Metabolism of the cellular slime mould <i>Dictyostelium discoideum</i> grown in axenic culture

Ashworth, J.; Watts, Donald J.

doi:10.1042/bj1190175

Cited by 129 publications

(52 citation statements)

References 14 publications

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“…In most experiments, cells were propagated on 15-cm GYP plates (11) with Escherichia coli B/r as the food source. Strain AX3k was grown on E. coli B/r or axenically in HL5 medium (12). Incubation was at 21°C in a humidified growth chamber or at 21°C in a water bath on a rotary shaker.…”

Section: Methodsmentioning

confidence: 99%

Electrophoretic karyotype for Dictyostelium discoideum.

Cox

Vocke

Walter

et al. 1990

Proc. Natl. Acad. Sci. U.S.A.

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This paper reports on the separation of the Dictyostelium discoideum chromosomes by pulse-field electrophoresis and the correlation of the electrophoretic pattern with linkage groups established by classical genetic methods. In two commonly used laboratory strains, five chromosome-sized DNA molecules have been identified. Although the majority of the molecular probes used in this study can be unambiguously assigned to established linkage groups, the electrophoretic karyotype differs between the closely related strains AX3k and NC4, suggesting that chromosomal fragmentation may have occurred during their maintenance and growth. The largest chromosome identified in this study is approximately 9 million base pairs. To achieve resolution with molecules of this size, programmed voltage gradients were used in addition to programmed pulse times.The cellular slime mold Dictyostelium discoideum is widely used in the study of morphogenesis, cell-cell interaction, and signal transduction (for recent reviews, see refs. 1 and 2). Extensive genetic studies have helped to define the genes underlying these processes, and many genes have been mapped to well-studied linkage groups (reviewed in ref.3). A considerable number have also been ordered by mitotic recombination (4, 5). Laboratory strains ofD. discoideum are thought to have seven chromosomes (6, 7), although only six linkage groups have been identified (3).To aid in the correlation of linkage group with physical structure and to develop a rapid and accurate method for studying genetic fine structure in D. discoideum, we have examined the conditions necessary for separation of D. discoideum chromosomal-sized DNA molecules by pulsefield gel electrophoresis (8, 9). We report here on the number ofelectrophoretic species detected by clamped homogeneous electric field (CHEF) electrophoresis (10) and show, using a variety of molecular probes, that it is possible to correlate the genetic and physical map, thus enlarging the genetic possibilities in this organism. MATERIALS AND METHODSStrains. D. discoideum AX3k and NC4 were received from R. Firtel (University of California, San Diego, CA) and J. T. Bonner (Princeton University, Princeton, NJ), respectively.Growth conditions. In most experiments, cells were propagated on 15-cm GYP plates (11) with Escherichia coli B/r as the food source. Strain AX3k was grown on E. coli B/r or axenically in HL5 medium (12). Incubation was at 21°C in a humidified growth chamber or at 21°C in a water bath on a rotary shaker. Cells were starved by plating well-washed cells at a density of approximately S x 104 amoebae per mm2 on 2% agar plates (20 g of agar per liter of distilled deionized water).Cell Preparation. Cells were harvested at various stages of growth and development, washed several times at room temperature in phosphate buffer (13), and mixed at 390C with an equal volume of FMC InCert agarose. For most of the experiments reported here, cells were allowed to develop to the loose-mound stage before they were harvested. As we will di...

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Section: Methodsmentioning

confidence: 99%

Electrophoretic karyotype for Dictyostelium discoideum.

Cox

Vocke

Walter

et al. 1990

Proc. Natl. Acad. Sci. U.S.A.

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“…Consequently, suspensions of washed cells, harvested from axenic cultures of D. discoideum strain ax-2 [13], were assayed for cyclic AMP phosphodiesterase in presence of excess inhibitor. Although the inhibitor is known to almost completely inactivate soluble cyclic AMP phosphodiesterase excreted by D. discoideum cells [9], a phosphodiesterase activity resistant to the inhibitor was found in the cell suspensions.…”

Section: N Vivo Hydrolysis Of Cyclic a M Pmentioning

confidence: 99%

Membrane‐Bound Cyclic AMP Phosphodiesterase in Chemotactically Responding Cells of Dictyostelium discoideum

Malchow

Nägele

Schwarz

et al. 1972

European Journal of Biochemistry

271

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I n cells of the cellular slime mold, Dictyostelium discoideum, that respond chemotactically to cyclic AMP, cell-bound cyclic AMP phosphodiesterase was studied under two conditions : in suspensions of living cells with external cyclic AMP as a substrate, and in the 27000 x g sediment of cell homogenates. Under both conditions a drastic increase of cell-bound phosphodiesterase activity in the developmental phase preceding cell aggregation was found. I n non-aggregating mutants this increase is smaller or absent. The particle-bound cyclic AMP phosphodiesterase has an optimum a t pH 7.0-8.0. I n vivo it is resistant to inhibition by external glutathione and to a macromolecular phosphodiesterase inhibitor from Dictyostelium discoideum ; in particle fractions enzyme activity is partially sensitive to these inhibitors. A possible function of this enzyme in the chemotactic receptor system is discussed.Development of the cellular slime mold, Dictyostelium discoideum, proceeds from single amoeboid cells to the formation of a multicellular organism by cell aggregation. Cells remain single during growth and differentiate later on within a preaggregation phase of several hours to aggregation-competent ones. These aggregation-competent cells are more sensitive to the chemotactic action of cyclic AMP than growth phase cells [I] ; they are expected, therefore, to possess either a greater number of, or more sensitive cyclic-AMP receptors on their surface. Precise chemotactic orientation over a period of time requires continuous re-evaluation of the concentration gradient of the chemotactic agent by the responding cells. The following receptor mechanisms would fulfil such a function. (1) The receptor is a cyclic-AMPbinding compound without catalytic activity. Continuous functioning of the receptor system requires in this case either (a) high turnover of receptor molecules, or (b) rapid readjustment of the equilibrium of free to receptor-bound cyclic AMP, or (c) it requires enzymatic conversion of the bound cyclic AMP into a nonbinding product, e.g. by phosphodiesterase action. (2) The receptor itself acts as a phosphodiesterase. I n this case the free energy change associated with hydrolysis of the phosphodiester ring would enable the cell to couple with hydrolysis of cyclic AMP a t the membrane an endergonic reaction, the product of which could mediate chemotactic motion. The AGO of cyclic AMP hydrolysis is -11.9 kcal/mol (pH 7.3; 25 "C; Mg2+ equimoles to nucleotide) [2]. If possibility 1 c or 2 is the case then a phosphodiesterase activity should be detectable on the cell surface, and this activity should be correlated with the responsiveness of the cells to cyclic AMP. I n this paper we study the properties of such an enzyme with regard to its possible function as part of the chemotactic receptor system. METHODS Cell CulturesAll Dictyostelium discoideum strains were cultivated in suspension on a gyratory shaker a t 23 "C. The ax-2 strain was grown, if not stated otherwise, in peptone-yeast extract medium supplemented with 1....

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“…The role of the nucleus in the differentiation of D. discoideum has, however, only recently received attention [6] due to the difficulties involved in isolating useful quantities of clean, intact nuclei and to the low DNA content of the organism [7,8]. The recent development of axenic strains [9,10] of D. discoideum has permitted the production of large quantities of amoebae in pure culture and facilitated the isolation of cellular components.…”

mentioning

confidence: 99%

The Isolation of Nuclei and Basic Nucleoproteins from the Cellular Slime Mold Dictyostelium discoideum

Charlesworth

Parish

1975

European Journal of Biochemistry

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A method is described for the isolation of nuclei from an axenic strain of Dictyostelium discoideum using a sorbitol/Ficoll solution and low concentration of Triton X-100.Basic proteins have been extracted from the nuclei and on polyacrylamide gel electrophoresis yield a consistent pattern in which five major groups or bands predominate. Four of these five fractions comigrate with calf thymus histones and one fraction seems to be unique to D. discoideum.The slowest moving of the five fractions is soluble in 0.5 M perchloric acid and comigrates with calf thymus histone F I . After recovery from the perchloric acid solution by precipitation with acetone this fraction yielded one major band on electrophoresis.The cellular slime mold Dictyostelium discoideum undergoes a period of morphogenesis during its life cycle which can be clearly distinguished from the phase of vegetative growth. Following a period of starvation the amoebae aggregate and differentiate to form only two major cell types, spore cells and stalk cells [l].This relatively uncomplicated system is a useful model for the study of the biochemical changes associated with cell differentiation and has been extensively investigated [2-51. The role of the nucleus in the differentiation of D. discoideum has, however, only recently received attention [6] due to the difficulties involved in isolating useful quantities of clean, intact nuclei and to the low DNA content of the organism [7,8]. The recent development of axenic strains [9,10] of D. discoideum has permitted the production of large quantities of amoebae in pure culture and facilitated the isolation of cellular components.The work described in this paper was undertaken firstly to develop a method for the isolation of clean, intact nuclei from D. discoideum; secondly, it was undertaken to establish whether the nuclei contained basic proteins and if so to what extent they resembled the histones isolated from other organisms. . The first of these methods gave variable results with rather low yields and nuclei of doubtful quality. Neither method was really suitable for dealing with the large quantities of cells required to obtain useful amounts of nuclear material (see Discussion).A paper by Coukell and Walker [6] appeared during the course of this work describing the isolation of nuclei using Triton X-100 and of basic nuclear proteins from D. discoideum. We also used their method but with varying success. As a result of work on the isolation of intact mitochondria and peroxisomes from D. discoideum using sorbitol/Ficoll medium [15,16] andTritonX-100, wemodifiedCoukel1 and Walker's method and developed the one described below. The method is suitable for the isolation of nuclei and other organelles from small or large quantities of cells and from non-axenic as well as from axenic strains of D . discoideum.Subsequent isolation and partial characterisation by polyacrylamide gel electrophoresis of basic nucleoproteins produced consistent results which differed somewhat from those of Coukell and Walker. Althoug...

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Metabolism of the cellular slime mould Dictyostelium discoideum grown in axenic culture

Cited by 129 publications

References 14 publications

Electrophoretic karyotype for Dictyostelium discoideum.

Electrophoretic karyotype for Dictyostelium discoideum.

Membrane‐Bound Cyclic AMP Phosphodiesterase in Chemotactically Responding Cells of Dictyostelium discoideum

The Isolation of Nuclei and Basic Nucleoproteins from the Cellular Slime Mold Dictyostelium discoideum

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