AU8150501A - Immunosuppressant target proteins - Google Patents

Description

-I-

AUSTRALIA

PATENTS ACT 1990 DIVISIONAL APPLICATION NAME OF APPLICANT: Ariad Pharmaceuticals, Inc.

ADDRESS FOR SERVICE: DAVIES COLLISON CAVE Patent Attorneys 1 Little Collins Street Melbourne, 3000.

INVENTION TITLE: "Immunosuppressant target proteins" The following statement is a full description of this invention, including the best method of performing it known to us: IP Australia 19 OCT 2001 M[ I 1A Immunosuppressant Target Proteins Background of the Invention Cyclosporin A, FK506, and rapamycin are microbial products with potent immunosuppressive properties that result primarily from a selective inhibition of T lymphocyte activation. Rapamycin was first described as an antifungal antibiotic extracted from a streptomycete (Streptomyces hygroscopicus) (Vezina et al. (1975) J. Antibiot., 28:721; Sehgal et al. (1975) J. Antibiot. 28:727; and Sehgal et al., U.S. Pat. No. 3,929.992).

Subsequently, the macrolide drug rapamycin was shown to exhibit immunosuppressive as well as antineoplastic and antiproliferative properties (Morris (1992) Transplant Res 6:39- 87).

Each of these compounds, cyclosporin A, FK506 and rapamycin, suppress the immune system by blocking distinctly different biochemical reactions which would ordinarily initiate the activation of immune cells. Briefly, cyclosporin A and FK506 act soon 15 after Ca 2 +-dependent T-cell activation to prevent the synthesis of cytokines important for the perpetuation and amplification of the immune response. Rapamycin acts later to block multiple affects of cytokines on immune cells including the inhibition of interleukin-2 (IL2)triggered T-cell proliferation, but its antiproliferative effects are not restricted solely to T and B cells. Rapamycin also selectively inhibits the proliferation of growth factor-dependent and growth factor-independent nonimmune cells. Rapamycin is generally believed to inhibit cell proliferation by blocking specific signaling events necessary for the initiation of S phase in a number of cell types, including lymphocytes (Bierer et al. (1990) PNAS 87:9231-9235; and Dumont et al. (1990) J. Immunol 144:1418-1424), as well as non-immune cells. such as hepatocytes (Francavilla et al. (1992) Hepatology 15:871-877; and Price et al. (1992) Science 257:973-977). Several lines of evidence suggest that the association of rapamycin with different members of a family of intracellular FK506/rapamycin binding proteins (FKBPs) is necessary for the inhibition of G I progression as mediated by rapamycin. For instance, the actions of rapamycin are reversed by an excess of the structurally FKBP-ligands FK506 or 506BD (Bierer et al. supra.; Dumont et al. supra.; and Bierer et al. (1990) Science 250:556- 559).

Cyclosporin A binds to a class of proteins called cyclophilins (Walsh et al. (1992) J Biol. Chem. 267:13115-13118), whereas the primary targets for both FK506 and rapamycin.

as indicated above, are the FKBPs (Harding et al. (1989) Nature 341:758-7601; Siekienka et al. (1989) Nature 341:755-757; and Soltoff et al. (1992 J. Biol. Chem. 267:17472-17477) Both the cyclophilin/cyclosporin and FKBPI2/FK506 complexes bind to a specific protein phosphatase (calcineurin) which is hypothesized to control the activity of IL-2 gene specific 2 transcriptional activators (reviewed in Schreiber (1991) Cell 70:365-368). In contrast, the downstream cellular targets for the rapamycin-sensitive signaling pathway have not been especially well characterized, particularly with regard to the identity of the direct target of the FKBP-rapamycin complex.

The TORI and TOR2 genes of S. cerevisiae were originally identified by mutations that rendered cells resistant to rapamycin (Heitman et al. (1991) Science 253:905-909) and there was early speculation that the FKBP/rapamycin complex might inhibit the cellular function of the TOR gene product by binding directly to a phosphoserine residue of either TORI or TOR2. Subsequently, however, new models for rapamycin drug interaction have been proposed which do not involve direct binding of the FKBP/rapamycin complex to the TOR proteins. For example, based on experimental data regarding cyclin-cdk activity in rapamycin treated cells, the Schreiber laboratory wrote in Albers et al. (1993) J Biol Chem.

268:22825-22829: "Although it is possible the TOR2 gene product is a direct target of the FKBP-rapamycin complex, a more likely explanation is that the TOR2 gene product lies downstream of the direct target of rapamycin and that the TOR2 mutation caused the protein to be constitutively active. If the latter model is correct, then the TOR2 gene product joins p70s 6 k, 20 cyclin-dependent kinases, and cyclin Dl as proteins that lie downstream of the direct target of the FKBP-rapamycin complex and have been shown to play important roles in cell cycle progression. The identification of the direct target of the 25 FKBP-rapamycin complex will likely reveal an upstream 25 component of the signal transduction pathway that leads to G progression and will help delineate the signal transduction pathways that link growth factor-mediated signaling events and cyclin-cdk activity required for cell cycle progression." Likewise, after studying the role of TORI and TOR2 mutations in rapamycinresistant yeast cells, Livi group wrote in Cafferkey et al. (1993) Mol. Cell Biol. 13:6012- S"6023: "Thus, the amino acid changes that we have identified in the rapamycin-released DRRI [TORI] protein may allow it to compensate for the loss of the proliferative signal inhibited by rapamycin by constitutively activating an alternative signal rather than by preventing its association with the FKBP12rapamycin complex. The positions of the mutations within the kinase domain, but in a region not shared by the PI 3-kinases, support this idea. Therefore, it is entirely possible that DRRI is not a component of the rapamycin-sensitive pathway in wildtype yeast cells. Instead, missense mutations in DRRI at Ser- 1972 may alter its normal activity and allow it to substitute for the function of an essential protein which is the true target of rapamnycin." Summary of the Invention The present invention relates to the discovery of novel proteins of mammalian origin which are immediate downstream targets for FKBP/rapamycin complexes. As described herein, a drug-dependent interaction trap assay was used to isolate a number of proteins which interact with an FK506-binding protein/rapamycin complex, and which are collectively referred to herein as "RAP-binding proteins" or "RAP-BPs". In particular several mammalian genes (orthologs) have been cloned for a protein referred to herein as "RAPTI which protein is apparently related to the yeast TORI and TOR2 gene products.

Furthermore, a novel ubiquitin-conjugating enzyme, referred to herein as "rap-UBC" has been cloned based on its ability to bind FKBP/rapamyin complexes. In addition, a RAPTI- Slike protein was cloned from the human pathogen Candida. The present invention, therefore, makes available novel proteins (both recombinant and purified forms), recombinant genes, antibodies to RAP-binding proteins, and other novel reagents and assays for diagnostic and 20 therapeutic use. s The present invention relates to the discovery in eukaryotic cells, particularly human cells. of novel protein-protein interactions between the FK506-binding protein/rapamycin complexes and certain cellular proteins, referred to hereinafter as "RAP-binding proteins" or

"RAP-BP".

25 In general, the invention features a mammalian RAPTI polypeptide, preferably a substantially pure preparation of a RAPTI polypeptide or a recombinant RAPTI polypeptide. In preferred embodiments the polypeptide has a biological activity associated with its binding to rapamycin, it retains the ability to bind to an FKBP/rapamycin complex, though it may be able to either agnoize or antagonize assembly of rapamycin dependent complexes. The polypeptide can be identical to a polypeptide shown in one of SEQ ID No: 2 or 12, or it can merely be homologous to that sequence. For instance, the polypeptide preferably has an amino acid sequence at least 70% homologous to the amino acid sequence of at least one of either SEQ ID No: 2 or 12, though higher sequence homologies of. for example, 80%, 90% or 95% are also contemplated, and will generally be preferred. The polypeptide can compi contemplated, an d w i ll generally b e 35 preferred. The poypeptide can comprise the full length protein, or a portion of a full length protein, such as the RAPTI polypetides represented in either SEQ ID No: 2 or 12, or an even 4 smaller fragment of that protein, which fragment may be. for instance, at least 5, 10, 20, 100, or 150 amino acids in length. As described below, the RAPTI polypeptide can be either an agonist mimics), or alternatively, an antagonist of a biological activity of a naturally occuring form of the protein, the polypeptide is able to modulate assembly of rapamycin complexes, such as complexes involving FK506-binding proteins, or cell cycle regulatory proteins.

In a preferred embodiment, a peptide having at least one biological activity of the subject RAPTI polypeptides may differ in amino acid sequence from the sequence in SEQ ID No: 2 or 12, but such differences result in a modified protein which functions in the same or similar manner as the native RAPTI protein or which has the same or similar characteristics of the native RAPTI protein. However, homologs of the naturally occuring protein are contemplated which are antagonistic of the normal cellular role of the naturally occurring protein.

In yet other preferred embodiments, the RAPTI protein is a recombinant fusion protein which includes a second polypeptide portion, a second polypeptide having an amino acid sequence unrelated to the RAPTI polypeptide portion, e.g. the second polypeptide portion is glutathione-S-transferase, e.g. the second polypeptide portion is a DNA binding domain of transcriptional regulatory protein, e.g. the second polypeptide portion is an RNA polymerase activating domain, e.g. the fusion protein is functional in a 20 two-hybrid assay.

Yet another aspect of the present invention concerns an immunogen comprising a .RAPTI peptide in an immunogenic preparation, the immunogen being capable of eliciting an immune response specific for the RAPTI polypeptide; e.g. a humoral response, e.g. an antibody response; e.g. a cellular response. In preferred embodiments, the immunogen comprising an antigenic determinant, e.g. a unique determinant, from a protein represented by SEQ ID No: 2 and/or 12.

A still further aspect of the present invention features an antibody preparation I specifically reactive with an epitope of the RAPTI immunogen.

In still another aspect, the invention features a RAPTI-like polypeptide from a Candida species (caRAPTI), preferably a substantially pure preparation of a caRAPTI polypeptide, or a recombinant caRAPTI polypeptide. As above, in preferred embodiments the caRAPTI polypeptide has a biological activity associated with its binding to rapamycin, it retains the ability to bind to a rapamycin complex, such as an FKBP/rapamycin complex. The polypeptide can be identical to the polypeptide shown in SEQ ID No: 14, or it can merely be homologous to that sequence. For instance, the caRAPTI polypeptide preferably has an amino acid sequence at least 60% homologous to the amino acid sequence in SEQ ID No: 14, though higher sequence homologies of. for example, 80%, 90% or are also contemplated. The caRAPTI polypeptide can comprise the entire polypeptide represented in SEQ ID No: 14. or it can comprise a fragment of that protein, which fragment may be, for instance, at least 5, 10, 20, 50 or 100 amino acids in length. The caRAPTI polypeptide can be either an agonist mimics), or alternatively, an antagonist of a biological activity of a naturally occuring form of the protein.

In a preferred embodiment, a peptide having at least one biological activity of the subject caRAPTI polypeptide may differ in amino acid sequence from the sequence in SEQ ID No: 14, but such differences result in a modified protein which functions in the same or similar manner as the native caRAPTI or which has the same or similar characteristics of the native protein. However, homologs of the naturally occuring caRAPTI protein are contemplated which are antagonistic of the normal cellular role of the naturally occurring protein.

In yet other preferred embodiments, the caRAPTI protein is a recombinant fusion protein which includes a second polypeptide portion, a second polypeptide having an amino acid sequence unrelated to the caRAPTI sequence, e.g. the second polypeptide portion is glutathione-S-transferase, e.g. the second polypeptide portion is a DNA binding domain of transcriptional regulatory protein, e.g. the second polypeptide portion is an RNA polymerase activating domain, e.g. the fusion protein is functional in a two-hybrid assay.

20 Yet another aspect of the presentinvention concerns an immunogen comprising a caRAPTI peptide in an immunogenic preparation, the immunogen being capable of eliciting an immune response specific for the caRAPTI polypeptide; e.g. a humoral response, e.g. an antibody response; e.g. a cellular response. In preferred embodiments, the immunogen comprising an antigenic determinant. e.g. a unique determinant, from a protein represented 25 by SEQ ID No: 14.

A still further aspect of the present invention features an antibody preparation specifically reactive with an epitope of the caRAPTI immunogen.

Still another embodiment of the present invention features fragments of a RAPT1, hRAPTI or mRAPTI, or other RAPTI-like polypeptide, caRAPTI, TORI or TOR2, which fragments retaing the ability to bind to an FK-binding protein in a rapamycin dependent manner. Accordingly. the present invention facilitates the generation of drug screening assays, particularly the high-throughout assays described below, for the identification immunosuppresants, anti-mycotic agents, and the like which act through the binding of the rapamycin-binding domain of the RAPTI-like proteins. For instance, the present invention provides portions of the RAPTI-like proteins which are easier to manipulate than the full length protein. The full length protein is, because of its size, more 6 difficult to express as a recombinant protein or a fusion protein which would retain rapamycin-binding activity, and may very well be insoluble. Accordingly, the present invention provides soluble polypeptides which include a soluble portion of a RAPTI-like polypeptide that binds to said FKBP/rapamycin complex, such as the rapamycin-binding domain represented by an amino acid sequence selected from the group consisting Val26of SEQ ID No. 2 (mRATPI), Va12012-Tyr2144 of SEQ ID No. 12 (hRAPTI), Val41-Tyrl73 of SEQ ID No. 14 (caRAPTI). Vall-Tyrl33 of SEQ ID No. 16 (TORI), and Vall-Argl33 of SEQ ID No. 18 (TOR2).

Another aspect of the present invention provides a substantially isolated nucleic acid having a nucleotide sequence which encodes a RAPTI polypeptide. In preferred embodiments: the encoded polypeptide specifically binds a rapamycin complexes and/or is able to either agnoize or antagonize assembly of rapamycin-containing protein complexes.

The coding sequence of the nucleic acid can comprise a RAPTI-encoding sequence which can be identical to the cDNA shown in SEQ ID No: 1 or 11. or it can merely be homologous to that sequence. For instance, the RAPTI-encoding sequence preferably has a sequence at least 70% homologous to one or both of the nucleotide sequences in SEQ ID No: 1 or 11, though higher sequence homologies of. for example, 80%, 90% or 95% are also contemplated. The nucleic acid can comprise the nucleotide sequence represented in SEQ ID 'No: 1, or it can comprise a fragment of that nucleic acid, which fragment may be, for o 20 instance, encode a fragment of which is, for example, at least 5, 10, 20, 50. 100 or 133 amino acids in length. The polypeptide encoded by the nucleic acid can be either an agonist (e.g.

mimics), or alternatively, an antagonist of a biological activity of a naturally occuring form of *the RAPTI protein, the polypeptide is able to modulate rapamycin-mediated protein complexes.

25 Furthermore, in certain preferred embodiments, the subject RAPTI nucleic acid will include a transcriptional regulatory sequence, e.g. at least one of a transcriptional promoter or transcriptional enhancer sequence, which regulatory sequence is operably linked to the RAPTI gene sequence. Such regulatory sequences can be used in to render the RAPTI gene sequence suitable for use as an expression vector.

In yet a further preferred embodiment, the nucleic acid hybridizes under stringent conditions to a nucleic acid probe corresponding to at least 12 consecutive nucleotides of SEQ ID No: 1 and/or 11; preferably to at least 20 consecutive nucleotides, and more preferably to at least 40 consecutive nucleotides. It yet another embodiment, the nucleic acid hybridizes to region of the human or mouse RAPTI genes corresponding to the binding domain for rapamycin.

7 Another aspect of the present invention provides a substantially isolated nucleic acid having a nucleotide sequence which encodes a caRAPTI polypeptide. In preferred embodiments: the encoded polypeptide specifically binds a rapamycin complexes and/or is able to either agnoize or antagonize assembly of rapamycin-containing protein complexes.

The coding sequence of the nucleic acid can comprise a caRAPTI-encoding sequence which can be identical to the cDNA shown in SEQ ID No: 13, or it can merely be homologous to that sequence. For instance, the caRAPTI-encoding sequence preferably has a sequence at least 60% homologous to the nucleotide sequences in SEQ ID No: 13, though higher sequence homologies of, for example, 80%, 90% or 95% are also contemplated. The nucleic acid can comprise the nucleotide sequence represented in SEQ ID No: 13, or it can comprise a fragment of that nucleic acid, which fragment may be. for instance, encode a fragment of which is, for example, at least 5, 10, 20, 50, 100 or 140 amino acids in length. The polypeptide encoded by the nucleic acid can be either an agonist mimics), or alternatively, an antagonist of a biological activity of a naturally occuring form of the caRAPTI protein, the polypeptide is able to modulate rapamycin-mediated protein complexes.

4Furthermore, in certain preferred embodiments, the subject caRAPTI nucleic acid will include a transcriptional regulatory sequence, e.g. at least one of a transcriptional promoter or transcriptional enhancer sequence, which regulatory sequence is operably linked to the caRAPTI gene sequence. Such regulatory sequences can be used in to render the caRAPTI gene sequence suitable for use as an expression vector.

In yet a further preferred embodiment, the nucleic acid hybridizes under stringent conditions to a nucleic acid probe corresponding to at least 12 consecutive nucleotides of SEQ ID No: 13; preferably to at least 20 consecutive nucleotides. and more preferably to at 25 least 40 consecutive nucleotides.

The invention also features transgenic non-human animals, e.g. mice. rats, rabbits or pigs, having a transgene, animals which include (and preferably express) a heterologous form of one of the RAP-BP genes described herein, e.g. a gene derived from humans, or which misexpress an endogenous RAP-BP gene, an animal in which expression of one or more of the subject RAP-binding proteins is disrupted. Such a transgenic animal can serve as an animal model for studying cellular disorders comprising mutated or mis-expressed RAP-BP alleles or for use in drug screening.

The invention also provides a probe/primer comprising a substantially purified oligonucleotide, wherein the oligonucleotide comprises a region of nucleotide sequence which hybridizes under stringent conditions to at least 10 consecutive nucleotides of sense or antisense sequence of one of SEQ ID Nos: 1, 11 or 13, or naturally occurring mutants .1 1 1 thereof. In preferred embodiments, the probe/primer further includes a label group attached thereto and able to be detected. The label group can be selected, from a group consisting of radioisotopes, fluorescent compounds, enzymes, and enzyme co-factors. Probes of the invention can be used as a part of a diagnostic test kit for identifying transformed cells, such as for detecting in a sample of cells isolated from a patient, a level of a nucleic acid encoding one of the subject RAP-binding proteins; e.g. measuring the RAP-BP mRNA level in a cell, or determining whether the genomic RAP-BP gene has been mutated or deleted. Preferably.

the oligonucleotide is at least 10 nucleotides in length, though primers of 20, 30, 50, 100, or 150 nucleotides in length are also contemplated.

In yet another aspect, the invention provides assay systems for screening test compounds for an molecules which induce an interaction between a RAP-binding protein and a rapamycin/protein complexes. An exemplary method includes the steps of combining a RAP-binding protein of the invention, an FK506-binding protein, and a test compound, e.g., under conditions wherein, but for the test compound, the FK506-binding protein and the RAP-binding protein are unable to interact; and (ii) detecting the formation of a drugdependent complex which includes the FK506-binding protein and the RAP-binding protein.

A statistically significant change, such as an increase, in the formation of the complex in the S0. presence of a test compound (relative to what is seen in the absence of the test compound) is S: indicative of a modulation, induction, of the interaction between the FK506-binding 20 protein and the RAP-binding protein. Moreover, primary screens are provided in which the FK506-binding protein and the RAP-binding protein are combined in a cell-free system and contacted with the test compound; i.e. the cell-free system is selected from a group consisting of a cell lysate and a reconstituted protein mixture. Alternatively, FK506-binding protein and the RAP-binding protein are simultaneously expressed, recombinantly, in a cell, and the cell is contacted with the test compound, e.g. as an interaction trap assay (two hybrid assay).

The present invention also provides a method for treating an animal having unwanted cell growth characterized by a loss of wild-type function of one or more of the subject RAPbinding proteins, comprising administering a therapeutically effective amount of an agent able to inhibit the interaction of the RAP-binding protein with other cellular or viral proteins.

30 In one embodiment, the method comprises administering a nucleic acid construct encoding a polypeptides represented in one of SEQ ID Nos: 2 or 12, under conditions wherein the construct is incorporated by cells deficient in that RAP-binding protein, and under conditions wherein the recombinant gene is expressed, e.g. by gene therapy techniques. In other embodiments, the action of a naturally-occurring RAP-binding protein is antagonized by therapeutic expression of a RAP-BP homolog which is an antagonist of, for example, assembly of rapamycin-mediated complexes, or by delivery of an antisense nucleic acid molecule which inhibits transcription and/or translation of the targeted RAP-BP gene.

I. 1 9 Another aspect of the present invention provides a method of determining if a subject, e.g. a human patient, is at risk for a disorder characterized by unwanted cell proliferation.

The method includes detecting, in a tissue of the subject, the presence or absence of a genetic lesion characterized by at least one of a mutation of a gene encoding a protein represented by one of SEQ ID Nos: 1 or 11, or a homolog thereof; (ii) the mis-expression of a gene encoding a protein represented by one of SEQ ID Nos: 1 or 11; or (iii) the mis-incorporation of a RAP-binding protein in a regulatory protein complex, e.g. a rapamycin-containing complex. In preferred embodiments: detecting the genetic lesion includes ascertaining the existence of at least one of: a deletion of one or more nucleotides from the RAP-BP gene; an addition of one or more nucleotides to the gene, an substitution of one or more nucleotides of the gene, a gross chromosomal rearrangement of the gene; an alteration in the level of a messenger RNA transcript of the gene; the presence of a non-wild type splicing pattern of a messenger RNA transcript of the gene; or a non-wild type level of the protein.

For example, detecting the genetic lesion can include providing a probe/primer including an oligonucleotide containing a region of nucleotide sequence which hybridizes to a sense or antisense sequence of one of SEQ ID Nos: 1 or 11, or naturally occurring mutants thereof or 5' or 3' flanking sequences naturally associated with the RAP-BP gene; (ii) exposing the probe/primer to nucleic acid of the tissue; and (iii) detecting, by hybridization of the probe/primer to the nucleic acid, the presence or absence of the genetic lesion; e.g.

20 wherein detecting the lesion comprises utilizing the probe/primer to determine the nucleotide sequence of the RAP-BP gene and, optionally, of the flanking nucleic acid sequences. For instance, the probe/primer can be employed in a polymerase chain reaction (PCR) or in a ligation chain reaction (LCR). In alternate embodiments, the level of the RAP-binding protein is detected in an immunoassay using an antibody which is specifically immunoreactive with a protein represented by one of SEQ ID Nos: I or 11.

In similar fashion, Candida infection can be detected by use of probes/primers which :"e hybridize to a Candida gene encoding a RAPTI-like protein. For instance, the method can include providing a probe/primer including an oligonucleotide containing a region of nucleotide sequence which hybridizes to a sense or antisense sequence of one of SEQ ID No: 30 13, or naturally occurring mutants thereof or 5' or 3' flanking sequences naturally associated with the caRAPTI gene; (ii) exposing the probe/primer to nucleic acid of a biological sample, tissue biopsy, fluid sample, stool, etc.; and (iii) detecting, by hybridization of the probe/primer to the nucleic acid, the presence or absence of a Candida organism.

Another aspect of the present invention concerns a novel in vivo method for the isolation of genes encoding proteins which physically interact with a "bait" protein/drug complex. The method relies on detecting the reconstitution of a transcriptional activator in the presence of the drug, particularly wherein the drug is a non-peptidyl small organic /0 molecule <2500K), e.g. a macrolide, e.g. rapamycin, FK506 or cyclosporin. In particular, the method makes use of chimeric genes which express hybrid proteins. The first hybrid comprises the DNA-binding domain of a transcriptional activator fused to the bait protein. The second hybrid protein contains a transcriptional activation domain fused to a "fish" protein, e.g. a test protein derived from a cDNA library. If the fish and bait proteins are able to interact in a drug-dependent manner, they bring into close proximity the two domains of the transcriptional activator. This proximity is sufficient to cause transcription of a reporter gene which is operably linked to a transcriptional regulatory site responsive to the transcriptional activator, and expression of the marker gene can be detected and used to score for the interaction of the bait protein/drug complex with another protein.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art.

Such techniques are explained fully in the literature. See, for example. Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II N. Glover ed., 1985); Oligonucleotide Synthesis J. Gait ed., 1984); Mullis et al. U.S. Patent No: 4,683,195; Nucleic Acid Hybridization D. Hames S. J. Higgins eds. 1984): Transcription And Translation D. Hames S. J. Higgins eds. 1984); Culture Of Animal Cells 1.

20 Freshney. Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B.

Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., Gene Transfer Vectors For Mammalian Cells H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 .and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker. eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV M. Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1986).

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

Description of the Figures Figure 1 illustrates the map of the pACT vector used to clone the human RAPTI clone. The RAPTI-containing version of pACT, termed "pIC524" has been deposited with the ATCC.

Figure 2 illustrates the interaction of FKBP12 and hRAPTI (rapamycin-binding domain) as a function of rapamycin concentration. INteraction is detected as P-galactosidase

I

activity. No interaction is detected if FK506 is used in place of rapamycin, or if lex.da (a control plasmid) replaces FKBP 12.

Figure 3 illustrates the relative strengths of interaction between pairs of FK506binding proteins and rapamycin-binding domain (BD) fusions in the presence of varying concentrations of rapamycin, measured by B-galactosidase expression (see Example The yeast reporter strain VBY567 was transformed with the indicated pairs of plasmids. LexA DNA-binding domain fusions to human FKBP12, yeast FKBPI2 and an unrelated sequence serving as negative control were used as "baits". The VP16 acidic activation domain fusions to human RAPTI BD, human RAPTI BD containing the serine to arginine substitution, yeast Torl BD, yeast Tor2 BD (not shown) and Candida albicans RAPTI BD were tested for interaction against the bait fusions. Transformants containing each pair of plasmids were tested for B-galactosidase expression on media containing the chromogenic substrate X-gal.

Colonies were scored as either white (open bars) or blue (solid bars) after growth at 30 0 C for 2 days. The levels of B-galactosidase expression were qualitatively scored by the intensity of the blue color, ranging from 1 (light blue) to 4 (deep blue).

Detailed Description of the Invention Recent studies have provided some remarkable insights into the molecular basis of eukaryotic cell cycle regulation. Passage of a mammalian cell through the cell cycle is regulated at a number of key control points. Among these are the points of entry into and exit from quiescence the restriction point, the GI/S transition, and the G./M transition (for review, see Draetta (1990) Trends Biol Sci 15:378-383; and Sherr (1993) Cell 73:1059- 1065). Ultimately, information from these check-point controls is integrated through the 25 regulated activity of a group of related kinases, the cyclin-dependent kinases (CDKs). For example, the Gi-to-S phase transition is now understood to be timed precisely by the transient assembly of multiprotein complexes involving the periodic interaction of a multiplicity of cyclins and cyclin-dependent kinases.

To illustrate, stimulation of quiescent T lymphocytes by cell-bound antigens triggers a complex activation program resulting in cell cycle entry (Go-to-G 1 transition) and the expression of high affinity interleukin-2 (IL-2) receptors. The subsequent binding of L-2 to its high affinity receptor drives the progression of activated T cells through a late GI-phase "restriction point" (Pardee (1989) Science 246:603-608), after which the cells are committed to complete a relatively autonomous program of DNA replication and, ultimately, mitosis.

1. P* 12 One important outcome of the information concerning eukaryotic cell cycle regulation is the delineation of a novel class of molecular targets for potential growth-modulatory drugs.

The macrolide ester, rapamycin, is a potent immunosuppressant whose mechanism of action is related to the inhibition of cytokine-dependent T cell proliferation (Bierer et al. (1990) PNAS 87:9231-9235; Dumont et al. (1990) J. Immunol 144:1418-1424; Sigal et al. (1991) Transplant Proc 23:1-5; and Sigal et al. (1992) Annu Rev Immunoll 0:519-560). Rapamycin specifically interferes with a late GI-phase event required for the progression of IL-2 stimulated cells into S-phase (Morice et al. (1993) J Biol Chem 268:3734-3738). The location of the cell cycle arrest point induced by rapamycin hints that this drug interferes with the regulatory proteins that govern the G -to-S phase transition, particularly in lymphocytes.

As described herein, the present invention relates to the discovery of novel proteins of mammalian origin which are immediate downstream targets for FKBP/rapamycin complexes.

As described below, a drug-dependent interaction trap assay was used to isolate a number of proteins which bind the FKBP12/rapamycin complex, and which are collectively referred to herein as "RAP-binding proteins" or "RAP-BPs". In particular, mouse and human genes have been cloned for a protein (referred to herein as "RAPTI") which is apparently related to the yeast TORI and TOR2 gene products. Furthermore, a novel ubiquitin-conjugating enzyme (referred to herein as "rap-UBC") has been cloned based on its ability to bind FKBP/rapamycin complexes. The present invention, therefore, makes available novel 20 proteins (both recombinant and purified forms), recombinant genes, antibodies to RAPbinding proteins, and other novel reagents and assays for diagnostic and therapeutic use.

Moreover, drug discovery assays are provided for identifying agents which can modulate the binding of one or more of the subject RAP-binding proteins with FK506-binding proteins.

Such agents can be useful therapeutically to alter the growth and/or differentiation of a cell.

but can also be used in vitro as cell-culture additives for controlling proliferation and/or differentiation of cultured cells and tissue. Other aspects of the invention are described below or will be apparent to those skilled in the art in light of the present disclosure.

For convience, certain terms employed in the specfication, examples, and appended claims are collected here.

As used herein, the term "nucleic acid" refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The term should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, singlestranded (such as sense or antisense) and double-stranded polynucleotides.

The term "gene" or "recombinant gene" refers to a nucleic acid comprising an open reading frame encoding a RAP-binding protein of the present invention, including both exon

S.

i3 and (optionally) intron sequences. A "recombinant gene" refers to nucleic acid encoding a RAP-binding protein and comprising RAP-BP encoding exon sequences, though it may optionally include intron sequences which are either derived frorn a chromosomal RAP-BP gene or from an unrelated chromosomal gene. Exemplary recombinant genes encoding illustrative RAP-binding proteins include a nucleic acid sequence represented by on of SEQ ID Nos: 1, 11, 13 or 23. The term "intron" refers to a DNA sequence present in a given RAP- BP gene which is not translated into protein and is generally found between exons.

As used herein, the term "transfection" refers to the introduction of a nucleic acid, an expression vector, into a recipient cell by nucleic acid-mediated gene transfer.

"Transformation", as used herein, refers to a process in which a cell's genotype is changed as a result of the cellular uptake of exogenous DNA or RNA, and, for example, the transformed cell expresses a recombinant form of the RAP-binding protein of the present invention or where anti-sense expression occurs from the transferred gene, the expression for a naturallyoccurring form of the RAP-binding protein is disrupted.

As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of preferred vector is an episome, a nucleic acid capable of extra-chromosomal replication. Preferred vectors are those capable of autonomous replication and/expression of nucleic acids to which they are linked. Vectors capable of directing the expression of genes to which they are operatively 20 linked are referred to herein as "expression vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form of "plasmids" which refer to circular double stranded DNA loops which, in their vector form are not bound to the chromosome. In the present specification, "plasmid" and "vector" are used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors which serve equivalent functions and which become known in the art subsequently hereto.

"Transcriptional regulatory sequence" is a generic term used throughout the specification to refer to DNA sequences, such as initiation signals, enhancers, and promoters, which induce or control transcription of protein coding sequences with which they are operably linked. In preferred embodiments, transcription of a recombinant RAP-BP gene is under the control of a promoter sequence (or other transcriptional regulatory sequence) which controls the expression of the recombinant gene in a cell-type in which expression is intended. It will also be understood that the recombinant gene can be under the control of transcriptional regulatory sequences which are the same or which are different from those sequences which control transcription of the naturally-occurring form of the RAP-binding protein.

I 11 /4 As used herein, the term "tissue-specific promoter" means a DNA sequence that serves as a promoter, regulates expression of a selected DNA sequence operably linked to the promoter, and which effects expression of the selected DNA sequence in specific cells of a tissue, such as cells of a lymphoid lineage, e.g. B or T lymphocytes. or alternatively, e.g. hepatic cells. In an illustrative embodiment, gene constructs utilizing lymphoid-specific promoters can be used as a pan of gene therapy to provide dominant negative mutant forms of a RAP-binding protein to render lymphatic cells resistant to rapamycin by directing expression of the mutant form of RAP-BP in only lymphatic tissue.

The term also covers so-called "leaky" promoters, which regulate expression of a selected DNA primarily in one tissue, but cause expression in other tissues as well.

As used herein, a "transgenic animal" is any animal, preferably a non-human mammal, a bird or an amphibian, in which one or more of the cells of the animal contain heterologous nucleic acid introduced by way of human intervention, such as by trangenic techniques well known in the art. The nucleic acid is introduced into the cell. directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus. The term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule. This molecule may be integrated within a chromosome, or it may be extrachromosomally replicating DNA. In the 20 typical transgenic animals described herein, the transgene causes cells to express a recombinant form of a subject RAP-binding protein, e.g. either agonistic or antagonistic forms. However, transgenic animals in which the recombinant RAP-BP gene is silent are also contemplated, as for example, the FLP or CRE recombinase dependent constructs described below. The "non-human animals" of the invention include vertebrates such as rodents, non-human primates, sheep, dog, cow, chickens, amphibians, reptiles, etc. Preferred non-human animals are selected from the rodent family including rat and mouse, most preferably mouse, though transgenic amphibians, such as members of the Xenopus genus, and transgenic chickens can also provide important tools for understanding, for example, embryogenesis and tissue patterning. The term "chimeric animal" is used herein to refer to 30 animals in which the recombinant gene is found, or in which the recombinant is expressed in some but not all cells of the animal. The term "tissue-specific chimeric animal" indicates that the recombinant RAP-BP gene is present and/or expressed in some tissues but not others.

As used herein, the term "transgene" means a nucleic acid sequence (encoding, a RAP-binding protein), which is partly or entirely heterologous, foreign, to the transgenic animal or cell into which it is introduced, or, is homologous to an endogenous gene of the transgenic animal or cell into which it is introduced, but which is designed to be inserted, or is inserted, into the animal's genome in such a way as to alter the genome of the cell into .4 which it is inserted it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout). A transgene can include one or more transcriptional regulatory sequences and any other nucleic acid. such as introns, that may be necessary for optimal expression of a selected nucleic acid.

As is well known, genes for a particular polypeptide may exist in single or multiple copies within the genome of an individual. Such duplicate genes may be identical or may have certain modifications, including nucleotide substitutions, additions or deletions, which all still code for polypeptides having substantially the same activity. The term "DNA sequence encoding a RAP-binding protein" may thus refer to one or more genes within a particular individual. Moreover, certain differences in nucleotide sequences may exist between individual organisms, which are called alleles. Such allelic differences may or may not result in differences in amino acid sequence of the encoded polypeptide yet still encode a protein with the same biological activity.

"Homology" refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences.

20 "Cells," "host cells" or "recombinant host cells" are terms used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications max occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

A "chimeric protein" or "fusion protein" is a fusion of a first amino acid sequence encoding one of the subject RAP-binding proteins with a second amino acid sequence defining a domain foreign to and not substantially homologous with any domain of the subject RAP-BP. A chimeric protein may present a foreign domain which is found (albeit in a different protein) in an organism which also expresses the first protein, or it may be an "interspecies", "intergeneric", etc. fusion of protein structures expressed by different kinds of organisms. For example, a fusion protein of the present invention can be represented by the general formula Z 1

-Z

2

-Z

3 wherein Z- represents all or a portion of a polypeptide sequence of a RAP-binding protein, and Z, and Z 3 each represent polypeptide sequences which are heterologous to the RAP-BP sequence, at least one of Z, and Z 3 being present in the fusion protein.

n I6 The term "evolutionarily related to", with respect to nucleic acid sequences encoding RAP-binding proteins, refers to nucleic acid sequences which have arisen naturally in an organism, including naturally occurring mutants. Moreover, the term also refers to nucleic acid sequences which, while initially derived from naturally-occurring isoforms of RAPbinding proteins, have been altered by mutagenesis, as for example, such combinatorial mutagenesis as described below, yet which still encode polypeptides that bind FKBP/rapamycin complexes, or that retain at least one activity of the parent RAP-binding protein, or which are antagonists of that protein's activities.

The term "isolated" as also used herein with respect to nucleic acids, such as DNA or RNA. refers to molecules separated from other DNAs, or RNAs, respectively, that are present in the natural source of the macromolecule. For example, an isolated nucleic acid encoding one of the subject RAP-binding proteins preferably includes no more than 10 kilobases (kb) of nucleic acid sequence which naturally immediately flanks that particular RAP-BP gene in genomic DNA, more preferably no more than 5kb of such naturally occurring flanking sequences. and most preferably less than 1.5kb of such naturally occurring flanking sequence.

The term isolated as used herein also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.

Moreover, an "isolated nucleic acid" is meant to include nucleic acid fragments which are not S 20 naturally occurring as fragments and would not be found in the natural state.

As used herein, an "rapamycin-binding domain" refers to a polypeptide sequence which confers a binding activity for specifically interacting with an FKBP/rapamycin complex. Exemplary rapamycin-binding domains are represented within the polypeptides defined by Val26-Tyrl60 of SEQ ID No. 2 (mRAPTI), Val2012-Tyr2144 of SEQ ID No. 12 25 (hRAPTI), Val41-Tyr173 of SEQ ID No. 14 (caRAPTI), Vall-Tyrl33 of SEQ ID No. !6 (TORI). and Vall-Argl33 of SEQ ID No. 18 (TOR2).

A "RAPTI-like polypeptide" refers to a eukaryotic cellular protein which is a direct binding target protein for an FKBP/rapamycin complex, and which shares some sequence homology with a mammalian RAPTI protein of the present invention. Exemplary RAPTIlike polypeptides include the yeast TORI and TOR2 proteins.

A "soluble protein" refers to a polypeptide which does not precipitate at least about 95-percent, more preferably at least 99-percent remains in the supernatant) from an aqueous buffer under physiologically isotonic conditions, as for example, 0.14M NaCI or sucrose, at a protein concentration of as much as 10 pM, more preferably as much as 10 mM.

These conditions specifically relate to the absence of detergents or other denaturants in effective concentrations.

n 17 As described below, one aspect of this invention pertains to an isolated nucleic acid comprising the nucleotide sequence encoding a RAP-binding protein, fragments thereof.

and/or equivalents of such nucleic acids. The term nucleic acid as used herein is intended to include such fragments and equivalents, the term equivalent is understood to include nucleotide sequences encoding functionally equivalent RAP-binding proteins or functionally equivalent peptides which, for example, retain the ability to bind to the FKBP/rapamycin complex, and which may additionally retain other activities of a RAP-binding protein such as described herein. Equivalent nucleotide sequences will include sequences that differ by one or more nucleotide substitutions, additions or deletions, such as allelic variants; and will also include sequences that differ from the nucleotide sequence of the mammalian RAPTI genes represented in SEQ ID No: 1 or SEQ ID No. 11, or the nucleotide sequence of the fungal RAPTI protein of SEQ ID No. 13, or the nucleotide sequence encoding the UBC enzyme represented in SEQ ID No. 23, due to the degeneracy of the genetic code. Equivalent nucleic acids will also include nucleotide sequences that hybridize under stringent conditions 1 5 equivalent to about 20-27 0 C below the melting temperature (Tm) of the DNA duplex formed in about IM salt) to a nucleotide sequence of a RAPTI protein comprising either the sequence shown in SEQ ID No: 2 or 12, or to a nucleotide sequence of the RAPTI gene insert of pIC524 (ATCC accession no. 75787). Likewise, equivalent nucleic acids encoding homologs of the subject rap-UBC enzyme include nucleotide sequences that hybridize under 20 stringent conditions to a nucleotide sequence represented in SEQ ID No. 23, or to a nucleotide sequence of the rap-UBC gene insert of SMR4-15 (ATCC accession no. 75786).

In one embodiment, equivalents will further include nucleic acid sequences derived from, and evolutionarily related to, a nucleotide sequence comprising that shown in either SEQ ID No.

1, or SEQ ID No. I1, or SEQ ID No. 13, or SEQ ID No. 23.

The amino acid sequence shown in SEQ ID No: 2. and the fragment represented in the Trr 7yo7 t h e A T C C c e 75 repr es t biolugically active portions of larger full-length forms of mammalian RAPTI proteins. In preferred embodiments, the RAPTI polypeptide includes a binding domain for binding to FKBP/rapamyin complexes, such as the rap-binding domains represented by residues 28-160 of SEQ ID No. 2, or residues 2012-2144 of SEQ ID No. 12.

30 In preferred embodiments, portions of the RAPTI protein isolated from the full-length form will retain a specfic binding affinity for an FKBP/rapamycin complex, e.g. an FKBP12/rapamycin complex, e.g. an affinity at least 50%, more prefereably at least and even more preferably at least 90% that of the binding affinity of a naturally-occurring form of RAPTI for such a rapamycin complex. A polypeptide is considered to possess a biological activity of a RAPTI protein if the polypeptide has one or more of the following properties: the ability to bind an FKBP/drug complex, an FKBP/macrolide complex, an FKBP/rapamycin complex; the ability to bind to an FKBP12/rapamycin complex; the ability to modulate assembly of FKBP/rapamycin-complexes; the ability to regulate cell is/8 proliferation, to regulate the cell-cycle, to regulate the progression of a cell through the G 1 phase. Moreover, based on sequence analysis, the biological function of the subject RAPTI proteins can include a phosphatidyl inositol-kinase activity, such as a PI-3-kinase activity. A protein is also considered bioactive with respect to RAPTI bioactivity if it is a specific agonist (mimetic) or antagonist of one of the above recited properties.

With respect to the rap-UBC enzyme, preferred embodiments of the subject the protein comprise at least a portion of the amino acid sequence of SEQ ID No. 24 (or of the rap-UBC gene insert of SMR4-15 described in Example 5) which possess either the ability to bind a FKBP/rapamycin complex or the ability to conjugating ubiquitin to a cellular protein, or both. Given that rapamycin causes a block in the cell-cycle during G I phase, it is probable that the spectrum of biological activity of the subject rap-UBC enzyme includes control of half-lives of certain cell cycle regulatory proteins, particularly relatively short lived proteins proteins which have half-lives on the order of 30 minutes to 2 hours). For example, the subject UBC may have the ability to mediate ubiquitination of, for example, p53. myc and/or cyclins. and therefore affects the cellular half-life of a cell-cycle regulatory protein in proliferating cells. The binding of the rap-UBC to the FKBP/rapamycin complex may result in sequestering of the enzyme away from its substrate proteins. Thus, rapamycin may intefere with the ubiquitin-mediated degradation of p53 in a manner which causes cellular p53 levels to rise which in turn inhibits progression of the GI phase.

20 Moreover, it will be generally appreciated that, under certain circumstances, it may be advantageous to provide homologs of the cloned RAP-binding proteins which function in a limited capacity as one of either a RAP-BP agonists or a RAP-BP antagonists, in order to either promote or inhibit only a subset of the biological activities of the naturally occurring form of the protein. Thus, specific biological effects can be elicited by treatment with a homolog of limited function, and with fewer side effects relative to treatment with agonists or antagonists which are directed to all RAP-BP related biological activities. For instance, RAPTI analogs and rap-UBC analogs can be generated which do not bind in any substantial fashion to an FKBP/rapamycin complex, yet which retain most of the other biological functions ascribed to the naturally-occurring form of the protein. For example, the RAPTI 30 homolog might retain a kinase activity, such as a phosphatidyl inositol kinase activity, e.g. a PI-3-kinase activity. Conversely, the RAPTI homolog may be engineered to lack a kinase activity, yet retain the ability to bind an FKBP/rapamycin complex. For instance, the FKBP/rapamycin binding portions of the RAPTI homologs, such as the rapamycin-binding domains represented in SEQ ID Nos. 2 or 12, can be used to competitively inhibit binding to rapamycin complexes by the naturally-occurring form of RAPTI.

Homologs of the subject RAP-binding proteins can be generated by mutagenesis, such as by discrete point mutation(s), or by truncation. For instance, mutation can give rise 19 to homologs which retain substantially the same, or merely a subset, of the biological activity of the RAP-BP from which it was derived. Alternatively, antagonistic forms of the protein can be generated which are able to inhibit the function of the naturally occurring form of the protein, such as by competitively binding to FKBP/rapamycin complexes.

The nucleotide sequence designated in SEQ ID No: 1 encodes a biologically active portion of the mouse RAPTI protein, and in particular, includes a rapamycin-binding domain. Accordingly, one embodiment of the present invention provides a nucleic acid encoding a polypeptide comprising an amino acid sequence substantially homologous to that portion of the RAPTI protein represented by SEQ ID No: 2. Preferably, the nucleic acid is a cDNA molecule comprising at least a portion of the nucleotide sequence shown in SEQ ID No: 1. Yet another embodiment of the present invention provides a nucleic acid encoding a polypeptide comprising an amino acid sequence substantially homologous to a portion of the RAPTI protein represented by SEQ ID No. 12 corresponding to a rapamycin-binding domain. e.g. Val2012 to Tyr 2144 of SEQ ID No: 12. In similar fashion, the present invention provides a nucleic acid encoding at least a portion, a rapamycin-binding portion, of the Candida RAPTI polypeptide of SEQ ID No. 14.

Preferred nucleic acids encode a polypeptide including an amino acid sequence which is at least 60% homologous, more preferably 70% homologous and most preferably :homologous with an amino acid sequence shown in one or more of SEQ ID Nos: 2, 12 or 14.

20 Nucleic acids encoding peptides, particularly peptides having an activity of a RAPTI protein, and comprising an amino acid sequence which is at least about 90%, more preferably at least about 95%, and most preferably at least about 98-99% homologous with a sequence shown in either SEQ ID No: 2, 12 or 14 are also within the scope of the invention, as of course are proteins which are identical to the aforementioned sequence listings. In one embodiment, the nucleic acid is a cDNA encoding a peptide having at least one activity of a subject RAPbinding protein. Preferably, the nucleic acid is a cDNA molecule comprising at least a portion of the nucleotide sequence represented in one of SEQ ID Nos: 2, 12 or 14. A preferred portion of these cDNA molecules includes the coding region of the gene. For instance, a recombinant RAP-BP gene can include nucleotide sequences of a PCR fragment 30 generated by amplifying the coding sequences for one of the RAP-BP clones of ATCC deposit No: 75787.

The nucleotide sequence shown in SEQ ID No: 23 encodes a biologically active human ubiquitin conjugating enzyme. Accordingly, in one embodiment of the present invention, the nucleic acid encodes a polypeptide including the rapamycin-binding domain of the rap-UBC protein represented by SEQ ID No: 24. Preferably, the nucleic acid is a cDNA molecule comprising at least a portion of the nucleotide sequence shown in SEQ ID No: 23.

Preferred nucleic acids encode a peptide comprising an amino acid sequence which is at least homologous, more preferably 70% homologous and most preferably 80% homologous with an amino acid sequence shown in SEQ ID No: 24. Nucleic acids encoding polypeptides.

particularly those having a ubiquitin conjugating activity, and comprising an amino acid sequence which is at least about 90%, more preferably at least about 95%. and most preferably at least about 98-99% homologous with a sequence shown in SEQ ID No: 24 are also within the scope of the invention.

In a further embodiment of the invention, the recombinant RAP-BP genes can further include, in addition to the amino acid sequence shown in in the appended sequence listing, additional nucleotide sequences which encode amino acids at the C-terminus and N-terminus of the protein though not shown in those sequence listings. For instance, the recombinant RAPTI gene can include nucleotide sequences of a PCR fragment generated by amplifying the RAPTI coding sequence of pIC524 using sets of primers such described in Example 4.

Additionally, in light of the present disclosure, it will be possible using no more than routine experimentation to isolate from, for example, a cDNA library, the remaining 5' sequences of RAPT1, such as by RACE PCR using primers designed from the sequences of the plC524 clone, to generate the full-length sequence of SEQ ID No: 12. In particular, the invention contemplates a recombinant RAPTI gene encoding the full-length RAPTI protein.

Yet another embodiment of the invention includes nucleic acids that encode isoforms of the mouse or human RAPTI, especially isoforms splicing variants, allelic variants, etc.) that S 20 are capable of binding with the FKBP12/rapamycin complex. Such isoforms. as well as other members of the larger family of RAP-binding proteins, can be isolated using the drugdependent interaction trap assays described in further detail below.

Another aspect of the invention provides a nucleic acid that hybridizes under high or low stringency conditions to a nucleic acid which encodes a peptide having at least a portion of an amino acid sequence represented bv one of SEO ID Nns.: 2 12 or 14 A ppropria stringency conditions which promote DNA hybridization, for example, 6.0 x sodium chloride/sodium citrate (SSC) at about 45°C, followed by a wash of 2.0 x SSC at 50 0 C, are known to those skilled in the art or can be found in Current Protocols in Molecular Biology.

John Wiley Sons, N.Y. (1989), 6.3.1-6.3.6. For example, the salt concentration in the 30 wash step can be selected from a low stringency of about 2.0 x SSC at 50°C to a high stringency of about 0.2 x SSC at 50 0 C. In addition, the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22*C, to high stringency conditions at about 65 0

C.

Nucleic acids having a sequence which differs from the nucleotide sequence shown in any of SEQ ID Nos: 1, 11 or 13 due to degeneracy in the genetic code are also within the scope of the invention. Such nucleic acids encode functionally equivalent peptides a peptide having a biological activity of a RAP-binding protein) but that differ in sequence

I

II

21 from the appended sequence listings due to degeneracy in the genetic code. For example, a number of amino acids are designated by more than one triplet. Codons that specify the same amino acid, or synonyms (for example, CAU and CAC each encode histidine) may result in "silent" mutations which do not affect the amino acid sequence of the RAP-binding protein.

However, it is expected that DNA sequence polymorphisms that do lead to changes in the amino acid sequences of the subject RAP-binding proteins will exist among vertebrates. One skilled in the art will appreciate that these variations in one or more nucleotides (up to about of the nucleotides) of the nucleic acids encoding polypeptides having an activity of a RAP-binding protein may exist among individuals of a given species due to natural allelic variation. Any and all such nucleotide variations and resulting arino acid polymorphisms are within the scope of this invention.

The present invention also provides nucleic acid encoding only a portion of a RAPTI protein, such as the rapamycin-binding domain. As used herein, a fragment of a nucleic acid encoding such a portion of a RAP-binding protein refers to a nucleotide sequence having fewer nucleotides than the nucleotide sequence encoding the entire amino acid sequence of a full-length RAP-binding protein, yet which still includes enough of the coding sequence so as to encode a polypeptide which is capable of binding to an FKBP/rapamycin complex.

Moreover, nucleic acid fragments within the scope of the invention include those fragments capable of hybridizing under high or low stringency conditions with nucleic acids from other 20 vertebrate species, particularly other mammals, and can be used in screening protocols to detect homologs, of the subject RAP-binding proteins. Nucleic acids within the scope of the invention may also contain linker sequences, modified restriction endonuclease sites and other sequences useful for molecular cloning, expression or purification of recombinant peptides derived from RAP-binding proteins.

As indicated by the examples set out below, a nucleic acid encodine a RAP-hinding protein may be obtained from mRNA present in any of a number of cells from a vertebrate organism, particularly from mammals, e.g. mouse or human. It should also be possible to obtain nucleic acids encoding RAP-binding proteins from genomic DNA obtained from both adults and embryos. For example, a gene encoding a RAP-binding protein can be cloned 30 from either a cDNA or a genomic library in accordance with protocols herein described, as well as those generally known in the art. For instance, a cDNA encoding a RAPTI protein, particularly other isoforms, e.g. paralogs or orthologs, of the RAPTI proteins represented by either SEQ ID No. 2 or 12, can be obtained by isolating total mRNA from a mammalian cell, e.g. a human cell, generating double stranded cDNAs from the total mRNA, cloning the cDNA into a suitable plasmid or bacteriophage vector, and isolating RAPTI clones using any one of a number of known techniques, e.g. oligonucleotide probes or western blot analysis.

Genes encoding proteins related to the subject RAP-binding proteins can also be cloned using 22 established polymerase chain reaction techniques in accordance with the nucleotide sequence information provided by the invention. The nucleic acid of the invention can be DNA or

RNA.

Another aspect of the invention relates to the use of the isolated nucleic acid in "antisense" therapy. As used herein, "antisense" therapy refers to administration or in situ generation of oligonucleotide probes or their derivatives which specifically hybridizes (e.g.

binds) under cellular conditions, with the cellular mRNA and/or genomic DNA encoding a RAP-binding protein so as to inhibit expression of that protein, as for example by inhibiting transcription and/or translation. The binding may be by conventional base pair complementarity, or, for example, in the case of binding to DNA duplexes, through specific interactions in the major groove of the double helix. In general, "antisense" therapy refers to the range of techniques generally employed in the art, and includes any therapy which relies on specific binding to oligonucleotide sequences.

An antisense construct of the present invention can be delivered, for example, as an expression plasmid which, when transcribed in the cell, produces RNA which is complementary to at least a unique portion of the cellular mRNA which encodes a RAPbinding protein. Alternatively. the antisense construct can be an oligonucleotide probe which is generated ex vivo and which, when introduced into the cell causes inhibition of expression by hybridizing with the mRNA and/or genomic sequences of a RAP-BP gene. Such 20 oligonucleotide probes are preferably modified oligonucleotides which are resistant to endogenous nucleases, e.g. exonucleases and/or endonucleases, and is therefore stable in vivo. Exemplary nucleic acid molecules for use as antisense oligonucleotides are phosphoramidate, phosphothioate and methylphosphonate analogs of DNA (see also U.S.

Patents 5.176,996: 5.264,564; and 5,256,775). Additionally, general approaches to constructing oligomers useful in antisense therapy have been revipwure for exap ,i der Krol et al. (1988) Biotechniques 6:958-976; and Stein et al. (1988) Cancer Res 48:2659- •2668.

Accordingly, the modified oligomers of the invention are useful in therapeutic.

diagnostic, and research contexts. In therapeutic applications, the oligomers are utilized in a manner appropriate for antisense therapy in general. For such therapy, the oligomers of the invention can be formulated for a variety of loads of administration, including systemic and topical or localized administration. Techniques and formulations generally may be found in Remmington's Pharmaceutical Sciences. Meade Publishing Co.. Easton, PA. For systemic administration, injection is preferred, including intramuscular, intravenous, intraperitoneal, and subcutaneuos for injection, the oligomers of the invention can be formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank's solution or Z3 Ringer's solution. In addition, the oligomers may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included.

Systemic administration can also be by transmucosal or transdermal means, or the compounds can be administered orally. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration bile salts and fusidic acid derivatives. In addition, detergents may be used to facilitate permeation. Transmucosal administration may be through nasal sprays or using suppositories. For oral administration, the oligomers are formulated into conventional oral administration forms such as capsules, tablets, and tonics. For topical administration, the oligomers of the invention are formulated into ointments, salves, gels, or creams as generally known in the art.

In addition to use in therapy, the oligomers of the invention may be used as diagnostic reagents to detect the presence or absence of the target DNA or RNA sequences to which they specifically bind. Such diagnostic tests are described in further detail below.

Likewise. the antisense constructs of the present invention, by antagonizing the normal biological activity of a RAP-binding protein, can be used in the manipulation of tissue, e.g. tissue proliferation and/or differentiation, both for in vivo and ex vivo tissue culture systems.

20 This invention also provides expression vectors containing a nucleic acid encoding a RAP-binding protein of the present invention, operably linked to at least one transcriptional regulatory sequence. Operably linked is intended to mean that the nucleotide sequence is linked to a regulatory sequence in a manner which allows expression of the nucleotide sequence. Regulatory sequences are art-recognized land are selected to direct expressinn of n 25 recombinant RAP-binding protein. Accordingly, the term transcriptional regulatory sequence *0 includes promoters, enhancers and other expression control elements. Such regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). For instance, any of a wide variety of expression control sequences-sequences that control the expression of a DNA sequence when operatively linked to it may be used in these vectors to express DNA sequences encoding the RAP-binding proteins of this invention. Such useful expression control sequences, include, for example, the early and late promoters of SV40. adenovirus or cytomegalovirus immediate early promoter, the lac system, the trp system, the TAC or TRC system, T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter regions of phage lambda, the control regions for fd coat protein, the promoter for 3 -phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, Pho5, the 24 promoters of the yeast a-mating factors, the polyhedron promoter of the baculovirus system and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof. It should be understood that the design of the expression vector may depend on such factors as the choice of the host cell to be transformed and/or the type of protein desired to be expressed. Moreover, the vector's copy number, the ability to control that copy number and the expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered. In one embodiment, the expression vector includes a recombinant gene encoding a polypeptide which mimics or otherwise agonizes the action of a RAP-binding protein, or alternatively, which encodes a polypeptide that antagonizes the action of an authentic RAP-binding protein. Such expression vectors can be used to transfect cells and thereby produce polypeptides, including fusion proteins, encoded by nucleic acids as described herein.

Moreover, the gene constructs of the present invention can also be used as a part of a gene therapy protocol to deliver nucleic acids encoding either an agonistic or antagonistic form of one or more of the subject RAP-binding proteins. Thus, another aspect of the invention features expression vectors for in vivo transfection and expression of a RAPbinding protein in particular cell types so as to reconstitute the function of, or alternatively, abrogate the function of one or more of the subject RAP-binding proteins in a cell in which that protein or other transcriptional regulatory proteins to which it bind are misexpressed.

20 For example, gene therapy can be used to deliver a gene encoding a rapamycin-insensitive RAP-binding protein in order to render a particulat tissue or cell-type resistant to rapamycin induced cell-cycle arrest.

Expression constructs of the subject RAP-binding proteins, and mutants thereof, may be administered in any biologically effective carrier, e.g. any formulation or composition capable of effectively delivering the RAP-BP gene to cells'in vivo. Approaches include insertion of the subject gene in viral vectors including recombinant retroviruses, adenovirus, adeno-associated virus, and herpes simplex virus-1, or recombinant bacterial or eukaryotic plasmids. Viral vectors transfect cells directly; plasmid DNA can be delivered with the help of, for example, cationic liposomes (lipofectin) or derivatized antibody conjugated).

30 polylysine conjugates, gramacidin S, artificial viral envelopes or other such intracellular carriers, as well as direct injection of the gene construct or CaPO 4 precipitation carried out in vivo. It will be appreciated that because transduction of appropriate target cells represents the critical first step in gene therapy, choice of the particular gene delivery system will depend on such factors as the phenotype of the intended target and the route of administration, e.g.

locally or systemically. Furthermore, it will be recognized that the particular gene construct provided for in vivo transduction of RAP-BP expression are also useful for in vitro transduction of cells, such as in diagnostic assays.

A preferred approach for in vivo introduction of nucleic acid into a cell is by use of a viral vector containing nucleic acid, e.g. a cDNA, encoding the particular form of the RAPbinding protein desired. Infection of cells with a viral vector has the advantage that a large proportion of the targeted cells can receive the nucleic acid. Additionally, molecules encoded within the viral vector, by a cDNA contained in the viral vector, are expressed efficiently in cells which have taken up viral vector nucleic acid.

Retrovirus vectors and adeno-associated virus vectors are generally understood to be the recombinant gene delivery system of choice for the transfer of exogenous genes in vivo, particularly into humans. These vectors provide efficient delivery of genes into cells, and the transferred nucleic acids are stably integrated into the chromosomal DNA of the host. A major prerequisite for the use of retroviruses is to ensure the safety of their use, particularly with regard to the possibility of the spread of wild-type virus in the cell population. The development of specialized cell lines (termed "packaging cells") which produce only replication-defective retroviruses has increased the utility of retroviruses for gene therapy.

and defective retroviruses are well characterized for use in gene transfer for gene therapy purposes (for a review see Miller, A.D. (1990) Blood 76:271). Thus, recombinant retrovirus can be constructed in which part of the retroviral coding sequence (gag, pol. env) has been replaced by nucleic acid encoding one of the subject receptors rendering the retrovirus i. replication defective.

20 The replication defective retrovirus is then packaged into virions which can be used to infect a target cell through the use of a helper virus by standard techniques. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such 0*00• viruses can be found in Current Protocols in Molecular Biology, Ausubel, F.M. et al. (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals. Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM which are well known to those skilled in the art. Examples of suitable packaging virus lines for preparing both ecotropic and amphotropic retroviral systems include yCrip, yCre, y2 and VAm.

Retroviruses have been used to introduce a variety of genes into many different cell types, including lymphocytes, in vitro and/or in vivo (see for example Eglitis, et al. (1985) Science 30 230:1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad Sci. USA 85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad Sci. USA 85:3014-3018; Armentano et al. (1990) Proc.

Nail. Acad. Sci. USA 87:6141-6145; Huber et al. (1991) Proc. Natl. Acad Sci. USA 88:8039- 8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; van Beusechem et al. (1992) Proc. Natl. Acad Sci. USA 89:7640- 7644; Kay et al. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Nail. Acad Sci. USA 89:10892-10895; Hwu et al. (1993) J. Immunol. 150:4104-4115; U.S. Patent No.

2& 4,868,116; U.S. Patent No. 4.980.286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; and PCT Application WO 92/07573).

Furthermore, it has been shown that it is possible to limit the infection spectrum of retroviruses and consequently of retroviral-based vectors, by modifying the viral packaging proteins on the surface of the viral particle (see, for example PCT publications W093/25234 and W094/06920). For instance, strategies for the modification of the infection spectrum of retroviral vectors include: coupling antibodies specific for cell surface antigens to the viral env protein (Roux et al. (1989) PNAS 86:9079-9083; Julan et al. (1992) J. Gen Virol 73:3251-3255; and Goud et al. (1983) Virology 163:251-254); or coupling cell surface receptor ligands to the viral env proteins (Neda et al. (1991) JBiol Chem 266:14143-14146).

Coupling can be in the form of the chemical cross-linking with a protein or other variety (e.g.

lactose to convert the env protein to an asialoglycoprotein), as well as by generating fusion proteins single-chain antibody/env fusion proteins). This technique, while useful to limit or otherwise direct the infection to certain tissue types, can also be used to convert an ecotropic vector in to an amphotropic vector.

Moreover, use of retroviral gene delivery can be further enhanced by the use of tissueor cell-specific transcriptional regulatory sequences which control expression of the RAP-BP gene of the retroviral vector.

Another viral gene delivery system useful in the present invention utilizes adenovirus- S 20 derived vectors. The genome of an adenovirus can be manipulated such that it encodes and expresses a gene product of interest but is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. See for example Berkner et al. (1988) BioTechniques 6:616; Rosenfeld et al. (1991) Science 252:431-434; and Rosenfeld et al. (1992) Cell 68:143-155.

Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 d1324 or other srains of adenouUvirus Ad2, Ad3, Ad7 etc.) are well known to those skilied in the art.

Recombinant adenoviruses can be advantageous in certain circumstances in that they are not capable of infecting nondividing cells and can be used to infect a wide variety of cell types.

Furthermore, the virus particle is relatively stable and amenable to purification and concentration, and as above, can be modified so as to affect the spectrum of infectivity.

30 Additionally, introduced adenoviral DNA (and foreign DNA contained therein) is not integrated into the genome of a host cell but remains episomal, thereby avoiding potential problems that can occur as a result of insertional mutagenesis in situations where introduced DNA becomes integrated into the host genome retroviral DNA). Moreover, the carrying capacity of the adenoviral genome for foreign DNA is large (up to 8 kilobases) relative to other gene delivery vectors (Berkner et al. cited supra; Haj-Ahmand and Graham (1986) J. Virol. 57:267). Most replication-defective adenoviral vectors currently in use and therefore favored by the present invention are deleted for all or parts of the viral El and E3 27 genes but retain as much as 80% of the adenoviral genetic material (see, Jones et al.

(1979) Cell 16:683; Berkner et al., supra; and Graham et al. in Methods in Molecular Biologv, E.J. Murray, Ed. (Humana. Clifton, NJ. 1991) vol. 7. pp. 109-127). Expression of the inserted RAP-BP gene can be under control of, for example, the ElA promoter, the major late promoter (MLP) and associated leader sequences, the E3 promoter, or exogenously added promoter sequences.

Yet another viral vector system useful for delivery of the subject RAP-BP gene is the adeno-associated virus (AAV). Adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle. (For a review see Muzyczka et al. Curr.

Topics in Micro. and Immunol. (1992) 158:97-129). It is also one of the few viruses that may integrate its DNA into non-dividing cells, and exhibits a high frequency of stable integration (see for example Flotte et al. (1992)Am. J. Respir. Cell. Mol. Biol. 7:349-356; Samulski et al.

(1989) J. Virol. 63:3822-3828: and McLaughlin et al. (1989) J. Virol. 62:1963-1973).

Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate.

Space for exogenous DNA is limited to about 4.5 kb. An AAV vector such as that described in Tratschin et al. (1985) Mol. Cell. Biol. 5:3251-3260 can be used to introduce DNA into cells. A variety of nucleic acids have been introduced into different cell types using AAV vectors (see for example Herrnonat et al. (1984) Proc. Nail. Acad Sci. USA 81:6466-6470; 20 Tratschin et al. (1985) Mol. Cell. Biol. 4:2072-2081; Wondisford et al. (1988) Mol.

91000" Endocrinol. 2:32-39; Tratschin et al. (1984)J. Virol. 51:611-619; and Flotte et al. (1993) J.

Biol. Chem. 268:3781-3790).

In addition to viral transfer methods, such as those illustrated above, non-viral methods can also be employed to cause expression of an RAP-binding protein in the tissue of an animal. Most nonviral methods of gene transfer rely on normal mechanisms used by mammalian cells for the uptake and intracellular transport of macromolecules. In preferred embodiments, non-viral gene delivery systems of the present invention rely on endocytic pathways for the uptake of the subject RAP-BP gene by the targeted cell. Exemplary gene delivery systems of this type include liposomal derived systems, poly-lysine conjugates, and S 30 artificial viral envelopes.

In a representative embodiment, a gene encoding one of the subject RAP-binding proteins can be entrapped in liposomes bearing positive charges on their surface lipofectins) and (optionally) which are tagged with antibodies against cell surface antigens of the target tissue (Mizuno et al. (1992) No Shinkei Geka 20:547-551; PCT publication W091/06309; Japanese patent application 1047381; and European patent publication EP-A- 43075). For example, lipofection of cells can be carried out using liposomes tagged with monoclonal antibodies against any cell surface antigen present on, for example. T-cells.

In clinical settings. the gene delivery systems for the therapeutic RAP-BP gene can be introduced into a patient by any of a number of methods, each of which is familiar in the art.

For instance, a pharmaceutical preparation of the gene delivery system can be introduced systemically, e.g. by intravenous injection, and specific transduction of the protein in the target cells occurs predominantly from specificity of transfection provided by the gene delivery vehicle, cell-type or tissue-type expression due to the transcriptional regulatory sequences controlling expression of the receptor gene, or a combination thereof. In other embodiments, initial delivery of the recombinant gene is more limited with introduction into the animal being quite localized. For example, the gene delivery vehicle can be introduced by catheter (see U.S. Patent 5,328,470) or by stereotactic injection Chen et al. (1994) PNAS 91: 3054-3057).

The pharmaceutical preparation of the gene therapy construct can consist essentially of the gene delivery system in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery system can be produced intact from recombinant cells, e.g. retroviral vectors, the pharmaceutical preparation can comprise one or more cells which produce the gene delivery system.

Another aspect of the present invention concerns recombinant RAP-binding proteins which are encoded by genes derived from eukaryotic cells, e.g. mammalian cells, e.g. cells 20 from humans, mice, rats, rabbits, or pigs. The term "recombinant protein" refers to a protein of the present invention which is produced by recombinant DNA techniques, wherein generally DNA encoding, for example, the RAPTI protein, is inserted into a suitable expression vector which is in turn used to transform a host cell to produce the heterologous protein. Moreover, the phrase "derived from", with respect to a recombinant gene encoding the recombinant RAP-binding protein, is meant to include within the meaning of "recombinant protein" those proteins having an amino acid sequence of a native RAP-binding protein, or an amino acid sequence similar thereto, which is generated by mutation so as to include substitutions and/or deletions relative to a naturally occurring form of the RAPbinding protein of a organism. Recombinant RAPTI proteins preferred by the present S" 30 invention, in addition to those having an amino acid sequence of a native RAPTI protein.

comprise amino acid sequences which are at least 70% homologous, more preferably homologous and most preferably 90% homologous with an amino acid sequence shown in one of SEQ ID No: 2, 12 or 14. A polypeptide having a biological activity of a RAPTI protein and which comprises an amino acid sequence that is at least about 95%, more preferably at least about 98%, and most preferably are identical to a sequence represented in one of SEQ ID No: 2, 12 or 14 are also within the scope of the invention.

2q Likewise, preferred embodiments of recombinant rap-UBC proteins include an amino acid sequence which is at least 70% homologous, more preferably 80% homologous, and most preferably 90% homologous with an amino acid sequence represented by SEQ ID No.

24. Recombinant rap-UBC proteins which are identical, or substantially identical 95 to 98% homologous) with an amino acid sequence of SEQ ID No. 24 are also specifically contemplated by the present invention.

In addition, the invention expressly encompasses recombinant RAPTI proteins produced from the ATCC deposited clones described in Example 4, e.g. from ATCC deposit number 75787, as well as recombinant ubiquitin-conjugating enzynes produced from ATCC deposit number 75786, described in Example The present invention further pertains to recombinant forms of the subject RAPbinding proteins which are evolutionarily related to a RAP-binding protein represented in one of SEQ ID No: 2 or 12, that is, not identical, yet which are capable of functioning as an agonist or an antagonist of at least one biological activity of a RAP-binding protein. The term "evolutionarily related to", with respect to amino acid sequences of recombinant RAPbinding proteins, refers to proteins which have amino acid sequences that have arisen naturally, as well as to mutational variants which are derived, for example, by recombinant mutagenesis.

Another aspect of the present invention pertains to methods of producing the subject 20 RAP-binding proteins. For example, a host cell transfected with a nucleic acid vector directing expression of a nucleotide sequence encoding the subject RAPTI protein or rap- UBC can be cultured under appropriate conditions to allow expression of the peptide to occur. The peptide may be secreted and isolated from a mixture of cells and medium containing the recombinant protein. Alternatively, the peptide may be retained cytoplasmically,. as the naturally occurring forms of the subjeci. RAP-binding proteins are believed to be, and the cells harvested, lysed and the protein isolated. A cell culture includes host cells, media and other byproducts. Suitable media for cell culture are well known in the art. The recombinant RAP-binding proteins can be isolated from cell culture medium, host cells, or both using techniques known in the art for purifying proteins including ion-exchange 30 chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification with antibodies specific for a RAP-binding protein. In one embodiment, the RAP-binding protein is a fusion protein containing a domain which facilitates its purification, such as a RAPTI-GST fusion protein or a rapUBC-GST fusion protein.

The present invention also provides host cells transfected with a RAP-BP gene for expressing a recombinant form of a RAP-binding protein. The host cell may be any prokaryotic or eukaryotic cell. Thus, a nucleotide sequence derived from the cloning of the RAP-binding proteins of the present invention, encoding all or a selected portion of a protein.

can be used to produce a recombinant form of a RAP-BP via microbial or eukaryotic cellular processes. Ligating a polynucleotide sequence into a gene construct, such as an expression vector, and transforming or transfecting host cells with the vector are standard procedures used in producing other well-known proteins, e.g. insulin, interferons, p53. myc, cyclins and the like. Similar procedures, or modifications thereof, can be employed to prepare recombinant RAP-binding proteins, or portions thereof, by microbial means or tissue-culture technology in accord with the subject invention. Host cells suitable for expression of a recombinant RAP-binding protein can be selected, for example, from amongst eukaryotic (yeast, avian, insect or mammalian) or prokaryotic (bacterial) cells.

The recombinant RAP-BP gene can be produced by ligating nucleic acid encoding a RAP-binding protein, or a portion thereof, into a vector suitable for expression in either prokaryotic cells, eukaryotic cells, or both. Expression vectors for production of recombinant forms of RAP-binding proteins include plasmids and other vectors. For instance, suitable vectors for the expression of a RAP-BP include plasmids of the types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmids for expression in prokaryotic cells, such as E. coli.

A number of vectors exist for the expression of recombinant proteins in yeast. For 20 instance, YEP24, YIP5, YEP51, YEP52. pYES2, and YRP17 are cloning and expression vehicles useful in the introduction of genetic constructs into S. cerevisiae (see, for example, Broach et al. (1983) in Experimental Manipulation of Gene Expression, ed. M. Inouye Academic Press, p. 83, incorporated by reference herein). These vectors can replicate in E coli due the presence of the pBR322 ori, and in S. cerevisiae due to the replication determinant of the yeast 2 micron plasmid. In addition, drug resistance markers such as ampicillin can be used.

Preferred mammalian expression vectors contain prokaryotic sequences to facilitate the propagation of the vector in bacteria, and one or more eukaryotic transcription regulatory sequences that cause expression of a recombinant RAP-BP gene in eukaryotic cells. The 30 pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples of mammalian expression vectors suitable for transfection of eukaryotic cells. Some of these vectors are modified with sequences from bacterial plasmids, such as pBR322, to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells. Alternatively, derivatives of viruses such as the bovine papilloma virus (BPV-1), or Epstein-Barr virus (pHEBo, pREPderived and p205) can be used for transient expression of proteins in eukaryotic cells.

Examples of other viral (including retroviral) expression systems can be found above in the description of gene therapy delivery systems.

In some instances, it may be desirable to express a recombinant RAP-binding protein by the use of a baculovirus expression system (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley Sons: 1992). Examples of such baculovirus expression systems include pVL-derived vectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derived vectors (such as the B-gal containing pBlueBac III).

The various methods employed in the preparation of the plasmids and transformation of host organisms are well known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells, as well as general recombinant procedures, see Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989) Chapters 16 and 17.

When expression of a portion of one of the subject RAP-binding proteins is desired.

i.e. a trunction mutant, such as the RAPTI polypeptides of SEQ ID Nos: 2, 12 or 14. it may be necessary to add a start codon (ATG) to the oligonucleotide fragment containing the :desired sequence to be expressed. It is well known in the art that a methionine at the Nterminal position can be enzymatically cleaved by the use of the enzyme methionine aminopeptidase (MAP). MAP has been cloned from E. coli (Ben-Bassat et al. (1987) 20 J. Bacteriol. 169:751-757) and Salmonella ryphimurium and its in vitro activity has been demonstrated on recombinant proteins (Miller et al. (1987) PNAS 84:2718-1722). Therefore.

removal of an N-terminal methionine. if desired, can be achieved either in vivo by expressing RAP-BP-derived polypeptides in a host which produces MAP E. coli or CM89 or S. cerevisiae), or in vitro by use of purified MAP procedure of Miller et.al:. supra).

Alternatively, the coding sequences for the polypeptide can be incorporated as a part of a fusion gene so as to be covalently linked in-frame with a second nucleotide sequence encoding a different polypeptide. This type of expression system can be useful, for instance, where it is desirable to produce an immunogenic fragment of a RAP-binding protein. For example, the VP6 capsid protein of rotavirus can be used as an immunologic carrier protein 30 for portions of the RAPTI polypeptide, either in the monomeric form or in the form of a viral particle. The nucleic acid sequences corresponding to the portion of the RAPTI protein to which antibodies are to be raised can be incorporated into a fusion gene construct which includes coding sequences for a late vaccinia virus structural protein to produce a set of recombinant viruses expressing fusion proteins comprising a portion of the protein RAPTI as part of the virion. It has been demonstrated with the use of immunogenic fusion proteins utilizing the Hepatitis B surface antigen fusion proteins that recombinant Hepatitis B virions 32 can be utilized in this role as well. Similarly, chimeric constructs coding for fusion proteins containing a portion of an RAPTI protein and the poliovirus capsid protein can be created to enhance immunogenicity of the set of polypeptide antigens (see, for example. EP Publication No. 0259149; and Evans et al. (1989) Nature 339:385; Huang et al. (1988) J. Virol. 62:3855; and Schlienger el al. (1992) J Virol. 66:2). The subject ubiquitin-conjugating enzyme can be manipulated as an immunogen in like fashion.

The Multiple Antigen Peptide system for peptide-based immunization can also be utilized, wherein a desired portion of a RAP-binding protein is obtained directly from organochemical synthesis of the peptide onto an oligomeric branching lysine core (see, for example, Posnett et al. (1988) JBC 263:1719 and Nardelli el al. (1992) J. Immunol. 148:914).

Antigenic determinants of the RAP-binding proteins can also be expressed and presented by bacterial cells.

In addition to utilizing fusion proteins to enhance immunogenicity, it is widely appreciated that fusion proteins can also facilitate the expression and purification of proteins.

such as any one of the RAP-binding proteins of the present invention. For example, a RAPbinding protein can be generated as a glutathione-S-transferase (GST) fusion protein. Such GST fusion proteins can simplify purification of a RAP-binding protein, as for example by affinity purification using glutathione-derivatized matrices (see, for example. Current Protocols in Molecular Biology, eds. Ausabel et al. John Wiley Sons, 1991)). In 20 another embodiment, a fusion gene coding for a purification leader sequence, such as a peptide leader sequence comprising a poly-(His)/enterokinase cleavage sequence, can be added to the N-terminus of the desired portion of a RAP-binding protein in order to permit purification of the poly(His)-fusion protein by affinity chromatography using a Ni 2 metal resin. The purification leader sequence can then be subsequently removed by treatment with enterokinase see Hochuli et al. (1987) J. Chromatography 411:177; and.Janknecht et al.

PNAS 88:8972).

Techniques for making fusion genes are known to those skilled in the art. Essentially, the joining of various DNA fragments coding for different polypeptide sequences is performed in accordance with conventional techniques, employing blunt-ended or stagger- 30 ended termini for ligation, restriction enzyme digestion to provide for appropriate termini.

filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which are subsequently annealed to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley Sons: 1992).

33 The present invention also makes available purified, or otherwise isolated forms of the subject RAP-binding proteins which is isolated from, or otherwise substantially free of other cellular proteins, especially FKBP or other rapamycin binding proteins, as well as ubiquitin and ubiquitin-dependent enzymes, signal transduction, and cell-cycle regulatory proteins, which may be normally associated with the RAP-binding protein. The term "substantially free of other cellular or viral proteins" (also referred to herein as "contaminating proteins") or "substantially pure or purified preparations" are defined as encompassing preparations of RAP-binding proteins having less than 20% (by dry weight) contaminating protein, and preferably having less than 5% contaminating protein. Functional forms of the subject RAP-binding proteins can be prepared, for the first time, as purified preparations by using recombinant proteins as described herein. Alternatively, the subject RAP-binding proteins can be isolated by affinity purification using, for example, matrix bound FKBP/rapamycin protein. By "purified", it is meant, when referring to a peptide or DNA or RNA sequence, that the indicated molecule is present in the substantial absence of other biological macromolecules, such as other proteins (particularly FK506 binding proteins, as well as other contaminating proteins). The term "purified" as used herein preferably means at least 80% by dry weight, more preferably in the range of 95-99% by weight, and most preferably at least 99% by weight, of biological macromolecules of the same type present (but water, buffers, and other small molecules, especially molecules 20 having a molecular weight of less than 5000, can be present). The term "pure" as used herein preferably has the same numerical limits as "purified" immediately above. "Isolated" and "purified" do not encompass either natural materials in their native state or natural materials that have been separated into components in an acrylamide gel) but not obtained either as pure lacking contaminating proteins, or chromatography reagents such as denaturing agents and polymers, e.g. acrylamide or agarose) substances or solutions.

F urthermore, isolated peJptJdyI prtions of e subject KAP-binding proteins cai also obtained by screening peptides recombinantly produced from the corresponding fragment of the nucleic acid encoding such peptides. In addition, fragments can be chemically synthesized using techniques known in the art such as conventional Merrifield solid phase f- 30 Moc or t-Boc chemistry. For example, a RAP-binding protein of the present invention may be arbitrarily divided into fragments of desired length with no overlap of the fragments, or preferably divided into overlapping fragments of a desired length. The fragments can be produced (recombinantly or by chemical synthesis) and tested to identify those peptidyl fragments which can function as either agonists or antagonists of a RAP-binding protein activity, such as by microinjection assays or in vitro protein binding assays. In an illustrative embodiment, peptidyl portions of a RAP-binding protein, such as RAPTI or rapUBC, can be tested for FKBP/rapamycin-binding activity.

S 4.

It will also be possible to modify the structure of a RAP-binding protein for such purposes as enhancing therapeutic or prophylactic efficacy, or stability ex vivo shelf life and resistance to proteolvtic degradation in vivo). Such modified peptides, when designed to retain at least one activity of the naturally-occurring form of the protein, are considered functional equivalents of the RAP-binding protein described in more detail herein. Such modified peptide can be produced, for instance, by amino acid substitution, deletion, or addition.

For example, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid conservative mutations) will not have a major effect on the folding of the protein, and may or may not have much of an effect on the biological activity of the resulting molecule. Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids are can be divided into four families: acidic aspartate, glutamate; basic lysine, arginine, histidine; nonpolar alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and uncharged polar glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine.

tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. In similar fashion, the amino acid repertoire can be grouped as acidic aspartate, glutamate; (2) 20 basic lysine, arginine histidine, aliphatic glycine, alanine, valine, leucine, isoleucine, S serine, threonine, with serine and threonine optionally be grouped separately as aliphatichydroxyl; aromatic phenylalanine, tyrosine, tryptophan; amide asparagine, glutamine; and sulfur -containing cysteine and methionine (see, for example, Biochemistry, 2nd ed., Ed. by L. Stryer, WH Freeman and Co.: 1981). Alternatively, amino 25 acid replacement can be based on steric criteria, e.g. isosteric replacements, without regard Sfor polarty or charge of amino acid sidechains. wheter a change.in the amino acid sequence of a peptide results in a functional RAP-BP homolog functional in the sense that it acts to mimic or antagonize the wild-type form) can be readily determined by assessing the ability of the variant peptide to produce a response in cells in a fashion similar to the 30 wild-type RAP-BP or competitively inhibit such a response. Peptides in which more than one replacement has taken place can readily be tested in the same manner.

This invention further contemplates a method of generating sets of combinatorial mutants of RAP-binding proteins, e.g. of RAPTI proteins and/or rap-UBC enzymes, as well as truncation mutants, thereof and is especially useful for identifying variant sequences (e.g RAP-BP homologs) that are functional in regulating rapamycin-mediated effects, as well as other aspects of cell growth or differentiation. In similar fashion, RAP-BP homologs can be generated by the present combinatorial approach which are antagonists in that they are able to interfere with the normal cellular functions of authentic forms of the protein.

One purpose for screening such combinatorial libraries is, for example, to isolate novel RAP-BP homologs from the library which function in the capacity as one of either an agonists or an antagonist of the biological activities of the wild-type ("authentic") protein, or alternatively, which possess novel biological activities all together. To illustrate, RAPTI homologs can be engineered by the present method to provide homologs which are unable to bind to the FKBP/rapamycin complex, yet still retain at least a portion of the normal cellular activity associated with authentic RAPTI. Thus, combinatorially-derived homologs.can be generated to provide rapamycin-resistance. Such proteins, when expressed from recombinant DNA constructs, can be used in gene therapy protocols.

Likewise, mutagenesis can give rise to RAP-BP homologs which have intracellular half-lives dramatically different than the corresponding wild-type protein. For example, the altered protein can be rendered either more stable or less stable to proteolytic degradation or other cellular process which result in destruction of, or otherwise inactivation of. the authentic RAP-binding protein. Such homologs, and the genes which encode them, can be utilized to alter the envelope of expression of a particular RAP-BP by modulating the half-life of the protein. For instance, a short half-life can give rise to more transient RAPTI biological effects and, when part of an inducible expression system, can allow tighter control of 20 recombinant RAPTI levels within the cell. As above, such proteins, and particularly their recombinant nucleic acid constructs, can be used in gene therapy protocols.

In an illustrative embodiment of this method, the amino acid sequences for a population of RAP-BP homologs, or other related proteins, are aligned, preferably to promote the highest homology possible. Such a population of variants can include, for example; RAPTI homologs from o.e or more species, e.g seque alignment f the mouse and human RAPT1 proteins represented by SEQ ID Nos. 2 and 12, or different RAP-BP isoforms from the same species, e.g. different human RAPTI isoforms. Amino acids which appear at each position of the sequence alignment can be selected to create a degenerate set of combinatorial sequences.

*o 30 In a preferred embodiment, the combinatorial RAP-BP library is produced by way of a degenerate library of genes encoding a library of polypeptides which each include at least a portion of potential RAP-BP sequences, e.g. the portion of RAPTI represented by SEQ ID No: 2 or 12, or the portion of rap-UBC represented by SEQ ID No. 24. A mixture of synthetic oligonucleotides can be enzymatically ligated into gene sequences such that the degenerate set of potential RAP-BP sequences are expressible as individual polypeptides, or 3 Lalternatively, as a set of larger fusion proteins for phage display) containing the RAP-BP sequence library therein.

There are many ways by which the library of RAP-BP homologs can be generated from a degenerate oligonucleotide sequence. For instance, chemical synthesis of a degenerate gene sequence can be carried out in an automated DNA synthesizer, and the synthetic genes then ligated into an appropriate gene for expression. The purpose of a degenerate set of RAP-BP genes is to provide, in one mixture, all of the sequences encoding the desired set of potential RAP-BP sequences. The synthesis of degenerate oligonucleotides is well known in the art (see, for example, Narang, SA (1983) Tetrahedron 39:3; Itakura et al.

(1981) Recombinant DNA, Proc 3rd Cleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp273-289; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura el al. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477. Such techniques have been employed in the directed evolution of other proteins (see, for example, Scott et al.

(1990) Science 249:386-390; Roberts et al. (1992) PNAS 89:2429-2433; Devlin el al. (1990) Science 249: 404-406; Cwirla et al. (1990) PNAS 87: 6378-6382; as well as U.S. Patents Nos.

5,223,409, 5,198,346, and 5,096,815).

Alternatively, other forms of mutagenesis can be utilized to generate a combinatorial library. For example, RAP-BP homologs (both agonist and antagonist forms) can be generated and isolated from a library generated by using, for example, alanine scanning 20 mutagenesis and the like (Ruf et al. (1994) Biochemistry 33:1565-1572; Wang et al. (1994) J Biol. Chem. 269:3095-3099; Balint et al. (1993) Gene 137:109-118; Grodberg et al. (1993) Eur. J Biochem. 218:597-601; Nagashima et al. (1993) J Biol. Chem. 268:2888-2892; Lowman et al. (1991) Biochemistry 30:10832-10838; and Cunningham et al. (1989) Science 244:1081-1085), by linker scanning mutagenesis (Gustin et al. (1993) Virology 193:653-660; S 25 Brown et al. (1992) Mol. Cell Biol. 12:2644-2652; McKnight et al. (1982) Science 232:316); by saturation mutagenesis (Meyers et al. (1986) Science 232:613); by PCR mutagenesis (Leung et al. (1989) Method Cell Mol Biol 1:11-19); or by random mutagenesis (Miller et al.

(1992) A Short Course in Bacterial Genetics, CSHL Press, Cold Spring Harbor, NY; and Greener et al. (1994) Strategies in Mol Biol 7:32-34).

30 A wide range of techniques are known in the art for screening gene products of variegated gene libraries made by combinatorial mutagenesis, especially for identifying individual gene products having a certain property. Such techniques will be generally adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of, for example, RAPTI homologs. The most widely used techniques for screening large gene libraries typically comprises cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity 1 37 facilitates relatively easy isolation of the vector encoding the gene whose product was detected. Each of the illustrative assays described below are amenable to high through-put analysis as necessary to screen large numbers of degenerate RAP-BP sequences created by combinatorial mutagenesis techniques.

In one screening assay, the candidate RAP-BP gene products are displayed on the surface of a cell or viral particle, and the ability of particular cells or viral particles to bind the FKBP12/rapamycin complex via this gene product is detected in a "panning assay". For instance, the degenerate RAP-BP gene library can be cloned into the gene for a surface membrane protein of a bacterial cell, and the resulting fusion protein detected by panning protocols (see, for example, Ladner et al., WO 88/06630; Fuchs el al. (1991) Bio/Technology 9:1370-1371: and Goward et al. (1992) TIBS 18:136-140). In a similar fashion, fluorescently labeled molecules which bind the RAP-binding protein, such as fluorescently labeled rapamycin or FKBP12/rapamycin complexes, can be used to score for potentially functional RAP-BP homologs. Cells can be visually inspected and separated under a fluorescence microscope, or, where the morphology of the cell permits, separated by a fluorescenceactivated cell sorter.

In an alternate embodiment, the gene library is expressed as a fusion protein on the surface of a viral particle. For instance, in the filamentous phage system, foreign peptide sequences can be expressed on the surface of infectious phage, thereby conferring two significant benefits. First, since these phage can be applied to affinity matrices at very high S* concentrations, a large number of phage can be screened at one time. Second, since each infectious phage displays the combinatorial gene product on its surface, if a particular phage is recovered from an affinity matrix in low yield, the phage can be amplified by another round of infection. The group of almost identical E.coli filamentous phages M13, fd. and fl are 25 most often used in phage display libraries, as either of the phage gill or gVIII coat proteins can be used to generate fusion proteins without disrupting the ultimate packaging of the viral particle (Ladner et al. PCT publication WO 90/02909; Garrard et al., PCT publication WO 92/09690; Marks et al. (1992) J. Biol. Chem. 267:16007-16010; Griffiths et al. (1993) EMBO J 12:725-734; Clackson et al. (1991) Nature 352:624-628; and Barbas et al. (1992) PNAS S 30 89:4457-4461). In an illustrative embodiment, the recombinant phage antibody system (RPAS, Pharmacia Catalog number 27-9400-01) can be easily modified for use in expressing and screening RAP-BP combinatorial libraries, and the RAP-BP phage library can be panned on glutathione-immobilized FKBP-GST/rapamycin complexes. Successive rounds of reinfection, phage amplification, and panning will greatly enrich for homologs which retain FKBP/rapamycin binding and which can be subsequently screened for further biological activities in order to discern between agonists and antagonists.

38 Homologs of the human and mouse RAP-binding proteins can also be generated through the use of interaction trap assays to screen combinatorial libraries of RAP-BP mutants. As described in Example 10 below, the same two hybrid assay used to screen cDNA libraries for proteins which interact with FK506-binding proteins in a drug-dependent manner can also be used to sort through combinatorial libraries of, for example, RAPTI mutants, to find both agonistic and antagonistic forms. By controlling the sensitivity of the assay for inteactions. e.g. through the manipulation of the strength of the promoter sequence used to drive expression of the reporter construct, the assay can be generated to favor agonistic forms of RAPTI with tighter binding affinities for rapamycin then the authentic form of the protein.

Alternatively, as described in Example 10, the assay can be used to select for RAPTI homologs which are now unable to bind rapamycin complexes and hence are versions of the RAPTI protein which can render a cell insensitive to treatment with that macrolide.

The invention also provides for reduction of the rapamycin-binding domains of the subject RAP-binding proteins to generate mimetics. e.g. peptide or non-peptide agents. which are able to disrupt binding of a polypeptide of the present invention with an FKBP/rapamycin complex. Thus, such mutagenic techniques as described above are also useful to map the determinants of RAP-binding proteins which participate in interactions involved in, for example, binding to an FKBP/rapamycin complex. To illustrate, the critical residues of a RAP-binding protein which are involved in molecular recognition of FKBP/rapamycin can 20 be determined and used to generate RAP-BP-derived peptidomimetics that competitively inhibit binding of the RAP-BP to rapamycin complexes. By employing, for example, scanning mutagenesis to map the amino acid residues of a particular RAP-binding protein involved in binding FKBP/rapamycin complexes, peptidomimetic compounds can be generated which mimic those residues in binding to the rapamycin complex. and which, by inhibiting binding of the RAP-BP to FKBP/rapamycin, can interfere with the function of rtnmrnvrin in rpll-rvrlp nrrpst Fnr inctnnrc nnnh xir-r.-,bll t;ro or.allr f .suc residues can be generated using retro-inverse peptides see U.S. Patents 5,116,947 and 5,218,089; and Pallai et al. (1983) Int J Pept Protein Res 21:84-92) benzodiazepine see Freidinger et al. in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: 30 Leiden, Netherlands, 1988), azepine see Huffman et al. in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), substituted gama lactam rings (Garvey et al. in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), keto-methylene pseudopeptides (Ewenson et al.

(1986) J Med Chem 29:295; and Ewenson et al. in Peptides: Structure and Function (Proceedings of the 9th American Peptide Symposium) Pierce Chemical Co. Rockland, IL, 1985), P-turn dipeptide cores (Nagai et al. (1985) Tetrahedron Lett 26:647; and Sato et al.

(1986) J Chem Soc Perkin Trans 1:1231), and P-aminoalcohols (Gordon et al. (1985) Biochem Biophys Res Communl26:419; and Dann et al. (1986) Biochem Biophys Res 3(i Commun 134:71). Utilizing side-by-side assays, peptidomimetics can be designed to specifically inhibit the interaction of human RAPTI (or other mammalian homologs) with the FKBP12/rapamycin complex in mammalian cells, but which do not substantially affect the interaction of the yeast protein TORI or TOR2 with the FKBI/rapamycin complex. Such a peptide analog could be used in conjunction with rapamycin treatment of mycotic infections to protect the host mammal from rapamycin side-effects, such as immunosuppression, without substantially reducing the efficacy of rapamycin as an antifungal agent.

Another aspect of the invention pertains to an antibody specifically reactive with one or more of the subject RAP-binding proteins. For example, by using immunogens derived from a RAP-binding protein, anti-protein/anti-peptide antisera or monoclonal antibodies can be made by standard protocols (See, for example, Antibodies: A Laboratory Manual ed. by Harlow and Lane (Cold Spring Harbor Press: 1988)). A mammal, such as a mouse, a hamster or rabbit can be immunized with an immunogenic form of the peptide a full length RAP-binding protein or an antigenic fragment which is capable of eliciting an antibody response). Techniques for conferring immunogenicity on a protein or peptide include conjugation to carriers or other techniques well known in the art. An immunogenic portion of the subject RAP-binding proteins can be administered in the presence of adjuvant.

2 The progress of immunization can be monitored by detection of antibody titers in plasma or 20 serum. Standard ELISA or other immunoassays can be used with the immunogen as antigen to assess the levels of antibodies. In a preferred embodiment, the subject antibodies are immunospecific for antigenic determinants of the RAP-binding proteins of the present invention, e.g. antigenic determinants of a protein represented in one of SEQ ID Nos: 2, 12 or a closely related human or non-human mammalian homolog thereof. For instance, a favored 25 anti-RAP-BP antibody of the present invention does not substantially cross react react specifically) with a protein which is less than 90 percent homologous to one of SEQ ID Nos: 2 or 12; though antibodies which do not substantially cross react with a protein which is less than 95 percent homologous with one of SEQ ID Nos: 2, 12 or 24, or even less than 98-99 percent homologous with one of SEQ ID Nos: 2 or 12, are specifically contemplated. By 30 "not substantially cross react", it is meant that the antibody has a binding affinity for a nonhomologous protein a yeast TORI or TOR2 protein) which is less than 10 percent, more preferably less than 5 percent, and even more preferably less than 1 percent, of the binding affinity for a mammalian RAPTI protein, such as represented one of SEQ ID Nos: 2 or 12.

Following immunization, anti-RAP-BP antisera can be obtained and, if desired, polyclonal anti-RAP-BP antibodies isolated from the serum. To produce monoclonal antibodies, antibody producing cells (lymphocytes) can be harvested from an immunized animal and fused by standard somatic cell fusion procedures with immortalizing cells such as myeloma cells to yield hybridoma cells. Such techniques are well known in the an, an include, for example, the hybridoma technique (originally developed by Kohler and Milstein, (1975) Nature, 256: 495-497), the human B cell hybridoma technique (Kozbar et al., (1983) Immunology Today, 4: 72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. pp. 77-96). Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with a RAP-binding protein of the present invention and monoclonal antibodies isolated from a culture comprising such hybridoma cells.

An antibody preparation of this invention prepared from a polypeptide as described above can be in dry form as obtained by lyophilization. However, the antibodies are normally used and supplied in an aqueous liquid composition in serum or a suitable buffer such as PBS.

The term antibody as used herein is intended to include fragments thereof which are also specifically reactive with one of the subject RAP-binding protein. Antibodies can be fragmented using conventional techniques, including recombinant engineering, and the fragments screened for utility in the same manner as described above for whole antibodies.

For example, F(ab') 2 fragments can be generated by treating antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' 20 fragments. The antibody of the present invention is further intended to include bispecific and chimeric molecules having an anti-RAP-BP portion.

Both monoclonal and polyclonal antibodies (Ab) directed against a RAP-binding protein can be used to block the action of that protein and allow the study of the role of a particular RAP-binding protein in, for example, cell-cycle regulation generally. or in the etiology of proliferative and/or differentiative disorders specifically, or in the mechanism of action of rapamycin, e.g. by microinjection of anti-RAP-BP antibodies into cells.

Antibodies which specifically bind RAP-BP epitopes can also be used in immunohistochemical staining of tissue samples in order to evaluate the abundance and pattern of expression of each of the subject RAP-binding proteins. Anti-RAP-BP antibodies 30 can be used diagnostically in immuno-precipitation and immuno-blotting to detect and evaluate RAP-BP levels in tissue or bodily fluid as part of a clinical testing procedure. For instance, such measurements as the level of free RAP-BP to RAP-BP/FKBP/drug complexes can be useful in predictive valuations of the efficacy of a particular rapamycin analog, and can permit determination of the efficacy of a given treatment regimen for an individual. The level of a RAP-binding protein can be measured in cells found in bodily fluid, such as in cells from samples of blood, or can be measured in tissue, such as produced by biopsy.

Another application of the subject antibodies is in the immunological screening of cDNA libraries constructed in expression vectors such as ?gtll1, Agt18-23. kZAP. and XORF8. Messenger libraries of this type. having coding sequences inserted in the correct reading frame and orientation, can produce fusion proteins. For instance, Xgtl I will produce fusion proteins whose amino termini consist of B-galactosidase amino acid sequences and whose carboxy termini consist of a foreign polypeptide. Antigenic epitopes of a RAPbinding protein can then be detected with antibodies, as, for example, reacting nitrocellulose filters lifted from infected plates with anti-RAP-BP antibodies. Phage, scored by this assay.

can then be isolated from the infected plate. Thus, the presence of RAP-BP homologs can be detected and cloned from other animals, and alternate isoforms (including splicing variants) can be detected and cloned from human sources.

Moreover, the nucleotide sequence determined from the cloning of the subject RAPbinding proteins from a human cell line will further allow for the generation of probes designed for use in identifying homologs in other human cell types, as well as RAP-BP homologs orthologs) from other mammals. For example, by identifying highly conserved nucleotides sequence through comparison of the mammalian RAPTI genes with the yeast TOR genes, it will be possible to design degenerate primers for isolating RAPT] homologs from virtually any eukaryotic cell. For instance, alignment of the mouse RAPTI gene sequence and the yeast DRR-1 and TOR2 sequences, we have determined that optimal primers for isolating RAPTI homologs from other mammalian homologs, as well as from pathogenic fungi, include the primers GRGAYTTRAWBGABGCHYAMGAWTGG, CAAGCBTGGGAYMTYMTYTAYTATMAYGTBTTCAG. and GAYYBGARTTGGCTG-

TBCCHGG.

Accordingly, the present invention also provides a probe/primer comprising a 25 substantially purified oligonucleotide, which oligonucleotide comprises a region of :nucleotide sequence that hybridizes under stringent conditions to at least 10 consecutive nucleotides of sense or anti-sense sequence of one of SEQ ID Nos: 1 or 11, or naturally occurring mutants thereof. In preferred embodiments, the probe/primer further comprises a label group attached thereto and able to be detected, e.g. the label group is selected from the 30 group consisting of radioisotopes, fluorescent compounds, enzymes, and enzyme co-factors.

Such probes can also be used as a part of a diagnostic test kit for identifying transformed cells, such as for measuring a level of a RAP-BP nucleic acid in a sample of cells from a patient; e.g. detecting mRNA encoding a RAP-BP mRNA level; e.g. determining whether a genomic RAP-BP gene has been mutated or deleted.

In addition, nucleotide probes can be generated which allow for histological screening of intact tissue and tissue samples for the presence of a RAP-BP mRNA. Similar to the diagnostic uses of anti-RAP-BP antibodies, the use of probes directed to RAP-BP 4Y2 mRNAs, or to genomic RAP-BP sequences, can be used for both predictive and therapeutic evaluation of allelic mutations which might be manifest in. for example. neoplastic or hyperplastic disorders unwanted cell growth) or abnormal differentiation of tissue.

Used in conjunction with an antibody immunoassays, the nucleotide probes can help facilitate the determination of the molecular basis for a developmental disorder which may involve some abnormality associated with expression (or lack thereof) of a RAP-binding protein. For instance, variation in synthesis of a RAP-binding protein can be distinguished from a mutation in the genes coding sequence.

Thus, the present invention provides a method for determining if a subject is at risk for a disorder characterized by unwanted cell proliferation or abherent control of differentiation. In preferred embodiments, the subject method can be generally characterized as comprising detecting, in a tissue sample of the subject a human patient), the presence or absence of a genetic lesion characterized by at least one of a mutation of a gene encoding one of the subject RAP-binding proteins or (ii) the misexpression of a RAP-BP gene. To illustrate, such genetic lesions can be detected by ascertaining the existence of at least one of a deletion of one or more nucleotides from a RAP-BP gene, (ii) an addition of one or more nucleotides to such a RAP-BP gene, (iii) a substitution of one or more nucleotides of a RAP-BP gene, (iv) a gross chromosomal rearrangement of one of the RAP-BP genes, a gross alteration in the level of a messenger 20 RNA transcript of a RAP-BP gene, (vi) the presence of a non-wild type splicing pattern of a messenger RNA transcript of a RAP-BP gene, and (vii) a non-wild type level of a RAPbinding protein. In one aspect of the invention there is provided a probe/primer comprising an oligonucleotide containing a region of nucleotide sequence which is capable of hybridizing to a sense or antisense sequence of one of SEQ ID Nos: 1 or 11. or naturally occurring mutants thereof, or 5' or 3' flanking sequences or intronic sequences naturally associated with the subject RAP-BP genes. The probe is exposed to nucleic acid of a tissue sample: and the hybridization of the probe to the sample nucleic acid is detected. In certain embodiments, detection of the lesion comprises utilizing the probe/primer in a polymerase chain reaction (PCR) (see, U.S. Patent Nos: 4,683,195 and 4,683,202) or, alternatively, 30 in a ligation chain reaction (LCR) (see, Landegran et al. (1988) Science, 241:1077- :1080: and NaKazawa et al. (1944) PNAS 91:360-364) the later of which can be particularly useful for detecting point mutations in the RAP-BP gene. Alternatively, immunoassays can be employed to determine the level of RAP-binding protein and/or its participation in protein complexes, particularly transcriptional regulatory complexes such as those involving FKBP/rapamycin.

Also, by inhibiting endogenous production of a particular RAP-binding protein, antisense techniques microinjection of antisense molecules, or transfection with plasmids .713 whose transcripts are anti-sense with regard to a RAP-BP mRNA or gene sequence) can be used to investigate role of each of the subject RAP-BP in growth and differentiative events, such as those giving rise to Wilm's tumor, as well as normal cellular functions of each of the subject RAP-binding proteins, e.g. in regulation of transcription. Such techniques can be utilized in cell culture, but can also be used in the creation of transgenic animals.

Furthermore, by making available purified and recombinant RAP-binding proteins, the present invention provides for the generation of assays which can be used to screen for drugs which are either agonists or antagonists of the cellular function of each of the subject RAP-binding proteins, or of their role in the pathogenesis of proliferative and differentiative disorders. For instance, an assay can be generated according to the present invention which evaluates the ability of a compound to modulate binding between a RAP-binding protein and an FK506-binding protein. In particular, such assays can be used to design and screen novel rapamycin analogs, as well as test completely unrelated compounds for their ability to mediate formation of FKBP/RAP-BP complexes. Such assays can be used to generate more potent anti-proliferative agents having a similar mechanism of action as rapamycin, e.g.

rapamycin analogs. A variety of assay formats will suffice and, in light of the present inventions, will be comprehended by skilled artisan.

ne aspect of the present invention which facilitates the generation of drug screening **assays, particularly the high-throughout assays described below, is the identification of the 20 rapamycin binding domain of RAPTI-like proteins. For instance, the present invention provides portions of the RAPTl-like proteins which are easier to manipulate than the full length protein. The full length protein is, because of its size, more difficult to express as a recombinant protein or a fusion protein which would retain rapamycin-binding activity, and may very well be insoluble. Accordingly, the present invention provides soluble 25 polypeptides which include a soluble portion of a RAPTI like polypeptide that binds to said FKBP/rapamycin complex, such as the rapamycin-binding domain represented by an amino acid sequence selected from the group consisting Val26-Tyrl60 of SEQ ID No. 2, Val2012- Tyr2144 of SEQ ID No. 12, Val41-Tyrl73 of SEQ ID No. 14, Vall-Tyr33 of SEQ ID No.

16, and Vall-Argl33 of SEQ ID No. 18.

0* 30 For instance, RAPTI polypeptides useful in the subject screening assays may be represented by the general formula X-Y-Z, Y represents an amino acid sequence of a rapamycin-binding domain within residues 2 0124o 2144 of SEQ ID No. 12, X is absent, or represents all or a C-terminal portion of the amino acid sequence between about residues 1700 and 2144 of SEQ ID No. 12 not represented by Y, and Z is absent, or represents all or an N-terminal portion of the amino acid sequence between residues 2012 and 2549 of SEQ ID No. 12 not represented by Y. Preferably, the polypeptide includes only about 50 to 200 residues of RAPTI protein sequence, which portion includes a rapamycin-binding domain.

Similar polypeptides can be generated for other RAPTI-like proteins.

In an alternative embodiment, the same formula can also be used to designate a bioactive fragment of the subject RAPTI protein, wherein Y represents a rapamycin-binding domain within residues 2012 to 2144 of SEQ ID No. 12, X is absent or represents a polypeptide from I to about 500 amino acid residues of SEQ ID No. 12 immediately Nterminal to the rapamycin-binding domain, and Z is absent or represents from I to about 365 amino acid residues of SEQ ID No. 2 immediately C-terminal to the selected rapamycinbinding domain.

In many drug screening programs which test libraries of compounds and natural extracts, high throughput assays are desirable in order to maximize the number of compounds surveyed in a given period of time. Assays which are performed in cell-free systems, such as may be derived with purified or semi-purified proteins, are often preferred as "primary" screens in that they can be generated to permit rapid development and relatively easy 1 5 detection of an alteration in a molecular target when contacted with a test compound.

Moreover, the effects of cellular toxicity and/or bioavailability of the test compound can be generally ignored in the in vitro system, the assay instead being focused primarily on the effect of the drug on the molecular target as may be manifest in an alteration of binding So. affinity with other proteins or change in enzymatic properties of the molecular target.

.20 Accordingly. in an exemplary screening assay of the present invention, the compound of "interest (the "drug") is contacted with a mixture generated from an isolated and purified RAPbinding protein, such as RAPTI or rapUBC, and an FK506-binding protein. Detection and quantification of drug-depedent FKBP/RAP-BP complexes provides a means for determining the compound's efficacy for mediating complex formation between the two proteins. The 25 efficacy of the comppund can be assessed by generating dose response curves, from data obtained using various concentrations of the test compound. Moreover, a control assay can also be performed to provide a baseline for comparison. In the control assay, isolated and purified RAP-BP is added to a composition containing the FK506-binding protein, and the formation of FKBPRAP-BP complexes is quantitated in the absence of the test compound.

S.

.o 30 Complex formation between the RAP-binding protein and an FKBP/drug complex may be detected by a variety of techniques. For instance, modulation in the formation of complexes can be quantitated using, for example, detectably labelled proteins (e.g.

radiolabelled, fluorescently labelled, or enzymatically labelled), by immunoassay. or by chromatographic detection.

Typically, it will be desirable to immobilize either the FK506-binding protein or the RAP-binding protein to facilitate separation of drug-dependent protein complexes from uncomplexed forms of one of the proteins, as well as to accommodate automation of the assay. In an illustrative embodiment, a fusion protein can be provided which adds a domain that permits the protein to be bound to an insoluble matrix. For example, glutathione-Stransferase/FKBP (FKBP-GST) fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtitre plates, which are then combined with the RAP-binding protein, e.g. an 35 S-labeled RAP-binding protein, and the test compound and incubated under conditions conducive to complex formation (see.

for instance, Example Following incubation, the beads are washed to remove any unbound RAP-BP, and the matrix bead-bound radiolabel determined directly beads placed in scintilant), or in the supemtantant after the FKBP/RAP-BP complexes are dissociated, e.g. when microtitre plates are used. Alternatively, after washing away unbound protein, the complexes can be dissociated from the matrix, separated by SDS-PAGE gel, and the level of RAP-BP found in the matrix-bound fraction quantitated from the gel using standard electrophoretic techniques.

Other techniques for immobilizing proteins on matrices are also available for use in the subject assay. For instance, the FK506-binding protein can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated FKBP can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well known in the art biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well 20 plates (Pierce Chemical). Alternatively, antibodies reactive with the FKBP can be derivatized to the wells of the plate, and FKBP trapped in the wells by antibody conjugation.

As above, preparations of a RAP-binding protein and a test compound are incubated in the FKBP-presenting wells of the plate, and the amount of FKBP/RAP-BP complex trapped in the well can be quantitated. Exemplary methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the RAP-binding protein, or which are reactive with the FK506-binding protein and compete for binding with the RAP-BP; as well as enzymelinked assays which rely on detecting an enzymatic activity associated with the RAP-binding protein. In the instance of the latter, the enzymatic activity can be endogenous, such as a 30 kinase (RAPTI) or ubiquitin ligase (rapUBC) activity, or can be an exogenous activity chemically conjugated or provided as a fusion protein with the RAP-binding protein. To illustrate, the RAP-binding protein can be chemically cross-linked with alkaline phosphatase, and the amount of RAP-BP trapped in the complex can be assessed with a chromogenic substrate of the enzyme, e.g. paranitrophenyl phosphate. Likewise, a fusion protein comprising the RAP-BP and glutathione-S-transferase can be provided, and complex formation quantitated by detecting the GST activity using 1-chloro-2,4-dinitrobenzene (Habig et al (1974) JBiol Chem 249:7130).

1 0 For processes which rely on immunodetection for quantitating one of the proteins trapped in the complex, antibodies against the protein, such as the anti-RAP-BP antibodies described herein, can be used. Alternatively, the protein to be detected in the complex can be "epitope tagged" in the form of a fusion protein which includes, in addition to the RAP-BP or FKBP sequence, a second polypeptide for which antibodies are readily available from commercial sources). For instance, the GST fusion proteins described above can also be used for quantification of binding using antibodies against the GST moiety. Other useful epitope tags include myc-epitopes see Ellison et al. (1991) J Biol Chem 266:21150-21157) which includes a 10-residue sequence from c-myc, as well as the pFLAG system (International Biotechnologies, Inc.) or the pEZZ-protein A system (Pharamacia, NJ).

Additionally, the subject RAP-binding proteins can be used to generate a drugdependent interaction trap assay, as described in the examples below, for detecting agents which induce complex formation between a RAP-binding protein and an FK506-binding protein. As described below, the interaction trap assay relies on reconstituting in vivo a functional transcriptional activator protein from two separate fusion proteins, one of which comprises the DNA-binding domain of a transcriptional activator fused to an FK506-binding protein (see also U.S. Patent No: 5,283,317; PCT publication W094/10300; Zervos et al.

(1993) Cell 72:223-232; Madura et al. (1993) J Biol Chem 268:12046-12054; Bartel et al.

(1993) Biotechniques 14:920-924; and Iwabuchi et al. (1993) Oncogene 8:1693-1696). The 20 second fusion protein comprises a transcriptional activation domain able to initiate RNA "polymerase transcription) fused to one of the subject RAP-binding proteins. When the FKBP and RAP-binding protein interact in the presence of an agent such as rapamycin, the two domains of the transcriptional activator protein are brought into sufficient proximity as to cause transcription of a reporter gene. In addition to the LexA interaction trap described in the examples below, yet another illustrative embodiment comprises Saccharomyces cerevisiae YPB2 cells transformed simultaneously with a plasmid encoding a GAL4db- FKBP fusion (db: DNA binding domain) and with a plasmid encoding the GAL4 activation domain (GAL4ad) fused to a subject RAP-BP. Moreover, the strain is transformed such that the GAL4-responsive promoter drives expression of a phenotypic marker. For example, the 30 ability to grow in the absence of histidine can depends on the expression of the HIS3 gene.

When the HIS3 gene is placed under the control of a GAL4-responsive promoter, relief of this auxotrophic phenotype indicates that a functional GAL4 activator has been reconstituted through the drug-dependent interaction of FKBP and the RAP-BP. Thus, agent able to promote RAP-BP interaction with an FKBP will result in yeast cells able to grow in the absence of histidine. Commercial kits which can be modified to develop two-hybrid assays with the subject RAP-binding proteins are presently available MATCHMAKER kit, ClonTech catalog number K1605-1, Palo Alto, CA).

c 7, In a preferred embodiment, assays which employ the subject mammalian RAPbinding proteins can be used to identify rapamycin mimetics that have therapeutic indexes more favorable than rapamycin. For instance, rapamycin-like drugs can be identified by the present invention which have enhanced tissue-type or cell-type specficity relative to rapamycin. To illustrate, the subject assays can be used to generate compounds which preferentially inhibit IL-2 mediated proliferation/activation of lymphocytes without substantially interfering with other tissues, e.g. hepatocytes. Likewise, similar assays can be used to identify rapamycin-like drugs which inhibit proliferation of yeast cells or other lower eukaryotes, but which have a substantially reduced effect on mammalian cells, thereby improving therapeutic index of the drug as an anti-mycotic agent relative to rapamycin.

In one embodiment, the identification of such compounds is made possible by the use of differential screening assays which detect and compare drug-mediated formation of two or more different types of FKBP/RAP-BP complexes. To illustrate, the assay can be designed for side-by-side comparison of the effect of a test compound on the formation of tissue-type specific FKBP/RAPT] complexes. Given the diversity of FKBPs. and the substantial likelihood that RAPTI represents a single member of a larger family of related proteins, it is probable that different functional FKBP/RAPTI complexes exist and, in certain instances, are localized to particular tissue or cell types. As described in PCT publication W093/23548, entitled "Method of Detecting Tissue-Specific FK506 Binding Protein Messenger RNAs and 20 Uses Thereof', the tissue distribution of FKBPs can vary from one species of the protein to the next. Thus; test compounds can be screened for agents able to mediate the tissue-specific formation of only a subset of the possible repertoire of FKBP/RAPTI complexes. In an exemplary embodiment, an interaction trap assay can be derived using two or more different bait proteins, e.g. FKBP12 (SEQ ID Nos. 5 and FKBP25 (GenBank Accession M90309), or FKBP52 (Genbank Accession M88279), while the fish protein is constant in each, e.g. a human RAPT1 construct. Running the.ITS side-by side permits the detection of agents which have a greater effect statistically significant on the formation of one of the FKBP/RAPTI complexes than on the formation of the other FKBP complexes.

In similar fashion, differential screening assays can be used to exploit the difference 30 in drug-mediated formation of mammalian FKBP/RAP-BP complexes and yeast FKBP/TOR complexes in order to identify agents which display a statistically significant increase in specificity for the yeast complexes relative to the mammalian complexes. Thus, lead compounds which act specifically on pathogens, such as fungus involved in mycotic infections, can be developed. By way of illustration, the present assays can be used to screen for agents which may ultimately be useful for inhibiting at least one fungus implicated in such mycosis as candidiasis, aspergillosis, mucormycosis, blastomycosis, geotrichosis.

cryptococcosis, chromoblastomycosis, coccidioidomycosis, conidiosporosis, histoplasmosis, 1 II 48 maduromycosis, rhinosporidosis, nocaidiosis, para-actinomycosis, penicilliosis. monoliasis, or sporotrichosis. For example, if the mycotic infection to which treatment is desired is candidiasis, the present assay can comprise comparing the relative effectiveness of a test compound on mediating formation of a mammalian FKBP/RAPTI complex with its effectiveness towards mediating such complexes formed from genes cloned from yeast selected from the group consisting of Candida albicans, Candida stellatoidea, Candida tropicalis. Candida parapsilosis. Candida krusei, Candida pseudorropicalis, Candida quillermondii, or Candida rugosa. Likewise. the present assay can be used to identify antifungal agents which may have therapeutic value in the treatment of aspergillosis by making use of the subject drug-dependent interaction trap assays derived from FKBP and TOR genes cloned from yeast such as Aspergillusfumigatus, Aspergillusflavus, Aspergillus niger, Aspergillus nidulans, or Aspergillus terreus. Where the mycotic infection is mucormycosis, the complexes can be derived from yeast such as Rhizopus arrhizus, Rhizopus oryzae, Absidia corymbifera, Absidia ramosa, or Mucor pusillus. Sources of other rapamycindependent complexes for comparison with a mammalian FKBP/RAPTI complex includes the pathogen Pneumocystis carinii. Exemplary FK506-binding proteins from human pathogens and other lower eukaryotes are provided by, for example, GenBank Accession numbers: M84759 (Candida albican); U01195, U01198, U01197, U01193, U01188, U01194. U01199 (Neisseria spp.); and M98428 (Streptomyces chrysomallus).

20 In an exemplary embodiment, the differential screening assay can be generated using at least the rapamycin-binding domain of the Candida albican RAPTI protein (see Example I1) and a Candida FK506-binding protein (such as RBPI, GenBank No. M84759. see also Ferrara et al. (1992) Gene 113:125-127), or a yeast FK506-binding protein (see Example 8 and Figure Comparison of formation of human RAPTI complexes and Candida RAPTI complexes provides a means for identifying agents which are more selective for the formation of caRAPTI complexes and, accordingly. likely to be more specific as anti-mycotic agents relative to rapamycin.

Another aspect of the present invention concerns transgenic animals which are comprised of cells (of that animal) which contain a transgene of the present invention and 30 which preferably (though optionally) express an exogenous RAP-binding protein in one or more cells in the animal. The RAP-BP transgene can encode the wild-type form of the protein, or can encode homologs thereof, including both agonists and antagonists, as well as antisense constructs designed to inhibit expression of the endogenous gene. In preferred embodiments, the expression of the transgene is restricted to specific subsets of cells, tissues or developmental stages utilizing, for example, through the use of cis-acting sequences that control expression in the desired pattern. In the present invention, such mosaic expression of the subject RAP-binding proteins can be essential for many forms of lineage analysis and can '/9 additionally provide a means to assess the effects of loss-of-function mutations, which deficiency might grossly alter development in small patches of tissue within an otherwise normal embryo. Toward this and, tissue-specific regulatory sequences and conditional regulatory sequences can be used to control expression of the transgene in certain spatial patterns. Moreover, temporal patterns of expression can be provided by, for example, conditional recombination systems or prokaryotic transcriptional regulatory sequences.

Genetic techniques which allow for the expression of transgenes can be regulated via site-specific genetic manipulation in vivo are known to those skilled in the art. For instance.

genetic systems are available which allow for the regulated expression of a recombinase that catalyzes the genetic recombination a target sequence. As used herein, the phrase "target sequence" refers to a nucleotide sequence that is genetically recombined by a recombinase.

The target sequence is flanked by recombinase recognition sequences and is generally either excised or inverted in cells expressing recombinase activity. Recombinase catalyzed recombination events can be designed such that recombination of the target sequence results in either the activation or repression of expression of a subject RAP-binding protein. For example, excision of a target sequence which interferes with the expression of a recombinant RAP-BP gene can be designed to activate expression of that gene. This interference with expression of the protein can result from a variety of mechanisms, such as spatial separation of the gene from a promoter element or an internal stop codon. Moreover, the transgene can 20 be made wherein the coding sequence of the gene is flanked by recombinase recognition sequences and is initially transfected into cells in a 3' to 5' orientation with respect to the promoter element. In such an instance, inversion of the target sequence will reorient the subject gene by placing the 5' end of the coding sequence in an orientation with respect to the promoter element which allow for promoter driven transcriptional activation.

In an illustrative embodiment, either the crelloxP recombinase system of:: bacteriophage PI (Lakso et al. (1992) PNAS 89:6232-6236; Orban et al. (1992) PNAS oo 89:6861-6865) or the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al.

(1991) Science 251:1351-1355; PCT publication WO 92/15694) can be used to generate in S: vivo site-specific genetic recombination systems. Cre recombinase catalyzes the site-specific 30 recombination of an intervening target sequence located between loxP sequences. loxP sequences are 34 base pair nucleotide repeat sequences to which the Cre recombinase binds and are required for Cre recombinase mediated genetic recombination. The orientation of loxP sequences determines whether the intervening target sequence is excised or inverted when Cre recombinase is present (Abremski et al. (1984) J. Biol. Chem. 259:1509-1514); catalyzing the excision of the target sequence when the loxP sequences are oriented as direct repeats and catalyzes inversion of the target sequence when loxP sequences are oriented as inverted repeats.

t. Accordingly, genetic recombination of the target sequence is dependent on expression of the Cre recombinase. Expression of the recombinase can be regulated by promoter elements which are subject to regulatory control, tissue-specific, developmental stage-specific, inducible or repressible by externally added agents. This regulated control will result in genetic recombination of the target sequence only in cells where recombinase expression is mediated by the promoter element. Thus, the activation expression of a RAPbinding protein can be regulated via regulation of recombinase expression.

Use of the cre/loxP recombinase system to regulate expression of a recombinant RAP-binding protein, such as RAPTI or rapUBC, requires the construction of a transgenic animal containing transgenes encoding both the Cre recombinase and the subject protein.

Animals containing both the Cre recombinase and the recombinant RAP-BP genes can be provided through the construction of "double" transgenic animals. A convenient method for providing such animals is to mate two transgenic animals each containing a transgene, e.g., the RAP-BP gene in one animal and recombinase gene in the other.

One advantage derived from initially constructing transgenic animals containing a .:transgene in a recombinase-mediated expressible format derives from the likelihood that the subject protein will be deleterious upon expression in the transgenic animal. In such an S.instance, a founder population, in which the subject transgene is silent in all tissues, can be propagated and maintained. Individuals of this founder population can be crossed with 20 animals expressing the recombinase in, for example, one or more tissues. Thus. the creation of a founder population in which, for example, an antagonistic RAP-BP transgene is silent will allow the study of progeny from that founder in which disruption of cell-cycle regulation in a particular tissue or at developmental stages would result in, for example, a lethal phenotype.

Similar conditional transgenes can be provided using prokaryotic promoter sequences which require prokaryotic proteins to be simultaneous expressed in order to facilitate expression of the transgene. Exemplary promoters and the corresponding trans-activating prokaryotic proteins are given in U.S. Patent No. 4,833,080. Moreover, expression of the conditional transgenes can be induced by gene therapy-like methods wherein a gene encoding the trans-activating protein, e.g. a recombinase or a prokaryotic protein, is delivered to the tissue and caused to be expressed using, for example, one of the gene therapy constructs described above. By this method, the RAP-BP transgene could remain silent into adulthood and its expression "turned on" by the introduction of the trans-activator.

In an exemplary embodiment, the "transgenic non-human animals" of the invention are produced by introducing transgenes into the germline of the non-human animal.

Embryonal target cells at various developmental stages can be used to introduce transgenes.

Different methods are used depending on the stage of development of the embryonal target cell. The zygote is the best target for micro-injection. In the mouse, the male pronucleus reaches the size of approximately 20 micrometers in diameter which allows reproducible injection of l-2pl of DNA solution. The use of zygotes as a target for gene transfer has a major advantage in that in most cases the injected DNA will be incorporated into the host gene before the first cleavage (Brinster et al. (1985) PNAS 82:4438-4442). As a consequence, all cells of the transgenic non-human animal will carry the incorporated transgene. This will in general also be reflected in the efficient transmission of the transgene to offspring of the founder since 50% of the germ cells will harbor the transgene. Microinjection of zygotes is the preferred method for incorporating transgenes in practicing the invention.

Retroviral infection can also be used to introduce a RAP-BP transgene into a nonhuman animal. The developing non-human embryo can be cultured in vitro to the blastocyst stage. During this time, the blastomeres can be targets for retroviral infection (Jaenich. R.

(1976) PNAS 73:1260-1264). Efficient infection of the blastomeres is obtained by enzymatic treatment to remove the zona pellucida (Manipulating the Mouse Embryo, Hogan eds. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1986). The viral vector system used to introduce the transgene is typically a replication-defective retrovirus carrying the transgene (Jahneretal. (1985) PNAS 82:6927-6931; Vander Putten etal. (1985) PNAS 82:6148-6152).

Transfection is easily and efficiently obtained by culturing the blastomeres on a monolayer of 20 virus-producing cells (Van der Putten, supra; Stewart et al. (1987) EMBO J 6:383-388).

Alternatively, infection can be performed at a later stage. Virus or virus-producing cells can be injected into the blastocoele (Jahner et al. (1982) Nature 298:623-628). Most of the founders will be mosaic for the transgene since incorporation occurs only in a subset of the cells which formed the transgenic non-human animal. Further, the founder may contain various retroviral insertions of the transgene at different positions in the genome which generally will segregate in the offspring. In addition, it is also possible to introduce transgenes into the germ line by intrauterine retroviral infection of the midgestation embryo (Jahner et al. (1982) supra).

A third type of target cell for transgene introduction is the embryonal stem cell (ES).

30 ES cells are obtained from pre-implantation embryos cultured in vitro and fused with embryos (Evans et al. (1981) Nature 292:154-156; Bradley et al. (1984) Nature 309:255-258; Gossler et al. (1986) PNAS 83: 9065-9069; and Robertson et al. (1986) Nature 322:445-448).

Transgenes can be efficiently introduced into the ES cells by DNA transfection or by retrovirus-mediated transduction. Such transformed ES cells can thereafter be combined with blastocysts from a non-human animal. The ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal. For review see Jaenisch, R.

(1988) Science 240:1468-1474.

C,

5-2 Methods of making knock-out or disruption transgenic animals are also generally known. See, for example, Manipulating the Mouse Embryo, (Cold Spring Harbor Laborator Press, Cold Spring Harbor, 1986). Recombinase dependent knockouts can also be generated, e.g. by homologous recombination to insert recombinase target sequences, such that tissue specific and/or temporal control of inactivation of a RAP-BP gene can be controlled as above.

Another aspect of the present invention concerns a novel in vivo method for the isolation of genes encoding proteins which physically interact with a "bait" protein/drug complex. The method relies on detecting the reconstitution of a transcriptional activator in the presence of the drug, particularly wherein the drug is a non-peptidyl small organic molecule <2500K), e.g. a macrolide, e.g. rapamycin, FK506 or cyclosporin. In particular, the method makes use of chimeric genes which express hybrid proteins. The first hybrid comprises the DNA-binding domain of a transcriptional activator fused to the bait protein. The second hybrid protein contains a transcriptional activation domain fused to a "fish" protein, e.g. a test protein derived from a cDNA library. If the fish and bait proteins are able to interact in a drug-dependent manner, they bring into close proximity the two domains of the transcriptional activator. This proximity is sufficient to cause transcription of a reporter gene which is operably linked to a transcriptional regulatory site responsive to the transcriptional activator, and expression of the marker gene can be detected and used to score .20 for the interaction of the bait protein/drug complex with another protein.

One advantage of this method is that a multiplicity of proteins can be simultaneously tested to determine whether any interact with the drug/protein complex. For example, a DNA fragment encoding the DNA-binding domain can be fused to a DNA fragment encoding the bait protein in order to provide one hybrid. This hybrid is introduced into the cells carrying the marker gene, and the cells are contacted with a drug which is known to bind the bait protein. For the second hybrid, a library' of plasmids'can be constructed which may include, eose for example, total mammalian complementary DNA (cDNA) fused to the DNA sequence encoding the activation domain. This library is introduced into the cells carrying the first hybrid. If any individual plasmid from the test library encodes a protein that is capable of 30 interacting with the drug/protein complex, a positive signal may be obtained by detecting expression of the reporter gene. In addition, when the interaction between the drug complex and a novel protein occurs, the gene for the newly identified protein is readily available.

As illustrated herein, the present interaction trap system is a valuable tool in the identification of novel genes encoding proteins which act at a point in a given signal transduction pathway that is directly upstream or downstream from a particular protein/drug complex. For example, the subject assay can be used to identify the immediate downstream targets of an FKBP/rapamycin complex, or of an FKBP/FK506 complex, or of a 53 cyclophilin/cyclosporin complex. Proteins that interact in a drug-dependent manner with one of such complexes may be identified, and these proteins can be of both diagnostic and therapeutic value.

A first chimeric gene is provided which is capable of being expressed in the host cell, preferably a yeast cell, most preferably Saccharomyces cerevisiae or Schizosaccharomyces pombe. The host cell contains a detectable gene having a binding site for the DNA-binding domain of the transcriptional activator, such that the gene expresses a marker protein when the marker gene is transcriptionally activated. Such activation occurs when the transcriptional activation domain of a transcriptional activator is brought into sufficient proximity to the DNA-binding domain of the transcriptional activator. The first chimeric gene may be present in a chromosome of the host cell. The gene encodes a chimeric protein which comprises a DNA-binding domain that recognizes the binding site on the marker gene in the host cell and a bait protein which is to be tested for drug-mediated interaction with a second test protein or protein fragment.

A second chimeric gene is provided which is capable of being expressed in the host cell. In one embodiment, both the first and the second chimeric genes are introduced into the host cell in the form ofplasmids. Preferably, however, the first chimeric gene is present in a o chromosome of the host cell and the second chimeric gene is introduced into the host cell as part of a plasmid. The second chimeric gene contains a DNA sequence that encodes a second S* 20 hybrid protein. The second hybrid protein contains a transcriptional activation domain. The second hybrid protein also contains a second test:protein or a.protein fragment which is to be tested for interaction with the first test protein or protein fragment. Preferably, the DNAeo *binding domain of the first hybrid protein and the transcriptional activation domain of the second hybrid protein are derived from transcriptional activators having separate DNAbinding and transcriptional activation domains.. These separate; DNA-binding and transcriptional activation domains are also known to be found in the yeast GAL4 protein, aid are also known to be found in the yeast GCN4 and ADRI proteins. Many other proteins involved in transcription also have separable binding and transcriptional activation domains which make them useful for the present invention. In another embodiment, the DNA-binding i 30 domain and the transcriptional activation domain may be from different transcriptional activators. The second hybrid protein is preferably encoded on a library of plasmids that contain genomic, cDNA or synthetically generated DNA sequences fused to the DNA sequence encoding the transcriptional activation domain.

The drug-mediated interaction between the first test protein and the second test protein in the host cell, therefore, causes the transcriptional activation domain to activate transcription of the detectable gene. The method is carried out by introducing the first chimeric gene and the second chimeric gene into the host cell, and contacting the cell with the drug of interest. The host cell is subjected to conditions under which the first hybrid protein and the second hybrid protein are expressed in sufficient quantity for the detectable gene to be activated. The cells are then tested for drug-dependent expression of the detectable gene.

Thus, interactions between a first test protein and a library of proteins can be tested in the presence of the drug of interest, in order to determine which members of the library are involved in the formation of drug-dependent complexes between the first and second protein.

For example, the bait protein may be a protein which binds FK506, rapamycin, or cyclosporin, e.g. can be an FKBP or cyclophilin. The second test protein may be derived from a cDNA library.

Exemplification The invention now being generally described, it will be more readily understood by reference to the following examples which are included merely for purposes of illustration of 5 certain aspects and embodiments of the present invention, and are not intended to limit the invention.

Example 1 Construction Of The Bait Plasmids For The 2-Hybrid Screen A. LexA-FKBP12 bait: The bait protein and fish protein constructs used in the present drug-dependent interaction trap are essentially the same as constructs used for other 2 hybrid assays (see, for example, U.S. Patent No. 5.283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al.

(1993) J:Biol Chem 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; and 25 Iwabuchi et al. (1993) Oncogene 8:1693-1696). Using the following olignucleotides: coding strand G GGT TTG G A A C CTA ATA ATG TCT GTA CAA GTA GAA ACC (SEQ ID No: 3) non-coding strand GGG TTT CGGGATCC GTC ATT CCA GTT TTA GAA G (SEQ ID No:4) PCR amplification was carried out from a lymphocyte cDNA library to isolated the coding sequence for the FKBPI2 protein. The sequence of the human FKBP12 cloned was confirmed as: .1 1. ATGTCCGTACAAGTAGAAACCATCTCCCCAGGAGACGGGCGCACCTTcCCCA AGCGCGGCCAGACCTGCGTGGTGCACTACACCGGgATGCTTGAGATGGA aACGAGGTCAGCGGA gGT~ CCAGATGAGTGTGG gTCAGCGTGCCA~aCTgACTAtAt CTCcAGaTtATgCcTATGgTGCCACTGG GCAcc CAGGCATCATCCCACCACATGCCACTCTCGTCTTCGATGTGGAGCTT CTAAAACTGGAATGA (SEQ ID The resulting PCR product containing the human FKBPI12 coding sequences was then digested with EcoRJ and BamnHI. and cloned into the EcoRl BainHl sites of pBTMI 116 creating an in-frame fusion between LexA and FKBP 12. The resulting plasmid is referred to below as plC5O4.

A. LexA-(gly) 6 -FKBPJ2 bait: In order to generate an in frame fusion between LexA and FKBP 12 separated by six 1 5 glycine residues, the coding sequence from human FKB3P12 was cloned by PCR as above, except that the sense oligonucleotide provided an additional 18 nucleotides which inserted 6 glycines in the open reading frame of the fusion protein. The oligos used for PCR were: ~Qodinf sztrand 20 TCG CCG GAA TIC GGG GGC GGA GGT GGA GGA GTA CAA GTA GAA ACC ATC (SEQ ID No: 7) nndignp trn GGG TTT CGG GALCC GTC ATT CCA GTT TTA GAA G 25 (SEQ ID No: 8) The PCR product containing the human FKBPI12 coding sequences was then digested with EcoRi and B.HI and cloned into the EcoRi B3amHl sites of pBTM1 116 as above.

The resulting plasrnid is referred to below as plC506.

o* 30 Examk Construction of the FKBPJ2 deletion strain A 1.8 kb Hindill-EcoRi yeast genomic fragment containing FKBI (the S Cerveisia homolog of FKBP 12) was cloned into the HindIll EcoRI sites of pSP72 (Promega) A one-step PCR strategy was used to create a precise deletion of the FKB I coding sequences extending from the ATG start codon to the TGA stop codon. Simultaneously a unique BamHI site was introduced in lieu of the FKB I coding sequences. The oligos used to generate the FKB I deletion and introduction of the unique BamHI site were: CGCGGATCCGCGCATTATTACTTGTTTTGATGATTT

TG

(SEQ ID No: 9)

CGCGGATCCGCGTAAAAGCAAAGTACTATCAATTGAGCCG

(SEQ ID No: The yeast ADE2 gene on a 3.6 kb BamHI fragment was then cloned into the unique BamHI site of the plasmid described above to generate the plasmid pVB172. Flanking the ADE2 disruption marker of pVB172 in the 5' and 3' noncoding sequence of FKBI are Xhol sites. pVB172 was digested with Xhol to release a linear fragment containing ADE2 flanked by FKBI noncoding sequences. This linear fragment was used to transform yeast strain (Mat a his3 A200 trpl-901 leu2-3,112 ade2 LYS2::(lexAop)4-HIS3 URA3::(lexAop) 8 -lacZ GAL4 gal80) selecting for adenine prototrophy.

ADE+ yeast transformants were tested for rapamycin resistance to confirm that the wild type FKB 1 allele was replaced by ADE2. This disruption allele of FKBI is designated L40-fkbl-2.

Example 3 *Cloning OfMammalian Rapamycin Target Genes We used the drug-dependent interaction trap described in Example 1 above, with the 20 LexA binding-domain fusion constructs as bait to detect interaction with clones from cDNA libraries containing VPI6 activation-domain fusions. The reporters used as "read-outs" signaling interaction in this system are the S. cerevisiae HIS3 and the E. coli LacZ genes. The yeast strain L40, the bait vector plasmid pBTM 16 and the mouse embryonic PCR library in the vector pVP16 were used to construct the cDNA fusion protein library The strain L40-fkbl-2, described above in Example 2, was transformed with each of two bait plasmids, plC504, encoding the LexA-FKBP12 fusion protein, or plC506, encoding the LexA-(gly)6-FKBP12 fusion protein. The transformants, L40-fkbl-2/plC504 (named ICY99) and L40-fkbl-2/plC506 (named ICY101) were maintained on yeast media lacking tryptophan which selects for cells harboring the bait plasmid.

A mouse embryo PCR library in pVP16 (designated pSH10.5), which was generated by standard protocols using random-primed synthesis of 10.5 day-post-coital CDI mouse embryo polyA+ RNA and size-selected for inserts between 350bp and 700bp in length, was used to transform the yeast ICY99 and ICY101. The transformed yeast cells were plated onto media lacking tryptophan and leucine. Approximately 107 transformants from each strain were pooled, thoroughly mixed, and stored frozen in aliquots in 50% glycerol at -80 0

C.

1 57 Prior to screening, cells were thawed, grown for 5 hours in liquid medium, and plated onto selective medium. Approximately 1.5xl07 ICY99/pSH10.5 cells were plated onto phosphate-buffered (pH7) synthetic agar medium containing all amino acids except tryptophan, leucine and histidine, (ii) Rapamycin at 125 ng/ml, (iii) the chromogenic substrate X-gal at 100 ng/ml, and (iv) 2% glucose as carbon source, at a plating density of approximately 106 per 15 cm plate. An identical protocol was used for screening ICY101/pSH10.5 transformants. except that a lower concentration of rapamycin was used, at 15.6 ng/ml.

Colonies which both grew on the selective medium and were blue were picked for further testing. These represent cells which do not require hisitidine for growth and which are expressing the P-galactosidase reporter. Candidate colonies appeared between 4-11 days after plating, and the blue color ranged from very light blue to deep blue. They were then subjected to the following tests.

i) Rapamycin-dependence Each candidate was streaked onto media lacking histidine and containing either 125ng/ml (for ICY99/pSH10.5 candidates), 15.6 ng/ml (for ICYI01/pSHIO.5 candidates) rapamycin, or no rapamycin (for both). Candidate clones which grew in the presence of S: rapamycin and failed to grow on media without rapamycin were chosen for the next test.

For the ICY99/pSHIO.5 screen, out of 107 His+ and LacZ+ candidates screened. 24 were rapamycin-dependent for growth on medium lacking hisitidine. For the ICYl01/pSH10.5 screen, 20 out of 101 His+ and LacZ+ candidates screened were Srapamycin-dependent.

ii) plasmid-linkage To eliminate false positives caused by chromosomal mutations, each candidate was 25 grown in non-selective medium (YPD) to permit loss of the bait (Trp+) and the cDNA (Leu+) plasmids. Cells which had lost the bait plasmid the cDNA plasmid (Leu-) or both plasmids (Trp- and Leu-), as well as those which had retained both plasmids (Trp+ and Leu+), were streaked onto media containing rapamycin but lacking histidine. Those candidates for which only the derivatives containing both plasmids (Trp+ and Leu+) grew, while the other three derivatives did not, were chosen for further analysis.

For the ICY99/pSH 10.5 screen. 23 out of 24 passed the test. For the ICYl 0/pSH 10.5 screen, all 20 passed the test.

iii) Positive and negative interaction with control baits 58 Whereas the previous test asked if the interaction disappears when either or both members of the interaction (bait and fish constructs) are lost, the present test asks if the candidate cDNA plasmid (Leu+) can confer interaction when transformed into yeast strains harboring various baits. DNA samples were prepared from each candidate and used to transform E. coli strain B290 (auxotrophic for trptophan and leucine). Since the yeast TRP I and LEU2 genes can complement the bacterial auxotrophies, respectively, B290 cells containing the bait plasmid are Trp+ and can grow on medium lacking tryptophan, while B290 cells containing the cDNA plasmid are Leu+ and can grow on medium lacking leucine.

Plasmid DNA samples were each containing a different bait: i) ICY99, the original strain used in the screen, containing the LexA::FKBP-2 bait fusion; ii) ICY101, containing the LexA::(gly) 6 ::FKBP12 bait fusion, and iii) ICY102, containing a LexA fusion bait irrelevant for the present study and which serves as a negative control. The ideal candidate clone should confer His+ and LacZ+ to ICY99 and ICY101 in a rapamycin-dependent manner, but not to ICY102.

For the ICY99/pSH10.5 screen, 11 out of the 23 candidates fulfilled the above criteria. For the ICYIOl/pSH0.5 screen, 10 out of the 20 candidates fulfilled the above S. criteria.

The cDNA inserts of these candidate clones were sequenced in both strands using the ABI fluorescent sequencing system. All 11 candidates from the ICY99/pSH10.5 screen, and 20 at least 4 out of 10 of the candidates from the ICYl01/pSHlO.5 screen contain overlapping fragments of an identical sequence. The 14 clones represent at least 5 independent cloning events from the library as judged by the insert/vector boundaries of each clone. The longest and the shortest inserts differ by approximately 70 bp at the amino-terminus and about 10 bp at the amino-terminus. The partial nucleotide sequence, and corresponding amino acid sequence, isolated from the mouse rapamycin/FKBP12 binding protein (RAPTI). is given in SEQ ID No: I and SEQ ID No: 2, respectively.

Surprisingly, a search of the GenBank database using the program BLAST, revealed that the peptide encoded by the above sequence shares some homology, though less than percent absolute homology, to the S. cerevisiae TORI (and DRRI) and TOR2 gene products 30 previously isolated from yeast.

Example 4 Cloning of Human Homologs ofRapamycin Target Genes Having isolated a partial sequence for the gene encoding a rapamycin-target-protein from a mouse library, we proceeded to isolate the human gene using the mouse sequence as a probe. The plasmid clone plC99.1.5, containing the longest insert of the RAPTI clone, was chosen as probe for hybridization. The insert (500 bp) was separated from plasmid DNA by digestion with Not I restriction endonuclease followed by agarose gel electrophoresis and fragment purification. The fragment was radiolabelled with aP 3 2 -labeled dCTP by randomincorporation with the Klenow fragment of DNA polymerase. The radiolabelled DNA probe was isolated away from free nucleotides by a G50 column, alkali-denatured, and added to the hybridization mix at 2xl0 6 cpm/ml.

Approximately 3xl06 phage of a human B cell cDNA library in X-pACT (Figure 1) were screened by filter hybridization using the probe described above, in 30% forrnamide, 5X Denhardts, 20 pg/ml denatured salmon sperm DNA, and I SDS, at 37C.

Following hybridization, the filters were washed at 0.5xSSC and 0.1% SDS, at 50 0 C. These represent conditions of medium stringency appropriate for mouse-to-human cross-species hybridizations. A number of positive plaques were obtained, and several were analyzed. A number of the isolated clones turned out to be various 3' fragments of the same gene. or very closely related genes, which, after sequence analysis, was determined to be the human RAPTI gene. The clone containing the longest coding sequence fragment, comprising what is believed to be roughly half the full-length protein (C-terminus) and including the FKBP/rapamycin binding site and the putative PI-kinase acitivity, is designated as plasmid pIC524. A deposit of the pACT plasmid form of pIC524 was made with the American Type Culture Collection (Rockville, MD) on May 27, 1994, under the terms of the Budapest Treaty. ATCC Accession number 75787 has been assigned to the deposit.

9.9o 20 Figure I is a map of the human RAPTI clone of plC524 (inserted at the Xhol site).

The insert is approximately 3.74 kb in length, and nucleotide RAPTI coding sequence from the insert has been obtained and is represented by nucleotide residues 4717-7746 of SEQ ID No. 11. The corresponding amino acid sequence is represented by residues His1541-Trp2549 of SEQ ID No. 12. The region of the human RAPTI clone corresponding to the mouse RAPTI fragment is greater than 95% homologous at the amino acid level and .homologous at the nucleotide level. In addition to the pIC524 clone, further 5' sequence of the human RAPTI gene was obtained from other overlapping clones, with the additional sequence of the 3'end of the -5.4kb partial gene given in SEQ ID No. 11. Furthermore,

SEQ

ID No. 19 provides additional 3' non-coding sequence (obtained from another clone) which 30 flanks the RAPTI coding sequence.

It will be evident to those skilled in the art that, given the present sequence information. PCR primers can be designed to amplify all, or certain fragments of the RAPTI gene sequence provided in pIC524. For example, the primers TGAAGATACCCCACCAA- ACCC (SEQ ID No. 21) and TGCACAGTTGAAGTGAAC (SEQ ID No. 22) correspond to pACT sequences flanking the Xhol site, and can be used to PCR amplify the entire RAPTI sequence from pIC524. Alternatively, primers based on the nucleic acid sequence of SEQ ID No. 11 can be used to amplify fragments of the RAPTI gene in plC524. The PCR primers can be subsequently sub-cloned into expression vectors, and used to produce recombinant forms of the subject RAPTI protein. Thus, the present provides recombinant

RAPTI

proteins encoded by recombinant genes comprising RAPTI nucleotide sequences from ATCC deposit number 75787. Moreover, it is clear that primer/probes can be generated which include even those portion of pIC524 not yet sequenced by simply providing

PCR

primers based on the known sequences.

Furthermore, our preliminary data indicate that other proteins which are related to RAPTI, e.g. RAPTI homologs, were also obtained from the present assay, suggesting that RAPTI is a member of a larger family of related proteins.

Cloning of Novel Human Ubiquitin Conjugating Enzyme Constructs similar to those described above for the drug-dependent interaction trap assay were used to screen a WI38 (mixed Go and dividing fibroblast) cDNA library (Clonetech, Palo Alto CA) in pGADGH (Xhol insert, Clonetech). Briefly, the two hybrid assay was carried out as above, using GAL4 constructs instead of LexA, and in an HF7C 0 yeast cell (Clonetech) in which FKBI gene was disrupted (see Example Of the clones isolated, a novel human ubiquitin-conjugating enzyme (rap-UBC) has been identified.

A

deposit of the pGADGH plasmid (clone "SMR4-15") was made with the American Type Culture Collection (Rovkville, MD) on May 27, 1994, under the terms of the Budapest' Treaty. ATCC Accession number 75786 has been assigned to the deposit. The insert is approximately IkB.

SThe sequence UBC-encoding portion of the SMR4-15 insert is given by SEQ ID No.

S23 (nucleotide) and SEQ ID No. 24 (amino acid). The sequence for the 3' portion of the 25 clone is provided by SEQ ID No. 25. As described above, priniers based on the nucleic acid sequence of SEQ ID No. 23 (and 25) can be used to amplify fragments of the rap-UBC gene from SMR4-15. The PCR primers can be subsequently sub-cloned into expression vectors, and used to produce recombinant forms of the subject enzyme. Thus, the present provides recombinant rap-UBC proteins encoded by recombinant genes comprising rap-UBC nucleotide sequences from ATCC deposit number 75786.

Examle 6 Construction of the Serine-to-Argenine RAPT1 mutation The smallest mRAPTI clone that interacted with the FKBPl2/rapamycin complex was 399 bp. defining a rapamycin binding domain. The RAPTI binding domain corresponds to a region in yeast TORI/TOR2 located immediately upstream, but outside of the lipid

(I

kinase consensus sequence. This region contains the serine residue which when mutated in yeast TORI confers resistance to rapamycin (Cafferkey et al. (1993) Mol Cell Biol 13:6012- 6023). Both a mouse and human RAPTI serine-to-argenine mutation was constructed by oligonucleotide mutagenesis. In the instance of the mRAPTI mutant, coding and noncoding strand oligonucleotides containing the mutations were: GAAGAGGCAAGACGCTTGTAC (SEQ ID NO:26) and GTACAAGCGTCTTGCCTCTTC (SEQ ID NO:27). PCR reactions were performed using these oligonucleotides in combination with oligonucleotides GAGTTTGAGCAGATGTTTA (SEQ ID NO:28) and the M13 universal primer which are sequences in the pVP16 vector, 5' and 3' of the mRAPTI insert, respectively. pVP16 containing mRAPTI was used as the template for PCR. The PCR product, digested with BamHI and EcoRI, was cloned into the BamHI and EcoRI sites in pVP16. The resulting clone was sequenced to verify that the clone contained the serine-to-argenine mutation and no others.

The smallest mRAPTI clone that interacted with the FKBP12/rapamycin complex was 399 bp. defining the RAPTI binding domain. The RAPTI binding domain corresponds to a region in yeast TOR located immediately upstream, but outside of the lipid kinase consensus sequence. This region contains the serine residue which when mutated in yeast TORI (also called DRRI) confers resistance to rapamycin (Cafferkey et al. (1993) Mol. Cell Biol. 13:6012-6023; Helliwell et al. (1994) Mol. Cell Biol. 5:105-118). The corresponding 20 mutation was constructed in mRAPTI. The serine-to-argenine mutation abolishes interaction of mRAPTI with the FKBP12/rapamycin complex (see Figure activating neither HIS3 nor lacZ expression on the two-hybrid assay, indicating that the serine is involved in the association of the FKBP 1 2 /rapamycin complex with mRAPT

S

Examle 7 "Northern Analysis S* The multiple tissue Northern blots (containing 2 jg of human RNA per lane) were obtained from Clonetech Labs., Inc. Hybridizations were at 420C in 5X SSPE, Denhardt's, 30% formamide, 1% SDS and 200 gg/ml denatured salmon sperm DNA. Washes 30 were at 0.1X SSC and 0.1% SDS at 55 0 C. The blot was exposed for 5 days prior to autoradiography. The levels of RNA loaded in each lane were independently monitored by hybridizing the same blots with a human G3PDH probe and were found to be similar in all lanes, with the exception of skeletal muscle, which had approximatelly 2-3 fold the signal.

RAPTI specifies a single transcript ofapproximatelly 9 kb that is present in all tissues examined, exhibiting the highest levels in testis. The transcript is sufficient to encode a protein equivalent to the size of yeast TOR which is 284 kDa. Assuming that RAPTI is of similar size, a small fragment of 133 amino acids has been cloned from within a large protein.

but which fragment is sufficient to bind FKBP12/rapamycin complex.

Example 8 High throughput assay based on the two-hybrid system for identifying novel rapamycin analogs.

To develop a high throughput screen based on the two-hybrid system, we devised a procedure to quantitate protein-protein interaction mediated by a small molecule. Since protein-protein interaction in the two-hybrid system stimulates transcription of the lacZ reporter gene, the assay utilizes a substrate of P-galactosidase (the lacZ gene product lacZ gene product) which when cleaved produces a chemiluminescent signal that can be quantitated. This assay can be performed in microtiter plates, allowing thousands of compounds to be screened per week. The assay includes the following steps: 1. Inoculate yeast cells from a single colony into 50 ml of growth medium, synthetic complete medium lacking leucine and tryptophan (Sherman, F. (1991) Methods Enzymol.

194:3-20). Incubate the flask overnight at 30 0 C with shaking (-200 rpm).

2. Dilute the overnight culture to a final A 600 of 0.02 in growth medium and incubate overnight as described in step 1.

3. Dilute the second overnight culture to a final A600 of 0.5 in growth medium. Using a Quadra 96 pipettor (TomTec, Inc.), dispense 135 p1 aliquots of the cell suspension into wells of a round bottom microtiter plate pre-loaded with 15 pl/well of the compound to be tested at various concentrations. (The compounds are dissolved in 5% dimethyl sulfoxide, so that the final DMSO concentration added to cells is 0.5% which does not perturb yeast cell growth.) Cover microtiter plates and incubate at 30*C for 4 hr with shaking at 300 rpm.

4. Centrifuge microtiter plate for 10 min at 2000 rpm. Remove the supematant with the Quadra 96 pipettor and wash with 225 pl phosphate buffered saline.

5. Dispense 100 pl of lysis buffer (100mM 2

HPO

4 pH 7.8; 0.2% Triton X-100; 1.0 mM ditiothriotol) into each well, cover, and incubate for 30 min at room temperature with shaking at 300 rpm.

6. Dispense into each well of a Microfluor plate (Dynatech Laboratories, Chantilly, VA), pl of the chemiluminescent substrate, Galacton PlusTM (Tropix. Inc.. Bedford. MA) in diluent (100 mM NaHPO 4 1 mM MgCl2, pH To these wells, transfer 20 pl of cell lysate and incubate in the dark for 60 min at room temperature.

63 7. Add to each well 75 pl of EmeralTM accelerator. Cover plate and count in a Topcount scintillation counter (Packard, Inc.) for 0.01 min/well.

The rapamycin target proteins, isolated as described above, were incorporated into the quantitative assay, as was a variety of FKBPs. The FKBPs included in the screen were human FKBP12 and that from pathogenic fungi, FKBP13 (Jin et al. (1991) Proc. Nail. Acad Sci. 88:6677) and FKBP25 (Jin et al. (1992) J Biol. Chem. 267:2942; Galat et al. (1992) Biochem. 31:2427-2434). Yeast strains containing different FKBP-target pairs can be tested against libraries of rapamycin and FK506 analogs. Such a screen can yield different classes of compounds including target-specific compounds, those that mediate interaction between a specific target and more than one FKBP, (ii) FKBP-specific compounds, those that mediate interaction between a particular FKBP and more than one target and, most ideally, (iii) FKBP/target-specific compounds, those that mediate interaction between a particular FKBP and target. The protein interactions mediated by the test compounds and measured in this assay can be correlated with immunosuppressive. antifungal, antiproliferative and toxicity profiles, as well as their Ki's for inhibition of FKBP PPlase activity.

Using the quantitative chemiluminescence assay described above, the interaction of human LexA-FKBP12 and VP16-RAPTI was analyzed in the presence and absence of rapamycin. Interaction between FKBP12 and RAPTI was measured as a function of drug concentration. Addition of rapamycin from 0 to 500 ng/ml increased P-galactosidase activity 20 approximately one thousand-fold. This effect was specific for rapamycin; FK506 over the same concentration range did not increase P-galactosidase activity significantly over background levels. If lexA-da, a control construct, is substituted for the lexA-FKBP12, p- S* galactosidase activity does not increase as a function of rapamycin addition. The basal levels of P-galactosidase in the negative controls are 0.1 per cent of the maximum levels detected in the yeast strain containing the FKBP12 and RAPTI constructs, grown in media containing 500 ng/ml rapamycin. These results, illustrated in Figure 2, indicate that protein interactions mediated by a small molecule in the two-hybrid system can be quantitated and assayed in a microtiter format that can be used for high throughput screening. Employing various FKBPs and RAPT1 proteins in the two-hybrid format (Figure 3) rapamycin-mediated interactions 30 were measured in this quantitative assay.

Example 9 In vitro protein interactions mediated by rapamycin Drug-mediated interactions of FK506-binding proteins and the RAPTI proteins is analyzed in vitro using purified FKBP12 fused to glutathione-S-transferase (GST) and 35

S

labeled RAPTI proteins prepared by in vitro transcription and translation. For this purpose FKBP12 is fused in the frame of GST in pGEX (Pharmacia, Piscataway, NJ). GST-FKBP12 fusion proteins are expressed and purified from E. coli (Vojtek et al. (1993) Cell 74:205-214).

RAPTI coding sequences are cloned behind the CMV and T7 promoters in the mammalian expression vector, pX (Superti-Furga et al. (1991) J. Immunol. Meths. 151:237-244). RAPTI sequences are transcribed from the T7 promoter and translated in vitro using commercially available reagents (Promega, Madison, WI) in a reaction containing 35 S-methionine. For in vitro binding (Toyoshima et al. (1994) Cell 78:67-74), 5 to 20 jl of the in vitro transcription/ translation reactions are added to 200 il of binding buffer (20mM HEPES[pH7.4]. 150 mM NaCI, 10% glycerol, 0.05% NP-40). After addition of 10 gl of GST-FKBPI2 bound to glutathione-agarose beads, the reaction is incubated at 4"C for 2 hr with rotation. Various contrations of drug are added to reactions, such as 0.1 to 10-fold that of FKBP12 on a molar basis. No drug is added to control reactions. The agarose beads are then precipitated and washed four times with binding buffer. Bound proteins iseluted by boiling in Laemmli sample buffer, resolved on 4-20% gradient SDS polyacrylamide gels, and visualized by autoradiography. Detection of 35 S-labelled RAPTI protein from binding reactions containing drug demonstrates direct binding to FKBP12 as a function of drug.

Example Effect of RAPT1 mutations on complex formation and rapamycin sensitivity To more particularly map the rapamycin-binding domain of RAPTI requires the 20 isolation of mutants that fail to bind to a FKBP/rapamycin complex. As described in the Examples above, association with the FKBP/rapamycin can be tested in the LexA two-hybrid system in which FKBP12 is expressed as a fusion to LexA and RAPTI proteins are expressed as fusions to the VP16 activation domain. Accordingly, a library of mutant RAPTI proteins is generated by mutagenizing coding sequences through PCR-generated random mutagenesis (Cadwell and Joyce (1992) PCR Methods Appl 2:28-33). The 5' and 3' oligos for PCR contain BamHl and EcoRI restriction sites, respectively, that allow subsequent cloning of the PCR products into pVP16 creating an in-frame fusion. In addition, the 3' oligo contains a 27 bp HA epitope sequence followed by an in frame stop codon. The addition of the HA epitope tag to the C-terminal end of the fusion proteins allows the characterization of the mutant RAPTI proteins (see below).

Upon completion of the mutagenesis, the EcoR1-BamHI digested PCR products are inserted into pVP16. The library of mutant RAPTI proteins is amplified by transformation into E. coli. To identify those mutations that impair the ability of a RAPT1 to interact with an FKBP/rapamycin complex, the mutagenized RAPTI library is introduced into a yeast strain containing the LexA-FKBP bait protein. Each transformed cell carries one individual mutant RAPTI fused to the transcriptional activator VP16. Interaction between the FKBP and wild type RAPT1 occurs when cells are grown in media containing rapamycin, inducing

II

lacZ expression and turning colonies blue on X-GAL indicator plates. Colonies in which the interaction between an FKBP/rapamycin complex and the RAPTI mutant is impaired are light blue or white. Two classes of mutations can produce this phenotype: nonsense mutations resulting in truncated version of RAPTI or sense mutations that affect the binding of RAPTI to the FKBP/rapamycin complex. To distinguish between these two types of mutations, total protein extracts made from these colonies is subjected to Western blot analysis using an anti-HA antibody. Nonsense mutations that give rise to shorter, truncated proteins do not contain the HA epitope at their C-terminus and thus are not be detected by the anti-HA antibody. Conversely, full-length proteins with an incorporated sense mutations are detected with this antibody.

The library plasmids from the light blue or white colonies that express full-length RAPTI protein with the HA epitope are rescued by retransformation into E Coli. The position of the mutation is determined by sequence analysis, and the phenotype verified by retransformation of these plasmids back into the yeast strain containing LexA-FKBP12.

Mutants that retest can also be cloned into the mammalian expression vector. pX. pX- RAPTI or pX lacking RAPTI sequences, are thenintroduced into the lymphoid (CTLL and Kit225) and nonlymphoid cells (MG63 and RH30) sensitive to rapamycin. The effect of the mutation on rapamycin sensitivity is measured in terms of inhibition of DNA synthesis monitored by BrdU incorporation. Mutants that confer resistance of rapamycin by virtue of 20 being unable to bind to the FKBP12/rapamycin complex indicate which mutations mediate drug sensitivity in lymphoid and nonlymphoid cells.' Of particular interest is whether different RAPT Is mediate drug sensitivity ir different cell types.

Example 11 Cloning of a RAPTI-like pr.lypeptide from Candida albican In order to clone homologs of the RAPTI genes from human pathogen Candida, degenerate oligonucleotides based on the conserved regions of the RAPTI and TOR proteins were designed and used to amplify C albicans cDNA in XZAP (strain 3153A). The amplification consisted of 30 cycles of 94 0 C for 1 minute, 55 0 C for 1 minute and 72*C for I minute with the PCR amplimers GGNAARGCNCAYCCNCARGC and ATNGCNGGRTAYTGYTGDATNTC. The PCR reactions were separated on a 2.5% low melting agarose gel, that identified a sizable fragment. The fragment was eluted and cloned into pCRII (TA cloning system, Invitrogen corporation).

The C albicans DNA probes were 32 p-labeled by nick translation and used on Southern blots to confirm the species identity of the fragments and to further screen C albicans cDNA libraries. Sequencing of the larger cDNAs confirmed the identity of the clones. The partial sequence of a C albicans RAPTI-like polypeptide. with the open-reading frame designated, is provided by SEQ ID Nos. 13 and 14.

All of the above-cited references and publications are hereby incorporated by reference.

Equivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

Page(s) 10S1I are claims pages they appear after the sequence listing 6'7 SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: Mitotix, Inc.

STREET: One Kendall Square, Building 600 CITY: Cambridge STATE: MA COUNTRY: USA POSTAL CODE (ZIP): 02139 TELEPHONE: (617) 225-0001 TELEFAX: (617) 225-0005 (ii) TITLE OF INVENTION: Immunosuppressant Target Proteins (iii) NUMBER OF SEQUENCES: (iv) COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: ASCII (text) (vi) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 08/250,795 FILING DATE: 27-MAY-1994 (vi) PRIOR APPLICATION DATA: S 30 APPLICATION NUMBER: US 08/250,795 FILING DATE: 20-DEC-1994 INFORMATION FOR SEQ ID NO:1: 35 SEQUENCE CHARACTERISTICS: S• LENGTH: 486 base pairs TYPE: nucleic acid STRANDEDNESS: both TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..486 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: CTC ACC CGT CAC AAT GCA GCC AAC AAG ATC TTG AAG AAC ATG TGT GAA 48 Leu Thr Arg His Asn Ala Ala Asn Lys Ile Leu Lys Asn Met Cys Glu 1 5 10 CAC AGC AAC ACG CTG GTC CAG CAG GCC ATG ATG GTG AGT GAA GAG CTG 96 His Ser Asn Thr Leu Val Gln Gln Ala Met Met Val Ser Glu Glu Leu 25 an 6-9 ATT CGiU (TA GCC ATC CTC TGG CAT GAG ATG TGG CAT GAA GGC CTG GAA Ile Arg Val Ala Ile Leu Trp His Glu Met Trp His Giu Gly Leu Giu

GAG

Giu

GAG

Glu

CTG

Leu

GCA

Ala

CTC

Leu

AAG

Lys

GCA

Al a s0

GTG

Val

AAG

Lys

CAA

Gin

ACG

Thr

CAG

Gin 130

TCT

Ser

CTG

Leu

GAA

Glu

GAA

Giu

CAA

Gin 115

CTA

Leu CGC TTG Arg Leu GAG CCC Glu Pro ACA TCC Thr Ser 85 TGG TGT Trp Cys 100 GCC TGG Ala Trp CCC CAG Pro Gin TAC TTT Tyr Phe 55 CTG CAT Leu His '70 TTT AAT Phe Asn CGA AAG Arg Lys GAC CTC Asp Leu CTC ACA Leu Thr 135 40

GGG

Gly

GCT

Aia

CAG

Gin

TAC

Tyr

TAC

Tyr 120

TCC

Ser

GAG

Giu

ATG

Met

GCA

Ala

ATG

Met 105

TAT

Tyr

CTG

Leu

AGG

Arg

ATG

Met

TAT

Tyr 90

AAG

Lys

CAC

His

GAG

Glu

AAC

Asn

GAA

Glu 75

GGC

Gly

TCG

Ser

GTG

Val1

CTG

Leu

GTG

Val

CGG

Arg

CGA

Arg

GGG

Gly

TTC

Phe

CAG

Gin 140

AAA

Lys

GGT

Gly

GAT

Asp

AAC

Asn

AGA

Arg 125

TAT

Tyr

GGC

Gly

CCC

Pro

TTA

Leu

GTC

Val 110

CGG

Axg

GTG

Val

ATG

Met

CGG

Arg

ATG

Met

AAG

Lys

ATC

Ile

TCC

Ser

TTT

Phe

ACT

Thr so

GAG

Giu

GAC

Asp

TCA

Ser

CCC

Pro 192 240 288 336 384 432 AAA CTT CTG ATG TGC CGA GAC CTT GAG TTG GCT GTG CCA GGA ACA TAC *n a .3v Lys 145

GAC

Asp Leu

CCC

Pro Leu Met Cys Arg Asp 1.50 Leu Glu Leu Ala 155 Val Pro Giy Thr INFORMATION FOR SEQ ID NO:2: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 162 amino acids TYPE: amino acid CD) TOPOLOGY: linear 45 (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Leu Thr Arg His Asn Ala Ala Asn Lys Ile Leu LYS Asn Met Cys Giu 50 1 5 10 His Ser Asn Thr Leu Vai Gin Gin Ala Met Met Vai Ser Giu Giu Leu 25 Ile Arg Val Ala Ile Leu Trp His Giu Met Trp His Giu Gly Leu Giu 40 Giu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Vai Lys Gly Met Phe Glu Val Leu Lys Leu Glu Pro Leu His Ala Met Met 70 Glu Gly Arg Gly Pro Arg Glu Thr Ser Phe Asn Gin Ala Arg Asp Leu Met Ala Gin Glu Leu Thr Gin 115 Lys Gin Leu 130 Trp 100 Ala Cys Arg Lys Tyr Ser Gly Asn Trp Asp Leu His Val Phe Val Lys Asp 110 Arg Ile Ser Val Ser Pro Pro Gin Leu Thr 135 Asp Leu Glu Leu Gin 140 Val Lys Leu Leu Met Cys 145 Asp Pro Arg 150 Leu Glu Leu Ala 155 Pro Gly Thr Tyr 160 0 0 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 40 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: GGGTTTGGAA TTCCTAATAA TGTCTGTACA AGTAGAAACC INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 34 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: GGGTTTCGGG ATCCCGTCAT TCCAGTTTTA CAAC INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 348 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 14..325 (xi) SEQUENCE DESCRIPTION: SEQ ID GGAATTCCTA ATA ATG TCC GTA CAA GTA GAA ACC ATC TCC CCA GGA GAC Met Ser Val Gin Val Glu Thr Ile Ser Pro Gly Asp GGG CGC ACC TTC Gly Arg Thr Phe CCC AAG CGC Pro Lys Arg CAG ACC TGC GTG GTG CAC TAC ACC Gin Thr Cys Val Val His Tyr Thr GGG ATG CTT Gly Met Leu GAA GAT GGA AAG AAA Glu Asp Gly Lys Lys 35 TTT GAT TCC TCC Phe Asp Ser Ser CGT GAC CGT AAC Arg Asp Arg Asn

AAG

Lys 45 CCC TTT AAG TTT Pro Phe Lys Phe

ATG

Met 50 CTA GGC AAG CAG Leu Gly Lys Gin

GAG

Glu 55 GTG ATC CGA GGC Val Ile Arg Gly

TGG

Tx-p

S

GAA GAA GGG GTT Glu Giu Gly Val CAG ATG AGT GTG Gin Met Ser Val CAG CGT GCC AAA Gin Arg Ala Lys CTG ACT Leu Thr 193 241 289 ATA TCT CCA GAT TAT GCC TAT GGT Ile Ser Pro Asp Tyr Ala Tyr Gly ACT GGG CAC CCA Thr Gly His Pro GGC ATC ATC Gly Ile Ile CCA CCA CAT GCC ACT CTC GTC Pro Pro His Ala Thr Leu Val TTC GAT GTG GAG CTT Phe Asp Val Giu Leu 100

CTAAAACTGG

335

S..

S

AATGACGGGA TCC INFORMATION FOR SEQ ID NO:6: Ci) SEQUENCE CHARACTERISTICS: CA) LENGTH: 104 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Met Ser Val Gin Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe 1 5 10 Pro Lys Arg Gly Gin Thr Cys Val Val His Tyr Thr Gly Met Leu Glu 25 Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys 40 Phe Met Leu Gly Lys Gin Glu Val Ile Arg Gly Trp Glu Glu Gly Val 50 55 Ala Gin Met Ser Val Gly Gin Arg Ala Lys Leu Thr Ile Ser Pro Asp 70 75 Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala 90 Thr Leu Val Phe Asp Val Glu Leu 100 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 48 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: o TCGCCGGAAT TCGGGGGCGG AGGTGGAGGA GTACAAGTAG AAACCATC 48 INFORMATION FOR SEQ ID NO:8: 40 SEQUENCE CHARACTERISTICS: LENGTH: 34 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid 50 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: GGGTTTCGGG ATCCCGTCAT TCCAGTTTTA GAAG 34 INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 41 base pairs TYPE: nucleic acid C (I 72 STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: CGCGGATCCG CGCATTATTA CTTGTTTTGA TTGATTTTTT G 41 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 40 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid 25 (xi) SEQUENCE DESCRIPTION: SEQ ID CGCGGATCCG CGTAAAAGCA AAGTACTATC AATTGAGCCG INFORMATION FOR SEQ ID NO:11: 3 SEQUENCE CHARACTERISTICS: LENGTH: 7824 base pairs TYPE: nucleic acid STRANDEDNESS: both TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 97..7743 45 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: AAGGCGGGCG GTGGGGCACG GGGCCTGAAG CGGCGGTACC GGTGCTGGCG GCGGCAGCTG 50 AGGCCTTGGC CGAAGCCGCG CGAACCTCAG GGCAAG ATG CTT GGA ACC GGA CCT 114 o Met Leu Gly Thr Gly Pro 1 GCC GCC GCC ACC ACC GCT GCC ACC ACA TCT AGC AAT GTG AGC GTC CTG 162 Ala Ala Ala Thr Thr Ala Ala Thr Thr Ser Ser Asn Val Ser Val Leu 10 s15 CAG CAG TTT GCC AGT GGC CTA AAG AGC CGG AAT GAG GAA ACC AGG GCC 210 Gln Gln Phe Ala Ser Gly Leu Lys Ser Arg Asn Glu Glu Thr Arg Ala t, AAA GCC GCC AAG GAG CTC CAG CAC TAT GTC ACC ATG GAA CTC CGA GAG Lys

ATG

Met

ATT

Ile

ATC

Ile

CGA

Arg

GAC

Asp

ATG

Met 135

CGA

Arg 35 OCA.

Al a

TTC

40 -Phe

TGG

Trp,

GCC

Al a 215

CCT

Pro Al a 40

AGT

Ser

TTT

Phe

TTG

Leu

ATT

Ile

CCA

Pro 120

GCA

Al a

GCC

Al a

GCT

Al a

TTC

Phe

GAC

Asp 200

TGT

Cys

CAG

Gin Al a

CAA

Gin

GAA

Glu

GCC

Ala

GGC

Gly 105

GTT

Val1

GGG

Gly

CTG

Leu

GTC

Val

CAG

Gin 185 ccc Pro

CTG

Leu

TGG

Trp Lys

GAG

Glu

TTG

Leu

ATA

Ile

AGA

Arg

GTC

Val1

GAC

Asp

GAA

Glu

CTG

Leu 170

CAA

Gin

AAA

Lys

ATT

Ile

TAC

Tyr Glu Leu GAG TCT Glu Ser 60 GTT TCC Vai Ser GCT AGC Ala Ser TTT GCC Phe Ala ATG GAA Met Giu ACT TTT Thr Phe 140 TGG CTG Trp Leu 155 GTT CTC Val Leu GTG CAA Val Gin CAG GCC Gin Ala CTC ACA Leu Thr 220 AGG CAC Arg His 235 Gin 45

ACT

Thr

AGC

Ser

CTC

Leu

AAC

Asn

ATG

Met 125

ACC

Thr

GGT

Gly

CGT

Arg

CCC

Pro

ATC

Ile 205

ACC

Thr

ACA

Thr His

CGC

Arg

TCA

Ser

ATA

Ile

TAT

Tyr 110

GCA

Ala

GCT

Al a

GCT

Ala

GAG

Glu

TTC

Phe 190

CGT

Arg

CAG

Gin

TTT

Phe Tyr

TTC

Phe

GAT

Asp

GGA

Gly 95

CTT

Leu 7CC Ser

GAG

Giu

GAC

Asp

CTG

Leu 175

TTT

Phe

GAG

Glu

CGT

Arg

GAA

Glu Val

TAT

Tyr

GCC

Ala 80

GTG

Val

CGG

Arg

AAG

Lys

TAC

Tyr

CGC

Arg 160

GCC

Ala

GAC'.

Asp

GGA

Gly

GAG

Gi u

GAA

Glu 240 Thr

GAC

Asp 65

AAT

Asn

GAA

Glu

AAC

Asn

GCC

Al a

GTG

Val 145

AAT

Asn

ATC

le

AAC

Asn

GCT

Al a

CCG

Pro 225

GCA

Ala Met

CAA

Gin

GAG

Glu

GGT

Gly

CTC

Leu

ATT

Ile 130

GAA

Glu

GAG

Glu

AGC

Ser

ATT

Ile

GTA

Val 210

AAG

Lys

GAG

Glu Glu

CTG

Leu

AGG

Arg

GGG

Gly

CTC

Leu 115

GGC

Gly

TTT

Phe

GGC

Gly

GTC

Val

TTT

Phe 195

CC

Al a

GAG

Clu

AAG

Lys Leu Arg Glu p

AAC

Asn

AAA

Lys

AAT

Asri 100

CCC

Pro

CGT

Arg

GAG

Cl u

CGG

Arg

CCT

Pro 180

GTG

Val1

GCC

Al a

ATG

Met

GGA

Gly

CAT

His

GGT

Gly

GCC

Al a

TCC

Ser

CTT

Leu

GTG

Val1

AGA

Arg 165

ACC

Thr

GCC

Ala*

CTT

Leu

CAG

Gin

TTT

Phe 245

CAC

His

GGC

Gi y

ACC

Thr

A.AT

Asn

CC

Al a

AAG

Lys 150

CAT

His

TTC

Phe

GTG

Val1

CGT

Arg

AAG

Lys 230

GAT

Asp 258 306 354 402 450 498 546 594 642 690 738 786 834 882 GAG ACC TTG GCC AAA GAG AAG GGC ATG AAT CGG GAT CAT CGG ATC CAT Glu Thr Leu Ala Lys Giu Lys Gly Met Asn Arg Asp Asp Arg Ile His 250 255 260 GGA GCC TTG TTG ATC CTT AAC GAG CTG GTC CGA ATC AGC AGC ATG GAG Giy Ala Leu Leu 265 Ile Leu Asn Giu 270 Leu Val Arg Ile Ser Met Giu GGA GAG Gly Giu 280 CGT CTG AGA GAA Arg Leu Arg Giu

GAA

Glu 285 ATG GAA GAA ATC Met Giu Glu Ile

ACA

Thr 290 CAG CAG CAG CTG Gin Gin Gin Leu

GTA

Val 295 CAC GAC AAG His Asp Lys TAC TGC Tyr Cys .300 AAA GAT CTC ATG Lys Asp Leu Met

GGC

Giy 305 TTC GGA AC.A AAA CCT Phe Giy Thr Lys Pro 310 1026 CGT CAC ATT ACC CCC TTC ACC AGT TTC Arg His Ile Thr Pro Phe Thr Ser Phe 315 TCA AAT GCC TTG GTG GGG CTG CTG GGG Ser Asn Aia Leu Vai Gly Leu Leu Gly 330 1 ;9 GCT GTA CAG CCC Ala Val Gin Pro CAG CAG Gin Gin 325 2074 TAC AGC TCT CAC Tyr Ser Ser His CAA GGC CTC Gin Gly Leu 340 CTG GTG GAG Leu Val Glu .1122 ATG GGA TTT Met Gly Phe 345 GGG ACC TCC CCC Giy Thr Ser Pro CCA GCT AAG TCC Pro Ala Lys Ser 1.170 1218 AGC CGG Ser Arg 360 TGT TGC AGA GAC Cys Cys Arg Asp

TTG

Leu 365 ATG GAG GAG AAA Met Glu Glu Lys GAT CAG GTG TGC Asp Gin Vai Cys

CAG

30 Gin 375 TGG GTG CTG AAA Trp Val Leu Lys TGC AGG AAT AGC AAG Cys Arg Asn Ser Lys 380 a a a. a..

a

AAC

Asn 385 TCG CTG ATC CAA Ser Leu Ile Gin 1266 1314 ACA ATC CTT AAT Thr Ile Leu Asn TTG CCC CGC TTG Leu Pro Arg Leu

GCT

Al a 400 GCA TTC CGA CCT Ala Phe Arg Pro TCT GCC Ser Ala 405 TTC ACA GAT ACC CAG TAT Phe Thr Asp Thr Gin Tyr 410 CTC CAA GAT ACC ATG AAC CAT GTC CTA AGC Leu Gin Asp Thr Met Asn His Val Leu Ser 415 '420 TGT GTC AAG Cys Val Lys 425 AAG GAG AAG GAA Lys Giu Lys Giu

CGT

Arg 430 ACA GCG GCC TTC Thr Ala Ala Phe

CAA

Gin 435 GCC CTG GGG Ala Leu Gly 1362 1410 1458 CTA CTT Leu Leu 440 TCT GTG GCT GTG Ser Val Ala Val

AGG

Arg 445 TCT GAG TTT AAG Ser Giu Phe Lys TAT TTG CCT CGC Tyr Leu Pro Arg

GTG

50 Val 455 CTG GAC ATC ATC Leu Asp Ile Ile

CGA

Arg 460 C GCC CTG CCC Ala Ala Leu Pro AAG GAC TTC CC Lys Asp Phe Ala

CAT

His 470 1506 1554 AAG AGG CAG, AAG Lys Arg Gin Lys

GCA

Ala 475 ATG CAG GTG GAC Met Gin Val Asp

GCC

Al a 480 ACA GTC TTC ACT Thr Val Phe Thr 7CC ATC Cys Ile 485 ACC ATG CTG GCT CGA GCA ATG CCC CCA GCC ATC CAG CAG GAT ATC AAG Ser Met Leu Ala Arg Ala Met Gly Pro Giy Ile Gin Gin Asp Ile Lys 1602 490 490 495 GAG CTG CTG Giu Leu Leu 505 GAG CCC ATG CTG Glu Pro Met Leu GCA GTG GGA CTA AGC CCT GCC CTC ACT Ala Val Gly Leu Ser Pro Ala Leu Thr 510 515 1650 GCA GTG CTC TAC GAC CTG AGC Ala Val 520 Leu Tyr Asp Leu Ser 525 CGT CAG ATT CCA Arg Gin Ile Pro ATG CTG TCC CTG Met Leu Ser Leu 545 CAG CTA AAG AAG GAC Gin Leu Lys Lys Asp 530 GTC CTT ATG CAC AAA Val Leu Met His Lys 550 CAA GAT GGG CTA CTG AAA Gin Asp Gly Leu Leu Lys 540 1698 1746 1794 CCC CTT CGC CAC Pro Leu Arg His

CCA

Pro 555 GGC ATG CCC AAG Gly Met Pro Lys CTG GCC CAT CAG Leu Ala His Gln CTG GCC Leu Ala 565 TCT CCT GGC Ser Pro Gly ACT CTT GCC Thr Leu Ala 585

CTC

Leu 570 ACG ACC CTC CCT Thr Thr Leu Pro

GAG

Giu 575 GCC AGC GAT GTG Ala Ser Asp Val GGC AGC ATC Gly Ser Ile 580 GGC C4C TCT Gly His Ser 1842 1890 CTC CGA ACG CTT Leu Arg Thr Leu

GGC

Gly 590 AGC TTT GAA TTT Ser Phe Giu Phe

GAA

Giu 595 CTG ACC Leu Thr 600 CAA TTT GTT CGC Gin Phe Val Arg TGT GCG GAT CAT Cys Ala Asp His CTG AAC AGT GAG Leu Asn Ser Giu 4 S S S. S

S

AAG GAG ATC CGC Lys Giu Ile Arg

ATG

Met 620 GAG GCT GCC CGC Giu Ala Ala Arg

ACC

Thr 625 TGC TCC CGC CTG Cys Ser Arg Leu

CTC

Leu 630 1938 1986 2034 2082 ACA CCC TCC ArC Thr Pro Ser Ile CTC ATC AGT GGC Leu Ile Ser Gly GCT CAT GTG GTT Ala His Val Val AGC CAG Ser Gin 645 ACC GCA GTG 40 Thr Ala Val GGG ATA ACA Gly Ile Thr 45 665

CAA

Gin 650 GTG GTG GCA GAT Val Val Ala Asp GTG CTT Val:Leu 655 AGC AAA CTG CTC GTA GTT Ser Lys Leu Leu Val Val 660 GAT CCT GAC CCT Asp Pro Asp Pro

GAC

Asp 670 ATT CGC TAC TGT Ile Arg Tyr Cys

GTC

Val 675 TTG GCG TCC Leu Ala Ser CTG GAC Leu Asp 680 GAG CGC TTT GAT Giu Arg Phe Asp

GCA

Ala 685 CAC CTG GCC CAG His Leu Ala Gin GAG AAC TTG CAG Giu Asn Leu Gin 2130 2178 2226 2274

GCC

Ala 695 TTG TTT GTG GCT Leu Phe Val Ala AAT GAC CAG GTG Asn Asp Gin Val

TTT

Phe 705 GAG ATC CGG GAG Giu Ile Arg Giu GCC ATC TGC ACT GTG GGC CGA CTC AGT AGC ATG AAC CCT GCC Ala Ile Cys Thr Val Gly Arg Leu Ser Ser Met Asn Pro Aia TTT GTC Phe Val 725 ATG CCT TTC CTG CGC AAG ATG CTC ATC CAG ATT TTG ACA GAG TTG GAG 2322 Met Pro Phe Leu Arg Lys Met Leu Ile Gin Ile Leu Thr Giu Leu Giu 730 735 740 CAC AGT GGG ATT GGA AGA ATC AAA GAG CAG AGT GCC CGC ATG CTG GGG 2370 His Ser Gly Ile Gly Arg Ile Lys Giu Gin Ser Ala Arg Met Leu Gly 745 750 755 CAC CTG GTC TCC AAT GCC CCC CGA CTC ATC CGC CCC TAC ATG GAG CCT 2418 His Leu Val Ser Asn Ala Pro Arg Leu Ile Arg Pro Tyr Met Glu Pro 760 765 770 ATT CTG AAG GCA TTA ATT TTG AAA CTG AAA GAT CCA GAC CCT GAT CCA 2466 Ile Leu Lys Ala Leu Ile Leu Lys Leu Lys Asp Pro.Asp Pro Asp Pro.

775 780 785 '190 AAC CCA GGT GTG ATC AAT AAT GTC CTG GCA ACA ATA GGA GAA TTG GCA 2514 Asn Pro Gly Val Ile Asn Asn Vai Leu Ala Thr Ile Gly Glu Leu Ala 795 800 805 CAG GTT AGT GGC CTG GAA ATG AGG AAA TGG GTT GAT GAA CTT TTT ATT 2562 Gin Vai Ser Giy Leu Giu Met Arg Lys Trp Val Asp Giu Leu Phe Ile 810 815 820 ATC ATC ATG GAC ATG CTC CAG GAT TCC TCT TTG TTG GCC AAA AGG C-AG 2610 Ile Ile Met Asp Met Leu Gin Asp Ser Ser Leu Leu Ala Lys Arg Gin 825 830 835 GTG GCT CTG TGG ACC CTG GGA CAG TTG GTG GCC AGC ACT GGC TAT GTA 2658 30...3 Vai Ala Leu Trp Thr Leu Gly Gin Leu Val Ala Ser Thr Gly Tyr Val *840 845 850 *GTA GAG CCC TAC AGG AAG TAC CCT ACT TTG CTT GAG GTG CTA CTG AAT 2706 Vai Giu Pro Tyr Arg Lys Tyr Pro Thr Leu Leu Giu Val Leu Leu Asn 855 860 865 870 *TTT CTG AAG ACT GAG CAG AAC CAG GGT ACA COC AGA GAG GCC ATC CGT 2754 Phe Leu Lys Thr Giu Gin Asn Gin Gly Thr Arg Arg Giu Ala Ile Arg -875 S80 885 GTG TTA GGG CTT TTA GGG GCT TTG GAT CCT TAC AAG CAC AAA GTG AAC 2802 Val Leu Gly Leu Leu Gly Ala Leu Asp Pro Tyr Lys His Lys Val Asn 890 895 900 AT' GGC ATG ATA GAC CAG TCC CGG GAT GCC TCT GCT GTC AGC CTG TCA 2850 I:..le Giy Met Ile Asp.Gin Ser Arg Asp Ala Ser Ala Val Ser Leu Ser 905 910 915 ~.:GAA TCC AAG TCA AGT CAG GAT TCC TCT GAC TAT AGC ACT ACT GAA ATG 2898 Giu Ser Lys Ser Ser Gin Asp Ser Ser Asp Tyr Ser Thr Ser Giu Met 920 925 930 *CTG GTC AAC ATG GGA AAC TTG CCT CTG CAT GAG TTC TAC CCA GCT GTG 2946 Leu Val Asn Met Cly Asn Leu Pro Leu Asp Giu Phe Tyr Pro Ala Val 935 940 945 950 TCC ATG GTG CCC CTG ATG CGG ATC TTC CCA GAC GAG TCA CTC TCT CAT 2994 Ser Met Vai Ala Leu Met Arg Ile Phe Arg Asp Gin Ser Leu Ser His CAT CAC ACC His His Thr GGA CTC AAA Gly Leu Lys 985 AAT GTC ATT Asn Val Ile 1000 CAG CTG GGA Gin Leu Gly 1015 ATG GAT GAA Met Asp Giu TCA ATT CAG

ATG

Met 970

TGT

Cys

CGA

Arg

ATG

Met

ATA

Ile

AGC

955 GTT GTC CAG GCC Val Vai Gin Ala GTG CAG TTC CTG Val Gin Phe Leu 990 GTC TGT GAT GGG Val Cys Asp Gly 1005 TTG GTG TCC TTT Leu Val Ser Phe 1020 GTC ACC CTC ATG Val Thr Leu Met 1035 ACG ATC AT? CTT 77 960 ATC ACC TTC ATC TTC AAG Ile Thr Phe Ile Phe Lys 975 980 CCC CAG GTC ATG CCC ACG Pro Gin Val Met Pro Thr 995 GCC ATC CGG GAA TTT TTG Ala Ile Arg Giu Phe Leu 1010 GTG AAG AGC CAC ATC AGA Vai Lys Ser His Ile Arg 1025 AGA GA.A TC TGG GTC ATG Arg Glu Phe Trp Val Met 1040 CTC AT? GAG CAA AT? GTG Leu Ile Giu Gin Ile Val 965 TCC CTG Ser Leu TC CTT Phe Leu TTC CAG Phe Gin CC? TAT Pro Tyr 1030 AAC ACC Asn Thr 1045 GTA GCT Val Ala 3042' 3090 3138 3186 3234 3282 Ser Ilie Gin Ser Thr Ile Ile Leu 1050 1055 1060 S S 5* S CT? GGG GGT GAA TTT AAG CTC TAC CTG CCC CAG CTG ATC CCA CAC ATG Leu Gly Gly Giu Phe Lys Leu Tyr Leu Pro Gin Leu Ile Pro His Met 1065 1070 1075 30 CTG CGT GTC TTC ATG CAT GAC AAC AGC CCA GGC CGC AT? GTC TCT ATC Leu Arg Vai Phe Met His Asp Asn Ser Pro Gly Arg Ile-Val Ser Ile- 1080 .1085 .1090 AAG TA CTG GCT GCA ATC CAG CTG TT GGC GCC AAC CTG CAT GAC TAC Lys Leu Leu Ala Ala Ile Gin Leu Phe Gly Ala Asn Leu Asp Asp Tyr 1095 1100 1105 1110 CTG CAT TTA CTG CTG CCT: CC? AT? GTT AAG-'TG TTT GA? GCC CCT GAA 40 Leu His Leu Leu Leu Pro Pro Ile Val Lys Leu Phe Asp Ala Pro Giu 1115 1120 1125 GCT CCA CTG CCA TCT CGA AAG GCA GCG CTA GAG ACT GTG GAC CGC CTG Ala Pro Leu Pro Ser Arg Lys Ala Ala Leu Giu Thr Val Asp Arg Leu 45 1130 1135 1140 ACG GAG TCC CTG GAT TTC ACT GAC TAT GCC TCC CGG ATC AT? CAC CC? Thr Giu Ser Leu Asp Phe Thr Asp Tyr Ala Ser Arg Ile Ile His Pro 1145 1150 1155 3330 3378 3426 3474 3522 3570 AT? GT? CGA ACA CTG Ile Val Arg Thr Leu 1160 GAC ACG CTG TCT TCA Asp Thr Leu Ser Ser 1175 GAC CAG AGC Asp Gin Ser 1165 CT? GT? 7?? Leu Val Phe 1180 CCA GAA Pro Giu CAG CTG Gin Leu CTG CGC TCC LeU Arg Ser 1170 GGG AAG AAG Gly Lys Lys 1185

ATG

Met

AT?

Ile 1190 3618 3666 11 .1 TTC ATT CCA Phe Ile Pro CAG CGC TAT Gin Arg Tyr ATG GTG AAT Met Vai Asn 1195 GAT GTG CTC Asp Val Leu 1210 79 AAA GTT CTG GTG CGA CAC Lys Val Leu Val Arg His 1200 CGA ATC AAT CAT Arg Ile Asn His 1205 ATC TGC AGA ATT Ile Cys Arg Ile 1215 GTC AAG GGA TAC ACA CTT Val Lys Gly Tyr Thr Leu 1220 37a4 3762 3810 GCT GAT GAA GAG Ala Asp Giu Giu 1225 GAG GAT CCT TTG ATT Glu Asp Pro Leu Ile 1230 TAC CAG CAT Tyr Gin His CGG ATG CTT AGG Arg Met Leu Arg 1235 AGT GGC CAA GGG Ser Giy Gin Gly 1240 GAT GCA TTG GCT AGT GGA Asp Ala Leu Ala Ser Giy 1245 CCA GTG GAA ACA GGA CCC Pro Val Glu Thr Giy Pro 1250 3858 ATG AAG AAA CTG CAC GTC AGC ACC ATC A.AC CTC CAA AAG GCC TGG GOC Met Lys Lys Leu His Val Ser Thr Ile Asn Leu Gin Lys Ala Trp Gly 1255 1260 1265 1270 3906 GCT GCC AGG AGG GTC TCC AAA GAT Aia Ala Arg Arg Val Ser Lys Asp 1275 GAC TGG CTG Asp Trp Leu 1280 GAA TGG CTG AGA CGG Giu Trp, Leu Arg Arg 1285 CTG AGC CTG Leu Ser Leu GAG CTG Giu Leu 1290 CTG AAG GAC TCA TCA TCG CCC TCC Leu Lys Asp Ser Ser Ser Pro Ser 1295 CTG CGC TCC Leu Arg Ser 1300 3954 4002 4050 4098 TGC TGG GCC CTG GCA 30 Cys Trp Ala Leu Ala 1305 CAG GCC TAC AAC CCG ATG Gin Ala Tyr Asn Pro Met 1310 9 9 0 0900 0@ 90 9

S

0*000@ 0 GCC AGG GAT CTC TTC Ala Arg Asp Leu Phe 1315 AAT GCT GCA Asn Ala Ala 35 1320 GAT GAG CTC Asp Giu Leu 133.5 GCT GAA GTC Ala Giu Val AGT GAC AAG Ser Asp Lys TTT GTG TCC Phe Val Ser ATC AGA AGC Ile Arg Ser 1340 ACA CAG ACC Thr Gin Thr 1355 GGC CCC CTG Gly Pro Leu 1370 TGC TGG-TCT GAA CTG AAT GAA GAT CAA CAG Cys Trp Ser Glu Leu Asn Giu Asp Gin Gin 1325 1330 ATC GAG TTG GCC CTC ACC TCA Ile Giu Leu Ala Leu Thr Ser 1345 CTC TTA AAC TTG GCT GAA TTC Leu Leu Asn Leu Ala Giu Phe 1360 CCA CTG AGA GAT GAC AAT GGC Pro Leu Arg Asp Asp Asn Gly 1375 CAA GAC ATC Gin Asp Ile 1350 ATG GAA CAC Met Giu His 1365 ATT GTT CTG Ile Vai Leu 1380 4146 4194 4242 CTG OCT GAG AGA GCT 50 Leu Gly Giu Arg Ala 1385 TAC AAA GAA CTG GAG Tyr Lys Giu Leu Giu 1400 GCC AAG TGC CGA GCA Ala Lys Cys Az-g Ala 1390 TTC CAG AAA GGC CCC Phe Gin Lys Gly Pro 1405 TAT GCC AAA GCA CTA CAC Tyr Ala Lys Ala Leu His 1395 ACC CCT GCC ATT CTA GAA Thr Pro Ala Ile Leu Giu 1410 4290 4338 TCT CTC ATC AGC ATT AAT AAT AAG CTA CAG CAG CCG GAG GCA GCG GCC Ser Leu Ilie Ser Ile Asn Asn Lys Leu Gin Gin Pro Giu Ala Ala Ala 4386 1415 2420 1425 2430 GGA GTG TTA GAA TAT GCC ATG AAA CAC TTT GGA GAG CTG GAG ATC CAG Gly Val Leu Glu Tyr Ala Met Lys His Phe Gly Giu Leu Glu Ile Gin 1435 1440 1445 4434 GCT ACC TGG TAT GAG AAA CTG CAC GAG TGG GAG Ala Thr Trp Tyr Glu Lys Leu His Glu Trp Glu GAT GCC CTT GTG GCC Asp Ala Leu Val Ala 1460 1450 1455 4482 4530 TAT GAC AAG AAA Tyr Asp Lys Lys 1465 GGC CGC ATG CGC Gly Arg Met Arg 1480

ATG

Met GAC ACC AAC AAG GAC GAC CCA GAG CTG ATO CTG Asp Thr Asn Lys Asp Asp Pro Glu Leu Met Leu 1470 1475 TGC CTC GAG GCC TTG GGG GAA TGG GGT CAA CTC CAC Cys Leu Glu Ala Leu Gly Glu Trp Gly Gin Leu His 1485 1490 4578 CAG CAG Gln Gin 1495 TGC TGT GAA Cys Cys Glu AAG TGG ACC CTG GTT Lys Trp Thr Leu Val 1500 AAT GAT GAG Asn Asp Glu 1505 AAG ATG GCC CGG Lys Met Ala Arg ATG GCT GCT Met Ala Ala 1515 GCA OCT GCA TGG GGT TTA Ala Ala Ala Trp Gly Leu 1520 ACC CAA GCC Thr Gin Ala 1510 GGT CAG TGG Gly Gin Trp 1525 ACC CAT GAT Thr His Asp 1540 4626 4674 GAC AGC ATG GAA GAA TAC ACC TOT ATO ATC CCT CGG GAC Asp Ser Met Glu Glu Tyr Thr Cys Met Ile Pro Arg Asp 1530 1535 0ff 0S es. OS e O bO 0* 0 SOS S *50000

S

55 *5 0*5555 0

SS

S

*5*5

S

0S@*SO

S

*5@5 J S.

S

GGG GCA TTT TAT Gly Ala Phe Tyr 1545 AGA GCT GTG Arg Ala Val CTG GCA CTG CAT Leu Ala Leu His 1550 CAG GAC CTC TTC TCC Gin Asp Leu Phe Ser 1555 4722 4770 4818 TTG GCA CAA Leu Ala Gin 1560 CAG TGC ATT Gin Cys Ile GAC AAG Asp Lys 1565 GCC AGG GAC Ala Arg Asp CTG CTG Leu Leu 1570 GAT GCT GAA Asp Ala Glu TTA ACT 40 Leu Thr 1575 GCA ATG GCA Ala Met Ala GGA GAG AGT Gly Giu Ser 1580 TAC AGT CGG GCA Tyr Ser Arg Ala 1585 TAT GGG GCC ATG Tyr Gly Ala Met 1590 ATC CAG TAC AAA Ile Gln Tyr Lys 1605 GTT TCT TGC Val Ser Cys CAC ATG CTG His Met Leu 1595 TCC GAG CTG Ser Glu Leu GAG GAG GTT Glu Glu Val 1600 4866 4914 4962 5010 OTT GTC CCC GAG CGA CGA GAG ATC Leu Val Pro Glu Arg Arg Olu Ile 1610 ATC CGC CAG Ile Arg Gin 1615 ATC TGG TGG GAG AGA Ile Trp Trp Giu Arg 1620 CAG AAA ATC CTT ATG Gin Lys Ile Leu Met 1635 CTG CAG GGC TGC CAG CGT Leu Gin Gly Cys Gin Arg 1625 GTG CGG TCC CTT GTG GTC Val Arg Ser Leu Val Val 1640 ATC GTA GAG GAC TGG Ile Val Giu Asp Trp 1630 AGC CCT CAT GAA GAC ATG AGA ACC TGG CTC Ser Pro His Glu Asp Met Arg Thr Trp Leu 5058 1645 1650 AAG TAT GCA AGC CTG TGC GGC AAG AGT GGC AGG CTG GCT CTT GCT CAT 5106 Lys Tyr Ala Ser Leu Cys Gly Lys Ser Gly Arg Leu Ala Leu Ala His 16s5 1660 1665 1670 AAA ACT TTA GTG TTG CTC CTG GGA GTT GAT CCG TCT CGG CAA CTT GAC 5154 Lys Thr Leu Val Leu Leu Leu Gly Val Asp Pro Ser Arg Gin Leu Asp 1675 1680 1685 CAT CCT CTG CCA ACA GTT CAC CCT CAG GTG ACC TAT GCC TAC ATG AAA 5202 His Pro Leu Pro Thr Val His Pro Gin Val Thr Tyr Ala Tyr Met Lys 1690 1695 1700 AAC ATG TGG AAG AGT GCC CGC AAG ATC GAT GCC TTC CAG CAC ATG CAG 5250 Asn Met Trp Lys Ser Ala Arg Lys Ile Asp Ala Phe Gin His Met Gin 1705 1710 1715 CAT TTT GTC CAG ACC ATG CAG CAA CAG GCC CAG CAT GCC ATC GCT ACT 5298 His Phe Val Gin Thr Met Gin Gin Gin Ala Gin His Ala Ile Ala Thr 1720 1725 1730 GAG GAC CAG CAG CAT AAG CAG GAA CTG CAC AAG CTC ATG GCC CGA TGC 5346 Glu Asp Gin Gin His Lys Gin Glu Leu His Lys Leu Met Ala Arg Cys 1735 1740 1745 1750 TTC CTG AAA CTT GGA GAG TGG CAG CTG AAT CTA CAG GGC ATC AAT GAG 5394 Phe Leu Lys Leu Gly Glu Trp Gin Leu Asn Leu Gin Gly Ile Asn Glu 1755 1760 1765 AGC ACA ATC CCC AAA GTG CTG CAG TAC TAC AGC GCC GCC ACA GAG CAC 5442 Ser Thr Ile Pro Lys Val Leu Gin Tyr Tyr Ser Ala Ala Thr Glu His 1770 1775 1780 GAC CGC AGC TGG TAC AAG GCC TGG CAT GCG TGG GCA GTG ATG AAC TTC 5490 Asp Arg Ser Trp Tyr Lys Ala Trp His Ala Trp Ala Val Met Asn Phe 1785 1790 1795 GAA GCT GTG CTA CAC TAC AAA CAT CAG AAC CAA GCC CGC GAT GAG AAG 5538 ***Glu Ala Val Leu His Tyr Lys His Gin Asn Gin Ala Arg Asp Glu Lys 1800 1805 181o AAG AAA CTG CGT CAT GCC AGC GGG GCC AAC ATC ACC AAC GCC ACC ACT 5586 Lys Lys Leu Arg His Ala Ser Gly Ala Asn Ile Thr Asn Ala Thr Thr es1815 1820 1825 1830 GCC GCC ACC ACG GCC GCC ACT GCC ACC ACC ACT GCC AGC ACC GAG GGC 5634 Ala Ala Thr Thr Ala Ala Thr Ala Thr Thr Thr Ala Ser Thr Glu Gly 1835 1840 1845 AGC AAC AGT GAG AGC GAG GCC GAG AGC ACC GAG AAC AGC CCC ACC CCA 5682 ser Asn Ser Glu Ser Glu Ala Glu Ser Thr Glu Asn Ser Pro Thr Pro 1850 1855 1860 TCG CCG CTG CAG AAG AAG GTC ACT GAG GAT CTG TCC AAA ACC CTC CTG 5730 Ser Pro Leu Gin Lys Lys Val Thr Glu Asp Leu Ser Lys Thr Leu Leu 1865 1870 1875 ATG TAC ACG GTG CCT GCC GTC CAG GGC TTC TTC CGT TCC ATC TCC TTG 5778 Met Tyr Thr Val Pro Ala Val Gin Gly Phe Phe Arg Ser Ile Ser Leu 1880 8801885 1890 TCA CGA GGC Ser Arg Gly 1895 TTT GAT TAT Phe Asp Tyr AAC AAC CTC CAG GAT Asn Asn Leu Gin Asp 1900 ACA CTC AGA Thr Leu Arg 1905 GTT CTC ACC TTA TGG Val Leu Thr Leu Trp 1910 GCC TTA GTG GAG GGG Ala Leu Val Glu Giy 1925 GTG AAA GCC Val Lys Ala GGT CAC TGG Gly His Trp 1915 ATC CAG ATT Ile Gin Ile 1930 CCA GAT GTC Pro Asp Val AAT GAG Asn Glu 1920 5826 5874 5922 GAT ACC TGG CTA CAG GTT ATA Asp Thr Trp Leu Gin Val Ile 1935 CC7 CAG CTC Pro Gin Leu 1940 ATT GCA AGA ATT Ile Ala Arg Ile 1945 CAG CTT CTC ACA Gin Leu iLeu Thr 1960 GAT ACG CCC AGA CCC TTG Asp Thr Pro Arg Pro Leu 1950 GTG GGA CGT CTC ATT CAC Val Gly Arg Leu Ile His 1955 5970 6018 GAC ATT GGT CGG Asp Ile Gly Arg 1.965 TAC CAC CCC CAG GCC CTC ATC TAC Tyr His Pro Gin Ala Leu Ile Tyr 1970 CCA CTG Pro Leu 19-75 ACA GTG GCT Thr Val Ala TCT AAG TCT Ser Lys Ser 1980 ACC ACG ACA GCC CGG CAC AAT Thr Thr Thr Ala Arg His Asn 1985

GCA

Ala 2990 6066 GCC AAC AAG ATT Ala Asn Lys Ile CTG AAG Leu Lys 1995 AAC ATG TGT Asn Met Cys GAG CAC AGC Giu His Ser 2000 CAG CAG GCC ATG ATG Gin Gin Ala Met Met 2010 GTG AGC GAG Val Ser Giu GAG CTG Glu Leu 2015 ATC GA Ile Arg AAC ACC CTG GTC Asn Thr Leu Val 2005 GTG GCC ATC CTC Val Ala Ile Leu 2020 TCT CGT TTG TAC Ser Arg Leu Tyr 2035 CTG GAG CCC-.TTG .eu Giu Pro Leu 6114 6162 6210 6258 TGG CAT GAG ATG Trp His Giu Met 2025 TGG CAT GAA Trp His Giu GGC CTG Gly Leu 2030 GAA GAG GCA Giu Giu Ala TTT GAG GTG Phe Giu Val 2050 TTT'GGG GAA 40 Phe Gly Giu 2040 AGG AAC GTG Arg Asn Val AAA GGC ATG Lys Gly Met 2045 CAT GCT His Ala 2055 ATG ATG GAA Met Met Giu CGG GGC CCC Arg Gly Pro 2060 CAG ACT CTG AAG Gin Thr Leu Lys 2065 GAA ACA TCC TTT Giu Thr Ser Phe 2070 6306 AAT CAG GCC Asn Gin Ala AAG TAC ATG Lys Tyr Met TAT GGT CGA Tyr Gly Arg 2075 AAA TCA GGG Lys Ser Gly 2090 GAT TTA ATG Asp Leu Met GAG GCC Giu Ala 2080 CAA GAG TGG Gin Giu Trp TGC AGG Cys Arg 2085 AAT GTC AAG GAC CTC Asn Val Lys Asp Leu 2095 CGA CGA ATC TCA AAG Arg Arg Ile Ser Lys 2110 ACC CAA GCC TGG GAC Thr Gin Ala Trp, Asp 2100 CAG CTG CCT CAG CTC Gin Leu Pro Gin Leu 2115 6354 6402 6450 CTC TAT TAT CAT GTG TTC Leu Tyr Tyr His Val Phe 2105 ACA TCC TTA GAG CTG CAA TAT GTT Thr Ser Leu Glu Leu Gin Tyr Val 2120 2125 GAC CTT GAA TTG GCT GTG CCA GGA Asp Leu Glu Leu Ala Val Pro Gly 2135 2140 TCC CCA AAA CTT CTG ATG TGC CGG Ser Pro Lys Leu Leu Met Cys Arg 2130 ACA TAT GAC CCC AAC CAG CCA ATC Thr Tyr Asp Pro Asn Gin Pro Ile 2145 2150 6498 6546 ATT CGC ATT Ile Arg Ile CAG AGG CCC Gin Arg Pro CAG TCC ATA GCA Gin Ser Ile Ala 2155 CCG TCT TTG CAA Pro Ser Leu Gin 2160 GTC ATC ACA TCC AAG Vai Ile Thr Ser Lys 2165 AAC GGA CAT GAG TTT Asn Giy His Giu Phe 21.80 CGG AAA Arg Lys 2170 TTG ACA CTT Leu Thr Leu ATG GGC AGC Met Gly Ser 2175 GTT TTC CTT CTA Val Phe Leu Leu 2185 ATG CAG CTC TTC Met Gin Leu Phe 2200 AAA GGC CAT Lys Gly His GAA GAT Giu Asp 2190 CTG CGC CAG Leu Arg Gin GAT GAG CGT GTG Asp Giu Arg Val 2195 6594 6642 6690 6738 6786 6834 GGC CTG GTT AAC Gly Leu Val Asn 2205 ACC CTT CTG Thr Leu Leu GCC AAT Ala Asn 2210 GAC CCA ACA Asp Pro Thr ATC CCT TTm Ile Pro Leu 2230 TCT CTT Ser Leu 2215 CGG AAA AAC A-rg Lys Asn CTC AGC Leu Ser 2220 ATC CAG AGA Ile Gin Arg TAC GCT GTC Tyr Ala Val 2225 TCG ACC AAC TCG Ser Thr Asn Ser GGC CTC Gly Leu 2235 ATT GGC TGG Ile Gly Trp GTT CCC Val Pro 2240 CAC TGT GAC His Cys Asp ACA CTG Thr Leu 2245 a a *aa.

CAC GCC CTC His Ala Leu ATC CGC Ile Arg 2250 GAC TAC AGG Asp Tyr Arg GAG AAG Ciu Lys 2255 AAG AAG ATC Lys Lys Ile CTT CTC AAC Leu Leu Asn 2260 ATC GAG CAT CC Ile Giu His Arg 2265 ATC ATG TTG Ile Met Leu CCC ATG Arg Met 2270 GCT CCG GAC Ala Pro Asp TAT GAC CAC TTG Tyr Asp His Leu 2275.

ACT CTG ATG Thr Leu Met 2280 CAG AAG GTG Gin Lys Val GAG GTG Ciu Val 2285 TTT GAG CAT Phe Giu His GCC GTC Ala Val 2290 AAT AAT ACA Asn Asn Thr 6882 6930 6978 7026 7 074 7122 45 GCT GGC Ala Gly 2295 GAC GAC CTG Asp Asp Leu GCC AAG Ala Lys 2300 CTG CTG TGG Leu Leu Trp CTG AAA Leu Lys 230.5 ACC CCC AGC Ser Pro Ser

TCC

Ser 2310 GAG GTG TGC TTT GAC CGA AGA Giu Vai Trp Phe Asp Arg Arg 2315 ATG TCA ATG CTT CCC TAT ATT Met Ser Met Vai Gly Tyr Ile 2330 ACC AAT TAT ACC CCT TCT TTA CC GTC Thr Asn Tyr Thr Arg Ser Leu Ala Val 2320 2325 TTA GGC CTC GCA GAT AGA CAC CCA TCC Leu Cly Leu Cly Asp Arg His Pro Ser 2335 2340 AAC CTG ATG CTC GAC CGT CTG ACT GGG AAC ATC CTC CAC ATT GAC TTT Asn Leu Met Leu Asp Arg Leu Ser Gly Lys Ile Leu His Ile Asp Phe 71-70D 83 2345 2350 2355 GGG GAC TGC Gly Asp Cys 2360 TTT GAG GTT GCT ATG Phe Giu Val Ala Met 2365 ACC CGA GAG AAG TTT CCA GAG AAG Thr Arg Glu Lys Phe Pro Giu Lys 2370 ATT CCA Ile Pro 2375 TTT AGA CTA Phe Arg Leu ACA AGA Thr Arg 2380 ATG TTG ACC Met Leu Thr AAT GCT Asn Ala 2385 ATG GAG GTT Met Glu Val

ACA

Thr 2390 7218 7266 7314 7362 GGC CTG GAT GGC Gly Leu Asp Gly AAC TAC Asn Tyr 2395 AGA ATC ACA Arg Ile Thr TGC CAC Cys His 2400 ACA GTG ATG Thr Val Met GAG GTG Glu Val 2405 -CTG CGA GAG Leu Arg Giu CAC AAG His Lys 2410 GAC AGT GTC ATG GCC GTG CTG Asp Ser Val Met Ala Val Leu 2415 GAA GCC TTT GTC Glu Ala Phe Val 2420 TAT GAC CCC TTG Tyr Asp Pro Leu 2425 CTG AAC TGG Leu Asn Trp AGG, CTG ATG Arg Leu Met 2430 GAC ACA AAT ACC AAA GGC Asp Thr Asn Thr Lys Gly 2435 AAC AAG CGA Asn Lys Arg 2440 TCC CGA ACG Ser Arg Thr AGG ACG Arg Thr 2445 GTC GAA Val Giu 2455 ATT TTG GAC Ile Leu Asp GGT GTG GAA Gly Val Glu 2460 GAT TCC TAC TCT Asp Ser Tyr Ser 2451 CTT GGA GAG CCA Leu Gly Glu Pro 2465 ATT CAT TCT TTC Ile His Ser Phe 2480 GCT GGC CAG TCA Ala Gly Gin Ser 0 GCC CAT AAG AAA Aia His Lys Lys 2470 ACG GGG ACC ACA GTG CCA GAA TCT Thr Gly Thr Thr Val Pro Glu Ser .2475 ATT GdA Ile Gly GAC GGT Asp Gly 2485 '7410 7458 7506 7554 7602 7698 TTG GTG AAA 1.eu Val Lys CCA GAG Pro Glu 2490 GCC CTA AAT AAG AAA Ala Leu Asn Lys Lys 2495 GCT ATC CAG Ala Ile Gin ATT AT? AAC Ile Ile Asn 2500 AGG GTT CGA GAT 40 Arg Val Arg Asp 2505 TTG GAT OTT CCA Leu Asp Val Pro 45 2520 AAO CTC ACT Lys Leu Thr G COG GAC Gly Arg Asp 2510 TTC TCT--CAT GAT GAC ACT Phe Ser His Asp Asp Thr 2515 ACO CAA GTT GAG Thr Gin Val Oiu 2525 CTG CTC ATC Leu Leu Ile AAA CAA Lys G In 2530 GCG ACA TCC Ala Thr Ser CAT GAA AAC His Giu Asn 2535 CTC TOC CAG TGC TAT Leu Cys Gin Cys Tyr 2540 ATT GGC TOG TGC CC? TTC TGG Ile Gly Trp, Cys Pro Phe Tr-p 2545 7743 TAACTGGAGG CCCAGATGTG CCCATCACGT TTTTTCTGAG GCTTTTGTAC

TTTAGTAAAT

GCTTCCACTA AACTGAAAAA A 7803 7824 INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 2549 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE Met Leu Gly Thr Gly DESCRIPTION: SEQ ID NO:12: Pro Ala Ala Ala Thr Thr Ala Ala Thr Thr Ser 1 Ser Asn Thr Asp Asn Glu ;sn Ala Val 145 ."Asn 40 Ile 45 Asn Ala Pro 225 Ala a Asn Glu Met Gin Glu Gly Leu Ile 130 Glu Glu Ser Ile Val 210 Lys 3lu Val Glu Glu Leu Arg Gly Leu 115 Gly Phe Gly Val Phe 195 Ala Glu Lys Ser Val Thr Arg Leu Arg Asn His Lys Gly 85 Asn Ala 100 Pro Ser Arg Leu Glu Val Arg Arg 165 Pro Thr 180 Val Ala Ala Leu Met Gln Gly Phe 245 Leu Ala Glu His 70 Gly Thr Asn Ala Lys 150 His' Phe Val Arg Lys 230 Asp Gin Lys Met 55 Ile Ile Arg Asp Met 135 Arg Ala Phe rrp Ala 215 Pro 3lu Gin Ala 40 Ser Phe Leu Ile Pro 120 Ala Ala Ala Phe Asp 200 Cys Gln Thr Phe 25 Ala Gin Glu Ala Gly 105 Val Gly Leu Va1 Gin 185 Pro Leu Trp Leu Ala Ser Lys Glu Glu Glu Leu Val 75 Ile Ala 90 Arg Phe Val. Met Asp '*hr Glu Trp 155 Leu Val 170 Gln Val Lys Gin Ile Leu Tyr Arg 235 Ala Lys 250 G1) Leu Ser Ser Ser Ala Glu Phe 140 Leu Leu Gin Ala Thr 220 His Glu Leu Gin Thr Ser Leu Asn Met 125 Thr Gly Arg Pro Ile 205 Thr Thr Lys Lys Ser His Tyr Arg Phe Ser Asp Ile Gly Tyr Leu 110 Ala Ser Ala Glu Ala Asp GIu; Leu 175 Phe Phe 190 Arg Glu Gin Arg 4 Phe Glu Gly Met Arg Val Tyr Ala Val Arg Lys Tyr Arg 160 Ala Asp Gly Glu G1u 240 ksn 255 Arg Asp Asp Arg Ile His Gly Ala Leu Leu Ile Leu Asn 260 265 Glu Leu Val 270 Arg Ile Ser Ser Met Giu Gly Giu Arg Leu Arg Giu Giu Met Glu Giu 275 280 285 Ile Thr Gin Gin Gin Leu Vai His Asp Lys Tyr Cys Lys Asp Leu Met 290 295 300 Giy Phe Gly Thr Lys Pro Arg His Ile Thr Pro Phe Thr Ser Phe Gin 305 310 315 320 I0 Aia Vai Gin Pro Gin Gin Ser Asn Aia Leu Vai Gly Leu Leu Giy Tyr 325 330 335 Ser Ser His Gin Gly Leu Met Giy Phe Giy Thr Ser Pro Ser Pro Aia 340 345 350 Lys Ser Thr Leu Val Giu Ser Arg Cys Cys Arg Asp Leu Met Glu Giu 355 360 365 Lys Phe Asp Gin Vai Cys Gin Trp Val Leu Lys Cys Arg Asn Ser Lys 370 375 380 Asn Ser Leu Ile Gin Met Thr Ile Leu Asn Leu Leu Pro Arg Leu Aia 385 390 395 400 Aia Phe Arg Pro Ser Aia Phe Thr Asp Thr Gin Tyr Leu Gin Asp Thr 405 410 415 Met Asn His Vai Leu Ser Cys Val Lys Lys Giu Lys Giu Arg Thr Aia 420 425 430 Aia Phe Gin Aia Leu Giy Leu Leu Ser Val Aia Val'Arg Ser Giu Phe 435 440 445 Lys Vai Tyr Leu Pro Arg Val Leu Asp Ile Ile Arg Aia Aia Leu Pro 450 455 460 Pro Lys Asp Phe Ala His Lys Arg Gin Lys Aia Met Gin Val Asp Ala* 465 470 475 480 Thr Val Phe Thr Cys Ile Ser Met Leu Ala Arg Ala Met Gly Pro Giy 485 490 495 .lie Gin Gin Asp Ile Lys Giu Leu Leu Giu Pro Met Leu Ala Val Gly 500 505 510 Leu Ser Pro Ala Leu Thr Ala VJai Leu Tyr Asp Leu Ser Arg Gin Ile 515 520 525 Pro Gin Leu Lys Lys Asp Ile Gin Asp Giy Leu Leu Lys Met Leu Ser 530 535 540 Leu Val Leu Met His Lys Pro Leu Arg His Pro Giy Met Pro Lys Gly *o545 550 55560 Leu Ala His Gin Leu Ala Ser Pro Gly Leu Thr Thr Leu Pro Glu Ala 565 570 575 Ser Asp Val Gly Ser Ile Thr Leu Ala Leu Arg Thr Leu Gly Ser Phe 580 595 590 Glu Phe Giu Gly His Ser Leu Thr Gin Phe Vai Arg His Cys Ala Asp 595 600 605 His Phe Leu Asn Ser Giu His Lys Giu Ile Arg Met Giu Ala Ala Arg 610 615 620 Thr Cys Ser Arg Leu Leu Thr Pro Ser Ile His Leu Ile Ser Gly His 625 630 635 640 Ala His Val Val Ser Gin Thr Ala Val Gin Vai Val Ala Asp Val Leu 645 650 655 Ser Lys Leu Leu Val Val Giy Ile Thr Asp Pro Asp Pro Asp Ile Arg 660 665 670 Tyr Cys Val Leu Ala Ser Leu Asp Giu Arg Phe Asp Ala His Leu Ala 675 680 685 Gin Al a Giu Asn Leu Gin Ala Leu Phe Val Aia Leu Asn Asp Gin Val 690 695 700 Phe Giu Ile Arg Giu Leu Ala Ile Cys Thr Val Gly Arg Leu Ser Ser 705 710 715 720 Met Asn Pro Ala Phe Val Met Pro Phe Leu Arg Lys Met Leu Ile Gin 725 730 735 Ile Leu Thr Giu Leu Giu His Ser Gly Ile Gly Arg Ile Lys Giu Gin 740 745 750 :::Ser Ala Arg Met Leu Gly His Leu Val Ser Asn Ala Pro Arg Leu Ile 755 760 765 :::Arg Pro Tyr Met Glu Pro Ile Leu Lys Ala Leu Ile Leu Lys Leu Lys 770 775 780 Asp Pro Asp Pro Asp Pro Asn Pro Gly Val Ile Asn Asn Val Leu Ala 785 790 795 800 Thr Ile Gly Giu Leu Ala Gin Vai Ser Gly Leu Giu Met Arg Lys Trp 805 810 815 Val Asp Giu Leu Phele Ile Ile Met Asp Met Leu Gin Asp Ser Ser 820 825 830 Leu Leu Ala Lys Arg Gin Val Ala Leu Trp Thr Leu Gly Gln Leu Val 835 840 845 Ala Ser Thr Gly Tyr Val Val Giu Pro Tyr Arg Lys Tyr Pro Thr Leu *850 855 860 Leu Giu Val Leu Leu Asn Phe Leu Lys Thr Giu Gin Asn Gin Gly Thr 865 870 875 880 Arg Arg Glu Ala Ile Arg Val Leu Gly Leu Leu Gly Ala Leu Asp Pro 8'7 885 890 895 Tyr Lys His Lys Val Asn Ile Gly Met Ile Asp Gin Ser Arg Asp Ala 900 905 910 Ser Ala Val Ser Leu Ser Giu Ser Lys Ser Ser Gin Asp Ser Ser Asp 915 920 925 Tyr Ser Thr Ser Giu Met Leu Vai Asn Met Giy Asn Leu Pro Leu Asp 930 935 940 Giu Phe Tyr Pro Aia Val Ser Met Vai Aia Leu Met Arg Ile Phe Arg 945 950 955 960 Asp Gin Ser Leu Ser His His His Thr Met Val Val Gin Ala Ile Thr 965 970 975 Phe Ile Phe Lys Ser Leu Gly Leu Lys Cys Val Gin Phe Leu Pro Gin 980 985 990 Val Met Pro Thr Phe Leu Asn Val Ile Arg Val Cys Asp Giy Aia Ile 995 1000 1005 Arg Giu Phe Leu Phe Gin Gin Leu Giy Met Leu Vai Ser Phe Val Lys 1010 1015 1020 Ser His Ile Arg Pro Tyr Met Asp Giu Ile Val Thr Leu Met Arg Giu 1025 1030 1035 1040 Phe Trp, Val Met Asn Thr Ser Ile Gin Ser Thr Ile Ile Leu Leu Ile 1045 1050 1055 Giu Gin Ile Val Val Ala Leu Gly Gly Giu Phe Lys Leu Tyr Leu Pro :::1060 1065 1070 Gin Leu Ile Pro His Met Leu Arg Val Phe met His Asp Asn Ser Pro :::1075 1080 1085 *.Gly Arg Ile Val Ser Ile Lys Leu Leu Ala Ala Ile Gin Leu Phe Gly 1090 1095 1100 Ala Asn Leu Asp Asp Tyr Leu His Leu Leu Leu Pro Pro Ile Val Lys 1105 1110 1115 1120 Leu Phe Asp Ala Pro Giu Ala Pro Leu Pro Ser Arg Lys Ala Ala Leu 1125 1130 1135 *Giu Thr Val Asp Arg Leu Thr Giu Ser Leu Asp Phe Thr Asp Tyr Ala 1140 1145 1150 Ser Arg Ile Ile His Pro Ile Val Arg Thr Leu Asp Gin Ser Pro Giu 1155 1160 1165 Leu Arg Ser Thr Ala Met Asp Thr Leu Ser Ser Leu Val Phe Gin Leu 1170 1175 1180 Gly Lys Lys Tyr Gin Ile Phe Ile Pro Met Val Asn Lys Val Leu Val 1185 1190 1195 1200 Arg His Arg Ile Asn His Gin Arg Tyr Asp Val Leu Ile Cys Arg Ile 1205 1210 1215 Vai Lys Gly Tyr Thr Leu Ala Asp Giu Giu Giu Asp Pro Leu Ile Tyr 1220 1225 1230 Gin His Arg Met Leu Arg Ser Gly Gin Gly Asp Ala Leu Ala Ser Gly 1235 1240 1245 Pro Val Giu Thr Gly Pro Met Lys Lys Leu His Val Ser Thr Ile Asn 1250 1255 1260 Leu Gin Lys Ala Trp Gly Ala Ala Arg Arg Val Ser Lys Asp Asp Trp, 1265 1270 1275 1280 Leu Giu Trp Leu Arg Arg Leu Ser Leu Giu Leu Leu Lys Asp Ser Ser 1285 1290 1295 Ser Pro Ser Leu Arg Ser Cys Trp Ala Leu Ala Gin Ala Tyr Asn Pro 1300 1305 1310 Met Ala Arg Asp Leu Phe Asn Ala Ala Phe Val Ser Cys Trp Ser Giu 1315 1320 1325 Leu Asn Giu Asp Gin Gin Asp Giu Leu Ile Arg Ser Ile Giu Leu Ala 1330 1335 1340 Leu Thr Ser Gin Asp Ile Ala Giu Val Thr Gin Thr Leu Leu Asn Leu 1345 1350 1355 1360 Ala Giu Phe Met Giu His Ser Asp Lys Gly Pro Leu Pro Leu Arg Asp 1365 1370 2375 Asp Asn Gly Ile Vai Leu Leu Giy Giu Arg Ala Ala Lys Cys Arg Ala 1380 1385 1390 Tyr Ala Lys Ala Leu His Tyr Lys Giu Leu Giu Phe Gin Lys Giy Pro 1395 1400 1405 *Thr Pro Ala Ile Leu Giu Ser Leu Ile Ser Ile Asn Asn Lys Leu Gin 1410 1415 1420 *.Gin Pro Giu Ala Ala Ala Gly Val Leu Giu Tyr Ala Met Lys His Phe 45 1425 1430 1435 1440 Gly Giu Leu Giu Ile Gin Ala Thr Trp Tyr Giu Lys Leu His Giu Trp 1445 1450 1455 50 Giu Asp Ala Leu Val Ala Tyr Asp Lys Lys Met Asp Thr Asn Lys Asp 1460 1465 1470 Asp Pro Giu Leu Met Leu Gly Arg Met Arg Cys Leu Gi u Ala Leu Gly 1475 1480 1485 Giu Trp Gly Gin Leu His Gin Gin Cys Cys Giu Lys Trp, Thr Leu Val 1490 1495 1500 Asn Asp Glu Thr Gin Ala Lys Met Ala Arg Met Ala Ala Ala Ala Ala 1505 1510 1515 1520 Trp Gly Leu Gly Gin Trp Asp Ser Met Glu Glu Tyr Thr Cys Met lle 1525 1530 1535 Pro Arg Asp Thr His Asp Gly Ala Phe Tyr Arg Ala Val Leu Ala Leu 1540 1545 1550 His Gin Asp Leu Phe Ser Leu Ala Gin Gin Cys Ile Asp Lys Ala Arg 1555 1560 1565 Asp Leu Leu Asp Ala Glu Leu Thr Ala Met Ala Gly Glu Ser Tyr Ser 1570 1575 1580 Arg Ala Tyr Gly Ala Met Val Ser Cys His Met Leu Ser Glu Leu Glu 1585 1590 1595 1600 Glu Val Ile Gin Tyr Lys Leu Val Pro Glu Arg Arg Glu Ile Ile Arg 1605 1610 1615 Gin Ile Trp Trp Glu Arg Leu Gin Gly Cys Gin Arg Ile Val Glu Asp 1620 1625 1630 Trp Gin Lys Ile Leu Met Val Arg Ser Leu Val Val Ser Pro His Glu 1635 1640 1645 Asp Met Arg Thr Trp Leu Lys Tyr Ala Ser Leu Cys Gly Lys Ser Gly 1650 1655 1660 Arg Leu Ala Leu Ala His Lys Thr Leu Val Leu Leu Leu Gly Val Asp 1665 1670 1675 1680 Pro Ser Arg Gin Leu Asp His Pro Leu Pro Thr Val His Pro Gin Val 35 1685 1690 1695 Thr Tyr Ala Tyr Met Lys Asn Met Trp Lys Ser Ala Arg Lys Ile Asp 1700 1705 1710 40 Ala Phe Gin His Met Gin His Phe Val Gin Thr Met Gin Gin Gln Ala 1715 1720 1725 Gn His Ala Ile Ala Thr Glu Asp Gin Gin His Lys Gin Glu Leu His 1730 1735 1740 Lys Leu Met Ala Arg Cys Phe Leu Lys Leu Gly Glu Trp Gin Leu Asn 1745 1750 1755 1760 Leu Gln Gly Ile Asn Glu Ser Thr Ile Pro Lys Val Leu Gin Tyr Tyr 50 1765 1770 1775 Ser Ala Ala Thr Glu His Asp Arg Ser Trp Tyr Lys Ala Trp His Ala 1780 1785 1790 55 Trp Ala Val Met Asn Phe Glu Ala Val Leu His Tyr Lys His Gln Asn 1795 1800 1805 Gin Ala Arg Asp Glu Lys Lys Lys Leu Arg His Ala Ser Gly Ala Asn 0. 1810 1815 1820 Ile Thr Asn Ala Thr Thr Ala Ala Thr Thr Ala Ala Thr Ala Thr Thr 1825 1830 1835 1840 Thr Ala Ser Thr Giu Gly Ser Asn Ser Glu Ser Giu Ala Giu Ser Thr 1845 1850 1855 Giu Asn Ser Pro Thr Pro Ser Pro Leu Gin Lys Lys Val Thr Giu Asp 1860 1865 1870 Leu Ser Lys Thr Leu Leu Met Tyr Thr Val Pro Ala Val Gin Gly Phe 1875 1880 1885 Phe Arg Ser Ile Ser Leu Ser Arg Gly Asn Asn .Leu Gin Asp Thr Leu 1890 1895 1900 Arg Val Leu Thr Leu Trp Phe Asp Tyr Gly His Trp Pro Asp Vai Asn 1905 1910 1915 1920 Giu Ala Leu Val Giu Gly Val Lys Ala Ile Gin Ile Asp Thr Trp Leu 1925 1930 1935 Gin Val Ile Pro Gin Leu Ile Ala Arg Ile Asp Thr Pro Arg Pro Leu 1940 1945 1950 Val Gly Arg Leu Ile His Gin Leu Leu Thr Asp Ile Gly Arg Tyr His 1955 1960 1965 Pro Gin Ala Leu Ile Tyr Pro Leu Thr Val Ala Ser Lys Ser Thr Thr 1970 1975 1980 Thr Ala Arg His Asn Ala Aia Asn Lys Ile Leu Lys Asn Met Cys'Giu ::*1985 1990 1995 2000 35 His Ser Asn Thr Leu Val Gin Gin Ala Met Met Val Ser Giu Giu Leu *2005 2010 2015 *le Arg Val Ala Ile Leu Trp His Glu.,.Met Trp His Giu-,Gly Leu Giu 2020 2025 '2030 Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys Gly Met Phe 2035 2040 2045 45 Glu Val Leu Giu Pro Leu His Ala Met Met Giu Arg Gly Pro Gin Thr 2050 2055 2060 Leu Lys Giu Thr Ser Phe Asn Gin Ala Tyr Gly Arg Asp Leu Met Glu 2065 2070 2075 2080 50 Al1a Gin Giti Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn Val Lys Asp 2085 2090 2095 *Leu Thr Gin Ala Trp Asp Leu Tyr Tyr His Vai Phe Arg Arg Ile Ser Lys Gin Leu Pro Gin Leu Thr Ser Leu Giu Leu Gin Tyr Vai Ser Pro 2115 2120 2125 Lys Leu Leu Met Cys Arg Asp Leu Giu Leu Ala Val Pro Gly Thr Tyr 2130 2.135 2140 Asp Pro Asn Gin Pro Ile Ile Arg Ile Gin Ser Ile Ala Pro Ser Leu 2145 2150 2155 2160 Gin Val Ile Thr Ser Lys Gin Arg Pro Arg Lys Leu Thr Leu Met Gly 102165 2170 2175 Ser Asn Giy His Giu Phe Val Phe Leu Leu Lys Gly His Glu Asp Leu 2180 2185 219 0 Arg Gin Asp Giu Arg Val Met Gin Leu Phe Gly Leu Val Asn Thr Leu 2195 2200 2205 Leu Ala Asn Asp Pro Thr Ser Leu Arg Lys Asn Leu Ser le Gin Arg 2210 2215 2220 Tyr Ala'Va Ile. Pro Leu Ser Thr Asn Ser Gly Leu Ile Gly Trp Val 2225 2230 2235 2240 Pro His Cys Asp Thr Leu His Ala Leu Ile Arg Asp Tyr Arg Giu Lys 2245 2250 2255 Lys Lys Ile Leu Leu Asn Ile Giu His Arg leMet Leu Arg Met Ala 2260 2265 2270 Pro Asp Tyr Asp His Leu Thr Leu Met Gin Lys Val Giu Val Phe Giu 2275 2280 2285 His Ala Val Asn Asn Thr Ala Gly Asp Asp Leu Ala Lys Leu Leu Trp, 2290 2295 2300 35 Leu -Lys Ser Pro Ser Ser Giu Vai Trp Phe Asp Arg Arg.Thr'Asn Tyr 2305 2310 2315 2320 *..Gly Asp Arg His Pro Ser Asn Leu Met Leu Asp Arg Leu Ser Gly Lys 2340 2345 2350 *le Leu His Ile Asp Phe Gly Asp Cys Phe Glu Val Ala Met Thr r 45 .2.3S5> .2360 2365 r Giu Lys Phe Pro Giu Lys Ile Pro Phe Arg Leu Thr Arg Met Leu Thr 2370 2352380 50 Asn Ala Met Glu Val Thr Giy Leu Asp Giy Asn Tyr Arg Ile Thr Cys 2385 2390 2395 2400 ***His Thr Val Met Giu Val Leu Arg Giu His Lys Asp Ser Val Met Ala ::2405 2410 2415 Val Leu Giu Ala Phe Val Tyr Asp Pro Leu Leu Asn Trp, Arg Leu Met 2420 2425 2430 '/2 Asp Thr Asn Thr Lys Gly Asn Lys Arg Ser Arg Thr Arg Thr Asp Ser 2435 2440 2445 Tyr Ser Ala Giy Gin Ser Val Glu Ile Leu Asp Giy Vai Glu Leu Gly 2450 2455 2460 Giu Pro Ala His Lys Lys Thr Gly Thr Thr Val Pro Giu Ser Ile His 2465 2470 2475 2480 Ser Phe Ilie Giy Asp Giy Leu Vai Lys Pro Giu Ala Leu Asn Lys Lys 2485 2490 2495 Ala Ile Gin Ile Ile Asn Arg Val Arg Asp Lys Leu Thr- Gly Arg Asp 2500 2505 2510 Phe Ser His Asp Asp Thr Leu Asp Val Pro Thr Gin Val Glu Leu Leu 2515 2520 2525 Ile Lys Gin Ala Thr Ser His Giu Asn Leu Cys Gin Cys Tyr Ile Giy 2530 2535 2540 Trp Cys Pro Phe Ti-p 2545 INFORMaATION FOR SEQ ID NO:i3: Ci) SEQUENCE

CHARACTERISTICS:

LENGTH: 1794 base pairs TYPE: nucleic acid STRANDEDNESS: both TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

FEATURE:

NAME/KEY:

CDS

LOCATION: 1. .1686 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:i3: .TTG GTT TAC CCT TTG ACA GTT GCT ATT ACT TCC GAA TCA ACG AGC CGT 48 Leu Val Tyr Pro Leu Thr Val Ala Ile Thr Ser Giu Ser Thr Ser Arg 1 5 10 AAA AAG GCA GCT CAA TCC ATT ATT GAA AAA ATG CGA GTA CAT TCT CCT 96 **Lys Lys Ala Ala Gin Ser Ile Ile Giu Lys Met Arg Val His Ser Pro 20 25 AGC TTG GTG GAT CAA GCA GAA TTA GTG AGT CGA GAA CTC ATC CGA CT? 144' .Ser Leu Val Asp Gin Ala Glu Leu Val. Ser Arg Glu Leu Ile Arg Vai 40 4 55 GCA GTT TTA TGG CAC GAA CAA TGG CAC CAT GCC TTG GAP, CAT GCT AGC 292 Ala Val Leu Ti-p His Giu Gin Trp His Asp Ala Leu Giu Asp Ala Ser 55 4.

AGG

Arg

GAA

Glu

CAA

Gin

TGG

TTT

Phe

CCA

Pro

GCC

Al a

GTG

TTC

Phe

TTA

Leu

TTT

Phe

CTC

TTT

Phe

CAT

His

GCA

Al a 100

AAC

GGT GAA Gly Glu '70 CAA ATG Gln met AAT GCT Asn Ala TTT AGA

CAC

His

TTG

Leu

TT

Phe AAC ACA Asn Thr CAA AAG Gin Lys GGC AGG Gly Arg 105 ACT AAA GAA AAG Glu Lays 75 GGA CCA Gly Pro 90 GAG TTG- Giu Leu

ATG

Met

GAA

Glu

ACA

Thr

TTT

Phe

ACG

Thr

GAT

Asp GAA ACA Glu Thr ATG AGG Met Arg GCA TAC Ala Tyr 110

TTG

Leu s0

GAA

Glu

GAG

Glu 240 288 336 384

AGA

GAC ATA ACC AAT TTG AAT *rrp VaI Leu Asn Phe Arg Arg Thr Lys Asp lie Thr Asn Leu Asn Gin 115 125 GCA TGG GAT ATA TAC TAC Ala Trp Asp Ile Tyr Tyr 130 0 0 0 0*0**0 0 *0 *0 00000G 0 0 0*0* 0*0* 0 *000 00000.

9

CAG

Gin 145

CAT

His

AAA

Lys

TCA

35 Ser

AAA

Lys 40

AAC

Asn 225 45

GAT

Asp 5 0 ATTC Ile I

CTG

Leu

GCT

Al a

CCT

Pro

TCT

Ser

GAC

Asp 210

TTA

Leu

CCG

Pro

CA

~ro

TTA

*Leu

CAA

Gin

GTG

Val

AAA

Lys 195

TAC

Tyr

GTG

Val1

GTA

Val

TTA

Leu

GCT

Al a

GAT

Asp

ATC

Ile 180.

CAA

Gin

CAA

Gin

ATG

Met

TGT

Cys

TCA

Ser

AGT

Ser

TTG

Leu 165

AGA

Arg

AGA

Arg

TAT

Tyr

CAA

Gin

TTC

Phe 245

CCA

Pro

*CTT

*Leu

GAA

Giu

ATA

Ile

CCG

Pro

GCG

Al a

TTG

Leu 230

AAG

Lys

AAA(

Lys

AAT

Asn 135

GAG

Giu

TTG

Leu

ATC

Ile

AGA

Arg

TTG

Leu 215

TTT

Phe

AGA

krg

GTC

Val2

TTG

Leu

GCT

Ala

AAA

Lys

AAA

Lys 200

AAA

Lys

GGT

Gly

CAT

His

TTT

Phe

CAG

Gin

GTA

Val

TTT

Phe 185

TTA

Leu

GGA

Gly

TTG

Leu

TTG

Leu

AGA

Arg

TAT

Tyr

AGA

Arg

GTA

AGC

Se r

CCG

Pro 155 CCA GGT ACT TAC CAA Pro 170

GAT

Asp

TCG

Ser

CAT

His

GTT

Val

GAT

Asp 250 Gly

CCT

Pro

TGC

Cys

GAA

Glu

AAT

Asn 235

ATA

Ile Thr

ACT

Thr

AGA

Arg

GAT

Asp 220

ACG

Thr

CAA

Gin Tyr

TTT

Phe

GGA

Gly 205

ATC

I le

TTG

Leu

CAA

Gin Gin

TOG

Ser 190

AGT

Ser

AGA

Arg

TTG

Leu

TAT

Tyr

AA;

Lys

GAC

Asp CAG GTG Gin Vai TTA GAG Leu Giu 160 GCA GGC Ala Gly 175 ATT ATT Ile Ile GAT GGT Asp Giy CAA GAT Gln' Asp GTA AAT Val Asn 240 OCT GCT Pro Ala 255 528 576 624 672 720 768 3TG GGA TTG CTT GGT TGG GTT CCA lal 260 GAC ACT TTC CAT GTA TTG Asp Thr Phe His Val Leu 275

ATO

Ile Gly

AAA

Lys Leu 265

GGC

Gly Leu

TAT

Gly

CGC

Arg Val

TCA

Ser 285 280 ATG TTG AAT ATT GAA CAC AGG CTT TTG TTG CAA ATG GCA OCT GAT TAT Met Leu Asn Ile Glu His Arg Lieu Leu Leu Gin Met Ala Pro Asp Tyr 290 295 300

GAT

Asp 305 TTC TTG ACA TTA TTG CA.A AAA GTT GAA GTG TTC ACA AGT Phe Leu Thr Leu Leu Gin Lys Val Glu Val Phe Thr Ser GCA ATG Ala Met 320 AAA TCT Lys Ser 335 GAT AAT TGT AAG GGA CAG GAT TTG TAC AAA GTG TTA TGG CTC Asp Asn Cys Lys AAA TCA TCC GAG Lys Ser Ser Giu Gly 325 Gin Asp Leu Tyr Lys Val Leu Trp Leu 960 1008 2056 1104 GCG TGG TTG GAC CGT Ala Trp Leu Asp Arg 345 AGA ACA ACA TAC Arg Thr Thr Tyr ACG AGA TCA Thr Arg'Ser' 350 GGG GAT AGG Gly Asp Arg TTA GCT GTA Leu Ala Val 355 ATG TCT ATG GTT Met Ser Met Val

GGG

Gly 360 TAT ATA TTA GGT Tyr Ile Leu Gly CAC CCA His Pro 370 TCA AAT TTG ATG Ser Asn Leu Met

TTG

Leu 375 GAC CGT ATT ACT Asp Arg Ile Thr AAA GTC ATC CAT Lys Val Ile His

ATT

Ile 385 GAT TTC GGA GAC Asp Phe Gly Asp TTT GAA GCA GCA Phe Giu Ala Ala

ATA

Ile 395 TTA CGT GAG AAG Leu Arg Glu Lys

TAT

Tyr 400 1152 1200 1248 CCA GAG AGA GTT Pro Giu Arg Val TTT AGA TTG ACG Phe Arg Leu Thr

AGA

Arg 410 ATG CTT AAT TAT Met Leu Asn Tyr GCC ATG Ala Met 415 0O a. @0Sg

S.

S 4

SS

5 0

S

0

S..

GAA GTT ACT Giu Val Ser ATG AGG GTG Met Arg Val 435

GGA

Gly 620 ATA GAG GGC TCG Ile Giu Gly Ser AGA ATC ACA TGT Arg Ile Thr Cys GAA CAT GTT Glu His Val 430 ATA TTA GAG Ile Leu Giu TTG CGT GAT AAT Leu Arg Asp Asn

A;A

Lys 440 GAG TCT TTA ATG Giu Ser Leu Met GCC TTT Ala Phe 450 GCT TAC GAT CCC Ala Tyr Asp Pro

TTG

Leu 455 ATA AAT TGG GGG Ile Asn Trp Giy GAT TTC CCA ACA Asp Phe Pro Thr 1296 1344 1392 1440 1488

AAG

45 Lys 465 GCG TTG GCT GAA Ala Leu Ala Giu

TCA

Ser 470 ACG GGT ATA CGT Thr Giy Ile Arg CCA CAA GTC AAC Pro Gin Val Asn GCA GAA TTA TTA Ala Giu Leu Leu AGA GGA CAG ATT Arg Gly Gin Ile

GAC

Asp 490 GAA AAA GAA GCT Glu Lys Giu Ala GTA AGA Val Arg 495 TTG CAA AAG CAA AAT GAA TTG GAA Leu Gin Lys Gin Asn Giu Leu Giu 500

ATA

Ile 505 AGA AAC GCT AGA Arg Asn Ala Arg GCT GCA TTA Ala Ala Leu 510 ATC AAA CGG Ile Lys Arg 1536 GTG TTG AAA Val Leu Lys 515 CGT ATT ACC GAT Arg Ile Thr Asp AAG TTA ACT GGT AAC GAT Lys Leu Thr Gly Asn Asp 520 525 158 4 TTG AGA GGA TTA GAT GTG CCT ACT CAA GTC GAT AAA TTG ATT CAA CAA 1632 Leu Arg Gay Leu Asp Val Pro Thr Gin Val Asp Lys Leu Ile Gin Gin 530 535 540 GCC ACC AGT GTT GAG AAT TTG TGT CAG CAT TAC ATT GGT TGG TGT TCG 1680 Ala Thr Ser Val Glu Asn Leu Cys Gin His Tyr Ile Gly Trp Cys Ser 545 550 555 560 TGT TGG TAGGTTGATT ATCGTCATGT GTCGATAAGT ATGGTATTGT GGTAACTATT 1736 Cys Trp TTATAAAGGG AAATATTAAA GAATTGTATA TTATT"A AAAAAAA AACTtGAG 17.94 INFORM4ATION FOR SEQ ID NO: 14: SEQUENCE CHARACTERISTICS: LENGTH: 562 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: Leu Val Tyr Pro Leu Thr Val Ala Ile Thr Ser Glu Ser Thr Ser Arg 1 510 Lys Lys Ala Ala Gin Ser Ile Ilie Giu !,ys Met Arg Val His Ser Pro 25 *Ser Leu Val Asp Gin Ala Giu Leu Val Ser Arg Glu Leu Ile Arg Val 35 40 Ala Val Leu Trp His Giu Gin Trp His Asp Ala Leu Giu Asp Ala Ser so* 5 55 Arg Phe Phe Phe Gly Gilu His Asn Thr Glu Lys Met Phe Glu Thr Leu 70 75 Giu Pro Leu His Gin Met Leu Gin Lys Gly Pro Glu Thr met Arg Giu 8S 90 Gin Ala Phe Ala Asn Ala Phe Gly Arg Glu LeU Thr Asp Ala Tyr Glu 100 105 110 Trp Val Leu Asn Phe Arg Arg Thr Lys Asp Ile Thr Azn Leu Asn Gin 13.5 120 125 Ala Trp Asp Ile Tyr Tyr Asn Val Phe Arg Arg Val Ser Lys Gin Val 130 135 140 Gin Leu Leu Ala Ser Leu Glu Leu Gin Tyr Val Ser Pro Asp Leu Glu 145 iso 1S5 160 His Aia Gin Asp Leu Giu Leu Ala Val Pro Gly Thr Tyr Gin Ala Gly Lys Ser Lys Asn 225 As p Ile Asp Met Asp 305 Asp Lys Leu His Ile 385 Pro Giu Met Al a Pro Val Ser Lys 195 Asp Tyr 210 Leu Val Pro Val Pro Leu Thr Phe 275 Leu Asn 290 Phe Leu Asn Cys Ser Ser Ala Val 355 Pro Ser 37 0 Asp Phe Giu Arg Val Ser Arg Val 435 Phe Ala 450 Ile 180 Gin Gin Met Cys Ser 260 His Ile Thr Lys Glu 340 Met Asn Gly Val Gly 420 Leu Tyr Arg Arg Tyr Gin Phe 245 Pro Val Giu Leu Gly 325 Al a Ser L Asp Pro 405 Ile Arg Asp Ile Ile Lys Pro Al a Leu 230 Lys Lys Leu His Leu 310 Gin Trp Met me": Cys 390 Phe Giu Asp Pro Lys 200 Lys Gly His Gly Lys 280 Leu Lys Leu Asp Gly 360 Asp Glu Leu Ser Lys 440 Ile Phe 185 Leu Gly Leu Leu Leu 265 Gly Leu Val Tyr Arg 345 Tyr Arg Ala Thr Phe 425 Giu Asn Pro Cys Glu Asn 235 Ile Gly Arg Gin Val 315 Val Thr Leu Thr Ile 395 Met Ile Leu Gly Thr Phe Arg Gly 205 Asp Ile 220 Thr .Leu Gin Gin Trp Val Glu Ser 285 Met Ala 300 Phe Thr Leu Trp *ThrTyr Gly Leu 365 Gly, Lys.

3F_ Leu Arg Leu Asn Thr Cys Met Ala 445 Phe Asp 460 Ser 190 Ser Arg Leu Tyr Pro 270 Arg Pro Ser Leu Thr 350 Gly -Val1 Glu Tyr Giu 430 Ile Phe 175 Ile Asp Gin Val Pro 255 Asn Ser Asp Al a Lys 335 Arg Asp .Il e Lys Al a 415 His Leu Pro Ile Giy Asp Asn 240 Al a Ser Ile Tyr Met 32*0 Ser Sex Arg His Tyr 400 Met Val Giu Thr Lys Aia Leu Ala Giu Ser Thr Gly Ile Arg Val Pro Gin Val Asn Thr 470 475 480 Ala Leu Val Leu Al a 545 Cys Giu Leu Leu Arg 485 Gin Lys Gin Asn 500 Leu Lys Arg Ile 515 Arg GJly Leu Asp 530 Thr Ser Val Giu Trp Arg Giu Thr Val Asn 550 Gin Ile Giu Ile 505 Lys Leu 520 Thr Gin Cys Gin 477 Asp Giu 490 Arg Asn Thr Gly Val Asp His Tyr.

555 Lys Giu Ala Ala Arg Ala 510 Asn Asp Ile 525 Lys Leu Ile 540.

Iie:Gly Trp Val 495 Ala Lys Gin .Cys Arg Leu Arg Gin Ser 560 0 90 0 0:0.

INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 399 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: ()NAME/KEY CDS LOCATION: 1.-399 (xi) SEQUENCE DESCRIPTION: SEQ ID GTT AGT CAC GAG TTG ATC AGA... GTA. GCC. GTT. T v Val Ser Hisi Glu Leu Ile Arg Val.Ale'Val Leu 1 5 TAT GAA GGA CTG GAA GAT GCG AGC CGC CAA TTT Tyr Giu Giy Leu Giu Asp Ala Ser Arg Gin Phe 45 20 25 ATA GAA AAA ATG TTT TCT ACT TTA GAA CCT TTA Ile Giu Lys Met Phe Ser Thr Leu Giu Pro Leu 40 AAT GAG CCT CAA ACG TTA AGT GAG GTA TCG TTT Asn Glu Pro Gin Thr Leu Ser Glu Val Ser Phe I 55 AGA CAT TTG AAC GAT GCC TAC GAA TGG TTG AAT Arg Asp Leu Asn Asp Ala Tyr Giu Trp Leu Asn 70 75

GAA

GIu

GAA

Glu

CAC

His

TCA

Ser

AAA

Lys

TGG

Trp

AAC

Asn

GGC

Gly

GGT

Gly

TCA

Ser 96 144 192 240 1. .1 1 AAA GAC ATC AAT AAT TTG AAC CAA GCT TGG Lys Asp Ile Asn Asn Leu Asn Gin Ala Trp 90 TTC AGA AA.A ATA ACA CGT CAA ATA CCA CAG Phe Arg Lys Ile Thr Arg Gin Ile Pro Gin 100 105 CAG CAT GTT TCT CCC CAG CTT CTG GCT ACT Gin His Val Ser Pro Gin Leu Leu Ala Thr 115 120 GTT CCT GGG ACA TAT Val Pro Giy Thr Tyr.

130 INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 133 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ IDI Val Ser His Giu Leu Ile Arg Val Ala Val I i510 Tyr Giu Gly Leu Giu Asp Ala Ser Arg Gin i 20 25 35 Ile Giu Lys Met Phe Ser Thr Leu Glu Pro 1 35 40 Asn Giu Pro Gin Thr Leu Ser Giu Val Ser P 50 Arg Asp Leu Asn Asp Ala Tyr Giu Trp Leu A 65 70 Lys Asp Ile Asn Asn Leu Asn Gin Ala Trp A 85 90 Phe Arg Lys Ile Thr Arg Gin Ile Pro Gin L 100 105 Gin His Val Ser Pro Gin Leu Leu Ala Thr H 115 120 Val Pro Giy Thr Tyr 130 INFORMATION FOR SEQ ID NO:17: GAT ATT TAT TAT AAC GTC Asp Ile Tyr Tyr Asn Val TTA CAA ACC TTA GAC TTA Leu Gin Thr Leu Asp Leu 110 CAT GAT CTC GAA TTG GCT His Asp Leu Giu Leu Ala 125 S 'JO: 16: eu Trp ~he Phe ~eu His ~he Gin sn Asn 75 .sp Ile eu Gin is Asp His Val1 Lys Lys Tyr Tyr Thr Leu 125 Giu Giu His Ser Lys Tyr Leu 110 Glu Leu Trp His Asn Leu Gly Phe Gly Lys Ser s0 Asn Val Asp Leu Leu Ala p 4.

Wi SEQUENCE CHARACTERISTICS: LENGTH: 399 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE:

NAME/KEY:

LOCATION:

CDS

1. .399 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: AGC CAC GAA TTG ATA CGT ATG GCG Ser His Glu Leu Ile Arg Met Ala CTT TGG CAT GAG CAA TGG Leu Trp His Glu Gin Trp TAT GAG GGT Tyr Glu Gly

CTG

Leu GAT GAC GCC ACT Asp Asp Ala Ser

AGG

Arg 25 CAG TTT TTT GGA Gin Phe Phe Gly GAA CAT AAT Glu His Asn ATG CTG AAG Met Leu Lys ACC GAA AAA Thr Glu Lys ATG TTT GCT GCT TTA GAG CCT CTG TAC Met Phe Ala Ala Leu Giu Pro Leu Tyr AGA GGA Arg Gly CCG GAA ACT TTG Pro Giu Thr Leu GAA ATA TCG TTC Giu Ile Ser Phe AAT TCT TTT GGT Asn Ser Phe Gly

AGG

Arg 35 65 GAC TTG AAT GAC Asp Leu Asn Asp

GCT

Ala 70 TAC GAA TGG CTG ATG AAT TAC AAA AAA Tyr G)lu Trp Leu Met Asn Tyr Lys Lys

TCT

Se r s0 240 288 AAA GAT Lys Asp GTT AGT AAT TTA Val Ser Asn Leu AAC CA.A GCG TGG Asn Gin Ala Trp GAC ATT TAC TAT AAT GTT Asp Ile Tyr Tyr Asn Val TTC AGG AAA ATT Phe Arg Lys Ile 100 GGT AAA CAG TTG CCA CAA TTA CAA ACT CTT GAA CTA Gly Lys Gin Leu Pro Gin Leu Gin Thr Leu Giu Leu CAA CAT GTG TCG CCA AAA Gin His Val Ser Pro Lys 115 GTC CCC GGG ACC CGT Val Pro Gly Thr Arg 130 CTA CTA Leu Leu 120 TCT GCG CAT GAT Ser Aia. His Asp

TTG

Leu 125 GAA TTG GCT Glu Leu Ala INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 133 amino acids TYPE: amino acid 1009 TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: Val Ser His Glu Leu Ile Arg Met Ala Val Leu Trp His Glu Gin Trp Tyr Glu Gly Thr Glu Lys Arg Gly Pro Asp Asp Ala Ser Arg is Phe Phe Gly Glu His Asn Leu Tyr Glu Met Leu Lys Phe Al a Al a Glu Pro Glu Thr Leu Arg Tyr Glu Ile Ser Phe Ser Phe Gly Lys Asp Leu Asn Asp Glu Trp, Leu Tyr Lys Lys Asp Val Ser Leu Asn Gin Ala Ile Tyr Tyr Phe Arg Lys Ile 100 Lys Gin Leu Leu Gin Thr Leu Giu Leu 110 Glu Leu Ala Gin His Val Ser Pro 115 Val Pro Gly Thr Arg 130 Lys Leu Leu Ser Ala His Asp 120 Leu 125 35 INFORMATION FOR SEQ ID NO: 29: SEQUENCE CHARACTERISTICS: LENGTH: 531 base pairs TYPE: nucleic acid STRAjqDEDNESS: both TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: TGACCCTCAC CCCTTCCACC TATCCCAAAA ACCTCACTGG GTCTGTGGAC AAACAACA14A AATNTTTTCC ANANAGGCCC CAAATGAGNC CCANGGGTCT NTCTTCCATC AGACCCAGTG ATTCTGCGAC TCACACNCTT CAATTCAAGA CCTGACCNCT AGTAGGGAGG TTTANTCAGA TCGCTGGCAN CCTCGGCTGA NCAGATNCAN AGNGGGGNTC GCTGTTCAGT GGGNCCACCC TCNCTGGCCT TCTTCANCAG GGGTCTGGGA TGTTTTCAGT GGNCCNAA2NA CNCTGTTTAG 1. 4.

AGCCAGGGCT CAGNAAACAG AAAANC TNT C NACATATTGG GGATTATGAN CAGNACCAAN AACCATNTCT ANACNCCATN TTNTATCAGN ANTTTNAACA TNAAAAAAAA AAAAAAAAA INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS LENGTH: 231 base pa TYPE: nucleic acid STRANDEDNESS: doubi TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA /c/ ATGGNGGTTC TGGACACAGG GNAGGTCTGG ACNCCACTAA ATNCCCCAAG NANAAAGTGT ANAAATTTTIN TTCCNATAAA TGACATCAGN AAAANAAAAA AAAAAAAAAA A 360 420 480 531 (ix) FEATURE: NAME/KEY: misc feature LOCATION: 128 OTHER INFORMATION: /label= XhoI (xi) SEQUENCE DESCRIPTION: SEQ ID GCGTATAACG CGTTTGGA .AT CACTACAGGG ATGTTTAATA CCACTACAAT GGATGATGTA TATAACTATC TATTCGATGA TGAAGATACC-CCACCAAACC CAAAAAAAGA GATCTGGAAT TCGGATCCTC GAGAGATCTA TGAATCGTAG ATACTGAAAA ACC CCGCAAG TX'CACTTCAA 35 CTGTGCATCG TGCACCATCT CAATTTCTTT CATTTATACA TCGTTTTGCC 71 INFORMATION FOR SEQ ID NO:21: Ci)SEQUENCE

CHARACTERISTICS:

LENGTH: 21 base pairs TYPE: nucleic acid STR.ANDEDNESS. single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: TGAAGATACC CCACCAAACC C INFORMATION FOR SEQ ID NO:22: Wi SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs 120 180 231 102 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: TGCACAGTTG

AAGTGAAC

INFORMATION FOR SEQ ID NO:23: SEQUEN~CE CHARACTERISTICS: LENGTH: 907 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: CA) NAME/KEY: CDS CB) LOCATION: 34. .507 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: GCCGGGGCTG CGGCCGCCCG, AGGGACTTTG AAC ATG TCG GGG ATC GCC CTC AGC Met Ser Gly Ile Ala Leu Ser AGA CTC GCC CAG GAG AGG AAA GCA TGG AGG AAA GAC CAC CCA Arg Leu Ala Gln Glu Arg Lys Ala Trp Arg Lys Asp His Pro TTT GGT Phe Gly TTC GTG Phe Val.

25 GCT GTC CCA ACA Ala Val Pro Thr AAA AAT CCC GAT GGC 'ACG ATG AAC CTC ATG Lys Asn Pro Asp Gly Thr Met Asn Leu Met 30 CCA GGA AAG AAA GGG ACT CCG TGG GAA OGA Pro Gly Lys Lys Gly Thr Pro Trp Glu Gly 54 102 150 198 246

AAC

Asn TGG GAG TOC GCC Trp Glu Cys Ala GGC TTG TTT AAA Gly Leu Phe Lys CGG ATG CTT TTC Arg Met Leu Phe GAT GAT TAT CCA Asp Asp Tyr Pro TCT TCG Ser Ser CCA CCA AAA Pro Pro Lys AAA TTC GAA CCA Lys Phe Glu Pro TTA TTT CAC CCG Leu Phe His Pro AAT GTG TAC Asn Val Tyr CCT TCG GGG ACA GTG TGC CTG Pro Ser Gly Thr Val Cys Leu TCC ATC TTA GAG GAG Ser Ile Leu Glu Glu GAC AAG GAC TGG Asp Lys Asp Tr-p 100 AGG CCA Arg Pro 105

CTA

Leu 120

ATT

Ile

AAT

Asn

TAC

Tyr GCC ATC ACA ATC AAA Ala Ile Thr Ile Lys 11.0 GAA CCA AAT ATC CAA Giu Pro Asn Ile Gin 125 TGC CAA AAC AGA GTG Cys Gin Asn Arg Val /0:3 CAG ATC CTA TTA GGA ATA CAG GAA CTT Gin Ile Leu Leu Giy Ile Gin Giu Leu 115 GAC CCA GCT CAA GCA GAG GCC TAC ACG Asp Pro Ala Gin Ala Giu Ala Tyr Thr 130 135 GAG TAC GAG AAA AGO GTC CGA GCA CAA Glu Tyr Glu Lys Arg Val Arg Aia Gin 145 150 TAAGCAGCGA CCTTGTGGCA TCGTCAAAAG GCC AAG AAG Ala Lys Lys GAAGGGATTG G ATACAATGAC TGTGCGGTCT

C

TGGTATTTTT

G

AACTGCTGTAA

CTCTGGGATG C

TGGAAATGCA

TTT GCG CCC TCA Phe Ala Pro Ser 155 ;TTTGGCAAG AACTTGTTTA 'AGTCACCTG

GGGGGGTTGG

GATTCGCTG

AATTGCCCGT

GATTGT'rAT GTAAAACTCG AATTATAAA CTTTTATACT AGGCATGCT TCTCACCGTG 390 438 486 537 597 657 717 77-7 8 37 8 97 907 CAACATTTTT

GGCAAATCTA

GCGGGCGCCA

TCTTCCATTG

TTCCATACAG GGTCTCTTcC CTTTTATTTT

AATATTGATG

GGGTAAGTCC

CCCAGGGGCG

CAGAGCTGCA CTTGNCCTCA

AAGTTGCTCC

CCGCCGCGGG

TTCGGTCTTT

TCAGTAT'TTC

AGTTNCCTCG

GCTGNtTGNA INFORMATION FOR -SEQ ID NO: 24: 35 Ci) SEQUENCE CHARACTERISTICS: CA) LENGTH: 158 amino acids CB) TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: Met Ser Giy Ile Ala Leu Ser Arg Leu Ala Gin Giu Arg Lys Aia Tr-p 1 5 10 Arg Lys A-sp His Pro Phe Gly Phe Val Ala Val Pro Thr Lys Asn Pro 25 Asp Gly Thr Met Asn Leu Met Asn Trp, Giu Cys Ala Ile Pro Gly Lys 35 40 Lys Gly Thr Pro Trp Glu Gly Gly Leu Phe Lys Leu Arg Met Leu Phe 55 Lys Asp Asp Tyr Pro Ser Ser Pro Pro Lys Cys Lys Phe Giu Pro Pro 70 75

C)*

Gly Thr Val Cys Leu Leu Phe His Pro Asn Val Tyr Pro SerGl Th Va Cy Le Ser Ile Leu Glu Glu Asp Lys 100 Leu Leu Gly Ile Gin 115 0 Ala Gin Ala Glu Ala 130 Glu Lys Arg Val Arg Asp Trp Arg Pro 105 Glu Leu Leu Asn 120 Tyr Thr Ile Tyr 135 Ala Gin Ala Lys 150 Ala Ile Thr Ile Lys Gin Ile 110 Giu Pro Asn Ile Gin Asp Pro 125 Cys Gin Asn Arg Val Glu Tyr 140 Lys Phe Ala Pro Ser 155 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 207 base pairs TYPE: nucleic acid STRANDEDNESS: both TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID CCCTCCCTCC TGCCGCTCCT CTCTAGAACC TTCTAGAACC TGGGCTGTGC TGCTTTTGAG CCTCAGACCC CAGGGCAGCA TCTCGGTTCT GCGCCACTTC CTTTGTGTTT ANAT .GGCGTT 35 TTGTCTGTGT TGCTGTTTAG AGTAGATNAA CTGTTTANAT AAAAAAA NAAAATTI7AC TNGAGGGGGC NTGNAGGCAT GCNNAAC 120 180 207

Claims (11)

1. A substantially pure preparation of an RAPT1 polypeptide, or a fragment thereof, having an amino acid sequence at least 70% homologous to SEQ ID NO. 2 or 12.

2. The polypeptide of claim 1, wherein said polypeptide binds to an FKBP/rapamycin complex.

3. The polypeptide of claim 1, having an amino acid sequence at least homologous to the amino acid sequence of SEQ ID No. 2 or 18.

4. The polypeptide of claim 1, wherein said polypeptide functions in one of either role of an agonist of rapamycin regulation of cell proliferation or an antagonist of rapamycin regulation of cell proliferation. The polypeptide of claim 1, wherein said polypeptide is a recombinant protein produced from a pIC524 clone of ATCC deposit 75787.

6. The polypeptide of claim 1, wherein polypeptide is of mammalian origin.

7. An antibody preparation specifically reactive with an epitope of the polypeptide of claim 1.

8. An isolated or recombinant polypeptide comprising a rapamycin-binding domain having an amino acid sequence at least 70% homologous to one or both of Val26- Tyrl 60 of SEQ ID No. 2 and Val2012-Tyr2144 of SEQ ID No. 12

9. A soluble polypeptide which specifically binds an FKBP/rapamycin complex, which binding is rapamycin-dependent.

10. The polypeptide of claim 9, which polypeptide comprises a soluble portion of a RAPTI-like polypeptide that binds to said FKBP/rapamycin complex. S 11. The polypeptide of claim 9, wherein said RAPTI-like polypeptide portion has an amino acid sequence identical or homologous with a rapamycin-binding domain represented by an amino acid sequence selected from the group consisting Val26-Tyr 160 of SEQ ID No. 2, Val2012-Tyr2144 of SEQ ID No. 12, Val41-Tyrl73 of SEQ ID No. 14, Vall- Tyrl33 of SEQ ID No. 16, and Vall-Arg133 of SEQ ID No. 18. 4 I

106- 12. The polypeptide of claim 1, which polypeptide is a fusion polypeptide comprising a first polypeptide portion for binding to said FKBP/rapamycin complex, and a second polypeptide portion having an amino acid sequence unrelated to said first polypeptide portion. 13. The polypeptide of claim 12, wherein said second polypeptide portion provides a detectable label for detecting the presence of said fusion protein. 14. The polypeptide of claim 12, wherein said second polypeptide portion provides a matrix-binding domain for immobilizing said fusion protein on an insoluble matrix. The polypeptide of claim 12, wherein said fusion polypeptide is functional in a rapamycin-dependent two-hybrid assay. 16. A soluble protein comprising a rapamycin-binding domain of a RAPTI-like polypeptide, which protein specifically binds an FKBP/rapamycin complex in a rapamycin-dependent manner. 17. The protein of claim 16, wherein said rapamycin-binding domain has an amino acid sequence identical or homologous with a rapamycin-binding domain represented by an amino acid sequence selected from the group consisting Val26-Tyrl 60 of SEQ ID No. 2, Val2012-Tyr2144 of SEQ ID No. 12, Val41-Tyrl73 of SEQ ID No. 14, Vall-Tyrl33 of SEQ ID No. 16, and Vall-Argl33 of SEQ ID No. 18. 18. A soluble polypeptide portion of a RAPTI protein, which polypeptide is represented by the general formula Zl-Z 2 -Z 3 wherein *9fl** Zi represents a rapamycin-binding domain within residues 1272 to 1444 of SEQ ID No. 12, Z 2 is absent or represents a polypeptide from 1 to about 500 amino acid residues of '*39 SEQ ID No. 12 immediately N-terminal to said rapamycin-binding domain, and Z is absent or represents from 1 to about 365 amino acid residues of SEQ ID No. 2 immediately C-terminal to said rapamycin-binding domain, wherein said polypeptide specifically binds an FKBP/rapamycin complex in a rapamycin-dependent manner. 19. A chimeric polypeptide represented by the general formula A-B-C, wherein -107- B represents a rapamycin-binding domain consisting essentially of amino acid residues 2012 to 2144 of SEQ ID No. 12, or a corresponding rapamycin-binding domain of a RAPTI-like protein homologous thereto, and X and Z are, seperately, absent or represent polypeptides having amino acid sequences unrelated to a RAPTI-like protein. A substantially pure nucleic acid having a nucleotide sequence which encodes RAPT1 protein, or a fragment thereof, having an amino acid sequence at least homologous to one or both of SEQ ID Nos: 2 or 12. 21. The nucleic acid of claim 20, wherein said RAPT1 protein binds to an FKBP/rapamycin complex. 22. The nucleic acid of claim 20, wherein said RAPT1 protein functions in one of either role of an agonist of rapamycin regulation of cell proliferation or an antagonist of rapamycin regulation of cell proliferation. 23. The nucleic acid of claim 20, wherein said RAPT1 protein has a phophatidylinositol kinase activity. C! 24. The nucleic acid of claim 20, comprising a RAPTI coding sequence from a pIC524 clone of ATCC deposit 75787. The nucleic acid of claim 20, which hybridizes under stringent conditions to a '2 2 nucleic acid probe corresponding to at least 12 consecutive nucleotides of SEQ ID No. 1 or 11. C 26. The nucleic acid of claim 20, further comprising a transcriptional regulatory sequence *tooperably linked to said nucleotide sequence so as to render said nucleotide sequence 6'30 suitable for use as an expression vector. 27. An expression vector, capable of replicating in at least one of a prokaryotic cell and eukaryotic cell, comprising the nucleic acid of claim 26. 28. A host cell transfected with the expression vector of claim 27 and expressing said polypeptide. -108- 29. A method of producing a recombinant RAPTI protein comprising culturing the cell of claim 28 in a cell culture medium to express said RAPTI protein and isolating said RAPTi protein from said cell culture. 30. A nucleic acid encoding a soluble polypeptide which specifically binds an FKBP/rapamycin complex, which binding is rapamycin-dependent. 31. The nucleic acid of claim 30, wherein said soluble polypeptide includes an amino acid sequence identical or homologous with a rapamycin-binding domain represented by an amino acid sequence selected from the group consisting Val26-Tyrl60 of SEQ ID No. 2, Val2012-Tyr2144 of SEQ ID No. 12, Val41-Tyrl73 of SEQ ID No. 14, Vall-Tyrl33 of SEQ ID No. 16, and Vall-Argl33 of SEQ ID No. 18. 32. The nucleic acid of claim 30, which nucleic acid encodes a fusion polypeptide comprising a first polypeptide portion for binding to said FKBP/rapamycin complex, and a second polypeptide portion having an amino acid sequence unrelated to said first polypeptide portion. S 33. The nucleic acid of claim 32, wherein said second polypeptide portion provides a 2 0 detectable label for detecting the presence of said fusion protein. 34. The nucleic acid of claim 32, wherein said second polypeptide portion provides a matrix-binding domain for immobilizing said fusion protein on an insoluble matrix. 35. The nucleic acid of claim 32, wherein said fusion polypeptide is functional in a rapamycin-dependent two-hybrid assay. 36. A nucleic acid encoding a polypeptide portion of a RAPTI polypeptide, which polypeptide specifically binds an FKBP/rapamycin complex in a rapamycin-dependent manner, and is represented by the general formula ZI-Z 2 -Z 3 wherein Z 1 represents a rapamycin-binding domain within residues 1272 to 1444 of SEQ ID No. 12, Z 2 is absent or represents a polypeptide from 1 to about 500 amino acid residues of SEQ ID No. 12 immediately N-terminal to said rapamycin-binding domain, and Z is absent or represents from 1 to about 365 amino acid residues of SEQ ID No. 2 immediately C-terminal to said rapamycin-binding domain. 37. A chimeric polypeptide represented by the general formula A-B-C, wherein

109- Y represents a rapamycin-binding domain consisting essentially of amino acid residues Val41-Tyrl73 of SEQ ID No. 14, Vall-Tyrl33 of SEQ ID No. 16, or Vall- Argl33 of SEQ ID No. 18, or a corresponding rapamycin-binding domain of a yeast or fungal RAPTl-like protein homologous thereto, and X and Z are, seperately, absent or represent polypeptides having amino acid sequences unrelated to a RAPT -like protein. 38. A recombinant RAPTi polypeptide, or a fragment thereof, having an amino acid sequence at least 70% homologous to SEQ ID NO. 2 or 12. 39. The polypeptide of claim 28, wherein said polypeptide binds to an FKBP/rapamycin complex. An assay for screening test compounds for agents which induce the binding of a RAP- binding protein with an FK506-binding protein, comprising i. combining a RAP-BP polypeptide comprising a rapamycin-binding domain represented by an amino acid sequence SEQ ID No. 2 or 12, and a FKBP polypeptide comprising a rapamycin-binding domain of an 2 0 FK506-binding protein under conditions wherein said RAP-BP and FKBP polypeptides are able to interact; ii. contacting said combination with a test compound; and iii. detecting the formation of a complex comprising said RAP-BP and FKBP polypeptides, wherein a statistically significant increase in the formation of said complex in the presence of said test compound, relative to the formation of said complex in the absence, is indicative of an inducer of the interaction between a RAP-binding protein with an FK506-binding protein. S0 41. An assay for screening test compounds for agents which induce the binding of a RAP- binding protein with an FK506-binding protein, comprising i. combining a RAP-BP polypeptide consisting essentially of a rapamycin-binding domain of a RAPTI or RAPTI-like protein, and a FKBP polypeptide comprising a rapamycin-binding domain of an FK506-binding protein -110- under conditions wherein said RAP-BP and FKBP polypeptides are able to interact; ii. contacting said combination with a test compound; and iii. detecting the formation of a complex comprising said RAP-BP and FKBP polypeptides, wherein a statistically significant increase in the formation of said complex in the presence of said test compound, relative to the formation of said complex in the absence, is indicative of an inducer of the interaction between a RAP-binding protein with an FK506-binding protein. 42. A method for screening test compounds for agents which induce the binding of a RAP-binding protein with an FK506-binding protein, comprising providing a host cell containing a detectable gene wherein the detectable gene expresses a detectable protein when the detectable gene is activated by an amino acid sequence including a transcriptional activation domain when the transcriptional activation domain is in sufficient proximity to the detectable gene; (ii) transforming the host cell with a first chimeric gene that is capable of being expressed in the host cell, the first chimeric gene comprising a DNA sequence 20 that encodes a first hybrid protein, the first hybrid protein comprising: a DNA-binding domain that recognizes a binding site on the detectable gene in the host cell; and oe: a rapamycin-binding domain of an FK506-binding protein; (iii) transforming the host cell with a second chimeric gene that is capable of being expressed in the host. cell, the second chimeric gene comprising a DNA sequence that encodes a second hybrid protein, the second hybrid protein comprising: the transcriptional activation domain; and a rapamycin-binding domain of a RAPT 1-like protein; "3 (iv) subjecting the host cell to conditions under which the first hybrid protein and the second hybrid protein are expressed in sufficient quantity for the detectable gene to be activated; contacting the host cell with a test agent; and (vi) determining whether the detectable gene has been expressed to a degree statistically significantly greater than expression in the absence of an interaction between the first test protein and the second test protein. 111 43. The method of claim 42, wherein the DNA-binding domain and transcriptional activation domain are derived from transcriptional activators having separable DNA- binding and transcriptional activation domains. 44. The method of claim 43, wherein the DNA binding domain and the transcriptional activation domain are selected from the group consisting of transcriptional activators GAL4, GCN4, LexA, VP16 and ADR1. The method of claim 42, wherein the rapamycin-binding domain of the FK506- binding protein is part of the second hybrid protein rather than the first hybrid protein and the rapamycin-binding domain of the RAPTI-like protein is part of the first hybrid protein rather than the second hybrid protein. 46. A probe/primer comprising a substantially purified oligonucleotide, said oligonucleotide containing a region of nucleotide sequence which hybridizes under stringent conditions to at least 20 consecutive nucleotides of sense or antisense sequence of nucleic acid selected from the group consisting of SEQ ID No. 1 or 11, or naturally occuring mutants thereof. 20 47. The probe/primer of claim 46, further comprising a label group attached thereto and able to be detected. 48. The probe/primer of claim 47, wherein said label group being selected from a group consisting of radioisotopes, fluorescent compounds, enzymes, and enzyme co-factors. 49. A method of determining if a subject is at risk for a disorder characterized by unwanted cell proliferation, comprising detecting, in a tissue of said subject, the presence or absence of a genetic lesion characterized by at least one of a mutation of a gene encoding a protein represented by SEQ ID No. 2 or 12, or a mammalian homolog thereof; and the mis-expression of said gene. S 50. The method of claim 49, wherein detecting said genetic lesion comprises o ascertaining the existence of at least one of i. a deletion of one or more nucleotides from said gene, ii. an addition of one or more nucleotides to said gene, iii. an substitution of one or more nucleotides of said gene, iv. a gross chromosomal rearrangement of said gene. -112- v. a gross alteration in the level of a messanger RNA transcript of said gene, vi. the presence of a non-wild type splicing pattern of a messanger RNA transcript of said gene, and vii. a non-wild type level of said protein. 51. The method of claim 49, wherein detecting said genetic lesion comprises i. providing a probe/primer comprising an oligonucleotide containing a region of nucleotide sequence which hybridizes to a sense or antisense sequence of a nucleic acid selected from a group consisting of SEQ ID No. 1 and 11, or naturally occuring mutants thereof, or 5' or 3' flanking sequences naturally associated with said gene; ii. exposing said probe/primer to nucleic acid of said tissue; and iii. detecting, by hybridization of said probe/primer to said nucleic acid, the presence or absence of said genetic lesion. 52. The method of claim 51, wherein detecting said lesion comprises utilizing said probe/primer to determine the nucleotide sequence of said gene and, optionally, of said flanking nucleic acid sequences. i.: 2 Q' 53. The method of claim 51, wherein detecting said lesion comprises utilizing said probe/primer to in a polymerase chain reaction (PCR). 54. The method of claim 51, wherein detecting said lesion comprises utilizing said probe/primer in a ligation chain reaction (LCR). *0.00* 55. The method of claim 51, wherein the level of said protein is detected in an immunoassay. DATED this 19 th day of October 2001 Ariad Pharmaceuticals, Inc. by DAVIES COLLISON CAVE Patent Attorneys for the Applicants