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Current Genetics

, Volume 52, Issue 2, pp 97–105 | Cite as

Six new amino acid-auxotrophic markers for targeted gene integration and disruption in fission yeast

  • Yan Ma
  • Reiko Sugiura
  • Mariko Saito
  • Atsushi Koike
  • Susie Ong Sio
  • Yasuko Fujita
  • Kaoru Takegawa
  • Takayoshi KunoEmail author
Technical Note

Abstract

Fission yeast Schizosaccharomyces pombe is amenable to genetics and is an excellent model system for studying eukaryotic cell biology. However, auxotrophic markers that can be used for both targeted gene integration and disruption are very limited. Here we performed a forward genetic screen in an effort to develop a new set of selectable markers for use in this yeast. Mutants that were auxotrophic for arginine, asparagine, cysteine, lysine, methionine and phenylalanine were isolated. Six genes were analyzed in detail and the mutations in the genes were identified. Among these six are three new genes: asn1 +, cys2 + and pha2 + were required for biosynthesis of asparagine, cysteine and phenylalanine, respectively. New alleles of arg1 +, lys3 + and met6 + were also identified. All of these genes proved to be suitable as selectable markers for targeted gene integration and disruption. We also showed that in Schizosaccharomyces pombe there are two apparent homologues of Saccharomyces cerevisiae MET2: the previously known met6 +, and SPBC106.17c (named cys2 +). The cys2 mutation required cysteine rather than methionine. These new tools, specifically, new selectable markers, will be useful in further genetic and biological studies in fission yeast.

Keywords

Pombe Auxotrophic mutant Genetic marker Amino acid 

Notes

Acknowledgments

This work was partly supported by twenty-first Century COE Program and research grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

References

  1. Apolinario E, Nocero M, Jin M, Hoffman CS (1993) Cloning and manipulation of the Schizosaccharomyces pombe his7 + gene as a new selectable marker for molecular genetic studies. Curr Genet 24:491–495PubMedCrossRefGoogle Scholar
  2. Bahler J, Wu JQ, Longtine MS, Shah NG, McKenzie A, Steever AB, Wach A, Philippsen P, Pringle JR (1998) Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe. Yeast 14:943–951PubMedCrossRefGoogle Scholar
  3. Banszky L, Simonics T, Maraz A (2003) Sulphate metabolism of selenate-resistant Schizosaccharomyces pombe mutants. J Gen Appl Microbiol 49:271–278PubMedCrossRefGoogle Scholar
  4. Baroni M, Livian S, Martegani E, Alberghina L (1986) Molecular cloning and regulation of the expression of the MET2 gene of Saccharomyces cerevisiae. Gene 46:71–78PubMedCrossRefGoogle Scholar
  5. Burke JD, Gould KL (1994) Molecular cloning and characterization of the Schizosaccharomyces pombe his3 gene for use as a selectable marker. Mol Gen Genet 242:169–176PubMedCrossRefGoogle Scholar
  6. Chen D, Toone WM, Mata J, Lyne R, Burns G, Kivinen K, Brazma A, Jones N, Bahler J (2003) Global transcriptional responses of fission yeast to environmental stress. Mol Biol Cell 14:214–229PubMedCrossRefGoogle Scholar
  7. Cottarel G (1995) The Saccharomyces cerevisiae HIS3 and LYS2 genes complement the Schizosaccharomyces pombe his5-303 and lys1-131 mutations, respectively: new selectable markers and new multi-purpose multicopy shuttle vectors, pSP3 and pSP4. Curr Genet 28:380–383PubMedCrossRefGoogle Scholar
  8. Dang VD, Valens M, Bolotin-Fukuhara M, Daignan-Fornier B (1996) Cloning of the ASN1 and ASN2 genes encoding asparagine synthetases in Saccharomyces cerevisiae: differential regulation by the CCAAT-box-binding factor. Mol Microbiol 22:681–692PubMedCrossRefGoogle Scholar
  9. Davis L, Smith GR (2001) Meiotic recombination and chromosome segregation in Schizosaccharomyces pombe. Proc Natl Acad Sci USA 98:8395–8402PubMedCrossRefGoogle Scholar
  10. Deng L, Sugiura R, Takeuchi M, Suzuki M, Ebina H, Takami T, Koike A, Iba S, Kuno T (2006) Real-time monitoring of calcineurin activity in living cells: evidence for two distinct Ca2+-dependent pathways in fission yeast. Mol Biol Cell 17:4790–4800PubMedCrossRefGoogle Scholar
  11. Fujita Y, Giga-Hama Y, Takegawa K (2005) Development of a genetic transformation system using new selectable markers for fission yeast Schizosaccharomyces pombe. Yeast 22:193–202PubMedCrossRefGoogle Scholar
  12. Fujita Y, Ukena E, Iefuji H, Giga-Hama Y, Takegawa K (2006) Homocysteine accumulation causes a defect in purine biosynthesis: further characterization of Schizosaccharomyces pombe methionine auxotrophs. Microbiology 152:397–404PubMedCrossRefGoogle Scholar
  13. Giga-Hama Y, Kumagai H (1999) Expression system for foreign genes using the fission yeast Schizosaccharomyces pombe. Biotechnol Appl Biochem 30(Pt 3):235–244PubMedGoogle Scholar
  14. Grimm C, Kohli J, Murray J, Maundrell K (1988) Genetic engineering of Schizosaccharomyces pombe: a system for gene disruption and replacement using the ura4 gene as a selectable marker. Mol Gen Genet 215:81–86PubMedCrossRefGoogle Scholar
  15. Guertin DA, Trautmann S, McCollum D (2002) Cytokinesis in eukaryotes. Microbiol Mol Biol Rev 66:155–178PubMedCrossRefGoogle Scholar
  16. Hayles J, Nurse P (1992) Genetics of the fission yeast Schizosaccharomyces pombe. Annu Rev Genet 26:373–402PubMedCrossRefGoogle Scholar
  17. Hentges P, Van Driessche B, Tafforeau L, Vandenhaute J, Carr AM (2005) Three novel antibiotic marker cassettes for gene disruption and marker switching in Schizosaccharomyces pombe. Yeast 22:1013–1019PubMedCrossRefGoogle Scholar
  18. Hoffman RL, Hoffman CS (2006) Cloning the Schizosaccharomyces pombe lys2 + gene and construction of new molecular genetic tools. Curr Genet 49:414–420PubMedCrossRefGoogle Scholar
  19. Hoheisel JD, Maier E, Mott R, McCarthy L, Grigoriev AV, Schalkwyk LC, Nizetic D, Francis F, Lehrach H (1993) High resolution cosmid and P1 maps spanning the 14 Mb genome of the fission yeast S. pombe. Cell 73:109–120PubMedCrossRefGoogle Scholar
  20. Ito H, Fukuda Y, Murata K, Kimura A (1983) Transformation of intact yeast cells treated with alkali cations. J Bacteriol 153:163–168PubMedGoogle Scholar
  21. Iwaki T, Takegawa K (2004) A set of loxP marker cassettes for Cre-mediated multiple gene disruption in Schizosaccharomyces pombe. Biosci Biotechnol Biochem 68:545–550PubMedCrossRefGoogle Scholar
  22. Kaur R, Ingavale SS, Bachhawat AK (1997) PCR-mediated direct gene disruption in Schizosaccharomyces pombe. Nucleic Acids Res 25:1080–1081PubMedCrossRefGoogle Scholar
  23. Keeney JB, Boeke JD (1994) Efficient targeted integration at leu1-32 and ura4-294 in Schizosaccharomyces pombe. Genetics 136:849–856PubMedGoogle Scholar
  24. Lorenz MC, Muir RS, Lim E, McElver J, Weber SC, Heitman J (1995) Gene disruption with PCR products in Saccharomyces cerevisiae. Gene 158:113–117PubMedCrossRefGoogle Scholar
  25. Ma Y, Kuno T, Kita A, Asayama Y, Sugiura R (2006) Rho2 Is a Target of the Farnesyltransferase Cpp1 and Acts Upstream of Pmk1 MAP Kinase Signaling in Fission Yeast. Mol Biol Cell 12:5028–5037CrossRefGoogle Scholar
  26. Maftahi M, Nicaud JM, Levesque H, Gaillardin C (1995) Sequencing analysis of a 24.7 kb fragment of yeast chromosome XIV identifies six known genes, a new member of the hexose transporter family and ten new open reading frames. Yeast 11:1077–1085PubMedCrossRefGoogle Scholar
  27. Masselot M, Robichon-Szulmajster H (1975) Methionine biosynthesis in Saccharomyces cerevisiae. I. Genetical analysis of auxotrophic mutants. Mol Gen Genet 139:121–132PubMedCrossRefGoogle Scholar
  28. Maundrell K (1993) Thiamine-repressible expression vectors pREP and pRIP for fission yeast. Gene 123:127–130PubMedCrossRefGoogle Scholar
  29. Millar JB, Buck V, Wilkinson MG (1995) Pyp1 and Pyp2 PTPases dephosphorylate an osmosensing MAP kinase controlling cell size at division in fission yeast. Genes Dev 9:2117–2130PubMedCrossRefGoogle Scholar
  30. Moreno S, Klar A, Nurse P (1991) Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. Methods Enzymol 194:795–823PubMedGoogle Scholar
  31. Mortimer RK, Hawthorne DC (1966) Genetic mapping in Saccharomyces. Genetics 53:165–173PubMedGoogle Scholar
  32. Rothstein RJ (1983) One-step gene disruption in yeast. Methods Enzymol 101:202–211PubMedCrossRefGoogle Scholar
  33. Shiozaki K, Russell P (1995) Cell-cycle control linked to extracellular environment by MAP kinase pathway in fission yeast. Nature 378:739–743PubMedCrossRefGoogle Scholar
  34. Sugiura R, Toda T, Shuntoh H, Yanagida M, Kuno T (1998) pmp1 +, a suppressor of calcineurin deficiency, encodes a novel MAP kinase phosphatase in fission yeast. EMBO J 17:140–148PubMedCrossRefGoogle Scholar
  35. Takahashi K, Yanagida M (2000) Cell cycle. Replication meets cohesion. Science 289:735–736PubMedCrossRefGoogle Scholar
  36. Thomas D, Surdin-Kerjan Y (1997) Metabolism of sulfur amino acids in Saccharomyces cerevisiae. Microbiol Mol Biol Rev 61:503–532PubMedGoogle Scholar
  37. Toda T, Dhut S, Superti-Furga G, Gotoh Y, Nishida E, Sugiura R, Kuno T (1996) The fission yeast pmk1 + gene encodes a novel mitogen-activated protein kinase homolog which regulates cell integrity and functions coordinately with the protein kinase C pathway. Mol Cell Biol 16:6752–6764PubMedGoogle Scholar
  38. Waddell S, Jenkins JR (1995) arg3 +, a new selection marker system for Schizosaccharomyces pombe: application of ura4 + as a removable integration marker. Nucleic Acids Res 23:1836–1837PubMedCrossRefGoogle Scholar
  39. Wood V, Gwilliam R, Rajandream MA, Lyne M, Lyne R, Stewart A, Sgouros J, Peat N, Hayles J, Baker S, Basham D, Bowman S, Brooks K, Brown D, Brown S, Chillingworth T, Churcher C, Collins M, Connor R, Cronin A, Davis P, Feltwell T, Fraser A, Gentles S, Goble A, Hamlin N, Harris D, Hidalgo J, Hodgson G, Holroyd S, Hornsby T, Howarth S, Huckle EJ, Hunt S, Jagels K, James K, Jones L, Jones M, Leather S, McDonald S, McLean J, Mooney P, Moule S, Mungall K, Murphy L, Niblett D, Odell C, Oliver K, O’Neil S, Pearson D, Quail MA, Rabbinowitsch E, Rutherford K, Rutter S, Saunders D, Seeger K, Sharp S, Skelton J, Simmonds M, Squares R, Squares S, Stevens K, Taylor K, Taylor RG, Tivey A, Walsh S, Warren T, Whitehead S, Woodward J, Volckaert G, Aert R, Robben J, Grymonprez B, Weltjens I, Vanstreels E, Rieger M, Schafer M, Muller-Auer S, Gabel C, Fuchs M, Dusterhoft A, Fritzc C, Holzer E, Moestl D, Hilbert H, Borzym K, Langer I, Beck A, Lehrach H, Reinhardt R, Pohl TM, Eger P, Zimmermann W, Wedler H, Wambutt R, Purnelle B, Goffeau A, Cadieu E, Dreano S, Gloux S, Lelaure V, Mottier S, Galibert F, Aves SJ, Xiang Z, Hunt C, Moore K, Hurst SM, Lucas M, Rochet M, Gaillardin C, Tallada VA, Garzon A, Thode G, Daga RR, Cruzado L, Jimenez J, Sanchez M, del Rey F, Benito J, Dominguez A, Revuelta JL, Moreno S, Armstrong J, Forsburg SL, Cerutti L, Lowe T, McCombie WR, Paulsen I, Potashkin J, Shpakovski GV, Ussery D, Barrell BG, Nurse P, Cerrutti L (2002) The genome sequence of Schizosaccharomyces pombe. Nature 415:871–880PubMedCrossRefGoogle Scholar
  40. Yoshida T, Toda T, Yanagida M (1994) A calcineurin-like gene ppb1 + in fission yeast: mutant defects in cytokinesis, cell polarity, mating and spindle pole body positioning. J Cell Sci 107(Pt 7):1725–1735PubMedGoogle Scholar
  41. Zhao Y, Lieberman HB (1995) Schizosaccharomyces pombe: a model for molecular studies of eukaryotic genes. DNA Cell Biol 14:359–371PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Yan Ma
    • 1
  • Reiko Sugiura
    • 2
  • Mariko Saito
    • 1
  • Atsushi Koike
    • 1
  • Susie Ong Sio
    • 4
  • Yasuko Fujita
    • 3
  • Kaoru Takegawa
    • 3
  • Takayoshi Kuno
    • 1
    Email author
  1. 1.Division of Molecular Pharmacology and Pharmacogenomics, Department of Biochemistry and Molecular BiologyKobe University Graduate School of MedicineKobeJapan
  2. 2.Laboratory of Molecular Pharmacogenomics, School of Pharmaceutical SciencesKinki UniversityHigashi-OsakaJapan
  3. 3.Department of Life Sciences, Faculty of AgricultureKagawa UniversityMiki-choJapan
  4. 4.Department of Pharmacology and Toxicology, College of MedicineUniversity of the Philippines ManilaManilaPhilippines

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