Electrophoretic karyotype for Dictyostelium discoideum.

Abstract

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...

Electrophoretic karyotyping from Tolypocladium inflatum and six related strains allows differentiation of morpologically similar species

“…This is most probably a result of hybridization of subsequences partially homologous with the fl-tubulin gene and chromosome II. Similar results have been described for D. discoideurn in which duplicated gene sequences or crosshybridization with related genes are believed to cause multiple hybridization signals (Cox et al 1990). The isolation and characterization of chromosome-specific probes as presented here enables us to identify homologous chromosomes in varying karyotypes.…”

“…Both hybridizing DNA bands may represent degradation products with homology to the rDNA probe. Alternatively, these bands consist of extrachromosomal circular rDNA molecules, as have been reported for Dictyostelium discoideum (Cox et al 1990). The fl-tubulin probe (43/2) hybridizes with the 4.8-Mb doublet, which represents the co-migrating chromosomes IV and V. In addition, a weak signal is observed in chromosome II for the fl-tubutin probe.…”

A High-Resolution HAPPY Map of Dictyostelium discoideum Chromosome 6

“…DNA was prepared according to the procedure of Cox et al (1990), with the modification that agarose strings were used instead of plugs. In brief, cells were suspended at density of 4 ‫ן‬ 10 8 cells/mL in KK2 buffer (16.6 mM KH 2 PO 4 , 3.8 mM K 2 HPO 4 at pH 6.2).…”

“…The genome of D. discoideum comprises six chromosomes, with a total size of ∼34 Mbp (Cox et al 1990;Loomis and Kuspa 1997). Because of inefficient sexual reproduction in this organism there are no highresolution genetic maps based on meiotic recombination.…”

Intracellular Growth ofLegionella pneumophilainDictyostelium discoideum, a System for Genetic Analysis of Host-Pathogen Interactions

“…The organism is haploid and has a relatively small genome of 34 Mb (10,30). It is possible to transform the cells by electroporation and to knock out genes by homologous recombination and marker replacement (12,32).…”