Search PubMed⌕ Search

Biomedical subjects

F Sprenger

Publications and source records attributed to F Sprenger.

At least 19 recordsLinked to original sources
PubMed2005

High-resolution dissociative recombination of cold H3+and first evidence for nuclear spin effects.

The energy-resolved rate coefficient for the dissociative recombination (DR) of H(3)(+) with slow electrons has been measured by the storage-ring method using an ion beam produced from a radiofrequency multipole ion trap, employing buffer-gas cooling at 13 K. The electron energy spread of the merged-beams measurement is reduced to 500 microeV by using a cryogenic GaAs photocathode. This and a previous cold- measurement jointly confirm the capability of ion storage rings, with suitable ion sources, to store and investigate H(3)(+) in the two lowest, (J,G) = (1,1) and (1,0) rotational states prevailing also in cold interstellar matter. The use of para-H(2) in the ion source, expected to enhance para-H(3)(+) in the stored ion beam, is found to increase the DR rate coefficient at meV electron energies.

Journal Article↗
PubMed2001

Mitotic degradation of cyclin A is mediated by multiple and novel destruction signals.

Exit from mitosis requires Cdk1 inactivation, with the most prominent mechanism of Cdk1 inactivation being proteolysis of mitotic cyclins [1]. In higher eukaryotes this involves sequential destruction of A- and B-type cyclins. CycA is destroyed first, and CycA/Cdk1 inactivation is required for the metaphase-to-anaphase transition [2]. The degradation of CycA is delayed in response to DNA damage but is not prevented when the spindle checkpoint is activated [3, 4]. Cyclin destruction is thought to be mediated by a conserved motif, the destruction box (D box). Like B-type cyclins, A-type cyclins contain putative destruction box sequences in their N termini [5]. However, no detailed in vivo analysis of the sequence requirements for CycA destruction has been described so far. Here we tested several mutations in the CycA coding region for destruction in Drosophila embryos. We show that D box sequences are not essential for mitotic destruction of CycA. Destruction is mediated by at least three different elements that act in an overlapping fashion to mediate its mitotic degradation.

Amino Acid Sequence↗
PubMed2001

The cyclin-dependent kinase inhibitor Roughex is involved in mitotic exit in Drosophila.

BACKGROUND: Exit from mitosis is a tightly regulated event. This process has been studied in greatest detail in budding yeast, where several activities have been identified that cooperate to downregulate activity of the cyclin-dependent kinase (CDK) Cdc28 and force an exit from mitosis. Cdc28 is inactivated through proteolysis of B-type cyclins by the multisubunit ubiquitin ligase termed the anaphase promoting complex/cyclosome (APC/C) and inhibition by the cyclin-dependent kinase inhibitor (CKI) Sic1. In contrast, the only mechanism known to be essential for CDK inactivation during mitosis in higher eukaryotes is cyclin destruction. RESULTS: We now present evidence that the Drosophila CKI Roughex (Rux) contributes to exit from mitosis. Observations of fixed and living embryos show that metaphase is significantly longer in rux mutants than in wild-type embryos. In addition, Rux overexpression is sufficient to drive cells experimentally arrested in metaphase into interphase. Furthermore, rux mutant embryos are impaired in their ability to overcome a transient metaphase arrest induced by expression of a stable cyclin A. Rux has numerous functional similarities with Sic1. While these proteins share no sequence similarity, we show that Sic1 inhibits mitotic Cdk1-cyclin complexes from Drosophila in vitro and in vivo. CONCLUSIONS: Rux inhibits Cdk1-cyclin A kinase activity during metaphase, thereby contributing to exit from mitosis. To our knowledge, this is the first mitotic function ascribed to a CKI in a multicellular organism and indicates the existence of a novel regulatory mechanism for the metaphase to anaphase transition during development.

Animals↗
PubMed2000

Cyclins: growing pains for Drosophila.

Tradition holds that cyclin D is required for the initiation of cell division; recent studies in Drosophila, however, suggest that cyclin D has a separate function in governing growth.

Animals↗
PubMed2000

Homeodomain position 54 specifies transcriptional versus translational control by Bicoid.

Bicoid (BCD), the anterior determinant of Drosophila, controls embryonic gene expression by transcriptional activation and translational repression. Both functions require the homeodomain (HD), which recognizes DNA motifs at target gene enhancers and a specific sequence interval in the 3' untranslated region of caudal (cad) mRNA. Here we show that the BCD HD is a nucleic acid-binding unit. Its helix III contains an arginine-rich motif (ARM), similar to the RNA-binding domain of the HIV-1 protein REV, needed for both RNA and DNA recognition. Replacement of arginine 54, within this motif, alters the RNA but not the DNA binding properties of the HD. Corresponding BCD mutants fail to repress cad mRNA translation, whereas the transcriptional target genes are still activated.

Amino Acid Motifs↗
PubMed1999

Rux is a cyclin-dependent kinase inhibitor (CKI) specific for mitotic cyclin-Cdk complexes.

BACKGROUND: Roughex (Rux) is a cell-cycle regulator that contributes to the establishment and maintenance of the G1 state in the fruit fly Drosophila. Genetic data show that Rux inhibits the S-phase function of the cyclin A (CycA)-cyclin-dependent kinase 1 (Cdk1) complex; in addition, it can prevent the mitotic functions of CycA and CycB when overexpressed. Rux has no homology to known Cdk inhibitors (CKIs), and the molecular mechanism of Rux function is not known. RESULTS: Rux interacted with CycA and CycB in coprecipitation experiments. Expression of Rux caused nuclear translocation of CycA and CycB, and inhibited Cdk1 but not Cdk2 kinase activity. Cdk1 inhibition by Rux did not rely on inhibitory phosphorylation, disruption of cyclin-Cdk complex formation or changes in subcellular localization. Rux inhibited Cdk1 kinase in two ways: Rux prevented the activating phosphorylation on Cdk1 and also inhibited activated Cdk1 complexes. Surprisingly, Rux had a stimulating effect on CycA-Cdk1 activity when present in low concentrations. CONCLUSIONS: Rux fulfils all the criteria for a CKI. This is the first description in a multicellular organism of a CKI that specifically inhibits mitotic cyclin-Cdk complexes. This function of Rux is required for the G1 state and male meiosis and could also be involved in mitotic regulation, while the stimulating effect of Rux might assist in any S-phase function of CycA-Cdk1.

Animals↗
PubMed1998

Exit from mitosis in Drosophila syncytial embryos requires proteolysis and cyclin degradation, and is associated with localized dephosphorylation.

The cyclin proteolysis that accompanies the exit from mitosis in diverse systems appears to be essential for restoration of interphase. The early syncytial divisions of Drosophila embryos, however, occur without detectable oscillations in the total cyclin level or Cdk1 activity. Nonetheless, we found that injection of an established inhibitor of cyclin proteolysis, a cyclin B amino-terminal peptide, prevents exit from mitosis in syncytial embryos. Similarly, injection of a version of Drosophila cyclin B that is refractory to proteolysis results in mitotic arrest. We infer that proteolysis of cyclins is required for exit from syncytial mitoses. This inference can be reconciled with the failure to observe oscillations in total cyclin levels if only a small pool of cyclins is destroyed in each cycle. We find that antibody detection of histone H3 phosphorylation (PH3) acts as a reporter for Cdk1 activity. A gradient of PH3 along anaphase chromosomes suggests local Cdk1 inactivation near the spindle poles in syncytial embryos. This pattern of Cdk1 inactivation would be consistent with local cyclin destruction at centrosomes or kinetochores. The local loss of PH3 during anaphase is specific to the syncytial divisions and is not observed after cellularization. We suggest that exit from mitosis in syncytial cycles is modified to allow nuclear autonomy within a common cytoplasm.

Animals↗
PubMed1997

S-phase function of Drosophila cyclin A and its downregulation in G1 phase.

BACKGROUND: Cyclin E is the normal inducer of S phase in G1 cells of Drosophila embryos. Stable G1 quiescence requires the downregulation both of cyclin E and of other factors that can bypass the normal regulation of cell cycle progression. RESULTS: High-level expression of cyclin A triggered the G1/S transition in wild-type embryos and in mutant embryos lacking cyclin E. Three types of control downregulated this activity of cyclin A. First, cyclin destruction limited the accumulation of cyclin A protein in G1. Second, inhibitory phosphorylation of cdc2, the kinase partner of cyclin A, reduced the S-phase promoting activity of cyclin A in G1. Third, rux, a protein with unknown biochemical function, limited cyclin A function in G1. Overexpression of rux blocked S phase induction by coexpressed cyclin A and promoted the degradation of cyclin A. Rux also prevented a stable cyclin A mutant from inducing S phase, indicating that inhibition does not require cyclin destruction, and drove the nuclear localization of cyclin A. CONCLUSIONS: Cyclin A can drive the G1/S transition, but this function is suppressed by three types of control: cyclin A destruction, inhibitory phosphorylation of cdc2, and inhibition by rux. The partly redundant contributions of these three inhibitory mechanisms safeguard the stability of G1 quiescence until the induction of cyclin E. The action of rux during G1 resembles the action of inhibitors of mitotic kinases present during G1 in yeast, although no obvious sequence similarity exists.

Animals↗
PubMed1996

The activity of D-raf in torso signal transduction is altered by serine substitution, N-terminal deletion, and membrane targeting.

The Raf family of serine/threonine kinases are essential components in many receptor tyrosine kinase-mediated signal transduction pathways. Here, we analyze the function of D-raf in the Torso (Tor) pathway required to specify cellular fates at the embryonic poles. Using mutant embryos lacking endogenous D-raf protein, we show that D-raf's serine/threonine kinase activity is essential for its role in Tor signal transduction and that human Raf-1 will substitute for D-raf in this pathway. After Tor activation, D-raf becomes hyperphosphorylated. We identified two putative serine phosphorylation sites (S388 and S743) in SF9 cells and demonstrate that S743 or its phosphorylation is essential for D-raf function in embryos. Alanine substitution at S388, N-terminal truncation, or targeted membrane association permits transmission of the Torso signal by D-raf, but these D-raf molecules differ in their rescuing potential and relative biological activity. Membrane-targeted D-raftor4021 showed the highest level of activity, followed by alanine-substituted D-rafS388A and N-terminal-truncated D-raf delta 445. Since the activity profiles for these altered forms of D-raf are distinct, these findings indicate that each structural modification differentially affects the regulation and/or propagation of the Tor signal by these mutant D-raf proteins.

Alanine↗
PubMed1996

Variation in the number of activated torso receptors correlates with differential gene expression.

Activation of receptor tyrosine kinases triggers many developmental decisions, yet we do not understand how activation of a single receptor can be transduced into different cell responses. The torso pathway in Drosophila provides a model to address this issue since it generates more than one response in the embryo. The torso receptor tyrosine kinase is activated at the embryonic poles under the control of trunk, a protein with similarities to several types of extracellular growth factors. Activation of torso is responsible for the development of a variety of structures, whose appearance can be correlated with activation of at least two different genes along the terminal region. In this study we have analyzed mutations in torso and trunk that express low levels of the respective proteins. We show that different amounts of torso or trunk molecules correlate with the expression of different zygotic genes, implicating changes in the number of activated torso molecules as one of the mechanisms defining differential gene expression. We suggest that variation in the number of activated receptors at the cell surface is a general mechanism that leads to differential gene expression and thus the generation of different cell responses.

Animals↗
PubMed1995

Drosophila Wee1 kinase rescues fission yeast from mitotic catastrophe and phosphorylates Drosophila Cdc2 in vitro.

Cdc2 kinase activity is required for triggering entry into mitosis in all known eukaryotes. Elaborate mechanisms have evolved for regulating Cdc2 activity so that mitosis occurs in a timely manner, when preparations for its execution are complete. In Schizosaccharomyces pombe, Wee1 and a related Mik1 kinase are Cdc2-inhibitory kinases that are required for preventing premature activation of the mitotic program. To identify Cdc2-inhibitory kinases in Drosophila, we screened for cDNA clones that rescue S. pombe wee1- mik1- mutants from lethal mitotic catastrophe. One of the genes identified in this screen, Drosophila wee1 (Dwee1), encodes a new Wee1 homologue. Dwee1 kinase is closely related to human and Xenopus Wee1 homologues, and can inhibit Cdc2 activity by phosphorylating a critical tyrosine residue. Dwee1 mRNA is maternally provided to embryos, and is zygotically expressed during the postblastoderm divisions of embryogenesis. Expression remains high in the proliferating cells of the central nervous system well after cells in the rest of the embryo have ceased dividing. The loss of zygotically expressed Dwee1 does not lead to mitotic catastrophe during postblastoderm cycles 14 to 16. This result may indicate that maternally provided Dwee1 is sufficient for regulating Cdc2 during embryogenesis, or it may reflect the presence of a redundant Cdc2 inhibitory kinase, as in fission yeast.

Amino Acid Sequence↗
PubMed1994

Distinct molecular mechanism regulate cell cycle timing at successive stages of Drosophila embryogenesis.

The conserved regulators of cell cycle progression--Cyclins, Cdc2 kinase, and String phosphatase (Cdc25)--accommodate multiple modes of regulation during Drosophila embryogenesis. During cell cycles 2-7, Cdc2/Cyclin complexes are continuously present and show little fluctuation in abundance, phosphomodification, or activity. This suggests that cycling of the mitotic apparatus does not require cytoplasmic oscillations of known regulatory activities. During cycles 8-13 a progressive increase in the degradation of Cyclins at mitosis leads to increasing oscillations of Cdc2 kinase activity. Mutants deficient in cyclin mRNAs suffer cell cycle delays during this period, suggesting that Cyclin accumulation times these cycles. During interphase 14, programmed degradation of maternal String protein leads to inhibitory phosphorylation of Cdc2 and cell cycle arrest. Subsequently, mitoses 14-16 are triggered by pulses of zygotic string transcription.

Amino Acid Sequence↗
PubMed1993

Biochemical analysis of torso and D-raf during Drosophila embryogenesis: implications for terminal signal transduction.

Determination of anterior and posterior terminal structures of Drosophila embryos requires activation of two genes encoding putative protein kinases, torso and D-raf. In this study, we demonstrate that Torso has intrinsic tyrosine kinase activity and show that it is transiently tyrosine phosphorylated (activated) at syncytial blastoderm stages. Torso proteins causing a gain-of-function phenotype are constitutively tyrosine phosphorylated, while Torso proteins causing a loss-of-function phenotype lack tyrosine kinase activity. The D-raf gene product, which is required for Torso function, is identified as a 90-kDa protein with intrinsic serine/threonine kinase activity. D-Raf is expressed throughout embryogenesis; however, the phosphorylation state of the protein changes during development. In wild-type embryos, D-Raf is hyperphosphorylated at 1 to 2 h after egg laying, and thereafter only the most highly phosphorylated form is detected. Embryos lacking Torso activity, however, show significant reductions in D-Raf protein expression rather than major alterations in the protein's phosphorylation state. This report provides the first biochemical analysis of the terminal signal transduction pathway in Drosophila embryos.

3T3 Cells↗
PubMed1992

Torso receptor activity is regulated by a diffusible ligand produced at the extracellular terminal regions of the Drosophila egg.

torso encodes a receptor tyrosine kinase (torso) required for anterior and posterior terminal development of the Drosophila embryo. Injecting eggs with in vitro synthesized torso mRNAs revealed that torso activation is governed by an extracellular molecule, presumably the torso ligand, produced at terminal regions of the egg during early embryogenesis. In the absence of torso, the ligand shows no apparent localization, indicating that it is diffusible and normally bound by an excess of torso receptor at the egg poles. Mutant ligand-binding torso proteins can suppress telson formation in a dominant negative manner, suggesting that the ligand is limited in amount. Analysis of torso mutations indicates that torso functions as a tyrosine kinase and that gain-of-function mutations causing ligand-independent activation are located in the extracellular domain.

Animals↗
PubMed1992

Raf functions downstream of Ras1 in the Sevenless signal transduction pathway.

Specification of the R7 cell fate in the developing Drosophila eye requires activation of the Sevenless (Sev) receptor tyrosine kinase, located on the surface of the R7 precursor cell, by its interaction with the Boss protein, expressed on the surface of the neighbouring R8 cell. Four genes that participate in the intracellular transmission of this signal have so far been identified and molecularly characterized: Ras1, Sos, Gap1 and sina (refs 4-8). The Drosophila homologue of the mammalian Raf-1 serine/threonine kinase, which has been implicated in signal transduction pathways activated by many receptor tyrosine kinases (reviewed in refs 9 and 10), is encoded by the raf locus (also known as l(1)polehole, Draf-1 or Draf). Here we show that the Drosophila Raf serine/threonine kinase also plays a crucial role in the R7 pathway: the response to Sev activity is dependent on raf function, and a constitutively activated Raf protein can induce R7 cell development in the absence of sev function. We also present genetic evidence suggesting that Raf acts downstream of Ras1 and upstream of Sina in this signal transduction cascade.

Animals↗
PubMed1992

Prepattern in the developing Drosophila eye revealed by an activated torso--sevenless chimeric receptor.

Induction of the R7 photoreceptor cell fate in the developing eye of Drosophila depends on the activation of the sevenless receptor tyrosine kinase in the R7 precursor cell. The sevenless protein is expressed transiently in 8 of the 20 precursors of an ommatidium. Activation of the sevenless kinase in these eight cells indicates that six of them are competent to become R7 cells. To test the competence of all 20 ommatidial precursors in a temporally unrestricted manner we have used a constitutively activated sevenless kinase created by fusing the extracellular domain of a mutant torso protein, another Drosophila receptor tyrosine kinase, to the sevenless kinase. Our results show that competence to develop as neuronal cells in response to sevenless activity is spatially and temporally limited to the cells expressing sevenless. Therefore, the expression of sevenless marks a preexisting pattern of developmental potential in the disc epithelium.

Animals↗
PubMed1989

The Drosophila gene torso encodes a putative receptor tyrosine kinase.

The maternal gene torso, required for determination of anterior and posterior terminal structures in the Drosophila embryo, was cloned using P-element tagging. Genetic evidence suggests that the action of the gene product is spatially restricted to the terminal regions; the torso messenger RNA, however, is evenly distributed. Structural similarities of the predicted torso protein with growth-factor receptor tyrosine kinases suggest that the spatial restriction of torso activity results from a localized activation of the torso protein at the anterior and posterior egg pole.

Animals↗