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The yeast two-hybrid system is a poau]werful molecular genetic tool conceived by Fields and Song (1). The article is a comprehensive set of methods designed to take the reader through a yeast two-hybrid analysis of your favorite gene (YFG). This article details the preparation for a screen, the screen itself, as well as the analysis of the positives identified. Using these methods, the readers should be able to successfully negotiate a yeast two-hybrid screen using the Fields' or related systems. [ABSTRACT FROM AUTHOR]

The technique for the transformation of Saccharomyces cerevisiae using the LiAc/SS Carrier DNA/PEG method is described. We describe a rapid method, for use when large numbers of transformants are not necessary. A high-efficiency method for the generation of large numbers of transformants is also given. A method for the transformation of plasmid libraries, which includes yeast two-hybrid applications, also is listed to aid the reader in generating transformants to effectively cover the library complexity. Finally, a protocol for transformation using a 96-well format is included for transformation applications that require it. [ABSTRACT FROM AUTHOR]

The two-hybrid system (THS) (1) is a molecular genetic screen that detects protein-protein interactions. The protein specified by the yeast GAL4 gene activates the transcription of genes involved in galactose metabolism. It has two functional domains, a DNA binding domain, Gal4BD, and a transcriptional activating domain, Gal4AD, which interact with DNA sequences in the promoter regions of GAL1, GAL2, and GAL7 to stimulate transcription. The screen involves two plasmids; one carries the GAL4BD sequence fused, in-frame, to a sequence coding for a "bait" protein, and the other carries GAL4AD sequences, fused to "prey" sequences from a cDNA library. The two plasmids are introduced, typically by transformation, into a yeast strain carrying a reporter gene coupled to a GAL1, GAL2, or GAL7 promoter. If the proteins encoded by the bait and prey sequences interact to allow correct positioning of the Gal4AD and GaL4BD moieties, the reporter gene is activated. Transformants are plated on medium that allows the detection of reporter gene activation. The plasmid carrying the GAL4AD:cDNA plasmid can be recovered, and the positive cDNA isolated and characterized. [ABSTRACT FROM AUTHOR]

The yeast two-hybrid system was first introduced by Fields and Song (1) in 1989. They showed that plasmids expressing the GAL4BD-SNF1 and GAL4AD-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 GAL4BD fused to sequences from the 3′ end of SIR4 and the other containing GAL4AD fused to fragments of yeast genomic DNA (2). The cotransformation was accomplished by the newly reported high-efficiency lithium acetate/single-stranded carrier DNA/polyethylene glycol (LiAc/ssDNA/PEG) protocol (3) 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) (3), and the frequency of cotransformation was shown to range from 30 to 40% (4). 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 (5). 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. [ABSTRACT FROM AUTHOR]