FB2026_02 , released June 18, 2026

CV ID	CV term	Synonyms	Definition
MI:0018	two hybrid	2 hybrid [EXACT] 2-hybrid [EXACT] 2H [EXACT] 2h [EXACT] classical two hybrid [EXACT] Gal4 transcription regeneration [EXACT] two-hybrid [EXACT] Y-2H Y2H [EXACT] yeast two hybrid [EXACT]	The classical two-hybrid system is a method that uses transcriptional activity as a measure of protein-protein interaction. It relies on the modular nature of many site-specific transcriptional activators (GAL 4) , which consist of a DNA-binding domain and a transcriptional activation domain. The DNA-binding domain serves to target the activator to the specific genes that will be expressed, and the activation domain contacts other proteins of the transcriptional machinery to enable transcription to occur. The two-hybrid system is based on the observation that the two domains of the activator need to be non-covalently brought together by the interaction of any two proteins. The application of this system requires the expression of two hybrid. Generally this assay is performed in yeast cell, but it can also be carried out in other organism. The bait protein is fused to the DNA binding molecule, the prey to the transcriptional activator.
MI:0398	two hybrid pooling approach	two hybrid pooling [EXACT]	In the pooling strategy sets of either both bait and prey hybrid vectors are mated or, more commonly, individual baits are mated against pools of preys. This approach required cloning baits and preys into both two-hybrid vectors, followed by pooling sets of transformants. The positive double hybrid clones are the interacting partners. The pooling of both baits and prey molecules is now a rarely used technique as the pooling of baits often leads to misleading results.
MI:0397	two hybrid array		Two-hybrid screening can be done in a colony array format, in which each colony expresses a defined pair of proteins. Because the particular protein pair expressed in each colony is defined by its position in the array, positive signals identify interacting proteins without further characterization, thus obviating the need for DNA purification and sequencing. The interrogation of a two-hybrid colony array usually involves a mating strategy in which every DNA binding domain hybrid (the bait) is tested against all activation domain hybrids (the preys) in a grid pattern. Arrays usually use full-length open reading frames.
MI:1113	two hybrid bait and prey pooling approach		def
MI:1111	two hybrid bait or prey pooling approach		Individual baits are mated against pools of preys, or pools of baits are mated against individual preys. This approach required cloning baits and preys into both two-hybrid vectors, followed by pooling sets of transformants. The positive double hybrid clones are the interacting partners.
MI:0399	two hybrid fragment pooling approach	2h fragment pooling [EXACT]	Individual baits are mated against pools of random fragmented preys. The usage of degenerated fragment allows identification of the minimal protein region required for the interaction. Since multiple clones that encode overlapping regions of protein are often identified, the minimal domain for interaction may be readily apparent from the initial screen.
MI:1112	two hybrid prey pooling approach		Individual baits are mated against pools of preys. This approach required cloning baits and preys into both two-hybrid vectors, followed by pooling sets of transformants. The positive double hybrid clones are the interacting partners.
MI:0655	lambda repressor two hybrid	BacterioMatch [EXACT] lambda two hybrid [EXACT]	A protein of interest (the bait) is fused to the full-length bacteriophage lambda repressor protein (lambdacI, 237 amino acids), containing the amino terminal DNA-binding domain and the carboxylterminal dimerization domain. The corresponding target (prey) protein is fused to the N-terminal domain of the alfa-subunit of RNA polymerase (248 amino acids). The bait is tethered to the lambda operator sequence upstream of the reporter promoter through the DNA-binding domain of lambdacI. When the bait and prey interact, they recruit and stabilize the binding of RNA polymerase at the promoter and activate the transcription of the HIS3 reporter gene. Due to the tendency of both the lambda repressor protein and the N-terminal domain of the alfa-subunit of RNA polymerase to dimerize, this system might not be optimal for the analysis of proteins that self-associate unless their interaction with other protein partners depends on the oligomerization.
MI:1320	membrane yeast two hybrid	MYTH [EXACT]	Traditional yeast two hybrid assays are not suitable for the analysis of membrane proteins, as they require the interactions to occur in the nucleus. Methods have been specifically developed to assay for interactions of membrane proteins with both cytosolic or membrane-bound partners.
MI:0726	reverse two hybrid		Yeast strains are generated in which expression of DB-X/AD-Y or DBPX hybrid proteins is toxic under particular conditions (negative selection). Under these conditions, dissociation of an interaction should provide a selective advantage thereby facilitating detection: a few growing yeast colonies in which DB-X/AD-Y (or DBPX/binding site) fail to interact should be identified among many nongrowing colonies containing interacting DB-X/AD-Y or DBPX/binding site.
MI:1356	validated two hybrid		The protein pairs, often initially identified by a separate 2-hybrid screening methodology, are subjected to a rigorous re-analysis which may include independent re-screening of the entire search space, retesting the assay in different strain backgrounds, and multiple retesting of identified protein pairs reversing bait-prey orientations. Orthogonal data from other experimental and bioinformatic approaches may also be used to support the identification of the final high-confidence protein pairs but the primary means of selection must be experimentally based. This method would be expected to identify protein pairs with a higher degree of confidence than any single protein complementation technique.
MI:0014	adenylate cyclase complementation	adenylate cyclase [EXACT] bacterial two-hybrid [EXACT]	Adenylate cyclase is encoded by the cyaA gene and contains a catalytic domain which can be proteolytically cleaved into two complementary fragments, T25 and T18, which remain associated in the presence of calmodulin in a fully active ternary complex. In the absence of calmodulin, the mixture of the two fragments does not exhibit detectable activity, suggesting that the two fragments do not associate. When expressed in an adenylate cyclase-deficient E. coli strain (E. coli lacks calmodulin or calmodulin-related proteins), the T25 and T18 fragments fused to putative interacting proteins are brought into close association which result in cAMP synthesis. The level of reconstructed adenylate cyclase can be estimated by monitoring the expression of a cAMP dependent reporter gene. The T25 tagged protein is generally regarded as the bait, the T18 as the prey.
MI:0728	gal4 vp16 complementation	gal4 vp16 complement [EXACT] karyoplasmic interaction ion strategy [EXACT] KISS [EXACT] mammalian two hybrid [EXACT]	A chimeric protein consisting of the GAL4 DNA-binding domain (aa 1-147 of GAL4) and a transcriptional activation domain from the herpes simplex virus protein VP16 (either aa 411-490 or aa 411-455) can specifically activate transcription of a reporter gene located downstream ofGAL4 DNA binding sites and the E1B minimal promoter. Similarly, two chimeric proteins, one encoding a chimeric GAL4 protein and the other encoding a chimeric VP16 protein, can activate the reporter gene, if the domains fused to the GAL4 and VP16 sequences can complex with appropriate conformation. However, if the domains fused to the GAL4 and VP16 sequences do not interact specifically to form a + complex that reconstitutes GAL4 function, the reporter gene cannot be activated.
MI:1321	ire1 reconstruction	endoplasmic reticulum membrane yeast-two hybrid ER-MYTH	Upon interaction of N-terminal generated Ire1p-fusions, the Ire1p kinase domains oligomerize, transphosphorylate and activate their C-terminal RNAseL domains, which specifically splice the mRNA of a transcriptional activator, Hac1. The expression of the mature form of Hac1p leads to the interaction-specific expression of a reporter. Specifically designed to identify interactors of proteins found in the endoplasmic reticulum.
MI:0112	ubiquitin reconstruction	membrane yeast two-hybrid myth split-ubiquitin ub reconstruction [EXACT]	The split-ubiquitin system provides a method for examining the interactions of membrane proteins. In the split-ubiquitin system, two integral membrane proteins to be studied are fused to two different ubiquitin moieties: a C-terminal ubiquitin moiety (\"Cub\", residues 35-76) and an N-terminal ubiquitin moiety (\"Nub\", residues 1-34). These fused proteins are called the bait and prey, respectively. In addition to being fused to an integral membrane protein, the Cub moiety is also fused to a transcription factor that can be cleaved off by ubiquitin specific proteases. Upon bait-prey interaction, Nub and Cub-moieties assemble, reconstituting the split-ubiquitin. The reconstituted split-ubiquitin molecule is recognized by ubiquitin specific proteases, which cleave off the reporter protein, allowing it to induce the transcription of reporter genes.

CV ID

CV term

Synonyms

Definition

MI:0018

2 hybrid [EXACT]
2-hybrid [EXACT]
2H [EXACT]
2h [EXACT]
classical two hybrid [EXACT]
Gal4 transcription regeneration [EXACT]
two-hybrid [EXACT]
Y-2H
Y2H [EXACT]
yeast two hybrid [EXACT]

The classical two-hybrid system is a method that uses transcriptional activity as a measure of protein-protein interaction. It relies on the modular nature of many site-specific transcriptional activators (GAL 4) , which consist of a DNA-binding domain and a transcriptional activation domain. The DNA-binding domain serves to target the activator to the specific genes that will be expressed, and the activation domain contacts other proteins of the transcriptional machinery to enable transcription to occur. The two-hybrid system is based on the observation that the two domains of the activator need to be non-covalently brought together by the interaction of any two proteins. The application of this system requires the expression of two hybrid. Generally this assay is performed in yeast cell, but it can also be carried out in other organism. The bait protein is fused to the DNA binding molecule, the prey to the transcriptional activator.

MI:0398

two hybrid pooling approach

two hybrid pooling [EXACT]

In the pooling strategy sets of either both bait and prey hybrid vectors are mated or, more commonly, individual baits are mated against pools of preys. This approach required cloning baits and preys into both two-hybrid vectors, followed by pooling sets of transformants. The positive double hybrid clones are the interacting partners. The pooling of both baits and prey molecules is now a rarely used technique as the pooling of baits often leads to misleading results.

MI:0397

two hybrid array

Two-hybrid screening can be done in a colony array format, in which each colony expresses a defined pair of proteins. Because the particular protein pair expressed in each colony is defined by its position in the array, positive signals identify interacting proteins without further characterization, thus obviating the need for DNA purification and sequencing. The interrogation of a two-hybrid colony array usually involves a mating strategy in which every DNA binding domain hybrid (the bait) is tested against all activation domain hybrids (the preys) in a grid pattern. Arrays usually use full-length open reading frames.

MI:1113

two hybrid bait and prey pooling approach

def

MI:1111

two hybrid bait or prey pooling approach

Individual baits are mated against pools of preys, or pools of baits are mated against individual preys. This approach required cloning baits and preys into both two-hybrid vectors, followed by pooling sets of transformants. The positive double hybrid clones are the interacting partners.

MI:0399

two hybrid fragment pooling approach

2h fragment pooling [EXACT]

Individual baits are mated against pools of random fragmented preys. The usage of degenerated fragment allows identification of the minimal protein region required for the interaction. Since multiple clones that encode overlapping regions of protein are often identified, the minimal domain for interaction may be readily apparent from the initial screen.

MI:1112

two hybrid prey pooling approach

Individual baits are mated against pools of preys. This approach required cloning baits and preys into both two-hybrid vectors, followed by pooling sets of transformants. The positive double hybrid clones are the interacting partners.

MI:0655

lambda repressor two hybrid

BacterioMatch [EXACT]
lambda two hybrid [EXACT]

A protein of interest (the bait) is fused to the full-length bacteriophage lambda repressor protein (lambdacI, 237 amino acids), containing the amino terminal DNA-binding domain and the carboxylterminal dimerization domain. The corresponding target (prey) protein is fused to the N-terminal domain of the alfa-subunit of RNA polymerase (248 amino acids). The bait is tethered to the lambda operator sequence upstream of the reporter promoter through the DNA-binding domain of lambdacI. When the bait and prey interact, they recruit and stabilize the binding of RNA polymerase at the promoter and activate the transcription of the HIS3 reporter gene. Due to the tendency of both the lambda repressor protein and the N-terminal domain of the alfa-subunit of RNA polymerase to dimerize, this system might not be optimal for the analysis of proteins that self-associate unless their interaction with other protein partners depends on the oligomerization.

MI:1320

membrane yeast two hybrid

MYTH [EXACT]

Traditional yeast two hybrid assays are not suitable for the analysis of membrane proteins, as they require the interactions to occur in the nucleus. Methods have been specifically developed to assay for interactions of membrane proteins with both cytosolic or membrane-bound partners.

MI:0726

reverse two hybrid

Yeast strains are generated in which expression of DB-X/AD-Y or DBPX hybrid proteins is toxic under particular conditions (negative selection). Under these conditions, dissociation of an interaction should provide a selective advantage thereby facilitating detection: a few growing yeast colonies in which DB-X/AD-Y (or DBPX/binding site) fail to interact should be identified among many nongrowing colonies containing interacting DB-X/AD-Y or DBPX/binding site.

MI:1356

validated two hybrid

The protein pairs, often initially identified by a separate 2-hybrid screening methodology, are subjected to a rigorous re-analysis which may include independent re-screening of the entire search space, retesting the assay in different strain backgrounds, and multiple retesting of identified protein pairs reversing bait-prey orientations. Orthogonal data from other experimental and bioinformatic approaches may also be used to support the identification of the final high-confidence protein pairs but the primary means of selection must be experimentally based. This method would be expected to identify protein pairs with a higher degree of confidence than any single protein complementation technique.

MI:0014

adenylate cyclase complementation

adenylate cyclase [EXACT]
bacterial two-hybrid [EXACT]

Adenylate cyclase is encoded by the cyaA gene and contains a catalytic domain which can be proteolytically cleaved into two complementary fragments, T25 and T18, which remain associated in the presence of calmodulin in a fully active ternary complex. In the absence of calmodulin, the mixture of the two fragments does not exhibit detectable activity, suggesting that the two fragments do not associate. When expressed in an adenylate cyclase-deficient E. coli strain (E. coli lacks calmodulin or calmodulin-related proteins), the T25 and T18 fragments fused to putative interacting proteins are brought into close association which result in cAMP synthesis. The level of reconstructed adenylate cyclase can be estimated by monitoring the expression of a cAMP dependent reporter gene. The T25 tagged protein is generally regarded as the bait, the T18 as the prey.

MI:0728

gal4 vp16 complementation

gal4 vp16 complement [EXACT]
karyoplasmic interaction ion strategy [EXACT]
KISS [EXACT]
mammalian two hybrid [EXACT]

A chimeric protein consisting of the GAL4 DNA-binding domain (aa 1-147 of GAL4) and a transcriptional activation domain from the herpes simplex virus protein VP16 (either aa 411-490 or aa 411-455) can specifically activate transcription of a reporter gene located downstream ofGAL4 DNA binding sites and the E1B minimal promoter. Similarly, two chimeric proteins, one encoding a chimeric GAL4 protein and the other encoding a chimeric VP16 protein, can activate the reporter gene, if the domains fused to the GAL4 and VP16 sequences can complex with appropriate conformation. However, if the domains fused to the GAL4 and VP16 sequences do not interact specifically to form a + complex that reconstitutes GAL4 function, the reporter gene cannot be activated.

MI:1321

ire1 reconstruction

endoplasmic reticulum membrane yeast-two hybrid
ER-MYTH

Upon interaction of N-terminal generated Ire1p-fusions, the Ire1p kinase domains oligomerize, transphosphorylate and activate their C-terminal RNAseL domains, which specifically splice the mRNA of a transcriptional activator, Hac1. The expression of the mature form of Hac1p leads to the interaction-specific expression of a reporter. Specifically designed to identify interactors of proteins found in the endoplasmic reticulum.

MI:0112

ubiquitin reconstruction

membrane yeast two-hybrid
myth
split-ubiquitin
ub reconstruction [EXACT]

The split-ubiquitin system provides a method for examining the interactions of membrane proteins. In the split-ubiquitin system, two integral membrane proteins to be studied are fused to two different ubiquitin moieties: a C-terminal ubiquitin moiety (\"Cub\", residues 35-76) and an N-terminal ubiquitin moiety (\"Nub\", residues 1-34). These fused proteins are called the bait and prey, respectively. In addition to being fused to an integral membrane protein, the Cub moiety is also fused to a transcription factor that can be cleaved off by ubiquitin specific proteases. Upon bait-prey interaction, Nub and Cub-moieties assemble, reconstituting the split-ubiquitin. The reconstituted split-ubiquitin molecule is recognized by ubiquitin specific proteases, which cleave off the reporter protein, allowing it to induce the transcription of reporter genes.