bluuebunny/crossref_metadata_2025 huggingface dataset:
Chromatin structure of transferred genes in transgenic plants
Authors: Weising Kurt , Bohn Horst , Kahl Günter
Published: May 2005

The chromatin structure of foreign genes in transgenic tobacco plants was investigated by digestion of nuclei with DNase I and micrococcal nuclease, respectively, followed by restriction and Southern analysis of the digestion products. The results were compared to the differential expression of the different transgenes. Two model systems were used: plants harbouring vector DNA derived from the disarmed vector pGV 3850 and plants harbouring the light‐regulated and organ‐specifically expressed potato ST‐LS1 gene and the cotransferred ncpaline synthase (nos) reporter gene. Our results show that transferred genes are located in DNase l‐sensitive domains in all transformants. Slight variations of DNase l‐sensitivity of the transferred ST‐LS1 constructs in different transformants neither reflected the between‐transformant variability of expression nor the organ‐specific activity of the transgenes. A deletion event was found responsible for silencing the ST‐LS1 gene but not the nos gene in one of the transformants. Whereas no DNase l‐hypersensitive sites were found within the 3850‐T‐DNA and the ST‐LS1 gene, one prominent site was mapped to the nos promoter within the ST‐LS1 construct in all transformants. Digestion of chromatin harbouring 3850‐T‐DNA with micrococcal nuclease resulted in a blurred nucleosomal pattern as compared to nucleolar and bulk chromatin, the extent of blurring being independent of the expression of transferred genes. The present results favour the “permissive domain” hypothesis which capitalizes on the chromatin surrounding the integration site as the determining factor for the chromatin structure of incoming alien genes. However, between‐transformant variability of expression is not reflected by differential sensitivity to DNase I. Hence, other factors than chromatin structure must be involved in creating “position effects”.
Dosage compensation in Drosophila melanogaster male and female embryos generated by segregation distortion of the sex chromosomes
Authors: Polito Catello , Pannuti Antonio , Lucchesi John C.
Published: May 2005

We report the first practical application of a genetic scheme devised for the purpose of obtaining large quantities of embryos of a specific sex. The scheme, which is based on the meiotic drive system Segregation Distorter, results in the production of populations of zygotes that are almost exclusively of one sex. We have used this scheme to determine that the steady‐state levels of transcripts of X‐linked genes are the same in early male and female embryos, establishing that these genes are dosage compensated.
Separate molecular mechanisms regulate CAT2 gene expression before and after glyoxysome maturation in two genetic lines of maize
Authors: Skadsen Ronald W. , Ruzsa Stephanie M. , Scandalios John G.
Published: May 2005

The temporal expression pattern of the CAT‐2 catalase isozyme in scutella of Zea mays seedlings normally coincides with that of other major glyoxysomal enzymes. In standard genetic lines (e.g., W64A), the CAT‐2 enzyme is synthesized de novo after imbibition, reaches a peak at approximately 4 days later, and then declines steadily. In a high CAT‐2 genetic line, R6‐67, the enzyme accumulates in a linear fashion for at least 8 days after imbibition and reaches a level 3‐fold higher than in W64A. During the first 9 days of early seedling growth in W64A, the correlation between Cat2 mRNA levels and CAT‐2 protein suggests that pretranslational control governs Cat2 gene expression. In R6‐67, the steady rise in CAT‐2 protein appears to result from a pretranslational control mechanism in which Cat‐2 mRNA apparently never declines to levels which would limit the rate of accumulation of CAT‐2 protein. In addition, the amount of Cat2 mRNA bound to polysomes is 3‐fold higher in R6‐67 at day 9 relative to W64A at day 9, reflecting a much greater capacity to synthesize CAT‐2 later in development. Despite substantial differences in Cat2 mRNA levels between genetic lines, early CAT‐2 protein accumulation is similar until day 5, when other glyoxysomal enzymes also attain maximal activity levels. The early increase in CAT‐2, between day 2 and day 5 post‐imbibition, occurs despite a sharp decline in polysomal Cat2 mRNA. This is related to a transient decline in total extractable polysomes which paradoxically coincides with the peak in glyoxysomal enzyme activities. Early Cat2 gene expression is likely controlled by the compartmentalization of CAT‐2 in glyoxysomes.
Actin‐Associated proteins in Dictyostelium discoideum
Authors: Luna Elizabeth J. , Condeelis John S.
Published: May 2005

The cellular slime mold Dictyostelium discoideum is becoming the premier system for the explication of the biochemical and cellular events that occur during motile processes. Proteins associated with the actin cytoskeleton, in particular, appear to play key roles in cellular responses to many external stimuli. This review summarizes our present understanding of the actin‐associated proteins in Dictyostelium, including their in vitro activities and their structural and/or functional analogues in mammalian cells.
Mechanisms of amoeboid chemotaxis: An evaluation of the cortical expansion model
Authors: Condeelis J. , Bresnick A. , Demma M. , Dharmawardhane S. , Eddy R. , Hall A. L. , Sauterer R. , Warren V.
Published: May 2005

In this work we evaluate the cartical expansion model for amoeboid chemo‐taxis with regard to new information about molecular events in the cytoskeleton following chemo‐tactic stimulation of Dictyostelium amoebae. A rapid upshift in the concentration of chemoattrac‐tant can be used to synchronize the motile behavior of a large population of cells. This synchrony presents an opportunity to study the biochemical basis of morphological changes such as pseudopod extension that are required for amoeboid chemotaxis. Changes in the composition and activity of the cytoskeleton following stimulation can be measured with precision and correlated with important morphological changes. Such studies demonstrate that activation of actin nucleation is one of the first and most crucial events in the actin cytoskeleton following stimulation. This activation is followed by incorporation of specific actin cross‐linking proteins into the cytoskeleton, which are implicated in the extension of pseudopods and filopods. These results, as well as those from studies with mutants deficient in myosin, indicate that cortical expansion, driven by focal actin polymerization, cross‐linking and gel osmotic swelling, is an important force for pseudopod extension.

It is concluded that whereas three forces, frontal sliding, tail contraction, and cortical expansion may cooperate to produce amoeboid movement, the cortical expansion model offers the simplest explanation of how focal stimulation with a chemoat‐tractant causes polarized pseudopod extension.
Intracellular vesicle movement, cAMP and myosin II in Dictyostelium
Authors: Soll David R. , Wessels Deborah , Murray John , Vawter Holly , Voss Edward , Bublitz Ann
Published: May 2005

Dictyostelium amoebae were analyzed before and after rapid addition of 10−6 M cAMP for cellular motility, dynamic shape changes, and intracellular particle movement. Before cAMP addition, amoebae moved in a persistent anterior fashion and were elongate with F‐actin localized predominantly in the anterior pseudopod. Intracellular particles moved rapidly and anteriorly. Within seconds after 10−6 M cAMP addition, cells stopped translocating, pseudopod formation ceased, intra‐cellular particle movement was depressed, and F‐actin was lost from the pseudopod and concomitantly relocalized in the cell cortex After 10 seconds, expansion zones reappeared but were small and no longer anteriorly localized. Vesicle movement partially rebounded but was no longer anteriorly directed. The myosin II null mutant HS2215 exhibited both depressed cellular translocation and vesicle movement. The addition of cAMP to HS2215 cells did not result in any detectable change in the random, depressed movement of particles. The results with HS2215 suggest that myosin II is essential for (1) rapid cellular translocation, (2) cellular polarity, (3) rapid particle movement, (4) anteriorly directed particle movement, and (5) the cAMP response. Electron micrographs suggest that at least half of the particles examined in this study contain in turn smaller membrane bound vesicles or multilameilar membrane bodies. The possible role of these vesicles is discussed.
Ponticulin, a developmentally‐regulated plasma membrane glycoprotein, mediates actin binding and nucleation
Authors: Luna Elizabeth J. , Wuestehube Linda J. , Chia Catherine P. , Shariff Aneesa , Hitt Anne L. , Ingalls Hilary M.
Published: May 2005

Ponticulin is a 17,000‐dalton transmembrane glycoprotein that is involved in the binding and nucleation of actin filaments by Dictyostelium discoideum plasma membranes. The major actin‐binding protein isolated from these membranes by F‐actin affinity chromatography, ponticulin also binds F‐actin on blot overlays. The actin‐binding activity of ponticulin in vitro is identical to that observed for purified plasma membranes: it resists extraction with 0.1 N NaOH, is sensitive to high salt concentrations, and is destroyed by heat, proteolysis, and thiol reduction and alkylation. A cytoplasmic domain of ponticulin mediates binding to actin because univalent antibody fragments directed against the cytoplasmic surface of this protein inhibit 96% of the actin‐membrane binding in sedimenlation assays. Antibody specific for ponticulin emoves both ponticu‐lin and the ability to reconstitute actin nucleation activity from detergent extracts of solubilized plasma membranes. Levels of plasma membrane ponticulin increase 2‐ to 3‐fold during aggregation streaming, when cells adhere to each other and are highly motile. Although present throughout the plasma membrane, ponticulin is preferentially localized to some actin‐rich membrane structures, including sites of cell‐cell adhesion and arched regions of the plasma membrane reminiscent of the early stages of pseudopod formation. Ponticulin also is present but not obviously enriched at phagocytic cups of log‐phase amebae. These results indicate that ponticulin may function in vivo to attach and nucleate actin filaments at the cytoplas‐mic surface of the plasma membrane. A 17,000‐dalton analogue of ponticulin has been identified in human polymorphonuclear leukocyte plasma membranes by immunoblotting and immunofluo‐rescence microscopy. These findings suggest that the structure and function of ponticulin in motile cells has been evolutionarily conserved.
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

The 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

The 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

Whereas 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

A 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

We 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

A 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

Vital 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

Dictyostelium 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

Dictyostelium 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

The 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

We 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

The 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

A 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

A 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

Cyclic 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

During 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.
Structure, expression, and regulation of members of the developmentally controlled V and H gene classes from Dictyostelium
Authors: Singleton Charles K. , Delude Russel L. , Ken Ruey , Manning Suzanne S. , McPherson Clifton E.
Published: May 2005

We have examined the expression and structure of vegetative specific genes belonging to the V and H gene classes. Both classes of genes are deactivated at the onset of development by a reduction in the rate of transcription. Thus, the genes must be reactivated when the terminally differentiated spores germinate and the resulting amebae return to the vegetative state. During germination, activation of expression of most members of the V gene class was found to parallel the emergence of amaebae from the spore coats. The activation of the V genes did not occur when protein synthesis was inhibited. The timing of activation of the H genes was more heterogeneous and did not parallel emergence. H gene activation occurred even when protein synthesis was inhibited. V4 was found to be the only vegetative specific gene that was responsive to the presence of bacteria. V4 expression was induced by 25–100 fold via transcriptional activation when bacteria were added to amebae growing axenically. Isolation and sequence analysis of the corresponding genomic clones revealed that two V genes, V18 and V1, encode ribosomal proteins. Promoter analysis has delineated the sequences necessary for expression and regulation for several of the V and H genes. In all cases, expression was determined by sequences within the first several hundred base pairs of the transcription start site. For V18 and V14, a positive constitutive element was identified in addition to the sequences involved in regulation. Finally, all of the characterizations and findings are discussed in terms of postulated models for V and H gene expression and regulation.
Cyclic nucleotide phosphodiesterase of Dictyostelium discoideum and its glycoprotein inhibitor: Structure and expression of their genes
Authors: Franke Jakob , Faure Michel , Wu Lin , Hall Anne L. , Podgorski Gregory J. , Kessin Richard H.
Published: May 2005

The genes coding for the cyclic nucleotide phosphodiesterase (PD) and the PD inhibitory glycoprotein (PDI) have been cloned and characterized. The PDI gene was isolated as a 1.6 kb genomic fragment, which included the coding sequence containing two small introns and 510 nucleotides of non‐translated 5′ sequence. From the deduced amino acid sequence we predict a protein with a molecular weight (MW) of 26,000 that, in agreement with previous data, contains 15% cysteine residues. Genomic Southern blot analysis indicates that only one gene encodes the inhibitor. Northern blot analysis shows a single transcript of 0.95 kb. The PDI gene is expressed early in development with little transcript remaining following aggregation. The appearance of PDI mRNA is prevented by the presence of cAMP, but when cAMP is removed the transcript appears within 30 minutes. When cAMP is applied to cells expressing PDI the transcript disappears with a half‐life of less than 30 minutes. The PD gene of D. discoideum is transcribed into three mRNAs: a 1.9 kb mRNA specific for growth, a 2.4 kb mRNA specific for aggregation, and a 2.2 kb mRNA specific for late development. The 2.2 kb mRNA is also specific for prestalk cells, and is induced by differentiation‐inducing factor. All three mRNAs contain the same coding sequence, and differ only in their 5′ non‐coding sequences. Each mRNA is transcribed from a different promoter, and by using the chloramphenicol acyltransferase gene as a reporter, we have shown that each promoter displays the same regulation as its cognate mRNA. Transformation of wild‐type strains with the PD gene causes PD overexpression which accelerates aggregation and blocks subsequent cell differentiation and pattern formation.
Coordinate regulation of the spore coat genes in Dictyostelium discoideum
Authors: Fosnaugh Kathy L. , Loomis William F.
Published: May 2005

Genomic clones of the genes coding for the three major spore coat proteins, SP60, SP70, and SP96, were used to measure the accumulation of their respective mRNAs in mutant and wild‐type cells allowed to develop under a variety of conditions. These prespore‐specific mRNAs were found to be both temporally and quantitatively coordinate under all conditions indicating that they may be subject to identical regulatory processes. Accumulation of the spore coat mRNAs is dependent upon the function of both cAMP receptors and Gα2 proteins during the aggregation stage as well as upon concomitant protein synthesis. When cells are dissociated from aggregates at 10 hr of development and rapidly shaken in 0.1 mM EDTA they form clumps but do not accumulate any of the prespore‐specific RNAs assayed. However, if either 0.1 rnM Ca++ or 20 μM cAMP is added to these cells, the spore coat mRNAs accumulate. Lower concentrations of either Ca++ or cAMP had no effect. These results suggest that expression of the spore coat genes normally involves a Ca++ ‐dependent process, but the Ca++ requirement can be overcome by adding Ca++ high concentrations of exogenous CAMP.Addition of 50 nM DIF to dissociated cell blocks the accu‐ mulation of the spore coat mRNAs even when cAMP or Ca++ is present. The upstream regions of the spore coat genes were compared to those of another gene, D19, that codes for the prespore‐specific protein SP29. Short sequences related to CACCCAC were found at about the same position relative to the transcriptional start sites of these co‐ ordinately regulated genes.
Ras‐related genes in Dictyostelium discoideum
Authors: Robbins Stephen M. , Khosla Meenal , Thiery Richard , Weeks Gerald , Spiegelman George B.
Published: May 2005

Dicfyostelium discoideum, like other eukaryotes, has been shown to express several ras‐related genes. Two gene products, Ddras and DdrasG, are highly conserved relative to the human ras proteins. Ddras is expressed at the pseudoplasmodial stage of development, whereas DdrasG is expressed in vegetative cells and during early development. In addition, Dicfyostelium possesses three ras‐related genes, SAS1, SAS2 and Ddrap1, whose gene products are only partially conserved relative to those of the ras genes. The expression of these three genes is also developmentally regulated.
Regulation of cysteine proteinases during different pathways of differentiation in cellular slime molds
Authors: North Michael J. , Cotter David A.
Published: May 2005

Cysteine proteinase activities have been determined using gelatin‐SDS‐PAGE analysis and assays based on peptide nitroanilides. Vegetative myxamoebae of all species examined contain high levels of cysteine proteinose activity present in multiple forms. In both Dictyostelium discoideum and Polysphondylium pallidum the proteinase content is dependent on whether the cells are grown axenically or in association with bacteria. In all instances development is accompanied by a decreased intracellular cysteine proteinase activity. This occurs during the formation of fruiting bodies in D. discoideum, microcysts in P. pallidum, and macrocysts in Dictyostelium mucoroides. Significant quantities of proteinase activity are always secreted by myxamoebae immediately on starvation. In D. mucoroides this leads to an almost total depletion of intracellular cysteine proteinases by the aggregation stage. As a consequence of this depletion it has been relatively easy to detect a developmentally regulated accumulation of cysteine proteinases at the enzyme activity level, something which has not yet proved possible with D. discoideum. Three cysteine proteinases are produced as D. mucoroides macrocysts develop and mature. In the case of microcyst formation in P. pallidum the proteinase contents of the developing cells and of the microcysts are dependent on how the myxamoebae are grown. In this developmental pathway at least, there is no absolute requirement for specific proteinases to be present (or absent) at a particular stage. The diversity of cysteine proteinases found in cellular slime molds and the variety of features apparent in their regulation suggest that they will prove to be very useful for investigating features of the structure/function relationships in this important group of enzymes.
Structure, expression, and inactivation by gene disruption of the Dictyostelium discoideum prespore gene EB4
Authors: Hildebrandt Martin , Saur Uschi , Nellen Wolfgang
Published: May 2005

We present the nucleotide sequence of the cell type specific prespore gene EB4 which encodes a protein containing a hydrophobic leader sequence and two distinct domains of amino acid repeats. By RNase protection experiments we have determined the genomic organization of the gene which differs substantially from the previously published data. An antibody directed against one of the repeat structures (hexamer repeat) specifically reacts with a developmentally regulated antigen of 58 kd. Gene disruption transformants were obtained by transformation with a genomic DNA fragment. The transformants express a 3′ truncated mRNA and do not react with the anti‐hexamer antibody. So far, we could not detect any phenotypic aberrations in the transformed cell line.
Lethal(1)aberrant immune response mutations leading to melanotic tumor formation in Drosophila melanogaster
Authors: Watson Kellie L. , Johnson Terrell K. , Denell Robin E.
Published: May 2005

Using P element‐mediated mutagenesis we have isolated 20 X‐linked lethal mutations, representing at least 14 complementation groups, which exhibit melanotic tumor phenotypes. We present the systematic analysis of this interesting group of lethal mutations that were selected for their visible melanotic or immune response. The lethal and melanotic tumor phenotypes of each lethal(1) aberrant immune response (air) mutation are pleiotropic effects of single genetic lesions. Lethality occurs throughout the larval and early pupal periods of development and larval development is extended in some air mutants. The air mutant lethal syndromes include abnormalities associated with the brain, haematopoietic organs, gut, salivary glands, ring glands, and imaginal discs. Additional characterization of the melanotic tumor mutations Tuml and tu(1)Szts have indicated that the melanotic tumor phenotype is similar to that observed in the air mutants. These studies have led to the proposal that two distinct classes of melanotic tumor mutations exist. Class 1 includes mutants in which melanotic tumors result from “autoimmune responses” or the response of an apparently normal immune system to the presence of abnormal target tissues. The Class 2 mutants display obvious defects in the haematopoietic organs or haemocytes, manifested as overgrowth, and the resulting aberrant immune system behavior may contribute to melanotic tumor formation.
Differential expression and isozymic composition of sn‐glycerol‐3‐phosphate dehydrogenase in tissues from variant lines of Drosophila melanogaster
Authors: Cook Julia L. , Shaffer Jacquelin B. , Bewley Glenn C.
Published: May 2005

The tissue‐specific expression and isozymic composition of Drosophila sn‐glycerol‐3‐phosphate dehydrogenase (GPDH) (EC 1.1.1.8) have been determined for a high‐activity control line and two variant lines that alter either the temporal or systemic expression of GPDH through a reduction in rates of polypeptide synthesis. The temporal variant exhibits a reduction in enzyme levels in all larval tissues and in the adult abdomen, while levels of activity in the adult thorax are equal to the control line. Isozymic analyses of these tissues demonstrate that it is the GPDH‐3 species that is reduced in a temporal and tissue‐specific manner. In contrast, the systemic variant demonstrates a uniform reduction of all isozymic species in each tissue and developmental stage. Analyses of the tissues of F1 hybrid offspring of each variant line and appropriately marked electrophoretic variants demonstrate that the tissue‐specific effects observed are due to cis‐acting elements that are tightly linked to the structural gene.
Maternal effect mutations affecting macronuclear determination and development in Paramecium tetraurelia
Authors: Berger James D. , Havelka Monika
Published: May 2005

Gene mutations that interfere with macronuclear development in Paramecium were obtained by selecting lines that failed to produce normal macronuclear anlagen following the second autogamy after mutagenesis. The mutants fell into several complementation groups. There was one case of apparent intragenic noncomplementation among the eight mutants examined. In the stronger mutants, macronuclear anlagen were not formed, and all four mitotic products of the posfzygotic divisions of the synkaryon remained as micronuclei. Under semirestrictive conditions, cells often contained a single anlage, suggesting that determination of anlagen was a discrete event for each nucleus. The missing anlagen trait was recessive and associated with a strong maternal effect. The phenocritical period of one of the stronger alleles, aala, began at the second postzygotic division and ended with the first morphological differentiation of macronuclear anlagen. Nuclear migration in this mutant was abnormal. Under restrictive conditions, the posterior products of the second postzygotic division reached a posterior‐most position, which was 8% of cell length more anterior than that of the most posterior nuclei in wild‐type cells. Under permissive conditions, the pattern of migration was intermediate between that of wild‐type cells and mutants under fully restrictive conditions. The patterns of nuclear migration were consistent with the nuclear growth kinetics.
Generating lineage‐specific markers to study Drosophila development
Authors: Perrimon Norbert , Noll Elizabeth , McCall Kimberly , Brand Andrea
Published: May 2005

To generate cell‐ and tissue‐specific expression patterns of the reporter gene lacZ in Drosophila, we have generated and characterized 1,426 independent insertion strains using four different P‐element constructs. These four transposons carry a lacZ gene driven either by the weak promoter of the P‐element transposase gene or by partial promoters from the even‐skipped, fushi‐tarazu, or engrailed genes. The tissue‐specific patterns of β‐galactosidase expression that we are able to generate depend on the promoter utilized. We describe in detail 13 strains that can be used to follow specific cell lineages and demonstrate their utility in analyzing the phenotypes of developmental mutants. Insertion strains generated with P‐elements that carry various sequences upstream of the lacZ gene exhibit an increased variety of expression patterns that can be used to study Drosophila development.