Human ADA2 polypeptides

This application is a divisional and claims priority under 35 U.S.C. §120 to application Ser. No. 08/705,771, filed Aug. 30, 1996, now U.S. Pat. No. 6,054,289, which claims priority under 35 U.S.C. §119(e) of Provisional Application Serial No. 60/002,993, filed Aug. 30, 1995, both herein incorporated by reference in their entirety.

This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. The invention also relates to inhibiting the action of such polypeptides.

Identification and sequencing of human genes is a major goal of modern scientific research. For example, by identifying genes and determining their sequences, scientists have been able to make large quantities of valuable human “gene products.” These include human insulin, interferon, Factor VIII, tumor necrosis factor, human growth hormone, tissue plasminogen activator, and numerous other compounds. Additionally, knowledge of gene sequences can provide the key to treatment or cure of genetic diseases (such as muscular dystrophy and cystic fibrosis).

In accordance with one aspect of the present invention, there are provided novel mature polypeptides, as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof. The polypeptides of the present invention are of human origin.

In accordance with another aspect of the present invention, there are provided isolated nucleic acid molecules encoding the polypeptides, including mRNAs, DNAs, cDNAs, genomic DNAs as well as analogs and biologically active and diagnostically or therapeutically useful fragments thereof.

In accordance with yet a further aspect of the present invention, there is provided a process for producing such polypeptides by recombinant techniques comprising culturing recombinant prokaryotic and/or eukaryotic host cells, containing a nucleic acid sequence of the present invention, under conditions promoting expression of said proteins and subsequent recovery of said proteins.

In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such polypeptides, or polynucleotides encoding such polypeptide for therapeutic and diagnostic purposes.

In accordance with yet a further aspect of the present invention, there is also provided nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to the nucleic acid sequences.

In accordance with yet a further aspect of the present invention, there are provided antibodies against such polypeptides.

In accordance with another aspect of the present invention, there are provided agonists to the polypeptides.

In accordance with yet another aspect of the present invention, there are provided antagonists to such polypeptides, which may be used to inhibit the action of such polypeptides, for therapeutic and diagnostic purposes.

In accordance with still another aspect of the present invention, there are provided diagnostic assays for detecting diseases related to the under-expression of the polypeptides of the present invention and mutations in the nucleic acid sequences encoding such polypeptides.

In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such polypeptides, or polynucleotides encoding such polypeptides, for in vitro purposes related to scientific research, synthesis of DNA and manufacture of DNA vectors.

In the case where the polypeptides of the present invention are receptors, there are provided processes for using the receptor to screen for receptor antagonists and/or agonists and/or receptor ligands.

These and other aspects of the present invention should be apparent to those skilled in the art from the teachings herein.

Table 1 sets forth information regarding identifying polynucleotide clone numbers, identification of the polynucleotide sequence which corresponds to the putative identification of the polypeptide encoded by the polynucleotide, and cross-referencing to the SEQ ID NOS. as set forth in the sequence listing.

Table 2 includes information regarding identifying polypeptide numbers, identification of the SEQ ID NOS. of the polypeptides, and cross-reference to the SEQ ID NO. which sets forth the amino acid sequence which corresponds to a given polypeptide in the sequence listing.

FIG. 1 shows the full length polynucleotide sequence (SEQ ID NO: 1) of the HBGBA67X clone and correlates the coding region with the derived amino acids (127 amino acids whose entire sequence (SEQ ID NO:12) is also shown for the full length amyloid-like protein present in breast tissue.

FIGS. 2A-2B show the complete nucleotide (SEQ ID NO:2) and amino acid sequences (SEQ ID NO:13) of the hADA2 gene and protein.

FIGS. 3A-3B show the full length sequence of the TFIId homolog clone (SEQ ID NO:3) including the full length sequence of the polynucleotide coding for TATA related factor (TRF, SEQ ID NO:14).

FIG. 4 shows full length cDNA (SEQ ID NO:4) and deduced amino acid sequence (SEQ ID NO:15) of hRPB 11.

FIGS. 5A-5B show the full nucleotide (SEQ ID NO:5) sequence of the IRF3 gene and amino acid sequence (SEQ ID NO:16) for the resulting protein. The predicted molecular weight of IRF3 is 47,087; the predicted isoelectric is 5.06; and the net charge equals −14.

FIG. 6 shows individually the full length sequence (SEQ ID NO:6) of the TM4SF gene, the coding region sequence portion and the amino acid sequence (SEQ ID NO:17) of the translation product TM4SF.

FIGS. 7A-7B show the full length nucleotide sequence (SEQ ID NO:7) of TNFR AF1 C1, the complete coding sequence region of the full length sequence and the derived amino acid sequence (SEQ ID NO:18) of the resulting protein.

FIG. 8 shows the full length sequence (SEQ ID NO:8), the coding region sequence and the derived amino acid sequence (SEQ ID NO:19) of the expression product protein of TM4SF (transmembrane 4 super family) CD53.

FIG. 9 shows the full length cDNA (SEQ ID NO:9) and the resulting expression of the product protein (SEQ ID NO:20) of its coding region for retenoid receptor gamma.

FIG. 10 shows the full length nucleotide sequence (SEQ ID NO:10) (1237 bp) and the translation product (412 amino acid, SEQ ID NO:21) resulting from the nucleotide sequence for the cytosolic resiniferatoxin binding protein RBP-26.

FIG. 11 shows the nucleotide sequence (SEQ ID NO:11) for the human protein (SEQ ID NO:22) kinase C inhibitor protein.

In accordance with an aspect of the present invention, there are provided isolated nucleic acids (polynucleotides) which code for mature polypeptides having the deduced amino acid sequences shown in the FIGS. 1-11 or for the mature polypeptides encoded by the cDNA of the clone deposited as ATCC Deposit No. 97242 on Aug. 15, 1995 with the ATCC, 10801 University Boulevard Manassas, Va. 20110-2209.

The polynucleotides of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA. The DNA may be double-stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand. The coding sequence which encodes the mature polypeptide may be identical to the coding sequence shown in SEQ ID NOS:1-11) or that of the deposited clone or may be a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same mature polypeptide as the DNA of SEQ ID NOS:1-11 or the deposited cDNA.

The polynucleotides which code for the mature polypeptides of FIGS. 1-11 or for the mature polypeptides encoded by the deposited cDNA may include, but are not limited to: only the coding sequence for the mature polypeptide; the coding sequence for the mature polypeptide and additional coding sequence such as a leader or secretory sequence or a proprotein sequence; the coding sequence for the mature polypeptide (and optionally additional coding sequence) and non-coding sequence, such as introns or non-coding sequence (SEQ ID NO:1) 5′ and/or 3′ of the coding sequence for the mature polypeptide.

Thus, the term “polynucleotide encoding a polypeptide” encompasses a polynucleotide which includes only coding sequence for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequence.

The present invention further relates to variants of the hereinabove described polynucleotides which code for fragments, analogs and derivatives of the polypeptide having the deduced amino acid sequences of FIGS. 1-11 or the polypeptides encoded by the cDNA of the deposited clone. The variant of the polynucleotide may be a naturally occurring allelic variant of the polynucleotide or a non-naturally occurring variant of the polynucleotide.

Thus, the present invention includes polynucleotides encoding the same mature polypeptides as shown in FIG. 1 or the same mature polypeptides encoded by the cDNA of the deposited clone as well as variants of such polynucleotides which variants code for a fragment, derivative or analog of the polypeptides of FIGS. 1-11 or the polypeptides encoded by the cDNA of the deposited clone. Such nucleotide variants include deletion variants, substitution variants and addition or insertion variants.

As hereinabove indicated, the polynucleotides may have a coding sequence which is a naturally occurring allelic variant of the coding sequences shown in FIGS. 1-11 or of the coding sequences of the deposited clone. As known in the art, an allelic variant is an alternate form of a polynucleotide sequence which may have a substitution, deletion or addition of one or more nucleotides, which does not substantially alter the function of the encoded polypeptide.

The present invention also includes polynucleotides, wherein the coding sequence for the mature polypeptide may be fused in the same reading frame to a polynucleotide sequence which aids in expression and secretion of a polypeptide from a host cell, for example, a leader sequence which functions as a secretory sequence for controlling transport of a polypeptide from the cell. The polypeptide having a leader sequence is a preprotein and may have the leader sequence cleaved by the host cell to form the mature form of the polypeptide. The polynucleotides may also code for a proprotein which is the mature protein plus additional 5′ amino acid residues. A mature protein having a prosequence is a proprotein and is an inactive form of the protein. Once the prosequence is cleaved an active mature protein remains.

Thus, for example, the polynucleotide of the present invention may code for a mature protein, or for a protein having a prosequence or for a protein having both a prosequence and a presequence (leader sequence).

The polynucleotides of the present invention may also have the coding sequence fused in frame to a marker sequence which allow s for purification of the polypeptide of the present invention. The marker sequence may be a hexa-histidine tag supplied by a pQE-9 vector to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell, 37:767 (1984)).

The term “gene” means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons).

Fragments of the full length genes of the present invention may be used as hybridization probes for a cDNA library to isolate the full length cDNA and to isolate other cDNAs which have a high sequence similarity to the gene or similar biological activity. Probes of this type preferably have at least 30 bases and may contain, for example, 50 or more bases. The probe may also be used to identify a cDNA clone corresponding to a full length transcript and a genomic clone or clones that contain the complete gene including regulatory and promotor regions, exons and introns. An example of a screen comprises isolating the coding region of one of the genes by using the known DNA sequence to synthesize an oligonucleotide probe. Labeled oligonucleotides having a sequence complementary to that of the gene of the present invention are used to screen a library of human cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.

The present invention further relates to polynucleotides which hybridize to the hereinabove-described sequences if there is at least 70%, preferably at least 90%, and more preferably at least 95% identity between the sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the hereinabove-described polynucleotides. As herein used, the term “stringent conditions” means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences. The polynucleotides which hybridize to the hereinabove described polynucleotides in a preferred embodiment encode polypeptides which either retain substantially the same biological function or activity as the mature polypeptide encoded by the cDNAs of FIGS. 1-11 (SEQ ID NOS:1-11) or the deposited cDNA(s).

Alternatively, the polynucleotides may have at least 20 bases, preferably 30 bases, and more preferably at least 50 bases which hybridize to a polynucleotide of the present invention and which have an identity thereto, as hereinabove described, and which may or may not retain activity. For example, such polynucleotides may be employed as probes for the polynucleotides any of SEQ ID NOS:1-11, for example, for recovery of the polynucleotide or as a diagnostic probe or as a PCR primer.

Thus, the present invention is directed to polynucleotides having at least a 70% identity, preferably at least 90% and more preferably at least 95% identity to a polynucleotides which encode the polypeptides of SEQ ID NOS: 12-22, as well as fragments thereof, which fragments have at least 30 bases and preferably at least 50 bases and to polypeptides encoded by such polynucleotides.

The deposit(s) referred to herein will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for purposes of Patent Procedure. These deposits are provided merely as convenience to those of skill in the art and are not an admission that a deposit is required under 35 U.S.C. §112. The sequence of the polynucleotides contained in the deposited materials, as well as the amino acid sequence of the polypeptides encoded thereby, are incorporated herein by reference and are controlling in the event of any conflict with any description of sequences herein. A license may be required to make, use or sell the deposited materials, and no such license is hereby granted.

The present invention further relates to polypeptides which have the deduced amino acid sequence of SEQ ID NOS. 12-22 or which have the amino acid sequences encoded by the deposited cDNAs, as well as fragments, analogs and derivatives of such polypeptides.

The terms “fragment”, “derivative” and “analog” when referring to the polypeptides of SEQ ID NOS. 12-22 or those encoded by the deposited cDNA, means polypeptides which retain essentially the same biological function or activity as such polypeptide. Thus, an analog and derivative includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.

The polypeptides of the present invention may be recombinant polypeptides, natural polypeptides or synthetic polypeptides, preferably recombinant polypeptides.

The fragments, derivatives or analogs of the polypeptides of SEQ ID NOS. 12-22 or those encoded by the deposited cDNAs may be (i) those in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, (ii) those in which one or more of the amino acid residues includes a substituent group, (iii) those in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol) or (iv) those in which the additional amino acids are fused to the mature polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.

The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.

The term “isolated” means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.

The polypeptides of the present invention include the polypeptides of SEQ ID NOS: 12-22 (in particular the mature polypeptides) as well as polypeptides which have at least 70% similarity (preferably a 70% identity) to the polypeptides of SEQ ID NOS: 12-22 and more preferably a 90% similarity (more preferably a 90% identity) to the polypeptides of SEQ ID NOS: 12-22 and still more preferably a 95% similarity (still more preferably a 95% identity) to the individual polypeptides of SEQ ID NOS: 12-22 and also include portions of such polypeptides with such portion of the polypeptides generally containing at least 30 amino acids and more preferably at least 50 amino acids.

As known in the art “similarity” between two polypeptides is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide.

Fragments or portions of the polypeptides of the present invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, the fragments may be employed as intermediates for producing the full-length polypeptides. Fragments or portions of the polynucleotides of the present invention may be used to synthesize full-length polynucleotides of the present invention.

The present invention also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.

Host cells are genetically engineered (transduced or transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector. The vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the genes of the present invention. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

The polynucleotides of the present invention may be employed for producing polypeptides by recombinant techniques. Thus, for example, the polynucleotides may be included in any one of a variety of expression vectors for expressing the corresponding polypeptide. Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies. However, any other vector may be used as long as it is replicable and viable in the host.

The appropriate DNA sequence may be inserted into the vector by a variety of procedures. In general, the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art.

The DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis. As representative examples of such promoters, there may be mentioned: LTR or SV40 promoter, the E. coli. lac or trp, the phage lambda P L promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also contains a ribosome binding site for translation initiation and a transcription terminator. The vector may also include appropriate sequences for amplifying expression.

In addition, the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.

The vector containing the appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein.

As representative examples of appropriate hosts, there may be mentioned: bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium; fungal cells, such as yeast; insect cells such as Drosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS or Bowes melanoma; adenoviruses; plant cells, etc. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.

More particularly, the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above. The constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available. The following vectors are provided by way of example; Bacterial: pQE70, pQE60, pQE-9 (Qiagen), pBS, pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia); Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid or vector may be used as long as they are replicable and viable in the host.

Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers. Two appropriate vectors are pKK232-8 and pCM7. Particular named bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P R , P L and trp. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.

In a further embodiment, the present invention relates to host cells containing the above-described constructs. The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation (Davis, Dibner and Battey, Basic Methods in Molecular Biology, (1986)).

The constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Alternatively, the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.

Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), the disclosure of which is hereby incorporated by reference.

Transcription of the DNA encoding the polypeptides of the present invention by higher eukaryotes is increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase its transcription. Examples including the SV40 enhancer on the late side of the replication origin bp 100 to 270, a cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence. Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), α-factor, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium. Optionally, the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.

Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter. The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host. Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.

As a representative but nonlimiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017). Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis., USA). These pBR322 “backbone” sections are combined with an appropriate promoter and the structural sequence to be expressed.

Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.

Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.

Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, such methods are well know to those skilled in the art.

Various mammalian cell culture systems can also be employed to express recombinant protein. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell, 23:175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5′ flanking nontranscribed sequences. DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.

The polypeptides can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.

The polypeptides of the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture). Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. Polypeptides of the invention may also include an initial methionine amino acid residue.

The amyloid-like gene and gene product may be employed as part of a diagnostic process for the early detection of pre-cancerous growth or cancer in the breast. The amyloid protein forms amyloid fibrils which in turn are capable of attracting calcium molecules leading to calcium deposition and calcification. Micro-fibrils and micro-calcification caused by microinjury in the breast tissue result in densified breast tissue which is an early symptom detectable by mammography. The amyloid-like protein gene according to the invention was isolated and recovered as a full length gene by computer-assisted analysis of expression sequence tag data basis from a primary breast cancer library, a normal breast library, an activated monocyte library and an embryonic library. The assembly of ESTs represents a full length gene which is illustrated in FIG. 1 . Full length human ADA2 nucleotide sequence was isolated from a 12 week old early stage human primary testes lambda library.

The expression levels of the amyloid-like protein according to the invention may be detectable in the serum and/or ductal fluid of the breast due to its secretory nature, thus the amyloid-like protein may be employed as a target for detection in such breast fluids. Further, examination of tissue samples from the breast for increased levels of the amyloid protein according to the invention may be helpful as part of an overall diagnostic regimen to screen for abnormal breast tissue growth or for breast cancer.

The amyloid-like protein according to the invention is toxic to surrounding breast cells which leads to apoptosis. The deposition of this protein in the breast tissue may be an early lesion for cancerous growth in the breast. Thus, this gene may be a target for breast cancer diagnosis.

Human transcriptional regulator hADA2 is the human homolog of yeast factor identified as being important for mediating the transcriptional activation properties of the Herpes Simplex transactivator VP16. It is possible that being able to control the activity of this factor (perhaps through anti-sense or screened antagonists) will allow the regulation of specific viral and human genes whose expression is controlled by this factor. This could lead to the controlled regulation of certain medically important genes. Furthermore, it is possible that disruption of this gene could result in unregulated transcription leading to cancer, in which case gene therapy would be medically important. Administration of HADA2 via gene therapy may be employed to treat cancer since disruption of the HADA2 gene results in unregulated transcription. We have recently mapped the chromosomal location of this gene to 17q12-21. The gene encoding the HADA2 protein was isolated from a 12 week old human primary testes library.

Modulating the activity of the human transcription regulator HADA2 may be employed to enhance or reduce the amount of a particular gene product produced. For example, in the case of an elevated level of a polypeptide the gene responsible may be down-regulated by inhibiting HADA2. Likewise, if an up-regulation of a gene product is desired, e.g., growth hormone, HADA2 may be stimulated.

Human transcription regulator factor (hTRF) is a homolog of the TATA Box Binding protein which plays a pivotal role in the expression of all genes. The full length cDNA of TRF was isolated by screening a human testes library. The hRPB11 gene was isolated from a subtracted human pituitary library. It is possible that lack or overexpression of this gene could lead to unregulated transcription leading to cancer. The human transcription factor hTRF may play a pivotal role in the expression of nearly all human genes since it is thought to bind to the “TATA box” upstream of all translated genes. Accordingly, modulation of hTRF, via gene therapy, stimulation and antagonism may be employed to control gene expression. Lack of hTRF may cause unregulated transcription which may lead to cancer. Accordingly administration of hTRF protein, or administration of the hTRF gene via gene therapy may be employed to treat cancer.

The human RNA polymerase subunits hRPB8, hRPB10 and hRPB11 play vital roles in mRNA synthesis since they possess the catalytic machinery for the formation of the 3′-5′ phosphodiester bonds between ribonucleoside triphosphates and respond to signals from the multiple factors involved in regulating their function during initiation and elongation of mRNA synthesis. These subunits are able to support normal yeast cell growth in vivo. The coding region in some flanking 5′ and 3′ UTR have been sequenced. The protein has a predicted molecular weight of 13,293; an isoelectric point of 5.73 and is 117 residues long.

Accordingly, since the subunits are vital to mRNA synthesis, their administration may be employed to up-regulate the expression of certain genes and to down-regulate others as needed. Administration may be via gene therapy. Abnormal cellular proliferation, e.g., cancer, may be treated with the subunits since lack of expression of these genes may lead to unregulated transcription.

The human interferon regulatory factor IRF3 gene shows strong homology to a group of transcription factors including IRF1 (Interferon Regulatory factor 1) and IRF2 (interferon Regulatory factor 2) which are important in mediating the transcriptional activation of interferon-alpha and -beta induced genes. It is possible that this gene also is important in mediating the transcriptional activation properties of interferon and that this factor may have some of the properties associated with interferon such as anti-viral activity. The human interferon regulatory factor IRF3 is potentially important in regulating the transcriptional activation of interferon-α) and -β genes. IRF3 may also be important in mediating the transcriptional activation properties of interferon. The IRF3 polypeptide may be employed as an anti-viral agent. The administration of the IRF3 gene and its gene product may be employed to enhance the expression of interferon which has many medically important uses. The IRF3 gene was isolated from a human adult retina library.

The TM4SF gene may be employed as a target for the development of compounds to treat human T-cell leukemia virus type I since several monoclonal antibodies inhibitory to syncytium formation targeted this TM4SF molecule.

The TM4SF gene may also be employed in the regulation of cell growth. This gene may also be employed as an immunogen or target to implement active and passive immunotherapy in patients with cancer. The gene encoding TM4SF was isolated from a human T-cell lymphoma library.

The TNFR-AF1, C1 gene and gene product may be employed to regulate B-lymphocyte proliferation, immunoglobulin class-switching and apoptosis. The TNFR-AF1 may also be employed to up-regulate the biological activity of TNF which is known to regress tumors. The gene encoding TNFR-AF1 C1 was isolated from an activated human nutrophil library.

The TM4SF, CD53 gene and gene product may be employed to regulate lymphoma cell growth and may also be employed to regulate cell growth. The gene encoding TM4SF (transmembrane 4 super family), CD53 was isolated from a human tumor pancreas library.

The retinoid X receptor γ may be employed to treat psoriasis and recalcitrincystic acne and cancer. This retinoid X receptor γ may also be employed to prevent a variety of pre-malignant lesions of skin and mucous membranes. The receptor may also be employed as a tumor suppressor. The receptor may also be employed to stimulate cell proliferation, differentiation and keratinization. The receptor may also be employed to treat X linked adrenal hypoplasia and hypogonatropic hypoglonatism. The gene encoding retinoid X receptor gamma was isolated from a human fetal lung III library.

The cytosolic resiniferatoxin binding protein (RBP-26) may be employed to reduce pain sensation due to its ability to selectively block mechanoheat nociceptors and warm receptors of the skin that are known to play a significant role in sensation of pain. The gene encoding RBP-26 was isolated from a human osteoclastoma stromal cell library.

The protein kinase C inhibitor protein has siginificant medical application uses such as inhibiting tumor cell growth and in regulating the many physiological functions that are mediated by the activation of protein kinase C. The gene encoding the protein kinase C inhibitor protein was isolated from a human corpus colosum library.

The polynucleotides and polypeptides of the present invention may be employed as research reagents and materials for discovery of treatments and diagnostics to human disease.

This invention provides a method for identification of the receptors for the polypeptides listed in Table 1. The gene encoding the receptor can be identified by numerous methods known to those of skill in the art, for example, ligand panning and FACS sorting (Coligan, et al., Current Protocols in Immun., 1(2), Chapter 5, (1991)). Preferably, expression cloning is employed wherein polyadenylated RNA is prepared from a cell responsive to the respective polypeptide, and a cDNA library created from this RNA is divided into pools and used to transfect COS cells or other cells that are not responsive to the proteins. Transfected cells which are grown on glass slides are exposed to labeled protein. The protein can be labeled by a variety of means including iodination or inclusion of a recognition site for a site-specific protein kinase. Following fixation and incubation, the slides are subjected to auto-radiographic analysis. Positive pools are identified and sub-pools are prepared and re-transfected using an iterative sub-pooling and re-screening process, eventually yielding a single clone that encodes the putative receptor.

As an alternative approach for receptor identification, labeled protein can be photoaffinity linked with cell membrane or extract preparations that express the receptor molecule. Cross-linked material is resolved by PAGE and exposed to X-ray film. The labeled complex containing the protein-receptor can be excised, resolved into peptide fragments, and subjected to protein microsequencing. The amino acid sequence obtained from microsequencing would be used to design a set of degenerate oligonucleotide probes to screen a cDNA library to identify the gene encoding the putative receptor.

This invention provides a method of screening compounds to identify those which enhance (agonists) or block (antagonists) interaction of protein to receptor. An agonist is a compound which increases the natural biological functions, while antagonists eliminate such functions. As an example, a mammalian cell or membrane preparation expressing the receptor would be incubated with labeled protein in the presence of the drug. The ability of the drug to enhance or block this interaction could then be measured.

Alternatively, the response of a known second messenger system following interaction of protein and receptor would be measured and compared in the presence or absence of the drug. Such second messenger systems include but are not limited to, cAMP guanylate cyclase, ion channels or phosphoinositide hydrolysis.

In the case where the polypeptides of the present invention are receptor polypeptides, the present invention also relates to methods for determining whether a ligand can bind to the receptor which comprises transfecting a cell population (one presumed not to contain a receptor) with the appropriate vector expressing the receptor, such that the cell will now express the receptor. A suitable response system is obtained by transfection of the DNA into a suitable host containing the desired second messenger pathways including cAMP, ion channels, psosphoinositide kinase, or calcium response. Such a transfection system provides a response system to analyze the activity of various ligands exposed to the cell. Ligands chosen could be identified through a rational approach by taking known ligands that interact with similar types of receptors or using small molecules, cell supernatants or extracts or natural products.

The present invention also relates to an assay for identifying potential antagonists. An example of such an assay combines the protein and a potential antagonist with membrane-bound receptors or recombinant receptors under appropriate conditions for a competitive inhibition assay. The protein can be labeled, such as by radio activity, such that the number of molecules bound to the receptor can determine the effectiveness of the potential antagonist.

The polypeptides listed in Table 1 of the present invention which have putatively been identified as receptors may be employed in a process for screening for antagonists and/or agonists for the receptor.

In general, such screening procedures involve providing appropriate cells which express the receptor on the surface thereof. In particular, a polynucleotide encoding the receptor of the present invention is employed to transfect cells to thereby express the receptor. Such transfection may be accomplished by procedures as hereinabove described.

One such screening procedure involves the use of melanophores which are transfected to express the receptor of the present invention. Such a screening technique is described in PCT WO 92/01810 published Feb. 6, 1992.

Thus, for example, such assay may be employed for screening for a receptor antagonist by contacting the melanophore cells which encode the receptor with both the receptor ligand and a compound to be screened. Inhibition of the signal generated by the ligand indicates that a compound is a potential antagonist for the receptor, i.e., inhibits activation of the receptor.

The screen may be employed for determining an agonist by contacting such cells with compounds to be screened and determining whether such compound generates a signal, i.e., activates the receptor.

Other screening techniques include the use of cells which express the receptor (for example, transfected CHO cells) in a system which measures extracellular pH changes caused by receptor activation, for example, as described in Science, volume 246, pages 181-296 (October 1989). For example, potential agonists or antagonists may be contacted with a cell which expresses the receptor and a second messenger response, e.g. signal transduction or pH changes, may be measured to determine whether the potential agonist or antagonist is effective.

Another such screening technique involves introducing RNA encoding the receptor into xenopus oocytes to transiently express the receptor. The receptor oocytes may then be contacted in the case of antagonist screening with the receptor ligand and a compound to be screened, followed by detection of inhibition of a calcium signal.

Another screening technique involves expressing the receptor in which the receptor is linked to a phospholipase C or D. As representative examples of such cells, there may be mentioned endothelial cells, smooth muscle cells, embryonic kidney cells, etc. The screening for an antagonist or agonist may be accomplished as hereinabove described by detecting activation of the receptor or inhibition of activation of the receptor from the phospholipase second signal.

Another method involves screening for antagonists by determining inhibition of binding of labeled ligand to cells which have the receptor on the surface thereof. Such a method involves transfecting a eukaryotic cell with DNA encoding the receptor such that the cell expresses the receptor on its surface and contacting the cell with a potential antagonist in the presence of a labeled form of a known ligand. The ligand can be labeled, e.g., by radioactivity. The amount of labeled ligand bound to the receptors is measured, e.g., by measuring radioactivity of the receptors. If the potential antagonist binds to the receptor as determined by a reduction of labeled ligand which binds to the receptors, the binding of labeled ligand to the receptor is inhibited.

The present invention also provides a method for determining whether a ligand not known to be capable of binding to a receptor can bind to such receptor which comprises contacting a mammalian cell which expresses a receptor with the ligand under conditions permitting binding of ligands to the receptor, detecting the presence of a ligand which binds to the receptor and thereby determining whether the ligand binds to the receptor. The systems hereinabove described for determining agonists and/or antagonists may also be employed for determining ligands which bind to the receptor.

In general, antagonists for receptors which are determined by screening procedures may be employed for a variety of therapeutic purposes. For example, such antagonists have been employed for treatment of hypertension, angina pectoris, myocardial infarction, ulcers, asthma, allergies, psychoses, depression, migraine, vomiting, and benign prostatic hypertrophy.

Agonists for receptors are also useful for therapeutic purposes, such as the treatment of asthma, Parkinson's disease, acute heart failure, hypotension, urinary retention, and osteoporosis.

Potential antagonists against the polypeptides of the present invention include an antibody, or in some cases, an oligopeptide, which binds to the polypeptide. Alternatively, a potential antagonist may be a closely related protein which binds to the receptors of the polypeptide, however, they are inactive forms of the polypeptide and thereby inhibit the action of the polypeptides.

Another potential antagonist is an antisense construct prepared using antisense technology. Antisense technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA. For example, the 5′ coding portion of the polynucleotide sequence, which encodes for the mature polypeptides of the present invention, is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix—see Lee et al., Nucl. Acids Res., 6:3073 (1979); Cooney et al, Science, 241:456 (1988); and Dervan et al., Science, 251: 1360 (1991)), thereby preventing transcription and the production of the polypeptide. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the polypeptide (Antisense—Okano, J. Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)). The oligonucleotides described above can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production of the protein.

Potential antagonists include a small molecule which binds to and occupies the active site of the polypeptide or to the receptor where the polypeptide of the present invention is a receptor, thereby making it inaccessible to substrate such that normal biological activity is prevented. Examples of small molecules include but are not limited to small peptides or peptide-like molecules.

Another potential antagonist includes a soluble form of the receptor polypeptides, e.g. a fragment of the receptor, which binds to the ligand and prevents the ligand from interacting with membrane bound receptors.

Antagonists to the human transcription regulator hADA2 may be employed to regulate the expression of Herpes simplex transactivator VP16, since hADA2 mediates its transcriptional activation properties. Many medically important genes may also be regulated by the antagonism of hADA2.

Antagonists to TATA related factor (TRF) may be employed to control general protein expression and for the regulation of the expression of specific important gene groups.

Antagonists to RNA polymerase subunits HRPB8, HRPB10 and HRPB11 may be employed to treat cancer since over expression of these subunits may lead to unregulated transcription.

Antagonists to interferon related factor-3 (IRF-3) may be employed to down regulate the overexpression of interferon with its adverse effects.

Antagonists to TM4SF may be employed to inhibit tumor growth.

Antagnoists to TNFR AF1, C1 may be employed to inhibit inflammation and apoptosis.

Antagnoists to TM4SF (transmembrane 4 super family) CD53 may be employed to inhibit certain leukemias.

Antagonists to the retinoid X receptor γ may be employed to treat psoriasis and inflammation. The antagonists may also be employed to prevent and/or treat hyperplasia and tumors in the lung, breast and other tissues.

Antagonists to protein kinase C inhibitor protein may be employed to inhibit the activation function of protein kinase C.

The antagonists may be employed therapeutically in a composition with a pharmaceutically acceptable carrier, e.g., as hereinafter described.

The polypeptides of the present invention and agonists and antagonists may be employed in combination with a suitable pharmaceutical carrier. Such compositions comprise a therapeutically effective amount of the polypeptide or agonist or antagonist, and a pharmaceutically acceptable carrier or excipient. Such a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should suit the mode of administration.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. In addition, the polypeptides of the present invention or agonists or antagonists may be employed in conjunction with other therapeutic compounds.

The pharmaceutical compositions may be administered in a convenient manner such as by the oral, topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes. The pharmaceutical compositions are administered in an amount which is effective for treating and/or prophylaxis of the specific indication. In general, they are administered in an amount of at least about 10 μg/kg body weight and in most cases they will be administered in an amount not in excess of about 8 mg/Kg body weight per day. In most cases, the dosage is from about 10 μg/kg to about 1 mg/kg body weight daily, taking into account the routes of administration, symptoms, etc.

The polypeptides and agonists and antagonists which are polypeptides may also be employed in accordance with the present invention by expression of such polypeptides in vivo, which is often referred to as “gene therapy.”

Thus, for example, cells from a patient may be engineered with a polynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide. Such methods are well-known in the art and are apparent from the teachings herein. For example, cells may be engineered by the use of a retroviral plasmid vector containing RNA encoding a polypeptide of the present invention.

Similarly, cells may be engineered in vivo for expression of a polypeptide in vivo by, for example, procedures known in the art. For example, a packaging cell is transduced with a retroviral plasmid vector containing RNA encoding a polypeptide of the present invention such that the packaging cell now produces infectious viral particles containing the gene of interest. These producer cells may be administered to a patient for engineering cells in vivo and expression of the polypeptide in vivo. These and other methods for administering a polypeptide of the present invention by such method should be apparent to those skilled in the art from the teachings of the present invention.

Retroviruses from which the retroviral plasmid vectors hereinabove mentioned may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, adenovirus, Myeloproliferative Sarcoma Virus, and mammary tumor virus. In one embodiment, the retroviral plasmid vector is derived from Moloney Murine Leukemia Virus.

The vector includes one or more promoters. Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoter described in Miller et al., Biotechniques, 7(9):980-990 (1989), or any other promoter (e.g., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol III, and β-actin promoters). Other viral promoters which may be employed include, but are not limited to, adenovirus promoters, thymidine kinase (TK) promoters, and B19 parvovirus promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein.

The nucleic acid sequence encoding the polypeptide of the present invention is under the control of a suitable promoter. Suitable promoters which may be employed include, but are not limited to, adenoviral promoters, such as the adenoviral major late promoter; or heterologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) promoter; inducible promoters, such as the MMT promoter, the metallothionein promoter; heat shock promoters; the albumin promoter; the ApoAI promoter; human globin promoters; viral thymidine kinase promoters, such as the Herpes Simplex thymidine kinase promoter; retroviral LTRs (including the modified retroviral LTRs hereinabove described); the β-actin promoter; and human growth hormone promoters. The promoter also may be the native promoter which controls the gene encoding the polypeptide.

The retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines. Examples of packaging cells which may be transfected include, but are not limited to, the PE501, PA317, ψ-2, ψ-AM, PA12, T19-14X, VT-19-17-H2, ψCRE, ψCRIP, GP+E-86, GP+envAm12, and DAN cell lines as described in Miller, Human Gene Therapy, 1:5-14 (1990), which is incorporated herein by reference in its entirety. The vector may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaPO 4 precipitation. In one alternative, the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then administered to a host.

The producer cell line generates infectious retroviral vector particles which include the nucleic acid sequence(s) encoding the polypeptides. Such retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vitro or in vivo. The transduced eukaryotic cells will express the nucleic acid sequence(s) encoding the polypeptide. Eukaryotic cells which may be transduced include, but are not limited to, embryonic stem cells, embryonic carcinoma cells, as well as hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial cells, and bronchial epithelial cells.

This invention is also related to the use of the DNA (RNA) sequences diagnostically. Detection of a mutated form of sequences will allow a diagnosis of a disease or a susceptibility to a disease which results from under-expression of the protein.

Individuals carrying mutations in a human gene of the present invention may be detected at the DNA level by a variety of techniques. Nucleic acids for diagnosis may be obtained from a patient's cells, such as from blood, urine, saliva, tissue biopsy and autopsy material. The genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR (Saiki et al., Nature, 324:163-166 (1986)) prior to analysis. RNA or cDNA may also be used for the same purpose. As an example, PCR primers complementary to the nucleic acid encoding the protein can be used to identify and analyze mutations. For example, deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to radiolabeled RNA or alternatively, radiolabeled antisense DNA sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting temperatures.

Sequence differences between the reference gene and genes having mutations may be revealed by the direct DNA sequencing method. In addition, cloned DNA segments may be employed as probes to detect specific DNA segments. The sensitivity of this method is greatly enhanced when combined with PCR. For example, a sequencing primer is used with double-stranded PCR product or a single-stranded template molecule generated by a modified PCR. The sequence determination is performed by conventional procedures with radiolabeled nucleotide or by automatic sequencing procedures with fluorescent-tags.

Genetic testing based on DNA sequence differences may be achieved by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Small sequence deletions and insertions can be visualized by high resolution gel electrophoresis. DNA fragments of different sequences may be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see, e.g., Myers et al., Science, 230:1242 (1985)).

Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and S1 protection or the chemical cleavage method (e.g., Cotton et al., PNAS, USA, 85:4397-4401 (1985)).

Thus, the detection of a specific DNA sequence may be achieved by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes, (e.g., Restriction Fragment Length Polymorphisms (RFLP)) and Southern blotting of genomic DNA.

In addition to more conventional gel-electrophoresis and DNA sequencing, mutations can also be detected by in situ analysis.

The present invention also relates to a diagnostic assay for detecting altered levels of the polypeptides of the present invention and soluble form of the receptor polypeptides of the present invention, in various tissues since an over-expression of the proteins compared to normal control tissue samples can detect the presence of a disease. Assays used to detect levels of protein in a sample derived from a host are well-known to those of skill in the art and include radioimmunoassays, competitive-binding assays, Western Blot analysis and preferably an ELISA assay. An ELISA assay initially comprises preparing an antibody specific to the antigen, preferably a monoclonal antibody. In addition a reporter antibody is prepared against the monoclonal antibody. To the reporter antibody is attached a detectable reagent such as radioactivity, fluorescence or in this example a horseradish peroxidase enzyme. A sample is now removed from a host and incubated on a solid support, e.g. a polystyrene dish, that binds the proteins in the sample. Any free protein binding sites on the dish are then covered by incubating with a non-specific protein such as bovine serum albumin. Next, the monoclonal antibody is incubated in the dish during which time the monoclonal antibodies attach to any proteins attached to the polystyrene dish. All unbound monoclonal antibody is washed out with buffer. The reporter antibody linked to horseradish peroxidase is now placed in the dish resulting in binding of the reporter antibody to any monoclonal antibody bound to the protein of interest. Unattached reporter antibody is then washed out. Peroxidase substrates are then added to the dish and the amount of color developed in a given time period is a measurement of the amount of protein present in a given volume of patient sample when compared against a standard curve.

A competition assay may be employed wherein antibodies specific to the protein is attached to a solid support and labeled protein and a sample derived from the host are passed over the solid support and the amount of label detected attached to the solid support can be correlated to a quantity of the protein in the sample.

The sequences of the present invention are also valuable for chromosome identification. The sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome. Moreover, there is a current need for identifying particular sites on the chromosome. Few chromosome marking reagents based on actual sequence data (repeat polymorphisms) are presently available for marking chromosomal location. The mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disease.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis of the 3′ untranslated region of the gene is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the primer will yield an amplified fragment.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome. Using the present invention with the same oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes or pools of large genomic clones in an analogous manner. Other mapping strategies that can similarly be used to map to its chromosome include in situ hybridization, prescreening with labeled flow-sorted chromosomes and preselection by hybridization to construct chromosome specific-cDNA libraries.

Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step. This technique can be used with cDNA having at least 50 or 60 bases. For a review of this technique, see Verma et al., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York (1988).

Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available on line through Johns Hopkins University Welch Medical Library). The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes).

Next, it is necessary to determine the differences in the cDNA or genomic sequence between affected and unaffected individuals. If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease.

With current resolution of physical mapping and genetic mapping techniques, a cDNA precisely localized to a chromosomal region associated with the disease could be one of between 50 and 500 potential causative genes. (This assumes 1 megabase mapping resolution and one gene per 20 kb).

The polypeptides, their fragments or other derivatives, or analogs thereof, or cells expressing them can be used as an immunogen to produce antibodies thereto. These antibodies can be, for example, polyclonal or monoclonal antibodies. The present invention also includes chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and fragments.

Antibodies generated against the polypeptides corresponding to a sequence of the present invention can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptides to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptides itself. In this manner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies binding the whole native polypeptides. Such antibodies can then be used to isolate the polypeptide from tissue expressing that polypeptide.

For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler and Milstein, Nature, 256:495-497, 1975), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., Immunology Today 4:72, 1983), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).

Techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce single chain antibodies to immunogenic polypeptide products of this invention. Also, transgenic mice may be used to express humanized antibodies to immunogenic polypeptide products of this invention.

The present invention will be further described with reference to the following examples; however, it is to be understood that the present invention is not limited to such examples. All parts or amounts, unless otherwise specified, are by weight.

In order to facilitate understanding of the following examples certain frequently occurring methods and/or terms will be described.

“Plasmids” are designated by a lower case p preceded and/or followed by capital letters and/or numbers. The starting plasmids herein are either commercially available, publicly available on an unrestricted bases, or can be constructed from available plasmids in accord with published procedures. In addition, equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.

“Digestion” of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA. The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinarily skilled artisan. For analytical purposes, typically 1 μg of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 μl of buffer solution. For the purpose of isolating DNA fragments for plasmid construction, typically 5 to 50 μg of DNA are digested with 20 to 250 units of enzyme in a larger volume. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37° C. are ordinarily used, but may vary in accordance with the supplier's instructions. After digestion the reaction is electrophoresed directly on a polyacrylamide gel to isolate the desired fragment.

Size separation of the cleaved fragments is performed using 8 percent polyacrylamide gel described by Goeddel, D. et al., Nucleic Acids Res., 8:4057 (1980).

“Oligonucleotides” refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized. Such synthetic oligonucleotides have no 5′ phosphate and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.

“Ligation” refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments (Maniatis, T., et al., Id., p. 146). Unless otherwise provided, ligation may be accomplished using known buffers and conditions with 10 units of T4 DNA ligase (“ligase”) per 0.5 μg of approximately equimolar amounts of the DNA fragments to be ligated.

Unless otherwise stated, transformation was performed as described in the method of Graham, F. and Van der Eb, A., Virology, 52:456-457 (1973).

2

AATTCCTGGG GGGTCTCGGC GAGGGAGTCA TCAAGCTTTG GTGTATGTGT TGGCCGGTTC 60

TGAAGTCTTG AAGAAGCTCT GCTGAGGAAG ACCAAAGCAG CACTCGTTGC CAATTAGGGA 120

ATG GAC CGT TTG GGT TCC TTT AGC AAT GAT CCC TCT GAT AAG CCA CCT 168

Met Asp Arg Leu Gly Ser Phe Ser Asn Asp Pro Ser Asp Lys Pro Pro

5 10 15

TGC CGA GGC TGC TCC TCC TAC CTC ATG GAG CCT TAT ATC AAG TGT GCT 216

Cys Arg Gly Cys Ser Ser Tyr Leu Met Glu Pro Tyr Ile Lys Cys Ala

20 25 30

GAA TGT GGG CCA CCT CCT TTT TTC CTC TGC TTG CAG TGT TTC ACT CGA 264

Glu Cys Gly Pro Pro Pro Phe Phe Leu Cys Leu Gln Cys Phe Thr Arg

35 40 45

GGC TTT GAG TAC AAG AAA CAT CAA AGC GAT CAT ACT TAT GAA ATA ATG 312

Gly Phe Glu Tyr Lys Lys His Gln Ser Asp His Thr Tyr Glu Ile Met

50 55 60

ACT TCA GAT TTT CCT GTC CTT GAT CCC AGC TGG ACT GCT CAA GAA GAA 360

thr Ser Asp Phe Pro Val Leu Asp Pro Ser Trp Thr Ala Gln Glu Glu

65 70 75 80

ATG GCC CTT TTA GAA GCT GTG ATG GAC TGT GGC TTT GGA AAT TGG CAG 408

Met Ala Leu Leu Glu Ala Val Met Asp Cys Gly Phe Gly Asn Trp Gln

85 90 95

GAT GTA GCC AAT CAA ATG TGC ACC AAG ACC AAG GAG GAG TGT GAG AAG 456

Asp Val Ala Asn Gln Met Cys Thr Lys Thr Lys Glu Glu Cys Glu Lys

100 105 110

CAC TAT ATG AAG CAT TTC ATC AAT AAC CCT CTG TTT GCA TCT ACC CTG 504

His Tyr Met Lys His Phe Ile Asn Asn Pro Leu Phe Ala Ser Thr Leu

115 120 125

CTG AAC CTG AAA CAA GCA GAG GAA GCA AAA ACT GCT GAC ACA GCC ATT 552

Leu Asn Leu Lys Gln Ala Glu Glu Ala Lys Thr Ala Asp Thr Ala Ile

130 135 140

CCA TTT CAC TCT ACA GAT GAC CCT CCC CGA CCT ACC TTT GAC TCC TTG 600

Pro Phe His Ser Thr Asp Asp Pro Pro Arg Pro Thr Phe Asp Ser Leu

145 150 155 160

CTT TCT CGG GAC ATG GCC GGG TAC ATG CCA GCT CGA GCA GAT TTC ATT 648

Leu Ser Arg Asp Met Ala Gly Tyr Met Pro Ala Arg Ala Asp Phe Ile

165 170 175

GAG GAA TTT GAC AAT TAT GCA GAA TGG GAC TTG AGA GAC ATT GAT TTT 696

Glu Glu Phe Asp Asn Tyr Ala Glu Trp Asp Leu Arg Asp Ile Asp Phe

180 185 190

GTT GAA GAT GAC TCG GAC ATT TTA CAT GCT CTG AAG ATG GCT GTG GTA 744

Val Glu Asp Asp Ser Asp Ile Leu His Ala Leu Lys Met Ala Val Val

195 200 205

GAT ATC TAT CAT TCC AGG TTA AAG GAG AGA CAA AGA CGA AAA AAA ATT 792

Asp Ile Tyr His Ser Arg Leu Lys Glu Arg Gln Arg Arg Lys Lys Ile

210 215 220

ATA AGA GAC CAT GGA TTA ATC AAC CTT AGA AAG TTT CAA TTA ATG GAA 840

Ile Arg Asp His Gly Leu Ile Asn Leu Arg Lys Phe Gln Leu Met Glu

225 230 235 240

CGG CGG TAT CCC AAG GAG GTC CAG GAC CTG TAT GAA ACA ATG AGG CGA 888

Arg Arg Tyr Pro Lys Glu Val Gln Asp Leu Tyr Glu Thr Met Arg Arg

245 250 255

TTT GCA AGA ATT GTG GGG CCA GTG GAA CAT GAC AAA TTC ATT GAA AGC 936

Phe Ala Arg Ile Val Gly Pro Val Glu His Asp Lys Phe Ile Glu Ser

260 265 270

CAT GCA TTG GAA TTT GAA CTC CGA AGG GAA ATC AAG AGG CTC CAA GAA 984

His Ala Leu Glu Phe Glu Leu Arg Arg Glu Ile Lys Arg Leu Gln Glu

275 280 285

TAC AGG ACA GCA GGC ATT ACC AAT TTT TGT AGT GCC AGA ACC TAC GAT 1032

Tyr Arg Thr Ala Gly Ile Thr Asn Phe Cys Ser Ala Arg Thr Tyr Asp

290 295 300

CAC CTC AAG AAG ACA CGG GAG GAA GAG CGC CTT AAA CGC ACT ATG CTC 1080

His Leu Lys Lys Thr Arg Glu Glu Glu Arg Leu Lys Arg Thr Met Leu

305 310 315 320

TCA GAA GTT CTC CAG TAT ATC CAG GAC AGT AGT GCT TGC CAG CAG TGG 1128

Ser Glu Val Leu Gln Tyr Ile Gln Asp Ser Ser Ala Cys Gln Gln Trp

325 330 335

CTC CGC CGG CAA GCT GAC ATT GAT TCC GGC CTG AGT CCT TCC ATT CCA 1176

Leu Arg Arg Gln Ala Asp Ile Asp Ser Gly Leu Ser Pro Ser Ile Pro

340 345 350

ATG GCT TCG AAT TCA GGT AGA CGG AGT GCA CCA CCC TTG AAC CTC ACT 1224

Met Ala Ser Asn Ser Gly Arg Arg Ser Ala Pro Pro Leu Asn Leu Thr

355 360 365

GGC CTC CCT GGC ACA GAG AAG CTG AAT GAA AAA GAA AAG GAG CTC TGT 1272

Gly Leu Pro Gly Thr Glu Lys Leu Asn Glu Lys Glu Lys Glu Leu Cys

370 375 380

CAG ATG GTG AGG TTG GTC CCT GGA GCC TAT TTA GAA TAC AAA TCT GCT 1320

Gln Met Val Arg Leu Val Pro Gly Ala Tyr Leu Glu Tyr Lys Ser Ala

385 390 395 400

CTA TTG AAC GAA TGT AAC AAG CAA GGA GGC TTA AGA CTG GCG CAG GCA 1368

Leu Leu Asn Glu Cys Asn Lys Gln Gly Gly Leu Arg Leu Ala Gln Ala

405 410 415

AGA GCA CTC ATC AAG ATA GAT GTG AAC AAA ACC CGG AAA ATC TAT GAT 1416

Arg Ala Leu Ile Lys Ile Asp Val Asn Lys Thr Arg Lys Ile Tyr Asp

420 425 430

TTC CTC ATC AGA GAA GGA TAC ATC ACT AAA GGC T AAGGCTCCAA 1460

Phe Leu Ile Arg Glu Gly Tyr Ile Thr Lys Gly

435 440

GAGCTTGGGA TCAGAAGTCA GAAGTTTGGA ATGTGGTGGG TCAAAGGACA ATATGGGTGG 1520

GCATTCTGGA GAGTTTGTTT TTCAGCTGAA TTCTCATGGT GAAAACAGGG GAAAGGACAA 1580

AGGAAACCTT AAGTTGTATT GTCTACTTTC TTCTCCATCC TGCTTTAAAA CACTCCTGTT 1640

GTTGGTATTA TGCTGCAGAG TTGTGTGCTA CATAAGCTAT TATTAAATGT GAGTGGGCAT 1700

TCAAAAAAAA AAAAAAAAAA 1720

1537 base pairs

nucleic acid

single

linear

3

GGCACGAGGC GCCCCCGGGG TCCCCCGGCC CCTGCAGGGC TACTTGGGCG CAGAGCCGCG 60

GAGGGTCTCC GTTCCTAGAG GTCCTCCTAT CCCGGGCTGC CTGAGTCCTC GCCAGCATCC 120

GCCCTCTCCC ACTCCCATCC TTCCTGGATC CGCCTCTCGG TTCCCGAGGG ACAGTCCCGA 180

CCGCAAACCC ACGTAGAGTA AGGAATGTGG GACAGGCGAC AGAAGTGGCA TACGGTCCTG 240

CGTTATCCCT CCGTCTCGCC ACACCTTGTG TCTCCATCTC TCCCCACTTC CTTCCCTCCG 300

TCTGTCATCT GTCATCCCCG GTCGCTCTAA GACCAGGATT CCAATTCGCC TAGTGAGGAA 360

TCTCACTAGG GGAATTTATC GCGACATCAT AAATTAACGG GTTCATTTTG ACTGAAAAGC 420

GAAGGACTTT TTTCAGGCAG AAAACAAGTC TCGTCTGGAC GGATGTGATC TTCGTGGTGG 480

AAAGCTAAAT TTTAAAACCA CCCCA ATG GAT GCA GAC AGT GAT GTT GCA TTG 532

Met Asp Ala Asp Ser Asp Val Ala Leu

1 5

GAC ATT CTA ATT ACA AAT GTA GTC TGT GTT TTT AGA ACA AGA TGT CAT 580

Asp Ile Leu Ile Thr Asn Val Val Cys Val Phe Arg Thr Arg Cys His

10 15 20 25

TTA AAC TTA AGG AAG ATT GCT TTG GAA GGA GCA AAT GTA ATT TAT AAA 628

Leu Asn Leu Arg Lys Ile Ala Leu Glu Gly Ala Asn Val Ile Tyr Lys

30 35 40

CGT GAT GTT GGA AAA GTA TTA ATG AAG CTT AGA AAA CCT AGA ATT ACA 676

Arg Asp Val Gly Lys Val Leu Met Lys Leu Arg Lys Pro Arg Ile Thr

45 50 55

GCT ACA ATT TGG TCC TCA GGA AAA ATT ATT TGC ACT GGA GCA ACA AGT 724

Ala Thr Ile Trp Ser Ser Gly Lys Ile Ile Cys Thr Gly Ala Thr Ser

60 65 70

GAA GAA GAA GCT AAA TTT GGT GCC AGA CGC TTA GCC CGT AGT CTG CAG 772

Glu Glu Glu Ala Lys Phe Gly Ala Arg Arg Leu Ala Arg Ser Leu Gln

75 80 85

AAA CTA GGT TTT CAG GTA ATA TTT ACA GAT TTT AAG GTT GTT AAC GTT 820

Lys Leu Gly Phe Gln Val Ile Phe Thr Asp Phe Lys Val Val Asn Val

90 95 100 105

CTG GCA GTG TGT AAC ATG CCA TTT GAA ATC CGT TTG CCA GAA TTC ACA 868

Leu Ala Val Cys Asn Met Pro Phe Glu Ile Arg Leu Pro Glu Phe Thr

110 115 120

AAG AAC AAT AGA CCT CAT GCC AGT TAC GAA CCT GAA CTT CAT CCT GCT 916

Lys Asn Asn Arg Pro His Ala Ser Tyr Glu Pro Glu Leu His Pro Ala

125 130 135

GTG TGC TAT CGG ATA AAA TCT CTA AGA GCT ACA TTA CAG ATT TTT TCA 964

Val Cys Tyr Arg Ile Lys Ser Leu Arg Ala Thr Leu Gln Ile Phe Ser

140 145 150

ACA GGA AGT ATC ACA GTA ACA GGG CCC AAT GTA AAG GCT GTT GCT ACT 1012

Thr Gly Ser Ile Thr Val Thr Gly Pro Asn Val Lys Ala Val Ala Thr

155 160 165

GCT GTG GAA CAG ATT TAC CCA TTT GTG TTT GAA AGC AGG AAA GAA ATT 1060

Ala Val Glu Gln Ile Tyr Pro Phe Val Phe Glu Ser Arg Lys Glu Ile

170 175 180 185

TTA T AATTCACCAC TTAATTGGTT AGAATCTCTA ACTGAGCACC TTTTAAACCT 1114

Leu

GCTGCACATT GGACTCAAAA GGAAAACTGG ACCAACAATA ATTGAGGAAA TAGACTTTTT 1174

TATTCATTCA CGGCTACAGT GTAAGCTCCA GTCCCTTTGG ATTTTATTCC AAACCTTGCT 1234

GTAATATAAA AGGAAGTTTA CAAGACATGA TATTGCTGCT TTTACAAAAG GACATTCTAT 1294

TTATTTTCGC AGTAATTCTC ATGTCCCCAT AAGCAGAGCT GTCACAGTGT GCACTACCTT 1354

AGATTGTTTT ATTGTCGTCA TTGTTATTTT TTTCCATTTG GAGCTAATGT GTTTTATTTG 1414

TGAATAGTCT TTTACATTTT TGTATGCTGA ATATGGGCAC CAAAGAACCT GTAAAAGTTA 1474

TCTTTTTCAA TTGAATGTGC ACAAATAAAA GTTTGGAAAG AAAAAAAAAA AAAAAAAAAA 1534

AAA 1537

555 base pairs

nucleic acid

single

linear

4

ACGAGCAACG GCGGCGGGAG C ATG AAC GCC CCT CCA GCC TTC GAG TCG TTC 51

Met Asn Ala Pro Pro Ala Phe Glu Ser Phe

1 5 10

TTG CTC TTC GAG GGC GAG AAG AAG ATC ACC ATT AAC AAG GAC ACC AAG 99

Leu Leu Phe Glu Gly Glu Lys Lys Ile Thr Ile Asn Lys Asp Thr Lys

15 20 25

GTA CCC AAT GCC TGT TTA TTC ACC ATC AAC AAA GAA GAC CAC ACA CTG 147

Val Pro Asn Ala Cys Leu Phe Thr Ile Asn Lys Glu Asp His Thr Leu

30 35 40

GGA AAC ATC ATT AAA TCA CAA CTC CTA AAA GAC CCG CAA GTG CTA TTT 195

Gly Asn Ile Ile Lys Ser Gln Leu Leu Lys Asp Pro Gln Val Leu Phe

45 50 55

GCT GGC TAC AAA GTC CCC CAC CCC TTG GAG CAC AAG ATC ATC ATC CGA 243

Ala Gly Tyr Lys Val Pro His Pro Leu Glu His Lys Ile Ile Ile Arg

60 65 70

GTG CAG ACC ACG CCG GAC TAC AGC CCC CAG GAA GCC TTT ACC AAC GCC 291

Val Gln Thr Thr Pro Asp Tyr Ser Pro Gln Glu Ala Phe Thr Asn Ala

75 80 85 90

ATC ACC GAC CTC ATC AGT GAG CTG TCC CTG CTG GAG GAG CGC TTT CGG 339

Ile Thr Asp Leu Ile Ser Glu Leu Ser Leu Leu Glu Glu Arg Phe Arg

95 100 105

GTG GCC ATA AAA GAC AAG CAG GAA GGA ATT GAG T AGGGGCCAGA 383

Val Ala Ile Lys Asp Lys Gln Glu Gly Ile Glu

110 115

GGGGGCTCTG CTCGGCCTGT GAGCCCCGTT CCTACCTGTG CCTGACCCTC CGCTCCAGGT 443

ACCACACCGA GGAGAGCGGC CAGTCCCAGC CATGGCCCGC CTTGTGGCCA CCCCTCACCC 503

TGACACCGAC GTGTCCTGTA CATAGATTAG GTTTTATATT CCTAATAAAG TA 555

1426 base pairs

nucleic acid

single

linear

5

GGTTCCAGCT GCCCGCACGC CCCGACCTTC CATCGTAGGC CGGACCATGG GAACCCCAAA 60

GCCACGGNTC CTGCCCTGGC TGGTGTCGCA GCTGGACCTG GGGCAACTGG AGGGCGTGGC 120

CTGGGTGAAC AAGAGCCGCA CGCGCTTCCG CATCCCTTGG AAGCACGGCC TACGGCAGGA 180

TGCACAGCAG GAGGATTTCG GAATCTTCCA GGCCTGGGCC GAGGCCACTG GTGCATATGT 240

TCCCGGGAGG GATAAGCCAG ACCTGCCAAC CTGGAAGAGG AATTTCCGCT CTGCCCTCAA 300

CCGCAAAGAA GGGTTGCGTT TAGCAGAGGA CCGGAGCAAG GACCCTCACG ACCCACATAA 360

AATCTACGAG TTTGTGAACT CAGGAGTTGG GGACTTTTCC CAGCCAGACA CCTCTCCGGA 420

CACCAATGGT GGAGGCAGTA CTTCTGATAC CCAGGAAGAC ATTCTGGATG AGTTACTGGG 480

TAACATGGTG TTGGCCCCAC TCCCAGATCC GGGACCCCCA AGCCTGGCTG TAGCCCCTGA 540

GCCCTGCCCT CAGCCCCTGC GGAGCCCCAG CTTGGACAAT CCCACTCCCT TCCCAAACCT 600

GGGGCCCTCT GAGAACCCAC TGAAGCGGCT GTTGGTGCCG GGGGAAGAGT GGGAGTTCGA 660

GGTGACAGCC TTCTACCGGG GCCGCCAAGT CTTCCAGCAG ACCATCTCCT GCCCGGAGGG 720

CCTGCGGCTG GTGGGGTCCG AAGTGGGAGA CAGGACGCTG CCTGGATGGC CAGTCACACT 780

GCCAGACCCT GGCATGTCCC TGACAGACAG GGGAGTGATG AGCTACGTGA GGCATGTGCT 840

GAGCTGCCTG GGTGGGGGAC TGGCTCTCTG GCGGGCCGGG CAGTGGCTCT GGGCCCAGCG 900

GCTGGGGCAC TGCCACACAT ACTGGGCAGT GAGCGAGGAG CTGCTCCCCA ACAGCGGGCA 960

TGGGCCTGAT GGCGAGGTCC CCAAGGACAA GGAAGGAGGC GTGTTTGACC TGGGGCCCTT 1020

CATTGTAGAT CTGATTACCT TCACGGAAGG AAGCGGACGC TCACCACGCT ATGCCCTCTG 1080

GTTCTGTGTG GGGGAGTCAT GGCCCCAGGA CCAGCCGTGG ACCAAGAGGC TCGTGATGGT 1140

CAAGGTTGTG CCCACGTGCC TCAGGGCCTT GGTAGAAATG GCCCGGGTAG GGGGTGCCTC 1200

CTCCCTGGAG AATACTGTGG ACCTGCACAT TTCCAACAGC CACCCACTCT CCCTCACCTC 1260

CGACCAGTAC AAGGCCTACC TGCAGGACTT GGTGGAGGGC ATGGATTTCC AGGGCCCTTC 1320

GGAGAGCTGA GCCCTCGCTC CTCATGGTGT GCCTCCAACC CCCCTGTTCC CCACCACCTC 1380

AACCAATAAA CTGGTTCCTG CTATGAAAAA AAAAAAAAAA AAAAAA 1426

1001 base pairs

nucleic acid

single

linear

6

AATTCGGCAG AGGCAGTTCC TAGCGAGGAG GCGCCGCGCA TTGCCGCTCT CTCGGTGAGC 60

GCAGCCCGCT CTCCGGGCCG GGCCTTCGCG GGCCACCGCG CC ATG GGC CAG TGC 114

Met Gly Gln Cys

1

GGC ATC ACC TCC TCC AAG ACC GTG CTG GTC TTT CTC AAC CTC ATC TTC 162

Gly Ile Thr Ser Ser Lys Thr Val Leu Val Phe Leu Asn Leu Ile Phe

5 10 15 20

TGG GGG GCA GCT GGC ATT TTA TGC TAT GTG GGA GCC TAT GTC TTC ATC 210

Trp Gly Ala Ala Gly Ile Leu Cys Tyr Val Gly Ala Tyr Val Phe Ile

25 30 35

ACT TAT GAT GAC TAT GAC CAC TTC TTT GAA GAT GTG TAC ACG CTC ATC 258

Thr Tyr Asp Asp Tyr Asp His Phe Phe Glu Asp Val Tyr Thr Leu Ile

40 45 50

CCT GCT GTA GTG ATC ATA GCT GTA GGA GCC CTG CTT TTC ATC ATT GGG 306

Pro Ala Val Val Ile Ile Ala Val Gly Ala Leu Leu Phe Ile Ile Gly

55 60 65

CTA ATT GGC TGC TGT GCC ACA ATC CGG GAA AGT CGC TGT GGA CTT GCC 354

Leu Ile Gly Cys Cys Ala Thr Ile Arg Glu Ser Arg Cys Gly Leu Ala

70 75 80

ACG TTT GTC ATC ATC CTG CTC TTG GTT TTT GTC ACA GAA GTT GTT GTA 402

Thr Phe Val Ile Ile Leu Leu Leu Val Phe Val Thr Glu Val Val Val

85 90 95 100

GTG GTT TTG GGA TAT GTT TAC AGA GCA AAG GTG GAA AAT GAG GTT GAT 450

Val Val Leu Gly Tyr Val Tyr Arg Ala Lys Val Glu Asn Glu Val Asp

105 110 115

CGC AGC ATT CAG AAA GTG TAT AAG ACC TAC AAT GGA ACC AAC CCT GAT 498

Arg Ser Ile Gln Lys Val Tyr Lys Thr Tyr Asn Gly Thr Asn Pro Asp

120 125 130

GCT GCT AGC CGG GCT ATT GAT TAT GTA CAG AGA CAG CTG CAT TGT TGT 546

Ala Ala Ser Arg Ala Ile Asp Tyr Val Gln Arg Gln Leu His Cys Cys

135 140 145

GGA ATT CAC AAC TAC TCA GAC TGG GAA AAT ACA GAT TGG TTC AAA GAA 594

Gly Ile His Asn Tyr Ser Asp Trp Glu Asn Thr Asp Trp Phe Lys Glu

150 155 160

ACC AAA AAC CAG AGT GTC CCT CTT AGC TGC TGC AGA GAG ACT GCC AGC 642

Thr Lys Asn Gln Ser Val Pro Leu Ser Cys Cys Arg Glu Thr Ala Ser

165 170 175 180

AAT TGT AAT GGC AGC TGG CCA CCC TTC CGA CTC TAT GCT GAG GGG TGT 690

Asn Cys Asn Gly Ser Trp Pro Pro Phe Arg Leu Tyr Ala Glu Gly Cys

185 190 195

GAG GCT CTA GTT GTG AAG AAG CTA CAA GAA ATC ATG ATG CAT GTG ATC 738

Glu Ala Leu Val Val Lys Lys Leu Gln Glu Ile Met Met His Val Ile

200 205 210

TGG GCC GCA CTG GCA TTT GCA GCT ATT CAG CTG CTG GGC ATG CTG TGT 786

Trp Ala Ala Leu Ala Phe Ala Ala Ile Gln Leu Leu Gly Met Leu Cys

215 220 225

GCT TGC ATC GTG TTG TGC AGA AGG AGT AGA GAT CCT GCT TAC GAG CTC 834

Ala Cys Ile Val Leu Cys Arg Arg Ser Arg Asp Pro Ala Tyr Glu Leu

230 235 240

CTC ATC ACT GGC GGA ACC TAT GCA TAG T TGACAACTCA AGCCTGAGCT 882

Leu Ile Thr Gly Gly Thr Tyr Ala

245 250

TTTTGGTCTT GTTCTGATTT GGAAGGTGAA TTGAGCAGGT CTGCTGCTGT TGGCCTCTGG 942

AGTTCATCTA GTTAAAGCAC ATGTACACTG GTGTTGGACA GAGCAGCTTG GCTTTTCAT 1001

2361 base pairs

nucleic acid

single

linear

7

GAGCCAGGAC TCCACAAGGC TGGTCCCCTG CCCTGGAGCA ACTTAAACAG GCCCTCTGGC 60

CAGCCTGGAA CCCTGAG ATG GCC TCC AGC TCA GGC AGC AGT CCT CGC CCG 110

Met Ala Ser Ser Ser Gly Ser Ser Pro Arg Pro

1 5 10

GCC CCT GAT GAG AAT GAG TTT CCC TTT GGG TGC CCT CCC ACC GTC TGC 158

Ala Pro Asp Glu Asn Glu Phe Pro Phe Gly Cys Pro Pro Thr Val Cys

15 20 25

CAG GAC CCA AAG GAG CCC AGG GCT CTC TGC TGT GCA GGC TGT CTC TCT 206

Gln Asp Pro Lys Glu Pro Arg Ala Leu Cys Cys Ala Gly Cys Leu Ser

30 35 40

GAG AAC CCG AGG AAT GGC GAG GAT CAG ATC TGC CCC AAA TGC AGA GGG 254

Glu Asn Pro Arg Asn Gly Glu Asp Gln Ile Cys Pro Lys Cys Arg Gly

45 50 55

GAA GAC CTC CAG TCT ATA AGC CCA GGA AGC CGT CTT CGA ACT CAG GAG 302

Glu Asp Leu Gln Ser Ile Ser Pro Gly Ser Arg Leu Arg Thr Gln Glu

60 65 70 75

AAG GTT CAG GCG GAG GTC GCT GAG GCT GGG ATT GGG TGC CCC TTT GCT 350

Lys Val Gln Ala Glu Val Ala Glu Ala Gly Ile Gly Cys Pro Phe Ala

80 85 90

GTT GTC GGC TGC TCC TTC AAG GGA AGC CCA CAG TTT GTG GAA GAG CAT 398

Val Val Gly Cys Ser Phe Lys Gly Ser Pro Gln Phe Val Glu Glu His

95 100 105

GAG GTC ACC TCC CAG ACC TCC CAC CTA AAC CTG CTG TTG GGG TTC ATG 446

Glu Val Thr Ser Gln Thr Ser His Leu Asn Leu Leu Leu Gly Phe Met

110 115 120

AAA CAG TGG AAG GCC CGG CTG GGC TGT GGC CTG GAT TCT GGG CCC ATG 494

Lys Gln Trp Lys Ala Arg Leu Gly Cys Gly Leu Asp Ser Gly Pro Met

125 130 135

GCC CTG GAG CAG AAC CTG TCA GAC CTG CAG CTG CAG GCA GCC GTG GAA 542

Ala Leu Glu Gln Asn Leu Ser Asp Leu Gln Leu Gln Ala Ala Val Glu

140 145 150 155

GTG GCG GGG GAC CTG GAG GTC GAT TGC TAC CGG GCA CCC TGC TCC GAG 590

Val Ala Gly Asp Leu Glu Val Asp Cys Tyr Arg Ala Pro Cys Ser Glu

160 165 170

AGC CAG GAG GAG CTG GCC CTG CAG CAC TTC ATG AAG GAG AAG CTT CTG 638

Ser Gln Glu Glu Leu Ala Leu Gln His Phe Met Lys Glu Lys Leu Leu

175 180 185

GCT GAG CTG GAG GGG AAG CTG CGT GTG TTT GAG AAC AAT GTT GCT GTC 686

Ala Glu Leu Glu Gly Lys Leu Arg Val Phe Glu Asn Asn Val Ala Val

190 195 200

CTC AAC AAG GAG GTG GAG GCC TCC CAC CTG GCC CTG GCC ACC TCT ATC 734

Leu Asn Lys Glu Val Glu Ala Ser His Leu Ala Leu Ala Thr Ser Ile

205 210 215

CAC CAG AGC CAG CTG GAC CGT GAG CGC ATC CTG AGC TTG GAG CAG AGG 782

His Gln Ser Gln Leu Asp Arg Glu Arg Ile Leu Ser Leu Glu Gln Arg

220 225 230 235

GTG GTG CAG GTT CAG CAG ACC CTG GCC CAG AAA GAC CAG GCC CTG GGC 830

Val Val Gln Val Gln Gln Thr Leu Ala Gln Lys Asp Gln Ala Leu Gly

240 245 250

AAG CTG GAG CAG AGC TTG CGC CTC ATG GAG GAG GCC TCC TTC GAT GGC 878

Lys Leu Glu Gln Ser Leu Arg Leu Met Glu Glu Ala Ser Phe Asp Gly

255 260 265

ACT TTC CTG TGG AAG ATC ACC AGT GTC ACC AGG CGG TGC CAT GAG TCG 926

Thr Phe Leu Trp Lys Ile Thr Ser Val Thr Arg Arg Cys His Glu Ser

270 275 280

GCC TGT GGC AGG ACC GTC AGC CTC TTC TCC CCA GCC TTC TAC ACT GCC 974

Ala Cys Gly Arg Thr Val Ser Leu Phe Ser Pro Ala Phe Tyr Thr Ala

285 290 295

AAG TAT GGC TAC AAG TTG TGC CTG CGG CTG TAC CTG ATT GGA GAT GGC 1022

Lys Tyr Gly Tyr Lys Leu Cys Leu Arg Leu Tyr Leu Ile Gly Asp Gly

300 305 310 315

ACT GGA AAG AGA ACC CAT CTT TCG CTC TTC ATC GTG ATC ATG AGA GGG 1070

Thr Gly Lys Arg Thr His Leu Ser Leu Phe Ile Val Ile Met Arg Gly

320 325 330

GAG TAT GAT GCG CTG CTG CCG TGG CCT TTC CGG AAC AAG GTC ACC TTC 1118

Glu Tyr Asp Ala Leu Leu Pro Trp Pro Phe Arg Asn Lys Val Thr Phe

335 340 345

ATG CTG CTG GAC CAG AAC AAC CGT GAG CAC GCC ATT GAC GCC TTC CGG 1166

Met Leu Leu Asp Gln Asn Asn Arg Glu His Ala Ile Asp Ala Phe Arg

350 355 360

CCT GAC CTA AGC TCA GCG TCC TTC CAG AGG CCC CAG AGT GAA ACC AAC 1214

Pro Asp Leu Ser Ser Ala Ser Phe Gln Arg Pro Gln Ser Glu Thr Asn

365 370 375

GTG GCC AGT GGA TGC CCA CTC TTC TTC CCC CTC AGC AAA CTG CAG TCA 1262

Val Ala Ser Gly Cys Pro Leu Phe Phe Pro Leu Ser Lys Leu Gln Ser

380 385 390 395

CCC AAG CAC GCC TAC GTA AGG ACG ACA CAA TGT TCC TCA AGT GCA TTG 1310

Pro Lys His Ala Tyr Val Arg Thr Thr Gln Cys Ser Ser Ser Ala Leu

400 405 410

TGG AGA CCA GCA CTT AGG GTGGGCGGGG CTCCTGAGGG AGTTCCAACT 1358

Trp Arg Pro Ala Leu Arg

415

CAGAAGGGAG CTAGCCAGAG GACTGTGATG CCCTGCCCTT GGCACCCAAG AACTCAGGGC 1418

ACAAAGATGG GTGAAGGCTG GCATGATCCA AGCAAGATGA GGGGTCGATT CGGGTGGCCA 1478

TCTGGTTAGA TGGCAGGACG TGGGTGGGCC CACAAAGGCA AAGGGTCCAG AAGGAGACAG 1538

GCAGAGCTGC TCCCCTCTGC ACGGACCATG CGACACTGGG AGGCCAGTGA GCCACTCCGG 1598

CCCCGAATGT TGAGGTGGAC TCTCACCAAA TGAGAAGAAA ATGGAACCAG GCTTGGAACC 1658

GTAGGACCCA AGCAGAGAAG CTCTCGGGCT AGGAAGATCT CTGCAGGGCC GCCAGGGAGA 1718

CCTGGACACA GGCCTGCTCT CTTTTTCTCC AGGGTCAGAA ACAGGACCGG GTGGAAGGGA 1778

TGGGGTGCCA GTTTGAATGC AGTCTGTCCA GGCTCGTCAT TGGAGGTGAA CAAGCAAACC 1838

CAGACGGCTC CACTAGGACT TCAAATTGGG GGTTGGATTT GAAGACTTTT AAGTTTCCTT 1898

CCAGCCCAGA AAGTCTCTCA TTCTAGCCTC CTGGCCCAGG TGAGTCCTAG AGCTACAGGG 1958

GTTCTGGAAA CATTCAGGAG CTTCCTGTCC TCCCAGCTCC TCACTCACCT TCAGTAACCC 2018

CCACTGGACT GACCTGGTCC ACAGGGCACC TGCCACCCTG GGCCTGGCAG CTCAGCTTCC 2078

CAACACGCAG GAGCACACCC AGCCCCCACA TCCTGTGCCT CCATCAGCTA AACACCACGT 2138

CACTTCATGC AGGTGAAACC CAGTCACTGT GAGCTCCCAG GTGCAGCCAG AGGCACCTCA 2198

AGAAGAAGAG GGGCATAAAC TTTCCTCTTC CTGCCTAGAG GCCCCACCTT TGGTGCTTTC 2258

CAGAATCCCG TAACACCTGA TTAACTGAGG CATCCACTTC TTTCAGCAGA CTGATCAGGA 2318

CCTCCAAGCC ACTGAGCAAT GTATAACCCC AAAGGGAATT CAA 2361

1344 base pairs

nucleic acid

single

linear

8

GGCGAGGCTG CTGGTGGCTG TGGAGAGCTT GGGGCTTCCT TGGTCGCACC CACCACCTGC 60

CTGCCCACTG GTCAGCCTTC AGGGAACCCT GAGCACCGCC TGGTCTCTTT CCTGTGGCCA 120

GCCCAGAACT GAAGCGCTGC GGC ATG GCG CGC GCC TGC CTC CAG GCC GTC 170

Met Ala Arg Ala Cys Leu Gln Ala Val

1 5

AAG TAC CTC ATG TTC GCC TTC AAC CTG CTC TTC TGG CTG GGA GGC TGT 218

Lys Tyr Leu Met Phe Ala Phe Asn Leu Leu Phe Trp Leu Gly Gly Cys

10 15 20 25

GGC GTG CTG GGT GTC GGC ATC TGG CTG GCC GCA CAA CAG GGG AGC TTT 266

Gly Val Leu Gly Val Gly Ile Trp Leu Ala Ala Gln Gln Gly Ser Phe

30 35 40

GCC ACG CTG TCC TCT TCC TTC CCG TCC CTG TGG GCT GCC AAC CTG CTC 314

Ala Thr Leu Ser Ser Ser Phe Pro Ser Leu Trp Ala Ala Asn Leu Leu

45 50 55

ATC ATC ACC GGC GCC TTT GTC ATG GCC ATC GGC TTC GTG GGC TGC CTG 362

Ile Ile Thr Gly Ala Phe Val Met Ala Ile Gly Phe Val Gly Cys Leu

60 65 70

GGT GCC ATC AAG GAG AAC AAG TGC CTC CTG CTC ACT TTC TTC CTG CTG 410

Gly Ala Ile Lys Glu Asn Lys Cys Leu Leu Leu Thr Phe Phe Leu Leu

75 80 85

CTG CTG CTG GTG TTC CTG CTG GAG GGC ACC ATC GCC ATC CTC TTC TTC 458

Leu Leu Leu Val Phe Leu Leu Glu Gly Thr Ile Ala Ile Leu Phe Phe

90 95 100 105

GCC TAC ACG GAC AAG ATT GAC AGG TAT GCC CAG CAA GAC CTG AAG AAA 506

Ala Tyr Thr Asp Lys Ile Asp Arg Tyr Ala Gln Gln Asp Leu Lys Lys

110 115 120

GGC TTG CAC CTG TAC GGC ACG CAG GGC AAC GTG GGC CTC ACC AAC GCC 554

Gly Leu His Leu Tyr Gly Thr Gln Gly Asn Val Gly Leu Thr Asn Ala

125 130 135

TGG AGC ATC ATC CAG ACC GAC TTC CGC TGC TGT GGC GTC TCC AAC TAC 602

Trp Ser Ile Ile Gln Thr Asp Phe Arg Cys Cys Gly Val Ser Asn Tyr

140 145 150

ACT GAC TGG TTC GAG GTG TAC AAC GCC ACG CGG GTA CCT GAC TCC TGC 650

Thr Asp Trp Phe Glu Val Tyr Asn Ala Thr Arg Val Pro Asp Ser Cys

155 160 165

TGC TTG GAG TTC AGT GAG AGC TGT GGG CTG CAC GCC CCG GCA CTG GTG 698

Cys Leu Glu Phe Ser Glu Ser Cys Gly Leu His Ala Pro Ala Leu Val

170 175 180 185

GAG GGC CGT GCT ACG AGA GGT GAA GGT GTG GCT TCA GGA GAA CTG CTG 746

Glu Gly Arg Ala Thr Arg Gly Glu Gly Val Ala Ser Gly Glu Leu Leu

190 195 200

GCT GTG GGC ATC TTT GGG CTG TGC ACG GCG CTG GTG CAG ATC CTG GGC 794

Ala Val Gly Ile Phe Gly Leu Cys Thr Ala Leu Val Gln Ile Leu Gly

205 210 215

CTG AAC TTC GCC ATG ACC ATG TAC TGG CAA GTG GTC AAG GCA GAC ACC 842

Leu Asn Phe Ala Met Thr Met Tyr Trp Gln Val Val Lys Ala Asp Thr

220 225 230

TAC TGT GCG TAG G CCCCCCACCG CCCGCTTCTC TTTCAAAAGG ACGCCCACGG 895

Tyr Cys Ala

235

GGAGATGGCC GCACCCACAG AGTGTCTTTC CCACCACCAG CCTCGGTGCT CTTTCCCATG 955

CTGGGAGGAG GGAGGGAGGG AAAGTTGCCT GGAGCCCCCG GAACCCTGTT TCTGGAAGGC 1015

CCTAGCTCAG GTGGCTTTCA GGGCCTCCGG ACCCCCCCTG GGAAGGGTGG CCACGTGCTG 1075

GCTTCGGAAC CCAGGGCAGG GGTGGGAGGG GCCTCCAGCA CTTTTTATAT TTACGTATTC 1135

TCCAAAACAG TGTTCACACG GGAGCCAACC TGTGGCCCCC AGCCTCCTGG AAAAAAGGTT 1195

GGCGCTGGAG GAACCGGGTC TTGGCATCCT GGAGGTGGCC CCACTGGTCC TGGTGCTCCA 1255

GGCGGGGCCG TGGACCCCTC ACCTACATTC CATAGTGGGC CCGTGGGGCT CCTGGTGCAT 1315

CTTAATAAAG TGTGAGCAGC AAAAAAAAA 1344

1641 base pairs

nucleic acid

single

linear

9

AGCCCAAGTT GAAGAAAGCC GGGCTGTGCC TGGAAGCCGA GAGAGGCGGT AATATTTAGA 60

AGCTGCACAG GAGAGGAACA TGAACTGACG AGTAAAC ATG TAT GGA AAT TAT TCT 115

Met Tyr Gly Asn Tyr Ser

1 5

CAC TTC ATG AAG TTT CCC GCA GGC TAT GGA GGC TCC CCT GGC CAC ACT 163

His Phe Met Lys Phe Pro Ala Gly Tyr Gly Gly Ser Pro Gly His Thr

10 15 20

GGC TCT ACA TCC ATG AGC CCA TCA GCA GCC TTG TCC ACA GGG AAG CCA 211

Gly Ser Thr Ser Met Ser Pro Ser Ala Ala Leu Ser Thr Gly Lys Pro

25 30 35

ATG GAC AGC CAC CCC AGC TAC ACA GAT ACC CCA GTG AGT GCC CCA CGG 259

Met Asp Ser His Pro Ser Tyr Thr Asp Thr Pro Val Ser Ala Pro Arg

40 45 50

ACT CTG AGT GCA GTG GGG ACC CCC CTC AAT GCC CTG GGC TCT CCA TAT 307

Thr Leu Ser Ala Val Gly Thr Pro Leu Asn Ala Leu Gly Ser Pro Tyr

55 60 65 70

CGA GTC ATC ACC TCT GCC ATG GGC CCA CCC TCA GGA GCA CTT GCA GCG 355

Arg Val Ile Thr Ser Ala Met Gly Pro Pro Ser Gly Ala Leu Ala Ala

75 80 85

CCT CCA GGA ATC AAC TTG GTT GCC CCA CCC AGC TCT CAG CTA AAT GTG 403

Pro Pro Gly Ile Asn Leu Val Ala Pro Pro Ser Ser Gln Leu Asn Val

90 95 100

GTC AAC AGT GTC AGC AGT TCA GAG GAC ATC AAG CCC TTA CCA GGG CTT 451

Val Asn Ser Val Ser Ser Ser Glu Asp Ile Lys Pro Leu Pro Gly Leu

105 110 115

CCC GGG ATT GGA AAC ATG AAC TAC CCA TCC ACC AGC CCC GGA TCT CTG 499

Pro Gly Ile Gly Asn Met Asn Tyr Pro Ser Thr Ser Pro Gly Ser Leu

120 125 130

GTT AAA CAC ATC TGT GCT ATC TGT GGA GAC AGA TCC TCA GGA AAG CAC 547

Val Lys His Ile Cys Ala Ile Cys Gly Asp Arg Ser Ser Gly Lys His

135 140 145 150

TAC GGG GTA TAC AGT TGT GAA GGC TGC AAA GGG TTC TTC AAG AGG ACG 595

Tyr Gly Val Tyr Ser Cys Glu Gly Cys Lys Gly Phe Phe Lys Arg Thr

155 160 165

ATA AGG AAG GAC CTC ATC TAC ACG TGT CGG GAT AAT AAA GAC TGC CTC 643

Ile Arg Lys Asp Leu Ile Tyr Thr Cys Arg Asp Asn Lys Asp Cys Leu

170 175 180

ATT GAC AAG CGT CAG CGC AAC CGC TGC CAG TAC TGT CGC TAT CAG AAG 691

Ile Asp Lys Arg Gln Arg Asn Arg Cys Gln Tyr Cys Arg Tyr Gln Lys

185 190 195

TGC CTT GTC ATG GGC ATG AAG AGG GAA GCT TGT GCA AAG AAG GAA AGA 739

Cys Leu Val Met Gly Met Lys Arg Glu Ala Cys Ala Lys Lys Glu Arg

200 205 210

CAG AGG AGC CGA GAG CGA GCT GAG AGT GAG GCA GAA TGT GCT ACC AGT 787

Gln Arg Ser Arg Glu Arg Ala Glu Ser Glu Ala Glu Cys Ala Thr Ser

215 220 225 230

GGT CAT GAA GAC ATG CCT GTG GAG AGG ATT CTA GAA GCT GAA CTT GCT 835

Gly His Glu Asp Met Pro Val Glu Arg Ile Leu Glu Ala Glu Leu Ala

235 240 245

GTT GAC CCA AAG ACA GAA TCC TAT GGT GAC ATG AAT ATG GAG AAC TCG 883

Val Asp Pro Lys Thr Glu Ser Tyr Gly Asp Met Asn Met Glu Asn Ser

250 255 260

ACA AAT GAC CCT GTT ACC AAC ATA TGT CAT GCT GCT GAC AAG CAG CTT 931

Thr Asn Asp Pro Val Thr Asn Ile Cys His Ala Ala Asp Lys Gln Leu

265 270 275

CAC ACC CTC GGT GAA TGG GCC AAG CGT ATT CCC CAC TTC TCT GAC CTC 979

His Thr Leu Gly Glu Trp Ala Lys Arg Ile Pro His Phe Ser Asp Leu

280 285 290

ACC TTG GAG GAC CAG GTC ATT GTG CTT CGG ACA GGG TGG AAT GAA TTG 1027

Thr Leu Glu Asp Gln Val Ile Val Leu Arg Thr Gly Trp Asn Glu Leu

295 300 305 310

CTG ATT GCC TCT TTC TCC CAC CGC TCA GTT TCC GTG GAG GAT GGC ATC 1075

Leu Ile Ala Ser Phe Ser His Arg Ser Val Ser Val Glu Asp Gly Ile

315 320 325

CCT CTG GCC ACG GGT TTA CAT GTC CAC CGG AGC AGT GCC CAC AG 1119

Pro Leu Ala Thr Gly Leu His Val His Arg Ser Ser Ala His

330 335 340

TGCTGGGGTC GGCTCCATCT TTGACAGAGC TCTAACTGAG CTGGTTTCCA AACTGAAAGA 1179

CATGCAGGTG GACAAGTCGG AACTGGGATG CCTGCGAGCC ATTGTTCTCT TTCAACCCCA 1239

GATGCCCAAG GGCCTGCCCA CCCCCTTTGA GGTGGAGACT CTGCGAAAGA AGGTTTATGC 1299

CACCCTTGAG GCCCACCACC AAGCAGAATA TCCGGAACAG CCAGGCAAGG TTTGCCAAGC 1359

TGCTGTGCGC CTCCCAGCTC TGCGTTCCAT TGGCTTGAAA TGCCTGGAGC ACCTCTTCTT 1419

CTTCAAGCTC ATCGGGGACA CCCCCATTGA CACCTTCCTC ATGGAGATGT TGGAGACCCC 1479

GCTGCAGATC ACCTGAGCCC CACCAGCCAA AGCCTCCCCA CCCAGGATGA CCCCTGGGCA 1539

GGTGTGTGTG GACCCCCACC CTGCACTTTC CTCCACCTCC CACCCTGACC CCCTTCCTGT 1599

CCCCAAAATG TGATGCTTAT AATAAAGAAA ACCTTTCTAC AA 1641

1185 base pairs

nucleic acid

single

linear

10

ATG GCG TTG GAG GTC GGC GAT ATG GAA GAT GGG CAG CTT TCC GAC TCG 48

Met Ala Leu Glu Val Gly Asp Met Glu Asp Gly Gln Leu Ser Asp Ser

1 5 10 15

GAT TCC GAC ATG ACG GTC GCA CCC AGC GAC AGG CCG CTG CAA TTG CCA 96

Asp Ser Asp Met Thr Val Ala Pro Ser Asp Arg Pro Leu Gln Leu Pro

20 25 30

AAA GTG CTA GGT GGC GAC AGT GCT ATG AGG GCC TTC CAG AAC ACG GCA 144

Lys Val Leu Gly Gly Asp Ser Ala Met Arg Ala Phe Gln Asn Thr Ala

35 40 45

ACT GCA TGT GCA CCA GTA TCA CAT TAT CGA GCT GTT GAA AGT GTG GAT 192

Thr Ala Cys Ala Pro Val Ser His Tyr Arg Ala Val Glu Ser Val Asp

50 55 60

TCA AGT GAA GAA AGT TTT TCT GAT TCA GAT GAT GAT AGC TGT CTT TGG 240

Ser Ser Glu Glu Ser Phe Ser Asp Ser Asp Asp Asp Ser Cys Leu Trp

65 70 75 80

AAA CGC AAA CGA CAG AAA TGT TTT AAC CCT CCT CCC AAA CCA GAG CCT 288

Lys Arg Lys Arg Gln Lys Cys Phe Asn Pro Pro Pro Lys Pro Glu Pro

85 90 95

TTT CAG TTT GGC CAG AGC AGT CAG AAA CCA CCT GTT GCT GGA GGA AAG 336

Phe Gln Phe Gly Gln Ser Ser Gln Lys Pro Pro Val Ala Gly Gly Lys

100 105 110

AAG ATT AAC AAC ATA TGG GGT GCT GTG CTG CAG GAA CAG AAT CAA GAT 384

Lys Ile Asn Asn Ile Trp Gly Ala Val Leu Gln Glu Gln Asn Gln Asp

115 120 125

GCA GTG GCC ACT GAA CTT GGT ATC TTG GGA ATG GAG GGC ACT ATT GAC 432

Ala Val Ala Thr Glu Leu Gly Ile Leu Gly Met Glu Gly Thr Ile Asp

130 135 140

AGA AGC AGA CAA TCC GAG ACC TAC AAT TAT TTG CTT GCC AAG AAA CTT 480

Arg Ser Arg Gln Ser Glu Thr Tyr Asn Tyr Leu Leu Ala Lys Lys Leu

145 150 155 160

AGG AAG GAA TCT CAA GAG CAT ACA AAA GAT CTA GAC AAG GAA CTA GAT 528

Arg Lys Glu Ser Gln Glu His Thr Lys Asp Leu Asp Lys Glu Leu Asp

165 170 175

GAA TAT ATG CAT GGT GGC AAA AAA ATG GGA TCA AAG GAA GAG GAA AAT 576

Glu Tyr Met His Gly Gly Lys Lys Met Gly Ser Lys Glu Glu Glu Asn

180 185 190

GGG CAA GGT CAT CTC AAA AGG AAA CGA CCT GTC AAA GAC AGG CTA GGG 624

Gly Gln Gly His Leu Lys Arg Lys Arg Pro Val Lys Asp Arg Leu Gly

195 200 205

AAC AGA CCA GAA ATG AAC TAT AAA GGT CGA TAC GAG ATC ACA GCG GAA 672

Asn Arg Pro Glu Met Asn Tyr Lys Gly Arg Tyr Glu Ile Thr Ala Glu

210 215 220

GAT TCT CAA GAG AAA GTG GCT GAT GAA ATT TCA TTC AGG TTA CAG GAA 720

Asp Ser Gln Glu Lys Val Ala Asp Glu Ile Ser Phe Arg Leu Gln Glu

225 230 235 240

CCA AAG AAA GAC CTG ATA GCC CGA GTA GTG AGG ATT ATT GGT AAC AAA 768

Pro Lys Lys Asp Leu Ile Ala Arg Val Val Arg Ile Ile Gly Asn Lys

245 250 255

AAG GCA ATT GAA CTT CTG ATG GAA ACC GCT GAA GTT GAA CAA AAT GGT 816

Lys Ala Ile Glu Leu Leu Met Glu Thr Ala Glu Val Glu Gln Asn Gly

260 265 270

GGT CTC TTT ATA ATG AAT GGT AGT CGA AGA AGA ACA CCA GGT GGA GTT 864

Gly Leu Phe Ile Met Asn Gly Ser Arg Arg Arg Thr Pro Gly Gly Val

275 280 285

TTT CTG AAT CTC TTG AAA AAC ACT CCT AGT ATC AGC GAG GAA CAA ATT 912

Phe Leu Asn Leu Leu Lys Asn Thr Pro Ser Ile Ser Glu Glu Gln Ile

290 295 300

AAG GAC ATT TTC TAC ATT GAA AAC CAA AAG GAA TAT GAA AAT AAA AAA 960

Lys Asp Ile Phe Tyr Ile Glu Asn Gln Lys Glu Tyr Glu Asn Lys Lys

305 310 315 320

GCT GCT AGG AAG AGG AGA ACA CAA GTG TTG GGG AAA AAG ATG AAA CAA 1008

Ala Ala Arg Lys Arg Arg Thr Gln Val Leu Gly Lys Lys Met Lys Gln

325 330 335

GCT ATT AAA AGT CTA AAT TTT CAA GAA GAT GAT GAT ACA TCA CGA GAA 1056

Ala Ile Lys Ser Leu Asn Phe Gln Glu Asp Asp Asp Thr Ser Arg Glu

340 345 350

ACT TTT GCA AGT GAC ACG AAT GAG GCC TTG GCC TCT CTT GAT GAG TCA 1104

Thr Phe Ala Ser Asp Thr Asn Glu Ala Leu Ala Ser Leu Asp Glu Ser

355 360 365

CAG GAA GGA CAT GCA GAA GCC AAG TTG GAG GCA GAG GAA GCC ATT GAA 1152

Gln Glu Gly His Ala Glu Ala Lys Leu Glu Ala Glu Glu Ala Ile Glu

370 375 380

GTT GAT CAT TCT CAT GAT TTG GAC ATC TTT TAA 1185

Val Asp His Ser His Asp Leu Asp Ile Phe

385 390

638 base pairs

nucleic acid

single

linear

11

CACCTGCGCA GGCTTGGCTG CGCCTCTCGC GCCGCACGCT CTGGGGTTCC TCCCTTCTTC 60

CGAGCCTCTC CTCTGGCCGC CGCGCGGGAG AGAGGCCGAG ATG GCA GAT GAG ATT 115

Met Ala Asp Glu Ile

1 5

GCC AAG GCT CAG GTC GCT CGG CCT GGT GGC GAC ACG ATC TTT GGG AAG 163

Ala Lys Ala Gln Val Ala Arg Pro Gly Gly Asp Thr Ile Phe Gly Lys

10 15 20

ATC ATC CGC AAG GAA ATA CCA GCC AAA ATC ATT TTT GAG GAT GAC CGG 211

Ile Ile Arg Lys Glu Ile Pro Ala Lys Ile Ile Phe Glu Asp Asp Arg

25 30 35

TGC CTT GCT TTC CAT GAC ATT TCC CCT CAA GCA CCA ACA CAT TTT CTG 259

Cys Leu Ala Phe His Asp Ile Ser Pro Gln Ala Pro Thr His Phe Leu

40 45 50

GTG ATA CCC AAG AAA CAT ATA TCC CAG ATT TCT GTG GCA GAA GAT GAT 307

Val Ile Pro Lys Lys His Ile Ser Gln Ile Ser Val Ala Glu Asp Asp

55 60 65

GAT GAA AGT CTT CTT GGA CAC TTA ATG ATT GTT GGC AAG AAA TGT GCT 355

Asp Glu Ser Leu Leu Gly His Leu Met Ile Val Gly Lys Lys Cys Ala

70 75 80 85

GCT GAT CTG GGC CTG AAT AAG GGT TAT CGA ATG GTG GTG AAT GAA GGT 403

Ala Asp Leu Gly Leu Asn Lys Gly Tyr Arg Met Val Val Asn Glu Gly

90 95 100

TCA GAT GGT GGA CAG TCT GTC TAT CAC GTT CAT CTC CAT GTT CTT GGA 451

Ser Asp Gly Gly Gln Ser Val Tyr His Val His Leu His Val Leu Gly

105 110 115

GGT CGG CAA ATG CAT TGG CCT CCT GGT TAA GCACGTTTTG GGGATAATTT 501

Gly Arg Gln Met His Trp Pro Pro Gly

120 125

TCTCTTCTTT AGGCAATGAT TAAGTTAGGC AATTTCCAGT ATGTTAAGTA ACACCTTAT 561

TTTGCCTGTG TATGGAGAGA TTCAAGAAAT AATTTTAAAA CCGCATACAT AATAAAAGA 621

ATTGTTGCAT GGCTTAT 638

127 amino acids

amino acid

linear

protein

12

Met Asp Val Phe Lys Lys Gly Phe Ser Ile Ala Lys Lys Gly Val

5 10 15

Val Gly Ala Val Glu Lys Thr Lys Gln Gly Val Thr Glu Ala Ala

20 25 30

Glu Lys Thr Lys Glu Gly Val Met Tyr Val Gly Ala Lys Thr Lys

35 40 45

Glu Asn Val Val Gln Ser Val Thr Ser Val Ala Glu Lys Thr Lys

50 55 60

Glu Gln Ala Asn Ala Val Ser Lys Ala Val Val Ser Ser Val Asn

65 70 75

Thr Val Ala Thr Lys Thr Val Glu Glu Ala Glu Asn Ile Ala Val

80 85 90

Thr Ser Gly Val Val Arg Lys Glu Asp Leu Arg Pro Ser Ala Pro

95 100 105

Gln Gln Glu Gly Glu Ala Ser Lys Glu Lys Glu Glu Val Ala Glu

110 115 120

Glu Ala Gln Ser Gly Gly Asp

125

443 amino acids

amino acid

linear

protein

13

Met Asp Arg Leu Gly Ser Phe Ser Asn Asp Pro Ser Asp Lys Pro

5 10 15

Pro Cys Arg Gly Cys Ser Ser Tyr Leu Met Glu Pro Tyr Ile Lys

20 25 30

Cys Ala Glu Cys Gly Pro Pro Pro Phe Phe Leu Cys Leu Gln Cys

35 40 45

Phe Thr Arg Gly Phe Glu Tyr Lys Lys His Gln Ser Asp His Thr

50 55 60

Tyr Glu Ile Met Thr Ser Asp Phe Pro Val Leu Asp Pro Ser Trp

65 70 75

Thr Ala Gln Glu Glu Met Ala Leu Leu Glu Ala Val Met Asp Cys

80 85 90

Gly Phe Gly Asn Trp Gln Asp Val Ala Asn Gln Met Cys Thr Lys

95 100 105

Thr Lys Glu Glu Cys Glu Lys His Tyr Met Lys His Phe Ile Asn

110 115 120

Asn Pro Leu Phe Ala Ser Thr Leu Leu Asn Leu Lys Gln Ala Glu

125 130 135

Glu Ala Lys Thr Ala Asp Thr Ala Ile Pro Phe His Ser Thr Asp

140 145 150

Asp Pro Pro Arg Pro Thr Phe Asp Ser Leu Leu Ser Arg Asp Met

155 160 165

Ala Gly Tyr Met Pro Ala Arg Ala Asp Phe Ile Glu Glu Phe Asp

170 175 180

Asn Tyr Ala Glu Trp Asp Leu Arg Asp Ile Asp Phe Val Glu Asp

185 190 195

Asp Ser Asp Ile Leu His Ala Leu Lys Met Ala Val Val Asp Ile

200 205 210

Tyr His Ser Arg Leu Lys Glu Arg Gln Arg Arg Lys Lys Ile Ile

215 220 225

Arg Asp His Gly Leu Ile Asn Leu Arg Lys Phe Gln Leu Met Glu

230 235 240

Arg Arg Tyr Pro Lys Glu Val Gln Asp Leu Tyr Glu Thr Met Arg

245 250 255

Arg Phe Ala Arg Ile Val Gly Pro Val Glu His Asp Lys Phe Ile

260 265 270

Glu Ser His Ala Leu Glu Phe Glu Leu Arg Arg Glu Ile Lys Arg

275 280 285

Leu Gln Glu Tyr Arg Thr Ala Gly Ile Thr Asn Phe Cys Ser Ala

290 295 300

Arg Thr Tyr Asp His Leu Lys Lys Thr Arg Glu Glu Glu Arg Leu

305 310 315

Lys Arg Thr Met Leu Ser Glu Val Leu Gln Tyr Ile Gln Asp Ser

320 325 330

Ser Ala Cys Gln Gln Trp Leu Arg Arg Gln Ala Asp Ile Asp Ser

335 340 345

Gly Leu Ser Pro Ser Ile Pro Met Ala Ser Asn Ser Gly Arg Arg

350 355 360

Ser Ala Pro Pro Leu Asn Leu Thr Gly Leu Pro Gly Thr Glu Lys

365 370 375

Leu Asn Glu Lys Glu Lys Glu Leu Cys Gln Met Val Arg Leu Val

380 385 390

Pro Gly Ala Tyr Leu Glu Tyr Lys Ser Ala Leu Leu Asn Glu Cys

395 400 405

Asn Lys Gln Gly Gly Leu Arg Leu Ala Gln Ala Arg Ala Leu Ile

410 415 420

Lys Ile Asp Val Asn Lys Thr Arg Lys Ile Tyr Asp Phe Leu Ile

425 430 435

Arg Glu Gly Tyr Ile Thr Lys Gly

440

186 amino acids

amino acid

linear

protein

14

Met Asp Ala Asp Ser Asp Val Ala Leu Asp Ile Leu Ile Thr Asn

5 10 15

Val Val Cys Val Phe Arg Thr Arg Cys His Leu Asn Leu Arg Lys

20 25 30

Ile Ala Leu Glu Gly Ala Asn Val Ile Tyr Lys Arg Asp Val Gly

35 40 45

Lys Val Leu Met Lys Leu Arg Lys Pro Arg Ile Thr Ala Thr Ile

50 55 60

Trp Ser Ser Gly Lys Ile Ile Cys Thr Gly Ala Thr Ser Glu Glu

65 70 75

Glu Ala Lys Phe Gly Ala Arg Arg Leu Ala Arg Ser Leu Gln Lys

80 85 90

Leu Gly Phe Gln Val Ile Phe Thr Asp Phe Lys Val Val Asn Val

95 100 105

Leu Ala Val Cys Asn Met Pro Phe Glu Ile Arg Leu Pro Glu Phe

110 115 120

Thr Lys Asn Asn Arg Pro His Ala Ser Tyr Glu Pro Glu Leu His

125 130 135

Pro Ala Val Cys Tyr Arg Ile Lys Ser Leu Arg Ala Thr Leu Gln

140 145 150

Ile Phe Ser Thr Gly Ser Ile Thr Val Thr Gly Pro Asn Val Lys

155 160 165

Ala Val Ala Thr Ala Val Glu Gln Ile Tyr Pro Phe Val Phe Glu

170 175 180

Ser Arg Lys Glu Ile Leu

185

117 amino acids

amino acid

linear

protein

15

Met Asn Ala Pro Pro Ala Phe Glu Ser Phe Leu Leu Phe Glu Gly

5 10 15

Glu Lys Lys Ile Thr Ile Asn Lys Asp Thr Lys Val Pro Asn Ala

20 25 30

Cys Leu Phe Thr Ile Asn Lys Glu Asp His Thr Leu Gly Asn Ile

35 40 45

Ile Lys Ser Gln Leu Leu Lys Asp Pro Gln Val Leu Phe Ala Gly

50 55 60

Tyr Lys Val Pro His Pro Leu Glu His Lys Ile Ile Ile Arg Val

65 70 75

Gln Thr Thr Pro Asp Tyr Ser Pro Gln Glu Ala Phe Thr Asn Ala

80 85 90

Ile Thr Asp Leu Ile Ser Glu Leu Ser Leu Leu Glu Glu Arg Phe

95 100 105

Arg Val Ala Ile Lys Asp Lys Gln Glu Gly Ile Glu

110 115

427 amino acids

amino acid

linear

protein

16

Met Gly Thr Pro Lys Pro Arg Xaa Leu Pro Trp Leu Val Ser Gln

5 10 15

Leu Asp Leu Gly Gln Leu Glu Gly Val Ala Trp Val Asn Lys Ser

20 25 30

Arg Thr Arg Phe Arg Ile Pro Trp Lys His Gly Leu Arg Gln Asp

35 40 45

Ala Gln Gln Glu Asp Phe Gly Ile Phe Gln Ala Trp Ala Glu Ala

50 55 60

Thr Gly Ala Tyr Val Pro Gly Arg Asp Lys Pro Asp Leu Pro Thr

65 70 75

Trp Lys Arg Asn Phe Arg Ser Ala Leu Asn Arg Lys Glu Gly Leu

80 85 90

Arg Leu Ala Glu Asp Arg Ser Lys Asp Pro His Asp Pro His Lys

95 100 105

Ile Tyr Glu Phe Val Asn Ser Gly Val Gly Asp Phe Ser Gln Pro

110 115 120

Asp Thr Ser Pro Asp Thr Asn Gly Gly Gly Ser Thr Ser Asp Thr

125 130 135

Gln Glu Asp Ile Leu Asp Glu Leu Leu Gly Asn Met Val Leu Ala

140 145 150

Pro Leu Pro Asp Pro Gly Pro Pro Ser Leu Ala Val Ala Pro Glu

155 160 165

Pro Cys Pro Gln Pro Leu Arg Ser Pro Ser Leu Asp Asn Pro Thr

170 175 180

Pro Phe Pro Asn Leu Gly Pro Ser Glu Asn Pro Leu Lys Arg Leu

185 190 195

Leu Val Pro Gly Glu Glu Trp Glu Phe Glu Val Thr Ala Phe Tyr

200 205 210

Arg Gly Arg Gln Val Phe Gln Gln Thr Ile Ser Cys Pro Glu Gly

215 220 225

Leu Arg Leu Val Gly Ser Glu Val Gly Asp Arg Thr Leu Pro Gly

230 235 240

Trp Pro Val Thr Leu Pro Asp Pro Gly Met Ser Leu Thr Asp Arg

245 250 255

Gly Val Met Ser Tyr Val Arg His Val Leu Ser Cys Leu Gly Gly

260 265 270

Gly Leu Ala Leu Trp Arg Ala Gly Gln Trp Leu Trp Ala Gln Arg

275 280 285

Leu Gly His Cys His Thr Tyr Trp Ala Val Ser Glu Glu Leu Leu

290 295 300

Pro Asn Ser Gly His Gly Pro Asp Gly Glu Val Pro Lys Asp Lys

305 310 315

Glu Gly Gly Val Phe Asp Leu Gly Pro Phe Ile Val Asp Leu Ile

320 325 330

Thr Phe Thr Glu Gly Ser Gly Arg Ser Pro Arg Tyr Ala Leu Trp

335 340 345

Phe Cys Val Gly Glu Ser Trp Pro Gln Asp Gln Pro Trp Thr Lys

350 355 360

Arg Leu Val Met Val Lys Val Val Pro Thr Cys Leu Arg Ala Leu

365 370 375

Val Glu Met Ala Arg Val Gly Gly Ala Ser Ser Leu Glu Asn Thr

380 385 390

Val Asp Leu His Ile Ser Asn Ser His Pro Leu Ser Leu Thr Ser

395 400 405

Asp Gln Tyr Lys Ala Tyr Leu Gln Asp Leu Val Glu Gly Met Asp

410 415 420

Phe Gln Gly Pro Gly Glu Ser

425

252 amino acids

amino acid

linear

protein

17

Met Gly Gln Cys Gly Ile Thr Ser Ser Lys Thr Val Leu Val Phe

5 10 15

Leu Asn Leu Ile Phe Trp Gly Ala Ala Gly Ile Leu Cys Tyr Val

20 25 30

Gly Ala Tyr Val Phe Ile Thr Tyr Asp Asp Tyr Asp His Phe Phe

35 40 45

Glu Asp Val Tyr Thr Leu Ile Pro Ala Val Val Ile Ile Ala Val

50 55 60

Gly Ala Leu Leu Phe Ile Ile Gly Leu Ile Gly Cys Cys Ala Thr

65 70 75

Ile Arg Glu Ser Arg Cys Gly Leu Ala Thr Phe Val Ile Ile Leu

80 85 90

Leu Leu Val Phe Val Thr Glu Val Val Val Val Val Leu Gly Tyr

95 100 105

Val Tyr Arg Ala Lys Val Glu Asn Glu Val Asp Arg Ser Ile Gln

110 115 120

Lys Val Tyr Lys Thr Tyr Asn Gly Thr Asn Pro Asp Ala Ala Ser

125 130 135

Arg Ala Ile Asp Tyr Val Gln Arg Gln Leu His Cys Cys Gly Ile

140 145 150

His Asn Tyr Ser Asp Trp Glu Asn Thr Asp Trp Phe Lys Glu Thr

155 160 165

Lys Asn Gln Ser Val Pro Leu Ser Cys Cys Arg Glu Thr Ala Ser

170 175 180

Asn Cys Asn Gly Ser Trp Pro Pro Phe Arg Leu Tyr Ala Glu Gly

185 190 195

Cys Glu Ala Leu Val Val Lys Lys Leu Gln Glu Ile Met Met His

200 205 210

Val Ile Trp Ala Ala Leu Ala Phe Ala Ala Ile Gln Leu Leu Gly

215 220 225

Met Leu Cys Ala Cys Ile Val Leu Cys Arg Arg Ser Arg Asp Pro

230 235 240

Ala Tyr Glu Leu Leu Ile Thr Gly Gly Thr Tyr Ala

245 250

417 amino acids

amino acid

linear

protein

18

Met Ala Ser Ser Ser Gly Ser Ser Pro Arg Pro Ala Pro Asp Glu

5 10 15

Asn Glu Phe Pro Phe Gly Cys Pro Pro Thr Val Cys Gln Asp Pro

20 25 30

Lys Glu Pro Arg Ala Leu Cys Cys Ala Gly Cys Leu Ser Glu Asn

35 40 45

Pro Arg Asn Gly Glu Asp Gln Ile Cys Pro Lys Cys Arg Gly Glu

50 55 60

Asp Leu Gln Ser Ile Ser Pro Gly Ser Arg Leu Arg Thr Gln Glu

65 70 75

Lys Val Gln Ala Glu Val Ala Glu Ala Gly Ile Gly Cys Pro Phe

80 85 90

Ala Val Val Gly Cys Ser Phe Lys Gly Ser Pro Gln Phe Val Glu

95 100 105

Glu His Glu Val Thr Ser Gln Thr Ser His Leu Asn Leu Leu Leu

110 115 120

Gly Phe Met Lys Gln Trp Lys Ala Arg Leu Gly Cys Gly Leu Asp

125 130 135

Ser Gly Pro Met Ala Leu Glu Gln Asn Leu Ser Asp Leu Gln Leu

140 145 150

Gln Ala Ala Val Glu Val Ala Gly Asp Leu Glu Val Asp Cys Tyr

155 160 165

Arg Ala Pro Cys Ser Glu Ser Gln Glu Glu Leu Ala Leu Gln His

170 175 180

Phe Met Lys Glu Lys Leu Leu Ala Glu Leu Glu Gly Lys Leu Arg

185 190 195

Val Phe Glu Asn Asn Val Ala Val Leu Asn Lys Glu Val Glu Ala

200 205 210

Ser His Leu Ala Leu Ala Thr Ser Ile His Gln Ser Gln Leu Asp

215 220 225

Arg Glu Arg Ile Leu Ser Leu Glu Gln Arg Val Val Gln Val Gln

230 235 240

Gln Thr Leu Ala Gln Lys Asp Gln Ala Leu Gly Lys Leu Glu Gln

245 250 255

Ser Leu Arg Leu Met Glu Glu Ala Ser Phe Asp Gly Thr Phe Leu

260 265 270

Trp Lys Ile Thr Ser Val Thr Arg Arg Cys His Glu Ser Ala Cys

275 280 285

Gly Arg Thr Val Ser Leu Phe Ser Pro Ala Phe Tyr Thr Ala Lys

290 295 300

Tyr Gly Tyr Lys Leu Cys Leu Arg Leu Tyr Leu Ile Gly Asp Gly

305 310 315

Thr Gly Lys Arg Thr His Leu Ser Leu Phe Ile Val Ile Met Arg

320 325 330

Gly Glu Tyr Asp Ala Leu Leu Pro Trp Pro Phe Arg Asn Lys Val

335 340 345

Thr Phe Met Leu Leu Asp Gln Asn Asn Arg Glu His Ala Ile Asp

350 355 360

Ala Phe Arg Pro Asp Leu Ser Ser Ala Ser Phe Gln Arg Pro Gln

365 370 375

Ser Glu Thr Asn Val Ala Ser Gly Cys Pro Leu Phe Phe Pro Leu

380 385 390

Ser Lys Leu Gln Ser Pro Lys His Ala Tyr Val Arg Thr Thr Gln

395 400 405

Cys Ser Ser Ser Ala Leu Trp Arg Pro Ala Leu Arg

410 415

236 amino acids

amino acid

linear

protein

19

Met Ala Arg Ala Cys Leu Gln Ala Val Lys Tyr Leu Met Phe Ala

5 10 15

Phe Asn Leu Leu Phe Trp Leu Gly Gly Cys Gly Val Leu Gly Val

20 25 30

Gly Ile Trp Leu Ala Ala Gln Gln Gly Ser Phe Ala Thr Leu Ser

35 40 45

Ser Ser Phe Pro Ser Leu Trp Ala Ala Asn Leu Leu Ile Ile Thr

50 55 60

Gly Ala Phe Val Met Ala Ile Gly Phe Val Gly Cys Leu Gly Ala

65 70 75

Ile Lys Glu Asn Lys Cys Leu Leu Leu Thr Phe Phe Leu Leu Leu

80 85 90

Leu Leu Val Phe Leu Leu Glu Gly Thr Ile Ala Ile Leu Phe Phe

95 100 105

Ala Tyr Thr Asp Lys Ile Asp Arg Tyr Ala Gln Gln Asp Leu Lys

110 115 120

Lys Gly Leu His Leu Tyr Gly Thr Gln Gly Asn Val Gly Leu Thr

125 130 135

Asn Ala Trp Ser Ile Ile Gln Thr Asp Phe Arg Cys Cys Gly Val

140 145 150

Ser Asn Tyr Thr Asp Trp Phe Glu Val Tyr Asn Ala Thr Arg Val

155 160 165

Pro Asp Ser Cys Cys Leu Glu Phe Ser Glu Ser Cys Gly Leu His

170 175 180

Ala Pro Ala Leu Val Glu Gly Arg Ala Thr Arg Gly Glu Gly Val

185 190 195

Ala Ser Gly Glu Leu Leu Ala Val Gly Ile Phe Gly Leu Cys Thr

200 205 210

Ala Leu Val Gln Ile Leu Gly Leu Asn Phe Ala Met Thr Met Tyr

215 220 225

Trp Gln Val Val Lys Ala Asp Thr Tyr Cys Ala

230 235

340 amino acids

amino acid

linear

protein

20

Met Tyr Gly Asn Tyr Ser His Phe Met Lys Phe Pro Ala Gly Tyr

5 10 15

Gly Gly Ser Pro Gly His Thr Gly Ser Thr Ser Met Ser Pro Ser

20 25 30

Ala Ala Leu Ser Thr Gly Lys Pro Met Asp Ser His Pro Ser Tyr

35 40 45

Thr Asp Thr Pro Val Ser Ala Pro Arg Thr Leu Ser Ala Val Gly

50 55 60

Thr Pro Leu Asn Ala Leu Gly Ser Pro Tyr Arg Val Ile Thr Ser

65 70 75

Ala Met Gly Pro Pro Ser Gly Ala Leu Ala Ala Pro Pro Gly Ile

80 85 90

Asn Leu Val Ala Pro Pro Ser Ser Gln Leu Asn Val Val Asn Ser

95 100 105

Val Ser Ser Ser Glu Asp Ile Lys Pro Leu Pro Gly Leu Pro Gly

110 115 120

Ile Gly Asn Met Asn Tyr Pro Ser Thr Ser Pro Gly Ser Leu Val

125 130 135

Lys His Ile Cys Ala Ile Cys Gly Asp Arg Ser Ser Gly Lys His

140 145 150

Tyr Gly Val Tyr Ser Cys Glu Gly Cys Lys Gly Phe Phe Lys Arg

155 160 165

Thr Ile Arg Lys Asp Leu Ile Tyr Thr Cys Arg Asp Asn Lys Asp

170 175 180

Cys Leu Ile Asp Lys Arg Gln Arg Asn Arg Cys Gln Tyr Cys Arg

185 190 195

Tyr Gln Lys Cys Leu Val Met Gly Met Lys Arg Glu Ala Cys Ala

200 205 210

Lys Lys Glu Arg Gln Arg Ser Arg Glu Arg Ala Glu Ser Glu Ala

215 220 225

Glu Cys Ala Thr Ser Gly His Glu Asp Met Pro Val Glu Arg Ile

230 235 240

Leu Glu Ala Glu Leu Ala Val Asp Pro Lys Thr Glu Ser Tyr Gly

245 250 255

Asp Met Asn Met Glu Asn Ser Thr Asn Asp Pro Val Thr Asn Ile

260 265 270

Cys His Ala Ala Asp Lys Gln Leu His Thr Leu Gly Glu Trp Ala

275 280 285

Lys Arg Ile Pro His Phe Ser Asp Leu Thr Leu Glu Asp Gln Val

290 295 300

Ile Val Leu Arg Thr Gly Trp Asn Glu Leu Leu Ile Ala Ser Phe

305 310 315

Ser His Arg Ser Val Ser Val Glu Asp Gly Ile Pro Leu Ala Thr

320 325 330

Gly Leu His Val His Arg Ser Ser Ala His

335 340

394 amino acids

amino acid

linear

protein

21

Met Ala Leu Glu Val Gly Asp Met Glu Asp Gly Gln Leu Ser Asp

5 10 15

Ser Asp Ser Asp Met Thr Val Ala Pro Ser Asp Arg Pro Leu Gln

20 25 30

Leu Pro Lys Val Leu Gly Gly Asp Ser Ala Met Arg Ala Phe Gln

35 40 45

Asn Thr Ala Thr Ala Cys Ala Pro Val Ser His Tyr Arg Ala Val

50 55 60

Glu Ser Val Asp Ser Ser Glu Glu Ser Phe Ser Asp Ser Asp Asp

65 70 75

Asp Ser Cys Leu Trp Lys Arg Lys Arg Gln Lys Cys Phe Asn Pro

80 85 90

Pro Pro Lys Pro Glu Pro Phe Gln Phe Gly Gln Ser Ser Gln Lys

95 100 105

Pro Pro Val Ala Gly Gly Lys Lys Ile Asn Asn Ile Trp Gly Ala

110 115 120

Val Leu Gln Glu Gln Asn Gln Asp Ala Val Ala Thr Glu Leu Gly

125 130 135

Ile Leu Gly Met Glu Gly Thr Ile Asp Arg Ser Arg Gln Ser Glu

140 145 150

Thr Tyr Asn Tyr Leu Leu Ala Lys Lys Leu Arg Lys Glu Ser Gln

155 160 165

Glu His Thr Lys Asp Leu Asp Lys Glu Leu Asp Glu Tyr Met His

170 175 180

Gly Gly Lys Lys Met Gly Ser Lys Glu Glu Glu Asn Gly Gln Gly

185 190 195

His Leu Lys Arg Lys Arg Pro Val Lys Asp Arg Leu Gly Asn Arg

200 205 210

Pro Glu Met Asn Tyr Lys Gly Arg Tyr Glu Ile Thr Ala Glu Asp

215 220 225

Ser Gln Glu Lys Val Ala Asp Glu Ile Ser Phe Arg Leu Gln Glu

230 235 240

Pro Lys Lys Asp Leu Ile Ala Arg Val Val Arg Ile Ile Gly Asn

245 250 255

Lys Lys Ala Ile Glu Leu Leu Met Glu Thr Ala Glu Val Glu Gln

260 265 270

Asn Gly Gly Leu Phe Ile Met Asn Gly Ser Arg Arg Arg Thr Pro

275 280 285

Gly Gly Val Phe Leu Asn Leu Leu Lys Asn Thr Pro Ser Ile Ser

290 295 300

Glu Glu Gln Ile Lys Asp Ile Phe Tyr Ile Glu Asn Gln Lys Glu

305 310 315

Tyr Glu Asn Lys Lys Ala Ala Arg Lys Arg Arg Thr Gln Val Leu

320 325 330

Gly Lys Lys Met Lys Gln Ala Ile Lys Ser Leu Asn Phe Gln Glu

335 340 345

Asp Asp Asp Thr Ser Arg Glu Thr Phe Ala Ser Asp Thr Asn Glu

350 355 360

Ala Leu Ala Ser Leu Asp Glu Ser Gln Glu Gly His Ala Glu Ala

365 370 375

Lys Leu Glu Ala Glu Glu Ala Ile Glu Val Asp His Ser His Asp

380 385 390

Leu Asp Ile Phe

126 AMINO ACIDS

<Unknown>

22

Met Ala Asp Glu Ile Ala Lys Ala Gln Val Ala Arg Pro Gly Gly

5 10 15

Asp Thr Ile Phe Gly Lys Ile Ile Arg Lys Glu Ile Pro Ala Lys

20 25 30

Ile Ile Phe Glu Asp Asp Arg Cys Leu Ala Phe His Asp Ile Ser

35 40 45

Pro Gln Ala Pro Thr His Phe Leu Val Ile Pro Lys Lys His Ile

50 55 60

Ser Gln Ile Ser Val Ala Glu Asp Asp Asp Glu Ser Leu Leu Gly

65 70 75

His Leu Met Ile Val Gly Lys Lys Cys Ala Ala Asp Leu Gly Leu

80 85 90

Asn Lys Gly Tyr Arg Met Val Val Asn Glu Gly Ser Asp Gly Gly

95 100 105

Gln Ser Val Tyr His Val His Leu His Val Leu Gly Gly Arg Gln

110 115 120

Met His Trp Pro Pro Gly

125