U.S. patent application number 10/810995 was filed with the patent office on 2004-09-16 for adjustable sensitivity, genetic molecular interaction systems, including protein-protein interaction systems for detection and analysis.
Invention is credited to Edwards, David N., Leon, Arlene, Ranney, David F..
Application Number | 20040180325 10/810995 |
Document ID | / |
Family ID | 32929975 |
Filed Date | 2004-09-16 |
United States Patent
Application |
20040180325 |
Kind Code |
A1 |
Edwards, David N. ; et
al. |
September 16, 2004 |
Adjustable sensitivity, genetic molecular interaction systems,
including protein-protein interaction systems for detection and
analysis
Abstract
A method for detecting interactions between first and second
interacting molecules a variable sensitivity. This variable
sensitivity may be obtained by providing for the overexpression of
either a bait hybrid protein containing a DNA binding domain
(desensitization) or a prey hybrid protein containing the DNA
activation domain for a reporter gene (enhanced sensitivity). The
use of exogenous activators of one or the other according to the
needs of a particular system is readily accomplished.
Inventors: |
Edwards, David N.; (Addison,
TX) ; Leon, Arlene; (Garland, TX) ; Ranney,
David F.; (Dallas, TX) |
Correspondence
Address: |
BAKER BOTTS L.L.P.
PATENT DEPARTMENT
98 SAN JACINTO BLVD., SUITE 1500
AUSTIN
TX
78701-4039
US
|
Family ID: |
32929975 |
Appl. No.: |
10/810995 |
Filed: |
March 26, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10810995 |
Mar 26, 2004 |
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09680738 |
Oct 6, 2000 |
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60158079 |
Oct 7, 1999 |
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Current U.S.
Class: |
435/4 ;
435/483 |
Current CPC
Class: |
C12Q 1/02 20130101; C12N
15/1055 20130101; G01N 33/743 20130101 |
Class at
Publication: |
435/004 ;
435/483 |
International
Class: |
C12Q 001/00; C12N
015/74 |
Claims
1. A method of detecting an interaction between a bait polypeptide
and a prey polypeptide comprising: introducing a first nucleic acid
encoding a first hybrid protein into a host cell, the first nucleic
acid having a first exogenously activatable promoter, and the first
hybrid protein having a DNA binding region and the bait
polypeptide; introducing a second nucleic acid encoding a second
hybrid protein into the host cell, the second nucleic acid having a
second exogenously activatable promoter different from the first
exogenously activatable promoter, and the second hybrid protein
having a transcriptional activation region and the prey
polypeptide; activating the first and second promoters using first
and second exogenous activators to induce expression of the first
and second hybrid proteins; and detecting an interaction between
the bait polypeptide and the prey polypeptide by activation of a
detectable reporter gene in the host cell, wherein the DNA binding
region binds near the reporter gene and the transcriptional
activation region activates transcription of the reporter gene when
brought into proximity to the reporter gene by an interaction
between the bait polypeptide and the prey polypeptide; wherein
sensitivity of detecting an interaction may be continuously
adjusted by altering the relative or absolute amount of at least
one of the first or second hybrid proteins in the host cell and
wherein amounts of the first and second hybrid proteins in the host
cell are independent of one another.
2. The method of claim 1, further comprising continuously adjusting
the amount of the first hybrid protein in the host cell through
activation of the first exogenous promoter.
3. The method of claim 1, further comprising continuously adjusting
the amount of the second hybrid protein in the host cell through
activation of the second exogenous promoter.
4. The method of claim 1, wherein the first nucleic acid further
comprises a plurality of exogenous promoters operable to induce
expression of the first hybrid protein in the host cell over a
wider continuous range of amounts than the range obtainable using
only one of the plurality of exogenous promoters.
5. The method of claim 1, wherein the second nucleic acid further
comprises a plurality of exogenous promoters operable to induce
expression of the second hybrid protein in the host cell over a
wider continuous range of amounts than the range obtainable using
only one of the plurality of exogenous promoters.
6. The method of claim 1, further comprising detecting a detectable
reporter protein produced by activation of the detectable reporter
gene.
7. The method of claim 1, wherein sensitivity of detecting an
interaction may be continuously adjusted on a dose-responsive
basis.
8. The method of claim 1, further comprising interfering with
activation of at least one of the first or second exogenously
activatable promoters by providing a modulatory agent to the host
cell.
9. The method of claim 1, wherein at least one of the first or
second exogenous activators comprises a natural or synthetic,
metabolically active or inactive steroid, steroid analogue or
steroid mimic.
10. The method of claim 1, further comprising at least one of the
first or second exogenous activators selected from the group
consisting of: cortisol, cortisone, hydrocortisone,
mineralcorticoids and mineralcorticoid analogues, dexamethasone
estrogen, estradiol, estrone, progesterone, androgens, ecdysone,
retinoid, steroids complementary to orphan receptors, other agent
operable to interact with steroid responsive elements, and any
combinations thereof.
11. The method of claim 1, wherein at least one of the first or
second exogenous activators comprises a membrane-active agent or
analog thereof selected from the group consisting of: ionophores,
anesthetic agents, detergents, amphoteric agents, hydrophobic
agents, lipid-active agents, solvents, transmembrane signaling
agents, intramembrane signaling agents, farnesylating agents, and
any combinations thereof.
12. The method of claim 1, wherein at least one of the first or
second exogenous activators comprises a small molecular
pharmaceutical agent selected from the group consisting of:
antimicrobial agents, anti-tumor agents, nucleic acid-binding
agents, cytoskeletal active agents, chelators, inducers,
co-repressors, agents affecting intracellular trafficking,
localization, protection and degradation of exogenous or endogenous
mediators, hormones, and any combinations thereof.
13. The method of claim 1, wherein at least one of the first or
second exogenous activators comprises a biomolecule or natural or
synthetic biopharmaceutical selected from the group consisting of:
growth factors, cytokines, hormones, their cellular receptors,
fragments thereof, mimics thereof, and any combination thereof.
14. A method of detecting an interaction between a bait polypeptide
and a prey polypeptide comprising: introducing a first nucleic acid
encoding a first hybrid protein into a host cell, the first nucleic
acid having an estrogen-sensitive promoter, and the first hybrid
protein having a GAL4 binding domain and the bait polypeptide;
introducing a second nucleic acid encoding a second hybrid protein
into the host cell, the second nucleic acid having a
glucocorticoid-sensitive promoter, and the second hybrid protein
having a GAL4 transcriptional activation domain and the prey
polypeptide; activating the promoters to induce expression of the
first and second hybrid proteins; and detecting an interaction
between the bait polypeptide and the prey polypeptide by activation
of a UAS.sub.g-LacZ reporter gene in the host cell; wherein
sensitivity of detecting an interaction may be continuously
adjusted by altering the relative or absolute amount of at least
one of the first or second hybrid proteins in the host cell and
wherein amounts of the first and second hybrid proteins in the host
cell are independent of one another.
15. The method of claim 14, wherein activating the promoters
further comprises supplying estrogen or an estrogen analogue and a
glucocoritcoid or a glucorticoid analog to the host cell.
16. The method of claim 14, wherein sensitivity of detecting an
interaction may be continuously adjusted by altering the amount of
estrogen or an estrogen analogue supplied to the host cell.
17. The method of claim 14, wherein sensitivity of detecting an
interaction may be continuously adjusted by altering the amount of
glucocorticoid or a glucocorticoid analogue supplied to the host
cell.
18. The method of claim 14, further comprising continuously
adjusting the amount of the first hybrid protein in the host cell
through activation of the estrogen-sensitive promoter.
19. The method of claim 14, further comprising continuously
adjusting the amount of the second hybrid protein in the host cell
through activation of the glucocorticoid-sensitive promoter.
20. The method of claim 14, further comprising detecting LacZ
produced by activation of UAS.sub.g-LacZ reporter gene.
21. The method of claim 20, further comprising detecting LacZ using
colorimetric analysis.
Description
RELATED PATENT APPLICATION
[0001] This Patent Application is a Continuation of U.S. patent
application Ser. No. 09/680,738 filed on Oct. 6, 2000, which claims
priority to U.S. Provisional Application Serial No. 60/158,079
filed on Oct. 7, 1999 and incorporated by reference herein.
BACKGROUND OF THE PRESENT INVENTION
[0002] Genetically-based interaction systems are commonly used in
scientific research and in commercial and therapeutic applications
derived from that research. Current genetically-based interaction
systems are severely limited by a fixed level of interaction
sensitivity which is either completely "on" or completely "off"
(Fields and Song, 1989; Bartel et al., 1993; Gyuris et al., 1993;
Mendelsohn and Brent, 1994; Phizicky and Fields, 1995; Bai and
Elledge, 1997; Brachmann and Boeke, 1997; Finley and Brent, 1997;
Young, 1998). This creates problems related to both the detection
of numerous biologically irrelevant interactions, as well as a
failure to detect relevant biological interactions. The
consequences of this problem may be either a complete inability or
prolonged time required to elucidate important biologically
relevant interactions, cellular pathways, and potentially related
modulatory agents and drugs.
[0003] Historically, the first description of a genetic system to
detect molecular interactions is the two-hybrid system (Fields and
Song, 1989; FIG. 1). This set forth the original concept and
practice of detecting protein-protein interactions in Saccharomyces
cerevisiae. This original system features detection of an in vivo
protein-protein interaction within the nucleus of the yeast cells.
These cells were engineered to express the visually detectable
bacterial gene lacZ in the presence of an interaction. Basically,
the host cells were transformed with an expressible gene coding for
a first hybrid protein composed of a DNA binding domain and a first
polypeptide. The host cells were additionally transformed with a
second hybrid protein consisting of a transcriptional activation
domain and a second polypeptide of stable interaction with the
first protein fragment. Finally, the cells were also transformed
with a lacZ reporter gene containing at least one DNA binding
sequence for the DNA binding domain of the first hybrid protein and
capable of being transcribed at increased and detectable levels
when the transcriptional activation domain of the second hybrid
protein was in close proximity. Field and Song demonstrated that
when the two hybrid proteins were expressed, levels of the LacZ
reporter protein dramatically increased in the host cell. This
indicated that the DNA binding domain in the first hybrid protein
was binding to the DNA binding sequence of the reporter gene and
that the first polypeptide of the first hybrid protein was
interacting with the second polypeptide of the second hybrid
protein in such a manner as to bring the transcriptional activation
domain of the second hybrid protein into proximity of the lacZ gene
and thus increase its transcription and subsequent expression.
[0004] This basic approach has been employed in all later
two-hybrid and three-hybrid systems. Extensions of this work
describe such detection capability in nuclear, cytoplasmic, or
membrane locations of eukaryotes (Aronheim et al., 1997; Gyuris et
al., 1997), as well as in prokaryotes (Bustos and Schleif, 1993;
Bunker and Kingston, 1995; Hays et al., 2000). The initial art has
also been subsequently extended to include multiple prokaryotic
(Bustos and Schleif, 1993; Bunker and Kingston, 1995; Hays et al.,
2000) and eukaryotic organisms (other fungal strains, arthropod,
plant, and mammalian cells) (e.g., Vasavada et al., 1991; Fearon et
al., 1992; Luo et al., 1997; Shoda et al., 2000).
[0005] Parallel approaches to genetic molecular interaction
detection have been described for detecting protein interactions
with RNA and DNA, as well as with small ligands, including peptides
and drugs (Li and Herskowitz, 1993; Yang et al., 1995; SenGupta et
al., 1996; Brachmann and Boeke, 1997; Young, 1998). All of these
systems work on the same basic concept of using the living cell as
a means of detecting the interaction between two intracellular
molecules.
[0006] Genetic molecular detection systems following the original
Fields two-hybrid system also usually include the additional
feature of genetic selection (Fields and Song, 1989). Selection
allows the detection of an interaction by choosing the phenotype of
survival; cells containing proteins that do not interact strongly
enough or at all are unable to grow, and are no longer considered.
The current methods of selection are limited to an "all or nothing"
auxotrophic nutrient, antibiotic selection or other means of
affecting survival (Fields and Song, 1989; Gyuris et al., 1993; Bai
and Elledge, 1997). Selection yields a great advantage for the
various detection systems, since cells containing potentially
irrelevant pairs of candidate interacting molecules are eliminated
without intervention from the scientist or other automated
analysis.
[0007] However, the introduction of genetic selection introduced a
new and severely limiting aspect to the in vivo genetic molecular
detection systems. All current methods of selecting for molecular
interactions in vivo must make a priori assumptions about the
strength of the interactions that they detect. The systems must be
constructed such that there is a threshold above which an
interaction will be detected, and below which it will not. That is,
there is an implicit assumption that very weak or transient
interactions are probably less likely to be real or important.
Systems are designed to exclude these interactions because, if
systems are too sensitive, they will detect too much background.
However, if the system is not sensitive at all, important
interactions will be missed. Those constructing these systems built
them and tested them, and then used the systems with the most
reasonable compromise of detection sensitivity. In short, they
chose the compositions that yielded, on average, a tolerable
background while missing a tolerable number of biologically
relevant interactions.
[0008] Early crude attempts to overcome this "all or nothing"
threshold of reporting output have included: (a) exposure of yeast
to toxic nutrient analogues at sub-lethal concentrations, for
example, 3-AT as a histidine synthesis inhibitor (Mangus et al.,
1998); and (b) the creation of complicated genetic modifications of
the reporter, which give several different fixed (nonadjustable)
levels of detection (James et al., 1996; Finley and Brent, 1997;
Serebriiskii et al., 1999). Such complicated modifications include
the use of (b.1.) variable numbers of reporter binding sites,
(exemplified by the use of multiple LexA binding sites (by, e.g.
multiple LexA binding sites for a Leucine reporter as described in
Finley and Brent, 1997), for a Leucine reporter as described in
Finley and Brent, 1997), and (b.2) variable distance between
reporter binding site and the transcriptional start site (West et
al., 1984).
[0009] A feature of current detection systems is the capacity to
turn the detection of protein interactions on or off completely by
providing for the expression or lack of expression of the
two-hybrid library fusion under standard nutrient conditions.
Gyuris et al. (1993) found that by being able to express one of the
two hybrid proteins at high levels or by being able to limit
expression of one such protein completely, it was possible to show
in vivo that the presence of both of the hybrid proteins were
necessary for activation of the reporter gene; in other words, they
added a switch enabling on or off control of one of the interacting
components. This control is useful and exerts its effects by
modulating reporter activity, but it does not provide for the
continuous adjustability of the sensitivity of a two-hybrid protein
interaction system. Thus, the Gyuris system further demonstrates
the limitation of the prior art: it is either on or off, above or
below the same detection threshold set by the reporters chosen when
the system was constructed.
[0010] The level of reporter gene expression that will result from
any given molecule-molecule interaction in a two-hybrid system is
uniform for those molecules used in combination with that reporter.
The Brent lab first demonstrated this in experiments using a
traditional two-hybrid protein-protein interaction system. The
experiments showed that output of the quantitative lacZ reporter
was directly proportional to the independently determined strength
(or Kd) of the protein-protein interaction for the protein
fragments used in the hybrid proteins. If the two proteins
interacted strongly in vitro, they gave robust expression from the
two-hybrid reporters and vice versa. Therefore, they also
demonstrated that the output of a given reporter is constant for a
given pair of interacting proteins. This is now generally accepted,
since many publications of genetic molecular interactions include
the quantitative reporter output from the interaction system as a
relative indication of the strength of the interaction itself
(Edwards et al., 1997).
[0011] The present invention yields surprising and unexpected
advantages relative to earlier systems in providing for
adjustability of the sensitivity of such detection systems.
SUMMARY OF THE INVENTION
[0012] The present invention comprises an improved two-hybrid or
three-hybrid detection method and a kit utilizing this method. The
method of the current invention may be used with any conventional
two-hybrid or three-hybrid methods, including inhibition or
competition two-hybrid methods, as well as any future variations of
those methods. In all embodiments of the present invention, the
sensitivity of a detectable reporter gene in a host cell is
continuously adjustable by altering the relative or absolute
amounts of interacting molecules provided to the host cell. The
method may be used to detect interactions between any types of
molecules including, but not limited to, proteins, polypeptides,
DNA molecules, RNA molecules, pharmaceutical agents, other
biological or chemical agents, and other small molecules or
macromolecules. The method may be used to detect interactions in
both prokaryotic and eukaryotic organisms or cells. The molecular
interactions may occur at various locations, including, but not
limited to, extracellular regions, the cell membrane, the
cytoplasm, the nuclear membrane, the nucleus, and other
intracellular regions.
[0013] In a preferred embodiment, the first chimeric gene and the
second chimeric gene are introduced into the host cell. The host
cell is then subjected to conditions under which a first hybrid
protein and a second hybrid protein are expressed in at least
sufficient quantities for the detectable reporter gene within the
host cell to be activated. The first chimeric gene contains a first
exogenously activatable promoter and a sequence encoding the first
hybrid protein. The first hybrid protein contains a DNA binding
domain capable of binding near the reporter gene and a first
interacting polypeptide(bait). The second chimeric gene contains a
second exogenously activatable promoter and a sequence encoding a
second hybrid protein. The second hybrid protein contains a
transcriptional activation domain capable of inducing or increasing
transcription of the reporter gene and a second interacting
polypeptide(prey). This second polypeptide may be derived from a
library.
[0014] The sensitivity of the reporter gene may be altered by
adding a first and/or second exogenous activator and thus, altering
the relative or absolute amounts of the first and/or second hybrid
proteins. These alterations affect the activity and thus
sensitivity of the reporter gene. The sensitivity of this
activation may be decreased by adding a first exogenous activator
capable of activating the first exogenous promoter. This results in
increased production of the first hybrid protein and raises its
level in the host cell relative to the level of the second hybrid
protein. Thus, after this increase, more DNA binding sites of the
reporter gene are occupied by the first hybrid proteins for which
there is not second hybrid protein available for interaction.
Therefore, less of the reporter genes are activated or activation
is weaker.
[0015] The sensitivity of the reporter activation may be increased
by adding a second exogenous activator capable of activating the
second exogenous promoter. This results in increased production of
the second hybrid protein and raises its level in the host cell
relative to the level of the first hybrid protein. Thus, after this
increase, more of the DNA binding sites of the reporter gene are
occupied by a first hybrid protein that is additionally interacting
with a second hybrid protein. Therefore, more of the reporter genes
are activated or activation is stronger. Subsequent to the hybrid
protein expressions, detectable reporter gene expression is
measured and compared to the amount of expression in the absence of
any interaction between the first test protein and the second test
protein.
[0016] A kit utilizing the method of this invention may also be
prepared. The kit may comprise any host cell described above, any
first or second chimeric genes described above, or any combination
thereof. The kit may also contain the first and second exogenous
activators and also chemicals or assays for detecting the
detectable reporter gene product.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 shows the Original Two-hybrid system relies on the
reconstitution of a functional transcription factor to report the
interaction of two proteins, depicted as X and Y. The Fields
two-hybrid system uses the DNA-binding domain and activation domain
from the Gal4p transcription factor (figure adapted from Fields and
Song, 1989).
[0018] FIG. 2 shows the Novel Library vector described herein is a
shuttle vector containing ampicillin and the colE1 origin of
replication for selection in E.coli as well as TRP1 and the 2
micron origin of replication for selection in yeast. Unknown cDNAs
are fused to the Gal4p activation domain, and continuously variable
expression is obtained by the induction of GRE upstream activating
element(s) attached to CYC1 promoter. AMP=Ampicillin, E.coli
selectable marker; ori=colE1 bacterial origin of replication;
TRP=TRP1 gene, yeast selectable marker; 2 um=origin of replication
for yeast; GRE=Glucocortocoid Response Element; CYC1p=CYC1 promoter
from yeast; Gal4AD=Gal4 activation domain; AdhT=Alcohol
dehydrogenase terminator.
[0019] FIG. 3 shows the novel Bait vector in a shuttle vector
containing kanamycin and the colE1 origin of replication for
selection in E.coli as well as URA3 and the 2 um origin of
replication for selection in yeast. A known cDNA is fused to the
Gal4p DNA binding domain, and continuously variable expression is
obtained by the induction of ERE element(s) attached to CYC1
promoter. KAN=Kanamycin, E.coli selectable marker; ori=colE1
bacterial origin of replication; URA=URA3 gene, yeast selectable
marker; 2 um=origin of replication for yeast; ERE=Estrogen Response
Element; CYC1p=CYC1 promoter from yeast; Gal4pBD=Gal4p DNA binding
domain; AdhT=Alcohol dehydrogenase terminator.
[0020] FIG. 4 shows Continuously Dose Responsive Expression of
proteins fused to Gal4pBD in the "Bait Vector" in yHYB001 strain.
Strain yHYB001 with the bait vector is grown in selective minimal
media with varying concentrations of estradiol. The bait vector
contains the Gal4pBD fused to the marker lacZ. .quadrature.-gal
expression assays are performed three times per estradiol
concentration; the data represents an averaging of three assays per
sample. Growth was overnight and strains were at OD.sub.600 ca. 0.8
when assayed. Strain yHYB001 is described in the text.
.quadrature.-gal expression assays are described in Guarente
(1983). The variable "n" in the x-axis label "Dose (10.sup.-n)
dexamethasone" represents any given number on the x-axis.
[0021] FIG. 5 shows Continuously Dose Responsive Expression of
proteins fused to Gal4pAD in the "Library Vector" in yHYB001
strain. Strain yHYB001 with the library vector is grown in
selective minimal media with varying concentrations of
dexamethasone. The library vector contains the Gal4pAD fused to the
marker lacZ. .quadrature.-gal expression assays are performed three
times per dexamethasone concentration; the data represents an
averaging of three assays per sample. Growth was overnight and
strains were at OD.sub.600 ca. 0.8 when assayed. Strain yHYB001 is
described in the text. .quadrature.-gal expression assays are
described in Guarente (1983). The variable "n" in the x-axis label
"Dose (10.sup.-n) dexamethasone" represents any given number on the
x-axis.
[0022] FIG. 6 shows the Principle of Variable Reporter Output with
changes in relative concentration of interactors in a novel
molecular genetic interaction detection system. The number of bars
represent relative levels of library fusion protein and bait fusion
protein present in the cell. At equilibrium, only a fraction of the
fusion proteins will be physically paired at any given time,
representing the Kd of the interaction (in this example, we assume
50% are bound at a given time.) A medium sensitivity assay results
when both fusion proteins are present in roughly equal amounts. At
equilibrium, half of them are interacting, resulting in an output
that is 1/2 of the theoretical maximum from the reporter. This is
true since half of the DNA-binding domain fusions at the reporter
will not be paired to an activating library fusion. Lower
sensitivity can be achieved by reducing the amount of library
fusion and/or increasing the amount of bait fusion. A two-fold
difference in levels yields, at equilibrium, 1/4 of the theoretical
maximum from the reporter. High sensitivity results from an
overabundance of library fusion; a two-fold excess at equilibrium
will yield an output close to the theoretical maximum for the
reporter.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Method to Adjust the Sensitivity of Genetic Macromolecular
Interaction Systems
[0023] This invention comprises novel compositions, methods and
uses for continuously and/or discontinuously adjusting the
sensitivity of genetic detection systems to enable significantly
improved detection and analysis of molecular interactions. The
molecular interactions include those at extracellular, membrane and
intracellular sites, and include but are not limited to
protein-protein interactions, protein-DNA interactions, protein-RNA
and protein-small molecule interactions in eukaryotic and
prokaryotic organisms.
[0024] The method of the present invention involves changing the
relative quantity of each macromolecular or small molecular
component provided within the system, such that the absolute or
relative amounts of actual interacting pairs changes within the
system. By altering the relative amounts of interacting molecules
(particularly the molecule bound to the detectable reporter gene),
the output of the system via reporter gene expression is also
altered.
[0025] In the method of the present invention, the host cell may be
provided with a detectable reporter gene. This reporter gene may be
provided before or after interacting molecules or other components
are provided. However, the reporter gene is preferably provided
first so that host cells that do not contain the reporter gene may
be eliminated before interacting molecules or other components are
introduced. The reporter gene may be provided through any method of
gene transfer currently known or later developed. In a preferred
embodiment, the reporter gene is provided through electroporation
of the host cell. The reporter gene may also be provided in any
form capable of transfer to the host cell using the selected
transfer method. For instance, it may be provided as a portion of a
plasmid.
[0026] Any detectable reporter gene that may be activated by an
interaction of the interacting molecules is appropriate for this
method. For instance, the reporter gene may produce a detectable
reporter protein or other detectable gene product. The method of
this invention can function with any detectable reporter gene
because the method involves changing the amounts of the interacting
macromolecules themselves. If there are more interaction to report,
any reporter gene will report this as a relatively stronger
activation of the reporter gene. If there are relatively fewer
interactions to report, any reporter system will report this as a
relatively weaker output.
[0027] In a preferred embodiment, the reporter gene comprises at
least one DNA binding site capable of interaction with a
polypeptide including a DNA binding domain or with another DNA
binding molecule, such as a small molecule or pharmaceutical agent.
This DNA binding site is located such that if a first interacting
molecule binds to the site and additionally interacts with a second
interacting molecule, the transcriptional activation domain of the
second molecule will be able to activate transcription of the
reporter gene.
[0028] A first interacting molecule which may be a macromolecule or
small molecule into a host cell or its extracellular region. This
molecule should contain a polypeptide containing a DNA binding
domain or it should contain another molecular region capable of
binding DNA. This first interacting molecule may be a protein, a
DNA, a RNA, or a pharmaceutical agent or any other molecule that
contains or may be bound to a molecule containing a DNA binding
region or domain.
[0029] In a preferred embodiment, the molecule is a protein, a DNA,
or a RNA. In such a preferred embodiment, the macromolecule is
provided by introducing into the host cell a first chimeric gene
capable of being transcribed in the host cell. This first chimeric
gene may include a first exogenously activatable promoter, a
sequence coding for a polypeptide, DNA, or RNA containing a DNA
binding region, and a sequence coding for the first interacting
macromolecule. In a more preferred embodiment, the first chimeric
gene comprises a first exogenously activatable promoter and a first
hybrid protein. This first hybrid protein comprises a DNA binding
polypeptide and a first interacting polypeptide (bait) capable of
interacting with at least one second interacting polypeptide
(prey).
[0030] A second interacting macromolecule or small molecule is also
introduced into the host cell or its extracellular region. This
molecule should contain a transcriptional activation domain. This
transcriptional activation domain may be a polypeptide or a region
of another molecule capable of activating transcription such as a
region of a pharmaceutical agent or a nucleic acid. In a preferred
embodiment, this second molecule may be a protein, a DNA, a RNA, a
pharmaceutical agent, or any other molecule meeting the
requirements stated above. In a more preferred embodiment it is a
protein, a DNA, or a RNA. In such a preferred embodiment, the
macromolecule may be produced in the host cell by introducing a
second chimeric gene capable of being transcribed in the host cell.
This second chimeric gene may include a second exogenously
activatable promoter, a sequence coding for a transcriptional
activation domain, and a sequence coding for the second
macromolecule. In a more preferred embodiment, the chimeric gene
may contain a second exogenously activatable promoter and a second
hybrid protein. This second hybrid protein may contain a
polypeptide containing a transcriptional activation domain and the
second interacting polypeptide (prey).
[0031] In all embodiments of the present invention, the sensitivity
of the detectable reporter gene in a host cell is continuously
adjustable by altering the relative or absolute amounts of the
first and/or second interacting molecules provided to the host
cell. In a preferred embodiment, the host cell itself has the
capacity to regulate the absolute of relative levels of the first
or second molecules. As indicated in the preferred embodiments
described above, this may be accomplished by introducing chimeric
genes encoding the first and second macromolecules and containing
first and second exogenously activatable promoters. These
exogenously activatable promoters may be activated by exogenous
activators.
[0032] While any promoters and activators may be used in the method
of the present invention, in a more preferred embodiment, the
activator is a natural or synthetic, metabolically active or
inactive steroid, steroid analogue or mimic and the promoter
induces transcription in response to the activator. The relative or
absolute amount of at least one of the hybrid proteins may then be
controlled in a manner responsive to the dose of one or more of
these activators, its analog, or its antagonist. Generally, if both
chimeric genes are under the control of exogenously activatable
promoters, the promoters will be different for each gene and will
be activated by different molecules.
[0033] By regulating the relative levels of the first and second
interacting molecules, it is possible to alter the sensitivity of
the reporter. For instance, if the system is flooded with one
component, usually the second molecule, it is possible to drive the
system towards interaction of a first molecule bound to the
reporter with a second molecule such that reporter activity is
increased. At maximum sensitivity, every first molecule binding the
reporter is involved in an interaction with a second molecule, and
therefore the reporter is activated more often or more
strongly.
[0034] It is also possible to dampen the output of the reporter by
increasing the relative amount of one of the two interacting
molecules, usually the first molecule. If this is done then most of
the first molecules bound to the reporter are not additionally
bound to a second molecule and thus the reporter activation is
lowered.
[0035] Since the strength of a given interaction between any two
molecules does not change, the capacity to regulate the relative or
total amounts of either of the two interacting molecules results in
a system that reports interaction at continuously adjustable levels
of sensitivity. Thus, if an interaction is weak, sensitivity may be
increased by increasing the relative number of interactions.
Because there are more interactions, the reporter will report more
strongly despite the weakness of the interactions. If an
interaction is strong, sensitivity may be decreased by decreasing
the relative number if interactions. Because there are fewer
interactions, the reporter will report less strongly. Thus,
interactions that might be deemed unimportant or undetectable using
a conventional two-hybrid system may be detected with the present
invention.
[0036] In another embodiment of the present invention, the cells
may additionally be provided with other macromolecules or small
molecules that mediate or interfere with the interaction between
the first and second interacting molecules. For instance, in a
three-hybrid system, a third macromolecule or small molecule may be
provided that facilitates or is required for the interaction of the
first and second molecules. This third molecule will most commonly
be a protein. It may exert its effect by stabilizing the
interaction between the first and second molecules or by forming a
connection between them when they otherwise would not interact.
[0037] In a preferred embodiment, this third molecule may also
interfere with the interaction of the first two. For, instance, if
all of the molecules are proteins, the third molecule may contain a
polypeptide that is identical or similar to the bait or prey
polypeptides. Thus, the third molecule will interfere with the
ability of the bait and prey to interact. This variation of the
two-hybrid assay is commonly known as a inhibition or competition
two-hybrid assay. It is especially amenable to the method of the
present invention because such assays do not currently provide
precise results. Thus, inhibition two-hybrid assays would benefit
greatly from the present invention because relative amounts of the
bait and prey polypeptides greatly influence the ability of the
third molecule to inhibit the bait-prey interaction and thus, the
sensitivity of the reporter system. The techniques for fine-tuning
and varying the amounts of the hybrid proteins of this method might
also be applied to regulate the relative or absolute amount of a
third inhibition polypeptide in an inhibition two-hybrid assay.
[0038] The interaction between the first and second interacting
molecules may take place and be detected anywhere within the cell.
For instance, it may occur in the nucleus, in the cytoplasm, at or
in the membrane, or in an organelle. The system would be expected
to work similarly in prokaryotes and eukaryotes, including
bacteria, yeast, plant, arthropod, and mammalian cells.
[0039] In one preferred embodiment of the present invention, the
method is applied to a genetic molecular interaction detection
system. Regulation of the amounts of hybrid proteins is
accomplished by using compositions comprising alternate promoters
for different intrinsic levels of expression of a downstream hybrid
protein or molecule. These promoters may be derived from natural or
synthetic, yeast or non-yeast sources. Regulation may be
accomplished by several methods, including, but not limited to: (a)
changing the promoter upstream of a hybrid protein, for instance a
hybrid in which the second interacting (prey) polypeptide is
derived from a library, to give different levels of expression, as
further exemplified below, using a GAL1/10 promoter, CYC1 promoter,
or ADH1 promoter, which exhibit different levels of expression
(Guarente and Ptashne, 1981; Guarente et al., 1982; Ammerer, 1983;
Guarente, 1983; Cantwell et al., 1986); or (b) modifying the
promoter itself, as further specified and exemplified below, using
a GAL1/10 or CYC1 promoter (or other promoters) with the upstream
activating sequences, UAS.sub.G or UAS.sub.C, respectively, or
other activating sequences, any of which may be positioned at
various distances from the transcriptional start sites (West et
al., 1984).
[0040] In another preferred embodiment of the present invention,
regulation of hybrid protein amounts is accomplished by using a
single promoter, for example, GAL 1/10, CYC1, ADH1 or other natural
or synthetic yeast or non-yeast promoters in combination with
different upstream enhancer sequences from yeast or non-yeast
sources.
[0041] In another preferred embodiment, the method of the present
invention discloses surprising and unexpected results applicable to
all known genetic systems for detecting and analyzing protein
interactions with other proteins and with any other classes of
molecules. It is clearly and categorically distinguished from the
prior art, based on its sensitivity being continuously adjustable;
that is, the sensitivity may be adjusted on a plural stepped
dose-responsive basis. This includes, in one preferred embodiment,
pharmacologically modifying the transcription or expression of the
fusion protein or molecule and detecting the various reporter gene
expression levels in a single screen.
[0042] This embodiment of the present invention gives major
technical advantages including, but not limited to: (a) detecting
and analyzing interactions of various strengths, without any prior
knowledge of even the range of such interaction strengths; (b)
avoidance of biologically non-relevant interactions; (c) the
detection of potentially very important but currently
systematically undetected, weak interactions; and (d) the potential
for actually quantifying the in vivo strength of intermolecular
binding, as characteristically defined by dissociation constant
(Kd) (Estojak et al., 1995). The practical implications of these
and related advantages, include but are not limited to: (a)
substantial acceleration of detecting and analyzing
protein-molecular interactions; (b) elimination of a large subset
of biologically irrelevant but previously detected interactions;
(c) detection of biologically important new interactions; (d) the
potential for true in vivo estimations and correlations of Kd; (e)
substantial enhancement of large-scale commercial screening; and
(f) substantially improved effectiveness and efficiency of
identifying and elucidating cellular pathways, potential drug
targets, potentially complementary drugs, and a variety of other
scientifically and commercially important molecular
interactions.
[0043] In one preferred embodiment of the invention, an
extracellular ligand binds and modulates the activity of a specific
transmembrane receptor to effect a dose-responsive change in
expression or activity of one or more interacting molecules.
Examples of such extracellular ligands include but are not limited
to growth factors, cytokines, hormones, synthetic agents and
biopharmaceuticals and their cellular receptors (Mercurio and
Manning, 1999; Baldwin, 1996; Mohal and Sternberg, 1999).
[0044] In another preferred embodiment, an intracellular ligand
interacts either cytoplasmically or within the nucleus to modulate
the expression or activity of the interacting molecules. Examples
of such intracellular ligands include but are not limited to small
molecular pharmaceutical agents and modulators, including but not
limited to antimicrobial agents, anti-tumor agents, nucleic
acid-binding agents, cytoskeletal active agents, chelating agents,
inducers, co-repressors, and agents affecting intracellular
trafficking, localization and protection or degradation (Schena et
al., 1991; Rossi and Blau, 1998).
[0045] In another preferred embodiment, a membrane-active agent
interacts to modulate the level of cellular activation or response
potential. Examples of such an agent include but are not limited to
ionophores, amphoteric and hydrophobic lipid-active agents and
detergents, various anesthetics and solvents, transmembrane and
intramembrane signaling agents, and farnesylating agents (Berridge
et al., 1999).
[0046] In a more preferred embodiment, the amounts of the hybrid
proteins containing the bait and/or prey or other proteins and
molecules are continuously varied or limited using exogenously
activated promoters, exogenous activating agents, and other
molecules including, but not limited to: (i) steroid responsive
elements (SRE's), including but not limited to those sensitive to
natural or synthetic estrogens (e.g., estradiol, estrone and
others), androgens, progesterones, glucocorticoids (e.g.,
dexamethasone, cortisone, hydrocortisone and cortisol, among
others), mineralocorticoids, ecdysones, metabolically inactive
corticoids, other steroids (e.g., ones complementary to orphan
receptors), and retinoids; and/or (ii) agonist and antagonist
agents in combination with (i); (iii) any other molecules,
receptors and response elements, in any or all combinations
effective to provide continuously variable amounts of (iii.a) a
hybrid protein containing the bait polypeptide, (iii.b) a hybrid
protein containing the prey polypeptide, which may have been
derived from a library, and/or (iii.c) generally any molecular
expression involved in genetic molecular interaction systems, such
as to enable the relevant detection and analysis of the preceding
biologically relevant interactions (Schena et al., 1988; Picard et
al., 1990; Kralli et al., 1995; Mangelsdorf and Evans, 1995;
Kliewer, 1999; Martinez et al., 1999).
[0047] Another more preferred embodiment, which forms the basis for
the Examples below, comprises a novel Interaction Hybrid System
(IHS) which is steroid-hormone-dependent, continuously adjustable,
and contains a traditional triple reporter in a Saccharomyces
cerevisiae two-hybrid system. This relates to and novelly extends
the principles and basic design of a yeast two-hybrid system as
first presented and patented by Stan Fields (U.S. Pat. No.
5,283,173), which is incorporated by reference herein.
[0048] Saccharomyces cerevisiae strain yHYB001 was constructed
containing auxotrophies for the selectable markers leu2, ade2,
trp1, ura3, and arg4. The strain is deltaGAL4 and deltaGAL80, so as
to enable the use of the GAL4 DNA-binding domain (GAL4bd) and the
GAL4 transcriptional activation domain (GAL4ad) as fusions for
two-hybrid interaction detection, exactly as used in the original
Fields two-hybrid system. The strain also contains integrated human
estrogen receptor and integrated rat glucocorticoid receptor genes,
expressed constitutively. The strain is lem1, which enables the use
of decreased concentrations of dexamethasone in yeast, presumably
by eliminating a membrane pump (Kralli et al., 1995). The strain
contains three integrated reporters for the detection of two-hybrid
interactions. The first is a UAS.sub.G-LacZ construct for
colorimetric and quantitative assays and screening. The second and
third are UAS.sub.G-ADE2 and UAS.sub.G-LEU2, respectively; these
each enable qualitative selection for yeast that contain
interacting hybrid proteins or other molecules based on rescue of
nutrient auxotrophies.
[0049] In this embodiment, in the first hybrid protein, the bait
may be fused to the carboxyl-terminal end of the GAL4bd, a DNA
binding domain (FIG. 3). This first hybrid protein may be
transcribed in a continuous range of amounts over up to five orders
of magnitude, and under the influence of an estrogen response
element (ERE) within a minimal promoter. This results in variable
expression of the bait first hybrid protein over a continuous range
of amounts in response to changing levels of estrogen or estrogen
antagonists in the yeast growth medium. This promoter-first hybrid
protein construct is provided on a two-micron plasmid either under
ARG4 or URA3 selection.
[0050] The second hybrid protein may be formed by fusion of the
prey polypeptide, which may be derived from a library, to the
carboxyl-terminal end of the GAL4ad, a transcriptional activation
domain (FIG. 2). This second hybrid protein may be transcribed in a
continuous range of amounts over up to five orders of magnitude and
under the influence of preferably one to six, and in the present
example, three, glucocorticoid response elements (GREs) within a
minimal promoter, for example, including but not limited to that
from CYC1. This results in variable expression of the second hybrid
protein over a continuous range of amounts in response to changing
levels of glucocorticoids or their antagonists, including but not
limited to dexamethasone, in the yeast growth medium. This
promoter--second hybrid protein construct is provided on a
two-micron plasmid under TRP1 selection. Both hybrid protein
plasmids are also shuttle vectors containing either ampicillin or
kanamycin resistance and a colE1 origin of replication, which
provide for manipulation in E. coli bacteria.
[0051] Wide ranges of estrogen and dexamethasone concentrations in
the yeast medium result in wide ranges of variable and relative
expression of the hybrid proteins. Estrogen is used over a
concentration range of at least about 10.sup.-12 to 10.sup.-8 M
while dexamethasone is used over a concentration range of at least
about 10.sup.-7 to 10.sup.-4M. Estradiol has some effect on yeast
growth above concentrations of 10.sup.-6.
[0052] Although only preferred embodiments of the invention are
specifically described above, it will be appreciated that
modifications and variations of the invention are possible without
departing from the spirit and intended scope of the invention.
[0053] The following non-limiting examples are provided to more
clearly illustrate the aspects of the invention and are not
intended to limit the scope of the invention.
EXAMPLES
Example 1
Interaction Hybrid System Adjusted to Give Variable Quantitative
Reporter Output without Modifying the Reporter System
[0054] As noted above, the Brent lab has shown that a given set of
two-hybrid protein interactors yield a uniform quantitative
reporter output directly proportional to their strength of
interaction (Estojak et al., 1995). Utilizing the novel adjustable
yeast interaction hybrid system (IHS), introduced and described as
a more preferred embodiment in the paragraphs above, three sets of
proteins pairs previously demonstrated to interact in a two-hybrid
system are demonstrated to give variable levels of reporter output
when expressed at different relative concentrations. The level of
expression of the first hybrid protein containing the bait is
proportional to the concentration of estradiol, and the level of
the second hybrid protein containing the prey derived from a
library is proportional to dexamethasone concentration (Kralli et
al., 1995; Gaido et al., 1997 (FIGS. 4 and 5)).
1TABLE 1 Quantitation of known interactors in a traditional
Two-Hybrid Screen (2HS) and the novel Interaction Hybrid System
(IHS) at various levels of sensitivity LOW MEDIUM HIGH BAIT LIBRARY
TRADITIONAL SENSITIVITY SENSITIVITY SENSITIVITY HYBRID HYBRID 2HS
IHS IHS IHS SNF1 SNF4 300 50 250 2000 Pelle Tube 250 20 150 1400
Pelle Dorsal 1300 100 1400 2500 *All quantitations of interactions
are in Miller units.
[0055] Table 1 shows a traditional two-hybrid system and the novel
Interaction Hybrid System were done using proteins previously
described in Edwards et al. (1997). Methods for analysis of the
two-hybrid screen are described in Edwards et al. (1997). Low
sensitivity assays in the IHS used 10.sup.-10M Estradiol and
10.sup.-7M Dexamethasone. Medium sensitivity assays used
10.sup.-10M Estradiol and 10.sup.-5M Dexamethasone. High
sensitivity assays used 10.sup.-12M Estradiol and 10.sup.-4M
Dexamethasone.
[0056] Table 1 demonstrates that different relative levels of
expression of a bait and of a library (prey) hybrid protein in the
context of the novel IHS system gives variable levels of reporter
activity. Since clearly the Kd of the interaction is not changing,
and the sensitivity of the reporter output has not been altered,
the quantitative level of expression from the lacZ reporter must be
altered by the change in relative concentrations of the hybrid
proteins themselves (Table 1).
[0057] FIG. 6 is an illustration of the principle of varying
reporter output in a genetic interaction system given a constant
reporter set. Yeast colonies containing identical hybrid proteins
in identical strains were observed to express reporter protein at
different levels when exposed to various steroid combinations.
Yeast cells containing SNF1 and SNF4 or pelle and tube constructs,
fused to the bait and prey vector (respectively, see Table 1), were
plated in the corresponding minimal media in the presence of
different concentrations of estradiol and/or dexamethasone. An
integrated copy of UAS.sub.G-LacZ was used as a reporter for the
interaction. UAS.sub.G-LacZ expression was detected by the
development of blue color in yeast colonies. Cells grown in plates
containing 10.sup.-12M estradiol and 10.sup.-4M dexamethasone
showed the highest expression of UAS.sub.G-LacZ. At concentrations
of 10.sup.-10M estradiol and 10.sup.-5M dexamethasone the level of
LacZ expression diminished, being at its lowest when the cells were
grown at 10.sup.-9M estradiol and 10.sup.-7M dexamethasone. The
different levels of LacZ expression observed corresponded with the
sensitivity of the assay, thus at high sensitivity the intensity of
the blue color was at its maximum. As the sensitivity of the assay
decreased the blue color became less intense. At the weakest level
of sensitivity, a light blue color was observed. No blue color
developed in yeast colonies containing the prey hybrid alone in the
presence 10.sup.-9M estradiol. Similarly, no blue color was
observed in yeast colonies containing the bait hybrid alone in the
presence of 10.sup.-4M dexamethasone or in yeast colonies with
neither prey hybrid nor bait hybrid when grown at in levels of
steroids sufficient to produce the highest sensitivity level when
both hybrid gene constructs were present.
Example 2
Interaction Hybrid System Adjusted to Low Sensitivity for
Promiscuous Bait
[0058] When low sensitivity is desired, as in the screening of the
mammalian baits IRAK kinase, its Drosophila homolog Pelle kinase,
or its plant kinase homologues, high expression of the bait hybrid
protein is achieved using 10.sup.-9 M estradiol (for example, as in
Table 2, below). Low relative expression of the library/prey hybrid
proteins is achieved only at 10.sup.-7 M dexamethasone (for
example, as in Table 2, below). The excess of bait hybrid protein
decreases the background expression of weak and irrelevant
interactions common to these kinases (refer to FIG. 6). This
enables the successful selection, screening and discovery of their
respective interactions with activators and scaffolds from a random
cDNA library. By muting the background signal, many irrelevant
interactions are reduced or eliminated which otherwise would
interfere with timely and cost-effective analysis of these
screening results.
2TABLE 2 Low-sensitivity Screen with Pelle as bait (promiscuous
bait) Total False Bait Estradiol Dexamethasone Positives Positives
Pelle 10.sup.-9M 10.sup.-7M 5-25 0-50%* Pelle 10.sup.-10M
10.sup.-6M 10-50 50-90% Pelle 10.sup.-11M 10.sup.-5M 100-500
90-95%** Pelle 10.sup.-12M 10.sup.-4M 10,000 >99% *Results are
those obtained uniquely in the present example and invention
involving promiscuous bait and employing the improved, continuously
adjustable system calibrated to low sensitivity in order to
decrease false positives. **Results are those comparable to ones
characteristically obtained using a standard nonadjustable
two-hybrid system with no difference in expression between bait and
library/prey hybrid proteins.
[0059] Table 2 shows utilizing the novel adjustable yeast
interaction hybrid system introduced and described as a more
preferred embodiment in the paragraphs above, a standard
interaction assay on yeast medium is modified to contain various
concentrations of steroid substances as shown in Table 2. The
readout is total positives comprising the number of colonies
surviving selection for interaction. The false positives are
colonies containing proteins not interacting with the bait Pelle as
determined by separate in vivo or in vitro confirmation assays,
including genetic analysis and immunoprecipitation. By comparing
the first and third lines of Table 2, it is evident that a maximum
of only 13 false positives are obtained in the adjusted low
sensitivity screen (see line 1), whereas there are at least 90
false positives observed under screening conditions where there is
no difference in expression between bait and library/prey hybrid
proteins (see line 3). There is no observed decrease in sensitivity
to true positives (data not shown). Note: There is no difference
between lem1-1 and wild-type yeast with regard to the response of
the estrogen class of steroids (Kralli et al., 1995).
Example 3
Interaction Hybrid System Adjusted to High Sensitivity for Poor
Quality Bait
[0060] If high sensitivity is desired, as in the screening of the
mammalian baits Interleukin-1 receptor, or its Drosophila homolog
Toll, or its plant or mammalian homologues, minimal expression of
the bait hybrid protein is achieved using 10.sup.-9M estradiol.
High relative expression of the library/prey hybrid protein is
achieved using 10.sup.-5M dexamethasone. The excess of library/prey
hybrid protein apparently drives the weak interaction equilibrium
toward forming heterodimers; more of the limiting bait hybrid
proteins are occupied at a given time by interaction with the
abundant library/prey hybrid proteins (FIG. 6). The system thereby
detects the weak (high Kd) interaction of these receptors with
their cytoskeletal adapter and cytoplasmic proteins.
3TABLE 3 High sensitivity Screen with toll Receptor intracellular
domain as bait Total False Bait Estradiol Dexamethasone Positives
Positives Toll 10.sup.-9M 10.sup.-7M 0 n/a Toll 10.sup.-10M
10.sup.-6M 0 n/a Toll 10.sup.-11M 10.sup.-5M 0-5 0-50%* Toll
10.sup.-12M 10.sup.-4M 10-30 0-50%** *Results are those comparable
to ones characteristically obtained using a standard nonadjustable
two-hybrid system with no difference in expression between bait and
library/prey hybrid proteins. **Results are those obtained uniquely
in the present example and invention involving poor quality bait
and employing the improved, continuously adjustable system
calibrated to high sensitivity in order to increase total
positives. n/a: denotes "not applicable" due to no colonies
surviving selection.
[0061] Table 3 shows using the same modified interaction hybrid
system as described above under Example 2, a standard interaction
assay on yeast medium is modified to contain various concentrations
of steroid substances. The readout is again total positives,
comprising the number of colonies surviving selection for
interaction. The false positives are colonies containing prey
hybrid proteins not likely to interact with the bait Toll based on
DNA sequence analysis. By comparing the third and fourth lines of
Table 3, it is evident that very few positives result from screens
equivalent to the nonadjustable two-hybrid assays characteristic of
the prior art (see line 3), whereas, there is a significant
increase in positives obtained from the uniquely adjustable high
sensitivity screen of the present example (line 4). There is no
observed increase in non-specific interactions for this
example.
[0062] Summarizing Examples 2 and 3, modulation, including
continuous and dose-responsive modulation of the expression of bait
and library/prey hybrid proteins, enables the present, novel
interaction hybrid system to detect both weak and strong
interactions without the necessity of changing the reporters within
the system itself. Strong interactions are not confounded by
background levels, and weak interactions are not missed entirely,
as characteristically occurs when using standard, nonadjustable
interaction hybrid systems.
Example 4
Advantages of Applying the Approach of Example 1 to Expedite the
Discovery of Novel Interactors with the Promiscuous Bait, Human
Irak1 Kinase
[0063] Example 4 demonstrates the markedly improved effectiveness
and efficiency of detecting known, functionally relevant and novel
interactions with promiscuous bait, including but not limited to
human Irak1 kinase bait. Two parallel sets of screens using Irak1
as bait, are initiated with either the Roger Brent LexA
nonadjustable yeast interaction trap (Gyuris et al., 1993) and with
the present adjustable interaction hybrid system. Using lymphocyte
cDNA libraries constructed for each system, 107 possible
interactions are placed under selection. Positives are yeast
colonies surviving selection and therefore containing putative
proteins that would interact with Irak1. Results of the Brent
system are ca. 5000 total positives, only 960 of which can be
accommodated for further analysis based on the practical
limitations of time and cost. By comparison, using the present
adjustable system and simultaneously screening at 5 levels of
sensitivity, 207 yeast colonies are selected as putative positives
at low sensitivity, all of which can be accommodated for further
analysis.
[0064] Upon analysis of the 960 colonies chosen for workup from the
Brent system, 48 represent multiple hits for two separate unique
proteins known to interact with Irak1. All other putative positives
are random and unrelated proteins designated as false positives. By
comparison, upon analysis of all of the 207 colonies passing
selection from the present adjustable system, 102 represent the
same two unique known proteins, and importantly, two additional
positives represent a single unknown protein presumed to be rare in
the cDNA library.
[0065] Screening and initial analysis using equivalent personnel
and materials requires 4 months using the Brent system, but only
4.5 weeks using the present adjustable system. Additionally, based
on materials cost alone, the present system affords a ca. five-fold
reduction in analytical costs. A parallel five-fold reduction of
personnel costs are also achieved based on the reduction in
technologist's time required for analytical steps.
[0066] Of greatest benefit, is the elimination of random weak
interactions, enabling detection of the rare and potentially
valuable unknown protein that may be involved in signal
transduction of inflammatory signal downstream of the Interleukin 1
receptor.
Example 5
Advantages of Applying the Approach of Example 2 to Expedite the
Discovery of Novel Interactors using a Poor Quality Bait
[0067] Example 5 elucidates the markedly improved effectiveness of
detecting novel interactions with the poor quality bait, human Fc
gamma receptor 1 intracellular domain (Fc gamma R1). Two parallel
sets of screens using Fc gamma R1 as bait, are initiated with the
Roger Brent LexA nonadjustable yeast interaction trap (Gyuris et
al., 1993) in comparison with the present adjustable interaction
hybrid system. Using lymphocyte cDNA libraries constructed for each
system, 108 possible interactions are placed under selection for
the standard Brent interaction system and 107 interactions are
placed under selection for the present adjustable system. Positives
are yeast colonies surviving selection and therefore containing
putative proteins that would interact with Fc gamma R1. Results of
the Brent system are zero positives, allowing no possibility of
further analysis. By comparison, using the present adjustable
system and simultaneously screening at 5 levels of sensitivity, 15
yeast colonies are selected as putative positives at highest
sensitivity only, all of which importantly are amenable to further
analysis. All 15 positives represent copies of a single putative
protein interactor, which are candidates for further biochemical
and genetic analysis as important modulators of B lymphocyte
activation.
Example 6
Commercial and Scientific Relevance
[0068] The present adjustable system provides a markedly improved
means to obtain cloned DNA sequences together with their
corresponding protein structural and functional information for new
and known proteins of known and novel functions that can serve as
candidate drugs and drug targets. Iterative use of this system
enables the improved and accelerated elucidation of entire
signaling pathways linking the cell membrane to the nucleus for use
in all scientifically and commercially relevant DNA-based
organisms. The increased effectiveness and efficiency of screening
for protein-protein and other relevant interactions exhibited by
the present adjustable interaction hybrid system is potentially
widely applicable to enable a markedly increased volume of screens
per unit time and cost, as well as mass screening entailing a
markedly reduced analytical load. This results in many fewer
biologically irrelevant interactors, but retains and increases
valuable and biologically important interactors. In turn, this
benefits scientific and commercial developments in the fields of
medicine, pharmaceutical and biopharmaceutical discovery,
agribusiness and bioinformatics, among others. Application of this
improved, novel technology can potentially markedly enhance the
bioinformation of cellular signaling pathways, knowledge of which
is becoming essential to the rational development of drugs,
antibiotics, biopharmaceuticals, diagnostics, medical interventions
and agricultural products, as well as the enhanced elucidation of
gene-based disease mechanisms. Hence, this technology potentially
provides extended benefits to diverse activities, which utilize
leading-edge interaction hybrid systems both academically and
commercially. These activities include the elucidation of genomic
functional pathways, the rapid correlation of such information with
gene sequencing and induced genomic (e.g., RNA expression) assays,
and acceleration of the commercial discovery and development of
numerous practical genomic products and future applications.
Example 7
Application to Other Molecular Genetic Detection Systems
[0069] The general principle of a reporter having a threshold of
detection given a constant level of expression, transcription, or
presence for the interactors in question implies the obvious
extension of the improvements described herein to all in vivo
molecular genetic detection systems. This system would be expected
to function identically whether the interactors are proteins, RNA,
DNA, carbohydrates, small molecules, drugs, or other potential
biological interactors. Extension is also obvious whether the
detection of an interaction occurs in the nucleus, in the
cytoplasm, within the membranes, or at the membrane of the cell.
Finally, the same modifications can be applied to prokaryotic as
well as other eukaryotic organisms, including mammalian cell-based
two-hybrid systems.
[0070] Citations in the following list of References are
incorporated in pertinent part by reference herein for the reasons
cited in the text.
* * * * *