Developmental control of the expression of the dihydroorotate dehydrogenase and UMP synthase genes in Dictyostelium discoideum

Faure, Michel; Kalékine, Monique; Boy‐Marcotte, Emmanuelle; Jacquet, Michel

doi:10.1016/0045-6039(88)90028-0

Cited by 9 publications

(1 citation statement)

References 12 publications

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“…Transcription of the pyr5-6 gene of Dictyostelium discoideum, which codes for OMP decarboxylase activity, is strongly regulated during development (10). The genera Myxococcus and Dictyostelium, although not evolutionarily related, both initiate development in response to nutrient deprivation (14).…”

Section: Resultsmentioning

confidence: 99%

Targeted disruption of the Myxococcus xanthus orotidine 5'-monophosphate decarboxylase gene: effects on growth and fruiting-body development

Kimsey

Kaiser

1991

J Bacteriol

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The Myxococcus xanthus gene coding for orotidine 5'-monophosphate (OMP) decarboxylase (EC 4.1.1.23) was cloned. The M. xanthus uraA gene efficiently complemented an Escherichia coli OMP decarboxylase mutant, permitting it to grow in the absence of uracil. Electroporation of M. xanthus with a circular plasmid carrying a selectable uraA::kan gene disruption resulted in homologous recombination at the chromosomal uraA locus. Chromosomal integration of the gene disruption plasmid created heterozygous (uraA+/uraA::kan) tandem duplications. These tandem duplications were unstable and segregated auxotrophic uraA::kan daughters at frequencies of 2 x 10(-4) to 8 x 10(-4) per viable cell. Rare uraA::kan segregants were easily obtained by selecting for resistance to the toxic analog 5-fluoroorotic acid. Our experiments suggest that the cloned uraA gene could facilitate the use of gene duplications in the genetic analysis of M. xanthus development. The uraA mutants could utilize uracil, uridine, or uridine 5'-phosphate for growth, indicating that M. xanthus has pyrimidine salvage pathways. During multicellular development, uraA::kan gene disruption mutants sporulated to wild-type levels but formed smaller and more numerous aggregates than did their uraA+ parent, regardless of whether uracil was added to the medium. Pyrimidine deprivation of uraA mutants, under conditions that otherwise supported vegetative growth, failed to induce fruiting-body development or sporulation.

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Section: Resultsmentioning

confidence: 99%

Targeted disruption of the Myxococcus xanthus orotidine 5'-monophosphate decarboxylase gene: effects on growth and fruiting-body development

Kimsey

Kaiser

1991

J Bacteriol

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

et al. 1991

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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.

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Sequence analysis of the DdPYR5-6gene coding for UMP synthase in Dictyostelium discoideum and comparison with orotate phosphoribosyl transferases and OMP decarboxylases

1988

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A Dictyostelium discoideum DNA fragment that complements the ura3 and the ura5 mutants of Saccharomyces cerevisiae has been sequenced. It contains an open reading frame of 478 codons capable of encoding a polypeptide of molecular weight 52475. This gene, named DdPYR5-6, encodes a bifunctional protein composed of the orotate phosphoribosyl transferase (OPRTase) and the orotidine-5'-phosphate decarboxylase (OMPdecase) domains described for UMP synthase in mammals. The existence of separate domains for the two activities was suspected because deletion of the N-terminal coding segment of the gene eliminated the ura5 but not the ura3 complementing activity. We have now confirmed that the two parts of the open reading frame share homology with known OPRTase and OMPdecase sequences. Several blocks of sequence are conserved among OPRTase from bacteria, fungi and slime mold and one of them corresponds to the consensus sequence for phosphoribosylbinding sites. The OMPdecase domain shows extensive similarity with the yeast and Neurospora crassa enzymes, suggesting that they have evolved from an ancestral gene which was fused to the OPRTase gene in D. discoideum. It is less related to the bacterial enzyme but all these sequences present conserved blocks of homology which could identify the active site. The codon usage is strongly biased in a manner similar to that found for other D. discoideum genes. The flanking DNA contains homopolymers of A and T and alternating sequences that are characteristic of the gene organization in D. discoideum.

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Developmental control of the expression of the dihydroorotate dehydrogenase and UMP synthase genes in Dictyostelium discoideum

Cited by 9 publications

References 12 publications

Targeted disruption of the Myxococcus xanthus orotidine 5'-monophosphate decarboxylase gene: effects on growth and fruiting-body development

Targeted disruption of the Myxococcus xanthus orotidine 5'-monophosphate decarboxylase gene: effects on growth and fruiting-body development

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

Sequence analysis of the DdPYR5-6gene coding for UMP synthase in Dictyostelium discoideum and comparison with orotate phosphoribosyl transferases and OMP decarboxylases

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