Current Genetics

, Volume 28, Issue 2, pp 138–149 | Cite as

As in Saccharomyces cerevisiae, aspartate transcarbamoylase is assembled on a multifunctional protein including a dihydroorotase-like cryptic domain in Schizosaccharomyces pombe

  • Marc Lollier
  • Laurence Jaquet
  • Triana Nedeva
  • François Lacroute
  • Serge Potier
  • Jean-Luc Souciet
Original Paper


The organisation of the URA1 gene of Schizosaccharomyces pombe was determined from the entire cDNA cloned by the transformation of an ATCase-deficient strain of Saccharomyces cerevisiae. The URA1 gene encodes the bifunctional protein GLNase/CPSase-ATCase which catalyses the first two steps of the pyrimidine biosynthesis pathway. The complete nucleotide sequence of the URA1 cDNA was elucidated and the deduced amino-acid sequence was used to define four domains in the protein; three functional domains, corresponding to GLNase (glutamine amidotransferase), CPSase (carbamoylphosphate synthetase) and ATCase (aspartate transcarbamoylase) activities, and one cryptic DHOase (dihydroorotase) domain. Genetic investigations confirmed that both GLNase/CPSase and ATCase activities are carried out by the same polypeptide. They are also both feedback-inhibited by UTP (uridine triphosphate). Its organization and regulation indicate that the S. pombe URA1 gene product appears very similar to the S. cerevisiae URA2 gene product.

Key words

Schizosaccharomyces pombe Pyrimidine pathway Feedback inhibition DHOase-like Multifunctional protein 


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  1. Bach ML (1987) Cloning and expression of the OMP decarboxylase gene URA4 from Schizasaccharomyces pombe. Curr Genet 12:527–534Google Scholar
  2. Becker DM, Guarente L (1991) High-efficency transformation of yeast by electroporation. Methods Enzymol 194:182–187Google Scholar
  3. Bradford M (1976) A rapid method for the quantification of Microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254Google Scholar
  4. Clewell DB, Helinski DR (1969) A supercoiled circular DNA-protein complex in Escherichia coli: purification and induced conversion to an opened circular DNA form. Proc Natl Acad Sci USA 62:1159–1166Google Scholar
  5. Davidson JN, Chen KC, Jamison RS, Musmanno LA, Kern CB (1993) The evolutionary history of the first three enzymes in pyrimidine biosynthesis. Bioessays 15:157–164Google Scholar
  6. Devereux J, Haeberli P, Smithies O (1984) A comprehensive set of sequence analyses programs for the VAX. Nucleic Acids Res 12:387–395Google Scholar
  7. Dower WJ, Miller JF, Ragsdale CW (1988) High-efficiency transformation of Escherichia coli by high-voltage electrophoresis. Nucleic Acids Res 16:6127–6145Google Scholar
  8. Faure M, Camonis JH, Jacquet M (1989) Molecular characterization of a Dictyostelium discoideum gene encoding a multifunctional enzyme of the pyrimidine pathway. Eur J Biochem 179:345–358Google Scholar
  9. Freund JN, Jarry BP (1987) The rudimentary gene of Drosophila melanogaster encodes four enzymatic functions. J Mol Biol 193:1–13Google Scholar
  10. Glansdorff N (1965) Topography of co-transductible arginine mutations in Escherichia coli K12. Genetics 51:167–179Google Scholar
  11. Guyonvarch A, Nguyen JM, Hubert JC, Lacroute F (1988) Structure of the Saccharomyces cerevisiae URA4 gene encoding dihydroorotase. Mol Gen Genet 212:134–141Google Scholar
  12. Gygax A, Thuriaux P (1984) A revised chromosome map of the fission yeast Schizosaccharomyces pombe. Curr Genet 8:85–92Google Scholar
  13. Hoffman CS, Winston F (1987) A ten-minute preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene 57:267–272Google Scholar
  14. Jaquet L, Lollier M, Souciet JL, Potier S (1993) Genetic analysis of yeast strains lacking negative feedback control — one-step method for positive selection and cloning of carbamoylphosphate synthetase-aspartate transcarbamoylase mutants unable to respond to UTP. Mol Gen Genet 241:81–88Google Scholar
  15. Jones ME (1980) Pyrimidine nucleotide biosynthesis in animals: genes, enzymes and regulation of UMP biosynthesis. Annu Rev Biochem 49:253–279Google Scholar
  16. Jund R, Lacroute F (1970) Genetic and physiological aspects of resistance to 5-fluoropyrimidines in Saccharomyces cerevisiae. J Bacteriol 102:607–615Google Scholar
  17. Kohli J (1987) Genetic nomenclature and gene list of the fission yeast Schizosaccharomyces pombe. Curr Genet 11:575–589Google Scholar
  18. Lacroute F (1968) Regulation of pyrimidine biosynthesis in Saccharomyces cerevisiae. J Bacteriol 95:824–832Google Scholar
  19. Lacroute F, Pierard A, Grenson M, Wiame JM (1965) The biosynthesis of carbamoylphosphate in Saccharomyces cerevisiae. J Gen Microbiol 40:127–142Google Scholar
  20. Loppes R, Michels R, Decroupette I, Joris B (1991) Sequence analysis of the ARG7 gene of Schizosaccharomyces pombe coding for arginosuccinate lyase: expression of the gene in Saccharomyces cerevisiae. Curr Genet 19:255–260Google Scholar
  21. Losson R, Fuchs RP, Lacroute F (1985) Yeast promoters URA1 and URA3. Examples of positive control. J Mol Biol 185: 65–81Google Scholar
  22. Megnet R (1958) Untersuchungen über die Biosynthese von Uracil bei Schizosaccharomyces pombe. Arch Julius Klaus-Stift XXXIII 3/4:299–334Google Scholar
  23. Minet M, Dufour ME, Lacroute F (1992) Complementation of Saccharomyces cerevisiae auxotrophic mutants by Arabidopsis thaliana cDNAs. The Plant Jour 2:417–422Google Scholar
  24. Mortimer RK, Hawthorne DC (1966) Genetic mapping in Saccharomyces cerevisiae. Genetics 53:165–173Google Scholar
  25. Nagy M, Lacroute F, Thomas D (1992) Divergent evolution of pyrimidine biosynthesis between anaerobic and aerobic yeasts. Proc Natl Acad Sci USA 89:8966–8970Google Scholar
  26. O'Donovan GA, Neuhard J (1970) Pyrimidine metabolism in microorganisms. Bacteriol Rev 34:278–343Google Scholar
  27. Padgett RA, Wahl GM, Starck GR (1982) Structure of the gene for CAD, the multifunctional protein that initiates UMP synthesis in Syrian hamster cells. Mol Cell Biol 209:283–289Google Scholar
  28. Potier S, Winsor B, Lacroute F (1982) Genetic selection for reciprocal translocation at chosen chromosomal sites in Saccharomyces cerevisiae. Mol Cell Biol 2:1025–1032Google Scholar
  29. Potier S, Souciet JL, Lacroute F (1987) Correlation between restriction map, genetic map and catalytic functions in the gene complex URA2. Mol Gen Genet 209:283–289Google Scholar
  30. Potier S, Lacroute F, Hubert JC, Souciet JL (1990) Studies on transcription of the yeast URA2 gene. FEMS Microbiol Lett 60:215–219Google Scholar
  31. Prescott LM, Jones ME (1969) Modified methods for the determination of carbamoylaspartate. Anal Biochem 32:408–419Google Scholar
  32. Russel P, Nurse P (1986) Schizosaccharomyces pombe and Saccharomyces cerevisiae: a look at yeasts divided. Cell 45:781–782Google Scholar
  33. Sakagushi J, Yamamoto M (1982) The cloned URA1 locus of Schizosaccharomyces pombe propagates autonomously in this yeast assuming a polymeric form. Proc Natl Acad Sci USA 79:7819–7823Google Scholar
  34. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, New YorkGoogle Scholar
  35. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467Google Scholar
  36. Sherman F, Hicks J (1991) Micromanipulation and dissection of asci. Methods Enzymol 194:21–37Google Scholar
  37. Souciet JL, Hubert JC, Lacroute F (1982) Cloning and restriction mapping of the yeast URA2 gene coding for the carbamylphosphate synthetase-aspartate transcarbamylase complex. Mol Gen Genet 186:385–390Google Scholar
  38. Souciet JL, Potier S, Hubert JC, Lacroute F (1987) Nucleotide sequence of the pyrimidine-specific carbamoylphosphate synthetase, a part of the yeast multifunctional protein encoded by the URA2 gene. Mol Gen Genet 207:314–319Google Scholar
  39. Souciet JL, Nagy M, Le Gouar GM, Lacroute F, Potier S (1989) Organization of the yeast URA2 gene: identification of a defective dihydroorotase-like domain in the multifunctional carbamoylphosphate synthetasé-aspartate transcarbamylase complex. Genetics 79:59–70Google Scholar
  40. Vissers S, Thuriaux P (1985) Genetical evidence of carbamoylphosphate compartmentation in Schizosaccharomyces pombe and Schizosaccharomyces japonicus. Curr Genet 9:561–565Google Scholar
  41. Wild JR, Wales ME (1990) Molecular evolution and genetic engineering of protein domains involving aspartate transcarbamoylase. Annu Rev Microbiol 44:193–218Google Scholar
  42. Winston F, Chumley F, Fink GR (1983) Eviction and transplacement of mutant genes in yeast. Methods Enzymol 101: 221–228Google Scholar
  43. Yamamoto M, Asakura Y, Yanagida M (1981) Cloning of a gene from the fission yeast S. pombe which complements E. coli pyrB, the gene for aspartate transcarbamylase. Mol Gen Genet 182:426–429Google Scholar
  44. Zimmermann BH, Evans DR (1993) Cloning, overexpression, and characterization of the functional dihydroorotase domain of the mammalian multifunctional protein CAD. Biochemistry 32:1519–1527Google Scholar

Copyright information

© Springer-Verlag 1995

Authors and Affiliations

  • Marc Lollier
    • 1
  • Laurence Jaquet
    • 1
  • Triana Nedeva
    • 2
  • François Lacroute
    • 3
  • Serge Potier
    • 1
  • Jean-Luc Souciet
    • 1
  1. 1.Laboratoire de Microbiologie et de GénétiqueURA n-1481 Université Louis-Pasteur/CNRSStrasbourg CedexFrance
  2. 2.Faculty of BiologyUniversity St Kliment OhridskiSofiaBulgaria
  3. 3.Centre de Génétique Moléculaire du CNRSGif-sur-Yvette CedexFrance

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