Note: the following is useful for background information. Please see Protocols Page for more updated, and recommended interactor hunt protocols.

Interaction trap cloning with yeast.

The following manuscript is a draft of a chapter appearing in "DNA Cloning 2: A Practical Approach, Expression Systems", 2nd Edition, eds D. M. Glover and B. D. Hames. Oxford University Press, 1995.
 
 

Russell L. Finley Jr. and Roger Brent

Department of Molecular Biology
Massachusetts General Hospital
and Department of Genetics
Harvard Medical School
Boston Massachusetts 02114
U.S.A.
 
 

Address:
Department of Molecular Biology
Massachusetts General Hospital
50 Blossom St.
Boston Massachusetts 02114
USA

Phone (617) 726-5925
FAX (617) 726-6893
e-mail brent@frodo.mgh.harvard.edu
finley@frodo.mgh.harvard,edu
 
 

Contents

1. Introduction
1.1 Background
1.2 The interaction trap
2. Making and testing baits
2.1 LexA fusion expression plasmids
2.2 Reporters and yeast strains
2.2.1 LEU2 reporter strains
2.2.2 lacZ reporters
2.3 Testing the bait protein
2.3.1 Testing whether the bait protein activates transcription of the
reporters

Protocol 1. Testing baits for transcription activation

2.3.2 Demonstrating that the bait enters the yeast nucleus and binds
operators
Protocol 2. The repression assay
2.3.3 Verifying that a full-length fusion protein is made.

3. Libraries
4. An interactor hunt
4.1 Introducing the library into the selection strain.
4.1.1 Selecting interactors from library transformants.
4.1.2 Performing a one step selection for interactors

Protocol 3. Transforming the selection strain with library DNA.

4.2 Isolating yeast with galactose dependent Leu+ and lacZ+
phenotypes

Protocol 4. Selecting interactors.

5. Verifying specificity

Protocol 5. Isolating and classifying library plasmids.
Protocol 6. Determining specificity of interactors.

6. Using a mating assay to verify specificity.
Figure 4a. Mating assay cartoon.
Figure 4b. Mating assay result.

Protocol 7. Mating assay.

7. Expected results.

Appendix
Sequencing and PCR primers for pEG202 and pJG4-5
Media recipes.
 
 
 

Interaction trap cloning with yeast.
 

1. Introduction
The interaction trap is a two-hybrid system for cloning cDNAs
that encode proteins that interact with a protein whose coding
sequences are known. The method uses the transcription of yeast
reporter genes as a synthetic phenotype to detect protein-protein
interactions. It can also be used to study interactions between
known proteins.

1.1 Background

The two-hybrid approach takes advantage of the modular
domain structure of eukaryotic transcription factors. Many
eukaryotic transcription activators have at least two distinct
functional domains, one that directs binding to specific DNA
sequences and one that activates transcription (1, 2). This modular
structure is best illustrated by yeast experiments showing that the
DNA-binding domains or activation domains can be exchanged from
one transcription factor to the next and retain function. For example,
when the DNA-binding domain of the yeast transcription factor Gal4
is replaced with the DNA binding domain of the bacterial repressor
LexA, the resulting hybrid protein activates transcription of genes
containing upstream LexA binding sites (3). Similarly, when the DNA
binding domain of Gal4, which by itself does not activate
transcription, is fused to activation domains from other proteins the
resulting hybrid proteins activate transcription of reporters with
upstream Gal4 binding sites (4-6). A crucial corollary of the modular
nature of transcription activators is that the DNA-binding and
activation domains need not be covalently attached to each other for
activation to occur. This was first demonstrated by Ma and Ptashne
(7) with a Gal4 derivative that contained the DNA-binding domain as
well as a domain that interacts with another yeast protein, Gal80, but
that lacked the activation domain. When this derivative was
expressed in yeast it did not activate transcription of a reporter gene
containing upstream Gal4 binding sites. However, when it was co-
expressed with a second, hybrid protein, consisting of Gal80 fused to
an activation domain, interaction between the Gal4 DNA-binding
derivative and the Gal80-activation domain hybrid resulted in
activation of the reporter gene.

The general utility of the modularity of transcription factors
was demonstrated by Fields and Song (8) who showed that yeast
transcription could be used to assay the interaction between two
proteins if one of them was fused to a DNA-binding domain and the
other was fused to an activation domain. In their experiment, one of
the hybrid proteins contained the DNA-binding domain of Gal4 fused
to the yeast protein Snf1, and the other contained the activation
domain of Gal4 fused to another yeast protein, Snf4. When Snf1 and
Snf4 interacted they brought together the DNA-binding and
activation domains, so that the two hybrid proteins bound to Gal4
binding sites upstream of a lacZ reporter gene and activated its
transcription. Thus, the interaction between Snf1 and Snf4 was
assayed as production of beta-galactosidase. The success of this
experiment prompted Fields and Song to make the seminal
suggestion that yeast transcription could be used in this way to clone
cDNAs encoding proteins that interact with a given known protein
(8). In their scheme, a known protein is expressed fused to the DNA-
binding domain of Gal4, and a cDNA library is expressed so that
proteins encoded by the cDNA are fused to an activation domain
(activation-tagged). Transcription of a reporter gene will be
activated in yeast containing activation-tagged cDNA-encoded
proteins that interact with the known protein.

Based on this suggestion, two-hybrid cloning systems have
been developed in several labs (9-13). All have three basic
components: Yeast vectors for expression of a known protein fused to
a DNA-binding domain, yeast vectors that direct expression of cDNA-
encoded proteins fused to a transcription activation domain, and
yeast reporter genes that contain binding sites for the DNA-binding
domain. These components differ in detail from one system to the
other. All systems utilize the DNA binding domain from either Gal4
or LexA. The Gal4 domain is efficiently localized to the yeast nucleus
where it binds with high affinity to well-defined binding sites which
can be placed upstream of reporter genes (14-16). LexA does not
have a nuclear localization signal, but enters the yeast nucleus and,
when expressed at a sufficient level, efficiently occupies LexA
binding sites (operators) placed upstream of a reporter gene (3, 17,
18). No endogenous yeast proteins bind to the LexA operators.
Different systems also utilize different reporters. Most systems use a
reporter that has a yeast promoter, either from the GAL1 gene or the
CYC1 gene, fused to lacZ (19, 20). These lacZ fusions either reside on
multicopy yeast plasmids or are integrated into a yeast chromosome.
To make the lacZ fusions into appropriate reporters, the GAL1 or
CYC1 transcription regulatory regions have been removed and
replaced with binding sites that are recognized by the DNA-binding
domain being used. A screen for activation of the lacZ reporters is
performed by plating yeast on indicator plates that contain X-Gal (5-
bromo-4-chloro-3-indolyl-b-D-galactoside); on this medium yeast in
which the reporters are transcribed produce beta-galactosidase and turn
blue. Some systems use a second reporter gene and a yeast strain
that requires expression of this reporter to grow on a particular
medium. These "selectable marker" genes usually encode enzymes
required for the biosynthesis of an amino acid. Such reporters have
the marked advantage of providing a selection for cDNAs that encode
interacting proteins, rather than a visual screen for blue yeast. To
make appropriate reporters from the marker genes their upstream
transcription regulatory elements have been replaced by binding
sites for a DNA-binding domain. The HIS3 and LEU2 genes have both
been used as reporters in conjunction with appropriate yeast strains
that require their expression to grow on media lacking either
histidine or leucine, respectively.

Finally, different systems use different means to express
activation-tagged cDNA proteins. In all current schemes the cDNA-
encoded proteins are expressed with an activation domain at the
amino terminus. The activation domains used include the strong
activation domain from Gal4, the very strong activation domain from
the Herpes simplex virus protein VP16, or a weaker activation
domain derived from bacteria, called B42. The activation-tagged
cDNA-encoded proteins are expressed either from a constitutive
promoter, or from a conditional promoter such as that of the GAL1
gene. Use of a conditional promoter makes it possible to quickly
demonstrate that activation of the reporter gene is dependent on
expression of the activation-tagged cDNA proteins.

Many of these systems now provide the investigator with a
relatively good chance to recover proteins that interact with other
proteins. Because most are based on the same concepts, some of their
components are often interchangeable. However, different systems
utilize the yeast selectable markers in different ways. Moreover,
systems that employ the DNA-binding domain of Gal4 must use a
yeast strain that lacks wild type Gal4; these system cannot use
library vectors that direct synthesis of the activation-tagged proteins
from the GAL1 promoter whose transcription requires Gal4.

1.2 The interaction trap

The interaction trap is an implementation of the two-hybrid
system developed by Gyuris et. al (11). It consists of three critical
components (see Figure 1). First, it uses a vector for expression of a
protein of interest fused to LexA. Because the goal of interaction
trap cloning is to find proteins that interact with the protein fused to
LexA, this hybrid is referred to as the "bait". Second, the trap uses a
yeast strain with two reporter genes. One reporter is a yeast LEU2
derivative that has its normal upstream regulatory sequences
replaced with LexA operators. Transcription of the LexA-operator-
LEU2 gene (LexAop-LEU2) can be measured by the ability of the
strain to grow in the absence of leucine, which requires the LEU2
gene product. The LexAop-LEU2 gene is integrated into the yeast
chromosome. The other reporter gene is lacZ, which provides a
secondary assay of activation by the bait and activation-tagged
proteins interacting with it, as well as some quantitative information
about the interaction. Third, the interaction trap uses a library
plasmid that directs the conditional expression of cDNA-encoded
proteins fused at their amino termini to a moiety containing three
domains: a nuclear localization signal, a transcription activation
domain, and an epitope tag. The activation-tagged cDNA-encoded
protein is expressed from the yeast GAL1 promoter, which is induced
by galactose and repressed by glucose.

The interaction trap is illustrated in Figure 1. The bait protein
is constitutively expressed. It binds to LexA operators upstream of
the reporter genes LEU2 and lacZ but does not activate their
transcription. The activation-tagged cDNA-encoded protein is
conditionally expressed from the GAL1 promoter. In glucose
medium the GAL1 promoter is repressed, no cDNA-encoded protein
is made, and the yeast does not grow in the absence of leucine.
When the yeast are grown on galactose medium, activation-tagged
cDNA-encoded proteins are expressed, and those that interact with
the bait activate transcription of the LEU2 and lacZ reporters. Thus,
cells containing activation-tagged cDNA proteins that interact with
the bait form colonies on galactose medium lacking leucine and form
blue colonies on galactose X-Gal plates.

(Figure 1. The interaction trap)

An outline of an interactor hunt is presented in Figure 2. The
protocols for using the interaction trap described below require
knowledge of a few basic yeast microbiological and genetic
techniques. A more detailed description of such techniques, together
with recipes for appropriate media can be found elsewhere (21-23).

(Figure 2. Flow chart of an interactor hunt.)

2. Making and testing baits

2.1 LexA fusion expression plasmids

To make a plasmid that directs the synthesis of the LexA fusion
or "bait" protein, the coding region for the protein of interest is
inserted into pEG202 or a related plasmid (11) (see Appendix).
pEG202 is a multicopy yeast plasmid containing the yeast 2 mm
origin of replication and the selectable marker gene HIS3, as well as
the full-length LexA coding region flanked by the yeast ADH1
promoter and terminator. Bait proteins expressed from this plasmid
contain amino acids 1 to 202 of LexA, which include the DNA-binding
and dimerization domains. Downstream of the LexA coding region in
pEG202 are unique EcoRI, BamHI, SalI, NcoI, NotI, and XhoI cloning
sites. The bait plasmid can be introduced and maintained in a his3
yeast strain (e.g. EGY48, see below) by selecting transformants on
media lacking histidine. Transformants will constitutively express
the protein of interest with LexA at its amino terminus. Although it
does not contain a yeast nuclear localization signal, LexA and most
LexA fusions will enter the nucleus (3, 17, 18, 24-29). The
expression levels afforded by the ADH1 promoter are generally
sufficient to provide occupancy of LexA operators upstream of the
reporter genes. For the rare bait that is excluded from the nucleus, a
pEG202 derivative can be used that directs expression of LexA
fusions that contain a nuclear localization signal (W. Breitwieser and
A. Ephrussi, personal communication).

2.2 Reporters and yeast strains

2.2.1 LEU2 reporter strains

An interactor hunt employs a selection for cDNAs encoding
interactors. The selection depends on a yeast strain, EGY48 (11), that
has an integrated LEU2 gene with its upstream regulatory region
replaced by LexA operators. The strain has no other LEU2 gene and
does not grow in the absence of leucine unless the LexAop-LEU2
gene is transcribed. The LEU2 reporter in EGY48 is very sensitive; it
is activated by even weak transcription activators fused to LexA, or
by activation-tagged proteins that interact weakly with LexA fusions.
The high sensitivity is due to the presence of three high affinity
LexA operators positioned near the LEU2 transcription start. The
operators are from the bacterial colE1 gene and each can potentially
bind two LexA dimers (30).

While the sensitivity of EGY48 is an advantage in that it
facilitates isolation of activation-tagged cDNA proteins that interact
weakly with the bait, this strain may be too sensitive for use with
baits that are themselves weak transcription activators. Many
proteins, including some that are not transcription factors, will
activate transcription of LEU2 in EGY48. For a bait to be used in an
interactor hunt it must not activate LEU2 transcription. For baits
that fail to meet this criterion, two approaches can be taken. First,
the sensitivity of the reporter strain can be reduced. One way to do
this is by using a strain containing fewer operators upstream of LEU2
(e.g., one operator instead of three; E. Golemis, D. Krainc, R.L.F.,
unpublished data). If a bait still activates transcription of LEU2 in a
strain with only one operator, the sensitivity can be reduced further
by using a diploid yeast strain, in which, for unknown reasons, LexA
fusions activate less transcription of the reporter genes (E. Golemis,
A. Mendelsohn, D. Krainc, R.L.F., unpublished data). A second, more
drastic, approach is to construct deletion derivatives of it that do not
activate. A good way to start, if prior knowledge of the precise
location of transcription activation domains is unavailable, is to
construct derivatives that lack highly acidic regions which are often
responsible for transcription activation in yeast (2, 4, 31). The
obvious disadvantage of this approach is that regions important for
interaction with other proteins may be removed.

In addition to the mutation in the endogenous LEU2 gene,
EGY48 and related strains carry mutations in three other marker
genes (his3, trp1, ura3) that are needed to allow selection of the
plasmids used in the interaction trap. The his3 mutation is
complemented by the HIS3 gene on the bait expression vector. The
trp1 and ura3 mutations are respectively complemented by the TRP1
gene on the library plasmid, and the URA3 gene on the lacZ reporter
plasmid (see below). The plasmids for the bait, library, and lacZ
reporter each contain the yeast 2 mm origin of replication so that
under continued selection they should be maintained at 20 to 100
copies per cell (32).
 

2.2.2 lacZ reporters

In addition to the LexAop-LEU2 reporter, an interactor hunt
employs a LexAop-lacZ reporter. The lacZ reporters contain the
GAL1 TATA, transcription start, and a small part of the GAL1 coding
sequence fused to lacZ (19, 33). The GAL1 upstream activating
sequences (UASg) have been replaced with an XhoI site into which
various numbers of LexA operators have been inserted (see
Appendix). In the absence of a LexA fusion, or interacting
activation-tagged protein, yeast bearing these reporters make no
detectable beta-galactosidase and appear white on X-Gal plates. Use of
the lacZ reporters provides two advantages in an interactor hunt.
First, false positives that may arise by activation of the LEU2 gene
due to a yeast mutation, or to binding of the activation-tagged cDNA
protein to the LEU2 promoter, can be identified because they will fail
to activate the lacZ reporter. Second, the lacZ reporters provide a
relative measure of the amount of transcription caused by the
interaction of an activation-tagged cDNA protein with a bait. The
phenotype measured with the LEU2 reporter, growth in the absence
of leucine, while very sensitive, is difficult to quantitate. In contrast,
the beta-galactosidase activity in a yeast is directly proportional to the
amount of lacZ transcription, and is easily measured (33). Careful
use of the lacZ reporters may even allow comparison of interaction
affinities between different baits and activation-tagged proteins (11).

The threshold affinity of protein-protein interactions to be
detected in an interactor hunt can be adjusted by choosing between
different lacZ reporters. The sensitivity of the lacZ reporter
phenotype depends on the number of LexA operators positioned
upstream of lacZ. Activation-tagged proteins that interact weakly
with a bait can be identified by using a more sensitive lacZ reporter
containing more operators. However, the search can be limited to
find only cDNA-encoded proteins that interact tightly with the bait
by using a less sensitive lacZ reporter. All lacZ reporters commonly
used are less sensitive than LexAop-LEU2 reporters. Because of this,
some LexA activators will activate LEU2 and allow EGY48 to grow in
the absence of leucine, but will not activate lacZ and cause the strain
to turn blue on X-Gal plates. The lacZ reporters reside on 2 mm
plasmids containing the URA3 gene (see appendix).

2.3 Testing the bait protein

Before conducting an interactor hunt, the bait should be tested
to ensure that the fusion protein enters the nucleus, binds LexA
operators, and does not activate transcription of the reporter genes.
This is done in two steps. First, a selection strain is made by
introducing the bait plasmid into EGY48 that contains a LexAop-lacZ
reporter. The resulting selection strain is used to show that the bait
protein does not activate transcription of LEU2 and lacZ (Protocol 1).
Eventually, the library will be introduced into this strain. Second,
the bait plasmid is introduced into a related strain that contains a
different lacZ reporter to verify that the bait protein enters the yeast
nucleus and binds LexA operators. This is done with a repression
assay (Protocol 2).

2.3.1 Testing whether the bait protein activates transcription of the
reporters

Protocol 1 describes how to verify that the bait does not
activate transcription of the LEU2 reporter. In addition to the bait
expression plasmid, this protocol uses three related HIS3 plasmids as
controls. The first is a plasmid that makes no LexA protein, or one
that makes LexA fused to a protein that does not activate
transcription. EGY48 derivatives that contain such plasmids fail to
grow on media lacking leucine. The second is a plasmid that makes
LexA fused to a transcription activation domain, like the activation
domain of Gal4. Such a plasmid will allow EGY48 to grow in the
absence of leucine. The third is the parent plasmid, pEG202. The
LexA protein encoded by pEG202 includes several amino acids
encoded by the polylinker which make the protein a weak
transcription activator. EGY48 containing pEG202 grows slowly on
medium lacking leucine and eventually forms colonies. A good
criterion for determining whether a bait plasmid can be used for an
interactor hunt is to show that it causes EGY48 to grow more slowly
on medium lacking leucine than pEG202.

Note on yeast transformation.
While only a handful of yeast transformants are needed in Protocol 1
and Protocol 2, the transformation efficiency (transformants per mg
of plasmid) must be very high for Protocol 3, in which the selection
strain is transformed with the library. To become familiar with high
efficiency yeast transformation is advisable to use a high efficiency
method for the transformations in Protocols 1 and 2. There are
several effective high efficiency yeast transformation protocols to
choose from including electroporation (34, 35) and those that employ
lithium salts (36). The method described in Protocol 3 results in
about 10e5 transformants per mg and may be scaled down to use in
Protocol 1 and 2. Two other considerations deserve mention. First,
once a plasmid has been introduced into a strain it must be
maintained by continued selection for its presence. Thus, a strain
that already contains the URA3 lacZ reporter plasmid should be
transformed with a second plasmid by first growing on media lacking
uracil (Glu ura-; see appendix for media designations). Although
strains that contain different plasmids can be constructed by
introducing more than one plasmid at a time, the transformation
efficiency will be lower than when strains are constructed by serial
transformation of one plasmid at a time. Second, each time a strain
is transformed, a control transformation should be performed using
no plasmid DNA.
 

______________________________________________________
Protocol 1. Testing baits for transcription activation

1. To construct the selection strain, transform EGY48 with a URA3
lacZ reporter plasmid and select transformants on Glu ura- plates
(see appendix for plate designations; see above note on
transformation). Combine three colonies from these plates, grow in
Glu ura- liquid, and transform them with the HIS3 bait plasmid (or
control plasmids). Select transformants on Glu ura-his- plates.

2. Pick four individual colonies from each transformation and use
each colony to inoculate 5 ml Glu ura-his- liquid cultures. At the
same time, streak the same four transformants to another Glu ura-
his- plate for storage and later recovery. All four should behave
identically in the tests below, in which case any one will serve as the
selection strain into which the library will be introduced. Incubate
these plates at 30oC until colonies form (about two days) and then
store at 4oC.

3. Grow the liquid cultures at 30oC shaking to OD600=0.5
(corresponding to about 10e7 cells/ml). This is mid-log phase. If the
overnight cultures grow to a density greater than OD600=0.5, dilute
to less than OD600=0.2 and then grow to OD600=0.5 so that the cells
are in mid-log phase when harvested.

4. Make 10e2- and 10e3 -fold dilutions of each culture in sterile
water. Spot 10 ml of the culture and 10 ml of the dilutions onto two
plates:
** Gal/Raf ura-his-
** Gal/Raf ura-his-leu-
Spot yeast containing the bait plasmid being tested and yeast
containing the control plasmids onto the same plates for side by side
comparison. Incubate at 30oC. Note: galactose is used in the media
because the actual selection will eventually be done on galactose
plates to induce expression of the activation-tagged cDNA protein.
Raffinose is added to aid yeast growth; it provides a better carbon
source than galactose alone but does not block the ability of galactose
to induce the GAL1 promoter (R.L.F., unpublished data).

5. Monitor the growth of the cells in the spots for several days. The
yeast in all of the spots should grow at a similar rate on the Gal/Raf
ura-his- plates. If growth on the Gal/Raf ura-his- plates is
reproducibly diminished for a given bait, relative to the controls, it
may indicate that its expression is toxic to yeast. Yeast with no LexA
or with a non-activating LexA fusion should not grow after several
days on Gal/Raf ura-his-leu- plates. Yeast containing LexA fused to a
protein that activates transcription may grow as fast on Gal/Raf ura-
his-leu- plates as on Gal/Raf ura-his- plates, depending on the
strength of the activator.

The above steps establish whether a bait can be used in an
interactor hunt. For a bait to be used, the selection strain containing
it must not grow on Gal/Raf ura-his-leu- plates for two to three days.
If yeast that contain a bait begin form visible colonies before three
days, it may be necessary to construct a less sensitive selection strain
(see section 2.2.1). However, if the yeast grow very slowly, e.g. form
colonies only after three days, it is still possible to do an interactor
hunt by selecting yeast with library plasmids that cause colonies to
form in one or two days.

6. Look for lacZ expression in the selection strain. Patch individual
transformants from step 1 to Glu ura-his- X-Gal plates and incubate
at 30oC. Yeast with the control LexA-activator fusion should turn
blue overnight while those lacking LexA or containing a
transcriptionally inert bait will remain white after many days. If a
bait activates the LEU2 gene but not the lacZ gene (which is
frequently observed because the LexAop-LEU2 reporter is more
sensitive than LexAop-lacZ reporter), it may be possible to perform
an interactor hunt by screening for yeast containing activation-
tagged cDNA proteins that interact with the bait and activate the lacZ
reporter; these yeast will be blue on X-Gal plates. However, this
method loses the advantage of a selection.
______________________________________________________
 

2.3.2 Demonstrating that the bait enters the yeast nucleus and binds
operators

Baits that do not activate the LEU2 reporter in the assay in
Protocol 1 should be tested to be sure they enter the nucleus and
bind to LexA operators. This can be done with a repression or
"blocking" assay. The repression assay is based on the observation
that LexA and non-activating LexA fusions can repress transcription
of a yeast reporter gene that has operators positioned between the
TATA and upstream activating sequence (UAS) (18). The mechanism
of this repression is not understood but presumably is not equivalent
to repression by repressor proteins native to yeast (37). While some
LexA fusions repress more than others (24), repression does depend
on the presence of operators in the reporter, and thus any repression
observed may be attributed to operator occupancy by the bait.

The reporter plasmid used for the repression assay (pJK101;
see Appendix) is similar to the plasmids used to test activation; it
contains the 2 mm origin, URA3, and a GAL1-lacZ fusion. Unlike the
plasmids used for testing activation, the GAL1-lacZ fusion in pJK101
contains most of the GAL1 upstream activating sequence, UASg. In
addition it contains one LexA operator positioned between UASg and
the TATA box. lacZ expression is induced by galactose and is
detectable in the presence of glucose, because negative regulatory
elements that normally keep GAL1 completely repressed in glucose
are not present (38). Transcriptionally inert LexA fusions that bind
to the operator in pJK101 repress lacZ expression from 2 to 20-fold
in the presence of galactose. Repression appears more profound
when the yeast are grown in glucose medium because there is less
lacZ expression to begin with. The repression assay can often be
done on X-Gal plates by looking for differences in blueness between
yeast with different baits. However, when looking for low levels of
lacZ expression in yeast grown in glucose, or when looking for slight
differences in lacZ expression (2 to 4-fold), more sensitive beta-
galactosidase assays may be necessary (39, 40).

______________________________________________________
Protocol 2. The repression assay

1. Transform EGY48 with pJK101 and select transformants on Glu
ura- plates. Combine three colonies from these plates and transform
them with the HIS3 bait plasmid (or HIS3 control plasmids). Select
transformants on Glu ura-his- plates.

2. Pick four individual colonies from each transformation and streak
a patch of them onto Glu ura-his- and Gal/Raf ura-his- plates
containing X-Gal. Incubate at 30oC.

3. Examine the X-Gal plates after 1, 2, and 3 days. Yeast lacking
LexA will begin to turn blue on the Gal/Raf plates after one day and
will appear light blue on the glucose plates after two or more days.
Yeast containing a bait that enters the nucleus and binds operators
turn blue more slowly than the yeast lacking LexA.

4. Baits that repress transcription of lacZ in pJK101 by 2-fold or less
may not cause a visible reduction in blue on X-Gal plates. If no
repression is observed on the X-Gal plates, perform beta-galactosidase
assays with transformants from step 1. Grow the transformants in 5
ml Glu ura-his- and Gal/Raf ura-his- liquid media, or on Glu ura-his-
and Gal/Raf ura-his- plates for 2 days, before doing beta-galactosidase
assays (39, 40).
______________________________________________________

2.3.3 Verifying that a full-length fusion protein is made.

Finally, it is usually good practice to demonstrate that the full-
length bait protein is made. This can be done by running extracts
from yeast cells that harbour the bait plasmid on an SDS
polyacrylamide gel, immunoblotting with either an antibody to LexA
or one specific to the protein fused to LexA (27, 29), and detecting a
fusion protein of the expected apparent molecular weight. Yeast cell
extracts can be prepared by growing yeast in liquid culture (lacking
histidine to maintain selection for the bait plasmid) to OD600 of 0.5,
spinning 1 ml of the culture to pellet the cells, and resuspending the
cells in 50 ml of 2X Laemmli sample buffer (41). The cells can then
be broken by freezing on dry ice followed by boiling for 5 minutes
prior to loading on an SDS polyacrylamide gel (about 15 ml/lane).
The proteins can then be transferred to a filter and blotted with
standard immunoblotting (western) methods (22, 42).
 

3. Libraries

In an interactor hunt the expressed cDNA-encoded proteins are
fused to an activation domain, as well as a nuclear localization signal
to increase their nuclear concentration, and an epitope tag so they
may be immunologically identified. The prototypical library plasmid
for expression of these activation-tagged cDNA proteins is pJG4-5
(11) (see Appendix). This is a 2 mm plasmid that contains the TRP1
marker and the GAL1 promoter. Downstream of the GAL1 promoter
there is an ATG followed by 105 codons. These encode 9 amino acids
from the SV40 Large T nuclear localization signal, 87 amino acids
that make up the activation domain called B42, followed by 9 amino
acids comprising the hemagluttinin (HA) epitope tag. The B42
domain is derived from E.coli and acts as a moderately strong
transcription activation domain in yeast (4). Use of this activation
domain avoids the possible toxic effects of overexpressing a strong
activation domain (43, 44). Downstream of the sequences that
encode the fusion moiety there are unique EcoRI and XhoI sites for
insertion of cDNAs. Proteins encoded by cDNAs that are in-frame
carry the fusion moiety at their amino terminus. The activation-
tagged cDNA-encoded proteins will be expressed in yeast grown on
galactose but not in yeast grown in glucose. Numerous libraries have
been made using pJG4-5. These include cDNA libraries made from
RNA derived from HELA cells (11), Drosophila, ovaries, discs, and 0 to
12 h embryos (Finley and Brent, in preparation), adult Drosophila
heads (J.Huang and M. Rosbach, personal communication), Drosophila
16 to 26 h embryos (V. Neel and M. Young, personal communication),
serum-starved WI38 cells (C. Sardet, J. Gyuris, R.B., unpublished
data), human brain (D. Krainc and R.B., unpublished data),
Arabidopsis (H.Zhang and H.Goodman, personal communication), and
a library made from yeast genomic DNA (P. Watt personal
communication). Construction of libraries is a complex topic beyond
the scope of this article, but is described elsewhere (11, 45, 46)
(Finley and Brent, in preparation).
 

4. An interactor hunt
4.1 Introducing the library into the selection strain.
4.1.1 Selecting interactors from library transformants.

To conduct an interactor hunt the library is introduced into the
selection strain in a large transformation so that many transformed
cells are obtained, each of which contains an individual library
plasmid. Those cells that contain library plasmids that encode
proteins that interact with the bait protein are then selected. To
date, the most frequently successful method has been to do this in
two steps. In the first step, yeast that already contain the bait and
lacZ plasmids are transformed with the library and library
transformants are isolated and frozen. In the second step, the
selection for interactors is applied to the library transformants. In
this approach yeast transformed with the TRP1 library plasmid are
selected by plating the transformation mix onto medium lacking
tryptophan (that also lacks uracil and histidine to maintain selection
for the bait and lacZ plasmids, i.e., Glu ura-his-trp- plates). The
transformants are then scraped from the plates and frozen for
storage. Aliquots are thawed and plated onto a medium containing
galactose and lacking leucine. This induces expression of the
activation-tagged cDNA proteins, and selects for transcription of the
LEU2 reporter. This two step approach results in a uniform increase
in the number of cells carrying each library plasmid; each cell
transformed with the library is allowed to multiply to form a colony,
and the colonies are harvested at approximately the same size. One
advantage to this approach is that the number of transformed yeast
is amplified before the synthesis of cDNA-encoded proteins is
induced. This ensures that yeast containing toxic or mildly toxic
cDNA-encoded proteins will not be depleted from the population.
This method is described in Protocol 3 and Protocol 4.

4.1.2 Performing one step selection for interactors

A simpler, but so far inferior, alternative approach is to
perform a one step selection for yeast containing activation-tagged
cDNA proteins that interact with the bait. This method involves
plating the library transformation mix directly on plates lacking
leucine (i.e. Gal/Raf ura-his-trp-leu- plates) to select for expression
of the LEU2 reporter, without first selecting cells transformed with
the library plasmid. Although it is easier that the two step method,
it suffers from some disadvantages. First, only a fraction of the yeast
containing activation-tagged proteins that interact with the bait
survive on the leu- selection plates, possibly because some cells die
in the time it takes to induce synthesis of the activation-tagged
protein, the LEU2 product, and leucine. This lower plating efficiency
on the leu- selection plates is particularly evident when the yeast
contain an activation-tagged cDNA-encoded protein that interacts
only weakly with the bait. Second, the one step approach may
reduce the probability of isolating cDNAs that encode proteins that
are somewhat toxic to yeast because these proteins will further
reduce the plating efficiency on leu- plates. Although both of these
disadvantages might be overcome by plating very large numbers of
library transformants, obtaining such numbers would require
exceptional yeast transformation efficiencies or huge amounts of
library DNA. Furthermore, if expression of an activation-tagged
cDNA protein causes yeast to plate at reduced efficiency, a much
larger number of yeast will need to be characterized to find them.
For these reasons we prefer the two step method. However, we also
present the best current version of the one step method, which may
eventually be improved to overcome the relative disadvantages
described above. To do the one step method follow Protocol 3 steps
1 to 10 and then skip to Protocol 4.

______________________________________________________
Protocol 3. Transforming the selection strain with library DNA.

The following protocol is a variation of the high efficiency lithium
acetate method developed by Geitz et al. (36). Before transforming
the strain with library DNA (which is usually fairly valuable),
perform pilot transformations of the strain with the TRP1 library
vector. A transformation efficiency of at least 10e5
transformants/mg DNA should be the goal. Other high efficiency
yeast transformation protocols, e.g. electroporation (34, 35), may be
substituted.

1. Grow yeast containing the bait and lacZ reporter plasmids in 400
ml of Glu ura-his- medium at 30oC, with shaking (~150 rpm) to an
OD600 of 1.0, corresponding to about 3 X 10e7 cells/ml. The
doubling time in this medium is rather slow (2 to 4 hours). For this
reason it is sometimes convenient to start a smaller, 50 ml culture,
grow it to OD600=2.0 or greater, and then dilute it into 400 ml. For
high transformation efficiencies it is important to start the 400 ml
culture at OD600=0.2 or less and allow it to grow to OD600=1.0 so
that the cells are in mid-log phase when harvested.

2. Spin culture at 2000 X g for 5 min and pour off supernate. Wash
the cells in 20 ml sterile water. These spins and all following
manipulations are done at 20 - 25oC.

3. Resuspend yeast in 5 ml of filter sterilized LiOAc/TE (10 mM Tris
HCl pH7.5, 1 mM EDTA, 100 mM LiOAc; make from a filter sterile
stock of 1 M LiOAc, pH 7.5). Pellet again and pour off supernate.

4. Resuspend yeast in 2.0 ml LiOAc/TE. 50 ml of this suspension
provides enough competent cells to be transformed with 1 mg of
DNA. Note: use of more than 1 mg DNA per 50 ml of cells can result
in introduction of multiple library plasmids in each yeast cell. This
should be avoided because of the large amount of additional work
that will be required to determine which of the library plasmids in a
cell expresses the cDNA-encoded protein that interacts with the bait
(see below).

5. Aliquot 100 ml of competent yeast into sterile eppendorf tubes.
To each tube add 2 mg of library DNA and 60 mg of carrier (single
stranded salmon sperm; (47)) in a total volume of 20 ml. The
number of tubes to be used depends on the desired number of
transformants and the expected transformation efficiency. If pilot
transformations resulted in efficiencies of 10e5 transformants per
mg, each tube should yield 200,000 transformants, all of which can
be plated on onto a single 24cm X 24cm plate (see below).

6. Add DMSO to 10% vol/vol. This improves transformation
efficiency by 3 to 5-fold (48) (R.L.F. and B. Cohen, unpublished data).

7. Add 600 ml of filter sterilized 40% PEG 4000 in LiOAc/TE (made
from stocks of 1 M LiOAc pH 7.5, filter sterile 50% PEG 4000 in water,
1 M Tris HCl pH 7.5, and 0.5 M EDTA). Gently invert tube several
times to mix.

8. Incubate at 30oC for 30 min. Agitation is not necessary.

9. Heat shock at 42oC for 15 min and return to room temperature.

10. Determine the total number of transformants by removing 10 ml
from each tube and making three dilutions (10-, 10e2-, and 10e3-
fold) in sterile water. Plate 100 ml of each dilution onto 100 mm Glu
ura-his-trp- plates and incubate at 30oC. You will be able to
calculate the total number of transformants from the number of
colonies on these dilution plates.

At this point the transformation mixes can be plated to first select
for all library transformants (as discussed in 4.1.1). This method is
described below starting with step 11. Alternatively, the
transformation mix can be used in a one step selection for interactors
(as discussed in 4.1.2); to perform this, proceed to Protocol 4.

Selecting library transformants.

11. Plate each transformation mix (less then 1 ml) onto a single 24cm
X 24cm Glu ura-his-trp- plate. There is no need to spin the cells or
remove the PEG. The media in these plates should be at least 0.6 cm
thick, level, and free of bubbles. To achieve an even distribution of
cells pour about 100 sterile glass beads (4 mm diameter, Fisher
Scientific; sterilized by autoclaving) on the plate with the cells.
Gently roll the beads around the plate to distribute the
transformation mix, then pour the beads off, or onto the next plate.
Alternatively, distribute the transformation mix with a sterile bent
glass rod. Both techniques work best when the surface of the plates
are not too wet so that the media absorbs the transformation mix. To
achieve this moisture content put newly solidified plates into a
laminar flow hood with the lids ajar for about two hours before
plating.

12. Incubate the plates at 30oC. Colonies should appear after about
24 hours. Continue incubation until colonies are 1 to 2 mm in
diameter, which should take a total of approximately 2 days.

Harvest the transformants.

13. Place the plates at 4oC for 2 to 4 hours to harden the agar.

14. Using the long edge of a sterile glass microscope slide (and
sterile technique) scrape the yeast from the plate. Try not to scrape
any agar as it will interfere with pipeting. Collect the yeast from the
glass slide by wiping it on the lip of a sterile 50 ml Falcon tube.

16. Wash the cells two times with 2 or 3 volumes of sterile TE. It is
best to pellet the cells each time in a sterile round bottom
polypropylene tube at 2000 X g for 4 min so they may be easily
resuspended. The pellet volume for 500,000 transformants will be
about 8 ml.

17. Resuspend the cells thoroughly by swirling in 1 pellet volume of
glycerol solution (65% glycerol (vol/vol), 0.1 M MgSO4, 25 mM Tris
pH 7.4). Mix well by vortexing on low speed. Freeze 1 ml aliquots at
-70oC.

18. Determine the plating efficiency by thawing an aliquot of library
transformants and making serial dilutions in sterile water. Plate 100
ml of each dilution onto 100 mm Gal/Raf ura-his-trp- plates. Count
the colonies that grow after 2 to 3 days at 30oC and represent the
plating efficiency in colony forming units (CFU) per unit volume of
frozen cells. The plating efficiency will be on the order of 10e8
CFU/100 ml.
______________________________________________________

4.2 Isolating yeast with galactose dependent Leu+ and lacZ+
phenotypes

Library transformants containing cDNAs that encode proteins
that interact with the bait will exhibit galactose-dependent growth
on media lacking leucine (Leu+), and galactose-dependent beta-
galactosidase activity (lacZ+). Isolation of these galactose-dependent
Leu+ and lacZ+ yeast is accomplished in two steps. First, library
transformants are plated on galactose medium lacking leucine (leu-)
to select yeast that are Leu+. Second, the Leu+ yeast are isolated,
placed on a glucose master plate, and then replica plated to four new
plates to test for lacZ expression and galactose dependence. These
four plates include two leu- plates and two X-Gal plates: one leu-
plate and one X-Gal plate contain galactose to induce cDNA
expression (plus raffinose to enhance growth), while the other leu-
plate and the other X-Gal plate contain glucose to repress cDNA
expression. Yeast that grow on leu- galactose medium but not on
leu- glucose medium, and that turn blue on galactose X-Gal plates but
remain white on glucose X-Gal plates (i.e. those that are galactose-
dependent Leu+ and lacZ+) are picked for further characterization.

In this procedure the yeast are grown on the glucose master
plates to shut off cDNA expression before replica plating so that the
galactose dependence of the Leu+ and lacZ+ phenotypes can be
assessed. This avoids a problem that can arise when the Leu+ yeast
are replica plated from galactose to glucose plates: There may be
sufficient message and protein product from the activation-tagged
cDNA protein to allow the yeast to grow on leu- glucose for several
generations and turn blue on glucose X-Gal without further cDNA
expression. This would mask the galactose-dependence of the Leu+
and lacZ+ phenotypes.

______________________________________________________
Protocol 4. Selecting interactors.

Note: If TRP+ library transformants were selected and stored frozen
follow "a" in the steps below. If library transformants were not
selected and stored, follow "b" in the steps below.

1. Induce synthesis of activation-tagged cDNA-encoded proteins.
a) If library transformants were selected and frozen, thaw an
aliquot of the cells and dilute 10-fold into Gal ura-his-trp- liquid
media.
b) If performing the one step method to select for interactors,
dilute the transformation mix from Protocol 3 step 9, 10-fold into Gal
ura-his-trp- media.

Incubate at 30oC with shaking for 4 hours to induce the GAL1
promoter. There is almost no increase in cell number during this
time, and any increase can be neglected when calculating the number
of colony forming units or transformants to plate onto leu- selection
plates.

3. Pellet cells at 2000 X g for 4 min at 20 - 25oC and resuspend in
sterile water.

4. Plate onto Gal/Raf ura-his-trp-leu- plates.
a) If library transformants were pre-selected and frozen, plate
10e6 CFU (determined from the plating efficiency test in Protocol 3
step 17) onto each 100 mm plate.
b)If performing the one step selection, plate an amount of the
transformation mix that should contain 10e6 transformants, based on
estimates from transformation efficiencies obtained in pilot
transformation experiments, onto each 100 mm plate. Determine the
actual number of library transformants plated by counting the
colonies that grow when dilutions of the transformation mix are
plated onto Gal/Raf ura-his-trp- as described in Protocol 3 step 10.

5. Incubate selection plates at 30oC. Colonies should appear in 2 to 5
days. To keep the plates from drying out after two days, it may be
helpful to put them in plastic bags or containers, or put parafilm
around each plate. Generally, there will be more galactose-
dependent Leu+ and lacZ+ yeast among the colonies that appear
sooner, and fewer among the colonies that appear later.

6. Pick colonies with sterile toothpicks or applicator sticks and patch,
or streak for single colonies, onto another Gal/Raf ura-his-trp-leu-
plate. Ideally the Leu+ yeast should be streaked for single colonies
to isolate them from contaminating Leu- yeast that were present
when the Leu+ colony was forming. However, when there are large
numbers of Leu+ colonies, it may be inconvenient to streak purify
every one; in this case growth of patches on a second selection plate
will at least enrich for the Leu+ cells.

7. To show that the Leu+ phenotype is galactose-dependent, patch
the Leu+ yeast onto Glu ura-his-trp- master plates to turn off the
GAL1 promoter and stop expression of the activation-tagged cDNA
protein. Grow at 30oC for about 24 hours.

8. Replica the master plates to the following five plates: 1. Glu ura-
his-trp- X-Gal; 2. Gal/Raf ura-his-trp- X-Gal; 3. Glu ura-his-trp-leu-;
4. Gal/Raf ura-his-trp-leu-. Incubate at 30oC and examine results
after 1, 2, and 3 days.

Pick yeast that are Leu+ and lacZ+ only on galactose (for example, see
Figure 3). Further characterize these by isolating the library plasmid
and determining the interaction specificity.
______________________________________________________

Three alternative procedures to the method described in
Protocol 4 have been used successfully. First, if a large number of
Leu+ yeast colonies appear on the initial leu- selection plates, those
that are lacZ+ can be quickly identified using a filter beta-galactosidase
assay. The filter assay, described in detail elsewhere (49), involves
lifting a replica of the yeast from the colonies on the leu- selection
plate with a nitrocellulose filter, lysing the yeast on the filter, and
exposing the filter to buffer and X-Gal. The filter is then examined
for blue spots corresponding to lacZ+ yeast. Leu+ yeast that
correspond to those that are also lacZ+ are then picked from the
original leu- selection plate, put onto glucose master plates, and
replica plated as described in Protocol 4, steps 7 and 8. The second
alternative is to include X-Gal in the initial leu- selection plates and
pick those that grow and turn blue. Again, the Leu+ lacZ+ yeast are
picked, patched onto glucose master plates, and replica plated to the
two sets of indicator plates. The disadvantage to this approach is
that yeast grow less well on X-Gal plates because these plates contain
a buffer to give them neutral pH; the reduced growth rate at this
higher pH reduces the plating efficiency during the selection. Finally,
the yeast colonies on the original leu- selection plate can be replica
plated directly to X-Gal plates to determine which are lacZ+ (A.
Mendelsohn, personal communication). Only those that are Leu+ and
lacZ+ are isolated and tested for galactose dependence.

Most activation-tagged proteins that interact strongly with the
bait will render the yeast containing them galactose-dependent Leu+
and lacZ+. However, yeast containing activation-tagged cDNA
proteins that interact only weakly with the bait may be galactose-
dependent Leu+, yet may appear light blue or even white on X-Gal
plates. This is due to the different sensitivities of the Leu and lacZ
phenotypes (see 2.2). Results from several interactor hunts have
shown that there are usually more biologically relevant interactors in
the class of yeast that are galactose-dependent Leu+ and lacZ+ than in
yeast that are lacZ- (R.L.F., unpublished data). However, the class of
yeast that are galactose-dependent Leu+ but lacZ- may also contain
biologically relevant interactors (Finley and Brent, in preparation).
For this reason the decision whether or not to further characterize
the latter class must be somewhat arbitrary.

5. Verifying specificity

A finding of galactose-dependent Leu+ and lacZ+ in a yeast
isolate can be considered a demonstration that the reporter genes are
activated due to expression of the activation-tagged cDNA-encoded
protein. However, it is important to determine that activation of the
reporters is due to specific interaction of this protein with the bait,
rather than to its nonspecific interaction with LexA, with the
promoters, or with some part of the transcription machinery. To
verify that the cDNA-encoded protein interacts specifically with the
bait, library plasmids are rescued from the galactose-dependent Leu+
lacZ+ yeast and re-introduced into the original selection strain and
into other strains containing different baits. Specific interactors
confer the galactose-dependent Leu+ and lacZ+ phenotype to yeast
containing the original bait, but not to yeast containing unrelated
baits. To test this, master plates are made from the new
transformants and replica plated onto four indicator plates as
described in Protocol 4 steps 7 and 8 (see Figure 3). Alternatively,
the specificity can be determined using a mating assay (Protocol 6).

Library plasmids are rescued from yeast by performing a quick
yeast plasmid miniprep and using the miniprep DNA to transform
E.coli (most yeast miniprep protocols do not provide enough clean
plasmid DNA for restriction analysis). If a large number of galactose-
dependent Leu+ lacZ+ yeast are obtained, it is useful to reduce the
number of library plasmids that need to be rescued by determining
which ones contain identical cDNAs. This can be done by comparing
restriction digests of PCR products containing the cDNA insert. In
this procedure, yeast miniprep DNA is used as template in PCR
reactions with primers derived from sequences in the library
plasmid flanking the cDNA insertion site. PCR products are then
digested with one or two restriction enzymes that cut frequently.
This procedure is described in Protocol 5.

Since the yeast contain three different plasmids, rescuing the
library plasmid depends on distinguishing it from the bait and lacZ
reporter plasmids. There are at least three ways to do this. In the
most efficient one, yeast minipreps are used to transform a strain of
E.coli that contains a mutation in the trpC gene. The inability of the
trpC- E.coli to grow in the absence of tryptophan is complemented by
the yeast TRP1 gene on the library plasmid (50). trpC E.coli
transformed with the TRP1 library plasmid are selected on minimal
plates lacking tryptophan and containing ampicillin. In another
method, yeast minipreps are used to transform E.coli, and E.coli
minipreps from several individual transformants are analyzed by
restriction digestion to determine which minipreps contain the
library plasmid. Alternatively, the E.coli transformations are plated
onto LB amp plates containing X-Gal; transformants that contain the
lacZ reporter plasmid form light blue colonies and are not picked.
Restriction analysis on minipreps from the white colonies is used to
distinguish between the library and bait plasmids.

______________________________________________________
Protocol 5. Isolating and classifying library plasmids.

Several effective methods are available for isolating plasmids from
yeast in amounts sufficient for E.coli transformation (e.g. the "smash
and grab" method of Hoffman and Winston (51)). The method
described below is quick, and yields plasmid DNA clean enough to
transform E.coli efficiently and to work as a template for PCR.

1. Scrape a large mass of yeast from a plate and resuspend in 1 ml
of TE in an eppendorf tube (the OD600 of this suspension should be
between 2 and 5). Yeast from a fresh, 2 to 3 day old plate work best.
The yeast can also be obtained from a 1 ml overnight liquid culture.

2. Spin briefly to pellet cells. Resuspend yeast in 0.5 ml S buffer (10
mM KPO4 pH 7.2, 10 mM EDTA, 50 mM 2-mercaptoethanol, 50
mg/ml zymolase).

3. Incubate at 37oC for 30 min.

4. Add 0.1 ml lysing solution (0.25 M Tris Cl pH7.5, 25 mM EDTA,
2.5% SDS). Vortex to mix.

5. Incubate at 65oC 30 min.

6. Add 166 ml 3 M KOAc. Chill on ice for 10 min.

7. Spin in an eppendorf centrifuge for 10 min. Pour supernate into a
new tube.

8. Precipitate DNA by adding 0.8 ml cold ethanol. Incubate on ice for
10 min, spin for 10 min, and pour off supernate.

9. Wash pellet with 0.5 ml 70% ethanol and dry pellet.

10. Disolve the pellet in 40 ml sterile water. Use 1 or 2 ml of this
crude yeast miniprep to transform E.coli by electroporation.

Before transforming E.coli with the crude yeast minipreps it is often
useful to determine which yeast minipreps contain the same library
plasmid so that fewer need to be characterized. This can be done by
restriction analysis of PCR products.

11. Set up a 0.5 ml tube for each yeast miniprep containing:
** 13 ml sterile water
** 2 ml 10X Taq polymerase buffer
** 2 ml dNTP mix (all 4 dNTPs at 2.5 mM each)
** 1 ml 5' primer (0.1 mg/ml) derived from the library vector fusion
moiety sequence (see appendix).
** 1 ml 3' primer (0.1 mg/ml) derived from the ADH1 terminator
sequence in the library vector (see appendix).
** 0.2 ml Taq polymerase (5 units/ml)

12. Add 1 ml of yeast miniprep DNA to each tube.

13. Incubate for 25 cycles of 92oC for 30 sec., 65oC for 2 min, 75oC
for 30 sec.

14. Set up two tubes for each PCR reaction, one for AluI digestion
and one for HaeIII digestion (these enzymes are recommended
because they work well in the presence of the PCR reactants). Add 8
ml of the PCR reaction to each tube. Save the remainder of the PCR
reaction for gel analysis of the full-length PCR product.

15. Add 1 ml of the appropriate 10X restriction enzyme buffer to the
8 ml. Add 1 ml of AluI to one tube and 1 ml of HaeIII to the other
tube. Incubate at 37oC for 2 hours.

16. Analyze AluI digests, HaeIII digests, and the uncut PCR products
on 1.5% agarose gels.

It should be readily apparent from this analysis which cDNAs are
identical. In some cases, only some of the restriction fragments will
appear identical between two plasmids, suggesting that these
plasmids contain different length cDNAs made from the same gene.
Rescue library plasmids from yeast minipreps that give different
restriction patterns.
______________________________________________________

______________________________________________________
Protocol 6. Determining specificity of interactors.

1. Use the rescued library plasmid DNA (from a miniprep of
transformed E. coli) to transform the original yeast strain containing
the bait plasmid and the lacZ reporter plasmid. Additionally, use
each library plasmid to transform one or more other strains
containing different baits. Select transformants on Glu ura-his-trp-
plates. As a control transform each different bait strain with the
library vector without a cDNA insert.

2. Use sterile toothpicks or applicator sticks to pick three to four
individual colonies from each transformation plate, and patch these
to Glu ura-his-trp- master plates. Patch control transformants
(library plasmid with no cDNA) onto each plate for side by side
comparison. Grow for one or two days at 30oC.

3. Replica plate from the master plates to four plates: 1. Glu ura-his-
trp- X-Gal, 2. Gal/Raf ura-his-trp- X-Gal, 3. Glu ura-his-trp-leu-, 4.
Gal/Raf ura-his-trp-leu-.

4. Incubate plates at 30oC and examine after 1, 2, and 3 days.

Figure 3 shows an example of the result obtained for three specific
interactors.
_____________________________________________________________

(Figure 3. Four plates showing specific interactors.)

6. Using a mating assay to verify specificity.

We have recently developed a mating assay as an alternative
way to test for interaction between a given activation-tagged protein
and a panel of LexA fusion proteins (baits). This scheme takes
advantage of the fact that haploid cells of the opposite mating type
will fuse to form diploids when brought into contact with each other
(52). In this mating assay, the activation-tagged protein is expressed
in one yeast strain and the bait is expressed in a second strain of the
opposite mating type. When the two strains are mixed on the same
plate, they form diploids in which the bait and activation-tagged
proteins have the opportunity to interact and activate the reporter
genes. As before, interaction is measured as activation of the
LexAop-LEU2 and LexAop-lacZ reporters. This technique can be
used to check the specificity of the cDNA-encoded proteins isolated in
the interaction trap to ensure that they interact with only the
original bait and not with unrelated LexA fusions. It can also be
used to examine interactions between a given set of activation-
tagged proteins, such as those isolated in an interactor hunt, and a
large panel of bait proteins without performing an unwieldy number
of yeast transformations. Finally, it could be used to conduct
interactor hunts; the library plasmids can be introduced in bulk into
EGY48, transformants can be stored frozen, and then thawed and
mated with a second strain that contains a bait plasmid. This would
enable several separate interactor hunts with different baits to be
done by performing a single large scale library transformation. The
disadvantage of the mating assay is that the sensitivity of the
reporters to activation by baits and activation-tagged proteins that
interact with them is generally less in diploid cells relative to haploid
cells. To minimize the effect of this difference, the most sensitive
LEU2 reporter is used.

As described in Protocol 7 and illustrated in Figure 4, the
activation-tagged protein is expressed in EGY48 (mating type a) and
the bait protein is expressed in a second strain, RFY206 (mating type
a) which may also contains the lacZ reporter plasmid. The EGY48
derivatives are streaked onto plates lacking tryptophan to maintain
selection for the library plasmid, and the RFY206 derivatives are
streaked onto plates lacking uracil and histidine to maintain selection
for the URA3 lacZ plasmid and the HIS3 bait plasmid. The two
strains are mated by applying them to the same replica velvet or
filter and lifting their "print" with a YPD plate. The YPD plate is
incubated overnight at 30oC to promote mating and then replica
plated to the same indicator plates used in Protocol 4 steps 7 and 8.
In the example shown in Figure 4 the lacZ reporter is not used. The
his-trp-plates will contain only diploids: TRP1 is provided by the
strain that contains the library plasmid while HIS3 is provided by
the strain that contains the bait plasmid. Only the diploids that
contain interacting pairs of activation-tagged protein and bait will
grow on the plates lacking leucine but containing galactose.

(Figure 4a. Mating assay cartoon.)
(Figure 4b. Mating assay result.)

______________________________________________________
Protocol 7. Mating assay.

1. Introduce TRP1 library plasmids into yeast strain EGY48 and select
transformants on Glu trp- plates. As a control, transform EGY48 with
a library plasmid that has no cDNA insert.

2. Introduce HIS3 bait plasmids into yeast strain RFY206 (or other
ura3 his3 trp1 leu2 MATa strain) along with a URA3 lacZ reporter
and select transformants on Glu ura-his- plates. Note: in the example
shown in Figure 4 the URA3 lacZ reporter is not used.

3. Use sterile toothpicks or applicator sticks to streak individual
EGY48 transformants onto standard 100 mm Glu trp- plates in
parallel lines, 6 or 7 to a plate (Figure 4). Include at least one streak
of the transformants with the control plasmid (no cDNA). Likewise,
streak individual RFY206 transformants onto Glu ura-his- plates in
parallel lines, 6 or 7 to a plate. Incubate at 30oC until heavy growth.
The lines of yeast should be at least 2 mm wide.

4. Onto the same replica filter or velvet, print the EGY48 derivatives
and the RFY206 derivatives so that the streaks from the two plates
are perpendicular to each other.

5. Lift the print of the two strains from the filter or velvet with a
YPD plate. Incubate the YPD plate at 30oC overnight. Diploids will
form where the two strains intersect. One strain may grow more
rapidly than the other during this time but this does not hinder
formation of diploids in the intersections.

6. Replica from the YPD plate to the following plates: 1. Glu ura-his-
trp- X-Gal, 2. Gal/Raf ura-his-trp- X-Gal, 3. Glu ura-his-trp-leu-, 4.
Gal/Raf ura-his-trp-leu-. Incubate at 30oC and examine after 1, 2,
and 3 days.

Only diploids will grow on the X-Gal plates and only interactors will
grow on galactose plates lacking leucine (for example, see Figure 4).
______________________________________________________
 

7. Expected results.

The two most critical parameters that determine whether an
interactor hunt will succeed are the quality of the library and the
nature of the bait. If the library contains cDNAs that encode proteins
that interact with the bait in the interaction trap, the bait becomes
the most critical parameter. The bait can affect the outcome of an
interactor hunt in two ways. First, the number of nonspecific cDNA-
encoded interactors obtained appears to depend on the bait. For
example, use of some baits can result in isolation of activation-tagged
cDNA proteins that seem to interact with many other LexA fusions
when the specificity determination is performed. It is unlikely that
these cDNA-encoded proteins interact with the LexA moiety or with
the reporters or transcription machinery directly because if they did,
they would be expected to arise in all interactor hunts. Rather, these
cDNA-encoded proteins may simply be "sticky", for example, because
they contain patches of hydrophobic or charged residues that
interact with corresponding regions on many baits. Second, the
number of spurious Leu+ yeast that arise independently of the
library plasmid differs from one bait to another. For example, when
some selection strains are transformed with library DNA and plated
onto media lacking leucine, hundreds of galactose-independent Leu+
colonies grow. This phenomenon is observed more frequently with
baits that activate a low level of transcription than with
transcriptionally inert baits, perhaps because a low level of
activation is readily enhanced in a fraction of cells in a population.
For example, a bait may gain the ability to activate the reporters in
some cells if there is an increase in the copy number of the bait
plasmid, which could result from natural variation in plasmid copy
number or from a mutation in the plasmid or a yeast gene that
affects plasmid copy number. This problem can be best addressed
by reducing the activation potential of a bait as described in section
4.2. It is worth noting that, in our experience, these problems with
baits are usually surmountable, and that most proteins can
eventually be used successfully in interactor hunts.

Conclusion
We have described detailed procedures for isolating proteins
using the interaction trap. These procedures provide a relatively
rapid, simple, and robust method for isolating proteins that interact
with known proteins, and for measuring protein-protein interactions
inside living cells.
 
 
 

Acknowledgment

We are very grateful to the numerous current and past lab members,
and to the many lab visitors whose efforts contributed to the
development of this interaction technology. We thank Tod Gulick,
Pierre Colas, and Andrew Mendelsohn for critcal readings of the
manuscript. RLF was supported by a postdoctoral fellowship from
the NIH. RB was supported by the Pew Scholar's program. Work was
supported by the HFSP.
 
 
 
 
 
 

Appendix

Figure of plasmids.
pEG202
pLR1d1 (pSH18-34, pJK103, pJK101)
pJG4-5

Sequencing and PCR primers for pEG202 and pJG4-5.
For sequencing inserts in the polylinker of pEG202 primers can
be derived from LexA coding sequences. The primer shown below,
LEX1, is derived from LexA coding sequences 40 bp upstream of the
EcoRI site in the polylinker (note: all eukaryotic LexA expression
plasmids lack the EcoRI site found in the wild-type LexA coding
region (18)). For sequencing from the 5' end of cDNA inserts in pJG4-
5, BCO1 can be used. It is derived from the coding sequence for the
B42 activation domain 70 bp upstream of the EcoRI site. For
sequencing from the 3' end of the cDNA insert in pJG4-5, BCO2 can be
used. It is derived from the sequence of the ADH1 terminator
approximately 40 bp downstream of the XhoI site. BCO1 and BCO2
can be used for PCR amplification of the cDNA insert as described in
Protocol 5.

LEX1 5' CGT CAG CAG AGC TTC ACC ATT G 3'
BCO1 5' CCA GCC TCT TGC TGA GTG GAG ATG 3'
BCO2 5' GAC AAG CCG ACA ACC TTG ATT GAA G 3'
 
 

Media recipes.
Dropout media.
Dropout media, also known as complete minimal (CM) dropout
media, contains a nitrogen base, a mixture of nutrients shown in
Table 1 with one or a few left out (dropped out), and a carbon source
(usually a sugar). It is convenient to make a dropout powder
corresponding to each of the dropout media that will be used, e.g. for
media lacking tryptophan (trp-), the dropout powder used would
contain all of the nutrients listed in Table 1 except for tryptophan.
Three separate stocks of the carbon sources (20% galactose, 20%
glucose, 20% raffinose) should be made and filter sterilized. As
needed, galactose (Gal) and glucose (Glu) are each added to 2 % final
concentration, raffinose (Raf) is added to 1 % final concentration.

Dropout plates
For 1 liter
Mix in 850 ml deionized H2O
** 6.7 g yeast nitrogen base (YNB) without amino acids (Difco)
** 2 g Dropout powder lacking the appropriate nutrient(s)
(Table 1)
** one pellet of NaOH (~ 0.1 g)
** 20 g agar (Difco Bacto-agar)
Autoclave
Add the appropriate carbon source from sterile stocks. For Gal/Raf
plates add galactose to 2% and raffinose to 1% final concentrations;
for Glu plates add glucose to 2 % final concentration.

Make liquid dropout media the same way as dropout plates, leaving
out the agar and NaOH pellet.

Table 1. Dropout powder.
 

Nutrient Amt in dropout powder (g)a Final conc. in media (ug/ml)
adenine 2.5 40
L-arginine (HCl) 1.2 20
L-aspartic acid 6.0 100
L-glutamic acid (monosodium) 6.0 100
L-histidine (his) 1.2 20
L-isoleucine 1.8 30
L-leucine (leu) 3.6 60
L-lysine 1.8 30
L-methionine 1.2 20
L-phenylalanine 3.0 50
L-serine 22.5 375
L-threonine 12.0 200
L-tryptophan (trp) 2.4 40
L-tyrosine 1.8 30
L-valine 9.0 150
uracil (ura) 1.2 20
a Combine all but the appropriate nutrients, e.g. if making media
lacking tryptophan (trp-) leave out tryptophan. Grind in a clean dry
mortar and pestle until homogeneous. Store at room temperature.

YPD (also known as YEPD) plates.
For 1 litre
Mix in 900 ml deionized H2O
** 10 g yeast extract (Difco Bacto-yeast extract)
** 20 g peptone (Difco Bacto-peptone
** one pellet NaOH (~0.1 g)
** 20 g agar (Difco Bacto-agar)
Autoclave
Add 100 ml sterile 20% glucose

X-Gal plates.

For 1 litre
Mix in 800 ml deionized H2O
** 6.7 g YNB without amino acids
** 1.5 g dropout powder
** 20 g agar (Difco Bacto-agar)
Autoclave
Immediately add the appropriate sterile 20% carbon
source(s)
Allow to cool to 65oC
** add 100 ml of 10X BU Salts (see below)
** add 4 ml of 20 mg/ml X-Gal dissolved in DMF dimethyl
formamide (stored at -20oC).

Note: adding salts while media is too hot causes the salts to
precipitate. Also, X-Gal is thermolabile and will be destroyed if
added to hot media.
 

10X BU Salts
For 1 liter
** 70 g Na2HPO4 7H2O
** 30 g NaH2PO4
Adjust pH to 7.0
Autoclave, store at room temp.

Figure Legends

Figure 1. The interaction trap.

a. Glucose. The LexA fusion protein (bait) is made and binds to LexA
operators (black box) upstream of the two reporter genes, LEU2 and
lacZ. The bait does not activate transcription of the reporters. The
activation-tagged cDNA-encoded protein is not expressed because the
GAL1 promoter on the library plasmid is repressed in the presence
of glucose. The cell does not form a colony on a medium lacking
leucine and forms a white colony on an X-Gal plate.

b. Galactose. Here, galactose induces expression of an activation-
tagged cDNA-encoded protein that does not interact with the bait.
The cell does not grow on a medium lacking leucine and forms a
white colony on X-Gal plates.

c. Galactose. Here, galactose induces expression of an activation-
tagged cDNA-encoded protein that interacts with the bait. The
activation domain activates transcription of LEU2 and lacZ. The cell
forms a colony on a medium lacking leucine and forms a colony that
turns blue on an X-Gal plate.
 
 

Figure 3. Specificity test.

Specificity of Drosophila Cdc2 kinase interactors (Cdis; Finley and
Brent, in preparation). Four replica plates made from a glucose
master plate (not shown) that contained EGY48 derivatives with
different bait plasmids and library plasmids. All strains contained
the lacZ reporter plasmid pJK103, and one of four bait plasmids
directing the synthesis of LexA fused to Drosophila proteins: Cdc2
kinase (Cdc2), a Cdc2 kinase analog (Cdc2c), a derivative of the
homeodomain protein Bicoid (BcdDC), or the helix-loop-helix protein
Hairy (Hairy). Each bait strain was transformed with the library
vector pJG4-5 (v), or with pJG4-5 that contained cDNAs that encode
Cdc2 kinase interactors: Cdi3 (3), Cdi2 (2), and Cdi7 (7). Four
individual colonies from each transformation were patched onto a
glucose master plate which was then replica plated to two ura-his-
trp-leu- plates (leu-), and two ura-his-trp- X-Gal plates (XGal). The
plates on the left have glucose and the plates on the right have
galactose and raffinose. Interaction is detected by growth of strains
on the galactose leu- plate; e.g., Cdc2 interacts with Cdi3, Cdi2 and
Cdi7; Cdc2c interacts with Cdi3 and Cdi2. The strength of the
interactions is suggested by the level of activation of lacZ as indicated
on the X-Gal plate; e.g., the interaction between Cdc2 and Cdi2
activates lacZ strongly, the interaction between Cdc2 and Cdi7
activates lacZ very weakly, and the other interactions activate lacZ
moderately.
 

Figure 4. The mating assay for specificity.

a. A typical mating assay (Finley and Brent, in preparation). The his-
glucose plate (top left) contains four RFY206 derivatives streaked
vertically. Each derivative contains a different HIS3+ bait plasmid.
The trp- glucose plate (top right) contains seven EGY48 derivatives
streaked horizontally. Each derivative contains a different TRP1
library plasmid. The RFY206 derivatives are MAT a, HIS3, trp1-, and
Leu-. The EGY48 derivatives are MAT a TRP1, his3-, and Leu-. The
two plates are pressed to the same replica velvet or filter and the
replica is lifted with a YPD plate (center). During overnight
incubation at 30oC the two strains grow. At the intersections on the
YPD plate the two strains mate and form His+ Trp+ diploids. The YPD
plate is then replica plated to three plates: 1. a his-trp- galactose
plate (with raffinose), on which diploids grow, but neither haploid
parent grows; 2. a his-trp-leu- glucose plate, on which the activation-
tagged cDNA encoded proteins are not expressed, LEU2 is not
transcribed, and no strains grow; 3. a his-trp-leu- galactose plate
(with raffinose), on which activation-tagged cDNA encoded proteins
are expressed, interact with the baits, activate the LEU2 gene, and
allow growth.

b. Results of a mating assay. The horizontally streaked strains are
RFY206 derivatives expressing LexA fusions to Drosophila Cdc2
kinase (Cdc2); the Cdc2 kinase analog, Cdc2c (Cdc2c); a Bicoid
derivative (BcdDC); Hairy (Hairy); the budding yeast Cdc2 kinase
homolog, Cdc28 (Cdc28), and two proteins isolated from a hunt for
Cdc2c interactors (Cdi5 and Cdi11) (Finley and Brent, in preparation).
The vertically streaked strains are EGY48 derivatives that contain
the pJG4-5 library vector without a cDNA insert (v) or with Cdc2
kinase interactor cDNAs, CDI2 (2), CDI3, (3), and CDI7 (7).
 
 

Appendix Figure legends.

pEG202.

pEG202 (11) is a yeast - E.coli shuttle vector that contains a yeast
expression cassette that includes the promoter from the yeast ADH1
gene (PADH1), sequences that encode amino acids 1 to 202 of the
bacterial repressor protein LexA, a polylinker, and the transcription
terminator sequences from the yeast ADH1 gene (TADH1). It also
contains a E.coli origin of replication (pBR ori), the ampicillin
resistance gene (AmpR), a yeast selectable marker gene (HIS3), and a
yeast origin of replication (2 mm ori). pEG202 confers upon a his3-
yeast strain the ability to grow in the absence of histidine and directs
the constitutive expression of LexA (fused to approximately 17
amino acids encoded by the polylinker). Protein coding sequences
can be inserted in-frame with LexA into the unique restriction sites
shown.
 

PJG4-5.

pJG4-5 (11) is a yeast - E.coli shuttle vector that contains a yeast
expression cassette that includes the promoter from the yeast GAL1
gene (PGAL1), followed by sequences that encode the 106 amino acid
fusion moiety or activation tag, and the transcription terminator
sequences from the yeast ADH1 gene (TADH1). cDNAs or other
protein coding sequences can be inserted into the unique EcoRI and
XhoI sites so that encoded proteins are expressed with the fusion
moiety at their amino terminus. The fusion moiety includes the
nuclear localization signal from the SV40 virus large T antigen
(PPKKKRKVA; (53)), the B42 transcription activation domain (4), and
the hemagglutinin (HA) epitope tag (YPYDVPDYA; (54)). The plasmid
also contains an E.coli origin of replication (pUC ori), the ampicillin
resistance gene (AmpR), a yeast selectable marker gene (TRP1), and
a yeast origin of replication (2 mm ori).
 
 

lacZ reporters.

The lacZ reporters are derived from a plasmid that contains the wild-
type GAL1 promoter fused to lacZ (19). Reporters for measuring
activation are derived from pLR1D1, in which the GAL1 upstream
activation sequences (UASg) have been deleted (33). Various
numbers and types of LexA operators have been inserted in place of
UASg to create lacZ reporters with different sensitivities. Two
examples are shown: pJK103 contains a single high affinity
overlapping type colE1 LexA operator which binds two LexA dimers;
pSH18-34 contains four of these colE1 operators and so is much more
sensitive than pJK103 to activation by LexA fusions and activation-
tagged proteins that interact with them. Derivatives with two, three,
or more than four colE1 operators have also been made (S. Hanes,
personal communication; (28)). A reporter less sensitive than
pJK103, pRB1840, contains one lower affinity LexA operator (3).
pJK101 is used to measure repression by LexA fusions (18; 55) It
contains most of UASg and one colE1 operator between UASg and the
GAL1 TATA . All of the lacZ reporters also contain a E.coli origin of
replication (pBR ori), the ampicillin resistance gene (AmpR), a yeast
selectable marker gene (URA3), and a yeast origin of replication (2
mm ori).
 

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