CA2236867A1 - Method to identify compounds for disrupting protein/protein interactions - Google Patents

Abstract

The present invention relates generally to materials and methods for identification of inhibitors of interactions between known binding partner proteins.

Description

CA 02236867 1998-0~-26 W O 98/135~2 PCTrUS97/17276 MET:HODS TO IDENTIFY COMPOUNDS FOR
DISRUPI'ING PROTEIN/PROTEIN INTERACTIONS

Back~round of the Invention The present invention relates to a novel method to identify 5 inhibitors of protein/protein interactions.

B~ck~round Modulation of protein/protein interactions is an attractive target for drug discovery and development. Potential methods by which drugs can regulate protein/protein interactions are numerous, including, for example, 10 regulation of expression of one or more of the binding proteins, modulation of post-translational modificatioll, and direct interference with thc capacity of one protein to bind to one or more binding partners. More importantly, recent observations make it increasingly clear that supramolecular protein complexes, involving two or more binding proteins, play an important and 15 essential roles in signal transduction, gene expression, cell proliferation and duplicatioll, and cell cycle progression. For example, in the repair of W
damaged DNA, a so-called "repairsome" that contains over ten individual proteins is assembled into a complex which can then carry out the necessary repair. Likewise, gene transcription occurs through the concerted action of 20 greater than twenty proteins. Signal transduction proteins, such as receptor protein kinases, are part of large complexes with many proteins. Contacts through Src homology type 2 (SH2) domains on the receptor kinases, for example, are noteworthy protein interaction which are part of one or more enzymatic cascade important for many metabolic processes. Disrupting the 25 binding capacity of one or more proteins which form any of these larger complex is therefore an important and untapped method to control action of the overall complex.
Protein/protein interactions have been discovered and characterized by a variety of methods: (i) standard biochemical afflnity CA 02236867 1998-0~-26 W O 98tl3502 PCTrUS97/17276 methods such as chromatography or co-immunoprecipitations; (ii) gel overlay ~, methods; (iii) co-purification by traditional biochemistry; and (iv) two-hybrid analysis [Fields and Song, Nature 340:245-246 (1989); Fields, Methods: A
Co npanion to Methods in Enzymology 5:1 16-124 (1993); U.S. Patent 5,283, 173 issued February 1, l9g4 to Fields, et al.]. The most recent of these approaches, the two hybrid method, has enjoyed broad application because of its relative ease of use for gene identification from cDNA fusion libraries.
[See Chien et al., Proc. Natl. Acad. Sci. (USA) 88:9578-9582 (1991); Dalton and Treisman, Cell 72:223-232 (1993); and Durfee, et al., Genes and Devel.
7:555-569 (1993)].
The two hybrid system is based on targeting and identifying a protein/protein interaction througl1 the use of a reporter system. The described two hybrid systems either use the yeast Gal4 DNA binding domain or the E. coli lexA DNA binding domain and couple this region to a transcriptional activator such as Gal4 or VP16 that drives a reporter like galactosidase or HIS3.
In principle the two hybrid assay could be used for drug screening. [See WO 96/03501 and WO 96/03499.] In such a scenario, loss of ,~ galactosidase or HIS3 activity would be identified after the yeast strain is treated with a compound. In practice, however, use of the two hybrid system is technically undesirable for several reasons. In in~t~nçes where the galactosidase or HIS3 protein are employed as the reporter protein, a loss of activity is particularly difficult to detect because the expressed reporter protcin is too long lived to be used in a high throughput mode. If a candidate binding inhibitor compound is metabolized faster than the previously expressed reporter protein is turned over, it is difficult to detect inhibitory action of the ~n~ te drug while a reporter protein is still active. In high throughput screening, the loss of a positive signal, for example"B galactosidase or HIS3 is impossible to detect. Present robotocized screening and detection methods are simply not suf~lciently sensitive or robust to detect loss of a signal.

CA 02236867 1998-0~-26 W O 98/13502 PCTrUS97/17276 ,. Thus there is a need in the art to develop a rapid screening method that gives a positive signal, as opposed to a negative signal, when a -protein/protein interaction is disrupted. Such a system must be capable of using protein interactions that are initially detected by any of the above 5 mentioned approaches and must be sumciently robust to detect a gain of function when a protein interaction is lost. In essence, the screening method must give a signal when an interaction is lost, not lose a signal when an interaction is lost. Such a system must be sensitive to subtle interactions, in particular ones that are caused by post-translational modification like protein 10 phosphorylation. Finally for large scale screening, such as high throughput screening, the system must be manipulable such that a large signal-to-noise ration can be easily detected.

Brief Summarv of the Invention In one aspect, the present invention provides materials that are 15 useful for the identification of compounds which inhibit interaction between known binding partner proteins. See Figure 1. The invention provides host cells transformed or transfected with DNA comprising: (i) a repressor gene encoding DNA binding protein that acts as a repressor protein, said repressor gene under transcriptional control of a promoter; (ii) a selectable marker gene 20 encoding a selectable marker protein; said selectable marker gene under transcriptional control of an operator; said operator regulated by interactio with said ,~r~ssor protein; (iii) a ~lrst recombinant fusion protein gene encoding a first binding protein or binding fragment thereof in frame with either a DNA binding domain of a transcriptional activating protein or a 25 transactivating domain of a transcriptional activating protein; and ~iv) a second recombinant fusion protein gene encoding a second binding protein or binding fragment thereof in frame with either a DNA binding domain of a tramscriptional activating protein or a transactivating domain of a transcriptional activating protein, whichever domain is not encoded by the first CA 02236867 1998-0~-26 WO 98113502 PCT~US97/17276 ~ 4 -fusion protein gene, said second binding protein or binding fragment thereof capable of interacting with said first binding protein or binding fragment thereof such that interaction of said second binding protein or binding fragment thereof and said ~1rst binding protein or binding fragment thereof 5 brings into proximity a DNA binding domain and a transactivating domain forming a functional transcriptional activating protein; said functional transcriptional activating protein acting on said promoter to increase expression of said repressor gene.
The invention comprehends host cells wherein the various genes 10 and regulatory sec~uences arc encoded on a single DNA molecule as well as host cells wherein one or more of the repressor gene, the selectab~e marker gene, the first recombinant fusion protein gene, and the second recombinant fusion protein gene are encoded on distinct DNA expression constructs. In a preferred embodiment, the host cells are transformed or transfected with DNA encoding the repressor gene, the selectable marker gene, the first recombinant fusion protein gene, and the second recombinant fusion protein gene, each encoded on a distinct expression construct. Regardless of the number of DNA expression constructs introduced, each transformed or transfectedDNA expression construct further comprises a selectable marker 20 genc sequence, the expression of which is used to confirm that transfection or transformation was, in fact, accomplislled. Seléctable marker genes encoded on individually transformed or transfected DNA expression constructs are distinguishable from the selectable marker under transcriptional regulation of the tCt operator in that expression of the selectable marker gene regulated by 25 the tCt operator is central to the plel~.led embodiment; i.e., regulated expression of the selectable marker gene by the tet operator provides a measurable phenotypic change in the host cell that is used to identify a bindingprotein inhibitor. Selectable nnarker genes encoded on individually transformed or transfected DNA expression constructs are provided as 30 determin~nt.~ of successful transfection or transformation of the individual CA 02236867 1998-0~-26 W O 98113~02 PCT~US97/17276 DNA expression constructs. Preferred host cells of the invention include transformed S. cerel~isiae strains ~lesign~ d YI596 and YI58~ which were deposited August 13, 1996 with the ~merican Type Culture Collection ~ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852, and assigned Accession Numbers ATCC 74384 and ATCC 74385, respectively.
The host cells of the invention include any cell type capable of expressing the heterologous proteins required as described above and which are capable of being transformed or transfected with functional promoter and operator sequences which regulate expression of the heterologous proteins also l O as described. In a preferred embodiment, the host cells are of either m~mm~l, insect or yeast origim Presently, the most preferred host cell is a yeast cell.
The prel~erred yeast cells of the invention can be selected from various strains, including the S. cerevisiae yeast transformants described in Table 1.
Alternative yeast specimens include S. pombe, K lactis, P. pastoris, S.carlsbergensis and C~.albicans. Preferred m~mm~ n host cells of the invention include Chinese hamster ovary ~CHO), COS, HeLa, 3T3, CVl, LTK, 293T3, Ratl, PC12 or any other transfectable cell line of human or rodent origin. Preferred insect cells lines include SF9 cells.
In a preferred embodiment, the selectable marker gene is regulated by an operator and encodes an enzyme in a pathway for synthesis ~ of a nutritional requirement for said host cell such that expression of said selectable marker protein is required for growth of said host cell on media lacking said nutritional requirement. Thus, as in a preferred embodiment where a repressor protein interacts with the operator, transcription of the selectable marker gene is down-regulated and the host cells are identi~led by an inability to grow on media lacking the nutritional requirement and an ability to grow on media cont~ining the nutritional requirement. In a most preferred embodiment, the selectable marker gene encodes the HIS3 protein, and host cells transformed or transfected with a ~S3-encoding DNA
expression construct are selected following growth on media in the presence CA 02236867 1998-0~-26 W O 98/135~2 PCTrUS97/17276 and absence of histidine. The invention, however, comprehends any of a number of alternative selectable marker genes regulated by an operator. Gene alternatives include, for example UR~3, LEU2, LYS2 or those encoding any of the multitude of enzymes required in various pathways for production of 5 a nutritional requirement which can be definitively excluded from the media of growth. In addition, conventional reporter genes such as chloramphenicol acetyltransferase (CAT), firefly luciferase, ~-galactosidase (,~-gal), secreted alkaline phosphatasc (SEAP), green fluorescent protein (GFP), human growth hormone (hGH), ~-glucuronidase, neomycin, hygromycin, thymidine kinase 10 (TK) and thc lil~e may be utilized in the invention.
In the preferred embodiment, the host cells include a repressor protein gene encoding the tetracycline resistance protein which acts on the tet operator to decrease expression of the selectable marker gene. The invention, however, also encompasses alternatives to the tet repressor and operator, for 15 example, E. coli trp repressor and operator, his repressor and operator, and ~ac operon repressor and operator.
The DNA binding domain and transactivating domain components of the fusion protein may be derived from the same transcription factor or from different transcription factors as long as bringing the two 20 domains into proximity permits formation of a functional transcriptional activity protein that increases expression of the repressor protein with high efficiency. A high efficiency transcriptional activating protein is defined as having both a DNA binding domain exhibiting high affinity binding for the recognized promoter sequence and a transactivating domain having high 25 affinity binding for transcriptional machinery proteins required to express repressor gene mRNA. The DNA binding domain component of a fusion protein of the invention can be derived from any of a number of diffe}ent proteins including, for example, LexA or C~al4. Similarly, the transactivating component of the invention's fusion proteins can be derived from a number 30 of different transcriptional activating proteins, including for example, Gal4 or CA 02236867 1998-0~-26 W O 98113502 PCTrUS97/17276 VP16. In one embodiment of the invention, polynucleotides encoding binding partner proteins CREB and CBD are inserted in plasmids pVP16-CREB and pLexA-CBD, respectively, which were deposited with the ATCC
and assigned Accession Numbers ATCC 98138 and ATCC 98139, 5 respectively.
The promoter sequence of the invention which regulates transcription of the repressor protein can be any sequence capable of driving transcription in the chosen host cell. The promoter may be a DNA sequence specifically recognized by the chosen DNA binding domain of the invention, 10 or any other DNA sequence with which the DNA binding domain of the fusion protein is capable of high affinity interaction. In a ,~,Ic;rellc;d embodiment of the invention, the promoter sequence of the invention is either a ~S3 or alcohol dehydrogenase (ADH3 promoter. In a presently most preferred embodiment, the ADH promotor is emp~oyed in the invention. The 15 invention, however, encompasses numerous alternative promoters, including, for example, those derived from genes encoding HIS3, ADH, URA3, LEU2 and the li~e.
In another aspect. the invention provides methods to identify molccules that inhibit interaction between known binding partner proteins. In 20 one embodiment, the invention provides a method to identify an inhibitor of binding between a first binding protein or binding fragment thereof and a second binding protein or binding fragment thereof comprising the steps of (a) growing host cells transfonned or transfected as described above in the absence of a test compound and under conditions which permit expression of 25 said first binding protein or binding fragment thereof and said second binding protein or binding fragment thereof such that said first binding protein or fragment thereof and second binding protein or binding fragment thereof interact bringing into proximity said DNA binding domain and said transactivating domain forming a functional transcriptional activating protein;
30 the transcriptional activating protein acting on said promoter to increase CA 02236867 l998-0~-26 W O 98/13502 PCTrUS97/17276 expression of said repressor protein; said repressor protein interacting with said operator such that said selectable marker protein is not expressed; (b~
confirming lack of expression of said selectable marker protein in said host cell; (c) growing said host cells in the presence of a test compound; and (d) 5 comparing expression of said selectable marker protein in the presence and absence of said test compound wherein increased expression of said selectable marker protein is indicative that thc test compound is an inhibitor of binding between said ~Irst binding protein or binding fragment thereof and said second binding protein or binding fragment thereof.
In a most preferred embodiment, thc invention provides a method to identify an inhibitor of binding between a first binding protein or binding fragment thereof and a second binding protein or binding fragment thereof comprising the steps of: (a) trans~orming or transfccting a host cell with a first DNA expression construct comprising a first selectable marker 15 gene encoding a first selectable marker protein and a repressor gene encodinga repressor protein, said repressor gene under transcriptional control of a promoter; (b) transforming or transfecting said host cell with a second DNA
expression construct comprising a second selectable marker gene encoding a second selcctable marker protein and a third selectable marker gene encoding 20 a third selectable marker protein, said third selectable marker gene under transcriptional control of an operator~ said operator specifically acted upon bysaid repressor protein such that interaction of said repressor protein with saidoperator decreases expression of said third selectable marker protein; ~c) transforming or transfecting said host cell with a third DNA e~pl c;ssion 25 construct comprising a fourth selectable marker gene encoding a fourth selectable marker protein and a first fusion protein gene encoding a first binding protein or binding fragment thereof in frame with either a DNA
binding domain of a transcriptional activation protein or a transactivating domain of said transcriptional activation protein; (d) transforming or 3û transfecting said host cell with a fourth DNA expression construct comprising CA 02236867 1998-0~-26 W O 98/13S02 PCTrUS97117276 a fifth selectable marker gene encoding a ~Ifth selectable marker protein and a second fusion protein gene encoding a second binding protein or binding fragment thereof in frame with either the DNA binding domain of said transcriptional activation protein or the transactivating domain of said 5 transcriptional activation protein, whichever is not included in first fusion protein gene; ~e) growing said host cell under conditions which permit expression of said first binding protein or fragment thereof and said second binding protein or fragment thereof such that said ~Irst binding protein or fragment thereof and second binding protein or binding fragment thereof 10 interact bringing into proximity said ~NA binding domain and said transactivating domain recons~ituting said transcriptional activating protein;
said transcriptional activating protein acting on said promoter to increase expression of said repressor protein; said repressor protein interacting with said operator such that said third selectable marker protein is not expressed;
lS (f~ detecting absence of expression of said selectable gene; (g) growing saidhost cell in the presence of a test compound of binding between said first protein or fragment thereof and said second binding protein or fragment thereof; and (h) comparing cxpression of said selectable marker protein in the presence and absence of said test compound wherein decreased expression of 20 said selectable marker protein is indicative of an ability of the test compound to inhibit binding between said first binding protein or binding fragment thereof and said second binding protein or binding fragrnent thereof such that said transcriptional activating protein is not reconstituted, expression of saidrepressor protein is not increased, and said operator increases expression of 25 said selectable marker protein.
The methods of the invention encompass any and all of the variations in host cells as described above. In particular, the invention encomp~.~ses a method wherein: the host cell is a yeast cell; the sPlect~hle marker gene encodes HIS3; transcription of the selectable marker gene is 30 regulated by the tet operator; the repressor protein gene encodes the CA 02236867 l998-0~-26 W O 98/13502 PCTrUS97/17276 r tetracycline resistance protein; transcription of the tetracycline resistance protein is regulated by the HIS3 promoter; the DNA binding domain is derived from LexA; and the transactivating domain is derived from VP16.
In another embodiment, the invention encompasses a method wherein: the host 5 cell is a yeast cell; the selectable marker gene encodes ~S3; transcription o~the selectable marker gene is regulated by the tet operator; the repressor protein gene encodes the tetracycline resistance protein; transcription of the tetracycline resistance protein is regulated by the alcohol dehydrogenase promoter; the DNA binding domain is derived from LexA; and the 10 transactivating domain is derived from VP16.
In alternative embodiments of the invention wherein the host cell is a m~mm~ n cell, variations include the use of mammalian DNA
expression constructs to encode the first and second recombinant fusion genes, the repressor gene, and the selectable marker gene, and use of selectable 15 marker genes encoding antibiotic or drug resistance markers (i.e., neomycin, hygromycin, thymidine kinase).
There are at least three different types of libraries used for the identification of small molecule modulators. These include: (1) chemical libraries, ~2) natural product libraries, and (3) combinatorial libraries 20 comprised of random peptides, oligonucleotides or organic molecules.
Chemical libraries consist of structural analogs of known compounds or compounds that are identified as "hits" via natural product screening. Natural product libraries are collections of microorg~nicms, animals plants or marine organisms which are used to create mixtures for 25 screening by: (1) ferrnentation and extraction of broths from soil, plant or marine microor~nismc or (2) extraction of plants or marine org~nicmc Combinatorial libraries are composed of large numbers of peptides, oligollucleotides or organic compounds as a mixture. They are relatively easy to prepare by traditional automated synthesis methods, PCR, cloning or 30 proprietary synthetic methods. Of particular interest are peptide and CA 02236867 1998-0~-26 W O 98/13502 PCTrUS97/17276 oligonucleotide combinatorial 1ibraries. ~till other libraries of interest include peptide, protein, peptidomimetic, multiparallel synthetic collection, rccombinatorial, polypeptide libraries.
The utility of the various aspects of the invention is manifest.
5 Host cells of the invention are useful to demonstrate in vivo binding capacity of both known and suspected binding partner proteins in a recombinant system. Such an expression system permits systematic analysis of the structure and function of a particular binding protein, thus permitting identification and/or synthesis of potential modulators of the physiological 10 activity of the binding proteins. The methods of the invention are particularly useful to identify and improve molecules which are capable of inhibiting specific and general protein/protein interactions. Inhibitors identified by the methods of the invention can then be examined for utility in vivo as therapeutic and/or prophylactic medicaments for conditions associated with 15 various protein/protein interactions.

Description of the Drawin~
Figure 1 describes the mechanics of the split hybrid assays.

Detailed Description of thc Invention The present invention relates generally to methods de~ign~te-1 20 split hybrid assays to identify inhibitors of protein/protein interactions and is illustrated by the following examples describing various methods for making and using the invention. In particular, Example 1 relates to construction of valious plasmids and expression constructs utilized in the invention. Example 2 described generation of various yeast transformants used to identify inhibitor25 compounds. Examples 3, 4, 5 and 6 address use of the split hybrid assay to examine CREB/CBD binding, Tax/SRF binding, CKI/CREB binding and AKAP 79 binding to various partner protein, respectively. Example 7 describe general application of the split hybrid assay. Example 8 relates to , W O 98/13~02 PCT~US97/17276 use of the split hybrid assay for weakly inleracting binding partners. Example 9 describes general assay methods. Example 10 addresses use of the split hybrids assay to identify agents that prevent receptor desensitization and drug tachypllylaxis.

Example 1 Plasmid Construction In the examples that follow, various plasmid constructs were utilized as described. To simplify discussion of the exempli~led assays, this example describes construction of the various plasmids uscd in the following examples. For clarity, the plasmids are grouped according common features relating to their applications in the assays later discussed.

I. Plasmids Encodin~ Reporter Gene ~IS3 A. pRS303/ 1 xtetop-Mlul One copy of the tet operator sequence was engineered into position -53 in the HIS3 promoter of pRS313 [Sikorski, R.S. et al., Genetics 122:19-27 (1989)] by using the polymerase chain reaction (PCR~. Two primary PCR reactions using pRS313 as a template were performed which utilized a 5'-terminal oligonucleotide de.cign~ted Eco4/111-5' and a 3'-inner oligonucleotide designated Tetop internal 3 ' to yield a primary 5 '-PCR productand a 5'-inner oligonucleotide designated Tetop internal 5 ' and a 3'-terminal oligonucleotide de~ign~tecl Nhe I 3' to yield a primary 3'-PCR product.

Eco47 m-5' SEQ ID NO: I
5 '-TTGGTGAGCGCTAGGAGTCACTGCCAG
Tetop int. 3' SEQ ID NO: 2 25 5 ' -TATACTCTATCAATGATAGAGTAATTCATTATGTGATAATGCC
Tetop int. 5' SEQ ID NO: 3 S'-ATTACTCTATCATTGATAGAGTATATAAAGTAATGTGATTTC) Nhe I 3' SEQ ID NO: 4 W O98/13502 PCT~US97/17276 S ' -AAl~CTGCTAGCCTCTGCAAAGC

5' and 3' inner oligonucleotides contain complementary sequence such that 3' sequence of the primary 5' PCR product overlaps with S' sequence of the primary 3 ' PCR product. The 5 ' terrninal oligonucleotide contains the 5 restriction site Eco47m while the 3' terminal oligonucleotide contains the restriction site N/leI in order to facilitate subsequent subcloning. The primaryPCR reactions were performed with P~ DNA polymerase ~Stratagene, La Jolla, CA) using reaction conditions described by the manufacturer. PCR
products were isolated hy Biolûl (Vista~ CA) Gene Clean m gel extraction.
10 The primary 5' and 3' PCR products were then combined in a second PCR
reaction and amplified using the 5'- and 3'- terminal oligonucleotides, Eco47m-s ' and Nhe I 3 ' . The second PCR reaction was performed with Vent DNA polymerase (New England Biolabs, Beverly, MA) using reaction conditions described by the manufacturer, except that the reactions were 15 supplemented with 4 mM Mg2+. The fimal PCR product contained one tet operator sequence inserted into position -53 of the HIS3 promoter and nucleotides 52-48 deleted in the construction. The final PCR product was isolated, digested with Eco47m and NlleI and cloned into pRS313 previously digested with Eco47m and lVheI. The resulting plasmid was ~lesign~e~
20 pRS3 13/1 xtetop. DNA sequencing confirmed the presence of one copy of the tet operator sequence in pRS3 13/1 xtetop and confirmed integrity of the ECO47m and N~neI junctions.
A MluI restriction enzyme site was engineered into position -22 in the HIS3 promoter of pRS313/lxtetop by ~Itili7ing PCR using Vent DNA
25 polymerase using pRS313/lxtetop as template. One PCR construct was amplified using the 5' terminal oligonucleotide Eco47 m-s~ (SEQ ID NO: 1) cont~3ining an Eco47m restriction site and a 3'-oligonucleotide design~t~cl Mlu I 3' cont~ining a MluI restriction site.

W O 98/13502 PCTrUS97/17276 Mlu I 3' SEQ ID N O: 5 5'-CGCACGCGTCGAAGAAATCACATTAC'l'l'l'ATATA

A second PCR product was amplified using the 3'-terrninal oligonucleotide Nhe I 3' (SEQ ID NO: 4) cont~ining a NqneI restriction site and a 5'-S oligonucleotide tlt sign~tP(l Mlu I 5' cont~ining a MIuI restriction site.

Mlu I 5' SEQ nD N O: 6 5' -CGCACGCGTATACTAAAAAATGAGCAGGCAAG

The Flrst PCR product was isolated and digested with Eco47m and MIuI, wllile the second PCR product was isolated and digested with MIuI and N~neI.
10 These digested products were isolated and ligated in a triple ligation with pRS313 previously digested with Eco47~ and N~neI. The resulting plasmid was designated pRS313/ 1 xtetop-MluI . DNA sequencing confirmed the presence of theMluI site in pRS313/lxtetop-MluI and confirrned that integrity of the Eco47rlI and NheI junctions were m~int~ined.
A pRS303/lxtetop-MluI plasmid was constructed by first removing the Eco47~1NheI fragment con~ining the altered HIS3 promoter from the pRS313/lxtetop-MluI vector and ligating the isolated fragment into pRS303 previously digested with Eco47rII and MheI. DNA sequencing conflnned proper insertion of the ~co47m/~eI fragment.

B. pRS303/2xtetop-LYS2 One copy each of the tet operator sequence was engineered into positions -53 and -22 in the HIS3 promoter of pRS303 [Sikorski, et al., Genetics 122:19-27 (1989)]. PCR was utilized to engineer one copy into position -53 which resulted in plasmid p3i~S303/lxtetop. To insert the second 25 copy, a MluI site was introduced at position -22 in the HIS3 promoter using PCR. The new plasmid was designated pRS303/lxtetop-MluI.
The tet operator was created by annealing two complementary W O 98/13502 PCT~US97/17276 oligonucleotides tetop-l and tetop-2.

tetop- l SEQ ID NO: 7 5 ' -CGCGTACTCTATCATTGATA(:~AGTA;
tetop-2 SEQ ID NO: 8 5 ' -ATGAGATAGTAACTATCTCATGCGC

When annealed, the tet operator sequence contains flanlc~ng MluI sites. Both oligonucleotides were phosphorylated using T4 polynucleotide kinase (Gibco BRL, Grand Island, NY) at 37~C for one hour and annealed by first heating at 70~C for 10 minutes and then cooling to room temperature. The annealed 10 oligonucleotides were isolated and ligated into pRS303/ 1 xtetop-MluI
previously digeste~l with MluI. The resulting plasmid was de~ign:~t~cl pRS303/2xtetop. DNA sequencing confirmed insertion of one copy of the tet operator sequence in the MluI site.
The LYS2 gene was digested from pLYS2 ~Hollenberg, S.M.
et al., Mol. Cell.Biol. 15:3813-3822 (1995)] with EcoRI and Hind~I and the isolated fragment blunt ended using the large fragment of DNA polymerase I (Gibco BRL, Grand Island, NY). Phosphorylated SstI linkers (New England Biolabs, Beverly, MA) were ligated to the fragment, the fragment digested with SstI, and the resulting fragment ligated into pRS313 previously digested 20 with SstI. The resulting plasmid was designated pRS313/LYS2.
TheLYS2 fragment was removed from pRS313/LYS2 with SstI
digestion and inserted into pRS303/2xtetop previously digested with SstI. The resulting plasmid was design:~ted pRS303/2xtetop-LYS2.
Similarly, the LYS2 Ssd fragment was inserted into 25 pRS303/ 1 xtetop-MluI previously digested with SstI yield pRS303/1 xtetop-- MluI-LYS2.

C. pRS303/3xtetop-LYS2 -CA 02236867 1998-0~-26 W O 98/13502 PCT~US97/17276 Two copies of the tet operator sequence were created by self-anncaling a palindromic oligonucleotide Tetop 2x with itself.

Tetop 2x SEQ ID NO: 9 5'-CGCGTACTCTATCATTGATAGAGTCTAGACTCTATCAATGATAGAGTA

5 The annealed oligonucleotide contained flanking MluI sites. The oligonucleotide was phosphorylated, annealed, and isolated as above. The isolated annealed and MluI-digested oligonucleotide was ligated into pRS303/ l xtetop-MluI-LYS2 previously digested with MluI to yield pRS303/3xtetop-LYS2. The presence of two copies of the tet operator lO sequcnce in the MluI site was confirmed by DNA sequencing.

D. pRS303/4xtetop-LYS2 and pRS303/8xtetop-LYS2 Three or seven copies of the tet operator were created using PCR with Vent DNA polymerase as described above. Plasmid pUHC-13-3 [Grossen and Bujarg, Proc. Natl. Acad. Sci. ~USA) 89:5547-5551 (1992)~ was 15 used as template DNA using 5'- and 3'- oligonucleotides, Mlu I/Sph I 5' and Mlu I Sph I 3', containing an exterior MluI restriction enzynle site nested internally by a SphI restriction enzyme site.
Mlu I/Sph 1 5' SEQ ID NO. 10 5 ' -GCGACGCGTGCATGCCGTCTTCAAGAATTCCTCGAG
Mlu I Sph I 3' SEQ ID NO: 11 5 ' -GCGACGCGTGCATGCCCACCGTACACGCCTACTCGA

The PCR products were separated on an agarose gel and the ladder of different sized DNA fragments was isolated, digested with MluI, and ligated into the MluI restriction site of pRS303/lxtetop-MluI-LYS2. DNA sequenc-ing rcvealed that either three or seven copies of tet operators were inserted into the Mlu site of pRS303/ 1 xtetop-MluI-LYS2 to provide either pRS303/4xtetop-LYS2 or pRS303/8xtetop-LYS2.

W O 98/13502 PCT~US97117276 E. pRS303/6xtetop-LYS2 and pRS303/lOxtetop-LYS2 A SphI restriction enzyme site was introduced at position -85 in the HIS3 promoter of pRS303/3xtetop-LYS2 using PCR with Vent DNA
polymerase as described. Plasmid pRS303/3xtetop-LYS2 was used as a S template DNA. A first fragment was amplified using the 5 ' -terminal oligonucleotide l~co47 m-5' (SEQ ID NO: 1) described above cont~ining an ~:co47rrl restriction site and a 3'-oligonucleotide Sph I 3' cont~ining a SphI
restriction site.

Sph I 3' S3~Q ID NO: 12 5 ' -CATGGCATGCAAAAAAAAAGAGTCATCCGCTAGG

A second PCR product was amplified using the 3'-terrninal oligonucleotide Nhe I 3' ~SEQ ID NO: 4) described above cont~ining a NheI restriction site and a 5'-oligonucleotide cont~inin~ a SphI restriction site.

Sph I 5' SEQ ID NO: 13 5 ' CATGGCATGCTTAGCGATTGGCATTATCACAT

The PCR products were isolated as described above. The first PCR product was digested with Eco47m and SphI, and the second PCR product was digested with SphI and NheI. Both digestion products were ligated in a triple ligation along with pRS303/3xtetop-LYS2 previously digested with both Eco47m and NheI. The resulting plasmid was c~e~ign~1ed pRS303/3xtetop-SphI-LYS2. The presence of the SphI site in pRS303/3xtetop-SphI-LYS2 was confirmed by DNA sequencing analysis.
Three copies of tet operators were isolated as a single fragment by digesting pRS303/4xtetop-LYS2 with SphI. The isolated fragment was ligated into the SphI site of pRS303/3xtetop-SphI-LYS2 to yield pRS303/6xtetop-LYS2. The presence of three additional copies of the tel operator in pRS303/6xtyetop-LYS2 at the SphI site was confirrned by DNA

CA 02236867 l998-05-26 W O 98/13502 PCTrUS97/17276 sequencing.
Seven copies of tet operators were isolated as a single fragment by digesting pRS303/8xtetop-LYS2 with SphI. The isolated fragment was ligated into the SphI site of pRS303/3xtetop-SphI-LYS2 to yield 5 pRS303/lOxtetop-LYS2. The presence of seven additional copies of the tet operator in pRS303/lOxtetop-LYS2 at the SphI site was confirmed by DNA
sequencing.

F. pRS313/MluI and pRS3Q3/MluI
A MluI restriction en~yme site was engineered into position -22 10 int}le HIS3 promoter of pRS313 lltili7.ing PCR and Vent DNA polymerase as noted above. Plasmid pRS313 was used as a template for these PCR
reactions. One PCR construct was amplified using the 5' terminal oligonucleotide Eco47 m-s~ ~SEQ ID NO: 1) cont~inin~ an Eco47m restriction sitc and a 3 ' oligonucleotide Mlu I 3' (SEQ ID NO: 5) cont~ining 15 a MluI restriction site. A second PCR product was amplified using the 3' tenninal oligonucleotide Nhe I 3 ' (SEQ ID NO: 4) cont~ining a N7neI
restriction site and the 5 ' oligonucleotide Mlu I 5' (SEQ ID NO: 6) cont;~ininga MluI restriction site. The first PCR product was isolated and digested with Eco47m and MluI, while the second PCR product was isolatcd and digested 20 with MluI and N~eI. The digested products were partially purified and joined in a triple ligation with pRS313 which had been previously digested with Eco47m and N7zeI. The resulting plasmid was ~l~sign~te(3 pRS313/MluI.
DNA se~uencing confirmed the presence of the MluI site in pRS313/MluI and to conf~n the integrity of the ~co47rll and I~neI Junctions.
pRS303/MluI was constructed in exactly the same manner as pRS313/MluI cxcept that pRS303 was used in place of pRS313.

G . pRS313/ 1 xtetop See above wherein pRS313/lxtetop is an intermediate in the CA 02236867 1998-0~-26 W O 98tl3502 PCTrUS97/17276 constmction of pRS303/1xtetop-MluI.

H. pRS313/MluI-lxtetop and pRS303/MluI-lxtetop One copy of the tet operator sequence was created by ~nne;~lin~
two complementary oligonucleotides tetop-l and tetop-2 (SEQ ID NO: 7 and 5 SEQ ID NO: 8). The annealed tet~operator sequence contains flanking MIuI
sites. The oligonucleotides were phosphorylated using T4 polynucleotide kinase (Gibco ~RL, Grand Island, NY) at 37~C for one hour and annealed by first heating at 70~C for 10 minutes followed by cooling to room temperature.
The annealed oligonucleotides were isolated and ligated separately into MIuI-digested pRS313/MluI and pRS303/MluI, the resulting plasmids being design~tPd pRS313/MluI- 1 xtetop and pRS303/MluI- 1 xtetop. DNA sequencing confirmed the presence of one copy of the tet operator in the MIuI sites of both plasmids.
In order to produce plasmids bearing multiple copies of the tet operator, annealed oligonucleotides described above were ligated together overnight at 1~~C. After isolation of the ligation products, they were inserted into the MIIlI of pRS313/MluI. DNA sequencing analysis confirrned that one clone, pRS313/MluI-4xtetop, was produced which contained four copies of tet operator in the MIuI site. However, upon further e~r~min~tion of this clone it was discovered that it had been subjected to a recombination event and was therefore not useful for further cloning steps. Continued alLe~ to insert multiple copies of the tet operator into the MIuI site of pRS313/MluI by ligating multimers of the tet operator have been unsuccessful.

I. pRS313/ 1 xtetop-MluI
See above wherein construction of pRS313/lxtetop-MluI was an intermediate in the construction of pRS303/lxtetop-MluI.

CA 02236867 l998-05-26 W O 98/13502 PCTrUS97/17276 J . pRS313/2xtetop One copy of the tet operator sequence was created using annealed complementary oligonucleotides tetop-1 and tetop-2 (SEQ ID NO:
7 and SEQ ID NO: 8). Annealed oligonucleotides were ligated into the MluI
site of pRS313/lxtetop-MluI to yield pRS313/2xtetop. DNA sequencing confinned the presence of two copies of the tet operator in the MluI site.

K. pRS303/2xtetop See above wherein pRS303/2xtetop was an intermediate in the constmction of pRS3V3/2x/tetop-LYS2.

L. pRS313/LYS2 and pRS313/LYS2 The LY52 gene was digested from pLYS2 with ~coRl and HindIII digestion. The EcoPllHin~m fragment was blunt ended using the large fragment of DNA polymerase I (Gibco BRL, Grand Island, NY) and ligated with phosphorylated SstI linkers (New F.ng?7~l(1 Biolabs, Beverly, MA).
The resulting fragment was digested with SstI and ligated into pRS313 previously digested with SstI. Thc resulting plasmid was designated pRS313/LYS2. Because the LYS2 fragment was shown to have inserted into pRS3I3 in both orientations, plasmids with theLYS2 gene in both orientations ~ were transfortned separately into the yeast strain SEY6210O~_(ll~To~_ leu2-3,112 IJ:ra3-52 his3-~200 trp1-~901 Iys2-801 suc2-~9 [Robinson et al., Mol.
Cell. Biol. 8 :4936-4948 (1988)~ . Both clones allowed the yeast to grow in the absence of lysine indicating that orientation of the LYS2 gene in pRS313 did not affect the expression o~ an active gene.
The LYS2 fragment was removed from pRS313/LYS2 with SstI
and ligated into the SstI site of:

pRS313/lxtetop-MluI giving plasmid pRS313/lxtetop-MluI-LYS2, pRS313/2xtetop giving plasmid pRS313/2xtetop-LYS2, CA 02236867 l998-05-26 W O 98/13502 PCTrUS97/17276 r .. -- 21 --pRS303/ 1 xtetop-MluI giving plasmid pRS303/ 1 xtetop-MluI-LYS2, and pRS303/2xtetop giving plasmid pRS303/2xtetop-LYS2.

II. Plasmids Encodin~ Reporter Gene TetR
A. pRS306/~S3:TetRlTerm 5The 5 ' promoter sequence of the yeast HIS3 gene, encompassing nucleotides -75 to +23, was ligated to the translational start of TetR. In addition, the DNA sequence encoding the simian virus 4U (SV40) large T antigen nuclear localization signal was ligated in frame with the nucleotide sequence encoding the last amino acid residue of TetR. The 10chimeric fragment was created by the same PCR strategy as described above.
The HIS3 promoter fragment, the primary 5'-PCR product, was amplified by PCR from plasmid p601 rGrueneberg,D.A., Science 257:1089-1095 (1992)] using a 5'-terminal oligonucleotide T7 Promoter primer and a 3'-inner oligonucleotide 3'-TetR inner primer.

T7 Promoter primer SEQ ID NO: 14 5 '-TAATACGACTCACTATATAGGG
3'-TetR inner primer SEQ ID NO: 15 5 '-TCTAGACTTTGCCTTCGITTATC

The primary 3 ' PCR product containing the TetR coding sequence was 20amplii~ied from pSLF104 [Forsburg, Nucl. Acid. Res. 21:2955-2956 ~1993)]
with a S ' -inner oligonucleotide 5 ' -TetR inner primer and a 3 ' -terminal oligonucleotide 3'-TetR terminal primer.

5'-TetR inner primer SEQ ID NO: 16 5'-CGAAGGCAAACATGTCTAGATTAGATAAAAG
3'-TetR terminal primer SEQ ID NO: 17 5'-CGCGGATCCGCTTTCTCTT~'l"ll"l"ll'GGAGACCCACTTTCACATTTAAG

W O 98113S02 PCTrUS97/17276 An EcoRI site derived from the p601 fragment and a BamHI site in the 3'-terminal oligonucleotide were used in subsequent subcloning. The PCR
products were gel-purified and amplified in a second PCR reaction with 5'-and 3-' tcrminal oligonucleotides, T7 Promoter primer (SEQ IDNO: 14) and 5 3'-TetR terminal primer (SEQ ID NO: 17). The secondary PCR product was isolated, digested with EcoRI and BamHI, and ligatcd into pRS306/Term previously digested with EcoRI and BamHI. The resulting plasmid was designated pRS306/HIS3:TetR/Term which comprises the complete TetR
coding sequence in framc with sequences encoding the nuclear localization lO signal of SV40 large T antigen.

B. pRS3 1 6/HIS3 :l'etR/Term The construction protocol for this plasmid was the same as described above for subcloning a HIS3 DNA into pRS306/Term except that the vector for subcloning was pRS316/Term described above.

C. pRS3()6/lxLexAop/HIS3:TetR
Oligonucleotides LexAop (lOOa) and LexAop (lOOb) cont~ining a single copy of LexA operator were phosphorylated with T4 polynucleotide kinase (Gibco BRL, Grand Island, rlY) at 37~C for one hour.

LexAop (lOOa) SEQ ID NO: 18 5 ' -AAl-rGCTCGAGTACTGTATGTACATACAGTAG
LexAop (lOOb) SEQ ID NO: l9 5 '-AATTCTACTGTATGTACATACAGTACTCGAGC

Following phosphorylation, the oligonucleotides were annealed by heating at 70~C for 10 minutes followed by cooling to room temperature. The annealed 25 oligonucleotide contzlining ~ and 3 EcoP;I overhanging ends was subcloned into pRS306/~S3:TetRlTerm previously digested with EcoP~. The number W O9S/13502 PCT~US97/17276 of copies of inserted oligonucleotide was confirmed by DNA sequencing. The plasmid cont~in;ng a single copy of the LexA operator was de~ign~t~d pRS306/ 1 xLexAop/HIS3 :TetR.

D . pRS316/2xLexAop/HIS3: TetR
The subcloning protocol for this construct was the same as described above for pRS306/ 1 xLexAop/~S3: TetR. The annealed oligomlcleotides encoding the LexA operator included overh~ngin~ EcoRI ends and during ligation, the individual annealed fragments were able to multimerize, inserting into the parental plasmid more than one copy of the desired LexA sequence. The mlmber of copies of inserted oligonucleotides was confirmed by DNA sequencing.

E. pRS306/2xLexAop/HIS3: TetR
A DNA fragment Cont~ining tWO copies of LexA operator and the chimeric HIS3:TetR reporter was excised from pRS316/2xLexAop/HIS3:TetR by digestion with KpnI and BamHI restriction enzymes. The fragment was gel-purified and subcloned into pRS306/Term previously digested with KpnI and BamHI and the resulting construct was sequenced to confirm the presence of two copies of the LexA operator.

F. pRS306/4xLexAop/HlS3: TetR
and pRS306/8xLexAop/HIS3:TetR
A pair of oligonucleotides SHlOlA and SHlOlB were utilized in PCR to amplify the LexA binding site multimer from the plasmid SH18-34~Spe [Hollenberg, S.M., el al., Mol.Cell.Biol. 15:3813-3822 (1995)~.
SHlOlA SEQ ID NO: 20 - 25 5'-CCGGAATTCTCGAGACATATCCATATCTAATC
SHlOlB SEQ ID NO: 21 5 '-CCGGAATTCACTAATCGCATTATCATC

W O 98113S02 PCT~US97/17276 The amplification product contz-ining four copies of LexA operator was gel-purified, digested with EcoRI, and subcloned into pRS306/HIS3:TetR/Term previously digested with EcoRI. The number of LexA operators were determined by DNA sequencing.

S G. pRS306/8xLex~op/HIS3::TetR
A PCR strategy was used to link the S' promoter sequence of the yeast HIS3 gene encomp~ing nucleotides-75 to +23 to the translational start of TetR. Sequences encoding the SV40 large T antigen nuclear local-ization signal were fused in frame with the nucleotide sequence encoding the last amino acid residue of TetR. The PCR product was digested with EcoRI
and BamHI and inserted into pRS306/Tenn previously digested with Eco~
and BamHI. The resulting plasmid was de~ign~tefl pRS306/HIS3:TetR/Term, and was shown to encode the complete TetR protein in frame with the nuclear localization signal of SV40 large T antigen. The fusion protein is followed by four amino acids generated by the vector backbone ~Arg-Ile-His-Asp).
The LexA binding site multimer from the plasmid pSH18-34/~Spe [Hollenberg, S.M. et al.. Mol.Cell.Biol. ~5:3813-3822 (1995)] was amplified by PCR, digested with EcoRI, and subcloned into the EcoRI site of pRS306/HIS3:TetR/Term resulting in plasmid pRS306/8xLexAop/TetR.

H. pADH/TetR
The DNA coding sequence of TetR was amplified by PCR from pSLF104 using two oligonucleotides, NcoI-TetR and 3'-TetR terminal primer (SEQ ID NO: 17).

NcoI-TetR SEQ ID NO: 22 5 ' -CATGCCATGGCCATGTCTAGATTAGATAAAAG

W O 98/13502 PCT~US97/17276 The resulting product was gel-purified, digested with NcoI and BamHI, and subcloned into a pBTM116 ~Bartel, et al., in Cellular Interactions in Dcvelopment: a Practical Approach, Hartley ~ed.), IRL Press; Oxford, pp.
153-179 (1993)] shuttle vector cont~ining an ADH promoter, previously S digested with NcoI and BamHI. For construction of this vector, DNA
generated by PCR and DNA obtained by restriction enzyme digestion of the polylinker region in plasmid pBluescript (Stratagene, La Jolla, California) were used to engineer additional restriction sites 5' and 3' of the ADH
promoter. The TetR protein encoded from this construct is expressed 0 cont~ining additional amino acids Met~2-Ala~l before the ini~i~tin~ methionineand also contains the nuclear localization signal of SV40 large T antigen located after the last amino acid of TetR as described above.

I. pRS306/ADH:TetR/Term A fragment encoding the ADH promoter and TetR was removed 15 from plasmid pADH/TetR with XhoI and blunted-ended with the large fragment of DNA polymerase I (Gibco BLR, Grand Island, NY). EcoRI
linkers (New Lngland BioLabs, Beverly, MA) were added and the fragment was digested with EcoRI and BamHI. The resulting fragment was gel-purified and ligated into pRS306/Term previously digested with EcoRI and BamHI.

J. pRS306/4xLexAop/ADH: :TetR
and pRS306/8xLexAop/ADH::TetR
The subcloning protocol used to insert multiple copies of the LexA operator into pRS306/ADH:TetRtTerm was the same as described previously for pRS306/4xLexAop/HIS3:TetR and pRS306/8xLexAop/HIS3 :TetR.

W O 98/13502 PCT~US97/17276 m. Plasmids ~ncodin~ Bindin~ Prote;ns A. pLexA-CBD
A DNA fragment contAining the CREB binding domain of CBP
(CBD), amino acids 461-682, was PCR ampli~led from plasmid CBP-0.8 5[Chrivia, J.C. et al., Nature 365:855-859 (1993)] using a pair of oligonucleotides designated 5' CBD primer and 3' CBD primer.

S' CBD primer SEQ ID NO: 23 5 ' -GCGAAl-rCGCCAGGGCAACAGAATGCCACT
3' CBD primer SEQ ID NO: 24 105 '-CGGGATCCTGGCTGGTIACCCAGGATGCCl~G

Following gel purirlcation, the ampli~lcatioll product was digested with EcoRI
and BamHI, and ligated into plasmid pBTMl 16 [Bartel, et al., in Cellular I~lteractions in Development: a Practical Approac~l, (ed) Hartley, D.A. (IRL
Press, Oxford), pp. 153-17g ~1993)] previously digested with EcoPU and BamHI.

B. pVPl 6-CBD
A DNA fragment encoding the CBP sequence was excised from pLexA-CBD by digestion with EcoRI and BaniHI. Plasmid pLexA-CBD was linearized with EcoRI digestion, the reslllting overh~nging ends blunt-ended 20 using the Klenow fragment of DNA polymerasc I, and the ends ligated with BamHI linkers. The resulting fragment was inserted into pVP16 [Hollenberg, et al., A~ol. Cell. Biol. 1~:3813-3822 (1995)] previously digested with into BamF~.

W O 98/13502 PCTrUS97/17276 C. pVP16 CRUEB
Plasmid pcDNA3/CREB283 [Sun and Maurer, J. Biol. Chem.
~ 270:7041-7044 (1995~], cont~ining the VP16 transactivation domain fused to sequences of the rat CREB transactivation domain (l to 283 aa) was linc~n7~
with XhoI and BamHI linkers (New P~ngl~ncl BioLab) ligated to the resulting blunt-ended ~hoI sites. DNA encoding the VP1 6/CREB chimeric protein was removed with Hind~I and BanlHI digestion and following gel purification, ligated into the Hindm and BamEI sites of pVPI 6 which encodes the LEU2 gene.

D. pVP16-CRE;B(B~lII-SacII)-LacZ
ADNAfragment encoding ~-galactosidase was PCR amplified from plasmid pSV-,B-galactosidase vector (Promega, Madison, WI) using a pair of oligonucleotides, S ~-gal prihner and 3 ,B-gal primer and inserted into the NotI site of pVP16 to produce pVP16-LacZ.

5',B-gal primer SLQ ID NO: 29 5-ATGGTACCAGCGGCCGCTAGTC~r~ l-lACAACGTCGTGAC
3 ,B-gal primer SEQ ID NO: 30 5-ATGGTACCGCGGCCGCTTAll-l-l-lGACACCAGACCAAC

A PCR fragment cont~ining CREB sequences encoding amino acid residues I to 283 was amplified from plasmid pRSV-CREB341 [Kwok, et al., Nan~re 380: 642-646 (1996)] using a pair of oiigonucleotides, 5 CREB 341 primer and 3 CREB 283 primer, and inserted into pVP16-LacZ vector at the BamHI
site.
5 CREB 341 primer SEQ ID NO: 25 - 25 5'-CGCGGATCCGGATGACCATGGACTCTGGAG
3' CREB 283 primer SEQ ID NO: 28 S'-CGCGGATCCGTGCTGCTTCTTCAGCAGGCTG

W O 98/13502 PCT~US97/17276 To generate a ç~et~ vector for producing and subcloning mutated CREB
sequences as described below, PC:R was used to engineer a Bgl~ site using oligonucleotides S' BglII primer and 3 Bgl~ primer, at nucleotides 273 to 278 and a SacII site using oligonucleotides 5' SacII primer and 3' Sac~
primer at nucleotides 500 to 505 of the CREB activation domain.

5' BglII primer SEQ ID NO: 31 5 -CGGAGATCTAAAGAGAcrT l-l CTCCGGAACTCAG
3 Bgm primer SEQ ID NO: 32 S -CGGAGATC l-l 1 ACAGGAAGACTGAACTGT
5 Sac~ primer SEQ ID NO: 33 S -CCACCGCGGCAGTGCCAACCCCGAl~AC
3 Sacll primer SEQ ID NO: 34 3 '-CATCCGCGGTG&TGATGGCAGGGGCTGA

E. pLexA-CREB 283 A DNA fragment cont~ining the rat CRE13 transactivation domain (amino acids 1 to 2~3) was excised from pcDNA/CRE13283 [Sun and Maurer, supra] with SmaI and XbaI digestion. The 5' XbaI site was b1unt ended with the large fragment of DNA polymerase I (Gibco BRL, Grand Island, NY) and SalI linkers (New England Biolabs, Beverly, MA) added.
The fragment was digested with SalI and subcloned into the SalI site of pBTMl 16.

F. pLexA-CREB 341 A DNA fragment Cont~ining the rat CREB 341 cDNA was amplified by PCR from pcDNA/CREB341 [Kwok, supra] using a pair of oligonucleotides, 5 CREB 341 primer (SEQ ID NO: 25) and 3 CREB 341 primer.

CA 02236867 l998-05-26 W O 98/13502 PCTnUS97/17276 3' CRE~ 341 primer SEQ ID NO: 26 S ' -CGCGGATCCrrAATCTGACTTGTGGCAGTA

After gel purification, the PCR product was digested with BamHI, and subcloned into the BamHI site of pBTMl 16.

G. pLexA-CREB 341-MI
A DNA fragment cont:lining the rat CREB sequence with a mutation ch~nging serine at position 133 to alanine was amplified by PCR
from plasmid Rc/RSV CRE~3-Ml [lKwok, et al., supra] using the same set of primers as described for pLexA-CREB 341, 5' CREB 341 primer (SEQ ID
NO: 25) and 3' CREB 341 primer (SEQ ID NO: 26). The resulting amplification product was gel-purified, digested with BamHI, and subcloned into the BamHI site of pBTMl 16.

H . pVPI 6-CRE13 M 1 A PCR fragment cont;~ining CREB sequences coding for amino acid residues 1 to 283 including the serine 133 mutation to alanine was amplified using a pair of oligonucleotides, S ' CREB 283 primer and 3 ' CREB
283 pr~mer (SEQ ID NO: 28). The PCR fragment was gel-purified, digested with BamHI and inserted into the BamHI site of pVP16.

S' CRLB 283 primer SEQ ID NO: 27 5'-CGCGGATCCCCATGACCATGGAATCTGGAGCC

I. pLexA-SRF
A DNA fragment cont:~ining human SRF was excised from plasmid pCGN-SRF [Grueneberg, D.A., et al., Science, 257:1089-1095 (1992)] with XhoI and BamXI digestion. The XhoI site of the fragment was 25 blunt-ended by the large fragment of DNA polymerase I (Gibco BRL, Grand Island, NY), ligated with BamHI linkers, digested with BamHI, and inserted CA 02236867 1998-0=,-26 W O98/13502 PCT~US97/17276 into pBTM116 previously digested with Bam~.

J. pVP16-Tax A DNA sequence encoding full length Tax protein was excised from pS~424 [Kwok, l~.P.S., et al., Nature 380:642-646 ~1996)~ with Barri~
5 digestion and was inserted into pVP16 previously digested with BamHI.

IV. Plasmids For Bin~in~ Protein Controls A. pLeu Plasmid pVP16 was digested with Hin~m and BamHI to remove the fragment encoding the VP16 transactivation domain. The digested 10 vector was blunt-ended and self-ligated.

B. pLexA-VP16 The VP16 transactivation donlain was PCR ampli~led from pGal-VP16 [Sadowski, et al., Natloe 335:563-564 (19g8~] witll a pair of oligonuclcotides, 5 '-VP16SH and 3 'VP16SH and the resulting ampli~lcation 15 product was digested with ClaI, blunt-endcd, and insertcd into pBTM116.

5 '-VP16SH SBQ ID NO: 35 GGCTATCGATACGGCCCCCCCGACCGAT
3'-VP16SH SEQ ID NO: 36 GCGTATCGATCTACCCACCGTACTCGTC

C. pLexA-Lamin See Hollenberg, S.M. et al., Mol. Cell.Biol. 15:3813-3822 (1995)] .

-V. Plasmids Encof~in~ Reporter Gene Controls A. pRS306/Tertn The alcohol dehydrogenase ~ADH) terminator sequence was excised from plasmid pBTM116 [Bartel, et al., in C~ellular Interactions in 5 Development: a Practical Approach, (ed) Hartley, D.A. (IRL Press, Oxford), pp. 153-179 (1993)] with SplzI and PstI restriction enzymes and both 3'-overhanging sequences were blunted by 7'4 DNA polymerase (Gibco BLR, Grand Island, NY). The fragment was gel-purified and subcloned into the blunt-ended NotI site in pRS306 [Sikorski and EIieter, Genetics:122:19-27 10 (1989)~. The orientation of inserted fragment was detertnined by DNA
sequencing.

B. pRS3 1 6/Term The subcloning protocol for inserting the ADH terminator sequence into pRS316 was the same as described for inserting the ADH
15 sequence in pRS306.

Example 2 Generation of Yeast Ass~y Transforrnant Selection of an appropriate yeast assay strain is an empirical determination based on growth characteristics of the transformed alternatives.
20 A general method to make the a~ u,uliale selection is described as follows.
Candidate yeast assay strains were transformed individually with reporter gene constructs and/or a plasmid encoding one of the experimental binding proteins. Assay strains thus transformed were then compared for relative differences in growth characteristics, with an optimal assay strain 25 showing negligible growth on media lacking histidine and vigorous growth on media containing histidine. In practical application of this first step in selection using various plasmids transformed into assay strain YI584, the following results were observed.

, CA 02236867 1998-0~-26 W O 98/13502 PCT~US97/17276 When the plasmid pLexA-VP16 encoding both the LexA DNA
binding domain and the VP16 transactivating domain as a single protein was introduced into the assay cells, growth in the absence of histidine in the mediawas significantly reduced three days after transformation.
In assays including transformation with plasmids encoding multiple copies of the tet operator upstream of the HIS3 gene, the following plasmids were separately lltili7ecl pRS303/lxtetop-HIS ~encoding a single tet operator sequence), pRS303/2xtetop-HIS (encoding two tet operator sequences)~
pRS303/3xtetop-HIS (encoding three tet operator sequences), pRS303/4xtetop-HIS (encoding four tet operator sequences), pRS303/6xtetop-HIS (encoding six tet operator sequences), pRS303/8xtetop-HIS (encoding eight tet operator sequences), or pRS303/lOxtetop-HIS (encoding ten tet operator sequences).

In the assay strains transforrned with plasmids encoding either one, two, or three copies of the tet operator upstream from the HIS3 gene, cells grew on media lacking histidine at a rate similar to cells grown on media containing histidine. In yeast assay strains transfonned with plasmids encoding either six, eight, or ten copies of the tet operator upstream from the HIS3 gene, cell growth was low suggesting that these strains would not be usefu1 in assays to examine binding and interrllption of binding between test proteins. These results suggested that, in assay strains transformed with a reporter plasmid having more than three tet operator sequences upstream from the HIS3 gene, normal activity of the HIS3 promoter is disrupted and that these plasmids would not be useful.
In assays wherein yeast cells were transformed with only reporter plasmids (and not plasmids encoding binding partner fusion proteins) encoding multiple copies of the LexA operator 5 of the TetR gene, the CA 02236867 1998-0~-26 W O 98/13502 PCT~US97/17276 following results were observed. Growtll of assay cells transformed with plasmids bearing one, two, four, and eight copies of the regulatory LexA
operator upstream of the TetR gene appeared to be "copy number" dependent.
Yeast cells transforrned with plasmids having two copies of the LexA operator S grew at a rate significantly higher than those assay cell transformed with a plasmid bearing only one copy of the operator. Cells transformed with plasmids encoding either four or eight LexA operators upstream of the TetR
gene grew at an approximately equal rate, and better than assay cells bearing a TetR gene driven by two copies of the operator.
When the alcohol dehydrogenase (ADH) promoter was included upstream of the LexA operator (plasmids encoding either four or eight LexA
operators) in the various reporter gene constructs, cell viability was the lowest.
The various cell lines constructed by the methods described 15 above are shown in Table 1, wherein various transforlned yeast strains are identified (Strain #) along with the number of LexA operator sequences in the plasmid encoding TetR, the number of tetracycline operator sequences regulating expression of HIS3, and relative growth rate of the transfonned strain on media cont~ining histidine. It is important to note that growth 20 variation of transformed cells in media containing histidine is observed, even in cell lines identically transformed. The number of " + " signs in Table 1 is indicative of the host cell's relative ability to grow on media lacking histidine in the absence of transformation with plasmids encoding potential binding proteins. Also in Table 1, a subscript "a" is indicative of transformation with 25 a plasmid bearing the alcohol dehydrogenase promoter; absence of a subscript "a" indicates use of the HIS3 promoter.

W O 98113502 PCT~US97/17276 Table 1 Va~ous Yeast Transfo~nants Dipl~-ids L40 Diploid6 L40 Strain # L~xA TetOp Hi~+ Strsin # LexA TetOp Hi6+
S Y1579 IX ZX t + + Y1602 4Xn 6X --Y1581 IX 2X + + + Y1607 4X 6X + + +
Y1628 4X 6X + +
Y1580 2X 2X + ++ Y1632 4X" 6X -?
Y15$. 2X ~X ++ t Y1605 4X~10X --Y1610 4X 10X +
Diploid6 L40 Y1622 4X 10X + ~
StrDin # L~xA T~tOp His+ Y1626 4X,,10X + +
Y1583 4X X + + I
Y1585 4X 2X +++ YIS9~ 8X 2X + + +
Y1587 4X 2X + + + Y1596 8XD 2X + ++
Y1589 4X 2X +++
Y1598 8X 4X +
Y1584 8X 2X ++-t Y1635 8X~ 4X +
Y1586 8X 2X +++ Y1637 8X 4X + +
Y1588 8X 2X +++ Y1601 8X 6X +
Y1590 8X2X + + t Y1608 8X~ 6X +
Y1629 8X~ 6X + + +
Y1631 8X 6X + + +
Diploid6 L40 Y1604 8X 10X +
strAil) # LexA TetOp His+ Y1611 8X~ 10X +
Y1591 X 2X +++ Y1623 8X"10X + +
Y15942X 2X + + + Y16Z5 8X 10X + +

Y~6332X 4X +
Y1631) X 4X + t- strDin# LexA T~tOp btrAin Hi6+
Y1664 4X~ 3X w303(50) + + +
Y16002X 6X -- Y1666 4X~ 3X w303(51) + + +
Y1606~X 6X +
Y16302X 6X + Y1668 4Xu 2X L40 (69) + + +
Y16 72X 6X + + + Y1670 4X~ X L40 (70) + + +
3~
v Y16032X lox t Y1665 8X, 3X w303(50) + + +
Y1621 2X 10X + + Y1667 8Xn 3X w303~51) + + +
Y1609 2X 10X + Y1671 8X~ 3X L40 (69) + +
Y1624 ~X 10X + +
Y1669 8Xn 2X L40 (69) + + +
Y1593 4Xc 2X +++ Y1671 8Xn 2X L40 (70) + + +
ylS95 4X 2X + ++
Y1671 8Xn 6X L40 (69) + + +
Y1599 4X~ 4X --Y1634 4X 4X +
Y1638 4X~ 4X +

W O 98/13502 PCTrUS97117276 ~ Example 3 CREB/CBP Binding Interaction Use of the split-hybrid assay for studies of proteinlprotein binding wherein one of the binding components is randomly mutagenized was carried out using CREL and CBP ~inding proteins. The binding of CREB to CBP has been shown to require the phosphorylation of the CREB serine residue at position 133 in a region ~iesign~ted the "kinase~inducihle domain"
(KID) [Chrivia, et al., Nature 365, 855-859 (1993); Kwok, et al., Nature 370, 223-226 (1994)]. Functionally, ch~n~ing serine at position 133 to alanine (a mutant de~ign~ted CREB-Ml) abolishes the ability of CBP to activate CRE~3-mediated transcription. Preliminary studies have indicated that the CREB-Ml mutant in the split-hybrid system prevents the interaction with CBP and subsequent growth of the yeast assay strain on media lacking histidine. Precisely what other portions of ~he KID of CRE33 are required for binding to CBP is unknown, however. To de~me other potentially important amino acid residues, the KID (amino acid residues 102 to 160) of CREB 341 was randomly mutagenized using PCR.

A. PCR Muta~enesis and Creation of Mutant Library The technique used for mutagenic PCR was a modification of that described by Uppaluri and Towle rMol. Cell. Biol. 15, 1499-1512 (1995)]. The reaction mixture contained 20 ng of pVP16-CREB(BglII-SacII)-LacZ, 16 mM (NH4)2SO4, 67 mM Tris-HCl, pH 8. 8, 6. 1 mM MgC12, 0.5 mM MnCl?, 6.7 ,uM EDTA, 10 mM ~-mercaptoethanol, 1 mM primers, lmM
each dGTP, dl-rP, and dCTP, 400 ,uM dATP, and 2.5 units of Taq DNA
polymerase (Promega, Madison, WI). After seven cycles of PCR (94~C ~or 40 sec, 5Q~C for 40 sec, and 72~C for 40 sec), the PCR product was amplified a second time using the same primers and Vent DNA polymerase (New Pn~l~n~1 BioLabs, Beverly, MA) under the same conditions for 25 cycles. The resultant PCR product was gel purified, digested with Bgm and CA 02236867 1998-0~-26 W O 9~113502 PCTrUS97/17276 Sacl~, and inserted into the Bgm and Sac~ sites of pVP1 6-CR~(BglII-SacII)-LacZ (construction of which is described above). The resulting plasmids were transformed into DH5O~ bacterial cells. Transforrnants were pooled and plasmid DNA was isolated by CsCI gradient centrifugation.
.

5 B~ Construction and Use of pVPI~-CRE13(L~lII-SacII)-LacZ
A DNA fragment encoding the ~-galactosidase gene was fused in frame to the carboxyl-terminal end of VP16-CRE33 as described above.
The carboxy-terminal tag allowed identification of clones that contain frame-shift and nonsense mutations; colonies that remain positive for ~-galactosidase 10 were presumed to contain an open reading frame throughout the mutated region. To facilitate the subcloning of mutated sequences, a cassette version of the CREL cDNA was generated that contained BglII and a SacII sites flanking the 5' and 3 ends of the KID, respectively. These modifications altered the amino acid residue at position 168 from valine to alanine. The 15 cDNA altered in this manner was indistinguishable from the original VP16-CREB and from VP16-CREB-LacZ when tested in the split hybrid assay.
Primers complementary to regions llanking the KID were used in mutagenic PC~ amplification reactions as described above under conditions which were optimized to achieve one to three mutations in the 177 bp region encoding the 20 KID. PCR products were introduced into pVPl 6-CRE~3(Bgm-SacII)-LacZ in place of wild-type sequence. A library of mutated sequences was trans~ormed into yeast assay strain YI584 expressing LexA-CBD. Approximately 27,000 yeast transformants were screened, yielding about 5,000 colonies that were capablc of growing on selective media supplemented with 10 ,~4g/ml of 25 tetracycline and I mM of 3AT, determined as described below.
Two screening steps were performed to elimin~te uninformative mutations and false positives. First, filter ,~-galactosidase assays were performed by standard methods [Vojtek, et al.~ Cell 74:205-214 (1993)] Oll the 5,000 colonies which exhibited positive growth on media lacking =

CA 02236867 l998-0~-26 W O 98/13502 PCT~US97/17276 tryptophan, histidine, uracil, leucine, and Iysine to elimin~te expressed proteins having frame-shift and nonsense mutations. Five hundred thirty si~c ~ colonies developed a dark blue color, whereas 412 colonies turned white and were presumed to express mutants cont?inin~ either frame-shift or nonsense 5 mutations. The other colonies developed a pale blue color, and control experiments suggested that these colonies may have expressed unstable lacZ
fusion proteins. Pale blue colonies were not analyzed further.
DNA from 536 dark blue colonies was isolated and transformed into E.coli MCl()66 cells. One hundred ninety three pVP16-CREB-(BglIl-10 SacII)-LacZ cDNAs were then isolated.
In a second screening step, the 193 cDNAs were separately re-transformed along with pLexA-CBD into the split-hybrid strain as well as into the two-hybrid L40 strain [Vojtek, et al., supra] in order to identify false positives and confirm that the mutant CREB proteins did not interact with CBP. Among the 193 cDNAs re-screened, 152 did not interact with CBP in the yeast two-hybrid system, 15 interacted weakly, and 26 interacted like wild type CREB.
Following these two screening steps, the 152 CREB mutants were sequenced. Seventy CRE.13 mutants were found to contain a single amino acid change. Sixty four CRE~3 mutants contained two amino acid residue mutations and 13 mutants contained more than two amino acid mutations. Mutants cont~ining more than one amino acid alteration were not analyzed further. The expression level of mutant proteins having one amino acid change were determined using a standard ,~-galactosidase assay.
The CR~B mutations identified in the split-hybrid screen were shown to carry atnino acid changes centered around the phosphorylation site at serine at position 133. No disrupting mutations were found to contain amino acid alterations outside of the region between amino acids 13û to 141.
Most of the mutations abrogated the PKA phosphorylation region, but others wefe identified at isoleucine position 137, leucine at position 138, and leucine CA 02236867 1998-0=,-26 W O 98/13S02 PCT~US97/17276 at position 141. The mutations at positions 137, 138, and 141 geneMlly changed the hydrophobic residues at these positions to polar residues. The ability of the split-hybrid system to detect only a limited number of CRE13 mutants, many of which have been proposed previously to disrupt CREB
association with CBP [Parker, et al., Mol. Cell. Biol. 16, 694-703. (1996)], indicates the specificity of the split-hybrid system.
These results lead to interesting suggestions. Various CRE13 mutations were identified which ~disrupt CREB-CBP interaction and the majority of disrupting mutations occurred in the CREB PKA phosphorylation 10 motif. This result was consistent with previous observations that nonphosphorylatcd CREB and CBP do not interact [Kwok, et al., Nature 370:223-226 (1994)]. The most common motif for PKA phosphorylation is an RRX(S/T)X amino acid sequence but RX(S/T)X and K~XX(S/T)X are also phosphorylated [Kemp and Pearson, T.I.B.S. lS, 342-346 (1990)]. The 15 argininc residues in the phosphorylation site are critical for electrostatic interactions with acidic amino acid residues in the catalytic subunit of PKA
[Knighton, et al., Science 253, 414-420 (1991)], and consistent with this observation, CRI~B mutants with changes at arginine residues 130 and 131 were identified in the split hybrid assay that did not interact with CBP.
Results also showed that CRE~3 mutations at amino acids proline at residue 132 and tyrosine 134 were unable to bind CBP. It is likely that the mutations at these residues adversely affect the structure of the phosphorylation motif, although these positions are generally thought to be less critical to CBP binding. It is possible that the substitution of proline at25 position 132 with threonine created a new phosphorylation site (RXIX) that interfered witll the critical phosphorylation of serine at position 133.
Although not generally thought to be part of the "classical" consensus PKA
phosphorylatioll motif, hydrophobic amino acids are commonly found carboxy-terminal to PKA sites [Kemp, et al., T.I.B.S. 19:440-444 (19~4)].
3n The importance of these flanking residues n~ay explain the fre~uent occurrence CA 02236867 1998-0~-26 W O 98/13502 PCTrUS97/17276 of disrupting mutations involving tyrosine at position 134. Further studies will be directed to determining if mutations of proline at position 134 and tyrosine at position 134 directly disrupt phosphorylation of serine at position 133 or disrupt binding of CREB to CBP by some other mechanism.
In addition, substitution of serine at position 133 with threonine also prevented the interaction of CREB and CBP. PKA protein substrates cont~ining a phosphorylatable threonine residue are known to exist in nature (i. e., protein phosphatase inhibitor I and myelin basic protein), although theyare less common than those with phosphorylatable serines [Zetterqvist, et al., 10 in Peptides and Protein Phosphorylation, (ed.) Kemp, B.E. (CRC Press, Boca Raton, FL), pp. 172-187 (1990)], and synthetic peptides cont~inin~ serine to threonine substitutions are relatively poor substrates for PKA phosphorylation [Zetterqvist, et al., supra]. In the split-hybrid assay, however, it is unclear whether the mutation of threonine at position 133 disrupts the CREB-CBP
15 interaction or if the mutant fails to become phosphorylated. Despite previousobservations that serine residue at position 133 of m~mm~ n CR~B can be phosphorylated by a variety of protein kinases other than PKA, for example calcium/calmodulin-dependent protein kinase II and IV, protein kinase C, and a nerve growth factor (NGF)-activated ~REB kinase [Sheng, et al., Neuron 20 4:571-582 (1990); Sheng, et al., Science 252:1427-1430 (1991); Xie and Rothstein, J. Immunol. 154:1717-1723 (1995); Ginty, et al., Cell 77:1-20 (1994)~, it is not known which, if any, of these particular protein kinases are able to phosphorylate CREB at the serine at position 133 in yeast. The requirement for integrity of the entire RRXSX amino acid sequence, however, 25 suggests that PKA is a reasonablc candidate.
The second category of mutations were identified adjacent the PKA phosphorylation motif. Amino acids isoleucine at position 137 and leucine at position 138 have previously been suggested to be important for hydrophobic interactions of CREB with CBP [Parker, et al., Mol. Cell. Biol.
16, 694-703 (1996)]. In this study, most of the mutations at position 137 and CA 02236867 l998-0~-26 W O 98/13502 PCTnUS97/17276 138 converted these hydrophobic residues to polar amino acids. Thus, another possibility for the failure of these ~utants to bind to CBP is that changes at these positions affect protein folding. Similarly, the mutation at position 141 substituted a polar residue for the wild-type hydrophobic leucine, and this 5 mutation also has the potential to affect protein folding.
Substitution of the isoleucine at position 137 with a hydrophobic phenyl~l~nin~ residue was found to disrupt the ;nteraction between CREB and CBP as well. This result could have been the result of a detrimental effect on folding because of the steric hindrance associated with the comparatively 10 largcr sizc of phenyl~l~nine. Alternatively, the proposed hydrophobic interactions between CREB and CBP are somewhat spccific. Structural studies will be directed to definitively determine how these mutations affect binding.
Perhaps most surprising was the finding that clitical mutations 15 were restricted to a small region in the KID sequence, even though the relatively low affinity of phosphorylated CREB and CBP, determined to be between 250 and 400 nM by fluorescence anisotropy measurements [Kwok, et al., l~atu~e 370, 223-226 (199~)], is consistent with a restricted protein binding domain. The capability of the split-hybrid system to screen for a 2û limited number of CREB mutants suggests that the system is highly speci~lc, and thus, should be useful to identify mutations which disrupt inte}acts between other pairs of binding proteins.

CA 02236867 1998-0~-26 W O 98/13502 PCTrUS97/17276 Example 4 Tax/SRF Binding Interaction To further investigate the feasibility of using the split-hybrid system to study protein-protein interactions, a pair of we~l characterized interacting proteins, SRF and Tax, was tested. Previous studies indicated that SRF and Tax interact in a standard yeast two-hybrid system suggesting that the proteins may be utilized in the split hybrid assay. Plasmid pLexA-SRF, conl~inin~ a human SRF cDNA fused to the LexA DNA binding domain, was transforrned into strain YI584 along with either pVP16-Tax or pVP16 alone.
10 As with the pLexA-VP16 transformation, the yeast strains co-expressing LexA-SRF and VP16-Tax failed to yield any colonies on medium lacking histidine. In contrast, when LexA-SRF was co-transformed with a vector encoding the VP16 activation domain alone, yeast growth occurred on medium lacking histidine, suggesting that TetR expression was not activated. These 15 results demonstrated that a protein-protein interaction in the split-hybrid system can effectively prevent yeast growth and further indicated the utility of thc assay for the study of various protein/protein interactions.

Example 5 Casein Kinase Binding Assays 20 Hrr25 In another example of use of the split hybrid assay to examine protein/protein interactions, Hrr25, a yeast casein kinase isoforln, or human casein kinase ~ isoforrn ~, was employed in the assay with a known binding partner protein.
Previous work using the two hybrid assay had identified three genes encoding proteins which interact with the yeast casein kinase isoforrn Hrr25. Proteins encoded by the genes were de~ign~t~cl TIH1, TIH2, and TIHE3. The Hrr25 expression construct which was generated for use in the two hybrid assay was used in combination with the individual TIH encoding 30 constructs in the split hybrid assay to determine if interaction between the CA 02236867 1998-0~-26 W O98113502 PCT~US97/17276 binding partners would decrease growth of assay yeast cells on media lacking histidine. Construction of the Hrr25 expression plasmid and isolation of plasmids encoding TIH proteins is discussed below.
In order to identify genes encoding proteins that interact with S 5. ce)evisiae HRR25 CKI protein kinase~ a plasmid library encoding ~ilsions between tlle yeast GAL4 activation domain and S. cerev~siae genomic fragments ("prey" components) was screened for interaction with a DNA
binding domain hybrid that contained the E. coli lexA gene fused to H}~R25 ("bait" component). The fusions were constructed in plasmid pBTM116 10 which contains the yeast TRPI gene, a 2,11 origin of replication, and a yeastADHI promoter driving expression of the E. coli lexA protein cont~ining a DNA binding domain (amino acids 1 to 202).
Plasmid pBTM116::HRR25 encoding the lexA::~lRR25 fusion protein was constructed in several steps. The DNA sequenGe encoding the 15 initiating methionine and second amino acid of HRR25 was changed to a SmaI
restriction site by site-directed mutagenesis using a MutaC~ene mutagenesis kit from BioRad (Richmond, California). Tlle DNA sequence of HRR25 is set out in SEQ ID NO: 39. The oligonucleotide used for the mutagenesis is set forth below? wherein the SmaI site is underlined.

20 5 '-CCTACTCTTAGGCCCGGGT~ l l l-l-l AATGTATCC-3 ' (SEQ ID NO: 37) After digestion with SmaI, the resulting altered HRR25 gene was ligated into plasmid pBTM116 at the S~laI site to create the lexA::HRR25 filsion construct.
Interactions between bait and prey fusion proteins were detected in yeast reportcr strain CTY10-5d (genotype=MATa ade2 t~p1-901 leu2-3,112 ~is 3-200 gal4 gal80 URA3::1exA op-lacZ.) [Luban, et al., Cell 73:1067-1078 (1993)] carrying a lexA binding site that directs transcription of CA 02236867 l998-05-26 lacZ. Strain CTY 1 0-5d was first transformed with plasmid pBTM116::HRR25 by lithium acetate-mediated transformation [Ito, et al., ~ J.Bacte~ol. 153:163-168 (1983)]. The resulting transformants were thentransformed with a prey yeast genomic library prepared as G,AL4 fusions in S the plasmid pGAD [Chien, et al., Proc.Natl.Acad.Sci (USA) 21:9578-9582 ( 1991 )] in order to screen the expressed proteins from the library for interaction with HRR25. A total of 500,000 double transformants were assayed for ~-galactosidase expression by replica plating onto nitrocellulose filters, Iysing the replicated colonies by quick-freezing the filters in liquid nitrogen, and incubating the Iysed colonies with the blue chromogenic substrate 5-bromo-4-chloro-3-indolyl-,~-D-galactoside (X-gal) . ~-galactosidase activity was measured using Z buffer (0.06 M Na2HPO4, 0.04 M NaH2P04, 0.01 M KCI, 0.001 M MgS04, 0.05 M ~-mercaptoethanol) cont~ining X-gal at a concentration of O.002 % [Guarente, Meth. Enzymol. 101 : 1 8 1 - 1 9 1(1983)] .
Reactions were termin~tecl by floating the filters on lM Na2CO3 and positive colonies were identified by their dark blue color.
Library fusion plasmids (prey constructs) that conferred blue color to the reporter strain co-dependent upon the presence of the HRR25/DNA binding domain fusion protein partner (bait construct) were identified. The sequence adjacent to the fusion site in each library plasmid was determined by extending DNA sequence from the GAL4 region. The sequencing primer utilized is set forth below.

5'-GGAATCACTACAGGGATG-3' (SEQ ID NO: 38 ) DNA sequence was obtained using a Sequenase version II kit (US
Biochemicals, Cleveland, Ohio) or by automated DNA sequencing with an ABI373A sequencer (Applied Biosystems, Foster City, California).
Four library clones were identified and the proteins they encoded are designated herein as TIH proteins 1 through 4 for Targets Interacting with W O 98/13S02 PCT~US97/17276 HRR25-like protein kinase isoforrns. The TIH1 portion of the TIH1 clone insert corresponds to nucleotides 1528 to 2580 of SEQ ID NO: 40; the TlH2 portion of the Tm2 clone insert corresponds to nucleotides 2611 to 4053 of SEQ ID NO: 41; and the 1'Ltl3 portion of thc 1~i3 clone insert corresponds to nucleotides 248 to 696 of SEQ ID NO: 42. Based on DNA sequence analysis of the 'l'~i genes, it was determined that TIH1 and TIH3 were novel sequences that were not representative of any protein motif present in the GenBank cl~t~b~ce (July 8, 1993). TIH2 sequences were identified in the database as similar to a yeast open reading frame having no identified fimction. (GenBank Accession No. Z23261, open reading frame YBL0506) When the various T~I proteins were used in the split hybrid assay in combination with Hrr25, it was observed that Hrr25/'1'1~3 binding, previously determined to be weaker than Hrr25/T~I2 or Hrr25/'l'l~l 1 interactions, produced the lowest level of growth in the transformed yeast strain .

CKI~
In order to isolate cDNAs which encode proteins that interact witll CKI~, the two hybrid assay was perforrned using a LexA-CKI~ fusion protein as bait. The coding region of CKI~ was subcloned into a BamHI site of pBTM116 and transfonned into a yeast strain designated CKI~/IA0 (MAT
a his3 ~\200 trpl-901 leu2-3 112 ade2 LYS::(lexAop)4HIS3 URA3::(1exAop)8-lcZ GAL 4). CKI~/L40 was subjccted to a large scale transformation with a cDNA library made from mouse embryos staged at days 9.5 and 10.5.
Approximately 40 million transformants were obtained. Eighty-eight million were plated onto selective media lacking leucine, tryptophan and histidine.
The ability of yeast transformants to grow in the absence of histidine suggested that there was an interaction between CKI~ and some library protein.
In a second screening, interaction of the two proteins was -CA 02236867 1998-0~-26 W O 98/13502 PCTrUS97tl7276 assayed by the ability of the interaction to activate transcription of ,B-galactosidase. Colonies that turned blue in the presence of X-gal were ~ streaked onto media lacking leucine, tryptophan and histidine, grown up in liquid culture and pooled for isolation of total DNA. Isolated DNA was used 5 to transforrn E. coli strain 600 which lacks the ability to grow on media lacking leucine. Colonies that grew were used for plasmid preparation and three classes of cDNA were identified. One class was closely related to a Drosop~zila transcription factor dCREBa.
When CKI~/CREB interaction was examined in the split hybrid 10 assay, cells were shown to grow on media con~ining histidine, but in the absence of histidine, growth was inhibited. Addition of small amounts of tetracycline tO the cell culture restored the cel~'s ability to grow, suggestingthat the interaction between CKI~ and CRE;Ba was very weak.

Example 6 AKAP 79 Binding Assays Expression Plasmid Utilized In still another example of use of the split hybrid assay to examine protein/protein interactions, an anchoring protein for the cAMP
dependent protein kinase, AKAP 79, was utilized separately with binding 20 partner proteins including the cAMP protein kinase regulatory subunit type I
(RI), the cAMP dependent protein kinase regulatory subunit type II (RII) or calcineurin (CaN). Plasmids used in the assay were constructed as described below.
A 1.3 kb NcoIlBamHI fragment cont~;ning the coding region 25 of AKAP 79 was isolated from a pETl ld backbone and ligated into plasmid pASl. Plasmid pASl is a 2 micron based plasmid with an ADH promoter linked to the Gal4 DNA binding subunit [amino acids 1-147 as described in Keegan et al., Science, 231:699-704 (1986)], followed by a hemagglutin (HA) tag, polyclonal site and an ADH terminator. The expressed protein was W O 98/13502 PCT~US97/17276 therefore a filsion between AKAP 79 and the DNA binding domain of Gal4.
Plasmids encoding RI, RII or CaN were isolated from a pACT
murine T cell library in a standard two hybrid assay using the AKAP 79 expression construct described above. Plasmid pACT is a leu2, 2 micron S based plasmid cont~ining an ADH promoter and terminator with the Gal4 transcription activation domain II ramino acids 768-881 as described in Ma and Ptashne, Cell, 48:847-853 (1987)], followed by a multiple cloning site.
RI, RII and CaN encoding plasmids were isolated as described below.
A 500 ml SC-Trp yeast cell culture (OD600 = 0.6-0.8) was harvested, washed with 100 ml distilled water, and repelleted. The pellet was brought up in 50 ml LiSORB (100 mM lithium aceta~e, 10 mM Tris pH8, 1 mM EDTA pH8, and 1 M Sorbitol), transferred to a 1 liter flask and shaken at 220 rpm during an incubation of 30 minutes at 30~C. The cells were pelleted, resuspended in 625 ,ul LiSORB, and held on ice wllile preparing the l 5 DNA.
The DNA was prepared for transformation by boiling 400 ,ul 10 mg/ml salmon sperm DNA for 10 minutes after which 500 ,ul LiSORB was added and the solution allowed to slowly cool to room temperature. DNA
from a Mu T ccll library was added (40-50 ,ug) from a 1 mg/nnl stock. The iced yeast cell culture was dispensed into 10 Eppendorf tubes with 120 ~l of prepared DNA. The tubes were incubated at 30~C with shaking at 220 RPM.
After 30 minutes, 900 ~1 of 40% PEG3350 in 100 mM Li acetate, 10 mM
Tris, pH 8, and l mM EDTA, pH 8, was mixed with each culture and incubation continued for an additional 30 minutes. The samples were pooled and a small aliquot (5 ,ul) was removed to test for transformation efficiency and plated on SC-Leu-Trp plates. The remainder of the cells were added to 100 ml SC-Leu-TIp-His media and grown for one hour at 30~C with .ch~king at 220 RPMS. Harvested cells were resuspended in 5.5 ml SC-Leu-Trp-His cont:~inin~ 50 mM 3AT (3-amino tliazole) media and 300 ~1 aliquots plated on l50 mm SC-Leu-Trp-His also conl;linin~ 50mM 3AT. Cell were left to CA 02236867 l998-0~-26 W O 98/13502 PCTrUS97/17276 grow for one week at 30~C.
After four days, titer plates were counted and 1. lx105 colonies ~ were screened. Large scale ,~-gal assays were performed on library plates and ten positive clones were isolated for single colonies. One of these colonies 5 grew substantially larger than the rest, and was termed clone 11.1. Sequence from clone 11.1 revealed an open reading frame 487 aa long which was correctly fused to the Gal-4 activation domain of pACT. The NrH sequence h~e was searched and the sequence was found to be closely homologous to the human calmodulin dependent protein phosphatase, calcineurin.
10Additional screening using pACT Mu T-cell library DNA and the pASI AKAP 79 bait strain was performed in order to identify other AKAP
79 binding proteins by the protocol described above. Results from screening approximately 211,000 colonies gave one positive clone ~le~ign~tp~l pACT 2-1.
Sequencing and a subsequent data base search indicated that the clone had 15 91% identity with rat type lo~ regulatory subunit of protein kinase A (RI).
The library was rescreened using the same AKAP 79 bait and fifte~n positives were detccted from approximately 520,000 transformants. Of these fifteen, eleven were found to be homologous to the rat regulatory subunit type I of PKA. Each of these isolates were fused to the 5 ' 20 untr~n~l~ted region of RI and remained open through the initi~ting methionine.

Split Hybrid Analysis ~ n split hybrid analysis of AKAP79 binding interactions, a plasmid was first constructed for expression of a LexA:AKAP 79 fusion protein. An AKAP 79 coding region was excised from pAS AKAP 79 as an 25 NcoI/BamHI fragment and inserted into pBTM1 16 previously digested with the sanne enzymes. The resulting plasmid was designated pBTM1 16-AKAP79.
Approximately 50,000 W303 yeast cells (strain YI665, see Table 1) in logarithmic growth were rinsed in media lacking hi~tiAinP, suspended in 100 ,~bl to 200 ~l of the same media, and plated on agar lacking CA 02236867 l998-0~-26 W O 98/13502 PCT~US97/17276 histidine (to select for absence of protein/protein interaction) and also lacking leucine and tryptophan (to select for transfonmants bearing expression constructs encoding AKAP 79 and its binding partner). When RII was employed as the AKAP 79 binding partner, 2 to 4 ,uM tetracycline and 5 mM
5 3AT were required to prevent the transformed host from growing under conditions where the expressed proteins interacted.
Once conditions were established under which growth of the transformed host was elimin~d, various candidate inhibitor compounds were separately added to the agar. It was presumed that if one of the candidate 10 compounds was capable of disrupting AKAP 79 interaction with the binding partner protein, growth of the transforrned host should be ~lçtçchhle in the vicinity of the compound on the agar. In the split hybrid assay wherein AKAP 79 and RII binding was examined, 2,u1 of a 30 mM stock solution of ICOS Compound 4273 in DMSO, 2 ,~bl of a 10 mM stock solution of ICOS
Compound 1062 in DMSO, and 2 ,hl DMSO alone (as a negative control) were spotted on to the plate which was incubated at 30~C for four to five days. For ICOS Compound 4273 a ring of growth was detected.
In order to determine an IC50 for an inhibitor identified as dcscribed above, alternative methods may be used. In one method, the inllibitor compound is added to the agar over a range of concentrations.
Ideally, the compound is diluted to the point that host cell growth is essentially not detectable.
In another method, a 96 well plate is used and the compounds of interest are serially diluted across one row of a 96 well plate, one compound per row. Media lacking histidine, tryptophan, and leucine is added (prçsl~min~ that the expression plasmids encoding the binding partners also encode trp and leu proteins) along with the appropriately transformed host yeast strain. Tetracycline and 3AT are added at concentration previously detennined to extinguish growth of the transformed host cell. After two to five days incubation at 30~C, the plate wells are read at ~ruxi~llately 600 WO 98/13502 PCT~US97/17276 nm using a plate reader. The concentration of inhibitor half way between zero and the lowest concentration that permits growth of the host cell to the level - observed on media cont~ining histidine is estim~ted to be ICso.
A modi~lcation of this second method is particularly amenable for use in a high throughput screen of large numbers of candidate inhibitors.
For example, rather than attempting to determine the IC50 for a previously identified inhibitor, separate candidate inhibitors are added to each well of a 96 well plate, preferably at more than one concentration, and host cell growth de~ennined after several days incubation. Inhibitory activity of compounds identified in this manner is confirmec~ on an agar plate and the IC50 determined on 96 well plates, each assay as described above.

~xample 7 General Application of The Split-Hybrid Screen In order to examine general utility of the split hybrid system, variolls experiments were conducted with binding proteins known to interact.
In addition, a number of control experiments were included in order to determine if the effects observed with the known binding partners were in fact due to protein/protein interaction.

A. Yeast Assay Strain Construction Yeast transfonnants used in assays indicated below were derived from LYS2-deficient strains AMR6g (Mat a his3 Iys2 leu2 trpl, URA3:LexA::LacZ) and AMR70 (Mat o~ his3 Iys2 trpl leu2, URA3:LexA::LacZ) [Hollenberg, et al., Mol. Cell. Biol. 15, 3813-3822 (1995); Chien, et al., Proc. Natl. Acad. Sci. (USA) 88:97578-9582 (1991);
Fields and Song, Nature 340:245-246 (1989)]. Yeast were grown in YEPD
~ or selective minim~l medium using standard conditions rSherrnan, F., et al., Methods in Yeast Genetics. Cold Spring Harbor Lab., Cold Spring Harbor, NY (1986); Methods in Enzymology, Vol. 194 Guide to Yeast Genetics and W O 98/13502 PCTrUS97/17276 Molccular Biology. Eds. Christine and Fink]. Derivatives of both AMR69 and AMR70 strains lacking URA3 were first generated by streaking cells on synthetic media cont~ining 5 mg/ml 5-fluoro-orotic acid (SFOA) ~Methods in ~n~mology, Vol. 194 Guide to Yeast Genetics and Molecular Biology. Eds.
5 Christine and Fink]. Two URA3 de~1cient mutants were required due to the fact that these strains were subsequently mated. URA3-def;cient colonies were confirmcd by testing ~or uracil auxotrophy and deletion of the URA:LexA::Lac~; locus was confirmed by an absence of ,5-galactosidase activity assayed by standard methods. The mutant strains selected were designated 69-4 and 70-1.
Targeted integration of pRS306/8xLexAop/TetR was carried out by trallsforming [Hollenberg, et al., Mol. ~ell. Biol. 15, 3813-3822 (1995)]
the 64-4 strain with plasmid lineaEized at a unique NcoI site. The reporter gene construct was constructed using parental plasmid pRS306 which encodes UR,43 as a selectable marker. Stably integrated plasmid thereby permitted selection on media lacking uracil. The positive uracil prototrophic strains were examined by Southem analysis to confirm insertion of the plasmid sequences.
Targeted integration of pRS303/2xtetop-LYS was carried out by transformation [Hollenberg, et al., supra] of strain 70-1 with plasmid linearized at a unique HpaI site. The resulting lysine pr~tot.ophic strains were examined by Southern analysis to confirm insertion of the plasmid DNA.
The AMR69 derivative strain (MAT o~) cont~ining the pRS303/2xtetop-LYS insertion was mated with the AMR70-derivative strain (MAT a) cont~ining pRS306/8xLexAop/TetR and mated cells were selected on media lacking both Iysine and uracil. Single colonies were grown up and tested for the ability to grow on media lacking histidine. The resulting strain was designated YI584. In instances where yeast strains were transforrned with other reporter gene pair combinations, the strains were uniquely deci~n~t~cl.
Yeast bearing integrated reporter gene constructs were -CA 02236867 1998-0~-26 W O 98/13502 PCTrUS97/17276 subsequently transformed [Hollenberg, et al., supra~ with plasmids encoding chimeric binding protein. Plasmids encoding the LexA DNA binding region - were generally derived from parental plasmid pBTM116 which also encodes TRPl as a selectable marker. Plasmids encoding the VP16 transactivating S domain were generally derived from parental plasmid pVP16 which also encodes LEU2 as a selectable marker. Yeast cells which were successfully transformed with the four exogenous plasmids were therefore selected by an ability to grow on media lacking Iysine, uracil, tryptophan, and leucine.
Plasmids encoding various binding proteins were transfonned into the yeast 10 assay strain as indicated below.

B. Liquid Assay After three days growth at 30~C on selection media as described above, a pool of colonies from each transformation was collected and diluted in 5 ml selective media. The mixture was vortexed and lS immediately sonicated for ten seconds. Cells in the resulting suspension werecounted and seeded at 1000 cells/ml in selective media, 2 ml per 15 ml tube.
Tetracycline, 3AT, and histidine were included as determined appropriate by the method described above. Each aliquot of cells was incubated with shaking for two days at 30~C and cell density measured at OD600.

20 C. Characterization of the Assay The utility of the split-hybrid assay was first determined using well characterized binding proteins and various controls.
In an initial study, YI584 cells were transformed with plasmids - pLexA-VPl6 and pLeu. While the expressed proteins from the two plasmids 25 do not interact, pLexA-VPl6 encodes a fusion protein cont~ining tlle VP16 activation domain fused directly to LexA which contains a DNA binding domain. The chimeric LexA-VP16 protein is a strong transactivator for a promoter cont~inin~ LexA operators. Plasmid pLeu is essentially a blank used CA 02236867 1998-0~-26 W O 98113502 PCT~US97tl7276 as a control co-transformation plasmid.
Yeast transformed with the LexA-VP16 plasmid were able to express TetR protein as indicated by gel shift analysis using a tet operator oligonucleotide. In addition, the cells were unable to grow on media in the 5 absence of histidine. Combined, these observations suggested that overexpressed TetR protein was capable of binding to tet operators and preventing the expression of HIS3. The transformed yeast grew on plates cont~ining histidine, further indicating that overexpression of TetR did not have a toxic effect on the assay cells.
The results were consistent with previous observations and supported the earlier suggestion that activation of TetR expression, either through a single transcription factor or association of individual transcriptionfactor domains, is capable of preventing assay cell growth on media lacking histidine, presumably by elimin~ting HIS3 production.

Example 8 Split-Hybrid Assay With Weakly Interacting Binding Proteins Protein/protein interaction was examined in the split-hybrid assay to determine utility of the system using two fusion proteins known to interact weakly. In this instance, the binding proteins were a 283 amino acid 20 fragment of a cAMP regulatory binding protein (C~EB283) fused to LexA
and a fragment of the CREB binding protein con~ ting of the CREB binding domain (CBD) fused to VP16.
In this assay, yeast strain YI584 described above was employed and transformation carried out as previously described. In a first assay, 25 plasnaids pLexA-CRE13 and pVP16-CBD were transformed into the cells and cell growth was observed in the absence of histidine in the media. Expression of the fusion proteins was confirmed by Western blotting. Attempts to decrease cell growth by titration with 3AT were unsuccessful in that the concentration of 3AT required to reduce growth in cells transforrned with CA 02236867 1998-0~-26 W O 98/13502 PCT~US97/17276 pLexA-CREB and pVP16-CBD also elimin~tecl growth in cells transformed with pLexA-CREB and the control plasmid pVP16.
In light of these results, two alternative approaches were taken in order to permit study of binding proteins wherein the interaction is relatively weak. Under the assumption that the system was failing at the level of TetR transcription, alternative approaches were taken in attempts to amplify the TetR effect on expression of HIS3 gene. To achieve this end, assay cells were transformed with reporter constructs which encoded multiple tet operator sequences upstream from the ~ i3 gene. In the second approach, the HIS3 10 promoter used to drive expression of the TetR gene was replaced with the stronger alcohol dehydrogenase (ADH) promoter.
In YIS96 cells wherein the ADH promoter replaced the HIS3 promoter to drive TetR expression, transformation with plasmids pLexA-CRE~ and pVP16-CBD showed substantially decreased growth on his- media 15 as compared to that in assay strain YI592 wherein the HIS3 promoter was used to drive Tet~ expression. ~Iowever, in cells transformed with plasmids pLexA-CREB 341-M1 and pVP16-CBD, no decrease in assay cell growth was detected on media lacking histidine. These results indicate that incorporation of the ADH promoter to drive TetR expression may be more useful in studies 20 involving ~inding proteins that have low afflnity.
When assay strains were utilized which incorporated plasmids wherein expression of the HIS3 gen~ was driven by multiple copies of the tet operator, transformed cell lines did not grow well enough to indicate potential utility in subsequent assays.

CA 02236867 1998-0~-26 W O98113502 PCT~US97/17276 .' - 54 -Example 9 General Assay Methods A. "Fine Tuning"
In instances where either of the test fusion proteins possesses 5 intrinsic capacity for transcriptional activation, TetR will be expressed and growth of the assay strain media lacking histidine will be depressed proportional to the level of TetR expression. In order to restore growth of these cells to approximately the level observed on media cont~;ning histidine, the initially transformed assay yeast strains are grown in the presence of 10 increasing concentrations of tetracycline which binds to the TetR gene product and prcvents TetR binding to the tet operator. Precise titration of expressed TetR with tetracycline, only to the point that growth of the assay strain is restored to the level detected in the presence of histidine, permits detection of subse4uent decreased growth of the assay strain following increased TetR
15 expression resulting from interaction of the test binding proteins. The empirically determined tetracycline concentration is therefore employed to increase "signal-to-noise" ratios under assay conditions.
After an a~pr~)l iate tetracycline concentration has been detennined for each of the candidate assay strains, the cells are transformed 20 witll the second plasmid encoding the second fusion binding protein. As before, growth of each candidate assay strain is examined on media in the presence and absence of histidine. A desirable yeast assay strain is chosen which shows vigorous growth in the presence of hi~ti~lin~, and negligible growth on media lacking histidine (indicative of the expected protein/protein 25 interaction and resultant decreased expression of HIS3).
In instances where binding between the two test proteins is comparatively weak, TetR expression may not be sufficiently increased to abolish HIS3 expression and cells expressing the resultant low levels of HIS3 will still grow on media which lacks histidine. Cells which show this low 30 level of viability are grown in the presence of increasing concentrations of 3-CA 02236867 1998-0~-26 W O 98/13502 PCT~US97/17276 aminotriazole (3AT), a competitive inhibitor in the hi~ti(1ine synthesis pathway, in order to reduce cell growth to negligible levels when plated on media lacking hi~ticline. As with titration of TetR with tetracycline, addition of 3AT to the media is designed to increase the signal-to-noise ratio by providing significant changes in growth in the presence and absence of histidine in the media.
In a practical apF)lication of the methods for fine tuning, binding between CREB and the CREB binding protein (CBP) is illustrative. Growth of the yeast strain YI584 transformed with p~exA-CBD, encoding the CREB
binding domain (CBD~ of CBP, and pVP16-CREB or pLexA-CBD and the control plasmid pVP16 was substantially decreased and virtually indistinguishable growth rates were ~1et~cted in both in~t~nces on media lacking histidine. This observation indicated that the LexA-CBD protein product possessed sufficient transactivating capacity to elimin~te H[S3 lS production. In order to distinguish growth differences between assay cells transformed with either pVP16 and pVP16-CREB, increasing amounts of tetracycline were added to the media lacking histidine.
In both transformants, tetracycline was able to relieve growth repression in a dose dependent manner, and at increasing concentrations of tetracycline, the difference in growth between the two colonies was increasingly magnified, with the most distinct growth difference observed following addition of tetracycline at 10 ,ug/ml. Addition of tetracycline was therefore able to overcome the intrinsic transactivating capability of the LexA-CBD fusion protein.
Because the ultim~e use of the split-hybrid system is for structure-function studies, mutagenesis studies, drug identification and libraryscreens, it is important to minimi7e background growth that might be confused with disrupted protein-protein associations. This can be accomplished by the addition of 3AT, a competitive inhibitor of the H153 gene product. For instance, in the presence of 10 ,ug/ml of tetracycline, the yeast strain CA 02236867 1998-0~-26 W O 98113502 PCT~US97/17276 transfonned with pLexA-CBD and pVPI 6-C}~EB still conferred approximately 12 % growth of that observed in the presence of his+ media. To ~limini~h this background, increasing concentrations of 3AT were added to the media in the presence of 10 ,ug/ml of tetracycline. At the 3AT concentration of 0.25 mM, the growth of the yeast strain expressing LexA-CBD and VP16-CREB was below 5 %, while the growth of the control strain was still m~int~in~d at 70%
of control levels. These results indicate that split-hybrid system can be modulated by 3AT in addition to tetracycline in order to effectively increase the signal-to-noise ratio.

10 B. Preparation of yeast extracts In order to assess the utility of various plasmids to function in the split-hybrid assay, a number of control experiments can be employed w}licll lend insight into expression of a desired protein from the transformed plasmid. For example, standard immunological methodologies, i. e., im munoprecipitation, ELISA, etc., can be used to determine to the extent to which a desired protein is expressed. Similarly, a variation of the gel shift assay (discussed immediately hereafter) can be used to determine both if a protein is expressed and if the expressed protein is capable of DNA binding.
In each of these control assays, a yeast extract is re~uired which can be 20 prepared as follows.
Extracts were prepared as described by Uppaluri and Towle [Mol. Cell. Biol. 15:1499-1512 (1995)] and were used for electrophoretic mobility shift assays as discussed below. The yeast cells transformed with pLexA-VP16 were grown in 100 ml of selective synthetic medium lacking 25 uracil, tryptophan, and Iysine to a density of A6"0 = 1. CeIls were harvested and washed with 5 ml of EB (con~ining 0.~ M Tris-HCI, pH 8.0, 400 mM
(NH4)2SO4, 10 mM MgCl~l 1 mM EDTA, 10% glycerol, and 7 mM ~-mercaptoethanol). Cells were transferred to microcentrifuge tubes and collected by centrifugation. After resuspending in 200 ,bl EB con~ining 1 W O 98/13502 PCT~US97117276 mM phenylmethylsulfonyl fluoride (PMSF), 1,ug/ml leupeptin, and 1,tbg/ml pepstatin, a one-half volume of glass beads was added. The suspension was ~ frozen in a -80~C freezer for 1 hour and thawed on ice. Thawed cells were vortexed at 4~C for 20 minutes, alFter which an additional 100 ,~1 E;E3 was 5 added, and cells were left on ice for 30 minutes. The suspension was centrifuged for 5 minutes, the supernatant was transferred to a new tube which was centrifuged for 1 hour in a microcentrifuge. The supeln~ t was then made to 40% with (NH4)2SO4 and gently rocked for 30 minutes. After a 10 minute centrifugation, the pellet was resuspended in 300 ,ul of 10 mM
10 HEPES, pH 8.0, 5 mM EDTA, 7 mM ,B-mercaptoethanol, I mM PMSF, 1 ~ug/ml leupeptin, and 1 ,llg/ml pepstatin, and 20% glycerol. The resulting suspension was dialyzed against the same buffer, and aliquots were stored at -80~C.

C. Electrophoretic mobility shift assays Electrophoretic mobility shift assays were performed as described by Shih and Towle [J. Biol. Che~l. 267:13222-13228 (1992)1. Double-stranded tet operator oligonucleotides were prepared by combining equivalent amounts of complementary singlc-stranded DNA ~SEQ ID NOS: 7 and 8) in a solution cont~ining 50 mM Tris-HCl, pH 8.0, 10 mM MgC12, and 50 mM NaCI2, 20 heating the mixture to 70~C for 10 minutes, and then cooling to room temperature. The annealed oligonucleotides were labeled by filling in overh~nging 5 ends using the Klenow fragment of E. coli DNA polymerase I with [o~-32P]dCTP. Binding reactions were carried out in 20 ,ul cont~inin~
10 mM Tris-HCl, pH 7.5, 50 mM NaCl, I mM EDTA, 1 mM dithiothreitol, 25 5% glycerol, and 2 mg of poly[d(I-C)]. A typical reaction contained 20,000 cpm (0.5-1 ng) of end-labeled D~A with 3-5 ,ug of yeast extract. Following incubation at 22~C for 30 minutes, samples were separated on a 4.5%
nondenaturing polyacrylamide gel cont~-ining 50 mM Tris, 384 mM glycine, and 2 mM EDTA, pH 8.3. For competition binding experiments, the CA 02236867 1998-0~-26 W O 98/13502 PCT~US97/17276 conditions were exactly as above except that speci~lc and nonspecific competitor DNAs were included in the binding mixture before the yeast extract was added. The concentration of tetracycline, a competitive inhibitor of TetRltet operator binding, was 1 ~M when utili7~d l~}.ample 10 Application of the Split-Hybrid Assay to Identify Agents That Prevent Receptor I)esen~ tion and Drug Tachyphylaxis Over half of the drugs that are used clinically affect the function of seven transmembrane receptors. Althougll many of the characteristics of these receptors are distinct, two general features appear to be conserved. One is the ability to signal through dissociation of h~ lhlleric G proteins. The second is the capacity to lose rcsponsiveness to ligand binding in a process termcd desensitization which is mediated by receptor phosphorylation and the subsequent binding of factors that recognize the phosphorylated state of the receptor which prevents continued cign~ling. Desensitization results in an intrinsic limitation to drug action imposed by the action of the drug itself, i. e., activation of a reccptor by a holmone or drug initiates mechz-nicmc that prevent subsequent responses to repeated a-lminictration of the same agent.
The coupled mech:~nicmc of activation and deactivation together have been termed "homologous desenciti7~tion7" while the inability of a drug to m~int~in its efficacy is known as "tachyphylaxis." Even though the mech~nicmc underlying homologous desensitization have been worked out in great detail over the past few years, there are currently no useful pharmacological approaches available that prevent the inactivation mechanism.
The potential clinical utility of agents that could prevent or modulate drug desen~iti7:ltion is enormous. Four examples where therapy is limited by the inability of receptors to m~intain responsiveness to drugs include: (i) asthma wherein desensitization of airway adrenergic receptors CA 02236867 1998-0~-26 W O98/13502 PCTrUS97117276 _ 59 renders epinephrine treabnent ineffective after a period of hours; (ii) congestive heart failure wherein desen~iti7~tion of adrenergic and VIP
receptors, coupled with an elevation of the ,B adrenergic receptor kinase (,BARK), prevents the inotropic effects of endogenous regulatory hormones;
S (iii) Parkinson's disease, wherein dopamine receptor desenciti7~tion limits the usefulness of agents like L-Dopa; and (iv) chronic pain wherein tolerance results from opiate receptor desensitization. Indeed, it is difficult to conceive of a pharmacological modality in use today that is not limited in its effectiveness by the phenomenon of desensitization.
The biochemical basis ~or G protein-coupled receptor desensiti-zation involves three classes of proteins including arrestins, kinases and G-proteins, all of which have been cloned ~eflcowitz, Nature Biotechnology 14:283-286 (1996)]. FoIlowing activation of a seven transmembrane receptor, a region is phosphorylated by one or more G protein-coupled receptor kinases (known as GRKs 1-6). For example, in the ,B-adrenergic receptor (,BAR) and rhodopsin, the cytoplasmic tail is phosphorylated IPremont, et al., J. biol.
Chem. 269:6832-6841 (1994); Free-lm~n, etal., J. Biol. Chem. 270:17953-17961 (19g5); Palczewski,et al., J. Biol. C~lem. 266:12949-12955 (1991);
Palczewski, et al., J. Biol. C~le~7l. 270: 15294-15298 (1995)] while in the m2 muscarinic receptor, the third cytoplasmic loop is phosphorylated [Nakata, et al., Eur. J. Biochent. 220:29-36 (1994)~. The best characterized members of the family of G protein receptor kinases are the ~AR kinase (,BARK) and rhodopsin kinase which are both membrane-associated. While rhodopsin kinase contains an intrinsic membrane targeting signal [Inglese, et al., Nat~re 359:147-150 ~1992)], ,BARK appears to be targeted to the membrane by association with G protein ~ y subunits [Pitcher, et al., Science 257: 126~-1267(1992); Inglese, et al., Natllre 3S9:147-150 (1992)]. Once the substrate receptor for each kinase is activated, presumably by ligand binding, the kinase associates and phosphorylates serine and threonine residues on the receptor.
The phosphorylated receptor then becomes a binding target for one or more W O 98/13502 PCT~US97/17276 other proteins. In the case of ,(~AR, for example, phosphorylation allows bindillg of arresting which prevents association with G proteins and promotes receptor sequestration and desen.siti7:~t;0n. Using the ~AR as an exemplary desensitization model, it becomes apparent that multiple steps in the pathway 5 appear to provide potential points of regulation each of which is amenable to the split-hybrid screen to identify molecules that can block the overall descnsitization pathway. Speci~lcally in the case of ,BAR, the split hybrid system can be used to identify small molecules that: (i) prevent interaction between ~ARK and the G protein ~ subunit; (ii) inhibit ~ARK activity; and 10 (iii) disrupt the,~A~K:arresting complex.

A. Plasmid Constructions The study of G-protein receptor kinases in the split-hybrid system involves three or more recombinant proteins or two or more recombinant proteins and a recombinant peptide library. In the split-hybAd 15 system discussed above, two yeast primary expression plasmids are employed:
pBTM116 [Bartel et al., Cellular Interactions in Developmenf: a Practical App~oac~l, (cd) Hartley, IRL Press, Oxford, pp. lS3-179 (1993)], which encodes the LexA-fusion protein and the TRPI selectable marker, and pVP16 [Hollenberg et al., Mol. Cell. Biol., 15:3813-3822 (1995)], which encodes the 20 VP16-fusion protein and the LElJ2 selectable marker. In order to study interactions involving more than two recombinant proteins in the split-hybrid system, however, additional selectable markers are employed. Construction of additional yeast expression plasmids which are used to examine interactions between more than two binding proteins is discussed below.
-W O 98/13502 PCTr~S97117276 1. Plasmid pDRM
A DNA fragment colllpli~ g the ADH promoter and LexA
sites, the TetR encoding gene, the nuclear loc~ ti-~n signal, and the ADH
terminator sequence are removed from pRS306/4xLexAop/ADH::TetR with SacI, blunt-ended, and digested with SalI. The fragment is isolated and ligated into pRS303/2xtetop-LYS2 which has previously been digested with NotI, blunt-endcd, and digested with ,SalI. The resulting plasmid, clesi,~n~teclpDRM, is integrated into the LYS2 locus in the yeast genome as described above, and the resulting strain designated YIDRM. Placing the repressor gene and selectable marker reporter gene in the r Y52 locus allows ERA3 to be used a selectable marker.

2. Plasmid pRSURA3 A modified version of the pRS306 vector ~Sikorski et al., Genetics, 122:19-27 (1989)] cont~ining the URA3 selectable marlcer gene is also used to encode additional recombinant proteins in the split-hybrid system.
The plasmid, pRS4~6, has the 2 micron origin of replication inserted into a unique AatI site of pRS306. Plasmid pRS426 is further modified in the foliowing manner:
(i) The ADH promoter sequence is amplifled by PCR from ~ 20 BTM116 using primers which incorporate into the ampli~lcation product the DNA sequence encoding the SV40 large T antigen nuclear localization signal (NLS~ and an initi~tin~ ATG sequence 3' to the ADH promoter. The ADH
promoter/NLS/ATG sequence is inserted into the polylinker of pRS426.
(ii) The ADH terminator sequence is amplified by PCR from - 25 BTM116 using primers which incorporate into the product a DNA sequenceencoding an antibody tag, for example, FLAG, hemagglutinin protein (~A), or thioredoxin (Thio) (FLAG, HA, and Thio antibodies are available through Santa Cruz Biotechnology, Santa Cruz, CA~ and DNA sequences encoding stop codons in all three frames to the ~ ' end of the ADH terminator sequence.

W O 98113502 PCTrUS97/17276 The antibody tag/stop codon/ADH terminator sequence is inserted into the polylinker of pRS426.

3. Plasmid pRS~DE2 PCR is used to engineer uni~ue restriction sites, including for 5 example, Bgm~ Eco47~I, MluI, NheI, and SphI, immediately adjacent the 5' and 3' ends of the URA3 cassette in pRSUR~3. The URA3 cassette is digested from pRSUl~A3 and replaced with the ADE2 cassette which is amplified by PCR.

4. Plasmid pBTM 116/AD4 A ~ragment containing th~ ADH promoter, polylinker, and ADH telminator is digested from pAD4 [Young et al., Proc. Nat'l. Acad. Sci.
(USA), 86:7989-7993 (1989)] with BamHI, blunt-ended and inserted into the blunt-ended PvuI site of BTM116 as described rKeegan et al., Oncogene, 12:1537-1544 (1996)], and the resulting vector design~ted pBTM116/AD4.
PCR is also used to engineer a nuclear localization signal 3' of the ADH
promoter as described above. This vector contains the 17~P1 selectable marker and can encode two recombinant proteins: (i) a LexA-fusion protein and (ii~ a protein expressed from the pAD4 region of the vector.

B. ~ARK and G Protein ~ Subunit Bindin~
In a ~lrst application of the split hybrid assay, disruption of binding between the carboxy-terminal domain of ,GARK, cont~ining the pleckstrin homology (P~I) domain, and the G protein ,B subunit (G,~2) is examined. Previous work indicates that the PH domain of ~BARK interacts directly with the ~By subunits of G proteins [Pitcher, J.A., et al. Science 257: 1264- 1267 (1992) and Touhara, K. et al., J.Biol. Chenl. 269: 10217-10220 (1994~]. C onsistent with this observation is work by pl-migli~, et al.
rPumiglia, K.M., et al., J.Biol.Chem. 270:14251-14254 ~1995)] which CA 02236867 l998-05-26 W O 98/13502 PCT~US97/17276 indicates that G~2 interacts with Rafl in yeast and that the interaction is disrupted by ~ARK in Vitro.
A DNA fragment con~ining the carboxy-terminal 222 amino acids (residues 467 to 689) of ,~ARK1, which includes the PH domain, is ampli~led by PCR from bovine ~BA~K1 [Pitcher et al., Science, 257:1264-1267 (1992)] and the gel-purified amplification product is inserted into pBTM116. The resulting plasmid is ~le.~ign~te~ LexA-COO~-,~ARK. A DNA
fragment cont7~ining the entire coding sequence of G~2 [Fong et al., Proc.
Nat'l. Acad. Sci. (USA), 84:3792-3796 (1987)~ is PCR amplified from pGEM-10 l lZf(-)G,B2 ~Inigez-Lluhi et al., JBC, 267:23409-23417 (1992)] and the gel-purified amplification product inserted into pVP16. The resulting plasmid is designated pVP16-G,B2. PCR is used in a similar manner to clone the carboxy-terminal domain of ,~ARK into pVP16 and G,~2 into pBTM116.
,BARK and G,~2 binding is first examined in the two-hybrid 15 system to determine if expression of either binding partner as a fusion protein in yeast affects protein/protein interaction. Binding of the two proteins is then examined in the split hybrid assay in order to deterrnine if protein/protein interaction is capable of abolishing growth of the assay yeast strain. As above, addition of tetracycline and/or 3-aminotriazole required to maximize 20 the difference in growth in the presence and absence of histidine is empirically determined.
Split-hybrid yeast strains cont:~ining ,BARK and G~2 subunits are used to screen libraries of small molecules. Several types of small molecule libraries can be examined in the split-hybrid assay, including for 25 example, chemical libraries, libraries of products naturally produced by microorg~ni.cmc, ~nim:~lc, plants and/or marine or~ni~mc, combinatorial, recombinatorial, peptidomimetic, multiparallel synthetic collection, protein, peptide and polypeptide libraries. A library of small peptides can be cloned into pRSURA3 as described [Yang et al., Nuc. Acids Res., 23:1152-1156 (1995~ and Colas et al., Nah~re, 380:548-550)] . Peptides corresponding to the W O 98/13502 PCTrUS97/17276 carboxy-tenninus of ,~ARK or other GRKs which have previously been shown to block calcium channel desen~iti7~tion in intact neurons, presumably by blocking ~BARK and G,B2 binding and subsequent trafrlcking of ~BARK to the cellular membrane [Diverse-Pierluissi, et al., Neuron 16:579-585 (199~] can 5 ~e identified in such a screen. Further, it is important to show that the molecules identified through the split hybrid selection affect ~ARK:G,B
interaction as opposed to, for example, tetracycline analogues identified in thescreen that would not be usefill to specifically modulate ,~ARK/G,~2 binding.

B. Identi~ication of ~ARK Inhibitors In a second approach, agents that directly inhibit ,BARK
function are identified in a modification of the split-hybrid system. While identification of specific ,BARK inhibitors may be difflcult, prcliminary data from split hybrid assays using CRE;13/CBP binding partners indicates that the system can be used to identify serine kinase inhibitors. The serine kinase results also suggest several approaches can be employed in attempts to overcome potential problems in identifying ~ARK inhibitors.
Briefly, binding between the phosphorylated G-protein couplcd receptor (P-GR) and arresting is e~mined first in the standard two hybrid assay, followed by identification of inhibitors of P-GR/arresting binding in thesplit hybrid assay. For these studies, fragments of three G protein-coupled receptors are examined: the carboxy-terminal tail of ,B2AR and the third cytoplasmic loop of the m2 muscarinic receptor. A DNA fragment cont~inin~
the carboxy-terminal tail of the ,B2AR (amino acids 330 to 413) is PCR
amplified [KolbiLka et al., JBC, 262:7321-7327 (1987)] and the gel purified product inserted into pBTM116/Ad4 to produce a LexA-,~2AR fusion gene.
The resulting plasmid is de~ign~ted pBTM-~B2AR/AD4. A DNA fragment cont~ining the third cytoplasmic loop of the human m2 muscarinic receptor (nucleotides 268-324) is amplified from pGEX-I3m2 [Haga et al., JBC, CA 02236867 1998-0~-26 W O 98/13502 PCTrUS97/17276 269:12594-12599 (1994)] by PCR and cloned into pBTM116/Ad4 creating a LexA-m2 fusion gene. The resulting plasmid is ~lesign~ted pBTM-m2/AD4.
The entire bovine ,~ARKl coding sequence [Benovic et al., Science, 246:235-240 (1989)] is PCR ampli~led and cloned into the polylinker region origin~tin~
5 from AD4 in pBTM-,~2AR/AD4 and pBTM-m2/AD4. The resulting plasmids are designated pBTM-~2AR/AD4-,~ARK and pBTM-m2/AD4-~ARK, respectively. PCR is used to amplify the DNA fragment contzlining bovine ~arresting-l (amino acids 1 to 437) [Lohse, et al., ~cience, 248:1547-1550 (199Q)]. This fragment is inserted into pVP16 and is decign~ted pVP16-10 ~arresting- 1. PCR is used to amplify the DNA fragment con~ining rat ~arresting-2 (amino acids 1 to 428) [Attramadal, et al., JBC, 267:17882-17890 (1992)] which is inserted into pVP16 to give plasmid pVP16-,Barresting-2. A PCR strategy is also used to clone arresting into the pBTM116/AD4-~ARK plasmid and the ,I~AR and m2 fragments into pVP16. As above, the 15 yeast split-hybrid YIDRM strain is transfonned with the P-GR-arresting along with peptide libraries (cloned into pRSURA3) or grown following transfonnation in the presence of combinatorial drug libraries.
Inhibitors identified in the split hybrid assay should effect dismption of protein/protein interaction either by: (i) inhibiting ,BARK
20 phosphorylation of the receptor, thus preventing recognition of the receptor by arresting, or (ii) by physical disruption of binding between the receptor andarresting. Agents that allow yeast growth for trivial reasons, i. e., tetracycline analogues, can be easi1y identified through use of simple controls.
A first potential problem to overcome in this study is that 25 cytoplasmic ,~ARK enzyme must be targeted to the substrate receptor and, ~ once targeted, must phosphorylate the receptor at appropriate sites. In normal cells, ~By association serves to target ~BARK to the cell membrane; the ,B
subunit binds to both the ~ARK PH domain and the isoprenylated 1~ subunit in association with the membrane. One possible means to encourage the 30 necessary specific interactions is to target the binding components in the assay CA 02236867 1998-0~-26 W O 98113S02 PCT~US97/17276 r ~ 66 ~
by tagging the proteins with nuclear localization signals, i.e."BARK, the receptor cytoplasmic tail, and arresting~ to the nucleus. The plasmids proposed for the study of the P-GR-arresting interaction all contain nuclear localization signal sequences adjacent to recombinant gene sequence.
A second problem is somewhat more difficult to approach. The current model is that receptors must be activated by ligand binding before being phosphorylated by ,BA}~K, i. e., targeting of ,BARK via ,l~ y is not sufficient for receptor phosphorylation. There are two possible explanations for this requiretnent. The first is that phosphorylation sites on the receptor are masked in tlle absence of ligand and ligand binding causes a conformational change which "llnm~kc" the phosphorylation sites. If this is the case, a fragment of the receptor cont~inin~ the immediate phosphorylation site may bc used as the ,BARK target. However, although peptides representing portions of the ,BAR cytoplasmic tail can be phosphorylated by ~ARK, the Km for the phosphorylation reaction is poor, suggesting that the kinase may require some other part of the receptor for binding and that the llnm~kin~ of this binding site by agonist is a critical step.
This problem is addressed in two ways. In the first, the m2 muscarinic receptor is used in place of th~ ~AR in view of previous results which indicate that the m2 protein is a good substrate for ~ARK. The third cytoplasmic loop of the m2 receptor serves as both the binding site and phosphorylation site for kinase and which should allow use of a Lex~/m2 receptor third cytoplasmic loop fusion gene as one component in the screening system.
An alternative approach is to artificially mimic the activated state of the receptor. Haga, e~ al. IJ. Biol. Chen~. 269:12594-12599 (1994)]
have shown that the activity of ,BARK can be stimulated in vitro in the presence of mastoporan, a bee venom peptide. Mastoporan is believed to mimic the cytoplasmic face of an activated receptor and has been shown to increase the affinity of ~BAR~ ~or a GST-m2 receptor fusion protein by over four orders of magnitude. The same effect can be seen by using peptides representing the flanking regions of the m2 third cytoplasmic loop. ~hus, mastoporan should also activate ,BARK in the two-hybrid yeast strains, allow phosphorylation of the receptor fusion protein, and promote interaction with 5 arresting. If mastoparan is needed, oligonucleotides cont~ining the coding and non-coding nucleotide sequences of the 14-merpeptide (INlliAl,~AI~KKlL-NH2, SEQ ID NO: 43) are annealed and ligated into prSADE2. The yeast split-hybrid strain YIDRM is transformed with pBTM-~ or m2)/AD4-,BARK, pVP16-arresting, pRSADE2-mastoparan, and a pRSURA3-peptide 10 library or combinatorial drug library.

Numerous modifications and variations in the invention as set forth in the above illustrative examples are expected to occur to those skilled in the art. Consequently only such limitations as appear in the appended claims should be placed on the invention.

WO 98/13502 PCTrUS97/17276 ~U~N~ LISTING
(1) GENERAL INFORMATION:
(i) APPLI Q NT: Hoekstra, Merl F.
(ii) TITLE OF lNv~NllON: Method~ to Identi~y Compound~ For Di~rupting Protein/Protein Interactions (iii) NUMBER OF SEQUENCES: 43 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Mar~hall, O'Toole, Gerstein, Murray & Borun (B) STREET: 6300 Sear~ Tower, 233 South Wacker Drive (C) CITY: Chicago (D) STATE: Illinois (E) COUNTRY: United States of America (F) ZIP: 60606-6402 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Relea~e #1.0, Version ~1.30 (vi) CURRENT APPhI QTION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFI QTION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER:
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~viii) ATTORNEY/AGENT INFORMATION:
(A) NAME:
(B) REGISTRATION NUMBER:
(C) REFERENCE/DOCKET NUMBER: 27866/33424 (ix) TEhECOMMUNICATION INFORMATION:
(A) TELEPHONE: 312/474-6300 (B) TELEFAX: 312/474-0448 (C) TELEX: 25-3856 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pair~
(B) TYPE: nucleic acid (C) STRANDEDNESS: ~ingle (D) TOPOLOGY: linear (ii) MOLECUhE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 ba8e pairs (B) TYPE: nucleic acid -CA 02236867 l998-0~-26 W O 98/13502 PCTrUS97/l7276 (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(Xi ) ~U~N~ DESCRIPTION: SEQ ID NO:3:

(2) INFORMATION FOR SEQ ID NO 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(xi) S~Ou~N~ DESCRIPTION: SEQ ID NO:5:
CGCACGCGTC GAAGA~ATCA CATTACTTTA TATA 34 (2) INFORMATION FOR SEQ ID NO:6:

(i) S~Qu~ CHARACTERISTICS:
(A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA

CA 02236867 1998-0~-26 W O 98/13502 PCT~US97/17276 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STR~NDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

(2) INFORWATION FOR SEQ ID NO:9:
(i) ~U~N~ CHARACTERISTICS:
(A) LENGTH: 48 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

(2) INFORMATION FOR SEQ ID NO:lO:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: 8ingle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l0:

, CA 02236867 1998-0~-26 WO 98/13502 PCT~US97/17276 r (2) INFORMATION FOR SEQ ID NO~
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3k base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: 8ingle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll:

(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) STRANDEDN~SS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
CATGGCATGC PPuu~L~AG AGTCATCCGC TAGG 34 (2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pair6 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear CA 02236867 l998-0~-26 WO 98/13502 PCTrUS97/l7276 (ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:

(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
CGAAGGCA~A GATGTCTAGA TTAGATA~AA G 31 (2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 49 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
CGCGGATCCG CTTTCTCTTC L~ L~llGGAG ACCCACTTTC ACATTTAAG 4g (2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pair8 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) ~OLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:

(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) STR~NDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:

CA 02236867 l998-0~-26 W O 98/135Q2 PCT~US97/17276 (2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 ba8e pair8 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(xi) SEQ~ENCE DESCRIPTION: SEQ ID NO:20:

(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQMENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRA~DEDNESS: single (D) TOPOLOGY: linear (ii) MO~ECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:

(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) STR~NDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:

(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
GCGA~TTCGC CAGGGCAACA GAATGCCACT 30 (2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 ba8e pair8 (B) TYPE: nucleic acid (C) STRA~DEDNESS: single (D) TOPOLOGY: linear CA 02236867 1998-0~-26 WO 98/135~2 PCTrUS97/17276 (ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:

(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQ~ENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single ( D ) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
CGCGGATCCG GATGACCATG GACTCTGGAG

(2) INFORMATION FOR SEQ ID NO:26:
(i) ~u~ CHARACTERISTICS:
(A) ~ENGTH: 30 ~ase pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
CGCGGATCCT TAATCTGACT TGTGGCAGTA

(2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
CGCGGATCCC CATGACCATG GA~TCTGGAG CC

(2) INFOR~ATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA

CA 02236867 l998-0~-26 W O 98/13502 PCTrUS97/17276 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
CGCGGATCCG TGCTGCTTCT TCAGCAGGCT G

(2) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 40 base pairs (B) TYPE: nucleic acid (C) STR~NDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DFSCRIPTION: SEQ ID NO:29:
ATGGTACCAG CGGCCGCTAG lC~ll~ ACA ACGTCGTGAC 40 (2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:
ATGGTACCGC GGCCGCTTAT TTTTGACACC AGACCAAC

(2~ INFORMATION FOR SEQ ID NO:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3~ base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(xi) SkQu~NC~ DESCRI~TION: SEQ ID NO:31:
CGGAGATCTA AAGAGACTTT TCTCCGGAAC TCAG

(2) INFORWATION FOR SEQ ID NO:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STR~NDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:
CGGAGATCTT TACAGGAAGA CTGAACTGT

CA 02236867 1998-0~-26 (2) INFORMATION FOR SEQ ID NO:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:
CCACCGCGGC AGTGCCAACC CCGATTTAC

(2) INFORMATION FOR SEQ ID NO:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: sinyle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:
C~TCCGCGGT GGTGATGGCA GGGGCTGA

(2) INFORMATION FOR SEQ ID NO:35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:
GGCTATCGAT ACGGCCCCCC CGACCGAT

(2) INFORMATION FOR SEQ ID NO:36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:
GCGTATCGAT CTACCCACCG TACTCGTC

(2) INFORMATION FOR SEQ ID NO:37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs WO 98/13502 PCT~US97/17276 (B) TYPE: nucleic acid (C) STR~NDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DWA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:
CCTACTCTTA GGCCCGGGTC TTTTTAATGT ATCC

(2) INFORMATION FOR SEQ ID NO:38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: l8 ba8e pair~
(B) TYPE: nucleic acid (C) STR~NDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:
GGAATCACTA CAGGGATG

(2) INFORMATION FOR SEQ ID NO:39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1485 ba~e pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA

CA 02236867 1998-0~-26 W098/13502 PCTrUS97/17276 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:

G~lG~l~lGG GAATCCCGTT Q TCAGATGG TTTGGCAGAG AGGGTGAATA TAATGCTATG 240 GT QTCGATC TTCTAGGCCC ATCTTTGGAA GATTTATTCA ACTACTGTCA ~AGAAGGTTC 300 TCCTTTAAGA CGGTTATCAT GCTGGCTTTG CA~ATGTTTT GCCGTATTCA GTATATACAT 360 TA~ ~A TCTATTTTTG TAAGGGTTCT TTGC QTGGC AGGGTTTGAA AGCAACCACC 660 AAGAAACA~A AGTATGATCG TAT QTGGAA AAGA~ATTAA ACGTTAGCGT GGA~ACTCTA 720 GATGAGAAGC CAGATTATTT GTTCTTGGCA AGG~ lA AAGATCTGAG TATTA~ACTA 840 GAGAAGCAAA GGGACCTCCT CATCGA~AAA GGTGATTTGA ACGCAAATAG CAATGCAGCA 960 AGTGCAAGTA ACAGCACAGA CAACAAGTCT GA~ACTTTCA ACAAGATTAA ACTGTTAGCC 1020 ATGAAGAAAT TCCCCACCCA TTTCCACTAT TACAAGAATG AAGACA~ACA TAAlC~ll~A 1080 CCAGAAGAGA TCA~ACAACA AACTATCTTG AATAATAATG CAGCCTCTTC TTTACCAGAG 1140 GAATTATTGA ACGCACTAGA TAAAGGTATG GA~AACTTGA GACAACAGCA GCCGCAGCAG 1200 CAGGTCCAAA GTTCGCAGCC ACAACCACAG CCCCAACAGC TACAGCAGCA ACCA~ATGGC 1260 CA~AGACCAA ATTATTATCC TGAACCGTTA CTACAGCAGC AA Q~AGAGA TTCTCAGGAG 1320 CAACAGCAGC AAGTTCCGAT GGCTACAACC AGGGCTACTC AGTATCCCCC ACAA~TAAAC 1380 (2) INFORMATION FOR SEQ ID NO:40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGT~: 2625 base pairs (B) TYPE: nucleic acid (C) STRA~DEDNESS: single (D) TOPOLOGY: line~r (ii) MOLECULE TYPE: DNA

CA 02236867 l998-0~-26 W O 98/13502 PCT~US97/17276 (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 796..2580 (~i) SEQUENCE DESCRIPTION: SEQ ID NO:40:
CAllll~l~l~A ATTCTTTTAT GTGCTTTTAC TA~lll~ AGTTCAAAAC AATAGTCGTT 60 ATTCTTAGGT ACTATAGCAT AAGACAAGAA AAGA~AAATA AGGGACAAAT AACATTAGCA 120 GATACGAATC ACGGCAAACT ATATTCA~G CTCATAGATA ATCGTCGTAA GGCTGACACT 300 GGTTACCTAA AGAGA~CATA AGTAATACTC ATGACAGAAT CAAAACACAA TACAAAATTT 420 ACCAGGAAAA ACAAATATTG AATCCTTGTG AAGGATTCCA CA~ll~ll~lA ATCCTCCTTA 720 Met Ser Leu Pro Leu Ary His Ala Leu Glu Asn Val Thr Ser Val Asp Arg Ile Leu Glu Asp Leu Leu Val Arg Phe Ile Ile Asn Cys Pro Asn Glu Asp Leu Ser Ser Val Glu Arg Glu Leu Phe His Phe Glu Glu Ala Ser Trp Phe Tyr Thr Asp Phe Ile Lys Leu Met Asn Pro Thr Leu Pro Ser Leu Lys Ile Lys Ser Phe Ala Gln Leu Ile Ile Lys Leu Cys Pro Leu Val Trp Lys Trp Asp Ile Arg Val Asp Glu Ala Leu Gln Gln Phe Ser Lys Tyr Lys Lys Ser Ile Pro Val Arg Gly Ala Ala Ile Phe Asn Glu Asn Leu Ser Lys Ile Leu Leu Val Gln Gly Thr CA 02236867 l998-0~-26 WO 98/13502 PCT~US97/17276 GAA TCG GAT TCT TTG TCA TTC CCA AGG GGG AAG ATA TCT A~A GAT GAA 1215 Glu Ser Asp Ser Leu Ser Phe Pro Arg Gly Lys Ile Ser Lys ABP Glu Asn Asp Ile Asp Cy6 Cys Ile Arg Glu Val Lys Glu Glu Ile Gly Phe Asp Leu Thr Asp Tyr Ile Asp Asp Asn Gln Phe Ile Glu Arg Asn Ile CAA GGT AAA AAT TAC A~A ATA TTT TTG ATA TCT GGT GTT TCA GAA GTC 1359 Gln Gly Lys Asn Tyr Lys Ile Phe Leu Ile Ser Gly Val Ser Glu Val TTC AAT TTT A~A CCT CAA GTT AGA AAT GAA ATT GAT AAG ATA GAA TGG 1407 Phe Asn Phe Lys Pro Gln Val Arg Asn Glu Ile Asp Lys Ile Glu Trp TTC GAT TTT AAG AAA ATT TCT A~A ACA ATG TAC AAA TCA AAT ATC AAG 1455 Phe Asp Phe Lys Lys Ile Ser Lys Thr Met Tyr Lys Ser Asn Ile Lys Tyr Tyr Leu Ile A~n Ser Met Met Arg Pro Leu Ser Met Trp Leu Arg 225 230 . 235 CAT CAG AGG CAA ATA A~A AAT GAA GAT CAA TTG AAA TCC TAT GCG GAA 1551 His Gln Arg Gln Ile Lys Asn Glu Asp Gln Leu Ly6 Ser Tyr Ala Glu Glu Gln Leu Lys Leu Leu Leu Gly Ile Thr Lys Glu Glu Gln Ile Asp Pro Gly Arg Glu Leu Leu Asn Met Leu His Thr Ala Val Gln Ala Asn Ser Asn Asn Asn Ala Val Ser Asn Gly Gln Val Pro Ser Ser Gln Glu Leu Gln His Leu Lys Glu Gln Ser Gly Glu His Asn Gln Gln Lys A6p Gln Gln Ser Ser Phe Ser Ser Gln Gln Gln Pro Ser Ile Phe Pro Ser Leu Ser Glu Pro Phe Ala A6n Asn Lys Asn Val Ile Pro Pro Thr Met Pro Met Ala Asn Val Phe Met Ser Asn Pro Gln Leu Phe Ala Thr Met =

CA 02236867 1998-0~-26 W O 98/13S02 PCT~US97/17276 Asn Gly Gln Pro Phe Ala Pro Phe Pro Phe Met Leu Pro Leu Thr Asn Asn Ser Asn Ser Ala Asn Pro Ile Pro Thr Pro Val Pro Pro Asn Phe AAT GCT CCT CCG AAT CCG ATG GCT TTT GGT GTT CCA AAC ATG CAT A~C 2031 Asn Ala Pro Pro Asn Pro Met Ala Phe Gly Val Pro Asn Met His A~n Leu Ser Gly Pro Ala Val Ser Gln Pro Phe Ser Leu Pro Pro Ala Pro Leu Pro Arg Asp Ser Gly Tyr Ser Ser Ser Ser Pro Gly Gln Leu Leu GAT ATA CTA AAT TCG AAA AAG CCT GAC AGC AAC GTG CAA TCA AGC A~A 2175 Asp Ile Leu Asn Ser Lys Lys Pro Asp Ser Asn Val Gln Ser Ser Lys Lys Pro Lys Leu Lys Ile Leu Gln Arg Gly Thr Asp Leu Asn Ser Leu Lys Gln Asn Asn Asn Asp Glu Thr Ala His Ser Asn Ser Gln Ala Leu Leu Asp Leu Leu Lys Lys Pro Thr Ser Ser Gln Lys Ile His Ala Ser Lys Pro Asp Thr Ser Phe Leu Pro Asn Asp Ser Val Ser Gly Ile Gln Asp Ala Glu Tyr Glu ABP Phe Glu Ser Ser Ser Asp Glu Glu Val Glu Thr Ala Arg Asp Glu Arg Asn Ser Leu Asn Val Asp Ile Gly Val Asn Val Met Pro Ser Glu Lys Asp Ser Arg Arg Ser Gln Lys Glu Lys Pro Arg Asn Asp Ala Ser Lys Thr Asn Leu Asn Ala Ser Ala Glu Ser Asn Ser Val Glu Trp Gly Ala Gly CA 02236867 l998-0~-26 WO 98/13502 PCT~US97/17276 (2) INFORMATION FOR SEQ ID NO:41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6854 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA

(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 2050..4053 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:

ATTTGCAGCT AGTTTGCAGT TCGTACAACC TCGCCTATTC TTGTAACGA~ GAAGAACGTA 120 TTTATAATAT TGGGCTGTAA ~ GAGT TTAGTAATAG ATA~AGTAGG ACAGAGTTCT 180 GT~~ Glll ATCTATGGGG TTCAGAGTGA TAAGGGGCAG GATAAGGAAG TTAA~ 240 A~AGGTTACG TTATATAACG AAAGAAAAGA A~CGAGCGAA GTGCCAACTA TAGCCCAATA 300 TCAAGAATGC AAGTCAGCAA AGTACAGTAA TCGTATGAAG ATACGCGATG CGTA~TATCC 360 GACTCGA~CC ACAGCTAACT TCTCGTGA~A AGATGGCTTC AACTTCGCTC TTGCAATAAC 480 TTTGA~ACAC ACGAACA~AG GTTTATTGCG CTTGATTAAC GTTGGAAGTA TATGATACTA 54Q

GACTCCTTAA TTTTATTCAA AATGGTAATT TTCCATTTAT CTAGTCTCAT A~AATTGTCA 780 TGTAAAGTTC TTGCAGCAGC GACTGCATCA GTAGCAGCTA GCTGACA~AG CCCll~ 960 TTGATA~AAC ACTTATTCGA TAATGCTACC GACTGGTCTT GGGCATACCA CTCACCAGCG 1140 AGCTCATAGC AATCTATAGC TTTTG QTAG TCATGCAAAT CATTTTCTAG AA~ CCA 1200 AGCTCAAACT TGA~ATTAGC ACCTCTCCGG AACTGCCCCC TATGAGTAAA AATTTGAATA 1260 GCCTCTACGT AGGTATTTCC TG~'l"l'C~'l'~l~ TCATTACCAG C~~ l'~ ATAGTCAGCA 1380 CA 02236867 l998-0~-26 W O 98113~02 PCTAUS97/17276 GCTTTCAAAA ACGAGTCTCC TGCCA~GTTT AA~~ C TTAGACGGTA AATGGTGGCT 1440 ~ TTCATGAAAC CCGATGAAGG AACACCCTTC TTCTCAGCCT TAACACAACG GGA~ATATCA 1560 llll~llCCT TTGATATATT TCCCTACTAC ATA~l~llll CAATAACTCT ACAGGGTCTG 1680 TTAGTTCAGA TAAACTGCTT CGGTGCTGCC CA~l~l~llAT CATTACTTCA ACTTTACCTT . 1800 CCCTATACCT GTGT&TCCTT ATTAATTCAA GTTAATCCGA GGTAATAGAT TAGGGTAACC 1860 TTCAATGATG TCACGA~ACA CGGATGCTGC AACTTTGCGA ll~llllCCTG GA~AAGAATA 1920 ACAATTAAAG GCAGCCTTTC AGCTGAGATT ACCAGCAGGT CTTTGGAGAT TAGCGCA~GA 1980 AGAAGTGTGA TATAGTACTC ATAGAGGCAG GCTACAGACT AGGGA~AGCG TGTTCAACAA 2040 Met Glu Thr Ser Ser Phe Glu Asn Ala Pro Pro Ala Ala Ile Asn Asp Ala Gln Asp A6n Asn Ile A6n Thr Glu Thr Asn Asp Gln Glu Thr Asn Gln Gln Ser Ile Glu Thr Arg Asp Ala Ile Asp Lys Glu 30 35 . 40 45 Asn Gly Val Gln Thr Glu Thr Gly Glu Asn Ser Ala Lys Asn Ala Glu Gln Asn Val Ser Ser Thr Asn Leu Asn Asn Ala Pro Thr Asn Gly Ala Leu Asp Asp Asp Val Ile Pro Asn Ala Ile Val Ile Lys Asn Ile Pro Phe Ala Ile Lys Lys Glu Gln Leu Leu Agp Ile Ile Glu Glu Met Asp CTT CCC CTT CCT TAT GCC TTC AAT TAC CAC TTT GAT AAC GGT ATT TTC 2g24 Leu Pro Leu Pro Tyr Ala Phe Asn Tyr His Phe Asp Asn Gly Ile Phe Arg Gly Leu Ala Phe Ala Asn Phe Thr Thr Pro G1U Glu Thr Thr Gln GTG ATA ACT TCT TTG ~AT GGA AAG GAA ATC AGC GGG AGG AAA TTG AAA 2520 Val Ile Thr Ser Leu Asn Gly Lys Glu Ile Ser Gly Arg Lys Leu Lys CA 02236867 l998-0~-26 WO 98/135Q2 PCTrUS97/17276 GTG GAA TAT AAA A~A ATG CTT CCC CAA GCT GAA AGA GAA AGA ATC GAG 2568Val Glu Tyr Lys Lys Met Leu Pro Gln Ala Glu Arg Glu Arg Ile Glu AGG GAG AAG AGA GAG A~A AGA GGA CAA TTA GAA GAA CAA CAC AGA TCG 2616 Arg Glu Lys Arg Glu Lys Arg Gly Gln Leu Glu Glu Gln His Arg Ser TCA TCT AAT CTT TCT TTG GAT TCT TTA TCT A~A ATG AGT GGA AGC GGA 2664 Ser Ser Asn Leu Ser Leu Asp Ser Leu Ser Lys Met Ser Gly Ser Gly Asn Asn Asn Thr Ser Asn Asn Gln Leu Phe Ser Thr Leu Met Asn Gly Ile Asn Ala Asn Ser Met Met Asn Ser Pro Met Asn Asn Thr Ile Asn Asn Asn Ser Ser Asn Asn Asn Asn Ser Gly Asn Ile Ile Leu Asn Gln Pro Ser Leu Ser Ala Gln His Thr Ser Ser Ser Leu Tyr Gln Thr Asn Val Asn Asn Gln Ala Gln Met Ser Thr Glu Arg Phe Tyr Ala Pro Leu Pro Ser Thr Ser Thr Leu Pro Leu Pro Pro Gln Gln Leu Asp Phe Asn Asp Pro Asp Thr Leu Glu Ile Tyr Ser Gln Leu Leu Leu Phe Lys Asp AGA GAA A~G TAT TAT TAC GAG TTG GCT TAT CCC ATG GGT ATA TCC GCT 30g8 Arg Glu Lys Tyr Tyr Tyr Glu Leu Ala Tyr Pro Met Gly Ile Ser Ala Ser His Lys Arg Ile Ile Asn Val Leu Cys Ser Tyr Leu Gly Leu Val Glu Val Tyr Asp Pro Arg Phe Ile Ile Ile Arg Arg Lys Ile Leu Asp His Ala Asn Leu Gln Ser His Leu Gln Gln Gln Gly Gln Met Thr Ser Ala His Pro Leu Gln Pro Asn Ser Thr Gly Gly Ser Met Asn Arg Ser Gln Ser Tyr Thr Ser Leu Leu Gln Ala His Ala Ala Ala Ala Ala Asn CA 02236867 l998-0~-26 AGT ATT AGC AAT CAG GCC GTT AAC A~T TCT TCC AAC AGC AAT ACT ATT 3336 Ser Ile Ser Asn Gln Al~ Val Asn Asn Ser Ser Asn Ser Asn Thr Ile Asn Ser Asn Asn Gly Asn Gly Asn Asn Val Ile Ile Asn Asn Asn Ser GCC AGC TCA ACA CCA A~A ATT TCT TCA CAG GGA CAA TTC TCC ATG CAA 3g32 Ala Ser Ser Thr Pro Lys Ile Ser Ser Gln Gly Gln Phe Ser Met Gln CCA ACA CTA ACC TCA CCT A~A ATG AAC ATA CAC CAT AGT TCT CAA TAC 3480 Pro Thr Leu Thr Ser Pro Lys Met Asn Ile His His Ser Ser Gln Tyr AAT TCC GCA GAC CAA CCG CAA CAA CCT CAA CCA CAA ACA CAG CAA A~T 3528 Asn Ser Ala Asp Gln Pro Gln Gln Pro Gln Pro Gln Thr Gln Gln Asn Val Gln Ser Ala Ala Gln Gln Gln Gln Ser Phe Leu Arg Gln Gln Ala Thr Leu Thr Pro Ser Ser Arg Ile Pro Ser Gly Tyr Ser Ala Asn His Tyr Gln Ile Asn Ser Val Asn Pro Leu Leu Arg Asn Ser Gln Ile Ser Pro Pro Asn Ser Gln Ile Pro Ile Asn Ser Gln Thr Leu Ser Gln Ala Gln Pro Pro Ala Gln Ser Gln Thr Gln Gln Arg Val Pro Val Ala Tyr Gln Asn Ala Ser Leu Ser Ser Gln Gln Leu Tyr Asn Leu Asn Gly Pro Ser Ser Ala Asn Ser Gln Ser Gln Leu Leu Pro Gln His Thr Asn Gly Ser Val His Ser Asn Phe Ser Tyr Gln Ser Tyr Hi~ Asp Glu Ser Met Leu Ser Ala His Asn Leu Asn Ser Ala Asp Leu Ile Tyr Lys Ser Leu Ser His Ser Gly Leu Asp Asp Gly Leu Glu Gln Gly Leu Asn Arg Ser Leu Ser Gly Leu Asp Leu Gln Asn Gln Asn Lys Lys Asn Leu Trp CA 02236867 l998-0~-26 W 098/13502 PCTrUS97/17276 TAATATATAC TTCCATTATT CTATGATTAT AGA~ll~ TGGTATTTGT ATATCGCACG 4113 ATACAAGTAA TGAGGGGTGC TTACACAAGA TAAAAGATAA A~AAATATAT ATATATAATA 4173 AAAACCATCA A~AACACCAT TGAU~ TATA~AAAAA AAUUUU9AATA ACCGAATATG 4233 AATATGA~AT TAATGATCAT GATGAAGTTA ATTTTTACTG AGAAACGTCA CCTA~TGTCG 4293 ATGA~ACGAT GATAATGAAT GAATGATGAG GCTACTTTAA GTAACGCAAT GTAATCAAGC 4353 CAAAATTATC C~l~ llll Tl~ CCCT CTTTTGAGAT TTTATTTTTA ACCTACTACT 4413 TA~lllllll TTTTGAACGT ~ CCCA CATACTTTTA TATATGGTAT TTATATGTAC 4473 GATGTTTAAT CACAGAGATG TTTCTACCTT ACTCGATATT ~lllll~GCAT TA~TTGATAT 4533 ATTGACATTT ~l"l"l~l"l"l'~C AATGATCAGA GA~GAGCAGA AAGTTTCATA GTCAAACGTT 4653 TATTTTGGTG ~ GATT AATTGGGGGC TTCATTGTTT GAAATAAAGA GTCGGGAAAA 4773 TAGCACAGAA ACA~AGCATA TTAAAAGAGG CAAAAGAAGA AAGAACGAAT ATA~AAGGTA 4833 AAAAAGGA~A AGCATTGCTA ll'~'ll"ll~lC ATAGGTGTTA TTCATACCGC C~l~l~l~ll~ 4893 CTTCCTTCTT CATTAATTAG TCTCCGTATA ATTTGCAGAT AATGTCATTA ACAGCA~ACG 4953 ACGAATCGCC AAAACCCAAA AAAAATGCAT TATTGA~AAA CTTAGAGATC GATGATCTGA 5013 TACATTCTCA A~ll~l~AGA AGCGATACAA ATGGACATAG AACTACAAGA CGACTATTCA 5073 ATA~AATAAA AAAAGGGTTG ATTTCCCAGC AGTCCA~ACT TGCGTCAGAA AAl~ C 5193 A~AATATCGT TAATAGGGAC AATAAGATGG GAGCAGTAAG TTTCCCCATT ATTGAACCTA 5253 ATATTGAAGT CAGCGAGGAG TTGAAGGTTA GAATTAAGTA TGATTCTATC AAAll~lll~ 5313 ATTTTGA~AG ACTAATATCT A~ATCTTCAG TCATAGCACC TTTAGTTAAC AAAAATATAA 5373 CATCATCCGG lC~l~lAATC GGGTTTCAAA GAAGAGTTAA CAGGTTA~AG CAAACATGGG 5433 ATCTAGCAAC CGA~AACATG GAGTACCCAT All~ll~ A TAATACGCCA TTCAGGGATA 5493 ACGATTCTTG GCAATGGTAC GTACCATACG GCGGAACAAT Pau~u~AaTG A~AGATTTCA 5553 GTACA~AAAG AACTTTACCC ACCTGGGAAG ATAAAATAAA ~~ ACA TTTTTAGAAA 5613 ACTCTAAGTC TGCAACGTAC ATTAATGGTA ACGTATCACT TTGCAATCAT AATGA~ACCG 5673 ATCAAGA~AA CGAAGATAGG AAAAAAAGGA AAGGGA~AGT ACCAAGAATC AAAAATA~AG S733 TGTGGTTTTC CCAGATAGAA TACATTGTTC TTCGA~ATTA TGA~ATTAAA C~ G~l~ATA 5793 CA~ C~l~ TCCGGAACAC ATCAACCAAA ATAAAATGGT TTTTATATGT GAGTTCTGCC 5853 TA~AATATAT GA~ll~lCGA TATACTTTTT ATAGACACCA ACTA~AGTGT CTAACTTTTA 5913 AGCCCCCCGG A~ATGA~ATT TATCGCGACG GTAAGCTGTC TGTTTGGGAA ATTGATGGGC 5973 CA 02236867 l998-0~-26 GGGAGAATGT CTTGTATTGT CAA~ATCTTT GCCTGTTGGC AAAA~ Lll ATCAATTCTA 6033 AGA~ l~l~ TTACGATGTT GAACC'L-lll~ TATTCTATAT TCTAACGGAG AGAGAGGATA 6093 AAAAATTCAA CTCCA~TGAC TATAACCTAA GTTGTATTTT AACTCTACCC ATATACCAGA 6213 GGAAGATAAA ATGTGCTGAA GTGCTATTAA AATTA~GAGA CAGTGCTAGA CGTCGATCAA 6393 ATAATAAAAA TGAAGATACT TTTCAGCAGG TTAGCCTA~A CGATATCGCT AAACTAACAG 6453 GAATGATACC AACAGACGTT ~~ lGGAT TGGAACAACT TCAAGTTTTG TATCGCCATA 6513 ACAGGATTGA AAATATTTAC A~AACTTGGA GCTCAAAAAA CTATCCTCGC GTCAAATATG 6633 ACTTAGAACC CACCGCATTA GCGGACGAAG CTCTTACAAA TGA~ACTATG GCTCCGGTAA 6753 GAAGAAGAAG AAGAAGTAGT GAGCACAaAA CATCCAAGCT T 6854 (2) INFORMATION FOR SEQ ID NO:42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2814 ~ase pair~
(B) TYPE: nucleic acid (C) STRANDEDNESS: 8ingle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA

(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..696 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
GAA TTC CAA TAC ACC A~A CAG CTG CAT TTC CCT GTG GGG CCC AAA TCC 48 Glu Phe Gln Tyr Thr Ly3 Gln Leu His Phe Pro Val Gly Pro Lys Ser Thr Asn Cys Glu Val Ala Glu Ile Leu Leu His Cys Asp Trp Glu Arg Tyr Ile Asn Val Leu Ser Ile Thr Arg Thr Pro Asn Val Pro Ser Gly Thr Ser Phe Ser Thr Ary Thr Arg Tyr Met Phe Arg Trp A6p Asp Gln _ CA 02236867 l998-0~-26 W 098/13502 PCT~US97/17276 Gly Gln Gly Cys Ile Leu Lys Ile Ser Phe Trp Val Asp Trp Asn Ala TCC AGT TGG ATC AAG CCA ATG GTA GAG AGC AAT TGT A~A AAT GGA CAA 288 Ser Ser Trp Ile Lys Pro Met Val Glu Ser Asn Cys Lys Asn Gly Gln Ile Ser Ala Thr Lys Asp Leu Val Lys Leu Val Glu Glu Phe Val Glu A~A TAC GTG GAA TTG AGC A~A GAA A~A GCA GAT ACA CTC A~G CCG TTG 384 Lys Tyr Val Glu Leu Ser Lys Glu Lys Ala Asp Thr Leu Lys Pro Leu Pro Ser Val Thr Ser Phe Gly Ser Pro Arg Lys Val Ala Ala Pro Glu Leu Ser Met Val Gln Pro Glu Ser Lys Pro Glu Ala Glu Ala Glu Ile TCA GAA ATA GGC AGC GAC AGA TGG AGG TTT A~C TGG GTG AAC ATA ATA 528 Ser Glu Ile Gly Ser Asp Arg Trp Arg Phe Asn Trp Val Asn Ile Ile Ile Leu Val Leu Leu Val Leu Asn Leu Leu Tyr Leu Met Ly6 Leu Asn Lys Lys ~et Asp Lys Leu Thr Asn Leu ~et Thr His Lys Asp Glu Val Val Ala His Ala Thr Leu Leu Asp Ile Pro Ala Gln Val Gln Trp Ser Arg Pro Arg Arg Gly A~p Val Leu AGGTTATGTA ~l~llC~L~TG GTATGGA~AA A~a~ALA AAAGGATGCT ATGTGGAGAA 786 ACTCTGAGAG CTTGCTCCGG TATTAAGTTG TGCGTTTGAA A~ GGA A~AAAGAAAT 906 TGATTGGTTG AAGCTATACT CGTCGAAAGA ~ C'llCGGC A~l~G~ll~ GCTCCACCTG 966 CACGGGAGTT ~l~lllGCGT TTATGTTCGG CTTGGCTATA TTATTAGCGA GTGATGTTTG 1026 CAATTTGCTG TATTGAGAAT CAATTTGGGT GCGTA~GCTT T Q ATAATTT TGCAGACCGC 1086 AGGCACTTCC AACTTTATGA GTTGCAGGTA 'l"l'C'l'~'l"l"l"l'A TGAATATACG ATGACGACGA 1146 TGACGACGAC GCATCCATGC GCAAAAGCTC AGG~l~l~l~A GATAGTTTGT TAGTCAATAA 1206 ATCCACATAT CTA~AATAAT A~ATAAACGA CAGCGA QAG l~C~llGGCCT GGAACGCA Q 1266 CA 02236867 1998-0~-26 W O 98/13502 PCTrUS97/17276 ~TATCTAACCA TTGATTTACG TATAAAACTG TCGATGCTCA TCGCCTAGCA ATGAAAAAAT 1566 TTTTTCTTTT ~ l'LATT A1'1"1'~1L11L' GTTGCGTACT '~ "l'~ATT GC~ C'GCG 1626 GCAAAAGCGA TTCGAGTTGA CTGGA~GTGT GTTATACTAT A~AAAGTGTA TATGCCTATT 1686 TTACGGCAGC GGTATTCGCC TCGGCATCAG CAGCCGCCCA CGGTAGAGTA G~~ ~llG 1806 AGCCCGCAGC AGCAT QGAG A~'11~'11L~'1'G TGTAAGGGAC ACCCCTCAAC TCCTTGACTC 1986 'l"l'L'l"l"l"l'~'l'G CACTTTGCCC TTTAAATGCG TTTTTAACGC TATAGCAGTC TCCATGTATT 2046 TGGCACAGTG TATGCAATAG TGCTGACCAA GGCCCGGTTT G~ll~ ~ATCC AATGGCTGGT 2106 TCAGAAGCTT CTGTACTGAT TC~ll~G~lGG ACAAATCGTT ATAGATCAGG TCCAAGTCTC 2166 ~l~ll~ll~Cl TTTAGTCTTG TA~ lCA CCGAATATCT ACCCATGATG CGCTATTGTT 2226 TTATCTTCAC 'l"l'~'l'L'l'~'l'~'l' GTTTAACTGC CTTTCA~TTC ACCTCATCTC ATCTCCCGCT 2286 ACTTTCCATA TATAAAAGCA AAATTAATTT G~~ lCCC CTGTCAGTAT AAAAAAATTT 2346 TCCGCAGGAT ATAGAAAAAA AAGA~ATGAA ATTATAGTAG CGGTTATTTC CGTGGGGTGC 2406 ~ lllACAC CTGTACATCT TTTCCCTCCG TACATTTTTT TTAl~ TTTGGGTTTT 2466 Tll~ lC~A TATTTTTCCC TCCGAAACTA GTTAGCACAA TAATGCTGAC TAAGGAAACT 2526 TTTCATCTCA GAATTGATGG TCAGTTTGGT 'l"l'~lC'l'AGAG AATAGTTTAT A~AAAGATGT 2586 ACTTTAAAGT ACAGTATATC AAATAACTAA TTCAAGATGG CTAGA~GACC AGCTAGATGT 2706 TCCAAGATCA GAATCTACGA TTTGGGTAAG AAGAAGGCTA CC~l'C~AT 2814 (2) INFORMATION FOR SEQ ID NO:43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
Ile A~n Leu Lys Ala Leu Ala Ala Leu Ala Lys Lys Ile Leu

Claims (19)

WHAT IS CLAIMED IS:

1. A host cell transformed or transfected with DNA
comprising:
a repressor gene encoding a repressor protein, said repressor gene under transcriptional control of a promoter;
a selectable marker gene encoding a selectable marker protein; said selectable marker gene under transcriptional control of an operator; said operator regulated by interaction with said repressor protein;
a first recombinant fusion protein gene encoding a first binding protein or binding fragment thereof in frame with either a DNA binding domain of a transcriptional activating protein or a transactivating domain of a transcriptional activating protein; and a second recombinant fusion protein gene encoding a second binding protein or binding fragment thereof in frame with either a DNA binding domain of a transcriptional activating protein or a transactivating domain of a transcriptional activating protein, whichever domain is not encoded by the first fusion protein gene, said second binding protein or binding fragment thereof capable of interacting with said first binding protein or binding fragment thereof such that interaction of said second binding protein or binding fragment thereof and said first binding protein or binding fragment thereof brings into proximity a DNA binding domain and a transactivating domain forming a functional transcriptional activating protein; said functional transcriptional activating protein acting on said promoter to increase expression of said repressor gene.

2. The host cell of claims 1 wherein said DNA binding domain and said transactivating domain are derived from a common transcriptional activating protein.

3. The host cell of claim 1 wherein one or more of the repressor gene, the selectable marker gene, the first recombinant fusion protein gene, and the second recombinant fusion protein gene are encoded on distinct DNA expression constructs.

4. The host cell of claim 1 wherein said selectable marker protein is an enzyme in a pathway for synthesis of a nutritional requirement for said host cell such that expression of said selectable marker protein is required for growth of said host cell on media lacking said nutritional requirement.

5. The host cell of claim 1 wherein said host cell is a yeast cell or a mammalian.

6. The host cell of claim 2 wherein said selectable marker gene encodes HIS3;

7. The host cell of claim 2 wherein said repressor protein gene encodes a tetracycline resistance protein;

8. The host cell of claim 2 wherein said operator is a tet operator.

9. The host cell of claim 2 wherein said promoter is selected from the group consisting of the LexA promoter, the alcohol dehydrogenase promoter, the Ga14 promoter.

10. The host cell of claim 2 wherein said DNA binding domain derived from a protein selected from the group consisting of LexA and Ga14.

11. The host cells of claim 2 wherein said transactivating domain is derived from a protein selected from the group consisting of VP16 and Ga14.

12. The host cell of claim 2 wherein the first binding protein is CREB and the second binding protein is CBP.

13. The host cell of claim 2 wherein the first binding protein is Tax and the second binding protein is SRF.

14. The host cell of claim 2 wherein the first binding protein is casein kinase I and the second binding protein is CREB.

15. The host cell of claim 2 wherein the first binding protein is AKAP 79 and the second binding protein is selected from the group consisting of RI, RII and calcineurin.

16. A method to identify an inhibitor of binding between a first binding protein or binding fragment thereof and a second binding protein or binding fragment thereof comprising the steps of:
a) growing host cells of any one of claims 1 through 15 in the absence of a test compound and under conditions which permit expression of said first binding protein or binding fragment thereof and said second binding protein or binding fragment thereof such that said first binding protein or fragment thereof and second binding protein or binding fragment thereof interact bringing into proximity said DNA binding domain and said transactivating domain forming said functional transcriptional activating protein; said transcriptional activating protein acting on said promoter to increase expression of said repressor protein; said repressor protein interacting with said operator such that said selectable marker protein is not expressed;
b) confirming lack of expression of said selectable marker protein in said host cell;
c) growing said host cells in the presence of a test compound; and d) comparing expression of said selectable marker protein in the presence and absence of said test compound wherein increased expression of said selectable marker protein is indicative that the test compound is an inhibitor of binding between said first binding protein or binding fragment thereof and said second binding protein or binding fragment thereof.

17. The method of claim 16 wherein the host cell is a yeast cell;
the selectable marker gene encodes HIS3;
transcription of the selectable marker gene is regulated by the tet operator;
the repressor protein gene encodes the tetracycline resistance protein;
transcription of the tetracycline resistance protein is regulated by the LexA promoter;
the DNA binding domain is derived from LexA; and the transactivating domain is derived from VP16.

18. The method of claim 16 wherein the host cell is a yeast cell;
the selectable marker gene encodes HIS3;
transcription of the selectable marker gene is regulated by the tet operator;
the repressor protein gene encodes the tetracycline resistance protein;
transcription of the tetracycline resistance protein is regulated by the alcohol dehydrogenase promoter;
the DNA binding domain is derived from LexA; and the transactivating domain is derived from VP16.

19. A kit to identify an inhibitor of binding between a first binding protein or binding fragment thereof and a second binding protein or binding fragment thereof, said inhibitor identified by the method of claim 16.