The research carried out in the subject application was supported in part by grants from the National Institutes of Health. The government may have rights in any patent issuing on this application.

INTRODUCTION Field of the Invention The field of this invention is transcription factor proteins involved in vascularization.

Background Roughly a dozen proteins classified as basic helix-loop-helix/PAS domain transcription factors have been described in both vertebrates and invertebrates. Members of this class derive their name from the shared presence of a basic helix-loop-helix (bHLH) motif that specifies sequence dependent recognition of DNA and a PAS domain composed of two imperfect repeats. PAS is an acronym derived from the first three proteins observed to contain this motif. These include the product of the period gene of Drosophila melanogaster (Jackson et al. 1986 ; Citri et al. 1987), the aryl hydrocarbon nuclear transporter gene (ARNT) of mammals (Burbach et al. 1992), and the product of the fruit fly single-minded gene (Nambu et al. 1991).

The imperfect, direct repeats within the PAS domain are approximately 50 amino acids in length and contain a signature His-X-X-Asp sequence in each repeat. Three biochemical functions have been assigned to the PAS domain. First, it acts in concert with the helix-loop-helix domain of bHLH/PAS proteins to form a dimerization surface (Reisz- Porszasz et al. 1994 ; Fukunaga et al. 1995 ; Lindebro et al. 1995). In the case of the period gene product, which lacks a bHLH domain, the PAS domain specifies heterodimerization with the product of the timeless locus (Gekakis et al. 1995 ; Myers et al. 1995). Interaction between the period and timeless gene products represents a crucial event in the control of circadian rhythm in fruit flies (Hunter-Ensor et al. 1996 ; Lee et al. 1996 ; Myers et al. 1996 ; Zeng et al 1996). In contrast, the aryl hydrocarbon receptor (AHR) heterodimerizes with

ARNT via PAS domain interactions (Fukunaga et al. 1995), producing a heterodimer that is competent for nuclear gene interaction. Second, the PAS domain mediates interaction with heat shock protein 90 (HSP-90). Several PAS domain proteins, including the single-minded gene product and the AHR, can be sequestered in the cytoplasm in an inactive state.

Maintenance of the inactive state involves interactions between the PAS domain and HSP-90 (Perdew, 1988 ; Chen and Perdew, 1994 ; Henry and Gasiewicz, 1993 ; McGuire et al. 1995).

Finally, the PAS domain of the AHR facilitates high affinity binding of certain xenobiotic compounds including dioxin (reviewed in Hankinson, 1995 ; Schmidt and Bradfield, 1996).

PAS domain transcription factors perform diverse functions in a variety of cell types and organisms. The period gene product helps regulate circadian rhythm in fruit flies (Konopka and Benzer, 1971), whereas the mammalian AHR provides response to xenobiotics by activating genes whose products facilitate detoxification (Schmidt and Bradfield, 1996). A more recently described member of the PAS domain family, hypoxia inducible factor (HIF- la), activates genes whose products regulate hematopoiesis in response to oxygen deprivation (Wang et al. 1995). In Drosophila, the single-minded gene product affects neurogenesis (Nambu et al. 1991) and the trachealess gene product controls the formation of tubular structures in the embryo (Wilk et al. 1996 ; Isaac and Andrew, 1996).

The utilization of bHLH/PAS domain proteins in diverse species and physiological processes raises the possibility that this family of transcription factors might consist of many undiscovered members. Here we report the initial characterization of new members of this protein family collectively designated endothelial PAS domain protein 1 (EPAS 1).

SUMMARY OF THE INVENTION The invention provides methods and compositions relating to endothelial PAS domain protein 1 (EPAS 1), related nucleic acids, and protein domains thereof having EPAS1-specific activity. EPAS 1 proteins can regulate specification of endothelial tissue, such as vasculature, the blood brain barrier, etc. The proteins may be produced recombinantly from transformed host cells from the subject EPAS 1 encoding nucleic acids or purified from mammalian cells.

The invention provides isolated EPAS1 hybridization probes and primers capable of specifically hybridizing with the disclosed EPAS1 gene, EPAS1-specific binding agents such as specific antibodies, and methods of making and using the subject compositions in diagnosis (e. g. genetic hybridization screens for EPAS 1 transcripts), therapy (e. g. gene

therapy to modulate EPAS1 gene expression) and in the biopharmaceutical industry (e. g. as immunogens, reagents for isolating B-cell specific activators or other transcriptional regulators, reagents for screening chemical libraries for lead pharmacological agents, etc.).

SEQ ID NO : LISTING SEQ ID NO : 1 : human EPAS 1 cDNA.

SEQ ID NO : 2 : murine EPAS1 cDNA.

SEQ ID NO : 3 : HIF-la binding site.

SEQ ID NO : 4 : human EPAS1 protein.

SEQ ID NO : 5 : murine EPAS1 protein.

SEQ ID NO : 6 : human HIF-laprotein.

SEQ ID NO : 7 : murine HIF-la protein DETAILED DESCRIPTION OF THE INVENTION The nucleotide sequence of a natural cDNA encoding a human and murine EPAS 1 proteins are shown as SEQ ID NOS : 1 and 2, respectively, and the full conceptual translates as SEQ ID NOS : 4 and 5, respectively. The EPAS 1 proteins of the invention include incomplete translates of SEQ ID NOS : 1 and 2 and deletion mutants of SEQ ID NOS : 4 and 5, which translates and deletion mutants have EPAS1-specific amino acid sequence and binding specificity or function. Such active EPAS 1 deletion mutants, EPAS 1 peptides or protein domains comprise at least 14, preferably at least about 16, more preferably at least about 20 consecutive residues of SEQ ID NO : 4 or 5. For examples, EPAS 1 protein domains identified below are shown to provide dimerization, protein-binding, and nucleic acid binding function.

Additional such domains are identified in and find use, inter alia, in solid-phase binding assays as described below.

EPAS 1-specific activity or function may be determined by convenient in vitro, cell- based, or in vivo assays : e. g. in vitro binding assays, cell culture assays, in animals (e. g. immune response, gene therapy, transgenics, etc.), etc. Binding assays encompass any assay where the molecular interaction of an EPAS 1 protein with a binding target is evaluated. The binding target may be a natural intracellular binding target such as another bHLH/PAS protein, a heat shock protein, or a nucleic acid sequence/binding site or other regulator that directly modulates EPAS1 activity or its localization ; or non-natural binding target such a

specific immune protein such as an antibody, or an EPAS 1 specific agent such as those identified in screening assays such as described below. EPAS 1-binding specificity may assayed by binding equilibrium constants (usually at least about 10'M-', preferably at least about 108 M-', more preferably at least about 109 M-'), by the ability of the subject protein to function as negative mutants in EPAS 1-expressing cells, to elicit EPAS 1 specific antibody in a heterologous host (e. g a rodent or rabbit), etc. In any event, the EPAS1 binding specificity of the subject EPAS1 proteins necessarily distinguishes HIF-la.

The claimed EPAS 1 proteins are isolated or pure : an"isolated"protein is unaccompanied by at least some of the material with which it is associated in its natural state, preferably constituting at least about 0. 5%, and more preferably at least about 5% by weight of the total protein in a given sample and a pure protein constitutes at least about 90%, and preferably at least about 99% by weight of the total protein in a given sample. The EPAS1 proteins and protein domains may be synthesized, produced by recombinant technology, or purified from mammalian, preferably human cells. A wide variety of molecular and biochemical methods are available for biochemical synthesis, molecular expression and purification of the subject compositions, see e. g. Molecular Cloning, A Laboratory Manual (Sambrook, et al. Cold Spring Harbor Laboratory), Current Protocols in Molecular Biology (Eds. Ausubel, et al., Greene Publ. Assoc., Wiley-Interscience, NY) or that are otherwise known in the art.

The invention provides natural and non-natural EPAS 1-specific binding agents, methods of identifying and making such agents, and their use in diagnosis, therapy and pharmaceutical development. For example, EPAS 1-specific agents are useful in a variety of diagnostic and therapeutic applications. Novel EPAS1-specific binding agents include EPAS1-specific receptors, such as somatically recombine protein receptors like specific antibodies or T-cell antigen receptors (see, e. g Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory) and other natural intracellular binding agents identified with assays such as one-, two-and three-hybrid screens, non-natural intracellular binding agents identified in screens of chemical libraries such as described below, etc. For diagnostic uses, the binding agents are frequently labeled, such as with fluorescent, radioactive, chemiluminescent, or other easily detectable molecules, either conjugated directly to the binding agent or conjugated to a probe specific for the binding agent. Agents of particular interest modulate EPAS 1 function, e. g. EPAS 1-dependent

transcriptional activation ; for example, isolated cells, whole tissues, or individuals may be treated with an EPAS 1 binding agent to activate, inhibit, or alter EPAS 1-dependent transcriptional processes.

The amino acid sequences of the disclosed EPAS1 proteins are used to back-translate EPAS 1 protein-encoding nucleic acids optimized for selected expression systems (Holler et al. (1993) Gene 136, 323-328 ; Martin et al. (1995) Gene 154, 150-166) or used to generate degenerate oligonucleotide primers and probes for use in the isolation of natural EPAS1- encoding nucleic acid sequences ("GCG"software, Genetics Computer Group, Inc, Madison WI). EPAS 1-encoding nucleic acids used in EPAS 1-expression vectors and incorporated into recombinant host cells, e. g. for expression and screening, transgenic animals, e. g. for functional studies such as the efficacy of candidate drugs for disease associated with EPAS 1- modulated transcription, etc.

The invention also provides nucleic acid hybridization probes and replication/ amplification primers having a EPAS 1 cDNA specific sequence contained in SEQ ID NO : 1 and sufficient to effect specific hybridization thereto (i. e. specifically hybridize with SEQ ID NO : 1 in the presence of endothelial cell cDNA). Such primers or probes are at least 12, preferably at least 24, more preferably at least 36 and most preferably at least 96 bases in length. Demonstrating specific hybridization generally requires stringent conditions, for example, hybridizing in a buffer comprising 30% formamide in 5 x SSPE (0. 18 M NaCI, 0. 01 M NaPO4, pH7. 7, 0. 001 M EDTA) buffer at a temperature of 42°C and remaining bound when subject to washing at 42°C with 0. 2 x SSPE ; preferably hybridizing in a buffer comprising 50% formamide in 5 x SSPE buffer at a temperature of 42°C and remaining bound when subject to washing at 42°C with 0. 2 x SSPE buffer at 42°C. EPAS1 cDNA homologs can also be distinguished from other protein using alignment algorithms, such as BLASTX (Altschul et al. (1990) Basic Local Alignment Search Tool, J Mol Biol 215, 403- 410).

The subject nucleic acids are of synthetic/non-natural sequences and/or are isolated, i. e. unaccompanied by at least some of the material with which it is associated in its natural state, preferably constituting at least about 0. 5%, preferably at least about 5% by weight of total nucleic acid present in a given fraction, and usually recombinant, meaning they comprise a non-natural sequence or a natural sequence joined to nucleotide (s) other than that which it is joined to on a natural chromosome. Nucleic acids comprising the nucleotide sequence of

SEQ ID NO : 1 or 2 or fragments thereof, contain such sequence or fragment at a terminus, immediately flanked by a sequence other than that which it is joined to on a natural chromosome, or flanked by a native flanking region fewer than 10 kb, preferably fewer than 2 kb, which is at a terminus or is immediately flanked by a sequence other than that which it is joined to on a natural chromosome. While the nucleic acids are usually RNA or DNA, it is often advantageous to use nucleic acids comprising other bases or nucleotide analogs to provide modified stability, etc.

The subject nucleic acids find a wide variety of applications including use as translatable transcripts, hybridization probes, PCR primers, diagnostic nucleic acids, etc. ; use in detecting the presence of EPAS 1 genes and gene transcripts and in detecting or amplifying nucleic acids encoding additional EPAS1 homologs and structural analogs. In diagnosis, EPAS1 hybridization probes find use in identifying wild-type and mutant EPAS1 alleles in clinical and laboratory samples. Mutant alleles are used to generate allele-specific oligonucleotide (ASO) probes for high-throughput clinical diagnoses. In therapy, therapeutic EPAS1 nucleic acids are used to modulate cellular expression or intracellular concentration or availability of active EPAS1.

The invention provides efficient methods of identifying agents, compounds or lead compounds for agents active at the level of a EPAS1 modulatable cellular function.

Generally, these screening methods involve assaying for compounds which modulate EPAS1 interaction with a natural EPAS 1 binding target. A wide variety of assays for binding agents are provided including labeled in vitro protein-protein binding assays, immunoassays, cell based assays, etc. The methods are amenable to automated, cost-effective high throughput screening of chemical libraries for lead compounds. Identified reagents find use in the pharmaceutical industries for animal and human trials ; for example, the reagents may be derivatized and rescreened in in vitro and in vivo assays to optimize activity and minimize toxicity for pharmaceutical development. Target indications include neoproliferative disease, inflammation, hypersensitivity, wound healing, immune deficiencies, infection etc.

In vitro binding assays employ a mixture of components including an EPAS 1 protein, which may be part of a fusion product with another peptide or polypeptide, e. g. a tag for detection or anchoring, etc. The assay mixtures comprise a natural intracellular EPAS 1 binding target. While native binding targets may be used, it is frequently preferred to use portions (e. g. peptides) thereof so long as the portion provides binding affinity and avidity to

the subject EPAS 1 protein conveniently measurable in the assay. The assay mixture also comprises a candidate pharmacological agent. Candidate agents encompass numerous chemical classes, though typically they are organic compounds ; preferably small organic compounds and are obtained from a wide variety of sources including libraries of synthetic or natural compounds. A variety of other reagents may also be included in the mixture. These include reagents like salts, buffers, neutral proteins, e. g. albumin, detergents, protease inhibitors, nuclease inhibitors, antimicrobial agents, etc. may be used.

The resultant mixture is incubated under conditions whereby, but for the presence of the candidate pharmacological agent, the EPAS 1 protein specifically binds the cellular binding target, portion or analog with a reference binding affinity. The mixture components can be added in any order that provides for the requisite bindings and incubations may be performed at any temperature which facilitates optimal binding. Incubation periods are likewise selected for optimal binding but also minimized to facilitate rapid, high-throughput screening.

After incubation, the agent-biased binding between the EPAS 1 protein and one or more binding targets is detected by any convenient way. For cell-free binding type assays, a separation step is often used to separate bound from unbound components. Separation may be effected by precipitation (e. g. TCA precipitation, immunoprecipitation, etc.), immobilization (e. g on a solid substrate), etc., followed by washing by, for examples, membrane filtration (e. g. Whatman's P-81 ion exchange paper, Polyfiltronic's hydrophobic GFC membrane, etc.), gel chromatography (e. g. gel filtration, affinity, etc.). For EPAS1- dependent transcription assays, binding is detected by a change in the expression of an EPAS 1-dependent reporter.

Detection may be effected in any convenient way. For cell-free binding assays, one of the components usually comprises or is coupled to a label. The label may provide for direct detection as radioactivity, luminescence, optical or electron density, etc. or indirect detection such as an epitope tag, an enzyme, etc. A variety of methods may be used to detect the label depending on the nature of the label and other assay components, e. g. through optical or electron density, radiative emissions, nonradiative energy transfers, etc. or indirectly detected with antibody conjugates, etc.

A difference in the binding affinity of the EPAS 1 protein to the target in the absence of the agent as compared with the binding affinity in the presence of the agent indicates that

the agent modulates the binding of the EPAS 1 protein to the EPAS 1 binding target.

Analogously, in the cell-based transcription assay also described below, a difference in the EPAS 1 transcriptional induction in the presence and absence of an agent indicates the agent modulates EPAS 1-induced transcription. A difference, as used herein, is statistically significant and preferably represents at least a 50%, more preferably at least a 90% difference.

The following experimental section and examples are offered by way of illustration and not by way of limitation.

EXPERIMENTAL cDNAs encompassing the coding region of the human EPAS 1 were isolated by screening a HeLa cell cDNA library with a radiolabeled probe derived from an expressed sequence tag (#T70415) obtained from the Genbank data base (see Materials and Methods). Multiple cDNA clones were isolated and subjected to DNA sequence analysis to derive the conceptually translated protein sequence of human EPAS 1 shown in Table 1. The predicted M, of the human EPAS1 was 96, 528. A termination codon was located 24 nucleotides 5'of the designated initiator methionine in the human sequence. cDNAs encoding the murine homologue were isolated from an adult mouse brain cDNA library using a probe obtained by reverse transcriptase polymerase chain reactions with oligonucleotide primers derived from the human EPAS 1 cDNA sequence (see Materials and Methods). The predicted protein sequence of murine EPAS1 is aligned and compared with the human sequence in Table 1. The two proteins share 88% sequence identity. Data base searches revealed that the human and murine EPAS 1 proteins share extensive primary amino acid sequence identity with hypoxia inducible factor-la (HIF-la), a member of the bHLH/PAS domain family of transcription factors (Wang et al. 1995 ; Wenger et al. 1995). EPAS1 and HIF-la share 48% primary amino acid sequence identity as revealed by the alignment shown in Table 1. Sequence conservation between the two proteins is highest in the basic-helix-loop-helix (85%), PAS A (68%) and PAS-B (73%) regions. A second region of sequence identity occurs at the extreme carboxy terminis of the EPAS1 and HIF-la proteins.

This conserved region in mHIFIa has been recently shown to contain a hypoxia response domain (Li et al., 1996). EPAS1 also shares sequence relatedness with other PAS domain proteins, however the degree of similarity between EPAS 1 and other family members is less striking than that between HIF-la and EPAS1.

Genomic clones encoding the human EPAS1 transcript were isolated by screening bacteriophage libraries of human DNA. The intron-exon structure of the gene was established

by comparison of DNA sequences obtained from the genomic DNA to that of the cDNA. The coding region of EPAS1 is specified by 15 exons. The exonic sequences mapped to six non-overlapping bacteriophage lambda clones whose average insert size was 20 kb, indicating that the EPAS 1 gene spans at least 120 kb of genomic DNA. A comparison of the EPAS 1 gene structure with that of the aryl hydrocarbon receptor (Schmidt et al. 1993) reveals that the positions of introns within the regions encoding the amino-terminal halves of the two proteins are highly conserved. In contrast, the portion of the EPAS 1 gene specifying the carboxy-terminal half of the protein is interrupted by seven introns, whereas the AHR gene contains only a single intron in this region. Thus the 5'-ends of the two genes may have arisen from an ancient gene duplication event, whereas the 3'-regions have a more recent evolutionary origin.

Two methods were used to determine the chromosomal location of the human EPAS 1 gene. Fluorescent in situ hybridization (FISH) analysis was performed using a biotinylated probe containing exons 8-14 of the EPAS 1 gene. This analysis revealed a single hybridization signal over chromosome 2, bands pl6-p21. As a second assay for gene localization, an oligonucleotide primer pair derived from exon 8 was used to amplify a segment of the EPAS1 gene from the genomic DNAs of a radiation hybrid panel. Computer-assisted analysis of the results indicated linkage of the EPAS 1 gene to the D2S288 marker on chromosome 2p with a LOD score of 8. 7 and a cR8000 value of 12. 96. Thus, the data obtained from two independent mapping methods consistently positioned the EPAS 1 gene on the short arm of chromosome 2 and indicate that the EPAS1 gene is non-syntenic with the HIF-la gene, which maps to chromosome 14q21-24 (Semenzaetal. 1996).

The high degree of sequence similarity between the EPAS 1 and HIF-la proteins raises the possibility that they share a common physiological function. To test this hypothesis, RNA blotting experiments were used to compare and contrast the distributions of EPAS1 and HIF-la mRNAs in a variety of human tissues. An EPAS 1 mRNA of approximately 5. 8 kb was detected in all tissues examined with the single exception of peripheral blood leukocytes. Among the positive tissues, highly vascularized organs such as the heart, placenta and lung showed the highest levels of EPAS 1 mRNA. A HIF-1 a mRNA of approximately 4. 4 kb was detected in all human tissues. In contrast to EPAS 1 mRNA, however, peripheral blood leukocytes contained very high levels of HIF-l a mRNA. Likewise, we observed no enrichment of HIF-la mRNA in highly vascularized tissues.

These RNA blotting data indicate that, with few exceptions, most tissues express both

EPAS 1 and HIF-1 a mRNAs. To determine if this overlap extended to the cellular level, in situ mRNA hybridization was used to determine the cell type specific expression patterns of the two gene products. Sections from day 11 and day 13 mouse embryos were examined first. In day 11 embryo sections, EPAS1 transcripts were observed almost exclusively in endothelial cells of the intersegmental blood vessels separating the somites, the atrial and ventricular chambers of the heart, and the dorsal aorta. Extra-embryonic membranes, such as the yolk sac, which are highly vascularized, also expressed abundant levels of EPAS 1 mRNA. In the developing brain of a day 13 embryo, endothelial cells of the highly vascularized choroid plexus contained abundant EPAS 1 transcripts. The brain section also revealed intense EPAS 1 mRNA hybridization in the endothelial cells of a blood vessel lying along the edge of post-mitotic neurons emanating from the lateral ventricle region. When a nearby section was hybridized with an anti-sense probe that was specific for the HIF-la mRNA, only a diffuse signal somewhat over background was detected, indicating a low level of HIF-la expression in many cell types. In contrast to the results with the EPAS1 probe, no concentration of HIF-la mRNA was detected in the endothelial cells of the adjacent blood vessel. A differential expression pattern between EPAS1 and HIF-la was also apparent in the region of the embryo containing the umbilicus.

EPAS1 transcripts were detected in the endothelium of blood vessels within this structure, whereas HIF-la mRNA was concentrated in the mesenchyme surrounding the vascular endothelium.

In tissues of adult mice, EPAS 1 mRNA was also detected at high levels in endothelial cells, yet was also present at lower levels in several additional cells types. For example, decidual cells of the placenta contained very high levels of EPAS 1 mRNA as did parenchymal tissue in the lung. The distinction between EPAS1 expressing cell types and HIF-la expressing cells was also apparent in adult tissues. A section through the cortex of the kidney showed EPAS1 expression in the mesangial cells. In contrast, HIF-la expression was found in the cells of the collecting ducts. Taken together, these in situ mRNA hybridization results reveal very divergent patterns of EPAS 1 and HIF-1 a mRNA distribution.

The presence of basic helix-loop-helix and PAS domain motifs in EPAS 1 raised the possibility that this protein might be capable of forming a complex with the aryl hydrocarbon receptor nuclear transport protein (ARNT) (Hoffman et al. 1991), and that the resulting heterodimer might exhibit sequence-specific DNA binding. To test these predictions, EPAS 1 and ARNT expression vectors were used to program a reticulocyte lysate. The EPAS 1 expression

vector was modified at its carboxy-terminus with a c-Myc epitope tag to facilitate immunological detection of the EPAS1 translation product. Radiolabeled methionine was included in the translation mix containing the ARNT mRNA, whereas unlabeled methionine was used in the EPAS 1 reaction. After translation, the two reactions were mixed and subsequently incubated with a monoclonal antibody that recognizes the c-Myc epitope present on the EPAS 1 protein.

Under these conditions the c-Myc antibody was capable of immunoprecipitating the radiolabeled ARNT protein only when EPAS 1-Myc protein was present in the reaction.

The bHLH domains of HIF-la and EPAS1 are nearly identical in primary amino acid sequence. Thus, to test for the ability of EPAS1 to form a functional heterodimer with ARNT, we used a HIF-la response element derived from the 3'-flanking region of the erythropoietin gene (Semenza and Wang, 1992) in gel mobility shift assays with in vitro translated proteins.

The data showed that a new complex was formed when both EPAS 1 and ARNT were included in the DNA binding reaction, and that this complex was specifically recognized by an anti-peptide antibody directed against the EPAS1 protein. Competition experiments using a 100-fold excess of unlabeled competitor DNA containing the HIF-la response element, or a response element with three point mutations in this sequence, indicated that EPAS 1 exhibited sequence-specific binding properties. Taken together, the data indicate that EPAS 1 is capable of binding the HIF-la response element in the presence of the ARNT protein.

The ability of EPAS 1 to trans-activate a reporter gene containing the HIF-I a response element was tested by transient transfection. Expression vectors in which either EPAS1, HIF-la, or ARNT were placed under the control of a cytomegalovirus promoter were constructed. Two luciferase reporter constructs were prepared. One contained nucleotides-105 through +58 of the herpes simplex virus thymidine kinase promoter (McKnight et al. 1981) linked to three copies of the HIF-la response element from the erythropoietin gene (pRE-tk-LUC). The other contained a TATA sequence from the adenovirus major late gene promoter (Lillie and Green, 1989) linked to the same three HIF-la response elements (pElB-LUC). Combinations of these plasmids were then transfected into cultured human embryonic kidney 293 cells and the expression of luciferase enzyme activity was monitored in cell lysates 16-20 hours post-transfection. The data showed that EPAS 1 induced a 12-fold increase in luciferase enzyme activity when transfected in the absence of the ARNT vector. Cotransfection of the ARNT expression vector with low levels of EPAS 1 expression vector did not increase the EPAS1-mediated induction of luciferase activity, suggesting that this cell line might contain

adequate amounts of endogenous ARNT to support heterodimer formation with EPAS1. A seven-fold stimulation of luciferase activity was also obtained when larger amounts of the HIF- la expression plasmid were introduced into 293 cells. The introduction of three point mutations into the core sequence of the hypoxia response element eliminated both EPAS 1-dependent and HIF-la-dependent activation of the reporter gene.

The potential of HIF-1 a to induce expression of target genes is increased by both hypoxia and pharmacological compounds that mimic hypoxia in cells, such as desferrioxamine (DFX) and cobalt chloride (CoCl,) (Wang et al. 1995). To determine if EPAS1 activity might also be stimulated by these agents, 293 cells were incubated under hypoxic conditions or treated with DFX or CoCl2 prior to transfection with the plasmids. Pretreatment of cells under conditions that mimic hypoxia increased expression from the luciferase construct in the absence of exogenous EPAS 1 or HIF-1 a. This trans-activation presumably arises from endogenous HIF-1 a or EPAS 1 proteins whose mRNAs are present in 293 cells. As noted above, introduction of the EPAS 1 expression vector led to 5-to 10 times higher levels of luciferase activity over those seen in mock-transfected cells. An extra 2 to 4-fold stimulation of luciferase expression was observed upon pretreatment with CoCl2, DF, or hypoxia relative to that measured in EPAS 1-transfected but untreated cells. Of the three conditions, pretreatment with Cocu. led to a slightly larger increase in EPAS 1 activity, resulting in a four-fold higher level of luciferase activity over that detected in untreated cells. As has been observed in previous studies (Jiang et al. 1996 ; Forsythe et al. 1996), hypoxic conditions also stimulated the ability of HIF-la to trans-activate the target gene containing the hypoxia response element.

The EPAS 1 expression vector was also tested for its ability to activate a reporter gene (pRE-Elb-LUC) following transfection into murine hepatoma cells (Hepalclc7) that express ARNT, as well as in a mutant line derived from these parental cells that does not express ARNT (c4 variant, Legraverend et al. 1982). Expression of EPAS1 in the Hepalclc7 cells led to a nine-fold increase in luciferase activity. Transfection of EPAS 1 alone into c4 cells increased luciferase enzyme activity only slightly (1. 8-fold) whereas cotransfection of EPAS1 and ARNT led to a 12-fold stimulation of activity. These findings are consistent with the interpretation that EPAS 1 forms an active heterodimeric transcription factor with ARNT, and they confirm the results showing heterodimerization of these two proteins obtained in coimmunoprecipitation and gel mobility shift assays.

The experiments demonstrating the functional activity of EPAS1 utilized a hypoxia

response element derived from the erythropoietin gene, which is a known target gene for HIF-la (Semenza and Wang, 1992). Despite the activity of EPAS 1 in these assays, as well as the high degree of sequence similarity between HIF-la and EPAS1, the in situ mRNA hybridization results indicate that the two proteins are expressed in different cell types and thus might activate different target genes. The high level of expression of EPAS 1 in endothelial cells raises the possibility that the EPAS1 protein might activate genes whose expression is limited to endothelial cells. To test this hypothesis, we transfected 293 cells with a c-Myc-tagged EPAS1 expression vector and a marker gene composed of the 5'-flanking region of the Tie-2 gene linked to p-galactosidase. Tie-2 encodes a tyrosine kinase receptor that is specifically expressed in cells of endothelial lineage (Dumont et al. 1992 ; Maison-Pierre et al. 1993 ; Sato et al. 1993 ; Schnurch and Risau, 1993). The data showed that EPAS1 potently stimulated expression of the Tie-2-driven reporter gene, and that the degree of stimulation correlated with the level of immunodetectable EPAS1 in the transfected cells. Surprisingly, little or no transcriptional activation of the Tie-2 reporter gene by HIF-la was detected, even though equivalent amounts of HIF-la and EPAS 1 proteins were expressed in the 293 cells.

These data reveal that EPAS 1 proteins and nucleic acids provide reagents to modulate the formation of the endothelial tissues including vasculature, the blood brain barrier, etc. and to modulate cellular or tissue responsiveness to oxygenation, hypoxia and other hemodynamic stimuli. cDNA and genomic cloning, chromosomal mapping In the course of screening for genes that are differentially expressed in prostate adenocarcinoma versus normal tissue, a cDNA encoding a bHLH/PAS domain protein was isolated. Data base searches generated several expressed-sequence tags that showed sequence similarity to this family of transcription factors. EPAS1 cDNAs correspond to the human expressed sequence tag #T70415 in the Genbank collection and were isolated by a combination of reverse transcriptase polymerase chain reactions and screening of a HeLa cell cDNA library (Yokoyama et al. 1993) using standard methods. Similar approaches were used to isolate the murine homologue from a commercially available mouse adult brain cDNA library (#837314, Stratagene Corp., La Jolla, CA). A human HIF-la cDNA was generated by ligation of an amplified cDNA fragment to expressed sequence tag hbc025 (Takeda et al. 1993). Bacteriophage . clones harboring genomic DNA inserts corresponding to the human EPAS 1 gene were isolated by screening a commercially available fibroblast genomic library (kFIXII vector, #946204,

Stratagene Corp.) Fluorescence in situ hybridization to identify the chromosomal localization of the human EPAS 1 gene was carried out as previously described (Craig and Bickmore, 1994). This analysis indicated hybridization to the short arm of chromosome 2, bands pl6-21. To confirm the assignment, a 269 bp segment of exon 8 from the EPAS1 gene was amplified from the 83 genomic DNAs of a radiation hybrid panel (Stanford G3 panel, Research Genetics, Huntsville, AL) using oligonucleotide primers and a thermocycler program consisting of 35 cycles of 94°C/1 min, 68°C/1 min. Analysis of the results via an e-mail server at Stanford University indicated linkage to the D2S288 marker (logarithm of the odds score of 8. 7, cR8000 value of 12. 96), which is located approximately 82 centimorgans from the telomere of the short arm of chromosome 2 (MIT Center for Genome Research).

RNA blotting and in situ hybridization Human multiple tissue RNA blots (Clontech Laboratories, Palo Alto, CA) were probed with EPAS1 and HIF-la cDNA probes using Rapid-Hyb from Amersham Corp. (Arlington Heights, IL). For in situ mRNA hybridization, mouse tissues were fixed in 4% paraformaldehyde, sectioned at 5 pm thickness, and subjected to in situ mRNA hybridization as described (Berman et al. 1995). A ["P]-labeled antisense RNA probe recognizing the EPAS1 mRNA was derived by in vitro transcription of an-300 bp DNA fragment encoding amino acids 225-327 of the sequence shown in Table 1. A segment of the murine HIF-la cDNA encoding amino acids 41-125 was isolated by reverse transcriptase-polymerase chain reactions using mRNA template isolated from embryonic day 10 mouse embryo.

Co-immunoprecipitation experiments Human EPAS 1 and mouse ARNT proteins were generated in vitro using a transcription-translation kit (TNT System, Promega Corp., Madison, WI). cDNAs encoding full- length proteins were subcloned into the pcDNA3 vector (Invitrogen Corp., San Diego, CA) prior to coupled transcription/translation. For immunoprecipitation, approximately 5 u. l of each reaction were transferred to a separate tube, mixed well and subsequently diluted by the addition of 500 1 of ice-cold buffer (20 mM Hepes-KOH, pH 7. 4/100 mM Cl/10% (v/v) glycerol/ 0. 4% (v/v) Nonidet P-40/5 mM EGTA/5 mM EDTA/100 pg/ml bovine serum albumin/1 mM dithiothreitol) (Huang et al. 1993). The diluted mixture was incubated with 1 il (0. 1 u. g) of anti-Myc monoclonal antibody 9E10 (Santa Cruz Biotechnology, Santa Cruz, CA) for 2 hours at 4°C. A 10 1ll aliquot of beads (-4 x 106 yin number, Dynal Corp., Lake Success, NY) coated

with rat anti-mouse IgGI antibody were then added followed by a further incubation for 1 hour at 4°C. Beads were washed three times with 1. 5 ml of the above buffer and bound proteins were subsequently analyzed by electrophoresis through 8% polyacrylamide gels containing SDS.

Gel retention assays EPAS 1 and ARNT cDNAs were translated in vitro as described above. Gel retention assays were performed as described previously (Semenza and Wang, 1992) using a double-stranded oligonucleotide probe radiolabeled with the Klenow fragment of E. coli DNA polymerase I and containing an HIF-la binding site (5'-GCCCTACGTGCTGTCTCA-3', SEQ ID NO : 3) from the erythropoietin gene (Semenza and Wang, 1992). For supershift assays, a polyclonal antibody was raised against residues 1 to 10 of the human EPAS 1 protein by standard methods and 1 u. l of serum was added to the gel retention reaction mixture prior to the 30 minute incubation at 4°C. A preimmune serum served as a negative antibody control.

Transient transfection assays The pTK-RE3-luc reporter plasmid was constructed by inserting three copies of a 50-nucleotide hypoxia-inducible enhancer from the erythropoietin gene (Semenza and Wang, 1992) into pGL3-TK. The Tie-2-p-galactosidase reporter gene pT2HLacZpAlI. 7, containing 10. 3 kb of 5'-flanking DNA from the murine Tie-2 gene was obtained from the Cardiovascular Division, Beth Israel Hospital, Boston, MA. Human embryonic kidney 293 cells (ATCC CRL#1573) were cultured in Dulbecco's modified Eagle's medium (DMEM, low glucose ; Gibco-BRL) supplemented with 10% fetal calf serum. The murine hepatoma cell line Hepalclc7 and the c4 ARNT deficient mutant derived from this line were maintained as described previously (Legraverend et al. 1982). Approximately 24 hours before transfection, cells were inoculated in 12-well plates at a density of 120, 000 cells per well. Plasmid DNA (1-10 u. g) was transfected into cells using a kit (MBS, Stratagene Corp., La Jolla, CA). Cells were allowed to recover for 3 hours at 35°C in a 3% CO2 atmosphere. Where indicated, 125 uM CoCl2 (#C3169, Sigma Chem. Corp., St. Louis, MO) or 130 I1M desferrioxamine (&num D9533, Sigma) were added to cells at this time and the incubation continued for an additional 16 hours in atmospheres containing 20% or 1% 02. Luciferase and p-galactosidase enzyme activities were determined according to the manufacturer's instructions (Tropix, Bedford, MA). Reporter gene expression was normalized by cotransfection of a p-galactosidase expression vector (pCMV-p-gal) and/or to expression obtained from the pGL3-Control plasmid (Promega Corp., Madison, WI). Levels of expressed c-Myc epitope-tagged EPAS1 or HIF-la were assessed by immunoblotting with

the anti-Myc monoclonal antibody 9E10 (Santa Cruz Biotechnology, Santa Cruz, CA) using a protocol supplied by the manufacturer.

References Antonsson, C., V. et al. 1995. J. Biol. Chem. 270 : 13968-13972.

Berman, D. M., H. Tian, and D. W. Russell 1995. Mol. Endocrinol. 9 : 1561-1570.

Brogi, E., G. et al. 1996. J. Clifi. Ifivest. 97 : 469-476.

Burbach, K. M., A. Poland, and C. A. Bradfield 1992. Proc. Natl. Acad. Sc. U. S. A. 89 : 8185- 8189.

Chen, H. S., and G. H. Perdew 1994. J. Biol. Chem. 269 : 27554-27558.

Citri, Y., et al. 1987. Nature 326 : 42-47.

Craig, C. M., and W. A. Bickmore 1994. Nature Genetics 7 : 376-382.

Dumont, D. J., et al. Genes & Dev. 8 : 1897-1909.

Dumont, D. J., et al. 1995. Developmental Dynamics 203 : 80-92.

Dumont, D. J., et al. 1992. Oncogene 7 : 1471-1480.

Fong, G-H., J. Reascend, M. Gertsenstein, and M. L. Breitman 1995. Nature 376 : 66-70.

Forsythe, J. A., et al. 1996. Molec. Cell. Biol. 16 : 4604-4613.

Fukunaga, B. N., et al. 1995. J Biol. Chem. 270 : 29270-29278.

Gekakis, N., et al. 1995. Science 270 : 811-815.

Hankinson, O. 1995. Ann. Rev. Pharmacol. Toxicol. 35 : 307-340.

Henry, E. C., and T. A. Gasiewicz 1993. Biochem. J. 294 : (Pt 1) 95-101.

Hirose, K., et al. 1996. Cell. Biol. 16 : 1706-1713.

Hoffman, E. C., et al. 1991. Science 252 : 954-958.

Huang, Z. J., 1. Edery, and M. Rosbash 1993. Nature 364 : 259-262.

Hunter-Ensor, M., A. Ousley, and A. Sehgal 1996. Cell 84 : 677-685.

Isaac, D. D., and D. J. Andrew 1996. Genes & Dev. 10 : 103-117.

Jackson, F. R., T. A. Bargiello, S. H. Yun, and M. W. Young 1986. Nature 320 : 185-188.

Jiang, B-H., E. Rue, G. L. Wang, R. Roe, and G. L. Semenza 1996. J. Biol. Chem. 271 : 17771- 1778.

Konopka, R. J., and S. Benzer 1971. Proc. Natl. Acad. Sci. U. S. A. 68 : 2112-2116.

Lee, C., V. Parikh, T. Itsukaichi, K. Bae, and 1. Edery 1996. Science 271 : 1740-1744 Legraverend, C., et al. 1982. J. Biol. Chem. 257 : 6402-6407.

Lillie, J. W., and M. R. Green 1989. Nature 338 : 39-44.

Lindebro, M. C., L. Poellinger, and M. L. Whitelaw 1995. EMBO J. 14 : 3528-3539.

Maison-Pierre, P. C., M. Goldfarb, G. D. Yancopoulos, and G. Gao 1993. Oncogene 8 : 1631-1637.

McGuire, J., et al. 1995. J. Biol. Chem. 270 : 31353-31357.

McKnight. S. L., E. R. Gavis, R. Kingsbury, and R. Axel 1981. Cell 25 : 385-398.

Myers, M. P., et al. 1995. Science 270 : 805-808.

Myers, M. P., et al. 1996. Science 271 : 1736-1740.

Nambu, J. R., J. O. Lewis, K. A. Wharton, and S. T. Crews 1991. Cell 67 : 1157-1167.

Namiki, A., etal. 1995. J. Biol. Chem. 270 : 31189-31195.

Perdew, G. H. 1988.. J. Biol. Chem. 263 : 13802-13805.

Reisz-Porszasz, S., et al. 1994. Mol. Cell. BioL 14 : 6075-6086.

Sato, T. N., et al. 1995. Nature 376 : 70-74.

Sato, T. N., et al. 1993. Proc. Natl. Acad. Sci. U. S. A. 90 : 9355-9358.

Schmidt, J. V., and C. A. Bradfield 1996. Ah. Annu. Rev. Cell. Dev. Biol. 12 : in press.

Schmidt, J. V., L. A. Carver, and C. A. Bradfield 1993.. J. Biol. Chem. 268 : 22203-22209.

Schnurch, H., and W. Risau 1993. Development 119 : 957-968.

Semenza, G. L., E. A. Rue, N. V. lyer, M. G. Pang, and W. G. Keams 1996. Genomics 34 : 437-439.

Semenza, G. L. 1994. Hematology-Oncology Clinics of North America 8 : 863-884.

Semenza, G. L., and G. L. Wang 1992. Mol. Cell. Biol. 12 : 5447-5454.

Shalaby, F., et al. 1995. Nature 376 : 62-66.

Sogawa, K., et al. 1995. Proc. Natl. Acad. Sci. U. S. A. 92 : 1936-1940.

Takeda, J., H. Yano, S. Eng, Y. Zeng, and G. I. Bell 1993. Hum Mol. Genet. 2 : 1793-1798/ Wang, G. L., and G. L. Semenza 1995. J. Biol. Chem. 270 : 1230-1237.

Wang, G. L., et al. 1995. Proc. Natl. Acad. Sci. U. S. A. 92 : 5510-5514.

Wenger, R. H., et al. 1996. Biochem. Biophys. Res. Commun. 223 : 54-59.

Wilk, R., 1. Weizman, and B-Z. Shilo 1996. Genes & Dev. 10 : 93-102.

Wood, S. M., et al. 1996. J. Biol. Chem. 271 : 15117-15123.

Yokoyama, C., et al. 1993. Cell 75 : 187-197.

Zeng, H., Z. Qian, M. P. Myers, and M. Rosbash 1996. Nature 380 : 129-135.

EXAMPLES 1. Protocol for high throughput EPAS 1-ARNT complex formation assay.

A. Reagents :

-Neutralite Avidin : 20 u. g/ml in PBS.

-Blocking buffer : 5% BSA, 0. 5% Tween 20 in PBS ; 1 hour at room temperature.

-Assav Buffer : 100 mM KCI, 20 mM HEPES pH 7. 6, 1 mM MgCl2, 1% glycerol, 0. 5% NP-40, 50 mM-mercaptoethanol, 1 mg/ml BSA, cocktail of protease inhibitors.

-3 P EPAS1 protein 10x stock : 10-$-10-6 M"cold"EPAS 1 supplemented with 200, 000- 250, 000 cpm of labeled EPAS1 (Beckman counter). Place in the 4°C microfridge during screening.

-Protease inhibitor cocktail (1000X): 10 mg Trypsin Inhibitor (BMB # 109894), 10 mg Aprotinin (BMB # 236624), 25 mg Benzamidine (Sigma # B-6506), 25 mg Leupeptin (BMB # 1017128), 10 mg APMSF (BMB &num 917575), and 2mM NaVo3 (Sigma &num S-6508) in 10 ml of PBS.

-ARNT : 10-'-10-5 M biotinylated ARNT in PBS.

B. Preparation of assay plates : -Coat with 120 ul of stock N-Avidin per well overnight at 4°C.

-Wash 2 times with 200 1ll PBS.

-Block with 150 µl of blocking buffer.

-Wash 2 times with 200 u. l PBS.

C. Assay : -Add 40 µl assay buffer/well.

-Add 10 µl compound or extract.

-Add 10 gl"P-EPAS 1 protein (20-25, 000 cpm/0. 1-10 pmoles/well =10-''-10-'M final conc).

-Shake at 25°C for 15 minutes.

-Incubate additional 45 minutes at 25°C.

-Add 40 ul biotinylated hTFII subunit (0. 1-10 pmoles/40 ul in assay buffer) -Incubate 1 hour at room temperature.

-Stop the reaction by washing 4 times with 200 µl PBS.

-Add 150 1ll scintillation cocktail.

-Count in Topcount.

D. Controls for all assays (located on each plate) : a. Non-specific binding b. Soluble (non-biotinylated EPAS1) at 80% inhibition.

2. Protocol for high throughput human EPAS 1/ARNT-DNA complex formation assay.

A. Reagents : -Neutralite Avidin : 20 µg/ml in PBS.

-Blocking buffer : 5% BSA, 0. 5% Tween 20 in PBS ; 1 hour at room temperature.

-Assav Buffer : 100 mM KCI, 20 mM HEPES pH 7. 6, 1 mM MgCl2, 1% glycerol, 0. 5% NP-40, 50 mM P-mercaptoethanol, 1 mg/ml BSA, cocktail of protease inhibitors.

-33P human EPAS1 protein 10x stock : 10-8-10-6M"cold"human EPAS1 subunit (plu5) supplemented with 200, 000-250, 000 cpm of labeled human EPAS1 (Beckman counter). Place in the 4°C microfridge during screening.

-Protease inhibitor cocktail (1000X) : 10 mg Trypsin Inhibitor (BMB # 109894), 10 mg Aprotinin (BMB # 236624), 25 mg Benzamidine (Sigma # B-6506), 25 mg Leupeptin (BMB # 1017128), 10 mg APMSF (BMB # 917575), and 2mM NaVo3 (Sigma # S-6508) in 10 ml of PBS.

-DNA : 10-7-10-4M biotinylated DNA (SEQ ID NO : 3) in PBS.

-ARNT protein : 10-'-10-'M ARNT in PBS.

B. Preparation of assay plates : -Coat with 120 u. l of stock N-Avidin per well overnight at 4°C.

-Wash 2 times with 200 pl PBS.

-Block with 150 u. l of blocking buffer.

-Wash 2 times with 200 ul PBS.

C. Assay : -Add 40 u. l assay buffer/well.

-Add 10 zip compound or extract.

-Add 10 p133P-h EPAS 1 protein (20-25, 000 cpm/0. 1-10 pmoles/well =10-'-lor'M final).

-Add 10p1 ARNT protein.

-Shake at 25°C for 15 minutes.

-Incubate additional 45 minutes at 25°C.

-Add 40 ul biotinylated DNA (0. 1-10 pmoles/40 ul in assay buffer) -Incubate 1 hour at room temperature.

-Stop the reaction by washing 4 times with 200 µl PBS.

-Add 150 u. l scintillation cocktail.

-Count in Topcount.

D. Controls for all assays (located on each plate) :

a. Non-specific binding b. Soluble (non-biotinylated EPAS1/ARNT combination) at 80% inhibition.

All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

TABLE 1<BR> 1 MTAD---KEKKRSSSERRKEKSRDAARCRRSKETEVFYELAHELPLPHSVSSHLDKASIM RLEISFLRTHKLLSSVCSENESEAEA<BR> 1 MTAD---KEKKRSSSELRKEKSRDAARCRRSKETEVFYELAHELPLPHSVSSHLDKASIM RLAISFLRTHKLLSSVCSENESEAEA<BR> 1 MEGAGGANDKKKISSERRKEKSRDAARCRRSKETEVFYELAHQLPLPHSVSSHLDKASIM RLAISFLRTHKLLDA--GDLDIEDD@<BR> 1 M------------SSERRKEKSRDAARSRRTKESEVFYELAHQLPLPHNVSSHLDKASVM RLTISYLRVRKLLDA--GGLDSEDEE@<BR> 88 DNLYLKALECFIAVVTQDGDMIFLSENISKFMGLTQVELTGHSIFDFTHPCDHEEIRENL SLKNGSGFGKKSKDMSTERDFFMRM@<BR> 88 DNLYLKALEGFIAVVTQDGDMIFLSENISKFMGLTQVELTGHSIFDFTHPCDHEEIRENL TLKNGSGFGKKSKDVSTERDFFMRM@<BR> 89 NCFYLKALEGFVMVLTQDGDMIYISENISKFMGLTQVELTGHSVFDFTHPCDHEEMREML THRNGLV--KKGKEQNTQRSFFLRM@<BR> 77 DCFYLKALEGFVMVLTQDGDMVYISENISKFMGLTQVELTGHSVFDFTHPCDHEEMREML THRNGPV--KKGKELNTQRSFFLRM@<BR> 178 NRGRTVNLKSATWK-VLHCTGQVKVYNNCPPHNSLCGYKEPLLSCLIIMCEPIQUPSHMD IPLDSKTFLSRHSMDMKFTYCDDRI@<BR> 178 NRGRTVNLKSATWKSVLHCTGQVKVYNNCPPHSSLCGYKEPLLSCLIIMCEPIQUPSHMD IPLDSKTFLSRHSMDMKFTTCDDRI@<BR> 177 SRGRTMNIKSATWK-VLHCTGHIHVYDT-NSNQPQCGYKKPPMTCLVLICEPIPHPSNIE IPLDSKTFLSRHSLDMKFSYCDERI@<BR> 165 SRGRTMNIKSATWK-VLHCTGHIHVYDT-NSNQPQCGYKKPPMTCLVLICEPIPHPSNIE IPLDSKTFLSRHSLDMKFSYCDERI@<BR> 267 YHPEELLGRSAYEFYHALDSENMTKSHQULCTKGQVVSGQYRMLAKHGGYVWLETQGTVI YNPRNLQPQCIMCVNYVLSEIEKND@<BR> 268 YHPEELLGRSAYEFYHALDSENMTKSHQULCTKGQVVSGQYRMLAKHGGYVWLETQGTVI YNPRNLQPQCIMCVNYVLSEIEKND@<BR> 265 YEPEELLGRSIYEYYHALDSDHLTKTHHDMFTKGQVTTGQYRMLAKRGGYVWVETQATVI YNTKNSQPQCIVCVNYVVSGIIQHD@<BR> 253 YEPEELLGRSIYEYYHALDSDHLTKTHHDMFTKGQVTTGQYRMLAKRGGYVWVETQATVI YNTKNSQPQCIVCVNYVVSGIIQHD@<BR> 357 DQTESLFKP---HLMAMNSIFDSSGKGAVSEKSNFLFTKLKWWPEELAQLAPTPGDAIIS LDFGN-------QNFEESSAYGKAII<BR> 358 DQTELSFKP---HLMAMNSIFDSSDDVAVTEKSNYLFTKLFEEPEELAQLAPTPGDAIIS LDFGS-------QNFDEPSAYGKAII<BR> 355 QQTECVLKPVESSDMKMTQLFTKVE----SEDTSSLFDKLFKEPDALTLLAPAAGDTIIS LDFGSNDTETDDQQLEEVPLYNDVMI<BR> 343 QQTECVLKPVESSDMKMTQLFTKVE----SEDTSSLFDKLFKEPDALTLLAPAAGDTIIS LDFGSDDTETEDQQLEDVPLYNDVMI<BR> 437 -------------PWATE---LRSHST-----------QSEAGSLP-AFTVPQAAAPGST TPSATSSSSSCSTPNSPEDYYTSLD@<BR> 438 -------------PWVSG---LRSHSA-----------QSESGSLP-AFTVPQADTPGNT TPSA-SSSSSCSTPSSPEDYYSSLE@<BR> 441 EKLQNINLAMSPLPTAETPKPLRSSADPALNQEVALKLEPNPESLELSFTMPQIQDQTPS PSDG-STRQSSPEPNSPSEYCFYVD@<BR> 429 EKL-NINLAMSPLPSSETPKPLRSSADPALNQEVALKLESSPESLGLSFTMPQIQDQPAS PSDG-STRQSSPEPNSPSEYCFDVS@ TABLE 1-continued<BR> 497 --KIEVIEKLFAMDTEAKDPGSTQTDFNELDLETLAPYIPMDGEDFQLSPICPEERLLAE NPQS---TPQHCFSA--MTNIFQPL-<BR> 497 --KIEVIEKLFAMDTEPRDPGSTQTDFNELDLETLAPYIPMDGEDFQLSPICPEERLMPE SPQP---TPQHCFSA--MTNIFQPL-<BR> 530 EFKLELVEKLFAEDTEAKNPFSTQD--TDLDLEMLAPYIPMD-DDGQLRSFDQLSPLESS SASPESASPQSTVTVFQQTQIQEPT-<BR> 517 VFKLELVEKLFAEDTEAKNPFSTQD--TDLDLEMLAPYIPMD-DDGQLRSFDQLSPLESS SASP---PSMSTVTGFQQTQIQKPTI<BR> 579 PHSPFLLDKRQQQLESKKTEPEHRPMSSIFFDAGSKASLPPCCGQASTPLSSMGGRSNTQ WPPDPPLHFGPTKWAVGDQRTEFLGA<BR> 579 THGPFFLDKYPQQLESRKTESEHWPMSSIFFDAGSKGSLSPCCGQASTPLSSMGGRSNTQ WPPDPPLHFGPTKWPVGDQSAESLGA<BR> 616 TTTA-------TTDELKTVTKDRMEDIKILIASPSPTHIH----KETTSATSSPYRDTQS RTASP-----------NRAGKGVIEQ<BR> 601 TTTA-------TTDESKTETKDRMEDIKILIASPSSTQVP----QETTTAKASAYSGTHS RTASP-----------DRAGKRVIEQ<BR> 669 P-----PVSPP-HVSTFKTRSAKGFGARGPDVLSPAMVALSNKLKLKRQLEYEEQAFQDL SGG---DPPG--GSTSHLMWKRMKNL<BR> 669 SWQLELPSAPL-HVSMFKMRSAKDFGARGPYMMSPAMIALSNKLKLKRQLEYEEQAFQDT SGG---DPPG--TSSSHLMWKRMKSL<BR> 684 H-----PRSPNVLSVALSQRTTVP-----EEELNPKILALQNAQR-KRKMEHDGSLFQAV GIGTLLQQPDDHAATTSLSWKRVKGC<BR> 669 H-----PRSLN-LSATLNQRNTVP-----EEEINPKTIASQNAQR-KRKMEHDGSLFQAV GIGTLLQQPGDCAPTMSLSWKRVKGF<BR> 748 CPLMPDKPLSANVPNDKFTQNPMRGLGHPLRHLPLPQPPSAISPGENSKSRFPPQCYATQ YQDYSLSSAHKVSGMASRLLGPSFES<BR> 753 CPLMPDKTISANMAPDEFTQKSMRGLGQPLRHLPPPQPPSTRSSGENAKTGFPPQCYASQ FQDYGPPGAQKVSGVASRLLGPSFEP<BR> 761 ---------------------------------------SEQNGMEQKTIILIP------ ------------SDLACRLLGQSMDE<BR> 745 ---------------------------------------SEQNGTEQKTIILIP------ ------------SDLACRLLGQSMDV<BR> 838 ELTRYDCEVNVPVLGSSTLLQCGDLLRALDQAT<BR> 843 ELTRYDCEVVPVPGSSTLLLQCRDLLRALDQAT<BR> 794 QLTSYDCEVNAPIQGSRNLLQCEELLRALDQVN<BR> 778 QLTSYDCEVNAPIQGSRNLLQCEELLRALDQVN

SEQUENCE LISTING (1) GENERAL INFORMATION : (i) APPLICANT : McKnight, Steven L.

Russell, David W.

Tian, Hui (ii) TITLE OF INVENTION : Endothelial PAS Domain Protein (iii) NUMBER OF SEQUENCES : 7 (iv) CORRESPONDENCE ADDRESS : (A) ADDRESSEE : SCIENCE & TECHNOLOGY LAW GROUP (B) STREET : 268 BUSH STREET, SUITE 3200 (C) CITY : SAN FRANCISCO (D) STATE : CALIFORNIA (E) COUNTRY : USA (F) ZIP : 94104 (v) COMPUTER READABLE FORM : (A) MEDIUM TYPE : Floppy disk (B) COMPUTER : IBM PC compatible (C) OPERATING SYSTEM : PC-DOS/MS-DOS (D) SOFTWARE : PatentIn Release #1. 0, Version #1. 30 (vi) CURRENT APPLICATION DATA : (A) APPLICATION NUMBER : US 08/785, 241 (B) FILING DATE : 17-JAN-1997 (C) CLASSIFICATION : (viii) ATTORNEY/AGENT INFORMATION : (A) NAME : OSMAN, RICHARD A (B) REGISTRATION NUMBER : 36, 627 (C) REFERENCE/DOCKET NUMBER : UTSD : 1229 (ix) TELECOMMUNICATION INFORMATION : (A) TELEPHONE : (415) 343-4341 (B) TELEFAX : (415) 343-4342 (2) INFORMATION FOR SEQ ID NO : 1 : (i) SEQUENCE CHARACTERISTICS : (A) LENGTH : 2816 base pairs (B) TYPE : nucleic acid (C) STRANDEDNESS : double (D) TOPOLOGY : linear (ii) MOLECULE TYPE : cDNA

(xi) SEQUENCE DESCRIPTION : SEQ ID NO : 1 : CCTGACTGCG CGGGGCGCTC GGGACCTGCG CGCACCTCGG ACCTTCACCA CCCGCCCGGG 60 CCGCGGGGAG CGGACGAGGG CCACAGCCCC CCACCCGCCA GGGAGCCCAG GTGCTCGGCG 120 TCTGAACGTC TCAAAGGGCC ACAGCGACAA TGACAGCTGA CAAGGAGAAG AAAAGGAGTA 180 GCTCGGAGAG GAGGAAGGAG AAGTCCCGGG ATGCTGCGCG GTGCCGGCGG AGCAAGGAGA 240 CGGAGGTGTT CTATGAGCTG GCCCATGAGC TGCCTCTGCC CCACAGTGTG AGCTCCCATC 300 TGGACAAGGC CTCCATCATG CGACTGGAAA TCAGCTTCCT GCGAACACAC AAGCTCCTCT 360 CCTCAGTTTG CTCTGAAAAC GAGTCCGAAG CCGAAGCTGA CCAGCAGATG GACAACTTGT 420 ACCTGAAAGC CTTGGAGGGT TTCATTGCCG TGGTGACCCA AGATGGCGAC ATGATCTTTC 480 TGTCAGAAAA CATCAGCAAG TTCATGGGAC TTACACAGGT GGAGCTAACA GGACATAGTA 540 TCTTTGACTT CACTCATCCC TGCGACCATG AGGAGATTCG TGAGAACCTG AGTCTCAAAA 600 ATGGCTCTGG TTTTGGGAAA AAAAGCAAAG ACATGTCCAC AGAGCGGGAC TTCTTCATGA 660 GGATGAAGTG CACGGTCACC AACAGAGGCC GTACTGTCAA CCTCAAGTCA GCCACCTGGA 720 AGGTCTTGCA CTGCACGGGC CAGGTGAAAG TCTACAACAA CTGCCCTCCT CACAATAGTC 780 TGTGTGGCTA CAAGGAGCCC CTGCTGTCCT GCCTCATCAT CATGTGTGAA CCAATCCAGC 840 ACCCATCCCA CATGGACATC CCCCTGGATA GCAAGACCTT CCTGAGCCGC CACAGCATGG 900 ACATGAAGTT CACCTACTGT GATGACAGAA TCACAGAACT GATTGGTTAC CACCCTGAGG 960 AGCTGCTTGG CCGCTCAGCC TATGAATTCT ACCATGCGCT AGACTCCGAG AACATGACCA 1020 AGAGTCACCA GAACTTGTGC ACCAAGGGTC AGGTAGTAAG TGGCCAGTAC CGGATGCTCG 1080 CAAAGCATGG GGGCTACGTG TGGCTGGAGA CCCAGGGGAC GGTCATCTAC AACCCTCGCA 1140 ACCTGCAGCC CCAGTGCATC ATGTGTGTCA ACTACGTCCT GAGTGAGATT GAGAAGAATG 1200 ACGTGGTGTT CTCCATGGAC CAGACTGAAT CCCTGTTCAA GCCCCACCTG ATGGCCATGA 1260 ACAGCATCTT TGATAGCAGT GGCAAGGGGG CTGTGTCTGA GAAGAGTAAC TTCCTATTCA 1320 CCAAGCTAAA GGAGGAGCCC GAGGAGCTGG CCCAGCTGGC TCCCACCCCA GGAGACGCCA 1380 TCATCTCTCT GGATTTCGGG AATCAGAACT TCGAGGAGTC CTCAGCCTAT GGCAAGGCCA 1440 TCCTGCCCCC GAGCCAGCCA TGGGCCACGG AGTTGAGGAG CCACAGCACC CAGAGCGAGG 1500 CTGGGAGCCT GCCTGCCTTC ACCGTGCCCC AGGCAGCTGC CCCGGGCAGC ACCACCCCCA 1560 GTGCCACCAG CAGCAGCAGC AGCTGCTCCA CGCCCAATAG CCCTGAAGAC TATTACACAT 1620 CTTTGGATAA CGACCTGAAG ATTGAAGTGA TTGAGAAGCT CTTCGCCATG GACACAGAGG 1680 CCAAGGACCA ATGCAGTACC CAGACGGATT TCAATGAGCT GGACTTGGAG ACACTGGCAC 1740 CCTATATCCC CATGGACGGG GAAGACTTCC AGCTAAGCCC CATCTGCCCC GAGGAGCGGC 1800 TCTTGGCGGA GAACCCACAG TCCACCCCCC AGCACTGCTT CAGTGCCATG ACAAACATCT 1860 TCCAGCCACT GGCCCCTGTA GCCCCGCACA GTCCCTTCCT CCTGGACAAG TTTCAGCAGC 1920 AGCTGGAGAG CAAGAAGACA GAGCCCGAGC ACCGGCCCAT GTCCTCCATC TTCTTTGATG 1980 CCGGAAGCAA AGCATCCCTG CCACCGTGCT GTGGCCAGGC CAGCACCCCT CTCTCTTCCA 2040 TGGGGGGCAG ATCCAATACC CAGTGGCCCC CAGATCCACC ATTACATTTT GGGCCCACAA 2100 AGTGGGCCGT CGGGGATCAG CGCACAGAGT TCTTGGGAGC AGCGCCGTTG GGGCCCCCTG 2160 TCTCTCCACC CCATGTCTCC ACCTTCAAGA CAAGGTCTGC AAAGGGTTTT GGGGCTCGAG 2220

GCCCAGACGT GCTGAGTCCG GCCATGGTAG CCCTCTCCAA CAAGCTGAAG CTGAAGCGAC 2280 AGCTGGAGTA TGAAGAGCAA GCCTTCCAGG ACCTGAGCGG GGGGGACCCA CCTGGTGGCA 2340 GCACCTCACA TTTGATGTGG AAACGGATGA AGAACCTCAG GGGTGGGAGC TGCCCTTTGA 2400 TGCCGGACAA GCCACTGAGC GCAAATGTAC CCAATGATAA GTTCACCCAA AACCCCATGA 2460 GGGGCCTGGG CCATCCCCTG AGACATCTGC CGCTGCCACA GCCTCCATCT GCCATCAGTC 2520 CCGGGGAGAA CAGCAAGAGC AGGTTCCCCC CACAGTGCTA CGCCACCCAG TACCAGGACT 2580 ACAGCCTGTC GTCAGCCCAC AAGGTGTCAG GCATGGCAAG CCGGCTGCTC GGGCCCTCAT 2640 TTGAGTCCTA CCTGCTGCCC GAACTGACCA GATATGACTG TGAGGTGAAC GTGCCCGTGC 2700 TGGGAAGCTC CACGCTCCTG CAAGGAGGGG ACCTCCTCAG AGCCCTGGAC CAGGCCACCT 2760 GAGCCAGGCC TTCTACCTGG GCAGCACCTC TGCCGACGCC GTCCCACCAG CTTCAC 2816 (2) INFORMATION FOR SEQ ID NO : 2 : (i) SEQUENCE CHARACTERISTICS : (A) LENGTH : 3031 base pairs (B) TYPE : nucleic acid (C) STRANDEDNESS : double (D) TOPOLOGY : linear (ii) MOLECULE TYPE : cDNA (xi) SEQUENCE DESCRIPTION : SEQ ID NO : 2 : CGACAGAGAG CTGCGGAGGG CCACAGCAAA GAGAGCGGCT GCAGCCCCTA CGGGGTTAAG 60 GAACCCAGGT GCTCCGGGTC TCGGAGGGCC ACGGCGACAA TGACAGCTGA CAAGGAGAAA 120 AAAAGGAGCA GCTCAGAGCT GAGGAAGGAG AAATCCCGTG ATGCCGCGAG GTGCCGGCGC 180 AGCAAGGAGA CGGAGGTCTT CTATGAGTTG GCTCATGAGT TGCCCCTGCC TCACAGTGTG 240 AGCTCCCACC TGGACAAAGC CTCCATCATG CGCCTGGCCA TCAGCTTCCT TCGGACACAT 300 AAGCTCCTGT CCTCAGTCTG CTCTGAAAAT GAATCTGAAG CTGAGGCCGA CCAGCAAATG 360 GATAACTTGT ACCTGAAAGC CTTGGAGGGT TTCATTGCTG TGGTGACCCA AGACGGTGAC 420 ATGATCTTTC TGTCGGAAAA CATCAGCAAG TTCATGGGAC TTACTCAGGT AGAACTAACA 480 GGACACAGCA TCTTTGACTT CACTCATCCT TGCGACCATG AAGAGATCCG TGAGAACCTG 540 ACTCTCAAAA ACGGCTCTGG TTTTGGGAAG AAGAGCAAAG ACGTGTCCAC CGAGCGTGAC 600 TTCTTCATGA GGATGAAGTG CACGGTCACC AACAGAGGCC GGACTGTCAA CCTCAAGTCG 660 GCCACCTGGA AGTCCGTCCT GCACTGCACC GGGCAAGTGA GAGTCTACAA CAACTGCCCC 720 CCTCACAGTA GCCTCTGTGG CTCCAAGGAG CCCCTGCTGT CCTGCCTTAT CATCATGTGT 780 GAGCCAATCC AGCACCCATC CCACATGGAC ATCCCCCTGG ACAGCAAGAC TTTCCTGAGC 840 CGCCACAGCA TGGACATGAA GTTCACCTAC TGTGACGACA GAATCTTGGA ACTGATTGGT 900 TACCACCCCG AGGAGCTACT TGGACGCTCT GCCTATGAGT TTTACCATGC CCTGGATTCG 960 GAGAACATGA CCAAAAGTCA CCAGAACTTG TGCACCAAGG GGCAGGTGGT ATCTGGCCAG 1020 TACCGGATGC TAGCCAAACA CGGAGGATAT GTGTGGCTGG AGACCCAGGG GACGGTCATC 1080 TACAACCCCC GCAACCTGCA GCCTCAGTGT ATCATGTGTG TCAACTATGT GCTGAGTGAG 1140

ATCGAGAAGA ACGACGTGGT GTTCTCCATG GACCAGACCG AATCCCTGTT CAAGCCACAC 1200 CTGATGGCCA TGAACAGCAT CTTTGACAGC AGTGACGATG TGGCTGTAAC TGAGAAGAGC 1260 AACTACCTGT TCACCAAACT GAAGGAGGAG CCCGAGGAAC TGGCCCAGTT GGCCCCCACC 1320 CCAGGAGATG CCATTATTTC TCTCGATTTC GGAAGCCAGA ACTTCGATGA ACCCTCAGCC 1380 TATGGCAAGG CCATCCTTCC CCCGGGCCAG CCATGGGTCT CGGGGCTGAG GAGCCACAGT 1440 GCCCAGAGCG AGTCCGGGAG CCTGCCAGCC TTCACTGTGC CCCAGGCAGA CACCCCAGGG 1500 AACACTACAC CCAGTGCTTC AAGCAGCAGT AGCTGCTCCA CGCCCAGCAG CCCTGAGGAC 1560 TACTATTCAT CCTTGGAGAA TCCCTTGAAG ATCGAAGTGA TTGAGAAGCT TTTCGCCATG 1620 GACACGGAGC CGAGGGACCC GGGCAGTACC CAGACGGACT TCAGTGAACT GGATTTGGAG 1680 ACCTTGGCAC CCTACATCCC TATGGACGGC GAGGACTTCC AGCTGAGCCC CATCTGCCCA 1740 GAGGAGCCGC TCATGCCAGA GAGCCCCCAG CCCACCCCCC AGCACTGCTT CAGTACCATG 1800 ACCAGCATCT TCCAGCCGCT CACCCCGGGG GCCACCCACG GCCCCTTCTT CCTCGATAAG 1860 TACCCGCAGC AGTTGGAAAG CAGGAAGACA GAGTCTGAGC ACTGGCCCAT GTCTTCCATC 1920 TTCTTTGATG CTGGGAGCAA AGGGTCCCTG TCTCCATGCT GTGGCCAGGC CAGCACCCCT 1980 CTCTCTTCTA TGGGAGGCAG ATCCAACACG CAGTGGCCCC CGGATCCACC ATTACATTTC 2040 GGCCCTACTA AGTGGCCTGT GGGTGATCAG AGTGCTGAAT CCCTGGGAGC CCTGCCGGTG 2100 GGGTCATGGC AGTTGGAACT TCCGAGCGCC CCGCTTCATG TCTCCATGTT CAAGATGAGG 2160 TCTGCAAAGG ACTTCGGGGC CCGAGGTCCA TACATGATGA GCCCAGCCAT GATCGCCCTG 2220 TCCAACAAGC TGAAGCTAAA GCGGCAGCTG GAGTATGAGG AGCAAGCCTT CCAAGACACA 2280 AGCGGGGGGG ACCCTCCAGG CACCAGCAGT TCACACTTGA TGTGGAAACG TATGAAGAGC 2340 CTCATGGGCG GGACCTGTCC TTTGATGCCT GACAAGACCA TCAGTGCGAA CATGGCCCCC 2400 GATGAATTCA CCCAAAAATC TATGAGAGGC CTGGGCCAGC CACTGAGACA CCTGCCACCT 2460 CCCCAGCCAC CATCTACCAG GAGCTCAGGG GAGAACGCCA AGACTGGGTT CCCGCCACAG 2520 TGCTATGCCT CCCAGTTCCA GGACTACGGT CCTCCAGGAG CTCAAAAGGT GTCAGGCGTG 2580 GCCAGTCGAC TGCTGGGGCC ATCGTTCGAG CCTTACCTGT TGCCGGAACT GACCAGATAT 2640 GACTGTGAGG TGAACGTGCC CGTGCCTGGA AGCTCCACAC TCCTGCAGGG GAGAGACCTT 2700 CTCAGAGCTC TGGACCAGGC CACCTGAGCC AGGGCCTCTG GCCGGGCATG CCCCTGCCTG 2760 CCCCGCCGTC TTGACCTGCC AGCTTCACTT CCATCTGTGT TGCTATTAGG TATCTCTAAC 2820 ACCAGCACAC TTCTTACGAG ATGTACTCAA CCTGGCCTAC TGGCCAGGTC ACCAAGCAGT 2880 GGCCTTTATC TGACATGCTC ACTTTATTAT CCATGTTTTA AAAATACATA GTTGTTGTAC 2940 CTGCTATGTT TTACCGTTGA TGAAAGTGTT CTGAAATTTT ATAAGATTTC CCCCTCCCTC 3000 CCTCCCTTGA ATTACTTCTA ATTTATATTC C 3031 (2) INFORMATION FOR SEQ ID NO : 3 : (i) SEQUENCE CHARACTERISTICS : (A) LENGTH : 18 base pairs (B) TYPE : nucleic acid (C) STRANDEDNESS- : double

(D) TOPOLOGY : linear (ii) MOLECULE TYPE : cDNA (xi) SEQUENCE DESCRIPTION : SEQ ID NO : 3 : GCCCTACGTG CTGTCTCA 18 (2) INFORMATION FOR SEQ ID NO : 4 : (i) SEQUENCE CHARACTERISTICS : (A) LENGTH : 870 amino acids (B) TYPE : amino acid (C) STRANDEDNESS : single (D) TOPOLOGY : linear (ii) MOLECULE TYPE : peptide (xi) SEQUENCE DESCRIPTION : SEQ ID NO : 4 : Met Thr Ala Asp Lys Glu Lys Lys Arg Ser Ser Ser Glu Arg Arg Lys 1 5 10 15 Glu Lys Ser Arg Asp Ala Ala Arg Cys Arg Arg Ser Lys Glu Thr Glu 20 25 30 Val Phe Tyr Glu Leu Ala His Glu Leu Pro Leu Pro His Ser Val Ser 35 40 45 Ser His Leu Asp Lys Ala Ser Ile Met Arg Leu Glu Ile Ser Phe Leu 50 55 60 Arg Thr His Lys Leu Leu Ser Ser Val Cys Ser Glu Asn Glu Ser Glu 65 70 75 80 Ala Glu Ala Asp Gln Gln Met Asp Asn Leu Tyr Leu Lys Ala Leu Glu 85 90 95 Gly Phe Ile Ala Val Val Thr Gln Asp Gly Asp Met Ile Phe Leu Ser 100 105 110 Glu Asn Ile Ser Lys Phe Met Gly Leu Thr Gln Val Glu Leu Thr Gly 115 120 125 His Ser Ile Phe Asp Phe Thr His Pro Cys Asp His Glu Glu Ile Arg 130 135 140 Glu Asn Leu Ser Leu Lys Asn Gly Ser Gly Phe Gly Lys Lys Ser Lys 145 150 155 160 Asp Met Ser Thr Glu Arg Asp Phe Phe Met Arg Met Lys Cys Thr Val 165 170 175 Thr Asn Arg Gly Arg Thr Val Asn Leu Lys Ser Ala Thr Trp Lys Val 180 185 190 Leu His Cys Thr Gly Gln Val Lys Val Tyr Asn Asn Cys Pro Pro His

195 200 205 Asn Ser Leu Cys Gly Tyr Lys Glu Pro Leu Leu Ser Cys Leu Ile Ile 210 215 220 Met Cys Glu Pro Ile Gln His Pro Ser His Met Asp Ile Pro Leu Asp 225 230 235 240 Ser Lys Thr Phe Leu Ser Arg His Ser Met Asp Met Lys Phe Thr Tyr 245 250 255 Cys Asp Asp Arg Ile Thr Glu Leu Ile Gly Tyr His Pro Glu Glu Leu 260 265 270 Leu Gly Arg Ser Ala Tyr Glu Phe Tyr His Ala Leu Asp Ser Glu Asn 275 280 285 Met Thr Lys Ser His Gln Asn Leu Cys Thr Lys Gly Gln Val Val Ser 290 295 300 Gly Gln Tyr Arg Met Leu Ala Lys His Gly Gly Tyr Val Trp Leu Glu 305 310 315 320 Thr Gln Gly Thr Val Ile Tyr Asn Pro Arg Asn Leu Gln Pro Gln Cys 325 330 335 Ile Met Cys Val Asn Tyr Val Leu Ser Glu Ile Glu Lys Asn Asp Val 340 345 350 Val Phe Ser Met Asp Gln Thr Glu Ser Leu Phe Lys Pro His Leu Met 355 360 365 Ala Met Asn Ser Ile Phe Asp Ser Ser Gly Lys Gly Ala Val Ser Glu 370 375 380 Lys Ser Asn Phe Leu Phe Thr Lys Leu Lys Glu Glu Pro Glu Glu Leu 385 390 395 400 Ala Gln Leu Ala Pro Thr Pro Gly Asp Ala Ile Ile Ser Leu Asp Phe 405 410 415 Gly Asn Gln Asn Phe Glu Glu Ser Ser Ala Tyr Gly Lys Ala Ile Leu 420 425 430 Pro Pro Ser Gln Pro Trp Ala Thr Glu Leu Arg Ser His Ser Thr Gln 435 440 445 Ser Glu Ala Gly Ser Leu Pro Ala Phe Thr Val Pro Gln Ala Ala Ala 450 455 460 Pro Gly Ser Thr Thr Pro Ser Ala Thr Ser Ser Ser Ser Ser Cys Ser 465 470 475 480 Thr Pro Asn Ser Pro Glu Asp Tyr Tyr Thr Ser Leu Asp Asn Asp Leu 485 490 495 Lys Ile Glu Val Ile Glu Lys Leu Phe Ala Met Asp Thr Glu Ala Lys

500 505 510 Asp Gln Cys Ser Thr Gln Thr Asp Phe Asn Glu Leu Asp Leu Glu Thr 515 520 525 Leu Ala Pro Tyr Ile Pro Met Asp Gly Glu Asp Phe Gln Leu Ser Pro 530 535 540 Ile Cys Pro Glu Glu Arg Leu Leu Ala Glu Asn Pro Gln Ser Thr Pro 545 550 555 560 Gln His Cys Phe Ser Ala Met Thr Asn Ile Phe Gln Pro Leu Ala Pro 565 570 575 Val Ala Pro His Ser Pro Phe Leu Leu Asp Lys Phe Gln Gln Gln Leu 580 585 590 Glu Ser Lys Lys Thr Glu Pro Glu His Arg Pro Met Ser Ser Ile Phe 595 600 605 Phe Asp Ala Gly Ser Lys Ala Ser Leu Pro Pro Cys Cys Gly Gln Ala 610 615 620 Ser Thr Pro Leu Ser Ser Met Gly Gly Arg Ser Asn Thr Gln Trp Pro 625 630 635 640 Pro Asp Pro Pro Leu His Phe Gly Pro Thr Lys Trp Ala Val Gly Asp 645 650 655 Gln Arg Thr Glu Phe Leu Gly Ala Ala Pro Leu Gly Pro Pro Val Ser 660 665 670 Pro Pro His Val Ser Thr Phe Lys Thr Arg Ser Ala Lys Gly Phe Gly 675 680 685 Ala Arg Gly Pro Asp Val Leu Ser Pro Ala Met Val Ala Leu Ser Asn 690 695 700 Lys Leu Lys Leu Lys Arg Gln Leu Glu Tyr Glu Glu Gln Ala Phe Gln 705 710 715 720 Asp Leu Ser Gly Gly Asp Pro Pro Gly Gly Ser Thr Ser His Leu Met 725 730 735 Trp Lys Arg Met Lys Asn Leu Arg Gly Gly Ser Cys Pro Leu Met Pro 740 745 750 Asp Lys Pro Leu Ser Ala Asn Val Pro Asn Asp Lys Phe Thr Gln Asn 755 760 765 Pro Met Arg Gly Leu Gly His Pro Leu Arg His Leu Pro Leu Pro Gln 770 775 780 Pro Pro Ser Ala Ile Ser Pro Gly Glu Asn Ser Lys Ser Arg Phe Pro 785 790 795 800 Pro Gln Cys Tyr Ala Thr Gln Tyr Gln Asp Tyr Ser Leu Ser Ser Ala

805 810 815 His Lys Val Ser Gly Met Ala Ser Arg Leu Leu Gly Pro Ser Phe Glu 820 825 830 Ser Tyr Leu Leu Pro Glu Leu Thr Arg Tyr Asp Cys Glu Val Asn Val 835 840 845 Pro Val-Leu Gly Ser Ser Thr Leu Leu Gln Gly Gly Asp Leu Leu Arg 850 855 860 Ala Leu Asp Gln Ala Thr 865 870 (2) INFORMATION FOR SEQ ID NO : 5 : (i) SEQUENCE CHARACTERISTICS : (A) LENGTH : 875 amino acids (B) TYPE : amino acid (C) STRANDEDNESS : single (D) TOPOLOGY : linear (ii) MOLECULE TYPE : peptide (xi) SEQUENCE DESCRIPTION : SEQ ID NO : 5 : Met Thr Ala Asp Lys Glu Lys Lys Arg Ser Ser Ser Glu Leu Arg Lys 1 5 10 15 Glu Lys Ser Arg Asp Ala Ala Arg Cys Arg Arg Ser Lys Glu Thr Glu 20 25 30 Val Phe Tyr Glu Leu Ala His Glu Leu Pro Leu Pro His Ser Val Ser 35 40 45 Ser His Leu Asp Lys Ala Ser Ile Met Arg Leu Ala Ile Ser Phe Leu 50 55 60 Arg Thr His Lys Leu Leu Ser Ser Val Cys Ser Glu Asn Glu Ser Glu 65 70 75 80 Ala Glu Ala Asp Gln Gln Met Asp Asn Leu Tyr Leu Lys Ala Leu Glu 85 90 95 Gly Phe Ile Ala Val Val Thr Gln Asp Gly Asp Met Ile Phe Leu Ser 100 105 110 Glu Asn Ile Ser Lys Phe Met Gly Leu Thr Gln Val Glu Leu Thr Gly 115 120 125 His Ser Ile Phe Asp Phe Thr His Pro Cys Asp His Glu Glu Ile Arg 130 135 140 Glu Asn Leu Thr Leu Lys Asn Gly Ser Gly Phe Gly Lys Lys Ser Lys 145 150 155 160

Asp Val Ser Thr Glu Arg Asp Phe Phe Met Arg Met Lys Cys Thr Val 165 170 175 Thr Asn Arg Gly Arg Thr Val Asn Leu Lys Ser Ala Thr Trp Lys Ser 180 185 190 Val Leu His Cys Thr Gly Gln Val Arg Val Tyr Asn Asn Cys Pro Pro 195 200 205 His Ser Ser Leu Cys Gly Ser Lys Glu Pro Leu Leu Ser Cys Leu Ile 210 215 220 Ile Met Cys Glu Pro Ile Gln His Pro Ser His Met Asp Ile Pro Leu 225 230 235 240 Asp Ser Lys Thr Phe Leu Ser Arg His Ser Met Asp Met Lys Phe Thr 245 250 255 Tyr Cys Asp Asp Arg Ile Leu Glu Leu Ile Gly Tyr His Pro Glu Glu 260 265 270 Leu Leu Gly Arg Ser Ala Tyr Glu Phe Tyr His Ala Leu Asp Ser Glu 275 280 285 Asn Met Thr Lys Ser His Gln Asn Leu Cys Thr Lys Gly Gln Val Val 290 295 300 Ser Gly Gln Tyr Arg Met Leu Ala Lys His Gly Gly Tyr Val Trp Leu 305 310 315 320 Glu Thr Gln Gly Thr Val Ile Tyr Asn Pro Arg Asn Leu Gln Pro Gln 325 330 335 Cys Ile Met Cys Val Asn Tyr Val Leu Ser Glu Ile Glu Lys Asn Asp 340 345 350 Val Val Phe Ser Met Asp Gln Thr Glu Ser Leu Phe Lys Pro His Leu 355 360 365 Met Ala Met Asn Ser Ile Phe Asp Ser Ser Asp Asp Val Ala Val Thr 370 375 380 Glu Lys Ser Asn Tyr Leu Phe Thr Lys Leu Lys Glu Glu Pro Glu Glu 385 390 395 400 Leu Ala Gln Leu Ala Pro Thr Pro Gly Asp Ala Ile Ile Ser Leu Asp 405 410 415 Phe Gly Ser Gln Asn Phe Asp Glu Pro Ser Ala Tyr Gly Lys Ala Ile 420 425 430 Leu Pro Pro Gly Gln Pro Trp Val Ser Gly Leu Arg Ser His Ser Ala 435 440 445 Gln Ser Glu Ser Gly Ser Leu Pro Ala Phe Thr Val Pro Gln Ala Asp 450 455 460

Thr Pro Gly Asn Thr Thr Pro Ser Ala Ser Ser Ser Ser Ser Cys Ser 465 470 475 480 Thr Pro Ser Ser Pro Glu Asp Tyr Tyr Ser Ser Leu Glu Asn Pro Leu 485 490 495 Lys Ile Glu Val Ile Glu Lys Leu Phe Ala Met Asp Thr Glu Pro Arg 500 505 510 Asp Pro Gly Ser Thr Gln Thr Asp Phe Ser Glu Leu Asp Leu Glu Thr 515 520 525 Leu Ala Pro Tyr Ile Pro Met Asp Gly Glu Asp Phe Gln Leu Ser Pro 530 535 540 Ile Cys Pro Glu Glu Pro Leu Met Pro Glu Ser Pro Gln Pro Thr Pro 545 550 555 560 Gln His Cys Phe Ser Thr Met Thr Ser Ile Phe Gln Pro Leu Thr Pro 565 570 575 Gly Ala Thr His Gly Pro Phe Phe Leu Asp Lys Tyr Pro Gln Gln Leu 580 585 590 Glu Ser Arg Lys Thr Glu Ser Glu His Trp Pro Met Ser Ser Ile Phe 595 600 605 Phe Asp Ala Gly Ser Lys Gly Ser Leu Ser Pro Cys Cys Gly Gln Ala 610 615 620 Ser Thr Pro Leu Ser Ser Met Gly Gly Arg Ser Asn Thr Gln Trp Pro 625 630 635 640 Pro Asp Pro Pro Leu His Phe Gly Pro Thr Lys Trp Pro Val Gly Asp 645 650 655 Gln Ser Ala Glu Ser Leu Gly Ala Leu Pro Val Gly Ser Trp Gln Leu 660 665 670 Glu Leu Pro Ser Ala Pro Leu His Val Ser Met Phe Lys Met Arg Ser 675 680 685 Ala Lys Asp Phe Gly Ala Arg Gly Pro Tyr Met Met Ser Pro Ala Met 690 695 700 Ile Ala Leu Ser Asn Lys Leu Lys Leu Lys Arg Gln Leu Glu Tyr Glu 705 710 715 720 Glu Gln Ala Phe Gln Asp Thr Ser Gly Gly Asp Pro Pro Gly Thr Ser 725 730 735 Ser Ser His Leu Met Trp Lys Arg Met Lys Ser Leu Met Gly Gly Thr 740 745 750 Cys Pro Leu Met Pro Asp Lys Thr Ile Ser Ala Asn Met Ala Pro Asp 755 760 765

Glu Phe Thr Gln Lys Ser Met Arg Gly Leu Gly Gln Pro Leu Arg His 770 775 780 Leu Pro Pro Pro Gln Pro Pro Ser Thr Arg Ser Ser Gly Glu Asn Ala 785 790 795 800 Lys Thr Gly Phe Pro Pro Gln Cys Tyr Ala Ser Gln Phe Gln Asp Tyr 805 810 815 Gly Pro Pro Gly Ala Gln Lys Val Ser Gly Val Ala Ser Arg Leu Leu 820 825 830 Gly Pro Ser Phe Glu Pro Tyr Leu Leu Pro Glu Leu Thr Arg Tyr Asp 835 840 845 Cys Glu Val Asn Val Pro Val Pro Gly Ser Ser Thr Leu Leu Gln Gly 850 855 860 Arg Asp Leu Leu Arg Ala Leu Asp Gln Ala Thr 865 870 875 (2) INFORMATION FOR SEQ ID NO : 6 : (i) SEQUENCE CHARACTERISTICS : (A) LENGTH : 826 amino acids (B) TYPE : amino acid (C) STRANDEDNESS : single (D) TOPOLOGY : linear (ii) MOLECULE TYPE : peptide (xi) SEQUENCE DESCRIPTION : SEQ ID NO : 6 : Met Glu Gly Ala Gly Gly Ala Asn Asp Lys Lys Lys Ile Ser Ser Glu 1 5 10 15 Arg Arg Lys Glu Lys Ser Arg Asp Ala Ala Arg Ser Arg Arg Ser Lys 20 25 30 Glu Ser Glu Val Phe Tyr Glu Leu Ala His s Gln Leu Pro Leu Pro His 35 40 45 Asn Val Ser Ser His Leu Asp Lys Ala Ser Val Met Arg Leu Thr Ile 50 55 60 Ser Tyr Leu Arg Val Arg Lys Leu Leu Asp Ala Gly Asp Leu Asp Ile 65 70 75 80 Glu Asp Asp Met Lys Ala Gln Met Asn Cys Phe Tyr Leu Lys Ala Leu 85 90 95 Asp Gly Phe Val Met Val Leu Thr Asp Asp Gly Asp Met Ile Tyr Ile 100 105 110 Ser Asp Asn Val Asn Lys Tyr Met Gly Leu Thr Gln Phe Glu Leu Thr

115 120 125 Gly His Ser Val Phe Asp Phe Thr His Pro Cys Asp His Glu Glu Met 130 135 140 Arg Glu Met Leu Thr His Arg Asn Gly Leu Val Lys Lys Gly Lys Glu 145 150 155 160 Gln Asn Thr Gln Arg Ser Phe Phe Leu Arg Met Lys Cys Thr Leu Thr 165 170 175 Ser Arg Gly Arg Thr Met Asn Ile Lys Ser Ala Thr Trp Lys Val Leu 180 185 190 His Cys Thr Gly His Ile His Val Tyr Asp Thr Asn Ser Asn Gln Pro 195 200 205 Gln Cys Gly Tyr Lys Lys Pro Pro Met Thr Cys Leu Val Leu Ile Cys 210 215 220 Glu Pro Ile Pro His Pro Ser Asn Ile Glu Ile Pro Leu Asp Ser Lys 225 230 235 240 Thr Phe Leu Ser Arg His Ser Leu Asp Met Lys Phe Ser Tyr Cys Asp 245 250 255 Glu Arg Ile Thr Glu Leu Met Gly Tyr Glu Pro Glu Glu Leu Leu Gly 260 265 270 Arg Ser Ile Tyr Glu Tyr Tyr His Ala Leu Asp Ser Asp His Leu Thr 275 280 285 Lys Thr His His Asp Met Phe Thr Lys Gly Gln Val Thr Thr Gly Gln 290 295 300 Tyr Arg Met Leu Ala Lys Arg Gly Gly Tyr Val Trp Val Glu Thr Gln 305 310 315 320 Ala Thr Val Ile Tyr Asn Thr Lys Asn Ser Gln Pro Gln Cys Ile Val 325 330 335 Cys Val Asn Tyr Val Val Ser Gly Ile Ile Gln His Asp Leu Ile Phe 340 345 350 Ser Leu Gln Gln Thr Glu Cys Val Leu Lys Pro Val Glu Ser Ser Asp 355 360 365 Met Lys Met Thr Gln Leu Phe Thr Lys Val Glu Ser Glu Asp Thr Ser 370 375 380 Ser Leu Phe Asp Lys Leu Lys Lys Glu Pro Asp Ala Leu Thr Leu Leu 385 390 395 400 Ala Pro Ala Ala Gly Asp Thr Ile Ile Ser Leu Asp Phe Gly Ser Asn 405 410 415 Asp Thr Glu Thr Asp Asp Gln Gln Leu Glu Glu Val Pro Leu Tyr Asn

420 425 430 Asp Val Met Leu Pro Ser Pro Asn Glu Lys Leu Gln Asn Ile Asn Leu 435 440 445 Ala Met Ser Pro Leu Pro Thr Ala Glu Thr Pro Lys Pro Leu Arg Ser 450 455 460 Ser Ala Asp Pro Ala Leu Asn Gln Glu Val Ala Leu Lys Leu Glu Pro 465 470 475 480 Asn Pro Glu Ser Leu Glu Leu Ser Phe Thr Met Pro Gln Ile Gln Asp 485 490 495 Gln Thr Pro Ser Pro Ser Asp Gly Ser Thr Arg Gln Ser Ser Pro Glu 500 505 510 Pro Asn Ser Pro Ser Glu Tyr Cys Phe Tyr Val Asp Ser Asp Met Val 515 520 525 Asn Glu Phe Lys Leu Glu Leu Val Glu Lys Leu Phe Ala Glu Asp Thr 530 535 540 Glu Ala Lys Asn Pro Phe Ser Thr Gln Asp Thr Asp Leu Asp Leu Glu 545 550 555 560 Met Leu Ala Pro Tyr Ile Pro Met Asp Asp Asp Phe Gln Leu Arg Ser 565 570 575 Phe Asp Gln Leu Ser Pro Leu Glu Ser Ser Ser Ala Ser Pro Glu Ser 580 585 590 Ala Ser Pro Gln Ser Thr Val Thr Val Phe Gln Gln Thr Gln Ile Gln 595 600 605 Glu Pro Thr Ala Asn Ala Thr Thr Thr Thr Ala Thr Thr Asp Glu Leu 610 615 620 Lys Thr Val Thr Lys Asp Arg Met Glu Asp Ile Lys Ile Leu Ile Ala 625 630 635 640 Ser Pro Ser Pro Thr His Ile His Lys Glu Thr Thr Ser Ala Thr Ser 645 650 655 Ser Pro Tyr Arg Asp Thr Gln Ser Arg Thr Ala Ser Pro Asn Arg Ala 660 665 670 Gly Lys Gly Val Ile Glu Gln Thr Glu Lys Ser His Pro Arg Ser Pro 675 680 685 Asn Val Leu Ser Val Ala Leu Ser Gln Arg Thr Thr Val Pro Glu Glu 690 695 700 Glu Leu Asn Pro Lys Ile Leu Ala Leu Gln Asn Ala Gln Arg Lys Arg 705 710 715 720 Lys Met Glu His Asp Gly Ser Leu Phe Gln Ala Val Gly Ile Gly Thr

725 730 735 Leu Leu Gln Gln Pro Asp Asp His Ala Ala Thr Thr Ser Leu Ser Trp 740 745 750 Lys Arg Val Lys Gly Cys Lys Ser Ser Glu Gln Asn Gly Met Glu Gln 755 760 765 Lys Thr Ile Ile Leu Ile Pro Ser Asp Leu Ala Cys Arg Leu Leu Gly 770 775 780 Gln Ser Met Asp Glu Ser Gly Leu Pro Gln Leu Thr Ser Tyr Asp Cys 785 790 795 800 Glu Val Asn Ala Pro Ile Gln Gly Ser Arg Asn Leu Leu Gln Gly Glu 805 810 815 Glu Leu Leu Arg Ala Leu Asp Gln Val Asn 820 825 (2) INFORMATION FOR SEQ ID NO : 7 : (i) SEQUENCE CHARACTERISTICS : (A) LENGTH : 810 amino acids (B) TYPE : amino acid (C) STRANDEDNESS : single (D) TOPOLOGY : linear (ii) MOLECULE TYPE : peptide (xi) SEQUENCE DESCRIPTION : SEQ ID NO : 7 : Met Ser Ser Glu Arg Arg Lys Glu Lys Ser Arg Asp Ala Ala Arg Ser 1 5 10 15 Arg Arg Thr Lys Glu Ser Glu Val Phe Tyr Glu Leu Ala His Gln Leu 20 25 30 Pro Leu Pro His Asn Val Ser Ser His Leu Asp Lys Ala Ser Val Met 35 40 45 Arg Leu Thr Ile Ser Tyr Leu Arg Val Arg Lys Leu Leu Asp Ala Gly 50 55 60 Gly Leu Asp Ser Glu Asp Glu Met Lys Ala Gln Met Asp Cys Phe Tyr 65 70 75 80 Leu Lys Ala Leu Asp Gly Phe Val Met Val Leu Thr Asp Asp Gly Asp 85 90 95 Met Val Tyr Ile Ser Asp Asn Val Asn Lys Tyr Met Gly Leu Thr Gln 100 105 110 Phe Glu Leu Ala Gly His Ser Val Phe Asp Phe Thr His Pro Cys Asp 115 120 125

His Glu Glu Met Arg Glu Met Leu Thr His Arg Asn Gly Pro Val Arg 130 135 140 Lys Gly Lys Glu Leu Asn Thr Gln Arg Ser Phe Phe Leu Arg Met Lys 145 150 155 160 Cys Thr Leu Thr Ser Arg Gly Arg Thr Met Asn Ile Lys Ser Ala Thr 165 170 175 Trp Lys Val Leu His Cys Thr Gly His Ile His Val Tyr Asp Thr Asn 180 185 190 Ser Asn Gln Pro Gln Cys Gly Tyr Lys Lys Pro Pro Met Thr Cys Leu 195 200 205 Val Leu Ile Cys Glu Pro Ile Pro His Pro Ser Asn Ile Glu Ile Pro 210 215 220 Leu Asp Ser Lys Thr Phe Leu Ser Arg His Ser Leu Asp Met Lys Phe 225 230 235 240 Ser Tyr Cys Asp Glu Arg Ile Thr Glu Leu Met Gly Tyr Glu Pro Glu 245 250 255 Glu Leu Leu Gly Arg Ser Ile Tyr Glu Tyr Tyr His Ala Leu Asp Ser 260 265 270 Asp His Leu Thr Lys Thr His His Asp Met Phe Thr Lys Gly Gln Val 275 280 285 Thr Thr Gly Gln Tyr Arg Met Leu Ala Lys Arg Gly Gly Tyr Val Trp 290 295 300 Val Glu Thr Gln Ala Thr Val Ile Tyr Asn Thr Lys Asn Ser Gln Pro 305 310 315 320 Gln Cys Ile Val Cys Val Asn Tyr Val Val Ser Gly Ile Ile Gln His 325 330 335 Asp Leu Ile Phe Ser Leu Gln Gln Thr Glu Ser Val Leu Lys Pro Val 340 345 350 Glu Ser Ser Asp Met Lys Met Thr Gln Leu Phe Thr Lys Val Glu Ser 355 360 365 Glu Asp Thr Ser Cys Leu Phe Asp Lys Leu Lys Lys Glu Pro Asp Ala 370 375 380 Leu Thr Leu Leu Ala Pro Ala Ala Gly Asp Thr Ile Ile Ser Leu Asp 385 390 395 400 Phe Gly Ser Asp Asp Thr Glu Thr Glu Asp Gln Gln Leu Glu Asp Val 405 410 415 Pro Leu Tyr Asn Asp Val Met Phe Pro Ser Ser Asn Glu Lys Leu Asn 420 425 430

Ile Asn Leu Ala Met Ser Pro Leu Pro Ser Ser Glu Thr Pro Lys Pro 435 440 445 Leu Arg Ser Ser Ala Asp Pro Ala Leu Asn Gln Glu Val Ala Leu Lys 450 455 460 Leu Glu Ser Ser Pro Glu Ser Leu Gly Leu Ser Phe Thr Met Pro Gln 465 470 475 480 Ile Gln Asp Gln Pro Ala Ser Pro Ser Asp Gly Ser Thr Arg Gln Ser 485 490 495 Ser Pro Glu Pro Asn Ser Pro Ser Glu Tyr Cys Phe Asp Val Asp Ser 500 505 510 Asp Met Val Asn Val Phe Lys Leu Glu Leu Val Glu Lys Leu Phe Ala 515 520 525 Glu Asp Thr Glu Ala Lys Asn Pro Phe Ser Thr Gln Asp Thr Asp Leu 530 535 540 Asp Leu Glu Met Leu Ala Pro Tyr Ile Pro Met Asp Asp Asp Phe Gln 545 550 555 560 Leu Arg Ser Phe Asp Gln Leu Ser Pro Leu Glu Ser Asn Ser Pro Ser 565 570 575 Pro Pro Ser Met Ser Thr Val Thr Gly Phe Gln Gln Thr Gln Leu Gln 580 585 590 Lys Pro Thr Ile Thr Ala Thr Ala Thr Thr Thr Ala Thr Thr Asp Glu 595 600 605 Ser Lys Thr Glu Thr Lys Asp Asn Lys Glu Asp Ile Lys Ile Leu Ile 610 615 620 Ala Ser Pro Ser Ser Thr Gln Val Pro Gln Glu Thr Thr Thr Ala Lys 625 630 635 640 Ala Ser Ala Tyr Ser Gly Thr His Ser Arg Thr Ala Ser Pro Asp Arg 645 65, 0 655 Ala Gly Lys Arg Val Ile Glu Gln Thr Asp Lys Ala His Pro Arg Ser 660 665 670 Leu Asn Leu Ser Ala Thr Leu Asn Gln Arg Asn Thr Val Pro Glu Glu 675 680 685 Glu Leu Asn Pro Lys Thr Ile Ala Ser Gln Asn Ala Gln Arg Lys Arg 690 695 700 Lys Met Glu His Asp Gly Ser Leu Phe Gln Ala Ala Gly Ile Gly Thr 705 710 715 720 Leu Leu Gln Gln Pro Gly Asp Cys Ala Pro Thr Met Ser Leu Ser Trp 725 730 735

Lys Arg Val Lys Gly Phe Ile Ser Ser Glu Gln Asn Gly Thr Glu Gln 740 745 750 Lys Thr Ile Ile Leu Ile Pro Ser Asp Leu Ala Cys Arg Leu Leu Gly 755 760 765 Gln Ser Met Asp Val Ser Gly Leu Pro Gln Leu Thr Ser Tyr Asp Cys 770 775 780 Glu Val Asn Ala Pro Ile Gln Gly Ser Arg Asn Leu Leu Gln Gly Glu 785 790 795 800 Glu Leu Leu Arg Ala Leu Asp Gln Val Asn 805 810