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DNA sequences in chromosomes 11 and VII code for pyruvate carboxylase isoenzymes in Saccharomyces cerevisiae: analysis of pyruvate carboxylase-deficient strains

Summary

A gene encoding pyruvate carboxylase has previously been isolated from Saccharomyces cerevisiae. We have isolated a second gene, PYC2, from the same organism also encoding a pyruvate carboxylase. The gene PYC2 is situated on the right arm of chromosome II between the DUR 1, 2 markers and the telomere. We localized the previously isolated gene, which we designate PYC1, to chromosome VII. Disruption of either of the genes did not produce marked changes in the phenotype. However, simultaneous disruption of both genes resulted in inability to grow on glucose as sole carbon source, unless aspartate was added to the medium. This indicates that in wild-type yeast there is no bypass for the reaction catalysed by pyruvate carboxylase. The coding regions of both genes exhibit a homology of 90% at the amino acid level and 85% at the nucleotide level. No appreciable homology was found in the corresponding flanking regions. No differences in the K m values for ATP or pyruvate were observed between the enzymes obtained from strains carrying inactive, disrupted versions of one or other of the genes.

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References

  1. Bennetzen JL, Hall BB (1982) Codon selection in yeast. J Biol Chem 257:3026–3031

  2. Broach JR, Strathern JN, Hicks JB (1979) Transformation in yeast: Development of a hybrid cloning vector and isolation of the CANT gene. Gene 8:121–133

  3. Buchman AR, Kimmerly WJ, Rine J, Kornberg RD (1988) Two DNA-binding factors recognize specific sequences at silencers, upstream activating sequences, autonomously replicating sequences, and telomeres in Saccharomyces cerevisiae. Mol Cell Biol 8:210–225

  4. Burgers PJM, Percival KJ (1987) Transformation of yeast spheroplasts without cell fusion. Anal Biochem 163:391–397

  5. Cazzulo JJ, Claisse LM, Stoppani AOM (1968) Carboxylase levels and carbon dioxide fixation in baker's yeast. J Bacteriol 96:623–628

  6. Chambers A, Stanway C, Tsang JSH, Henry Y, Kingsman AJ, Kingsman SM (1990) ARS binding factor 1 binds adjacent to RAP1 at the UASs of the yeast glycolytic genes PGK and PYK1. Nucleic Acids Res 18:5393–5399

  7. Chasman DI, Lue NF, Buchman AR, La Pointe JW, Lorch Y, Kornberg RD (1990) A yeast protein that influences the chromatin structure of UASG and functions as a powerful auxiliary gene activator. Genes Dev 4:503–514

  8. Cohen R, Holland JP, Yokoi T, Holland MJ (1986) Identification of a regulatory region that mediates glucose-dependent induction of the Saccharomyces cerevisiae enolase gene ENO2. Mol Cell Biol 6:2287–2297

  9. Dorsman JC, van Heerswijk WC, Grivell LA (1990) Yeast general transcription factor GF1: sequence requirements for binding to DNA and evolutionary conservation. Nucleic Acids Res 18:2769–2766

  10. Eigel A, Olah J, Feldmann H (1981) Structural comparison of two yeast tRNAGlu3 genes. Nucleic Acids Res 9:2961–2970

  11. Entian KD, Fröhlich KU (1984) Saccharomyces cerevisiae mutants provide evidence of hexokinase PII as a bifunctional enzyme with catalytic and regulatory domains for triggering carbon catabolite repression. J Bacteriol 158:29–35

  12. Feinberg AP, Vogelstein B (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6–13

  13. Genbauffe FS, Chisholm GE, Cooper TG (1984) Tau, sigma and delta. A family of repeated elements in yeast. J Biol Chem 259:10518–10525

  14. Glass CK, Holloway JM, Devary OV, Rosenfeld MG (1988) The thyroid hormone receptor binds with opposite transcriptional effects to a consensus sequence motif in thyroid hormone and estrogen response elements. Cell 54:313–323

  15. Haarasilta S, Oura E (1975) On the activity and regulation of anapleurotic and gluconeogenetic enzymes during the growth process of baker's yeast. Eur J Biochem 52:1–7

  16. Haarasilta S, Taskinen L (1977) Location of three key enzymes of gluconeogenetis in baker's yeast. Arch Microbiol 113:159–161

  17. Harbury PAB, Struhl K (1989) Functional distinctions between yeast TATA elements. Mol Cell Biol 9:5298–5304

  18. Hauber J, Stucka R, Krieg R, Feldmann H (1988) Analysis of yeast chromosomal regions carrying members of the glutamate tRNA family: various transposable elements are associated with them. Nucleic Acids Res 16:10623–10634

  19. Heinisch J, Ritzel RG, von Borstel RC, Aguilera A, Rodicio R, Zimmermann FK (1989) The phosphofructokinase genes of yeast evolved from two duplication events. Gene 78:309–321

  20. Hill DE, Hope IA, Macke JP, Struhl K (1986a) Saturation mutagenesis of the his3 regulatory site: requirements for transcriptional induction and for binding by GCN4 activator protein. Science 234:451–457

  21. Hill JE, Myers AM, Koerner TJ, Tzagoloff A (1986b) Yeast/E. coli shuttle vectors with multiple unique restriction sites. Yeast 2:163–167

  22. Hohmann S, Cederberg H (1990) Autoregulation may control the expression of yeast pyruvate decarboxylase structural genes PDC1 and PDC5. Eur J Biochem 188:615–621

  23. Ito H, Fukuda Y, Murata K, Kimura A (1983) Transformation of intact yeast cells treated with alkali cations. J Bacteriol 153:163–168

  24. Kim KS, Rosenkrantz MS, Guarente L (1986) Saccharomyces cerevisiae contains two functional citrate synthase genes. Mol Cell Biol 6:1936–1942

  25. Lim F, Rohde M, Morris CP, Wallace JC (1987) Pyruvate carboxylase in the yeast pyc mutant. Arch Biochem Biophys 258:259–264

  26. Lim F, Morris CP, Occhiodoro F, Wallace JC (1988) Sequence and domain structure of yeast pyruvate carboxylase. J Biol Chem 263:11493–11497

  27. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurements with the Folin phenol reagent. J Biol Chem 193:265–275

  28. McAlister L, Holland MJ (1982) Targeted deletions of a yeast enolase structural gene. Identification and isolation of yeast enolase isoenzymes. J Biol Chem 257:7181–7188

  29. McAllister L, Holland MJ (1985) Differential expression of the three yeast glyceraldehyde-3-phosphate dehydrogenase genes. J Biol Chem 260:15019–15027

  30. Martin BR, Denton RM (1970) The intracellular localization of enzymes in white-adipose-tissue-fat-cells and permeability properties of fat-cell mitochondria. Transfer of acetyl units and reducing power between mitochondria and cytoplasm. Biochem J 117:861–877

  31. Mellor J, Jiang W, Funk M, Rathjen J, Barnes CA, Hinz T, Hegemann JH, Philippsen P (1990) CPF1, a yeast protein which functions in centromeres and promoters. EMBO J 9:4017–4026

  32. Moore PA, Bettany AJE, Brown AJP (1990) Expression of a yeast glycolytic gene is subject to dosage limitation. Gene 89:85–92

  33. Morris CP, Lim F, Wallace JC (1987) Yeast pyruvate carboxylase: gene isolation. Biochem Biophys Res Commun 145:390–396

  34. Nishizawa MR, Araki R, Teranishi Y (1989) Identification of an upstream activating sequence and an upstream repressible sequence of the pyruvate kinase gene of the yeast Saccharomyces cerevisiae. Mol Cell Biol 9:442–451

  35. Privalsky ML, Sharif M, Yamamoto KR (1990) The viral erbA oncogene protein, a constitutive repressor in animal cells, is a hormone-regulated activator in yeast. Cell 63:1277–1286

  36. Rickey TM, Lewin AS (1986) Extramitochondrial citrate synthase activity in baker's yeast. Mol Cell Biol 6:488–493

  37. Rhode PR, Sweden KS, Oegema KF, Campbell JLK (1989) The gene encoding ARS-binding factor I is essential for the viability of yeast. Genes Dev 3:1926–1939

  38. Rothstein R (1985) Cloning in yeast. In: Glover DM (ed) DNA cloning. A practical approach, Vol II. IRL Press, Oxford Washington, pp 45–65

  39. Ruiz-Amil M, De Torróntegui G, Palacián E, Losada M (1965) Properties and function of yeast pyruvate carboxylase. J Biol Chem 240:3485–3492

  40. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor NY

  41. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74:5463–6467

  42. Scrutton MC (1978) Fine control of the conversion of pyruvate (phosphoenolpyruvate) to oxaloacetate in various species. FEBS Lett 89:1–9

  43. Sharp PM, Tuohy TMF, Mosursky KR (1986) Codon usage in yeast: cluster analysis clearly differentiates highly and lowly expressed genes. Nucleic Acids Res 14:5125–5143

  44. Sikorski S, Hieter P (1989) A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122:19–27

  45. Stucka R, Hauber J, Feldmann H (1986) Conserved and non-conserved features among the yeast Ty elements. Curr Genet 11:193–200

  46. Utter MF, Barden RE, Taylor BL (1975) Pyruvate carboxylase: an evaluation of the relationships between structure and mechanism and between structure and catalytic activity. Adv Enzymol 42:1–72

  47. Van Urk H, Schipper D, Breveld GJ, Mak PR, Scheffers WA, van Dijken JP (1989) Localization and kinetics of pyruvate-metabolizing enzymes in relation to aerobic alcoholic fermentation in Saccharomyces cerevisiae CBS 8066 and Candida utilis CBS 621. Biochim Biophys Acta 992:78–86

  48. Verdier JM (1990) Regulatory DNA-binding proteins in yeast: an overview. Yeast 6:271–297

  49. Vezinhet F, Blondin B, Hallet JN (1990) Chromosomal DNA patterns and mitochondrial DNA polymorphism as tools for identification of enological strains of Saccharomyces cerevisiae. Appl Microbiol Biotechnol 32:568–571

  50. Vieira J, Messing J (1982) The pUC plasmids, a M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:259–268

  51. Wills C, Melham T (1985) Pyruvate carboxylase deficiency in yeast: a mutant affecting the interaction between the glyoxylate and Krebs cycle. Arch Biochem Biophys 236:782–791

  52. Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vector and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103–119

  53. Young MR, Tolbert B, Utter MF (1969) Pyruvate carboxylase from Saccharomyces cerevisiae. Methods Enzymol 13:250–258

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Author information

Correspondence to Carlos Gancedo.

Additional information

A preliminary report of this work was presented at the 15th International Conference on Yeast Genetics and Molecular Biology, The Hague, Netherlands. Abstract appeared in Yeast 6, S-240 (1990)

Communicated by C.P. Hollenberg

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Stucka, R., Dequin, S., Salmon, J. et al. DNA sequences in chromosomes 11 and VII code for pyruvate carboxylase isoenzymes in Saccharomyces cerevisiae: analysis of pyruvate carboxylase-deficient strains. Molec. Gen. Genet. 229, 307–315 (1991). https://doi.org/10.1007/BF00272171

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Key words

  • Pyruvate carboxylase
  • Chromosome II
  • Chromosome VII
  • Saccharomyces
  • PYC1, PYC2