AU2003200898B2 - Phosphodiesterase 8A - Google Patents

Description

P/00/011 28/5/91 Regulation 3.2

AUSTRALIA

Patents Act 1990

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT Name of Applicant: Actual Inventor: Address for service is: ICOS Corporation Kate LOUGHNEY WRAY ASSOCIATES Level 4, The Quadrant 1 William Street Perth, Western Australia Australia Attorney code: WR Invention Title: "Phosphodiesterase 8A" This application is a Divisional Application by virtue of Section 39 of Australian Patent Application 10974/99.

The following statement is a full description of this invention, including the best method of performing it known to me:- PHOSPHODIESTERASE 8A This application is a continuation-in-part of U.S. Patent Application Serial No. 08/951,646, filed October 16, 1997, which is pending.

FTELD OF THE INVENTION The present invention relates generally to a family ofphosphodiesterases designated PDE8A and uses thereof BACKGROUND OF THE INVENTION Phosphodiesterases (PDEs) hydrolyze 5' cyclic nucleotides to their respective nucleoside 5 monophosphates. The cyclic nucleotides cAMP and cGMP are synthesized by adenylyl and guanylyl cyclases, respectively, and serve as second messengers in a number of cellular signaling pathways The duration and strength of the second messenger signal is a function of the rate of synthesis and the rate of hydrolysis of the cyclic nucleotide.

Multiple families ofPDEs have been identified The nomenclature system includes first a number that indicates the PDE family. To date, seven families (PDE1-7) are known which are classified by: primary structure; (ii) substrate preference; (iii) response to different modulators; (iv) sensitivity to specific inhibitors; and modes of regulation [Loughney and Ferguson, in Phosphodiesterase Inhibitors, Schudt, el al. (Eds), Academic Press: New York, New York (1996) pp. 1-19]. The number indicating the family is followed by a capital letter, indicating a distinct gene, and the capital letter followed by a second number, indicating a specific splice variant or a specific transcript which utilizes a unique transcription initiation site.

The amino acid sequences of all mammalian PDEs identified to date include a highly conserved region of approximately 270 amino acids located in the carboxy terminal half of the protein [Charbonneau. el Proc. Nail. Acad. Sci. (USA) 83:9308- 9312 (1986)]. The conserved domain includes the catalytic site for cAMP and/or cGMP hydrolysis and two putative zinc binding sites as well as family specific determinants [Beavo, Physiol. Rev. 75:725-748 (1995); Francis. et al., J Biol. ChemI. 269:22477- 22480 (1994)]. The amino terminal regions of the various PDEs are highly variable and include other family specific determinants such as: calmodulin binding sites (PDE1); (ii) non-catalytic cyclic GMP binding sites (PDE2, PDE5, PDE6); (iii) membrane targeting sites (PDE4); (iv) hydrophobic membrane association sites (PDE3); and (v) phosphorylation sites for either the calmodulin-dependent kinase II (PDE1), the cAMPdependent kinase (PDEI, PDE3, PDE4), or the cGMP dependent kinase (PDE5) [Beavo, Physiol. Rev. 75:725-748 (1995); Manganiello, et al., Arch. Biochem. Acta 322:1-13 (1995); Conti, el al., Physiol Rev. 75:723-748 (1995)].

Members of the PDEI family are activated by calcium-calmodulin. Three genes have been identified: PDEIA and PDEIB preferentially hydrolyze cGMP while PDE1C has been shown to exhibit a high affinity for both cAMP and cGMP. The PDE2 family is characterized as being specifically stimulated by cGMP [Loughney and Ferguson, supra]. Only one gene has been identified, PDE2A, the enzyme product of which is specifically inhibited by erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA). Enzymes in the PDE3 family are specifically inhibited by cGMP. Two genes are known, PDE3A and PDE3B, both having high affinity for both cAMP and cGMP, although the V. for cGMP hydrolysis is low enough that cGMP functions as a competitive inhibitor for cAMP hydrolysis. PDE3 enzymes are specifically inhibited by milrinone and enoximone [Loughney and Ferguson, supra]. The PDE4 family effects cAMP hydrolysis and includes four genes, PDE4A, PDE4B, PDE4C. and PDE4D, each having multiple splice variants.

Members of this family are specifically inhibited by the anti-depressant drug rolipram.

Members of PDE5 family bind cGMP at non-catalytic sites and preferentially hydrolyze cGMP. Only one gene. PDE5A, has been identified. The photoreceptor PDE6 enzymes specifically hydrolyze cGMP [Loughney and Ferguson, supra]. Genes include PDE6A and PDE6B (the protein products of which dimerize and bind two copies of a smaller y inhibitory subunit to form rod PDE). in addition to PDE6C which associates with three smaller proteins to form cone PDE. The PDE7 family effects cAMP hydrolysis but, in contrast to the PDE4 family, is not inhibited by rolipram [Loughney and Ferguson, supra].

Only one gene, PDE7A, has been identified.

1. Given the importance of cAMP and cGMP in intracellular second messenger signaling, there thus exists an ongoing need in the art to identify addition PDE species. Identification of heretofore unknown families of PDEs, and genes and splice variants thereof, will provide additional pharmacological approaches to treating conditions in which cyclic nucleotide pathways are aberrant as well as conditions in which modulation of intracellular cAMP and/or cGMP levels in certain cell types is desirable.

SUMMARY OF THE INVENTION In brief, the present invention provides polypeptides and underlying polynucleotides for a novel PDE family designated PDE8. The invention includes both naturally occurring and non-naturally occurring PDE8 polynucleotides and polypeptide products thereof. Naturally occurring PDE8 products include distinct gene and polypeptide species within the PDE8 family PDESA); these species include those which are expressed within cells of the same animal and well as corresponding species homologs expressed in cells of other animals. Within each PDE8 species, the invention further provides splice variants encoded by the same polynucleotide but which arise from distinct mRNA transcripts PDE8AI and PDE8A2). Non-naturally occurring PDE8 products include variants of the naturally occurring products such as analogs wherein one or more amino acids are added, substituted, or deleted) and those PDE8 products which include covalent modifications fusion proteins, glycosylation variants, Met 'PDE8s, Met-'-Lys 1 PDE8s, Gly-PDESs and the like). The PDE8 family is distinguished from previously known PDE families in exhibiting high affinity for hydrolysis of both cAMP and cGMP but relatively low sensitivity to enzyme inhibitors specific for other PDE families. In a preferred embodiment, the invention provides a polynucleotide comprising the sequence set forth in SEQ ID NO: 1. The invention also embraces polynucleotides encoding the amino acid sequence set out in SEQID NO: 2. A presently preferred polypeptide of the invention comprises the amino acid sequence set out in SEQ ID NO: 2. The invention provides two splice variant cDNAs which give rise to two polypeptides designated PDE8A1 and PDE8A2 PDE8A1 and PDE8A2 polypeptides, and the polynucleotides encoding the polypeptides, are discussed herein as representative of the PDE8 enzyme family embraced by the invention.

The present invention provides novel purified and isolated polynucleotides DNA sequences and RNA transcripts, both sense and complementary antisense strands, including splice variants thereof) encoding the human PDE8s. DNA sequences of the invention include genomic and cDNA sequences as well as wholly or partially chemically synthesized DNA sequences. "Synthesized," as used herein and is understood in the art, refers to purely chemical, as opposed to enzymatic, methods for producing polynucleotides. "Wholly" synthesized DNA sequences are therefore produced entirely by chemical means, and "partially" synthesized DNAs embrace those wherein only portions of the resulting DNA were produced by chemical means A preferred DNA sequence encoding a human PDE8 polypeptide is set out in SEQ ID NO: 1. Also preferred are polynucleotides encoding the PBE8 polypeptide of SEQ ID NO: 2 and the PDE8A1 and PDE8A2 splice variant polypeptides set out in SEQ ID NOs: 6 and 4, respectively. Preferred polynucleotides encoding PDESAI and PDE8A2 are set out in SEQ ID NOs: 5 and 3, respectively The invention further embraces species, preferably mammalian, homologs of the human PDE8 DNA The invention also embraces DNA sequences encoding PDE8 species which hybridize under moderately stringent conditions to the non-coding strands, or complements, of the polynucleotides in SEQ ID NOs: 1, 3 and 5. DNA sequences encoding PDESA polypeptides which would hybridize thereto but for the redundancy of the genetic code are contemplated by the invention. Exemplary moderate hybridization conditions are as follows: hybridization at 65°C in 3X SSC, 0.1% sarkosyl, and 20 mM sodium phosphate, pH 6.8, and washing at 65°C in 2X SSC with 0.1% SDS. It is understood in the art that conditions of equivalent stringency can be achieved through variation of temperature and buffer, or salt concentration as described Ausebel, el al.

Protocols in Molecular Biology, John Wiley Sons (1994), pp. 6.0.3 to 6.4.10.

Modifications in hybridization conditions can be empirically determined or precisely calculated based on the length and the percentage of guanosine/cytosine (GC) base pairing of the probe. The hybridization conditions can be calculated as described in Sambrook, et al., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York (1989), pp. 9.47 to 9.51.

Autonomously replicating recombinant expression constructions such as plasmid and viral DNA vectors incorporating PDE8 sequences are also provided.

Expression constructs wherein PDES-encoding polynucleotides are operatively linked to an endogenous or exogenous expression control DNA sequence and a transcription terminator are also provided.

According to another aspect of the invention, host cells are provided, including procaryotic and eukaryotic cells, either stably or transiently transformed with DNA sequences of the invention in a manner which permits expression of PDE8 polypeptides of the invention. lost cells of the invention are a valuable source of immunogen for development of antibodies specifically immunoreactive with PDE8. Host cells of the invention are also conspicuously useful in methods for large scale production of PDE8 polypeptides wherein the cells are grown in a suitable culture medium and the desired polypeptide products are isolated from the cells or from the medium in which the cells are grown by, for example, immunoaffinity purification.

Knowledge of PDE8 DNA sequences allows for modification of cells to permit, or increase, expression of endogenous PDE8. Cells can be modified by homologous recombination) to provide increased PDE8 expression by replacing, in whole or in part, the naturally occurring PDE8 promoter with all or part of a heterologous promoter so that the cells express PDE8 at higher levels The heterologous promoter is inserted in such a manner that it is operatively-linked to PDE8 encoding sequences. See, for example, PCT International Publication No. WO 94/12650, PCT International Publication No WO 92/20808. and PCT International Publication No. 91/09955. The invention also contemplates that, in addition to heterologous promoter DNA, amplifiable marker DNA ada, dhfr, and the multifunctional CAD gene which encodes carbamyl phosphate synthase, asparate transcarbamylase, and dihydroorotase) and/or intron DNA may be inserted along with the heterologous promoter DNA. If linked to the PDE8 coding sequence, amplification of the marker DNA by standard selection methods results in co-amplification of the PDE8 coding sequences in the cells.

The DNA sequence information provided by the present invention also makes possible the development through, e.g homologous recombination or "knock-out" strategies [Capecchi, Science 244-:1288-1292 (1989)], of animals that fail to express functional PDE8 or that express a variant of PDE8. Such animals are useful as models for studying the in vivo activities of PDES and modulators of PDE8.

The invention also provides purified and isolated mammalian PDE8 polypeptides. Presently preferred PDESA polypeptides are set out in SEQ ID NOs: 4 and 6. Most preferred is a PDE8 polypeptide comprising the amino acid sequence set out in SEQ ID NO: 2. PDE8 polypeptides of the invention may be isolated from natural cell sources or may be chemically synthesized, but are preferably produced by recombinant procedures involving host cells of the invention. Use of mammalian host cells is expected to provide for such post-translational modifications glycosylation, truncation, lipidation, and phosphorylation) as may be needed to confer optimal biological activity on recombinant expression products of the invention. PDE8 products of the invention may be full length polypeptides, biologically active fragments, or variants thereof which retain specific PDE8 biological activity. Variants may comprise PDE8 polypeptide analogs wherein one or more of the specified naturally encoded) amino acids is deleted or replaced or wherein one or more non-specified amino acids are added: without loss of one or more of the biological activities or immunological characteristics specific for PDE8; or with specific disablement of a particular biological activity of PDE8.

Variant products of the invention include mature PDE8A products, i.e., PDE8 products wherein leader or signal sequences are removed, having additional amino terminal residues. PDES products having an additional methionine residue at position -1 (Met-'-PDE8) are contemplated, as are PDEB products having additional methionine and lysine residues at positions -2 and -1 (Met--Lys-'-PDES). Variants of these types are particularly useful for recombinant protein production in bacterial cell types.

The invention also embraces PDE8 variants having additional amino acid residues which result from use of specific expression systems. For example, use of commercially available vectors that express a desired polypeptide such as a glutathione-Stransferase (GST) fusion product provide the desired polypeptide having an additional glycine residue at position -l as a result of cleavage of the GST component from the desired polypeptide. Variants which result from expression in other vector systems are also contemplated.

The invention further embraces PDE8 products modified to include one or more water soluble polymer attachments. Particularly preferred are PDE8 products covalently modified with polyethylene glycol (PEG) subunits Water soluble polymers may be bonded at specific positions, for example at the amino terminus of the PDE8 products, or randomly attached to one or more side chains of the polypeptide.

Also comprehended by the present invention are antibodies monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, CDRgrafted antibodies and the like) and other binding proteins specific for PDE8 products or fragments thereof Specific binding proteins can be developed using isolated or recombinant PDE8 products, PDES variants, or cells expressing such products. Binding proteins are useful for purifying PDE8 products and detection or quantification of PDE8 products in fluid and tissue samples using known immunological procedures. Binding proteins are also manifestly useful in modulating blocking, inhibiting or stimulating) biological activities of PDE8, especially those activities involved in signal iransduction.

Anti-idiotypic antibodies specific for anti-PDE8 antibodies are also contemplated.

The scientific value of the infrnnation contributed through the disclosures ofDNA and amino acid sequences of the present invention is manifest. As one series of examples, knowledge of the sequence of a cDNA for PDE8A makes possible through use of Southern hybridization or polymerase chain reaction (PCR) the identification of genomic DNA sequences encoding PDE8 and PDE8 expression control regulatory sequences such as promoters, operators, enhancers, repressors, and the like DNA/DNA hybridization procedures carried out with DNA sequences of the invention under moderately to highly stringent conditions are likewise expected to allow the isolation of DNAs encoding allelic variants of PDESA; allelic variants are known in the art to include structurally related proteins sharing one or more of the biochemical and/or immunological properties specific to PDE8A. Similarly, non-human species genes encoding proteins homologous to PDESA can also be identified by Southern and/or PCR analysis. As an alternative, complementation studies can be useful for identifying other human PDE8 products as well as non-human proteins, and DNAs encoding the proteins, sharing one or more biological properties of PDE8A.

Polynucleotides of the invention are also useful in hybridization assays to detect the capacity of cells to express PDE8. Polynucleotides of the invention may also be the basis for diagnostic methods useful for identifying a genetic alteration(s) in a PDE8 locus that underlies a disease state or states.

Also made available by the invention are anti-sense polynucleotides which recognize and hybridize to polynucleotides encoding PDES Full length and fragment anti-sense polynucleotides are provided. Anti-sense polynucleotides are particularly relevant to regulating expression of PDES by those cells expressing PDE8 mRNA.

The DNA and amino acid sequence information provided by the present invention also makes possible the systematic analysis of the structure and function of PDESs. DNA and amino acid sequence information for PDE8 also permits identification of molecules with which PDESA will interact. Agents that modulate increase, decrease, or block) PDE8 activity may be identified by incubating a putative modulator with PDE8 and determining the effect of the putative modulator on PDE8 phosphodiesterase activity. The selectivity of a compound that modulates the activity of the PDE8 can be evaluated by comparing its activity on the PDE8 to its activity on other PDE enzymes. Cell based methods, such as di-hybrid assays and split hybrid assays, as well as in vitro methods, including assays wherein a polypeptide or its binding partner are immobilized, and solution assays are contemplated by the invention.

Selective modulators may include, for example, antibodies and other proteins or peptides which specifically bind to the PDE8 or PDE8 nucleic acid.

oligonucleotides which specifically bind to tihe PDE8 or PDE8 nucleic acid, and other nonpeptide compounds isolated or synthetic organic molecules) which specifically react with PDE8 or PDES-encoding nucleic acid. Mutant forms of PDE8 which affect the enzymatic activity or cellular localization of the wild-type PDE8 are also contemplated by the invention. Presently preferred targets for the development of selective modulators include, for example: regions of the PDES which contact other proteins and/or localize the PDE8 within a cell, regions of the PDE8 which bind substrate, (3) allosteric cyclic nucleotide-binding site(s) of PDE8, phosphorylation site(s) of PDE8 and regions of the PDE8 which are involved in multimerization of PDE8 subunits.

Modulators of PDE8 activity may be therapeutically useful in treatment of a wide range of diseases and physiological conditions in which PDE activity is known to be involved.

The invention further contemplates small molecule modulators of PDE8A enzyme activity. There are at least three different types of libraries used for the identification of small molecule modulators. These include chemical libraries. (2) natural product libraries, and combinatorial libraries comprised of random peptides, oligonucleotides or organic molecules.

Chemical libraries consist of structural analogs of known compounds or compounds that are identified as "hits" or "leads" via natural product screening. Natural product libraries are collections of microorganisms, animals, plants. or marine organisms which are used to create mixtures for screening by: fermentation and extraction of broths from soil, plant or marine microorganisms or extraction of plants or marine organisms. Combinatorial libraries are composed of large numbers of peptides, oligonucleotides or organic compounds as a mixture They are relatively easy to prepare by traditional automated synthesis methods, PCR, cloning or proprietary synthetic methods. Of particular interest a:e peptide and oligonucleotide combinatorial libraries.

Still other libraries of interest include peptide. protein, peptidomimetic, multiparallel synthetic collection. recombinatorial, and polypeptide libraries. For a review of combinatorial chemistry and libraries created therefrom, see Myers, Curr. Opion Biotechnol. 8:701-707 (1997).

Identification of modulators through use of the various libraries described herein permits modification of the candidate "hit" (or "lead") to optimize the capacity of the "hit" to modulate activity.

The invention further provides methods to identify a specific binding partner compound of a PDE8A polypeptide of the invention comprising the steps of: a) contacting the PDESA polypeptide with a compound under conditions which permit binding between the compound and the PDE8A polypeptide; b) detecting binding of the compound to the PDE8A polypeptide, and c) identifying the compound as a specific binding partner of the PDE8A polypeptide. Binding partner identified in the methods of the invention preferably modulate PDE8A enzyme activity, either through inhibition or activation, or enhancement, of the enzyme.

The invention also provides methods to identify a specific binding partner compound of a PDE8A polynucleotide of the invention comprising the steps of a) contacting the PDE8A polynucleotide with a compound under conditions which permit binding between the compound and the PDESA polvnucieotide: b) detecting binding of the compound to the PDESA polynucleotide; and c) identifying the compound as a specific binding partner of the PDESA polynucleotide. The binding partner of the PDE8A polynucleotide preferably modulates expression of the PDE8A polypeptide encoded by the PDE8A polynucleotide, either through inhibiting expression or enhancing expression.

The invention also provides compounds identified by a method of the invention, as well as compositions comprising a compound identified and a pharmaceutically acceptable carrier.

DETAILED DESCRIPTION OF THE INVENTION The present invention is illustrated by the following examples which relate to the isolation of polynucleotides encoding PDE8 polypeptides as well as expression and characterization of the encoded polypeptides. Example 1 describes methods for searching expressed sequence tag (EST) databases in order to identify probes potentially useful for isolating DNAs of the invention. Example 2 relates to identification of PDESA-encoding polynucleotides. Example 3 addresses sequence analysis of the isolated polynucleotides.

Example 4 describes analysis of polypeptides encoded by the PDE8A polynucleotides.

Example 5 addresses expression of recombinant PDESA polypeptides. Example 6 relates to Northern analysis of PDESA expression. Example 7 describes chromosome mapping of the gene encoding PDESA Example 8 describes confirmation that PDE8A1 and PDE8A2 are splice variants. Example 9 addresses expression and characterization of recombinant PDE8A. Example 10 details production of anti-PDESA monoclonal antibodies. Example 11 describes an analysis of PDE8A expression by in situ hybridization.

Example 1 Identification of an EST Related to a Human PDE Using the sequences of known human, 5' cyclic nucleotide phosphodiesterases, a search of the National Center for Biotechnology Information (NCBI) Expressed Sequence Tags (EST) database was undertaken in order to identify cDNA fragments that could potentially be useful for the identification of novel phosphodiesterase (PDE) genes This database contains DNA sequences representing one or both ends of cDNAs collected from a variety of tissue sources. A single sequencing run is performed on one or both ends of the cDNA and the quality of the DNA sequence varies tremendously. At the time the PDE searches were performed, the EST sequence database contained more than 600.000 cDNA sequences from a variety of organisms.

The search for novel PDE sequences included three steps First the BLASTN program available through NCBI was used to identify DNA sequences in the EST sequence database with homology to cDNA sequences encoding known human PDEs. The program compares a nucieotide query sequence against a nucleotide sequence database. The cDNA sequences of the fifteen known human PDEs were submitted and fifteen BLASTN searches were performed the query PDE sequences included PDE1A3 [Loughney, et al., J. Biol. Chemn. 271:796-806 (1996)], PDE1B1 [Yu, et al., Cell Signaling, in press (1997)], PDEIC2 [Loughney, el al., J. Biol. Chem. 271:796-806 (1996)], PDE2A3 [Rosman, ef Gene 191:89-95 (1997)], PDE3A [Meacci, et al., Proc. Natl. Acad. Sci. (USA) 89:3721-3725 (1992)], PDE3B [Miki et al., Genomics 36:476-485 (1996)], PDE4A5 [Bolger.e/ Mol. Cell. Biol. 13:6558-6571 (1993)], PDE4B2 [Bolger, etal., ol. (Cell. Riol. 13:6558-6571 (1993)], PDE4C [Bolger, el a., Mol. Cell. Biol. 13:6558-6571 (1993)]. PDE4DI and PDE4D3 [Bolger, el al., Mol. Cell.

Biol. 13:6558-6571 (1993)], PDE5A, PDE6A [Pittler, el al., Genomics 6:272-283 (1990)], PDE6B [Collins, etal., (;Genncsc 13:698-704 (1992)], PDE6C [Piriev, etal., Genomics 28:429-435 (1995), and PDE7A1 [Michaeli, et J. Biol. Chem. 17:12925- 12932 (1993)]. The BLASTN results were examined and EST sequences that were judged as corresponding to each of the fifteen known PDE cDNAs were identified and collected into a table. The PDE6A and PDE6B sequences used as queries were truncated at 3' end (removing a portion of the 3' untranslated region) due to the presence of repetitive elements in the 3' untranslated region of the cDNAs.

Secondly, the NCBI TBLASTN program was used to examine the homology between the protein sequence of the fifteen known human PDEs (as above) and the six different possible proteins encoded by each of the EST DNA sequences. In this search, the EST sequences are translated in six frames and the amino acid sequences generated are compared to the quei' PDE amino acid sequences. Sequences identified as homologous at the amino acid level were examined and any EST sequences positively identified as corresponding to a known PDE during the BLASTN search described above were discarded.

The third step of the search involved analyzing the sequences that were not known PDEs. These amino acid sequences were homologous to a known PDE but were not identified as one of the 15 known PDE genes during the BLASTN searches.

The BLAST searches identified an EST sequence (designated W04835) from a human fetal lung cDNA library as encoding an amino acid sequence having homology to the catalytic region ofPDE2A, PDE3A. PDE3B. PDE4A. PDE4B, PDE4C, rod alpha PDE6A, rod beta PDE6B, cone alpha PDE6C, and PDE7A. The database sequence for W04835 is set out in SEQ ID NO: 7. Results from the database analysis as discussed below are exemplified using the PDE4D sequence.

W04835 cDNA was obtained from American Type Culture Collection (Rockville, MD) which maintains and makes publicly available deposits ofESTs identified and sequenced by Lawrence Livermore National Laboratory, Livermore, CA). The WO4835 DNA was sequenced upon receipt to confirm its identity and determined to be consistent with SEQ ID NO: 7.

The amino acid sequence encoded by the -1 reading frame of EST sequence W04835 was recognized by all of the PDE query cDNA sequences except 13 PDE1A, 1B and 1C. Using the TBLASTN results with PDE4D3 as an example, two regions of similarity were detected. The first region showed 15/37 exact matches or identity (19/37 similar amino acids) and included the HD(X),HXG(X),,A (SEQ ID NO: 8) motif found in all of the query sequences [Charboneau, Mol. Pharmacol. Cell Regul.

2:267-298 (1990)]. The second region showed 9/20 exact matches or 45% identity and included the YHNxxHA motif found in most of the query sequences. BLASTN analysis of the W04835 sequence revealed that it was unique in that it was not identical to any other human DNA sequences in the Genbank database. The EST database entry for W04835 identified the sequence as being similar to PIR:A48719, the bovine cGMP binding, cGMP hydrolyzing PDE5AI sequence Comparison of the protein sequence of W04835 frame -1 to the bovine PDE5Ai sequence revealed 58/153 matches for an overall identify of 38%. Within this region were small regions of greater homology; one region showed a 12/14 identical amino acids. Given the unique nature of the W04835 sequence, its relatively low homology to bovine PDE5A1, and the presence of the amino acid motifs found in most other known human PDE amino acid sequences, W04835 represents a novel human PDE cDNA.

Example 2 Isolation of Putative PDE cDNA W04835 cDNA insert was digested from the pT7T3D vector into two fragments with the restriction enzymes EcoRI and HindIll and the two fragments were purified using two sequential low melting agarose gels. Both fragments were used as probes to screen cDNA libraries derived from human heart (Stratagene, La Jolla, CA), and human fetal brain (Stratagene) using procedures routinely practiced in the art.

Approximately 5 x 10' phage from each library were screened. Hybridization was carried out overnight in buffer containing 3X SSC. 0.1% Sarkosyl, 20 mM sodium phosphate, pH 6.8, 10X Denhardt's solution, and 50 Lig/ml salmon sperm DNA at 65°C. The filters were washed at 65 C in buffer containing 2X SSC and 0.1% SDS prior to autoradiography.

Nine clones from the fetal brain cDNA library and two from the heart cDNA library hybridized to the WO4835 probe. Partial sequencing and mapping led to 14the selection of one clone from the fetal brain library designated FB66a for further characterization.

A second screening of approximately 7.5 x 10 s phage from the fetal brain cDNA library under conditions used in the first screening using the 1.3 kb EcoRUHiTdIII fragment from the 5' portion of W04835 yielded nineteen additional cDNA clones. Six of these cDNAs also hybridized to a HindlII/KnmI fragment ofW04835 which includes a 256 nucleotide region at the 5' end ofW04835. Partial sequencing and mapping of five of the clones led to the selection of a second clone designated FB85c-2 for further analysis.

Example 3 DNA Sequence Analysis of FB66a and FB85c-2 The DNA sequence of FB66a was determined for both strands using DNA oligonucleotide primers set out below in SEQ ID NOs: 9 to 31 and a Perkin Elmer Applied Biosystems Division 373A DNA Sequencer according to the maunfacturer's suggested protocol The amount of PCR product used as template was calculated based on the size of the PCR product and was sequenced using ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit with ApliTaq DNA Polymerase, FS (Perkin Elmer, Foster City. CA) and asymmetric PCR The reaction product was purified on a AGCT spin column (Advanced Genetic Technologies Corp., Gaithersburg, MD) and dried Loading buffer was added to each purified sample and the mixture heated at 90'C for two minutes. The solution was transferred to ice until being loaded onto a 4% polyacrylamide gel. Data was automatically collected once the Data Collection program was initiated and was automatically analyzed and read by the Sequence Analysis program. All editing was performed manually and the resulting sequences were aligned where the consensus sequence was determined.

M13Rev.1 GGAAACAGCTATGACCATG SEQ ID NO: 9 W48A2 ACTCTCCAAGGAAATACAG SEQ ID NO: W48A9 CTGTCTCTGCACTAACAC SEQ ID NO: 11 W48A4 TTGGCAAGGCCTCTGCAT SEQ ID NO: 12 W48S1 CCTCTATGAACTGAGCAG SEQ ID NO: 13 15 W48AI W48S6 W48A5 W48S7 W48A6 W48S2 W48S3 W48A8 W48S4 W48S5 W48S8 W48A7 W48S9 W4S 0 M13 W48AI] W48A1 0 W48S11

GAAGGCACTGCCACTGAT

TCGAGCTGTATCGGCACT

AGCGTGTGATTGTTCTGAA

TGCTGGCCAAGTAGCAAG

AAGGTCACAGGCAGTCAT

GAAGAGTGGCAAGGTCTC

TCATGACCTGGACCACCAG

CCTTCTTGAAGAGGTTTGC

ATGACTGCCTGTGACCTT

CT GCTA TAC AAC CCTTAC C GCTAt"rATTGCTGAGGCC

TAAGTGAGAGGTGACTGC

CCTAAAGGGCTGAGATCA

CGCAGTCACCTCTCACTT

TGTAAAACGACGGCCAGT

ACAAA C GCCT ATGGTGG

TTGATCTCAGCCCTTTAGC

TCATGTGGCAGGAAACTG

SEQ ID NO: 14 SEQ MI NO: SEQ lID NO: 16 SEQ ED NO: 17 SEQ ED NO: 18 SEQ ED NO: 19 SEQ ID NO: SEQ lID NO: 21 SEQ lID NO: 22 SEQ lID NO: 23 SEQ EiD NO: 24 SEQ ID NO: SEQ EiD NO: 26 SEQ ED NO: 27 SEQ ID NO: 28 SEQ ID NO: 29 SEQ ED NO: SEQ ED NO. 31 The FB66a cDNAk set out in SEQ ID NO: is 4389 nucleotides in length and, from nucleotide 3 to nucleotide 241 I, encodes a protein of 803 amino acids with a predicted molecular weight of approximatelY 90,775 Da The deduced amino acid sequence for FB66a is set out in SEQ ID NO: 4. 'The first mnethionine is encoded at nucleotide 45; the absence of an upstreamn in frame stop codon makes it unclear whether this residue is an internal methionine or the beginning of the open reading frame.

The DNA sequence of FB8 5c-2 (SEQ lID NO: 5) was similarly determined using primers Ml3Rev. 1, W48A2, V48A9, W48A4. W4851. W48A1, W48S6, W48A5, W48A6, W48S2, W48S3, W48S4, W485. W48S7, W48A8, and M13. FB85c-2 appeared to include two distinct DNA inserts, univ one of which was homologous to W04835. The region homologous to W04835 was approximately 2.8 kb in leno-th. The precise sequence at the 5' end of the insert could not be determined and thus a few hundred bases of sequence in what iiay be a 5 '-untranslated region are not included in the 2573 nucleotide sequence set out in SEQ ID NO: 5. Nucleotide 67 to nucleoride 2406 encodes a protein having 779 ainon acid protein (SEQ ID NO: 6) having a predicted -16molecular weight of 88,353 Da. An in frame upstream stop codon makes it likely that the methionine encoded at nucleotide position 67 is the initiation methionine.

The proteins encoded by FB66a and FB85c-2 have different amino terminal sequences which may be due to alternative splicing. The DNA sequences diverge from each other 5' of nucleotide 112 in FB66a and nucleotide 104 in FB85c-2. Thus, FB85c-2 has 13 amino acids at the amino terminus that are not found in the FB66a protein. The FB66a protein includes 23 unique amino terminal residues if the initiating methionine at presumed to be encoded at nucleoride 35; the protein includes more than 37 unique amino terminal residues if the open reading frame in the FB66a clone is incomplete.

BLASTN analysis, wherein a query nucleotide sequence is compared against a nucleotide sequence database, of the FB66a sequence revealed no identity with sequences in Genbank, NCBI STS. NCB1 HTGS, or NCBI GSS databases. However, two identical sequences were identified in the NCBI EST database.

One sequence was the W04835 EST which was used to identify the cDNA clone The second. AA307865 (SEQ ID NO: 32), derived from a colon cancer cell line KM12C (HCC) showed sequence identity with the 3' untranslated region of the FB66a and FB85c-2 clones. During the search in which AA307865 was identified, additional EST DNAs were identified presumably encoding putative mouse (EST AA386789. SEQ ID NO: 38) and rat (EST H327347 SEQ ID NO: 33) homologs to the human proteins encoded by FB66a and FB85c-2 The mouse sequence was S6% identical to the human sequences and the rat sequence was 8 1%.

Example 4 Analysis FB85c-2 and FB66a Protein The PDEs encoded by clones FB85c-2 and FB66a were designated PDE8AI and PDE8A2, respectively. Both PDESA proteins, having complete amino acid sequence identity beyond the point of divergence discussed above, are most similar to human PDE2A, PDESA, PDE6A, PDE6B. and PDE6C. Tables 1 and 2 show percent amino acid identity between PDE8A and PDE2A, PDE5A and PDE6A,

I

PDESA1 and PDESA2 share homnology with other PDEs over the catalytic region (amino acids 492 through 748 in PDE8AI) andwihtepaivcivbndg domain conserved in the amino term-inal reuion of the PDE2A, PDE5A, PDE6A, PDE6B, AN"D PDE6C. The potential cGMP binding domnain of PDESA extends from amnino acids 75 to amnino acid 445 in the PDE8A I polypeptidec. Within the cGMP binding domains of PDE2A, PDE5A, PDE6A, PDEGB, and PDE6C, there are two internal repeats designated and and each repeat contains a seties of conserved amnino acids [McAllister-Lucas, et al., J. Bio. (2hein. 268:228_763-22S7 3 993)], In the corresponding repeat region of PDE8A, all of the conserved amnino acids are found:, in the corresponding repeat region, only some of the conserved residues were detected. An aspartate residue, shown to be essential for the cGMP bindiroc by bovine PDE5A [M cAl 1'ster- Lucas, et J. Bio!.

Chein. 270: 1-9 (1995)] Is not presen in the irepeat rciort of PDE8A It is therefore uncertain whether this region in PDESA functions to bind cGNIP.

Table 1 PDE8A Identitv in the Entire Protein PDE 2A -5A 6A 8A 2A 100 19 16 28 100 23 28 6A 100 21 8A 100 Table 2 PDESA Identity in the Catalytic Domain PDE 2A ,A 6A 8A 2A 100 _39 33 41 5A 100 42 46 6A 100 37 SA 100 18- Example Expression of Recombinant PDE8A An expression construct for PDE8A was generated that included DNA sequences 3' from the point of divergence of PDESA1 and PDE8A2 through the stop codon. The expression construction included DNA encoding an eight amino acid epitope tag. The so-called "FLAG tag," comprising the peptide sequence set out in SEQ ID NO: 34, was added to the amino terminus in order that the protein could be identified by Western blotting techniques using an anti-FLAG M2 antibody (Eastman Kodak, Rochester, NY) which specifically recognized the peptide of SEQ ID NO: 34.

SEQ ID NO: 34 Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys Sequences encoding an initiating methionine at the proteins amino terminus was also added.

As a first step in constructing the expression plasmid, PCR was performed using FB66a DNA as a template using primers set out in SEQ ID NOs: 35 (below) and W48A2 (SEQ ID NO: 10, p. 14) in a reaction mixture containing 2 iil each primer (stock 100 ig/ml), 2 pl 10X PCR buffer II (Perkin Elmer). 2 pl 1OX stock of each nucleotide (stock 2 mM), 1.2 p1 MgCl, (stock 25 mM). 0.09 1 5 Units/pl faq polymerase (Perkin Elmer), FB66a DNA and water to bring the reaction mixture to 20 pl. In the 5' primer (SEQ ID NO: 35), an Ncol site is in bold and the FLAG tag encoding region is underlined.

SEQ ID NO:

CAGTCAGCTAGCCGCCATGGACTACAAGGAC-

GACGATGACCAAGTTGACTGATGAAAAGGTG

PCR was carried out in a Perkin Elmer DNA Thermal Cycler under the following conditions: 94'C for 4 minutes followed by 30 cycles of94°C for one minute, 50 0 C for one minute, and 72 C for two minutes.

The resulting PCR product was digested with NcoI and KpnI, gel purified, and subcloned into Bluescript SKII vector previously digested with the same enzymes.

The Bluescript vector had previously been modified to include a Sac/ANcol alcohol dehydrogenase 2 (ADH2) promoter fragment removed from a YEpC-PADH2d vector [Price, et al., Meth. Enzymol. 185:308-315 (1990)] The resulting plasmid was designated W48pcrl.

AKpnIISstI fragment containing the 3' portion of the open reading frame was isolated from a FB66a cDNA and inserted into W48pcrl previously digested with KpnI and EcoRV. The resulting plasmid was designated W485.1.

A Sacl/Kpnl fragment containing the ADH2 promoter and the 5' portion of the PDE8A gene was isolated from W49pcr A KpnI/Sall fragment containing the 3' region of PDE8A was isolated from W485.1. The two fragments were ligated into the yeast expression vector YEpC-PADH2d that had been previously digested with SacI and Sail. The resulting plasmid was designated W48-2ADH2 and was deposited on October 2, 1997 under the terms of the Budapest Treaty with the American Type Culture Collection 12301 Parklawn Drive, Rockville, MD 20852. The bacterial strain bearing plasmid W48-2ADH2 was assigned accession number ATCC 98552. The DNA sequences generated by PCR and the DNA sequences at the PDE8/vector junctions were determined to insure proper plasmid construction. Upon confirmation of the sequence, the plasmid was transformed into a yeast strain BJ2-54 lacking endogenous PDE activity (ura3-52,trpl; leu2;cir ';gal2:pep4-3,prh l- 122:.prcl-402;APDE]:: U4RA3;HIS3; API)E2::.-TRPI).

The host cells were grown overnight in SC-leu selective media including 2% glucose, diluted to 1-2 x 10' cells/ml and subsequently grown to a density of 107 cells/ml in the same media. The presence of the expression plasmid appeared to increase the doubling time for cell growth two- to three-fold even under non-inducing conditions The cells were collected by centrifugation, washed with YEP media including 3% glycerol, resuspended in YEP/3% glycerol at a density of 107 cells/ml, and grown for 24 hours prior to harvest. Cells were frozen until use.

Frozen cell pellets (0.06 ml) were thawed and suspended in 0.2 ml lysis buffer containing 100 mM MOPS, pH 8.0. 200 mM NaC1, 2 pM ZnSO,, 2 mM dithiothreitol, and 10 pg/ml each protease inhibitors pepstatin, leupeptin, and aprotinin.

Approximately 0.2 ml of 0.5 mm glass beads were added to the cells which were then lysed with four 30-second cycles ofvortexing. The lysate was aspirated and the beads were washed twice with 0.3 ml lysis buffer. The lysate was combined with the washes to generate the yeast extract. In some experiments the lysate was fractionated by centrifugation at 105,000 x g for thirty minutes.

Western analysis was carried out on yeast extract containing the recombinant protein as follows. Proteins were first separated on SDS-PAGE and transferred to Immobilon-P (Millipore) using standard methods. The protein blots were blocked using 5% non-fat dry milk in 20 mM Tris-HC1, pH 7.4, 150 mM NaCI, 0.05%/ (TBST buffer plus milk) for one hour at room temperature. The blots were incubated with anti-FLAG M2 antibody (discussed above) at a concentration of 1 pg/ml in TBST buffer plus milk for one hour, after which the blots were washed four times with TBST buffer. The blots were then incubated for one hour with blotting grade affinity purified goat anti-mouse IgG antibody conjugated to horse radish peroxidase (HRP) (BioRad). The goat IgG was previously diluted 1:10,000 in TBST buffer plus milk. The blots were washed four times with TBST and treated, according to the manufacturer's suggested protocol, with the Renaissance® system (New England Nuclear Life Sciences Products) for enhanced chemiluminescence prior to autoradiography. The majority of the protein detected by the antibody was the size expected for the recombinant protein.

PDE activity was assayed by detection of 3 P-phosphate released from 3

"P-

cAMP or 3 "P-cGMP as described previously [Loughney et J Bio. Chem. 271:796- 806 (1996)]. The yeast extract was diluted in 0.5X lysis buffer also containing 0.5 mg/ml bovine serum albumin. Twenty ul of the yeast extract, or diluted yeast extract, was assayed in a 100 ul reaction volume which included an additional 50 mM Tris-HCl (pH 5 mM MgC,1 1 pM Zn SO,, and 0.1 mg/ml bovine serum albumin. Protein concentration was assayed by the method ofBradford.

PDE8A was observed to hydrolyze both cAMP and cGMP. In unfractionated lysates, the specific activity for cAMP was 3.9 nmol/min/mg and for cGMP was 7.6 nmol/min/mg. Fractionation revealed that 20-40% of the total activity was associated with the high speed supernatant fraction. Kinetic analysis of the activity with cAMP as substrate suggested the presence of both low and high K. forms of the enzyme -21 in a 1:1 activity ratio. The estimated values were 0.2 uM and 350 iM. Analysis of the high speed pellet suggested that the same species were present but in a high K,:low K, activity ratio of 1:4. Kinetic analysis with cGMP as substrate also suggested the presence of low and high forms of the enzyme. In these analyses, K, values were estimated to be 3 pM and 300 pM.

The ICo values for inhibition of PDE8A activity were determined using a set of isozyme-selective PDE inhibitors and the non-selective inhibitor isomethyl butyl xanthine (IBMX). Since these assays were performed at a cAMP concentration of 60 nM, the ICs 0 values reflect inhibition of the low K, form only. The results are set out in Table 3 with values shown in micromolar units.

Table 3 PDE8 Inhibition with Isozyme-specific PDE Inhibitors Compound Target PDE IC, for IC, 0 for Fold Family Target Family PDE8 Difference IC224 PDEI 0.08-0.008 2.7 38-338 EHNA PDE2 2 65 31 Cilostamide PDE3 0.02 12 750 IC197 PDE4 0.02 14 714 DMPPO PDE5 0.016 1.1 66 IBMX Non-selective 1-40 4.6 0.12-4.6 The ICs 0 values for each of the selective inhibitors were at least 30 times higher against PDE8 than against their target isozymes which suggests that the inhibitory profile of PDE8 is distinct from that of PDEs 1-5. The hydrolysis of cAMP and cGMP clearly distinguishes the enzymatic activity of PDE8A from that of PDE6 and PDE7A. The ICso of the non-selective inhibitor IBMX for PDE8 was in the range observed for known human PDEs suggesting that the catalytic site of PDE8 resembles those of other human and mammalian PDEs and is distinct from lower eukaryotic forms that are insensitive to IBMX.

22- Example 6 Northern Analysis of PDE8A Expression Northern analysis of PDE8A expression was carried out using a human multiple tissue blot (Clontech, Palo Alto, CA). The 327 base probe was extended from nucleotide 1767 to nucleotide 2293 in SEQ ID NO: 3. Riboprobe preparation and hybridization conditions were as previously described [Loughney, et al. supra].

Results showed a 9.5 kb mRNA in all tissues examined but band intensity varied. The signal was strongest in heart, brain, and kidney; the signal was weaker in liver, placenta, pancreas, and skeletal muscle. The signal was weakest in lung.

Example 7 Chromosome Mapping of Human PDE8A Yeast artificial chromosomes (YACs) containing the human PDE8A gene were isolated from a panel of human YACs purchased from Research Genetics and screened by PCR as follows.

The YAC super-pools were screened with two nested pairs of primers.

In the first screening reaction, sense primer W48S8 (SEQ ID NO: 36) was paired with the anti-sense primer W48A10 (SEQ ID NO: 37). PCR was carried out with 10 mM Tris-HCl, pH 8.3, 50 mM KC1, 2 mM MgSO 4 0.2 mM of each dNTP, 10 ~g/ml of each primer, 0.5 units of Taq polymerase (Perkin-Elmer) and 1.5 Fxl of YAC pool DNA as template. Reactions were carried out for 30 cycles, each cycle consisting of one minute at 94'C, two minutes at 60"C, and four minutes at 72"C. After the first round of amplification, the reaction products were reamplified with the internal pair of primers W48S12 (SEQ ID NO: 36) and W48A12 (SEQ ID NO: 37).

W48S12 SEQ ID NO: 36

CCAGAAGGGGTACTITTCC

W48A12 cr\ Tr Q L A/ J. N

CATTGTCCTGAGGCTGTGG

-23 The reactions were carried out as described above except that the template was 1 1 l of a 1:10 dilution (in water) of the first round reaction. Super-pools yielding the correct size PCR product were identified and the corresponding sub-pools were screened with the same nested pairs of primers under the same conditions to identify unique addresses for YACs containing PDE8A.

Yeast strains harboring the relevant YACs were purchased from Research Genetics. In order to verify the presence of the PDE8A gene in the various YACs, DNA was prepared from each strain and analyzed by PCR with primers W48S8 and W48A10. DNA was prepared from each strain according to a method previously described [Hoffman and Winston, Gene 57:267-272 (1987)] but modified as follows.

Strains were grown overnight at 30 0 C in YEP media containing glucose. Ten ml of culture was pelleted by centrifugation and resuspended in 200 Fl of aqueous buffer containing 10 mM Tris-HCI, pH 8.0, 100 mM NaCI, 1 mM NaEDTA, 1% SDS, and 2% Triton-X100. The cells were lysed by vortexing in the presence of 200 Al of phenol/chloroform (1:1 mixture) and 100 lI of glass beads (425-600 Following lysis, 200 ul of TE Buffer (10 mM Tris, pH 8.0, 1 mM NaEDTA) was added and the sample was centrifuged to separate the phases. The organic phase was extracted again with 200 fl of aqueous buffer. The pooled aqueous phase was treated with 100 units of bovine pancreatic RNase (Boehringer Mannheim) for 1 hour at 37 C and the sample was extracted with phenol/chloroform, re-extracted with chloroform, and ethanol precipitated according to established methods. The resultant pellet was resuspended in 50 zl TE Buffer. PCR was carried out as described above except that the reaction volume was 25 pl and the template consisted of 1 ul of the relevant yeast DNA preparation.

Three human YACs containing the PDE8 gene were identified with addresses 805B6, 919H10 and 920A3 (as per the CEPH designation). According to information in the Center for Genome Research database (Whitehead), the three YACs overlap one another and are part of a singly-linked contig (WC6.16) on human chromosome 6. Two sequence tagged sites within this contig (D6S305 and D6S411) -24have been placed on the chromosomes 6 genetic map at a position 167 cM from the end of 6p in work at the Center for Genome Research; D6S305 has been mapped to a position 173 cM from the end of 6p in work at CEPH-Genethon. Three other YACs within the WC6.16 contig (932F1, 956B1 and 947D5) have been mapped by florescence in situ hybridization at CEPH-Genethon. The hybridization signals fall between 0.94 and 0.99 fractional length units from the end of 6p. According to the CEPH integrated summary map [Chumakov et al., Nature 377 (Supp):175-297 (1995)], this region corresponds to the cytogenetic region 6q26-27.

Heritable defects that have been associated with this region of the human genome include retinal cone degeneration (OMIM database), Insulin-dependent diabetes mellitus [Davies et al. Nature 371:130-136 (1994); Luo et al. Am. J. Hum. Genet.

57:911-919 (1995)] and juvenile onset parkinsonism [Matsumine et al. Am. J. Hum.

Genet. 60:588-596 (1997)]. In addition, loss of heterozygosity (LOH) is frequently observed in this region in a variety of different cancer cells, including Burkitt's lymphoma [Parsa et al. Genes, Chromosomes Cancer 9:13-18 (1994)], astrocytoma [Liang et al. Neurology 44:533-536 (1994)], gastric carcinoma [Queimado et al.

Genes, Chromosomes Cancer 14:28-34 (1995)], parathyroid adenoma [Tahara et al.

Cancer Res. 56:599-605 (1996)] and ovarian carcinoma [Cooke et al. Genes, Chromosomes Cancer 15:223-233 (1996); Saito et al. Cancer Res. 56:5586-5589 (1996)]. LOH has been suggested to indicate the presence of a tumor suppressor gene in the affected region [Weinberg, Science 254:1138-1146 (1991)]. Due to its widespread expression, it is possible that mutation of the PDE8 gene may be involved in all or some of these genetic abnormalities.

Example 8 Verification that PDE8AI and PDE8A2 Represent Splice Variants and Efforts to Extend the 5' Sequence of PDE8A2 To verify that PDE8AI and PDE8A2 represent 5'splice variants, two approaches were taken. First, PCR analysis revealed that, in genomic DNA, neither PDE8A nor PDE8A2 sequences were adjacent the DNA sequence of the common region. The genomic sequences upstream of the common region were present in a third PDE8A cDNA, FB74b, which was identified in the group of six original clones that hybridized to the 5' end of probe W04835 described in Example 2. The partial sequence (755 nucleotides at the 3' end) of clone FB74b is set out in SEQ ID NO: 39. The FB74b cDNA diverged from FB85c-2 and FB66a at the same position as FB85c-2 and FB66a diverged from each other, but the FB74b clone did not maintain the open reading frame.

In the FB74b sequence 5' to the point of sequence divergence from the FB66a and FB85c-2 clones, an in-frame stop codon was closer to the point of divergence than an initiating methionine codon indicating that, if FB74b represented a cDNA rather than an unspliced precursor, the initiating methionine would necessarily be located in the sequence common to both FB66a and FB85c-2.

PCR analysis was performed using one primer designated FB74bSI (SEQ ID NO: 40) within the FB74b upstream sequences and a second primer designated W48A9 (SEQ ID NO: 11) within the sequences common to FB74b, FB66a, and FBS5c-2.

FB74bS 1 GTTAGATGAGAGGTTGCTGG SEQ ID NO: Using 1 iug of human genomic DNA as template, a band was amplified having the same size as the one amplified using FB74b as template, indicating that the sequences unique to FB74b and the common region were adjacent in genomic DNA. Thus, the FB74b sequence may represent an unspliced intron or may represent a third splice variant that would encode a protein with an initiating methionine within the common region. In either case, the FB85c-2 and FB66a sequences are presumably generated by splicing.

Secondly, 5' RACE analysis was performed using RNA isolated from human cortex, cerebellum, heart, liver and lung tissues. RNA was isolated from frozen tissue fragments as described [Loughney et al, Biol. Chem. 271: 796-806 (1996)] and poly A' mRNA was selected using the Fast Track TM mRNA isolation system (Invitrogen).

Double stranded cDNA was prepared using 5 pg poly A' mRNA and a cDNA synthesis -26 kit (Boehringer Mannheim). The cDNA was ligated to a linker formed by annealing oligonucleotides L15 (SEQ ID NO: 41) and L30 (SEQ ID NO: 42), GTATGCTAATCTCAG SEQ ID NO: 41 CAACTCGAATTCCTTGACAGATTAGCATAC SEQ ID NO:42 For the 5' RACE, the linker-ligated cDNA was amplified by PCR using oligonucleotides LIS (SEQ ID NO: 43) and W48A13 (SEQ ID NO: 44).

LI8 CAACTCGAATTCCTTGAC SEQ ID NO: 43 W48AI3 GTTGTTCTTCCTCTTCAGCC SEQ ID NO: 44 The reaction contained 10 mM Tris-HCI, pH 8.3, 50 mM KCI, 1.5 mM MgCI,, 0.2 mM of each dNTP, 10 pg/ml of each primer and 1 ul of linker-ligated cDNA in a reaction volume of 25 pl. Following heating step at 94°C, PCR was initiated by the addition of 0.1 unit of Taq polymerase (Boehringer Mannheim) and continued with 30 cycles of one minute at 94°C, two minutes at 60°C, and four minutes at 72"C.

The products of the PCR reaction were diluted ten-fold with water and used as template in a second PCR reaction with oligonucleotides L21 (SEQ ED NO: and W48A9S (SEQ ID NO: 46) under the same conditions described above.

L21 CAACTCGAATTCCTTGACAGA SEQ ID NO: W48A9S GATCGTCGACCTGTCTCTGCACTAACAC SEQ ID NO: 46 DNA amplified in the second PCR reaction was cleaved with EcoRI and Sall and ligated into the vector Bluescript (Stratagene) previously digested with the same enzymes.

Initially, DNA sequences in five plasmids from each tissue source were examined and both PDESA1 and PDESA2 5' sequences were found among the cDNAs isolated. FB74b 5Ssequences were also obtained, as were several sequences, each isolated only once, that could represent yet additional splice variants or unrelated DNA sequences.

-27- Because none of the PDE8A2-like cDNAs extended further 5' than did the original FB66a cDNA, additional PDESA2 RACE clones were analyzed in an attempt to extend the 5' end sequence. An additional five lung PDE8A2 cDNAs were identified and sequenced, but none extended the PDE8A2 sequence.

A second round of RACE PCR was repeated using the L21 primer (SEQ ID NO: 45) with primer W48A14S (SEQ ID NO: 47).

W48A14S SEQ ID NO: 47

GATCGTCGACAAGCACTCGGTCAGCCTTCG

The resultant clones were screened by PCR and the longest ones were chosen for sequencing. Only two clones were longer than the original FB66a cDNA and they extended the 5 sequence 8 and 12 bp, respectively, in the untranslated region. The FB66a sequences were extended with 5 '-CCCAGGGCGCCA. The extreme 5' end of FB66a is very GC rich which may contribute to the difficulty in isolating full length cDNAs.

Example 9 Expression and Characterization of PDE8A The recombinant PDE8A described in Example 5 existed in both low affinity and high affinity forms in yeast extract. Because of the possibility that the low affinity form represented partially inactive enzyme, PDE8A expression was carried out in sf9 and COS cells in an attempt to either obtain a homogeneous enzyme or determine if the two kinetic forms are always expressed from the cDNA.

The PDE8 sf9 expression construct was generated with a 3' Kpni-SaH fragment from plasmid W485. (described in Example 5) and a 5' fragment generated by PCR as follows. The primers FLAG-1 (SEQ ID NO: 48) and W48A4 (SEQ ID NO:12) were used in PCR with PDE8 COS-1 DNA (described below) as template.

FLAG-1 GATCGGATCCACCATGGACTACAAGG SEQ ID NO: 48 28 PCR was performed as described in Example 8 except that 2 mM MgSO 4 was used in place of MgCI, and 0.02 U Taq polymerase was used. Following a four minute initial incubation at 94'C, 30 cycles were performed with one minute at 94°C, one minute at 50'C, and two minutes at 72 0 C. The 5' amplification product was cleaved with Ban2HI and KpnI, gel purified, and ligated with the 3' fragment into vector pFASTBAC (Gibco BRL, Gaithersburg, MD) previously digested with BamHI and SalI. The resulting plasmid was designated pFBRPDES. All PCR amplification products and all new junctions were verified by sequencing.

Recombinant viral stocks were produced using the FastBac system (Gibco BRL) according to the manufacturer's suggested protocol and protein expression was carried out as follows. Sf9 cells were grown at 27°C in CCM3 media (Hyclone, Logan, UT) containing 50 U/mi penicillin and 50 jIg/ml streptomycin sulfate (Gibco).

Exponentially growing cells were infected at a multiplicity of approximately two virus per cell and incubated for 48 hours. Cells were collected by centrifugation, washed with CMF-PBS (2.7 mM KCI, 1.5 mM KHPO,, 137 mM NaC1, 8.1 mM Na 2

PO)

and the pellets were frozen and stored at -80°C until use. Cells were lysed in buffer mM MOPS pH 7.2, 10 pM zinc sulfate, 1 mM DTT, 2 mM benzamidine, zg/ml each pepstatin, leupeptin, and aprotinin, and 20 pg/ml each calpain I and calpain II inhibitors) by vortexing in the presence of an equal volume of glass beads (acid washed, 0.5 mm, Sigma) and PDE activity was determined as described in Example In the sf9 extract, 45.4 nmol/min/mg PDE activity was detected for cAMP hydrolysis (100 pM substrate) and 69.4 nmol/min/mg for cGMP hydrolysis (100 aM substrate). The background PDE activity was negligible. The PDE8A activity appeared to be a mixture of high and low affinity forms as detected in yeast extracts as described in Example For expression in COS cells, PDE8 COS-1 was generated by combining a 3' KpnIlSail fragment from plasmid W485.1 (Example 5) and a NheIlKpnI fragment obtained by cleavage of a PCR amplification product from a reaction including FB66a -29cDNA as a template with primers W48A2 (SEQ ID NO: 10) and ATG (SEQ ID NO: Conditions for the PCR included an initial incubation for four minutes at 94 C followed by 30 cycles of one minute at 94°C, one minute at 50'C and two minutes at 72°C in a Perkin Elmer Cetus DNA thermal cycler. The resulting 5' fragment and the 3 fragment described above were ligated into vector pClneo (Promega, Madison, WI) which had been previously digested with NheI and Sall.

Semi-confluent COS cells growing in 15 cm dishes were washed once with 25 ml DMEM (Dulbecco's Modified Eagle Media, 100 U/ml penicillin and 100 zg/ml streptomycin sulfate, GIBCO), after which 14 ml of DMEM/DEAEdextran/chloroquine was added per plate. DMEM/DEAE dextran/chloroquine is comprised of 75 ml DMEM and 30 I 0.25 M chloroquine in PBS (2.7 mM KCI, mM KH 2

PO

4 137 mM NaCI, 8.1 mM Na,PO,. 0.9 mM CaCI, 0.5 mM MgC), together with 0.75 ml 50 kg/ml DEAE-dextran (Pharmacia, Uppsala, Sweden).

Twenty pg of plasmid DNA in 135 Al Tris/EDTA buffer (TE) was added per plate and the plates were incubated for two hours at 37°C in 5 CO,. The media was removed and 12 ml of 10% DMSO/PBS was added for one minute and removed. The cells were washed once with 25 ml DMEM, after which another 25 ml of DMEM containing fetal calf serum (Hyclone, Logan, UT) was added and the cells were incubated overnight at 37°C in 5% CO,. The media was removed and the monolayer was washed with 25 ml of CMF-PBS. Six ml of a solution containing 0.05 mM EDTA (Gibco) was added and the cells were incubated five minutes at 37'C.

Cells were removed from the plates by trituration and transferred to conical centrifuge tubes. The plates were washed with six ml of complete DMEM to harvest any remaining cells and the wash solution was added to the centrifuge tubes. Cells were pelleted by centrifugation for five minutes at approximately 340 x g, resuspended in five ml complete DMEM, removed to a 15 cm tissue culture dish containing 20 ml complete DMEM, and incubated overnight in 5% CO,.

The monolayer was washed two times with CMF-PBS, incubated five minutes at 37 0 C in versene (0.5 mM NaEDTA-2HO, 137 mM NaC1, 2.68 mM KC1, 8.1 mM NaHPO 4 1.1 mM glucose, pH and harvested as described above.

Pelleted cells were washed with CMF-PBS, frozen in dry ice, and stored at -80"C until use. Cells were lysed in buffer (50 mM MOPS, pH 7.2, 10 tM zinc sulfate, 1 mM DTT, 2 mM benzamidine, 10 Ag/ml each pepstatin, leupeptin, and aprotinin, and pg/ml each calpain I and calpain II inhibitors) by passage through a French pressure cell (SLM Instruments) at 20,000 psi and PDE activity was determined as described in Example PDESA expression was low in the COS cell extract and could not be accurately characterized due to the high level of background activity from endogenous PDEs. In order to more fully characterize the COS cell expression product, the enzyme including a FLAG tag at the amino terminus (Example 5) is purified from a 100,000 x g supernatant of cell extract using an anti-FLAG M2 affinity column (Sigma) according to the manufacturer's suggested protocol. In order to more accurately characterize yeast PDE8A activity, expression of a recombinant protein that is truncated at the amino terminus but retains the catalytic region is carried out as described in Example 5 in an attempt to obtain a homogenous protein.

Example Production of Anti-PDE8A Antibodies A GST fusion protein was produced in E. coli to provide an antigen for generation of monoclonal antibodies to PDE8A. An EcoRI fragment from FB70a (a PDESA cDNA that includes nucleotides 182-1330 of FB85c-2 and which was one of the nine clones originally identified which hybridized to the full length W04835 probe described in Example 2) was inserted into the EcoRI site of pGEX5X1 (Pharmacia) and the resultant construct was transformed in the E. coli strain XL1 Blue. A GST-PDE8A fusion protein including 382 amino acids from PDESA was produced from this construct following induction with IPTG. The fusion protein was isolated using SDS-PAGE, the band of appropriate size excised from the gel following staining with cold 0.4 M KCI, and the protein obtained from the acrylamide by electroelution. The -31 elution product was dialyzed against PBS and concentrated using Centriprep 10 and Centricon columns (Amicon, Beverly MA) prior to being injected into mice.

On day 0, four Balb/c mice were pre-bled and injected subcutaneously with a panel of antigens including 30 g/mouse GST-PDE8 fusion protein in complete Freund's adjuvant in 200 tl total volume. The same injections were repeated at weeks three and nine in incomplete Freund's adjuvant. Ten days after the last immunization, test bleeds were obtained and screened by antigen capture ELISA and Western analysis.

In the ELISA, Immulon 4 plates (Dynex, Cambridge, Massachusetts) were coated at 4°C with 50 l/well of a solution containing 2 tg/ml GST-PDE8 in mM carbonate buffer, pH 9.6. Plates were blocked with 0.5% fish skin gelatin (Sigma) for 30 minutes and 50 ul serum diluted in PBS with 0.5 Tween 20 (PBST) was added. Serum dilutions ranged from 1:100 to 1:102,400 and were obtained by a series of doubling dilutions. After incubation at 37°C for 30 minutes and washing three times with PBST, 50 Ml of horseradish peroxidase-conjugated goat anti-mouse IgG(fc) antibody (Jackson) (diluted 1:10000 in PBST) was added. Plates were incubated as above and washed four times with PBST. Antibody was detected with addition of tetramethyl benzidine (Sigma Chemical, St. Louis, Missouri) and the color reaction was stopped after five minutes with the addition of 50 u 1 of 15% H 2

SO

Absorbance at 450 nM was measured on a plate reader.

For Western analysis, SDS-PAGE gels were run with approximately tg yeast PDE8 extract and approximately 200 ng of gel-purified GST-PDE8 and the proteins were transferred to Immobilon-PVDF. A standard enhanced chemiluminescence (ECL) Western blot protocol was performed using BioRad goat anti-mouse IgG horseradish peroxidase as the secondary antibody.

In preparation of hybridomas, splenocytes from mice giving a positive result from the ELISA and/or Western blotting protocols above, were fused to NS-1 cells in a ratio of 5:1 by standard methods using polyethylene glycol 1500 (Boehringer Mannheim) (Harlow and Lane, Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory, 1988). The fused cells were resuspended in 200 ml RPMI containing -32- FBS, 100 mM sodium hypoxanthine, 0.4 mM aminopterin, 16 mM thymidine (HAT) (Gibco), 25 units/ml IL-6 (Boehringer Mannheim) and 1.5 x 106 murine thymocytes/ml and dispensed into ten 96-well flat bottom tissue culture plates (Corning, United Kingdom) at 200 Al/welI. Cells were fed on days 2, 4, and 6 days post fusion by aspirating approximately 100 .l from each well with an 18 G needle (Becton Dickinson) and adding 100 /l/well plating medium described above except containing units/ml IL-6 and lacking thymocytes. On days 9 to 12, supernatants from the fusion wells were screened by antigen capture ELISA using GST and GST-PDE8 and by ECL Western analysis as described above.

A positive signal of the expected size was obtained on both lanes of the Western blot using mouse blood and a monoclonal antibody with very weak reactivity to the yeast recombinant protein was obtained in the subsequent fusion. The entire procedure is repeated using 50 yg antigen/mouse to obtain more strongly immunoreactive monoclonal antibodies.

Example 11 Analysis of PDE8A Expression by in situ Hybridization Expression of PDE8A was examined in tissue sections by in situ hybridization as described below.

Preparation of probe An Xhol/EcoRI restriction enzyme fragment from the cDNA (corresponding to nucleotides 571 to 1226 of SEQ ID NO: 1) was subcloned into a Bluescript vector (Stratagene, La Jolla, CA) to generate an expression plasmid designated PDE8XR2A. The plasmid was cleaved with Xhol and transcribed (see below) with T3 polymerase to generate an antisense probe. A sense probe was generated by cleaving PDE8XR2A with EcoRI and transcribing with T7 polymerase.

The PDE8A templates were transcribed using a RNA Transcription kit (Stratagene, La Jolla, CA) in a reaction containing 5 tl of 5X transcription buffer (Stratagene), 30 mM DTT (Stratagene), 0.8 mM each ATP, CTP, GTP (10 mM (Stratagene), 40 U RNase -33 Block II (Stratagene), 12.5 U T3 or T7 polymerase (Stratagene), and 300 ng linearized plasmid template, 50 ACi "S-UTP (greater than 1000 Ci/mmol, Amersham, Arlington Heights, IL). The mixture was incubated at 37°C for one hour after which the template DNA was removed by addition of 1 pl of RNase-free DNase I (Stratagene) and incubation for 15 minutes at 37°C. The probe was hydrolyzed by adding 4 pl 1 M NaHCO 3 and 6 l 1 M Na 2 CO for 22 minutes at 60°C and the reaction mixture was neutralized by addition of 25 ul of a solution containing 100 l 3 M sodium acetate, il acetic acid (VWR, So. Plainfield, NJ), and 395 ,l dH 2 0. A Quick Spin G50 RNA column Inc., Boulder, CO) was prepared according to the manufacturer's suggested protocol. The probe was placed in the center of the column and the column centrifuged for four minutes at 1,000 rpm in a desk top centrifuge. The column flowthrough was mixed with 50 il dH 2 O, 2 pl of a 10 mg/ml tRNA solution, 10 l1 3 M sodium acetate, and 200 ul 100% ethanol (VWR) and the resulting mixture was incubated at -20°C overnight. The probe solution was microfuged for 15 minutes at 4°C, the supernatant was removed, and the pellet was resuspended in 40 /x IX TBE containing Ipl of 0.1 M DTI. The probe was stored at -70'C until the in situ hybridization assay was performed.

Preparation of tissue samples and in situ hybridization Tissues (National Disease Research Interchange, Philadelphia, PA and Cooperative Human Tissue Network, Philadelphia, PA) were sectioned at 6 pm and placed on Superfrost Plus slides (VWR). Sections were fixed for 20 minutes at 4°C in 4% paraformaldehyde (Sigma, St. Louis, MO). The slides were rinsed in three changes of IX CMF-PBS, dehydrated with three successive washes with 70 ethanol, ethanol and 100% ethanol and dried for 30 minutes at room temperature. The slides were placed in 70% formamide Baker) in 2X SSC for two minutes at 700C, rinsed in 2X SSC at 4°C, dehydrated through 70%, 95% and 100% ethanol washes, and dried for 30 minutes at room temperature.

-34- A prehybridization step was performed by placing the slides in an airtigh box cootsining a Piece Of filter paper saturated with box buffer containing 50 forinamide Btaker) in 4X SSC. Each section was covered with 100 yd of rHB2 buffer consisting of 10% dextrari sulfate (Sigma), 50% fonnamide Baker, Philipsburg, NJ), 100 mM DTI (Boehringer Mannheim, Indianapolis, IN), 0.3 M NaCI (Sigma), 20 mM Tris, PH 7.5. 5 mM HDTA (Sigma), and IX Denhardt's solution (Sigma) and the slides were incubated at 42 0 C for 1 hour. The probe, as described above, was prepared by mixing 4 x l D' cpni/tissue section with 5 jul of a mgfml tINA solution per section and hetating the mixture at 95 0 C for three minutes.

Ice cold rHB2 buffer was added to bring the final volume to 20 iz1/section. The probecontaining solution (20 pll/section) \N as addcd to 100 a1 rHB2 buffer previously applied. The slides were incuiud at 55'C for 12 to 16 hours. Following hybridization, the slides were washed once in 4X SSC containing 10 mM DIT for one hour at room temperature, once in deionized formamnide Baker), IX SSC, and I mM Dtr for 40 minutes at 00(-C. once in 2X SSC for 30 minutes at room tenpratre; and once in 0.lX SSC for 30 minutes at room temperature. Thbe section: were dehydrated through 70%, 95%. and 100% ethanol washes and air dried for Minute.. The slides were dipped in Kodak NT32 nuclea~r emulsion, dried for one to three hours at room temperatttre. in ;he dark and stored in the dark at V~C with desicantuntil time of development.Th slides were developed in V 0 C Kodak Dehol developer for four minutes, dipped four times in V'C dH 2 Q, and placed in VC Kodak fixe for fou minutes. The slides were rinsed in dH.O and a standard H&B stain was performed as follows.

The slides Wer rinsed in cH,O and stained with hematoxylin and eosin by transfer of the slides through a series of the following step: five minutes in formadehydlfcohol (100 ml forntldkehydle. 900 ml 80% ethanol); three rinses in wa o oa ftomnts five minutes in 0. 75 Harris heniatoxylin (Sigma); three rim~es in water for a total of two innutes; one dip in 1 IICI/5O ethanol; one rins in walr; four dips in I lithlium carbonate; ten minutes in tap water; two minutes in 0.5 eosin (Sigma); three rinses in water for a total of two minutes; two 00 S minutes in 70% ethanol; three one minute rinses in 95 ethanol; two one minute rinses in 100% ethanol; and two two minutes rinses in xylene. Slides were mounted with 00 0C cytoseal 60 (Stephens Scientific, Riverdale, NJ).

00 SThe signals obtained with an antisense PDE8A probe were compared to the control signals generated by a sense PDE8A probe and any signal specific to the antisense probe was assumed to represent PDE8A expression. PDE8A signal was detected throughout much of the cerebellum, in a subset of cells in the seminiferous tubules of the testes, on scattered cells of yet undetermined origin in skeletal muscle, in granulosa cells and ovarian stroma in the ovary, in epithelial cells in the loop of Henle in the kidney and on the smooth muscle of some arterioles in the heart.

These results differ from those obtained by Northern blotting and described in Example 6 in that a moderate signal was detected in heart by Northern blot while the in siru data using this heart sample gave a weak signal. The inconsistency could reflect differences in the tissues from different individuals or level of detection differences inherent in the two methods. The signal in the ovary and the signal in the kidney may indicate that PDE8A is involved in ovulation or in salt and/or water homeostasis. respectively.

Numerous modifications and variations in the invention as set forth in the above illustrative examples are expected to occur to those skilled in the art.

Consequently only such limitations as appear in the appended claims should be placed on the invention.

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

Claims (20)

1. A purified and isolated PDES polypeptide. 00 00 2. The polypeptide according to claim 1 comprising the amino acid sequence Sset out in SEQ ID NO: 2, o 5 3. The polypeptide according to claim 2 comprising the amino acid sequence C set forth in SEQ ID NO: 4.

4. The polypeptide according to claim 2 comprising the amino acid sequence set forth in SEQ ID NO: 6. A purified and isolated polypeptide encoding the polypeptide according to claim 1, 2, 3 or 4.

6. The polynucleotide according to claim 5 comprising the sequence set forth in SEQ ID NO: 1.

7. The polynucleotide according to claim 5 comprising the sequence set forth in SEQ ID NO: 3.

8. The polynucleotide according to claim 5 comprising the sequence set forth in SEQ ID NO:

9. A purified and isolated polynucleotide encoding a human PDE8 polypeptide selected from the group consisting of: the polynucleotide according to claim 5; and a DNA which hybridizes under stringent conditions, including a final wash at 65'C, to the polynucleotide of(a). A purified and isolated polynucleotide encoding a human PDE8 polypeptide selected from the group consisting of: the polynucleotide according to any one of claims 6, 7, and 8; and T 3. MAY. 2006 9:03- WRAY AND ASSOCIATES NO. 816 P. 0 0 -37- a DNA which hybridizes under stringent conditions, including a final wash at 65°C, to the polynucleotide of(a).

11. The polynucleotide of claim 5 which is a DNA molecule. 00 00 12. The DNA of claim 11 which is a cDNA molecule. O O e 5 13. The DNA of claim 11 which is a genomic DNA molecule. 0 CI 14. The DNA of claim 11 which is a wholly or partially chemically synthesized DNA molecule. An anti-sense polynucleotide which specifically hybridizes with the complement of the polynucleotide of claim

16. A expression construct comprising the polynucleotide according to claim 38

17. A host cell transformed or transfected with the polynucleotide according to claim 16.

18. A method for producing a PDE8 polypeptide comprising the steps of: a) growing the host cell according to claim 17 under conditions appropriate for expression of the PDE8 polypeptide and b) isolating the PDE8 polypeptide from the host cell of the medium of its growth.

19. An antibody specifically immunoreactive with the polypeptide according to claim 1, 2, 3, or 4. The antibody according to claim 19 which is a monoclonal antibody.

21. A hybridoma which secretes the antibody according to claim

22. An anti-idiotype antibody specifically immunoreactive with the antibody according to claim 19. tn -39-

23. A method to identify a specific binding partner compound of a PDE8A C polypeptide comprising the steps of: contacting the PDE8A polypeptide with a compound under conditions 00 which permit binding between the compound and the PDE8A polypeptide; 00 detecting binding of the compound to the PDE8A polypeptide; and 00 identifying the compound as a specific binding partner of the PDE8A polypeptide. cN 24. The method according to claim 23 wherein the specific binding partner modulates activity of the PDE8A polypeptide. The method according to claim 24 wherein the compound inhibits activity of the PDE8A polypeptide.

26. The method according to claim 24 wherein the compound enhances activity of the PDE8 polypeptide.

27. A method to identify a specific binding partner compound of a PDE8A polynucleotide comprising the steps of: contacting the PDE8A polynucleotide with a compound under conditions which permit binding between the compound and the PDE8A polynucleotide; detecting binding of the compound to the PDE8A polynucleotide; and identifying the compound as a specific binding partner of the PDE8A polynucleotide.

28. The method according to claim 27 wherein the specific binding partner modulates expression of a PDE8A polypeptide encoded by the PDE8A polynucleotide.

29. The method according to claim 28 wherein the compound inhibits expression of the PDE8A polypeptide. tn The method according to claim 28 wherein the compound enhances expression of the PDE8 polypeptide. 00 31. A purified and isolated polynucleotide according to claim 1 as herein before described with reference to the examples. 00 C 5 DATED this EIGHTEENTH day of FEBRUARY 2005. 00 C ICOS Corporation Applicant 0 10 Wray Associates CN Perth, Western Australia Patent Attorneys for the Applicant. -1I- SEQUENCE LISTING <110> Loeghney, Kate <120> Phesnhodiesterase SA <130> 27866/35047 <140> <141> <150> 08/351,648 <151> 1997-10-16 <160> 48 <170> Peteotlfl Var. <210> 1 <211> 2298 <212> DNA <213> None sapiens <220> <221> CDS <222> (2299) <220> <221> <222> <223> misc feature (S68) (870) The amino acid encoded by nucleotides 868-8-7D is either Pro or Lee <400> 1 ttg ace pat pea ace peg apr gca cat Led Eth Asp Gic Lye Val LVs Ala Tyrc 7cc Ct Cc retc SCe Leu His ro rap pta 91:n -Val tte gat gee Lee Asp Gle cgg cog aag Trp Lee Lye et-a tot pea ego got ago gre gap ace Val 5cr Glu See Val See Ale Olu The gtea gap aec Val Glu Lye egg ap at at Arp Lye Asm Aen t-a ens gat gee org gCt ct aep Set C-lu Ace3 Gle Sec Ala Pro Lye gee gt apt egg tat ccc Gle Val Set Arp l-Tye Gin s0 acp act cog tag ET', .sn Met Glin gga gtt 'fly Val gee tat gee Val Tye Olu 192 ec at ago Lee Asm Set tat ate pea cac Eyr Ile Gle Gin 70 tgg top per Arg Lee Asp ace gga ggs pat ac tag 240 The G 1 y Sly Asp Acm Gin 75 s0 eta eec eec tat yaa ety age aye Len Len Leu Tyrr Gin Len See See ate ate aaa ata gee aca aaa le Ile Lys Ile Ala The Lys gat gga Asp Gly ace tee Ile Fle get gyg Ala Sly 130 tee app See Ary 145 eea aye Pro Arg eye tta Cys Len ety tat Leu Tyr gct pee Val Ala 210 cay yea Gin Val 225 etc gee Len Asp te etc Len Len ceg tat Len Tyr ec ggy Pro Sly act cay Thr Gin ctg eta Len Len 150 yga ety Sly Len 165 gte! ace Vai The tyg gyc Trp Sly eat ett Ase Len pyc ett Giy Len 230 aaa aca Lys The 245 sea acy ile Metc tee: cay Flee Gin ttc etc ge Phe Len Sly 105 ata aay ya Ile Lys Gin yye ace ace Sly The The 135 pta yaa par Val Glnu A sp:: pea eca g Gln Ser 0-iy yea etc ype Ala ile Giy 185 aea gaa per Lys Gin -Ala 230 pee egg yet Al a Trp Al 1a 215 pee eca cay Ala Ly s G In eat tc gat Tr Ple Asp ate eat pea Tie Thyr Ala 265 ytg yac eat Val Asp Hi-s 28D eat Asn ccc Pee Ala 140 ypa Sly ate Tie ert Ile gee .Al a Len gte Val cC p Lenl aap Lys eye tyt pea A ry Cys Ala 275 gao cyr ttt gat att gge gag Aso Xac Pie Asp Ile Sly Gin pea ap pea pga aaa rot ate tre aeg Sin Lys Sinu Sly Lye Pro Vel Pie Lys gaa grc erg ac Sin Tal Len Aso gaa gta gee erg Si I Vl AspD Leu 345 Coo arc gro age Pro :ls Val Ser 360 aaa ate apt ge Lye lie Sec Sly 375 atg ttt gee gte Mer Pie Al a Val 390 cat age art ego His Amg Tie Ar9 etg tee tar ear Len Set 'Tye His 425 rae Ore arc rtr Sin Pi e Tie Lee 440 rte gee art ggr Pie Ase Ile GSl'v 455 cog ptt eat egg Met Val His -Arg 470 art pet Ile Ala pee tar Ala Tyr tar ac '1y r Thr 350 pop ara va, Ie 365 ret aec Sec 14s tea pee Len Ala tpe at 53's Ile ecr ta Thr Sac 430 etc rge teu tee 445 eac atg Asn Mat ace tee Tic Sec ge rae Sly Gin 320 pea gae Ala Asp 335 erg egg Tic Acg Sly Val aea get Tir Aep Len His 400 tee egg T-yc Aeg 415 pea gag Sin Sin a pa Lye Sin rpp rot Tcp Pro rge tt Sys Pie 480 912 960 1008 1056 1104 1152 1200 1248 1296 1344 1392 1440 14B8 gag etr pee ap Org opt ego err art erg Ocr prg ap ap ac oat Sin Len Sln Lys Len Cys Acg Pie lIe Met Sec Tel Lys Lys Aso Tyr 4- ogg cpp prt cct tat coc aec tag Arp Arg Vol Pro Tyr His Act T=p cat gcg ptt His Ala Val act gta poa c Tnr Val1 Ala His 510 tpc atg tat pcc Cys mot Tyr Ala 515 ota ott cap aac oat coc aop Ott Ile Len Gin Asn Act His Thr Lou 52D ace gac Ott Thr Asp Lou 1536 1584 1632 pop cgo Pin Arp 530 oaa pga ctp cop ott pop tqt ctg tpt cat gao ctg pa: coo Lys Ply Len Lou Ile Ala Cys Leu Cys His Asp Len Asp His 535 540 pgc ttc apt aac apc tac otg cap aap Ply Phe Sec Asn Sot Tyr Len Pln Lys 550 to pa: The Asp 555 coo oct org His Pro Len pct ctc tao too Ala Len Tyr Ser tOO 000 arg pop Set Thr Hot P7-n coo coo tto too His His ?he Set oap oct Gln Thy 575 gtg too ato oto cap trp gao gp ceo oat 000 tto too Vel Sec Ile Len Gln Lou Pin Ply His Asn Ile The Sec An 585 act ctp ago The Len Set 590 go: at: att Ala Ile Ile too apt ga Ser Let Pin 5;95 tat pap cop ptp ott gap arc arc opo Tyr Pin Gin Vol Leu Pln lie le Ar; 600 p00 aca Ala Thr 610 gao ot-t got tto to: rtt ppa ac app Asp Len Ale Len ITLr e Ply Acen Arg 615 cog trp gaa gag Gin Len Pin Pin too cop aoc gpo T-yr Gin Thr Ply ota ooc ott ar Len Asn Len Aso raa tca oat Gin Ser His aga gao At; Asp 640 gtg 000 Vol Thr 655 1728 1776 1824 1272 1920 195e 2016 2064 2112 opt pta art ppt A:!rp Vol Ile Ply org at; act gco Met Met Thr Ala gec ott opgt rt A-s Len Cx's Set a cotp tpp coo gtt 000 000 ttg Lye -ten Tr Pro Vol Thr LYS LSn 660 Poe at cot otoa Ala Aces Asp Ile tat P00 ga0 Tyjr Ala Gln 67 0 cap crt ott Pin Pro Ile tto tgg pot Phe Top Ala 675 ct Otg org Pro Hot Mot 690 gap ggt got gas Pin Ply ASP Pln sag as tog 99a Lys Lye Len Ply pat coo p00 coo Asp Pln '1a1 Pro 700 g00 age gac aa sag Asp Atg Asp Lye Lys 695 coo gpo cop ct Pin Ply Gin Len ggg ttc tao aeat gC0 grg 900 at Sly Phe Thrr Ace Ala Val Ala le 705 710 etc Cto COt COO ap pap cot Ott ile Len Pro Pro Thr Sin Pro Leu '72 5 apt cap tyg pap asp ptg att cga Gin Trp Gin Lys Val Ile Arp 740 toe too oca tcc pro got cap aag Ser Oar Pro Oar Val Ala Gin Lys 755 760 coo tqo tat eta aoo ctt act cap Pro Cys Tfyr Thr Thr Len Thr Gin 715 720 org ace. poe too aog oat eat oto Len Lys Al a Cys Acg Asp Ace Len 730 735 pgp pap cap act poe aoo rpg at Sly Sln Gin Thr Ala Thc Tp Ile '745 750 poe gct poe tot gas get Ala Ala Ale Sec Sin Asp 765 2160 2208 2256 2298 <210> 2 <211> 766 <212> PPM' <213> Home sapiens <400D> 2 Leu Thr Asp Gin Ly5 I Len Asp Gin Pho Va TPro Len Lys Arp Ly Gln Val Ser Acp Ty 0 Len Ace Ser r~yr Tl Len Len Len Tyr 01' 8 Asp Giy Phe Ala La 100 Ile Phe Thr Pro Pr 115 Ala Sly Pro Ile Th 130 Oar Arp Lye Thr Le 145 s Ace c Gin 70 u Len S Ty r o Sly Sin u Len ISO Lye Ale tIr Sbu Sec Val 25 Ace Lye Sec 40 Aso Thr Ass 55 Sin Acg Lau Oar Sec ile Phe Len G 1 y 105 Ile Lys Sinj 120 Sly Thr Tmc 135 Val C-lu Asp S !u Thc Gly Val G lv SlIy Ile Ala Pro Gin Vel Val Gin Lys A la Pro Lys Val T-.c Sin Asp Ace Gin Tho Lye Ale 3cr Len Cyc 110 Len le Pro Val Aie Lys Gin Acp Phe 160 Cys Asn Ace Lve Pro Arg 125 Per Ale Tryc 140 Len Sly Asp 155 Pro Arg Cys Leu 6 Gly Thr Gly Leu Glu Ser Gly Thr 165 170 Pro Ile Val Thr Ala Ile Gly Asp Ile Gin Ile Gly 180 185 Leu Tyr Arg His Trp Gly Lys Glu Ala Phe Cys 195 Val Ala Thr Ala Asn 210 Gin Val Cys Arg Gly 225 Leu Asp Val Ser Lys 245 Leu Leu Glu His Ile 260 Arg Cys Ala Leu Phe 275 Asp Xaa Phe Asp Ile 290 Lys Thr Lys Glu Ile 305 Val Ala Arg Thr Gly 325 Pro Arg Phe Asn Arg 340 Asn Ile Leu Cys Met 355 Val Gin Met Val Asn 370 Glu Asn Asn Phe Lys 385 Cys Ala Asn Met Tyr 405 Val Thr Met Glu Lys 420 200 Ala Trp 215 Ala Lys Tyr Phe Ile Tyr. Val Asp 280 Glu Glu 295 Phe Ser Val Leu Val Asp Ile Val 360 Ile Ser 375 Phe Ala Arg Ile Ser Tyr Ala Ser Val Leu Ser His Gin Giu 205 Ala Ile His Gin Val 220 Gin Thr Glu Leu Asn Asp Phe Leu 235 Ile Val Asn Leu Asn Lys Gly Lys 300 Lys Gly 315 Pro Asp Thr Gly Gly Ser Ala Phe 380 Cys Ala 395 Ser Glu Ala Ile Asp 255 Val Asn Ala 270 Glu Leu Tyr 285 Pro Val Phe Ile Ala Gly Ala Tyr Ala 335 Tyr Thr Thr 350 Val Ile Gly 365 Ser Lys Thr Leu Ala Leu Cys Ile Tyr 415 240 Ser Asp Ser Lys Gin 320 Asp Arg Val Asp His 400 Arg His Ser lie Cys Thr Ser Glu Glu 425 430 Trp Gin Gly Leu Met Gin Phe Thr 435 440 Leu Pro Val Arg Cys Lys Glu lie Glu Leu Phe His Phe Asp I 450 455 Gly Ile Phe Val Tyr Met Val H 465 470 Glu Leu Glu Lys Leu Cys Arg P 485 Arg Arg Val Pro Tyr His Asn T 500 Cys Met Tyr Ala Ile Leu Gin 515 Glu Arg Lys Gly Leu Leu Ile I 530 535 Arg Gly Phe Ser Asn Ser Tyr 545 550 Ala Leu Tyr Ser Thr Ser Thr 565 Val Ser Ile Leu Gin Leu Glu 580 Ser Ser Glu Tyr Glu Gin Val 595 Ala Thr Asp Leu Ala Leu Tyr 610 615 Met Tyr Gin Thr Gly Ser Leu 625 630 Arg Val Ile Gly Leu Met Met 645 Lys Leu Trp Pro Val Thr Lys 660 Phe Tro Ala Glu Gly Asp Gllu 675 Pro Met Met Asp Arg Asp Lys 690 695 Gly Phe Tyr Asn Ala Val Ala 705 710 Ile Leu Pro Pro Thr Glu Pro 725 7 le Gly Pro Phe Glu Asn Met Trp Pro 460 is Arg Ser Cys Gly Thr Ser Cys Phe 475 480 he Ile Met Ser Val Lys Lys Asn Tyr 490 495 rp Lys His Ala Val Thr Val Ala His 505 510 Asn Asn His Thr Leu Phe Thr Asp Leu 520 525 la Cys Leu Cys His Asp Leu Asp His 540 Leu Gin Lys Phe Asp His Pro Leu Ala 555 560 Met Glu Gin His His Phe Ser Gin Thr 570 575 Gly His Asn Ile Phe Ser Thr Leu Ser 585 590 Leu Glu Ile Ile Arg Lys Ala Ile Ile 600 605 Phe Gly Asn Arg Lys Gin Leu Glu Glu 620 Asn Leu Asn Asn Gin Ser His Arg Asp 635 640 Thr Ala Cys Asp Leu Cys Ser Val Thr 650 655 Leu Thr Ala Asn Asp Ile Tyr Ala Glu 665 670 Met Lys Lys Leu Gly Ile Gin Pro Ile 680 685 Lys Asp Glu Val Pro Gin Gly Gin Leu 700 Ile Pro Cys Tyr Thr Thr Leu Thr Gin 715 720 Leu Leu Lys Ala Cys Arg Asp Asn Leu 730 735 Set Gin Trp Gin Lye Val Ile Arg Sly Gin Sin Tin Ala Thr Tp Ile 740 745 750 Set Sat Pro Set Val Ala Sir- Lys Al a Ala Ala Set Gin Asp 755 760 765 <210> 3 <211> 4389 <212> DNA <21-3> h-omo sapiens <220> <221> CDS <222> (2411) <400> 3 pa ttc paz ata pro paz pzg gta gap ztg cto ttc gpa tar gaz atp Phs Ala Len Ala Ala Ala Ala Ala Len Len Ohs Sly Sec Asp Met 1 5 10 pa pat ga act tat sat sat Gin Asp Sly Pro Ser Aen Aen 7gc ttz aug apr arc apt org tys Phe Len Set Pro Ser Len rat at: car cc zag pta toe Set Len His Pro Sln Val Len qca gag aos ptausag aa tap Ala Sin Thr Val Sin Lys Pro 70 pat pea tag pat cr0 aspg gas Asp Gin Set Ala-Pro Lye Sin cap pga pin pta rat pea ataa Gin Sly Val Val Tyr in Len 100 ace ppa ge pac sa cap ota Thr Sly Sly Asp Aen Gin Len 115 ago tgz ttc cpa app rtp 25 pat gaa Aso Gin 43 paa tt aep app Lys -kug ago app Set Arg spc tat Sert rr 105 zrz tat Len Tyt 120 pug ap Val Lys tat ga Set Sin aso aa A-sn A-sn aaa pat Sin Asp gas cas Sin Gin ctp apr Len Set Tht tat Tyr p3tt Psi ucaL Set ast Asn- ttg Leu 110 son Ie aaa eta pro aca Lye Ile Ala Tho 130 aas paz pat ppa ttt Lye Ala Asp Sly Phe 1.35 ga ctq tat Ala Len Tyr att gpa pep Len Sly Sin opo at eat ago ctp opt eta too aop oca. oct ggg ate ap pea gpa Cye Ast Act Set Len ye Ile Phe Thr Pro Pro Sly Ile Lye Sln Sly 145 150 coo cpa otc etc cot pct ppp Pro Arp Len Ilie Pro Ala Sly 165 000 etc act Pro Il1e Thr 170 cag gpo aco co Sin Six', Thr Thr tot got tat ptp Set Ala Tyr Mel gcc ap too agg eee ace 009 ote pta Ale Lye 5cr Arp Lys Thr Leu Len Vel 180 185 ote ttt cca age gpo act ppa cog pee. Arg Woe Pro Arg Sly Thr Sly Len 0-in ctt ppa pet Len Sly Asp opt etc cep Arp Ile Sin 210 pee gao etc Sin Asp Ile 190 toe ppg act- Seo Sly Thr 205 etc pgt gao Ile Sly Asp tot pot ott opo Ser Vel Len Cye ccc att ptc Pro Ile Vel eot poe Thr Ale 220 tog ett Len Ile 225 ggt cOO oto gap Sly ile Len Sin tat opp oec 099 Tyr Arp His Org Lye 0-lu Ala The ott apt ceo cap pep pot poe ace poe eat ott 9OO 099 900 toe Len Per His Sin Sin Vel le Thr Ale Act Len Ala Trp Ale Per 245 250 255 gte Poe ata cat Mel Ale Ile His pop cap pta Mel Sin Mel tgo age Lye Arp 265 gpo cot p00 ace Sly Len Ale Lye oeg ace Sin Tho 270 pee top eat Sin Leu Let too te coo ao pta toe ccc ace oat Phe Len Let Asp Mel Ser Lye Thr Tyvr 230 ott pat ac Phe Len Act 285 tat goe cee rlyr Ale Lye ate ptt poe eta. pet tot ota ctt pea ceo soc cop ate Ile Val Ale Ile Asp Ser Leu Len Gin His Ile Met lie 290 295 300 eec ctp Act Len 305 pop eat g00 gat Mel Act Ale As; tpt pta ott too oep pop uao cat ap Lye Ala Len The 5"7n Mel Asp H4 s Lye 315 aco ap gag rt t Len Lye Sin Len TPyr 320 ppa ace cot p00 tOO Sly Lye Pro Mel Phe 340 toe gao Oct Set Asp Pro 325 tot pat ett ppe peg pea ap pee The Asp Ile Sly Sin Sin Lye Sin I 33S 1007 1055 ap aeg eco eec pep ate ape Ott toe L~ye Lye Thr Lye ln Ile Arg The 5cr at!: pap Ile Sin 350 10 aaa gga stt got ggo osa gos goa aga 505 99g Lys Gly Ile Ala Gly Gin Val Ala Arg Thr Gly 355 360 gaa gtc otg sac att Gin Val Len Asn Ile 365 oca gat goc tat goa gac Pro Asp Ala Tyr Ala Asp 3-10 oca ogo Pro Ar; 3.75 tot sac aga gas Phe Aso Ar; Gin gac: tO; tao Asp Len Tyr 1103 1151 11.99 aca ggo tao aoo ac; og; sac soc cog tgo at; coo Thr Gly Tyr Thr Thr Ar; Asn Ile Len Cys Met Pro 385 390 395 ato ;to ago oga le Val Sec- Ar; ggo syc go; Gly Ser Val 400 ata ggt go; Ile Gly Val 405 go; cag at; gto sac ass ato ago ggo Val Gin -Met Val Asn Lys Ile Sec Sly goc tOO tot ass sca gat gaa sac aso Ala Phe Sec Lys Thr Asp Gin Aso Asn 420 as at; ott cc Lys Met Pbs Ala gzc ,ttt Hal The 430 tgt got Ota Cys Ala Len Oca gag ogo Sec Gin Cys 450 bta oao ogo got Len His tys Ala at; tat cat aoa Met TIvo His Acg att ogo cac Ile Ar; HIs 445 tao cat ago Tyrr His Sec st tao ogg gts Ile Tyr Ac; Vsi at; gsa as; cog Mat Gln Lys Len sot ot Ilie Cys 465 act -c gaa gag Thr Sec Gin Gin osa ott sop Gin Sly Len Met tOO 500 Ott 000 Phs Thc L=an Pro gtg ogo coo ogo ass gsa sot gas tos too Vai Ac; T-ao Cys Lys Gin lie Ginu >eu ?he 480 485 tot gas s~ac at; tg; cot ggs sot Ott goc Phs Gin Aso Met Tcp Pro Giy Ile Pheta 'al 505 coot gao sot ogo Phe Asp lie Sly 1295 1343 1391 1439 1487 1535 1583 1631 1679 c so; got cat Ty-r Mat Vai His cog too Ac; Sec 510 tgt ggg sos too ogo Ott gag ott- ga Cys Gly Thr Sec Cys ?he Gin Len Gin3 515 529 asp top tgt cgt Lys Len tys Ar; Ott ato atg The Ile Met 525 tgg sag cat Trp Lys His tot go; sag Sec Val Lys 530 sag sac Oat Lys Asn Tyr ogg go-t cot Oat Ar; Ac;r Vsal Pro Tyc 535 tcc sog tat goc ate tys Met c-yr Ala Ile 550 oat sac His Asn 540 go; ;to Ala Val 545 sot gta. gos tao Thin Val Aia His ott cap sac sat cat Len Gli Asn Asfl His ttc ace gao ott Phe 71hr Asp Len 565 gao otp gao cao Asp LeU Asp His 580 1~1 :Tag 090 aaa ppe otg 3!u Arg Lys Gly Leo 570 app gpo too apt eec Arp Gly ?he Ser Ago 5B5 ctg ett pog 590. ctg Len Ile Ala Cvs Len 575 agc tao cop oap Rag Per Tyr Len Sin Lys 590 too gao cao cot Phe Asp His Pro 595 ceo ceo too too His His Phe Par 610 etc ttc too act Ile Phe Per Thr etc ogo aee 900 Ile Arg Lys Ala 640 egg eep cap tp A-rp Lys Gin Len eat cee toe oat Asn Gin Per His 675 gec ott opt tot As Ln Cys Ser 690 eat pat eta tat Aen ASp Ile Tyc 705 ttp ppa ate 0S9 Len Giy Ilie 01n 720 pto ccc cee p90 Val Pro Gin Sly tat ace eco Olt Tyvr Thr Thr Len otp g Len Gin3 otp !'en atoC gee 660 age Arp Val Ala Oct Pro cap Gin 740 eco: T1hr co got 020 tao too act too eco atp peg Per 'nor Met C;in C cap ia Ale Len Tor Per 600 ot gtp too etc oto hr Vel Per Ile Len 6!5 ,too apt pee tat er Per Per Gin Tnyr 630 to 900 ace gar- Ott le Aia Th r Asp Len 4 5 ;ag atp tec oag eco flu Met Tyr Gin Thr 665 3ao opt gta act opt :ep Arg Vel Ile Gly 680 ate aaa c-9 t99 cOO :rhr Lys ten Pr- Pro 695 ttg Leu cap Gin 635 1te Len tca Per at p Met aca Th: gae Gin 620 ptp Vel tao Tyr cte Leu atp Met Lys 700 gaz Gi'- Ab9c u Prc 605 999 Gly Ott Len tt ?he aec Ago act Thr 68B5 ttp ten atp iMet g ap sLy s c t ale t ott o Len 765 fin pag Gly ct Len 670 p00 Al a aop Thr ap Lys pat AsoD ccc Pro 750 cog Len Ret ksn etc Ile eec Asn 655 eat Ago t Cys poe Abs aaa Gin 735 090 dye Lys 1727 1775 1823 18,71 1919s 1967 2015 2063 2111 2159 2207 2255 2303 pot gap gpo get Ala Giu Ply Aso 715 atp gao epa gac Met Asp) Arp As; 730 tao eat 900 gt Ttr r Aso Ala Va 745 oct coO acp pea Pro Pro Thr dii 12 pca tgc app pat aat ctc apt cap tgp gag aap ptp att cpa gp gag 2351 Ala Cys Arg Asp Asn Len Set Gin Trp Glu Lys Val Ie Arp Sly Gin 770 775 780 gag act pra arc rpp att rca tcC cca tcc ptp gct cap aag gca pct Gin Thr Ala Thr Tr Ile Ser Ser pro Set Val Ala Gin Lye Ala Ala 785 790 795 pra tcr gaa pat tpapcactpp tcaccctpac acpctptccc acctacapat Ala Ser Gin Asp Boo 2451 cctcatcttg cctpptaacr appacttcct ccacpttcap ataggctaat tttccattpt crrtaacatc ar-tpt gaaS p agcctcappa cttatttcap apppctpapa patgagprat ttttaaaatt aaataaaagc atttttcara attcacaatg raaacat raa crrCacttatt agaaacapas crcacagatc ~ttpgttgE cttcrttpac gppptpt~aaa pccaatcrt aaatCagctp attpctgapp atrtttctaa aa~:tgttt ptpaatpttr caaatptcpt- agcttcatga tcaataaat atpttacaat- pttttrpttp S cppcat-tgt atpat tc crt aaaaptttar *actataaaaa ccttcracpt cagaaaprap -apatrartc Iccaptattat attcttttcc cctrtttaag tctptpagpt pccaaptpac cc-ttaaagga taapggttta tgatpttgpz atptargarc taagttaca atrtapttap taacartaga aaatctrape ctgtctrgp aatt ttttta tatatttca rtraaacatt ptpta-tttp actpcacpgt tacaatcatg atapaagcct catacaatpt rtrttttpg aapptaarat ppppgtgggP gpaacttgca 2511 caaataaata aptcaapcag 2571 atcatapara ctpatcaacc tccatrtgac tt9Caaacca aarppacaaa aattas crap aaatgptatt attarpptat ppwataaat apgaattttt tactgtaat taarttpagt gagtaayaaa tparppcaaa acraaarata atzttlaap cpaaptapat trcctp=tt acttcaapra pcagttteaaa agttt-tactg aacccztttgt atppacaapt crratppata appazcatcr 2631 pcctzttcta 2691 aaggptart 2751 tpractppp 2811 acarattact 2871 rptaptccac 2931 acpt-caaact 2991 trcaacctaa 3051 ttpgapaapa 3111 apatgtctpt 3171 ataaapaaca 3231 rgaagpptat 329: i rtttptptCa taattt ta-Ct catattt car tpgaccttc prppptttcp aagttttta ttgcagaatt ttagactctc aaraccaztac crgacctgcc pagCtttctP tcrt rttct- atptttctt aaaaa-tacga aprtctc-ttt gcagtcacct tccatagcac ctttactctt ctataracct aagr:aatact 3411 3471 3591 3651 3711 ctgcaccoct toctacaaag agatttttag tgcaatggca gaeagtgacag gcocatcata atatogoott oaataaaatg gtatatga gcctggtttg atgttgtagc aaagttggct grtcagtttt crtgtgggct tettcan:t atacactatg ggctagatat azacacacac acagatgtar ataggataca acatacacat acacacacac :aggaaaaaa tataaazc <210> 4 <211> 803 <212> PRT <213> Porno sap~iens <403> 4 atgataaagZ: cggtaacctc tcaaataact gaaaaatatg tgcaaaatta ctacatasac S ctgaaagcr aggtaaaatg atatatat-at cact oatata 13 agreacttct ggcgcatzttt aataa-tttaa caaacctgeia tragacagag actcactcag tattcacttt ocatgoggca agaaactgga ctrctacata tttaccacca taggogottto aatcagaggc ttctacaaca tggtt-tattt attgttctta tcttaaaatt aacactatgt gccatcttoc ctccgtggag ccccatttgc taataacaaa agagagagaa gtaccactyt pgatggacgt arttaogta gaacacacac tgzaaacgta tecatatgtg tatatatgat 3 721 3831 3891 2951 4011 4071 4131 4191 4251 4311 gagagacaga aggaaagaga ggaagagaga agcaaacatg 4371 4389 The Ala Leu Ala Ala Ala Ala Ala 1 5 Asp 017 Pro Sar A sn Asn Ale Ser >,en Pne Gly Sac Asp Hat Gin Phe Lan Ser Pro 2cr Len Thr Asn 40 Cys The Acg Arg LnThr Ginj Cys 3 0 Gin Lye Val Lye Ala T-yr Len Sec P he Val Per C-lu Sec Val Par Ala e0 Acp Lys Aen Asn Lys Ser Gi Asp Lou His so Gin Thr Pro Gin Val Len Asp Gin 55 Val Gin Lye Trp Leni Lye Gin Per Ala Pro Lys C-lu Val Per Arg Tlyr Gin Asp Thc Asn Gly Val Val T-yr Gin Len Asn Per 'Tyc 100 105 ile Glu Gln Acg Len Asp Thr 110 Gly Gly Asp Asn Gin Leu 115 Ile Ala Thr Lys Ala Asp 130 Asn Asn Ser Leu Cys Ile 145 150 Pro Arg Leu Ile Pro Ala 165 Ala Tyr Val Ala Lys Ser 180 Gly Asp Glu Arg Phe Pro 195 Ile Gin Ser Val Leu Cys 210 Ile Gly Ile Leu Glu Leu 225 230 Leu Ser His Gin Glu Val 245 Ala Ile His Gln Val Gin 260 Leu Asn Asp Phe Leu Leu 275 Val Ala Ile Asp Ser Leu 290 Leu Val Asn Ala Asp Arg 305 310 Lys Glu Leu Tyr Ser Asp 325 Lys Pro Val Phe Lys Lys 340 Gly Ile Ala Gly Gin Val 355 Asp Ala Tyr Ala Asp Pro 370 Gly Tyr Thr Thr Arg Asn 385 390 Leu Leu Tyr Glu Leu Ser Ser Ile Ile Lys 120 Gly Phe Ala 135 Phe Thr Pro Gly Pro Ile Arg Lys Thr 185 Arg Gly Thr 200 Leu Pro Ile 215 Tyr Arg His Ala Thr Ala Val Cys Arg 265 Asp Val Ser 280 Leu Glu His 295 Cys Ala Leu Pro Phe Asp Thr Lys Glu 345 Ala Arg Thr 360 Arg Phe Asn 375 Ile Leu Cys 125 Leu Tyr Phe Leu Gly 140 Pro Gly Ile Lys Glu 155 Thr Gin Gly Thr Thr 170 Leu Leu Val Glu Asp 190 Gly Leu Glu Ser Gly 205 Val Thr Ala Ile Gly 220 Trp Gly Lys Glu Ala 235 Asn Leu Ala Trp Ala 250 Gly Leu Ala Lys Gin 270 Lys Thr Tyr Phe Asp 285 Ile Met Ile Tyr Ala 300 Phe Gln Val Asp His 315 Ile Gly Glu Glu Lys 330 Ile Arg Phe Ser Ile 350 Gly Glu Val Leu Asn 365 Arg Glu Val Asp Leu 380 Met Pro Ile Val Ser 395 Glu Cys Gly Lys 160 Val Ser 175 Ile Leu Thr Arg Asp Leu Phe Cys 240 Ser Val 255 Thr Glu Asn Ile Lys Asn Lys Asn 320 Glu Gly 335 Glu Lys Ile Pro Tyr Thr Arg Gly 400 Scr Val. Ile SWy Phe Scr Lys Thr 420 Ala Leo Ala Lena 435 Gin Cys Ile Tyr 450 Cys Thr Ser Gin 465 Arg Len Cys Lys Gin Asn Met Tro S00 Sly Thr Ser tys 515 Val. Lys Lye Aso 530 Val Thr Val Alea 545 Len Phe Thr Asp His A-sp Leu A4sp 589 Ago His Pro Leu His Phe 5cr Gin 610 The 3cr Thr Len 025 Arg Lye Ala Ile Lys Gin Leo Gin 660 Gin 5cr His Arg 675 i5 Vai Val Gin Me: Pal Aen 410 Asp Gli Asn Asn Phe Lys 425 His Cys Ala Asn Me: Tyr 440 Arg Val Thr Met Gin Lye 455 GS 1n Trm Gin Sly Leo Her

41-70 Gin ice Sbu Len Phe His 485 490 Pro Sly ie Phe Pal Tyr 505 Phe Gin Len Gin Lye Len 520 Tyr Arg Arg Pal Pro Tr 533 His Cys Met Tlyr Ale Tic 550 Len Gin Arg Lye Sly Len 505 570 His Trg Sl-y Phe S -cr Aen 535 A l a Al a Len Pyvr 3c-r The 1 600 Thr Pal 3cr Ile Le'n SW1 615 Scr 3cr Ser Gin Tyr GW 630 7Me Ale Thr Aso Len Als 045 ESC Gin Met Tyr Sin Thr Gi1 665 Asp Arg Pal Ile S-ly LeL i6 Len Cye Ser Val Thr Lys Len Trm Pro Val Thr Lye Len Thr Ala Aen 690 695 700 Asp Ile Tyr Ala Gin Phe fl-p Ala Gin ely Asp Gin Met Lys Lys Len 705 710 715 720 ely Ile Gin Pro Tie Pro Met Met Asp Arg Asp Lys Lys Asp Gin Val 725 730 735 Pro Gin Gly Gin Len Sly Phe Tyr Aen Ala Vel Ala lie Pro Inr Tyr 740

74.5 750z Thr Thr Len Thr Gin Ie Len Pro Pro Thr Gin Pro Leu Len Lys Ala 755 760 765 lye Arp Asp Aen Len Ser Gin Tmo Sin Lys Val Ie Arp Sly Gi Sin Gl 770 775 780 Thr Al a Thr To Ilie Ser Ser Pro Set Val Ala Gin Lye Ala Al a Ala 785 790 795 BOO Set Gin Asp <210> <211> 3195 <212> DNA <213> Mama sapiens <220> <221> 115 <222> -(2403) <400> retodecape gagttapa Ilgeeaart gggoe:gap apacoartga apocceagee G0 tepcg atp app ate gas pep app sea tot teaa tat ts ace ppt ttp 108 Met Ar; Ile Gin Gin Ary Lye Set SIn His Len, Thr Sly Len 1 510 eaa pat pea ace pop aep pta tat ott loct ott oat coo cap gza o-te Thr Asp Gin Lye Val Lys Al Le e en His Pro Gin Vel Lenl 20 25 pat pea tot poe tot gee apt pot agt poe pap acs pta pep eec tpp 204 Asp Sin Phe Tel Per Gin Set Vel Per Aia Thr Tel Sinu Lye T= 40 alp ap egg sag at at ace toe sea pat pea top pot cot ap pea 252 Len Lys Arp Lys Aen Aen Lys Set Sin Asp Gin Per Ala Pro Lye Sin s0 55 17 atg cag Met Gin gtcaego egg tao cee gat eog eat Val Ser Arg Tyr Gin Asp Thr PAn gga gtt gta tat pea ca 5-ly VPal Val TIyr Gin Len 300 eec ago tat ate gee oaa an Ser Tvr Ile Gin Gin so top gao ace gga g Len Asp Thr SlyC ;ga fly gcC oto oto tat gee oty ego ego etc att eee eta Len Len ge ttt Giy Phe to aog The Thr pgg ccc Sly Pro app aa Arg Lys 160 apa gpo Arg Sly 17 5 toe cca Len Pro tat Cug Tyr Arg gce ace A Ul Tho gta t Val Cvs 240 gac pta Asp Val 255 Ile le poe gag Sly Gin gee. gpe Sbi Sly 135 aco pro Thr Val 150 patc eta Asp Ile ggp act Sly Thr pgt gao Sly Asp Ile Ala 105 eat eat Asn Aso =~cc cgo Pro Arg go-t tat Ale Tyr pga get Sly Asp 170 eto cap Ile Sin 185 cot gpt le Siy ott apt Leu Per S -a eta Ala Ile t to eat Len Asn 250 got Poe Val Ala 2 gao: Asp ace Thr ecc Lei- gtp Vel 155 gee tott Per Ile cao oat 235 gac Asp gee gao Sin Alea tgg got Trp Ala 230 eee cap Lys Gin 245 to-t olat Phe Asp ate pat tan Ile Asp Per is ott gaa tao Len Gin His ata atg Ile Me: 275 ata tat gta. ass. sac tup gcp sat got gat opt Ile Tvr Ala Lye Asn Len Val Asa Ala Asp Arp 290 285 924 972 tgt gta ott ttc tag pty pat oat tys Ala Len The Gin Vsi Aso His 230 sac asy pap tta Asn Lys Sin Len tat tos gac Tyr Set Asp 300 tot ttt pat Len Phe Asp 305 att pys gag gaa aap gsa ppa as ott ptt ttc asp sag Ile Sly Gin Sin Lys Gin Sly Lys Pro Vsi Phe Lys Lys 310 315 ato saa gag sos ags. ttt Tho Lys Gin Ile Arp Ohs 320 sot pap as pga sot act gpo osa pta Ile Glu Lye- le Ala Sip Sin Val spa acs ppp gsa pt tp at att 005 Sat Occ tat pta gao Arp Tho Sly Gin Psi Len Aen ile Pro Asp Ale Tyr Ala Asp opt ttt at spa gas gta, gao ttp tat Arg Phs ken Arp Gin Pal Asp Len Tyr 355 ggo tao act sop Slyi Tyr Thr Thr ogp sac Arp ken 365S stt tp ot Ile Len Cys cap cog gt Gin Meto Psi 385 too st ptt spo Pro Ile Val Set gpo ago pop ats Sly, Set Pal Ile Sly Pal Psi 380 at see aco apt gpoco aoo ot tzcc tcct arc acccq gas ken Lye Ile Set Sly Set Ala ?he Set Lys Thr Aso Gin 1020 1058 1!'16 1164 1212 1269 1230P 1356 140)4 1452 1500 390 395 ao 550 Asn Ace 400 tto 555 SOP Ott Qcoopto tot tgt got roea pc ta 05 05 go- The Lys Met The Ala Pal Ohs tys Ala Len Ala Len Hie Cys 405 410 sat sop tat cat Ace Haet Z-yr His at: opt oat toe Qcg 0:90 cto tao ap gta Ils Arp His Sec Sln Cy 75 i Tyr Arg Psi 425 430 tat tat apt sot Opt act oca gas gag o-g Set Ise Cys Tat Set Gin Gin The sop sop gas asp Thr Het Sin Lye ttg tot Len Set 435 440 445 taa ggt to atg 5aa ttt 500 tO Sin Sly Len Met Sin Phe Thr Len 450 pop opt ott tpo Pal Az-p Len u ass osa act Lye Sln Ile 460 tgp tot gga Trn Pro Sly gaa tts to Sin Len. Phe 465 050 ttt eat His Ohe Asp sot ppt lie Sly 470 Cto tt gas at Pro Phe Sin ken ato ott goc tac at; gto- cat cgg tcc tgt ggg aca lie Pita Val Tyr Mao Val His Ar; Set Cts Gly iTtr 480 485 490 ccc tyc ott g~ag Set Cys Pita Gin cot gae eag t Len Gin Ly's Len 4.95 cgg gtt cct tat Arg Val Pro Pl'yt tgt cgt ttt atO at; oct gO; aeg eag aat tat cgg Cys Ar Pita Ilie Hat Sat Vci Lys Lys Asn iTtv Arg 500 505 510 aac ogg eag cat Aen Top Lys His gt act gta gte Val Tht Val Ala cac tot His lye 525 at; tat gct Met Tyr Ala cgc aaa gga Ar; Lys Gly 545 ctt cag at act Lena Gin Asn Asn at; cot tt aca iTtr Len The ThIr gec tt gag Aen Len Gin 540 get tat egg Ace Etc Arg ctg cog ato ;cg Len Len le Ala ctg tgt cat gec Lela Cyc Eic ASP g9Cc toc Gly The 550 ago at agc oat Ser Ace Set Tyr tag as; tt get Sin Lvcs Phe Asp tct cog qcc gct Pro Len Ale Ala o:actoct att tcc PTr Ser iTtr Set at; gag tag cat Met Gin Gin His ttc ztt tag act ;ita Set Gin iTt 1548 159G 16644 1692 174 0 1788 1836 1884 -1932 1980 2028 2076 2124 Oct etc ct tag Ser ile Len Gin gee. ggq cat eat Gin Gly His Ace. ttc at cog Pha Set PThr Len agt tt Set Set 505 agO gee oat Set Gin Tyr ace get coo Phr Asp Lenu 625 tag gt; Cto gag etc act tgt aea gct Gin Pal Len Sin ile 7le Acg Lys Ala E55 etc att gtt Ile Ile Ala 620 gee gag at; Gin,, Gn eat gct toe tat tt Aila Len Tyr Pita at egg cc; tag APsn Arg Lys Gile eat eat tee tca Ace Ace Gin Ser 550 tat tag act gge tca cta at ct Tyr Gin Phr O- 1 y Ser Len Aen Len 84n 645 tat age get cgt His Arg Asp Arg gta ct Pal Ile 655 ggo otg at; Gly Len eat at; act Met Phr 660 gttc tgt: get Ala tys Asp tgt Oct gtg CIrs Set Pal ace aee Thr Lye 670 ctg tgg ccc gtt ace eea tog at; gte Len Trp Pro Pal Pttr Lye Len Phr Ala 675 eat get ata oat gce gee, ttt ken Asp Ile rt Ala Gin ?ita 680 685 20 tgg got gag Trp Ala G9 atg atg gao Net Met Aso, 705 to tao aao Phe Tyr Asn 720 OtO 000 000 Leo Pro Pro got Gly 690 aga Arg g900r Ala aog gat gaa atg aag Asp Glu Net Lys gac aag aag gat Asp Lys Lys Asp 710 gog gao att coo Val Ala Ile Pro 725 gag oot ott cog Gin Pro L-eo- Leu aaa tog gga ata. oag 000 ato oot Lys Leu Gly Ile Gin Pro Ile Pro 695 702) gaa 900 000 oaa 990 oag 000 ggg Gin Val Pro Sin Gly Gin Leu Gly 715 tgo tat aoa. aoo Ott aoo zag ato Cys rr Thm Thr Len Thr Gin Ile 2172 2220 2268 2316 aaa goa 090 Lys Ala Cys 745 agg gat aat 000 Aog Asp Asn Leo tg9 gag aag9 Trp Gin Lys att oga ggg gag ie Ara Sly Sin act oca acc 099 Thr Ala Thm Tr-p art rca Ile Ser 765 too oca too Ser Pro Ser 909 900 cag aag goa got wca tot gaa- gat rgaooaorgg Val Ala Gin Lys Ala Ala Ala Gao Glu Asp 770 775 toaccctoac aogo-tgtooo tttttttggg gggggtgggg aaggtaaoar- caaataaata tzggyaggot gaggtgggrg atggtgaaao tctgtcota. tgoatgooog tagttocggga orgaaotgag ceaggoogo oroaaaaaaa aaaaaaagra rggtttarca ataagaaaga oatgoor~gtg atorocagoac ogagacoato ctragooaaoa otgggogtgg zgogoatagt ogottgaacc caggaggoag aootacagat ggaacctgca agtcaagcag gatoaoo~tga otaaaaatao ggczgaggr-a gtcagtgcac tarootacaa tgttaaaaaa tttgggaggc tggtgaaaoo ggrgraatto aggttgcagr cto-atottg ottotttgac at ortotrcc rctggraaor aggarro cot ggto-tagagt aaaaateoaa ggagaacac zoccagooteg4 atgctaatta rgatgaoaaa ogaggogggt oogtottao oagotacot gagcoaggat ggggrgoaaa ccctcaag ggaarrooaa totoaaoacr- rogagacogq aot-gggoaag aattagorgg gtgrggtggt ttgaaootgg ggggtggagg aoaacagaao aegao'rorgt atttttococ aoregotaar tgoagtoggt taoegtggot ggatcatgag gtoaagagat tgaaaaaaaa aaaaaattag ggaggotgag goaggagaat ggtoggaato otgoagcocg 2413 2473 2533 2593 2653 2713 2773 2833 2893 2953 3013 3073 3133 3193 3195 <210> 6 21 <211> 779 <212> PRT <213> Homo sapiens <400> 6 Met Arg Ile Glu Gil 1 Glu Lys Val Lys Al Phe Val Ser Glu Se: Arg Lys Asn Asn Lv Arg Tyr Gin Asp Th Tyr Ile Glu Gin Ar Tyr Glu Leu Ser Se 100 Ala Leu Tyr Phe Le 115 Pro Pro Gly Ile Ly 130 1le Thr Gin Gly Th 145 Thr Leu Leu Val Gl 16 Thr Gly Leu Glu Se 180 Ile Val Thr Ala Ti 195 His Trp Gly Lys G! 210 Ala Asn Leu Ala Tr 225 Arg Gly Leu Ala Ly 24 u Arg Lys a Tyr Leu r Val Ser s Ser Glu r Asn Met 70 g Leu Asp r Ile Ile u Gly Glu s Glu Gly 135 r Thr Val 150 u Asp Ile 5 r Gly Thr e Gly Asp u Ala Phe 215 p Ala Ser 230 s Gln Thr 5 Ser Gln His Leu Thr Gly Leu Thr Asp 10 Ser Leu His Pro 25 Ala Glu Thr Val 40 Asp Glu Ser Ala Gin Gly Val Val Thr Gly Gly Asp 90 Lys Ile Ale Thr 105 Cys Asn Asn Ser 120 Lys Pro Arg Leu Ser Ala Tyr Val 155 Leu Gly Asp Giu 170 Arg Ile Gln Ser 185 Leu Ile Gly Ile 200 Cys Leu Ser His Val Ala Ile His 235 Glu Leu Asn Asp 250 Val Leu Asp Lys Trp Leu Lys Glu Val Glu Leu Asn Gin Leu Leu Ala Asp Gly 110 Cys Ile Phe 125 Pro Ala Gly Lys Ser Arg Phe Pro Arg 175 Leu Cys Leu 190 Glu Leu Tyr 205 Giu Val Ala Val Gln Val Leu Leu Asp 255 2 Thr Tyr Phe Asp Asn Ile Val 260 265 Met Ile Tyr Ala Lys Asn Leu 275 280 Gin Val Asp His Lys Asn Lys 295 Gly Glu Glu Lys Glu Gly Lys 2 Ala Val Glu Pro Ile Asp Asn Ala Leu Tyr 300 Val Phe Leu Leu 270 Arg Cys Asp Leu Lys Thr 310 315 Glu Iie Arg Phe Ser Ile Glu Lys Gly Ile Ala Gly Gin Val Ala Arg 325 330 Thr Gly Glu Val Leu Asn Iie Pro Asp Ala Tyr Ala 340 Val Asp Leu Ile Val Ser Ile Ser Gly 390 Phe Ala Val 405 Arg Ile Arg 420 Ser Tyr His Phe Thr Leu Asp Ile Gly 470 Val His Arg 485 Arg Phe Ile 500 345 Thr Gly Tyr Thr Thr 360 Gly Ser Val Ile Gly 380 Ala Phe Ser Lys Thr 395 Cys Ala Leu Ala Leu 410 Ser Glu Cys le Tyr 425 Tie Cys Thr Setr Gl 440 Val Arg Leu Cys Lys 460 Phe Glu Asn Met Trp 475 Cys Gly Thr Ser Cys 490 Ser Val Lys Lys Asn 505 Asp Arg 365 Val Asp His Arg Glu 445 Glu Pro Phe yT His 335 Pro Arg Phe 350 Asn Ile Leu Val Gin Met Glu Asn Asn 400 Cys Ala Asn 415 Val Thr Met 430 Trp Gin Gly ,ie Glu Leu Gly Iie Phe 480 Glu Leu Glu 495 Arg Arg Val 510 Cys Met Tyr Pro Tyr His Asn Trp Lys His Ala Val Thr 515 520 525 Leu Glu Arg Lys Ala Ile Leu Gin 530 Asn Asn His Thr Leu Phe Thr Asp 535 540 Gly Leu Leu Ile Ala Cvs L 545 550 Ser Asn Ser Tyr Leu Gin I 565 Ser Thr Ser Thr Met Glu G 580 Leu Gin Leu Glu Gly His I 595 Tyr Glu Gin Val Leu Glu 610 Leu Ala Leu yr Phe Gly 625 630 Thr Gly Ser Leu Asn Leu 645 Gly Leu Met Met Thr Ala 660 Pro Val Thr Lys Leu Thr 675 Glu Gly Asp Glu Met Lys 690 Asp Arg Asp Lys Lys Asp 705 710 Asn Ala Val Ala Ile Pro 725 Pro Thr Glu Pro Leu Leu 740 Glu Lys Val Ile Arg Gly 755 Ser Val Ala Gin Lys la 770 23 eu Cys His Asp ys Phe Asp His 570 1n His His Phe 585 Asn lie Phe Ser 600 ile Ile Arg Lys 615 Asn Arg Lys Gin Asn Asn Gin Ser 650 Cys Asp Leu Cys 665 Ala Asn Asp Ile 680 Lys Leu Gly Ile 695 Glu Val Pro Gin Cys Tyr Thr Thr 730 Lys Aia Cys Ar9 745 Glu Glu Thr Ala 760 Ala Ala Ser Glu 775 Leu Asp His Arg Gly Phe 555 560 Pro Leu Ala Ala Leu 'yr 575 Ser Gin Thr Val Ser Ile 590 Thr Leu Ser Ser Ser Glu 605 Ala Ile ie Ala Thr Asp 620 Leu Glu Glu Met T yr Gin 635 640 His Arg Asp Arg Val Ile 655 Ser Val Thr Lys Leu Trp 670 Tiyr Ala Glu Phe Trp Ala 685 Gin Pro Ile Pro Met Met 700 Gly Gin Leu Gly Phe Tyr 715 720 Leu Thr Gin Ile Leu Pro 735 Asp Asn Leu Ser Gin Trp 750 Thr Trp Ile Ser Ser Pro 765 1 Asp <210> 7 <211> 458 <212> DNA <213> Homo sapiens <400> 7 attcggcacg ggctgcggce agagggtggt cgaacttctg caggtaactg ttactgagcc 24 ccrgnpgtcc aggtcatgac acagacacyc aatcagcagt cctttgtgdt caagu-tct gt 120 gaaaagcgtg tgattgttct gaagtatggc atacatgcag tgtectacag rgaccgcatg 180 cztccagttg tgataaggaa cccgccgata gztcttcttc acagacataa taaaacg5ca 240 caacttttca agctcasegc aggatgtcct acaggecqca :gaaccatgt agacaaaaat 300 I-ccaggccac atgttttcaa aaggaccaat nrtcaaagtgg aataatrcaa tttc'-ttggc 360 agagazgcac cgggaaaggg cgaazrtgca rgagaccttt agccactctt ctgraasgtac 420 aaatgctatg gtaggacagt tttt-tccgzc ggt-taccO 456 <210> 8 <211> 21 <212> PRT <213> Homo sapiens <400> 8 His Aso Xaa XAr His as Gly Xaa Yarc Xas XAs as ass ass As Ass 1 5 10 Ass XAss. As Xaa Ala <210> 9 <21L> 19 <212> DNA <213> Artificial Sequence <220> <223> Descrieititn ef Artif4icial Sequencee: priere <400> 9 gceaacsgct atgactaty 19 <210> <211> 18 <212> D)NA <212> Artificial Sequence <220> <223> Descriptionl of Artificial Sequence: cromer <400> 10 actctccssg gaatacag 18 <210> 11 <211> 18 2 '212> DNA <213> Artificial Sequence <220> ,223> Description of Artificial sequence :primer <400> 11 1 ctgtctctgc actaacac 1 <210> 12 <211> 18 <212> D NA <213> Artificial Sequence <220> <223> Description. of Arifcial Seqiuence:pr.mer <400> 12 ttgqc-aaggc ctctgcat 1B <210> 13 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Descri~tion of Artificial Sequence:primfer <400> 13 cctctatgaa ctgagcag 18 <210> 14 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequience:primrn< <400> 14 gaaggcactg ccactgat 18 <210> <211> 18 <212> DNA <213> Artificial Sectience <220> <223> Descrintion of Artificial Secaueflce::Orimer <400> 15 1B tcgagctgta tcggcact <210> 16 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:primer <400> 16 agcgtgtgat tgttctgaa <210> 17 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:primer <400> 17 tgctggccaa gtagcaag <210> 18 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:primer <400> 18 aaggtcacag gcagtcat <210> 19 <211> 1B <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:primer <400> 19 gaagagtggc aaggtctc <210> <211> 19 27 <212> DNA <213> Artificial Sequence <220> <223> Descripotion of Artificial Sequence: primter <400> tcatgaCctg gaccaccag <210> 21 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial sequence:primfer <400> 21 catocttgaa gaggtttgo <210> 22 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Descriptnf of Artificial Sequence: primer <400> 22 atgactgcct gtgacctt <210> 23 <211> 19 <212> DNA <213> Artificial Secuence <220> <223> Description of Artificial Secujefle:primfer <400> 23 ctgctaca acccztcc <210> 24 <211> 18 <212> DNA <213> Artificial Seauence <220> <223> Description of Artificial Seq-Lence:primer 28 <400> 24 18 gczaatattg ctgaggcc <210> <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:primer <400> taagcgagag gtgactgc 1 <210> 26 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:primer <400> 26 cctaaagggc tgagatca 18 <210> 27 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:primer <400> 27 cgcagtcacc cctcactt 18 <210> 28 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:primer <400> 28 tgtaaaacga cggccagt 18 <210> 29 <211> 18 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:primfer <400> 29 ecaaaacgcc tatys:tgg: <210> <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Descrip~tion, of Artificial2 Seaueoce:primer <400> tcgatctcag ccctttagc 19 <210> 31 <211> 18 <212> DNA <213> Artificial Sequence <22 0> <223> Description of Artificial Seqaece: primerC1 <400> 31 tcatgtggca ggaaactg 18 <210> 32 <211> 484 <212> DNA <212> Homo sapiens <400> 32 gcacgagacc agtat-tacte tacaatgoca gtgtt:7aaa aaatacgaaa gtaatactct gcaccccttc ctacaaagat gataaagcag tcactt-cg cgcttaa a taaag 120 atttt:agtg caatzggcacg gtaaccz~cca aacctgaatt agacagagac tcactcagga 180 agtgecaggc ccatcatatc aaataactca tccaczttcc atgtggcagg aaactggaat 240 atcgctttca ataaaatgga aaaatasgct tctacaztt caccaccata ggc-gttttgt 300 ccatacgagc csggtccgcg caaaatcaaa ccageggctt ctacacatgg tttattztc 360 ttgtagcaaa gttggctcta cataaacat: gttcctcatct cacatteac actatgtgtt 420 30 cgtttooct gcgggcttot gagttgcc stcttccto ogtggagcttc catttpctat 480 tto 434 <210> 33 <z211> 404 <212> DNA <213> Home sapiens <400> 33 cttgaacaca tcosgatata tgoaeaaaat ctagtgaacg oogaoogctg cgcgc--cttc G0 caggtgpaoc acaagaaoea ggagotgtaO toggacotg7 ttgaoattpg ggaggagaag 120 gegggago cogttttcaa caagacosag gagatoepet iottocattga gaasgggazt 130 gctggtoaag tggcaagaac gggagaagtc otgaacsttc crgatgoota ogoagacoog 240 ogottaaca pggaggtgga ootgtcaaa ggctataoca ogopgaacat tottgtatg 300 coataguga gcogoggoet ttgattoggt gtggzgoaaa rggtttaaca agatcagogg 36D caggootttc oaagacggat gagaacaaot toasgargtr t-tga 404 <210> 34 <211> 7 <212> PET <213> Artificial Seq-uence <220> <223> Description of Artificial S;ecoenoe:c ag <400> 34 Asp Lys Asp Asp Asp Asp Lys <210> <211> 62 <212> DNA <213> Home sapiens <400> cagtoagota gccgocatgg aotaoaagga opsogargac caagttgaot getgeaaagg G0 tg 32 <210> 36 <211> 19 <212> DNA 31 <213> Artificial Sequence <220> <223> Description of Artificial Sequence:primer <400> 36 ccagaagpgp tactttncc <210> 37 <211> 19 <212> DNA <213> Artificial Seorusoce <220> <223> Description of -Artificial Seoruance:primer <400> 37 catrgcccg aqqctgtgg <210> 3S <211> 477 <212> DNA <213> Homo sapiens <400> 38 tatcccaasa gccgacgc tgtgttcara ccecccgc caccagggc accaccat ggatatcctt ggggatga ccagrczgtt cttogctt sctgtscagg cactgggs caatcctgct tgggcttc acagaocgss crgaatgs a7r ttgcactgza rsa tgaagqaapg :ct C--gcctac-gr L5c gaccttcct cg 9-o costIlpocac ca aagaqgcctt cg tagosatace ct tcctactcga Cttccttgge ocaaccccgg 40Crepgt Ct appoactgpa tpccatzga ctpcctcagc ecappopoag cpta"ccaap gsptqcaara appaapacgt ct ppaat cap catcappapp acatactttg arapoc: pcp cppppcccaz tgttpprapa praccopoat peat ccttpa ttpcaacsgc gtctcpccae ataacat- <210> 39 <211> 755 <212> DNA <213> Homo sapiens <400> 39 gaattccttt tptatcatt tcttctctaa cttactttat ctttttaaa: ggaatasctc G0 tgatagaatt atgcttagtc cttaccttct actttcagat ttcatctgtc atatcaatga aacggagag& tctactygga aatatacgra cattttocat tgcagttagt gcaataaatt acagtgttaa aatgccgta gat togt tgt acaaattcat aaacctgta& aaggtatagt gggczggaaa gogacagaa atgccactaa atatacgtgt ttagcaattt tcagagttac tatoogtcta agatagaaa& tctctctt ctaatacctg aagaattga arcacaacac gagcggg&&g agttttggEa attgtacgct gzgtgtgtgt taaactacat atatgaacca 32 ctttatattt agcotggotta cagccatttc ggtgaaga aagaat&&Ba arttatgctaa gcaagctac ctagacaggt taaaacagca gogrgogtgt atattacatc cacactctag aatatattaa aaatgagato cacaagagac tctactgtct co.gcactaac gaaagatatg ccttcacttt orcacctata caacarcCtC gacccagcaa octtcatct tgcttagtgg otacagagt gaatactgcc caacattg9g octaaaatgg taaaaaacao atgaccaca& oaaaaaagaa atgtracata gotgtcta <210> <211> <212> DNA <213> Artificial Secuence <220> <222> Description of Artificial Sequence:orimer <400> gttagatgag aggO-tgotgg <210> 41 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 41 gtaogctaat cocag <210> 42 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:primer 33 <400> 42 caactcgaat tccttgacag attagcatac <210> 43 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Secuence:Drimer <400> 43 caactcgaat tccttgac 18 <210> 44 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Seouence:primer <400> 44 gttgttcttc ctcttcagcc <210> <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Secuence:Drimer <400> caactcgaat tccttgacag a 21 <210> 46 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:primer <400> 46 gatcgtcgac ctgtctctgc actaacac 28 <210> 47 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Secuence:crimer 34 <400> 47 gatcgtcgac aagcactcgg tCagccttcg <210> 48 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Descriptionl of Artificial Sequence~primer <400> 48 gatcggatcc accatggact acaagg 26