STUDIES ON THE TRANSFORMATION OF INTACT YEAST-CELLS BY THE LIAC S-DNA/PEG PROCEDURE/

An improved lithium acetate (LiAc)/single-stranded DNA (SS-DNA)/polyet hylene glycol (PEG) protocol which yields >1 x 10(6) transformants/mu g plasmid DNA and the original protocol described by Schiestl and Giet z (1989) were used to investigate aspects of the mechanism of LiAc/SS- DNA/PEG transformation. The highest transformation efficiency was obse rved when 1 x 10(8) cells were transformed with 100 ng plasmid DNA in the presence of 50 mu g SS carrier DNA. The yield of transformants inc reased linearly up to 5 mu g plasmid per transformation. A 20-min heat shock at 42 degrees C was necessary for maximal yields. PEG was found to deposit both carrier DNA and plasmid DNA onto cells. SS carrier DN A bound more effectively to the cells and caused tighter binding of P- 32-labelled plasmid DNA than did double-stranded (DS) carrier. The LiA c/SS-DNA/PEG transformation method did not result in cell fusion. DS c arrier DNA competed with DS vector DNA in the transformation reaction. SS plasmid DNA transformed cells poorly in combination with both SS a nd DS carrier DNA. The LiAc/SS-DNA/PEG method was shown to be more eff ective than other treatments known to make cells transformable. A mode l for the mechanism of transformation by the LiAc/SS-DNA/PEG method is discussed.