Protein tyrosine kinase, JAK3

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to a protein tyrosine kinases and specifically to JAK3, a novel protein tyrosine kinase of the JAK family.
2. Description of Related Art
Proliferation and differentiation of hematopoietic cells is dependent upon the binding of hematopoietic growth factors and cytokines to their respective cell surface receptors (Cross, et al., Cell, 64:271, 1991; Ogawa, M., Blood, 81:2844, 1993; Heimfeld, S., et al., Proc. Natl. Acad. Sci. USA, 88:9902, 1991). Some of these receptors transduce the signal at the cell surface to the cytoplasm through the activation of a tyrosine kinase domain in the cytoplasmic portion of the receptor (e.g., CSF1, c-kit, STK-1/FLT3/FLK2-) (Boyle, W. J., Current Opinion in Oncology, 4:156, 1992, Chiba, T., et al, Nature, 362:646, 1993, Schlessinger, J., et al., Neuron, 9:383, 1992, Ullrich, A. and Schlessinger, J., Cell, 61:203, 1990). Another group of hematopoietic receptors lack intrinsic kinase catalytic domains (e.g., IL-3, GM-CSF, G-CSF, and EPO receptors) (Miyajima, A., et al., Blood, 82:1960, 1993, Fukunaga, R., et al., EMBO, 10:2855, 1991, Wojchowski, D. M., et al., Stem Cells, 11:381, 1993), however, upon binding of their ligands, these receptors activate protein tyrosine phosphorylation of second messengers and the subsequent signal pathways to the cell's nucleus (Kishimoto, T. et al, Science, 258:593, 1992, Stahl, N., et al., Cell, 74:587, 1993).
Tyrosine kinases often play pivotal roles in the proliferation and differentiation of many cell types. Many growth factor receptors contain a tyrosine kinase domain as part of their cytoplasmic tail such that binding by ligand directly activates their tyrosine kinase activity. However, many other receptors do not contain a tyrosine kinase domain in their cytoplasmic tail. Addition of ligand to many cell types expressing these receptors still results in increased levels of phosphotyrosine. The JAK family, a series of related intracellular tyrosine kinases, has recently been shown to link these receptors and other members of the signal transduction pathway.
The JAK family members contain the highly conserved catalytic domain found in other tyrosine kinases (Firmbach-Kraft, I., et al., Oncogene, 5:1329, 1990, Hanks, S. K., et al., Methods in Enzymology, 200: 38, 1991, Hunter, T., Methods in Enzymology, 200:3, 1991, Wilks, A. F., Proc. Natl. Acad. Sci. USA, 86:1603, 1989). One feature that distinguishes the JAK family from other tyrosine kinases is that each member also contains a second kinase-like domain of unknown function (Harpur, A. G., et al., Oncogene, 7:1347, 1992). In addition, the JAK family members do not contain SH2 or SH3 domains, signal peptide sequences, or transmembrane domains, and are localized in the cytoplasm (Wilks, A. F., et al., Molecular and Cellular Biology, 11:2057, 1991).
Three members of the JAK family, JAK1, JAK2, an TYK-2, have been functionally described. The first two members were isolated by a PCR approach utilizing degenerate oligonucleotide primers and TYK-2 was isolated by screening with a tyrosine kinase probe at reduced stringency (Silvennoinen, O. et al., Proc. Natl. Acad. Sci. USA, 90:8429,1993). To date, the JAK family members have been shown to be involved with the receptors for numerous cytokines and growth factors, including IFN .alpha..beta. and .gamma., IL-3, GM-CSF, EPO, GH, CNTF, LIF, OSM, IL-6, and PRL (Argetsinger, L. S., et al., Cell, 74:237, 1993, Luttichen, C., et al., Science, 263:89, 1994, Muller, M., et al., Nature, 366:129, 1993, Stahl, N., et al., Science, 263:92, 1994, Velazquez, L., et al., Cell, 70:313, 1992, Watling, D., et al., Nature, 366:166, 1993, Witthuhn, B. A., et al., Cell, 74:227, 1993, Rui, H., et al., The Journal of Biological Chemistry, 269:5364, 1994). In most cases, the JAK family members seem to associate with the proximal membrane portion of the cytoplasmic domain of the receptor (e.g., gp130, LIFR.beta., EPO) as a constitutive complex (Narazaki, M., et al., Proc. Natl. Acad. Sci. USA, 91:2285, 1994). In other cases, the association is not evident until ligand binding takes place (e.g., GH receptor). In either case, ligand binding results in increased JAK kinase activity.
The first evidence for the functional role of JAK family members was provided when it was shown that TYK-2 could rescue IFN.alpha./.beta. responsiveness in a cell line that had become unresponsive. In a similar fashion, JAK1 and JAK2 have been shown to function in the signalling of interferon pathways, as well. In each case, two different JAKS have been found to act with each type of IFN receptor; JAK2 and TYK-2 are involved exclusively with IFN .gamma. and IFN .alpha./.beta., respectively, whereas JAK1 is involved with both receptors. Stimulation of the IFN.alpha./.beta. receptors by the binding of their respective ligands results in the phosphorylation of p91 (STAT1) and p113 (STAT2), which are subunits of the ISGF3 transcription complex that binds the interferon-stimulated response element (ISRE). In the case of IFN.gamma., p91 alone is phosphorylated, which then binds gamma-activated sequences (GAS) of IFN.gamma. activated genes (Shual, K., et al., Nature, 366:580, 1993, Ihle, J. N., et al., Trends in Biological Science, 19:222, 1994). Because each of these receptors associate with JAK1 it has been suggested that JAK1 may directly phosphorylate p91 (Loh, J. E., et al., Molecular and Cellular Biology, 14:2170, 1994). It has been recently reported that IL-6 (via gp130), which associates with JAK1 and TYK-2, also triggers the activation of p91 (STAT1) (Yuan, J., et al., Molecular and Cellular Biology, 14:1657, 1994). The EPO, and IL-3 receptors are also believed to similarly activate STAT family members. As all of the hematopoietic receptors seem to utilize certain common proteins in their signal transduction pathways, some of the specificity of the pathways may reside in the cell specific expression of STAT family members and their activation by JAK family members (Metcalf, D., Blood, 82:3515, 1993, Darnell, J. E., et al., Science, 264:1415, 1994).
Additional pairs of JAK family members have been found to associate with other receptors (e.g., CNTF, LIF, IL-6) and both become tyrosine phosphorylated upon the stimulation of these receptors (Silvennoinen, O., et al., Nature, 366:583, 1993). It is possible that reciprocal tyrosine phosphorylation between two JAKs is required as phosphorylation of both associating JAKs is necessary for signal transduction to occur. Thus, JAK family members may act in pairs, possibly as heterodimers.
Recently a Drosophila JAK family member, hop, was shown to be required maternally for normal embryonic development (Binari, et al., Genes & Dev., 8:300, 1994). Mutants in hop showed defects in the expression of several paired-rule and segment polarity genes, implicating it in the control of transcription of these genes, a role that could be analogous to the defect in TYK-2, JAK1, or JAK2 in several cell lines that lost IFN responsiveness.
The present invention provides a new member of the JAK protein tyrosine kinase family. The structural homology between the JAK3 of this invention and the other members of JAK family, indicates that JAK3 is a new member of this family of non-receptor tyrosine kinases. In analogy to the other JAK family members, JAK3 is likely involved in the signal transduction pathway of already characterized receptors which lack intrinsic activity. Because of its strong expression in the fraction enriched for CD34+ normal human bone marrow, JAK3 is likely to be important in stem/progenitor cell growth and/or differentiation, by transducing the signals of receptors which modulate these processes. In addition, JAK3 may also be involved in the signal transduction pathways of any of several non-tyrosine kinase receptors with which the other JAK members have not been shown to associate (e.g. IL-2, IL-4, IL-11).
SUMMARY OF THE INVENTION
The present invention provides a novel protein tyrosine kinase JAK3, a polynucleotide sequence which encodes JAK3 and antibodies which are immunoreactive with the protein. The amino acid sequence of JAK3 indicates that it is a new member of the JAK family of non-receptor tyrosine kinases. JAK3 is highly expressed in the CD34+/lin- fraction in normal human bone marrow which is highly enriched in hematopoietic stem/progenitor cells. Therefore, by analogy to other JAK family member, it is likely that JAK3 plays a role in the growth factor modulated differentiation/proliferation of the stem/progenitor cells.
JAK3 is expressed in mammalian tissues, and particularly human tissue. For example, JAK3 is expressed in human hematopoietic tissues, (e.g., bone marrow), and non-hematopoietic human tissues, such as liver, lung, kidney, spleen and intestine. In particular, JAK-3 is most highly expressed in the stem/progenitor cell enriched fraction of normal human bone marrow. JAK-3 is further expressed in a wide range of leukemic derived cell lines including AMLs (KG1, TF-1, HEL), B lineage ALLs (PB697, Nalm-16, and Nalm-6), and T-ALLs (Molt-16, and Molt-3).
JAK3 is localized to chromosome 19, band p12-13.1, where the another member of the JAK family, TYK-2 is co-localized. Several other genes containing tyrosine kinase domains are tandemly linked and may have evolved by cis duplications. Examples include the genes for the receptors of c-fms (CSF-1 receptor) and PDGFR .beta. on chromosome 5 bands q31-q33, c-kit and PDGFR .alpha. on chromosome 4 bands q11-q13, as well as FLT1 and STK-1/FLT3/FLK2 on chromosome 13 band q12.
In another embodiment, the invention provides a method for ameliorating a cell proliferative disorder associated with JAK-3. In another embodiment, the invention provides a method for stimulating stem/progenitor cell proliferation/survival and differentiation in vitro.

BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows nucleotide (cDNA) and predicted amino acid sequence of JAK3, (SEQ ID NO:1 and NO:2, respectively. The predicted amino acids are numbered on the left of each column with the nucleotides of the largest open reading frame numbered on the right, starting with the initiating methionine of JAK3. The conserved tyrosine kinase motifs GXGXXG and DFG are shown boxed. The highly conserved peptide regions chosen for the design of the degenerate oligo nucleotides used for the initial PCR �VHRDLA & DVWSFG! are shown in ovals. Also shown are 167 bases of the 5' untranslated region and 394 bases of the 3' untranslated region. Potential polyadenylation signals are underlined.
FIG. 2 (SEQ ID NO:5-9) shows an amino acid comparison between JAK3 and other JAK family members. The numbering system begins with the initiating methionine of the JAK family members. The numbering system does not take into account the insertion of gaps and, therefore, should be only regarded as a relative measure of location. The consensus sequence (CONS) is derived if three out of four JAK family members have the identical amino acid in that position. The conserved kinase domain of all tyrosine kinases, JAK homology domain 1 (JH1), and the putative second kinase domain, JAK homology domain 2 (JH2) are designated with arrows. With the exception of JAK2, (murine), all sequences are human.
FIG. 3 (SEQ ID NO: 10-12) shows an amino acid comparison between human JAK3 and rat JAK3. The amino acid residues of each member are numbered beginning with the initiating methionine. The consensus sequence (CONS) of the two JAK family members are listed below the compared sequences when identical. Pluses (+) denote conservative amino acid substitutions.
FIG. 4a shows an RNAse protection analysis of JAK3 expression in leukemic derived cell lines. On the left side the undigested full-length JAK3 and actin probes are denoted. The RNA sources are labeled above each lane. To show the specificity of the protected bands, reactions with no RNA and with tRNA (tRNA) were also conducted. The position of the protected JAK3 and actin species are denoted on the right side.
FIG. 4b shows a northern blot analysis of JAK3 expression in leukemic derived cell lines. (Upper half) A Northern blot of poly A+ RNA from the leukemic-derived cell lines noted above each lane was hybridized with a randomly primed .sup.32 P .alpha.-dCTP labeled probe corresponding to a 1.8 kb fragment of JAK3. The relative mobilities of the 28S ribosomal RNA and RNA markers are denoted on the right. The JAK3 band is indicated by an arrow. (Lower half) The blot was stripped and reprobed with actin.
FIG. 4c shows a northern blot analysis of JAK3 expression in non-hematopoietic tissues. A multiple tissue Northern blot (Clonetech, Palo Alto, Calif.) containing 2 ug of poly A+ RNA from human heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas was hybridized with the same probe as in FIG. 4b. The relative mobilities of the RNA markers are denoted on the left. The JAK3 band is indicated by an arrow.
FIG. 5a shows an RNAse protection analysis of JAK3 expression in normal bone marrow fractions. On the left side the undigested, full-length, JAK3 and actin probes are denoted. The RNA sources are labeled above each lane. To show the specificity of the protected bands, reactions with no RNA (None) and with tRNA (tRNA) were also conducted. The protected JAK3 and actin species are denoted on the right side. The unlabelled band that migrates between the JAK3 and actin bands is present in all lanes, including the no RNA and tRNA lanes, and is a result of incomplete digestion of the probe.
FIG. 5b shows a phosphorimage analysis of bone marrow fractions. Following exposure to film, the gel shown in FIG. 5a was exposed to a phosphorimage screen (Molecular Dynamics, Sunnyvale, Calif.). Bands were quantified using the ImageQuantify program and normalized relative to the actin signals.
FIG. 6a shows fluorescence in situ hybridization (FISH) used to identify the localization of JAK3. A plasmid containing approximately 80 kb of the JAK3 genomic DNA was labelled with biotin-14 dATP and hybridized to chromosome spreads made from normal human male lymphocytes cultured with BrdU. Analysis of 36 metaphase cells showed 20 cells (56%) had at least one pair of signals (involving both chromatids of a single chromosome), an example of which is shown. Paired signals are indicated by arrows.
FIG. 6b shows G-banding of chromosome spreads. The same metaphase spread shown in FIG. 6a was G-banded by fluorescence plus Giemsa (FPG) after hybridization, photographed and aligned with the color FISH slides. The position of the paired FISH signals on the G-banded chromosomes are indicated by arrows.
FIG. 6c shows an ideogram of human chromosome 19, revealing localization of JAK3 to 19p12-13.1. Each dot represents a paired signal seen on metaphase chromosomes. Signals clearly located on a single band are diagrammed to the right of the ideogram; those which could not be sublocalized to a single band are assigned to regions diagrammed to the left (brackets).

DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a novel protein tyrosine kinase, JAK3, and a polynucleotide sequence encoding JAK3 polypeptide. The amino acid sequence of JAK3 indicates that it is a new member of the JAK family of non-receptor tyrosine kinases. In normal human bone marrow, JAK3 is highly expressed in the CD34+/lin- fraction which is enriched in hematopoietic stem/progenitor cells. As JAK kinases have been shown to be involved in the signal transduction pathways of various hematopoietic growth factors, it is likely that JAK3 plays a role in the growth factor modulated differentiation/proliferation/survival of hematopoietic stem/progenitor cells.
In a first embodiment, the invention provides a substantially pure JAK3 polypeptide consisting essentially of the amino acid sequence of SEQ ID NO:2. The full-length JAK3 polypeptide sequence has 1082 amino acids with a molecular weight of approximately 121 kD. JAK3 has 48% identity and 67% similarity with JAK2 (murine), 41% identity and 61% similarity with JAK1 (human), and 40% identity and 60% similarity with TYK-2 (human). Comparison of human JAK3 with the rat JAK3 shows 77% identity and 84% similarity (Takahashi, T. and Shirasawa, T., FEBS Letters, 342:124, 1994).
The term "substantially pure" or "isolated" as used herein, refers to JAK3 polypeptide which is substantially free of other proteins, lipids, carbohydrates, nucleic acids, or other materials with which it is naturally associated. One skilled in the art can purify JAK3 using standard techniques for protein purification. The substantially pure polypeptide will yield a single major band on a non-reducing polyacrylamide gel. The purity of the JAK3 polypeptide can also be determined by amino-terminal amino acid sequence analysis.
The invention includes a functional polypeptide, JAK3, and functional fragments thereof. As used herein, the term "functional polypeptide" refers to a polypeptide which possesses a biological function or activity which is identified through a defined functional assay and
The invention includes a functional polypeptide, JAK3, and functional fragments thereof. As used herein, the term "functional polypeptide" refers to a polypeptide whic possesses a biological function or activity which is identified through a defined functional assay and which is associated with a particular biologic, morphologic, or phenotypic alteration in the cell. Functional fragments of the JAK3 polypeptide, includes fragments of JAK3 as long as the activity, e.g., protein tyrosine kinase activity, of JAK3 remains. Smaller peptides containing the biological activity of JAK3 are included in the invention. The biological function, for example, can vary from a polypeptide fragment as small as an epitope to which an antibody molecule can bind to a large polypeptide which is capable of participating in the characteristic induction or programming of phenotypic changes within a cell. An enzymatically functional JAK3 polypeptide or fragment thereof possesses JAK3 tyrosine kinase activity. A "functional polynucleotide" denotes a polynucleotide which encodes a functional polypeptide as described herein.
Minor modifications of the JAK3 primary amino acid sequence may result in proteins which have substantially equivalent activity as compared to the JAK3 polypeptide described herein. Such modifications may be deliberate, as by site-directed mutagenesis, or may be spontaneous. All of the polypeptides produced by these modifications are included herein as long as the tyrosine kinase activity of JAK3 is present. Further, deletion of one or more amino acids can also result in a modification of the structure of the resultant molecule without significantly altering its kinase activity. This can lead to the development of a smaller active molecule which may have broader utility. For example, it is possible to remove amino or carboxyl terminal amino acids which may not be required for JAK3 kinase activity.
The JAK3 polypeptide of the invention also includes conservative variations of the polypeptide sequence. The term "conservative variation" as used herein denotes the replacement of an amino acid residue by another, biologically similar residue. Examples of conservative variations include the substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, and the like. The term "conservative variation" also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid provided that antibodies raised to the substituted polypeptide also immunoreact with the unsubstituted polypeptide.
The invention also provides an isolated polynucleotide sequence consisting essentially of a polynucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO:2. As used herein, "polynucleotide" refers to a polymer of deoxyribonucleotides or ribonucleotides, in the form of a separate fragment or a larger construct. The term "isolated" as used herein includes polynucleotides substantially free of other nucleic acids, proteins, lipids, carbohydrates or other materials with which it is naturally associated. Polynucleotide sequences of the invention include DNA, cDNA and RNA sequences which encode JAK3. It is understood that all polynucleotides encoding all or a portion of JAK3 are also included herein, as long as they encode a polypeptide with JAK3 kinase activity. Such polynucleotides include naturally occurring, synthetic, and intentionally manipulated polynucleotides. For example, JAK3 polynucleotide may be subjected to site-directed mutagenesis. The polynucleotide sequence for JAK3 also includes antisense sequences. The polynucleotides of the invention include sequences that are degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included in the invention as long as the amino acid sequence of JAK3 polypeptide encoded by the nucleotide sequence is functionally unchanged. In addition, the invention also includes a polynucleotide consisting essentially of a polynucleotide sequence encoding a polypeptide having an amino acid sequence of SEQ ID NO:2 and having at least one epitope for an antibody immunoreactive with JAK3 polypeptide.
Specifically disclosed herein is a cDNA sequence which encodes JAK3 which comprising a 3,807 base pair (bp) predicted coding region for JAK3, 167 base pairs of 5' untranslated and 394 base pairs of 3' untranslated sequence (SEQ. ID NO: 1). The cDNA includes an open reading frame of 3,246 base pairs encoding a protein of about 1082 amino acids, having a molecular weight of about 121 kD. The putative initiating methionine shows the strongest homology with the Kozak consensus sequence (Kozak, M., Nucleic Acids Research, 15:8125, 1987). At the 3' end, an in frame stop codon defines the C-terminus of the JAK3 protein at position 3242.
The polynucleotide encoding JAK3 includes the nucleotide sequence in FIG. 1 (SEQ ID NO: 1), as well as nucleic acid sequences complementary to that sequence. A complementary sequence may include an antisense nucleotide. When the sequence is RNA, the deoxynucleotides A, G, C, and T of FIG. 1 are replaced by ribonucleotides A, G, C, and U, respectively. Also included in the invention are fragments of the above-described nucleic acid sequences that are at least 15 bases in length, which is sufficient to permit the fragment to selectively hybridize to DNA that encodes the protein of FIG. 1 (SEQ ID NO: 2) under physiological conditions.
DNA sequences of the invention can be obtained by several methods. For example, the DNA can be isolated using hybridization techniques which are well known in the art. These include, but are not limited to: 1) hybridization of genomic or cDNA libraries with probes to detect homologous nucleotide sequences; 2) antibody screening of expression libraries to detect cloned DNA fragments with shared structural features; and 3) PCR amplification of a desired nucleotide sequence using oligonucleotide primers.
Preferably the JAK3 polynucleotide of the invention is derived from a mammalian organism, and most preferably from human. Screening procedures which rely on nucleic acid hybridization make it possible to isolate any gene sequence from any organism, provided the appropriate probe is available. Oligonucleotide probes, which correspond to a part of the sequence encoding the protein in question, can be synthesized chemically. This requires that short, oligopeptide stretches of amino acid sequence must be known. The DNA sequence encoding the protein can be deduced from the genetic code, however, the degeneracy of the code must be taken into account. It is possible to perform a mixed addition reaction when the sequence is degenerate. This includes a heterogeneous mixture of denatured double-stranded DNA. For such screening, hybridization is preferably performed on either single-stranded DNA or denatured double-stranded DNA. Hybridization is particularly useful in the detection of cDNA clones derived from sources where an extremely low amount of mRNA sequences relating to the polypeptide of interest are present. In other words, by using stringent hybridization conditions directed to avoid non-specific binding, it is possible, for example, to allow the autoradiographic visualization of a specific cDNA clone by the hybridization of the target DNA to that single probe in the mixture which is its complete complement (Wallace, et al., Nucl. Acid Res., 9:879, 1981).
The development of specific DNA sequences encoding JAK3 can also be obtained by: 1) isolation of double-stranded DNA sequences from the genomic DNA; 2) chemical manufacture of a DNA sequence to provide the necessary codons for the polypeptide of interest; and 3) in vitro synthesis of a double-stranded DNA sequence by reverse transcription of mRNA isolated from a eukaryotic donor cell. In the latter case, a double-stranded DNA complement of mRNA is eventually formed which is generally referred to as cDNA.
Of the three above-noted methods for developing specific DNA sequences for use in recombinant procedures, the isolation of genomic DNA isolates is the least common. This is especially true when it is desirable to obtain the microbial expression of mammalian polypeptides due to the presence of introns.
The synthesis of DNA sequences is frequently the method of choice when the entire sequence of amino acid residues of the desired polypeptide product is known. When the entire sequence of amino acid residues of the desired polypeptide is not known, the direct synthesis of DNA sequences is not possible and the method of choice is the synthesis of cDNA sequences. Among the standard procedures for isolating cDNA sequences of interest is the formation of plasmid- or phage-carrying cDNA libraries which are derived from reverse transcription of mRNA which is abundant in donor cells that have a high level of genetic expression. When used in combination with polymerase chain reaction technology, even rare expression products can be cloned. In those cases where significant portions of the amino acid sequence of the polypeptide are known, the production of labeled single or double-stranded DNA or RNA probe sequences duplicating a sequence putatively present in the target cDNA may be employed in DNA/DNA hybridization procedures which are carried out on cloned copies of the cDNA which have been denatured into a single-stranded form (Jay, et al., Nucl. Acid Res., 11:2325, 1983).
A preferred method for obtaining genomic DNA for example is Polymerase Chain Reaction (PCR), which relies on an in vitro method of nucleic acid synthesis by which a particular segment of DNA is specifically replicated. Two oligonucleotide primers that flank the DNA fragment to be amplified are utilized in repeated cycles of heat denaturation of the DNA, annealing of the primers to their complementary sequences, and extension of the annealed primers with DNA polymerase. These primers hybridize to opposite strands of the target sequence and are oriented so that DNA synthesis by the polymerase proceeds across the region between the primers. Since the extension products themselves are also complementary to and capable of binding primers, successive cycles of amplification essentially double the amount of the target DNA synthesized in the previous cycle. The result is an exponential accumulation of the specific target fragment, approximately 2.sup.n, where n is the number of cycles of amplification performed (see PCR Protocols, Eds. Innis, et al., Academic Press, Inc., 1990, incorporated herein by reference).
A cDNA expression library, such as lambda gt11, can be screened indirectly for JAK3 peptides having at least one epitope, using antibodies specific for JAK3. Such antibodies can be either polyclonally or monoclonally derived and used to detect expression product indicative of the presence of JAK3 cDNA.
The polynucleotide sequence for JAK3 also includes sequences complementary to the polynucleotide encoding JAK3 (antisense sequences). Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific mRNA molecule (Weintraub, Scientific American, 262:40, 1990). The invention embraces all antisense polynucleotides capable of inhibiting production of JAK3 polypeptide. In the cell, the antisense nucleic acids hybridize to the corresponding mRNA, forming a double-stranded molecule. The antisense nucleic acids may interfere with the translation of the mRNA since the cell will not translate a mRNA that is double-stranded. Antisense oligomers of about 15 nucleotides are preferred, since they are easily synthesized and are less likely to cause problems than larger molecules when introduced into the target JAK3-producing cell. The use of antisense methods to inhibit the translation of genes is well known in the art (Marcus-Sakura, Anal. Biochem., 172:289, 1988).
In addition, ribozyme nucleotide sequences for JAK3 are included in the invention. Ribozymes are RNA molecules possessing the ability to specifically cleave other single-stranded RNA in a manner analogous to DNA restriction endonucleases. Through the modification of nucleotide sequences which encode these RNAs, it is possible to engineer molecules that recognize specific nucleotide sequences in an RNA molecule and cleave it (Cech, J. Amer. Med. Assn., 260:3030, 1988). A major advantage of this approach is that, because they are sequence-specific, only mRNAs with particular sequences are inactivated.
There are two basic types of ribozymes namely, tetrahymena-type (Hasselhoff, Nature, 334:585, 1988) and "hammerhead"-type. Tetrahymena-type ribozymes recognize sequences which are four bases in length, while "hammerhead"-type ribozymes recognize base sequences 11-18 bases in length. The longer the recognition sequence, the greater the likelihood that sequence will occur exclusively in the target mRNA species. Consequently, hammerhead-type ribozymes are preferable to tetrahymena-type ribozymes for inactivating a specific mRNA species and 18-based recognition sequences are preferable to shorter recognition sequences.
DNA sequences encoding JAK3 can be expressed in vitro by DNA transfer into a suitable host cell. "Host cells" are cells in which a vector can be propagated and its DNA expressed. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term "host cell" is used. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host, are known in the art.
In the present invention, the JAK3 polynucleotide sequences may be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to a plasmid, virus or other vehicle known in the art that has been manipulated by insertion or incorporation of the JAK3 genetic sequences. Such expression vectors contain a promoter sequence which facilitates the efficient transcription of the inserted genetic sequence of the host. The expression vector typically contains an origin of replication, a promoter, as well as specific genes which allow phenotypic selection of the transformed cells. Vectors suitable for use in the present invention include, but are not limited to the T7-based expression vector for expression in bacteria (Rosenberg, et al., Gene, 56:125, 1987), the pMSXND expression vector for expression in mammalian cells (Lee and Nathans, J. Biol. Chem., 263:3521, 1988) and baculovirus-derived vectors for expression in insect cells. The DNA segment can be present in the vector operably linked to regulatory elements, for example, a promoter (e.g., T7, metallothionein I, or polyhedrin promoters).
Polynucleotide sequences encoding JAK3 can be expressed in either prokaryotes or eukaryotes. Hosts can include microbial, yeast, insect and mammalian organisms. Methods of expressing DNA sequences having eukaryotic or viral sequences in prokaryotes are well known in the art. Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host are known in the art. Such vectors are used to incorporate DNA sequences of the invention.
Methods which are well known to those skilled in the art can be used to construct expression vectors containing the JAK3 coding sequence and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination/genetic techniques. See, for example, the techniques described in Maniatis, et al., 1989 Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y.
A variety of host-expression vector systems may be utilized to express the JAK3 coding sequence. These include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing the JAK3 coding sequence; yeast transformed with recombinant yeast expression vectors containing the JAK3 coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the JAK3 coding sequence; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the JAK3 coding sequence; or animal cell systems infected with recombinant virus expression vectors (e.g., retroviruses, adenovirus, vaccinia virus) containing the JAK3 coding sequence, or transformed animal cell systems engineered for stable expression. Since JAK3 has not been confirmed to contain carbohydrates, both bacterial expression systems as well as those that provide for translational and post-translational modifications may be used; e.g., mammalian, insect, yeast or plant expression systems.
Depending on the host/vector system utilized, any of a number of suitable transcription and translation elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector (see e.g., Bitter, et al., 1987, Methods in Enzymology, 153:516-544). For example, when cloning in bacterial systems, inducible promoters such as pL of bacteriophage .gamma., plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used. When cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the retrovirus long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter) may be used. Promoters produced by recombinant DNA or synthetic techniques may also be used to provide for transcription of the inserted JAK3 coding sequence.
In bacterial systems a number of expression vectors may be advantageously selected depending upon the use intended for the expressed. For example, when large quantities of JAK3 are to be produced, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Those which are engineered to contain a cleavage site to aid in recovering are preferred. Such vectors include but are not limited to the E. coli expression vector pUR278 (Ruther, et al., EMBO J., 2:1791, 1983), in which the JAK3 coding sequence may be ligated into the vector in frame with the lac Z coding region so that a hybrid -lac Z protein is produced; pIN vectors (Inouye and Inouye, Nucleic Acids Res., 13:3101, 1985; Van Heeke and Schuster, J. Biol. Chem. 264:5503, 1989) and the like.
In yeast, a number of vectors containing constitutive or inducible promoters may be used. For a review see, Current Protocols in Molecular Biology, Vol. 2, 1988, Ed. Ausubel, et al., Greene Publish. Assoc. & Wiley Interscience, Ch. 13; Grant, et al., 1987, Expression and Secretion Vectors for Yeast, in Methods in Enzymology, Eds. Wu and Grossman, 31987, Acad. Press, N.Y., Vol. 153, pp. 516-544; Glover, 1986, DNA Cloning, Vol. II, IRL Press, Wash., D.C., Ch. 3; and Bitter, 1987, Heterologous Gene Expression in Yeast, Methods in Enzymology, Eds. Berger and Kimmel, Acad. Press, N.Y., Vol. 152, pp. 673-684; and The Molecular Biology of the Yeast Saccharomyces, 1982, Eds. Strathern, et al., Cold Spring Harbor Press, Vols. I and II. A constitutive yeast promoter such as ADH or LEU2 or an inducible promoter such as GAL may be used (Cloning in Yeast, Ch. 3, R. Rothstein In: DNA Cloning Vol.11, A Practical Approach, Ed. D M Glover, 1986, IRL Press, Wash., D.C.). Alternatively, vectors may be used which promote integration of foreign DNA sequences into the yeast chromosome.
In cases where plant expression vectors are used, the expression of the JAK3 coding sequence may be driven by any of a number of promoters. For example, viral promoters such as the 35S RNA and 19S RNA promoters of CaMV (Brisson, et al., Nature, 310:511, 1984), or the coat protein promoter to TMV (Takamatsu, et al., EMBO J., 6:307, 1987) may be used; alternatively, plant promoters such as the small subunit of RUBISCO (Coruzzi, et al., EMBO J., 3:1671-1680, 1984; Broglie, et al., Science, 224:838, 1984); or heat shock promoters, e.g., soybean hsp17.5-E or hsp17.3-B (Gurley, et al., Mol. Cell. Biol., 6:559, 1986) may be used. These constructs can be introduced into plant cells using Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, etc. For reviews of such techniques see, for example, Weissbach and Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp. 421-463; and Grierson and Corey, 1988, Plant Molecular Biology, 2d Ed., Blackie, London, Ch. 7-9.
An alternative expression system which could be used to express is an insect system. In one such system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The JAK3 coding sequence may be cloned into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter). Successful insertion of the JAK3 coding sequence will result in inactivation of the polyhedrin gene and production of non-occluded recombinant virus (i.e., virus lacking the proteinaceous coat coded for by the polyhedrin gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed. (e.g., see Smith, et al., J. Viol., 46:584, 1983; Smith, U.S. Pat. No. 4,215,051).
Eukaryotic systems, and preferably mammalian expression systems, allow for proper post-translational modifications of expressed mammalian proteins to occur. Eukaryotic cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, phosphorylation, and advantageously, secretion of the gene product may be used as host cells for the expression of JAK3. Mammalian cell lines may be preferable. Such host cell lines may include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, -293, and WI38.
Mammalian cell systems which utilize recombinant viruses or viral elements to direct expression may be engineered. For example, when using adenovirus expression vectors, the JAK3 coding sequence may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing the protein in infected hosts (e.g., see Logan and Shenk, Proc. Natl. Acad. Sci. USA, 81:3655, 1984). Alternatively, the vaccinia virus 7.5K promoter may be used. (e.g., see, Mackett, et al., Proc. Natl. Acad. Sci. USA, 79:7415, 1982; Mackett, et al., J. Virol., 49: 857, 1984; Panicali, et al., Proc. Natl. Acad. Sci. USA, 79:4927, 1982). Of particular interest are vectors based on bovine papilloma virus which have the ability to replicate as extrachromosomal elements (Sarver, et al., Mol. Cell. Biol, 1:486, 1981). Shortly after entry of this DNA into mouse cells, the plasmid replicates to about 100 to 200 copies per cell. Transcription of the inserted cDNA does not require integration of the plasmid into the host's chromosome, thereby yielding a high level of expression. These vectors can be used for stable expression by including a selectable marker in the plasmid, such as, for example, the neo gene. Alternatively, the retroviral genome can be modified for use as a vector capable of introducing and directing the expression of the JAK3 gene in host cells (Cone and Mulligan, Proc. Natl. Acad. Sci. USA, 81:6349, 1984). High level expression may also be achieved using inducible promoters, including, but not limited to, the metallothionine IIA promoter and heat shock promoters.
For long-term, high-yield production of recombinant proteins, stable expression is preferred. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with the JAK3 cDNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. For example, following the introduction of foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler, et al., Cell, 11: 223, 1977), hypoxanthine-guanine phosphoribosyltransferase (Szybalska and Szybalski, Proc. Natl. Acad. Sci. USA, 48:2026, 1962), and adenine phosphoribosyltransferase (Lowy, et al., Cell, 22: 817, 1980) genes can be employed in tk.sup.-, hgprt.sup.- or aprt.sup.- cells respectively. Also, antimetabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (Wigler, et al., Natl. Acad. Sci. USA, 77: 3567, 1980; O'Hare, et al., Proc. Natl. Acad. Sci. USA, 78: 1527, 1981); gpt, which confers resistance to mycophenolic acid (Mulligan and Berg, Proc. Natl. Acad. Sci. USA, 78: 2072, 1981; neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin, et al., J. Mol. Biol., 150:1, 1981); and hygro, which confers resistance to hygromycin (Santerre, et al., Gene, 30:147, 1984) genes. Recently, additional selectable genes have been described, namely trpB, which allows cells to utilize indole in place of tryptophan; hisD, which allows cells to utilize histinol in place of histidine (Hartman and Mulligan, Proc. Natl. Acad. Sci. USA, 85:8047, 1988); and ODC (ornithine decarboxylase) which confers resistance to the ornithine decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue L., 1987, In: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory ed.).
Transformation of a host cell with recombinant DNA may be carried out by conventional techniques as are well known to those skilled in the art. Where the host is prokaryotic, such as E. coli, competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCl.sub.2 method using procedures well known in the art. Alternatively, MgCl.sub.2 or RbCl can be used. Transformation can also be performed after forming a protoplast of the host cell if desired.
When the host is a eukaryote, such methods of transfection of DNA as calcium phosphate co-precipitates, conventional mechanical procedures such as microinjection, electroporation, insertion of a plasmid encased in liposomes, or virus vectors may be used. Eukaryotic cells can also be cotransformed with DNA sequences encoding the JAK3 of the invention, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thyridine kinase gene. Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein. (see for example, Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982).
Isolation and purification of microbial expressed polypeptide, or fragments thereof, provided by the invention, may be carried out by conventional means including preparative chromatography and immunological separations involving monoclonal or polyclonal antibodies.
The invention includes antibodies immunoreactive with or which bind to JAK3 polypeptide or functional fragments thereof. Antibody which consists essentially of pooled monoclonal antibodies with different epitopic specificities, as well as distinct monoclonal antibody preparations are provided. Monoclonal antibodies are made from antigen containing fragments of the protein by methods well known to those skilled in the art (Kohler, et al., Nature, 256:495, 1975). The term antibody as used in this invention is meant to include intact molecules as well as fragments thereof, such as Fab and F(ab').sub.2, which are capable of binding an epitopic determinant on JAK3. The antibodies of the invention include antibodies which bind to the polypeptide of SEQ ID NO:2 and which bind with immunoreactive fragments of SEQ ID NO:2.
The term "antibody" as used in this invention includes intact molecules as well as fragments thereof, such as Fab, F(ab').sub.2, and Fv which are capable of binding the epitopic determinant. These antibody fragments retain some ability to selectively bind with its antigen or receptor and are defined as follows:
(1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain;
(2) Fab', the fragment of an antibody molecule can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule;
(3) (Fab').sub.2, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab').sub.2 is a dimer of two Fab' fragments held together by two disulfide bonds;
(4) Fv, defined as a genetically engineered fragment containing the variable genetically fused single chain molecule.
Methods of making these fragments are known in the art. (See for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1988), incorporated herein by reference).
As used in this invention, the term "epitope" means any antigenic determinant on an antigen to which the paratope of an antibody binds. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.
Antibodies which bind to the JAK3 polypeptide of the invention can be prepared using an intact polypeptide or fragments containing small peptides of interest as the immunizing antigen. The polypeptide such as SEQ ID NO:2 used to immunize an animal can be derived from translated cDNA or chemical synthesis which can be conjugated to a carrier protein, if desired. Such commonly used carriers which are chemically coupled to the peptide include keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid. The coupled peptide is then used to immunize the animal (e.g., a mouse, a rat, or a rabbit).
If desired, polyclonal or monoclonal antibodies can be further purified, for example, by binding to and elution from a matrix to which the polypeptide or a peptide to which the antibodies were raised is bound. Those of skill in the art will know of various techniques common in the immunology arts for purification and/or concentration of polyclonal antibodies, as well as monoclonal antibodies (See for example, Coligan, et al., Unit 9, Current Protocols in Immunology, Wiley Interscience, 1991, incorporated by reference).
The invention also provides a method for detecting a cell proliferative disorder associated with JAK3 in a subject, comprising contacting a target cellular component containing JAK3, with a reagent which detects JAK3. The target cell component can be nucleic acid, such as DNA or RNA, or it can be protein. When the component is nucleic acid, the reagent is a nucleic acid probe or PCR primer. When the cell component is protein, the reagent is an antibody probe. The probes can be detectably labeled, for example, with a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelator, or an enzyme. Those of ordinary skill in the art will know of other suitable labels for binding to the antibody, or will be able to ascertain such, using routine experimentation.
For purposes of the invention, an antibody or nucleic acid probe specific for JAK3 may be used to detect the presence of JAK3 polypeptide (using antibody) or polynucleotide (using nucleic acid probe) in biological fluids or tissues. Oligonucleotide primers based on any coding sequence region in the JAK3 sequence are useful for amplifying DNA, for example by PCR. Any specimen containing a detectable amount of polynucleotide or antigen can be used. A preferred sample of this invention is blood or a tissue of liver, lung, kidney, spleen and intestine. Preferably the subject is human. When the cell proliferative disorder associated with JAK3 is a hematopoietic cell disorder, it may include leukemia, myelodysplasia, polyethemia vera, thrombocytosis and aplastic anemia, for example.
Monoclonal antibodies used in the method of the invention are suited for use, for example, in immunoassays in which they can be utilized in liquid phase or bound to a solid phase carrier. In addition, the monoclonal antibodies in these immunoassays can be detectably labeled in various ways. Examples of types of immunoassays which can utilize monoclonal antibodies of the invention are competitive and non-competitive immunoassays in either a direct or indirect format. Examples of such immunoassays are the radioimmunoassay (RIA) and the sandwich (immunometric) assay. Detection of the antigens using the monoclonal antibodies of the invention can be done utilizing immunoassays which are run in either the forward, reverse, or simultaneous modes, including immunohistochemical assays on physiological samples. Those of skill in the art will know, or can readily discern, other immunoassays formats without undue experimentation.
The term "immunometric assay" or "sandwich immunoassay", includes simultaneous sandwich, forward sandwich and reverse sandwich immunoassays. These terms are well understood by those skilled in the art. Those of skill will also appreciate that antibodies according to the present invention will be useful in other variations and forms of assays which are presently known or which may be developed in the future. These are intended to be included within the scope of the present invention.
Monoclonal antibodies can be bound to many different carriers and used to detect the presence of JAK3 polypeptide. Examples of well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses and magnetite. The nature of the carrier can be either soluble or insoluble for purposes of the invention. Those skilled in the art will know of other suitable carriers for binding monoclonal antibodies, or will be able to ascertain such using routine experimentation.
In performing the assays it may be desirable to include certain "blockers" in the incubation medium (usually added with the labeled soluble antibody). The "blockers" are added to assure that non-specific proteins, proteases, or anti-heterophilic immunoglobulins to anti-JAK3 immunoglobulins present in the experimental sample do not crosslink or destroy the antibodies on the solid phase support, or the radiolabeled indicator antibody, to yield false positive or false negative results. The selection of "blockers" therefore may add substantially to the specificity of the assays described in the present invention.
It has been found that a number of nonrelevant (i.e., nonspecific) antibodies of the same class or subclass (isotype) as those used in the assays (e.g., IgG1, IgG2a, IgM, etc.) can be used as "blockers". The concentration of the "blockers" (normally 1-100 .mu.g/.mu.l) may be important, in order to maintain the proper sensitivity yet inhibit any unwanted interference by mutually occurring cross reactive proteins in the specimen.
In using a monoclonal antibody for the in vivo detection of antigen, the detectably labeled monoclonal antibody is given in a dose which is diagnostically effective. The term "diagnostically effective" means that the amount of detectably labeled monoclonal antibody is administered in sufficient quantity to enable detection of the site having the JAK3 antigen for which the monoclonal antibodies are specific.
The concentration of detectably labeled monoclonal antibody which is administered should be sufficient such that the binding to those cells having JAK3 is detectable compared to the background. Further, it is desirable that the detectably labeled monoclonal antibody be rapidly cleared from the circulatory system in order to give the best target-to-background signal ratio.
As a rule, the dosage of detectably labeled monoclonal antibody for in vivo diagnosis will vary depending on such factors as age, sex, and extent of disease of the individual. The dosage of monoclonal antibody can vary from about 0.001 mg/m.sup.2 to about 500 mg/m.sup.2, preferably 0.1 mg/m.sup.2 to about 200 mg/m.sup.2, most preferably about 0.1 mg/m.sup.2 to about 10 mg/m.sup.2. Such dosages may vary, for example, depending on whether multiple injections are given, tumor burden, and other factors known to those of skill in the art.
For in vivo diagnostic imaging, the type of detection instrument available is a major factor in selecting a given radioisotope. The radioisotope chosen must have a type of decay which is detectable for a given type of instrument. Still another important factor in selecting a radioisotope for in vivo diagnosis is that the half-life of the radioisotope be long enough so that it is still detectable at the time of maximum uptake by the target, but short enough so that deleterious radiation with respect to the host is minimized. Ideally, a radioisotope used for in vivo imaging will lack a particle emission, but produce a large number of photons in the 140-250 keV range, which may be readily detected by conventional gamma cameras.
For in vivo diagnosis, radioisotopes may be bound to immunoglobulin either directly or indirectly by using an intermediate functional group. Intermediate functional groups which often are used to bind radioisotopes which exist as metallic ions to immunoglobulins are the bifunctional chelating agents such as diethylenetriaminepentacetic acid (DTPA) and ethylenediaminetetraacetic acid (EDTA) and similar molecules. Typical examples of metallic ions which can be bound to the monoclonal antibodies of the invention are .sup.111 In, .sup.97 Ru, .sup.67 Ga, .sup.68 Ga, .sup.72 As, .sup.89 Zr, and .sup.201 Tl.
A monoclonal antibody useful in the method of the invention can also be labeled with a paramagnetic isotope for purposes of in vivo diagnosis, as in magnetic resonance imaging (MRI) or electron spin resonance (ESR). In general, any conventional method for visualizing diagnostic imaging can be utilized. Usually gamma and positron emitting radioisotopes are used for camera imaging and paramagnetic isotopes for MRI. Elements which are particularly useful in such techniques include .sup.157 Gd, .sup.55 Mn, .sup.162 Dy, .sup.52 Cr, and .sup.56 Fe.
The present invention also provides a method for treating a subject with a cell proliferative disorder associated with JAK3 comprising administering to a subject with the disorder a therapeutically effective amount of reagent which modulates JAK3. In hematopoietic cancers, for example, the JAK3 nucleotide sequence may be under-expressed as compared to expression in a normal cell, therefore, it is possible to design appropriate therapeutic or diagnostic techniques directed to this sequence. Thus, where a cell-proliferative disorder is associated with the expression of JAK3 associated with malignancy, nucleic acid sequences that modulate JAK3 expression at the transcriptional or translational level can be used. In cases when a cell proliferative disorder or abnormal cell phenotype is associated with the under expression of JAK3, for example, nucleic acid sequences encoding JAK3 (sense) could be administered to the subject with the disorder.
The term "cell-proliferative disorder" denotes malignant as well as non-malignant cell populations which often appear to differ from the surrounding tissue both morphologically and genotypically. Such disorders may be associated, for example, with absence of expression of JAK3. Essentially, any disorder which is etiologically linked to expression of JAK3 could be considered susceptible to treatment with a reagent of the invention which modulates JAK3 expression.
The term "modulate" envisions the suppression of JAK3 gene expression when JAK3 is over-expressed. When JAK3 is over-expressed, an antisense polynucleotide for JAK3 can be introduced into the cell. Alternatively, when a cell proliferative disorder is associated with under-expression of JAK3 polypeptide, a sense polynucleotide sequence (the DNA coding strand) encoding JAK3 polypeptide can be introduced into the cell. The term "therapeutically effective" amount refers to that amount of reagent includes that amount which modulates JAK3 expression o kinase activity such that the symptoms of the disorder are reduced.
The present invention also provides gene therapy for the treatment of cell proliferative disorders which are mediated by JAK3. Such therapy would achieve its therapeutic effect by introduction of the appropriate JAK3 polynucleotide which contains a JAK3 gene (sense), into cells of subjects having the proliferative disorder. Delivery of sense JAK3 polynucleotide constructs can be achieved using a recombinant expression vector such as a chimeric virus or a colloidal dispersion system. An expression vector including the JAK3 polynucleotide sequence could be introduced to the subject's cells ex vivo after removing, for example, stem cells from a subject's bone marrow. The cells are then reintroduced into the subject, (e.g., into subject's bone marrow).
Various viral vectors which can be utilized for gene therapy as taught herein include adenovirus, herpes virus, vaccinia, or, preferably, an RNA virus such as a retrovirus. Preferably, the retroviral vector is a derivative of a murine or avian retrovirus. Examples of retroviral vectors in which a single foreign gene can be inserted include, but are not limited to: Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV), and gibbon ape leukemia virus (GaLV), which provides a broader host range than many of the murine viruses. A number of additional retroviral vectors can incorporate multiple genes. All of these vectors can transfer or incorporate a gene for a selectable marker so that transduced cells can be identified and generated. By inserting a JAK3 sequence (including promoter region) of interest into the viral vector, along with another gene which encodes the ligand for a receptor on a specific target cell, for example, the vector is now target specific. Preferred targeting is accomplished by using an antibody to target the retroviral vector. Those of skill in the art will know of, or can readily ascertain without undue experimentation, specific polynucleotide sequences which can be inserted into the retroviral genome to allow target specific delivery of the retroviral vector containing the JAK3 sense or antisense polynucleotide.
Since recombinant retroviruses are defective, they require assistance in order to produce infectious vector particles. This assistance can be provided, for example, by using helper cell lines that contain plasmids encoding all of the structural genes of the retrovirus under the control of regulatory sequences within the LTR. These plasmids are missing a nucleotide sequence which enables the packaging mechanism to recognize an RNA transcript for encapsidation. Helper cell lines which have deletions of the packaging signal include but are not limited to .PSI.2, PA317 and PA12, for example. These cell lines produce empty virions, since no genome is packaged. If a retroviral vector is introduced into such cells in which the packaging signal is intact, but the structural genes are replaced by other genes of interest, the vector can be packaged and vector virion produced.
Another targeted delivery system for JAK3 polynucleotide is a colloidal dispersion system. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. The preferred colloidal system of this invention is a liposome. Liposomes are artificial membrane vesicles which are useful as delivery vehicles in vitro and in vivo. It has been shown that large unilamellar vesicles (LUV), which range in size from 0.2-4.0 um can encapsulate a substantial percentage of an aqueous buffer containing large macromolecules. RNA, DNA and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley, et al., Trends Biochem. Sci., 6:77, 1981). In addition to mammalian cells, liposomes have been used for delivery of polynucleotides in plant, yeast and bacterial cells. In order for a liposome to be an efficient gene transfer vehicle, the following characteristics should be present: (1) encapsulation of the genes of interest at high efficiency while not compromising their biological activity; (2) preferential and substantial binding to a target cell in comparison to non-target cells; (3) delivery of the aqueous contents of the vesicle to the target cell cytoplasm at high efficiency; and (4) accurate and effective expression of genetic information (Mannino, et al., Biotechniques, 6:682, 1988).
The composition of the liposome is usually a combination of phospholipids, particularly high-phase-transition-temperature phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids may also be used. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations.
Examples of lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides. Particularly useful are diacylphosphatidylglycerols, where the lipid moiety contains from 14-18 carbon atoms, particularly from 16-18 carbon atoms, and is saturated. Illustrative phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.
The targeting of liposomes has been classified based on anatomical and mechanistic factors. Anatomical classification is based on the level of selectivity, for example, organ-specific, cell-specific, and organelle-specific. Mechanistic targeting can be distinguished based upon whether it is passive or active. Passive targeting utilizes the natural tendency of liposomes to distribute to cells of the reticulo-endothelial system (RES) in organs which contain sinusoidal capillaries. Active targeting, on the other hand, involves alteration of the liposome by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein, or by changing the composition or size of the liposome in order to achieve targeting to organs and cell types other than the naturally occurring sites of localization.
The surface of the targeted delivery system may be modified in a variety of ways. In the case of a liposomal targeted delivery system, lipid groups can be incorporated into the lipid bilayer of the liposome in order to maintain the targeting ligand in stable association with the liposomal bilayer. Various linking groups can be used for joining the lipid chains to the targeting ligand.
In general, the compounds bound to the surface of the targeted delivery system will be ligands and receptors which will allow the targeted delivery system to find and "home in" on the desired cells. A ligand may be any compound of interest which will bind to another compound, such as a receptor.
In general, surface membrane proteins which bind to specific effector molecules are referred to as receptors. In the present invention, antibodies are preferred receptors. Antibodies can be used to target liposomes to specific cell-surface ligands. For example, certain antigens expressed specifically on tumor cells, referred to as tumor-associated antigens (TAAs), may be exploited for the purpose of targeting JAK3 antibody-containing liposomes directly to the malignant tumor. Since the JAK3 gene product may be indiscriminate with respect to cell type in its action, a targeted delivery system offers a significant improvement over randomly injecting non-specific liposomes. Preferably, the target tissue is human brain, colon, lung, and renal cancers. A number of procedures can be used to covalently attach either polyclonal or monoclonal antibodies to a liposome bilayer. Antibody-targeted liposomes can include monoclonal or polyclonal antibodies or fragments thereof such as Fab, or F(ab').sub.2, as long as they bind efficiently to an antigenic epitope on the target cells. Liposomes may also be targeted to cells expressing receptors for hormones or other serum factors.
For use in the diagnostic research and therapeutic applications suggested above, kits are also provided by the invention. The invention provides a diagnostic kit useful for the detection of a target cellular component indicative of a cell proliferative disorder associated with JAK3 comprising carrier means being compartmentalized to receive in close confinement therein one or more containers comprising a first container containing a probe for detection of JAK3 nucleic acid. Such a kit may comprise a carrier means being compartmentalized to receive in close confinement one or more container means such as vials, tubes, and the like, each of the container means comprising one of the separate elements to be used in the method.
For example, one of the container means may comprise a probe which is or can be detectably labelled. Such probe may be an antibody or nucleotide specific for a target protein or a target nucleic acid, respectively, wherein the target is indicative, or correlates with, the presence of JAK3 of the invention. Where the kit utilizes nucleic acid hybridization to detect the target nucleic acid, the kit may also have containers containing nucleotide(s) for amplification of the target nucleic acid sequence and/or a container comprising a reporter-means, such as a biotin-binding protein, such as avidin or streptavidin, bound to a reporter molecule, such as an enzymatic, florescent, or radionucleotide label.
A number of embodiments of the present invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, it is to be understood that the invention is not to be limited by the specific illustrated embodiment, but only by the scope of the appended claims.
EXAMPLE 1
Cloning and Sequencing of JAK3
In order to clone the cDNA for a new member of the JAK family of non-receptor protein tyrosine kinases, degenerate oligonucleotides corresponding to parts of the highly conserved tyrosine kinase domain were used to amplify first strand cDNA from oligo (dT) primed, reverse transcribed, CD34+ total RNA from normal human bone marrow.
1. Bone Marrow Fractions. Iliac crest bone marrow was aspirated from consenting adult volunteers under an IRB approved protocol. Mononuclear cells were separated by Ficoll-Hypaque (Pharmacia, Piscataway, N.J.) density gradient separation. Cell subsets were purified by immunomagnetic separation (Strauss, L. C., et al., The American Journal of Pediatric Hematology/Oncology, 13:217, 1991, Civin, C. I., et al, Bone Marrow Purging and Processing, 1990). Positive selection of the CD34+ fraction was done by incubation of the mononuclear fraction with 0.5 ug CD34 (HPCA-1, Becton Dickinson, San Jose, Calif.) antibody per 10.sup.6 cells for 30 minutes at 4.degree. C. Cells were washed twice in RPMI 1640 (Sigma, St. Louis, Mo.) then resuspended in RPMI-1640 containing 1% human serum albumin at 5.times.10.sup.7 cells/ml and incubated with sheep anti-mouse IgG1 conjugated immunomagnetic microspheres for 30 minutes at 4.degree. C. The CD34+ bound cells were released from the microspheres by treatment with chymopapain (Chymodiactin TM, Boots USA, Lincolnshire Ill.; final concentration 200 U/ml, 15 min., RT). The microspheres were removed from the free (CD34+ enriched) cells using a magnetic particle concentrator (Dynal, Great Neck, N.Y.). CD34+ cells were further purified to obtain CD34+/Lin- cells by negative selection as described by Gore (Gore, S. D. et al., Blood, 8:1681, 1991).
2. Isolation of RNA. Poly A+ RNA was isolated from human hematopoietic cell lines using the Mini Ribosep mRNA isolation kit (Becton Dickinson, San Jose, Calif.). Total RNA from bone marrow cells and hematopoietic cell lines was extracted using the guanidium thiocyanate method (Chomczynski, P. and Sacchi, N., Anal Biochem., 162:156, 1987).
3. Cloning of JAK3. Total RNA isolated from CD34+ cells (see above) was reverse transcribed with Superscript Moloney murine-leukemia-virus reverse transcriptase (BRL, Gaithersburg, Md.) using oligo d(T) (Boehringer Mannheim, Germany) for printing. PCR amplification was carried out using degenerate oligonucleotides based on the highly conserved sequence motifs VHRDLA (5' GTNCA(T,C)(T,C)(C,A) GNGA(T,C)(TN GC3') AND DVWSYG (5' CCC(G,A)TAN(G,C)(A,T) CCA NAC (G,A)TC3') from the PTK catalytic domain (Wilks, A. F., et al., Gene, 85:67, 1989, Wilks, A. F., Methods in Enzymology, 200: 533, 1991). To facilitate subcloning of the amplified PCR products Not 1 and Sal 1 sites were included as part of the PCR primers.
The resultant 226 bp bands were isolated after electrophoresis in agarose gels and cloned into the Not 1/Sal 1 sites of pBluescript II KS- (Stratagene, La Jolla, Calif.). After sequencing, products containing known tyrosine kinase motifs were compared to reported sequences using the NCBI BlastN program (Altschul, S. F., et al., Journal of Molecular Biology, 215:403, 1990). The fragment did not match any other sequences in the databases but was most closely related to members of the JAK family of tyrosine kinases at 65-70% nucleic acid identity.
The conditions for RT-PCR and thermal RACE were carried out as described by Frohman (Frohman, M. A., Methods in Enzymology, 218:340, 1993). KG1a poly A+RNA was used as the substrate for RACE. The 5' and 3' ends of JAK3 were also amplified from normal human bone marrow cDNA isolated from a .lambda.gt10 human bone marrow library (Clonetech, Palo Alto, Calif.) using primers specific for JAK3 with primers specific for the arms of .lambda.gt10 under the same PCR conditions used for RACE.
4. Sequencing of JAK3. To correct for PCR errors, multiple overlapping partial clones of JAK3 isolated from KG1a and bone marrow cells were sequenced and compared using the dideoxy DNA sequencing method (USB, Cleveland, Ohio) (Sanger, F., et al., Proc. Natl. Acad. Sci. USA, 74:5463, 1977). To verify some regions, RT PCR amplified fragments from normal human bone marrow and exon containing portions of normal human JAK3 P1 genomic clones (see below) were also sequenced.
5. RNAse Protection Assays. Efforts to obtain the full-length JAK3 clone by screening of several libraries proved unsuccessful. Therefore, RNAse protection assay were developed utilizing the initial PCR amplified kinase domain fragment to screen for leukemic derived cell lines expressing JAK3. RNAse protection assays were carried out using the MAXIscript T3 in vitro Transcription Kit (Ambion, Austin, Tex.). Briefly, an anti-sense RNA probe was synthesized by runoff transcription using Bacteriophage T3 RNA polymerase on a pBluescript II KS- (Stratagene, La Jolla, Calif.) template linearized downstream of the JAK3 207 nucleotide PTK domain fragment. The resulting .sup.32 P.alpha.-UTP labelled 249 base RNA probe was hybridized with approximately 5 .mu.g of total RNA from hematopoietic cell lines and RNA from approximately equal numbers of cells from normal human bone marrow sub-fractions. RNA-RNA hybrids were treated with RNAse A and T, denatured and separated on an 8 M-Urea, 6% acrylamide gel and exposed to film (Kodak X-OMAT) (Melton, D. A., et al., Nucleic Acids Research, 12:7035, 1984). As an internal standard, a .beta.-actin probe was also included with each hybridization reaction.
6. JAK3 nucleotide and predicted amino acid sequence: Of the cell lines tested in this initial screening, JAK3 was most highly expressed by the myeloblastic cell line KG1a. Thermal RACE and PCR was employed to cone the full-length cDNA of JAK3 from KG1a and normal human bone marrow cells (Frohman, M. A., Therman RACE, Methods in Enzymology, 218:340, 1993). Using several rounds of RACE we isolated 3,807 bp of JAK3 cDNA, a region which covers the entire predicted coding region for JAK3, 167 bases of 5' untranslated and 394 bases of 3' untranslated sequence. FIG. 1 shows the nucleotide and predicted amino acid sequence of JAK3. The cDNA includes an open reading frame of 3,246 bases that predicts a protein of 1082 amino acids with a molecular weight of 121 kD. The putative initiating methionine shows the strongest homology with the Kozak consensus sequence (Kozak, M., Nucleic Acids Research, 15:8125,1987). At the 3' end an in frame stop codon defines the C terminus of the JAK3 protein at position 3242.
EXAMPLE 2
Sequence Comparison Between JAK3 and Other JAK Family Member
1. Amino acid comparison between JAK3 and other JAK family members: The initial identification of JAK3 as the fourth member of the JAK family was based on a database search using the 207 bp PCR fragment. The comparison of full-length-JAK3 with the other JAK family members is shown in FIG. 2. Sequences of JAK family members were aligned using the Pileup program (GCG Company, Madison, Wis.). The numbering system begins with the initiating methionine of the JAK family members. The numbering system does not take into account the insertion of gaps and, therefore, should be only regarded as a relative measure of location. The fifth line in the figure shows a consensus sequence derived if three out of four JAK family members have the identical amino acid in that position. Full-length JAK3 has 48% identity and 67% similarity with JAK2 (murine), 41% identity and 61% similarity with JAK1, and 40% identity and 60% similarity with TYK-2. In addition, recently, small fragments of tyrosine kinases by PCR approaches from a human breast cancer cell line (TK5) and rat brain (Ptk-2) have been isolated (Cance, W. G., et al., Int. J. Cancer, 54:571, 1993, Sanchez, M. P., et al., Proc. Natl. Acad. Sci. USA, 91:1819, 1994). Both of these TK's show 93% identity with JAK3 in this short region, while rat Jak3 shares 99% identity in this region. How JAK3 relates to these PTKs must await the isolation of their full coding regions.
2. Amino acid comparison between JAK3 and rat JAK3: FIG. 3 shows the comparison of JAK3 with the recently reported rat Jak3. The sequences of human JAK3 and rat Jak3 were aligned using the Pileup program (GCG Company, Madison, Wis.). The amino acids of each member are numbered beginning with the initiating methionine. The comparison shows 77% identity and 84% similarity making it likely that these genes are homologies (Takahashi, T. and Shirasawa, T., FEBS Letters, 342:124, 1994).
EXAMPLE 3
Characterization of JAK3 Expression
1. RNAse protection analysis of JAK3 expression in leukemic derived cell lines: To investigate the hematopoietic expression of JAK3, the RNAse protection assay was used utilizing the 206 bp PCR kinase domain fragment of JAK3 to screen leukemic derived cell lines (see above). Briefly, a 32P .alpha.-UTP labelled anti-sense RNA probe to the kinase domain of JAK3 was hybridized with 5 ugs total RNA from hematopoietic cell lines. A .beta.-actin probe was also included with each reaction as an internal standard, with the exception of the bone marrow and ML-1 populations, which were separately assayed for JAK3 and actin. As shown in FIG. 4a, a protected band migrating at the expected size is seen in a number of lanes. Positive signals were discernable for the Molt-16, Molt-3, KG1, KG1a, PB697, Nalm-16, Nalm-6, and TF-1 cell lines. These positive cell lines represent various forms of leukemia; the Molt lines were derived from T-ALL, the KG1 lines from AML, PB697 and the Nalm lines are B lineage ALL, and TF-1 was established from an erythroleukemia. No signals were seen from RNA derived from the ML-1, HL60, K562, or Daudi cell lines representing additional AML, APL, CML, and Burkitt's leukemia lines, respectively.
2. Northern blot analysis of JAK3 expression in leukemic derived cell lines: Although the coding region of the cDNAs for the JAK family are .about.3400 bp, the 5' and 3' untranslated regions and polyadenylation result in transcripts ranging from 4.4 kbp for JAK1, 4.8 kbp for JAK2, 5.4 kbp for TYK-2 and 4.0 kbp for rat Jak3. To investigate the size of JAK3 message, a Northern blot with poly A+ RNA isolated from a number of hematopoietic cell lines was probed with a 1.8 kbp JAK3 fragment. 5 .mu.g of poly A+ RNA samples from hematopoietic cell lines were incubated at 55.degree. C. for 15 minutes with 50% formamide, 6.5% formaldehyde, and 1.times. MOPS. Following the addition of formaldehyde loading buffer and ethidium bromide, RNA samples were electrophoresed in a 1.2% agarose gel containing 1.times. MOPS and 11% formaldehyde.
Following electrophoresis, gels were transferred by capillary action to nitrocellulose 47. Sambrook J., Fritsch E. F., Maniatis T.: Molecular Cloning. A Laboratory Manual. 1989). As is evident from FIG. 4b (upper half), JAK3 is not a very highly expressed message. Even after an exposure of 17 days at -80.degree. C. with two intensifying screens, signals were barely visible in the lanes containing RNA from the HEL, REH, KG1, and KG1a cell lines (HEL represents an erythroleukemia, REH is derived from a B-ALL, and the KG1 and KG1a cell lines are myeloblastic). RNA markers give an estimate of 5.8 kbp for the JAK3 transcript in these cells.
3. Northern blot analysis of JAK3 expression in non-hematopoietic tissues: To assess the expression of JAK3 in non-hematopoietic normal adult tissues, a Northern blot containing 2 .mu.g of poly A+ RNA from human heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas was screened. (Clonetech, Palo Alto, Calif.). Northern blots were prehybridized for 2 hrs in 50% formamide, 5.times. SSPE, 10.times. Denhardt's, 2% SDS, and 100 ug/ml denatured salmon sperm DNA (Clonetech). Blots were hybridized with a randomly primed .sup.32 P-dCTP labeled probe corresponding to a 1.8 kbp fragment of JAK3 cDNA (Feinberg, A. P. and Vogelstein, B., Anal. Biochem., 132:6, 1983). The blots were exposed to film (Kodak x-OMAT) for 1'/days at -80.degree. C. between two intensifying screens.
When the same JAK3 fragment was also used to probe a Northern blot containing RNA from non-hematopoietic human tissues (FIG. 4c), signals are seen from placenta, lung, liver, kidney, and pancreas, all with a similar message size of 5.8 kbp with possibly an additional less distinct band at -7.5 kbp. Unlike rat JAK3, which is expressed in rat heart and brain, no signals were seen from the RNA representing heart, brain, or skeletal muscle.
4. RNAse Protection of JAK3 expression in normal bone marrow fractions: Although the initial JAK3 fragment was generated by PCR amplification of CD34+ enriched bone marrow RNA, it remained a possibility that JAK3 expression was restricted to contaminating CD34- cells. To determine which populations of normal bone marrow express JAK3, fractions representing whole BM, CD34+, CD34- (i.e. depleted of CD34+ cells), CD34+/lin-, as well as peripheral blood were isolated. RNA was then extracted and used to perform the RNAse protection assay. The same probe used in FIG. 4a was hybridized with approximately 1-5 .mu.gs of RNA from normal total bone marrow, bone marrow subfractions, and from peripheral blood. As an internal standard, a .beta.-actin probe was also included with each reaction as a standard for the amount and quality of RNA loaded in each sample. The presence of a band that migrates between the JAK3 and actin bands in all lanes, including the no RNA and tRNA control lanes, is a result of incomplete digestion of the probe. All of the sample lanes give a protected JAK3 band migrating at the expected size. However, probably because of the limited amounts of RNA obtained from several fractions, the actin bands indicate a variation in total RNA loaded for each sample.
To determine the relative expression of JAK3 in these different populations, the bands were quantified by phosphorimager scanning and normalized relative to the actin signal. FIG. 5b shows the phosphorimage analysis of bone marrow fractions. Following exposure to film, the gel shown in FIG. 5b was exposed to a phosphor-image screen (Molecular Dynamics, Sunnyvale, Calif.). Bands were quantified using the ImageQuantify program and normalized relative to the actin signals. FIG. 5b shows the strongest relative signals result from the CD34+ RNA and the even more stem cell enriched CD34+/lin- RNA sample. Thus, JAK3 is most highly expressed in this primitive population of cells and may play a role in transducing the signal of receptor functioning in the proliferative and/or developmental pathways of these cells. JAK3 is also expressed in the CD34- and peripheral blood fractions and is thus likely to be involved with a subset of receptors involved in differentiated cell signalling, in analogy to JAK1, JAK2, and TYK-2.
EXAMPLE 4
Chromosomal Localization of the JAK3 Gene
1. Somatic cell hybrid analysis. To determine the chromosomal localization of the JAK3 gene, a human/rodent somatic cell hybrid mapping panel, NIGMS #2, which included human, mouse and hamster genomic DNA controls was screened by PCR (Drwinga, H., et al., Genomics, 16:311, 1993, Dubois, B. and Naylor, S., Genomics, 16:315, 1993). In this panel, most of the somatic cell hybrid samples contained DNA from a single specific human chromosome in a rodent background. To preclude cDNA contamination problems, a primer pair was selected that resulted in a PCR product from genomic DNA that was larger than the produce resulting from cDNA due to the presence of intronic sequence. The plus strand oligo 5'AGCCGCCTCCTTCTCT3' (SEQ ID NO:3) and minus strand oligo 5'CGGCAGCAGCTTAGCTAGG3' (SEQ ID NO:4) amplify an approximate 410 base pair product from human genomic DNA and a 156 base pair product from JAK3 cDNA.
For PCR, 100 ngs of genomic DNA from each hybrid cell line were used as the target for amplification. PCR amplification was performed using the following parameters: (94.degree. C., 1'.fwdarw.55.degree. C., 1'.fwdarw.72.degree. C., 2').times.30.fwdarw.72.degree. C., 15'. The final concentrations of reagents were 0.2 mM dNTP, 50 mM KCL, 3.0 mM Mg, 0.1 U Taq Polymerase/ml, and 2.5 mM each primer. The results from the PCR amplification were confirmed by Southern transfer and hybridization with a .sup.32 P .gamma.-ATP kinase labelled oligo internal to the primers used for amplification.
Using the primer pair results in a PCR product only with DNA from human cells and not from mouse or hamster DNA. These oligonucleotides were then used on DNA samples from the library representing each of the human chromosomes. The amplified DNA was electrophoresed and after transfer to nitrocellulose was hybridized to a radiolabelled oligonucleotide internal to the other oligonucleotides used for the PCR. Only the DNA from a cell line containing human chromosome 19 gave a significant signal.
2. Fluorescence in situ hybridization: TYK-2 has also been mapped to chromosome 19 (JAK1 and JAK2 have been mapped to 1p31.3 and 9p24, respectively).sup.55,56 Several pairs of tyrosine kinases (e.g. PDGFR.beta. and c-fms, PDGF.alpha. and c-kit, FLT3 and FLT1) have been shown to be closely linked, leading to the hypothesis that these receptor tyrosine kinases evolved by a trans duplication followed by a cis duplication..sup.57-60 In order to confirm the location of the gene on chromosome 19, to sublocalize the gene to a specific band, and to investigate the possibility that JAK3 and TYK-2 were linked, FISH experiment was carried out.
First clone containing approximately 80 kbp of the JAK3 gene was isolated by PCR screening of a P1 library using the same oligonucleotides used above. Briefly, P1 genomic clones of JAK3 were obtained by PCR screening of the Du Pont Merck Pharmaceutical Company Human Foreskin Fibroblast P1 Library #1 (DMPC-HFF#1)--(Genome Systems, St. Louis, Mo.). The clones were designated DMPC-HFF#1-1441, DMPC-HFF#1-1442, DMPC-HFF#1-1443 and identified using the same primer pairs and PCR conditions used for the screening of the human/rodent somatic cell hybrid mapping panel (see above). Partial sequencing of these P1 clones has confirmed that they represent genomic JAK3 DNA.
The P1 vector containing the approximate 80 kbp genomic clone 1441 of JAK3 was nick-translated with biotin-14 dATP (BRL, Gaithersburg, Md.), with 30% incorporation determined by tritium tracer incorporation. Slides with chromosome spreads were made from normal male lymphocytes cultured with BrdU (Bhatt, B., et al., Nucleic Acids Res., 16:3951, 1988). Fluorescence in situ hybridization was performed as described by Lichter, et. al., (53. Lichter, P., Tang, C., Call, K., Hermanson, G., Evans, G., Housman, D., Ward, D.: High resolution mapping of human chromosome 11 by in situ hybridization with cosmid clones. Science 247:64, 1990) with modifications. Probe mix (2.times.SSCP, 60% formamide, 10% dextran sulfate, 4 ng/ul biotinylated probe, 300 ng/ul Cot-1 DNA (to suppress repeated sequences) and 150 ng/ul salmon sperm DNA was denatured at 70.degree. C. for 5 minutes, preannealed at 37.degree. C. for 40 minutes, placed on slides and hybridized at 37.degree. C. overnight. Slides were washed in 70% formamide/2.times.SSC at 43.degree. C. for 20 minutes, and 2 changes of 2.times.SSC at 37.degree. C. for 5 minutes each. Biotinylated probe was detected with FITC-avidin and amplified with biotinylated anti-avidin, using reagents from an in situ hybridization kit (Oncor Inc., Gaithersburg, Md.), following the manufacturer's instructions.
Analysis of 36 metaphase cells showed 20 cells (56%) had at least one pair of signals (involving both chromatids of a single chromosome). These 20 metaphases were photographed on color slide film (Kodak Ekttachrome 400HC) and 33 paired signals were seen, with all but one located on the proximal short arm of an F-group (chr. 19 or 20) chromosome, an example of which is shown in FIG. 6a. To determine the specific chromosome and band location of the signals, the hybridized slides were G-banded by FPG (fluorescence plus Giemsa), photographed, and aligned with the color slides to determine the subband location. FIG. 6b shows the position of the paired FISH signals on the G-banded chromosomes. All 33 signals were analyzable after banding and all were on chromosome 19, with most on bands p12-13.1 (FIG. 6c). Thus JAK3 may be located near TYK-2, which has been localized to 19p13.2. FIG. 6c is the ideogram of human chromosome 19, showing localization of JAK3 to 19p12-13.1. Each dot represents a paired signal seen on metaphase chromosomes. Signals clearly located on a single band are diagrammed to the right of the ideogram; those which could not be sublocalized to a single band are assigned to regions diagrammed to the left (brackets).
The foregoing is meant to illustrate, but not to limit, the scope of the invention. Indeed, those of ordinary skill in the art can readily envision and produce further embodiments, based on the teachings herein, without undue experimentation.
__________________________________________________________________________# SEQUENCE LISTING- (1) GENERAL INFORMATION:- (iii) NUMBER OF SEQUENCES: 12- (2) INFORMATION FOR SEQ ID NO:1:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 3807 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:- AAACAGTTAA TACATATTTT TTATGTTACG TGTATTCTGT ACAACAAAGT AA - #GCTAGACA 60- AAAGAAAATG TTTTCTCCTT CCTGTGTGGG ACTTTCCTCT CGCTGCCTCC CG - #GCTCTGCC 120#GCA CCT 176CCAGGG TCCCTGCCCG CTAGGCAAGT TGCACTC ATG#Ala Pro Met#1- CCA AGT GAA GAG ACG CCC CTG ATC CCT CAG CG - #T TCA TGC AGC CTC TTG 224Pro Ser Glu Glu Thr Pro Leu Ile Pro Gln Ar - #g Ser Cys Ser Leu Leu# 15- TCC ACG GAG GCT GGT GCC CTG CAT GTG CTG CT - #G CCC GCT CGG GGC CCC 272Ser Thr Glu Ala Gly Ala Leu His Val Leu Le - #u Pro Ala Arg Gly Pro# 35- GGG CCC CCC CAG CGC CTA TCT TTC TCC TTT GG - #G GAC CAC TTG GCT GAG 320Gly Pro Pro Gln Arg Leu Ser Phe Ser Phe Gl - #y Asp His Leu Ala Glu# 50- GAC CTG TGC GTG CAG GCT GCC AAG GCC AGC GC - #G ATC CTG CCT GTG TAC 368Asp Leu Cys Val Gln Ala Ala Lys Ala Ser Al - #a Ile Leu Pro Val Tyr# 65- CAC TCC CTC TTT GCT CTG GCC ACG GAG GAC CT - #G TCC TGC TGG TTC CCC 416His Ser Leu Phe Ala Leu Ala Thr Glu Asp Le - #u Ser Cys Trp Phe Pro# 80- CGA GCC ACA TCT TCT CCG TGG AGG ATG CCA GC - #A CCC CAA GTC CTG CTG 464Arg Ala Thr Ser Ser Pro Trp Arg Met Pro Al - #a Pro Gln Val Leu Leu# 95- TAC AGG ATT CGC TTT TAC TTC CCC AAT TGG TT - #T GGG CTG GAG AAG TGC 512Tyr Arg Ile Arg Phe Tyr Phe Pro Asn Trp Ph - #e Gly Leu Glu Lys Cys100 1 - #05 1 - #10 1 -#15- CAC CGC TTC GGG CTA CGC AAG GAT TTG GCC AG - #T GCT ATC CTT GAC CTG 560His Arg Phe Gly Leu Arg Lys Asp Leu Ala Se - #r Ala Ile Leu Asp Leu# 130- CCA GTC CTG GAG CAC CTC TTT GCC CAG CAC CG - #C AGT GAC CTG GTG AGT 608Pro Val Leu Glu His Leu Phe Ala Gln His Ar - #g Ser Asp Leu Val Ser# 145- GGG CGC CTC CCC CGT GGC CTC AGT CTC AAG GA - #G CAG GGT GAG TGT CTC 656Gly Arg Leu Pro Arg Gly Leu Ser Leu Lys Gl - #u Gln Gly Glu Cys Leu# 160- AGC CTG GCC GTG TTG GAC CTG GCC CGG ATG GC - #G CGA GAG CAG GCC CAG 704Ser Leu Ala Val Leu Asp Leu Ala Arg Met Al - #a Arg Glu Gln Ala Gln# 175- CGG CGG GGA GAG CTG CTG AAG ACT GTC AGC TA - #C AAG GCC TGC CTA CCC 752Arg Arg Gly Glu Leu Leu Lys Thr Val Ser Ty - #r Lys Ala Cys Leu Pro180 1 - #85 1 - #90 1 -#95- CCA AGC CTG CGC GAC CTG ATC CAG GGC CTG AG - #C TTC GTG ACG GGG AGG 800Pro Ser Leu Arg Asp Leu Ile Gln Gly Leu Se - #r Phe Val Thr Gly Arg# 210- CGT ATT CGG AGG ACG GTG GAG AGC CCC CTG CG - #C CGG GTG GCC GCC TGC 848Arg Ile Arg Arg Thr Val Glu Ser Pro Leu Ar - #g Arg Val Ala Ala Cys# 225- CAG GCA GAC CGG CAC TCG CTC ATG GCC AAG TA - #C ATC ATG GAC CTG GAG 896Gln Ala Asp Arg His Ser Leu Met Ala Lys Ty - #r Ile Met Asp Leu Glu# 240- CGG CTG GAT CCA GCC GGG GCC GCC GAG ACC TT - #C CAC GTG GGC CTC CCT 944Arg Leu Asp Pro Ala Gly Ala Ala Glu Thr Ph - #e His Val Gly Leu Pro# 255- GGG GCC CTT GGT GGC CAC GAC GGG CTG GGG CT - #C GTC CGC GTG GCT GGT 992Gly Ala Leu Gly Gly His Asp Gly Leu Gly Le - #u Val Arg Val Ala Gly260 2 - #65 2 - #70 2 -#75- GAC GGC GGC ATC GCC TGG ACC CAG GGA GAA CA - #G GAG GTC CTC CAG CCC1040Asp Gly Gly Ile Ala Trp Thr Gln Gly Glu Gl - #n Glu Val Leu Gln Pro# 290- TTC TGC GAC TTT CCA GAA ATC GTA GAC ATT AG - #C ATC AAG CAG GCC CCG1088Phe Cys Asp Phe Pro Glu Ile Val Asp Ile Se - #r Ile Lys Gln Ala Pro# 305- CGC GTT GGC CCG GCC GGA GAG CAC CGC CTG GT - #C ACT GTT ACC AGG ACA1136Arg Val Gly Pro Ala Gly Glu His Arg Leu Va - #l Thr Val Thr Arg Thr# 320- GAC AAC CAG ATT TTA GAG GCC GAG TTC CCA GG - #G CTG CCC GAG GCT CTG1184Asp Asn Gln Ile Leu Glu Ala Glu Phe Pro Gl - #y Leu Pro Glu Ala Leu# 335- TCG TTC GTG GCG CTC GTG GAC GGC TAC TTC CG - #G CTG ACC ACG GAC TCC1232Ser Phe Val Ala Leu Val Asp Gly Tyr Phe Ar - #g Leu Thr Thr Asp Ser340 3 - #45 3 - #50 3 -#55- CAG CAC TTC TTC TGC AAG GAG GTG GAC CCG AG - #G CTG CTG GAG GAA GTG1280Gln His Phe Phe Cys Lys Glu Val Asp Pro Ar - #g Leu Leu Glu Glu Val# 370- GCC GAG CAG TGC CAC GGC CCC ATC ACT CTG GA - #C TTT GCC ATC AAC AAG1328Ala Glu Gln Cys His Gly Pro Ile Thr Leu As - #p Phe Ala Ile Asn Lys# 385- CTC AAG ACT GGG GGC TCA CGT CCT GGC TCC TA - #T GTT CTC CGC CGC ATC1376Leu Lys Thr Gly Gly Ser Arg Pro Gly Ser Ty - #r Val Leu Arg Arg Ile# 400- CCC CAG GAC TTT GAC AGC TTC CTC CTC ACT GT - #C TGT GTC CAG AAC CCC1424Pro Gln Asp Phe Asp Ser Phe Leu Leu Thr Va - #l Cys Val Gln Asn Pro# 415- CTT GGT CCT GAT TAT AAG GGC TGC CTC ATC CG - #G CGC AGC CCC ACA GGA1472Leu Gly Pro Asp Tyr Lys Gly Cys Leu Ile Ar - #g Arg Ser Pro Thr Gly420 4 - #25 4 - #30 4 -#35- ACC TTC CTT CTG GTT GGC CTC AGC CGA CCC CA - #C AGC AGT CTT CGA GAG1520Thr Phe Leu Leu Val Gly Leu Ser Arg Pro Hi - #s Ser Ser Leu Arg Glu# 450- CTC CTG GCA ACC TGC TGG GAT GGG GGG CTG CA - #C GTA GAT GGG GTG GCA1568Leu Leu Ala Thr Cys Trp Asp Gly Gly Leu Hi - #s Val Asp Gly Val Ala# 465- GTG ACC CTC ACT TCC TGC TGT ATC CCC AGA CC - #C AAA GAA AAG TCC AAC1616Val Thr Leu Thr Ser Cys Cys Ile Pro Arg Pr - #o Lys Glu Lys Ser Asn# 480- CTG ATT GTG GTC CAG AGA GGT CAC AGC CCA CC - #C ACA TCA TCC TTG GTT1664Leu Ile Val Val Gln Arg Gly His Ser Pro Pr - #o Thr Ser Ser Leu Val# 495- CAG CCC CAA TCC CAA TAC CAG CTG AGT CAG AT - #G ACA TTT CAC AAG ATC1712Gln Pro Gln Ser Gln Tyr Gln Leu Ser Gln Me - #t Thr Phe His Lys Ile500 5 - #05 5 - #10 5 -#15- CCT GCT GAC AGC CTG GAG TGG CAT GAG AAC CT - #G GGC CAT GGG TCC TTC1760Pro Ala Asp Ser Leu Glu Trp His Glu Asn Le - #u Gly His Gly Ser Phe# 530- ACC AAG ATT TAC CGG GGC TGT CGC CAT GAG GT - #G GTG GAT GGG GAG GCC1808Thr Lys Ile Tyr Arg Gly Cys Arg His Glu Va - #l Val Asp Gly Glu Ala# 545- CGA AAG ACA GAG GTG CTG CTG AAG GTC ATG GA - #T GCC AAG CAC AAG AAC1856Arg Lys Thr Glu Val Leu Leu Lys Val Met As - #p Ala Lys His Lys Asn# 560- TGC ATG GAG TCA TTC CTG GAA GCA GCG AGC TT - #G ATG AGC CAA GTG TCG1904Cys Met Glu Ser Phe Leu Glu Ala Ala Ser Le - #u Met Ser Gln Val Ser# 575- TAC CGG CAT CTC GTG CTG CTC CAC GGC GTG TG - #C ATG GCT GGA GAC AGC1952Tyr Arg His Leu Val Leu Leu His Gly Val Cy - #s Met Ala Gly Asp Ser580 5 - #85 5 - #90 5 -#95- ACC ATG GTC GAG GAA TTT GTA CAC CTG GGG GC - #C ATA GAC ATG TAT CTG2000Thr Met Val Glu Glu Phe Val His Leu Gly Al - #a Ile Asp Met Tyr Leu# 610- CGA AAA CGT GGC CAC CTG GTG CCA GCC AGC TG - #G AAG CTG CAG GTG GTC2048Arg Lys Arg Gly His Leu Val Pro Ala Ser Tr - #p Lys Leu Gln Val Val# 625- AAA CAG CTG GCC TAC GCC CTC AAC TAT CTG GA - #G GAC AAA GGC CTG TCC2096Lys Gln Leu Ala Tyr Ala Leu Asn Tyr Leu Gl - #u Asp Lys Gly Leu Ser# 640- CAT GGC AAT GTC TCT GCC CGG AAG GTG CTC CT - #G GCT CGG GAG GGG GCT2144His Gly Asn Val Ser Ala Arg Lys Val Leu Le - #u Ala Arg Glu Gly Ala# 655- GAT GGG AGC CCG CCC TTC ATC AAG CTG AGT GA - #C CCT GGG GTC AGC CCC2192Asp Gly Ser Pro Pro Phe Ile Lys Leu Ser As - #p Pro Gly Val Ser Pro660 6 - #65 6 - #70 6 -#75- GCT GTG TTA AGC CTG GAG ATG CTC ACC GAC AG - #G ATC CCC TGG GTG GCC2240Ala Val Leu Ser Leu Glu Met Leu Thr Asp Ar - #g Ile Pro Trp Val Ala# 690- CCC GAG TGT CTC CGG GAG GCG CAG ACA CTT AG - #C TTG GAA GCT GAC AAG2288Pro Glu Cys Leu Arg Glu Ala Gln Thr Leu Se - #r Leu Glu Ala Asp Lys# 705- TGG GGC TTC GGC GCC ACG GTC TGG GAA GTG TT - #T AGT GGC GTC ACC ATG2336Trp Gly Phe Gly Ala Thr Val Trp Glu Val Ph - #e Ser Gly Val Thr Met# 720- CCC ATC AGT GCC CTA GAT CCT GCT AAG AAA CT - #C CAA TTT TAT GAG GAC2384Pro Ile Ser Ala Leu Asp Pro Ala Lys Lys Le - #u Gln Phe Tyr Glu Asp# 735- CGG CAG CAG CTG TCG GCC CCC AAG TGG ACA GA - #G CTG GCC CTG CTG ATT2432Arg Gln Gln Leu Ser Ala Pro Lys Trp Thr Gl - #u Leu Ala Leu Leu Ile740 7 - #45 7 - #50 7 -#55- CAA CAG TGC ATG GCC TAT GAG CCG GTC CAG AG - #G CCC TCC TTA CGA GCC2480Gln Gln Cys Met Ala Tyr Glu Pro Val Gln Ar - #g Pro Ser Leu Arg Ala# 770- GTC ATT CGT GAC CTC AAT AGT CTC ATC TCT TC - #A GAC TAT GAG CTC CTC2528Val Ile Arg Asp Leu Asn Ser Leu Ile Ser Se - #r Asp Tyr Glu Leu Leu# 785- TCA GAC CAC ACC TGG TGC CCT GGC ACT CGT GA - #T GGG CTG TGG AAT GGT2576Ser Asp His Thr Trp Cys Pro Gly Thr Arg As - #p Gly Leu Trp Asn Gly# 800- GCC CAG CTC TAT GCC TGC CAA GAC CCC ACG AT - #C TTC GAG GAG AGA CAC2624Ala Gln Leu Tyr Ala Cys Gln Asp Pro Thr Il - #e Phe Glu Glu Arg His# 815- CTC AAG TAC ATC TCA CAG CTG GGC AAG GGC TT - #C TTT GGC AGC GTG GAG2672Leu Lys Tyr Ile Ser Gln Leu Gly Lys Gly Ph - #e Phe Gly Ser Val Glu820 8 - #25 8 - #30 8 -#35- CTG TGC CGC TAT GAC CCG CTA GGC GAC AAT AC - #A GGT GCC CTG GTG GCC2720Leu Cys Arg Tyr Asp Pro Leu Gly Asp Asn Th - #r Gly Ala Leu Val Ala# 850- GTG AAA CAG CTG CAG CAC AGC GGG CCA GAC CA - #G CAG AGG GAC TTT CAG2768Val Lys Gln Leu Gln His Ser Gly Pro Asp Gl - #n Gln Arg Asp Phe Gln# 865- CGG GAG ATT CAG ATC CTC AAA GCA CAG CAC AG - #T GAT TTC ATT GTC AAG2816Arg Glu Ile Gln Ile Leu Lys Ala Gln His Se - #r Asp Phe Ile Val Lys# 880- TAT CGT GGT GTC AGC TAT GGC CCG GGC CGC CA - #G AGC CCT GCG CTG GTC2864Tyr Arg Gly Val Ser Tyr Gly Pro Gly Arg Gl - #n Ser Pro Ala Leu Val# 895- ATG GAG TAC CTG CCC AGC GGC TGC TTG CGC GA - #C TTC CTG CAG CGG CAC2912Met Glu Tyr Leu Pro Ser Gly Cys Leu Arg As - #p Phe Leu Gln Arg His900 9 - #05 9 - #10 9 -#15- CGG GGC CTC GAT GCC AGC CGC CTC CTT CTC TA - #T TCC TCG CAG ATC TGC2960Arg Gly Leu Asp Ala Ser Arg Leu Leu Leu Ty - #r Ser Ser Gln Ile Cys# 930- AAG GGC ATG GAG TAC CTG GGC TCC CGC CGC TG - #C GTG CAC CGC GAC CTG3008Lys Gly Met Glu Tyr Leu Gly Ser Arg Arg Cy - #s Val His Arg Asp Leu# 945- GCC GCC CGA AAC ATC CTC GTG GAG AGC GAG GC - #A CAC GTC AAG ATC GCT3056Ala Ala Arg Asn Ile Leu Val Glu Ser Glu Al - #a His Val Lys Ile Ala# 960- GAC TTC GGC CTA GCT AAG CTG CTG CCG CTT GA - #C AAA GAC TAC TAC GTG3104Asp Phe Gly Leu Ala Lys Leu Leu Pro Leu As - #p Lys Asp Tyr Tyr Val# 975- GTC CGC GAG CCA GGC CAG AGC CCC ATT TTC TG - #G TAT GCC CCC GAA TCC3152Val Arg Glu Pro Gly Gln Ser Pro Ile Phe Tr - #p Tyr Ala Pro Glu Ser980 9 - #85 9 - #90 9 -#95- CTC TCG GAC AAC ATC TTC TCT CGC CAG TCA GA - #C GTC TGG AGC TTC GGG3200Leu Ser Asp Asn Ile Phe Ser Arg Gln Ser As - #p Val Trp Ser Phe Gly# 10105- GTC GTC CTG TAC GAG CTC TTC ACC TAC TGC GA - #C AAA AGC TGC AGC CCC3248Val Val Leu Tyr Glu Leu Phe Thr Tyr Cys As - #p Lys Ser Cys Ser Pro# 10250- TCG GCC GAG TTC CTG CGG ATG ATG GGA TGT GA - #G CGG GAT GTC CCC CGC3296Ser Ala Glu Phe Leu Arg Met Met Gly Cys Gl - #u Arg Asp Val Pro Arg# 10405- CTC TGC CGC CTC TTG GAA CTG CTG GAG GAG GG - #C CAG AGG CTG CCG GCG3344Leu Cys Arg Leu Leu Glu Leu Leu Glu Glu Gl - #y Gln Arg Leu Pro Ala# 10550- CCT CCT TGC TGC CCT GC TGAGGTGAGT TGCTACAGTG GCT - #GGAGAGA3391Pro Pro Cys Cys Pro1060- CGACATCTGC CTGCCTGCTG AGTGAGTTGC TACAGTGGCT GAGAGACGAC AT - #CTGCTCCA3451- TGGCTGGTGG CCGACAGTAA TCTCACGCCG GACCTGCCGC AGCCCCTGCC CC - #AGACCTCT3511- CACCATCACC GCCACCACCG TGCAGCTGCC ACCAACCCTG CACGCTACTG CT - #GCCTCAGT3571- GGCTGTACCC AACAAGACCT GCTGACCCTC TGTCCCTACT GATTCCTCCT TG - #GGTGCAGC3631- CTCAGAGTGC CTGAGGCCCA GAGGGTCTGG TCTGGTGAGC TCCTGAGGCC AC - #ACAGCACC3691- ATAAAGTCTC GCATCTACAG GCCTTTGATT ACCTCCTGGG ATGGGTGCTC AC - #TATCTACC3751- CCAGACCAAC GCCACCTGCA GCCTGTGGAG TCAACTGCAG AATAAATCAC AC - #CCTA3807- (2) INFORMATION FOR SEQ ID NO:2:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 1064 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:- Met Ala Pro Pro Ser Glu Glu Thr Pro Leu Il - #e Pro Gln Arg Ser Cys# 15- Ser Leu Leu Ser Thr Glu Ala Gly Ala Leu Hi - #s Val Leu Leu Pro Ala# 30- Arg Gly Pro Gly Pro Pro Gln Arg Leu Ser Ph - #e Ser Phe Gly Asp His# 45- Leu Ala Glu Asp Leu Cys Val Gln Ala Ala Ly - #s Ala Ser Ala Ile Leu# 60- Pro Val Tyr His Ser Leu Phe Ala Leu Ala Th - #r Glu Asp Leu Ser Cys# 80- Trp Phe Pro Arg Ala Thr Ser Ser Pro Trp Ar - #g Met Pro Ala Pro Gln# 95- Val Leu Leu Tyr Arg Ile Arg Phe Tyr Phe Pr - #o Asn Trp Phe Gly Leu# 110- Glu Lys Cys His Arg Phe Gly Leu Arg Lys As - #p Leu Ala Ser Ala Ile# 125- Leu Asp Leu Pro Val Leu Glu His Leu Phe Al - #a Gln His Arg Ser Asp# 140- Leu Val Ser Gly Arg Leu Pro Arg Gly Leu Se - #r Leu Lys Glu Gln Gly145 1 - #50 1 - #55 1 -#60- Glu Cys Leu Ser Leu Ala Val Leu Asp Leu Al - #a Arg Met Ala Arg Glu# 175- Gln Ala Gln Arg Arg Gly Glu Leu Leu Lys Th - #r Val Ser Tyr Lys Ala# 190- Cys Leu Pro Pro Ser Leu Arg Asp Leu Ile Gl - #n Gly Leu Ser Phe Val# 205- Thr Gly Arg Arg Ile Arg Arg Thr Val Glu Se - #r Pro Leu Arg Arg Val# 220- Ala Ala Cys Gln Ala Asp Arg His Ser Leu Me - #t Ala Lys Tyr Ile Met225 2 - #30 2 - #35 2 -#40- Asp Leu Glu Arg Leu Asp Pro Ala Gly Ala Al - #a Glu Thr Phe His Val# 255- Gly Leu Pro Gly Ala Leu Gly Gly His Asp Gl - #y Leu Gly Leu Val Arg# 270- Val Ala Gly Asp Gly Gly Ile Ala Trp Thr Gl - #n Gly Glu Gln Glu Val# 285- Leu Gln Pro Phe Cys Asp Phe Pro Glu Ile Va - #l Asp Ile Ser Ile Lys# 300- Gln Ala Pro Arg Val Gly Pro Ala Gly Glu Hi - #s Arg Leu Val Thr Val305 3 - #10 3 - #15 3 -#20- Thr Arg Thr Asp Asn Gln Ile Leu Glu Ala Gl - #u Phe Pro Gly Leu Pro# 335- Glu Ala Leu Ser Phe Val Ala Leu Val Asp Gl - #y Tyr Phe Arg Leu Thr# 350- Thr Asp Ser Gln His Phe Phe Cys Lys Glu Va - #l Asp Pro Arg Leu Leu# 365- Glu Glu Val Ala Glu Gln Cys His Gly Pro Il - #e Thr Leu Asp Phe Ala# 380- Ile Asn Lys Leu Lys Thr Gly Gly Ser Arg Pr - #o Gly Ser Tyr Val Leu385 3 - #90 3 - #95 4 -#00- Arg Arg Ile Pro Gln Asp Phe Asp Ser Phe Le - #u Leu Thr Val Cys Val# 415- Gln Asn Pro Leu Gly Pro Asp Tyr Lys Gly Cy - #s Leu Ile Arg Arg Ser# 430- Pro Thr Gly Thr Phe Leu Leu Val Gly Leu Se - #r Arg Pro His Ser Ser# 445- Leu Arg Glu Leu Leu Ala Thr Cys Trp Asp Gl - #y Gly Leu His Val Asp# 460- Gly Val Ala Val Thr Leu Thr Ser Cys Cys Il - #e Pro Arg Pro Lys Glu465 4 - #70 4 - #75 4 -#80- Lys Ser Asn Leu Ile Val Val Gln Arg Gly Hi - #s Ser Pro Pro Thr Ser# 495- Ser Leu Val Gln Pro Gln Ser Gln Tyr Gln Le - #u Ser Gln Met Thr Phe# 510- His Lys Ile Pro Ala Asp Ser Leu Glu Trp Hi - #s Glu Asn Leu Gly His# 525- Gly Ser Phe Thr Lys Ile Tyr Arg Gly Cys Ar - #g His Glu Val Val Asp# 540- Gly Glu Ala Arg Lys Thr Glu Val Leu Leu Ly - #s Val Met Asp Ala Lys545 5 - #50 5 - #55 5 -#60- His Lys Asn Cys Met Glu Ser Phe Leu Glu Al - #a Ala Ser Leu Met Ser# 575- Gln Val Ser Tyr Arg His Leu Val Leu Leu Hi - #s Gly Val Cys Met Ala# 590- Gly Asp Ser Thr Met Val Glu Glu Phe Val Hi - #s Leu Gly Ala Ile Asp# 605- Met Tyr Leu Arg Lys Arg Gly His Leu Val Pr - #o Ala Ser Trp Lys Leu# 620- Gln Val Val Lys Gln Leu Ala Tyr Ala Leu As - #n Tyr Leu Glu Asp Lys625 6 - #30 6 - #35 6 -#40- Gly Leu Ser His Gly Asn Val Ser Ala Arg Ly - #s Val Leu Leu Ala Arg# 655- Glu Gly Ala Asp Gly Ser Pro Pro Phe Ile Ly - #s Leu Ser Asp Pro Gly# 670- Val Ser Pro Ala Val Leu Ser Leu Glu Met Le - #u Thr Asp Arg Ile Pro# 685- Trp Val Ala Pro Glu Cys Leu Arg Glu Ala Gl - #n Thr Leu Ser Leu Glu# 700- Ala Asp Lys Trp Gly Phe Gly Ala Thr Val Tr - #p Glu Val Phe Ser Gly705 7 - #10 7 - #15 7 -#20- Val Thr Met Pro Ile Ser Ala Leu Asp Pro Al - #a Lys Lys Leu Gln Phe# 735- Tyr Glu Asp Arg Gln Gln Leu Ser Ala Pro Ly - #s Trp Thr Glu Leu Ala# 750- Leu Leu Ile Gln Gln Cys Met Ala Tyr Glu Pr - #o Val Gln Arg Pro Ser# 765- Leu Arg Ala Val Ile Arg Asp Leu Asn Ser Le - #u Ile Ser Ser Asp Tyr# 780- Glu Leu Leu Ser Asp His Thr Trp Cys Pro Gl - #y Thr Arg Asp Gly Leu785 7 - #90 7 - #95 8 -#00- Trp Asn Gly Ala Gln Leu Tyr Ala Cys Gln As - #p Pro Thr Ile Phe Glu# 815- Glu Arg His Leu Lys Tyr Ile Ser Gln Leu Gl - #y Lys Gly Phe Phe Gly# 830- Ser Val Glu Leu Cys Arg Tyr Asp Pro Leu Gl - #y Asp Asn Thr Gly Ala# 845- Leu Val Ala Val Lys Gln Leu Gln His Ser Gl - #y Pro Asp Gln Gln Arg# 860- Asp Phe Gln Arg Glu Ile Gln Ile Leu Lys Al - #a Gln His Ser Asp Phe865 8 - #70 8 - #75 8 -#80- Ile Val Lys Tyr Arg Gly Val Ser Tyr Gly Pr - #o Gly Arg Gln Ser Pro# 895- Ala Leu Val Met Glu Tyr Leu Pro Ser Gly Cy - #s Leu Arg Asp Phe Leu# 910- Gln Arg His Arg Gly Leu Asp Ala Ser Arg Le - #u Leu Leu Tyr Ser Ser# 925- Gln Ile Cys Lys Gly Met Glu Tyr Leu Gly Se - #r Arg Arg Cys Val His# 940- Arg Asp Leu Ala Ala Arg Asn Ile Leu Val Gl - #u Ser Glu Ala His Val945 9 - #50 9 - #55 9 -#60- Lys Ile Ala Asp Phe Gly Leu Ala Lys Leu Le - #u Pro Leu Asp Lys Asp# 975- Tyr Tyr Val Val Arg Glu Pro Gly Gln Ser Pr - #o Ile Phe Trp Tyr Ala# 990- Pro Glu Ser Leu Ser Asp Asn Ile Phe Ser Ar - #g Gln Ser Asp Val Trp# 10050- Ser Phe Gly Val Val Leu Tyr Glu Leu Phe Th - #r Tyr Cys Asp Lys Ser# 10205- Cys Ser Pro Ser Ala Glu Phe Leu Arg Met Me - #t Gly Cys Glu Arg Asp# 10401030 - # 1035- Val Pro Arg Leu Cys Arg Leu Leu Glu Leu Le - #u Glu Glu Gly Gln Arg# 10550- Leu Pro Ala Pro Pro Cys Cys Pro 1060- (2) INFORMATION FOR SEQ ID NO:3:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 16 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:# 16- (2) INFORMATION FOR SEQ ID NO:4:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 19 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:# 19 AGG- (2) INFORMATION FOR SEQ ID NO:5:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 1082 amino (B) TYPE: amino acid (C) STRANDEDNESS: Not R - #elevant (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:- Met Ala Pro Pro Ser Glu Glu Thr - # Pro Leu Ile Pro Gln Arg SerCys# 15- Ser Leu Leu Ser Thr Glu Ala Gly - # Ala Leu His Val Leu Leu ProAla# 30- Arg Gly Pro Gly Pro Pro Gln Arg - # Leu Ser Phe Ser Phe Gly AspHis# 45- Leu Ala Glu Asp Leu Cys Val Gln - # Ala Ala Lys Ala Ser Ala IleLeu# 60- Pro Val Tyr His Ser Leu Phe Ala - # Leu Ala Thr Glu Asp Leu SerCys# 80- Trp Phe Pro Arg Ala Thr Ser Ser - # Pro Trp Arg Met Pro Ala ProGln# 95- Val Leu Leu Tyr Arg Ile Arg Phe - # Tyr Phe Pro Asn Trp Phe GlyLeu# 110- Glu Lys Cys His Arg Phe Gly Leu - # Arg Lys Asp Leu Ala Ser AlaIle# 125- Leu Asp Leu Pro Val Leu Glu His - # Leu Phe Ala Gln His Arg SerAsp# 140- Leu Val Ser Gly Arg Leu Pro Arg - # Gly Leu Ser Leu Lys Glu GlnGly# 160- Glu Cys Leu Ser Leu Ala Val Leu - # Asp Leu Ala Arg Met Ala ArgGlu# 175- Gln Ala Gln Arg Arg Gly Glu Leu - # Leu Lys Thr Val Ser Tyr LysAla# 190- Cys Leu Pro Pro Ser Leu Arg Asp - # Leu Ile Gln Gly Leu Ser PheVal# 205- Thr Gly Arg Arg Ile Arg Arg Thr - # Val Glu Ser Pro Leu Arg ArgVal# 220- Ala Ala Cys Gln Ala Asp Arg His - # Ser Leu Met Ala Lys Tyr IleMet# 240- Asp Leu Glu Arg Leu Asp Pro Ala - # Gly Ala Ala Glu Thr Phe HisVal# 255- Gly Leu Pro Gly Ala Leu Gly Gly - # His Asp Gly Leu Gly Leu ValArg# 270- Val Ala Gly Asp Gly Gly Ile Ala - # Trp Thr Gln Gly Glu Gln GluVal# 285- Leu Gln Pro Phe Cys Asp Phe Pro - # Glu Ile Val Asp Ile Ser IleLys# 300- Gln Ala Pro Arg Val Gly Pro Ala - # Gly Glu His Arg Leu Val ThrVal# 320- Thr Arg Thr Asp Asn Gln Ile Leu - # Glu Ala Glu Phe Pro Gly LeuPro# 335- Glu Ala Leu Ser Phe Val Ala Leu - # Val Asp Gly Tyr Phe Arg LeuThr# 350- Thr Asp Ser Gln His Phe Phe Cys - # Lys Glu Val Asp Pro Arg LeuLeu# 365- Glu Glu Val Ala Glu Gln Cys His - # Gly Pro Ile Thr Leu Asp PheAla# 380- Ile Asn Lys Leu Lys Thr Gly Gly - # Ser Arg Pro Gly Ser Tyr ValLeu# 400- Arg Arg Ile Pro Gln Asp Phe Asp - # Ser Phe Leu Leu Thr Val CysVal# 415- Gln Asn Pro Leu Gly Pro Asp Tyr - # Lys Gly Cys Leu Ile Arg ArgSer# 430- Pro Thr Gly Thr Phe Leu Leu Val - # Gly Leu Ser Arg Pro His SerSer# 445- Leu Arg Glu Leu Leu Ala Thr Cys - # Trp Asp Gly Gly Leu His ValAsp# 460- Gly Val Ala Val Thr Leu Thr Ser - # Cys Cys Ile Pro Arg Pro LysGlu# 480- Lys Ser Asn Leu Ile Val Val Gln - # Arg Gly His Ser Pro Pro ThrSer# 495- Ser Leu Val Gln Pro Gln Ser Gln - # Tyr Gln Leu Ser Gln Met ThrPhe# 510- His Lys Ile Pro Ala Asp Ser Leu - # Glu Trp His Glu Asn Leu GlyHis# 525- Gly Ser Phe Thr Lys Ile Tyr Arg - # Gly Cys Arg His Glu Val ValAsp# 540- Gly Glu Ala Arg Lys Thr Glu Val - # Leu Leu Lys Val Met Asp AlaLys# 560- His Lys Asn Cys Met Glu Ser Phe - # Leu Glu Ala Ala Ser Leu MetSer# 575- Gln Val Ser Tyr Arg His Leu Val - # Leu Leu His Gly Val Cys MetAla# 590- Gly Asp Ser Thr Met Val Glu Glu - # Phe Val His Leu Gly Ala IleAsp# 605- Met Tyr Leu Arg Lys Arg Gly His - # Leu Val Pro Ala Ser Trp LysLeu# 620- Gln Val Val Lys Gln Leu Ala Tyr - # Ala Leu Asn Tyr Leu Glu AspLys# 640- Gly Leu Ser His Gly Asn Val Ser - # Ala Arg Lys Val Leu Leu AlaArg# 655- Glu Gly Ala Asp Gly Ser Pro Pro - # Phe Ile Lys Leu Ser Asp ProGly# 670- Val Ser Pro Ala Val Leu Ser Leu - # Glu Met Leu Thr Asp Arg IlePro# 685- Trp Val Ala Pro Glu Cys Leu Arg - # Glu Ala Gln Thr Leu Ser LeuGlu# 700- Ala Asp Lys Trp Gly Phe Gly Ala - # Thr Val Trp Glu Val Phe SerGly# 720- Val Thr Met Pro Ile Ser Ala Leu - # Asp Pro Ala Lys Lys Leu GlnPhe# 735- Tyr Glu Asp Arg Gln Gln Leu Ser - # Ala Pro Lys Trp Thr Glu LeuAla# 750- Leu Leu Ile Gln Gln Cys Met Ala - # Tyr Glu Pro Val Gln Arg ProSer# 765- Leu Arg Ala Val Ile Arg Asp Leu - # Asn Ser Leu Ile Ser Ser AspTyr# 780- Glu Leu Leu Ser Asp His Thr Trp - # Cys Pro Gly Thr Arg Asp GlyLeu# 800- Trp Asn Gly Ala Gln Leu Tyr Ala - # Cys Gln Asp Pro Thr Ile PheGlu# 815- Glu Arg His Leu Lys Tyr Ile Ser - # Gln Leu Gly Lys Gly Phe PheGly# 830- Ser Val Glu Leu Cys Arg Tyr Asp - # Pro Leu Gly Asp Asn Thr GlyAla# 845- Leu Val Ala Val Lys Gln Leu Gln - # His Ser Gly Pro Asp Gln GlnArg# 860- Asp Phe Gln Arg Glu Ile Gln Ile - # Leu Lys Ala Gln His Ser AspPhe# 880- Ile Val Lys Tyr Arg Gly Val Ser - # Tyr Gly Pro Gly Arg Gln SerPro# 895- Ala Leu Val Met Glu Tyr Leu Pro - # Ser Gly Cys Leu Arg Asp PheLeu# 910- Gln Arg His Arg Gly Leu Asp Ala - # Ser Arg Leu Leu Leu Tyr SerSer# 925- Gln Ile Cys Lys Gly Met Glu Tyr - # Leu Gly Ser Arg Arg Cys ValHis# 940- Arg Asp Leu Ala Ala Arg Asn Ile - # Leu Val Glu Ser Glu Ala HisVal# 960- Lys Ile Ala Asp Phe Gly Leu Ala - # Lys Leu Leu Pro Leu Asp LysAsp# 975- Tyr Tyr Val Val Arg Glu Pro Gly - # Gln Ser Pro Ile Phe Trp TyrAla# 990- Pro Glu Ser Leu Ser Asp Asn Ile - # Phe Ser Arg Gln Ser Asp ValTrp# 10050- Ser Phe Gly Val Val Leu Tyr Glu - # Leu Phe Thr Tyr Cys Asp LysSer# 10205- Cys Ser Pro Ser Ala Glu Phe Leu - # Arg Met Met Gly Cys Glu ArgAsp# 10405- Val Pro Arg Leu Cys Arg Leu Leu - # Glu Leu Leu Glu Glu Gly GlnArg# 10550- Leu Pro Ala Pro Pro Cys Cys Pro - # Ala Glu Val Ser Cys Tyr SerGly# 10700 - # 1065- Trp Arg Asp Asp Ile Cys Leu Pro - # Ala Glu# 1080- (2) INFORMATION FOR SEQ ID NO:6:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 1129 amino (B) TYPE: amino acid (C) STRANDEDNESS: Not R - #elevant (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:- Met Gly Met Ala Cys Leu Thr Met - # Thr Glu Met Glu Ala Thr SerThr# 15- Ser Pro Val His Gln Asn Gly Asp - # Ile Pro Gly Ser Ala Asn SerVal# 30- Lys Gln Ile Glu Pro Val Leu Gln - # Val Tyr Leu Tyr His Ser LeuGly# 45- Gln Ala Glu Gly Glu Tyr Leu Lys - # Phe Pro Ser Gly Glu Tyr ValAla# 60- Glu Glu Ile Cys Val Ala Ala Ser - # Lys Ala Cys Gly Ile Thr ProVal# 80- Tyr His Asn Met Phe Ala Leu Met - # Ser Glu Thr Glu Arg Ile TrpTyr# 95- Pro Pro Asn His Val Phe His Ile - # Asp Glu Ser Thr Arg His AspIle# 110- Leu Tyr Arg Ile Arg Phe Tyr Phe - # Pro His Trp Tyr Cys Ser GlySer# 125- Ser Arg Thr Tyr Arg Tyr Gly Val - # Ser Arg Gly Ala Glu Ala ProLeu# 140- Leu Asp Asp Phe Val Met Ser Tyr - # Leu Phe Val Gln Trp Arg HisAsp# 160- Phe Val His Gly Trp Ile Lys Val - # Pro Val Thr His Glu Thr GlnGlu# 175- Glu Cys Leu Gly Met Ala Val Leu - # Asp Met Met Arg Ile Ala LysGlu# 190- Lys Asp Gln Thr Pro Leu Ala Val - # Tyr Asn Ser Val Ser Tyr LysThr# 205- Phe Leu Pro Lys Cys Val Arg Ala - # Lys Ile Gln Asp Tyr His IleLeu# 220- Thr Arg Lys Arg Ile Arg Tyr Arg - # Phe Arg Arg Phe Ile Gln GlnPhe# 240- Ser Gln Cys Lys Ala Thr Ala Arg - # Asn Leu Lys Leu Lys Tyr LeuIle# 255- Asn Leu Glu Thr Leu Gln Ser Ala - # Phe Tyr Thr Glu Gln Phe GluVal# 270- Lys Glu Ser Ala Arg Gly Pro Ser - # Gly Glu Glu Ile Phe Ala ThrIle# 285- Ile Ile Thr Gly Asn Gly Gly Ile - # Gln Trp Ser Arg Gly Lys HisLys# 300- Glu Ser Glu Thr Leu Thr Glu Gln - # Asp Val Gln Leu Tyr Cys AspPhe# 320- Pro Asp Ile Ile Asp Val Ser Ile - # Lys Gln Ala Asn Gln Glu CysSer# 335- Asn Glu Ser Arg Ile Val Thr Val - # His Lys Gln Asp Gly Lys ValLeu# 350- Glu Ile Glu Leu Ser Ser Leu Lys - # Glu Ala Leu Ser Phe Val SerLeu# 365- Ile Asp Gly Tyr Tyr Arg Leu Thr - # Ala Asp Ala His His Tyr LeuCys# 380- Lys Glu Val Ala Pro Pro Ala Val - # Leu Glu Asn Ile His Ser AsnCys# 400- His Gly Pro Ile Ser Met Asp Phe - # Ala Ile Ser Lys Leu Lys LysAla# 415- Gly Asn Gln Thr Gly Leu Tyr Val - # Leu Arg Cys Ser Pro Lys AspPhe# 430- Asn Lys Tyr Phe Leu Thr Phe Ala - # Val Glu Arg Glu Asn Val IleGlu# 445- Tyr Lys His Cys Leu Ile Thr Lys - # Asn Glu Asn Gly Glu Tyr AsnLeu# 460- Ser Gly Thr Asn Arg Asn Phe Ser - # Asn Leu Lys Asp Leu Leu AsnCys# 480- Tyr Gln Met Glu Thr Val Arg Ser - # Asp Ser Ile Ile Phe Gln PheThr# 495- Lys Cys Cys Pro Pro Lys Pro Lys - # Asp Lys Ser Asn Leu Leu ValPhe# 510- Arg Thr Asn Gly Ile Ser Asp Val - # Gln Ile Ser Pro Thr Leu GlnArg# 525- His Asn Asn Val Asn Gln Met Val - # Phe His Lys Ile Arg Asn GluAsp# 540- Leu Ile Phe Asn Glu Ser Leu Gly - # Gln Gly Thr Phe Thr Lys IlePhe# 560- Lys Gly Val Arg Arg Glu Val Gly - # Asp Tyr Gly Gln Leu His LysThr# 575- Glu Val Leu Leu Lys Val Leu Asp - # Lys Ala His Arg Asn Tyr SerGlu# 590- Ser Phe Phe Glu Ala Ala Ser Met - # Met Ser Gln Leu Ser His LysHis# 605- Leu Val Leu Asn Tyr Gly Val Cys - # Val Cys Gly Glu Glu Asn IleLeu# 620- Val Gln Glu Phe Val Lys Phe Gly - # Ser Leu Asp Thr Tyr Leu LysLys# 640- Asn Lys Asn Ser Ile Asn Ile Leu - # Trp Lys Leu Gly Val Ala LysGln# 655- Leu Ala Trp Ala Met His Phe Leu - # Glu Glu Lys Ser Leu Ile HisGly# 670- Asn Val Cys Ala Lys Asn Ile Leu - # Leu Ile Arg Glu Glu Asp ArgArg# 685- Thr Gly Asn Pro Pro Phe Ile Lys - # Leu Ser Asp Pro Gly Ile SerIle# 700- Thr Val Leu Pro Lys Asp Ile Leu - # Gln Glu Arg Ile Pro Trp ValPro# 720- Pro Glu Cys Ile Glu Asn Pro Lys - # Asn Leu Asn Leu Ala Thr AspLys# 735- Trp Ser Phe Gly Thr Thr Leu Trp - # Glu Ile Cys Ser Gly Gly AspLys# 750- Pro Leu Ser Ala Leu Asp Ser Gln - # Arg Lys Leu Gln Phe Tyr GluAsp# 765- Lys His Gln Leu Pro Ala Pro Lys - # Trp Thr Glu Leu Ala Asn LeuIle# 780- Asn Asn Cys Met Asp Tyr Glu Pro - # Asp Phe Arg Pro Ala Phe ArgAla# 800- Val Ile Arg Asp Leu Asn Ser Leu - # Phe Thr Pro Asp Tyr Glu LeuLeu# 815- Thr Glu Asn Asp Met Leu Pro Asn - # Met Arg Ile Gly Ala Leu GlyPhe# 830- Ser Gly Ala Phe Glu Asp Arg Asp - # Pro Thr Gln Phe Glu Glu ArgHis# 845- Leu Lys Phe Leu Gln Gln Leu Gly - # Lys Gly Asn Phe Gly Ser ValGlu# 860- Met Cys Arg Tyr Asp Pro Leu Gln - # Asp Asn Thr Gly Glu Val ValAla# 880- Val Lys Lys Leu Gln His Ser Thr - # Glu Glu His Leu Arg Asp PheGlu# 895- Arg Glu Ile Glu Ile Leu Lys Ser - # Leu Gln His Asp Asn Ile ValLys# 910- Tyr Lys Gly Val Cys Tyr Ser Ala - # Gly Arg Arg Asn Leu Arg LeuIle# 925- Met Glu Tyr Leu Pro Tyr Gly Ser - # Leu Arg Asp Tyr Leu Gln LysHis# 940- Lys Glu Arg Ile Asp His Lys Lys - # Leu Leu Gln Tyr Thr Ser GlnIle# 960- Cys Lys Gly Met Glu Tyr Leu Gly - # Thr Lys Arg Tyr Ile His ArgAsp# 975- Leu Ala Thr Arg Asn Ile Leu Val - # Glu Asn Glu Asn Arg Val LysIle# 990- Gly Asp Phe Gly Leu Thr Lys Val - # Leu Pro Gln Asp Lys Glu TyrTyr# 10050- Lys Val Lys Glu Pro Gly Glu Ser - # Pro Ile Phe Trp Tyr Ala ProGln# 10205- Ser Leu Thr Glu Ser Lys Phe Ser - # Val Ala Ser Asp Val Trp SerPhe# 10405- Gly Val Val Leu Tyr Glu Leu Phe - # Thr Tyr Ile Glu Lys Ser LysSer# 10550- Pro Pro Val Glu Phe Met Arg Met - # Ile Gly Asn Asp Lys Gln GlyGln# 10700 - # 1065- Met Ile Val Phe His Leu Ile Glu - # Leu Leu Lys Ser Asn Gly ArgLeu# 10850- Pro Arg Pro Glu Gly Cys Pro Asp - # Glu Ile Tyr Val Ile Met ThrGlu# 11005- Cys Trp Asn Asn Asn Val Ser Gln - # Arg Pro Ser Phe Arg Asp LeuSer# 11205- Phe Gly Trp Ile Lys Cys Gly Thr - # Val# 1125- (2) INFORMATION FOR SEQ ID NO:7:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 1154 amino (B) TYPE: amino acid (C) STRANDEDNESS: Not R - #elevant (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:- Met Gln Tyr Leu Asn Ile Lys Glu - # Asp Cys Asn Ala Met Ala PheCys# 15- Ala Lys Met Arg Ser Ser Lys Lys - # Thr Glu Val Asn Leu Glu AlaPro# 30- Glu Pro Gly Val Glu Val Ile Phe - # Tyr Leu Ser Asp Arg Glu ProLeu# 45- Arg Leu Gly Ser Gly Glu Tyr Thr - # Ala Glu Glu Leu Cys Ile ArgAla# 60- Ala Gln Ala Cys Arg Ile Ser Pro - # Leu Cys His Asn Leu Phe AlaLeu# 80- Tyr Asp Glu Asn Thr Lys Leu Trp - # Tyr Ala Pro Asn Arg Thr IleThr# 95- Val Asp Asp Lys Met Ser Leu Arg - # Leu His Tyr Arg Met Arg PheTyr# 110- Phe Thr Asn Trp His Gly Thr Asn - # Asp Asn Glu Gln Ser Val TrpArg# 125- His Ser Pro Lys Lys Gln Lys Asn - # Gly Tyr Glu Lys Lys Lys IlePro# 140- Asp Ala Thr Pro Leu Leu Asp Ala - # Ser Ser Leu Glu Tyr Leu PheAla# 160- Gln Gly Gln Tyr Asp Leu Val Lys - # Cys Leu Ala Pro Ile Arg AspPro# 175- Lys Thr Glu Gln Asp Gly His Asp - # Ile Glu Asn Glu Cys Leu GlyMet# 190- Ala Val Leu Ala Ile Ser His Tyr - # Ala Met Met Lys Lys Met GlnLeu# 205- Pro Glu Leu Pro Lys Asp Ile Ser - # Tyr Lys Arg Tyr Ile Pro GluThr# 220- Leu Asn Lys Ser Ile Arg Gln Arg - # Asn Leu Leu Thr Arg Met ArgIle# 240- Asn Asn Val Phe Lys Asp Phe Leu - # Lys Glu Phe Asn Asn Lys ThrIle# 255- Cys Asp Ser Ser Val Ser Thr His - # Asp Leu Lys Val Lys Tyr LeuAla# 270- Thr Leu Glu Thr Leu Thr Lys His - # Tyr Gly Ala Glu Ile Phe GluThr# 285- Ser Met Leu Leu Ile Ser Ser Glu - # Asn Glu Met Asn Trp Phe HisSer# 300- Asn Asp Gly Gly Asn Val Leu Tyr - # Tyr Glu Val Met Val Thr GlyAsn# 320- Leu Gly Ile Gln Trp Arg His Lys - # Pro Asn Val Val Ser Val GluLys# 335- Glu Lys Asn Lys Leu Lys Arg Lys - # Lys Leu Glu Asn Lys Asp LysLys# 350- Asp Glu Glu Lys Asn Lys Ile Arg - # Glu Glu Trp Asn Asn Phe SerPhe# 365- Phe Pro Glu Ile Thr His Ile Val - # Ile Lys Glu Ser Val Val SerIle# 380- Asn Lys Gln Asp Asn Lys Lys Met - # Glu Leu Lys Leu Ser Ser HisGlu# 400- Glu Ala Leu Ser Phe Val Ser Leu - # Val Asp Gly Tyr Phe Arg LeuThr# 415- Ala Asp Ala His His Tyr Leu Cys - # Thr Asp Val Ala Pro Pro LeuIle# 430- Val His Asn Ile Gln Asn Gly Cys - # His Gly Pro Ile Cys Thr GluTyr# 445- Ala Ile Asn Lys Leu Arg Gln Glu - # Gly Ser Glu Glu Gly Met TyrVal# 460- Leu Arg Trp Ser Cys Thr Asp Phe - # Asp Asn Ile Leu Met Thr ValThr# 480- Cys Phe Glu Lys Ser Glu Gln Val - # Gln Gly Ala Gln Lys Gln PheLys# 495- Asn Phe Gln Ile Glu Val Gln Lys - # Gly Arg Tyr Ser Leu His GlySer# 510- Asp Arg Ser Phe Pro Ser Leu Gly - # Asp Leu Met Ser His Leu LysLys# 525- Gln Ile Leu Arg Thr Asp Asn Ile - # Ser Phe Met Leu Lys Arg CysCys# 540- Gln Pro Lys Pro Arg Glu Ile Ser - # Asn Leu Leu Val Ala Thr LysLys# 560- Ala Gln Glu Trp Gln Pro Val Tyr - # Pro Met Ser Gln Leu Ser PheAsp# 575- Arg Ile Leu Lys Lys Asp Leu Val - # Gln Gly Glu His Leu Gly ArgGly# 590- Thr Arg Thr His Ile Tyr Ser Gly - # Thr Leu Met Asp Tyr Lys AspAsp# 605- Glu Gly Thr Ser Glu Glu Lys Lys - # Ile Lys Val Ile Leu Lys ValLeu# 620- Asp Pro Ser His Arg Asp Ile Ser - # Leu Ala Phe Phe Glu Ala AlaSer# 640- Met Met Arg Gln Val Ser His Lys - # His Ile Val Tyr Leu Tyr GlyVal# 655- Cys Val Arg Asp Val Glu Asn Ile - # Met Val Glu Glu Phe Val GluGly# 670- Gly Pro Leu Asp Leu Phe Met His - # Arg Lys Ser Asp Val Leu ThrThr# 685- Pro Trp Lys Phe Lys Val Ala Lys - # Gln Leu Ala Ser Ala Leu SerTyr# 700- Leu Glu Asp Lys Asp Leu Val His - # Gly Asn Val Cys Thr Lys AsnLeu# 720- Leu Leu Ala Arg Glu Gly Ile Asp - # Ser Glu Cys Gly Pro Phe IleLys# 735- Leu Ser Asp Pro Gly Ile Pro Ile - # Thr Val Leu Ser Arg Gln GluCys# 750- Ile Glu Arg Ile Pro Trp Ile Ala - # Pro Glu Cys Val Glu Asp SerLys# 765- Asn Leu Ser Val Ala Ala Asp Lys - # Trp Ser Phe Gly Thr Thr LeuTrp# 780- Glu Ile Cys Tyr Asn Gly Glu Ile - # Pro Leu Lys Asp Lys Thr LeuIle# 800- Glu Lys Glu Arg Phe Tyr Glu Ser - # Arg Cys Arg Pro Val Thr ProSer# 815- Cys Lys Glu Leu Ala Asp Leu Met - # Thr Arg Cys Met Asn Tyr AspPro# 830- Asn Gln Arg Pro Phe Phe Arg Ala - # Ile Met Arg Asp Ile Asn LysLeu# 845- Glu Glu Gln Asn Pro Asp Ile Val - # Ser Arg Lys Lys Asn Gln ProThr# 860- Glu Val Asp Pro Thr His Phe Glu - # Lys Arg Phe Leu Lys Arg IleArg# 880- Asp Leu Gly Glu Gly His Phe Gly - # Lys Val Glu Leu Cys Arg TyrAsp# 895- Pro Glu Asp Asn Thr Gly Glu Gln - # Val Ala Val Lys Ser Leu LysPro# 910- Glu Ser Gly Gly Asn His Ile Ala - # Asp Leu Lys Lys Glu Ile GluIle# 925- Leu Arg Asn Leu Tyr His Glu Asn - # Ile Val Lys Tyr Lys Gly IleCys# 940- Thr Glu Asp Gly Gly Asn Gly Ile - # Lys Leu Ile Met Glu Phe LeuPro# 960- Ser Gly Ser Leu Lys Glu Tyr Leu - # Pro Lys Asn Lys Asn Lys IleAsn# 975- Leu Lys Gln Gln Leu Lys Tyr Ala - # Val Gln Ile Cys Lys Gly MetAsp# 990- Tyr Leu Gly Ser Arg Gln Tyr Val - # His Arg Asp Leu Ala Ala ArgAsn# 10050- Val Leu Val Glu Ser Glu His Gln - # Val Lys Ile Gly Asp Phe GlyLeu# 10205- Thr Lys Ala Ile Glu Thr Asp Lys - # Glu Tyr Tyr Thr Val Lys AspAsp# 10405- Arg Asp Ser Pro Val Phe Trp Tyr - # Ala Pro Glu Cys Leu Met GlnSer# 10550- Lys Phe Tyr Ile Ala Ser Asp Val - # Trp Ser Phe Gly Val Thr LeuHis# 10700 - # 1065- Glu Leu Leu Thr Tyr Cys Asp Ser - # Asp Ser Ser Pro Met Ala LeuPhe# 10850- Leu Lys Met Ile Gly Pro Thr His - # Gly Gln Met Thr Val Thr ArgLeu# 11005- Val Asn Thr Leu Lys Glu Gly Lys - # Arg Leu Pro Cys Pro Pro AsnCys# 11205- Pro Asp Glu Val Tyr Gln Leu Met - # Arg Lys Cys Trp Glu Phe GlnPro# 11350- Ser Asn Arg Thr Ser Phe Gln Asn - # Leu Ile Glu Gly Phe Glu AlaLeu# 11500 - # 1145- Leu Lys- (2) INFORMATION FOR SEQ ID NO:8:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 1187 amino (B) TYPE: amino acid (C) STRANDEDNESS: Not R - #elevant (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:- Met Pro Leu Arg His Trp Gly Met - # Ala Arg Gly Ser Lys Pro ValGly# 15- Asp Gly Ala Gln Pro Met Ala Ala - # Met Gly Gly Leu Lys Val LeuLeu# 30- His Trp Ala Gly Pro Gly Gly Gly - # Glu Pro Trp Val Thr Phe SerGlu# 45- Ser Ser Leu Thr Ala Glu Glu Val - # Cys Ile His Ile Ala His LysVal# 60- Gly Ile Thr Pro Pro Cys Phe Asn - # Leu Phe Ala Leu Phe Asp AlaGln# 80- Ala Gln Val Trp Leu Pro Pro Asn - # His Ile Leu Glu Ile Pro ArgAsp# 95- Ala Ser Leu Met Leu Tyr Phe Arg - # Ile Arg Phe Tyr Phe Arg AsnTrp# 110- His Gly Met Asn Pro Arg Glu Pro - # Ala Val Tyr Arg Cys Gly ProPro# 125- Gly Thr Glu Ala Ser Ser Asp Gln - # Thr Ala Gln Gly Met Gln LeuLeu# 140- Asp Pro Ala Ser Phe Glu Tyr Leu - # Phe Glu Gln Gly Lys His GluPhe# 160- Val Asn Asp Val Ala Ser Leu Trp - # Glu Leu Ser Thr Glu Glu GluIle# 175- His His Phe Lys Asn Glu Ser Leu - # Gly Met Ala Phe Leu His LeuCys# 190- His Leu Ala Leu Arg His Gly Ile - # Pro Leu Glu Glu Val Ala LysLys# 205- Thr Ser Phe Lys Asp Cys Ile Pro - # Arg Ser Phe Arg Arg His IleArg# 220- Gln His Ser Ala Leu Thr Arg Leu - # Arg Leu Arg Asn Val Phe ArgArg# 240- Phe Leu Arg Asp Phe Gln Pro Gly - # Arg Leu Ser Gln Gln Met ValMet# 255- Val Lys Tyr Leu Ala Thr Leu Glu - # Arg Leu Ala Pro Arg Phe GlyThr# 270- Glu Arg Val Pro Val Cys His Leu - # Arg Leu Leu Ala Gln Ala GluGly# 285- Glu Pro Cys Tyr Ile Arg Asp Ser - # Gly Val Ala Pro Thr Asp ProGly# 300- Pro Glu Ser Ala Ala Gly Pro Pro - # Thr His Glu Val Leu Val ThrGly# 320- Thr Gly Gly Ile Gln Trp Trp Pro - # Val Glu Glu Glu Val Asn LysGlu# 335- Glu Gly Ser Ser Gly Ser Ser Gly - # Arg Asn Pro Gln Ala Ser LeuPhe# 350- Gly Lys Lys Ala Lys Ala His Lys - # Ala Phe Gly Gln Pro Ala AspArg# 365- Pro Arg Glu Pro Leu Trp Ala Tyr - # Phe Cys Asp Phe Arg Asp IleThr# 380- His Val Val Leu Lys Glu His Cys - # Val Ser Ile His Arg Gln AspAsn# 400- Lys Cys Leu Glu Leu Ser Leu Pro - # Ser Arg Ala Ala Ala Leu SerPhe# 415- Val Ser Leu Val Asp Gly Tyr Phe - # Arg Leu Thr Ala Asp Ser SerHis# 430- Tyr Leu Cys His Glu Val Ala Pro - # Pro Arg Leu Val Met Ser IleArg# 445- Asp Gly Ile His Gly Pro Leu Leu - # Glu Pro Phe Val Gln Ala LysLeu# 460- Arg Pro Glu Asp Gly Leu Tyr Leu - # Ile His Trp Ser Thr Ser HisPro# 480- Tyr Arg Leu Ile Leu Thr Val Ala - # Gln Arg Ser Gln Ala Pro AspGly# 495- Met Gln Ser Leu Arg Leu Arg Lys - # Phe Pro Ile Glu Gln Gln AspGly# 510- Ala Phe Val Leu Glu Gly Trp Gly - # Arg Ser Phe Pro Ser Val ArgGlu# 525- Leu Gly Ala Ala Leu Gln Gly Cys - # Leu Leu Arg Ala Gly Asp AspCys# 540- Phe Ser Leu Arg Arg Cys Cys Leu - # Pro Gln Pro Gly Glu Thr SerAsn# 560- Leu Ile Ile Met Arg Gly Ala Arg - # Ala Ser Pro Arg Thr Leu AsnLeu# 575- Ser Gln Leu Ser Phe His Arg Val - # Asp Gln Lys Glu Ile Thr GlnLeu# 590- Ser His Leu Gly Gln Gly Thr Arg - # Thr Asn Val Tyr Glu Gly ArgLeu# 605- Arg Val Glu Gly Ser Gly Asp Pro - # Glu Glu Gly Lys Met Asp AspGlu# 620- Asp Pro Leu Val Pro Gly Arg Asp - # Arg Gly Gln Glu Leu Arg ValVal# 640- Leu Lys Val Leu Asp Pro Ser His - # His Asp Ile Ala Leu Ala PheTyr# 655- Glu Thr Ala Ser Leu Met Ser Gln - # Val Ser His Thr His Leu AlaPhe# 670- Val His Gly Val Cys Val Arg Gly - # Pro Glu Asn Ser Met Val ThrGlu# 685- Tyr Val Glu His Gly Pro Leu Asp - # Val Trp Leu Arg Arg Glu ArgGly# 700- His Val Pro Met Ala Trp Lys Met - # Val Val Ala Gln Gln Leu AlaSer# 720- Ala Leu Ser Tyr Leu Glu Asn Lys - # Asn Leu Val His Gly Asn ValCys# 735- Gly Arg Asn Ile Leu Leu Ala Arg - # Leu Gly Leu Ala Glu Gly ThrSer# 750- Pro Phe Ile Lys Leu Ser Asp Pro - # Gly Val Gly Leu Gly Ala LeuSer# 765- Arg Glu Glu Arg Val Glu Arg Ile - # Pro Trp Leu Ala Pro Glu CysLeu# 780- Pro Gly Gly Ala Asn Ser Leu Ser - # Thr Ala Met Asp Lys Trp GlyPhe# 800- Gly Ala Thr Leu Leu Glu Ile Cys - # Phe Asp Gly Glu Ala Pro LeuGln# 815- Ser Arg Ser Pro Ser Glu Lys Glu - # His Phe Tyr Gln Arg Gln HisArg# 830- Leu Pro Glu Pro Ser Cys Pro Gln - # Leu Ala Thr Leu Thr Ser GlnCys# 845- Leu Thr Tyr Glu Pro Thr Gln Arg - # Pro Ser Phe Arg Thr Ile LeuArg# 860- Asp Leu Thr Arg Val Gln Pro His - # Asn Leu Ala Asp Val Leu ThrVal# 880- Asn Arg Asp Ser Pro Ala Val Gly - # Pro Thr Thr Phe His Lys ArgTyr# 895- Leu Lys Lys Ile Arg Asp Leu Gly - # Glu Gly His Phe Gly Lys ValSer# 910- Leu Tyr Cys Tyr Asp Pro Thr Asn - # Asp Gly Thr Gly Glu Met ValAla# 925- Val Lys Ala Leu Lys Ala Asp Cys - # Gly Pro Gln His Arg Ser GlyTrp# 940- Lys Gln Glu Ile Asp Ile Leu Arg - # Thr Leu Tyr His Glu His IleIle# 960- Lys Tyr Lys Gly Cys Cys Glu Asp - # Gln Gly Glu Lys Ser Leu GlnLeu# 975- Val Met Glu Tyr Val Pro Leu Gly - # Ser Leu Arg Asp Tyr Leu ProArg# 990- His Ser Ile Gly Leu Ala Gln Leu - # Leu Leu Phe Ala Gln Gln IleCys# 10050- Glu Gly Met Ala Tyr Leu His Ala - # His Asp Tyr Ile His Arg AspLeu# 10205- Ala Ala Arg Asn Val Leu Leu Asp - # Asn Asp Arg Leu Val Lys IleGly# 10405- Asp Phe Gly Leu Ala Lys Ala Val - # Pro Glu Gly His Glu Tyr TyrArg# 10550- Val Arg Glu Asp Gly Asp Ser Pro - # Val Phe Trp Tyr Ala Pro GluCys# 10700 - # 1065- Leu Lys Glu Tyr Lys Phe Tyr Tyr - # Ala Ser Asp Val Trp Ser PheGly# 10850- Val Thr Leu Tyr Glu Leu Leu Thr - # His Cys Asp Ser Ser Gln SerPro# 11005- Pro Thr Lys Phe Leu Glu Leu Ile - # Gly Ile Ala Gln Gly Gln MetThr# 11205- Val Leu Arg Leu Thr Glu Leu Leu - # Glu Arg Gly Glu Arg Leu ProArg# 11350- Pro Asp Lys Cys Pro Cys Glu Val - # Tyr His Leu Met Lys Asn CysTrp# 11500 - # 1145- Glu Thr Glu Ala Ser Phe Arg Pro - # Thr Phe Glu Asn Leu Ile ProIle# 11650- Leu Lys Thr Val His Glu Lys Tyr - # Gln Gly Gln Ala Pro Ser ValPhe# 11805- Ser Val Cys 1185- (2) INFORMATION FOR SEQ ID NO:9:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 498 amino (B) TYPE: amino acid (C) STRANDEDNESS: Not R - #elevant (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:- Met Pro Leu Gly Ala Glu Glu Cys - # Ala Ala Ile Pro His Asn LeuPhe# 15- Ala Leu Trp Pro Pro Asn Leu Tyr - # Arg Ile Arg Phe Tyr Phe AsnTrp# 30- Gly Leu Leu Asp Glu Tyr Leu Phe - # Gln Asp Val Glu Gln Glu CysLeu# 45- Gly Met Ala Val Leu Ala Glu Ser - # Tyr Lys Pro Arg Ile Leu ThrArg# 60- Arg Ile Arg Phe Phe Leu Phe Leu - # Lys Lys Tyr Leu Leu Glu LeuGlu# 80- Phe Val Val Val Thr Gly Gly Gly - # Ile Gln Trp Glu Phe Cys AspPhe# 95- Pro Ile Ile Lys Val Gln Asp Asn - # Lys Leu Glu Leu Ser Glu AlaLeu# 110- Ser Phe Val Ser Leu Val Asp Gly - # Tyr Phe Arg Leu Thr Ala AspHis# 125- Tyr Leu Cys Val Ala Pro Pro Ile - # Cys His Gly Pro Ile Phe AlaIle# 140- Lys Leu Gly Tyr Val Leu Arg Trp - # Ser Asp Phe Leu Thr Val ValLys# 160- Ile Gly Leu Gly Arg Phe Ser Leu - # Arg Phe Cys Cys Pro Pro SerAsn# 175- Leu Leu Val Gln Ser Gln Phe His - # Ile Leu Glu Leu Gly Gly ThrThr# 190- Ile Tyr Gly Asp Val Leu Lys Val - # Leu Asp His Phe Glu Ala AlaSer# 205- Met Ser Gln Val Ser His His Leu - # Val Gly Val Cys Val Glu AsnVal# 220- Glu Phe Val Gly Leu Asp Arg Trp - # Lys Val Ala Lys Gln Leu AlaAla# 240- Leu Tyr Leu Glu Asp Leu Leu His - # Gly Asn Val Cys Asn Ile LeuLeu# 255- Ala Arg Glu Gly Pro Phe Ile Lys - # Leu Ser Asp Pro Gly Val LeuSer# 270- Glu Arg Ile Pro Trp Ala Pro Glu - # Cys Asn Leu Ser Ala Asp LysTrp# 285- Phe Gly Thr Leu Trp Glu Cys Gly - # Pro Leu Lys Phe Tyr Glu LeuPro# 300- Glu Leu Ala Leu Cys Met Tyr Glu - # Pro Gln Arg Pro Phe Arg AlaArg# 320- Asp Leu Asn Leu Pro Asp Pro Thr - # Phe Glu Arg Leu Lys Ile LeuGly# 335- Gly Phe Gly Val Glu Leu Cys Arg - # Tyr Asp Pro Asp Asn Thr GlyGlu# 350- Val Ala Val Lys Leu Ser Gly His - # Asp Glu Ile Ile Leu Leu HisIle# 365- Val Lys Tyr Lys Gly Cys Gly Leu - # Met Glu Tyr Leu Pro Gly SerLeu# 380- Arg Asp Tyr Leu Ile Leu Leu Leu - # Tyr Leu Leu Gln Ile Cys LysGly# 400- Met Tyr Leu Gly Tyr His Arg Asp - # Leu Ala Ala Arg Asn Leu ValGlu# 415- Glu Val Lys Ile Gly Asp Phe Gly - # Leu Lys Pro Asp Lys Glu TyrTyr# 430- Val Glu Gly Ser Pro Phe Trp Tyr - # Ala Pro Glu Leu Leu Phe AlaSer# 445- Asp Val Trp Ser Phe Gly Val Leu - # Tyr Glu Leu Thr Tyr Cys AspSer# 460- Ser Pro Phe Leu Met Ile Gly Gly - # Gln Met Val Arg Leu Glu LeuLeu# 480- Gly Arg Leu Pro Pro Cys Pro Glu - # Val Tyr Leu Met Cys Trp SerArg# 495- Phe Leu- (2) INFORMATION FOR SEQ ID NO:10:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 1082 amino (B) TYPE: amino acid (C) STRANDEDNESS: Not R - #elevant (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:- Met Ala Pro Pro Ser Glu Glu Thr - # Pro Leu Ile Pro Gln Arg SerCys# 15- Ser Leu Leu Ser Thr Glu Ala Gly - # Ala Leu His Val Leu Leu ProAla# 30- Arg Gly Pro Gly Pro Pro Gln Arg - # Leu Ser Phe Ser Phe Gly AspHis# 45- Leu Ala Glu Asp Leu Cys Val Gln - # Ala Ala Lys Ala Ser Ala IleLeu# 60- Pro Val Tyr His Ser Leu Phe Ala - # Leu Ala Thr Glu Asp Leu SerCys# 80- Trp Phe Pro Arg Ala Thr Ser Ser - # Pro Trp Arg Met Pro Ala ProGln# 95- Val Leu Leu Tyr Arg Ile Arg Phe - # Tyr Phe Pro Asn Trp Phe GlyLeu# 110- Glu Lys Cys His Arg Phe Gly Leu - # Arg Lys Asp Leu Ala Ser AlaIle# 125- Leu Asp Leu Pro Val Leu Glu His - # Leu Phe Ala Gln His Arg SerAsp# 140- Leu Val Ser Gly Arg Leu Pro Arg - # Gly Leu Ser Leu Lys Glu GlnGly# 160- Glu Cys Leu Ser Leu Ala Val Leu - # Asp Leu Ala Arg Met Ala ArgGlu# 175- Gln Ala Gln Arg Arg Gly Glu Leu - # Leu Lys Thr Val Ser Tyr LysAla# 190- Cys Leu Pro Pro Ser Leu Arg Asp - # Leu Ile Gln Gly Leu Ser PheVal# 205- Thr Gly Arg Arg Ile Arg Arg Thr - # Val Glu Ser Pro Leu Arg ArgVal# 220- Ala Ala Cys Gln Ala Asp Arg His - # Ser Leu Met Ala Lys Tyr IleMet# 240- Asp Leu Glu Arg Leu Asp Pro Ala - # Gly Ala Ala Glu Thr Phe HisVal# 255- Gly Leu Pro Gly Ala Leu Gly Gly - # His Asp Gly Leu Gly Leu ValArg# 270- Val Ala Gly Asp Gly Gly Ile Ala - # Trp Thr Gln Gly Glu Gln GluVal# 285- Leu Gln Pro Phe Cys Asp Phe Pro - # Glu Ile Val Asp Ile Ser IleLys# 300- Gln Ala Pro Arg Val Gly Pro Ala - # Gly Glu His Arg Leu Val ThrVal# 320- Thr Arg Thr Asp Asn Gln Ile Leu - # Glu Ala Glu Phe Pro Gly LeuPro# 335- Glu Ala Leu Ser Phe Val Ala Leu - # Val Asp Gly Tyr Phe Arg LeuThr# 350- Thr Asp Ser Gln His Phe Phe Cys - # Lys Glu Val Asp Pro Arg LeuLeu# 365- Glu Glu Val Ala Glu Gln Cys His - # Gly Pro Ile Thr Leu Asp PheAla# 380- Ile Asn Lys Leu Lys Thr Gly Gly - # Ser Arg Pro Gly Ser Tyr ValLeu# 400- Arg Arg Ile Pro Gln Asp Phe Asp - # Ser Phe Leu Leu Thr Val CysVal# 415- Gln Asn Pro Leu Gly Pro Asp Tyr - # Lys Gly Cys Leu Ile Arg ArgSer# 430- Pro Thr Gly Thr Phe Leu Leu Val - # Gly Leu Ser Arg Pro His SerSer# 445- Leu Arg Glu Leu Leu Ala Thr Cys - # Trp Asp Gly Gly Leu His ValAsp# 460- Gly Val Ala Val Thr Leu Thr Ser - # Cys Cys Ile Pro Arg Pro LysGlu# 480- Lys Ser Asn Leu Ile Val Val Gln - # Arg Gly His Ser Pro Pro ThrSer# 495- Ser Leu Val Gln Pro Gln Ser Gln - # Tyr Gln Leu Ser Gln Met ThrPhe# 510- His Lys Ile Pro Ala Asp Ser Leu - # Glu Trp His Glu Asn Leu GlyHis# 525- Gly Ser Phe Thr Lys Ile Tyr Arg - # Gly Cys Arg His Glu Val ValAsp# 540- Gly Glu Ala Arg Lys Thr Glu Val - # Leu Leu Lys Val Met Asp AlaLys# 560- His Lys Asn Cys Met Glu Ser Phe - # Leu Glu Ala Ala Ser Leu MetSer# 575- Gln Val Ser Tyr Arg His Leu Val - # Leu Leu His Gly Val Cys MetAla# 590- Gly Asp Ser Thr Met Val Glu Glu - # Phe Val His Leu Gly Ala IleAsp# 605- Met Tyr Leu Arg Lys Arg Gly His - # Leu Val Pro Ala Ser Trp LysLeu# 620- Gln Val Val Lys Gln Leu Ala Tyr - # Ala Leu Asn Tyr Leu Glu AspLys# 640- Gly Leu Ser His Gly Asn Val Ser - # Ala Arg Lys Val Leu Leu AlaArg# 655- Glu Gly Ala Asp Gly Ser Pro Pro - # Phe Ile Lys Leu Ser Asp ProGly# 670- Val Ser Pro Ala Val Leu Ser Leu - # Glu Met Leu Thr Asp Arg IlePro# 685- Trp Val Ala Pro Glu Cys Leu Arg - # Glu Ala Gln Thr Leu Ser LeuGlu# 700- Ala Asp Lys Trp Gly Phe Gly Ala - # Thr Val Trp Glu Val Phe SerGly# 720- Val Thr Met Pro Ile Ser Ala Leu - # Asp Pro Ala Lys Lys Leu GlnPhe# 735- Tyr Glu Asp Arg Gln Gln Leu Ser - # Ala Pro Lys Trp Thr Glu LeuAla# 750- Leu Leu Ile Gln Gln Cys Met Ala - # Tyr Glu Pro Val Gln Arg ProSer# 765- Leu Arg Ala Val Ile Arg Asp Leu - # Asn Ser Leu Ile Ser Ser AspTyr# 780- Glu Leu Leu Ser Asp His Thr Trp - # Cys Pro Gly Thr Arg Asp GlyLeu# 800- Trp Asn Gly Ala Gln Leu Tyr Ala - # Cys Gln Asp Pro Thr Ile PheGlu# 815- Glu Arg His Leu Lys Tyr Ile Ser - # Gln Leu Gly Lys Gly Phe PheGly# 830- Ser Val Glu Leu Cys Arg Tyr Asp - # Pro Leu Gly Asp Asn Thr GlyAla# 845- Leu Val Ala Val Lys Gln Leu Gln - # His Ser Gly Pro Asp Gln GlnArg# 860- Asp Phe Gln Arg Glu Ile Gln Ile - # Leu Lys Ala Gln His Ser AspPhe# 880- Ile Val Lys Tyr Arg Gly Val Ser - # Tyr Gly Pro Gly Arg Gln SerPro# 895- Ala Leu Val Met Glu Tyr Leu Pro - # Ser Gly Cys Leu Arg Asp PheLeu# 910- Gln Arg His Arg Gly Leu Asp Ala - # Ser Arg Leu Leu Leu Tyr SerSer# 925- Gln Ile Cys Lys Gly Met Glu Tyr - # Leu Gly Ser Arg Arg Cys ValHis# 940- Arg Asp Leu Ala Ala Arg Asn Ile - # Leu Val Glu Ser Glu Ala HisVal# 960- Lys Ile Ala Asp Phe Gly Leu Ala - # Lys Leu Leu Pro Leu Asp LysAsp# 975- Tyr Tyr Val Val Arg Glu Pro Gly - # Gln Ser Pro Ile Phe Trp TyrAla# 990- Pro Glu Ser Leu Ser Asp Asn Ile - # Phe Ser Arg Gln Ser Asp ValTrp# 10050- Ser Phe Gly Val Val Leu Tyr Glu - # Leu Phe Thr Tyr Cys Asp LysSer# 10205- Cys Ser Pro Ser Ala Glu Phe Leu - # Arg Met Met Gly Cys Glu ArgAsp# 10405- Val Pro Arg Leu Cys Arg Leu Leu - # Glu Leu Leu Glu Glu Gly GlnArg# 10550- Leu Pro Ala Pro Pro Cys Cys Pro - # Ala Glu Val Ser Cys Tyr SerGly# 10700 - # 1065- Trp Arg Asp Asp Ile Cys Leu Pro - # Ala Glu# 1080- (2) INFORMATION FOR SEQ ID NO:11:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 1100 amino (B) TYPE: amino acid (C) STRANDEDNESS: Not R - #elevant (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:- Met Ala Pro Pro Ser Glu Glu Thr - # Pro Leu Ile Ser Gln Arg SerCys# 15- Ser Leu Ser Ser Ser Glu Ala Gly - # Ala Leu His Val Leu Leu ProPro# 30- Arg Gly Pro Gly Pro Pro Gln Arg - # Leu Ser Phe Ser Phe Gly AspTyr# 45- Leu Ala Glu Asp Leu Cys Val Arg - # Ala Ala Lys Ala Cys Gly IleLeu# 60- Pro Val Tyr His Ser Leu Phe Ala - # Leu Ala Thr Glu Asp Leu SerCys# 80- Trp Phe Pro Pro Ser His Ile Phe - # Ser Ile Glu Asp Val Asp ThrGln# 95- Val Leu Val Tyr Arg Leu Arg Phe - # Tyr Phe Pro Gly Trp Phe GlyLeu# 110- Glu Thr Cys His Arg Phe Gly Leu - # His Lys Asp Leu Thr Ser AlaIle# 125- Leu Asp Val His Val Leu Glu His - # Leu Phe Ala Gln His Arg SerAsp# 140- Leu Val Ser Gly Arg Leu Pro Val - # Gly Leu Ser Leu Lys Asp GlnGly# 160- Glu Phe Leu Ser Leu Ala Val Leu - # Asp Leu Ala Gln Met Ala ArgLys# 175- Gln Ala Gln Arg Pro Gly Glu Leu - # Leu Lys Ser Val Ser Tyr LysAla# 190- Cys Leu Pro Pro Ser Leu Arg Asp - # Leu Ile Gln Gly Gln Ser PheVal# 205- Thr Arg Arg Arg Ile Arg Arg Thr - # Val Val Gln Ala Leu Ala ProCys# 220- Ser Ser Leu Pro Ser Arg Pro Tyr - # Ala Leu Met Ala Lys Tyr IleLeu# 240- Asp Leu Glu Arg Leu His Pro Ala - # Ala Thr Thr Glu Ser Phe LeuVal# 255- Gly Leu Pro Gly Ala Gln Glu Glu - # Pro Gly Cys Leu Arg Val ThrGly# 270- Asp Asn Gly Ile Ala Trp Ser Ser - # Lys Asp Gln Glu Leu Phe GlnThr# 285- Phe Cys Asp Phe Pro Glu Ile Val - # Asp Val Ser Ile Lys Gln AlaPro# 300- Arg Val Gly Pro Ala Gly Glu His - # Arg Leu Val Thr Ile Thr ArgMet# 320- Asp Gly His Ile Leu Glu Ala Glu - # Phe Pro Gly Leu Pro Glu AlaLeu# 335- Ser Phe Val Ala Leu Val Asp Gly - # Tyr Phe Arg Leu Ile Cys AspSer# 350- Arg His Phe Phe Cys Lys Glu Val - # Ala Pro Pro Arg Leu Leu GluGlu# 365- Glu Ala Glu Leu Cys His Gly Pro - # Ile Thr Leu Asp Phe Ala IleHis# 380- Lys Leu Lys Ala Ala Gly Ser Leu - # Pro Gly Ser Tyr Ile Leu ArgArg# 400- Ser Pro Gln Asp Tyr Asp Ser Phe - # Leu Leu Thr Ala Cys Val GlnThr# 415- Pro Leu Gly Pro Asp Tyr Lys Gly - # Cys Leu Ile Arg Gln Asp ProSer# 430- Gly Ala Phe Ser Leu Val Gly Leu - # Ser Gln Leu His Arg Ser LeuGln# 445- Glu Leu Leu Thr Ala Cys Trp His - # Ser Gly Leu Gln Val Asp GlyThr# 460- Ala Leu Asn Leu Thr Ser Cys Cys - # Val Pro Arg Pro Lys Glu LysSer# 480- Asn Leu Ile Val Val Arg Arg Gly - # Arg Asn Pro Thr Pro Ala ProGly# 495- His Ser Pro Ser Cys Cys Ala Leu - # Thr Lys Leu Ser Phe His ThrIle# 510- Pro Ala Asp Ser Leu Glu Trp His - # Glu Asn Leu Gly His Gly SerPhe# 525- Thr Lys Ile Phe His Gly His Arg - # Arg Glu Val Val Asp Gly GluThr# 540- His Asp Thr Glu Val Leu Leu Lys - # Val Met Asp Ser Arg His GlnAsn# 560- Cys Met Glu Ser Phe Leu Glu Ala - # Ala Ser Leu Met Ser Gln ValSer# 575- Tyr Pro His Leu Val Leu Leu His - # Gly Val Cys Met Ala Gly AspSer# 590- Ile Met Val Gln Glu Phe Val Tyr - # Leu Gly Ala Ile Asp Thr TyrLeu# 605- Arg Lys Arg Gly His Leu Val Pro - # Ala Ser Trp Lys Leu Gln ValThr# 620- Lys Gln Leu Ala Tyr Ala Leu Asn - # Tyr Leu Glu Asp Lys Gly LeuPro# 640- His Gly Asn Val Ser Ala Arg Lys - # Val Leu Leu Ala Arg Glu GlyVal# 655- Asp Gly Asn Pro Pro Phe Ile Lys - # Leu Ser Asp Pro Gly Val SerPro# 670- Thr Val Leu Ser Leu Glu Met Leu - # Thr Asp Arg Ile Pro Trp ValAla# 685- Pro Glu Cys Leu Gln Glu Ala Gly - # Thr Leu Asn Leu Glu Ala AspLys# 700- Trp Gly Phe Gly Ala Thr Thr Trp - # Glu Val Phe Ser Gly Ala ProMet# 720- His Ile Thr Ser Leu Glu Pro Ala - # Lys Lys Leu Lys Phe Tyr GluAsp# 735- Arg Gly Gln Leu Pro Ala Leu Lys - # Trp Thr Glu Leu Glu Gly LeuIle# 750- Ala Gln Cys Met Ala Tyr Asp Pro - # Gly Arg Arg Pro Ser Phe ArgAla# 765- Ile Leu Arg Asp Leu Asn Gly Leu - # Ile Thr Ser Asp Tyr Glu LeuLeu# 780- Ser Asp Pro Thr Pro Gly Ile Pro - # Asn Pro Arg Asp Glu Leu CysGly# 800- Gly Ala Gln Leu Tyr Ala Cys Gln - # Asp Pro Ala Ile Phe Glu GluArg# 815- His Leu Lys Tyr Ile Ser Leu Leu - # Gly Lys Gly Asn Phe Gly SerVal# 830- Glu Leu Cys Arg Tyr Asp Pro Leu - # Gly Asp Asn Thr Gly Pro LeuVal# 845- Ala Val Lys Gln Leu Gln His Ser - # Gly Pro Glu Gln Gln Arg AspPhe# 860- Gln Arg Glu Ile Gln Ile Leu Lys - # Ala Leu His Cys Asp Phe IleVal# 880- Lys Tyr Arg Gly Val Ser Tyr Gly - # Pro Gly Arg Gln Glu Leu ArgLeu# 895- Val Met Glu Tyr Leu Pro Ser Gly - # Cys Leu Arg Asp Phe Leu GlnArg# 910- His Arg Ala Arg Leu His Asn Asp - # Arg Leu Leu Leu Phe Ala TrpGln# 925- Ile Cys Lys Gly Met Glu Tyr Leu - # Gly Ala Arg Arg Cys Val HisArg# 940- Asp Leu Ala Ala Arg Asn Ile Leu - # Val Glu Ser Glu Ala His ValLys# 960- Ile Ala Asp Phe Gly Leu Ala Lys - # Leu Leu Pro Leu Gly Lys AspTyr# 975- Tyr Val Val Arg Val Pro Gly Gln - # Ser Pro Ile Phe Trp Tyr AlaPro# 990- Glu Ser Leu Ser Asp Asn Ile Phe - # Ser Arg Gln Ser Asp Val TrpSer# 10050- Phe Gly Val Val Leu Tyr Glu Leu - # Phe Thr Tyr Ser Asp Lys SerCys# 10205- Ser Pro Ser Thr Glu Phe Leu Arg - # Met Ile Gly Pro Glu Arg GluGly# 10405- Ser Pro Leu Cys His Leu Leu Glu - # Leu Leu Ala Glu Gly Arg ArgLeu# 10550- Pro Pro Pro Ser Thr Cys Pro Thr - # Glu Val Gln Glu Leu Met GlnLeu# 10700 - # 1065- Cys Trp Ser Pro Asn Pro Gln Asp - # Arg Pro Ala Phe Asp Thr LeuSer# 10850- Pro Gln Leu Asp Ala Leu Trp Arg - # Gly Ser Pro Gly# 11005- (2) INFORMATION FOR SEQ ID NO:12:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 846 amino (B) TYPE: amino acid (C) STRANDEDNESS: Not R - #elevant (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:- Met Ala Pro Pro Ser Glu Glu Thr - # Pro Leu Ile Gln Arg Ser CysSer# 15- Leu Ser Glu Ala Gly Ala Leu His - # Val Leu Leu Pro Arg Gly ProGly# 30- Pro Pro Gln Arg Leu Ser Phe Ser - # Phe Gly Asp Leu Ala Glu AspLeu# 45- Cys Val Ala Ala Lys Ala Ile Leu - # Pro Val Tyr His Ser Leu PheAla# 60- Leu Ala Thr Glu Asp Leu Ser Cys - # Trp Phe Pro Gln Val Leu TyrArg# 80- Arg Phe Tyr Phe Pro Trp Phe Gly - # Leu Glu Cys His Arg Phe GlyLeu# 95- Lys Asp Leu Ser Ala Ile Leu Asp - # Val Leu Glu His Leu Phe AlaGln# 110- His Arg Ser Asp Leu Val Ser Gly - # Arg Leu Pro Gly Leu Ser LeuLys# 125- Gln Gly Glu Leu Ser Leu Ala Val - # Leu Asp Leu Ala Met Ala ArgGln# 140- Ala Gln Arg Gly Glu Leu Leu Lys - # Val Ser Tyr Lys Ala Cys LeuPro# 160- Pro Ser Leu Arg Asp Leu Ile Gln - # Gly Ser Phe Val Thr Arg ArgIle# 175- Arg Arg Thr Val Leu Leu Met Ala - # Lys Tyr Ile Asp Leu Glu ArgLeu# 190- Pro Ala Glu Phe Val Gly Leu Pro - # Gly Ala Gly Arg Val Gly AspGly# 205- Ile Ala Trp Gln Glu Gln Phe Cys - # Asp Phe Pro Glu Ile Val AspSer# 220- Ile Lys Gln Ala Pro Arg Val Gly - # Pro Ala Gly Glu His Arg LeuVal# 240- Thr Thr Arg Asp Ile Leu Glu Ala - # Glu Phe Pro Gly Leu Pro GluAla# 255- Leu Ser Phe Val Ala Leu Val Asp - # Gly Tyr Phe Arg Leu Asp SerHis# 270- Phe Phe Cys Lys Glu Val Pro Arg - # Leu Leu Glu Glu Ala Glu CysHis# 285- Gly Pro Ile Thr Leu Asp Phe Ala - # Ile Lys Leu Lys Gly Ser ProGly# 300- Ser Tyr Leu Arg Arg Pro Gln Asp - # Asp Ser Phe Leu Leu Thr CysVal# 320- Gln Pro Leu Gly Pro Asp Tyr Lys - # Gly Cys Leu Ile Arg Pro GlyPhe# 335- Leu Val Gly Leu Ser His Ser Leu - # Glu Leu Leu Cys Trp Gly LeuVal# 350- Asp Gly Ala Leu Thr Ser Cys Cys - # Pro Arg Pro Lys Glu Lys SerAsn# 365- Leu Ile Val Val Arg Gly Pro Thr - # Ser Leu Phe His Ile Pro AlaAsp# 380- Ser Leu Glu Trp His Glu Asn Leu - # Gly His Gly Ser Phe Thr LysIle# 400- Gly Arg Glu Val Val Asp Gly Glu - # Thr Glu Val Leu Leu Lys ValMet# 415- Asp His Asn Cys Met Glu Ser Phe - # Leu Glu Ala Ala Ser Leu MetSer# 430- Gln Val Ser Tyr His Leu Val Leu - # Leu His Gly Val Cys Met AlaGly# 445- Asp Ser Met Val Glu Phe Val Leu - # Gly Ala Ile Asp Tyr Leu ArgLys# 460- Arg Gly His Leu Val Pro Ala Ser - # Trp Lys Leu Gln Val Lys GlnLeu# 480- Ala Tyr Ala Leu Asn Tyr Leu Glu - # Asp Lys Gly Leu His Gly AsnVal# 495- Ser Ala Arg Lys Val Leu Leu Ala - # Arg Glu Gly Asp Gly Pro ProPhe# 510- Ile Lys Leu Ser Asp Pro Gly Val - # Ser Pro Val Leu Ser Leu GluMet# 525- Leu Thr Asp Arg Ile Pro Trp Val - # Ala Pro Glu Cys Leu Glu AlaThr# 540- Leu Leu Glu Ala Asp Lys Trp Gly - # Phe Gly Ala Thr Trp Glu ValPhe# 560- Ser Gly Met Ile Leu Pro Ala Lys - # Lys Leu Phe Tyr Glu Asp ArgGln# 575- Leu Ala Lys Trp Thr Glu Leu Leu - # Ile Gln Cys Met Ala Tyr ProArg# 590- Pro Ser Arg Ala Arg Asp Leu Asn - # Leu Ile Ser Asp Tyr Glu LeuLeu# 605- Ser Asp Thr Pro Arg Asp Leu Gly - # Ala Gln Leu Tyr Ala Cys GlnAsp# 620- Pro Ile Phe Glu Glu Arg His Leu - # Lys Tyr Ile Ser Leu Gly LysGly# 640- Phe Gly Ser Val Glu Leu Cys Arg - # Tyr Asp Pro Leu Gly Asp AsnThr# 655- Gly Leu Val Ala Val Lys Gln Leu - # Gln His Ser Gly Pro Gln GlnArg# 670- Asp Phe Gln Arg Glu Ile Gln Ile - # Leu Lys Ala His Asp Phe IleVal# 685- Lys Tyr Arg Gly Val Ser Tyr Gly - # Pro Gly Arg Gln Leu Val MetGlu# 700- Tyr Leu Pro Ser Gly Cys Leu Arg - # Asp Phe Leu Gln Arg His ArgAla# 720- Leu Arg Leu Leu Leu Gln Ile Cys - # Lys Gly Met Glu Tyr Leu GlyArg# 735- Arg Cys Val His Arg Asp Leu Ala - # Ala Arg Asn Ile Leu Val GluSer# 750- Glu Ala His Val Lys Ile Ala Asp - # Phe Gly Leu Ala Lys Leu LeuPro# 765- Leu Lys Asp Tyr Tyr Val Val Arg - # Pro Gly Gln Ser Pro Ile PheTrp# 780- Tyr Ala Pro Glu Ser Leu Ser Asp - # Asn Ile Phe Ser Arg Gln SerAsp# 800- Val Trp Ser Phe Gly Val Val Leu - # Tyr Glu Leu Phe Thr Tyr AspLys# 815- Ser Cys Ser Pro Ser Glu Phe Leu - # Arg Met Gly Glu Arg Leu CysLeu# 830- Leu Glu Leu Leu Glu Gly Arg Leu - # Pro Pro Cys Pro Glu Val# 845__________________________________________________________________________

Protein tyrosine kinase, JAK3

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