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Science is international, but its funding and administration are usually a matter for governments of nation states. In Europe, this balkanization leads to problems; perhaps the worst is finding peer reviewers when, in a small country, applicants for support may have few peers. Of course, the definition of "peer" is not always clear, and it can be difficult to identify real experts in a field unless one is in it. That creates a problem, because supporting anything less than superior research wastes taxpayers' money. In a small country, identifying quality depends on the fair and accurate opinions of foreign experts.

Tipin and its interacting partner Tim1 (Timeless) form a complex at replication forks that plays an important role in the DNA damage checkpoint response. Here we identify Xenopus laevis Tipin as a substrate for cyclin E/cyclin-dependent kinases 2 that is phosphorylated in interphase and undergoes further phosphorylation upon entry into mitosis. During unperturbed DNA replication, the Tipin/Tim1 complex is bound to chromatin, and we were able to detect interactions between Tipin and the MCM helicase. Depletion of Tipin from Xenopus extracts did not significantly impair normal replication but substantially blocked the ability of stalled replication forks to recover after removal of a block imposed by aphidicolin. Tipin-depleted extracts also showed defects in the activation of Chk1 in response to aphidicolin, probably because of a failure to load the checkpoint mediator protein Claspin onto chromatin.

In vertebrates Cdk1 is required to initiate mitosis; however, any functionality of this kinase during S phase remains unclear. To investigate this, we generated chicken DT40 mutants, in which an analog-sensitive mutant cdk1 as replaces the endogenous Cdk1, allowing us to specifically inactivate Cdk1 using bulky ATP analogs. In cells that also lack Cdk2, we find that Cdk1 activity is essential for DNA replication initiation and centrosome duplication. The presence of a single Cdk2 allele renders S phase progression independent of Cdk1, which suggests a complete overlap of these kinases in S phase control. Moreover, we find that Cdk1 inhibition did not induce re-licensing of replication origins in G2 phase. Conversely, inhibition during mitosis of Cdk1 causes rapid activation of endoreplication, depending on proteolysis of the licensing inhibitor Geminin. This study demonstrates essential functions of Cdk1 in the control of S phase, and exemplifies a chemical genetics approach to target cyclin-dependent kinases in vertebrate cells.

The circadian clock, which governs metabolic and physiological rhythms in diverse organisms, shares common features with the cell cycle. Yet, these two oscillatory systems seem to be fully independent of each other. Recent studies now reveal that some essential regulatory elements are common to both the cell cycle and circadian clock.

Fertilization induces a transient increase in cytoplasmic Ca2+ concentration in animal eggs that releases them from cell cycle arrest in the second meiotic metaphase. In frog eggs, Ca2+ activates Ca2+/calmodulin-activated kinase, which inactivates cytostatic factor, allowing the anaphase-promoting factor to turn on and ubiquitinate cyclins and securin, which returns the cell cycle to interphase. Here we show that the calcium-activated protein phosphatase calcineurin is also important in this process. Calcineurin is transiently activated after adding Ca2+ to egg extracts, and inhibitors of calcineurin such as cyclosporin A (ref. 8) delay the destruction of cyclins, the global dephosphorylation of M-phase-specific phosphoproteins and the re-formation of a fully functional nuclear envelope. We found that a second wave of phosphatase activity directed at mitotic phosphoproteins appears after the spike of calcineurin activity. This activity disappeared the next time the extract entered M phase and reappeared at the end of mitosis. We surmise that inhibition of this second phosphatase activity is important in allowing cells to enter mitosis, and, conversely, that its activation is required for a timely return to interphase. Calcineurin is required to break the deep cell cycle arrest imposed by the Mos-MAP (mitogen-activated protein) kinase pathway, and we show that Fizzy/Cdc20, a key regulator of the anaphase-promoting factor, is an excellent substrate for this phosphatase.

Clinical guidance on the management of pain in elderly patients is presented. The way in which pain presents in elderly people and how it differs form other segments of the population are discussed.

Degradation of specific protein substrates by the anaphase-promoting complex/cyclosome (APC) is critical for mitotic exit. We have identified the protein Xenopus nuclear factor 7 (Xnf7) as a novel APC inhibitor able to regulate the timing of exit from mitosis. Immunodepletion of Xnf7 from Xenopus laevis egg extracts accelerated the degradation of APC substrates cyclin B1, cyclin B2, and securin upon release from cytostatic factor arrest, whereas excess Xnf7 inhibited APC activity. Interestingly, Xnf7 exhibited intrinsic ubiquitin ligase activity, and this activity was required for APC inhibition. Unlike other reported APC inhibitors, Xnf7 did not associate with Cdc20, but rather bound directly to core subunits of the APC. Furthermore, Xnf7 was required for spindle assembly checkpoint function in egg extracts. These data suggest that Xnf7 is an APC inhibitor able to link spindle status to the APC through direct association with APC core components.

This paper describes the application of two novel screening technologies, i.e. Domain Scan (24- and 30-mer peptides) and Matrix Scan (24-mer peptides) technology, in the mapping of a discontinuous epitope on FSH-beta for a series of 20 monoclonal antibodies. 11 out of 20 mAb's, mapping of which was not successful by conventional Pepscan technology (12-mer peptides), showed selective binding to peptide-constructs corresponding to the beta3-loop of FSH in the Domain and/or Matrix Scan. Systematic replacement analysis studies with peptide-construct 57VYETVRVPGCAC-SAc-ADSLYTYPVATQ81 revealed that for most mAb's the amino acids R62, A70, D71, and L73 form the core of the epitope. A Domain Scan performed in the C-O format showed highly selective binding for mAb's 1 and 2 with only three beta1-beta3 peptide-constructs covering the residues 60TVRVPGCAHHADSLY74 in combination with 10IAIEKEECRFAI21, while for mAb 10 binding was observed with peptide-constructs containing the C-terminal residues 97RGLGPSYCSFGEMKE114 in combination with the residues 10IAIEKEECRFAI21. A Matrix Scan of mAb 17 showed that peptides from four different regions on FSH (1st strand beta3-loop, alpha 1-loop, long alpha2-loop, det. loop) showed enhanced binding in combination with several 70ADSL73-containing peptides. BIACORE measurements with mAb's 1, 2, 13, and 17 using a set of 21 different peptide(-construct)s partially confirmed the Domain and Matrix Scan screening results. Only 24- and 33-mer peptides covering both the 1st and 2nd strand of the beta3-loop showed measurable binding. Cyclic beta3-loop peptide mimics were found to bind significantly stronger (Kd approximately 5 microM) than the lineair analogues, in agreement with the fact that the discontinuous epitope is part of a loop structure. Coupling of the lineair beta1-peptide 1oIAIEKEECRFAI21 to the linear beta3-peptide *52TFKELVYETVRVPGCAHHADSLYTYPVATQAH83# via disulfide bond formation showed a 2-3 fold increase in Kd, thus conforming participation of the beta 1-loop in antibody binding for these mAb's.

International audience; Oocyte maturation and early development have been intensively studied for well over 100 years. The earliest theory proposed that after fertilisation and during cell division determinants were unequally distributed to control cell fate; experimental proof came from using frog eggs (Roux, 1888). After understanding the contribution of the nucleus and the chromosomes into cell cycle progression using sea urchin eggs (Boveri, 1902), it was the discovery of the cytoplasm contribution to the G2/M transition that led the cell cycle community in search of the "mitosis-inducing factor", MPF. Yoshio Masui was the first to experimentally demonstrate that few nanoliters of cytoplasm taken from a metaphase-arrested oocyte and microinjected in a G2-arrested oocyte was able to trigger the G2 to metaphase transition (Masui and Markert, 1971). Although the way to identify the mitotic factor seemed obvious, it proved very hard and was not purified until 1988 (Lohka et al, 1988), then work from a variety of organisms including Xenopus, starfish, clams, sea urchins and yeast converged on the identification of MPF as a complex of cdc2 and cyclin B (Dunphy et al, 1988; Gautier et al., 1988; Draetta et al., 1889; Meijer et al., 1989; Labbé et al., 1989; Gautier et al., 1990). Since then, the oocyte and egg extracts developed by Lohka and Masui have often been used to study cell cycle events such as nuclear envelop formation, chromatin condensation, DNA replication, repair, and recombination, Golgi formation, microtubule dynamics, spindle assembly, chromosome segregation as well as cell cycle controls.

Two multiprotein E3 (ubiquitin-protein ligase) ubiquitin ligases, the SCF (Skp1-Cullin-1-F-box) and the APC/C (anaphase promoting complex/cyclosome), are vital in ensuring the temporal order of the cell cycle. Particularly, timely destruction of cyclins via these two E3s is essential for down-regulation of cyclin-dependent kinase. In general, G(1) and S phase cyclins are ubiquitylated by the SCF, whereas ubiquitylation of mitotic cyclins is catalyzed by the APC/C. Here we show that fission yeast S phase cyclin Cig2 is ubiquitylated and degraded via both the SCF and the APC/C. Cig2 instability during G(2) and M phase is dependent upon the SCF complex, whereas the APC/C is responsible for Cig2 destruction during anaphase and G(1), thereby ensuring a spike pattern of Cig2 levels, peaking only at S phase. Two F-box/WD proteins Pop1 and Pop2, homologues of budding yeast Cdc4 and human Fbw7, are responsible for Cig2 instability. Pop1 binds Cig2 in vivo. An in vitro binding assay shows that an internal 93 amino acid residues comprising a part of the cyclin box are necessary and sufficient for this binding. Cig2 phosphorylation is also required for interaction with Pop1. We previously showed that transcriptional oscillation of cig2(+) requires Pop1 and Pop2 function. SCF(Pop1/Pop2) therefore regulates Cig2 levels in a dual manner, transcriptionally and post-translationally. Our results also highlight a collaborative action of the APC/C and the SCF toward the common substrate Cig2. This type of composite degradation control may be more general as the regulatory mechanism in other complex systems.

This article reports on the development of an Interprofessional Capability Framework that articulates the learning outcomes that students need to achieve and continue to develop in order to become capable interprofessional workers. Although there tends to be general agreement around the subject matter to be included under the rubric of interprofessional learning, little information is available regarding the learning outcomes students need to achieve in order to become effective interprofessional workers. The Interprofessional Capability Framework defines capabilities that underpin interprofessional working and are relevant to all health and social care professions. The categories of the Framework were generated utilising grounded theory strategies in the analysis of the Quality Assurance Agency benchmark statements that inform the undergraduate curricula of all health and social care courses in the United Kingdom. This resulted in the conceptualisation of four key domains in which capabilities and learning levels have been articulated: Knowledge in Practice, Ethical Practice, Interprofessional Working and Reflection (learning).

How to Win the Nobel Prize—An Unexpected Life in Science J. Michael Bishop Harvard University Press, London, UK 320 pages, $18.50 ISBN 0 674 00880 4 ![][1] I first met J. Michael Bishop at a Gordon Conference in the late 1970s, just after Ray Erikson had discovered that Src was a protein kinase, and before Tony Hunter had realized that it transferred phosphate to tyrosine residues. It was an unnerving experience, because although I had had some experience of protein phosphorylation and protein kinases, whereas Mike had little or none, he had read far, wide and deep, and hadn‘t missed a trick. I felt myself to be in the presence of a superior being. I had a similar experience years later with Bishop's colleague Harold Varmus, this time in connection with protein synthesis. Together, Mike and Harold made a formidable team, as was confirmed in 1989 when they were jointly awarded the Nobel Prize for their discovery of the cellular origin of retroviral oncogenes. Years after our first meeting, I found myself serving under Mike on the Scientific Advisory Board of the Research Institute of Molecular Pathology in Vienna. Once, while we were chatting in a room in the Opera House, he recalled his first inkling of his Nobel Prize, which had come in that very room. “Where are you going to be in the second week … [1]: /embed/graphic-1.gif

Cyclin-dependent kinase (Cdk)2/cyclin E is imported into nuclei assembled in Xenopus egg extracts by a pathway that requires importin-α and -β. Here, we identify a basic nuclear localization sequence (NLS) in the N-terminus ofXenopus cyclin E. Mutation of the NLS eliminated nuclear accumulation of both cyclin E and Cdk2, and such versions of cyclin E were unable to trigger DNA replication. Addition of a heterologous NLS from SV40 large T antigen restored both nuclear targeting of Cdk2/cyclin E and DNA replication. We present evidence indicating that Cdk2/cyclin E complexes must become highly concentrated within nuclei to support replication and find that cyclin A can trigger replication at much lower intranuclear concentrations. We confirmed that depletion of endogenous cyclin E increases the concentration of cyclin B necessary to promote entry into mitosis. In contrast to its inability to promote DNA replication, cyclin E lacking its NLS was able to cooperate with cyclin B in promoting mitotic entry.

The idea that Cdc2 and cyclins play a key role in the control of the G2/M transition of the cell cycle came largely from genetic analysis of fission yeast and physiological studies of clam, frog, sea urchin and starfish eggs and oocytes. However, it took a long time to realise that Cdc2 and cyclins form a stoichiometric complex and that a cyclin subunit is necessary for the Cdc2 subunit to gain its protein kinase activity. Cyclins were first recognized as proteins whose abundance oscillates during the early cell cycles of marine invertebrate eggs and their connection with MPF (maturation-promoting factor), the entity defined in frog and starfish oocytes whose activity controls entry into M phase, was far from clear at first. Indeed, it was a long time before MPF was shown to be a protein kinase,and direct proof that MPF is a heterodimer comprising one molecule of cyclin and one molecule of Cdc2 was finally obtained only when the Cdc2-associated component of purified starfish MPF was sequenced and found to be cyclin B. When this fundamental discovery was confirmed in vertebrates and mammalian members of the Cdc2 family were also shown to bind cyclins, Cdc2 became Cdk1,the first cyclin-dependent protein kinase.

We describe a novel set of oscillation mechanisms for the fission yeast S phase cyclin Cig2, which contains an authentic destruction box and is destroyed at anaphase via the APC/cyclosome (APC/C). Unlike the mitotic cyclin Cdc13, however, Cig2 mRNA and protein peak at the G1/S boundary and decline to low levels in G2 and M phases. We show here that SCF(Pop1, Pop2) plays a role in transcriptional periodicity, as pop mutations result in constitutive cig2(+) transcripts. The instability of Cig2 during G2 and M is independent of either the APC/C or Pop1/Pop2, but requires Skp1, a core component of SCF. These data indicate that the APC/C and SCF control Cig2 levels differentially at different stages of the cell cycle.

Recent drilling activity in the War-Wink area of west Texas has established a new play in submarine channel and levee/overbank sandstones of the middle and lower Cherry Canyon Formation, Delaware Mountain Group (Guadalupian). Previous exploration and development, spanning the 1960s, 1970s, and early 1980s, had focused on the upper part of the formation. The new play, located a few miles west of the Central Basin platform, suggests the existence of multiple channel trends not yet explored in the Delaware basin. Entrapment in the War-Wink area is related to both structural and stratigraphic factors. Lower Cherry Canyon reservoirs have porosities ranging from 18 to 24% and permeabilities ranging from 10 to 60 md and thus are higher in quality than reservoirs of the underlying Brushy Canyon Formation. Core data indicate that permeabilities increase rapidly above a threshold porosity value of about 18%. Lower Cherry Canyon wells are completed by fracture stimulation in one or two main reservoir zones, with several secondary zones commonly behind pipe. Wells usually flow at rates of 100-200 bbl oil per day for up to 1 yr before being put on pump. Main producing zones are capable of recoveries ranging from 50,000 to 120,000 bbl. Attempts at using horizontal drilling in place of fracture stimulation have proved highly successful in certain cases. Use of this approach for Cherry Canyon development, guided by good geologic control, may provide an excellent technique applicable to many portions of the Delaware basin.

To the Editor, Cyclin-dependent kinases (CDKs) are protein kinases involved in critical cellular processes, such as cell cycle or transcription, whose activity requires association with specific cyclin subunits. Based on sequence similarity, the human genome contains 21 genes encoding CDKs and five additional genes encoding a more distant group of proteins known as CDK-like (CDKL) kinases. The current nomenclature for CDK proteins includes 11 classical CDKs (CDK1–11), two newly proposed family members (CDK12 and 13) and additional proteins whose names are based on the presence of a cyclin-binding element (PFTAIRE and PCTAIRE proteins) or simply based on a sequence relationship with the original CDKs, such as CDC2-like kinases (CDC2L) or cell cycle-related kinases (CCRK)

Ever since it emerged that cyclin-dependent protein kinases catalyzed cell cycle transitions and with cancer seen as "a disease of the cell cycle," people have pursued the aim of testing kinase inhibitors as anti-cancer drugs. Quite early on, Laurent Meijer and his colleagues discovered roscovitine as a potent inhibitor of Cdk1 and the compound went into clinical trials (as CYC202 or Seliciclib) whose outcomes are awaited. It was never clear to me that cancer was really a disease of the cell cycle (strictly speaking--considering that cancer cells have no trouble dividing), or how inhibiting cell cycle progression could reveal a window of therapeutic advantage between normal and neoplastic cells. Everyone knows what happens if you permanently block cell division in humans: they die. Yet, at the same time as harboring doubts about the rationale for using anti-Cdk drugs for cancer therapy, I would also be the first to admit that our understanding of cell cycle control is so far from complete, that, given the relative ease of developing specific protein kinase inhibitors, it is not a bad idea to try and see if they have selective effects on tumors. You never know.

The location of origins of DNA replication within the Saccharomyces cerevisiae genome is primarily determined by the origin recognition complex (ORC) interacting with specific DNA sequences. The analogous situation in vertebrate cells is far less clear, although ORC subunits have been identified in several vertebrate organisms including Xenopus laevis. Monoclonal antibodies were raised against Xenopus Orc1p and used for single-step immunoaffinity purification of the entire ORC from an egg extract. Six polypeptides ( approximately 110, 68, 64, 48, 43, and 27 kDa) copurified with Xenopus Orc1p. Protein sequencing also showed the 64-kDa protein to be the previously identified Xenopus Orc2p. Microsequencing of the 43- and 48-kDa proteins that copurified with Orc1p and Orc2p led to their identification as the Orc4p and Orc5p subunits, respectively. Peptide sequences from the 43-kDa protein also allowed the isolation of cDNAs encoding the Xenopus, mouse, and human ORC4 subunits. Human ORC5 was also cloned; its sequence displayed extensive homology to both Drosophila and yeast ORC5. Surprisingly, comparison of the amino acid sequences of Orc1p, Orc4p, and Orc5p suggests that they are structurally related to each other and to the replication initiation protein, Cdc6p. Finally, we present the sequence of the putative Xenopus and human Orc3p.

Background: Cyclins play a regulatory role in cell cycle progression, associated with cyclin-dependent kinases. We have investigated the structure–function relationships of cyclin A, mainly using Xenopus egg extracts in vitro. To further analyse the function and structure of cyclin A in vivo, we expressed Xenopus cyclin A1 in the budding yeast Saccharomyces cerevisiae. Results: We herein show that vertebrate cyclin A1 can associate with endogenous Cdc28 to form histone H1 kinase. The growth of the yeast was inhibited by the expression of indestructible cyclin A1, but not by a non-Cdk binding cyclin A1 mutant. The induction of cyclin A1 expression in yeast caused cell cycle arrest with an abnormal distribution of nuclear DNA to the daughter bud. Suppressors of the cyclin A1-mediated growth arrest were identified as new alleles of the cdc28 mutation that reduced the binding of cyclin A1 and possessed different affinities for the cyclin–Cdc28 complexes. The temperature-sensitivity of the cdc28 mutation was thus preferentially suppressed by the endogenous cyclins CLN2 and CLB2. Conclusions: These results suggest that the Cdc28 protein kinase activity mediated by vertebrate cyclin A1 may be involved in the process of nuclear movement in the yeast, and thereby affect the dependence of the M phase on the completion of the S phase through a preferential binding affinity of the cyclin–Cdc28 complex.

A cell-free system derived from Xenopus eggs was used to identify the 'destruction box' of the Schizosaccharomyces pombe B-type cyclin, Cdc13, as residues 59-67: RHALDDVSN. Expression of indestructible Cdc13 from a regulated promoter in S.pombe blocked cells in anaphase and inhibited septation, showing that destruction of Cdc13 is necessary for exit from mitosis, but not for sister chromatid separation. In contrast, strong expression of a polypeptide comprising the N-terminal 70 residues of Cdc13, which acts as a competitive inhibitor of destruction box-mediated proteolysis, inhibited both sister chromatid separation and the destruction of Cdc13, whereas an equivalent construct with a mutated destruction box did not. Appropriately timed expression of this N-terminal fragment of Cdc13 overcame the G1 arrest seen in cdc10 mutant strains, suggesting that proteins required for the initiation of S phase are subject to destruction by the same proteolytic machinery as cyclin.

We have studied how the cell cycle-specific oscillations of mitotic B-type cyclins are generated in mouse fibroblasts. A reporter enzyme comprising the N-terminus of a B-type cyclin fused to bacterial chloramphenicol acetyl transferase (CAT) was degraded at the end of mitosis like endogenous cyclins. Point mutations in the destruction box of this construct completely abolished its mitotic instability. When the destructible reporter was driven by the cyclin B2 promoter, CAT activity mimicked the oscillations in the level of the endogenous cyclin B2. These oscillations were largely conserved when the reporter was transcribed constitutively from the SV40 promoter. Pulse-chase experiments or addition of the proteasome inhibitors lactacystin and ALLN showed that cyclin synthesis continued after the end of mitosis. The destruction box-specific degradation of cyclins normally ceases at the onset of S phase, and is active in fibroblasts arrested in G0 and in differentiated C2 myoblasts. We were able to reproduce this proteolysis in vitro in extracts of synchronized cells. Extracts of G1 cells degraded cyclin B1 whereas p27Kip1 was stable, in contrast, cyclin B1 remained stable and p27Kip1 was degraded in extracts of S phase cells.

Dog thyroid epithelial cells in primary culture constitute a model of positive control of DNA synthesis initiation and G0-S prereplicative phase progression by cyclic AMP as a second messenger for TSH. In tis early steps, this mitogenic control is quite distinct from cyclic AMP-independent mitogenic cascades elicited by growth factors. We demonstrate here that TSH (cyclic AMP) and EGF+serum (cyclic AMP-independent) stimulations cooperate and finally converge on proteins that control the cell cycle machinery. This convergence included a common induction of the expression of cyclin A and p34cdc2, and to a lesser extent of p33/38cdk2, which was already expressed in quiescent thyroid cells, and common changes of cdc2 and CDK2 phosphorylations as evidenced by electrophoretic mobility shifts. Kinetic differences in these processes after stimulation by TSH or EGF+serum or by these factors in combination correlated with differences in cell cycle kinetics. Moreover, an immunofluorescence analysis of these proteins using the double labeling of PCNA as a marker of each cell cycle phase shows: (1) a previously undescribed nuclear translocation of CDK2 before S phase initiation; (2) a sudden increase of cdc2 nuclear immunoreactivity at G2/mitosis transition. These data support the roles of CDK2 and cdc2 at G1/S and G2/mitosis transitions, respectively. (3) We were unable to demonstrate in individual cells a strict association between the nuclear appearance of cyclin A and G1/S transition, and an association of cyclin A and CDK2 with PCNA-stained DNA replication sites. On the other hand, the lengthening of G2 phase in the TSH/cyclic AMP-dependent thyroid cell cycle was associated with a stabilization of Tyr15 inhibitory phosphorylation of cdc2 and an especially high nuclear concentration of cyclin A and CDK2. We hypothesize that high nuclear accumulation of cyclin A and CDK2 during G2 phase could be causative in the cyclic AMP-dependent delay of mitosis onset.

The meiotic maturation of Xenopus oocytes triggered by progesterone requires new protein synthesis to activate both maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAP kinase). Injection of mRNA encoding mutant p34cdc2 (K33R) that can bind cyclins but lacks protein kinase activity strongly inhibited progesterone-induced activation of both MPF and MAP kinase in Xenopus oocytes. Similar results were obtained by injection of GST-p34cdc2 K33R protein or by injection of a monoclonal antibody (A17) against p34cdc2 that blocks its activation by cyclins. Both the dominant-negative p34cdc2 and monoclonal antibody A17 blocked the accumulation of p39mos and activation of MAP kinase in response to progesterone, as well as blocking the appearance of MPF, although they did not inhibit the translation of p39mos mRNA. These results suggest that: (i) activation of free p34cdc2 by newly made proteins, probably cyclin(s), is normally required for the activation of both MPF and MAP kinase by progesterone in Xenopus oocytes; (ii) the activation of translation of cyclin mRNA normally precedes, and does not require either MPF or MAP kinase activity; and (iii) de novo synthesis and accumulation of p39mos is probably both necessary and sufficient for the activation of MAP kinase in response to progesterone.

Background: Cip1 is a 21 kD protein that interacts with and inhibits cyclin-dependent kinases (cdks). Expression of Cip1 is induced by the tumour suppressor p53, and tumour cells have greatly reduced levels of Cip1. As cdks are required for normal progression through the cell cycle, their inhibition by Cip1 may mediate the ability of p53 to block cell proliferation. Cip1 has also been shown to inhibit the DNA polymerase δ auxiliary factor PCNA (proliferating cell nuclear antigen), which is required for replication-fork elongation, and this could be an alternative mechanism by which p53-induced Cip1 blocks cell proliferation. Results We have investigated the effect of Cip1 protein on chromosomal DNA replication, using cell-free extracts of Xenopus eggs that initiate and complete chromosome replication under normal cell-cycle control. Cip1 protein strongly inhibited an early stage of DNA replication in this system, and this inhibition was not complemented by extracts that had been affinity-depleted of cdks. In contrast, Cip1 did not inhibit the elongation of replication forks that had accumulated in the presence of aphidicolin. Cip1 inhibition of DNA replication was fully rescued by addition of cyclins A or E, but not cyclin B, cdk2 or PCNA. Conclusion Our results suggest that Cip1 specifically blocks the initiation of DNA replication by inhibition of a cyclin-dependent kinase (cdk2), but has no major effect on the elongation of preassembled replication forks. The ability of cyclin A or cyclin E to rescue the Cip1 inhibition suggests that these cyclins may play a direct role in the initiation of replication in the Xenopus system.

Phosphorylation of the neurofilament proteins of high and medium relative molecular mass, as well as of the Alzheimer's tau protein, is thought to be catalysed by a protein kinase with Cdc2-like substrate specificity. We have purified a novel Cdc2-like kinase from bovine brain capable of phosphorylating both the neurofilament proteins and tau. The purified enzyme is a heterodimer of cyclin-dependent kinase 5 (Cdk5) and a novel regulatory subunit, p25 (ref. 8). When overexpressed and purified from Escherichia coli, p25 can activate Cdk5 in vitro. Unlike Cdk5, which is ubiquitously expressed in human tissue, the p25 transcript is expressed only in brain. A full-length complementary DNA clone showed that p25 is a truncated form of a larger protein precursor, p35, which seems to be the predominant form of the protein in crude brain extract. Cdk5/p35 is the first example of a Cdc2-like kinase with neuronal function.

Early in the development of many animals, before transcription begins, any change in the pattern of protein synthesis is attributable to a change in the translational activity or stability of an mRNA in the egg. As a result, translational control is critical for a variety of developmental decisions, including axis formation in Drosophila and sex determination in Caenorhabditis elegans. Previous work demonstrated that increases in poly(A) length can activate translation, whereas removal of poly(A) can prevent it. In this report we focus on the control of c-mos and cyclin A1, B1, and B2 mRNAs during meiotic maturation and after fertilization of frog eggs. We show that addition and removal of poly(A) from these mRNAs is extensively regulated: The time at which each mRNA receives or loses poly(A), as well as the number of adenosines it gains or loses, differ substantially. Signals in the 3'-untranslated region (UTR) of each mRNA are sufficient to reconstitute both the temporal and quantitative control of poly(A) addition: Chimeric mRNAs in which a luciferase-coding region is joined to the 3' UTRs of cyclin A1, cyclin B1, or c-mos mRNA, receive poly(A) of the same length and at the same time as do the endogenous mRNAs. Moreover, each 3' UTR also regulates translation of the chimeric mRNAs, determining when and how much translation of the luciferase reporter is stimulated during maturation. The magnitude of stimulation in luciferase activity varies from 5- to 100-fold, depending on the 3' UTR. Translational stimulation by each 3' UTR requires poly(A) lengthening, as it is prevented by mutations that prevent that process. These results suggest that the 3' UTRs of cyclin and c-mos mRNAs control not only whether or not an mRNA is turned on during maturation, but when that activation occurs and to what extent. Translational control of c-mos mRNA, which may be achieved through regulation of poly(A) length, may be critical in the activation of maturation, and in the onset of cleavage divisions. Our findings, as well as those of others, suggest that even quite complex patterns of translational activation in the early embryo can be attained through the differential control of a common mechanism.

The specific and rapid destruction of cyclins A and B during mitosis is their most remarkable property. A short peptide motif of approximately 10 amino acids near the N-terminus, known as the destruction box, is absolutely required for programmed proteolysis. In this paper we show that although the destruction box is necessary for the degradation of cyclin A, it is not sufficient. Mutant versions of cyclin A that cannot form complexes with p34cdc2 are stable, which we interpret to mean that this cyclin must bind to p34cdc2 in order to undergo programmed proteolysis. Thus, N-terminal fragments of cyclin A containing little more than the destruction box and its surroundings are indestructible. p34cdc2 binding also appears to be required for the destruction of cyclin B2. In contrast, cyclin B1 does not require p34cdc2 binding for specific proteolysis. The systems for the proteolysis of cyclins A, B1 and B2 thus appear to show important differences in the way they recognize their substrates.

Fv fragments whose genes have been cloned using common PCR primers carry identical peptide motifs at their termini. We have raised antibodies against the C-terminal motif of the VH chain GQGTTVTVSS and evaluated their utility as reagents for the assay and purification of Fvs in the fermenter culture. Three different Fvs were included in the investigation. We found that the motif was exposed and available for capture when Fv fragments were blotted onto nitrocellulose paper or adsorbed directly onto microtiter plates. In contrast, the motif was either partially or totally obscured when the Fv was complexed with immobilised antigen or when free in solution. This reactivity profile enabled us to develop a general-purpose assay for Fv protein, but not a general-purpose assay for monitoring active Fv. The apparent inaccessibility of the C-terminus of VH conflicts with currently held views on the three-dimensional structure of these molecules.

The binding of cyclin A to p34cdc2 and p32cdk2 and the protein kinase activity of the complexes has been measured by cell-free translation of the corresponding mRNA in extracts of frog eggs, followed by immunoprecipitation. A variety of mutant cyclin A molecules have been constructed and tested in this assay. Small deletions and point mutations of highly conserved residues in the 100-residue "cyclin box" abolish binding and activation of both p34cdc2 and p32cdk2. By contrast, large deletions at the N-terminus have no effect on kinase binding and activation, until they remove residues beyond 161, where the first conserved amino acids are found in all known examples of cyclin A. At the C-terminus, removal of 14 or more amino acids abolishes activity. We also demonstrate that deletion of, or point mutations, in the cyclin A homologue of the 10-residue "destruction box," previously described in cyclin B (Glotzer et al., 1991) abolish cyclin proteolysis at the transition from M-phase to interphase.

A system is described in which transcription of cDNA clones by bacteriophage T7 RNA polymerase is coupled to translation in the micrococcal nuclease treated rabbit reticulocyte lysate in a single reaction of coupled transcription-translation. The monovalent and divalent cation requirements for translation are dominant for optimum expression in this coupled system, so that transcription is relatively inefficient. Nevertheless, the use of appropriate DNA concentrations leads to the synthesis of sufficient RNA to saturate the protein synthesis capacity of the system. The fidelity and efficiency of expression in this coupled system are high, and the degree of purification of the plasmid DNA is relatively uncritical. The system therefore offers very considerable advantages for rapid screening of 'mini-preparations' of cDNA plasmid constructs for retention of the correct reading frame and expression of the desired protein product.

Fertilization of clam oocytes initiates a series of cell divisions, of which the first three--meiosis I, meiosis II, and the first mitotic division--are highly synchronous. After fertilization, protein synthesis is required for the successful completion of every division except meiosis I. When protein synthesis is inhibited, entry into meiosis I and the maintenance of M phase for the normal duration of meiosis occur normally, but the chromosomes fail to interact correctly with the spindle in meiosis II metaphase. By contrast, inhibition of protein synthesis immediately after completion of meiosis or mitosis stops cells entering the next mitosis. We describe the behavior of cyclins A and B in relation to these "points of no return." The cyclins are synthesized continuously and are rapidly destroyed shortly before the metaphase-anaphase transition of the mitotic cell cycles, with cyclin A being degraded in advance of cyclin B. Cyclin destruction normally occurs during a 5-min window in mitosis, but in the monopolar mitosis that occurs after parthenogenetic activation of clam oocytes, or when colchicine is added to fertilized eggs about to enter first mitosis, the destruction of cyclin B is strongly delayed, whereas proteolysis of cyclin A is maintained in an activated state for the duration of metaphase arrest. Under either of these abnormal conditions, inhibition of protein synthesis causes a premature return to interphase that correlates with the time when cyclin B disappears.

Richard Nixon was not my favourite President of the United States of America, but he declared his famous War on Cancer while I was a postdoctoral fellow at the Albert Einstein College of Medicine. I was then working in the Department of Medicine in the laboratory of Irving London, who was Head of that department. This was a remarkable privilege for a young scientist: to be a first‐hand witness of the emerging interface between medicine and molecular biology. By 1970, haematology had long been a molecular business and was a specialty in which—for a variety of simple practical reasons—the distance between basic and applied research was very short. But other specialties, of which perhaps virology would be a …

Progesterone induces fully grown, stage VI, Xenopus oocytes to pass through meiosis I and arrest in metaphase of meiosis II. Protein synthesis is required twice in this process: in order to activate maturation promoting factor (MPF) which induces meiosis I, and then again after the completion of meiosis I to reactivate MPF in order to induce meiosis II. We have used antisense oligonucleotides to destroy maternal stores of cyclin mRNAs, and demonstrate that new cyclin synthesis is not required for entry into either meiosis I or II. This finding is consistent with the demonstration that stage VI oocytes contain a store of B-type cyclin polypeptides (Kobayashi, H., J. Minshull, C. Ford, R. Golsteyn, R. Poon, and T. Hunt. 1991. J. Cell Biol. 114:755-765). Although approximately 70% of cyclin B2 is destroyed at first meiosis, the surviving fraction, together with a larger pool of surviving cyclin B1, must be sufficient to allow the reactivation of MPF and induce entry into second meiotic metaphase. Since stage VI oocytes do not contain any cyclin A, our results show that cyclin A is not required for meiosis in Xenopus. We discuss the possible nature of the proteins whose synthesis is required to induce meiosis I and II.

Clam oocytes display a striking difference in the pattern of protein synthesis on fertilization , which is maintained when cell-free extracts are assayed in the rabbit reticulocyte lysate. The mRNAs encoding ribonucleotide reductase and cyclin A (the major masked messages in the oocyte extracts) can be translational ly activated by gel filtration in 0.5 M KCl, presumably by removal of repressor protein(s). When synthetic RNAs corresponding to different segments of ribonucleotid e reductase and cyclin A mRNAs were added to oocyte extracts in 0.5 M KCl in a "competitive unmasking" assay, specific and complete unmasking of these mRNAs occurred on addition of antisense 3'-noncoding transcripts. The unmasking region in ribonucleotid e reductase mRNA maps to a region of 134 nucleotides centered one-third of the way down the 3'-noncoding region.

THE cell cycles of early Xenopus embryos consist of a rapid succession of alternating S and M phases1. These cycles are controlled by the activity of a protein kinase complex (cdc2 kinase) which contains two subunits. One subunit is encoded by the frog homologue of the fission yeast cdc2+ gene, p34cdc2 (ref. 2) and the other is a cyclin3. The concentration of cyclins follows a sawtooth oscillation because they accumulate in interphase and are destroyed abruptly during mitosis3. The association of cyclin and p34cdc2 (refs 4–7) is not sufficient for activation of cdc2 kinase, however; dephosphorylation of key tyrosine and threonine residues of p34cdc2 is necessary to turn on its kinase activity8–11. The activity of cdc2 kinase is thus regulated by a combination of translational and post-translational mechanisms. The loss of cdc2 kinase activity at the end of mitosis depends on the destruction of the cyclin subunits3,4,12,13. It has been suggested that this destruction is induced by cdc2 kinase itself, thereby providing a negative feedback loop to terminate mitosis14,15. Here we report direct experimental evidence for this idea by showing that cyclin proteolysis can be triggered by adding cdc2 kinase to a cell-free extract of interphase Xenopus eggs.

Highly purified maturation-promoting factor (MPF) from Xenopus eggs contains both cyclin B1 and cyclin B2 as shown by Western blotting and immunoprecipitation using Xenopus anti-B-type cyclin antibodies. Immunoprecipitates with these antibodies display the histone H1 kinase activity characteristic of MPF, for which exogenously added B1 and B2 cyclins are both substrates. Protein kinase activity against cyclin oscillates in maturing oocytes and activated eggs with the same kinetics as p34cdc2 kinase activity. These data indicate that B-type cyclin is the other component of MPF besides p34cdc2.

The pattern of protein synthesis changes soon after fertilization of clam oocytes. The most abundant of the mRNAs whose translation increases at this time encode ribonucleotide reductase and the A- and B-type cyclins. These mRNAs have been cloned and sequenced, yet their sequences do not show regions of similarity that could explain the masking mechanism. However, these mRNAs retain their 'masked' state in cell-free translation assays and their translation can be activated by gel filtration in high salt, which probably removes repressor proteins. A 'competitive unmasking' assay was used to identify the protein-binding regions of each mRNA. This involved adding short segments of antisense RNA that annealed to the mRNA and displaced the repressors. The unmasking regions in ribonucleotide reductase and cyclin A mRNAs revealed by this assay are 120-140 nt long and are located in the central portions of the 3' non-coding regions.