Abstract:
This invention relates to novel nucleic acid sequences encoding three novel human phosphodiesterase (hPDE IV) isozymes. It also relates to polypeptides encoded by such sequences.  
     This invention also relates to an assay method for detecting the presence of such novel isozymes in human cells, and to a method of identifying compounds or other substances that inhibit or modify the activity of such isozymes.

Description:
BACKGROUND OF THE INVENTION  
         [0001]    This invention relates to novel nucleic acid sequences encoding three novel human phosphodiesterase IV (hPDE IV) isozymes.  
           [0002]    Cyclic nucleotide phosphodiesterases (PDE&#39;s) are a family of enzymes that catalyze the degradation of cyclic nucleotides. Cyclic nucleotides, particularly cAMP, are important intracellular second messengers, and PDEs are one cellular component that regulates their concentration. In recent years, five PDE enzymes (PDE I-PDE V), as well as many subtypes of these enzymes, have been defined based on substrate affinity and cofactor requirements (Beavo J A and Reifsnyder D H,  Trends Pharmacol. Sci.  11:150 [1990]; Beavo J, in:  Cyclic Nucleotide Phosphodiesterases: Structure, Regulation and Drug Action.  Beavo J and Housley M D (Eds.). Wiley: Chichester, pp. 3-15 [1990]).  
           [0003]    Theophylline, a general PDE inhibitor, has been widely used in the treatment of asthma. It has been speculated that selective inhibitors of PDE isozymes and their subtypes (particularly the cAMP-specific PDE IV) will lead to more effective therapy with fewer side effects (for reviews, see Wieshaar R E et al.,  J. Med. Chem.,  28:537 [1985] and Giembycz M A,  Biochem. Pharm.,  43:2041 [1992], Lowe J A and Cheng J B,  Drugs of the Future,  17:799-807 [1992]). However, even PDE IV selective drugs such as rolipram suffer from emetic side effects that limit their use. An even more selective approach is to inhibit individual subtypes of PDE IV, each one of which is expected to have its own tissue distribution. If the PDE IV isozyme responsible for efficacy is different from that causing side effects, an isozyme selective drug could separate therapeutic and side effects. The cloning and expression of the human PDE IVs would greatly aid the discovery of isozymeselective inhibitors by providing purified isoenzymes to incorporate into drug assays.  
           [0004]    Mammalian PDE IV, the homologue of the Drosophila Dunce gene (Chen C N et al.,  Proc. Nat. Acad. Sci.  (USA) 83:9313 [1986]), is known to have four isoforms in the rat (Swinnen J V et al.,  Proc. Nat. Acad. Sci.  (USA) 86:5325 [1989]). The cloning of one human isoform of PDE IV from monocytes was reported in 1990 (Livi G P et al.,  Mol. Cell. Bio.,  10:2678 [1990]). From Southern blot data, the authors concluded that this enzyme was probably the only PDE IV gene in humans, with the possible exception of one other isozyme. The same group has recently published the sequence of a second human isoform isolated from brain that they designate hPDE IV-B to distinguish it from the monocyte form, which they designate as hPDE IV-A (McLaughlin M M et al.,  J. Biol. Chem.  268:6470 [1993]). For clarity, we will use this nomenclature as well.  
           [0005]    Our invention relates to the nucleic acid sequences encoding three novel human PDE IV isozymes generated by differential splicing from a single gene. We designate these isoforms as hPDE IV-B1, hPDE IV-B2 and hPDE IV-B3. The hPDE IV-B2 sequence encodes a polypeptide nearly identical to that reported for hPDE IV-B (McLaughlin M M et al.,  J. Biol. Chem.  268:6470 [1993]), and the hPDE IV-B2 splice variant represents the unspliced genomic sequence with respect to the differential splice site. Of the two other splice variants, hPDE IV-B1 encodes the longest polypeptide chain, as well as the N-terminal sequence homologous to its rat homologue, DPD (Colicelli J, et al.,  Proc. Nat. Acad. Sci.  (USA) 86:3599 [1989]).  
           [0006]    The novel human PDE IV DNA sequences and their encoded peptides may be used to screen for drugs that are selective for a particular human PDE IV isozyme. Such novel DNA sequences may also be used in assays to detect the presence of a particular PDE IV isozyme in human cell lines, thus providing information regarding the tissue distribution of each isozyme and its biological relevance with respect to particular disease states.  
           [0007]    The following abbreviations are used throughout this patent:  
                                       BAL   bronchoalveolar lavage       bp   base pair(s)       cAMP   cyclic adenosine 3′,5′-monophosphate       dNTP   2′-deoxynucleoside-5′-triphosphate       dATP   2′-deoxyadenosine-5′-triphosphate       dCTP   2′-deoxycytidine-5′-triphosphate       dGTP   2′-deoxyguanidine-5′-triphosphate       dTTP   2′-deoxythymidine-5′-triphosphate       hPDE IV-A   human monocyte PDE IV       hPDE IV-B   human brain PDE IV       hPDE IV-B1   human brain PDE IV, splice variant 1       hPDE IV-B2   human brain PDE IV, splice variant 2       hPDE IV-B3   human brain PDE IV, splice variant 3       kb   kilobase(s)       PCR   polymerase chain reaction       PDE   cyclic nucleotide phosphodiesterase       PDE I   Ca 2+ /Calmodulin-dependent PDE       PDE II   cGMP stimulated PDE       PDE III   cGMP inhibited PDE       PDE IV   high affinity cAMP-specific PDE       PDE V   cGMP specific PDE       RACE     R apid  A mplification of  c DNA  E nds       RT   avian myeloblastosis virus (AMV) reverse transcriptase       RT-PCR   PCR of RT-transcribed mRNA       SSC   1 × SSC = 0.15 M NaCl, 0.015 Na 3  citrate pH 7.0                  
 
           [0008]    The nucleotides and amino acids represented in the various sequences contained herein have their usual single letter designations used routinely in the art.  
         SUMMARY OF THE INVENTION  
         [0009]    This invention relates to novel nucleic acid sequences encoding the novel hPDE IV isozymes hPDE IV-B1, hPDE IV-B2 and hPDE IV-B3. More specifically, it relates to DNA segments comprising, respectively, the DNA sequences of SEQUENCE ID NO. 1, SEQUENCE ID NO. 2 and SEQUENCE ID NO. 3, as defined below, or alleleic variations of such sequences. It also relates to polypeptides produced by expression in a host cell into which has been incorporated one of the foregoing DNA sequences or an alleleic variation of such sequence.  
           [0010]    This invention also relates to an isolated polypeptide comprising the amino acid sequence of SEQUENCE ID NO. 4, SEQUENCE ID NO. 5 or SEQUENCE ID NO. 6.  
           [0011]    This invention also relates to recombinant DNA comprising the DNA sequence of SEQUENCE ID NO. 1, SEQUENCE ID NO. 2 or SEQUENCE ID NO. 3, or an alleleic variations of such sequence.  
           [0012]    This invention also relates to an isolated DNA segment comprising the genomic promoter region that regulates transcription or translation of the DNA sequence of SEQUENCE ID NO. 1, SEQUENCE ID NO. 2 or SEQUENCE ID NO. 3, or an alleleic variation of such sequence.  
           [0013]    This invention also relates to an assay method for detecting the presence of hPDE IV-B1, hPDE IV-B2 or hPDE IV-B3 in human cells, comprising: (a) performing a reverse transcriptase-polymerase chain reaction on total RNA from such cells using a pair of polymerase chain reaction primers that are specific for, respectively, hPDE IV-B1, hPDE IV-B2 or hPDE IV-B3, as determined from, respectively: (i) the DNA sequence of SEQUENCE ID NO. 1 or an alleleic variation thereof; (ii) the DNA sequence of SEQUENCE ID NO. 2 or an alleleic variation thereof; or (iii) the DNA sequence of SEQUENCE ID NO. 3 or an alleleic variation thereof, and (b) assaying the appearance of an appropriately sized PCR fragment by agarose gel electrophoresis.  
           [0014]    This invention also relates to a method of identifying compounds or other substances that inhibit or modify the activity of hPDE IV-B1, hPDE IV-B2 or hPDE IV-B3, comprising measuring the activity of, respectively, hPDE IV-B1, hPDE IV-B2 or hPDE IV-B3, in: (a) a cell line into which has been incorporated recombinant DNA comprising the DNA sequence of SEQUENCE ID NO. 1, SEQUENCE ID NO. 2 or SEQUENCE ID NO. 3, or an alleleic variation thereof, or (b) a cell line that naturally selectively expresses hPDE IV-B1, hPDE IV-B2 or hPDE IV-B3, as determined by the assay method described above.  
           [0015]    This invention also relates to an isolated DNA segment comprising a DNA sequence that is a subset of SEQUENCE ID NO. 1, SEQUENCE ID NO. 2 or SEQUENCE ID NO. 3, or an alleleic variation thereof, and that is capable of hybridizing to, respectively, SEQUENCE ID NO. 1, SEQUENCE ID NO. 2 or SEQUENCE ID NO. 3, or an alleleic variation thereof, when used as a probe, or of amplifying all or part of such sequence when used as a polymerase chain reaction primer.  
           [0016]    As used herein, the term “functionally equivalent DNA segment” refers to a DNA segment that encodes a polypeptide having an activity that is substantially the same as the activity of the polypeptide encoded by the DNA to which such segment is said to be functionally equivalent.  
           [0017]    As used herein, the term “subset of a DNA sequence” refers to a nucleotide sequence that is contained in and represents part, but not all of such DNA sequence, and is sufficient to render it specific to such sequence when used as a PCR primer and to render it capable of hybridizing to such sequence when used as a probe at high stringency.  
           [0018]    The term “functionally equivalent polypeptide” refers to a polypeptide that has substantially the same activity as the polypeptide to which it is said to be functionally equivalent.  
           [0019]    The term “subset of a polypeptide” refers to a peptide sequence that is contained in and represents part, but not all of such polypeptide. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0020]    [0020]FIG. 1. hPDE IV-B1, hPDE IV-B2 and hPDE IV-B3 Restriction Map and Clone Diagram. This figure shows the relationship between the cDNA sequences encoding the three splice variants. Black boxes indicate protein coding regions and open boxes indicate untranslated regions.  
         [0021]    [0021]FIG. 2. hPDE IV-B1, hPDE IV-B2 and hPDE IV-B3 DNA and Translated Amino Acid Sequences. (+) Numbering begins with the “A” of the ATG start codon in hPDE IV-B3. Four stop codons are designated by “***”. These include the protein translation stop (1,552), and the stop codons that prevent the coding region from continuing further in the 5′ direction in each splice variant: hPDE IV-B1 (−630), hPDE IV-B2 (−270) and hPDE IV-B3 (−89). The alternate splice junction is between nucleotides −23 and −24, and the putative splice acceptor sequence in hPDE IV-82 (−33 to −24) is underlined.  
         [0022]    [0022]FIG. 3. Alternative Splice Junction. This figure is a close-up view of the splice junction between −24 and −23, showing the three aligned sequences hPDE IV-B1, hPDE IV-B2 and hPDE IV-B3. The putative splice acceptor sequence in hPDE IV-B2 (−33 to −24) is underlined.  
         [0023]    [0023]FIG. 4. Amino Acid Sequence Comparison: hPDE IV-B1, hPDE IV-B2, hPDE IV-B3. and Rat DPD. Identity with the hPDE IV-B1 sequence is indicated with a dash. A translation of the region upstream of the hPDE IV-B3 start codon is shown in parenthesis to highlight the complete sequence divergence of hPDE IV-B2 and hPDE IV-B3 from hPDE IV-B1 at amino acid 196.  
         [0024]    [0024]FIG. 5. Restriction MaR of the hPDE IV-B Genomic Locus. Transcriptional orientation (5′-3′) of hPDE IV-B is from left to right, with the approximate positions of exons known by partial sequence analysis indicated by solid boxes (coding). The position of the stop codon is indicated by an asterisk, followed by a segment of a 3′ untranslated region (open box). Regions hybridizing strongly to the 308 bp probe, as described in the text, are indicated by a dark hatched box, while weakly hybridizing regions are shown as lighter hatched regions. It is because of weak hybridization between the EcoRI and HindIII sites in λ11.1 that we position an exon (with a “?”) in that interval. The hybridizing restriction fragments seen in genomic blots with the 308 bp probe are indicated below the figure.  
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0025]    The procedures by which the DNA sequences encoding for novel isozymes hPDE IV-B1, hPDE IV-B2 and hPDE IV-B3 were identified and isolated as described below.  
         [0026]    Discovery of PDE IV-B Using Degenerate PCR: The degenerate PCR primers (5′-Deg and 3′-Deg, as described below in the section labelled Materials and Methods) were designed against amino acid sequences that were conserved (with one exception) between the six published PDE IV sequences from human, rat, and Drosophila (Livi G P et al.,  Mol. Cell. Biol.  10:2678 [1990], Swinnen J V et al.,  Proc. Nat. Acad. Sci.  (USA) 86:5325 [1989], and Chen C N et al..  Proc. Nat. Acad. Sci.  (USA) 83:9313 [1986]). These primers were expected to amplify 308 bp of PDE IV sequence from any human isoform mRNA that also conserved those amino acids. The RT-PCR was done on human BAL sample total RNA as described below in the section labelled Materials and Methods, and a fragment of the correct size was obtained. Sequence analysis of this fragment showed it to be different from hPDE IV-A (Livi G R et al., [1990]). This fragment of hPDE IV-B corresponds to bp 1,575 to 1,882 in SEQUENCE ID NO. 1. This fragment was isolated from several independent PCR reactions and sequenced to confirm that no apparent differences were due to PCR artifacts.  
         [0027]    Isolation of a cDNA Clone for hPDE IV-B: The human medulla cDNA library was screened as described below in the section labelled in Materials and Methods, and a single cDNA clone was obtained. The insert sequence corresponds to bp 924 to 2,554 of SEQUENCE ID NO. 1, and was clearly not full length in the coding region by comparison with the known PDE IV sequences. Also, since no polyA tract was found at the 3′ end of this clone, we do not believe that the 3′ untranslated region is complete; however, this is of no functional significance with respect to producing a hPDE protein. There was one nucleotide difference between the cDNA sequence and the PCR fragment sequence. SEQUENCE ID NO. 1 contains a C at bp 1792, the nucleotide seen in the cDNA sequence, rather than the T that has been seen at this position in PCR isolations. We believe that this difference, which changes an amino acid, is real, and represents an alleleic difference in the human population.  
         [0028]    Completion of the cDNA Sequence using the RACE Method: The RACE method showed that there was not just a single 5′ end to the hPDE IV-B cDNA, but at least three. Fragments of different sizes were obtained, all beginning at the GSi oligonucleotide primer site and extending towards the 5′ end of the cDNA. The three fragments that were successfully sequenced had a variable length of non-homologous sequence at the 5′ end that joins the hPDE IV sequence at the same point in all three cases. These different 5′ ends, when joined to the rest of the cDNA sequence, make three forms of the hPDE IV-B gene that we designate hPDE IV-B1 (SEQUENCE ID NO. 1), hPDE IV-B2 (SEQUENCE ID NO. 2), and hPDE IV-B3 (SEQUENCE ID NO. 3). The three hPDE IV-B isoforms make polypeptides of different lengths. From the cDNA sequences, hPDE IV-B1 is predicted to encode a protein of 721 amino acids (SEQUENCE ID NO. 4), hPDE IV-32 a protein of 564 amino acids (SEQUENCE ID NO. 5), and hPDE IV-B3 a protein of 517 amino acids (SEQUENCE ID NO. 6). The three isoforms are shown diagrammatically in FIG. 1, and the DNA sequence and amino acid translation of the three isoforms of hPDE IV-B is shown in FIG. 2.  
         [0029]    The most logical explanation for the three hPDE IV-B isoforms is that they are If generated by alternative splicing of 5′ exons onto the shared 3′ sequence. The putative alternative splice junction is shown at −23 bp in FIG. 2. To test this hypothesis, we amplified PCR fragments from human genomic DNA using primers on either side of the putative splice junction. hPDE IV-B1 and hPDE IV-B3 specific 5′ primers did not give amplified fragments, indicating that the sequences on either side of the putative splice lie further than 2 kb apart in genomic DNA (the practical limit for PCR amplification). Primers specific for the hPDE IV-B2 isoform gave the identically sized fragment as predicted from the cDNA (data not shown), indicating that at least with respect to the putative splice junction at −23 bp, this is the unspliced genomic sequence. Indeed, examination of the sequence of hPDE IV-B2 at this location (underlined bp −33 to −24 in FIGS. 2 and 3) reveals an excellent match for a splice acceptor sequence (Breathnach R and Chambon P,  Ann. Rev. Biochem.  50:349 [1981]).  
         [0030]    hPDE IV-B is very similar to one of the known rat isozymes, DPD (Colicelli J. et al.,  Proc. Nat. Acad. Sci.  (USA) 86:3599 [1989]), with 96.3% amino acid identity in the regions that can be aligned, as compared to only a 74.6% identity with hPDE IV-A. However, of the three splice variants, only hPDE IV-B1 continues to have homology to rat DPD 5′ of the putative splice junction (FIG. 4). Indeed, hPDE IV-B1 extends much further 5′ than rat DPD, and the homology between the two continues to the 5′ end of rat DPD. The fact that the hPDE IV-B1 sequence has been conserved in evolution is strong evidence that this sequence is functional and is translated into protein in vivo. We cannot be sure that the other two splice variants are functional in vivo, although the recent paper (McLaughlin M M et al.,  J. Biol. Chem.  268:6470 [1993]) reporting the hPDE IV-B2 sequence has shown by expression cloning that this isoform can produce enzymatically active protein in a yeast expression system.  
         [0031]    Mammalian Expression Clones for hPDE IV-B1, B-2, -B3: The hPDE IV-B1, -B2, -B3 cDNA sequences were subcloned into the mammalian expression vector pcDNA1-amp, a vector that is suitable for transiently expressing these genes in COS cells and that was constructed by replacing the 950 bp NheI fragment of pcDNA1 (Invitrogen) with a 1.2 kb PCR fragment from pUC18 (Sigma) containing the Amp resistance gene. The resulting expression clones are designated pc-hPDE IV-B1, pc-hPDE IV-B2, and pc-hPDE IV-B3. All three clones have been shown to direct the expression of proteins that catalyze the degradation of cAMP when transiently transfected into COS cells.  
         [0032]    Genomic Sequences for hPDE IV-B: Overlapping genomic clones define “26 kb of genomic sequence encoding at least the 3′ half of the hPDE IV-B gene (FIG. 5). Limited DNA sequencing of these genomic clones confirms that the SaII restriction site in clone λK2.1 is contained in an exon, and corresponds to the unique SaII site seen in the cDNA sequence (1,235-1,240 in Sequence ID No. 1). Hybridization data (FIG. 5) defines the orientation of the gene, and confirms the hybridizing fragment sizes seen in genomic Southern blots hybridized at high stringency with the 308 bp PCR fragment (bp 1,575-1,882 in SEQUENCE ID NO. 1) from hPDE IV-B: EcoRI6.6 kb, HindIII-4.4 kb, BamHI-4.2 kb.  
         [0033]    Deposits  
         [0034]    Three cDNA clones (pc-hPDE IV-B1, pc-hPDE IV-B2, and pc-hPDE IV-B3) are being deposited with the American Type Culture Collection, Rockville, Md. U.S.A. (ATCC).  
         [0035]    Assays  
         [0036]    Using the DNA sequence of hPDE IV-B and hPDE IV-A, one could make a large number of isoenzyme specific PCR primer pairs. We have made and tested the following hPDE IV-B and hPDE IV-A specific primer pairs. The sequences 5′B(5′-CGAAGAAAGTTACAAGTTC-3′) and 3′B(5′-AACCTGGGATTTTTCCACA-3′) are a pair of 19-mer primers that specifically amplify a 245 bp fragment from hPDE IV-B, and the sequences 5′A(5′-CACCTGCATCATGTACATG-3′) and 3′A(5′-TCCCGGTTGTCCTCCAAAG-3′) are 19-mers that amplify an 850 bp fragment specifically from hPDE IV-A. In addition, one skilled in the art could easily design a pair of PCR primers specific for each of the hPDE IV-B splice variants by using the unique 5′ sequences. Using these primers, one can sensitively assay the presence of these isozymes in any tissue from which total RNA can be isolated (e.g., by the method of Chomcynski P and N Sacchi,  Anal. Biochem.  162:156 1987) by performing an RT-PCR reaction on such total RNA using the specific primers and then assaying the amount of the appropriately sized DNA PCR product by agarose gel electrophoresis. The RT-PCR conditions are identical to those described in Materials and Methods, except that the thermocycling parameters are as follows: Denature-94° C., 30 sec; Anneal-55° C. 30 sec; Polymerize-72° C., 60. Amplify for at least 30 cycles.  
         [0037]    The claimed DNA sequences of this invention can be reproduced by one skilled in the art by either PCR amplification of the coding region using PCR primers designed from the sequences or by obtaining the described cDNA clones from ATCC directly.  
         [0038]    Utility of the Invention  
         [0039]    A general utility of the novel human PDE IV genes and their encoded peptides is to allow screening for human PDE IV isozyme specific/selective drugs that may be improved therapeutics in the areas of asthma and inflammation. The cloned genes make it possible, by expression cloning methods familiar to those skilled in the art, to produce active, purified isoenzymes that can be used in PDE IV activity assays (e.g., Davis C W, and Daly J W,  J. Cyclic Nucleotide Res.  5:65 [1979], Torphy T J and Cielinski L B,  Mol. Pharm.  37:206 [1990]) to measure the potency of inhibitors against individual isoenzymes. This is true both for distinguishing hPDE IV-A and hPDE IV-B selective inhibitors and for distinguishing inhibitors selective between hPDE IV-B1, hPDE IV-B2, or hPDE IV-B3. Since the hPDE IV-B splice variants may each have their own tissue distribution and may be pharmacologically separable from each other, it may be valuable to screen for inhibitors specific for individual splice variants.  
         [0040]    Genomic sequences are also of utility in the context of drug discovery. It may be valuable to inhibit the mRNA transcription of a particular isoform rather than to inhibit its translated protein. This is particularly true with hPDE IV-B, since the different splice variants may be transcribed from different promoters. There is precedent for multiple promoters directing the transcription of a mouse brain 2′,3′-cyclic-nucleotide 3′ phosphodiesterase (Kurihara T et al.,  Biochem. Biophys. Res. Comm.  170:1074 [1990]). This invention would provide the means for one skilled in the art to locate multiple promoters. Isolation of genomic clones containing the promoter(s) and the 5′-most exons of hPDE IV-B1, hPDE IV-B2, and hPDE IV-B3 may be accomplished by screening a human genomic library with the unique 5′ sequences. Such promoters could then be linked to a convenient reporter gene such as firefly luciferase (de Wet J R et al.,  Mol. Cell. Biol.  7:725 [1987]), transfected into a mammalian cell line, and used to screen for agents that inhibit the activity of the promoter of interest while having minimal effect on other promoters.  
         [0041]    Another utility of the invention is that the DNA sequences, once known, give the information needed to design assays to specifically detect each isoenzyme or splice variant. Isozyme-specific PCR primer pairs are but one example of an assay that depends completely on the knowledge of the specific DNA sequence of the isozyme or splice variant. Such an assay allows detection of mRNA for the isozyme to access the tissue distribution and biological relevance of each isozyme to a particular disease state. It also allows identification of cell lines that may naturally express only one isozyme—a discovery that might obviate the need to express recombinant genes. If specific hPDE IV isozymes are shown to associated with a particular disease state, the invention would be valuable in the design of diagnostic assays to detect the presence of isozyme mRNA.  
         [0042]    Materials and Methods  
         [0043]    (a) Cells/Reagents  
         [0044]    Cells from a human bronchoalveolar lavage (BAL) were purchased from the Johns Hopkins University (Dr. M. Uu). Human brainstem tissue was purchased from the International Institute for the Advancement of Medicine. Unless noted below, all restriction endonucleases and DNA modifying enzymes were from Boehringer-Mannheim.  
         [0045]    (b) Degenerate RT-PCR  
         [0046]    Total RNA was isolated from human tissue as previously described (Chomcynski P and Sacchi N,  Anal. Biochem.  162:156 [1987]). To prepare an 80 μl reverse transcriptase (RT) reaction, 4 μg total RNA and 4 μg random hexamer primers (Pharmacia/LKB) were heated to 90° C. for 5 min in 60 μl RNase free water. After chilling on ice, the reaction was brought to 80 μl and the following conditions by the addition of concentrated stocks: 1× RT buffer (50 mM Tris pH 8.3, 6 mM magnesium chloride, 40 mM KCl); 1 mM each dATP, dGTP, dCTP, and dTTP; 1 mM dithiothreitol; 25 U/mi RNasin (Promega); and 900 U/ml AMV reverse transcriptase (RT). Incubate at 42° C. for 1 hour, then boil for 5 minutes to inactivate the RT.  
         [0047]    A 50 μl PCR reaction was set up by using 3.25 μl of the above reaction mix. Final buffer conditions were (including carryover from RT): 10 mM Tris pH 8.3, 50 mM potassium chloride, 1.5 μM magnesium chloride, 10 μg/ml bovine serum albumin, 2.5% (v/v) Formamide, 200 μM each dNTP, 0.5 pmol/μl each degenerate primer (5′-Deg=5′-CAGGATCCAAPACNATGGTNGAPAC-3′, 3′-Deg=5′-GCTCTAGATCNGCCCANGTYTCCCA-3′, where N=A, G, C, or T, P=A or G and Y=C or T) and 0.05 U/μl Amplitaq polymerase (Perkin Elmer). Amplification was done in a Perkin Elmer 9600 PCR thermocycler using the following parameters: denature—94° C., 30 sec; anneal-37° C.+0.5° C./cycle, 60 sec+1 sec/cycle; polymerize—72° C., 60 sec. Amplify for 35 cycles.  
         [0048]    (c) Library Screening  
         [0049]    8×10 5  clones from a commercially available human medulla cDNA library (Clontech # HL 1089a) were screened with an 857 bp DNA fragment containing the entire conserved catalytic domain of hPDE IV-A. This fragment was generated by RT-PCR amplification from the Jurkat T-cell line mRNA using unique primers to amplify bp 573-1430 from the PDE IV-A sequence (Livi G P, et al.,  Mol. Cell. Bio.,  10:2678 [1990]). The fragment was labeled to a specific activity&gt;5×10 8  cpm/μg, and hybridized under the following conditions: 6× SSC, 5× Denhardt&#39;s Solution (1× Denhardt&#39;s=0.02% each of Ficoll, polyvinylpyrrolidone, and bovine serum albumin), 0.1% sodium dodecyl sulfate (SDS), 100 μg/ml yeast tRNA. Probe concentration was 4×10 5  cpm/ml. Filters were hybridized at 65° C. for &gt;16 hours, and then washed to a final stringency of 1× SSC at 55° C.  
         [0050]    1×10 6  clones from a commercially available human genomic library (Clontech #HL1111j) were screened with the 308 bp PCR fragment of hPDE IV-B (bp 1,575 to 1,882 in SEQUENCE ID NO. 1) and the homologous fragment from hPDE IV-A. The screening conditions were as follows: 5× SSC, 5× Denhardts solution (see above), 40% formamide, 0.5% sodium dodecyl sulfate, and 20 μg/ml herring sperm DNA. Probe concentration was 4×10 5  cpm/ml. The filters were hybridized at 42° C. for &gt;16 hours, and then washed to a final stringency of 0.5× SSC at room temperature. A genomic library was also constructed in the vector LambdaGEM12 (Promega) using the XhoI half-site method, and 1×10 8  clones screened under the same hybridization conditions used for the previous genomic library.  
         [0051]    (d) DNA Sequencing  
         [0052]    All DNA sequencing was done using an ABI model 373A DNA sequencer on DNA fragments cloned into various pGEM vectors (Promega). Sequencing reactions were done using the Taq sequencing method.  
         [0053]    (e) RACE Method  
         [0054]    The RACE method (Rapid Amplification of cDNA Ends) was adapted from a published method (Frohman M A and Martin G R, In:  Technique—a Journal of Methods in Cell and Molecular Biology,  Vol. 1, No. 3, pp. 165-170 [1989]). In order to produce the 5′ end of the cDNA, an RT reaction was performed on human brainstem total RNA as above with the exception that the gene specific RT primer (GS-RT: 5′-GCAAGTTCTGAATTTGT-3′) was at a concentration of 0.1 pmol/μl. The reaction was incubated at 42° C. for 1 hour and then shifted to 52° C. for 30 min. This higher temperature seems to be critical to avoiding a premature truncation product presumably caused by a sequence that AMV RT has difficulty reading through.  
         [0055]    After removing buffers using a Centricon 30 filtration device and concentrating in a speedvac, one tails the cDNA with dATP using terminal transferase (TdT) in a 20 μl reaction volume. Final conditions are: 1× TdT buffer (40 mM K-Cacodylate pH 6.8, 0.1 mM dithiothreitol), 0.75 mM CoCl 2 , 0.2 mM dATP, 1,250 U TdT/ml. Incubate 37° C. for 5 min, inactivate TdT at 65° C. 5 min. This reaction is diluted with water to 500 μl and used as a template in a series of nested PCR reactions.  
         [0056]    The first PCR amplification (50 ml) uses the same PCR buffer conditions as above, but uses three primers: the Primer/Adaptor (Ro-dT 17 : 5′-AAGCATCCGTCAGCATCGGCAGGACAAC(T 17 )-3′) at 0.2 pmol/μl, the Forward Outside Primer (Ro: 5′-AAGCATCCGTCAGCATC-3′) at 0.5 pmol/μl, and the Gene-Specific Reverse Outside Primer (GSo: 5′-ATGGCAGCCAGGATTTC-3′) at 0.5 pmol/μl. Taq DNA polymerase is only added after denaturing the reaction to 95° C. for 5 min and equilibrating to 72° C. For the first cycle, the annealing step is 10 min at 55° C., and the extension is at 72° C. for 40 min. After that, cycling parameters (PE 9600 machine) are: Denature 94° C., 30 sec; Anneal 53° C., 30 sec; Polymerize 72° C., 45 sec. Amplify 28 cycles. Dilute this product 20× to serve as template for a second PCR reaction using primers nested just inside those used in the first PCR reaction. This greatly increases the specificity of the final PCR products.  
         [0057]    The second 50 μl PCR reaction uses identical buffer conditions to the first, and uses 1 μl of the 20× diluted product from the first PCR reaction as template DNA. The primers are the Forward Inside Primer (Ri: AGCATCGGCAGGACAAC-3′) and Gene-Specific Inside Primer (GSi: 5′-GGTCGACTGGGCTACAT-3′) both at 0.5 pmol/μl. For 12 cycles, the parameters are the same as the final 28 cycles of the previous amplification. The annealing temperature is then raised to 60° C. for another 18 cycles. Products are then analyzed on an agarose gel. They should extend from the GSi primer to the 5′ end of the mRNA(s).  
         [0058]    Sequence ID Summary  
         [0059]    1. hPDE IV-B1 cDNA sequence. 2,554 bp.  
         [0060]    2. hPDE IV-B2 cDNA sequence. 2,246 bp.  
         [0061]    3. hPDE IV-B3 cDNA sequence. 2,045 bp.  
         [0062]    4. Predicted amino acid sequence of hPDE IV-B1. 721 amino acids.  
         [0063]    5. Predicted amino acid sequence of hPDE IV-B2. 564 amino acids.  
         [0064]    6. Predicted amino acid sequence of hPDE IV-B3. 517 amino acids.   
     
       
       
         1 
         
           
             
6 
 
           
           
             
               2554 base pairs  
               nucleic acid  
               double  
               linear  
             
             
               cDNA  
             
              1 

TGGATGGTGA AAGCTAGCAC TCCTTACAAG ATATGACAGC AAAAGATTCT TCAAAGGAAC     60 

TTACTGCTTC TGAACCTGAG GTTTGCATAA AGACTTTCAA GGAGCAAATG CATTTAGAAC    120 

TTGAGCTTCC GAGATTACCA GGAAACAGAC CTACATCTCC TAAAATTTCT CCACGCAGTT    180 

CACCAAGGAA CTCACCATGC TTTTTCAGAA AGTTGCTGGT GAATAAAAGC ATTCGGCAGC    240 

GTCGTCGCTT CACTGTGGCT CATACATGCT TTGATGTGGA AAATGGCCCT TCCCCAGGTC    300 

GGAGTCCACT GGATCCCCAG GCCAGCTCTT CCGCTGGGCT GGTACTTCAC GCCACCTTTC    360 

CTGGGCACAG CCAGCGCAGA GAGTCATTTC TCTACAGATC AGACAGCGAC TATGACTTGT    420 

CACCAAAGGC GATGTCGAGA AACTCTTCTC TTCCAAGCGA GCAACACGGC GATGACTTGA    480 

TTGTAACTCC TTTTGCCCAG GTCCTTGCCA GCTTGCGAAG TGTGAGAAAC AACTTCACTA    540 

TACTGACAAA CCTTCATGGT ACATCTAACA AGAGGTCCCC AGCTGCTAGT CAGCCTCCTG    600 

TCTCCAGAGT CAACCCACAA GAAGAATCTT ATCAAAAATT AGCAATGGAA ACGCTGGAGG    660 

AATTAGACTG GTGTTTAGAC CAGCTAGAGA CCATACAGAC CTACCGGTCT GTCAGTGAGA    720 

TGGCTTCTAA CAAGTTCAAA AGAATGCTGA ACCGGGAGCT GACACACCTC TCAGAGATGA    780 

GCCGATCAGG GAACCAGGTG TCTGAATACA TTTCAAATAC TTTCTTAGAC AAGCAGAATG    840 

ATGTGGAGAT CCCATCTCCT ACCCAGAAAG ACAGGGAGAA AAAGAAAAAG CAGCAGCTCA    900 

TGACCCAGAT AAGTGGAGTG AAGAAATTAA TGCATAGTTC AAGCCTAAAC AATACAAGCA    960 

TCTCACGCTT TGGAGTCAAC ACTGAAAATG AAGATCACCT GGCCAAGGAG CTGGAAGACC   1020 

TGAACAAATG GGGTCTTAAC ATCTTTAATG TGGCTGGATA TTCTCACAAT AGACCCCTAA   1080 

CATGCATCAT GTATGCTATA TTCCAGGAAA GAGACCTCCT AAAGACATTC AGAATCTCAT   1140 

CTGACACATT TATAACCTAC ATGATGACTT TAGAAGACCA TTACCATTCT GACGTGGCAT   1200 

ATCACAACAG CCTGCACGCT GCTGATGTAG CCCAGTCGAC CCATGTTCTC CTTTCTACAC   1260 

CAGCATTAGA CGCTGTCTTC ACAGATTTGG AAATCCTGGC TGCCATTTTT GCAGCTGCCA   1320 

TCCATGACGT TGATCATCCT GGAGTCTCCA ATCAGTTTCT CATCAACACA AATTCAGAAC   1380 

TTGCTTTGAT GTATAATGAT GAATCTGTGT TGGAAAATCA TCACCTTGCT GTGGGTTTCA   1440 

AACTGCTGCA AGGAGAACAC TGTGACATCT TCATGAATCT CACCAAGAAG CAGCGTCAGA   1500 

CACTCAGGAA GATGGTTATT GACATGGTGT TAGCAACTGA TATGTCTAAA CATATGAGCC   1560 

TGCTGGCAGA CCTGAAGACA ATGGTAGAAA CGAAGAAAGT TACAAGTTCA GGCGTTCTTC   1620 

TCCTAGACAA CTATACCGAT CGCATTCAGG TCCTTCGCAA CATGGTACAC TGTGCAGACC   1680 

TGAGCAACCC CACCAAGTCC TTGGAATTGT ATCGGCAATG GACAGACCGC CTCATGGAGG   1740 

AATTTTTCCA GCAGGGAGAC AAAGAGCGGG AGAGGGGAAT GGAAATTAGC CCAATGTGTG   1800 

ATAAACACAC AGCTTCTGTG GAAAAATCCC AGGTTGGTTT CATCGACTAC ATTGTCCATC   1860 

CATTGTGGGA GACATGGGCA GATTTGGTAC AGCCTGATGC TCAGGACATT CTCGATACCT   1920 

TAGAAGATAA CAGGAACTGG TATCAGAGCA TGATACCTCA AAGTCCCTCA CCACCACTGG   1980 

ACGAGCAGAA CAGGGACTGC CAGGGTCTGA TGGAGAAGTT TCAGTTTGAA CTGACTCTCG   2040 

ATGAGGAAGA TTCTGAAGGA CCTGAGAAGG AGGGAGAGGG ACACAGCTAT TTCAGCAGCA   2100 

CAAAGACGCT TTGTGTGATT GATCCAGAAA ACAGAGATTC CCTGGGAGAG ACTGACATAG   2160 

ACATTGCAAC AGAAGACAAG TCCCCCGTGG ATACATAATC CCCCTCTCCC TGTGGAGATG   2220 

AACATTCTAT CCTTGATGAG CATGCCAGCT ATGTGGTAGG GCCAGCCCAC CATGGGGGCC   2280 

AAGACCTGCA CAGGACAAGG GCCACCTGGC CTTTCAGTTA CTTGAGTTTG GAGTCAGAAA   2340 

GCAAGACCAG GAAGCAAATA GCAGCTCAGG AAATCCCACG GTTGACTTGC CTTGATGGCA   2400 

AGCTTGGTGG AGAGGACTGA AGCTGTTGCT GGGGGCCGAT TCTGATCAAG ACACATGGCT   2460 

TGTAAATGGA AGACACAACA CTGAGAGATC ATTCTGCTCT AAGTTTCGGG AACTTATCCC   2520 

CGACAGTGAC TGAACTCACT GACTAATAAC TTCC                               2554 

 
           
           
             
               2246 base pairs  
               nucleic acid  
               double  
               linear  
             
             
               cDNA  
             
              2 

CATTTATGCA GATGAGCTTA TAAGAGACCG TTCCCTCCGC CTTCTTCCTC AGAGGAAGTT     60 

TCTTGGTAGA TCACCGACAC CTCATCCAGG CGGGGGGTTG GGGGGAAACT TGGCACCAGC    120 

CATCCCAGGC AGAGCACCAC TGTGATTTGT TCTCCTGGTG GAGAGAGCTG GAAGGAAGGA    180 

GCCAGCGTCC AAATAATGAA GGAGCACGGG GGCACCTTCA GTAGCACCGG AATCAGCGGT    240 

GGTACGGGTG ACTCTGCTAT GGACAGCCTG CAGCCGCTCC AGCCTAACTA CATGCCTGTG    300 

TGTTTGTTTG CAGAAGAATC TTATCAAAAA TTAGCAATGG AAACGCTGGA GGAATTAGAC    360 

TGGTGTTTAG ACCAGCTAGA GACCATACAG ACCTACCGGT CTGTCAGTGA GATGGCTTCT    420 

AACAAGTTCA AAAGAATGCT GAACCGGGAG CTGACACACC TCTCAGAGAT GAGCCGATCA    480 

GGGAACCAGG TGTCTGAATA CATTTCAAAT ACTTTCTTAG ACAAGCAGAA TGATGTGGAG    540 

ATCCCATCTC CTACCCAGAA AGACAGGGAG AAAAAGAAAA AGCAGCAGCT CATGACCCAG    600 

ATAAGTGGAG TGAAGAAATT AATGCATAGT TCAAGCCTAA ACAATACAAG CATCTCACGC    660 

TTTGGAGTCA ACACTGAAAA TGAAGATCAC CTGGCCAAGG AGCTGGAAGA CCTGAACAAA    720 

TGGGGTCTTA ACATCTTTAA TGTGGCTGGA TATTCTCACA ATAGACCCCT AACATGCATC    780 

ATGTATGCTA TATTCCAGGA AAGAGACCTC CTAAAGACAT TCAGAATCTC ATCTGACACA    840 

TTTATAACCT ACATGATGAC TTTAGAAGAC CATTACCATT CTGACGTGGC ATATCACAAC    900 

AGCCTGCACG CTGCTGATGT AGCCCAGTCG ACCCATGTTC TCCTTTCTAC ACCAGCATTA    960 

GACGCTGTCT TCACAGATTT GGAAATCCTG GCTGCCATTT TTGCAGCTGC CATCCATGAC   1020 

GTTGATCATC CTGGAGTCTC CAATCAGTTT CTCATCAACA CAAATTCAGA ACTTGCTTTG   1080 

ATGTATAATG ATGAATCTGT GTTGGAAAAT CATCACCTTG CTGTGGGTTT CAAACTGCTG   1140 

CAAGGAGAAC ACTGTGACAT CTTCATGAAT CTCACCAAGA AGCAGCGTCA GACACTCAGG   1200 

AAGATGGTTA TTGACATGGT GTTAGCAACT GATATGTCTA AACATATGAG CCTGCTGGCA   1260 

GACCTGAAGA CAATGGTAGA AACGAAGAAA GTTACAAGTT CAGGCGTTCT TCTCCTAGAC   1320 

AACTATACCG ATCGCATTCA GGTCCTTCGC AACATGGTAC ACTGTGCAGA CCTGAGCAAC   1380 

CCCACCAAGT CCTTGGAATT GTATCGGCAA TGGACAGACC GCCTCATGGA GGAATTTTTC   1440 

CAGCAGGGAG ACAAAGAGCG GGAGAGGGGA ATGGAAATTA GCCCAATGTG TGATAAACAC   1500 

ACAGCTTCTG TGGAAAAATC CCAGGTTGGT TTCATCGACT ACATTGTCCA TCCATTGTGG   1560 

GAGACATGGG CAGATTTGGT ACAGCCTGAT GCTCAGGACA TTCTCGATAC CTTAGAAGAT   1620 

AACAGGAACT GGTATCAGAG CATGATACCT CAAAGTCCCT CACCACCACT GGACGAGCAG   1680 

AACAGGGACT GCCAGGGTCT GATGGAGAAG TTTCAGTTTG AACTGACTCT CGATGAGGAA   1740 

GATTCTGAAG GACCTGAGAA GGAGGGAGAG GGACACAGCT ATTTCAGCAG CACAAAGACG   1800 

CTTTGTGTGA TTGATCCAGA AAACAGAGAT TCCCTGGGAG AGACTGACAT AGACATTGCA   1860 

ACAGAAGACA AGTCCCCCGT GGATACATAA TCCCCCTCTC CCTGTGGAGA TGAACATTCT   1920 

ATCCTTGATG AGCATGCCAG CTATGTGGTA GGGCCAGCCC ACCATGGGGG CCAAGACCTG   1980 

CACAGGACAA GGGCCACCTG GCCTTTCAGT TACTTGAGTT TGGAGTCAGA AAGCAAGACC   2040 

AGGAAGCAAA TAGCAGCTCA GGAAATCCCA CGGTTGACTT GCCTTGATGG CAAGCTTGGT   2100 

GGAGAGGACT GAAGCTGTTG CTGGGGGCCG ATTCTGATCA AGACACATGG CTTGTAAATG   2160 

GAAGACACAA CACTGAGAGA TCATTCTGCT CTAAGTTTCG GGAACTTATC CCCGACAGTG   2220 

ACTGAACTCA CTGACTAATA ACTTCC                                        2246 

 
           
           
             
               2045 base pairs  
               nucleic acid  
               double  
               linear  
             
             
               cDNA  
             
              3 

GTGGAAGCAA ACAGCGGAGG CAAGGGGTTG TTTCGGACAC ACTAGAGAGT AAGTCAGAGA     60 

ATCTTCGTGT TGAGGCAGCA TTGCAAAATT GAAGATGAAG AAAGGAAGGA AGAAGAATCT    120 

TATCAAAAAT TAGCAATGGA AACGCTGGAG GAATTAGACT GGTGTTTAGA CCAGCTAGAG    180 

ACCATACAGA CCTACCGGTC TGTCAGTGAG ATGGCTTCTA ACAAGTTCAA AAGAATGCTG    240 

AACCGGGAGC TGACACACCT CTCAGAGATG AGCCGATCAG GGAACCAGGT GTCTGAATAC    300 

ATTTCAAATA CTTTCTTAGA CAAGCAGAAT GATGTGGAGA TCCCATCTCC TACCCAGAAA    360 

GACAGGGAGA AAAAGAAAAA GCAGCAGCTC ATGACCCAGA TAAGTGGAGT GAAGAAATTA    420 

ATGCATAGTT CAAGCCTAAA CAATACAAGC ATCTCACGCT TTGGAGTCAA CACTGAAAAT    480 

GAAGATCACC TGGCCAAGGA GCTGGAAGAC CTGAACAAAT GGGGTCTTAA CATCTTTAAT    540 

GTGGCTGGAT ATTCTCACAA TAGACCCCTA ACATGCATCA TGTATGCTAT ATTCCAGGAA    600 

AGAGACCTCC TAAAGACATT CAGAATCTCA TCTGACACAT TTATAACCTA CATGATGACT    660 

TTAGAAGACC ATTACCATTC TGACGTGGCA TATCACAACA GCCTGCACGC TGCTGATGTA    720 

GCCCAGTCGA CCCATGTTCT CCTTTCTACA CCAGCATTAG ACGCTGTCTT CACAGATTTG    780 

GAAATCCTGG CTGCCATTTT TGCAGCTGCC ATCCATGACG TTGATCATCC TGGAGTCTCC    840 

AATCAGTTTC TCATCAACAC AAATTCAGAA CTTGCTTTGA TGTATAATGA TGAATCTGTG    900 

TTGGAAAATC ATCACCTTGC TGTGGGTTTC AAACTGCTGC AAGGAGAACA CTGTGACATC    960 

TTCATGAATC TCACCAAGAA GCAGCGTCAG ACACTCAGGA AGATGGTTAT TGACATGGTG   1020 

TTAGCAACTG ATATGTCTAA ACATATGAGC CTGCTGGCAG ACCTGAAGAC AATGGTAGAA   1080 

ACGAAGAAAG TTACAAGTTC AGGCGTTCTT CTCCTAGACA ACTATACCGA TCGCATTCAG   1140 

GTCCTTCGCA ACATGGTACA CTGTGCAGAC CTGAGCAACC CCACCAAGTC CTTGGAATTG   1200 

TATCGGCAAT GGACAGACCG CCTCATGGAG GAATTTTTCC AGCAGGGAGA CAAAGAGCGG   1260 

GAGAGGGGAA TGGAAATTAG CCCAATGTGT GATAAACACA CAGCTTCTGT GGAAAAATCC   1320 

CAGGTTGGTT TCATCGACTA CATTGTCCAT CCATTGTGGG AGACATGGGC AGATTTGGTA   1380 

CAGCCTGATG CTCAGGACAT TCTCGATACC TTAGAAGATA ACAGGAACTG GTATCAGAGC   1440 

ATGATACCTC AAAGTCCCTC ACCACCACTG GACGAGCAGA ACAGGGACTG CCAGGGTCTG   1500 

ATGGAGAAGT TTCAGTTTGA ACTGACTCTC GATGAGGAAG ATTCTGAAGG ACCTGAGAAG   1560 

GAGGGAGAGG GACACAGCTA TTTCAGCAGC ACAAAGACGC TTTGTGTGAT TGATCCAGAA   1620 

AACAGAGATT CCCTGGGAGA GACTGACATA GACATTGCAA CAGAAGACAA GTCCCCCGTG   1680 

GATACATAAT CCCCCTCTCC CTGTGGAGAT GAACATTCTA TCCTTGATGA GCATGCCAGC   1740 

TATGTGGTAG GGCCAGCCCA CCATGGGGGC CAAGACCTGC ACAGGACAAG GGCCACCTGG   1800 

CCTTTCAGTT ACTTGAGTTT GGAGTCAGAA AGCAAGACCA GGAAGCAAAT AGCAGCTCAG   1860 

GAAATCCCAC GGTTGACTTG CCTTGATGGC AAGCTTGGTG GAGAGGACTG AAGCTGTTGC   1920 

TGGGGGCCGA TTCTGATCAA GACACATGGC TTGTAAATGG AAGACACAAC ACTGAGAGAT   1980 

CATTCTGCTC TAAGTTTCGG GAACTTATCC CCGACAGTGA CTGAACTCAC TGACTAATAA   2040 

CTTCC                                                               2045 

 
           
           
             
               721 amino acids  
               amino acid  
               single  
               linear  
             
             
               peptide  
             
              4 

Met Thr Ala Lys Asp Ser Ser Lys Glu Leu Thr Ala Ser Glu Pro Glu 
1               5                   10                  15 

Val Cys Ile Lys Thr Phe Lys Glu Gln Met His Leu Glu Leu Glu Leu 
            20                  25                  30 

Pro Arg Leu Pro Gly Asn Arg Pro Thr Ser Pro Lys Ile Ser Pro Arg 
        35                  40                  45 

Ser Ser Pro Arg Asn Ser Pro Cys Phe Phe Arg Lys Leu Leu Val Asn 
    50                  55                  60 

Lys Ser Ile Arg Gln Arg Arg Arg Phe Thr Val Ala His Thr Cys Phe 
65                  70                  75                  80 

Asp Val Glu Asn Gly Pro Ser Pro Gly Arg Ser Pro Leu Asp Pro Gln 
                85                  90                  95 

Ala Ser Ser Ser Ala Gly Leu Val Leu His Ala Thr Phe Pro Gly His 
            100                 105                 110 

Ser Gln Arg Arg Glu Ser Phe Leu Tyr Arg Ser Asp Ser Asp Tyr Asp 
        115                 120                 125 

Leu Ser Pro Lys Ala Met Ser Arg Asn Ser Ser Leu Pro Ser Glu Gln 
    130                 135                 140 

His Gly Asp Asp Leu Ile Val Thr Pro Phe Ala Gln Val Leu Ala Ser 
145                 150                 155                 160 

Leu Arg Ser Val Arg Asn Asn Phe Thr Ile Leu Thr Asn Leu His Gly 
                165                 170                 175 

Thr Ser Asn Lys Arg Ser Pro Ala Ala Ser Gln Pro Pro Val Ser Arg 
            180                 185                 190 

Val Asn Pro Gln Glu Glu Ser Tyr Gln Lys Leu Ala Met Glu Thr Leu 
        195                 200                 205 

Glu Glu Leu Asp Trp Cys Leu Asp Gln Leu Glu Thr Ile Gln Thr Tyr 
    210                 215                 220 

Arg Ser Val Ser Glu Met Ala Ser Asn Lys Phe Lys Arg Met Leu Asn 
225                 230                 235                 240 

Arg Glu Leu Thr His Leu Ser Glu Met Ser Arg Ser Gly Asn Gln Val 
                245                 250                 255 

Ser Glu Tyr Ile Ser Asn Thr Phe Leu Asp Lys Gln Asn Asp Val Glu 
            260                 265                 270 

Ile Pro Ser Pro Thr Gln Lys Asp Arg Glu Lys Lys Lys Lys Gln Gln 
        275                 280                 285 

Leu Met Thr Gln Ile Ser Gly Val Lys Lys Leu Met His Ser Ser Ser 
    290                 295                 300 

Leu Asn Asn Thr Ser Ile Ser Arg Phe Gly Val Asn Thr Glu Asn Glu 
305                 310                 315                 320 

Asp His Leu Ala Lys Glu Leu Glu Asp Leu Asn Lys Trp Gly Leu Asn 
                325                 330                 335 

Ile Phe Asn Val Ala Gly Tyr Ser His Asn Arg Pro Leu Thr Cys Ile 
            340                 345                 350 

Met Tyr Ala Ile Phe Gln Glu Arg Asp Leu Leu Lys Thr Phe Arg Ile 
        355                 360                 365 

Ser Ser Asp Thr Phe Ile Thr Tyr Met Met Thr Leu Glu Asp His Tyr 
    370                 375                 380 

His Ser Asp Val Ala Tyr His Asn Ser Leu His Ala Ala Asp Val Ala 
385                 390                 395                 400 

Gln Ser Thr His Val Leu Leu Ser Thr Pro Ala Leu Asp Ala Val Phe 
                405                 410                 415 

Thr Asp Leu Glu Ile Leu Ala Ala Ile Phe Ala Ala Ala Ile His Asp 
            420                 425                 430 

Val Asp His Pro Gly Val Ser Asn Gln Phe Leu Ile Asn Thr Asn Ser 
        435                 440                 445 

Glu Leu Ala Leu Met Tyr Asn Asp Glu Ser Val Leu Glu Asn His His 
    450                 455                 460 

Leu Ala Val Gly Phe Lys Leu Leu Gln Gly Glu His Cys Asp Ile Phe 
465                 470                 475                 480 

Met Asn Leu Thr Lys Lys Gln Arg Gln Thr Leu Arg Lys Met Val Ile 
                485                 490                 495 

Asp Met Val Leu Ala Thr Asp Met Ser Lys His Met Ser Leu Leu Ala 
            500                 505                 510 

Asp Leu Lys Thr Met Val Glu Thr Lys Lys Val Thr Ser Ser Gly Val 
        515                 520                 525 

Leu Leu Leu Asp Asn Tyr Thr Asp Arg Ile Gln Val Leu Arg Asn Met 
    530                 535                 540 

Val His Cys Ala Asp Leu Ser Asn Pro Thr Lys Ser Leu Glu Leu Tyr 
545                 550                 555                 560 

Arg Gln Trp Thr Asp Arg Leu Met Glu Glu Phe Phe Gln Gln Gly Asp 
                565                 570                 575 

Lys Glu Arg Glu Arg Gly Met Glu Ile Ser Pro Met Cys Asp Lys His 
            580                 585                 590 

Thr Ala Ser Val Glu Lys Ser Gln Val Gly Phe Ile Asp Tyr Ile Val 
        595                 600                 605 

His Pro Leu Trp Glu Thr Trp Ala Asp Leu Val Gln Pro Asp Ala Gln 
    610                 615                 620 

Asp Ile Leu Asp Thr Leu Glu Asp Asn Arg Asn Trp Tyr Gln Ser Met 
625                 630                 635                 640 

Ile Pro Gln Ser Pro Ser Pro Pro Leu Asp Glu Gln Asn Arg Asp Cys 
                645                 650                 655 

Gln Gly Leu Met Glu Lys Phe Gln Phe Glu Leu Thr Leu Asp Glu Glu 
            660                 665                 670 

Asp Ser Glu Gly Pro Glu Lys Glu Gly Glu Gly His Ser Tyr Phe Ser 
        675                 680                 685 

Ser Thr Lys Thr Leu Cys Val Ile Asp Pro Glu Asn Arg Asp Ser Leu 
    690                 695                 700 

Gly Glu Thr Asp Ile Asp Ile Ala Thr Glu Asp Lys Ser Pro Val Asp 
705                 710                 715                 720 

Thr 

 
           
           
             
               564 amino acids  
               amino acid  
               single  
               linear  
             
             
               peptide  
             
              5 

Met Lys Glu His Gly Gly Thr Phe Ser Ser Thr Gly Ile Ser Gly Gly 
1               5                   10                  15 

Ser Gly Asp Ser Ala Met Asp Ser Leu Gln Pro Leu Gln Pro Asn Tyr 
            20                  25                  30 

Met Pro Val Cys Leu Phe Ala Glu Glu Ser Tyr Gln Lys Leu Ala Met 
        35                  40                  45 

Glu Thr Leu Glu Glu Leu Asp Trp Cys Leu Asp Gln Leu Glu Thr Ile 
    50                  55                  60 

Gln Thr Tyr Arg Ser Val Ser Glu Met Ala Ser Asn Lys Phe Lys Arg 
65                  70                  75                  80 

Met Leu Asn Arg Glu Leu Thr His Leu Ser Glu Met Ser Arg Ser Gly 
                85                  90                  95 

Asn Gln Val Ser Glu Tyr Ile Ser Asn Thr Phe Leu Asp Lys Gln Asn 
            100                 105                 110 

Asp Val Glu Ile Pro Ser Pro Thr Gln Lys Asp Arg Glu Lys Lys Lys 
        115                 120                 125 

Lys Gln Gln Leu Met Thr Gln Ile Ser Gly Val Lys Lys Leu Met His 
    130                 135                 140 

Ser Ser Ser Leu Asn Asn Thr Ser Ile Ser Arg Phe Gly Val Asn Thr 
145                 150                 155                 160 

Glu Asn Glu Asp His Leu Ala Lys Glu Leu Glu Asp Leu Asn Lys Trp 
                165                 170                 175 

Gly Leu Asn Ile Phe Asn Val Ala Gly Tyr Ser His Asn Arg Pro Leu 
            180                 185                 190 

Thr Cys Ile Met Tyr Ala Ile Phe Gln Glu Arg Asp Leu Leu Lys Thr 
        195                 200                 205 

Phe Arg Ile Ser Ser Asp Thr Phe Ile Thr Tyr Met Met Thr Leu Glu 
    210                 215                 220 

Asp His Tyr His Ser Asp Val Ala Tyr His Asn Ser Leu His Ala Ala 
225                 230                 235                 240 

Asp Val Ala Gln Ser Thr His Val Leu Leu Ser Thr Pro Ala Leu Asp 
                245                 250                 255 

Ala Val Phe Thr Asp Leu Glu Ile Leu Ala Ala Ile Phe Ala Ala Ala 
            260                 265                 270 

Ile His Asp Val Asp His Pro Gly Val Ser Asn Gln Phe Leu Ile Asn 
        275                 280                 285 

Thr Asn Ser Glu Leu Ala Leu Met Tyr Asn Asp Glu Ser Val Leu Glu 
    290                 295                 300 

Asn His His Leu Ala Val Gly Phe Lys Leu Leu Gln Gly Glu His Cys 
305                 310                 315                 320 

Asp Ile Phe Met Asn Leu Thr Lys Lys Gln Arg Gln Thr Leu Arg Lys 
                325                 330                 335 

Met Val Ile Asp Met Val Leu Ala Thr Asp Met Ser Lys His Met Ser 
            340                 345                 350 

Leu Leu Ala Asp Leu Lys Thr Met Val Glu Thr Lys Lys Val Thr Ser 
        355                 360                 365 

Ser Gly Val Leu Leu Leu Asp Asn Tyr Thr Asp Arg Ile Gln Val Leu 
    370                 375                 380 

Arg Asn Met Val His Cys Ala Asp Leu Ser Asn Pro Thr Lys Ser Leu 
385                 390                 395                 400 

Glu Leu Tyr Arg Gln Trp Thr Asp Arg Leu Met Glu Glu Phe Phe Gln 
                405                 410                 415 

Gln Gly Asp Lys Glu Arg Glu Arg Gly Met Glu Ile Ser Pro Met Cys 
            420                 425                 430 

Asp Lys His Thr Ala Ser Val Glu Lys Ser Gln Val Gly Phe Ile Asp 
        435                 440                 445 

Tyr Ile Val His Pro Leu Trp Glu Thr Trp Ala Asp Leu Val Gln Pro 
    450                 455                 460 

Asp Ala Gln Asp Ile Leu Asp Thr Leu Glu Asp Asn Arg Asn Trp Tyr 
465                 470                 475                 480 

Gln Ser Met Ile Pro Gln Ser Pro Ser Pro Pro Leu Asp Glu Gln Asn 
                485                 490                 495 

Arg Asp Cys Gln Gly Leu Met Glu Lys Phe Gln Phe Glu Leu Thr Leu 
            500                 505                 510 

Asp Glu Glu Asp Ser Glu Gly Pro Glu Lys Glu Gly Glu Gly His Ser 
        515                 520                 525 

Tyr Phe Ser Ser Thr Lys Thr Leu Cys Val Ile Asp Pro Glu Asn Arg 
    530                 535                 540 

Asp Ser Leu Gly Glu Thr Asp Ile Asp Ile Ala Thr Glu Asp Lys Ser 
545                 550                 555                 560 

Pro Val Asp Thr 

 
           
           
             
               517 amino acids  
               amino acid  
               single  
               linear  
             
             
               peptide  
             
              6 

Met Glu Thr Leu Glu Glu Leu Asp Trp Cys Leu Asp Gln Leu Glu Thr 
1               5                   10                  15 

Ile Gln Thr Tyr Arg Ser Val Ser Glu Met Ala Ser Asn Lys Phe Lys 
            20                  25                  30 

Arg Met Leu Asn Arg Glu Leu Thr His Leu Ser Glu Met Ser Arg Ser 
        35                  40                  45 

Gly Asn Gln Val Ser Glu Tyr Ile Ser Asn Thr Phe Leu Asp Lys Gln 
    50                  55                  60 

Asn Asp Val Glu Ile Pro Ser Pro Thr Gln Lys Asp Arg Glu Lys Lys 
65                  70                  75                  80 

Lys Lys Gln Gln Leu Met Thr Gln Ile Ser Gly Val Lys Lys Leu Met 
                85                  90                  95 

His Ser Ser Ser Leu Asn Asn Thr Ser Ile Ser Arg Phe Gly Val Asn 
            100                 105                 110 

Thr Glu Asn Glu Asp His Leu Ala Lys Glu Leu Glu Asp Leu Asn Lys 
        115                 120                 125 

Trp Gly Leu Asn Ile Phe Asn Val Ala Gly Tyr Ser His Asn Arg Pro 
    130                 135                 140 

Leu Thr Cys Ile Met Tyr Ala Ile Phe Gln Glu Arg Asp Leu Leu Lys 
145                 150                 155                 160 

Thr Phe Arg Ile Ser Ser Asp Thr Phe Ile Thr Tyr Met Met Thr Leu 
                165                 170                 175 

Glu Asp His Tyr His Ser Asp Val Ala Tyr His Asn Ser Leu His Ala 
            180                 185                 190 

Ala Asp Val Ala Gln Ser Thr His Val Leu Leu Ser Thr Pro Ala Leu 
        195                 200                 205 

Asp Ala Val Phe Thr Asp Leu Glu Ile Leu Ala Ala Ile Phe Ala Ala 
    210                 215                 220 

Ala Ile His Asp Val Asp His Pro Gly Val Ser Asn Gln Phe Leu Ile 
225                 230                 235                 240 

Asn Thr Asn Ser Glu Leu Ala Leu Met Tyr Asn Asp Glu Ser Val Leu 
                245                 250                 255 

Glu Asn His His Leu Ala Val Gly Phe Lys Leu Leu Gln Gly Glu His 
            260                 265                 270 

Cys Asp Ile Phe Met Asn Leu Thr Lys Lys Gln Arg Gln Thr Leu Arg 
        275                 280                 285 

Lys Met Val Ile Asp Met Val Leu Ala Thr Asp Met Ser Lys His Met 
    290                 295                 300 

Ser Leu Leu Ala Asp Leu Lys Thr Met Val Glu Thr Lys Lys Val Thr 
305                 310                 315                 320 

Ser Ser Gly Val Leu Leu Leu Asp Asn Tyr Thr Asp Arg Ile Gln Val 
                325                 330                 335 

Leu Arg Asn Met Val His Cys Ala Asp Leu Ser Asn Pro Thr Lys Ser 
            340                 345                 350 

Leu Glu Leu Tyr Arg Gln Trp Thr Asp Arg Leu Met Glu Glu Phe Phe 
        355                 360                 365 

Gln Gln Gly Asp Lys Glu Arg Glu Arg Gly Met Glu Ile Ser Pro Met 
    370                 375                 380 

Cys Asp Lys His Thr Ala Ser Val Glu Lys Ser Gln Val Gly Phe Ile 
385                 390                 395                 400 

Asp Tyr Ile Val His Pro Leu Trp Glu Thr Trp Ala Asp Leu Val Gln 
                405                 410                 415 

Pro Asp Ala Gln Asp Ile Leu Asp Thr Leu Glu Asp Asn Arg Asn Trp 
            420                 425                 430 

Tyr Gln Ser Met Ile Pro Gln Ser Pro Ser Pro Pro Leu Asp Glu Gln 
        435                 440                 445 

Asn Arg Asp Cys Gln Gly Leu Met Glu Lys Phe Gln Phe Glu Leu Thr 
    450                 455                 460 

Leu Asp Glu Glu Asp Ser Glu Gly Pro Glu Lys Glu Gly Glu Gly His 
465                 470                 475                 480 

Ser Tyr Phe Ser Ser Thr Lys Thr Leu Cys Val Ile Asp Pro Glu Asn 
                485                 490                 495 

Arg Asp Ser Leu Gly Glu Thr Asp Ile Asp Ile Ala Thr Glu Asp Lys 
            500                 505                 510 

Ser Pro Val Asp Thr 
        515