HUMAN GLUTAMATE RECEPTOR PROTEINS

The invention relates to the novel human metabotropic glutamate receptors 3 and 4 (human mGluR3 and human mGluR4) and polypeptide fragments thereof, DNA coding therefor and oligonucleotide fragments thereof, antibodies specific for the receptors and a method for screening for compounds capable of binding to or modulating the activity of the receptors.

In the mammalian central nervous system, L-glutamate serves as a major excitatory neurotransmitter. The interaction of glutamate with its membrane bound receptors is believed to play a role in many important neuronal processes including fast synaptic transmission, synaptic plasticity, and long term potentiation. These processes are fundamental to the maintenance of life and normal human abilities such as learning and memory (Monaghan, D.T. et al. , 8 Neuron 267 (1992)) .

In addition to its role in normal human physiology, interaction of L-glutamate with its receptors is believed to play a key role in many neurological disorders such as stroke, epilepsy, and head trauma, as well as neurodegenerative processes such as

Alzheimer' s disease (Olney, R.W. , 17 Drug Dev.Res. 299 (1989)) .

For this reason, understanding the molecular structure of human L-glutamate receptors is important for understanding these disease processes as well as for searching for effective therapeutic agents. To date, the search for therapeutic agents which will bind and modulate the function of human glutamate receptors has been hampered by the unavailability of homogeneous sources of receptors. The brain tissues commonly used by pharmacologists are derived from experimental animals

(non-human) and furthermore contain mixtures of various types of glutamate receptors.

In searching for drugs for human therapy, it is desirable to use receptors which are more analogous to those in the intact human brain than are the rodent receptors employed to date.

The discovery of human glutamate receptors, therefore, provides a necessary research tool for the development of selective pharmaceutical agents.

Pharmacological characterisation of receptors for L-glutamate has led to their classification into two families based on their biological function: the ionotropic receptors which.are directly coupled to cation channels in the cell membrane, and the metabotropic receptors which function through coupling to G-proteins. To date seven different rat mGluRs and three different human mGluRs have been cloned. These are: rat mGluRl, (Masu et al, Nature, Vol 349, 760 - 765, (1991) , Houamed, K.M., et al . Science, Vol 252, 1318-1321, (1991)) ; rat mGluR2, (Tanabe, Y. et al. , Neuron 8, 166-179, (1992)) ; rat mGluR3, ibid.; rat mGluR4, ibid.; rat mGluR5, (Abe, T. et al., J. Biol. Chem. , 267, 13361-13368, (1992)) ; rat mGluR6, (Nakajima, Y. et al. , J. Biol. Chem., 268, 11868-11873, (1993)) ; rat mGluR7 (Okamoto, N et al, J. Biol. Chem. vol 269, 1231-1236 (1994) , Saugstad, J.A. et al, Mol. Pharmacol vol 45, 367-372 (1994)) ; human mGluRl (WO 94/29449 and EP-A-569 240) ; human mGluR2 (WO 94/29449) ; human mGluR3 (WO 94/29449) and human mGluR5 (Minakami, R. et al Biochem. Biophys. Res. Comm. vol 199, 1136-1143 (1994) and WO 94/29449) . Types 1 and 5 are coupled to phosphoinositol metabolism via phospholipase C. The other five mGluRs are all negatively coupled to adenylate cyclase (Nakanishi, S., Science 258, 597-603, (1992); Nakajima, Y. , Iwakabe, H. , Akazawa, C, Nawa, H., Shigemoto, R. , Mizuno, N. and Nakanishi, S., J. Biol. Chem. 268, 11868-11873 (1993) ; Okamoto, N. et al, J. Biol. Chem. vol 269, 1231-1236 (1994) ; Saugstad, J.A. et al, Mol. Pharmacol, vol 45, 367-372 (1994)) . Because the mGluRs have only been recently described, they are poorly-defined pharmacologically. However, they appear to fall into three groups by their agonist selectivity: mGluRl and 5 (guisqualate) , mGluR2 and 3 [(2S,1'R,2'R,3'R)2- (2,3-dicarboxycyclopropyl)glycine, DCG-IV] and mGluR4, 6 and 7 (2-amino-4-phosphonobutyrate, AP4) (Nakanishi, S., Science 258, 597-603, (1992) ; Nakajima, Y. ,

Iwakabe, H., Akazawa, C, Nawa, H. , Shigemoto, R. , Mizuno, N. and Nakanishi, S., J. Biol. Chem. 268, 11868-11873. (1993) , Hayashi, Y. , Momiyama, A., Takahashi, T., Ohishi, H., Ogawa-Meguro, R., Shigemoto, R. , Mizuno, N. and Nakanishi, S., Nature 366, 687-690 (1993), Okamoto, N. et al, J. Biol. Chem. vol 269, 1231-1236 (1994), Saugstad, J.A. et al, Mol. Pharmacol, vol 45, 367-372 (1994)) . These groups also reflect the relative sequence homologies of each of the receptors.

The present invention provides a DNA isolate encoding human mGluR3 or human mGluR4. The DNA isolate may encode human mGluR3 having an amino acid sequence at least 97% (e.g. at least 98% or at least 99%) identical to the sequence of SEQ ID NO:2. The DNA isolate may alternatively encode human mGluR4 having an amino acid sequence at least 97% (e.g. at least 98% or at least 99%) identical to the sequence of SEQ ID NO:4. Preferably, the DNA isolate encodes human mGluR3 having exactly the amino acid sequence of SEQ ID NO:2 or human mGluR4 having exactly the amino acid sequence of SEQ ID NO:4.

The nucleotide sequence of the DNA isolate may be at least 90% identical (e.g. at least 95%, at least 96%, at least 97% at least 98% or at least 99% identical) to the human mGluR3 coding sequence set forth from position 253 or 259 to position 2889 of SEQ ID NO:l. The nucleotide sequence may alternatively be at least 90% identical (e.g. at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical) to the human mGluR4 coding sequence set forth from position 171 to position 2906 of SEQ ID NO:3.

In general, the nucleotide sequence of the DNA isolate is exactly the sequence from position 253 or 259 to position 2889 of SEQ ID NO:l or exactly the nucleotide sequence from position 171 to position 2906 of SEQ ID NO:3. However, these sequences from SEQ ID NOs:3 and 4 may be changed by nucleotide substitution, deletion, insertion or extension. Such changed sequences should still encode a protein which retains mGluR3 or

mGluR4 activity, e.g. L-glutamate binding and/or negative coupling to adenylate cyclase. A substitution, deletion or insertion may involve one or more nucleotides, typically from one to five, one to ten or one to twenty nucleotides.

The human mGluR3 nucleotide and amino acid sequences set forth in present SEQ ID NO:l contain three differences compared to the human mGluR3 sequences set forth in WO 94/29449 (see SEQ ID NO:5) , as follows:

1. There are two internal nucleotide change differences which both result in amino acid changes. Amino acid Gly 334

(GGC) in the WO 94/29449 sequence is Asp (GAC) in the present sequence and Glu 374 (GAA) in the WO 94/29449 sequence is Asp (GAC) in the present sequence.

2. The mGluR3 nucleotide sequence contains two ATG codons in the same reading frame at the beginning of the translated sequence. In WO 94/29449, the earlier of the two codons is assumed to be the start codon so the translated sequence begins MetLysMet.... However, the sequence surrounding the second ATG codon is much more favourable for initiation of translation and it is therefore likely that this codon initiates translation giving a translated sequence which begins MetLeuThr.... It is possible that two protein products are produced from the two initiation codons and both products are included in the invention.

3. The final 17 nucleotides of the 3' untranslated end of the WO 94/29449 sequence differ completely from the corresponding region of present SEQ ID NO:l.

The invention includes an oligonucleotide fragment having a sequence of a portion of a DNA isolate encoding human mGluR3 or mGluR4 of the invention. The fragment may have a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to sequence encoding human mGluR3

or human mGluR4 of the invention, for example sequence shown ih SEQ ID NO:l or SEQ ID NO:3. The fragment is at least 12 nucleotides in length, e.g. at least 15, at least 18, at least 30, at least 60, at least 180, at least 360 or at least 720 nucleotides in length. The fragment may be single or double stranded. When the fragment is single stranded, it may have a sequence from either a sense or antisense strand of a DNA molecule encoding a human mGluR3 or a human mGluR4 of the invention. .An antisense fragment may be useful in the therapeutic treatment of a disease involving over-expression of human mGluR3 or human mGluR4.

The present invention also provides a human mGluR3 protein and a human mGluR4 protein, such as those having the amino acid sequences of SEQ ID NOs:2 and 4. Alternatively, there is provided a human mGluR3 or mGluR4 protein having a sequence which is at least 96% identical to the amino acid sequence of SEQ ID NO:2 or 4, for example a sequence at least 97%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO: 2 or 4.

The invention further includes a polypeptide fragment of a human mGluR3 or mGluR4 protein of the invention, which fragment is at least 8 amino acids in length, for example at least 12, at least 24, at least 48 or at least 96 amino acids in length.

The human mGluR3 or mGluR4 protein will usually be obtained by recombinant DNA techniques. However, the protein may be obtained using biochemical purification of the protein from its natural origin.

The human mGluR3 or mGluR4 is generally expressed on the surface of a host cell, but may be in purified or isolated form. Preferably, the mGluR3 or mGluR4 in purified form comprises a preparation in which at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% of the weight of protein in the preparation is human mGluR3 or mGluR4.

According to a further embodiment, there is also provided a DNA isolate encoding a fragment of the amino acid sequence of the human mGluR3 or mGluR4 protein. DNA sequences encoding fragments of the human mGluR3 or mGluR4 protein preferably encode those parts of the amino acid sequence which characterise the receptor, i.e. those parts which are most distinct from other human mGlu receptor proteins.

A person of ordinary skill in the art would by reference to the sequences disclosed herein know how to obtain a DNA isolate according to the invention using methodologies and techniques well known to the skilled person. The various means include for example, DNA synthesis, or more preferably, recombinant DNA techniques . Techniques for constructing recombinant isolates are described by Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) .

Human mGluR3 or mGluR4 protein and fragments thereof may be obtained by expression of DNA isolates of the present invention in appropriate expression vectors. A library of replicable expression vectors may be created by cloning genomic DNA or, more preferably, cDNA into a parent vector. The library is screened for clones containing the desired nucleic acid sequence, e.g. by means of a DNA probe. Probes having consensus sequences for specific regions of various human mGluR proteins may be used. For example, the following consensus primers designed from the published sequences of rat mGluRl to 5 proteins may be used:

ACAACGACACACCCGTGGTCAA (SEQ ID NO:5) ; TCCGGTCGGGAGCTCTGCTA (SEQ ID NO:6); GCTACTCTGCCCTGCTGACCAAGAC (SEQ ID NO:7) ; GCAATGCGGTTGGTCTTGGT (SEQ ID NO:8) ; AAGTTCATCGGCTTCACCATGTACAC (SEQ ID NO:9);

GATGCAGGTGGTGTACATGGTGAA (SEQ ID NO:10) ; and TCTCCGGTTGGAAGAGGATGATGT (SEQ ID NO:11) .

A replicable expression vector is a vector which contains the appropriate origin of replication sequence for replication of the vector and the appropriate sequences for expression of the foreign nucleotide sequence in the vector. The sequences for expression of a foreign sequence will generally include a transcription promoter operably linked to the foreign sequence. The term "operably linked" refers to a linkage in which the promoter and foreign sequence are connected in such a way to permit expression of the foreign sequence. The transcription promoter sequence may be part of the parent vector sequence into which the foreign sequence is inserted. Alternatively, the promoter sequence may be a native promoter sequence of a gene encoding human mGluR3 or mGluR4 of the invention. A vector may be, for example, a plasmid, virus or phage vector. A vector may contain one or more selectable markers, for example an ampicillin resistance gene in the case of a bacterial vector or a neomycin resistance gene in the case of a mammalian vector. A foreign gene sequence inserted into a vector may be transcribed in vitro or the vector may be used to transform or transfect a host cell.

According to a further aspect of the invention, there is provided a host cell transformed or transfected with a vector into which there has been inserted a DNA isolate (or a fragment thereof) encoding human mGluR3 or mGluR4 protein. A vector and host cell will be chosen so as to be compatible with each other, and may be prokaryotic or eukaryotic. A prokaryotic host may, for example, be E. coli, in which case the vector may, for example, be a bacterial plasmid or a phage vector. A eukaryotic host may, for example, be a yeast (e.g. S.cerevisiae) cell, a Chinese hamster ovary (CHO) cell, BHK cell, oocyte or an insect cell (e.g. Spodoptera frugiperda) . When the host is an insect cell, the vector is generally a baculovirus vector (reviewed by Luckow and Summers in BIO/TECHNOLOGY, Vol. 6, 47-55 (1988)) . When the host is an oocyte mRNA encoding human mGluR3 or mGluR4 protein can be injected into the oocyte for expression of the protein.

A host cell expressing human mGluR3 or a human mGluR4 according to the invention may be produced by a method comprising

(a) transforming or transfecting cells with a vector comprising a DNA isolate (or fragment thereof) encoding human mGluR3 or mGluR4 ;

(b) culturing the cells under conditions in which the mGluR3 or mGluR4 is expressed; and

(c) recovering a host cell expressing the mGluR3 or mGluR4 from the culture.

An oligonucleotide fragment according to the invention will generally be DNA, although other types of nucleic acid may be used, for example RNA or modified DNA. A number of different types of nucleic acid modification are known in the art . These include methylphosphonate and phosphorothioate backbones, and addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule.

Oligonucleotide fragments may be useful in the detection of nucleic acid sequences. An oligonucleotide fragment corresponding to a portion of the human mGluR3 or mGluR4 coding sequence may be an oligonucleotide probe or an oligonucleotide primer (e.g. a polymerase chain reaction (PCR) primer) , which will hybridise to a nucleic acid molecule (e.g. a DNA or RNA molecule) encoding a human mGluR3 or mGluR4 protein of the invention. The probe or a pair of primers may be used to detect or quantitatively determine the nucleic acid sequence. This has diagnostic utility in detecting and quantitatively determining human mGluR3 or mGluR4 mRNA associated with a disease state, for example disorders of the central nervous system.

A fragment which is a probe or primer may carry a revealing label, such as 32P, digoxigenin or biotin. Preferably, the probe or primer will specifically hybridise only to its target sequence, e.g. a portion of the sequence disclosed in SEQ ID

NO:l or SEQ ID NO:3, and not to other sequences. A probe or primer which hybridises only to its target sequence will generally be exactly complementary to the target sequence whereas a probe or primer which is only selective may have a sequence which is, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% complementary to the target sequence. A probe which is not exactly complementary to its target sequence has utility in the identification of new human mGluR coding nucleotide sequences. A fragment which is a probe or primer may have from 12 to 60 nucleotides, e.g. from 12 to 40 nucleotides, or from 15 to 30 nucleotides.

Primers for PCR are generally provided as a pair. A first primer hybridises to a sense sequence 3' to the sequence to be amplified and a second primer hybridises to an antisense sequence 5' to the sequence to be amplified. This allows synthesis of double stranded DNA representing the region between the two primers.

Thus, the invention provides a method of amplifying a target nucleic acid sequence present in a nucleic acid encoding a human mGluR3 or mGluR4 protein of the invention, which method comprises carrying out PCR employing a primer of the invention. Such a method generally comprises carrying out cycles of

(a) denaturing double stranded DNA containing the target sequence to obtain single stranded DNA;

(b) hybridizing a first primer to a sense strand 3' to the target sequence, and hybridising a second primer to an antisense strand 5' to the target sequence; and

(c) synthesising DNA from the first and second primers.

The number of cycles is suitably from 10 to 50, preferably 20 to 40, more preferably 25 to 35. The method may be carried out starting from a double stranded nucleic acid (e.g. dsDNA) or a single stranded nucleic acid (e.g. mRNA) . The target sequence

may be a complete human mGluR3 or mGluR4 protein encoding sequence or a partial human mGluR3 or mGluR4 protein encoding sequence.

As will be appreciated by a person skilled in the art, the method described above is based upon the well-known polymerase chain reaction (PCR) method. A skilled person would know of detailed protocols for carrying out PCR and reverse transcriptase-PCR (RT-PCR) . Reviews of PCR are provided by Mullis Cold Spring Harbor Symp. Quant. Biol. 51, 263-273 (1986) ; Saiki et al. Bio/Technology 3, 1008-1012 (1985) ; and Mullis et al Meth. enzymol 155, 335-350 (1987) .

An oligonucleotide probe according to the invention has utility in detecting or quantitatively determining a nucleic acid (e.g. a DNA or RNA) encoding a human mGluR3 or mGluR4 protein according to the invention. Conventional methods for detecting or quantitatively determining a nucleic acid may be used, for example in situ hybridization, Southern blotting or Northern blotting. Accordingly, there is provided a method of detecting or quantitatively determining in a sample a target nucleic acid encoding human mGluR3 or mGluR4 according to the invention, which method comprises

(a) contacting the probe with the sample; and

(b) detecting or quantitatively determining hybridization of the probe with any target nucleic acid present in the sample.

The sample containing the target nucleotide sequence may, for example, be a tissue specimen, a tissue extract or cell extract from a patient suffering from a disease associated with abnormal human mGluR3 or mGluR4 protein activity such as a disorder of the central nervous system. Alternatively, the sample may, for example, be a sample produced as a result of a recombinant DNA procedure, in which case the sample may be recombinant cells. The target nucleic acid sequence may be a

complete human mGluR3 or mGluR4 protein coding sequence or a partial human mGluR3 or mGluR4 protein coding sequence.

A preferred method of detecting or quantitatively determining a target nucleic acid sequence in a sample comprises

(i) subjecting the sample to gel electrophoresis to separate the nucleic acids; (ii) transferring the separated nucleic acids onto a solid support (e.g. a nitrocellulose support) by blotting; and (iii) hybridising a probe according to the invention to the target nucleic acid sequence.

A probe can be used in an in situ hybridization procedure to locate a nucleic acid sequence encoding a human mGluR3 or mGluR4 protein of the invention. This can be done to determine the spatial distribution of human mGluR3 or mGluR4 protein coding mRNA sequences in a cell or tissue. For mRNA detection, the tissue is gently fixed so that its RNA is retained in an exposed form and the tissue is then incubated with a labelled complementary probe.

The invention includes an antibody specific for human mGluR3 or mGluR4 protein according to the invention. The antibody has utility in detecting and quantitatively determining human mGluR3 or mGluR4 protein, and hence is useful in diagnosis of diseases associated with human mGluR3 or mGluR4 protein, such as the diseases listed hereinabove. The antibody also has utility in production of human mGluR3 or mGluR4 protein by recombinant DNA procedures, for example in detection of positive clones containing a target sequence.

The antibody is preferably monoclonal, but may also be polyclonal. The antibody may be labelled. Examples of suitable antibody labels include radiolabels, biotin (which may be detected by avidin or streptavidin conjugated to

peroxidase) , alkaline phosphatase and fluorescent labels (e.g. fluorescein and rhodamine) . The term "antibody" is used herein to include both complete antibody molecules and fragments thereof. Preferred fragments contain at least one antigen binding site, such as Fab and F(ab')2 fragments. Humanised antibodies and fragments thereof are also included within the term "antibody" .

The antibody is produced by raising antibody in a host animal against a human mGluR3 or mGluR4 protein according to the invention or an antigenic epitope (e.g. a peptide) thereof

(hereinafter "the immunogen") . Methods of producing monoclonal and polyclonal antibodies are well-known. A method for producing a polyclonal antibody comprises immunising a suitable host animal, for example an experimental animal, with the immunogen and isolating immunoglobulins from the serum. The animal may therefore be inoculated with the immunogen, blood subsequently removed from the animal and the IgG fraction purified. A method for producing a monoclonal antibody comprises immortalising cells which produce the desired antibody. Hybridoma cells may be produced by fusing spleen cells from an inoculated experimental animal with tumour cells (Kohler and Milstein, Nature 256, 495-497, (1975)) . The antibody may also be produced by recombinant DNA technology, for example as described in Skerra et al (1988) Science 240 , 1038-1041.

An immortalized cell producing the desired antibody may be selected by a conventional procedure. The hybridomas may be grown in culture or injected mtraperitoneally for formation of ascites fluid or into the blood stream of an allogenic host or immunocompromised host. Human antibody may be prepared by in vitro immunisation of human lymphocytes, followed by transformation of the lymphocytes with Epstein-Barr virus.

For the production of both moncolonal and polyclonal antibodies, the experimental animal is suitably a goat, rabbit,

rat or mouse. If desired, the immunogen may be administered as a conjugate in which the immunogen is coupled, for example via a side chain of one of the amino acid residues, to a suitably carrier. The carrier molecule is typically a physiologically acceptable carrier. The antibody obtained may be isolated and, if desired, purified.

The invention provides a method of detecting or quantitatively determining in a sample a human mGluR3 or mGluR4 protein of the invention, which method comprises

(a) contacting the sample with an antibody of the invention; and

(b) detecting or quantitatively determining the binding of the antibody.

A preferred method for detecting or quantitatively determining a human mGluR3 or mGluR4 protein is Western blotting. Such a method can comprise the steps of

(i) subjecting a sample containing a target human mGluR3 or mGluR4 protein to gel electrophoresis to separate the proteins in the sample;

(ii) transferring the separated proteins onto a solid support (e.g. a nitrocellulose support) by blotting; and (iii) allowing an antibody according to the invention which has been labelled to bind to the target human mGluR3 or mGluR4 protein.

Preferred methods of quantitative determination are ELISA (enzyme-linked immunoassay) methods such as non-competitive ELISA methods. Typically, an ELISA method comprises the steps of

(i) immobilising on a solid support an unlabelled antibody according to the invention;

(ii) adding a sample containing ihe target human mGluR3 or mGluR4 protein such that the human mGluR3 or mGluR4 protein is captured by the unlabelled antibody; (iii) adding an antibody according to the invention which has been labelled; and

(iv) quantitatively determining the amount of bound labelled antibody.

.An antibody of the invention may be employed histologically for in situ detection of a human mGluR3 or mGluR4 protein, e.g. by immunofluorescence or immunoelectron micropsy. In situ detection may be accomplished by removing a histological specimen from a patient, and allowing a labelled antibody to bind to the specimen. Through use of such a procedure, it is possible to determine not only the presence of a human mGluR3 or mGluR4 protein but also its distribution.

.An antibody of the invention may be used to purify a target human mGluR3 or mGluR4 protein. Conventional methods of purifying an antigen using an antibody may be used. Such methods include immunoprecipitation and immunoaffinity column methods. In an immunoaffinity column method, an antibody in accordance with the invention is coupled to the inert matrix of the column and a sample containing the target human mGluR3 or mGluR4 protein is passed down the column, such that the target human mGluR3 or mGluR4 protein is retained. The human mGluR3 or mGluR4 protein is then eluted.

The sample containing the target mGluR3 or mGluR4 protein used in the detection, determination and purification methods may be a tissue specimen, a tissue extract or a cell extract from a patient suffering from a disease associated with human mGluR3 or mGluR4 protein such as a disease listed hereinabove. Alternatively, the sample may be one produced as a result of recombinant DNA procedures, e.g. an extract of host cells.

The invention provides a pharmaceutical composition containing a pharmaceutically acceptable carrier or diluent and, as active ingredient, an antibody or polypeptide fragment of the invention.

A human mGluR3 or mGluR4 protein of the invention is useful for screening for drugs which modulate (e.g. inhibit or stimulate) the receptor. The invention includes a method for identifying a compound which binds to or modulates the activity of the human mGluR3 or mGluR4 protein, which method comprises one or more of the following steps:

(a) incubating whole cells and/or membranes prepared from cells containing the human mGluR3 or mGluR4 protein with the compound;

(b) measuring the binding of the compound to human mGluR3 or mGluR4 protein;

(c) comparing the binding with that of known ligands;

(d) measuring the effect of the compound on basal/stimulated (e.g. forskolin stimulated) adenylate cyclase activity ;

(e) comparing the ability of the compound to inhibit/stimulate human IP3 production;

(f) comparing the effect of the compound using other functional models of mGluR3 or mGluR4 activity as appropriate (e.g. microphysiometry) ;

(g) measuring G protein coupling by other (standard) means (e.g. binding/functional studies) .

The invention also extends to agents (compounds) identified by use of a particular screen. Preferably, the agent is a chemical molecule of relatively low molecular weight, for example less than about 1000D.

Methods for identifying compounds which bind to human mGluR3 or human mGluR4 receptor generally comprise the following steps :

(a) contacting a compound with the receptor, and

(b) measuring any binding of the compound to the receptor.

Such methods for identifying compounds which bind to receptors are well-known in the art. A competitive binding assay may, for example, be used. A competitive binding assay may comprise

(a) contacting a test compound and a labelled ligand for the receptor (e.g. [ ³ H] glutamate) with the receptor,

(b) measuring the amount of the labelled ligand bound to the receptor, and

(c) comparing the amount of the labelled ligand bound to the receptor measured in step (b) with the amount of the labelled ligand that binds to the receptor in the absence of the test compound.

If less labelled ligand binds to the receptor in the presence of the test compound than in the absence of the test compound, this indicates that the test compound is competing for binding to the receptor with the ligand. The amount of the ligand bound to the receptor in step (b) may be measured indirectly; the amount of bound ligand may be determined by extrapolation from the amount of unbound ligand.

Methods for identifying compounds which modulate the activity of human mGluR3 or human mGluR4 receptor generally comprise

(a) contacting a compound with the receptor, and (b) measuring any change in second messenger receptor activity.

The second messenger activity measured in step (b) may be basal or stimulated adenylate cyclase activity. Adenylate cyclase activity may be measured by measuring the level of cAMP, which is the product of adenylate cyclase. Typically, adenylate cyclase is stimulated using forskolin and any decrease in

adenylate cyclase activity caused or prevented by the presence of the test compound is measured. A suitable adenylate cyclase assay is described in Tanabe et al (1992) Neuron 8 _. , 169-179.

The invention includes a combination (or complex) of human mGluR3 or human mGluR4 receptor and an agent (compound) which modulates the second messenger activity of the receptor. Examples of such agents are L-glutamate (mGluR3 and mGluR4) ; (2S, l'R,2'R,3'R) 2- (2, 3-dicarboxycyclopropyl)glycine, DCG-IV (mGluR3) ; L-2-amino-4-phosphonobutyrate, L-AP4 (mGluR4) ; trans- l-aminocyclopentane-l,3-dicarboxylate, t-ACPD (mGluR4) ; and quisqualate (mGluR4) .

The invention also includes a method of modulating the second messenger activity of human mGluR3 or human mGluR4 receptor, which method comprises contacting the receptor with a compound which modulates the second messenger activity of the receptor. Examples of suitable compounds which modulate receptor activity are given above. The method may be carried out either on the human/animal body or outside the human or animal body (e.g. in vitro) .

Brief Description of the Drawings

Figure 1 shows a comparison between human and rat mGluR3 protein sequences. The human amino acid sequence (SEQ ID NO:2) is given with differences in the rat sequence shown underneath. The seven putative transmembrane domains (TMD-I to VII) are underlined.

Figure 2 shows a comparison between human and rat mGluR4 protein sequences. The human amino acid sequence (SEQ ID NO:4) is given with differences in the rat sequence shown underneath. The seven putative transmembrane domains (TMD-I to VII) are underlined.

Figure 3 shows the effect of mGluR4 ligands on the accumulation

of forskolin-stimulated cyclic AMP. Dose response curves are given for L-amino-4-phosphonobutyrate (L-AP4) , glutamate, trans-1-aminocyclopentane-l, 3-dicarboxylate (t-ACPD) and quisqualate.

The following Examples illustrate the invention.

EXAMPLE It CLONING OF mGluR3 DNA Construction of a Human cDNA Library

Human brain material from the median frontal cortex and amygdala from one individual was used as the source of mRNA using the Fast Track mRNA Isolation Kit (Invitrogen) . The mRNA was used to construct a library containing a mixture of oligo-dT and random hexamer-primed cDNA, using the Timesaver cDNA Synthesis Kit (Pharmacia) and λZAP II arms (Stratagene) .

Source of Probes

Specific regions of various mGluRs were amplified by the polymerase chain reaction (PCR) using either human brain cDNA prepared as above or in Example 2 or human genomic DNA

(Stratagene) and the following consensus primers designed from the published sequences of rat mGluRl to 5

[ACAACGACACACCCGTGGTCAA (BN29, SEQ ID NO:5) TCCGGTCGGGAGCTCTGCTA (BN30, SEQ ID NO:6) GCTACTCTGCCCTGCTGACCAAGAC (BN31, SEQ ID NO:7) GCAATGCGGTTGGTCTTGGT (BN32, SEQ ID NO:8) AAGTTCATCGGCTTCACCATGTACAC (BN33, SEQ ID NO:9)

GATGCAGGTGGTGTACATGGTGAA (BN34, SEQ ID NO:10) and TCTCCGGTTGGAAGAGGATGATGT (BN35, SEQ ID NO:11)] .

A computer-generated alignment of rat sequences of mGluR cDNAs 1 to 5 revealed a number of regions of high homology. Seven regions were used to design oligonucleotide primers such that

(a) each primer contained a consensus sequence for all five

genes, (b) the highest homology of each primer was towards its 3' end and (c) the most 3' base was never a mismatch. Four primers (BN29, 30, 31 and 33) were from the coding strand and three primers (BN32, 34 and 35) were from the other strand. All seven primers came from the region of each gene which encodes the seven transmembrane domains.

PCR amplifications were performed with all nine combinations of the primers using either human genomic or human brain cDNA. Analysis of these PCR products showed that with several primer pairs, more than one mGluR subtype was amplified. Between the primer pairs all five human genes homologous to the five rat mGluR protein genes used to generate the primers were represented (Table 1) . There was generally a strong bias in favour of the human mGluR3 gene. Since mGluR3 was also over-represented in PCRs involving genomic DNA, it may reflect a bias in the primers rather than the relative abundance of mGluR3 mRNA in the brain samples. When the annealing temperature in the PCR conditions was lowered to 38°C the distribution of mGluRs among the clones analysed was more even.

Unless otherwise stated the PCR conditions were: 35 cycles of 96°C for 35 sec, 56°C for 2 min and 72°C for 2 min. The PCR products were separated by electrophoresis, the bands excised and the isolated fragments cloned into pT7Blue T-vector (Novagen) . The products were identified by sequencing of the cloned fragments.

Isolation of Clones

The cDNA library was screened using a mixed probe derived from inserts from cloned PCR products specific to each of human mGluRl to 5. Hybridization was at 65°C in 10% dextran sulphate, 1% SDS, 0.1% sodium pyrophosphate, 1M Tris-Cl (pH 7.5) and lOOμg/ml single stranded salmon testis DNA. The filters were washed in 1 x SSC and 1% SDS at 65oC followed by 0.1 x SSC at room temperature. Three phage clones were

isolated and stored in SM buffer (Sambrook et al, ibid) . The largest of these extended from the 3' end to within 500 bp of the initiating ATG codon.

Alternative Method of Isolating Clones

One of the plates, the filter of which did not yield any of the three clones, was re-examined since its autoradiograph had a very high background. The phage from the entire plate were taken up in SM buffer and stored at 4°C in the presence of chloroform. An aliquot was used as a target for PCR using one of the pairs of consensus primers (BN30 and BN34) . Electrophoresis showed that a product of the expected size (510bp) had been amplified. Phage taken from the other plates after the three mGluR3 positive phage clones had been removed did not generate this PCR product.

The PCR on the amplified phage was repeated but with samples of increasing dilution until the signal was lost. From this titration, it could be calculated that the PCR signal was derived from approximately 1 per 10 ⁵ phage, consistent with one positive phage on the original plate of approximately 80,000 independent clones.

The positive clone was recovered from the amplified mixture using PCR after each of several rounds of dilution and replication as described by Israel , D.I., Nucl. Acids Res., 21, 2627-2631, (1993) . This fourth clone was found to contain the complete coding sequence of human mGluR3. The sequence was determined for both strands of the entire insert and is shown in SEQ ID N0:1.

Seguencing of cDNA Clone

Plasmids containing the inserts were excised using helper phage R408 (Stratagene) . Some sequencing was performed on such plasmid clones using flanking or internal primers. Most was on

sub-clones generated by limited exonuclease III digestion using the Nested Deletion Kit (Pharmacia) .

Table 1. Distribution of human mGluRs amplified by different consensus primer pairs, The predicted size of each PCR product is given in brackets. a annealing at 50°C b annealing at 38°C

EXAMPLE 2; CLONING OF mGluR4 DNA Construction of a Human cDNA Library

A human cDNA library was constructed in the same way as Example 1, except that human brain material from the cerebellum was used as the source of mRNA.

Source of Probes

The probes generated in Example 1 were used.

Isolation of Clones

The cDNA library was screened using an mGluR4 probe derived from inserts from cloned PCR products specific to human mGluR4.

Hybridization was at 65°C in 10% dextran sulphate, 1% SDS, 0.1% sodium pyrophosphate, 1M Tris-Cl (pH 7.5) and 100 μg/ml single stranded salmon testis DNA. The filters were washed in 1 x SSC and 1% SDS at 65°C followed by 0.1 x SSC at room temperature.

Phage clones were stored in SM buffer (Sambrook et al, ibid) .

Several clones were isolated, all at the 3' end, of which the largest extended to within 900bp of the start of the coding sequence (clone 17) .

Alternative Method of Isolating Clones

New PCR primers (AP43 and AP44) (see herebelow) were designed from the published rat mGlu4 sequence. These were located in order to generate a product immediately downstream of the start of the coding sequence.

Another clone was recovered from the same cerebellum cDNA library using PCR using the new primers (AP43 and AP44 see herebelow) after each of several rounds of dilution and replication as described by Israel, D.I., Nucl. Acids Res., 21, 2627-2631, (1993) . This second clone (clone 31) was found to contain the 5' end of the coding sequence of human mGluR4, but did not overlap the 3' clone (clone 17) .

5' - CTCTGCCTACTCCTCAGCCTTTA - 3' AP43, SEQ ID NO:12 5' - GCACAAAGGTCAGTGACTGCTC - 3' AP44, SEQ ID NO:13

Another pair of primers (CA396 and 400) were designed using the 3' end of clone 31 and the rat sequence in the gap between

clones 31 and 17.

AGCTCGGTCTCCATCAT CA396, SEQ ID NO:14 TGATGTCATCCTCGTTGGC CA400, SEQ ID NO:15

These primers were used to screen the same cerebellum library, again using the method of Israel. This generated clone 32 which overlapped both clones 31 and 17. Clones 17 and 32 were completely sequenced on both strands. Part of clone 31 was also sequenced on both strands to generate the coding sequence and some of the 5' untranslated sequence.

Sequencing of cDNA Clone

Sequencing was carried out in the same way as Example 1.

EXAMPLE 3; EXPRESSION OF HUMAN mGluR3

The sequence GTACAG (SEQ ID NO:l, positions 236-241) upstream of both alternative initiating ATG codons was mutated to CTCGAG

(Xhol recognition sequence) by PCR. The sequence TGTGAA (SEQ

ID NO:l, positions 2894-2899) downstream of the termination codon was mutated to TCTAGA (Xbal recognition sequence) by PCR.

The primers AP47 and AP48 were designed for this purpose.

5' - AGAGCTCGAGAAACAGGATTCATGAAGATG - 3ΑP47 (SEQ ID NO:16) 5' - GCAATCTAGAATCACAGAGATGAGGTG - 3ΑP48 (SEQ ID NO:17)

The entire coding sequence of human mGluR3 was cloned between the Xhol and Xbal sites of the expression vector pSVL

(Pharmacia) in which transcription of mGluR3 is under the control of the SV40 late promoter. The resulting vector is pSVLHMGR3.

DHfr ^" CHO cells were grown at 37°C in an atmosphere containing 5% C0 ₂ in glutamate-free Dulbecco's modified Eagles medium supplemented with 5% dialysed foetal calf serum, 2mM glutamine,

ImM proline, lOOμM hypoxanthine and 16μM thymidine. Petri dishes (10cm) were seeded with 10 ⁶ cells and incubated for 24 hours. The cells were washed with serum-free medium and replaced by fresh serum-free medium containing lOμg pSVLHMGR3 and lμg pSV2 dhfr [Subramani, S. et al. Mol. Cell. Biol. Vol 1, 854 (1981)] in the presence of Transfectam (Promega) . After 5 hours the medium was replaced by complete medium. After 48 hours the cells were detached from the plates with trypsin and seeded in flasks in medium without hypoxanthine/thymidine. After 1-2 weeks surviving colonies which were dhfr ⁺ were pooled and clonal colonies were obtained by dilution cloning.

Several clones were selected and transferred to individual flasks. Each culture was assayed for mGluR3 receptor activity by measuring the inhibition of forskolin-stimulated cyclic .AMP in response to the agonist trans-1-aminocyclopentane-l, 3- dicarboxylate (t-ACPD) .

Forskolin-stimulated cyclic AMP assay: Approximately 2 x 10 ⁵ cells were seeded in each well of a 12 well microtitre plate. After incubation at 37°C for 24 hours the cells were exposed to 100μ 3-isobutyl-l-methylxanthine (phosphodiesterase inhibitor) at 37°C for 20 minutes followed by both the ligand and lOμM forskolin for 10 minutes. The medium was removed and ice-cold ethanol was added to the cells. After 2 hours at room temperature the supernatant was transferred to fresh tubes and the ethanol was removed by evaporation. The accumulation of cyclic AMP was measured in each tube using a scintillation proximity assay (Amersham) .

EXAMPLE 4; EXPRESSION OF HUMAN mGluR4

The complete mGluR4 coding sequence was constructed from clones 17, 31 and 32, utilising the restriction endonucleases EcoRI (SEQ ID NO:3, position 290-295) , Fspl (SEQ ID NO:3, position 1615-1620) and Ncol (SEQ ID N0:3, position 2915-2920) . A unique BamHI site was introduced downstream of both the TAG

stop codon and of the Ncol site by subcloning the EcoRI-FspI fragment from clone 32 and the Fspl-Ncol fragment from clone 17 into pSL1190 (Pharmacia) . The sequence TCTAGG in clone 31 (SEQ ID NO:3, positions 155-160) upstream of the initiating ATG codon was mutated to TCTAGA (Xbal recognition sequence) by PCR using primers CA587 and CA565.

5' - GGGTGTCTAGAGATTTCCGAG - 3' CA587 (SEQ ID NO:18) 5' - AGAGGCGAAGGATGTTG - 3' CA565 (SEQ ID NO:19)

The entire coding sequence was cloned as an Xbal-BamHI fragment into the expression vector pSVL (Pharmacia) in which transcription of mGluR4 is under the control of the SV40 late promoter. The resulting vector is pSVLHMGR4.

Dhrf ^" CHO cells were grown at 37°C in an atmosphere containing 5% C0 ₂ in glutamate-free Dulbecco's modified Eagles medium supplemented with 5% dialysed foetal calf serum, 2mM glutamine, ImM proline, lOOμM hypoxanthine and 16μM thymidine. Petri dishes (10cm) were seeded with 10 ⁶ cells and incubated for 24 hours. The cells were washed with serum-free medium and replaced by fresh serum-free medium containing lOμg pSVLHMGR4 plus pSV2 dhfr [Subramani S. et al Mol. Cell. Biol. Vol 1, 854

(1981)] in a range between lOng and lμg in the presence of Transfeetarn (Promega) . After 5 hours the medium was replaced by complete medium. After 48 hours the cells were detached from the plates with trypsin and seeded in 15cm petri dishes in medium without hypoxanthine/thymidine. After 1-2 weeks surviving colonies which were dhfr ⁺ were selected and transferred to individual flasks. Each selected culture was assayed for mGluR4 receptor activity by measuring the inhibition of forskolin-stimulated cyclic AMP in response to the agonist L-2-amino-4-phosphonobutyrate (L-AP4) . Cyclic AMP assays were performed as for mGluR3. Expressing colonies were then dilution cloned to obtain clonal cell lines.

EXAMPLE 5: INTERACTION BETWEEN HUMAN mGluR4 AND LIGANDS

The effect of several ligands on human mGluR4 was investigated by measuring the accumulation of forskolin-stimulated cyclic AMP. Dose response curves are given in Figure 3 for L-2-amino- 4-phosphonobutyrate (L-AP4), glutamate, trans-1- aminocyclopentane-1,3-dicarboxylate (t-ACPD) and quisqualate.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

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(ii) TITLE OF INVENTION: HUMAN GLUTAMATE RECEPTOR PROTEINS (iii) NUMBER OF SEQUENCES: 19

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

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(D) SOFTWARE: Patentln Release #1.0, Version #1.30 (EPO)

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3410 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION:259..2889

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

CTTTTGTGTC GGATGAGGAG GACCAACCAT GAGCCAGAGC CCGGGTGCAG GCTCACCGCC 60

GCCGCTGCCA CCGCGGTCAG CTCCAGTTCC TGCCAGGAGT TGTCGGTGCG AGGAATTTTG 120

TGACAGGCTC TGπAGTCTG TTCCTCCCTT ATTTGAAGGA CAGGCCAAAG ATCCAGTTTG 180

GAAATGAGAG AGGACTAGCA TGACACATTG GCTCCACCAT TGATATCTCC CAGAGGTACA 240

GAAACAGGAT TCATGAAG ATG πG ACA AGA CTG CAA Gπ Cπ ACC πA GCT 291

Met Leu Thr Arg Leu Gin Val Leu Thr Leu Ala 1 5 10 πG πr TCA AAG GGA ΉT πA CTC TCT πA GGG GAC CAT AAC πr CTA 339

Leu Phe Ser Lys Gly Phe Leu Leu Ser Leu Gly Asp His Asn Phe Leu 15 20 25

AGG AGA GAG Aπ AAA ATA GAA GGT GAC CTT Gπ πA GGG GGC CTG Tπ 387 Arg Arg Glu He Lys He Glu Gly Asp Leu Val Leu Gly Gly Leu Phe 30 35 40

CCT Aπ AAC GAA AAA GGC ACT GGA ACT GAA GAA TGT GGG CGA ATC AAT 435 Pro He Asn Glu Lys Gly Thr Gly Thr Glu Glu Cys Gly Arg He Asn 45 50 55

GAA GAC CGA GGG Aπ CAA CGC CTG GAA GCC ATG πG πT GCT Aπ GAT 483 Glu Asp Arg Gly He Gin Arg Leu Glu Ala Met Leu Phe Ala He Asp 60 65 70 75

GAA ATC AAC AAA GAT GAT TAC πG CTA CCA GGA GTG AAG πG GGT Gπ 531 Glu He Asn Lys Asp Asp Tyr Leu Leu Pro Gly Val Lys Leu Gly Val 80 85 90

CAC Aπ πG GAT ACA TGT TCA AGG GAT ACC TAT GCA πG GAG CAA TCA 579 His He Leu Asp Thr Cys Ser Arg Asp Thr Tyr Ala Leu Glu Gin Ser 95 100 105

CTG GAG TTT GTC AGG GCA TCT πG ACA AAA GTG GAT GAA GCT GAG TAT 627 Leu Glu Phe Val Arg Ala Ser Leu Thr Lys Val Asp Glu Ala Glu Tyr UO 115 120

ATG TGT CCT GAT GGA TCC TAT GCC Aπ CAA GAA AAC ATC CCA Cπ CTC 675 Met Cys Pro Asp Gly Ser Tyr Ala He Gin Glu Asn He Pro Leu Leu 125 130 135

Aπ GCA GGG GTC Aπ GGT GGC TCT TAT AGC AGT Gπ TCC ATA CAG GTG 723 He Ala Gly Val He Gly Gly Ser Tyr Ser Ser Val Ser He Gin Val 140 145 150 155

GCA AAC CTG CTG CGG CTC πc CAG ATC CCT CAG ATC AGC TAC GCA TCC 771 Ala Asn Leu Leu Arg Leu Phe Gin He Pro Gin He Ser Tyr Ala Ser 160 165 170

ACC AGC GCC AAA CTC AGT GAT AAG TCG CGC TAT GAT TAC TTT GCC AGG 819 Thr Ser Ala Lys Leu Ser Asp Lys Ser Arg Tyr Asp Tyr Phe Ala Arg 175 180 185

ACC GTG CCC CCC GAC πC TAC CAG GCC AAA GCC ATG GCT GAG ATC πG 867 Thr Val Pro Pro Asp Phe Tyr Gin Ala Lys Ala Met Ala Glu He Leu 190 195 200

CGC πc πC AAC TGG ACC TAC GTG TCC ACA GTA GCC TCC GAG GGT GAT 915 Arg Phe Phe Asn Trp Thr Tyr Val Ser Thr Val Ala Ser Glu Gly Asp 205 210 215

TAC GGG GAG ACA GGG ATC GAG GCC πC GAG CAG GAA GCC CGC CTG CGC 963 Tyr Gly Glu Thr Gly He Glu Ala Phe Glu Gin Glu Ala Arg Leu Arg 220 225 230 235

AAC ATC TGC ATC GCT ACG GCG GAG AAG GTG GGC CGC TCC AAC ATC CGC 1011 Asn He Cys He Ala Thr Ala Glu Lys Val Gly Arg Ser Asn He Arg 240 245 250

AAG TCC TAC GAC AGC GTG ATC CGA GAA CTG πG CAG AAG CCC AAC GCG 1059 Lys Ser Tyr Asp Ser Val He Arg Glu Leu Leu Gin Lys Pro Asn Ala 255 260 265

CGC GTC GTG GTC CTC πc ATG CGC AGC GAC GAC TCG CGG GAG CTC Aπ 1107 Arg Val Val Val Leu Phe Met Arg Ser Asp Asp Ser Arg Glu Leu He 270 275 280

GCA GCC GCC AGC CGC GCC AAT GCC TCC πc ACC TGG GTG GCC AGC GAC 1155 Ala Ala Ala Ser Arg Ala Asn Ala Ser Phe Thr Trp Val Ala Ser Asp 285 290 295

GGC TGG GGC GCG CAG GAG AGC ATC ATC AAG GGC AGC GAG CAT GTG GCC 1203 Gly Trp Gly Ala Gin Glu Ser He He Lys Gly Ser Glu His Val Ala 300 305 310 315

TAC GGC GCC ATC ACC CTG GAG CTG GCC TCC CAG CCT GTC CGC CAG πC 1251 Tyr Gly Ala He Thr Leu Glu Leu Ala Ser Gin Pro Val Arg Gin Phe 320 325 330

GAC CGC TAC πC CAG AGC CTC AAC CCC TAC AAC AAC CAC CGC AAC CCC 1299 Asp Arg Tyr Phe Gin Ser Leu Asn Pro Tyr Asn Asn His Arg Asn Pro 335 340 345

TGG πC CGG GAC πC TGG GAG CAA AAG TTT CAG TGC AGC CTC CAG AAC 1347 Trp Phe Arg Asp Phe Trp Glu Gin Lys Phe Gin Cys Ser Leu Gin Asn 350 355 360

AAA CGC AAC CAC AGG CGC GTC TGC GAC AAG CAC CTG GCC ATC GAC AGC 1395 Lys Arg Asn His Arg Arg Val Cys Asp Lys His Leu Ala He Asp Ser 365 370 375

AGC AAC TAC GAG CAA GAG TCC AAG ATC ATG TTT GTG GTG AAC GCG GTG 1443 Ser Asn Tyr Glu Gin Glu Ser Lys He Met Phe Val Val Asn Ala Val 380 385 390 395

TAT GCC ATG GCC CAC GCT πG CAC AAA ATG CAG CGC ACC CTC TGT CCC 1491 Tyr Ala Met Ala His Ala Leu His Lys Met Gin Arg Thr Leu Cys Pro 400 405 410

AAC ACT ACC AAG OT TGT GAT GCT ATG AAG ATC CTG GAT GGG AAG AAG 1539 Asn Thr Thr Lys Leu Cys Asp Ala Met Lys He Leu Asp Gly Lys Lys 415 420 425 πG TAC AAG GAT TAC πG CTG AAA ATC AAC πC ACG GCT CCA πC AAC 1587 Leu Tyr Lys Asp Tyr Leu Leu Lys He Asn Phe Thr Ala Pro Phe Asn 430 435 440

CCA AAT AAA GAT GCA GAT AGC ATA GTC AAG Tπ GAC ACT TTT GGA GAT 1635 Pro Asn Lys Asp Ala Asp Ser He Val Lys Phe Asp Thr Phe Gly Asp 445 450 455

GGA ATG GGG CGA TAC AAC GTG πC AAT πC CAA AAT GTA GGT GGA AAG 1683 Gly Met Gly Arg Tyr Asn Val Phe Asn Phe Gin Asn Val Gly Gly Lys 460 465 470 475

TAT TCC TAC πG AAA Gπ GGT CAC TGG GCA GAA ACC πA TCG CTA GAT 1731 Tyr Ser Tyr Leu Lys Val Gly His Trp Ala Glu Thr Leu Ser Leu Asp 480 485 490

GTC AAC TCT ATC CAC TGG TCC CGG AAC TCA GTC CCC ACT TCC CAG TGC 1779 Val Asn Ser He His Trp Ser Arg Asn Ser Val Pro Thr Ser Gin Cys 495 500 505

AGC GAC CCC TGT GCC CCC AAT GAA ATG AAG AAT ATG CAA CCA GGG GAT 1827 Ser Asp Pro Cys Ala Pro Asn Glu Met Lys Asn Met Gin Pro Gly Asp 510 515 520

GTC TGC TGC TGG Aπ TGC ATC CCC TGT GAA CCC TAC GAA TAC CTG GCT 1875 Val Cys Cys Trp He Cys He Pro Cys Glu Pro Tyr Glu Tyr Leu Ala 525 530 535

GAT GAG TTT ACC TGT ATG GAT TGT GGG TCT GGA CAG TGG CCC ACT GCA 1923 Asp Glu Phe Thr Cys Met Asp Cys Gly Ser Gly Gin Trp Pro Thr Ala 540 545 550 555

GAC CTA ACT GGA TGC TAT GAC Cπ CCT GAG GAC TAC ATC AGG TGG GAA 1971 Asp Leu Thr Gly Cys Tyr Asp Leu Pro Glu Asp Tyr He Arg Trp Glu 560 565 570

GAC GCC TGG GCC Aπ GGC CCA GTC ACC Aπ GCC TGT CTG GGT TTT ATG 2019 Asp Ala Trp Ala He Gly Pro Val Thr He Ala Cys Leu Gly Phe Met 575 580 585

TGT ACA TGC ATG Gπ GTA ACT Gπ πT ATC AAG CAC AAC AAC ACA CCC 2067 Cys Thr Cys Met Val Val Thr Val Phe He Lys His Asn Asn Thr Pro 590 595 600 πG GTC AAA GCA TCG GGC CGA GAA CTC TGC TAC ATC πA πG πT GGG 2115 Leu Val Lys Ala Ser Gly Arg Glu Leu Cys Tyr He Leu Leu Phe Gly 605 610 615

Gπ GGC CTG TCA TAC TGC ATG ACA πC πC πC Aπ GCC AAG CCA TCA 2163 Val Gly Leu Ser Tyr Cys Met Thr Phe Phe Phe He Ala Lys Pro Ser 620 625 630 635

CCA GTC ATC TGT GCA πG CGC CGA CTC GGG CTG GGG AGT TCC πC GCT 2211 Pro Val He Cys Ala Leu Arg Arg Leu Gly Leu Gly Ser Ser Phe Ala 640 645 650

ATC TGT TAC TCA GCC CTG CTG ACC AAG ACA AAC TGC Aπ GCC CGC ATC 2259 He Cys Tyr Ser Ala Leu Leu Thr Lys Thr Asn Cys He Ala Arg He 655 660 665

πC GAT GGG GTC AAG AAT GGC GCT CAG AGG CCA AAA πC ATC AGC CCC 2307 Phe Asp Gly Val Lys Asn Gly Ala Gin Arg Pro Lys Phe He Ser Pro 670 675 680

AGT TCT CAG Gπ πC ATC TGC CTG GGT CTG ATC CTG GTG CAA Aπ GTG 2355 Ser Ser Gin Val Phe He Cys Leu Gly Leu He Leu Val Gin He Val 685 690 695

ATG GTG TCT GTG TGG CTC ATC CTG GAG GCC CCA GGC ACC AGG AGG TAT 2403 Met Val Ser Val Trp Leu He Leu Glu Ala Pro Gly Thr Arg Arg Tyr 700 705 710 715

ACC Cπ GCA GAG AAG CGG GAA ACA GTC ATC CTA AAA TGC AAT GTC AAA 2451 Thr Leu Ala Glu Lys Arg Glu Thr Val He Leu Lys Cys Asn Val Lys 720 725 730

GAT TCC AGC ATG πG ATC TCT CTT ACC TAC GAT GTG ATC CTG GTG ATC 2499 Asp Ser Ser Met Leu He Ser Leu Thr Tyr Asp Val He Leu Val He 735 740 745 πA TGC ACT GTG TAC GCC πC AAA ACG CGG AAG TGC CCA GAA AAT πC 2547 Leu Cys Thr Val Tyr Ala Phe Lys Thr Arg Lys Cys Pro Glu Asn Phe 750 755 760

AAC GAA GCT AAG πC ATA GGT Tπ ACC ATG TAC ACC ACG TGC ATC ATC 2595 Asn Glu Ala Lys Phe He Gly Phe Thr Met Tyr Thr Thr Cys He He 765 770 775

TGG πG GCC πC CTC CCT ATA TTT TAT GTG ACA TCA AGT GAC TAC AGA 2643 Trp Leu Ala Phe Leu Pro He Phe Tyr Val Thr Ser Ser Asp Tyr Arg 780 785 790 795

GTG CAG ACG ACA ACC ATG TGC ATC TCT GTC AGC CTG AGT GGC TTT GTG 2691 Val Gin Thr Thr Thr Met Cys He Ser Val Ser Leu Ser Gly Phe Val 800 805 810

GTC πG GGC TGT πG πr GCA CCC AAG Gπ CAC ATC ATC CTG TTT CAA 2739

Val Leu Gly Cys Leu Phe Ala Pro Lys Val His He He Leu Phe Gin 815 820 825

CCC CAG AAG AAT Gπ GTC ACA CAC AGA CTG CAC CTC AAC AGG πC AGT 2787 Pro Gin Lys Asn Val Val Thr His Arg Leu His Leu Asn Arg Phe Ser 830 835 840

GTC AGT GGA ACT GGG ACC ACA TAC TCT CAG TCC TCT GCA AGC ACG TAT 2835 Val Ser Gly Thr Gly Thr Thr Tyr Ser Gin Ser Ser Ala Ser Thr Tyr 845 850 855

GTG CCA ACG GTG TGC AAT GGG CGG GAA GTC CTC GAC TCC ACC ACC TCA 2883 Val Pro Thr Val Cys Asn Gly Arg Glu Val Leu Asp Ser Thr Thr Ser 860 865 870 875

TCT CTG TGAπGTGAA πGCAGπCA GπCTTGTGT TTTTAGACTG πAGACAAAA 2939 Ser Leu

GTGCTCACGT GCAGCTCCAG AATATGGAAA CAGAGCAAAA GAACAACCCT AGTACCTTTT 2999

TTTAGAAACA GTACGATAAA πAππTGA GGACTGTATA TAGTGATGTG CTAGAACTπ 3059

CTAGGCTGAG TCTAGTGCCC CTAπAπAA CAAπCCCCC AGAACATGGA AATAACCAπ 3119

GπTACAGAG CTGAGCAπG GTGACAGGGT CTGACATGGT CAGTCTACTA AAAAACAAAA 3179

AAAAAAAACA AAAAAAAAAA AACAAAAGAA AAAAATAAAA ATACGGTGGC AATAπATGT 3239

AACC111111 CCTATGAAGT TTTTTGTAGG TCCTTGπGT AACTAAHTA GGATGAGTTT 3299

CTATGπGTA TAπAAAGπ ACAπATGTG TAACAGAπG AπTTCTCAG CACAAAATAA 3359

AAAGCATCTG TAπAATGTA AAGATACTGA GAATAAAACC πCAAGGπT T 3410

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 877 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE πPE: protein

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

Met Leu Thr Arg Leu Gin Val Leu Thr Leu Ala Leu Phe Ser Lys Gly 1 5 10 15

Phe Leu Leu Ser Leu Gly Asp His Asn Phe Leu Arg Arg Glu He Lys 20 25 30

He Glu Gly Asp Leu Val Leu Gly Gly Leu Phe Pro He Asn Glu Lys 35 40 45

Gly Thr Gly Thr Glu Glu Cys Gly Arg He Asn Glu Asp Arg Gly He 50 55 60

Gin Arg Leu Glu Ala Met Leu Phe Ala He Asp Glu He Asn Lys Asp 65 70 75 80

Asp Tyr Leu Leu Pro Gly Val Lys Leu Gly Val His He Leu Asp Thr 85 90 95

Cys Ser Arg Asp Thr Tyr Ala Leu Glu Gin Ser Leu Glu Phe Val Arg 100 105 UO

Ala Ser Leu Thr Lys Val Asp Glu Ala Glu Tyr Met Cys Pro Asp Gly 115 120 125

Ser Tyr Ala He Gin Glu Asn He Pro Leu Leu He Ala Gly Val He 130 135 140

Gly Gly Ser Tyr Ser Ser Val Ser He Gin Val Ala Asn Leu Leu Arg 145 150 155 160

Leu Phe Gin He Pro Gin He Ser Tyr Al a Ser Thr Ser Al a Lys Leu 165 170 175

Ser Asp Lys Ser Arg Tyr Asp Tyr Phe Al a Arg Thr Val Pro Pro Asp 180 185 190

Phe Tyr Gin Al a Lys Al a Met Al a Glu He Leu Arg Phe Phe Asn Trp 195 200 205

Thr Tyr Val Ser Thr Val Al a Ser Gl u Gly Asp Tyr Gly Gl u Thr Gly 210 215 220

He Glu Ala Phe Gl u Gin Glu Al a Arg Leu Arg Asn He Cys He Al a 225 230 235 240

Thr Ala Glu Lys Val Gly Arg Ser Asn He Arg Lys Ser Tyr Asp Ser 245 250 255

Val He Arg Glu Leu Leu Gin Lys Pro Asn Ala Arg Val Val Val Leu 260 265 270

Phe Met Arg Ser Asp Asp Ser Arg Glu Leu He Ala Ala Ala Ser Arg 275 280 285

Ala Asn Ala Ser Phe Thr Trp Val Al a Ser Asp Gly Trp Gly Ala Gin 290 295 300

Glu Ser He He Lys Gly Ser Glu His Val Ala Tyr Gly Ala He Thr 305 310 315 320

Leu Glu Leu Al a Ser Gin Pro Val Arg Gin Phe Asp Arg Tyr Phe Gin 325 330 335

Ser Leu Asn Pro Tyr Asn Asn His Arg Asn Pro Trp Phe Arg Asp Phe 340 345 350

Trp Gl u Gin Lys Phe Gin Cys Ser Leu Gin Asn Lys Arg Asn His Arg 355 360 365

Arg Val Cys Asp Lys His Leu Ala He Asp Ser Ser Asn Tyr Glu Gin 370 375 380

Glu Ser Lys He Met Phe Val Val Asn Al a Val Tyr Ala Met Ala His 385 390 395 400

Al a Leu His Lys Met Gin Arg Thr Leu Cys Pro Asn Thr Thr Lys Leu 405 410 415

Cys Asp Ala Met Lys He Leu Asp Gly Lys Lys Leu Tyr Lys Asp Tyr 420 425 430

Leu Leu Lys He Asn Phe Thr Al a Pro Phe Asn Pro Asn Lys Asp Al a 435 440 445

Asp Ser He Val Lys Phe Asp Thr Phe Gly Asp Gly Met Gly Arg Tyr 450 455 460

Asn Val Phe Asn Phe Gin Asn Val Gly Gly Lys Tyr Ser Tyr Leu Lys 465 470 475 480

Val Gly His Trp Al a Gl u Thr Leu Ser Leu Asp Val Asn Ser He His 485 490 495

Trp Ser Arg Asn Ser Val Pro Thr Ser Gin Cys Ser Asp Pro Cys Ala 500 505 510

Pro Asn Glu Met Lys Asn Met Gin Pro Gly Asp Val Cys Cys Trp He 515 520 525

Cys He Pro Cys Gl u Pro Tyr Glu Tyr Leu Al a Asp Gl u Phe Thr Cys 530 535 540

Met Asp Cys Gly Ser Gly Gin Trp Pro Thr Ala Asp Leu Thr Gly Cys 545 550 555 560

Tyr Asp Leu Pro Gl u Asp Tyr He Arg Trp Glu Asp Al a Trp Al a He 565 570 575

Gly Pro Val Thr He Ala Cys Leu Gly Phe Met Cys Thr Cys Met Val 580 585 590

Val Thr Val Phe He Lys His Asn Asn Thr Pro Leu Val Lys Al a Ser 595 600 605

Gly Arg Gl u Leu Cys Tyr He Leu Leu Phe Gly Val Gly Leu Ser Tyr 610 615 620

Cys Met Thr Phe Phe Phe He Al a Lys Pro Ser Pro Val He Cys Al a 625 630 635 640

Leu Arg Arg Leu Gly Leu Gly Ser Ser Phe Al a He Cys Tyr Ser Al a 645 650 655

Leu Leu Thr Lys Thr Asn Cys He Ala Arg He Phe Asp Gly Val Lys 660 665 670

Asn Gly Ala Gin Arg Pro Lys Phe He Ser Pro Ser Ser Gin Val Phe 675 680 685

He Cys Leu Gly Leu He Leu Val Gin He Val Met Val Ser Val Trp 690 695 700

Leu He Leu Glu Ala Pro Gly Thr Arg Arg Tyr Thr Leu Ala Glu Lys 705 710 715 720

Arg Gl u Thr Val He Leu Lys Cys Asn Val Lys Asp Ser Ser Met Leu 725 730 735

He Ser Leu Thr Tyr Asp Val He Leu Val He Leu Cys Thr Val Tyr 740 745 750

Al a Phe Lys Thr Arg Lys Cys Pro Glu Asn Phe Asn Glu Al a Lys Phe 755 760 765

He Gly Phe Thr Met Tyr Thr Thr Cys He He Trp Leu Ala Phe Leu 770 775 780

Pro He Phe Tyr Val Thr Ser Ser Asp Tyr Arg Val Gin Thr Thr Thr 785 790 795 800

Met Cys He Ser Val Ser Leu Ser Gly Phe Val Val Leu Gly Cys Leu 805 810 815

Phe Ala Pro Lys Val His He He Leu Phe Gin Pro Gin Lys Asn Val 820 825 830

Val Thr His Arg Leu His Leu Asn Arg Phe Ser Val Ser Gly Thr Gly 835 840 845

Thr Thr Tyr Ser Gin Ser Ser Ala Ser Thr Tyr Val Pro Thr Val Cys 850 855 860

Asn Gly Arg Glu Val Leu Asp Ser Thr Thr Ser Ser Leu 865 870 875

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3884 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION:171..2906

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

CCGAGTGACA AGGAGGTGGG AGAGGGTAGC AGCATGGGCT ACGCGGπGG CTGCCCTCAG 60

TCCCCCTGCT GCTGAAGCTG CCCTGCCCAT GCCCACCCAG GCCGTGGGGC CAGGGGCCTG 120

CCAGGGCTAG GAGTGGGCCT GCCGπCATG GGTCTCTAGG GATTTCCGAG ATG CCT 176

Met Pro

GGG AAG AGA GGC πG GGC TGG TGG TGG GCC CGG CTG CCC CTT TGC CTG 224

Gly Lys Arg Gly Leu Gly Trp Trp Trp Ala Arg Leu Pro Leu Cys Leu 880 885 890 895

CTC CTC AGC Cπ TAC GGC CCC TGG ATG CCT TCC TCC CTG GGA AAG CCC 272

Leu Leu Ser Leu Tyr Gly Pro Trp Met Pro Ser Ser Leu Gly Lys Pro 900 905 910

AAA GGC CAC CCT CAC ATG AAT TCC ATC CGC ATA GAT GGG GAC ATC ACA 320 Lys Gly His Pro His Met Asn Ser He Arg He Asp Gly Asp He Thr

915 920 925

CTG GGA GGC CTG πC CCG GTG CAT GGC CGG GGC TCA GAG GGC AAG CCC 368 Leu Gly Gly Leu Phe Pro Val His Gly Arg Gly Ser Glu Gly Lys Pro 930 935 940

TGT GGA GAA Cπ AAG AAG GAA AAG GGC ATC CAC CGG CTG GAG GCC ATG 416 Cys Gly Glu Leu Lys Lys Glu Lys Gly He His Arg Leu Glu Ala Met 945 950 955

CTG πC GCC CTG GAT CGC ATC AAC AAC GAC CCG GAC CTG CTG CCT AAC 464 Leu Phe Ala Leu Asp Arg He Asn Asn Asp Pro Asp Leu Leu Pro Asn 960 965 970 975

ATC ACG CTG GGC GCC CGC Aπ CTG GAC ACC TGC TCC AGG GAC ACC CAT 512 He Thr Leu Gly Ala Arg He Leu Asp Thr Cys Ser Arg Asp Thr His 980 985 990

GCC CTC GAG CAG TCG CTG ACC TTT GTG CAG GCG CTC ATC GAG AAG GAT 560 Ala Leu Glu Gin Ser Leu Thr Phe Val Gin Ala Leu He Glu Lys Asp 995 1000 1005

GGC ACA GAG GTC CGC TGT GGC AGT GGC GGC CCA CCC ATC ATC ACC AAG 608 Gly Thr Glu Val Arg Cys Gly Ser Gly Gly Pro Pro He He Thr Lys 1010 1015 1020

CCT GAA CGT GTG GTG GGT GTC ATC GGT GCT TCA GGG AGC TCG GTC TCC 656 Pro Glu Arg Val Val Gly Val He Gly Ala Ser Gly Ser Ser Val Ser 1025 1030 1035

ATC ATG GTG GCC AAC ATC CTT CGC CTC πC AAG ATA CCC CAG ATC AGC 704 He Met Val Ala Asn He Leu Arg Leu Phe Lys He Pro Gin He Ser 1040 1045 1050 1055

TAC GCC TCC ACA GCG CCA GAC CTG AGT GAC AAC AGC CGC TAC GAC πC 752 Tyr Ala Ser Thr Ala Pro Asp Leu Ser Asp Asn Ser Arg Tyr Asp Phe 1060 1065 1070 πC TCC CGC GTG GTG CCC TCG GAC ACG TAC CAG GCC CAG GCC ATG GTG 800 Phe Ser Arg Val Val Pro Ser Asp Thr Tyr Gin Ala Gin Ala Met Val 1075 1080 1085

GAC ATC GTC CGT GCC CTC AAG TGG AAC TAT GTG TCC ACA GTG GCC TCG 848 Asp He Val Arg Ala Leu Lys Trp Asn Tyr Val Ser Thr Val Ala Ser 1090 1095 1100

GAG GGC AGC TAT GGT GAG AGC GGT GTG GAG GCC πC ATC CAG AAG TCC 896 Glu Gly Ser Tyr Gly Glu Ser Gly Val Glu Ala Phe He Gin Lys Ser 1105 1110 1115

CGT GAG GAC GGG GGC GTG TGC ATC GCC CAG TCG GTG AAG ATA CCA CGG 944 Arg Glu Asp Gly Gly Val Cys He Ala Gin Ser Val Lys He Pro Arg 1120 1125 1130 1135

GAG CCC AAG GCA GGC GAG πc GAC AAG ATC ATC CGC CGC CTC CTG GAG 992 Glu Pro Lys Ala Gly Glu Phe Asp Lys He He Arg Arg Leu Leu Glu

1140 1145 1150

ACT TCG AAC GCC AGG GCA GTC ATC ATC TTT GCC AAC GAG GAT GAC ATC 1040 Thr Ser Asn Ala Arg Ala Val He He Phe Ala Asn Glu Asp Asp He 1155 1160 1165

AGG CGT GTG CTG GAG GCA GCA CGA AGG GCC AAC CAG ACA GGC CAT πC 1088 Arg Arg Val Leu Glu Ala Ala Arg Arg Ala Asn Gin Thr Gly His Phe 1170 1175 1180 πC TGG ATG GGC TCT GAC AGC TGG GGC TCC AAG Aπ GCA CCT GTG CTG 1136 Phe Trp Met Gly Ser Asp Ser Trp Gly Ser Lys He Ala Pro Val Leu 1185 1190 1195

CAC CTG GAG GAG GTG GCT GAG GGT GCT GTC ACG ATC CTC CCC AAG AGG 1184 His Leu Glu Glu Val Ala Glu Gly Ala Val Thr He Leu Pro Lys Arg 1200 1205 1210 1215

ATG TCC GTA CGA GGC πC GAC CGC TAC πC TCC AGC CGC ACG CTG GAC 1232 Met Ser Val Arg Gly Phe Asp Arg Tyr Phe Ser Ser Arg Thr Leu Asp 1220 1225 1230

AAC AAC CGG CGC AAC ATC TGG TTT GCC GAG πC TGG GAG GAC AAC πC 1280 Asn Asn Arg Arg Asn He Trp Phe Ala Glu Phe Trp Glu Asp Asn Phe 1235 1240 1245

CAC TGC AAG CTG AGC CGC CAC GCC CTC AAG AAG GGC AGC CAC GTC AAG 1328 His Cys Lys Leu Ser Arg His Ala Leu Lys Lys Gly Ser His Val Lys 1250 1255 1260

AAG TGC ACC AAC CGT GAG CGA Aπ GGG CAG GAT TCA GCT TAT GAG CAG 1376 Lys Cys Thr Asn Arg Glu Arg He Gly Gin Asp Ser Ala Tyr Glu Gin 1265 1270 1275

GAG GGG AAG GTG CAG TTT GTG ATC GAT GCC GTG TAC GCC ATG GGC CAC 1424 Glu Gly Lys Val Gin Phe Val He Asp Ala Val Tyr Ala Met Gly His 1280 1285 1290 1295

GCG CTG CAC GCC ATG CAC CGT GAC CTG TGT CCC GGC CGC GTG GGG CTC 1472 Ala Leu His Ala Met His Arg Asp Leu Cys Pro Gly Arg Val Gly Leu 1300 1305 1310

TGC CCG CGC ATG GAC CCT GTA GAT GGC ACC CAG CTG CTT AAG TAC ATC 1520 Cys Pro Arg Met Asp Pro Val Asp Gly Thr Gin Leu Leu Lys Tyr He 1315 1320 1325

CGA AAC GTC AAC πC TCA GGC ATC GCA GGG AAC CCT GTG ACC πC AAT 1568 Arg Asn Val Asn Phe Ser Gly He Ala Gly Asn Pro Val Thr Phe Asn 1330 1335 1340

GAG AAT GGA GAT GCG CCT GGG CGC TAT GAC ATC TAC CAA TAC CAG CTG 1616 Glu Asn Gly Asp Ala Pro Gly Arg Tyr Asp He Tyr Gin Tyr Gin Leu 1345 1350 1355

CGC AAC GAT TCT GCC GAG TAC AAG GTC Aπ GGC TCC TGG ACT GAC CAC 1664 Arg Asn Asp Ser Ala Glu Tyr Lys Val He Gly Ser Trp Thr Asp His

1360 1365 1370 1375

CTG CAC Cπ AGA ATA GAG CGG ATG CAC TGG CCG GGG AGC GGG CAG CAG 1712 Leu His Leu Arg He Glu Arg Met His Trp Pro Gly Ser Gly Gin Gin 1380 1385 1390

CTG CCC CGC TCC ATC TGC AGC CTG CCC TGC CAA CCG GGT GAG CGG AAG 1760 Leu Pro Arg Ser He Cys Ser Leu Pro Cys Gin Pro Gly Glu Arg Lys 1395 1400 1405

AAG ACA GTG AAG GGC ATG CCT TGC TGC TGG CAC TGC GAG CCT TGC ACA 1808 Lys Thr Val Lys Gly Met Pro Cys Cys Trp His Cys Glu Pro Cys Thr 1410 1415 1420

GGG TAC CAG TAC CAG GTG GAC CGC TAC ACC TGT AAG ACG TGT CCC TAT 1856 Gly Tyr Gin Tyr Gin Val Asp Arg Tyr Thr Cys Lys Thr Cys Pro Tyr 1425 1430 1435

GAC ATG CGG CCC ACA GAG AAC CGC ACG GGC TGC CGG CCC ATC CCC ATC 1904 Asp Met Arg Pro Thr Glu Asn Arg Thr Gly Cys Arg Pro He Pro He 1440 1445 1450 1455

ATC AAG Cπ GAG TGG GGC TCG CCC TGG GCC GTG CTG CCC CTC πC CTG 1952 He Lys Leu Glu Trp Gly Ser Pro Trp Ala Val Leu Pro Leu Phe Leu 1460 1465 1470

GCC GTG GTG GGC ATC GCT GCC ACG πG πC GTG GTG ATC ACC TTT GTG 2000 Ala Val Val Gly He Ala Ala Thr Leu Phe Val Val He Thr Phe Val 1475 1480 1485

CGC TAC AAC GAC ACG CCC ATC GTC AAG GCC TCG GGC CGT GAA CTG AGC 2048 Arg Tyr Asn Asp Thr Pro He Val Lys Ala Ser Gly Arg Glu Leu Ser 1490 1495 1500

TAC GTG CTG CTG GCA GGC ATC πC CTG TGC TAT GCC ACC ACC πC CTC 2096 Tyr Val Leu Leu Ala Gly He Phe Leu Cys Tyr Ala Thr Thr Phe Leu 1505 1510 1515

ATG ATC GCT GAG CCC GAC CTT GGC ACC TGC TCG CTG CGC CGA ATC πc 2144 Met He Ala Glu Pro Asp Leu Gly Thr Cys Ser Leu Arg Arg He Phe 1520 1525 1530 1535

CTG GGA CTA GGG ATG AGC ATC AGC TAT GCA GCC CTG CTC ACC AAG ACC 2192 Leu Gly Leu Gly Met Ser He Ser Tyr Ala Ala Leu Leu Thr Lys Thr 1540 1545 1550

AAC CGC ATC TAC CGC ATC πc GAG CAG GGC AAG CGC TCG GTC AGT GCC 2240 Asn Arg He Tyr Arg He Phe Glu Gin Gly Lys Arg Ser Val Ser Ala 1555 1560 1565

CCA CGC πC ATC AGC CCC GCC TCA CAG CTG GCC ATC ACC πC AGC CTC 2288 Pro Arg Phe He Ser Pro Ala Ser Gin Leu Ala He Thr Phe Ser Leu 1570 1575 1580

ATC TCG CTG CAG CTG CTG GGC ATC TGT GTG TGG TTT GTG GTG GAC CCC 2336 He Ser Leu Gin Leu Leu Gly He Cys Val Trp Phe Val Val Asp Pro

1585 1590 1595

TCC CAC TCG GTG GTG GAC πC CAG GAC CAG CGG ACA CTC GAC CCC CGC 2384 Ser His Ser Val Val Asp Phe Gin Asp Gin Arg Thr Leu Asp Pro Arg 1600 1605 1610 1615 πC GCC AGG GGT GTG CTC AAG TGT GAC ATC TCG GAC CTG TCG CTC ATC 2432 Phe Ala Arg Gly Val Leu Lys Cys Asp He Ser Asp Leu Ser Leu He 1620 1625 1630

TGC CTG CTG GGC TAC AGC ATG CTG CTC ATG GTC ACG TGC ACC GTG TAT 2480 Cys Leu Leu Gly Tyr Ser Met Leu Leu Met Val Thr Cys Thr Val Tyr 1635 1640 1645

GCC ATC AAG ACA CGC GGC GTG CCC GAG ACC πC AAT GAG GCC AAG CCC 2528 Ala He Lys Thr Arg Gly Val Pro Glu Thr Phe Asn Glu Ala Lys Pro 1650 1655 1660

Aπ GGC πC ACC ATG TAC ACC ACT TGC ATC GTC TGG CTG GCC πC ATC 2576 He Gly Phe Thr Met Tyr Thr Thr Cys He Val Trp Leu Ala Phe He 1665 1670 1675

CCC ATC πC πT GGC ACC TCG CAG TCG GCC GAC AAG CTG TAC ATC CAG 2624 Pro He Phe Phe Gly Thr Ser Gin Ser Ala Asp Lys Leu Tyr He Gin 1680 1685 1690 1695

ACG ACG ACG CTG ACG GTC TCG GTG AGT CTG AGC GCC TCG GTG TCC CTG 2672 Thr Thr Thr Leu Thr Val Ser Val Ser Leu Ser Ala Ser Val Ser Leu 1700 1705 1710

GGA ATG CTC TAC ATG CCC AAA GTC TAC ATC ATC CTC πC CAC CCG GAG 2720 Gly Met Leu Tyr Met Pro Lys Val Tyr He He Leu Phe His Pro Glu 1715 1720 1725

CAG AAC GTG CCC AAG CGC AAG CGC AGC CTC AAA GCC GTC Gπ ACG GCG 2768 Gin Asn Val Pro Lys Arg Lys Arg Ser Leu Lys Ala Val Val Thr Ala 1730 1735 1740

GCC ACC ATG TCC AAC AAG πC ACG CAG AAG GGC AAC πC CGG CCC AAC 2816 Ala Thr Met Ser Asn Lys Phe Thr Gin Lys Gly Asn Phe Arg Pro Asn 1745 1750 1755

GGA GAG GCC AAG TCT GAG CTC TGC GAG AAC CTT GAG GCC CCA GCG CTG 2864 Gly Glu Ala Lys Ser Glu Leu Cys Glu Asn Leu Glu Ala Pro Ala Leu 1760 1765 1770 1775

GCC ACC AAA CAG ACT TAC GTC ACT TAC ACC AAC CAT GCA ATC 2906

Ala Thr Lys Gin Thr Tyr Val Thr Tyr Thr Asn His Ala He 1780 1785

TAGCGAGTCC ATGGAGCTGA GCAGCAGGAG GAGGAGCCGT GACCCTGTGG AAGGTGCGTC 2966

GGGCCAGGGC CACACCCAAG GGCCCAGCTG TCTTGCCTGC CCGTGGGCAC CCACGGACGT 3026

GGCTTGGTGC TGAGGATAGC AGAGCCCCCA GCCATCACTG CTGGCAGCCT GGGCAAACCG 3086

GGTGAGCAAC AGGAGGACGA GGGGCCGGGG CGGTGCCAGG CTACCACAAG AACCTGCGTC 3146 πGGACCAπ GCCCCTCCCG GCCCCAAACC ACAGGGGCTC AGGTCGTGTG GGCCCCAGTG 3206

CTAGATCTCT CCCTCCCπC GTCTCTGTCT GTGCTGπGG CGACCCCTCT GTCTGTCTCC 3266

AGcccTGTCT πcTGπcTc πATCTcπr GπrcAccπ πcccTCTCT GGCGTCCCCG 3326

GCTGCπGTA CTCπGGCCT TπCTGTGTC TCCTTTCTGG CTCπGCCTC CGCCTCTCTC 3386

TCTCATCCTC TTTGTCCTCA GCTCCTCCTG CTTTCπGGG TCCCACCAGT GTCACTITTC 3446

TGCCGlTπC πTCCTGπC TCCTCTGCπ CAπCTCGTC CAGCCAπGC TCCCCTCTCC 3506

CTGCCACCCT TCCCCAGπC ACCAAACCπ ACATGπGCA AAAGAGAAAA AAGGAAAAAA 3566

AATCAAAACA CAAAAAAGCC AAAACGAAAA CAAATCTCGA GTGTGπGCC AAGTGCTGCG 3626

TCCTCCTGGT GGCCTCTGTG TGTGTCCCTG TGGCCCGCAG CCTGCCCGCC TGCCCCGCCC 3686

ATCTGCCGTG TGTCπGCCC GCCTGCCCCG CCCGTCTGCC GTCTGTCπG CCCGCCTGCC 3746

CGCCTGCCCC TCCTGCCGAC CACACGGAGT TCAGTGCCTG GGTGTTTGGT GATGGπAπ 3806

GACGACAATG TGTAGCGCAT GAπGππT ATACCAAGAA CATTTCTAAT AAAAATAAAC 3866

ACATGGTHT GCAAAAAA 3884

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 912 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

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

Met Pro Gly Lys Arg Gly Leu Gly Trp Trp Trp Ala Arg Leu Pro Leu 1 5 10 15

Cys Leu Leu Leu Ser Leu Tyr Gly Pro Trp Met Pro Ser Ser Leu Gly 20 25 30

Lys Pro Lys Gly His Pro His Met Asn Ser He Arg He Asp Gly Asp 35 40 45

He Thr Leu Gly Gly Leu Phe Pro Val His Gly Arg Gly Ser Glu Gly 50 55 60

Lys Pro Cys Gly Glu Leu Lys Lys Glu Lys Gly He His Arg Leu Glu 65 70 75 80

Ala Met Leu Phe Ala Leu Asp Arg He Asn Asn Asp Pro Asp Leu Leu 85 90 95

Pro Asn He Thr Leu Gly Ala Arg He Leu Asp Thr Cys Ser Arg Asp 100 105 UO

Thr His Ala Leu Gl u Gin Ser Leu Thr Phe Val Gin Al a Leu He Glu 115 120 125

Lys Asp Gly Thr Glu Val Arg Cys Gly Ser Gly Gly Pro Pro He He 130 135 140

Thr Lys Pro Gl u Arg Val Val Gly Val He Gly Al a Ser Gly Ser Ser 145 150 155 160

Val Ser He Met Val Ala Asn He Leu Arg Leu Phe Lys He Pro Gin 165 170 175

He Ser Tyr Ala Ser Thr Ala Pro Asp Leu Ser Asp Asn Ser Arg Tyr 180 185 190

Asp Phe Phe Ser Arg Val Val Pro Ser Asp Thr Tyr Gin Al a Gin Ala 195 200 205

Met Val Asp He Val Arg Al a Leu Lys Trp Asn Tyr Val Ser Thr Val 210 215 220

Al a Ser Glu Gly Ser Tyr Gly Gl u Ser Gly Val Glu Ala Phe He Gin 225 230 235 240

Lys Ser Arg Gl u Asp Gly Gly Val Cys He Ala Gin Ser Val Lys He 245 250 255

Pro Arg Glu Pro Lys Al a Gly Glu Phe Asp Lys He He Arg Arg Leu 260 265 270

Leu Glu Thr Ser Asn Ala Arg Al a Val He He Phe Al a Asn Glu Asp 275 280 285

Asp He Arg Arg Val Leu Glu Al a Al a Arg Arg Al a Asn Gin Thr Gly 290 295 300

His Phe Phe Trp Met Gly Ser Asp Ser Trp Gly Ser Lys He Ala Pro 305 310 315 320

Val Leu His Leu Glu Glu Val Ala Glu Gly Ala Val Thr He Leu Pro 325 330 335

Lys Arg Met Ser Val Arg Gly Phe Asp Arg Tyr Phe Ser Ser Arg Thr 340 345 350

Leu Asp Asn Asn Arg Arg Asn He Trp Phe Al a Glu Phe Trp Glu Asp 355 360 365

Asn Phe His Cys Lys Leu Ser Arg His Ala Leu Lys Lys Gly Ser His 370 375 380

Val Lys Lys Cys Thr Asn Arg Gl u Arg He Gly Gin Asp Ser Al a Tyr 385 390 395 400

Gl u Gin Glu Gly Lys Val Gi n Phe Val He Asp Al a Val Tyr Al a Met 405 410 415

Gly His Al a Leu His Al a Met His Arg Asp Leu Cys Pro Gly Arg Val 420 425 430

Gly Leu Cys Pro Arg Met Asp Pro Val Asp Gly Thr Gin Leu Leu Lys 435 440 445

Tyr He Arg Asn Val Asn Phe Ser Gly He Ala Gly Asn Pro Val Thr 450 455 460

Phe Asn Glu Asn Gly Asp Ala Pro Gly Arg Tyr Asp He Tyr Gin Tyr 465 470 475 480

Gi n Leu Arg Asn Asp Ser Al a Glu Tyr Lys Val He Gly Ser Trp Thr 485 490 495

Asp His Leu His Leu Arg He Glu Arg Met His Trp Pro Gly Ser Gly 500 505 510

Gin Gin Leu Pro Arg Ser He Cys Ser Leu Pro Cys Gin Pro Gly Glu 515 520 525

Arg Lys Lys Thr Val Lys Gly Met Pro Cys Cys Trp His Cys Gl u Pro 530 535 540

Cys Thr Gly Tyr Gi n Tyr Gin Val Asp Arg Tyr Thr Cys Lys Thr Cys 545 550 555 560

Pro Tyr Asp Met Arg Pro Thr Glu Asn Arg Thr Gly Cys Arg Pro He 565 570 575

Pro He He Lys Leu Glu Trp Gly Ser Pro Trp Al a Val Leu Pro Leu 580 585 590

Phe Leu Al a Val Val Gly He Al a Ala Thr Leu Phe Val Val He Thr 595 600 605

Phe Val Arg Tyr Asn Asp Thr Pro He Val Lys Ala Ser Gly Arg Glu 610 615 620

Leu Ser Tyr Val Leu Leu Ala Gly He Phe Leu Cys Tyr Al a Thr Thr 625 630 635 640

Phe Leu Met He Ala Gl u Pro Asp Leu Gly Thr Cys Ser Leu Arg Arg 645 650 655

He Phe Leu Gly Leu Gly Met Ser He Ser Tyr Al a Al a Leu Leu Thr 660 665 670

Lys Thr Asn Arg He Tyr Arg He Phe Glu Gin Gly Lys Arg Ser Val 675 680 685

Ser Al a Pro Arg Phe He Ser Pro Ala Ser Gin Leu Al a He Thr Phe 690 695 700

Ser Leu He Ser Leu Gin Leu Leu Gly He Cys Val Trp Phe Val Val 705 710 715 720

Asp Pro Ser His Ser Val Val Asp Phe Gin Asp Gi n Arg Thr Leu Asp 725 730 735

Pro Arg Phe Al a Arg Gly Val Leu Lys Cys Asp He Ser Asp Leu Ser 740 745 750

Leu He Cys Leu Leu Gly Tyr Ser Met Leu Leu Met Val Thr Cys Thr 755 760 765

Val Tyr Al a He Lys Thr Arg Gly Val Pro Glu Thr Phe Asn Glu Ala 770 775 780

Lys Pro He Gly Phe Thr Met Tyr Thr Thr Cys He Val Trp Leu Ala 785 790 795 800

Phe He Pro He Phe Phe Gly Thr Ser Gin Ser Al a Asp Lys Leu Tyr 805 810 815

He Gin Thr Thr Thr Leu Thr Val Ser Val Ser Leu Ser Al a Ser Val 820 825 830

Ser Leu Gly Met Leu Tyr Met Pro Lys Val Tyr He He Leu Phe His 835 840 845

Pro Gl u Gin Asn Val Pro Lys Arg Lys Arg Ser Leu Lys Al a Val Val 850 855 860

Thr Al a Ala Thr Met Ser Asn Lys Phe Thr Gin Lys Gly Asn Phe Arg 865 870 875 880

Pro Asn Gly Gl u Al a Lys Ser Glu Leu Cys Gl u Asn Leu Glu Al a Pro 885 890 895

Al a Leu Ala Thr Lys Gin Thr Tyr Val Thr Tyr Thr Asn His Al a He 900 905 910

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: ACAACGACAC ACCCGTGGTC AA 22

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: TCCGGTCGGG AGCTCTGCTA 20

(2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: GCTACTCTGC CCTGCTGACC AAGAC 25

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: GCAATGCGGT TGGTCπGGT 20

(2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: AAGπCATCG GCπCACCAT GTACAC 26

(2) INFORMATION FOR SEQ ID NO: 10:

(1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: GATGCAGGTG GTGTACATGG TGAA 24

(2) INFORMATION FOR SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: TCTCCGGπG GAAGAGGATG ATGT 24

(2) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: CTCTGCCTAC TCCTCAGCCT πA 23

(2) INFORMATION FOR SEQ ID NO: 13:

(1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: GCACAAAGGT CAGTGACTGC TC 22

(2) INFORMATION FOR SEQ ID NO: 14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: AGCTCGGTCT CCATCAT 17

(2) INFORMATION FOR SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: TGATGTCATC CTCGπGGC 19

(2) INFORMATION FOR SEQ ID NO: 16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: AGAGCTCGAG AAACAGGATT CATGAAGATG 30

(2) INFORMATION FOR SEQ ID NO: 17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: GCAATCTAGA ATCACAGAGA TGAGGTG 27

(2) INFORMATION FOR SEQ ID NO: 18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: GGGTGTCTAG AGATTTCCGA G 21

(2) INFORMATION FOR SEQ ID NO: 19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: AGAGGCGAAG GATGπG 17