AU780470B2 - Inhibitor of osteoclast precursor formation - Google Patents

Description

1 INHIBITOR OF OSTEOCLAST PRECURSOR FORMATION This invention relates to a polypeptide factor which is able to inhibit the formation of osteoclasts. In particular, the invention relates to a factor which inhibits the differentiation of haematopoietic precursor cells into mononucleate osteoclast precursors. In a preferred form of the invention, the factor is a type II membrane polypeptide expressed on the osteoblast cell surface, which we have designated osteoclast inhibitory lectin (OCIL) BACKGROUND OF THE INVENTION All references, including any patents or patent 15 applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and 20 pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents •forms part of the common general knowledge in the art, in Australia or in any other country.

In normal adults, the processes of bone formation and resorption are balanced in order to maintain a normal healthy bone mass. With the onset of the menopause in females and with ageing in both sexes, the rate of bone resorption exceeds that of bone formation, resulting in net bone loss, and ultimately in osteoporosis.

Osteoblasts are the bone cells responsible for bone formation, while osteoclasts are responsible for resorption of bone. Our understanding of the factors that regulate the formation and function of osteoclasts has been greatly enhanced by laboratory methods that have enabled us to isolate and grow these cells in culture. It is now well \\melbeiles\hom$\cintae\Keep\speci\57975.OO.doc 10/02/03 la established that the development of active osteoclasts in vitro requires intimate contact between osteoblastic stromal cells and precursors of osteoclasts which are derived from haematopoietic cells belonging to the monocyte/macrophage lineage (Takahashi et al, 1988). This process is influenced by a variety of factors, including 1,25-dihydroxyvitamin D3, parathyroid hormone, prostaglandin E2, and interleukins 6, 11 and 17, all of which enhance osteoclast formation. In contrast, cytokines such as interleukins 4, 10, 13 and 18 are inhibitory (Suda et al, 1995; Martin and Udagawa, 1998).

All factors which stimulate osteoclast formation

S

\\melbfiles\home$\cinta\Keep\speci\57975.00.do 10/02/03 WO 01/05964 PCT/AU00/00864 2 act directly on the osteoblast population and not on the osteoclast precursors, leading to the proposal that osteoblasts or stromal cells express a membrane-associated peptide that regulates the formation of functional multinucleate osteoclasts. A factor, termed "Osteoclast Differentiation Factor" (ODF), that fulfils the functions of such a putative membrane-associated peptide has recently been cloned. ODF encodes a 316 amino acid type II transmembrane protein, and is a member of the TNF ligand family (Yasuda et al, 1998). Recombinant protein corresponding to the extracellular domain of ODF stimulates the formation of active, bone-resorbing osteoclasts from haematopoietic cells within the spleen, even in the absence of stromal cells. A peptide identical to ODF has also been cloned from T cells, and designated Tumour Necrosis Factorrelated activation-induced cytokine (TRANCE; Wong et al, 1997), or receptor activator of NF-KB ligand (RANKL; Anderson et al, 1997). When released by T cells following activation of the T cell receptor, it mediates the interaction of T cells and dendritic cells, resulting in stimulation as well as increased survival of the naive T cells. RANK, another member of the TNF-receptor family, has been identified on dendritic cells, and acts as the receptor for TRANCE/RANKL (Wong et al, 1997; Anderson et al, 1997).

Osteoprotegerin (OPG) is a soluble factor that belongs to the Tumour Necrosis Factor (TNF) receptor family. This factor is also known as Osteoclastogenesis Inhibitory Factor (OCIF). It has been shown to bind to TRANCE/RANKL/ODF, resulting in the inhibition of formation of functional multinucleate osteoclasts in vitro. OPG is a 401 amino acid polypeptide. Overexpression of OPG in transgenic mice results in severe osteopetrosis, with a loss of bone marrow cavities and profound depletion of osteoclasts. The same effects were observed upon administration of OPG to normal mice. Furthermore, OPG blocked ovariectomy-associated bone loss in rat. OPG mRNA 3 transcripts have been identified within bone and cartilage, vascular structures, midgut and kidney, and in several osteoblast cell lines. Current data suggest that OPG blocks the terminal stages of osteoclast differentiation, but not the formation of mononuclear osteoclast precursors (Simonet et al, 1997; Tsuda et al, 1997). The nomenclature adopted thoughout this specification is RANKL, OPG and RANK, in accordance with that proposed by Suda et al., (1999).

The interaction between RANKL and OPG in the formation of osteoclasts is illustrated in Figure 1.

Osteoclasts are derived from haematopoietic stem cells that differentiate along the monocyte/macrophage lineage.

Mononuclear precursors of osteoclasts are required to come into direct or close contact with osteoblasts to be rendered capable of differentiating into mature, functional, multinucleate osteoclasts. Osteoblasts express RANKL, a membrane-bound protein that stimulates the differentiation and formation of multinucleate osteoclasts S 20 from mononuclear precursors when it binds to its receptor, RANK. RANKL expression is stimulated by bone-resorbing factors such as PTH, PGE2, 1,25-dihydroxyvitamin D3 and interleukins 6 and 11. The action of RANKL is antagonised by Osteoprotegerin, a soluble factor secreted by 25 osteoblastic stromal cells. It binds to RANKL to inhibit the formation of differentiated multinucleate osteoclasts, but does not prevent the formation of mononuclear osteoclast precursors.

We have now identified a polypeptide factor which is able to inhibit formation of mononuclear osteoclast precursors from haematopoietic stem cells, and which is expressed at least on the cell membranes of osteoblasts.

7 9 7 5.OO.doc 2/02/05 4 Received 30 October 2001 It appears that when the molecule is expressed on the osteoblast cell membrane it is not secreted. Preventing expression of the factor results in increased formation of mononuclear precursors of osteoclasts.

SUMMARY OF THE INVENTION In a first aspect, the invention provides a nucleic acid molecule which comprises a sequence encoding a protein which inhibits osteoclast differentiation from haematopoietic cell precursors, selected from the group consisting of osteoclast inhibitory lectin (OCIL) and OCIL-related protein in which the nucleic acid molecule has a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 44, SEQ ID NO: 45 or SEQ ID NO: 46 a sequence able to hybridize to one or more of the sequences defined in under conditions of moderate to high stringency; and a sequence having greater than sequence identity with one or more of the sequences set out in Preferably the protein is expressed at least by osteoblasts.

Suitable conditions of moderate to high stringency are well known in the art. See the well-known textbook by Sambrook et al (1989), and Example 2 herein.

The nucleic acid may be cDNA, genomic DNA or messenger RNA. Preferably the nucleic acid molecule is a cDNA. More preferably the cDNA comprises a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46.

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1 1 -V w w V Received 2001 4A Preferably the protein inhibits differentiation of haematopoietic cells to osteoclast cells. In a particularly preferred embodiment, the nucleic acid molecule of the invention comprises a 110 base pair sequence as set out in SEQ ID NO: 2.

This aspect of the invention also encompasses anti-sense sequences directed against the nucleic acid molecule defined above, and particularly encompasses an anti-sense sequence directed against SEQ ID NO: Preferably the anti-sense sequence is SEQ ID NO: 24 or SEQ ID NO: In a second aspect, the invention provides a polypeptide encoded by the nucleic acid molecule of the \\melb_files\homeS\WendyS\Keep\species\ST VINCENTS osteoclast final.doc 27/02/01 AMENDED

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C I/AUUU/UUZ04 Received 24 September 2001 5 invention. Preferably the polypeptide is encoded by the human cDNA sequence. More preferably the polypeptide comprises an amino acid sequence encoded by SEQ ID NO: In a third aspect, the invention provides an antibody directed against a polypeptide of the invention.

Preferably the antibody is directed against an epitope present in a sequence selected from the group consisting of Cys-Met-Ala-Gln-Glu-Ala-Gln-Leu-Ala-Arg-Phe-Asp-Asn-Gln- Asp-Glu-Leu-Asn-Phe (SEQ ID NO: 26), Cys-Val-Thr-Lys-Ala-Ser-Leu-Pro-Met-Leu-Ser-Pro-Thr- Gly- Ser-Pro-Gln-Glu (SEQ ID NO: 48), and Cys-Val-Gln-Lys-Pro-Glu-Glu-Gly-asn-Gly-Pro-Leu-Gly-Thr- Gly-Asp (SEQ ID NO: 49).

The antibody may be polyclonal or monoclonal, but is preferably monoclonal. Suitable methods for generating either polyclonal or monoclonal antibodies are very well known in the art. It will be clearly understood that the invention encompasses biologically-active fragments and analogues of such antibodies, including but not limited to ScFv fragments, trimeric antibodies, humanised antibodies and the like. Again, methods for producing such active fragments and analogues are well known in the art. See for example PCT/AU93/00491 and PCT/AU97/00212 and references cited therein.

In a fourth aspect, the invention provides a composition comprising a polypeptide or an antibody of the invention, together with a pharmaceutically-acceptable carrier.

Methods and pharmaceutical carriers for preparation of pharmaceutical compositions are well known in the art, for example as set out in textbooks such as Remington's Pharmaceutical Sciences, 17th Edition, Mack Publishing Company, Easton, Pennsylvania, USA.

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WO 01/05964 PCT/AU00/00864 6 In a fifth aspect, the invention provides a method of treatment of a condition characterised by abnormal bone resorption, comprising the step of administering an effective amount of a modulator of expression or function of the polypeptide of the invention.

Where the condition involves excessive bone resorption, the method will comprise administration of the polypeptide of the invention or the nucleic acid encoding this polypeptide, or a biologically-active fragment or analogue thereof. Such conditions include, but are not limited to, osteoporosis, primary hyperparathyroidism, Paget's disease, rheumatoid arthritis, renal osteodystrophy, humoral hypercalcaemia of malignancy, and conditions where cancer has metastasised to bone.

Conditions characterised by deficient bone resorption include osteopetrosis. Antibodies directed against the polypeptide of the invention or anti-sense oligonucleotides directed against the nucleic acid of the invention may be used to inhibit the function of the polypeptide and thus to increase bone resorption.

It is also contemplated that the polypeptide of the invention may be used to promote healing of bone fractures, particularly in individuals where fracture healing is delayed or deficient. These include individuals suffering from osteoporosis or diabetes mellitus.

Factors which influence bone resorption, such as parathyroid hormone-related protein and RANKL, affect breast development by altering apoptosis of cells. The OCIL factor of the invention also appears to alter apoptosis of cells, and may therefore participate in breast and lymph node development, similarly to other agents which modulate bone resorption.

Thus in a sixth aspect, the invention provides a method of modulating breast and lymph node development, comprising the step of administering an effective amount of a modulator of expression or function of the polypeptide of the invention to a subject in need of such treatment.

7 In a seventh aspect, the invention provides a diagnostic kit, comprising a reagent selected from the group consisting of a nucleic acid of the invention or a fragment thereof capable of hybridising to a nucleic acid of the invention; an anti-sense nucleic acid of the invention; a polypeptide of the invention, and an antibody of the invention. For example, diagnostic kits for use in methods such as polymerase chain reaction, fluorescent in situ hybridisation, immunoassay, and the like are contemplated. Where appropriate, the molecule of the invention which is used may be labelled with a detectable marker, such as a radioactive, fluorescent, chemiluminescent or enzymic marker. Such diagnostic kits are useful for detection of abnormalities in the structure, expression or control of the factor of the invention, which may lead to increased bone resorption and concomitant pathological manifestations. They are also useful for ;*"-screening of candidate drugs to assess their ability to modulate expression or function of the polypeptide of the 20 invention.

In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

BRIEF DESCRIPTION OF THE FIGURES :vl Figure 1 summarises the factors and mechanisms involved in control of osteoclast differentiation and development, as understood before the date of the present invention.

Figure 2 shows detection by Northern blotting of a 780 base pair mRNA species using rOCIL323 (SEQ. ID NO: 2) as a probe in a variety of rat clonal osteoblast-like cell lines, which were either untreated or subjected to treatment with 10 6 M retinoic acid for 24 hours.

Figure 3 shows the results of Northern blot analysis of rat clonal osteoblast-like cell lines treated H:\cintae\Keep\speci\57975.OO.doc 2/02/05 WO 01/05964 PCT/AUOO/00864 8 with 10-6 M retinoic acid, using rOCIL402 (SEQ. ID NO: 4), a 402 base pair fragment obtained by screening of a rat ROS 17/2.8 cDNA library using the polymerase chain reaction.

Similar results were obtained using rOCIL323 fragment as a probe.

Figure 4 shows the comparison of rOCIL323 and rOCIL402 probes in Northern blotting of mRNA from 1,25dihydroxyvitamin D 3 -treated rat UMR 106-06 cells. The results showed that both fragments detected the same species of mRNA.

Figure 5 shows the results of Northern blot analysis of UMR 201 mRNA using mOCIL 2kb (SEQ ID NO: showing that this probe detected the same 780 bp species as rOCIL323 and rOCIL402.

Figure 6 shows the intron-exon structures of the mOCIL gene (SEQ ID NO: 10) and of the mOCILrP1 gene ((SEQ ID NO: 11).

Figure 7 summarises the homology between mOCIL, mOCILrP1 and mOCILrP2.

Figure 8 shows the deduced protein sequences corresponding to mOCIL (Figure 8a), mOCILrP1 (Figure 8b), and mOCILrP2 (Figure 8c), illustrating the domain structure of each protein.

Figure 9 shows a comparison between the deduced protein sequences of mOCIL, mOCILrP1, and mOCILrP2, as generated using the program Clustal W.

Figure 10 shows the results of treatment of cocultures of primary mouse calvarial osteoblasts and mouse bone marrow cells with an anti-sense oligonucleotide, 323 A/S (SEQ ID NO: 22) and 402 A/S (SEQ ID NO: 23), directed against the C-type lectin region of OCIL, antisense oligonucleotide, 474 A/S (SEQ ID NO: 25), which was directed against the sequence in the open reading frame but outside the C-type lectin region, antisense oligonucleotide, 439 A/S (SEQ ID NO: 24), which was directed against the sequence upstream of the open reading.

a: cocultures treated with anti-sense WO 01/05964 PCT/AU00/00864 9 oligonucleotide under basal conditions.

b-d: cocultures stimulated with anti-sense oligonucleotides 323 A/S (SEQ ID NO: 22), 402 A/S (SEQ ID NO: 23), 439 A/S (SEQ ID NO: 24) and 474 A/S (SEQ ID NO: 25), in the presence of 1,25-dihydroxyvitamin D 3 and PGE 2 Figure 11 shows the results of Northern blot analysis of mRNA from UMR106 parental cells, demonstrating upregulation of expression of OCIL by retinoic acid, PTH, IL-la, IL-1P, IL-11, IL-17, TNFa, TGFO, M-CSF, GM-CSF,

PGE

2 1,25-dihydroxy-vitamin D 3 1,25-dihydroxyvitamin D 3 plus PGE 2 and PGE 2 plus dexamethasone.

Figure 12A shows the results of a time-course study, showing upregulation of OCIL by PTHrP.

Figure 12B shows that the upregulation could be detected using either rOCIL402 or mOCIL 2kb as the probe.

Figure 13 shows upregulation of expression of OCIL in primary mouse calvarial osteoblasts by IL-la, IL-1p, IL-11, dexamethasone, and 1,25-dihydroxyvitamin D 3 Figure 14A shows upregulation of expression of OCIL in ST2 mouse stromal cells by PGE 2 dexamethasone, 1,25-dihydroxyvitamin D 3 IL-11, PTH, and 1,25-dihydroxyvitamin D 3 plus PGE 2 Figure 14B shows the time course of upregulation of OCIL expression in ST2 cells by dexamethasone.

Figure 15 shows the constitutive expression of mOCIL and mOCILrPl/rP2 mRNA during osteoclast formation in mouse bone marrow cell cultures.

Figure 16 shows the results of Northern blot analysis of adult mouse tissues (left panel) and adult rat tissues (right panel), demonstrating expression of OCIL.

Figure 17 shows a schematic comparison between the sequences of rOCIL402, rOCIL1.3kb, rOCIL323 and rOCIL, illustrating differences between the 3' and 5' end regions.

Figure 18a compares the exon structures of four different hOCIL clones.

Figure 18b shows the deduced protein sequences corresponding to hOCIL clone 3, illustrating the domain WO 01/05964 PCT/AUOO/00864 10 structure of the protein.

Figure 19 is a schematic representation of plasmid constructs used for recombinant expression of soluble mOCIL (Figure 19a) and soluble rOCIL (Figure 19b).

Figure 20 shows the effect of recombinant rOCIL (Figure 20a) protein or mOCIL protein (Figure 20b) on formation of multinucleate osteoclasts from mouse calvarial osteoblast-spleen cell cocultures.

Figure 21 shows the effects of hM-CSF and sRANKL in the absence or presence of mOCIL protein on osteoclast formation in mouse spleen cell cultures.

Figure 22 is a schematic representation of a plasmid construct used for recombinant expression of an MBP-mOCIL fusion protein.

Figure 23 is a schematic representation of a plasmid construct used for recombinant expression of an MBP-mOCILrP1 fusion protein.

Figure 24 is a schematic representation of a plasmid construct used for recombinant expression of an MBP-mOCILrP2 fusion protein.

Figure 25 is a schematic representation of a plasmid construct used for recombinant expression of an MBP-hOCIL fusion protein.

Figure 26 shows the effects of hM-CSF and sRANKL in the absence or presence of MBP or MBP-mOCIL fusion protein on osteoclast formation in total spleen cell (Figure 26a) or T cell-depleted mouse spleen cell cultures (Figure 26b).

Figure 27 shows the effects of hM-CSF and sRANKL in the absence or presence of the fusion proteins MBPmOCILrPI (Figure 27a) and MBP-mOCILrP2 (Figure 27b) on osteoclast formation in T cell-depleted mouse spleen cell cultures.

DETAILED DESCRIPTION OF THE INVENTION The invention will now be described in detail by way of example only, with reference to the following non- WO 01/05964 PCT/AUOO/00864 11 limiting examples and drawings.

We set out to clone a gene encoding a peptide that would function to prevent osteoclast formation. It is known that mature osteoblasts have limited potential to support osteoclast formation, and we postulated that mature osteoblasts might express osteoclastogenic inhibitors. The pre-osteoblastic cell line UMR201 can be differentiated to a more mature osteoblastic phenotype by treatment with 6 M retinoic acid for 24 hr (Ng et al, 1988). mRNA species differentially expressed between mature osteoblasts (retinoic acid-treated UMR201 cells) and immature osteoblasts (untreated UMR201 cells) were identified using an array of oligonucleotide primers in reverse transcription PCR, where products amplified from RNA from the two cellular populations were compared. We characterised products which were elevated in mature osteoblasts as candidates for osteoclastogenic inhibitory molecules.

Abbreviations used herein are as follows:

GM-CSF

hPTH

IGF

IL

LIF

M-CSF

1,25(OH)

OCIF

ODF

OPG

PCR

PGE2

PTH

PTHrP

RANK

RANKL

TGF

granulocyte/macrophage colony stimulating factor human parathyroid hormone insulin like growth factor interleukin leukaemia inhibitory factor macrophage colony stimulating factor (CSF-1) 2D3 1,25-dihydroxyvitamin D 3 osteoclastogenesis inhibitory factor osteoclast differentiation factor osteoprotegerin polymerase chain reaction prostaglandin E 2 parathyroid hormone parathyroid hormone-related protein Receptor activator of NF-KB Receptor activator of NF-KB ligand transforming growth factor WO 01/05964 PCT/AUGO/00864 12

TNF

TRAP

tumour necrosis factor tartrate-resistant acid phosphatase.

13 Throughout this specification amino acids are represented using the conventional single-letter code.

Example 1: Isolation of Rat cDNA Encoding the Inhibitory Factor Total RNA was isolated from retinoic acid-treated preosteoblastic UMR201 cells using guanidine thiocyanate (Chomczynski et al, 1987). First strand cDNA was synthesised from 2 pg of total RNA by incubating for 1 h at 42 0 C with 15 units of AMV reverse transcriptase (Promega, Madison, WI) following oligo priming with the 3'adaptor primer 5'-GGC CAC GCG TCG ACT AGT ACT TTT TTT TTT TTT TTT (Clontech, California, USA). A sense primer that was complementary to rat calcitonin cDNA, designated primer 15 CT1: *i CT1 5'-ATG CTG GGC ACG TAC ACA CAA-3' (SEQ ID NO:1) and 3'UAP GGC CAC GCG TCG ACT AGT' AC-3' (Clontech, 20 California, USA) were used as primers in the polymerase Schain reaction (PCR). The PCR conditions utilised a touchup PCR protocol with denaturation at 94 0 C for 5 min, S: and then 5 cycles at 94 0 C for 1 min, 37 0 C for 1 min and 72 0 C for 1 min, followed by 35 cycles of 94 0 C for 1 min, 49 0 C for 1 min and 72 0 C for 1 min. For these experiments, Expand High Fidelity PCR System (Boehringer Mannheim) was used in a Perkin Elmer Cetus 480 thermal cycler. A 321 bp PCR product was obtained. This 321 bp fragment, which we designated rOCIL323 (SEQ ID NO: was used as a probe in Northern blots. As shown in Figure 2, it hybridised to a 780 bp mRNA species in UMR 201, UMR 201-10B, UMR 106-06, UMR 106-01 and ROS 17/2.8 cells, all of which are rat clonal osteoblast-like cell lines.

Since retinoic acid and PTH enhance OCIL mRNA expression dramatically in UMR 106-06 and UMR 106 parental cells, a similar RT-PCR procedure was carried out using RNA isolated from retinoic acid or hPTH 1-34 treated UMR 106 \\melb-files\homeS\cintaeKeep\speci\5195.00.doc 10/02/03 14 parental cells. A PCR product identical to the 321 bp fragment for rOCIL323 was obtained, and its expression was found to be upregulated in UMR 106 cells treated with either retinoic acid or hPTH 1-34.

To extend the sequence of OCIL, anchored PCR was used to screen a rat ROS 17/2.8 cDNA library with Xgtll arms. An antisense 25 bp primer, designated OCILrl: OCILrl 5'-TGA GTG TTG TCT GTC CAC TTC CAA G-3' (SEQ ID NO: 3) complementary to a sequence in the 321 bp fragment, was used with either the Xgtll forward primer S5'-GGT GGC GAC GAC TCC TGG AGC C-3' or Xgtll reverse primer 15 5'-GAC ACC AGA CCA ACT GGT AAT G-3' (Clontech) to amplify an aliquot (106 plaque forming units) of the recombinant library. Cycling parameters were 94 0 C for 5 min, then cycles of 94 0 C for 30 sec, 60 0 C for 30 sec, and 72 0 C for 2 min, followed by a final extension step of 72 0 C for min. A 402 bp fragment was obtained with Xgtll reverse primer as the anchored primer. Sequencing of this 402 bp fragment showed 88.6% identity over a length of 97 bp with rOCIL323 (SEQ ID NO: The 402 bp fragment, designated rOCIL402, whose sequence is set out in SEQ ID NO: 4, was 25 used to probe Northern blots obtained from the rat osteoblast-like cell line. It hybridised to the same 780 bp mRNA species observed with the rOCIL323 probe.

These results are shown in Figures 3 and 4. The same results were obtained in both the presence and absence of 1,25-dihydroxyvitamin D 3 A 3' Rapid Amplification of cDNA Ends (3'-RACE) strategy was used to obtain the 3' ends of the cDNA of interest. First-strand cDNA was synthesised from total RNA isolated from hPTH 1-34 treated UMR 106 parental cells by incubating for 1 h at 42 0 C with 15 units of AMV reverse transcriptase (Promega, Madison, WI) following priming with the 3'adaptor primer 5'-GGC CAC GCG TCG ACT AGT ACT TTT \\mebfiles\homS\cinOae\Ke ep\speci\57975.OO.doc 10/02/03 15 TTT TTT TTT TTT (Clontech, California, USA) according to the manufacturer's instructions. The sense specific primers used were OCILrll (SEQ ID NO: OCILrll 5'-GAA ACA TCC CCC TGG AGT ATC C-3' and OCILrl2 (SEQ ID NO: 6) OCILrl2 5'-CCA AGT AAC TGG ACA TTG AGC CAG A-3' complementary to sequences within rOCIL402 (SEQ ID NO: 4).

First-strand cDNA was synthesised from total RNA isolated from hPTH 1-34 treated UMR 106 parental cells, using the oligo dT-anchor primer. The cDNA was further amplified by PCR using OCILrll or OCILrl2 and 3'UAP 5'-GGC CAC GCG TCG ACT AGT AC-3' (Clontech, California, USA) PCRs were run at 94°C for 5 min, then 35 cycles of 94°C for 30 s, 62 0 C for 30s, and 72 0 C for 2 min, followed by a final extension step of 72 0 C for 10 min. Three different polyadenylated 3' 20 sequences were obtained, designated rOCIL1.3kb (SEQ ID NO: rOCIL738bp (SEQ ID NO: 8) and rOCIL620bp (SEQ ID NO: 9) respectively. The region of sequence identity between rOCIL323 and rOCIL402 was found to extend to 117 bp.

Example 2: Isolation of Mouse cDNA and gDNA Encoding the Inhibitory Factor rOCIL402 was labelled with 32 P] a-dCTP by using the Random Primer labelling kit (Boehringer Mannheim), and a mouse liver cDNA library was subjected to hybridisation screening at 65 0 C in a hybridisation buffer containing 4 x SSPE (SSPE contains 0.15 M NaC1, 0.01 M NaH 2

PO

4 and 0.001 M EDTA), 5 x Denhardt's solution, 0.5% sodium dodecyl sulfate (SDS) for 24 hr. The filters were then washed sequentially in 2 x SSC at 65 0 C for 15 min, 2 x SSC with 0.1% SDS at 65 0 C for 30 min, and finally 0.1 x SSC at for 10min. We obtained a 1907bp mouse cDNA, designated 7 975.00.doc 2/02/05 16 mOCIL2kb (SEQ ID NO: 10). The sequence of mOCIL2kb shows identity over a length of 461 bp to that of rOCIL1.3kb.

When used as an antisense riboprobe in Northern blot analysis, mOCIL2kb hybridised to a 780 bp mRNA species in UMR201 as detected by rOCIL323 and rOCIL402,as shown in Figure A cDNA fragment corresponding to the nucleotides 58-776 of mOCIL2kb was used as a cDNA probe to screen a genomic BAC Mouse I Hybridisation library. The screening was performed under contract by Genome Systems, Inc.

According to their protocol, the cDNA fragment was labelled with 32 P] a-dCTP by random primer labelling and the library was screened under the hybridisation conditions of 55 0 C in a hybridisation buffer containing 5.5 x SSC, 5 x 15 Denhardt's solution, 0.5% SDS and 0.5 x HEPES buffer for 18 hr. The filters were then washed sequentially in 1mM Tris- SHC1 pH 8.0 and 1% sarkosyl for 15 min, and 3 times in 1mM S' Tris-HCl pH 8.0 for 15 min washes at room temperature.

Eight positive clones were isolated, of which seven positive clones were screened. After the genomic DNA was digested with HindIII or BamHI, Southern blot analyses were carried on with the same cDNA probe. Four clones (db.20147, db.20149, db.20151 and db.20152) were related, and other clones (db.20150, db.20153 and db.20154) are yet to be extensively analysed. The clones db.20147 and db.20149 were identical, and these differed from the other two identical clones db. 20151 and db.20152. These four clones were sequenced by subcloning into the pBS vector and by direct sequencing of the genomic clone using cycle sequencing. Sequencing of 8622 bp of the genomic clone (db.20152) of mOCIL was completed (SEQ ID NO: 37). It contains 5 exons, as shown in Figure 6. The 5' flanking region adjacent to exon I contains an A/T-rich motif, AATAAA, as TATA box gene promoter. Sequencing of 9862 bp of the genomic clone (db. 20149) was also completed (SEQ ID NO: 11). It contains 6 exons, as shown in Figure 6. The \\melbfiles\hoeS\cintae\Keep\speci\579 7 5.OO.doc 10/02/03 PCT/AU00/00864 Received 24 September 2001 17 sequences of exons I and II were completely different to that of mOCIL exon 1. The sequence of exons III to VI was 90.28% identical to that of mOCIL from exon II. The flanking region adjacent to exon I is a GC-rich region, containing a Sp 1 binding site. In combination, these features indicated that in fact this was a different gene.

A search of the GenBank database showed that exons I and II showed 100% identity to a cDNA clone encoding a C-type lectin expressed in mouse bone marrow-derived dendritic cells, which was deposited in the GenBank data base on January 1999 (Accession No. AF121352) and released on June 1999. However, exons III to VI are 92% identical to AF121352 cDNA. The sequence of this genomic clone is redesignated as mOCIL-related protein 1 (mOCILrP1) gene (SEQ ID NO:11). The full length mOCILrP1 cDNA was originally thought to be a splice variant of mOCIL, and is 990 bp in length (SEQ ID NO: 12).

To confirm the mOCIL (SEQ ID NO: 36) and mOCILrP1 (SEQ ID NO:12) cDNA sequences, RT-PCR was carried out using total RNA isolated from ST2 mouse stromal cells, primary mouse calvarial osteoblasts and mouse liver tissue. The sense primer represented nucleotides 18-36 of mOCILrP1 (SEQ ID NO:12), and is designated as primer OCILm47 (SEQ ID NO: 13), OCILm47 TCC CAT GCC AGA TTG CTT G-3' The antisense primer, which was originally designed from mOCIL2kb (SEQ ID NO: 10) nucleotides 136-157, represented nucleotides 746-725 of mOCIL (SEQ ID NO: 36) and is designated primer OCILml2 (SEQ ID NO: 14), OCILml2 5'-GGG ACC ATA GGG GAA AGA GTA G-3' The PCR was run at 94 0 C for 5 min, then 35 cycles of 94 0 C for 30s, 60 0 C for 30s, and 72 0 C for 1 min, followed by a final extension step of 72 0 C for 10 min. Seven clones \\melb_files\homeS\evonee\Keep\Speci\PCT-AUOO-00864.doc 24/09/01 AMEND:ED SHEET

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18 containing a 721 bp fragment were obtained from three sources, ST2 mouse stromal cells, primary mouse calvarial osteoblasts and mouse liver tissue. In 2 of the 7 clones, there was 100% identity to mOCILrP1 sequence, and 92.2% identity to mOCIL after the first 115 bp. In the other clones, when compared to the mOCILrP1 sequence, there was 100% identity in the first 106 bp (exons I and II), but only 90.5% identity in the remaining 615 bp. This 721 bp fragment, originally designated as mOCIL47, was redesignated as mOCILrP2 (SEQ ID NO: 15). MOCILrP2 is related to, but distinct from, mOCIL (SEQ ID NO: 36) and mOCILrP1 (SEQ ID NO: 12).

A sense primer representing nucleotides 343-364 of mOCIL2kb (SEQ ID NO:10) and representing nucleotides 34- 15 57 of mOCIL (SEQ ID NO:36), designated as OCILml7 (SEQ ID NO: 16), OCILml7 5'-TGG AAA CTC AGC TCC TCA GCT CTG-3' and antisense primer OCILml2 were also used to carry out RT-PCR with RNA from three sources, ST2 mouse stromal cells, primary mouse calvarial osteoblasts and mouse liver tissue, as above. PCR was run under the same conditions as above. Ten clones were obtained, each containing a 713 bp 25 fragment. This sequence is designated mOCIL17 (SEQ ID NO: 17), and is 100% identical to mOCIL (SEQ ID NO: 36).

RT-PCR was also carried out using a sense primer corresponding to the region located at the junction of exons II and III, representing nucleotides 245-269 of mOCIL (SEQ ID NO: 36) and at the junction of exon III and exon IV, representing nucleotides 243-267 of mOCILrP1 (SEQ ID NO: 12), and designated primer OCILm32 (SEQ ID NO: 18), OCILm32 TTT GTC AGC AAC AAA GAC AGA ACA G-3' The primer oligonucleotide OCILm32 has 24 of 25 bp complementary to mOCILrP1: \\melb-files\homeS\cintae\Keep\speci\ 51 91 5 .OO-doc 10/02/03 Received 24 September 2001 19 5'-TTTGTCAGCAACAAAGACAGAACAG-3' Primer I I I l I I I I I I I I I I I I I lI I I mOCILrP1 (267) Strand Primer OCILml2 was used as an antisense primer.

RT-PCR was carried out with RNA from three sources, ST2 mouse stromal cells, primary mouse calvarial osteoblasts and mouse liver tissue, as above. PCR was run under the same conditions. Four clones were obtained, each containing a 502 bp fragment. Three of the four clones have 100% identity to mOCIL (SEQ ID NO: 36) and one of the four clones is 100% identical to mOCILrP1 (SEQ ID NO: 12).

RT-PCR was also carried out using the sense primer OCILm47 (SEQ ID NO: 13) and an antisense primer representing nucleotides 855-874 of mOCILrP1 (SEQ ID NO: 12), designated primer OCILm49 (SEQ ID NO: 38), OCILm49 5'-GTG GTT GCT CAG ATG TGA AC-3' RT-PCR was carried out with RNA from the same three sources, ST2 mouse stromal cells, primary mouse calvarial osteoblasts and mouse liver tissue, as above. PCR was run under the same conditions. Two clones were obtained, each containing a 856 bp fragment with 100% identity to AF121352 and in which the first 721 bp are 100% identical to mOCILrP2 (SEQ ID NO: To further confirm that mOCILrp2 is AF121352, an antisense primer was designed based on the sequence of AF121352 (nucleotides 908-929), designated as primer OCILm48 (SEQ ID NO: 39), OCILm48 5'-TTC ACA CAT CCC AGA AGA GGA C-3' OCILm47 (SEQ ID NO: 13) was used as sense primer. RT-PCR was carried out under same conditions as above. Two clones were obtained, each containing a 916 bp fragment which has \\melbfiles\homeS\evonnee\Keep\Speci\PCT-AUOO-00864.doc 24/09/01 AMENDED SHEET

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A IV VIVV UV Received 24 September 2001 20 100% identity to AF121352 and in which the first 721 bp is 100% identical to mOCILrP2 (SEQ ID NO: The full length mOCILrP2 cDNA is 988 bp in length. Its first 123 bp is 100% identical to mOCILrP1, but only 91.7% identical in the remaining 865 bp. Figure 7 summarises the homology between mOCIL, mOCILrPl and mOCILrP2. The three different sequences (SEQ ID NO: 12, and 36), which overall have 87% identity, may represent gene duplications, where either one or all three sequences may have similar biological outcomes. The functional data we have to date, relating to the inhibition of osteoclast formation from haemopoietic precursor cells using antisense oligonucleotides (SEQ ID NO: 24 and 25), have been obtained mainly with mOCIL17 (SEQ ID NO: 17), although experiments with recombinant protein (see below) indicate that the extracellular domains of mOCIL, mOCILrP1 and mOCILrP2 respectively can inhibit osteoclast formation.

mOCIL has an open reading frame encoding a 207 amino acid protein. As shown in Figure 8a, its putative protein structure is typical of a type II membrane protein, with a predicted 143 amino acid extracellular domain, a 21 amino acid transmembrane domain, and a 43 amino acid cytoplasmic domain. The extracellular domain has 5 cysteine residues. There are three potential N-linked glycosylation sites at residues 74, 100 and 158, all of which are in the extracellular domain. The putative protein sequence for mOCIL is designated mOCIL protein (SEQ ID NO: Comparison of the putative protein sequences derived from the rOCIL323, rOCIL1.3kb and mOCIL cDNA sequences with the SwissProt protein database indicated that the mOCIL protein sequence included a 113 amino acid C-lectin type motif, from positions 80 to 192 in the mOCIL protein sequence (SEQ ID NO: 40). This C-lectin motif is similar to that of CD69, a membrane-bound lectin expressed by bone marrow haematopoietic cells, and thought to be involved in monocyte differentiation. C-lectin motifs are \\melbtiles\homSS\evonnee\Keep\Speci\PCT-AUOO-00864.doc 24/09/01 AMENDED SHEEk

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Received 24 September 2001 21 also involved in cell-cell contact and lipid binding (Sharon and Lis, 1995; Gabius 1997; Kieda, 1998).

mOCILrP1 has an open reading frame encoding a 218 amino acid protein. The putative protein sequence for mOCILrP1 is designated mOCILrP1 protein (SEQ ID NO: 41).

Its structure is also typical of a type II membrane protein, with a predicted 142 amino acid extracellular domain, a 21 amino acid transmembrane domain, and a amino acid cytoplasmic domain. The mOCILrP1 protein sequence also has a 113 amino acid C-lectin type motif, from positions 92 to 204 in the mOCILrP1 protein sequence (Figure 8b). The extracellular domain has 6 cysteine residues. There are three potential N-linked glycosylation sites at residues 86, 112 and 207, all of which are in the extracellular domain. There is a myristylation motif in the intracellular domain.

MOCILrP2 has an open reading frame encoding a 217 amino acid protein. The putative protein sequence for mOCILrP2 is designated mOCILrP2 protein (SEQ ID NO: 42).

Its structure is also that of a type II membrane protein, with a predicted 141 amino acid extracellular domain, a 21 amino acid transmembrane domain, and a 55 amino acid cytoplasmic domain. Similarly to mOCIL and mOCILrP1, the mOCILrP2 protein sequence has an 113 amino acid C-lectin type motif, from positions 92 to 204 in the mOCILrP2 protein sequence (Figure 8c). The extracellular domain has cysteine residues. There are four potential N-linked glycosylation sites at residues 86, 95, 112 and 165, all of which are in the extracellular domain.

The three different mouse protein sequences (SEQ ID NO: 40, 41 and 42) overall have 89% identity as shown in Figure 9. There are differences in the intracelullar domains between mOCIL and mOCIL-related proteins, and these domains may have different functional roles. If the C-type lectins act as receptors, the intracellular domains may confer different properties as a result of signal transduction. Comparison of the protein sequences in the \\melbtiles\home$\evonnee\Keep\Speci\PCT-AUOO-00864.doc 24/09/01 AMENDED H-ET

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WO 01/05964 PCT/AUOO/00864 22 intracellular domain against the PROSITE database, using the ScanProsite program, showed that mOCIL protein has a Casein Kinase II (CK2) phosphorylation site at position 16-19 in SEQ ID NO: 40. In contrast, mOCILrP1 protein has two Protein Kinase C (PKC) phosphorylation sites at positions 42-44 and 51-53 (SEQ ID NO: 41), while mOCILrP2 (SEQ ID NO: 42) has no phosphorylation sites.

The three different proteins, mOCIL, mOCILrPl and mOCILrP2, may be distinguished by several criteria: Nucleotide sequence: mOCIL, mOCILrP1 and mOCILrP2 appear to be derived from a common ancestral gene; however there are nucleotide differences which permit identification of the three molecules using specific oligonucleotide primers in RT-PCR, as described in Example 5, Figure Gene structure: The promoter of mOCIL is a TATA promoter (SEQ. ID. No. 37), while the promoter for mOCILrP1 is a GC-rich region containing a SP 1 binding site (SEQ. ID. No. 11).

mOCIL expression is regulated by PTH, while the expression of mOCILrPl and mOCILrP2 is not (see example The polypeptide products of mOCIL, mOCILrP1 and mOCILrP2 can be distinguished using antibodies directed against peptide fragments of mOCIL (SEQ. ID. No. 48) and mOCILrPl/rP2 (SEQ. ID. No. 49) based on the intracellular domains of the respective proteins. These have been used in tissue localisation studies, as described in Example 6.

Example 3: Isolation of Human cDNA Encoding the Inhibitory Factor a-dCTP labelled rOCIL402 was used to probe a human fetal cDNA library under low stringency hybridisation conditions at 55 0 C in a hybridisation buffer containing 4 x SSPE, 5 x Denhardt's solution, 0.5% SDS for 24 hr, and then washed with low stringency at 2 x SSC with 0.1% SDS at 40 0 C for 15 min, and 1 x SSC/0.1% SDS at 40 0

C

r I UVIVV0+ Received 24 September 2001 23 for 15min. Eight positive clones were obtained after tertiary screening. Clone No. 6 is a 1.3 kb cDNA segment, whose sequence was designated hOCIL clone 6 (SEQ ID NO: 19). The putative protein sequence encoded by bp883- 1059 was a C-type lectin moiety, which showed 73% homology to the C-type lectin sequence previously demonstrated in rOCIL323, rOCIL1.3kb and mOCIL2kb. However, regions of amino acid sequence 5' and 3' to this C-type lectin domain were different from those of the mouse and rat sequences, as shown in Figure 6.

Clone No. 8 is 960 bp long. It has 64% identity over a length of 145 bp with rOCIL402. A search of the EST database showed that clone No. 8 has 99.5% sequence identity with an EST clone of unknown function from human pregnant uterus, Accession No. AA029932, over the published length of this EST 209 bp. This EST clone was ordered and further sequenced. The EST clone is 680 bp in length, and has 64% identity with rOCIL1.3kb over a length of 343 bp.

It also has 64% identity over a length of 346 bp compared to mOCIL. RT-PCR showed that clone No 8 and AA029932 represent overlapping clones, which are contiguous, and combine to represent a human OCIL clone 1 of 1305 bp in length (SEQ ID NO: The deduced protein sequence has 56% homology to the deduced protein sequence of rOCIL1.3kb, and 62% homology to that of mOCIL. These differences are principally at the N-terminus. Although there is homology between the mouse and human OCIL proteins incertain regions, this indicates that the mouse cDNA could not reliably be used to isolate a human genomic DNA encoding hOCIL.

In order to obtain the hOCIL gene, the 680 bp cDNA insert of clone AA029932 was isolated and screened by Genome Systems, Inc. against the genomic BAC Human Release II Hybridisation library, as described in Example 2. One positive clone was obtained. This genomic sequence, corresponding to the sequence from 654bp-1304bp of hOCIL, \\melbiles\homeS\evonee\Keep\Speci\PCT-AUOO-00864.doc 24/09/01 AMENDED ;-ii

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I A W r- Received 24 September 2001 24 clone has 100% identity to a sequence segment within a human genomic clone of 178,607 bp, which was deposited in the GenBank database on April 6, 1999 (Accession No.

AC007068), and the 5' flanking region, promoter region and the first 654 bp of cDNA sequences are represented in the 74,801 bp sequence deposited in the GenBank database on December 9, 1999 (Accession No. AC010186).

The hOCIL gene is located in chromosome 12p.

Chromosome 12 and chromosome 11 are considered to be evolutionarily related. There are several examples of evolutionarily related proteins whose genes are located on chromosome 12 and chromosome 11, such as PTH and PTHrP, IGF and IGF I Harvey ras sarcoma 1, and Kisten ras sarcoma 2, etc. (Martin et al., 1991). Thus chromosome 11 and chromosome 12 share genes of similar biological characteristics with redundant function.

Example 4: Effect of Anti-Sense Oligonucleotides on Osteoclast Formation Primary mouse calvarial osteoblasts were cocultured with mouse bone marrow cells to generate mononuclear and multinucleate osteoclasts. Staining for tartrate-resistant acid phosphatase (TRAP), performed using a commercial leukocyte acid phosphatase kit from Sigma Diagnostics (St. Louis, MO, USA; Katsogiannis et al, 1998), identified these cells as osteoclasts. Under normal conditions, multinucleate functional osteoclasts are not formed unless the cocultures are stimulated with 1,25dihydroxyvitamin D3 and PGE2.

Experiments were carried out to block translation of OCIL mRNA in order to determine the function of its translated product. Antisense oligonucleotides may also down-regulate mRNA levels, and thus may effectively decrease transcription as well as translation. Primary mouse calvarial osteoblasts were treated with antisense oligonucleotides. Four antisense oligonucleotide sequences were designed. Two of these antisense oligonucleotide \\elb_files\hoeS\evonnee\Keep\Speci\PCT-AU0O-0086 4 .doc 24/09/01 AMENDED SHEET

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25 sequences were complementary to the C-type lectin region, and designated 323 (SEQ ID NO: 22) and 402 (SEQ ID NO: 23) respectively: 323 5'-GAG TGT TGT CTG TCC ACT TCC-3' 402 5'-TTT CCA ACT CCA ATC CAG TTT-3' The 323 antisense oligonucleotide has 19 of 21 bp complementary to mOCILrP2 (SEQ ID NO: 5'-GAGTGTTGTCTGTCCACTTCC-3' 323 antisense (SEQ ID NO: 22) 153'-GTCACAACAAACAGGTGAAGG-5' mOCILrP2 Strand and has 20 of 21 bp complementary to mOCILrP1 (SEQ ID NO: 12) :5'-GAGTGTTGTCTGTCCACTTCC-3' 323 antisense (SEQ ID NO: 22) 2 0 1 1 1 1 1 1 1 1 1 1 3' -GTCACAACAGACAGGTGAAGG-5' mOCILrPl Strand :and 100% to mOCIL (SEQ ID NO: 36) 5'-GAGTGTTGTCTGTCCACTTCC-3' 323 antisense (SEQ ID NO: 22) 5 3' -GTCACAACAGACAGGTGAAGG- 5' mOCIL Strand The 402 antisense oligonucleotide (SEQ ID NO: 23) has 100% complementarity to mOCILrP1 (SEQ ID NO: 12) and has 20 of 21 bp complementary to mOCILrP2 (SEQ ID NO: 15) and mOCIL (SEQ ID NO: 36): 51-TTTCCAACTCCAATCCAGTTT-3' 402 antisense (SEQ ID NO: 23) II I I1111111 11 11I 1 II 31-AAAGGTTGAGGTCAGGTCAAA-51 mOCILrP2 Strand lb..iles\home$\Ciftae\Keep\gpeci\579?S .OO.doc 10/02/03 26 3 402 antisense (SEQ ID NO: 23) I I I lI I II I I I I I II I I I I I mOCIL Strand The other two antisense oligonucleotide sequences, respectively designated 439 (SEQ ID NO: 24) and 474 (SEQ ID NO: 25), specifically inhibit the translation of mOCIL (SEQ ID NO: 36) but not mOCILrPl (SEQ ID NO: 12) and mOCILrP2 (SEQ ID NO: The oligonucleotide 439 (SEQ ID NO: 24) is antisense to the sense primer OCILml7 and located upstream of the open reading frame: 439 5' GAG GAG CTG AGT TTC CAC TAC-3' *o o S" The antisense oligonucleotide 474 (SEQ ID NO: Sis complementary to a region in mOCIL17 (SEQ ID NO: 17) in the open reading frame in the intracellular domain but outside the C-type lectin region: 474 5'-GGT AGG GAA GCC TTT GTG AC-3'.

Under basal conditions, ie. in the absence of stimulation with 1,25-dihydroxyvitamin D3 and PGE2, there S: 25 was a 3- to 5-fold increase in the number of mononucleate TRAP-positive cells in the cocultures treated with the 323 (SEQ ID NO: 22) and 474 (SEQ ID NO: 25) antisense oligonucleotides over the period from 3 to 7 days. -With the 402 antisense oligonucleotide (SEQ ID NO: 23), a 4.5-fold increase in the formation of mononucleate

TRAP

positive cells was observed after 7 days treatment.

Multinucleate TRAP-positive cells (323 (SEQ ID NO: 22); 2, 474 (SEQ ID NO: 25); 4.25 1.25) were also observed in cocultures treated with both 323 (SEQ ID NO: 22) and 474 (SEQ ID NO: 25) antisense oligonucleotides at a concentration of 5pM, whereas none were observed in the control. These experiments were performed three times, and \\melb-files\hom S\cintae\ePep\ eci\57975O0doc 10/02/03 WO 01/05964 PCT/AUOO/00864 27 a representative result is shown in Figure When the cocultures were stimulated with 1,25-dihydroxyvitamin D 3 and PGE 2 multinucleate TRAPpositive osteoclasts were formed after 7 days. Treatment with 5 pM 323 antisense oligonucleotide (SEQ ID NO: 22) resulted in a seven-fold increase in the number of multinucleate osteoclasts, as shown in Figure Treatment with 10 pM 402 (SEQ ID NO: 23), 5 pM 439 (SEQ ID NO: 24) and 474 (SEQ ID NO: 25) antisense oligonucleotide resulted in a 2 to 3-fold increase in the formation of multinucleate osteoclasts after 7 days, as shown in Figures 10c and These TRAP-positive cells were further characterized as osteoclasts by the presence of receptors for calcitonin, demonstrated using autoradiography and immunostaining, and by the ability of these cells to form resorption pits in bone slices.

Effects of mOCIL antisense oligonucleotides on the three phases of osteoclast formation were also investigated. Mouse bone marrow and primary osteoblastic cells were cocultured in the absence of 1,25-dihydroxyvitamin D 3 and PGE 2 for a 7 day culture period. 323 (SEQ ID NO: 22) and 474 (SEQ ID NO: antisense oligonucleotides were added for the 3 phases of culture: the first phase (0-3 days), in which there is proliferation of osteoclast progenitors, the second phase days) and the final phase (5-7 days), in which these cells differentiate into mature osteoclasts. TRAP-positive osteoclasts were counted. In order to examine the role of OCIL on the bone resorptive activity of mature osteoclastic cells, the cells were also cultured on dentine slices under the same culture conditions as above, and resorption pits formed on dentine slices were quantitated. The results are shown in Table 1, and indicate that the OCIL acted at an early stage in osteoclast formation.

0 Table 1 Effects of mOCIL Antisense Oligonucleotides on the Three Phases of Osteoclast Development TIME TREATMENT MONO MNC PITS 7 days control 3596 511.5 0 10 4.4 0-3 days 323 6880 674 7.7 5.3 34 474 6893 429.6 8.7 1.7 20 days 323 2840 197.6 0 14 6 474 3110 334 4.3 2 24 2.5 5-7 days 323 4236 518.6 0 10.6 0.79 474 3363 139.8 0 3.3 2 0-7 days 474 5223 571 3.3 1 20.3 5.9 mono mononuclear osteoclast precursors MNC multinucleate osteoclasts pits resorption pits formed on dentine slices *p 0.05 vs. control 0.01 vs. control WO 01/05964 PCT/AU00/00864 29 Example 5: Regulation of expression of OCIL mRNA The regulation of OCIL mRNA expression was examined in the UMR106 parental osteoblast-like cell line using rOCIL402 as a probe. As shown in Figure 11, expression of the mRNA was upregulated by retinoic acid parathyroid hormone parathyroid hormone related protein TNF-a, interleukin la (IL-1 IL- 13, IL-11, IL-17, GM-CSF, M-CSF, TGFP, dexamethasone, 1,25dihydroxyvitamin D3 and prostaglandin E2. A time course study, illustrated in Figure 12, showed that parathyroid hormone-related protein (1-34) increased levels of OCIL mRNA as early as 1 hour, peaking at 4 hours and maintaining the high level of expression until 48 hours.

As shown in Figure 13, in primary mouse calvarial osteoblasts, OCIL mRNA was upregulated by IL-1 a, IL-1j IL-11, 1,25-dihydroxyvitamin D3 and retinoic acid. In ST2 mouse stromal cells, OCIL mRNA was upregulated by dexamethasone, 1,25- dihydroxyvitamin D3 and IL-11. The time course also showed that dexamethasone increased OCIL mRNA at 1 hour, peaking at 2 hours and returning at basal level at 24 hours. These results are illustrated in Figures 14A and 14B, respectively.

Example 6: OCIL mRNA expression during osteoclast formation in mouse marrow cultures OCIL mRNA expression during osteoclast formation in mouse marrow cultures was investigated by RT-PCR. The mouse bone marrow cells were prepared and cultured for 8 days in the presence of 1,25-dihydroxyvitamin D3, as described by Ikegame et al (1995). At each time point, total RNA was isolated. RT-PCR was carried out using OCILml7 (SEQ ID NO: 16) and OCILml2 (SEQ ID NO: 14) as sense and antisense primers respectively. The PCR was run at 94 0 C for 5 min, then 30 cycles of 94°C for 30s, 60°C for 30s, and 720C for 1 min, followed by a final extension step of 72 0 C for 10 min. RT-PCR was also carried out to investigate mOCILrP1/rP2 mRNA expression during such WO 01/05964 PCT/AUOO/00864 30 cocultures. Primers specific for mOCILrPl/rP2, which distinguish these from mOCIL, were OCILm47 (SEQ ID NO: 13), a sense primer located on the intracelullar domain of mOCILrP1 and mOCILrP2, and OCILml2 (SEQ ID NO: 14) as an antisense primer. The PCR was run at 94°C for 5 min, then cycles of 940C for 30s, 60°C for 30s, and 720C for 1 min, followed by a final extension step of 72°C for 10 min.

Southern blot analysis was carried out as described by Zhou et al (1994); 20 pl of each PCR reaction mixture was run on a 2% agarose gel, transferred to nylon membranes, and the products authenticated by probing with an internal antisense strand oligonucleotide, OCILrl (SEQ. ID NO: 3).

OCILrl, which has 24 of 25 bp complementary to mOCIL and mOCILrP1 and 23 of 25 bp complementary to mOCILrP2, was labelled with digoxigenin-dUTP using a 3'-tailing kit (Boehringer Mannheim). Hybridisation was carried out with 2 pmol/ml labeled oligonucleotides in a buffer containing x SSC, 0.02% SDS, 0.1% sarcosine and 100 ng/ml poly A, at for 14 h. Detection was by chemiluminescence using CDP-star (Boehringer Mannheim), according to the manufacturer's instructions.

For comparison with another osteoclast inhibitor, OPG mRNA expression was also investigated. A set of sense and antisense primers was used as described by Horwood et al. (1998), having nucleotide sequences represented by OPG- 7 (5'-TGAGTGTGAGGAAGGGCGTTAC-3', nucleotides 405-426) and OPG-3 (5'-TTTCTCGTTCTCTCAATCTC-3', nucleotides 1021-1040), respectively. The PCR was run at 94°C for 5 min, then cycles of 94 0 C for 30s, 57 0 C for 30s, and 72°C for 1 min, followed by a final extension step of 72°C for 10 min.

Hybridisation was carried out using digoxigenin labeled internal sense strand oligonucleotide, OPG-1 ACCAAAGTGAATGCCGAG-3') under the same conditions described above. To ensure equal starting quantities of RNA in each sample, the reverse transcribed material was also amplified using oligonucleotide primers specific for rat GAPDH (39).

A 414 bp fragment was amplified using a WO 01/05964 PCT/AU00/00864 31 oligonucleotide, GAPDH-4 (5'-CATGGAGAAGGCTGGGGCTC-3', representing nucleotides 306-325 of rat GAPDH) and a 3'specific oligonucleotide, GAPDH-5 representing nucleotides 701-720). Products were verified with a digoxigenin-labelled internal sense strand oligonucleotide, GAPDH-1 (5'-GCTGTGGGCAAGGTCATCCC-3', representing nucleotides 640-659) using the hybridisation conditions described above.

As shown in Figure 15, OCIL mRNA was constitutively expressed in fresh bone marrow cells at a high level. When cultures were stimulated by 1,25dihydroxyvitamin D3, a time-dependent decrease in OCIL mRNA relative to GAPDH mRNA occurred. In contrast, OPG mRNA was constitutively expressed at a low level in fresh bone marrow cells, and this level was increased by treatment with 1,25-dihydroxyvitamin D3 after 4 days. This increased mRNA level was maintained for up to 8 days culture in the presence of 1,25-dihydroxyvitamin D3. As reported by Romas et al. (1996), in this system, multinucleate osteoclast formation was observed after day 5, correlated with the decrease in OCIL mRNA expression, and an increase in mRNA for IL-11Ra as well as calcitonin receptor.

PTH (1-34) or PTHrP which influence bone resorption, have been shown to induce osteoclast formation in the coculture of primary mouse calvarial osteoblasts and mouse bone marrow cells. To investigate whether PTH 1-34 regulates mRNA expression for mOCIL and the related proteins, mOCILrP1 and mOCILrP2, RT-PCR was carried out using RNA isolated from primary mouse calvarial osteoblast cells which were treated with 100ng/ml hPTH-(1-34) over a time course of 0.5 to 72 hours. OCILml7 (SEQ ID NO: 16) and OCILml2 (SEQ ID NO: 14) were used as sense and antisense primers, respectively, to determine mOCIL mRNA expression. Oligonucleotides OCILm47 (SEQ ID NO: 13) and OCILml2 (SEQ ID NO: 14) were used as sense and antisense primers, respectively, to determine mOCILrPl/rp2 mRNA expression. PCR and Southern blot analyses were carried WO 01/05964 PCT/AUOO/00864 32 out under the same conditions as described above. The results showed that mOCIL mRNA expression was upregulated five-fold by PTH at 1 hour, peaking at 2 hours and returning to basal levels by 4 hours treatment: levels were unchanged over the remainder of the experiment (24 hours).

In contrast, mOCILrP1/rP2 mRNA was not regulated by PTH.

This indicates that mOCIL is differentially regulated, while mOCILrP1 and mOCILrP2 are not.

Example 7: Localization of OCIL mRNA mRNA encoding OCIL was localised in fetal, newborn and adult mouse tissues by in situ hybridisation using the rOCIL 402 antisense probe, using a method described previously (Katsogiannis et al, 1997 and 1998).

Plasmid cDNA was labelled with digoxigenin (DIG) using an RNA labelling kit (Boehringer-Mannheim, Mannheim GmbH, Germany). Hybridisation signals were detected by alkaline phosphatase staining with BCIP/NBT after incubation with an anti-digoxigenin antibody coupled to alkaline phosphatase.

The mRNA is expressed in a range of tissues, as summarized in Table 2.

WO 01/05964 PCT/AU00/00864 33 Table 2 Adult Rat Tissues Probed with rOCIL402 Tissue OCIL mRNA Calvaria Osteoblasts Marrow hematopoietic cells Megakaryocytes Kidney Medulla (collecting tubules) Outer cortex (collecting tubules only) Glomeruli (endothelial -ve cells only weakly positive) Proximal/distal tubules -ve Lung pneumocytes bronchial epithelium Brain Neurones in cerebral cortex, cerebellar cortex, hippocampus; choroid plexus Heart Cardiac muscle Spleen White pulp Cortex Red pulp -ve Gut Luminal epithelium Liver Hepatocytes -ve In situ hybridization was also carried out to detect OCIL mRNA localisation in adult murine tissue and human skin, using the same method, and the results are summarized in Table 3.

WO 01/05964 WO 0105964PCT/AUOO/00864 34 Table 3 Normal Murine Tissues Expressing OCIL mRNA Tissues IFetal Newborn lAdult I_ (day 15) (day 1) 1(5-8 weeks) Ext raskel etal tissues Brain Lung Heart Kidney (collecting tubules) Small Intestine Liver Skeletal muscle Skin Spleen nd nd Skeletal tissues/cells Long bone chondrocytes osteoblasts na osteoclasts nd or orperichondriun/ periosteun marrow/megakaryocytes na Calvarial bone osteoblasts osteoclasts periosteum nd 1++ (na) denotes weak signal denotes moderate signal denotes strong signal denotes absence of signal not applicable WO 01/05964 PCT/AU00/00864 35 (nd) not determined.

megakaryoblast of fetal liver.

OCIL mRNA localization in human skin probed with OCIL402 (SEQ ID NO: 4): Epidermis (all layers) **Basal layer slightly weaker signal In Northern blot analyses of adult mouse tissues using mOCIL2kb (SEQ ID NO: 10) as the probe, OCIL mRNA was shown to be expressed in heart, skin, lung, liver, kidney, gut and brain. In adult rat, OCIL mRNA was found to be expressed in brain, bone, lung, liver, gut, kidney, mouse, skin and heart. These results are illustrated in Figure 16.

Since the nucleotides 900-1907 of mOCIL2kb (SEQ ID NO: 10) were not part of the mOCIL sequence, the Northern blot analysis was performed using mOCIL17 (SEQ ID NO: 17) as probe, which detected the same 780 bp species of mRNA. Northern blot analysis was also carried out using plasmid containing the nucleotides 900-1907 of mOCIL2kb (SEQ ID NO: 10) only as a probe. This probe failed to hybridize with any mRNA.

Example 8: Confirmation of full length sequences Rat OCIL A 5'-Rapid Amplification of cDNA Ends strategy was used to obtain the 5' ends of the rOCIL cDNA, using the SMART RACE cDNA Amplification Kit (Clontech, California, USA). The antisense primer used was (SEQ ID NO: 32) 5'-CTC AGT GTT GTC TGT CCA CTT CCA AGG G-3' complementary to sequences within rOCIL402 (SEQ ID NO: 4).

First-strand cDNA was synthesised from total RNA isolated WO 01/05964 PCT/AUOO/00864 36 from hPTH 1-34 treated UMR 106 parental cells according to the manufacturer's instructions. The cDNA was further amplified by PCR using OCILr25 and UNP primer as the anchored primer. The PCR conditions utilised a touchdown PCR protocol with denaturation at 94 0 C for 1 min, then cycles at 94 0 C for 30 sec, 72 0 C for 1 min, and then cycles of 94 0 C for 30 sec, 70 0 C for 30 sec and 72 0 C for 1 min, followed by 50 cycles of 94 0 C for 30 s, 65 0 C for and 72 0 C for 1 min. An extension of 398 bp of sequence of rOCIL1.3kb (SEQ ID NO: 7) was obtained. The full length rat OCIL sequence is 1628 bp, designated rOCIL (SEQ ID NO: 33). Figure 17 summarises the sequence of 402 (SEQ ID NO: rOCIL1.3 (SEQ ID NO:7), rOCIL323 and rOCIL (SEQ ID NO: 33).

Mouse OCIL A 5'-RACE strategy was used to confirm the mOCIL2kb sequence. The antisense primers used were (SEQ ID NO: 32), which was 100% identical to mOCIL2kb (SEQ ID NO: 10), and a specific primer OCILm75 (SEQ ID NO: 34).

5'-CAG TTT TGC GGG CAA GCA GCA TAG-3' complementary to sequences within mOCIL2kb (SEQ ID NO: First-strand cDNA was synthesised from total RNA isolated from mouse spleen cells according to the manufacturer's instructions. The cDNA was further amplified by PCR using or OCILm75 and UNP as the 5' anchored primer, in a touchdown PCR protocol with denaturation at 94 0 C for 1 min, then 5 cycles at 94 0 C for 30 sec, 72 0 C for 1 min, and then cycles of 94 0 C for 30 sec, 70 0 C for 30 sec and 72 0 C for 1 min, followed by 40 cycles of 94 0 C for 30 s, 65 0 C for and 720C for 1 min.

A 3'-RACE strategy was also used to obtain the 3' ends of the mOCIL cDNA. The sense specific primer used was OCILm76 (SEQ ID NO: WO 01/05964 PCT/AU00/00864 37 OCILm76 5'-AGG CAG CCC GCA GGA GGT AGA AG-3' complementary to sequences within mOCIL2kb (SEQ ID NO: First-strand cDNA was synthesised from total RNA isolated from mouse spleen cells according to the manufacturer's instructions. The cDNA was further amplified by PCR using OCILm76 and UNP primer as the 3' anchored primer in a touchdown PCR protocol with denaturation at 940C for 1 min, then 5 cycles at 94°C for 30 sec, 720C for 1 min, and then 5 cycles of 94 0 C 30 sec, 70 0 C for 30 sec and 72 0 C for 1 min, followed by 30 cycles of 940C for 30 s, 650C for and 72°C for 1 min.

A full length mOCIL cDNA sequence of 1206 bp was obtained and designated mOCIL (SEQ ID NO: 36). This sequence confirmed that nucleotides 1-320 of the original mOCIL2kb represented an inverted repeat of the 3' end of the sequence, and that nucleotides 900-1907 of mOCIL2kb were not part of the mOCIL sequence.

Human OCIL A 5'-RACE strategy was used to confirm the hOCIL sequence. The specific antisense primer used was OCILhl (SEQ ID NO: 43), OCILh: 5'-CTC TGC TCA GCC CAA TCC AGT GAT CAG-3' complementary to sequences within hOCIL clone 1 (SEQ ID NO: According to the manufacturer's instructions, firststrand cDNA was synthesised using human placental total RNA, which is included in the SMART RACE cDNA Amplification Kit (Clontech, California, USA). The cDNA was further amplified by PCR using OCILhl and UNP primer as the anchored primer using a touchdown PCR protocol with denaturation at 94 0 C for 1 min, then 5 cycles at 94 0 C for 30 sec, 720C for 1 min, and then 5 cycles of 94 0 C for sec, 70 0 C for 30 sec and 720C for 1 min, followed by cycles of 94 0 C for 30 s, 650C for 30s, and 72 0 C for 1 min.

38 Three different 5' end sequences were obtained, designated hOCIL clone 2 (SEQ ID NO: 44), hOCIL clone 3 (SEQ ID NO: 45) and hOCIL clone 4 (SEQ ID NO: 46).

The length of hOCIL clone 2 (SEQ ID NO: 44), hOCIL clone 3 (SEQ ID NO: 45) and hOCIL clone 4 (SEQ ID NO: 46) is 820, 845 and 937 bp, respectively. Minor differences in the nucleotide sequences were noted between hOCIL clones 1, 2, 3 and 4. These were: 1) At position 545 in human OCIL clone 1 a was called and a was at this equivalent position in clones 2 and 3, whilst in clone 4 (at position 117), a was called.

At position 649 in human OCIL clone 1 a was called, and a was called at this position in human OCIL clone 4, whilst a was called in the equivalent positions for clones 2 (at position 164) and 3 (at position 189).

At position 835 in human OCIL clone 1 and at equivalent positions for hOCIL clones 2 and 3, a was called, whilst at an equivalent position for human OCIL clone 4 (at position 467), an was called.

Soo: Of these clones, only hOCIL clone 3 predicted a protein sequence, while hOCIL clones 1, 2 and 4 did not possess a Kozak sequence predicting an initiating methionine residue. Analysis of the genomic structure of human OCIL (below) predicts that hOCIL clones 1 and 2 result from read-through into intron II, and therefore, represent prespliced mRNA species (Figure 18a). HOCIL clone 4 appears to represent an alternatively spliced mRNA transcript that does not encode a full length protein.

HOCIL clone 3 predicts a protein of 191 amino acids which is a C-lectin type II membrane-bound protein. The protein is predicted to have an intracellular domain of 30 amino acids, transmembrane domain of 29 amino acids and an extracellular domain of 132 amino acids. Within the extracellular domain is a C-lectin domain of 112 amino acids (amino acids 75 to 186; Figure 18b). The sequence \\mlbpfile\ho$eS\cintae\XOep\peci\5797S 0.Odoc 10/02/03 WO 01/05964 PCT/AU00/00864 39 from 13 bp to 845 bp of hOCIL clone 3 is identical to that from 7 bp to 850 bp of LLT1, a cDNA of 850 bp encoding a C-type lectin expressed by Natural Killer (NK) cells, T cells, and B cells (GenBank database Accession No.

AF133299). The features of hOCIL clone 3 identify this as encoding the human equivalent of mouse and rat OCIL.

The hOCIL gene is 46.5kb in length (SEQ ID NO: 21). The hOCIL gene is composed of 6 exons, and may be alternatively spliced at the 5' end, as illustrated in Figure 18a. hOCIL clone 1 (SEQ ID NO: 20), as well as hOCIL clone 2 (SEQ ID NO: 44) contains exons III, IV,V and VI. hOCIL clone 3 (SEQ ID NO: 45) contains exons II, III, IV, V and VI. hOCIL clone 4 (SEQ ID NO: 46) contains exons I, III, IV, V and VI.

A 3-RACE strategy was also used to obtain the 3' ends of the cDNA using the SMART RACE cDNA Amplification Kit (Clontech, California, USA). The sense specific primer used was OCILh3'-1 (SEQ ID NO: 47) OCILh3'-1 5'-GCTGATCTTGCTCAGGTTGAAAGCTTCC-3' complementary to sequences within hOCIL (SEQ ID NO: First-strand cDNA was synthesised from total RNA isolated from MG63 cells, a human osteosarcoma cell line, according to the manufacturer's instructions. The cDNA was further amplified by PCR using OCILh3'-1 and UNP primer as the 3' anchored primer. The PCR conditions utilised a touchdown PCR protocol with denaturation at 94 0 C for 1 min, then cycles at 94 0 C for 30 sec, 72 0 C for 1 min, and then cycles of 94°C 30 sec, 700C for 30 sec and 72°C for 1 min, followed by 30 cycles of 94°C for 30 s, 650C for 30s, and 72 0 C for 1 min.

3' RACE confirmed the 3'end sequence of the hOCIL clones.

Received 24 September 2001 40 Example 9: Antibodies Directed Against OCIL The following peptide fragment of the deduced amino acid sequence derived from the cDNA sequence of mOCILl7 (SEQ ID NO: 17) was synthesized, and was used to immunize rabbits, using standard protocols.

Cys -Met-Ala-Gln-Glu-Ala-Gln-Leu-Ala-Arg-Phe-Asp-Asn-Gln- Asp-Glu--Leu-Asn-Phe (SEQ ID NO: 26) This

(SEQ

peptide sequence showed a high homology to mOCILrPl ID NO: 41) and mOCILrP2 (SEQ ID NO: 42), as follows: 1 CMAQEAQLARFDNQDELN II 1111111111111111 108 CMAQEAQLARFDNQDELN 1 CMAQEAQLARFDNQDELN 120 CIMAQEAQLARFDNEKELN 1 CMAQEAQLARFDNQDELN 1 1 1 1 1 1 1 1 1 1 1 1 120 CMAQEAQLARFDNEEELI mOCIL (SEQ ID NO: mOCILrPl (SEQ ID NO: 41) mOCILrP2 (SEQ ID NO: 42) Two specific peptide fragments of the deduced amino acid sequence derived from the cDNA sequence of mOCIL (SEQ ID NO: 36) and mOCILrPl/mOCILrP2 (SEQ ID NO: 12 and in the intracellular domain were synthesised, and-were also used to raise antibodies: Antibody MOCIL-3 is specific for an epitope in the following sequence of mOCIL: Cys-Val -Thr-Lys-Ala-Ser-Leu-Pro-Met-Leu-Ser-Pro-Thr- Gly-Ser-Pro-Gln-Glu (SEQ ID NO: 48) \melb..f i es hooe\ ewnee \Keep\ Spec i\ PCT-A7OO -00864. doc 24/09/01 AMENDED SH2- 1PEN1AU rk- I uVVu/VVuuout Received 24 September 2001 41 Antibody MOCIL-RP-1 is specific for an epitope in the following sequence of mOCILrPl/mOCILrP2: Cys-Val-Gln-Lys-Pro-Glu-Glu-Gly-Asn-Gly-Pro-Leu- Gly-Thr-Gly-Asp (SEQ ID NO: 49) The antibodies raised may be used to detect the OCIL protein, using standard immunohistochemical methods, or to neutralize OCIL activity in murine co-cultures to stimulate osteoclast formation.

Example 10: Immunohistochemistry Rabbit polyclonal antibodies prepared as described in Example 7 were used for immunohistochemistry.

A kit for the standard peroxidase-labelled streptavidinbiotin detection method (DAKO, Boenisch, 1989) was used according to the manufacturer's instructions, with minor modifications. The dilution of the antiserum used was optimised in preliminary experiments. Incubation of tissue sections with a 1:100 dilution of the primary antiserum was carried out overnight at 4°C in a humidified chamber.

Peroxidase activity was detected with 3'-3'-diaminobenzidine tetrahydrochloride (Sigma) and 0.15% H20 2 Slides were counterstained with haematoxylin, dehydrated and mounted on a coverslip. The tissue expression of mOCIL, mOCILrP1 and mOCILrP2 proteins as detected using the 3 antibodies raised against the sequences SEQ ID NO: 26; SEQ ID NO: 48 and SEQ ID NO: 49 was identical. The results are summarized in Table 4.

\\melb_.iles\hom$S\evomee\Keep\Speci\PCT-AUOO-008 6 4.doc 24/09/01 AMENDED SHEL

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WO 01/05964 WO 0105964PCT/AUGO/00864 42 Table 4 Normal Murine Tissues Expressing mOCIL or mOCILrP protein Tissues Fetal Newborn Adult (day 15) (day 1) (5-8 weeks) Extraskeletal tissues Brain nd Lung nd Heart nd Kidney nd (collecting tubules) Small Intestine nd nd nd Liver nd nd nd Skeletal muscle nd Skin nd Spleen nd nd nd Skeletal tissues/cells Long bone chondrocytes nd osteoblasts na osteoclasts nd nd nd perichondrium./periosteum, nd marrow/megakaryocytes na 1++ denotes weak signal; denotes moderate signal; denotes strong signal; -)denotes absence of signal; (na) not applicable; (nd) not determined.

Received 24 September 2001 43 Example 11: Production of Recombinant OCIL protein in a mammalian expression system OCIL proteins were prepared by recombinant DNA technology to allow more extensive laboratory studies of their actions on osteoclast formation as well as osteoblast function. Soluble mouse and rat OCIL cDNA tagged at the Nterminus with the FLAG epitope were constructed in the pEF- BOS Mammalian expression vector (Mizushima Nagata 1990), which had been modified to contain an in-frame IL-3 signal sequence and FLAG peptide coding sequence (gift of Dr. D Hilton).

In order to obtain a RT-PCR product encoding the mOCIL (SEQ ID NO: 36) extracellular domain (amino acids 63- 207) to clone into the MluI site of the vector, as shown in Figure 19a, the RT-PCR was carried out using total RNA isolated from primary mouse calvarial osteoblasts, which support osteoclast differentiation in coculture. A sense primer, OCILm33, comprising OCILm32 representing nucleotides 245-269 of mOCIL (SEQ ID NO: 36) and containing a MluI site, designated primer OCILm33 (SEQ ID NO: 27): OCILm33 5'-GCC ACG CGT TTG TCA GCA ACA AAG ACA GAA CAG-3' and an antisense primer representing nucleotides 746-725 of mOCIL (SEQ ID NO: 36) and containing a MluI site, designated primer OCILm46 (SEQ ID NO: 28), OCILm46 5'-GCC ACG CGT GGG ACC ATA GGG GAA AAA GTA G-3' were used as primers in the PCR. PCR was run at 94 0 C for min, then 35 cycles of 94 0 C for 30 s, 60 0 C for 30s, and 72 0 C for 1 min, followed by a final extension step of 72 0

C

for 10 min. A 501 bp fragment was obtained and further cloned into the expression vector pEF-BOS. Sequencing confirmed the open reading frame and FLAG fusion (bp 1-132) and the 501 bp fragment sequence (bp133-633) (SEQ ID NO: 29), which was confirmed to be identical to mOCIL17 \\melb_files\homeS\evonnee\Keep\Speci\PCT-AUOO-00864 .doc 24/09/01 AMENDED SHEEi

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Received 24 September 2001 44 (SEQ ID NO: 17). The sequencing results also showed that the primer OCILm46 had 22 of 23 bp complementary to mOCIL17 (SEQ ID NO: 17) and mOCIL (SEQ ID NO: 36): 5'-GCCACGCGTGGGACCATAGGGGAAAAAGTAG-3' Primer OCILm46 i I I I! l ll l l l I l I I I I I I mOCIL (725) Strand To obtain a PCR product encoding the rOCIL1.3kb (SEQ ID NO: 7) extracellular domain (amino acids 40-179), a sense primer to represent nucleotides 126-146 of rOCIL1.3kb with the Mlul site, designated primer OCILr22 (SEQ ID NO: OCILr22 GCC ACG CGT TCA GTA AAA AAG ACA GCC AAG-3' and an antisense primer representing nucleotides 544-526 of rOCIL1.3kb with the Mlul site, and designated primer OCILr23 (SEQ ID NO: 31), OCILr23 5'-GCC CAG CGT AAC TAC AGG CAC TGT GAG G-3' were used as primers in a PCR. PCR was carried using rOCILl.3 kb plasmid as a template and run at 940C for min, then 35 cycles of 94°C for 30 s, 60°C for 30s, and 72°C for 1 min, followed by a final extension step of 72°C for min. A 421 bp fragment was obtained and cloned into the expression vector pEF-BOS, as shown in Figure 19b. The open reading frame and FLAG fusion were confirmed by sequencing.

HEK 293 cells were transfected with both mouse and rat expression constructs using Lipofectamine (Life Technologies, Inc). Supernatant was harvested after 72 hours. The recombinant protein was purified by incubation with the anti-FLAG M2 affinity gel (Kodak), and eluted with the FLAG peptide (Kodak) as outlined in the manufacturer's protocol. The purified protein was used to H:\evomee\Keep\Speci\PCT-A1 00-00864.doc 24/09/01 AMENDED SHE=

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WO 01/05964 PCT/AU00/00864 45 study its effects on osteoclast formation in murine cocultures.

An experiment was also carried out to determine the action of rOCIL protein in osteoclast formation.

Primary mouse calvarial osteoblasts were cocultured with spleen or bone marrow cells obtained from 6 week-old mice and stimulated with 1,25-dihydroxyvitamin D3 and PGE2 in the presence of rOCIL or mOCIL protein (15 ng/ml) for days. A negative control was carried out with carrier buffer alone. As shown in Figure 20, both rOCIL protein (Figure 20a) and mOCIL protein (Figure 20b) significantly reduced the number of osteoclasts formed when compared to the presence of carrier buffer alone.

Example 12: Effect of mOCIL Protein on Osteoclast Formation To determine the action of mOCIL protein on osteoclast formation, mouse spleen cells were obtained from 6-week old mice and cultured in medium containing 10% FCS, 25ng/ml hM-CSF and 50ng/ml murine soluble RANKL in the absence or presence of mOCIL protein at a concentration of 12.5 ng/ml. mOCIL protein treatment resulted in a inhibition of sRANKL and hM-CSF stimulated osteoclast formation, as illustrated in Figure 21.

Example 13: Production of Recombinant OCIL protein in an E.coli expression system In order to increase the expression level for mOCIL protein, an E.coli expression system was used. A DNA fragment encoding the extracellular domain (residues 76- 207) of mOCIL was obtained by PCR and cloned into the EcoRI and HindIII site of pMAL-c2 (New England Biolabs Inc.), creating a gene fusion with the MBP (maltose binding protein)-encoding malE gene. PCR was performed using a plasmid which contained mOCIL17 cDNA sequence (SEQ ID NO: 17) as a template. The reaction used a sense primer representing nucleotides 285-303 of mOCIL (SEQ ID NO: 36) WO 01/05964 PCT/AU00/00864 46 encoding amino acids 76-81, TYAACP, in SEQ ID NO: 39 with an EcoRI site, designated primer OCILm65 (SEQ ID NO: 5'-TCAGAATTCACCTATGCTGCTTGCCCGC-3' and an antisense primer representing nucleotides 711-690 of mOCIL after the stop codon in SEQ ID NO: 36 with a HindIII site, and designated primer OCILm66 (SEQ ID NO: 51): OCILm66 5'-GGTTAAGCTTCAGGCTAAAAAGCGTCTCTTGG-3'.

PCR was run at 94 0 C for 5 min, then 30 cycles of 94°C for s, 60°C for 30s, and 72 0 C for 1 min, followed by a final extension step of 72 0 C for 10 min. The PCR product was then digested with EcoRI and HindIII, and cloned into pMALc2, as shown in Figure 22. The open reading frame and MBP fusion were confirmed by sequencing.

Example 14: Recombinant mOCIL-related protein constructs To determine whether mOCIL-related proteins also have an inhibitory effect on osteoclast formation, the sequences encoding the extracellular domains of mOCILrP1 and mOCILrP2 were also inserted into the EcoRI and HindIII sites of pMAL-c2 (New England Biolabs Inc.), as described above.

PCR was performed using a plasmid which contained mOCILrP1 cDNA sequence (SEQ ID NO: 12) as a template. A sense primer representing nucleotides 283-302 of mOCILrP1 (SEQ ID NO: 12) encoding amino acids 88-93, TYAACP, in SEQ ID NO: 40 with an EcoRI site, designated primer OCILm88 (SEQ ID NO: 52), OCILm88 5'-TCAGAATTCACCTATGCTGCTTGCCCGAA-3' and an antisense primer representing nucleotides 742-720 of mOCILrP1 (SEQ ID NO: 12) and 739-717 of mOCILrP2 (SEQ ID NO: 15), with a HindIII site, and designated primer OCILm87 WO 01/05964 PCT/AU00/00864 47 (SEQ ID NO: 53) OCILm87 5'-GGTTAAGCTTGGGACCATAGGGGAAAAAGTAG-3' were constructed. PCR was run at 94°C for 5 min, then cycles of 94 0 C for 30 s, 60 0 C for 30s, and 72 0 C for 1 min, followed by a final extension step of 72 0 C for 10 min. The PCR product was then digested with EcoRI and HindIII and cloned into pMAL-c2 (Figure 23). The open reading frame and MBP fusion were confirmed by sequencing.

For the mOCILrP2 construct, PCR was performed using a plasmid which contained mOCILrP2 cDNA sequence (SEQ ID NO: 15) as a template. A sense primer, representing nucleotides 283-302 of mOCILrP2 (SEQ ID NO: 15) encoding amino acids 88-93, TYAACS of SEQ ID NO: 41 with an EcoRI site, designated primer OCILm89 (SEQ ID NO: 54), OCILm89 5'-TCAGAATTCACCTATGCTGCTTGCTCAAA-3' and antisense primer OCILm87 (SEQ ID NO: 53) were used for PCR. PCR and cloning procedures were carried out under the same conditions as above, as shown in Figure 24.

Example 15: Recombinant hOCIL protein construct A recombinant hOCIL protein construct was also made using the same system. A sense primer, representing nucleotides 694-711 of hOCIL clone 1 (SEQ ID NO: 20) with an EcoRI site, designated primer hpMAL-1 (SEQ ID NO: hpMAL-1 5'-GCGGAATTCCTTCAAGCTGCATGCCC-3' and an antisense primer representing nucleotides 1034-1055 of hOCIL clone 1 (SEQ ID NO: 20) with a BamHI site, and designated primer hpMAL-2 (SEQ ID NO: 56) hpMAL-2 5'-CCTGGGATCCGCTTTGCTGTAACATCTAGAC-3' WO 01/05964 PCT/AUOO/00864 48 were used to run PCR under the same conditions as above.

The PCR product was then digested with EcoRI and BamHI, and cloned into pMAL-c2. The open reading frame and MBP fusion was confirmed by sequencing, as shown in Figure Example 16: Expression and purification of mOCILrP and hOCIL Competent strain BL21 E.coli cells were transformed with the constructs produced in the preceding two examples, and the corresponding fusion protein was induced with IPTG (isopropyl-1-thio-P-D-galactopyranoside) according to the manufacturer's instructions. The MBP-OCIL fusion protein was isolated from the soluble bacterial fraction using affinity chromatography as outlined in the manufacturer's instructions. The eluant fractions were subjected to SDS-PAGE, and transferred to membranes [PVDF (polyvinylidene difluoride) Western blotting membranes (Roche Molecular Biochemicals)]. Western blot analyses were performed with a rabbit anti-MBP serum (New England Biolabs Inc.) and a BM chemiluminescence blotting substrate (POD) detection system (Roche Molecular Biochemicals).

Fractions containing the MBP-OCIL fusion protein were pooled and concentrated using an Amicon ultrafiltration membrane (Millipore, Bedford, MA). The protein concentration was ascertained in a BCA protein assay (Pierce). The estimated yield of MBP-mOCIL ranged between 0.1-0.4 mg/L.

Example 17: Inhibition of osteoclast formation Experiments were carried out to determine the action of MBP fusion OCIL proteins on osteoclast formation.

Mouse culture system Mouse spleen cells obtained from 6 week old adult mice were cultured in medium containing 10% FCS and M-CSF and 50ng/ml sRANKL in the absence or presence of MBP or MBP-mOCIL fusion protein at various concentrations.

After 7 days, cells were fixed and subjected to TRAP WO 01/05964 PCT/AU00/00864 49 staining. As shown in Figure 26a, MBP-mOCIL fusion protein significantly reduced the number of osteoclasts formed when compared to MBP protein alone, and this effect was shown in a dose-dependent manner.

It has been reported that IL-18 inhibits osteoclast formation mediated by T cells (Horwood et al., 1998). To further investigate whether the mOCIL inhibition of osteoclast formation is a T cell-dependent effect, T cell-depleted mouse spleen cell cultures were carried out as reported by Horwood et al., (1998). T cells were depleted with CD3 antibody from spleen cells and the remaining cells were cultured in medium containing 10% FCS, M-CSF and 50ng/ml sRANKL in the absence or presence of MBP or MBP-mOCIL fusion protein at a concentration of 500ng/ml. The results, summarised in Figure 26b, showed that mOCIL inhibited osteoclast formation, implying that its actions were T-cell independent.

The effect of MBP-mOCILrP1 and MBP-mOCILrP2 fusion protein on osteoclast formation was also examined in the T-cell depleted mouse spleen cell culture system. The results are shown in Figure 27. Both MBP-mOCILrP1 (Figure 27a) and MBP-mOCILrP2 fusion proteins (Figure 27b), like mOCIL, inhibited osteoclast formation in a T cell independent fashion.

Human monocyte culture Monocyte cultures were prepared as described by Quinn et al., (1998). Monocytes were isolated from the peripheral blood of normal healthy subjects. Human PBMCs were prepared from diluted blood (1:1 in Hanks Balanced Salt Solution (HBSS; Life Technologies, Grand Island, NY) which was layered over Ficoll-Paque® solution (Pharmacia Biotech, Uppsala, Sweden), centrifuged (693g), then washed and resuspended in MEM medium containing 10% FCS. Monocyte cultures were prepared by adding 106 PBMCs to 6mm diameter culture wells containing bovine cortical bone slices in MEM medium containing 10% FCS; after 1 hour, coverslips and bone slices were removed, vigorously rinsed to remove non- WO 01/05964 PCT/AU00/00864 50 adherent cells, and placed in 10mm diameter culture wells.

Monocyte cultures were maintained in these culture wells in 0.4ml MEM medium containing 10% FCS, recombinant human M- CSF (25ng/ml) and recombinant human sRANKL (30 ng/ml) in the absence or presence of MBP (500 ng/ml) or MBP-hOCIL (500ng/ml) fusion protein for 21 days. Medium and added factors were entirely replaced every 3 days. After 21 days, bone slices were removed for TRAP staining and bone resorption pit analysis. The multinucleate osteoclasts were counted, and the results are shown in Table 5. Human OCIL inhibited osteoclast formation from human monocytic cells.

Table Effects of MBP-hOCIL fusion protein on osteoclast formation in human Inonocyte cultures Well Control(RANKL+M-CSF) MBP (500 ng/ml) MBP-hOCTL (500 ng/ml) 1 205 40 6 2 146 180 16 3 37 17 34 4 66 42 9 66 22 73 6 71 63 14 7 3*8 54 8 25 29 34 Mean ±SEN 81.7 ±22.0 55.9 ±18.6 26.12 ±7.62 WO 01/05964 PCT/AU00/00864 52

DISCUSSION

We conclude that in osteoblasts OCIL and OCIL related proteins are expressed on the cell surface as type II membrane peptides. Contact with haematopoietic precursor cells prevents further differentiation into mononucleate osteoclast precursors, and ultimately into functional multinucleate osteoclasts.

Without wishing to limit the scope of the invention by any proposed mechanism, we consider that upregulation of OCIL mRNA expression by the same osteotropic factors that increase expression of RANKL is consistent with the hypothesis that regulation of bone resorption by osteoclasts is tightly regulated. According to this hypothesis, stimulation of multinucleate osteoclast formation through RANKL would simultaneously prevent the generation of new osteoclasts through the action of OCIL.

If this system is operative under normal physiological conditions, then bone resorption becomes a self-limiting process.

Notwithstanding the above, mOCILrP1 and mOCILrP2 are not regulated by osteotropic agents that regulate mOCIL, but like mOCIL, both mOCILrP1 and mOCILrP2 have the capacity to inhibit osteoclast formation. Thus each of the three polypeptides, mOCIL, mOCILrP1 and mOCILrP2 is equally useful for therapy to limit osteoclast formation or to promote osteoclast formation through blockade of their actions. Given the degree of homology between these molecules, each may substitute for one another. However, each can be distinguished by several criteria. These include: Nucleotide sequence: mOCIL, mOCILrP1 and mOCILrP2 appear to be derived from a common ancestral gene; however, there are nucleotide differences which permit identification of the three molecules using specific oligonucleotide primers in RT-PCR.

Gene structure: The promoter of mOCIL is a TATA promoter, while the promoter for mOCILrP1 is a GC-rich WO 01/05964 PCT/AUOO/00864 53 region containing an SP 1 binding site.

The expression of mOCIL is regulated by PTH, while the expression of mOCILrP1 and mOCILrP2 is not.

The polypeptide products of mOCIL, mOCILrP1 and mOCILrP2 can be distinguished using antibodies directed against peptides fragments of mOCIL and mOCILrPl/rP2 based on the intracellular domains of the respective proteins.

In vivo, OCIL and OCILrP have the potential to be used as therapeutic agents in the treatment of conditions which are characterised by excessive bone resorption, such as osteoporosis, primary hyperparathyroidism, Paget's disease, rheumatoid arthritis, renal osteodystrophy, and humoral hypercalcaemia of malignancy, as well as metastatic bone disease. Modulation of the expression or function of the factor may also be useful in the treatment of disorders involving extra-skeletal calcification.

It will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding, various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this specification.

References cited herein are listed on the following pages, and are incorporated herein by this reference.

WO 01/05964 PCT/AUOO/00864 54

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Proc. Natl. Acad. Sci. USA, [1998] 95 3597-3602.

WO 01/05964 PCT/AUOO/00864 57 Zhou et al J. Biol.Chem., 269 22433-22439.

EDITORIAL NOTE APPLICATION NUMBER 57975/00 The following Sequence Listing pages are part of the description.

The claims pages follow on pages 58 to 62.

Received 30 October 2001 SEQUENCE LISTING <110> St Vincent's Institute of Medical Research <120> Inhibitor of Osteoclast Precursor Formation <130> FP13129 <140> PCT/AU00/00864 <141> 2000-07-19 <150> AU PQ1675 <151> 1999-07-19 <160> 56 <170> PatentIn version <210> 1 <211> 21 AMEND7I41 I 1PUIvu1Vuuout Received 30 October 2001 <212> DNA <213> Artificial <220> <223> sense primer complementary to rat calcitonin cDNA <400> 1 atgctgggca cgtacacaca a <210> <211> <212> <213> 2 321

DNA

Rattus rattus <400> 2 cgctctagcc cctcctgtta catgcattct tttgattact aacactcagt ttgttgaggt cggccacgcg tctctagagg cggctttttg ggatcggcct ataactactc cttgtgtgta tcgactagta aagctgtgga tgttttatta gcacagagag gtatgtt tca c cagctccaaa gagattccag cagaatttct tcctcagagc caatgttttt tctgtgcccc gatcatctga taagcagata acccttggaa tcttctactg tcagttcctc aacagagaca caaagggagt gtggacagac tgttcatgtc 120 180 240 300 321 <210> 3 2 AMENDED~ SKEEF I P F-VA U Received 30 October 2001 <211> <212> <213>

DNA

Artificial <220> <223> antisense primer <400> 3 tgagtgttgt ctgtccactt ccaag <210> <211> <212> <213> 4 402

DNA

Rattus rattus <400> 4 acagtaaaat cttactgctg ctttgtcagt gaaac tggat cattgagcca aggagc tgaa gagagtcatc gctccaagga ctacgtagtg aaaaaagaca tggagttgga gaccctctgt tttcctaagg agcgcaccct aagcttccca atcattgtcc gccaagatct aataaatgtt aaggaacaag agatacaaag tggaagtgga gaaacatccc tcagtgttag caaccataaa tttattttaa gggccgagc t ggagttcagg cagacaacac cc tggagtat ctgtagttct tacttatgct tgaaatacca agcacgattt ttactggtcc tc cctgctgggc ctttctgttg gcttgcccga agtaactgga gacaccgagg ggtctgcaca 120 180 240 300 360 402 AMEN SH- Received 30 October 2001 <210> <211> <212> <213> 22

DNA

Artificial <220> <223> sense specific primer complementary to SEQ ID NO:4 <400> gaaacatccc cctggagtat cc <210> <211> <212> <213> 6

DNA

Artificial <220> <223> sense specific primer complementary to SEQ ID NO:4 <400> 6 ccaagtaact ggacattgag ccaga <210> 7 <211> 1302 c- Received 30 October 2001 <212> DNA <213> Rattus rattus <400> 7 acagtaaaat cttactgctg ctttgtcagt gaaactggat cattgagcca aggagc tgaa gagagtcatc ccatcggagg gttatatagt agttttgtgt tctacagtgc gacatctaag aaataggtat tattagttat aaataccgtc tacttctgaa gactgtacag gc tccaagga ctacgtagtg aaaaaagaca tggagttgga gaccctctgt tttcctaagg agcgcaccct agatgaaaaa gaggaagtcg ccttggttga ctgaatcatg ataacaactg tcaggtatga ctctactttc CCCtCCttgt gagatgttcc gattaggtgc aagcttccca atcattgtcc gccaagatct aataaatgtt aaggaacaag agatacaaag tggaagtgga catggcttcc atttgtagga gactttgtcc aacaatctgc aggcatattt gctggttctc ttttttctct tCCCCCttCt cccaccccac atcctctcat gaaacatccc tcagtgttag caaccataaa tttattttaa gggccgagct ggagttcagg cagacaacac tgagtgacaa agcccaacag taacagtcat taaaatcatc tgcttgggag acatcttaaa catttaaatt agagttgttc tctgagtatt agtgaggcca cctggagtat ctgtagttct tacttatgct tgaaatacca agcacgattt ttactggttc tgagtataac tgggttcagc ctacacctca gaggaacaca ttcaattcat atcatgaatt cataaactga atattattta actccatacc tCCCttCtCt actgtaggga cctgctgggc ctttctgttg gcttgcccga agtaac tgga gacaccgagg ggtctgcaca aactcggttt agtggcaggg cagtgcctgt gaacatggta aatgtgtggt gttctatatt atcatgtcag tttatattcc ccttcatctt tggactttag gctgcgacat 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020

IPENAU

Received 30 October 2001 gccgtgcctc aaaatggtgc tggtttccgc gtagtaaaca agtccttatt tgactatgcc agcctgtctg catgacccat gtggcacgtt atgtagggca ttcccctggg gtagtagatg gaagaaaaaa aaaaaaaaaa aagtactagt cttccaccct cccaacagtg agcgctcctt tgcctggcct gctaggttca gcatagtgac ggggttggtt ggtgttggat acataagctg attgtatcaa ggttcctgaa taaactgctt cgacgcgtgg cc 1080 1140 1200 1260 1302 <210> <211> <212> <213> 8 738

DNA

Artificial Sequence <400> 8 agtaaaatgc tactgctgct ttgtcagtaa aactggattg ttgagccaga gagctgaatt gagtcatcag ctcagatggg gattttgatg cattcggaac tccaaggaaa acgtagtgat aaaagacagc gagttggaaa ccctctgtaa tcctaaggag cgcacccttg gagccgggac tccactattt ctcctttaaa gcttcccaga cattgtcctc caagatctca taaatgtttt ggaacaaggg atacaaaggg gaagtggaca tctgaaatcc gcatggctgc gttttgtcat aacatccccc agtgttagct accataaata tattttaatg gccgagctag agttcaggtt gacaacactc cagaaagcca acctgttcag cacagagcac 6 tggagtatcc gtagttctct cttatgctgc aaataccaag cacgatttga actggtccgg agtataacta ctgcagaact gaaagcagag ccaaaacagt tgctgggcct ttctgttgct ttgcccgaga taactggaca caccgaggag tctgcacaga ctcacagagc gcaagcctga attttaagga cctcgaatca 120 180 240 300 360 420 480 540 600 AM HEET

IPEA/AU

r I I f-IA.J1J'J1U- Received 30 October 2001 caggcccagt cccatccacc gttaaagcac ctttgagcaa tttaataaga cccatgtgta aaatgaataa aaacagaatt ggaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaa agtgcgtgtt aaaaaaaaaa 660 720 738 <210> <211> <212> <213> 9 620

DNA

Rattus rattus <400> 9 agtaaaatgc tactgctgct ttgtcagtaa aactggattg ttgagccaga gagctgaatt gagtcatcag atccggggag tatgcagaca cctttttctc aaaaaaaaaa tccaaggaaa acgtagtgat aaaagacagc gagt tggaaa ccctctgtaa tcctaaggag cgcacccttg tggaaagata aaagatggag ctatgtagct aaaaaaaaaa gc ttcccaga cattgtcctc caagatctca taaatgtttt ggaacaaggg atacaaaggg gaagtggaca tgcctacctg ctgtagcaaa tttgatcaag aacatccccc agtgttagct accataaata tattttaatg gccgagctag agttcaggtt gacaacactc aacgacatcg cttaacagct agagatgctt tggagtatcc gtagttctct cttatgctgc aaataccaag cacgatttga actggtccgg agtataacta ggatcagcag atagcctcca tttagtctgc tgc tgggcc t ttctgttgct ttgcccgaga taactggaca caccgaggag tctgcacaga c tcgc tt tcc tgccagggtc atgcaaaact taaaaaaaaa 120 180 240 300 360 420 480 540 600 620 AMEND.ED loHEE-FT

IPE&VAU

rl.~ I t~uuu/uuout* Received 30 October 2001 <210> <211> 1907 <212> DNA <213> Mus ntusculus <400> ccgaatgttt aggc tgaggc gtttgatagc agcgtctctt gttgagcttg gttgttcagg gttgtctgtc agtggaaact tagtcccaca aaccatctcc cactgtagct agtcaacaag ttatttttct ggcccaacta gaattttgat cctgcaacac aaagatgaca accccagcct gccaccattt gaaaggecag catgtgcacc actttgggac ggtaaatgct ctacagatcc taggcaaatc cacttccaag cagctcctca ggcagcccgc cctgagtctt gtaattgctc acctatgctg gaatacccaa gctcggtttg tcctggattg ttccatttca tttctgatgt cataggggaa agagtagcac aggaaggaaa aaaaggagtt acatccgaag tgaatagatc tttcctctcc ccggatggga ggtgctcttt ggcctctcag gctctgagat gtgtgtcaca aggtagaagt gggtaaaatt gtgctaagct ttactgctac tttctgttgc tttgtcagca cttgcccgca aaactggatt gtaactggac attcgcccag acaaccagga tgagctgaat gcctgcacag agagtcgtca 8 AMEN-ID-D SHEET IP2,VAU taagaaatat ccacagataa tggcagtgga ctggtactgc atcgtgttgt ctttcaagtt aaggcttccc ctccaaggaa tatggagtga acaaagacag ggagttgaaa gccttctgca ttcctaatga gagcaccctt tctctatctg caggctaaaa ggctatagct tgatcccgtt tatactcagt tcaatcctgt tacctatgct aaaggcacgg tcatggtcct aacagatccc ataaatgttt tgcgcacaga gatacaaggc ggaagtggac 120 180 240 300 360 420 480 540 600 660 720 780 840 900 If-tUVVIVVOV-t Received 30 October 2001 agacaacact gagtataaca acacgattcc atccggggag aggaaagatt aacaacaacg attcatgaga tgcttcatct tgt tcaaagc ggcagcgtgt ggggaaggga tttgtggttt ttatttaaag gtttcactta ttcaaggtgg aataatttga aaattgtgct ggatcaggga aaactgtctt gaactgatct caagctcttt ctcctcagtc aagatagagg catgtttggg aaacgagtgc aatacattta tggttgtatt ctgcagggaa ctgcctccac attccgggac tctactccag gaattgaggc cccttctagg tggctattgt cctaaggtga tgcaattgtt tctggcatta tataaagagc ggaacaagtg gttgacagaa ccatgtggtg gggaatggac ccatactgtg ccctgctgga catttctaaa acccgtcagc tgctctcagt aaatgtaggg tgcc tgggtc gatctttccc aattttaaaa cttgagacaa ttaaataaac caatgttaaa ttctttctgt agcattactt gtttgaatga ctgtttgata gatggccttt taatggagta aaaaaaaaaa attcctggag gaccaatggc ttggcttcct tagtgcacat atagaaagag aactcaatct aagtagaact ctaatgtaag agtgttatgg cagctagatt ctgtatgcta aaatgtggcc ggat tagaag gcctagtctg accctctgaa aaaaaag tgcctacctg aaaattcggc tactgagtgc gcaggaagac tagtcttgtt tcaggaacga gttggtttga ttgaaatact tctatgaaga ataataattc cctiggtataa caacccttta atagtctcat gattggaggc tcaaccccag actgtaagca 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1907 atttggatgt ttaatcacta gaggcctatt agttttcatg agctcctgat ggaggaagtg cctgagtgct taaatgcttt <210> 11 <211> 9862 9 AMENL).W :VA IPE VIAiJ A I I r- ~lM w Received 30 October 2001 <212> <213> <220> <221> <222> <223>

DNA

Mus musculus Unsure (636)..(636) unknown <400> 11 tgcattacac attaatccca tc tacagagt tactttttgt aacatgctcc ccaatctctg atagagggaa tgcatagcct aatgctaact agcctgccat ctctgctgtg ttaaatacgt acacacacac gcactgggga gagttccagg accttgaaat cccccccccc gcagcttatc gacagcgggg cccatgccag aaactaaagt agtgggtcta gtcggaaagt t tgggaggag acacacacac ggcagagaca atatccaggg ctaaaatatg ccgccgccgc aagtcagctg aaaaaggggc attgcttgga gggggaggc t tatagaccag accgatgcca tgcaaccttt acacaaggcc ggcagatttc ctacacagag tctcaactct agtttttcag atgtaaaaat gggctcattg gacaggagaa tcctcagggg ctgaggctgg c tctgngct t tgagtttgta gggcagtggt tgagttcaag aaaccctgtt gtttgtttct ttccagatct aatcccacaa cttcagcaag aaactgtacg agctggatct ggtggggtgg tctggtatgg aataaaagca ggtgcacacc gccagcctgg tcaaaaaagt tttacagtat aggtaggcac ctcacaaaat aagatagtgg tatttaatga tgctcctgtt atggtgggag ccaatgttac ggtgcccaga 120 180 240 300 360 420 480 540 600 660 720 AMEND.ED IPEiVAL' K--II .U vv o t Received 30 October 2001 ttcctggagg ataccttaag CtCCCCCttt atcaggtaat ttttccagta ttcactgggt ctgttgagcc ttcttgaaaa taccaggcaa ctgggaggtg agggccctac gtcccttaaa ggtatgattg ctcctcacag ctttgttctt aagggacata tctctagaag gaaaggtcat gtctagatga ttctaaaatg aaacaggcct attgactgga tgCCCCtCCt cccaaacctc actctttctg gggtgggggt gc tgggaatg atgactttac gcagagttca gctccttcag ggggacaggt tccatgccat cagaccctta gcctgctgcc taagccatag cagacttttc aagcaac tga ccaccgcagc gagtaatact acac tagagg gcgacttttt gtgcagcctt ggacc ttggg acacacctac caagcttttg gccttaaatg gggagattta aaccaagtcc ccccacttta caattgttgt agtggacagc cgaggaagag cc tgtgcaca atcagagtgt tttgactgtg cgcctgttgt tatgggttaa atttggtgaa ttctctaatg actgtttctc gccttaagag tcatcacctt tctcctctca ggtgctctgg ctacaacttt gaatttctct actcttgtgg tcccatctac tctctctgca atacttttgt tagatcaatg ctagctgcta ggagacc taa cctaaagtac agaacagggt agc tatagcc gttgggaaaa aaatggcagc agttactgta ttttcacctc agataagcca agtccatgac ccggacctga ttggtgCCCC cacaccctgc ct tt tcaggc ctcttcccaa caccagggaa aaatccatct agagcagcaa ttaggaataa gcctagtggc agatgattgg ccatgcctcc cctcctccac cttcttgagg aggttccacc cttagaaaag caggtcctac tctgctgtct ctctgactca tgtgcttctg tgagcaataa gaacaaatct tgccagtgag cacccagccc tcccacagta ggacacgggt ggatcagttt acagttttag gctcccttcc aatatcaatt agcc tgagga aaatactgtt cttcaaccct ggctatcctg cagagtagca aagtcccata taaagatttc agcagtggcc cacagtggtc tacccaaaga agggcaagta aatggtgagg tggctactta caaatccaaa 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 AMEMDEIFI, SHEET IPE,4/AU Received 30 October 2001 cagttcaagg tctttgtttc cattgttgtg aaagaaatta cttgcttgtg ttaataagcc cagggcaagg aaggtgtttt ccagagcagg attatggtga agcagtattc ttatctgttc tgtttacaat aactcgagct tcaccagtct tatagattgt agtcccagcg tggtcaccta ctgggctttg gctccaacca tactcccata tggctggacc tcatcagttg gatttggaaa ctcctttaca gaggagggc t agcacaggcg ctgggagttc cctaaggctc ggtgccaggg aacagacatc tatactggaa tgggtgacat tagaggggtt gatttttgaa atctgaaggg gaggagggct aatttattat tgtgtggatc CtttCCCtCC atgtggtttt atatattcaa atcctactgt tgtctctact agccagcaca gaattaaaag cagggcacaa tggtgaagga ggc tggggac ctggggtggt agcatctatc tactatgtac gctgagacaa tgttagctaa atcacagtgc gtttagaaat cccagcagca gctatcattt caaaatgcaa ttgttaatgt tgattgacac aatcccttgg gcctttgaac ccccacagcc tggaacacac tagaaaagca ggtggaagtc aaatgacc tg aaggttgcta gggaggtgtg agtggatgag tagtaagata attagtcagg aagcagacag agaacctgaa ctcccaagac catggctgag tatattctgt aagctcagtg aatagaagct agtaagatga gctagcttta aacagatctg cctggtaaga tagtaggctt tatttatttc ctgcccaggc tctgccacaa cagctaccat gggtatgggc tgtggaaaac ggatgtcttt cactgaaagg gagtttacac atcacaatga ttcctttcta aggtgctggg agaac tagaa cttaaagcct ttctggtatt acgatcatgt aaattcggta atatcacttt gatattttta ctctgctgac agaacagttc tatggcaggg gctgggcttg gaatgtggaa caagtcggag ctgtgataca tgtgacaaca ccaacattgt tcttttcttc aaccaaacca tagggagtgt gctggaaatg agaattaagg tctttctaat ttaggtgtgc 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 12 gaaaatgcac actcaataaa gtgttcttca tgctcttgct cctaattcca tgtagtacac caaagcactc gaccccctac cacagccatg cccacaagag tgccaatcag atagcatcgc aaatactatg gaagtgaata tctgaccttt aaaatgcaag attaattcat ttaagataca gaggac tgag attccaggga tacatacata ttaaatgtaa cctacctgtt agtagtcacc ttctttcagc tagtgtgtca ctacacagaa caaagaggtt gtggcagttg gcatgagctt gattaaagtc ttggtataca taggggc tgg gtttcactcc tccaatatat catacataca aataaataag tccccgttgt tttccagatg ttgtcacagc gaaacatgca tcatgctctg gtgaaaagct ggggtctctt actttcgggt aagtggcatg gtttaccttg gatccttaaa agcatgggaa gtagaagctg catcaatgta agtgaaaac t tttctgtgaa agacttggct cagcacacat tttctaaatt ttacattcac actaaaacag ctgctgcaga acttaaaatg aatgccttct ctgctggtga agttcagaaa gtcaggaaac cacaaaggaa tagtttaggg agataaggca tcatcagctt gctgctgcaa tatgtgggta taaaatttag taaccatcct 13 FUL I /AUUU/UU04 Received 30 October 2001 ctgtaaaaac tataattagc 3240 ttttgaaaac atataatcag 3300 caaccactga gagcatttat 3360 atggtggctc aacacccacc 3420 cctctaacag taatcatgca 3480 acattcttac atttagctga 3540 tcattttaaa aatatataca 3600 cactctcacc actcctccgc 3660 ggtccatgaa gcagagaagt 3720 gctcatcact cacagtgcag 3780 gatgctgagg gatcataccc 3840 tttttaagaa tctcaccagc 3900 ttctagagaa gtgataggag 3960 actgggactt cctgtagctc 4020 acactttggg gaagaagccc 4080 agcacaggca tgtgttccaa 4140 ggggatattt taatggtcac 4200 atattcacat aaagatgtct 4260 ggatgggagc atatcacatt 4320 ccttaagaag ttgttagttc 4380 gttatgcaga cagtgatttt 4440 ttgggacatt tggcctgtta gttgtttctt aaatgtgtca tgcaacttga ttacacttac tacctcaaca gtgttgagag ttttcacagg attcctttcc aaacagcttt tggacatcca

'~HEET

Received 30 October 2001 gctcttaaat cgaagatgat tttgCCCaag acaagttcac aaacattccc aaaaatcaaa gactgtagaa ggtgtttcag atctctggtt ttttctgctg cccatgagaa ttctcagaat atacaacttg acacatatat atgtgtttag ttcgcctgca gttctcagac atttaaccca ttttatttct ttagatttgt caggacaata tttttttttt tagaaccctg tgactttatg atttatgcct tagcacacac aagtggtctc tgttgtttca catgagacat aatttttgac gtgctatgat gtgtgt tcag cgctgccttg tagatatcta ttcctgaata ggtcctctga ttatatattt aattttatgt caactgattt ttttttacat tcatc tgtga atattaagtt atttatttat ggggagtgat ttgaccaccc gtgtctactc caggtcaggc tattaatttt tttttttttt aaattgttga atttctttct tggattgtgt tggatttaca aagatcaaca acatgtagtt ttttttggca taccatgctg ccaacggtct aaagaaacac ttgtgcttgg ttatttattt gccatctttt acatgtgttt tgatgctagc cttgtgatct tgactctttt c tgaaggcaa catgagcaat ttatttcaaa gcattcctag gaggcttgtg aatgttcgaa tttgttatgc ttactgtcta tcaccctgtc tttgtagatt tttcacctgt gatctctatg ttttaggaga ccctgtctaa gacctcattg accaggcttt ttctgatttt taattttcat ctacatctct atggagtgtt aatgtaacat tgcccaccaa agctcttata accactgagc tgactatgaa agatattaat ctgcagtttt tgtttggatt ttctgagaag ttacttttct tctacaaatg cagatggaag agactggttc tggagtcctg cattcctgtt gacctcttca attacttctc ggaatatgtt ctagttcatt gtacgagtgc ggttaggaaa ttggttctag caactttcct catcctattt ttccacttga ataaactcat tttatgtaga cataaacctc 4500 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220 5280 5340 5400 5460 5520 5580 5640 ctttcgttct ttaagagact cattttatgt atgtggctgt ttgcctgcag gtttgtacat 14 aacatgaatg cccagtgtgt acagaagcca gaagagggCa atggacccct ggtgtgcaat taacagctga tgggaaaagt acacattcat ttttaatgga ttttgttgtt attacatcaa gagccaaaac gctcttccat tccatcctct aaagggttgg attttatttt agataaaatt ttactgctgc tttgtcaggt cagttatgct actggaaggt cagaggcttt gcaggggaca tccaagtaga gttgtaagct gccattctcc ctacctggag aatggcacca tgaattttgt tattgagata tttattttct cagatttttg tattcaccct ctagacttct cagaagcctt attttattta c tccaaagaa tatggagtga aagtgacata ttctaagcac cctgttctct gatggggaca taggtcccct aacttcaaag accttcaatt caagtcctct agacacctga ccctatttct taatgagcag acatctgact ttgagagtgc agctgactga agggttgaag aagttccttt tttctaattt ttcctctttt agtgtttcta caactccttc ttgtttgcat gtcactggga gagttcagtg atcagaagtc ttctaccatg gacatccttt tcaggaactg ataggcaatt agaaattctt gaaattcgca aaagtctcag agccatctcc tcatggtcct cactgtagct ccctccaaat tctgtgacac tgtgatccag gcactgtggc gagaatttag gttccaacag tccccgggaa gatgacatcc tgggggaaca attcaaggca gtgaggccag gaaggtacag AMENDEfv) SHEE-T

IPEEA/AU

ggaagagc tg ttgtcctggc cttgctcctg agttggactt cacttccatt cttattttgt tagccagggc tctccactcc tccctggcag gtggaccagg gtcaagtggg ttcttatatt cctgagtctt gtagttgctc tctgtccata aagggctcta gaagatgcag agcaagctct gagaataaca tgcctttgac PCT/AUOO/00864 Received 30 October 2001 tggaactgga 5700 aaagagatct 5760 gagcacctcc 5820 attgctggga 5880 actgctttgt 5940 ttccacatat 6000 aatacatcta 6060 gctttttatg 6120 gtccattttt 6180 aagttctgtt 6240 gccttctcta 6300 ttttgtggtt 6360 catattttac 6420 ctgctaagct 6480 tttctgttgc 6540 ttcacattgc 6600 gaggaaacac 6660 tgaaggaaca 6720 agacagagat 6780 agagggaatc 6840 catgacccat 6900 rk, I I tUVUIVUOO'-t Received 30 October 2001 gagtttagat agaaatttat tcctcttaag acccaaaaat gagtgcttgc gatgctgtta atgggacaca attcctacag agagtgtggc agtaacaaag gattggagtt ccagaccttc ggtiaagcaat agatagagag gggaatgtga caagaaacat ctgggatacc gagacaactg atgtgaccct ccaggatcat accaggctca aggattttag tatttgaagt tctcatagaa tgctgtagca tagaagtgca ggcccacggt atcagttagg aaaacgagag acagaacaga ggaaataaat tgcatggcac gggcagggat ttacagatga gtgtgtgcca tctctcatga tggtgaccat ataataactg cagttacatc ctgaaacaaa actctatttt taccacaata catcctttag atctcaatta atccccactg ctccctggga gggaggcctt aaagctacca aagacctgct tcctaatcaa gtttttattt aagaggccca tggtttgtct ggcccgagga tttgctgatg agtgctcata gagtgttttc cagtggaagg ctcctgttat tagacacatg gaaagtatta ttcagaatag tgcaaggtgt tcaggctggg tagtcaatgg gccctactga tagttaaagg tcagattcac tgctatgatc caagacctat ttctgaatac actagctcgg gtctgttctg agggatccca cttgacttct gtcagctgga ccatatgctg ttaacccaga ctctagagaa tattcttgac aatggaaagt tgcaatacaa ctgcaccgta tgtcagtagg tcatccaaag cagtgagcac cacatctcga acctcacagc catcatattc gctgcttgcc acaagtaact tttgacaacg ttgaatatta cccaagcaca gactggagcc aggtcaaata gcatatgttg actgtccaaa aggtgtggag ttttttgtgt tcctgaagta tcttgcactg tatacgacc t tgtccctaca ctcagaaagt C tgagagaga tcaggagagg tgagctcagg tctacatttt cgaaaaactg ggacatttgc agaaggagc t tattgccttg tggagacata ctgagatagt tgccatttta ggtacagaag ccacagagga tggagagac t tttatgacag 6960 7020 7080 7140 7200 7260 7320 7380 7440 7500 7560 7620 7680 7740 7800 7860 7920 7980 8040 8100 16 AM END EDC- H E ZT

IPEAVAU

rk-. I/u1uuVUU6O0I Received 30 October 2001 aatttcctaa tgagatacaa ggcaaatttt gattcctgga ttggactgca tcagagcacc atgtttttcc tggaagtcaa tgcctggagg aagctctaca tttcaaaacc agagtagcta gtgagggata ctgcagtatc ggaaagcaga cccaaaacag atccatcact taaaacagaa tggcagcgag gacaaaggag gtgattcctc tcattgcagg ggattcccat ccaggcac ta gcccaactcc cttggaagtg ttctattatg tgtcatgtga tgtggctcag cacccacaaa tttgcatcat ttgtttgctc tagacagagc tgcaagcctg gactctaagt tcttgggttt gaagccctta ttgaaaatca caccagccca tggtgttcaa agggactttt agcaaatgca ccagggagtg tatacctcgg tgttcctgtc gacagacaac ttcatgtgtt agccaactgt gggtagagag tttaaatact gtcattttac tggctccaac cttagatggg agattcagct acatttggaa gagctgtttt gagcaattta CCCttggtgg atgccttgta atcctgagaa tgattcatat gttctgggct gaaacatgtg atatggatct tagcatttac actgagtata gtgatatgtg actgggaaga tgtgtataaa tcagaggttt tcaaaatatt ttaaacattt cagtgggggc ttccactatt cctcctctaa actgggatgg ctaagtgggc ggtcactcat Cacacattac tatgagacag taccaaaaat ctgtaggctt cctacctgag gtagcaagct caagagac tc 17 acaacatgta tgtgtcgtgg aagaaaaaaa tgcaatatcc tcctgtttat taccataatg ctgttgttga tctggaatcc tgcatgtctg agtctcgtca taaatcacag gtccccatat ggctgcagtt aggattcacc taggtgtaaa tagtggagac actt tt t tgg cggcaatggg taacaactat ttcctagcct cagagagtcg tgttttcacg ctatgagaga aatgaaccct aatccccaga tgaccgtcat atggtgtctg taaatgtcct cagaaagcca cacctgttca tcactgagca actcagtcac ataaaatgcc catttgaaca atggacaaat actaatgcag tggtgagatt tttcttttca atcagcagtt agcctccact gttatctatg 8160 8220 8280 8340 8400 8460 8520 8580 8640 8700 8760 8820 8880 8940 9000 9060 9120 9180 9240 9300 9360 IPEAiAu I AV/0U Received 30 October 2001 ggtgctactt aacgtcgcaa gc tc ttct gc taatgacatc gataaataag aggattacaa agtacaacta ttcttttgaa atgaaagcag tttcccctat atgaaaacca atgtcccgag ttatgtcctc taaatcttaa gaccatagat atgttaggga tttatgagac aatggctggg ggtcccacag tcaaggctgg ttcacatctg ttCtgtggtg aaaagaaaaa gagttttaaa accccacttt cacaaaaatg at tgctatcaaa agagattgct agcaaccaca tgtgaaaaca gaaaaccacc tagatgtcag tatgatatta agtcccccaa cgggattgag ccgtagttaa tggtgtctta gctacactat ttagagaggt cactcatacc aggttttgtg catgggtgta aatatttttt gagactgact caaacatctg acctacatat gcacacatgg ttaagcctaa cagagaattc acctttaata 9420 9480 9540 9600 9660 9720 9780 9840 9862 <210> <211> <212> <213> 12 990

DNA

Mus musculus <400> 12 gatagtggtg cagagcctcc catgccagat tgcttggaga caggagaaaa actgtttgta cataacatga atgcccagtg tgtacagaag ccagaagagg gcaatggacc ccttggaact ggagataaaa ttctccaaag aaaaagtctc agagccatct cccctgagtc ttctgctaag CtttactgCt gctatggagt gatcatggtc ctcactgtag ctgtagttgc tctttctgtt gctttgtcag taacaaagac agaacagatc ctaatcaaca agacctatgc tgcttgcccg 120 180 240 300 18 AMENDE E IPEAiALI rt. I iA~UViuvoU', Received 30 October 2001 aaaaac tgga acatttgccc aaggagc tga agagagtcgt cccatccagg cactatatac actcctgttc tactttttcc cgcaaatgaa tctgcatgtc acatcttatg ataagtaaat ttggagttgg agaccttctg atttcctaat cagagcaccc gagtggaaac ctcggatatg ctgtctagca cctatggtcc aaccatcaag ccgagttcac tcctcttctg cttaaaaaaa aaataaatgt catggcacaa gagatacaag ttggaagtgg atgtgcctac gatctgtagc tttaccaaga cacagtgcta gc tggagaga atctgagcaa tggtgtgtga aaaaaaaaaa ttttattttt gaggcccaac gcaaattttg acagacaaca ctgagcggca aagcttaaca gactcttcct tcaaacggga ttgctccgta ccacatlggtg aaacagctac ctgaatacac tagctcggtt attcctggat ctgagtataa atgggatcag actatagcct agcctgttat ttgagaatat gttaagagac tcttacaaac actataccta aagtaactgg tgacaacgag tggactgcac caacatgatt cagttccagg ccactgccca ctatgggtgc tttttaacgt tgactgctct atctgtaatg catatgataa 360 420 480 540 600 660 720 780 840 900 960 990 <210> <211> <212> 13 19

DNA

<213> Artificial <220> <223> sense primer <400> 13 tcccatgcca gattgcttg 19

%SHIEE=T

IPEA/AU

r- I I rIJ 'Iu u~uvjvr Received 30 October 2001 <210> 14 <211> 22 <212> DNA <213> Artificial <220> <223> antisense primer <400> 14 gggaccatag gggaaagagt ag 22 <210> <211> 721 <212> DNA <213> Mus musculus <400> tcccatgcca gattgcttgg agacaggaga aaaactgttt gtacataaca tgaatgccca gtgtgtacag aagccagaag agggcaatgg accccttgga actggaggta aaattgtcca 120 aggaaaatgt ttcagaatca tctccactgt gtctcctgtt aaactttact gctgctatgg 180 agtgatcatg gtcctcactg tagctgtaat tgctctttct gttgctttgt caacaaaaaa 240 gacagaacag atcataatca acaagaccta tgctgcttgc tcaaaaaact ggactggagt 300 !.E"rFDSHEET

IPENIAU

tggaaataaa ctgcatggca aaagagattc cccttggaag aagatatgcc gtggatctgt cttaccaaga tgtttttatt caagaggccc aagggggat t tggacaaaca tacctgagca agcaagctca gactcttctt tttctggata aactagctcg ttgattgctg acactgagta gcgataggat acaactataa agcctgttat cccacgtaac gtttgacaac gattggcctg taacaacatg cagcagttcg ccttcattgc ctatgggtgc tggacatttg gaggaggagc cacagagagt aatcccatcc aggagctata caaactcctc tactttttcc I Received 30 October 2001 cccaggcctt 360 tgattttcct 420 cgtcagagca 480 taggagtggg 540 taaatcggat 600 ctigtctagca 660 cctatggtcc 720 721 <210> 16 <211> 24 <212> DNA <213> Artificial <220> <223> sense primer <400> 16 tggaaactca gctcctcagc tctg <210> 17 <211> 713 <212> DNA 21 AMENDL'r! z:r~

IPEAIAU

FL; I AUUU/UU864 Received 30 October 2001 <213> Mus musculus <400> 17 tggaaactca gtcccacagg gaaccatctc tcactgtagc cagtcaacaa tttatttttc aggcccaact cgaattttga cagacaacac tgaacaacaa agctcaacag gagacgcttt gctcctcagc cagcccgcag ccctgagtct tgtaattgct gacctatgct tgaataccca agctcggttt ttcctggatt tgagtataac cgggatcagc ctatagcctc ttagcctgtt tctgagatgt gaggtagaag tgtgctaagc ctttctgttg gcttgcccgc agtaactgga gacaaccagg ggcctgcaca aacacgattc agtaccagga cactgccaaa atctgtgggt gtgtcacaaa tgggtaaaat t ttac tgc ta ctttgtcagc aaaactggat cattcgccca atgagctgaa gagagtcgtc ccatccgggg tctattcact ctcctttttt gctacttttt ggcttcccta tctccaagga ctatggagtg aacaaagaca tggagt tgaa ggccttctgc tttcctaatg agagcaccct agaggaaaga tcggatgtgg tccttcctag cccctatggt cctatgctta aaaaggcacg atcatggtcc gaacagatcc aataaatgtt atggcacaag agatacaagg tggaagtgga tttgcctacc atctgtagca catttaccaa ccc 120 180 240 300 360 420 480 540 600 660 713 <210> <211> <212> <213> 18

DNA

Artif icial 22 AMENC.iZ. :A iEY IPEAiU r%..I/ItAuuuiuuou'-t Received 30 October 2001 <220> <223> sense primer <400> 18 tttgtcagca acaaagacag aacag <210> 19 <211> 1229 <212> DNA <213> Homo sapiens <400> 19 cgggtgggcg CCtCCCtCCg gtcacctgga gccagggcct gtgcgatgcc aactttgggg agggcttttg ccacgggccc ttgtggatgc gcaactggtc ggtgccactg cgagagagcc tgCCtCCCCg gctccgaaaa tctcggttgg tccccacatg gacctgagag gaagggcgga tcccccaacc tg tgac ctc a ctgagcccct tttttgtaac tagaagccca ccttttctgc aagcagaaga gtgagcactc tcacgggact gggagtgccc gagagggaaa tctctctgat agaagacttg attttcctcc atgttgctcc tgtagccgcg agagctccgc aggcgct tt t tctctgacca ctgacttgcc cctggacggg cggcgtccta ccaacttgtt gcattttatt cacctttctt tagctcttag aatcccgcag cctggagtga tatttagcca ggccatgaaa tttgtcgtca ccacggctgt gtggcctgct tttccagcct tggaagatag ggggaaactt cattcatggt ccccagtlaca aggaggagcc gtgtgacccc agaaaaatct gagtttgcag ctgtggctta tcagggccac agttggaaac acatctattt gtttttaagg actgttgtaa 120 180 240 300 360 420 480 540 600 660 23 AMENDfza 'HEET atggagaaag tctagcactt cagtcctgac tacaaaggcc aaatggacag gccttcctga tgtagcaagc tagtagagct cagcttccac atctaaaaaa agtaattcac atatctccag tacaagtgtg cttctgacca acaacatgga atgacaatgg caaacaatta gtgagaagag catgcccctg aaaaaaaaaa gcagagccgg taactcctgt attgcacttt ttggattggc atataataac agtcaacagt tgtctacagt ggccaccatc gaaaaac tgc aaaaaaaaa ctttgcagat caaggtttat ctattgtttt cttagaagag atgcttgcta ggcagaatct tgccagttat ctccagactc aagtaacaga aaaactctgc tgttgcttgt gtcagaattt aatcatccca tcagaggaag acatgaacag gtccccac tg cagaatggtg cctgcacatg FL I/AUUUIUUStb4 Received 30 October 2001 aaagacaatg 720 tcatccattg 780 cctgaaaaga 840 tcgcatttgg 900 tggagaatgt 960 aaaatggatt 1020 ggatactacc 1080 gaatcatcag 1140 tatcccctac 1200 1229 <210> <211> <212> <213> 1305

DNA

Homo sapiens <400> cgggacaatg ccttccaaga cttttttcct atcatttaaa atttccagtt ttatgtggct ataacactaa ccaaaacaat aatatttctt acttggattt cagaggccct ccatgtattc tttgacctct acaataatca gccttttaag tctattttaa tacattttta gacaggaatc tgttgatttc attcctgtgg 24 AMENDED) 2HEEST IPENiAU ttaactattc ctttatcact tcgatagatt agaataattt tttagttgac actctcctat ccagttttgc tcctcttaat gctatgttga atgaatctct mL I /Auvv/uVVOD, Received 30 October 2001 ccaattgaaa tggaaaatga gcatccttct ttctggcagt ttgtctgcat aatcatgttt taactgccat ttttcaaaga gttttgtgac cctgttgaga accatggaaa agagtgtgcc gtggatttgt tttagaagca taccaacagg aatcatagta atgacatacc gggtaacttg ttaaattctg agtggaaaag attatgtctg tcaaaagagc ctgacaatca caagagccat aagtgtttct tcacaagatg tataaaggcc tggataaatg tatttgaatg tccaaatcag tattggaact tatatgaaaa aaatattacc cgagactgag aatatggtag gagatagaat cgaaggaat t tatttaattg attctattaa tagtgtgtgg cagtatgtct atttttctga c tga tc ttgc catctgatca gtactgaatg acaaaggtgc atatacatgt gataactcca tatgctcaat tgttttcatg taatttataa ctggaaagtt aggtaaggtt aaataaaaat ttaaaatgtt agctacctta aatggttgct tcaagctgca tgacaccaag tcaggttgaa ctggattggg gacaagacag cagtagtgcc ctagatgtta ttttaaaatg atcactaata gtgctaatat ataaagagat aaaatcaatt cataagatga aacactttga tttcaataat atttggcgct gctttaagtg tgcccagaaa aactggacat agcttccagg ctgagcagag tttcctatcc aggcactaca cagcaaagcc agcaaagaat ac tgggaaaa tacctgttct ttaat cttttaactt caggtcattt tgcttaatgt tttttccagg tatttttctt caataagagc gctggattgg caagtcagag aactgaattt aacaaggcca tgggagcagg caaagaggaa ccaactaatc ttatttctta tacaaatcaa cccactgcta 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1305 <210> <211> 21 10221 AMENDEID SH-EET

IPENIAU

rk.I /PiUVUUU04 Received 30 October 2001 <212> DNA <213> Hojmo sapiens <400> 21 gaattccttc cgtacctctg aggctattaa tcttcctggt cctgggtatc gaagcttcat ctatcaaccc tgaaaaggca tttcagagct tttttctatt gtagaattcg ttactgcctc ttagtcttgg accagcaaag cccagagggg ctgctaggag gtc t ttccc t ggc tggcagg gtttggaata gcagtgcatt agtttcagaa gagggtgtat gc tgaagaaa cacgcaccag ttgtctccca cagaagagc t aatgtttaag agggagataa tgCCCtgCCC tggctttgct cactgtgagg caagcttgag agtgggtctt tccctggctt tccactgggg atttcagaag tgtctggaca cctgttattg gtgtccagga agcaaagat t atgccagctg gtcaggaggc cgagcactgt tctcctgaag gagttttatc agagaggagg gcactgtggt ggaaaatcac catctggggt agcttgctgg cagccccctt tatgaaaaaa gaatggtacc tgggcttgtt gtctattcag atttatccat gctgcctgct agctgtcctg atgggggtca gc tgggagat ccgtgaccac tataagcccc aatctagaga gggctccgcc ctactcaagc ccacttcaga gctctgtggg tccagcagag actcctgcag agctcctctt ttggttgggt gaatctgatt ttcttccttg ccttcctctg tatgaggtgc gggacccact ccactgctct agccaccctt tgactggggc ggcagtccgg cagtccgaac ctcagtaatg ctgctgtgct gattggaccc tgaatgattc ttatcttggt 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 tcccccaggt gctctgtccc tgc tgcc t tt ctgcagtggc ttcccccagg gcggatgcac ggcagcaaga actgagcaag tgtctcagtg ctttcagaga tttgctgcac gctttgttta ctcccctcac atttccagcc accacttggc ggttccaggc AMENDED SHEET

IPEAIAU

Received 30 October 2001 gactgcccaa ctccagagaa ccagatagca cccacccctt gggctgcacc tgcagaaatc taatcagcca cagtgatagc ttcaagaaat agatacaata tcataactaa tgatagtaat attgagttga tcagtgacaa cticctgatta aagtgtagaa tttcatattt tcacataagg caaccttgtt aaggaatggt ttattacatt atcgccaccc tctcctggtc cctcacagca gtgcttcctg cacttgtgta acctgccttc tCttgCCCtC acaacaaata tacattttaa cacgtttgcc gagtgatttc gattctatga aggtagaagg ctatctggag tataagaagt aacctcacta atgatagtat catctctatc ggcatgaggg ttggggataa gtgtaataat *agttttgtgc tgtgggttgc cagtccctca ggtgaagcag accagtccca tgcattggtc tctggtctgg tgcacagatt tagttataat tcatgttatg tcatctcaca atacccagca tggttatata agacattcat gggtcagaaa aatctcctta ttagacatat ctgcccttca accagttttg tttaagtgca atataataaa ttgaaaccca aaaaaccgtg caacttccct cgccccaccc gtgagatgat tcactgggaa tcgttttctt tggggaaagt ttggtatcac atttgttatt agagccaaat ttctggtcta atagaagatg tcatctcgat agcctgtcca gtggaaggaa acaaggcttt ttttaccaag tgaaagacaa ttacttttat ataattatac gggttctggt ggaaaagcgt tggctagggg tgcttctgct cctggtacct ctgcagacca taaattggtt catcctgcat cttgtttgtg cagattacac ccaaggataa tcatagacac aactggtagc cccacatgaa gtgaagtatt gttcactgta ttcacatcaa tcatctggag cgttttcatg tgtgcccttt aactcaccat agtgt tggca agtatctggg agggagttct tgccctctgt Cagttggaaa gagctgtttc gatacaggag tatggtc tgg ataccaaacc cagttattat tggtgccaat tttcagaacc tactaggggc gagagcattt gctgccttca caacaactta gaaaccttat cagcaatcgc gactggggtc atttccatta aatgtagaat 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 AMENDL:r Aq-=-T

IPEA/AU

a a~ IlfJ'J WWI WWWWl Received 30 October 2001 Cagtgggaac gacagtgaca catgagaagt acaggaggtg agcttcactt atggaccagt aactatctgt agggtaacac aactcctttt tgctgtgtaa atacatttaa tacaatataa agctaactgc tggttttcaa gaggttttgt aagaaaatag aattatctaa ttttttgcat attaccttaa gacttgtgag cctgagctag gttcatcagg tcccaatagg gagctcatga actcattcgc actgatccat tc tccaaagc tgttgagagc tcagctcacc tgtatgcaat tacttaacag aagtaagaaa catcaagagc agaaagtgtt gactcacaag ttctggccag gaggtaggtt aacgaaagag tattcgtggc ccagaattgt tttttctgaa tattagattc gtttgccctc ggtaatgtga tgcttacctc ggcctagggg tgatcaaatg caaatagata ccctgctgaa tatatttagc gagtgtcaga tttttatttt catcagtatg tctatttttc atgctgatct aatcaaagat tagacatctg taacctagca aggaacaggc gcaaataagg agtagatggt tcacaaggag c tatgagaat gtgatgggga ctgctgtgca ttggggaccc ctatcattaa catggcccag ggcatgagtt atcatatttc aagtatatta atttattttt tcttcaagct tgatgacacc tgctcaggtt tcagccctac cttttacatt tgtattatat ccagagggca attagaaaag accatctgtg cccacaacct Ctaatgccac gtggctgtaa gcctgcttcc ctaatctaga tgtatctaat agcaagctta tgaatctcag tcacttgaaa gcacttggta attttatttt gcatgcccag aagaac tgga gaaagcttcc aaggatatgt gat tt t ttt t tttacagtga agcaagccag tattggtaga *agtgaiggga agattcctca tgctgatctg atacagatga tgactcatc gcacttggag attttaagaa agttactaat ttttgccatt aatgaaaata atttatacaa cagcaataag aaagctggat catcaagtca aggaactggt tttcctgtga tttttttttt accatctaaa agccttcttt aacccagttt 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 AM E N D E D HEET7

IPEA'/ALJ

ri I /AUUU/UU50 4 Received 30 October 2001 taagtttgta ggcacatgcc ggagtttgag attagctggg gaattgcttg cagcctaaca caagaatgat agaaaactat gttaaacact tgttggtgaa gggatccatg ataagcacct gggcagacac aggatttaaa ttttattaga caacagagaa ccaagatttg gtattcataa cattcacagg aaatatgagt tgaagttagc tgtaatccca atcagcctgg catggtggca aacctgggag gagtgagact gtcatagagg tgaactgtca ggaagcatca tttcagcctt ggaactgctg ttttgtccaa agaagtgggc taaatagtat taactggctg taaaaatagt acaatccctg tgtccagttc aagaaaaagt caaaaatgtt aacattgttt gcactttggg ccaacatggt tacgcctgta gtggaggcct ctatctcaaa ttggtagact aaaactgtct agggaagagt tccactcaat cccatgttct atgtcagggt tatcattgca gtgtttttcc acagcagaga tgggaaaaaa aagagcaaaa tcaaaaaaaa aatctgacag aaatcagctg caaaataaat aggccaaggc gaaaccccat gttccagcta acagttagct aaaataataa aaaaagc tac gaagtaacta ttgataaaga aataactatt tgtaatgaat tattgctgat tcagtcctca tccctttagg taacagaaca tcatgaccaa taattaagtt attacaaaac aaactatccc tcttaagtat caaacaaggc gggtgtatca ctcaactaaa c tcaggaggc gaaatcatgc aataaaaaca agaaatctgt t t ttggaat t ggatgataaa ttccataccc tcctgcagcc ttctgccttt tcagc tgaag aagcagtcat gagatttcaa atgactctga accagagtta atgcaaagaa tgaagaggct aaccaatgag caagagcagt cttgaggtca aaatacaaaa tgaggcagca tactgtactc ataagtcaag tcctccactg ctcgagtcta ttttggttaa cattattgca agggtgaaca gaatgctgag tggcttccca ttaagacaat tgaccatgca gccacaacaa ccacaacata aagtatggtt cagatattaa t taaaggaaa 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 4740 ctagacaaaa agctaaagga aaccgaaaac ataataaatg 29 A M E N i D 'ESZX. 1Em

IPEA/AU

aacaaaatta gaatatcaat E 4 1 1 IrkLjU'..IU1o'Jt Received 30 October 2001 ataaaggtag actgaaattt gtaagagatc agaaaaagaa acatatgtac aagaaataat aggctcaatg caagttgaca ttacataaga aaggtagtgg ttctatgtat accaataatt ccttttaggc attcataaaa gtttataact aaatgaataa acagtcttat gctatgttga aggtaaccac ctataaaaaa aaattgtaaa aaaaataatt agaaaacttg tgaagataaa tgtagaaatc gaacaaaact aactccaaaa aaatccacag ccctgaataa aatggcatat agcaaagttg aagggatttt tgaactgaaa gtaacaatag tctcctattt aaattataaa gaagcaggga tattatgaaa taataaaatt ccaattaaat aaagaaccaa t taaaaactc aaaataagat taaattctaa caggaaagag t tccaaaatc gggtaaactt agagaatttt aattaatagc ttaaatgtct tccttcaaga at taccagta gtacactaga aggtaaatat atagctttct tatgatagtg tgaagacata ttatattgtt accaaaatta acaaaaaaag gcaaaaattc aatgaagaag aat tgaaaca ggaacctgta aagagaaaga tgaggaaata aaagagatc t gaaagcagcc tga t tttc tc gaaagaaaaa atgaaggaaa gacatgtgct cagcaattca aaaacccaga atatttatat gtcatataat taggatcaaa acaagtttaa tgcagaaaag cagagctgaa ttcaacagca atccagacta ggacatcagc gaagcagaga cataaatata acattgagac agaatgaagc tgagaaacca ataaaactgc aagtaacaca acagaaaatg gagcctccaa attactacat ttatctataa gtataaagat atgtttgcat gatgctaatt gaaaaaagaa aagtacaatg gatttgagaa agaaaaacaa aaacatacta aatacactta tacatccaag aaaatatagt aactcatcat tggagatcag caaccatgaa ttttcagata ctaaaggaaa aataaagaat gtgtcatata cttcataggc gcaatctgtg agttattgaa ataattctca aaacaataca 4800 4860 4920 4980 5040 5100 5160 5220 5280 5340 5400 5460 5520 5580 5640 5700 5760 5820 5880 5940 tcagtaacag acaacttgag aaacaaagac AMENDED SHJEET

IPEAIAU

rL. II/AUVVIUU6O' Received 30 October 2001 atataagata tagagaaaac aaatgattaa atggcaaaag taaatcttgt acattaaata aaaactaaaa tgccttagat acaaacagta aaagtaaaat ccaaataatt aataatitgct attccacttt ttgaacaaca agagcagaat cacaaaacaa acaatggaat aaatttaaaa gaaaatacc t acatagttct tcaaatccat gtcatcttta aataaagaaa aggacagtac gtgaggataa gaaaagga tg taaattaaaa ataaggacac gtaaccaaga ttacaagaga atgttataaa ataatttaag caacatgaga ctataaacca attaattctt gtgttagtaa gaagctagaa acccgctctt tgagacaaat aagagggaaa agcctaactg tgatttgaaa attgtttata acat tgacaa tttggtacac aagccatcct agaaaaacaa aaagaagttg tagtgccgag aaaagaaggg atatatgtac ggagaaaaga ttaaacaact attatcccta ctcaaatgca gtgtgaaaat atcaataact caacagccat gaagtaaaaa t tta tagc tg tacactgtaa gagtaaaaga tatttaattg ggaaacacag ttacattagt attaaagggc gactcatcta aaagtaaaag tggctatatt cattatgcat ttaataatac acagttctat agacataaga acaggcatat catggaacat ttgaagtcat agaaaaacca tggtcaaaga tacaaatagc tgagcagtta ggaactaaaa tttacctaat cgttaaaatt caaaggaaac ttggtgtctt 31 tgagactgac catgctgtct gactgaaaaa tttgtcaaac tgacaaaaat agcctcaaaa gaaaagttag tcaataagga acagaagaat tcttaaacca aaaaagtatc gaaaagtcac agaaatcaca acgtttatgg actaaaaaag aaagtaaaac aagtccctaa cagaacttgt cagcctatgc ttctttctct tttagtaatc aagttggcta ataagagact gatattccat aaaataaact tttgacatag tatatgaagc agaatgaaat aatagaaaat ctiacccaaca tatgttaggc ttttgcaatt gcatatgtag agggacatta tatacactga aagaaagatc aaaaatagaa atttactaat aatcataaaa tgctgctgtt ttctttcttt 6000 6060 6120 6180 6240 6300 6360 6420 6480 6540 6600 6660 6720 6780 6840 6900 6960 7020 7080 7140 7200

AMENDED)$HE

IPEA/AU

Received 30 October 2001 ctttctttct tctattgccc aggttcaaat tcacgcccag agcctagtct ggattacagg gggcc taaaa acttgataaa aaaggagtac attatcttct ctttctttcg aggctggagt gattctcatg ctaatttttg caaactcttg tgcttggcct attctattct accctggtgt aatacagaaa tttgatatgt ttcgttcgtt gcagtggcgt cctcagcctc tatttttttt gcctcaggtg ggtggtgtca aggacaggtg caaccgatag caaaagtaac atgataatat aaaattatga tgttagatta ggcccatctg aatggtactg aatttacttt ttaaggttag cctcgaatga cttatctatg tgttccttac tgtgaggaac cgttcgtttc gatcttggct ccaaatagct aatagagagg atccgcctgc t ttc ttaaag ccaagaatgt tataatagat caaattatca agcacaccta gtagttaaga atgaatatca atcactggat aatggacaag gcattaaatc tcatgaaaga atccaagacc tcattttcaa ctccacaaca agaggttttg tttttgagac cactacaact gggattacag gggcttcatc CtCggCCtCC ttgacaaaaa catagtagca aaattggaga acaatttctc ttctgtatgt atatagcata tctctgtgtt tgggctgagc acagtaagtt tgaagtgttc agatggtgcc tgtcatggtg acactttcat aaattaaatt atgtatcagg agaatctcac tttgtctccc gtgcatgcca atgttggcca caaagtactg gcatatcctg tacattccaa agagtcatac tcagttttaa attactaaac gcagcaacat ttctgaaaga agagaacaag ctaaaaatct tctagttaca aacttgtatg aggtagactg tttaagcaaa gcacttgtcc gaaagattat 7260 7320 7380 7440 7500 7560 7620 7680 7740 7800 7860 7920 7980 8040 8100 8160 8220 8280 8340 8400 aatacaaaat caaaaggaag ttctgggaga atttcctgtt gccaaccatg ggcagtaata tgctttaaaa ttgcaaaggt actgtgaact ccatacaata tcctgatttc ggatgggtta gagatataaa gaaatggata tttgtatttg aattctcatt ttcacaagtt tggctccagg tctttaagtc acagggttga A.MENDED SK;F-= IPE-kA' gagtgacagc aattatacct attatttaaa ataagacaat agttttaaaa ggtaaagttt ctaaaaagat tctgagagat aatataactt tttttctcat tacatttata tagaattaac tcttaaaatg aggctggagt gagcctccca cttttttttt atcttgaact caggtgtgag ctaaccagtt gtctaacaag aagaggaaaa tcagctaaat tcaagcatgg gggaaac tgt gtacttgaaa ggcac tagaa ttgagataat gagacagttt tatttaataa tagcttttga cattgttgta ttttatttct gt tt t ttgt t gcagtagtac tctcagcctc tttttttttt cctgagctca ccaccatgcc tttggaaact tttcaacaat atttataatc agcactgcag tttttagaaa gtaaagaaaa gaatattatg aatttaatct gctggaaaaa tgaaaagc ta aaagagcccc ccttggatgg tctacactgc tattttacaa tgtttgtatt catcgtggct ctgagtagct t t tt tt tcag agcaatcctc tggcctctca gctcatcact tagcaaatta caaatgctga tttcagtact tagctgctga cgtgtcagac cttacagaca caattgtata ttggattcac ctttattgta agactggaca gatggtaagt cttgcacatt tacggaagta tttgagagac cac tgcagcc gggactacag cgatggggtc ccacc tcggc aaatatttta taaagaaatg tatcatagat cctaaaatat aaaaccacca gtcttcagtt ttctcccatc tgaaataggt 33 tttataggat acaatttcac atacattcat ttggcaggtt tttagaaatc gtgactgctt gtaaattttc agggtcttac ttgacttccc gtgtgtgcca tcactatgtt ctcccaaaat aggatcaaat taaaatatta gatagtgatt ctgtgccaag gggaagtagg atttaaggaa agccagctaa ttgattcagg rL, I I AUUUUOU't Received 30 October 2001 ttttaaaatg 8460 cttcagatta 8520 tcaatgtttg 8580 caatattgga 8640 tgaaatgagt 8700 agagaacatg 8760 cataaatatc 8820 tctacctaat 8880 tctgttaccc 8940 tggctcaagt 9000 ccttgcttgg 9060 gcctgggctg 9120 gttggaatta 9180 atattattaa 9240 tatgattaag 9300 ccaatgagca 9360 ccatctaaac 9420 aggaataaaa 9480 gcaaaatatt 9540 ggctttggat 9600 actttgcagt 9660 tAMENDr0 H2Em (PE4 AU ML I /AUUU/UU50O4 Received 30 October 2001 attcctatag cactacacaa ttctcatctt ttctcttgca gccagtagtg gtctagatgt cattttaaaa atatcactaa tggtgc taat aaataaaaga ttgatttata ggaaaaacag cattctctct ggtttcctat ccaggcacta tacagcaaag tgagcaaaga taactgggaa attacctgtt gatttaattg acatctcctg cattattttt gctgttgaag cctgggagca cacagagagg ccccaactaa atttatttct aatacaaatc ctcccactgc a ctaagcaaag agaggctgaa aaatgttcag ggagagtgtg aagtggattt tctttagaag tataccaaca aaaatcatag taatgacata cccactgact ttaatgttag tggccaactg cctatttgaa gttccaaatc catattggaa ggtatatgaa taaaatatta cccgagactg aat tag tcac ttttctcatt attctgcttc tgacaaaggt agatatacat ctgataactc aatatgctca cctgttttca agtaatttat 9720 9780 9840 9900 9960 10020 10080 10140 10200 10221 <210> 22 <211> 21 <212> DNA <213> Artificial <220> <223> antisense to C-type lectin region of OCIL <400> 22 gagtgttgtc tgtccacttc c 34 AMENEDM sH=F~ Received 30 October 2001 <210> <211> <212> <213> 23 21

DNA

Artificial <220> <223> antisense to C-type lectin region of OCIL <400> 23 tttccaactc caatccagtt t <210> <211> <212> <213> 24 21

DNA

Artificial <220> <223> antisense to sequence upstream of open reading frame of OCIL <400> 24 gaggagctga gtttccacta c <210> <211> AEND~~~: PCT/AUOO/00864 Received 30 October 2001 <212> DNA <213> Artificial <220> <223> antisense to sequence in open reading frame of OCIL but outside C -type lectin regio <400> ggtagggaag cctttgtgac <210> 26 <211> 19 <212> PRT <213> Artificial <220> <223> polypeptide fragment deduced from cDNA sequence of mOCL17 <400> 26 Cys Met Ala Gln Glu Ala Gln Leu Ala Arg Phe Asp Asn Gln Asp Glu 1 5 10 Leu Asn Phe <210> 27 <211> 33 36 AMENDED SHEE1

IPEA/AU

Fu. I /AUUUfuuboD4 Received 30 October 2001 <212> DNA <213> Artificial <220> <223> sense primer <400> 27 gccacgcgtt tgtcagcaac aaagacagaa cag 33 <210> 28 <211> 31 <212> DNA <213> Artificial <220> <223> antisense primer <400> 28 gccacgcgtg ggaccatagg ggaaaaagta g 31 <210> 29 <211> 633 <212> DNA <213> Mus musculus 37

IPEAIAU

Received 30 October 2001 <400> 29 acaatggttc ctcttccacc gacaagacgc gcttgcccgc agtaactgga gacaaccagg ggcctgcaca aacacgattc agtaccagga cactgccaaa atctgtgggt ttgccagctc tgggactcca gtttgtcagc aaaactggat cattcgccca atgagc tgaa gagagtcgtc ccatccgggg tctattcact ctcctttttt gc tact ttt t taccaccagc agcttcaatc aacaaagaca tggagttgaa ggccttctgc tttcctaatg agagcaccct agaggaaaga tcggatgtgg tccttcctag cccctatggt atccacacca tcggcgcgcc gaacagatc aataaatgtt atggcacaag agatacaagg tggaagtgga tttgcctacc atctgtagca catttaccaa ccc tgctgctcct aggactacaa cagtcaacaa tttatttttc aggcccaact cgaattttga cagacaacac tgaacaacaa agctcaacag gagacgcttt gctcctgatg ggacgacgat gacctatgct tgaataccca agctcggttt ttcctggatt tgagtataac cgggatcagc ctatagcctc ttagcctgtt 120 180 240 300 360 420 480 540 600 633 <210> <211> <212> <213> <220> <223> <400>

DNA

Arti fic ial sense primer 38 AMENDED SH!I-r-.

!PEA/AL1 rL, I /AUUU/UU7S4 Received 30 October 2001 gccacgcgtt cagtaaaaaa gacagccaag <210> 31 <211> 28 <212> DNA <213> Artificial <220> <223> antisense primer <400> 31 gcccagcgta actacaggca ctgtgagg 28 <210> 32 <211> 28 <212> DNA <213> Artificial <220> <223> antisense primer <400> 32 ctcagtgttg tctgtccact tccaaggg 28 <210> 33 39

AMENED-~~E

IPEA/AU

Received 30 October 2001 <211> 1628 <212> DNA <213> Rattus rattus <400> 33 cggccctact acccttacac gctgcctctg taatcaatgc acacagaatg tctgcctctg caaggaaagc gtagtgatca aagacagcac gttggaaata ctctgtaagg ctaaggagat cacccttgga gaaaaacatg aagtcgattt ggttgagact aaatgccatc agaatgaagg cctctgagtc cgtccagtgc aaggacagac agtcattgtc ttcccagaaa ttgtcctcag agatctcaac aatgttttta aacaaggggc acaaagggag agtggacaga gcttcctgag gtaggaagcc ttgtcctaac cagtgcacac acagacctcc attgtcaggt acacctacag ctccttgagg aggttccaca catccccctg tgtagc tgta cataaatact ttttaatgaa cgagctagca ttcaggttac caacactgag tgacaatggg caacagc tac agtcatgagg ctacaggatc ttgaggcaga tccacagagt gatcctcccc cagagtagca gagtcaagaa gagtaticctg gttgctcttt tatgctgctt ataccaagta cgatttgaca tggttcggtc tataacaact ttcagcagtg acctcacagt ctcccccact gtagcagctg caagaattcc cactcctctc gctgtggtcc ttcctcacag ctgggcttta ctgttgcttt gcccgagaaa actggacatt ccgaggagga tgcacagaga cggtttccat gcaggggtta gcctgtagtt cctctccagg tggtctgtct tcacaggacg caggaccctt gtctgctgcc taaaatgctc ctgctgctac gtcagtaaaa ctggattgga gagccagacc gctgaatttc gtcatcagcg cggaggagat tatagtgagg ttgtgtcctt 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 aacacagaac atggtatcta cagtgcctga AMENJm jyF HE IP2AVAU atcatgaaca caactgaggc gtatgagctg actttctttt CCttgttCCC tgttccccca aggtgcatcc tggtgctggt cttatttgac acccatgtgg cctggggtag aaaaaaaa atctgctaaa atattttgct gttctcacat ttctctcatt ccttctagag ccccactctg tctcatagtg ttccgccttc tatgcctgcc cacgttgggg atcatcttca tgggagatca cttaaacata taaattatat ttgttcactc agtatttccc aggccaac tg caccctccca tggcctgcta ttggttggtg attcataatg tgaattgttc aac tgaatca tatttattta catacccctt ttctcttgga tagggagc tg acagtgagcg ggttcagcat ttggatacat tgtggtgaca tatattaaat tgtcagtatt tattccaaat catctttact ctttaggact cgacatgccg ctccttgtag agtgacagcc aagctgatgt PCT/AUOO/00864 Received 30 October 2001 tctaagataa 1020 aggtattcag 1080 agttatctct 1140 accgtcccct 1200 tctgaagaga 1260 gtacaggatt 1320 tgcctcaaaa 1380 taaacaagtc 1440 tgtictgcatg 1500 agggcattcc 1560 tagatgattg tatcaaggtt cctgaataaa ctgcttgaag aaaaaaaaaa 1620 1628 <210> <211> <212> <213> 34 24

DNA

Arti ficial <220> <223> antisense primer <400> 34 cagttttgcg ggcaagcagc atag AMENEE!, c,+E-rT IPEA/AtJ I ,ujuiujo0ri Received 30 October 2001 <210> <211> 23 <212> DNA <213> Artificial <220> <223> sense specific primer <400> aggcagcccg caggaggtag aag 23 <210> 36 <211> 1206 <212> DNA <213> Mus musculus <400> 36 gtgcctctca gctttcaagt ttcaatcctg tagtggaaac tcagctcctc agctctgaga tgtgtgtcac aaaggcttcc ctacctatgc ttagtcccac aggcagcccg caggaggtag 120 aagtgggtaa aattctccaa ggaaaaaggc acggaaccat ctcccctgag tcttgtgcta 180 agctttactg ctactatgga gtgatcatgg tcctcactgt agctgtaatt gctctttctg 240 ttgctttgtc agcaacaaag acagaacaga tcccagtcaa caagacctat gctgcttgcc 300 42 AMENDEED: suEET

;PEA/AL#

Received 30 October 2001 cgcaaaactg gattggagtt gaaaataaat gtttttattt ttctgaatac ggacattcgc aggatgagct acagagagtc ttcccatccg ggatctattc aaactccttt ggtgctactc aacatcagaa actgactact acatccgtaa caatttaaag ggacgaaata gtctttttaa gagagggaca tactac ccaggccttc gaatttccta gtcagagcac gggagaggaa acttcggatg ttttccttcc tttcccctat atgaaaacca cttccgaagg tgagatcttc catgagggac acaggc taaa agaccaggtc tgattggggg tgcatggcac atgagataca ccttggaagt agatttgcct tggatctgta tagcatttac ggtcccaaag tcatttcatt tcctgagttc tgaggtgtat ataggagagt aagcctctct gc tgaggaga aggggaggga aagaggccca aggcgaattt ggacagacaa acctgaacaa gcaagctcaa caagagacgc tgctatcaaa tcatgcagag acatctgagc gaaaacagct tagctacccc tgaactcttc aagagatgga agggaaatta actagctcgg tgattcctgg cactgagtat caacgggatc cagctatagc tttttagcct ccagatagag attgttcagt aaccacatgg acactgtact acactgatga atcctttctt gaaatggggg ataaaaaaat ccaagtaact tttgacaacc attggcctgc aacaacacga agcagtacca ctccactgcc gttatctgtg aatatttctt ggttaaaatc tggc tcacaa ttatactctg gtcccaaaaa CtCCCtcttg aagggaaggg aaaaccaaaa 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1206 <210> <211> <212> <213> 37 6622

DNA

Mus musculus AMENDED SHEEF IPEA4A

U

PCT/AUOO/00864 Received 30 October 2001 <220> <221> <222> <223> Unsure (332)..(332) unknown <400> 37 agatattgaa cttccacttt Ctctcctctc ttctttcttt tccttccttc tctttcagat gc tggagtta aattctaatt gaaaaagtgc cagcagctgg gtggatatta tgaaactgaa acaatcatca atctagagac catgctccaa ttcctctttc CtCCCCtCCC ctttctttct cttccttcct ttatgtgtat taggcagtta gctgatctag taatatttta tgaggtaaca gcccagaaac gaagaacgaa atggaaggat ttgccatatc agatgattaa ttCCCtCCCt CtCCCCttCC ttctttcttt tccttccttc gtgggtgcct tgagctacac gaagtcctag taaagaaata caatcacaaa t taggatacc gaccaaagtg tacagagaca cagggatcca cttatgcagg ccctcctttt tttCCCtCCC ctttctttct cttcctttct tnagacagca agtgtggttc ttttgaaaaa ctcctatatt agaac tcaaa caagatataa tggacacttt aagtttggag tcccataatt tattctcttt CtttCtCtCt ttccttactc ttctttcttt ttcttctttc gaggcaacac ccaggaacag gtagtttcta tgcatacgtt tgatatgtac gatacaattt gCCCCt tCt t ctgagagaaa agcctccaaa CtCtCttCCC CtCtCCtCCC CttCCCtCtC ctttcttcct tttctttctt gtcctctgca aacccagggg ctcagaagtt aaagagttga tcactgataa gcaaaacaca agaat tggaa aggatggacc caatgacagc 120 180 240 300 360 420 480 540 600 660 720 780 840 44 AMENDED HE

IPEA/AG~

attgcataca taggccgggg agggccccca gtaggtgcaa atcaaaagat tatatgcccc gtggggggca tgtaataaaa gtaagttaca taatcctgac tacacatatg ccaaatgggt tattacgcag actgttgcag tcatgaaatt aatcaggaaa cagaatcctt ctagcaagcg cctagcaaac a tggaggagc cattatgaac ggcctggtcg agtacagggg gggtgtgggg aaaagagttg gttgtatttt catctggcct ctgtggcata aactctccag taaaatctgt gcctgtgata cttagacaaa gaacacacat t ttagaaggg tttgctgcaa acataagtgg tagagaaagt taaccagtac gccatcac tg aatgccaggg gacttttggg acagctttct ttcaaaggaa ggatcacagg gatatgcccc tgtttcaaat ttggtttttt agaaagggta tggatggatc gatatgcact ggaacaaaat gagactgcca cactagcaga tggcagatac gaagcagcta ggaacctgat atgctctcag atccaaggag cccggagctc gaaagagagg ccaaaaaaat ctagcattgg ttcaaaactt tggactcagg agaacagggc gctcaagaaa agttgaatgt tgatctcccc agaggttcaa tggaggatat cactggtaag acccatggaa gagactgccc tgccaacaag agaagtagat gagaaagtac PCT/AUO0/00864 Received 30 October 2001 atagctgtct cttgtgagac 900 tcagctattg gatggatcac 960 ctaaagagat ctgcaaccct 1020 ttgactctag ctgcatatgt 1080 cccattggac acgcaaactt 1140 gggaatgggt gggtaggaaa 1200 aaatataatt gaggaaaata 1260 taaccaagac aaattaataa 1320 gcttaaaagc tcttgccaca 1380 agcaggagag gacagactcc 1440 taaagtagtt ttttaatggg 1500 gactattgga tataaatatt 1560 agggttctct gtttcccaga 1620 agctgttaaa aacaatgaat 1680 catcttgagt aaggtaaccc 1740 tggacattag cccagaagct 1800 ggagttacag agacaaagtt 1860 cacttgggga tccattccat 1920 agcctgctat agctgtctcc 1980 gctcacagtc attcattgga 2040 ccagggagct aaagggatct 2100 tggagcagag cctgaaggaa aaacaaccac tgagggcctt cagagcacaa caaacccaga tgtcagtgcc agtccccaat AMENDE~D SIz-j)

W&VMALI

rk- I /AUUU/UU~sb4 Received 30 October 2001 gcaaccctat gctgcatatg cttgcaaact gggcagggga aatgaggaaa tcagtaaggt acaaagtaaa gaccttgaaa gtatgtacag gtgcctctca tgtgtgtcac aagtgggtaa agggcaggaa tgagcccgtc gttacattag gagcagcgag agcttgctag attgctgtag gcaatgtgca ttgtaattaa aggtggaacc tatcaaaaga ttatatgccc gtgggggggg atacctaata cagcttgaat ccagaagcaa tgcgtttccc aaaggaggag gctttcaagt aaaggcttcc gtattcaata ggcaaaagag actgtgggtc ttattctatt tcaccttcta tccactcttc taaacgtttg tagtggcaga aatagaattg tcattatgaa tggcctagtc cagtacagga ggagggtatg aaaaatatta tatgtttcta acaggaaagt aatgagattt ctaaaggata ttcaatcctg ctacctatgc gtatttgaac ttgaaccttg tgcagagcag ggagcataca actgccagag ccagggatct ctttaagtcc agttgcctgg taagggggtg ctaaccagta ggccatcact gaacgccagg ggggact tt t aaaaaaaaaa aatcagagtg cagtctccag tgttaggccc aaataaatac tagtggaaac ttagtcccac caatgggagg aacaaaagat gagaatgcag atactcgaat ctgtagccac ggtaagttac ctgagtgaag gaagc ttgga gcttggggtg ccccggagc t gtaaagagag gccaaaagtg ggaataacat aaaagaaaat tgctgaaaga atggcgccag tgagccaacc tgaaacc tcc tcagctcctc aggcagcccg ggcagagagg taagaacaga tcgggattag agttctcagg agcgttctcg agtctggtgg gaaactcaga gcttgtgttt gggtgggggt cttgactcta gcccattgga ggaaatgggt tggaaatgta ccctgtgacc gaaacgaaac tgtggctcct aagcgtgtgt ccacgtattt agctctgaga caggaggtag agtttcaaac agggcgtctg ctatgaggtt caagagaaat ctttgtactt attacatcaa caacagcttt tgcagatcca ggggggcgct 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 46 AMENDED PH42SrT IPEANAu Received 30 October 2001 gaacctactc tataatgggg caaactgtct tttcttcaca aagattgaca caagtattct actgaggatg ctcatacttt tttttaaaat ttctgtctca aatgtggagc ttcactcctt accaggccag ttaaataaag ggagatctgt cagaattata aagaaatgct ccaataacca ctaatattat ccattagtta ctaggttctc aggaccaaat tttcctactt atgctccatt ttaatttttt tctatcaaaa gaaataataa atgatgactg accccaagct caaatacaca agtgcagctg tcctgtctca tgaagcagta caatacttca acaggaaagt gtgtatacag ctggtgtaaa ggtgtagaac taaatgtgct ttttttcaca tgtcagtgtg tgagactgat cctttctgtt aattcttcag atattgtgtt tacaaaattc taataataat actgactgat ggcc tggaac aatatttagc ttgcatagct agtgcagctg gcttgtttta tttctttggg tatgttcaaa caaaattcct cacagaggat tctaaataaa cctttaattc tgattttggt ttctattaca ccttacatag ttgagctctt aaaggaagca cctttatttt tgtggaaaga ccaacaaatg aataataata tgacctgatt tCCtgCtCCC cattggaaac agctgcaggc ttgtataact tcattgaaac ttttttgaga ttgtgctatg gtttacatat tatctgtaaa taaaattacc caacttgcta aagcttgtta tttatgtgcc gataagttac aaattgtgtt CCttttCCCC ctactgaatt ctaatgttta aattataata gattgattct tctgcctcag atttcctgag attttgaagc agctgcaggc agtttttaag gatcatttcc gaacacattc tagaaggaaa tcttactctt attctgagtt agagttcttg aaatgctccc tttattaatt agaaaaagaa tcttgtgttt ctcattcctg ttgagaaagt tcacttaaac aattattatt ttggcaaagt cagggt tgac aatgtggagc ctgtcttgtg attacacaac taagc taaaa aacatitactt gaatttagaa cagacagtat aatatcatat tttgaaatgc ttattttaga atatttttat tatttactga 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 4560 tccaggttga gttcaaactt ctaatgtagc AMENDED SIHEET fPEAfAU PCT/AUOO/00864 Received 30 October 2001 caaggctagt gtgtaccaac cccctttagg cttaactcct actcagtgtt ttggatgttg ccctttgttc tagacatcat caaatc taca attttcaggt taagctttac tgttgctttg caaggtcagt aatcacactg ggaacacaca ttcaataagc cagagaataa aatatggcca tgaattttca cttgtactcc ctgtttgtct agctgagatt cagccatctc attctcagtc tttgctttca aatgactgga ttataaacca gataattctt aaaattctcc tgctactatg tcaggtaagt tatacttact gaaattcctg gtctgtggtg tctcaac tga agagaggaaa ggaagataca aagtattaaa tgactccagc cgttgactgg taggagc taa tgcagcctcc ctagccagtc gcctgatctt agactccatg ggaccttctg tttctttaaa aaggaaaaag gagtgatcat gacttattct gaccactgtg ttctctggga tacacaggag gatgtcaggg tttcaaagta gtgcctccca tggaaagttt ttctgcctcc agcaggagtt ctcctgtcct tgctgatccc tattcttagg cactc t ttc t tttccctttc tgaaggggt t tgttttttga gcacggaacc ggtcctcact ccaaattatg acccaggcat acttaggttc tcttggcttg acataaatct ggaacctcaa ccatgaccta ctgaagtaag ctagcactgg acacaggtgg c tagaagagc agtctgtccc gagcaaaatc gtttcttgtt atctagtctt tgcaatgggt gattggcgtc atctcccctg gtagctgtaa tgacactttg tgtgggaagg tagc tggaag gcatctgtga ccttggggaa aggtgaggac gtttagttac aaatttatag aaatataaaa ttatgaggtg aacaattgat catccttggc tatgaatagc tcttcattgg c tgtgagcat gaatacaagc tcatatttat agtcttgtgc ttgctctttc tccacattca gctctggaga gtgcagtgaa gaagatgaca ctgttcaagg agggaagagt caggctaaac gattttagtg 4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220 5280 5340 5400 5460 5520 5580 5640 5700 5760 ccacaatgtc agaatagtgc aatacaatct tgcactgtcc tcttaagtat ttgaagtcat 48 1PEA/AU Received 30 October 2001 cctttagtgc aatgtgtctg caccgtatat actacctaca caaaagttct cacagcaatc 5820 tcaattatca cctactgtag atagaagtgc aggcccacgg cagattcaca gccatgatcc aagacctatg tctgaatacc ctagctcggt tctgttctgt ggatcccatc t tgac ttc tg tcagttggaa atatgctggc aacccagaac gtggagtgga gtgtctgatg tgcacagaga cgtatgtttt gtggctatgg ggctgggtgt tcaatggtca acttcctggg tgggaggcc t cctcacagct attgtattct CtgCttgCCC caagtaactg ttgacaacca tgaatattat ccaagcacat actggagccc ggtcagatat acttgttgtg tgagtaatgt gagattccag acagaatttc gtcgtcagag cacaaagttt gagatgaaag cagt tcca agc c ttag gagc ctac gcaa gaca ggat attg ggag tgag gccai tacac gacc gatc ctaai cacc ttCCt gcagt aggtgt ccctacagag aaagct cagaaagtga ctactg acagtgagca ttaaag gcccatcaga tcagga gggtgtgcca atttta gcaacaaaga aactgg attggagttg ttcgcc caggccttct gagctg gtaagcaatg ccttga gatagagagt acatag ggaatgtgag acagtc aagaaacttt ttttac tggatacctg gaagga agcaactgtt ctcagt tacaccctcc atctga aacaaagaga tgagat acaaggcgaa ::ttgga agtggacaga ttctat tatgttcatg :gtcat gtgaagccaa 49 AMENDED SHEET I PErAAU tgcttgctgc tatagaagtg cctgagagag tcagttagga aaacgagaga cagaacagat aaaataaatg gcatggcaca ggcagggat t tacagatgag tgtgtgccat ctctcatgaa gtggtcatca aataactgca tgttatctct cacatgtatt ttttgattcc caacactgag tgttgtgata ttgtactggg tggagcaatc atatagtgtt aatgggacac aagctatcat agacctgctt cccagtcaac tttttatttt agaggcccaa ggtttgtctg gcctgaggaa ttgctgatgc gtgttcatag gtgttttccc atgggaggtt agaggaatct cttggtcttt tggattggcc tataacaaca tgtgtgagtt aaggaagaaa 5880 5940 6000 6060 6120 6180 6240 6300 6360 6420 6480 6540 6600 6660 6720 6780 6840 6900 6960 7020 Received 30 October 2001 aaagaaaatg acagccaatc gtttattggc ctaatgatgg tgttgataaa aaatcccaga tgtctgcacc tcatcatcac atcacagact gtccccatat tggctgcagt caggattcac gtaggtgtaa tcagtggaga tactgttttt aacaacaacg ctcaacagct gacgcttttt tcaaaccaga aacccttgca cccagaaaac tgtcattttc tgtctgggag tgtcctgtga aagccactgc tgttcaggaa tgagctccca cagtcacatc ataaaatgcc tcatttgaac catggacaaa aactaatgca ctggtgagat ggtttctttt ggatcagcag atagcctcca agcctgttat tagagaatat tggaggtgtg tccacattcc aaaacttcca tagctattgt gggatataga agtatctgca agcagagact aaacagttct catcac tgaa taaaaacaga atggcagcga tgacaaagga ggtgattcct ttcattgcag caggattccc taccaggatc ctgccaaact ctgtgggtgc ttcttaacat gctcagaggt cacaaactta cttagtgttg ttgctctggc cagagcctta aggctgagat ctaagtacat tgggtttgag gcccttagag attgaaaatc gcaccagccc gtggtgtaca cagggacttt gagcaaatgc atccggggag tattcacttc cctttttttc tactctttcc cagaaatgaa agagattgtg aatgcttcag ttttactcaa tccaacttaa gatgggcagt tcagctttcc ttggaacctc ctgttttcct ccatttatta acccttagtc aatgccttgt aatcctgaga ttgattcata agttctgggt aggaaagatt ggatgtggat cttcctagca cctatggtcc aaccatcatt tttacatgca aggttttcct aatctttact acatttctgt gggggctctg actatttgca ctctaaagtc gggatggtaa agaagtgggc gggtcac tca acacacatta atatgagaca t taccaaaaa tctgcaggct tgcctacctg ctgtagcaag tttaccaaga caaagtgcta tcatttcatg 7080 7140 7200 7260 7320 7380 7440 7500 7560 7620 7680 7740 7800 7860 7920 7980 8040 8100 8160 8220 cagagattgt tcagtggtta aaatcactga ctactcttcc gaaggtcctg agttcacatc AMENDED SHEET

IPEA/AU

tgagcaacca cagctacact accccacact tcttcatcct atggagaaat aattaataaa cttgtatggt catggtggct gtactttata gatgagtccc ttcttctccc gggggaaggg aaaataaaac tcgtatatat cacaaacatc ctctgcaatt aaaaaggacg tcttggtctt aaggggagag caaaatacta ttaccccacc cgtaatgaga taaagcatga aaataacagg tttaaagacc ggacatgatt catttgtacg tgtgctcgag tcttctgagg gggacatagg ctaaaaagcc aggtcgctga gggggagggg gac tt catt t ca FU I /AUUU/00864 Received 30 October 2001 tgtatgaaaa 8280 agagttagct 8340 tctcttgaac 8400 ggagaaagag 8460 agggaaggga 8520 atgcttattg 8580 8622 <210> <211> <212> <213> 38

DNA

Artificial <220> <223> antisense primer <400> 38 gtggttgctc agatgtgaac <210> <211> <212> <213> 39 22

DNA

Artif icial AM E NDE D S H1E7

IPEA/AU

PCT/AUOO/00864 Received 30 October 2001 <220> <223> antiense primer <400> 39 ttcacacatc ccagaagagg ac <210> <211> 207 <212> PRT <213> Mus musculus <400> 4 Met Cys 1 Pro Gin 0 Val Thr Lys 5 Giu Val Giu Ala Ser Leu Pro Leu Ser Pro Thr Gly Ser Val Gly Lys Pro Ile 25 Lys Gin Gly Lys Thr Ile Ser Glu Ser Cys Ala 40 Val1 Leu Tyr Cys Tyr Val1 Arg is Gly Tyr Gly Val Aia Leu Ser Ile Met Val Leu Ala Thr Lys Thr Pro Gin Asn Trp Tyr Pro Ser Asn Thr Val Ile Ala Leu Giu Ile Trp Gin 70 Gly Pro Val Asn Lys 75 Cys Tyr Aia Aia Cys Giu Val Giu Asn Lys 90 Phe Tyr Phe Ser Thr Phe Aia Gin Ala Phe Cys Met Aia Gin Giu AMEND2LiD SE-i

IPEAVAU

A 1 1 ,rJ'J,1J'Ju'Jrv Received 30 October 2001 Ala Gin Leu 115 Arg Tyr Lys Ala Arg Phe Asp Asn 120 Ser Asp Giu Leu Asn 125 His 110 Phe Leu Met Arg Glu Ser Ala Asn Phe 130 Ser Glu Asp 135 Trp Trp Ile Gly Leu 140 Glu His Pro Trp 145 Ile Lys 150 Thr Asp Asn Thr 155 Tyr Tyr Asn Asn Thr 160 Pro Ile Arg Ile Ser Ser Leu Asn Ser 195 Thr 180 Tyr Gly Giu 165 Arg Ile Ser Leu Giu Arg Phe Tyr Ser Leu 185 His Cys Gin 200 Ala 170 Arg Leu Asn Asn Asn Gly 175 Ser Lys Met Trp Ile Cys 190 Pro Ser Thr Pro Phe Phe 205 <210> <211> <212> <213> 41 218

PRT

Mus musculus <400> 41 Met Pro Asp Cys Leu Giu Thr Gly Giu Lys Leu Phe Val His Asn Met 1 5 10 Asn Ala Gin Cys Val Gin Lys Pro Giu Glu Gly Asn Gly Pro Leu Gly 25 Thr Gly Asp Lys Ile Leu Gin Arg Lys Ser Leu Arg Ala Ile Ser Pro 40 53 AMEN.D SHEET

IPENVAU

Received 30 October 2001 Giu Ser Ser Ala Lys Leu Tyr Cys Cys Tyr Gly Val Ile Met Val Leu Thr Val Ala Val Val Ala 70 Leu Ser Val Ala Leu 75 Ser Val Thr Lys Thr Glu Gin Ile Leu Ile Asn Lys Thr Tyr Ala 90 Ala Cys Pro Lys Asn Trp Ile Gly Val Trp Thr Phe 115 Asn Lys Cys Phe Tyr 105 Phe Ser Giu Tyr Thr Ser Asn 110 Gin Leu Ala Ala Gin Thr Phe Met Ala Gin Giu Ala 125 Arg Phe 130 Asp Asn Glu Lys Glu 135 Leu Asn Phe Leu Arg Tyr Lys Ala Asn 145 Trp Phe Asp Ser Trp Lys Trp Thr Asp 165 Ile 150 Giy Leu His Arg Giu 155 Ser Ser Giu His Pro 160 Asn Thr Giu Tyr Asn 170 Asn Met Ile Pro Ile Gin 175 Gly Val Glu Arg His Tyr 195 Thr 180 Cys Ala Tyr Leu Gly Asn Gly Ile Ser Ser Ser 190 Asn Asn Tyr Ile Pro Arg Ile Trp 200 Ile Cys Ser Lys Leu 205 Ser Leu 210 His Cys Pro Thr Pro 215 Val Pro Val <210> 42 <211> 217 <212> PRT <213> Mus musculus AMEND&

IPENAA

PCT/AUOO/00864 Received 30 October 2001 <400> 42 met Pro A~ 1 Asn Ala G sp Cys Leu 5 ln Cvs Val Giu Thr Gly Glu Lys 10 Giu Leu Phe Val His Gin Lys Pro Giu 25 Lys Gly Asn Gly Pro Ile Asn Met Leu Giy Ser Thr Thr Gly Giy Vai Ser Pro Lys Ile Val Gin Giy 40 Cys Cys Phe Arg Ile Ile Vai Lys Leu Thr Val Tyr 55 Leu Cys Tyr Gly Val1 Ser Met Vai Leu Aia Val Ile Ser Val Ala Glu Leu 75 Ala Thr Lys Lys Thr Gin Ile Ile Ile Asn Lys Thr Tyr Aia 90 Phe Cys Ser Lys Asn Trp Thr Giy Vai Trp Thr Phe Arg Phe Asp Giy 100 Ala Lys Cys Phe Ser Giy Tyr Gin Ala Phe Cys 120 Leu Ala Gin Giu Ala 125 Arg Pro Arg Asn 110 Gin Leu Ala Phe Lys Gly Asn Giu Giu 130 Asp Phe Giu 135 Gly Ile Phe Leu Lys 140 Ser Asp Cys Trp 145 Trp Ile 150 Asn Leu His Arg Giu 155 Asn Ser Giu His Lys Trp Thr Asn 165 Tyr Thr Giu Tyr Gly Val Gly Arg Ser Tyr Ala Tyr Leu Ser 185 Ile Asn 170 Ser Cys Met Asn Pro Ile Leu 175 Asp Arg Ile Ser Lys Leu Ser Ser Ser 190 Asn Asn Tyr Asn Arg Met Trp AMENDE-Ep 'SHEED' IPEAu Received 30 October 2001 195 200 Asn Leu His Cys Gin Thr Pro Pro Val 210 215 <210> 43 <211> 27 <212> DNA <213> Artificial <220> <223> antisense primer <400> 43 ctctgctcag cccaatccag tgatcag <210> <211> <212> <213> 44 820

DNA

Homno sapiens <400> 44 gcagtattat gtctgtattt tgcattcaaa agagcattct tgtttctgac aatcatagtg gccatcaaga gccatcagta aattgttaaa atgtttttca ataatttttt ccaggttgtc attaaagcta ccttaatttg gcgcttattt ttcttaatca tgtggaatgg ttgctgcttt aagcgcaata agagctaact tgtcttcaag ctgcatgccc agaaagctgg attggttttc 56 AMENWDD~f

IPENAL

120 180 240 PCT/AUOO/00864 Received 30 October 2001 aaagaaagtg tttctatttt tctgatgaca ccaagaactg gacatcaagt cagaggtttt 300 gtgac tcaca tgagatataa ggaaatggat gtgcctattt tttgttccaa aagcatattg acaggtatat tagtaaaata atacccgaga agatgctgat aggcccatct aaatggtact gaatgacaaa atcagatata gaactgataa gaaaatatgc ttacctgttt ctgagtaatt cttgctcagg gatcactgga gaa tggacaa ggtgccagta catgtctaga ctccatttta tcaatatcac tcatggtgct tataaataaa ttgaaagctt ttgggctgag gacagtttcc gtgccaggca tgttacagca aaatgagcaa taataactgg aatattacct gagatttaat ccaggaactg cagagaacaa tatcctggga ctacacaaag aagccccaac agaatttatt gaaaatacaa gttctcccac aatttcctgt ggccaaccat gcaggagagt aggaagtgga taatctttag tcttatacca atcaaaatca tgctaatgac 360 420 480 540 600 660 720 780 820 <210> <211> <212> <213> 845

DNA

Homo sapiens <400> atagaaactg gaggcaaaat gcatgacagt aacaatgtgg agaaagacat tacaccatct gaattgcctg caaacccagg ttgtctgcat tcaaaagagc attctattaa agctacctta atttggcgct tatttttctt aatcatgttt ctgacaatca tagtgtgtgg aatggttgct gctttaagcg caataagagc taactgccat caagagccat cagtatgtct tcaagctgca 120 180 240 A ME N D S H E

IPEA/AUJ

r4- 1 Received 30 October 2001 tgcccagaaa gctggattgg ttttcaaaga aagtgtttct atttttctga tgacaccaag 300 aactggacat agcttccagg c tgagcagag tttcctatcc aggcactaca cagcaaagcc agcaaagaat actgggaaaa tacctgttct ttaat caagtcagag aactgaattt aacaaggcca tgggagcagg caaagaggaa ccaactaatc ttatttctta tacaaatcaa cccactgcta gttttgtgac cctgttgaga accatggaaa agagtgtgcc gtggatttgt tttagaagca taccaacagg aatcatagta atgacatacc tcacaagatg tataaaggcc tggataaatg tatttgaatg tccaaatcag tattggaact tatatgaaaa aaatattacc cgagactgag ctgatcttgc catctgatca gtactgaatg acaaaggtgc atatacatgt gataactcca tatgctcaat tgttttcatg taatttataa tcaggttgaa ctggattggg gacaagacag cagtagtgcc ctagatgtta ttttaaaatg atcactaata gtgctaatat ataaagagat 360 420 480 540 600 660 720 780 840 845 <210> <211> <212> <213> 46 937

DNA

H-omo sapiens <400> 46 gatggaatta ctagaaggct ttatcatagg tcctaggaca aactagaaat gatgaaatag taaagaaaaa gatatataaa atcttacaga aactggaact cagtcctaat gcaacttcat ttctatttga taaaggcaat agctgtccaa tctggaactt atttcttaca ggttgtgtgc attcaaaaga gcattctatt aaagctacct taatttggcg cttatttttc ttaatcatgt 58 AMENDED SHEET

IPEA!U

120 180 240 rk- I itiuuuiuvaoq Received 30 October 2001 ttctgacaat catagtgtgt ggaatggttg ctgctttaag tgcaataaga atcaagagcc gaaagtgttt ac tcacaaga gatataaagg aatggataaa cctatttgaa gttccaaatc catattggaa ggtatatgaa taaaatatta cccgagactg atcagtatgt ctatttttct tgctgatctt cccatctgat tggtactgaa tgacaaaggt agatatacat ctgataactc aatatgctca cctgttttca agtaatttat cttcaagctg gatgacacca gctcaggttg cactggattg tggacaagac gccagtagtg gtctagatgt cattttaaaa atatcactaa tggtgctaat aaataaagag catgcccaga agaactggac aaagcttcca ggctgagcag agtttcctat ccaggcacta tacagcaaag tgagcaaaga taactgggaa attacctgtt atttaat aagctggatt atcaagtcag ggaactaaat agaacaaggc cctgggagca cacaaagagg ccccaactaa atttatttct aatacaaatc ctcccactgc gctaactgcc ggttttcaaa aggttttgtg ttcctgttga caaccatgga ggagagtgtg aagtggattt tctttagaag tataccaaca aaaatcatag taatgacata 300 360 420 480 540 600 660 720 780 840 900 937 <210> <211> <212> <213> <220> <223> <400> 47 28

DNA

Artificial sense specific primer 47 59 AMENDED SH-EE-

IPEAIAU

Received 30 October 2001 gctgatcttg ctcaggttga aagcttcc 28 <210> 48 <211> 18 <212> PRT <213> Artificial <220> <223> pepetide epitope recognised by antibody MOCIL-3 <400> 48 Cys Val Thr Lys Ala Ser Leu Pro Met Leu Ser Pro Thr Gly Ser Pro 1 5 10 Gln Glu <210> 49 <211> 16 <212> PRT <213> Artificial <220> <223> peptide epitope recognised by antibody MOCIL-RP-1 <400> 49 AMENDE: r.

IPEVAAU

PCT/AUOO/o0864 Received 30 October 2001 Cys Val Gin Lys Pro Glu Glu Gly Asn Gly Pro Leu Gly Thr Gly Asp 1 5 10 <210> <211> 28 <212> DNA <213> Artificial <220> <223> sense primer <400> tcagaattca cctatgctgc ttgcccgc 28 <210> 51 <211> 32 <212> DNA <213> Artificial <220> <223> antisense pimer <400> 51 ggttaagctt caggctaaaa agcgtctctt gg 32 <210> 52 61 AMENDED, sxEi

IPEVAL

PCT/AUO0086 Received 30 October 2001 <211> 29 <212> DNA <213> Artificial <220> <223> sense primer <400> 52 tcagaattca cctatgctgc ttgcccgaa 29 <210> 53 <211> 32 <212> DNA <213> Artificial <220> <223> antisense primer <400> 53 ggttaagctt gggaccatag gggaaaaagt ag 32 <210> 54 <211> 29 <212> DNA 62 AMEWoEL) SliEET1

IPEA,'A'J

rk- I II1UUU/UU54 Received 30 October 2001 <213> Artificial <220> <223> sense primer <400> 54 tcagaattca cctatgctgc ttgctcaaa <210> <211> <212> <213> 26

DNA

Artif icial <220> <223> sense primer <400> gcggaattcc ttcaagctgc atgccc <210> 56 <211> 31 <212> DNA <213> Artificial 63 AMENDED cIHC-j

IP&-A'AU

PCT/AUOO/00864 Received 30 October 2001 <220> <223> antisense primer <400> 56 cctgggatcc gctttgctgt aacatctaga c AM2~:~ ED SHEET Ii~EA!A!1

Claims (43)

1. An isolated nucleic acid molecule which comprises a sequence encoding a protein which inhibits osteoclast differentiation from haematopoietic cell precursors, selected from the group consisting of osteoclast inhibitory lectin (OCIL) and OCIL-related protein, and which is either able to hybridize under conditions of moderate to high stringency to one or more sequences selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46 or selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 21, and SEQ ID NO: 37; or (ii) has greater than 80% sequence identity with one or more of the sequences set out in

2. A nucleic acid molecule according to claim 1, which encodes a type II membrane protein.

3. A nucleic acid molecule according to claim 1 or claim 2, which is expressed at least by osteoblasts.

4. A nucleic acid molecule according to any one of claims 1 to 3, which is of human, mouse or rat origin. A nucleic acid molecule according to any one of claims 1 to 3, which is a cDNA.

6. A nucleic acid molecule according to claim 5, in which the cDNA comprises a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46.

7. A nucleic acid molecule according to any one of claims 1 to 4, which is a gDNA.

8. A nucleic acid molecule according to any one of claims 1 to 4, in which the gDNA comprises a sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID \\elbtiles\homeS\evonnoe\Kaep\Speci\PCT-AUOO-00864.doc 24/09/01 AMENDED SHEE' IPEA/AU Received 24 September 2001 59 NO: 21, and SEQ ID NO: 37, or which hybridises to said nucleic acid molecule under stringent conditions.

9. A nucleic acid molecule according to any one of claims 1 to 8, which encodes an extracellular domain of an OCIL or of an OCIL-related protein. A nucleic acid molecule according to any one of claims 1 to 9, which encodes a protein which inhibits differentiation of haematopoietic stem cells to osteoclast progenitor cells.

11. A nucleic acid molecule according to any one of claims 1 to 10, which comprises a 110 base pair sequence as set out in SEQ ID NO: 2.

12. An anti-sense sequence directed against a nucleic acid molecule according to any one of claims 1 to 11.

13. An anti-sense sequence according to claim 12, directed against SEQ ID NO:

14. An anti-sense sequence according to claim 12 or claim 12, which is SEQ ID NO: 24 or SEQ ID NO: An isolated polypeptide encoded by a nucleic acid molecule according to any one of claims 1 to 11.

16. A polypeptide according to claim 15, which is encoded by the human cDNA or gDNA sequence.

17. A polypeptide according to claim 16, which is encoded by the mouse cDNA or gDNA sequence.

18. A polypeptide according to claim 17, comprising a sequence selected from the group consisting of SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42.

19. A polypeptide according to claim 15, which comprises an amino acid sequence encoded by SEQ ID NO:

20. An isolated polypeptide selected from the group consisting of a C-lectin motif, an extracellular domain, a transmembrane domain, or a cytoplasmic domain of a polypeptide according to any one of claims 15 to 19.

21. An antibody directed against a polypeptide according to any one of claims 15 to H:\evonnee\Keep\Speci\PCT-AU00-00864.doc 24/09/01 AMENDED SHEL. IPEA/AU I A I r-l;JVIJVJO'JT Received 24 September 2001 60

22. An antibody according to claim 21, which is directed against an epitope present in a sequence selected from the group consisting of Cys-Met-Ala-Gln-Glu-Ala-Gln-Leu-Ala-Arg-Phe-Asp-Asn-Gln- Asp-Glu-Leu-Asn-Phe (SEQ ID NO: 26). Cys-Val-Thr-Lys-Ala-Ser-Leu-Pro-Met-Leu-Ser-Pro-Thr- Gly- Ser-Pro-Gln-Glu (SEQ ID NO: 48), and Cys-Val-Gln-Lys-Pro-Glu-Glu-Gly-asn-Gly-Pro-Leu-Gly-Thr- Gly-Asp (SEQ ID NO: 49).

23. An antibody according to claim 21 or claim 22, which is monoclonal.

24. A composition comprising a polypeptide according to any one of claims 15 to 20, together with a pharmaceutically-acceptable carrier.

25. A composition comprising an antibody according to any one of claims 21 to 23, together with a pharmaceutically-acceptable carrier.

26. A method of treatment of a condition characterised by abnormal bone resorption, comprising the step of administering an effective amount of a modulator of expression or function of a polypeptide according to any one of claims 15 to

27. A method according to claim 26, in which the condition involves excessive bone resorption, and the method comprises administration of a polypeptide according to any one of claims 15 to 19, or a nucleic acid encoding this polypeptide, or encoding a biologically-active fragment or analogue thereof.

28. A method according to claim 27, in which the condition is selected from the group consisting of osteoporosis, primary hyperparathyroidism, Paget's disease, rheumatoid arthritis, renal osteodystrophy, humoral hypercalcaemia of malignancy, and conditions where cancer has metastasised to bone.

29. A method according to claim 26, in which the condition involves deficient bone resorption, and the method comprises administration of an antibody according to \\mlb_ile\homS\evonnee\Keep\Spec\PCT-AUOO-00864.doc 24/09/01 AMENDED SHEEt IPEA/AU r% I UUIVVUUOUt Received 24 September 2001 61 any one of claims 21 to 23 or an anti-sense oligonucleotide according to any one of claims 11 to 13. A method according to claim 28, in which the condition is osteopetrosis.

31. A method of promoting healing of bone fractures, particularly in an individual in whom fracture healing is delayed or deficient, comprising the step of administering an effective amount of a polypeptide according to any one of claims 15 to

32. A method according to claim 31, in which the individual is suffering from osteoporosis or diabetes mellitus.

33. A method of modulating breast and/or lymph node development, comprising the step of administering an effective amount of a modulator of expression or function of a polypeptide according to any one of claims 15 to 20 to a subject in need of such treatment.

34. A diagnostic kit for detection of abnormalities in the structure, expression or control of a type II membrane polypeptide expressed on the osteoblast cell surface, selected from the group consisting of osteoclast inhibitory lectin (OCIL) and OCIL-related protein, comprising a reagent selected from the group consisting of a nucleic acid according to any one of claims 1 to 11, or a fragment thereof capable of hybridising to a nucleic acid according to any one of claims 1 to 11; an anti-sense nucleic acid according to any one of claims 12 to 14; a polypeptide according to any one of claims to 20, and an antibody according to any one of claims 21 to 23. A diagnostic kit according to claim 34, in which the reagent is labelled with a detectable marker.

36. A method of screening of candidate agents for treatment of a condition characterised by abnormal bone H:\evonee\Keep\Speci\PCT-AUOO-00864.doc 24/09/01 AMENDED SHEET IPEA/AU 62 resorption, comprising the step of assessing the ability of each agent to modulate expression or function of a polypeptide according to any one of claims 15 to 19.

37. An oligonucleotide primer selected from the group consisting of sense primers having the sequence set out in SEQ.ID. NO:5, 6, 30, 35, 13, 14, 16, 18, 27, 47, 52, 54, or 55, and antisense primers having the sequence set out in SEQ.ID. NO: 3, 31, 32, 14, 28, 34, 38, 39,51, 53, 22, 23, 24, 25, 43 or 56.

38. A polypeptide according to claim 18, comprising the sequence set out in SEQ ID

39. An isolated polypeptide according to any one of claims 15 to 20, whose expression is differentially regulated by PTH or PTHrP.

40. A method according to claim 27 which comprises administration of a polypeptide according to claim 15, or a nucleic acid encoding this polypeptide, or encoding a biologically-active fragment or analogue thereof. S 20 41. A method according to claim 29, which comprises administration of an antibody according to claim 21.

42. A method according to claim 29, which comprises administration of an anti-sense oligonucleotide according to claim 11.

43. A diagnostic kit for detection of abnormalities in the structure, expression or control of a type II membrane polypeptide expressed on the osteoblast cell surface, selected from the group consisting of osteoclast inhibitory lectin (OCIL) and OCIL-related protein, comprising an anti-sense nucleic acid according to claim 12.

44. A diagnostic kit according to claim 43, in which the anti-sense nucleic acid is labelled with a detectable marker. A diagnostic kit for detection of abnormalities in the structure, expression or control of a H:\cintae\Keep\speci\57975.OO.doc 2/02/05 63 type II membrane polypeptide expressed on the osteoblast cell surface, selected from the group consisting of osteoclast inhibitory lectin (OCIL) and OCIL-related protein, comprising a polypeptide according to claim

46. A diagnotic kit according to claim 45, in which the polypeptide is labelled with a detectable marker.

47. A diagnostic kit for detection of abnormalities in the structure, expression or control of a type II membrane polypeptide expressed on the osteoblast cell surface, selected from the group consisting of osteoclast inhibitory lectin (OCIL) and OCIL-related protein, comprising an antibody according to claim 21.

48. A diagnotic kit according to claim 47, in which the antibody is labelled with a detectable marker.

49. An isolated nucleic acid molecule according .to claim 1, substantially as herein described with reference to the examples and drawings.

50. An oligonucleotide primer according to claim 37, substantially as herein described with reference to the 20 examples and drawings. Dated this 2nd day of February 2005 ST VINCENT'S INSTITUTE OF MEDICAL RESEARCH By their Patent Attorneys 25 GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia H:\cintae\Keep\speci\57975.00.doc 2/02/05