Visualization of the synergistic effect of lithium acetate and single-stranded carrier DNA on Saccharomyces cerevisiae transformation

Curr Genet. 2011 Aug;57(4):233-9. doi: 10.1007/s00294-011-0341-7. Epub 2011 May 12.

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.

MeSH terms

  • Acetates / pharmacology*
  • DNA, Single-Stranded / metabolism*
  • DNA, Single-Stranded / ultrastructure
  • Microscopy, Electron, Transmission
  • Saccharomyces cerevisiae / drug effects*
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae / ultrastructure
  • Transgenes / drug effects*

Substances

  • Acetates
  • DNA, Single-Stranded
  • lithium acetate