Using the Yeast Two-Hybrid System to Identify Protein–Protein Interactions - Plant Proteomics: Methods and Protocols - page 233

DNA-binding domain (DNA-BD), while another gene or cDNA is
expressed as a fusion to the GAL4 activation domain (AD) [ 1 , 6 ] .
Protein interaction is detected by activation of reporter genes
under the control of distinct GAL4 upstream activating sequences
(UASs). In yeast, galactose metabolism is controlled by two regu-
latory proteins, GAL4 and GAL80, as well as by the carbon source
in the medium. When galactose is present, the GAL4 protein binds
to the GAL-responsive elements within the UAS of at least 20
known galactose-responsive genes (including GAL1). In the
absence of galactose, Gal80p binds to Gal4p and this interaction
blocks transcriptional activation of the galactose-responsive genes.
Furthermore, in the presence of glucose, transcription of galactose
genes is immediately repressed. To avoid interference by endoge-
neous GAL4 and GAL80 proteins, the yeast host strains used in
the GAL4 based two-hybrid system must carry deletions of the
GAL4 and GAL80 genes. Due to the deletion of these two genes,
the yeast cells grow more slowly as compared to yeast containing
the wild type version of these genes. In the GAL4-based two-
hybrid system, either the intact GAL1 UAS, which contains four
GAL4-binding sites, or an artifi cially constructed UAS consisting
of three copies of the 17-mer consensus binding sequence are used
to control de expression of the reported genes.

2 Materials

2.1 Plasmids

A large number of different DNA-binding domain (BD) and tran-
scription activation domain (AD) containing vectors have been
successfully used. In our experience, pGBKT7 and pGADT7,
designed to obtain a high-level of protein expression, gave the best
results. In these vectors, bait and prey/library inserts are expressed
as GAL4 fusions with c-Myc and hemagglutinin (HA) epitope
tags, respectively. The epitope tags eliminate the need to generate
specifi c antibodies to each new protein and allow convenient iden-
tifi cation of the fusion proteins. Otherwise anti-GAL4 BD and AD
monoclonal antibodies can be used (Clontech Cat. No 630402
and 630403). Plasmid information is provided in Table 1 . A com-
plete list of other plasmids can be found in http://www.bioteach.
ubc.ca/Journal/V01I01/8188 yeast.pdf.

2.2 Yeast Strains

Several yeast strains containing different reporter genes are avail-
able. Among them, PJ69-4A and AH109 strains use three report-
ers— ADE2 , HIS3, and lacZ (or MEL1)— under the control of
distinct GAL4 UASs. These promoters yield strong and specifi c
responses to GAL4.
The ADE2 reporter alone provides strong nutritional selec-
tion, however using also the HIS3 selection reduces the incidence
of false positives and allows control of the stringency of selection