Positive selection for Dictyostelium discoideum mutants lacking UMP synthase activity based on resistance to 5-fluoroorotic acid.
In the cellular slime mould Dictyostelium discoideum the two enzymatic activities of the pyrimidine pathway, orotidine-5'-phosphate decarboxylase (EC 4.1.1.23; OMPdecase) and orotate phosphoribosyl transferase (EC 2.4.2.10; OPRTase), are encoded by a single gene (DdPYR5-6). As in higher eukaryotes the bifunctional enzyme is referred to as UMP synthase. Here we present a method that allows efficient generation and selection of mutants lacking UMP synthase. D. discoideum cells are transformed with either of two different types of plasmids. One plasmid type contains no sequences homologous to the UMP synthase gene whereas the other type contains at least parts of this gene. UMP synthase- mutants, which were positively selected for in the presence of 5-fluoroorotic acid (5-FOA), were obtained with both plasmids. However, mutation rates were at least one order of magnitude higher if plasmids containing various portions of the UMP synthase gene were used as opposed to plasmids that lack any homology to the UMP synthase locus. Several mutant strains were extensively characterized. These strains lack OMPdecase activity and exhibit in addition to 5-FOA resistance a ura- phenotype. All mutants carry UMP synthase loci with deletions of various extents but integration of transforming plasmids was not detected. This efficient generation of 5-FOA resistance is part of a proposed complex selection scheme which allows multiple rounds of transformation of D. discoideum.
Kalpaxis
D
D
Institut für Biochemie der Medizinischen Fakultät, Universität Erlangen-Nürnberg, Federal Republic of Germany.
Zündorf
I
I
Werner
H
H
Reindl
N
N
Boy-Marcotte
E
E
Jacquet
M
M
Dingermann
T
T
D015139:Blotting, Southern; D004023:Dictyostelium / Q000187:drug effects / Q000201:enzymology / Q000235:genetics* / Q000302:isolation & purification; D004352:Drug Resistance, Microbial / Q000235:genetics; D005800:Genes, Fungal; D005838:Genotype; D007700:Kinetics; D009097:Multienzyme Complexes / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D009962:Orotate Phosphoribosyltransferase / Q000235:genetics* / Q000378:metabolism; D009963:Orotic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D009964:Orotidine-5'-Phosphate Decarboxylase / Q000235:genetics* / Q000378:metabolism; D010641:Phenotype; D010957:Plasmids; D015183:Restriction Mapping; D014170:Transformation, Genetic
D009097:Multienzyme Complexes; D009963:Orotic Acid; C027157:uridine 5'-monophosphate synthase; C001242:5-fluoroorotic acid; D009962:Orotate Phosphoribosyltransferase; D009964:Orotidine-5'-Phosphate Decarboxylase