MXPA00005180A - Novel i(dkr) polypeptides - Google Patents

Abstract

Disclosed are nucleic acid molecules encoding novel i(DKR) polypeptides. Also disclosed are methods of preparing the nucleic acid molecules and polypeptides, and methods of using these molecules.

Description

NOVEDOUS DKR POLYPEPTIDES Field of the Invention This invention relates generally to novel genes that code for proteins that have use as anti-cancer therapeutic agents.

BACKGROUND Related Technique One of the contrasting brands of cells that have become cancerous is the change in the pattern of gene expression in those cells compared to the normal, non-cancerous cells. An intricate series of cell signaling events leads to what is known as "differential gene expression," which results in the conversion of a normal cell to a cancer cell (also known as "oncogenesis" or "cell transformation"). ). Numerous cell signaling pathways have been implicated in the cell transformation process, such as, for example, the cadherin pathway, the tailed pathway, the sonic urchin pathway, and the wnt / wingless pathway (Hunter , Cell, 88: 333-346 - [1997]; Currie, Cf. ol. Med., 76: 421-433 [1988]; Peifer, Science, 275: 1752-1753 REF. : 120642 [1977]. Interestingly, those same pathways are involved in cell morphogenesis, or cell differentiation, during embryo development (Hunter, supra; Cadigan et al., Genes and Devolp., 11: 3286-3305 [1977]). The wnt genes code for glycoproteins that are secreted from the cell. These glycoproteins are found in both vertebrate and invertebrate organisms. Currently, there are at least 20 members of the wnt family, and it is believed that these members function in different ways in controlling growth and tissue differentiation.

Recently, the discovery of a novel gene in Xenopus and mouse was identified and has been called dickkopf-1 ("dkk-1"). This gene is presumed to be a potent antagonist of wnt-8 signaling (Glinka et al., Nature, 391: 357-362 [1998]). Interestingly, it is also assumed that this gene is involved in morphogenesis in the developing embryo (Glinka, et al., Supra). This gene thus represents the novel growth factor, which can be useful in tissue regeneration, and also represents means to potentially inhibit cellular transformation via wnt signaling. The Frzb proteins and the Cerberus protein are examples of secreted proteins that supposedly inhibit wnt signaling (Brown, Curr, Opinion Cell Biol., 10: 182-187 [1998]. PCT WO 98/35043, published on August 13, 1998 describes human SDF proteins which are supposedly useful in regulating the binding of wnt polypeptides to their receptors. PCT WO 98/23730, published June 4, 1998, describes tumor cells transfectant with wnt-5a that supposedly decrease tumorigenicity. The wnt-5 a claim to be antagonists of other wnt. In view of the devastating effects of cancer, there is a need in the art to identify additional genes that can serve as antagonists of the proteins involved in cell transformation. Accordingly, an object of this invention is to provide nucleic acid molecules and polypeptides that may be useful as anti-cancer compounds. A further object is to provide methods for altering the level of expression and / or activity of such polypeptides in the human body. Other related objects will be readily apparent from reading this description.

BRIEF DESCRIPTION OF THE INVENTION In one embodiment, the present invention provides an isolated nucleic acid molecule that encodes a biologically active DKR polypeptide selected from the group consisting of: (a) the nucleic acid molecule comprising SEQ ID NO: 1; (b) the nucleic acid molecule comprising SEQ ID NO: 2; (c) the nucleic acid molecule comprising the SEQ ID NO: 3; (d) the nucleic acid molecule comprising SEQ ID NO: 4; (e) the nucleic acid molecule comprising SEQ ID NO: 5; (f) the nucleic acid molecule comprising SEQ ID NO: 6; (g) the nucleic acid molecule comprising SEQ ID NO: 7; (h) the nucleic acid molecule comprising the SEQ ID NO: 75; (i) the nucleic acid molecule comprising SEQ ID NO: 76; (j) the nucleic acid molecule comprising SEQ ID NO: 77; (k) the nucleic acid molecule comprising SEQ ID NO: 78; (1) the nucleic acid molecule encoding the polypeptide of SEQ ID NO: 8; (m) a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 9; (n) a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 10, or a biologically active fragment thereof; (o) a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 11, or a biologically active fragment thereof; (p) a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 12, or a biologically active fragment thereof; (q) a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 13, or a biologically active fragment thereof; (r) a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 14, or a biologically active fragment thereof; (s) a nucleic acid molecule encoding a polypeptide that is at least 85 percent identical to the polypeptide of SEQ ID NOs: 10, 11, 12, 13 or 14; (t) a nucleic acid molecule encoding a biologically active DKR polypeptide having 1-100 amino acid substitutions and / or deletions compared to the polypeptide of any of SEQ ID NOs: 8, 9, 10, 11 , 12, 13 or 14; and (u) a nucleic acid molecule that hybridizes under very stringent conditions to any of (c), (d), (e), (f), (g), (h), (i), (k), (1), (), (n), (o), (p), (q), (r), (s), and (t) above. In another embodiment, the invention provides an isolated nucleic acid molecule that is the complement of any of the above nucleic acid molecules. In still another embodiment, the invention provides an isolated nucleic acid molecule encoding a biologically active DKR polypeptide selected from the group of: amino acids 16-350, 21-350, 22-350, 23-350, 33-350, or 42-350, 21-145, 40-145, 40-150, 45-145, 45-145, 145-290, _L50-290, 300-350, or 310-350 of SEQ ID NO: 9; amino acids 15-266, 24-266, or 32-266 of SEQ ID NO: 10; amino acids 17-259, 26-259, or 34-359 of SEQ ID NO: 12; amino acids 19-224, 20-224, 21-224, or 22-224 of SEQ ID NO: 14. In other embodiments, the invention provides vectors comprising the nucleic acid molecules, and host cells comprising the vectors.

In yet another embodiment, the invention provides a process for producing a biologically active DKR polypeptide comprising the steps of: (a) expressing a polypeptide encoded by any of the nucleic acid molecules herein in a suitable host; and (b) isolating the polypeptide. In yet another embodiment, the invention provides a biologically active DKR polypeptide selected from the group consisting of: (a) the polypeptide of SEQ ID NO: 8; (b) the polypeptide of SEQ ID NO: 9; (c) the polypeptide of SEQ ID NO: 10; (d) the polypeptide of SEQ ID NO: 11; (e) the polypeptide of SEQ ID NO: 12; (f) the polypeptide of SEQ ID NO: 13; (g) the polypeptide of SEQ ID NO: 14; (h) a polypeptide having 1-100 substitutions or deletions of amino acids compared to the polypeptide of any of (a) - (g) above; and (i) a polypeptide that is at least 85 percent identical to any of the polypeptides of (c) - (h) above. In still another embodiment, the invention provides the following polypeptides: a polypeptide consisting of amino acids 16-350, 21-350, 22-350, 23-350, 33-350, or 42-350, 21-145, 40 -145, 40-150, 45-145, 45-145, 145-290, 145-300, 150-290, 300-350, or 310-350 of Figure 9; a polypeptide consisting of amino acids 15, 266, 24-266, or 32-266 of Figure 10; a polypeptide consisting of amino acids 17-259, 26-259, or 34-359 of Figure 12; and a polypeptide consisting of amino acids 19-224, 20-224, 21-224, or 22-224 of Figure 14.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 (SEQ ID NO: 1) describes the sequence of the mouse DKR-3 cDNA. Figure 2 (SEQ ID NO: 2) describes the sequence of the human DKR-3 cDNA. Figure 3 (SEQ ID NO: 3) describes the sequence of the CDNA of human DKR-1. Figure 4 (SEQ ID NO: 4) describes the sequence of the mouse DKR-2 cDNA. Figure 5 (SEQ ID NO: 5) describes the sequence of the human DKR-2 cDNA. Figure 6 (SEQ ID NO: 6) describes the sequence of the human DKR-2a cDNA, a splice variant of the DKR-2 gene. Figure 7 (SEQ ID NO: 7) describes the sequence of the human DKR-4 cDNA.

Figure 8 (SEQ ID NO: 8) describes the amino acid sequence of translated mouse DKR-3 from the corresponding cDNA. Figure 9 (SEQ ID NO: 9) describes the amino acid sequence of the translated human DKR-3 of the corresponding cDNA. Figure 10 (SEQ ID NO: 10) describes the amino acid sequence of human DKR-1 translated from the corresponding cDNA. Figure 11 (SEQ ID NO: 11) describes the amino acid sequence of mouse DKR-2 translated from the corresponding cDNA. Figure 12 (SEQ ID NO: 12) describes the amino acid sequence of human DKR-2 translated from the corresponding cDNA. Figure 13 (SEQ ID NO: 13) describes the amino acid sequence of human DKR-2 translated from the corresponding cDNA. Figure 14 (SEQ ID NO: 14) describes the amino acid sequence of human DKR-4 translated from the corresponding cDNA. Figures 15A-15D are photographs of spots Northern which were probed with human DKR-3. The Figure 15A shows the level of transcription of DKR-3 in several normal (lanes 1-2) and immortal human (lanes 3-4) cell lines, and in human breast cancer cell lines with higher estrogen receptor ("ER +"; Lanes 5-9) and lower estrogen receptor ("ER-"; Lanes 10-16). Figure 15B shows the level of transcription of human DKR-3 in human normal lung cells (Lane 1), and in several non-small cell lung cancer cell lines ("NSCLC", Lanes 2-9) and lung cancer. small cell phone ("SCLC"; Lanes 10-15).

Figure 15C shows the amount of transcription of human DKR-3 in five glioblastoma cell lines; three of these lines (SNB-19, U-87MG, and U-373MG) are capable of forming tumors in nude mice, while the other two lines (Hs 683 and A 172) are not. Figure 15D shows the transcription level of human DKR-3 in immortal (non-cancerous) and normal human cervical cells, and in human cervical cancer cell lines (indicated as "tumor cells"). Figure 16 is a photograph of gel electrophoresis with SDS. The content of the lanes is shown in the Examples here. Figure 17 is a photograph of gel electrophoresis with SDS. The content of the lanes is shown in the Examples here.

Figure 18 is a photograph of gel electrophoresis with SDS. The content of the lanes is shown in the Examples here. Figure 19 is a photograph of gel electrophoresis with SDS. The content of the lanes is shown in the Examples here. Figure 20 is a photograph of gel electrophoresis with SDS. The content of the lanes is shown in the Examples here. Figure 21 is a photograph of a Western stain. The content of the lanes is indicated in the Examples here. Figure 22 (SEQ ID NO: 75) is a nucleic acid sequence of human DKR-1 with codons optimized for expression in E. coli. Figure 23 (SEQ ID NO: 76) is a nucleic acid sequence of human DKR-2 with codons optimized .., for expression in E. coli. Figure 24 (SEQ ID NO: 77) is a human DKR-3 nucleic acid sequence with codons optimized for expression in E. coli. Figure 25 (SEQ ID NO: 78) is a nucleic acid sequence of human DKR-4 with codons optimized for expression in E. coli.

DESCRIPTION OF ALLADA OF THE INVENTION Included within the scope of this invention are the DKR polypeptides such as the polypeptides of SEQ ID NOs: 8-14, and biologically active, related, polypeptide fragments, variants and derivatives thereof. Also included within the scope of the present invention are nucleic acid molecules that encode DKR polypeptides such as the nucleic acid molecules of SEQ ID NOs: 1-7. Additionally included within the scope of the present invention are non-human mammals such as mice, rats, rabbits, goats or sheep in which the gene (or genes) encoding a native DKR polypeptide has been altered ("knocked out") , so that the level of expression of this gene or genes decreases significantly or is completely abolished. Such mammals can be prepared using techniques and methods such as those described in U.S. Patent No. 5,557,032. The present invention further includes non-human mammals such as mice, rats, rabbits, goats or sheep in which the gene (or genes) encoding DKR polypeptides in which either the native form of the gene for that mammal or a gene of the The heterologous DKR polypeptide is overexpressed by the mammal, thereby creating a "transgenic" mammal. Such transgenic mammals can be prepared using well known methods such as those described in U.S. Patent No. 5,489,743 and PCT patent application no. 94/28122, published on December 8, 1994. The present invention further includes non-human mammals in which the promoter for one or more of the DKR polypeptides of the present invention is activated or inactivated (using homologous recombination methods as described below) to alter the level of expression of one. or more of the native DKR polypeptides. The DKR polypeptides of the present invention are expressed to be useful as anticancer therapeutic agents for those cancers such as mammary tumors, proliferative cell tumors, or other cancers in which the wnt and / or hedgehog signal transduction pathways are activated. sonic (shh) Specific wnt members can transform breast tissue ('Hunter, supra) and are expressed abnormally in many human tumors (Huguet, Cancer Res., 54: 2615: 2621 [1994]; Dale, Cancer Res., 56: 4320-4323 [ 1996], see also PCT WO 97/39357). Such activity is expected in view of the data presented here in which the level of transcription of DKR-3 decreased or is not detectable at all in the cell lines of many cancers compared to normal cell lines. Furthermore, such an activity is expected in view of the relationship of the genes and polypeptides of the present invention to the dickkopf-1 gene (which, as mentioned above, is assumed to be a potent wnt-8 antagonist). DKR-1, a novel gene of the present invention is an ortholog of dkk-1, DKR-2, DKR-3, and DKR-4, each related to the DKR-1 by your cysteine pattern. In particular, these DKR polypeptides can be used for the treatment of tumors of proliferating cells, mammary tumors, and other cancers in which the wnt genes are expressed, and in cancers where wnt and / or shh signaling is activated. klO The DKR polypeptides of the present invention can also be administered as agents that can induce and / or increase tissue differentiation, such as bone formation, cartilage formation, muscle tissue formation, nervous tissue formation and formation of hematopoietic cells. Such activities are expected in view of the fact that a) the dkk-1 Xenopus supposedly promotes the induction of the head, heart formation, and ) differentiation or development of the CNS (Glinka, supra); and b) certain wnt polypeptides appear to work in the development embryonic (Cadigan, Genes and Devel., 11: 3286-3305 [1997]), specifically the development of the pituitary (Treier, Genes and Devel., 12: 1691-1704 [1998]), myogenesis (Munsterberg et al. ., Genes and Devel., 9: 2911-2922 [1995]), "osteogenesis (PCT WO 95/17416; PCT W098 / 16641), kidney development (Stark et al., Nature 372: 679-683 [1994] ), development of the SNC (Dickinson et al., Development, 120: 1453-1471 [1994]), and hematopoiesis (PCT WO 98/06747). Thus, the addition of certain DKR polypeptides in such cell or tissue cultures can serve to modify the activity of various wnt polypeptides in cell differentiation processes. The DKR polypeptides herein can be used in either an in vivo manner or an ex vivo manner for such applications. For example, one or more DKR polypeptides of the present invention can be added to a culture of cartilage tissue or nerve tissue, or hematopoietic proliferating cells, either alone or in combination with other growth factors and / or other tissue differentiating factors, to induce or increase the regeneration of such tissues. Alternatively, such DKR polypeptides of the present invention can, for example, be injected directly into a binding that requires cartilage, in the spinal cord where the marrow has been damaged, in damaged brain tissue or in the bone marrow to increase the hematopoiesis. The term "DKR polypeptides" as used herein refers to any protein or any polypeptide having the properties described herein for the DKR polypeptides. The DKR polypeptides may or may not have terminal amino methionines, depending on the form in which they are prepared. By way of illustration, the DKR polypeptides refer to (1) a biologically active polypeptide encoded by any of the nucleic acid molecules for the DKR polypeptides as defined in any of (a) - (f) below.; (2) natural allelic variants and synthetic variants of any DKR polypeptide in which one or more substitutions, deletions and / or amino acid insertions are present in comparison with the DKR polypeptides SEQ ID NOs: 8-14, and / or (3) ) biologically active polypeptides, or fragments or variants thereof, that have been chemically modified. As used herein, the term "DKR polypeptide fragment" refers to a peptide or polypeptide that is less than the full-length amino acid sequence of a natural DKR polypeptide but having the biological activity of any of the "DKR polypeptides" provided Here, such a fragment can be truncated at the amino terminus, the carboxy terminal and / or internally (such as by natural splicing), and can be a variant or a derivative of any of the DKR polypeptides. In addition, "the DKR polypeptide fragments can be natural fragments such as the splice variants of the DKR polypeptide (SEQ ID NO: 13), other splice variants, and resulting fragments of protease activity in live natural. Preferred DKR polypeptide fragments include amino acids 16-350, 21-350, 22-350, 23-350, 33-350, 42-350, 21-145, 40-145, 40-150, 45-145, 145 -290, 145-300, 145-350, 150-290, 300-350, and 310-350, of SEQ ID NO: 9; amino acids 15-266, 24-266, or 32-266 of SEQ ID NO: 10; amino acids 17-259, 26-259, or 34-359 of SEQ ID NO: 12; and amino acids 19-224, 20,224, 21,224, or 22-224 of SEQ ID NO: 14. As used herein, the term "DKR polypeptide variants" refers to DKR polypeptides whose amino acid sequences contain one or more substitutions. , deletions and / or insertions in the amino acid sequence compared to the amino acid sequences of DKR polypeptides set forth in SEQ ID NOS: 8-14. Such variants of the DKR polypeptides can be prepared from the variants of the nucleic acid molecules of the corresponding DKR polypeptides, which have a DNA sequence that varies according to the DNA sequences for the natural DKR polypeptides as disclosed. in SEQ ID NOS: 7-14. Preferred variants of the human DKR polypeptides include alanine substitutions at one or more amino acid positions. Other preferred substitutions include conservative substitutions at the amino acid positions indicated in the Examples herein, as well as those encoded by DKR nucleic acid molecules as described below. As used herein, the term "DKR polypeptide derivatives" refers to DKR polypeptides, variants, or fragments thereof, which have been chemically modified, such as, for example, by the addition of one or more polyethylene glycol molecules, sugars, phosphates and / or other such molecules, where the molecule or molecules are not naturally bound to natural DKR polypeptides. As used herein, the terms "biologically active DKR polypeptides", "biologically active DKR polypeptide fragments", "biologically active DKR polypeptide variants", and "biologically active DKR polypeptide derivatives" refer to DKR polypeptides having the ability to decrease the proliferation of cancer cells in the Anchorage Independent Growth Assay of Example 12 herein, or in the Live Jn Tumor Test of Example 13 herein, or in both assays. As used herein, the term "DKR polypeptide nucleic acid" when used to describe a nucleic acid molecule refers to a nucleic acid molecule or fragment of -the same which - (a) has • the nucleotide sequence that is set forth in any of SEQ ID NOS: 1-7; (b) it has a nucleic acid sequence that codes for a polypeptide that is at least 85 percent identical, but that can be more than 85 percent, that is, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent identical to the polypeptide encoded by any of the SEQ ID NOS: 10-14; (c) is a natural allelic variant or alternative splice variant of (a) or (b); (d) is a variant of the nucleic acid of (a) - (c) produced according to the provisions herein; (e) has a sequence that is complementary to (a) - (d); (f) hybridizes to any of (a) - (e) under highly stringent conditions and / or (g) has a nucleic acid sequence that encodes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or up to 100 substitutions and / or amino acids of any mature DKR polypeptide (i.e., a DKR polypeptide with its endogenous signal peptide removed). The percent identity of the sequence can be determined by standard methods that are commonly used to compare the similarity in the position of the amino acids of two polypeptides. By way of example, using a computer algorithm such as the GAP (Genetic Computing Group, University of Wisconsin, Madison, Wl), or two polypeptides for which the percent identity of the sequence is to be determined are aligned to optimally compare your amino acids (the "compared space", as determined by the algorithm). A gap opening penalty (which is calculated as 3x the average diagonal, the "average diagonal" is the average of the diagonal of the comparison matrix that is being used, the "diagonal" is the value or number assigned to each perfect amino acid by the particular comparison matrix) and a vacuum extension penalty (which is usually 1/10 times the vacuum opening penalty), as well as a comparison matrix such as the PAM 250 or BLOSUM 62 are used in set with the algorithm. A standard comparison matrix is also used (see Dayhoff et al., In: Atlas of Protein Sequence and Structure, vol.5, supp.3 [1978] for the PAM250 comparison matrix, see Henikoff et al., Proc. Nati Acad Sci USA, 89: 10915-10919 [1992] _for the BLOSUM comparison matrix 62) by the algorithm. The identity percent is then calculated by the algorithm by determining the identity percent as follows: Total number of identical pairs in the compared stretch X 100 [length of the longest sequence within the compared stretch] + [number of empties entered in the longest sequence to align the two sequences] Polypeptides that are at least 85 percent identical will typically have one or more substitutions, deletions and / or amino acid insertions compared to any of the natural DKR polypeptides. Usually, the substitutions of the native residue will be either alanine, or a conservative amino acid having little or no effect on the total net charge, polarity or hydrophobicity of the protein. Conservative substitutions are set forth in Table I below.

Table I Conservative Substitutions of Basic Amino Acids: arginine lysine histidine Acids: glutamic acid aspartic acid - Polar not charged: glutamine asparagine Table I (continued) Conservative Amino Acid Substitutions Serine Threonine Tyrosine Non Polar: Phenylalanine Tryptophan Cysteine Glycine Alanine Valine Proline Methionine Leucine Isoleucine The term "highly stringent conditions" refers to hybridization and washing under conditions that allow the binding of a nucleic acid molecule used for selection, such as an oligonucleotide probe or cDNA molecule probe, to highly homologous sequences. An exemplary highly stringent wash solution is 0.2 X SSC and 0.1 percent SDS used at a temperature between 50 ° C-65 ° C. Where oligonucleotide probes are used to select cDNA or genomic libraries, one of the following two highly stringent solutions may be used. The first of these is 6 X SSC with 0.05 percent sodium pyrophosphate at a temperature of 35 ° C-62 ° C, depending on the length of the oligonucleotide probe. For example, probes of 14 base pairs were washed at 35-40 ° C, probes of 17 base pairs were washed at 45-50 ° C, probes of 20 base pairs were washed at 52-57 ° C, and washed probes of 23 base pairs at 57-63 ° C. The temperature can be increased 2-3 ° C, where the binding does not specify antecedent seems to be high. A second highly stringent solution uses tetramethylammonium chloride (TMAC) to wash oligonucleotide probes. A strict wash solution is TMAC 3M, 50mM Tris-HCl, pH 8.0, and 0.2 percent SDS__ The wash temperature using this solution depends on the length of the probe. For example, a probe of 17 base pairs was washed at about 45-50 ° C. As used herein, the terms "effective amount" and "therapeutically effective amount" refer to the amount of a DKR polypeptide necessary to support one or more biological activities of the DKR polypeptides as set forth above.

A full-length DKR polypeptide or fragment thereof can be prepared using well known recombinant DNA technology methods such as those set forth in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY [1989]) and / or Ausubel et al., Eds., (Current Protocols in Molecular Biology, Green Publishers Inc. and Wiley and Sons, Ny [1994]). A gene or cDNA encoding a DKR polypeptide or a fragment thereof can be obtained for example by selecting a genomic or cDNA library, or by PCR amplification. Waves or polymers useful for selecting the library can be generated based on the sequence information for other genes or fragments of known genes thereof or a related gene family, such as, for example, conserved motifs found in other DKR polypeptides such as the cysteine pattern. In addition, where a gene encoding the DKR polypeptide of a species has been identified, all or a portion of that gene can be used as a probe to identify homologous genes from other species. The probes or primers can be used to select cDNA libraries from various tissue sources that are believed to express the DKR gene. Typically, highly stringent selection conditions will be employed to minimize the number of false positives obtained from the selection.

Other means for preparing a gene encoding a DKR polypeptide or fragment thereof is to employ chemical synthesis using methods well known to those skilled in the art such as those described by Engels et al. (Angew. Chem. Intl. Ed., 28: 716-734

[1989]), such methods include, inter alia, phosphotriester, phosphoramidite, and H-phosphonate methods for the synthesis of nucleic acid. A preferred method for such chemical synthesis is polymer-supported synthesis using the chemistry of the standard phosphoramidite. Typically, the DNA encoding the DKR polypeptide will be several hundred nucleotides in length. Nucleic acids of more than about 100 nucleotides can be synthesized as several fragments using those methods. The fragments can then be ligated to form the full-length DKR polypeptide. Usually, the DNA fragment encoding the amino terminus of the polypeptide will have an ATG, which codes for a methionine residue. This methionine may or may not be present in the mature form of the DKR polypeptide, depending on whether the polypeptide produced in the host cell is designed to be selected from that cell. In some cases, it may be desirable to prepare nucleic acid and / or amino acid variants of the natural DKR polypeptides. Nucleic acid variants can be produced using site-directed mutagenesis, PCR amplification, or other appropriate methods, where the primers have the desired point mutations (see Sambrook et al. , supra, and Ausubel et al. , supra, for descriptions of mutagenesis techniques). The chemical synthesis using the methods described by Engels et al. , supra, can also be used to prepare such variants. Other methods known to those skilled in the art can also be used. Preferred nucleic acid variants are those containing nucleotide substitutions that contribute to the codon preference in the host cell to be used to produce the DKR polypeptides. 'Such "codon optimization" can be determined via computer algorithms which incorporate codon frequency tables such as the "Ecohigh.CoD" for the codon preference of highly expressed bacterial genes as provided by the Package of the University of Wisconsin Version 9.0, Genetics Computer Group, Madison, Wl. Other useful codon frequency tables include "Celegans_high. Cod", "Celegans_low. Cod", "Drosophila_high.cod", "Human_high. Cod", _ "Maize_high. Cod" and "Yeast__high.cod". Other preferred variants are those which code for conservative amino acid changes as described above (eg, where the charge or polarity of the natural amino acid side chain is not substantially altered by substitution with a different amino acid) as compared to natural and / or those designed to generate a novel glycosylation and / or phosphorylation site, or those designed to suppress an existing glycosylation and / or phosphorylation site. The gene, cDNA, or fragment thereof encoding the DKR polypeptide can be inserted into a vector of expression or appropriate amplification using the technique of • standard ligation. The vector is typically selected so that it is functional in the particular host cell employed (i.e., that the vector is compatible with the machinery of the host cell so that the gel amplification and / or gene expression). The gene, cDNA or fragment thereof encoding the DKR polypeptide can be amplified / expressed in prokaryotic, yeast, insect (baculovirus systems) and / or eukaryotic host cells. The selection of the cell The host will depend in part on whether the DKR polypeptide or fragment thereof is to be glycosylated and / or phosphorylated. If so, yeast, insect or mammalian host cells are preferable. Typically, the vectors used in either of the host cells will contain 5 'flanking sequences (also known as "promoters") and other regulatory elements as well as an amplifier, an origin of the duplication element, a transcriptional termination element, a complete intron sequence containing a site of donor and receptor junction, a signal peptide sequence, an element of the ribosomal binding site, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, and a selectable marker element. Each of these elements is discussed later. Optionally, the vector may contain a "tag" sequence, i.e., an oligonucleotide molecule located at the 5 'or 3' end of the sequence encoding the DKR polypeptide; the oligonucleotide molecule encoding polyHis (such as hexaHis) or another "tag" such as FLAG (FLAG); HE HAS (Hemagglutinin Influenza virus), or myc for which there are commercially available antibodies. This tag typically fuses to the polypeptide after expression of the polypeptide, and may serve as a means for affinity purification of the DKR polypeptide of the host cell. Affinity purification can be effected, for example, by column chromatography using anti-brand antibodies as an affinity matrix. Optionally, the tag can be subsequently removed from the purified DKR polypeptide by various means such as using certain peptidases. The human immunoglobulin hinge and the Fc region could be fused at any N-terminal or C-terminal DKR polypeptide by one skilled in the art. The subsequent Fc fusion protein could be purified by the use of a protein A affinity column. It is known that Fc exhibits a prolonged pharmacokinetic half-life in vivo and it has been found that proteins fused to Fc exhibit a substantially higher half-life. in vivo compared to the non-merged counterpart. Also, fusion to the Fc region allows the dimerization / multimerization of the molecule that may be useful for the bioactivity of some molecules. The 5 'flanking sequence can be homologous (i.e. of the same species and / or strain as the host cell), heterologous (i.e., of a different species from that of the species or strain of the host cell), hybridized (i.e. say, a combination of the 5 'flanking sequences from more than one source), synthetic, or it may be the 5' flanking sequence of the native DKR polypeptide gene. Therefore, the source of the 5 'flanking sequence can be any unicellular prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, as long as the 5' flanking sequence is functional, and can be activated by, the machinery of the host cell. The 5 'flanking sequences useful in the vectors of this invention can be obtained by any of several methods well known in the art. Typically the 5 'flanking sequences useful here different from the flanking sequence of the DKR gene will have been previously identified by mapping and / or by restriction endonuclease digestion and thus can be isolated from the appropriate tissue source using the restriction endonucleases appropriate. In some cases, the full length nucleotide sequence of the 5 'flanking sequence may be known. Here, the 5 'flanking sequence can be synthesized using the methods described above for nucleic acid synthesis or cloning. Where all or only a portion of the 5 'flanking sequence is known, it can be obtained using PCR and / or by cloning a PCR library and / or selecting a genomic library with the appropriate oligonucleotide and / or fragments of the 5' flanking sequence of the same or another species. Where the 5 'flanking sequence is not known, a DNA fragment containing a 5' flanking sequence of a larger piece of DNA that may contain, for example, a coding sequence or even another gene or genes can be isolated. Isolation can be effected by restriction endonuclease digestion using one or more carefully selected enzymes to isolate - the appropriate DNA fragment. After digestion, the desired fragment can be isolated by agarose gel purification, column Qiagen® or other methods known to those skilled in the art. The selection of suitable enzymes to carry out this purpose will be readily apparent to one skilled in the art. The origin of the duplication element is typically a part of the commercially obtained prokaryotic expression vectors, and aids in the amplification of the vector in a host cell. Amplification of the vector to a certain number of copies may, in some cases, be important for optimal expression of the DKR polypeptide. If the vector of choice does not contain an origin of the duplication site, one can be synthesized chemically based on a known sequence, and ligated into the vector. The transcription termination element is typically located at the 3 'position of the end of the sequence encoding the DKR polypeptide and serves to terminate the transcription of the DKR polypeptide. Usually, the transcription termination element in prokaryotic cells is a fragment rics in GC followed by a poly T sequence. Although the element is easily cloned from a library or even commercially obtained as part of a vector, it can also be easily synthesized using methods for nucleic acid synthesis such as those described above. An element of the selectable marker gene encodes a protein necessary for the survival and growth of a host cell growing in a selective culture medium. Typical selection marker genes encode proteins that (a) confer resistance to antibiotics or other toxins, eg, ampicillin, tetracycline, or kanamycin for prokaryotic host cells, (b) complement auxotrophic deficiencies of the cell; or (c) provide critical nutrients not available from complex media. Preferred selectable markers are the __a kanamycin resistance gene, the ampicillin resistance gene, and the tetracycline resistance gene. The ribosomal binding element, commonly known as Shine-Dalgarno sequence (prokaryotes) or Kozak sequence (eukaryotes), is usually necessary for the initiation of mRNA translation. The element is typically located 3 'to the promoter and 5' to the coding sequence of the DKR polypeptide to be synthesized. The Shine-Dalgarno sequence varies but is typically a polypurine (ie, having a high content of A-G). Many Shine-Dalgarno sequences have been identified, each of which can be easily synthesized using the methods discussed above and used in a prokaryotic vector. In those cases where it is desirable for a DKR polypeptide to be secreted from the host cell, a signal sequence can be used to direct the DKR polypeptide outside the host cell where it is synthesized, and the carboxyterminal part of the protein can be deleted to prevent the walking of the membrane. Typically, the signal sequence is located in the coding region of the DKR gene or cDNA, or directly at the 5 'end of the coding region of the DKR gene. Many signal sequences have been identified, and some of them that are functional in the host cell can be used in conjunction with the DKR gene or cDNA. Therefore, the signal sequence can be homologous or heterologous to the DKR cDNA gene, and can be homologous or heterologous to the gene or cDNA of the DKR polypeptides. Additionally, the signal sequence can be synthesized cjuimicamente using the methods discussed above.

In many cases, selection of the polypeptide from the host cell via the presence of a signal peptide will result in the removal of the amino terminal methionine from the polypeptide. In many cases, the transcription of the DKR gene or cDNA is increased due to the presence of one or more introns in the vector, this is particularly true where the DKR polypeptide is produced in eukaryotic host cells, especially mammalian host cells. The introns used can naturally be found within the DKR gene, especially where the gene used is a full length genomic sequence or a fragment thereof. Where the intron is not naturally found within the gene (as for most cDNAs), introns can be obtained from another source. The position of the intron with respect to the 5 'flanking sequence and the DKR gene is generally important, since the intron can be transcribed to be effective. Therefore, where the DKR gene is inserted into the expression vector is a cDNA molecule, the preferred position for the intron is 3 'to the transcription start site, the transcription termination sequence of poly A. Preferably for the DKR cDNA, the intron or introns will be located on one side - or another (ie, 5 'or 3f) of the cDNA, so as not to interrupt this coding sequence. Any intron can be used from any source, including any viral, prokaryotic and eukaryotic organisms (plant or animal) to practice this invention, as long as they are compatible with the host cells into which they are inserted. Synthetic introns are also included here. Optionally, more than one intron can be used in the vector. Where one or more of the above-mentioned elements are not already present in the vector to be used, they can be obtained individually and bound in the vector. The methods used to obtain each of the elements are well known to those skilled in the art and are comparable to the methods set forth above (ie, DNA synthesis, library selection, and the like.) The final vectors used to practice this invention are typically constructed from initial vectors such as a commercially available vector.Such vectors may or may not contain some of the elements to be included in the complete vector.If none of the desired elements are present in the initial vector, each element may be ligated individually into the vector by cutting the vector with the appropriate restriction endonucleases, so that the ends of the element are ligated into and the ends of the vector are compatible for ligation, In some cases, it may be necessary to "blunt" the ends to be linked to obtain a satisfactory ligation. The blunting is achieved by filling prime the "adhesive ends" using Klenow DNA polymerase or T4 DNA polymerase in the presence of the four nucleotides.

This method is well known in the art and is described, for example, in Sambrook et al. , supra. Alternatively, two or more of the elements to be inserted in the vector may be first linked together (if they are to be placed adjacent to each other) and then linked in the vector. Another method to construct the vector to construct all the ligations of the different elements simultaneously in a reaction mixture. Here, many non-functional or non-functional vectors will be generated due to the ligation or inappropriate insertion of the elements, however the functional vector can be identified and selected by digestion with restriction endonuclease. Preferred vectors for practicing this invention are those that are compatible with host cells of bacteria, insects and mammals. Such vectors include, inter alia, pCRII, pCR3, and pcDN3.1 (Invitrogen Company, San Diego, CA), pBSII (Stratagene Company, La Jolla, CA), pET15b (Novagen, Madison, Wl), pGEX (Pharmacia Biotech, Piscataway, NJ), pEGFP-N2 (Clontech, Palo Alto, CA), pETL (BlueBacII; Invitrogen), and pFastBacDual (Gibco / BRL, Great Island, NY). After the vector has been constructed and a nucleic acid molecule encoding the full-length or truncated DKR polypeptide has been inserted at the appropriate site of the vector, the entire vector can be inserted into a suitable host cell for amplification and / or expression of the polypeptide. The host cells can be prokaryotic host cells (such as E. coli) or eukaryotic host cells (such as a yeast cell, an insect cell or a vertebrate cell). The host cell, when cultured under appropriate conditions, can synthesize the DKR polypeptide, which can then be harvested from the culture medium (if the host cell secretes it into the medium) or directly from the host cell that produces it (if it is not secreted). After harvesting, the DKR polypeptide can be purified using methods such as molecular sieve chromatography, affinity chromatography and the like. The selection of the host cell for the production of DKR polypeptide will depend in part on whether the DKR polypeptide is to be glycosylated or phosphorylated (in which case eukaryotic host cells are preferred), and the manner in which the host cell is capable of "folding" the protein into its native tertiary structure (eg, proper orientation of the disulfide bridges, etc.) so that the biologically active protein is prepared by the DKR polypeptide having biological activity, the polypeptide DKR can be "folded" after synthesis using appropriate chemical conditions as discussed below. Suitable cells or cell lines can be mammalian cells, such as hamster ovary cells Chinese (CHO), 293 or 293T human embryonic kidney cells (KEH), or 3T3 cells. The selection of suitable mammalian host cells and methods for transformation, culture, amplification, selection, production and purification of the product are known in the art. Other suitable mammalian cell lines are the COS-1 and COS-7 monkey cell lines, and the CV-1 cell line. Additional exemplary mammalian host cells include primate cell lines and rodent cell lines, including transformed cell lines. Normal diploid cells, strains of cells derived from primary tissue culture, as well as primary explants, are also suitable. The candidate cells may be genotypically deficient in the selection gene, or they may contain a dominantly active selection gene. Other suitable mammalian cell lines include, but are not limited to, mouse neuroblastoma N2A cells, HeLa or mouse L-929 cells, 3T3 lines derived from Swiss, Balb-c or NIH mice, hamster DHK or HaK cell lines. Equally useful as host cells suitable for the present invention are bacterial cells. For example, different species of E. coli (eg, HB101, DH5, DH10, and MC1061) are well known as host cells in the field of biotechnology. Various strains of B. subtilis, Pseudomonas spp. , other Bacillus spp. , Streptomyces spp. , and similar in this method. Many strains of yeast cells known to those skilled in the art as host cells for the expression of the polypeptides of the present invention are also available. Additionally, where desired, insect cell systems may be utilized in the methods of the present invention. Such systems are described, for example, in Kitts et al. (Biotechniques, 14: 810-817 [1993]), Lucklow (Curr Opin. Biotechnol '., 4: 564-572 [1993]) and Lucklow et al. (J. Virol., '67: 4566-4579 [1993]). The preferred insect cells are Sf-9 and Hi5 (Invitrogen, Carlsbad, CA).

The insertion (also known as "transformation" or "transfection") of the vector in the selected host cell can be carried out using method such as the calcium chloride method, electroporation, microinjection, lipofection or DEAE-dextran. The selected method will be partly a function of the type of host cell to be used. These methods and other methods are well known to those skilled in the art, and are set forth, for example, in Sambrook et al., Supra. Host cells that contain the vector (ie, transformed or transfected) can be cultured using standard means well known to those skilled in the art. The media will usually contain all the nutrients necessary for the growth and survival of the cells. Suitable means for culturing E. coli cells are, for example, Luria Broth (LB) and / or Terfific Broth (TB). Suitable means for cultivating incariate cells are RPMI 1640, MEM, DMEM, all of which can be supplemented with serum and / or growth factors according to what is required by the particular cell line being cultivated. A suitable medium for insect cultures is Grace's medium supplemented with yeastolato, lactoalbumin hydrolyzate and / or fetal sheep serum as necessary.

Typically, an antibiotic or other compound useful for the selective growth of the transformed cell alone is added as a supplement to the media. The compound to be used will be dictated by the selectable marker element present in the plasmid with which the host cell was transformed. For example, where the selectable marker element is kanamycin resistance, the compound added to the culture medium will be kanamycin. The amount of DKR polypeptide produced in the host cell can be evaluated using standard methods well known in the art. Such methods include, without limitation, Western blot analysis, electrophoresis on SDS-polyacrylamide gel, electrophoresis on non-denaturing gel, separation by CLAP, immunoprecipitation, and / or activity assays such as the gene deviation assay that binds to the DNA If the DKR polypeptide has been designed to be secreted from the host cells, the majority of the polypeptide can be found in the cell culture medium. Polypeptides prepared in this manner will typically not possess an amino terminal methionine, since it is removed during cell secretion. If, however, the DKR polypeptide is not secreted from the host cell, it will be present in the cytoplasm and / or the nucleus (for eukaryotic host cells) or in the cytosol (for host cells of large negative bacteria) and may have a amino terminal methionine. For the DKR polypeptide located in the cytoplasm of the host cell and / or the nucleus, the host cells are typically first mechanically disrupted or with detergent to release the intracellular content in a buffered solution. The DKR polypeptide can then be isolated from this solution. Purification of the DKR polypeptide from the solution can be achieved using a variety of techniques. If the polypeptide has been synthesized so as to contain a label such as Hexahistidine (Z? FCR / hexaHis polypeptide) or other pecjuene peptide such as FLAG (Eastman Kodak Co., New Haven, CT), or myc (Invitrogen, Carlsbad, CA) at any of its carboxy or amino termini, can be essentially purified in a one step process by passing the solution through an affinity column where the matrix the column has high efficiency by the label or by the polypeptide directly (i.e., a monoclonal antibody that specifically recognizes the DKR polypeptide). For example, polyhistidine binds with great affinity and specificity to nickel, thus a nickel affinity column (such as Qiagen nickel columns) can be used for the purification of the IFCR / polyHis polypeptide. (See, for example, Ausubel et al., Eds., Current Protocols in Molecular Biology, Section 10.11.8, John Wiley & Sons, New York [1993]). Where the DKR polypeptide is prepared without a bound label, and antibodies are not available, other well known methods for purification may be used. Such methods include, without limitation, ion exchange chromatography, molecular sieve chromatography, CLAP, native gel electrophoresis in combination with gel elution, and preparative isoelectric focusing (machine / "Isoprime" technique, Hoefer Scientific).

In some cases, two or more of these techniques can be combined to achieve greater purity. If it is anticipated that the DKR polypeptide will be found mainly within the cell, the 'intracellular material (including the inclusion bodies of gram negative bacteria) can be extracted from the host cell using any standard technique known to those skilled in the art. For example, host cells can be used to release the periplasmic / cytoplasmic contents by means of a French press, homogenization and / or sonication followed by centrifugation. If the DKR peptide has formed inclusion bodies in the cytosol, the inclusion bodies can often bind to the internal and / or external cell membranes and thus will be found mainly in the sedimented material after centrifugation. The settled material can then be treated at extreme pH or with a chaotropic agent such as a detergent, guanidine, guanidine derivatives, urea or urea derivatives in the presence of a reducing agent such as dithiothreitol at alkaline pH or tris carboxyethyl phosphine at pH acid to release, break and solubilize the inclusion bodies. The DKR polypeptide is now in its soluble form and can then be analyzed using gel electrophoresis, immunoprecipitation or the like. If it is desired to isolate the DKR polypeptide the isolation can be carried out, using standard method such as those set out below in Marston et al. (Meth. Enz., 182: 264-275 [1990]). In some cases, the DKR polypeptide may not be biologically active after isolation. Various methods can be used to "redouble" or convert the polypeptide to its tertiary structure and generate disulfide bonds, to restore biological activity. Such methods include exposing the solubilized polypeptide to a pH usually greater than 7 and in the presence of a particular concentration of a chaotrope. The selection of the chaotrope is very similar to the choice used for solubilization of inclusion bodies but usually at a lower concentration and is not necessarily the same chaotrope used for solubilization. In most cases the redoubling / oxidizing solution will also contain a reducing agent or reducing agent plus its oxidized form in a specific ratio to generate a particular redox potential that allows disulfide distribution to occur in the formation of the bridges. protein cysteine. Some of the commonly used redox pairs include cysteine / cystamine, glutathione (GSH) / dithiobis GSH, cupric chloride, dithiothreitol (DTT) / dithian DTT, 2-mercaptoethanol (bME) / dithio-b (ME). In many cases a co-solvent is necessary to increase the efficiency of the redoubling and the most common reagents used for this purpose include glycerol, polyethylene glycol of various molecular weights and arginine. If inclusion bodies of the DKR polypeptide are not formed to a significant degree in the host cell, the DKR polypeptide will mainly be found in the supernatant after centrifugation of the cell homogenate, and the DKR polypeptide can be isolated from the supernatant using such methods. as those discussed later. In those situations where it is preferable to partially or completely isolate the DKR polypeptide, the purification can be carried out using standard methods well known to those skilled in the art. Such methods include, without limitation, separation by electrophoresis followed by electroelution, various types of chromatography (immunoaffinity, molecular sieve, and / or ion exchange), and / or high pressure liquid chromatography. In some cases, it may be preferable to use more than one of these methods to complete the purification. In addition to preparing and purifying the DKR polypeptide using recombinant DNA techniques, the DKR polypeptides, fragments, and / or derivatives thereof can be prepared by chemical synthesis methods (such as solid phase peptide synthesis) using methods known in the art. the technique such as those exposed by Merrifield et al. , (J. Am. Chem. Soc., 85: 2149 [1963]), Houghten et al. ,. { Proc Nati Acad. Sci. USA, 82: 5132 [1985]), and Stewart and Young (Solid Phase Peptide Synthesis, Pierce Chemical Co. , Rockford, IL [1984]). Such polypeptides can be synthesized with or without a methionine on the amino terminus. The DKR polypeptides or fragments can be oxidized using the methods set forth in those references to form disulfide bridges. It is expected that DKR polypeptides or fragments thereof have comparable biological activity with DKR polypeptides produced recombinantly or purified from natural sources, and thus can be used interchangeably with recombinant or natural DKR polypeptides. Compositions of chemically modified DKR polypeptides in which the DKR polypeptide is linked to a polymer are included within the scope of the present invention. The selected polymer is typically soluble in water, so that the protein to which it is attached does not precipitate in an aqueous environment, such as a physiological environment. The polymer selected is usually modified to have a single reactive group, such as a > active ester for acylation or an aldehyde for alkylation, so that the degree of polymerization can be controlled according to the provisions of the methods herein. The polymer can be of any molecular weight, and can be branched or unbranched. Included within > Scope of the DKR polypeptide polymers is a mixture of polymers. Preferably, for the therapeutic use of the final product preparation, the polymer will be pharmaceutically acceptable. The water soluble polymer or mixture thereof may be selected from the group consisting of, for example, polyethylene glycol (PEG), monomethoxy polyethylene glycol, dextran, cellulose, or other polymers based on carbohydrate, poly- (N-vinyl pyrrolidone) polyethylene glycol, homopolymers of propylene glycol, a copolymer of polypropylene oxide / ethylene oxide, polyoxyethylated polyols (eg, glycerol) and polyvinyl alcohol. For acylation reactions, the selected polymers should have a single reactive ester group. For reductive alkylation, the selected polymers should have a single reactive aldehyde group. A preferred reactive aldehyde is polyethylene glycol propionaldehyde, which is stable in water, or C1-C10 mono alkoxy or aryloxy derivatives thereof (see U.S. Patent No. 5,252,714). PEGylation of the DKR polypeptides can be carried out by any of the pegylation reactions known in the art, as described for example in the following references: Focus on Growth Factors 3: 4-10 (1992); EP 0 154 316; and EP 0 401 384. Preferably, the pegylation is carried out via an acylation reaction or an alkylation reaction with an active polyethylene glycol molecule (or a water soluble polymer, reagent, analog) as described further ahead. A particularly preferred water-soluble polymer for use herein is polyethylene glycol, abbreviated as PEG. As used herein, polyethylene glycol means that it encompasses any of the PEG forms that have been used to derive other proteins, such as (C 1 -C 10) monoalkoxy or aryloxy polyethylene glycol.

In general, the chemical derivatization can be effected under any suitable conditions used to react a biologically active substance with an activated polymer molecule. Methods for preparing PEGylated DKR polypeptides will generally comprise the steps of (a) reacting the polypeptide with polyethylene glycol (such as a reactive ester or aldehyde derivative of the PEG) under conditions whereby the DKR polypeptide binds to one or more PEG groups, and (b) obtain the reaction products. In general, the optimal reaction conditions for the acylation reactions will be determined based on known parameters and the desired result. For example, the higher the PEG: protein ratio, the greater the percentage of polypeglylated product. Generally, conditions that can be alleviated or modulated by the administration of the polymer / polypeptides herein include those described herein for the DKR polypeptide molecules. However, the DKR polymer / polypeptide molecules described herein may have additional activities, increased or reduced biological activity, or other characteristics, such as increased or decreased half-life, as compared to non-derived molecules. The DKR polypeptides, fragments thereof, variants and derivatives, can be used alone, together, or in combination with other pharmaceutical compositions. The DKR polypeptides, fragments, variants and derivatives can be used in combination with cytokines, growth factors, antibiotics, anti-inflammatories and / or chemotherapeutic agents as appropriate for the indication being treated. DKR nucleic acid molecules, fragments, and / or derivatives that do not themselves encode polypeptides that are active in activity assays can be useful as hybridization probes in diagnostic assays to test, either qualitatively or quantitatively, the presence of DKR DNA or the corresponding RNA in tissue samples or mammalian body fluid. DKR polypeptide fragments, variants, and / or derivatives that are not active in activity assays themselves may be useful for preparing antibodies that recognize DKR polypeptides. - The DKR polypeptides, fragments, variants, and / or derivatives can be used to prepare antibodies using standard methods. Thus, antibodies that react with the DKR polypeptides, as well as the reactive fragments of such antibodies, are also contemplated within the scope of the present invention. The antibodies can be polyclonal, monoclonal, recombinant, chimeric, single chain and / or biospecific. Typically, the antibody or fragment thereof will be of human origin, or will be "humanized", that is, prepared to prevent or minimize a reaction immune with the antibody when administered to a patient.

The antibody fragment can be any fragment that reacts with the DKR polypeptides of the present invention, such as, Fab, Fab ', etc. Also provided by this invention are the hybridomas generated by presenting any DKR polypeptide or fragments thereof as an antigen to selected cells of a mammal, followed by fusion (e.g., spleen cells) of the mammal with certain cancer cells to create immortalized cell lines by known techniques. The methods used to generate such cell lines and antibodies directed against all or portions of a human DKR polypeptide of the present invention are also encompassed by this invention. - The antibodies can be used therapeutically, to inhibit the binding of the DKR polypeptide to your union pattern. The antibodies can also be used for diagnostic purposes in vivo and in vi tro, such as in labeled form to detect the presence of the DKR polypeptide in a body fluid or cell sample.

Preferred antibodies are human antibodies, either polyclonal or monoclonal.

Therapeutic Compositions and Administration The therapeutic compositions of DKR polypeptides are within the scope of the present invention. Such compositions may comprise a therapeutically effective amount of the polypeptide or fragments, variants or derivatives in admixture with a pharmaceutically acceptable carrier. He The carrier material can be water for injection, preferably supplemented with other common materials in solution for administration to mammals. Typically, a DKR polypeptide therapeutic compound will be administered in the form of a composition comprising polypeptide Purified, fragment, variant or derivative thereof in conjunction with one or more physiologically acceptable carriers, excipients or diluents. Saline solution or neutral buffered mixed saline solution with serum albumin are exemplary suitable carriers. Preferably, the The product is formulated as a lyophilisate using appropriate excipients (e.g., sucrose). Can be included if other carriers, diluents and * standard excipients Other exemplary compositions comprise Tris buffer with a pH of about 7.0-8.5, or acetate buffer with a pH of about 4.0-5.5, which may also include sorbitol or a suitable substitute thereof. The DKR polypeptide compositions can be administered parenterally. Alternatively, the compositions may be administered intravenously or subcutaneously. When administered systemically, the therapeutic compositions for use in this invention may be in the form of a parenterally acceptable, pyrogen-free aqueous solution. The preparation of such pharmaceutically acceptable protein solutions, with respect to the proper pH, isotonicity, stability and the like, is within the skill of the art. Therapeutic formulations of the DKR polypeptide compositions useful for practicing the present invention can be prepared for storage by mixing the selected composition to the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences, 18th Edition, AR Gennaro, ed., Mack Publishing Company [1990]) in the form of a lyophilized cake or an aqueous solution. Acceptable carriers, excipients or stabilizers are not toxic to the receptors and are preferably inert at the doses and concentrations employed, and include buffers such as phosphate, citrate or other organic acids; antioxidants such as ascorbic acid; low molecular weight polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions that form salts such as sodium; and / or nonionic surfactants, such as Tween, pluronic or polyethylene glycol (PEG). An effective amount of the DKR polypeptide compositions to be employed therapeutically will depend, for example, on therapeutic objectives such as the indication for which the DKR polypeptide is being used, the route of administration, and the condition of the patient. Consequently, it will be necessary for a therapist to determine the dose and modify the route of administration as required to obtain the optimal therapeutic effect. A typical daily dose can range from about 0.1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. Typically, a clinician will administer the composition until a dose is reached that achieves the desired effect. The composition can, therefore, be administered as a single dose, or as two or more doses (which may or may not contain the same amount of polypeptide DKR) over time, or as a continuous infusion via an implantation device or catheter. As additional studies are conducted, information will emerge regarding the appropriate dose levels for the treatment of various conditions in several patients, and a person skilled in the art, considering the therapeutic context, the type of disorder under treatment, the age and general health of the recipient will be able to determine the appropriate dose. The DKR polypeptide composition to be used for in vivo administration must be sterile. This is easily achieved by filtration through sterile filtration membranes. Where the composition is lyophilized, sterilization using those methods can be conducted either before, or after, lyophilization-and reconstitution. The composition for parenteral administration will commonly be stored in lyophilized form or in solution. Therapeutic compositions are generally placed in a container having a sterile access door, for example, a bag or vial of intravenous solution having a stopper pierceable by a hypodermic injection needle.

The route of administration of the composition is according to known methods, for example, oral, injection or infusion by intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial or intralesional routes, or by delivery systems. sustained or an implantation device that may optionally involve the use of a catheter. Where desired, the compositions can be administered continuously by infusion, injection of a bolus or by an implantation device. Alternatively or additionally, the composition can be administered locally via implantation in the affected area of a membrane, sponge or other appropriate material on which the DKR polypeptide has been absorbed. Where an implantation device is used, the device can be implanted in any suitable tissue or organ, and the release of the DKR polypeptide can be directly through the device via a bolus, or via continuous administration, or via a catheter using continuous infusion . The DKR polypeptide can be administered in a sustained release formulation or formulation. Suitable examples of sustained-release preparations include polymeric and impermeable matrices in the form of formed articles, for example, films or microcapsules.

Sustained-release matrices include polyesters, hydrogels, polylactides (U.S. 3,773,919, EP 58,881), L-glutamic acid copolymers and ethyl-L-glutamate range (Sidman eta al, Biopolimers, 22: 547-556 [1983]), poly (2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed.

Res., 15: 167-277 [1981] and Langer, Chem. Tech., 12: 98-105

[1982]), ethylene vinyl acetate (Langer et al., Supra) or poly-D (poly-D) (-) -3-hydroxybutyric acid (Ep 133,988). The release compositions may also include liposomes which may be prepared by any of the various methods known in the art (e.g., Eppstein et al., Proc Nati Acad Sci USA, 82: 3688-3692 [1985] EP 36,676, EP 88,046, EP 143,949). In some cases, it may be desirable to use DKR polypeptide compositions in an ex vivo form. Here, cells, tissues or organs that have been removed from the patient are exposed to DKR polypeptide compositions after which the cells, tissues and / or organs are subsequently implanted back into the patient. In other cases, the DKR polypeptide can be released through the patient's implant of certain cells that have been genetically engineered, using methods such as those described herein, to express and secrete the polypeptides, fragments, variants or derivatives thereof. Such cells can be animal or human cells and can be derived from the patient's own tissue or from another source, be it human or non-human. Optionally, the cells can be immortalized. However, to decrease the possibility of an immune response, it is preferred that the cells are encapsulated to avoid infiltration of the surrounding tissues. The encapsulation materials are typically semi-permeable, biocompatible, polymeric membranes or membranes, which allow the release of the protein product but prevent the destruction of the cells by the patient's immune system or by other damaging factors of the surrounding tissues. The methods used for the encapsulation of cells by means of a membrane are familiar to those skilled in the art, and the preparation of encapsulated cells and their implantation in patients can be achieved without undue experimentation. See, for example, U.S. Nos. 4,892,538; 5,011,472; and 5,106,627 _._ A system for encapsulating living cells is described in PCT WO 91/10425 (Aebischer et al.). Techniques for formulating a variety of other sustained or controlled release media, such as liposome carriers, particles or bioerodible beads, they are also known to those skilled in the art and are described, for example, in U.S. Pat. No. 5,653,975 (Baetge et al., CytoTherapeutics, Inc.). The cells, with or without encapsulation, can be implanted in suitable tissues or organs of the patient. As discussed above, it may be desirable to treat populations of isolated cells such as proliferating cells, lymphocytes, red blood cells, chondrocytes, neurons, and the like with one or more polypeptides.

DKR, variants, derivatives and / or fragments thereof. This can be done by exposing the isolated cells to the polypeptide, variant, derivative or fragment thereof directly, where it is in a form that is permeable to the cell membrane. Alternatively, genetic therapy can be employed as described below. One way in which genetic therapy can be applied to the DKR gene (either genomic DNA or cDNA and / or synthetic DNA encoding a DKR polypeptide, or a fragment, or variant, or derivative thereof), which can be operably delivered to a constitutive or inducible promoter to form a "DNA construct for gene therapy". The promoter can be homologous or heterologous to the endogenous DKR gene, provided that it is active in the cell or tissue type in which the construct will be inserted. Optionally, as required for other components of the DNA construct for gene therapy, DNA molecules designed for integration to a specific site (eg, endogenous flanking sequences useful for homologous recombination), a tissue-specific promoter, amplifier or silencer, DNA molecules capable of providing a selective advantage over the stem cell, DNA molecules, useful as tags to identify transformed cells, negative selection systems, cell-specific binding agents (such as, for example, cell targets) of internalization specific to the cell, and transcription factors to increase the expression by a vector as well as factors to allow the manufacture of the vector. This DNA construct for gene therapy can then be introduced into the patient's cells (either ex vivo or in vivo). The means for introducing a genetic therapy DNA construct is via viral vectors. Suitable viral vectors typically used in gene therapy to deliver DNA constructs for gene therapy, include without limitation, adenoviruses, adeno-associated viruses, herpes simplex virus, lentivirus, papilloma virus and retroviral vectors. Some of these vectors, such as retroviral vectors, will release the DNA construct for genetic therapy to the chromosomal DNA of patient cells, and the DNA construct for gene therapy can be integrated into the chromosomal DNA; other vectors will function as episomes and the DNA construct for gene therapy will remain in the cytoplasm. The use of vectors for gene therapy is described, for example, in U.S. Pat. Nos. 5,672,344 (September 30, 1997; Kelly et al., University of Michigan), 5,399,246 (March 21, 1995, Anderson et al., Department of Health and Humanitarian Services of the United States), 5,631,236 (May 20, 1997; Woo et al., College Baylor of Medicine), and ,635,399 (June 3, 1997; Kriegler et al., Chiron Corp.) Alternative means to deliver constructs of DNA for gene therapy to cells of a patient without the use of viral vectors include, without limitation, liposome-mediated transfer, direct injection of pure DNA, receptor-mediated transfer (ligand-DNA complex), electroporation, calcium phosphate precipitation and bombardment of microparticles (for example, "genetic cannon"). See U.S. Pat. Nos. 4,970,154 (November 13, 1990; Chang, Baylor College of Medicine), Wo 96/40958 (December 19, 1996; Smith et al., Baylor College of Medicine), 5,679,559 (October 21, 1997; Kim et al. ., University of Utah) 5,676,954 (October 14, 1997; Brigham, Vanderbilt University) and, 5,593,875 January 14, 1997; Wurm et al., Genetech). Other means for increasing the expression of endogenous DKR polypeptide in a cell via gene therapy is inserted one or more amplifying elements into the DKR polypeptide promoter where the amplifying element can serve to increase the transcriptional activity of the DKR polypeptide gene. The amplifier element used will be selected based on the fabric in which the | you want to activate the gene; the amplifying elements that are knows confer activation of the promoter in that tissue will be selected. For example, if a DKR polypeptide is going to be "activated" in T cells, the amplifier element of the lck promoter can be used. Here, the functional portion of the transcriptional element to be added may be inserted into a DNA fragment containing the promoter > of the DKR polypeptide (and optionally, a vector, a 5 'and / or 3' flanking sequence, etc.) using standard cloning techniques. This construct, known as a "homologous recombination constrict" can then be introduced into the desired cells either ex vivo or in vivo. Genetic therapy can be used to decrease the expression of the DKR polypeptide by modifying "the nucleotide sequence of the endogenous promoters." Such modification is typically achieved via methods of homologous recombination. For example, a DNA molecule containing all or a portion of the promoter of the DKR genes selected for inactivation can be modified to remove and / or replace pieces of the promoter that regulate transcription. Here, the TATA box and / or the binding site of a promoter transcriptional promoter can be deleted using standard molecular biology techniques; such suppression can inhibit the activity of the promoter, thereby oppressing the transcription of the corresponding DKR gene. The deletion of the TATA box or the binding site of the transcription activator in the promoter can be achieved by generating a DNA construct comprising all or a relevant portion of the DKR polypeptide promoters (from the same or related species as the genes from DKR to be regulated) in which one or more of the nucleotides of the TATA box and / or transcriptional activator site are mutated via substitution, deletion and / or insertion of one or more nucleotides so that the TATA box and / or Activator site has less activity or becomes completely inactive. This construct, which will typically also contain at least 500 DNA bases corresponding to the 5 'and 3' (endogenous) flanking regions native to the promoter segment that has been modified, can be introduced into the appropriate cells (either ex vivo or in vivo) either directly or via a viral vector as described above. Typically, the integration of the genomic DNA construct of the cells will be via homologous recombination, where the 5 'and 3' flanking DNA sequences in the promoter construct can serve to help integrate the modified promoter region via hybridization to the endogenous chromosomal DNA . Other methods of gene therapy may also be employed where it is desirable to inhibit one or more DKR polypeptides. For example, molecular of antisense DNA or RNA may be introduced which have a sequence that is complementary to at least a portion of the selected DKR polypeptide genes in the cell. Typically, each such antisense molecule will be complementary to the start site (5 'end) of each selected DKR gene. When the antisense molecule is then hybridized to the mRNA of the corresponding DKR polypeptides, translation of the mRNA is prevented. Alternatively, genetic therapy can be employed to create a dominant negative inhibitor of one or more of the DKR polypeptides. In this situation, the DNA encoding a full length or truncated mutant polypeptide of each selected DKR polypeptide can be prepared and introduced into the cells of a patient using viral or non-viral methods as described above. Each such mutant is typically designed to compete with the endogenous polypeptide in its biological role.

The cell line samples of E coli GM121 and GM94 have been deposited with the American Type Culture Collection, 10801 University Blvd., Manassas, VA, United States United States on September 22, 1998 as access numbers 202173 and 202174, respectively. The following examples are intended to be for illustrative purposes only, and should not be construed as limiting the scope of the invention in any way.

EXAMPLES Example 1: Cloning of the Mouse DKR-3 Gene Approximately 120 adult mice with an average body weight of about 18 grams were each injected intraperitoneally with a solution of kainate (prepared as a standard solution of about 1 mg / ml of kainate in Sterile PBS) at a dose of approximately 25 mg of kainatee per kilogram of body weight. Approximately six hours after the injection, the mice were sacrificed, and the hippocampus was dissected from each mouse. Total RNA was extracted from hippocampal tissue using the Trizol method (Gibco BRL, Gran Island, NY). The fraction of poly (A +) mRNA was isolated from the total RNA using Message Maker (Gibco BRL, Gran Island, NY) according to the procedure recommended by the manufacturer. Hippocampal tissue was also obtained from control mice (which received an injection of PBS only), and poly (A +) was obtained from this tissue also using the same procedure. Two primed cDNA libraries were prepared • randomly; one of the poly (A +) mRNA treated with kainate and one of the control using the Superscript® plasmid system (Gibco BRL; Gaithersburg, MD). 'A random cDNA primer containing an internal NotI restriction site was used to initiate synthesis of the first strand and had the following sequence: • GGAAGGAAAAAAGCGGCCGCAACANNNNT? SINNN (SEQ ID NO: 15) where N is A, G, C or T. 15 Both of the synthesis of the first strand of cDNA and the synthesis of the second strand of cDNA were carried out according to the protocol recommended by the manufacturer. After the synthesis of the second strand, the reaction products > were extracted with phenol: chloroform: isoamyl alcohol (in a volume ratio of 25: 24: 1) followed by ethanol precipitation. The double-stranded cDNA products were achieved using standard ligation procedures up to the following double-stranded oligonucleotide adapter (obtained from Gibco BRL, Gran Island, NY): TCGACCCACGCGGTCCG (SEQ ID NO: 16) GGGTGCGCAGGC (SEQ ID NO: 17) After ligation, the cDNA was digested to completion with Notl, and fractionated by size on a 1 percent agarose gel. CDNA products between about 250 and 800 base pairs were selected and gel purified using Qiagen® gel extraction equipment (Qiagen, Chatsworth, CA). The purified cDNA products were directionally ligated into vector pYY41L (American Type Culture Collection, "ATCC" 1081 University Blvd., Manassas, VA, USA, accession number 209636), which had previously been digested with NotJ and Sali. The ligated AD? C was then introduced into electrocompetent E. coli ElectroMax® DH108 cells (Gibco-BRL, Gran Island,? Y) via standard electroporation techniques. The library was then titrated by a serial dilution of the transformation cell mixture. Approximately one million primary clones were divided into 20 groups (50,000 clones per group) and each group was grown on 245mm x 245 mm square plates containing MR2001 medium (MacConnel Research, San Diego, CA) and approximately 60 ug / ml of carbonylin. After incubation overnight at 37 ° C, the colonies were detached from the plate in approximately 20 ml of SOC (the SOC contains approximately 2 percent Bactotriptone, 0. 5 percent yeast extract, 10 mM sodium chloride, 2.5 mM potassium chloride, and 10 mM magnesium sulfate) and were pelleted by centrifugation at approximately 6000 rpm for approximately 10 minutes. The plasmids were then recovered from the cells using Qiagen® prep maxi columns (Qiagen, Chatsworth, CA) according to the protocol suggested by the manufacturer. Approximately two hundred and fifty thousand clones (50 ug of total plasmids / 10 ug of each group) were used to transform the yeast strain YPH499 (accession number ATCC 90834) and an amylase-based signal trap assay was conducted as follows ( see the co-pending USSN 09 / 026,959 filed on February 20, 1998 for a detailed description of this technique). About 1000 transformants were cultured on a single selection plate containing starch (15 cm in diameter with a medium containing approximately 0.6 percent of yeast nitrogen base, 2 percent glucose, 0.1 percent CAA, 1.0 X drip trp solution, 0.7 percent potato starch, and 1.5 percent agarose). The plates were incubated at about 30 C for 4-5 days until the total development of halos was observed. The colonies in the center of the halo were removed and placed again on a fresh plate to form isolated colonies. The colonies isolated with halos were then detached and arranged in 96-well microtiter plates containing approximately 100 μl of water per well, thereby generating the "solutions of yeast colonies". Approximately ten microliters of each well of each solution of yeast colonies was used as a standard to recover the cDNA fragment from that colony through PCR. Therefore, ninety-six PCR reactions were performed independently using Beads® Ready for PCR (96-well format, Amersham-Pharmacia Biotech, Pistcataway, NJ) and the following oligonucleotides according to the manufacturer's protocol: ACTAGCTCCAGTGATCTC (SEQ ID NO: 18] CGTCATTGTTCTCGTTCC (SEQ ID NO: 19) The PCR was conducted using a Perkin-Elmer 9600 thermal cycler with the following cycle conditions: 94C for 10 minutes followed by 35 cycles of 94C for 30 seconds, 55C for 30 seconds and 72C. for 1 minute, after which a final extension cycle of 72C was conducted for 10 minutes. Most PCR reactions contained a single PCR product. The amplified cDNA products were purified using the Qiagen® PCR purification kit (Qiagen, Chatsworth, CA). These products were sequenced in an automated Applied Biosystems 373A DNA sequencer using the following oligonucleotide primer: GCTATACCAAGCATACAATC (SEQ ID NO: 35) Sequencing reactions were conducted with the Taq dye terminator (Applied Biosystems, Foster City, CA) following the procedures recommended by the manufacturer. Each fragment of the PCR was translated in all six possible ways to identify those fragments that (1) had a potential signal peptide in the same direction as the reporter gene; (2) had a high-current codon above the start site of the putative methionine translation; and (3) they seemed to lack transmembrane domain. A clone that met those criteria, called "ymrs2-00009-c4", was selected for further analysis. This clone contained the 5 'sequence, including a putative signal sequence, but lacked the 3' sequence. To obtain the 3 'sequence of this clone, a 3' RACE reaction was carried out using group 4 of the YmHK2 cDNA library as a standard. This library of YmHK2 was prepared as follows: First the synthesis of the cDNA strand was performed using approximately 2 micrograms of the RNA obtained from the hippocampus of the mice treated with kainate and about 1 ug of primer-adapter Not I having the following sequence: GACTAGTTCTAGATCGCGAGCGGCCGCCCTTTTTTTTTTTTTTT (SEQ ID NO: 42) Both synthesis reactions of the first and second cDNA strands were performed using the Superscript® plasmid system (Gibco BRL, Grand Island, MD). After synthesis of the second strand, the double-stranded cDNA products were ligated into the double-stranded adapters of SEQ ID NOs: 16 and 17. After ligation, the cDNA was completely digested with Not I, and fractionated by size on a 0.8 percent agarose gel. The AD? C products of more than about 800 base pairs were selected and purified from the gel using the Qiagen® gel extraction equipment (Qiagen, Chatsworth, CA). The purified AD? C products were ligated directly into the pSport® vector digested with Sal I and Not I (Gibco BRL, Grand Island,? Y). The bound AD? C products were then introduced into cells of. Electrocompetent E. coli called ElectroMax® DH10B (Gibco BRL, Grand Island, NY).

Then the library was titled. Approximately twelve million primary clones were obtained, and expanded into approximately 250 ml of LB containing 100 ug / ml of ampicillin. After incubation overnight at 37C, the plasmids were recovered using the Qiagen® maxi-prep kit (Qiagen, Chatsworth, CA). Approximately 20 ng of the plasmid library was used to transform ElectroMax® DH10B electrocompetent E. coli cells using standard electroporation techniques. About two million transformants were divided into 40 groups (containing approximately 50,000 plasmids / group). Each group was then expanded into approximately 3 ml of LB medium containing approximately 100 ug / ml of ampicillin. After incubation overnight at 37C, the plasmids were recovered using the Qiagen® mini-prep kit. The DNA of each group was then stored at approximately 20C for future use. The RACE 3 'reaction was performed using approximately 1.5 ng of group # 4 from the YmHK2 library as standard, and using the Advantage® cDNA PCR kit.

(Clontech, Palo Alto, CA) with the following oligonucleotides: CCAGCTGCTCTGTGGCAGCCCAG (SEQ ID NO: 20) CCCAGTCACGACGTTGTAAAACGACGGCC SEQ ID N0: 21) The reaction was conducted in a standard thermal cycler (Perkin-Elmer 9600) for thirty-five cycles under the following conditions: 94 C for 1 minute; 94 C for 5 seconds, and 72 C for 5 minutes. This was followed by a final extension at 72C for 10 minutes. Approximately 1 microliter of the reaction products was diluted to 50 ul using TE buffer (10 mM TRIS at pH 8.0 and 1 mM EDTA). To enrich the RACE reaction for the gene of interest, nested PCR reactions were conducted using approximately five microliters of the TE solution (containing the RACE reaction products according to that described in the preceding paragraph) together with the following oligonucleotides: AACATGCAGCGGCTCGGGGG (SEQ ID NO: 22) GGTGACACTATAGAAGAGCTATGACGTCGC (SEQ ID NO: 23) The nested PCR reaction was incubated in a thermal cycler (Perkin-Elmer 9600) using the following protocol: 94 C for one minute; five cycles of 94 C for 5 seconds followed by 72 C for 5 minutes; five cycles of 94C for 5 seconds, followed by 70C for 5 minutes; and 20-25 cycles of 94C for 5 minutes followed by 68C for 5 minutes. After this PCR, the products RACE 3 'and the nested PCR products were analyzed using standard agarose gel electrophoresis. A PCR product of approximately 3.3 kb of the nested PCR reaction was identified. This fragment was purified using the Qiagen® Gel Extraction Kit (Qiagen, Chatsworth, CA) and ligated into the pCRII-TOPO vector (Invitrogen, Carlsbad, CA) according to the procedures recommended by the manufacturer. After ligation, the products were transformed into E. coli One Shot® cells (Invitrogen, Carlsbad, CA) and cultured on an LB plate (Luria broth) containing approximately 100 ug / ml ampicillin and approximately 1.6 mg of X-gal. After incubation overnight at 37C, 12 white colonies and one blue colony were selected and selected using Beads® Ready for PCR (Amersham-Pharmacia Biotech, Pistcataway, NJ) according to the protocol recommended by the manufacturer using the oligonucleotide. of SEQ ID NO: 20 together with the following primer: GTGCTGAGTGTCTTCCATCAGC [SEQ ID NO: 24) Two colonies were separated that produced PCR products of the expected size of approximately 192 base pairs. These colonies were inoculated into approximately 3 ml of LB medium containing approximately 100 ug / ml of ampicillin, and incubated at 37C. The cultures were placed on a shaker for approximately 16 hours, and the plasmids were coated using Qiagen® mini-prep colonies (Qiagen, Chatsworth, CA) according to the manufacturer's protocol. The plasmid DNA was then sequenced as described above. The continuous stretch of the DNA of approximately 3366 nucleotides was assembled by combining the sequence of the clone and mrs2-0009-c4 (containing the 5 'sequence) together with the fragment of the 3.3 kb nested PCR containing the 3' sequence. Within this contiguous sequence is an open reading frame of 349 amino acids. The nucleotide sequence of this novel mouse gene, known as DKR-3, is set forth in Figure 3. The putative amino acid sequence, translated from the DNA sequence, is set forth in Figure 8. A BLAST search (QUICK ) from the Genbank database using the amino acid sequence of DKR-3 revealed that this open reading frame has homology with a gene known as mRNA 7-1 similar to the human rig (accession number of Genbank AF034208; see also Ligon et al. al., J NeuroVirology, 4: 217-226 [1998]). DKR-3 also has homology with the clfest4 chicken lens fiber protein gene (accession number of Genbank D26311); the total identity with this protein is approximately 50 percent with the highest homology in the middle part of the protein.

Example 2: Cloning of the Human DKR-3 Gene The DNA of the mouse DKR-3 can be used to search a public EST database for human homologs, resulting in the identification of the following access numbers of the Genbank: AA628979 AA349552 AA633061 AA351624 W61032 T30923 AA683017 AA324686 T08793 T31076 R14945 AA226979 W45085 AA424460 R58671 R58671 R57834 AF034208 These EST sequences were analyzed and assembled to create a putative sequence for human DKR-3. Based on this putative sequence, two oligonucleotides were designed for use in PCR in an attempt to clone the human DKR-3 gene. The sequence of these nucleotides is: GAGATGCAGCGGCTTGGGGCCACCC (SEQ ID NO: 25) GCCTGGTCAGCCCACGCCTAAAG (SEQ ID NO: 26) PCR was performed using the Advantage® cDNA PCR kit (Clontech, Palo Alto, CA) together with Quick-Clone® human fetal brain cDNA (Clontech). The PCR was assembled in a thermal cycler (Perki? -Elmer 9600) under the following conditions of the cycle: 94C for 2 minutes; 94C for 30 seconds, and 72C for 2 minutes. They were conducted for thirty-five cycles after which the samples were treated at 72 ° C for 10 minutes. A single fragment of approximately 1150 base pairs was visible when the PCR products were visualized on a 1 percent agarose gel. This fragment was purified using the Qiagen® Gel Extraction Kit (Qiagen, Chatsworth, CA) and ligated into the pCRII-TOPO vector (Invitrogen, Carlsbad, CA). After ligation, the products were transformed into E. coli One cells Shoot® (Invitrogen, Carlsbad, CA) and were grown on an LB plate containing approximately 100 ug / ml of | ampicillin and approximately 1.6 mg of X-gal. After 5 overnight incubation at 37 ° C, 2 white colonies were separated and approximately 3 ml of LB medium containing approximately 100 μg / ml of ampicillin was inoculated. The cultures were maintained in a shaker at about 37 ° C for about 16 hours. The plasmids were isolated using Qiagen® mini-prep columns (Qiagen, Chatsworth, CA) according to the protocol recommended by the manufacturer, and the inserts were then sequenced using the methods described above. The cloned fragment is 1141 bp in length and contains an open reading frame of 350 amino acids. The nucleotide sequence is as shown in Figure 2, and the putative amino acid sequence, translated from "the DNA sequence, is set forth in Figure 9. This amino acid sequence is approximately 80 percent identical to the gene mouse DKR-3. In addition, human DKR-3 is identical to the protein fragment similar to the human rig described by Lignon et al. , supra between amino acids 157 and 308 of DKR-3. Significantly, the rig-like protein has an amino terminal start corresponding to amino acid 156 of the DKR-3. The rig-like protein does not appear to be a secreted protein, and the carboxy-terminal region of the rig-like protein has no homology to human DKR-3. As for the mouse DKR-3, the human DKR-3 is approximately 54 percent identical to the elfest chicken lens fiber protein. Human DKR-3 appears to be secreted, with a site of cleavage of the signal peptide after either amino acid 20 or 21. Other potential cleavage sites (due to signal peptides or other endogenous processing sites are found after amino acid 16). , 22, 32 and / or 41). There appear to be N-linked glycosylation sites at amino acids 96, 106, 121 and 204, which would become preferred sites for generating substitution mutants. The amino acid sequences of human DKR-3 and mouse DKR-3 differ in amino acids at positions 6, "7, 11, 24, 27, 29, 30, 32, 33, 39, 81, 89, 93, 99, 101, 103, 109, 113, 115, 123, 126, 142, 156, 157, 162, 165, 173, 175, 191, 197, 198, _201, 203, 245, 247, 259, 283, 287, 292, 294, 295, 296, 298, 299, 304, 310, 311, 312, 314 , 315, 329, 330, 334, 335, 336, 339, 340, 341, 342, 343, 345 and 347 (all with respect to the sequence of the human DKR-3), which makes those positions preferable to generate variants of substitution or deletion of human DKR-3. Based on a computer analysis of the amino acid sequence of DKR-3, significant regions of the molecule include the stretch around amino acids 21-145 (a potential alpha helical region and a potential ligated N glycosylation region) such as , for example, amino acids 21-145, 40-145, 40-150, 45-145, and 45-150, and the stretch around amino acids 145-350, such as for example 145-290, 145-300 and 145-350 and the stretch around 300-350 (a second potential helical alpha region), such as for example amino acids 310-350. Such regions would be the appropriate fragments of full-length DKR-3. A Northern blot analysis was conducted to evaluate the specific expression in woven of human DKR-3.

A probe was prepared for use in Northern blot analysis by PCR of human fetal brain cDNA Quick-Clone® (Clont Palo Alto, CA) using the following oligonucleotides: CCTGCTGCTGGCGGCGGCGGTCCCCACGGC (SEQ ID NO: 27 i GCCTGGTCAGCCCACGCCTAAAG (SEQ ID NO: 28) The PCR reaction was conducted in a thermal cycler (Perkin-Elmer 9600). The PCR conditions were: 94C for 2 minutes; 94C for 30 seconds, and 72C for 2 and 1/2 minutes. They were conducted for thirty-five cycles followed by a final extension treatment at 72 ° C for 10 minutes. The PCR products were tested on a 1 percent agarose gel, a gel band of approximately 1100 bp was purified using the Extraction Equipment Qiagen® gel (Qiagen, Chatsworth, CA), was cloned into the pCRII-TOPO vector (Invitrogen, Carlsbad, CA) and sequenced to confirm that the band contained the open reading frame of human DKR-3 minus the 10 amino acids. terminals. Approximately twenty-five nanograms of this probe were denatured by heating at about 100 ° C for about 5 minutes, followed by placing on ice, and then radioactively labeled with alpha-32P-dCTP using the Rediprime® marking equipment.

(Amersham, Arlington Heights, IL) and following the manufacturer's instructions. A human multiple tissue Northern blot (Clont Palo Alto, CA) was purchased and first prehybridized in approximately 5 ml of ClontExpress® hybridization buffer at about 68 C for 30-60 minutes. After prehybridization, the labeled probe was added to the solution and allowed to hybridize for about 60 minutes. After hybridization, the spot was first washed with 2xSSC plus 0.05 percent SDS at room temperature for about 30 minutes, then washed with O.lxSSC plus 0.1 percent SDS at about 65C for about 30 minutes. The stain was briefly dried and then exposed to a Phosphorimager screen (Molecular Dynamics, Sunnyvale, CA). After exposing during the night, the image of the stain was analyzed on a Storm 820 machine (Molecular Dynamics, Sunnyvale, CA) with the Imagequat fe program (Molecular Dynamics, Sunnyvale, CA). 5 The size of the human DKR-3 RNA transcript is approximately 2.6.kb. The results of the Northern blot analysis indicate that the DKR-3 is highly • expressed in the adult heart and brain, although a weak expression is also evident in the placenta, adult lung, skeletal muscle, kidney and pancreas. A second, smaller transcript is evident in the adult pancreas, and could be the result of degradation of the full-length transcript. To evaluate the role of this gene in cancer, analyzed a variety of human cancer cell lines to determine the presence or absence of the DKR-3 RNA transcript. . Glioblastoma cell lines Hs 683; A 172; SNB-19; U-87MG; and U-373MG are all from the ATCC, and they were grown in the media recommended by the ATCC. Normal human mammary epithelial cells (NMEC) derived from breast reduction mammoplasties were purchased.

Clonetics Corp. (San Diego, CA) and the Corriel Institute (Camden, N.J.). The breast epithelial cell line immortalized MCF-10 and cell line ER + MCF-7 can be obtained from the American Type Culture Collection. The cells ER + BT20T were provided by Dr. K. Keyomarsi (Department of State Health of N.Y.). 184A1 breast cancer cells immortalized and others including T47-D, ZR75-1, and BT474, MDA-MB-157, MDA-MB-231, MDA-MB-361, MDA-MB-453, MD-MBA-468, HS578T and SKBr3 were all obtained from the American Type Culture Collection, (10801 University Blvd., Manassas, VA). NMEC, 184A1 and MCF10 cells were cultured in a modified DME / F12 medium (Gibco / BRL, Grand Island, NY) supplemented with 10 mM Hepes, 2 mM glutamine, 0.1 mM non-essential amino acids, 0.5 mM ethanolamine, 5 mg / ml transferrin. , 1 mg / ml Bovine serum albumin, 5.0 ng / ml sodium selenite, 20 ng / ml triiodothyronine, 10 ng / ml EGF, 5 μg / ml insulin and 0.5 μg / ml hydrocortisone (DMEM / F12C) ( Ethier et al., Cancer Letters, 74: 189-195 [1993]). ER + breast cancer ER + cells were cultured in Alpha or Richter enhanced minimum essential medium (MEM) (Gibco / BRL) supplemented with 10 mM Hepes, 2 mM glutamine, 0.1 mM non-essential amino acids, 10 percent fetal serum albumin and 1 μg / ml of insulin. Human bronchial and cervical epithelial cells were obtained from Clonetics Corp. ("San Diego, CA.) Normal cervical epithelial cells were grown in KBM2 (Clonetics Corp. San Diego, CA) supplemented with 13 mg / ml pituitary extract. bovine, 0.5 μg / ml hydrocortisone, 2 mg / ml EGF, 0.5 mg / ml epinephrine, 0.1 ng / ml retinoic acid, 5 μg / ml transferrin, 6.5 ng / ml triiodothyronine and 5 μg / ml Insulin: Normal bronchial epithelial cells were grown in BEBM (Clonetics Corp. San Diego, CA) supplemented with 0.5 mg / ml hydrocortisone, 0.5 ng / ml EGF, 0.5 μg / ml epinephrine, 10 μg / ml transferrin, 5 μg / ml insulin, 0.1 ng / ml retinoic acid and 5.5 ng / ml triiodothyronine, lung cancer cell lines H1299, H23, H358, H441, H460, H520, H522, H727, H146, H209 , H446, H510A, H526, and H889 and cervical cancer cells Caski, C-5-I, MS751, SiHa and C-33-A were all obtained from the American Type Cul Ture Collection Lung cancer cells were cultured in RPMI (MEM) (Gibco / BRL) supplemented with 10 mM Hepes, 2 mM glutamine, 0.1 mM non-essential amino acids and 10 percent fetal serum albumin (FBS). The cervical cancer cells were cultured in Eares MEM supplemented with 0.1 mM non-essential amino acids, 1 mM sodium pyruvate and 10 percent FBS. All cells were routinely selected for contamination by mycoplasmas and maintained at approximately 37 ° C in an atmosphere of approximately 6.5 percent C02. Total RNA was prepared using monolayers of cells in guanidinium isothiocyanate and centrifuged on a 5.7 M CsCl cushion as described above (Gudas, Proc. Nati. Acad. Sci USA, 85: 4705-4709

[1988]). The RNA (approximately 20 ug) was subjected to electrophoresis on denaturing formaldehyde gels, transferred to MagnaNT membranes (Micron Separatoins, Inc., Westboro, MA) and cross-linked with UV irradiation. The spots were prehybridized, probed, and washed under the same conditions as previously exposed for the tissue stain. The spots were briefly dried and then exposed to a Phosphorimager screen (Molecular Dynamics, Sunnyvale, CA). After exposing during the night, the image of the spot was analyzed on a Storm 820 machine with the Imagequat program both from (Molecular Dynamics). The results are shown in Figures 15A-15D. As can be seen in Figure 15A, expression of DKR-3 decreased in most of the breast cancer cell lines compared to normal cell lines. Figure 15B indicates that the expression of DKR-3 decreased in the lines of non-small cell lung cancer cells, and in most small cell lung cancer cell lines as well. Figure 15C indicates that the expression of DKR-3 diminished in the three glioblastoma cell lines (SNB-19, U-87MG, and U-373MG) and that they are capable of forming tumors in nude mice (the other two cell lines, Hs 683 and A 172 do not form tumors in nude mice). Figure 15D indicates that the expression of DKR-3 was reduced in cervical cancer cell lines compared to normal and immortalized cells.

Example 3: Gene Cloning DKR-1 Human cDNA sequences and amino acid DKR-3 human and mouse to search the Genbank using the BLAST program in an attempt to identify related DKR-3 genes were used. A number of ESTs (expressed sequence tags) were found and analyzed to determine if the sequences overlapped. Using the following human EST accesses, a novel gene, called DKR-1, was predicted.

AA336797 AA043027 R27865 W39690 N94525 HUM517H04B AA143670 AA137219 AA641247 AA115249 AA031969 • AA136192 AA032060 AA035583 AA207078 AA371363 AA037322 AA088618 AA115337 AA693679 W46873 W30750 H83554 A PCR was conducted in an attempt to clone the full-length gene, and the following two oligonucleotides were used for PCR: CCCGGACCCTGACTCTGCAGCCG (SEQ ID NO: 29) GAGGAAAAATAGGCAGTGCAGCACC (SEQ ID NO: 30) PCR was performed using the Advantage® cDNA PCR kit (Clontech, Palo Alto, CA) containing the oligonucleotides listed above and the human placenta cDNA Quick-Clone® (Clontech, Palo Alto, CA). The reaction was conducted according to the manufacturer's recommendations. Thirty-five PCR cycles were conducted in a thermal cycler (Perkin-Elmer 9600) under the following conditions: 94C for 2 minutes; 94C for 30 seconds, and 72C for 1-1 / 2 minutes, followed by a final extension at 72C for 10 minutes. After the cycle, the PCR products were dialyzed on a 1 percent agarose gel. A single band of approximately 1200 base pairs in length was detected after electrophoresis on agarose gel. This fragment was purified using Qiagen® gel extraction equipment (Qiagen, Chatsworth, CA) and ligated into the pCRII-TOPO vector (Invitrogen, Carlsbad, CA) using standard ligation procedures. After ligation, the products were transformed into One Shoot® competent E. coli cells according to the procedures recommended by the manufacturer (Invitrogen, Carlsbad, CA). Transformed E. coli cells were grown on an LB plate containing approximately 100 ug / ml ampicillin and approximately 1.6 mg X-gal. After incubating overnight at about 37 C, two white colonies were removed and inoculated with approximately 3 ml of TB containing 100 ug / ml of ampicillin. The culture was incubated at 37 C for approximately 16 hours, then the plasmids were recovered using Qiagen® mini-prep columns (Qiagen, Chatsworth, CA and were sequenced. Both colonies contained the same insert. The insert is 1193 base pairs, and is known as human DKR-1. The sequence of this gene is shown in Figure 3. This gene contains an open reading frame of 266 amino acids. The amino acid sequence is set forth in Figure 10. A stop codon is present upstream of the first methionine, indicating that the first methionine is probably the amino terminus of the protein. The DKR-1 human has a predicted signal peptide cleavage site of predicted signal peptide between amino acids 19 and 20. The gene has about 80 percent homology to dkk-1 mouse gene (Glinka et al., Supra ), however, the mouse dkk-1 gene is 272 amino acids in length, while the human DKR-1 is 266 amino acids in length. Human DKR-1 differs from mouse dkk-1 in amino acids at positions 3, 4, 5, 7, 8, 10, 12, 13, 14, 15, 16, 17, 18, 19, 22, 23, 24, 29, 53, 55, 62, 66, 69, 77, 93, 98, 101, 105, 106, 123, 139, 140, 143, 144, 153, 155, 157, 158, 163, 164, 165, 169, 175, 178, 197, 224, and 244. In addition, the alignment of human DKR-1 and mouse dkk-1 • shows a vacuum in human DKR-1 between amino acids 37 and 38, and two vacuums between 103 and 104, 146 and 147, and 165 and 166.

Glinka et al. , states on page 362 of his article that "Coordinated depositions of members of the Xenopus dkk family have been deposited at the Genbank with the following access numbers ... hdkk-1 AA207078". Nevertheless, the following three translations of the AA207078 frame by the inventors herein showed no homology to the published Xenopus mouse • dkk-1 sequences, or the human DKR-1 sequence, except at the 3 'end of this access, which exhibits 95 percent identity with the human DKR-1 of amino acids 81-179, indicating - that AA207078 does not code for full-length human dkk-1. Significantly, AA207078 lacks amino acids 1-90 and 180-350 of human DKR-1 including the signal peptide and the. second right domain of cysteine, respectively.

Example: Cloning of the Mouse Gene DKR-2 The accession number of Genbank AA265561 (a mouse sequence) has homology with both the human DKR-1 and the human DKR-3 at the amino acid level based mainly on its cysteine To extend this EST sequence in both 5 'and 3' directions, the following oligonucleotides were designed: GCCACAGTCCCCACCAAGGATCATC (SEQ ID N0: 31) GATGATCCTTGGTGGGGACTGTGGC (SEQ ID NO: 32] CTGCAAACCAGTGCTCCATCAGGG (SEQ ID NO: 33] CCCTGATGGAGCACTGGTTTGCAG (SEQ ID NO: 34) RACE 5 'and RACE 3' reactions were performed separately, according to the manufacturer's protocol using Marathon-Ready® mouse DNA and the Advantage® cDNA PCR kit (both from Clontech, Palo, CA) and using the oligonucleotides from SEQ ID NOs: 31 and 34. The RACE reactions were incubated in a thermal cycler (Perkin-Elmer 9600) using the following cycle conditions: 94C for 1 minute; five cycles of 94C for 5 seconds followed by 72C for 5 minutes; five -cycles of 94C for five seconds, followed by 70C for 5 minutes; and 20-25 cycles of 94C for 5 seconds followed by 68C for 5 minutes. To enrich each RACE reaction in the desired product, about one microliter of each of the RACE PCR products was added, and the mixture was diluted to about 50 ul using TE buffer. Approximately five microliters of this solution was used to drive nested PCR reactions. The Advantage® cDNA PCR kit (Clontech, Palo Alto, CA) and the oligonucleotide of SEQ ID NOs: 32 and 33 were used for the nested 5 'and 3' reactions, respectively. Nested PCR reactions were incubated in a thermal cycler (Perkin-Elmer 9600) using the following program for thirty-five cycles: 94C for 1 minute; 94C for 5 seconds; 72C for 2 minutes. A final extension at 72C was then conducted for 10 minutes. The PCR products were analyzed using a 1 percent agarose gel. Several fragments ranging from about 500 bp to about 1500 base pairs of the 5 'nested PCR reaction were obtained, and two fragments of approximately 1900 bp and 450 bp of the 3' nested PCR reaction were obtained. These PCR products were purified using the Qiagen® PCR purification kit (Qiagen, Chatsworth, CA) and then ligated into the vector PCRII-TOPO (Invitrogen). The ligation products were transformed into E. coli OneShot® cells (Invitrogen, Carlsbad, CA), and the cells were then placed on two plates containing X-gal (one per reaction) as described above. Eight white colonies were detached from each plate and selected by PCR via RACE reactions using the Clontech primer AP2 and the oligonucleotide of SEQ ID NO: 32 (for the 5 'RACE) or the oligonucleotide of SEQ ID NO: 33 ( for RACE 3 '). Three colonies from each plate containing the correct sized fragments were cultured, and the plasmids were isolated and sequenced using the procedures described above. Three clones, 9813302, 981304 and 9813305 had the sequence that extended the EST sequence in the 5 'direction.

One clone, 9813308, had the sequence extending the EST in the 3 direction. In this way a continuous sequence of 2678 base pairs was assembled using the sequences of the clones 9813308, 9813304, and the EST Añ.265561. This full length DNA has been called DKR-2, and the sequence is set forth in Figure 4. The corresponding amino acid sequence is set forth in Figure 11. Within the amino acid sequence is an open reading frame of 259 amino acids . This protein has an identity of about 38 percent with the mouse dkk-1 at the amino acid level. Mouse DKR-2 has a signal peptide predicted with a signal peptide decision site between amino acids 33 and 34.

Example 5: Cloning of the Human DKR-2 Gene We searched the EST Genbank database using the BLAST program with both DNA and amino acid sequences of human DKR-1 and human DKR-3, and identified a human EST, W55979 , which showed homology with both human DKR-1 and human DKR-3 at the amino acid level based on their cysteine pattern. W55979 is approximately 88 percent identical to mouse DKR-2 at the DNA level, approximately 93 percent identical to mouse DKR-2 at the amino acid level. A BLAST search of W55979 at Genbank indicated that W55979 has homology to clone B284B3 (Genbank accession number AC003099). The BAC B284B3 clone has a length of 95129 base pairs. Three portions of W55979 are homologous to three different regions of clone B284B3 indicating that human DKR-2 has at least three exons. A 3 'sequence of 556 bp in length was assembled based on the sequences of both clones B284B3 and W55979 and it was determined that these sequences give the 3"portion of the human ortholog of the mouse DKR-2 Within these 3' sequence of the DKR -2 human is an open reading frame of 174 amino acids, and a stop codon present after amino acid 174. This 3 'sequence of human DKR-2 is approximately 97 percent identical to mouse DKR-2. 5 'end sequence of the mouse DKR-2, a 5' RACE reaction was performed using human mouse DNA from Clontech Marathon-Ready® and the Advantage® cDNA PCR kit, together with the polygonucleotide of SEQ ID NO: 34. The RACE reaction was carried out according to the manufacturer's protocol The products of the 5 'RACE reaction were then subjected to nested PCR to enrich the 5' sequence using the Advantage® cDNA PCR kit and the oligonucleotide of the ESQ ID NO: 32. The conditions The PCR reactions for both of the 5 'RACE reaction and the nested PCR reaction were the same as described in Example 4. The nested PCR products were purified using the Qiagen® PCR purification kit (Qiagen, Chatsworth, CA) and ligated into the Zero-Blunt® vector (Invitrogen, San Diego CA) according to the procedures recommended by the manufacturer. The ligation products were transformed into OneShot® E. coli cells which were then cultured on plates containing X-gal as described above.

After overnight culturing, three white colonies were removed and approximately 3 ml of TB containing approximately 100 ug / ml ampicillin was inoculated. The cultures were allowed to grow for approximately 16 hours, after which the plasmids were isolated using Qiagen® mini-prep columns (Qiagen, Chatsworth, CA) according to the manufacturer's protocol. Next, the sequence of each insert was obtained. One of the 5 'RACE clones, called 9812826, extended the sequence of human DKR-2 5' terminally. A continuous sequence of 1531 bp in length was assembled using this clone 9812826 together with the 3 'sequence of human DKR-2. Within this contiguous sequence is an open reading frame of 259 amino acids. The human DKR-2 gene has a predicted signal peptide of about 33 amino acids, with a predicted cleavage site between amino acids 33 and 34, and is approximately 95 percent identical to mouse DKR-2 at the amino acid level. The positions of the amino acids that differ in human and mouse DKR-2 include (with respect to the numbering of the human sequence) 7, 12, 28, 48, 50, 58, 71, 102, 119, 170, 173, and 191, making those preferable positions to generate substitution or amino acid deletion variants. An alternative spliced isoform of human DKR-2 was discovered when a PCR was conducted using Marathon-Ready® human heart cDNA (Clontech, Palo Alto, CA) and the Advantage® cDNA PCR kit (Clontech, Palo Alto, CA) together with the following oligonucleotides: GGGTTGAGGGAACACAATCTGCAAG (SEQ ID NO.36) GRCTGCAATTGATGATGTTCCTCAATGG (SEQ ID NO: 37) PCR was conducted using the parameters set forth in the manufacturer's protocol. The products of the PCR were analyzed by electrophoresis on agarose gel, and two PCR products were obtained. The bands corresponding to these products were gel purified as described above, amplified and purified as described above, and then sequenced. One product corresponds to full length DKR-2, however, the other band corresponds to 'an isoform of DKR-2. This isoform has an open reading frame of 207 amino acids, and appears to lack an exon. This isoform is known as human DKR-2a. The DNA sequence of human DKR-2a is set forth in Figure 6, and the translated amino acid sequence of DNA is set forth in Figure 13.

Example 6: Cloning of the Human DKR-4 Gene A human EST was identified in the Genbank that showed significant homology with the human DKR-1 and the human DKR-3 at the protein level. This sequence, accession number of Genbank AA565546, has a cysteine pattern that is similar to that of human DKR-1 and human DKR-3. A BLAST search of Genbank showed that human ESTs do not overlap with AA565546. Therefore, it extends the EST sequence in the 5 'direction, a 5' RACE reaction was performed using Marathon-Ready® human heart cDNA (Clontech, Palo Alto, CA) and the PCR equipment for cDNA.

Advantage (Clontech, Palo Alto, CA) the following oligonucleotide: CCAGGGCCACAGTCGCAACGCTGG (SEQ ID NO: 38) The RACE reaction is performed according to the protocol provided with the Advantage® device. After RACE 5 ', the products were stained to enrich the desired 5' sequence using the Advantage cDNA PCR kit according to the manufacturer's recommendations, along with the following oligonucleotide: CTCCCTCTTGTCCCTTCCTGCCTTG (SEQ ID NO: 39) After the nested PCR reaction, the products were purified using the Qiagen® PCR purification product (Qiagen, Chatsworth, CA) ligated into the pCRII-TOPO vector (Invitrogen, Carlsbad, CA), and transformed into E. coli OneShot® cells as described above. After transformation, the cells were grown on an LB plate containing approximately 100 ug / ml ampicillin and approximately 1.6 mg X-gal. After overnight incubation at 37 ° C, four white colonies were removed from the plate and inoculated into approximately 3 ml of TB containing approximately 100 μg / ml ampicillin. The cultures were incubated at about 37 C for about 16 hours. The plasmids were then recovered using Qiagen® mini-prep columns (Qiagen, Chatsworth, CA) and sequenced. It was found that two clones, called 9813563 and 9853564, contained the 5 'sequence of human DKR-4. To obtain the 3 'sequence of human DKR-4, a 3' RACE reaction was performed using Marathon-Ready® human uterine cDNA (Clontech, Palo Alto, CA) together with the Advantage® cDNA for Cloning (Clontech) and The following oligonucleotide: CAAGGCAGGAAGGGACAAGAGGGAG (SEQ ID NO: 40; The RACE 3 'reaction is carried out according to the manufacturer's recommendations. After the RACE reaction, the products were nested using the Advantage® cDNA PCR kit and the following oligonucleotide: CCAGCGTTGCGACTGTGGCCCTGG '(SEQ ID NO: 41) The parameters by PCR were 94C for one minute followed by thirty-five cycles of 94C for 5 seconds and then 72C after which a final extension of 70C was conducted for 10 minutes. After the nesting reaction, the products were analyzed on a 1% agarose gel. A single band of approximately 1200 bp in length was observed. This band was purified from the gel using the methods described above, and then cloned into the vector pCR2.1-TOPO (Invitrogen, Carlsbad, CA) and sequenced. The sequence of this band indicated that it contained the 3 'sequence of human DKR-4, and this sequence was assembled together with the 5' sequence (of clones 9813563 and 9853564) to the full-length sequence generated from human DKR-4. This sequence is set forth in Figure 7 (DNA sequence) and 14 (translated amino acid sequence) the polypeptide is 224 amino acids in length and is approximately 34 percent identical to human DKR-1 at the level of the amino acid sequence.

Example 7: Expression of Human DKR-1 in Bacteria PCR amplification using the primer pairs and the pattern described below were used to generate a recombinant of human DKR-1. A primer from each pair introduces a high TAA codon and a unique BamHI site after the carboxy terminus of the gene. The other primer of each pair introduces a unique N-site, an N-terminal methionine, and codons optimized for the amino terminal portion of the gene. PCR and thermocycling were carried out using the standard recombinant DNA methodology. The PCR products were purified, restriction digested, and inserted into the Ndel and BamHi sites and unique to the pAMG21 vector (accession number ATCC 98113) and transformed into prototype host E. coli GM121 (deposited with the American Type Culture Collection on September 22, 1998 as access number 202174). Other expression vectors and commonly used E. coli host cells are also suitable for expression by one skilled in the art. After transformation, the positive clones were selected and examined to determine the expression of the recombinant gene product. The pAMG21-DKR-human-l-24-266 construct was designed to be 244 amino acids long and to have the following N-terminal and C-terminal residues, respectively: Met-His-Pro-Leu-Leu-Gly (SEQ ID NO: 3) Thr-Cys-Gln-Arg-His (SEQ ID NO: 44) The pattern used for PCR was the human DKR-1 cDNA and the following oligonucleotides were the pair of primers used for PCR and cloning of this genetic construct: GTTCTCCTCATATGCATCCATTATTAGGCGTAAGTGCCACCTTGAACTCGGTTCTCAAT (SEQ ID NO: 45) TACGCACTGGATCCTTAGTGTCTCTGACAAGTGTGAAG (SEQ ID NO: 46) The transformed E. coli GM121 containing pAMG21-DKR-1-human-l-24-266 was grown in 2x YT media containing 20 micrograms / ml kanamycin at 30C until the culture reached an optimum density of approximately 600 nm of approximately 0.5. Induction of DKR-1 protein expression was achieved by the addition of Vü io fischeri synthetic autoinducer at 100 ng / ml final and incubation of the culture at 30 ° C or 37 ° C for approximately 9 hours with further agitation. Furthermore, as an induced control, for each culture no autoinducer was added to an aliquot of the culture, but the culture was also incubated for an additional 9 hours around 30 ° C with agitation along with the induced cultures. After about 9 hours, the optical densities of the cultures were measured at 600 nm, an aliquot of cultures was examined by oil immersion microscopy at an amplification of 1600X, aliquots of cultures were fed by centrifugation. Bacterial procedures of the cultures were processed by electrophoresis on SDS-polyacrylamide gel at examination levels of 14 percent protein gelt produced in crude lysates and for confirmation of the N-terminal sequencing of the recombinant gene product. The gel was stained with Coomassie blue. The results are shown in the photo of Figure 16. Lane 1 contains molecular weight markers; lanes 2 and 5 contain crude lysates of induced control cells incubated at 30 ° C; lanes 3 and 6 are crude lysates of induced cells cultured at 30C; lanes 4 and 7 are crude lysates of induced cells cultured at 37C. The arrow to the left of lane 1 'indicates the location of the human DKR-1-24-266. As can be seen, large quantities of recombinant proteins were observed in the crude leaves of the induced cultures at both 30 ° C and 37 ° C (lanes 3 and 6, and 4 and 7). Microscopic analysis of the bacterial cells revealed that most of the cells contained at least one inclusion body, suggesting that at least some protein can be produced in the insoluble fraction of E. coli.

Example 8: Expression of DKR-2 in Bacteria PCR amplification using the primer pairs and patterns described below were used to generate the different forms of DKR-2. A primer from each pair introduces a high TAA codon and a unique BamHI site after the carboxy terminus of the gene. The other primer of each pair introduces a site. Ndel-, an N-terminal methionine, and codons optimized for the amino terminal portion of the gene. PCR and thermocycling were carried out using the standard recombinant DNA methodology. The PCR products were purified, restriction digested, and inserted into the unique Ndel and BamHi sites of the vector pAMG21 (accession number ATCC 98113) and transformed into host prototype E. coli GM121 or GM94 (the GM94 was deposited with the ATCC on September 22, 1998 as access number 202173). Other expression vectors and commonly used E. coli host cells are also suitable for expression. After the transformation, the positive clones were selected and examined for the expression of the recombinant gene product. The human pAMG21-DKR-2-2-26-259 construct was designed to be 235 amino acids long and have the following N-terminal and C-terminal amino acids, respectively: Met-Ser-Gln-Ile-Gly-Ser ( SEQ ID NO: 47) Val-Cys-Gln-Lys-Ile (SEQ ID NO: 48) The pattern used for PCR was the human DKR-2 cDNA and the following oligonucleotides were the pair of primers used for PCR and cloning of this genetic construct: GTTCTCCTCATATGTCTCAAATTGGTAGTTCTCGTGCCAAACTCAACTCCATCAAG (SEQ ID NO: 49) TACGCACTGGATCCTTAAATTTTCTGACACACATGGAGT (SEQ ID NO: 48) The pAMG21 DKR-2-mouse pattern-26-259 was designed to be 235 amino acids in length and to have the following N-terminal and C-terminal residues, respectively: Met-Set-Gln-Leu-Gly-Ser (SEQ ID NO: 51) Val-Cys-Gln-Lys-Ile (SEQ ID NO: 52) The pattern used for the PCR was the mouse DKR-2 cDNA and the following oligonucleotides were the pair of primers used for PCR and cloning of this genetic construct: GTTCTCCTCATATGTCTCAATTAGGTAGCTCTCGTGCTAAACTCAACTCCATCAAGTCC (SEQ ID NO: 53) TACGCACTGGATCCTTAGATCTTCTGGCATACATGGAGT (SEQ ID NO: 54) Transformed GM121 or GM94 E. coli containing either plasmid pAMG21-DKR-2-human-26-259 or pAMG21-DKR-2-mouse-26-259 were grown in 2x YT media containing 20 μg / ml of anamicin-a at 30 ° C until the culture reached an optical density at 600 nm of about 0.5. The induction of DKR-2 protein expression was achieved by the addition of Vibrio fischeri synthetic autoinducer at 100 ng / ml final and incubation of the culture at 30 C or 37 C for approximately 5 or 9 additional hours with agitation. In addition, as an induced control, for each culture no autoinducer was added to an aliquot of the culture, but the culture was also incubated for approximately 5 or 9 more hours at 30 ° C with shaking together with the induced cultures. After 5 or 9 hours of incubation, the optical densities of the cultures were measured at approximately 600 nm, an aliquot of cultures was examined by oil immersion microscopy at an amplification of 1600X, and the aliquots of the cultures were pelleted by centrifugation . The bacterial sediments of the cultures were processed by electrophoresis-on SDS-polyacrylamide gel on a 14 percent gel to examine protein levels produced in crude lysates and for confirmation of the N-terminal sequencing of the recombinant gene product. The gel was stained with Coomassie blue. The results are shown in Figure 16, lanes 8-10 (human DKR-2 polypeptide) in Figure 17 (mouse DKR-2 polypeptide). In Figure 16, lane 8 contains crude lysate of induced control cells; lane 9 contains crude lysate of induced cells cultured at 30C; and lane 10 contains crude lysate of induced cells cultured at 37C. The arrows to the left of lane 10 indicate the expected location of the DKR-2-human-26-259. As can be seen, significant amounts of polypeptide were generated in the induced cultures and grown at 30 C or 37 C, while the induced cells did not produce a large amount of polypeptide. Figure 17 shows the results of the production ^ of DKR-2-mouse polypeptide-26-259. Lane 1 is that of molecular weight markers. Lanes 2-4 are a clone of E. coli cells transfected with the plasmid DKR-2, while lanes 5-7 are a second clone transfected with the same plasmid. Lanes 2 and 5 are crude lysates of non-induced control cells; The rails 3 and 6 are crude lyses of induced cultivated at 30C; and lanes 4 and 7 are crude lysates of cells cultured at 37C. The arrows to the left of lanes 4 and 7 indicate the expected location of the DKR-2 polypeptide. As can be observed, large amounts of recombinant proteins were observed in the crude lysates of induced cultures. 37C but not at 30C. Microscopic analysis of the bacterial cells revealed that the majority of the cells contained at least one inclusion body, suggesting that at least some of the proteins can be produced in the insoluble fraction of E. coli.

Example 9: Expression of DKR-3 in Bacteria PCR amplification using the primer pairs and the patterns described below were used to generate the different forms of DKR-3. A primer from each pair introduces a high TAA codon and a unique Sacll site after the carboxy terminus of the gene. Other primers of each pair introduce a unique N-site, an N-terminal methionine, and codons optimized for the amino terminal portion of the gene. PCR and thermocycling were carried out using the standard "" recombinant DNA methodology. The PCR products were purified, restriction digested, and inserted into the unique Ndel and Sacll sites of the pAMG21 vector (accession number ATCC 98113) and transformed into the prototrophic E. coli host GM121. Other expression vectors and commonly used E. coli host cells are also suitable for expression by one skilled in the art. After the transformation, positive clones were selected, plasmid DNA was isolated and the insert sequence of the gene was confirmed DKR-3. The pAMG21-DKR-3-human-3-23-350 construct was designed to be 329 amino acids in length and to have the following N-terminal and C-terminal residues, respectively: Met-Pro-Ala-Pro-Thr-Ala (SEQ ID NO: 55) Gly-Gly-Glu-Glu-Ile (SEQ ID NO: 56) The pattern used for PCR was the human DKR-3 cDNA and the following oligonucleotides were the pair of primers used for PCR and cloning of this genetic construct: GTTCTCCTCATATGCCTGCTCCAACTGCAACTTCGGCTCCAGTCAAGCCCGGCC [SEQ ID NO: 57) TACGCACTCCGCGGTTAAATCTCTTCCCCTCCAGCA (SEQ ID NO: 58) The pAMG21-DKR-3-human-33-350 construct was designed to be 319 amino acids in length and to have the following N-terminal and C-terminal residues, respectively: Met-Lys-Pro-Gly-pro-Ala (ESQ ID NO.59) Gly-Gly-Glu-Glu-Ile (SEQ ID NO: 60) The pattern used for PCR was the human DKR-3 cDNA and the following oligonucleotides were the pair of primers used for PCR and cloning of this genetic construct: GTTCTCCTCATATGAAACCAGGTCCAGCCTTAAGCTACCCGCAGGAGGAGGCCA (SEQ ID NO: 61) TACGCACTCCGCGGTTAAATCTCTTCCCCTCCCAGCA (SEQ ID NO: 62; The pAMG21-DKR-3-human-33-350 construct was designed to be 319 amino acids in length and to have the following N-terminal and C-terminal residues, respectively: Met-Gln-Glu-Glu-Ala-Thr (SEQ ID NO: 63) Gly-Gly-Glu-Glu-Ile (SEQ ID NO: 64) The pattern used for PCR was the human DKR-3 cDNA and the following oligonucleotides were the pair of primers used for PCR and cloning. this genetic construct: GTTCTCCTCATATGCAAGAAGAAGCTACTCTGAATGAGATGTTCCGCGAGGTT (SEQ ID NO: 65) TACGCACTCCGCGGTTAAATCTCTTCCCCTCCAGCA (SEQ ID NO: 66) The pAMG21-DKR-3-human-33-349 construct was designed to be 318 amino acids in length and to have the following N-terminal and C-terminal residues, respectively: Met-Glu-pro-Gly-Pro-Ala (SEQ ID NO: 67) Gly-Glu-Glu-Glu-Ile (SEQ ID NO: 68) The pattern used for the PCR was the cDNA_del Human DKR-3 and the following oligonucleotides were the pair of primers used for PCR and cloning of this genetic construct: GTTCTCCTCATATGGAACCAGGTCCAGCTTTAAACTACCCTCAGGAGGAAGCTA (SEQ ID NO: 69) TACGCACTCCGCGGTTAAATCTCCTCCTCTCCGCCTA (SEQ ID NO: 70; Transformed GM121 E. coli containing the different pAMG21 plasmids of DKR-3 described above were grown in 2X YT media containing 20 micrograms / ml kanamycin at 30 ° C until the culture reached an optical density at 600 nm of approximately 0.5 Induction of DKR-3 polypeptide expression was achieved by the addition of synthetic Vibrio fischeri autoinducer at a final concentration of 100 ng / ml and incubation of the culture at 30 or 37C for approximately an additional 6 hours with agitation. In addition, as an uninduced control, an aliquot of the culture was added for each non-autoinducer culture, but the culture was also incubated for an additional 6 hours at 30C with shaking together with the induced cultures. After about 6 hours, the optical densities of the cultures were measured at approximately 600 nm, aliquots of the cultures were examined by oil immersion microscopy at an amplification of 1600X, and aliquots of the cultures were pelleted by centrifugation. The bacterial sediments of the cultures were processed by SDS-polyacrylamide gel electrophoresis to examine the protein levels produced in the crude lysates, or the bacterial pellets were processed to determine if the recombinant protein was in the insoluble or insoluble fraction of the E coli and for the confirmation of the N terminal sequencing of the recombinant gene product. The results are shown as photos in gels SDS in Figures 18 and 19. In Figure 18, Lane 10 is that of the molecular weight markers, and Lanes 1-9 are crude lyses of bacterial cells. Lane 1 is crude lysate of non-induced control cells; Lanes 2, 4, 6 and 8 are crude lysates of induced cells cultured at 30C; Lanes 3, 5, 7 and 9 are induced cells cultured at 37C. Lanes 1-5 contain lysates of cells transfected with the construct pAMG21-DKR-3-human-23-350; and Lanes 6-9 contain lysates of cells transfected with the pAMG21-DKR-3-human-33-350 construct. The arrows to the left of Lane 2 and to the right of Lane 9 indicate the expected location of the DKR-3 polypeptides. Figure 19 contains markers for molecular weight in Lane 10; lanes 1-5 are crude lysates of cultured cells transfected with the pAMG21-DKR-3-human-42-350 construct; lanes 6-9 are crude lysates of cells transfected with mouse construct pAMG21-DKR-3-33-349. Lanes 1 and 6 are non-induced controls; Lanes 2, 4, 7 and 8 are crude lyses of cells induced at 30C (two different clones of each construct); Lanes 3, 5 and 9 are crude lysates of induced cells cultured at 37C (two separate clones of the human DKR-3-42-350 construct in Lanes 3 and 5). The arrow to the right of Lane 9 indicates the expected location of the mouse DKR-3 polypeptides; the arrow to the left of Lane 4 indicates the expected location of the human DKR-3 polypeptide. As can be seen, all the DKR-3 constructs produced large amounts of recombinant protein in E. coli. Inclusion bodies could not be detected by oil immersion microscopy, and the recombinant polypeptides were found mostly in the soluble fraction of the cells.

Example 10: Expression of DKR-4 in Bacteria PCR amplification using the primer pairs and pattern described below was used to generate a recombinant form of human DKR-4. A primer from each pair introduces a high TAA codon and a unique BamHI site after the carboxy terminus of the gene. The other primers of each pair introduce a unique N-site - an N-terminal methionine, and codons optimized for the amino terminal portion of the gene. PCR and thermocycling were carried out using the standard recombinant DNA methodology. The PCR products were purified, restriction digested, and inserted into the unique N eX and BamH1 sites of the pAMG21 vector (accession number ATCC 98113) and transformed into the photomicrophic E. coli host GM94. The other expression vectors and commonly used E. coli host cells are also suitable for expression. After transformation, the positive clones were selected and examined for the expression of the recombinant gene product. The pAMG21-DKLR-4-human-19-224 construct was designed to be 207 amino acids in length and to have the following N terminal and C terminal residues, respectively: Met-Leu-Val-Leu-Asp-Phe (SEQ ID N0: 7i; Lys-Ile-Glu-Lys-Leu (SEQ ID NO: 72) The pattern used for PCR was the human DKR-4 cDNA and the following oligonucleotides were the pair of primers used for PCR and cloning of this genetic construct: GTTCTCCTCATATGTTAGTTTTGGATTTCAACAACATCAGGAGCTCT (SEQ ID NO: 73) TACGCACTGGATCCTTACAGTTTTTCTATTTTTTGGCATACTCTTAATC (SEQ ID NO: 74) It was anticipated that the DKR-4 polypeptide could be prepared using the PCR product as described above for the other DKR polypeptides.

EXAMPLE 11 Production and Purification of the DKR-3 Polypeptide in Mammalian Cells The human DKR-3 cDNA was cloned into the mammalian expression vector pCDNA3.1 (-) mycHis (Invitrogen, Carlsbad, CA) and the vector construct it was amplified using the standard ligation techniques of the Qiagen maxi-prep kit (Qiagen, Chatsworth, CA). 293T human kidney embryonic cells (American Type Culture Collection) were grown on 10 cm discs, and were grown to approximately an 80 percent confluence. Cells were then transfected with the vector construct using the DMRIE-C® liposome formulation (Gibco BRL, Grand Island, NY) as follows. Approximately 240 microliters of DMRIE-C® was added to 8 ml of Optimem medium. Then approximately 40 ul (equivalent to about 56 micrograms) of purified vector construct was added to another 8 ml of Optimem. After mixing and incubating at room temperature for approximately 15 minutes, 2 ml of this solution was added to each of the 8 plates. After about 5 hours, the medium was aspirated and 10 ml of DME medium containing approximately 10 percent fetal sheep serum was added. The cells were incubated 16-18 hours after which the medium was removed and approximately 10 ml of Optimem SF medium were added per well without phenol red. After approximately 24 hours, this medium, the "conditioned medium" was harvested, passed over a 0.22 micron filter and stored at 4 ° C. The cells were then incubated in another 10 ml of SF Optimem per plate. After 24 hours, this medium was collected, filtered and also stored at 4 ° C. The conditioned media was added to a buffer containing 50 mM NaP04, pH8, and 250 mM sodium chloride, and passed over a nickel-Sephadex column (Qiagen, Chatsworth, CA). No unbound proteins were specifically eluted using the same buffer containing 10 mM imidazole, followed by the same buffer containing 20 mM imidazole. The DKR-3 was then eluted using 125 mM-250 mM imidazole. The fractions of the column were subjected to electrophoresis on a gel with 12% SDS and stained with silver. The results are shown in Figure 20. Lane 2 contains material that was loaded onto the gel. Lane 3 contains the flow through the fraction after loading the column with conditioned media, Lanes 4, 5, 6 and 7 contain fractions from the column after treatment with imidazole 10, 20, 125 and 250 mM. The standard molecular weights are shown in Lane 8. As can be seen, there is a single protein band of the correct molecular weight in the Lanes 5 and 6, indicating that this procedure resulted in the generation of purified DKR-3 protein (bound to the myc and His tags). The dispersion of the protein band may be due to glycosylation. Separately, a Western blot test was performed to confirm that the verified protein was in fact His-labeled (indicating that the mycHis fusion protein of DKR-3 was produced). The Western blot was prepared using standard procedures and tested with a polyclonal anti-His-HRP antibody.

(Invitrogen, Carlsbad, CA). A photo of the Western blot is shown in Figure 21; the Lanes correspond to those of the gel (described immediately above). As can be seen, there is antibody binding in Lanes 2, 5 and 6, indicating that the mycHis that was loaded on the column and was eluted in the washes with imidazole 20 and 125 mM.

Example 12: Independent Growth Test of the Walking A distinctive feature of many cancer cell lines is their ability to grow in a manner independent of anchoring. While normal cells will only grow and divide until they come into contact with their neighbors, the. Cancer cells continue to grow and divide after contact, thus forming tumors. In this way, a test of the compounds cancer cell growth inhibitors measures the ability of cancer cells to grow and divide in the presence of the compound. There are many ways known to those skilled in the art in which this assay can be conducted, however the two preferred methods are those that are exposed below.

A. Assay of Stably Transfected Cells i In this procedure, a lineage of human cancer cells that does not express the DKR gene to be tested (either the human DKR-1, 2, 3, 4 or a fragment or variant thereof) is transfected with the DKR gene under evaluation, where the gene DKR is inserted into a vector such as pcDNA3.1 (Invitrogen, Carlsbad, CA) or other suitable mammalian expression vector. Transfection can be conducted as describes here. Transfected cancer cells are cultured to generate a stably transfected cell line. Once a stably transfected cell line has been established, the cells are added to Noble agar or equivalent (approximately 0.35 percent) prepared in culture medium of standard mammalian cells such as RPMI. The cell / agar solution is poured onto petri dishes containing solidified agar (approximately 0.5 percent agar).

Colony formation is evaluated daily to determine the growth rate of the cells, and culture medium is added to each plate as necessary. Separately, the same cells are transfected with vector alone (which does not contain DKR gene). Those "control" cells They are then treated in an identical way with the cells that contain the DKR gene and can be used as a comparison standard for the cells that contain the gene DKR Examples of cancer cell lines suitable for conducting this assay include, without limitation, the human breast cancer cell line MCF7 and the glioblastoma cell line U-87MG.

B. Protein Assay An alternative or additional assay for measuring the growth of cancer cell lines treated with a DKR polypeptide is as follows. A human cancer cell line that does not express the DKR polypeptide under evaluation can be cultured and prepared with an agar solution as described above. The cells can then be cultured as described, and a solution of DKR polypeptide (either full-length, or a fragment or variant thereof) can be added in culture medium to the agar daily, or every other day, or once per day. week for three weeks. Typically, a concentration of approximately 10 nM will be added, although a series of dilutions ranging from 1 M to 1 mM can be used. The control plates will receive a solution of culture medium only. The plates can be checked daily for up to about three weeks to evaluate the formation of cell colonies. After three weeks, the control and experimental plates can be compared to determine the number and size of cell colonies. It was anticipated that those plaques that harbor DKR polypeptide that is biologically active will have fewer cell colonies, and the colonies will be smaller, compared to the control plaques.

Example 13: Jn Live Tumor Test The ability of each DKR polypeptide to inhibit tumor growth in vivo can be evaluated as follows.

Tumor cells which do not express the DKR gene under equalization can be transfected using the methods described herein with a DKR nucleic acid construct encoding a full-length DKR gene, or a fragment or variant thereof. The transfected cells can be kept in culture (as described here) until ready to be used. Nude male and female nude mice (Charles River Labs, Boston MA) were kept in a sterile environment. The mice were then injected with approximately 2 x 10 6 cells (cells transfected with DKR or cells "transfected with" vector alone ", control) in a total volume of approximately 0.1 ml, which can be injected subcutaneously. The mice can then be examined daily to determine the appearance of (a) tumors and to determine the size of the tumor. Preferably, the mice will be examined for up to about six months to allow time for tumor growth (and regression where the DKR polypeptides are effective in shrinking the tumor). Tumors, where present, can then be removed, weighed and examined to (1) determine the presence of DKR polypeptide, and (2) determine the morphology.

Tumors of mice containing cells transfected with the DKR construct can be compared to the tumors of mice containing cells transfected with vector alone. It was anticipated that DKR polypeptides, due to their similarity to dkk-1, a potent wntd antagonist, will be able to decrease tumor size compared to controls.

LIST OF SEQUENCES < 110 > Amgen Inc. < 120 > DKRiNOVEDOSOS POLIPEPTIDES < 130 > A-548 < 140 > 09 / 161,241 < 141 > 1998-09-25 < 160 > 78 < 170 > Patentln Ver. 2.0 < 210 > 1 < 211 > 1050 < 212 > AD < 213 > Ratór. < 400 > 1 atgcagcggc tcgggggtat tttgctgtgt acactgctgg cggcggcggt ccccactgct 60 cctgctcctt ccccgacggt cacttggact ccggcggagc cgggcccagc tctcaactac 120 cctcaggagg aagctacgct caatgagatg tttcgagagg tggaggagct gatggaagac 180 actcagcaca aactgcgcag tgccgtggag gagatggagg cggaagaagc agctgctaaa 240 acgtcctctg aggtgaacct ggcaagctta cctcccaact atcacaatga gaccagcacg 300 gagaccaggg tgggaaataa cacagtccat gtgcaccagg aagttcacaa gataaccaac 360 aaccagagtg gacaggtggt cttttctgag acagtcatta catctgtagg ggatgaagaa 420 ggcaagagga gccatgaatg tatcattgat gaagactgtg ggcccaccag gtactgccag 480 ttctccagct tcaagtacac ctgccagcca tgccgggacc agcagatgct atgcacccga 540 gacagtgagt gctgtggaga ccagctgtgt gcctggggtc actgcaccca aaaggccacc 600 aaaggtggca atgggaccat ctgtgacaac cagagggatt gccagcctgg cctgtgttgt 660 gccttccaaa gaggcctgct gttccccgtg tgcacacccc tgcccgtgga gggagagctc 720 tgccatgacc ccaccagcca gctgctggat ctcatcacct gcctgaagga gggaactgga 780 gctttggacc gatgcccctg cgccagtggc ctcctatgcc agccacacag ccacagtctg 840 gtgtacatgt gcaagcca gc cttcgtgggc agccatgacc acagtgagga gagccagctg 900 cccagggagg ccccggatga gtacgaagat gttggcttca taggggaagt gcgccaggag 960 ctggaagacc tggagcggag cctagcccag gagatggcat ttgaggggcc tgcccctgtg 1020 gagtcactag gcggagagga ggagatttag 1050 < 210 > 2 < 211 > 1053 < 212 > DNA < 213 > Human < 400 > 2 atgcagcggc ttggggccac cctgctgtgc ctgctgctgg cggcggcggt ccccacggcc 60 cccgcgcccg ctccgacggc gacctcggct ccagtcaagc ccggcccggc tctcagctac 120 ccgcaggagg aggccaccct caatgagatg ttccgcgagg ttgaggaact gatggaggac 180 acgcagcaca aattgcgcag cgcggtggaa gagatggagg cagaagaagc tgctgctaaa 240 gcatcatcag aagtgaacct ggcaaactta cctcccagct gaccaacaca atcacaatga 300 gacacgaagg ttggaaataa taccatccat gtgcaccgag aaattcacaa gataaccaac 360 aaccagactg gacaaatggt cttr.tcagag acagttatca catctgtggg agacgaagaa 420 ggcagaagga gccacgagtg catcatcgac gaggactgtg ggcccagcat gtactgccag 480 tttgccagct tccagtacac ctgccagcca tgccggggcc agaggatgct ctgcacccgg 540 gacagtgagt gctgtggaga ccagctgtgt gtctggggtc actgcaccaa aatggccacc 600 aggggcagca atgggaccat ctgtgacaac cagagggact gccagccggg gctgtgctgt 660 gccttccaga gaggcctgct gttccctgtg tgcacacccc tgcccgtgga gggcgagctt 720 tgccatgacc ccgccagccg gcttctggac ctcatcacct gggagctaga gcctgatgga 780 gccttggacc gatgcccttg tgccagtggc ctcctctgcc agccccacag ccacagcctg 840 gtgtatgtgt gcaagccgac cttcgtgggg agccgtgacc aagatgggga gatcctgctg 900 cccagagagg tccccgatga gtatgaagtt ggcagcttca tggaggaggt gcgccaggag 960 ctggaggacc tggagaggag cctgactgaa gagatggcgc tgggggagcc tgcggctgcc 1020 gccgctgcac tgctgggagg ggaagagatt tag 1053 < 210 > 3 < 211 > 801 < 212 > DNA < 213 > Human < 400 > 3 atgatggctc tgggcgcagc gggagctacc cgggtctttg tcgcgatggt agcggcggct 60 ctcggcggcc accctctgct gggagtgagc gccaccttga actcggttct caattccaac 120 gctatcaaga acctgccccc accgctgggc ggcgctgcgg ggcacccagg ctctgcagtc 180 agcgccgcgc cgggaatcct gtacccgggc gggaataagt accagaccat tgacaactac 240 cagccgtacc cgtgcgcaga ggacgaggag tgcggcactg atgagtactg cgctagtccc 300 acccgcggag gggacgcggg cgtgcaaatc tgtctcgcct gcaggaagcg ccgaaaacgc 360 tgcatgcgtc acgctatgtg ctgccccggg aattactgca aaaatggaat atgtgtgtct 420 tctgatcaaa atcatttccg aggagaaatt gaggaaacca tcactgaaag ctttggtaat 480 gateatagca ccttggatgg gtattccaga agaaccacct tgtcttcaaa aatgtatcac 540 accaaaggac aagaaggttc tgtttgtctc cggtcatcag actgtgcctc aggattgtgt 600 tgtgctagac acttctggtc caagatctgt aaacctgtcc tgaaagaagg tcaagtgtgt 660 accaageata ggagaaaagg ctctcatgga ctagaaatat tccagcgttg ttactgtgga 720 gaaggtctgt cttgccggat acagaaagat caccatcaag ecagtaatte ttctaggctt 780 801 agagacacta cacacttgtc < 210 > 4 < 211 > 780 < 212 > DNA < 213 > Mouse < 400 > 4 atggccgcgc tgatgcgggt caa ^ gattea tcccgctgcc ttctcctact ggccgcggtg 60 ctgatggtgg agagetcaca gctaggcagc tcgcgggcca aactcaactc catcaagtcc 120 tctctaggag gggagactcc tgctcagtca gccaaccgat ctgcaggcat gaaccaagga 180 ctggctttcg gcggcagtaa gaagggcaaa agcctggggc aggcctaccc ttgcagcagt 240 gataaggaat gtgaagttgg aagatactgc cacagtcccc accaaggatc atcagcctgc 300 ggaggaaaaa atgctctgta gaaacgatgc cacagagatg ggatgtgttg ccctggtacc 360 cgctgcaata atggaatctg catcccagtc actgagagca tcctcacccc acatatccca 420 gctctggatg gcacccggca tagagatege aaccatggtc actattecaa ccatgacctg 480 ggatggcaga atctaggaag gccacactcc aagatgeetc atataaaagg acatgaagga 540 gacccatgcc tacggtcatc agactgeatt gatgggtttt gttgtgctcg ccacttctgg 600 accaaaatct gcaaaccagt gctccatcag ggggaagtct gtaccaaaca acgcaagaag 660 ggttcgcacg ggctggagat tttccagagg tgtgactgtg caaagggcct gtcctgcaaa 720 atgccaccta gtgtggaaag ctcttccaaa atgtatgcca gccagactcc gaagatctga 780 < 210 > 5 < 211 > 780- < 212 > DNA < 213 > Human < 400 > 5 atggccgcgt tgatgcggag caaggattcg tcctgctgcc tgctcctact ggccgcggtg 60 ctgatggtgg agagetcaca gatcggcagt tcgcgggcca aactcaactc catcaagtcc 120 tctctgggcg gggagacgcc tggtcaggcc gccaatcgat ctgcgggcat gtaccaagga 180 ctggcattcg gcggcagtaa gaagggcaaa aacctggggc aggcctaccc ttgtagcagt 240 gataaggagt gtgaagttgg gaggtattgc cacagtcccc accaaggatc atcggcctgc 300 atggtgtgtc ggagaaaaaa gaagcgctgc cacegagatg gcatgtgctg ccccagtacc 360 cgctgcaata atggcatctg tateccagtt actgaaagca tcttaacccc tcacatcccg 420 gctctggatg gtactcggca cagagatega aaccacggtc attactcaaa ccatgacttg 480 ggatggcaga atctaggaag accacacact aagatgtcac atataaaagg gcatgaagga 540 gacccctgcc taegatcate agactgeatt gaagggtttt gctgtgctcg tcatttctgg 600 accaaaatct gcaaaccagt gctccatcag ggggaagtct gtaccaaaca acgcaagaag 660 ggttctcatg ggctggaaat tttccagcgt tgcgactgtg cgaagggcct gtcttgcaaa 720 atgccaccta gtatggaaag ctcctccaaa atgtgtgtca gccagactcc gaaaatttga 780 < 210 > 6 < 211 > 624 < 212 > DNA < 213 > Human < 400 > 6 atggccgcgt tgatgcggag caaggattcg tcctgctgcc tgctcctact ggccgcggtg 60 ctgatggtgg agagetcaca gatcggcagt tcgcgggcca aactcaactc catcaagtcc 120 tctctgggcg gggagacgcc tggtcaggcc gccaatcgat ctgcgggcat gtaccaagga 180 ctggcattcg gcggcagtaa gaagggcaaa aacctggggc aggcctaccc ttgtagcagt 240 gataaggagt gtgaagttgg gaggtattgc cacagtcccc accaaggatc atcggcctgc 300 atggtgtgtc ggagaaaaaa gaagcgctgc caccgagatg gcatgtgctg ccccagtacc 360 atgggcatga cgctgcaata aggagacccc tgcctacgat catcagactg cattgaaggg 420 ttttgctgtg ctcgtcattt ctggaccaaa atctgcaaac cagtgctcca tcagggggaa 480 gtctgtacca aacaacgcaa gaagggttct catgggctgg aaattttcca gcgttgcgac 540 tgtgcgaagg gcctgtcttg caaagtatgg aaagatgcca .cctactcctc caaagccaga 600 ctccatgtgt gtcagaaaat ttga 624 < 210 > 7 < 211 > 675 < 212 > DNA < 13 > Human < 400 > 7 atggtggcgg ccgtcctgct ggggctgagc tggctctgct ctcccctggg agctctggtc 60 ctggacttca acaacatcag gagctctgct gacctgcatg gggcccggaa gggctcacag 120 tgcctgtctg acacggactg caataccaga aagttctgcc tccagccccg cgatgagaag 180 ccgttctgtg ctacatgtcg tgggttgcgg aggaggtgcc agcgagatgc catgtgctgc 240 cctgggacac tctgtgtgaa cgatgtttgt actaegatgg aagatgcaac eccaatatta 300 gaaaggcagc ttgatgagca agatggcaca catgeagaag gaacaactgg gcacccagtc 360 caggaaaacc aacccaaaag gaagccaagt attaagaaat cacaaggcag gaagggacaa 420 gagggagaaa gttgtctgag aacttttgac tgtggccctg gactttgctg tgctcgtcat 480 aaatttgtaa ttttggacga gccagtcctt ttggagggac aggtctgctc cagaagaggg 540 cataaagaca ctgctcaagc tecagaaate ttccagcgtt gcgactgtgg ccctggacta 600 ctgtgtcgaa gccaattgac cagcaatcgg cagcatgctc gattaagagt a gccaaaaa 660 atagaaaagc tataa 675 < 210 > 8 < 211 > 349 < 212 > PRT < 213 > Mouse < 400 > 8 Met Gln Arg Leu Gly Gly lie Leu Leu Cye Thr Leu Leu Wing Wing Ala 1 5 10 15 Val Pro Thr Wing Pro Wing Pro Pro Thr Val Thr Trp Thr Pro Wing 20 25 30 Glu Pro Gly Pro Wing Leu Asn Tyr Pro Gln Glu Glu Wing Thr Leu Asn 35 40 45 Glu Met Phe Arg Glu Val Glu Glu Leu Met Glu Asp Thr Gln His Lys 50 55 60 Leu Arg Ser Wing Val Glu Glu Met Glu Ala Glu Glu Ala Ala Ala Lys 65 70 75 80 Thr Ser Ser Glu Val Asn Leu Wing Ser Leu Pro Pro Asn Tyr His Asn 85 90 95 Glu Thr Ser Thr Glu Thr Arg Val Gly Asn Asn Thr Val His Val His 100 105 110 Gln Glu Val His Lys He Thr Asn Asn Gln Ser Gly Gln Val Val Phe 115 120 125 Ser Glu Thr Val He Thr Ser Val Gly Asp Glu Glu Gly Lys Arg Ser 130 135 140 His Glu Cys He He Asp Glu Asp.Cys Gly Pro Thr Arg Tyr Cys Gln 145 150 155 160 Phe Ser Ser Phe Lys Tyr Thr Cys Gln Pro Cys Arg Asp Gln Gln Met 165 170 175 Leu Cys Thr Arg Asp Ser Glu Cys Cys Gly Asp Gln Leu Cys Wing Trp 180 185 190 Gly His Cys Thr Gln Lys Wing Thr Lys Gly Gly Asn Gly Thr He Cys 195 200 205 Asp Asn Gln Arg Asp Cys Gln Pro Gly Leu Cys Cys Wing Phe Gln Arg 210 215 220 Gly Leu Leu Phe Pro Val Cys Thr Pro Leu Pro Val Glu Gly Glu Leu 225 230 235 240 Cys His Asp Pro Thr Ser Gln Leu Leu Asp Leu He Thr Trp Glu Leu 245 250 255 Glu Pro Glu Gly Wing Leu Asp Arg Cys Pro Cys Wing Ser Gly Leu Leu 260 265 270 Cys Gln Pro His Ser His Ser Leu Val Tyr Met Cys Lys Pro Wing Phe 275 280 285 Val Gly Ser His Asp His Ser Glu Glu Ser Gln Leu Pro Arg Glu Wing 290 295 300 Pro Asp Glu Tyr Glu Asp Val Gly Phe He Gly Glu Val Arg Gln Glu 305 310 315 320 Leu Glu Asp Leu Glu Arg Ser Leu Wing Gln Glu Met Wing Phe Glu Gly 325 330 335 Pro Wing Pro Val Glu Ser Leu Gly Gly Glu Glu Glu He 340 345 < 210 > 9 < 211 > 350 < 212 > PRT < 213 > Human < 400 > 9 Met Gln Arg Leu Gly Ala Thr Leu Leu Cys Leu Leu Leu Ala Ala Ala 1 5 10 15 Pro Pro Wing Wing Pro Wing Pro Pro Wing Wing Pro 'Val 20 25 30 Lys Pro Gly Pro Wing Leu Ser Tyr Pro Gln Glu Glu Wing Thr Leu Asn 35 40 45 Glu Met Phe Arg Glu Val Glu Glu Leu Met Glu Asp Thr Gln His Lys 50 55 60 Leu Arg Ser Wing Val Glu Glu.Met Glu Wing Glu Glu Wing Wing Ala Lys 65 70 75 80 Wing Being Ser Glu Val Asn Leu Wing Asn Leu Pro Pro Ser Tyr His Asn 85 90 95 Glu Thr Asn Thr Asp Thr Lys Val Gly Asn Asn Thr He His Val His 100 105 110 Arg Glu He His Lys He Thr Asn Asn Gln Thr Gly Gln Met Val Phe 115 120 125 Ser Glu Thr Val He Thr Ser Val Gly Asp Glu Glu Gly Arg Arg Ser 130 135 140 His Glu Cys He He Asp Glu Asp Cys Gly Pro Ser Met Tyr Cys Gln 145 150 155 160 Phe Wing Being Phe Gln Tyr Thr Cys Gln Pro Cys Arg Gly Gln Arg Met 165 170 175 Leu Cys Thr Arg Asp Ser Glu Cys Cys Gly Asp Gln Leu Cys Val Trp 180 185 190 Gly His Cys Thr Lys Met Wing Thr Arg Gly Ser Asn Gly Thr He Cys 195 200 205 Asp Asn Gln Arg Asp Cys Gln Pro Gly Leu Cys Cys Wing Phe Gln Arg 210 215 220 Gly Leu Leu Phe Pro Val Cys Thr Pro Leu Pro Val Glu Gly Glu Leu 225 230 235 240 Cys His Asp Pro Wing Ser Arg Leu Leu Asp Leu He Thr Trp Glu Leu 245 250 255 Glu Pro Asp Gly Ala Leu Asp Arg Cys Pro Cys Wing Ser Gly Leu Leu 260 265 270 Cys Gln Pro His Ser His Ser Leu Val Tyr Val -Cys Lys Pro Thr Phe 275. 280 285 Val Gly Ser Arg Asp Gln Asp Gly Glu He Leu Leu Pro Arg Glu Val 290 295 300 Pro Asp Glu Tyr Glu Val Gly Ser Phe Met Glu Glu Val Arg Gln Glu 305 * 310 315 320 Leu Glu Asp Leu Glu Arg Ser Leu Thr Glu Glu Met Wing Leu Gly Glu 325 330 335 Pro Ala Ala Ala Ala Ala Ala Leu Leu Gly Gly Glu Glu He 340 345 350 < 210 > 10- < 211 > 266 < 212 > PRT < 213 > Human < 400 > 10 Met Met Ala Leu Gly Ala Ala Gly Ala Thr Arg Val Phe Val Ala Met 1 5 10 15 Val Ala Ala Ala Leu Gly Gly His Pro Leu Leu Gly Val Ser Wing Thr 20 25 30 Leu Asn Ser Val Leu Asn Ser Asn Wing He Lys Asn Leu Pro Pro Pro 35 40 45 Leu Gly Gly Wing Wing Gly His Pro Gly Ser Wing Val Be Wing Wing Pro 50 55 60 Gly He Leu Tyr Pro Gly Gly Asn Lys Tyr Gln Thr He Asp Asn Tyr 65 70 75 80 Gln Pro Tyr Pro Cys Wing Glu Asp Glu Glu Cys Gly Thr Asp Glu Tyr 85 90 95 Cys Wing Pro Thr Arg Gly Gly Asp Wing Gly Val Gln He Cys Leu 100 105 110 Wing Cys Arg Lys Arg Arg Lys Arg Cys Met Arg His Wing Met Cys Cys 115 120 125 Pro Gly Asn Tyr Cys Lys Asn Gly He Cys Val Ser Ser Asp Gln Asn 130 135 140 His Phe Arg Gly Glu lie Glu Glu Thr He Thr Glu Ser Phe Gly Asn 145 150 155 160 Asp His Ser Thr Leu Asp Gly Tyr Ser Arg Arg Thr Thr Leu Ser Ser 165 170 175 Lys Met Tyr His Thr Lys Gly dln Glu Gly Ser Val Cys Leu Arg Ser 180 185 190 Ser Asp Cys Ala Ser Gly Leu Cys Cys Ala Arg His Phe Trp Ser Lys 195 200 205 He Cys Lys Pro Val Leu Lys Glu Gly Gln Val Cys Thr Lys His Arg 210 215 220 Arg Lys Gly Ser His Gly Leu Glu He Phe Gln'Arg Cys Tyr Cys Gly 225 230 235 240 Glu Gly Leu Ser Cys Arg lie Gln Lys Asp His His Gln Wing Ser Asn 245 250 255 Being Ser Arg Leu His Thr Cys Gln Arg His 260 265 < 210 > 11 < 211 > 259 < 212 > PRT < 213 > Mouse < 400 > 11 Met Ala 'Ala Leu Met Arg Val Lys Asp Ser Ser Arg Cys Leu Leu Leu 1 5 10 15 Leu Ala Ala Val Leu Met Val Glu Be Ser Gln Leu Gly Be Ser Arg 20 25 30 Wing Lys Leu Asn Ser He Lys Ser Ser Leu Gly Glu Thr Pro Pro Wing 35 40 45 Gln Ser Wing Asn Arg Ser Wing Gly Met Asn Gln Gly Leu Ala Phe Gly 50 55 60 Gly Ser Lys Lys Gly Lys Ser Leu Gly Gln Wing Tyr Pro Cys Ser Ser 65 70 75 80 Asp Lys Glu Cys Glu Val Gly Arg Tyr Cys His Ser Pro His Gln Gly 85 90 95 Be Being Wing Cys Met Leu Cys Arg Arg Lys Lys Lys Arg Cys His Arg 100 105 110 Asp Gly Met Cys Cys Pro Gly Thr Arg Cys Asn Asn Gly He Cys He 115 120 125 Pro Val Thr Glu Ser He Leu Thr Pro His He Pro Ala Leu Asp Gly 130 135 140 Thr Arg His Arg Asp Arg Asn His Gly His Tyr Ser Asn Hie Asp Leu 145 150 155 160 Gly Trp Gln Asn Leu Gly Arg Pro His Ser Lys Met Pro His He Lys 165 170 175 Gly His Glu Gly Asp Pro Cys Leu Arg Ser As Asp Cys He Asp Gly 180 185 190 Phe Cys Cys Ala Arg His Phe Trp Thr Lys He Cys Lys Pro Val Leu 195 200 205 His Gln Gly Glu Val Cys Thr Lys Gln Arg Lys Lys Gly Ser His Gly 210 215 - 220 Leu Glu He Phe Gln Arg Cys Asp Cys Ala Lys Gly Leu Ser Cys Lys 225 230 235 240 Val Trp Lys Asp Wing Thr Tyr Ser Ser Lys Wing Arg Leu His Val Cys 245 250 255 Gln Lys He < 210 > 12 < 211 > 259 < 212 > PRT < 213 > Human < 400 > 12 Met Ala Ala Leu Met Arg Ser Lys Asp Ser Ser Cys Cys Leu Leu Leu 1 '5 10 15 Leu Ala Ala Val Leu Met Val Glu Be Ser Gln He Gly Ser Ser Arg 20 25 30 Ala Lys -Leu Asn Ser He Lys Ser Ser Leu Gly Glu Glu Thr Pro Gly 35 40 45 Gln Ala Ala Asn Arg Ser Ala Gly Met Tyr Gln Gly Leu Wing Phe Gly 50 55 60 Gly Ser Lys Lys Gly Lys Asn Leu Gly Gln Wing Tyr Pro Cys Ser Ser 65 70 75 80 Asp Lys Glu Cys Glu Val Gly Arg Tyr Cys His Ser Pro His Gln Gly 85 90 95 Be Being Wing Cys Met Val Cys Arg Arg Lys Lys Lys Arg Cys His Arg 100 105 110 Asp Gly Met Cys Cys Pro Ser Thr Arg Cys Asn As Gly He Cys He 115 120 125 Pro Val Thr Glu Ser He Leu Thr Pro His He Pro Ala Leu Asp Gly 130 - 135 140 Thr Arg His Arg Asp Arg Asn His Gly His Tyr Ser Asn His Asp Leu 145 150 155 160 Gly Trp Gln Asn Leu Gly Arg Pro His Thr Lys Met Ser His He Lys 165 170 175 Gly His Glu Gly Asp Pro Cys Leu Arg Ser As Asp Cys He Glu Gly 180 185 190 Phe Cys Cys Ala Arg His Phe Trp Thr Lys He Cys Lys Pro Val Leu 195 200 205 His Gln Gly Glu Val Cys Thr Lys Gln Arg Lys Lys Gly Ser His Gly 210 215 220 Leu Glu He Phe Gln Arg Cys Asp Cys Ala Lys Gly Leu Ser Cys Lys 225 230 235 240 Val Trp Lys Asp Ala Thr Tyr - Ser Ser Lys Ala Arg Leu His Val Cys 245 250 255 Gln Lys He < 210 > 13 < 211 > 207 < 212 > PRT < 213 > Human < 400 > 13 Met Ala Ala Leu Met Arg Ser Lys Asp Ser Ser Cys Cys Leu Leu Leu 1 5 10 15 Leu Ala Ala Val Leu Met Val Glu Be Ser Gln He Gly Ser Ser Arg 20 25 30 Wing Lys Leu Asn Ser He Lvs Ser Ser Leu Gly Glu Glu Thr Pro Gly 35 40 45 Gln Wing Wing Asn Arg Wing Wing Gly Met Tyr Gln Gly Leu Ala Phe Gly 50 55 60 Gly Ser Lys Lys Gly Lys Asn Leu Gly Gln Wing Tyr Pro Cys Ser Ser 65 70 75 80 Asp Lys Glu Cys Glu Val Gly Arg Tyr Cys His Ser Pro His Gln Gly 85 90 95 Be Being Wing Cys Met Val Cys Arg Arg Lys Lys Lys Arg Cys His Arg 100 105 110 Asp Gly Met Cys Cys Pro Ser Thr Arg Cys Asn Asn Gly His Glu Gly 115 120 125 Asp Pro Cys Leu Arg Ser Ser Asp Cys He Glu Gly Phe Cys Cys Wing 130 135 140 Arg His Phe Trp Thr Lys He Cys Lys Pro Val Leu His Gln Gly Glu 145 150 155 160 Val Cys Thr Lys Gln Arg Lys Lys Gly Ser His Gly Leu Glu He Phe 165 170 175 Gln Arg Cys Asp Cys Wing Lys Gly Leu Ser Cys Lys Val Trp Lys Asp 180 185 190 Wing Thr Tyr Ser Ser Lys Wing Arg Leu His Val Cys Gln Lys He 195 200 205 < 210 > 14 < 211 > 224 < 212 > PRT < 213 > Human < 400 > 14 Met Val Ala Ala Val Leu Leu Gly Leu Ser Trp Leu Cys Ser Pro Leu 1 5 10 15 Gly Ala Leu Val Leu Asp Phe Asn Asn He Arg Ser Be Wing Asp Leu 20 25 30 His Gly Wing Arg Lys Gly Ser Gln Cys Leu Ser Asp Thr Asp Cys Asn 35 40 45 Thr Arg Lys Phe Cys Leu Gln Pro Arg Asp Glu Lys Pro Phe Cys Ala 50 55 60 Thr Cys Arg Gly Leu Arg Arg Arg Cys Gln Arg Asp Ala Met Cys Cys 65 70 75 80 Pro Gly Thr Leu Cys Val Asn. Asp Val Cys Thr Thr Met Glu Asp Wing 85 90 95 Thr Pro He Leu Glu Arg Gln Leu Asp Glu Gln Asp Gly Thr His Wing 100 105 110 Glu Gly Thr Thr Gly His Pro Val Gln Glu Asn Gln Pro Lys Arg Lys 115 120 125 Pro Ser He Lys Lys Ser Gln Gly Arg Lys Gly Gln Glu Gly Glu Ser 130 135 140 Cys Leu Arg Thr Phe Asp Cys Gly Pro Gly Leu Cys Cys Ala Arg His 145 150 155 160 Phe Trp Thr Lys He Cys Lys Pro Val Leu Leu Glu Gly Val Val Cys 165 170 175 Be Arg Arg Gly His Lys Asp Thr Ala Gln Ala Pro Glu He Phe Gln 180 185 190 Arg Cys Asp Cys Gly Pro Gly Leu Leu Cys Arg Ser Gln Leu Thr Ser 195 200 205 Asn Arg Gln His Wing Arg Leu Arg Val Cys Gln Lys He Glu Lys Leu 210 215 220 < 210 > 15 211 > 33 < 212 > DNA '< 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Oligonucleotide Primer < 400 > 15 ggaaggaaaa aagcggccgc aacannnnnn nnn 33 < 210 > 16 < 211 > 16 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Oligonucleotide adapter < 400 > 16 tcgacccacg cgtccg 16 < 210 > 17 < 211 > 12 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Oligonucleotide adapter < 400 > 17 gggtgcgcag ge 12 < 210 > 18 < 211 > 18 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Oligonucleotide Primer < 400 > 18 actagctcca gtgatctc 18 < 210 > "19 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of the Artificial Sequence: Oligonucleotide Primer <400> 19 cgtcattgtt ctcgttcc 18 < 210 > 20 < 211 > 23 < 212 > DNA < 213 > < Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Oligonucleotide Primer < 400 > 20 ccagctgctc tgtggcagcc cag 23 < 210 > 21 < 211 > 29 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Oligonucleotide Primer < 400 > 21 cccagtcacg acgttgtaaa acgacggcc 29 < 210 > 22 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence - < 220 > < 223 > Description of the Artificial Sequence: Oligonucleotide Primer < 400 > 22 aacatgcagc ggctcggggg 20 < 210 > 23 < 211 > 30 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Oligonucleotide Primer < 400 > 23 ggtgacacta tagaagagct atgacgtcgc 30 < 210 > 24 < 211 > 22 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Oligonucleotide Primer < 400 > .24 gtgctgagtg tcttccatca ge 22 < 210 > 25 < 211 > 25 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Oligonucleotide primers < 400 > 25 gagatgcagc ggcttggggc cacee 25 < 210 > 26 < 211 > 23 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Oligonucleotide primers < 400 > 26 gcctggtcag cccacgccta aag 23 < 210 > 27 < 211 > 30 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Oligonucleotide primers < 400 > 27 cctgctgctg gcggcggcgg tccccacggc 30 < 210 > 28 < 211 > 23 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Oligonucleotide primers < 400 > 28 gcctggtcag .cccacgccta aag 23 < 210 > 29 < 211 > 23 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Oligonucleotide primers < 400 > 29 cccggaccct gactctgcag ccg 23 < 210 > 30 < 211 > 25 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description..of the Artificial Sequence: Oligonucleotide primers < 400 > 30 gaggaaaaat aggcagtgca gcacc 25 < 210 > 31 < 211 > 25 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Oligonucleotide primers < 400 > 31 gccacagtcc ccaccaagga tcatc 25 < 210 > 32 < 211 > 25 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Oligonucleotide primers < 400 > 32 gatgatcctt ggtggggact gtggc _ '25 < 210 > 33 < 211 > 24 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Oligonucleotide primers < 400 > 33 ctgcaaacca gtgctccatc aggg 24 < 210 > 34 < 211 > 24 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Oligonucleotide primers < 400 > 34 ccctgatgga gcactggttt gcag 24 < 210 > 35 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Oligonucleotide Primer < 400 > 35 gctataccaa gcatacaatc 20 < 210 > 36 < 211 > 25 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Oligonucleotide Primer < 400 > 36 gggttgaggg aacacaatct gcaag 25 < 210 > 37 < 211 > 28 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Oligonucleotide Primer < 400 > 37 gtctgcaatt gatgatgttc ctcaatgg 28 110 < 210 > 38 < 211 > 24 < 212 > DNA < 213 > Artificial Sequence - < 220 > < 223 > Description of the Artificial Sequence: Oligonucleotide Primer < 400 > 38 ccagggccac agtcgcaacg ctgg 24 < 210 > 39 < 211 > 25 15 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Oligonucleotide Primer < 400 > 39 ctccctcttg tcccttcctg ccttg 25 < 210 > 40 < 211 > 25 < 212 > DNA 20 < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Oligonucleotide Primer < 400 > 40 caaggcagga agggacaaga gggag 25 < 210 > 41 < 211 > 24 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Oligonucleotide Primer < 400 > 41 ccagcgttgc gacfcgtggcc ctgg 24 < 210 > 42 < 211 > 44 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer / oligonucleotide adapter < 400 > 42 gactagttct agatcgcgag cggccgccct tttttttttt tttt 44 < 210 > 43 < 211 > 6 < 212 > PRT < 213 > Human < 400 > 43 Met His Pro Leu Leu Gly - - 1 5 - < 210 > 44 < 211 > 5 < 212 > PRT < 213 > Human < 400 > 44 Thr Cys Gln Arg His 1 5 < 210 > 45 < 211 > 59 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Oligonucleotide Primer < 400 > 45 gttctcctca tatgcatcca ttattaggcg taagtgccac ettgaacteg gttctcaat 59 < 210 > 46 < 211 > 38 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Oligonucleotide Primer < 400 > 46 tacgcactgg atccttagtg tctctgacaa gtgtgaag 38 < 210 > 47 < 211 > 6 < 212 > PRT < 213 > Human < 400 > 47 Met Ser Gln He Gly Ser 1 5. < 210 > 48 < 211 > 5 < 12 > PRT < 213 > Human < 400 > 48 Val Cys Gln Lys He 1 5 < 210 > 49 < 211 > 56 < 212 > DNA < 213 > Artificial Sequence, 10 • < 220 > < 223 > Description of the Artificial Sequence: Primer oligonucleotídicß "<400> 49 gttctcctca tatgtctcaa attggtagtt ctcgtgccaa actcaactcc atcaag 56 <210> 50 <211> 39 <212> DNA <213> Artificial Sequence < 220 > 15 < 223 > Description of the Artificial Sequence: Oligonucleotide Primer <400> 50 tacgcactgg atccttaaat tttctgacac acatggagt 39 <210> 51 <211> 6 &<212> PRT <213 >Mouse <400> 51 Met Ser Gln Leu Gly Ser 20 1 5 < 210 > 52 < 211 > 5 < 212 > PRT < 213 > Mouse < 400 > 52 Val Cys Gln Lys He 1 5 < 210 > 53 < 211 > 59 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Oligonucleotide Primer < 400 > 53 gttctcctca tatgtetcaa ttaggtaget ctcgtgctaa actcaactcc atcaagtcc 59 < 210 > 54 < 211 > 39 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: IgG nucleotide primer < 400 > 54 tacgcactgg atccttagat cttctggcat acatggagt 39 < 210 > 55 < 211 > 6 < 212 > PRT < 213 > Human - < 400 > 55 Met Pro Ala Pro Thr Ala 1 5 < 210 > 56 < 211 > 5 < 212 > PRT < 213 > Human < 400 > 56 Gly Gly Glu Glu He 1 5 < 210 > 57 < 211 > 54 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Oligonucleotide Primer < 400 > 57 gttctcctca tatgcctgct ccaactgcaa cttcggctcc agtcaagccc ggcc 54 < 210 > 58 < 211 > 37 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Oligonucleotide Primer < 400 > 58 tacgcactcc gcggttaaat ctcttcccct cccagca 37 < 210 > 59 < 211 > 6 < 212 > PRT < 213 > Human < 400 > 59 Met Lys Pro Gly Pro Wing 1 5 < 210 > 60 < 211 > 5 < 212 > PRT < 213 > Human < 400 > 60 Gly Gly Glu Glu He 1 5 < 210 > 61 < 211 > 54 < 212 > DNA < 213 > Artificial Sequence - < 220 > < 223 > Description of the Artificial Sequence: Oligonucleotide Primer < 400 > 61 gttctcctca tatgaaacca ggtccagcct taagctaccc gcaggaggag gcca 54 < 210 > 62 < 211 > 37 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of 'Artificial Sequence: Oligonucleotide Primer < 400 > 62 tacgcactcc gcggttaaat ctcttcccct cccagca 37 < 210 > 63 < 211 > 6 < 212 > PRT < 213 > Human < 400 > 63 Met Gln Glu Glu Ala Thr 1 5 < 210 > 64 < 211 > 5 < 212 > PRT < 213 > Human < 400 > 64 Gly Gly Glu Glu He 1 5 < 210 > 65 < 211 > 53 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: - Oligonucleotide tester < 400 > 65 gttctccfcca tatgcaagaa gaagctactc tgaatgagat gttccgcgag gtt 53 < 210 > 66 < 211 > 37 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Oligonucleotide Primer < 400 > 66 tacgcactcc gcggttaaat ctcttcccct cccagca 37 < 210 > 67"< 211 > 6 < 212 > PRT < 213 > Mouse < 400 > 67 Met Glu Pro Gly Pro Wing 1 5 < 210 > 68 < 211 > 5 < 212 > PRT < 213 > Mouse < 400 > 68 Gly Glu Glu Glu He 1 5 < 210 > 69 < 211 > 54 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Oligonucleotide Primer < 400 > '69 gttctcctca tatggaacca ggtccagctt taaactaccc tcaggaggaa gcta 54 < 210 > 70 < 211 > 37 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Oligonucleotide Primer < 400 > 70 tacgcactcc gcggttaaat ctcctcctct ccgccta 37 < 210 > 71 < 211 > 6 < 212 > PRT < 213 > Human < 400 > 71 Met Leu Val Leu Asp Phe 1 5 < 210 > 72 < 211 > 5 < 212 > PRT < 213 > Human < 400 > 72 Lys He Glu Lys Leu 1 5 < 210 > 73 < 211 > 47 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Oligonucleotide primer. < 400 > 73 gttctcctca tatgttagtt ttggatttca acaacatcag gagctct 47 < 210 > 74 < 211 > 49 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Oligonucleotide Primer < 400 > 74 tacgcactgg atccttacag tttttctatt ttttggcata ctcttaatc 49 < 210 > 75 • < 211 > 798 < 212 > DNA < 213 > Human < 400 > 75 atgatggctc tgggtgctgc tggtgctacc cgtgttttcg ttgctatggt tgctgctgct 60 ctgggtggtc acccgctgct gggtgtttcc gctaccctga actccgttct gaactccaac 120 gctatcaaaa acctgccgcc gccgctgggt ggtgctgctg gtcacccggg ttccgctgtt 180 tccgctgctc cgggtatcct gtacccgggt ggtaacaaat accagaccat cgacaactac 240 cagccgtacc cgtgcgctga agacgaagaa tgcggtaccg acgaatactg cgcttccccg 300 acccgtggtg gtgacgctgg tgttcagatc tgcctggctt gccgtaaacg tcgtaaacgt 360 tgcatgcgtc acgctatgtg ctgcccgggt aactactgca aaaacggtat ctgcgtttcc 420 tccgaccaga accacttccg tggtgaaatc gaagaaacca tcaccgaatc cttcggtaac 480 gaccactcca ccctggacgg ttactcccgt cgtaccaccc tgtcctccaa aatgtaccac 540 accaaaggtc aggaaggttc cgtttgcctg cgttcctccg actgcgcttc cggtctgtgc 600 tgcgctcgtc acttctggtc caaaatctgc aaaccggttc tgaaagaagg tcaggtttgc 660 accaaacacc gtcgtaaagg ttcccacggt ctggaaatct tccagcgttg ctactgcggt 720 gaaggtctgt cctgccgtat ccagaaagac caccaccagg cttccaactc ctcccgtctg 780 798 cacacctgcc agcgtcac • < 210 > 76 < 211 > 777 < 212 > DNA < 213 > Human < 400 > 76 atggctgctc tgatgcgttc caaagactcc tcctgctgcc tgctgctgct ggctgctgtt 60 ctgatggttg aatcctccca gatcggttcc tcccgtgcta aactgaactc catcaaatcc 120 tccctgggtg gtgaaacccc gggtcaggct gctaaccgtt ccgctggtat gtaccagggt 180 ctggctttcg gtggttccaa aaaaggtaaa aacctgggtc aggcttaccc gtgctcctcc 240 gacaaagaat .gcgaag tgg tcgttactgc cactccccgc accagggttc ctccgcttgc 300 atggtttgcc gtcgtaaaaa aaaacgttgc caccgtgacg gtatgtgctg cccgtccacc 360 cgttgcaaca acggtatctg catcccggtt accgaatcca tcctgacccc gcacatcccg 420 gctctggacg gtacccgtca ccgtgaccgt aaccacggtc actactccaa ccacgacctg 480 ggttggcaga acctgggtcg tccgcacacc aaaatgtccc acatcaaagg tcacgaaggt 540 gacccgtgcc tgcgttcctc cgactgcatc gaaggtttct gctgcgctcg tcacttctgg 600 • accaaaatct gcaaaccggt tctgcaccag ggtgaagttt gcaccaaaca gcgtaaaaaa 660 ggttcccacg gtctggaaat cttccagcgt tgcgactgcg ctaaaggtct gtcctgcaaa 720 gtttggaaag acgctaccta ctcctccaaa gctcgtctgc acgtttgcca gaaaatc 777 < 210 > 77 < 211 > 1050 < 212 > DNA < 213 > Human < 400 > 77 atgcagcgtc tgggtgctac cctgctgtgc ctgctgctgg ctgctgctgt tccgaccgct 60 ccggctccgg ctccgaccgc tacctccgct ccggttaaac cgggtccggc tctgtcctac 120 ccgcaggaag aagctaccct gaacgaaatg ttccgtgaag ttgaagaact gatggaagac 180 acccagcaca aactgcgttc cgctgttgaa gaaatggaag ctgaagaagc tgctgctaaa 240 gcttcctccg aagttaacct ggctaacctg ccgccgtcct accacaacga aaccaacacc 300 gacaccaaag ttggtaacaa caccatccac gttcaccgtg aaatccacaa aatcaccaac 360 aaccagaccg gtcagatggt tttctccgaa accgttatca cctccgttgg tgacgaagaa 420 ggtcgtcgtt cccacgaatg catcatcgac gaagactgcg gtccgtccat gtactgccag 480 ttcgcttcct tccagtacac ctgccagccg tgccgtggtc agcgtatgct gtgcacccgt 540 gactccgaat gctgcggtga ccagctgtgc gtttggggtc actgcaccaa aatggctacc 600 cgtggttcca acggtaccat ctgcgacaac cagcgtgact gccagccggg tctgtgctgc 660 gctttccagc gtggtctgct gttcccggtt tgcaccccgc tgccggttga aggtgaactg 720 tgccacgacc cggcttcccg tctgctggac ctgatcacct gggaactgga accggacggt 780 gctctggacc gttgcccgtg cgcttccggt ctgctgtgcc agccgcactc ccactccctg 840 gtttacgttt gcaaaccgac cttcgttggt tcccgtgacc aggacggtga aatcctgctg 900 ccgcgtgaag ttccggacga atacgaagtt ggttccttca tggaagaagt tcgtcaggaa 960 ctggaagacc tggaacgttc cctgaccgaa gaaatggctc tgggtgaacc ggctgctgct 1020 gctgctgctc tgctgggtgg tgaagaaatc 1050 < 210 > 78 < 211 > 672 < 212 > DNA < 213 > Human < 400 > 78 atggt'tgctg ctgttctgct gggtctgtcc tggctgtgct ccccgctggg tgctctggtt 60 ctggacttca acaacatccg ttcctccgct gacctgcacg gtgctcgtaa aggttcccag 120 tgcctgtccg acaccgactg caacacccgt aaattctgcc tgcagccgcg tgacgaaaaa 180 ccgttctgcg ctacctgccg tggtctgcgt cgtcgttgcc agcgtgacgc tatgtgctgc 240 ccgggtaccc tgtgcgttaa cgacgtttgc accaccatgg aagacgctac cccgatcctg 300 tggacgaaca gaacgtcagc ggacggtacc cacgctgaag gtaccaccgg tcacccggtt 360 caggaaaacc agccgaaacg taaaccgtcc atcaaaaaat cccagggtcg taaaggtcag 420 gaaggtgaat cctgcctgcg taccttcgac tgcggtccgg gtctgtgctg cgctcgtcac 480 aaatctgcaa ttctggacca accggttctg ctggaaggtc aggtttgctc ccgtcgtggt 540 cacaaagaca ccgctcaggc tccggaaatc ttccagcgtt gcgactgcgg tccgggtctg 600 ctgtgccgtt cccagctgac ctccaaccgt cagcacgctc gtctgcgtgt ttgccagaaa 660 atcgaaaaac tg 672 It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is the conventional one for the manufacture of the objects to which it relates.

Claims (20)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. An isolated nucleic acid molecule that encodes a biologically active DKR polypeptide, characterized in that it is selected from the group consisting of: (a) the nucleic acid molecule comprising the SEQ ID NO: l; (b) the nucleic acid molecule comprising SEQ ID NO: 2; (c) the nucleic acid molecule comprising SEQ ID NO: 3; (d) the nucleic acid molecule comprising SEQ ID NO: 4; (e) the nucleic acid molecule comprising the SEQ ID NO: 5; (f) the nucleic acid molecule comprising the SEQ ID NO: 6; (g) the nucleic acid molecule comprising SEQ ID NO: 7; (h) the nucleic acid molecule comprising SEQ ID NO: 75; (i) the nucleic acid molecule comprising SEQ ID NO: 76; (j) the nucleic acid molecule comprising SEQ ID NO: 77; (k) the nucleic acid molecule comprising SEQ ID NO: 78; (1) the nucleic acid molecule encoding the polypeptide of SEQ ID NO: 8; (m) a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 9; (n) a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 10, or a biologically active fragment thereof; (o) a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 11, or a biologically active fragment thereof; (p) a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 12, or a biologically active fragment thereof; (q) a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 13, or a biologically active fragment thereof; (r) a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 14, or a biologically active fragment thereof; (s) a nucleic acid molecule encoding a polypeptide that is at least 85 percent identical to. polypeptide of SEQ ID NOs: 10, 11, 12, 13 or 14; (t) a nucleic acid molecule encoding a biologically active DKR polypeptide having 1-100 amino acid substitutions and / or deletions compared to the polypeptide of any of SEQ ID NOs: 8, 9, 10, 11 , 12, 13 or 14; and (u) a nucleic acid molecule that hybridizes under very stringent conditions to any of (c), (d), (e), • (f), (g), (h), (i), (k), (1), (m), (n), (o), (p), (q), (r), ( s), and (t) above.

2. An isolated nucleic acid molecule, characterized in that it is the complement of the molecule of The nucleic acid according to claim 1.

3. An isolated nucleic acid molecule, characterized in that it comprises SEQ ID NO: 1, SEQ ID NO: 2, • SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7. 20

4. An isolated nucleic acid molecule, characterized in that it encodes the polypeptide of the SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14.

5. A nucleic acid molecule isolated, Characterized in that it encodes a biologically active DKR polypeptide selected from the group consisting of: amino acids 16-350, 21-350, 22-350, 23-350, 33-350, or 42-. 350, 21-145, 40-145, 40-150, 45-145, 45-145, 145-290, 145-300, 145,300, 150-290, 300-350, or 310-350 of SEQ ID NO. : 9; amino acids 15-266, 24-266, or 32-266 of SEQ ID NO: 10; amino acids 17-259, 26-259, or 34-359 of SEQ ID NO: 12; amino acids 19-224, 20-224, 21-224, or 22-224 of SEQ ID NO: 14.

6. A vector, characterized in that it comprises the The OR nucleic acid molecule according to claim 1.

7. A vector, characterized in that it comprises the nucleic acid molecule according to claim 2.

8. A vector, characterized in that it comprises the nucleic acid molecule according to claim 3.

9. A vector, characterized in that it comprises the nucleic acid molecule according to Claim 4.

A vector, characterized in that it comprises the nucleic acid molecule according to claim 5.

11. A host cell, characterized in that 25 comprises the vector according to claim 6.

12. A host cell, characterized in that it comprises the vector according to claim 7.

13. A host cell, characterized in that it comprises the vector according to claim 8.

14. A host cell, characterized in that it comprises the vector according to claim 9.

15. A host cell, characterized in that it comprises the vector according to claim 10.

16. A process for producing a DKR? biologically active, characterized in that it comprises the steps of: (a) expressing a polypeptide encoded by the nucleic acid according to claim 1 in a suitable host; and 5 (b) isolating the "polypeptide

17. The process" according to claim 16, characterized in that the polypeptide is SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14. 0

18. A biologically active DKR polypeptide characterized in that it is selected from the group consisting of: (a) the polypeptide of SEQ ID NO: 8; (b) the polypeptide of SEQ ID NO: 9; (c) the polypeptide of SEQ ID NO: 10; 5 (d) the polypeptide of SEQ ID NO: 11; (e) the polypeptide of SEQ ID NO: 12, (f) the polypeptide of SEQ ID NO: 13, (g) the polypeptide of SEQ ID NO: 14, (h) a polypeptide having 1-100 substitutions or amino acid deletions compared to the polypeptide of any of (a) - (g) above; and (i) a polypeptide that is at least 85 percent identical to any of the polypeptides of (c) - (h) above.

19. The polypeptide according to claim 18, characterized in that it does not possess an endogenous signal peptide.

20. A polypeptide, characterized in that it is selected from the group consisting of amino acids 16-350, 21-350, 22-350, 23-350, 33-350, 42-350, 21-145, 40-145, 40- 150, 45-145, 45-145, 145-290, 145-300, 145-350, 150-290, 300-350, or 310-350 of SEQ ID NO: 9; amino acids 15-266 ~ 24-266, or 32-266 of SEQ ID NO: 10; amino acids 17-259, 26-259, or 34-359 of SEQ ID NO: 12; amino acids 19-224, 20-224, 21-224, or 22-224 of SEQ ID N0: 14.