Abstract
Transformation is an indispensable method for the genetic manipulation of cells. Saccharomyces cerevisiae can be transformed by incubating intact cells and plasmid DNA in the presence of polyethylene glycol alone. Lithium acetate (LiAc) and single-stranded carrier DNA (ssDNA) enhance the transformation efficiency, but the mechanism underlying this enhancement has remained elusive. In this study, we first confirmed that LiAc and ssDNA synergistically improve the transformation efficiency of S. cerevisiae intact cells. We then used transmission electron microscopy to observe the cell walls of yeast incubated with both LiAc and ssDNA in the presence of negatively charged Nanogold (in this context, a mimic of DNA). Under these conditions, the cell walls exhibited protruded, loose, and porous structures. The Nanogold was observed within the cell wall, rather than on the surface. We also made observations using YOYO-1, a fluorescent DNA probe. Based on the transmission electron microscopy and fluorescence data, we speculated that ssDNA covers the whole cell and enters, at least partially, into the cell wall structure, causing the cell wall to become protruded, loose, and porous; meanwhile, LiAc produces effect on the cell wall. Together, the two compounds synergistically enhance the transformation efficiency and frequency.
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References
Baba M (2008) Electron microscopy in yeast. Methods Enzymol 451:133–149
Broach JR, Strathern JN, Hicks JB (1979) Transformation in yeast: development of a hybrid cloning vector and isolation of the CAN1 gene. Gene 8:121–133
Bruschi CV, Comer AR, Howe GA (1987) Specificity of DNA uptake during whole cell transformation of S. cerevisiae. Yeast 3:131–137
Brzobohaty B, Kovac L (1986) Factors enhancing genetic transformation of intact yeast cells modify cell wall porosity. J Gen Microbiol 132:3089–3093
Chen P, Liu HH, Cui R, Zhang ZL, Pang DW, Xie ZX, Zheng HZ, Lu ZX, Tong H (2008) Visualized investigation of yeast transformation induced with Li+ and polyethylene glycol. Talanta 77:262–268
Gietz RD, Woods RA (2001) Genetic transformation of yeast. Biotechniques 30:816–820 (822-816, 828 passim)
Gietz RD, Woods RA (2002) Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods Enzymol 350:87–96
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
Gietz RD, Schiestl RH, Willems AR, Woods RA (1995) Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure. Yeast 11:355–360
Gurrieri S, Wells KS, Johnson ID, Bustamante C (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 249:44–53
Hayama Y, Fukuda Y, Kawai S, Hashimoto W, Murata K (2002) Extremely simple, rapid and highly efficient transformation method for the yeast Saccharomyces cerevisiae using glutathione and early log phase cells. J Biosci Bioeng 94:166–171
Hinnen A, Hicks JB, Fink GR (1978) Transformation of yeast. Proc Natl Acad Sci USA 75:1929–1933
Ito H, Fukuda Y, Murata K, Kimura A (1983) Transformation of intact yeast cells treated with alkali cations. J Bacteriol 153:163–168
Kawai S, Pham TA, Nguyen HT, Nankai H, Utsumi T, Fukuda Y, Murata K (2004) Molecular insights on DNA delivery into Saccharomyces cerevisiae. Biochem Biophys Res Commun 317:100–107
Kawai S, Hashimoto W, Murata K (2010) Transformation of Saccharomyces cerevisiae and other fungi: methods and possible underlying mechanism. Bioeng Bugs 1:395–403
Pham TA, Kawai S, Kono E, Murata K (2011) The role of cell wall revealed by the visualization of Saccharomyces cerevisiae transformation. Curr Microbiol 62:956–961
Rye HS, Yue S, Wemmer DE, Quesada MA, Haugland RP, Mathies RA, Glazer AN (1992) Stable fluorescent complexes of double-stranded DNA with bis-intercalating asymmetric cyanine dyes: properties and applications. Nucleic Acids Res 20:2803–2812
Schiestl RH, Gietz RD (1989) High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr Genet 16:339–346
Sherman F (2002) Getting started with yeast. Methods Enzymol 350:3–41
Sikorski RS, 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
Yamakawa M, Hishinuma F, Gunge N (1985) Intact cell transformation of Saccharomyces cerevisiae by polyethylene glycol. Agric Biol Chem 49:869–871
Zheng HZ, Liu HH, Chen SX, Lu ZX, Zhang ZL, Pang DW, Xie ZX, Shen P (2005) Yeast transformation process studied by fluorescence labeling technique. Bioconjug Chem 16:250–254
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Communicated by P. Sunnerhagen.
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Pham, T.A., Kawai, S. & Murata, K. Visualization of the synergistic effect of lithium acetate and single-stranded carrier DNA on Saccharomyces cerevisiae transformation. Curr Genet 57, 233–239 (2011). https://doi.org/10.1007/s00294-011-0341-7
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DOI: https://doi.org/10.1007/s00294-011-0341-7