GB2377940A

GB2377940A - Identification and use of molecules implicated in pain

Info

Publication number: GB2377940A
Application number: GB0118354A
Authority: GB
Inventors: Kevin Lee; Alistair Kerr Dixon; Robert Alan Brooksbank; Robert Denham Pinnock
Original assignee: Warner Lambert Co LLC
Current assignee: Warner Lambert Co LLC
Priority date: 2001-07-27
Filing date: 2001-07-27
Publication date: 2003-01-29
Also published as: GB0118354D0

Abstract

The use in the screening of compounds that are effective in the treatment of pain, or in the diagnosis of pain, of: <SL> <LI>(a) an isolated gene sequence that is differentially expressible in neuronal tissue in response to a nociceptive stimulus; <LI>(b) an isolated gene sequence having at least 80% sequence identity with the nucleic acids of tables I-X; <LI>(c) an isolated nucleic acid comprising a sequence that is hybridizable to the above gene sequence under stringent hybridisation conditions; <LI>(d) a recombinant vector comprising the above gene sequence; <LI>(e) a host cell containing the vector; <LI>(f) an animal having in its genome an introduced gene sequence that is differentially expressed in neuronal tissue in response to a nociceptive stimulus; <LI>(g) an isolated polypeptide containing an amino acid sequence at least 90% identical to a sequence encoded by the above gene sequences, or a variant thereof <LI>(h) an isolated antibody that binds specifically to the isolated polypeptide. </SL>

Description

IDENTIFICATION AND USE OF MOLECULES IMPLICATED IN PAIN FIELD OF THE INVENTION The present invention relates to nucleic acids, their expression products and pathways involved in pain, and their use in screening for molecules that can alleviate pain. The invention further relates to methods for the assay and diagnosis of pain in patients.

BACKGROUND TO THE INVENTION Pain is currently classified into four general types. Post-operative acute pain can be successfully treated with existing pain medications of e. g. the opioid and nonsteroidal anti-inflammatory (NSAID) types, and is usually short-term and selflimiting. A second type of pain, present e. g. in cancer and arthritis, is also responsive to medication initially with a NSAID and in its later stages with opioids. Neuropathic pain arises from damage to the central or peripheral nerve systems, and is more effectively treated with antidepressants or anticonvulsants. A fourth type of pain called central sensitization results from changes in the central nervous system as a result of chronic pain, these changes often being irreversible and difficult to treat.

Nerve pain from shingles or diabetes falls into this and the neuropathic category.

Changes occur where pain is at first poorly controlled and gradually progress to the point where a person is sensitive to stimuli which would not normally cause pain, for example a light touch. People with pain of this kind often describe a widening of the pain area to include areas which had originally not been injured or which were thought not to be involved in pain. This classification is, however based on clinical symptoms rather than on the underlying pain mechanisms.

Opiates such as morphine belong to a traditional class of pain-relieving compounds that are now recognized as binding to opiate receptors. Naturally

occurring polypeptides have also been found to have opiate-like effects on the central nervous system, and these include p-endorphin, met-enkephalin and leu-enkephalin.

Salicin was isolated at the beginning of the 19th century, and from that discovery a number of NSAIDs such as aspirin, paracetamol, ibuprofen, flurbiprofen and naproxen were developed. NSAIDs are by far the most widely used painrelieving compounds, but can exhibit side effects, in particular irritation of the GI tract that can lead to the formation of ulcers, gastrointestinal bleeding and anemia.

Interest in the neurobiology of pain is developing: see a colloquium sponsored by the US National Academy of Sciences in December 1998 concerning the neurobiology of pain and reviewed in The Scientist 13[1], 12,1999. Many pain mechanism were discussed including the role of the capsaicin receptors in pain, (M. J.

Caterina et al., Nature, 389,816-824, 1997). Large dosages of capsaicin were reported to disable that receptor, (W. R. Robbins et al., Anesthesia and Analgesia, 86, 579-583,1998). Additionally, a tetrodotoxin-resistant sodium channel found in small diameter pain-sensing neurons (PN3) was discussed (A. N. Akopian et al., Nature, 379,257-262, 1986) and L. Sangameswaran et al., Journal of Biological Chemistry, 271,953-956, 1996). Its involvement in transmission and sensitisation to pain signals has been reported, (S. D. Novakovic et al., Journal of Neuroscience, 18,2174-2187, 1998). A further TTX-resistant sodium channel has been reported (S. Tate et al., Nature Neuroscience, 1,653-655 1998).

Second messenger systems have also been shown to be important since knockout-mice lacking protein kinase C (PKC) y were reported to respond to acute pain e. g. from a hot surface, but not to respond to neuropathic pain when their spinal nerves are injured (Malmberg et al., Science, 278,279-283 (1997).

Present methods for identifying novel compounds that relieve pain of one or more of the types indicated above suffer from the defect that they are dependent either on the relatively limited number of receptors known to be involved in pain or

on the empirical discovery of new receptors which is an uncertain process. In relation to known receptors, for example the opioid receptor, research directed to improved compounds offers the possibility of discovering compounds that have a better therapeutic ratio and fewer side effects. It does not lead naturally to compounds for different pain receptors that have new modes of action and new and qualitatively different benefits. Even when recent discoveries of additional receptors are taken into account, known receptors revolve around tens of gene products. However, there are between 30,000 and 40,000 genes in the genome of an animal and more of them are concerned with nervous system function than with peripheral function. We therefore concluded that a large number of receptors and pathways are important to the transduction of pain, but up to now have remained unknown.

SUMMARY OF THE INVENTION It is an object of the invention to provide sequences of genetic material for which no role in pain has previously been disclosed, and which are useful, for example, in:

'identifying metabolic pathways for the transduction of pain * identifying from said metabolic pathways compounds having utility in the diagnosis or treatment of pain * producing proteins and polypeptides related to pain; producing genetically modified animals that are useful in the screening of compounds for the treatment or diagnosis of pain.

Identifying ligand molecules for said metabolic pathways having utility in the treatment of pain.

It is yet a further object of the invention to provide animals and microorganisms that can be used in screening compounds for pharmacological activity, especially pain-reducing activity.

In one aspect, the invention relates to the use in the screening of compounds that are effective in the treatment of pain, or in the diagnosis of pain, of : (a) an isolated gene sequence that is differentially expressible in neuronal tissue in response to a nociceptive stimulus; (b) an isolated gene sequence having at least 80% sequence identity with the nucleic acids of tables I-X (c) an isolated nucleic acid comprising a sequence that is hybridizable to the above gene sequence under stringent hybridisation conditions; (d) a recombinant vector comprising the above gene sequence; (e) a host cell containing the vector; (f) an animal having in its genome an introduced gene sequence or a removed or down-regulated sequence that is differentially expressed in neuronal tissue in response to a nociceptive stimulus; (g) an isolated polypeptide containing an amino acid sequence at least 90% identical to a sequence encoded by the above gene sequences, or a variant thereof with sequential amino acid deletions from either the C terminus or the Nterminus; or (h) an isolated antibody that binds specifically to the isolated polypeptide.

The invention also relates to the use of naturally occurring compounds such as peptide ligands of the expression products of the above gene sequences and their associated signal transduction pathways for the treatment of pain.

Other aspects of the invention are to be found in the accompanying claims, to which attention is hereby directed.

DESCRIPTION OF PREFERRED EMBODIMENTS DEFINITIONS Within the context of the present invention: . "Comprising" means consisting of or including. Thus nucleic acid comprising a defined sequence includes nucleic acid that may contain a full-length gene or full- length cDNA. The gene may include any of the naturally occurring regulatory sequence (s), such as a promoter, mRNA start site, TATA box and terminational regulatory sequences. Further, nucleic acid comprising cDNA or gene may

include any appropriate regulatory sequences for the efficient expression thereof in vitro.

. "Isolated" requires that the material be removed from its original environment (e. g. the natural environment if it is naturally occurring). For example, a naturally occurring polynucleotide or a peptide present in a living animal is not isolated, but the same polynucleotide or peptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotide can be part of a vector and/or such polynucleotide or peptide can be part of a composition, and still be isolated in that the vector or composition is not a part of its natural environment.

. Purified" does not require absolute purity; instead it is intended as a relative definition. Purification of starting materials or natural materials to at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated.

"Nucleic acid or gene"means a nucleotide sequence characterized by function, any variant or homologue thereof, or truncated or extended sequence thereof, and is preferably indicated by a Genebank accession number. The terms"nucleic

acid (s), nucleic acid sequence (s) or gene (s) refer interchangeably, without bias to polynucleotide sequence (s). Also within the scope of the present invention are nucleic acids which encode expression products of signaling pathways of the identified up or down regulated nucleic acids. This includes any variant or homologue thereof or truncated or extended sequence thereof. Further, within the scope of the present invention, the term nucleic acid (s) product, or expression product or gene product or a combination of terms refers without being biased to any, protein (s), polypeptide (s), peptide (s) or fragment (s) encoded by nucleic acids, as indicated above or fragments thereof.

*"Operably linked"refers to a linkage of polynucleotide elements in a functional relationship. For instance, a promoter or an enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. In particular, two DNA molecules (such as a polynucleotide containing a promoter region and a polynucleotide encoding a desired polypeptide or polynucleotide) are said to be"operably linked"if the nature of the linkage between the two polynucleotides does not (1) result in the introduction of a frame-shift mutation and (2) interfere with the ability of the polynucleotide containing the promoter to direct the transcription of the coding polynucleotide.

. "Pain" includes chronic pain and in particular diabetic pain.

."Stringent hybridization conditions"is a recognized term in the art and for a given nucleic acid sequence refers to those conditions which permit hybridization of that sequence to its complementary sequence and not to a substantially different sequence. Conditions of high stringency may be illustrated in relation to filter-bound DNA as e. g. 2X SCC, 65 C (where SSC = 0. 15M sodium chloride, 0. 015M sodium citrate, pH 7.2), or as 0. 5M NaHP04, 7% sodium dodecyl sulfate

(SDS), ImM EDTA at 65 C, and washing in 0. 1xSCC/0. 1% SDS at 680C (Ausubel F. M. et al., eds, 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc., and John Wiley & Sons Inc. , New York, at p.

2.10. 3; incorporated herein by reference in its entirety). Hybridization conditions can be rendered highly stringent by raising the temperature and/or by the addition of increasing amounts of formamide, to destabilize the hybrid duplex of non- homologous nucleic acid sequence relative to homologous nucleic acid sequences. Thus, particular hybridisation conditions can be readily manipulated, and will generally be chosen depending on the desired results.

"Variants or homologues"include (a) sequence variations of naturally existing gene (s) resulting from polymorphism (s), mutation (s), or other alteration (s) as compared to the above identified sequences, and which do not deprive the encoded protein of function (b) recombinant DNA molecules, such as cDNA molecules encoding genes indicated by the relevant Genebank accession numbers and (c) any sequence that hybridizes with the above nucleic acids under stringent conditions and encodes a functional protein or fragment thereof.

IDENTIFIED SEQUENCES The inventors have identified nucleic acid sequences that give rise to expression products listed in the tables below, that become differentially expressed in the spinal cord in response to a pain stimulus and that are believed to be involved in the transduction of pain. In the tables below, * denotes more preferred nucleic acids and ** denotes most preferred nucleic acids. These nucleic acids have not previously been implicated in the transduction of pain.

The proposition that the identified sequences are involved in the transduction of pain is supported by the fact that the experimental procedure used is capable of simultaneously identifying and validating nucleic acid sequences. Further conformation of the utility of this process arises from the fact that nucleic acid sequences were obtained whose function in the transmission of pain has been previously confirmed. These nucleic acid sequences are not part of this invention.

Any of the nucleic acid sequences and expression products, can be used to develop

screening technologies for the identification of novel molecules for the prevention or treatment of pain. These screening technologies could also be used to ascribe new pain therapeutic indications to molecules, which have not previously been ascribed for the prevention or treatment of pain. Furthermore, the said nucleic acid sequences can be used as diagnostic tools and for the development of diagnostic tools.

Table I-Sequences whose expression products are kinases

Expression Rat Mouse Human Direction of Kinase reaction product or name Accession accession Accession Modulation Number number Number Phosphofructokin U25651 AF249894 Y00698 Both Sugar kinase ase, muscle (PFK-M) Putative DNA D87521 Down Ser/Thr kinase dependent protein kinase catalytic subunit Putative protein U38894 Down Receptor tyrosine kinase tyrosine kinase t-Rorl Pyruvate kinase, M24359 X97047 X56494 Down Carbohydrate MI and M2 kinase subunit s Janus protein AJ000556 S63728 Up Non-receptor tyrosine kinase 1 Tyr kinase (JAKI) Phosphatidyl- D84667 Up Phospholipid inositol 4-kinase kinase Rho kinase U3 8481 U58513 D8793 1 Up Ser/Thr kinase Alpha Diacylglycerol D78588 U51477 Down lipid kinase kinase (DGK-IV) * Elk protein X 13411 Down receptor tyrosine kinase *Tyrosine kinase Casein kinase II L15619 Up Ser/Thr kinase beta subunit * ERK3, protein M64301 X80692 Up Ser/Thr kinase serine/threonine kinase, extracellular * Neural receptor M55291 XI7647 Up Receptor Tyr protein-tyrosine kinase kinase (trkB) * A-raf oncogene, X06942 U01337 Up Ser/Thr kinase liver expressed **

Table II-Sequences whose expression products are phosphatases

Expression Rat Mouse Human Direction of Phosphatase product or name accession accession Accession Modulation Reaction number number Number PP-I alpha for D00859 U25809 X70848 Down Ser/Thr catalytic subunit of protein phosphatase type I alpha Protein tyrosine S49400 U28216 U27831 Down Non-receptor phosphatase, Tyr striatum enriched Protein D90164 X80910 Up Ser/Thr phosphatase 1 Protein tyrosine Ai007016 AF035644 U48297 Up Tyr phosphatase * Putative receptor U55057 Up Receptor Tyr tyrosine phosphatase * Calcineurin, A M31809 M29550 Up N/A subunit, beta isoform * Table III-Sequences whose expression products are phosphodiesterases

EXPRESSION RAT MOUSE HUMAN DIRECTION OF PRODUCT OR ACCESSION ACCESSION ACCESSION MODULATION NAME NUMBER NUMBER NUMBER Alkaline D28560 API 23542 D4542 ! Up phosphodiesterase 1 * Phosphodiesterase, L27058 L20966 Up cAMP-specific (PDE4D) ** Table IV-Sequences whose expression products are ion channel proteins

EXPRESSION RAT MOUSE HUMAN DIRECTION OF PRODUCT ACCESSION ACCESSION ACCESSION MODULATION NUMBER NUMBER NUMBER Voltage dependent AF268468 U30838 L06328 Down anion channel 2 *

Table V-Sequences whose expression products are receptors

Expression Rat Mouse Human Direction of Receptor Product or Accession accession Accession Modulation Function Name Number Number Number Neurotensin X97121 Up N/A receptor type 2 (NTR2) Dopamine 158000 Down Dopamine receptor D. sub. l * Neonatal glycine X57281 Down Glycine receptor NglyR * Syntaxin 13 * AF044581 Down SNARE Dopamine 158000 Down receptor D. sub. l ** Putative GABA-AFH4168 Down GABA B I a receptor ** Putative X85999 Up IL-I interleukin 1 receptor accessory protein ** Table VI-Sequences whose expression products are transporters

Expression Rat Mouse Human Direction of Transporter Product or Accession Accession Accession Modulation Function Name Number Number Number Adenine D 12770 Down Nucleotide nucleotide translocator, mitochondrial ATPase, calcium, J03753 Down Calcium plasma membrane, isoform 1 Calcium AF209196 Down Calcium transporter CaT2 Camitine/acyl- X97831 Down Camitine camitine carrier protein Differentiation-AF271235 Down Phosphate associated Nadependent inorganic phosphate cotransporter Na+, K±ATPase D00189 Down Na+/K+ alpha-subunit

Putative potent AB012808 AB040056 Down Organic ions brain type organic ion transporter Synaptic vesicle L01788 Down N/A transmembrane transporter UDP-galactose D87991 Down Nucleotide-sugar transporter transporter Amino acid AF249673 Up Amino acids system A transporter Digoxin carrier U88036 Up Organic anions protein and cardiac glycosides Na+K+ ATPase M14512 Up Na+/K+ alpha+ isoform catalytic subunit Putative vacuolar U13837 AF 113129 Up H+ ATP synthase subunit A Synaptic vesicle L01788 Up Neurotransmembrane transmitters transporter Choline X66494 Down Choline transporter CHOTI ** Glycine LI 3600 Up Glycine transporter ** Famesyl M34477 Down diphosphate synthetase ** Glutamine M91652 Down synthetase (EC 6.3. 1.2.) ** Delta-D86297 Up aminolevulinate synthase, erythroid-specific ** Polypeptide U35890 Up GaINAc transferase ! ** Putative NAD+ AJ007780 Up ADPribosyltransferase.

**

Table VII-Sequences whose expression products are G-protein coupled receptor proteins

Expression Rat Muse Human Direction of Receptor product or Accession Accession Accession Modulation Function Name Number Number Number Rab GDI, alpha X74402 Down G-protein species, ras related GTPase Putative K-ras M54968 Up N/A Putative ras- Down Small GTPrelated protein binding protein Rab-5c * ARF-like protein X78604 Up Ras-related 5 ARL5 *

Table VIII-Sequences whose expression products are DNA-binding proteins

Expression Rat Mouse Human Direction of DNA-binding Product or Accession Accession Accession modulation protein Name Number Number Number function Histone, core, M99065 Down DNA binding H2A. 1 Hypoxia- AF057308 AFOO3695 U2243 1 Both DNA binding inducible factor-1 alpha (Hifla) Putative histone X91866 Ml 1354 Down DNA binding H3. 3A Putative signal U06924 Down DNA binding transducer and regulator transcription I (Statl) Transcription L28801 Down DNA binding factor IIIC, alpha subunit Putative Up Helicase chromodomainhelicase-DNAbinding protein Table IX-Sequences whose expression products are proteases

Expression Rat Mouse Human Direction of Protease Product or Accession Accession Accession Modulation function Name Number Number Number Putative Lon X76040 Down Endopeptidase proteinase Carboxypeptidase U62897 D85391 U65090 Up Metallocarboxy D precursor peptidase Caspase 2 (Ich-1) U77933 Up Cysteine protease Cathepsin B X82396 M14222 Up Thiol protease Cathepsin L (EC Y00697 M20495 Up Cysteine 34. 22.15) protease Dipeptidylpeptida M76427 AF092507 M96859 Up Inactive se (dpp6) aminopeptidase family member Proteasome M58593 AF055983 D00762 Up Endoprotease subunit C8 Putative AF057146 D29956 Up N/A deubiquitinating enzyme 8

Table X-Sequences whose expression products are other enzymes

Expression Rat Mouse Human Direction of Function product or Accession Accession Accession Modulation Name Number Number Number 3-Hydroxy3-X52625 Down Ligase methylglutaryl coenzyme A synthase, cytosolic* Acyl-CoA D360666 Down Ligase synthetase II, brain Famesyl M34477 Ligase diphosphate synthase fatty acid X62888 Down Ligase synthase GM3 synthase AB018049 Down Ligase Glutamine M91652 Ligase synthetase (EC 6.3. 1.2) Putative seryl- X91257 Down Ligase tRNA synthetase Putative E12457 Down Ligase ubiquitinconjugating enzyme (EC 6.3. 2.-) E2 Putative X69656 Up Ligase tryptophanyltRNA synthetase Putative Y 17267 Up Ligase ubiquitinconjugating enzyme Aldolase A M12919 Y00516 MI 1560 Both Lyase Enolase, alpha X02610 X52379 M 14328 Down Lyase alpha, nonneuronal (NNE) Enolase, neuron-MI 1931 Down Lyase specific Putative carbonic Down Lyase anhydrase XIV Aldose reductase, X05884 Down Oxidoreductase lens Cytochrome c X14848 Both Oxidoreductase oxidase polypeptide III

Cytochrome-c M64496 Up Oxidoreductase oxidase 11, mitochondrial Cytochrome-c S79304 Down Oxidoreductase oxidase 1, mitochondrial Cytosolic malate AF093773 M29462 D55654 Down Oxidoreductase dehydrogenase (Mdh) * Hem-binding D30035 Down Oxidoreductase protein (HBP23) Lactate U07181 X51905 Y00711 Down Oxidoreductase dehydrogenase-B (LDH-B) Putative X 13 923 Down Oxidoreductase cytochrome c oxidase VIB (EC 1.9. 3. 1) Putative NADH-U59509 Down Oxidoreductase ubiquinone oxidoreductase MLRQ subunit Putative NADH: AF044953 Down Oxidoreductase ubiquinone oxidoreductasePG IV subunit Peptidylglycine M25719 U79523 m37721 Down Oxidoreductase alpha-amidating monooxygenase * Putative succinate AF095938 Ah 171022 Down Oxidoreductase dehydrogenase flavoprotein Putative J04973 Down Oxidoreductase Pbiquinolcytochrome-c reductase (EC 1. 10.2. 2) core protein ! ! Squalene D37920 Down Oxidoreductase epoxidase * Stearoyl-coA AB032243 M26270 Down Oxidoreductase desaturase 2 * Superoxide M21060 X05634 K00065 Down Oxidoreductase dismutase, copper-zinc (SOD-I) 3-Hydroxy-3-M29249 MH058 Up Oxidoreductase methylglutaryl coenzyme A reductase NADH subunit 5, X14848 Up Oxidoreductase mitochondrial Aspartate 104171 Down Transferase aminotransferase, cytosolic *

Branched-chain AF165887 Down Transferase aminotransferase, cytosolic, brain Diacylglycerol D78588 Down Transferase kinase (DGK-IV) * Dihydrolipoamide D9040 1 Down Transferase succinyltransferase Phosphofructokin U25651 AF249894 Y00698 Down Transferase ase, muscle (PFKM) Ribophorin I * X05300 Down Transferase RNA ABO 17711 Down Transferase polymerase II Squalene M95591 Down Transferase synthetase, hepatic Sulfotransferase- AF188699 Down Transferase like protein UDP-Gal : AF048687 Down Transferase glucosylceramide beta-1, 4galactosyl transferase Delta-amino- D86297 Transferase levulinate synthase, erythroid-specific Polypeptide U35890 Transferase GalNAc transferase T1 Putative NAD+ AJ007780 Transferase ADP-ribosyltransferase ATP synthase, X56133 Down Hydrolase H+, alpha subunit, mitochondrial (EC 3.6. 1.34) FIFO ATPase U00926 Down Hydrolase delta subunit Putative D78013 Down Hydrolase dihydropyrimidin ase related protein * Putative AF116910 Down Hydrolase ribonuclease III arginase II U90887 U90886 Up Hydrolase ATP synthase L19927 D16563 Up Hydrolase gamma chain, mitochondrial

N-G, N-G- D86041 Up Hydrolase dimethylarginine dimethylaminhydrolase Putative gluco- M24119 Up Hydrolase cerebrosidase Putative N-acetyl- Z12173 Up Hydrolase glucosamin-6sulfatase Round spermatid U97667 Up Hydrolase protein RSP29 putative AB024303 Down activation of Sentrin/SUMO protein AOS1 PRODUCTION OF POLYPEPTIDES AND NUCLEIC ACIDS Vectors Recombinant expression vectors comprising a nucleic acid can be employed to express any of the nucleic acid sequences of the invention. These expression products can be used to develop screening technologies for the identification of molecules that can be used to prevent or treat pain, and in the development of diagnostic tool for the identification and characterization of pain. The expression vectors may also be used for constructing transgenic animals.

Expression requires that appropriate signals be provided in the vectors, said signals including various regulatory elements such as enhancers/promoters from both viral and mammalian sources that drive expression of the genes of interest in host cells. The regulatory sequences of the expression vectors used in the invention are operably linked to the nucleic acid encoding the pain-associated proteins of interest or a peptide fragment thereof.

Generally, recombinant expression vectors include origins of replication, selectable markers, permitting transformation of the host cell, and a promoter derived

from a highly expressed gene to direct transcription of a downstream structural sequence. The heterologous structural sequence is assembled in an appropriate frame with the translation, initiation and termination sequences, and if applicable a leader sequence capable of directing sequences of the translated protein into the periplasmic space, the extra-cellular medium or cell membrane.

In a specific embodiment wherein the vector is adapted for transfecting and expressing desired sequences in mammalian host cells, preferred vectors will comprise an origin of replication from the desired host, a suitable promoter and an enhancer, and also any necessary ribosome binding sites, polyadenylation site, transcriptional termination sequences, and optionally 5'-flanking non-transcribed sequences. DNA sequences derived from the SV 40 or CMV viral genomes, for example SV 40 or CMV origin, early promoters, enhancers, and polyadenylation sites may be used to provide the required non-transcribed genetic elements.

A recombinant expression vector used in the invention advantageously also comprises an untranscribed polynucleotide located at the 3'end of the coding sequence (ORF), this 3'-UTR polynucleotide being useful for stabilizing the corresponding mRNA or for increasing the expression rate of the vector insert if this 3'-UTR harbours regulation signal elements such as enhancer sequences.

Suitable promoter regions used in the expression vectors are chosen taking into account the host cell in which the heterologous nucleic acid sequences have to be expressed. A suitable promoter may be heterologous with respect to the nucleic acid sequence for which it controls the expression, or alternatively can be endogenous to the native polynucleotide containing the coding sequence to be expressed. Additionally, the promoter is generally heterologous with respect to the recombinant vector sequences within which the construct promoter/coding sequence has been inserted. Preferred bacterial promoters are the Lad, LacZ, T3 or T7 bacteriophage RNA polymerase promoters, the lambda PR, PL and trp promoters (a EP-0 036 776),

the polyhedrin promoter, or the plO protein promoter from baculovirus (kit Novagen; Smith et al., (1983); O'Reilly et al. (1992).

Preferred selectable marker genes contained in the expression recombinant vectors used in the invention for selection of transformed host cells are preferably dehydrofolate reductase or neomycin resistance for eukaryotic cell culture, TRP I for

S. cerevisiae or tetracycline, rifampicin or ampicillin resistance in E. coli, or Levamsaccharase for Mycobacteria, this latter marker being a negative selection marker.

Preferred bacterial vectors are listed hereafter as illustrative but not limitative examples: pQE70, pQE60, pQE-9 (Quiagen), pDIO, phagescript, psiX174, p. Bluescript SK, pNH8A, pNH16A, pNH18A, pNH46A (Stratagene); pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia) ; pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene); pSVK3, pBPV, pMSG, pSVL (Pharmacia); pQE-30 (QIA express).

Preferred bacteriophage recombinant vectors of the invention are PI bacteriophage vectors such as described by Stemberg N. L. (1992; 1994).

A suitable vector for the expression of any of the pain associated polypeptides used in the invention or fragments thereof, is a baculovirus vector that can be propagated in insect cells and in insect cell-lines. A specific suitable host vector system is the pVL 1392/1393 baculovirus transfer vector (Pharmingen) that is used to transfect the SF9 cell line (ATCC N CRL 1711) that is derived from spodoptera jrugiperda.

The recombinant expression vectors used in the invention may also be derived from an adenovirus. Suitable adenoviruses are described by Feldman and Steig (1996) or Ohno et al. (1994). Another preferred recombinant adenovirus is the human

adenovirus type two or five (Ad 2 or Ad 5) or an adenovirus of animal origin (Patent Application WO 94/26914) Particularly preferred retrovirus as for the preparation or construction of retroviral in vitro or in vivo gene delivery vehicles include retroviruses selected from the group consisting of Mink-Cell Focus Inducing Virus, murine sarcoma virus, and Ross Sarcoma Virus. Other preferred retroviral vectors are those described in Roth et al. (1996), in PCT Application WO 93/25234, in PCT Application WO 94/06920, and also in Roux et al. (1989), Julan et al. (1992) and Nada et al. (1991).

Yet, another viral vector system that is contemplated consist in the adeno associated viruses (AAV) such as those described by Flotte et al. (1992), Samulski et al. (1989) and McLaughlin et al. (1996).

Host cells expressing pain associated polypeptides Host cells that endogenously express pain associated polypeptides or have been transformed or transected with one of the nucleic acid ssequences described herein, or with one of the recombinant vector described above, particularly a recombinant expression vector, can be used in the present invention. Also included are host cells that are transformed (prokaryotic cells) or are transected (eukaryotic cells) with a recombinant vector such as one of those described above.

Preferred host cells used as recipients for the expression vectors used in the invention are the following: (a) prokaryotic host cells: Escherichia coli, strains. (i. e. DH5-a, strain) Bacillus subtilis, Salmonella typhimurium and strains from species like Pseudomonas, Streptomyces and Staphylococcus for the expression of up and down regulated nucleic acids modulated by pain, characterized by having at least 80% sequence identity with any of the nucleic acid sequences of Tables I-X. Expression

in these host cells can provide plasmids for transfecting other cells transiently e. g. using electroporation or stably by incorporation into cellular DNA.

(b) eukaryotic host cells: HeLa cells (ATCC N CCL2 ; N CCL2. 1; N CCL2. 2), Cv 1 cells (ATCC N CCL70), COS cells (ATCC N CRL 1650 ; N CRL 1651), Sf-9 cells (ATCC N CRL 1711), C127 cells (ATCC N CRL-1804), 3T3 cells (ATCC N CRL-6361), CHO cells (ATCC N CCL-61), human kidney 293 cells (ATCC ? 45504; N CRL-1573), BHK (ECACC N084100 501; N084111301), PC12 (ATCC NO CRL-1721), NT2, SHSY5Y (ATCC ? CRL-2266), NG108 (ECACC N088112302) and F11, SK-N-SH (ATCC NO CRL-HTB-l1), SK-N-BE (2) (ATCC NO CRL-2271), IMR-32 (ATCC NO CCL-127). A preferred system to which the nucleic acids of the invention can be expressed are neuronal cell lines such as PC12, NT2, SHSY5Y, NG108 and Fll, SK-N-SH, SK-N-BE (2), IMR-32 cell lines, COS cells, 3T3 cells, HeLa cells, 292 cells and CHO cells. The above cell lines could be used for the expression of any of the nucleic acid sequences of Tables I-X.

When a nucleic acid sequence of any of Tables I-X is expressed using a neuronal cell line, the sequence can be expressed through an endogenous promoter or native neuronal promoter, or through an exogenous promoter. Suitable exogenous promoters include SV 40 and CMV and eukaryotic promoters such as the tetracycline promoter. The preferred promoter when pain associated molecules are endogenously expressed is an endogenous promoter. A preferred promoter in a recombinant cell line is the CMV promoter.

In a specific embodiment of the host cells described above, these host cells have also been transfected or transformed with a polynucleotide or a recombinant vector allowing the expression of a natural ligand of any of the nucleic acid sequences of tables I-X or a modulator of these expression products.

PROTEINS, POLYPEPTIDES AND FRAGMENTS The expression products of the nucleic acid sequences of tables I-X or fragment (s) thereof can be prepared using recombinant technology, cell lines or chemical synthesis. Recombinant technology and chemical synthesis of the proteins or fragments thereof can allow the modification of the gene encoding the proteins to include such features as recognition tags, cleavage sites and other modifications of the expression products or fragments thereof. For efficient polypeptide production, the endogenous expression system or recombinant expression system should allow the expression products to be expressed in a manner that will allows the production of a functional protein or fragment thereof which can be purified. Preferred cell lines are those that allow high levels of expression of polypeptide or fragments thereof.

Such cell lines include cell lines which naturally express any of the nucleic acid sequences of tables I, II, III or IV or common mammalian cell lines such as CHO cells and COS cells, etc, or more specific neuronal cell lines such as PC 12. However, other cell types that are commonly used for recombinant protein production such as insect cells, amphibian cells such as oocytes, yeast and procaryotic cell lines such as

E. coli can also be considered.

The expression products of tables I-X or fragments thereof can be utilized in screens to identify potential therapeutic ligands, either as a purified protein, as a protein chimera such as those described in phage display, as a cell membrane (lipid or detergent) preparation, or in intact cells.

The invention also relates generally to the use of proteins, peptides and peptide fragments for the development of screening technologies for the identification of molecules for the prevention or treatment of pain, and the development of diagnostic tools for the identification and characterization of pain.

These peptides include expression products of the nucleic acid sequences of Tables I - X and purified or isolated polypeptides or fragments thereof having at least 90%, preferably 95%, more preferably 98% and most preferably 99% sequence identity

with the any of the expression products of nucleic acid sequences of Tables I-X.

Expressed peptides and fragments of any of these nucleic acid sequences can be used to develop screening technologies for the identification of novel molecules for the prevention or treatment of pain. These screening technologies could also be used to ascribe new pain therapeutic indications to molecules, which have not previously been ascribed for the prevention or treatment of pain. Furthermore the said expressed peptides and fragments can be used as diagnostic tools and for the development of diagnostic tools.

SCREENING METHODS As discussed above, we have identified nucleic acid sequences whose expression is regulated by pain, particularly chronic pain and more particularly diabetic pain. The expression products of these nucleic acids can be used for screening ligand molecules for their ability to prevent or treat pain, and particularly, but not exclusively, chronic pain. The main types of screens that can be used are indicated below. The test compound can be a peptide, protein or chemical entity, either alone or in combination (s), or in a mixture with any substance. The test compound may even represent a library of compounds.

The expression products of any of the nucleic acid sequences of Tables I-X or fragments thereof can be utilized in a ligand binding screen format, a functional screen format or invivo format. Examples of screening formats are provided.

A) Ligand binding screen In ligand binding screening a test compound with is contacted with an expression product of one of the sequences above, and the ability of said test compound to interact with said expression product is determined, e. g. the ability of the test compound (s) to bind to the expression product is determined. The expression

product can be a part of an intact cell, lipidic preparation or a purified polypeptide (s), optionally attached to a support, such as beads, a column or a plate etc.

Binding of the test compound is preferably performed in the presence of a ligand to allow an assessment of the binding activity of each test compound. The ligand may be contacted with the expression product either before, simultaneously or after the test compound. The ligand should be detectable and/or quantifiable. To achieve this, the ligand can be labelled in a number of ways, for example with a chromophore, radioactive, fluorescent, phosphorescent, enzymatic or antibody label.

Methods of labelling are known to those in the art. If the ligand is not directly detectable it should be amenable to detection and quantification by secondary detection, which may employ the above technologies. Alternatively the expression product or fragment thereof can be detectable or quantifiable. This can be achieved in a similar manner to that described above.

Binding of the test compound modifies the interaction of the ligand with its binding site and changes the affinity or binding of the ligand for/to its binding site.

The difference between the observed amount of ligand bound relative to the theoretical maximum amount of ligand bound (or to the ligand bound in the absence of a test compound under the same conditions) is a reflection of the binding ability (and optionally the amount and/or affinity) of a test compound to bind the expression product.

Alternatively, the amount of test compound bound to the expression product can be determined by a combination of chromatography and spectroscopy. The amount of test compound bound to the expression product can also be determined by direct measurement of the change in mass upon compound or ligand binding to the expression product. This can be achieved with technologies such as Biacore (Amersham Pharmacia). Alternatively, the expression product, compound or ligand can be fluorescently labelled and the association of expression product with the test compound can be followed by changes in Fluorescence Energy Transfer (FRET).

The invention therefore includes a method of screening for pain alleviating compounds, comprising: a) contacting a test compound or test compounds in the presence of a ligand with an expression product of any of the above nucleic acid sequences or with cell expressing at least one copy of the expression product or with a lipidic matrix containing at least one copy of the expression product; b) determining the binding of the test compound to the expression product, and c) selecting test compounds on the basis of their binding abilities.

In the above method, the ligand may be added prior to, simultaneously with or after contact of the test compound with the expression product. Non limiting examples and methodology can be gained from the teachings of the Molecular Probes handbook and references therein (Molecular Probes, Inc. , 4849 Pitchford Ave, Eugene, USA), Methods in neurotransmitter receptor analysis (Yamamura HI. , Enna, SJ. , and Kuhar, MJ. , Raven Press New York, the Glaxo Pocket Guide to

Pharmacology, Dr. Michael Sheehan, Glaxo Group Research Ltd, Ware, Herts SG12 ODP), Bylund DB and Murrin LC (2000, Life Sciences, 67 (24) 2897-911), Owicki JC (2000, J biomol Screen (5) 297-306), Alberts et al (1994, Molecular Biology of the Cell, 3rd Edn, Garland Publication Inc), Butler JE, (2000 Methods 22 (1) : 4-23, Sanberg SA (2000, Curr Opin Biotechnol 11 (1) 47-53), and Christopoulos A (1999, biochem Pharmacol 58 (5) 735-48).

B) Functional screening (a) Kinase assays The expression products of any of the above nucleic acid sequences which encodes a kinase, and in particular the nucleic acid sequences listed in Table X, are amenable to screening using kinase assay technology.

Kinases have the ability to add phosphate molecules to specific residues in ligands such as binding peptides in the presence of a substrate such as adenosine triphosphate (ATP). Formation of a complex between the kinase, the ligand and substrate results in the transfer of a phosphate group from the substrate to the ligand.

Compounds that modulate the activity of the kinase can be determined with a kinase functional screen. Functional screening for modulators of kinase activity therefore involves contacting one or more a test compounds with an expression product of one of the above nucleic acid sequences which encodes a kinase, and determining the ability of said test compound to modulate the transfer of a phosphate group from the substrate to the ligand.

The expression product can be part of an intact cell or of a lipidic preparation or it can be a purified polypeptide (s), optionally attached to a support, for example beads, a column, or a plate. Binding is preferably performed in the presence of ligand and substrate to allow an assessment of the binding activity of each test compound.

The ligand should contain a specific kinase recognition sequence and it should not be phosphorylated at its phosphoryation site. The ligand and/or substrate may be contacted with the kinase either before, simultaneously or after the test compound.

Optionally the substrate may be labelled with a kinase transferable labelled phosphate. The assay being monitored by the phosphorylation state of the substrate and/or the ligand. The ligand should be such that its phosphorylation state can be determined. An alternative method to do this is to label the ligand with a phosphorylation-state-sensitive molecule. To achieve this, the ligand can be labelled in a number of ways, for example with a chromophore, radioactive, fluorescent, phosphorescent, enzymatic or antibody label. If the ligand is not directly detectable it should be amenable to detection and quantification by secondary detection, which may employ the above technologies. Such technologies are known to those in the art.

Binding of the test compound to the kinase modifies its ability to transfer a phosphate group from the substrate to the ligand. The difference between the

observed amount of phosphate transfer relative to the theoretical maximum amount of phosphate transfer is a reflection of the modulatory effect of the test compound. Alternatively, the degree of phosphate transfer can be determined by a combination of chromatography and spectroscopy. The extent of phosphorylation of the ligand peptide or dephosphorylation of the substrate can also be determined by direct measurement. This can be achieved with technologies such as Biacore (Amersham Pharmacia).

The invention also provides a method for screening compounds for the ability to relieve pain, which comprises: (a) contacting at one or more test compounds in the presence of ligand and substrate with an expression product of any of the sequences set out above which is a kinase or with a cell containing at least 1 copy of the expression product or with a lipidic matrix containing at least 1 copy of an expression product; (b) determining the amount of phosphate transfer from the substrate to the ligand; and (c) selecting test compounds on the basis of their capacity to modulate phosphate transfer.

Optionally ligand, substrate and/or other essential molecules may be added prior to contacting the test compound with expression product of step (a) or after step (a).. Non limiting examples and methodology can be gained from the teachings of the Molecular Probes handbook and references therein (Molecular Probes, Inc. , 4849 Pitchford Ave, Eugene, USA), Methods in Molecular Biology 2000 ; 99: 191-201, Oncogene 2000 20; 19 (49): 5690-701, and FASAB Journal, (10,6, P55, P1458,1996, Pocius D Amrein K et al).

b) Phosphatase assays The expression products of any of the above nucleic acid sequences which encodes a phosphatase, and in particular the nucleic acid sequences listed in Table X, are amenable to screening using phosphatase assay technology.

Phosphatase enzymes have the ability to remove phosphate molecules from specific residues in ligands such as peptides. This reaction takes place in the presence of a substrate such as Adenosine Diphosphate (ADP). The complexing of the phosphatase polypeptide, ligand and substrate results in the transfer of a phosphate group from the ligand to substrate. Compounds that modulate the activity of the Phosphatase can be determined with a Phosphatase functional screen. This screen detects the reverse of the Kinase functional screen outlined above.

The invention also provides a method for screening compounds for the ability to relieve pain, which comprises : (a) contacting at one or more test compounds in the presence of ligand and substrate with an expression product of any of the sequences set out above which is a phosphatase or with a cell containing at least 1 copy of the expression product or with a lipidic matrix containing at least 1 copy of an expression product ; (b) determining the amount of phosphate transfer from the ligand to the substrate; and (c) selecting test compounds on the basis of their capacity to modulate phosphate transfer.

Optionally ligand, substrate and/or other essential molecules may be added prior to contacting the test compound with expression product of step (a) or after step (a).. Non limiting examples and methodology can be gained from the teachings of the Molecular Probes handbook and references therein (Molecular Probes, Inc., 4849 Pitchford Ave, Eugene, USA), and FASAB Journal, (10,6, P55, P1458, 1996, Pocius D Amrein K et ao.

c) Phosphodiesterase assays An expression product of any of the above nucleic acid sequences which encodes a phosphodiesterase, and in particular any of the nucleic acid sequences listed in Table III, is amenable to screening using phosphodiesterase assay technology.

Phosphodiesterases have the ability to cleave cyclic nucleotides cAMP (cyclic adenosine monophosphate) and/or cGMP (cyclic guanosine monophosphate) (substrate) at their 3'phosphatase bond to form 5'AMP and 5'GMP. Functional screening for modulators of phosphodiesterase polypeptide comprises contacting one or more test compounds with an expression product as set out above which is a phosphodiesterase and determining the ability of said test compound (s) to modulate the cleavage of cyclic nucleotides cAMP and/or cGMP at their 3'phosphatase bond to form 5'AMP and 5'GMP. The expression product can be part of an intact cell or lipidic preparation or a purified polypeptide (s), optionally attached to a support, for example beads, a column, or a plate. Binding is preferably performed in the presence of cAMP or cGMP to allow an assessment of the binding activity of each test compound. The cAMP orcGMP and other essential molecules may be contacted with the phosphodiesterase peptide either before, simultaneously or after the test compound (s).

A characteristic of the cAMP or cGMP is that it can readily be radio labelled (Thompson et al, Advances in cyclic nucleotide research, 10,69-92 (1974) ). The conversion of cAMP or cGMP to 5'AMP or 5'GMP can be detected with the use of chromatography and separation technologies. Such technology is known to those in the art. Binding of the test compound to the phosphodiesterase polypeptide modifies its ability to convert cAMP or cGMP to 5'AMP or 5'GMP. The difference between the observed amount of conversion relative to the theoretical maximum amount of conversion is a reflection of the modulatory effect of the test compound (s).

A non limiting general understanding of how to assay for the activity of Phosphodiesterases can be gained from Horton, JK. And Baxendale, PM (Methods in molecular Biology, 1995,41, p 91-105, Eds. Kendall, DA. and Hill, SJ, Humana Press, Towota, NJ) and Molecular Probes handbook and references therein (Molecular Probes, Inc. , 4849 Pitchford Ave, Eugene, USA).

The invention also provides a methods of screening for pain alleviating compounds, comprising; a) contacting one or more test compounds in the presence of cAMP and/or cGMP with an expression product as set out above which is a phosphodiesterase or with a cell expressing at least 1 copy of a expression product or with a lipidic matrix containing at least 1 copy of an expression product; b) determining the amount of cAMPand/or cyclic Guanosine Mono Phosphate convereted to 5'AMP and/or 5'GMP, and c) selecting test compounds on the basis of their ability to modulate said conversion. d) Ion Channel Protein assays An expression product of any of the above nucleic acid sequences which encodes an ion channel protein, and in particular any of the nucleic acid sequences listed in Table IV, is amenable to screening using ion channel protein assay technology.

Ion channels are membraneous proteins. They are divided into three main groups: (1) ligand gated ion channels; (2) voltage gated ion channels; and (3) mechano gated ion channels.

Ion channels allow the passage of ions through cellular membranes upon stimulation by ligand, change in membrane potential or physical changes in environment such as temperature and pH. Compounds that modulate the activity of ion channels can be determined with an ion channel functional screen.

Functional screening for modulators of ion channels involves contacting a test compound with an expression product as aforesaid which is an ion channel protein or a fragment thereof and determining the ability of said test compound to modulate the activity of said expression product or fragment thereof. The expression product can be a part of an intact cell, membrane preparation or lipidic preparation, optionally attached to a support, for example beads, a column, or a plate. The ligand may be contacted with the ion channel peptide before, simultaneously with or after the test compound. Optionally other molecules essential for the function of the ion channel may be present. Ion channel opening is detectable with the addition of Ion channel sensitive dye, such dyes are known to those in the art. Examples are provided in Molecular Probes handbook and references therein (Molecular Probes, Inc. , 4849 Pitchford Ave, Eugene, USA) and Glaxo Pocket Guide to Pharmacology (Dr Michael Sheenal Pharmacology Division Staff, Glaxo Group Research Ltd. , Ware, Herts SG12 ODP). Binding of the test compound to the ion channel protein modifies its ability to allow ion molecules across a membrane. The difference between the observed amount of movement of ions across a membrane relative to the theoretical maximum amount of ions that can move across the membrane is a reflection of the modulatory effect of the test compound.

The invention therefore also relates to a method of compounds for their ability to alleviate pain, which method comprises: (a) contacting at least one test compound in the presence of voltage potential sensing dye with a cell containing at least 1 copy of an expression product as aforesaid which is an ion channel protein or with a lipidic matrix containing at least one copy of the expression product; a) applying a ligand or stimulus; b) determining the opening of the ion channel, and

c) selecting a test compound on the basis of its ability to modulate movement of ions across the membrane. e) Receptor assays An expression product of any of the above nucleic acid sequences which encodes a receptor, and in particular any of the nucleic acid sequences listed in Table V, is amenable to screening using receptor assay technology.

Receptors are membraneous proteins that initiate intracellular signalling upon ligand binding. Therefore, the identification of molecules for the prevention and treatment of pain can be achieved with the use of a ligand binding assay, as outlined above. Such an assay would utilize an endogenous or non-endogenous ligand as a component of the ligand binding assay. The binding of this ligand to the receptor in the presence of one or more test compounds would be measures as described above.

Such is the nature of receptors that the assay is usually, but not exclusively performed with a receptor as an intact cell or membraneous preparation.

The invention therefore includes a method of screening for pain alleviating compounds, comprising: a) contacting a test compound or test compounds in the presence of a ligand with cell expressing at least one copy of the expression product or with a lipidic matrix containing at least one copy of the expression product; b) determining the binding of the test compound to the expression product, and c) selecting test compounds on the basis of their binding abilities.

f) Transporter protein assays An expression product of any of the above nucleic acid sequences which encodes a transporter protein, and in particular any of the nucleic acid sequences listed in Table VI, is amenable to screening using transporter protein assay technology. Non limiting examples of technologies and methodologies are given by

Carroll FI, et al (1995, Medical Research Review, Sepl5 (5) p419-444), Veldhuis JD and Johnson Ml (1994, Neurosci. Biobehav Rep. , winter 18 (4) 605-12), Hediger MA and Nussberger S (1995, Expt Nephrol, July-Aug 3 (4) p211-218, Endou Hand Kanai Y, (1999, Nippon Yakurigaku Zasshi, Oct. 114 Suppll : Ip-16p), Olivier B et al (2000, Prog. Drug Res. , 54,59-119), Braun A et al (2000, Eur J Pharm Sci, oct 11, Suppl 2 S51-60) and Molecular Probes handbook and references therein (Molecular Probes, Inc. , 4849 Pitchford Ave, Eugene, USA).

The main function of transporter proteins is to facilitate the movement of molecules across a cellular membrane. Compounds that modulate the activity of transporter proteins can be determined with a transporter protein functional screen.

Functional screening for modulators of transporter proteins comprises contacting at least one test compound with an expression product as aforesaid which is a transporter protein and determining the ability of said test compound to modulate the activity of said transporter protein. The expression product can be part of an intact cell, or lipidic preparation, optionally attached to a support, for example beads, a column or a plate. Binding is preferably performed in the presence of the molecule to be transported, which should only able to pass through a cell membrane or lipidic matrix with the aid of the transporter protein. The molecule to be transported should be able to be followed when it moves into a cell or through a lipidic matrix.

Preferably the molecule to be transported is labelled to aid in characterization, e. g. with a chromophore, radioactive, fluorescent, phosphorescent, enzymatic or antibody label. If the molecule to be transported is not directly detectable it should be amenable to detection and quantification by secondary detection, which may employ the above technologies. The molecule to be transported may be contacted with the

transporter protein before, simultaneously with or after the test compound. Binding of the test compound to the transporter protein modifies its ability to transport molecules through a membraneous or lipidic matrix. The difference between the observed amount of transported molecule in a cell/or through a lipidic matrix relative to the theoretical maximum amount is a reflection of the modulatory effect of the test compound.

The invention further provides a method for screening compounds for their ability to relieve pain, comprising a) contacting at least one test compounds in the presence of transporter molecules with a cell containing at least one copy of an expression product as aforesaid which is a transporter protein or with a lipidic matrix containing at least one copy of the expression product; c) measuring the movement of transporter molecules into or from the cell, or across the lipidic matrix; and d) selecting test compounds on the basis of their ability to modulate the movement of transporter molecules. g) G-protein coupled receptor protein assays An expression product of any of the above nucleic acid sequences that encodes a G-protein coupled receptor protein, and in particular any of the nucleic acid sequences listed in Table VII, is amenable to screening using G-protein coupled receptor protein assay technology.

G-protein coupled receptor proteins (GPCRs) are membranous proteins whose main function is to transduce a signal through a cellular membrane. Upon ligand binding, GPCRs undergoes a conformational change that allows complexing of the GPCRs with a G-protein. G-proteins possess a GTP/GDP binding site. The formation of the G-protein/ligand complex allows exchange of GTP for GDP,

resulting in confonnational change of the G-protein. This conformational change initiates signal transduction.

Functional screening for modulators of GPCRs comprises contacting at least one test compound with an expression product as aforesaid which is a G-protein coupled receptor protein, and assessing the ability of the test compound (s) to modulate the exchange of GTP for GDP or the modulation of the GPCR signal transduction pathway. The expression product can be part of an intact cell or lipidic preparation, optionally attached to a support, for example beads, a column or a plate.

Binding is preferably performed in the presence of a ligand, G-protein and GTP/GDP to allow an assessment of the binding activity of each test compound. Alternatively components of the signaling pathway are also included. In particular, in a preferred embodiment, a labeled GTP is used and the ability of the test compound (s) to modulate the exchange of GTP to GDP is determined. A further optional characteristic of the assay can be the inclusion of a reporter molecule that enables monitoring the regulation of the signaling pathway. The ligand may be contacted with the GPCR before, simultaneously with or after the test compound. Binding of the test compound to the GPCR modifies its ability to modulate the exchange of GTP for GDP and hence the modulation of signal transduction. The difference between the observed amount of GTP exchanged for GDP relative to the theoretical maximum amount of GTP is a reflection of the modulatory effect of the test compound.

Likewise the relative activities of signal transduction reporter molecules are also a reflection of the modulatory effect of the test compound. Non limiting examples of technologies and methodologies can be found in Molecular Probes handbook and references therein (Molecular Probes, Inc. , 4849 Pitchford Ave, Eugene, USA), Glaxo Pocket Guide to Pharmacology, (Michael Sheehan, Pharmacology Division staff, Glaxo Group Research Ltd. , Ware, Herts SC12 ODP) and Xing et al (2000, J.

Recept. Signal. Transduct. Res. 20 (40 189-210).

The invention provides a method of screening compounds for their ability to relieve pain, comprising:

a) contacting at least one test compound in the presence of a ligand, GTP/GDP, G-protein with a cell containing at least one copy of an expression product of a sequence as aforesaid which is a G-protein coupled receptor protein or with a lipidic matrix containing at least one copy of the expression product; b) measuring the exchange of GTP for GDP, and c) selecting test compounds on the basis of their ability to modulate said exchange.

In the above method, the ligand, GTP/GDP, G-protein and other essential molecules can be added before, simultaneously with or after the contacting of the test compound (s) with the cell line or lipidic matrix in step a). h) DNA-binding protein assays An expression product of any of the above nucleic acid sequences that encodes a DNA-binding protein, and in particular any of the nucleic acid sequences listed in Table VIII, is amenable to screening using DNA-binding protein assay technology.

DNA binding proteins are proteins that are able to complex with DNA. The complexing of the DNA binding protein with the DNA in some instances requires specific nucleic acid sequence. Screens can be developed in a similar manner to ligand binding screens as previously indicated and will utilise DNA as the ligand. DNA-binding protein assays can be carried using similar principles described in ligand binding assays as described above. Non limiting examples of methodology

and technology can be found in the teachings of Haukanes BI and Kvam C (Biotechnology, 1993 Jan 11 60-63), Alberts B et al (Molecular Biology of the Cell, 1994, 3rd Edn., Garland Publications Inc, Kirigiti P and Machida CA (2000 Methods Mol Biol, 126,431-51) and Molecular Probes handbook and references therein (Molecular Probes, Inc. , 4849 Pitchford Ave. , Eugene, USA).

The invention therefore includes a method of screening for pain alleviating compounds, comprising:

a) contacting a test compound or test compounds in the presence of a plurality of nucleic acid sequences with an expression product of any of the above nucleic acid sequences or with cell expressing at least one copy of the expression product or with a lipidic matrix containing at least one copy of the expression product ; b) determining the binding of the test compound to the expression product, and c) selecting test compounds on the basis of their binding abilities.

In the above method, the plurality of nucleic acid sequence may be added prior to, simultaneously with or after contact of the test compound with the expression product. i) Protease assays An expression product of any of the above nucleic acid sequences that encodes a protease protein, and in particular any of the nucleic acid sequences listed in Table IX, is amenable to screening using protease protein assay technology. Non limiting examples of such technologies are illustrated in Molecular Probes handbook and references therein (Molecular Probes, Inc. , 4849 Pitchford Ave, Eugene, USA), Cook et al (1992, Structure and function of the aspartic proteinases, Ed. Dunn BM, Plenum Press, New York, 525-528) and Peranteau AG et al (1995, Anal Biochem, 1, 227 (1) 242-5).

Protease proteins have the property of being able to cleave peptide sequences.. In brief, one characteristic of the protease screening assay is the peptide ligand, which may contains at least one copy of a protease protein recognition sequence. The cleavage of this peptide by the protease results in the fragmentation of the peptide ligand. The propensity of the protease to fragment the peptide ligand in the presence of a candidate compound (s) is a measure of the inhibitory qualities of the candidate compound (s).

The protease screening assay may be performed in the presence of at least a plurality of ligand binding peptide and a protease.. These screening assays can be performed in the presence of isolated expression product, expression product in cells or expression product in a lipidic matrix. The invention therefore includes a method of screening for pain alleviating compounds, comprising: (a) contacting one or more test compounds in the presence of peptide ligand with an expression product of a sequence as described above which is a protease or with a lipidic matrix containing at least one copy of the expression product; (b) determining the amount of cleavage of peptide ligand; and (c) selecting test compounds on the basis of their ability to modulate the amount of ligand clevage. j) Assays using other enzymes Expression products of any of the above nucleic acid sequences that encode other enzymes, e. g. ligases, lyases, oxidoreductases, transferases and hydrolases, and in particular any of the nucleic acid sequences listed in Table IX, is amenable to screening using appropriate assay technology.

Each class of enzyme has a defined function. Ligases have the property of being able to splice molecules together. This is achieved with the conversion of ATP substrate to AMP. Therefore, the activity of a ligase can be followed by monitoring the conversion of ATP to AMP. Such technologies are known to those in the art.

Non limiting examples and methodologies can are illustrated by Ghee. T. Tan et al (1996, Biochem J. 314, 993-1000, Yang SW et al (1992,15 : 89 (6) 2227-31 and refrences therein, and in Molecular Probes handbook and references therein (Molecular Probes, Inc. , 4849 Pitchford Ave, Eugene, USA).

The invention also provides a methods of screening for pain alleviating compounds, comprising ;

a) contacting one or more test compounds in the presence of ATP with a ligase expression product or with a cell expressing at least 1 copy of a expression product or with a lipidic matrix containing at least 1 copy of an expression product; b) determining the amount of ATP converted to AMP, and c) selecting test compounds on the basis of their ability to modulate said conversion.

Lyases are enzymes which catalyse the cleavage of by reactions other than hydrolysis. These enzymes can be grouped into seven groups according to type of bond cleaved. These groups are carbon-carbon lyases (E. C. No 4.1), carbon-oxygene lyases (E. C. No 4.2), carbon-nitrogen lyases (E. C. No 4.3), carbon-sulphur lyase (E. C. No 4.4) carbon-halide lyases (E. C. No 4.5), phosphorous-oxygene lyases (E. C.

No 4.6) and other lyases (E. C. No 4.99) (Analytical Biochemistry 3rd Edn, David J.

Holme and Hazel Peck, Longman press). The enzyme commission number (E. C. ) of the International Union of Biochemistry relates to the type of reaction catalysed by

the enzyme. Further teachings on how to develop assays and screens for lyases can be obtainedftom Methods in Enzymology (Academic Press).

Oxidoreductases are enzymes that catalyse the transfer of hydrogen or oxygen atoms or electrons. These enzymes can by sub-grouped into twenty categories according to their specific mode of action. These groups are oxidoreductases acting on the CH-OH group of donors (E. C. No 1. 1), oxidoreductases acting on the aldehyde or oxo group of donors (E. C. No 1.2), oxidoreductases acting on the CH-CH group of the donor (E. C. No 1.3), oxidoreductases acting on the CH-NH2 group of donors (E. C. 1.4), oxidoreductases acting on the CH-NH group of donor (E. C. 1.5), oxidoreductases acting on the NADH or NADPH (E. C. No 1.6), oxidoreductases acting on other nitrogen compounds as donors (E. C. No 1.7), oxidoreductases acting on a sulphure group of donors (E. C. No 1.8), oxidoreductases acting on a haem group of donors (E. C. Nol. 9), oxidoreductases acting on diphenols and related substances as donors (E. C. 1.10), oxidoreductases acting on hydrogen peroxide as acceptor (E. C.

No 1. 11), oxidoreductases acting on hydrogen as donor (E. C. No 1. 12), oxidoreductases acting on single doners with incorporation of molecular oxygen (E. C. No 1. 13), oxidoreductases acting on paired donors with incorporation of molecular oxygene (E. C. No 1. 14), oxidoreductases acting on superoxide radicals as acceptors (E. C. 1. 15), oxidoreductases oxidizing metal ions (E. C. No 1. 16), oxidoreductases acting on-CH2 groups (E. C. No 1. 17), oxidoreductases acting on reduced ferredoxin as donor (E. C. No 1. 18), oxidoreductases acting on reduced flavodoxin as donor (E. C. No 1. 19) and other oxidoreductases (E. C. No 1. 97) (Analytical Biochemistry 3rd Edn, David J. Holme and Hazel Peck, Longman Press).

The enzyme commission number (E. C.) of the International Union of Biochemistry relates to the type of reaction catalysed by the enzyme. Further teachings on how to develop assays and screens for oxidoreductases can be obtained from Methods in Enzymology (Academic Press) with special reference to volume 249.

Transferases are enzymes that catalyse the transfer of specific groups. They are classified into eight sub groups according to function, transferring one-carbon group (E. C. No 2. 1), Transfering aldehyde or ketonic residues (E. C. No 2. 2), Acetyltransferases (E. C. 2. 3), glycosyltransferases (E. C. No 2. 4), transferring alkyl or aryl groups other than methyl groups (E. C. No 2. 5), transferring nitrogeneous groups (E. C. No 2. 6), transferring phosphorous-containing groups (E. C. No 2. 7) and transferring sulphur-containing groups (E. C. No 2. 8) (Analytical Biochemistry 3rd Edn, David J. Holme and Hazel Peck, Longman Press). The enzyme commission number (E. C.) of the International Union of Biochemistry relates to the type of reaction catalysed by the enzyme. Further teachings on how to develop assays and screens for transferases can be obtained from Methods in Enzymology (Academic Press).

Hydrolases are enzymes that catalyse hydrolytic reactions and are subgrouped into eleven classes according to the type of reaction they carry out.

Hydrolases acting on ester bonds (E. C. No 3. 1), hydrolases acting on glycosyl compounds (E. C. No 3. 2), hydrolases acting on ether bonds (E. C. No 3. 3),

hydrolases acting on peptide bonds (E. C. No 3.4), hydrolases acting on carbonnitrogen bonds, other than peptide bonds (E. C. No 3.5), hydrolases acting on acid anhydrides (E. C. No 3.6), hydrolases acting on acid anhydrides (E. C. No 3.6), hydrolases acting on carbon-carbon bonds (E. C. No 3.7), hydrolases acting on halide bonds (E. C. No 3.8), hydrolases acting on phosphorous-nitrogen bonds (E. C. No 3.9), hydrolases acting on sulphur-nirtgen bonds (E. C. No 3.10) and hydrolases acting on carbon-phosphorous bonds (Analytical Biochemistry 3rd Edn, David J. Holme and Hazel Peck, Longman Press). The enzyme commission number (E. C.) of the International Union of Biochemistry relates to the type of reaction catalysed by the enzyme. Further teachings on how to develop assays and screens for hydrolases can be obtained from Methods in Enzymology (Academic Press) with special reference to volume 249.

C) In vivo functional screen Any of the genes described above or homologues thereof may be inserted by means of an appropriate vector into the genome of a lower vertebrate or of an invertebrate animal or may be inactivated or down regulated in the genome of said animal. The resulting genetically modified animal may be used for screening compounds for effectiveness in the regulation of pain. The invertebrate may, for example, be a nematode e. g. Caenorhabditis elegans, which is a favourable organism for the study of response to noxious stimuli. Its genome sequence has been determined, see Science, 282,2012 (1998), it can be bred and handled with the speed of a micro-organism (it is a self-fertilizing hermaphrodite) and can therefore be used in a high throughput screening format (WO 00/63424, WO 00/63425, WO 00/63426 and WO 00/63427), and it offers a full set of organ systems, including a simple nervous system and contains many similarly functioning genes and signaling pathways to mammals. A thermal avoidance model based on a reflexive withdrawal reaction to an acute heat stimulus has been described by Wittenburg et al, Proc. Natl.

Acad. Sci. USA, 96, 10477-10482 (1999), and allows the screening of compounds for the treatment of pain with the modulation of pain sensation as an endpoint.

The genome of C. elegans can be manipulated using homologous recombination technology which allows direct replacement of nucleic acids encoding C. elegans with their identified mammalian counterpart. Replacement of these nucleic acids with those nucleic acids outlined above would allow for the direct screening of test compound (s) with their expression products. Any of the pain-related genes described above may be ligated into a plasmid and introduced into oocytes of the worm by microinjection to produce germline transformants. Successful plasmid injection into C. elegans and expression of inserted sequences has been reported by Devgen B. V. , Ghent, Belgium. It is also possible to produce by routine methods worms in which the target sequences are down-regulated or not expressed (knock-out worms). Further non limiting examples of methodology and technology can be found in the teachings of Hazendonk et al (1997, Nat genet. 17 (1) 119-21), Alberts et ai, (1994, Molecular Biology of the Cell 3rd Ed. Garland Publishing Inc, Caenorhabditis elegans is anatomically and genetically simple), Broverman S et al, (1993, PNAS USA 15; 90 (10) 4359-63) and Mello et al (1991,10 (12) 3959-70).

A further method for screening compounds for ability to modify response to pain, e. g. relieve pain, comprises : (a) contacting one or more test compounds with at least one C elegans containing at least one copy of a sequence as set out above; (b) subjecting the C. elegans to a nociceptive stimulus; (c) observing the response of the C. elegans to said stimulus; and d) selecting test compounds on the basis of their ability to modify the response of C. elegans to said stimulus.

DIAGNOSTIC TOOLS AND KITS Affinity peptides, ligands and substrates Pain associated polypeptides and fragments thereof can be detected at the tissue and cellular levels with the use of affinity peptides, ligands and substrates, which will enable a skilled person to define more precisely a patient's ailment and help in the prescription of a medicament. Such affinity peptides are characterized in that firstly they are able to bind specifically to a pain associated polypeptide, and secondly that they are capable of being detected. Such peptides can take the form of a linear peptide for example a linear antibody or ligand, substrate (e. g. an enzyme substrate such as NAD (H)), or a multimeric peptide complex such as an antibody.

The preparation of such linear and multimeric peptides are known to those in the art. Antibodies, these may be polyclonal or monoclonal, and include antibodies derived from immunized animals or hybridomas, or derivatives thereof such as humanized antibodies, Fab or F (ab') 2 antibody fragments or any other antibody fragment retaining the antigen specificity.

Antibodies directed against the gene products of pain associated molecules may be produced according to conventional techniques, including the immunization of a suitable mammal with the peptides or polypeptides or fragment thereof.

Polyclonal antibodies can be obtained directly from the serum of immunized animals.

Monoclonal antibodies are usually produced from hybridomas, resulting from a fusion between splenocytes of immunized animals and an immortalized cell line (such as a myeloma). Fragments of said antibodies can be produced by protease cleavage, according to known techniques. Single chain antibodies can be prepared according to the techniques described in US 4,946, 778. Detection of these affinity peptides could be achieved by labeling. technologies which allow detection of peptides, such as enzymatic labeling, fluorescence labeling or radio-labeling are well known to those in the art. Optionally these affinity peptides, ligands and substrates

could themselves be detected with the use of a molecule that has specific affinity to the peptides, ligands and substrates and is itself labeled.

The invention further provides a kit which comprises; (1) a plurality of affinity peptides, ligands or substrates for an expression product outlined above; and (2) a defined quantity of an expression product outlined above.

Complimentary nucleic acids Pain associated nucleic acids can be characterized at the tissue and cellular levels with the use of complimentary nucleic acids. Detection of the level of expression of pain associated nucleic acids can help in the prognosis of a pain condition and the prescription of a medicament. These complimentary nucleic acids are characterized in that they can specifically bind to a pain associated nucleic acid sequence and their presence can be detected through various techniques. Such techniques are known to those in the art and may include detection by polymerase chain reaction or detection by labeling of complimentary nucleic acids by enzymatic labeling, affinity labeling fluorescent labeling or radio labeling. Complimentary strand nucleic acids of this invention are 10 to 50 bases long, more preferably 15 to 50 bases long and most preferably 15 to 30 bases long, and hybridize to the coding sequence of the nucleic acid.

A further aspect of this invention is a kit that comprises; (a) a plurality of complimentary nucleic acids which possesses affinity to any one of the nucleic acid sequences outlined above; and (b) a defined quantity of one or more complimentary nucleic acids that possesses affinity to any one of the nucleic acid sequences outlined above.

IDENTIFICATION AND VALIDATION Subtractive hybridization enables the identification of nucleic acid sequences whose expression profiles are modified by a stimulus. Upon stimulation of a system (in the case of this invention a nociceptive stimulus on an animal model) all observed changes in the level of nucleic acid sequence expression are due to the reaction of the system to the stimulus. Characterization of these changes in expression by way of identification of nucleic acid sequence and level of expression is both identification and validation.

The inventors have developed a four step process which allows for the simultaneous identification and validation of nucleic acid sequences whose expression are regulated by a pain stimulus, preferably a chronic pain stimulus, and more preferably a diabetic pain stimulus. This process may comprise the following steps: (a) induction of a nociceptive stimulus in test animals; (b) extraction of nucleic acids from specific neuronal tissue of test and control animals; (c) selective amplification of differentially expressed nucleic acids; and (d) identification and characterization of differentially expressed gene products that are modulated by a nociceptive stimulus.

The above process is described in more detail below.

(a) Induction of nociceptive stimulus The effect of the selected nociceptive stimulus on the test animal needs to be confirmable. The test subjects are therefore a species that has a"developed"nervous system, preferably similar to that of humans, most preferably rats or mice. Advantageously, the nociceptive stimulus is analogous to known pain paradigms in humans. One such paradigm of pain is the pain associated with diabetes, which can

be induced in rodents with the use of streptozotocin (STZ). However, other pain paradigms could be used.

Streptozotocin (STZ) induces hyperglycemia and Type 1 diabetes mellitus in rats. In particular, STZ contains a glucose analogue that allows it to be taken up by the glucose transporter 2 present on the surface of pancreatic cells, the site of insulin synthesis. Once inside the cell, STZ causes a reduction in the level of nicotinamide adenine dinucleotide (NAD+). The decrease in NAD+ levels eventually

leads to necrosis of the pancreatic cell, causing a reduction in insulin levels and then diabetes, leading to neuropathy (diabetic) and neuropathic pain (R. B. Weiss, Cancer Treat. Rep., 66, 427-438 1982, Guy et al, Diabetologica, 28, 131-137 1985 ; Ziegler et al, Pain, 34, 1-10 1988 ; Archeret al, J Neurol. Neurosurg. Psychiatry, 46, 491-499 1983). The diabetic rat model has been shown to be a reliable model of hyperalgesia. We have used an STZ-induced diabetic rat model to create a state of hyperlagesia that can be compared with control animals (Courteix et al, Pain, 53,81- 88 1993).

(b) Extraction of nucleic acids from neuronal tissue of test and control animals the inventors have determined that RNA extraction of whole spinal cord nervous tissue would provide a way of identifying nucleic acid sequences whose expression is regulated by streptozotocin induced pain. Test (subjected to the nociceptive stimulus) and control animals were sacrificed, and the tissue to be studied e. g. neural tissue separated. Techniques for so doing vary widely from animal to animal and will be familiar to skilled persons.

Extraction ofmRNA from neural tissue of the test and control animals and its separation from other forms of RNA can be carried out by extracting total RNA from the tissue sample and to creating a cDNA library directly from the total RNA. Where possible, however, it is preferred to isolate the mRNA from the total RNA of the test and control animals and then reverse transcribe the mRNA, and from the test and

control animals to give test and control cDNA. Converting mRNA from the test and control animals to corresponding cDNA may be carried out by any suitable reverse transcription method, e. g. a method as described by Gubler & Hoffman, Gene, 25, 263-269 (1983). If desired a proprietary kit may be used e. g. the CapFinder PCR cDNA Library Construction Kit (Life Technologies) which is based on long-distance PCR and permits the construction of cDNA libraries from nanograms of total RNA.

(c) Selective amplification of differentially expressed nucleic acids The reverse transcribed cDNA of the test and control animals is subjected to subtractive hybridisation and amplification so that differentially expressed sequences become selectively amplified and commonly expressed sequences become suppressed, so as to produce DNA associated with said nociceptic stimulus. A wide range of subtractive hybridisation methods can be used, but the preferred method is so-called suppression subtractive hybridisation, see US-A-5565340 and Diatchenko

et al, Proc. Nat. Acad. Sci. USA, 93, 6025-6030 (1996), the disclosures of which are herein incorporated by reference. Kits for carrying out this method are available from CLONTECH Laboratories, Inc.

(d) Cloning and Sequencing the differentially expressed cDNA The differentially expressed cDNA was ligated to a cloning vector, after which cells of E. coli are transformed with the vector and cultured. Positive clones are selected and burst to release plasmids containing the cDNA insert. The plasmids are primed using forward and reverse primers to either side of the cloning site and the cDNA insert is sequenced. Vector and adaptor sequences are then removed from the output data from the sequencer, leaving only the nucleotide sequence of the differentially expressed gene. The sequence is then checked against data held in a database for homology to known genes, expressed sequence tags (ESTs) and proteins.

(e) Validation of the above method The importance of the sequences that we have identified in pain is confirmed by the fact that genes that represent nucleic acid sequences which have previously been implicated in pain, including Calmodulin (pRCMl, Genebank X13933), Enkephalin (Genebank Y07503) and Neurotensin receptor type 2 (Genebank X97121).

The inventors have identified nucleic acids of the MAP kinase pathway, a previously non pain-associated biological pathway. The inventors have subsequently shown that intra-spinal injection of a MEK inhibitor (MEK is part of the MAP kinase pathway) produces a powerful inhibition of pain (Patent application No US 60/144292). Subsequently, it has been shown that the MAP kinase is also implicated in acute inflammatory pain (Woolf et al, Nature Neuroscience 1999).

The invention will now be further described in the following Example.

EXAMPLE Induction of diabetes Diabetes was induced in adult (150-200g) male Sprague-Dawley rats (n=6) as described by Courteix et alet al (supra). Animals were injected intraperitoneally with streptozotocin (STZ) (50 mg/kg) dissolved in distilled water. Control or sham animals (age-matched animals, n=6) were injected with distilled water only.

Nociceptive testing Static allodynia (a form of hyperlagesia) was measured using a method described by Chaplan let al,"Quantitative assessment of tactile allodinya in the rat paw", J. Neurosci. Methods, 53,55-63 (1994). A series of von Frey filaments of

different buckling weight (i. e. the load required for the filament to bend) were applied to the plantar surface of the right hand paw. The starting filament had a buckling weight of 20g. Lifting of the paw was taken to be a positive result, in which case a filament with the next lowest buckling weight was used for the next measurement. The test was continued until a filament was found for which there was an absence of response for longer than 5 seconds whereas a re-test with the next heaviest filament gave a positive response. Animals were considered hyperalgesic if their thresholds were found to be < 4g of those of comparable untreated rats, see Calcutt & Chaplan,"Spinal pharmacology of tactile allodynia in diabetic rats", British J. Pharmacol, 122,1478-1482 (1997).

Tissue Extraction STZ-treated and control animals were anaesthetized with 4% halothane and perfused with ice-cold 0.9% saline containing 1% citric acid (pH adjusted to 7.4 with NaOH). The animals were decapitated and the lumbar spinal cord exposed. A 2centimetre length of spinal cord ending at L6 (lmbar-6 forward) was removed from the spinal column. Attached dorsal root ganglia and contaminating spinal connective tissues were removed. The spinal cord tissue was snap frozen in dry ice and isopentane.

Total RNA Extraction Total RNA was extracted from the above pooled male rat tissues using the TRIZOL Reagent Kit (Life Technologies). Briefly, tissue samples were homogenised fully using a Polytron homogenizer in 1ml of TRIZOL reagent per 50-lOOmg of tissue. Homogenized samples were then incubated at room temperature for 5 minutes and phase separated using 0.2ml chloroform per 1ml TRIZOL reagent followed by centrifugation at 3,000g. The aqueous phase was transferred to a fresh tube and the RNA precipitated out with an equal volume of isopropyl alcohol and centrifugation at

10, 000g. The RNA pellet was washed in 75% ethanol and re-centrifuged. The pellet was then air dried and re-suspended in water. mRNA Extraction In contrast to ribosomal RNA and transfer RNA, the vast majority of mRNAs of mammalian cells carry tracts of poly (A+) at their 3'termini. mRNAs can therefore be separated from the bulk of cellular RNA by affinity chromatography on oligo (dT)-cellulose. mRNA was extracted from Total RNA using the MESSAGEMAKER Kit (Life Technologies) in which mRNA (previously heated to 65 C in order to disrupt secondary structures and so expose the poly (A+) tails) was bound to oligo (dT) cellulose under high salt concentrations (0.5M NaCl) in a filter syringe.

Unbound RNA was then washed away and the poly (A) mRNA eluted in distilled water. A tenth of the volume of 7. 5 M Ammonium Acetate, 500g of glycogen/ml mRNA sample and 2 volumes of absolute alcohol were then added to the samples and the tubes placed at-20 C overnight. Following precipitation, the mRNA was spun down at 12,000g for 30 minutes at 4 C. RNAase free water was used to resuspend the pellets, which were then and stored at-80 C.

PCR SELECT The technique used was based on that of the CLONTECH PCR Select Subtraction Kit. The following protocol was performed using STZ-treated lumbar spinal cord Poly A + RNA as the'Tester'and Sham lumbar spinal cord poly A RNA as the'Driver' (Forward Subtraction). A second subtraction experiment using the Sham lumbar spinal cord mRNA as the'Tester'and STZ treated lumbar spinal cord mRNA as the'Driver' (Reverse Subtraction) was performed in parallel using the same reagents and protocol. As a control for both experiments, the subtraction was also carried out using human skeletal muscle mRNA that had been provided by the manufacturer.

First-Strand cDNA Synthesis 2 ug of PolyA+ RNA and 1 al of cDNA synthesis primer (10 OM) were combined in a 0. 5ml Eppendorf tube and sterile H2O was added where necessary to achieve a final volume of 5 Ill. The contents were mixed gently and incubated in a thermal cycler at 70 C for 2 min. The tubes were then cooled on ice for two minutes, after which 2 {il of 5X First-strand buffer, 1 al of dNTP mix (10 mM each), sterile H20 and 1 III of AMV reverse transcriptase (20 units/ill) was also added. The tubes were then placed at 42 C for 1.5 hr in an air incubator. First-strand cDNA synthesis was terminated by placing the tubes on ice. (The Human skeletal muscle cDNA produced by this step was used as the'control driver'in later steps).

Second-Strand cDNA Synthesis

48. 4 III of Sterile H2O, 16. 0 al of 5X Second-strand buffer, 1. 6 III of dNTP mix (10 mM) and 4. 0 al of 20X Second-strand enzyme cocktail were added to each of the first-strand synthesis reaction tubes. The contents were then mixed and incubated at 16 C in a thermal cycler for 2 hr. 6 units (2 Ill) ofT4 DNA polymerase was then introduced and the tubes were incubated for a further 30 min at 16 C. In order to terminate second-strand synthesis, 4 al of 20X EDTA/glycogen mix was added. A phenol : chloroform extraction was then carried out using the following protocol :- 100 III of phenol : chloroform : isoamyl alcohol (25: 24: 1) was added to the tubes which were then vortexed thoroughly and centrifuged at 14,000 rpm for 10 min at room temperature. The top aqueous layer was removed and placed in a fresh tube. 100 ul of chloroform : isoamyl alcohol (24: 1) was then added to the aqueous layer and

the tubes were again vortexed and centrifuged at 14, 000 rpm for 10 min. 40 III of 4 M NtLtOAc and 300 ul of 95% ethanol were then added and the tubes centrifuged at 14,000 rpm for 20 min. The supernatant was removed carefully, then 500 III of 80% ethanol was added to the pellet. The tubes were centrifuged at 14,000 rpm for 10 min

and the supernatant was removed so that the pellet could be air-dried. The precipitate was then dissolved in 50 ul of H2O. 6 ul was transferred to a fresh microcentrifuge tube. The remainder of the sample was stored at-20 C until needed.

Rsa I Digestion All products of the above procedures were subjected to a restriction digest, using the restriction endonuclease Rsa I, in order to generate ds cDNA fragments that are short and thus are optimal for subtraction hybridisation due to the standard kinetics of the hybridisation. Also, as Rsa I makes a double stranded cut in the middle of a recognition sequence,'blunt ends'of a known nucleotide sequence are produced allowing ligation of adaptors onto these ends in a later step. The following reagents were added to the 6 III product of the second hybridisation (see above) : 43. 5 ul of ds cDNA, 5. 0 III I OX Rsa I restriction buffer and 1. 5 ul of Rsa I (10 units/ul). The reaction mixture was incubated at 37 C for 1. 5 hr. 2. 5 III of 20X EDTA/glycogen mix was used to terminate the reaction. A phenol : chloroform extraction was then performed as above (second-strand cDNA synthesis section). The pellet produced was then dissolved in 5. 5 ul of H20 and stored at-20 C until needed. The preparation of the experimental'Driver'cDNAs and the control skeletal muscle cDNA was thus completed.

Adaptor Ligation The adaptors were not ligated to the driver cDNA.

1 III of each Rsa 1-digested experimental cDNA (from the Rsa I Digestion above) was diluted with 5 u. t of sterile water. Preparation of the control skeletal muscle tester cDNA was then undertaken by briefly centrifuging the tube containing control DNA (Hae III-digest of OX174 DNA [3 ng/u)]) and diluting 2 ui of the DNA with 38 u ! of sterile water (to 150 ng/ml). 1 u) of control skeletal muscle cDNA (from the Rsa I Digestion) was then mixed with 5 ut of the diluted X174/

Hae III DNA (150 ng/ml) in order to produce the control skeletal muscle tester cDNA.

Preparation of the adaptor-ligated tester cDNA A ligation master mix was prepared by combining 3, ut of Sterile water, 2 j. ! 1 of 5X ligation buffer and 1 l T4 DNA ligase (400 units/ul) per reaction. 2 j. ! 1 of

adaptor 1 (10 uM) was then added to 2 j. ! 1 of the diluted tester cDNA. To this, 6 j. ! 1 of the ligation master mix was also added. The tube was therefore labelled Tester 1-1. In a separate tube, 2 ul of the adaptor 2R (10 uM) was mixed with 2 j. ! 1 of the diluted tester cDNA and 6 u ! of the master mix. This tube was named Tester 1-2.

2 l of Tester 1-1 and 2 j. of Tester 1-2 were then placed into fresh tubes. These would later be used as the unsubtracted tester control. The remainder of the contents of Tester 1-1 and Tester 1-2 tubes were then centrifuged briefly and incubated at 16 C overnight. The ligation reaction was stopped by adding 1l J of EDTA/glycogen mix and the samples were heated at 72 C for 5 min in order to inactivate the ligase.

In doing so, preparation of the experimental and control skeletal muscle adaptorligated tester cDNAs was complete.

1 l from each Unsubtracted tester control was then removed and diluted into 1 ml of water. These samples were set aside as they were to be used later for PCR (see below). All of the samples were stored at -20 C Analysis of Ligation efficiency 1 l of each ligated cDNA was diluted into 200 l of water and the following reagents were then combined in four separate tubes:

Component Tube: 1 2 3 4 Tester 1-1 (ligated to Adaptor 1) 1 1 - Tester 1-2 (ligated to Adaptor 2R)--11 G3PDH 3'primer (10 1 1 1 1 G3PDH 5' primer (10 M) - 1 - 1 PCR primer 1 (10 M) 1 - 1 Total volume l 3 3 3 3

A master mix for all of the reaction tubes plus one additional tube was made up by adding 18. 5 J. ! I of sterile H20, 2. 5 J. ! I of 10X PCR reaction buffer, 0. 5 ) of dNTP mix (10 mM), and 0. 5 III of 50X Advantage cDNA Polymerase Mix, per reaction, into a fresh tube. 22 ul of this master mix was then aliquotted into each of the 4 reaction tubes prepared above. The contents of the tubes were overlaid with 50 III of mineral oil. The reaction mix was incubated in a thermal cycler at 75 C for 5 min in order to extend the adaptors. The following protocol was then carried out immediately in a thermal cycler (Perkin-Elmer GeneAmp PCR Systems 2400) : 94 C for 30 sec (1 cycle), 94 C 10 sec, 65 C 30 sec and then 68 C 2. 5 min (25 cycles) First Hybridisation 1. 5 III of the Adaptor I-ligated Tester 1-1 was combined with 1. 5 u. i of the Rsa 1-digested driver cDNA, prepared earlier and I 11 of 4X Hybridisation buffer.

This process was then repeated combining the Adaptor 2R-ligated Tester 1-2 with the Rsa 1-digested driver cDNA and 4X Hybridisation buffer. The samples were incubated in a thermal cycler at 98 C for 1. 5 min followed by incubation at 68 C for 8 hr.

Second Hybridisation 1 ut of Driver cDNA (i. e. the Rsa I-digested cDNA (see above)), 1 ut 4X Hybridisation buffer and 2 n I Sterile H2O were all combined in a fresh tube. 1 1 of this mix was then removed and placed in a new tube, overlaid with 1 drop of mineral oil and incubated at 98 C for 1. 5 min in order to denature the driver. The following procedure was used to simultaneously mix the driver with hybridisation samples 1 and 2 (prepared in the first hybridisation), thus ensuring that the two hybridisation samples were mixed together only in the presence of freshly denatured driver: A micropipettor was set at 15 u. L The pipette tip was then touched onto the mineral oil/sample interface of the tube containing hybridisation sample 2. The entire sample was drawn partway into the tip before it was removed from the tube in order to draw a small amount of air into the tip. The pipette tip was then touched onto the interface of the tube containing the freshly denatured driver (i. e. the tip contained both samples separated by a small pocket of air) before the entire mixture was transferred to the tube containing hybridisation sample 1. The reaction was then incubated at 68 C overnight. 200 ut of dilution buffer was added to the tube, which was then heated in a thermal cycler at 68 C for 7 min. The product of this second hybridisation was stored at-20 C.

PCR Amplification Seven PCR reactions were set up: (1) The forward-subtracted experimental cDNA, (2) the unsubtracted tester control (see preparation of the adaptor ligated tester cDNA), (3) the reverse-subtracted experimental cDNA, (4) the unsubtracted tester control for the reverse subtraction, (5) the subtracted control skeletal muscle cDNA, (6) the unsubtracted tester control for the control subtraction, and (7) the PCR control subtracted cDNA (provided in the kit). The PCR control subtracted cDNA was required to provide a positive PCR control as it contained a successfully subtracted mixture of Hae III-digested #X174 DNA.

The PCR templates were prepared by aliquotting I pu of each diluted cDNA (i. e. , each subtracted sample from the second hybridisation and the corresponding diluted unsubtracted tester control produced by the adaptor ligation see above) into an appropriately labelled tube. 1 l of PCR control subtracted cDNA was placed into a fresh tube. A master mix for all of the primary PCR tubes, plus one additional

reaction, was then prepared by combining 19. 5 III of Sterile water, 2. 5 III of 10X PCR reaction buffer, 0. 5 III of dNTP Mix (10 mM), 1. 0 III of PCR primer 1 (10 uM) and 0. 5 III of 50X Advantage cDNA Polymerase Mix. 24 III of Master Mix was then aliquotted into each of the 7 reaction tubes prepared above and the mixture was overlaid with 50 24 l mineral oil, before being incubated in a thermal cycler at 75 C for 5 min in order to extend the adaptors. Thermal cycling was then immediately

started using the following protocol : 94 C 25 sec (I cycle), 94 C 10 sec, 66 C 30 sec and 72 C 1. 5 min (32 cycles).

3 III of each primary PCR mixture was then diluted in 27 III of HzO, 1 III or each of these dilutions was then placed into a fresh tube.

A master mix for the secondary PCRs, (plus an additional reaction) was set up by combining 18. 5 ut of sterile water, 2. 5 ut of 10X PCR reaction buffer, 1. 0 III of Nested PCR primer 1 (10 hum), 1. 0 III of Nested PCR primer 2R (10 pom), 0. 5 PI of dNTP Mix (10 mM) and 0. 5 III of 50X Advantage cDNA Polymerase Mix per reaction. l of the III of this Master Mix was then added into each reaction tube containing the 1 l diluted primary PCR mixture. The following PCR protocol was then carried out: 94 C 10 sec, 68 C 30 sec and 72 C 1.5 min (12 cycles). The reaction products were then stored at-20 C.

Ligation into a Vector/Transformation & PCR The products of the PCR amplification (enriched for differentially expressed cDNAs) were ligated into the pCR2. 1-TOPO vector using a T/A cloning kit (Invitrogen), transformed into TOPO One Shot competent cells according to the manufacturers protocol and grown up on LB (Luria-Bertani) Agar plates overnight at 37 C. 1,000 colonies were then individually picked (using fresh sterile tips) and dipped into 5 III of sterile water which had been aliquotted previously into 96 well PCR plates. The water/colonies were heated in a thermal cycler at I OOIC for 10 minutes in order to burst the cells, thus releasing the plasmids containing a differentially expressed cDNA insert. The 5 ul of water/plasmid was then used as a template in a PCR reaction (see below) using M13 Forward and Reverse primers (10 ng/Ill), complementary to the M13 site present on either side of the cloning site on the vector. 5 III of the PCR product was then run on a 2% agarose gel and stained by ethidium bromide. PCR products of an amplified insert were identified and 5 III of the remainder of the PCR product (i. e. from the 15 III that had not been run on the gel) was diluted 1/10 with water. 5 u. l of the diluted PCR product was then used as a template in a sequencing reaction.

Sequencing A sequencing reaction containing M13 primer (3.2 pmol/u. l),'BigDye' reaction mix (i. e. AmpliTaq < S) DNA polymerase, MgC12, buffer and fluorescent dNTPs [each of the four deoxynucleoside triphosphates is linked to a specific fluorescent donor dye which in turn is attached to a specific acceptor dye]) and cDNA template (diluted PCR product) was set up. The reaction was carried out on a

thermal cycler for 25 cycles of 10 seconds at 96 C, 20 seconds at 50 C and 4 minutes at 60 C. Each reaction product was then purified through a hydrated Centri-Sep column, and lyophilised. The pellets were resuspended in Template Supression Reagent and sequenced on an ABI Prism 310 Genetic Analyser. The analyser uses an

ion laser to excite the specific donor dye that transfers its energy to the acceptor dye, which emits a specific energy spectrum that can be read by the sequencer.

The first 150 potentially upregulated genes were sequenced at Parke-Davis, Cambridge. The remaining 877 from both the forward and back subtractions were sequenced at the applicant's core sequencing facility in Ann Arbor, MI, USA.

Bioinformatics The sequencing results were analysed using the computer program CHROMAS in which the vector and adaptor sequences were clipped off, leaving only the nucleotide sequence of the differentially expressed gene. Each sequence was then checked for homology to known genes, Expressed Sequence Tags (ESTs) and Proteins using various Basic Local Alignment Search Tool (BLAST) searches against the Genbank sequence database at the National Centre for Biotechnology Information, Bethesda, Maryland, USA (NCBI).

Two lists were derived called STZup and STZdown that contain the nucleic acids from the forward and back subtracted libraries respectively. In each list there are given accession numbers and descriptions for the known rat genes identified, and where available corresponding mouse or human genes. Sequences that are considered to be of interest are listed in Tables I-X above.

Claims

1. Use in the screening of compounds that are effective in the treatment of pain, or in the diagnosis of pain, of : (a) an isolated gene sequence that is differentially expressible in neuronal tissue in response to a nociceptive stimulus; (b) an isolated gene sequence having at least 80% sequence identity with the nucleic acids of tables I-X ; (c) an isolated nucleic acid comprising a sequence that is hybridizable to the above gene sequence under stringent hybridisation conditions; (d) a recombinant vector comprising the above gene sequence; (e) a host cell containing the vector; (f) an animal having in its genome an introduced gene sequence or a removed or down-regulated sequence that is differentially expressed in neuronal tissue in response to a nociceptive stimulus; (g) an isolated polypeptide containing an amino acid sequence at least 90% identical to a sequence encoded by the above gene sequences, or a variant thereof with sequential amino acid deletions from either the C terminus or the Nterminus ; or (h) an isolated antibody that binds specifically to the isolated polypeptide.

2. Use according to claim I concerning an isolated gene sequence that is differentially expressed in neuronal tissue of a mammal.

3. Use according to claim 1 or 2 concerning an isolated gene sequence that is differentially expressed in the spinal cord.

4. Use according to claim 1,2 or 3 concerning an isolated gene sequence that is differentially expressed in response to a chronic pain stimulus.

5. Use according to any preceding claim concerning an isolated gene sequence that encodes a kinase.

6. Use according to claim 5, wherein the isolated gene sequence encodes an expression product or fragment thereof of phosphofructokinase, muscle (PFK-M) (U25651, AF249894, Y00698), putative DNA dependent protein kinase catalytic subunit (D87521), putative protein tyrosine kinase t-Rorl (U38894), pyruvate kinase, MI and M2 subunit s (M24359, X97047, X56494), Janus protein tyrosine kinase I (JAKI), (AJ000556, S63728), phosphatidylinositol 4-kinase, (D84667) and Rho kinase alpha (U38481, U58513, D87931).

7. Use according to claim 5, wherein the isolated gene sequence encodes an expression product or fragment thereof of diacylglycerol kinase (DGK-IV) (D78588, U51477), elk protein tyrosine kinase (XI 3411), putative cAMP-dependent protein kinase regulatory subunit RIalpha (AJ278429), casein kinase II beta subunit (L15619), ERK3, protein serine/threonine kinase, extracellular (M64301, X80692), and neural receptor protein-tyrosine kinase (trkB), (M55291, X17647).

8. Use according to claim 5, wherein the isolated gene sequence encodes an expression product or fragment thereof of A-raf oncogene, liver expressed (X06942, U01337).

9. Use according to any of claims 1-4 concerning an isolated gene sequence that encodes a phosphatase.

10. Use according to claim 9, wherein the isolated gene sequence encodes an expression product or fragment thereof of PP-1 alpha for a catalytic subunit of protein phosphatase type 1 alpha (D00859, U25809, X70848).

11. Use according to claim 9, wherein the isolated gene sequence encodes an expression product or fragment thereof of protein tyrosine phosphatase, striatum enriched, (S49400, U28216, U27831), protein phosphatase 1 (D90164, X80910),

protein tyrosine phosphatase (AJ007016, AF035644, U48297), putative receptor tyrosine phosphatase (U55057) and calcineurin, A subunit, beta isoform (M31809, M29550).

12. Use according to any of claims 1-4 concerning an isolated gene sequence that encodes a phosphodiesterase.

13. Use according to claim 12, wherein the isolated gene sequence encodes an expression product or fragment thereof of alkaline phosphodiesterase 1 ( (D28560, AF123542, D45421).

14. Use according to claim 12, wherein the isolated gene sequence encodes an expression product or fragment thereof of phosphodiesterase, cAMP-specific (PDE4D) (L27058, L20966).

15. Use according to any of claims 1-4 concerning an isolated gene sequence that encodes an ion channel protein.

16. Use according to claim 15, wherein the isolated gene sequence encodes an expression product or fragment thereof of voltage dependent anion channel 2 (AF268468, U30838, L06328).

17. Use according to any of claims 1-4 concerning an isolated gene sequence that encodes a receptor protein.

18. Use according to claim 17, wherein the isolated gene sequence encodes an expression product or fragment thereof of putative GABA-B1a receptor (AF114168), syntaxin 13 (AF044581) or neurotensin receptor type 2 (NTR2) (X97121).

19. Use according to claim 17, wherein the isolated gene sequence encodes an expression product or fragment thereof of neonatal glycine receptor NglyR (X57281) or syntaxin 13 (AF044581).

20. Use according to claim 17, wherein the isolated gene sequence encodes an expression product or fragment thereof of dopamine receptor D. sub. 1 (158000),

putative GABA-B1a receptor (AF114168) or putative interleukin 1 receptor accessory protein (X85999).

21. Use according to any of claims 1-4 concerning an isolated gene sequence that encodes a transporter protein.

22. Use according to claim 21, wherein the isolated gene sequence encodes an expression product or fragment thereof of adenine nucleotide translocator, mitochondrial (D12770), ATPase, calcium, plasma membrane, isoform 1 (J03753), calcium transporter CaT2 (AF209196) carnitine/acylcarnitine carrier protein (X97831), differentiation-associated Na-dependent inorganic phosphate cotransporter (AF271235), Na+, K±ATPase alpha-subunit (D00189), putative potent brain type organic ion transporter (AB012808, AB040056), synaptic vesicle transmembrane transporter (L01788), UDP-galactose transporter (D87991), amino acid system A transporter (AF249673), digoxin carrier protein (U88036), glycine transporter (L13600), putative vacuolar ATP synthase (U13837, AF113129) or synaptic vesicle transmembrane transporter (L01788).

23. Use according to claim 21, wherein the isolated gene sequence encodes an expression product or fragment thereof of choline transporter CHOT1 (X66494) or Na+K+ ATPase alpha+ isofonn catalytic subunit (M14512).

24. Use according to any of claims 1-4 concerning an isolated gene sequence that encodes a G-protein coupled receptor protein.

25. Use according to claim 24, wherein the isolated gene sequence encodes an expression product or fragment thereof of rab GDI, alpha species, ras related GTPase (X744020) and putative K-ras (M54968).

26. Use according to claim 25, wherein the isolated gene sequence encodes an expression product or fragment thereof of putative ras-related protein Rab-5c or ARF-like protein 5 ARL5 (X78604).

27. Use according to any of claims 1-4 concerning an isolated gene sequence that encodes a DNA binding protein.

28. Use according to claim 27, wherein the isolated gene sequence encodes an expression product or fragment thereof of histone, core, H2A. 1 (M99065), hypoxiainducible factor-l alpha (Hifla) (AF057308, AF003695, U22431), putative histone H3.3A (X91866, M11354), putative signal transducer and regulator transcription 1 (Statl) (U06924), transcription factor IIIC, alpha subunit (L28801) or putative chromodomain-helicase-DNA-binding protein.

29. Use according to any of claims 1-4 concerning an isolated gene sequence that encodes a protease protein.

30. Use according to claim 29, wherein the isolated gene sequence encodes an expression product or fragment thereof of putative Lon proteinase (X76040), carboxypeptidase D precursor (U62897, D85391, U65090), caspase 2 (Ich-l) (U77933), cathepsin B (X82396, M14222), cathepsin L (EC 34.22. 15) (Y00697, M20495), dipeptidylpeptidase (dpp6) (M76427, AF092507, M96859), proteasome subunit C8 (M58593, AF055983, D00762) or putative deubiquitinating enzyme 8 (AF057146 D29956).

31. Use according to any of claims 1-4 concerning an isolated gene sequence that encodes a ligase, lyase, oxidoreductase, transferase or hydrolase.

32. Use according to claim 31, wherein the isolated gene sequence encodes an expression product or fragment thereof of acyl-CoA synthetase II, brain (D360666), aldolase A (M12919, Y00516, M11560), aldose reductase, lens (X05884), aspartate aminotransferase, ATP synthase, H+, alpha subunit, mitochondrial (EC 3.6.

1.34) (X56133), branched-chain aminotransferase, cytosolic, brain (AF165887), cytochrome c oxidase polypeptide III (X14848), cytochrome-c oxidse II, mitochondrial (M64496), cytochrome-c oxidase I, mitochondrial (S79304),

dihydrolipoamide succinyltransferase (D90401), enolase, alpha alpha, non-neuronal (NNE) (X02610, X52379, M14328), enolase, neuron-specific (MI1931), FIFO ATPase delta subunit (U00926), fatty acid synthase (X62888), GM3 synthase (ABO 18049), heme-binding protein (HBP23) (D30035), lactate dehydrogenase-B (LDH-B) (U07181, X51905, Y00711), phosphofructokinase, muscle (PFK-M) (U25651, AF249894, Y00698), putative activation of Sentrin/SUMO protein AOS1 (AB024303), putative carbonic anhydrase XIV, putative cytochrome c oxidase VIB

(EC 1. 9. 3. 1) (X139230), putative NADH-ubiquinone oxidoreductase MLRQ subunit (U59509), putative NADH : ubiquinone oxidoreductasePGIV subunit (AF044953), putative ribonuclease III (AF116910), putative seryl-tRNA synthetase (X91257), putative succinate dehydrogenase flavoprotein (AF095938, AF171022), putative ubiquinol-cytochrome-c reductase (EC 1.10. 2.2) core protein II (J04973), putative ubiquitin-conjugating enzyme (EC 6.3. 2.-) E2 (E 12457), RNA polymerase II (AB017711), squalene synthetase, hepatic (M95591), sulfotransferase-like protein (AF188699), superoxide dismutase, copper-zinc (SOD-1) (M21060, X05634, K00065), UDP-Gal: glucosylceramide beta-1, 4-galactosyltransferase (AF048687), 3hydroxy-3-methylglutaryl coenzyme A reductase (M29249, M11058), arginase II (U90887, U90886), ATP synthase gamma chain, mitochondrial (LI 9927, D 16563), N-G, N-G-dimethylarginine dimethylaminohydrolase (D86041), NADH subunit 5, mitochondrial (X14848), putative glucocerebrosidase (M24119), putative Nacetylglucosamine-6-sulfatase (Z 12173), putative tryptophanyl-tRNA synthetase (X69656), putative ubiquitin-conjugating enzyme (Y17267) or round spennatid protein RSP29 (U97667).

33. Use according to claim 31, wherein the isolated gene sequence encodes an expression product or fragment thereof of 3-hydroxy 3-methylglutaryl coenzyme A synthase, cytosolic (X52625), aspartate aminotransferase, cytosolic (J04171), cytosolic malate dehydrogenase (Mdh) (AF093773, M29462, D55654), diacylglycerol kinase (DGK-IV) (D78588), putative dihydropyrimidinase related protein (D78013), ribophorin I (X05300), Peptidylglycine alpha-amidating

monooxygenase (M25719, U79523, M37721), squalene epoxidase (D37920), stearoyl-coA desaturase 2 (AB032243, M26270), erythroid-specific (D86297).

34. Use according to claim 31, wherein the isolated gene sequence encodes an expression product or fragment thereof of famesyl diphosphate synthetase (M34477),

glutamine synthetase (EC 6. 3. 1. 2.) (M91652), delta-aminolevulinate synthase, polypeptide GalNAc transferase Tl (U35890), and putative NAD+ ADP- ribosyltransferase (AJ007780).

35. An animal for use in the screening of compounds that are effective in the treatment of pain, or in the diagnosis of pain and having in its genome an introduced gene sequence or a removed or down-regulated sequence that is differentially expressed in neuronal tissue in response to a nociceptive stimulus.

36. The animal of claim 35, wherein the introduced gene sequence is as defined in any of claims 2-34.

37. The animal of claim 35 or 36 which is C. elegans.

38. A kit which comprises; (a) a plurality of affinity peptides, ligands or substrates for an expression product of a gene sequence that is differentially expressable in neuronal tissue in response to a nociceptive stimulus; and (b) a defined quantity of an expression product of a gene sequence that is differentially expressable in neuronal tissue in response to a nociceptive stimulus.

39. The kit of claim 38, wherein the gene sequence is as defined in any of claims 1-34.

40. A kit that comprises:

(a) a plurality of complimentary nucleic acids which possesses affinity to a the nucleic acid sequence that is differentially expressable in neuronal tissue in response to a nociceptive stimulus; and (b) a defined quantity of one or more complimentary nucleic acids that possesses affinity to any one of the nucleic acid sequences outlined above.

41. The kit of claim 40, wherein the gene sequence is as defined in any of claims 1-34.

42. A compound that is useful in the treatment or diagnosis of pain and that modulates the action of an expression product of a gene sequence that is differentially expressible in neuronal tissue in response to a nociceptive stimulus.

43. The compound of claim 42 that modulates the action of an expression product of a gene sequence as defined in any of claims 2-34.

44. A pharmaceutical composition comprising an effective amount of the compound of claim 42 or 43 and a pharmaceutically acceptable carrier or diluent.

45. Use of a compound as defined in claim 42 or 43 for the manufacture of a medicament for the treatment or diagnosis of pain.

46. Use of a compound as defined in claim 42 or 43 for the manufacture of a medicament for the treatment or diagnosis of chronic pain.

47. A method of treatment of pain, which comprises administering to a patient an effective amount of a compound as defined in claim 42 or 43.