From the Division of Basic Science, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111
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ABSTRACT |
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Biological regulatory systems require the
specific organization of proteins into multicomponent complexes. Two
hybrid systems have been used to identify novel components of signaling
networks based on interactions with defined partner proteins. An
important issue in the use of two-hybrid systems has been the degree to which interacting proteins distinguish their biological partner from
evolutionarily conserved related proteins and the degree to which
observed interactions are specific. We adapted the basic two-hybrid
strategy to create a novel dual bait system designed to allow
single-step screening of libraries for proteins that interact with
protein 1 of interest, fused to DNA binding domain A (LexA), but do not
interact with protein 2, fused to DNA binding domain B ( To understand and manipulate the function of a particular protein
of biological interest, it is generally useful to identify other
proteins with which it associates. Although identification of protein
interactions initially proceeded almost solely by technically difficult
biochemical methods, in recent years yeast two-hybrid systems (1) have
developed as a powerful genetic tool to rapidly select previously
uncharacterized proteins that specifically interact with a target
protein of interest from a suitable library (2-5). In this schema, a
protein of interest is synthesized in yeast as a fusion to a DNA
binding domain (DBD),1 which
is typically the bacterial repressor protein LexA or the amino-terminal
end of the yeast transcription factor GAL4. Interaction of this DBD
protein fusion (a "bait") with a transcriptional activation domain-fused partner protein (either a defined partner or a novel protein screened from a library) allows the activation of reporter genes (lacZ, HIS3, LEU2) responsive to
the cognate DBD. More recently, interest has focused on expanding the
utility of two-hybrid systems to enable the detection of interactions
between proteins and RNA (6, 7), proteins and nonprotein ligands (8),
proteins and peptides (9, 10), and proteins and multiple partners (11,
12). A second thrust has been to enable whole-genome applications
(13-15), leading to the generation of maps of protein interaction
networks with the potential to complement the vast resource of sequence
information now being developed as part of the Genome Project. Finally,
there has been interest in developing two-hybrid systems as tools in
high throughput drug discovery screening strategies to identify agents
regulating the activity of biologically important target proteins.
As two-hybrid technologies have evolved to more complex applications, a
question of mounting importance has been the degree to which library
screens performed in these systems yield partners specific for the
utilized bait, as opposed to proteins of broad interaction capability
("false positives"). Although the large number of published
two-hybrid papers indicates that many specific partners are obtained, a
recent survey has suggested that the majority of library screens
isolate at least some cDNAs that are nonspecific.2 As a related
issue, it is clear that many biologically important proteins are
organized into families of evolutionarily related members that conserve
substantial sequence similarity (e.g. Refs. 17-19). Thus,
the degree to which two-hybrid systems isolate proteins partners
absolutely specific for individual baits, rather than those that
interact generally with a class of protein ("familial positives"),
is also an issue. Although existing two-hybrid systems allow
discrimination of uniquely specific interactors from false positives or
familial positives through use of various methods of specificity
testing performed subsequent to a screen (20), these methods are
frequently laborious, particularly when many possible interactors must
be tested. For this reason, it has been of considerable interest to
devise a method to eliminate such clones before selection.
In this report, we describe a novel adaptation of the two-hybrid system
designated the dual bait system. This system incorporates controls for
false positive or nonspecific interactions in a single step and allows
the simultaneous assay of a protein interaction with two related or
unrelated partners in a single cell, which should also be useful for a
variety of high throughput and genome-oriented studies. We demonstrated
that these reagents are effective at selectively identifying two
discrete sets of interacting proteins against an extensive background
population of nonspecifically interacting proteins, supporting the idea
of reagent appropriateness for large scale genomic applications.
Molecular Biology and Genetic Techniques--
DH5 Dual Bait System Reagents--
Relevant properties of all
strains and plasmids are described in the text. The bacteriophage cI-responsive LacZ Reporters--
A 68-base pair fragment of the
cI-responsive LYS2 Yeast Strains--
An EcoRI
fragment containing a minimal GAL1 promoter, cI
operator cassette, and the translational start of the GAL1
gene was excised from cIop-lacZA and inserted
into pRFiLYS8 (a gift of R. Finley) to generate pCIL-1. In this
construct, the GAL1 promoter-cI operator cassette
directs the expression of a fusion protein in which the first 31 amino
acids of the GAL1 gene are fused to LYS2-coding sequences. The yeast strain RFY206 (13) (MATa trp1
ura3-52 his3200 leu2-3 lys2
The yeast strain SK01was crossed to EGY48 (MAT
As a separate approach, as SK10 proved refractory to deletion of the
URA3 gene, the PstI (475045)-XbaI
(474175) fragment of Saccharomyces cerevisiae chromosome II,
located 295 base pairs upstream of the wild-type LYS2 gene,
and a PstI-HindIII fragment of pcIL-1,
encompassing the cI-responsive cassette, were assembled on a pUC-based
plasmid to yield pcIL-2. pcIL-2 reconstructed about 6 kilobases of
yeast genomic sequence with the native LYS2 promoter replaced by GAL1 promoter/cI operator sequence.
This construct was used for integrative transformation of the EGY48 (4)
and EGY191 (23) yeast strains, followed by Lys selection (ability to
grow on 0.2% DL( cI-responsive gusA Reporters--
The novel yeast vector pRG00
was used as the basis of cIoperator-gusA reporter
vector construction. The detailed map and sequence files for pRG00 are
available upon request; this vector contains the colE1
origin of replication and kanamycin resistance gene for selection in
bacteria, a 2-µm origin and URA3 gene for selection in
yeast, and the complete coding sequence for E. coli
cI Fusion Bait Vectors--
A DNA fragment containing the
complete coding sequence (with no stop codon) for bacteriophage
Subsequently, a BsaBI-EcoRI fragment of pGKS3
(encompassing cI) was used to replace the lexA gene in the
plasmid pLexZeo (Invitrogen), which had been digested with
HpaI-EcoRI. The resulting plasmid, pGKS6, used
the ADH1 promoter to express a cI fusion. It had a 2-µm
origin of replication and used Zeocin (Invitrogen) resistance for
selection in yeast and bacteria. Expression of proteins was assayed by
standard lysis of cells expressing appropriate constructs (20) followed
by SDS-polyacrylamide gel electrophoresis and Western analysis with
antibodies to Krev-1 (Transduction Labs, Inc.), LexA, or cI repressor
(a gift of G. Kalmar).
cI Fusion Bait/cIop-gusA Reporter Dual Purpose Vectors--
To
allow introduction of all system components into a single yeast strain,
a combined cI bait expression/cIop-gusA reporter with a single
selectible marker (ZeoR) was constructed. To create this
plasmid, pGKS8, the 3cIop-gusA reporter cassette was excised
from pRG2 as a KpnI-KpnI fragment and inserted
into the BsrGI site of pGKS6, destroying both restriction sites. The map of pGKS8 is available on request; this plasmid retained
EcoRI, SacI, BglII, PvuII,
KpnI, SacII, and NotI sites for the
insertion of coding sequences for expression as cI fusion proteins.
Baits and Preys--
To create transcriptionally activating
DBD-fused bait plasmids, the full-length Krit1 gene (30) was inserted
into EcoRI-XhoI-digested pGKS3, pGKS6, pGKS8, and
pEG202, as noted in the text. Nonactivating bait fusions were
constructed by cloning the full-length Krev-1 gene (31) into the
EcoRI-XhoI sites of pGKS3 or into
EcoRI-SacII sites of pGKS8 and by cloning the Ras
gene into the EcoRI-XhoI sites of pEG202.
Activation domain fusion plasmids were obtained by cloning Krit1
(full-length) and Raf1 ( Assaying Reporter Activation--
Activation of LacZ
reporters was assessed qualitatively by on-plate overlay assays (24)
using the substrates X-Gal or Magenta-Gal (Diagnostic Chemicals Ltd)
and quantitatively using
Activation of LYS2 or LEU2 reporters was accomplished by streaking or
replica-plating yeast to plates lacking leucine or lysine and
monitoring growth over 4 days. In assessing activation of the LYS2
reporter by bait-prey combinations (as in Fig. 5), this was done either
on medium lacking uracil, histidine, tryptophan, and lysine in the
presence of zeocin, or alternatively, on medium lacking only uracil,
histidine, and lysine in the absence of zeocin, with the selection for
growth on lysine medium providing positive selective force for the
retention of cI bait and activation domain-fused prey. Qualitatively
identical patterns of protein interaction are obtained with both
media; however, because of minor nonspecific growth inhibitory effects
of zeocin, growth is faster (~24 h to point of large colonies) with
zeocin omitted.
For mixing experiments, yeast were pregrown in medium selective for
plasmid retention and inducing for activation domain fusion expression
(ura-his-trp-galactose/raffinose + zeocin), diluted as described in
results, and plated to selective medium; colony outgrowth was
monitored over 4 days.
Outline of Strategy--
The general approach taken with a dual
bait selection strategy is outlined in Fig.
1. In the interaction trap two-hybrid
system (Fig. 1A (4)), a LexA-fused bait (expressed from
plasmid pEG202 or a derivative) interacts with a galactose-inducible
B42 "acid blob" activation domain-fused partner (expressed from
plasmid pJG4-5) to induce the expression of two reporter genes under
transcriptional control of lexA operator (op)
sites. These are (lexAop)n-lacZ (borne on
plasmid pSH18-34 (n = 8), pJK103 (n = 2), or pRB1840 (n = 1)) and an integrated
(lexAop)n-LEU2 (in yeast strain EGY48
(n = 6) or EGY191(n = 2)).
In the dual bait system described here, three further components are
added (Fig. 1B). The first of these is a cI-fused alternate bait, expressed from the novel ZeoR, 2-µm plasmid, pGKS8.
The second is a (cIop)n-gusA
(
As a first example, in a library screen, if an activation domain-fused
interacting protein associates uniquely with a LexA-fused primary bait
but not with a cI-fused alternate bait, SKY48 or SKY191 yeast
containing the appropriate bait and reporter constructs would turn blue
on medium containing X-Gal but not on medium containing X-Gluc, and
grow on medium lacking leucine, but fail to grow on medium lacking
lysine; in contrast, promiscuously interacting clones would be revealed
by their growth on medium lacking both leucine and lysine and blue
color with both X-Gal and X-Gluc. False positives would be eliminated
simultaneously with isolation of true positive clones. As a second
example, in targeted examination of the interaction of a single
activation domain-fused protein with two defined partners (for example,
interaction of activation domain-fused cyclin D with LexA-fused CDK4
and cI-fused CDK6), a randomly mutagenized pool of activation
domain-fused partners could be screened to identify mutations that
disrupt interaction with either one or both of the partner proteins. As
a third example, one source of interest in two-hybrid systems is their
use in drug screening approaches to identify compounds that disrupt
interactions between discrete pairs of interacting proteins (8, 32,
33); dual bait reagents would apply a simultaneous control to the
specificity of such interactions.
Parallel Performance of LexA and cI Expression and Reporter
Systems--
Given that assessment of protein interactions in
two-hybrid systems is a factor of bait expression levels (34) and
stringency of reporter systems (23), for these hypothetical uses to be practicable, the two-bait-reporter combinations utilized in the dual
bait system must express respective baits to similar levels and possess
similar sensitivities to transcriptional activation. Therefore, an
initial step was to carefully measure these parameters. To this end, we
constructed equivalent pEG202 (LexA) and pGKS3 (cI) fusions to the
protein Krev-1/rap1A (31), a human Ras-family GTPase. These and parent
vectors were transformed in parallel into EGY48 yeast, and expression
of the synthesized proteins was assayed by Western analysis using
antibodies to Krev-1, LexA, or cI (Fig.
2). Expression of the two Krev-1 fusion
constructs was found to be comparable in 4 randomly chosen colonies,
with slightly higher levels (~2-3-fold) in the cI constructs.
Furthermore, expression of the fusion protein was in each case similar
to the matching unfused DNA binding domain, indicating that cI
tolerated attachment of a fusion domain without loss of stability.
Finally, essentially identical expression levels were observed using
pGKS6-Krev-1, a ZeoR instead of HIS3 version of
pGKS3 (not shown), indicating the selectable marker could be exchanged
without gross alteration of plasmid copy number.
We next determined the degree to which activation through cI
operators was comparable with activation through lexA
operators. As a conservative first step, we constructed analogous
fusions of pGKS3 and pEG202 to Krit1 (a Krev-1-interacting protein (30) that fortuitously functions as a transcriptional activator of moderate
strength) and assayed activation of the closely related cIop- and lexAop-lacZ reporters.
Parallel transformations were performed with pGKS3-Krit1 (cI-Krit1)
plus cIop-lacZA and
cIop-lacZB, which contained the three naturally
occurring cI operators OR1-OR3 of
Finally, we compared direct activation of the LEU2
versus LYS2 auxotrophy reporters, again using
analogous LexA- and cI-fused Krit1(Fig.
4). Using SKY48 and SKY191 as hosts, we
determined that LexA-Krit1 activates the LEU2 reporter of
these strains, whereas cI-Krit1 does not (Fig. 4, second panel
from top). Conversely, cI-Krit1 is capable of activating the
LYS2 reporter of SKY strains, whereas LexA-Krit1 is not
(Fig. 4, third panel from top). Finally, each fusion
activated the appropriate lacZ reporter to comparable degrees, independent of growth properties on Leu or Lys medium (Fig. 4,
bottom panel, middle two rows). Notably, positive
growth dependent on activation of the LEU2 and
LYS2 reporters could be assessed in similar time frame, with
results detectable at 24-48 h after plating yeast on selective media.
Based on visual estimation of growth rate, the sensitivity of the
cIop-LYS2 reporter in these strains appears to be
intermediate between that of the LEU2 reporters in EGY48
and EGY191.
Cumulatively, these results indicated that the cI- and LexA-based
expression and reporter constructs yielded results in a similar
sensitivity range, making them suitable for comparative purposes. One
point remaining was the development of reagents suitable for expressing
all baits and reporters in the same strain, to allow simultaneous
assay. SKY strains (MAT Specificity of the Dual Bait System in Controlled Two-hybrid
Assay--
The major criterion for effective use of a dual bait system
is that it should effectively discriminate interactions of a partner with related but distinct proteins. Ras and Krev-1 possess 56% amino
acid identity and are known to interact with an overlapping set of
protein partners (35-37). In experiments described elsewhere, we
determined that Raf preferentially interacts with Ras by a two-hybrid
system assay, whereas Krit1 preferentially interacts with Krev-1 (30).
The Ral guanine nucleotide dissociation stimulator RalGDS interacts
with both Krev-1 and Ras (36). Neither Ras nor Krev-1 activates
transcription when expressed as a DNA binding domain fusion.
The strain SKY191 with the plasmid pSH18-34 was used as a host for
transformation by pEG202-Ras (LexA-Ras) and pGKS8-Krev-1 (cI-Krev-1).
We then super-transformed the SKY191/pEG202-Ras/pGKS8-Krev-1 combination in parallel with each of the galactose-inducible expression plasmids pJG4-5-Raf (AD-Raf), pJG4-5-Krit1 (AD-Krit1), or
pYesTrp2-RalGDS (AD-RalGDS) or with empty AD vector and assayed for
reporter activation/growth properties on selective medium. As noted
above, activation through a LexA fusion permits growth on Leu medium
and production of LacZ (cleaves X-Gal, Magenta-Gal, etc. to produce
colored products); activation through a cI fusion permits growth on Lys
medium and production of
All yeast grew on nonselective plates (ura
The results of X-Gal and X-Gluc assay on the plates are in good
correspondence with the auxotrophic selection assay, with Raf-Ras
positive with X-Gal (Fig. 5, panel C), Krev1-Krit1 positive with X-Gluc (Fig. 5, panel B), and Ral-GDS positive with
both (Fig. 5, panel B and C). Note: using a
complementary set of color-producing substrates (Magenta-Gal + X-Gluc),
both LacZ and GusA activities can also be assayed on a single plate
(Fig. 5, panel D). These results paralleled those previously
obtained using a conventional two-hybrid selection (30) and confirmed
that the dual bait system can be used to distinguish interactions
between two closely related potential partner proteins. We note that
although the Selection of Specifically Interacting Protein Pairs from a
Nonspecific Pool--
The previous results demonstrated that direct
streaking of uniform populations of yeast containing predetermined
combinations of baits, activation domain fusions, and reporters yields
expected results. A more rigorous test of the power of the ability of
these reagents to discriminate specific interactions was performed
using a mixing experiment, as outlined in Fig.
6. Four populations of SKY191 yeast were
generated. Each expressed LexA-Ras and cI-Krev-1 and contained
lexAop-lacZ and cIop-gusA reporters and an
activation domain fused to either 1) Raf, 2) RalGDS, 3) Krit1, or 4)
nonspecific (a fragment of hsp90). 10-100 colony forming units
(~30-300 cells), each of populations 1-3, were mixed together with
2 × 106 cells containing the nonspecific control and
parallel samples of the pooled cells plated to media selective for the
lexAop-LEU2 (ura-his-trp-leu-Zeo) or
cIop-LYS2 (ura-his-trp-lys-) reporters.
Approximately 50 colonies arose on each of these plates, in good accord
with the number anticipated based on the seed. Of these, 24 were chosen
from each of the Leu and Lys plates and transferred to a master plate,
then retested for growth on both leu In this report, we describe the development and characterization
of novel dual bait reagents that can be used to study the interaction
of a protein with two distinct partners in a single yeast cell. The cI
repressor/cI operator system utilized in the SKY yeast
strains and cIop-LacZ plasmids is demonstrated to
function with a sensitivity range closely comparable with the
pre-existing LexA repressor/lexA operator system in the
interaction trap, facilitating their combined use. In a model system
assaying the interaction of the related GTPases Ras and Krev-1 with
their preferred partners Raf and Krit1 and their shared partner RalGDS,
the dual bait system clearly differentiates higher affinity
versus lower affinity interactions. In addition to
effectiveness in discriminating interactions in grids of yeast streaked
to selective plates, the discrimination observed is sufficiently robust
to allow the isolation of yeast containing specifically interacting
protein pairs against a vast excess of noninteracting pairs. These
properties support the idea that these reagents will be useful in
library screening and genome-scale applications. The reagents described
here offer the option of performing two independent, simultaneous
screens in a single yeast, with one set of positives registering
through lexAop activation of LEU2 and
lacZ and a second set through cIop activation of
LYS2 and gusA, both negatively controlled against
each other. Together, these developments have the potential to greatly
expand two-hybrid system contributions to studies of biological interactions.
Several groups have recently described the use of two simultaneously
expressed baits to identify mutations that selectively affect
interactions of an activation domain-fused protein with one of two
partners (38-40). In each case, introduction of a second bait-reporter
system was obtained by eliminating one of the two reporters used for
the primary bait, greatly reducing the screening power available to the
system. However, in these novel reagents, both baits retain two
distinct reporters, greatly facilitating the screening process. The
value of having two separate reporters is evident even in the mixing
experiment performed here (Fig. 6), as their use allowed the immediate
discrimination of the noninteracting background clones from specific
partners, which activated both reporters. The dual bait reagents
described here can be similarly used for mutational analysis and have
been recently used to successfully identify mutations in Pak1 kinase,
which selectively reduce interaction affinity for either of two
partners, the Cdc42 or Rac
GTPases.3 Finally, there is
preliminary evidence that these reagents possess the power necessary to
perform library screens in organisms with complex genomes; as in
several recent library screens using the above reagents, cI- and the
LexA-fused baits have yielded specific partners.4
On a more basic level, the system allows considerable savings in the
effort required by individual investigators wishing to perform multiple
two-hybrid screens without invoking the dual bait selectivity function.
Instead of having to perform two separate library transformations or
matings (generally the most laborious step in a screening process) and
subsequent selection of positive clones, only one such step is required
for any two baits. Furthermore, in the case where only one-half of a
dual bait screen of a previously untested cDNA library is positive
(e.g. if a LexA bait yields interactors but a cI bait does
not), the fact that positive interactors are obtained for at least one
bait will provide useful information toward the determination of
whether the library utilized was of acceptable quality. Finally, it has
previously been noted that some proteins of interest for cDNA
library screening perform better with particular fusion domains
(i.e. are productively utilized as LexA fusions but not as
GAL4 fusions; or vice versa (23)). In cases where specific
DNA binding domain/fusion domain problems are suspected, an
investigator could express a bait of interest both as a LexA and as a
cI fusion and screen with the bait in both configurations to maximize
chances of obtaining valid positive interacting partners.
The dual bait reagents are built upon the interaction trap form of
two-hybrid system (4). cI and LexA are similar in size (237 versus 202 amino acids) and structure (41) and use related amino-terminal helix-turn-helix domains to bind palindromic operator sites with similar Km values, ranging from 200 pM to 20 nM for LexA (discussed in Estojak
et al. (23)) similar to cI (42). Because of these many
similarities, it is clear that the LexA and cI systems are well
matched. However, in addition to use in the current interaction trap
shell, the cI "add-on" parts of this system have been constructed
to potentially supplement any of the currently existing two-hybrid
variants. Thus, the reporter system developed in this study purposely
uses a DNA binding domain (cI), reporter genes (gusA and
LYS2), and plasmid marker (zeocin resistance) not in use in
any other two-hybrid-based system (2, 3, 5), including the recently
described membrane-based Sos recruitment system (16). Thus, these
reagents could readily be integrated with any of the other screening
systems operating on two-hybrid principles; in the case of the Sos
recruitment system, this raises the possibility that with minor
modifications of the library vector, a single bait could be
simultaneously used to identify interactors using either a
membrane-based or a transcriptional activation-based selection
strategy, enlarging the potential pool of interacting proteins
obtained. An additionally useful feature of the gusA
reporter is that it is assayed using similar protocols even on the same
plates as the lacZ reporter standardly used in two-hybrid
systems, again contributing to ease of use. Finally, although the dual
bait reagents here described have been optimized for use in conjunction
with LexA fusions, parameters have been previously established to test
and vary sensitivity levels (23), making merging of two-hybrid systems
a relatively simple and certainly useful effort that should contribute
to efforts to understand complex protein-protein interactions on the
genome scale.
cI). Using
the selective interactions of Ras and Krev-1(Rap1A) with Raf, RalGDS,
and Krit1 as a model, we systematically compared LexA- and cI-fused
baits and reporters. The LexA and cI baitr reporter systems are well
matched for level of bait expression and sensitivity range for
interaction detection and allow effective isolation of specifically
interacting protein pairs against a nonspecific background. These
reagents should prove useful to refine the selectivity of library
screens, to reduce the isolation of false positives in such screens,
and to perform directed analyses of sequence elements governing the
interaction of a single protein with multiple partners.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Escherichia coli was grown on LB medium (21)where
appropriate, and antibiotics were added at the concentrations
recommended by the suppliers. Standard DNA manipulation techniques were
as described in (21). Yeast were grown on yeast-peptone-glucose or
minimal medium and manipulated using standard techniques (22). Two-hybrid experiments and
-galactosidase assays were performed as
described (23), with six independent colonies assayed for each value
presented; for sensitive plate-based X-Gal and X-Gluc assays, the
procedure of Duttweiler (24) was used.
repressor protein cI (25) was used as the basis of reagent development,
as its size, structure, and DNA binding properties suggested it might
behave comparably as a DNA binding domain to the pre-existing
two-hybrid system DBD LexA (26-28).
bacteriophage genome (LAMCG, nucleotides 37950-38018) containing 3 naturally occurring cI operators was amplified, and
XhoI ends were added by PCR. The resulting product was
digested with XhoI and inserted into the XhoI
site of the plasmid LR1
1 (parent of all currently utilized interaction trap lexAop-lacZ reporters (20)) in
either orientation upstream of a basal GAL1 promoter
directing expression of the lacZ gene. The resulting
plasmids pcIop-LacZA and pcIop-LacZB have a
2-µm origin of replication and use a URA3 marker for
selection in yeast; they differ only in the orientation of the
cI operator cassette.
201 trp1::hisG) was
transformed with ApaI-digested pCIL-1 (targeting integration
to URA3 gene), and stable integrants were selected on ura-
dropout medium. One of these strains, SK01 (MATa trp1
ura3-52 his3200 leu2-3 lys2
201 trp1::hisG
URA3:cIop-LYS2) was taken for subsequent characterizations.
trp1 ura3 his3
lexAop-LEU2) (4), and the resulting diploid was sporulated. The
strain SK10, with the genotype (MATa trp1 ura3 his3 lexAop-LEU2 lys2
201 URA3:cIop-LYS2) was obtained following tetrad dissection.
) aminoadipic acid (a toxic metabolite of the LYS2
protein (29)) as the sole source of nitrogen. 24 stable integrants in
each of the parent strains were selected and confirmed to be
Lys-Leu-His-Ura-Trp-; representative integrants into each parent have
been designated SKY48 and SKY191 and have the genotype (MAT
trp1 ura3 his3 lexAop-LEU2 cIop-LYS2), with 6 or 2 lexA operators upstream of LEU2, respectively.
-glucuronidase (gusA; a gift of J. Vossen, University of
Amsterdam). An EcoRI fragment containing a minimal
GAL1 promoter, cI operator cassette (containing
three naturally occurring cI operators), and the translational start of
the GAL1 gene was excised from
cIop-lacZA and recloned immediately upstream of
the gusA sequences in pRG00 to create the plasmid pRG2
(cIop-gusA).
cI
repressor protein (LAMCG, nucleotides 37230-37940) was amplified by
PCR and cloned into the plasmid pUC19 to yield pUC-cI. Separately, a
HIS3-containing fragment of pEG202 was removed by
AatII-ClaI digestion and replaced by a synthetic
AatII-ClaI linker, to create pGK202. Subsequent HindIII digestion, fill-in reaction, and EcoRI
digestion were used to remove the lexA gene from pGK202,
followed by replacement with the cI gene on a BglII
(filled-in)-EcoRI fragment excised from pUC-cI. The
resulting plasmid pGK302, was digested with BamHI and
AatII and ligated to a BamHI-AatII
fragment of pEG202 to create pGKS3, a pEG202 "sibling" with the cI
gene exactly replacing the lexA gene. pGKS3 has a 2-µm
origin of replication, carries a HIS3 marker for yeast
selection, and was used in control experiments.
amino acids 1-56) genes into the
EcoRI-XhoI sites of the plasmid pJG4-5 (4) and
RalGDS (amino acids 767-848) (a gift of A. Vojtek) into
BamH-EcoRI sites of pYesTrp2 (Invitrogen).
-galactosidase assays as described in (21).
Activation of gusA reporters was assessed qualitatively
using the same overlay procedure as for LacZ but with X-Gluc
(Diagnostic Chemicals Ltd) as a substrate. A quantitative assay was
performed as for a
-galactosidase assay but using
4-nitrophenyl-
-D-glucuronic acid instead of
2-nitrophenyl-
-D-galactopyranoside as the substrate. For
both assays, determination was made in a plate reader; in a standard
procedure, 100 µl of exponential phase yeast cultures were harvested
by centrifugation in a microtiter plate. Yeast pellets were resuspended
in 50 µl of Z-buffer, and the A590 was
determined in a plate reader, after which the plate was frozen at
70 °C. After thawing, 50 µl of
4-nitrophenyl-
-D-glucuronic acid (2 mg/ml) or
2-nitrophenyl-
-D-galactopyranoside (2 mg/ml) in
Z-buffer, as appropriate, was added to each well. Immediately, the
A405 was measured, and the plate was then placed
in a 30 °C incubator. Additional A405
readings were taken between 5 and 45 min, depending on the intensity of
the reaction. To calculate
-glucuronidase or
-galactosidase
activity, the following formula was used:
where v is volume in ml, t is time, and
(A405t
(Eq. 1)
A4050) is a net increase of
optical density at 405 nm between zero time and time t, and
A590 is optical density at 590 nm.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Interaction trap and dual bait systems.
A, an activation (Act.) domain-fused prey
interacts with a LexA-fused bait to drive transcription of
lexAop-responsive LEU2 and lacZ reporters.
B, an activation domain-fused prey interacts with a
LexA-fused bait to drive transcription of lexAop-responsive
LEU2 and lacZ reporters but does not interact with a
cI-fused bait and, thus, does not turn on transcription of
cIop-responsive LYS2 and gusA reporters. Note, as
shown here, that cI bait is drawn as representing a nonspecific
partner; the system can also be configured so prey interacts with both
baits; see text.
-glucuronidase) (n = 3) reporter gene cassette,
additionally borne on the plasmid GKS8. The third is an additional
integrated reporter system in which cI operators direct the
expression of the LYS2 gene;
(cIop)n-LYS2 (n = 3) in
the yeast strains SKY48 or SKY191 (derivatives of EGY48 and EGY191,
respectively). These reagents can be utilized in multiple ways to
enhance measurement of protein interactions over currently existing approaches.
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Fig. 2.
LexA and cI expression vectors synthesize
comparable levels of fusion protein. Whole cell extracts from
yeast expressing either pEG202-Krev-1 (LexA-Krev-1),
pGKS3-Krev-1 (cI-Krev-1), or parental vectors pEG202 or
pGKS3 were examined by protein immunoblot with the antibodies to Krev-1
(top panel); the blot was then stripped and reprobed with
antibodies to LexA (bottom left) or cI (bottom
right). Before transfer to membrane, the blot was stained with
aqueous Coomassie to confirm equivalent protein load in all
lanes (not shown).
phage in either orientation (25). In addition, for comparison, yeast were transformed with pEG202-Krit1 (LexA-Krit1) plus pRB1840, pJK103, or pSH18-34 (1, 2, or 8 lexAop-lacZ)
(23), and as a negative control, with pEG202-Krit1 plus
cIop-lacZA.
-Galactosidase assays were used to
measure activation of the lacZ reporters (Fig. 3). In these tests, the cI-Krit1 fusion
protein activated the two cIop-lacZ constructs to
equivalent levels, which were closely comparable with that obtained
using the combination of LexA-Krit1 and pJK103. As a negative control,
the LexA-Krit1 construct was also shown not to activate the
cIop-lacZ reporters, as expected. Based on this
result, we next used the cIop/minimal promoter cassette to
develop a cIop-gusA reporter, pRG2, to be used in
conjunction with the standard lexAop-lacZ reporters. Yeast
were transformed with the reporter and an activating cI-Krit1 fusion
protein or LexA-fused Ras (which does not activate transcription) as a
negative control, and gusA transcription was assessed with a
quantitative
-glucuronidase assay, analogous to a
-galactosidase
assay (see "Experimental Procedures"); as with the
cIop-lacZ reporters, a high degree of specific activation
was observed against a much lower background (6300
-glucuronidase
units for cI-Krit1 versus 85 for the negative control, a
75-fold difference), although overall the gusA reporter was
more sensitive than lacZ, as reflected in the higher units
(700
-galactosidase units for cI-Krit1). In plate overlay assays,
positive and negative were similarly clearly distinct, with cI-Krit1
medium to dark blue, whereas the negative LexA-fused control was pale
blue or white (not shown; also, see below).
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Fig. 3.
Proportional activation of lacZ
reporters by cI or LexA-fused activators. A,
comparative activation of lacZ reporters, lexA-op
versus cI. Values shown reflect proportional
enhancement of activation in -galactosidase assays; the degree of
activation obtained from LexA-Krit1 through a 2 lexAop-lacZ
reporter is arbitrarily set to 1.
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Fig. 4.
Comparative activation of yeast reporter
plasmids and strains through LexA versus cI
operators. Strains SKY48 and SKY191 were transformed with the
pairwise combinations of either pEG202-Krit1 or pGKS3-Krit1,
transcriptionally activating fusions to LexA and cI, respectively, and
either pJK103 or cIop-lacZA, reporters with
lacZ transcriptionally responsive to LexA or
cI operators, respectively. Three independent transformants
were replica-plated either on nonselective medium (top) or
medium selecting for activation of LEU2 (leucine,
second panel), LYS2 (lysine, third
panel), and LacZ (with X-Gal, fourth panel)
reporters.
trp1 ura3 his3 lexAop-LEU2
cIop-LYS2) utilize the LEU2 and LYS2 markers
for reporter genes. The activation domain fusion expression plasmid
(pJG4-5) uses a TRP1 marker; the lexAop-LacZ
reporter (pJK103) uses a URA3 marker; and the LexA fusion
bait plasmid (pEG202) uses a HIS3 marker; the cI fusion bait
plasmid (pGKS6) utilizes a ZeoR marker. To introduce the
cIop-gusA reporter, we took advantage of the fact that the
cIop-gusA cassette is only 2.6 kilobases, whereas the
plasmid backbone for the cI fusion plasmid pGKS3 is unusually small, as
the ZeoR marker is used for both bacterial and yeast
selection. The cIop-gusA cassette was introduced into pGKS6,
resulting in a new plasmid, pGKS8, which encompassed both cI-bait and
cIop-gusA reporter. Control experiments similar to those
outlined above demonstrated that this bait-reporter-combined plasmid
yielded results similar to those obtained with the combination of pGKS3
and pRG2 (not shown). This construct was used for the experiments
described in the following sections.
-glucuronidase (cleaves X-Gluc, etc., to
produce colored products).
his
trp
, glucose, or
galactose, Fig. 5, panel A).
No strains grew on either Leu or Lys plates when glucose was present as
the carbohydrate source. However, under galactose induction, strains
containing pJG4-5-Raf were able to grow preferentially on Leu medium
(Fig. 5, panel F) but only minimally on Lys medium, based on
the association between Raf and LexA-Ras; conversely, strains
containing pJG4-5-Krit1 grew well on Lys medium but only weakly on Leu
medium, based on the interaction between Krit1 and cI-Krev-1 (Fig. 5,
panel E). Strains containing pYesTrp2-RalGDS grew well on
both Lys and Leu medium (Fig. 5, panels E and F),
whereas a negative control (strains containing empty plasmid pJG4-5)
did not grow on any selective plates (Fig. 5, panels E and
F). Interaction of RalGDS with both baits could be also
detected on the double auxotrophic Lys-Leu plate, where this was the
sole plasmid combination resulting in growth (Fig. 5, panel
G). Interactors that associated with only the cI-fused bait or
nonselectively, with both the cI- and LexA-fused baits, could be
counterselected by inclusion of
-aminoadipate in the growth medium
as the sole source of nitrogen (Fig. 5, panel H).
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Fig. 5.
Discrimination of interaction specificity by
dual bait. All yeast colonies shown express LexA-Ras (from pEG202)
and cI-Krev-1 (from pGKS8) and contain lexAop-lacZ (pJK103)
and cIop-gusA (pGKS8) reporters and integrated
lexAop-LEU2 and cIop-LYS2 (SKY191) reporters.
Yeast additionally contain the AD fusion vector (top row),
AD-Krit1 (second row), AD-RalGDS (third row),
AD-Raf (fourth row). Panel A, growth on
nonselective medium; panel B, growth on plate assay for
-glucuronidase activity with X-Gluc; panel C, growth on
plate assay for
-galactosidase activity with X-Gal; panel
D, dual assay for for
-glucuronidase and
-galactosidase
activity with X-Gluc and Magenta-Gal; panel E, growth on
plates lacking lysine; panel F, growth on plates lacking
leucine; panel G, growth on plates lacking both leucine and
lysine; panel H, growth on plates lacking leucine but
containing
-aminoadipic acid (
AA) (counterselective
for LYS2). Note: less yeast are generally plated when assaying for
-glucuronidase than for
-galactosidase because of the increased
sensitivity of the gusA reporter; similarly, when performing
dual assays, an initial overlay is done with agar containing
Magenta-Gal, followed by a subsequent addition of X-Gluc.
-aminoadipate counterselection works well in the
controlled situation shown above, over time background colonies arose
on the counterselected plates, suggesting this particular application
may be more useful in targeted disruption of known interactions than in
conjunction with library screens.
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Fig. 6.
Selection of specific interacting pairs from
a mixed pool. Yeast were mixed in noted ratios, then plated to
medium selective for activation of the lexAop-LEU2
(UHWL Zeo) or cIop-LYS2 (UHK)
reporters. Colonies arising on selective plates were tested for
activation of LEU2, LYS2, gusA, and lacZ
reporters; 5 colonies, predicted to contain Krit1, Raf, or RalGDS based
on their reporter activation profile, were used for PCR amplification
with library vector-based primers, and amplified product was compared
with similarly amplified control plasmids as a size measure for
respective inserts. M, marker lane; Ral, Krit, Raf in
top line are amplified control plasmid.
and lys
medium, as well as
activation of lacZ or gusA reporters. 43 of the
48 analyzed colonies were specific for growth on Leu or Lys medium,
whereas 5 of the 48 total colonies analyzed grew on both Leu and Lys
medium. In tests with the colorigenic reporters, 45 of the 48 displayed
expected patterns for lacZ and gusA; LEU+ colonies were blue with X-Gal, LYS+ colonies were blue with X-Gluc, LEU+LYS+ colonies were blue with both; the remaining 3 colonies were
white with both substrates. Finally, 5 colonies for each group (LEU+
LacZ+ LYS-gusA
, LEU+ LacZ+ LYS+
gusA+, LEU
LacZ-LYS+ gusA+) were
selected at random and used for PCR using primers containing sequences
complementary to library vector sequence-flanking inserts to identify
inserts based on characteristic product size (Fig. 6,
bottom). Separately, each individual library plasmid was
amplified to provide a size standard for Krit1, Raf, and RalGDS). As
shown, each of the selected LEU+LYS
colonies contained Raf, each of
the LEU-LYS+ colonies contained Krit1, and each of the LEU+LYS+
colonies contained RalGDS as predicted, based on their reporter
activation profile. In contrast, PCR from the three colonies with an
inappropriate pattern of activation (e.g. LYS+ but
gusA
) revealed the presence of the hsp90 fragment (data
not shown). In numerous tests of simple two-hybrid system reagents,
such as the interaction trap, we previously found that a common source of false positives is selection of strains mutated so as to transcribe the auxotrophic reporter (LEU2) in the absence of contribution from
either bait or prey; these false positives are standardly detected
because of their inability to additionally activate the second
chromogenic reporter (20). The detection of the nonspecific hsp90
fragment in the LYS+ gusA
cells suggests that these colonies represent similar such selected mutant strains and supports the idea
that the presence of two distinct reporters continues to provide a
useful specificity for the system.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Russ Finley, Anne Vojtek, Jack
Vossen, and Gabe Kalmar for their gifts of reagents, Michelle Berman
for excellent technical assistance, Joe Hurley for yeast photography,
and Melissa Reeder and Marijane Russell for their efforts in testing the dual bait system. We are grateful to Jonathon Chernoff,
Randy Strich, Garabet Toby, Russ Finley, and Bob Perry for helpful
critique of the manuscript.
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FOOTNOTES |
---|
* This work was supported by an award from the Merck Genome Research Institute, by National Institutes of Health Core Grant CA06927, and by a Small Business Innovative Research program grant subcontract from Invitrogen.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Current address: Small Molecule Therapeutics, 11 Deer Park Dr.,
Monmouth Junction, NJ 08852.
§ To whom correspondence should be addressed: Fox Chase Cancer Center, 7701 Burholme Ave., Philadelphia, PA 19111. Tel.: 215-728-2860; Fax: 215-728-3616; E-mail: EA_Golemis{at}fccc.edu.
2 I. Serebriiskii and E. A. Golemis, E. A. (1996) http://www.fccc.edu/ research/labs/golemis/interactiontrapinwork.html.
3 M. Reeder, J. Chernoff, E. A. Golemis, and I. Serebriiskii, unpublished data.
4 M. J. Russell, personal communication; V. Khazak, unpublished data; Y. Z. Zhang and E. Golemis, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are:
DBD, DNA binding
domain;
X-Gal, 5-bromo-4-chloro-3-indolyl
-D-galactopyranoside;
PCR, polymerase chain reaction;
AD, activation domain;
X-Gluc, 5-bromo-4-chloro-3-indolyl
-D-glucuronic acid, sodium
salt;
Magenta-Gal, 5-bromo-6-chloro3-indolyl
-D-galactopyranoside.
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REFERENCES |
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