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.

1 - 107 . (Canceled).  
     
     
         108 . A yeast cell comprising: 
 a) a nucleotide sequence encoding a first heterologous fusion protein comprising a first peptide of a known peptide binding pair that bind through extracellular interaction in their natural environment, or a segment thereof, joined to a transcriptional activation protein DNA binding domain;    b) a nucleotide sequence encoding a second heterologous fusion protein comprising a second peptide of the known peptide binding pair, or a segment thereof, joined to a transcriptional activation protein transcriptional activation domain;    wherein binding of the first peptide or segment thereof and the second peptide or segment thereof reconstitutes a transcriptional activation protein; and    c) a reporter gene activated under positive transcriptional control of the reconstituted transcriptional activation protein, wherein expression of the reporter gene produces a selected phenotype.    
     
     
         109 . The yeast cell of  claim 108  further comprising at least one endogenous nucleotide sequence selected from the group consisting of a nucleotide sequence encoding the transcriptional activation protein DNA binding domain, a nucleotide sequence encoding the transcriptional activation protein transcriptional activation domain, and a nucleotide sequence encoding the reporter gene, wherein at least one of the endogenous nucleotide sequences is inactivated by mutation or deletion.  
     
     
         110 . The yeast cell of  claim 108  wherein the peptide binding pair comprises a ligand and a receptor to which the ligand binds.  
     
     
         111 . The yeast cell of  claim 108  wherein the transcriptional activation protein is Gal4, Gcn4, Hap1, Adr1, Swi5, Ste12, Mcm1, Yap1, Ace1, Ppr1, Arg81, Lac9, Qa1F, VP16, or a mammalian nuclear receptor.  
     
     
         112 . The yeast cell of  claim 108  wherein at least one of the heterologous fusion proteins is expressed from an autonomously-replicating plasmid.  
     
     
         113 . The yeast cell of  claim 108  wherein the DNA binding domain is a heterologous transcriptional activation protein DNA binding domain.  
     
     
         114 . The yeast cell of  claim 108  wherein the reporter gene is selected from the group consisting of lacZ, a gene encoding luciferase, a gene encoding green fluorescent protein, and a gene encoding chloramphenicol acetyltransferase.  
     
     
         115 . The yeast cell of  claim 108  wherein the peptide binding pair is other than an antigen and a corresponding antibody.  
     
     
         116 . The yeast cell of  claim 108  wherein the yeast cell is  Saccharomyces cerevisiae, Schizosaccharomyces pombe , or  Pichia pastoris.    
     
     
         117 . The yeast cell of  claim 108  wherein the yeast cell is  Saccharomyces cerevisiae.    
     
     
         118 . The yeast cell of  claim 112  wherein at least one peptide of the peptide binding pair is selected from the group consisting of a cytokine, an interleukin, a hematopoietic growth factor, insulin, an insulin-like growth factor, a growth hormone, prolactin, an interferon, a growth factor, a ligand for G-protein coupled receptors, a ligand for guanylyl cyclase receptors, a ligand for tyrosine phosphatase receptors, and a ligand for tyrosine kinase receptors.  
     
     
         119 . The yeast cell of  claim 113  wherein the DNA binding protein is selected from the group consisting of a mammalian steroid receptor and bacterial LexA protein.  
     
     
         120 . The yeast cell of  claim 118  wherein the peptide is a growth factor selected from the group consisting of epidermal growth factor, nerve growth factor, leukemia inhibitory factor, fibroblast growth factor, platelet-derived growth factor, vascular endothelial growth factor, tumor necrosis factor, oncostatin M, ciliary neurotrophic factor, erythropoietin, steel factor, placental lactogen, and transforming growth factor β (TGF).  
     
     
         121 . A yeast cell comprising: 
 a) a nucleotide sequence encoding a first heterologous fusion protein comprising a first peptide of a known peptide binding pair, or a segment thereof, wherein the peptide binging pair comprises a ligand and a receptor for the ligand, joined to a transcriptional activation protein DNA binding domain;    b) a nucleotide sequence encoding a second heterologous fusion protein comprising a second peptide of the known peptide binding pair, or a segment thereof, joined to a transcriptional activation protein transcriptional activation domain;    wherein binding of the first peptide or segment thereof and the second peptide or segment thereof reconstitutes a transcriptional activation protein, and    c) a reporter gene activated under positive transcriptional control of the reconstituted transcriptional activation protein, wherein expression of the reporter gene prevents exhibition of a selected phenotype.    
     
     
         122 . The yeast cell of  claim 121  further comprising at least one endogenous nucleotide sequence selected from the group consisting of a nucleotide sequence encoding the transcriptional activation protein DNA binding domain, a nucleotide sequence encoding the transcriptional activation protein transcriptional activation domain, and a nucleotide sequence encoding the reporter gene, wherein at least one of the endogenous nucleotide sequences is inactivated by mutation or deletion.  
     
     
         123 . The yeast cell of  claim 121  wherein the ligand and receptor for the ligand bind through extracellular interaction in their natural environment.  
     
     
         124 . The yeast cell of  claim 121  wherein the transcriptional activation protein is Gal4, Gcn4, Hap1, Adr1, Swi5, Ste12, Mcm1, Yap1, Ace1, Ppr1, Arg81, Lac9, Qa1F, VP16, or a mammalian nuclear receptor.  
     
     
         125 . The yeast cell of  claim 121  wherein at least one of the heterologous fusion proteins is expressed from an autonomously-replicating plasmid.  
     
     
         126 . The yeast cell of  claim 121  wherein at least one peptide of the peptide binding pair is selected from the group consisting of a cytokine, an interleukin, a hematopoietic growth factor, insulin, an insulin-like growth factor, a growth hormone, prolactin, an interferon, a growth factor, a ligand for G-protein coupled receptors, a ligand for guanylyl cyclase receptors, a ligand for tyrosine phosphatase receptors, and a ligand for tyrosine kinase receptors.  
     
     
         127 . The yeast cell of  claim 121  wherein the reporter gene is selected from the group consisting of a gene that prevents growth on cycloheximide and a gene that prevents growth on canavanine.  
     
     
         128 . The yeast cell of  claim 121  wherein the peptide binding pair is other than an antigen and a corresponding antibody.  
     
     
         129 . The yeast cell of  claim 121  wherein the yeast cell is  Saccharomyces cerevisiae, Schizosaccharomyces pombe , or  Pichia pastoris.    
     
     
         130 . The yeast cell of  claim 121  wherein the yeast cell is  Saccharomyces cerevisiae.    
     
     
         131 . The yeast cell of  claim 126  wherein the peptide is a growth factor selected from the group consisting of epidermal growth factor, nerve growth factor, leukemia inhibitory factor, fibroblast growth factor, platelet-derived growth factor, vascular endothelial growth factor, tumor necrosis factor, oncostatin M, ciliary neurotrophic factor, erythropoietin, steel factor, placental lactogen, and TGF.  
     
     
         132 . The yeast cell of  claim 131  wherein the DNA binding domain is a heterologous transcriptional activation protein DNA-binding domain.  
     
     
         133 . The yeast cell of  claim 132  wherein the DNA binding protein is selected from the group consisting of a mammalian steroid receptor and bacterial LexA protein.  
     
     
         134 . A yeast cell comprising: 
 a) a nucleotide sequence encoding a first heterologous fusion protein comprising first peptide of a peptide binding pair, or a segment thereof, joined to a transcriptional activation protein DNA binding domain;    b) a nucleotide sequence encoding a second heterologous fusion protein comprising a second peptide of the peptide binding pair, or a segment thereof, joined to a transcriptional activation protein transcriptional activation domain;    wherein the nucleotide sequence encoding either the first or second heterologous fusion protein is present in an effective copy number of at least 5 copies per yeast cell and the nucleotide sequence encoding the other heterologous fusion protein is present at a copy number of 1 or 2 per yeast cell; and    wherein binding of the first peptide or segment thereof and the second peptide or segment thereof reconstitutes a transcriptional activation protein; and    c) a reporter gene activated under positive transcriptional control of the reconstituted transcriptional activation protein, wherein expression of the reporter gene prevents exhibition of a selected phenotype.    
     
     
         135 . The yeast cell of  claim 134  further comprising at least one endogenous nucleotide sequence selected from the group consisting of a nucleotide sequence encoding the transcriptional activation protein DNA binding domain, a nucleotide sequence encoding the transcriptional activation protein transcriptional activation domain, and a nucleotide sequence encoding the reporter gene, wherein at least one of the endogenous nucleotide sequences is inactivated by reconstitution or deletion.  
     
     
         136 . The yeast cell of  claim 134  wherein the peptide binding pair comprises a ligand and a receptor for the ligand.  
     
     
         137 . The yeast cell of  claim 134  wherein the transcriptional activation protein is Gal4, Gcn4, Hap1, Adr1, Swi5, Ste12, Mcm1, Yap1, Ace1, Ppr1, Arg81, Lac9, Qa1F, VP16, or a mammalian nuclear receptor.  
     
     
         138 . The yeast cell of  claim 134  wherein at least one of the heterologous fusion proteins is expressed from an autonomously-replicating plasmid.  
     
     
         139 . The yeast cell of  claim 134  wherein the DNA binding domain is a heterologous transcriptional activation protein DNA-binding domain.  
     
     
         140 . The yeast cell of  claim 134  wherein the DNA binding protein is selected from the group consisting of a mammalian steroid receptor and bacterial LexA protein.  
     
     
         141 . The yeast cell of  claim 134  wherein the reporter gene is selected from the group consisting of a gene that prevents growth on cycloheximide and a gene that prevents growth on canavanine.  
     
     
         142 . The yeast cell of  claim 134  wherein the peptide binding pair is other than an antigen and a corresponding antibody.  
     
     
         143 . The yeast cell of  claim 134  wherein the yeast cell is  Saccharomyces cerevisiae, Schizosaccharomyces pombe , or  Pichia pastoris.    
     
     
         144 . The yeast cell of  claim 134  wherein the yeast cell is  Saccharomyces cerevisiae.    
     
     
         145 . The yeast cell of  claim 138  wherein at least one peptide of the peptide binding pair is selected from the group consisting of a cytokine, an interleukin, a hematopoietic growth factor, insulin, an insulin-like growth factor, a growth hormone, prolactin, an interferon, a growth factor, a ligand for G-protein coupled receptors, a ligand for guanylyl cyclase receptors, a ligand for tyrosine phosphatase receptors, and a ligand for tyrosine kinase receptors.  
     
     
         146 . The yeast cell of  claim 145  wherein the peptide is a growth factor selected from the group consisting of epidermal growth factor, nerve growth factor, leukemia inhibitory factor, fibroblast growth factor, platelet-derived growth factor, vascular endothelial growth factor, tumor necrosis factor, oncostatin M, ciliary neurotrophic factor, erythropoietin, steel factor, placental lactogen, and TGF.  
     
     
         147 . A yeast cell comprising: 
 a) a nucleotide sequence encoding a first heterologous fusion protein comprising a first peptide of a peptide binding pair, or a segment thereof, wherein the peptide binding pair comprises a ligand and a receptor for the ligand, joined to a transcriptional activation protein DNA binding domain;    b) a nucleotide sequence encoding a second heterologous fusion protein comprising a second peptide of the binding pair, or a segment thereof, joined to a transcriptional activation protein transcriptional activation domain;    wherein binding of the first peptide or segment thereof and the second peptide or segment thereof reconstitutes a transcriptional activation protein; and    c) a reporter gene activated under positive transcriptional control of the reconstituted transcriptional activation protein, wherein when the reporter gene is expressed the yeast cell does not grow on a selective medium.    
     
     
         148 . The yeast cell of  claim 147  further comprising at least one endogenous nucleotide sequence selected from the group consisting of a nucleotide sequence encoding the transcriptional activation protein DNA binding domain, a nucleotide sequence encoding the transcriptional activation protein transcriptional activation domain, and a nucleotide sequence encoding the reporter gene, wherein at least one of the endogenous nucleotide sequences is inactivated by mutation or deletion.  
     
     
         149 . The yeast cell of  claim 147  wherein the ligand and receptor for the ligand bind through extracellular interaction in their natural environment.  
     
     
         150 . The yeast cell of  claim 147  wherein the transcriptional activation protein is Gal4, Gcn4, Hap1, Adr1, Swi5, Ste12, Mcm1, Yap1, Ace1, Ppr1, Arg81, Lac9, Qa1F, VP16, or a mammalian nuclear receptor.  
     
     
         151 . The yeast cell of  claim 147  wherein at least one of the heterologous fusion proteins is expressed from an autonomously-replicating plasmid.  
     
     
         152 . The yeast cell of  claim 147  wherein the DNA binding domain is a heterologous transcriptional activation protein DNA-binding domain.  
     
     
         153 . The yeast cell of  claim 147  wherein the peptide binding pair is other than an antigen and a corresponding antibody.  
     
     
         154 . The yeast cell of  claim 147  wherein the yeast cell is  Saccharomyces cerevisiae, Schizosaccharomyces pombe , or  Pichia pastoris.    
     
     
         155 . The yeast cell of  claim 147  wherein the yeast cell is  Saccharomyces cerevisiae.    
     
     
         156 . The yeast cell of  claim 151  wherein at least one peptide of the peptide binding pair is selected from the group consisting of a cytokine, an interleukin, a hematopoietic growth factor, insulin, an insulin-like growth factor, a growth hormone, prolactin, an interferon, a growth factor, a ligand for G-protein coupled receptors, a ligand for guanylyl cyclase receptors, a ligand for tyrosine phosphatase receptors, and a ligand for tyrosine kinase receptors.  
     
     
         157 . The yeast cell of  claim 152  wherein the DNA binding protein is selected from the group consisting of a mammalian steroid receptor and bacterial LexA protein.  
     
     
         158 . The yeast cell of  claim 156  wherein the peptide is a growth factor selected from the group consisting of epidermal growth factor, nerve growth factor, leukemia inhibitory factor, fibroblast growth factor, platelet-derived growth factor, vascular endothelial growth factor, tumor necrosis factor, oncostatin M, ciliary neurotrophic factor, erythropoietin, steel factor, placental lactogen, and TGF.  
     
     
         159 . A yeast cell comprising: 
 a) a nucleotide sequence encoding a first heterologous fusion protein comprising a first peptide of a peptide binding pair, or a segment thereof, joined to a transcriptional activation protein DNA binding domain;    b) a nucleotide sequence encoding a second heterologous fusion protein comprising a second peptide of the binding pair, or a segment thereof, joined to a transcriptional activation protein transcriptional activation domain;    wherein the nucleotide sequence encoding either the first or second heterologous fusion protein is present in an effective copy number of at least 5 copies per yeast cell and the nucleotide sequence encoding the other heterologous fusion protein is present at a copy number of 1 or 2 per yeast cell; and    wherein binding of the first peptide or segment thereof and the second peptide or segment thereof reconstitutes a transcriptional activation protein; and    c) a reporter gene activated under positive transcriptional control of the reconstituted transcriptional activation protein, wherein when the reporter gene is expressed the yeast cell does not grow on a selective medium.    
     
     
         160 . The yeast cell of  claim 159  further comprising at least one endogenous nucleotide sequence selected from the group consisting of a nucleotide sequence encoding the transcriptional activation protein DNA binding domain, a nucleotide sequence encoding the transcriptional activation protein transcriptional activation domain, and a nucleotide sequence encoding the reporter gene, wherein at least one of the endogenous nucleotide sequences is inactivated by mutation or deletion.  
     
     
         161 . The yeast cell of  claim 159  wherein the peptide binding pair comprises a ligand and a receptor for the ligand.  
     
     
         162 . The yeast cell of  claim 159  wherein the transcriptional activation protein is Gal4, Gcn4, Hap1, Adr1, Swi5, Ste12, Mcm1, Yap1, Ace1, Ppr1, Arg81, Lac9, Qa1F, VP16, or a mammalian nuclear receptor.  
     
     
         163 . The yeast cell of  claim 159  wherein at least one of the heterologous fusion proteins is expressed from an autonomously-replicating plasmid.  
     
     
         164 . The yeast cell of  claim 159  wherein the DNA binding domain is a heterologous transcriptional activation protein DNA-binding domain.  
     
     
         165 . The yeast cell of  claim 159  wherein the DNA binding protein is selected from the group consisting of a mammalian steroid receptor and bacterial LexA protein.  
     
     
         166 . The yeast cell of  claim 159  wherein the peptide binding pair is other than an antigen and a corresponding antibody.  
     
     
         167 . The yeast cell of  claim 159  wherein the yeast cell is  Saccharomyces cerevisiae, Schizosaccharomyces pombe , or  Pichia pastoris.    
     
     
         168 . The yeast cell of  claim 159  wherein the yeast cell is  Saccharomyces cerevisiae.    
     
     
         169 . The yeast cell of  claim 163  wherein at least one peptide of the peptide binding pair is selected from the group consisting of a cytokine, an interleukin, an hematopoietic growth factor, insulin, an insulin-like growth factor, a growth hormone, prolactin, an interferon, a growth factor, a ligand for G-protein coupled receptors, a ligand for guanylyl cyclase receptors, a ligand for tyrosine phosphatase receptors, and a ligand for tyrosine kinase receptors.  
     
     
         170 . The yeast cell of  claim 169  wherein the peptide is a growth factor selected from the group consisting of epidermal growth factor, nerve growth factor, leukemia inhibitory factor, fibroblast growth factor, platelet-derived growth factor, vascular endothelial growth factor, tumor necrosis factor, oncostatin M, ciliary neurotrophic factor, erythropoietin, steel factor, placental lactogen, and TGF.  
     
     
         171 . A yeast cell comprising: 
 a) a nucleotide sequence encoding a first heterologous fusion protein comprising a first peptide of a known peptide binding pair that bind through extracellular interaction in their natural environment, or a segment thereof, joined to a transcriptional activation protein DNA binding domain;    b) a nucleotide sequence encoding a second heterologous fusion protein comprising a second peptide of the known peptide binding pair, or a segment thereof, joined to a transcriptional activation protein transcriptional activation domain;    wherein binding of the first peptide or segment thereof and the second peptide or segment thereof reconstitutes a transcriptional activation protein; and    c) a reporter gene activated under positive transcriptional control of the reconstituted transcriptional activation protein, wherein expression of the reporter gene prevents exhibition of a selected phenotype.    
     
     
         172 . The yeast cell of  claim 171  further comprising at least one endogenous nucleotide sequence selected from the group consisting of a nucleotide sequence encoding the transcriptional activation protein DNA binding domain, a nucleotide sequence encoding the transcriptional activation protein transcriptional activation domain, and a nucleotide sequence encoding the reporter gene, wherein at least one of the endogenous nucleotide sequences is inactivated by mutation or deletion.  
     
     
         173 . The yeast cell of  claim 171  wherein the peptide binding pair comprises a ligand and a receptor for the ligand.  
     
     
         174 . The yeast cell of  claim 171  wherein the transcriptional activation protein is Gal4, Gcn4, Hap1, Adr1, Swi5, Ste12, Mcm1, Yap1, Ace1, Ppr1, Arg81, Lac9, Qa1F, VP16, or a mammalian nuclear receptor.  
     
     
         175 . The yeast cell of  claim 171  wherein at least one of the heterologous fusion proteins is expressed from an autonomously-replicating plasmid.  
     
     
         176 . The yeast cell of  claim 171  wherein at least one peptide of the peptide binding pair is selected from the group consisting of a cytokine, an interleukin, a hematopoietic growth factor, insulin, an insulin-like growth factor, a growth hormone, prolactin, an interferon, a growth factor, a ligand for G-protein coupled receptors, a ligand for guanylyl cyclase receptors, a ligand for tyrosine phosphatase receptors, and a ligand for tyrosine kinase receptors.  
     
     
         177 . The yeast cell of  claim 171  wherein the reporter gene is selected from the group consisting of a gene that prevents growth on cycloheximide and a gene that prevents growth on canavanine.  
     
     
         178 . The yeast cell of  claim 171  wherein the peptide binding pair is other than an antigen and a corresponding antibody.  
     
     
         179 . The yeast cell of  claim 171  wherein the yeast cell is  Saccharomyces cerevisiae, Schizosaccharomyces pombe , or  Pichia pastoris.    
     
     
         180 . The yeast cell of  claim 176  wherein the peptide is a growth factor selected from the group consisting of epidermal growth factor, nerve growth factor, leukemia inhibitory factor, fibroblast growth factor, platelet-derived growth factor, vascular endothelial growth factor, tumor necrosis factor, oncostatin M, ciliary neurotrophic factor, erythropoietin, steel factor, placental lactogen, and TGF.  
     
     
         181 . The yeast cell of  claim 179  wherein the yeast cell is  Saccharomyces cerevisiae.    
     
     
         182 . The yeast cell of  claim 180  wherein the DNA binding domain is a heterologous transcriptional activation protein DNA-binding domain.  
     
     
         183 . The yeast cell of  claim 180  wherein the DNA binding protein is selected from the group consisting of a mammalian steroid receptor and bacterial LexA protein.  
     
     
         184 . A yeast cell comprising: 
 a) a nucleotide sequence encoding a first heterologous fusion protein comprising a first peptide of a peptide binding pair that bind through extracellular interaction in their natural environment, or a segment thereof, joined to a transcriptional activation protein DNA binding domain;    b) a nucleotide sequence encoding a second heterologous fusion protein comprising a second peptide of the binding pair, or a segment thereof, joined to a transcriptional activation protein transcriptional activation domain;    wherein binding of the first peptide or segment thereof and the second peptide or segment thereof reconstitutes a transcriptional activation protein; and    c) a reporter gene activated under positive transcriptional control of the reconstituted transcriptional activation protein, wherein when the reporter gene is expressed the yeast cell does not grow on a selective medium.    
     
     
         185 . The yeast cell of  claim 184  further comprising at least one endogenous nucleotide sequence selected from the group consisting of a nucleotide sequence encoding the transcriptional activation protein DNA binding domain, a nucleotide sequence encoding the transcriptional activation protein transcriptional activation domain, and a nucleotide sequence encoding the reporter gene, wherein at least one of the endogenous nucleotide sequences is inactivated by mutation or deletion.  
     
     
         186 . The yeast cell of  claim 184  wherein the peptide binding pair comprises a ligand and a receptor for the ligand.  
     
     
         187 . The yeast cell of  claim 184  wherein the transcriptional activation protein is Gal4, Gcn4, Hap1, Adr1, Swi5, Ste12, Mcm1, Yap1, Ace1, Ppr1, Arg81, Lac9, Qa1F. VP16, or a mammalian nuclear receptor.  
     
     
         188 . The yeast cell of  claim 184  wherein at least one of the heterologous fusion proteins is expressed from an autonomously-replicating plasmid.  
     
     
         189 . The yeast cell of  claim 184  wherein the DNA binding domain is a heterologous transcriptional activation protein DNA-binding domain.  
     
     
         190 . The yeast cell of  claim 184  wherein the peptide binding pair is other than an antigen and a corresponding antibody.  
     
     
         191 . The yeast cell of  claim 184  wherein the yeast cell is  Saccharomyces cerevisiae, Schizosaccharomyces pombe , or  Pichia pastoris.    
     
     
         192 . The yeast cell of  claim 188  wherein at least one peptide of the peptide binding pair is selected from the group consisting of a cytokine, an interleukin, a hematopoietic growth factor, insulin, an insulin-like growth factor, a growth hormone, prolactin, an interferon, a growth factor, a ligand for G-protein coupled receptors, a ligand for guanylyl cyclase receptors, a ligand for tyrosine phosphatase receptors, and a ligand for tyrosine kinase receptors.  
     
     
         193 . The yeast cell of  claim 189  wherein the DNA binding protein is selected from the group consisting of a mammalian steroid receptor and bacterial LexA protein.  
     
     
         194 . The yeast cell of  claim 191  wherein the yeast cell is  Saccharomyces cerevisiae.    
     
     
         195 . The yeast cell of  claim 192  wherein the peptide is a growth factor selected from the group consisting of epidermal growth factor, nerve growth factor, leukemia inhibitory factor, fibroblast growth factor, platelet-derived growth factor, vascular endothelial growth factor, tumor necrosis factor, oncostatin M, ciliary neurotrophic factor, erythropoietin, steel factor, placental lactogen, and TGF.