AU777192B2 - Immunosuppressant target proteins - Google Patents

Description

C.

AUSTRALIA

PATENTS ACT 1990 DIVISIONAL APPLICATION NAME OF APPLICANT: Ariad cPharmaeutic SEC I P 113 4 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

ME

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 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 mul.iple affects of Cv okines Von immune cells inciuding 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 25 257:973-977). 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 ofS. 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 TOWR gene product joins p7 U JUI.1 p2V

V

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 FKBP-rapamycin complex will likely reveal an upstream 25 component of the signal transduction pathway that leads to G I progression and will help delineate the signal transduction pathways that link growth factor-mediated signaling events and Scyclin-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- 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 rapamycin." 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/rapamycin complexes. In addition, a RAPTI- .like protein was cloned from the human pathogen Candida. The present invention, therefore.

.makes available novel prtens (h r nbinant and purified forms), recombinant genes, S antibodies to RAP-binding proteins, and other novel reagents and assays for diagnostic and 20 therapeutic use.

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 rapamycindependent 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 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 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 "1 A ubuding 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 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 o o 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 transcripticnl regultory 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.

Yet another aspect of the present invention 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 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 C j o 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 (mRATP1), Val2012-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 SF'Q I n No: 1, or it can comprise a fragment of that nucleic acid, which fragment may be, for 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 S• the RAPTI protein, the polypeptide is able to modulate rapamycin-mediated protein complexes.

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: I 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.

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.

Furthermore, in certain preferred embodiments, the subject caRAPTI nucleic acid *e will include a transcriptional regulatory sequence, e.g. at least one of a transcriptin a 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 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 I 'I 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 presence of a test compound (relative to what is seen in the absence of the test compound) is 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.

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.

*i 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: 1 or 11.

In similar fashion, Candida infection can be detected by use of probes/primers which oo" 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 ed. 1984): Transcr.,-:, And Translation D. Hames S. J. Higgins eds. 1984); Culture Of Animal Cells I.

i 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

SI

activity. No interaction is detected if FK506 is used in place of rapamycin. or if lex.da (a control plasmid) replaces FKBP12.

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 FKBP12 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 I (light blue) to 4 (deep blue).

Detailed Description of the Invention S* 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 2 /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 i transition) and the expression of high affinity interleukin-2 (IL-2) receptors. The subsequent binding ofIL-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.

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 Immunoll0:519-560). 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 gover the G l-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 hin 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 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 from 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 naturallvoccurring 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 s c.pa ,1 Oi auionoituuus replication and/expression of nucleic acids to which they are linked. Vectors capable of directing the expression of genes to which they are operatively 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 o*o *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.

/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 moleci_!e 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 S 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 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 tat posi,i,,. A delgrce of homoiogy between sequences is a function of the number of matching or homologous positions shared by the sequences.

"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 may 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 ofZ, and Z 3 being present in the fusion protein.

/4c 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 chernirn!lv csnthesized.

Moreover, an "isolated nucleic acid" is meant to include nucleic acid fragments which are not 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 (hRAPTI). Val41-Tyrl73 of SEQ ID No. 14 (caRAPTI), Vall-Tyrl33 of SEQ ID No. 16 (TOR1). 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 RAPTI like 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.

/7 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*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 S. oogs of the subjct rap-U,,, LC.. enzIyme include nucieotide sequences that hybridize under stringent conditions to a nucleotide sequence represented in SEQ ID No. 23, or to a S.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 ATCC clone 75787 represent biologically active portions of larger full-length forms of mammalian RAPTI proteins. In preferred embodiments, the RAPT1 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.

S 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 FKBPI2/rapamycin complex; the ability to modulate assembly of FKBP/rapamycin-complexes; the ability to regulate cell

IS

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 ubiauitin-mediated degrdation of p53 in a m r hich causs celluiar p53 levels torise 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 RAPT1.

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 Q J a O t

F

iq 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 S. s a t a t u homologous, mure preferabiy 70% homologous and most preferably homologous with an amino acid sequence shown in one or more of SEQ ID Nos: 2, 12 or 14.

S. 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 S•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 nurlecp acids that enco"e iscforms of .th 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 by one of SEQ ID Nos.: 2, 12 or 14. Appropriate 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 0 C, to high stringency conditions at about 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 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 amino 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 encoding a RAP-binding 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 s g~n~rated eA vivu 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 reviewed, for example, by van der Krol et al. (1988) Biotechniques 6:958-976; and Stein et al. (1988) Cancer Res 48:2659- So 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 I I O

I

23 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 maninnultion 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 and are selected to direct expression of a 25 recombinant RAP-binding protein. Accordingly, the term transcriptional regulatory sequence 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 nrtein. in a ce!! 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 particular 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).

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 retrovims replication defective.

So* 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 *ooo 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 WCrip, WCre, Y2 and 4Am.

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.

Natl. 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. Natl. Acad Sci. USA 89:10892-10895; Hwu et al. (1993) J. Immunol. 150:4104-4115; U.S. Patent No.

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) J Biol 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 C L IMJ ILIVaI VeaUI.

Another viral gene delivery system useful in the present invention utilizes adenovirusderived 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 strains of adenovirus Ad2, Ad3, Ad7 etc.) are well known to those skilled 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 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 Biology, 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 EIA 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 o cells. A variety of nucleic acids have been introduced into different cell types usinp AAV vectors (see for example Hermonat 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 !i 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 Spathways 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 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 an.

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.

uiotuihci aspi;i 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 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 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 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 subject 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 S. 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 pAcUWI). 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 ,he at that a nethionine a 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) J. Bacteriol. 169:751-757) and Salmonella typhimurium 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).

25 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 et 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 affi purif.ic.on using glutathIIIUicUne-ivatized matrices (see, for example, Current Protocols in Molecular Biology, eds. Ausabel et al. John Wiley Sons. 1991)). In 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 preent (but water, ulTfers, and other small molecules, especially molecules i: 20 having a molecular weight of less than 5000. can be present). The term "pure" as used herein Spreferably 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 25 agents and polymers, e.g. acrylamide or agarose) substances or solutions.

Furthermore, isolated peptidyl portions of the subject RAP-binding proteins ca also be 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 virro protein binding assays. In an illustrative embodiment, peptidyl portions of a RAP-binding protein, such as RAPT) or rapUBC. can be tested for FKBP/rapamycin-binding activity.

0 34 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 proteolytic 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, 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 for polarity or charge of amino acid sidechains. Whether 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 nrotein. For instanre a shrt half-life cn give rise t tranient RAPT1 iolgica effects and, when part of an inducible expression system, can allow tighter control of S 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 i 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 one or more species, e.g. a sequence alignment of the mouse and human RAPTI 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.

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 c alternatively, 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 et 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 et 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 lihrary. For example, RA1P-B hcmrlcgs (bt agonisi and antagonist forms) can be Sgenerated 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.

SBiol. 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: 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 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 el 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 rface nf a virl nart;l, Fnr ;ntn in the filen u jJ85u 3yLzItL% l IV-II PCPLIUC 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 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 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 25 inhibiting binding of the RAP-BP to FKBP/rapamycin, can interfere with the function of rapamycin in cell-cycle arrest. For instance, non-hydrolyzable peptide analogs of such S 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 0-aminoalcohols (Gordon et al. (1985) Biochem Biophys Res Communl26:419; and Dann et al. (1986) Biochem Biophys Res 0 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 FKBl/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.

IT..he 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 S 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 art, 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 .ex pic. F(ab') 2 ,fagments can be generated by treating antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' S 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 Xgtll, .gt18-23. 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 1 will produce fusion proteins whose amino termini consist of 1-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 RAPTI 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 20 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 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 -92 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 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 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.

One aspec of th present inventioi whichi faciiiiates the generation ol drug screening assays, particularly the high-throughout assays described below, is the identification of the S 20 rapamycin binding domain of 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 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 RAPT1-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-Tyrl33 of SEQ ID No.

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

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 2012 ,o 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 mnifest in an aratin of binding affinity with other proteins or change in enzymatic properties of the molecular target.

Accordingly. in an exemplary screening assay of the present invention, the compound of S• 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 5 efficacy of the compound can be assessed by generating dose response curves from data obtained using various concentrations of the test compound. Moreover, a control assay can 0• 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.

4, 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.

rierce 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 I-chloro- 2 ,4-dinitrobenzene (Habig et al (1974) JBiol Chem 249:7130).

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 WO94/10300; 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 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- :i 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 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).

47.

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/RAPTI 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. FKBPI2 (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 RAPTI 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, 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 siellatoidea, 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, Aspergillusniger, 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 oi c.ro n AOOA"10 v r.f, -UA WCIy ct LiryyumaiI us).

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 11) 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 cre/loxP recombinase system of bacteriophage PI (Lakso et al. (1992) PNAS 89:6232-6236; Orban et al. (1992) PNAS *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 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.

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 iimsaniuc, a founder popuiation, in which the subject transgene is silent in all tissues, can be :propagated and maintained. Individuals of this founder population can be crossed with 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 prorriter sequences which require prokaryotic proteins to be simultaneous expressed in order to facilitate expression of the transgene. Exemplary promoters and the corresponding trans-activating prokarvotic 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 (Jahner et al. (1985) PNAS 82:6927-6931; Van der Putten et al. (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 Puten, 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.

1-2 Methods of making knock-out or disruption transgenic animals are also generally known. See, for example, Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory 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 transcriptonal 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, 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 of plasmids. Preferably. however, the first chimeric gene is present in a chromosome of the host cell and the secnnd chimerie opnp i intrr- t--l i;nt-- t )Ct ,-oI part of a plasmid. The second chimeric gene contains a DNA sequence that encodes a second 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 DNAbinding 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, and 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 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 -54 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 15 certain aspects and embodiments of the present invention, and are not intended to limit the i. nvention.

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) JBiol Chem 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; and Iwabuchi et al. (1993) Oncogene 8:1693-1696). Using the following olignucleotides: coding strand G GGT TTG GAA T T C CTA ATA ATG TCT GTA CAA GTA GAA ACC (SEQ ID No: 3) non-coding strand GGG TTT CGG GATCCC 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 FKBP12 protein. The sequence of the human FKBP12 cloned was confirmed as: ATGTCCGTACAAGTAGAAACCATCTcCCCAGGAGACGGGCGCACCTTCCCCA AG CGCGGCCAGACCTGCGTGGTGCACTACACCGGgATG

CTTGAAGATGGAAA~

GAATTTGATTCCTCCCGTGACCGTAACAAGCCCTTTAGTTtATgCTAGGC aAGCAGGAGGTGATCCGAGGCTGGGAAGAagGGGTTGc

CCAGATGAGTGTGG

gTCAGCGTGCCAAa CTgACTAtAt CTCcAGaTtATgCcTATGgTGCCACTGG GCAcc CAGGCATCATCCCACCACATGCCACTCTCGTCTTCGATGTGGAGCTT CTAAAACTGGAATGA (SEQ ID The resulting PCR product containing the human FKBP 12 coding sequences was then digested with EcoRJ and BamHI. and cloned into the EcoRI BamHI sites of pBTMI 116 creating an in-frame fusion between LexA and FKBP 12. The resulting plasmid is referred to below as plC5O4.

B. LexA-(gly) 6 -FKBPJ2 bait.

In order to generate an in frame fusion between LexA and FKBPI2 separated by six 1 5 glycine residues, the coding sequence from human FKBP1 2 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: 20 TCG CCG GAA IhC GGG GGC GGA GGT GGA GGA GTA CAA GTA GAA ACC ATC (SEQ ID No: 7) non-coding strad GOG TTr CGG GALCC GTC ATT CCA GT f T TA GAA G S 25 (SEQ ID No: 8) The PCR product containing the human FKBP 12 coding sequences was then digested with EcoRi and Bkm.HI and cloned into the EcoRi BamHI sites of pBTMI 116 as above.

The resulting plasrnid is referred to below as plC506.

30Exml2 Construction of the FKBPJ2 deletion strain A 1.8 kb HindIIl-EcoRJ yeast genomic fragment containing FKBI (the S Cen'eisia hornolog of FKBP 12) was cloned into the Hindll 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 Bani-H] site were: CGCGGATCCGCGCATTATTACTTGTTTTGATTGATT 1 1 1 G (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 pVB 172 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)g-lacZ GAL4 gal80) selecting for adenine prototrophy.

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

Example 3 Cloning Of Mammalian Rapamycin Target Genes We used the drug-dependent interaction trap described in Example I above, with the 20 LexA binding-domain fusion constructs as bait to detect interaction with clones from cDNA libraries containing VP16 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 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 ICYI01) were maintained on yeast media lacking tryptophan which selects for cells harboring the bait plasmid.

A mouse embryo PCR library in pVPI6 (designated pSHIO.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.

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 ICYl01/pSHl0.5 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 1-galactosidase reporter. Candidate colonies appeared between 4-1 1 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/pSHI0.5 candidates), 15.6 ng/ml (for ICYI01/pSHIO.5 candidates) rapamycin. or no rapamycin (for both). Candidate clones which grew in the presence of 'rapamycin and failed to grow on media without rapamycin were chosen for the next test.

For the ICY99/pSHI0.5 screen, out of 107 His+ and LacZ+ candidates screened. 24 were rapamycin-dependent for growth on medium lacking hisitidine. For the ICY101/pSHl0.5 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 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 0* 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 ICY 01/pSH 10.5 screen, all 20 passed the test.

iii) Positive and negative interaction with control baits 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 prpared .ro 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::FKBP12 bait fusion; ii) ICYI01, 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 ICY 01 in a rapamycin-dependent manner, but not to ICY102.

For the ICY99/pSHI0.5 screen, 11 out of the 23 candidates fulfilled the above criteria. For the ICYI01/pSH10.5 screen, 10 out of the 20 candidates fulfilled the above S. criteria.

The h ,.,aniuate lus weic Ysquenced 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/pSHI0.5 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: 1 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 pIC99.1.5, 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 32 -labeled dCTP by randomincorporation with the Klenow fragment of DNA polymerase. The radiolabelled DNA probe was isolated away from free nuc!eotides by a G50 column, alkali-denatured, and added to the hybridization mix at 2x 10 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 37 0

C.

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 plC524. 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 Budanest .:Treaty. ATCC Accession number 75787 has been assigned to the deposit.

S. 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 His 1541-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 plC524 clone, further 5' sequence of the human RAPTI gene was obtained from other overlapping clones, with the additional sequence of the 3end 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 plC524. For example, the primers TGAAGATACCCCACCAA- ACCC (SEQ ID No. 21) and TGCACAGTTGAAGTGAAC (SEQ ID No. 22) correspond to pACT sequences flanking the XhoI 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 depo..sit mbr 75 Moreover, ii 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.

Example 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 yeast cell (Clonetech) in which FKBI gene was disrupted (see Example Of the clones at a o v i el urnan ubiquitin-conjugating enzyme (rap-UBC) has been identified.

A

deposit of the pGADGH plasmid (clone "SMR4-15") was made with the American Type 20 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.

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

23 (nucleotide) and SEQ ID No. 24 (amino acid). The sequence for the 3' portion of the clone is provided by SEQ ID No. 25. As described above, primers 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.

Example Construction of the Serine-to-Argenine RAPTI mutation The smallest mRAPTI clone that interacted with the FKBP12/rapamycin complex was 399 bp. defining a rapamycin binding domain. The RAPTI binding domain corresponds to a region in yeast TORU/TOR2 located immediately upstream, but outside of the lipid 6/ 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 FKBPl2/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 *0 consensus sequence. This region contains the serine residue which when mutated in yeast •TORI (also called DRRI) confers resistance to rapamvin (taffe.e, t 1993) inn l. Ce 7a Y IV I. L el 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 FKBP12/rapamycin complex with mRAPT to Examl 7 Northern Analysis The multiple tissue Northern blots (containing 2 pg 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 pg/ml denatured salmon sperm DNA. Washes were at 0.1X SSC and 0.1% SDS at 550C. 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 of approximatelly 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 S 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 i.

3. Dilute the second overnight culture to a final A0 0 o of 0.5 in growth medium. Using a Quadra 96 pipettor (TomTec, Inc.), dispense 135 pl aliquots of the cell suspension into wells of a round bottom microtiter plate pre-loaded with 15 jAl/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 0 C for 4 hr with S 25 shaking at 300 rpm.

S4. Centrifuge microtiter plate for 10 min at 2000 rpm. Remove the supernatant with the Quadra 96 pipenor and wash with 225 gl phosphate buffered saline.

5. Dispense 100 .tl 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 ofa Microfluor plate (Dynatech Laboratories, Chantilly, VA), gl of the chemiluminescent substrate, Galacton PlusTM (Tropix. Inc.. Bedford. MA) in diluent (100 mM Na2HPO4, 1 mM MgCI2, pH To these wells, transfer 20 gl 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, FKBPI3 (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 FKBF12 and KATII 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, Pgalactosidase 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 RAPTI 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 FKBPI2 is fused in the frame of GST in pGEX (Pharmacia, Piscataway, NJ). GST-FKBP12 6:4 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 pi of the in vitro transcription/ translation reactions are added to 200 pl of binding buffer (20mM HEPES[pH7.4]. 150 mM NaCI, 10% glycerol, 0.05% NP-40). After addition of 10 pl of GST-FKBP12 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 RAPTI 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 RAPT1 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 30 RAPTI proteins (see below).

Upon completion of the mutagenesis, the EcoRl-BamHI digested PCR products are S: 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 RAPTI 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 RAPTI occurs when cells are grown in media containing rapamycin. inducing 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 result.... in u.rcated version uIf PT. i 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-FKBP 2.

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 ;I1~IIviL is masured in terms of inhibition of DNA synthesis monitored by BrdU incorporation. Mutans that confer resistance of rapamycin by virtue 2C being unable to bind to the FKBPl2/rapai. 'cin complex indicate which mutations media.

drug sensitivity in lymphoid and nonlymphoid cells. Of particular interest is whethe different RAPTls mediate drug sensitivity in different cell types.

Example 11 2 5 Cloning ofa RAPTI-like polypeptidefrom 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 0 C for I S 30 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.

EDITORIAL NOTE APPLICATION NUMBER 81505/01 The following Sequence Listing pages 67 to 104 are part of the description. The claims pages follow on pages 105 to 115.

67 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: 30 APPLICATION NUMBER: US 08/250,795 FILING DATE: 20-DEC-1994 INFORMATION FOR SEQ ID NO:1: 35 SEQUENCE CHARACTERISTICS: LENGTH: 486 base pairs TYPE: nucleic acid STRANDEDNESS: both TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: 45 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 69 ATG TGG ATT CGG GTA GCC ATC CTC TGG CAT GAG CAT GAA GGC CTG GAA Ile Arg Vai Ala Ile Leu Trp His Giu Met Trp His Glu Gly Leu Giu 40

GAG

Glu

GAG

Glu

CTG

Leu

GCA

Ala

CTC

Leu

AAG

Lys

GCA

Al a

GTG

Val

AAG

Lys

CAA

Gin

ACG

Thr

CAG

Gin 130

TCT

CTG

Leu

GAA

Glu

GAA

Giu

CAA

Gin 115

CTA

Leu

CGC

Arg

GAG

Giu

ACA

Thr

TGG

Trp, 100

GCC

Al a

CCC

Pro

TTG

Leu Ccc Pro

TCC

Ser 85

TGT

Cys

TGG

Trp

CAG

Gln

TAC

CTG

Leu 70

TTT

Phe

CGA

Arg

GAC

Asp

CTC

Leu

TTT

55

CAT

His

AAT

Asn

AAG

Lys

CTC

Leu

ACA

Thr

GGG

Gly

GCT

Ala

CAG

Gin

TAC

Tyr

TAC

Tyr 120

TCC

Ser

GAG

Giu

ATG

Met

GCA

Al a

ATG

Met 105

TAT

Tyr

CTG

Leu

AGG

Arg

ATG

Met

TAT

Tyr 90

AAG

Lys

CAC

His

GAG

Glu

AAC

Asn

GAA

Giu 75

GGC

Gly

TCG

Ser

GTG

Val1

CTG

Leu

GTG

Val

CGG

Arg

CGA

Arg

GGG

Gly

TTC

Phe

CAG

Gin

AAA

Lys

GGT

Gly

GAT

Asp

AAC

Asn

AGA

Arg 12

TAT

Tyr

GGC

Gly

CCC

Pro

TTA

Leu

GTC

Val 110

CGG

Arg

GTG'

Val

ATG

Met

CGG

Arg

ATG

Met

AAG

Lys

ATC

Ile

TCC

TTT

Phie

ACT

Thr s0

GAG

Glu

GAC

Asp

TCA

Ser

CCC

Pro 192 240 288 336 384 432 135 140 AAA CTT CTG ATG TGC CGA GAC CTT GAG TTG GCT GTG CCA GGA ACA TAC Leu

CCC

Pro Leu Met Cys Arg I50 Asp Lieu Giu Leu Ala 155 Val Pro Gly Thr Tyr 160 INFORMATION FOR SEQ ID NO:2: 40 SEQUENCE CHARACTERISTICS: LENGTH: 162 amino acids TYPE: amino acid TOPOLOGY: linear (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 Val Gin Gin Ala Met Met Val Ser Glu Clii Leu 25 Ile Arg Val Ala Ile Leu Trp His Glu Met Trp His Glu Gly Leu Giu 40 Giu Ala Ser Arg Leu Tyr Phe Gly Giu Arg Asn Val Lys Gly Met Phe 55 Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly Pro Arg Thr 70 75 Leu Lys Glu Thr Ser Phe Asn Gin Ala Tyr Gly Arg Asp Leu Met Glu 90 Ala Gin Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn Val Lys Asp 100 105 110 Leu Thr Gin Ala Trp Asp Leu Tyr Tyr His Val Phe Arg Arg Ile Ser 115 120 125 Lys Gin Leu Pro Gin Leu Thr Ser Leu Glu Leu Gin Tyr Val Ser Pro 130 135 140 Lys Leu Leu Met Cys Arg Asp Leu Glu Leu Ala Val Pro Gly Thr Tyr 145 150 155 160 Asp Pro INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 40 base pairs TYPE: nucleic acid STRANiED:NES: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: GGGTTTGGAA TTCCTAATAA TGTCTGTACA AGTAGAAACC 40 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 34 base pairs TYPE: nucleic acid 45 STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: GGGTTTCGGG ATCCCGTCAT TCCAGTTTTA CAAC 34 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 348 base pairs TYPE: nucleic acid STR.ANDEDNESS: single TOPOLOGY: linear (ii) MOLECULITE TYPE. c-A (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 Giu Thr Ile Ser Pro Gly Asp 1 5 GGG CGC ACC TTC Ccc AAG CGC GGC CAG ACC TGC GTG GTG CAC TAC ACC Gly Arg Thr Phe Pro Lys Arg Gly Gin Thr Cys Vai Val His Tyr Thr 49 97 145

GGG

Gly

AAG

Lys 45 ATG CTT GAA GAT GGA AAG AAA TTT GAT TCC TCC CGT GAC CGT AAC Met Leu Giu Asp Giy Lys ~35 Lys Phe Asp Ser Ser Arg Asp Arg Asn CCC TTT AAG TIT Pro Phe Lys Phe CTA GGC AAG Le-u Gly Lys CAG GAG Gin Giu GGT CAG Gly Gin 70 GTG ATC CGA GGC Vai Ile Arg Gly GAA GAA GGG GTT Giu Giu Giy Val ATA TCT CCA GAT Ile Ser Pro Asp so

GCC

Al a CAG ATG ACT GTG Gin Met Ser Val CGT GCC AAA CTG ACT Arg Ala Lys Leu Thr 241 289 TAT GCC TAT GGT TIyr Ala Tyr Gly ACT GGG CAC Thr Giy His CCA GGC ATC ATC Pro Gly Ile Ile CCA CCA CAT GCC ACT CTC Pro Pro His Ala Thr Leu GTC TTC CAT GTG GAG CTT CTAAAACTGG Val Phe Asp Val Glu Leu 100 AATGACGGGA TCC INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: 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 Cly Arg Thr Phe 1 5 Pro Lys Arg Gly Gin Thr Asp Gly Lys Lys Phe Asp 10 Cys Val Val His Tyr Phe Met Leu Gly Lys Gin Ser Ser 40 Glu Val 55 Gin Arg Arg Asp Arg Ile Arg Gly Ala Lys Leu 75 Thr Gly Met Leu Glu Asn Lys Pro Phe Lys Trp Glu Glu Gly Val Thr Ile Ser Pro Asp Ile Pro Pro His Ala Ala Tyr Gin Met Ser Val Gly 70 Ala Tyr Gly Ala Thr Gly His Pro Ile 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 iD) TOPULGY: linear (ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: TCGCCGGAAT TCGGGGGCGG AGGTGGAGGA GTACAAGTAG AAACCATC INFORMATION FOR SEQ ID NO:8: 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:8: GGGTTTCGGG ATCCCGTCAT TCCAGTTTTA GAAG INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 41 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: CGCGGATCCG CGCATTATTA CTTGTTTTGA TTGATTTTT G 41 INFORMATION FOR SEQ ID NO:l0: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 40 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID CGCGGATCCG CGTAAAAGCA AAGTACTATC AATTGAGCCG INFORMA.TION FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: CA) LENGTH: 7824 base pairs CB) TYPE: nucleic acid CC) STRANDEDNESS: both TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS C(B) LOCATION: 97. .7743 (xi) SEQUENCE DESCRIPTION:'SEQ ID NO:11: *.AAGGCGGGCG GTGGGGCACG GGGCCTGAAG CGGCGGTACC GGTGCTGGCG GCGGCAGCTG *AGGCCTTGGC CGAAGCCGCG CGAACCTCAG GGCAAG ATG CTT GGA ACC GGA CCT 114 50 Met Leu Gly Thr Gly Pro 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 20 15 CAG CAG TTT GCC AGT GGC CTA AAG AGC CGG A.AT GAG GAA ACC AGO GCC 210 Gln Gln Phe Ala Ser Gly Leu Lys Ser Arg Asn Glu Glu Thr Arg Ala AAA GCC GCC AAG GAG CTC CAG CAC TAT Lys

ATG

Met

ATT

Ile

ATC

Ile

CGA

Arg

GAC

Asp

ATG

135

CGA

Arg

GCA

Al a

TTC

40 Phe

TGG

Trp

GCC

Al a 215

CCT

Pro Ala 40

AGT

Ser

TTT

Phe

TTG

Leu

ATT

Ile

CCA

Pro 120

GCA

GCC

Al a

GCT

Al a

TTC

Phe

GAC

ASP

200

TGT

cys

CAG

Gin Al a

CAA

Gin

GAA

Glu

GCC

Al a

GGC

Gly 105

GTT

Val1

GGG

C- Iy

CTG

Leu

GTC

Vai

CAG

Gin 185

CCC

Pro

CTG

Leu

TGG

Trp Lys Glu Leu C

GAG

Giu

TTG

Leu

ATA

Ile

AGA

Arg

GTC

Val1

GAC

GAA

Giu

CTG

Leu 170

CAA

GIn

AAA

Lys

ATT

Ile

TAC

Tyr

GAG

Gi u

GTT

Val

GCT

Al a

TTT

Phe

ATG

Met

ACT

TGG

Ti-p 155

GTT

Vai

GTG

Val

CAG

Gin

CTC

Leu

AGG

Arg 235

TCT

Ser 60

TCC

Ser

AGC

Ser

GCC

Aila

GAA

Giu

TTT

140

CTG

Leu

CTC

Leu

CAA

GIn

GCC

Ala

ACA

Thr 220

CAC

His

'I

C

L

A

A

A

1

A

G

G

C

A

C

p

A

1 2

A'

TJ

A4

TI

;In His 45 ~CT CGC 'hr Arg LGC TCA er Ser ~TC ATA *eu Ile .AC TAT 1 sn Tyr 110 .TG GCA let Ala 25 CC GCT GT GCT iy Ala GT GAG rg Giu CC TTC ro Phe 190 TC CGT le Arg 05 CC CAG hr Gin CA =T hir Phe Tyr

TTC

Phe

GAT

Asp

GGA

Gly 95

CTT

Leu

TCC

Se r

GAG

GAC

Asp

CTG

Leu 175

TTT

Phe

GAG

Glu

CGT

Arg GAA4 Glu

GTC

Val

TAT

Tyr

GCC

Aia 80

GTG

Val

CGG

Arg

AAG

Lys

TAC

CGC

Arg 160

GCC

Ala

GAC

Asp

GGA

Gly

GAG

Glu

GAA

Glu 240 Thr

GAC

Asp 65

AAT

Asn

GAA

Giu

AAC

Asn

GCC

Ala

GTG

S0 .1 145

AAT

Asn

ATC

Ile

AAC

Asn

GCT

Al a

CCG

Pro 225

GCA

Ala Met

CAA

Gin

GAG

Giu

GGT

Gly

CTC

Leu

ATT

Ile 130

GAA

'34. U

GAG

Giu

AGC

Ser

ATT

Ile

GTA

Val 210

AAG

Lys

GAG

Glu ACC ATG GAA CTC CGA GAG Glu Leu Arg Glu

CTG

Leu

AGG

Arg

GGG

Gly

CTC

Leu 115

GGC

Gly

TTT

pije

GGC

Gly

GTC

Val

TTT

Phe 195

GCC

Al a

GAG

Glu

AAG

Lys

AAC

Asn

AAA

Lys

AAT

Asn 100

CCC

Pro

CGT

Axg

GAG

'34.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

AGA

Arg 165

ACC

Thr

GCC

Ala

CTT

Leu

CAG

Gin

TTT

Phe 245

*CAC

His

GGC

Gly

ACC

Thr

AAT

Asn

GCC

Ala

AAG

150

CAT

His

TTC

Phe

GTG

Val1

CGT

Arg

AAG

Lys 230

GAT

Asp

S

S

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 GAT CGG ATC CAT Giu Thr Leu Ala Lys Giu Lys Gly Met Asn Arg Asp Asp Arg Ile His 250 255 r, n 7 GTC CGA ATC AGC AGC GGA GCC TTG Gly Ala Leu 265 TTG ATC CTT AAC Leu Ile Leu Asn GAG CTG Giu Leu 270 Val ArgIle Ser ATG GAG Met Giu 930 978 GGA GAG Gly Glu 290 CGT CTG AGA GAA Axg Leu Arg Giu

GAA

Giu 285 ATG GAA GAA ATC Met Glu 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 GGC TTC GGA ACA AAA Lys Asp Leu Met Gly Phe Giy Thr Lys 305

CC.'

Pro 310 1026 CGT CAC ATT ACC Arg His Ile Thr TTC ACC AGT TTC Phe Thr Ser Phe GCT GTA CAG CCC Ala Vai Gin Pro CAG CAG Gin Gin 325 1074 TCA AAT GCC Ser Asn Ala ATG GGA TTT Met Giy Phe 345

TTG

Leu 330 GTG GGG CTG CTG Val Giy Leu Leu TAC AGC TCT CAC Tyr Ser Ser His CAA GGC CTC Gin Gly Leu 340 CTG GTG GAG Leu Val Glu 1122 1170 GGG ACC TCC CCC Gly Thr Ser Pro

AGT

Ser 350 CCA GCT AAG TCC Pro Aia Lys Ser AGC CGG Ser Arg 360 TOT TGC AGA GAC Cys Cys Arg Asp

TTG

Leu 365 ATG GAG GAG AAA Net Giu Giu Lys

TTT

Phe 370 GAT CAG GTG TGC Asp Gin Val Cys 1218

CAG

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

AAC

Asn 385 TCG CTG ATC CAA Ser Leu Ile Gin

ATG!

Met 390 4 ACA ATC CT.' AAT Thr Ile Leu Asn

TTG

Leu 395 TTG CCC CCC TTG Leu Pro Arg Leu GCA TTC CGA CC.' Ala Phe Arg Pro TCT CC- Ser Ala 405 1266 1314 1362 1410 TTC ACA GAT Phe Thr Asp TGT GTC AAG Cys Val Lys 425

ACC

Thr 410 CAC TAT CTC CAA Gin Tyr Leu Gin

GAT

Asp 415 ACC ATG AAC CAT Thr Met Asn His GTC CTA AGC Val Leu Ser.

420 AAC GAG AAC Lys Ciu Lys

CCT

Arg 430 ACA GCG GCC TTC CAA GCC CTG CCC Thr Ala Ala Phe Gin Ala Leu Cly 435 CTA CTT Leu Leu 440 TCT GTG GCT GTG Ser Val Ala Val

AGG

Arg 445 TCT GAG TTT AAG Ser Giu Phe Lys

GTC

Vai 450 TAT TTG CCT CC Tyr Leu Pro Arg 1458

GTG

50 Val 455 CTG CAC ATC ATC Leu Asp Ile Ile

CGA

Arg 460 GCG GCC CTG CCC Ala Ala Leu Pro

CCA

Pro 465 AAG GAC TTC GCC Lys Asp Phe Ala

CAT

His 47D 1506 1554 AAG AGO CAG AAO Lys Arg Gin Lys

GCA

Al a 475 ATG CAG GTG CAC Met Gin Val Asp ACA OTC TTC ACT Thr Vai Phe Thr TOC ATC Cys Ile 485 AGC ATO CTO GCT CGA CCA ATG CGG CCA GGC ATC CAG CAC GAT ATC AAG Ser Met Leu Ala Arg Ala Net Cly Pro Giy Ile Gin Gin Asp Ile Lys 1602 490 495 Soo GAG CTG CTG GAG CCC ATG CTG GCA GTG GGA CTA AGC CCT GCC CTC ACT Giu Leu Leu Glu Pro Met Leu Ala Val. Gly Leu Ser Pro Ala Leu Thr 505 510 515 1650 GCA GTG Ala Val 520 CTC TAC GAC CTG Leu Tyr Asp Leu

AGC

Ser 525 CGT CAG ATT CCA Arg Gin Ile Pro CTA AAG AAG GAC Leu Lys Lys Asp CAA GAT GGG CTA Gin Asp Gly Leu

CTG

Leu 540 AAA ATG CTG TCC Lys Met Leu Ser

CTG

Leu 545 GTC CTT ATG CAC Val Leu Met His

AAA

Lys 550 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 Gin 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

Gi u 575 GCC AGC GAT GTG Ala Ser Asp Val GGC AGC ATC Gly Ser Ile 580 GGC CAC 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

Glu 595 CTG ACC CAA TTT GTT CGC Leu Thr 600 n Phe I? m 2 A---g

CAC

Ui 605 TGT GCG GAT CAT Cy a A LS p n--.L

TTC

ene 610 CTG AAC AGT GAG Leu Asn Ser .Giu AAG GAG ATC CGC Lys Giu Ile Arg

ATG

Met 620 GAG GCT GCC CGC Glu Ala Ala Arg

ACC

Thr 625 TGC TCC CGC CTG Cys Ser Arg Leu 1938 1986 2034 ACA CCC TCC ATC Thr Pro Scr Ile

CAC

His 635 CTC ATC AGT Leu Ile Ser GGC CAT Gly His 640 GTG CTT Val 'Leu 655 GCT CAT GTG GTT Ala His Val Val AGC CAG Ser Gin 645 ACC GCA GTG 40 Thr Ala Val GTG GTG GCA GAT Val Val Ala Asp AGC AAA CTG Ser Lys Leu CTC GTA GTT Leu Val Val 660 TTG GCG TCC Leu Ala Ser GOG ATA ACA Giy Ile Thr 665 GAT CCT GAC CCT Asp Pro Asp Pro

GAC

Asp 670 ATT CGC TAC TGT Ile Arg Tyr Cys

GTC

Val1 675 CTG GAC Leu Asp 680 GAG CGC TTT GAT Giu Arg Phe Asp

GCA

Al a 685 CAC CTG GCC CAG His Leu Ala Gin

GCG

Ala 690 GAG AAC TTG CAG Giu Asn Leu Gin 2082 2130 2 1 78 2226 2274 TTG TTT GTG GCT Leu Phe Val Ala

CTG

Leu 700 AAT GAC CAG GTG Asn Asp Gin Val CGA CTC ACT AGC Arg Leu Ser Ser 720 rT Phe 705 GAG ATC CGG GAG Giu Ile Arg Giu

CTG

Leu 710 GCC ATC TGC ACT Ala Ile Cys Thr GTG GGC Val Gly 715 ATG AAC CCT GCC TTT GTC Met Asn Pro Ala Phe Val 725 7Z CAG ATT TTG ACA GAG TTG GAG ATG CCT TTC CTG CGC AAG ATG CTC ATC 2322 Met Pro Phe CAC AGT GGG His Ser Gly 745 Leu 730 Arg Lys Met Leu Gin Ile Leu Thr Giu Leu Giu 740 ATT GGA AGA ATC AAA GAG CAG AGT GCC CGC ATG CTG GGG 2370 Ile Gly Arg Ile Lys 750 Giu Gin Ser Ala Arg Met Leu Gly 755 TAC ATG GAG CCT Tyr Met Glu Pro CAC CTG His Leu 760 GTC TCC A.AT GCC Val Ser Asn Ala CGA CTC ATC CGC CCC 2418 Arg Leu Ile Arg Pro 770

ATT

Ile 775 CTG AAG GCA TTA Leu Lys Ala Leu

ATT

Ile 780 TTG AAA CTG AAA Leu Lys Leu Lys CCA GAC CCT GAT Pro Asp Pro Asp

CCA

Pro 790 2466 2514 AAC CCA GGT GTG Asn Pro Gly Val

ATC

Ile 795 AAT AAT GTC CTG Asn Asn Val Leu ACA ATA GGA GAA Thr Ile Gly Glu TTG GCA Leu Aia 805 GTT AGT GGC Val Ser Gly 820 CTG GAA ATG AGG Leu Giu Met Arg

AAA

Lys 815 TGG GTT GAT GAA Trp Vai Asp Giu CT? TTT ATT Leu Phe Ile 820 AAA AGG CAG Lys Arg Gin 2562 2610 ATC ATC ATG Ile Ile Met 825 GAC ATG CTC CAG Asp Met Leu Gin TCC TCT TTG TTG Ser Ser Leu Leu

GCC

Ala 835 4* a..a a GTG GCT 30 Val Ala 840 CTG TGG ACC CTG Leu Trp Thr Leu

GGA

Gi y 845 CAG TTG GTG GCC AGC ACT GGC TAT GTA Gin Leu Val Ala Ser Thr Gly Tyr Val.

850 2658 2706

GTA

Val 855 GAG CCC TAC AGG Giu Pro Tyr Arg

AAG

Lys 860 TAC CCT ACT TTG Tyr Pro Thr Leu

CTT

Leu 865 GAG GTG CTA CTG Glu Vai Leu Leu

AAT

Asn 870 TTT CTG AAG ACT Phe Leu Lys Thr CAG AAC CAG GGT Gin Asn Gin Gly CGC AGA GAG GCC Arg Arg Glu Ala ATC CGT Ile Arg 685 GTG TTA GGG Val Leu Gly AT? GGC ATG Ile Gly Met

CT?

Leu 890 TTA GGG GCT TTG Leu Gly Ala Leu

GAT

Asp 895 CCT TAC AAG CAC Pro Tyr Lys His AAA GTG AAC Lys Val Asn 900 AGC CTG TCA Ser Leu Ser 2754 2802 2850 ATA GAC CAG TCC Ile Asp.Gin Ser

CGG

Arg 910 GAT GCC TCT GCT Asp Ala Ser Ala

GTC

Val 915 GAA TCC 50 Giu Ser 920 AAG TCA AGT CAG Lys Ser Ser Gin

GAT

Asp 925 TCC TCT GAC TAT AGC ACT ACT GAA ATG Ser Ser Asp Tyr Ser Thr Ser Giu Met 930 2898

CTG

Leu 935 CTC AAC ATG GGA AAC TG CCT CTG GAT GAG TTC TAC CCA GCT CTG Val Asn Met Gly Asn Leu Pro Leu Asp Giu Phe Tyr Pro Ala Val 940 945 950 2946 TCC ATG GTG GCC CTG ATG CGG ATC TTC CGA GAC CAG TCA CTC TCT CAT Ser Met Val Ala Leu Met Arg Ile Phe Arg Asp Gin Ser Leu Ser His 2994 77 955 960 965 CAT CAC ACC ATG OTT GTC CAG GCC ATC ACC TTC ATC TTC AAO TCC CTG 3042 His His Thr Met Val Vai Gin Ala Ile Thr Phe Ile Phe Lys Ser Leu 970 975 980 GGA CTC AAA TGT GTG CAG TTC CTG CCC CAG GTC ATG CCC ACG TTC CTT 3090 Giy Leu Lys Cys Vai Gin Phe Leu Pro Gin Val Met Pro Thr Phe Leu 985 990 995 I0 AAT GTC ATT CGA GTC TGT GAT 000 GCC ATC COG GAA TTT TTG TTC CAG 3138 Asn Vai Ile Arg Vai Cys Asp Gly Ala Ile Arg 0Th Phe Leu Phe Gin 1000 1005 1010 CAG CTO GGA ATG TTG GTG TCC TTT GTG AAG AGC CAC ATC AGA CCT TAT 3186 Gin Leu Giy Met Leu Vai Ser Phe Val Lys Ser His Ile Arg Pro Tyr 1015 1020 1025 1030 ATG GAT GAA ATA GTC ACC CTC ATG AGA GAA TTC TGG GTC ATG AAC ACC 3234 Met Asp Giu Ile Val Thr Leu Met Arg Oiu Phe Trp Val Met Asn Thr 1035 1040 1045 TCA ATT CAG AOC ACG ATC ATT CTT CTC ATT GAG CAA ATT GTG GTA OCT 3282 Ser Ile Gin Ser Thr Ile Ile Leu Leu Ile Giu Gin Ile Val Val Aia 1050 1055 1060 CTT 000 GOT GAA TTT AAG CTC TAC CTG CCC CAG CTO ATC CCA CAC ATG 3330 Leu~I y Ow OiGhlisT~ TrL~ r T Leu iie Pro His Met 1065 1070 1075 :::CTG COT GTC TTC ATG CAT GAC AAC AOC CCA GGC COC ATT OTC TCT ATC 3378 Leu Arg Val Phe Met His Asp Asn Ser Pro Oiy Arg Ile Vai Ser Ile 9..1080 -1085 .1090 35 AAG TTA CTO OCT OCA ATC CAO CTG TTT GOC 0CC AAC CTO OAT GAC TAC 3426 *Lys Leu Leu Aia Ala Ile Gin Leu Phe Gly Ala Asn Leu Asp Asp Tyr *.*1095 1100 1105 1110 CTG CAT TTA CTO CTG CCT. CCT ATT OTT AAO -TTG TTT OAT GCC CCT GAA.....3474 40 Leu His Leu Leu Leu Pro Pro Ile Vai Lys Leu Phe-Asp Ala Pro Clii ills 11 1120 1125 OCT CCA CTO CCA TCT CGA AAG OCA OCO CTA GAO ACT OTO GAC CCC CTO 3522 Ala Pro Leu Pro Ser Arg Lys Ala Aia Leu Oiu Thr Val Asp Arg Leu 1130 1135 1140 ACO GAO TCC CTO OAT TTC ACT GAC TAT 0CC TCC COO ATC ATT CAC CCT 3570 Thr Giu Ser Leu Asp Phe Thr Asp Tyr Ala Ser Arg Ile Ile His Pro 2 145 1150 1155 9 AT OTT COA ACA CTO GAC CAO AOC CCA OAA CTO COC TCC ACA 0CC ATO 3618 Ile Val Arg Thr Leu Asp Gin Ser Pro Oiu Leu Arg Ser Thr Ala Met 1160 1165 1170 GAC ACO CTO TCT TCA CTT OTT TTT CAC CTG 0CC AAC AAO TAC CAA ATT 3666 Asp Thr Leu Ser Ser Leu Val Phe Oln Leu Cly Lys Lys Tyr Gin Ile 1175 1180 1185 1190 TTC ATT CCA ATG GTG AAT AAA GTT CTG GTG CGA CAC CGA ATC AAT CAT 3714 Phe Ile Pro met Val Asn Lys Val Leu Val Arg His Arg Ile Asri His 1195 1200 1205 CAG CGC TAT GAT GTG CTC ATC TGC AGA ATT GTC AAG GGA TAC ACA CTT 3762 Gin ArS Ty Asp Val. Lau lie Cys Arg lie Val. Lys Gly Tyr Thr Leu 1210 1215 1220 GCT GAT GAA GAG GAG GAT CCT TTG ATT TAC CAG CAT CGG ATG CTT AGG 3810 Ala Asp Giu Glu Giu Asp Pro Leu Ile Tyr Gin His Arg Met Leu Arg 1225 1230 1235 AGT GGC CAA GGG GAT GCA TTG GCT ACT GGA CCA GTG GAA ACA GGA CCC 3858 Ser Gly Gin Gly Asp Ala Leu Ala Ser Gly Pro Val Glu Thr Gly Pro 1240 1245 1250 ATG AAG AAA CTG CAC GTC AGC ACC ATC AAC CTC CAA AAG GCC TGG GGC 3906 Met Lys Lys Leu His Val Ser Thr Ile Asn Leu Gin Lys Ala Trp Gly 1255 1260 1265 1270 GCT GCC AGG AGG GTC TCC AAA GAT GAC TGG CTG GAA TGG CTG AGA CGG 3954 Ala Ala Arg Arg Val Ser Lys Asp Asp Trp Leu Giu Trp Leu Arg Arg 1275 1280 1285 CTG AGC CTG GAG CTG CTG AAG GAC TCA TCA TCG CCC TCC CTG CGC TCC 4002 Leu Ser Leu Giu Leu Leu Lys Asp Ser Ser Ser Pro Ser Leu Arg Ser 1290 1295 1300 TGC TCC CCC CTG GCA CAG CCC TAC AAC CCG ATG GCC AGC GAT CTC TTC 4050 Cys Trp, Ala Leu Ala Gin Ala Tyr Asn Pro Met Ala Arg Asp Leu Phe 1305 1310 1315 AAT GCT OCA TTT GTG TCC TGC TGG TCT GAA CTG AAT CAA CAT CAA CAG 4098 :::Asn Ala Ala Phe Val Ser Cys Trp Ser Ciu Leu Asn Giu Asp Gin Gin 35 1320 1325 1330 GAT GAG CTC ATC AGA AGC ATC GAG TTG GCC CTC ACC TCA CAA CAC ATC 4146 Asp Glu Leu Ile Arg Ser Ile Ciu Leu Ala Leu Thr Ser Gin Asp Ile 1335 -1340.. 1345 1350 GCT GAA GTC ACA CAG ACC CTC TTA AAC TTG GCT GAA TTC ATG GAA CAC 4194 *Ala Giu Val Thr Gin Thr Leu Leu Asn Leu Ala Giu Phe Met Glu His 1355 1360 1365 45 ACT GAC AAG GGC CCC CTG CCA CTC AGA GAT GAC AAT GGC ATT GTT CTG 4242 Ser Asp Lys Gly Pro Leu Pro Leu Arg Asp Asp Asn Gly Ile Val Leu *C1370 1375 1380 *CTG GGT GAG AGA GCT GCC AAG TCC CGA GCA TAT CCC AAA GCA CTA CAC 4290 50 Leu Giy Giu Arg Ala Ala Lys Cys Arg Ala Tyr Ala Lys Ala Leu His 1385 1390 1395 TAC AAA GAA CTG GAG TTC CAC AAA GGC CCC ACC CCT GCC ATT CTA GAA 4338 Tyr Lys Clu Leu Ciu Phe Gin Lys Cly Pro Thr Pro Ala Ile Leu Giu 1400 1405 1410 TCT CTC ATC ACC ATT AAT AAT AAC CTA CAC CAG CCC GAG GCA CC CC 4386 Ser Leu Ile Ser Ile Asn Asn Lys Leu Gin Gin Pro Ciu Ala Ala Ala 1415 1420 1425 1430 GGA GTG TTA GAA TAT GCC ATG AA.A CAC TTT GGA GAG CTG GAG ATC CAG 4434 Gly Val Leu Giu Tyr Ala Met Lys His Phe Gly Giu Leu Glu Ile Gin 1435 1440 1445 GCT ACC TGG TAT GAG AAA CTG CAC GAG TGG GAG GAT GCC CTT GTG GCC 4482 Ala Thr Trp Tyr Glu Lys Leu His Giu Trp Giu Asp Ala Leu Vai Aia 1450 1455 1460 TAT GAC AAG AAA ATG GAC ACC AAC AAG GAC GAC CCA GAG CTG ATG CTG 4530 Tyr Asp Lys Lys Met Asp Thr Asn Lys Asp Asp Pro Giu Leu Met Leu 1465 1470 1475 GGC CGC ATG CGC TGC CTC GAG GCC TTG GGG GAA TGG GGT CAA CTC CAC 4578 Gly Arg Met Arg Cys Leu Giu Ala Leu Giy Giu Trp Gly Gin Leu His 1480 1485 1490 CAG CAG TGC TGT GAA A.AG TGG ACC CTG GTT AAT GAT GAG ACC CAA GCC 4626 Gln Gin Cys Cys Glu Lys Trp, Thr Leu Vai Asn Asp Giu Thr Gin Ala 1495 1500 1505 1510 AAG ATG GCC CGG ATG GCT GCT GCA. GCT GCA TGG GGT TTA GGT CAG TGG 4674 Lys Met Ala Arg Met Ala Ala Ala Ala Ala Trp Gly Leu Gly Gin Trp 1515 1520 1525 GAC AGC ATG GAA GAA TAC ACC TGT ATG ATC CCT CGG GAC ACC CAT GAT 4722 Asp Ser Met Glu Glu Tvr Thr ryz T Arg AsY Tiris ASP 1530 1535 1540 GGG GCA TTT TAT AGA GCT GTG CTG GCA CTG CAT CAG GAC CTC TTC TCC 4 77 0 0eeGly Ala Phe Tyr Arg Ala Val Leu Ala Leu His Gin Asp Leu Phe Ser 1545 1550 1555 ses: 35 TTG GCA CAA CAG TGC ATT GAC AAG GCC AGG GAC CTG CTG GAT GCT GAA 4818 Leu Ala Gln Gin Cys Ile Asp Lys Ala Arg Asp Leu Leu Asp Ala Glu *.*1560 2565 1570 TTA ACT GCA ATG GCA GGA GAG AGT TAC ACT CGG GCA TAT GGG 6CC ATG 4866 40 Leu Thr Ala Met Ala Gly Glu Ser Tyr Ser Arg Ala Tyr Gly Ala Met 1575 1580 1585 1590 GTT TCT TGC CAC ATG CTG TCC GAG CTG GAG GAG GT T ATC CAG TAC AAA 4914 Val Ser Cys His Met Leu Ser Glu Leu Giu Giu Val Ile Gin Tyr Lys 1595 1600 1605 *CCTT GTC CCC GAG CGA CGA GAG ATC ATC CGC CAG ATC TGG TGG GAG AGA 4962 Leu. Val Pro Giu Arg Arg Gu Ile Ile Arg Gin Ile Trp, Trp Giu Arg 501610 1615 1620 CTG CAG GGC TGC CAG CGT ATC GTA GAG GAC TGG CAG AAA ATC CTT ATG 5010 Leu Gin Gly Cys Gin Arg Ile Val Giu Asp Trp Gin Lys Ile Leu Met 1625 1630 1635 GTG CGG TCC CTT GTG GTC AGC CCT CAT GAA GAC ATG AGA ACC TGG CTC 5058 Val Axg Ser Leu Val Val Ser Pro His Glu Asp Met Arg Thr Trp Leu 1640 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 .165S5 1660 1665 1670 AAA ACT TTA GTG TTG CTC CTG GGA GTT GAT CCG TCT CGC CAJA CTT GAC 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 CAT 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 CCA TGC 5346 Glu Asp Gin Gin His Lys Gin Clu Leu His Lys Leu Met Ala Arg Cys 1735 1740 1745 17 5 0 TTC CTG AAA CTT GGA GAG TGG CAG CTG AAT CTA CAG GCC ATC AAT GAG 5394 Phe Leu Lys Leu Gly Giu Trp, Gin Leu Asn Leu Gin Gly Ile Asn Clu 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 Clu His 17'70 1775 1780 *GAC CGC ACC TGG TAC AAG GCC TGG CAT GCG TGG GCA CTG ATG AAC TTC 5490 *Asp Arg Ser Trp Ty r 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 1B05 1810 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 Aia Thr Thr 1815 1820 1825 1830 GCC GCC ACC ACG CCC GCC ACT GCC ACC ACC ACT CCC 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 ACT GAG AGC GAG GCC GAG AGC ACC GAG AAC AGC CCC ACC CCA 5682 50 Ser Asn Ser Glu Ser Glu Ala Glu Ser Thr Giu Asn Ser Pro Thr Pro 1850 1855 1860 TCG CCC CTG CAG AAG AAG GTC ACT GAG CAT 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 ATC TAC ACG GTG CCT CCC GTC CAG GGC TTC TTC CGT TCC ATC TCC TTG 577 8 Met Tyr Thr Val Pro Ala Val Gin Cly Phe Phe Arg Ser Ile Ser Leu 1880 1880e188 1890 TCA C-GA GGC Ser Axg Gay 1895 AAC AAC CTC CAG GAT Asn Asn Leu Gin Asp 1900 ACA CTC AGA GTT Thr Leu Arg Val 1905 GTC AAT GAG GC-C Val Asn Glu Ala 1920 CTC ACC TTA TGG Leu Thr Leu Trp, TTA GTG GAG 000 Leu Val Giu Gly 1925 5826 TTT GAT TAT GGT C-AC TGG CC-A GAT Phe Asp Tyr Gly His Trp Pro Asp 1915 GTG AAA GCC ATC C-AG ATT GAT ACC Val Lys Ala Ile Gin Ile Asp Thr 1930 5874 5922 TGG C-TA CAG GTT ATA C-C7 CAG CTC Trp Leu Gin Val Ile Pro Gin Leu 1935 1940 ATT GCA AGA ATT GAT Ile Ala Arg Ile Asp 1945 AC-G CC-C AGA CCC TTG Thr Pro Arg Pro Leu 1950 GTG GGA CGT CTC AT? C-AC Val Giy Arg Leu Ile His 1955 5970 6018 C-AG CTT C-TC Gin Leu 1,eu 1960 AC-A GAC ATT Thr Asp Ile GGT CGG Gl y Arg 1965 TAC CAC CCC C-AG GC-C CTC ATC TAC Tyr His Pro Gin Ala Leu Ile Tyr 1970 C-C-A C-TG Pro Leu 19-75 AC-A GTG GCT Thr Val Ala TC-T AAG Ser Lys 1980 TCT ACC ACG Ser Thr Thr ACA GC-C C-GO Thr Ala Arg 1985 C-AC AAT CA His Asn Ala 1990 6066 GCC AAC AAG AT? C-TG AAG AAC ATG TGT I Azn Lys lie JLeu Ly's Asn Met Cys GAG C-AC AGC Giu His Ser 2000 AAC ACC CTG GTC Asn Thr Leu Val 2005 6114 C-AG C-AG GCC Gin Gin Ala ATG ATG GTG Met Met Val 2010 AGC GAG GAG C-TO ATC Ser Giu Giu Leu Ile 203.5 C-GA GTG 0CC ATC C-TC Arg Val Ala Ile Leu 2020 TGG C-AT GAG ATG Trp His Giu Met 2025 TGG C-AT GAA Trp His Glu GGC C-TG Oly Leu 2030 GAA GAG GCA Glu Glu Ala TCT C-GT TTG TAC Ser Arg Leu Tyr 2035 6162 6210 6258 6306 TTT GGG GAA 40 Phe Giy Glu 2040 C-AT GCT ATG His Ala Met 2055 AGO AAC GTG Arg Asn Val AAA GGC ATG Lys Giy Met 2045 TTT GAG GTG CTG GAG CCC-TTG Phe Glu Val Leu Glu Pro Leu 2050 ATG GAA COG GGC Met Giu Arg Oly 2060 C-C-C C-AG ACT C-TG AAG Pro Gin Thr Leu Lys 2065 GAA ACA TCC TTT Giu Thr Ser Phe 2070 AAT C-AG GC-C Asn Gin Ala AAG TAC ATG Lys Tyr Met TAT GOT C-GA Tyr Gly Arg 2075 AAA TCA GG Lys Ser Gly 2090 OAT TTA ATG Asp Leu Met GAG GCC Glu Ala 2080 C-AA GAG TGG Gin Glu Trp TOC AGO Cys Arg 2085 AAT GTC AAG GAC CTC ACC Asn Val Lys Asp Leu Thr 2095 C-AA GCC TOG GAC Gin Ala Trp Asp 2100 6354 6402 6450 C-TC TAT TAT C-AT GTO TTC C-GA C-GA ATC TCA AAG C-AG C-TO C-CT C-AG C-TC Leu Tyr Tyr His Val Phe Arg Arg Ile Ser Lys Gin Leu Pro Gin Leu 2105 2110 2115 ACA TCC TTA GAG CTG CAA TAT GTT Thr Ser Leu Giu Leu Gin Tyr Val 2120 2125 GAC CTT GAA TTG GCT GTG CCA GGA Asp Leu Giu Leu Ala Val Pro Gly 2135 2140 82 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 TCC ATA Gin Ser Ile 2155 GCA CCG TCT Ala Pro Ser TTG CAA GTC Leu Gin Vai 2160 ATC ACA TCC AAG Ile Thr Ser Lys 2165 6594 CAG AGG CCC Gin Arg Pro CGG AAA Arg Lys 2170 TTG ACA CTT Leu Thr Leu ATG GGC Met Gly 2175 AGC AAC OGA Ser Asn Gly CAT GAG TTT His Giu Phe 2180 GTT TTC CTT CTA Val Phe Leu Leu 2185 AAA GGC CAT Lys Gly His GAA GAT Glu Asp 2190 CTG CGC CAG Leu Arg Gin GAT GAG CGT GTG Asp Giu Arg Val 2195 6642 6690 6738 6786 ATG CAG CTC Met Gin Leu 2200 TCT CTT CGG Ser Leu Arg 2215 TTC GGC CTG Phe Gly Leu GTT AAC ACC CTT CTG Val Asn Thr Leu Leu 2205 GCC AAT GAC CCA ACA Ala Asn Asp Pro Thr 2210 AAA AAC CTC AGC ATC Lys Asn Leu Ser Ile 2220 CAG AGA TAC OCT Gin Arg Tyr Ala 2225 GTC ATC CCT Val Ile Pro

TTA

Leu 2230 TCG ACC AAC TCG ser Thr Asn Ser GGC CTC Gly Leu 2235 ATT GGC TGG lie Gly Trp GTT CCC Val Pro 2240 CAC TGT GAC His Cys Asp ACA CTG Thr Leu 2245 6834 CAC GCC CTC His Ala Leu ATC CGG Ile Arg 2250 GAC TAC AGO Asp Tyr Arg GAG AAG AAG Glu Lys Lys 2255 AAG ATC CTT CTC AAC Lys Ile Leu Leu Asn 2260 ATC GAG CAT CGC ATC lie Giu His Arg lle 2265 ATG TTG CGG ATO Met Leu 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 Glu Val 2285 TTT GAG CAT Phe Giu His GCC GTC Ala Val 2290 AAT AAT ACA Asn Asn Thr 6882 6930 6978 7026 70'74 7122 OCT GGG Ala Gly 2295 GAC GAC CTG Asp Asp Leu GCC AAG Ala Lys 2300 CTG CTG TGG Leu Leu Trp CTG AAA Leu Lys 2305 AGC CCC AGC Ser Pro Ser

TCC

Ser 2310 GAG GTG TG TTT GAC CGA AGA ACC AAT Giu Val Trp Phe Asp Arg Arg Thr Asn 2315 TAT ACC Tyr Thr 2320 CGT TCT TTA Arg Ser Leu GCG GTC Ala Val 2325 ATG TCA ATG Met Ser Met OTT GGG TAT ATT Val Gly Tyr Ile 2330 TTA GGC CTG GGA Leu Gly Leu Oly 2335 OAT AGA CAC CCA TCC Asp Arg His Pro Ser 2340 AAC CTG ATG CTG GAC COT CTG AGT GGG AAG ATC CTG CAC ATT GAC TTT Asn Leu Met Leu Asp Arg Leu Ser Gly Lys Ile Leu His Ile Asp Phe 7170 2345 2350 GGG GAC TGC TTT GAG GTT GCT ATG ACC 83

CGA

2355 GAG AAG TTT CCA GAG AAG Gly Asp Cys Phe Giu Val Ala Met Thr Arg Giu Lys Phe Pro Giu Lys 2360 2365 AT? CCA TTT AGA CTA ACA AGA ATG Ile Pro Phe Arg Leu Thr Arg Met 2370

TTG

Leu 2375 2380 GGC CTG GAT GGC AAC TAC AGA ATC ACA Gly Leu Asp Gly Asn Tyr Arg Ile Thr 2395 CTG CGA GAG CAC AAG GAC ACT GTC ATG Leu Arg Giu His Lys Asp Ser Val Met 2410 2415 TAT GAC CCC TTG CTG AAC TGG AGG CTG Tyr Asp Pro Leu Leu Asn Trp Arg Leu1 2425 2430 AAC AAG CGA TCC CGA ACG AGG ACG GAT Asn Lys Arg Ser Arg Thr Axg Thr Asp 2440 2445 GTC GAA AT? TTG GAC GGT GTG GAA CTT C Val Glu Ile Leu ASv Glv Val GC11, L.oi" r 2455 2460 ACG GGG ACC AC'A GTG CCA GAA TCT AT? Thr Gly Thr Thr Vai Pro Giu Ser Ile H~ 2475 2 TTG CTG AA.A CCA GAG GCC CTA AAT AAG iLeu Val Lys Pro Giu Ala Leu Asn Lys L ACC AAT C? Thr Asn Ala 2385 TGC CAC ACA Cys His Thr 2400 GCC GTG CTG

ATG

Met

CTG

Val

GAG

Clu

ATG

Met GTT ACA Val Thr 2390 GAG CTC Giu Val 2405 7218 7266 7314 7362 Ala Val Leu Ciu Ala Phe Val 2420 kTG CAC ACA AAT ACC AAA GGC ~4et Asp Thr Asn Thr Lys Gly 2435 rCC TAC TCT GCT GGC CAG TCA ~er Tyr Ser Ala Gly Gin Ser 2450 ;GA GAG CCA GCC CAT AAG AAA u" rc Ala HI'S Lys Lys5 2465 2470 AT TCT TTC AT? GGA GAC GGT [is Ser Phe Ile Gly Asp Gly 480 2485 AA GCT ATC CAG AT? AT? AAC ys Ala Ile Gin Ile Ile Asn 2500 AC TTC TCT-CAT GATr GAC ACT '7410 '745 8 7506 7554 7602 7650.

2490 2495 GG G AGG GT? Arg Val CGA GA? Arg Asp 2505 AAG CTC ACT GGT Lys Leu Thr Gly 2510 Arg Asp Phe Ser His Asp Asp Thr 2515 TTG CAT CT? CCA ACG CAA CTT GAG CTG CTC ATC AAA CAA GCG ACA TCC Leu Asp Val Pro Thr Gin Val Ciu Leu Leu Ile Lys Gin Ala Thr Ser 2520 2525 2530 CAT GAA AAC CTC TGC CAG TCC TAT ATT GGC TGG TGC CC? TTC TGG His Giu Asn Leu Cys Gin Cys Tyr Ile Gly Trp Cys Pro Phe Trp 2535 2540 2545 TAACTGGAGG CCCAGATGTG CCCATCACGT TTTTTCTGAG GCTTTTGTAC TTTAGTAAAT GCTTCCACTA AACTGAAAAA A INFORMATION FOR SEQ ID NO:12: Ci) SEQUENCE CHARACTERISTICS: 7698 7743 7803 7824 LENGTH: 2549 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein Met 1 Ser Asr Thr Asp Asn Glu xAsn Ala Val 14 5 Ash Ile Asn Ala Pro 225 Ala (xi) SEQUENCE Leu Gly Thr Gly 5 Asn Val Ser Val Glu Giu Thr Arg Met Glu Leu Arg Gln Leu Asn His Glu Arg Lys Gly 85 Gly Gly Asn Ala 100 Leu Leu Pro Ser 115 Ile Gly Arg Leu 130 Glu Phe Glu Val Glu Gly Arg Arg 165 Ser Val Pro Thr Ile Phe Val Ala 195 Val Ala Ala Leu 210 Lys Glu Met Gln Glu Lys Gly Phe 245 DESCRIPTION: SEQ ID NO: 12: Pro Ala Ala Ala Thr Thr Ala Ala Thr Thr Ser 10 Leu Ala Glu His 70 Gi y Thr Asn Al a Lys 150 His Phe Val1 Arg Lys 230 ksp Gln Lys Met 55 Ile Ile Arg Asp Met 135 Arg Al a Phe Trp Al a 215 Pro Glu Glr Ala 40 Ser Phe Leu Ile Pro 120 Al a Al a Al a Phe Asp 200 Cys Gin Thr a 1Ph( 25 Ala *Glr Glu *Ala Gly 105 Val Gly Leu Val Gin 185 Pro Leu Tx-p Leu Ala Ser Lys Giu Glu Glu Leu Val 75 Ile Ala 90 Arg Phe Val Met Asp -hr Glu Trp 155 LetuiVa 1 170 Gin Val Lys Gin Ile Leu Tyr Arg 1 235 Ala Lys C 250 Gl~ Let Ser Ser Ser Ala Giu Phe 140 Leu Leu Gln k.la rhr 220 is flu eLeu aGin -Thr *Ser *Leu Asn Met 125 Thr Gly Arg Pro Ile 205 Thr4 Thr Lys Lys 3C His Arg Ser Ile T1yr 110 Al a Ala Al a G1l4 Phe 190 Arg Gln Phe Ser Phe Asp Gly Leu Ser Gl u Asp Leu 175 Phe Glu Arg Glu Met3 255 Arg Val Tyr Al a ValI Arq Lys Tyr Arg 160 Ala A*sp 3ly Glu 1lu 240 Lsn Arg Asp Asp Arg Ile His Gly Ala Leu Leu Ile Leu Asn Glu Leu Val 260 265 270 Leu Arg Giu Giu Met Glu Giu 285 Arg Ile Ser Ser Met Glu Gly Giu Arg 275 280 Ile Thr Cin Gin Gin Leu xVal Hi s Asp Lys 'r Cys Lys Asp 290 295 Gly Phe Gly Thr Lys Pro Arg His Ile 305 310 I0 Ala Val GIn Pro Gin Gin Ser Asn Ala 325 5cr His Gin Gly Leu Met Gly Phe 340 345 Lys 5cr Thr Leu Val Giu Ser Arg Cys 355 360 Lys Phe Asp Gin Vai Cys Gin Tr-p Val 370 375 Asn Ser Leu Ile Gin Met Thr Ile Leu 385 390 Ala Phe Arg Pro Ser Ala Phe Thr Asp 405 Met Asn His Vai Leu Ser Cys Val Lys Th Lei 334 G1~ Cy..

Lei.

Asn Thr 410 Lys Val Ile Lys .l a 190 3lu ryr ;iy r

LI

0 e, Pro 315 Val Thr Arg Lys Leu 395 Gin Giu 300 *Phe Gly Ser Asp Cys 380 Leu Tyr Lys Thr Leu Pro Leu 365 Arg Pro Leu Giu S er Leu Ser 350 Met Asn Arg Gin Arg Leu Phe Gly 335 Pro Gi u Ser Le u Asp 435 Thr meL Gin 320 Tyr Ala Gi u Lys Al a 400 rhr Al a A. A*.

A A Ala Lys' Pro 465 40 Thr N Ilie C Leu S Pro G Phe Val1 450 ys Pal ;In er In Gin 435 Tyr Asp Phe Gin Pro 515 Leu 420 Ala Leu Phe Thr Asp 500 Al a Lys Leu Pro Al a Cys 485 Ile I Leu 'I Lys Gi y Arg iis 470 Ilie .ys 'hr xsp Leu Val1 455 Lys Ser Giu Ala Ile 535 Pro 425 Leu Ser 440 Leu Asp Arg Gin Met Leu Leu Leu 505 Val Leu 520 Gin Asp

G

Ala Val' Arg 445 Ile Arg Ai a 460 Ala Met Gin 475 Arg Ala Met Pro Met Leu Asp Leu Ser 525 Leu Leu Lys 540 Pro Gly Met 5cr Al a Val2 Gi y Al a 510 Arg Met Pro Giu Leu Asp Pro 495 Val Gin Leu Lys Phe Pro Ala 4 Gly Gly Ile Gly 430 530 Leu Vai Leu Met His Lys 545 550 Leu Arg His 560 Leu Ala His Gin Leu 565 Ala Ser Pro Giy Leu 570 Thr Thr Leu Pro Giu Ala 575 Ser Asp Val Gly Ser Ile Thr Leu Ala Leu Arg Thr Leu Gly Ser Phe 580 585 590 Giu Phe Giu Gly His Ser Leu Thr Gin Phe Val Arg His Cys Ala Asp 595 600 605 His Phe Leu Asn Ser Glu 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 Aia Val Gin Val Vai Aia Asp Val Leo 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 Aia His Leu Ala 675 680 685 Gin Ala Giu Asn Leu Gin Ala Leo Phe Val Ala Leu Asn Asp Gin Val 690 695 700 Phe Gu Ile Arg Giu Leu Ala Ile Cys Thr Val Gly Arg Leu Ser Ser 705 710 715 720 Met Asn Pro Ala Php V-1 MAot- -h ~Ag Lys Mt=L Leu lie u.in 725 730 735 Ile Leu Thr Giu Leu Giu His Ser Gly Ile Gly Arg Ile Lys Glu Gin 740 745 750 Ala Arg Met Leu Gly His Leu Val Ser Asn Ala Pro Arg Leo Ile 35 755 760 765 *..Arg Pro Tyr Met Glu Pro Ile Leo Lys Ala Leo Ile Leu Lys Leo Lys 770 775 780 Asp Pro Asp Pro Asp Pro Asn Pro Gly Val Ile Asn Asn Val Leu Ala 785 790 795 B0o Thr Ile Gly Glu Leo Ala Gin Val Ser Gly Leo Glu met Arg Lys Trp 805 810 815 Vai Asp Glu Leo Phele Ile Ile Met Asp met Leo Gin Asp Ser Ser 820 825 830 Leo Leu Ala Lys Arg Gin Val Ala Leo Trp Thr Leo Gly Gin Leo Val 835 840 845 Ala Ser Thr Gly Tyr Val Val Glu Pro Tyr Arg Lys Tyr Pro Thr Leu 850 855 860 Leo Giu Val Leu Leu Asn Phe Leo Lys Thr Gbu Gin Asn Gin Gly Thr 865 870 875 880 Arg Arg G2.o Ala Ile Arg Val Leu Gly Leo Leu Gly Ala Leu Asp Pro 87 885 890 895 Tyr Lys His Lys Vai An 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 Gly Asn Leu Pro Leu Asp 930 935 940 Giu Phe Tyr Pro Aia Vai Ser met Val Aia Leu Met Arg Ile Phe Arg 945 950 955 960 Asp Gin Ser Leu Ser His His His Thr Met Val Val Gin Aia Ile Thr 965 970 975 Phe Ile Phe Lys Ser Leu Gly Leu Lys Cys Vai Gin Phe Leu Pro Gin 980 985 990 Val Met Pro Thr Phe Leu Asn Val Ile Arg Val Cys Asp Gly Ala Ile 995 1000 1005 Arg Glu Phe Leu Phe Gin Gin Leu Gly Met Leu Val Ser Phe Val Lys 1010 1015 1020 Ser His Ile Arg Pro Tyr Met Asp Giu Ile Vai Thr Leu Met Arg Giu 1025 1030 1nArInAf 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 *.Giy Arg Ile Val Ser Ile Lys Leu Leu Ala Ala Ile Gin Leu Phe Gly 40 1090 1095 1100 Ala Asn Leu Asp Asp Tyr Leu His Leu Leu Leu Pro Pro Ile Val Lys 1105 1110 1115 1120 45 Leu Phe Asp Aia Pro Giu Ala Pro Leu Pro Ser Arg Lys Ala Ala Leu 1125 1130 1135 too. Giu Thr Val Asp Arg Leu Thr Glu Ser Leu Asp Phe Thr Asp Tyr Ala 1140 1145 1150 Ser Arg Ile Ile His Pro Ile Vai 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 Vai 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 Glu Glu Asp Pro Leu Ile Tyr i220 i2.25 1230 Gin His Arg Met Leu Arg Ser Gly Gin Giy Asp Ala Leu Ala Ser Gly 1235 1240 1245 Pro Vai Giu Thr Gly Pro Met Lys Lys Leu His Val Ser Thr Ile Asri 1250 1255 1260 Leu Gin Lys Ala Trp, Gly Ala Ala Arg Arg Val Ser Lys Asp Asp Trp 1265 1270 1275 1280 Leu Glu Trp Leu Arg Arg Leu Ser Leu Glu 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 Glu 1315 1320 1325 Leu Asn Giu Asp Gin Gin Asp Giu Leu Ile Arg Ser Ile Glu 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 1375 S 35 Asp Asn Gly Ile Val Leu Leu Gly Giu Arg Ala Ala Lys Cys Arg Ala 1390 1385 1390 Tyr Ala Lys Ala Leu His Tyr Lys Giu Leu Giu Phe Gin Lys Gly Pro 1395 1400 1405 *Thr Pro Ala Ile Leu Glu Ser Leu Ile Ser Ile Asn Asn Lys Leu Gin 1410 1415 1420 *.Gin Pro Giu Ala Ala Ala Gly Vai Leu Glu Tyr Ala Met Lys His Phe 45 1425 1430 1435 1440 Gly Giu Leu Glu Ile Gin Ala Thr Trp Tyr Giu Lys Leu His Giu Trp 1445 1450 1455 50 Glu 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 Gl u Ala Leu Gly 1475 1480 1485 *4 Giu Trp, Gly Gin Leu His Gin Gin Cys Cys Giu Lys Trp, Thr Leu Val 1490 1495 1500

I

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 Ile 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 Lvs 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 S* Pro Ser Arg Gin Leu Asp His Pro Leu Pro Thr Val His Pro Gin Val 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 Gln His Ala Ile Ala Thr Glu Asp Gin Gln 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 Gin Gly Ile Asn Glu Ser Thr Ile Pro Lys Val Leu Gin Tyr Tyr 50 1765 1770 1775

S

er 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 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 Glu Gly Ser Asn Ser Glu Ser Glu Ala Glu Ser Thr 1845 1850 1855 Glu Asn Ser Pro Thr Pro Ser Pro Leu Gin Lys Lys Val Thr Glu Asp 1860 1865 1870 Leu Ser Lys Thr Leu Leu Met Tyr Thr Val Pro Ala Val G1n 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 Val Asn 2 1905 1910 1915 1920 Glu Ala Leu Val Glu 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 1 9 5 5 o6u 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 Ala Asn Lys Ile Leu Lys Asn Met Cys Glu 1985 1990 1995 2000 35 His Ser Asn Thr Leu Val Gin Gin Ala Met Met Val Ser Glu Glu Leu 2005 2010 2015 40 Ile Arg Val Ala Ile Leu Trp His Glu Met Trp His Glu-Gly Leu Glu 40 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 Glu Pro Leu His Ala Met Met Glu Arg Gly Pro Gin Thr 2050 2055 2060 Leu Lys Glu Thr Ser Phe Asn Gin Ala Tyr Gly Arg Asp Leu Met Glu 2065 2070 2075 2080 5 0 Ala Gin Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn Val Lys Asp 2085 2090 2095 Leu Thr Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg Arg Ile Ser 2100 2105 2110 Lys Gin Leu Pro Gin Leu Thr Ser Leu Glu Leu Gin Tyr Val Ser Pro 2115 2120 2125 9/ Lys Leu Leu Met Cys Arg Asp Leu Giu Leu Ala Val Pro Gly Thr Tyr 2130 2135 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 2165 2170 2175 Ser Asn Gly His Glu Phe Val Phe Leu Leu Lys Gly His Glu Asp Leu 2180 2185 2190 Arg Gin Asp Glu Arg Vai Met Gin Leu Phe Giy Leu Val Asn Thr Leu 2195 2200 2205 Leu Ala Asn Asp Pro Thr Ser Leu Arg Lys Asn Leu Ser Ile Gin Arg 2210 2215 2220 Tyr Ala'Val le. Pro Leu Ser Thr Asn Ser Gly Leu Ile Giy Trp Vai 2225 2230 2235 2240 Pro His Cys Asp Thr Leu His Ala Leu Ile Arg Asp Tyr Arg Giu Lys 252245 2250 2255 Lys Lys Ile Leu Leu Asn Ile Giu His Arg leMet Leu Arg Met Ala 2260 2265 2 270 Pro Asp Tyr Asp His Leu Thr Leu Met Gin Lys Vai Giu Vai Phe Giu 2275 2280 2285 His Ala Val Asn Asn Thr Ala Gly Asp Asp Leu Ala Lys Leu Leu Trp 2290 2295 2300 Leu *Lys Ser -Pro' Ser Ser Giu Val Trp Phe Asp Arg Arg.Thr Asn Tyr 2305 2310 2315 2320 ThrArgSerLeu Ala Vai Met Ser Met Val Gly Tyr le Leu Giy e 232523025 23023 *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 Giy Asp Cys Phe Giu Val Ala Met Thr Arg 45 2355 2360 2365 Giu Lys Phe Pro Giu Lys Ile Pro Phe Arg Leu Thr Arg Met Leu Thr 2370 2375 2380 50 Asn Ala Met Giu Val Thr Gly Leu Asp Gly 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 Vai Tyr Asp Pro Leu Leu Asn Ti-p Arg Leu Met 2420 2425 2430 1/2 Asp Thr Asn Thr Lys Gly Asn Lys Arg Ser Arg Thr Arg Thr Asp Ser 2435 2440 2445 Tyr Ser Ala Gly Gin Ser Val Glu Ile Leu Asp Gly Val Glu Leu Gly 2450 2455 2460 Glu Pro Ala His Lys Lys Thr Gly Thr Thr Val Pro Giu Ser Ile His 2465 24'70 2475 2480 Ser Phe Ile Gly Asp Gly Leu Val Lys Pro Glu 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 Giu Leu Leu 2515 2520 2525 Ile Lys Gin Ala Thr Ser His Giu Asn Leu Cys Gin Cys Tyr Ile Gly 2530 2535 2540 Trp Cys Pro Phe Trp 2545 INFORMATION FOR SEQ ID NO:13: (i SEOtJRNCrF Oy'*v,-CTIR S 7 .C LENGTH: 1794 base pairs TYPE: nucleic acid STRANDEDNESS: both TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY:

CDS

LOCATION: 1. .1686 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: TTG GTT TAC CCT TTG ACA GTT GCT ATT ACT TCC GAA TCA ACG AGC CGT Leu Val Tyr Pro Leu Thr Val Ala Ile Thr Ser Glu Ser Thr Ser Arg 1 5 10 AAA AAG GCA GCT CAA TCC ATT ATT GAA AAA ATG CGA GTA CAT TCT CCT 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 GTT Ser Leu Val Asp Gin Ala Giu Leu Val Ser Arg Glu Leu Ile Arg Val 35 40 :55 GCA GTT TTA TGG CAC GAA CAA TGG CAC GAT OCT TTG GAA GAT GCT AGC Ala Val Leu Trp His Civ Gin Trp His Asp Ala Leu Giu Asp Ala Ser 55 48 96 144 192 AGG TTT TTC TTT GGT GAA CAC AAC ACA GAA AAG ATG TTT GAA ACA TTG 240 Arg Phe Phe Phe Gay Glu His Asn Thr Giu Lys Met Phe Giu Thr Leu 70 75 GAA CCA TTA CAT CAA ATG TTG CAA AAG GGA CCA GAA ACG ATG AGG GAA 288 Giu Pro Leu His Gin Met Leu Gin Lys Gly Pro Glu Thr Met Arg Giu 90 CAA GCC TTT GCA AAT GCT TTT GGC AGG GAG TTG ACA GAT GCA TAC GAG 336 Gin Ala Phe Ala Asn Ala Phe Gly Arg Giu Leu Thr Asp Ala Tyr Glu 100 105 110 TGG GTG CTC AAC TTT AGA AGA ACT AAA GAC ATA ACC AAT TTG AAT CAA 384 Trp Val Leu Asn Phe Arg Arg Thr Lys Asp Ile Thr Asn Leu Asn Gin 115 120 125 GCA TGG GAT ATA TAC TAC AAT GTC T-,T AGA AGA GTA AGC AAA CAG GTG 432 Ala Trp Asp Ile Tyr Tyr Asn Val Phe Arg Arg Val Ser Lys Gin Val 130 135 140 CAG CTG TTA GCT AGT CTT GAG TTG CAG TAT GTA TCT CCG GAC TTA GAG 480 Gin Leu Leu Ala Ser Leu Glu Leu Gin Tyr Val Ser Pro Asp Leu Glu 145 150 155 160 CAT GCT CAA GAT TTG GAA TTG GCT GTA CCA GGT ACT TAC CAA GCA GGC 528 His Ala Gin Asp Leu Glu Leu Ala Val Pro Gly Thr Tyr Gln Ala Giy I G 1-lu175 AAA CCT GTG ATC AGA ATA ATC AAA TTT GAT CCT ACT TTT TCG ATT ATT 576 Lys Pro Val Ile Arg Ile Ile Lys Phe Asp Pro Thr Phe Ser Ile Ile :180 185 190 :::TCA TCT AAA CAA AGA CCG AGA AAA TTA TCG TGC AGA GGA AGT GAT GGT 624 35 Ser Ser Lys Gin Arg Pro Arg Lys Leu Ser Cys Arg Gly Ser Asp Gly 195 200 205 **see: AAA GAC TAC CAA TAT GCG TTG AAA GGA CAT GAA GAT ATC AGA CAA GAT 672 *.Lyz Asp Tyr Gin Tyr Ala Leu Lys Gly His Glu Asp Ile Arg Gin-Asp 40 210 215 220 AAC TTA GTG ATG CAA TTG TTT GGT TTG GTT AAT ACG TTG TTG GTA AAT 720 As n Leu Val Met Gin Leu Phe Gly Leu Vai Asn Thr Leu Leu Val Asn *225 230 235 240 GAT CCG GTA TGT TTC AAG AGA CAT TTG GAT ATA CAA CAA TAT CCT GCT 768 Asp Pro Val Cys Phe Lys Arg His Leu Asp Ile Gin Gin TIyr Pro Ala *245 250 255 50 ATT CCA TTA TCA CCA AAA GTG GGA TTG CTT GGT TGG GTT CCA AAT AGT 816 Ile Pro Leu Ser Pro Lys Val Gly Leu Leu Gly Trp Val Pro Asn Ser **260 265 270 GAC ACT TTC CAT GTA TTG ATC AAA GGC TAT CGC GAA TCA AGA AGT ATA 864 55 Asp Thr Phe His Val Leu Ile Lys Gly Tyr Arg Glu Ser A-rg Ser Ile 275 280 285 ATG TTG AAT ATT GAA CAC AGG CTT TTG TTG CAA ATG GCA CCT CAT TAT 912 IV I/ Met Leu Asn Ile Giu His Arg Leu Leu Leu Gin Met Aia Pro Asp Tyr 290 295 300 GAT T7C TTG ACA TTA TTG CAA AAA GT7 GAA GTG TTC ACA AGT GCA ATG Asp 305 Phe Leu Thr Leu Leu Gin 310 Lys Val Glu Va) Phe Thr Ser Ala Met 315 320 GAT AAT TGT AAG Asp Asn Cys Lys

GGA

Gi y 325 CAG GAT TTG TAC Gin Asp Leu Tyr

AAA

Lys 330 GTG TTA TGG CTC Val Leu Trp Leu AAA TCT Lys Ser 335 1008 AAA TCA Lys Ser 7CC GAG GCG TGG TTG Ser Giu 340 Ala Trp Leu GAC CGT 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 Giy

TTG

Leu 365 1056 1104 1152 CAC CCA His Pro 370 TCA AAT TTG ATG Ser Asn Leu Met

TTG

Leu 375 GAC CGT ATT ACT Asp Arg Ile Thr

GGG

Gly 380 AAA GTC ATC CAT Lys Val Ile His

ATT

Ile 385 GAT TTC GGA GAC Asp Phe Gly Asp

TGT

Cys 390 TTT GAA GCA GCA Phe Giu Ala Aia

ATA

Ile 395 TTA CGT GAG AAG Leu Arg Giu Lys

TAT

T

yr 400 1200 1248 CCA GAG AGA r Pro Giu Arg V'al

CCG

Pro 405 TTT AGA TTG ACG Phe Arg Leu Thr ATG CTT AAT TAT GCC ATG Met Leu Asr. Tyr Ala Met 415

S.

S. emS *5*5 0

S.

S 0 es. S

S.

S S

S.

GAA GTT ACT Giu Val Ser ATG AGG GTG Met Arg Val 435 ATA GAG GGC TCG Ile Glu Gly Ser

TTC

Phe 425 AGA ATC ACA TGT Arg Ile Thr Cys GAA CAT GTT Glu His Vai 430 TTG CGT GAT AAT Leu Arg Asp Asn

AAA

Lys 440 GAG TCT TTA ATG GCA ATA TTA GAG Giu Ser Leu Met Ala Ile Leu Giu 1296 1344 1392 GCC 777 Ala Phe 450 GCT TAC CAT CCC Ala Tyr Asp Pro

TTG

Leu 455 ATA AAT TGG GGG Ile Asn Trp Giy

TTT

Phe 460 GAT TTC CCA ACA Asp Phe Pro Thr

AAG

Lys 465 GCG TTG GCT GAA Ala Leu Ala Giu GAA TTA TTA CC Giu Leu Leu Arg 485

TCA

Ser 470 ACG GGT ATA CGT Thr Gly Ile Arg CCA CAA GTC AAC Pro Gin Vai Asn 1440 1488 Of 0:0

GCA

Ala AGA GGA CAG ATT Arg Giy Gin Ile GAA AAA GAA GC7 Giu Lys Ciu Ala GTA AGA Vai Arg 495 TTG CAA AAC CAA AAT GAA TTC GAA Leu Gin Lys Gin Asn Giu Leu Giu 500 GTG TTG AA.A CGT ATT ACC CAT AAG Val Leu Lys Arg Ile Thr Asp Lys 515 S2 0

ATA

Ile 505 AGA AAC GCT AGA Arg Asn Aia Arg CCT GCA TTA Ala Ala Leu 510 1536 1584 TTA ACT CCT AAC CAT ATC AAA CCC Leu Thr Gly Asn Asp Ile Lys Arg 525 1 TTG AGA GGA TTA GAT GTG CCT ACT CAA GTC GAT AAA TTG AT? CAA CAA Leu Arg Gly 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 Ala Thr Ser Val Giu Asn Leu Cys Gin His Tyr Ile Gly Trp Cys Ser 545 550 555 560 TGT TGG TAGGTTGATT ATCGTCATGT GTCGATAAGT ATGGTATTGT GGTAACTATT Cys Trp TTATAAAGGG AAATATTAAA GAA'rTGTATA TTATTAAAAA AAAAAA AACTCGAG INFOR-MATION 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 Tvr Pro Th 1- c: Thr Gei G!u Ser Thy ser Arg 1 510 3 0 Lys Lys Ala Ala Gin Ser Ile Ile Giu :ys Met Arg Val Hi~s Ser Pro 25 Js0 Ser Leu Val Asp Gin Ala Glu Leu Val Sar Arg Giu Leu Ile Arg Val 35 35 40 Ala Val Leu Trp His Glu Gin Trp His Asp Ala Leu Giu Asp Ala Ser 55 Arg Phe Phe 1lne Gly Glu His Asn Thr G2.u Lys Met Phe Glu *rhr Leu 70 75 so Giu Pro Leu His Gin Met Leu Gin Lys Gly Pro Giu Thr Met Arg Giu 90 Gin Ala Phe Ala Asn Ala Phe Gly Arg Glu Leu Thr Asp Ala Tyr Giu 100 105 110 Trp Val Leu Asn Phe Arg Arg Thr Lys Asp Ile Thr Asn Leu Asn Gin 013.5 120 125 Ala T-p, Asp Ile Tyr Tyr Asn Val Phe Arg Arg Val Ser Lys Gin Val 130 135 140 Gin Leu Leu Ala Ser Leu Giu Leu Gin Tyr Vai Ser Pro Asp Leu Glu 145 ISO i55 160 His Ala Gin Asp Leu Giu Leu Ala Val Pro Gly Thr Tyr Gin Ala Giy 1632 1680 1736 1794 165 170 175 Lys Pro Val Ile Arg Ile Ile Lys Phe Asp Pro Thi- Phe Ser Ile Ile 185 190 Gar Ser Lys Gin Arg Pro Arg Lys Leu Ser Cys Arg Gly Ser Asp Gly 195 200 205 Lys Asp Tyr Gin Tyr Ala Leu Lys Gly His Giu Asp Ile Arg Gin Asp 210 215 220 Asn Leu Val Met Gin Leu Phe Gly Leu Val Asn Thr Leu Leu Vai Asn 225 230 235 240 Asp Pro Val Cys Phe Lys Arg His Leu Asp Ile Gin Gin Tyr Pro Aia 245 250 255 Ile Pro Leu Ser Pro Lys Val Gly Leu Leu Gly Ti-p Val Pro Asn Ser 260 265 270 Asp Thr Phe His Val Leu Ile Lys Gly Tyr Arg Giu Ser Arg Ser Ile 275 280 285 Met Leu Asn Ile Giu His Arg Leu Leu Leu Gin Met Ala Pro Asp Tyr 290 295 300 Asp Phe Leu Thr Leu Le Gin Lys Val Giu Val Phe Thr Ser Ala Met 305 310 315 Asp Asn Cys Lys Gly Gin Asp Leu Tyz Lys Val Leu Ti-p Leu Lys Sea- 325 330 335 Lys S-ar Ser Giu Ala Ti-p Leu Asp Arg Arg Thr-Thr.Tyr Thr Arg Ser 340 345 350 ***Leu Ala Val Met Ser Met Val Gly Tyr Ile Leu Gly Leu Giy Asp Arg 355 360 365 .*His Pro Ser Asn L-eu MeZ Leu Asp Arg I2- Thi- Gly Lys Val Ile His 37 0 375 Ile Asp Phe Gly Asp Cys Phe Giu Ala Ala Ile Leu Arg Giu Lys Tyr 385 390 395 400 Pro Giu Arg Val Pro Phe Arg Leu Thr Arg Met Leu Asn Tyr Ala Met 405 410 415 Giu Val Ser Gly Ile Giu Gly Ser Phe Arg Ile Thr Cys Giu His Val 420 425 430 *Met Arg Val Leu Arg Asp Asn Lys Giu Ser Leu Met Ala Ile Leu Giu 435 440 445 Ala Phe Ala Tyr Asp Pro Leu Ile Asn Ti-p Gly Phe Asp Phe Pro Thr 450 455 460 Lys Ala Leu Ala Giu Ser Thi- Gly Ile Arg Val Pro Gin Val Asn Thr 470 475 Ala Leu Val Leu Al a 545 Cys Giu Gin Leu Arg 530 Thr Trp Leu Lys Lys 515 Gi y Ser Leu Gin 500 Arg Leu Val Arg 485 Asn Ile Asp Giu Arg Gly Glu Leu Thr Asp Vai Pro 535 Asn Leu 550 Gin Gi u Lys 520 Thr Cys Ile Ile 505 Leu Gin Gin 91'7 Asp Giu 490 Arg Asn Thr Gly Val Asp His Tyr 555 Lys Giu Aia Arg Asn .Asp 525 Lys Leu 540.

Ile Giy Aila Al a 5 Ile Ile Trp Vai 495 Ala Lys Gin Cys Arg Leu Arg Gin Ser 560 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 399 base pairs TYPE: nucleic acid STRANDEDNESS: singie TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATUVRE: (IA) NAME/KEY: CDS CB.) LOCATION: 1. .399 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1S: GTT ACT CAC GAG TTG ATC AGA.GTA GCC GTT. CTA TG Val Ser HisE Giu Leu Ile Arg Vai Ala Vai Leu Trp 1 5 10 TAT GAA GGA CTG GAA GAT GCG AGC CGC CAA TTT TTC Tyr Giu Giy Leu Giu Asp Aia Ser Arg Gin Phe Phe 20 25 ATA GAA AAA ATG TTT TCT ACT TTA GAA CCT TTA CAT Ile Giu Lys Met Phe Ser Thr Leu Giu Pro Leu His 40 AAT GAG CCT CAA ACG TTA ACT GAG GTA TCG TTT CAG Asn Giu Pro Gin Thr Leu Ser Giu Val Ser Phe Gin s0 55 AGA GAT TTG AAC GAT GCC TAC GAA TGG TTG AAT AAC Arg Asp Leu Asn Asp Aia Tyr Giu Trp Leu Asn Asn 70 75

CAC

His

GTT

Val

AAA

Lys

AAA

Lys

TAC

Tyr GAA TTA Glu Leu GAA CAT Giu His CAC 7TA His Leu TCA TTT Ser Phe AAA AAG Lys Lys

TGG

Trp

AAC

Asn

GGC

Gly

GGT

Gly

TCA

Ser s0 48 96 144 :192 240 18 AAA GAC ATC AAT AAT TTG AAC CAA GCT TOG GAT Ar Lys Asp Ile Asn Asn Leu Asn Gin Ala Trp Asp 11 90 TTC AGA AAA ATA ACA CGT CAA ATA CCA CAG TTACA Phe Arg Lys Ile Thr Arg Gin Ile Pro Gin Leu Gli 100 105 CAG CAT GTT TCT CCC CAG CTT CTG GCT ACT CAT GAI Gin His Val Ser Pro Gin Leu Leu Ala Thr His Asj 115 120 GTT CCT GGG ACA TAT Val Pro Gly 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 ID NO:16: Val Ser His Giu Leu Ile Arg Val Ala Val Leu Trp, 5~ 10 Tyr Glu Gly Leu Giu Asp Ala Ser'Arg Gin Phe Phe 20 25 35 le Glu Lys Met Phe Ser Thr Leu Glu Pro Leu His 35 40 Asn Giu Pro Gin Thr Leu Ser Giu Val Ser Phe Gin .5 60 Arg Asp Leu Asn Asp Ala Tyr Giu Trp Leu Asn Asn 65 70 75 Lys Asp Ile Asn Asn Leu Asn Gin Ala Trp Asp Ile 585 90 Phe A-rg Lys Ilie Thr Axg Gin Ile Pro Gin Leu Gin 100 105 0 Gin His Val Ser Pro Gin Leu Leu Ala Thr His Asp 115 120 Val Pro Gly Thr Tyr 130 INFORMATION FOR SEQ ID NO:17: ITAT TAT AAC GTC e Tyr Tyr Asn Val ~ACC TTA GAC TTA a Thr Leu Asp Leu 110 CTC GAA TTG GCT Leu Giu Leu Ala 125 288 336 384 399 His Val1 Lys Lys Tyr Tyr Thr Leu 125 Giu Giu His Se r Lys Tyr Leu 110 Glu Leu Trp His Asn Leu Gly Phe Gly Lys Ser Asn Val Asp Leu leu Ala Wi 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 DESCR IPTION: SEQ ID NO:17: GTC AGC CAC GAA TTG ATA CGT ATG GCG GTG CTT TGG CAT GAG CAA TGG Val Ser His Glu Leu Ile

S

S

TAT GAG Tyr Glu ACC GAA Thr Glu AGA GGA Arg Gly AGG GAC Arg Asp 35 65 AAA GAT Lys Asp TTC AGG Phe Arg CAA CAT Gin His GTC CCC Val Pro 130

GGT

Gi y

AAA

Lys

CCG

Pro

TTG

Leu

GTT

Val1

AAA

Lys

GTG

Val1 115

GGG

Gly

CTG

Leu

ATG

Met

GAA

Glu

AAT

Asn

AGT

Ser

AT?

Ile 100

TCG

Ser

ACC

Thr

GAT

Asp

TTT

Phe

ACT

Thr

GAC

Asp

AAT

Asn

GGT

Gly

CCA

Pro

CGT

Arg

GAC

Asp

GCT

Ala

TTG

Leu

C

Ala 70

TTA

Leu

AAA

Lys

AAA

Lys Arg

CC

Al a

GCT

Al a

AG

Arg 55

TAC

Tyr

AAC

Asn

CAG

Gin

AG?

Ser

TTA

Leu 40

CAA

Glu

GAA

Gi u

CAA

Gin

TTG

Leu

AGG

Arg 25

GAG

Glu

ATA

Ile

TGG

Trp

CCG

Al a

CCA

Pro 105 10

CAG

Gin

CC?

Pro

TCG

Ser

CTG

Leu

TGG

Trp 90

CAA

Gln

TTT

Phe

CTG

Leu

TTC

Phe

ATG

Met 75

CAC

Asp

TTA

Leu

TT

Phe

TAC

Tyr

CAA

Gin

AAT

Asn

AT?

Ile

CAA

Gin

GGA

Gly

GAA

Glu

AAT

Asn

TAC

Tyr

TAC

Tyr

ACT

Thr

GAA

Glu

ATG

Met

TCT

Ser

AAA

Lys

TAT

Tyr

CTT

Leu 110

CAT

His

CTG

Leu

TTT

Phe

AAA

Lys

AAT

Asn

GAA

Giu

AAT

Asn

AAG

Lys

GGT

Gi y

TCT

Ser s0

GTT

Val1

CTA

Leu 96 144 192 Met Ala Val Leu Trp His Glu Gin Trp 288 CTA CTA TCT GCG CAT GAT TTG GAA TTG GCT Leu Leu Ser Ala His Asp Leu Glu Leu Ala 120 125 INFORMATION FOR SEQ ID NO:1B: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 133 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:i8: Val Ser His Glu Leu Ile Arg Met Ala Val Leu Trp His Giu

I

Tyr Gin Trp Giu Gly Leu Asp Asp Glu Lys Met Phe Ala Ala Ser Arg 25 Ala Leu Giu Gin Phe Phe Pro Leu Tyr Thr Gly Giu His Asn Giu Met Leu Lys Asn Ser Phe Gly Arg Giy Pro 40 Giu Thr Leu Arg 55 Tyr Glu Ile Ser Phe Gin Lys Asp Leu Asn Asp Giu Trp Leu Asn Tyr Lys Lys Asp Val Ser Asn Gly Leu Asn Gin Ala Asp Ile Tyr Tyr Asn Val Glu Leu Phe Arg Lys Gin His Val 115 Lys Gin Leu Leu Gin Thr Leu 110 Pro Lys Leu Leu Ser Ala His A~qn T.-i G1- Ie U 120 12c; Vai Pro 130 Gly Thr Arg 35 INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 531 base pairs TYPE: nucleic acid STRANDEDNESS: both TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: TGACCCTCAC CCCTTCCACC TATCCCAAAA ACCTCACTGG GTCTGTGGAC AAACAACA1RA AATNTTTTCC ANANAGGCCC CAAATGAGNC CCANGGGTCT NTCTTCCATC AGACCCAGTG ATTCTGCGAC TCACACNCTT CAATTCAAGA CCTGACCNCT AGTAGGGAGG TTTANTCAGA TCGCTGGCAN CCTCGGCTGA NCAGATNCAN AGNGGGGNTC GCTGTTCAGT GGG.NCCACCC WCNCTGGCCT TCTTCANCAG GGGTCTGGGA TGTTTTCAGT GGNCCNAANA CNCTGTTTAG AGCCAGGGCT CAGNAAACAG AAAANCTNTC NACATATTGG GGATTATGAN CAGNACCAAN AACCATNTCT ANACNCCATN TTNTATCAGN AN7TTTNAACA TNAA.AAAAAA AAAAAA INFORM~ATION FOR SEQ ID SEQUENCE CHARACTERISTICS CA) LENGTH: 231 base pa CE) TYPE: nucleic acid STRANDEDNESS: doubl TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

/C/

ATGGNGGTTC TGGACACAGG GNAGGTCTGG ACNCCACTAA ATNCCCCAAG NANAAAGTGT ANAATTTTN TTCCNATAAA TGACATCAGN AAAANAAAAA AAAAAA A 360 420 480 531 (ix) FEATURE: NAME/KEY: misc feature CB) LOCATION: 128 CD) OTHER INFORMATION: /label= Xhol Cxi) SEQUENCE DESCRIPTION: SEQ ID NO:2O: GCGTATAACG CGTTTGGAAT CACTACAGGG ATGTTTAATA CCACTACAAT GGATGATGTA TATAACTATC TATTCGATGA TGAAGATACC CCACCAAACC CAAAAAAAGA GATCTGGAAT TCGGATCCTC GAGAGATCTA TGAATCGTAG ATACTGAAAA ACCCCGCAAG TTCACTICAA 35 CTGTGCATCG TGCACCATCT CAATTTCTTT CATTTATACA TCGTTrrGCC T INFORMATION FOR SEQ ID NO:21: Ci) SEQUENCE CHARACTERISTICS: CA) LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear Cii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: TGAAGATACC CCACCAAACC C INFORMATION FOR SEQ ID NO:22: 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: SEQUENCE CHARACTERISTICS: LENGTH: 907 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/K.EY: CDS 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 351 AGA CTC GCC CAG GAG AGG AAA GCA TGG AGG AAA GAC CAC CCA TTT GGT Arg Leu Ala Gln Glu Arg Lys Ala Trp Arg Lys Asp His Pro Phe Gly 10 15 TTC GTG GCT GTC CCA ACA AAA AAT CCC GAT GCC ACG ATG AAC CTC ATG Phe Val Ala Val Pro Thr Lys Asn Pro Asp Gly Thr Met Asn Leu Met 102 150S

AAC

Asn TGC GAG TCC GCC ATT CCA GCA Trp Glu Cys Ala Ile Pro Gly AAC AAA GGG Lys Lys Cly so ACT CCC TGC GAA Thr Pro T-p, Glu a GGC TTG TTT AAA Cly Leu Phe Lys CGG ATG CTT TTC Arg Met Leu Phe CAT GAT TAT CCA Asp Asp Tyr Pro TCT TCG Ser Ser 198 246 294 342 CCA CCA AAA TCT AAA TTC GAA CCA Pro Pro Lys Cys Lys Phe Glu Pro

CCA

Pro TTA TTT CAC CCG Leu Phe His Pro AAT GTG TAC Asn Val Tyr as AAC GAC TGG Lys Asp Tx-p CCT TCG GGC ACA GTC TGC CTG TCC ATC TTA GAG Pro Ser Gly Thr Val Cys Leu Ser Ile Leu Glu 95 GAG GAC Clu Asp 100 /0 AGG CCA GCC ATC ACA ATC AAA CAG ATC CTA TTA GGA ATA CAG GAA CTT Arg Pro Ala Ile Thr Ile Lys Gin Ile Leu LeU Giy Ile Gin Glu Leu 105 110 115 CTA 111T GAA C--A -T ATC CAAGAC CCA GCT CAAGCA GAGGCC TACACG Leu Asn GJlu Pro Asn Ile Gin Asp Pro Ala Gin Ala Glu Ala Tyr Thr 120 125 130 135 ATT TAC TGC CAA AAC AGA GTG GAG TAC GAG AAA AGG GTC CGA GCA CAA Ile Tyr Cys Gin Asn Arg Val Glu Tyr Glu Lys Arg Vai Arg Ala Gin 140 145 IS0 GCC AAG AAG TTT GCG CCC TCA TAAGCAGCGA CCTTGTGGCA

TCGTCAAAAG

Ala Lys Lys Phe Ala Pro Ser 155 GAAGGGATTG GTTTGGCAAG AACTTGTTTA CAACATTTTT GGCAAATCTA AAGTTGCTC ATACAATGAC TAGTCACCTG GGGGGGTTGG GCGGGCGCCA TCTTCCATTG

CCGCCGCGG

TGTGCGGTCT CGATTCGCTG AATTGCCCGT TTCCATACAG GGTCTCTTCC

TI'CGGTCTT,

TGGTAT TTT T GGATTGTTAT GTAAAACTCG CTTTTATTTT AATATTGATG TCAGTA rT AACTGCTGTA AAATTATAAA CTTTTATACT GGGTAAGTCC CCCAGGGGCG

AGTTNCCTCC

CTCTGGGATG CAGGCATGCT TCTCACCGTG cAAmar~crt 390 438 486 537 597 6 57 717 777 837 907

C

G

TGGAAATGCA

A %J14J.

INFORMATION FOR-SEQ ID NO:24: 35 Ci) SEQUENCE CHARACTERISTICS: LENGTH: 158 amino acids TYPE: amino acid TOPOLOGY: linear (ii) .MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: Met Ser Gly Ile Ala Leu Ser Arg Leu Ala Gin Glu Arg Lys Ala Trp i 5 10 Arg Lys Asp His Pro Phe Giy Phe Val Ala Vai Pro Thr Lys Asn Pro 20 25 Asp Gly Thr Met Asn Leu Met Asn Trp Glu Cys Ala Ile Pro Gly Lys 35 40 Lys Giy Thr Pro Trp Giu Giy Gly Leu Phe Lys Leu Arg Met Leu Phe s0 55 Lys Asp Asp Tyr Pro Ser Ser Pro Pro Lys Cys Lys Phe Glu Pro Pro 70 75 Leu Phe His Pro Asn Vai Tyr Pro Ser Leu Giu Glu Asp Lys Asp 100 Leu Leu Gly Ile Gin Giu 11.5 Ala Gin Ala Glu Ala Tyr 130 Giu Lys Arg Val Arg Ala 145 150 Trp Arg Pro 105 Leu Leu Asn 120 Thr Ile Tyr 135 Gin Ala Lys Gly Thr Val Cys Ala Ile Thr Ile Giu Pro Asn Ile 125 Leu Ser Ile Lys Gin Ile 110 Gin Asp Pro Cys Gin Asn Arg Val 140 Lys Phe Aia Pro Ser Giu Tyr INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LFlNGTH: 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 GCGCCAC7TC CTTTGTGTTT ANATGGCGTT 35 TTGTCTGTGT TGCTGTTTAG AGTAGATNA-A CTGTTTANAT AAAAAA NAAAATTNAC TNGAGGGGGC NTGNAGGCAT GCNNAAC 120 180 207

Claims (5)

105- The claims defining the invention are as follows: 1. A substantially pure preparation of an RAPTI polypeptide, or a fragment thereof, wherein said polypeptide comprises 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 95% 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 rapmycin regulation Cf ell proliferation or an antagonist ofrapamycin regulation of cell proliferation. 5. 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 said polypeptide is of mammalian origin. 7. An antibody preparation specifically reactive with an epitope of the polypeptide of claim 1. 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-Tyrl60 of SEQ ID No. 2 and Va12012-Tyr2144 of SEQ ID No. 12 9. The polypeptide of claim 1, wherein said polypeptide is a soluble polypeptide which specifically binds an FKBP/rapamycin complex, and which binding is rapamycin-dependent. P %OPER\PxhSI 505-01 clauns 167 doc- I S-604

106- 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 of Val26-Tyrl60 of SEQ ID No. 2, Val2012-Tyr2144 of SEQ ID No. 12, Val41-Tyrl73 of SEQ ID No. 14, Vall-Tyr133 of SEQ ID No. 16, and Vall-Argl33 of SEQ ID No. 18. 11. 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. 12. The polypeptide of claim 11, wherein said second polypeptide portion provides a tt.U .%.LUrV tflV lA A UY .fl 6 Sfl% j~JC C. .3t44t LA« %AltIU J A1 L/A t_'ktVtliAJ 13. The polypeptide of claim 11, wherein said second polypeptide portion provides a matrix-binding domain for immobilizing said fusion protein on an insoluble matrix. 14. The polypeptide of claim 11, wherein said fusion polypeptide is functional in a rapamycin-dependent two-hybrid assay. 15. 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, and wherein said RAPTI-like polypeptide comprises an amino acid sequence at least 70% identical to all or a portion of SEQ ID NO: 2 or SEQ ID NO: 12. 16. The protein of claim 15, 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 of Val26-Tyrl60 of SEQ ID No. 2, Val20 12- 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. P OPER\Pxk\8150501 cldim 167 doc-150604 -107- 17. A soluble polypeptide portion of a RAPTI protein, which polypeptide is represented by the general formula ZI-Z 2 -Z 3 wherein 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 SEQ ID No. 12 immediately N-terminal to said rapamycin-binding domain, and Z 3 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- ronorlont rvnner 18. A chimeric polypeptide represented by the general formula X-Y-Z, wherein Y 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, separately, absent or represent polypeptides having amino acid sequences unrelated to a RAPTI-like protein. c** 19. A substantially pure nucleic acid, comprising a nucleotide sequence which encodes a RAPTI protein, or a fragment thereof, wherein said protein comprises an amino acid sequence at least 70% homologous to one or both of SEQ ID Nos: 2 or 12. The nucleic acid of claim 19, wherein said RAPTI protein binds to an FKBP/rapamycin complex. P \OPERPxkS 1505.01 rclim 167.doc.-06/04 -108- 21. The nucleic acid of claim 19, wherein said RAPTI 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. 22. The nucleic acid of claim 19, wherein said RAPTI protein has a phophatidylinositol kinase activity. 23. The nucleic acid of claim 19, comprising a RAPTI coding sequence from a pIC524 clone of ATCC deposit 75787. 24. The nucleic acid of claim 19, which hybridizes under stringent conditions to a nucleic cPid probe cepnng to at Ips 12 ncutive UL Arlc.tidcU of12 NA. U or 1j1. The nucleic acid of claim 19, further comprising a transcriptional regulatory sequence operably linked to said nucleotide sequence so as to render said nucleotide sequence suitable for use as an expression vector. 26. An expression vector, capable of replicating in at least one of a prokaryotic cell and eukaryotic cell, comprising the nucleic acid of claim 27. A host cell transfected with the expression vector of claim 26 and expressing said polypeptide. 28. A method of producing a recombinant RAPT1 protein comprising culturing the cell of claim 27 in a cell culture medium to express said RAPTI protein and isolating said RAPTI protein from said cell culture. 29. The nucleic acid of claim 19, wherein said nucleic acid encodes a soluble polypeptide which specifically binds an FKBP/rapamycin complex, and which binding is rapamycin- P:OPER\PxklIS-O dl daimsJ67 .ISM6M4

109- dependent. The nucleic acid of claim 29, 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 of 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. 31. The nucleic acid of claim 29, 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 32. The nucleic acid of claim 31, wherein said second polypeptide portion provides a detectable label for detecting the presence of said fusion protein. 33. The nucleic acid of claim 31, wherein said second polypeptide portion provides a matrix-binding domain for immobilizing said fusion protein on an insoluble matrix. 34. The nucleic acid of claim 31, wherein said fusion polypeptide is functional in a rapamycin-dependent two-hybrid assay. 35. 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 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 SEQ ID No. 12 immediately N-terminal to said rapamycin-binding domain, and laims 67 doc-1S064

110- Z 3 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. 36. A chimeric polypeptide represented by the general formula X-Y-Z, wherein 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 RAPTI-like protein homologous thereto, and X and Z are, separately, absent or represent polypeptides having amino acid sequences unrelated to a RAPTI-like protein. 37. A recombinant RAPTI polypeptide, or a fragment thereof, having an amino acid sequence at least 70% homologous to SEQ ID NO. 2 or 12. 38. The polypeptide of claim 37, wherein said polypeptide binds to an FKBP/rapamycin complex. 39. 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 according to S: claim 8, and a FKBP polypeptide comprising a rapamycin-binding domain of an 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, P \OPER\Pxt\ 1505)I clums 167 doc- 0604 11 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. 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 according to claim 8, and a FKBP polypeptide comprising a rapamycin-binding domain of an FK506- binding protein under conditions wherein said RAP-BP and FKBP nnlvnpntierlp ar ~habl to intveract r-1 J[ 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. 41. 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 that encodes a first hybrid protein, the first hybrid protein comprising: P \OPER\ IP\ 05l-OI clms 167 doc-1 AM4 -112- a DNA-binding domain that recognizes a binding site on the detectable gene in the host cell; and 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 RAPTI-like protein according to claim 8; (iv) subjecting the host cell to conditions under which the first hybrid protein and the second hybrid protein are epr VesOO in u f 4r.f n .quInt frt-. -c^t^LI gene to be activated; contacting the host cell with a test agent; and "g (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. 42. The method of claim 41, wherein the DNA-binding domain and transcriptional activation domain are derived from transcriptional activators having separable DNA-binding and transcriptional activation domains. 43. The method of claim 42, 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. P \OPER\Pxk150)I clms 167 d I S6D4 -113- 44. The method of claim 41, 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. 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 occurring mutants thereof. 46 The nroenhprimpr nfrlaim 45 filrtr irmnrr;nn la l nrrn,,r ot,rhr th~r-to, able to be detected. o. 47. The probe/primer of claim 46, wherein said label group being selected from a group consisting of radioisotopes, fluorescent compounds, enzymes, and enzyme co-factors. 48. 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. 49. The method of claim 48, wherein detecting said genetic lesion comprises 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[.], PAOPER\Pxk2II05-0I claims 167 doc- 15/004

114- v. a gross alteration in the level of a messenger RNA transcript of said gene, vi. the presence of a non-wild type splicing pattern of a messenger RNA transcript of said gene, and vii. a non-wild type level of said protein. The method of claim 48, 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 occurring mutants thereof, or 5' or 3' flanking sequences naturally associatdcu wiluai gcnc; 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. 51. The method of claim 50, 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. o* 52. The method of claim 50, wherein detecting said lesion comprises utilizing said *c probe/primer to in a polymerase chain reaction (PCR). 53. The method of claim 50, wherein detecting said lesion comprises utilizing said probe/primer in a ligation chain reaction (LCR). P NOPER\k%2 1505-01 dis 67 d.516)04 -115 54. The method of claim 50, wherein the level of said protein is detected in an immunoassay. DATED this 1 5 'h day of June, 2004 ARIAD PHARMACEUTICALS, INC. by its Patent Attorneys DAVIES COLLISON CAVE