AU731968B2 - A novel haemopoietin receptor and genetic sequences encoding same - Google Patents

Description

67074.599 A NOVEL HAEMOPOIETIN RECEPTOR AND GENETIC SEQUENCES ENCODING SAME The present invention relates generally to a novel haemopoietin receptor or derivatives thereof and to genetic sequences encoding same. Interaction between facilitates proliferation, differentiation and survival of a wide variety of cells. The novel receptor and its derivatives and the genetic sequences encoding same of the present invention are useful in the development of a wide range of agonists, antagonists, therapeutics and diagnostic reagents based on ligand interaction with its receptor.

Bibliographic details of the publications numerically referred to in this specification are collected at the end of the description. Sequence Identity Numbers (SEQ ID NOs.) for the nucleotide and amino acid sequences referred to in the specification are defined following the bibliography.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

The rapidly increasing sophistication of recombinant DNA techniques is greatly facilitating research into the medical and allied health fields. Cytokine research is of particular importance, especially as these molecules regulate the proliferation, differentiation and function of a wide variety of cells. Administration of -o ICIoOS~ 2 recombinant cytokines or regulating cytokine function and/or synthesis is becoming increasingly the focus of medical research into the treatment of a range of disease conditions.

Despite the discovery of a range of cytokines and other secreted regulators of cell function, comparatively few regimens. One reason for this is the pleiotropic nature of many cytokines. For example, interleukin (IL)-11 is a functionally pleiotropic molecule initially characterized by its ability to stimulate proliferation of the IL-6-dependent plasmacytoma cell line, T11 Other biological actions of IL-11 include induction of multipotential haemopoietin progenitor cell proliferation enhancement of megakaryocyte and platelet formation stimulation of acute phase protein synthesis (11) and inhibition of adipocyte lipoprotein lipase activity (12, 13).

Other important cytokines in the IL-11 group include IL- 6, leukaemia inhibitory factor (LIF), oncostatin M (OSM) and CNTF. All these cytokines exhibit pleiotropic properties with significant activities in proliferation, differentiation and survival of cells. Members of the haemopoietin receptor family are defined by the presence of a conserved amino acid domain in their extracellular region. However, despite the low level of amino acid sequence conservation between other haemopoietin receptor domains of different receptors, they are all predicted to assume a similar tertiary structure, centred around two fibronectin-type III repeats (18,19).

The size of the haemopoietin receptor family has now become extensive and includes the cell surface receptors for may cytokines including interleukin-2 IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-11, IL-12, IL-13, IL- AMENDED

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granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage-CSF (GM-CSF), erythropoietin, thrombopoietin, leptin, leukaemia inhibitory factor, oncostatin-M, ciliary neurotrophic factor, cardiotrophin, growth hormone and prolactin. Although most of the members of the haemopoietin receptor family act as classic cell surface receptors, binding their intracellular signal transduction, some receptors are also produced in naturally occuuring soluble forms.

These soluble receptors can either act as cytokine antagonists, by binding to cytokines and inhibiting productive interactions with cell surface receptors (eg LIF binding protein; (20) or as agonists, binding to cytokine and potentiating interaction with cell surface receptor components (eg soluble interleukin-6 receptor a-chain; Still other members of the family appear to be produced only as secreted proteins, with no evidence of a cell surface form. In this regard, the IL-12 p40 subunit is a useful example. The cytokine IL- 12 is secreted as a heterodimer composed of a subunit which shows similarity to cytokines such as IL-6 (22) and a p40 subunit which shares similarity with the IL-6 receptor a-chain In this case the soluble receptor acts as part of the cytokine itself and essential to formation of an active protein. In addition to acting as cytokines (eg IL-12p40), cytokine agonists (eg IL-6 receptor a-chain) or cytokine antagonists (LIF binding protein), members of the haemopoietin receptor have been useful in the discovery of small molecule cytokine mimetics. For example, the discovery of peptide mimetics of two commercially valuable cytokines, erythropoietin and thrombopoietin, centred on the selection of peptides capable of binding to soluble versions of the erythropoietin and thrombopoietin receptors (24,25). Due to the importance and multifactorial nature of these cytokines, there is a AIMAENDEO SHEET 4 need to identify receptors, including both cell bound and soluble, for pleiotropic cytokines. Identification of such receptors permits the identification of pleiotropic cytokines and the development of a range of therapeutic and diagnostic agents.

Accordingly, one aspect of the present invention relates nucleotides encoding or complementary to a sequence encoding a novel haemopoietin receptor or a derivative thereof.

More particularly, the present invention provides a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a novel haemopoietin receptor or a derivative thereof having the motif: Trp Ser Xaa Trp Ser [SEQ ID NO:1], wherein Xaa is any amino acid and is preferably Asp or Glu.

Even more particularly, the present invention is directed to a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a novel haemopoietin receptor or a derivative thereof, said receptor comprising the motif: Trp Ser Xaa Trp Ser [SEQ ID NO:1] wherein Xaa is any amino acid and is preferably Asp or Glu, said nucleic acid molecule is identifiable by hybridisation to said molecule under low stringency conditions at 42 0 C with (A/G)CTCCA(A/G)TC(A/G)CTCCA 3' [SEQ ID NO:7] and (A/G)CTCCA(C/T)TC(A/G)CTCCA 3' [SEQ ID NO:8].

AMENDED SHEET 5 Still more particularly, the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in SEQ ID NO:12 or a nucleotide sequence having at least similarity to the nucleotide sequence set forth in SEQ ID NO:12 or a nucleotide sequence capable of hybridising thereto under low stringency conditions at 42 0 C and haemopoietin receptor or a derivative thereof.

In a related embodiment, the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in SEQ ID NO:14 or a nucleotide sequence having at least similarity to the nucleotide sequence set forth in SEQ ID NO:14 or a nucleotide sequence capable of hybridising thereto under low stringency conditions at 42 0 C and wherein said nucleotide sequence encodes a novel haemopoietin receptor or a derivative thereof.

In another related embodiment, the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in SEQ ID NO:16 or a nucleotide sequence having at least 60% similarity to the nucleotide sequence set forth in SEQ ID NO:16 or a nucleotide sequence capable of hybridising thereto under low stringency conditions at 42 0 C and wherein said nucleotide sequence encodes a novel haemopoietin receptor or a derivative thereof.

In a further related embodiment, the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in SEQ ID NO:18 or a nucleotide sequence having at least 60% similarity to the nucleotide sequence set forth in SEQ ID NO:18 or a nucleotide sequence capable of hybridising thereto under low stringency conditions at 1 AMENDED SHEET 6 42 0 C and wherein said nucleotide sequence encodes a novel haemopoietin receptor or a derivative thereof.

In yet a further related embodiment, the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in SEQ ID NO:24 or a nucleotide sequence having at I-ast 60z; -z sequence set forth in SEQ ID NO:24 or a nucleotide sequence capable of hybridising thereto under low stringency conditions at 42 0 C and wherein said nucleotide sequence encodes a novel haemopoietin receptor or a derivative thereof.

Still yet a further embodiment of the present invention is directed to a sequence of nucleotides substantially as set forth in SEQ ID NO:28 or a nucleotide sequence having at least 60% similarity to the nucleotide sequence set forth in SEQ ID NO:28 or a nucleotide sequence capable of hybridising thereto under low stringency conditions at 42 0 C and wherein said nucleotide sequence encodes a novel haemopoietin receptor or a derivative thereof.

In still yet another embodiment, the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides substantially set forth in SEQ ID NO:38 or a nucleotide sequence having at least similarity to the nucleotide sequence set forth in SEQ ID NO:38 or a nucleotide sequence capable of hybridising thereto under low stringency conditions at 42 0 C and wherein said nucleotide sequence encodes a novel haemopoietin receptor or a derivative thereof.

The term "receptor" is used in its broadest sense and includes any molecule capable of binding, associating or otherwise interacting with a ligand. Generally, the AMENDED

SHEET

7 interaction will have a signalling effect although the present invention is not necessarily so limited. For example, the "receptor" may be in soluble form, often referred to as a cytokine binding protein. A receptor may be deemed a receptor notwithstanding that its ligand or ligands has or have not been identified.

or a species of bird. Particularly, preferred mammals include humans, primates, laboratory test animals (e.g.

mice, rats, rabbits, guinea pigs), livestock animals sheep, horses, pigs, cows), companion animals dogs, cats) or captive wild animals deer, foxes, kangaroos). Although the present invention is exemplified with respect to mice, the scope of the subject invention extends to all animals and in particular humans.

The present invention is predicated in part on an ability to identify members of the haemopoietin receptor family with limited sequence similarity. Based on this approach, a genetic sequence has been identified in accordance with the present invention which encodes a novel receptor. The expressed genetic sequence is referred to herein as "NR6". Different forms of NR6 are referred to as, for example, NR6.1, NR6.2 and NR6.3.

The nucleotide and corresponding amino acid sequences for these molecules are represented in SEQ ID NOs:12, 14 and 16, respectively.

Preferred human and murine nucleic acid sequences for NR6 or its derivatives include sequences from brain, liver, kidney, neonatal, embryonic, cancer or tumourderived tissues.

Reference herein to a low stringency at 42 0 C includes and encompasses from at least about 1% v/v to at least AMENDED

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8 about 15% v/v formamide and from at least about 1M to at least about 2M salt for hybridisation, and at least about 1M to at least about 2M salt for washing conditions. Alternative stringency conditions may be applied where necessary, such as medium stringency, which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide and from at last aiou: 0.5.4 co a: ieas: abou: salt f J O L.I.OUZ 0 i'4'l CO- a C -L -0 -c a uiic .j'i 5dLt L hybridisation, and at least about 0.5M to at least about 0.9M salt for washing conditions, or high stringency, which includes and encompasses from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.01M to at least about 0.15M salt for hybridisation, and at least about 0.01M to at least about 0.15M salt for washing conditions.

The nucleic acid molecules contemplated by the present invention are generally in isolated form and are preferably cDNA or genomic DNA molecules. In a particularly preferred embodiment, the nucleic acid molecules are in vectors and most preferably expression vectors to enable expression in a suitable host cell.

Particularly useful host cells include prokaryotic cells, mammalian cells, yeast cells and insect cells.

The cells may also be in the form of a cell line.

Accordingly, another aspect of the present invention provides an expression vector comprising a nucleic acid molecule encoding the novel haempoietin receptor or a derivative thereof as hereinbefore described, said expression vector capable of expression in a selected host cell.

Another aspect of the present invention contemplates a method for cloning a nucleotide sequence encoding NR6 or a derivative thereof, said method comprising searching a nucleotide data base for a sequence which encodes the AMEND 0 SH-wC, 9 amino acid sequence set forth in SEQ ID NO:1, designing one or more oligonucleotide primers based on the nucleotide sequence located in the search, screening a nucleic acid library with said one or more oligonucleotides and obtaining a clone therefrom which encodes said NR6 or part thereof.

indicated above encoding NR6, oligonucleotides may be designed which bind cDNA clones with high stringency.

Direct colony hybridisation may be employed or PCR amplification may be used. The use of oligonucleotide primers which bind under conditions of high stringency ensures rapid cloning of a molecule encoding the novel NR6 and less time is required in screening out cloning artefacts. However, depending on the primers used, low or medium stringency conditions may also be employed.

Alternatively, a library may be screened directly such as using oligonucleotides set forth in SEQ ID NO:7 or SEQ ID NO:8 or a mixture of both oligonucleotides may be used. In addition, one or more of oligonucleotides defined in SEQ ID NO:2 to 11 may also be used.

Preferably, the nucleic acid library is a cDNA, genomic, cDNA expression or mRNA library.

Preferably, the nucleic acid library is a cDNA expression library.

Preferably, the nucleotide data base is of human or murine origin and of brain, liver, kidney, neo-natal tissue, embryonic tissue, tumour or cancer tissue origin.

Preferred percentage similarities to the reference nucleotide sequences include at least about 70%, more SmFN.D)YJ SHkE'l preferably at least about 80%, still more preferably at least about 90% and even more preferably at least about or above.

Another aspect of the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding a novel haemopoietin receptor or r-i-vrti a heraoz having an amin.n acaid sequence as set forth in SEQ ID NO:13 or having at least about similarity to all or part thereof.

Still yet another aspect of the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding a novel haemopoietin receptor or derivative thereof having an amino acid sequence as set forth in SEQ ID NO:15 or having at least about 50% similarity to all or part thereof.

Even yet another aspect of the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding a novel haemopoietin receptor or derivative thereof having an amino acid sequence as set forth in SEQ ID NO:17 or having at least about 50% similarity to all or part thereof.

A further aspect of the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding a novel haemopoietin receptor or derivative thereof having an amino acid sequence as set forth in SEQ ID NO:19 or having at least about similarity to all or part thereof.

Even yet a another aspect of the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding a novel haemopoietin receptor or derivative thereof having an amino acid 9 ,AL sequence as set forth in SEQ ID NO:25 or having at least

LL-

'N______MENDED SHAEEt 11 about 50% similarity to all or part thereof.

Another aspect of the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding a novel haemopoietin receptor or derivative thereof having an amino acid sequence as set forth in one or more of SEQ ID NOs:29 or having at least Preferably, the percentage amino acid similarity is at least about 60%, more preferably at least about even more preferably at least about 80-85% and still even more preferably at least about 90-95% or greater.

The NR6 polypeptide contemplated by the present invention includes, therefore, derivatives which are components, parts, fragments, homologues or analogues of the novel haemopoietin receptors which are preferably encoded by all or part of a nucleotide sequences substantially set forth in SEQ ID NO:12 or 14 or 16 or 18 or 25 or 20 or 24 or 28 or 38 or a molecule having at least about 60% nucleotide similarity to all or part thereof or a molecule capable of hybridising to the nucleotide sequence set forth in SEQ ID NO:12 or 14 or 16 or 18 or 20 or 24 or 28 or 38 or a complementary form thereof. The NR6 molecule may be glycosylated or nonglycosylated. When in glycosylated form, the glycosylation may be substantially the same as naturally occurring haemopoietin receptor or may be a modified form of glycosylation. Altered or differential glycosylation states may or may not affect binding activity of the novel receptor.

The NR6 haemopoietin receptor may be in soluble form or may be expressed on a cell surface or conjugated or fused to a solid support or another molecule.

AMENDED SHEET -12 As stated above, the present invention further contemplates a range of derivatives of NR6. Derivatives include fragments, parts, portions, mutants, homologues and analogues of the NR6 polypeptide and corresponding genetic sequence. Derivatives also include single or multiple amino acid substitutions, deletions and/or additions to NR6 or single or multiple nucleotide SituS i- Litionis, elet2-iClA. a Ui rC.QJ CadGii>5 C he genIecJ.L.

sequence encoding NR6. "Additions" to amino acid sequences or nucleotide sequences include fusions with other peptides, polypeptides or proteins or fusions to nucleotide sequences. Reference herein to "NR6" includes reference to all derivatives thereof including functional derivatives or NR6 immunologically interactive derivatives.

Analogues of NR6 contemplated herein include, but are not limited to, modification to side chains, incorporating of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecule or their analogues.

Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH 4 amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH 4 -13 The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.

The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by corresponding amide.

Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4-chloromercuribenzoate, 4chloromercuriphenylsulphonic acid, phenylmercury chloride, 2-chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified by, for example, oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.

Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6- S v t JJ 14 aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4 -amino-3-hydroxy- 6-methylheptanoic acid, 2-thienyl alanine and/or Disomers of amino acids. A list of unnatural amino acid, contemplated herein is shown in Table 1.

These types of modifications may be important to use as a diagnostic reagent.

Crosslinkers can be used, for example, to stabilise 3D conformations, using homo-bifunctional crosslinkers such as the bifunctional imido esters having (CH2)n spacer groups with n=l to n=6, glutaraldehyde, Nhydroxysuccinimide esters and hetero-bifunctional reagents which usually contain an amino-reactive moiety such as N-hydroxysuccinimide and another group specificreactive moiety such as maleimido or dithio moiety (SH) or carbodiimide (COOH). In addition, peptides can be conformationally constrained by, for example, incorporation of Ca and N a-methylamino acids, introduction of double bonds between Ca and C3 atoms of amino acids and the formation of cyclic peptides or analogues by introducing covalent bonds such as forming an amide bond between the N and C termini, between two side chains or between a side chain and the N or C terminus.

AMENDED SHEET i5 TABLE 1 N'on-conventional amino acid Code Non-conventional amino acid Code aminobutyric acid Amino -a-methylbutyrate pzoparne carboxylate aminoisobutyric acid aminonorbornyl carboxylate cyclohexylalanine cyc lopentylalanine D-alanine D-arginine D-aspartic acid D-cysteine D-glut amine D-glutamic acid D -his tidine D-isoleucine D-leucine D-lysine D-methionine D-ornithine D -phenylalanine D-proline D-serine D-threonine D -tryptophan D-tyrosine D-valine D-cr-methylalanine D-a-methylarginine D -a -methylasparagine D-a-methylaspartate Abu Mgabu Aib Norb Cpen Dal Darg Dasp Dcys Dgln Dglu Dhis Di le Dleu Dlys Dmet Dorn Dphe Dpro Dser Dthr Dt rp Dtyr Dva 1 Dmala Dmarg Dmasn Dmasp L-N-methylalanine Nmala L-N-methylarginine Nmarg L-N-methylaspartic acid Nmasp L-N-methylcysteine Nmcys L-N-methylglutamine Nmgln L-N-methylglutamic acid Nmglu ChexaL-N'-methylhistidine Nmhis L-N-methylisolleucine Nmile L-N-methylleucine Nmleu L-N-methyllysine Nmlys L-N-methylmethionine Nmmet L-N-methylnorleucine mnnie L-N.-methylnor-valine Nmnva L-N-methylornithine Nmorn L-N-methylphenylalanine Nmphe L-N-methylproline Ninpro L-N-methylserine Nmser L-N-methylthreonine Nmthr L-N-methyltryptophan Nmtrp L-N-methyltyrosine Nmtyr L-N-methylvaline Ninval L-N-methylethylglycine N'metg L-N-methyl-t-butylglycine Nmtbug L-norleucine Nie L-norvaline Nva a-methyl-aminoisobutyrate Maib a-methyl -y-aminobutyrate Mgabu a-methylcyclohexylalanine Mchexa a-methylcylcopentylalanine Mcpen a-methyl -a-napthylalanine Manap a-methylpenicillamine Mpen 16 D-a-methylcysteine D -a-methylglutamine D -a-methylhistidine D-a-methylisoleucine D-a-methylleucine D-cx-methyllysine D -c-methylmethionine D -a-methylphenylalanine D-a-methylproline D-a-methylserine Da-methylthreonine D-ca-methyltryptophan D-cz-methyltyrosine D-cx-methylvaline D-N-methylalanine D -N-methylarginine D -N-methylasparagine D -N-methylaspartate D-N-methylcysteine D-N-methylglutamine D -N-methylglutamate D-N-methylhistidine D-N-methylisoleucine D-N-methylleucine D-N-methyllysine N-methylcyclohexylalanine D -N-methylornithine N-methylglycine N-methylaminoisobutyrate N- Ci-methyipropyl) glycine N- (2-methyipropyl) glycine D-N-methyltryptophan D-N-methyltyrosine D-N-methylvaline y-aminobutyric acid L- t-butylglycine Dmcys Dmgln Dmhis Dmile Dmleu Dmlys Dnimet Dmphe Dmpro Dmser Dmthr Dintrp Dinty Dinval Dnmal1a Dnmarg Dnmasn Dninasp Dnmcys Dnmgln Dnmglu Dnmhi s Dnmile Dnml eu Dnmlys Ninchexa Dnmorn Nal a Nmaib Nile Ni eu Dnmtrp Dnrntyr Dnmval Gabu Tbug N- (4-aminobutyl)glycine Nglu N- (2-aminoethyl)glycine Naeg N- (3-arinopropyl)glycine Norn N-amino-ci-methylbutyrate Nmaabu c-napthylalanine Anap N-benzylglycine Nphe N- (2-carbamylethyl) glycine Ngln N- (-caboxythy~glyineNgl N- (2carboxyethyl)glycine Nasp N-(ccloutglcine i Ncbu N-cyclohetylglycine Nchep N-cyclohexylglycine, Nchex N-cyclodecylglycine Ncdec N-cyclodecylglycine Ncded N-cycoocylglycine Ncot N-cyclopotylglycine Ncpo N-cycloundecylglycine Ncund N- (2,2 -diphenylethyl) glycine Nbhm N- 3-diphenylpropyl)glycine Nbhe N- (3-guanidinopropyl)glycine Narg N- (l-hydroxyethyl)glycine Nthr N- (hydroxyethyl) )glycine Nser N- (imidazolylethyl) )glycine Nhis N- (3-indolylyethyl)glycine Nhtrp N-methyl -y-aminobutyrate Nmgabu D-N-methylmethionine Dnmmet N-methylcyclopentylalanine Nmcpen D-N-methylphenylalanine Dnmphe D-N-methylproline Dnmpro D-N-methylserine Dnmser D-N-methylthreonine Dnmthr N- (l-methylethyl)glycine Nval N-methyla-napthylalanine Ninanap N-methylpenicillamine Ninpen N- (P-hydroxyphenyl) glycine Nhtyr N- (thiomethyl) glycine Ncys 17 L-ethylglycine L -homophenylalanine L -a-methylarginine L -a-methylaspartate L-a-methylcysteine L-a-methylglutamine L-a-methylhistidine L-a-methylleucine L-ca-methylmethionine L-c-methylnorvaline L -a-methylphenylalanine L-a-methylserine L-a-methyltryptophan L-a-methylvaline N- (2,2-diphenylethyl) carbamylmethyl) glycine Etg Hphe Marg Masp

MCYS

Mgln Mhis Ml eu Mmet Mnva Mphe Mser Mtrp Mval Nnbhm penicillamine Pen L-a-methylalanine Mala L-c-methylasparagine Masn L-ct-methyl- t-butylglycine Mtbug L-methylethylglycine Metg L-ca-methylglutamate MglU L-c-methylhomophenylalanine Mhphe L-ci-methyllysine Mlys L-a-methylnorleucine Mnle L-ct-methylornithine Morn L-ct-methylproline Mpro L-cx-methylthreonine Mthr L-ci-methyltyrosine Mtyr L-N'-methylhorophenylalanine Nmhphe N- (3,3-diphenyipropyl) Nnbhe carbamylmethyl) glycine 1-carboxy-l- 2-dipheftyl- Nmbc ethylamino) cyclopropane The present invention further contemplates chemical analogues of NRG capable of acting as antagonists or agonists of NRE or which can act as functional analogues of NRE. Chemical analogues may not necessarily be derived from NR6 but may share certain conformational similarities. Alternatively, chemical analogues may be specifically designed to mimic certain physiochemical properties of NR6. Chemical analogues may be chemical ly synthesised or may be detected following, for example, natural product screening.

The identification of NR6 permits the generation of a range of therapeutic molecules capable of modulating expression of NR6 or modulating the activity of NR6.

Modulators contemplated by the present invention includes agonists and antagonists of NR6 expression.

AMENDED SHEET 18 Antagonists of NR6 expression include antisense molecules, ribozymes and co-suppression molecules.

Agonists include molecules which increase promoter ability or interfere with negative regulatory mechanisms. Agonists of NR6 include molecules which overcome any negative regulatory mechanism. Antagonists of NR6 include antibodies and inhibitor peptide fragmen;-s.

Other derivatives contemplated by the present invention include a range of glycosylation variants from a completely unglycosylated molecule to a modified glycosylated molecule. Altered glycosylation patterns may result from expression of recombinant molecules in different host cells.

Another embodiment of the present invention contemplates a method for modulating expression of NR6 in a subject such as a human or mouse, said method comprising contacting the genetic sequence encoding NR6 with an effective amount of a modulator of NR6 expression for a time and under conditions sufficient to up-regulate or down-regulate or otherwise modulate expression of NR6. Modulating NR6 expression provides a means of modulating NR6-ligand interaction or NR6 stimulation of cell activities.

Another aspect of the present invention contemplates a method of modulating activity of NR6 in a human, said method comprising administering to said mammal a modulating effective amount of a molecule for a time and under conditions sufficient to increase or decrease NR6 activity. The molecule may be a proteinaceous molecule or a chemical entity and may also be a derivative of NR6 or its ligand or a chemical analogue or truncation mutant of NR6 or its ligand.

AMENDED SHEET 19 The present invention, therefore, contemplates a pharmaceutical composition comprising NR6 or a derivative thereof or a modulator of NR6 expression or NR6 activity and one or more pharmaceutically acceptable carriers and/or diluents. These components are referred to as the "active ingredients".

include sterile aqueous solutions (where water soluble) and sterile powders for the extemporaneous preparation of sterile injectable solutions. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dilution medium comprising, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.

The proper fluidity can be maintained, for example, by the use of superfactants. The preventions of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thirmerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of AMENDED SHEET 20 preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.

When the active ingredients are suitably protected they may be orally administered, for example, with an inert iZ~t ith an a ii:'Z3aZZ e~ibla arr, r i: ay be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.

Such compositions and preparations should contain at least 1% by weight of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit. The amount of active compound in such therapeutically useful compositions in such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.1 ug and 2000 mg of active compound. Alternative dosage amounts include from about 1 Ag to about 1000 mg and from about 10 Ag to about 500 mg.

The tablets, troches, pills, capsules and the like may also contain the components as listed hereafter: A binder such as gum, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such a sucrose, lactose or saccharin may be added or a AMENDED SHEET 21 flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, ~sugia oA In. syup or e ikliaye acziVe compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound(s) may be incorporated into sustained-release preparations and formulations.

The present invention also extends to forms suitable for topical application such as creams, lotions and gels as well as a range of "paints" which are applied to skin and through which the active ingredients are absorbed.

Pharmaceutically acceptable carriers and/or diluents include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art and except insofar as any conventional media or agent is incompatible with the active ingredient, their use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit AMENDED SHEET 4* 22 form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the novel dosage unit forms of the invention are characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active material for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired as herein disclosed in detail.

The principal active ingredient is compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in dosage unit form as hereinbefore disclosed.

A unit dosage form can, for example, contain the principal active compound in amounts ranging from 0.5 pg to about 2000 mg. Expressed in proportions, the active compound is generally present in from about 0.5 gg to about 2000 mg/ml of carrier. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose'and manner of administration of the said ingredients.

Dosages may also be expressed per body weight of the recipient. For example, from about 10 ng to about 1000 mg/kg body weight, from about 100 ng to about 500 mg/kg body weight and for about 1 Ag to above 250 mg/kg body weight may be administered.

The pharmaceutical composition may also comprise genetic molecules such as a vector capable of transfecting '111FAIrt (Nr 23 target cells where the vector carries a nucleic acid molecule capable of modulating NR6 expression or NR6 activity. The vector may, for example, be a viral vector.

Still another aspect of the present invention is directed to antibodies to NR6 and its derivatives. Such ant.j-ibo-ies may e ronoc i.onai or polyc.Lornaj an(d liay i selected from naturally occurring antibodies to NR6 or may be specifically raised to NR6 or derivatives thereof. In the case of the latter, NR6 or its derivatives may first need to be associated with a carrier molecule. The antibodies and/or recombinant NR6 or its derivatives of the present invention are particularly useful as therapeutic or diagnostic agents.

For example, NR6 antibodies or antibodies to its ligand may act as antagonists.

For example, NR6 and its derivatives can be used to screen for naturally occurring antibodies to NR6. These may occur, for example in some autoimmune diseases.

Alternatively, specific antibodies can be used to screen for NR6. Techniques for such assays are well known in the art and include, for example, sandwich assays and ELISA. Knowledge of NR6 levels may be important for diagnosis of certain cancers or a predisposition to cancers or for monitoring certain therapeutic protocols.

Antibodies to NR6 of the present invention may be monoclonal or polyclonal. Alternatively, fragments of antibodies may be used such as Fab fragments.

Furthermore, the present invention extends to recombinant and synthetic antibodies and to antibody hybrids. A "synthetic antibody" is considered herein to include fragments and hybrids of antibodies. The antibodies of this aspect of the present invention are particularly useful for immunotherapy and may also be 24 used as a diagnostic tool for assessing apoptosis or monitoring the program of a therapeutic regimen.

For example, specific antibodies can be used to screen for NR6 proteins. The latter would be important, for example, as a means for screening for levels of NR6 in a cell extract or other biological fluid or purifying NR6 m1aze by recqr;Zi,:;z ean3 zr ce se-rnazcrai fluid. Techniques for the assays contemplated herein are known in the art and include, for example, sandwich assays and ELISA.

It is within the scope of this invention to include any second antibodies (monoclonal, polyclonal or fragments of antibodies or synthetic antibodies) directed to the first mentioned antibodies discussed above. Both the first and second antibodies may be used in detection assays or a first antibody may be used with a commercially available anti-immunoglobulin antibody. An antibody as contemplated herein includes any antibody specific to any region of NR6.

Both polyclonal and monoclonal antibodies are obtainable by immunization with the enzyme or protein and either type is utilizable for immunoassays. The methods of obtaining both types of sera are well known in the art.

Polyclonal sera are less preferred but are relatively easily prepared by injection of a suitable laboratory animal with an effective amount of NR6, or antigenic parts thereof, collecting serum from the animal, and isolating specific sera by any of the known immunoadsorbent techniques. Although antibodies produced by this method are utilizable in virtually any type of immunoassay, they are generally less favoured because of the potential heterogeneity of the product.

The use of monoclonal antibodies in an immunoassay is AMENDED SHEET particularly preferred because of the ability to produce them in large quantities and the homogeneity of the product. The preparation of hybridoma cell lines for monoclonal antibody production derived by fusing an immortal cell line and lymphocytes sensitized against the immunogenic preparation can be done by techniques which are well known to those who are skilled in the Another aspect of the present invention contemplates a method for detecting NR6 in a biological sample from a subject said method comprising contacting said biological sample with an antibody specific for NR6 or its derivatives or homologues for a time and under conditions sufficient for an antibody-NR6 complex to form, and then detecting said complex.

The presence of NR6 may be accomplished in a number of ways such as by Western blotting and ELISA procedures.

A wide range of immunoassay techniques are available as can be seen by reference to US Patent Nos. 4,016,043, 4, 424,279 and 4,018,653. These, of course, includes both single-site and two-site or "sandwich" assays of the non-competitive types, as well as in the traditional competitive binding assays. These assays also include direct binding of a labelled antibody to a target.

Sandwich assays are among the most useful and commonly used assays and are favoured for use in the present invention. A number of variations of the sandwich assay technique exist, and all are intended to be encompassed by the present invention. Briefly, in a typical forward assay, an unlabelled antibody is immobilized on a solid substrate and the sample to be tested brought into contact with the bound molecule. After a suitable period of incubation, for a period of time sufficient to allow formation of an antibody-antigen complex, a second antibody specific to the antigen, labelled with a AMEN~IUh: F 26 reporter molecule capable of producing a detectable signal is then added and incubated, allowing time sufficient for the formation of another complex of antibody-antigen-labelled antibody. Any unreacted material is washed away, and the presence of the antigen is determined by observation of a signal produced by the reporter molecule. The results may either be signal, or may be quantitated by comparing with a control sample containing known amounts of hapten.

Variations on the forward assay include a simultaneous assay, in which both sample and labelled antibody are added simultaneously to the bound antibody. These techniques are well known to those skilled in the art, including any minor variations as will be readily apparent. In accordance with the present invention, the sample is one which might contain NR6 including cell extract, tissue biopsy or possibly serum, saliva, mucosal secretions, lymph, tissue fluid and respiratory fluid. The sample is, therefore, generally a biological sample comprising biological fluid but also extends to fermentation fluid and supernatant fluid such as from a cell culture.

In the typical forward sandwich assay, a first antibody having specificity for the NR6 or antigenic parts thereof, is either covalently or passively bound to a solid surface. The solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. The solid supports may be in the form of tubes, beads, discs of microplates, or any other surface suitable for conducting an immunoassay.

The binding processes are well-known in the art and generally consist of cross-linking covalently binding or physically adsorbing, the polymer-antibody complex is washed in preparation for the test sample. An aliquot AUm ff wT 27 of the sample to be tested is then added to the solid phase complex and incubated for a period of time sufficient 2-40 minutes or overnight if more convenient) and under suitable conditions from about room temperature to about 37 0 C) to allow binding of any subunit present in the antibody. Following the incubation period, the antibody subunit solid phase is wasi-.e acw dr-ed anid incazz5 d wi-;a secc;- c anr_ specific for a portion of the hapten. The second antibody is linked to a reporter molecule which is used to indicate thebinding of the second antibody to the hapten.

An alternative method.involves immobilizing the target molecules in the biological sample and then exposing the immobilized target to specific antibody which may or may not be labelled with a reporter molecule. Depending on the amount of target and the strength of the reporter molecule signal, a bound target may be detectable by direct labelling with the antibody. Alternatively, a second labelled antibody, specific to the first antibody is exposed to the target-first antibody complex to form a target-first antibody-second antibody tertiary complex. The complex is detected by the signal emitted by the reporter molecule.

In another alternative method, the NR6 ligand is immobilised to a solid support and a biological sample containing NR6 brought into contact with its immobilised ligand. Binding between NR5 and its ligand can then be determined using an antibody to NR6 which itself may be labelled with a reporter molecule or a further antiimmunoglobulin antibody labelled with a reporter molecule could be used to detect antibody bound to NR6.

By "reporter molecule" as used in the present specification, is meant a molecule which, by its AMENDED SHEET -28 chemical nature, provides an analytically identifiable signal which allows the detection of antigen-bound antibody. Detection may be either qualitative or quantitative. The most commonly used reporter molecules in this type of assay are either enzymes, fluorophores or radionuclide containing molecules (i.e.

radioisotopes) and chemiluminescent molecules.

conjugated to the second antibody, generally by means of glutaraldehyde or periodate. As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan. Commonly used enzymes include horseradish peroxidase, glucose oxidase, betagalactosidase and alkaline phosphatase, amongst others.

The substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable colour change.

Examples of suitable enzymes include alkaline phosphatase and peroxidase. It is also possible to employ fluorogenic substrates, which yield a fluorescent product rather than the chromogenic substrates noted above. In all cases, the enzyme-labelled antibody is added to the first antibody hapten complex, allowed to bind, and then the excess reagent is washed away. A solution containing the appropriate substrate is then added to the complex of antibody-antigen-antibody. The substrate will react with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of hapten which was present in the sample.

"Reporter molecule" also extends to use of cell agglutination or inhibition of agglutination such as red blood cells on latex beads, and the like.

Alternately, fluorescent compounds, such as fluorescein S. 29 and rhodamine, may be chemically coupled to antibodies without altering their binding capacity. When activated by illumination with light of a particular wavelength, the fluorochrome-labelled antibody adsorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic colour visually detectable with a light antibody is allowed to bind to the first antibody-hapten complex. After washing off the unbound reagent, the remaining tertiary complex is then exposed to the light of the appropriate wavelength the fluorescence observed indicates the presence of the hapten of interest.

Immunofluorescene and EIA techniques are both very well established in the art and are particularly preferred for the present method. However, other reporter molecules, such as radioisotope, chemiluminescent or bioluminescent molecules, may also be employed.

The present invention also contemplates genetic assays such as involving PCR analysis to detect the NR6 gene or its derivatives. Alternative methods or methods used in conjunction include direct nucleotide sequencing or mutation scanning such as single stranded conformational polymorphisms analysis (SSCP) as specific oligonucleotide hybridisation, as methods such as direct protein truncation tests.

The nucleic acid molecules of the present invention may be DNA or RNA. When the nucleic acid molecule is in a DNA form, it may be genomic DNA or cDNA. RNA forms of the nucleic acid molecules of the present invention are generally mRNA.

Although the nucleic acid molecules of the present invention are generally in isolated form, they may be integrated into or ligated to or otherwise fused or AMENDED SHEET associated with other genetic molecules such as vector molecules and in particular expression vector molecules.

Vectors and expression vectors are generally capable of replication and, if applicable, expression in one or both of a prokaryotic cell or a eukaryotic cell.

Preferably, prokaryotic cells include E. coli, Bacillus sp and Pseudomonas sp. Preferred eukaryotic cells Accordingly, another aspect of the present invention contemplates a genetic construct comprising a vector portion and a mammalian and more particularly a human NR6 gene portion, which NR6 gene portion is capable of encoding an NR6 polypeptide or a functional or immunologically interactive derivative thereof.

Preferably, the NR6 gene portion of the genetic construct is operably linked to a promoter on the vector such that said promoter is capable of directing expression of said NR6 gene portion in an appropriate cell.

In addition, the NR6 gene portion of the genetic construct may comprise all or part of the gene fused to another genetic sequence such as a nucleotide sequence encoding maltose binding protein or glutathione-Stransferase or part thereof.

The present invention extends to such genetic constructs and to prokaryotic or eukaryotic cells comprising same.

The present invention also extends to any or all derivatives of NR6 including mutants, part, fragments, portions, homologues and analogues or their encoding genetic sequence including single or multiple nucleotide or amino acid substitutions, additions and/or deletions to the naturally occurring nucleotide or amino acid AMENDED SHEET 31 sequence.

NR6 may be important for the proliferation, differentiation and survival of a diverse array of cell types. Accordingly, it is proposed that NR6 or its functional derivatives be used to regulate development, maintenance or regeneration in an array of different NR6 is contemplated to be useful in modulating neuronal proliferation, differentation and survival.

Soluble NR6 polypeptides are also contemplated to be useful in the treatment of a range of diseases, injuries or abnormalities.

Membrane bound or soluble NR6 may be used in vitro on nerve cells or tissues to modulate proliferation, differentiation or survival, for example, in grafting procedures or transplantation.

As stated above, the NR6 of the present invention or its functional derivatives may be provided in a pharmaceutical composition comprising the NR6 together with one or more pharmaceutically acceptable carriers and/or diluents. In addition, the present invention contemplates a method of treatment comprising the administration of an effective amount of a NR6 of the present invention. The present invention also extends to antagonists and agonists of NR6s and their use in therapeutic compositions and methodologies.

A further aspect of the present invention contemplates the use of NR6 or its functional derivatives in the manufacture of a medicament for the treatment of NR6 mediated conditions defective or deficient.

Still a further aspect of the present invention AMNDED SHEET 32 contemplates a ligand for NR6 preferably, in isolated or recombinant form or a derivative of said ligand.

The present invention further contemplates knockout animals such as mice or other murine species for the NR6 gene including homozygous and heterozygous knockout animals. Such animals provide a particularly useful well as screening for agents capable of acting as agonists or antagonists of NR6.

According to this embodiment there is provided a transgenic animal comprising a mutation in at least one allele of the gene encoding NR6. Additionally, the present invention provides a transgenic animal comprising a mutation in two alleles of the gene encoding NR6. Preferably, the transgenic animal is a murine animal such as a mouse or rat.

The present invention is further described by the following non-limiting Figures and Examples.

In the Figures: Figure 1 is a diagrammatic representation showing expansion of sequenced region of the mouse NR6 gene indicating splicing patterns seen in the three forms of NR6 cDNA, NR6.1, NR6.2 and NR6.3.

Figure 2 is a representation of the nucleotide sequence of the mouse NR6 gene, containing exons encoding the cDNA from nucleotide 148 encoding D50 of the cDNAs shown in SEQ ID NOs:12 and 14 to the end of the 3' untranslated region shared by both NR6.1, NR6.2 and NR6.3. In this figure, this region encompasses nucleotides g1182 to g6617. This sequence is also defined in SEQ ID NO:28.

MENOED SH ET 33 Figure 3 is a representation of the nucleotide sequence of the mouse genomic NR6 gene with additional sequences. The coding exons of NR6 span approximately 11kb of the mouse genome. There are 9 coding exons separated by 8 introns: exonl at least 239nt intronl 5195nt exon 2 282nt intron2 214nt exon4 170nt intron4 1372nt exon5 158nt intron5 68nt exon6 169nt intron6 2020nt exon6 188nt intron7 104nt exon8 43nt intron8 181nt exon9 252nt Exon 1 encoding the signal sequence, exon 2 the Ig-like domain, exons 3 to 6 the hemopoietin domain. Exons 7, 8 and 9 are alternatively spliced.

Figure 4 is a diagrammatic representation showing the genomic structure of murine NR-6.

Figure 5 is a diagrammatic representation showing targetting of the NR6 locus by homologous recombination.

4MEND7 SENEE; 34 Single and three letter abbreviations for amino acid residues used in the specification are summarised in Table 2: TABLE 2 Amino Acid Three-letter One-letter Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamine Gln

Q

Glutamic acid Glu E Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V Any residue Xaa X 7P:# 35 TABLE 3 SUMMARY OF SEQ ID NO.

Sequence.

Amino acid sequence WSXWS Oligonucleotide primers and probes listed in Example 1 Amino acid sequence of NR6.1 Nucleotide sequence of NR6.2 2 Amino acid sequence of NR6.2 Nucleotide sequence of NR6.3 3 Amino acid sequence of NR6.3 Nucleotide sequence of products generated by 5' RACE of brain cDNA using NR6 specific primers 4 Amino acid sequence of SEQ ID NO:18 Nucleotide sequence unique to 5' RACE of brain cDNA Amino acid sequence for SEQ ID NO:20 Unspliced murine NR6 nucleotide sequence PCR product for human NR6 Nucleotide sequence of clone HFK-66 encoding human NR6 Amino acid sequence of SEQ ID NO:24 Oligonucleotide sequences UP1 and LP1, respectively Genomic nucleotide sequence of murine NR6 Amino acid sequence of SEQ ID NO:28 Murine NR6.1 oligonucleotide primers Murine IL-3 signal sequence Linker sequence for mouse IL-3 signal sequence and FLAG epitope Genomic nucleotide sequence of murine NR6 containing additonal 5' sequence Oligonucleotide 2199 and 2200, respectively N-terminal region of NR6 SEQ ID NO.

1 2-11 13 14 16 17 18 19 21 22 23 24 26-27 28 29 30, 31 32 33-35 38 36, 37 39 AMFNnFn

C'LJC,.

-36 iThe polyadenylation signal AATAAATAAA is at nucleotide position 1451 to 1460; NR6.1 (SEQ ID NO:12) and NR6.2 (SEQ ID NO:14) are identical to nucleotide 1223 encoding Q407, the represents the end of an exon. NR6.1 splices out an exon present only in NR6.2 and uses a different reading frame for the final exon which is shared with NR6.2; this corresponds to amino acids VLPAKL at amino untranslated DNA shared by NR6.1, NR6.2 and NR6.3 is from nucleotide 1240 to 1475. The WSXWS motif is at amino acid residues 330 to 334.

2 The polyadenylation signal AATAAA is at nucleotide positions 1494 to 1503. The WSXWS motif is at amino acid residues 330 to 334. NR6.1 and NR6.2 are identical to nucleotide 1223 encoding Q407 which represents the end of an exon. NR6.2 splices in an exon beginning at amino acid residue D408, nucleotide 1224 and ends at residue G422, nucleotide 1264. The region of 3' untranslated DNA shared by NR6.1, NR6.2 and NR6.3 is from nucleotide position 1283 to 1517.

3 The nucleotide and amino acid numbering corresponds to SEQ ID NO:12 and 14. The WSXWS motif is at amino acid residues 330 to 334. The polyadenylation signal AATAAATAAA is from nucleotide 1781 to 1780. NR6.1, NR6.2 and NR6.3 are identical to nucleotide 1223 encoding Q407, this represents the end of an exon.

NR6.3 fails to splice from this position and, therefore, translation continues through the intron, giving rise to the C-terminal protein region from amino acid residues 408 to 461. The region of 3' untranslated DNA shared by NR6.1, NR6.2 and NR6.3 is from nucleotide 1469 to 1804.

4 The nucleotide sequence is identical to NR6.1, NR6.2 and NR6.3 from nucleotide C151, the first nucleotide for ALPro51. The numbering from this nucleotide is the same 37 as for SEQ ID NO:14 and 16. The 5' of this point is unique to the products generated by 5' RACE not being found in NR6.1, NR6.2 and NR6.3 and is represented in SEQ ID NOs:20 and 21.

Structure of the murine genomic NR6 locus. The coding exons of NR6 span approximately 11kb of the mouse introns: exon exon exon exon exon exon exon exon exon at least 239nt 282nt 130nt 170nt 158nt 169nt 188nt 43nt 252nt intronl intron2 intron3 intron 4 intron5 intron6 intron7 intron8 5195nt 214nt 107nt 1372nt 68nt 2020nt 104nt 181nt Exon 1 encodes the signal sequence, exon 2 the Ig-like domain, exons 3 to 6 the hemopoietin domain. Exons 7, 8 and 9 are alternatively spliced.

The NRG molecules of the present invention have a range of utilities referred to in the subject specification.

Additional utilities include: 1. Identification of molecules that interact with NR6.

These may include a) a corresponding ligand using standard orphan receptor techniques (26), b) monoclonal antibodies that act either as receptors antagonists or agonists, AMEN!D~Fl .g;;r P:\opr\jh\amaded\2149090.amn dcdclaims.doc-18/09/00 -38c) mimetic or antagonistic peptides isolated using phage display technology (27,28), d) small molecule natural products that act either as antagonists or agonists.

2. Development of diagnostics to detect deletions/ rearrangements in the NR6 gene.

10 The NR6 knock-out mice studies described herein provide a useful model for this utility. There are also applications in the field of reproduction. For example, people can be tested for their NR6 status. NR6 carriers might be expected to give rise to offspring with developmental 15 problems.

Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common 20 general knowledge in Australia or any other country.

39 EXAMPLE 1 Oligonucleotides M116: M108: M159: M242:

WSDWS

WSEWS

1944 2106 2120 ACTCGCTCCAGATTCCCGCCTTTT 3' [SEQ ID NO:2] TCCCGCCTTTTTCGACCCATAGAT 3' [SEQ ID NO:3] GGTACTTGGCTTGGAAGAGGAAAT 3' [SEQ ID NO:4] CGGCTCACGTGCACGTCGGGTGGG 3' [SEQ ID 1S£Q ±U ii:j (A/G)CTCCA(A/G)TC(A/G)CTCCA 3' [SEQ ID NO:7] (A/G)CTCCA(C/T)TC(A/G)CTCCA 3' [SEQ ID NO:8] AAGTGTGACCATCATGTGGAC 3' [SEQ ID NO:9] GGAGGTGTTAAGGAGGCG 3' [SEQ ID ATGCCCGCGGGTCGCCCG 3' [SEQ ID NO:11] EXAMPLE 2 Isolation of initial NR6 cDNA clones using oligonucleotides designed against the conserved WSXWS motif found in members of the haemopoietin receptor family A commercial adult mouse testis cDNA library cloned into the UNI-ZAP bacteriophage (Stratagene, CA, USA; Catalogue numbers 937 308) was used to infect Escherichia coli of the strain LE392. Infected bacteria were grown on twenty 150 mm agar plates, to give approximately 50,000 plaques per plate. Plaques were then transferred to duplicate 150 mm diameter nylon membranes (Colony/Plaque Screen, NEN Research Products, MA, USA), bacteria were lysed and the DNA was denatured and fixed by autoclaving at 100 0 C for 1 min with dry exhaust. The filters were rinsed twice in 0.1%(w/v) sodium dodecyl sulfate (SDS), 0.1 x SSC (SSC is 150 mM sodium chloride, 15 mM sodium citrate dihydrate) at room temperature and pre-hybridized overnight at 42 0 C in 6 x SSC containing 2 mg/ml bovine serum albumin, 2 mg/ml Ficoll, 2 mg/ml polyvinylpyrrolidone, 100 mM ATP, mg/ml tRNA, 2 mM sodium pyrophosphate, 2 mg/ml salmon ,,JIntiu tEt: sperm DNA, 0.1% SDS and 200 mg/ml sodium azide.

The pre-hybridisation buffer was removed. 1.2 pg of the degenerate oligonucleotides for hybridization (WSDWS; Example 1) were phosphorylated with T4 polynucleotide kinase using 960 mCi of y 32 P-ATP (Bresatec, S.A., Australia). Unincorporated ATP was separated from the labelled oligonucleotide using a pre-packed gel Filters were hybridized overnight at 42 0 C in 80 ml of the prehybridisation buffer containing SDS, rather than NP40, and 106 10 7 cpm/ml of labelled oligonucleotide. Filters were briefly rinsed twice at room temperature in 6 x SSC, SDS, twice for min at 45 0 C in a shaking waterbath containing 1.5 1 of the same buffer and then briefly in 6 x SSC at room temperature. Filters were then blotted dry and exposed to autoradiographic film at -70 0 C using intensifying screens, for 7 14 days prior to development.

Plaques that appeared positive on orientated duplicate filters were picked, eluted in 1 ml of 100 mM NaC1, mM MgC12, 10 mM Tris.HC1 pH7.4 containing gelatin and 0.5% chloroform and stored at 4 0

C.

After 2 days LE392 cells were infected with the eluate from the primary plugs and replated for the secondary screen. This process was repeated until hybridizing plaques were pure.

Once purified, positive cDNAs were excised from the ZAP II bacteriophage according to the manufacturer's instructions (Stratagene, CA, USA) and cloned into the plasmid pBluescript. A CsCl purified preparation of the DNA was made and this was sequenced on both strands.

Sequencing was performed using an Applied Biosystems automated DNA sequencer, with fluorescent dideoxynucleotide analogues according to the manufacturer's instructions. The DNA sequence was analysed using software supplied by Applied Biosystems.

V AMENDED SHEET -41 Two clones isolated from the mouse testis cDNA library shared large regions of nucleotide sequence identity 68- 1 and 68-2 and appeared to encode a novel member of the haemopoietin receptor family and the inventors gave the putative receptor the working name "NR6".

(ii) In a parallel series of experiments, a commercial tnouse bra±Lii cDNA library (STRATAGENE #9673i1, Baib/c whole brain cDNA/Uni-ZAP XR Vector) was used to infect E.coli strain XL1-Blue MRF'. Infected bacteria were grown on 90x135mm square agar plates to give about 25,000 plaques per plate. Plaques were then transferred to positively charged nylon membranes, Hybond-N(+) (Amersham RPN 203B), bacteria were lysed and the DNA was denatured with denaturing 0.5 M NaOH, 1.5 M NaCl at room temperature for 7 min. The membranes were neutralized with 0.5 M Tris-HCL pH7.2, 1.5 M NaC1, 1 mM EDTA at room temperature for 10 min before the DNA fixation by UV crosslinking.

A mixture of WSDWS and WSEWS oligonucleotide probes (SEQ ID NOs: 7 and 8) were labelled with a [a-32P]-ATP (TOYOBO #PNK-104 Kination kit). The membranes from the mouse brain cDNA library were then hybridized with the mixture of WSDWS and WSEWS oligonucleotide probes in the Rapid Hybridization Buffer (Amersham, RPN1636) at 42 0

C

for 16 hours. Filters were washed with 1xSSC/0.1% (w/v) SDS at 42 0 C before autoradiography. Plaques that appeared positive on orientated duplicate filters were picked and replated on E. coli, XL1-Blue MRF' with the process of immobilisation on nylon membranes, hybridization of membranes with oligonucleotide probes, washing and autoradiography repeated until pure plaques had been obtained.

The cDNA fragment from pure positively hybridizing A- plaques was isolated by excision with the helper phage ^2 s -42strain ExAssist according to the manufacturer=s instructions (Stratagene, #967319). Sequencing was performed after the amplification with Ampli-Taq DNA polymerase and Taq dideoxy terminator cycle sequencing kit (Perkin Elmer, #401150) by 25 cycles of 96 0 C for sec, 50 0 C for 5 sec, 60 0 C for 4 min followed by 600C for min with the sequencing primers on an ABI model 377 One clone, MBC-8, from the mouse brain library shared large regions of nucleotide sequence identity with both the 68-1 and 68-2 clones isolated from the mouse testis cDNA library.

(iii) In a third series of experiments, total RNA was prepared from the mouse osteoblastic cell line, KUSA, according to the method of Chirgwin et al. and poly(A)+RNA was further purified by oligo(dT)-cellulose chromatography (Pharmacia Biotech). Complementary DNA was synthesized by oligo(dT) priming, inserted into the UniZAP XR directional cloning vector (Stratagene), and packaged into X phage using Gigapack Gold (Stratagene), yielding 1.25 x 107 independent clones.

Approximately 106 clones were screened essentially as described in (ii) above. Briefly, probes were labeled with 32 P using T4 polynucleotide kinase and prehybridization was performed for 4 hr in the Rapid hybridization buffer (Amersham LIFE SCIENCE) at 42 0

C.

Filters (Hybond Amersham) were then hybridized for 19 hr under the same condition with the addition of 32p_ labeled WSXWS mix oligonucleotides and washed 3 times.

The final wash was for 30 min in 1 x SSPE, 0.1% (w/v) SDS at 42 0 C. Filters were then exposed with an intensifying screen to Kodak X-OMAT AR film for 5 days.

41711111n H ET 43 Isolated clones were subjected to the in vivo excision of pBluescript phagemid (Stratagene), and plasmid DNA was prepared by the standard method. DNA sequences were determined using an ABI PRISM 377 DNA Sequencer (Perkin Elmer) with appropriate synthetic oligonucleotide primers. A clone pKUSA166 shared large regions of nucleotide sequence identity with the MBC-8, oo CAl oC-2 1i00 CCies i6raLed ZrOiti iii i .A ajil iand testis cDNA libraries.

EXAMPLE 3 Isolation of further NR6 cDNA clones using probes specific for NR6 In order to identify other cDNA libraries containing cDNA clones for NR6, the inventors performed PCR upon 1 Al aliquots of X-bacteriophage cDNA libraries made from mRNA from various human tissues and using oligonucleotides 2070 and 2057, designed from the sequence of 68-1 and 68-2, as primers. Reactions contained 5 tl of 10 x concentrated PCR buffer (Boehringer Mannheim GmbH, Mannheim, Germany), 1 pl of mM dATP, dCTP, dGTP and dTTP, 2.5 Al of the oligonucleotides HYB2 and either T3 or T7 at a concentration of 100 mg/ml, 0.5 pl of Taq polymerase (Boehringer Mannheim GmbH) and water to a final volume of 50 1l. PCR was carried out'in a Perkin-Elmer 9600 by heating the reactions to 96 0 C for 2 min and then for cycles at 96 0 C for 30 sec, 55 0 C for 30 sec and 72 0 C for 2 min. PCR products were resolved on an agarose gel, immobilized on a nylon membrane and hybridized with 32p_ labelled oligonucleotide 1943 (SEQ ID NO:42).

In addition to the original library, a mouse brain cDNA library appeared to contain NR6 cDNAs. These were screened using a 32 P-labelled oligonucleotides 1944, 2106, 2120 (Example 1) or with a fragment of the .Y~hJUlJ -44 original NR6 cDNA clone from 68-1 (nucleotide 934 to the end of NR6.1 in Figure 1) labelled with 32 P using a random decanucleotide labelling kit (Bresatec).

Conditions used were similar to those described in (i) above except that for the labelled oligonucleotides, filters were washed at 55 0 C rather than 45 0 C, while for the NR6 cDNA fragment prehybridization and hybridization x SSC at 65 0 C. Again, as described in above, positively hybridising plaques were purified, the cDNAs were recovered and cloned into plasmids pBluescript II or pUC19. Independent cDNA clones were sequenced on both strands.

Using this procedure, 6 further clones, 68-5, 68-35, 68- 41, 68-51, 68-77 and 73-23, contained large regions of sequence identity with 68-1, 68-2, MBC-8 and pKUSA166.

In a parallel series of experiments, further screening was performed with hybridization probes prepared from the 1.7 kbp EcoRI-XhoI fragment excised from pKUSA166.

This fragment was excised and labeled with 32 P by using T7QuickPrime Kit (Pharmacia Biotech). Approximately 6x10 5 clones were screened. Hybond N+ filters (Amersham) were first prehybridized for 4hr at 42 0 C in formamide, 5xSSPE, 5xDenhardt's solution, 0.1% SDS, and 0.1mg/ml denatured salmon sperm DNA.

Hybridization was for 16 hours under the same conditions with the addition of 32 P- labelled NR6- cDNA fragment probes. Finally the filters were washed once for lhr in 0.2xSSC, 0.1% SDS at 68 0 C. Eight clones were isolated, and phage clones were subjected to the in vivo excision of the pBluescript phagemid (Stratagene).

The plasmid DNAs were prepared by the standard method.

DNA sequences were determined by an ABI PRISM 377 DNA Sequencer using appropriate synthetic oligonucleotide primers.

AMENDED SHEET 45 Using this procedure 8 further clones from the KUSA library contained large regions of sequence identity with 68-1, 68-2, MBC-8, pKUSA166, 68-5, 68-35, 68-41, 68-51, 68-77 and 73-23 were isolated.

EXAMPLE 4 Isolation of genomic DNA encoding NR6 DNA encoding the murine NR6 genomic locus was also isolated using the 68-1 cDNA as a probe. Two positive clones, 2-2 and 57-3, were isolated from a mouse 129/Sv strain genomic DNA library cloned into X FIX. These clones were overlapping and the position of the restriction sites, introns and exons were determined in the conventional manner. The region of the genomic clones containing exons and the intervening introns were sequenced on both strands using an Applied Biosystems automated DNA sequencer, with fluorescent dideoxynucleotide analogues according to the manufacturer's instructions. Figure 2 shows the nucleotide sequence and corresponding amino acid sequence of the translation regions. This is also shown in SEQ ID NOs:30 and 31. Figure 3 provides the genomic NR6 gene sequence but with additional 5' sequence. This is also represented in SEQ ID NO:38 in relation to this sequence. The coding exons of NR6 span approximately 11kb of the mouse genome. There are 9 coding exons separated by 8 introns: exonl at least 239nt intronl 5195nt exon2 282nt intron2 214nt exon3 130nt intron3 107nt exon4 170nt intron4 1372nt 158nt intron5 68nt exon6 169nt intron6 2020nt exon7 188nt intron7 104nt exon8 43nt intron8 181nt AMENDED

SHEET

46 exon9 252nt Exon.l encodes the signal sequence, exon 2 the Ig-like domain, exons 3 to 6 the hemopoietin domain. Exons 7, 8 and 9 are alternatively spliced.

EXAMPLE 51 R7 an--.s of 5'-RACE was used to investigate the nature of the sequence 5' of nucleotide 960, encoding Ile321 of NR6.1, 2 and 3. The nucleotide and corresponding amino acid sequences are shown in SEQ ID NOs:12, 14 and 16, respectively. 5'-RACE was performed using Advantage KlenTaq polymerase (CLONTECH, CAT NO. K1905-1) on mouse brain Marathon-ready cDNA (CLONTECH, CAT NO. 7450-1) according to the manufacturer's instructions. Briefly, the first rounds of amplification were performed using 5p1 of cDNA in a total volume of 501, with ImM each of the primers AP1&M116 [SEQ ID NO:2] or AP1&M159 [SEQ ID NO:4] by 35 cycles of 94 0 C x 0.5min, 68 0 C x 2.0min on GeneAmp 2400 (Perkin-Elmer). An amount of 5I1 of fold diluted product from the first amplification was then re-amplified for the products generated with primers AP1 and M116 [SEQ ID NO:2] 'in the first amplification, 1 mM of the primers AP2&M108 [SEQ ID NO:3] were used in the second amplification. For the products generated with primers AP1 and M116 [SEQ ID NO:2] in the first amplification, two separate secondary reactions were performed, one reaction with 1 mM primers AP2&M242 [SEQ ID NO:5] and the other with 1 mM primers AP2&M112 [SEQ ID NO:6]. Amplification was achieved using 25 cycles of 94 0 C x 0.5min, 68 0 C x 2.0min. These samples were analyzed by agarose gel electrophoresis.

When a single ethidium bromide staining amplification product was observed, it was purified by QIAquick PCR purification kit according to the manufacturer's ,_AMENDED SHEET 47 instructions (QIAGEN, CAT NO. DG-0281) and its sequence was directly determined using both primers used in the secondary amplification step, that is AP2 and either M108 [SEQ ID NO:3], M242 [SEQ ID NO:5] or M112 [SEQ ID NO:6].

EXAMPLE 6 Cloning of NR6 From the initial screens of mouse brain and testis cDNA libraries with the degenerate WSXWS oligonucleotides and subsequent screening of cDNA libraries from mouse testis, mouse brain and the KUSA osteoblastic cells line a total of 18 NR6 cDNAs have been isolated. Nucleotide sequence of NR6 was also determined from 5'RACE analysis of brain cDNA. Additionally, two murine genomic DNA clones encoding NR6 have also been isolated.

Comparison of the NR6 cDNA clones revealed a common region of nucleotide sequence which included a 123 base pairs 5'-untranslated region and 1221 base pairs open reading frame, stretching from the putative initiation methionine, Metl to Gln407 (SEQ ID NOs:12, 14 and 16, respectively). Within this common open reading frame, a haemopoietin receptor domain was observed which contained the four conserved cysteine residues and the five amino acid motif WSXWS typical of members of the haemopoietin receptor family, was observed.

Further analyses revealed that after nucleotide 1221, three different classes of NR6 cDNAs could be found, these were termed NR6.1, NR6.2 and NR6.3 (SEQ ID NOs:12, 14 and 16, respectively). Each encoded a receptor that appeared to lack a classical transmembrane domain and, would, therefore be likely to be secreted into the extracellular environment. Although the putative Cterminal region of the three classes of NR6 proteins AMENDED SHEET 48 appear to be different, the cDNAs encoding them also had a common region of 3'-untranslated region.

With regard to SEQ ID NOs:12, 14 and 16, the number of both nucleotides and amino acids begins at the putative initiation methione. NR6.1 and NR6.2 are identical to nucleotide 1223 encoding Q407, this represents the end of an exon. NR6.1 splices out an exon present only in NR6.2 and uses a different reading frame for the final exon which is shared with NR6.2. The 3N-untranslated region is shared by NR6.1, NR6.2 and NR6.3, NR6.2 splices in an exon starting with nucleotide 1224 encoding D408 and ending with nucleotide 1264 encoding the first nucleotide in the codon for G422 and uses a different reading frame for the final exon which is shared with NR6.2 (see Figure NR6.3 fails to splice from position nucleotide 1224, therefore, translation continues through the intron, giving rise to the Cterminal protein region.

The sequence of NR6 cDNA products generated by amplification from mouse brain cDNA preparation is shown in SEQ ID NO:18. The nucleotide sequence identified using 5'-RACE appeared to be identical to the sequence of cDNAs encoding NR6.1, NR6.2, and NR6.3 from nucleotide C151, the first nucleotide for the codon for Pro51. 5' of this nucleotide, the sequences diverged and the sequence is unique not being found in NR6.1, NR6.2 or NR6.3. Additionally, there is a single nucleotide difference, with the sequence from the RACE containing an G rather than an A at nucleotide 475, resulting in Thrl59 becoming Ala.

Analysis of the genomic clones, revealed that they were overlapping and contained exons encoding the majority of the coding region of the three forms of NR6 (Figures 1, 2 and These genomic clones, contained exons AMENDED)

*HEET

49 encoding from Asp50 (nucleotide 148) of the NR6 cDNAs.

Sequence 5' of this in the cDNAs, including the untranslated region and the region encoding Metl to Gln49 (SEQ ID NOs:12, 14 and 16), and the 5' end predicted from analysis of 5' RACE products (SEQ ID NO:18) were not present in the two genomic clones isolated.

Analysis of the NR6 genomic DNA clones also provided an explanation of the three classes of NR6 cDNAs found. It is likely that NR6.1, NR6.2 and NR6.3 arise through alternative splicing of NR6 mRNA (Figure The last amino acid residue that these different NR6 proteins are predicted to share is Gln407. SEQ ID NO:18 shows that Gln407 is the last amino acid encoded by the exon thatcovers nucleotides g5850 to g6037 (see Figure 2).

Alternative splicing from the end of this exon (Figure 1) accounts for the generation of cDNAs encoding NR6.1 (SEQ ID NO:12), NR6.2 (SEQ ID NO:14) and NR6.3 (SEQ ID NO:16). In the case of NR6.1, the region from g6038 to g6425 is spliced out, leading to juxtaposition of g6037 and g6426. In the case of NR6.2, the region from g6038 to 6141 is spliced out, an exon from 6142 to g6183 is retained and then this is followed by splicing out of the region from g6183 to g6425. NR6.3 appears to arise when there is no splicing from nucleotide g6038. For all three forms, a secreted rather then transmembrane form is generated, these differ however in their predicted C-terminal region. The genomic NR6 sequence with additional 5' sequence is shown in Figure 3.

EXAMPLE 7 ESTs Databases were searched with the murine NR6 corresponding to the unspliced version shown in SEQ ID NO:16. The murine NR6 sequence used is shown in SEQ ID AMENDED SHEET 50 NO:22.

The databases searched were: dbEST Database of Expressed Sequence Tags National Center for Biotechnology Information National Library of Medicine, 38A, 8N8058600 Rockville Pike, Bethesda, MD 20894 Phone: 0011-1-301-496-2475 Fax: (ii) DNA Data Bank of Japan DNA Database Release 3689.

Prepared by: Sanzo Miyazawa Manager/Database Administrator HidenoriHayashida Scientific Reviewer Yukiko Yamazaki/Eriko Hatada/Hiroaki Serizawa Annotators/reviewers Motono Horie/Shigeko Suzuki/Yumiko SataoSecretaries/typists DNA Data Bank of JapanNationa-l Institute of Genetics Center for Genetic Information research Laboratory of Genetic Information Analyses 1111 YataMishima, Shizuoka 411 Japan.

(iii) EMBL Nucleic Acid Sequence Data Bank Release 47.0.

(iv) EMBL Nucleic Acid Sequence Data Bank Weekly Updates Since Release 44.

Genetic Sequence Data Bank NCBI-GenBank Release 94 National Center for Biotechnology Information National Library of Medicine, 38A, 8N805 8600 Rockville Pike, Bethesda, MD 20894 Phone: 0011-1-301-495-2475 Fax: 0015-1-301-480-9241 USA.

(vi) Cumulative Updates since NCBI-GenBank Release 88 National Center for Biotechnology Information National Library of Medicine, 38A, 8N805 8600 Rockville Pike, Bethesda, MD 20894 USA.

51 The search of the databases with the murine probe identified several EST's having sequence similarity to the probe. The EST's were: W66776 (murine sequence) MM5839 (murine sequence) AA014965 (murine sequence) W46603 (human sequence) H14009 (human sequence) N78873 (human sequence) R87407 (human sequence).

EXAMPLE 8 Isolation of 3' cDNA clones encoding human NR6 PCR products encoding human NR6 were generated using oligonucleotides UP1 and LP1 (see below) based on human ESTs (Genbank Acc:H14009, Genbank Acc:AA042914) that were identified from databases searched with murine NR6 sequence (SEQ ID NO:22). PCR was performed on a human fetal liver cDNA library (Marathon ready cDNA CLONTECH #7403-1) using Advantage Klen Taq Polymerase mix (CLONTECH #8417-1) in the buffer supplied at 94 0 C for 30s and 68 0 C for 3 min for 35 cycles followed by 68°C for 4 min and then stopping at 15 0 C. A standard PCR programme for the Perkin-Elmer GeneAmp PCT system 2400 thermal cycle was used. The PCR yielded a prominent product of approximately 560 base pairs (bp; SEQ ID NO:18), which was radiolabelled with [a- 32 P] dCTP using a random priming method (Amersham, RPN, 1607, Mega prime kit) and used to screen a human fetal kidney PLUS cDNA library (CLONTECH #HL1150x). Library screens were performed using Rapid Hybridisation Buffer (Amersham, RPn 1636) according to manufacturer's instructions and membranes washed at 65 0 C for 30 min in 0.1xSSC/0.1% SDS. Two independent cDNA clones AMENDED SHEET -52 were obtained as lambda phage and subsequently subcloned and sequenced. Both clones (HFK-63 and HFK-66) contained 1.4 kilobase (kb) inserts that showed sequence similarity with murine NR6. The sequence and corresponding amino acid translation of HFK-66 is shown in SEQ ID NO:24.

The t-c1y !t ion protein. eP-nce' of clone F-.F shc..

a high degree of sequence similarity with the mouse NR6.

OLIGONUCLEOTIDES

UP1: 5'TCC AGG CAG CGG TCG GGG GAC AAC 3' [SEQ ID NO:26] LP1: 5' TTG CTC ACA TCG TCC ACC ACC TTC 3' [SEQ ID NO:27] EXAMPLE 9 Genomic Structure of Human NR6 Human genomic DNA clones encoding human NR6 was isoloated by screening a human genomic library (Lambda FIXJII Stratagene 946203) with radiolabelled oligonucleotides, 2199 and 2200 (see below). These oligonucleotides were designed based on human ESTs (Genbank Acc:R87407, Genbank Acc:H14009) that were identified from databases searched with murine NR6.

Filters were hybridised overnight at 37 0 C in 6xSSC containing 2 mg/ml bovine serum albumin, 2 mg/ml Ficoll, 2mg/ml polyvinylpyrrolidone, 100 mM ATP, 10 mg/ml tRNA, 2 mM sodium pyrophosphate, 2 mg/ml salmon sperm DNA, 0.1% SDS and 200 mg/ml sodium azide and washed at 0 C in 6 x SSC/0.1% SDS. Five independent genomic clones were obtained and sequenced. The extend of sequence obtained has determined that the clones overlap and exhibit a similar genomic structure to murine NR6.

Exon coding regions are almost identical over the region covered by the genomic clones while intron coding regions differ, although the size of the introns are 53 comparable. The extent of known overlap is shown in Fig.

OLIGONUCLEOTIDES:

2199: 5' CCC ACG CTT CTC ATC GGA TTC TCC CTG 3' [SEQ ID NO:36] 22"?0: C TCr A mC T Tfre,( 7C'm r' 1'7r TM- I f rr-- NO:37] EXAMPLE Northern Blot Analysis of Human NR6 mRNA Expression Clontech Multiple Tissue Northern Blots (Human MTN Blot, CLONTECH #7760-1, Human MTN Blot IV, CLONTECH #7766-I, Human Brain MTN Blot II, CLONTECH #7755-1, Human Brain MTN Blot III, CLONTECH #7750) were probed with a radiolabelled 3' human NR6 cDNA clone, HFK-66 (SEQ ID NO:24). The clone was labelled with [a- 32 P] dCTP using a random priming method (Amersham, RPN 1607, Mega prime kit). Hybridisation was performed in Express Hybridisation Solution (CLONTECH H50910) for 3 hours at 67 0 C and membranes were washed in 0.1xSSC/0.1% w/v SDS at 50 0

C.

A 1.8 kb transcript was detected in a variety of human tissues encompassing reproductive, digestive and neural tissues. High levels were observed in the heart, placenta, skeletal muscle, prostate and various areas of the brain, lower levels were observed in the testis, uterus, small intestine and colon. Photographs showing these Northern blots are available upon request. This expression pattern differs from the expression pattern observed with murine NR6.

AMENOED SVAEET 54 EXAMPLE 11 Mouse NR6 Expression Vectors pEF-FLAG/mNR6.1 The mature coding region of mouse NR6.1 was amplified using the PCR to introduce an in-frame Asc I restriction sit2 at the 5' se cf t!7 cc-_ 777 and an Mlu I site at the 3' end, using the following oligonucleotides:oligo 5'-AGCTGGCGCGCCTCCCGGGCGGATCGGGAGCCCAC-3'

[SEQ

ID 3' oligo 5'-AGCTACGCGTTTAGAGTTTAGCCGGCAG-3' [SEQ ID NO:31] The resulting PCR derived DNA fragment was then digested with Asc I and Mlu I and cloned into the Mlu I site of pEF-FLAG. Expression of NR6 is under the control of the polypeptide chain elongation factor la promoter as described (16) and results in the secretion, using the IL3 signal sequence from pEF-FLAG, of N-terminal FLAGtagged NR6 protein.

pEF-FLAG was generated by modifying the expression vector pEF-BOS as follows:pEF-BOS (16) was digested with Xba I and a linker was synthesized that encoded the mouse IL3 signal sequence (MVLASSTTSIHTMLLLLLMLFHLGLQASIS) and the FLAG epitope (DYKDDDDK). Asc I and Mlu I restriction enzyme sites were also introduced as cloning sites. The sequence of the linker is as follows:- M V L A S S T T S I H T M

CTAGACTAGTGCTGACACAATGGTTCTTGCCAGCTCTACCACCAGCATCCACACCATG

TGATCACGACTGTGTTACCAAGAACGGTCGAGATGGTGGTCGTAGGTGTGGTAC

55 L L L L L M L F H L G L Q A S I S Asc I

CTGCTCCTGCTCCTGATGCTCTTCCACCTGGGACTCCAAGCTTCAATCTCGGCGCGCC

GACGAGGACGAGGACTAGCAGAAGGTGGACCCTGAGGTTCGAAGTTAGAGCCGCGCGG

D Y K D D D D K Mlu I

AGGACTACAAGGACGACGATGACAAGACGCGTGCTAGCACTAGT

TCCT0TCTTCT7CTit"TCTTTCTC CCTrCCT. TCAm 7 The two oligonucleotides were annealed together and ligated into the Xba I site of pEF-BOS to give pEF-FLAG.

pCOS1/FLAG/mNR6 pCHO1/FLAG/mNR6 A DNA fragment containing the sequences encoding IL3 signal sequence/Flag/mNR6 and the poly(A) adenylation signal from human G-CSF cDNA, was excised from pEF- FLAG/mNR6 using the restriction enzyme EcoR I. This DNA fragment was then inserted into the EcoR I cloning site of pCOS1 and pCHO1.

The pCOSI and pCHO1 vectors were constructed as follows.

pCHO1 is also described in reference (17) but with a different selectable marker.

pCOS1 was prepared by digesting HEF-12h-gal (see Figure 24 of International Patent Publication No. WO 92/19759) with EcoRI and SmaI and ligating the digesting product iwht an EcoRI-NotI-BamHI adaptor (Takara 4510). The resulting plasmid comprises an EFIa promoter/enhancer, Ncor marker gene, SV40E, ori and an Amp' marker gene.

pCHO1 was constructed by digesting DHFR-PMh-grl (see Figure 25 of International Patent Publication No. WO 92/19759) with PvuI and Eco47III and ligating same with pCOSI digested with PvuI and Eco47III. The resulting vector, pCHO1, comprises an EFIc promoter/enhancer, an AMENDED

SHEET

56 DHFR marker gene, SV40E, Ori and a Ampr gene.

EXAMPLE 12 mRN6 has been expressed as an N' Flag tagged protein following transfection of CHO cells and as a C' Flag tagged protein following transfection of KUSA cells in I-ses~ v e f diri were secreted.

EXAMPLE 13 Murine NR6 expression NR6 expression studies were conducted in murine Northern Blots. At the level of sensitivity used in the adult mouse, NR6 expression was detected in salivary gland, lung and testis. During embryonic development, NR6 is expressed in fetal tissues from day 10 of gestation through to birth. In cell lines, NR6 expression has been observed in the T-lymphoid line CTLL-2 as well as in FD-PyMT (FDC-P1 myeloid cells expressing polyoma midle T gene), and fibroblastoid cells including bone marrow and fetal liver stromal lines.

EXAMPLE 14 Expression, purification and characterisation of CHO and KUSA mNR6 The methods provide for the production of a dimeric form of CHO derived N' FLAG-mNR6 without refolding. All other methods are capable of producing NR6 and are encompassed by the present invention.

SAMFNDFn czTrrT 57 A. Production of CHO derived N' FLAG-mNR6 (dimeric form) Protein Production To analyse structure and functional activity, a cDNA fragment containing the entire coding sequence of murine NR6 with an N-terminal FLAG FLAG) sequence was cl- cd in-^ te c0 -i nt t l ^the pCHO1. For stable production of N-terminal FLAG-tagged NR6 the vector contains the DHFR (dihydrofolate reductase) gene as a selective marker with the NR6 gene under the control of an EFla promoter. CHO cells were transfected with the construct using a polycationic liposome transfection reagent (Lipofectamine, GibcoBRL) (ii) Lipofectamine transfection method Using six well tissue culture plates either 2 x 10 5

KUSA

cells in 2ml IMDM 10% FCS or 2 x 10 5 CHO cells were cultured in 2ml a-MEM 10% FCS until confluent. 2Ag DNA diluted in 100l OPTI-MEM I (Gibco BRL, USA) was mixed gently with 12Al lipofectamine diluted in 100il OPTI-MEM I and incubated at room temperature for 30min to allow DNA complex formation.

DNA complexes were gently diluted in a total volume of 1ml of OPTI-MEM I and overlaid onto washed KUSA or CHO cell monolayers. A further 1ml IMDM 20% FCS (KUSA cells) or 1ml a-MEM 20% FCS (CHO cells) was added to transfected cells after 5 hours. At 24 hours, the culture medium was replaced with fresh complete growth medium. At 48 hours after transfection, selection was applied. A methotrexate resistant clone secreting comparatively high levels of NR6 was selected and expanded for further analysis.

Wr.NCEDD SHEET 58 (iii) Protein expression CHO cells were grown to confluence in roller bottles in nucleoside free a-MEM 10% FCS. Selection was maintained by using 100 ng/ml Methotrexate in the conditioned media according to manufacturer instructions. Expression was monitored by Biosensor and B. Protein Analysis Biosensor analysis Expression and purification was monitored by Biosensor analysis (BiaCoreTM, Sweden) where anti FLAG peptide M2 antibody (Kodak Eastman, USA), specific for the FLAG peptide sequence was bound to the sensorchip. Fractions were analysed for binding to the sensor surface (resonance units) and the sample then removed from the surface using 50 mM Diethylamine pH 12.0 prior to analysis of the next fraction. Immobilisation and running conditions of the Biosensor follow the manufacturer's instructions.

(ii) Protein Production In order to generate and characterise NR6, conditioned media (2 L) produced by CHO cells was harvested after day 3, post confluence. Conditioned media was concentrated using diafiltration with a 10,000 molecular weight cut-off. (Easy flow, Sartorius, Aus). At a volume of 200 ml 10 x concentrated) the sample was buffer exchanged into 20 mM Tris, 0.15M NaC1, 0.02% Tween pH 7.5 (Buffer A).

4iDf\c- i 59 (iii) Immunoprecipitation and Western Blot analysis of mNR6 Concentrated conditioned media (1ml) was immunoprecipitated with M2 affinity resin (201, Kodak Eastman). To examine the structural characterisation of mNR6 SDS PAGE was performed under reducing and nonreducing conditions. Separation was performed on NOVEX 4-20% Tris/glycine gradient gels and protein transfered on PVDF membrane. Western blots were probed with biotinylated M2 antibody (primary, 1:500) and then streptavidin peroxidase (secondary, 1:3000). Samples were visualised by autoradiography using electrochemiluminescence (ECL, Dupont, USA).

By regressional analysis of prestained standards (BIORAD, Aus.) the molecular weight of the monomeric unit was calculated to be 65,000 daltons. Under nonreducing conditions the molecular weight was calculated to be 127,000 indicating that NR6 is a disulphide linked dimer. A tetrameric complex running at approximately 250,000 daltons was also observed. Although a band running at approximately 50,000 daltons was observed, no monomeric NR6 was detected under non-reducing conditions indicating that the majority of NR6 expressed in this system is disulphide linked.

(iv) Affinity Chromatography of mNR6 Concentrated conditioned media (200 ml) was applied to M2 affinity resin (5ml) under gravity. To enhance recovery the unbound fraction was reapplied to the column four times prior to extensive washing of the column with 200 volumes of Buffer A. Biosensor analysis indicates that approximately 20% of the M2 binding originally present in the concentrate remains in the unbound fraction. The bound fraction was eluted from the Ak4FA.1ncSHEEr_ n. 60 column using an immunodesorbant (50 ml actisep (Sterogene Labs, USA).

Ion exchange and Desalting of mNR6 In order to buffer exchange mNR6 prior to anion chromatography, 10 ml batches of the eluted fraction ml) were applied to an XK column (400 x 26 mm T.D containing G25 sepharose (Pharmacia, Sweden).

Chromatography was developed at 4 ml/min using an FPLC (Pharmacia, Sweden) equipped with an online UV280 and conductivity monitor. The mobile phase was 10 mM Tris, 0.1M NaCl, 0.02% v/v Tween, pH 8.0. 10 ml fractions were collected between 12.5 min and 25 min to optimise recovery and removal of salt. Fractions were analysed by Biosensor analysis and pooled according to binding.

All pooled active fractions were diluted with an equal volume of 20 mM Tris, 0.02% Tween, pH 8.5 (Buffer B) and then loaded onto a Mono Q 5/5 (Pharmacia, Sweden) at a flow rate of 2 ml/min. The column was washed with buffer B. Elution was performed using a linear gradient between buffer B and buffer B containing 0.6M NaCl over min at a flow rate of 1 ml/min. Fractions (1 minute) were collected and analysed on the Biosensor and also by SDS PAGE and Western blot analysis. Fractions 15 to 26 (approximately 0.4M NaC1) appear to contain the majority of mNR6 as indicated by the Biosensor.

C. Production of CHO derived N' FLAG-mNR6 (monomeric form) Protein Production A cDNA fragment containing the entire coding sequence of murine NR6 with an N-terminal FLAGTM sequence was cloned into the expression vector pCHO1 for production of N- AMENDED SHEET 61 terminal FLAG-tagged protein. This vector contains a neomycin resistance gene with expression of the NR6 gene under the control of an EFla promoter. This expression construct was transfected into CHO cells using Lipofectamine (Gibco BRL, USA) according to the manufacturer instructions. Transfected cells were cultured in IMDM 10% FCS with resistant cells selected in geneticin (600ug/ml, Gibco BRL, USA) A neomycin resistant clone, secreting comparatively high' levels of NR6 was selected and expanded for further analysis.

(ii) Protein expression N' FLAG-NR6 expressed in serum free conditioned media litre) was harvested from transfected CHO and cells.

Collected media was concentrated using a CH2 ultrafiltration system equipped with a S1Y10 cartridge (Amicion molecular weight cut-off 10,000). Preliminary examination of the expressed product under reducing and non-reducing SDS PAGE followed by western blot analysis was performed. Visualisation of the protein on Westerns was specific to the primary antibody anti FLAG M2. Under reducing conditions a band approximately at 65,000 daltons was observed. Under non-reducing conditions, dimer and larger molecular weight aggregates were observed. These are disulphide linked monomers as they are not present in the reducing gel. Small amounts of monomer appear to be present in non-reducing gels.

(iii) Affinity Chromatography of NR6 Concentrated conditioned media was applied to an anti FLAG M2 affinity resin (100 x 16 mm After washing the unbound proteins off the column, the bound proteins were eluted using FLAG peptide (60Ag/ml) in PBS.

62 (iv) Ion Exchange Chromatography of NR6 Eluted fractions from affinity column were dialysed overnight against 20 mM Tris-HC pH 8.5 (buffer C) containing 50 mM Dithiothretol (DTT) using 25,000 cutoff dialysis tubing (Spectra/Por7, Spectrum). The dialysed fractions were loaded onto Mono Q (Pharmacia. Sweden) previnoisl~y eihli-brated .rith biffa C containing 5 mM DTT. Chromatography was developed using a linear gradient between buffer C and buffer C containing 1.0 M NaC1 at a flow rate of 0.5 ml min.

Refolding of NR6 Fractions containing NR6 from the Mono Q were adjusted to 50 mM DTT and left overnight at 4 0 C. To initiated refolding the sample was then dialysed against 50 mM Tris-HCl (pH 2 M Urea, 0.1% Tween 20, 10 mM Glutathione (reduced) and 2 mM Glutathione (oxidised) at a final protein concentration of 100 Ag/ml. Folding was carried out at ambient temperature with one change of the buffer over 24 hours.

Reversed Phase High Performance Liquid Chromatography (RP-HPLC) The folded product was further purified by RP-HPLC using a Vydac C4 resin (250 x 4.6 mm previously equilibrated with 0.1% Trifluoroacetic acid (TFA).

Elution was carried out using a linear gradient from 0 to 80% acetonitrile 0.1% TFA at a flow rate of 1 ml per minute.

D. pCHO1/NR6/FLAG In order to determine the native N termini of NR6, a C terminal FLAG NR6 CHO cell line was established.

AMENCF- SHEET 63 The plasmid pKUSA166 (murine NR6 cDNA cloned into the EcoR I site of pBLUESCRIPT) was digested with BamH I to remove the sequences encoding the last 15 amino acids of murine NR6. Synthetic oligonucleotides which encode the 3' end of mouse NR6 followed by the FLAG peptide tag were annealed and ligated into the BamH I site of pKUSA166. The sequence of the oligonucleotides was as follows:- I L P S G R R G A A R G P A G D Y K D D D D K [SEQ ID NO:34]

GATCTTGCCCTCGGGCAGACGGGGTGCGGCGAGAGGTCCTGCCGGCGACTACAAGG

ACGACGATGACAAGTA G [SEQ ID NO:33]

AACGGGAGCCCGTCTGCCCCACGCCGCTCTCCAGGACGGCCGCTGATGTTCCTGCT

GCTACTGTTCATCCTAG [SEQ ID The 5' end of the linker introduces a silent mutation (CTG TTG), to destroy the 5' BamH I site upon insertion of the linker. The NR6 cDNA (with native signal sequence) with the C-terminal FLAG was cut out of pKUSA166 with EcoR I and BamH I and cloned into the EcoR I BamH I cloning sites of pCHO-1. This vector results in the secretion of NR6 protein with a C-terminal flag tag FLAG-mRN6).

This vector results in the secretion of NR6 protein from KUSA cells. The vector pCHO1 has been previously described in (17) although with a different secretable marker.

Production of polyclonal NR6 antiserum The following peptide from the N terminal area of NR6 was chosen for production of polyclonal antiserum to NR6 VISPQDPTLLIGSSLQATCSIHGDTP [SEQ ID NO:39] CSH~r 64 The peptide was conjugated to KLH and injected into rabbits. Production and purification of the polyclonal antibody specific to the NR6 peptide sequence follows standard methods.

(ii) Protein expression KUSA cells transfected with cDNA of C terminal taaad mNR6 were grown to confluence in flasks (800ml) using IMDM media containing 10% FBS. Conditioned media (100 ml) was harvested 3 -4 days post confluence.

(iii) Characterisation of NR6 by Immunoprecipitation and Western blotting In order to establish that NR6 with the predicted sequence is produced in KUSA cells transfected with the cDNA, western blot analysis using both M2 antibody and purified NR6 specific rabbit antibody were performed.

Conditioned media (1 to 5 ml) was immunoprecipitated with M2 affinity resin (10-20 A1). Then after sufficient time for binding, the beads were washed with MT-PBS and subsequently NR6 eluted with 100 Ag/ml FLAG peptide pl, 5 minute incubation). The sample was then subjected to reducing and non reducing SDS PAGE followed by western blot analysis. Both purified NR6 polyclonal antibody (purified by protein G) and M2 antibody recognise a band under reducing conditions of a molecular weight size approximately 65,000 daltons.

Since the two antibodies reconising resides at the N terminus and C terminus it is reasonable to assume that full length NR6 is produced. Biotinylation of the respective antibodies by standard methods reduces the background. Under non-reducing conditions polyclonal NR6 bind antibodies to a band of a molecular weight of approximately 127,000, consistent with a dimeric NR6 disulphide linked form. Minor components of tetrameric 65 NR6 are present, no monomeric NR6 is evident using polyclonal NR6 antibodies.

EXAMPLE Generation of NR6 knockout mice To construct the NR6 targeting vector, 4.1kb of genomic T\Pn DNA containing exon 2 through tn wa deleted rnd replaced with G418-resistance cassette, leaving 5' and 3' NR6 arms of 2.9 and 4.5 kb respectively. A 4.5 kb Xhol fragment of the murine genomic NR6 clone 2.2 (Figure 3) containing exons 7, 8 and 3' flanking sequence was subcloned into the XhoI site of pBluescript generating pBSNR6Xho4.5. A 2.9kb NotI-Stul fragment within NR6 intron 1 from the same genomic clone was inserted into NotI and EcoRV digested pBSNR6Xho4.5 creating pNR6-Ex2-6. This plasmid was digested with ClaI, which was situated between the two NR6 fragments, and following blunt ending, ligated with a blunted 6kb HindIII fragment from placZneo, which contains the lacZgene and a PGKneo cassette, to generate the final targeting vector, pNR61acZneo. pNR61acZneo was linearised with NotI and electroporated into embryonic stem cells. After 48 hours, transfected cells were selected in 175 Ag/ml G418 and resistant clones picked and expanded after a further 8 days.

Clones in which the targetting vector had recombined with the endogenous NR6 gene were identified by hybridising Spel-digested genomic DNA with a 0.6 kb XhoI-StuI fragment from genomic NR6 clone 2.2. This probe (probe A, Figure which is located 3' to the NR6 sequences in the targeting vector, distinguished between the endogenous (9.9 kb) and targeted (7.1 kb) NR6 loci (Figure AMENDED

SHEET

66 Genomic DNA was digested with Spel for 16hrs at 37 0

C,

electrophoresed through 0.8% agarose, transferred to nylon membranes and hybridised to "P-labelled probe in a solution containing 0.5M sodium phosphate, 7% (w/v) SDS, ImM EDTA and washed in a solution containing sodium posphate, 1% SDS at 65 0 C. Hybridising bands were visualised by autoradiography for 16 hours at 0 C using Kodak XAR-5 film and intensifyina screenc Two targeted ES cell clones, W9.5NR6-2-44 and W9.5NR6-4- 2, were injected into C57B1/6 blastocysts to generate chimeric mice. Male chimeras were mated with C57B1/6 females to yield NR6 heterozygotes which were subsequently interbred to produce wild-type (NR6' heterozygous (NR6' and mutant (NR6 mice. The genotypes of offspring were determined by Southern Blot analysis of genomic DNA extracted from tail biopsies.

Genotyping of mice at weaning from matings between NR'/heterozygous mice derived from both targated ES cell clones revealed an absence of homozygous NR6 mutants.

As no unusual loss of mice was observed between birth and weaning, this suggest that lack of NR6 is lethal during embryonic development or immediately after birth.

Genotyping of embryonic tissues at various stages of development suggests that death occurs late in gestation (beyond day 16) or at birth.

EXAMPLE 16 Oligonucleotides 1943: GTC CAA GTG CGT TGT AAC CCA 3' 2070: GCT GAG TGT GCG CTG GGT CTC ACC 3' 2057: 5' GGC TCC ACT CGC TCC AGA 3' AMuE1.pC SlIEC 67 Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in specif ica i.dividuall or c ollecti, any and all combinations of any two or more of said steps or features.

3 l~L~)F; 68

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AMENDED

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70- SEQUENCE LISTING GENERAL INFORMATION: APPLICANT:(Other than US) AMRAD OPERATIONS PTY

LTD

(US only) Douglas James HILTON, Nicos Antony NICOLA, Alison FARLEY, Tracey WILLSON, Jian-Guo ZHANG, Warren ALEXANDER, Steven RAKAR, Louis FABRI, Tetsuo KOJIMA, Masatsugu MAEDA, Yasumfumi KIKUCHI, Andrew

NASH

(ii) TITLE OF INVENTION: A NOVEL HAEMOPOIETIN RECEPTOR AND GENETIC SEQUENCES ENCODING SAME (iii) NUMBER OF SEQUENCES: 39 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: DAVIES COLLISON CAVE STREET: 1 LITTLE COLLINS STREET CITY: MELBOURNE STATE: VICTORIA COUNTRY: AUSTRALIA ZIP: 3000 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: PCT INTERNATIONAL APPLICATION AMENDED

SHEET

71 FILING DATE: 11-SEP-1997 (vi) PRIOR APPLICATION DATA: APPLICATION NUMBER: P02246/96 FILING DATE: 11-SEP-1996 (viii) ATTORNEY/AGENT INFORMATION: NAME- HUGH ES DR, E JOHN L REFERENCE/DOCKET NUMBER: EJH/AF (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: +61 3 9254 2777 TELEFAX: +61 3 9254 2770 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: Trp Ser Xaa Trp Ser INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear AMF~rhICFD ;H~7Ett 72 (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: ACTCGCTCCA GATTCCCGCC TTTT INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: TCCCGCCTTT TTCGACCCAT AGAT INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA 73 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: GGTACTTGGC TTGGAAGAGG AAAT 24 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TVDYPE: ncloc -id STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID CGGCTCACGT GCACGTCGGG TGGG 24 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: AGCTGCTGTT AAAGGGCTTC TC 22 AMENDED SHEET 74 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 15 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE-DESCRIPTION: SEQ ID NO:7: (A/G)CTCCA(A/G)TC(A/G) CTCCA INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 15 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: (A/G)CTCCA(C/T)TC(A/G) CTCCA INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs AMENDED

SHEET

75 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: AAGTGTGACC ATCATGTGGA C INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID GGAGGTGTTA AGGAGGCG INFORMATION FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid

SNEDSHEET

76 STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA C ~WTPMCv 7)L!PTDTCrn,. CLI Trn V :-1 ATGCCCGCGG GTCGCCCG INFORMATION FOR SEQ ID NO:12: Wi SEQUEN CE CHARACTERISTICS: LENGTH: 1506 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: NAM4E/KEY:

LOCATION:

CDS

1. .1242 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: GGCACGAGCT TCGCTGTCCG CGCCCAGTGA CGCGCGTGCG GACCCGAGCC CCAATCTGCA -64 CCCCGCAGAC TCGCCCCCGC CCCATACCGG CGTTGCAGTC ACCGCCCGTT GCGCGCCACC -4 ccc -3 ATG CCC GCG GGT CGC CCG GGC CCC GTC GCC CAA TCC GCG CGG CGG CCG 48 ~AENDED SHEE 77 Met Pro Ala Gly Arg Pro Gly Pro Val Ala Gin Ser Ala Arg Arg Pro is CCG CGG CCG CTG SPro Arg Pro Leu Gly Val Pro Arg 0 35 TCC TCG CTG TGG TCG CCT CTG TTG CTC TGT GTC CTC Ser Ser Leu Trp Ser Pro Leu Leu Leu Cys Val Leu 25 Gly Gly Ser Gly Ala His Thr Ala Val Ile Ser Pro 40 CAG GAC Gin Asp ATA CAT Ile His CCC ACC CTT CTC ATC Pro Thr Leu Leu Ile 55 GGC*TCC TCC CTG tAA GCT ACC TGC TCT Gly Ser Ser Leu Gin Ala Thr Cys Ser GGA GAC ACA Gly Asp Thr CCT GGG GCC ACC GCT GAG Pro Gly Ala Thr Ala Glu 70 75 CTG CCC TCT GAG CTG TCC Leu Pro Ser Glu Leu Ser 90 GGG CTC TAC TGG ACC Gly Leu Tyr Trp Thr CGC CTC CTT AAC ACC Arg Leu Leu Asn Thr CTC AAT GGT CGC CGC Leu Asn Gly Arg Arg TCC ACC CTG GCC CTG Ser Thr Leu Ala Leu 100 240, 288 336 GCC CTG GCT AAC Ala Leu Ala Asn 105 CTT AAT GGG TCC AGG CAG CAG Leu Asn Gly Ser Arg Gin Gin 110 TCA GGA GAC Ser Gly Asp 115 AAT CTG GTG TGT CAC Asn Leu Val Cys His 120 GCC CGA GAC GGC AGC Ala Arg Asp Gly Ser .125 ATT CTG OCT Ile Leu Ala GGC TCC Gly Ser 130 TGC CTC TAT GTT Cys Leu Tyr Val

GGC

Gly 135 TTG CCC CCT GAG AAG Leu Pro Pro Giu Lys 140 CCC TTT AAC ATC 432 Pro Phe Asn 7le* -78 AGC TGC TGG TCC CGG AAC ATG AAG GAT CTC ACG Ser Cys Trp Ser Arg Asn Met Lys Asp Leu Thr 145 150 155 GGT GCA CAC GGG GAG ACA TTC TTA CAT ACC AAC Gly Ala His Gly Glu Thr Phe Leu His Thr Asn 165 170 AAG CTG AGG TGG TAC GGT CAG GAT AAC ACA TGT Lys Leu Arg Trp Tyr Gly Gin Asp Asn Thr Cys 180 185 GTG GGC CCT CAC TCA TGC CAT ATC CCC AAG GAC Val Gly Pro His Ser Cys His Ile Pro Lys Asp 195 .200 CCC TAT GAG ATC TGG GTG GAA GCC ACC AAT CGC Pro Tyr Glu Ile Trp Val Glu Ala Thr Asn Arg 210 215 TCT GAT GTC CTC ACA CTG GAT GTC CTG GAC GTG Ser Asp Val Leu Thr Leu Asp Val Leu Asp Val 225 230 235 TGC CGC TGG ACA CCG Cys Arg Trp Thr Pro 160 TAC TCC CTC AAG TAC Tyr Ser Leu Lys Tyr 175 GAG GAG TAC CAC ACT Glu Glu Tyr His Thr 190 CTG GCC Leu Ala 205 CTC TTC ACT 624 Leu Phe Thr CTA GGC TCA GCA AGA Leu Gly Ser Ala Arg 220 GTG ACC ACG GAC CCC Val Thr Thr Asp Pro 240 CCA CCC GAC GTG CAC Pro Pro Asp Val His 245 GTG AGC CGC GTT Val Ser Arg Val TCA CCA CCA GCT Ser Pro Pro Ala 265 GGG GGC CTG GAG GAC CAG CTG Gly Gly Leu Glu Asp Gin Leu 250 255 CTC AAG GAT TTC CTC TTC CAA Leu Lys Asp Phe Leu Phe Gin 270 768 816 AGT GTG CGC TGG GTC Ser Val Arg Trp Val 260 GCC AAG TAC Ala Lys Tyr 275 CAG ATC CGC TAC CGC Gin Ile Arg Tyr Arg 280 GTG GAG GAC Val Glu Asp AGC GTG GAC TGG AAG Ser Val Asp Trp Lys 285 79 GTG GTG Vai Val 290 GAT GAC GTC AGC Asp Asp Val Ser CAG ACC TCC Gin Thr Ser TGC CGT CTC GCG GGC CTG Cys Arg Leu Ala Gly Leu 300

AAG

Lys 305 CCC GGC ACC GTT TAC Pro Gly Thr Val Tyr 310 TTC GTC CAA GTG CGT Phe Val Gin Val Arg 315 TGT AAC CCA TTC GGG Cys Asn Pro Phe Gly 320 ATC TAT GGG TCG AAA AAG GCG GGA ATC TGG AGC GAG TGG AGC Ile Tyr Gly Ser Lys Lys Ala Gly Ile Trp Ser.Glu Trp Ser 325 330 CAC CCC His Pro 335 1008 ACC GCT GCC TCC Thr Ala Ala Ser 340.

ACC CCT CGA AGT Thr Pro Arg Ser GAG CGC CCG GGC CCG GGC GGC GGG Giu Arg Pro Gly Pro Gly Giy Gly 345 350 CCC AGC. TCG GGC CCG GTG CGG CGC Pro Ser Ser Gly Pro Vai Arg Arg 365 1056 1104 GTG TGC GAG Val Cys Glu 355 CCG CGG GGC GGC GAG Pro Arg Gly Gly Giu 360 GAG CTC Glu Leu .370 AAG CAG TTC CTC GGC Lys Gin Phe Leu Gly 375 TGG CTC AAG AAG CAC GCA TAC TGC TCG Trp Leu Lys Lys His Ala Tyr Cys Ser 380 1152

AAC

Asn 385 CTT AGT TTC CGC CTG Leu Ser Phe Arg Leu 390 TAC GAC CAG, TGG CGT Tyr Asp Gin Trp Arg 395 GCT TGG ATG CAG AAG Ala Trp Met Gin Lys 400 1200 TCA CAC AAG ACC CGA AAC CAG Ser His Lys Thr Arg Asn Gin' 405 GTC CTG CCG GCT AAA CTC Val Leu Pro Ala Lys Leu 410 TAAGGATAGG 1249 CCATCCTCCT GCTGGGTCAG ACCTGGAGGC TCACCTGAAT TGGAGCCCCT CTGTACCATC 1309 TGGGCAACAA AGAAACCTAC CAGAGGCTGG GGCACAATGA GCTCCCACAA CCACAGCTTT 1369 GGTCCACATG ATGGTCACAC TTGGATATAC CCCAGTGTGG GTAAGGTTGG GGTATTGCAG 1429 GGCCTCCCAA CAATCTCTTT AAATAAATAA AGGAGTTGTT CAGGTAAAAA AAAAAAAAAA 1489 AAAAAA AAA 1506 INFORMATION FOR SEQ ID NO:i3: LENGTH: 413 amino acids TYPE: amino acid TOPOLOGY: linear (xi)

MOLECULE

SEQUENCE

TYPE: protein DESCRIPTION: SEQ 1s met Pro

ID

Ser Pro Ala Gly Arg Pro Gly Pro Val Ala Gin Arg Pro Leu Ser Ser Leu Trp Ser 25 Ala Pro Leu Leu NO: 13: Ala Arg Arg Pro Leu Cys Vai Leu Val Ile Ser Pro Ala Thr Cys Ser Gly Vai Pro Gin Asp Pro Arg Thr Gly Giy Ser Leu Leu Ile Thr Pro Gly His Thr Ala Gly Ser Ser Leu Ile His 65 Gly Asp Ala Thr Ala Giu 75 Leu Tyr Trp Thr Leu Asn Giy Arg Arg Leu Pro Ser Giu Leu .90 Ser Arg Leu Leu Asn Thr Gin Gin Ser Thr Leu Leu Ala Leu Ala As n 105 Leu Asn Giy Ser AMENDED

SHEET

81 Ser Gly Asp 115 Asn Leu Val Cys His 120 Ala Arg Asp Gly Ile Leu Ala Gly Ser 130 Cys Leu Tyr Val Gly 135 Leu Pro Pro Gl u Lys Pro Phe Asn Ile 140 Ser Cys Trp Ser Arg Asn Met Lys Asp Leu Thr Cys Arg Trp Thr Pro Gly Ala His Giy Thr Phe Leu His Thr 170 Asn Tyr Ser Leu Lys Tyr 175 Lys Leu Arg Val Gly Pro 195 Tyr Gly Gin Asp Asn 185 Thr Cys Glu Glu Tyr His Thr 190 Leu Phe Thr His Ser Cys His Ile 200 Pro Lys Asp Leu Pro Tyr 210 Glu Ile Trp Val Ala Thr Asn Arg Gly Ser Ala Arg Ser 225 Asp Vai Leu Thr Leu 230 Asp Val Leu Asp Val1 235 Val Thr Thr Asp Pro 240 Pro Pro Asp Val His 245 Val Ser Arg Val Gly 250 Gly Leu Glu Asp Gin Leu 255 Ser Val Arg Ala Lys Tyr Val Ser Pro Pro Al a 265 Leu Lys Asp Phe Leu Phe Gin 270 Asp Trp Lys Gin Ile Arg Tyr Arg 280 Val Giu Asp Ser Val 285 Val Val 290 Asp Asp Val Ser As n 295 Gin Thr Ser Cys Arg 300 Leu Ala Gly Leu AMENDED

SHEET

82- Lys 305 Pro Gly Thr Val Tyr Phe Val Gin Val 310 Arg 315 Cys Asn Pro Phe Ile Tyr Giy Ser Lys 325 Lys Ala Gly Ile Trp 330 Ser Glu Trp Ser His Pro 335 Thr.Ala Ala Ser Thr Pro Arg Ser Giu Arg Pro Gly Pro 345 Gly Gly Gly Val Arg Arg Val Cys Giu 355 Pro Arg Gly Gly Giu 360 Pro Ser Ser Gly Giu Leu 370 Lys Gin Phe Leu Gly 375 Trp Leu Lys Lys His Aia Tyr Cys Ser 380 Asn 385 Leu Ser Phe Arg Leu Tyr Asp Gin Trp, .390 Arg 395 Ala Trp Met Gin Lys 400 Ser His Lys Thr Arg 405 Asn Gin Val Leu Pro 410 Ala Lys Leu INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 1549 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: A) NAME/KEY:

LOCATION:

CDS

1. .1278 AMENDE L 83 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: GGOAOGAGCT TCGCTGTOOG CGCCCAGTGA CGCGCGTGCG GAOOCGAGCC CCAATCTGCA CCCGCAGAC TCGCCCOGO CCOATAOCGG CGTTGCAGTC ACCGCCCGTT GCGCGCCACC

ATG

Met 1.

CCC GCG Pro Ala GGT CGC Gly Arg CCG GGC CCC GTC Pro Gly Pro Val GCC CAA TCC GCG OGG CGG COG Ala Gin Ser Ala Arg Arg Pro 210 OCG CGG COG Pro Arg Pro GGG GTG COT Gly Val Pro OTG TOO TCG CTG TGG TOG OCT OTG TTG CTO TGT GTC CTC Leu Ser Ser Leu Trp Ser Pro Leu Leu Leu Cys Val Leu 25 OGG GGO GGA TOG GGA GOC CAC ACA GOT GTA ATO AGO 000 Arg Gly Gly Ser Gly Ala His Thr Ala Val Ile Ser Pro.

40 CAG GAO Gin Asp ATA OAT Ile His COO ACC OTT OTO ATO Pro Thr Leu Leu Ile 155 GGO TOO TOO CTG CAA GOT ACC TGO TOT Giy Ser Ser Leu Gin Aia Thr Cys Ser GGA GAO ACA OCT Gly Asp Thr Pro 70 GGG GOC ACC GOT GAG GGG Gly Ala Thr Ala Glu Gly 75F OTO TAO TGG ACC Leu Tyr Trp Thr s0

OTO

Leu AAT GGT OGO OGO Asn Gly Arg Arg CTG CCC TOT GAG CTG Leu Pro Ser Glu Leu 90 TOO OGO OTO OTT AAC ACC Ser Arg Leu Leu Asn Thr AAT GGG TOO AGG CAG CAG Asn Gly Ser Arg Gin Gin 110

TOO

Ser ACC CTG GC Thr Leu Ala 1.00 OTG GOC CTG GOT Leu Ala Leu Ala AAO OTT Asn Leu 105 84 GGC AGC ATT CTG GCT Gly Ser Ile Leu Ala 125 384 TCA GGA GAC AAT CTG GTG TGT CAC GCC CGA GAC Ala Arg Asp Ser Gly Asp 115 Asn Leu Val Cys GGC TCC Gly Ser 130 TGC CTC TAT GTT GGC Cys Leu Tyr Val Gly 135 TTG CCC CCT GAG AAG CCC TTT AAC ATC Leu Pro Pro Glu Lys Pro Phe Asn Ile 140 432

AGC

Ser 145 TGC TGG TCC CGG AAC Cys Trp Ser Arg Asn 150 ATG AAG GAT CTC ACG Met Lys Asp Leu Thr 155 TGC CGC TGG ACA CCG Cys Arg Trp Thr Pro 160 480 GGT GCA CAC GGG GAG Gly Ala His Gly Glu 165 AAG CTG AGG TGG TAC Lys Leu Arg Trp Tyr 180.

GTG GGC CCT CAC TCA Val Gly Pro His Ser' 195 ACA TTC TTA CAT ACC Thr Phe Leu His Thr 170 GGT CAG GAT AAC ACA Gly Gin Asp Asn Thr 185 AAC TAC TCC CTC AAG TAC Asn Tyr Ser Leu Lys Tyr 175 TGT GAG GAG TAC CAC ACT Cys Giu Giu Tyr His Thr 190 TGC CAT ATC Cys His Ile 200 CCC AAG GAC CTG GCC CTC TTC ACT Pro Lys Asp Leu Ala Leu Phe Thr 205 CCC TAT Pro Tyr 210 GAG ATC TGG GTG GAA Glu Ile Trp Val Glu 215 GCC ACC AAT CGC CTA GGC TCA GCA AGA Ala Thr Asn Arg Leu Giy Ser Aia Arg 220

TCT

Ser 225 GAT GTC CTC ACA CTG Asp Vai Leu Thr Leu 230 GAT GTC CTG GAC GTG GTG ACC ACG GAC CCC Asp. Val Leu Asp Vai Val Thr Thr Asp Pro 235 240 CCA CCC GAC GTG Pro Pro Asp Val

CAC

His 245 GTG AGC CGC GTT Val Ser Arg Val

GGG

Gly 250 GGC CTG GAG GAC CAG CTG Gly Leu Giu Asp Gin Leu 255 .i -1 r I too

I..

S.

85 AGT GTG Ser Val CGC TGG Arg Trp, 260 GTC TCA CCA CCA Val Ser Pro Pro

GCT

Al a 265 CTC AAG GAT TTC CTC TTC CAA Leu Lys Asp Phe Leu Phe Gin 270 816 GCC AAG TAC Ala Lys Tyr 275 GTG GTG GAT Val Val Asp 290 CAG ATC CGC TAC CGC GTG GAG GAC AGC GTG GAC TGG AAG Gin Ile Arg Tyr Arg Vai Giu Asp Ser Val Asp Trp Lys 280 285 GAC GTC AGC AAC CAG ACC TCC TGC CGT CTC GCG GGC CTG Asp Vai Ser Asn Gin Thr Ser Cys Arg Leu Ala Gly Leu 295 300

AAG

Lys 305 CCC GGC ACC GTT TAC Pro Gly Thr Val Tyr 310 TTC GTC CAA GTG CGT Phe Val Gin Val Arg 315 TGT AAC CCA TTC GGG Cys Asn Pro Phe Gly 320 960 ATC TAT GGG.TCG AAA Ile Tyr Gly Ser Lys S325 AAG GCG GGA ATC TGG Lys Ala Gly Ile Trp 330 AGC GAG TGG AGC CAC CCC Ser Glu Trp Ser His Pro 335 1008 ACC GCT GCC TCC Thr Ala Ala Ser 340 ACC CCT CGA AGT GAG Thr Pro Arg Ser Giu 345 CGC CCG GGC Arg Pro Gly CCG GGC GGC GGG Pro Gly Gly G ly 350 1056 GTG TGC GAG Vai Cys Glu 355 GAG CTC AAG Giu Leu Lys 370 CCG CGG GGC GGC GAG CCC AGC TCG GGC CCG GTG CGG CGC Pro Arg Gly Gly Giu Pro Ser Ser Gly Pro Val Arg Arg 360 365 CAG TTC CTC GGC TGG CTC AAG AAG, CAC GCA TAC TGC TCG Gin Phe Leu Gly Trp Leu Lys Lys His Ala Tyr Cys Ser 375 380 1104 1152

AAC

Asn 385 CTT AGT TTC CGC CTG TAC Leu Ser Phe Arg Leu Tyr 390 GAC CAG TGG Asp Gin Trp

CGT

Arg 395 GCT TGG ATG CAG AAG Ala Trp Met Gln Lys 400 1200 AMENDED

SHEET

-86- TCA CAC AAG ACC CGA AAC CAG GAC GAG GGG ATC CTG CCT TCG GGC AGA Ser His Lys Thr Arg Asn Gin Asp Glu Gly Ile Leu Pro Ser Giy Arg 405 410 415 CGG GGT GCG GCG AGA GGT CCT GCC GGT TAAACTCTAA GGATAGGCCA Arg Gly Ala Ala Arg Gly Pro Ala Gly 420 425 1248 1295

TCCTCCTGCT

GCAACAAAGA

CCACATGATG

CTCCCAACAA

G(3(TCAGACC TGGAGGCTCA CCTGAATTGG AGCCCCTCTG TACCATCTGG AACCTACCAG AGGCTGGGGC ACAATGAGCT CCCACAACCA CAGCTTTGGT GTCACACTTG GATATACCCC AGTGTGGGTA AGGTTGGGGT ATTGCAGGGC TCTCTTTAAA TAAATAAAGG AGTTGTTCAG GTAAAAAAAA ~AAAAnAA

AAA

1355 1415 1475 1535 1549 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 425 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Met Pro Ala Gly Arg Pro Gly Pro Val Ala Gin Ser Ala Arg Arg Pro 1 5 10 Pro Arg Pro Leu Ser Ser Leu Trp Ser Pro Leu Leu Leu cys Val Leu 25 p.ATKwED SHEET 87 Gly Val Pro Gin Asu Pro Arg Gly Gly Ser Gly Ala 40 Gly Ser Thr Leu Leu Ile His Ile Gly His Thr Ala Ser Leu Gin Ala Glu Gly Val Ala Ile Ser Pro Thr Cys Ser Gly Asp Thr Ala Thr Leu Tyr Trp, Leu Asn Gly Arg Arg Leu Pro Ser Glu Leu 90 Leu Arg Leu Leu Asn Thr Ser Thr Leu Ala Leu Ala Asn 105 Ala Asn Gly Ser Ser Gly Asp 115 Gly Ser Cys 130 Leu ValCys Arg Asp Gly Ser 125 Pro Arg Gin Gin 110 Ile Leu Ala Phe Asn Ile Leu Tyr Val Gly 135 Met Pro Pro Giu Ser 145 Giy Cys Trp Ser Arg As n 150 Thr Lys Asp Leu Thr 155 As n Arg Trp Thr Pro 160 Ala His Gly Giu 165 Tyr Phe Leu His Thr 170 Thr Tyr Ser Leu Lys Leu Arg Giy Gin Asp Asn 185 Pro Cys Giu Glu Tyr 19.0 Leu Lys T-yr 175 His Thr Phe Thr Val Gly Pro 195 Ser Cys His Ile 200 Lys Asp Leu Pro Tyr Giu Ile Trp Val Glu Ala Thr Asn Arg Leu Giy Ser A.1a Arg 210 3 10215 220 88 Leu Asp Val Val Thr Thr Ser Asp Val Leu Thr Leu Asp Val Pro Asp Val 230 His Val Ser Arg Val 245 Val Ser Pro Pro Ala Asp Pro 240 Gin Leu 255 Phe Gin Gly 250 Leu Gly Leu Glu Asp Ser Val Arg Trp Gin Lys Asp Phe Leu Ala Lys Tyr 275 Val Val Asp Ile Arg Tyr Val Glu Asp Ser Val1 285 Leu Asp Trp Lys Ala Gly Leu Asp Val Ser As n 295 Phe Thr Ser Cys Lys 305 Ile Gly Thr Val Val Gin Val Arg 315 Ser Asn Pro Phe Gly 320 Tyr Gly Ser Lys Lys Ala Gly 325 Thr Pro Arg Ser Ile Trp 330 Arg Glu Trp Ser His Pro 335 Thr Ala Ala Glu 345 Pro Gly Pro Val Cys Glu 355 Glu Leu Lys Arg Gly Gly Pro Ser Ser Gly Pro 365 Al a Gly Gly Gly 350 Val Arg Arg Tyr Cys Ser Gin Phe Leu 370 Leu Gly 375 Tyr Leu Lys Lys Asn 385 Ser Ser Phe Arg Asp Gin Trp Arg 395 Ile Trp Met Gin Lys 400 Arg His Lys Thr Arg 405 Gin Asp Glu Gly 410 Leu Pro Ser Gly 415 -89 Arg Gly Ala Ala Arg Gly Pro Ala Gly 420 425 INFORMATION FOR SEQ I'D NO:16: LENGTH: 938 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE:

NAME/KEY:

LOCATION:

CDS

l. .468 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l6:

GGC

Gly

GGG

Gly ACC GTT TAG TTC GTC CAA GTG CGT TGT AAC CCA TTC GGG ATC TAT Thr Val Tyr Phe Val Gin ValArg Cys Asn Pro Phe Gly Ile Tyr 10 TCG AAA AAG GCG GGA ATC TGG AGC GAG TGG AGC CAC CCC ACC GCT Ser Lys Lys Ala Gly :2 5 Ile Trp Ser Giu Trp Ser His Pro Thr Ala Gcc Tcc ACC Ala Ser Thr ccT CGA Pro Arg AGT GAG cGIc Ser Giu Arg 40 ccG GGC ccG GGC GGC GGG GTG TGC Pro Gly Pro Gly Gly Gly Val cys 144 GAG CCG Glu Pro CGG GGC GGC GAG CCC Arg Gly Gly Glu Pro 55 90 AGC TCG GGC Ser Ser Gly CCG GTG Pro Val CGG CGC GAG CTC Arg Arg Glu Leu

AAG

Lys

AGT

Ser

CAG

Gin TTC CTC GGC Phe Leu Gly TTC CGC CTG TAC Phe Arg Leu Tyr TGG CTC AAG AAG Trp Leu Lys Lys 70* GAC CAG TGG CGT Asp Gin Trp Arg GTA GGA AAG TTG Val Gly Lys Leu 105 GAG AGA GAC CCG Glu Arg Asp Pro CAC GCA His Ala 75

TAC

Tyr TOC TCG AAC CTT Cys Ser Asn Leu AAG ACC CGA AAC Lys Thr Arg Asn 100 AAA GGA GCA GAG Lys Gly Ala Glu 115 CGC ACT CTT CTT Arg Thr Leu Leu 130 GAC GGG GTG CGG Asp Gly Val Arc

CAG

Gin GCT TGG ATG CAG AAG TCA CAC Ala Trp Met Gin Lys Ser His 90 GGG GAG GCT TGC GTG GGG GGT Gly Glu Ala Cys Val Gly Gly 110 GGT GAG CAG CCT CCA CAA CAC Gly"Glu Gin Pro Pro Gin His 125 AGG GGA TCC TGC CCT CGG GCA Arg Gly Ser Cys Pro Arg Ala

GAA

Glu 192 240 288 336 384 432 485 545 605 665 725 785 120 TCC AAG CAC AGG Ser Lys His Arg 135

ACG

Thr 140 CGA GAG GTA Ara Glu Val 145

CTAGATG;

CACCCACI

TAATGTGC

TCCTGCTC

AACAAAG;

150 LGG CCCTTTCCCC T( .GC AAAGAGCCCC A( ;CC TCTTTTCTGC C( ;GG TCAGACCTGG A LAA CCTACCATGA G(

CCTTC

3GTTT

CTCAG

3GCTC .7CTGG AGG GGG TCT GGG Arg Gly Ser Gly 155 GGTG TTGCTCAAAG, TACT GCATCATCAA GTCC TdCCGGCTA.A ACCT GAATTGGAGC GGCA CAATGAGCTC TGAGTGGGGC CTACAGCAGT

GGATCTCTTA

GTTGCTGAAG

ACTCTAAGGA

CQCTCTGTAC

CCACAACCAC

GTGCTCATTT

GGTCCAGGCT

TAGGCCATCC

CTATCTGGGC

AGCTTTGGTC

91- CACATGATGG TCACACTTGG ATATACCCCA GTGTGGGTAA GGTTGGGGTA TTGCAGGGCC TCCCAACAAT CTCTTTAAAT AAATAAAGGA GTTGTTCAGG TAAAAAAAAA AAAAA AAAAA AAA 0 ()SEQUENCE CHARACTERISTICS: LENGTH: 155 amino acids TYPE: amino acid TOPOLOGY: linear 845 905 938 (xi) Thr Val

MOLECULE

SEQUENCE

Tyr Phe Val 5 Lys Ala Gly TYPE: protein DESCRIPTION: SEQ ID NO: 17: Gly 1 Gly Gin Val Arg Ser Lys Ile Trp Ser 25 Pro cys Asn Pro 10 Giu Trp Ser Gly Pro Gly His Pro Thr Ala Gly Val Cys Phe Gly Ile Tyr is Ala Ser Thr Giu Pro Arg Pro Arg Ser Giu Gly Gly Gly Glu Gin Pro Ser Gly Pro Lys Phe Leu Gly Leu Lys Lys His Arg Arg Glu Leu cys Ser Asn Leu Gin Lys Ser His Ser Phe Arg Leu Tyr Asp Gin Trp Arg Ala 90 Trp Met

S

0*tS Z 92 Lys Thr Arg Asn 100 Gin Val Gly Lys Leu Gly Glu 105 Lys Gly Ala Glu Glu Glu Arg 115 Pro Gly Glu Ala Cys Val Gly Gly 110 Gin Pro Pro Gin His 125 Ser Cys Pro Arg Ala 14t) Arg Thr 130 Leu Leu Ser Lys His Arg Thr Arg Gly Asp 145 Gly Val Arg Arg Val Arg Gly Ser Gly 155 INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 834 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE:

NAME/KEY:

LOCATION:

CDS

834 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: ACC CTT CTC ATC GGC TCC TCC CTG CAA GCT ACC TGC TCT ATA CAT Thr Leu Leu Ile Gly Ser Ser Leu Gin Ala Thr Cys Ser Ile His

CCC

Pro 51 r ,i 93

GGA

Gly GAC ACA CCT Asp Thr Pro GGG GCC ACC GCT GAG GGG Gly Ala Thr Ala Glu Gly 75 CTC TAC TGG ACC CTC AAT Leu Tyr Trp Thr Leu Asn GGT CGC CGC Gly Arg Arg CTG CCC TCT GAG CTG Leu Pro Ser Giu Leu 90 TCC CGC CTC CTT AAC ACC TCC ACC Ser Arg Leu Leu Asn Thr Ser Thr CTG GCC CTG GCC CTG GCT AAC CTT AAT GGG TCC AGG CAG CAG TCA GGA Leu Ala Leu Ala Leu Ala Asn Leu Asn Gly Ser Arg Gin Gin Ser Gly 100 105 110

GAC

Asp 115 AAT CTG GTG TGT CAC AsnLeu Val Cys His 120 GCC CGA GAC GGC AGC Ala Arg Asp Gly Ser 125 ATT CTG GCT GGC TCC Ile Leu Ala Gly Ser 130 290 TGC CTC TAT GTT GGC Cys Leu Tyr Val Gly 135 TTG CCC CCT GAG AAG CCC TTT AAC ATC AGC TGC Leu Pro Pro Glu Lys Pro Phe Asn Ile Ser Cys 140 145 338 TGG TCC CGG Trp Ser Arg CAC GGG GAG His Gly Glu 205 AAC ATG AAG GAT Asn Met Lys Asp 150 ACA TTC TTA CAT Thr Phe Leu His CTC ACG Leu Thr 155 TGC CGC TGG ACA CCG GGT GCA Cys Arg Trp Thr Pro Gly Ala 200 ACC AAC TAC TCC CTC AAG TAC AAG CTG Thr Asn Tyr Ser Leu Lys Tyr Lys Leu 210 215 ACA TGT GAG GAG TAC CAC ACT GTG GGG Thr Cys Glu Giu Tyr His Thr Val Gly 230 AGG TGG Arg Trp 220 TAC GGT CAG GAT AAC Tyr Gly Gin Asp Asn 225

CCC

Pro 235 CAC TCA TGC CAT ATC His Ser Cys His Ile 240 CCC AAG GAC CTG GCC CTC TTC ACT CCC TAT Pro Lys Asp Leu Ala Leu Phe Thr Pro Tyr 245 250 94 GAG ATC TGG GTG GAA Glu Ile Trp Val Glu 255 GCC ACC AAT CGC CTA Ala Thr Asn Arg Leu 260 GGC TCA GCA AGA TCT GAT Gly Ser Ala Arg Ser Asp 265 GTC CTC ACA CTG Val Leu Thr Leu 270 GAT GTC CTG GAC GTG GTG ACC ACG GAC CCC CCA CCC Asp Val Leu Asp Val Val Thr Thr Asp Pro Pro Pro 275 280 GAC GTG CAC Asp Val His 285 GTG AGC CGC GTT GGG Val Ser Arg Val Gly 290 GGC CTG GAG GAC CAG CTG AGT GTG Gly Leu Glu Asp Gin Leu Ser Val 295 CGC TGG Arg Trp 300 GTC TCA CCA CCA GCT Val Ser Pro Pro Ala 305 CTC AAG GAT TTC CTC TTC CAA GCC AAG Leu Lys Asp Phe Leu Phe Gin Ala Lys 310 TAC CAG ATC CGC TAC Tyr Gin Ile Arg Tyr 315 GAT GAC GTC AGC AAC Asp Asp Val Ser Asn 335 CGC GTG GAG GAC AGC Arg Val Glu Asp Ser 320 CAG ACC TCC TGC CGT Gin Thr Ser Cys Arg 340 GTG GAC TGG AAG GTG GTG Val Asp Trp Lys Val Val 325 330 CTC GCG GGC CTG AAG CCC Leu Ala Gly Leu Lys Pro 345 AAC CCA TTC GGG ATC TAT Asn Pro Phe Gly Ile Tyr 360 GGC ACC GTT TAC Gly Thr Val Tyr 350 TTC GTC CAA GTG Phe Val Gin Val CGT TGT Arg Cys 355 GGG TCG AAA AAG GCG GGA Gly Ser Lys Lys Ala Gly 365 INFORMATION FOR SEQ ID NO:19: 894 SEQUENCE CHARACTERISTICS: LENGTH: 278 amino acids

EDSHEET

95 TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: Pro 51 Thr Leu Gly Asp Thr Gly Arg Arg Leu Ala Leu Leu Ile 55C Pro Gly Leu Pro Gly Ser Ser Leu Ala Thr Cys Ser Ile His Ala Thr Ala Glu 75 Ser Leu Tyr Trp Ser Glu Leu 90 Leu Arg Leu Leu Asn Gin Thr Leu Asn Thr Ser Thr Gin Ser Gly Ala Leu Ala Asn Gly Ser 100 Asn Asp 115 Cys Leu Val Cys Arg Asp Gly Ser 125 Pro Leu Ala Gly Ser 130 Leu Tyr Val Gly 135 Met Pro Pro Glu Phe Asn Ile Trp Ser Arg His Gly Giu 205 Arg Trp Tyr 220 Asn 150 Thr Lys Asp Leu Thr 155 Asn Arg Trp Thr Ser Cys 145 Gly Ala Lys Leu Phe Leu His Thr 210 Thr Tyr Ser Leu Lys 215 His Gly Gin Asp Cys Glu Giu Thr Val Gly Pro 235 His Ser Cys His Pro Lys Asp Leu Ala Leu Phe Thr Pro 245 Tyr 250 AmiNDE 0

SH-EET

Giu Ile Trp Val Giu Ala Thr Asn 255 96 Arg Leu 260 Val Val Gly Ser Ala Arg Ser Asp 265 hr Asp Pro Pro Pro Val Leu Thr Leu 270 Val Asp Val Leu Asp Thr T 275 Gly 280 Leu Ser Val Asp Val His Ser Arg Val Gly 290 Leu Giu Asp Arg Trp 300 Tyr Gin Val Ser Pro Pro Leu Lys Asp Phe Phe Gin Ala Lys Ile Arg Tyr 315 Asp Arg 320 Gin Val Giu Asp Thr Ser Cys Ser Val 325 Arg Leu 340 Trp Lys Val Asp Val Ser Asn 335 Ala Gly Leu Lys Pro 345 Ile Tyr Gly Thr Val Tyr-Phe Val 350 Gly Ser Lys'Lys Ala Gly 365 Gin Val Arg 355 Cys Asn Pro Phe Gly 360 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 143 base pairs TYPE: nucleic acids TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Amun'thD SH'ET 97 S GGCATGAAGG CTTAGGGTGG GGATCGGTAG GACCCATGCA CCCAGAGAAA GGGACTGGTG GCAACTTTCA AACTCTCTGG GGAAGGAAGA AGGGCTGAAA GAGG 104 ATG AAC GGG CTC AGA CAC AGC TGT AAT CAG CCC CCA GGA 143 Met Asn Gly Leu Arg His Ser Cys Asn Gin Pro Pro Gly INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 13 amino acids TYPE: amino acids TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2i: Met Asn Gly Leu Arg His Ser Cys Asn Gin Pro Pro Gly INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 1930 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA 311 98 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:22: GGCACGAGCT TCdCTGTCCG CCCCGCAGAC TCGCCCCCGC CCCAATGCCC GCGGGTCGCC GCTGTCCTCG CTGTGGTCGC GGGAGCCCAC ACAGCTGTAA GCAAGCTACC TGCTCTATAC GACCCTCAAT -GGTCGCCGCC GGCCCTGGCC CTGGCTAACC TCACGCCCGA GACGGCAGCA GAAGCCCTTT AACATCAGCT ACCGGGTGCA CACGGGGAGA GTGGTACGGT CAGGATAACA TATCCCCAAG GACCTGGCCC CCTAGGCTCA GCAAGATCTG CCCCCCACCC GACGTGCACG CTGGGTCTCA CCACCAGCTC CCGCGTGGAG GACAGCGTGG CCGTCTCGCG GGCCTGAAGC

CGCCCAGTGA

CCCATACCGG

CGGGCCCCGT

CTCTGTTGCT

TCAGCC CC CA

ATGGAGACAC

TGCCCTCTGA

TTAATGGGTC

TTCTGGCTGG

GCTGGTCCCG

CATTCTTACA

CATGTGAGGA

TCTTCACTCC

ATGTCCTCAC

TGAGCCGCGT

TCAAGGATTT

ACTGGAAGGT

CCGGCACCGT

CGCGCGTGCG

CGTTGCAGTC

CGCCCAATCC

CTGTGTCCTC

GGACCCCACC

ACCTGGGGCC

GCTGTCCCGC

CAGGCAGCAG

CTCCTGCCTC

GAACATGAAG

TACCAACTAC

GTACCACACT

CTATGAGATC

ACTGGATGTC

TGGGGGCCTG

CCTCTTCCAA

GGTGGATGAC

TTACTTCGTC

GACCCGAGCC

ACCGCCCGTT

GCGCGGCGGC

GGGGTGCCTC

CTTCTCATCG

ACCG.CTGAGG

CTCCTTAACA

TCAGGAGACA

TATGTTGGCT

GATCTCACGT

TCCCTCAAGT

GTGGGCCCTC

TGGGTGGAAG

CTGGACGTGG

GAGGACCAGC

GCCAAGTACC

GTCAGCAACC

CAAGTGCGTT

CCAATCTGCA

GCGCGCCACC

CGCCGCGGC

GGGGCGGATC

GCTCCTCCCT

GGCTCTACTG

CCTCCACCCT

ATCTGGTGTG

TGCCCCCTGA

GCCGCTGGAC

ACAAGCTGAG

ACTCATGCCA

CCACCAATCG

TGACCACGGA

TGAGTGTGCG

AGATCCGCTA

AGACCTCCTG

GTAACCCATT

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 99 TGGAGCCACC CCACCGCTGC

CGGGATCTAT

CTCCACCCCT

C GAGCCCAGC

GCACGCATAC

GAAGTCACAC

AGGAGCAGAG

CAAGCACAGG

GTCTGGGTGA

CTCAAAGGGA

TCATCAAGTT

CGGCTAAACT

TTGGAGCCCC

TGAGCTCCCA

TGGGTAAGGT

GTTCAGGTAA

GGGTCGAAAA

CGAAGTGAGC

TCGGGCCCGG

TGCTCGAACC

AAGACCCGAA

GAAGAGAGAG

ACGAGGGGAT

GTGGGGCCTA

TCTCTTAGTG

GCTGAAGGGT

CTAAGGATAG

TCTGTACCTA

CAACCACAGC

TGGGGTATTG

AGGCGGGAAT

GCCCGGGCCC

TGCGGCGCGA

TTAGTTTCCG

ACCAGGTAGG

ACCCGGGTGA

CCTGCCCTCG

CAGCAGTCTA

CTCATTTCAC

CCAGGCTTAA

GCCATCCTCC

TCTGGGCAAC

TTTGGTCCAC

CAGGGCCTCC

CTGGAGCGAG

GGGCGGCGGG.

GCTCAAGCAG

CCTGTACGAC

AAAGTTGGGG

GCAGCCTCCA

GGCAGACGGG

GATGAGGCCC

CCACTGCAAA

TGTGGCCTCT

TGCTGGGTCA

AAAGAAACCT

ATGATGGTCA

'CAACAATCTC

GTGTGCGAGC

TTCCTCGGCT

CAGTGGCGTG

GAGGCTTGCG

CAACACCGCA

GTGCGGCGAG

TTTCCCCTCC

GAGCCCCAGG

TTTCTGCCCT

GACCTGGAGG

ACCATGAGGC

CACTTGGATA

TTTAAATAAA

CGCGGGGCGG

GGCTCAAGAA

CTTGGATGCA

TGGGGGGTAA

CTCTTCTTTC

AGGTAAGGGG

TTCGGTGTTG

TTTTACTGCA.

CAGGTCCTGC

CTCACCTGAA

TGGGGCACAA

TACCCCAGTG

TAAAGGAGTT

1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1930 INFORMATION FOR SEQ ID NO:23: i)SEQUENCE CHARACTERISTICS: LENGTH: 560 base pairs TYPE: nucleic acid -100- STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:

TCCAGGCAGC

GGCTCCTGCC

AAGAACATGA

CACACCAACT

GAGTACCACA

CCCTATGAGA

ACGCTGGATA

GTCGGGGGCC

TTCCTTTTTC

GTGGTGGACG

GGTCGGGGGA

TCTATGTTGG

AGGACTTGAC

ACTCCCTCAA

CAGTGGGGCC

TCTGGGTGGA

TCCTGGATGT

TGGAGGACCA

AAGCCAAATA

ATGTGAGCAA

CAACCTCGTG

CCTGCCCCCA

CTGCCGCTGG

GTACAAGCTT

CCACTCCTGC

GGCCACCAAC

GGTGACCACG

GCTGAGCGTG

CCAGATCCGC

TGCCACGCCC

GAGAAACCCG

ACGCCAGGGG

AGGTGGTATG

CACATCCCCA

CGCCTGGGCT

GACCCCCCGC

CGCTGGGTGT

TACCGAGTGG

GTGACGGCAG

TCAACATCAG

CCCACGGGGA

GCCAGGACAA

AGGACCTGGC

CTGCCCGCTC

CCGACGTGCA

CGCCACCCGC

AGGACAGTGT

CATCCTGGCT

CTGCTGGTCC

GACCTTCCTC

CACATGTGAG

TCTCTTTACG

CGATGTACTC

CGTGAGCCGC

CCTCAAGGAT

GGAATGGAAG

120 180 -240 300 360 420 *480 540 560 INFORMATION FOR SEQ ID NO:24: Wi SEQUENCE CHARACTERISTICS: LENGTH: 1391 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear AMENDED

SHEET

101 (ii) MOLECULE TYPE: DNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1. .1053 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:

ACC

Thr 1 CTC AAC GGG CGC CGC CTG CCC CCT GAG CTC Leu Asn Gly Arg Arg Leu Pro Pro Glu Leu TCC CGT GTA CTC AAC Ser Arg Val Leu Asn

GCC

Ala TCC ACC TTG Ser Thr Leu GCT CTG GCC CTG GCC AAC CTC AAT GGG TCC AGG CAG Ala Leu Ala Leu Ala Asn Leu Asn Gly Ser Arg Gin 25 CGG TCG GGG GAC AAC CTC GTG TGC CAC GCC CGT GAC GGC AGC ATC CTG Arg Ser Gly Asp Asn Leu Val Cys His Ala Arg Asp Gly Ser Ile Leu 40

GCT

Al a TCC TGC CTC TAT GTT Ser Cys Leu Tyr Val GGC CTG CCC CCA GAG AAA CCC GTC AAC Gly Leu Pro Pro Glu Lys Pro Val Asn

ATC

Ile AGC TGC TGG TCC AAG Ser Cys Trp Ser Lys 70 AAC ATG AAG GAC TTG Asn Met Lys Asp Leu 75 TGC CGC TGG ACG Cys Arg Trp Thr CCA GGG GCC CAC GGG Pro Giy Ala.His Gly GAG ACC TTC CTC Glu Thr Phe Leu CAC ACC AAC TAC TCC CTC AAG, His Thr Asn Tyr Ser Leu Lys 90 AAC ACA TGT GAG GAG TA C CAC Asn Thr Cys Glu Glu Tyr His 110 240 288 336 TAC AAG CTT AGG Tyr Lys Leu Arg 100 TGG TAT GGC CAG Trp Tyr Gly Gin

GAC

Asp 105 S EE1 102

ACA

Thr GTG GGG CCC Val Gly Pro S115 CAC TCC TGC His Ser Cys CAC ATC CCC AAG GAC CTG GCT CTC TTT His Ile Pro Lys Asp Leu Ala Leu Phe 120 125 GAG GCC ACC AAC CGC CTG GGC TCT GCC Giu Ala Thr Asn Arg Leu Gly Ser Ala 140 ACG CCC Thr Pro 130 TAT GAG ATC TGG GTG Tyr Giu Ile Trp Val 135

CGC

Arg 145 TCC GAT GTA CTC ACG Ser Asp Val Leu Thr 150 CTG GAT ATC CTG GAT GTG Leu Asp Ile Leu Asp Val 155 GTG ACC ACG GAC Val Thr Thr Asp 160 CCC CCG CCC GAC GTG Pro Pro Pro Asp Val 165 CAC GTG AGC CGC GTC His Val Ser Arg Val 170 GGG GGC CTG GAG GAC CAG Gly Gly Leu Giu Asp Gin 175' CTG AGC GTG CGC Leu Ser Vai.Arg 180 CAA GCC AAA TAC Gin Ala Lys Tyr 195.

TGG GTG TCG CCA CCC Trp Val Ser Pro Pro 185 GCC CTC AAG GAT Ala Leu Lys Asp TTC CTC TTT Phe Leu Phe 190 CAG, ATC CGC TAC Gin Ile Arg Tyr 200 CGA GTG GAG GAC AGT GTG GAC TGG Arg Val Giu Asp Ser Val Asp Trp 205 AAG GTG Lys Val 210 GTG GAC GAT GTG AGC Val Asp Asp Val Ser 215 AAC CAG ACC TCC TGC Asn Gin Thr Ser Cys 220 CGC CTG GCC CC Arg Leu Ala Gly

CTG

Leu 225 AAA CCC GGC ACC GTG Lys Pro Gly Thr Vai 230 TAC TTC GtG CAA GTG Tyr Phe Val Gin Val 235 CGC TGC AAC CCC TTT Arg Cys Asn Pro Phe 240 GGC ATC TAT GGC Gly Ile Tyr Gly TCC AAG AAA GCC GGG Ser Lys Lys Ala Giy 245

ATC

Ile 250 TGG AGT GAG TGG AGC CAC Trp Ser Giu Trp Ser His zczZ AMENDIE0 -MKcT CCC ACA GCC Pro Thr Ala GCC TCC ACT CCC Ala Ser Thr Pro 260 GAA CCG CGG GGC Glu Pro Ara Glv -103 CGC AGT GAG Arg Ser Glu 265 GGA GAG CCG Gly Glu Pro CGC CCG GGC CCG ~rg Pro Gly Pro 270 GGC GGC Gly Gly GGG GCG TGC Gly Ala Cys 275 280 AGC TCG GGG CCG GTG CGG Ser Ser Gly Pro Val Arg 285 AAG AAG CAC GCG TAC TGC Lys Lys His Ala Tyr-Cys 816 864 912 CGC GAG Arg Glu 290 TCC AAC Ser Asn 305

CTC

Leu AAG CAG TTC CTG Lys Gin Phe Leu 295 AGC TTC CGC CTC Ser Phe Arg Leu

GGC

Gly TGG CTC Trp Leu 300

CTC

Leu 310 TAC GAC CAG TGG Tyr Asp Gin Trp 315

AAG,

Lys

CGG

Arg

GGCC

ACCC

CGGC

TGTG

GGGG

GCTA

TCG CAC AAG ACC CGC AAC CAG CAC AGG ACG Ser His Lys Thr Arg Asn Gin His Arg Thr 325 330 GCA GAC GGG GCA CGG CGA GAG GTC CTG CCA Ala Asp Gly Ala Arg Arg Glu Val Leu Pro 340 345 'ACCCTC CCTGCCACGT GGAGACGCAG AGGCCGAACC TCACTT CAGGGCACCT GAGCCCCTCA GCAGGAGCTG 'CATAAC AGCTCTGACT CCCACGTGAG GCCACCTTTG TGTGTG TGAGGGTTGG TTGAGTTGCC TAGAACCCCT AGTCAT TACTCCCCAT TACCTAGGGC CCCTCCAAAA CGA GCC TGG ATG CAG 960 Arg Ala Trp Met Gin 320 AGG GGA TCC TGC CCT 1008 Arg Gly Ser Cys Pro 335 GAT AAG CTG TAGGGGCTCA 1060 Asp Lys Leu 350

CAAACTGGGG

GGGTGGCCCC

GGTGCACCCC

GCCAGGGCTG

GAGTCCTTTT

:CACCTCTGT

TGAGCTCCAA

PGTGGGTGTG

GGGGTGAGAA

A.AATAAATGA

1120 1180 1240 1300 1360 1391 .TTTAGG TGCAAAAAAA AlAAtAAAA A -104 INFORMATION FOR SEQ ID SEQUENCE CH ARACT ERISTICS: LENGTH: 350 amino acids TYPE: amino acid TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID Thr Leu Asn Gly Arg Arg Leu Pro Pro Glu Leu Ser Arg Val Leu Asn 1 510 Ala Ser Thr Leu Ala Leu Ala Leu Ala Asn Leu Asn Gly Ser Arg Gin 25 Arg Ser Giy Asp Asn Leu Vai Cys His Ala Arg Asp Giy Ser Ile Leu 40 Ala Gly Ser Cys Leu Tyr Val Giy Leu Pro Pro Giu Lys Pro Val Asn 55 Ile Ser Cys Trp Ser Lys Asn Met Lys Asp Leu Thr Cys Arg Trp Thr 65 70 75 Pro Gly Ala His. Gly Glu Thr Phe Leu His Thr Asn Tyr Ser Leu Lys 90 Tyr Lys Leu Arg Trp Tyr Gly Gin Asp Asn Thr Cys Giu Giu Tyr His 100 105 1.10 Thr Val Gly Pro His Ser Cys His Ile Pro Lys Asp Leu Ala Leu Phe 115 120 125 Thr Pro Tyr Giu Ile Trp Val Giu Ala Thr Asn Arg Leu Giy Ser Aia 130 135 140 105 Arg 145 Ser Asp Val Leu Leu Asp Ile Leu Asp 155 Val Val Thr Thr Asp 160 Pro Pro Pro Asp Val1 165 His Val Ser Arg Val1 170 Gly Gly Leu Giu Asp Gin 175 Leu Ser Val Arg Trp Val Ser Pro Pro Ala Leu Lys Asp Phe Leu Phe Gin Ala Lys 195 Tyr Gin Ile Arg Tyr 200 Arg Val Giu Asp Ser 205 Val Asp Trp Lys Val 210 Val Asp Asp Val Ser 215 Asn Gin Thr Ser' Arg Leu Ala Gly Leu 225 Lys Pro Gly Thr Tyr Phe Val Gin Vai 235 Arg Cys Asn Pro Gly Ile Tyr Gly Ser 245 Lys Lys Ala Giy Ile 250 Trp Ser Glu Trp Ser His 255 Pro Thr Ala Gly Ala Cys 275 Al a 260 Ser Thr Pro Arg Ser 265 Giu Arg Pro Giy Pro Gly Gly 270 Pro Vai Arg Giu Pro Arg Gly Gly 280 Giu Pro Ser Ser Gly 285 Arg Giu 290 Leu Lys Gin Phe Leu Ser Phe Arg 310 Leu 295 Gly Trp Leu Lys His Ala Tyr Cys Ser A 305 ~sn Leu Tyr Asp Gin Trp 315 Arg Ala Trp Met Gin 320 Lys Ser His Lys Thr 325 Arg.Asn Gin His Arg 330 Thr Arg. Gly Ser *Cys Pro 335 I A, t 106 Arg Ala Asp Gly Ala Arg Arg Glu Val Leu Pro Asp Lys Leu 340 345 350 INFORMATION FOR SEQ ID NO:26: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid .0 STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: TCCAGGCAGC GGTCGGGGGA CAAC INFORMATION FOR SEQ ID NO:27: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: TTGCTCACAT CGTCCACCAC CTTC AMENDED SHEET -107- INFORMATION FOR SEQ ID NO:28: SEQUENCE CHARACTERISTICS: LENGTH: 6663 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID N0:28:

CCCAGAACTC

GGTAATGAGA

TGTGAGACAG

CCCTAGGAAT

GCTCCAATGT

TTGAGTAGGC

TGTTTGTATT

TAGTCCTGGC

CGCCTGCTTG

GCATACTTTA

GCCAAGGATA

TTTGTGCTTG

TTGGACGCTG

CCCTGTCAAG

CTTGGTGGGT

CCATGGTAGA

GCACAcACAC

ATTTATGACT

TGGTTTGGTT

TGTCCTTGGA

TGCTTCCCAA

ACCCCAGTAT

CAGAGTGAGA

TGTATGTACA

AGGCAGGAGG

AAAAGAAAAG

AAGGGGCACT

AGGAGAAAGC

AGGGAGACAT

GATGTTTTAA

TGGTTTGAGT

ACTCACTCTG

GTGCTTAGAT

TTGGGAGGCA

CCCTATTCTT

TGTGTGTTGC

ATTCCCAAGT

AAATAAAGAG

TGCCTCCAAT

AAACTCGCAG

AATCAATTAA

AATTTTTATT

TTTGTTTATT

TAGACCAGGC

TAAAGGTGTG

GAGGCAGACT

ACCCTCCCCC

AGCACGTAAA

TTCAAGACAG

ACAAGAAAAT

CAAGATGACC

CTGCTGACCT

TAGGATGTAT

TGATTTTATG

TGAGACAGGG

TGGCCTTGAA

CACTGCCATT

AATGTGTGAA

CCCAAAACCC

TGTCCAAGGA

TGTGTTTCTA

GTTTATAGGC

TCAGCCCCAT

CCATACATGT

TTGCTTAGAT

AAAATATACC

CTTCTcTGTG

CTCAGAAATC

CAGCAAAATT

T7CCAGGCTA

CAAAATCTAT

.CAA=GTTAG

120 180 240 300 360 420 480 540 600 660 720 AMENDED

SHEET

108 AAGTTCTCTC CGTTCACAGT CTAAGTCCTG AATTCAAACT

CAGTCTTCTT

TGAGATAAGG

GAGCTGCTGG

CTGGGGAAGG

TCTCGTTGTT

TCTTGAAACA

TGTCGTCTTT

GCTCCTCCCT

GGCTCTACTG

CCTCCACCCT

ATCTGGTGTG

GTAAGTGGGG

TGGGTCTTCT

CCAGCACAGG

CAGCTTTAGA

AGCTGCTGGT

GAGACATTCT

GCCTTGCTGT

TATGTACTGA

TCTCTTGTAG

TACTCTTGCT

GGCTGGCCTT

TCTTTTCTTT

TCCAGGCAGG

GACCCCAGAC

GCAAGCTACC

GACCTTCAAT

GGCCCTGGCC

TCACGCCCGA

CCCCAGACAC

GTCCTGGGGC

CATTGCAACT

AAAGCTGTCA

CCCGGAACAT

TACATACCAA

GTGACTTCTG

GCCATTTCAC

CTCTAGCTAG

TCCACCCCAA

GGCCTTGATT

ATCTGTGAAA

GTGAGGGACT

ACAGCTGTAA

TGCTCTATAC

GGTCGCCGCC

CTGGCTAACC

GACGGCAGCA

TCAGAGATAG

AGAGCCATGG

CTAGGGACAG

TGTTTTCCTT

GAAGGATCTC

CTACTCCCTC

GCAATACTTA

TGGCCCTGGA

GCTCAAACTA

GTGGTGGAAT

TTGTTGCCTC

TGGGTGAACA

TGAAGTGGGC

TCAGCCCCCA

ATGGAGACAC

TGCCCTCTGA

TTAATGGGTC

TTCTGGCTGG

ATGGGGGTTG

GCTCTCACTT

CTGTGGCTGC

GTAGTGCCCC

ACGTGCCGCT

AAGTACAAGC

CCTTCTCTGA

AAGGTCCTCA

TTGACTGATG

TGAACTCCCA

GATACTCAGG

AGCTTCAATG

CCTGTTCAAG

TCATCCCATG

GGACCCCACC

ACCTGGGGCC

GCTGTCCCGC

CAGGCAGCAG

CTCCTGCCTC

GCAATGACAG

GCATGCAGGC

ACTGTCCCCT

CTGAGAAGCC

GGACACCGGG

TGAGGTTGGT

TCAAATATGT

GGCTTAGCCA

AATTAATTTT

AGGTCATCTT

CAGCACTTCT

AGTGCTTGGG

ACTTCCTGAC

CCTAACAAAG

CTTCTCATCG

ACCGCTGAGG

CTCCTTAACA

TCAGGAGACA

TATGTTGGCT

ATTTAGAGCC

ATGGTCATAC

GTGTACCCCA

CTTTAACATC

TGCACACGGG

ACCCAGCCAA

TCCTGTTTAT

780 840 900 .960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860

AMD'DEDSH-FET

109 GAACTCAAAA GGGACTCTCG CACCTCCACA GGTGGTACGG TCAGGATAAC ACATGTGAGG

AGTACCACAC

CCTATGAGAT

CACTGGATGT

TATAGGGCGC

AGCCACGGTG

ACAGTCAAGA

AACACCTGGC

GGACTGAGAG

GGGGGGGGCG

TAATTAGGGT

GAAGACTCAG

GTGCCCCTGG

TTGTGCTCTG

GAGGTGGACA

GATATACAAT

GTTGACATCA

AGAGTCAGCT

CCTCACTCCC

TGTGGGCCCT

CTGGGTGGAA

.CCTGGACGTG

CTCCCCCCCA

GTTGCAGGAC

TTTTTCCCCT

CTGACCACCC

GAGG.CGCCCA

AGGGTTGGAG

GTTCCCAGCC

GGGAGAGATC

CTCATTCCCA

TGGCTGGCAC

TGGGATGGGG

AAAGCTTGTC

CTCTGGGACA

TGTGACTTAA

TGTTTAGTGA

CACTCATGCC

GCCACCAATC

GGTGAGCCCC

TCCCCCCAGA

AGTGGTTGTT

CCCCACCCCC

TCCCTCTCTA

GGTCTGAAGG

GCACGAACTG

CAAAGCAGCC

AGCTTGTACT

CATCCAGAGG

AGCTGCCCCG

ATACATAGGG

ACCCTGACGC

TGTAGTGAGA

TACTGGAACT

GATCTCTGCG

ATATCCCCAA

GCCTAGGCTC

CAGTGTCCAC

CTTTTTGGTT

CATAACTTAA

AACACACACA

CAGCCCAGGT

CGCCCCAGGA

GATGATCCCT

TGGGCCATTT

CTCTCCATGG

TTTTGTGTCT

TGGAGGCTCT

ATGGAGCCAA

TCAGAAAGCC

CCCTAGCTCA

CAGGGCCTAA

CTAATCTCCA

GGACCTGGCC

AGCAAGATCT

CTGTGTTCTG

CTTCTAGAGG

TGCAAAGACT

TACACACACA

GTTCAGAAGG

AGCCGAGGCC

GAGCACAACT

AACCCTTCAA

TCCCCCAGGA

TCCTGGCATC

TGGTAATGTA

ATAGCACCTC

TACTCATGAT

AAACACAGAC

TAGGTGCTGG

CCC CAGCTGG

CTCTTCACTC

GATGTCCTCA

CCCTAGACCT

TCTTAGCCAC

TTCCCCCAAG

CTCTGCAGAG

GAGTCCTAGG

TTGAGCTGGG

GGGCCTAATC

GTGCCTCACT

GGGTTCCTGG

TAACCCTCAG

CAAGGCATCA

AAGGTGGGGT

GATCACAATT

AGTAGCTTTA

GTGATGCTCG

GTGGGCTGCT

1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 110 CTGTCCCCTT GAGGGCAGGA ATGTGTGTCT TCCATCAGAG ATAGGACCCG TGGTAGCAGC

AACTGCTGCT

AGCTAACAGG

GCCTAGATTA

ACTGGGAGCA

GAGGCAGAAG

CAGCCAGGGT

TGCTCCACCC

CCTGGAGGAC

CCAAGCCAAG

CCCGCCCCGG

GTGGTGGATG

GTTTACTTCG

ATCTGGAGCG

TCCAGGGCTG

TTTTGAGACA

TGACCTCGAG

ACCTTTGGGA

AACAGTGTGA

GGCTGTTTCT

GGTGGGGGCG

CACCACTGGG

CTGTTGCCAG

GATCTCTCTG

GCGCAGTAAA

CCAGTGACCA

CAGCTGAGTG

TACCAGATCC

ACCCGCCCCT

ACGTCAGCAA

TCCAAGTGCG

AGTGGAGCCA

GCTGGCCCAT

GCGTCTTCAG

CTCCTGGTCT

GACTAGCCAT

CCTCTTGTAA

GGAATATTAA

TGGTCTGGAA

TGTTCTGTCA

.CATTTAATGC

AGTTCAAGGC

ACCTTGTCTC

CGGACCCCCC

TGCGCTGGGT

GCTACCGCGT

GACCCCGCCC

CCAGACCTCC

TTGTAACCCA

CCCCACCGCT

GGAATCCCCA

GTAGCGCATG

TTTTGTCTCC

GGAGTCTATT

GAGAACTGAA

ATGACAGTAA

AACGCAGATA

CTAGGCCATT

CAGCATTTAA

CATCCTGAAT

AAAAAACAAA

ACCCGACGTG

CTCACCACCA

GGAGGACAGC

CCCGCATCTG

TGCCGTCTCG

TTCGGGATCT

GCCTCCACCC

ATCCATCCTG

CTGGCCTTAA

ACTTAGAGAC

TAGCCTGTCA

GACAGGCTGT

TCTATCAGGC

GGGTCATAGG

CTCACCAAGC

TGCCAGCATT

TTACATAAAG

GCATCTTTAG

CACGTGAGCC

GCTCTCAAGG

GTGGACTGGA

ACTCCTCCCT

CGGGCCTGAA

ATGGGTCGAA

CTCGAAGTGG

TTCCTTCCCC

ATTCAGTATG

AATGGCCAGT

TTTGGTGACA

TTTTAACCCC

CTGGGTGAGT

AGCCACTGCA

AGTCCTCAGA

AGGGGAGGCA

AGCTCCAGGC

TGACCAGGCT

GCGTTGGGGG

ATTTCCTCTT

AGGTGCCCGT

CACCGTGCAG

GCCCGGCACC

AAAGGCGGGA

TGAGCACCTC

CCCACCCTTT

TAGTCAAGGA

GGCCATCACC

GATGGAGTAC

AATATCCTAG

3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 *n ill, GCTCTCTAGA GGTTAACTTT ATATAAAATA GAGACTATTA

CCACAGAACC

TCTCTACTGC

GCAATCCTCC

TTCTCTGGGT

CTCTCCGAGC

CTCAGTTTCC

GCAGGCAGTA

ATACTTCTAC

ATAATGGGGA

GGAAGCTCTC

AGCTGGGAGG

CTTGCACGGG

GGTTGGAGGG

AGTCCCCAGT

TGGCTGCGGC

CATGTCACAC

TCTGTCTCTA

TACGTTTTAA

TTTTGTCACA

TGGCCCACCC

TACCTCAGCC

CCCTTTCTTA

CCATGGATCT

CCACCTGTCA

TAAGAAGAAG

ACTGAAACTG

AACTGAGGCT

CAGCCCCCAT

GGCTGGAGCC

AGCCAGCAGG

AGGTAAGCAG

TTTATTTATG

TGGGGATTGG

CCGTGCATTC

TTTCTGTCAT

AAATTGCTTT

CAACCTATAG

CTCCAACCCT

TCTTGAATGC

AGGTGGGAGG

GCACTCTCTA

GGTTTAGGCA

CTCCCATCCC

AACTCTCGCA

CCGAGAGATT

CCGGGCCTCT

TGGGAGCTTT

CGGCTGCGTC

GGGCTGTGGG

GCGTGAGGCC

ACCCAAGGGC

TCTGAGGCTT

TCACTTTCCC

TGTATAATGT

ACCACAGTGC

TAAAAGGTAA

TCAGAAACCA

GCCTAAAGAT

ATATGAAATA

GCACAGTCGG

CCACCCGCTT

GACGCATATG

CCTGGAGGAA

CCAGGTTCTG

GGCCCTTGCT

CGCCCGAGAG

GGCCGAAGCT

GATGTCCTTA

TGGCTTCCCA

ATCTTGGGAA

AGAGCCTTTT

GTGTGCCTTC

CAGCCAGTTA

CTGTGCCTAC

CCTAGGCAGC

GGCATTAACC

GACTTCCTTT

TATTGCATAA

TCCAAGACAC

CCTCCGGTCC

CTCACTTTAA

GAGGGTCAAA

GGCTTGGCGG

CGTGCCCAGC

ACTGAAGAAG

TGTGCCAGGG

TCCGCTGGCC

CTCAGTCCTC

CCCGCCCTTG

TTTTATGCTT

GTGAGCGTGC

TCACATGGTC.

CACATAAGGG

CTTAATATTT

CAAGTTTCTC

GTCCTGAAGA

AATGTCTGGC

TTCATTATTT

CTAAGACAGA

TGATGATGAA

ACCAGCTCCA

GAGTGAACAC

ACCTGCGATT

CCGGGGGTAG

CCTGTCAGCG

TGCTGGGGGA

CAGCCCACTC

TTCTGTGCTG

TTAATATAAC

GTGCCACAAC

4200 4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220 5280 I I t,* 9.

112 ACACACGTGA AGGTTAGAGA ACTTTGTTGA GTAGGCTCCT

CTGGCGACAA

TGTTTGGATA

GCCTCAGCCT

TGGGGGTGAC

CTTTGTCCGT

CGAATACTCG

TGGCATTGCC

CCAGCCGAAG

GCCCCAACAC

AGCCGCGGGG

GCTGGCTCAA

GTGCTTGGAT

GCGTGGGGGG

GCACTCTTCT

GAGAGGTAAG

TCCTTCGGTG

AGGTTTTACT

CCTCAGGTCC

GAGCAATTAC

GTCATAGGTA

ACCAAGTGCT

ACAGTCCCAA

GTCCCTAGAG

GTTTTACCTC

ATTCCTCTGG

CTGGTCTGGT

TGCCGCTCCA

CGGCGAGCCC

GAAGCACGCA

GCAGAAGTCA

TAAAGGAGCA

TTCCAAGCAC

GGGGTCTGGG

TTGCTCAAAG

GCATCATCAA

TGCCGGCTAA

TGAGTCATCT

ATCGAAGGTA

GTGCTACCAC

GATCTCTGCT

TCTCCGGCCC

CCACTGATTT

GTGACTCTGG

ATGGGAGGCC

TTCTCTTTAG

AGCTCGGGCC

TACTGCTCGA

CACAAGACCC

*GAGGAAGAGA

AGGACGAGGG

TGAGTGGGGC

GGATCTCTTA

GTTGCTGAAG

ACTCTAAGGA

CGCCAGCCCC

AATCGCTGGC

GTTTGTGGGA

TTCTAGGTCt

CACTTATCCA

GACTCCCTCC

GTCCACACCT

GCCGTCCCGC

AGCGCCCGGG

CGGTGCGGCG

ACCTTAGTTT

GAAACCAGGT

GAGACCCGGG

GATCCTGCCC

CTACAGCAGT

GTGCTCATTT

GGTCCAGGCT

TAGGCCATCC

TCCACCATGT

TCACCCCTCA

TTTAATTTCG

GGGGCTCTCC

TTGTCTTAGT

TTGACTGGTC

TTTGCTTGTC

GACACCTTTC

GCGCGCCTCC

CCCGGGCGGC

CGAGCTCAAG

CCGCCTGTAC

AGGAAAGTTG

TGAGCAGCCT

TCGGGCAGAC

CTAGATGAGG

CACCCACTGC

TAATGTGGCC

TCCTGCTGGG

GGGACTAGGG

CTTCCCATCC

TAGCTATCCT

TCCCAGTGTC

TTGCCCCTTG

TTTCCTTTAC

TCCATCGCCG

CCAACTTTCC

TGCTGGCCGC

GGGGTGTGCG

CAGTTCCTCG

GACCAGTGGC

GGGGAGGCTT

CCACAA CACC

GGGGTGCGGC

CC CTTT CCCC

AAAGAGCCCC

7TCTTTTCTGC

TCAGACCTGG

5340 5400 5460 5520 5580 5640 5700 5760 5820 5880 5940 6000 6060 6120 6180 6240 6300 6360 6420 MAEmH~t~sEET -113 AGGCTCACCT GAATTGGAGC CCCTCTGTAC CATCTGGGCA CTGGGCACAA TGAGCTCCCA CAACCACAGC TTTGGTCCAC TACCCCAGTG TGGGTAGGGT TGGGGTATTG CAGGGCCTCC TAAAGGAGTT GTTCAGGTCC CGATGGCCAG TGTGTTTGGG

GGA

ACAAAGAAAC

ATGATGGTCA

CAAGAGTCTC

GCCTATGTGC

CTACCAGAGG

CACTTGGATA

TTTAAATAAA

TGGGGTGGGG

6480 6540 6600 6660 6663 INFORMATION FOR SEQ ID NO:29: Wi SEQUENCE CHARACTERISTICS: LENGTH: 186 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Asp Pro Thr Leu Leu Ile Gly Ser Ser Leu Gin Ala 10 His Gly Asp Thr Pro Gly Ala Thr Ala Glu Gly Leu N1O: 2 9: rhr cys Ser Ile 25 Asn Gly Arg Thr Leu Ala so Arg Leu Pro Ser Leu Ser Arg Leu T'yr Trp Thr Phe Leu Asn Thr Ser Arg Gin Gin Ser Leu Ala Leu Ala Asn Leu Asn Gly AMOED

SHEET

Gly Ser Asp Asn Leu Val -114 Cys His Ala Arg Asp Gly 70 75 Ser lie Ala Gly Ile Ser Cys Leu Tyr Gly Leu Pro Pro Lys Pro Phe Asn Cys Trp Ser Ala His Gly 115 Arg on00 Asn Met Lys Asp Leu 1OF; His Thr 120 Thr Cys Arg Trp Thr Pro Gly Lys Tyr Lys Lys Tyr Lys Glu Thr Phe Leu Asn Tyr Ser Leu 125 Leu Arg 130 Leu Val Arg Ser Gly 135 His Met Val Pro His Cys Gly 145 Pro Ser Leu Met Tyr Pro Gin Gly Pro 155 Pro Leu His Leu Asp Leu Gly 165 Ser His Gin Ser 170 Pro Arg Leu Ser Lys Ile 175 Cys Pro Thr Gly Cys Pro Gly Arg 185 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 35 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID i~~s~Er;ii.i~yj SHEtlT C 115 AGCTGGCGCG CCTCCCGGGC GGATCGGGAG CCCAC INFORMATION FOR SEQ ID NO:31: SEQUENCE CHARACTERISTICS: a LENGTH: 28 b?.

0 e kE -ir_ TYPE: nucleic acid 0 STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: AGCTACGCGT TTAGAGTTTA GCCGGCAG INFORMATION FOR SEQ ID NO:32: SEQUENCE CHARACTERISTICS: LENGTH: 30 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: Met Val Leu Ala Ser Ser Thr Thr Ser Ile His Thr Met Leu Leu Leu itSc~ri~' j~ E~ 116 Leu Leu Met Leu Phe His Leu Gly Leu Gin Ala Ser Ile Ser 25 INFORMATION FOR SEQ ID NO:33: i4 N C~CTTr Tc rLTC r 7U T 'o ?vmrTICiT r' C! LENGTH: 30 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: Ile Lys Pro Ser Gly Arg Arg Gly Ala Ala Arg Gly Pro Ala Gly Asp Tyr Lys Asp Asp Asp Asp Lys INFORMATION FOR SEQ ID NO:34: SEQUENCE CHARACTERISTICS: LENGTH: 73 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA y.

St 54 *4 117 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: GATCTTGCCC TCGGGCAGAC GGGGTGCGGC GAGAGGTCCT GCCGGCGACT ACAAGGACGA CGATGACAAG TAG 73 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 73 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID AACGGGAGCC CGTCTGCCCC ACGCCGCTCT CCAGGACGGC CGCTGATGTT CCTGCTGCTA CTGTTCATCC TAG INFORMATION FOR SEQ ID NO:36: SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear 118 (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: CCCACGCTTC TCATCGGATT CTCCCTG INFORMATION FOR SEQ ID NO:37: SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: CAGTCCACAC TGTCCTCCAC TCGGTAG INFORMATION FOR SEQ ID NO:38: SEQUENCE CHARACTERISTICS: LENGTH: 11832 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA 119 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:

GCGGCCGCTG

TTTTTCCGTG

GGAATGCAGG

AAGGGCTCCC

TGAGAAGGGA

ACGAAACGAG

GACCCATGCA

AGGGCTGAAA

TGGGTATGGG

GGAGGGGATC

ACAGGATCCC

CACGGGCTGG

GTAACTGGGC

GTGCGGGGCC

CGGGGCGAGC

CGCTGTCCGC

CGCCCCCGCC

GGGTCGCCCG

CAGTGATTAC TCACCGCGTG

GGGGGATGTG

GTTCGGTCCC

TGCACGCGCT

CCAGAGGCCG

ACTACAGCGA

CCCAGAGAAA

GAGGATGAAC

GGCCCCGTAA

CTGGAAAAGC

AGATGAGGGG

TGGGGAAAGA

GGAGGCCGGC

CACGATCAAC

GGCGCATTAG

GCCCAGTGAC

CCATACCGGC

GGCCCCGTCG

AAGAAGTTTA

GTTCCCCAAA

CCGGGACATC

GAGACTCCCT

TGGGAGAGGT

GGGACTGGTG

GGGCTCAGGT

GAGGGGCGGG

ACCAGGGCTG

GTGGGAAGCC

GTGGGGGGCT

CGGGCGGGGC

CCCCCCCCAG

CGCCTTGTCA

GCGCGTGAGG

GTTGCAGTCA

CCCAATCCGC

GCGCACCCCA

GGGAGAACTC

GGACACACCT

CCCATATCCA

CCCTGCCTTC

GGCATGAAGG

GCAACTTTCA

ACTGCTCAAT

GAAGGTGGAT

CGAGCTAGGA

TGGGACGGGC

TCGCGCAGGA

GCGCGGTGCC

GGGCCGGGCC

ATTTCGGCTG

ACCCGAGCCC

CCGCCCGTTG

GCGGCGGCCG

CCCGCGGGCC

TTCTGCACCG

CTCCCCATAA

ATACCCGCAG

TGGCTTTCCC

CTTAGGGTGG

AACTCTCTGG

GTGTGTGTGG

AGGAAGGATC

ACCCATTCGG

GGGACCAGAG

GGATGGGACG

CGiCGGGCGGT

GGGCCGGGGG

CTCAGACTTG

CAATCTGCAC

CGCGCCACCC

CCGCGGCCGC

GCTGAGTGGA

ATGGGAACTA

GCCCACTCAT

ATATGATAGT

CCCCCCCTGC

GGATCGGTAG

GGAAGGAAGA

C.GGACCAAAG

CCGGTAGACT

AGTTAAGGGT

AGGGAGGTCC

TTCAGGAGTG

GGGAAGGCCG

CGGGGCCGGG

CTCCGGCCTT

CCCGCAGACT.

CCATGCCCGC

TGTCCTCGCT

120 180 240 300 360 420 480 Y~540 600 660 720 780 840 900 960 1020 1080

GTGGTCGCCT

GTACCGTGCG

AGTCGCGGGG

GGCGGCCCTC

AGTACCCCGT

AGGCTCAGTT

GCTTCGGGGC

GGGCGCACGC

GAAGTGATGA

.ATGCGGCCCG

CTATAGCAGA

CGGTCTCATT

CGAGAGCAAG

GGGGGTCAGC

ATCACCCAAC

CACACCCAAA

CGCGCGCTGC

ACACACACAC

ACACGCACGC

CTGTTGCTCT

CCCTGCTCCC

GATGGAAGAA

.GGGGCGCCCT

TATACATCAG

TGAAGGACAT

GCACGCCTGT

TTGGGTGCGT

TCCCCGGGGG

GCGTCCCTCG

CTCCATGCTT

CAGGCTGCGC

CGTGTCCGGG

TGCCGAGAGA

GCACACATCC

GACACACAAA

AGCCCAGATG

ACACACACAC

ACACACACGC

GTGTCCTCGG

CACCTCCCCA

GGGGCGCGAG

CACCTGTGGG

AGGCCTCTTA

CGCAGTGTCC

GTCTTGGATA

TGGGTTGGGT

GAGGGTGGGG

GGACTTGCCT

TGGTATCCTC

TGGGTTGAGA

CACCGCGAGC

ATCCCACTGT

CCGCCAGGAT

AGAGCCCCAC

CGTATTCGCA

ACACACACAC

ACGCCCGCAC

120

GGTGCCTCGG

GGGAAGCCGG

CGCCACCTGG

GCTCATGGCA

TCTGTATCCC

TGGGACCCCC

TCAGAGCGGA

GCTGGCGCAA

CGTTATCGTG

CTCCGTGGGG

GAAGTCCTCT

GCCTCTAGCG

CCAGACTTCA

CCCAGGAGGA

GCGGTCTCCA

TGGCTTATGT

CACCATCGCG

GGCGGATCGG

GATCCGGCGC

ACGTCCCGGG

CCACCACCCA

CTTTGCGAGG

CTCCTTCAGG

AGGGAAGCCT

AGTGGGGTCC

AGCCCTCCTG

TCGGCGCCGC

CCACTGGTGG

ACTGAAATTT

TTGTCTAAGG

ACTCCTGGCC

CATCCAGACC

CCCGTCACCC

GCGCTCGCAT

GAGCCCGTGA

CCCGGGGGGT

AACAAAGGAA

GCCTCCCAAG

CTGTCTGGCC

GTGCTGGGAC

CCCTGGCCGG

CCTCCCCCAT

TCCGCCTGGC

CCCCTCCCCC

GGCTCACAAC

CGGTGAGGAG

GGCACCCAGT

TTGAGCCCCC

CTCTCTGGGA

TGCCCTCCGA

TCCATCCTCT

ACGCACACAC

CACAGGGGAG

1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 ACACACACAC ACACACAGAC TCGTGGTCCC ACATTTATTT 121

GCAACACCGG

ACCCCATCCG

TAGTAGTCTT

ACAGGAACCT

GACCTTTCCG

GCGCTAAGCT

TGGCGCGTGT

TGCAATCTGT

AAAGTGTAT.G

GAGGCCACCT

GTGTTCTTTT

GGCCCCGGCT

GGCTTAGGGG

AGTGGCTTTG

TACTCCAGAG

GAATCAGGGA

AAGGAGAAAG

TAGTGTGGTA

GGTACGCATA

GAGACACAGG

GTGCAGTTTG

ACACTCCTGC

GGGAGTTGGT

TTGTTTCCGG

GTTTTTTCTT

TTGTACTTAC

CAGGTACCAG

TCCCGTTGGC

TAATAACGGC

TTGTGGAAAG

GCTGTCAGCT

GCCCATTGTT

TCAGGCTTCT

AGGGGGTGCC

CTTGGGCTTG

AAAGGGCATT

TGGTTGAGTG

CCACACCGCA

TCCGCGGTGT

TTGCCCAAGG

GTTGCTGCCA

GCGGGCTGCA

TTAAGGGGGA

CGTGTGTCTT

CGGGACAGGA

CTTTCAGGGA

AGCAACTCCG

GAGGGGAAGA

GCTGCTCTGT

TGTGGAAGCC

CAGTCCGAGC

AGGTGGACTA

CCCCCCTCCC

ACTCCCCAGC

CACTGGAGAT

GGGGCACCAC

CTGTGGACGC

CGGCTGGGCA

AGCCTGGGTA

GAGCAACAGG

GAGAAATTAA

AACACCTGAC

GATGGGGGCC

ATCTCACACT

CATTGGGAAA

GGGAAGAAAA

CTAGCTTGGC

AAGAGGGAGA

CCAGAGAACG

CGTTCTGCTG

CCCTCAAGCC

CAGGACCCCC

CTTTCCCCAC

GCTGCGCTGC

CCTCCCGCTC

GGTGATGTGG

GTTTTTGAAT

CGAAGGTGGC

ATAAGAGGTT

CAGCCAGCCG

CCTGGGGTAT

TTTCCCTTTT

GGGGGAAATA

AAGGAGGGGT

ATGTGTGTGC

CTGGAGTCCT

TCTTCCCTGT

AGGACTGTAC

ACGAAGGGCA

CAGAGAGTCC

CACTCTCAGG

TGCTCTGGGC

TTGTCAGGGG

TGACACCCGG

GCCACCAATA

GGAGTGGGGG

CTCACACCTC

GTGGGTCGTA

GGCTGGGATG

AAAACACATG

AGCTTGTATA

GTCTCCTCCA

CCCAGTCCCC

CTATCTCTGG

TTTATGGAGG

CAGTCGCTCG

GCTGCTAGGC

CCTTCCTGGC

2280 2340 2400 2460 2520 2580 2640 2700 2.760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 CAGACAAATG CTGGGGAGGG ACAGAGGGGT GTGATCATTG CCCAGGAGTG CAGACAGTGG AMENDED SHEET 122

GGTCCCGGGT

GGGTGGGCCG

GCGGGCGGCT

GCCTGCTCCT

CCCAAATGCA

CCTGGGAGAA

GGGCATGAAG

GCAGCCTCTG

AATACTCTTT

TGCAGTCTTC

CCTCTCCCCT

CAGGGCCTCT

CTCTTTTGCT

TCTCTGTACA

ACAAACCTAC

AGTAGTTAAG

TAGGCGATGG

CTTGAATCGT

GGCAGCCTGG

CGGGCAGTGC

GGGTAGAGAC

GGCTGCCTGG

CCTGCTCCTT

ACTGCGATTG

GTCATTCAGG

GACCGTCCAG

TTCTCCGAGC

TCCTCTCATC

CCTAACCTTT

TGCCCAACTG

CTGACACACA

TCTGAGACTT

GCCCTGGCTG

CTGCCTCTGC

TGTTTTGCTG

ATGGATGAAT

CCTGAGTGAA

CCTGCTGGTC

CTCCCACCCT

GCTGGdACGT

GACCTCCGGG

CGCACGGACG

CAGGCTTCGC

GCCCAGACTA

GGCTGCAGTT

CTCTTTGGAA

CCATCCCGGG

TCTTTGCTTC

GGGCTCCAGC

GGGTTGTAGC

AATTTTTTTC

CCCTGGCACT

CTTTCCAGTG

TGTCTTTATT

GGATGGATGG

AAAAGAGACC

TCATGGGAGC

GCTGAGGGGG

CCCAGTTCAT

GCGGCCCCCT

CTGAGACCTC

AAGACCCGCC

GAACCATGTT

TAGCTTCTTA

ATCGGTTTTG

ACTGTTTTCC

TACCCCAGGG

CTTACTGCAT

CCCAGCTCCC

TTTTTCTTTT

CATTCTGTAG

CTGGCACTAA

CCTATAGTGA

ATGGATGGAT

TCAGAGAACT

TCCCTGTGAA

GCGCCCAGGC

GCCGAAGGAA

GGCCCCCGCC

CGCTGAGCCC

TCCTCCCAAG

GGTGCCACCT

ATAGGAACCT

TTTTTGTTTT

TCCCTAAGGG

CCTTTGCACA

TTGGCTCTTG

TCTCTTCTCC

TGGCTTTTTG

ACCAGGCTAG

AGATGTGGGC

CCTCAGTTCC

GGATGGTTGG

GAATGGAGTT

ACTTCCCCCA

AGGAAGCGGT

TTCTGAATTA

GCTCCGTCTG

TGGGACAAGC

GCCAAATTTG

CATCCATCTG

GGGGGTGGGT

.TGTTTTTTCC

TTGAGAGCCC

TGGAGTCCCA

GTAACTGTCC

TCCCCCCTTT

AGACAGGGTT

CCTCAAACTC

CACCACAACT

TGGCATATTG

ATOGGAGCAAG

AGGTTC-CCAG

CACZC-CCAC

3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 AMENDED SIHfhT

CACCCTGCCA

AGGGAAGCTT

CCAGCCTATG

GTCCCTCAGG

AGGAAATGAT

GCTTCTGTGG

TGTGAGGAGG

CCAACAGGGC

TTTGATTCCC

TTTTAGATAT

ACCACCAGGA

TGGCTTATGT

CAGTGTGTTC

ATGTTTATAG

CCTCAGCCCC

TCCATACATG

TTTGCTTAGA

GAAAATATAC

GCTTCTCTGT

TCCTGTGTGG

GGAATATGTT

AGTAGGGCAG

GTGGGTCACA

TGTGGAGAGT

CTGTCCCTTC

GCACGGGGAA

TCACCTCTCC

TTCCTTTGGT

GTCCATTCTC

CAGACAAAGA

GTAATCCCAG

TAGGTAATGA

GCTGTGAGAC

ATCCCTAGGA

TGCTCCAATG

TTTGAGTAGG

CTGTTTGTAT

GTAGTCCTGG

CTGACAAGAA

CCCCTCCTCA

CTGTGGGCTG

GGATTGAGGT

CAGAACTCCT

TCTTGTGGTC

AATGAAGGCT

TCTGGACAGG

CTCCTGGGAT

CAGAAACACA

ATTGGAGAGG

AACTCTGGAC

GACCCTGTCA

AGCTTGGTGG

ATCCATGGTA

TGCACACACA

CATTTATGAC

TTGGTTTGGT

CTGTCCTTGG

123

AGGCCAATGG

TATCCTAGGC

CCCTAAGGTT

CATTTCCAAA

GTTGGGAGTT

CTTTGCACAG

CAGCCCCTCA

CTCTCACTGT

GACAAACATT

CTTGTGAGGT

AAGGAAATTG

GCTGAGGCAG

AGAAAAGAAA

GTAAGGGGCA

GAAGGAGAAA

CAGGGAGACA

TGATGTTTTA

TTGGTTTGAG

AACTCACTCT

CCAGATGGGG

CTTGTTGTCC

GGGTAGGCATA

GTGGCCATCA

GTAGAGGGCC

TCCCCTCGTG

GCTTGCCCTT

ATGCACAGAT

TACCAGGGTA

TAGGGTATCA

GTAAGCCAGG

GAGGATTCCA

AGAAATAAAG

CTTGCCTCCA

GCAAACTCCA

TAATCAATTA

AAATTTTTAT

TTTTGTTTAT

GTAGACCAGG

ACACAGACTC

CCCTGAGGGC

GAAGGGGGTG

CAGTGGCCCT

TTGCATGTGG

TGTGCTGGGA

CACGGTTCAC

TGGCCTCACA

GGATTTTACA

GTGAAAGGAC

CCATGCTTGA

AGTTTCAAGA

AGACAAGAAA

ATCAAGATGA

GCTGCTGACC*

ATAGGATGTA

TTGATTTTAT

TTGAGACAGG

CTGGCCTTGA

4560 4620 4680 4740 4800 4860 4920 4980 5040.

5100 5160 5220 5280 5346- 5400 5460 5520 5580 5640

~P

ACTCAGAAAT

TCAGCAAAAT

ATTCCAGGCT

CCAAAATGTA

ACAACTTGTA

AGGCTTAGCC

GAATTAATTT

AAGGTCATCT

GCAGCACTTC

GAGTGCTTGG

GACTTCCTGA

GCCTAACAAA

CCTTCTCATC

CACCGCTGAG

CCTCCTTAAC

GTCAGGAGAC

CTATGTTGGC

GATTTAGAGC

CATGGTCATA

CCGCCTGCTT

TGCATACTTT

AGCCAAGGAT

TTTTGTGCTT

GAAGTTCTCT

ACAGTCTTCT

TTGAGATAAG

TGAGCTGCTG

TCTGGGGAAG

GTCTCGTTGT

CTCTTGAAAC

GTGTCGTCTT

GGCTCCTCCC

GGGCTCTACT

ACCTCCACCC

AATCTGGTGT

TGTAAGTGGG

CTGGGTCTTC

CCCAGCACAG

GTGCTTCCCA

AACCCCAGTA

ACAGAGTGAG

GTGTATGTAC

CCGTTCACAG

TTATGTACTG

GTCTCTTGTA

GTACTCTTGC

GGGCTGGCCT

TTCTTTTCTT

ATCCAGGCAG

TGACCCCAGA

TGCAAGCTAC

GGACCTTCAA

TGGCCCTGGC

GTCACGCCCG

GCCCCAGACA

TGTCCTGGGG

GCATTGCAAC

124- AGTGCTTAGA TTAAAGGTGT TTTGGGAGGC AGAGGCAGAC ACCCTATTCT TACCCTCCCC ATGTGTGTTG CAGCACGTAA TCTAAGTCCT GAATTCAAAC AGCCATTTCA CTGGCCCTGG GCTCTAGCTA GGCTCAAACT TTCCACCCCA AGTGGTGGAA TGGCCTTGAT TTTGTTGCCT TATCTGTGAA ATGGGTGAAC GGTGAGGGAC TTGAAGTGGG CACAGCTGTA ATCAGCCCCC CTGCTCTATA CATGGAGACA TGGTCGCCGC CTGCCCTCTG CCTGGCTAAC CTTAATGGGT AGACGGCAGC ATTCTGGCTG CTCAGAGATA GATGGGGGTT CAGAGCCATG GGCTCTCACT

GCACTGCCAT

TAATGTGTGA

CCCCAAAACC

ATGTCCAAGG

TAAGGTCCTC

ATTGACTGAT

ATGAACTCCC

TGATACTCAG

CAGCTTCAAT

ACCTGTTCAA

CTCATCCCAT

AGGACCCCAC

CACCTGGGGC

AGCTGTCCCG

CCAGGCAGCA

GCTCCTGCCT

GGCAATGACA

TGCATGCAGG

5700 5760 5820 5880 5940 6000 6060 6120 6180 6240 6300 6360 6420 64'80 6540 6600 6660 6720 6780 TCTAGGGACA GCTGTGGCTG CACTGTCCCC

TGTGTACCCC

CCTTTAACAT

GTGCACACGG

TACCCAGCCA

TTCCTGTTTA

CACATGTGAG

CCTCTTCACT

TGATGTCCTC

GCCCTAGACC

GTCTTAGCCA

TTTCCCCCAA

CTCTGCAGAG

GAGTCCTAGG

TTGAGCTGGG

GGGCCTAATC

GTGCCTCACT

GGGTTCCTGG

TAACCCTCAG

ACAGCTTTAG

CAGCTGCTGG

GGAGACATTC

AGCCTTGCTG

TGAACTCAAA

GAGTACCACA

CCCTATGAGA

ACACTGGATG

TTATAGGGCG

CAGCCACGGT

GACAGTCAAG

AACACCTGGC

GGACTGAGAG

GGGGGGGGCG

TAATTAGGGT

GAAGACTCAG

GTGCCCCTGG

TTGTGCTCTG

AAAAGCTGTC

TCCCGGAACA

TTACATACCA

TGTGACTTCT

AGGGACTCTC

CTGTGGGCCC

TCTGGGTGGA

TCCTGGACGT

CCTCCCCCCC

GGTTGCAGGA

ATTTTCCCCT

CTGACCACCC

GAGGCGCCCA

AGGGTTGGAG

GTTCCCAGCC

GGGAGAGATC

CTCATTCCCA

TGGCTGGCAC

125

ATGTTTTCCT

TGAAGGATCT

ACTACTCCCT

GGCAATACTT

GCACCTCCAC

TCACTCATGC

AGCCACCAAT

GGGTGAGCCC

ATCCCCCCAG

CAGTGGTTGT

CCCCACCCCC

TCCCTCTCTA

GGTCTGAAGG

GCACGAACTG

CAAAGCAGCC

AGCTTGTACT

CATCCAGAGG

AGCTGCCCCG

TGTAGTGCCC

CACGTGCCGC

CAAGTACAAG

ACCTTCTCTG

AGGTGGTACG

CATATCCCCA

CGCCTAGGCT

CCAGTGTCCA

ACTTTTTGGT

TCATAACTTA

AACACACACA

CAGCCCAGGT

CGCCCCAGGA

GATGATCCCT

TGGGCCATTT

CTCTCCATGG

TTTTGTGTCT

TGGAGGCTCT

CCTGAGAAGC

TGGACACCGG

CTGAGGTTGG

ATCAAATATG

GTCAGGATAA

AGGACCTGGC

CAGCAAGATC

CCTGTGTTCT

TCTTCTAGAG

ATGCAAAGAC

TACACACACA

GTTCAGAAGG

AGCCGAGjGCC

GAGCACAACT

AACCCTTCAA

TCCCCCAGGA

TCCTGGCATC

TGGTAATGTA

6840 6900 6960 7020 7080 7140 7200 7260 7320 7380 7440 7500 7560 7620 7680 7740 7800 7860 CAAGGCATCA GAGGTGGACA TGGGATGGGG ATACATAGGG ATGGAGCCAA ATAGCACCTC 72 7920 126

AAGGTGGGGT

GATCACAATT

AGTAGCTTTA

GTGATGCTCG

GTGGGCTGCT

TGGTAGCAGC

CTGGGTGAGT

AGCCACTGCA

AGTCCTCAGA

AGGGGAGGCA

AGCTCCAGGC

TGACCAGGCT

GCGTTGGGGG

ATTTCCTCTT

AGGTGCCCGT

CACCGTGCAG

GCCCGGCACC

AAAGGCGGGA

TGAGCACCTC

GATATACAAT

GTTGACATCA

AGAGTCAGCT

CCTCACTCCC

CTGTCCCCTT

AACTGCTGCT

AGCTAACAGG

GCCTAGATTA

ACTGGGAGCA

GAGGCAGAAG

CAGCCAGGGT

TGCTCCACCC

CCTGGAGGAC

CCAAGCCAAG

CCCGCCCCGG

GTGGTGGATG

GTTTACTTCG

ATCTGGAGCG

TCCAGGGCTG

AAAGCTTGTC

CTCTGGGACA

TGTGACTTAA

TGTTTAGTGA

GAGGGCAGGA

GGCTGTTTCT

GGTGGGGGCG

CACCACTGGG

CTGTTGCCAG

GATCTCTCTG

GCGCAGTAAA

CCAGTGACCA

CAGCTGAGTG

TACCAGATCC

ACCCGCCCCT

ACGTCAGCAA

TCCAAGTGCG

AGTGGAGCCA

GCTGGCCCAT

ACCCTGACGC

TGTAGTGAGA

TACTGGAACT

GATCTCTGCG

ATGTGTGTCT

GGAATATTAA

TGGTCTGGAA

TGTTCTGTCA

CATTTAATGC

AGTTCAAGGC

ACCTTGTCTC

CGGACCCCCC

TGCGCTGGGT

6CTACCGCGT

GACCCCGCCC

CCAGACCTCC

TTGTAACCCA

CCCCACCGCT

GGAATCCCCA

TCAGAAAGCC

CCCTAGCTCA

CAGGGCCTAA

CTAATCTCCA

TCCATCAGAG

ATGACAGTAA

AACGCAGATA

CTAGGCCATT

CAGCATTTAA

CATCCTGAAT

AAAAAACAAA

ACCCGACGTG

CTCACCACCA

GGAGGACAGC

CCCGCATCTG

TGCCGTCTCG

TTCGGGATCT

GCCTCCACCC

ATCCATCCTG

TACTCATGAT

AAACACAG AC

TAGGTGCTGG

CCCCAGCTGG

ATAGGACCCG

TCTATCAGGC

GGGTCATAGG

CTCACCAAGC

TGCCAGCATT

TTACATAAAG

GCATCTTTAG

CACGTGAGCC

GCTCTCAAGG

GTGGACTGGA

ACTCCTCCCT

CGGGCCTGAA

ATGGGTCGAA

CTCGAAGTGG

TTCCTTCCCC

7980 8040 8100 8160 8220 8280 8340 8400 8460 8520 8580 8640 8700 8760 8820 8880 8940 9000 9060

CCCACCCTTT

TAGTCAAGGA

GGCCATCACC

GATGGAGTAC

AATATCCTAG

TCACATGGTC

CACATAAGGG

CTTAATATTT

CAAGTTTCTC

GTCCTGAAGA

AATGTCTGGC

TTCATTATTT

CTAAGACAGA

TGATGATGAA

ACCAGCTCCA

GAGTGAACAC

ACCTGCGATT

CCGGGGGTAG

CCTGTCAGCG

TTTTGAGACA

TGACCTCGAG

ACCTTTGGGA

AACAGTGTGA

GCTCTCTAGA

CCACAGAACC

TCTCTACTGC.

GCAATCCTCC

TTCTCTGGGT

CTCTCCGAGC

CTCAGTTTCC

GCAGGCAGTA

ATACTTCTAC

ATAATGGGGA

GGAAGCTCTC

AGCTGGGAGG

CTTGCACGGG

GGTTGGAGGG

AGTCCCCAGT

GCGTCTTCAG

CTCCTGGTCT

GACTAGCCAT

CCTCTTGTAA

GGTTAACTTT

TTTTGTCACA

TGGCCCACCC

TACCTCAGCC

CCCTTTCTTA

CCATGGATCT

CCACCTGTCA

TAAGAAGAAG

ACTGAAACTG

AACTGAGGCT

CAGCCC.CCAT

GGCTGGAGCC

AGCCAGCAGG

AGGTAAGCAG

TTTATTTATG

127

GTAGCGCATG

TTTTGTCTCC

GGAGTCTATT

GAGAACTGAA

ATATAAAATA

CAACCTATAG

CTCCAACCCT

TCTTGAATGC

AGGTGGGAGG

GCACTCTCTA

GGTTTAGGCA

CTCCCATCCC

AACTCTCGCA

CCGAGAGATT

CCGGGCCTCT

TGGGAGCTTT

CGGCTGCGTC

GGGCTGTGGG

GCGTGAGGCC

CTGGCCTTAA

ACTTAGAGAC

TAGCCTGTCA

GACAGGCTGT

GAGACTATTA

ACCACAGTGC

TAAAAGGTAA

TCAGAAACCA

GCCTAAAGAT

ATATGAAATA

GCACAGTCGG

CCACCCGCTT

GACGCATATG

CCTGGAGGAA

CCAGGTTCTG

GGCCCTTGCT

CGCCCGAGAG

GGCCGAAGCT

ATTCAGTATG 9120 AATGGCCAGT 9180 TTTGGTGACA 9240 TTTTAACCCC 9300 CAGCCAGTTA 9360 CTGTGCCTAC 9420 CCTAGGCAGC 9480 GGCATTAACC 9540 GACTTCCTTT 9600 TATTGCATAA 9660 TCCAAGACAC 9720 CCTCCGGTCC 9780 CTCACTTTAA 9840 GAGGGTCAAA 9900 GGCTTGGCGG 9960 CGTCCCCAGC 10020 ACT C;-A;GAAG 10080 TG7-JCCAGGG. 10140 GATGTCCTTA TCC-JCrTGCC 10200 12 8

TGCTGGGGGA

CAGCCCACTC

TTCTGTGCTG

TTAATATAAC

GTGCCACAAC

GGGACTAGGG

CTTCCCATCC

TAGCTATCCT

TCCCAGTGTC

TGCCCCTTGC

CTTTCTGACt

CCATCGCCGT

CAACTTTCCC

GCTGGCCGCG

GGGTGTGCGA

AGTTCCTCGG

ACCAGTGGCG

GGGAGGCTTG

CACAACACCG

TGGCTGCGGC

CATGTCACAC

TCTGTCTCTA

TACGTTTTAA

ACACACGTGA

CTGGCGACAA

TGTTTGGATA

GCCTCAGCCT

TGGGGGTACA

TTTGTCCGTG

GAATACTCGG

GGCATTGCCA

CAGCCGAAGC

CCCCAACACT

GCCGCGGGGC

CTGGCTCAAG

TGCTTGGATG

CGTGGGGGGT

CACTCTTCTT

TGGGGATTGG

CCGTGCATTC

TTTCTGTCAT

AAATTGCTTT

AGGTTAGAGA

GAGCAATTAC

GTCATAGGTA

ACCAAGTGCT

CAGTCCCAAG

TCCCTAGAGT

TTTTACCTCC

TTCCTCTGGG

TGGTCTGGTA

GCCGCTCCAT

GGCGAGCCCA

AAGCACGCAT

CAGAAGTCAC

AAAGGAGCAG

TCCAAGCACA

ACCCAAGGGC

TCTGAGGCTT

TCACTTTCCC

TGTATAATGT

ACTTTGTTGA

TGAGTCATCT

ATCGAAGGTA

GTGCTACCAC

ATCTCTGCTT

CTCCGGCCCC

CACTGATTTG

TGACTCTGGG

TGGGAGGCCG

TCTCTTTAGA

GCTCGGGCCC

ACTGCTCGAA

ACAAGACCCG

AGGA.AGAGAG

GGACGAGGGG

TGGCTTCCCA

ATCTTGGGAA

AGAGCCTTTT

GTGTGdCTTC

GTAGGCTCCT

CGCCAGCCCC

AATCGCTGGC

GTTTGTGGGA

TCTAGGTCTT

ACTTAGTCTC

ACTCCCTCCT

TCCACACCTG

CCGTCCCGCG

GCGCCCGGGC

GGTGCGGCGC

CCTTAGTTTC

AAACCAGGTA

AGACCCGGGT

ATCCTGCCCT

CTCAGTCCTC

CCCGCCCTTG

TTTTATGCTT

GTGAGCGTGC

TCCACCATGT

TCACCCCTCA

TTTAATTTCG

GGGGCTCTCC

TGTCTTAGTT

CATTGATTTC

TTGCTTGTCT

ACACCTTTCC

CGCGCCTCCT

CCGGGCGGCG

GAGCTCAAGC

CGCCTGTACG

GGAAAGTTGG

GAGCAGCCTC

CGGGCAGACG

10260 10320 10380 10440 10500 10560 10620' 10680 10740 10800 10860 10920 10980 11040 11100 11160 11220 11280 11340

GGGTGCGGCG

CCTTTCCCCT

AAGAGCCCCA

CTTTTCTGCC

CAGACCTGGA

TACCAGAGGC

ACTTGGATAT

TTAAATAAAT

GGGGTGGGGG

AGAGGTAAGG

CCTTCGGTGT

GGTTTTACTG

CTCAGGTCCT

GGCTCACCTG

TGGGCACAAT

ACCCCAGTGT

AAAGGAGTTG

GA

GGGTCTGGGT

TGCTCAAAGG

CATCATCAAG

GCCGGCTAAA

AATTGGAGCC

GAGCTCCCAC

GGGTAGGGTT

TTCAGGTCCC

129

GAGTGGGGCC

GATCTCTTAG

TTGCTGAAGG

CTCTAAGGAT

CCTCTGTACC

AACCACAGCT

GGGGTATTGC

GATGGCCAGT

TACAGCAGTC

TGCTCATTTC

GTCCAGGCTT

AGGCCATCCT

ATCTGGGCAA

TTGGTCCACA

AGGGCCTCCC

GTGTTTGGGG

TAGATGAGGC 11400 ACCCACTGCA 11460 AATGTGGCCT 11520 CCTGCTGGGT 11580 CAAAGAA-ACC 11640 TGATGGTCAC 11700 AAGAGTCTCT 11760 CCTATGTGCT 11820 11832 INFORMATION FOR SEQ ID NO:39: SEQUENCE CHARACTERISTICS: LENGTH: 26 amino acids TYPE: amino acids STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: Val Ile Ser Pro Gin Asp Pro Thr Leu Leu Ile Giy Ser Ser Leu Gin Ala Thr Cys Ser 10 15 Ile His Gly Asp Thr Pro AMEN~DED

SHEET

Claims (10)

1. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a novel haemopoietin receptor or derivative thereof wherein said nucleic acid molecule comprises a sequence of nucleotides substantially as set forth in SEQ ID NO:12 or a nucleotide sequence having at least 60% similarity to the nucleotide sequence set forth in SEQ ID NO:12 or a nucleotide sequence capable of hybridising thereto or its complementary sequence under low stringency conditions at 42 0 C. *o

2. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a novel haemopoietin receptor or derivative thereof wherein said nucleic acid molecule *comprises a sequence of nucleotides substantially as set forth in SEQ ID NO:14 or a nucleotide sequence having at least 60% similarity to the nucleotide sequence set forth in SEQ ID NO:14 or a nucleotide sequence capable of hybridising thereto or its complementary sequence under low stringency conditions at 42 0 C.

3. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a novel haemopoietin receptor or derivative thereof wherein said nucleic acid molecule comprises a sequence of nucleotides substantially as set forth in SEQ ID NO:16 or a nucleotide sequence having at S least 60% similarity to the nucleotide sequence set forth in SEQ ID NO:16 or a nucleotide sequence capable of hybridising P:\Op\Ejh.mdod\2149090.nd.mmdodclims.d-l9IO2/OI 131 thereto or its complementary sequence under low stringency conditions at 42 0 C.

4. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a novel haemopoietin receptor or derivative thereof wherein said nucleic acid molecule comprises a sequence of nucleotides substantially as set forth in SEQ ID NO:18 or a nucleotide sequence having at least 60% similarity to the nucleotide sequence set forth in SEQ ID NO:18 or a nucleotide sequence capable of hybridising thereto or its complementary sequence under low stringency conditions at 42 0 C. *o

5. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a novel haemopoietin receptor or derivative thereof wherein said nucleic acid molecule comprises a sequence of nucleotides substantially as set forth in SEQ ID NO:24 or a nucleotide sequence having at least 60% similarity to the nucleotide sequence set forth in SEQ ID NO:24 or a nucleotide sequence capable of hybridising thereto or its complementary sequence under low stringency conditions at 42°C.

6. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a novel haemopoietin receptor or derivative thereof wherein said nucleic acid molecule comprises a sequence of nucleotides substantially as set forth in SEQ ID NO:28 or a nucleotide sequence having at least 60% similarity to the nucleotide sequence set forth in P:\OM\Ejb. mcodcd\ 149090.amradmmdsdcaimsdw- 19/02/01 -132- SEQ ID NO:28 or a nucleotide sequence capable of hybridising thereto or its complementary sequence under low stringency conditions at 420C.

7. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a novel haemopoietin receptor or derivative thereof wherein said nucleic acid molecule comprises a sequence of nucleotides substantially as set forth in SEQ ID NO:38 or a nucleotide sequence having at least 60% similarity to the nucleotide sequence set forth in SEQ ID NO:38 or a nucleotide sequence capable of hybridising thereto or its complementary sequence under low stringency conditions at 42 0 C.

8. An isolated nucleic acid molecule according to claim 1 or 2 or 3 or 4 or 6 or 7 wherein said haemopoietin receptor is of murine origin.

9. An isolated nucleic acid molecule according to claim 5 wherein said haemopoietin receptor is of human origin. An expression vector comprising a nucleic acid molecule selected from the list consisting of:- a nucleotide sequence as set forth in SEQ ID NO:12; (ii) nucleotide sequence as set forth in SEQ ID NO:14; (iii) a nucleotide sequence as set forth in SEQ ID NO:16; PA\pcr\Ej'h.=dcdU 149090.amra&=mdchamsdm

133- (iv) a nucleotide sequence as set forth in SEQ ID NO:18; a nucleotide sequence as set forth in SEQ ID NO:24; (vi) a nucleotide sequence as set forth in SEQ ID NO:28; and (vii) a nucleotide sequence as set forth in SEQ ID NO:38. 11. A method for cloning a nucleotide sequence encoding an NR6 haemopoietin receptor or a derivative Sthereof, said method comprising searching a nucleotide database for a sequence as set forth in SEQ ID NO:8 or a nucleotide sequence which would hybridise to SEQ ID NO:7 and/or SEQ ID NO:8 or their complementary forms, designing one or more oligonucleotide primers based on the nucleotide sequence located in said search, screening a nucleic acid library with said one or more oligonucleotides and obtaining a clone therefore which encodes NR6 or a part or derivative thereof. 12. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding a haemopoietin receptor or derivative thereof having an amino acid sequence substantially as set forth in SEQ ID NO:13 or having at least about 50% similarity thereto. 13. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding a haemopoietin receptor or derivative thereof having an amino acid sequence substantially as set forth in SEQ ID NO:15 or having at least about 50% similarity thereto. P:\Opa\bhnmded\21 4900m dedclaims.do-l19/02/01 -134- 14. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding a haemopoietin receptor or derivative thereof having an amino acid sequence substantially as set forth in SEQ ID NO:17 or having at least about 50% similarity thereto. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding a haemopoietin receptor or derivative thereof having an amino acid sequence substantially as set forth in SEQ ID NO:19 or having at least about 50% similarity thereto. 16. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding a haemopoietin receptor or derivative thereof having an amino acid sequence substantially as set forth in SEQ ID NO:25 or having at least about 50% similarity thereto. 17. An isolated nucleic acid molecule comprising a a sequence of nucleotides encoding a haemopoietin receptor or derivative thereof having an amino acid sequence substantially as set forth in SEQ ID NO:29 or having at least about 50% similarity thereto. 18. An isolated haemopoietin receptor comprising the amino acid sequence substantially as set forth in SEQ ID NO:13. 19. An isolated haemopoietin receptor comprising the amino acid sequence substantially as set forth in SEQ ID P: po\Ej'h.mded\2I49090.amraLmddcaimsdo 9/2/01 -135- An isolated haemopoietin receptor comprising the amino acid sequence substantially as set forth in SEQ ID NO:17. 21. An isolated haemopoietin receptor comprising the amino acid sequence substantially as set forth in SEQ ID NO:19. 22. An isolated haemopoietin receptor comprising the amino acid sequence substantially as set forth in SEQ ID *9 NO:29. 24. A method for modulating expression of NR6 in a mammal, said method comprising contacting a genetic sequence encoding said NR6 with an effective amount of a modulator of :29. NR6 expression for a time and under conditions sufficient to up-regulate or down-regulate or otherwise modulate expression of NR6, wherein the genetic sequence encoding said NR6 is selected from the nucleotide sequence set forth in SEQ ID NO:12 or 14 or 16 or 18 or 24 or 28 or 38 or is a sequence having at least about 60% similarity to at least one of SEQ ID NO:12 or 14 or 16 or 18 or 24 or 28 or 38 and is capable of hybridising thereto or a complementary sequence under low stringency conditions at 42 0 C. A method of modulating activity of NR6 in a Smammal, said method comprising administering to said mammal, seuneudrlwsrnecNodtosa 2C P:\O \Ejh. wmdcd\2149090.dramdc.902/01 -136- a modulating effective amount of a molecule for a time and under conditions sufficient to increase or decrease NR6 activity wherein said NR6 comprises an amino acid sequence: encoded by a nucleotide sequence selected from the nucleotide sequence set forth in SEQ ID NO:12 or 14 or 16 or 18 or 24 or 28 or 38 or a nucleotide sequence having at least 60% similarity to the nucleotide sequence set forth in SEQ ID NO:12 or 14 or 16 or 18 or 24 or 28 or 38 and which is capable of hybridising thereto or a complementary sequence under low stringency conditions at 42 0 C; and i (ii) substantially as set forth in SEQ ID NO:12 or 14 or 16 or 18 or 32 or 30 or a sequence having at least 50% similarity thereto. comprises the amino acid sequence: 26. A pharmaceutical composition comprising an NR6 receptor in soluble form and one or more pharmaceutically acceptable carriers and/or diluents wherein said NR6 comprises the amino acid sequence: encoded by a nucleotide sequence selected from the nucleotide sequence set forth in SEQ ID NO:12 or 14 or 16 or 18 or 24 or 28 or 38 or a nucleotide sequence having at least 60% similarity to the nucleotide sequence set forth in SEQ ID NO:12 or 14 or 16 or 18 or 24 or 28 or 38 and which is capable of hybridising thereto or a PA\Om\Ejh.mncd\2149090 -d mdeddimc 11010 -137- complementary sequence under low stringency conditions at 42 0 C; and (ii) substantially as set forth in SEQ ID NO:12 or 14 or 16 or 18 or 32 or 30 or a sequence having at least 50% similarity thereto. 26. An isolated antibody or a preparation of antibodies specific for NR6 receptor, said NR6 receptor comprising the amino acid sequence: encoded by a nucleotide sequence selected from the nucleotide sequence set forth in SEQ ID NO:12 or 14 or 16 or 18 or 24 or 28 or 38 or a nucleotide sequence having at least 60% similarity to the nucleotide sequence set forth in SEQ ID NO:12 or 14 or 16 or 18 or 24 or 28 or 38 and which is capable of hybridising thereto or a complementary sequence under low stringency conditions at 42 0 C; and (ii) substantially as set forth in SEQ ID NO:12 or 14 or 16 or 18 or 24 or 28 or 38 or a sequence having at least 50% similarity thereto. 27. A trangenic non-human animal comprising a mutation in at least one allele of the gene encoding NR6. 28. A transgenic non-human animal according to claim 28 L comprising a mutation in two alleles of the gene encoding AC, NR6. P:\Opcr\Ejh uwdcd\249090. a&dedc,,sdc -l9/2IOI -138- 29. A transgenic non-human animal according to claim 28 or 29 wherein said animal is a murine animal. An isolated nucleic acid molecule according to any one of claims 1-9 or 12-23 or an expression vector according to claim 10 or a method according to any one of claims 11, 24 or 25 or a pharmaceutical composition according to claim 26 or an isolated antibody according to claim 27 or a transgenic non-human animal according to any one of claims 28-30 substantially as hereinbefore defined with reference to the Figures and/or Examples. S