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:
This application is a continuation of application Ser. No. 08/472,600, filed Jun. 7, 1995, now U.S. Pat. No. 6,323,041 which is a divisional of application Ser. No. 08/432,327, filed May 1, 1995, now abandoned, which is a continuation of application Ser. No. 08/075,450, filed Jun. 11, 1993, now abandoned. 
    
    
     BACKGROUND OF THE INVENTION 
     This invention relates to novel nucleic acid sequences encoding three novel human phosphodiesterase IV (hPDE IV) isozymes. 
     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]). 
     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 isozyme-selective inhibitors by providing purified isoenzymes to incorporate into drug assays. 
     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. 
     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]). 
     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. 
     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 Rapid Amplification of cDNA Ends 
     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 
     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 
     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. 
     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. 
     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. 
     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. 
     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. 
     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. 
     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. 
     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. 
     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. 
     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. 
     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 
     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. 
     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-B2 (−33 to −24) is underlined. 
     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. 
     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. 
     FIG.  5 . Restriction Map 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 
     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. 
     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. 
     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. 
     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-B2 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 . 
     The most logical explanation for the three hPDE IV-B isoforms is that they are 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]). 
     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. 
     Mammalian Expression Clones for hPDE IV-B1, -B2, -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. 
     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 Sall restriction site in clone λK2.1 is contained in an exon, and corresponds to the unique Sall 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: EcoRI-6.6 kb, HindIII-4.4 kb, BamHI-4.2 kb. 
     Deposits 
     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). 
     Assays 
     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. 
     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. 
     Utility of the Invention 
     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. 
     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. 
     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. 
     Materials and Methods 
     (a) Cells/Reagents 
     Cells from a human bronchoalveolar lavage (BAL) were purchased from the Johns Hopkins University (Dr. M. Liu). 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. 
     (b) Degenerate RT-PCR 
     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/ml 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. 
     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. 
     (c) Library Screening 
     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. 
     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 Xhol half-site method, and 1×10 6  clones screened under the same hybridization conditions used for the previous genomic library. 
     (d) DNA Sequencing 
     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. 
     (e) RACE Method 
     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. 
     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. 
     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. 
     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). 
     Sequence ID Summary 
     1. hPDE IV-B1 cDNA sequence. 2,554 bp. 
     2. hPDE IV-B2 cDNA sequence. 2,246 bp. 
     3. hPDE IV-B3 cDNA sequence. 2,045 bp. 
     4. Predicted amino acid sequence of hPDE IV-B1. 721 amino acids. 
     5. Predicted amino acid sequence of hPDE IV-B2. 564 amino acids. 
     6. Predicted amino acid sequence of hPDE IV-B3. 517 amino acids. 
     
       
         
           
             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