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The invention relates to a gene, mammalian Additional Sex Combs (niammalian Asx), implicated in proliferative disorders, including malignancies, and in developmental processes.

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Cancer and malignancy therapies have included treatment with chemical toxins, radiation, and surgery. Genes known to be over-expressed or underexpressed in cancer are used for diagnosis of the disease and evaluation of a patient's progression with the disease and treatment.

The study of transcription has provided information about cell differentiation: early in the development of a cell lineage, transcription factors direct development along a particular pathway by activating genes of a differentiated phenotype.

Differentiation can involve not only changes in patterns of expressed genes, but also involve the maintenance of those new patterns.

The genetic basis of niainmalian development, and the genetic link between development and cancer has not been fully elucidated. There is a need in the art for knowledge of the key genes underlying niammalian cancer, particularly those also implicated in normal mammalian developmental processes.

.Summary nf thι> Invention

In one embodiment of the invention an isolated mammalian Asx (mammalian Asx) polypeptide is provided. The polypeptide comprises a sequence of at least 18 consecutive amino acids of the sequence of SEQ ID NO: 2.

In another embodiment of the invention an isolated nucleic acid molecule is provided. The nucleic acid molecule encodes a polypeptide having the sequence of SEQ ID NO: 2.

According to yet another embodiment, an isolated nucleic acid molecule is provided which comprises at least 13 contiguous nucleotides selected from the sequence of SEQ ID NO: 1.

In another embodiment of the invention, an antibody preparation is provided. The antibodies specifically bind to an mammalian Asx polypeptide, and do not bind specifically to other mammalian proteins. According to yet another aspect of the invention a pharmaceutical composition is provided. The composition comprises an effective amount of a therapeutic agent comprising a mammalian Asx polypeptide which comprises the sequence of SEQ ID NO: 2 and a pharmaceutically acceptable carrier.

Another aspect of the invention is a method of diagnosing neoplasia. The method comprises: contacting (a) a tissue sample suspected of neoplasia isolated from a patient with (b) an mammalian Asx gene probe comprising at least 13 nucleotides of the sequence of SEQ ID NO: 1, wherein a tissue which overexpresses mammalian Asx or expresses a variant mammalian Asx is categorized as neoplastic. According to another embodiment of the invention a method of diagnosing neoplasia is provided. The method comprises: contacting PCR primers which specifically hybridize with an mammalian Asx gene sequence of SEQ ID NO: 1, with nucleic acids isolated from a tissue suspected of neoplasia; amplifying mammalian Asx sequences in the nucleic acids of the tissue; and detecting a mutation in the amplified sequence, wherein a mutation is identified when the amplified sequence differs from a sequence similarly amplified from a normal human tissue.

In yet another embodiment of the invention a method of diagnosing neoplasia is provided. The method comprises: contacting a bDNA probe with nucleic acids isolated from a tissue suspected of

neoplasia, wherein the bDNA probe specifically hybridizes with a mammalian Asx gene sequence of SEQ ID NO: 1; detecting hybrids formed between the bDNA probe and nucleic acids isolated from the tissue; and identifying a mutation in the nucleic acids isolated from the tissue by comparing the hybrids formed with hybrids similarly formed using nucleic acids from a normal human tissue.

According to still another aspect of the invention a method of diagnosing neoplasia is provided. The method comprises: contacting a tissue sample suspected of being neoplastic with an antibody selected from the group consisting of: one which specifically binds to wild-type mammalian Asx as shown in SEQ ID NO:2, or one which specifically binds to an expressed mammalian Asx variant; detecting binding of the antibody to components of the tissue sample, wherein a difference in the binding of the antibody to components of the tissue sample, as compared to binding of the antibody to a normal human tissue sample indicates neoplasia of the tissue.

Another aspect of the invention is yet another method of diagnosing neoplasia. The method comprises: contacting RNA from a tissue suspected of being neoplastic with PCR primers which specifically hybridize to an mammalian Asx gene sequence as shown in SEQ ID NO: 1 or a bDNA probe which specifically hybridizes to said sequence; deteπnining quantitative levels of mammalian Asx RNA in the tissue by PCR amplification or bDNA probe detection, wherein higher levels of mammalian Asx RNA as compared to a normal human tissue indicate neoplasia.

According to another embodiment a method of diagnosis of neoplasia is provided. The method comprises: contacting a tissue sample suspected of neoplasia isolated from a patient with an niammalian Asx gene probe comprising at least 13 contiguous nucleotides of the sequence of SEQ ID NO: 1, wherein a tissue which overexpresses mammalian Asx or expresses a variant mammalian Asx is categorized as neoplastic.

In yet another aspect of the invention a method of inducing cell de- differentiation is provided. The method comprises: contacting a differentiated cell with a mammalian Asx polypeptide which comprises the sequence of SEQ ID NO: 2, whereby de-differentiation of the cell is induced.

In still another aspect of the invention a method of dysregulating cell growth is provided. The method comprises: contacting a cell whose growth is controlled with a mammalian Asx polypeptide which comprises the sequence of SEQ ID NO: 2, whereby growth of the cell is dysregulated.

According to still another aspect of the invention a method of diagnosing neoplasia is provided. The method comprises: contacting RNA from a tissue suspected of being neoplastic with PCR primers which specifically hybridize to an mammalian Asx gene sequence as shown in SEQ ID NO: 1, or a bDNA probe which specifically hybridizes to said sequence; deteπnining quantitative levels of mammalian Asx RNA in the tissue by PCR amplification or bDNA probe detection, wherein higher levels of mammalian Asx RNA as compared to a normal human tissue indicate neoplasia.

In yet another aspect of the invention a method of identifying modulators of mammalian Asx function is described. The method comprises: contacting a test substance with a mammalian cell which comprises an mammalian Asx gene or a reporter construct comprising an mammalian Asx promoter and a reporter gene; quantitating transcription of mammalian Asx or the reporter gene transcription in the presence and absence of the test substance, wherein a test substance which decreases transcription is a candidate drug for anti-neoplastic therapy.

Also provided are nucleic acid molecules which can be used in regulating a heterologous coding sequence coordinately with mammalian Asx. These sequences include the 5' untranslated region of an mammalian Asx gene, the 3' untranslated region of an mammalian Asx gene, the promoter region of an mammalian Asx gene, and an intron of an mammalian Asx gene.

Also provided by the present invention is a method of identifying modulators of mammalian Asx function comprising: contacting a mammalian cell which comprises an mammalian Asx gene or a reporter construct comprising an mammalian Asx promoter and a reporter gene with a test substance; quantitating transcription of mammalian Asx or the reporter gene in the presence and absence of the test substance, wherein a test substance which decreases transcription is a candidate drug for anti-neoplastic therapy. Detailed Ttesmptinn The inventors have discovered a gene, the mammalian Additional Sex Combs

(mammalian Asx), that operates to regulate protein expression in mammals, particularly humans. Mammalian Asx may operate by controlling homeotic gene expression. Although the invention is not limited by any theory or mechanism of how the invention works, it is believed that control by this gene involves multiprotein complexes capable of negative regulation of transcription.

The polypeptides of the invention according to SEQ ID NO: 2 and 4 contain various domains of the mammalian Asx gene. The nucleic acid molecules according to SEQ ID NO: 1 and 3 encode the mammalian Asx polypeptides and have been cloned from mammalian cells. The polynucleotide of SEQ ID NO: 1 encodes the polypeptide of SEQ ID NO: 2; the polynucleotide of SEQ ID NO: 3 encodes the polypeptide of SEQ ID NO: 4. Polypeptides comprising at least 6, 10, 18, 20, 30, 40, 50, 54, 60, 65, or 75 amino acids of mammalian Asx are useful as immunogens for raising antibodies and as competitors in immunoassays. They can also be used to purify antibodies. Nucleic acid molecules of at least 12, 13, 15, 20, 30, 40, or 50 contiguous nucleotides are useful as probes for use in diagnostic assays.

Both human and murine Asx, and their coding sequences, are provided herein. There is sequence conservation between murine and human Asx. They are 84 % similar and 75 % identical at the amino acid level. Other mammalian Asx proteins and genes can be obtained by screening of cDNA libraries of a mammalian species with a probe derived from the murine or human sequences. Similar levels of identity and similarity are expected with other mammals. Such techniques are well known in

the art, and can be employed by those of skill in the art.

The domains of mammalian Asx protein which appear to be most conserved are those found in the following locations in the human protein. The conserved domains are at aa 250-356 and aa 1501-1536. In addition, there is a lysine rich putative nuclear translocation sequence from amino acid 2 to 11.

The human Asx gene has been mapped to chromosome 20ql 1. This was accomplished by FISH mapping. Intriguingly, there are many chromosomal aberrations associated with a variety of cancers which also map to this chromosomal segment. Mammalian Asx is implicated in development, by contributing to the activation or repression of certain genes during development. Thus mammalian Asx can be used therapeutically to change the gene expression pattern and thus the phenotype of a cell. Thus, for example, mamrnalian Asx can be used to direct de-differentiation of a differentiated cell. Similarly, inhibition of mammalian Asx will direct a differentiated cell to become less differentiated, i.e. , to alter its pattern of gene expression.

Proliferative indications for which an mammalian Asx-based therapeutic agent can be used include, restinosis, benign prostatic hyperplasia, uterine fibroids, retinopathy, psoriasis, keloids, arthritis, wound healing, and premalignant lesions including for example, intestinal polyps, cervical dysplasia, and myeloid dysplasia. Neoplasias that may be treatable with an mammalian Asx-based therapeutic agent, include, but are not limited to, carcinoma, colorectal adenocarcinoma, leukemia, Burkitt's lymphoma and melanoma.

The coding region of mammalian Asx can be used for expression of mammalian Asx and for development of mammalian Asx variants for therapeutic applications. Mammalian Asx coding sequence can be used as a probe for diagnosis of disease or biological disorder where overexpression of mammalian Asx occurs, such as, for example, in cancers such as carcinoma, colorectal adenocarcinoma, lympha ~ cancer, promyelocytic leukemia, Burkitt's lymphoma, melanoma, and myeloma. l ..i 5' untranslated and 3' untranslated regions of mammalian Asx can also be used diagnostically to the same effect as the mammalian Asx coding sequence, for example, the 5' untranslated region can be isolated and used to probe tissue, for example, colon

tissue, where colon carcinoma is suspected. Because mammalian Asx has been shown to be upregulated in colon carcinoma, probing with any portion of the mammalian Asx gene can identify the upregulation of mammalian Asx in the tissue, as an aid to making a diagnosis. Such diagnostic probes may also be used for continued monitoring of a diagnosed patient, for signs of improvement after and during treatment, and for indications of progression of the disease.

Mammalian Asx genes can be cloned and isolated by probing genomic DNA with the coding region of mammalian Asx, or by probing genomic DNA with any probe-length piece (at least 12 nucleotides) of mammalian Asx DNA. A PI clone of genomic DNA containing ΛAsx (ATCC #98426, CMCC #4738) has been deposited at the American Type Culture Collection, Rockville, MD. The genomic DNA can be subcloned into a cloning vector, for example a cosmid vector, for sequencing and assembly of the entire gene sequence. The promoter region of mammalian Asx is useful for expression of mammalian Asx in a gene therapy protocol, and for further analysis of mammalian Asx gene function and regulatory control. Knowledge of promoter region sequences specific for binding transcriptional activators that activate the mammalian Asx promoter can facilitate improved expression of mammalian Asx for therapeutic purposes. The mammalian Asx promoter region may be useful for tissue specific expression of heterologous genes, such as, for treatment of colon carcinoma. The region immediately 5' of the coding region of mammalian Asx can be used, for example, as a diagnostic probe for cancer or a developmental disorder associated with aberrant mammalian Asx activity. The full length gene, or such non- coding regions of it as the promoter and the 5' or 3' untranslated regions can be isolated by probing genomic DNA with a probe comprising at least about 12 nucleotides of mammalian Asx cDNA, and retrieving a genomic sequence that hybridizes to one of these sequences. The 5' untranslated end and the promoter regions, for example, can be cloned by PCR cloning with random oligonucleotide and a 5' portion of the known coding sequence.

The polypeptides of the invention can further be used to generate monoclonal or polyclonal antibodies. Monoclonal antibodies, are prepared using the method of Kohler and Milstein, as described in Nature (1975) 256: 495-96, or a modification

thereof. Antibodies to mammalian Asx or fragments thereof, or fusion proteins thereof, either polyclonal or monoclonal, can be used therapeutically. They are desirably compatible with the host to be treated. For example, for treatment of humans, the antibodies can be human monoclonal antibodies or humanized antibodies, as the term is generally known in the art. Alternatively, single chain antibodies may be used for therapy. Antibodies may act to antagonize or inhibit the polypeptide activity of mammalian Asx, and are also useful in diagnosing a condition characterized by mammalian Asx expression or over-expression, such as, for example, a malignancy condition. Useful antibodies bind specifically to mammalian Asx but not to other human proteins. More preferred is the situation where the antibodies are human species niammalian Asx-specific.

Expression of mammalian Asx can be accomplished by any expression system appropriate for the purpose and conditions presented. Some exemplary expression systems are listed below. Where mammalian Asx itself is used as a therapeutic, the polypeptide can be expressed and subsequently administered to a patient.

Alternatively a gene encoding at least a functional portion of mammalian Asx can be administered to a patient for expression in the patient.

Recombinant mammalian Asx may be used as a reagent for diagnostic methods for diagnosis of cancer or a developmental disorder. It may also be used as a therapeutic for inducing de-differentiation in a population of differentiated cells. Recombinant mammalian Asx can also be used to develop modulators of mammalian Asx for achieving a desired therapeutic effect. Construction and expression of any of the recombinant molecules of the invention can be accomplished by any expression system most appropriate for the task, including, for example, an expression system described below.

Fτpτe5iRinn Systems

Although the methodology described below is believed to contain sufficient details to enable one skilled in the art to practice the present invention, other constructs can be constructed and purified using standard recombinant DNA techniques as described in, for example, Sambrook et al. (1989), MOLECULAR

CLONING: A LABORATORY MANUAL, 2nd ed. (Cold Spring Harbor Press, Cold Spring Harbor, New York); and under current regulations described in United States Dept. of Health and Human Services , National Institutes of Health (NLH) Guidelines for Recombinant DNA Research. The polypeptides of the invention can be expressed in any expression system, including, for example, bacterial, yeast, insect, amphibian and mammalian systems. Expression systems in bacteria include those described in Chang et al, Nature (1978) 275: 615, Goeddel et al, Nature (1979) 281: 544, Goeddel et al, Nucleic Acids Res. (1980) 8: 4057, EP 36,776, U.S. 4,551,433, deBoer et al., Proc. Natl Acad. Sci. USA (1983) 80. 21-25, and Siebenlist et al. Cell (1980) 20. 269. Expression systems in yeast include those described in Hinnen et al. , Proc.

Natl. Acad. Sci. USA (1978) 75: 1929; Ito et al, J. Bacteriol. (1983) 153: 163; Kurtz et al., Mol Cell Biol (1986) 6: 142; Kunze et al, J. Basic Microbiol (1985) 25: 141; Gleeson et al., J. Gen. Microbiol (1986) 132: 3459, Roggenkamp et al., Mol Gen. Genet. (1986) 202 :302) Das et al., J. Bacteriol (1984) 158: 1165; De Louvencourt et al, J. Bacteriol (1983) 154: 737, Van den Berg et al.,

Bio/Technology (1990) 8: 135; Kunze et al., J. Basic Microbiol (1985) 25: 141; Cregg et al., Mol. Cell Biol (1985) 5: 3376, U.S. 4,837,148, US 4,929,555; Beach and Nurse, Nature (1981) 300. 706; Davidow et al., Curr. Genet. (1985) 10. 380, Gaillardin et al., Curr. Genet. (1985) 10. 49, Ballance et al., Biochem. Biophys. Res. Commun. (1983) 112: 284-289; Tilburn et al, Gene (1983) 26: 205-221, Yelton et al., Proc. Natl. Acad. Sci. USA (1984)_8i: 1470-1474, Kelly and Hynes, EMBO J. (1985) 4: 475479; EP 244,234, and WO 91/00357. Expression of heterologous genes in insects can be accomplished as described in U.S. 4,745,051, Friesen et al. (1986) "The Regulation of Baculovirus Gene Expression" in: THE MOLECULAR BIOLOGY OF BACULOVIRUSES (W. Doerfler, ed.), EP 127,839, EP 155,476, and Vlak et al. , J. Gen. Virol (1988) 69: 765-776, Miller et al, Ann. Rev. Microbiol (1988) 42: 111, CarboneU et al., Gene (1988) 73: 409, Maeda et al., Nature (1985) 315: 592-594, Lebacq-Verheyden et al., Mol. Cell. Biol. (1988) 8: 3129; Smith et al, Proc. Natl. Acad. Sci. USA (1985) 82: 8404, Miyajima et al, Gene (1987) 58: 273; and Martin et al., DNA (1988) 7:99. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts are described in Luckow et al ,

Bio/Technology (1988) 6: 47-55, Miller et al, in GENERIC ENGINEERING (Setlow, J.K. et al. eds.), Vol. 8 (Plenum Publishing, 1986), pp. 277-279, and Maeda et al , Nature, (1985) 315L 592-594. Mammalian expression can be accomplished as described in Dijkema et al, EMBO J. (1985) 4: 761, Gorman et al. , Proc. Natl Acad. Sci. USA (1982b) 79: 6777, Boshart et al, Cell (1985) 41: 521 and U.S. 4,399,216. Other features of mammalian expression can be facilitated as described in Ham and Wallace, Meth. Enz. (1979) 58: 44, Barnes and Sato, Anal Biochem. (1980) 102L 255, U.S. 4,767,704, US 4,657,866, US 4,927,762, US 4,560,655, WO 90/103430, WO 87/00195, and U.S. RE 30,985. Constructs including an mammalian Asx coding sequence or constructs including coding sequences for modulators of mammalian Asx can be administered by a gene therapy protocol, either locally or systemically. These constructs can utilize viral or non-viral vectors and can be delivered in vivo or ex vivo or in vitro. Expression of such coding sequence can be driven by endogenous mammalian or heterologous promoters. Expression of the coding sequence in vivo can be either constitutive or regulated.

Gene delivery vehicles (GDVs) are available for delivery of polynucleotides to cells, tissue, or to a the mammal for expression. For example, a polynucleotide sequence of the invention can be administered either locally or systemically in a GDV. These constructs can utilize viral or non-viral vector approaches in in vivo or ex vivo modality. Expression of such coding sequence can be induced using endogenous mammalian or heterologous promoters. Expression of the coding sequence in vivo can be either constitutive or regulated. The invention includes gene delivery vehicles capable of expressing the contemplated polynucleotides. The gene delivery vehicle is preferably a viral vector and, more preferably, a retroviral, adenoviral, adeno-associated viral (AAV), herpes viral, or alphavirus vectors. The viral vector can also be an astrovirus, coronavirus, orthomyxovirus, papovavirus, paramyxovirus, parvovirus, picornavirus, poxvirus, togavirus viral vector. See generally, Jolly, Cancer Gene Therapy 1:51-64 (1994); Kimura, Human Gene Therapy 5:845-852 (1994), Connelly, Human Gene Therapy 6:185-193 (1995), and Kaplitt, Nature Genetics 6: 148-153 (1994). Retroviral vectors are well known in the art and we contemplate that any retroviral gene therapy

vector is employable in the invention, including B, C and D type retroviruses, xenotropic retroviruses (for example, NZB-X1, NZB-X2 and NZB9-1 (see O'Neill, J. Vir. 53:160, 1985) polytropic retroviruses (for example, MCF and MCF-MLV (see Kelly, J. Vir. 45:291, 1983), spumaviruses and lentiviruses. See RNA Tumor Viruses, Second Edition, Cold Spring Harbor Laboratory, 1985.

Portions of the retroviral gene therapy vector may be derived from different retroviruses. For example, retroviral LTRs may be derived from a Murine Sarcoma Virus, a tRNA binding site from a Rous Sarcoma Virus, a packaging signal from a Murine Leukemia Virus, and an origin of second strand synthesis from an Avian Leukosis Virus. These recombinant retroviral vectors may be used to generate transduction competent retroviral vector particles by introducing them into appropriate packaging cell lines (see U.S. Serial No. 07/800,921, filed November 29, 1991). Retrovirus vectors can be constructed for site-specific integration into host cell DNA by incorporation of a chimeric integrase enzyme into the retroviral particle. See, U.S. Serial No. 08/445,466 filed May 22, 1995. It is preferable that the recombinant viral vector is a replication defective recombinant virus. Packaging cell lines suitable for use with the above-described retrovirus vectors are well known in the art, are readily prepared (see U.S. Serial No. 08/240,030, filed May 9, 1994; see also WO 92/05266), and can be used to create producer cell lines (also termed vector cell lines or "VCLs") for the production of recombinant vector particles. Preferably, the packaging cell lines are made from human parent cells (e.g. , HT1080 cells) or mink parent cell lines, which eliminates inactivation in human serum. Preferred retroviruses for the construction of retroviral gene therapy vectors include Avian Leukosis Virus, Bovine Leukemia, Virus, Murine Leukemia Virus, Mink-Cell Focus-Inducing Virus, Murine Sarcoma Virus, Reticuioendotheliosis Virus and Rous Sarcoma Virus. Particularly preferred Murine Leukemia Viruses include 4070A and 1504A (Hartley and Rowe, J. Virol. 19:19-25, 1976), Abelson (ATCC No. VR-999), Friend (ATCC No. VR-245), Graffi, Gross (ATCC No. VR-590), Kirsten, Harvey Sarcoma Virus and Rauscher (ATCC No. VR-998) and Moloney Murine Leuicemia Virus (ATCC No. VR-190). Such retroviruses may be obtained from depositories or collections such as the American Type Culture Collection ("ATCC") in Rockville, Maryland or isolated from known sources using

commonly available techniques. Exemplary known retroviral gene therapy vectors employable in this invention include those described in GB 2200651; EP No. 415,731; EP No. 345,242; PCT Publication Nos. WO 89/02468, WO 89/05349, WO 89/09271, WO 90/02806, WO 90/07936, WO 90/07936, WO 94/03622, WO 93/25698, WO 93/25234, WO 93/11230, WO 93/10218, and WO 91/02805, in U.S. Patent Nos. 5,219,740, 4,405,712, 4,861,719, 4,980,289 and 4,777,127, in U.S. Serial No. 07/800,921 and in Vile, Cancer Res. 53:3860-3864 (1993); Vile, Cancer Res 53:962-967 (1993); Ram, Cancer Res 53:83-88 (1993); Takamiya, J. Neurosci. Res. 33:493-503 (1992); Baba, J Neurosurg 79:729-735 (1993); Mann, Cell 33:153 (1983); Cane, Proc Natl Acad Sci 81:6349 (1984) and Miller, Human Gene Therapy 1 (1990). Human adenoviral gene therapy vectors are also known in the art and employable in this invention. See, for example, Berkner, Biotechniques 6:616 (1988), and Rosenfeld, Science 252:431 (1991), and PCT Patent Publication Nos. WO 93/07283, WO 93/06223, and WO 93/07282. Exemplary known adenoviral gene therapy vectors employable in this invention include those described in the above-referenced documents and in PCT Patent Publication Nos. WO 94/12649, WO 93/03769, WO 93/19191, WO 94/28938, WO 95/11984, WO 95/00655, WO 95/27071, WO 95/29993, WO 95/34671, WO 96/05320, WO 94/08026, WO 94/11506, WO 93/06223, WO 94/24299, WO 95/14102, WO 95/24297, WO 95/02697, WO 94/28152, WO 94/24299, WO 95/09241, WO 95/25807, WO 95/05835, WO 94/18922 and WO 95/09654. Alternatively, administration of DNA linked to killed adenovirus as described in Curiel, Hum. Gene Ther. 3:147-154 (1992) may be employed. The gene delivery vehicles of the invention also include adenovirus associated virus (AAV) vectors. Leading and preferred examples of such vectors for use in this invention are the AAV-2 basal vectors disclosed in Srivastava, PCT Patent Publication No. WO 93/09239. Most preferred AAV vectors comprise the two AAV inverted terminal repeats in which the native D-sequences are modified by substitution of nucleotides, such that at least 5 native nucleotides and up to 18 native nucleotides, preferably at least 10 native nucleotides up to 18 native nucleotides, most preferably 10 native nucleotides are retained and the remaining nucleotides of the D-sequence are deleted or replaced with non-native nucleotides. The native D-sequences of the AAV inverted terminal repeats are sequences of 20 consecutive

nucleotides in each AAV inverted terminal repeat (i.e., there is one sequence at each end) which are not involved in HP formation. The non-native replacement nucleotide may be any nucleotide other than the nucleotide found in the native D-sequence in the same position. Other employable exemplary AAV vectors are pWP-19, pWN-1 , both of which are disclosed in Nahreini, Gene 124:257-262 (1993). Another example of such an AAV vector is psub201. See Samulski, J. Virol. 61:3096 (1987). Another exemplary AAV vector is the Double-D LTR vector. How to make the Double D ΪTR vector is disclosed in U.S. Patent No. 5,478,745. Still other vectors are those disclosed in Carter, U.S. Patent No. 4,797,368 and Muzyczka, U.S. Patent No. 5,139,941, Chartejee, U.S. Patent No. 5,474,935, and Kotin, PCT Patent PubUcation No. WO 94/288157. Yet a further example of an AAV vector employable in this invention is SSV9AFABTKneo, which contains the AFP enhance and albumin promoter and directs expression predominantly in the liver. Its structure and how to make it are disclosed in Su,Human Gene Therapy 7:463-470 (1996). Additional AAV gene therapy vectors are described in U.S. Patent Nos. 5,354,678; 5,173,414; 5,139,941; and 5,252,479. The gene therapy vectors of the invention also include herpes vectors. Leading and preferred examples are herpes simplex virus vectors containing a sequence encoding a thymidine kinase polypeptide such as those disclosed in U.S. Patent No. 5,288,641 and EP No. 176,170 (Roizman). Additional exemplary herpes simplex virus vectors include HFEM/ICP6-LacZ disclosed in PCT Patent No. WO 95/04139 (Wistar Institute), pHSVlac described in Geller, Science 241:1667-1669 (1988) and in PCT Patent PubUcation Nos. WO 90/09441 and WO 92/07945, HSV Us3::pgC-lacZ described in Fink, Human Gene Therapy 3:11-19 (1992) and HSV 7134, 2 RH 105 and GAL4 described in EP No. 453,242 (Breakefield), and those deposited with the ATCC as accession numbers ATCC VR-977 and ATCC VR-260. Alpha virus gene therapy vectors may be employed in this invention. Preferred alpha virus vectors are Sindbis viruses vectors. Togaviruses, Semtiki Forest virus (ATCC VR-67; ATCC VR-1247), Middleberg virus (ATCC VR-370), Ross River virus (ATCC VR-373; ATCC VR-1246), Venezudan equine encephaUtis virus (ATCC VR923; ATCC VR-1250; ATCC VR-1249; ATCC VR-532), and those described U.S. Patent Nos. 5,091,309 and 5,217,879, and PCT Patent PubUcation No. WO 92/10578. More particularly, those alpha virus vectors described in U.S. Serial No. 08/405,627,

filed March 15, 1995, and U.S. Serial No. 08/198,450 and in PCT Patent PubUcation Nos. WO 94/21792, WO 92/10578, and WO 95/07994, and U.S. Patent Nos. 5,091,309 and 5,217,879 are employable. Such alpha viruses may be obtained from depositories or coUections such as the ATCC in Rockville, Maryland or isolated from known sources using commonly available techniques. Preferably, alphavirus vectors with reduced cytotoxidty are used (see co-owned U.S. Serial No. 08/679640). DNA vector systems such as eukaryotic layered expression systems are also useful for expressing the nucleic acids of the invention. See PCT Patent PubUcation No. WO 95/07994 for a detaUed description of eukaryotic layered expression systems. Preferably, the eukaryotic layered expression systems of the invention are derived from alphavirus vectors and most preferably from Sindbis viral vectors. Other viral vectors suitable for use in the present invention include those derived from potiovirus, for example ATCC VR-58 and those described in Evans, Nature 339:385 (1989), and Sabin, J. Biol. Standardization 1:115 (1973); rhinovirus, for example ATCC VR-1110 and those described in Arnold, J CeU Biochem (1990) L401; pox viruses such as canary pox virus or vaccinia virus, for example ATCC VR-111 and ATCC VR-2010 and those described in Fisher-Hoch, Proc Natl Acad Sci 86 (1989) 317, Flexner, Ann NY Acad Sci 569:86 (1989), Flexner, Vaccine 8:17 (1990); in U.S. Patent Nos. 4,603,112 and 4,769,330 and in WO 89/01973; SV40 virus, for example ATCC VR-305 and those described in MulUgan, Nature 277:108 (1979) and Madzak, J Gen Vir 73:1533 (1992); influenza virus, for example ATCC VR-797 and recombinant influenza viruses made employing reverse genetics techniques as described in U.S. Patent No. .5,166,057 and in Enami, Proc. Natl. Acad. Sd. 87:3802-3805 (1990); Enami and Palese, J. Virol. 65:2711-2713 (1991); and Luyties, CeU 59:110 (1989), (see also McMicheal., New England J. Med. 309:13 (1983), and Yap, Nature 273:238 (1978) and Nature 277:108, 1979); human immunodeficiency virus as described in EP No. 386,882 and in Buchschacher, J. Vir. 66:2731 (1992); measles virus, for example, ATCC VR-67 and VR-1247 and those described in EP No. 440,219; Aura virus, for example, ATCC VR-368; Bebaru virus, for example, ATCC VR-600 and ATCC VR-1240; Cabassou virus, for example, ATCC VR-922; Chikungunya virus, for example, ATCC VR-64 and ATCC VR-1241; Fort Morgan Virus, for example, ATCC VR-924; Getah virus, for example, ATCC VR-369

and ATCC VR-1243; Kyzylagach virus, for example, ATCC VR-927; Mayaro virus, for example, ATCC VR-66; Mucambo virus, for example, ATCC VR-580 and ATCC VR-1244; Ndumu virus, for example, ATCC VR-371; Pixuna virus, for example, ATCC VR-372 and ATCC VR-1245; Tonate virus, for example, ATCC VR-925; Triniti virus, for example ATCC VR-469; Una virus, for example, ATCC VR-374; Whataroa virus, for example ATCC VR-926; Y-62-33 virus, for example, ATCC VR-375; O'Nyong virus, Eastern encephaUtis virus, for example, ATCC VR-65 and ATCC VR-1242; Western encephaUtis virus, for example, ATCC VR-70, ATCC VR-1251, ATCC VR-622 and ATCC VR-1252; and coronavirus, for example, ATCC VR-740 and those described in Hamre, Proc. Soc. Exp. Biol. Med. 121:190 (1966). Delivery of the compositions of this invention into ceUs is not timited to the above mentioned viral vectors. Other deUvery methods and media may be employed such as, for example, nucldc add expression vectors, polycationic condensed DNA linked or unlinked to killed adenovirus alone, for example see U.S. Serial No. 08/366,787, filed December 30, 1994, and Curid, Hum Gene Ther 3: 147-154 (1992) Ugand linked DNA, for example, see Wu, J. Biol. Chem. 264:16985-16987 (1989), eukaryotic ceU deUvery vehicles ceUs, for example see U.S. Serial No. 08/240,030, filed May 9, 1994, and U.S. Serial No. 08/404,796, deposition of photopolymerized hydrogel materials, hand-held gene transfer particle gun, as described in U.S. Patent No. 5,149,655, ionizing radiation as described in U.S. Patent No. 5,206,152 and in PCT Patent Publication No. WO 92/11033, nucldc charge neutralization or fusion with ceU membranes. Additional approaches are described in PhiUp, Mol. CeU. Biol. 14:2411-2418 (1994) and in Woffendin, Proc. Natl. Acad. Sci. 91:1581-585 (1994). Particle mediated gene transfer may be employed, for example see U.S. provisional application No. 60/023,867. Briefly, the sequence can be inserted into conventional vectors that contain conventional control sequences for high level expression, and then be incubated with synthetic gene transfer molecules such as polymeric DNA-binding cations like polylysine, protamine, and albumin, linked to ceU targeting tigands such as asialoorosomucoid, as described in Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987), insulin as described in Hucked, Biochem. Pharmacol. 40:253-263 (1990), galactose as described in Plank, Bioconjugate Chem 3:533-539 (1992), lactose or transferrin. Naked

DNA may also be employed. Exemplary naked DNA introduction methods are described in PCT Patent PubUcation No. WO 90/11092 and U.S. Patent No. 5,580,859. Uptake efficiency may be improved using biodegradable latex beads. DNA coated latex beads are efficiently transported into ceUs after endocytosis initiation by the beads. The method may be improved further by treatment of the beads to increase hydrophobidty and thereby faciUtate disruption of the endosome and release of the DNA into the cytoplasm. Liposomes that can act as gene deUvery vehicles are described in U.S. Patent No. 5,422,120, PCT Patent PubUcation Nos. WO 95/13796, WO 94/23697, and WO 91/144445, and EP No. 524,968. As described in co-owned U.S. provisional application No. 60/023,867, on non-viral deUvery, the nucleic acid sequences can be inserted into conventional vectors that contain conventional control sequences for high level expression, and then be incubated with synthetic gene transfer molecules such as polymeric DNA-binding cations like polylysine, protamine, and albumin, linked to ceU targeting Ugands such as asialoorosomucoid, insulin, galactose, lactose, or transferrin. Other deUvery systems include the use of Uposomes to encapsulate DNA comprising the gene under the control of a variety of tissue-specific or ubiquitously-active promoters. Further non-viral deUvery suitable for use includes mechanical deUvery systems such as the approach described in Woffendin et al., Proc. Natl. Acad. Sci. USA 91(24):11581-U585 (1994). Moreover, the coding sequence and the product of expression of such can be deUvered through deposition of photopolymerized hydrogel materials. Other conventional methods for gene deUvery that can be used for deUvery of the coding sequence include, for example, use of hand-hdd gene transfer particle gun, as described in U.S. Patent No. 5,149,655; use of ionizing radiation for activating transferred gene, as described in U.S. Patent No. 5,206,152 and PCT Patent PubUcation No. WO 92/11033. Exemplary Uposome and polycationic gene deUvery vehicles are those described in U.S. Patent Nos. 5,422,120 and 4,762,915, in PCT Patent PubUcation Nos. WO 95/13796, WO 94/23697, and WO 91/14445, in EP No. 524,968 and in Stryer, Biochemistry, pages 236-240 (1975) W.H. Freeman, San Frandsco, Szoka, Biochem. Biophys. Acta. 600:1 (1980); Bayer, Biochem. Biophys. Acta. 550:464 (1979); Rivnay, Meth. Enzymol. 149:119 (1987); Wang, Proc. Natl. Acad. Sci. 84:7851 (1987); and Plant, Anal. Biochem. 176:420 (1989).

Test compounds can be tested as candidate modulators by testing the abitity to increase or decrease the expression of mammalian Asx. The candidate modulators can be derived from any of the various possible sources of candidates, such as for example, libraries of peptides, peptoids, smaU molecules, polypeptides, antibodies, polynucleotides, smaU molecules, antisense molecules, ribozymes, cRNA, cDNA, polypeptides presented by phage display. Described below are some exemplary and possible sources of candidates, including synthesized Ubraries of peptides, peptoids, and smaU molecules. The exemplary expression systems can be used to generate cRNA or cDNA Ubraries that can also be screened for the ability to modulate mammaUan Asx activity or expression. Candidate molecules screened for the abitity to agonize mammaUan Asx expression or activity may be useful for inducing differentiation in a population of progenitor ceUs. SmaU molecules can be screened for the abiUty to either affect mammaUan Asx expression or affect mammaUan Asx function by enhancing or interfering in mammaUan Asx's abiUty to interact with other molecules that mammaUan Asx normaUy interacts with in mammaUan Asx's normal function.

MammaUan Asx peptide modulators are screened using any available method. The assay conditions ideaUy should resemble the conditions under which the mammaUan Asx modulation is exhibited in vivo, that is, under physiologic pH, temperature, ionic strength, etc. Suitable antagonists will exhibit strong inhibition of mammaUan Asx expression or activity at concentrations that do not cause toxic side effects in the subject. A further alternative agent that can be used herein as a modulator of mammaUan Asx is a smaU molecule antagonist. SmaU molecules can be designed and screened from a pool of synthetic candidates for abiUty to modulate mammaUan Asx. There exist a wide variety of smaU molecules, including peptide analogs and derivatives, that can act as inhibitors of proteins and polypeptides. Libraries of these molecules can be screened for those compounds that inhibit the activity or expression of mammaUan Asx. Similarly, ribozymes can be screened in assays appropriate for ribozymes, taking into account the special biological or biochemical nature of ribozymes. Assays for affecting mammaUan Asx expression can measure mammaUan Asx message or protein directly, or can measure a reporter gene expression which is under the control of an mammaUan Asx promoter and/or 5'

untranslated region (UTR).

MammaUan Asx or a modulator of mammaUan Asx can be administered to a patient exhibiting a condition characterized by abnormal ceU proUferation, in which aberrant mammaUan Asx gene expression is impUcated, particularly excessive mammaUan Asx activity, or excessive activity controlled or induced by mammalian Asx activity. The modulator can be incorporated into a pharmaceutical composition that includes a pharmaceutically acceptable carrier for the modulator. Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycoUc adds, polymeric amino adds, amino add copolymers, and inactive virus particles. Such carriers are weU known to those of ordinary skLU in the art. PharmaceuticaUy acceptable salts can be used therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the Like. A thorough discussion of pharmaceuticaUy acceptable excipients is available in REMINGTON'S PHARMACEUΗCAL SCIENCES (Mack Pub. Co., NJ. 1991). PharmaceuticaUy acceptable carriers in therapeutic compositions may contain tiquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. TypicaUy, the therapeutic compositions are prepared as injectables, either as Uquid solutions or suspensions; sotid forms suitable for solution in, or suspension in, Uquid vehicles prior to injection may also be prepared.

Liposomes are included within the definition of a pharmaceuticaUy acceptable carrier. The term "Uposomes" refers to, for example, the Uposome compositions described in U.S. Patent NO: 5,422,120, WO 95/13796, WO 94/23697, WO 91/14445 and EP 524,968 Bl. Liposomes may be pharmaceutical carriers for the peptides, polypeptides or polynucleotides of the invention, or for combination of these therapeutics.

Any therapeutic of the invention, including, for example, polynucleotides for expression in the patient, or ribozymes or antisense oUgonucleotide, can be formulated into an enteric coated tablet or gel capsule according to known methods in the art.

These are described in the foUowing patents: US 4,853,230, EP 225,189, AU 9,224,296, AU 9,230,801, and WO 92144,52. Such a capsule is administered oraUy to be targeted to the jejunum. At 1 to 4 days foUowing oral administration expression of the polypeptide, or inhibition of expression by, for example a ribozyme or an antisense oUgonucleotide, is measured in the plasma and blood, for example by antibodies to the expressed or non-expressed proteins.

Adniinistration of a therapeutic agent of the invention, including for example an mammaUan Asx modulator, includes administering a therapeutically effective dose of the therapeutic agent by a means considered or empiricaUy deduced to be effective for inducing the desired effect in the patient. Both the dose and the administration means can be determined based on the specific quaUties of the therapeutic, the condition of the patient, the progression of the disease, and other relevant factors. Administration of the therapeutic agents of the invention can include, local or systemic administration, including injection, oral administration, particle gun or catheterized administration, and topical administration. The therapeutics of the invention can be administered in a therapeuticaUy effective dosage and amount, in the process of a therapeuticaUy effective protocol for treatment of the patient. The initial and any subsequent dosages administered wiU depend upon the patient's age, weight, condition, and the disease, disorder or biological condition being treated. Depending on the therapeutic, the dosage and protocol for administration will vary, and the dosage wiU also depend on the method of administration selected, for example, local or systemic adniinistration.

For polypeptide therapeutics, for example, a dominant negative mammaUan Asx polypeptide or a polypeptide modulator of mammaUan Asx, the dosage can be in the range of about 5 μg to about 50 μg/kg of patient body weight, also about 50 μg to about 5 mg/kg, also about 100 μg to about 500 μg/kg of patient body weight, and about 200 to about 250 μg/kg.

For polynucleotide therapeutics, depending on the expression of the polynucleotide in the patient, for tissue targeted adniinistration, vectors containing expressible constructs including mammaUan Asx coding sequences or modulator coding sequences, or non-coding sequences can be administered in a range of about

100 ng to about 200 mg of DNA for local administration in a gene therapy protocol, also about 500 ng to about 50 mg, also about 1 ug to about 2 mg of DNA, about 5 ug of DNA to about 500 ug of DNA, and about 20 ug to about 100 ug during a local administration in a gene therapy protocol, and for example, a dosage of about 500 ug, per injection or administration.

Non-coding sequences that act by a catalytic mechanism, for example, catalyticaUy active ribozymes may require lower doses than non-coding sequences that are hdd to the restrictions of stoichiometry, as in the case of, for example, antisense molecules, although expression Umitations of the ribozymes may again raise the dosage requirements of ribozymes being expressed in vivo in order that they achieve efficacy in the patient. Factors such as method of action and efficacy of transformation and expression are therefore considerations that wϋl effect the dosage required for ultimate efficacy for DNA and nucldc adds. Where greater expression is desired, over a larger area of tissue, larger amounts of DNA or the same amounts readministered in a successive protocol of administrations, or several administrations to different adjacent or close tissue portions of for example, a tumor site, may be required to effect a positive therapeutic outcome.

For administration of smaU molecule modulators of mammaUan Asx polypeptide activity, depending on the potency of the small molecule, the dosage may vary. For a very potent inhibitor, microgram (μg) amounts per kilogram of patient may be sufficient, for example, in the range of about 1 μg/kg to about 500 mg/kg of patient weight, and about 100 μg/kg to about 5 mg/kg, and about 1 μg/kg to about 50 μg/kg, and, for example, about 10 ug/kg. For administration of peptides and peptoids the potency also affects the dosage, and may be in the range of about 1 μg/kg to about 500 mg/kg of patient weight, and about 100 μg/kg to about 5 mg/kg, and about 1 μg/kg to about 50 μg/kg, and a usual dose might be about 10 ug/kg.

In aU cases, routine experimentation in clinical trials wiU determine specific ranges for optimal therapeutic effect, for each therapeutic, each administrative protocol, and administration to specific patients wiU also be adjusted to within effective and safe ranges depending on the patient condition and responsiveness to initial administrations.

Diagnostic assays for Asx sequence can be appUed to cancers demonstrating upregulationof Asx transcript, particularly to cancers of lymphoma, melanoma, and adenocarcinoma. Such diagnostics can be accompUshed using any portion of the Asx gene, including the 3' and 5' untranslated regions of the gene, to probe patient tissues ^• to determine an upregulation of an Asx transcript.

Administration of a therapeutic agent for a condition in which increased expression of mammaUan Asx is impUcated, for example, in the case of promyelocytic leukemia, chronic myelogenous leukemia, lymphoblastic leukemia, Burkitt's lymphoma, colorectal adenocarcinoma, melanoma, and lymphoma, can be preceded by diagnosis of the condition using an mammaUan Asx probe, generated from any portion of the mammaUan Asx gene, and probing the suspect tissue. bDNA technology using bDNA probes to mammaUan Asx gene sequences or mammaUan Asx mRNA sequences may be used, as described in WO 92/02526 or U.S. 5,451,503, and U.S. 4,775,619. Once diagnosis is complete, treatment can include administration of mammaUan Asx polynucleotides or anti-sense oUgonucleotide by a gene therapy protocol, or by adniinistration by other means including local or systemic administration, of an mammaUan Asx modulator, for example an mammaUan Asx- spedfic ribozyme, or a geneticaUy altered mammaUan Asx variant, for example a dominant negative mammaUan Asx, or a smaU molecule or peptide or peptoid mammaUan Asx modulator, or any combination of these potential therapeutics. The patient can be subsequently monitored by periodic reprobing of the affected tissue with an mammaUan Asx probe.

Even in cancers where mammaUan Asx mutations are not impUcated, mammaUan Asx downregulation or inhibition of mammaUan Asx function may have therapeutic appUcation. In these cancers, decreasing mammaUan Asx expression or inhibiting mammaUan Asx function may help to suppress the tumors. Similarly, even in tumors where mammaUan Asx expression is not aberrant, effecting mammaUan Asx downregulation or diminution of mammaUan Asx activity may suppress metastases. Further objects, features, and advantages of the present invention wiU become apparent from the detatied description. It should be understood, however, that the

detatied description, while indicating preferred embodiments of the invention, is given by way of iUustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. Definitions

A "nucleic acid molecule" or a "polynucleotide," as used herein, refers to either RNA or DNA molecule that encodes a specific amino acid sequence or its complementary strand. Nucleic acid molecules may also be non-coding sequences, for example, a ribozyme, an antisense oUgonucleotide, or an untranslated portion of a gene. A "coding sequence" as used herein, refers to either RNA or DNA that encodes a specific amino add sequence or its complementary strand. A polynucleotide may include, for example, an antisense oUgonucleotide, or a ribozyme, and may also include such items as a 3' or 5' untranslated region of a gene, or an intron of a gene, or other region of a gene that does not make up the coding region of the gene. The DNA or RNA may be single stranded or double stranded. Synthetic nucleic adds or synthetic polynucleotides can be chemicaUy synthesized nucleic acid sequences, and may also be modified with chemical moieties to render the molecule resistant to degradation. Synthetic nucleic acids can be ribozymes or antisense molecules, for example. Modifications to synthetic nucleic acid molecules include nucleic add monomers or derivative or modifications thereof, including chemical moieties. For example, phosphothioates can be used for the modification. A polynucleotide derivative can include, for example, such polynucleotides as branched DNA (bDNA). A polynucleotide can be a synthetic or recombinant polynucleotide, and can be generated, for example, by polymerase chain reaction (PCR) amptification, or recombinant expression of complementary DNA or RNA, or by chemical synthesis. MammaUan Asx polynucleotides share at least 70 % and preferably at least 75, 80, 85, 90, or 95 % identity with either mouse or human ΛAsx sequences. These can be obtained, inter alia, by hybridization of mouse or human Asx probes under conditions of stringent hybridization. Encompassed within the definition of mammaUan, human, and mouse Asx are sequences which contain aUetic variants, as weU as sequences which differ due to the degeneracy of the genetic code.

The term "functional portion of as used herein refers to a portion of an mammaUan Asx wild-type molecule which retains at least 50% of activity of mammaUan Asx. It also encompasses a portion of an mammaUan Asx gene having single base substitutions, deletions, or insertions that have no adverse effect on the activity of the molecule. Truncations of mammaUan Asx, fragments of Asx, and combinations of fragments of Asx, which retain at least 50% activity are contemplated. Such portions of hAsx may also be fused to other proteins, such as in a gene fusion.

The term "functional" as used herein refers to a gene functional in cancer or differentiation. A molecule is functional if its expression causes, directly or indirectly, an event specificaUy associated with differentiation, mitosis, oncogenesis, metastasis, or the like.

The term "modulate" as used herein refers to the abiUty of a molecule to alter the function or expression of another molecule. Thus, modulate could mean, for example, inhibit, antagonize, agonize, upregulate, downregulate, induce, or suppress. A modulator has the capability of altering function of its target. Such alteration can be accompUshed at any stage of the transcription, translation, expression or function of the protein, so that, for example, modulation of mammaUan Asx can be accompUshed by modulation of the DNA, RNA, and protein products of the gene. It assumed that modulation of the function of the target, for example, mammaUan Asx, will in turn modulate, alter, or affect the function or pathways leading to a function of genes and proteins that would otherwise associate, and interact, or respond to, mammaUan Asx.

A "maUgnancy" includes any proUferative disorder in which the ceUs protiferating are ultimately harmful to the host. Cancer is an example of a proUferative disorder that manifests a maUgnancy. Neoplasia is the state of ceUs which experience uncontroUed ceU growth, whether or not maUgnant.

The term "regulatory sequence" as used herein refers to a nucleic acid sequence encoding one or more elements that are capable of affecting or effecting expression of a gene sequence, including transcription or translation thereof, when the gene sequence is placed in such a position as to subject it to the control thereof. Such

a regulatory sequence can be, for example, a minimal promoter sequence, a complete promoter sequence, an enhancer sequence, an upstream activation sequence ("UAS"), an operator sequence, a downstream termination sequence, a polyadenylation sequence, an optimal 5' leader sequence to optimize initiation of translation, and a Shine-Dalgarno sequence. Alternatively, the regulatory sequence can contain a combination enhancer/promoter element. The regulatory sequence that is appropriate for expression of the present construct differs depending upon the host system in which the construct is to be expressed. Selection of the appropriate regulatory sequences for use herein is within the capabiUty of one skilled in the art. For example, in prokaryotes, such a regulatory sequence can include one or more of a promoter sequence, a ribosomal binding site, and a transcription termination sequence. In eukaryotes, for example, such a sequence can include one or more of a promoter sequence and/or a transcription termination sequence. If any necessary component of a regulatory sequence that is needed for expression is lacking in the polynucleotide construct, such a component can be suppUed by a vector into which the polynucleotide construct can be inserted for expression. Regulatory sequences suitable for use herein may be derived from any source including a prokaryotic source, an eukaryotic source, a virus, a viral vector, a bacteriophage or from a linear or circular plasmid. An example of a regulatory sequence is the human immunodeficiency virus ("HIV") promoter that is located in the U3 and R region of the HIV long teπrdnal repeat ("LTR"). Alternatively, the regulatory sequence herein can be a synthetic sequence, for example, one made by combining the UAS of one gene with the remainder of a requisite promoter from another gene, such as the GADP/ADH2 hybrid promoter. The terms "protein", "polypeptide", "polypeptide derivatives" and modifications and variants thereof refer herein to the expression product of a polynucleotide construct of the invention as defined above. The terms further include truncations, variants, aUeles, analogs and derivatives thereof. Unless specificaUy mentioned otherwise, such mammaUan Asx polypeptides possess one or more of the bioactivities of the mammaUan Asx protein, such as those discovered herein. This term is not Umited to a specific length of the product of the mammaUan Asx gene. Thus,

polypeptides that are identical or contain at least 70 % , and more preferably 75 % , and most preferably 80, 85, 90, or 95 % identity with the mammaUan Asx protein or the mature mammaUan Asx protein, wherever derived, from human or nonhuman sources are included within this definition of the mammaUan Asx polypeptide. Also included, therefore, are aUeles and variants of the product of the mammaUan Asx gene that contain amino acid substitutions, deletions, or insertions. The amino acid substitutions can be conservative amino acid substitutions or substitutions to eUminate non-essential amino acid residues such as to alter a glycosylation site, a phosphorylation site, an acetylation site, or to alter the folding pattern by altering the position of the cysteine residue that is not necessary for function, etc. Conservative amino acid substitutions are those that preserve the general charge, hydrophobicity/hydrophiUdty and/or steric bulk of the amino acid substituted, for example, substitutions between the members of the foUowing groups are conservative substitutions: Gly/ Ala, Val/Ile/Leu, Asp/Glu, Lys/ Arg, Asn/Gln, Ser/Thr/Cys and Phe/Trp/Tyr. Analogs include peptides having one or more peptide mimics, also known as peptoids, that possess mammaUan Asx protein-like activity. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), polypeptides with substituted linkages, as weU as other modifications known in the art, both naturaUy occurring and nonnaturaUy occurring. The term "mammaUan Asx" also may include post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations, myrstylations, farnesylations, palmitoylations and the like.

The term "polypeptide fragment" as used herein refers to a polypeptide sequence that does not encode the full length of a protein but that is identical to a region of the protein. The fragment is designed to retain the functional aspect of the region of the polypeptide from which it is derived. Two fragments can cooperate to provide function. Two distinct polypeptide fragments of the same gene may represent expressed sptice variants of that gene, although functionaUty and expression of the polypeptide sptice variant products may occur in similar biological conditions, and may be related, at least in part, in function.

The term "derivative" as used herein in reference to a polypeptide or a polynucleotide means a polypeptide or polynucleotide that retains at least 50% of the functionaUty of the polypeptide or polynucleotide to which it is a derivative. They may be variously modified by nucleotide or amino acid deletions, substitutions, insertions or inversions by, for example, site directed mutagenesis of the underlying nucleic acid molecules. Derivatives of a polypeptide or polynucleotide may also be fragments or combinations of fragments thereof. In any case, a derivative, or a fragment, retains at least some, and preferably aU of the function of the polypeptide from which it is derived. An "isolated polypeptide" or "isolated polynucleotide" as used herein refers to a polypeptide or polynucleotide, respectively, produced in vivo or in vitro in an environment manipulated by humans using state of the art techniques of molecular biology, biochemistry and gene therapy. For example, an isolated polypeptide can be produced in a cell free system by automated peptide or polypeptide synthesis, in heterologous host ceUs transformed with the nucleic add sequence encoding the polypeptide and regulatory sequences for expression in the host ceUs, and in an animal into which the coding sequence of the polypeptide has been introduced for expression in the animal. A polypeptide or polynucleotide is "isolated" for purposes herein to the extent that it is not present in its natural state inside a ceU as a product of nature. For example, such isolated polypeptides or polynucleotides can be 10% pure, 20% pure, or a higher degree of purity, such as 50% , 75 % , 85 % , or 90% .

The term "condition" as used herein in terms of "a patient having a condition" refers to a particular state of molecular and ceUular systems in a biological context. A biological context includes any organism considered to have life, and for the purposes of this invention includes but is not timited the foUowing organisms or groups: animals, mammals, humans, and vertebrates. A biological condition can include, for example, a disease or a medical condition that may or may not be characterized by identifiable symptoms or indicators. A "condition characterized by abnormal ceU proUferation" is most likely a cancer condition, but may also be a condition arising in the development of an organism.

The term "modulator" as used herein describes any moiety capable of changing

the endogenous activity or a polypeptide. Modulatory activities can include, for example, modulation at the level of transcription, translation, expression, secretion, or modulation of polypeptide activity inside or outside a cell. Modulation can include, for example, inhibition, antagonism, and agonism, and modulation can include, for example, modulation of upstream or downstream effects that effect the ultimate activities in a pathway, or modulation of the configuration of a polypeptide such that its activity is altered. Modulation can be transitory or permanent, and may be a dose dependent effect.

The term "inhibitor" for use herein can be any inhibitor of a polypeptide activity. The category includes but is not Umited to any of the herein described antagonists of mammaUan Asx. The inhibitor of mammaUan Asx can be an antibody- based mammaUan Asx antagonist, or a polypeptide fragment thereof, a peptide mammaUan Asx antagonist, a peptoid mammaUan Asx antagonist, or a smaU molecule mammaUan Asx antagonist. The polypeptide inhibitor can be one screened from a cDNA, cRNA, or phage display tibrary of polypeptides. The inhibitor can be a polynucleotide, such as, for example a ribozyme or an antisense oUgonucleotide, or can be derivatives of these. It is expected that some inhibitors wiU act at transcription, some at translation, and some on the mature protein. However, the use and appropriateness of such inhibitors of mammaUan Asx for the purposes of the invention are not Umited to any theories of mechanism of action of the inhibitor. It is sufficient for purposes of the invention that an inhibitor inhibit the activity of mammaUan Asx.

The term "antagonist" as used herein refers to a molecule that inhibits or blocks the activity of a polypeptide, either by blocking the polypeptide itself, or by causing a reduced expression of the polypeptide by either blocking transcription of the gene encoding the polypeptide, or by interfering with or destroying a transcription or translation product of the gene. An antagonist may be, for example, a smaU molecule, peptide, peptoid, polypeptide, or polynucleotide. The polynucleotide may be, for example, a ribozyme, an antisense oUgonucleotide, or a coding sequence. The term "agonist" as used herein refers to a molecule that mimics the activity of the target polypeptide. For example, in the case of mammaUan Asx, an agonist

could mimic the transcriptional negative regulation capability of mammaUan Asx. An agonist may be, for example a smaU molecule, peptide, peptoid, polypeptide, or polynucleotide.

The term "pharmaceutical composition" refers to a composition for administration of a therapeutic agent, such as antibodies or a polypeptide, or inhibitors or genes and other therapeutic agents Usted herein, in vivo, and refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity. The term "an effective amount" as used herein refers to an amount that is effective to induce a desired effect. Where the effect is a therapeutic effect, the effective amount is that amount that wiU accompUsh a therapeutic goal, for example, tumor regression, tumor marker reduction, or a positive indication from other indicia of cancer that indicates a reduction or growth slowing of cancer ceUs. Where the therapeutic agent is, for example, an antagonist of mammaUan Asx, the effective amount of the antagonist would be an amount that antagonizes mammaUan Asx activity among a population of ceUs. The amount that is effective depends in part upon the indicia selected for determining effectiveness, and depends upon the effect sought. An administration of a therapeutic agent of the invention includes adrninistration of a therapeuticaUy effective amount of the agent of the invention. The term "therapeuticaUy effective amount" as used herein refers to an amount of a therapeutic agent to treat or prevent a condition treatable by administration of a composition of the invention. That amount is the amount sufficient to exhibit a detectable therapeutic or preventative or ameUorative effect. The effect may include, for example, treatment or prevention of the conditions Usted herein. The predse effective amount for a subject wtil depend upon the subject's size and health, the nature and extent of the condition being treated, recommendations of the treating physician, and the therapeutics or combination of therapeutics selected for adrninistration. Thus, it is not useful to specify an exact effective amount in advance. However, the effective amount for a given situation can be determined by routine

experimentation. Administration can include administration of a polypeptide, and causing the polypeptide to be expressed in an animal by administration of the polynucleotide encoding the polypeptide.

A "recombinant vector" herein refers to any vector for transfer or expression of the polynucleotides herein in a ceU, including, for example, viral vectors, non-viral vectors, plasmid vectors and vectors derived from the regulatory sequences of heterologous hosts and expression systems.

The term "in vivo administration" refers to administration to a mammal of a polynucleotide encoding a polypeptide for expression in the mammal. In particular, direct in vivo adrninistration involves transfecting a mammal's ceU with a coding sequence without removing the ceU from the mammal. Thus, direct in vivo adrninistration may include direct injection of the DNA encoding the polypeptide of interest in the region afflicted by the maUgnancy or proUferative disorder, resulting in expression in the mammal's ceUs. The term "ex vivo administration" refers to transfecting a ceU, for example, a cell from a population of ceUs that are maUgnant or proUferating, after the cell is removed from the mammal. After transfection the ceU is then replaced in the mammal. Ex vivo administration can be accompUshed by removing ceUs from a mammal, optionaUy selecting for cells to transform, (i.e. ceUs that are maUgnant or proUferating) rendering the selected ceUs incapable of repUcation, transforming the selected ceUs with a polynucleotide encoding a gene for expression, (i.e. mammaUan Asx), including also a regulatory region for facilitating the expression, and placing the transformed cells back into the mammal for expression of the mammaUan Asx.

"BiologicaUy active" refers to a molecule that retains a specific activity. A biologicaUy active mammaUan Asx polypeptide, for example, retains the activity including for example the control of a homeotic gene or group of homeotic genes.

"MammaUan ceU" as used herein refers to a subset of eukaryotic ceUs useful in the invention as host ceUs, and includes human ceUs, and animal cells such as those from dogs, cats, cattle, horses, rabbits, mice, goats, pigs, etc. The cells used can be geneticaUy unaltered or can be geneticaUy altered, for example, by transformation with appropriate expression vectors, marker genes, and the like. MammaUan cells

suitable for the method of the invention are any mammaUan ceU capable of expressing the genes of interest, or any mammaUan ceUs that can express a cDNA tibrary, cRNA tibrary, genomic DNA library or any protein or polypeptide useful in the method of the invention. MammaUan ceUs also include ceUs from ceU Unes such as those immortalized ceU Unes available from the American Type Culture CoUection (ATCC). Such ceU Unes include, for example, rat pheochromocytoma ceUs (PC 12 ceUs), embryonal carcinoma ceUs (P19 ceUs), Chinese hamster ovary (CHO) ceUs, HeLa ceUs, baby hamster kidney (BHK) ceUs, monkey kidney ceUs (COS), human hepatoceUular carcinoma ceUs (e.g., Hep G2), human embryonic kidney ceUs, mouse sertoli ceUs, canine kidney ceUs, buffalo rat Uver ceUs, human ceUs, human Uver ceUs, mouse mammary tumor ceUs, as weU as others. Also included are hematopoetic stem ceUs, neuronal stem ceUs such as neuronal sphere ceUs, and embryonic stem cells (ES cells).

The present invention wtil now be iUustrated by reference to the foUowing examples which set forth particularly advantageous embodiments. However, it should be noted that these embodiments are iUustrative and are not to be construed as restricting the invention in any way.

Example 1 A smaU molecule modulator of mammaUan Asx is identified and incorporated into a pharmaceutical composition including a Uposomal-based pharmaceuticaUy acceptable carrier for adniinistration to a cancer patient for controlling the expression or activity of mammaUan Asx in the patient. Adrninistration the composition is achieved by injection into the tumor tissue. The patient is monitored for reduction of mammaUan Asx activity as a diagnostic marker evaluating the effectiveness of the treatment.

Example 2 A population of differentiated ceUs are treated with a functional portion of recombinant mammaUan Asx polypeptide and induced to de-differentiate to a less diffeεntiated state. The process is reversed by administering to the population of ceUs an inhibitor of mammaUan Asx activity. The process can be monitored by differential display of mRNA transcripts of the ceUs. Example 3

Northern blots of mRNA isolated from various tissues were probed with a 4.5 kb HindDI fragment of mammaUan Asx cDNA for an analysis of the expression differential of mammaUan Asx in normal and cancerous tissues, using standard techniques for accompUshing the hybridizations. Poly A ⁺ RNA was isolated from normal ceUs and cancer ceU Unes. The mRNA was dectrophoreticaUy fractionated and transferred to a nylon filter. The mRNA on the filter was immobilized by UV crosslinking. A labeled probe was prepared from the sequence of SEQ ID NO: 1, labeled with ³² P radionucleotide, and used in a hybridization reaction with the RNA on the filter under stringent conditions. The filter was aUowed to hybridize to the probe, and the unbound probe was washed from the filter. The hybridization was conducted using standard techniques for Northern hybridizations, for example, as described in Sambrook et al (1989), MOLECULAR CLONING: A LABORATORY MANUAL, 2nd ed. (Cold Spring Harbor Press, Cold Spring Harbor, New York). Exposure of the filter to X-ray film showed pronounced bands in the cancer cell Unes, and very tittle activity in the normal cell lines. Beta actin was used as a

S97/07806

control to normalize expression levels in the ceU Unes. The normal tissues probed were human adult heart, skeletal muscle, pancreas, prostate, testes, ovary, colon, thymus, brain, placenta, lung, Uver, kidney, peripheral leukocytes, and spleen. The tissue specific expression of hAsx in normal human adult tissue indicated moderate hAsx transcript in human testes, ovary and thymus. Nondetectable or very low quantities of transcript was present in human prostate, colon, brain, placenta, lung, Uver, and kidney, leukocytes, and spleen.

Two transcripts, one at about 7.5 kUobases, and one at about 5.5 kUobases were observed in the testes, and only the larger transcript was observed in the ovary and thymus tissues.

By contrast, hAsx transcripts were present at a very high level in the foUowing human cancer ceU Unes: promydocytic leukemia HL-60, HeLa ceU S3, chronic myelogenous leukemia K-562, lymphoblastic leukemia MOLT-4, Burkitt's lymphoma Raji, colorectal adenocarcinoma SW480, and melanoma G361. In addition, hAsx transcript was also abundantly expressed in other colorectal adenocarcinoma tissue, and lymphocytic cancer tissues. Expression was very low in the lung cancer tine A549. The hAsx transcripts were about 8.5 kilobases and about 6.5 kUobases in aU these ceU Unes for aU hybridizations. Hybridizations were conducted using stringent conditions and a standard hybridization protocol for accompUshing Northern blot hybridizations.

Transcript levels were controUed for by probing with actin probe on the same blots probed with hAsx coding sequence.

F.τamp1e 5 A human PI genomic clone was obtained from a PI filter tibrary using an hAsx cDNA as a probe. After DNA preparation , the identity of the clone was confirmed by sequencing a PCR product generated from the Asx PI clone. Sequence matching hAsx exon sequence was interrupted by nonexonic sequence. Consensus sptice donor and acceptor sites were present at the intron-exon boundaries. The hAsx genomic clone was labeled and used as a probe against human metaphase chromosomes. FISH mapping showed that hAsx maps to 20qll.

The description of the invention draws on previously pubUshed work and, at times, on pending patent applications. By way of example, such work consists of sdentific papers, abstracts, or issued patents, and pubUshed patent appUcations. AU pubUshed work cited herein are hereby incorporated by reference. The foUowing sequences are described below:

SEQ ID NO: 1 is the human cDNA sequence for Asx SEQ ID NO: 2 is the translated human amino acid sequences for Asx SEQ ID NO: 3 is the mouse cDNA for Asx SEQ ID NO: 4 is the translated mouse amino acid sequence for Asx

SEQUENCE LISTING

( 1 ) GENERAL INFORMATION:

( i ) APPLICANT : Randazzo , Filippo

(ii) TITLE OF INVENTION: Mammalian Additional Sex Combs

(Asx) Acts as an Oncogene

(iii) NUMBER OF SEQUENCES: 4

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Chiron Corporation (B) STREET: 4560 Horton Street

(C) CITY: Emeryville

(D) STATE: California

(E) COUNTRY: U.S.A.

(F) ZIP: 94608

(V) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.30

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE: (C) CLASSIFICATION:

(Viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Guth, Joseph H.

(B) REGISTRATION NUMBER: 31,261 (C) REFERENCE/DOCKET NUMBER: 1228.003

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (510) 923-3888

(B) TELEFAX: (510) 655-3542

(2) INFORMATION FOR SEQ ID Nθ:l:

( i ) SEQUENCE CHARACTERISTICS :

(A) LENGTH : 4926 base pairs

(B) TYPE : nucleic acid

( C ) STRANDEDNESS : single (D) TOPOLOGY : linear

( ii) MOLECULE TYPE : DNA (genomic )

(xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:l:

CGCCCGGGCC CCCCTCGCGG GGGACCGTGC CCCCGCCGCC GGGGCGAAGG ATGAAGGACA 60

AACAGAAGAA GAAGAAGGAG CGCACGTGGG CCGAGGCCGC GCGCCTGGTA TTAGAAAACT 120

ACTCGGATGC TCCAATGACA CCAAAACAGA TTCTGCAGGT CATAGAGGCA GAAGGACTAA 180

AGGAAATGAG AAGTGGGACT TCCCCTCTCG CATGCCTCAA TGCTATGCTA CATTCCAATT 240

CAAGAGGAGG AGAGGGGTTG TTTTATAAAC TGCCTGGCCG AATCAGCCTT TTCACGCTCA 300

AGAAGGATGC CCTGCAGTGG TCTCGCCATC CAGCTACAGT GGAGGGAGAG GAGCCAGAGG 360

ACACGGCTGA TGTGGAGAGC TGTGGGTCTA ATGAAGCCAG CACTGTGAGT GGTGAAAACG 420

ATGTATCTCT TGATGAAACA TCTTCGAACG CATCCTGTTC TACAGAATCT CAGAGTCGAC 480

CTCTTTCCAA TCCCAGGGAC AGCTACAGAG CTTCCTCACA GGCGAACAAA CAAAAGAAAA 540

AGACTGGGGT GATGCTGCCT CGAGTTGTCC TGACTCCTCT GAAGGTAAAC GGGGCCCACG 600

TGGAATCTGC ATCAGGGTTC TCGGGCTGCC ACGCCGATGG CGAGAGCGGC AGCCCGTCCA 660

GCAGCAGCAG CGGCTCTCTG GCCCTGGGCA GCGCTGCTAT TCGTGGCCAG GCCGAGGTCA 720

CCCAGGACCC TGCCCCGCTC CTGAGAGGCT TCCGGAAGCC AGCCACAGGT CAAATGAAGC 780

GCAACAGAGG GGAAGAAATA GATTTTGAGA CACCTGGGTC CATTCTTGTC AACACCAACC 840

TCCGTGCCCT GATCAACTCT CGGACCTTCC ATGCCTTACC ATCACACTTC CAGCAGCAGC 900

TCCTCTTCCT CCTGCCTGAA GTAGACAGAC AGGTGGGGAC GGATGGCCTG TTGCGTCTCA 960

GCAGCAGTGC ACTAAATAAC GAGTTTTTTA CCCATGCGGC TCAGAGCTGG CGGGAGCGCC 1020

TGGCTGATGG TGAATTTACT CATGAGATGC AAGTCAGGAT ACGACAGGAA ATGGAGAAGG 1080

AAAAGAAGGT GGAACAATGG AAAGAAAAGT TCTTTGAAGA CTACTATGGA CAGAAGCTGG 1140

GTTTGACCAA AGAAGAGTCA TTGCAGCAGA ACGTGGGCCA GGAGGAGGCT GAAATCAAAA 1200

GTGGCTTGTG TGTCCCAGGA GAATCAGTGC GTATACAGCG TGGTCCAGCC ACCCGACAGC 1260

GAGATGGGCA TTTTAAGAAA CGCTCTCGGC CAGATCTCCG AACCAGAGCC AGAAGGAATC 1320

TGTACAAAAA ACAGGAGTCA GAACAAGCAG GGGTTGCTAA GGATGCAAAA TCTGTGGCCT 1380

CAGATGTTCC CCTCTACAAG GATGGGGAGA CTAAGACTGA CCCAGCAGGG CTGAGCAGTC 1440

CCCATCTGCC AGGCACATCC TCTGCAGCAC CCGACCTGGA GGGTCCCGAA TTCCCAGTTG 1500

AGTCTGTGGC TTCTCGGATC CAGGCTGAGC CAGACAACTT GGCACGTGCC TCTGCATCTC 1560

CAGACAGAAT TCCTAGCCTG CCTCAGGAAA CTGTGGATCA GGAACCCAAG GATCAGAAGA 1620

GGAAATCCTT TGAGCAGGCG GCCTCTGCAT CCTTTCCCGA AAAGAAGCCC CGGCTTGAAG 1680

ATCGTCAGTC CTTTCGTAAC ACAATTGAAA GTGTTCACAC CGAAAAGCCA CAGCCCACTA 1740

AAGAGGAGCC CAAAGTCCCG CCCATCCGGA TTCAACTTTC ACGTATCAAA CCACCCTGGG 1800

TGGTTAAAGG TCAGCCCACT TACCAGATAT GCCCCCGGAT CATCCCCACC ACGGAGTCCT 1860

CCTGCCGGGG TTGGACTGGC GCCAGGACCC TCGCAGACAT TAAAGCCCGT GCTCTGCAGG 1920

TCCGAGGGGC GAGAGGTCAC CACTGCCATA GAGAGGCGGC CACCACTGCC ATCGGAGGGG 1980

GGGGTGGCCC GGGTGGAGGT GGCGGCGGGG CCACCGATGA GGGAGGTGGC AGAGGCAGCA 2040

GCAGTGGTGA TGGTGGTGAG GCCTGTGGCC ACCCTGAGCC CAGGGGAGGC CCGAGCACCC 2100

CTGGAAAGTG TACGTCAGAT CTACAGCGAA CACAACTACT GCCGCCTTAT CCTCTAAATG 2160

GGGAGCATAC CCAGGCCGGA ACTGCCATGT CCAGAGCTAG GAGAGAGGAC CTGCCTTCTC 2220

TGAGAAAGGA GGAAAGCTGC CTACTACAGA GGGCTACAGT TGGACTCACA GATGGGCTAG 2280

GAGATGCCTC CCAACTCCCC GTTGCTCCCA CTGGGGACCA GCCATGCCAG GCCTTGCCCC 2340

TACTGTCCTC CCAAACCTCA GTAGCTGAGA GATTAGTGGA GCAGCCTCAG TTGCATCCGG 2400

ATGTTAGAAC TGAATGTGAG TCTGGCACCA CTTCCTGGGA AAGTGATGAT GAGGAGCAAG 2460

GACCCACCGT TCCTGCAGAC AATGGTCCCA TTCCGTCTCT AGTGGGAGAT GATACATTAG 2520

AGAAAGGAAC TGGCCAAGCT CTTGACAGTC ATCCCACTAT GAAGGATCCT GTAAATGTGA 2580

CCCCCAGTTC CACACCTGAA TCCTCACCGA CTGATTGCCT GCAGAACAGA GCATTTGATG 2640

ACGAATTAGG GCTTGGTGGC TCATGCCCTC CTATGAGGGA AAGTGATACT AGACAAGAAA 2700

ACTTGAAAAC CAAGGCTCTC GTTTCTAACA GTTCTTTGCA TTGGATACCC ATCCCATCGA 2760

ATGATGAGGT AGTGAAACAG CCCAAACCAG AATCCAGAGA ACACATACCA TCTGTTGAGC 2820

CCCAGGTTGG AGAGGAGTGG GAGAAAGCTG CTCCCACCCC TCCTGCATTG CCTGGGGATT 2880

TGACAGCTGA GGAGGGTCTA GATCCTCTTG ACAGCCTTAC TTCACTCTGG ACTGTGCCAT 2940

CTCGAGGAGG CAGTGACAGC AATGGCAGTT ACTGTCAACA GGTGGACATT GAAAAGCTGA 3000

AAATCAACGG AGACTCTGAA GCACTGAGTC CTCACGGTGA GTCCACGGAT ACAGCCTCTG 3060

ACTTTGAAGG TCACCTCACG GAGGACAGCA GTGAGGCTGA CACTAGAGAA GCTGCAGTGA 3120

CAAAGGGATC TTCGGTGGAC AAGGATGAGA AACCCAATTG GAACCAATCT GCCCCACTGT 3180

CCAAGGTGAA TGGTGACATG CGTCTGGTTA CAAGGACAGA TGGGATGGTT GCTCCTCAGA 3240

GCTGGGTGTC TCGAGTATGT GCGGTCCGCC AAAAGATCCC AGATTCCCTA CTGCTGGCCA 3300

GTACTGAGTA CCAGCCAAGA GCCGTGTGCC TGTCCATGCC TGGGTCCTCA GTGGAGGCCA 3360

CTAACCCACT TGTGATGCAG TTGCTGCAGG GTAGCTTGCC CCTAGAGAAG GTTCTTCCAC 3420

CAGCCCACGA TGACAGCATG TCAGAATCCC CACAAGTACC ACTTACAAAA GACCAGAGCC 3480

ATGGCTCGCT ACGCATGGGA TCTTTACATG GTCTTGGAAA AAACAGTGGC ATGGTTGATG 3540

GAAGCAGCCC CAGTTCTTTA AGGGCTTTGA AGGAGCCTCT TCTGCCAGAT AGCTGTGAAA 3600

CAGGCACTGG TCTTGCCAGG ATTGAGGCCA CCCAGGCTCC TGGAGCACCC CAAAAGAATT 3660

GCAAGGCAGT CCCAAGTTTT GACTCCCTCC ATCCAGTGAC AAATCCCATT ACATCCTCTA 3720

GGAAACTGGA AGAAATGGAT TCCAAAGAGC AGTTCTCTTC CTTTAGTTGT GAAGATCAGA 3780

AGGAAGTCCG TGCTATGTCA CAGGACAGCA ATTCAAATGC TGCTCCAGGA AAGAGCCCAG 3840

GAGATCTTAC TACCTCGAGA ACACCTCGTT TCTCATCTCC AAATGTGATC TCCTTTGGTC 3900

CAGAGCAGAC AGGTCGGGCC CTGGGTGATC AGAGCAATGT TACAGGCCAA GGGAAGAAGC 3960

TTTTTGGCTC TGGGAATGTG GCTGCAACCC TTCAGCGCCC CAGGCCTGCG GACCCGATGC 4020

CTCTTCCTGC TGAGATCCCT CCAGTTTTTC CCAGTGGGAA GTTGGGACCA AGCACAAACT 4080

CCATGTCTGG TGGGGTACAG ACTCCAAGGG AAGACTGGGC TCCAAAGCCA CATGCCTTTG 4140

TTGGCAGCGT CAAGAATGAG AAGACTTTTG TGGGGGGTCC TCTTAAGGCA AATGCCGAGA 4200

ACAGGAAAGC TACTGGGCAT AGTCCCCTGG AACTGGTGGG TCACTTGGAA GGGATGCCCT 4260

TTGTCATGGA CTTGCCCTTC TGGAAATTAC CCCGAGAGCC AGGGAAGGGG CTCAGTGAGC 4320

CTCTGGAGCC TTCTTCTCTC CCCTCCCAAC TCAGCATCAA GCAGGCATTT TATGGGAAGC 4380

TTTCTAAACT CCAACTGAGT TCCACCAGCT TTAATTATTC CTCTAGCTCT CCCACCTTTC 4440

CCAAAGGCCT TGCTGGAAGT GTGGTGCAGC TGAGCCACAA AGCAAACTTT GGTGCGAGCC 4500

ACAGTGCATC ACTTTCCTTG CAAATGTTCA CTGACAGCAG CACGGTGGAA AGCATCTCGC 4560

TCCAGTGTGC GTGCAGCCTG AAAGCCATGA TCATGTGCCA AGGCTGCGGT GCGTTCTGTC 4620

ACGATGACTG TATTGGACCC TCAAAGCTCT GTGTATTGTG CCTTGTGGTG AGATAATAAA 4680

TTATGGCCAT GGGAAACGTT GTATATTTAG TGTGTGTATT TTGATAATGA TTGATCTTAA 4740

ATCTGTATAC AGAATATCAT TGATACAATA CTCTTTAGGC AGGAGCACTC TTGCCTTCCC 4800

CCAAAATTTA CACTGCTAAA GCCCTCTGTC ACTTGGCGAC CCTTCTGGTC TTGCTGGAGG 4860

GGTTTCCTGG GTATAACCCA TTGGGCTGCC CAAGGCCAGC CAGCCTGAGC TCTCCTGCAA 4920

GACAGG 4926

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 903 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Val Gin His Asp Trp Asn Phe Gly Asp lie Lys Leu Ser Ser Ser Gin 1 5 10 15

Ser Ser Gly Asp Gin Gin Arg Asn Leu Ser His Glu Ala lie Asp Leu 20 25 30

Met Asp Val Val Gin Asp Ala Asp Val lie Asp Asp He Met His Asn 35 40 45

Asp Val Cys His Asp Val Leu Gly Asp Glu Asp Glu Gly Asp Gin Glu 50 55 60

Glu Asp Glu Asp Asp Glu Val Val Glu Cys Met Thr Glu Glu Gin Gin

65 70 75 80

Leu He Asp Glu Asp Ser Glu Ala Val Arg Glu He Val Asp Lys Leu 85 90 95

Gin Gin His Gin Gin Gin Gin Asn Gin Gin Gin His His Gin Gin Leu 100 105 110

His He Gin Asp Val Val Gin Leu Ala Gin His Ser Phe Met Pro Gin 115 120 125

Ala His Ser Glu Phe Gly Asn Asp He Gly Gin Glu Met Leu Cys Asp 130 135 140

Ala Val Pro Met Ser Ala Ala Glu Met Glu Val Ser Ser Thr Val He 145 150 155 160

Thr Asn Ser Ser Asn Ser Asn Asp Ser Ser Asn Asn He Ser Leu Cys 165 170 175

Ser Ser Thr Asn Ser Leu Thr He Asn Gin Met Pro His Gin Ala Ser 180 185 190

Gin Gin Pro Gin Gin Asn Ala Gin Ser Asn Ala Gin Gin Gin Arg Gin 195 200 205

He Leu Val Asp Ser Asn Gly Gin He He Gly Asn Phe Leu Leu Gin 210 215 220

Gin Gin Arg Gin Gin Gin Gin Gin Gin Leu Leu Gin Gin Phe Thr Leu 225 230 235 240

Gin Ala Ala Ala Ala Gin Gin Gin Gin Gin Gin Gin Gin Gin His Gin 245 250 255

Gin Gin Gin Gin Gin Gin Gin Gin Ala Thr Ser Ser Asn Ser Leu Gly 260 265 270

Lys Thr Leu Pro Val Ala Leu Arg Asn Gly Thr Gin Gin Phe Leu Ser 275 280 285

Pro Asn Leu He Ala Gin Gin His Gin Gin Gin Gin Gin Gin Gin Leu 290 295 300

Glu Gin His Gin Gin Gin Ala Thr Ala Gin Gin Lys His Gin Gin He 305 310 315 320

Gin Gin Phe Ala Leu Gin Gin Ala Gin Leu His Gin Arg Gin Leu Leu 325 330 335

Ala Gin Ala Ala Asn Asn Asn Leu Leu Gin Gin Gin Gin Gin Gin Gin 340 345 350

Gin Asn Val Ala Leu Pro Thr Thr Gin Ala Lys Phe He Ala Lys Pro 355 360 365

Leu Asn He He Ser Met Thr Arg Pro Ala Asn Ala Ser Pro Thr Thr 370 375 380

Ala Ala Thr Thr Ala Asn Thr Ala Ser He Pro Ser Ala Tyr Ala Asn 385 390 395 400

Val Val Ala Val Thr Gly Ala Gin Gin Gin Gin Ser Pro Pro Val Pro 405 410 415

Ala Pro Gin Gin Gin Thr Val Gin Gin Gin Gin Leu Ala Asn His Asn 420 425 430

Ser Asn Met Gin Gin Leu Pro Asn Val Leu Thr Met Lys Thr Leu Pro 435 440 445

Pro Ser Gly Val Pro Thr Thr He Ala Gin Gin Arg Leu Gin Pro Lys 450 455 460

Met Pro Thr Gly Lys Gly Arg Lys Ala Thr Ser Asn Arg Leu Pro Pro 465 470 475 480

Gly Ala Val Asn Leu Glu Arg Thr Tyr Gin He Cys Gin Ala Val He 485 490 495

Gin Asn Ser Pro Asn Arg Glu Asn Leu Lys Ala Gin Leu Arg Pro Pro 500 505 510

Ala Ala He Leu Asn Gin His Gin Pro Thr Thr Thr Thr Ala Pro Ala 515 520 525

Pro He Asn Pro Val Thr Leu Asn Val Ser Thr Val Ala Ala Thr Pro 530 535 540

Met Ser Asn He Thr Thr Ala Thr Gly Ser Met Ala Ala Ala Val Ala 545 550 555 560

Ala Ala Pro Pro Gin Asn Val Leu Lys Gin Glu Glu Leu Leu Val Ser 565 570 575

Gly Ala Val Gly Ala Gly Ala Leu Pro Ala Gly Leu Pro Pro Asn Val 580 585 590

Met Gly Val Gly Arg Pro Gly Val Tyr Lys Val He Gly Pro Arg Met 595 600 605

Ser Gly Phe Pro Arg Lys Lys Tyr Val Gin Arg Lys Pro Ser Pro Thr 610 615 620

Thr Leu He Arg His Val Phe Ser Pro Gly Pro Gly Gly Ala Thr Ala 625 630 635 640

Thr Ala Gin Gin Leu Gin Met Leu Gin Gin His His Gin Ser Thr Thr 645 650 655

Ser Pro Val Pro Val Gin Asn Pro Gin Gin Pro Ala Pro Glu Gin Leu 660 665 670

He His Gin Asn Gly Asn Gly Gin Tyr Val Leu Val His Arg Ala Asn 675 680 685

Val Gly Ala Ala Asp Asn Gin Ala Pro Arg Ala Ser Ser Ala Pro Pro 690 695 700

Met His Gin Asn Gin Phe Val Thr Val Gin Asn Pro Leu His Ser He 705 710 715 720

Asn Gly He Pro Met Gly Gly Arg Gly Arg Pro Ala Ser Val Asp Thr 725 730 735

Thr Ala Gly Ser Gly Asn Val He Ala Pro Pro He Ser Ala Thr Asp 740 745 750

Ala Leu His His His His Glu Met Gin Gin Gin Gin Gin His Gin Gin 755 760 765

Pro Gin Pro Leu Gly Asn Val Gly Ala Ala Ala Asn He Val Arg Arg 770 775 780

Asn He Ala Ala Gly Pro Asn He Ala Tyr He Asp Gly Ser Asn Thr 785 790 795 800

Asn Ser Ser Ala Val Ala Leu Met Glu Ala Gly Asn Asn Tyr He Val 805 810 815

Thr Thr Asn Ala Ser Pro Thr Ala Ala Pro Ser Pro He Asn Gin Gin

820 825 830

Pro Gin Ser Gin Pro Thr Gly Thr Gin His Gin His Pro Leu Leu Gin 835 840 845

Leu His Gin Thr Gly Glu Asn Thr Pro Pro Gly Asn Glu Ala Thr Ala 850 855 860

Thr Ala Asn Asn Cys Ala Cys Ser Leu Asn Ala Met Val He Cys Gin 865 870 875 880

Gin Cys Gly Ala Phe Cys His Asp Asp Cys He Gly Ala Ala Lys Leu 885 890 895

Cys Val Ala Cys Val He Arg

900

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5362 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

AAGCTTGATA TCGAATTCGG CACGAGACCG CCCCAGTCCG CCCCGCCCGA AGGACCCGCG 60

TGGAGCCGCC ACCGCCGCCG CGGAGGAGGA GGATGAAGGA CAAACAGAAG AGGAAGAAGG 120

AGCGCACGTG GGCCGAGGCC GCGCGCCTGG TGTTAGAAAA CTACTCAGAT GCTCCAATGA 180

CACCAAAACA GATTCTGCAG GTCATAGAGG CAGAAGGACT GAAGGAAATG AGAAGTGGGA 240

CATCCCCTCT TGCGTGCCTC AATGCCATGC TACATTCCAA CTCAAGAGGA GGAGAAGGGC 300

TGTTTTATAA ATTACCTGGC CGCATTAGTC TTTTCACACT CAAGAAAGAT GCAGTGCAGT 360

GGTCTAGAAA TGCAGCTACA GTGGATGGAG ACGAGCCAGA GGACTCCGCT GATGTGGAAA 420

GCTGTGGGTC TAATGAAGCC AGCACTGTGA GTGGTGAAAA TGATGTATCT CTGGATGAAA 480

CATCTTCAAA TGCATCCTGC TCTACAGAGT CTCAGAGCCG ACCCCTCTCC AATCCCAGGG 540

ACAGCCACAG GGCTTCCTCA CAGGCAAACA AACAGAAGAA AAGGACTGGG GTTATGCTAC 600

CTCGTGTTGT CCTGACTCCT CTGAAGGTAA ACGGGGCCCA CGTGGAACCT GCGTCAGGAT 660

TCTCAGGCCG CCACGCAGAT GGCGAGAGTG GCAGTCCATC GAGCAGCAGC AGCGGTTCTC 720

TGGCCTTGGG CAACAGTGCC ATTCGAGGCC AGGCCGAGGT CACTCGGGAC CCTGCCCCCC 780

TCTTAAGAGG CTTCCGGAAG CCAGCCACAG GGCAAATGAA GCGCAACAGA GGGGAAGAGG 840

TAGATTTTGA GACGCCTGGG TCCATTCTTG TTAACACCAA CCTCCGTGCT CTGATAAACT 900

CTCGGACCTT CCATGCCCTG CCACTACACT TCCAGCAGCA ACTCCTCCTC CTCCTGCCTG 960

AAGTGGACAG ACAGGTGGGG ACAGATGGCC TGCTGCGCCT CAGCGGCAGT GCACTCAATA 1020

ATGAGTTTTT CACCCATGCA GCTCAGAGCT GGCGAGAACG CCTTGCTGAT GGTGAATTCA 1080

CTCATGAGAT GCAAGTCAGG CTAAGACAGG AAATGGAAAA GGAGAAGAAG GTGGAACAAT 1140

GGAAGGAAAA GTTCTTTGAA GATTACTACG GACAGAAATT GGGTTTGACC AAAGAAGAAT 1200

CACTGCAGCA GAAAGAGGTC CAGGAGGAGG CCAAAGTCAA GAGTGGTTTA TGTGTCTCTG 1260

GAGAGTCTGT GCGGCCGCAG CGTGGGCCCA ACACCCGTCA ACGGGACGGA CATTTTAAGA 1320

AACGTTCTCG GCCAGATCTC CGAACCAGAT CCAGAAGGAA TATATACAAA AAACAGGAGC 1380

CAGAACAAGC AGGGGTTGCT AAAGATGCAA GTGCTGCACC AGACGTCTCA CTCTCTAAAG 1440

ATACTAAAAC CGACTTAGCA GGGGTGAACA GTACCCCTGG GCCAGATGTG TCCTCAGCAA 1500

CATCTGGACA GGAGGGTCCC AAGTGTCCCA GTGAACCTGT GGCTTCCCAG ATCCAAGCAG 1560

AAAGGGACAA CTTGGCATGT GCCTCTGCAT CTCCAGACAG AATCCCTACC TTACCTCAGG 1620

ACACTGTGGA TCAAGAGACA AAGGATCAGA AGAGAAAATC CTTTGAGCAG GAAGCCTCTG 1680

CATCCTTTCC CGAAAAGAAA CCCCGGCTTG AAGATCGTCA GTCCTTTCGT AACACAATTG 1740

AAAGTGTTCA CACCGAAAAG CCACAGCCCA CTAAAGAGGA GCCCAAAGTC CCGCCCATCC 1800

GGATTCAACT TTCACGTATC AAACCACCCT GGGTGGCTAA AGGTCGGCCC ACTTACCAGA 1860

TATGCCCCCG GATCGTCCCC ATCACGGAGT CCTCCTGCCG GGGTTGGACT GGTGCCAGGA 1920

CCCTCGCAGA CATTAAAGCC CGTGCTTTGC AGGCCCGAGG GGCGAGAGGT TACCACTGCA 1980

ATCGAGAGAC GGCCACCACT GCCATCGGAG GGGGGGGTGG CCCGGGTGGA GGTGGCAGTG 2040

GGGCCATCGA TGAGGGAGGT GGCAGAGACA GCAGCAGTGG TGATGGTAGT GAGGCCTGTG 2100

GCCACCCTGA GCCCAGGGGA GCCCCAAGCA CCTCTGGAGA GAGTGCGTCA GATCTACAGC 2160

GAACACAACT ACTGCCGCCT TGTCCTCTGA ATGGAGAGCA CACTCCAGCT GAAGCTGCCA 2220

TGCCCAGAGC CAGAAGAGAA GACTCAGCTT CTCTCAGAAA GGAAGAGAGC TGCCTGTTGA 2280

AGAGGGTCCC AGGTGTGCTT ACAAGTGGGC TGGAAGATGC CTCTCAACCC CCTATTGCTC 2340

CCACTGGAGA CCAGCCGTGT CAGGCTTTGC CCCCTCTGTC CTCCCAAACT CCAGTGGCCG 2400

AGATGTTAAC AGAGCAGCCT AAGTTGCTTC TAGATGATAG AACTGAGTGT GAATCTAGTA 2460

GAGAAGATCA AGGACCCACC ATTCCCTCAG AGAGTAGTTC TGGACGGTTT CCATTGGGAG 2520

ATCTATTAGG AGGAGGAAGT GACCAGGCCT TTGATAATAT GAAGGAGCCT GTAAGTATGA 2580

CACCTACTTT TATATCTGAA TTGTCATTAG CTAACTACCT ACAGGATAGG CCTGATGATG 2640

ATGGATTAGG GCTTGGTGCC ACAGGCCTAC TCATAAGGGA AAGTAGTAGA CAAGAAGCTT 2700

TGACTGAGGC TTTTGCATCT GGCAGTCCTA CCTCCTGGGT ACCCATTCTG TCAAATTATG 2760

AGGTAATAAA AACATCTGAT CCAGAATCCA GAGAAAACAT ACCATGTCCG GAGCCCCAGG 2820

ATGAAAAAGA GTGGGAGAGA GCTGTTCCTC TCATTGCAGC AACAGAAAGT GTGCCCCAAC 2880

CTGAGAGCTG CATTTCACAT TGGACACCTC CTCCAGCAGC TGTGGGCAGC ACTGGCAGTG 2940

ACAGTGAGCA AGTGGACCTT GAAAGACTGG AAATGAATGG CATCTCTGAA GCACCAAGTC 3000

CTCACAGTGA ATCCACAGAT ACAGCCTCTG ACTCCGAAGG CCATCTCTCT GAGGACAGCA 3060

GTGAGGTTGA TGCAAGTGAA GTCACAGTGG TAAAAGGGTC ATTAGGTGGG GATGAAAAGC 3120

AAGACTGGGA CCCATCTGCC TCACTGTCCA AGGTGAACAA TGACCTAAGT GTGCTTACAA 3180

GGACAGGAGG GGTGGCTGCT TCTCAGAGCT GGGTGTCTAG AGTATGTTCA GTCCCACACA 3240

AGATCCCAGA CTCTCTGTTG CTGTCCAGTA CTGAGTGCCA GCCGAGGTCT GTGTGCCCAC 3300

TGAGGCCTGG CTCTTCAGTG GAGGTTACCA ACCCACTTGT GATGCACCTG CTGCATGGTA 3360

ATTTGCCCTT GGAGAAGGTT CTTCCTCCAG GTCACAGAAG CAGCCGACTA GAGTCATCAC 3420

AGCTGCCACT TAGAGAACAG AGCCAGGATA GAGGCACTCT ACAAGGTACA GGGGAAAACA 3480

ATCGCCTAGC TGCCAGAATC AACCCTGGTT CTGCACAAAC ATTGAAAGAG TCTATTCTGG 3540

CCCAGAGCTA TGGAGCAAGT GCTGGTCTTG TCAGGGCAAT GGCCTCCAAG GCTCCTGCAA 3600

TGTCCCAGAA GATTGCGAAG ATGGTTACAA GTTTAGACTC ACAGCATCCA GAGACAGAAC 3660

TGACACCTTC CTCTGGCAAT CTGGAAGAAA TAGATTCCAA AGAGCATCTC TCTTCCTTCC 3720

TTTGTGAAGA GCAGAAAGAA GGCCATTCCC TGTCTCAAGG CAGTGATCCA GGTGCGGCCC 3780

CAGGCCAATG TCTAGGAGAT CACACTACCT CCAAAGTGCC ATGTTTCTCC TCCACAAATG 3840

TGAGCCTCTC CTTTGGATCT GAGCAGACAG ATGGGACCCT GAGTGATCAG AACAATGCTG 3900

GTGGTCATGA AAAGAAACTA TTTGGTCCCG GGAATACAGT TACCACCCTT CAGTGCCCCA 3960

GGTCTGAAGA GCAGACACCA CTACCTGCTG AGGTCCCTCC AGTGTTTCCC AGTAGGAAGA 4020

TAGAACCAAG CAAAAACTCT GTGTCTGGTG GTGTGCAAAC TACAAGGGAA AACAGGATGC 4080

CCAAACCACC TCCTGTCTCT GCTGACAGCA TCAAGACAGA GCAGACATTT TTGAGGGATC 4140

CTATTAAGGC AGATGCAGAG AACAGAAAAG CTGCAGGGTA CAGTTCTCTG GAACTAGTGG 4200

GTCACTTGCA AGGGATGCCT TTTGTTGTGG ATCTGCCTTT CTGGAAGTTA CCCAGAGAGC 4260

CAGGGAAAGG GTTCAGTCAA CCCCTGGAGC CTTCTTCCAT CCCTTCCCAA CTCAACATCA 4320

AGCAGGCCTT GTATGGGAAG TTGTCTAAAC TTCAGCTCAG TCCCACCAGC TTTAATTACT 4380

CCTCTAGCTC TGCTACCTTT CCCAAAGGCC TTGCTGGTGG TGTGGTGCAG CTGAGCCACA 4440

AAGCCAGCTT TGGTACAGGC CACACTGCAT CACTGTCCTT ACAAATGTTC GCTGACAGCA 4500

GTGCAGTAGA AAGCATCTCT CTCCAATGTG CATGCAGCCT GAAAGCCATG ATCATGTGCC 4560

AAGGCTGCGG AGCATTCTGC CATGATGACT GCATTGGACC TTCAAAGCTC TGTGTATTGT 4620

GCCTTGTGGT GAGATAATAA ATTATGGCCA TTGGAAACAT TGTACATTTA GTGTGTGTAT 4680

TTTAATAATG GTTGATCTTA AATCTGTATA CAAAATATCA CTGATATAAT GAACTCTCTC 4740

TCTCTAGACA AGATAAATTT TGCCTCCCCA TGAGATTTAT AGTGCTGAAG CCCTCTGTCA 4800

CTTGACACCC TTCTAGCCTT GTTGGAAGGG TTTTCAGGGA GATGGGGGCA CTATGGTTGC 4860

CCAAGACCAT AAACCCTCTT GTAGTCAGAC AGTATAGTGT AGCAGGGCAA TCTGTCTGAC 4920

ACCTAAATGG ACTTGAAATT GAAGCAGGAA GGTTGGGTTC TCCATGGATG GAACTCACCT 4980

GCCTGAACTG AGCAGGAATG TCAGTCTTCC ACTGCCCCTC CCTGCCATCT TCTGCTACTT 5040

AGCTTGGGAG TTGATGGTTG CAGAAGCCAC ACAGGGTTAA AGTAAATTCT GTCTTTGCCC 5100

ACCAGGGGAT CAAACCCCTG CTGATCTTGA TATCATATTT CTGTCATTTG CCAGTTGATG 5160

GAGCCAAGTT GACCTTTGGT TCTGGTGCTT CACCCAGTTT GGAACTTTAA TCTGTAACCC 5220

ATGGATCCAC AGATTTTCTT GGGAGCTTGA ATAGCCCTTC TTGGACAATG GGGTCTGGAA 5280

ATAGGGCTGT CTGCTTATGG AAATGCCATC TGTAGACCTT GAGAGTCAAC TGTACAGATG 5340

TTTGCAGGTG ACTCCTCGTG CC 5362

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1514 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Met Lys Asp Lys Gin Lys Arg Lys Lys Glu Arg Thr Trp Ala Glu Ala 1 5 10 15

Ala Arg Leu Val Leu Glu Asn Tyr Ser Asp Ala Pro Met Thr Pro Lys 20 25 30

Gin He Leu Gin Val He Glu Ala Glu Gly Leu Lys Glu Met Arg Ser 35 40 45

Gly Thr Ser Pro Leu Ala Cys Leu Asn Ala Met Leu His Ser Asn Ser 50 55 60

Arg Gly Gly Glu Gly Leu Phe Tyr Lys Leu Pro Gly Arg He Ser Leu 65 70 75 80

Phe Thr Leu Lys Lys Asp Ala Val Gin Trp Ser Arg Asn Ala Ala Thr 85 90 95

Val Asp Gly Asp Glu Pro Glu Asp Ser Ala Asp Val Glu Ser Cys Gly 100 105 110

Ser Asn Glu Ala Ser Thr Val Ser Gly Glu Asn Asp Val Ser Leu Asp 115 120 125

Glu Thr Ser Ser Asn Ala Ser Cys Ser Thr Glu Ser Gin Ser Arg Pro 130 135 140

Leu Ser Asn Pro Arg Asp Ser His Arg Ala Ser Ser Gin Ala Asn Lys 145 150 155 160

Gin Lys Lys Arg Thr Gly Val Met Leu Pro Arg Val Val Leu Thr Pro 165 170 175

Leu Lys Val Asn Gly Ala His Val Glu Pro Ala Ser Gly Phe Ser Gly 180 185 190

Arg His Ala Asp Gly Glu Ser Gly Ser Pro Ser Ser Ser Ser Ser Gly 195 200 205

Ser Leu Ala Leu Gly Asn Ser Ala He Arg Gly Gin Ala Glu Val Thr 210 215 220

Arg Asp Pro Ala Pro Leu Leu Arg Gly Phe Arg Lys Pro Ala Thr Gly 225 230 235 240

Gin Met Lys Arg Asn Arg Gly Glu Glu Val Asp Phe Glu Thr Pro Gly 245 250 255

Ser He Leu Val Asn Thr Asn Leu Arg Ala Leu He Asn Ser Arg Thr 260 265 270

Phe His Ala Leu Pro Leu His Phe Gin Gin Gin Leu Leu Leu Leu Leu 275 280 285

Pro Glu Val Asp Arg Gin Val Gly Thr Asp Gly Leu Leu Arg Leu Ser 290 295 300

Gly Ser Ala Leu Asn Asn Glu Phe Phe Thr His Ala Ala Gin Ser Trp 305 310 315 320

Arg Glu Arg Leu Ala Asp Gly Glu Phe Thr His Glu Met Gin Val Arg 325 330 335

Leu Arg Gin Glu Met Glu Lys Glu Lys Lys Val Glu Gin Trp Lys Glu 340 345 350

Lys Phe Phe Glu Asp Tyr Tyr Gly Gin Lys Leu Gly Leu Thr Lys Glu 355 360 365

Glu Ser Leu Gin Gin Lys Glu Val Gin Glu Glu Ala Lys Val Lys Ser 370 375 380

Gly Leu Cys Val Ser Gly Glu Ser Val Arg Pro Gin Arg Gly Pro Asn 385 390 395 400

Thr Arg Gin Arg Asp Gly His Phe Lys Lys Arg Ser Arg Pro Asp Leu 405 410 415

Arg Thr Arg Ser Arg Arg Asn He Tyr Lys Lys Gin Glu Pro Glu Gin 420 425 430

Ala Gly Val Ala Lys Asp Ala Ser Ala Ala Pro Asp Val Ser Leu Ser 435 440 445

Lys Asp Thr Lys Thr Asp Leu Ala Gly Val Asn Ser Thr Pro Gly Pro 450 455 460

Aβp Val Ser Ser Ala Thr Ser Gly Gin Glu Gly Pro Lys Cys Pro Ser 465 470 475 480

Glu Pro Val Ala Ser Gin He Gin Ala Glu Arg Asp Asn Leu Ala Cys 485 490 495

Ala Ser Ala Ser Pro Asp Arg He Pro Thr Leu Pro Gin Asp Thr Val 500 505 510

Asp Gin Glu Thr Lys Asp Gin Lys Arg Lys Ser Phe Glu Gin Glu Ala 515 520 525

Ser Ala Ser Phe Pro Glu Lys Lys Pro Arg Leu Glu Asp Arg Gin Ser 530 535 540

Phe Arg Asn Thr He Glu Ser Val His Thr Glu Lys Pro Gin Pro Thr 545 550 555 560

Lys Glu Glu Pro Lys Val Pro Pro He Arg He Gin Leu Ser Arg He 565 570 575

Lys Pro Pro Trp Val Ala Lys Gly Arg Pro Thr Tyr Gin He Cys Pro

580 585 590

Arg He Val Pro He Thr Glu Ser Ser Cys Arg Gly Trp Thr Gly Ala 595 600 605

Arg Thr Leu Ala Asp He Lys Ala Arg Ala Leu Gin Ala Arg Gly Ala 610 615 620

Arg Gly Tyr His Cys Asn Arg Glu Thr Ala Thr Thr Ala He Gly Gly 625 630 635 640

Gly Gly Gly Pro Gly Gly Gly Gly Ser Gly Ala He Asp Glu Gly Gly 645 650 655

Gly Arg Asp Ser Ser Ser Gly Asp Gly Ser Glu Ala Cys Gly His Pro

660 665 670

Glu Pro Arg Gly Ala Pro Ser Thr Ser Gly Glu Ser Ala Ser Asp Leu 675 680 685

Gin Arg Thr Gin Leu Leu Pro Pro Cys Pro Leu Asn Gly Glu His Thr 690 695 700

Pro Ala Glu Ala Ala Met Pro Arg Ala Arg Arg Glu Asp Ser Ala Ser 705 710 715 720

Leu Arg Lys Glu Glu Ser Cys Leu Leu Lys Arg Val Pro Gly Val Leu 725 730 735

Thr Ser Gly Leu Glu Asp Ala Ser Gin Pro Pro He Ala Pro Thr Gly

740 745 750

Asp Gin Pro Cys Gin Ala Leu Pro Pro Leu Ser Ser Gin Thr Pro Val 755 760 765

Ala Glu Met Leu Thr Glu Gin Pro Lys Leu Leu Leu Asp Asp Arg Thr 770 775 780

Glu Cys Glu Ser Ser Arg Glu Asp Gin Gly Pro Thr He Pro Ser Glu 785 790 795 800

Ser Ser Ser Gly Arg Phe Pro Leu Gly Asp Leu Leu Gly Gly Gly Ser

805 810 815

Asp Gin Ala Phe Asp Asn Met Lys Glu Pro Val Ser Met Thr Pro Thr 820 825 830

Phe He Ser Glu Leu Ser Leu Ala Asn Tyr Leu Gin Asp Arg Pro Asp 835 840 645

Asp Asp Gly Leu Gly Leu Gly Ala Thr Gly Leu Leu He Arg Glu Ser 850 855 860

Ser Arg Gin Glu Ala Leu Thr Glu Ala Phe Ala Ser Gly Ser Pro Thr 865 870 875 880

Ser Trp Val Pro He Leu Ser Asn Tyr Glu Val He Lys Thr Ser Asp

885 890 895

Pro Glu Ser Arg Glu Asn He Pro cys Pro Glu Pro Gin Asp Glu Lys 900 905 910

Glu Trp Glu Arg Ala Val Pro Leu He Ala Ala Thr Glu Ser Val Pro 915 920 925

Gin Pro Glu Ser Cys He Ser His Trp Thr Pro Pro Pro Ala Ala Val 930 935 940

Gly Ser Thr Gly Ser Asp Ser Glu Gin Val Asp Leu Glu Arg Leu Glu 945 950 955 960

Met Asn Gly He Ser Glu Ala Pro Ser Pro His Ser Glu Ser Thr Asp

965 970 975

Thr Ala Ser Asp Ser Glu Gly His Leu Ser Glu Asp Ser Ser Glu Val 980 985 990

Asp Ala Ser Glu Val Thr Val Val Lys Gly Ser Leu Gly Gly Asp Glu 995 1000 1005

Lys Gin Asp Trp Asp Pro Ser Ala Ser Leu Ser Lys Val Asn Asn Asp 1010 1015 1020

Leu Ser Val Leu Thr Arg Thr Gly Gly Val Ala Ala Ser Gin Ser Trp 1025 1030 1035 1040

Val Ser Arg Val Cys Ser Val Pro His Lys He Pro Asp Ser Leu Leu 1045 1050 1055

Leu Ser Ser Thr Glu Cys Gin Pro Arg Ser Val Cys Pro Leu Arg Pro 1060 1065 1070

Gly Ser Ser Val Glu Val Thr Asn Pro Leu Val Met His Leu Leu His 1075 1080 1085

Gly Asn Leu Pro Leu Glu Lys Val Leu Pro Pro Gly His Arg Ser Ser 1090 1095 1100

Arg Leu Glu Ser Ser Gin Leu Pro Leu Arg Glu Gin Ser Gin Asp Arg 1105 1110 1115 1120

Gly Thr Leu Gin Gly Thr Gly Glu Asn Asn Arg Leu Ala Ala Arg He 1125 1130 1135

Asn Pro Gly Ser Ala Gin Thr Leu Lys Glu Ser He Leu Ala Gin Ser 1140 1145 1150

Tyr Gly Ala Ser Ala Gly Leu Val Arg Ala Met Ala Ser Lys Ala Pro 1155 1160 1165

Ala Met Ser Gin Lys He Ala Lys Met Val Thr Ser Leu Asp Ser Gin 1170 1175 1180

His Pro Glu Thr Glu Leu Thr Pro Ser Ser Gly Asn Leu Glu Glu He

1185 1190 1195 1200

Asp Ser Lys Glu His Leu Ser Ser Phe Leu Cys Glu Glu Gin Lys Glu 1205 1210 1215

Gly His Ser Leu Ser Gin Gly Ser Asp Pro Gly Ala Ala Pro Gly Gin 1220 1225 1230

Cys Leu Gly Asp His Thr Thr Ser Lys Val Pro Cys Phe Ser Ser Thr 1235 1240 1245

Asn Val Ser Leu Ser Phe Gly Ser Glu Gin Thr Asp Gly Thr Leu Ser 1250 1255 1260

Asp Gin Asn Asn Ala Gly Gly His Glu Lys Lys Leu Phe Gly Pro Gly 1265 1270 1275 1280

Asn Thr Val Thr Thr Leu Gin Cys Pro Arg Ser Glu Glu Gin Thr Pro 1285 1290 1295

Leu Pro Ala Glu Val Pro Pro Val Phe Pro Ser Arg Lys He Glu Pro 1300 1305 1310

Ser Lys Asn ser Val ser Gly Gly Val Gin Thr Thr Arg Glu Asn Arg 1315 1320 1325

Met Pro Lys Pro Pro Pro Val Ser Ala Asp Ser He Lys Thr Glu Gin 1330 1335 1340

Thr Phe Leu Arg Asp Pro He Lys Ala Asp Ala Glu Asn Arg Lys Ala 1345 1350 1355 1360

Ala Gly Tyr Ser Ser Leu Glu Leu Val Gly His Leu Gin Gly Met Pro 1365 1370 1375

Phe Val Val Asp Leu Pro Phe Trp Lys Leu Pro Arg Glu Pro Gly Lys 1380 1385 1390

Gly Phe Ser Gin Pro Leu Glu Pro Ser Ser He Pro Ser Gin Leu Asn 1395 1400 1405

He Lys Gin Ala Leu Tyr Gly Lys Leu Ser Lys Leu Gin Leu Ser Pro 1410 1415 1420

Thr Ser Phe Asn Tyr Ser Ser Ser Ser Ala Thr Phe Pro Lys Gly Leu 1425 1430 1435 1440

Ala Gly Gly Val Val Gin Leu Ser His Lys Ala Ser Phe Gly Thr Gly 1445 1450 1455

His Thr Ala Ser Leu Ser Leu Gin Met Phe Ala Asp Ser Ser Ala Val 1460 1465 1470

Glu Ser He Ser Leu Gin Cys Ala Cys Ser Leu Lys Ala Met He Met 1475 1480 1485

Cys Gin Gly Cys Gly Ala Phe Cys His Asp Asp Cys He Gly Pro Ser 1490 1495 1500

Lys Leu Cys Val Leu Cys Leu Val Val Arg 1505 1510