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integration into the yeast genome. Integrations are generally done sequentially,
either by first integrating the DNA bait::HIS3 or the DNA bait::LacZ construct,
and following with the other. However, it is possible to integrate both constructs
simultaneously, but the efficiency will be much lower and only a handful of colonies
is usually obtained (unpublished data).
After picking integrant colonies, they need to be tested for background reporter
gene expression (auto-activation). Levels of auto-activation can differ between
integrants from the same DNA bait::reporter construct, most likely because of
differences in copy number (
Deplancke et al., 2004
). The degree of auto-activation
of DNA bait::HIS3 strains is determined by plating the colonies on media lacking
histidine, and with increasing concentrations of 3AT (5, 10, 20, 40, 60, and 80 mM).
Preferably colonies are selected that do not confer growth on low concentrations
(5-40 mM) of 3AT. The degree of auto-activation of DNA bait::LacZ strains is
determined by a colorimetric assay where white indicates no expression and
darker shades of blue indicate increasing induction of
b
Galactosidase. Colonies
with little or no blue should be selected where possible. In our hands 10-20% of
all DNA baits exhibit high levels of auto-activation. These baits are difficult to use
in Y1H assays although interacting TFs can sometimes be detected, particularly in
directed Y1H assays (
Ve rme i r s s e n et al., 2007b
).
After obtaining double integrant DNA bait strains that exhibit the lowest possible
levels of auto-activation, the actual Y1H experiment can be performed to detect
interacting TFs. In Y1H assays, TFs are fused to the transcription activation domain
(AD) of the yeast Gal4 protein. This ensures that both activators and repressors of
transcription can be detected. In other words, only physical protein-DNA interac-
tions are examined in Y1H assays. AD-TF clones can be obtained from different
sources and can be introduced into the DNA bait strain in different ways (
Fig. 4
). In
our Y1H system, AD-TF clones carry wild-type yeast TRP1 gene and, therefore,
colonies containing the plasmid are selected on media lacking tryptophan.
The most commonly used method is by transforming an AD-cDNA library into
haploid DNA bait strains (
Arda et al., 2010; Deplancke et al., 2004, 2006a, 2006b;
Martinez et al., 2008a; Vermeirssen et al.,2007a
). Another source for such haploid
transformations was created by cherry-picking relevant clones from the ORFeome,
transferring them to the AD Y1H Destination vector by Gateway cloning, and com-
bining them into a single AD-TF mini library (
Deplancke et al.,2004
). This library
consists of
650 full-length TFs. Screening such a mini library enables the detection
of TFs that are underrepresented in non-normalized cDNA libraries. Since TFs are
often of low abundance, this can be very useful. In library screens, interacting TFs
are identified by yeast colony PCR and sequencing. We have also developed mini
pools of individual AD-TF clones that can be introduced into DNA bait strains by
transformation (
Vermeirssen et al., 2007b
). These pools are designed using a ''Smart
pool'' strategy, based on a Steiner Triple System that is used in combinatorial math-
ematics. We have generated these pools as well as the scripts to deconvolute the
resulting interactions. This method is useful for higher throughput, cost-effective Y1H
experiments because it does not rely on extensive prey sequencing. Single AD-TF
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