A positive selection for mutants lacking orotidine-5′-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance

@article{Boeke2004APS,
  title={A positive selection for mutants lacking orotidine-5′-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance},
  author={Jef D. Boeke and Françoise Croute and Gerald R. Fink},
  journal={Molecular and General Genetics MGG},
  year={2004},
  volume={197},
  pages={345-346}
}
SummaryMutations at the URA3 locus of Saccharomyces cerevisiae can be obtained by a positive selection. Wild-type strains of yeast (or ura3 mutant strains containing a plasmid-borne URA3+ gene) are unable to grow on medium containing the pyrimidine analog 5-fluoro-orotic acid, whereas ura3− mutants grow normally. This selection, based on the loss of orotidine-5′-phosphate decarboxylase activity seems applicable to a variety of eucaryotic and procaryotic cells. 

Tables from this paper

Cloning and sequencing of the ura3 locus of the methylotrophic yeast Hansenula polymorpha and its use for the generation of a deletion by gene replacement
TLDR
The results show that gene replacement can be achieved in H. polymorpha, a yeast with a high level of non-homologous integration.
Cloning of orotidine-5′-phosphate decarboxylase (URA3) gene from sourdough yeast Candida milleri CBS 8195
TLDR
The cloned URA3 gene successfully complemented the ura3 mutation in S. cerevisiae, indicating that it encodes a functional OMPDCase in C. millieri CBS 8195.
Isolation of temperature-sensitive Saccharomyces cerevisiae with a mutation in erg25 for C-4 sterol methyl oxidase.
TLDR
Sequence analysis of the mERG25 mutant indicated three amino acid substitutions in ERG25p, namely N48D, V133A, and F135S, which indicate that the ERG 25 gene product is a new antifungal target.
Cloning of the PYR4 gene encoding orotidine-5′-phosphate decarboxylase in Cephalosporium acremonium
TLDR
The C. acremonium pyr4 gene complements an E. coli pyrF mutant lacking orotidine-5′-phosphate decarboxylase (OMPdecase), and most probably does not contain introns.
Use of synthetic lethal mutants to clone and characterize a novel CTP synthetase gene in Saccharomyces cerevisiae
TLDR
The present paper describes the selection of synthetic lethal mutants in the CTP biosynthetic pathway that led us to clone a second gene, named URA8, which also encodes a CTP synthetase, and based on the codon bias values for the two genes and the intracellular concentrations of CTP in strains deleted for one of theTwo genes, relative to the wild-type level, URA7 appears to be the major gene for C TP biosynthesis.
Adaptation of the Yeast URA3 Selection System to Gram-Negative Bacteria and Generation of a ΔbetCDE Pseudomonas putida Strain
TLDR
The versatility of pyrF as a selection system, allowing both positive and negative selection of the marker, and the robustness of the selection, make this setup a powerful tool for efficient homologous gene replacement in gram-negative bacteria.
Transformation of Aspergillus niger using the homologous orotidine-5′-phosphate-decarboxylase gene
TLDR
Analysis of the DNA of the A. niger PyrA+ transformants showed that transformation resulted in integration of the vector DNA into the genome by homologous recombination, and both gene replacements and integration of one or more copies of the complete vector have been observed.
Cloning of the C-URA3 gene and construction of a triple auxotroph (his5, ade1, ura3) as a useful host for the genetic engineering of Candida maltosa
TLDR
The C-URA3 gene of the n-alkane assimilating-yeast Candida maltosa was cloned by complementation of the ura3 mutation of Saccharomyces cerevisiae by selecting for spontaneous 5-fluoro-orotic acid (5FOA) resistance.
Selection and characterization of pyrG mutants of Penicillium chrysogenum lacking orotidine-5′-phosphate decarboxylase and complementation by the pyr4 gene of Neurospora crassaa
TLDR
Pyrimidine auxotrophs of Penicillium chrysogenum have been isolated at a high frequency among mutants resistant to 5-fluoroorotic acid, and a radiometric assay based on the conversion of OMP-decarboxylase into (6-14C)orotidine 5′-monophosphate (OMP) into ( 6- 14C)uridine5′-Monophosphates (UMP).
Isolation and characterization of temperature-sensitiveplc1 mutants of the yeastSaccharomyces cerevisiae
TLDR
The PLC1 gene of the yeast Saccharomyces cerevisiae has been discovered to encode a homolog of mammalian phosphoinositide-specific phospholipase C (PLC), indicating that PLC activity itself is important for cell growth.
...
1
2
3
4
5
...

References

SHOWING 1-10 OF 12 REFERENCES
Regulation of Orotidylic Acid Pyrophosphorylase in Saccharomyces cerevisiae
TLDR
Yeast mutants deficient in orotidine-5'-phosphate (OMP) pyrophosphorylase activity have been obtained and this enzyme appears to escape the scheme of regulation by mixed repression and induction controlling the other enzymes of the pyrimidine pathway.
Evidence for transcriptional regulation of orotidine-5'-phosphate decarboxylase in yeast by hybridization of mRNA to the yeast structural gene cloned in Escherichia coli.
TLDR
Data support the hypothesis that regulation of the ura3 gene in yeast is at the level of transcription, as a coordinate variation in proportion of labeled RNA complementary to the hybrid plasmid was found.
Mutations affecting Ty-mediated expression of the HIS4 gene of Saccharomyces cerevisiae.
TLDR
Seven unlinked genes (SPT genes) that affect the phenotypes of Ty and delta insertion mutations in the 5' noncoding region of the HIS4 gene of S. cerevisiae are identified.
Selection of lys2 Mutants of the Yeast SACCHAROMYCES CEREVISIAE by the Utilization of alpha-AMINOADIPATE.
TLDR
The use of alpha-aminoadipate medium has considerable utility as a selective medium for lys2 and lys5 mutants as well as other possible auxotrophic requirements.
Direct selective techniques for the isolation of pyrimidine auxotrophs in yeast
SummarySelective techniques have been developed to obtain auxotrophic mutants in four of the five loci controlling the enzymes involved in Yeast pyrimidine biosynthesis. These loci are thus available
Genetic and physiological characterization of met15 mutants of Saccharomyces cerevisiae: a selective system for forward and reverse mutations.
TLDR
It was established that a variety of mutational types, including missense, nonsense, and deletions, are recovered with this unique system in which both forward and reverse mutations can be selected on the basis of methyl mercury resistance and methionine requirement of the met15 mutants.
Eviction and transplacement of mutant genes in yeast.
Identification of a Ty insertion within the coding sequence of the S. cerevisiae URA3 gene
TLDR
Experiments presented here demonstrate that the ura3-52 mutation, a non-reverting ura 3 mutation, is caused by a Ty insertion mutation within the coding region of the URA3 gene.
Structure and function of the yeast URA3 gene: expression in Escherichia coli.
...
1
2
...