U.S. patent application number 10/921581 was filed with the patent office on 2005-03-10 for reverse two-hybrid systems.
This patent application is currently assigned to The Johns Hopkins University, a Maryland corporation. Invention is credited to Boeke, Jef D., Harlow, Ed, Vidal, Marc.
Application Number | 20050053913 10/921581 |
Document ID | / |
Family ID | 23666820 |
Filed Date | 2005-03-10 |
United States Patent
Application |
20050053913 |
Kind Code |
A1 |
Vidal, Marc ; et
al. |
March 10, 2005 |
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.
Inventors: |
Vidal, Marc; (Boston,
MA) ; Boeke, Jef D.; (Baltimore, MD) ; Harlow,
Ed; (Boston, MA) |
Correspondence
Address: |
FISH & RICHARDSON PC
225 FRANKLIN ST
BOSTON
MA
02110
US
|
Assignee: |
The Johns Hopkins University, a
Maryland corporation
The General Hospital Corporation, a Massachusetts
corporation
|
Family ID: |
23666820 |
Appl. No.: |
10/921581 |
Filed: |
August 18, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10921581 |
Aug 18, 2004 |
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10180235 |
Jun 26, 2002 |
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10180235 |
Jun 26, 2002 |
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10027219 |
Dec 21, 2001 |
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10027219 |
Dec 21, 2001 |
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09620680 |
Jul 20, 2000 |
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09620680 |
Jul 20, 2000 |
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09300839 |
Apr 28, 1999 |
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09300839 |
Apr 28, 1999 |
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08923274 |
Sep 4, 1997 |
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5955280 |
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08923274 |
Sep 4, 1997 |
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08420525 |
Apr 11, 1995 |
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Current U.S.
Class: |
435/4 ;
435/254.2; 435/483 |
Current CPC
Class: |
G01N 33/5023 20130101;
C12Q 2522/101 20130101; C12Q 2565/201 20130101; C12Q 1/6897
20130101; G01N 33/502 20130101; G01N 33/505 20130101; G01N 33/53
20130101; G01N 33/5011 20130101; C12Q 1/6897 20130101; G01N 33/5008
20130101; C12N 15/1055 20130101; G01N 2333/39 20130101 |
Class at
Publication: |
435/004 ;
435/483; 435/254.2 |
International
Class: |
C12Q 001/68; C12N
001/18; C12N 015/74 |
Goverment Interests
[0001] This invention was made at least in part with funds from the
Federal government, and the government therefor has certain rights
in the invention.
Claims
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 .beta. (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.
Description
BACKGROUND OF THE INVENTION
[0002] This invention relates to in vivo methods for characterizing
interactions between molecules (e.g., protein and/or RNA
molecules).
[0003] Numerous biologically important functions involve transient
interactions between DNA molecules and proteins, RNA molecules and
proteins, two or more proteins or RNA molecules, or ligands and
receptors. For example, during most of the cell cycle, the tumor
suppressor gene product pRb binds to the transcription factor E2F
and represses its activity. E2F activity is provided by a family of
at least seven proteins. The members of one subfamily (E2F-1, -2,
-3, -4, and -5) form heterodimers with the members of another
subfamily (DP-1 and -2). These heterodimers bind to the promoter of
target genes and activate their transcription at certain stages of
the cell cycle.
[0004] The transcriptional activity of the E2F/DP complexes can be
repressed by any of several functionally related proteins termed
the "pocket" proteins. Included in this category are proteins
termed p107, p130, and pRb (the retinoblastoma protein). The pocket
proteins exert their transcriptional inhibitory activity by
directly interacting with the E2F/DP complexes. At the G1/S
transition of the cell cycle, where E2F activity is required, the
pocket proteins are phosphorylated which causes pRb and E2F to
dissociate, leading to activation of the E2F transcription
factor.
[0005] The physiological relevance of the interactions between E2F
and the pocket proteins and between E2F and DP family members is
supported by several observations: (i) in a variety of tumors, both
copies of the RB gene contain loss of function mutations, and
reintroduction of the wild-type RB gene reduces tumorigenicity;
(ii) overexpression of E2F-1 in an experimental system can lead to
neoplastic transformation; (iii) PRAD1, the gene which encodes
cyclin D, a positive regulatory subunit of the pRb kinases, is, as
the result of a chromosomal rearrangement, overexpressed in
numerous tumors; (iv) disruption of the interaction of E2F with
proteins is required for the oncogenic activity of certain DNA
tumor viruses. Oncogenic proteins such as E1A of adenoviruses, the
large T antigen of SV40, and E7 of Human Papilloma Viruses can
abrogate pRb-mediated repression of E2F, causing the host cell to
enter the cell cycle inappropriately. Compounds which can
destabilize the interaction of an oncogenic viral protein with pRb
without affecting the interaction of pRb with E2F can be used
therapeutically to treat or prevent cancers associated with these
viruses.
[0006] Previous studies of interactions between regulatory proteins
have revealed important paradigms about how proteins interact with
each other. For example, studies of protein/protein interactions
have led to the identification of several structural motifs (e.g.,
the helix-loop-helix motif, SH2 and SH3 domains, and the leucine
zipper). The primary amino acid sequences of E2Fs, DPs, and the
pocket proteins do not resemble any of the known motifs. Thus, a
convenient method which permits a detailed study of the
protein/protein interactions involved in this novel family of
regulatory proteins may reveal new motifs for protein/protein
interactions. The E2F-1/DP-1 interaction domain has been mapped to
amino acids 120-310 of E2F-1 and amino acids 205-277 of DP-1. In
contrast, the E2F-1/pRb interaction domain has been mapped to amino
acids 409-427 of E2F-1. Thus, the DP-1 and pRb binding sites on
E2F-1 do not overlap. Accordingly, certain mutations may affect the
ability of E2F-1 to bind to DP-1 without affecting the ability of
E2F-1 to bind to pRb. Similarly, certain compounds may affect the
ability of E2F-1 to bind to DP-1 without affecting its ability to
bind to pRb.
[0007] Counterselectable Markers: While selectable markers have
been used to, under certain conditions, promote the growth of only
those cells which express the selectable markers, counterselectable
marker have been used, under certain conditions, to promote the
growth of only those cells which have lost the counterselectable
marker. Counterselectable markers when present on plasmids can be
used to select for cells that have lost the plasmid, a process
called plasmid "shuffling" (see, e.g., Sikorski and Boeke, 1991,
Meth. in Enzymol. 194: 302). For example, expression of the URA3
gene, which encodes orotidine-5'-phosphate, is lethal in the
presence of a medium containing 5-fluoro-orotic acid (5-FOA). Cells
expressing URA3 can also be positively selected for by growing them
on uracil-free media; thus, depending on the growth conditions,
URA3 can be used either for positive or negative conditions. The
LYS2 gene, which encodes .alpha.-aminoadipate reductase, can also
be used for counterselection; yeast cells which express LYS2 will
not grow on a medium containing .alpha.-aminoadipate as a primary
nitrogen source. Similarly, expression of LYS5 on a medium
containing .alpha.-aminoadipate is lethal. These genes, which are
involved in lysine biosynthesis, can be selected in a positive
fashion on a lysine-free medium. Another counterselectable reporter
gene is the CAN1 gene which encodes an arginine permease.
Expression of this gene in the absence of arginine and in the
presence of canavanine is lethal. Similarly, expression of the
counterselectable gene CYH2 is lethal in the presence of
cycloheximide. Expression of a counterselectable reporter gene has
been used to identify mutations in the activation domain of
estrogen receptor which inhibit its ability to activate
transcription (pierrat et al., 1992, Gene 119: 237-245).
SUMMARY OF THE INVENTION
[0008] We have discovered that a genetic screening system which
employs counterselection provides a convenient method for
characterizing molecular interactions in a bidirectional manner.
Thus, the invention can be used to determine whether two molecules
(e.g., proteins, RNA molecules, or DNA molecules) interact. In
addition, by using counterselection and by measuring the level of
expression of a reporter gene, the invention can be used to
determine how well two molecules interact. Thus, each of the
methods of the invention employs counterselection, and most
embodiments of the invention employ at least two hybrid proteins;
thus, the methods have been termed reverse two-hybrid systems. The
invention provides methods for (i) determining whether a first test
protein is capable of interacting with a second test protein, where
the proteins can be expressed from two separate nucleic acid
libraries (i.e., bidirectional combinatorial libraries); in
principle, this approach allows the identification all
proten/protein interactions in a given genome; (ii) determining
whether a compound can disrupt a protein/protein interaction; (iii)
determining whether a first test protein is capable of interacting
with a second test protein and incapable of interacting with a
third test protein; (iv) determining whether a test protein is
capable of interacting with a test RNA molecule; (iv) determining
whether a first test RNA molecule is capable of interacting with a
second test RNA molecule; (vi) identifying mutations which affect
protein/protein, interactions (two-step selection); (vii)
identifying a conditional allele of a protein which afects
protein/protein interactions; (viii) identifying compensatory
mutations which affect protein/protein interactions (bivalent
genetics), and (ix) identifying protein/DNA interactions. The
invention also features yeast strains and several genetic
constructs which are useful for identifying molecular interactions
with the disclosed methods.
[0009] The invention features, in one aspect, a method for
determining whether a first test protein is capable of interacting
with a second test protein. The method involves the following
steps:
[0010] (a) providing a first population of mating competent cells,
in which a plurality of the cells of the first 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 protein; the first
hybrid protein includes the first test protein covalently bonded to
a DNA-binding moiety which is capable of specifically binding to
the DNA-binding-protein recognition site;
[0011] (b) providing a second population of mating competent cells,
in which a plurality of the cells of the second population contain:
(i) a second selectable/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; the
second hybrid protein includes the second test protein covalently
bonded to a gene activating moiety;
[0012] (c) maintaining the first and the second populations of
mating competent cells, independently, under conditions such that
expression of the counterselectable reporter genes inhibits the
growth of said cells;
[0013] (d) mixing the first and the second populations of mating
competent cells under conditions conducive to formation of mated
cells; and
[0014] (e) detecting expression of a reporter gene as a measure of
the ability of the first test protein to interact with the second
test protein, where the reporter gene is the first or the second
reporter gene or another reporter gene included in the first or the
second mating competent cells or the mated cells, and is operably
linked to either the first of the second DNA-binding-protein
recognition sites.
[0015] In this aspect of the invention, the peptide sequences of
the first and second test proteins can be intentionally designed or
randomly generated. If desired, the sequence of one of the two test
proteins can be intentionally designed while the other is randomly
generated. In yet another embodiment of the invention, one part of
the protein is intentionally designed, and a second part is
randomly generated. Preferably, the selectable/counterselectable
reporter genes used in this aspect of the invention selected from
the group including URA3, LYS2, and GAL1. If desired, the first and
second counterselectable genes can be identical (e.g., both
counterselectable genes can be URA3 genes), or two different
counterselectable genes can be used (e.g., URA3 and LYS2).
[0016] In a second aspect, the invention features a method for
determining whether a test compound is capable of disrupting or
preventing binding between a first test protein and a second test
protein. The method involves the following steps:
[0017] (a) providing a cell containing:
[0018] (i) a counterselectable reporter gene operably linked to a
DNA-binding-protein recognition site;
[0019] (ii) a first fusion gene expressing a first hybrid protein
which includes the first test protein covalently bonded to a
DNA-binding moiety which is capable of specifically binding to the
DNA-binding-protein recognition site; and
[0020] (iii) a second fusion gene expressing a second hybrid
protein which includes the second test protein covalently bonded to
a gene activating moiety; the second test protein being one which
binds the first test protein in the absence of the test
compound;
[0021] (b) contacting the cell with the test compound under
conditions such that expression of counterselectable reporter gene
inhibits cell growth;
[0022] (c) detecting inhibition of expression of the
counterselectable reporter gene as a measure of the ability of the
compound to disrupt or prevent binding between the first and the
second test proteins.
[0023] In this aspect of the invention, the first and second test
proteins should be known to interact with each other in the absence
of the test compound. Suitable pairs of test proteins include, for
example, cFos and cJun, cJun and cJun, and E2F1 and pRb. The test
compound can be any molecule, such as a small, organic molecule or
a protein (e.g., a protein which is encoded by a nucleic acid of a
nucleic acid library, or a protein of a randomly generated peptide
sequence). Examples of preferred proteins to be used as test
compounds include E1A of adenovirus, large T antigen of SV40, and
E7 of a Human Papilloma Virus. Inhibition of expression of the
counterselectable reporter gene can be detected by assaying for
growth of the cell in the presence of a compound that normally is
toxic to the cell when the counter selectable reporter gene is
expressed. In this embodiment of the invention, suitable
counterselectable reporter genes include URA3, LYS2, GAL1, CYH2,
and CAN1.
[0024] The invention also features a method for determining whether
a first test protein is capable of interacting with a second test
protein and incapable of interacting with a third test protein. The
method involves:
[0025] (a) providing a cell which contains:
[0026] (i) a first fusion gene which expresses a first hybrid
protein; the first hybrid protein includes the first test protein
covalently bonded to a gene activating moiety;
[0027] (ii) a reporter gene which is operably linked to a first
DNA-binding-protein recognition site;
[0028] (iii) a second fusion gene which expresses a second hybrid
protein, the second hybrid protein includes the second test protein
covalently bonded to a DNA-binding moiety which is capable of
specifically binding to the first DNA-binding-protein recognition
site and which is incapable of specifically binding to a second
DNA-binding-protein recognition site;
[0029] (iv) a counterselectable reporter gene operably linked to
the second DNA-binding protein recognition site; and
[0030] (v) a third fusion gene which expresses a third hybrid
protein; the third hybrid protein includes the third test protein
covalently bonded to a second DNA-binding-moiety which is capable
of specifically binding to the second DNA-binding-protein
recognition site and incapable of binding to the first
DNA-binding-protein recognition site;
[0031] (b) maintaining the cell under conditions such that
expression of the reporter gene is detectable and does not inhibit
the growth of the cell, and expression of the counterselectable
reporter gene inhibits the growth of the cell; and
[0032] (c) detecting growth of the cell and expression of the
selectable reporter gene as a measure of the ability of the first
test protein to interact with the second test protein, and as a
measure of the inability of the first test protein to interact with
the third test protein.
[0033] If desired, the ability of the first test protein to
interact with the second test protein and not with the third test
protein can be measured in the presence of a test compound, such as
a polypeptide, a nucleic acid, or a small organic molecule. Where a
polypeptide acts as the test compound, the polypeptide can be of a
randomly generated peptide sequence, of an intentionally designed
peptide sequence, or encoded by a nucleic acid contained within a
nucleic acid library. In addition, any of the test proteins can
comprise a randomly generated peptide sequence or be mutagenized
versions of preferred proteins. Useful counterselectable reporter
genes include URA3, LYS2, GAL1, CYH2, and CAN1. Preferred reporter
genes include LEU2, TRP1, HIS3, and LacZ.
[0034] The invention further features a method for determining
whether a test RNA molecule is capable of interacting with a test
protein. The method involves:
[0035] (a) providing a first population of mating competent cells
in which a plurality of the cells of the population contain:
[0036] (i) a first selectable/counterselectable reporter gene
operably linked to a first DNA-binding-protein recognition
site;
[0037] (ii) a first fusion gene which expresses a first hybrid RNA
molecule in which the test RNA molecule is covalently bonded to a
non-random RNA molecule; and
[0038] (iii) a second fusion gene which expresses a first hybrid
protein having a DNA-binding moiety which is capable of
specifically binding to the first DNA-binding-protein recognition
site, the DNA-binding moiety being covalently bonded to an
RNA-binding moiety, and the RNA-binding moiety being capable of
specifically binding to the non-random RNA molecule;
[0039] (b) providing a second population of mating competent cells,
in which a plurality of the cells of the population contain:
[0040] (i) a second selectable/counterselectable reporter gene
operably linked to a second DNA-binding-protein recognition site;
and
[0041] (ii) a third fusion gene which expresses the test protein
covalently bonded to a gene activating moiety; and
[0042] (c) maintaining the first and the second populations of
mating competent cells, independently, under conditions such that
expression of the selectable/counterselectable reporter genes
inhibits growth of the cells of the populations;
[0043] (d) mixing the first and the second populations of mating
competent cells under conditions conducive to formation of mated
cells; and
[0044] (e) detecting expression of a selectable/counterselectable
reporter gene as a measure of the ability of the test RNA molecule
to interact with the test protein.
[0045] If desired, the test RNA molecule and/or test protein can
include a randomly-generated nucleotide or amino acid sequence;
alternatively, the test RNA molecule and/or test protein can be
intentionally designed. Optionally, the ability of the test RNA
molecule and test protein to interact can be measured in the
presence of a test compound (e.g., a dissociator or stabilizer of
the interaction), such as a protein (e.g., an intentionally
designed protein or a randomly generated protein such as a protein
encoded by a nucleic acid contained within a nucleic acid library).
Preferred selectable/counterselectable reporter genes include URA3,
LYS2, and GAL1.
[0046] An additional feature of the invention is a method for
determining whether a first test RNA molecule is capable of
interacting with a second test RNA molecule. The method
involves:
[0047] (a) providing a first population of mating competent cells
in which a plurality of the cells of the population contain.
[0048] (i) a first selectable/counterselectable reporter gene
operably linked to a first DNA-binding-protein recognition
site;
[0049] (ii) a first fusion gene which expresses a first hybrid RNA
molecule; the first hybrid RNA molecule includes the first test RNA
molecule covalently bonded to a first non-random RNA molecule;
and
[0050] (iii) a second fusion gene which expresses a first hybrid
protein; the first hybrid protein includes a DNA-binding moiety
which is capable of specifically binding to the first
DNA-binding-protein recognition site, and the DNA-binding moiety is
covalently bonded to a first RNA-binding moiety which is capable of
specifically binding to the first non-random RNA molecule;
[0051] (b) providing a second population of mating competent cells
in which a plurality of the cells of the population contain:
[0052] (i) a second selectable/counterselectable reporter gene
operably linked to a second DNA-binding-protein recognition
site;
[0053] (ii) a third fusion gene which expresses a second hybrid RNA
molecule; the second hybrid RNA molecule includes the second test
RNA molecule covalently bonded to a second non-random RNA molecule;
and
[0054] (iii) a fourth fusion gene which expresses a gene-activating
moiety covalently bonded to a second RNA-binding moiety which is
capable of specifically binding to the second non-random RNA
molecule;
[0055] (c) maintaining the first and the second populations of
mating competent cells, independently, under conditions such that
expression of the selectable/counterselectable reporter genes
inhibits growth of the cells;
[0056] (d) mixing the first and the second populations of mating
competent cells under conditions conducive to formation of mated
cells; and
[0057] (e) detecting expression of a counterselectable reporter
gene as a measure of the ability of the first test RNA molecule to
interact with the second test RNA molecule.
[0058] If desired, the first and/or second test RNA molecule can
include a randomly generated RNA sequence. The amino acid or RNA
sequence of a protein or RNA molecule used as a test compound can
be intentionally designed or randomly generated (e.g., be encoded
by a nucleic acid contained within a nucleic acid library).
Preferred selectable/counterselectable reporter genes in this
aspect of the invention include URA3, LYS2, and GAL1. Preferably,
the first RNA-binding moiety does not bind to the second non-random
RNA molecule, and the second RNA-binding moiety does not bind to
the first non-random RNA molecule.
[0059] In another aspect, the invention features a method for
determining whether a test DNA molecule is capable of interacting
with a test protein. The method involves:
[0060] (a) providing a cell which contains (i) a counterselectable
reporter gene operably linked to the test DNA molecule; and (ii) a
fusion gene which expresses the test protein covalently bonded to a
gene activating moiety; and
[0061] (b) detecting expression of said counterselectable reporter
gene as a measure of the ability of said test DNA molecule to
interact with said test protein.
[0062] If desired, the DNA can be randomly generated and/or the
protein include a randomly generated peptide sequence.
[0063] In yet another aspect, the invention features a method for
identifying a mutation in a reference protein which affects the
ability of the reference protein to interact with a test protein.
The method involves:
[0064] (a) providing a cell which contains:
[0065] (i) a counterselectable reporter gene operably linked to a
DNA-binding-protein recognition site;
[0066] (ii) a selectable reporter gene operably linked to a
DNA-binding-protein recognition site;
[0067] (iii) a first fusion gene expressing a first hybrid protein,
where the first hybrid protein includes the first test protein;
and
[0068] (iv) a second fusion gene expressing a second hybrid
protein, the second hybrid protein includes a candidate mutated
reference protein, and the second test protein is encoded within a
nucleic acid library of mutant alleles of the gene encoding the
reference protein; and one of the first and the second hybrid
proteins also includes a DNA-binding moiety which is capable of
specifically binding to the DNA-binding-protein recognition site,
and the other of the first and the second hybrid proteins also
includes a gene activating moiety;
[0069] (b) maintaining the cell under conditions such that
expression of the counterselectable reporter gene at a level equal
to or greater than the level of expression obtained with the
reference protein inhibits growth of the cell, and such that
expression of the counterselectable reporter gene at a level less
than the level of expression obtained with the reference protein
does not inhibit growth of the cell;
[0070] (c) in a separate step, maintaining the cell under
conditions such that expression of the counterselectable reporter
gene does not inhibit growth of the cell, and detecting expression
of the selectable reporter gene as a measure of the ability of the
first test protein to interact with the candidate mutated reference
protein.
[0071] If desired, the method can include comparing the sequence of
the candidate mutated protein with the sequence of the reference
protein as an indicator of a mutation in the reference protein
which affects the ability of the reference protein to interact with
the first test protein. If desired, the second fusion gene can
encode a functional c-term tag, and, as is described herein, the
presence of the functional C-term tag, indicating the presence of
the C-terminus of the candidate mutated protein, can be measured by
detecting expression of the selectable reporter gene or with other
methods (e.g., detection of GFP with UV light).
[0072] In another aspect, the invention features a method for
identifying a conditional mutant of a reference protein which has a
decreased ability to interact with a second protein under a first
set of conditions and which is capable of interacting with the
second protein under a second set of conditions. The method
involves:
[0073] (a) providing a cell which contains:
[0074] (i) a counterselectable reporter gene operably linked to a
DNA-binding-protein recognition site;
[0075] (ii) a selectable reporter gene operably linked to a
DNA-binding-protein recognition site;
[0076] (iii) a first fusion gene expressing a first hybrid protein,
where the first hybrid protein includes the candidate mutated
reference protein, and the candidate mutated reference protein is
encoded within a nucleic acid library of mutant alleles of the gene
encoding the reference protein; and
[0077] (iv) a second fusion gene expressing a second hybrid
protein, where the second hybrid protein includes a second protein,
and
[0078] one of the first or second hybrid proteins also includes a
DNA-binding moiety which is capable of specifically binding to the
DNA-binding-protein recognition site, and
[0079] the other of the first or second hybrid proteins also
includes a gene activating moiety;
[0080] (b) maintaining the cell under conditions in which
expression of the counterselectable reporter gene at a level equal
to or greater than the level of expression obtained with the
reference protein inhibits growth of the cell, and such that
expression of the counterselectable reporter gene at a level less
than the level of expression obtained with the reference protein
does not inhibit growth of the cell;
[0081] (c) in a separate step, maintaining the cell under
conditions such that expression of the counterselectable reporter
gene does not inhibit growth of the cell, and detecting expression
of the selectable reporter gene as a measure of the ability of the
candidate mutant protein to interact with the second protein;
and
[0082] (d) in a separate step, maintainng the cells under
conditions identical to those in step (c) except for one parameter,
and detecting expression of the selectable reporter gene as a
measure of the ability of the candidate mutant protein to interact
with the second protein, (expression of the selectable reporter
gene under step (c) conditions but not under step (d) conditions is
indicative of the conditional mutant).
[0083] If desired, the method can also include comparing the
sequence of the candidate mutant protein with the sequence of the
reference protein as a means for identifying a mutant of the
reference protein which has a decreased ability to interact with
the second protein under a first set of conditions and which is
capable of interacting with the second protein under a second set
of conditions.
[0084] The conditions under which the cell is maintained in step
(b) and the conditions under which the cell is maintained in step
(c) can differ in any way desired by the practitioner. For example,
the first and second growth conditions can differ in temperature
and/or by the presence of a drug (e.g., formamide or
deuterium).
[0085] The invention also features 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 the second and the first
reference proteins, respectively. The method involves:
[0086] (a) providing a first population of mating competent cells
in which a plurality of the cells of the population contain:
[0087] (i) a first counterselectable reporter gene operably linked
to a DNA-binding-protein recognition site;
[0088] (ii) a first selectable reporter gene operably linked to a
DNA-binding-protein recognition site;
[0089] (iii) a first fusion gene which expresses a first hybrid
protein, where the first hybrid protein includes a first candidate
mutant reference protein covalently bonded to a gene activating
moiety, and where the first candidate mutant protein is encoded
within a nucleic acid library of mutant alleles of the first
reference protein; and
[0090] (iv) a plasmid containing a first counterselectable marker,
and a second fusion gene which expresses a second hybrid protein,
where the second hybrid protein includes the second reference
protein covalently bonded to a DNA-binding moiety;
[0091] (b) providing a second population of mating competent cells
in which a plurality of the cells of the population contain:
[0092] (i) a second counterselectable reporter gene operably linked
to a DNA-binding-protein recognition site;
[0093] (ii) a second selectable reporter gene operably linked to a
DNA-binding-protein recognition site;
[0094] (iii) a third fusion gene which expresses a third hybrid
protein, where the third hybrid protein includes the second
candidate mutant reference protein covalently bonded to a
DNA-binding moiety, and where the second candidate mutant protein
is encoded within a nucleic acid library of mutant alleles of the
second reference protein; and
[0095] (iv) a plasmid containing a second counterselectable marker
and a fourth fusion gene which expresses a fourth hybrid protein,
where the hybrid protein includes the first reference protein
covalently bonded to a gene activating moiety;
[0096] (c) maintaining the first and the second populations of
mating competent cells, independently, under conditions such that
expression of the counterselectable reporter genes at a level equal
to or greater than the level of expression obtained with the first
and second reference proteins inhibits growth of the cells;
[0097] (d) maintaining the first and the second populations of
mating competent cells under conditions such that expression of the
counterselectable marker inhibits growth of the cells;
[0098] (e) maintaining the first and the second populations of
mating competent cells under conditions conducive to formation of
mated cells;
[0099] (f) detecting expression of the selectable reporter genes as
a measure of the ability of the first and the second candidate
proteins to interact with each other and not with the second and
the first reference proteins.
[0100] If desired, the method can also include comparing the
sequences of the first and the second candidate mutant proteins
which interact with each other with the sequences of the first and
the second reference proteins as a means for identifying
compensatory mutations in the first and the second reference
proteins.
[0101] The invention further features several genetic constructs
which are useful in practicing various aspects of the invention. In
one aspect, the genetic construct includes: (i) a yeast origin of
replication; (ii) a selectable marker; (iii) a yeast promoter; (iv)
a nuclear localization coding signal sequence; and (v) a bacterial
origin of replication. A preferred nuclear localization coding
signal sequence is the nuclear localization coding signal sequence
of SV40 large T antigen. A preferred promoter is the ADH1 promoter,
and a preferred genetic construct is the plasmid p2.5.
[0102] In another aspect, the genetic construct includes: (i) a
yeast origin of replication; (ii) a selectable marker; (iii) a
promoter; (iv) a bacterial origin of replication; (v) a
counterselectable marker; and (vi) a sequence which expresses a
DNA-binding moiety. Preferably, the genetic construct is
p97.CYH2.
[0103] In still another aspect, the genetic construct includes: (i)
a yeast origin of replication; (ii) a selectable marker; (iii) a
promoter; (iv) a bacterial origin of replication; (v) a
counterselectable marker; and (vi) a sequence which expresses a
gene activating moiety. Preferably, the genetic construct is
pMV257.
[0104] More generally, the invention features any genetic construct
(e.g., a plasmid or a chromosome) having a counterselectable
reporter gene operably-linked to a promoter which contains an
upstream repressing sequence and a DNA-binding-protein recognition
site for a DNA-binding moiety which can mediate transcription of
the counterselectable reporter gene (e.g., an intact or a
reconstituted transcription factor). Included in the preferred
promoters is a SPO13 promoter, and a preferred counterselectable
reporter gene is the URA3 gene. A preferred DNA-binding-protein
recognition site is the binding site for Gal4. Thus, a preferred
genetic construct is SPAL:URA3.
[0105] In addition, the invention features a yeast cell having
integrated into its genome a counterselectable reporter gene which
is operably linked to a promoter which includes
[0106] (i) an upstream repressing sequence, and
[0107] (ii) a DNA-binding-protein recognition site, wherein the
yeast cell lacks
[0108] (i) a naturally-occurring protein which is substantially
identical to the protein encoded by the counterselectable reporter
gene, and
[0109] (ii) at least one naturally-occurring protein which, when it
is expressed, confers a growth advantage on a cell containing it.
Such a yeast cell can contain a SPO13 promoter which includes a
DNA-binding-protein recognition site for a protein selected from
the group which includes GAL4, LexA, and Ace1. Preferred yeast
cells include MaV103, MaV203, and MaV99.
[0110] In preferred embodiments of each of the aforementioned
aspects of the invention, the cells of the populations of cells are
yeast cells; preferably, the yeast is saccharomyces cerevisiae. If
desired, the ability of two or more molecules to interact can be
measured in the presence of a test compound in a method of
identifying compounds which dissociate or stabilize the interaction
of two molecules of interest. The test compound can be expressed
within the cell by employing conventional methods for gene
expression, or the test compound can simply be added to the growth
medium. Yeast strains employed in the invention can be chemically
treated (e.g., with polymixin B nonapeptide) to increase the uptake
of compounds (see, e.g., Boguslawski et al., Mol. Gen. Genet. 199:
401-405 and Antimicrob. Agents and Therapies. 29: 330-332). Where
the test compound is added to the growth medium, yeast mutants
which have relatively high uptake levels of extraneous compounds,
such as the erg6, ise1, ISE2, and srb1 mutants of S. cerevisiae,
are particularly useful. Where two populations of mating competent
yeast cells are used to produce mated cells, the two populations
must include mating competent cells of compatible mating types
(e.g., MATa and MAT.alpha.).
[0111] If desired, the methods of the invention can be coupled with
methods for mutagenizing proteins or RNA molecules. In order to
identify amino acid residues or nucleotides-responsible for the
interaction of proteins and/or RNA molecules. For example,
mutations in one or both of two proteins which prevent two proteins
from interacting indicate that amino acids at those positions
contribute to the ability of the wild-type proteins to interact.
Similarly, compensatory mutations in two interacting proteins
define critical amino acids which contribute to the ability of the
corresponding wild-type proteins to interact. The invention also
provides methods for identifying conditional alleles that affect
protein/protein, protein/RNA, protein/DNA interactions, or RNA/RNA
interactions. Once identified, a conditional allele provides a
detectable phenotype that can be used to characterize the function
of a protein or RNA molecule. Such alleles can be identified by
mutating one of the interacting molecules and identifying those
mutants which can interact with its wild-type partner under certain
(i.e., permissive), but not other (i.e., restrictive),
conditions.
[0112] Preferably, each of the reporter genes is operably linked to
a promoter which carries a repressing sequence which prevents
transcription in the absence of a gene activating moiety. Thus, the
reporter gene should be positioned such that its expression is
highly responsive to the presence or absence of a transcription
factor. For example, it is preferred that where a URA3 allele is
used, the allele confers a Ura.sup.- Foa.sup.r phenotype in the
absence of a transcription factor, and it confers a
Ura.sup.+Foa.sup.s phenotype in the presence of a transcription
factor. Certain promoters, such as the SPO13 promoter, naturally
contain an upstream repressing sequence. Other promoters can be
engineered with conventional cloning methods to contain such
sequences. Where a counterselectable reporter gene is used,
expression of the gene can be detected by detecting inhibition of
cell growth.
[0113] Where more than one reporter gene is employed, the reporter
genes can be connected to promoters which are identical to each
other only at their DNA-binding-protein recognition sites, if
desired. Preferably, the reporter gene is one which allows for
titratable selection; thus, cell growth can be measured over a
range of conditions (e.g., 5-FOA concentrations).
[0114] A variety of DNA-binding moieties and gene activating
moieties are suitable for use in the various aspects of the
invention. Generally, the DNA-binding domain or gene activating
domain of any transcription factor can be used. If desired, the
gene activating domain of VP16 can be used. The DNA-binding-protein
recognition site and the gene activating and DNA-binding moieties
all can correspond to identical transcription factors, or they can
correspond to different transcription factors. Useful binding sites
include those for the yeast protein GAL4, the bacterial protein
LexA, the yeast metal-binding factor Ace1. These binding sites can
readily be used with a repressed promoter (e.g., a SPO13 promoter
can be used as the basis for SPAL, SPEX and SPACE promoters,
respectively, for a SPO13 promoter combined with GAL, LEX, and ACE1
DNA binding sites). Other useful transcription factors include the
GCN4 protein of S. cerevisiae (see, e.g., Hope and Struhl, 1986,
Cell 46: 885-894) and the ADR1 protein of S. cerevisiae (see, e.g.,
Kumar et al., 1987, Cell 51: 941-951). The DNA-binding-protein
recognition site should include at least one binding site for the
DB of the transcription factor that is used. While the number of
DNA-binding-protein recognition sites that can be used is
unlimited, the number of binding sites is preferably between 1 and
100, more preferably 1 and 20; still more preferably, the number of
binding sites is between 1 and 16. The number of binding sites can
be adjusted to account for factors such as the desired sensitivity
of the assay.
[0115] If desired, the allele for the reporter gene (e.g.,
SPALX:URA3) can be integrated into the genome of a haploid or
diploid cell. If desired, a combination of alleles can be used; for
example, SPALX:URA3 can be chromosomally located and SPEX:URA3 can
be located on a plasmid; SPALX:URA3 can be expressed from a plasmid
and SPACEX:URA3 can be located on a chromosome.
[0116] By "dissociator compound" is meant any molecule which
disrupts or prevents binding of two molecules. Examples of
dissociator compounds (also referred to herein as "dissociators")
are polypeptides, nucleic acids, and small, organic molecules
(i.e., molecules having a molecular weight of less than 1 kD).
[0117] By "reporter gene" is meant a gene whose expression can be
assayed as a measure of the ability of two test molecules to
interact (i.e., as a measure of protein/protein, protein/RNA,
RNA/RNA, or protein/DNA interactions). A useful reporter gene has
in its promoter a DNA-binding-protein recognition site to which a
reconstituted transcription factor or DNA-binding protein of
interest binds. Such genes include, without limitation, lacZ, amino
acid biosynthetic genes (e.g., the yeast LEU2, HIS3, LYS2, or
TRP1), URA3 genes, nucleic acid biosynthetic genes, the bacterial
chloramphenicol transacetylase (cat) gene, and the bacterial gus
gene. Also included are those genes which encode fluorescent
markers, such as the Green Fluorescent Protein gene. Certain
reporter genes are considered to be "selectable,"
"counterselectable," or "selectable/counterselectable" reporter
genes, as is described below.
[0118] By "test" protein, RNA molecule, or DNA molecule is meant a
molecule whose function (i.e., ability to interact with a second
molecule) is being characterized with the methods of the
invention.
[0119] By "DNA-binding" protein is meant any of numerous proteins
which can specifically interact with a nucleic acid. For example, a
DNA-binding protein used in the invention can be the portion of a
transcription factor which specifically interacts with a nucleic
acid sequence in the promoter of a gene. Alternatively, the
DNA-binding protein can be any protein which specifically interacts
with a sequence which is naturally-occurring or artificially
inserted into the promoter of a reporter gene. Where protein/DNA
interactions are characterized, the DNA-binding protein can be
covalently bonded to a gene-activating moiety such that binding of
the DNA-binding protein to a site located within the promoter of a
chosen reporter gene activates transcription of the reporter
gene.
[0120] By "selectable" marker is meant a gene which, when it is
expressed, confers a growth advantage on a cell containing it.
Examples of selectable markers include, without limitation, LEU2,
TRP1, and HIS3. Certain selectable markers described herein can be
used to promote the growth of cells containing a plasmid containing
a selectable marker. A promoter which is operably linked to a
selectable marker located on a plasmid can be the
naturally-occurring promoter for the marker, or the marker can be
engineered to be operably linked to a promoter other than the one
to which it is naturally operably linked. Generally, a promoter
which is operably linked to a selectable marker located on a
plasmid (e.g., a plasmid used to express an interacting molecule or
dissociator) used in the invention does not contain a
DNA-binding-protein recognition site(s) which is functionally
identical to a DNA-binding-protein recognition site contained
within the promoter of the reporter gene which is used to measure
the molecular interaction of interest. In other words, the
DNA-binding-protein which mediates transcription of the reporter
gene should not also mediate transcription of the selectable
marker, and the DNA-binding-protein which mediates transcription of
the selectable marker should not also mediate transcription of the
reporter gene.
[0121] By "screenable" reporter gene is meant a gene whose
expression can be detected in a cell by a means other by conferring
a selective growth advantage on a cell. An example of a screenable
reporter gene is the lacZ gene. If desired, a screenable reporter
gene can be integrated into the genome of a yeast cell. It is
preferred, though not essential, that the promoter of the
screenable reporter gene be distinct from the promoters of any
other reporter genes used in the cell. A screenable reporter gene
can be used in the invention to measure the ability of two
molecules to interact and reconstitute a transcription factor.
Thus, the promoter which is operably linked to a screenable
reporter gene should contain a DNA-binding-protein-recognition
site(s) to which a reconstituted transcription factor, or to which
a DNA-binding protein fused to a gene-activating moiety, can
bind.
[0122] By "counterselectable" marker is meant a gene which, when it
is expressed, prevents the growth of a cell containing it. Examples
of counterselectable reporter genes include URA3, LYS2, GAL1, CYE2,
and CAN1. These markers can be used to select for plasmid
elimination.
[0123] By "selectable" reporter gene is meant a reporter gene
which, when it is expressed under a certain set of conditions,
confers a growth advantage on cells containing it.
[0124] By "counterselectable" reporter gene is meant a reporter
gene which, when it is expressed under a certain set of conditions,
prevents the growth of a cell containing it. Examples of
counterselectable reporter genes include URA3, LYS2, GAL1, CYH2,
and CAN1.
[0125] By "selectable/counterselectable" reporter gene is meant a
reporter gene which, when it is expressed under a certain set of
conditions, is lethal to a cell containing it, and when it is
expressed a different set of conditions, confers a selective growth
advantage on cells containing it. Thus, a single gene can be used
as both a selectable reporter gene and a counterselectable reporter
gene. Examples of selectable/counterselectable reporter genes
include URA3, LYS2, and GAL1. In each aspect of the invention where
a selectable/counterselectable reporter gene is employed, a
combination of a selectable reporter gene and a counterselectable
reporter gene can be used in lieu of a single
selectable/counterselectable reporter gene. For example, in the
first aspect of the invention, each mating competent cell can be
provided with (i) a selectable reporter gene, and (ii) a
counterselectable reporter gene. Where two such genes substitute
for a single selectable/counterselectable gene, it is preferred
that the reporter genes be operably linked to identical promoters.
In particular, it is preferred that the reporter genes be operably
linked to promoters that have identical DNA-binding-protein
recognition site.
[0126] By "DNA-binding-protein recognition" site is meant a segment
of DNA that is necessary and sufficient to specifically interact
with a given polypeptide (i.e., the DNA-binding-protein).
[0127] By "covalently bonded" is meant that two molecules (e.g.,
RNA molecules or proteins) are joined by covalent bonds, directly
or indirectly. For example, the "covalently bonded" proteins or
protein moieties may be immediately contiguous, or they may be
separated by stretches of one or more amino acids within the same
hybrid protein.
[0128] By "protein" is meant a sequence of amino acids,
constituting all or a part of a naturally-occurring polypeptide or
peptide, or constituting a non-naturally-occurring polypeptide or
peptide.
[0129] By "DNA-binding moiety" is meant a stretch of amino acids
which is capable of directing specific polypeptide binding to a
particular DNA sequence (i.e., a DNA-binding-protein recognition
site).
[0130] By "RNA-binding moiety" is meant a stretch of amino acids
which is capable of directing specific polypeptide binding to a
particular RNA sequence (i.e., an RNA-binding-protein recognition
site).
[0131] By "hybrid" protein, RNA molecule, or DNA molecule is meant
a chimera of at least two covalently bonded polypeptides, RNA
molecules, or DNA molecules.
[0132] By "gene activating moiety" is meant a stretch of amino
acids which is capable of inducing the expression of a gene to
whose control region (i.e., promoter) it is bound.
[0133] By "operably linked" is meant that a gene and a regulatory
sequence(s) (e.g., a promoter) are connected in such a way as to
permit gene expression when the appropriate molecules (e.g.,
transcriptional activator proteins or proteins which include
transcriptional activation domains) are bound to the regulatory
sequence(s).
[0134] By "randomly generated" sequence is meant a sequence having
no predetermined sequence; this is contrasted with "intentionally
designed" sequences which have a DNA, RNA, or protein sequence or
motif which is determined prior to their synthesis. Randomly
generated sequences can be derived from a nucleic acid library.
[0135] By "mutated" is meant altered in sequence, either by
site-directed or random mutagenesis. Mutated sequences include
those sequences which have point mutations, insertions, deletions,
or rearrangements.
[0136] By "promoter" is meant minimal sequence sufficient to direct
transcription; such elements can be located in the 5' or 3' regions
of the native gene.
[0137] By "repressing" sequence is meant a DNA sequence which,
under certain conditions, inhibits expression of a gene to which it
is connected.
[0138] By nucleic acid "library" is meant a set of 5 or more DNA
molecules. Such a library can have hundreds, thousands, or even
millions of different DNA molecules.
[0139] By "bidirectional combinatorial library" is meant a very
large set of pairs of interacting hybrid molecules generated from
two separate, parental expression libraries. Typically, the size of
the set is approximately the product of the complexities of each
parental library.
[0140] By "compensatory" mutations is meant mutations in a pair of
interacting molecules (e.g., proteins) which allow the molecules to
interact with each other but not with wild-type molecules.
[0141] By "mass mating" is meant the mixing of suspensions of
mating competent yeast cells of complementary mating types so as to
generate a very large number of mated cells. Typically, 10.sup.10
or even 10.sup.12 mated cells are generated. Preferably, the
suspensions of cells are mixed at a 1:1 ratio (number of
cells:number of cells).
[0142] By "functional C-term tag" is meant a stretch of amino acids
located at the C-terminus of a test protein, the presence of which
can be assayed to confirm that the carboxyl terminus of the test
protein is intact, indicating that a full-length protein is
expressed at detectable levels. For example, the functional C-term
tag can be a sequence (e.g., the pocket binding domain of E2F1)
which can interact with a second protein (e.g., pRb, p107, or
p130). If desired, the functional C-term tag can be a sequence
which can be detected without binding a second protein. For
example, GFP (green fluorescent protein) can serve as a functional
C-term tag, and it can be detected with UV light.
[0143] The present invention offers several features and
advantages. For example, the invention allows one to screen two
libraries of cDNA clones encoding peptides or RNA molecules
simultaneously. Using the "mass mating" methods, the reaction
testing the functional relationship of the various molecules is
performed only once, and under identical conditions for all
combinations of molecules in a given system. In addition, it is not
necessary to have previously identified any of the molecules which
interact. The present invention facilitates generation and
screening of as many as 1.times.10.sup.13 interactions. Thus, the
invention facilitates screening of a large number of combinations
of molecules, increasing the probability of detecting relatively
rare association or dissociation events. The invention can be used,
on a large scale, to generate protein/protein linkage maps of most
or all interactions that occur with two libraries of interest.
Yeast cells containing each of the possible pairs of interacting
molecules can be organized on plates in a method of cataloging the
molecular interactions. For example, DNA encoding a protein of
interest can be used as a probe in a DNA hybridization against DNA
extracted from yeast colonies organized on a solid support (e.g., a
nitrocellulose filter). By identifying a yeast colony to which the
DNA of interest hybridizes, one immediately has identified a yeast
strain containing a molecule which interacts with the protein of
interest encoded by the DNA of interest. The gene encoding the few
interacting molecule can then be cloned from a yeast cell derived
from a hybridization positive colony.
[0144] The invention can also be used with great sensitivity to
detect relatively rare association events. Accordingly, the
invention addresses one of the most significant challenges in the
construction of combinatorial libraries: identification of the few
pairs of interacting molecules from a large population of
potentially interacting molecules.
[0145] The invention also permits the identification of molecules
which dissociate or prevent undesired interactions but which do not
dissociate or prevent desired interactions. For example, the
invention facilitates the identification of compounds which
dissociate or prevent binding of viral proteins to molecules in a
host cell but which do not affect binding of the host cell molecule
to preferred molecules. In addition, the invention allows these
dissociator compounds to be identified on a single medium (i.e., a
single plate), making the screening of therapeutic compounds a
rapid and convenient process. Compounds which stabilize molecular
interactions can also be identified rapidly and conveniently by
assaying for increased expression of a reporter gene in the
presence of the compound.
[0146] The invention can also be used to identify the targets of a
drug of interest (e.g., a dissociator or a stabilizer) for which
the relevant molecular interaction is unknown. This method employs
a collection of yeast cells, where each cell of the collection
contains a pair of interacting molecules from a bidirectional
combinatorial library. Each cell in the collection is exposed to
the drug of interest, and colonies which express the reporter gene
at an altered level (e.g., higher or lower) in the presence of the
drug represent cells containing hybrid proteins which are targets
of the drug of interest. The hybrid proteins encoded within these
cells can be identified with conventional methods.
[0147] Because low-copy plasmids can be used in the invention, the
proteins and RNA molecules of interest can be expressed at
physiologically relevant levels. Expression of the molecules of
interest from low-copy plasmids should allow a practitioner to
detect subtle differences between various pairs of interacting
molecules. When genes are overexpressed from high-copy plasmids,
differences between pairs of proteins tend to be more difficult to
detect as dissimilar pairs of interacting molecules can sometimes
cause apparently similar levels of expression of the reporter gene.
Reproducibility in the levels of expression of hybrid proteins in
different yeast cells can be optimized with the use of low-copy
plasmids.
[0148] Certain embodiments of the invention reduce the occurrence
of four types of false positives (relative to their incidence
obtained with other systems). Interactions classified as false
positives include interactions between:
[0149] (i) proteins which obviously could not interact under
physiological conditions because they are not expressed (a) in the
same cell-type, (b) in the same cellular compartment, or (c) at the
same stage of development;
[0150] (ii) proteins which are not biologically relevant and which
may result from expression of the incorrect open reading frame;
or
[0151] (iii) proteins which mediate transcription of the reporter
gene by themselves, without requiring a specific interaction
partner. The appearance of these false positives is highly
promoter-dependent (Bartel et al., 1993, Biofeedback 14: 920-924).
In addition, it has been suggested that 0.1% of random sequences
from E. coli can activate transcription (i.e., function as an AD)
when fused to a DB in a eukaryotic cell (Ma and Ptashne, 1987, Cell
51: 113-119).
[0152] By maintaining the level of expression of the hybrid
proteins at physiologically relevant levels, the invention inhibits
the recovery of the first two classes of false positives. If
desired, the chances of obtaining false positives can also be
decreased by using a "triple selection method" in practicing the
invention. For triple selection, three reporter genes are operably
linked to promoters which have different sequences, with the
exception of the DNA-binding-protein recognition sequence (FIG. 1).
By employing three reporter genes which are operably linked to
three different promoters, the likelihood of recovering the third
class of false positives is diminished.
[0153] Where the invention is used to detect binding of a
monoclonal antibody to an antigen, the invention offers the
following features. Like the immune system, the invention is
combinatorial in nature, and thus the mass mating method used in
the invention facilitates analysis of large numbers of combinations
of interacting molecules. In addition, the somatic refinement
capability of the immune system can be reproduced synthetically
with the use of the invention and the PCR mutagenesis method and
titratable selection method described herein.
[0154] The invention also provides a convenient method for
isolating mutant alleles of a protein or RNA molecule. While
conventional methods of isolating mutant alleles are based on a
previous implication of a particular region of a molecule (e.g., a
domain which is conserved among related molecules), the invention
permits large numbers of mutant alleles to be generated and
screened in a manner without prior knowledge of the molecule and
without bias in the mutagenesis method.
[0155] The invention can be used as a tool for providing
information regarding the structure and regulation of molecular
(e.g., protein/protein) interactions. Particularly interesting
molecular interactions that can be examined with the invention
include protein/protein interactions between a virus and components
of a host cell. Dissociator compounds which can disrupt or prevent
these interactions can be used therapeutically to decrease viral
pathogenicity.
DETAILED DESCRIPTION
[0156] The drawings will first be briefly described.
[0157] FIG. 1 is a schematic representation of three reporter genes
that are operably linked to promoters having different sequences
with the exception of the DNA-binding-protein recognition
sequences.
[0158] FIG. 2 is a map of the plasmid p2.5.
[0159] FIG. 3 is a photograph of yeast cells which demonstrates
that expression of a SPAL5:URA3 allele can be induced in cells and
confer a Foa.sup.s phenotype on cells. Control strains are
wild-type URA3 (two patches on right side of each panel) and
ura3-52 mutant strains (two patches on left side of each panel).
The cells were grown on synthetic complete medium lacking leucine
and tryptophan (Sc-L-T), synthetic complete medium lacking uracil
(Sc-ura), or synthetic complete medium lacking leucine and
tryptophan and containing 5-FOA (Sc-L-T+FOA), as indicated.
[0160] FIG. 4 is a schematic representation of the genetic
constructs used to express DB-cFos, AD-cJun, DB-pRb, and
AD-E2F1.
[0161] FIG. 5 is a photograph of yeast cells in which a GAL4
transcription factor was reconstituted with various interacting
proteins. Reconstitution induces expression of the SPAL5:URA3
alleles and confers Foa.sup.s on the cells. Control strains are
wild-type URA3 (two patches on right side of each panel) and
ura3-52 mutant strains (two patches on left side of each panel).
These experiments employ the yeast strain MaV103 which includes the
counterselectable reporter gene SPAL9:URA3. The cells were grown on
synthetic complete medium lacking leucine and tryptophan (Sc-L-T),
synthetic complete medium lacking uracil (Sc-ura), or synthetic
complete medium lacking leucine and tryptophan and containing 5-FOA
(Sc-L-T+FOA), as indicated.
[0162] FIG. 6 is a photograph of yeast cells which define the limit
of growth threshold on 5-FOA for various interacting proteins which
reconstitute a transcription factor: cFos/cJun (0.05%), pRb/E2F1
(0.1%), and cJun/cJun (0.2%). Control strains are wild-type URA3
(two patches on right side of each panel) and ura3-52 mutant
strains (two patches on left side of each panel). The cells were
grown on synthetic complete medium lacking leucine and tryptophan
(Sc-L-T), or synthetic complete medium lacking leucine and
tryptophan and containing 5-FOA (Sc-L-T+FOA), with 5-FOA at the
indicated concentrations.
[0163] FIG. 7 is a photograph of yeast cells which indicates that
the plasmid p2.5 can be used to express dissociator compounds in
cells expressing molecules which, in the absence of a dissociator,
would reconstitute a transcription factor. Control strains are
wild-type URA3 (two patches on right side of each panel) and
ura3-52 mutant strains (two patches on left side of each panel).
The cells were grown on synthetic complete medium lacking leucine
and tryptophan (Sc-L-T), synthetic complete medium lacking uracil
(Sc-ura), or synthetic complete medium lacking leucine and
tryptophan and containing 5-FOA (Sc-L-T+FOA), as indicated. Rb#1
and Rb#2 are two independent isolates of the construct encoding
Rb.
[0164] FIG. 8 is a photograph which shows the various phenotypes of
the MaV103 strain of yeast expressing any of a variety of hybrid
proteins under several different growth conditions. Plates
designated as 3AT are Sc-L-T-H (lack leucine, tryptophan, and
histidine), and contain 10 mM 3-amino triazole (3AT). Plates
designated as X-gal contain Sc-L-T medium and contain 20 mg/ml
5-bromo-4-chloro-3-indolyl-.beta.-D-galactopyranosid- e (X-gal)
which serves as substrate for .beta.-galactosidase.
[0165] FIG. 9 is a schematic representation of an example of the
reverse two-hybrid method used to generate a collection of
interacting molecules (i.e., a bidirectional combinatorial library
(BCL)).
[0166] FIG. 10A is a schematic representation of plasmids into
which the CYH2 counterselectable marker was inserted. FIG. 10B is a
schematic representation of the plasmids used to create hybrid
proteins with the GAL4-AD or GAL4-DB.
[0167] FIG. 11 is a chart summarizing the results of a
unidirectional (i.e., classical) two-hybrid screen performed with
MaV103. When compared to conventional two-hybrid systems, the
number of positives was relatively low. "Retested" refers to clones
that score positive for the three phenotypes. X->Y refers to the
number of X clones identifying Y proteins.
[0168] FIG. 12 is a photograph of yeast cells containing synthetic
libraries which contain two self-activating clones. The bottom left
panel is a photograph of a plate containing a Sc-L-T-H medium and
which contains 3AT. The cells growing on the plate in the
bottom-right panel were replica-plated from Sc-L to Sc-l+5-FOA to
Sc-L-T-H+3AT. As a negative control, the Sc-L plate was also
directly replica-plated onto 3AT plates lacking histidine, and the
resulting cells are shown in the bottom left panel. The large
patches on the right side of each plate represent control cells.
From top to bottom, the controls are pPC97/pPC86, Db-pRb/AD-E2F1,
Fos/Jun, and intact Gal4.
[0169] FIG. 13 is a chart which summarizes the interactions
observed with the synthetic libraries.
[0170] FIG. 14 is a photograph of yeast cells in which E1A is
overexpressed in cells which expressed either AD-E2F1 and DB-pRb,
or AD-E2F1 and DB-p107 hybrid molecules. Control strains are
wild-type URA3 (two patches on right side of each panel) and
ura3-52 mutant strains (two patches on left side of each panel).
The cells were grown on synthetic complete medium lacking leucine
and tryptophan (Sc-L-T), synthetic complete medium lacking uracil
(Sc-ura), or synthetic complete medium lacking leucine and
tryptophan and containing 5-FOA (Sc-L-T+FOA), as indicated. E1a#2
and E1a#4 refer to amino acids 30-132, and amino acids 30-86 and
120-139, respectively.
[0171] FIG. 15 is a photograph of yeast cells indicating that the
inability of the mutant, pRb.DELTA.22, to interact with E2F1 can be
detected with the invention. Control strains are wild-type URA3
(patch on left side of each panel) and ura3-52 mutant strains
(patch on right side of each panel). The cells were grown on
synthetic complete medium lacking leucine and tryptophan (Sc-L-T),
synthetic complete medium lacking uracil (Sc-ura), or synthetic
complete medium lacking leucine and tryptophan and containing 5-FOA
(Sc-L-T+FOA), as indicated.
[0172] FIG. 16 is a schematic representation of a two-step
selection method used to identify residues in E2F1 which mediate
its ability to interact with DP1.
[0173] FIG. 17 is a photograph of yeast cells indicating that the
GAL1:HIS3 and the SPAL9:URA3 reporter genes confer "titratable"
phenotypes.
[0174] FIGS. 18A and 18B are schematic representations of the
strategies used for PCR mutagenesis and in vivo gap repair.
[0175] FIG. 19 is a series of photographs showing growth of yeast
cells in the first and second steps of the two-step selection
method. At each step, surviving colonies were transferred by
replica-plating (RP). Control strains are wild-type URA3 (two
patches on right side of each panel) and ura3-52 mutant strains
(two patches on left side of each panel). The cells were grown on
synthetic complete medium lacking leucine and tryptophan (Sc-L-T),
synthetic complete medium lacking uracil (Sc-ura), or synthetic
complete medium lacking leucine and tryptophan and containing 5-FOA
(Sc-L-T+FOA), as indicated.
[0176] FIG. 20 is a series of photographs which display the
phenotypes of the E2F1 alleles obtained in the second step of the
two-step selection method.
[0177] FIG. 21 is a schematic representation of the Marked Box 2
domain and the mutations obtained with the two-step selection
method.
[0178] FIG. 22 is a schematic representation of E2F1 and its
previously described functional domains.
[0179] FIG. 23A is a chart summarizing a two-step selection method.
FIG. 23B is a schematic representation of a two-step method for
identifying conditional alleles (i.e., CATS).
[0180] FIG. 24 is a series of photographs of yeast cells expressing
DB-Fos and conditional alleles of AD-Jun. This figure indicates
that a conditional allele of Jun prevents AD-Jun and DB-Fos from
interacting at 30.degree. C. but not at 36.degree. C.
[0181] FIG. 25 is a schematic representation of a strategy useful
for identifying antigen/antibody interactions.
ABBREVIATIONS
[0182] Abbreviations used herein include:
[0183] AA amino acid
[0184] AD activation domain
[0185] DB, DBD DNA-binding domain
[0186] 5-FOA 5-fluoro-orotic acid
[0187] GBS GAL4 binding sequence
[0188] ORF open reading frame
[0189] URS upstream repressing sequence
[0190] Prom promoter
[0191] Term terminator
[0192] CEN centromere
[0193] ARS yeast origin of replication
[0194] RP replica-plate
[0195] 2 mu yeast 2 micron plasmid origin of replication
[0196] ORI bacterial origin of replication
[0197] 3AT 3-amino triazole
[0198] Before providing detailed examples of the invention, several
parameters of the invention are described.
[0199] Standard Two-hybrid System: The yeast two-hybrid system has
been used to detect the association of pairs of proteins (see,
e.g., Fields et al., U.S. Pat. No. 5,283,173). This method involves
in vivo reconstitution of two separable domains of a transcription
factor. The DNA binding domain (DB) of the transcription factor is
required for recognition of a chosen promoter. The activation
domain (AD) is required for contacting other components of the
cell's transcriptional machinery. In this system, the transcription
factor is reconstituted through the use of hybrid proteins. One
hybrid is composed of the AD and a first protein of interest. The
second hybrid is composed of the DB and a second protein of
interest. In cases where the first and second proteins of interest
interact with each other, the AD and DB are brought into close
physical proximity, thereby reconstituting the transcription
factor. Association of the proteins can be measured by assaying the
ability of the reconstituted transcription factor to activate
transcription of a reporter gene.
[0200] Useful reporter genes are those which are operably linked to
a promoter that is specifically recognized by the DB. Typically,
the two-hybrid system employs the yeast Saccharomyces cerevisiae
and reporter genes whose expression can be selected under
appropriate conditions. The two-hybrid system provides a convenient
method for cloning a gene encoding a protein which interacts with a
second, preselected protein. In such an experiment, a cDNA library
is constructed in order to fuse randomly generated sequences fused
to the AD, and the protein of interest is fused to the DB. In this
"unidirectional" screening method, proteins expressed from one
library of clones are tested for their ability to interact with one
pre-selected protein of interest. Methods employing two libraries
of clones (one fused to the AD and one fused to the DB) have not
been described.
[0201] Reporter Genes: The reporter genes described herein can be
located on a plasmid or can be integrated into the genome of a
haploid or diploid cell. The reporter gene whose expression is to
be assayed is operably linked to a promoter which has sequences
that direct transcription of the reporter gene. The reporter gene
is positioned such that it is expressed when a gene activating
moiety of a transcription factor is brought into close proximity to
the gene (e.g., by using hybrid proteins to reconstitute a
transcription factor, or by covalently bonding the gene-activating
moiety to a DNA-binding protein). The reporter gene can also be
operably linked to regulatory sequences which render it highly
responsive to the presence or absence of a transcription factor.
For example, in the absence of a specific transcription factor, a
highly responsive URA3 allele confers a Ura.sup.- Foa.sup.r
phenotype on the cell. In the presence of a specific transcription
factor, a highly responsive URA3 allele confers a Ura.sup.+
Foa.sup.s phenotype on the cell. Where the cell carrying the
reporter gene (i.e., a transformed yeast cell) normally contains a
wild-type copy of the gene (e.g., the URA3 gene), the exogenous
reporter gene can be integrated into the genome and replace the
wild-type gene. Conventional methods and criteria can be used to
connect a reporter gene to a promoter and to introduce the reporter
gene into a cell.
[0202] Promoters: Suitable promoters for expression of a reporter
gene are those which, when linked to the reporter gene, can direct
transcription of it in the presence of appropriate molecules (i.e.,
proteins having transcriptional activation domains), and which, in
the absence of a transcriptional activation domain, do not direct
transcription of the reporter gene. An example of a useful promoter
is the yeast SPO13 promoter. Other useful promoters include those
promoters which contain upstream repressing sequences (see, e.g.,
Vidal et al., 1995, Proc. Natl. Acad. Sci. USA 92: 2370-2374) and
which inhibit expression of the reporter gene in the absence of a
transcriptional activation domain. The ability of a promoter to
direct transcription of a reporter gene can be measured with
conventional methods of assaying for gene expression (e.g.,
detection of the gene product or its mRNA, or detection of cell
growth under conditions where expression of the reporter gene is
required for growth of a cell).
[0203] Conventional molecular biology techniques can be used to
construct derivatives of promoters which include one or more
DNA-binding-protein recognition sites. For example, the SPO13
promoter can be engineered to include one or more copies of the
GAL4 binding sequence (GBS). The DNA binding sites in natural
promoters for GAL4 have been extensively characterized, allowing
the creation of a synthetic sequence to which GAL4 binds with
relatively high affinity. URA3 alleles that are operably linked to
a SPO13 promoter are referred to as SPALX:URA3, for 13/GAL/URA3; X
represents the number of GBSs present in the promoter. Other useful
DNA-binding-protein recognition sites include the LexA and Ace1
binding sites. In addition, where the ability of a protein to bind
to a DNA sequence is measured, the DNA-binding-protein recognition
site can be a wild-type DNA-binding-protein recognition site, or it
can be any intentionally-designed or randomly-generated sequence of
interest in order to test the ability of the DNA sequence to
interact with a protein.
[0204] Yeast Strains: The yeast strains used in the invention can
be grown and maintained with standard methods. Saccharomyces
cerevisiae are particularly useful in the invention. In certain
aspects of the invention, mating of two mating competent yeast
cells is desired. For example, in certain methods, a hybrid protein
which includes an activation domain is expressed in one mating
competent cell, and a hybrid protein which includes a DNA-binding
domain is expressed in a second mating competent cell. In such a
case, the transcription factor is reconstituted by mating the first
and second mating competent cells. Obviously, the two mating
competent cells should be of compatible mating types. For example,
one mating competent cell can be of the MATa mating type, and the
other mating competent cell can be of the MAT.alpha. mating type.
It is inconsequential which hybrid protein is expressed in which
cell type.
[0205] A preferred yeast cell for characterizing molecular
interactions has, integrated into its genome, a counterselectable
reporter gene which is operably linked to a promoter which has (i)
an upstream repressing sequence, and (ii) a DNA-binding-protein
recognition site. The preferred yeast cell lacks (i) a
naturally-occurring protein which is substantially identical to the
protein encoded by the counterselectable reporter gene, and (ii) at
least one naturally-occurring protein which, when it is expressed
(e.g., from a plasmid), confers a growth advantage on a cell
containing it. In addition, a yeast cell can contain, integrated
into its genome, a selectable marker (e.g., HIS3) and/or a gene
whose expression can be screened (e.g., lacZ) Where three such
genes (i.e., a counterselectable reporter gene, a selectable
marker, and a screenable marker) are integrated into the genome of
a cell, it is preferred that the promoters of the three genes be
distinct with the exception of the DNA-binding-protein recognition
site (FIG. 1). The use of distinct promoters decreases the
likelihood of obtaining false positives.
[0206] We have constructed a set of yeast strains having the
following features: (i) a set of non-reverting auxotrophic
mutations for selection of the two plasmids expressing the
two-hybrids and dependence upon GAL1:HIS3 expression on medium
lacking histidine: leu2, trp1, and his3; (ii) two recessive drug
resistance mutations (can1 and cyh2) to facilitate plasmid
shuffling; and (iii) three integrated GAL4-inducible reporter genes
(Gal1:HIS3, Gal1:lacZ, and SPAL:URA3; FIG. 1). Yeast strains of
both mating types (MAT.alpha. and MATa) having these features were
constructed.
[0207] Of particular use in the invention are the yeast strains
MaV103 and MaV203, described below. Where uptake of a test compound
(e.g., a potential dissociator) is desired, the erg6 mutant strain
is particularly useful because of its relatively high ability to
take up compounds. Other methods of permeabilizing the yeast cell
may also be employed; these include treatment with chemicals such
as polymixin B nonapeptide.
[0208] Construction of Plasmid p2.5: We have designed a novel
plasmid, termed p2.5, which is useful for synthesizing dissociator
compounds (e.g., proteins or RNA molecules) that can be tested in
the invention (FIG. 2). More generally, this plasmid can be used to
express preferred genes in yeast cells. This plasmid allows for the
creation of cDNA libraries encoding dissociator compounds, and it
offers the following features: (i) a 2 .mu.m sequence which allows
the plasmid to be maintained at high copy numbers; (ii) a
selectable marker which, preferably, allows the plasmid to be
selected for independently of the genetic constructs (i.e.,
plasmids) encoding the hybrid proteins or hybrid RNA molecules used
in the invention; (iii) a yeast ADH1 promoter, which is a strong
constitutive promoter; (iv) a GAL4 recognition site; (v) a nuclear
localization signal located upstream of the polylinker,
facilitating transport of the encoded polypeptide to the nucleus of
the host cell; and (vi) a bacterial origin of replication. Plasmid
p2.5 was generated by inserting the XhoI-XhoI fragment of pPC86,
which contained the ADH1 promoter, into the XhoI site of pRS323,
and subsequently the SalI-BamHI fragment of pPC86 containing the
polylinker and the ADH1 terminator was inserted into the SalI-BamHI
sites of the pRS323 (Sikorski et al., 1989, Genetics 122:
19-27).
[0209] Construction of Plasmids for Producing Hybrid Proteins:
Plasmids p97.CYH2 and pMV257 are useful in the invention for
producing hybrid proteins having a GAL4-DB or AD, respectively,
fused to a potential interacting molecule of interest (FIG. 10B).
These plasmids are produced by inserting a sequence encoding CYH2
into pPC97 (for DB plasmids) or pPC97' (for AD plasmids) (FIG.
10A). Both p97.CYH2 and pMV257 have (i) a yeast ARS4 origin of
replication; (ii) a yeast CEN6 centromeric sequence; (iii) a
selectable marker (e.g., LEU2 for pPC97, and TRP1 for pPC86); (iv)
a yeast ADE1 promoter and terminator; (v) a GAL4-DB (for pPC97) or
a GAL4-AD (for pPCB6); (vi) an SV40 large T antigen sequence
encoding a nucleolar signal sequence positioned in frame with the
DB or AD domain; (vii) a bacterial origin of replication; and (vii)
a CYH2 counterselectable marker. Those skilled in the art recognize
that numerous similar plasmids can be used to produce hybrid
proteins. For example, hybrid proteins that include the DB or AD of
VP16 (from Herpes Simplex Virus or Ace1 can be produced with
plasmids having, in place of the GAL4-DB or -AD, sequences encoding
the VP16 or Ace1 DB or Ace1 AD. Similarly selectable markers other
than Leu2 and Trp1 can be used. These plasmids can be constructed
with conventional molecular biology methods. Generally, in order to
select for a yeast cell containing one of these plasmids, the yeast
cell should not, in the absence of the plasmid, express a
functional gene product which corresponds to the selectable marker.
For example, a yeast cell into which p97.CYH2 is transformed should
have a leu2 mutation; thus, a transformant containing p97.CYH2 can
be selected on a medium which lacks leucine. The yeast strains
MaV103 and MaV203 are particularly useful in conjunction with
p97.CYR2 and pMV257.
[0210] Assay of Protein/Protein Interactions: The invention
provides a convenient method for identifying protein/protein
interactions. This method employs two populations of mating
competent cells (e.g., yeast cells). Conventional cloning
techniques can be used to operably link a
selectable/counterselectable reporter gene (e.g., a URA3 gene) to a
promoter (e.g., a SPO13 promoter) which contains at least one
recognition site for a DNA-binding-protein (e.g., a transcriptional
factor such as GAL4). If desired, conventional methods can be used
to integrate the selectable/counterselectable reporter gene into
the genome of a yeast cell.
[0211] Assay of Protein/RNA Interactions: Conventional cloning
methods can be used to express a variety of protein or RNA
molecules in yeast cells. The RNA-binding moieties and the
non-random RNA molecules to which they bind are unlimited.
Generally, it is preferable that the RNA-binding moiety be composed
of fewer than 50 amino acids. Preferably, the non-random RNA
molecule is between 10 and 1,000 nucleotides in length; more
preferably, the non-random RNA molecule is between 10 and 100
nucleotides in length. An example of a suitable RNA-binding moiety
and the non-random RNA molecule to which it binds is the iron
response element binding protein and the iron response element.
[0212] Assay of RNA/RNA Interactions: Numerous RNA/RNA interactions
can be identified with the reverse two-hybrid system of the
invention. Construction of appropriate expression plasmids for use
in this aspect of the invention can be accomplished with
commonly-known cloning methods. Non-random RNA molecules and
RNA-binding moieties which are useful in identifying protein/RNA
interactions are also useful for identifying RNA/RNA
interactions.
[0213] Assay of DNA/Protein Interactions: The invention can also be
used to characterize protein/DNA interactions. In this aspect of
the invention, the DNA sequence of interest (the "test DNA
sequence") is contained within a promoter which is operably linked
to a counterselectable reporter gene. In this sense, the test DNA
sequence serves as the DNA-binding-protein recognition site. The
protein of interest (the "test protein") is examined for its
ability to bind the test DNA sequence. In this aspect of the
invention, the "test protein" is produced as a hybrid protein with
a gene activating moiety, and binding of the hybrid protein to the
test DNA sequence activates transcription of the counterselectable
reporter gene. If desired, the test DNA sequence and/or the
sequence of the test protein can be intentionally designed,
randomly generated, or composed of both intentionally designed and
randomly generated sequences. If desired, the test DNA sequence
and/or the gene encoding the test protein can be derived from a
nucleic acid library. Thus, a bidirectional combinatorial library
can be created and screened in this aspect of the invention. The
methods described herein for characterizing protein/protein
interactions and for identifying compounds and mutations which
affect protein/protein interactions can, with appropriate
modifications, be used to characterize protein/DNA
interactions.
[0214] Identification of Dissociator Compounds: Potential
dissociator compounds can be introduced into cells by simply adding
them to cultures. Many potential dissociator compounds are small
enough that they will be taken up by a cell by endocytosis.
Alternatively, if the dissociator compound is an RNA molecule or a
protein, it can be produced in a cell by transforming the cell with
a DNA construct expressing the desired RNA or protein. Dissociator
compounds can be identified rapidly by first plating cells
harboring a reconstituted transcription factor onto a solid medium
under conditions such that the reconstituted transcription factor
directs expression of a counterselectable reporter gene. This
procedure creates a lawn of non-growing cells on the medium.
[0215] The compounds to be tested are then deposited in an ordered
fashion (e.g., to form a pattern, such as a grid) onto the lawn of
non-growing cells. Compounds that are added in solution to the
solid medium will diffuse slowly throughout the medium, creating a
gradient in the concentration of the compound in the medium.
Dissociator compounds can be identified by a growth of cells at the
site at which the compound was deposited because dissociation of
the transcription factor inhibits expression of the
counterselectable reporter gene which prevents cell growth. Cells
which grow in response to the addition of a dissociator compound
will also form a gradient; the largest number of cells likely will
grow at the position on the plate at which the dissociator compound
was added. At the very center of a growing colony of cells, there
may be a ring of non-growth due to toxicity of the compound at high
concentrations. The diameter of the ring of growth will reflect the
strength of the dissociator compound and reflect the concentration
of compound required for dissociation.
[0216] Optimization of Sensitivity: Typically, before a dissociator
is identified as such, its relative affinity for either partner of
an interacting pair of molecules is unknown. Thus, the preferred
conditions for identifying dissociators should permit recognition
of even small decreases in the transcriptional activity of reporter
genes. Conditions of maximum sensitivity can be established by
minimizing the number of DNA-binding-protein recognition sites in
the promoters of the reporter genes, and by using the lowest
concentration of a drug (e.g., 5-FOA) sufficient to confer a
drug-sensitive (e.g., Foa.sup.s) phenotype on the host cell.
[0217] We describe below several examples of various aspects of the
invention which provide guidance for practicing other embodiments
of the invention.
[0218] Inducible Expression of a Reporter Gene: To demonstrate that
expression of a reporter gene used in the invention can be induced
with a transcription factor, we measured the ability of a
reconstituted GAL4 protein to induce expression of a SPALX:URA3
allele. In this example, we employed the SPAL5:URA3 allele, which
carries 5 GBSs. We analyzed the Ura and 5-FOA phenotypes conferred
in the presence of (i) the full-length, wild-type GAL4 protein, or
(ii) the GAL4-DB (amino acids 1-147) and the GAL4-AD (amino acids
768-881), expressed as two separate molecules in the same cell.
Transformants that expressed the full-length GAL4 transcription
factor exhibited strong, tightly regulated Ura.sup.+ and Foa.sup.s
phenotypes, while transformants which expressed GAL4-DB and GAL4-AD
as two separate molecules exhibited strong and tightly regulated
Ura.sup.- and Foa.sup.r phenotypes because the cells lacked a
molecule capable of reconstituting the transcription factor. The
strength of the Foa.sup.s phenotype was comparable to the phenotype
exhibited by an untransformed wild-type control strain (FIG. 3). As
was expected, none of the proteins (GAL4, GAL4-DB, or GAL4-AD) had
any effect in cells containing a null allele of URA3 (ura3-52)
(FIG. 3).
[0219] Use of Two Hybrid Molecules to Reconstitute a Transcription
Factor: Here, we show that two hybrid molecules can be used to
induce expression of a reporter gene. We demonstrate this with two
different pairs of proteins; the proteins in each pair are known to
interact. The first pair of proteins, cFos and cJun, interact with
relatively high affinity. The second pair of proteins, pRb and
E2F1, interact with relatively low affinity. We have used these two
pairs of proteins and SPALX:URA3 alleles to demonstrate
reconstitution of the GAL4 transcription factor. In these
experiments, a total of four hybrid molecules were used. For the
first pair of proteins, the interaction domain of cFos was
covalently bonded (i.e., fused) to GAL4-DB, and the interaction
domain of cJun was covalently bonded to GAL4-AD. For the second
pair of proteins, the interaction domain of pRb was fused to the
GAL4-DB, and the interaction domain of E2F1 was fused to the
GAL4-AD (FIG. 4).
[0220] DNA molecules encoding these fusion proteins each were
constructed with a centromeric plasmid carrying an ADH1 promoter
and a selectable marker. In this case, plasmids expressing the DBs
carried the yeast LEU2 gene as a selectable marker; plasmids
expressing the ADs carried the yeast TRP1 gene as a selectable
marker. As negative controls, the GAL4-DB and GAL4-AD were
expressed separately and without the interaction domains of cFos,
cJun, pRb, or E2F1. To demonstrate that the Foa.sup.s phenotype
provides a sensitive measure of transcription, we compared the
ability of the proteins to induce a Foa.sup.s phenotype with their
ability to induce expression of .beta.-galactosidase activity from
a GAL4-inducible GAL1:lacZ reporter gene.
[0221] We found that the cFos and cJun interaction domains, and the
interaction domains of pRb and E2F1 were able to reconstitute the
GAL4 transcription factor in vivo. Cell cultures which expressed
the DB-cFos hybrid and the AD-cJun hybrid also produced significant
levels of .beta.-galactosidase activity from GAL1:lacZ. Similarly,
cell cultures which expressed the GAL4-DB-pRb hybrid and the
GAL4-AD-E2F1 hybrid produced significant levels of
.beta.-galactosidase activity from GAL1:lacZ. To provide a
quantitative assessment of the ability of DB-cFos and AD-cJun and
of DB-E2F1 and AD-pRb to reconstitute a transcription factor, the
.beta.-galactosidase levels obtained by reconstituting GAL4 with
these hybrid molecules was compared with the level obtained with an
intact, full-length GAL4 protein (FIG. 5). Transcription of the
GAL1:lacZ reporter gene induced by the intact GAL4 protein produced
3,000 .beta.-galactosidase-specific units. The GAL4 protein
reconstituted with DB-cFos and AD-cJun gave 100
.beta.-galactosidase-specific units. Transcription induced by
reconstitution of GAL4 with DB-pRb and AD-E2F1 produced only 0.5
.beta.-galactosidase-specific units. These data indicate that the
relatively strong interaction of cFos and cJun, and even the
relatively weak interaction of pRb and E2F1, can be detected in the
assay (FIG. 5).
[0222] Determination of the Limit of Growth Threshold: It is
useful, though not necessary, to determine the "limit of growth
threshold" in order to perform the counterselection methods under
the ideal conditions for detecting compounds or mutations that may
only weakly affect the interaction of two molecules. The limit of
growth threshold is the minimum concentration of a drug (e.g.,
5-FOA), in combination with the minimum number of GBSs, required to
prevent growth of a cell. The higher the required concentration of
the drug, the stronger the interaction between the two molecules
responsible for reconstituting the transcription factor. The number
of GBSs used in the invention can vary, if desired.
[0223] We defined the limit of growth threshold for three different
pairs of interacting proteins which reconstitute the GAL4
transcription factor: (i) cFos/cJun, (ii) cJun/cJun, and (iii)
pRb/E2F1. Control cells which lacked a GBS in the SPO13:URA3
promoter were not sensitive to 5-FOA, even in the presence of a
GAL4 protein. Similarly, cells which expressed the GAL4-DB or
GAL4-AD in the absence of a polypeptide which enabled them to
associate (i.e., an interaction domain) also were resistant to
5-FOA, irrespective of the number of GBS. In contrast, cells in
which GAL4 was reconstituted with cFos/cJun, cJun/cJun, or pRb/E2F1
displayed a 5-FOA sensitive phenotype.
[0224] In this example, the relative strengths of the interactions
responsible for reconstituting the transcription factors are:
cFos/cJun>cJun/cJun>pRb/E2F1. A gradient of 5-FOA sensitivity
was observed on varying concentrations of 5-FOA in the context of
increasing numbers of GBSs over a range of concentrations of 5-FOA
for each interaction that was tested. These data indicate that the
limit of growth threshold is 0.05% 5-FOA for cFos/cJun, 0.1% 5-FOA
for pRb/E2F1, and 0.2% for cJun/cJun (FIG. 6).
[0225] Assay of Plasmid p2.5: To provide evidence of the
operability of the plasmid p2.5, we confirmed that this plasmid
does not erroneously affect transcription. We constructed
derivatives of p2.5 which expressed pRb (p2.5pRB) without
expressing an AD. When p2.5pRB was introduced into yeast cells that
expressed intact CAL4, the plasmid did not affect the Ura or Foa
phenotype of the host cell, indicating that the plasmid did not
affect GAL4-dependent transcriptional function. This result
indicates that pRb did not have a positive effect on expression of
SPAL:URA3. This plasmid did produce significant quantities of pRb,
as expression of this plasmid in cells conferred an Foa.sup.s
phenotype on cells expressing DB-pRb and AD-E2F1 (FIG. 7). We have
shown by Western blot analysis that the expression levels of the
hybrid molecule was unchanged in cells harboring the p2.5pRB
plasmids. These findings indicate that the p2.5 plasmids are useful
for expressing potential dissociator compounds to be tested with
the invention.
[0226] Construction of Yeast Strains Containing SPAL:URA3 Alleles:
A SPO13:URA3 construct was obtained from plasmid pPL128 (from R.
Strich and R. Esposito PUBLISHED????). This construct includes a
fully functional SPO13 promoter and an ORF encoding a fusion
protein having the first 15 amino acids of SPO13 fused to the
full-length Ura3 protein, excluding the first methionine codon.
Prior to insertion of the GAL4 binding sites (GBSS), the SPO13:URA3
fragment was excised from pPL128 with a SmaI-BamHI double digestion
and cloned into a pBSK plasmid (Stratagene) which had been digested
with ClaI, treated with Klenow, and subsequently digested with
BamHI. The resulting plasmid, pMV252, contains within the SPO13
promoter, two EcoRI sites at nucleotides -170 and -368, and a
unique HindIII site at -213. The GBSs were derived from plasmid
GAL4-5/E1bCAT (Lillie et al., 1989, Nature 338: 39-44). A fragment
containing 5 GBSs was excised from this plasmid with a HindIII-XbaI
double-digestion, and the fragment was subsequently blunt-ended
with Klenow. The resulting fragment was cloned into pMV252 which
had been digested with EcoRI and treated with Klenow. By sequence
and PCR analysis, we identified two plasmids, pMV262-11 and
pMV262-12, that contain 5 and 15 GBSs, respectively.
[0227] The SPAL:URA3 constructs were introduced into the yeast
genome by integrative recombination at the ura3-52 locus by
homologous recombination of the product of a polymerase chain
reaction (i.e., by the gap repair method), generating the
respective SPAL:URA3 alleles. The 5' primer was JB516 which
contains 40 nucleotides of the URA3 sequence upstream of its
promoter (-257 to -218) fused to 20 nucleotides of the SPO13
promoter (-370 to -351)
(5'-GAAGGTTAATGTGGCTGTGGTTTCAGGGTCCATAAAGCT-
GTCCTGGAAGTCTCATGGAG-3'; SEQ ID NO: 1) (Rose et al., 1984 Gene 29:
113-124; Buckingham et al., 1990, Proc. Natl. Acad. Sci. USA 87:
9406-9410). The 3' primer was 3'URA3 (nucleotides +656 to +632 of
URA3) (5'-TCAGGATCCCTAGGTTCCTTTGTTACTTCTTCCG-3'; SEQ ID NO: 2)
(Rose et al., 1984 Gene 29: 113-124). Standard PCR reaction
conditions using pMV262-11 or pMV262-12 as templates generated
either a product of the expected size (1,000 bp) or a mixture of
products ranging from 1,000 to 1,300 bp, respectively.
[0228] The PCR products were transformed directly into the yeast
strain MaV82, and transformants were selected on a medium which
lacked uracil. The yeast strain MaV82 is MaV52 transformed with
pCL1, a plasmid expressing GAL4 (Fields, et al., 1989, Nature 340:
245-246). MaV52 (MATa ura3-52 leu2-3, 112 trp1-901 his3.DELTA.200
ade2-101 gal4.DELTA.gal80.DELTA. GAL1:lacZ GAL1:HIS3@lys2 can1R
cyh2R) was obtained by 5-FOA selection (to eliminate
GAL1:lacZ@URA3) and subsequent Can selection of Y153 (Boeke et al.,
1984, Mol. Gen. Gen. 197: 345-346; and Durfee et al., 1993, Genes
and Development 7: 555-569). A double homologous recombination
event or a gene conversion event at the ura3-52 locus is expected
using the 40 nucleotides in the 5' end of the PCR product, and the
320 nucleotides between the Ty insertion of ura3-52 and the 3' end
of the PCR product (Rothstein, 1983, Methods Enzymol. 101: 202-211;
Baudin et al., 1993, Nucleic Acids Research 21: 3329-3330; and Rose
et al., 1984, Mol. Gen. Genet. 193: 557-560).
[0229] Approximately 50% of the transformants exhibited the
expected GAL4-dependent Ura.sup.+ phenotype as tested by pCL1
plasmid loss. Integration of the SPAL:URA3 alleles was confirmed,
and the number of GBSs was estimated in a PCR reaction using
genomic DNA as a template. Of the different transformants, MaV99
contained 10 GBSs and is therefor SPAL10:URA3. The 5' primer was
JB536 (nucleotides -298 to -276 of the URA3 sequence;
5'-GCGAGGCATATTTATGGTGAAGG-3; SEQ ID NO: 3). The 3' primer was 13-5
(nucleotides -124 to -145 of the SPO13 antisense sequence;
5'-CATTTCCGTGCAAGGTACTAAC-3'; SEQ ID NO: 4) (Buckingham et al.,
1990, Proc. Natl. Acad. Sci. USA 87: 9406-9410). Strains MaV10
(MATa, lacks the GAL1:HIS3 fusion) and MaV103 (MATa, contains the
GAL1:HIS3 fusion) and MaV203 (MAT.alpha., contains the GAL1:HIS3
fusion). MaV103 and MaV203 are meiotic segregants of a cross
between MaV99 and PCY2 (Chevray et al., 1992, Proc. Natl. Acad.
Sci. USA 89: 5789-5793).
[0230] Plasmid Constructions: The cFos and cJun hybrid proteins
(DB-cFos, AA 132-211 (pPC76); DB-Jun, AA 250-334 (pPC75); AD-cJun,
AA 250-334 (pPC79)) have previously been described (Chevray et al.,
1992, Proc. Natl. Acad. Sci. USA 89: 5789-5793). Other proteins
were generated by cloning PCR products so that they are in frame
with the GAL4-DB (AA 1-147) or the GAL4-AD (AA 768-881) with
plasmids pPC97 (for GAL4-DB) (pPC97 is pPC62 containing the pPC86
polylinker), or pPC86 (for GAL4-AD) (Chevray et al., 1992, Proc.
Natl. Acad. Sci. USA 89: 5789-5793). To produce proteins having
wild-type sequences, the PCR products were also cloned into
p97.CYR2. The CYH2 gene on this plasmid facilitates plasmid
shuffling and removal of the plasmid from a cell. DB-pRb included
AA 302-928 of pRb; DB-pRb.DELTA.22 included AA 281-894 of a mutant
pRb having a deletion of exon 22; DB-p107 included AA 372-1068 of
p107; AD-E2F1 included AA 342-437 of E2F1; AD-E2F1Y411C included AA
342-437 of mutant E2F1 having a tyrosine to cysteine change at AA
411; and AD-E2F4 included AA 1-413 of E2F4 (Hiebert et al., 1992,
Genes & Development 6: 177-185; Whyte et al., 1988, Nature 334:
124-129; Helin et al., 1993, Mol. Cell. Biol. 13: 6501-6508; Sardet
et al., 1995, Proc. Natl. Acad. Sci).
[0231] The p2.5 derivatives were generated by cloning PCR products
into p2.5: E1A#2 included AA 30-132 of E1A; E1A#4 included AA 30-86
and 120-139 of E1A; E1A-CR1 included AA 1-120 of E1A; pRB included
AA 302-928 of pRb; and E1A-CR2 included AA 76-139 of E1A. To
isolate an AD-E2F1 hybrid which is capable of interacting with
DB-DP1 without being toxic to the host cell, we screened a cDNA
library in yeast cells expressing the DB-DP1 hybrid. Among other
potential interacting molecules, we isolated an AD-E2F1 fusion
which included AA 159-437 of E2F1.
[0232] Mutagenesis Gap Repair Method: The polymerase chain reaction
(PCR) mutagenesis gap repair method provides a convenient means for
mutagenizing a chosen sequence (Muhlrad et al., 1992, Yeast 8:
79-82). In this method, DNA encoding the sequence to be mutated is
amplified in a PCR reaction under conditions which favor
incorporation of incorrect nucleotides into the DNA molecule. Such
conditions include relatively high manganese levels and/or a
unequal mixture of the various nucleotides. The PCR primers which
are used in this method generate linear PCR products which have at
their ends sequences which are homologous to portions of a
linearized expression plasmid. Yeast cells then are co-transformed
with the linearized plasmid and the PCR products. At a high
frequency, repair of the linearized plasmid in vivo results in the
formation of stable circular plasmids containing the mutagenized
sequence.
[0233] Compensatory Mutations: Compensatory mutations are mutations
in pairs of interacting molecules (e.g., RNA molecules or proteins)
which allow the mutated molecules to interact with each other but
not with the corresponding wild-type proteins or RNA molecules.
Examples of compensatory mutations include mutations which result
in a reversal of charged residues that contact each other. For
example, in two wild-type proteins (X and Y), a positively charged
residue in the interacting molecule X contacts a negatively charged
residue in interacting molecule Y. Compensatory mutations in X and
Y may mutate X so that it contains a negatively charged residue,
and mutate Y so that it contains a positively charged residue as a
site of interaction. Compensatory mutations may also involve
alterations in the sizes of interacting domains of the molecules.
For example, if a portion of interacting partner X fits into a
cavity of interacting molecule Y, compensatory mutations in X may
render the interacting domain larger in size, and compensatory
mutations in Y may render the interacting cavity larger in size to
accommodate the larger interacting domain of X.
[0234] Knowledge of compensatory mutations in interacting molecules
is of value to scientists because often these mutations are located
at sites which are critical for interaction of two molecules.
Compensatory mutations are thought to define key residues involved
in molecular interactions, such as contact residues or amino acids
or ribonucleotides which are responsible for proper folding of the
interacting molecules. To date, in the instances where compensatory
mutations have been identified in a protein and the protein's X-ray
crystal structure is known, there has been a significant
correlation between the interacting residues identified by the
crystal structure and the interacting residues identified with
compensatory mutations. The identification of residues which play
such a vital role in the function of a molecule is critical for the
rational design of therapeutic compounds which function by
disrupting undesired (i.e., disease-related) interactions between
proteins and/or RNA molecules.
[0235] Conditional Mutants: The study of the structure and function
of proteins and RNA molecules is facilitated by the identification
of conditional mutants of the molecules of interest. These
conditional alleles allow wild-type function under permissive
conditions, yet, when the cells are shifted to restrictive
conditions, there is a detectable change in the ability of a
molecule to function. The isolation of conditional alleles is
complicated by the fact that they occur at relatively low frequency
due to the fact that the resulting structural and/or functional
alterations are often subtle. In many classical methods, the genes
encoding interacting molecules are modified in vitro with methods
directed to creating either large deletions or site-directed
mutations. Such methods can be time-consuming. In addition,
classical methods do not enable one to select alleles that are (i)
functional under conditions that have been designated permissive
and (ii) non-functional under conditions that have been designated
restrictive.
Identification of Protein/Protein Interactions with Proteins
Encoded within Synthetic Libraries
[0236] Construction of Yeast Strains Containing Synthetic
Libraries: We have characterized the phenotype of the yeast strain
MaV103, and tested the reverse two-hybrid system with this strain
and with MaV203 and various hybrid proteins (FIG. 8). To
demonstrate the operability of the reverse two-hybrid method of the
invention, we used two synthetic libraries having a limited number
of unknown parameters to carry out reconstruction (i.e.,
reconstitution) experiments designed to determine (i) whether it is
possible to use the mass mating method to identify interactions at
a frequency of 10.sup.-6 in a bidirectional library, and (ii) the
efficiency of the counterselection method used to eliminate
self-activating mating competent clones prior to formation of mated
cells. The strategy used to create this "Bidirectional
Combinatorial Library" (BCL) is outlined in FIG. 9.
[0237] Construction of Synthetic Libraries: For the library of
clones having a polypeptide fused to a DNA binding moiety, the
GAL4-DB, was used (FIG. 10). We used the GAL4-DB vector to create
plasmids encoding 15 hybrid proteins which included various forms
of pRb, p107, p130, p21, cyclin D2, cFos, cJun, DCC1, or dE2F (FIG.
11). To dilute the plasmids encoding the 15 hybrid proteins, we
prepared a DNA mixture which contained 1 ng of each of the various
plasmids and 1 .mu.g of a plasmid which expressed the GAL4-DB alone
(i.e., not as a hybrid protein with another polypeptide). Because
each they contain an endogenous AD, both of the hybrid proteins
encoded by DB-DCC1 and dE2F are sufficient to activate
transcription of the reporter genes in the absence of any
polypeptide fused to GAL4-AD. Both of the hybrids are sufficient to
confer a 3AT resistant (in the absence of histidine) and 5-FOA
sensitive phenotype to the MaV103 cells. In this assay, these
hybrid proteins served as controls for the ability of the method to
detect and eliminate these false positives.
[0238] The GAL4-AD vector was used to assemble a synthetic library
of hybrid proteins having a polypeptide fused to an activation
domain (FIG. 10). The 15 polypeptides used to create the library of
hybrid proteins included various forms of cdk2, cJun, E2F-1, E2F-2,
E2F-3, or E2F-4 (FIG. 11). The library of AD hybrid proteins did
not include any self-activating clones (i.e., false positives). To
dilute the plasmids encoding the various hybrid proteins, we
prepared a DNA mixture which contained 1 ng of each of the various
plasmids and 1 .mu.g of a plasmid which expressed the GAL4-AD alone
(i.e., not as a hybrid protein with another polypeptide).
[0239] The mixtures of plasmids encoding the AD and the DB
molecules were separately transformed into yeast strains which
contained identical sets of reporter genes. One synthetic library
of plasmids was transformed into MaV203, a MAT.alpha. strain. The
other synthetic library of plasmids was transformed into MaV103, a
MATa strain. Which library is transformed into cells of which
mating type does not matter, provided that yeast of two compatible
mating types are used for the two libraries. The transformed yeast
cells were plated onto an agar medium lacking either leucine or
tryptophan, using either the LEU2 or the TRP1 marker, respectively,
to select for transformants. MATa Leu.sup.+ transformants were
haploid clones obtained with the library of polypeptides fused to
the GAL4-DB, and MATA Trp.sup.+ transformants were haploid clones
obtained with the library of polypeptides fused to the GAL4-AD.
[0240] Counterselection: Counterselection was used to eliminate the
mating competent clones which could independently activate
transcription. The Leu.sup.+ and Trp.sup.+ colonies obtained in the
first selection step were directly replica-plated, separately, to a
medium which included 0.2% 5-FOA (FIG. 12). On this medium, only
the colonies corresponding to the non-activator clones grew
further. If desired, the counterselection step can be repeated, and
in this case, the step was performed twice. As is shown in FIG. 12,
all of the clones which improperly activated transcription were
completely eliminated by counterselection on 5-FOA (the large
patches of cells on the right side of the plates represent controls
used in the experiment; compare the number of colonies recovered in
the absence of 5-FOA counterselection (bottom left panel) with the
number obtained with 5-FOA counterselection (bottom right panel).
After two rounds of 5-FOA counterselection, no self-activating
clones were detected on a medium lacking histidine and containing
3AT.
[0241] Mass Mating Method: Cells which survived the
counterselection step, indicating that they contained the
non-activator clones, were harvested and resuspended in liquid
media. Approximately 10.sup.10 cells from each of the two strains
of cells were resuspended, separately, in 10 mL of media, giving a
concentration of 10.sup.9 cells/mL. The two cell suspensions were
subsequently mixed together and incubated overnight under
conditions that favor formation of mated cells (i.e., mating). In
this case, the mixture of mating competent cells was spread onto a
15 cm plate containing YEPD, a rich medium, and the resulting mated
cells were re-plated on a medium which lacked both leucine and
tryptophan. Our data indicate that the efficiency of mating was
approximately 10%. Based on these data, we conclude that, if the
volume of the suspensions is increased up to a few liters, up to
10.sup.13 mated cells can be selected with the mass mating method.
These data suggest that by scaling up the reaction to a volume of a
few liters, as many as 10.sup.13 pairs of interacting proteins can
be generated and screened.
[0242] Selection: The mated cells which result from the mass mating
method were plated onto a solid medium that selects for the
presence of the plasmids encoding the AD and the DB. Here, a medium
lacking both leucine and tryptophan was used. The colonies which
grew on these plates were replica-plated onto a medium which lacked
leucine, tryptophan, and histidine, and which contained 20 mM
3AT.
[0243] For a negative control, we induced formation of diploid
cells from haploid cells that had been transformed exclusively with
plasmids encoding GAL4-DB or GAL4-AD without being fused to another
polypeptide. Of 5.times.10.sup.5 diploid cells generated from the
negative control, none of the diploids was able to survive on a
medium that lacked both leucine and tryptophan, indicating that no
false positives were obtained.
[0244] For a positive control, we constructed two synthetic
libraries of cells expressing either DB-cFos or AD-cJun hybrid
proteins. These libraries were diluted 1:100, and diploid cells
were formed and selected on plates lacking leucine, tryptophan, and
histidine. Under these conditions, surviving cells were obtained at
the expected frequency of approximately 10.sup.-4 (twelve
3AT-resistant colonies were obtained from approximately 50,000
diploids).
[0245] In contrast, cells containing the synthetic libraries give
rise to positive growing colonies on medium containing 3AT using
this procedure. Among, 5.times.10.sup.6 diploid tested, we
recovered 400 3AT-resistant colonies. The diploid cells in this
example were plated onto a medium lacking leucine and tryptophan
and then plated onto a medium lacking leucine, histidine, and
tryptophan, and containing 3AT. If desired, the mated cells can be
plated directly onto a medium containing 3AT and lacking leucine,
histidine, and tryptophan.
[0246] The 400 colonies that were recovered were tested for their
sensitivity to 5-FOA as a measure of the expression of the URA3
gene. They also were tested for .beta.-galactosidase activity on a
medium containing X-gal. Approximately 95% of the clones that were
tested expressed the URA3 and lacZ genes. Of these colonies, 120
were analyzed further. Plasmids were extracted from these colonies
and amplified in, and then extracted from, E. coli. We identified
by sequence analysis the inserts in plasmids encoding 80 pairs of
interacting proteins. The data obtained from the sequence analysis
(FIG. 13) indicate that (i) most of the expected interactions were
detected with the method; and (ii) the cFos/cJun interaction is
reconstituted at a high frequency, possibly due to the relatively
small sizes of the DNA encoding these polypeptides. Accordingly,
the invention provides a convenient and efficient method for
identifying protein-protein interactions.
Identification of Compounds which Disrupt Molecular
Interactions
[0247] Dissociation of a Reconstituted Transcription Factor: We
have tested the ability of the invention to detect inhibition of
transcription of a reporter gene where inhibition is caused by a
compound which disrupts (i.e., prevents or causes dissociation of)
the interaction of two molecules. This method can be used to
identify compounds (i.e., dissociators) which disrupt the ability
of two hybrid molecules to interact and mediate transcription.
Effective compounds cause a decrease in expression of the reporter
gene (e.g., SPALX:URA3). For example, where the reporter gene is
URA3, dissociator compounds confer a Foa.sup.r phenotype on the
host cell. Thus, the invention provides a convenient means for
identifying molecules which disrupt a protein/protein
interaction.
[0248] We have found that transcription can be blocked in this
system by overexpressing in a cell either one of the two
interacting proteins which lacks a DB or an AD. The overexpressed
interacting protein, which lacks a DB or AD, can compete with the
two hybrid molecules and prevent activation of transcription of the
reporter gene. These data provide evidence that dissociator
compounds can be produced in the cell and be identified with the
invention.
[0249] As another example of the ability of the invention to detect
dissociation of two interacting molecules, we overexpressed a third
protein, E1A, in cells which expressed either AD-E2F and DB-pRb, or
AD-E2F and DB-p107 hybrid molecules. We measured the ability of
adenovirus E1A protein to bind to pRb and p107 and cause
dissociation of pRb/E2F and p107/E2F4. In these studies, E1A was
expressed in yeast cells expressing AD-E2F and either DB-pRb or
DB-p107 by employing conventional cloning methods to insert the E1A
coding sequence into the polylinker of the plasmid p2.5. We found
that expression of EA in the yeast strains rescued the Foa.sup.s
phenotype (FIG. 14), indicating that the invention can detect
dissociation of both DB-pRb/AD-E2F and DB-p107/AD-E2F
interactions.
[0250] Several observations suggest that dissociation mediated by
E1A is specific: (i) overexpression of E1A did not affect the
steady-state levels of the various hybrid proteins; (ii) E1A
protein expression had no effect on the Foa.sup.5 phenotype
resulting from DB-DP1I/AD-E2F interactions; (iii) conserved region
II (CR2), known to be essential for pRb/E2F dissociation in
mammalian cells, was required for the Foa.sup.s phenotype; and (iv)
overexpression of pRb in the absence of any DB sequences rescued,
to the same extent as E1A, the Foa.sup.s phenotype in cells
expressing DB-pRb/AD-E2F1, but not the Foa.sup.s phenotype of
DB-p107/AD-E2F4 (FIG. 14).
[0251] Increasing the Strength of a Dissociator Compound: If
desired, the strength of a dissociator compound can be
characterized by examining the ability of the compound to
dissociate two interacting hybrid molecules (e.g., proteins) over a
range of drug (e.g., 5-FOA) concentrations that cause lethality.
For example, the first round of analysis can be performed with a
relatively low 5-FOA concentration (i.e., a concentration which is
close to the growth threshold) and with a low number of GBSs in
order to identify relatively weak dissociator compounds. In the
second round of analysis, the 5-FOA concentration and/or the number
of GBSs is increased, and more potent dissociators are identified.
The analysis can be repeated. This method is also useful in the
design of dissociator compounds. Weak dissociator compounds, once
identified, can be modified (e.g., by amino acid, nucleotide, or
chemical group substitution accomplished with standard techniques)
and then tested in subsequent rounds of analysis. Dissociator
compounds that have been rendered more potent by the modification
can be identified by their ability to promote cell growth (i.e.,
inhibit the interaction) under more stringent conditions (e.g., a
higher concentration of 5-FOA) than could the parental
molecule.
[0252] Use of a Diploid Yeast Strain to Identify Dissociator
Compounds: If desired, diploid strains of yeast carrying two copies
of a reporter gene can be used to identify dissociator compounds.
For example, the use of diploid strains carrying two copies of
SPALX:URA3 can reduce the probability that the appearance of an
Foa.sup.r clone is due to a spontaneous reversion of the Foa.sup.s
phenotype. Accordingly, the use of diploid strains increases the
sensitivity of the method. While dissociator compounds can be
identified in haploids or diploids, the use of diploids is
preferred.
[0253] We have found that mutations responsible for reversion of
the Foa.sup.s phenotype represented cis-acting mutations linked to
the SPAL:URA3 reporter genes. Theoretically, both cis- and
trans-acting mutations can lead to reversion of the Foa.sup.s
phenotype. Cis-acting mutations are likely to involve deletion of
the repeated GBSs in the promoters of the SPALX:URA3 allele, or
mutation of the URA3 ORF itself, while trans-acting mutations are
likely to represent gene conversion events between plasmid
sequences, or knockout mutations in the coding sequences of the
interacting molecules.
[0254] To characterize the nature of spontaneous mutations leading
to reversion of the Foa.sup.s phenotype, we assayed whether
expression of two reporter genes (GAL1:HIS3 and GAL1:lacZ) was
altered in the Foa.sup.r colonies (i.e., spontaneous mutants). Our
data indicate that expression of HIS3 and lacZ was not affected in
these cells, suggesting that the reversions represented cis-acting
mutations linked to the SPALX:URA3 promoter. Accordingly, diploid
strains of yeast, containing two copies of the SPALX:URA3 reporter
genes will decrease the frequency with which spontaneous revertants
appear. The frequency is calculated to be
10.sup.-6.times.10.sup.-6=10.sup.-12. The frequency of spontaneous
reversion can also be determined experimentally by comparing the
ratio of Foa.sup.r colonies arising from haploid cells expressing
the cFos/cJun hybrid proteins with that of diploid cells.
Use of Mutagenesis to Characterize Molecular Interactions
[0255] Identification of Mutant Interacting Molecules: We have also
tested the ability of the invention to detect physiologically
relevant mutations which abrogate interactions. An important
precept of the invention is that a mutation which dissociates the
interacting molecules should be able to reduce, to a detectable
extent, expression of the reporter gene to which the
DNA-binding-protein recognition site is operably linked. For
examples a mutation in the retinoblastoma protein of a pRb/E2F1
interacting pair should result in a Foa.sup.r phenotype in cells,
provided that the mutation involves a residue which participates in
the interaction of the two molecules. To test the ability of the
invention to detect decreases in transcription of the reporter
gene, we utilized a pRb allele that, due to a deletion of exon 22,
fails to associate with E2F1. We expressed this form of pRb as a
hybrid protein with the GAL4-DB and termed the hybrid protein
DB-pRb.DELTA.22. E2F1 was expressed as a hybrid protein with
GAL4-AD. We found that expression of these proteins in yeast
resulted in a Foa.sup.r phenotype even though the level of
expression of DB-pRb.DELTA.22 was comparable to the level of
expression of the wild-type pRb (FIG. 15). We also performed the
reciprocal experiment, which involves a hybrid protein having a
mutated allele of E2F1 (AD-E2FY411C) which fails to bind pRb.
Expression of this mutant allele also resulted in a Foa.sup.r
phenotype (FIG. 15). These data provide further evidence that the
reverse two-hybrid system of invention can be used to detect
mutations which prevent two molecules from associating.
[0256] Use of a Two-Step Selection Method to Identify Subtle
Mutations Which Define Structurally and Functionally Significant
Residues: We have used a two-step selection method to identify
residues in E2F1 which mediate its ability to interact with DP1.
This method relies upon the strategy outlined in FIG. 16. We first
identified mutations which affect the ability of DP1 and E2F1 to
bind to each other, and, in a second step, identified those which
do not completely abrogate interaction between the proteins. This
strategy was based on the premise that mutations which completely
destroy the ability of E2F1 to interact with DP1 may represent
uninformative mutations, such as those which alter the size of the
protein (e.g., non sense mutations, deletions, or insertions). This
method facilitates the identification of alleles (e.g., alleles
selected from a library of alleles) which mildly affect the
protein/protein interaction.
[0257] In this example of the two-step selection method, we used a
GAL1:HIS3 reporter gene (Durfee et al., 1993, Genes & Dev. 7:
555-569). This reporter gene is particularly well-suited for this
method because the His phenotype is titratable, i.e., the His
phenotype can be measured over a range of concentrations of 3AT, a
specific inhibitor of HIS3 enzymatic activity (FIG. 17). Cells in
which GAL1:HIS3 is expressed grow on a medium lacking histidine and
containing high concentrations of 3AT. In the present case,
expression of DB-DP1/AD-E2F1 allowed the cells to grow on a medium
containing up to 100 mM 3AT (FIG. 17). In this two-step selection
method, the first selection was performed with 0.1% 5-FOA, and the
second selection was performed with 10 mM 3AT (on a medium lacking
histidine).
[0258] In these experiments, a plasmid encoding the DB-DP1 hybrid
protein was transformed into the yeast strain MaV103 which contains
a SPAL10:URA3 allele. Transformants were selected on a medium which
lacked leucine. The E2F1 sequence was amplified by PCR, with a
plasmid encoding AD-E2F1 (AA 159-437 of E2F1) serving as a
template. The 5' primer which was used corresponded to a sequence
located in the coding sequence for AD. The sequence of the primer
was located approximately 100 bp upstream of the junction of AD and
the first amino acid (AA 159) of E2F1. The 3' primer that was used
corresponded to the sequence immediately adjacent to the stop codon
of the E2F1 ORF. Using these primers and this E2F1 template,
several PCR amplifications reactions were performed over a range of
conditions that are conducive to mutagenesis of the amplified
sequence. In these several reactions, the concentration of
manganese and/or the relative concentrations of nucleotides varied
according to conventional methods for using PCR to introduce
mutations in a sequence. While the optimal conditions for
mutagenesis depend on the length and sequence of the fragment being
amplified, suitable conditions give a mutagenesis frequency which
is high enough so that mutants can be detected among a number of
yeast colonies that can be practically screened on a single petri
plate, and yet the frequency is low enough to avoid multiple
mutations in the amplified sequence.
[0259] Gap Repair Method: The gap repair method was used to
incorporate the mutagenized sequences into a plasmid. (FIGS. 18A
and 18B). In this case, the AD-E2F1 plasmid was linearized by
digestion at a unique BglII site located in the middle of the E2F1
sequence. As an alternative, an "empty" AD plasmid that is
linearized in its polylinker can be used, provided that the PCR
primers for amplification of E2F1 correspond to plasmid sequences
and sequences in the PCR fragment.
[0260] For gap repair, 100 ng of the amplified PCR fragment and 100
ng of the linearized plasmid were co-transformed by the lithium
acetate method into yeast cells which expressed DB-DP1. In this
example, the transformants were selected on a growth medium which
lacked leucine and tryptophan. After two days of growth on a rich
growth medium, the first step of selection was performed by
replica-plating the transformants onto a medium which lacked
leucine and tryptophan and which included 0.1% 5-FOA (Sc-L-T+5FOA
medium) (FIG. 19). We detected a correlation between the number of
colonies on the plate and the concentration of manganese and the
composition of the nucleotides (i.e., the extent of mutagenesis).
Colonies which grew on a medium which included 5-FOA and which
lacked leucine and tryptophan were replica-plated onto plates
lacking leucine and tryptophan in order to allow recovery (FIG.
19).
[0261] For the second step in the selection, the colonies on these
plates were replica-plated onto plates which lacked leucine,
tryptophan, and histidine, and which contained low concentrations
of 3AT. Colonies which grew on these plates were expected to
contain a mutation in E2F1 which weakly affected the ability of
E2F1 to interact with DP-1 (FIG. 19). Data which are representative
of the data obtained with the two-step selection method are
provided in the Table 1.
1 TABLE 1 Number of Number of Number of Transformants 5-Foa.sup.R
3AT.sup.R no DNA 0 nt nt AD-E2F1 circular 10,000 2-3 0 AD empty
(pPC86) 10,000 10,000 0 PCR fragment alone 0 nt nt Linearized
plasmid alone 500 50 0 PCR + plasmid 10,000 500 20-30
[0262] To confirm the phenotype of the colonies which grew in the
second step of the selection process, the colonies were first
purified by picking them and streaking them for single colonies on
Sc-L-T plates. Four purified colonies were then patched onto Sc-L-T
plates, then replicated onto a medium lacking histidine and
containing 0.1% 5-FOA, 10 mM 3AT, and X-gal. Only the colonies were
still able to grow under these conditions were analyzed further.
Approximately 90% of the initially selected colonies passed this
additional test. DNA extracted from these cells was used to
transform E. coli cells, and transformed cells were selected on a
medium that included ampicillin. The resulting colonies contained
plasmids encoding either DB-DP1 or AD-E2F1 hybrid proteins.
Plasmids encoding AD-E2F1 were identified by restriction digest
analysis of DNA obtained from the transformed E. coli cells.
[0263] Plasmids encoding AD-E2F1 were re-introduced into yeast
cells containing the GAL1:HIS3 and SPAL10:URA3 alleles and which
expressed DB-DP1. Transformed cells were selected on Sc-L-T media.
Four transformants were patched onto a Sc-L-T medium then
replica-plated onto a medium lacking leucine, tryptophan, and
histidine, and containing 0.1% 5-FOA, 10 mM 3AT, and X-gal (FIG.
20). As a positive control, the wild-type DB-E2F1 allele was
reintroduced into the cells containing the GAL1:HIS3 and
SPAL10:URA3 alleles (FIG. 20, bottom row), and pPC86, an empty AD
plasmid (i.e., a plasmid lacking E2F1), served as a negative
control.
[0264] The AD-E2F1-34 allele provides an example of a plasmid which
does not retest the phenotypes expected of a mutant allele. In
other words, the growth and P-gal phenotypes of AD-E2F1-34 were
indistinguishable from wild-type AD-E2F1. The hypothesis that
AD-E2F1-34 was identical to the wild-type allele was confirmed by
sequence analysis of AD-E2F1-34 which did not reveal any mutations
in the sequence AD-E2F1-34. Although some wild-type alleles were
recovered in the shuttling process to E. coli, approximately 90% of
the recovered alleles were mutants, as is desired.
[0265] We sequenced 12 AD-E2F1 alleles, and in 11 of these 12
alleles, we detected a single nucleotide change in the 1.2 kb of
sequence encoding E2F1. In six of the alleles, the mutation mapped
to a domain that is termed the Marked Box 2 (MB2) domain (FIG. 21).
The MB2 domain is represented by a stretch of 18 amino acids. The
fact that the mutations are clustered within this 18 amino acid
region suggests that the MB2 domain is required for binding of E2F1
to DP1. Further support for the suggested role of the MB2 domain
comes from the observation that, between the five human E2F
proteins, there is a high degree of homology in this region of the
proteins (FIG. 21, top).
[0266] Additional support for the value of the two-step selection
method comes from the observation that there is a correlation
between (i) the various mutations that were produced and identified
with this method and (ii) the various phenotypes that were detected
(FIG. 20). For example, the E2F1-31 allele, which strongly affected
the interaction between E2F1 and DP1 (i.e., cells expressing this
allele exhibited a high level of resistance to 5-FOA (FIG. 20)),
was associated with a small in-frame deletion of the MB2 domain
(FIG. 21). In contrast, the allele containing two mutations,
E2F1-30, affected the interaction relatively mildly; cells
containing this allele grew poorly on 5-FOA. Although two mutations
were found in this allele, both mutations were at positions in the
MB2 domains which are not completely conserved between different
members of the E2F family (FIG. 21, top and bottom), suggesting
that these residues are less critical for the interaction. In
accordance with these data is the fact that the alleles which had
conservative mutations affected the interaction and the growth
phenotype to an intermediate extent. In these alleles (E2F1-20,
-32, and -65), the mutations replaced the isoleucine at amino acid
284 with either threonine or asparagine. If desired, these mutant
alleles can be reintroduced into yeast cells in order to examine
the function of the mutant gene products further.
[0267] Isolation of Relatively Strong Mutations by a Two-Step
Selection Method: We have isolated and sequenced eight alleles of
E2F1 which lacked the ability to interact with DP1 in the first
step of the two-step selection procedure (FIG. 19). Sequence
analysis of each of those alleles revealed a nonsense mutation,
deletion, or insertion which would result in truncation of the E2F1
protein. To avoid selection of truncated mutants, we used a
variation of the two-step selection method to identify mutant
alleles of E2F1 which are defective in their ability to bind to
DP1, but which retain their ability to interact with pRb. The
rationale underlying this approach is that, because the pRb binding
site is located at the C-terminal domain of the E2F1 allele (the
binding site is composed of amino acids 409-427 of amino acids
159-437 of E2F1), mutations which abrogate binding of E2F1 to DP1
without truncating the protein (i.e., affecting binding to pRb) can
easily be identified (FIG. 22). We have constructed a plasmid which
expresses a DB-pRb hybrid protein (amino acids 302-928 of pRb were
used).
[0268] For the first step of the selection method, cells are grown
on a Sc-L-T medium for two days, then replica-plated onto a
Sc-L-T+5-FOA (0.1%) medium. (as in FIG. 19). The plasmid expressing
DB-DP1 can be eliminated by growing the cells on non-selective
media, and cells that have lost the DB-DP1 plasmid while keeping
the AD-E2F1 plasmid can be identified by assaying for their ability
to grow on the appropriate selective media after replica plating.
An alternative method for identifying colonies that have lost the
DB-DP1 plasmid is to express a counterselectable marker on the
DB-DP1 plasmid and to grow the cells on a medium where expression
of the counterselectable marker is lethal (plasmid shuffling). For
example, the plasmid encoding DB-DP1 can be engineered to express a
CYH2 gene, and cells expressing DB-DP1 can be eliminated on a
medium containing cycloheximide. In the second step of the
selection, cells containing AD-E2F1 are mated with cells which form
a lawn on agar plates and which contain the DB-pRb plasmid, and
expression of the selectable reporter gene is measured. The
resulting mated cells are then tested on a medium lacking
histidine, leucine, and tryptophan and containing 10 mM 3AT. The
positive clones in this assay are representative of mutated, but
not truncated, E2F1 alleles. Among 350 Foa.sup.r colonies tested,
12 colonies scored positive after mating with cells containing
pRb.
[0269] In alternative embodiments of this method, a protein other
than E2F1 can be fused to the AD with conventional methods. If
desired, the protein to be mutagenized can be fused to the DB
instead of the AD. The transcription factor which is reconstituted
in this method can be one other than GAL4 (e.g., LexA or Ace1 can
be used). In addition, reporter genes other than URA3 and HIS3 can
be used, provided that combination of reporter genes allows for
counterselection in the first step and positive selection
(preferably with a titratable phenotype) in the second step.
[0270] Functional C-term Tag: To ensure that the mutant proteins
characterized in this two-step selection method do not simply
represent truncations of the wild-type protein, a functional C-term
tag can be covalently bonded to the C-terminal end of any protein
which can be expressed in the above clone. Such a functional C-term
tag would function like the pRb binding domain in the
above-disclosed example. A functional C-term tag is a stretch of
amino acids which includes a binding domain for a protein. The pRb
binding domain is particularly useful because, at 18 amino acids in
length, it is unlikely to dramatically alter the structure of the
protein being characterized. To assay for the presence of the
carboxyl terminus of the mutated protein, a protein which
specifically binds the functional C-term tag is introduced into the
cell as a hybrid protein with a DB (or an AD if the mutated protein
is fused to the DB). One can then assay the ability of the hybrid
protein expressed from the plasmid and the mutated protein present
as a hybrid to reconstitute a transcription factor. Positive
selection on an appropriate medium can be used to select for cells
which retain the full-length protein.
[0271] An alternative, but similar, method for identifying strong
mutations in the two-step selection method involves constructing a
tribrid protein consisting of GAL4-AD-E2F1-GFP (green fluorescent
protein) (Chalfie et al., 1994, Science 263: 802-805). In this
method, the green fluorescent protein serves as a functional C-term
tag, and alleles of the resulting fusion protein, AD-E2F1-Green,
can be assayed for their ability to interact with DB-DP1. Cells
express green fluorescent protein and in which hybrid proteins
interact can be identified by their 3AT-resistant, Foa-resistant,
.beta.-gal positive phenotype. In addition, cells expressing the
green fluorescent protein fluoresce under UV light. Thus, the green
fluorescent protein can be used in the selection of mutant alleles.
In the selection of strong and weak mutations, expression of normal
levels of the full-length interacting protein (e.g., E2F1) can be
confirmed by western blot analysis of cell extracts.
[0272] To determine whether the newly isolated alleles exhibit
similar phenotypes, protein binding assays can be used. For
example, each E2F allele can be tested in an in vitro binding assay
that involves amplifying, in a PCR reaction, the sequences encoding
the various E2F alleles. An example of an appropriate 5' primer is
one which has 25 nucleotides corresponding the phage T7 RNA
polymerase promoter sequence and 20 nucleotides that correspond to
the activation domain near the junction of the activation domain
and amino acid 159 of E2F1 (i.e., the first E2F1 amino acid). A
suitable 3' primer is one which corresponds to the 3' end of the
E2F1 sequence. The PCR products from amplification of this sequence
can be used in an in vitro transcription/translation system to
generate the corresponding proteins. The mutant proteins can be
bound to hybrid proteins having wild-type DP1 bound to
glutathione-S-transferas- e. Interacting pairs of proteins can be
purified with glutathione agarose beads, released from the beads,
and analyzed by SDS-polyacrylamide gel electrophoresis.
[0273] Identification of Compensatory Mutations: Additional
information about the mutations identified in the two-step
selection method can be gained by creating and identifying
mutations in the wild type partner (DP-1 in the example) that
restore interaction of the two proteins (here, E2F1 and DP-1). For
example, in this method, the sequence of DP-1 which encodes the
E2F1-binding domain is amplified and mutagenized by PCR. In
accordance with the gap repair method, the PCR products are then
co-transformed into yeast cells containing specific AD-E2F1 mutant
plasmids along with the DB-DP-1 plasmid linearized in the
corresponding region. The transformants then are replica-plated
onto a medium containing 3AT and lacking histidine, and the
surviving colonies are analyzed further. Each allele can be
amplified in E. Coli, sequenced, and re-introduced into yeast to
retest its phenotype to ensure that the pairs of mutants interact.
By carrying out this process for a number of alleles having a
variety of mutations, a genetic map representing the
protein/protein interactions can be constructed.
[0274] Isolation of a Relatively Large Set of Pairs of compensatory
Mutations by "Bivalent Genetics": The two-step selection methds and
the scheme leading to the construction of bidirectional
combinatorial libraries suggest the feasibility of a genetic method
referred to here as "bivalent genetics," by which it is possible to
select for large numbers of pairs of compensatory mutations in
genes encoding interacting molecules. In two independent
experiments, performed in yeast strains of different mating type,
libraries of mutations affecting an interaction are furst generated
according to the "two-step selection" procedure. In a second step,
these two libraries of mutant alleles are challenged with each
other by mass mating, and compensatory mutations (where the
interaction is restored) are selected in a set of steps similar to
the ones involved in the constrution of combinatorial libraries. In
particular, by "bivalent genetics" is meant a method by which
relatively large sets of pairs of compensatory mutations may be
recovered, and, by "two-step selection" is meant a method by which
informative mutations that affect moleular interactions in a
defined manner may be recovered.
[0275] Isolation of Conditional Alleles: The invention also
facilitates the production and identification of conditional
alleles of interacting molecules. Because the invention provides a
convenient method for screening a large number of mutant alleles
(approximately 10.sup.10), the invention facilitates the detection
of relatively rare conditional alleles. In this method, termed
Conditional Alleles in a Two-Step Selection (CATS), one of the two
interacting molecules is mutagenized in order to isolate
conditional mutant alleles that interact with the other, wild-type,
allele under certain conditions (i.e., permissive conditions) but
not under other conditions (i.e., restrictive conditions). Any of
numerous conditions, selected by the practitioner, can be used as
the permissive or restrictive conditions. Commonly, a difference in
temperature characterizes the distinction between permissive and
restrictive conditions, although the invention is not limited to
the use of alterations in temperature. For example, the presence of
absence of a drug can define the difference between a permissive
and a restrictive condition.
[0276] The CATS method relies upon the use of counterselection with
a selectable/counterselectable reporter gene and the method
resembles the more general two-step selection method described
above. A schematic representation of the strategy used for CATS is
provided in FIG. 23B. In this method, the desired interacting
molecules are fused, separately, to the DB and AD of a
transcription factor, and the employed yeast strain contains a
selectable/counterselectable reporter gene (e.g., a URA3 gene). PCR
mutagenesis methods (as described above) are used to mutate one of
the interacting partners, and the PCR products are introduced into
the cell with conventional methods for gap repair. Selectable
markers on the plasmids expressing the AD and the DB can be used to
select for repair of the gap and for maintenance of the plasmid
encoding the wild-type interacting molecule.
[0277] The resulting transformants then are replica-plated onto a
medium containing a drug (e.g., 5-FOA) which inhibits the growth of
cells expressing the counterselectable reporter gene, and the
transformants then are incubated under restrictive conditions. Of
the various transformants, only the cells which contain mutant
alleles affecting the interaction of the molecules of interest will
be selected for in this first (negative) selection step.
[0278] The second selection step selects for mutant alleles which
are functional under permissive conditions. The cells which
survived the first step are transferred (e.g., by replica-plating)
to a medium which positively selects for cells expressing the
selectable/counterselectable gene; these cells are incubated under
permissive conditions. Cells containing a conditional allele(s) of
one of the interacting molecules will grow.
[0279] The mutant alleles can then be recovered and characterized
by extracting the plasmid DNA and amplifying it in bacteria, then
characterizing the DNA and the encoded protein with conventional
methods. The conditional alleles identified with the invention
affect the ability of two molecules to interact, and thus these
conditional alleles point to residues or nucleotides that are
critical for interaction. As was described above, the
identification of the interaction domain of a molecule is critical
for the rational design of therapeutics and for a detailed
understanding of biological processes.
[0280] We have used CATS to isolate a conditional allele of cJun
which interacts with cFos at 36.degree. C. but not at 30.degree. C.
(FIG. 24). These data indicate that at 36.degree. C. in, cFos and
the mutant cJun reconstitute the GAL4 transcription factor, leading
to expression of URA3 and resulting in lethality when the cells are
grown on 5-FOA. In contrast, when the cells expressing the
conditional allele are grown at the restrictive temperature, the
interaction is prevented and the cells survive growth on 5-FOA.
Thus, these data indicate that the invention provides a convenient
method for isolating and identifying conditional alleles of
molecules which can be further characterized with conventional
techniques.
Other Embodiments
[0281] The interaction of numerous types of RNA molecules, DNA
molecules, or proteins can be measured in the invention. For
example, interactions which can be assayed in the invention include
interactions between antibodies and antigens, receptors and
ligands, a restriction enzyme and the DNA site it cleaves, and
viral proteins and host proteins. For example, the invention allows
for the identification of protein/protein interactions which occur
in the HIV provirus. In this method, HIV proteins are separately
expressed in the form of AD and DB hybrid proteins, and the ability
of the HIV proteins to reconstitute the intact transcription
factors is assayed. Thus, the invention provides a convenient
method for identifying all of the protein/protein interactions
encoded within an entire genome. The identification of HIV
protein/protein interactions facilitates the discovery of compounds
which exert a therapeutic activity by disrupting protein/protein
interactions. In a similar method, the invention can be used to
identify interactions between HIV proteins and proteins of
activated human T-cells.
[0282] The invention can also be used to isolate and characterize
monoclonal antibodies. In this method, an antigen/antibody binding
reaction is used to reconstitute a transcription factor. In this
method, an antigen and a DNA-binding moiety (e.g., the DB of GAL4)
are expressed as a hybrid protein; the immunoglobulin heavy chain
and a gene activating moiety (e.g., the AD of GAL4) are produced as
a hybrid protein; and an immunoglobulin light chain is expressed as
a fusion protein with a nuclear localization sequence (FIG. 25).
The ability of the antibody to bind to the antigen can be assayed
by detecting expression of the reporter gene(s). In view of the
combinatorial nature of the immune system, and the somatic
refinement capabilities of the immune system, the invention, which
is combinatorial in nature and capable of refinement, is
particularly well-suited for identifying antibody/antigen
interactions.
[0283] If desired, plasmids encoding self-activating hybrid
proteins can be eliminated from cells by using DB and AD vectors
which contain "shuffling" counterselectable markers. These genes
allow for selection of cells that have lost either the DB or AD
plasmid with integration of the gene encoding the hybrid protein.
For shuffling, expression of the counterselectable reporter gene
can be tested under conditions which select against the DB or AD
plasmid, and clones that score positive in this assay are
eliminated from further steps in the analysis. The plasmids used to
express the proteins and RNA molecules employed in the invention
can employ selectable markers to ensure that the plasmids are
maintained in the cell.
Sequence CWU 1
1
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