Home LiteratureArticle Details
PMID: 2017144 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

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

Molecular & general genetics : MGG ·Vol. 225 ·No. 3 ·1991-03-00 ·Pages 492-500

Kalpaxis D, Zündorf I, Werner H, Reindl N, Boy-Marcotte E, Jacquet M, Dingermann T

Abstract

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.

Related Genes
DdPYR5-6
MeSH Terms
Blotting, Southern Dictyostelium/drug effects,enzymology,genetics,isolation & purification Drug Resistance, Microbial/genetics Genes, Fungal Genotype Kinetics Multienzyme Complexes/genetics,metabolism Mutation Orotate Phosphoribosyltransferase/genetics,metabolism Orotic Acid/analogs & derivatives,pharmacology Orotidine-5'-Phosphate Decarboxylase/genetics,metabolism Phenotype Plasmids Restriction Mapping Transformation, Genetic
Chemicals
Multienzyme Complexes Orotic Acid uridine 5'-monophosphate synthase 5-fluoroorotic acid Orotate Phosphoribosyltransferase Orotidine-5'-Phosphate Decarboxylase
Authors & Affiliations
7 authors, click to expand affiliations / ORCID
Kalpaxis D
Institut für Biochemie der Medizinischen Fakultät, Universität Erlangen-Nürnberg, Federal Republic of Germany.
Zündorf I
Werner H
Reindl N
Boy-Marcotte E
Jacquet M
Dingermann T
References (40)
40 references, click to expand
  1. DNA-mediated transformation in Dictyostelium discoideum: regulated expression of an actin gene fusion.
    Mol Cell Biol. 1984 Dec;4(12):2890-8 PMID: 6098825
  2. Genetics of the cellular slime molds.
    Annu Rev Genet. 1978;12:69-93 PMID: 371534
  3. Two vectors which facilitate gene manipulation and a simplified transformation procedure for Dictyostelium discoideum.
    Gene. 1987;59(1):99-106 PMID: 3436528
  4. Developmental control of the expression of the dihydroorotate dehydrogenase and UMP synthase genes in Dictyostelium discoideum.
    Cell Differ. 1988 Jan;22(2):159-64 PMID: 2830032
  5. Biochemical characterization of fluoropyrimidine-resistant murine leukemic cell lines.
    Cancer Res. 1982 Mar;42(3):965-73 PMID: 7059992
  6. Homologous recombination in the Dictyostelium alpha-actinin gene leads to an altered mRNA and lack of the protein.
    EMBO J. 1987 Dec 20;6(13):4143-8 PMID: 2832153
  7. Inactivation of the alpha-actinin gene in Dictyostelium.
    Dev Genet. 1988;9(4-5):531-8 PMID: 3243033
  8. Linkage Analysis of Nystatin Resistance Mutations in DICTYOSTELIUM DISCOIDEUM.
    Genetics. 1986 May;113(1):53-62 PMID: 17246327
  9. A DNA sequence from Dictyostelium discoideum complements ura3 and ura5 mutations of Saccharomyces cerevisiae.
    Mol Gen Genet. 1984;193(3):406-13 PMID: 6323917
  10. The discoidin I gene family of Dictyostelium discoideum is linked to genes regulating its expression.
    Genetics. 1988 Jul;119(3):571-8 PMID: 3402731
  11. Growth of myxameobae of the cellular slime mould Dictyostelium discoideum in axenic culture.
    Biochem J. 1970 Sep;119(2):171-4 PMID: 5530748
  12. Interconversion of different molecular weight forms of the orotate phosphoribosyltransferase.orotidine-5'-phosphate decarboxylase enzyme complex from mouse Ehrlich ascites cells.
    J Biol Chem. 1979 Feb 25;254(4):1143-50 PMID: 762120
  13. Regulatory sequences in the promoter of the Dictyostelium Actin 6 gene.
    EMBO J. 1986 Dec 1;5(12):3367-72 PMID: 3816763
  14. Conversion of uracil and orotate to uridine 5'-phosphate by enzymes in lactobacilli.
    J Biol Chem. 1957 Jun;226(2):1093-101 PMID: 13438895
  15. Cyclic AMP and other signals controlling cell development and differentiation in Dictyostelium.
    Annu Rev Biochem. 1987;56:853-79 PMID: 3039908
  16. Dictyostelium discoideum: a useful model system to evaluate the function of queuine and of the Q-family of tRNAs.
    FEBS Lett. 1982 Sep 20;146(2):311-4 PMID: 7140979
  17. A Dictyostelium discoideum DNA fragment complements a Saccharomyces cerevisiae ura3 mutant.
    Gene. 1982 Dec;20(3):433-39 PMID: 6762322
  18. RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination.
    Biochemistry. 1977 Oct 18;16(21):4743-51 PMID: 911786
  19. Coordination of the synthesis of the enzymes in the pyrimidine pathway of E. coli.
    J Mol Biol. 1962 Dec;5:618-34 PMID: 13970180
  20. Developmental regulation of Dictyostelium discoideum actin gene fusions carried on low-copy and high-copy transformation vectors.
    Mol Cell Biol. 1986 Nov;6(11):3973-83 PMID: 3025622
  21. Isolation of developmentally regulated genes from Dictyostelium.
    Dev Biol. 1980 Oct;79(2):409-18 PMID: 7429011
  22. pISAR, a tool for cloning genomic sequences adjacent to the site of vector integration.
    Nucleic Acids Res. 1989 Jun 26;17(12):4894 PMID: 2748348
  23. High-copy-number transformants and co-transformation in Dictyostelium.
    Gene. 1985;39(2-3):155-63 PMID: 4092928
  24. A defined minimal medium for axenic strains of Dictyostelium discoideum.
    Proc Natl Acad Sci U S A. 1977 May;74(5):2157-61 PMID: 17120
  25. Transformation of yeast.
    Proc Natl Acad Sci U S A. 1978 Apr;75(4):1929-33 PMID: 347451
  26. A defined, synthetic growth medium for Dictyostelium discoideum, strain Ax-2.
    Hoppe Seylers Z Physiol Chem. 1977 Apr;358(4):527-9 PMID: 558953
  27. Disruption of the Dictyostelium myosin heavy chain gene by homologous recombination.
    Science. 1987 May 29;236(4805):1086-91 PMID: 3576222
  28. Auxotrophic mutants of Dictyostelium discoideum.
    Nature. 1978 Apr 6;272(5653):537-8 PMID: 29228
  29. Gene replacement in Dictyostelium: generation of myosin null mutants.
    EMBO J. 1989 Mar;8(3):923-32 PMID: 2721503
  30. Inheritance of extrachromosomal ribosomal DNA during the asexual life cycle of Dictyostelium discoideum: examination by use of DNA polymorphisms.
    Mol Cell Biol. 1985 Feb;5(2):273-80 PMID: 2983186
  31. Molecular complementation of a genetic marker in Dictyostelium using a genomic DNA library.
    Proc Natl Acad Sci U S A. 1989 Oct;86(20):7966-70 PMID: 2813371
  32. Complementation of a Dictyostelium discoideum thymidylate synthase mutation with the mouse gene provides a new selectable marker for transformation.
    Nucleic Acids Res. 1989 May 25;17(10):3655-61 PMID: 2734099
  33. Chromosomal mapping of tRNA genes from Dictyostelium discoideum.
    Mol Gen Genet. 1987 Apr;207(1):176-87 PMID: 3474495
  34. A positive selection for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance.
    Mol Gen Genet. 1984;197(2):345-6 PMID: 6394957
  35. PATHWAY OF PYRIMIDINE BIOSYNTHESIS IN PREMETAMORPHIC TADPOLES.
    J Biol Chem. 1964 Jul;239:2239-45 PMID: 14209953
  36. Homologous recombination and the repair of double-strand breaks during cotransformation of Dictyostelium discoideum.
    Mol Cell Biol. 1988 Jul;8(7):2779-86 PMID: 2841587
  37. Regulation of dictyostelium discoideum mRNAs specific for prespore or prestalk cells.
    Cell. 1983 Apr;32(4):1139-48 PMID: 6301681
  38. Hygromycin resistance as a selectable marker in Dictyostelium discoideum.
    Mol Cell Biol. 1989 May;9(5):1965-8 PMID: 2546056
  39. Sequence analysis of the DdPYR5-6 gene coding for UMP synthase in Dictyostelium discoideum and comparison with orotate phosphoribosyl transferases and OMP decarboxylases.
    Mol Gen Genet. 1988 Mar;211(3):441-5 PMID: 2835631
  40. A genetic map of Dictyostelium discoideum based on mitotic recombination.
    Genetics. 1982 Dec;102(4):691-710 PMID: 7187362
Article Info
Journal
Molecular & general genetics : MGG
Abbr.
Mol Gen Genet
ISSN
0026-8925
DOI
10.1007/BF00261692
Published
1991-03-00
Pages
492-500
Language
English
Region
Germany
NLM ID
0125036
Subset
IM
External Links
PubMed source DOI: 10.1007/BF00261692
Analysis Services
Analysis Services

Contact

No. 2 Wenbo Road, Zhangqiu District, Jinan, Shandong

Qilu Normal University · Genelibs Bioinformatics Lab

750 Shunhua Rd, Jinan

2F, Bldg F, University Science Park

Tel: 0531-88819269

WeChat Official Account

Follow our WeChat subscription account for real-time updates and the latest in medical and biological research.


Business Email

E-mail: [email protected]

Copyright © 2026.Genelibs Jinan Honghui Biotechnology Co., Ltd. 鲁ICP备20007159号-2