BE1014938A4 - Sequence dna encoding protein sodium channel, its production and use. - Google Patents

Abstract

The invention describes the isolation of a novel nucleic acid sequence encoding a potential-dependent mammalian sodium channel, preferably resistant to tetrodotixin. It also describes polypeptide products of recombinant expression of these sequences, expression vectors comprising the DNA sequence and host cells transformed by these expression vectors. Other aspects of the present invention are peptides whose sequences are based on the amino acid sequences deduced from these DNA sequences, antibodies specific for these proteins and peptides, methods for detection and quantitative determination such proteins and corresponding nucleic acids. Another aspect of the invention is the use of this potential-dependent sodium channel, preferably resistant to tetrodotoxin, as a therapeutic target for compounds.

Description

       

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  DNA sequence coding for a sodium channel protein, its production and use
The present invention relates generally to sodium channel proteins and more particularly to a novel nucleic acid sequence encoding a mammalian subunit of a sodium channel protein of nervous tissue, dependent on the potential, preferably resistant. with tetrodotoxin. The present invention further relates to its production by the recombination technique.



   The basic unit of information transmitted from one part of the nervous system to another is a single action potential or nerve impulse. The "transmission line" for these impulses is the axon or nerve fiber. The electrical excitability of the nerve membrane has been shown to depend on the tension-sensitive ion permeability system of the membrane, which allows it to use the energy stored in ion concentration gradients. The electrical activity of the nerve is triggered by a depolarization of the membrane, which opens through the membrane channels which are highly selective for sodium ions, which are then driven inward by the electrochemical gradient. Among the many ion channels, the potential dependent or potential sensitive sodium channel is one of the most studied.

   It's about

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 of a transmembrane protein which is essential for the production of action potentials in excitable cells. An excellent review article on ion channels is given by Catterall, TINS 16 (12), 500-506 (1993).



   The cDNAs for several Na + channels have been cloned and sequenced. Numa et al., Annals of the New York Academy of Sciences 479,338-355 (1986), describe one cDNA from the electrical organ of the eel and two different cDNAs from the rat brain. Rogart, US-A-5 380 836, describes a cDNA from rat heart tissue; also Rogart et al., Proc. Natl.



  Acad. Sci. 86.8170-8174 (1989). The sequences of PN1 and its orthologs in humans (hNE) and rabbits (Na +) have been published [see for example Klugbauer et al., EMBO J. 14, 1084-1090 (1995) and Belcher et al., proc.



  Natl. Acad. Sci. USA 923, 11034-11038 (1995)]. The PN1 sequence of rat cloned from dorsal root ganglia (GRD) and its expression of function have been described [see for example Sangameswaran et al., J.



  Biol. Chem. 272.14805-14809 (1997)]. Other cloned sodium channels include types I and II [Noda et al., Nature 320,188-192 (1986)], IIa [Auld et al., Neuron 1,449-461 (1988)] and III [Kayano et al., FEBS Lett. 228,187-194 (1988)] of rat brain, skeletal muscle sodium channels (SkMl) [Trimmer et al., Neuron 3, 33-49 (1989)], Rat NaCh6 [Schaller et al., J. Neurosci . 15, 3231-3242 (1995)], the sodium channel type III of the peripheral nerve (rPN3) [Sangameswaran et al., J. Biol. Chem. 271.5953-5956 (1996), also known as SNS, Akopian et al., Nature 379,257-262 (1996)], the atypical rat channel [Felipe et al., J.



  Biol. Chem. 269.30125-30131 (1994)] and the rat glial sodium channel [Akopian et al., FEBS Lett. 400, 183-187 (1997)].

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   These studies showed that the amino acid sequence of the sodium channel had been preserved for a long period of evolution. These studies also revealed that the channel was a single polypeptide containing four internal repeat sequences, or homologous domains (domains I-IV) having similar amino acid sequences. Each domain folds into six helical and predicted transmembrane segments: five are hydrophobic segments and one is a highly charged segment, with many positively charged lysine and arginine residues. This highly charged segment is the fourth transmembrane segment in each domain (the S4 segment) and is likely to be involved in activation by potential.

   The side chains positively charged on the S4 segment are likely to pair with the side chains negatively charged on the other five segments, so that depolarization of the membrane can displace the position of a helix relative to the other, which opens the channel. Accessory sub-units can modify the function of the channel.



   The therapeutic utility of recombinant materials from the DNA of many sodium channels has been discovered.



  For example, US-A-5,132,296 (Cherksey) describes purified sodium channels which have been found useful as diagnostic and therapeutic tools.



   Sodium channel isoforms are divided into "subfamilies". The term "isoform" is used to denote proteins of distinct but closely related sodium channels, ie those having an amino acid homology of about 60-80%. The latter also show a strong homology of functions. The term "subfamilies" is used to denote distinct sodium channels which have an amino acid homology of about 80-95%. Combinations of multiple factors are used to determine distinctions within

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 of a subfamily, for example, the speed of a channel, the chromosomal location, the expression data, the homology with other channels within the same species, and the homology with a channel of the same subfamily between different species.

   Another consideration is an affinity for tetrodotoxin ("TTX"). TTX is a very powerful toxin from the tetrodon, or puffer fish, which blocks the transmission of nerve impulses along the axons and in the excitable membranes of nerve fibers. TTX attaches to the sodium channel and stops the flow of sodium ions.



   Studies using TTX as a probe have shed much light on the mechanism and structure of sodium channels.



  There are three subtypes of sodium channels which are defined by the affinity for TTX, which can be measured by the values of the IC 50: sodium channels sensitive to TTX (CI50 = 1-30 nM), sodium channels insensitive TTX (Ci 50 = 1-5 M and sodium channels resistant to TTX (Ci 50 50 M).



   The action potentials insensitive to TTX were first studied in rat skeletal muscle (Redfern et al., Acta Physiol. Scand. 82, 70-78 (1971)].



  These action potentials have then been described in other mammalian tissues, including newborn mammalian skeletal muscle, mammalian heart muscle, mouse dorsal root ganglion cells in vitro and in culture, L6 cells and mammalian skeletal muscle in culture [see Rogart, Ann. Rev. Physiol. 43, 711-725 (1980)].



   The neurons of the rat dorsal root ganglia have both sodium channel currents sensitive to TTX (CI 50 ¯ 0.3 nM) and sodium channel currents resistant to TTX (CI 50 ¯ 100 M), as described by Roy et al. in J. Neurosci. 12, 2104-2111 (1992)]. We also measured sodium currents

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 resistant to TTX in petrous ganglia and plexiform ganglia of rat (see Ikeda et al., J. Neurophysiol. 55, 527-539 (1986) and Stea et al., Neurosci.



  47,727-736 (1992). Electrophysiologists believe that another sodium channel resistant to TTX remains to be discovered.



   Although cDNAs from brain, heart, and rat skeletal muscle are known, the identification and isolation of cDNA from peripheral sensory nerve tissue, such as root ganglia dorsal, were hampered by the difficulty of working with such a fabric.



   The present invention provides novel, purified and isolated nucleic acid sequences encoding sodium channel proteins from nerve tissue, preferably TTX-resistant, which are highly expressed in plexiform ganglia and root ganglia. dorsal fin of the adult, less strongly expressed in the brain, the spinal cord and the superior cervical ganglia, and are not expressed in the scientific nerve, the heart or the skeletal muscle. In currently preferred forms, the new DNA sequences include cDNA sequences encoding the sodium channel protein of rat nerve tissue. One aspect of the present invention is the α subunit of this sodium channel protein.



   A subject of the invention is also DNA, cDNA and mRNA derived from the nucleic acid sequences of the invention, and cRNA derived from mRNA. In particular, two cDNA sequences together encode the entire sodium channel of rat nerve tissue.



   The present invention also encompasses other forms of DNA, such as genomic DNA, DNA prepared by partial or total chemical synthesis from nucleotides, and DNA having deletions or

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 mutations.



   Yet another aspect of the invention is the novel rat sodium channel protein, resistant to TTX, and fragments thereof encoded by the DNA of the present invention.



   Another aspect of the present invention consists of recombinant polynucleotides and oligonucleotides comprising a nucleic acid sequence derived from the DNA sequence of the present invention.



   Another aspect of the invention is a method of stabilizing the whole cDNA which codes for the protein sequence of the invention.



   Another aspect of the invention includes expression vectors comprising the DNA of the invention, host cells transformed or transfected with these vectors and a cDNA library of these host cells.



   The present invention also encompasses an assay for the detection of inhibitors of the potassium channel protein, comprising contacting a compound, suspected to be an inhibitor, with an expressed sodium channel, and measuring the activity of the channel. sodium.



   A method of inhibiting the activity of the TTX-resistant sodium channel is further provided, comprising administering an effective amount of a compound having an IC50 value of 10 M or less.



   In addition, there are provided methods of using DNA for the production of monoclonal and polyclonal antibodies, for use as molecular targets for drug discovery, as highly specific markers of specific antigens, as detecting molecules, in diagnostic tests, and for therapeutic uses, such as pain relief, as a PN5 channel probe in other mammalian tissue, in the design of therapeutic agents and in the screening of

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 therapies.



   The present invention is illustrated with the aid of the accompanying drawings. In these drawings:
FIGS. 1A-E represent the native cDNA sequence of 5,908 nucleotides encoding the rat type 5 sodium channel ("PN5") (SEQ ID No. 1), derived from two overlapping cDNA clones, designated by 26. 2 and 1.18.



   FIGS. 2A-F represent the amino acid sequence deduced from PN5 (SEQ ID No. 2, represented in the three-letter amino acid code). Figures 2G-H, showing the amino acid sequence deduced from PN5 in the single letter amino acid code, also show the homologous domains (I-IV); the supposed transmembrane segments (S1-S6); the amino acid conferring resistance to TTX (4); N-glycosylation sites (#); the cAMP-dependent protein kinase A (PKA) phosphorylation site and the termination codon (*).



   Figure 3A shows a sequence of 856 base pairs for human PN5 (SEQ ID No. 3). Figure 3B shows the amino acid sequence comparison of the hPN5 fragment with the rat PN5.



   FIG. 4 represents the sequence of the new sodium channel IV domain probe (SEQ ID No. 4).



   Figures 5A-E show the sequence of 5,334 nucleotides modified for stability and expression (SEQ ID No. 5). Nucleotides 24 to 5,518 constitute the region of 5,295 bp encoding a protein with 1,765 amino acids.



   Figure 6 shows the PNS cloning map.



   The present invention relates to a purified and isolated nucleic acid sequence encoding a novel mammalian sodium channel protein, preferably resistant to TTX. The term "purified and isolated DNA" means DNA which is essentially free, i.e. contains less than about 30, preferably less than about

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 ron 10%, and even better preferably less than about 1%, of the DNA with which the DNA of interest is associated in the natural state. Techniques for determining purity are well known in the art and include, for example, restriction mapping, agarose gel electrophoresis and CsCl gradient centrifugation.



   The term "DNA" is intended to include cDNA, or complementary DNA, which consists of single-stranded or double-stranded DNA sequences produced by reverse transcription of mRNA isolated from a donor cell or by synthesis chemical. For example, treatment of mRNA with a reverse transcriptase, such as AMV reverse transcriptase or M-MuLV reverse transcriptase, in the presence of an oligonucleotide primer, will give an RNA-DNA duplex which can be treated with RNase H, DNA polymerase and DNA ligase, to give double stranded cDNA. If desired, the double-stranded cDNA can be denatured by conventional techniques, such as heating, to give single-stranded cDNA.

   The term "cDNA" includes DNA which is a complementary copy of naturally occurring mRNA, as well as complementary copies of naturally occurring variants of mRNA, which have the same biological activity. Variants include, for example, insertions, deletions, degenerate codon sequences and alleles.



   A "cRNA" corresponding to the mRNA transcribed from a DNA sequence coding for the α subunit of a new sodium channel protein, preferably resistant to TTX, is envisaged by the present invention. .



  The term "cRNA" refers to RNA which is a copy of the mRNA that is cut by a cell.



   In particular, the invention encompasses N comprising the native versions of the nucleotide sequences indicated in FIGS. 1A-E (SEQ ID No. 1), designated here by type 5 sodium channel (PN5). Figures 1A-E show-

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 sense the 5,908 nucleotide cDNA construct, comprising an open reading frame of 5,298 bases (including the stop codon) (SEQ ID No. 1).



  Nucleotide residue 79 represents the site of initiation and translation, and residue 5367 represents the end of the stop codon.



   The invention also encompasses modified versions of PN5, and in particular the version shown in Figures 5A-E (SEQ ID No. 5). The SalI-XbaI clone of 5,334 nucleotides lacks most of the untranslated sequences, from the open reading frame of 5,298 nucleotides starting from nucleotide 24 and ending at nucleotide 5321. The initiation codons and d termination is emphasized, as are translationally silent mutations at nucleotides 3932,3935, 3941, 3944 and 3947, which have been introduced to block a rearrangement in this region during growth in E. coli.



   The nucleotide sequence of SEQ ID No. 1 (Figures 1A-E) corresponds to rat cDNAs. Homology research showed that the most closely related sodium channel was in the rat heart channel, with 72.5% homology. The most closely related channels that follow are rPNl, with 72%, and types I and III of rat brain, with 71.8% and 71.3%, respectively. Homologies with rPN3a, hPN3, rPN4, rPN4a, rat brain type II and rat skeletal muscle each range from about 70 to 71%.



   In addition, a clone of 856 base pairs (SEQ ID No. 3), as represented in FIG. 3A, was isolated from a cDNA library of human sleeping root ganglia (GRD), and is closely related to the rat PN5 amino acid sequence, with 79% identity and 86% homology. The human PN5 sequence covers the region between IIIS1 and interdomain III / IV, which

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 includes the rapid inactivation valve (i.e. IFM) which is located in the interdomain III / IV.



   The term "cDNA library" refers to a collection of clones, usually in a bacteriophage or, less commonly, in bacterial plasmids, containing cDNA copies of mRNA sequences from a donor cell or tissue giver.



   Additional counterparts of the new TTX-resistant rat sodium channel described herein are also believed to be expressed in other mammalian tissue.



   Northern blot analysis (Example 5) indicates that PN5 is coded by a transcript of - 6.5 kb.



   The amino acid sequence deduced from PN5, represented in FIGS. 2A-F (SEQ ID No. 2), manifests the primary structural characteristics of an α subunit of a potential-dependent sodium channel resistant to TTX . Figures 2G-H show the homologous domains (I-IV); the supposed transmembrane segments (S1-S6); the amino acid conferring resistance to TTX (#); N-glycosylation sites (#); and cAMP-dependent PKA phosphorylation sites (o). DNA sequences coding for identical sodium channel protein polypeptides, analogs, or allelic variants of the nervous system, by use, at least in part, of degenerate codons, are also contemplated by the present invention.



   An interesting feature of this deduced amino acid sequence is that the amino acid which is most responsible for sensitivity to TTX is located at position 355 and is not aromatic. In the human brain and rat-type sodium channels, in the skeletal muscle channel, and in PN1 and PN4, this amino acid is tyrosine or phenylalanine, and these channels are all sensitive to TTX. In PN3 and PN5, the amino acid is serine. Since PN3 is very resistant to TTX, the implication is that PN5 is equally

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 a TTX resistant channel. The heart channel has a cysteine residue at this position and is "insensitive" to TTX.



   Although PN5 contains all of the distinguishing features of a potential-dependent sodium channel, it has specific structural characteristics that distinguish it from other sodium channels. For example, DIIS4 contains 5 basic amino acids conserved in all sodium channels which could play a significant role in the potential detection aspects of channel function. In PN5, the first basic amino acid is replaced by an alanine residue. Likewise, in DIIIS4, PN5 has 5 basic amino acids instead of the six that are present in other sodium channel sequences, the last arginine residue being replaced by a glutamine residue. In DIIIS3, the transmembrane segment contains only 18 amino acids, as opposed to 22 amino acids in the other channels.

   In addition, the short link loop (4 amino acids) between S3 and S4 in DIII is even shorter, due to a deletion of 3 amino acids. This shortening of S3 and the binding loop was confirmed by the design of primers in the appropriate region of the sequence for an RT-PCR (polymerase chain reaction-reverse transcription) assay from of rat GRD, and sequencing of the amplified DNA fragment. Such an assay was performed to confirm the sequence of another region of PN5, in the DIVS5-S6 loop, where there was a deletion of an 8 amino acid peptide.



   An RNA tissue distribution analysis was performed, by a polymerase-reverse transcription amplification chain reaction (RT-PCR primed by oligonucleotide), from the peripheral and central nervous systems of rats, in particularly from rat GRD. Eight main types of tissue were studied for

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 expression of the specific PN5 genes corresponding to positions 5651-5903 of SEQ ID No. 1 (FIGS. 1A-E). PN5 mRNA was present in five of the tissues studied: the brain, spinal cord, GRD, plexiform ganglia and upper cervical ganglia. PN5 was not present in the remaining tissues studied: sciatic nerve tissue, skeletal or cardiac muscle tissue.



  PN5 was found to be the strongest in GRD and plexiform glands, which led applicants to believe that GRD was enriched in PN5. PN5 has considerable differences in abundance among a set of tissues. PN5 has an expression gradient with strong expression in the GRDs. PN5 has an expression gradient like the other channels, but a more limited distribution.



   The invention not only includes the whole protein expressed by the cDNA sequences of SEQ ID Nos. 1, 2 and 3, but also includes protein fragments. These fragments can be obtained by cutting whole proteins or by using shorter DNA sequences or shorter polynucleotides to express the desired fragment.



   As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, whether ribonucleotides or deoxyribonucleotides. This term designates only the primary structure of the molecule. Thus, this term includes single strand DNA and double strand DNA, as well as double strand RNA and single strand RNA. It also includes modified forms, for example by methylation and / or styling, and unmodified forms of the polynucleotide.



   Furthermore, the term "polynucleotide" and intended to include a recombinant polynucleotide which is of genomic, cDNA, semi-synthetic or synthetic origin, which,

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 by virtue of its origin or of manipulation, is not associated with all or part of the polynucleotide with which it is associated in nature and / or is linked to a polynucleotide other than that to which it is linked in nature.



   Accordingly, the invention also includes polynucleotides which can be used to produce polypeptides having a length of about 10 to 1,500, preferably 10 to 100 amino acids. The isolation and purification of such recombinant polypeptides can be carried out by techniques which are well known in the art, for example preparative chromatographic separations or affinity chromatography.

   In addition, polypeptides can also be produced by synthetic means which are well known in the art.
The invention allows the manipulation of genetic materials by recombinant techniques, to produce polypeptides which have the structural and functional characteristics of the new potential-dependent, TTX-resistant sodium channel subunit a found in sensitive nerves. Site-directed mutagenesis can be used to obtain such recombinant polypeptides. For example, synthetic oligonucleotides can be specifically inserted, or placed specifically in place of existing oligonucleotides, in the segment of the gene of interest, to produce genes encoding and expressing a specific mutant.

   It is also possible to insert degenerate oligonucleotides at random and phage visualization techniques can be used to identify and isolate polypeptides having a functional property of interest.



   In addition, the present invention contemplates recombinant polynucleotides having a length of about 15 to 20 kb, preferably 10 to 15 kb, comprising a

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 nucleic acid sequence derived from the DNA of the invention.



   The term "derived" from a designated sequence refers to a nucleic acid sequence which is composed of a sequence of at least 6 to 8 nucleotides, preferably at least 10 to 12 nucleotides and particular of at least 15 to 20 nucleotides which correspond to a region of the designated sequence, that is to say are homologous or complementary thereto. The derived sequence is not necessarily physically derived from the indicated nucleotide sequence, but may be derived in some other way, including, for example, chemical synthesis or DNA replication or reverse transcription, techniques which are based on the information provided by the basic sequences in the region or regions from which the polynucleotide is derived.



   A neonatal expression test was carried out with Fil, a fusion cell line designed from GRD of newborn rats, welded to a mouse cell line, N18TG, from Massachusetts General Hospital. Fil responds to trophic agents, such as NGF (neuron growth factor), by extension of dendrites. PN5 was found to be present in both native wire and NGF-treated wire, which led applicants to believe that the sodium channel was expressed natively in wire.



   In situ hybridization of PN5 mRNA with rat GRD tissue gives localization mainly in small and medium neurons, with an absence in large neurons.



   PN5 was also mapped to its cytogenetic localization on mouse chromosome preparations. PN5 is located on the same chromosome as the heart channel and PN3.



   In general, the sodium channels include a subunit a and two subunits. The sub-units (3

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 can modulate the function of the channel. However, since the α subunit is all that is required for the channel to be fully functional, expression of the cDNA in SEQ ID No. 1 (Figures 1A-E) will give a fully functional protein. The gene encoding the ss1 subunit in peripheral nerve tissue has been shown to be identical to that found in the rat heart, brain and skeletal muscle. The subunit 1 cDNA is not described here, since it is well known in the art [see Isom et al., Neuron 12, 1183-1194 (1994)].

   However, it should be understood that by combining the known sequence coding for the / 3 subunit with the sequence of the a subunit described herein, it is possible to obtain a full PN5 sodium channel, dependent on the potential, preferably resistant to TTX.



   The present invention also includes expression vectors comprising the DNA or cDNA described above, host cells transformed by these expression vectors, capable of producing the sodium channel of the invention, as well as libraries cDNA comprising such host cells.



   The term "expression vector" means any genetic element, for example a plasmid, a chromosome, a virus, behaving as an autonomous expression unit of polynucleotide in a cell or being made capable of replication by insertion into a chromosome of a host cell, and to which another polynucleotide segment is attached, so as to cause replication and / or expression of the attached segment. Suitable vectors include, but are not limited to, plasmids, bacteriophages and cosmids. The vectors will contain the polynucleotide sequences which are necessary to perform ligation or insertion of the vector into a desired host cell and to effect expression of the attached segment.

   Such sequences differ

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 function according to the host organism or will include promoter sequences to effect transcription, activator sequences to enhance transcription, ribosome binding site sequences and transcriptional and translational termination sequences.



   The term "host cell" generally refers to prokaryotic or eukaryotic organisms and includes any organism capable of transformation or transfection, which is capable of expressing a protein and can be, or has been, used as recipient of expression vectors or other transferred DNA. Host cells can also be forced to express a protein by direct injection of exogenous cRNA, which can be translated into the protein of interest. A preferred host cell is the Xenopus oocyte.



   The term "transformed" refers to any known method for the insertion of foreign DNA or RNA sequences into a prokaryotic host cell. The term "transfected" refers to any known method for the insertion of foreign DNA or RNA sequences into a eukaryotic host cell. Such transformed or transfected cells include transformed or stably transfected cells, in which the inserted DNA is made capable of replication in the host cell. They also include temporarily expressing cells, which express inserted DNA or RNA for limited periods of time. The technique of transformation or transfection depends on the host cell that is transformed.

   It can include the encapsidation of the polynucleotide in a virus as well as the direct introduction of the polynucleotide, such as for example by lipofection or micro-injection. Transformation or transfection can result in the incorporation of DNA inserted into the host cell genome or the maintenance of DNA inserted in the host cell under

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 form of plasmid. Transformation methods are well known in the art and include, but are not limited to, viral infection, electroporation, lipofection and direct introduction caused by calcium phosphate.



   It should be understood that the present invention is intended to include other forms of expression vectors, host cells and transformation techniques serving equivalent functions and well known in the art.



   The invention also relates to a test for inhibitors of the new sodium channel protein resistant to TTX, comprising contacting a compound, suspected of being an inhibitor, with an expressed sodium channel, and the measurement of the sodium channel activity. The compound can be a practically pure compound of synthetic origin, brought into contact in an aqueous medium, or the compound can be a substance existing in nature, so that the test medium is an extract of origin biological, such as an extract from plant, animal or microbial cells.

   PN5 activity can be measured by methods such as electrophysiology (energizing using two electrodes or energizing by patch clamp of whole cells using a single electrode), tests guanidinium ion flow and toxin binding assays. An "inhibitor" is generally defined by the amount causing a decrease of more than 50% in PNS activity, preferably a decrease of more than 70% in PN5 activity, even better a decrease of more than 90 % of PN5 activity.



   There are many uses of the invention, some of which are described below: 1. Probe for mammalian canals
As mentioned above, additional homologues of the new rat sodium channel are believed to be

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 resistant to TTX, described here, are also expressed in mammalian tissue, particularly in human tissue. Whole cDNAs of rat PN5 sodium channels of the present invention can be used as a probe to discover whether there are additional potential-dependent, preferably TTX-resistant, new sodium channels in human tissue and, if exist to facilitate the isolation of cDNAs encoding human protein.



   Human counterparts of rat PN5 channels, resistant to TTX, can be cloned using a human GRD cDNA library. Human GRDs are obtained during an autopsy. The frozen tissue is homogenized and the RNA is extracted with guanidine isothiocyanate [Chirgwin et al., Biochemistry 18, 5294-5299 (1979)].



  The RNA is fractionated according to the size on a sucrose gradient, for enrichment in large mRNA, because the a subunits of the sodium channel are encoded by large transcripts (7-11 kb ). Double stranded cDNA is prepared using the SuperScript Choice cDNA kit (GIBCO BRL) with oligo (dT) or random hexamer primers. The EcoRI linkers are ligated onto the double stranded cDNA, which is then phosphorylated. The cDNA library is constructed by ligation of the double-stranded cDNA into the bacteriophage X ZAP II vector (Stratagene), followed by packaging into phage particles.



   The phages are spread on 150 mm plates, on a carpet of XLI-Blue MRF 'bacteria (Stratagene) and the plates are replicated on Nylon Hybond N membranes (Amersham). The filters are hybridized with rat PN5 cDNA probes, by conventional techniques, and detected by autoradiography or chemiluminescence. The signal produced by rat PN5 probes hybridizing with positive human clones, at a high

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 stringency, should be stronger than those obtained with rat brain sodium channel probes hybridizing with these clones. The positive plaques are further purified by limiting dilution and screened again by hybridization or PCR.

   Restriction mapping and polymerase chain reaction will identify overlapping clones which can be assembled by standard techniques in the complete human homolog of rat PNS. The human clone can be expressed by injection of cut-off cRNA in vitro from the complete cDNA clone into Xenopus oocytes, or by transfection of a mammalian cell line with a vector containing the cDNA linked to an appropriate promoter.



  2. Antibodies to PN5
The polypeptides of the invention are very useful for the production of antibodies against PNS. These antibodies can be used in affinity chromatography to purify recombinant proteins or recombinant sodium channel polypeptides, or they can be used as a research tool. For example, antibodies attached to a reporter molecule can be used in histochemical staining techniques, to identify other tissues and other cell types in which PN5s are present, or they can be used to identify functional regions or epitopes of the sodium channel protein of the invention.



   Antibodies can be monoclonal or polyclonal and can be produced by techniques which are well known in the art. Polyclonal antibodies are produced as follows: uses an immunogenic conjugate, comprising PN5 or a fragment thereof, optionally linked to a carrier protein, to immunize a selected mammal, such as mouse, rat, goat, etc. . We collect and process according to known techniques

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 serum from the immunized mammal, to separate the immunoglobulin fraction.



   Monoclonal antibodies are produced by the classical hybridoma technique, based on that reported by Kohler and Milstein in Nature 256,495-497 (1975). Spleen cells are obtained from a host animal immunized with the PN5 protein or a fragment thereof, possibly linked to a carrier substance. Hybrid cells are formed by fusion of these spleen cells with an appropriate myeloma line, and cultured. The antibodies produced by the hybrid cells are screened for their ability to bind to expressed PN5 proteins.



   A number of screening techniques well known in the art can be used, such as, for example, direct or indirect immunoenzymatic screening methods using an adsorbed body. The hybrid cells producing such antibodies are then subjected to recloning or to highly diluted conditions, in order to select a hybrid cell secreting a homogeneous population of antibodies specific for one or other protein of PN5.



   In addition, antibodies can be raised by cloning and expression of nucleotide sequences or mutated versions thereof, coding at least for the amino acid sequences required for the specific binding of natural antibodies, and these expressed proteins can be used as immunogens. Antibodies may include the complete immunoglobulin or a fragment thereof. Antibodies can be linked to a reporter group, as described above with regard to polynucleotides.



   Example 10 illustrates the practice of producing an antibody.

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  3. Therapeutic targets for compounds for the treatment of disorders and tests thereof
The present invention also encompasses the use of the novel potential-dependent sodium channel a subunit, preferably resistant to TTX, as a therapeutic target for compounds for the treatment of nervous system disorders, based on RT-PCR location data. The disorders include, but are not limited to, epilepsy, brain injury, brain injury, diabetic neuropathy, traumatic injury, chronic neuropathic pain and neuropathy associated with AIDS.



  4. Design of therapeutic agents based on inhibitor PN5 and tests thereof
The present invention is also directed to the inhibition of PN5 activity in tissues of the brain, spinal cord, dorsal root ganglia, plexiform ganglia and upper cervical ganglia. However, it should be understood that further studies may reveal that PN5 is present in other tissues, and, as such, these tissues may also be target areas. For example, detection of PN5 mRNA in plexiform lymph nodes suggests that PN5 may conduct TTX-resistant sodium currents in these lymph nodes and other sensitive ganglia in the nervous system.



   In addition, it has been found that proteins which are not normally expressed in certain tissues are expressed in a pathological condition. Accordingly, the present invention is intended to encompass the inhibition of PN5 in tissues and cell types in which the protein is normally expressed, and in tissues and cell types in which the protein is expressed only during a medical condition.

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   For example, sodium channels resistant to TTX are thought to play a key role in the transmission of nerve impulses in connection with sensory signals such as pain and pressure. This information will facilitate the design of therapeutic agents that can be targeted to a specific area, such as peripheral nervous tissue.



   The recombinant protein of the present invention can be used for the testing of potential therapeutic agents having the ability to inhibit the sodium channel of interest. In particular, it would be useful for selectively inhibiting the function of sodium channels in peripheral nerve tissues responsible for the transmission of pain and pressure signals without at the same time affecting the function of sodium channels in other tissues, such as the heart and the muscle. Such selectivity would allow the treatment of pain without causing side effects due to cardiac or neuromuscular complications. Consequently, it would be useful to have DNA sequences encoding sodium channels selectively expressed in peripheral nervous tissue.



  5. Analgesic
The sodium channels in the peripheral nervous tissue play an important role in the transmission of nerve impulses, and are therefore a tool for understanding the transmission of neuropathic pain. Neuropathic pain is divided into two components: allodynia, in which a normally non-painful stimulus becomes it, and hyperalgesia, in which a normally normally painful stimulus becomes extremely painful.



   In tissue localization studies, PN5 mRNA is found in small and medium-sized GRD neurons. PN5 mRNA is also found in the brain and spinal cord. The inhibition of its activi-

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 tees can help prevent disorders such as headaches and migraines. The ability to inhibit the activity of these sodium channels, that is, to reduce the conduction of nerve impulses, will affect the ability of the nerve to transmit painful impulses. Selective inhibition of the sodium channels in sensitive neurons such as GRD will allow the arrest of painful impulses without undesirable side effects caused by the inhibition of the sodium channels in other tissues such as the brain and the heart.

   In addition, some diseases are caused by sodium channels which produce influxes at an extremely high frequency. The possibility of reducing the activity of the canal can then eliminate or alleviate the disease. Consequently, potential therapeutic compounds can be screened by methods well known in the art, to discover whether they can inhibit the activity of the recombinant sodium channel of the invention [Barram M. et al. , Naun-Schmiedeberg's Archives of Pharmacology 347, 125-132 (1993), and McNeal ET et al., J. Med. Chem. 28, 381-388 (1985)]. For similar studies with the acetylcholine receptor, see Claudio et al., Science 238, 1688-1694 (1987).



   For example, pain can be relieved by inhibiting the activity of the new sodium channel, preferably resistant to TTX, by administering a therapeutically effective amount of a compound having a CI 50 of about 10 M or less, preferably 1 M. Potential therapeutic compounds are identified based on their ability to inhibit PN5 activity. Therefore, the above-mentioned assay can be used to identify compounds having a therapeutically effective IC 50.



   The term "IC50" designates the concentration of a compound which is required to inhibit the expressed PN5 activity by 50%, when the activity is measured by electro-

  <Desc / Clms Page number 24>

 physiology, flow tests and toxin binding tests, as mentioned above.



  6. Diagnostic tests
The basic molecular biology techniques used in the practice of the present invention, such as the isolation of RNA, DNA and plasmids, restriction enzyme digestion, the construction and probing of cDNA library, sequencing of clones, construction of expression vectors, transformation of cells, maintenance and development of cell cultures and other general techniques are well known in the art , and descriptions of these techniques can be found in general laboratory manuals, such as Molecular Cloning: A Laboratory Hanual by Sambrook et al. (Cold Spring Harbor Laboratory Press, 2c edition, 1989).



   For example, the polynucleotides of the invention can be linked to a "reporter molecule", for the formation of a polynucleotide probe useful for northern and southern blot analyzes and in situ hybridizations.



   The expression "reporter molecule" designates a chemical entity which can be detected by an appropriate detection means, comprising, but not limited to these, spectrophotometric, chemiluminescent, immunochemical or radiochemical means. The polynucleotides of the present invention can be conjugated to a reporter molecule by techniques well known in the art. Normally, the reporter molecule contains a functional group suitable for attachment to, or incorporation into, the polynucleotide. Functional groups suitable for attachment of the reporter group are usually activated esters or alkylating agents. Technical details for attaching reporter groups are well known in the art.

  <Desc / Clms Page number 25>

 domain; see for example Matthews J.

   A., Batki A., Hynds C. and Kricka L.J., Anal. Biochem. 151, 205-209 (1985) and Engelhardt et al., EP-A-0 302 175.



   The present invention is illustrated by the following descriptive and nonlimiting examples.



   Abbreviations
The following abbreviations are used in the examples and have the meanings given below.



  BSA: bovine serum albumin EDTA: ethylenediaminetetraacetic acid, tetrasodium salt GRD: ganglia of the dorsal root MEN: MOPS 20 mM, EDTA 1 mM, sodium acetate 5 mM, pH 7.0 MOPS: acid 3- (N- morpholino) propanesulfonic (Sigma
Chemical Company) PN5: peripheral nerve sodium channel 5 SDS: sodium dodecyl sulfate SNP: peripheral nervous system Denhardt solution: 0.02% BSA, 0.02% polyvinylpyrrolidone, 0.02% Ficoll (0 , 1 g of ASB, 0.1 g of
Ficoll, 0.1 g polyvinylpyrrolidone per 500 ml) SSC: 150 mM NaCl, 15 mM sodium citrate, pH 7.0 SSPE:

   80 mM NaCl, 10 mM sodium phosphate, 1 mM ethylenediaminetetraacetate, pH 8.0 TEV: tension clamp with two electrodes TTX: tetrodotoxin (Sigma Chemical Company).



   EXAMPLES
The following examples illustrate the practice of the invention.



  materials
The plasmid pBK-CMV was obtained from Stratagene (La Jolla, CA, USA); the plasmid pBSTA is described by Goldin et al. in Hethods in Enzymology (Rudy & Iverson editors) 207, 279-297; the plasmid pCInéo

  <Desc / Clms Page number 26>

 was obtained from Promega (Madison, WI, USA); and the plasmid pCRII was obtained from Invitrogen (Carlsbad, CA, USA).



   The expression vector plasmid in pBSTAcIIr oocytes was constructed from pBSTA by insertion of a synthetic oligonucleotide linker; the plasmid pKK232-8 was obtained from Pharmacia Biotech (Piscataway, NJ, USA); the plasmid pCRII was obtained from Invitrogen (San Diego, CA, USA). The competent E. coli cell lines STBL2TM and SURE 'were obtained from Gibco / BRL and Stratagene, respectively.



   EXAMPLE 1 Obtaining of RNA from GRD, from the brain and spinal cord of rats
Under a dissecting microscope, lumbar GRDs No. 4 and No. 5 (L4 and L5), the brains and the spinal cord of adult male Sprague-Dawley rats were removed. The tissues were frozen in carbon ice and homogenized using a Polytron homogenizer; the RNA was extracted by the guanidine isothiocyanate method [see Chomczynksi et al., Anal.



  Biochemistry 162, 156-159 (1987)]. The total RNA (5 g of each sample) was dissolved in MEN buffer containing 50% formamide, 6.6% formaldehyde and denatured at 65 C for 5-10 minutes. The RNA was electrophoresed on a 0.8% agarose gel containing 8.3% formaldehyde in MEN buffer. The electrode buffer was MEN buffer containing 3.7% formaldehyde; the gel was operated at 50 V for 12-18 hours.



   Molecular size markers including 18S and 28S ribosomal RNAs and RNA markers (GIBCO BRL) were electrophoresed in parallel columns of the gel. Their positions were determined

  <Desc / Clms Page number 27>

 born by staining with ethidium bromide (0.5 gg / ml) of the excised column, followed by photography under UV light.



   After electrophoresis, the gel was rinsed in SSC 2x and the RNA was transferred to a Duralose membrane (Stratagene) with SSC 20x, by capillary action; the membrane was heated under vacuum for 1 hour at 80 C.



   EXAMPLE 2 Probe from IIA of rat brain
A 32P-labeled cRNA probe, complementary to nucleotides 4637-5868 of the rat brain sodium channel IIA subunit sequence, was synthesized in vitro with T7 RNA polymerase (Pharmacia). using template DNA from pEAF8 (Noda et al., Nature 320, 188-192 (1986)] which had been linearized using BstEII.



   Protocols for each technique mentioned above can be found in Molecular Cloning: A Laboratory Manual by Sambrook et al. (Cold Spring Harbor Laboratory Press, 2nd edition, 1989).



   EXAMPLE 3 Hybridization of RNA with the Probe from IIA of Rat Brains
The membrane of Example 1 was prehybridized for 16 hours at 42 ° C. in 50% formamide, SSC 5x, 50 mM sodium phosphate, pH 7.1, Denhardt lx solution, 0.5% SDS and 1 mg / ml DNA from sheared salmon sperm, heat denatured. The membrane was hybridized for 18 hours at 42 ° C. in 50% formamide, SSC 5x, 50 mM sodium phosphate, pH 7.1, Denhardt solution 1x, 0.5% SDS and 200 gg / ml d The DNA of sheared salmon spermatozo, denatured by heat, with the cDNA probe labeled with 32P (approximately 1-3 × 10 6 cpm / ml) described in Example 2.

  <Desc / Clms Page number 28>

 



   The membrane was rinsed for 20 minutes with 2x SSC, 0.1% SDS, at room temperature, and then washed successively with 2x SSC, 0.1% SDS at 55 ° C for 30 minutes; 0.2x, 0.1% SDS at 65 C for 30 minutes; SSC 0.2x, 0.1% SDS at 70 C for 30 minutes and SSC 0.2x, 0.1% SDS, 0.1% sodium pyrophosphate at 70 C for 20 minutes. The filter was exposed against Kodak X-omat AR film at -80 C, with amplifier screens, for up to 2 weeks.



   The pEAF8 probe hybridized with the mRNAs in the GRD sample, with sizes of 11 kb, 9.5 kb, 7.3 kb and 6.5 kb, estimated on the basis of their positions relative to the stallions.



   EXAMPLE 4 New Sodium Channel Domain IV Probe The probe was obtained as follows: carried out an RT-PCR on RNA isolated from rat GRD, using degenerate oligonucleotide primers which were designed on the basis of homologies between the sodium channels known in the field IV. Domain IV products were cloned into a plasmid vector, transformed into E. coli, and individual colonies were isolated. Domain IV specific PCR products obtained from several of these colonies were sequenced individually.

   The new sequence of domain IV cloned as follows (SEQ ID NO 4): 1 CTCAACATGG TTACGATGAT GGTGGAGACC GACGAGCAGG GCGAGGAGAA 51 GACGAAGGTT CTGGGCAGAA TCAACCAGTT CTTTGTGGCC GTCTTCACGG 101 GCGAGTGTGT GATGAAGATG TTCGCCCTGC GACAGTACTA TTTCACCAAC 151 GGCTGGAACG TGTTCGACTT CATAGTGGTG ATCCTGTCCA TTGGGAGTCT 201 GCTGTTTCT GCAATCCTTA AGTCACTGGA AAACTACTTC TCCCCGACGC 251 TCTTCCGGGT CATCCGTCTG GCCAGGATCG GCCGCATCCT CAGGCTGATC 301 CGAGCAGCCA AGGGGATTCG CACGCTGCTC Incl.

  <Desc / Clms Page number 29>

 451 ATCGACGACA TGTTCAACTT CAAGACCTTT GGCAACAGCA TGCTGTGCC1 501 GTTCCAGATC ACCACCTCGG CCGGCTGGGA CGGCCTCCTC AGCCCCATCC 551 TCAACACGGG GCCTCCCTAC TGCGACCCCA ACCTGCCCAA CAGCAACGC 60

  TCCCGGGGGA ACTGCGGGAG CCCGGCGGTG GGCATCATCT TCTTCACCAC 651 CTACATCATC ATCTCCTTCC TCATCGTGGT CAACATGTAT ATCGCAGTCA 701 TC This sequence was marked with 32P by random priming.



   EXAMPLE 5 Hybridization of RNA with the New 3'-UTR Sodium Channel Probe
A northern blot was prepared with 10 g of total RNA from brain, spinal cord and GRD from rats. The fingerprint was hybridized with a cRNA probe from the 3 'untranslated region (3'-UTR).



  The 3'-UTR was cloned into the vector pSP73, and the cRNA was transcribed using a Trans Probe T kit (Pharmacia Biotech) and [32P] -UTP. The impression was prehybridized for 2 hours at 65 ° C. in a solution containing SSC 5x, Denhardt solution 1x, 0.5% SDS, 50 mM sodium phosphate, pH 7.1.1 mg / ml of Salmon sperm matozoids DNA and 50% formamide. The hybridization was carried out at 45 ° C. for 18 hours in the above solution, except that the DNA of salmon spermatozoa was included at a concentration of 200 g / ml and that the 32P labeled probe at 7.5x10 cpm / ml solution.

   The impression was then washed three times with 2x SSC and 0.1% SDS at room temperature, once with 0.2x SCC and 0.1% SDS at 65 ° C for 20 minutes, and once with 0.2x SSC, 0.1% SDS and 0.1% sodium pyrophosphate at 65 C for 20 minutes. The impression was analyzed on a Phospholmager device (BioRad), after a 2-day exposure.



  The results indicated that there was a band signal of - 6.5 kb, present in the brain, only in the

  <Desc / Clms Page number 30>

 column containing RNA from GRD. Due to the low abundance of PN5 RNA, as shown by the RT-PCR test, the 6.5 kb band was not detectable in the brain or spinal cord.



   EXAMPLE 6 Construction and screening of a cDNA library derived from rat GRD
An EcoRI-adapted cDNA library was prepared from poly (A) + RNA from normal adult male Sprague-Dawley rat GRD, using the SuperScript Choice system (GIBCO BRL). The cDNA (> 4 kb) was selected by sucrose gradient fractionation, as described by Kieffer, Gene 109, 115-119 (1991). The cDNA was then ligated into the Zap Express vector (Stratagene) and packaged using the packaging extract in X Gigapack II XL (Stratagene). Likewise, a GRD cDNA library of> 2 kb was synthesized.



   The phages (3.5x10) were screened by hybridization on a filter with a 32P-labeled probe [rBIIa, bases 4637-5868 as follows, d'Auld et al., Neuron 1, 449-461 (1988)]. Filters were hybridized in 50% formamide, SSPE5x, Denhardt 5x solution, 0.5% SDS, 250 gg / ml DNA from sheared, denatured salmon sperm, and 50 mM sodium phosphate, at 42 ° C , and washed in SSC 0.5x / 0.1% SDS, at 50 C.



   Southern blot prints of EcoRI digested plasmids were hybridized with the DNA probe labeled with 32P (SEQ ID No. 4). The filters were then hybridized at 42 ° C. in 50% formamide, SSC 6x, Denhardt 5x solution, 0.5% SDS and 100 gg / ml DNA of sheared, denatured salmon spermatozoa, and were washed in SSC 0, 1 # / 0.1% SDS, at 65 C.



   Positive clones were excised in vivo in pBK-CMV, using the ExAssist / XLOLR system (Stratagene).

  <Desc / Clms Page number 31>

 



   EXAMPLE 7 Clones and nucleotide analysis
CDNA clones, 26.2 and 25.1 were isolated from the cDNA library, and clone 1.18 was isolated from the> 2 kb GRD cDNA library. By sequence analysis, 26. 2 appeared to be a complete cDNA coding for a new sodium channel, and 25. 1 extended from domain II to 3'-UTR. However, each had a deletion which truncated the coding region. Clone 1.18 had the 3 'untranslated region, in addition to the C-terminus of the amino acid sequence deduced from PN5. The construct in the expression vector pBSTACIIr consisted of sequences from 26.2 and 1.18.



   The homology of PN5 was obtained with the other known sodium channels using the GAP / Best Fit (GCG) program:
 EMI31.1
 
 <tb> Channel <SEP>% <SEP> from <SEP> similarity <SEP>% <SEP> identity <September>
 <Tb>
 <tb> PN3a <SEP> 71 <SEP> 54
 <Tb>
 <tb> hPN3 <SEP> 71 <SEP> 55
 <Tb>
 <tb> PN4 <SEP> 71 <SEP> 53
 <Tb>
 <tb> PN4a <SEP> 71 <SEP> 53
 <Tb>
 <tb> PN1 <SEP> 72 <SEP> 55
 <Tb>
 <tb> Type <SEP> I <SEP> from <SEP> brain <SEP> from <SEP> rat <SEP> 72 <SEP> 55
 <Tb>
 <tb> Type <SEP> II <SEP> from <SEP> brain <SEP> from <SEP> rat <SEP> 71 <SEP> 54
 <Tb>
 <tb> Type <SEP> III <SEP> from <SEP> brain <SEP> from <SEP> rat <SEP> 71 <SEP> 54
 <Tb>
 <tb> Channel <SEP> cardiac <SEP> from <SEP> rat <SEP> 73 <SEP> 56
 <Tb>
 <tb> Channel <SEP> from <SEP> muscle <SEP> skeleton-
 <Tb>
 <tb> tick <SEP> from <SEP> rat <SEP> 71 <SEP> 53
 <Tb>
 Stabilization of the complete DNA of PN5 A.

   Media, E. coli cell lines and culture conditions
The culture of PN5 fragments could be carried out under conventional conditions, but the culture of plasmids containing complete constructs of

  <Desc / Clms Page number 32>

 PN5 (in pCInéo, pBSTAcIIr and other vectors) could not be carried out without the use of special culture media, special conditions and special strains of E. coli.

   The following conditions have been found to be optimal: use of E. coli STBL2 for primary transformation after ligation reactions and for large-scale culture; (2) the solid medium was FM 0.5x (see below) + LB lx (1% tryptone, 0.5% yeast extract, 0.5% NaCl) + 15 g / 1 agar , or FM lx + LB 0.5x; (3) the liquid medium was optimally FM lx + LB 0.5x; (4) 100 g / ml of carbenicillin was used for all media, since it was less rapidly metabolized than ampicillin;

   (5) the temperature for growth should not be higher than 30 C, normally 24-26 C; this required longer cultivation times than those normally used, ranging from 24 to 72 hours.
 EMI32.1
 
 <Tb>



  Middle <SEP> from <SEP> freezing <SEP> 2x <SEP> (FM <SEP> 2x):
 <Tb>
 <Tb>
 <Tb>
 <tb> K2HP04 <SEP> 12.6 <SEP> g
 <Tb>
 <Tb>
 <Tb>
 <tb> Citrate <SEP> trisodium <SEP> 0.9 <SEP> g
 <Tb>
 <Tb>
 <Tb>
 <Tb>
 <tb> MgS04.7H20 <SEP> 0.18 <SEP> g
 <Tb>
 <Tb>
 <Tb>
 <tb> (NH4) 2S04 <SEP> 1.8 <SEP> g
 <Tb>
 <Tb>
 <Tb>
 <Tb>
 <tb> KH2PO4 <SEP> 3.6 <SEP> g
 <Tb>
 <Tb>
 <Tb>
 <tb> Glycerol <SEP> 88 <SEP> g
 <Tb>
 <Tb>
 <Tb>
 <tb> H20 <SEP> complement <SEP> to <SEP> 1 <SEP> 1
 <Tb>
 FM 2x and the remaining components of the medium are prepared separately, sterilized by autoclaving, cooled to at least 60 ° C, and added together to form the final medium. Carbenicillin is prepared at a rate of 25 mg / ml of H 2 O and sterilized by filtration.



  FM 2x was first described for the preparation of frozen basic suspensions of bacterial cells [Practical Methods in Molecular Biology, Schleif R.F. and Wensing P.C., Springer-Verlag, New York (1981), p. 201-202].

  <Desc / Clms Page number 33>

 



  B. Expression vectors
In order to obtain increased stability of the complete cDNA, the expression vector pBSTAcIIr of oocytes was modified to reduce the number of plasmid copies during culture in E. coli and to reduce the continuation transcription from vector sequences, which could lead to toxic cryptic expression of the PN5 protein [Brosius J., Gene 27, 151-160 (1984)]. PBSTAcIIr was digested with PvuII. The 755 bp fragment, containing the T7 promoter, the ss-globin 5'-UTR, the multiple cloning site, the ± -globin 3'-UTR and the T3 promoter, was ligated with the 3.6 kb fragment containing the origin of replication, the ampicillin resistance gene,

   the transcription terminators rrnBT and rrnBT1T2 of pKK232-8, which had been completely digested with SmaI and partially digested with PvuII and treated with intestinal shrimp phosphatase to prevent self-ligation. The resulting plasmid, in which the orientation of the pBSTA fragment was such that the T7 promoter was closest to the rrnBT terminator, was identified by restriction mapping, is called pHQ8.

   As in the case of pBSTA, the direction of transcription of the ampicillin resistance gene and of the origin of replication of pHQ8 is opposite to that of the gene expression cassette, and the presence of the terminator rrnBTl should reduce any further reading continuation from the vector in the expression cassette driven by the T7 promoter.



  C. Complete cDNA assembly for expression
Given that pBK-CMV.26.2 has a deletion of 58 bp (corresponding to pb 4346 to 4403 of SEQ ID n 1) and that the sequence of pBK-CMV.1.18 begins at the level of pb 4180 of SEQ ID n 1, pBK-CMV. 1.18 could be used to "fix" pBK-CMV.26.2. We have developed a strategy to assemble a complete cDNA from

  <Desc / Clms Page number 34>

 clones pBK-CMV.26.2 and pBK-CMV.1.18 into three segments, truncating 5'-UTR and 3'-UTR and introducing unique restriction sites at the 5 'and 3' ends in the process. The 5 'end was generated by PCR from 26.2, by truncating the 5'-UTR, by incorporation of a SalI site just upstream of the initiation codon. The central segment was a restriction fragment from 26.2.

   The 3 'end was prepared by overlapping PCR from 26. 2 and 1.18, and incorporation of an XbaI site just downstream of the stop codon. These segments were digested at unique restriction sites, and assembled in pBSTAcIIr. This construct appeared to have a correct sequence, but after recloning as a SalI-XbaI fragment in pCInéo, two types of isolates were found, one with a deletion and one with an insertion of 8 bp. Further examination of the pBSTAcIIr clone showed that the sequence was "mixed" in this region, so that the clone must have been rearranged. It was found that the 8 bp insertion was a repeat of one of the members of an 8 bp duplication in the native sequence, forming a triple repetition of 8 bp in the rearranged isolate.

   Numerous attempts at cloning have inevitably given rise to this rearrangement. Overlapping PCR was used to introduce silent mutations into one of the 8 bp repeats, and a fragment containing this region was included when the region encoding PN5 was assembled in HQ8, the low-number version copies of pBSTAcIIr, to give the plasmid HR-1.



  This sequence was found to be stable (see Figures 5A-E, SEQ ID No. 5).



   The 5 'end fragment was prepared by PCR, using pBK-CMV DNA as a template. 26.2 and primers 4999 (CTTGGTCGACTCTAGATCAGGGTGAAGATGGAGGAG; soiled site underlined, homology with PN5 in italics, correspon-

  <Desc / Clms Page number 35>

 dant at bp 58-77 of SEQ ID No. 1; initiation codon in bold) and 4927 (GTCTTCTAGATGAGGGTTCAGTCATTGTG, corresponding to bp 1067 to 1047 of SEQ ID N 1), followed by gel purification, digestion with SalI and KpnI (KpnI site at bp 1003-1008, SEQ ID n 1) and gel purification.



   The 3.1 kb central fragment was prepared by digestion of pBK-CMV DNA. 26.2 by KpnI and AatII (AatII site at 4133-4138), followed by gel purification.



   The fragment of the 3 ′ end was prepared as follows: the PCR using primers 4837 (TCTGGGAAGTTTGGAAG, corresponding to pb 3613 to 3629 of SEQ ID No. 1) and 4931 (GACCACGAAGGCTATGTTGAGG, corresponding to pb 4239 to 4218 of SEQ ID No. 1) on the DNA of pBK-CMV. 26.2 as a matrix gave a fragment of 0.6kb. The PCR, using primers 4930 (CCTCAACATAGCCTTCGTGGTC, corresponding to pb 4218 to 4239 of SEQ ID n 1) and 4929 (GTCTTCTAGATGAGGGTTCAGTCATTGTG, XbaI site underlined, homologation with PNR in italics, corresponding to pb 5386 to 5365 of SEQ ID # 1, stop codon in bold) on pBK-CMV.1.18 DNA as a template, gave a 1.2 kb fragment, introducing an XbaI site at 7 bp from the stop codon.



  The 3 'end of fragment 4837-4931 therefore exactly completes the 5' end of fragment 4930-4929. These two fragments were purified in gel, and a fraction of each was combined as a matrix in a PCR reaction, using primers 4928 (CAAGCCTTTGTGTTCGAC, corresponding to bp 4084 to 4101 of SEQ ID No. 1) and 4929, to give a 1.3 kb fragment. This fragment was gel purified, digested with AatII and XbaI, and the 1.2 kb fragment was gel purified.



   The 3 'end fragment was cloned into pBSTAcIIr digested with AatII and XbaI. An isolate was digested with SalI and KpnI and ligated with the 5 'end fragment. The resulting plasmid, after verification of the sequence, was digested with KpnI and AatII and ligated with

  <Desc / Clms Page number 36>

 the central fragment of 3.1 kb, to give pBSTAcIIr.PNS (clone 21). pBSTAcIIr.PNS (clone 21) was digested with salI and XbaI, to release the 5.3 kb PN5 fragment, which was cloned in pCInéoII digested with SalI and XbaI. Multiple isolates were found, of which GPII-1, which was completely sequenced, was characteristic and contained an 8 bp insert.

   This CAGAAGAA insert, after pb 3994 of SEQ ID No. 1, converted the direct repetition of this sequence at this location into a direct triple repetition, causing a displacement in the reading frame. In an attempt to repair this defect, pBSTAcIIr.PN5 (clone 21) was digested by NheI (pb 2538-2543, SEQ ID n 1) and XhoI (pb 4828-4833, SEQ ID n 1), to obtain a fragment 6.2 kb, and with AatII and XhoI, to obtain a 0.7 kb fragment which has been ligated with the 1.6 kb fragment resulting from the digestion of pBK-CMV. 26.2 by AatII and NheI. Although no fully correct isolate was found, one isolate, HA-4, only had one base change, a deletion from C at bp 4827 (SEQ ID n 1), adjacent to the XhoI site .



   In order to prevent the 8 bp insertion rearrangement from occurring, three silent mutations were introduced into the 5 'repeat, and two additional mutations in a T series would also be introduced, as shown below. below (bp 3982 to 4014, SEQ ID n 1; mutation sites underlined, repetitions of 8 bp in the native sequence in italics):

   native sequence GAC ATT TTT ATG ACA GAA GAA CAG AAG AAA TA
Asp Ile Phe Met Thr Glu Glu Gln Lys Lys Ty mutant sequence GAC ATC TTC ATG ACT GAG GAG CAG AAG AAA T
Since the HA-4 isolate had the native direct repeat sequence (as opposed to, for example, pBSTAcIIr.PNS (clone 21)) and that the region around the XhoI site defect was not involved, we used it as template DNA for the following PCR reactions

  <Desc / Clms Page number 37>

 lowing. The primer P5-3716S (CCGAAGCCAATGTAACATTAGTAATTACTCGTG, corresponding to bp 3684 to 3716, SEQ ID n 1) was paired with the primer P5-3969AS (GCTCCTCAGTCATGAAGATGTCTTGGCCACCTAAC, corresponding to pb 4003 to 3969, bases ID n mutated are underlined), to give a product of 320 bp.



  The primer P5-4017S (GGCCAAGACATCTTCATGACTGAGGAGCAGAAGAAATATTAC, corresponding to pb 3976 to 4017, SEQ ID n 1; the mutated bases are underlined) was paired with the primer P5-4247AS (CTCAAAGCAAAGACTTTGATGAGACACTCTATGG, corresponding to pb 42, 42 1), to give a product of 305 bp. The 3 'end of the 320 bp fragment therefore had a 28 bp segment corresponding exactly to the 5' end of the 305 bp fragment. The two bands were purified in gel, and a fraction of each was combined, in a new PCR reaction, with the primers P5-3716S and P5-4247AS, to give a product of 597 bp, which was cloned T / A in the vector pCRII. We found the HO-7 isolate, which had the desired sequence.

   We performed a quadruple ligation to assemble the complete modified PN5:
The expression vector HQ-8 was digested with SalI and XbaI in oocytes, to obtain a vector fragment of 4.4 kb, GPII-1; GPII-1 was digested with SalI and MluI, to obtain a 3.8 kb fragment containing the 5 'half of PN5; HO-7 was digested with MluI (bp 3866 to 3871, SEQ ID No. 1) and AatII, to obtain a 0.3 kb fragment containing the repeating region of 8 bp mutant of PN5; GPII-1 was digested with AatII and XbaI, to obtain the remaining 3 ′ segment of 1.3 kb of PNS. Part of the ligation reaction mixture was transformed into Stable 2 E. coli cells.

   Among the 9.6 kb isolates containing all four fragments, HR-1 was sequenced and found to have the desired 5.4 kb sequence. These isolates developed well and did not

  <Desc / Clms Page number 38>

 there was no tendency to rearrange. The sequence of this constructed version of PN5 is shown in Figures 5A-E (SEQ ID No. 5).



   EXAMPLE 8 Human PNR
A 856 bp clone (Figure 3A, SEQ ID No. 3) was isolated from a human dorsal root ganglion (GRD) cDNA library, which is very closely related to rat PN5, with identity 79% for the amino acid sequence. The human PN5 sequence covers the region between IIIS1 and interdomain III / IV, which includes the rapid inactivation valve (i.e. IFM), which is located in interdomain III / IV.



   The GRD cDNA library was constructed from randomly primed GRD 4 and 5 lumbar total RNA. First strand cDNA was synthesized using SuperScript II reverse transcriptase (GIBCO BRL), and second strand synthesis was performed using T4 DNA polymerase. EcoRI connector segments were ligated to the ends of the double-stranded cDNAs, and the fragments were cloned into the ZAP II vector (Stratagene). The library was screened using digoxigenin-labeled rat PN3, rat PN1 and human heart hH1 probes. The positive clones were sequenced and compared to known sequences of human and rat sodium channels. Only the above clone has been identified as a human PN5 sequence.
 EMI38.1
 
 <Tb>



  Channel <SEP>% <SEP> from <SEP> similarity <SEP>% <SEP> identity
 <Tb>
 <tb> Brain <SEP> human <SEP> (HBA) <SEP> 76 <SEP> 69
 <Tb>
 <tb> Heart <SEP> human <SEP> (hHl) <SEP> 81 <SEP> 74
 <Tb>
 <tb> Heart <SEP> atypical <SEP> human <SEP> 60 <SEP> 52
 <Tb>
 <tb> Muscle Skeletal <SEP> <SEP> human <SEP> 80 <SEP> 71
 <Tb>
 <tb> Neuroendocrine <SEP> human <SEP> 78 <SEP> 71
 <Tb>
 <tb> PN3 <SEP> human <SEP> 77 <SEP> 70
 <Tb>
 <tb> PN1 <SEP> from <SEP> rat <SEP> 79 <SEP> 72
 <Tb>
 

  <Desc / Clms Page number 39>

 
 EMI39.1
 
 <tb> Channel <SEP>% <SEP> from <SEP> similarity <SEP>% <SEP> identity
 <Tb>
 <tb> PN3 <SEP> from <SEP> rat <SEP> 78 <SEP> 71
 <Tb>
 <tb> PN4 <SEP> from <SEP> rat <SEP> 78 <SEP> 70
 <Tb>
 <tb> PN5 <SEP> from <SEP> rat <SEP> 86 <SEP> 79
 <Tb>
 
FIG. 3B compares the amino acid sequence of the hPN5 fragment with the amino acid sequence of rat PN5,

   in the appropriate region.



   EXAMPLE 9 Tissue distribution by RT-PCR
It was isolated from normal adult, anesthetized male Sprague-Dawley rats, brain tissue, spinal cord, GRD, plexiform ganglia, upper cervical ganglia, sciatic nerve, heart and skeletal muscle, and we kept them at -80 C.



  The RNA was isolated from each tissue using RNAzol (Tel-Test Inc.). The randomly primed cDNA was transcribed by reverse transcription from 500 mg of RNA from each tissue. The direct primer (CAGATTGTGTTCTCAGTACATTCC) and the reverse primer (CCAGGTGTCTAACGAATAAATAGG) were designed from the 3 'untranslated region, to give a fragment of 252 base pairs. The cycle parameters were 94 C / 2 minutes (denaturation), 94 C / 30 s, 65 C / 30 s and 72 C / 1 min (35 cycles) and 72 C / 4 min. The reaction products were analyzed on a 4% agarose gel.



   A positive control and a control without a matrix were also included. CDNA from each tissue was also amplified by pCR, using primers specific for glyceraldehyde-3-phosphate dehydrogenase, to show the viability of the template, as described by Tso et al., Nucleic. Acid Res. 13.2485-2502 (1985).



   The tissue distribution profile of rPN5 by RT-PCR analysis of RNA from selected rat tissues was as follows:

  <Desc / Clms Page number 40>

 
 EMI40.1
 
 <tb> Fabric <SEP> RT-PCR <SEP> (35 <SEP> cycles)
 <tb> Brain <SEP> +
 <Tb>
 <tb> Marrow <SEP> spinal <SEP> +
 <Tb>
 <tb> GRD <SEP> +++
 <Tb>
 <tb> Lymph nodes <SEP> plexiform <SEP> +++
 <Tb>
 <tb> Lymph nodes <SEP> cervical <SEP> superiors <SEP> +
 <Tb>
 <tb> Nerf <SEP> sciatica-
 <Tb>
 <tb> Heart-
 <Tb>
 <tb> Muscle <SEP> skeletal-
 <Tb>
 <tb> Wire <SEP> no <SEP> processed <SEP> +
 <Tb>
 <tb> Wire <SEP> processed <SEP> +
 <Tb>
 
PN5 was also detected after only 25 cycles (24 + 1) in the same five tissues as above, in the same relative abundance.



   EXAMPLE 10 Antibodies
A synthetic peptide (26 amino acids in interdomains II and III - residues 977 to 1002) was conjugated with KLH and the antibody was raised in rabbits.



  The antiserum was then purified by affinity. PN5 is a subfamily of new sodium channel genes; these genes are different from those detectable with other probes (for example the PEAF8 and PN3 probes).



   It should be understood that the foregoing description has been given by way of illustration and not limitation, and that any variations or modifications may be made thereto without departing from the general scope of the present invention, as defined in the appended claims.

  <Desc / Clms Page number 41>

 



   LIST OF SEQUENCES (1) GENERAL INFORMATION: (I) APPLICANT: (A) NAME: HOFFMANN-LA ROCHE AG (B) STREET: 124 (C) CITY: Basel (D) STATE: (E) COUNTRY: (F) CODE POSTAL: (G) TELEPHONE: (H) FAX: 061-6881395 (I) TELEX: 962292/965542 hlr ch (II) TITLE OF THE INVENTION: DNA coding for a sodium channel protein, its production and its use (III) NUMBER OF SEQUENCES: (IV) FORM READABLE BY COMPUTER: (A) TYPE OF MEDIA: soft discs (B) COMPUTER: compatible PC (C) OPERATING SYSTEM: (D) SOFTWARE: PatentIn Release n 1. 0, version
1. 30 (2) INFORMATION FOR SEQ ID # 1:

   (I) CHARACTERISTICS OF THE SEQUENCE: '(A) LENGTH: 908 base pairs (B) TYPE: nucleic (C) TYPE OF STRAND: (D) TOPOLOGY: linear (II) TYPE OF MOLECULE: cDNA (III) HYPOTHETICAL: (IV) ANTISENSE: (VI) SOURCE OF ORIGIN: (A) ORGANISM: (B) TYPE OF FABRIC: from the golden root

  <Desc / Clms Page number 42>

   (C) TYPE OF CELL: peripheral (XI) DESCRIPTION OF THE SEQUENCE:

   ID # 1:
1 GAAGTCACAG GAGTGTCTGT CAGCGAGAGG AAGAAGGGAG AGTTTACTGA
51 GTGTCTTCTG CCCCTCCTCA GGGTGAAGAT GGAGGAGAGG TACTACCCGG
101 TGATCTTCCC GGACGAGCGG AATTTCCGCC CCTTCACTTC CGACTCTCTG
151 GCTGCCATAG AGAAGCGGAT TGCTATCCAA AAGGAGAGGA AGAAGTCCAA
201 AGACAAGGCG GCAGCTGAGC CCCAGCCTCG GCCTCAGCTT GACCTAAAGG
251 CCTCCAGGAA GTTACCTAAG CTTTATGGTG ACATTCCCCC TGAGCTTGTA
301 GCGAAGCCTC TGGAAGACCT GGACCCATTC TACAAAGACC ATAAGACATT
351 CATGGTGTTG AACAAGAAGA GAACAATTTA TCGCTTCAGC GCCAAGCGGG
401 CCTTGTTCAT TCTGGGGCCT TTTAATCCCC TCAGAAGCTT AATGATTCGT
451 ATCTCTGTCC ATTCAGTCTT TAGCATGTTC ATCATCTGCA CGGTGATCAT
501 CAACTGTATG TTCATGGCGA ATTCTATGGA GAGAAGTTTC GACAACGACA
551 TTCCCGAATA CGTCTTCATT GGGATTTATA TTTTAGAAGC TGTGATTAAA
601 ATATTGGCAA GAGGCTTCAT TGTGGATGAG TTTTCCTTCC TCCGAGATCC
651 GTGGAACTGG CTGGACTTCA TTGTCATTGG AACAGCGATC

  GCAACTTGTT 701 TTCCGGGCAG CCAAGTCAAT CTTTCAGCTC TTCGTACCTT CCGAGTGTTC 751 AGAGCTCTGA AGGCGATTTC AGTTATCTCA GGTCTGAAGG TCATCGTAGG
801 TGCCCTGCTG CGCTCGGTGA AGAAGCTGGT AGACGTGATG GTCCTCACTC
851 TCTTCTGCCT CAGCATCTTT GCCCTGGTCG GTCAGCAGCT GTTCATGGGA
901 ATTCTGAACC AGAAGTGTAT TAAGCACAAC TGTGGCCCCA ACCCTGCATC
951 CAACAAGGAT TGCTTTGAAA AGGAAAAAGA TAGCGAAGAC TTCATAATGT 1001 GTGGTACCTG GCTCGGCAGC AGACCCTGTC CCAATGGTTC TACGTGCGAT 1051 AAAACCACAT TGAACCCAGA CAATAATTAT ACAAAGTTTG ACAACTTTGG 1101 CTGGTCCTTT CTCGCCATGT TCCGGGTTAT GACTCAAGAC TCCTGGGAGA 1151 GGCTTTACCG ACAGATCCTG CGGACCTCTG GGATCTACTT TGTCTTCTTC

  <Desc / Clms Page number 43>

 1201 TTCGTGGTGG TCATCTTCCT GGGCTCCTTC TACCTGCTTA ACCTAACCCT 1251 GGCTGTTGTC ACCATGGCTT ATGAAGAACA GAACAGAAAT GTAGCTGCTG 1301 AGACAGAGGC

  CAAGGAGAAA ATGTTTCAGG AAGCCCAGCA GCTGTTAAGG 1351 GAGGAGAAGG AGGCTCTGGT TGCCATGGGA ATTGACAGAA GTTCCCTTAA 1401 TTCCCTTCAA GCTTCATCCT TTTCCCCGAA GAAGAGGAAG TTTTTCGGTA 1451 GTAAGACAAG AAAGTCCTTC TTTATGAGAG GGTCCAAGAC GGCCCAAGCC 1501 TCAGCGTCTG ATTCAGAGGA CGATGCCTCT AAAAATCCAC AGCTCCTTGA 1551 GCAGACCAAA CGACTGTCCC AGAACTTGCC AGTGGATCTC TTTGATGAGC 1601 ACGTGGACCC CCTCCACAGG CAGAGAGCGC TGAGCGCTGT CAGTATCTTA 1651 ACCATCACCA TGCAGGAACA AGAAAAATTC CAGGAGCCTT GTTTCCCATG 1701 TGGGAAAAAT TTGGCCTCTA AGTACCTGGT GTGGGACTGT AGCCCTCAGT 1751 GGCTGTGCAT AAAGAAGGTC CTGCGGACCA TCATGACGGA TCCCTTTACT 1801 GAGCTGGCCA TCACCATCTG CATCATCATC AATACCGTTT TCTTAGCCGT 1851 GGAGCACCAC AACATGGATG ACAACTTAAA GACCATACTG AAAATAGGAA 1901 ACTGGGTTTT CACGGGAATT TTCATAGCGG AAATGTGTCT CAAGATCATC 1951 GCGCTCGACC CTTACCACTA CTTCCGGCAC GGCTGGAATG TTTTTGACAG 2001 CATCGTGGCC CTCCTGAGTC TCGCTGATGT GCTCTACAAC

  ACACTGTCTG 2051 ATAACAATAG GTCTTTCTTG GCTTCCCTCA GAGTGCTGAG GGTCTTCAAG 2101 TTAGCCAAAT CCTGGCCCAC GTTAAACACT CTCATTAAGA TCATCGGCCA 2151 CTCCGTGGGC GCGCTTGGAA ACCTGACTGT GGTCCTGACT ATCGTGGTCT 2201 TCATCTTTTC TGTGGTGGGC ATGCGGCTCT TCGGCACCAA GTTTAACAAG 2251 ACCGCCTACG CCACCCAGGA GCGGCCCAGG CGGCGCTGGC ACATGGATAA
2301 TTTCTACCAC TCCTTCCTGG TGGTGTTCCG CATCCTCTGT GGGGAATGGA
2351 TCGAGAACAT GTGGGGCTGC ATGCAGGATA TGGACGGCTC CCCGTTGTGC
2401 ATCATTGTCT TTGTCCTGAT AATGGTGATC GGGAAGCTTG TGGTGCTTAA 2451 CCTCTTCATT GCCTTGCTGC TCAATTCCTT CAGCAATGAG GAGAAGGATG

  <Desc / Clms Page number 44>

 2501 GGAGCCTGGA AGGAGAGACC AGGAAAACCA AAGTGCAGCT AGCCCTGGAT 2551 CGGTTCCGCC GGGCCTTCTC CTTCATGCTG CACGCTCTTC AGAGTTTTTG 2601 TTGCAAGAAA TGCAGGAGGA AAAACTCGCC AAAGCCAAAA GAGACAACAG 2651

  TGGTGAGAAT AAAGACTCAA TCCTCCCGGA TGCGAGGCCC 2701 TGGAAGGAGT ATGATACAGA CATGGCTTTG TACACTGGAC AGGCCGGGGC 2751 TCCGCTGGCC CCACTCGCAG AGGTAGAGGA CGATGTGGAA TATTGTGGTG 2801 AAGGCGGTGC CCTACCCACC TCACAACATA GTGCTGGAGT TCAGGCCGGT 2851 GACCTCCCTC CAGAGACCAA GCAGCTCACT AGCCCGGATG ACCAAGGGGT 2901 TGAAATGGAA GTATTTTCTG AAGAAGATCT GCATTTAAGC ATACAGAGTC 2951 CTCGAAAGAA GTCTGACGCA GTGAGCATGC TCTCGGAATG CAGCACAATT 3001 GACCTGAATG ATATCTTTAG AAATTTACAG AAAACAGTTT CCCCCAAAAA 3051 GCAGCCAGAT AGATGCTTTC CCAAGGGCCT TAGTTGTCAC TTTCTATGCC 3101 ACAAAACAGA CAAGAGAAAG TCCCCCTGGG TCCTGTGGTG GAACATTCGG 3151 AAAACCTGCT ACCAAATCGT GAAGCACAGC TGGTTTGAGA GTTTCATAAT 3201 CTTTGTTATT CTGCTGAGCA GTGGAGCGCT GATATTTGAA GATGTCAATC 3251 TCCCCAGCCG GCCCCAAGTT GAGAAATTAC TAAGGTGTAC CGATAATATT 3301 TTCACATTTA TTTTCCTCCT GGAAATGATC CTGAAGTGGG TGGCCTTTGG 3351 ATTCCGGAGG TATTTCACCA GTGCCTGGTG CTGGCTTGAT TTCCTCATTG 3401

  TGGTGGTGTC TGTGCTCAGT CTCATGAATC TACCAAGCTT GAAGTCCTTC
3451 CGGACTCTGC GGGCCCTGAG ACCTCTGCGG GCGCTGTCCC AGTTTGAAGG
3501 AATGAAGGTT GTCGTCTACG CCCTGATCAG CGCCATACCT GCCATTCTCA
3551 ATGTCTTGCT GGTCTGCCTC ATTTTCTGGC TCGTATTTTG TATCTTGGGA
3601 GTAAATTTAT TTTCTGGGAA GTTTGGAAGG TGCATTAACG GGACAGACAT
3651 AAATATGTAT TTGGATTTTA CCGAAGTTCC GAACCGAAGC CAATGTAACA
3701 TTAGTAATTA CTCGTGGAAG GTCCCGCAGG TCAACTTTGA CAACGTGGGG
3751 AATGCCTATC TCGCCCTGCT GCAAGTGGCA ACCTATAAGG GCTGGCTGGA

  <Desc / Clms Page number 45>

 3801 AATCATGAAT GCTGCTGTCG ATTCCAGAGA GAAAGACGAG CAGCCGGACT 3851 TTGAGGCGAA CCTCTACGCG TATCTCTACT TTGTGGTTTT TATCATCTTC 3901 GGCTCCTTCT TTACCCTGAA CCTCTTTATC GGTGTTATTA TTGACAACTT 3951 CAATCAGCAG CAGAAAAAGT TAGGTGGCCA AGACATTTTT ATGACAGAAG 4001 AACAGAAGAA ATATTACAAT GCAATGAAAA AGTTAGGAAC

  CAAGAAACCT 4051 CAAAAGCCCA TCCCAAGGCC CCTGAACAAA TGTCAAGCCT TTGTGTTCGA 4101 CCTGGTCACA AGCCAGGTCT TTGACGTCAT CATTCTGGGT CTTATTGTCT 4151 TAAATATGAT TATCATGATG GCTGAATCTG CCGACCAGCC CAAAGATGTG 4201 AAGAAAACCT TTGATATCCT CAACATAGCC TTCGTGGTCA TCTTTACCAT 4251 AGAGTGTCTC ATCAAAGTCT TTGCTTTGAG GCAACACTAC TTCACCAATG 4301 GCTGGAACTT ATTTGATTGT GTGGTCGTGG TTCTTTCTAT CATTAGTACC 4351 CTGGTTTCCC GCTTGGAGGA CAGTGACATT TCTTTCCCGC CCACGCTCTT 4401 CAGAGTCGTC CGCTTGGCTC GGATTGGTCG AATCCTCAGG CTGGTCCGGG 4451 CTGCCCGGGG AATCAGGACC CTCCTCTTTG CTTTGATGAT GTCTCTCCCC 4501 TCTCTCTTCA ACATCGGTCT GCTGCTCTTC CTGGTGATGT'TCATTTACGC 4551 CATCTTTGGG ATGAGCTGGT TTTCCAAAGT GAAGAAGGGC TCCGGGATCG 4601 ACGACATCTT CAACTTCGAG ACCTTTACGG GCAGCATGCT GTGCCTCTTC 4651 CAGATAACCA CTTCGGCTGG CTGGGATACC CTCCTCAACC CCATGCTGGA 4701 GGCAAAAGAA CACTGCAACT CCTCCTCCCA AGACAGCTGT CAGCAGCCGC 4751 AGATAGCCGT CGTCTACTTC GTCAGTTACA

  TCATCATCTC CTTCCTCATC 4801 GTGGTCAACA TGTACATCGC TGTGATCCTC GAGAACTTCA ACACAGCCAC 4851 GGAGGAGAGC GAGGACCCTC TGGGAGAGGA CGACTTTGAA ATCTTCTATG 4901 AGGTCTGGGA GAAGTTTGAC CCCGAGGCGT CGCAGTTCAT CCAGTATTCG 4951 GCCCTCTCTG ACTTTGCGGA CGCCCTGCCG GAGCCGTTGC GTGTGGCCAA 5001 GCCGAATAAG TTTCAGTTTC TAGTGATGGA CTTGCCCATG GTGATGGGCG 5051 ACCGCCTCCA TTGCATGGAT GTTCTCTTTG CTTTCACTAC CAGGGTCCTC

  <Desc / Clms Page number 46>

 5101 GGGGACTCCA GCGGCTTGGA TACCATGAAA ACCATGATGG AGGAGAAGTT 5151 TATGGAGGCC AACCCTTTTA AGAAGCTCTA CGAGCCCATA GTCACCACCA 5201 CCAAGAGGAA GGAGGAGGAG CAAGGCGCCG CCGTCATCCA GAGGGCCTAC 5251 CGGAAACACA TGGAGAAGAT GGTCAAACTG AGGCTGAAGG ACAGGTCAAG 5301 TTCATCGCAC CAGGTGTTTT GCAATGGAGA CTTGTCCAGC TTGGATGTGG 5351 CCAAGGTCAA GGTTCACAAT GACTGAACCC TCATCTCCAC CCCTACCTCA 5401 CTGCCTCACA GCTTAGCCTC CAGCCTCTGG CGAGCAGGCG

  GCAGACTCAC 5451 TGAACACAGG CCGTTCGATC TGTGTTTTTG GCTGAACGAG GTGACAGGTT 5501 GGCGTCCATT TTTAAATGAC TCTTGGAAAG ATTTCATGTA GAGAGATGTT 5551 AGAAGGGACT GCAAAGGACA CCGACCATAA CGGAAGGCCT GGAGGACAGT 5601 CCAACTTACA TAAAGATGAG AAACAAGAAG GAAAGATCCC AGGAAAACTT 5651 CAGATTGTGT TCTCAGTACA TCCCCCAATG TGTCTGTTCG GTGTTTTGAG 5701 TATGTGACCT GCCACATGTA GCTCTTTTTT GCATGTACGT CAAAACCCTG 5751 CAGTAAGTTG ATAGCTTGCT ACGGGTGTTC CTACCAGCAT CACAGAATTG 5801 GGTGTATGAC TCAAACCTAA AAGCATGACT CTGACTTGTC AGTCAGCACC 5851 CCGACTTTCA GACGCTCCAA TCTCTGTCCC AGGTGTCTAA CGAATAAATA @ 5901 GGTAAAAG (3) INFORMATION FOR SEQ ID # 2:

   (I) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 765 amino acids (B) TYPE: (C) TYPE OF STRAND: - (D) TOPOLOGY: unrelated (II) TYPE OF MOLECULE: protein (III) HYPOTHETIC: yes (VI ) SOURCE OF ORIGIN: (A) ORGANISM: rat (B) TYPE OF TISSUE: from the golden root (C) TYPE OF CELL: peripheral nerve

  <Desc / Clms Page number 47>

   (XI) DESCRIPTION OF THE SEQUENCE:

   ID # 2:
Met Glu Glu Arg Tyr Tyr Pro Val Ile Phe Pro Asp Glu Arg Asn Phe
1 5 '10 15
Arg Pro Phe Thr Ser Asp Ser Leu Ala Ala Ile Glu Lys Arg Ile Ala
20 25 30
Ile Gln Lys Glu Arg Lys Lys Ser Lys Asp Lys Ala Ala Ala Glu Pro
35 40 45
Gln Pro Arg Pro Gln Leu Asp Leu Lys Ala Ser Arg Lys Leu Pro Lys
50 55 60
Leu Tyr Gly Asp Ile Pro Pro Glu Leu Val Ala Lys Pro Leu Glu Asp
65 70 75 80
Leu Asp Pro Phe Tyr Lys Asp His Lys Thr Phe Met Val Leu Asn Lys
85 90 95
Lys Arg Thr Ile Tyr Arg Phe Ser Ala Lys Arg Ala Leu Phe lie Leu
100 105 110
Gly Pro Phe Asn Pro Leu Arg Ser Leu Met Ile Arg Ile Ser Val His
115 120 125
Ser Val Phe Ser Met Phe Ile Cys Island Thr Val Ile Ile Asn Cys Met
130 135 140
Phe Met Ala Asn Ser Met Glu Arg Ser Phe Asp Asn Asp Ile Pro Glu
145 150 155 160
Tyr Val Phe Ile

  Gly Ile Tyr Ile Leu Glu Ala Val Ile Lys Ile Leu
165 170 175
Ala Arg Gly Phe Ile Val Asp Glu Phe Ser Phe Leu Arg Asp Pro Trp
180 185 190
Asn Trp Leu Asp Phe Ile Val Ile Gly Thr Ala Ile Ala Thr Cys Phe
195 200 205
Pro Gly Ser Gln Val Asn Leu Ser Ala Leu Arg Thr Phe Arg Val Phe
210 215 220
Arg Ala Leu Lys Ala Ile Ser Val Ile Ser Gly Leu Lys Val Ile Val
225 230 235 240
Gly Ala Leu Leu Arg Ser Val Lys Lys Leu Val Asp Val Met Val Leu
245 250 255
Thr Leu Phe Cys Leu Ser Ile Phe Ala Leu Val Gly Gln Gln Leu Phe
260 265 270

  <Desc / Clms Page number 48>

 
Met Gly Ile Leu Asn Gln Lys Cys Ile Lys His Asn Cys Gly Pro Asn
275 280 285
Pro Ala Ser Asn Lys Asp Cys Phe Glu Lys Glu Lys Asp Ser Glu Asp
290 295 300
Phe Ile Met Cys Gly Thr Trp Leu Gly Ser Arg Pro Cys Pro Asn Gly
305 310 315 320
Ser Thr Cys Asp Lys Thr Thr Leu Asn Pro Asp

  Asn Asn Tyr Thr Lys
325 330 335
Phe Asp Asn Phe Gly Trp Ser Phe Leu Ala Met Phe Arg Val Met Thr
340 345 350
Gin Asp Ser Trp Glu Arg Leu Tyr Arg Gln Ile Leu Arg Thr Ser Gly
355 360 365
Ile Tyr Phe Val Phe Phe Phe Val Val Val Ile Phe Leu Gly Ser Phe
370 375 380
Tyr Leu Leu Asn Leu Thr Leu Ala Val Val Thr Met Ala Tyr Glu Glu
385 390 395 400
Gln Asn Arg Asn Val Ala Ala Glu Thr Glu Ala Lys Glu Lys Met Phe
405 410 415
Gln Glu Ala Gln Gln Leu Leu Arg Glu Glu Lys Glu Ala Leu Val Ala
420 425 430
Met Gly Ile Asp Arg Ser Ser Leu Asn Ser Leu Gln Ala Ser Ser Phe
435 440 445
Ser Pro Lys Lys Arg Lys Phe Phe Gly Ser Lys Thr Arg Lys Ser Phe
450 455 460
Phe Met Arg Gly Ser Lys Thr Ala Gln Ala Ser Ala Ser Asp Ser Glu
465 470 475 480
Asp Asp Ala Ser Lys Asn Pro Gln Leu Leu Glu Gln Thr Lys Arg Leu
485 490 495
Ser Gin Asn Leu Pro Val Asp Leu Phe Asp Glu His Val Asp Pro Leu
500 505

  510
His Arg Gln Arg Ala Leu Ser Ala Val Ser Ile Leu Thr Ile Thr Met
515 520 525
Gln Glu Gln Glu Lys Phe Gln Glu Pro Cys Phe Pro Cys Gly Lys Asn
530 535 540

  <Desc / Clms Page number 49>

 Leu Ala Ser Lys Tyr Leu Val Trp Asp Cys Ser Pro Gln Trp Leu Cys 545 550 555 560 Ile Lys Lys Val Leu Arg Thr Ile Met Thr Asp Pro Phe Thr Glu Leu
565 570 575 Ala Ile Thr Ile Cys Ile Ile Asn Thr Val Phe Leu Ala Val Glu
580 585 590 His His Asn Met Asp Asp Asn Leu Lys Thr Ile Leu Lys Ile Gly Asn
595 600 605 Trp Val Phe Thr Gly Ile Phe Ile Ala Glu Met Cys Leu Lys Ile Ile
610 615 620 Ala Leu Asp Pro Tyr His Tyr Phe Arg His Gly Trp Asn Val Phe Asp 625 630 635 640 Ser

    Ile Val Ala Leu Leu Ser Leu Ala Asp Val Leu Tyr Asn Thr Leu
645 650 655 Ser Asp Asn Asn Arg Ser Phe Leu Ala Ser Leu Arg Val Leu Arg Val
660 665 670 Phe Lys Leu Ala Lys Ser Trp Pro Thr Leu Asn Thr Leu Ile Lys Ile
675 680 685 Ile Gly His Ser Val Gly Ala Leu Gly Asn Leu Thr Val Val Leu Thr
690 695 700 Ile Val Val Phe Ile Phe Ser Val Val Gly Met Arg Leu Phe Gly Thr 705 710 715 720 Lys Phe Asn Lys Thr Ala Tyr Ala Thr Gln Glu Arg Pro Arg Arg Arg
725 730 735 Trp His Met Asp Asn Phe Tyr His Ser Phe Leu Val Val Phe Arg Ile
740 745 750 Leu Cys Gly Glu Trp Ile Glu Asn Met Trp Gly Cys Met Gin Asp Met
755 760 765 Asp Gly Ser Pro Leu Cys Ile Ile Val Phe Val Leu! Le blet Val Ile
770 775 780 Gly Lys Leu Val Val Leu

  Asn Leu Phe lie Ala Leu Leu Leu Asn Ser 785 790 795 800 Phe Ser Asn Glu Glu Lys Asp Gly Ser Leu Glu Gly Glu Thr Arg Lys
805 810 815

  <Desc / Clms Page number 50>

 Thr Lys Val Gln Leu Ala Leu Asp Arg Phe Arg Arg Ala Phe Ser Phe
820 825 830 Met Leu His Ala Leu Gln Ser Phe Cys Cys Lys Lys Cys Arg Arg Lys
835 840 845 Asn Ser Pro Lys Pro Lys Glu Thr Thr Glu Ser Phe Ala Gly Glu Asn
850 855 860 Lys Asp Ser Ile Leu Pro Asp Ala Arg Pro Trp Lys Glu Tyr Asp Thr 865 870 875 880 Asp Met Ala Leu Tyr Thr Gly Gln Ala Gly Ala Pro Leu Ala Pro Leu
885 890 895 Ala.Glu Val Glu Asp Asp Val Glu Tyr Cys Gly Glu Gly Gly Ala Leu
900 905 910 Pro Thr Ser Gln His Ser Ala Gly Val Gln Ala Gly Asp Leu Pro Pro
915 920 925 Glu Thr Lys Gln Leu Thr Ser Pro Asp Asp Gln Gly Val Glu Met Glu
930 935 940 Val Phe Ser Glu Glu Asp Leu His Leu Ser Ile Gln Ser Pro Arg Lys

  945 950 955 960 Lys Ser Asp Ala Val Ser Met Leu Ser Glu Cys Ser Thr Ile Asp Leu @
965 970 975 Asn Asp Ile Phe Arg Asn Leu Gln Lys Thr Val Ser Pro Lys Lys Gin
980 985 990 Pro Asp Arg Cys Phe Pro Lys Gly Leu Ser Cys His Phe Leu Cys His
995 1000 1005 Lys Thr Asp Lys Arg Lys Ser Pro Trp Val Leu Trp Trp Asn Ile Arg
1010 1015 1020 Lys Thr Cys Tyr Gln Ile Val Lys His Ser Trp Phe Glu Ser Phe Ile 1025 1030 1035 1040! Le Phe Val Ile Leu Leu Ser Ser Gly Ala Leu Ile Phe Glu Asp Val
1045 1050 1055 Asn Leu Pro Ser Arg Pro Gin Val Glu Lys Leu Leu Arg Cys Thr Asp
1060 1065 1070 Asn Ile Phe Thr Phe Ile Phe Leu Leu Glu Met Ile Leu Lys Trp Val
1075 1080 1085

  <Desc / Clms Page number 51>

 Ala Phe Gly Phe - Arg Arg Tyr Phe Thr Ser Ala Trp Cys Trp Leu Asp
1090 1095 1100 Phe Leu Ile Val Val Val Ser Val Leu

  Ser Leu Met Asn Leu Pro Ser 1105 1110 1115 1120 Leu Lys Ser Phe Arg Thr Leu Arg Ala Leu Arg Pro Leu Arg Ala Leu
1125 1130 1135 Ser Gln Phe Glu Gly Met Lys Val Val Val Tyr Ala Leu Ile Ser Ala
1140 1145 1150 Ile Pro Ala Ile Leu Asn Val Leu Leu Val Cys Leu Ile Phe Trp Leu
1155 1160 1165 Val Phe Cys Ile Leu Gly Val Asn Leu Phe Ser Gly Lys Phe Gly Arg
1170 1175 1180 Cys Ile Asn Gly Thr Asp Ile Asn Met Tyr Leu Asp Phe Thr Glu Val 1185 1190 1195 1200 Pro Asn Arg Ser Gln Cys Asn Ile Ser Asn Tyr Ser Trp Lys Val Pro
1205 1210 1215 Gln Val Asn Phe Asp Asn Val Gly Asn Ala Tyr Leu Ala Leu Leu Gln
1220 1225 1230 Val Ala Thr Tyr Lys Gly Trp Leu Glu Ile Met Asn Ala Ala Val Asp
1235 1240 1245 Ser Arg Glu Lys Asp Glu Gln Pro Asp Phe Glu Ala Asn Leu Tyr Ala
1250 1255 1260 Tyr Leu Tyr Phe Val Val Phe Ile Ile Phe Gly Ser Phe Phe Thr Leu 1265 1270 1275 1280 Asn Leu Phe Ile Gly Val Ile Ile Asp

  Asn Phe Asn Gln Gln Gln Lys
1285 1290 1295 Lys Leu Gly Gly Gln Asp Ile Phe Met Thr Glu Glu Gln Lys Lys Tyr
1300 1305 1310 Tyr Asn Ala Met Lys Lys Leu Gly Thr Lys Lys Pro Gln Lys Pro Ile
1315 1320 1325 Pro Arg Pro Leu Asn Lys Cys Gln Ala Phe Val Phe Asp Leu Val Thr
1330 1335 1340 Ser Gln Val Phe Asp Val Ile Ile Leu Gly Leu Ile Val Leu Asn Met 1345 1350 1355 1360

  <Desc / Clms Page number 52>

 Ile Ile Met MétAla Glu Ser Ala Asp Gln Pro Lys Asp Val Lys Lys
1365 1370 1375 Thr Phe Asp Ile Leu Asn Ile Ala Phe Val Val Ile Phe Thr Ile Glu
1380 1385 1390 Cys Leu Ile Lys Val Phe Ala Leu Arg Gln His Tyr Phe Thr Asn Gly
1395 1400 1405 Trp Asn Leu Phe Asp Cys Val Val Val Val Leu Ser Ile Ile Ser Thr
1410 1415 1420 Leu Val Ser Arg Leu Glu Asp Ser Asp Ile Ser Phe Pro Pro Thr Leu 1425 1430 1435 1440 Phe Arg Val Val Arg Leu Ala Arg Ile Gly Arg Ile Leu Arg Leu Val
1445 1450 1455

    Arg Ala Ala Arg Gly Ile Arg Thr Leu Leu Phe Ala Leu Met Met Ser
1460 1465 1470 Leu Pro Ser Leu Phe Asn Ile Gly Leu Leu Leu Phe Leu Val Met Phe
1475 1480 1485 Tyr Ala Island Phe Gly Island Met Ser Trp Phe Ser Lys Val Lys Lys Gly
1490 1495 1500 Ser Gly Ile Asp Asp Ile Phe Asn Phe Glu Thr Phe Thr Gly Ser Met 1505 1510 1515 1520 Leu Cys Leu Phe Gln Ile Thr Thr Ser Ala Gly Trp Asp Thr Leu Leu
1525 1530 1535 Asn Pro Met Leu Glu Ala Lys Glu His Cys Asn Ser Ser Ser Gln Asp
1540 1545 1550 Ser Cys Gln Gln Pro Gln Ile Ala Val Val Tyr Phe Val Ser Tyr Ile
1555 1560 1565 Ile Ser Phe Leu Ile Val Val Asn Met Tyr Ile Ala Val Ile Leu
1570 1575 1580 Glu Asn Phe Asn Thr Ala Thr Glu Glu Ser Glu Asp Pro Leu Gly Glu 1585 1590 1595 1600 Asp Asp Phe Glu Ile Phe Tyr Glu Val Trp Glu Lys Phe Asp Pro Glu
1605 1610 1615 Ala Ser Gln Phe Ile Gln Tyr Ser Ala Leu Ser Asp Phe Ala Asp Ala

  
1620 1625 1630 Leu Pro Glu Pro Leu Arg Val Ala Lys Pro Asn Lys Phe Gln Phe Leu

  <Desc / Clms Page number 53>

 
1635 1640 1645
Val Met Asp Leu Pro Met Val Met Gly Asp Arg Leu His Cys Met Asp
1650 1655 1660
Val Leu Phe Ala Phe Thr Thr Arg Val Leu Gly Asp Ser Ser Gly Leu
1665 1670 1675 1680
Asp Thr Met Lys Thr Met Met Glu Glu Lys Phe Met Glu Ala Asn Pro
1685 1690 1695
Phe Lys Lys Leu Tyr Glu Pro Ile Val Thr Thr Thr Lys Arg Lys Glu
1700 1705 1710
Glu Glu Gin Gly Ala Ala Val Ile Gln Arg Ala Tyr Arg Lys His Met
1715 1720 1725
Glu Lys Met Val Lys Leu Arg Leu Lys Asp Arg Ser Ser Ser Ser His
1730 1735 1740
Gin Val Phe Cys Asn Gly Asp Leu Ser Ser Leu Asp Val Ala Lys Val
1745 1750 1755 1760
Lys Val His Asn Asp
1765 (4) INFORMATION FOR SEQ ID # 3:

   (I) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: base pairs (B) TYPE: nucleic (C) STRAND TYPE: (D) TOPOLOGY: (II) TYPE OF MOLECULE: cDNA (III) HYPOTHETIC: (IV) ANTISENSE: (VI) SOURCE OF ORIGIN: (A) ORGANISM: human (B) TYPE OF TISSUE: from the golden root (C) TYPE OF CELL: nerve peripheral (XI) DESCRIPTION OF THE SEQUENCE:

   ID # 3: GCTGAGCAGT GGGGCACTGA TATTTGAAGA TGTTCACCTT GAGAACCAAC

  <Desc / Clms Page number 54>

 
51 CCAAAATCCA AGAATTACTA AATTGTACTG ACATTATTTT TACACATATT
101 TTTATCCTGG AGATGGTACT AAAATGGGTA GCCTTCGGAT TTGGAAAGTA
151 TTTCACCAGT GCCTGGTGCT GCCTTGATTT CATCATTGTG ATTGTCTCTG
201 TGACCACCCT CATTAACTTA ATGGAATTGA AGTCCTTCCG GACTCTACGA
251 GCACTGAGGC CTCTTCGTGC GCTGTCCCAG TTTGAAGGAA TGAAGGTGGT
301 GGTCAATGCT CTCATAGGTG CCATACCTGC CATTCTGAAT GTTTTGCTTG
351 TCTGCCTCAT TTTCTGGCTC GTATTTTGTA TTCTGGGAGT ATACTTCTTT
401 TCTGGAAAAT TTGGGAAATG CATTAATGGA ACAGACTCAG TTATAAATTA
451 TACCATCATT ACAAATAAAA GTCAATGTGA AAGTGGCAAT TTCTCTTGGA
501 TCAACCAGAA AGTCAACTTT GACAATGTGG GAAATGCTTA CCTCGCTCTG
551 CTGCAAGTGG CAACATTTAA GGGCTGGATG GATATTATAT ATGCAGCTGT
601 TGATTCCACA GAGAAAGAAC AACAGCCAGA GTTTGAGAGC AATTCACTCG
651 GTTACATTTA CTTCGTAGTC TTTATCATCT

  TTGGCTCATT CTTCACTCTG 701 AATCTCTTCA TTGGCGTTAT CATTGACAAC TTCAACCAAC AGCAGAAAAA 751 GTTAGGTGGC CAAGACATTT TTATGACAGA AGAACAGAAG AAATACTATA
801 ATGCAATGAA AAAATTAGGA TCCAAAAAAC CTCAAAAACC CATTCCACGG
851 CCCGTT (5) INFORMATION FOR SEQ ID NO 4: (I) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: base pairs (B) TYPE: nucleic (C) TYPE OF STRAND: (D) TOPOLOGY: (II) TYPE OF MOLECULE: (A) DESCRIPTION: = "DNA probe / domain IV" (III) HYPOTHETIC: no

  <Desc / Clms Page number 55>

 (IV) ANTISENSE: (VI) SOURCE OF ORIGIN: (A) ORGANISM: rat (B) TYPE OF TISSUE: from the golden root (C) TYPE OF CELL: peripheral (XI) DESCRIPTION OF THE SEQUENCE:

   ID 4: 1 CTCAACATGG TTACGATGAT GGTGGAGACC GACGAGCAGG 'GCGAGGAGAA 51 GACGAAGGTT CTGGGCAGAA TCAACCAGTT CTTTGTGGCC GTCTTCACGG 101 GCGAGTGTGT GATGAAGATG TTCGCCCTGC GACAGTACTA TTTCACCAAC 151 GGCTGGAACG TGTTCGACTT CATAGTGGTG ATCCTGTCCA TTGGGAGTCT 201 GCTGTTTCT GCAATCCTTA AGTCACTGGA AAACTACTTC TCCCCGACGC 251 TCTTCCGGGT CATCCGTCTG GCCAGGATCG GCCGCATCCT CAGGCTGATC 301 CGAGCAGCCA AGGGGATTCG CACGCTGCTC TTCGCCCTCA TGATGTCCCT 351 GCCCGCCCTC TTCAACATCG GCCTCCTCCT CTTCCTCGTC ATGTTCATCT 401 ACTCCATCTT CGGCATGGCC AGCTTCGCTA ACGTCGTGGA CGAGGCCGGC 451 ATCGACGACA TGTTCAACTT CAAGACCTTT GGCAACAGCA TGCTGTGCCT 501 GTTCCAGATC ACCACCTCGG CCGGCTGGGA CGGCCTCCTC AGCCCCATCC 551 TCAACACGGG GCCTCCCTAC TGCGACCCCA ACCTGCCCAA CAGCAACGGC 601 TCCCGGGGGA ACTGCGGGAG CCCGGCGGTG GGCATCATCT TCTTCACCAC 651 CTACATCATC ATCTCCTTCC TCATCGTGGT CAACATGTAT ATCGCAGTCA TC 701 (6)

   INFORMATION FOR SEQ ID NO 5: (I) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 334 base pairs (B) TYPE: nucleic (C) TYPE OF STRAND: (D) TOPOLOGY: (II) TYPE OF MOLECULE: ( A) DESCRIPTION: cDNA (III) HYPOTHETIC: (IV) ANTISENSE: (VI) SOURCE OF ORIGIN: (A) ORGANISM:

  <Desc / Clms Page number 56>

 (B) TYPE OF FABRIC: (C) TYPE OF CELL: (XI) DESCRIPTION OF THE SEQUENCE:

   ID # 5:
1 GTCGACTCTA GATCAGGGTG AAGATGGAGG AGAGGTACTA CCCGGTGATC
51 TTCCCGGACG AGCGGAATTT CCGCCCCTTC ACTTCCGACT CTCTGGCTGC 101 CATAGAGAAG CGGATTGCTA TCCAAAAGGA GAGGAAGAAG TCCAAAGACA 151 AGGCGGCAGC TGAGCCCCAG CCTCGGCCTC AGCTTGACCT AAAGGCCTCC 201 AGGAAGTTAC CTAAGCTTTA TGGTGACATT CCCCCTGAGC TTGTAGCGAA 251 GCCTCTGGAA GACCTGGACC CATTCTACAA AGACCATAAG ACATTCATGG 301 TGTTGAACAA GAAGAGAACA ATTTATCGCT TCAGCGCCAA GCGGGCCTTG 351 TTCATTCTGG GGCCTTTTAA TCCCCTCAGA AGCTTAATGA TTCGTATCTC 401 TGTCCATTCA GTCTTTAGCA TGTTCATCAT CTGCACGGTG ATCATCAACT 451 GTATGTTCAT GGCGAATTCT ATGGAGAGAA GTTTCGACAA CGACATTCCC 501 GAATACGTCT TCATTGGGAT TTATATTTTA GAAGCTGTGA TTAAAATATT 551 GGCAAGAGGC TTCATTGTGG ATGAGTTTTC CTTCCTCCGA GATCCGTGGA 601 ACTGGCTGGA CTTCATTGTC ATTGGAACAG CGATCGCAAC TTGTTTTCCG 651 GGCAGCCAAG TCAATCTTTC AGCTCTTCGT ACCTTCCGAG TGTTCAGAGC 701

  TCTGAAGGCG ATTTCAGTTA TCTCAGGTCT GAAGGTCATC GTAGGTGCCC 751 TGCTGCGCTC GGTGAAGAAG CTGGTAGACG TGATGGTCCT CACTCTCTTC 801 TGCCTCAGCA TCTTTGCCCT GGTCGGTCAG CAGCTGTTCA TGGGAATTCT 851 GAACCAGAAG TGTATTAAGC ACAACTGTGG CCCCAACCCT GCATCCAACA 901 AGGATTGCTT TGAAAAGGAA AAAGATAGCG AAGACTTCAT AATGTGTGGT 951 ACCTGGCTCG GCAGCAGACC CTGTCCCAAT GGTTCTACGT GCGATAAAAC

  <Desc / Clms Page number 57>

 1001 CACATTGAAC CCAGACAATA ATTATACAAA GTTTGACAAC TTTGGCTGGT 1051 CCTTTCTCGC CATGTTCCGG GTTATGACTC AAGACTCCTG GGAGAGGCTT 1101 TACCGACAGA TCCTGCGGAC CTCTGGGATC TACTTTGTCT TCTTCTTCGT 1151 GGTGGTCATC TTCCTGGGCT CCTTCTACCT GCTTAACCTA ACCCTGGCTG 1201 TTGTCACCAT GGCTTATGAA GAACAGAACA GAAATGTAGC TGCTGAGACA 1251 GAGGCCAAGG AGAAAATGTT TCAGGAAGCC CAGCAGCTGT TAAGGGAGGA 1301 GAAGGAGGCT CTGGTTGCCA TGGGAATTGA

  CAGAAGTTCC CTTAATTCCC 1351 TTCAAGCTTC ATCCTTTTCC CCGAAGAAGA GGAAGTTTTT CGGTAGTAAG 1401 ACAAGAAAGT CCTTCTTTAT GAGAGGGTCC AAGACGGCCC AAGCCTCAGC 1451 GTCTGATTCA GAGGACGATG CCTCTAAAAA TCCACAGCTC CTTGAGCAGA 1501 CCAAACGACT GTCCCAGAAC TTGCCAGTGG ATCTCTTTGA TGAGCACGTG 1551 GACCCCCTCC ACAGGCAGAG AGCGCTGAGC GCTGTCAGTA TCTTAACCAT 1601 CACCATGCAG GAACAAGAAA AATTCCAGGA GCCTTGTTTC CCATGTGGGA 1651 AAAATTTGGC CTCTAAGTAC CTGGTGTGGG ACTGTAGCCC TCAGTGGCTG 1701 TGCATAAAGA AGGTCCTGCG GACCATCATG ACGGATCCCT TTACTGAGCT 1751 GGCCATCACC ATCTGCATCA TCATCAATAC CGTTTTCTTA GCCGTGGAGC 1801 ACCACAACAT GGATGACAAC TTAAAGACCA TACTGAAAAT AGGAAACTGG 1851 GTTTTCACGG GAATTTTCAT AGCGGAAATG TGTCTCAAGA TCATCGCGCT 1901 CGACCCTTAC CACTACTTCC GGCACGGCTG GAATGTTTTT GACAGCATCG 1951 TGGCCCTCCT GAGTCTCGCT GATGTGCTCT ACAACACACT GTCTGATAAC 2001 AATAGGTCTT TCTTGGCTTC CCTCAGAGTG CTGAGGGTCT TCAAGTTAGC 2051 CAAATCCTGG CCCACGTTAA

  ACACTCTCAT TAAGATCATC GGCCACTCCG 2101 TGGGCGCGCT TGGAAACCTG ACTGTGGTCC TGACTATCGT GGTCTTCATC 2151 TTTTCTGTGG TGGGCATGCG GCTCTTCGGC ACCAAGTTTA ACAAGACCGC 2201 CTACGCCACC CAGGAGCGGC CCAGGCGGCG CTGGCACATG GATAATTTCT 2251 ACCACTCCTT CCTGGTGGTG TTCCGCATCC TCTGTGGGGA ATGGATCGAG 2301 AACATGTGGG GCTGCATGCA GGATATGGAC GGCTCCCCGT TGTGCATCAT 2351 TGTCTTTGTC CTGATAATGG TGATCGGGAA GCTTGTGGTG CTTAACCTCT

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 2401 TCATTGCCTT GCTGCTCAAT TCCTTCAGCA ATGAGGAGAA GGATGGGAGC 2451 CTGGAAGGAG AGACCAGGAA AACCAAAGTG CAGCTAGCCC TGGATCGGTT 2501 CCGCCGGGCC TTCTCCTTCA TGCTGCACGC TCTTCAGAGT TTTTGTTGCA 2551 AGAAATGCAG GAGGAAAAAC TCGCCAAAGC CAAAAGAGAC AACAGAAAGC 2601 TTTGCTGGTG AGAATAAAGA CTCAATCCTC CCGGATGCGA GGCCCTGGAA 2651 GGAGTATGAT ACAGACATGG CTTTGTACAC TGGACAGGCC GGGGCTCCGC 2701 TGGCCCCACT CGCAGAGGTA GAGGACGATG TGGAATATTG TGGTGAAGGC 2751

  GGTGCCCTAC CCACCTCACA ACATAGTGCT GGAGTTCAGG CCGGTGACCT 2801 CCCTCCAGAG ACCAAGCAGC TCACTAGCCC GGATGACCAA GGGGTTGAAA 2851 TGGAAGTATT TTCTGAAGAA GATCTGCATT TAAGCATACA GAGTCCTCGA 2901 AAGAAGTCTG ACGCAGTGAG CATGCTCTCG GAATGCAGCA CAATTGACCT 2951 GAATGATATC TTTAGAAATT TACAGAAAAC AGTTTCCCCC AAAAAGCAGC 3001 CAGATAGATG CTTTCCCAAG GGCCTTAGTT GTCACTTTCT ATGCCACAAA 3051 ACAGACAAGA GAAAGTCCCC CTGGGTCCTG TGGTGGAACA TTCGGAAAAC 3101 CTGCTACCAA ATCGTGAAGC ACAGCTGGTT TGAGAGTTTC ATAATCTTTG 3151 TTATTCTGCT GAGCAGTGGA GCGCTGATAT TTGAAGATGT CAATCTCCCC 3201 AGCCGGCCCC AAGTTGAGAA ATTACTAAGG TGTACCGATA ATATTTTCAC 3251 ATTTATTTTC CTCCTGGAAA TGATCCTGAA GTGGGTGGCC TTTGGATTCC 3301 GGAGGTATTT CACCAGTGCC TGGTGCTGGC TTGATTTCCT CATTGTGGTG 2251 GTGTCTGTGC TCAGTCTCAT GAATCTACCA AGCTTGAAGT CCTTCCGGAC 3401 TCTGCGGGCC CTGAGACCTC TGCGGGCGCT GTCCCAGTTT GAAGGAATGA 3451 AGGTTGTCGT CTACGCCCTG ATCAGCGCCA

  TACCTGCCAT TCTCAATGTC 3501 TTGCTGGTCT GCCTCATTTT CTGGCTCGTA TTTTGTATCT TGGGAGTAAA 3551 TTTATTTTCT GGGAAGTTTG GAAGGTGCAT TAACGGGACA GACATAAATA 3601 TGTATTTGGA TTTTACCGAA GTTCCGAACC GAAGCCAATG TAACATTAGT 3651 AATTACTCGT GGAAGGTCCC GCAGGTCAAC TTTGACAACG TGGGGAATGC 3701 CTATCTCGCC CTGCTGCAAG TGGCAACCTA TAAGGGCTGG CTGGAAATCA 3751 TGAATGCTGC TGTCGATTCC AGAGAGAAAG ACGAGCAGCC GGACTTTGAG

  <Desc / Clms Page number 59>

 3801 GCGAACCTCT ACGCGTATCT CTACTTTGTG GTTTTTATCA TCTTCGGCTC 3851 CTTCTTTACC CTGAACCTCT TTATCGGTGT TATTATTGAC AACTTCAATC 3901 AGCAGCAGÀA ÀAAGTTAGGT GGCCAAGACA TCTTCATGAC TGAGGAGCAG 3951 AAGAAATATT ACAATGCAAT GAAAAAGTTA GGAACCAAGA AACCTCAAAA 4001 GCCCATCCCA AGGCCCCTGA ACAAATGTCA AGCCTTTGTG TTCGACCTGG 4051 TCACAAGCCA

  GGTCTTTGAC GTCATCATTC TGGGTCTTAT TGTCTTAAAT 4101 ATGATTATCA TGATGGCTGA ATCTGCCGAC CAGCCCAAAG ATGTGAAGAA 4151 AACCTTTGAT ATCCTCAACA TAGCCTTCGT GGTCATCTTT ACCATAGAGT 4201 GTCTCATCAA AGTCTTTGCT TTGAGGCAAC ACTACTTCAC CAATGGCTGG 4251 AACTTATTTG ATTGTGTGGT CGTGGTTCTT TCTATCATTA GTACCCTGGT 4301 TTCCCGCTTG GAGGACAGTG ACATTTCTTT CCCGCCCACG CTCTTCAGAG 4351 TCGTCCGCTT GGCTCGGATT GGTCGAATCC TCAGGCTGGT CCGGGCTGCC 4401 CGGGGAATCA GGACCCTCCT CTTTGCTTTG ATGATGTCTC TCCCCTCTCT 4451 CTTCAACATC GGTCTGCTGC TCTTCCTGGT GATGTTCATT TACGCCATCT 4501 TTGGGATGAG CTGGTTTTCC AAAGTGAAGA AGGGCTCCGG GATCGACGAC 4551 ATCTTCAACT TCGAGACCTT TACGGGCAGC ATGCTGTGCC TCTTCCAGAT 4601 AACCACTTCG GCTGGCTGGG ATACCCTCCT CAACCCCATG CTGGAGGCAA 4651 AAGAACACTG CAACTCCTCC TCCCAAGACA GCTGTCAGCA GCCGCAGATA 4701 GCCGTCGTCT ACTTCGTCAG TTACATCATC ATCTCCTTCC TCATCGTGGT 4751 CAACATGTAC ATCGCTGTGA TCCTCGAGAA CTTCAACACA

  GCCACGGAGG 4801 AGAGCGAGGA CCCTCTGGGA GAGGACGACT TTGAAATCTT CTATGAGGTC 4851 TGGGAGAAGT TTGACCCCGA GGCGTCGCAG TTCATCCAGT ATTCGGCCCT 4901 CTCTGACTTT GCGGACGCCC TGCCGGAGCC GTTGCGTGTG GCCAAGCCGA 4951 ATAAGTTTCA GTTTCTAGTG ATGGACTTGC CCATGGTGAT GGGCGACCGC 5001 CTCCATTGCA TGGATGTTCT CTTTGCTTTC ACTACCAGGG TCCTCGGGGA 5051 CTCCAGCGGC TTGGATACCA TGAAAACCAT GATGGAGGAG AAGTTTATGG 5101 AGGCCAACCC TTTTAAGAAG CTCTACGAGC CCATAGTCAC CACCACCAAG 5151 AGGAAGGAGG AGGAGCAAGG CGCCGCCGTC ATCCAGAGGG CCTACCGGAA

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 5201 ACACATGGAG AAGATGGTCA AACTGAGGCT GAAGGACAGG TCAAGTTCAT 5251 CGCACCAGGT GTTTTGCAAT GGAGACTTGT CCAGCTTGGA TGTGGCCAAG 5301 GTCAAGGTTC ACAATGACIG AACCCTCATC TAGA


    

Claims (4)

 CLAIMS 1. Isolated DNA sequence, characterized in that it comprises the nucleotide sequence indicated in SEQ ID No. 1 and SEQ ID No. 3.  2. DNA according to claim 1, characterized in that said DNA sequence codes for a sodium channel protein or a fragment thereof.  3. DNA according to claim 2, characterized in that said sodium channel protein is the a subunit or a fragment thereof.  4. DNA according to claim 3, characterized in that said sodium channel protein is resistant to tetrodotoxin.  5. DNA according to claim 3 or 4, characterized in that said sodium channel protein is found in mammals.  6. DNA according to claim 3 or 4, characterized in that said sodium channel protein is found in rats.  7. DNA according to claim 3 or 4, characterized in that said sodium channel protein is found in humans.  8. DNA according to claim 1, characterized in that said DNA is cDNA.  9. DNA according to claim 1, characterized in that said DNA is synthetic DNA.  10. Expression vectors, characterized in that they comprise the DNA of claim 8.  11. Expression vectors, characterized in that they comprise the synthetic DNA of claim 9.  12. Host cells transformed by the expression vectors of claim 10.  13. Host cells transformed by the expression vectors of claim 11.  <Desc / Clms Page number 62>    14. Recombinant polynucleotide, characterized in that it comprises a nucleic acid sequence originating from the DNA sequence of claim 1.  15. Sodium channel protein encoded by a DNA according to claims 1 to 9 or allelic variants thereof.  16. Sodium channel protein resistant to tetrodotoxin, encoded by DNA according to claims 1 to 9 or allelic variants thereof.  17. Protein according to claim 16, characterized in that it comprises the amino acid sequence indicated in SEQ ID No. 2.  18. A method of identifying inhibitors of the sodium channel protein resistant to tetrodotoxin, characterized in that it comprises bringing a compound, suspected to be such an inhibitor, into contact with the sodium channel protein of claim 16, and measuring the activity of said expressed sodium channel protein.  19. Poly- and / or monoclonal antibodies raised against a sodium channel protein resistant to tetrodotoxin, encoded by a DNA of claims 1 to 9 or allelic variants thereof.  20. A diagnostic kit, characterized in that it comprises a polynucleotide according to claim 14, capable of specifically hybridizing with a sodium channel protein resistant to tetrodotoxin or a fragment of such a protein.  21. Use of an isolated DNA sequence according to claims 1 to 9, for the identification of a compound suspected of being an inhibitor of sodium channel protein resistant to tetrodotoxin.  <Desc / Clms Page number 63>    Figure 1 A: SEQ IDNO: 1 1 GAAGTCACAG GAGTGTCTGT CAGCGAGAGG AAGAAGGGAG AGTTTACTGA 51 GTGTCTTCTG: CCCCTCCTCA GGGTGAAGAT GGAGGAGAGG TACTACCCGG 101 TGATCTTCCC GGACGAGCGG AATTTCCGCC CCTTCACTTC CGACTCTCTG 151 GCTGCCATAG AGAAGCGGAT TGCTATCCAA AAGGAGAGGA AGAAGTCCAA 201 AGACAAGGCG GCAGCTGAGC CCCAGCCTCG GCCTCAGCTT GACCTAAAGG 251 CCTCCAGGAA GTTACCTAAG CTTTATGGTG ACATTCCCCC TGAGCTTGTA 301 GCGAAGCCTC TGGAAGACCT GGACCCATTC TACAAAGACC ATAAGACATT 351 CATGGTGTTG AACAAGAAGA GAACAATTTA TCGCTTCAGC GCCAAGCGGG 401 CCTTGTTCAT TCTGGGGCCT TTTAATCCCC TCAGAAGCTT AATGATTCGT 451 ATCTCTGTCC ATTCAGTCTT TAGCATGTTC ATCATCTGCA CGGTGATCAT 501 CAACTGTATG TTCATGGCGA ATTCTATGGA GAGAAGTTTC GACAACGACA 551 TTCCCGAATA CGTCTTCATT GGGATTTATA TTTTAGAAGC TGTGATTAAA 601 ATATTGGCAA GAGGCTTCAT TGTGGATGAG TTTTCCTTCC TCCGAGATCC 651 GTGGAACTGG CTGGACTTCA TTGTCATTGG AACAGCGATC GCAACTTGTT 701 TTCCGGGCAG CCAAGTCAAT CTTTCAGCTC TTCGTACCTT CCGAGTGTTC 751 AGAGCTCTGA AGGCGATTTC AGTTATCTCA GGTCTGAAGG TCATCGTAGG 801 TGCCCTGCTG CGCTAGTCGTGA 851 TCTTCTGCCT CAGCATCTTT GCCCTGGTCG GTCAGCAGCT GTTCATGGGA 901 ATTCTGAACC AGAAGTGTAT TAAGCACAAC TGTGGCCCCA ACCCTGCATC 951 CAACAAGGAT TGCTTTGAAA AGGAAAAAGA TAGCGAAGAC TTCATAATGT 1001 GTGGTACCTG GCTCGGCAGC AGACCCTGTC CCAATGGTTC TACGTGCGAT 1051 AAAACCACAT TGAACCCAGA CAATAATTAT ACAAAGTTTG ACAACTTTGG 1101 CTGGTCCTTT CTCGCCATGT TCCGGGTTAT GACTCAAGAC TCCTGGGAGA 1151 GGCTTTACCG ACAGATCCTG CGGACCTCTG GGATCTACTT TGTCTTCTTC 1201 TTCGTGGTGG TCATCTTCCT GGGCTCCTTC TACCTGCTTA ACCTAACCCT  <Desc / Clms Page number 64>   Figure 1B:  SEQ ID No. 1 1251 GGCTGTTGTC ACCATGGCTT ATGAAGAACA GAACAGAAAT GTAGCTGCTG 1301 AGACAGAGGC CAAGGAGAAA ATGTTTCAGG AAGCCCAGCA GCTGTTAAGG 1351 GAGGAGAAGG AGGCTCTGGT TGCCATGGGA ATTGACAGAA GTTCCCTTAA 1401 TTCCCTTCAA GCTTCATCCT TTTCCCCGAA GAAGAGGAAG TTTTTCGGTA 1451 GTAAGACAAG AAAGTCCTTC TTTATGAGAG GGTCCAAGAC GGCCCAAGCC 1501 TCAGCGTCTG ATTCAGAGGA CGATGCCTCT AAAAATCCAC AGCTCCTTGA 1551 GCAGACCAAA CGACTGTCCC AGAACTTGCC AGTGGATCTC TTTGATGAGC 1601 ACGTGGACCC CCTCCACAGG CAGAGAGCGC TGAGCGCTGT CAGTATCTTA 1651 ACCATCACCA TGCAGGAACA AGAAAAATTC CAGGAGCCTT GTTTCCCATG 1701 TGGGAAAAAT TTGGCCTCTA AGTACCTGGT GTGGGACTGT AGCCCTCAGT 1751 GGCTGTGCAT AAAGAAGGTC CTGCGGACCA TCATGACGGA TCCCTTTACT 1801 GAGCTGGCCA TCACCATCTG CATCATCATC AATACCGTTT TCTTAGCCGT 1851 GGAGCACCAC AACATGGATG ACAACTTAAA GACCATACTG AAAATAGGAA 1901 ACTGGGTTTT CACGGGAATT TTCATAGCGG AAATGTGTCT CAAGATCATC 1951 GCGCTCGACC CTTACCACTA CTTCCGGCAC GGCTGGAATG TTTTTGACAG 2001 CATCGTGGCC CTCCTGAGTC TCGCTGATGT GCTCTACAAC ACACTGTCTG 2051 ATAACAATAG GTCTTTCTTG GCTTCCCTCA GAGTGCTGAG GGTCTTCAAG 2101 TTAGCCAAAT CCTGGCCCAC GTTAAACACT CTCATTAAGA TCATCGGCCA 2151 CTCCGTGGGC GCGCTTGGAA ACCTGACTGT GGTCCTGACT ATCGTGGTCT 2201 TCATCTTTTC TGTGGTGGGC ATGCGGCTCT TCGGCACCAA GTTTAACAAG 2251 ACCGCCTACG CCACCCAGGA GCGGCCCAGG CGGCGCTGGC ACATGGATAA 2301 TTTCTACCAC TCCTTCCTGG TGGTGTTCCG CATCCTCTGT GGGGAATGGA 2351 TCGAGAACAT GTGGGGCTGC ATGCAGGATA TGGACGGCTC CCCGTTGTGC 2401 ATCATTGTCT TTGTCCTGAT AATGGTGATC GGGAAGCTTG TGGTGCTTAA  <Desc / Clms Page number 65>   Figure 1 C:  SEQ ID NO: 2451 CCTCTTCATT-GCCTTGCTGC TCAATTCCTT CAGCAATGAG GAGAAGGATG 2501 GGAGCCTGGA AGGAGAGACC AGGAAAACCA AAGTGCAGCT AGCCCTGGAT 2551 CGGTTCCGCC GGGCCTTCTC CTTCATGCTG CACGCTCTTC AGAGTTTTTG 2601 TTGCAAGAAA TGCAGGAGGA AAAACTCGCC AAAGCCAAAA GAGACAACAG 2651 AAAGCTTTGC TGGTGAGAAT AAAGACTCAA TCCTCCCGGA TGCGAGGCCC 2701 TGGAAGGAGT ATGATACAGA CATGGCTTTG TACACTGGAC AGGCCGGGGC 2751 TCCGCTGGCC CCACTCGCAG AGGTAGAGGA CGATGTGGAA TATTGTGGTG 2801 AAGGCGGTGC CCTACCCACC TCACAACATA GTGCTGGAGT TCAGGCCGGT 2851 GACCTCCCTC CAGAGACCAA GCAGCTCACT AGCCCGGATG ACCAAGGGGT 2901 TGAAATGGAA GTATTTTCTG AAGAAGATCT GCATTTAAGC ATACAGAGTC 2951 CTCGAAAGAA GTCTGACGCA GTGAGCATGC TCTCGGAATG CAGCACAATT 3001 GACCTGAATG ATATCTTTAG AAATTTACAG AAAACAGTTT CCCCCAAAAA 3051 GCAGCCAGAT AGATGCTTTC CCAAGGGCCT TAGTTGTCAC TTTCTATGCC 3101 ACAAAACAGA CAAGAGAAAG TCCCCCTGGG TCCTGTGGTG GAACATTCGG 3151 AAAACCTGCT ACCAAATCGT GAAGCACAGC TGGTTTGAGA GTTTCATAAT 3201 CTTTGTTATT CTGCTGAGCA GTGGAGCGCT GATATTTGAA GATGTCAATC 3251 TCCCCAGCCG GCCCCAAGTT GAGAAATTAC TAAGGTGTAC CGATAATATT 3301 TTCACATTTA TTTTCCTCCT GGAAATGATC CTGAAGTGGG TGGCCTTTGG 3351 ATTCCGGAGG TATTTCACCA GTGCCTGGTG CTGGCTTGAT TTCCTCATTG 3401 TGGTGGTGTC TGTGCTCAGT CTCATGAATC TACCAAGCTT GAAGTCCTTC 3451 CGGACTCTGC GGGCCCTGAG ACCTCTGCGG GCGCTGTCCC AGTTTGAAGG 3501 AATGAAGGTT GTCGTCTACG CCCTGATCAG CGCCATACCT GCCATTCTCA 3551 ATGTCTTGCT GGTCTGCCTC ATTTTCTGGC TCGTATTTTG TATCTTGGGA 3601 GTAAATTTAT TTTCTGGGAA GTTTGGAAGG TGCATTAACG GGACAGACAT  <Desc / Clms Page number 66>   Figure 1D: SEQID NO:  3651 AAATATGTAT TTGGATTTTA CCGAAGTTCC GAACCGAAGC CAATGTAACA 3701 TTAGTAATTA CTCGTGGAAG GTCCCGCAGG TCAACTTTGA CAACGTGGGG 3751 AATGCCTATC TCGCCCTGCT GCAAGTGGCA ACCTATAAGG GCTGGCTGGA 3801 AATCATGAAT GCTGCTGTCG ATTCCAGAGA GAAAGACGAG CAGCCGGACT 3851 TTGAGGCGAA CCTCTACGCG TATCTCTACT TTGTGGTTTT.TATCATCTTC 3901 GGCTCCTTCT TTACCCTGAA CCTCTTTATC GGTGTTATTA TTGACAACTT 3951 CAATCAGCAG CAGAAAAAGT TAGGTGGCCA AGACATTTTT ATGACAGAAG 4001 AACAGAAGAA ATATTACAAT GCAATGAAAA AGTTAGGAAC CAAGAAACCT 4051 CAAAAGCCCA TCCCAAGGCC CCTGAACAAA TGTCAAGCCT TTGTGTTCGA 4101 CCTGGTCACA AGCCAGGTCT TTGACGTCAT CATTCTGGGT CTTATTGTCT 4151 TAAATATGAT TATCATGATG GCTGAATCTG CCGACCAGCC CAAAGATGTG 4201 AAGAAAACCT TTGATATCCT CAACATAGCC TTCGTGGTCA TCTTTACCAT 4251 AGAGTGTCTC ATCAAAGTCT TTGCTTTGAG GCAACACTAC TTCACCAATG 4301 GCTGGAACTT ATTTGATTGT GTGGTCGTGG TTCTTTCTAT CATTAGTACC 4351 CTGGTTTCCC GCTTGGAGGA CAGTGACATT TCTTTCCCGC CCACGCTCTT 4401 CAGAGTCGTC CGCTTGGCTC GGATTGGTCG AATCCTCAGG CTGGTCCGGG 4451 CTGCCCGGGG AATCAGGACC CTCCTCTTTG CTTTGATGAT GTCTCTCCCC 4501 TCTCTCTTCA ACATCGGTCT GCTGCTCTTC CTGGTGATGT TCATTTACGC 4551 CATCTTTGGG ATGAGCTGGT TTTCCAAAGT GAAGAAGGGC TCCGGGATCG 4601 ACGACATCTT CAACTTCGAG ACCTTTACGG GCAGCATGCT GTGCCTCTTC 4651 CAGATAACCA CTTCGGCTGG CTGGGATACC CTCCTCAACC CCATGCTGGA 4701 GGCAAAAGAA CACTGCAACT CCTCCTCCCA AGACAGCTGT CAGCAGCCGC 4751 AGATAGCCGT CGTCTACTTC GTCAGTTACA TCATCATCTC CTTCCTCATC 4801 GTGGTCAACA TGTACATCGC TGTGATCCTC GAGAACTTCA ACACAGCCAC  <Desc / Clms Page number 67>   Figure lE:    SEQ ID NO: 1 4851 GGAGGAGAGC GAGGACCCTC TGGGAGAGGA CGACTTTGAA ATCTTCTATG 4901 AGGTCTGGGA-GAAGTTTGAC CCCGAGGCGT CGCAGTTCAT CCAGTATTCG 4951 GCCCTCTCTG ACTTTGCGGA CGCCCTGCCG GAGCCGTTGC GTGTGGCCAA 5001 GCCGAATAAG TTTCAGTTTC TAGTGATGGA CTTGCCCATG GTGATGGGCG 5051 ACCGCCTCCA TTGCATGGAT GTTCTCTTTG CTTTCACTAC CAGGGTCCTC 5101 GGGGACTCCA GCGGCTTGGA TACCATGAAA ACCATGATGG AGGAGAAGTT 5151 TATGGAGGCC AACCCTTTTA AGAAGCTCTA CGAGCCCATA GTCACCACCA 5201 CCAAGAGGAA GGAGGAGGAG CAAGGCGCCG CCGTCATCCA GAGGGCCTAC 5251 CGGAAACACA TGGAGAAGAT GGTCAAACTG AGGCTGAAGG ACAGGTCAAG 5301 TTCATCGCAC CAGGTGTTTT GCAATGGAGA CTTGTCCAGC TTGGATGTGG 5351 CCAAGGTCAA GGTTCACAAT GACTGAACCC TCATCTCCAC CCCTACCTCA 5401 CTGCCTCACA GCTTAGCCTC CAGCCTCTGG CGAGCAGGCG GCAGACTCAC 5451 TGAACACAGG CCGTTCGATC TGTGTTTTTG GCTGAACGAG GTGACAGGTT 5501 GGCGTCCATT TTTAAATGAC TCTTGGAAAG ATTTCATGTA GAGAGATGTT 5551 AGAAGGGACT GCAAAGGACA CCGACCATAA CGGAAGGCCT GGAGGACAGT 5601 CCAACTTACA TAAAGATGAG AAACAAGAAG GAAAGATCCC AGGAAAACTT 5651 CAGATTGTGT TCTCAGTACA TCCCCCAATG TGTCTGTTCG GTGTTTTGAG 5701 TATGTGACCT GCCACATGTA GCTCTTTTTT GCATGTACGT CAAAACCCTG 5751 CAGTAAGTTG ATAGCTTGCT ACGGGTGTTC CTACCAGCAT CACAGAATTG 5801 GGTGTATGAC TCAAACCTAA AAGCATGACT CTGACTTGTC AGTCAGCACC 5851 CCGACTTTCA GACGCTCCAA TCTCTGTCCC AGGTGTCTAA CGAATAAATA 5901 GGTAAAAG  <Desc / Clms Page number 68>    EMI68.1  Figure 2A:  SEQ ID NO: 2 Met Glu Glu Arg Tyr Tyr Pro Val Ile Phe Pro Asp Glu Arg Asn Phe 1 5 - '10 1 Arg Pro Phe Thr Ser Asp Ser Leu Ala Ala Ile Glu Lys Arg Ile Ala 20 25 30 Ile Gln Lys Glu Arg Lys Lys Ser Lys Asp Lys Ala Ala Ala Glu Pro 35 40 45 Gln Pro Arg Pro Gln Leu Asp Leu Lys Ala Ser Arg Lys Leu Pro Lys 50 55 60 Leu Tyr.Gly Asp Ile Pro Pro Glu Leu Val Ala Lys Pro Leu Glu Asp 65 70 75 80 Leu Asp Pro Phe Tyr Lys Asp His Lys Thr Phe Met Val Leu Asn Lys 85 90 95 Lys Arg Thr Ile Tyr Arg Phe Ser Ala Lys Arg Ala Leu Phe Ile Leu 100 105 110 Gly Pro Phe Asn Pro Leu Arg Ser Leu Met Ile Arg Ile Ser Val His 115 120 125 Ser Val Phe Ser Met Phe Ile Ile Cys Thr Val Ile Ile Asn Cys Met 130 135 140 Phe Met Ala Asn Ser Met Glu Arg Ser Phe Asp Asn Asp Ile Pro Glu 145 150 155 160 Tyr Val Phe Ile Gly Ile Tyr Ile Leu Glu Ala Val Ile Lys Ile Leu 165 170 175 Ala Arg Gly Phe Ile Val Asp Glu Phe Ser Phe Leu Arg Asp Pro Trp 180 185 190 Asn Trp Leu Asp Phe Ile Val Ile Gly Thr Ala Ile Ala Thr. Cys Phe 195 200 205 Pro Gly Ser Gin Val Asn Leu Ser Ala Leu Arg Thr Phe Arg Val Phe 210 215 220 Arg Ala Leu Lys Ala Ile Ser Val Ile Ser Gly Leu Lys Val Ile Val 225 230 235 240 Gly Ala Leu Leu Arg Ser Val Lys Lys Leu Val Asp Val Met Val Leu 245 250 255 Thr Leu Phe Cys Leu Ser Ile Phe Ala Leu Val Gly Gin Gln Leu Phe 260 265 270 Met Gly Ile Leu Asn Gin Lys Cys Ile Lys His Asn Cys Gly Pro Asn 275 280 285 Pro Ala.Ser Asn Lys Asp Cys Phe Glu Lys Glu Lys Asp Ser Glu Asp 290 295 300 Phe Ile Met Cys Gly Thr Trp Leu Gly Ser Arg Pro Cys Pro Asn Gly 305 310 315 320  <Desc / Clms Page number 69>   Figure 2B:  SEQ ID NO- 2 Ser Thr Cys Asp Lys-Thr'Thr Leu Asn Pro Asp Asn Asn Tyr Thr Lys 325 330 335 Phe Asp Asn Phe Gly Trp Ser Phe Leu Ala Met Phe Arg Val Met Thr 340 345 350 Gln Asp Ser Trp Glu Arg Leu Tyr Arg Gln Ile Leu Arg Thr Ser Gly 355 360 365 Ile Tyr Phe Val Phe Phe Phe Val Val Val Ile Phe Leu Gly Ser Phe 370 375 380 Tyr Leu Leu Asn Leu Thr Leu Ala Val Val Thr Met Ala Tyr Glu Glu 385 390 395 400 Gln Asn Arg Asn Val Ala Ala Glu Thr Glu Ala Lys Glu Lys Met Phe 405 410 415 Gln Glu Ala Gln Gln Leu Leu Arg Glu Glu Lys Glu Ala Leu Val Ala 420 425 430 Met Gly Ile Asp Arg Ser Ser Leu Asn Ser Leu Gln Ala Ser Ser Phe 435 440 445 Ser Pro Lys Lys Arg Lys Phe Phe Gly Ser Lys Thr Arg Lys Ser Phe 450 455 460 Phe Met Arg Gly Ser Lys Thr Ala Gln Ala Ser Ala Ser Asp Ser Glu 465 470 475 480 Asp Asp Ala Ser Lys Asn Pro Gln Leu Leu Glu Gin Thr Lys Arg Leu 485 490 495 Ser Gln Asn Leu Pro Val Asp Leu Phe Asp Glu His Val Asp Pro Leu 500 505 510 His Arg Gln Arg Ala Leu Ser Ala Val Ser Ile Leu Thr Ile Thr Met 515 520 525 Gln Glu Gln Glu Lys Phe Gln Glu Pro Cys Phe Pro Cys Gly Lys Asn 530 535 540 Leu Ala Ser Lys Tyr Leu Val Trp Asp Cys Ser Pro Gln Trp Leu Cys 545 550 555 560 Ile Lys Lys Val Leu Arg Thr Ile Met Thr Asp Pro Phe Thr Glu Leu 565 570 575 Ala Ile Thr Ile Cys Ile Ile Asn Thr Val Phe Leu Ala Val Glu 580 585 590 His His Asn Met Asp Asp Asn Leu Lys Thr Ile Leu Lys Ile Gly Asn 595 600 605 Trp Val Phe Thr Gly Ile Phe Ile Ala Glu Met Cys Leu Lys Ile Ile 610 '' 615 620  <Desc / Clms Page number 70>   Figure 2C:  SEQ ID NO: 2 Ala Leu Asp Pro Tyr His Tyr Phe Arg His Gly Trp Asn Val Phe Asp 625 630 635 640 Ser Ile Val Ala Leu Leu Ser Leu Ala Asp Val Leu Tyr Asn Thr Leu 645 650 655 Ser Asp Asn Asn Arg Ser Phe Leu Ala Ser Leu Arg Val Leu Arg Val 660 665 670 Phe Lys Leu Ala Lys Ser Trp Pro Thr Leu Asn Thr Leu Ile Lys Ile 675 680 685 Ile Gly His Ser Val Gly Ala Leu Gly Asn Leu Thr Val Val Leu Thr 690 695 700 Ile Val Val Phe Ile Phe Ser Val Val Gly Met Arg Leu Phe Gly Thr 705 710 715 720 Lys Phe Asn Lys Thr Ala Tyr Ala Thr Gin Glu Arg Pro Arg Arg Arg 725 730 735 Trp His Met Asp Asn Phe Tyr His Ser Phe Leu Val Val Phe Arg Ile 740 745 750 Leu Cys Gly Glu Trp Ile Glu Asn Met Trp Gly Cys Met Gln Asp Met 755 760 765 Asp Gly Ser Pro Leu Cys Ile Ile Val Phe Val Leu Ile Met Val Ile 770 775 780 Gly Lys Leu Val Val Leu Asn Leu Phe Ile Ala Leu Leu Leu Asn Ser 785 790 795 800 Phe Ser Asn Glu Glu Lys Asp Gly Ser Leu Glu Gly Glu Thr Arg Lys 805 810 815 Thr Lys Val Gln Leu Ala Leu Asp Arg Phe Arg Arg Ala Phe Ser Phe 820 825 830 Met Leu His Ala Leu Gln Ser Phe Cys Cys Lys Lys Cys Arg Arg Lys 835 840 845 Asn Ser Pro Lys Pro Lys Glu Thr Thr Glu Ser Phe Ala Gly Glu Asn 850 855 860 Lys Asp Ser Ile Leu Pro Asp Ala Arg Pro Trp Lys Glu Tyr Asp Thr 865 870 875 880 Asp Met Ala Leu Tyr Thr Gly Gln Ala Gly Ala? Ro Leu Ala Pro Leu 885 890 895 Ala Glu Val Glu Asp Asp Val Glu Tyr Cys Gly Glu Gly Gly Ala Leu 900 905 910  <Desc / Clms Page number 71>   Figure 2D:    SEQ ID NO: 2 Pro Thr Ser Gln His Ser Ala Gly Val Gln Ala Gly Asp Leu Pro Pro 915 920 925 Glu Thr Lys Gln Leu Thr Ser Pro Asp Asp Gln Gly Val Glu Met Glu 930 935 940 Val Phe Ser Glu Glu Asp Leu His Leu Ser Ile Gln Ser Pro Arg Lys 945 950 955 960 Lys Ser Asp Ala Val Ser Met Leu Ser Glu Cys Ser Thr Ile Asp Leu 965 970 975 Asn Asp Ile Phe Arg Asn Leu Gln Lys Thr Val Ser Pro Lys Lys Gln 980 985 990 Pro Asp Arg Cys Phe Pro Lys Gly Leu Ser Cys His Phe Leu Cys His 995 1000 1005 Lys Thr Asp Lys Arg Lys Ser Pro Trp Val Leu Trp Trp Asn Ile Arg 1010 1015 1020 Lys Thr Cys Tyr Gln Ile Val Lys His Ser Trp Phe Glu Ser Phe Ile 1025 1030 1035 1040 Ile Phe Val Ile Leu Leu Ser Ser Gly Ala Leu Ile Phe Glu Asp Val 1045 1050 1055 Asn Leu Pro Ser Arg Pro Gln Val Glu Lys Leu Leu Arg Cys Thr Asp 1060 1065 1070 Asn Ile Phe Thr Phe Ile Phe Leu Leu Glu Met Ile Leu Lys Trp Val 1075 1080 1085 Ala Phe Gly Ph Arg Arg Tyr Phe Thr Ser Ala Trp Cys Trp Leu Asp 1090 1095 1100 Phe Leu Ile Val Val Val Ser Val Leu Ser Leu Met Asn Leu Pro Ser 1105 1110 1115 1120 Leu Lys Ser Phe Arg Thr Leu Arg Ala Leu Arg Pro Leu Arg Ala Leu 1125 1130 1135 Ser Gln Phe Glu Gly Met Lys Val Val Val Tyr Ala Leu Ile Ser Ala 1140 1145 1150 Ile Pro Ala Ile Leu Asn Val Leu Leu Val Cys Leu Ile Phe Trp Leu 1155 1160 1165 Val Phe Cys Ile Leu Gly Val Asn Leu Phe Ser Gly Lys Phe Gly Arg 1170 1175 1180 Cys Ile Asn Gly Thr Asp Ile Asn Met Tyr Leu Asp Phe Thr Glu Val 1185 1190 1195 1200 Pro Asn Arg Ser Gln Cys Asn Ile Ser Asn Tyr Ser Trp Lys Val Pro 1205 1210 1215  <Desc / Clms Page number 72>   Figure 2E:  SEQ ID NO: 2 Gln Val Asn Phe Asp Asn Val Gly Asn Ala Tyr Leu Ala Leu Leu Gln 1220 1225 1230 Val Ala Thr Tyr Lys Gly Trp Leu Glu Ile Met Asn Ala Ala Val Asp 1235 1240 1245 Ser Arg Glu Lys Asp Glu Gln Pro Asp Phe Glu Ala Asn Leu Tyr Ala 1250 1255 1260 Tyr Leu Tyr Phe Val Val Phe Ile Ile Phe Gly Ser Phe Phe Thr Leu 1265 1270 1275 1280 Asn Leu Phe Ile Gly Val Ile Ile Asp Asn Phe Asn Gln Gln Gln Lys 1285 1290 1295 Lys Leu Gly Gly Gln Asp Ile Phe Met Thr Glu Glu Gln Lys Lys Tyr 1300 1305 1310 Tyr Asn Ala Met Lys Lys Leu Gly Thr Lys Lys Pro Gln Lys Pro Ile 1315 1320 1325 Pro Arg Pro Leu Asn Lys Cys Gln Ala Phe Val Phe Asp Leu Val Thr 1330 1335 1340 Ser Gln Val Phe Asp Val Ile Ile Leu Gly Leu Ile Val Leu Asn Met 1345 1350 1355 1360 Ile Ile Met Met Ala Glu Ser Ala Asp Gln Pro Lys Asp Val Lys Lys 1365 1370 1375 Thr Phe Asp Ile Leu Asn Ile Ala Phe Val Val Ile Phe Thr Ile Glu 1380 1385 1390 Cys Leu Ile Lys Val Phe Ala Leu Arg Gln His Tyr Phe Thr Asn Gly 1395 1400 1405 Trp Asn Leu Phe Asp Cys Val Val Val Val Leu Ser Ile Ile Ser Thr 1410 1415 1420 Leu Val Ser Arg Leu Glu Asp Ser Asp Ile Ser Phe Pro Pro Thr Leu 1425 1430 1435 1440 Phe Arg Val Val Arg Leu Ala Arg Ile Gly Arg Ile Leu Arg Leu Val 1445 1450 1455 Arg Ala Ala Arg Gly Ile Arg Thr Leu Leu Phe Ala Leu Met Met Ser 1460 1465 1470 Leu Pro Ser Leu Phe Asn Ile Gly Leu Leu Leu Phe Leu Val Met Phe 1475 1480 1485 Tyr Ala Island Phe Gly Island Met Ser Trp Phe Ser Lys Val Lys Lys Gly 1490 1495 1500 Ser Gly Ile Asp Asp Ile Phe Asn Phe Glu Thr Phe Thr Gly Ser Met 1505 .- 1510 1515 1520  <Desc / Clms Page number 73>   Figure 2F:    SEQIDNO: 2 Leu Cys Leu Phe Gln Ile Thr Thr Ser Ala Gly Trp Asp Thr Leu Leu 1525 1530 1535 Asn Pro Met Leu Glu Ala Lys Glu His Cys Asn Ser Ser Ser Gln Asp 1540 1545 1550 Ser Cys Gln Gln Pro Gln Ile Ala Val Val Tyr Phe Val Ser Tyr Ile 1555 1560 1565 Ile Ser Phe Leu Ile Val Val Asn Met Tyr Ile Ala Val Ile Leu 1570 1575 1580 Glu Asn Phe Asn Thr Ala Thr Glu Glu Ser Glu Asp Pro Leu Gly Glu 1585 1590 1595 1600 Asp Asp Phe Glu Ile Phe Tyr Glu Val Trp Glu Lys Phe Asp Pro Glu 1605 1610 1615 Ala Ser Gln Phe Ile Gln Tyr Ser Ala Leu Ser Asp Phe Ala Asp Ala 1620 1625 1630 Leu Pro Glu Pro Leu Arg Val Ala Lys Pro Asn Lys Phe Gln Phe Leu 1635 1640 1645 Val Met Asp Leu Pro Met Val Met Gly Asp Arg Leu His Cys Met Asp 1650 1655 1660 Val Leu Phe Ala Phe Thr Thr Arg Val Leu Gly Asp Ser Ser Gly Leu 1665 1670 1675 1680 Asp Thr Met Lys Thr Met Met Glu Glu Lys Phe Met Glu Ala Asn Pro 1685 1690 1695 Phe Lys Lys Leu Tyr Glu Pro Ile Val Thr Thr Thr Lys Arg Lys Glu 1700 1705 1710 Glu Glu Gln Gly Ala Ala Val Ile Gln Arg Ala Tyr Arg Lys His Met 1715 1720 1725 Glu Lys Met Val Lys Leu Arg Leu Lys Asp Arg Ser Ser Ser Ser His 1730 1735 1740 Gln Val Phe Cys Asn Gly Asp Leu Ser Ser Leu Asp Val Ala Lys Val 1745 1750 1755 1760 Lys Val His Asn Asp 1765  <Desc / Clms Page number 74>   Figure 2G:  SEQ ID N0: 2 1 MEERYYPVIF PDERNFRPFT SDSLAAIEKR IAIQKERKKS KDKAAAEPQP 0 51 RPQLDLKASR KLPKLYGDIP PELVAKPLED LDPFYKDHKT FMVLNKKRTI 0 101 YRFSAKRALF ILGPFNPLRS LMIRISVHSV FSMFIICTVI INCMFMANSM # ------------ IS1 ------------ # 151 ERSFDNDIPE YVFIGIYILE AVIKILARGF IVDEFSFLRD PWNWLDFIVI # --------- -IS2 --------- # # ------ IS3 201 GTAIATCFPG SQVNLSALRT FRVFRALKAI SVISGLKVIV GALLRSVKKL --------- #.    # -------- IS4 --------- # 251 VDVMVLTLFC LSIFALVGQQ LFMGILNQKC IKHNCGPNPA SNKDCFEKEK # ---------- IS5 ----------- # 301 DSEDFIMCGT WLGSRPCPNG STCDKTTLNP DNNYTKFDNF GWSFLAMFRV 351 MTQDSWERLY RQILRTSGIY FVFFFVWIF LGSFYLLNLT LAVVTMAYEE # # # ---- ------- ---------- IS6 # 401 QNRNVAAETE AKEKMFQEAQ QLLREEKEAL VAMGIDRSSL NSLQASSFSP 451 KKRKFFGSKT RKSFFMRGSK TAQASASDSE DDASKNPQLL EQTKRLSQNL 0 501 PVDLFDEHVD PLHRQRALSA VSILTITMQE QEKFQEPCFP CGKNLASKYL 551 VWDCSPQWLC IKKVLRTIMT DPFTELAITI CIIINTVFLA VEHHNMDDNL # ----------- IIS1 -------------- # 601 KTILKIGNWV FTGFFYWALLFV  EMI74.1  1- - - --- --- II52 ------- ---- 1 1 ------ --- - -lIS3- 651 VLYNTLSDNN RSFLASLRVL RVFKLAKSWP TLNTLIKIIG HSVGALGNLT ---- #. # ---------- IIS4 ------- # ## - 701 WLTIWFIF SWGMRLFGT KFNKTAYATQ ERPRRRWHMD NFYHSFLWF ------ IIS5 ---------- # # 751 RILCGEWIEN MWGCMQDMDG SPLCIIVFVL IMVIGKLWL NLFIALLLNS # ----------- IIS6 ------------- # 801 FSNEEKDGSL EGETRKTKVQ LALDRFRRAF SFMLHALQSF CCKKCRRKNS 0 851 PKPKETTESF AGENKDSILP DARPWKEYDT DMALYTGQAG APLAPLAEVE 901 DDVEYCGEGG ALPTSQHSAG VQAGDLPPET KQLTSPDDQG VEMEVFSEED 951 LHLSIQSPRK KSDAVSMLSE CSTIDLNKKKFK 0 1001 LSCHFLCHKT DKRKSPWVLW WNIRKTCYQI VKHSWFESFI IFVILLSSGA # ---------- IIIS1 ----- 1051 LIFEDVNLPS RPQVEKLLRC TDNIFTFIFL LEMILKWVAF GFRRYFTSAW - # # ---------- IIIS2 -------------- # # - 1101 CWLDFLIVW SVLSLMNLPS LKSFRTLRAL RPLRALSQFE GMKVVVYALI  EMI74.2  ¯¯¯¯IIIS3-¯¯¯¯¯¯¯ (¯¯¯¯¯¯¯¯IIIS9-¯¯¯¯¯¯¯¯¯ 1151 SAIPAILNVL LVCLIFWLVF CILGVNLFSG KFGRCINGTD INMYLDFTEV # -------- IIIS5- --------- # # 1201 PNRSQCNISN YSWKVPQVNF DNVGNAYLAL LQVATYKGWL EIMNAAVDSR # # # 1251 EKDEQPDFEA NLYAYLYFW FIIFGSFFTL NLFIGVIIDN FNQQQKKLGG # ------------ IIIS6 - #  <Desc / Clms Page number 75>   Figure 2H:  SEQ ID NO: 2 1301 QDIFMTEEQK KYYNAMKKLG TKKPQKPIPR PLNKCQAFVF DLVTSQVFDV # -------- 1351 IILGLIVLNM IIMMAESADQ PKDVKKTFDI LNIAFVVIFT IECLIKVFAL  EMI75.1  IVS1 ------------ 1 1 --------- IVS2 ------------ 1 1401 ROHYFTNGWN LFDCVVVVVLS IISTLVSRLE DSDISFPPTL FRVVRLARIG # ----- ----- IVS3 ------------ # # -------- 1451 RILRLVRAAR GIRTLLFALM MSLPSLFNIG LLLFLVMFIY AIFGMSWFSK IVS4 ---------- # # --- ------- IVS5 --------- 1501 VKKGSGIDDI FNFETFTGSM LCLFOITTSA GWDTLLNPML EAKEHCNSSS # o # 1551 QDSCQQPQIA VVYFVSYIII SFLIVVNMYI AVILENFNTA TEESEDPLGE # ----------- IVS6 ----- -------- # 1601 DDFEIFYEVW EKFDPEASOF IQYSALSDFA DALPEPLRVA KPNKFQFLVM 1651 DLPMVMGDRL HCMDVLFAFT TRVLGDSSGL DTMKTMMEEK FMEANPFKKL 1701 YEPIVTTTKR KEEEQGAAVI QRAYRKHMEK MVKLSLKDRS SSSHQVFCNG 1751 DLSSLDVAKV KVHND '  <Desc / Clms Page number 76>   Figure3A: SEQIDN0: 3 1 GCTGAGCAGT GGGGCACTGA TATTTGAAGA TGTTCACCTT GAGAACCAAC 51 CCAAAATCCA:  AGAATTACTA AATTGTACTG ACATTATTTT TACACATATT 101 TTTATCCTGG AGATGGTACT AAAATGGGTA GCCTTCGGAT TTGGAAAGTA 151 TTTCACCAGT GCCTGGTGCT GCCTTGATTT CATCATTGTG ATTGTCTCTG 201 TGACCACCCT CATTAACTTA ATGGAATTGA AGTCCTTCCG GACTCTACGA 251 GCACTGAGGC CTCTTCGTGC GCTGTCCCAG TTTGAAGGAA TGAAGGTGGT 301 GGTCAATGCT CTCATAGGTG CCATACCTGC CATTCTGAAT GTTTTGCTTG 351 TCTGCCTCAT TTTCTGGCTC GTATTTTGTA TTCTGGGAGT ATACTTCTTT 401 TCTGGAAAAT TTGGGAAATG CATTAATGGA ACAGACTCAG TTATAAATTA 451 TACCATCATT ACAAATAAAA GTCAATGTGA AAGTGGCAAT TTCTCTTGGA 501 TCAACCAGAA AGTCAACTTT GACAATGTGG GAAATGCTTA CCTCGCTCTG 551 CTGCAAGTGG CAACATTTAA GGGCTGGATG GATATTATAT ATGCAGCTGT 601 TGATTCCACA GAGAAAGAAC AACAGCCAGA GTTTGAGAGC AATTCACTCG 651 GTTACATTTA CTTCGTAGTC TTTATCATCT TTGGCTCATT CTTCACTCTG 701 AATCTCTTCA TTGGCGTTAT CATTGACAAC TTCAACCAAC AGCAGAAAAA 751 GTTAGGTGGC CAAGACATTT TTATGACAGA   AGAACAGAAG AAATACTATA 801 ATGCAATGAA AAAATTAGGA TCCAAAAAAC CTCAAAAACC CATTCCACGG 851 CCCGTT  <Desc / Clms Page number 77>   Figure3B: SEQ IDN0: 3 (Upper line: human PN5) (Lower line: rat PN5) 1 ............................................. LSSGA 5 1001 LSCHFLCHKTDKRKSPWVLWWNIRKTCYQIVKHSWFESFIIFVILLSSGA 1050 6 LIFEDVHLENQPKIQELLNCTDIIFTHIFILEMVLKWVAFGFGKYFTSAW 55 1051 LIFEDVNLPSRPQVEKLLRCTDNIFTFIFLLEMILKWVAFGFRRYFTSAW 1100 56 CCLDFIIVIVSVTTLINLMELKSFRTLRALRPLRALSQFEGMKVWNALI 105  EMI77.1  1101 CWLDFLIVWSVLSLMNLPSLKSFRTLRALRPLRALSQFEGMKVVVYALI 1150 106 GAIPAILNVLLVCLIFWLVFCILGVYFFSGKFGKCINGTD..SVINYTII 153 1151 SAIPAILNVLLVCLIFWLVFCILGVNLFSGKFGRCINGTDINMYLDFTEV 1200 154 TNKSQCESGNFSWINQKVNFDNVGNAYLALLQVATFKGWMDIIYAAVDST 203 1201 PNRSQCNISNYSWKVPQVNFDNVGNAYLALLQVATYKGWLEIMNAAVDSR 1250  EMI77.2  204 EKEQQPEFESNSLGYIYFWFIIFGSFFTLNLFIGVIIDNFNQQQKKLGG 253 1251 EKDEQPDFEANLYAYLYFWFIIFGSFFTLNLFIGVIIDNFNQQQKKLGG 1300 254 QDIFMTEEQKKYYNAMKKLGSKKPQKPIPRPV .................. 285 1301 QDIFMTEEQKKYYNAMKKLGTKKPQKPIPRPLNKCQAFVFDLVTSQVFDV 1350  <Desc / Clms Page number 78>   Figure4: SEQ ID N0: April 1 CTCAACATGG TTACGATGAT GGTGGAGACC GACGAGCAGG GCGAGGAGAA 51 GACGAAGGTT CTGGGCAGAA TCAACCAGTT CTTTGTGGCC GTCTTCACGG 101 GCGAGTGTGT GATGAAGATG TTCGCCCTGC GACAGTACTA TTTCACCAAC 151 GGCTGGAACG TGTTCGAcTT CATAGTGGTG ATCCTGTCCA TTGGGAGTCT 201 GCTGTTTCT GCAATCCTTA AGTCACTGGA AAACTACTTC TCCCCGACGC 251 TCTTCCGGGT CATCCGTCTG GCCAGGATCG GCCGCATCCT CAGGCTGATC 301 CGAGCAGCCA AGGGGATTCG CACGCTGCTC TTCGCCCTCA TGATGTCCCT 351 GCCCGCCCTC TTCAACATCG GCCTCCTCCT CTTCCTCGtC ATGTTCATCT 401 ACTCCATCTT CGGCATGGCC AGCTTCGCTA ACGTCGTGGA CGAGGCCGGC 451 ATCGACGACA TGTTCAACTT CAAGACCTTT GGCAACAGCA TGCTGTGCCT 501 GTTCCAGATC ACCACCTCGG CCGGCTGGGA CGGCCTCCTC AGCCCCATCC 551 TCAACACGGG GCCTCCCTAC TGCGACCCCA ACCTGCCCAA CAGCAACGGC 601 TCCCGGGGGA ACTGCGGGAG CCCGGCGGTG GGCATCATCT TCTTCACCAC 651 CTACATCATC ATCTCCTTCC TCATCGTGGT CAACATGTAT ATCGCAGTCA 701 TC  <Desc / Clms Page number 79>   Figure5A:  SEQ ID NO: 5 1 GTCGACTCTA GATCAGGGTG AAGATGGAGG AGAGGTACTA CCCGGTGATC 51 TTCCCGGACG AGCGGAATTT CCGCCCCTTC ACTTCCGACT CTCTGGCTGC 101 CATAGAGAAG CGGATTGCTA TCCAAAAGGA GAGGAAGAAG TCCAAAGACA 151 AGGCGGCAGC TGAGCCCCAG CCTCGGCCTC AGCTTGACCT AAAGGCCTCC 201 AGGAAGTTAC CTAAGCTTTA TGGTGACATT CCCCCTGAGC TTGTAGCGAA 251 GCCTCTGGAA GACCTGGACC CATTCTACAA AGACCATAAG ACATTCATGG 301 TGTTGAACAA GAAGAGAACA ATTTATCGCT TCAGCGCCAA GCGGGCCTTG 351 TTCATTCTGG GGCCTTTTAA TCCCCTCAGA AGCTTAATGA TTCGTATCTC 401 TGTCCATTCA GTCTTTAGCA TGTTCATCAT CTGCACGGTG ATCATCAACT 451 GTATGTTCAT GGCGAATTCT ATGGAGAGAA GTTTCGACAA CGACATTCCC 501 GAATACGTCT TCATTGGGAT TTATATTTTA GAAGCTGTGA TTAAAATATT 551 GGCAAGAGGC TTCATTGTGG ATGAGTTTTC CTTCCTCCGA GATCCGTGGA 601 ACTGGCTGGA CTTCATTGTC ATTGGAACAG CGATCGCAAC TTGTTTTCCG 651 GGCAGCCAAG TCAATCTTTC AGCTCTTCGT ACCTTCCGAG TGTTCAGAGC 701 TCTGAAGGCG ATTTCAGTTA TCTCAGGTCT GAAGGTCATC GTAGGTGCCC 751 TGCTGCGCTC GGTGAAGAAG CTGGTAGACG TGATGGTCCT CACTCTCTTC 801 TGCCTCAGCA TCTTTGCCCT GGTCGGTCAG CAGCTGTTCA TGGGAATTCT 851 GAACCAGAAG TGTATTAAGC ACAACTGTGG CCCCAACCCT GCATCCAACA 901 AGGATTGCTT TGAAAAGGAA AAAGATAGCG AAGACTTCAT AATGTGTGGT 951 ACCTGGCTCG GCAGCAGACC CTGTCCCAAT GGTTCTACGT GCGATAAAAC 1001 CACATTGAAC CCAGACAATA ATTATACAAA GTTTGACAAC TTTGGCTGGT 1051 CCTTTCTCGC CATGTTCCGG GTTATGACTC AAGACTCCTG GGAGAGGCTT 1101 TACCGACAGA TCCTGCGGAC CTCTGGGATC TACTTTGTCT TCTTCTTCGT  <Desc / Clms Page number 80>   Figure5B:    SEQ ID NO: 5 1151 GGTGGTCATC TTCCTGGGCT CCTTCTACCT GCTTAACCTA ACCCTGGCTG 1201 TTGTCACCAT GGCTTATGAA GAACAGAACA GAAATGTAGC TGCTGAGACA 1251 GAGGCCAAGG AGAAAATGTT TCAGGAAGCC CAGCAGCTGT TAAGGGAGGA 1301 GAAGGAGGCT CTGGTTGCCA TGGGAATTGA CAGAAGTTCC CTTAATTCCC 1351 TTCAAGCTTC ATCCTTTTCC CCGAAGAAGA GGAAGTTTTT CGGTAGTAAG 1401 ACAAGAAAGT CCTTCTTTAT GAGAGGGTCC AAGACGGCCC AAGCCTCAGC 1451 GTCTGATTCA GAGGACGATG CCTCTAAAAA TCCACAGCTC CTTGAGCAGA 1501 CCAAACGACT GTCCCAGAAC TTGCCAGTGG ATCTCTTTGA TGAGCACGTG 1551 GACCCCCTCC ACAGGCAGAG AGCGCTGAGC GCTGTCAGTA TCTTAACCAT 1601 CACCATGCAG GAACAAGAAA AATTCCAGGA GCCTTGTTTC CCATGTGGGA 1651 AAAATTTGGC CTCTAAGTAC CTGGTGTGGG ACTGTAGCCC TCAGTGGCTG 1701 TGCATAAAGA AGGTCCTGCG GACCATCATG ACGGATCCCT TTACTGAGCT 1751 GGCCATCACC ATCTGCATCA TCATCAATAC CGTTTTCTTA GCCGTGGAGC 1801 ACCACAACAT GGATGACAAC TTAAAGACCA TACTGAAAAT AGGAAACTGG 1851 GTTTTCACGG GAATTTTCAT AGCGGAAATG TGTCTCAAGA TCATCGCGCT 1901 CGACCCTTAC CACTACTTCC GGCACGGCTG GAATGTTTTT GACAGCATCG 1951 TGGCCCTCCT GAGTCTCGCT GATGTGCTCT ACAACACACT GTCTGATAAC 2001 AATAGGTCTT TCTTGGCTTC CCTCAGAGTG CTGAGGGTCT TCAAGTTAGC 2051 CAAATCCTGG CCCACGTTAA ACACTCTCAT TAAGATCATC GGCCACTCCG 2101 TGGGCGCGCT TGGAAACCTG ACTGTGGTCC TGACTATCGT GGTCTTCATC 2151 TTTTCTGTGG TGGGCATGCG GCTCTTCGGC ACCAAGTTTA ACAAGACCGC 2201 CTACGCCACC CAGGAGCGGC CCAGGCGGCG CTGGCACATG GATAATTTCT 2251 ACCACTCCTT CCTGGTGGTG TTCCGCATCC TCTGTGGGGA ATGGATCGAG 2301 AACATGTGGG GCTGCATGCA GGATATGGAC GGCTCCCCGT TGTGCATCAT  <Desc / Clms Page number 81>   Figure 5C:  SEQ ID NO: 2351 TGTCTTTGTC CTGATAATGG TGATCGGGAA GCTTGTGGTG CTTAACCTCT 2401 TCATTGCCTT GCTGCTCAAT TCCTTCAGCA ATGAGGAGAA GGATGGGAGC 2451 CTGGAAGGAG AGACCAGGAA AACCAAAGTG CAGCTAGCCC TGGATCGGTT 2501 CCGCCGGGCC TTCTCCTTCA TGCTGCACGC TCTTCAGAGT TTTTGTTGCA 2551 AGAAATGCAG GAGGAAAAAC TCGCCAAAGC CAAAAGAGAC AACAGAAAGC 2601 TTTGCTGGTG AGAATAAAGA CTCAATCCTC CCGGATGCGA GGCCCTGGAA 2651 GGAGTATGAT ACAGACATGG CTTTGTACAC TGGACAGGCC GGGGCTCCGC 2701 TGGCCCCACT CGCAGAGGTA GAGGACGATG TGGAATATTG TGGTGAAGGC 2751 GGTGCCCTAC CCACCTCACA ACATAGTGCT GGAGTTCAGG CCGGTGACCT 2801 CCCTCCAGAG ACCAAGCAGC TCACTAGCCC GGATGACCAA GGGGTTGAAA 2851 TGGAAGTATT TTCTGAAGAA GATCTGCATT TAAGCATACA GAGTCCTCGA 2901 AAGAAGTCTG ACGCAGTGAG CATGCTCTCG GAATGCAGCA CAATTGACCT 2951 GAATGATATC TTTAGAAATT TACAGAAAAC AGTTTCCCCC AAAAAGCAGC 3001 CAGATAGATG CTTTCCCAAG GGCCTTAGTT GTCACTTTCT ATGCCACAAA 3051 ACAGACAAGA GAAAGTCCCC CTGGGTCCTG TGGTGGAACA TTCGGAAAAC 3101 CTGCTACCAA ATCGTGAAGC ACAGCTGGTT TGAGAGTTTC ATAATCTTTG 3151 TTATTCTGCT GAGCAGTGGA GCGCTGATAT TTGAAGATGT CAATCTCCCC 3201 AGCCGGCCCC AAGTTGAGAA ATTACTAAGG TGTACCGATA ATATTTTCAC 3251 ATTTATTTTC CTCCTGGAAA TGATCCTGAA GTGGGTGGCC TTTGGATTCC 3301 GGAGGTATTT CACCAGTGCC TGGTGCTGGC TTGATTTCCT CATTGTGGTG 2251 GTGTCTGTGC TCAGTCTCAT GAATCTACCA AGCTTGAAGT CCTTCCGGAC 3401 TCTGCGGGCC CTGAGACCTC TGCGGGCGCT GTCCCAGTTT GAAGGAATGA 3451 AGGTTGTCGT CTACGCCCTG ATCAGCGCCA TACCTGCCAT TCTCAATGTC 3501 TTGCTGGTCT GCCTCATTTT CTGGCTCGTA TTTTGTATCT TGGGAGTAAA  <Desc / Clms Page number 82>   Figure5D:    SEQ ID NO: 5 3551 TTTATTTTCT GGGAAGTTTG GAAGGTGCAT TAACGGGACA GACATAAATA 3601 TGTATTTGGA TTTTACCGAA GTTCCGAACC GAAGCCAATG TAACATTAGT 3651 AATTACTCGT GGAAGGTCCC GCAGGTCAAC TTTGACAACG TGGGGAATGC 3701 CTATCTCGCC CTGCTGCAAG TGGCAACCTA TAAGGGCTGG CTGGAAATCA 3751 TGAATGCTGC TGTCGATTCC AGAGAGAAAG ACGAGCAGCC GGACTTTGAG 3801 GCGAACCTCT ACGCGTATCT CTACTTTGTG GTTTTTATCA TCTTCGGCTC 3851 CTTCTTTACC CTGAACCTCT TTATCGGTGT TATTATTGAC AACTTCAATC 3901 AGCAGCAGAA AAAGTTAGGT GGCCAAGACA TCTTCATGAC IGAGGAGCAG 3951 AAGAAATATT ACAATGCAAT GAAAAAGTTA GGAACCAAGA AACCTCAAAA 4001 GCCCATCCCA AGGCCCCTGA ACAAATGTCA AGCCTTTGTG TTCGACCTGG 4051 TCACAAGCCA GGTCTTTGAC GTCATCATTC TGGTGATATTATTGA AACÇTTTGAT ATCCTCAACA TAGCCTTCGT GGTCATCTTT ACCATAGAGT 4201 GTCTCATCAA AGTCTTTGCT TTGAGGCAAC ACTACTTCAC CAATGGCTGG 4251 AACTTATTTG ATTGTGTGGT CGTGGTTCTT TCTATCATTA GTACCCTGGT 4301 TTCCCGCTTG GAGGACAGTG ACATTTCTTT CCCGCCCACG CTCTTCAGAG 4351 TCGTCCGCTT GGCTCGGATT GGTCGAATCC TCAGGCTGGT CCGGGCTGCC 4401 CGGGGAATCA GGACCCTCCT CTTTGCTTTG ATGATGTCTC TCCCCTCTCT 4451 CTTCAACATC GGTCTGCTGC TCTTCCTGGT GATGTTCATT TACGCCATCT 4501 TTGGGATGAG CTGGTTTTCC AAAGTGAAGA AGGGCTCCGG GATCGACGAC 4551 ATCTTCAACT TCGAGACCTT TACGGGCAGC ATGCTGTGCC TCTTCCAGAT 4601 AACCACTTCG GCTGGCTGGG ATACCCTCCT CAACCCCATG CTGGAGGCAA 4651 AAGAACACTG CAACTCCTCC TCCCAAGACA GCTGTCAGCA GCCGCAGATA 4701 GCCGTCGTCT ACTTCGTCTCTCCG  <Desc / Clms Page number 83>   Figure5E:    SEQ ID NO: 5 4751 CAACATGTAC ATCGCTGTGA TCCTCGAGAA CTTCAACACA GCCACGGAGG 4801 AGAGCGAGGA CCCTCTGGGA GAGGACGACT TTGAAATCTT CTATGAGGTC 4851 TGGGAGAAGT TTGACCCCGA GGCGTCGCAG TTCATCCAGT ATTCGGCCCT 4901 CTCTGACTTT GCGGACGCCC TGCCGGAGCC GTTGCGTGTG GCCAAGCCGA 4951 ATAAGTTTCA GTTTCTAGTG ATGGACTTGC CCATGGTGAT GGGCGACCGC 5001 CTCCATTGCA TGGATGTTCT CTTTGCTTTC ACTACCAGGG TCCTCGGGGA 5051 CTCCAGCGGC TTGGATACCA TGAAAACCAT GATGGAGGAG AAGTTTATGG 5101 AGGCCAACCC TTTTAAGAAG CTCTACGAGC CCATAGTCAC CACCACCAAG 5151 AGGAAGGAGG AGGAGCAAGG ATCCAGAGGG CCTACCGGAA 5 ACACATGGAG AAGATGGTCA AACTGAGGCT GAAGGACAGGTCATCGTAGTAGTGTAGTAGT