Abstract
In the pyrimidine biosynthetic pathway, CTP synthetase catalyses the conversion of uridine 5′-triphosphate (UTP) to cytidine 5′-triphosphate (CTP). In the yeast Saccharomyces cerevisiae, the URA7 gene encoding this enzyme was previously shown to be nonessential for cell viability. 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. Comparison of the predicted amino acid sequences of the products of URA7 and URA8 shows 78% identity. Deletion of the URA8 gene is viable in a haploid strain but simultaneous presence of null alleles both URA7 and URA8 is lethal. Based on the codon bias values for the two genes and the intracellular concentrations of CTP in strains deleted for one of the two genes, relative to the wild-type level, URA7 appears to be the major gene for CTP biosynthesis. Nevertheless, URA8 alone also allows yeast growth, at least under standard laboratory conditions.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
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 117:645–655
Basson ME, Thorsness M, Finer-Moore J, Stroud R, Rine J (1988) Structural and functional conservation between yeast and human 3-hydroxy-3-methylglutaryl coenzyme A reductases, the rate-limiting enzyme of sterol biosynthesis. Mol Cell Biol 8:3797–308
Bennetzen JL, Hall BD (1982) Codon selection in yeast. J Biol Chem 257:3026–3031
Boeke J, Lacroute F, Fink GR (1984) A positive selection for mutants lacking orotidine-5′ phosphate decarboxylase activity in yeast: 5-fluoroorotic acid resistance. Mol Gen Genet 197:345–346
Bonneaud N, Ozier-Kalogeropoulos O, Li G, Labouesse M, Minvielle-Sebastia L, Lacroute F (1991) A family of low and high copy replicative, integrative and single-stranded S. cerevisiae/E. coli shuttle vectors. Yeast 7:609–615
Bradford M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
de Korte D, Haverkort WA, Roos D, van Gennip AH (1985) Anion-exchange high performance liquid chromatography method for the quantification of nucleotides in human blood cells. Clin Chim Acta 148:185–196
de Montigny J, Kern L, Hubert J, Lacroute F (1990) Cloning and sequencing of URA10, a second gene encoding orotate phosphoribosyl transferase in Saccharomyces cerevisiae. Curr Genet 17:105–111
Elledge SJ, Davis RW (1990) Two genes diffentially regulated in the cell cycle and by DNA-damaging agents encode alternative regulatory subunits of ribonucleotide reductase. Genes Dev 4:740–751
Gietz D, St Jean A, Woods RA, Schiestl RH (1992) Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res 20:1425
Hartig A, Simon MM, Schuster T, Daugherty JR, Sook Yoo H, Cooper TG (1992) Differentially regulated malate synthase genes participate in carbon and nitrogen metabolism of S. cerevisiae. Nucleic Acids Res 20:5677–5686
Henikoff S (1984) Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28:351–359
Herbert CJ, Dujardin G, Labouesse M, Slonimski PP (1988) Divergence of the mitochondrial leucyl tRNA synthetase genes in two closely related yeasts Saccharomyces cerevisiae and Saccharomyces douglasii: A paradigm of incipient evolution. Mol Gen Genet 213:297–309
Jund R, Lacroute F (1970) Genetic and physiological aspects of resistance to 5-fluoropyrimidines in Saccharomyces cerevisiae. J Bacteriol 102:607–615
Klig LS, Homann MJ, Carman GM, Henry SA (1985) Coordinate regulation of phospholipid biosynthesis in Saccharomyces cerevisiae: pleiotropically constitutive opil mutant. J Bacteriol 162:1135–1141
Lacroute F, Piérard A, Grenson M, Wiame JM (1965) The biosynthesis of carbamoyl phosphate in Saccharomyces cerevisiae. J Gen Microbiol 40:127–142
Lalo D, Stettler S, Mariotte S, Slonimski PP, Thuriaux P (1993) Two yeast chromosomes are related by a fossil duplication of their centromeric region. C R Acad Sci [III] 316:1–6
Liberman I (1956) Enzymatic amination of uridine triphosphate to cytidine triphosphate. J Biol Chem 222:765–775
Long CW, Pardee AB (1967) Cytidine triphosphate synthetase of Escherichia coli B. Purification and kinetics. J. Biol Chem 242:4715–4721
Mark C (1988) “Strider strider”: a “C” program for analysis of DNA and protein sequences on the Apple Macintosh family of computers. Nucleic Acids Res 16:1829–1836
Mortimer RK, Hawthorne DC (1966) Genetic mapping in Saccharomyces cerevisiae. Genetics 53:165–173
Mossé M-O, Linder P, Lazowska J, Slonimski PP (1992) A comprehensive compilation of 1001 nucleotide sequences encoding for proteins from the yeast Saccharomyces cerevisiae (=List A2). Curr Genet 21:66–91
Orr-Weaver TL, Szostak JW, Rothstein R (1983) Genetic application of yeast transformation with linear and gapped plasmids. Methods Enzymol 101:228–245
Ozier-Kalogeropoulos O, Fasiolo F, Adeline M, Collin J, Lacroute F (1991) Cloning, sequencing and characterization of the Saccharomyces cerevisiae URA7 gene encoding CTP synthetase. Mol Gen Genet 231:7–16
Rose MD, Winston F, Hieter P (1990) Methods in yeast genetics: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, second edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Schricker R, Magdolen V, Kaniak A, Wolf K, Bandlow W (1992) The adenylate kinase family in yeast: identification of URA6 as a multicopy suppressor of deficiency in major AMP kinase. Gene 122:111–118
Sherman F, Fink GR, Hicks JB (1986) Laboratory course manual for methods in yeast genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
Stettler S, Chiannilkulchai N, Herman-Le-Denmat S, Lalo D, Lacroute F, Sentenac A, Thuriaux P (1993) A general suppressor of RNA polymerase I, II, III mutations in Saccharomyces cerevisiae. Mol Gen Genet 239:169–176
Snow R (1966) An enrichment method for auxotrophic yeast mutants using the antibiotic Nystatin. Nature 211:206–207
Trach K, Chapman JW, Piggot P, Lecoq D, Hoch JA (1988) Complete sequence and transcriptionnal analysis of the spo0F region of the Bacillus subtilis chromosome. J Bacteriol 170:4194–4208
Weng M, Makaroff CA, Zalkin H (1986) Nucleotide sequence of Escherichia coli pyrG encoding CTP synthetase. J Biol Chem 261:5568–5574
Yamauchi M, Yamauchi N, Meuth M (1990) Molecular cloning of the CTP synthetase gene by functional complementation with purified human metaphase chromosomes. EMBO J 9:2095–2099
Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequence of the M 13 mp18 and pUC19 vectors. Gene 33:103–119
Author information
Authors and Affiliations
Additional information
Communicated by C. Hollenberg
Rights and permissions
About this article
Cite this article
Ozier-Kalogeropoulos, O., Adeline, MT., Yang, WL. et al. Use of synthetic lethal mutants to clone and characterize a novel CTP synthetase gene in Saccharomyces cerevisiae . Molec. Gen. Genet. 242, 431–439 (1994). https://doi.org/10.1007/BF00281793
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00281793