate DNA replication.
Subject(s)
DNA Replication , DNA-Binding Proteins/metabolism , DNA/metabolism , Ovum/metabolism , Replicon , Animals , Cell Cycle Proteins/metabolism , Chromatin/metabolism , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/pharmacology , Cytarabine/pharmacology , Enzyme Inhibitors/pharmacology , Female , Origin Recognition Complex , Replication Origin , S Phase , Xenopus laevisABSTRACT
In metazoan cells, DNA replication during the S phase of the cell cycle takes place at discrete locations within the nucleus. These sites, or foci, appear to participate in clustering many replicons together and synchronously regulating the activation of these replicon units. Consistent with this role. many replication proteins have been observed to attach to foci during the S phase of the cell cycle. Recently, cell-free replication extracts have been used both to characterize the events that are involved in either the formation of these sites or in the regulation of foci assembly, and to purify candidate proteins which may be integral core structural proteins responsible for forming foci. These advances provide a foundation for investigating foci structure and function at a biochemical level.
Subject(s)
Cell Cycle/genetics , DNA Replication/genetics , Replicon/genetics , AnimalsABSTRACT
We have analyzed cyclin E1, a protein that is essential for the G1/S transition, during early development in Xenopus embryos. Cyclin E1 was found to be abundant in eggs, and after fertilization, until the midblastula transition (MBT) when levels of cyclin E1 protein, and associated kinase activity, were found to decline precipitously. Our results suggest that the reduced level of the cyclin E1 protein detected after the MBT does not occur indirectly as a result of degradation of the maternally encoded cyclin E1 mRNA. Instead, the stability of cyclin E1 protein appears to play a major role in reduction of cyclin E1 levels at this time. Cyclin E1 protein was found to be stable during the cleavage divisions but degraded with a much shorter half-life after the MBT. Activation of cyclin E1 protein turnover occurs independent of cell cycle progression, does not require ongoing protein synthesis, and is not triggered as a result of the ratio of nuclei to cytoplasm in embryonic cells that initiates the MBT. We therefore propose that a developmental timing mechanism measures an approximately 5-hr time period, from the time of fertilization, and then allows activation of a protein degradative pathway that regulates cyclin E1. Characterization of the timer suggests that it might be held inactive in eggs by a mitogen-activated protein kinase signal transduction pathway.
Subject(s)
Blastocyst/metabolism , CDC2-CDC28 Kinases , Cyclins/metabolism , Amino Acid Sequence , Animals , Cyclin E , Cyclin-Dependent Kinase 2 , Cyclin-Dependent Kinases/metabolism , Cyclins/genetics , Gene Expression Regulation, Developmental , Molecular Sequence Data , Peptides/chemistry , Protein Serine-Threonine Kinases/metabolism , RNA, Messenger/genetics , Xenopus Proteins , Xenopus laevisABSTRACT
In higher eukaryotes, Cdk2 kinase plays an essential role in regulating the G1-S transition. Here, we use cycling Xenopus egg extracts to examine the requirement of Cdk2 kinase on progression into mitosis. Interestingly, when Cdk2 kinase activity is inhibited by the Cdk-specific inhibitor, p21Cip1, a block to mitosis occurs, and inactive Cdc2-cyclin B accumulates. This block occurs in the absence of nuclei and is not due to direct inhibition of Cdc2 by Cip. Importantly, this block to mitosis is reversible by restoring Cdk2-cyclin E kinase activity to a Cip-treated cycling extract. Moreover, immunodepletion of Cdk2 from interphase extracts prevents activation of Cdc2 upon the addition of exogenous cyclin B. Thus, our data show that Cdk2 kinase is a positive regulator of Cdc2-cyclin B complexes and establish a link between Cdk2 kinase and cell cycle progression into mitosis.
Subject(s)
CDC2 Protein Kinase/metabolism , CDC2-CDC28 Kinases , Cyclin-Dependent Kinases/physiology , Cyclins/metabolism , Mitosis/physiology , Protein Serine-Threonine Kinases/physiology , Animals , CDC2 Protein Kinase/antagonists & inhibitors , Cell Cycle/physiology , Cell Extracts , Cyclin-Dependent Kinase 2 , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/pharmacology , DNA Replication/physiology , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Ovum/cytology , Ovum/enzymology , S Phase/physiology , Xenopus , Xenopus ProteinsABSTRACT
In the nuclei of eukaryotic cells, initiation of DNA replication occurs at a discrete number of foci. One component of these foci is the DNA replication factor RP-A. Here, the process leading to the association of RP-A with foci was reconstituted with cytosolic fractions derived from Xenopus eggs. With the use of this fractionated system, a 170-kilodalton protein required for the assembly of RP-A into foci was identified and purified. The protein appears to be an integral component of the foci at which replication of DNA is initiated in eukaryotic nuclei.
Subject(s)
Cell Nucleus/metabolism , DNA Replication , DNA-Binding Proteins/metabolism , Proteins/metabolism , Animals , Cell-Free System , Cytosol/chemistry , DNA, Single-Stranded/metabolism , Female , Male , Molecular Weight , Oocytes , Orientation , Proteins/chemistry , Replication Protein A , XenopusABSTRACT
Assembly and disassembly of the nucleus at mitosis in eukaryotes involves the reversible interaction of chromatin with the nuclear membrane. Previously we have shown that this interaction is regulated by the antagonistic activities of a kinase and a phosphatase. The kinase promotes membrane release while the phosphatase stimulates binding. In this report we describe four steps in the purification of the kinase needed for release of membranes from chromatin. We also show that the release kinase and the mitotic initiation kinase, cdc2, are distinct and are separated from each other during the second purification step. Reconstitution experiments using these two kinases demonstrate that the release kinase and cdc2 kinase work in concert to cause membrane release from chromatin. In phosphorylation experiments, protein targets that are substrates for the regulatory release kinase are identified on the membranes. These phosphorylated proteins ae candidates for regulated proteins mediating membrane-chromatin interaction. Finally, we find that membrane release activity can also be extracted from membranes by high salt treatment, indicating a possible dual localization of this activity.