MXPA00009172A - Mammalian blood loss-induced gene, kd312 - Google Patents

Abstract

Disclosed are nucleic acids encoding novel proteins, designated kd312. Also disclosed are amino acid sequences for kd312 polypeptides, methods for preparing kd312 polypeptides, and other related aspects.

Description

GENE THAT INDUCES THE BLOOD LOSS IN MAMMALS, KD312 BACKGROUND Field of the Invention This invention relates to a new polypeptide designated kd312 and the related polypeptides that have an effect on apoptosis, to new nucleic acid molecules that encode such polypeptides and to other related aspects.

Description of Related Art Apopt os i s Normal development and tissue homeostasis in animals require that the total number of cells be kept in an appropriate range. This is achieved through several highly regulatory processes that involve cell proliferation, survival and elimination through programmed cell death (apoptosis). An imbalance between cell production rates and cell loss can result in serious diseases in humans, such as cancer, systemic afflictions REF .: 122761 immune and neurodegenerations (reviewed by Rudin, C. M., And Thompson, CB Ann Rev. Med. 48 .: 267-81 (1997)).

Apoptosis seems to be a highly organized, evolutionarily conserved program of active cell destruction (reviewed by Miura, M. and Yuan, J., Curr. Topics, Dev. Biol. 32: 139-174 (1996); , DL and Strasser, A., Proc. Nati, Acad. Sci. USA 93: 2239-2244 (1996)). In the Caenorhabditis elegans nematode, 14 genes involved in apoptosis have been identified. Among these, the ced-3 gene encodes a cysteine protease of the capsase family and is a key effector in the path of cell death. The product of the ced-4 gene seems to be an adapter protein that activates ced-3 due to the reception of the signals of apoptosis (Vaux, DL, Cell 90: 389-390 (1997).) The ced-3 gene, a potent suppressor of programmed cell death, downregulates the activity of ced-3, probably by means of ced-4. of mammals, multiple capsases have been identified and shown to be part of the machinery of cell death (Henkart, PA, Immunity 4_: 195-201 (1996).) The proto-oncogene blc-2 seems to be the prototype of mammalian homologs of ced-9 (Vaux, DL, Cory, S., and Adams, J.M., Nature 335: 440-442 (1998); Vaux, DL, Weissman, IL, and Kim, SK, Science 258 : 1955-1957 (1992), Hengartner, MO, and Horvitz, HR, Cell 16: 665-676 (1994)).

Other members of the bcl-2 family consist of those (such as bcl-XL) functionally similar to bcl-2 that can block apoptosis; and others (bax, for example) that have opposite activity (Boise, LH, Gonzale z-Garcia, M., Postema, CE, Ding, L., Lidsten, T., Turka, LA, Mao, X., Nunez, G., and Thompson, CB, Cell 74: 597-608 (1993), Oltvai, ZN, Milliman, CL, and Korsmeyer, SJ, Cell 7_4_: 609-619 (1993)). Although the molecular mechanism is not yet clear, recent evidence showed that bcl-2 can block the release of cytochrome c from the mitochondria (Kluck, RM, Bos systi zel, E., Green, DR, and Newmeyer, DD , Science 275: 1132-1136 (1997)). In addition, bcl-2 appears to directly inhibit the activation of capsase by binding to mammalian ced-4 homologue (Zou, H., Henzel, WJ, Liu, X., Lutzchg, A., and Wang, X., Cell 0 .: 405-413 (1997)). Other genes besides the blc-2 family have also been implicated in programmed cell death. For example, transcription factors c-myc and NF-? B could be involved in translation signals for cell death or survival (Aske, DS Ashmun, RA, Simmons, BC, and Cleveland, JL, Oncogene 6: 1915 -1922 (1991); Evan GI, Wyllie, AH, Gilbert, CS, Littlewood, TD, Land, H., Brooks, M., Waters, CM, Penn, LZ, and Mancock, DC, Cell £ 9: 119- 128 (1992); Hsu, H., Xiong, J., and Goeddel, DV, Cell 8.1: 495-504 (nineteen ninety five); Beg, A.A., and Baldwin, A.S., Science 274: 782-784 (nineteen ninety six); Wang, C-Y., Mayo, M.W., and Baldwin, A.S., Science 27: 784-787 (1996)). The p53 tumor suppressor gene, which is mutated in approximately 50% of cancers in humans, plays an essential role in radiation-induced apoptosis in a wide variety of cell types (reviewed by Carson, D.A., Lancer 346: 1009-1011 (1995)).

Loss of blood and Apoptosis Massive blood loss could deprive animal organs of most of their oxygen supply and lead to cellular damage and necrotic cell death and apoptosis. It is known that many proteins are synthesized in response to low oxygen tensions (hypoxia). Among these proteins, a few with known functions, such as erythropoietin (to stimulate erythroid progenitors), vascular endothelial growth factor (for angiogenesis) or HAP1 protein (for DNA repair), it is known that cell survival during times of hypoxia. Some of the proteins induced by hypoxia could play important roles in cell survival also by means of the reduction or inhibition of apoptosis. From the point of view of the recent evidence, which indicates that alterations in the threshold of apoptosis contribute to pathological cell death or growth in a number of diseases in humans, such as neurodegenerative diseases, ischemic injury, AIDS and cancers ( Thompson, CB, Science, 267: 1456-1462 (1995)), it is important to identify the key factors that protect cells from apoptotic death.

Although a number of genes and proteins related to cell death are now known, a need remains to identify such additional proteins and genes and to determine their biological activity.

Therefore, it is an object of the present invention to provide new compounds that are associated with cell death, especially when they cause hypoxia, in mammals.

It is a further object of the invention to provide a method for treating diseases associated with cell death, such as those set forth herein.

These and other objects will be apparent to an expert in the art of the present disclosure.

BRIEF DESCRIPTION OF THE INVENTION To better understand the molecular cases that govern apoptosis, we carried out the screening of genes whose level of expression is significantly altered during hypoxia induced by blood loss.

One gene, kd312, has been isolated from the kidneys of rats and identified as a highly induced gene after severe blood loss. This gene was also found to be induced in the liver and thymus of the same animal. It was present in the brain, but was not detected in the bone marrow, heart or spleen of this animal. The levels of induction in the kidneys can be correlated with respect to the severity of blood loss. The human homolog of this gene was also isolated. The kd312 protein is well conserved between rats and humans. The deduced amino acid sequence of the rat kd312 protein (280 amino acids) shares 97.5% identity with that of the human counterpart (281 amino acids). The kd312 protein is distantly related to the Ras protein family and the human kd312 is more homologous (33.8%) with the R-Ras member of the Ras family of human. Similar to Ras proteins, kd312 carries a C terminal CAAX region and a GTP binding site near the amino terminus. Unlike the Ras genes, neither the rat or human kd312 gene induces the development of focus after expression in NIH3T3 cells. The expression of kd312 in the cell line of the embryonic kidney of human 293 protects the cells from apoptosis, similar to the effect observed with the expression of the bcl-2 gene in this cell line.

The present invention models several aspects, as established in the following: In a first embodiment, the present invention provides a nucleic acid molecule that encodes a polypeptide selected from the group consisting of: (a) the nucleic acid molecule of SEQ ID NO: 1 or SEQ ID NO: 3; (b) a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 2 or the biologically active fragment thereof; (c) a nucleic acid molecule encoding a polypeptide that is at least 85 percent identical to the polypeptide of SEQ ID: 2; (d) a nucleic acid molecule that hybridizes under stringent conditions to any of (a) - (c) above; Y (e) a nucleic acid molecule that is the complement of any of (a) - (d) above.

In another embodiment, the present invention provides a nucleic acid molecule that encodes a polypeptide selected from the group consisting of (a ') the nucleic acid molecule of SEQ ID NO: 4 or SEQ ID NO: 6; (b ') a nucleic acid molecule encoding the polypeptide of SEQ ID: 5 or a biologically active fragment thereof; (c ') a nucleic acid molecule encoding a polypeptide that is at least 85 percent identical to the polypeptide of SEQ ID NO: 5; (d ') a nucleic acid molecule that hybridizes under stringent conditions to any of (a') - (c ') above; Y (e ') a nucleic acid molecule that is the complement of any of (a') - (d ') above.

In another embodiment, the invention provides vectors comprising nucleic acid molecules, and host cells, either prokaryotic or eukaryotic, comprising the vectors.

The invention further provides a kd312 polypeptide selected from the group consisting of: (a) the polypeptide of SEQ ID NO: 2; (b) a polypeptide that is at least 85 percent identical to the polypeptide of (a); Y (c) a biologically active fragment of any of (a) - (b).

The invention further provides a kd312 polypeptide selected from the group consisting of: (a ') a polypeptide of SEQ ID NO: 5; (b ') a polypeptide that is at least 85 percent identical to the polypeptide of (a'); Y (c ') a biologically active fragment of any of (a') - (b ').

In another embodiment, the invention provides a process for producing a kd312 polypeptide, wherein the polypeptide could be SEQ ID NO: 2 or SEQ ID NO: 4 or a biologically active fragment thereof, and wherein the process comprises: (a) expressing a polypeptide encoding a kd312 nucleic acid molecule in an appropriate host; Y b) isolating the polypeptide The invention also provides anti-kd312 antibodies The above related and additional aspects of the invention will be better appreciated with reference to the figures described in the following section BRIEF DESCRIPTION OF THE DRAWINGS Figures 1 (A) and (B) represent the Northern blot analysis and RT-PCR of rat total RNA for the induction of blood loss of the kd312 and Epo genes. (A) Northern blot of the total RNA of a normal animal (lanes 2, 4, 6) and an animal whose hematocrit reading was 39% of the normal level after bleeding (lanes 1, 3, 5). Lanes 1 and 2, the RNA profile (6 μg) in 1% denatured agarose gel. Lanes 3 and 4, the same RNA as in lanes 1 and 2 hybridized with a fragment of the mouse β-actin gene of 500 base pairs. Lanes 5 and 6, the same RNA as in lanes 3 and 4 hybridized with a 391 bp rat kd312 cDNA fragment corresponding to a 31 untranslated region. The amount of RNA loaded in lane 1 differs slightly from that loaded in lane 2 as indicated by the amounts of RNAs and β-actin detected in each lane. (B) RT-PCR analysis of the same RNA preparation as in (A) for the presence of the Epo message. PCR was carried out following the synthesis of RNA cDNA from a normal animal (lanes 2 and 4) and from an animal that suffers blood loss (lanes 1 and 3).

Lanes 1 and 2, PCR synthesis (30 cycles) of a fragment of the rat Epo gene of 190 base pairs. Lanes 3 and 4, PCR synthesis (30 cycles) of a fragment of rat ß-tubulin gene of 267 base pairs. Lane 5, molecular weight marker. The amounts of RNA used in the two cDNA synthesis reactions differ slightly as indicated by the amounts of the β-tubulin gene fragment synthesized by PCR.

Figure 2 shows a comparison of the sequences of rat and human kd312 proteins (SEQ ID NOS: 2 and 5), respectively). The GTP binding site is highlighted near the CAAX terminal C and N terminal region.

Figure 3 shows a comparison of human kd312 and R-Ras proteins (SEQ ID NOS: 2 and 7, respectively). Only the regions of both proteins that share homology are shown.

Figure 4 depicts the inhibition of apoptosis by human kd312 as compared to other proteins (as indicated) . The graphs shown are the results of the FACS analysis of apoptosis induced by geranylgeraniol in 293 cells transferred with several genes. The cells were treated with 10 μM geranylgeraniol for several times as indicated. Subsequently, the cells were washed and stained with 50 μg / ml of propidium iodide in 3.8 m sodium citrate, pH 7.0 and analyzed using a FACScan flow cytometer. The degree of apoptosis was determined by measuring the proportion of cells representing hypo-diploid DNA content. The results represent the average of 2 independent experiments.

Figure 5 shows the genomic sequence and structure of kd312 of human (SEQ ID NO: 3). The two coding regions (exons) and the amino acid residues they encode are shown. The GC-rich stretch recognized by the SPI transcription factor and the TATA box, both upstream of the coding region and the downstream coding sequence for the polyadenylation signal are underlined. The arrows mark the 5 'and 3' end of the kd312 cDNA and the dotted lines mark two segments homologous to the two adjacent sequences essential for the induction of hypoxia within the promoter gene region of the Epo gene (Fig. 6).

Figure 6 shows the sequences homologous with the Epo promoter (SEQ ID NO: 8) and the upstream region kd312 of human (SEQ ID NO: 9). P-Epo indicates a segment of the minimal sequence within the Epo promoter region that is essential for a portion of the Epo hypoxia induction. The two short homologous sequences are placed one line higher than the middle sites of the response element of the streptococcus / thyroid hormone receptor. Immediately upstream of the kd312 coding region is a region designated 5'-kd312, which contains two sequences highly homologous to the P-Epo sequence as shown. The numbers indicate the base positions relative to the Epo transcription start site and the approximate kd312 transcription start site (Fig. 5) respectively, both of which are defined as +1.

Figure 7 shows the genomic sequence and structure of rat kd312 (SEQ ID NO: 6). The two coding regions (exons) and the amino acid residues they encode are shown. A line is placed to the TATA box, upstream of the coding region and the downstream coding sequence for the polyadenylation signal. Arrows mark the 5 'and 3' end of the kd312 cDNA.

Figure 8 shows the cDNA sequence and the kd312 structure of human (SEQ ID NO: 1).

Figure 9 shows the cDNA sequence and structure of rat kd312 (SEQ ID NO: 4).

Figure 10 shows in Western analysis of the human kd312 protein. Embryonic human 293 cells transferred with the pCEP4 or pCEP4 vector carrying the kd312 cDNA, were grown and processed for Western blotting with anti-kd312 antiserum as described in the Examples section. Lane 1, step of the Benchmark pre-stained protein (GibcoBRL). Lane 2, 293 cells transferred with pCEP4. Lane 3, 293 cells transferred with pCEP4 carrying the human kd312 cDNA insert. Lane 4, human kd312 and the thioredoxin fusion protein of partially purified E. coli from E. coli cells carrying the human kd312 insert in the expression vector pET-32a (+) (Materials and methods) . In lanes 2 and 3, the proteins of 150-200 μg and in lane 4, the protein of 5 ng were loaded into the gel. The anticipated molecular weight for the human kd312 protein is -32,000 and that of the kd312-thioredoxin fusion protein of pET-32a (+) is -51,000.

Figure 11 shows the SDS-PAGE analysis of the human kd312 protein produced by E. coli. The human kd312 gene was expressed from the pET-30a (+) - 2 vector and the kd312 protein was partially purified from E. coli cells using the Novagen His-Bind resin as described in the Examples section. The conditions for SDS-PAGE analysis were essentially the same as described in connection with Western analysis. The cell lysates were prepared from 400 ml of induced cells (OD600 = 0.6) and the kd312 protein was eluted from the His-Bind resin in 12 ml of elution buffer according to the protocol supplied by Novagen. In lane 2, 25 μl cell lysates and in lane 3, 5 μl of the protein eluted from the His-Bind resin was loaded onto the gel. Lane 1 is the standard molecular weight purchased from BIO-RAD.

Figure 12 shows two ideograms illustrating the chromosomal position of the human kd312 gene at 17pll.2 (See examples). Both ideograms are from International System for Human Cytogenetic Nomenclature (1995).

DETAILED DESCRIPTION OF THE INVENTION Included within the scope of this invention are the kd312 polypeptides, such as the polypeptides of SEQ ID NO: 2 (kd312-l of human) or SEQ ID NO: 5 (rat kd312-l), and fragments and derivatives of the same from related biologically active polypeptides. In addition, nucleic acid molecules encoding these polypeptides, methods for the preparation of the polypeptides and other related aspects are included within the scope of the present invention.

I. Proteins / Polypeptides kd312-1, Fragment s and Derivatives of the same s The term "kd312 protein" or "kd312 polypeptide" as used herein refers to any protein or polypeptide having the properties described herein for kd312. The kd312 polypeptide could or could not have an amino terminal methionine, depending, for example, on the manner in which it is prepared. By way of illustration, the kd312 protein or the kd312 polypeptide refer to: (1) an amino acid sequence encoded by the kd312 nucleic acid molecules as defined in any of the following points: (a) the nucleic acid molecules of SEQ ID NOS: 1, 3, 4 or 6; (b) the nucleic acid molecules encoding the polypeptides of SEQ ID NOS: 2 or 5, or the biologically active fragments thereof; (c) the nucleic acid molecules encoding the polypeptides that are at least 85 percent identical to the polypeptides of SEQ ID NOS: 2 or 5; (d) nucleic acid molecules that hybridize under stringent conditions to any of (a) - (c) above; Y (e) the nucleic acid molecules that are the complement of any of (a) - (d) above. (2) naturally occurring allelic variants of the kd312 gene (eg, human and rat kd312 genes; SEQ ID NOS: 1 and 4, respectively) resulting in one or more amino acid substitutions, deletions and / or insertions as compare with the kd312-l polypeptides of SEQ ID NO: 2 or SEQ ID NO: 5, and / or (3) chemically modified derivatives as well as nucleic acids and / or variants of amino acid sequences thereof as provided herein.

The kd312 polypeptides which have use in the practice of the present invention could be naturally occurring full-length polypeptides, or truncated polypeptides or peptides (i.e., "fragments").

The polypeptides could start at any of the three Met residues at positions 1, 6 and 10 of SEQ ID NOS: 2 and 5. Depending on which one is determined to be the first amino acid of the protein, the sequence could be numbered conveniently assigning the number 1 to the first Met residue thereof.

The polypeptides or fragments could be chemically modified, i.e., glycosylated, phosphorylated and / or linked to a polymer, as described below, and could have an amino terminal methionine, depending on how they are prepared. In addition, the polypeptides or fragments could be variants of naturally occurring kd312 polypeptides (ie, they could contain one or more deletions, insertions and / or amino acid substitutions as compared to naturally occurring kd312, eg, kd312-l) .

As used herein, the term "kd312 fragment" refers to a peptide or polypeptide that is less than the full-length amino acid sequence of the naturally occurring kd312 protein, but qualitatively has a substantially similar type of biological activity as the kd312 polypeptide or the kd312 protein described above. Such a fragment could be truncated at the amino terminus, the carboxy terminus or both, and could be modified chemically. Such kd312 fragments could be prepared with or without an amino terminal methionine. The activity of the fragments could be greater than, the same as, or less than the full-length kd312 (mature) polypeptide. Preferably, the activity of the fragment is = 50%, more preferably > 65%, more preferably > 80% of the activity of the total length polypeptide, as measured by a standard activity test, such as that established in the Examples section. Exemplary fragments of this invention include polypeptides wherein from 1 to 20 amino acids are removed from the C-terminus, the N-terminus or both terms of the kd312 polypeptide.

As used herein, the term "derivative of kd312" or "kd312 variant" refers to a kd312 polypeptide, protein or fragment that 1) has been chemically modified, such as, for example, by the addition of one or more molecules of polyethylene glycol, sugars, phosphates or other such unbound molecules naturally to the wild-type kd312 polypeptide, and / or 2) contains one or more substitutions, deletions and / or insertions of nucleic acid or amino acid sequences, as compared to the amino acid sequence kd312, such as those set forth in Figure 2.

As used herein, the terms "biologically active polypeptide" and "biologically active fragment" refer to a peptide or polypeptide according to the description above for kd312, wherein kd312 is capable of prolonging the survival of cells (eg, neutral or immunological cells).

Fragments and / or derivatives of kd312 that are not active in the activity tests, could be useful as modulators (e.g., inhibitors or stimulants) of the kd312 receptors or prepare vi antibodies for the kd312 polypeptides.

The amino acid variants of kd312 of this invention are preferably at least 85% identical to SEQ ID NO: 2 or SEQ ID NO: 5, more preferably at least about 90% identical, even more preferably at least about 95% identical .

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 program such as BLAST or FASTA, the two polypeptides to which the percent identity of the sequence is to be determined are aligned for optimal matching of their respective amino acids (the "matched distance", which may include the total length of one or both sequences, or a predetermined portion of one or both sequences). Each computer program provides a "non-identical" opening penalty and a "non-identical" separation penalty, and a registration matrix such as PAM 250. A standard registration matrix (see Dayhoff et al., In: At of Pro tein Se qu en ceand S tructu re, vol 5, suppl 3 (1978)) can be used in conjunction with the computer program. The identity percent can then be calculated using an algorithm contained in a program such as FASTA as: number of spaces introduced in the second section to align the two sections Polypeptides that are at least 85 percent identical will have one or more substitutions, deletions and / or amino acid insertions as compared to wild-type kd312. Usually, the substitutions will be conservative to have little or no effect on the overall net charge, polarity or hydrophobicity of the protein, but they could optionally increase the activity of kd312. Conservative substitutions are set forth in Table I below.

Table I Conservative amino acid substitutions The invention also encompasses homologous species of kd312; for example, kd312 homologs of a species of mammals, such as dog, cat, mouse, rat, monkey, horse, pig, goat, rabbit, calf and the like, in addition the human is also contemplated. The sequence of the exemplary rat protein, kd312-l, is provided as SEQ ID NO: 5.

The invention further encompasses the chimeric polypeptides, i.e., kd312 linked to all or a portion of another polypeptide. Preferably the chimeric polypeptide comprises kd312 linked to all or a portion of another factor. The polypeptides could be linked to the N to C terminus, C to C terminus or N to N terminus. They could also be linked directly, or they could be connected via a linker, such as a polyamino acid linker (e.g., poly-Gly). 11. Nucleic acids As used herein, the term "kd312" when used to describe a nucleic acid molecule refers to a nucleic acid molecule or fragment thereof, as set forth above.

The term "astringent conditions" refers to the conditions of hybridization or washing that allow only the binding of a nucleic acid molecule, such as an oligonucleotide or probe of cDNA molecule to the highly homologous sequences. An astringent wash solution is 0.015 M NaCl, 0.005 M Na citrate and 0.1 percent SDS used at a temperature of 55-65 ° C. Another astringent wash solution is 0.2 X SSC and 0.1 percent SDS used at a temperature between 50-65 ° C. When oligonucleotide probes are used to screen cDNA or genomic libraries, the following astringent washing conditions could be used. One protocol uses 6 X SSC with 0.05 percent sodium pyrophosphate at a temperature of 35-62 ° C, depending on the length of the oligonucleotide probe. For example, probes of 14 base pairs are washed at 35-40 ° C, probes of 17 base pairs are washed at 45-50 ° C, probes of 20 base pairs are washed at 52-57 ° C, and probes 23 base pairs are washed at 57-63 ° C. The temperature can be increased 2-3 ° C where the non-specific binding of origin seems high. A second protocol uses tetramethylammonium chloride (TMAC) for the washing of the oligonucleotide probes. An astringent wash solution is TMAC 3 M, 50 mM Tris-HCl, pH 8.0, and 0.2 percent SDS. The wash temperature used by this solution is a function of the length of the probe. For example, a probe of 17 base pairs is washed at about 45-50 ° C.

Molecules, fragments and / or nucleic acid derivatives of kd312 that do not encode polypeptides that are active in the activity tests, could be useful as hybridization probes in diagnostic tests to be tested, qualitatively or quantitatively, for the presence of kd312 DNA or RNA in mammalian tissue or body fluid samples.

Kd312 nucleic acid molecules that encode kd312 polypeptides linked to a chimeric polypeptide as described hereinabove are also included within the scope of this invention.

III Methods for the preparation of kd312 polypeptides A Recombinant methods The full-length kd312 polypeptide or fragment thereof can be prepared using known recombinant DNA technology methods, such as those set forth in Sambrook et al. . { Mo l C u l a r C on i n g: 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 ans Sons, NY [1994]). A gene or cDNA encoding the kd312 protein or fragment thereof could be obtained, for example, by screening a genomic or cDNA library or by PCR amplification. Alternatively, a gene encoding the kd312 polypeptide or fragment, could be prepared by 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]). These methods include, inter alia, the phosphorus phosphate method, phosphoramidite and H-phosphonate for the synthesis of nucleic acids. A preferred method for such chemical syntheses is the synthesis supported on polymers using the chemistry of the standard phosphoramidite. Typically, the DNA encoding the kd312 polypeptide will be several hundred nucleotides in length. Nucleic acids greater than about 100 nucleotides can be synthesized as several fragments using these methods.

The fragments can then be ligated together to form the full length kd312 polypeptide. Usually, the DNA fragment encoding the amino terminus of the polypeptide will have an ATG, which encodes a methionine residue. This methionine may or may not be present in the mature form of the kd312 polypeptide, depending on whether the polypeptide produced in the host cell is secreted from such a cell.

In some cases, it may be desirable to prepare naturally occurring kd312 nucleic acid and / or amino acid variants. Nucleic acid variants (wherein one or more nucleotides are designed to differ from naturally occurring or wild-type kd312) could be produced using site-directed mutagenesis or PCR amplification, 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, could also be used to prepare such variants. Other methods known to the person skilled in the art could also be used. Preferred nucleic acid variants are those containing the nucleotide substitutions that account for the codon preference in the host cell to be used to produce kd312. Other preferred variants are those that encode the conservative amino acid changes as described above (eg, wherein the charge or polarity of the naturally occurring amino acid side chain is substantially not altered by substitution with a different amino acid) as compared to the wild type, and / or those designed to generate a new glycosylation and / or phosphorylation site in kd312, or those designed to eliminate a glycosylation site and / or existing phosphorylation in kd312.

The kd312 gene or cDNA can be inserted into an expression vector appropriate for expression in a host cell. The vector is selected to be functional in the particular host cell employed (i.e., the vector is compatible with the machinery of the host cell, so that amplification of the kd312 gene and / or expression of the gene can occur). The kd312 polypeptide or fragment thereof could be amplified and / or expressed in prokaryotic host cells, yeast, insects (baculovirus systems) and / or eukaryotes. The selection of the host cell will depend at least in part on whether the kd312 polypeptide or fragment thereof will be glycosylated. If so, yeast, insect or mammalian host cells are preferable; the yeast cells will glycosylate the polypeptide, and insect or mammalian cells can glycosylate and / or phosphorylate the polypeptide as naturally occurring in the kd312 polypeptide (i.e., "native" glycosylation and / or phosphorylation).

Typically, the vectors used in any of these host cells will contain the 5 'flanking sequence (also referred to as a "promoter") and other regulatory elements, as well as an enhancer, an origin of replication element, a transcriptional termination element, a complete intron sequence that contains a donor and acceptor binding site, a peptide sequence signal, a ribosome binding site element, a polyadenylation sequence, a polylinker region to insert the nucleic acid encoding the polypeptide that goes to be expressed, and a selectable marker element. Each of these elements is discussed later. Optionally, the vector could contain a "tag" sequence, i.e., an oligonucleotide sequence located at the 51 or 31 end of the coding sequence of kd312 encoding polyHis (such as hexaHis) or another small immunogenic sequence. This tag will be expressed together with the protein, and may serve as an affinity tag for the purification of the kd312 polypeptide from the host cell. Optionally, the tag can be subsequently removed from the purified kd312 polypeptide by various means, such as using for example, a selected peptidase.

The 5 'flanking sequence could be homologous (ie, of the same species and / or strain as the host cell), heterologous (ie, of a species other than the host cell species or strain), hybridized (ie, a combination of the flanking sequences 5 'of more than one source), synthetic, or could be the flanking sequence 5' of native kd312. As such, the source of the flanking sequence 5 'could be any prokaryotic or eukaryotic unicellular organism, any vertebrate or invertebrate organism or any plant, with the proviso that the flanking sequence 5' is functional in, and can be activated by, the machinery of the host cell.

The flanking sequences 51 used in the vectors of this invention could be obtained by any of the methods well known in the art. Typically, the 5 'flanking sequences used here to part of the 5' flanking sequence of kd312 will have to have been previously identified by map formation and / or by restriction endonuclease digestion and thus, can be isolated of the appropriate tissue source using the appropriate restriction endonucleases. In some cases, the complete nucleotide sequence of the 5 'flanking sequence could be known. Here, the flanking sequence 5 'could be synthesized using the methods described above for the synthesis or cloning of nucleic acids.

When all or only a portion of the 5 'flanking sequence is known, it could be obtained using PCR and / or by screening a genomic library with the fragments of the appropriate oligonucleotide and / or 5' flanking sequence of the same or another species .

When the 5 'flanking sequence is not known, a DNA fragment containing a flanking sequence 'could be isolated from a larger piece of DNA that could contain, for example, a coding sequence or even another gene or genes. Isolation could be performed by restriction endonuclease digestion using one or more carefully selected enzymes to isolate the appropriate DNA fragment. After digestion, the desired fragment could be isolated by agarose gel purification, Qiagen® column or other methods known to the person skilled in the art. The selection of the appropriate enzymes to accomplish this purpose will be readily apparent to one skilled in the art.

The origin of the replication element is typically a part of the commercially acquired prokaryotic expression vectors, and aids in the amplification of the vector in a host cell. The amplification of the vector up to a certain number of copies, in some cases, may be important for the optimal expression of the kd312 polypeptide. If the vector of choice does not contain a replication site origin, it could be synthesized chemically based on a known sequence, and ligated into the vector.

The transcription termination element is typically located 3 'from the end of the coding sequence of the kd312 polypeptide and serves to terminate the construction of the kd312 polypeptide. Usually, the transcription termination element in prokaryotic cells is a GC-rich fragment followed by a poly T sequence. While the element is easily cloned from a library or commercially purchased as part of a vector, it can also be easily synthesized using the methods for the synthesis of nucleic acids such as those described above.

An element of the selected marker gene encodes a protein necessary for the survival and growth of a host cell that grows in a selective culture medium. Genes of the typical selection marker encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, tetracycline or kanamycin for prokaryotic host cells, (b) complement the auxotrophic deficiencies of the cell; or (c) provide critical nutrients not available from the complex medium. Preferred selected labels are the kanamycin resistance gene, the ampicillin resistance gene and the tetracycline resistance gene.

The ribosome binding element, commonly called the Shine-Dalgarno sequence (prokaryotes) or the Kozak sequence (eukaryotes), is necessary for the initiation of mRNA translation. The element is typically located 3 'to the promoter and 51 to the coding sequence of the kd312 polypeptide to be synthesized. The Shine-Dalgarno sequence is varied, but is typically a polypurine (i.e., which has a high content of A-G). Many Shine-Dalgarno sequences have been identified, each of which can be easily synthesized using the methods set forth above and used in a prokaryotic vector.

In many cases, the transcription of the kd312 polypeptide is increased by the presence of one or more introns in the vector; this is particularly true when kd312 is produced in eukaryotic host cells, especially in mammalian host cells. The introns used could naturally occur within the kd312 nucleic acid sequence, especially when the kd312 sequence used is a full length genomic sequence or a fragment thereof. When the intron does not occur naturally within the DNA sequence of kd312 (as for most cDNAs), the introns could be obtained from another source. The position of the intron with respect to the flanking sequence 51 and the coding sequence kd312 is important, as the intron could be transcribed to be effective. As such, when the kd312 nucleic acid sequence is a DNA sequence, the preferred position for the intron is 3 'with respect to the transcription start site, and 5' with respect to the polyA transcription termination sequence. Preferably, for kd312 cDNAs, the intron will be located on one side or the other (i.e., 5 'or 3') of the coding sequence of kd312, so as not to interrupt this coding sequence. Any intron of any source, including any viral, prokaryotic and eukaryotic organism (plant or animal), could be used for the practice of this invention, with the proviso that it is compatible with the host cells into which it is inserted. Synthesis introns are also included here. Optionally, more than one intron could be used in the vector.

When one or more of the elements set forth above are not present in the vector to be used, they could be obtained and ligated individually into the vector. The methods used to obtain each of the elements are well known to those skilled in the art and are comparable with the methods set forth above (i.e., DNA synthesis, screening of the library and the like).

The final vectors used for the practice of this invention are typically constructed from a start vector, such as a commercially available vector. Such vectors may or may not contain some elements to be included in the completed vector. If none of the desired elements are present in the start vector, each element could be linked individually in the vector by cutting the vector with the appropriate restriction end point (s), so that the ends of the element that is going to ligate and at the ends of the vector are compatible for ligation. In some cases, it may be necessary to "truncate" the ends to be ligated together to obtain a satisfactory ligation. The truncation is carried out first filling in "adhered ends" using the Klenow DNA polymerase or the T4 DNA polymerase in the presence of four nucleotides. This method is well known in the art and is described, for example, in Sambrook et al. , upra.

Alternatively, two or more of the elements to be inserted in the vector could first be ligated together (if they are placed adjacent to each other) and then ligated into the vector.

Another method for constructing the vector is to carry out all the ligations of the various elements simultaneously in a reaction mixture. Here, many non-sense or non-functional vectors will be generated due to the ligation or improper insertion of the elements, however, the functional vector could be identified and selected for the digestion of the restriction endonuclease.

Preferred vectors for the practice of this invention are those which are compatible with bacterial, insect and / or mammalian host cells. Such vectors include, inter alia, pCRII (Invitrogen Company, San Diego, CA), pBSII (Stratagene Company, LaJolla, CA) and pETL (BlueBacII; Invitrogen).

After the vector has been constructed and a kd312 nucleic acid has been inserted into the appropriate site of the vector, the entire vector could be inserted into a suitable host cell for the amplification and / or expression of the kd312 polypeptide.

The host cells could 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 kd312 protein that can be subsequently collected from the culture medium (if the host cells are secreted in the culture medium) or directly from the host cell that produces it (if not). secret). After the collection, the kd312 protein can be purified using the methods, such as chromatography of a molecular sieve, affinity and pure chromatography.

The selection of the host cells will depend in part on whether the kd312 protein will glycosylate or phosphorylate (in this case, eukaryotic host cells are preferred), and the manner in which the host cell is able to "fold" the protein in its native tertiary structure. { e. g. , the proper orientation of the disulfide bridges, etc.), so that the biologically active protein is prepared by the cell. However, when the host cell does not synthesize biologically active kd312, the kd312 could be "folded" after the synthesis using the appropriate chemical conditions as discussed below.

Appropriate cells or cell lines could be mammalian cells, such as Chinese hamster ovary (CHO) cells or 3T3 cells. The selection of mammalian host cells and the methods for transformation, culture, amplification, screening and production and purification of the product are known in the art. Other appropriate mammalian cell lines are the COS-1 and COS-7 monkey cell lines, and the CV-1 cell line. Examples of additional mammalian host cells include primate cell lines and rodent cell lines, including transformed cell lines. Normal diploid cells, cell strains derived from the in vitro culture of primary tissue, as well as primary explants, are also appropriate. The candidate cells could be genotically different in the selection gene, or they could contain a dominantly acting selection gene. Other appropriate mammalian cell lines include, but are not limited to, HeLa, mouse L-929 cells, 3T3 lines derived from Swiss, Balb-c or NIH mice, BHK or HaK hamster cell lines.

Similarly, useful as host cells suitable for the present invention are bacterial cells. For example, the various strains of E. coli (e.g., HB101, DH5a, DH10, DH125 and MC1061) are well known as host cells in the field of biotechnology. Several strains of B. subtilis, Pseudommonas ssp., Others Bacillus ssp. , Streptomyces ssp. , and the like could also be employed in this method.

Many strains of yeast cells known to those skilled in the art are also available as host cells for the expression of the polypeptides of the present invention. Additionally, when desired, insect cells could be used as host cells in the method of the present invention (Miller et al., Genetic Engineering 8: 277-298 [1986] The insertion (also referred to as "transformation" or "transfection") of the vector in the selected host cell could be performed using such methods as calcium phosphate, electroporation, microinjection, lipofection or the DEAE-dextran method. The selected method will be in part a function of the type of host cell to be used. These methods and other suitable methods are well known to those skilled in the art, and are established, for example, in Sambrook et al. , supra.

The host cells containing the vector (i.e., transformed or transferred) could be cultured using the standard medium well known to the person skilled in the art. The medium will usually contain all the nutrients necessary for the growth and survival of the cells. The appropriate medium for the culture of E cells. coli are for example, Luria broth (LB) and / or Terrific broth (TB). Suitable media for the culture of eukaryotic cells are RPMI 1640, MEM, DMEM, all of which could be supplemented with serum and / or growth factors as required by the cell line that is cultured. An appropriate medium for insect cultures is Grace's medium supplemented with yeastolato, hydrolysed lactalbumin and / or fetal calf serum as needed.

Typically, an antibiotic or other compound useful for the selective growth of the transformed cells is only added as a supplement to the medium. The compound to be used will be dictated by the selected marker element present in the plasmid with which the host cell is transformed. For example, when the selected marker element is resistant to kanamycin, the compound added to the culture medium will be kanamycin.

The amount of the kd312 polypeptide produced in the host cell can be evaluated using standard methods known in the art. Such methods include, without limitation, Western blot analysis, SDS-polyacrylamide gel electrophoresis, undenatured gel electrophoresis, HPLC separation, immunoprecipitation and / or activity tests.

If the kd312 polypeptide is designed to be secreted from the host cells, the majority of the polypeptide could be in the cell culture medium. The polypeptide prepared in this manner will typically not possess an amino terminal methionine as it is removed during cell secretion. If, however, the kd312 polypeptide is not secreted from host cells, it will be present in the cytoplasm (for host cells of eukaryotic, gram-positive and insect bacteria) or in the periplasm (for host cells of gram-negative bacteria) and could have an amino terminal methionine.

For the intracellular kd312 protein, the host cells are typically disrupted first mechanically or osmotically to release the contents of the cytoplasm in a buffer solution. The kd312 polypeptide can then be isolated from this solution.

Purification of the kd312 polypeptide from the solution can be performed using a variety of techniques. If the polypeptide has been synthesized, so that it contains a label such as Hexahistidine (kd312 / hexaHi s) or another small peptide, in either its carboxyl or amino terminus, it could be purified essentially in a one-step process by passing the solution through of an affinity column, wherein the column matrix has a high affinity for the tag or for the polypeptide directly (ie, a monoclonal antibody that specifically recognizes kd312). For example, pol ihi st idina binds with great affinity and specifically nickel, thus, a nickel affinity column (such as the nickel Qiagen columns) can be used for the purification of kd312 / polyHi s. (See, for example, Ausubel et al., Eds., Curren t Pro t olol i Mol ecu l a r Bi olgy, Section 10.11.8, John Wiley &Sons, New York [1993]).

When the kd312 polypeptide is unlabeled and antibodies are not available, other well-known methods for purification can be used. Such methods include, without limitation, ion exchange chromatography, molecular screening chromatography, HPLC, native gel electrophoresis in combination with gel elution, and preparative isoelectric focusing ("Isoprime" machine / technique, Hoefer Scientific). In some cases, two or more of these techniques could be combined to achieve increased purity. Preferred methods for purification include poly iHi st idine labeling and ion exchange chromatography in combination with preparative isoelectric focusing.

If it is anticipated that the kd312 polypeptide will be found primarily in the periplasmic space of bacteria or the cytoplasm of eukaryotic cells, the contents of the periplasm or cytoplasm, including inclusion bodies (eg, gram negative bacteria), if the processed polypeptide has formed such complexes can be extracted from the host cell using any standard technique known to the person skilled in the art. For example, host cells can be used to release the contents of the periplasm by French press, homogenization and / or sonication. The homogenate can then be centrifuged.

If the kd312 polypeptide has formed inclusion bodies in the periplasm, the inclusion bodies can frequently bind to the inner and / or outer cell membranes and thus will be found mainly in the agglomerated material after centrifugation. The agglomerated material can then be treated with a chaotropic agent, such as guanidine or urea to liberate, break and solubilize the inclusion bodies. The kd312 polypeptide in its now soluble form can then be analyzed using gel electrophoresis, immunoprecipitation or the like. If it is desired to isolate the kd312 polypeptide, isolation could be carried out using standard methods, such as those set forth below and in Mardston et al. . { Me th In z. , 182: 264-275 [1990]).

If the inclusion bodies of the kd312 polypeptide are not formed to a significant degree in the periplasm of the host cell, the kd312 polypeptide will mainly be found in the supernatant after centrifugation of cellular homogenate, and the kd312 polypeptide can be isolated from the supernatant using the methods, such as those established later.

In situations where it is preferable to partially or completely isolate the polypeptide, the purification can be performed using standard methods well known to the person skilled in the art. Such methods include, without limitation, separation by electrophoresis followed by elect roelution, various types of chromatography (immunoaffinity, molecular screening, 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.

Chemical synthesis methods In addition to the preparation and purification of the kd312 polypeptide using recombinant DNA techniques, the kd312 polypeptides, fragments and / or derivatives thereof could be prepared by chemical synthesis methods (such as solid-phase peptide synthesis) using the methods known in the art. art, such as those established by Merrifield et al. , (J Am. Ch em. So c., 85: 2149 [1964]), Houghten et al. (Pro c Na t i Acad. S ci USA, 82: 5132 [1985]), and Stewart and Young (Solid Phase Peptide Synthesis, Pierce Chem Co, Rockford, IL [1984]). Such polypeptides could be synthesized with or without a methionine at the amino terminus. Chemically modified kd312 polypeptides or fragments could be oxidized using the methods set forth in these references to form disulfide bridges. The kd312 polypeptides or fragments could be used as biologically active or immunological substitutes for the natural, purified kd312 polypeptides in the therapeutic and immunological processes.

IV. Chemically modified kd312 derivatives The chemically modified kd312 compositions. { i. e. , "derivatives") wherein the kd312 polypeptide is linked to a polymer ("kd312 polymers") are included within the scope of the present invention. The selected polymer is typically soluble in water, so that the protein to which it binds does not precipitate in an aqueous environment, such as a physiological environment. The selected polymer is usually modified to have a simple reactive group, such as an active ester for acylation or an aldehyde for alkylation, so that the degree of polymerization could be controlled as provided in the present methods. The polymer could be of any molecular weight, and could be branched or unbranched. A mixture of polymers is included within the scope of the kd312 polymers. Preferably, for the therapeutic use of the preparation of the terminal product, the polymer will be pharmaceutically acceptable.

The water-soluble polymer or mixture thereof could be selected from the group consisting of, for example, polyethylene glycol (PEG), mono-toxy-polyalkylene glycol, dextran, cellulose or other polymers based on carbohydrates, poly i- (N -vini 1-pyrrolidone) polyethylene glycol, homopolymers of propylene glycol, a copolymer of propylene oxide / ethylene oxide, polyoxyethylated polyols (eg, glycerol) and polyvinyl alcohol.

For the acylation reactions, the selected polymer should have a simple reactive ester group. For reductive alkylation, the selected polymers should have a simple reactive aldehyde group. A preferred reactive aldehyde is polyethylene glycol propionaldehyde, which is stable in water, or monoalkoxy or C1-C10 aryloxy derivatives thereof (see U.S. Patent 5, 252, 714).

The pegylation of kd312 could be carried out by any pegylation reaction known in the art, as described for example in the following references: Foc u s on Gro wth Fa c t ors 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 a reactive polyethylene glycol molecule (or a reactive water-soluble polymer) as described below.

A water soluble polymer particularly preferred for use herein, is polyethylene glycol, abbreviated PEG. As used herein, polyethylene glycol means that it encompasses any of the forms of PEG that have been used to derive other proteins, such as mono (C! -C10) alkoxy or ary loxi-polyethanol glycol.

In general, the chemical derivation could be carried out under any appropriate condition used to react a biologically active substance with an activated polymer molecule. Methods for the preparation of pegylated kd312, in general, will comprise the steps of (a) reacting a kd312 polypeptide with polyethylene glycol (such as a reactive ester or PEG aldehyde derivative) under the conditions whereby kd312 becomes bound to one or more PEG groups and (b) obtain the reaction product or products. In general, the optimal reaction conditions for acylation will be determined based on the known parameters and the desired result. For example, the highest ratio of PEG: prot ein, the highest percentage of the poly-pegylated product.

In general, the conditions that could be reduced or modulated by the administration of the present polymer / kd312 generally include those described herein for the kd312 molecules. However, the polymer / kd312 molecules described herein could have additional activities, improved or reduced activities or other characteristics, compared to non-derived molecules.

V Combinations The kd312 polypeptides and fragments thereof, either chemically or unmodified, could be employed alone or in combination with other pharmaceutical compositions such as, for example, cytokines, interferons, interleukins, growth factors, antibiotics, anti-inflammatories, chemotherapeutic agents , in the treatment of various conditions, such as cancer, immunodeficiency and neurodegeneration.

SAW . Antibodies The kd312 polypeptides, fragments and / or derivatives thereof could be used to prepare antibodies generated by standard methods. In this manner, antibodies that react with kd312 polypeptides, as well as reactive fragments of such antibodies, are also contemplated within the scope of the present invention. The antibodies could be polyclonal, monoclonal, recombinant, chimeric, single chain and / or bispecific. Typically, the antibody or fragment thereof will be "humanized", i.e., prepared to prevent or minimize an immune reaction to the antibody when administered to a patient. The antibody fragment could be any fragment that is reactive with kd312 of the present invention, such as, Fab, Fab ', etc. Hybridomas generated by the presence of kd312 or a fragment thereof as an antigen for a selected mammal are also provided by this invention, followed by fusion of the cells (eg, spleen cells) of a mammal with certain cancer cells, to create cell lines immortalized by known techniques. The methods employed to generate such cell lines and antibodies directed against all or portions of a human kd312 polypeptide of the present invention are also encompassed by this invention.

The antibodies could be used therapeutically, such as to inhibit the binding of kd312 to their substrates. The antibodies could be used additionally for diagnostic purposes in vivo and in vivo, such as in the labeled form for detecting the presence of the kd312 polypeptide in a tissue.

Antibodies against kd312-1 are preferred, particularly active and human fragments thereof.

VII. Therapeutic compositions and administration of the same As used herein, the terms "effective amount" and "therapeutically effective amount" refer to the amount of kd312 necessary to support one or more biological activities of kd312 as set forth herein.

Therapeutic compositions for treating various conditions or diseases associated with cell death are within the scope of the present invention.

Tale compositions could comprise a therapeutically effective amount of a kd312 polypeptide, a fragment thereof (any of which could be chemically modified) or a modulator of the activity of kd312, (collectively, a "kd312 therapeutic compound) in admixture with a pharmaceutically vehicle. The material of the vehicle could be water for injection, preferably supplemented with other materials common in solutions for administration to mammals Typically, a kd312 therapeutic compound will be administered in the form of a composition comprising the therapeutic compound kd312 in conjunction with one or more physiologically acceptable carriers, excipients or diluents.Saline solutions or neutral buffered saline solutions mixed with whey albumin are examples of suitable vehicles.Preferably, the product is formulated as a lyophilizate using the appropriate excipients (eg, sucrose). Standard excipients, diluents and excipients could be included as desired. An example composition comprises citrate buffer of about pH 4.0-4.5, which could also include NaCl.

The kd312 compositions can be administered systemically parenterally. Alternatively, the compositions could be administered intravenously or subcutaneously. When administered systemically, the therapeutic compositions for use in this invention could be in the form of a parenterally acceptable, pyrogen-free aqueous solution. The preparation of such pharmaceutically acceptable protein solutions, with due consideration to pH, isotonicity, stability and the like, is within the skill of the art.

Therapeutic formulations of the kd312 compositions useful for the practice of the present invention, could be prepared for storage by mixing the selected composition having the desired degree of purity with the optional physiologically acceptable carriers, excipients or stabilizers. { Rem i n gt on 's Ph a rm a ce u t i ca l S ci ces, 18th edition, A.R. Gennaro, ed., Mack Publishing Company [1990]) in the form of a lyophilized cake or an aqueous solution. Acceptable vehicles, excipients or stabilizers are not toxic to the recipients and are preferably inert to the dosages 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 and other carbohydrates including glucose, mannose or dextrins; chelating agents, such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions, such as sodium; and / or nonionic surfactants such as Tween, Pluronics or polyethylene glycol (PEG).

The composition of kd312 to be used for the administration should be sterile. This is easily done by filtration through sterile filtration membranes. When the composition of kd312 is lyophilized, sterilization could be carried out using these methods either before or after lyophilization and reconstitution. The composition for parenteral administration will ordinarily be stored in the lyophilized form or in solution The therapeutic compositions, in general, are placed in a container having a sterile access port, for example, an intravenous solution bag or ampoule having a plug pierceable by a hypodermic injection needle.

The route of administration of the composition is in accordance with known methods, e.g. oral routes, injection or infusion by intravenous, intraperitoneal, intracerebral (intraparenchymal), int racerebrovent ri cular, intramuscular, intraocular, int r aart er 1 or int lesional, or by prolonged release systems or implantation device that could optionally involve the use of a catheter.

When desired, the compositions could be administered continuously by infusion, bolus injection or by implantation devices. Alternatively or additionally, kd312 could be administered locally via implantation in the affected area of a membrane, sponge or other appropriate material in which the polypeptide has been absorbed.

When an implantation device is used, the device could be implanted in any tissue or organ, such as, for example, in a cerebral ventricle or cerebral parenchyma and the release of kd312 could be directly through the device via administration of bolus or continuous, or via a catheter using continuous infusion.

The kd312 polypeptide could be administered in a formulation or prolonged release preparation. Suitable examples of sustained release preparations include semipermeable polymer matrices in the form of molded articles, e.g. films or microcapsules. Prolonged release matrices include polyesters, hydrogels, polylactides (US 3,773,919, EP 58,881), copolymers of L-glutamic acid and gamma et i 1-L-glutamine (Sidman et al, Biopol imers, 22: 547-556 [1983] ), poly (2-hydroxyethyl and 1-methylacrylate) (Langer et al., J. Biomed, Mater. Res., 15: 167-277 [1981] and Langer, Chem. Tech., 12: 98-105 [ 1982]), ethylene vinyl acetate (Langer et al., Supra) or poly-D- (-) -3-hydroxybutyric acid (EP 133,988). Sustained-release compositions may also include liposomes, which may be prepared by any of the methods known in the art (eg, DE 3,218,121; Epstein et al., Proc. Nati. Acad. Sci. USA, 82: 3688-3692 [1985 ]); Hwang et al. , Pro c. Na t i. Aca d. S ci. USA, 77: 4030-4034 [1980]; EP 52,322; EP 36,676; EP 88,043; EP 143,949).

In some cases, it may be desirable to use kd312 compositions in an ex vivo manner, i.e., to treat cells or tissues that have been removed from the patient and then subsequently implanted back into the patient.

In other cases, kd312 could be released through the implantation in cells of certain patients who have been genetically engineered to express and secrete the kd312 polypeptide. Such cells could be cells of animals or humans and could be derived from the patient's own tissue or from another source, be it human or non-human. Optionally, the cells could be immortalized. The cells could be implanted in the brain, adrenal gland or in other tissues or appropriate body organs of the patient.

In certain situations, it may be desirable to use gene therapy methods for the administration of kd312 to patients suffering from certain conditions (e.g., neurological, immunological and others). In these situations, genomic DNA, cDNA and / or synthetic DNA encoding kd312 or a fragment or variant thereof, could be operably linked to a constitutive or inducible promoter that is active in the tissue in which the composition will be injected. This DNA construct of kd312, either inserted into a vector, or alone without a vector, can be injected or otherwise delivered directly to the brain, heart or other tissue, whether neuronal or non-neuronal.

An effective amount of the composition of kd312 to be used therapeutically will depend, for example, of the therapeutic objectives, such as the indication for which kd312 is being used, the route of administration, and the condition of the patient. Therefore, it will be necessary for the titular therapist to dose and modify the route of administration as required to obtain the optimal therapeutic effect. A typical dosage daily could be in the range of about 0.1 μg / kg up to 10 mg / kg or more, depending on the factors mentioned above. Typically, a clinician will administer the kd312 composition until a dosage is reached that achieves the desired effect.

The composition of kd312 could therefore be administered as a single dosage, or as two or more dosages (which may or may not contain the same amount of kd312) all the time, or as an implantation device via continuous infusion or catheter .

As more studies are carried out, the information will emerge considering the appropriate dosage levels for the treatment of several conditions in several patients, and the worker skilled in the art, considering the therapeutic context, the type of condition under treatment, age and The general health of the recipient will be able to verify the appropriate dosage.

VIII Diagnostic uses RNA levels of kd312 and proteins could be measured for diagnostic purposes. Such levels could be indicative of the presence or progression of various diseases, such as cancer, immunodeficiency diseases (eg, AIDS), seizures, heart attacks, head trauma and neurodegenerative diseases (eg, Parkinson's disease and Alzheimer's disease), preferably cancer.

IX. Conditions that are going to be treated with kd312 The kd312 proteins, fragments and / or derivatives thereof could be used to treat diseases and conditions associated with alterations in cell proliferation / death that could benefit exposure to kd312 or anti-kd312 antibodies.

The kd312 protein and / or fragments or derivatives thereof, could be used directly to treat patients suffering from cancer, immunodeficiency diseases (eg, AIDS), seizures, heart attacks, head trauma and neurodegenerative conditions (eg, Parkinson's and Alzheimer's disease).

X modulators of the kd312 levels In some situations, such as cancer treatment, it may be desirable to inhibit or significantly decrease the level of activity or expression of kd312. Compounds that inhibit the activity / expression of kd312 could be administered either in an ex vivo manner, or in a viral manner by local or intravenous (iv) injection, or by oral delivery, implantation device or the like. Exemplary inhibitory compounds are antisense oligonucleotides against kd312 genes, antibodies against kd312 proteins, and small molecule inhibitors of kd312 expression or activity.

In other situations, such as in cases where cell survival is desirably increased (eg, AIDS, attack, neurodegenerative diseases, head / brain trauma and heart attack, etc.), it may be desirable to improve or significantly increase the level of the activity or expression of kd312. Compounds that increase the activity or expression of kd312 could be administered either in an ex vivo manner or in a viral manner by local or iv injection, or by oral delivery, implantation device or the like.

The tests described below provide examples of the methods used for the identification of compounds that could inhibit or improve the activity of kd312.

For ease of reading, the following definition is used here to describe the tests: Test molecules "refers to a molecule or molecules that are under evaluation as a modulator of kd312, e.g., by virtue of its potential ability to block or improve the biological activity of kd312.

Several types of tests that use the purified protein could be carried out to identify the compounds that affect the function of kd312. Such an effect could be performed by a compound that typically inhibits or improves the level of activity or expression of kd312.

Two easily performed tests can be used to screen the test molecules for inducers or inhibitors of genomic kd312. 1. RT-PCR can be performed to estimate the message level of kd312 in cells that normally express kd312 in the presence and absence of several test molecules. 2. A reporter gene can be fused to the genomic kd312 and the fusion gene can then be cloned into a vector and introduced into the cells that normally ess kd312. The reporter protein activities can be measured to estimate the ession levels of kd312 in response to a test molecule.

For example, a fusion gene between rat genomic kd312 and firefly luciferase gene has been constructed for this purpose since a luciferase assay system is commercially available (PROMEGA) and is sensitive and precise. In this construct the kd312 coding region for the CAAX domain of membrane blank was removed to ensure that the luciferase protein is freely available within the cells for detection. The rat KD12 coding region was replaced between the ATG triplet specifying the initiation codon for translation and the Sacll restriction site within the first exon in frame with the total coding region of the firefly luciferase gene. The nucleotide sequences potentially important for the regulation of kd312, which include the transcribed region of the flanking sequences at the 5 'and 3' end and the simple intron sequence, are not completely altered in the fusion gene.

Typically, the test molecule will be tested over a range of concentrations, and a series of controls that lack one or more elements of the tests can be used for accuracy in the evaluation of the results.

In some cases, it may be desirable to evaluate two or more test molecules together for use in decreasing or increasing the activity of kd312. In these cases, the tests set forth above can be easily modified by adding such additional test molecules either simultaneously with, or subsequently to, the first test molecule. The rest of the stages in the test may be as stated above.

XI. Transgenic mammals Also 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 the human equivalent of kd312 (eg, kd312-l) ) has been interrupted ("deleted") so that the ession level of this gene is significantly decreased or completely removed. Such mammals could be prepared using techniques and methods, such as those described in U.S. Pat. No. 5,557,032. The present invention also includes non-human mammals such as mice, rats, rabbits, goats or sheep in which the gene (or genes) encoding kd312 (either the native form of kd312 for the mammal or a heterologous kd312 gene) is over essed by the mammal, whereby a "transgenic" mammal is created. Such transgenic mammals could be prepared using well known methods such as those described in Patent No. 5,489,743 and PCT patent application no. W094 / 28122, published December 8, 1994.

The examples are intended for illustration purposes only, and should not be construed as limiting the scope of the invention in any way.

EXAMPLES EXAMPLE 1 Animal Experiments Sprague-Dawley rat blood was withdrawn by cardiac puncture at 0, 3 and 8 h. Immediately after each blood removal, the animals were injected with an equal volume of 0.9% NaCl to avoid hypovolume shock. Three hours after the final blood removal, a small blood sample was taken and the hematocrit reading was determined. The animals were then sacrificed and the internal organs removed for future experiments. Organ samples were also obtained from normal animals for control experiments.

Molecular biology Total RNA and polyA + RNA were isolated using Fasttrack isolation kits from Invitrogen Corporation. The cDNA libraries were constructed in the plasmid pSPORT 1 (for rat polyA + RNA with blood loss) and in the plasmid pSPORT 2 (for polyA + RNA of normal rats) using the GIBCO BRL SuperScript ™ plasmid system for the synthesis of cDNA and the cloning of the plasmid. Subtractive hybridization to find the genes that induce the bleeding of rat kidneys, was performed according to Gruber et al. 1993, Focus 15, Number 3, pp. 59-65. After two rounds of subtractive hybridization, the individual colonies were poured and their DNA was isolated and analyzed on agarose gel. A major contaminant band was found in the gel and was identified as an elimination product of the vector pSPORTl after the determination of its nucleotide sequence. The larger circular DNA of the contaminating molecule was extracted from the gel and used in the transformation of the E host. co l i DH12S (GIBCO BRL) to contract a stolen library. Individual colonies from this library were screened for genes that induce bleeding by DNA sequencing on each isolated plasmid and RT-PCR on polyA RNA from the kidneys of normal rats and rats with severe blood loss, based on nucleotide sequences of inserts in the plasmids The nature that induces bleeding of each candidate gene was confirmed by Northern analysis The nucleotide sequences of all the clones derived in this project were determined by the Amgen sequencing group RT-PCR was performed using the SuperScript Pre-amplification system for the single-stranded DNA synthesis of GIBCO BRL and the central PCR kit from Boehringer Mannheim The oligonucleotides were synthesized by Amgen-Boulder Northern analysis was performed using the NouthernMax Northern Ambion kit .

The rat kd312 cDNA originally isolated after subtractive hybridization lacked its 5 'end (encoding amino acids 232 of 280). The 5 * end of the molecule was obtained using the 5 'RACE system for the rapid amplification of the Gibco BRL cDNA ends and assembled with the 3' portion of the molecule. A fragment of the human -500 base pair kd312 gene was isolated from cDNA synthesized from human brain polyA + RNA using the 5 'RACE method and a primer from the rat kd312 sequence. Based on the nucleotide sequence of this fragment and the published nucleotide sequence of the R-ras gene, the biotinylated oligonucleotide probes kd312 and R-ras from human (18-22 bases) were synthesized and the cDNA molecules of R-ras and kd312 of human total length were isolated from a human kidney cDNA library (Gibco BRL) using the Gene Trapper cDNA Positive Selection System from Gibco BRL. Genomic DNA from rat and human kd312 were isolated from a human placenta EMBL3 library (Clontech) and a Sprague-Dawley rat liver EMBL3 library (Clontech) by a plaque hybridization method according to Sambrook et al. (1989). In this experiment, the rat and human kd312 cDNA were used as templates to synthesize the 32P labeled probes with Amersham 32P-dCTP labeling system.

EXAMPLE 2 Kd312 cDNA from human and rat were expressed in E. co l i and in the 293 human embryonic cell line. See Figs. 10 and 11. The expression vectors pET (acquired in Novagen), in which the expression of the gene is controlled by the strong transcription of the bacteriophage T7 and the cDNA translation signals of kd312 of E. co l i. An additional expression vector, pET-3 Oa (+) -2, appropriate for this purpose was constructed from the commercially available vector pET-30a (+) (de Novagen). The restriction site Ndel in the expression vector pET-30a (+) was removed by cutting with Ndel followed by the end filling and the ligation of the ends. The Ncol site in the resulting molecule was then replaced with an Ndel site by replacing the small Asp718-BamHI fragment with a synthesis oligonucleotide with appropriate base changes to construct pET-3 Oa (+) -2. The tRNA nucleotide of ATG plus 5 'and its upstream binding t-nucleucleotide within the coding regions of rat and human kd312 were converted to an Ndel site with similar procedures and the coding sequence of human kd312 cDNA. and rat from the Ndel site were cloned into Ndel and another cloning site downstream of pET-30a (+) - 2 for expression. This construction of the kd312 coding sequence fused at the 5 'end with two short sequences encode the S mark and the His mark respectively. Both brands make it possible to purify the kd312 protein in one step using affinity resins. The rat and human kd312 cDNA bearing Ndel were also fused to the coding sequences for the S-tag, His tag and the thioredoxin protein of E. co l i in the vector pET-32a (+) and expressed as a fusion protein.

The recombinant constructs were introduced into a lysogen of strain BL21 of E. co l i by transformation and the resulting strains were grown at 30 ° C and induced with IPTG in LB for 3 hours according to the protocol supplied by Novagen. The kd312 proteins expressed from both systems were purified in amounts per milligram under denaturing conditions in the presence of 6M urea using the His-Bind resin, the His-Bind buffer kit and the protocol supplied by Novagen.

The human kd312 protein bearing the S-mark and the His-tag was used in the generation of the antiserum against the kd312 protein in rats. To express the kd312 proteins in mammalian cells, human and rat kd312 cDNAs were cloned into the multiple cloning site of the mammalian episomal expression pCEP4 vector (Invitrogen) and the resulting plasmids were introduced into the 293 cells for constitutive expression of the kd312 gene of the late cytomegalovirus (CMV) operator / promoter. The expression of the kd312 proteins was confirmed by Western Blotting which was carried out according to the protocol provided by Pierce Chemical Company. In these experiments, cell lysates were prepared by sonicating the cells in 6M urea in Tris buffer (pH 7.5, 20 mM) for 10-20 seconds after washing the cells in Dulbecco's phosphate-buffered saline (GibcoBRL). Cell lysates and the equal volume (15 μl) of the loaded buffer (50 mM Tris-Cl, pH 6.8, 100 mM dithiothreitol, 2% SDS, 0.1% bromophenol blue, 10% glycerol) were heated to 100 ° C. C for 5 minutes and the mixture was loaded on 12% polyacrylamide gel for separation and staining of the protein. The anti-kd312 antiserum was diluted 500-fold and the goat anti-rat IgG antibody labeled HRP (Pierce) was diluted 500-fold before use.

Transfers of 293 cells with the pCEP4 plasmid or its derivatives were performed using the LipofectAMINE reagent and the protocol supplied therewith by Gibco BRL. The transferred cells were grown in high glucose D-MEM medium from Gibco BRL lacking sodium pyruvate and supplemented with 10% PBS and 300 μg / ml hygromycin. The fluorescence activated cell sorting (FACS) for the apoptosis tests was performed according to Polverino and Patterson, J. Biol. Chem. 272, No. 11: 7013-7021 (1997).

EXAMPLE 3 Chromosomal localization of clone F457 by fluorescence of hibr idi z ation i n s i t u The DNA of ATCC 98666 was labeled with digoxigenin dUTP by shear translation. The labeled probe was combined with human cut DNA and hybridized to normal metaphase chromosomes derived from peripheral blood lymphocytes stimulated by PHA in a solution containing 50% formamide, 10% dextran sulfate and 2X SSC. Specific hybridization signals were detected by incubation of the hybridized slides in anti-idigoxigenin fluorescence antibodies followed by counterstaining with DAPI. The initial experiment resulted in the specific labeling of the short near arm of a group of chromosome E that was believed to be chromosome 17 on the basis of size, morphology and band pattern. A second experiment was carried out, in which a genomic probe that had been mapped previously to the long arm of chromosome 17 and confirmed by co-hybridization with a centromere probe of chromosome 17 was co-hybridized with the clone. F457 This experiment resulted in the specific labeling of the distal long arm and the short arm of chromosome 17. Measurements of the 10 specifically labeled chromosomes 17 demonstrated that ATCC DNA 98666 is located at a position that is 16% of the centromere distance towards the telomere of the arm of chromosome 17p, an area corresponding to the band 17pll.2. A total of 80 metaphase cells were analyzed with 41 that exhibit specific labeling.

Discussion A novel blood-inducing gene, kd312, was isolated from a cDNA library prepared from the kidneys of Sprague-Dawley rats which was recovered from severe blood loss by subtractive hybridization. A major volume (-60%) of the blood was first removed from the rats by cardiac puncture and replaced with saline. The kidneys and other organs were then removed several hours after the operation of the animals that recovered from the loss of blood. The polyA + RNAs were isolated from the kidneys as well as the kidneys of normal animals and converted into complementary DNA (cDNA). The DNA sequences common to both populations of DNA were removed by the technique of subtractive hybridization. It involved the preparation of the biotinylated RNA driver, hybridization with a single-stranded cDNA target, streptavidin binding and the removal of RNA-DNA hybrids by phenol extraction. The remaining unhybridized cDNA was converted to a double-stranded DNA library and the library was screened for genes that induce bleeding. The partial DNA sequences of the individual clones were determined and the sequences that induce bleeding were identified by RT-PCR on polyA + RNA from normal and anemic animals. Approximately 400 clones were screened from a cDNA library derived from a rat that was sacrificed 8 hours after the operation when the hematocrit reading was 39% of the normal level. A partial cDNA molecule lacking the 51 end was identified as a highly induced gene (designated kd312) after severe blood loss. The 5 'end of kd312 cDNA was isolated by the 5' RACE method (rapid amplification of the cDNA ends).

The nature that induces blood loss of kd312 was confirmed by Northern blotting experiments. To avoid cross-hybridization with related sequences, a kd312 cDNA fragment corresponding to a 3 'untranslated region was used to probe the total RNA prepared from the kidneys of the animals that gave rise to kd312 and also the kidneys of a normal animal. For comparison, the same RNA preparations were also analyzed by RT-PCR for the presence of Epo message. As shown in Fig. 1 both kd312 and Epo messages were highly induced when the animal recovered from severe blood loss. Neither the message was detectable in the kidneys of the normal animal. The same results were obtained in RNAs prepared from different animals suffering from blood loss (results not shown). In addition, kd312 was induced in the livers and thymuses of the same anemic rats tested (results not shown).

The human homolog of kd312 was isolated from a kidney cDNA library. Comparison of deduced amino acid sequences of rat and human kd312 proteins indicates that this protein is well conserved between rats and humans (Fig. 2). The rat kd312 protein (280 amino acids) shares 97.5% identity with that of the human counterpart (281 amino acids). GenBank and EMBL seek to reveal that the kd312 protein is distantly related to the Ras protein family. Human kd312 is more homologous to the Ras member of the Ras family of humans and shares 33.8% identity of the amino acid sequence with the human R-Ras (Fig. 3). Similar to Ras proteins, kd312 carries a GTP binding site close to the amino terminus and a CAAX region for the target of the membrane at the C terminus (Fig. 2). These characteristics suggest that kd312 functions as a GTP-dependent membrane protein.

The function of kd312 was investigated first by testing its ability to transform the cells and their ability to promote or inhibit apoptosis. The transformation of NIH373 cells was tested after expression of the kd312 gene. Under the experimental conditions, the expression of the murine H-ras gene (v-ras or c-ras), but not kd312 of human or rat of the vector pEV7 led to the formation of focus in NIH3T3 cells.

The presence of kd312 in apoptosis was tested in the human embryonic kidney 293 cell line after the expression of this episomal expression vector gene pCEP4. The 293 cell derivatives constitutively expressing the kd312 genes of human, blc-2, R-ras and murine H-ras of pCEP4 were established. The expression of each gene was confirmed by Western blotting experiments (results not shown). A cell line expressing the murine bax gene could not be established and the expression of this gene in 293 cells invariably leads to cell death. As shown in Fig. 4, the expression of 293 cells protected with kd312 from apoptosis induces geranylgeraniol as well as the expression of bcl-2 which encodes a potent inhibitor of apoptosis. It has been shown that R-ras promotes apoptosis after the expression of 32, D3, FL5.12 and NIH3T3 cells. However, the expression of R-ras or H-ras in 293 cells inhibited apoptosis (Fig. 4). The levels of inhibition after expression of the ras genes, particularly R-ras, were unmistakably lower than those resulting from the expression of kd312.

To define the way that kd312 exerts its function, its interaction with human Raf, H-Ras and bcl-2 was tested using a yeast two-hybrid system. No interaction was detected between kd312 and any of these proteins. Work is in progress to look for the interaction of kd312 proteins using the same yeast system.

As a first step to find the kd312 inducer, the human kd312 genomic DNA was isolated from a human placental genomic library and its nucleotide sequence was determined. The kd312 gene consists of two exons separated by a simple intron of 211 base pairs (Fig. 5). Only two short sequences that share significant homology with a promoter region of the human erythropoietin (Epo) gene essential for a portion of the hypoxia induction were identified upstream of the coding region of kd312 and within the transcribed region (Fig. 5 and 6). No additional upstream, a TATA box and a binding site for the SPI transcription factor were identified, none of which is present in the Epo promoter region (Fig. 5). The binding site for hypoxia inducible factor 1 (HIF-1) can not be recognized either upstream or downstream of the kd312 coding region.

A new kd312 gene was isolated and showed that this gene is highly induced i vi vi as is the Epo gene after severe blood loss. It is likely that improved synthesis of the kd312 protein, such as erythropoietin, helps the human body to survive severe stress such as massive blood loss. It has been shown that kd312 protects 293 cells from chemical induced apoptosis. kd312 contains a CAAX region and more likely functions as a membrane protein. The inducers or inhibitors of kd312 as well as the proteins that interact with kd312 are candidates for pharmaceutical products or therapeutic targets DNA deposit The following E. coli cells have been deposited with the ATCC (American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD, USA) on February 19, 1998 and assigned the access numbers: No. ATCC SCS110 strain of E. coli carrying the plasmid 98664 pRkd312, EcY534 E. coli strain SCS110 carrying the plasmid 98665 pHkd312, EcY5441 HB101 strain of E. coli that carries the plasmid 98666 pHkd312G, EcY5545 E. coli strain DH125 carrying the plasmid 98667 pRkd312G, EcY5602 LIST OF SEQUENCES (1. GENERAL INFORMATION: (i) APPLICANT: YEN, KWANG-MU. (ii) TITLE OF THE INVENTION: GEN INDUCING BLOOD LOSS IN MAMMALS, KD312 (iii) NUMBER OF SEQUENCES: 9 (iv) ADDRESS OF THE CORRESPONDENCE: (A) ADDRESS: AMGEN INC. (B) STREET: ONE AMGEN CENTER DRIVE (C) CITY: THOUSAND OAKS (D) STATE: CA (E) COUNTRY: US (F) C. P.: 91320 (v) AVAILABLE COMPUTER FORM: (A) TYPE OF MEDIA: Flexible disk (B) COMPUTER: IBM PC Compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) PROGRAMMING ELEMENTS: Patentln Relay # 1.0, Version # 1.30 (vi) CURRENT APPLICATION DATE: (A) APPLICATION NUMBER: US (B) SUBMISSION DATE: (C) CLASSIFICATION: (viii) INFORMATION FROM THE LAWYER / REPRESENTATIVE (A) NAME: COOK, ROBERT R. (B) REGISTRATION NUMBER: 31,602 (C) REFERENCE NUMBER / REGISTRATION: A-514 (2) INFORMATION FOR SEQ ID NO: 1: i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1841 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA ix) FEATURE: (A) NAME / KEY: CDS (B) LOCATION: 255..1097 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1: ACGCCTGCAG GTACCGGTCC GGAATTCCCG GGTCGACCAC GCGTCCGGCG GCCTGTGCCC 60 AGATCCTGGG AGAACCCCAG CCGAGCCCAG CCTAGCCCGA GCCCAGCCCG AGCGAAGCCG 120 GAGCCCCAAG CCCGAGCCGC GCCCAGCCCG AGCAGAGCCC TCCAGCCGCT CACCCCGCGT 180 GCCACCCCAG CGACCCTCAG CCGCTCTCTG CCCTTCTCTC GGCCCCGCGC CCGCCCTCGC 240 GGCCCCTCTG CCCA ATG AAA CTG GCC GCG ATG ATC AAG AAG ATG TGC CCG 290 Met Lys Leu Ala Ala Met lie Lys Met Cys Pro 1 5 10 AGC GAC TCG GAG CTG AGT ATC CCG GCC AAG AAC TGC TAT CGC ATG GTC 338 Ser Asp Ser Glu Leu Ser lie Pro Ala Lys Asn Cys Tyr Arg Met Val 15 20 25 ATC CTC GGC TCG TCC AAG GTG GGC AAG ACG GCC ATC GTG TCG CGC TTC 386 lie Leu Gly Ser Ser Lys Val Gly Lys Thr Ala lie Val Ser Arg Phe 30 35 40 CTC ACC GGC GCC TTC GAG GAC GCC TAC ACG CCT ACC ATC GAG GTC TTC 434 Leu Thr Gly Arg Phe Glu Asp Wing Tyr Thr Pro Thr He Glu Asp Phe 45 '50 55 60 CAC CGC AAG TTC TAC TCC ATC CGC GGC GAG GTC TAC CAG CTC GAC ATC 482 His Arg Lys Phe Tyr Ser He Arg Gly Glu Val Tyr Gln Leu Asp He 65 70 75 CTC GAC ACG TCC GGC AAC CAC CCG TTC CCC GCC ATG CGG CGC CTC TCC 530 Leu Asp Thr Ser Gly Asp His Pro Phe Pro Wing Met Arg Arg Leu Ser 80 85 90 ATC CTC ACA GGA GAC GTT TTC ATC CTG GTG TTC AGT CTG GAC AAC CGC 578 He Leu Thr Gly Asp Val Phe He Leu Val Phe Ser Leu Asp Asn Arg 95 100 105 GAC TCC TTC GAG GAG GTG CAG CGG CTC AGG CAG CAG ATC CTC GAC ACC 626 Asp Ser Phe Glu Glu Val Gln Arg Leu Arg Gln Gln He Leu Asp Thr 110 115 120 AAG TCT TGC CTC AAG AAC AAA ACC AAG GAG AAC GTG GAC GTG CCC CTG 674 Lys Ser Cys Leu Lys Asn Lys Thr Lys Glu Asn Val Asp Val Pro Leu 125 130 135 140 GTC ATC TGC GGC AAC AAG GGT GAC CGC GAC TTC TAC CGC GAG GTG GAC 722 Val He Cys Gly Asn Lys Gly Asp Arg Asp Phe Tyr Arg Glu Val Asp 145 150 155 CAG CGC GAG ATC GAG CAG CTG GTG GGC GAC GAC CCC CAG CGC TGC GCC 770 Gln Arg Glu He Glu Gln Leu Val Gly Asp Asp Pro Gln Arg Cys Wing 160 135 170 TAC TTC GAG ATC TCG GCC AAG AAG AAC AGC AGC CTG GAC CAG ATG TTC 818 Tyr Phe Glu He Be Wing Lys Lys Asn Ser Ser Leu Asp Gln Met Phe 175 180 185 CGC GCG CTC TTC GCC ATG GCC AAG CTG CCC AGC GAG ATG AGC CCA GAC 866 Arg Ala Leu Phe Ala Met Ala Lys Leu Pro Ser Glu Met Ser Pro Asp 190 195 200 CTG CAC CGC AAG GTC TCG GTG CAG TAC TGC GAC GTG CTG CAC AAG AAG 914 Leu His Arg Lys Val Ser Val Gln Tyr Cys Asp Val Leu His Lys Lys 205 210 215 220 GCG CTG CGG AAC AAG AAG CTG CTG CGG GCC GGC AHC GGC GGC GGC GGC 962 Wing Leu Arg Asn Lys Lys Leu Leu Arg Wing Gly Ser Gly Gly Gly Gly 225 230 235 GGC GAC CCG GGC GAC GCC TTT GGC ATC GTG GCA CCC TTC GCG CGC CGG 1010 Gly Asp Pro Gly Asp Wing Phe Gly He Val Wing Pro Phe Wing Arg Arg 240 245 250 CCC AGC GTA CAC AGC GAC CTC ATG TAC ATC CGC GAG AAG GCC AGC GCC 1058 Pro Ser Val His Ser Asp Leu Met Tyr He Arg Glu Lys Wing Ser Wing 255 260 265 GGC AGC CAG GCC AAG GAC AAG GAG CGC TGC GTC ATC AGC TAGGAGCCCC 1107 Gly Ser Gln Ala Lys Asp Lys Glu Arg Cys Val He Ser 270 275 280 GCCGCGCTGG CGACACAACC TAAGGAGGAC CTTTTTGTTA AGTCAAATCC AACGGCCCGG1167 TGCGCCCCAG GCCGGGAGCG CGCGCGGACT GGCGTCTCCC CTCCCGGCGA TCCGCCCCCA1227 GCACTGGGGA GGCGCCACTG AACCGAGAAG GGATGGTCAT CTGCTCCGGA AGGAAAGAGA1287 ACGGGCCAAG ACTGGGACTA TTCCCCACCC CCGGTCCCCA TTGAGGCCCG CCACCCCCAT1347 AACTTTGGGA GCGAGGGCCC AGCCGAGGGT GGATTTATCT TCTCAAAGAC CTAAGAGTGA1 07 GCGCGGGGTG GGGGAGGGAT GTGAAGTTAT CCAGCCTCTG CTAGGCTTCA AGAAACCGTC1467 ATGCCCGCTT GAGGGTCAGG ACCCACGGGG CATTATCTTG TCTGTGATTC CGGGTTGCTG1527 TGACAGCCGG TAGAGCCTCT GCCCTCCCGA AACTAAGCGG GGGGGCGTGG GTCAAATCAT1587 AGCCAAGTGA CTTGTTTACA TGTGAGTGAA ACTGCACAAA GGAACACAAA ACAAAACTTG1647 CACTTTAACG GTAGTTCCGG TGTCAACATG GACACGAACA AAACCTTACC CAGGTGTTTA1707 TACTGTGTGT GTGTGAGGTC TTTAAAGTTA TTGCTTTATT TGGTTTTTTA ATATACAATA1767 AAATAATTTA AAATGGAAAA AAAAAAAAAA AGGGCGGCCG CTCTAGAGGA TCCCTCGAGG1827 GCCCAAGCTT ACGC 1841 (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 281 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2: Met Lys Leu Ala Ala Met He Lys Lys Met Cys Pro Ser Asp Ser Glu 1 5 10 15 Leu Ser He Pro Ala Lys Asn Cys Tyr Arg Met Val He Leu Gly Ser 20 25 30 Ser Lys Val Gly Lys Thr Wing He Val Ser Arg Phe Leu Thr Gly Arg 35 40 45 Phe Glu Asp Ala Tyr Thr Pro Thr He Glu Asp Phe His Arg Lys Phe 50 5 - 60 Tyr Ser He Arg Gly Glu Val Tyr Gln Leu Asp He Leu Asp Thr Ser 65 70 75 80 Gly Asn His Pro Phe Pro Wing Met Arg Arg Leu Ser He Leu Thr Gly 85 90 95 Asp Val Phe He Leu Val Phe Ser Leu Asp Asn Arg Asp Ser Phe Glu 100 105 110 Glu Val Gln Arg Leu Arg Gln Gln He Leu Asp Thr Lys Ser Cys Leu 115 120 125 Lys Asn Lys Thr Lys Glu Asn Val Asp Val Pro Leu Val He Cys Gly 130 135 140 Asn Lys Gly Asp Arg Asp Phe Tyr Arg Glu Val Asp Gln Arg Glu He 145"150 155 160 Glu Gln Leu Val Gly Asp Asp Pro Gln Arg Cys Wing Tyr Phe Glu He 165 170 175 Be Ala Lys Lys Asn Be Ser Leu Asp Gln Met Phe Arg Ala Leu Phe 180 185 190 Wing Met Wing Lys Leu Pro Ser Glu Met Ser Pro Asp Leu His Arg Lys 195 200 205 Val Ser Val Gln Tyr Cys Asp Val Leu His Lys Lys Ala Leu Arg Asn 210 215 220 Lys Lys Leu Leu Arg Wing Gly Ser Gly Gly Gly Gly Gly Asp Pro Gly 225 230 235 240 Asp Wing Phe Gly He Val Wing Pro Phe Wing Arg Arg Pro Ser Val His 245 250 255 Being Asp Leu Met Tyr He Arg Glu Lys Wing Being Wing Gly Ser Gln Wing 260 265 270 Lys Asp Lys Glu Arg Cys Val He Ser 275 280 (2) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 3986 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3: TCCGCGCCTG AGGCCCTGAA ACCCCGAGTC CGCCCGGCGG TCGCCTCCCG GGAACAAGAG 60 CCCGGCTGGG GACCGGAGCG GAAGGGGGCT GGGGCTGGGG CTGTGCTCTG AGGACTGCAA 120 TATACGGTCC GCGCATGCAC TCAGCAAACG CTGCTGCGCT TACTGGGTTA CTTACTAGAT 180 TCCTATTCTC TGGGGAAACT GAGAACCAAA GAAAATAAGA GTACGCGCGC GGGAGGTGCA 240 GGAATGGGGG TCCTTGCCCG AAGTCGCAGA GGGACAGGGG CACCGCCGGG ACCAGAACCC 300 CGACGCCCCT GCGGCCGCCG AGCCCGCGGC AGTGGAAAAG CGGAGTCCGA GCGCCTCCAG 360 CCTCAGCCCG ACCCTGGACT GCTCCCCCCA GCCCCCGCGC CCAGAGAGCA GGAGCCCGGC 420 AGCGGGTGAC GAGGTCGCCG GGACTGGGAG CCGGTGCGGG GGAGGCGGGC CCCGCGGGGC 480 GTGACGCACC GAGCTGGGAG GGCCGGGGCG GGGCAGCCGA GCAGGCTGCA TATAAGGGCG 540 GCGGCCGGGC GCCAAAGCCA GAGCAAGCGG CCTGTGCCCA GATCCTGGGA GAACCCCAGC 600 CGAGCCCAGC CTAGCCCGAG CCCAGCCCGA GCGAAGCCGG AGCCCCAAGC CCGAGCCGCG 660 CCCAGCCCGA GCAGAGCCCT CCAGCCGCTC ACCCCGCGTG CCACCCCAGC GACCCTCAGC 720 CGCTCTCTGC CCTTCTCTCG GCCCCGCGCC CGCCCTCGCG GCCCCTCTGC CCAATGAAAC 780 TGGCCGCGAT GATCAAGAAG ATGTGCCCGA GCGACTCGGA GCTGAGTATC CCGGCCAAGA 840 ACTGCTATCG CATGGTCATC CTCGGCTCGT CCAAGGTGGG CAAGACGGCC ATCGTGTCGC 900 GCTTCCTCAC CGGCCGCTTC GAGGACGCCT ACACGCCTAC CATCGAGGAC TTCCACCGCA 960 AGTTCTACTC CATCCGCGGC GAGGTCTACC AGCTCGACAT CCTCGACACG TCCGGCAACC 1020 ACCCGTTCCC CGCCATGCGG CGCCTCTCCA TCCTCACAGG TGAGCCGGGG GCCGGGCAGG 1080 TGCGGGAGGG AAGGGCGGGG AACCCTCGGC CAGGGCGCCC CGCGAGCGCC GGTCCGGCTG 1140 CTGGCGCGCCG AGTAGTGCGC TTGCGCTTA GAGAGGCTAG CGCGCCCCGC GCGGCCTCAA1200 AGTCAGCCCG ACTTGTCCCC TGGGCGGCCA CCCTCACCTT CTCCTTTTCT GCTCTCTGTG1260 CCCCCTCTAG GAGACGTTTT CATCCTGGTG TTCAGTCTGG ACAACCGCGA CTCCTTCGAG 1320 GAGGTGCAGC GGCTCAGGCA GCAGATCCTC GACACCAAGT CTTGCCTCAA GAACAAAACC 1380 AAGGAGAACG TGGACGTGCC CCTGGTCATC TGCGGCAACA AGGGTGACCG CGACTTCTAC 1440 CGCGAGGTGG ACCAGCGCGA GATCGAGCAG CTGGTGGGCG ACGACCCCCA GCGCTGCGCC 1500 TACTTCGAGA TCTCGGCCAA GAAGAACAGC AGCCTGGACC AGATGTTCCG CGCGCTCTTC 1560 GCCATGGCCA AGCTGCCCAG CGAGATGAGC CCAGACCTGC ACCGCAAGGT CTCGGTGCAG 1620 TACTGCGACG TGCTGCACAA GAAGGCGCTG CGGAACAAGA AGCTGCTGCG GGCCGGCAGC 1680 GGCGGCGGCG GCGGCGACCC GGGCGACGCC TTTGGCATCG TGGCACCCTT CGCGCGCCGG 1740 CCCAGCGTAC ACAGCGACCT CATGTACATC CGCGAGAAGG CCAGCGCCGG CAGCCAGGCC 1800 AAGGACAAGG AGCGCTGCGT CATCAGCTAG GAGCCCCGCC GCGCTGGCGA CACAACCTAA 1860 GGAGGACCTT TTTGTTAAGT CAAATCCAAC GGCCCGGTGC GCCCCAGGCC GGGAGCGCGC 1920 GCGGACTGGC GTCTCCCCTC CCGGCGATCC GCCCCCAGCA CTGGGGAGGC GCCACTGAAC 1980 CGAGAAGGGA TGGTCATCTG CTCCGGAAGG AAAGAGAACG GGCCAAGACT GGGACTATTC 2040 CCCACCCCCG GTCCCCCATT GAGGCCCGCC ACCCCCATAA CTTTGGGAGC GAGGGCCCAG 2100 CCGAGGGTGG ATTTATCTTC TCAAAGACCT AAGAGTGAGC GCGGGGTGGG GGAGGGATGT 2160 GAAGTTATCC AGCCTCTGCT AGGCTTCAAG AAACCGTCAT GCCCGCTTGA GGGTCAGGAC 2220 CCACGGGGCA TTATCTTGTC TGTGATTCCG GGTTGCTGTG ACAGCCGGTA GAGCCTCTGC 2280 CCTCCCGAAA CTAAGCGGGG GGGCGTGGGT CAAATCATAG CCAAGTGACT TGTTTACATG 2340 TGAGTGAAAC TGCACAAAGG AACACAAAAC AAAACTTGCA CTTTAACGGT AGTTCCGGTG 2400 TCAACATGGA CACGAACAAA ACCTTACCCA GGTGTTTATA CTGTGTGTGT GTGAGGTCTT 2460 TAAAGTTATT GCTTTATTTG GTTTTTTAAT ATACAATAAA ATAATTTAAA ATGGAAAACC 2520 GGTTTTTTTT TTTTTTTTTT TTTTTTTTGC TTTTAGAGAT GGCTGGAGTG GGGAAGGGTG 2580 GGGAGAAGGA AAGGGCTGGG CTTTGACTTA GGTGGAACTA GAACTTACCT TCCCCAGAAC 2640 TGGAAAATAA CCCTGGCCTT CTGAAGGCAG CTTCAGCTGC CAGAAAAGCC CCAGATGCCT 2700 GGGGCATCTA TGTAGGGGAT GGTTCCCTAG AAAACCGGA AGAATATAAA GGATTTCAGG 2760 GTCTCCCCTG GAGATGAACT CTTTCTAGCC ATCCACCCGC TTAATTTTCT TTGGGTTAGA 2820 TGACAAAAGG CCTCATTTTC TGAGAGAATG TTCTGAATTC TTCAGCGTAA AAGCCACTGG 2880 AACTGTGCCT AACCATTTTG TCACCAGACT CAGTGTGGGC CCAGGCAAAC TTTCGGACTG 2940 TTGGAGGCAT CAGTCAGGCC CTGGGGAAAG AGCCTGAGAC CCCATCTGGA AACAGGACCA 3000 TCCTGGCGCG CCCCCACCAC CCGCTCACTC CAGGGTGCCA CCCTGTCTGG AAACAGCTAA 3060 CTCCTCAGCC TCTGCTCCCC TCTAGCTCCA GGAAGTGCTC CTGGCCAGGT GTAGAGCCCC 3120 ATCCCCCTTC AGCCTTGCTG TCTCGGTCTC ATGGCTAAGG CACCCCAGAA CACCAATCTC 3180 TCTGCCACTA GTACTGCAAA CCTGTTGGTG GGTGACACCT GCCAAGCCTC TAATTCTTCA 3240 CCCCGGGAAG AGAGAACACC CTCGGCATGG GCTCACTGTG GGGATTAAGT GTGATGTTTG 3300 AAAAGTACTT AGCATAAATG CCGGCCACCC AGTAATCCAA GTAATTGGTG GCTTTCAGAG 3360 GACGCTCAGC CCTGTGAGAG ACACTCAAAA TTGTCCTAGA AGGATTTCAA CCCTGCTCTG 3420 GTGAGGGCGG CTCCCCACAG GACTGAACCT CCTCGAGTCA CCAAAAGGCA CCCCCCACCT 3480 CCCCCCTCCA AAAATAAAAG GCAACTAAGG ACAGCCCAGG GGCCCGTGAC AGGCAGGGGC 3540 AGGGATGACC CGCCAGGCAA ACTGCCCTTG AGGCCAGCCG GGAGAGGAGT TCCTGTTCCA 3600 CACTGGTTCA GCGGGTGTGT GTGCTGGGGC GGGTGTGTGT GCTGGGGCGG AGGGAGTGAG 3660 GAGCAGAGGT GTGGTTGTGT CTGAAGAGAC TGAGAGAAAC ATTTTCCTCT CAACTATCTG 3720 AGAGCCATCC CACTATGAAT TTCTCAGTAC AAAAAGCATT ATGTCCTGAG ACAGCAGAGC 3780 ATAAGTCCTT TTAATTATGT GTTTGAAAAA TGTCACAAGT CAAAAAAGGA ACACAAGGCA 3840 GGCTCCGGCT CCCTCCACCC CCGTGAGGAG CCCTTGTCCA TTTCAGCCTT GCACTCAGAA 3900 AGACCCCGGG GGTCTTGTAG TTCCACGTGC TTCATGTTTC GTGGTATCTG TCAGAGCCTT 3960 AAAACAGGCC CACCCACTAC TGTGAA 3986 (2) INFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1689 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (ix) FEATURE: (A) NAME / KEY: CDS (B) LOCATION: 132..971 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4: CCCGCTGCCT GTACTCAAGA TTCCAGGCCA GCTCGCGCGG TCCCGAAGCC AAACTCTTCC 60 ACCACTCCGG CGCCCTCTGC AGCCCTCTAC CTTCTCTCAG CCACGCATCT GCCCTGGGGC 120 CCCTCTGCCC A ATG AAA CTG GCC GCG ATG ATC AAG AAG ATG TGC CCA AGC 170 Met Lys Leu Ala Wing Met He Lys Lys Met Cys Pro Ser 1 5 10 GAC TCT GAA CTG AGT ATC CCG GCC AAG AAC TGC TAC AGG ATG GTC ATC 21? Asp Ser Glu Leu Ser He Pro Wing Lys Asn Cys Tyr Arg Met Val He 15 20 25 CTC GGC TCA TCC AAA GTG GGC AAG ACG GCC ATC GTG TCG CGC TTC CTC 266 Leu Gly Ser Ser Lys Val Gly Lys Thr Ala He Val Ser Arg Phe Leu 30 35 40 45 ACG GGC CGC TTC GAG GAC GCT TAC ACC CCT ACC ATT GAA GAC TTC CAC 314 Thr Gly Arg Phe Glu Asp Wing Tyr Thr Pro Thr He Glu Asp Phe His 50 55 60 CGA AAG TTT TAC TCG ATC CGC GGC GAA GTC TAC CAG TTG GAC ATA CTG 362 Arg Lys Phe Tyr Ser He Arg Gly Glu Val Tyr Gln Leu Asp He Leu 65 70 75 GAC ACT TCT GGC AAT CAT CCG TTT CCC GCC ATG CGG CGC CTC TCT ATC 10 Asp Thr Ser Gly Asn His Pro Phe Pro Wing Met Arg Arg Leu Ser He 80 85 90 CTC ACA GGA GAC GTT TTC ATT CTG GTG TTC AGC TTA GAC AAC CGC GAC 458 Leu Thr Gly Asp Val Phe He Leu Val Phe Ser Leu Asp Asn Arg Asp 95 100 105 TCC TTC GAG GG GTG CAA AGG CTC AAA CAG CAG ATC CTA GAC ACC AAG 506 Ser Phe Glu Glu Val Gln Arg Leu Gln Gln He Leu Asp Thr Lys 110 115 120 125 TCC TGT CTC AAG AAA AAA AAA AAA GAG GTG GAC GTA CCG CTG GTC 554 Ser Cys Leu Lys Asn Lys Thr Lys Glu Asn Val Asp Val Pro Leu Val 130 135 140 ATT TGC GGT AAC AAA GGG GAC CGG GAC TTC TAC CGC GAA GTG GAG CAG 602 He Cys Gly Asn Lys Gly Asp Arg Asp Phe Tyr Arg Glu Val Glu Gln 145 150 155 CGG GAG ATT GAG CAG CTG GTG GGC GAT GAC CCT CAG CGT TGT GCC TAC 650 Arg Glu He Glu Gln Leu Val Gly Asp Asp Pro Gln Arg Cys Ala Tyr 160 165 170 TTC GAG ATC TCG GCC AAG AAG AAT AGC AGC CTG GAC CAG ATG TTC CGT 698 Phe Glu He Ser Wing Lys Lys Asn Ser Ser Leu Asp Gln Met Phe Arg 175 180 185 GCG CTC TTT GCC ATG GCC AAG CTG CCT AGC GAG ATG AGC CCT GAC TTG 746 Ala Leu Phe Ala Met Ala Lys Leu Pro Ser Glu Met Ser Pro Asp Leu 190 195 200 205 CAC CGC AAG GTG TCT GTG CAG TAC TGT GAC GTG CTG CAC AAA AAG GCT 794 His Arg Lys Val Ser Val Gln Tyr Cys Asp Val Leu His Lys Lys Ala 210 215 220 CTG AGG AAC AAG AAG CTT CTG CGT GCG GGC AGC GGA GGT GGG GGC GAC 842 Leu Arg Asn Lys Lys Leu Leu Arg Wing Gly Ser Gly Gly Gly Gly Asp 225 230 235 CAC GGA GAT GCC TTT GGC ATC TTG GCG CCC TTT GCT CGC AGA CCT AGC 890 His Gly Asp Wing Phe Gly He Leu Wing Pro Phe Wing Arg Arg Pro Ser 240 245 250 GTG CAT AGC GAC CTC ATG TAC ATT CGT GAG AAA ACC AGT GTC AGC AGC 938 Val His Ser Asp Leu Met Tyr He Arg Glu Lys Thr Ser Val Ser Ser 255 260 265 CAG GCT AAG GAC AAG GAG CGC TGT GTC ATC AGT TAGGAGCCCC CAGGGTCAGT 991 Gln Ala Lys Asp Lys Glu Arg Cys Val He Ser 270 275 280 CAGCCACACA ACCTGAGGAC CTTTTTTTGTT CAAAAGTCAA ATCGGTTTCC CAGGCTAACC1051 TGTGCACTCC GTGCCCCAAG AGCGCCAGCT GGCCTCCTCC CTCCCTCCCT GAGACCCAGCHH CCTGTGCACC AGGGAGATGC TGCCAAGACA GTAAGGGACA GTCATCTGCT GTGAGAGGAAH71 AGAACTAGCT AAGACTGGGA CTTTCGCCTC CGATTCTGGG ATGCCAGGAC CCAGCAGAGG1231 GTTAGTTGGC GTTTTTCTCA GAGACTTTGA GAGTGTGTGA AGGGCTTCGG CCTCTGAGAC1291 TTCAAGTAAC TGTGCGGCTT GCTGTGGGGC CAGGACTAAC AGGGCATTAT CTCGTCTGTG1351 ATTGGTGTTG CCATGACCGC TGTCAGCCAC CTCTGTCCTC AGCAAACTGG AAACTTTGGC1411 TCGAGGTGGG GGTTCAATCA TAGCCAGACA ACTTGTTTAC ATGTGTGTGT GTGTGTAATT1 71 ACCCAAAAGG AAAACAAAAC ACAAAACTTG CACTTTAACA GTTCCAGTGT CAACGTGACA1531 TGAACAAAAT CTCTACATTT CTATTGTGTG AGGTCTTTAT TATTTTTTTT AATTTAAAAT1591 AAAATAATTT TAAAATGGAA AAAAAAAAAA AAAAAAAAAA AAGGGCGGCC GCTCTAGAGG1651 ATCCAAGCTT ACGTACGCGT GCATGCGACG TCATACTC 1689 (2) INFORMATION FOR SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 280 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linearF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5: Met Lys Leu Ala Ala Met He Lys Lys Met Cys Pro Ser Asp Ser Glu 1 5 10 15 Leu Ser He Pro Ala Lys Asn Cys Tyr Arg Met Val He Leu Gly Ser 20 25 30 Ser Lys Val Gly Lys Thr Wing He Val Ser Arg Phe Leu Thr Gly Arg 35 40 45 Phe Glu Asp Wing Tyr Thr Pro Thr He Glu Asp Phe His Arg Lys Phe 50 55 60 Tyr Ser He Arg Gly Glu Val Tyr Gln Leu Asp He Leu Asp Thr Ser 65 70 75 80 Gly Asn His Pro Phe Pro Wing Met Arg Arg Leu Ser He Leu Thr Gly 85 90 95 Asp Val Phe He Leu Val Phe Ser Leu Asp Asn Arg Asp Ser Phe Glu 100 105 110 Glu Val Gln Arg Leu Lys Gln Gln He Leu Asp Thr Lys Ser Cys Leu 115 120 125 Lys Asn Lys Thr Lys Glu Asn Val Asp Val Pro Leu Val He Cys Gly 130 135 140 Asn Lys Gly Asp Arg Asp Phe Tyr Arg Glu Val Glu Gln Arg Glu He 145 150 155 160 Glu Gln Leu Val Gly Asp Asp Pro Gln Arg Cys Wing Tyr Phe Glu He 165 170 175 Be Ala Lys Lys Asn Be Ser Leu Asp Gln Met Phe Arg Ala Leu Phe 180 185 190 Wing Met Wing Lys Leu Pro Ser Glu Met Ser Pro Asp Leu His Arg Lys 195 200 205 Val Ser Val Gln Tyr Cys Asp Val Leu His Lys Lys Ala Leu Arg Asn 210 215 220 Lys Lys Leu Leu Arg Wing Gly Ser Gly Gly Gly Gly Asp His Gly Asp 225 230 235 240 Wing Phe Gly He Leu Wing Pro Phe Wing Arg Arg Pro Ser Val His Ser 245 250 255 Asp Leu Met Tyr He Arg Glu Lys Thr Ser Val Ser Ser Gln Ala Lys 260 265 270 Asp Lys Glu Arg Cys Val He Ser 275 280 INFORMATION FOR SEQ ID NO: 6 i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 3079 base pairs (B) TYPE: nucleic acid C) HEBRA: simple D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6: CCAGCGCACG TAGGTCTGGA GCACAGCCTC AGGCTCCAAG GCGG GGTCA CTGCGTCTAG_60_GAGGAGCCCG GAGCGTCCGG GGGCGGGACG TGACGCACCT TGGCTGGGAG GTGCCAGCCC 120 AGGCTTCGGT CAGCTGCATA TAAGAGTGGT GTGAGGCGCG GAAAGCCTGA GCCCGCTGCC 180 TGTACTCAAG ATTCCAGGCC AGCTCGCGCG GTCCCGAAGC CAAACTCTTC CACCACTCCG 240 GCGCCCTCTG CAGCCCTCTA CCTTCTCTCA GCCACGCACT TGCCCTGGGG CCCCTCTGCC 300 CAATGAAACT GGCCGCGATG ATCAAGAAGA TGTGCCCAAG CGACTCTGAA CTGAGTATCC 360 CGGCCAAGAA CTGCTACAGG ATGGTCATCC TCGGCTCATC CAAAGTGGGC AAGACGGCCA 420 TCGTGTCGCG CTTCCTCACG GGCCGCTTCG AGGACGCTTA CACCCCTACC ATTGAAGACT 480 TCCACCGAAA GTTTTACTCG ATCCGCGGCG AAGTCTACCA GTTGGACATA CTGGACACAT 540 CTGGCAATCA TCCGTTTCCC GCCATGCGGC GCCTCTCTAT CCTCACAGGT GAGTGGGGGA 600 CCGACAGGGA CCGTGGGGAG GGAATCTGCG GGGAGCGGAT GGGGCGGTGT GTTGTGCTTG 660 GGGCTGTGCT GTCTGCTGCT CCGTGCTTGG CAGCTGCCCT CACCTTTCCA CTCGTTCCCT 720 TGTAGGAGAC GTTTTCATTC TGGTGTTCAG CTTAGACAAC CGCGACTCCT TCGAGGAGGT 780 GCAAAGGCTC AAACAGCAGA TCCTAGACAC CAAGTCCTGT CTCAAGAACA AAACCAAAGA 840 GAATGTGGAC GTGCCGCTGG TCATTTGCGG TAACAAAGGG GACCGGGACT TCTACCGCGA 900 AGTGGAGCAG CGGGAGATTG AGCAGCTGGT GGGCGATGAC CCTCAGCGTT GTGCCTACTT 960 CGAGATCTCG GCCAAGAAGA ATAGCAGCCT GGACCAGATG TTCCGTGCGC TCTTTGCCAT1020 GGCCAAGCTG CCTAGCGAGA TGAGCCCTGA CTTGCACCGC AAGGTGTCTG TGCAGTACTG1080 TGACGTGCTG CACAAAAAGG CTCTGAGGAA CAAGAAGCTT CTGCGTGCGG GCAGCGGAGGH40 TGGGGGCGAC CACGGAGATG CCTTTGGCAT CTTGGCGCCC TTTGCTCGCA GACCTAGCGT1200 GCATAGCGAC CTCATGTACA TTCGTGAGAA AACCAGTGTC AGCAGCCAGG CTAAGGACAA1260 GGAGCGCTGT GTCATCAGTT AGGAGCCCCC AGGGTCAGTC AGCCACACAA CCTGAGGACC1320 TTTTTTGTTC AAAAGTCAAA TCGGTTTCCC AGGCTAACCT GTGCACTCCG TGCCCCAAGA1380 GCGCCAGCTG GCCTCCTCCC TCCCTCCCTG AGACCCAGCC CTGTGCACCA GGGAGATGCT1 40 GCCAAGACAG TAAGGGACAG TCATCTGCTG TGAGAGGAAA GAACTAGCTA AGACTGGGAC1500 TTTCGCCTCC GATTCTGGGA TGCCAGGACC CAGCAGAGGG TTAGTTGGCG TTTTTCTCAG1560 AGACTTTGAG AGTGTGTGAA GGGCTTCGGC CTCTGAGACT TCAAGTAACT GTGCGGCTTG1620 CTGTGGGGCC AGGACTAACA GGGCATTATC TCGTCTGTGA TTGGTGTTGC CATGACTGCT1680 GTCAGCCACC TCTGTCCTCA GCAAACTGGA AACTTTGGCT CGAGGTGGGG GTTCAATCAT1740 AGCCAGACAA CTTGTTTACA TGTGTGTGTG TGTGTAATTA CCCAAAAGGA AAACAAAACA1800 CAAAACTTGC ACTTTAATAG TTCCAGTGTC AACGTGACAT GAACAAAATC TCTACATTTC1860 TATTGTGTGA GGTCTTTATT ATTTTTTTTA ATTTAAAATA AAATAATTTT AAAATGGAAA1920 ATGGTGCTTC GCTTTGCTTT TGCTTTTAGG CTTCCTGCCT CGGTGGCAGT GGCCAAGAAC1980 TGGAAAAGGA CCTGGCTTTC AGAATATGGT CTCCCACTTC CAAGTGGGAC CTTCTGGCTT2040 TCTGTCTACA CTCTGCCCGG CCTGGCCTGT AACAGAGGGC CTTGTTTTAG AGAATATTCA2100 TACTCTCCTC CACACAGCCC ATCTGTTACT CATCATAGAA GGCAACAGAA AGCTGCCACA2160 CTTGAAACGC TAACCTTGAT TACCACAAAC ATGGAGGCTG AGGTGGAGAG GTGTGGCAGG2220 GAAGAGGCCT GCACTTAACG TTCATTCCTT GCCCCAGGGC TGCCGCAGGA CCTGGACAGG2280 GAAAGTACAG ATGGGTGGAG TGCAGCTCCC AGAAGCTCTC GAGCAGGTGG GGCCCACCTC23 0 CTCTGCACCT TCCTAACTCC CTGTGGCTAA GGGCTCATAG TTTGTGACCC AGATCTCCTT2400 GCCACTCCTA CGGTCAACTT AGGGCAAGTG TCGCCTTCCA AGTCTCCAAT TCTGCAGCTG2460 AGAAATCGAG GCACTCTGTG CAGGGGTCAA CATGGTCTCC AAGGAGTCAG CACAACTGCT2520 GCAGCCGCCC AGCCAAATAC TTGGTTTTTC CAGGGTCTTG ACCTTCGGGG ATGCTCAAAA2580 TTGTCTTGGG ACCGGGGGAG GAAGGGTCTT GTCAACCCTG CTTTGGGAAG GGCCACTTGC2640 AGGGAACTGT ACCTCCTCAA ATCTCAGAAA GGCACTCACT TCTCAACAAT GAATGGCTGG2700 GCTCCCCAGG GTCCCCTGCA GGAGACGTGT AGGCTTTGTG CTCATTTAAC AGATGTACAT2760 GCTGGGCGGA AGGAGGAGCG CAGTGAACAT TTTTGCCTCT ACACCACCCA CTCAACACAC2820 CACTCTAATT CTTCATTTTA AAGACCTGCA ATCCCAGGGT CTGACTGCTA GCCCTGAGAG2880 AGACAGAGAC AACAGGCAAA CCATGTGTCC TGAGAACAGA ACATACTTTT AATTACATAT2940 GTGAAAACAT GACAGGTCAA CACACTGGAA CAGAGAAGCC TCCGAGCCTC CAGCCCTTCG3000 AGGAAGCCTT CTTTCTTCCC TGTCAGGAAG ATCCGAAGCT CCTGCATTTC ACGTGCTCTG3060 CCCTTACAGG AACTGGGCA 3079 (2) INFORMATION FOR SEQ ID NO: 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 210 amino acids (B) TYPE: amino acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7: Thr Gly Arg Gly Arg Pro Arg Gly Gly Gly Pro Gly Pro Gly Asp Pro 1 5 10 15 Pro Pro Ser Glu Thr His Lys Leu Val Val Val Gly Gly Gly Val 20 25 30 Gly Lys Ser Ala Leu Thr He Gln Phe He Gln Ser Tyr Phe Val Ser 35 40 45 Asp Tyr Asp Pro Thr He Glu Asp Ser Tyr Thr Lys He Cys Ser Val 50 55 60 Asp Gly He Pro Wing Arg Leu Asp He Leu Asp Thr Wing Gly Gln Glu 65 70 75 80 Glu Phe Gly Wing Met Arg Glu Gln Tyr Met Arg Ala Gly His Gly Phe 85"90 95 Leu Leu Val Phe Ala He Asn Asp Arg Gln Ser Phe Asn Glu Val Gly 100 105 110 Lys Leu Phe Thr Gln He Leu Arg Val Lys Asp Arg Asp Asp Phe Pro 115 120 125 Val Val Leu Val Gly Asn Lys Wing Asp Leu Glu Ser Gln Arg Gln Val 130 135 140 Pro Arg Ser Glu Wing Be Wing Phe Gly Wing His His Val Val Wing Tyr 145 150 155 160 Phe Glu Wing Being Wing Lys Leu Arg Leu Asn Val Asp Glu Wing Phe Glu 165 170 175 Gln Leu Val Arg Wing Val Arg Lys Tyr Gln Glu Gln Glu Leu Pro Pro 180 185 190 Be Pro Pro Be Ala Pro Arg Lys Lys Gly Gly Gly Cys Pro Cys Val 195 200 205 Leu Leu 210 (2) INFORMATION FOR SEQ ID NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 53 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8: CCTGCTCTGA CCCCGGGTGG CCCCTACCCC TGGCGACCCC TCACGCACAC AGC 53 2) INFORMATION FOR SEQ ID NO: 9 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 9 CGCTCACCCC GGGTGCCACC CCCTGGCGGC CCCTC 35 It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, the content of the following is claimed as property.

Claims (19)

1. A nucleic acid molecule, characterized in that it encodes a polypeptide selected from the group consisting of: (a) the nucleic acid molecule of SEQ ID NO: 1 or SEQ ID NO: 3; (b) a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 2 or the biologically active fragment thereof; (c) a nucleic acid molecule encoding a polypeptide that is at least 85 percent identical to the polypeptide of SEQ ID: 2; (d) a nucleic acid molecule that hybridizes under stringent conditions to any of (a) - (c) above; Y (e) a nucleic acid molecule that is the complement of any of (a) - (d) above.

2. A nucleic acid molecule, characterized in that it encodes a polypeptide p.o selected from the group consisting of: (a) the nucleic acid molecule of SEQ ID NO: 4 or SEQ ID NO: 6; (b) a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 5, or a biologically active fragment thereof; (c) a nucleic acid molecule encoding a polypeptide that is at least 85 percent identical to the polypeptide of SEQ ID NO: 5; (d) a nucleic acid molecule that hybridizes under stringent conditions to any of (a) - (c) above; Y (e) a nucleic acid molecule that is the complement of any of (a) - (d) above.

3. The nucleic acid molecule, characterized in that it is SEQ ID NO: 1 or SEQ ID NO: 34.

The nucleic acid molecule, characterized in that it is SEQ ID NO: 4 or SEQ ID NO: 6.

5. A nucleic acid molecule, characterized in that it encodes the polypeptide of SEQ ID NO: 2.

6. A nucleic acid molecule, characterized in that it encodes the polypeptide of SEQ ID NO: 5.

7. A vector, characterized in that it comprises a nucleic acid molecule of any of claims 1 to 6.

8. A host cell, characterized in that it comprises a vector of claim 7.

9. A process for producing a kd312 polypeptide, characterized in that it comprises the steps of: (a) expressing a polypeptide encoded by a nucleic acid of any of claims 1-6 in a host cell; Y (b) isolating the polypeptide.

10. A kd312 polypeptide selected from the group consisting of (a) the polypeptide of SEQ ID NO: 2; (b) a polypeptide that is at least 85 percent identical to the polypeptide of (a); Y (c) a biologically active fragment of any of (a) - (b).

11. A kd312 polypeptide selected from the group consisting of: (a ') a polypeptide of SEQ ID NO: 5; (b ') a polypeptide that is at least 85 percent identical to the polypeptide of (a'); Y (c ') a biologically active fragment of any of (a') - (b ').

12. A kd312 polypeptide, characterized in that it is the polypeptide of SEQ ID NO: 2 or a biologically active fragment thereof.

13. A kd312 polypeptide, characterized in that it is the polypeptide of SEQ ID NO: 5 or a biologically active fragment thereof.

14. A kd312 polypeptide, characterized in that it consists of amino acids 6-281 of SEQ ID NO: 2.

15. A kd312 polypeptide, characterized in that it consists of amino acids 10-281 of SEQ ID NO: 2.

16. A kd312 polypeptide, characterized in that it consists of amino acids 6-280 of SEQ ID NO: 5.

17. A kd312 polypeptide, characterized in that it consists of amino acids 10-280 of SEQ ID NO: 5.

18. An antibody or fragment thereof, characterized in that it binds specifically to a polypeptide according to any of claims 10-17.

19. The antibody of claim 18, characterized in that it is a monoclonal antibody.