High-Efficiency Transformation of Plasmid DNA into Yeast

Abstract

The yeast two-hybrid system was first introduced by Fields and Song () in 1989. They showed that plasmids expressing the GAL4 BD-SNF1 and GAL4 AD-SNF4 fusion proteins activated a GALl-lacZ reporter gene. The first application of the system involved cotransformation of two plasmids into the strain GGY1:: 171—one plasmid containing the GAL4 BD fused to sequences from the 3′ end of SIR4 and the other containing GAL4 AD fused to fragments of yeast genomic DNA (). The cotransformation was accomplished by the newly reported high-efficiency lithium acetate/single-stranded carrier DNA/polyethylene glycol (LiAc/ssDNA/PEG) protocol () to generate 220,000 transformants and identified two positive clones. The efficiency of this transformation protocol was 2×104 transformants/(g of plasmid·108 cells) (), and the frequency of cotransformation was shown to range from 30 to 40% (). Screening a mam-malian cDNA library by the yeast two-hybrid system requires a large number of transformants. For example, the detection of proteins that interacted with huntingtin, the Huntington disease protein, involved a screen of 4×107 transformants from an adult human brain cDNA library (). We have reported several modifications and improvements to the LiAc/ssDNA/PEG protocol so that it can easily generate the numbers of transformants required for such screens.