A Two-hybrid Dual Bait System to Discriminate Specificity of Protein Interactions

Biological regulatory systems require the specific organization of proteins into multicomponent complexes. Two hybrid systems have been used to identify novel components of signaling networks based on interactions with defined partner proteins. An important issue in the use of two-hybrid systems has been the degree to which interacting proteins distinguish their biological partner from evolutionarily conserved related proteins and the degree to which observed interactions are specific. We adapted the basic two-hybrid strategy to create a novel dual bait system designed to allow single-step screening of libraries for proteins that interact with protein 1 of interest, fused to DNA binding domain A (LexA), but do not interact with protein 2, fused to DNA binding domain B (λ cI). Using the selective interactions of Ras and Krev-1(Rap1A) with Raf, RalGDS, and Krit1 as a model, we systematically compared LexA- and cI-fused baits and reporters. The LexA and cI baitr reporter systems are well matched for level of bait expression and sensitivity range for interaction detection and allow effective isolation of specifically interacting protein pairs against a nonspecific background. These reagents should prove useful to refine the selectivity of library screens, to reduce the isolation of false positives in such screens, and to perform directed analyses of sequence elements governing the interaction of a single protein with multiple partners. Biological regulatory systems require the specific organization of proteins into multicomponent complexes. Two hybrid systems have been used to identify novel components of signaling networks based on interactions with defined partner proteins. An important issue in the use of two-hybrid systems has been the degree to which interacting proteins distinguish their biological partner from evolutionarily conserved related proteins and the degree to which observed interactions are specific. We adapted the basic two-hybrid strategy to create a novel dual bait system designed to allow single-step screening of libraries for proteins that interact with protein 1 of interest, fused to DNA binding domain A (LexA), but do not interact with protein 2, fused to DNA binding domain B (λ cI). Using the selective interactions of Ras and Krev-1(Rap1A) with Raf, RalGDS, and Krit1 as a model, we systematically compared LexA- and cI-fused baits and reporters. The LexA and cI baitr reporter systems are well matched for level of bait expression and sensitivity range for interaction detection and allow effective isolation of specifically interacting protein pairs against a nonspecific background. These reagents should prove useful to refine the selectivity of library screens, to reduce the isolation of false positives in such screens, and to perform directed analyses of sequence elements governing the interaction of a single protein with multiple partners. To understand and manipulate the function of a particular protein of biological interest, it is generally useful to identify other proteins with which it associates. Although identification of protein interactions initially proceeded almost solely by technically difficult biochemical methods, in recent years yeast two-hybrid systems (1Fields S. Song O. Nature. 1989; 340: 245-246Crossref PubMed Scopus (4822) Google Scholar) have developed as a powerful genetic tool to rapidly select previously uncharacterized proteins that specifically interact with a target protein of interest from a suitable library (2Chien C.T. Bartel P.L. Sternglanz R. Fields S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9578-9582Crossref PubMed Scopus (1222) Google Scholar, 3Durfee T. Becherer K. Chen P.L. Yeh S.H. Yang Y. Kilburn A.E. Lee W.H. Elledge S.J. Genes Dev. 1993; 7: 555-569Crossref PubMed Scopus (1297) Google Scholar, 4Gyuris J. Golemis E.A. Chertkov H. Brent R. Cell. 1993; 75: 791-803Abstract Full Text PDF PubMed Scopus (1319) Google Scholar, 5Vojtek A.B. Hollenberg S.M. Cooper J.A. Cell. 1993; 74: 205-214Abstract Full Text PDF PubMed Scopus (1656) Google Scholar). In this schema, a protein of interest is synthesized in yeast as a fusion to a DNA binding domain (DBD), 1The abbreviations used are: DBD, DNA binding domain; X-Gal, 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside; PCR, polymerase chain reaction; AD, activation domain; X-Gluc, 5-bromo-4-chloro-3-indolylβ-d-glucuronic acid, sodium salt; Magenta-Gal, 5-bromo-6-chloro3-indolylβ-d-galactopyranoside1The abbreviations used are: DBD, DNA binding domain; X-Gal, 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside; PCR, polymerase chain reaction; AD, activation domain; X-Gluc, 5-bromo-4-chloro-3-indolylβ-d-glucuronic acid, sodium salt; Magenta-Gal, 5-bromo-6-chloro3-indolylβ-d-galactopyranoside which is typically the bacterial repressor protein LexA or the amino-terminal end of the yeast transcription factor GAL4. Interaction of this DBD protein fusion (a “bait”) with a transcriptional activation domain-fused partner protein (either a defined partner or a novel protein screened from a library) allows the activation of reporter genes (lacZ, HIS3, LEU2) responsive to the cognate DBD. More recently, interest has focused on expanding the utility of two-hybrid systems to enable the detection of interactions between proteins and RNA (6SenGupta D.J. Zhang B. Kraemer B. Pochart P. Fields S. Wickens M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8496-8501Crossref PubMed Scopus (435) Google Scholar, 7Wang Z.F. Whitfield M.L. Ingledue III, T.C. Dominski Z. Marzluff W.F. Genes Dev. 1996; 10: 3028-3040Crossref PubMed Scopus (215) Google Scholar), proteins and nonprotein ligands (8Licitra E.J. Liu J.O. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12817-12821Crossref PubMed Scopus (268) Google Scholar), proteins and peptides (9Colas P. Cohen B. Jessen T. Grishina I. McCoy J. Brent R. Nature. 1996; 380: 548-550Crossref PubMed Scopus (375) Google Scholar, 10Yang M. Wu Z. Fields S. Nucleic Acids Res. 1995; 23: 1152-1156Crossref PubMed Scopus (130) Google Scholar), and proteins and multiple partners (11Osborne M.A. Zenner G. Lubinus M. Zhang X. Songyang Z. Cantley L.C. Majerus P. Burn P. Kochan J.P. J. Biol. Chem. 1996; 271: 29271-29278Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar,12Tirode F. Malaguti C. Romero F. Attar R. Camonis J. Egly J.M. J. Biol. Chem. 1997; 272: 22995-22999Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). A second thrust has been to enable whole-genome applications (13Finley R. Brent R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12980-12984Crossref PubMed Scopus (240) Google Scholar, 14Bartel P.L. Roecklein J.A. SenGupta D. Fields S. Nat. Genet. 1996; 12: 72-77Crossref PubMed Scopus (272) Google Scholar, 15Fromont-Racine M. Rain J.-C. Legrain P. Nat. Gen. 1997; 16: 277-282Crossref PubMed Scopus (702) Google Scholar), leading to the generation of maps of protein interaction networks with the potential to complement the vast resource of sequence information now being developed as part of the Genome Project. Finally, there has been interest in developing two-hybrid systems as tools in high throughput drug discovery screening strategies to identify agents regulating the activity of biologically important target proteins.As two-hybrid technologies have evolved to more complex applications, a question of mounting importance has been the degree to which library screens performed in these systems yield partners specific for the utilized bait, as opposed to proteins of broad interaction capability (“false positives”). Although the large number of published two-hybrid papers indicates that many specific partners are obtained, a recent survey has suggested that the majority of library screens isolate at least some cDNAs that are nonspecific. 2I. Serebriiskii and E. A. Golemis, E. A. (1996)http://www.fccc.edu/ research/labs/golemis/interactiontrapinwork.html. 2I. Serebriiskii and E. A. Golemis, E. A. (1996)http://www.fccc.edu/ research/labs/golemis/interactiontrapinwork.html. As a related issue, it is clear that many biologically important proteins are organized into families of evolutionarily related members that conserve substantial sequence similarity (e.g. Refs. 17Alt F.W. DePinho R. Zimmerman K. LeGouy E. Hatton K. Ferrier P. Tesfaye A. Yancopoulos G. Nisen P. Cold Spring Harbor Symp. Quant. Biol. 1986; 51: 931-941Crossref PubMed Google Scholar, 18Meyerson M. Enders G.H. Wu C.-L. Su L.-K. Gorka C. Nelson C. Harlow E. Tsai L.-H. EMBO J. 1992; 11: 2909-2917Crossref PubMed Scopus (783) Google Scholar, 19Shalloway D. S.J. Biol. 1997; 7: Full Text PDF PubMed Scopus Google Scholar). the degree to which two-hybrid systems isolate proteins partners specific for that interact generally with a of protein is Although two-hybrid systems allow of specific from false positives or positives use of of performed to a E.A. Serebriiskii I. J. Brent R. in Scholar), these are many this it has been of interest to a to such this we a novel of the two-hybrid system the dual bait system for false or nonspecific interactions in a single and allows the of a protein interaction with related or partners in a single which should useful for a of high throughput and We that these reagents are effective at of interacting proteins against of interacting the of for large this we the and of novel dual bait reagents that used to the interaction of a protein with partners in a single yeast The cI system utilized in the yeast and is to function with a sensitivity range with the LexA system in the interaction their In a system the interaction of the related Ras and with their partners and Krit1 and their partner RalGDS, the dual bait system In to in interactions in of yeast to selective the observed is to allow the isolation of yeast specifically interacting protein pairs against a vast of These the that these reagents useful in library screening and The reagents the of screens in a single with of positives activation of and a second activation and against these have the potential to two-hybrid system to of biological have the use of baits to identify that interactions of activation domain-fused protein with of partners C. T. J. 1997; PubMed Google Scholar, R. M. Genes Dev. 1996; 10: PubMed Scopus (240) Google Scholar, Brent R. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar). In of a second system by of the used for the bait, the screening to the in these novel baits the screening The of is in the performed as their use the of the from specific which reporters. The dual bait reagents used for and have been used to identify in which reduce interaction for of the or J. E. A. Golemis, and I. Finally, there is that these reagents the to perform library screens in with complex as in recent library screens the and the baits have specific partners. J. Y. Z. Zhang and E. Golemis, a more basic the system allows in the by to perform multiple two-hybrid screens the dual bait selectivity of to perform library or the in a screening and of such is for in the of a dual bait of a previously library is (e.g. a LexA bait but a cI bait the that are for at least bait useful information the of the library utilized of Finally, it has previously been that some proteins of interest for library screening perform with particular fusion are utilized as LexA but not as or J. Brent R. Golemis E.A. Cell. Biol. 1995; PubMed Scopus Google In specific DNA binding domain are a bait of interest as a LexA and as a cI fusion and with the bait in to of interacting dual bait reagents are the interaction of two-hybrid system J. Golemis E.A. Chertkov H. Brent R. Cell. 1993; 75: 791-803Abstract Full Text PDF PubMed Scopus (1319) Google Scholar). cI and LexA are in and M. Nature. PubMed Scopus Google Scholar) and use related amino-terminal to with from to for LexA in J. Brent R. Golemis E.A. Cell. Biol. 1995; PubMed Scopus Google to cI Cell. Full Text PDF PubMed Scopus Google Scholar). of these many it is clear that the LexA and cI systems are well in to use in the interaction the cI of this system have been to of the two-hybrid the reporter system developed in this a DNA binding domain reporter genes and not in use in other system (2Chien C.T. Bartel P.L. Sternglanz R. Fields S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9578-9582Crossref PubMed Scopus (1222) Google Scholar, 3Durfee T. Becherer K. Chen P.L. Yeh S.H. Yang Y. Kilburn A.E. Lee W.H. Elledge S.J. Genes Dev. 1993; 7: 555-569Crossref PubMed Scopus (1297) Google Scholar, 5Vojtek A.B. Hollenberg S.M. Cooper J.A. Cell. 1993; 74: 205-214Abstract Full Text PDF PubMed Scopus (1656) Google Scholar), the system A. E. H. Elledge S.J. M. Cell. Biol. 1997; PubMed Scopus Google Scholar). these reagents with of the other screening systems on two-hybrid in the of the this the that with of the library a single bait used to identify a or a transcriptional the potential of interacting proteins An useful of the is that it is on the as the reporter used in two-hybrid to of Finally, the dual bait reagents have been for use in with LexA have been previously to and sensitivity J. Brent R. Golemis E.A. Cell. Biol. 1995; PubMed Scopus Google Scholar), of two-hybrid systems a and useful that should to to understand complex interactions on the To understand and manipulate the function of a particular protein of biological interest, it is generally useful to identify other proteins with which it associates. Although identification of protein interactions initially proceeded almost solely by technically difficult biochemical methods, in recent years yeast two-hybrid systems (1Fields S. Song O. Nature. 1989; 340: 245-246Crossref PubMed Scopus (4822) Google Scholar) have developed as a powerful genetic tool to rapidly select previously uncharacterized proteins that specifically interact with a target protein of interest from a suitable library (2Chien C.T. Bartel P.L. Sternglanz R. Fields S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9578-9582Crossref PubMed Scopus (1222) Google Scholar, 3Durfee T. Becherer K. Chen P.L. Yeh S.H. Yang Y. Kilburn A.E. Lee W.H. Elledge S.J. Genes Dev. 1993; 7: 555-569Crossref PubMed Scopus (1297) Google Scholar, 4Gyuris J. Golemis E.A. Chertkov H. Brent R. Cell. 1993; 75: 791-803Abstract Full Text PDF PubMed Scopus (1319) Google Scholar, 5Vojtek A.B. Hollenberg S.M. Cooper J.A. Cell. 1993; 74: 205-214Abstract Full Text PDF PubMed Scopus (1656) Google Scholar). In this schema, a protein of interest is synthesized in yeast as a fusion to a DNA binding domain (DBD), 1The abbreviations used are: DBD, DNA binding domain; X-Gal, 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside; PCR, polymerase chain reaction; AD, activation domain; X-Gluc, 5-bromo-4-chloro-3-indolylβ-d-glucuronic acid, sodium salt; Magenta-Gal, 5-bromo-6-chloro3-indolylβ-d-galactopyranoside1The abbreviations used are: DBD, DNA binding domain; X-Gal, 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside; PCR, polymerase chain reaction; AD, activation domain; X-Gluc, 5-bromo-4-chloro-3-indolylβ-d-glucuronic acid, sodium salt; Magenta-Gal, 5-bromo-6-chloro3-indolylβ-d-galactopyranoside which is typically the bacterial repressor protein LexA or the amino-terminal end of the yeast transcription factor GAL4. Interaction of this DBD protein fusion (a “bait”) with a transcriptional activation domain-fused partner protein (either a defined partner or a novel protein screened from a library) allows the activation of reporter genes (lacZ, HIS3, LEU2) responsive to the cognate DBD. More recently, interest has focused on expanding the utility of two-hybrid systems to enable the detection of interactions between proteins and RNA (6SenGupta D.J. Zhang B. Kraemer B. Pochart P. Fields S. Wickens M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8496-8501Crossref PubMed Scopus (435) Google Scholar, 7Wang Z.F. Whitfield M.L. Ingledue III, T.C. Dominski Z. Marzluff W.F. Genes Dev. 1996; 10: 3028-3040Crossref PubMed Scopus (215) Google Scholar), proteins and nonprotein ligands (8Licitra E.J. Liu J.O. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12817-12821Crossref PubMed Scopus (268) Google Scholar), proteins and peptides (9Colas P. Cohen B. Jessen T. Grishina I. McCoy J. Brent R. Nature. 1996; 380: 548-550Crossref PubMed Scopus (375) Google Scholar, 10Yang M. Wu Z. Fields S. Nucleic Acids Res. 1995; 23: 1152-1156Crossref PubMed Scopus (130) Google Scholar), and proteins and multiple partners (11Osborne M.A. Zenner G. Lubinus M. Zhang X. Songyang Z. Cantley L.C. Majerus P. Burn P. Kochan J.P. J. Biol. Chem. 1996; 271: 29271-29278Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar,12Tirode F. Malaguti C. Romero F. Attar R. Camonis J. Egly J.M. J. Biol. Chem. 1997; 272: 22995-22999Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). A second thrust has been to enable whole-genome applications (13Finley R. Brent R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12980-12984Crossref PubMed Scopus (240) Google Scholar, 14Bartel P.L. Roecklein J.A. SenGupta D. Fields S. Nat. Genet. 1996; 12: 72-77Crossref PubMed Scopus (272) Google Scholar, 15Fromont-Racine M. Rain J.-C. Legrain P. Nat. Gen. 1997; 16: 277-282Crossref PubMed Scopus (702) Google Scholar), leading to the generation of maps of protein interaction networks with the potential to complement the vast resource of sequence information now being developed as part of the Genome Project. Finally, there has been interest in developing two-hybrid systems as tools in high throughput drug discovery screening strategies to identify agents regulating the activity of biologically important target proteins. As two-hybrid technologies have evolved to more complex applications, a question of mounting importance has been the degree to which library screens performed in these systems yield partners specific for the utilized bait, as opposed to proteins of broad interaction capability (“false positives”). Although the large number of published two-hybrid papers indicates that many specific partners are obtained, a recent survey has suggested that the majority of library screens isolate at least some cDNAs that are nonspecific. 2I. Serebriiskii and E. A. Golemis, E. A. (1996)http://www.fccc.edu/ research/labs/golemis/interactiontrapinwork.html. 2I. Serebriiskii and E. A. Golemis, E. A. (1996)http://www.fccc.edu/ research/labs/golemis/interactiontrapinwork.html. As a related issue, it is clear that many biologically important proteins are organized into families of evolutionarily related members that conserve substantial sequence similarity (e.g. Refs. 17Alt F.W. DePinho R. Zimmerman K. LeGouy E. Hatton K. Ferrier P. Tesfaye A. Yancopoulos G. Nisen P. Cold Spring Harbor Symp. Quant. Biol. 1986; 51: 931-941Crossref PubMed Google Scholar, 18Meyerson M. Enders G.H. Wu C.-L. Su L.-K. Gorka C. Nelson C. Harlow E. Tsai L.-H. EMBO J. 1992; 11: 2909-2917Crossref PubMed Scopus (783) Google Scholar, 19Shalloway D. S.J. Biol. 1997; 7: Full Text PDF PubMed Scopus Google Scholar). the degree to which two-hybrid systems isolate proteins partners specific for that interact generally with a of protein is Although two-hybrid systems allow of specific from false positives or positives use of of performed to a E.A. Serebriiskii I. J. Brent R. in Scholar), these are many this it has been of interest to a to such In this we a novel of the two-hybrid system the dual bait system for false or nonspecific interactions in a single and allows the of a protein interaction with related or partners in a single which should useful for a of high throughput and We that these reagents are effective at of interacting proteins against of interacting the of for large this we the and of novel dual bait reagents that used to the interaction of a protein with partners in a single yeast The cI system utilized in the yeast and is to function with a sensitivity range with the LexA system in the interaction their In a system the interaction of the related Ras and with their partners and Krit1 and their partner RalGDS, the dual bait system In to in interactions in of yeast to selective the observed is to allow the isolation of yeast specifically interacting protein pairs against a vast of These the that these reagents useful in library screening and The reagents the of screens in a single with of positives activation of and a second activation and against these have the potential to two-hybrid system to of biological have the use of baits to identify that interactions of activation domain-fused protein with of partners C. T. J. 1997; PubMed Google Scholar, R. M. Genes Dev. 1996; 10: PubMed Scopus (240) Google Scholar, Brent R. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar). In of a second system by of the used for the bait, the screening to the in these novel baits the screening The of is in the performed as their use the of the from specific which reporters. The dual bait reagents used for and have been used to identify in which reduce interaction for of the or J. E. A. Golemis, and I. Finally, there is that these reagents the to perform library screens in with complex as in recent library screens the and the baits have specific partners. J. Y. Z. Zhang and E. Golemis, a more basic the system allows in the by to perform multiple two-hybrid screens the dual bait selectivity of to perform library or the in a screening and of such is for in the of a dual bait of a previously library is (e.g. a LexA bait but a cI bait the that are for at least bait useful information the of the library utilized of Finally, it has previously been that some proteins of interest for library screening perform with particular fusion are utilized as LexA but not as or J. Brent R. Golemis E.A. Cell. Biol. 1995; PubMed Scopus Google In specific DNA binding domain are a bait of interest as a LexA and as a cI fusion and with the bait in to of interacting dual bait reagents are the interaction of two-hybrid system J. Golemis E.A. Chertkov H. Brent R. Cell. 1993; 75: 791-803Abstract Full Text PDF PubMed Scopus (1319) Google Scholar). cI and LexA are in and M. Nature. PubMed Scopus Google Scholar) and use related amino-terminal to with from to for LexA in J. Brent R. Golemis E.A. Cell. Biol. 1995; PubMed Scopus Google to cI Cell. Full Text PDF PubMed Scopus Google Scholar). of these many it is clear that the LexA and cI systems are well in to use in the interaction the cI of this system have been to of the two-hybrid the reporter system developed in this a DNA binding domain reporter genes and not in use in other system (2Chien C.T. Bartel P.L. Sternglanz R. Fields S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9578-9582Crossref PubMed Scopus (1222) Google Scholar, 3Durfee T. Becherer K. Chen P.L. Yeh S.H. Yang Y. Kilburn A.E. Lee W.H. Elledge S.J. Genes Dev. 1993; 7: 555-569Crossref PubMed Scopus (1297) Google Scholar, 5Vojtek A.B. Hollenberg S.M. Cooper J.A. Cell. 1993; 74: 205-214Abstract Full Text PDF PubMed Scopus (1656) Google Scholar), the system A. E. H. Elledge S.J. M. Cell. Biol. 1997; PubMed Scopus Google Scholar). these reagents with of the other screening systems on two-hybrid in the of the this the that with of the library a single bait used to identify a or a transcriptional the potential of interacting proteins An useful of the is that it is on the as the reporter used in two-hybrid to of Finally, the dual bait reagents have been for use in with LexA have been previously to and sensitivity J. Brent R. Golemis E.A. Cell. Biol. 1995; PubMed Scopus Google Scholar), of two-hybrid systems a and useful that should to to understand complex interactions on the In this we the and of novel dual bait reagents that used to the interaction of a protein with partners in a single yeast The cI system utilized in the yeast and is to function with a sensitivity range with the LexA system in the interaction their In a system the interaction of the related Ras and with their partners and Krit1 and their partner RalGDS, the dual bait system In to in interactions in of yeast to selective the observed is to allow the isolation of yeast specifically interacting protein pairs against a vast of These the that these reagents useful in library screening and The reagents the of screens in a single with of positives activation of and a second activation and against these have the potential to two-hybrid system to of biological have the use of baits to identify that interactions of activation domain-fused protein with of partners C. T. J. 1997; PubMed Google Scholar, R. M. Genes Dev. 1996; 10: PubMed Scopus (240) Google Scholar, Brent R. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar). In of a second system by of the used for the bait, the screening to the in these novel baits the screening The of is in the performed as their use the of the from specific which reporters. The dual bait reagents used for and have been used to identify in which reduce interaction for of the or J. E. A. Golemis, and I. Finally, there is that these reagents the to perform library screens in with complex as in recent library screens the and the baits have specific partners. J. Y. Z. Zhang and E. Golemis, a more basic the system allows in the by to perform multiple two-hybrid screens the dual bait selectivity of to perform library or the in a screening and of such is for in the of a dual bait of a previously library is (e.g. a LexA bait but a cI bait the that are for at least bait useful information the of the library utilized of Finally, it has previously been that some proteins of interest for library screening perform with particular fusion are utilized as LexA but not as or J. Brent R. Golemis E.A. Cell. Biol. 1995; PubMed Scopus Google In specific DNA binding domain are a bait of interest as a LexA and as a cI fusion and with the bait in to of interacting partners. The dual bait reagents are the interaction of two-hybrid system J. Golemis E.A. Chertkov H. Brent R. Cell. 1993; 75: 791-803Abstract Full Text PDF PubMed Scopus (1319) Google Scholar). cI and LexA are in and M. Nature. PubMed Scopus Google Scholar) and use related amino-terminal to with from to for LexA in J. Brent R. Golemis E.A. Cell. Biol. 1995; PubMed Scopus Google to cI Cell. Full Text PDF PubMed Scopus Google Scholar). of these many it is clear that the LexA and cI systems are well in to use in the interaction the cI of this system have been to of the two-hybrid the reporter system developed in this a DNA binding domain reporter genes and not in use in other system (2Chien C.T. Bartel P.L. Sternglanz R. Fields S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9578-9582Crossref PubMed Scopus (1222) Google Scholar, 3Durfee T. Becherer K. Chen P.L. Yeh S.H. Yang Y. Kilburn A.E. Lee W.H. Elledge S.J. Genes Dev. 1993; 7: 555-569Crossref PubMed Scopus (1297) Google Scholar, 5Vojtek A.B. Hollenberg S.M. Cooper J.A. Cell. 1993; 74: 205-214Abstract Full Text PDF PubMed Scopus (1656) Google Scholar), the system A. E. H. Elledge S.J. M. Cell. Biol. 1997; PubMed Scopus Google Scholar). these reagents with of the other screening systems on two-hybrid in the of the this the that with of the library a single bait used to identify a or a transcriptional the potential of interacting proteins An useful of the is that it is on the as the reporter used in two-hybrid to of Finally, the dual bait reagents have been for use in with LexA have been previously to and sensitivity J. Brent R. Golemis E.A. Cell. Biol. 1995; PubMed Scopus Google Scholar), of two-hybrid systems a and useful that should to to understand complex interactions on the We and for their of for for yeast and and for their in the dual bait We are to and for of the