Current Microbiology

, Volume 62, Issue 3, pp 956–961 | Cite as

The Role of Cell Wall Revealed by the Visualization of Saccharomyces cerevisiae Transformation

  • Tuan Anh Pham
  • Shigeyuki Kawai
  • Emi Kono
  • Kousaku Murata


Transformation is an indispensable method for the manipulation of Saccharomyces cerevisiae cell. The spf1 cell, in which the gene encoding an endoplasmic reticulum-located P-type ATPase is deleted, has been known to show the high-transformation phenotype. In this study, fluorescent microscopic observation of transformation process of S. cerevisiae using plasmid DNA labelled with fluorescent DNA probe, YOYO-1, suggested that the spf1 cell absorbed more plasmid DNA on cellular surface than did the wild-type cell and the unwashed cell did more plasmid DNA than the washed cell. The amounts of the absorbed DNA correlated with the transformation efficiency (number of transformants per μg plasmid DNA) and frequency (transformation efficiency per viable cell number). The high-transformation phenotype of spf1 cell and the effect of heat shock, which effectively induces the transformation of intact cell, disappeared upon cell wall digestion. Electron microscopic observation of the transformation process using negatively charged Nanogold as a mimic of plasmid DNA supported the result obtained using YOYO-1 and implied that plasmid DNA enters into cell together with membrane structure. These data strongly suggest that during the transformation of intact cell, plasmid DNA is initially absorbed on the cell wall, passes through the cell wall with the aid of heat shock, reaches to the membrane, and enters into the cell together with the membrane structure and that the capacity of the cell wall to absorb DNA is at least one of the determinants of transformation efficiency and frequency.


  1. 1.
    Baba M (2008) Electron microscopy in yeast. Methods Enzymol 451:133–149PubMedCrossRefGoogle Scholar
  2. 2.
    Becker DM, Guarente L (1991) High-efficiency transformation of yeast by electroporation. Methods Enzymol 194:182–187PubMedCrossRefGoogle Scholar
  3. 3.
    Burgers PM, Percival KJ (1987) Transformation of yeast spheroplasts without cell fusion. Anal Biochem 163:391–397PubMedCrossRefGoogle Scholar
  4. 4.
    Chen P, Liu HH, Cui R et al (2008) Visualized investigation of yeast transformation induced with Li+ and polyethylene glycol. Talanta 77:262–268PubMedCrossRefGoogle Scholar
  5. 5.
    Cronin SR, Rao R, Hampton RY (2002) Cod1p/Spf1p is a P-type ATPase involved in ER function and Ca2+ homeostasis. J Cell Biol 157:1017–1028PubMedCrossRefGoogle Scholar
  6. 6.
    Durr G, Strayle J, Plemper R et al (1998) The medial-Golgi ion pump Pmr1 supplies the yeast secretory pathway with Ca2+ and Mn2+ required for glycosylation, sorting, and endoplasmic reticulum-associated protein degradation. Mol Biol Cell 9:1149–1162PubMedGoogle Scholar
  7. 7.
    Gietz RD, Woods RA (2001) Genetic transformation of yeast. Biotechniques 30: 816–820, 822–826, 828 passimGoogle Scholar
  8. 8.
    Gietz RD, Woods RA (2002) Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods Enzymol 350:87–96PubMedCrossRefGoogle Scholar
  9. 9.
    Gietz RD, Schiestl RH, Willems AR et al (1995) Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure. Yeast 11:355–360PubMedCrossRefGoogle Scholar
  10. 10.
    Griffith J, Reggiori F (2009) Ultrastructural analysis of nanogold-labeled endocytic compartments of yeast Saccharomyces cerevisiae using a cryosectioning procedure. J Histochem Cytochem 57:801–809PubMedCrossRefGoogle Scholar
  11. 11.
    Gurrieri S, Wells KS, Johnson ID et al (1997) Direct visualization of individual DNA molecules by fluorescence microscopy: characterization of the factors affecting signal/background and optimization of imaging conditions using YOYO. Anal Biochem 249:44–53PubMedCrossRefGoogle Scholar
  12. 12.
    Hayama Y, Fukuda Y, Kawai S et al (2002) Extremely simple, rapid and highly efficient transformation method for the yeast Saccharomyces cerevisiae using glutathione and early log phase cells. J Biosci Bioeng 94:166–171PubMedCrossRefGoogle Scholar
  13. 13.
    Hinnen A, Hicks JB, Fink GR (1978) Transformation of yeast. Proc Natl Acad Sci USA 75:1929–1933PubMedCrossRefGoogle Scholar
  14. 14.
    Ito H, Fukuda Y, Murata K et al (1983) Transformation of intact yeast cells treated with alkali cations. J Bacteriol 153:163–168PubMedGoogle Scholar
  15. 15.
    Jigami Y, Odani T (1999) Mannosylphosphate transfer to yeast mannan. Biochim Biophys Acta 1426:335–345PubMedGoogle Scholar
  16. 16.
    Kawai S, Pham TA, Nguyen HT et al (2004) Molecular insights on DNA delivery into Saccharomyces cerevisiae. Biochem Biophys Res Commun 317:100–107PubMedCrossRefGoogle Scholar
  17. 17.
    Lentz BR (2007) PEG as a tool to gain insight into membrane fusion. Eur Biophys J 36:315–326PubMedCrossRefGoogle Scholar
  18. 18.
    Prescianotto-Baschong C, Riezman H (2002) Ordering of compartments in the yeast endocytic pathway. Traffic 3:37–49PubMedCrossRefGoogle Scholar
  19. 19.
    Rye HS, Yue S, Wemmer DE et al (1992) Stable fluorescent complexes of double-stranded DNA with bis-intercalating asymmetric cyanine dyes: properties and applications. Nucl Acids Res 20:2803–2812PubMedCrossRefGoogle Scholar
  20. 20.
    Sherman F (2002) Getting started with yeast. Methods Enzymol 350:3–41PubMedCrossRefGoogle Scholar
  21. 21.
    Stateva LI, Oliver SG, Trueman LJ et al (1991) Cloning and characterization of a gene which determines osmotic stability in Saccharomyces cerevisiae. Mol Cell Biol 11:4235–4243PubMedGoogle Scholar
  22. 22.
    Suzuki C, Shimma YI (1999) P-type ATPase spf1 mutants show a novel resistance mechanism for the killer toxin SMKT. Mol Microbiol 32:813–823PubMedCrossRefGoogle Scholar
  23. 23.
    Tomlin GC, Hamilton GE, Gardner DC et al (2000) Suppression of sorbitol dependence in a strain bearing a mutation in the SRB1/PSA1/VIG9 gene encoding GDP-mannose pyrophosphorylase by PDE2 overexpression suggests a role for the Ras/cAMP signal-transduction pathway in the control of yeast cell-wall biogenesis. Microbiology 146(Pt 9):2133–2146PubMedGoogle Scholar
  24. 24.
    Yamakawa M, Hishinuma F, Gunge N (1985) Intact cell transformation of Saccharomyces cerevisiae by polyethylene glycol. Agric Biol Chem 49:869–871Google Scholar
  25. 25.
    Zheng HZ, Liu HH, Chen SX et al (2005) Yeast transformation process studied by fluorescence labeling technique. Bioconjug Chem 16:250–254PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Tuan Anh Pham
    • 1
  • Shigeyuki Kawai
    • 1
  • Emi Kono
    • 1
  • Kousaku Murata
    • 1
  1. 1.Laboratory of Basic and Applied Molecular Biotechnology, Graduate School of AgricultureKyoto UniversityUjiJapan

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