US 5,965,368 A · Reverse two-hybrid systems

Abstract

Disclosed are methods for identifying molecular interactions (e.g., protein/protein, protein/DNA, protein/RNA, or RNA/RNA interactions). All of the methods within the invention employ counterselection and at least two hybrid molecules. Molecules which interact reconstitute a transcription factor and direct expression of a reporter gene, the expression of which is then assayed. Also disclosed are genetic constructs which are useful in practicing the methods of the invention.

Claims105

Dependent claims

A method for determining whether a first test protein interacts with a second test protein, said method comprising:

(b) providing a second population of mating competent cells, wherein a plurality of the cells of said second population contain:
(i) a second counterselectable reporter gene operably linked to a second DNA-binding-protein recognition site;
and
(ii) a second fusion gene which expresses a second hybrid protein, said second hybrid protein comprising said second test protein covalently bonded to a DNA-binding moiety which specificallly binds to said DNA-binding-protein recognition site;
(b) providing a second population of mating competent cells, wherein a plurality of the cells of said second population contain:
(i) a second counterselectable reporter gene operably linked to a second DNA-binding-protein recognition site;
and
(ii) a second fusion gene which expresses a second hybrid protein, said hybrid protein comprising said second test protein covalently bonded to a gene activating moiety;
(c) maintaining said first and said second populations of mating competent cells, independently, under conditions such that expression of said counterselectable reporter genes inhibits the growth of said cells;
(d) mixing said first and said second populations of mating competent cells under conditions conducive to formation of mated cells;
and
(e) detecting expression of a reporter gene as a measure of the ability of said first test protein to interact with said second test protein, wherein said reporter gene is said first or said second reporter gene or another reporter gene included in said first or said second mating competent cells or said mated cells, and is operably linked to either said first or second DNA-binding-protein recognition sites.

9 more elements

A method for determining whether a first test protein interacts with a second test protein and does not interact with a third test protein, said method comprising:

(a) providing a cell which contains:
(i) a first fusion gene which expresses a first hybrid protein, said first hybrid protein comprising said first test protein covalently bonded to a gene activating moiety;
(ii) a reporter gene operably linked to a first DNA-binding-protein recognition site;
(iii) a second fusion gene which expresses a second hybrid protein, said second hybrid protein comprising said second test protein covalently bonded to a first DNA-binding moiety which specifically binds to said first DNA-binding-protein recognition site and which does not specifically bind to a second DNA-binding-protein recognition site;
(iv) a counterselectable reporter gene operably linked to said second DNA-binding-protein recognition site;
and
(v) a third fusion gene which expresses a third hybrid protein, said third hybrid protein comprising said third test protein covalently bonded to a second DNA-binding-moiety which specifically binds to said second DNA-binding-protein recognition site and which does not bind to said first DNA-binding-protein recognition site;
(b) maintaining said cell under conditions such that expression of said reporter gene does not inhibit growth of said cell and expression of said counterselectable reporter gene inhibits growth of said cell;
and
(c) detecting growth of said cell and expression of said reporter gene as a measure of the ability of said first test protein to interact with said second test protein and the inability of said first test protein to interact with said third test protein.

7 more elements

A method for determining whether a first test RNA molecule interacts with a test protein, said method comprising:

(a) providing a first population of mating competent cells, wherein a plurality of the cells of said population contain:
(i) a first selectable/counterselectable reporter gene operably linked to a first DNA-binding-protein recognition site;
(ii) a first fusion gene which expresses a first hybrid RNA molecule, said RNA molecule comprising said test RNA molecule covalently bonded to a first non-random RNA molecule;
and
(iii) a second fusion gene which expresses a first hybrid protein, said first hybrid protein comprising a DNA-binding moiety which specifically binds to said DNA-binding-protein recognition site, said DNA-binding moiety being covalently bonded to an RNA-binding moiety, wherein said RNA-binding moiety specifically binds to said non-random RNA molecule;
(b) providing a second population of mating competent cells, wherein a plurality of the cells of said population contain:
(i) a second selectable/counterselectable reporter gene operably linked to a second DNA-binding-protein recognition site;
and
(ii) a third fusion gene which expresses said test protein covalently bonded to a gene activating moiety;
and
(c) maintaining said first and said second populations of mating competent cells, independently, under conditions such that expression of said selectable/counterselectable reporter genes inhibits growth of the cells of said populations;
(d) mixing said first and said second populations of mating competent cells under conditions conducive to formation of mated cells;
and
(e) detecting expression of said selectable/counterselectable reporter genes as a measure of the ability of said test RNA molecule to interact with said test protein.

11 more elements

A method for determining whether a first test RNA molecule interacts with a second test RNA molecule, said method comprising:

(a) providing a first population of mating competent cells, wherein a plurality of the cells of said population contain:
(i) a first selectable/counterselectable reporter gene operably linked to a first DNA-binding-protein recognition site;
(ii) a first fusion gene which expresses a first hybrid RNA molecule, wherein said first hybrid RNA molecule comprises said first test RNA molecule covalently bonded to a first non-random RNA molecule;
and
(iii) a second fusion gene which expresses a first hybrid protein, said first hybrid protein comprising a DNA-binding moiety which specifically binds to said DNA-binding-protein recognition site, said DNA-binding moiety being covalently bonded to a first RNA-binding moiety which specifically binds to said first non-random RNA molecule;
(b) providing a second population of mating competent cells, wherein a plurality of the cells of said population contain:
(i) a second selectable/counterselectable reporter gene operably linked to a second DNA-binding-protein recognition site;
(ii) a third fusion gene which expresses a second hybrid RNA molecule wherein said second hybrid RNA molecule comprises said second test RNA molecule covalently bonded to a second non-random RNA molecule;
and
(iii) a fourth fusion gene which expresses a gene activating moiety covalently bonded to a second RNA-binding moiety which specifically binds to said second non-random RNA molecule;
and
(c) maintaining said first and said second populations of mating competent cells, independently, under conditions such that expression of said selectable/counterselectable reporter genes inhibits growth of said cells;
(d) mixing said first and said second populations of mating competent cells under conditions conducive to formation of mated cells;
and
(e) detecting expression of said selectable/counterselectable reporter genes as a measure of the ability of said first test RNA molecule to interact with said second test RNA molecule.

12 more elements

A method for determining whether a candidate mutated reference protein interacts with a test protein, said method comprising:

(a) providing a cell containing:
(i) a counterselectable reporter gene operably linked to a DNA-binding-protein recognition site;
(ii) a selectable reporter gene operably linked to a DNA-binding-protein recognition site;
(iii) a first fusion gene expressing a first hybrid protein, said first hybrid protein comprising said test protein;
and
(iv) a second fusion gene expressing a second hybrid protein, said second hybrid protein comprising said candidate mutated reference protein, wherein said candidate protein is encoded within a nucleic acid library of mutant alleles of the gene encoding said reference protein, and
wherein one of said first and said second hybrid proteins further comprises a DNA-binding moiety which specifically binds to said DNA-binding-protein recognition site, and the other of said first and said second hybrid proteins further comprises a gene activating moiety;
(b) maintaining said cell under conditions such that expression of said counterselectable reporter gene at a level equal to or greater than the level of expression obtained with said reference protein inhibits growth of said cell, and such that expression of said counterselectable reporter gene at a level less than the level of expression obtained with said reference protein does not inhibit growth of said cell;
and
(c) in a separate step, maintaining said cell under conditions such that expression of said counterselectable reporter gene does not inhibit growth of said cell, and detecting expression of said selectable reporter gene as a measure of the ability of said test protein to interact with said candidate mutated reference protein.

7 more elements

A method for identifying a conditional mutant of a reference protein with decreased ability to interact with a second protein under a first set of conditions and which interacts with said second protein under a second set of conditions, said method comprising:

(a) providing a cell containing:
(i) a counterselectable reporter gene operably linked to a DNA-binding-protein recognition site;
(ii) a selectable reporter gene operably linked to a DNA-binding-protein recognition site;
(iii) a first fusion gene expressing a first hybrid protein, said first hybrid protein comprising a candidate mutated reference protein, wherein said candidate protein is encoded within a nucleic acid library of mutant alleles of the gene encoding said reference protein;
and
(iv) a second fusion gene expressing a second hybrid protein, said second hybrid protein comprising said second protein, wherein:
one of said first or said second hybrid proteins comprises a DNA-binding moiety which specifically binds to said DNA-binding-protein recognition site, and
the other of said first or said second hybrid proteins comprises a gene activating moiety;
(b) maintaining said cell under conditions in which expression of said counterselectable reporter gene at a level equal to or greater than the level of expression obtained with said reference protein inhibits growth of said cell, and such that expression of said counterselectable reporter gene at a level less than the level of expression obtained with said reference protein does not inhibit growth of said cell;
(c) in a separate step, maintaining said cell under conditions such that expression of said counterselectable reporter gene does not inhibit growth of said cell, and detecting expression of said selectable reporter gene as a measure of the ability of said candidate mutant protein to interact with said second protein;
and
(d) in a separate step, maintaining the cells under conditions identical to those in step (c) except for one parameter, and detecting expression of said selectable reporter gene as a measure of the ability of said candidate mutant protein to interact with said second protein, said expression of said selectable reporter gene under step (c) conditions but not under step (d) conditions being indicative of said conditional mutant.

9 more elements

A method for identifying compensatory mutations in a first and a second reference protein which allow a first and a second mutant reference protein to interact with each other but not with said second and said first reference proteins, respectively, said method comprising:

(a) providing a first population of mating competent cells, wherein a plurality of the cells of said population contain:
(i) a first counterselectable reporter gene operably linked to a DNA-binding-protein recognition site;
(ii) a first selectable reporter gene operably linked to a DNA-binding-protein recognition site;
(iii) a first fusion gene which expresses a first hybrid protein, said first hybrid protein comprising said first candidate mutant protein covalently bonded to a gene activating moiety, wherein said first candidate mutant protein is encoded within a nucleic acid library of mutant alleles of said first reference protein;
and
(iv) a plasmid containing a first counterselectable marker, and a second fusion gene which expresses a second hybrid protein, said hybrid protein comprising said second reference protein covalently bonded to a DNA-binding moiety;
(b) providing a second population of mating competent cells, wherein a plurality of the cells of said population contain:
(i) a second counterselectable reporter gene operably linked to a DNA-binding-protein recognition site;
(ii) a second selectable reporter gene operably linked to a DNA-binding-protein recognition site;
(iii) a third fusion gene which expresses a third hybrid protein, said third hybrid protein comprising said second candidate mutant reference protein covalently bonded to a DNA-binding moiety, wherein said second test protein is encoded within a nucleic acid library of mutant alleles of said second reference protein;
and
(iv) a plasmid containing a second counterselectable marker, and a fourth fusion gene which expresses a fourth hybrid protein, said hybrid protein comprising said first reference protein covalently bonded to a gene activating moiety;
(c) maintaining said first and said second populations of mating competent cells, independently, under conditions such that expression of said counterselectable reporter genes at a level equal to or greater than the level of expression obtained with said first and second reference proteins inhibits growth of said cells;
(d) maintaining said first and said second populations of mating competent cells under conditions such that expression of said counterselectable marker inhibits growth of said cells;
(e) maintaining said first and said second populations of mating competent cells under conditions conducive to formation of mated cells;
(f) detecting expression of said selectable reporter genes as a measure of the ability of said first and said second candidate mutant proteins to interact with each other and not with said second and said first reference proteins.

13 more elements

Dependencies are inferred from the claim text.

Facts

Application

08/959,536Filed 24 Oct 1997 · continuation

Priority

11 Apr 1995

Granted

12 Oct 1999

Term ends

Not estimatedThe source records don’t support an estimate.

Classification12 codes

G01N 33/5023
For testing non-proliferative effects: on expression patterns
C12N 15/1055
Isolating an individual clone by screening libraries: protein x Protein interaction, e.g. two hybrid selection
C12Q 1/6897
Involving nucleic acids: involving reporter genes operably linked to promoters
G01N 33/5008
Involving human or animal cells: for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics

Abstract

Claims105

Dependent claims

A method for determining whether a first test protein interacts with a second test protein, said method comprising:

(b) providing a second population of mating competent cells, wherein a plurality of the cells of said second population contain:
(i) a second counterselectable reporter gene operably linked to a second DNA-binding-protein recognition site;
and
(ii) a second fusion gene which expresses a second hybrid protein, said second hybrid protein comprising said second test protein covalently bonded to a DNA-binding moiety which specificallly binds to said DNA-binding-protein recognition site;
(b) providing a second population of mating competent cells, wherein a plurality of the cells of said second population contain:
(i) a second counterselectable reporter gene operably linked to a second DNA-binding-protein recognition site;
and
(ii) a second fusion gene which expresses a second hybrid protein, said hybrid protein comprising said second test protein covalently bonded to a gene activating moiety;
(c) maintaining said first and said second populations of mating competent cells, independently, under conditions such that expression of said counterselectable reporter genes inhibits the growth of said cells;
(d) mixing said first and said second populations of mating competent cells under conditions conducive to formation of mated cells;
and
(e) detecting expression of a reporter gene as a measure of the ability of said first test protein to interact with said second test protein, wherein said reporter gene is said first or said second reporter gene or another reporter gene included in said first or said second mating competent cells or said mated cells, and is operably linked to either said first or second DNA-binding-protein recognition sites.

9 more elements

A method for determining whether a first test protein interacts with a second test protein and does not interact with a third test protein, said method comprising:

(a) providing a cell which contains:
(i) a first fusion gene which expresses a first hybrid protein, said first hybrid protein comprising said first test protein covalently bonded to a gene activating moiety;
(ii) a reporter gene operably linked to a first DNA-binding-protein recognition site;
(iii) a second fusion gene which expresses a second hybrid protein, said second hybrid protein comprising said second test protein covalently bonded to a first DNA-binding moiety which specifically binds to said first DNA-binding-protein recognition site and which does not specifically bind to a second DNA-binding-protein recognition site;
(iv) a counterselectable reporter gene operably linked to said second DNA-binding-protein recognition site;
and
(v) a third fusion gene which expresses a third hybrid protein, said third hybrid protein comprising said third test protein covalently bonded to a second DNA-binding-moiety which specifically binds to said second DNA-binding-protein recognition site and which does not bind to said first DNA-binding-protein recognition site;
(b) maintaining said cell under conditions such that expression of said reporter gene does not inhibit growth of said cell and expression of said counterselectable reporter gene inhibits growth of said cell;
and
(c) detecting growth of said cell and expression of said reporter gene as a measure of the ability of said first test protein to interact with said second test protein and the inability of said first test protein to interact with said third test protein.

7 more elements

A method for determining whether a first test RNA molecule interacts with a test protein, said method comprising:

(a) providing a first population of mating competent cells, wherein a plurality of the cells of said population contain:
(i) a first selectable/counterselectable reporter gene operably linked to a first DNA-binding-protein recognition site;
(ii) a first fusion gene which expresses a first hybrid RNA molecule, said RNA molecule comprising said test RNA molecule covalently bonded to a first non-random RNA molecule;
and
(iii) a second fusion gene which expresses a first hybrid protein, said first hybrid protein comprising a DNA-binding moiety which specifically binds to said DNA-binding-protein recognition site, said DNA-binding moiety being covalently bonded to an RNA-binding moiety, wherein said RNA-binding moiety specifically binds to said non-random RNA molecule;
(b) providing a second population of mating competent cells, wherein a plurality of the cells of said population contain:
(i) a second selectable/counterselectable reporter gene operably linked to a second DNA-binding-protein recognition site;
and
(ii) a third fusion gene which expresses said test protein covalently bonded to a gene activating moiety;
and
(c) maintaining said first and said second populations of mating competent cells, independently, under conditions such that expression of said selectable/counterselectable reporter genes inhibits growth of the cells of said populations;
(d) mixing said first and said second populations of mating competent cells under conditions conducive to formation of mated cells;
and
(e) detecting expression of said selectable/counterselectable reporter genes as a measure of the ability of said test RNA molecule to interact with said test protein.

11 more elements

A method for determining whether a first test RNA molecule interacts with a second test RNA molecule, said method comprising:

(a) providing a first population of mating competent cells, wherein a plurality of the cells of said population contain:
(i) a first selectable/counterselectable reporter gene operably linked to a first DNA-binding-protein recognition site;
(ii) a first fusion gene which expresses a first hybrid RNA molecule, wherein said first hybrid RNA molecule comprises said first test RNA molecule covalently bonded to a first non-random RNA molecule;
and
(iii) a second fusion gene which expresses a first hybrid protein, said first hybrid protein comprising a DNA-binding moiety which specifically binds to said DNA-binding-protein recognition site, said DNA-binding moiety being covalently bonded to a first RNA-binding moiety which specifically binds to said first non-random RNA molecule;
(b) providing a second population of mating competent cells, wherein a plurality of the cells of said population contain:
(i) a second selectable/counterselectable reporter gene operably linked to a second DNA-binding-protein recognition site;
(ii) a third fusion gene which expresses a second hybrid RNA molecule wherein said second hybrid RNA molecule comprises said second test RNA molecule covalently bonded to a second non-random RNA molecule;
and
(iii) a fourth fusion gene which expresses a gene activating moiety covalently bonded to a second RNA-binding moiety which specifically binds to said second non-random RNA molecule;
and
(c) maintaining said first and said second populations of mating competent cells, independently, under conditions such that expression of said selectable/counterselectable reporter genes inhibits growth of said cells;
(d) mixing said first and said second populations of mating competent cells under conditions conducive to formation of mated cells;
and
(e) detecting expression of said selectable/counterselectable reporter genes as a measure of the ability of said first test RNA molecule to interact with said second test RNA molecule.

12 more elements

A method for determining whether a candidate mutated reference protein interacts with a test protein, said method comprising:

(a) providing a cell containing:
(i) a counterselectable reporter gene operably linked to a DNA-binding-protein recognition site;
(ii) a selectable reporter gene operably linked to a DNA-binding-protein recognition site;
(iii) a first fusion gene expressing a first hybrid protein, said first hybrid protein comprising said test protein;
and
(iv) a second fusion gene expressing a second hybrid protein, said second hybrid protein comprising said candidate mutated reference protein, wherein said candidate protein is encoded within a nucleic acid library of mutant alleles of the gene encoding said reference protein, and
wherein one of said first and said second hybrid proteins further comprises a DNA-binding moiety which specifically binds to said DNA-binding-protein recognition site, and the other of said first and said second hybrid proteins further comprises a gene activating moiety;
(b) maintaining said cell under conditions such that expression of said counterselectable reporter gene at a level equal to or greater than the level of expression obtained with said reference protein inhibits growth of said cell, and such that expression of said counterselectable reporter gene at a level less than the level of expression obtained with said reference protein does not inhibit growth of said cell;
and
(c) in a separate step, maintaining said cell under conditions such that expression of said counterselectable reporter gene does not inhibit growth of said cell, and detecting expression of said selectable reporter gene as a measure of the ability of said test protein to interact with said candidate mutated reference protein.

7 more elements

(a) providing a cell containing:
(i) a counterselectable reporter gene operably linked to a DNA-binding-protein recognition site;
(ii) a selectable reporter gene operably linked to a DNA-binding-protein recognition site;
(iii) a first fusion gene expressing a first hybrid protein, said first hybrid protein comprising a candidate mutated reference protein, wherein said candidate protein is encoded within a nucleic acid library of mutant alleles of the gene encoding said reference protein;
and
(iv) a second fusion gene expressing a second hybrid protein, said second hybrid protein comprising said second protein, wherein:
one of said first or said second hybrid proteins comprises a DNA-binding moiety which specifically binds to said DNA-binding-protein recognition site, and
the other of said first or said second hybrid proteins comprises a gene activating moiety;
(b) maintaining said cell under conditions in which expression of said counterselectable reporter gene at a level equal to or greater than the level of expression obtained with said reference protein inhibits growth of said cell, and such that expression of said counterselectable reporter gene at a level less than the level of expression obtained with said reference protein does not inhibit growth of said cell;
(c) in a separate step, maintaining said cell under conditions such that expression of said counterselectable reporter gene does not inhibit growth of said cell, and detecting expression of said selectable reporter gene as a measure of the ability of said candidate mutant protein to interact with said second protein;
and
(d) in a separate step, maintaining the cells under conditions identical to those in step (c) except for one parameter, and detecting expression of said selectable reporter gene as a measure of the ability of said candidate mutant protein to interact with said second protein, said expression of said selectable reporter gene under step (c) conditions but not under step (d) conditions being indicative of said conditional mutant.

9 more elements

(a) providing a first population of mating competent cells, wherein a plurality of the cells of said population contain:
(i) a first counterselectable reporter gene operably linked to a DNA-binding-protein recognition site;
(ii) a first selectable reporter gene operably linked to a DNA-binding-protein recognition site;
(iii) a first fusion gene which expresses a first hybrid protein, said first hybrid protein comprising said first candidate mutant protein covalently bonded to a gene activating moiety, wherein said first candidate mutant protein is encoded within a nucleic acid library of mutant alleles of said first reference protein;
and
(iv) a plasmid containing a first counterselectable marker, and a second fusion gene which expresses a second hybrid protein, said hybrid protein comprising said second reference protein covalently bonded to a DNA-binding moiety;
(b) providing a second population of mating competent cells, wherein a plurality of the cells of said population contain:
(i) a second counterselectable reporter gene operably linked to a DNA-binding-protein recognition site;
(ii) a second selectable reporter gene operably linked to a DNA-binding-protein recognition site;
(iii) a third fusion gene which expresses a third hybrid protein, said third hybrid protein comprising said second candidate mutant reference protein covalently bonded to a DNA-binding moiety, wherein said second test protein is encoded within a nucleic acid library of mutant alleles of said second reference protein;
and
(iv) a plasmid containing a second counterselectable marker, and a fourth fusion gene which expresses a fourth hybrid protein, said hybrid protein comprising said first reference protein covalently bonded to a gene activating moiety;
(c) maintaining said first and said second populations of mating competent cells, independently, under conditions such that expression of said counterselectable reporter genes at a level equal to or greater than the level of expression obtained with said first and second reference proteins inhibits growth of said cells;
(d) maintaining said first and said second populations of mating competent cells under conditions such that expression of said counterselectable marker inhibits growth of said cells;
(e) maintaining said first and said second populations of mating competent cells under conditions conducive to formation of mated cells;
(f) detecting expression of said selectable reporter genes as a measure of the ability of said first and said second candidate mutant proteins to interact with each other and not with said second and said first reference proteins.

13 more elements

Dependencies are inferred from the claim text.

Reverse two-hybrid systems

Life of this patent

Abstract

Claims105

Reverse two-hybrid systems

Life of this patent

Abstract

Claims105