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Positive selection for Dictyostelium discoideum mutants lacking UMP synthase activity based on resistance to 5-fluoroorotic acid. 1991

D Kalpaxis, and I Zündorf, and H Werner, and N Reindl, and E Boy-Marcotte, and M Jacquet, and T Dingermann
Institut für Biochemie der Medizinischen Fakultät, Universität Erlangen-Nürnberg, Federal Republic of Germany.

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.

UI MeSH Term Description Entries
D007700 Kinetics The rate dynamics in chemical or physical systems.
D009097 Multienzyme Complexes Systems of enzymes which function sequentially by catalyzing consecutive reactions linked by common metabolic intermediates. They may involve simply a transfer of water molecules or hydrogen atoms and may be associated with large supramolecular structures such as MITOCHONDRIA or RIBOSOMES. Complexes, Multienzyme
D009154 Mutation Any detectable and heritable change in the genetic material that causes a change in the GENOTYPE and which is transmitted to daughter cells and to succeeding generations. Mutations
D009962 Orotate Phosphoribosyltransferase The enzyme catalyzing the formation of orotidine-5'-phosphoric acid (orotidylic acid) from orotic acid and 5-phosphoribosyl-1-pyrophosphate in the course of pyrimidine nucleotide biosynthesis. EC 2.4.2.10. Orotidine-5'-Phosphate Pyrophosphorylase,Orotidylic Acid Phosphorylase,OMPase,Orotidine-5'-Monophosphate Phosphohydrolase,Orotidine 5' Monophosphate Phosphohydrolase,Orotidine 5' Phosphate Pyrophosphorylase,Phosphohydrolase, Orotidine-5'-Monophosphate,Phosphoribosyltransferase, Orotate,Phosphorylase, Orotidylic Acid,Pyrophosphorylase, Orotidine-5'-Phosphate
D009963 Orotic Acid An intermediate product in PYRIMIDINE synthesis which plays a role in chemical conversions between DIHYDROFOLATE and TETRAHYDROFOLATE. Potassium Orotate,Sodium Orotate,Zinc Orotate,Acid, Orotic,Orotate, Potassium,Orotate, Sodium,Orotate, Zinc
D009964 Orotidine-5'-Phosphate Decarboxylase Orotidine-5'-phosphate carboxy-lyase. Catalyzes the decarboxylation of orotidylic acid to yield uridylic acid in the final step of the pyrimidine nucleotide biosynthesis pathway. EC 4.1.1.23. Orotidine Phosphate Carboxy-Lyase,Orotidylate Decarboxylase,OMP Decarboxylase,Orotidine 5 Phosphate Decarboxylase,Orotidine 5' Phosphate Decarboxylase,Orotidine-5-Phosphate Decarboxylase,Carboxy-Lyase, Orotidine Phosphate,Decarboxylase, OMP,Decarboxylase, Orotidine-5'-Phosphate,Decarboxylase, Orotidine-5-Phosphate,Decarboxylase, Orotidylate,Orotidine Phosphate Carboxy Lyase,Phosphate Carboxy-Lyase, Orotidine
D010641 Phenotype The outward appearance of the individual. It is the product of interactions between genes, and between the GENOTYPE and the environment. Phenotypes
D010957 Plasmids Extrachromosomal, usually CIRCULAR DNA molecules that are self-replicating and transferable from one organism to another. They are found in a variety of bacterial, archaeal, fungal, algal, and plant species. They are used in GENETIC ENGINEERING as CLONING VECTORS. Episomes,Episome,Plasmid
D004023 Dictyostelium A genus of protozoa, formerly also considered a fungus. Its natural habitat is decaying forest leaves, where it feeds on bacteria. D. discoideum is the best-known species and is widely used in biomedical research. Dictyostelium discoideum,Dictyostelium discoideums,Dictyosteliums,discoideum, Dictyostelium
D004352 Drug Resistance, Microbial The ability of microorganisms, especially bacteria, to resist or to become tolerant to chemotherapeutic agents, antimicrobial agents, or antibiotics. This resistance may be acquired through gene mutation or foreign DNA in transmissible plasmids (R FACTORS). Antibiotic Resistance,Antibiotic Resistance, Microbial,Antimicrobial Resistance, Drug,Antimicrobial Drug Resistance,Antimicrobial Drug Resistances,Antimicrobial Resistances, Drug,Drug Antimicrobial Resistance,Drug Antimicrobial Resistances,Drug Resistances, Microbial,Resistance, Antibiotic,Resistance, Drug Antimicrobial,Resistances, Drug Antimicrobial

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