Molecular and General Genetics MGG

, Volume 231, Issue 1, pp 7–16

Cloning, sequencing and characterization of the Saccharomyces cerevisiae URA7 gene encoding CTP synthetase

  • Odile Ozier-Kalogeropoulos
  • Franco Fasiolo
  • Marie-Therèse Adeline
  • Jocelyne Collin
  • François Lacroute
Article

Summary

The URA7 gene of Saccharomyces cerevisiae encodes CTP synthetase (EC 6.3.4.2) which catalyses the conversion of uridine 5′-triphosphate to cytidine 5′-triphosphate, the last step of the pyrimidine biosynthetic pathway. We have cloned and sequenced the URA 7 gene. The coding region is 1710 by long and the deduced protein sequence shows a strong degree of homology with bacterial and human CTP synthetases. Gene disruption shows that URA7 is not an essential gene: the level of the intracellular CTP pool is roughly the same in the deleted and the wild-type strains, suggesting that an alternative pathway for CTP synthesis exists in yeast. This could involve either a divergent duplicated gene or a different route beginning with the amination of uridine mono- or diphosphate.

Key words

Saccharomyces cerevisiae CTP synthetase Pyrimidine pathway Intracellular pyrimidine pool Non-essential gene 

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References

  1. Amuro N, Paluh JL, Zalkin H (1985) Replacement by site-directed mutagenesis indicates a role for histidine 170 in the glutamine amide transfer function of anthranilate synthase. J Biol Chem 260:14844–14849Google Scholar
  2. Basson ME, Moore RL, O'Rear J, Rine J (1987) Identifying mutations in duplicated functions in Saccharomyces cerevisiae: recessive mutations in HMG-CoA reductase genes. Genetics 117:645–655Google Scholar
  3. Beggs JD (1978) Transformation of yeast by a replicating hybrid plasmid. Nature 275:104–109Google Scholar
  4. Berk AJ, Sharp PA (1977) Sizing and mapping of early adenovirus mRNA3 by gel electrophoresis of S1 endonuclease digested hybrids. Cell 12:721–732Google Scholar
  5. Birnboim HC, Doty J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523Google Scholar
  6. Bonneaud N, Ozier-Kalogeropoulos O, Li G, Labouesse M, Minvielle-Sebastia L, Lacroute F (1991) A family of low and high copy replicatine, integrative and single-stranded S. cerevisiae/E. coli shuttle vectors. Yeast 7:609–615Google Scholar
  7. Bradford M (1976) A rapid and sensitive method for the quantitation of μg quantities of protein utilizing the principle of proteindye binding. Anal Biochem 72:248–254Google Scholar
  8. Cherest H, Surdin-Kerjan Y, Exinger F, Lacroute F (1978) S-Adenosyl methionine requiring mutants in Saccharomyces cerevisiae: Evidence for the existence of two methionine adenosyl transferases. Mol Gen Genet 163:153–167Google Scholar
  9. Chevallier MR, Bloch J-C, Lacroute F (1980) Transcriptional and translational expression of a chimeric bacterial-yeast plasmid in yeast. Gene 11:11–19Google Scholar
  10. de Montigny J, Belarbi A, Hubert J-C, Lacroute F (1989) Structure and expression of the URA5 gene of Saccharomyces cerevisiae. Mol Gen Genet 215:455–462Google Scholar
  11. de Montigny J, Kern L, Hubert J, Lacroute F (1990) Cloning and sequencing of URAJO, a second gene encoding orotate phosphoribosyl transferase in Saccharomyces cerevisiae. Curr Genet 17:105–111Google Scholar
  12. Elledge SJ, Davis RW (1990) Two genes differentially regulated in the cell cycle and by DNA-damaging agents encode alternative regulatory subunits of ribonucleotide reductase. Genes Dev 4:740–751Google Scholar
  13. Freeze E, Olempska-Beer Z, Eisenberg M (1984) Nucleotide composition of cell extracts analysed by full-spectrum recording in high-performance liquid chromatography. J Chromatogr 284:125–142Google Scholar
  14. Higgins DG, Sharp PM (1988) CLUSTAL: a package for performing multiple sequence alignments on a microcomputer. Gene 73:237–244Google Scholar
  15. Hinnen A., Hicks JB, Fink GR (1978) Transformation of yeast. Proc Natl Acad Sci USA 75:1929–1933Google Scholar
  16. Hoffman NE, Liao JC (1977) Reversed phase high performance liquid chromatographic separations of nucleotides in the presence of solvophobic ions. Anal Chem 49:2231–2234Google Scholar
  17. Jund R, Lacroute F (1970) Genetic and physiological aspects of resistance to 5-fluoropyrimidines in Saccharomyces cerevisiae. J Bacteriol 102:607–615Google Scholar
  18. Kaplan JC, Merkel WK, Nichols BP (1985) Evolution of glutamine amidotransferase genes. Nucleotide sequences of the pabA genes from Salmonella typhimurium, Klebsiella aerogenes and Serratia marcescens. J Mol Biol 183:327–340Google Scholar
  19. Kelsall A, Meuth M (1988) Direct selection of Chinese hamster ovary strains deficient in CTP synthetase activity. Somat Cell Mot Genet 14:149–154Google Scholar
  20. Lepesant-Kejzlarova J, Lepesant J-A, Walle J, Billault A, Dedonder R (1975) Revision of the linkage map of Bacillus subtilis 168: Indications for circularity of the chromosome. J Biol Chem 259:2355–2359Google Scholar
  21. Liberman I (1956) Enzymatic amination of uridine triphosphate to cytidine triphosphate. J Biol Chem 222:765–775Google Scholar
  22. Liljelund P, Lacroute F (1986) Genetic characterization and isolation of the Saccharomyces cerevisiae gene coding for uridine monophosphokinase. Mol Gen Genet 205:74–81Google Scholar
  23. Liljelund P, Sanni A, Friesen JD, Lacroute F (1989) Primary structure of the S. cerevisiae gene encoding uridine monophosphokinase. Biochem Biophys Res Commun 165:464–473Google Scholar
  24. Linder P, Slonimski PP (1989) An essential yeast protein, encoded by duplicated genes TIFJ and homologous to the mammalian translation initiation factor eIF-4A, can suppress a mitochondrial missense mutation. Proc Natl Acad Sci USA 86:2286–2290Google Scholar
  25. Mark C (1988) “DNA strider”: a “C” program for the fast analysis of DNA and protein sequences on the Apple Macintosh family of computers. Nucleic Acids Res 16:1829–1836Google Scholar
  26. McPartland RP, Weinfeld H (1979) Cooperative effects of CTP on calf liver CTP synthetase. J Biol Chem 4:1792–1799Google Scholar
  27. Meuth M (1989) The molecular basis of mutations induced by deoxyribonucleoside triphosphate pool imbalances in mammalian cells. Exp Cell Res 181:305–316Google Scholar
  28. Montgomery DL, Leung DW, Smith M, Shalit P, Faye G, Hall BD (1980) Isolation and sequence of the gene for iso-2-cytochrome c in Saccharomyces cerevisiae. Proc Natl Acad Sci USA 77:541–545Google Scholar
  29. Mortimer RK, Hawthorne DC (1966) Genetic mapping in Saccharomyces cerevisiae. Genetics 53:165–173Google Scholar
  30. Neuhard J (1983) Utilization of preformed pyrimidine bases and nucleotides. In: Munch-Petersen A (ed) Metabolism of nucleotides, nucleosides and nucleobases in microorganisms. Academic Press, London, pp 95–148Google Scholar
  31. Nichols BP, Miozzari GF, van Cleemput M, Bennett GN, Yanofsky C (1980) Nucleotide sequences of the trpG regions of Escherichia, Shigella dysenteriae, Salmonella typhimurium and Serratia marcescens. J Mol Biol 142:503–517Google Scholar
  32. Olempska-Beer Z, Bautz Freeze E (1984) Optimal extraction conditions for high-performance liquid chromatographic determination of nucleotides in yeast. Anal Biochem 140:236–245Google Scholar
  33. Orlean P (1987) Two chitin synthases in Saccharomyces cerevisiae. J Biol Chem 262:5732–5739Google Scholar
  34. Orr-Weaver TL, Szostak JW, Rothstein R (1983) Genetic application of yeast transformation with linear and gapped plasmids. Methods Enzymol 101:228–245Google Scholar
  35. Paluh JL, Zalkin H, Betsch D, Weith HL (1985) Study of anthranilate synthase function by replacement of cysteine 84 using sitedirected mutagenesis. J Biol Chem 260:1889–1894Google Scholar
  36. Pearson WR, Lipman DJ (1988) Improved tools for biological sequence comparison. Proc Natl Acad Sci USA 85:2444–2448Google Scholar
  37. Perret D (1987) Nucleotides, nucleosides and bases. In: Lim CK (ed) Hplc of small molecules, a practical approach. IRL Press, Oxford Washington DC, pp 221–259Google Scholar
  38. Pierard A, Glansdoff N, Gigot D, Crabeel M, Halleux P, Thiry L (1976) Repression of Escherichia coli carbamoylphosphate synthetase: relationships with enzyme synthesis in the arginine and pyrimidine pathways. J Bacteriol 127:291–301Google Scholar
  39. Piette J, Nyunoya C, Lusty CJ, Cunin R, Weyens G, Crabeel M, Charlier D, Glansdorff N, Piérard A (1984) DNA sequence of the carA gene and the control region of carAB. Tandem promoters, respectively controlled by arginine and the pyrimidines, regulate the synthesis of carbamoyl-phosphate synthetase in Escherichia coli K-12. Proc Natl Acad Sci USA 81:4134–4138Google Scholar
  40. Randerath K, Randerath E (1967) Thin-layer separation methods for nucleic acid derivatives. Methods Enzymol 12:323–347Google Scholar
  41. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, New YorkGoogle Scholar
  42. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Sci SA 74:5463–5467Google Scholar
  43. Sharp PM, Li WH (1987) The codon adaptation index: a measure of directional synonymous codon usage bias, and its potential applications. Nucleic Acids Res 15:1281–1295Google Scholar
  44. Sherman F, Fink GR, Hicks JB (1986) Laboratory course manual for methods in yeast genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New YorkGoogle Scholar
  45. Souciet JL, Potier S, Hubert JC, Lacroute F (1987) Nucleotide sequence of the pyrimidine specific carbamoylphosphate syn thetase, a part of the yeast multifunctional protein encoded by the URA2 gene. Mol Gene Genet 207:314–319Google Scholar
  46. Trach K, Chapman JW, Piggot P, Lecoq D, Hoch JA (1988) Complete sequence and transcriptional analysis of the spoOF region of the Bacillus subtilis chromosome. J Bacteriol 170:4194–4208Google Scholar
  47. Waleh NS, Ingraham JL (1976) Pyrimidine ribonucleoside monophosphokinase and the mode of RNA turnover in Bacillus subtilis. Arch Microbiol 110:49–54Google Scholar
  48. Weng M, Makaroff CA, Zalkin H (1986) Nucleotide sequence of Escherichia coli pyrG encoding CTP synthetase. J Biol Chem 261:5568–5574Google Scholar
  49. Williams JC, Kizaki H, Weiss E, Weber G (1978) Improved radioisotopic assay for cytidine 5′-triphosphate synthetase (EC 6.3.4.2). Anal Biochem 91:46–59Google Scholar
  50. Yamauchi M, Yamauchi N, Meuth M (1990) Molecular cloning of the CTP synthetase gene by functional complementation with purified human metaphase chromosomes. EMBO J 9:2095–2099Google Scholar
  51. Yanisch-Perron C, Viera J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequence of the Ml3mp18 and pUC19 vectors. Gene 33:103–119Google Scholar
  52. Zalkin H, Argos P, Narayana SVL, Tiedeman AA, Smith JM (1985) Identification of a trpG-related glutamine amide transfer domain in Escherichia coli GMP synthetase. J Biol Chem 260:3350–3354Google Scholar
  53. Zaret SK, Sherman F (1982) DNA sequence required for efficient transcription termination in yeast. Cell 28:563Google Scholar

Copyright information

© Springer-Verlag 1991

Authors and Affiliations

  • Odile Ozier-Kalogeropoulos
    • 1
  • Franco Fasiolo
    • 2
  • Marie-Therèse Adeline
    • 3
  • Jocelyne Collin
    • 1
  • François Lacroute
    • 1
  1. 1.Centre de Génétique Moléculaire du C.N.R.S.Laboratoire propre associé à l'Université Pierre et Marie CurieGif-sur-Yvette CedexFrance
  2. 2.I.B.M.C. du C.N.R.S.Strasbourg CedexFrance
  3. 3.Institut de Chimie des Substances Naturelles du C.N.R.S.Gif-sur-Yvette CedexFrance

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