bluuebunny/crossref_metadata_2025_split huggingface dataset:
Localization, expression, evolutionary conservation, and structure of the 30,000 dalton actin bundling protein of Dictyostelium discoideum
Authors: Furukawa Ruth , Fechheimer Marcus 
Published: May 2005

AbstractThe Dictyostelium discoideum 30,000 dalton actin‐binding protein is an actin cross‐linking protein that organizes formation of parallel bundles of actin filaments in vitro, and is present in filopodia in living cells. This protein binds calcium directly and exhibits a decreased affinity for actin filaments in the presence of micromolar calcium. In this work, the existence of antigenic homologs of the 30,000 dalton protein in Physarum polycephalum, Schistosoma mansoni, Chara carolina, and Drosophila melanogaster is detected by use of affinity purified antibody and electrophoretic blotting methods. The expression of this protein during development of Dictyostelium is also analyzed, revealing a progressive 3‐fold decrease in the level of this protein in amoebae between the vegetative and slug stages. A highly ordered structure of bundles of actin and the 30,000 dalton protein formed in vitro is inferred from the presence of transverse striations on the bundles with a minor periodicity at 11.4 nm and a major periodicity at 33.9 nm. Finally, we propose a working model of the interaction of this actin cross‐linking protein with actin filaments to form bundles.
Heterodimeric capping proteins constitute a highly conserved group of actin‐binding proteins
Authors: Hartmann Herbert , Schleicher Michael , Noegel Angelika A. 
Published: May 2005

AbstractThe two subunits of the het‐erodimeric protein cop32/34, an actin‐binding protein, are encoded by separate single‐copy genes. We have established the genomic structure of both genes. A sequence comparison of cap32/34 with capZ from chicken skeletal muscle and two partially known sequences from Saccharomyces cerevisiae and Xenopus laevis show that heterodimeric capping proteins belong to a highly conserved group of actin‐binding proteins. This conclusion is supported by the cross‐reaction of polyclonal antibodies against cap32 and cap34 with proteins from lower and higher eukaryotes. In addition, a system is presented that allows the expression of truncated cap34 polypeptides under the control of the cap34 promoter.
Positive selection for Dictyostelium mutants lacking uridine monophosphate synthase activity based on resistance to 5‐fluoro‐orotic acid
Authors: Kalpaxis Dimitrios , Werner Herbert , Marcotte Emmanuell Boy , Jacquet Michel , Dingermann Theodor 
Published: May 2005

AbstractWhereas transformation ofDictyostelium discoideum now can be done routinely and reliably, there is increasing demand for a system that allows selection of cells that have gone through repeated transformation cycles. Such a system is presented here. Selection is based on resistance to 5‐fluoro‐orotic‐acid (5‐FOA). D. discoi‐deum is highly sensitive to this drug, and cells can survive only in the presence of 5‐FOA if they carry a defective UMP‐synthase gene. After transformation with a plasmid carrying a cloned version of the UMP‐synthase gene, 5‐FOA resistant cells are obtained at high frequency. Because 5‐FOA‐resistant cells depend on exogenously added uracil, these cells can be taken through a second round of transformation. A plasmid‐borne UMP‐synthase gene renders 5‐FOA‐resistant cells phenotypically ura +. Finally, cells can be further transformed using the standard G418 selection system.
Quantification of transformation efficiency using a new method for clonal growth and selection of axenic Dictyostelium cells
Authors: Knecht David A. , Jung Jinha , Matthews Lisa 
Published: May 2005

AbstractA new method for clonal growth of Dictyostlium axenic amoebae has been developed. Cells are plated in growth medium containing 1% ultra‐low gelling temperature agarose. Cells grow normally in the agarose and form colonies up to several millimeters in diameter. When the colonies have grown to a sufficient size, they begin multicellular development. Pseudoplasmodia are formed, migrate to the surface of the agar, and then undergo fruiting body formation. Cells can be removed from the soft agarose colonies with a toothpick or by picking spores from the fruiting bodies. This method should be useful for drug, auxotrophic, and temperature selections where clonal maintenance of axenic colonies is important.This method has been used in combination with a selection for resistance to G418 to isolate independent colonies following DNA‐mediated transformation. Several parameters in the calcium phosphate and electroporation transformation protocols have been optimized and the transformation frequency qualified. Independent transformed colonies are obtained at a frequency of 1 in 104 to 1 in 105 cells when integrating plasmids are introduced using calcium phosphate coprecipitation. The frequency is about tenfold higher when extrachromosomal shuttle vectors are introduced into cells.
Nonsense suppression in Dictyostelium discoideum
Authors: Dingermann Theodor , Reindl Norbert , Brechner Thomas , Werner Herbert , Nerke Käthe 
Published: May 2005

AbstractWe describe the generation of Dictyostelium discoideum cell lines that carry different suppressor †RNA genes. These genes were constructed by primer‐directed mutagenesis changing a †RNATrp(CCA) gene from D. discoideum to a †RNATrp(amber) gene and changing a †RNAGlu(UUC) gene from D. discoideum to a †RNAGlu(ochre) as well as a †RNAGlu(amber) gene. These genes were stably integrated into the D. discoideum genome together with a reporter gene. An actin 6::lacZ gene fusion carrying corresponding translational stop signals served as a reported. Active β‐galactosidase is expressed only in D. discoideum strains that contain, in addition to the reporter, a functional suppressor †RNA. Both amber suppressors are active in D. discoideum without interfering significantly with cell growth and development. We failed, however, to establish cell lines containing a functional †RNAGlu(ochre) suppressor. This may be due to the fact that nearly every message from D. discoideum known so far terminates with UAA. Therefore a †RNA capable of reading this termination codon may not be compatible with cell growth.
Analysis of developmentally expressed antigens in Polysphondylium pallidum
Authors: Vocke Cathy D. , Cox Edward C. 
Published: May 2005

AbstractA series of monoclonal antibodies were previously raised against developing Polysphondylium pallidum cells. In this work, six of these antibodies have been used as probes to identify and characterize antigens regulated during development. Soluble and membrane fractions of P. pallidum cells at six stages of development or three stages of cyclic adenosine monophosphate (cAMP)‐induced development were run in sodium dodecyl sulfate (SDS)‐polyacrylamide gels and subjected to Western blot analysis. Three of the monoclonals, anti‐Tp200, anti‐Tp423, and antiPg 101, stain sorogen tips. Tp423 and Tp200 are membrane‐associated antigens; both are stable to urea extraction, and Tp200 remains in the membrane after NaOH extraction. Tp423 is present in starved cells but is more prominent in sorogens and particularly in cAMP‐developed cells. In contrast, Tp200 is first detected in early to mid‐aggregation and is more abundant late in development. Pg101, which is expressed as a gradient with its highest concentration in tips, first appears in tight aggregates but is much more abundant in sorogens; unlike the Tp antigens, Pg101 is not greatly induced in cAMP‐developed cells. All three of these antigens undergo changes in apparent molecular weight at the tight aggregate or sorogen stage: The gel mobilities of Tp200 and Pg101 increase, whereas that of Tp423 decreases. In addition to the tip‐specific monoclonals, two monoclonals that stain all but the tips of sorogens have been used for analysis. One of these, anti‐3D 10Pnk stains most cells within secondary tips, whereas anti‐3D 10Dif does not. 3D 10Dif is membrane associated; it is present very early in development, increasing two‐ to threefold through the sorogen stage and diminishing in late cAMP‐developed cells. 3D 10Pnk is a mostly soluble species first detected in late streaming. Anti‐1c3, a sixth monoclonal, which stains nuclei uniformly throughout sorogens, is also developmentally expressed. 1c3 is mainly membrane associated and is expressed from late streaming through the sorogen stage.
Timing of the formation of the prestalk and prespore zones in Dictyostelium discoideum
Authors: Bonner J. T. , Feit I. N. , Selassie A. K. , Suthers H. B. 
Published: May 2005

AbstractVital dyes have been extensively used in many laboratories to distinguish the prestalk and prespore zones of a migrating slug in Dictyostelium discoideum. Here we present evidence that the two zones do not form immediately following aggregation, as is sometimes assumed, but only after 1–4.5 hr of migration. When the transition to the two‐toned state occurs, it changes very quickly, in a matter of approximately 10 min. Furthermore, it can be rapidly induced by submerging the slug in mineral oil. The time of appearance of the zones is correlated with previously reported changes in the distribution of cyclic AMP secretion and with changes in regulation properties along the axis of the slug.
Biochemical and genetic analysis of the biosynthesis, sorting, and secretion of Dictyostelium lysosomal enzymes
Authors: Cardelli James A. , Schatzle John , Bush John M. , Richardson Jan , Ebert David , Freeze Hudson 
Published: May 2005

AbstractDictyostelium discoideum is a useful system to study the biosynthesis of lysosomal enzymes because of the relative ease with which it can be manipulated genetically and biochemically. Previous studies have revealed that lysosomal enzymes are synthesized in vegetatively growing amoebae as glycosylated precursor polypeptides that are phosphorylated and sulfated on their N‐linked oligosaccharide side‐chains upon arrival in the Golgi complex. The precursor polypeptiaes are membrane associated until they are proteolytically processed and deposited as soluble mature enzymes in lysosomes. In this paper we review biochemical experiments designed to determine the roles of post‐translational modification, acidic pH compartments, and proteolytic processing in the transport and sorting of lysosomal enzymes. We also describe molecular genetic approaches that are being employed to study the biosynthesis of these enzymes. Mutants altered in the sorting and secretion of lysosomal enzymes are being analyzed biochemically, and we describe recent efforts to clone the genes coding for three lysosomal enzymes in order to better understand the molecular mechanisms involved in the targeting of these enzymes.
Biochemical and genetic analysis of an antigenic determinant found on N‐linked oligosaccharides in Dictyostelium
Authors: Freeze Hudson H. , Bush John M. , Cardelli James 
Published: May 2005

AbstractDictyostelium discoideum synthesizes many highly immunogenic carbohydrates of unknown structure and function. We have used monoclonal antibodies prepared against one of these called CA1 to investigate its structure and the consequences of its loss. CA1 is preferentially expressed on lysosomal enzymes as a specific arrangement of mannose‐6‐SO4 residues on N‐linked oligosaccharides. Mutant strains HL241 and HL243 do not express CA1, and synthesize a truncated lipid‐linked oligosaccharide (LLO) precursor that lacks the critical mannose residues needed for expression. The lesion appears to result from the loss of mannosyl transferase activity involved in LLO biosynthesis. The truncated LLO is poorly transferred to an artificial peptide acceptor in a cell‐free N‐glycosylation assay, and this appears to result from improper topological localization of the LLO or to a lower affinity of the LLO for the oligosaccharyl transferase. Although both mutants share these lesions, they are biochemically and genetically distinct. Only HL243 is lower in N‐glycosylation in intact cells, and this is not a result of an altered structure of the LLO. There are other differences between the strains. HL241 can form fruiting bodies at a slower rate than normal while HL243 cannot aggregate. Genetic analysis of defects shows that the CA1 lesion in HL241 is recessive, while the lesions in both CA1 and in development are dominant and co‐segregate in HL243 and are, therefore, likely to be in the same gene. Lysosomal enzyme targeting is normal but enzyme processing proceeds at a 2–3 fold slower rate in HL241 and HL243 compared to wild‐type. Strain HL244 does not express CA1 since it completely lacks protein sulfation, but lysosomal enzyme targeting and processing proceeds at a normal rate, showing that sulfate is not essential for these processes. Alterations in oligosaccharide structure can have individualized effects on the biosynthesis of lysosomal enzymes. The results presented here illustrate how this approach can be used to study both the structure and function of carbohydrate epitopes.
Identification and purification of the cell surface glycoprotein homologous to PsA in different species of cellular slime mold on the basis of a shared carbohydrate epitope
Authors: Gooley Andrew A. , Zhou‐Chou Ti , Bernstein R. L. , Smith Elizabeth , Williams Keith L. 
Published: May 2005

AbstractThe monoclonal antibodies MUD1 and MUD50 recognize peptide and carbohydrate epitopes, respectively, on the Dictyoste‐lium discoideum developmentally regulated cell surface glycoprotein PsA. PsA is a putative pre‐spore cell adhesion molecule during the slug stage and is anchored to the cell membrane via a phos‐phatidylinositol glycan. Here we report on the presence of a PsA‐like homolog in several Dictyo‐stelium species. The MUD1 epitope is mostly confined to the D. discoideum complex, and only one non‐D. discoideum strain reacted strongly with this antibody. By contrast, the mAb MUD50 recognises a homologous molecule in several Dictyostelium species that do not react with MUD1. With mAb MUD50 immunoaffinity chromatography, it was possible to purify and obtain an amino‐terminal sequence for the PsA homolog from the Dictvoste‐lium sp. strain ZA3A and from one strain of D. pur‐pureum.
Regulation of protein phosphorylation in Dictyostelium discoideum
Authors: Anschutz Alison , Um Hong‐Duck , Tao Young‐Ping , Klein Claudette 
Published: May 2005

AbstractWe have examined the phosphorylation of the cyclic adenosine 3′:5′ monophosphate (cAMP) cell surface chemotactic receptor and a 36 kDa membrane‐associated protein (p36) in Dictyostelium discoideum. The activity of CAR‐kinase, the enzyme responsible for the phosphorylation of the cAMP receptor, was studied in plasma membrane preparations. It was foud that, as in intact cells, the receptor was rapidly phosphorylated in membranes incubated with [γ32P] adenosine triphosphate (ATP) but only in the presence of cAMP. This phosphorylation was not observed in membranes prepared from cells which did not display significant cAMP binding activity. cAMP could induce receptor phosphorylation at low concentrations, while cyclic guanosine 3′:5′ monophosphate (cGMP) could elicit receptor phosphorylation only at high concentrations. Neither ConA, Ca2+, or guanine nucleotides had an effect on CAR‐kinase. It was also observed that 2‐deoxy cAMP but not dibutyryl cAMP induced receptor phosphorylation. The data suggest that the ligand occupied form of the cAMP receptor is required for CAR‐kinase activity. Although the receptor is rapidly dephosphorylated in vivo, we were unable to observe its dephosphorylation in vitro. In contrast, p36 was rapidly dephosphorylated. Also, unlike the cAMP receptor, the phosphorylation of p36 was found to be regulated by the addition of guanine nucleotides. Guanosine diphosphate (GDP) enhanced the phosphorylation while guanosine triphosphate (GTP) decreased the radiolabeling of p36 indicating that GTP can compete with ATP for the nucleotide triphosphate binding site of p36 kinase. This was verified using radiolabeled GTP as the phosphate donor. Competition experiments with GTPγS, ATP, GTP, CTP, and uridine triphosphate (UTP) indicated that the phosphate donor site of p36 kinase is relatively non‐sepcific. The mechanism(s) by which GDP functions to alter p36 phosphorylation and the physiological significance of this event are currently under investigation.
Dynamics and function of the inositolcycle in Dictyostelium discoideum
Authors: Bominaar Anthony A. , Van Der Kaay Jeroen , Van Haastert Peter J. M. 
Published: May 2005

AbstractThe inositolcycle in Dictyostelium discoideum was studied under several conditions both in vitro and in vivo. The results are compared with the inositolcycle as it is known from higher eukaryotes: although there is a strong resemblance both cycles are different at some essential points.
Control of cAMP‐induced gene expression by divergent signal transduction pathways
Authors: Peters Dorien J. M. , Cammans Mariska , Smit Steven , Spek Wouter , Van Lookeren Campagne Michiel M. , Schaap Pauline 
Published: May 2005

AbstractA compilation of literature data and recent experiments led to the following conclusions regarding cyclic adenosine 3′:5′ monophosphate (cAMP) regulation of gene expression. Several classes of cAMP‐induced gene expression can be discriminated by sensitivity to stimulation kinetics. The aggregation‐related genes respond only to nanomolar cAMP pulses. The prestalk‐related genes respond both to nano‐molar pulses and persistent micromolar stimulation. The prespore specific genes respond only to persistent micromolar stimulation.The induction of the aggregation‐ and prestalk‐related genes by nanomolar cAMP pulses may share a common transduction pathway, which does not involve cAMP, while involvement of the inositol 1,4,5‐trisphosphate (IP3)/Ca2+ pathway is unlikely. Induction of the expression of prespore and prestalk‐related genes by micromolar cAMP stimuli utilizes divergent signal processing mechanisms. cAMP‐induced prespore gene expression does not involve cAMP and probably also not cyclic guanosine 3′.5′ monophosphate (cGMP) as intracellular intermediate. Involvement of cAMP‐induced phospholipase C (PLC) activation in this pathway is suggested by the observation that IP3 and 1,2‐diacylglycerol (DAG) can induce prespore gene expression, albeit in a somewhat indirect manner and by the observation that Li+ and Ca2+ antagonists inhibit prespore gene expression. Cyclic AMP induction of prestalk‐related gene expression is inhibited by IP3 and DAG and promoted by Li+, and is relatively insensitive to Ca2+ antagonists, which indicates that PLC activation does not mediate prestalk‐related gene expression. Neither prespore nor prestalk‐related gene expression utilizes the sustained cAMP‐induced pHi increase as intracellular intermediate.
cDNA sequence of cyclophilin from Dictyostelium discoidem
Authors: Barisic Karmela , Mollner Stefan , Noegel Angelika A. , Gerisch Guenther , Segall Jeffrey E. 
Published: May 2005

AbstractA cDNA encoding a protein homologous to cyclophilins from other species has been isolated from a Dictyostelium discoideum cDNA library. From the deduced amino acid sequence a protein with a molecular mass of 19 kD and 64% identity with human cyclophilin is predicted. Southern blot analysis indicates that there is one cyclophilin gene in the D. discoideum genome. The mRNA is present in all developmental stages.
Cell‐cell contact mediates cAMP secretion in Dictyostelium discoideum
Authors: Fontana Donna R. , Price Pamela L. , Phillips John C. 
Published: May 2005

AbstractCyclic adenosine 3′:5′ monophosphate (cAMP) and cell‐cell contact regulate developmental gene expression in Dictyostelium discoideum. Developing D. discoideum amoebae synthesize and secrete cAMP following the binding of cAMP to their surface cAMP receptor, a response called cAMP signaling. We have demonstrated two responses of developing D. discoideum amoebae to cell‐cell contact. Cell‐cell contact elicits cAMP secretion and alters the amount of cAMP secreted in a subsequent cAMP signaling response. Depending upon experimental conditions, bacterial‐amoebal contact and amoebal‐amoebal contact can enhance or diminish the amount of cAMP secreted during a subsequent cAMP signaling response. We have hypothesized that cell‐cell contact regulates D. discoideum development by altering cellular and extracellular levels of cAMP. To begin testing this hypothesis, these responses were further characterized.The two responses to cell‐cell contact are independent, i.e., they can each occur in the absence of the other. The responses to cell‐cell contact also have unique temperature dependences when compared to each other, cAMP signaling, and phagocytosis. This suggests that these four responses have unique steps in their transduction mechanisms.The secretion of cAMP in response to cell‐cell contact appears to be a non‐specific response; contact between D. discoideum amoebae and Enterobacter aerogenes, latex beads, or other amoebae elicits cAMP secretion. Despite the apparent similarities of the effects of bacterial‐amoebal and amoebal‐amoebal contact on the cAMP signaling response, this contact‐induced response appears to be specific. Latex beads addition does not alter the magnitude of a subsequent cAMP signaling response. A mutant, DV212, does not alter the magnitude of its cAMP signaling response following bacterial‐amoebal contact but alters the magnitude of its cAMP signaling response following amoebal‐amoebal contact. Thus, amoebae can differentiate between bead‐amoebal contact, bacterial‐amoebal contact, and amoebal‐amoebal contact.
Induction of gene expression in Dictyostelium by prestarvation factor, a factor secreted by growing cells
Authors: Rathi Asha , Kayman Samuel C. , Clarke Margaret 
Published: May 2005

AbstractDuring growth, Dictyostelium cells continuously secrete a factor, PSF, that accumulates in proportion to cell density. At sufficient concentration, it triggers the production of discoidin I and certain lysosomal enzymes. Our earlier studies demonstrated these effects of PSF on protein and enzyme levels [Clarke et al., Differentiation 34:79–87, 1987; Clarke et al., Dev Genet 9:315–326, 1988]. In the present study, we have examined whether PSF induces increased mRNA levels. By Northern blot analysis, we have found that discoidin I mRNA accumulates in exponentially growing NC4 cells as the cells reach high density; significant levels of mRNA are detectable in cells growing either on plates or in suspension, beginning about four generations before the end of exponential growth. High levels of discoidin I mRNA are also found in low‐density cells grown in the presence of buffer conditioned by high‐density cells. These results indicate that PSF induces the accumulation of discoidin I mRNA. Other “early developmental” genes, pCZ22 and the early I genes (16, 18, and 111), are also expressed in exponentially growing cells at high density or in the presence of conditioned buffer. We conclude that several genes previously found to be preferentially expressed very early in development are actually induced during late exponential growth by PSF.