MURINE GUANINE NUCLEOTIDE EXCHANGE FACTOR - (MNGEF) AND HUMAN HOMOLOGUES THEREOF

Abstract: The present invention concerns the cloning of a murine guanine nucleotide exchange factor designated MNGEF and a human homologue thereof. Polynucleotide probes derived from the nucleotide sequence of MNGEF and antibodies that recognise MNGEF are also provided.

Description: MURINE GUANINE NUCLEOTIDE EXCHANGE FACTOR - (MNGEF) AND HUMAN HOMOLOGUES THEREOF Field of the Invention The present invention relates to MNGEF, a member of the family of regulators of small GTP-binding proteins, and homologues of MNGEF. Background to the invention The superfamily of low molecular mass GTP-binding proteins (also known as G proteins), for which ras proteins are prototypes, has been implicated in the regulation of diverse biological activities. In addition to their involvement in regulating many aspects of growth and differentiation, members of this superfamily play an important role in the control of the cytoskeleton and in the regulation of protein trafficking between various membrane-bound compartments in the cell. These proteins function as binary switches, being 'on' in the GTP -bound state and 'off in the GDP-bound state. Cycling between these two forms is controlled by various accessory proteins. The guanine nucleotide exchange factors (GEFs), promote the exchange of GDP for GTP, thus activating the proteins whereas, the GTPase-activating proteins (GAPS) and GDP-dissociation inhibitory factors (GDIs) are negative modulators. The Ras-like proteins are divided into six main families, based on their sequences: Rab, Arf, Sar, Ran, Rho and Ras. Until recently, the Rho GTPases (such as Rac, Rho, Cdc42) were thought to be primarily involved in the organisation of the actin cytoskeleton. However, it has become evident that they play a critical role in controlling cell proliferation and progress has been made in identifying signalling cascades involving the Rho family members. A family of cell growth regulatory proteins and oncogene products have been discovered for which the Dbl oncoprotein is a prototype (Eva and Aaronson (1985) Nature 316, 273-275). These proteins are putative guanine nucleotide exchange factors for the Rho GTPases. They all contain a Dbl homology domain (DH) in tandem with a pleckstrin homology domain (PH), and seem to activate specific members of the Rho family to elicit a variety of biological functions in the cell. The DH domain is responsible for binding and activating the G proteins thus mediating downstream signalling events, whereas the PH domain is thought to play a role in targeting these guanine nucleotide exchange factors to specific cellular locations in order to carry out the signalling function. Since the initial identification of Dbl as a GEF for Rho GTPases, an increasing number of oncogene products and growth regulatory molecules have been shown to contain those two domains in tandem. Many of them, such as Bcr which is involved in the chromosomal rearrangements in chronic myelogenous leukaemia, Cdc24, Ras guanine nucleotide release factor and Vav have been implicated in cell growth regulation. Others, including Ect-2, Tim, Ost and Lbc were discovered, by virtue of their transforming capability, through gene transfer methods. Disclosure of the Invention Here, we report the isolation and preliminary characterisation of 3 overlapping mouse cDNAs (designated MNGEF 1, MNGEF2 and MNGEF3), which show homology to the TIM gene (Transforming Immortalized Mammary, Chan et al, (1994) Oncogene 9, 1057-1063) of the family of regulators of small GTP-binding proteins. The homology is observed at both the amino acid and nucleotide levels. However, the size of the transcript observed by Northern analysis and the expression pattern of MNGEF2 is markedly different to that of TLM, suggesting that this is a novel, neuronal-specific member of the above family of genes. In addition, MNGEF 1 and MNGEF2 contain a trinucleotide repeat. Together with the high expression pattern of MNGEF2 in brain, the presence of the triplet repeat and the homology to TIM, these cDNAs present potential candidates for disease related genes. We also report the cloning and sequencing of a fragment of the human homologue of MNGEF. Substantial homology is observed at both the amino acid and nucleotide levels between murine MNGEF and its human homologue NGEF. The MNGEF3 clone is 1.35 kb and is contained completely within the MNGEF1 cDNA which is 2.3 kb. MNGEF2 is the longest clone (2.8kb) but contains a 92bp unspliced intron within it (from nucleotides 1816 to 1907 of SEQ. LD No. 3), resulting in a premature termination codon. MNGEF 1 does not contain this intron and therefore its ORF extends beyond the stop codon of MNGEF2. From the sequences of MNGEF 1 and MNGEF2 we conclude that the cDNA designated MNGEF consists of 2741 bp (2833 bp minus 92 bp) which results in an ORF of 554 amino acids. The murine MNGEF cDNA sequence is set out as SEQ. LD No. 1. The amino acid sequence of the ORF from nucleotides 343 to 2004 is set out as SEQ. ID No. 2. The murine MNGEF2 cDNA sequence, which includes the 92 bp intron, is set out as SEQ ID No. 3. The amino acid sequence of the ORF from nucleotides 343 to 1860 is set out as SEQ. ID No. 4. The murine MNGEFl cDNA sequence is set out as SEQ. ID No. 5. The amino acid sequence of the ORF from nucleotides 2 to 1609 is set out as SEQ. ID No. 6. The partial human NGEF cDNA sequence is set out as SEQ. ID No. 7. The amino acid sequence of the ORF from nucleotides 3 to 803 is set out as SEQ LD No. 8. Thus the invention provides a murine guanine nucleotide exchange factor designated MNGEF, a human homologue thereof designated human NGEF or other mammalian homologue thereof which guanine nucleotide exchange factor is encoded by a cDNA sequence obtainable from a mammalian brain cDNA library, said DNA sequence being selectively detectable with a murine DNA sequence as shown in SEQ ID Nos. 1 , 3 or 5 or a human DNA sequence as shown in SEQ LD No. 7. The protein preferably has one or more of the additional features: (1) it comprises a Dbl homology domain having substantial homology to amino acids 124 to 306 of SEQ LD No. 2; (2) it comprises a pleckstrin homology domain having substantial homology to amino acids 333 to 445 of SEQ LD No. 2; (3) it comprises an SH3 domain (Src homology 3 domain) having substantial homology to amino acids 456 to 517 of SEQ LD No. 2 (4) it is found predominantly in neuronal cell types; (5) it is encoded by an mRNA of approximately 2.7 kb; (6) it promotes the exchange of GDP for GTP by low molecular mass GTP- binding proteins; and (7) it comprises a polyglutamine region. The term "selectively detectable" means that the cDNA used as a probe is used under conditions where a target cDNA of the invention is found to hybridize to the probe at a level significantly above background. The background hybridization may occur because of other cDNAs present in the brain cDNA library. In this event background implies a level of signal generated by interaction between the probe and a non-specific cDNA member of the library which is less than 10 fold, preferably less than 100 fold as intense as the specific interaction observed with the target cDNA. The intensity of interaction may be measured, for example, by radiolabelling the probe, e.g. with 32 P. Suitable conditions may be found by reference to the Examples. Accordingly, in a first aspect, the invention provides the MNGEF protein of SEQ ID. 2, 4, 6 and homologues thereof, polypeptide fragments thereof, as well as antibodies capable of binding the MNGEF protein or polypeptide fragments thereof. The invention also provides the human NGEF protein of SEQ. ID. No. 8 and homologues thereof, polypeptide fragments thereof, as well as antibodies capable of binding the human NGEF protein or polypeptide fragments thereof. Human NGEF proteins, homologues and fragments thereof, are also included in references below to polypeptides of the invention. In another aspect, the present invention provides a polynucleotide in substantially isolated form capable of hybridising selectively to any one of SEQ ID Nos. 1, 3, 5 or 7 or to the complement (i.e. opposite strand) thereof. The present invention also provides a polynucleotide in substantially isolated form capable of hybridising selectively to any one of SEQ LD Nos. 1, 3, 5 or 7 or to the complement (i.e. opposite strand) thereof. Also provided are polynucleotides encoding polypeptides of the invention. Such polynucleotides will be referred to as a polynucleotide of the invention. A polynucleotide of the invention includes DNA of SEQ ID Nos. 1, 3, 5 and fragments thereof capable of selectively hybridising to the gene encoding MNGEF. A polynucleotide of the invention also includes DNA of SEQ LD No 7 and fragments thereof capable of selectively hybridising to the gene encoding human NGEF. In a further aspect, the invention provides recombinant vectors carrying a polynucleotide of the invention, including expression vectors, and methods of growing such vectors in a suitable host cell, for example under conditions in which expression of a protein or polypeptide encoded by a sequence of the invention occurs. In an additional aspect, the invention provides kits comprising polynucleotides, polypeptides or antibodies of the invention and methods of using such kits in diagnosing the presence of absence of MNGEF, human NGEF and their homologues, or variants thereof, including deleterious MNGEF and human NGEF mutants. Detailed description of the invention. A. Polynucleotides. In the following description, it should be understood that references to MNGEF refer additionally to MNGEFl, MNGEF2, MNGEF3 and human NGEF. Polynucleotides of the invention may comprise DNA or RNA. They may be single or double stranded. They may also be polynucleotides which include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. For the purposes of the present invention, it is to be understood that the polynucleotides described herein may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of polynucleotides of the invention. Polynucleotides of the invention capable of selectively hybridising to the DNA of SEQ LD No. 1 will be generally at least 70%, preferably at least 80 or 90% and more preferably at least 95% homologous to the corresponding DNA of SEQ LD No. 1 over a region of at least 20, preferably at least 25 or 30, for instance at least 40, 60 or 100 or more contiguous nucleotides. Preferred polynucleotides of the invention will comprise regions homologous to the DH domain of MNGEF, from nucleotides 712 to 1260 of SEQ LD No. 1, preferably at least 80 or 90% and more preferably at least 95% homologous to the DH domain of MNGEF. Preferred polynucleotides of the invention will also comprise regions homologous to the PH domain of MNGEF, from nucleotides 1339 to 1677 of SEQ LD No. 1, preferably at least 80 or 90% and more preferably at least 95% homologous to the PH domain of MNGEF. Preferred polynucleotides of the invention will further comprise regions homologous to the SH3 domain of MNGEF, from nucleotides 1708 to 1893 of SEQ ID No 1, preferably at least 80 or 90% and more preferably at least 95% homologous to the SH3 domain of MNGEF It is to be understood that skilled persons may, using routine techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides of the invention to reflect the codon usage of any particular host organism in which the polypeptides of the invention are to be expressed. Any combination of the above mentioned degrees of homology and minimum sizes may be used to define polynucleotides of the invention, with the more stringent combinations (i.e. higher homology over longer lengths) being preferred. Thus for example a polynucleotide which is at least 80% homologous over 25, preferably 30 nucleotides forms one aspect of the invention, as does a polynucleotide which is at least 90% homologous over 40 nucleotides. Polynucleotides of the invention may be used to produce a primer, e.g. a PCR primer, a primer for an alternative amplification reaction, a probe e.g. labelled with a revealing label by conventional means using radioactive or non-radioactive labels, or the polynucleotides may be cloned into vectors. Such primers, probes and other fragments will be at least 15, preferably at least 20, for example at least 25, 30 or 40 nucleotides in length, and are also encompassed by the term polynucleotides of the invention as used herein. Polynucleotides such as a DNA polynucleotide and primers according to the invention may be produced recombinantly, synthetically, or by any means available to those of skill in the art. They may also be cloned by standard techniques. In general, primers will be produced by synthetic means, involving a step wise manufacture of the desired nucleic acid sequence one nucleotide at a time. Techniques for accomplishing this using automated techniques are readily available in the art. Longer polynucleotides will generally be produced using recombinant means, for example using a PCR (polymerase chain reaction) cloning techniques. This will involve making a pair of primers (e.g. of about 15-30 nucleotides) to a region of the MNGEF gene which it is desired to clone, bringing the primers into contact with mRNA or cDNA obtained from an animal or human cell (e.g. a brain cell), performing a polymerase chain reaction under conditions which bring about amplification of the desired region, isolating the amplified fragment (e.g. by purifying the reaction mixture on an agarose gel) and recovering the amplified DNA. The primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable cloning vector. Such techniques may be used to obtain all or part of the MNGEF sequence described herein. Genomic clones containing the MNGEF gene and its introns and promoter regions may also be obtained in an analogous manner, starting with genomic DNA from an animal or human cell, e.g. a brain cell. Although in general the techniques mentioned herein are well known in the art, reference may be made in particular to Sambrook et al, Molecular Cloning, A Laboratory Manual (1989) and Ausubel et al, Current Protocols in Molecular Biology (1995), John Wiley & Sons, Inc. Polynucleotides which are not 100% homologous to the sequences of the present invention but fall within the scope of the invention can be obtained in a number of ways. Other murine allelic variants of the MNGEF sequence described herein may be obtained for example by probing genomic DNA libraries made from a range of individuals, for example individuals from different populations. In addition, other animal, particularly mammalian (e.g. rat or rabbit, more particularly primate), homologues of MNGEF may be obtained and such homologues and fragments thereof in general will be capable of selectively hybridising to SEQ LD No. 1. Such sequences may be obtained by probing cDNA libraries made from dividing cells or tissues or genomic DNA libraries from other animal species, and probing such libraries with probes comprising all or part of SEQ LD. 1 under conditions of medium to high stringency (for example 0.03M sodium chloride and 0.03M sodium citrate at from about 50°C to about 60°C). Nucleic acid probes comprising all or part of SEQ LD No. 7 may be used to probe cDNA libraries from primate species, preferably humans, to obtain homologues of MNGEF. In particular nucleic acid probes comprising all or part of SEQ YD No. 7 may be used to probe cDNA libraries from humans, to obtain the full-length cDNA encoding human NGEF or a homologue thereof. Allelic variants and species homologues may also be obtained using degenerate PCR which will use primers designed to target sequences within the variants and homologues encoding conserved amino acid sequences. Conserved sequences can be predicted from aligning the MNGEF amino acid sequence with that of TIM. The primers will contain one or more degenerate positions and will be used at stringency conditions lower than those used for cloning sequences with single sequence primers against known sequences. In particular, primers can be designed to target the DH, PH and SH3 domains described above. Alternatively, such polynucleotides may be obtained by site directed mutagenesis of the MNGEF sequences or allelic variants thereof. This may be useful where for example silent codon changes are required to sequences to optimise codon preferences for a particular host cell in which the polynucleotide sequences are being expressed. Other sequence changes may be desired in order to introduce restriction enzyme recognition sites, or to alter the property or function of the polypeptides encoded by the polynucleotides. Further changes may be desirable to represent particular coding changes found in MNGEF 5 which give rise to mutant MNGEF genes which have lost their regulatory function. Probes based on such changes can be used as diagnostic probes to detect such MNGEF mutants. The invention further provides double stranded polynucleotides comprising a polynucleotide of the invention and its complement. Polynucleotides or primers of the invention may carry a revealing label. Suitable 0 labels include radioisotopes such as 32 P or 35 S, enzyme labels, or other protein labels such as biotin. Such labels may be added to polynucleotides or primers of the invention and may be detected using by techniques known per se. Polynucleotides or primers of the invention or fragments thereof labelled or unlabelled may be used by a person skilled in the art in nucleic acid-based tests for detecting 5 or sequencing MNGEF and its homologues in the human or animal body. Such tests for detecting generally comprise bringing a biological sample containing DNA or RNA into contact with a probe comprising a polynucleotide or primer of the invention under hybridising conditions and detecting any duplex formed between the probe and nucleic acid in the sample. Such detection may be achieved using techniques such as o PCR or by immobilising the probe on a solid support, removing nucleic acid in the sample which is not hybridised to the probe, and then detecting nucleic acid which has hybridised to the probe. Alternatively, the sample nucleic acid may be immobilised on a solid support, and the amount of probe bound to such a support can be detected. Suitable assay methods of this any other formats can be found in for example WO89/03891 and WO90/13667. 5 Tests for sequencing MNGEF and its homologues include bringing a biological sample containing target DNA or RNA into contact with a probe comprising a polynucleotide or primer of the invention under hybridising conditions and determining the sequence by, for example the Sanger dideoxy chain termination method (see Sambrook et al). o Such a method generally comprises elongating, in the presence of suitable reagents, the primer by synthesis of a strand complementary to the target DNA or RNA and selectively terminating the elongation reaction at one or more of an A, C, G or T U residue; allowing strand elongation and termination reaction to occur; separating out according to size the elongated products to determine the sequence of the nucleotides at which selective termination has occurred. Suitable reagents include a DNA polymerase enzyme, the deoxynucleotides dATP, dCTP, dGTP and dTTP, a buffer and ATP. Dideoxynucleotides are used for selective termination. Tests for detecting or sequencing MNGEF, or its homologue, in a biological sample may be used to determine MNGEF sequences within cells in individuals who have, or are suspected to have, an altered MNGEF gene sequence, for example within cancer cells including leukaemia cells and solid tumours such as breast, ovary, lung, colon, pancreas, testes, liver, brain, muscle and bone tumours or within cells from the nervous system of individuals suffering from neurological disorders. In addition, the discovery of MNGEF will allow the role of this gene in hereditary diseases to be investigated. In general, this will involve establishing the status of MNGEF, or its homologue (e.g. using PCR sequence analysis), in cells derived from animals or humans with, for example, neurological disorders or neoplasms. The probes of the invention may conveniently be packaged in the form of a test kit in a suitable container. In such kits the probe may be bound to a solid support where the assay format for which the kit is designed requires such binding. The kit may also contain suitable reagents for treating the sample to be probed, hybridising the probe to nucleic acid in the sample, control reagents, instructions, and the like. The present invention also provides polynucleotides encoding the polypeptides of the invention described below. Because such polynucleotides will be useful as sequences for recombinant production of polypeptides of the invention, it is not necessary for them to be selectively hybridisable to the sequence of any one of SEQ ID Nos. 1, 3, 5 or 7 although this will generally be desirable. Otherwise, such polynucleotides may be labelled, used, and made as described above if desired. Polypeptides of the invention are described below. B. Polypeptides. Polypeptides of the invention include polypeptides in substantially isolated form which comprise the sequence set out in SEQ ID Nos. 2, 4, 6 or 8. Polypeptides further include variants of such sequences, including naturally occurring allelic variants and synthetic variants which are substantially homologous to said polypeptides. In this context, substantial homology is regarded as a sequence which has at least 70%, e.g. 80% or 90% amino acid homology (identity) over 30 amino acids with the sequence of SEQ LD No. 2. Polypeptides also include other those encoding MNGEF homologues, and variants thereof as defined above, from other species including animals such as mammals (e.g. mice, rats or rabbits), especially primates, more especially humans. MNGEF homologues include human NGEF. Polypeptides of the invention also include fragments of the above mentioned full length polypeptides and variants thereof, including fragments of the sequences set out in SEQ ID Nos. 2, 4, 6 or 8. Preferred fragments include those which include an epitope. Suitable fragments will be at least about 5, e.g. 10, 12, 15 or 20 amino acids in size. Polypeptide fragments of the MNGEF and human NGEF proteins and allelic and species variants thereof may contain one or more (e.g. 2, 3, 5, or 10) substitutions, deletions or insertions, including conserved substitutions. Conserved substitutions may be made according to the following table which indicates conservative substitutions, where amino acids on the same block in the second column and preferably in the same line in the third column may be substituted for each other: Epitopes may be determined, for example, by techniques such as peptide scanning techniques as described by Geysen et al, 1986. Polypeptides of the invention may be in a substantially isolated form. It will be understood that the polypeptide may be mixed with carriers or diluents which will not interfere with the intended purpose of the polypeptide and still be regarded as substantially isolated. A polypeptide of the invention may also be in a substantially purified form, in which case it will generally comprise the polypeptide in a preparation in which more than 90%, e.g. 95%), 98% or 99% of the polypeptide in the preparation is a polypeptide of the invention. Polypeptides of the invention may be modified for example by the addition of histidine residues to assist their purification or by the addition of a signal sequence to promote their secretion from a cell. A polypeptide of the invention may be labelled with a revealing label. The revealing label may be any suitable label which allows the polypeptide to be detected. Suitable labels include radioisotopes, e.g. 125 I, enzymes, antibodies, polynucleotides and linkers such as biotin. Labelled polypeptides of the invention may be used in diagnostic procedures such as immunoassays in order to determine the amount of a polypeptide of the invention in a sample. Polypeptides or labelled polypeptides of the invention may also be used in serological or cell mediated immune assays for the detection of immune reactivity to said polypeptides in animals and humans using standard protocols. A polypeptide or labelled polypeptide of the invention or fragment thereof may also be fixed to a solid phase, for example the surface of an immunoassay well or dipstick. Such labelled and/or immobilised polypeptides may be packaged into kits in a suitable container along with suitable reagents, controls, instructions and the like. Such polypeptides and kits may be used in methods of detection of antibodies to the MNGEF or human NGEF proteins or their allelic or species variants by immunoassay. Immunoassay methods are well known in the art and will generally comprise: (a) providing a polypeptide comprising an epitope bindable by an antibody against said protein; (b) incubating a biological sample with said polypeptide under conditions which allow for the formation of an antibody-antigen complex; and (c) determining whether antibody-antigen complex comprising said polypeptide is formed. Polypeptides of the invention may be may by synthetic means (e.g. as described by Geysen et al, 1996) or recombinantly, as described below. Particularly preferred polypeptides of the invention include those spanning or within 5 the DH, PH or DH3 homology domains or sequences substantially homologous thereto. Preferred polypeptides comprise regions showing substantial homology to the DH domain of MNGEF represented as amino acids 124 to 306 of SEQ ID No. 2. Preferred polypeptides will also comprise regions showing substantial homology to the PH domain of MNGEF represented as amino acids 333 to 445 of SEQ ID No. 2. Preferred polypeptides will further 0 comprise regions showing substantial homology to the SH3 domain of MNGEF represented as amino acids 456 to 517 of SEQ ID No. 2. Fragments as defined above from this region are particularly preferred. The polypeptides and fragments thereof may contain amino acid alterations as defined above. Polypeptides of the invention may be used in in vitro or in vivo cell culture systems s to study the role of MNGEF, human NGEF and their homologues in disease. For example, truncated or modified MNGEF may be introduced into a cell to disrupt the normal functions which occur in the cell. The polypeptides of the invention may be introduced into the cell by in situ expression of the polypeptide from a recombinant expression vector (see below). The expression vector optionally carries an inducible promoter to control the expression of o the polypeptide. The use of mammalian host cells is expected to provide for such post-translational modifications (e.g. myristolation, glycosylation, truncation, lapidation and tyrosine, serine or threonine phosphorylation) as may be needed to confer optimal biological activity on recombinant expression products of the invention. Such cell culture systems in which 5 polypeptide of the invention are expressed may be used in assay systems to identify candidate substances which interfere with or enhance the functions of the polypeptides of the invention in the cell. C. Vectors. o Polynucleotides of the invention can be incorporated into a recombinant replicable vector. The vector may be used to replicate the nucleic acid in a compatible host cell. Thus in a further embodiment, the invention provides a method of making polynucleotides of the invention by introducing a polynucleotide of the invention into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about replication of the vector. The vector may be recovered from the host cell. Suitable host cells are described below in connection with expression vectors. Expression Vectors. Preferably, a polynucleotide of the invention in a vector is operably linked to a regulatory sequence which is capable of providing for the expression of the coding sequence by the host cell, i.e. the vector is an expression vector. The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A regulatory sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under condition compatible with the control sequences. The term "regulatory sequences" includes promoters and enhancers and other expression regulation signals. These may be selected to be compatible with the host cell for which the expression vector is designed. For example, yeast regulatory sequences include S. cerevisiae GAL4 and ADH promoters, S. pombe nmtl and adh promoters. Mammalian promoters, such as α-actin promoters, may be used. Mammalian promoters also include the metallothionein promoter which can upregulate expression in response to heavy metals such as cadmium and is thus an inducible promoter. Tissue-specific promoters, for example neuronal cell specific may be used. Viral promoters may also be used, for example the Moloney murine leukaemia virus long terminal repeat (MMLV LTR), the promoter rous sarcoma virus (RSV) LTR promoter, the SV40 promoter, the human cytomegalovirus (CMV) IE promoter, herpes simplex virus promoters or adeno virus promoters. All these promoters are readily available in the art. Such vectors may be transformed into a suitable host cell as described above to provide for expression of a polypeptide of the invention. Thus, in a further aspect the invention provides a process for preparing polypeptides according to the invention which comprises cultivating a host cell transformed or transfected with an expression vector as described above under conditions to provide for expression by the vector of a coding sequence encoding the polypeptides, and recovering the expressed polypeptides. The vectors may be for example, plasmid, virus or phage vectors provided with an origin of replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter. The vectors may contain one or more selectable marker genes, for example an ampicillin resistance gene in the case of a bacterial plasmid or a neomycin resistance gene for a mammalian vector. Vectors may be used in vitro, for example for the production of RNA or used to transfect or transform a host cell. The vector may also be adapted to be used in vivo, for example in a method of gene therapy. A further embodiment of the invention provides host cells transformed or transfected with the vectors for the replication and expression of polynucleotides of the invention. The cells will be chosen to be compatible with the said vector and may for example be bacterial, yeast, insect or mammalian. Polynucleotides according to the invention may also be inserted into the vectors described above in an antisense orientation in order to provide for the production of antisense RNA. Antisense RNA or other antisense polynucleotides may also be produced by synthetic means. Such antisense polynucleotides may be used in a method of controlling the levels of MNGEF or its variants or species homologues. D. Antibodies. The invention also provides monoclonal or polyclonal antibodies to polypeptides of the invention or fragments thereof. The invention further provides a process for the production of monoclonal or polyclonal antibodies to polypeptides of the invention. Monoclonal antibodies may be prepared by conventional hybridoma technology using the polypeptides of the invention or peptide fragments thereof, as immunogens. Polyclonal antibodies may also be prepared by conventional means which comprise inoculating a host animal, for example a rat or a rabbit, with a polypeptide of the invention or peptide fragment thereof and recovering immune serum. In order that such antibodies may be made, the invention also provides polypeptides of the invention or fragments thereof haptenised to another polypeptide for use as immunogens in animals or humans. For the purposes of this invention, the term "antibody", unless specified to the contrary, includes fragments of whole antibodies which retain their binding activity for a tumour target antigen. Such fragments include Fv, F(ab') and F(ab') 2 fragments, as well as single chain antibodies. Furthermore, the antibodies and fragments thereof may be humanised antibodies, e.g. as described in EP-A-239400. Antibodies may be used in method of detecting polypeptides of the invention present in biological samples by a method which comprises: (a) providing an antibody of the invention; (b) incubating a biological sample with said antibody under conditions which allow for the formation of an antibody-antigen complex; and (c) determining whether antibody-antigen complex comprising said antibody is formed. Suitable samples include extracts from brain tissue, both normal and neoplastic. Suitable samples may also include extracts from other tissues such as breast, ovary, lung, colon, pancreas, testes, liver, muscle and bone tissues or from neoplastic growths derived from such tissues. Antibodies of the invention may be bound to a solid support and/or packaged into kits in a suitable container along with suitable reagents, controls, instructions and the like. E. Therapeutic uses G-protein mediated signal transduction pathways have been shown to be involved in the control of cell division and growth. Many of the gene products involved in such pathways are proto-oncogenes i.e. they are capable of causing cellular transformation if mutated or aberrantly expressed, for example over-expressed. Therefore, mutations in MNGEF or its homologues may be a cause of cellular transformation, especially in the case of tumours associated with neuronal tissue, more particularly brain tissue. It may be possible to treat tumours that arise as a result by restoring normal MNGEF/NGEF function. This may be performed by means of gene therapy. Alternatively, it may be possible to raise antibodies that recognise specifically, mutated regions of the MNGEF protein, or its human homologue, NGEF. Such antibodies could be linked to therapeutic agents which would then target specifically cancer cells containing the mutated form of MNGEF/NGEF. Thus the polypeptides, polynucleotides and antibodies of the invention may be used in as compounds for treating neoplasms in animals or humans. Typically the compounds are formulated for clinical administration by mixing them with a pharmaceutically acceptable carrier or diluent. For example they can be formulated for topical, parenteral, intravenous, intramuscular, subcutaneous, intraocular or transdermal administration. Preferably, the compound is used in an injectable form. Direct injection into the patient's 5 tumour is advantageous because it makes it possible to concentrate the therapeutic effect at the level of the affected tissues. It may therefore be mixed with any vehicle which is pharmaceutically acceptable for an injectable formulation, preferably for a direct injection at the site to be treated. The pharmaceutically carrier or diluent may be, for example, sterile or isotonic solutions. 0 The dose of compound used may be adjusted according to various parameters, especially according to the compound used, the age, weight and condition of the patient to be treated, the mode of administration used, pathology of the tumour and the required clinical regimen. As a guide, the amount of compound administered by injection is suitably from 0.01 mg/kg to 30 mg/kg, preferably from 0.1 mg/kg to 10 mg/kg. 5 The routes of administration and dosages described are intended only as a guide since a skilled practitioner will be able to determine readily the optimum route of administration and dosage for any particular patient and condition. Compounds to be administered may include polypeptides, nucleic acids or antibodies. The nucleic acids may encode polypeptides or they may encode antisense o constructs that inhibit expression of a cellular gene. Nucleic acids may be administered by, for example, lipofection or by viral vectors. For example, the nucleic acid may form part of a viral vector such as an adenovirus. When viral vectors are used, in general the dose administered is between 10 4 and 10' 4 pfu/ml, preferably 10 6 to 10 10 pfu/ml. The term pfu ("plaque forming unit") corresponds to the infectivity of a virus solution and is determined 5 by infecting an appropriate cell culture and measuring, generally after 48 hours, the number of plaques of infected cells. The techniques for determining the pfu titre of a viral solution are well documented in the literature. Any cancer types may be treated by these methods, for example leukaemias, and solid tumours such as breast, ovary, lung, colon, pancreas, testes, liver, brain, muscle and o bone tumour. Preferably, the tumour is a tumour of the nervous system, in particular the central nervous system, for example the brain. The observation that MNGEF is expressed predominantly in brain tissue and that expression levels vary during foetal brain development (see Example 2) also suggest that MNGEF plays a role in neurological function, in particular neurological development. Thus it may be possible to diagnose, in particular prenatally, neurological conditions in which MNGEF and its human homologues are implicated using the detection methods discussed above. It may also be possible to treat such disorders by, in particular, gene therapy. Mapping data indicate that MNGEF maps to mouse chromosome 1 within a region syntenic to human chromosome 2q. NGEF maps to human chromosome 2 by hybridisation to a panel of mono-chromosomal somatic cell hybrids. A form of the neurological disorder dystonia also maps to the long arm of human chromosome 2. Thus, human NGEF may be implicated in this disease. Therefore the above-mentioned probes and DNA sequences may be used to detect and diagnose dystonia in humans by, for example, determining the presence of mutant human NGEF sequences as described above. Alternatively, the gene encoding human NGEF may lie in close proximity to the gene implicated in a form of dystonia which maps to the long arm of human chromosome 2. Therefore the above- mentioned probes and DNA sequences may be used to detect and diagnose dystonia in humans by, for example, genetic linkage analysis using techniques well-known in the art including analysis of restriction fragment length polymorphisms associated with the human NGEF locus. Detection and diagnosis in both cases outlined above may be carried out pre- natally using foetal tissue, or extracts thereof, or post-natally. Detection and diagnosis may also be carried out on germline tissue or extracts thereof. The following examples illustrate the invention: EXAMPLE 1 - Isolation of MNGEF2 and overlapping clones MNGEF2 and the overlapping clones were isolated from an adult mouse brain cDNA library (lzap Stratagene) cloned into the EcoRI and Xhol site of the vector pBluescript KS. Approximately, 10 6 plaques were screened using a oligonucleotide designated M3/6T7 Forward from the M3/6 gene (5'GCAGGAAAGCTGGGCAGCT 3' - SEQ ID No. 9). The probe was end-labelled with γ- 32 P dCTP (3000 Ci/mmol) using Promega kinase. The MNGEFl, MNGEF2 and MNGEF3 cDNA clones were isolated from the host bacteriophage using a standard in vivo excision protocol. The three inserts were released from the vector by digestion with the restriction enzymes EcoRI and Xhol. The sizes of the MNGEFl, MNGEF2 and MNGEF3 clones were approximately 2.3, 2.8 and 1.35 kb respectively. The clones were sequenced using a standard sequencing protocol from USB (Amersham). The full length cDNAs were digested using Taql restriction enzyme and the resulting fragments were subcloned into the CM site of the vector pBluescript KS to facilitate sequencing. Full length sequencing in one direction was obtained by carrying out sequential walks using insert specific oligonucleotides. Sequence analysis was done using the GCG Wisconsin package version 8. Results Approximately one million plaques from an adult mouse brain cDNA library were screened with an ohgonucleotide (M3/6T7 Forward) from the M3/6 cDNA sequence. Five positives clones were identified, three of which appeared to be the same transcript of varying length. Sequencing of these cDNA clones demonstrated that they showed significant homology to TLM, a transforming gene, whose sequence is related to regulators of small GTP-binding proteins. 60% homology was observed on the nucleotide level between the MNGEF2 and TIM. The homology extended over the region known as DH domain, which plays an important role in mediating cellular transformation. Sequencing also revealed that two of these cDNA clones (MNGEFl and MNGEF2) contained the following trinucleotide repeat (AGG) 8 GAG(AGG) 3 (SEQ ID No. 10). In addition it was observed that the longer of these cDNAs, MNGEF2, contained an extra 92bp sequence, which was not present in MNGEFl and MNGEF3, although the flanking sequence of the region was identical. This 92 bp fragment comprises an unspliced intron which results in a premature termination codon as shown in SEQ LD NO. 3. EXAMPLE 2 - Expression of MNGEF2 in mouse and human tissues To determine the pattern of expression of MNGEF, the cDNA clone MNGEF2 was hybridised to Northern blots of poly(A)+ RNA derived from a selection of adult mouse tissues and human foetal brain tissues. Northern analysis RNA was extracted from mouse tissue and poly(A)+ RNA was prepared from 100 μg of total RNA using the Dynabeads mRNA purification kit (Dynal). Northern blots were prepared according to Current Protocols in Molecular Biology, with each lane containing 2 μg of poly(A)+ RNA. The human foetal brain Northern blot and the mouse foetal developmental Northern blot were obtained from Clontech. The blots were hybridised at 42°C in standard formamide buffer and washed to a stringency of O.lxSSC, 0.1% SDS at 65°C. The blots were visualised by autoradiography after exposure for one or two days at -70°C. Results The MNGEF2 cDNA clone detected a transcript of approximately 3 kb predominantly in mouse brain and a faint one of the same size in mouse eye. In addition, a shorter transcript (approximately 2.2 kb) of less intensity was seen in the brain. A faint slightly larger transcript (about 3.5 kb) was also observed in small intestine and liver. Hybridisation of the MNGEF2 cDNA clone to a Northern blot of human brain tissues (Clontech), detects a 3 kb transcript expressed predominantly in the caudate nucleus, but also in the amygdala and the hippocampus. The same sized transcript, albeit much fainter, was observed in all the remaining tissues. A similar 3 kb transcript was seen when the MNGEF2 cDNA clone was used as a probe on a whole mouse embryo developmental Northern (Clontech). The strongest signal was observed in day 7 of embryonic development. Weaker signals of the same size were seen in days 11, 15 and 17. EXAMPLE 3 - Partial cloning of human NGEF To isolate the human homologue of MNGEF, primers m32bt7f and m32bt3f were used to amplify cDNA from human foetal brain. The sequences of the primers used are shown below: 3.2AT3F: 5'-CAAGAGAGGCTGGCAGAGGCAC-3' - SEQ ID No. 11 3.2AT7F 5'-GGACCAAGTTTGTATCCTTCAC-3' - SEQ ID No. 12 3.2BT7F 5'-GGACATCTGCTGCAGCTCACC-3' - SEQ ID No. 13 3.2BT3F 5'-GGAGAGCTCTGCCTCAGATCTG-3' - SEQ ID No. 14 An 803 bp product was amplified and cloned into the pGEMT vector (Promega). The clone HFB32 was sequenced and the sequence is shown as SEQ ID No. 7. The translated protein sequence is shown as SEQ ID No. 8. A comparison between mouse and the human nucleotide sequence indicates 87.8% homology. A comparison between the protein sequence of the two species indicates 97% homology. A search of the Yeast Genome database with the DH region of MNGEF showed homology to an open reading frame (ORF) from Chromosome XII (figure 6). This ORF corresponds to a yeast protein called ROM2 which is a GDP-GTP exchange protein for Rholp containing the DH domains and the pleckstrin domains. The RHO1 gene encodes a homologue of the mammalian RhoA small GTP binding protein in yeast. Rholp is localised at the growth site and required for bud formation. Disruption of ROM2 results in a temperature-sensitive growth phenotype. These mutants offer an attractive system to study activation of Rho. SEQUENCE LISTING (m) NUMBER OF SEQUENCES 8 (2) INFORMATION FOR SEQ ID NO 1 (i) SEQUENCE CHARACTERISTICS (A) LENGTH 2741 base pairs (B) TYPE nucleic acid (C) STRANDEDNESS both (D) TOPOLOGY linear (n) MOLECULE TYPE cDNA (ix) FEATURE (A) NAME/KEY CDS (B) LOCATION 343 2004 (xi) SEQUENCE DESCRIPTION SEQ ID NO 1 GCGCTCTACA GCAGCGGCGG CGGCAGCTCC GGCTTGAGCC GCGCGCGCTG CGACCTCACT 60 CAGAGCCCGC GCATTGCCCC CGGCTGGGCC CTGGGCCCCG CGCGGCTCCC CACCAGCCCC 120 TGAGCCTACC CGGTCGCTGG TCCCCATGGA GCTGCTGGCT GCAGCCTTCA GCGCCGCCTG 180 CGCCGTGGAC CACGACAGCT CCACCTCGGA GAGCGACACG CGCGACTCGG CGGCGGGACA 240 CCTGCCGGGC AGCGAGTCAT CCTCCACCCC TGGAAATGGA ACCACACCCG AGGAGTGCCC 300 AGCCCTCACC GACAGCCCCA CCACTCTCAC GGAGCCCTGC AG ATG ATC CAT CCC 354 1 ATT CCC GCC GAC TCC TGG AGA AAC CTC ATT GAA CAA ATA GGG CTC CTG 402 He Pro Ala Asp Ser Trp Arg Asn Leu He Glu Gin He Gly Leu Leu 5 10 15 20 TAT CAA GAG TAT AGA GAC AAA TCG ACT CTC CAA GAA ATT GAA ACA CGG 450 Tyr Gin Glu Tyr Arg Asp Lys Ser Thr Leu Gin Glu He Glu Thr Arg 25 30 35 AGG CAG CAG GAT GCA GAA ATC CAA GGC AAC TCC GAT GGG TCC CAG GTT 498 Arg Gin Gin Asp Ala Glu He Gin Gly Asn Ser Asp Gly Ser Gin Val 40 45 50 GGG GAA GAC GCT GGA GAG GAG GAG GAG GAG GAG GAG GAG GGA GAG GAG 546 Gly Glu Asp Ala Gly Glu Glu Glu Glu Glu Glu Glu Glu Gly Glu Glu 55 60 65 GAG GAG CTG GCC AGC CCT CCT GAG AGG AGA GCT CTG CCT CAG ATC TGC 594 Glu Glu Leu Ala Ser Pro Pro Glu Arg Arg Ala Leu Pro Gin He Cys 70 75 80 CTG CTC AGC AAC CCC CAC TCC AGG TTC AAC CTC TGG CAA GAC CTT CCT 642 Leu Leu Ser Asn Pro His Ser Arg Phe Asn Leu Trp Gin Asp Leu Pro 85 90 95 100 GAG ATC CAG AGC AGT GGC GTG CTG GAC ATT CTC CAG CCG GAG GAG ATC 690 Glu He Gin Ser Ser Gly Val Leu Asp He Leu Gin Pro Glu Glu He 105 110 115 AGG CTG CAG GAG GCC ATG TTT GAG TTG GTT ACC TCT GAG GCC TCC TAC 738 Arg Leu Gin Glu Ala Met Phe Glu Leu Val Thr Ser Glu Ala Ser Tyr 120 125 130 TAT AAG AGC CTG AAC CTG CTG GTG TCG CAC TTC ATG GAG AAC GAG CGT 786 Tyr Lys Ser Leu Asn Leu Leu Val Ser His Phe Met Glu Asn Glu Arg 135 140 145 CTG AAG AAG ATC CTG CAT CCA TCT GAG GCC CAC ATC CTC TTT TCC AAT 834 Leu Lys Lys He Leu His Pro Ser Glu Ala His He Leu Phe Ser Asn 150 155 160 GTC CTG GAT GTC ATG GCT GTC AGT GAG CGG TTT πG CTG GAG CTA GAG 882 Val Leu Asp Val Met Ala Val Ser Glu Arg Phe Leu Leu Glu Leu Glu 165 170 175 180 CAC CGC ATG GAG GAG AAC ATT GTT ATC TCG GAT GTG TGC GAC ATC GTG 930 His Arg Met Glu Glu Asn He Val He Ser Asp Val Cys Asp He Val 185 190 195 TAC CGT TAC GCA GCT GAT CAC TTC TCG GTC TAT ATC ACT TAC GTC AGT 978 Tyr Arg Tyr Ala Ala Asp His Phe Ser Val Tyr He Thr Tyr Val Ser 200 205 210 AAC CAG ACC TAC CAG GAA AGG ACA TAC AAG CAG CTC CTA CAG GAG AAG 1026 Asn Gin Thr Tyr Gin Glu Arg Thr Tyr Lys Gin Leu Leu Gin Glu Lys 215 220 225 GCC GCT TTC CGG GAA CTG ATC GCA CAG TTG GAG CTG GAC CCC AAA TGC 1074 Ala Ala Phe Arg Glu Leu He Ala Gin Leu Glu Leu Asp Pro Lys Cys 230 235 240 AAG GGC CTG CCT TTC TCC TCC TTC CTC ATC πG CCT πC CAG AGG ATC 1122 Lys Gly Leu Pro Phe Ser Ser Phe Leu He Leu Pro Phe Gin Arg He 245 250 255 260 ACG AGA CTC AAG CTG CTG GTC CAG AAT ATC CTG AAG AGA GTG GAG GAG 1170 Thr Arg Leu Lys Leu Leu Val Gin Asn He Leu Lys Arg Val Glu Glu 265 270 275 AGG TCT GAA CGT GAA GGC ACC GCC TTG GAT GCC CAC AAG GAG CTA GAA 1218 Arg Ser Glu Arg Glu Gly Thr Ala Leu Asp Ala His Lys Glu Leu Glu 280 285 290 ATG GTG GTA AAG GCA TGC AAT GAG GGT GTC CGG AAG ATG AGC CGC ACA 1266 Met Val Val Lys Ala Cys Asn Glu Gly Val Arg Lys Met Ser Arg Thr 295 300 305 GAA CAG ATG ATC AGC ATT CAG AAG AAG ATG GAG TTC AAG ATC AAG TCG 1314 Glu Gin Met He Ser He Gin Lys Lys Met Glu Phe Lys He Lys Ser 310 315 320 GTA CCC ATC ATC TCA CAC TCC CGG TGG CTG CTG AAG CAG GGT GAG CTG 1362 Val Pro He He Ser His Ser Arg Trp Leu Leu Lys Gin Gly Glu Leu 325 330 335 340 CAG CAG ATG TCC GGC CCC AAG ACC TCC CGC ACC CTG CGG ACC AAG AAG 1410 Gin Gin Met Ser Gly Pro Lys Thr Ser Arg Thr Leu Arg Thr Lys Lys 345 350 355 CTC TTC AGA GAA ATT TAC CTC TTC CTC TTC AAT GAC CTG CTG GTG ATC 1458 Leu Phe Arg Glu He Tyr Leu Phe Leu Phe Asn Asp Leu Leu Val He 360 365 370 TGC CGG CAG ATC CCT GGA GAC AAG TAC CAG GTG πT GAT TCG GCC CCA 1506 Cys Arg Gin He Pro Gly Asp Lys Tyr Gin Val Phe Asp Ser Ala Pro 375 380 385 AGG GGC CTG CTT CGA GTG GAG GAG CTG GAG GAC CAG GGT CAA ACA CTG 1554 Arg Gly Leu Leu Arg Val Glu Glu Leu Glu Asp Gin Gly Gin Thr Leu 390 395 400 GCT AAT GTG TTC ATC CTG CGG CTG CTG GAA AAT GCA GAT GAC CGA GAG 1602 Ala Asn Val Phe He Leu Arg Leu Leu Glu Asn Ala Asp Asp Arg Glu 405 410 415 420 GCC ACC TAT ATG CTG AAG GCA TCC TCC CAG AGC GAG ATG AAG CGC TGG 1650 Ala Thr Tyr Met Leu Lys Ala Ser Ser Gin Ser Glu Met Lys Arg Trp 425 430 435 ATG ACC TCA CTG GCC CCC AAC AGG AGG ACC AAG TTT GTA TCC TTC ACA 1698 Met Thr Ser Leu Ala Pro Asn Arg Arg Thr Lys Phe Val Ser Phe Thr 440 445 450 ^692 111V31V1W V39WW9V91111991V31991V111331 V3913V319911W91W9V rVX 3911119399 V3339V991V 913333999133131919113V31V1V9113999W9113 frK 3139V13133 V3391V311999V9139V1139VW33311399V39V31333913339W 17IS2 3393V39113131339V33913133919333319333913391333V919919V39V91V Ψftl 91119V999V 9333913333933991V9V3 V3V399V33V 1319V9W119139W9911 ^682 13V1333113319193V3913W9133331 V99V31913V 33W199W99199V399V3 ttZZ 9V39913331999W3V3V33399V9W9913V9199331133V9V9V331391V399V3 VLZZ 99W9W9V199V9V3131319V311333133V33V3V11 V19V399V113V19W139V IZZ WV919913V 3V3911391119113V33991331919V13139W99191391V333913 WIZ 3319131391339931139919V99V39V399V3131133919V3191W 9W999V113 ^602 3999V9V311 V9V3V933393V9V9V39933399V39V99991V999V3999191999W 059 U[9 β JV usv β JV J9S ^L9 VWZ 999W9133V 399V3139V3333133W91 W31931W 99339V 399 SfrS 0i?S 9S9 ΠΘI s i β jy βj y dsy sλη usv UL9 JΘS βJV LU9 ou dsv rug q.9W sλη 986T 9139W 99V 3933V99VV 1W 9V339V 3939V31333V9 VV991V 9W 085 S2S 029 SLH [BΛ β JV θL| sλ " 3 n 9 sλη naη usv t-Uø J9S β ^V 9 LI sλη oud usv 8861 1V3 V19993311191 W99W 3133W 9V333139331V 9W 3333W SIS 015 SOS naη ΘLI rU9 rU9 IU law J9S -J9S ou 3i|d dui Λ ' LD β JV rug UL9 dsv 0681 91331V 9V99V9 V3V 91V 33119V 333311991399 V9V 9V99V33V9 SLH naη 6JV ΠL9 dsv rug Ji|i s/Cη ΠL9 naη ΘLI 2V8I 1V39139939V919911131V 9919991V99V9 V3V 9W 9V991331V usv nai ΘLI dsv BLV naη ΠLD nan jμi naη n LD dsv QJd u[ø u g Bv t76ZT 3VV 91331V 1V9 V39913 W991393V 913 W91V91339V39V3339 LBΛ Jλl OJ SLH LBΛ sλO UL9 L Λ iU9 oud sΛO dsv naη naη βj v J3S 917ZI 919 IVl 9333V39191919V33199V33331913V9911913993131 ■ PZ - zoz o/Lβao/iod wi£zm OΛV TTGGCTGCAT TAAAAGAAGC ATCCTATATA AAAAAAAAAA AAAAAAA 2741 (2) INFORMATION FOR SEQ ID NO 2 (i) SEQUENCE CHARACTERISTICS (A) LENGTH 554 amino acids (B) TYPE amino acid (D) TOPOLOGY linear (n) MOLECULE TYPE protein (xi) SEQUENCE DESCRIPTION SEQ ID NO 2 Met He His Pro He Pro Ala Asp Ser Trp Arg Asn Leu He Glu Gin 1 5 10 15 He Gly Leu Leu Tyr Gin Glu Tyr Arg Asp Lys Ser Thr Leu Gin Glu 20 25 30 He Glu Thr Arg Arg Gin Gin Asp Ala Glu He Gin Gly Asn Ser Asp 35 40 45 Gly Ser Gin Val Gly Glu Asp Ala Gly Glu Glu Glu Glu Glu Glu Glu 50 55 60 Glu Gly Glu Glu Glu Glu Leu Ala Ser Pro Pro Glu Arg Arg Ala Leu 65 70 75 80 Pro Gin He Cys Leu Leu Ser Asn Pro His Ser Arg Phe Asn Leu Trp 85 90 95 Gin Asp Leu Pro Glu He Gin Ser Ser Gly Val Leu Asp He Leu Gin 100 105 110 Pro Glu Glu He Arg Leu Gin Glu Ala Met Phe Glu Leu Val Thr Ser 115 120 125 Glu Ala Ser Tyr Tyr Lys Ser Leu Asn Leu Leu Val Ser His Phe Met 130 135 140 Glu Asn Glu Arg Leu Lys Lys He Leu His Pro Ser Glu Ala His He 145 150 155 160 Leu Phe Ser Asn Val Leu Asp Val Met Ala Val Ser Glu Arg Phe Leu 165 170 175 Leu Glu Leu Glu His Arg Met Glu Glu Asn He Val He Ser Asp Val 180 185 190 Cys Asp He Val Tyr Arg Tyr Ala Ala Asp His Phe Ser Val Tyr He 195 200 205 Thr Tyr Val Ser Asn Gin Thr Tyr Gin Glu Arg Thr Tyr Lys Gin Leu 210 215 220 Leu Gin Glu Lys Ala Ala Phe Arg Glu Leu He Ala Gin Leu Glu Leu 225 230 235 240 Asp Pro Lys Cys Lys Gly Leu Pro Phe Ser Ser Phe Leu He Leu Pro 245 250 255 Phe Gin Arg He Thr Arg Leu Lys Leu Leu Val Gin Asn He Leu Lys 260 265 270 Arg Val Glu Glu Arg Ser Glu Arg Glu Gly Thr Ala Leu Asp Ala His 275 280 285 Lys Glu Leu Glu Met Val Val Lys Ala Cys Asn Glu Gly Val Arg Lys 290 295 300 Met Ser Arg Thr Glu Gin Met He Ser He Gin Lys Lys Met Glu Phe 305 310 315 320 Lys He Lys Ser Val Pro He He Ser His Ser Arg Trp Leu Leu Lys 325 330 335 Gin Gly Glu Leu Gin Gin Met Ser Gly Pro Lys Thr Ser Arg Thr Leu 340 345 350 Arg Thr Lys Lys Leu Phe Arg Glu He Tyr Leu Phe Leu Phe Asn Asp 355 360 365 Leu Leu Val He Cys Arg Gin He Pro Gly Asp Lys Tyr Gin Val Phe 370 375 380 Asp Ser Ala Pro Arg Gly Leu Leu Arg Val Glu Glu Leu Glu Asp Gin 385 390 395 400 Gly Gin Thr Leu Ala Asn Val Phe He Leu Arg Leu Leu Glu Asn Ala 405 410 415 Asp Asp Arg Glu Ala Thr Tyr Met Leu Lys Ala Ser Ser Gin Ser Glu 420 425 430 Met Lys Arg Trp Met Thr Ser Leu Ala Pro Asn Arg Arg Thr Lys Phe 435 440 445 Val Ser Phe Thr Ser Arg Leu Leu Asp Cys Pro Gin Val Gin Cys Val 450 455 460 His Pro Tyr Val Ala Gin Gin Pro Asp Glu Leu Thr Leu Glu Leu Ala 465 470 475 480 Asp He Leu Asn He Leu Glu Lys Thr Glu Asp Gly Trp He Phe Gly 485 490 495 Glu Arg Leu His Asp Gin Glu Arg Gly Trp Phe Pro Ser Ser Met Thr 500 505 510 Glu Glu He Leu Asn Pro Lys He Arg Ser Gin Asn Leu Lys Glu Cys 515 520 525 Phe Arg Val His Lys Met Glu Asp Pro Gin Arg Ser Gin Asn Lys Asp 530 535 540 Arg Arg Lys Leu Gly Ser Arg Asn Arg Gin 545 550 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS. (A) LENGTH: 2833 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS . both (D) TOPOLOGY- linear (n) MOLECULE TYPE: cDNA (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 343..1860 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3. GCGCTCTACA GCAGCGGCGG CGGCAGCTCC GGCTTGAGCC GCGCGCGCTG CGACCTCACT 60 CAGAGCCCGC GCAπGCCCC CGGCTGGGCC CTGGGCCCCG CGCGGCTCCC CACCAGCCCC 120 TGAGCCTACC CGGTCGCTGG TCCCCATGGA GCTGCTGGCT GCAGCCTTCA GCGCCGCCTG 180 CGCCGTGGAC CACGACAGCT CCACCTCGGA GAGCGACACG CGCGACTCGG CGGCGGGACA 240 CCTGCCGGGC AGCGAGTCAT CCTCCACCCC TGGAAATGGA ACCACACCCG AGGAGTGCCC 300 AGCCCTCACC GACAGCCCCA CCACTCTCAC GGAGCCCTGC AG ATG ATC CAT CCC 354 1 ATT CCC GCC GAC TCC TGG AGA AAC CTC ATT GAA CAA ATA GGG CTC CTG 402 He Pro Ala Asp Ser Trp Arg Asn Leu He Glu Gin He Gly Leu Leu 5 10 15 20 TAT CAA GAG TAT AGA GAC AAA TCG ACT CTC CAA GAA ATT GAA ACA CGG 450 Tyr Gin Glu Tyr Arg Asp Lys Ser Thr Leu Gin Glu He Glu Thr Arg 25 30 35 AGG CAG CAG GAT GCA GAA ATC CAA GGC AAC TCC GAT GGG TCC CAG GTT 498 Arg Gin Gin Asp Ala Glu He Gin Gly Asn Ser Asp Gly Ser Gin Val 40 45 50 GGG GAA GAC GCT GGA GAG GAG GAG GAG GAG GAG GAG GAG GGA GAG GAG 546 Gly Glu Asp Ala Gly Glu Glu Glu Glu Glu Glu Glu Glu Gly Glu Glu 55 60 65 GAG GAG CTG GCC AGC CCT CCT GAG AGG AGA GCT CTG CCT CAG ATC TGC 594 Glu Glu Leu Ala Ser Pro Pro Glu Arg Arg Ala Leu Pro Gin He Cys 70 75 80 CTG CTC AGC AAC CCC CAC TCC AGG πC AAC CTC TGG CAA GAC CTT CCT 642 Leu Leu Ser Asn Pro His Ser Arg Phe Asn Leu Trp Gin Asp Leu Pro 85 90 95 100 GAG ATC CAG AGC AGT GGC GTG CTG GAC ATT CTC CAG CCG GAG GAG ATC 690 Glu He Gin Ser Ser Gly Val Leu Asp He Leu Gin Pro Glu Glu He 105 110 115 AGG CTG CAG GAG GCC ATG TTT GAG πG GTT ACC TCT GAG GCC TCC TAC 738 Arg Leu Gin Glu Ala Met Phe Glu Leu Val Thr Ser Glu Ala Ser Tyr 120 125 130 TAT AAG AGC CTG AAC CTG CTG GTG TCG CAC πC ATG GAG AAC GAG CGT 786 Tyr Lys Ser Leu Asn Leu Leu Val Ser His Phe Met Glu Asn Glu Arg 135 140 145 CTG AAG AAG ATC CTG CAT CCA TCT GAG GCC CAC ATC CTC πT TCC AAT 834 Leu Lys Lys He Leu His Pro Ser Glu Ala His He Leu Phe Ser Asn 150 155 160 GTC CTG GAT GTC ATG GCT GTC AGT GAG CGG Tπ πG CTG GAG CTA GAG 882 Val Leu Asp Val Met Ala Val Ser Glu Arg Phe Leu Leu Glu Leu Glu 165 170 175 180 CAC CGC ATG GAG GAG AAC ATT GTT ATC TCG GAT GTG TGC GAC ATC GTG 930 His Arg Met Glu Glu Asn ie Val He Ser Asp Val Cys Asp He Val 185 190 195 TAC CGT TAC GCA GCT GAT CAC TTC TCG GTC TAT ATC ACT TAC GTC AGT 978 Tyr Arg Tyr Ala Ala Asp His Phe Ser Val Tyr He Thr Tyr Val Ser 200 205 210 AAC CAG ACC TAC CAG GAA AGG ACA TAC AAG CAG CTC CTA CAG GAG AAG 1026 Asn Gin Thr Tyr Gin Glu Arg Thr Tyr Lys Gin Leu Leu Gin Glu Lys 215 220 225 GCC GCT TTC CGG GAA CTG ATC GCA CAG TTG GAG CTG GAC CCC AAA TGC 1074 Ala Ala Phe Arg Glu Leu He Ala Gin Leu Glu Leu Asp Pro Lys Cys 230 235 240 AAG GGC CTG CCT TTC TCC TCC TTC CTC ATC TTG CCT TTC CAG AGG ATC 1122 Lys Gly Leu Pro Phe Ser Ser Phe Leu He Leu Pro Phe Gin Arg He 245 250 255 260 ACG AGA CTC AAG CTG CTG GTC CAG AAT ATC CTG AAG AGA GTG GAG GAG 1170 Thr Arg Leu Lys Leu Leu Val Gin Asn He Leu Lys Arg Val Glu Glu 265 270 275 AGG TCT GAA CGT GAA GGC ACC GCC πG GAT GCC CAC AAG GAG CTA GAA 1218 Arg Ser Glu Arg Glu Gly Thr Ala Leu Asp Ala His Lys Glu Leu Glu 280 285 290 ATG GTG GTA AAG GCA TGC AAT GAG GGT GTC CGG AAG ATG AGC CGC ACA 1266 Met Val Val Lys Ala Cys Asn Glu Gly Val Arg Lys Met Ser Arg Thr 295 300 305 GAA CAG ATG ATC AGC ATT CAG AAG AAG ATG GAG TTC AAG ATC AAG TCG 1314 Glu Gin Met He Ser He Gin Lys Lys Met Glu Phe Lys He Lys Ser 310 315 320 GTA CCC ATC ATC TCA CAC TCC CGG TGG CTG CTG AAG CAG GGT GAG CTG 1362 Val Pro He He Ser His Ser Arg Trp Leu Leu Lys Gin Gly Glu Leu 325 330 335 340 CAG CAG ATG TCC GGC CCC AAG ACC TCC CGC ACC CTG CGG ACC AAG AAG 1410 Gin Gin Met Ser Gly Pro Lys Thr Ser Arg Thr Leu Arg Thr Lys Lys 345 350 355 CTC TTC AGA GAA ATT TAC CTC TTC CTC TTC AAT GAC CTG CTG GTG ATC 1458 Leu Phe Arg Glu He Tyr Leu Phe Leu Phe Asn Asp Leu Leu Val He 360 365 370 TGC CGG CAG ATC CCT GGA GAC AAG TAC CAG GTG TTT GAT TCG GCC CCA 1506 Cys Arg Gin He Pro Gly Asp Lys Tyr Gin Val Phe Asp Ser Ala Pro 375 380 385 08^2 99V39V39913331999W3 V3V33399V9 W9913V9199331133V9V 9V331391V3 0Z82 99V399W9V V9V199V9V3131319V311333133V33V 3V11V19V399V113V19W 0182 139WW919913V3V3911391119113V 33991331919V13139W99191391V33 0S22 39133319131391339931139919V99V 39V399V3131133919V3191W9W999 0612 V1133999V9 V311V9V3V933393V9V9V 39933399V39V99991V999V39991919 08T2 99W999W9133V399V3139V3333133 W91W31931W99339V3999139W99 0Z02 V3933V99W 1W9V339V3939V31333V 9W991V9W 1V3V19993311191W99V 0102 V3133W9V333139331V9 W3333W91331V9V99V9 V3V91V33119V33331199 0S6I 1399V9V9V99V33V91V39139939V919911131V991999V39V119 V391991V9V SOS * oud UL9 λ " L9 na SLH 0681 13V99V191V 9V133991V13V99V9V9V9 V91 V339V33991131V3 nai JL|1 λ " L9 sλi uu. BJV oud ΠL9 λ " L9 dsv n[9 Ji|i sλi ΠLD nai ΘLI 28I 91313V 9999W 33V 3933339V91991V99V9 V3V 9W 9V991331V usv a 3LI sv LV nai nL9 nai JU.1 nai L9 dsv oud ULS UL9 BLV 176ZT 3W 91331V 1V9 V39913 VV991393V 913 W91V91339V39V3339 L Λ Jλl oud SLH LBΛ sλo UL9 LB UL9 oud SXQ dsv nai nai BJV aas 9PLI 919 IVl 9333V39191919V33199V33331913V9911913993131 j μi aμd J9S L ρ Λ θu,d sη JU,χ β uv β JV usv oud V nai J3S JU.119W 8691 V3V 311331 V191119W 33V 99V 99V 3W 333339913 V3133V 91V dJi J|I BLV 0S9I 9913939W 91V 9V939V 9V3331331 V399W 91391V IVl 33V 339 nL9 BJV dsv dsv BLV USV L9 nai nai β uv nai ΘLI aμd LBΛ USV BLV 209T 9V9 V933V91V9 V39 IW VV991391399391331V 311919 IW 139 nai Ji|i UL9 λ9 UL9 dsv nL9 nai n[9 n[9 LBΛ βJV nai nai /19 βuv frQSI 913 V3V W31999V33V99V99139V99V9919 V9311391339999V -oε- Z0££0/L6SO/lDd £P £Z/S6 OΛV CAGGTGGAAG GTAACCACTG TCAGGATCCC CTGAACTGCA CGTGTCCTTC CCTACTTTGG 2490 AAGCTGTTAA GAGTCTACCA GGCACACAGA TGGCCGCCCC TGCCCGAGGG AGTTTGATGA 2550 GCAGTGGTGA CCCTGCCTGC CCGTCCCCGT GCCTCTGCCA GCCTCTCTTG CACGCCAAGC 2610 CCTGCCCTCA GCAGGCπCC CAAAGCTTAG CTGAGGGTTC ATGCCACCTC TAGCTCCTTG 2670 AAGGGCTTGA TATCACTTGT GTCTCCTGGG CCCCTGATGG AGCCCAGGCG TTTTGCAGAA 2730 TGAATTGGTC ACTGCATCCT TTATGGTCAT GGTTπGAGA AAAGCAAATA TCATTTTTGG 2790 CTGCAπAAA AGAAGCATCC TATATAAAAA AAAAAAAAAA AAA 2833 (2) INFORMATION FOR SEQ ID NO: 4 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 505 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (n) MOLECULE TYPE: protein (xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 4 Met He His Pro He Pro Ala Asp Ser Trp Arg Asn Leu He Glu Gin 1 5 10 15 He Gly Leu Leu Tyr Gin Glu Tyr Arg Asp Lys Ser Thr Leu Gin Glu 20 25 30 He Glu Thr Arg Arg Gin Gin Asp Ala Glu He Gin Gly Asn Ser Asp 35 40 45 Gly Ser Gin Val Gly Glu Asp Ala Gly Glu Glu Glu Glu Glu Glu Glu 50 55 60 Glu Gly Glu Glu Glu Glu Leu Ala Ser Pro Pro Glu Arg Arg Ala Leu 65 70 75 80 Pro Gin He Cys Leu Leu Ser Asn Pro His Ser Arg Phe Asn Leu Trp 85 90 95 Gin Asp Leu Pro Glu He Gin Ser Ser Gly Val Leu Asp He Leu Gin 100 105 110 Pro Glu Glu He Arg Leu Gin Glu Ala Met Phe Glu Leu Val Thr Ser 115 120 125 Glu Ala Ser Tyr Tyr Lys Ser Leu Asn Leu Leu Val Ser His Phe Met 130 135 140 Glu Asn Glu Arg Leu Lys Lys He Leu His Pro Ser Glu Ala His He 145 150 155 160 Leu Phe Ser Asn Val Leu Asp Val Met Ala Val Ser Glu Arg Phe Leu 165 170 175 Leu Glu Leu Glu His Arg Met Glu Glu Asn He Val He Ser Asp Val 180 185 190 Cys Asp He Val Tyr Arg Tyr Ala Ala Asp His Phe Ser Val Tyr He 195 200 205 Thr Tyr Val Ser Asn Gin Thr Tyr Gin Glu Arg Thr Tyr Lys Gin Leu 210 215 220 Leu Gin Glu Lys Ala Ala Phe Arg Glu Leu He Ala Gin Leu Glu Leu 225 230 235 240 Asp Pro Lys Cys Lys Gly Leu Pro Phe Ser Ser Phe Leu He Leu Pro 245 250 255 Phe Gin Arg He Thr Arg Leu Lys Leu Leu Val Gin Asn He Leu Lys 260 265 270 Arg Val Glu Glu Arg Ser Glu Arg Glu Gly Thr Ala Leu Asp Ala His 275 280 285 Lys Glu Leu Glu Met Val Val Lys Ala Cys Asn Glu Gly Val Arg Lys 290 295 300 Met Ser Arg Thr Glu Gin Met He Ser He Gin Lys Lys Met Glu Phe 305 310 315 320 Lys He Lys Ser Val Pro He He Ser His Ser Arg Trp Leu Leu Lys 325 330 335 Gin Gly Glu Leu Gin Gin Met Ser Gly Pro Lys Thr Ser Arg Thr Leu 340 345 350 Arg Thr Lys Lys Leu Phe Arg Glu He Tyr Leu Phe Leu Phe Asn Asp 355 360 365 Leu Leu Val He Cys Arg Gin He Pro Gly Asp Lys Tyr Gin Val Phe 370 375 380 Asp Ser Ala Pro Arg Gly Leu Leu Arg Val Glu Glu Leu Glu Asp Gin 385 390 395 400 Gly Gin Thr Leu Ala Asn Val Phe He Leu Arg Leu Leu Glu Asn Ala 405 410 415 Asp Asp Arg Glu Ala Thr Tyr Met Leu Lys Ala Ser Ser Gin Ser Glu 420 425 430 Met Lys Arg Trp Met Thr Ser Leu Ala Pro Asn Arg Arg Thr Lys Phe 435 440 445 Val Ser Phe Thr Ser Arg Leu Leu Asp Cys Pro Gin Val Gin Cys Val 450 455 460 His Pro Tyr Val Ala Gin Gin Pro Asp Glu Leu Thr Leu Glu Leu Ala 465 470 475 480 Asp He Leu Asn He Leu Glu Lys Thr Glu Asp Gly Glu Pro Arg Thr 485 490 495 Lys Gly Thr Leu His Leu Gly Gin Pro 500 505 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2343 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS : both (D) TOPOLOGY, linear (n) MOLECULE TYPE: cDNA (lx) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 2..1609 (xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 5 : C CTG TAT CAA GAG TAT AGA GAC AAA TCG ACT CTC CAA GAA ATT GAA 46 Leu Tyr Gi n Gl u Tyr Arg Asp Lys Ser Thr Leu Gi n Gl u H e Gl u 1 5 10 15 ACA CGG AGG CAG CAG GAT GCA GAA ATC CAA GGC AAC TCC GAT GGG TCC 94 Thr Arg Arg Gi n Gi n Asp Al a Gl u H e Gi n Gly Asn Ser Asp Gly Ser 20 25 30 CAG GTT GGG GAA GAC GCT GGA GAG GAG GAG GAG GAG GAG GAG GAG GGA 142 Gin Val Gly Glu Asp Ala Gly Glu Glu Glu Glu Glu Glu Glu Glu Gly 35 40 45 GAG GAG GAG GAG CTG GCC AGC CCT CCT GAG AGG AGA GCT CTG CCT CAG 190 Glu Glu Glu Glu Leu Ala Ser Pro Pro Glu Arg Arg Ala Leu Pro Gin 50 55 60 ATC TGC CTG CTC AGC AAC CCC CAC TCC AGG TTC AAC CTC TGG CAA GAC 238 He Cys Leu Leu Ser Asn Pro His Ser Arg Phe Asn Leu Trp Gin Asp 65 70 75 Cπ CCT GAG ATC CAG AGC AGT GGC GTG CTG GAC Aπ CTC CAG CCG GAG 286 Leu Pro Glu He Gin Ser Ser Gly Val Leu Asp He Leu Gin Pro Glu 80 85 90 95 GAG ATC AGG CTG CAG GAG GCC ATG Tπ GAG TTG GTT ACC TCT GAG GCC 334 Glu He Arg Leu Gin Glu Ala Met Phe Glu Leu Val Thr Ser Glu Ala 100 105 110 TCC TAC TAT AAG AGC CTG AAC CTG CTG GTG TCG CAC TTC ATG GAG AAC 382 Ser Tyr Tyr Lys Ser Leu Asn Leu Leu Val Ser His Phe Met Glu Asn 115 120 125 GAG CGT CTG AAG AAG ATC CTG CAT CCA TCT GAG GCC CAC ATC CTC Tπ 430 Glu Arg Leu Lys Lys He Leu His Pro Ser Glu Ala His He Leu Phe 130 135 140 TCC AAT GTC CTG GAT GTC ATG GCT GTC AGT GAG CGG Tπ πG CTG GAG 478 Ser Asn Val Leu Asp Val Met Ala Val Ser Glu Arg Phe Leu Leu Glu 145 150 155 CTA GAG CAC CGC ATG GAG GAG AAC Aπ GTT ATC TCG GAT GTG TGC GAC 526 Leu Glu His Arg Met Glu Glu Asn He Val He Ser Asp Val Cys Asp 160 165 170 175 ATC GTG TAC CGT TAC GCA GCT GAT CAC TTC TCG GTC TAT ATC ACT TAC 574 He Val Tyr Arg Tyr Ala Ala Asp His Phe Ser Val Tyr He Thr Tyr 180 185 190 GTC AGT AAC CAG ACC TAC CAG GAA AGG ACA TAC AAG CAG CTC CTA CAG 622 Val Ser Asn Gin Thr Tyr Gin Glu Arg Thr Tyr Lys Gin Leu Leu Gin 195 200 205 GAG AAG GCC GCT TTC CGG GAA CTG ATC GCA CAG TTG GAG CTG GAC CCC 670 Glu Lys Ala Ala Phe Arg Glu Leu He Ala Gin Leu Glu Leu Asp Pro 210 215 220 AAA TGC AAG GGC CTG CCT TTC TCC TCC TTC CTC ATC TTG CCT TTC CAG 718 Lys Cys Lys Gly Leu Pro Phe Ser Ser Phe Leu He Leu Pro Phe Gin 225 230 235 AGG ATC ACG AGA CTC AAG CTG CTG GTC CAG AAT ATC CTG AAG AGA GTG 766 Arg He Thr Arg Leu Lys Leu Leu Val Gin Asn He Leu Lys Arg Val 240 245 250 255 GAG GAG AGG TCT GAA CGT GAA GGC ACC GCC TTG GAT GCC CAC AAG GAG 814 Glu Glu Arg Ser Glu Arg Glu Gly Thr Ala Leu Asp Ala His Lys Glu 260 265 270 CTA GAA ATG GTG GTA AAG GCA TGC AAT GAG GGT GTC CGG AAG ATG AGC 862 Leu Glu Met Val Val Lys Ala Cys Asn Glu Gly Val Arg Lys Met Ser 275 280 285 CGC ACA GAA CAG ATG ATC AGC ATT CAG AAG AAG ATG GAG TTC AAG ATC 910 Arg Thr Glu Gin Met He Ser He Gin Lys Lys Met Glu Phe Lys He 290 295 300 AAG TCG GTA CCC ATC ATC TCA CAC TCC CGG TGG CTG CTG AAG CAG GGT 958 Lys Ser Val Pro He He Ser His Ser Arg Trp Leu Leu Lys Gin Gly 305 310 315 GAG CTG CAG CAG ATG TCC GGC CCC AAG ACC TCC CGC ACC CTG CGG ACC 1006 Glu Leu Gin Gin Met Ser Gly Pro Lys Thr Ser Arg Thr Leu Arg Thr 320 325 330 335 AAG AAG CTC πC AGA GAA ATT TAC CTC πC CTC πC AAT GAC CTG CTG 1054 Lys Lys Leu Phe Arg Glu He Tyr Leu Phe Leu Phe Asn Asp Leu Leu 340 345 350 GTG ATC TGC CGG CAG ATC CCT GGA GAC AAG TAC CAG GTG TTT GAT TCG 1102 Val He Cys Arg Gin He Pro Gly Asp Lys Tyr Gin Val Phe Asp Ser 355 360 365 GCC CCA AGG GGC CTG Cπ CGA GTG GAG GAG CTG GAG GAC CAG GGT CAA 1150 Ala Pro Arg Gly Leu Leu Arg Val Glu Glu Leu Glu Asp Gin Gly Gin 370 375 380 ACA CTG GCT AAT GTG πC ATC CTG CGG CTG CTG GAA AAT GCA GAT GAC 1198 Thr Leu Ala Asn Val Phe He Leu Arg Leu Leu Glu Asn Ala Asp Asp 385 390 395 CGA GAG GCC ACC TAT ATG CTG AAG GCA TCC TCC CAG AGC GAG ATG AAG 1246 Arg Glu Ala Thr Tyr Met Leu Lys Ala Ser Ser Gin Ser Glu Met Lys 400 405 410 415 CGC TGG ATG ACC TCA CTG GCC CCC AAC AGG AGG ACC AAG TTT GTA TCC 1294 Arg Trp Met Thr Ser Leu Ala Pro Asn Arg Arg Thr Lys Phe Val Ser 420 425 430 TTC ACA TCT CGG CTG TTG GAC TGT CCC CAG GTC CAG TGT GTG CAC CCG 1342 Phe Thr Ser Arg Leu Leu Asp Cys Pro Gin Val Gin Cys Val His Pro 435 440 445 TAT GTG GCC CAG CAG CCT GAT GAA CTG ACG CTG GAA CTG GCA GAT ATC 1390 Tyr Val Ala Gin Gin Pro Asp Glu Leu Thr Leu Glu Leu Ala Asp He 450 455 460 CTG AAC ATC CTG GAG AAG ACA GAG GAT GGG TGG ATC TTT GGT GAG CGG 1438 Leu Asn He Leu Glu Lys Thr Glu Asp Gly Trp He Phe Gly Glu Arg 465 470 475 CTG CAT GAC CAG GAG AGA GGC TGG TTC CCC AGT TCC ATG ACA GAG GAG 1486 Leu His Asp Gin Glu Arg Gly Trp Phe Pro Ser Ser Met Thr Glu Glu 480 485 490 495 ATC CTG AAC CCC AAG ATC CGC TCC CAG AAC CTC AAG GAA TGT TTC CGG 1534 He Leu Asn Pro Lys He Arg Ser Gin Asn Leu Lys Glu Cys Phe Arg 500 505 510 GTA CAT AAG ATG GAA GAC CCT CAG CGC AGC CAG AAT AAG GAC CGC AGG 1582 Val His Lys Met Glu Asp Pro Gin Arg Ser Gin Asn Lys Asp Arg Arg 515 520 525 AAG CTG GGC AGC CGG AAT CGT CAA TGA ACCTCCCCAG CTCAGGCACC 1629 Lys Leu Gly Ser Arg Asn Arg Gin * 530 535 TGAAGGGAAG GGTGTGGGCA GGGATGGGGA GCAGGCCCGG CAGAGACGCC CGACAGAπC 1689 AGAGGGCCTT AGGGAAGAAT GTCAGTGCCT TCTCAGGCAG CAGGAGTGGC TTCGGCCTGC 1749 TCTGTCCCTG CCCATGCTGT GGAAGCTCTA GTGTCCTGGC CACTTGTTTG CTTGCACACT 1809 GGTGAAAAGC TAAGTACTTA GGCAGTATTA CACCACCTCC CTTCAGTCTC TCAGAGGTAG 1869 AAGAAGGCAG GCATGCTCCA GAGACCTTCC GGTGACTGGA AGAGGCCCAC ACAAGGGTCC 1929 CTGGCAGCAG GCAGGTGGAA GGTAACCACT GTCAGGATCC CCTGAACTGC ACGTGTCCTT 1989 CCCTACπTG GAAGCTGTTA AGAGTCTACC AGGCACACAG ATGGCCGCCC CTGCCCGAGG 2049 GAGTTTGATG AGCAGTGGTG ACCCTGCCTG CCCGTCCCCG TGCCTCTGCC AGCCTCTCTT 2109 GCACGCCAAG CCCTGCCCTC AGCAGGCTTC CCAAAGCTTA GCTGAGGGTT CATGCCACCT 2169 CTAGCTCCTT GAAGGGCTTG ATATCACTTG TGTCTCCTGG GCCCCTGATG GAGCCCAGGC 2229 GTTTTGCAGA ATGAATTGGT CACTGCATCC TTTATGGTCA TGGTπTGAG AAAAGCAAAT 2289 ATCATTTTTG GCTGCATTAA AAGAAGCATC CTATATAAAA AAAAAAAAM AAAA 2343 (2) INFORMATION FOR SEQ ID NO: 6. 0) SEQUENCE CHARACTERISTICS: (A) LENGTH: 535 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (n) MOLECULE TYPE: protein (xι) SEQUENCE DESCRIPTION- SEQ ID NO: 6: Leu Tyr Gin Glu Tyr Arg Asp Lys Ser Thr Leu Gin Glu He Glu Thr 1 5 10 15 Arg Arg Gin Gin Asp Ala Glu He Gin Gly Asn Ser Asp Gly Ser Gin 20 25 30 Val Gly Glu Asp Ala Gly Glu Glu Glu Glu Glu Glu Glu Glu Gly Glu 35 40 45 Glu Glu Glu Leu Ala Ser Pro Pro Glu Arg Arg Ala Leu Pro Gin He 50 55 60 Cys Leu Leu Ser Asn Pro His Ser Arg Phe Asn Leu Trp Gin Asp Leu 65 70 75 80 Pro Glu He Gin Ser Ser Gly Val Leu Asp He Leu Gin Pro Glu Glu 85 90 95 He Arg Leu Gin Glu Ala Met Phe Glu Leu Val Thr Ser Glu Ala Ser 100 105 110 Tyr Tyr Lys Ser Leu Asn Leu Leu Val Ser His Phe Met Glu Asn Glu 115 120 125 Arg Leu Lys Lys He Leu His Pro Ser Glu Ala His He Leu Phe Ser 130 135 140 Asn Val Leu Asp Val Met Ala Val Ser Glu Arg Phe Leu Leu Glu Leu 145 150 155 160 Glu His Arg Met Glu Glu Asn He Val He Ser Asp Val Cys Asp He 165 170 175 Val Tyr Arg Tyr Ala Ala Asp His Phe Ser Val Tyr He Thr Tyr Val 180 185 190 Ser Asn Gin Thr Tyr Gin Glu Arg Thr Tyr Lys Gin Leu Leu Gin Glu 195 200 205 Lys Ala Ala Phe Arg Glu Leu He Ala Gin Leu Glu Leu Asp Pro Lys 210 215 220 Cys Lys Gly Leu Pro Phe Ser Ser Phe Leu He Leu Pro Phe Gin Arg 225 230 235 240 He Thr Arg Leu Lys Leu Leu Val Gin Asn He Leu Lys Arg Val Glu 245 250 255 Glu Arg Ser Glu Arg Glu Gly Thr Ala Leu Asp Ala His Lys Glu Leu 260 265 270 Glu Met Val Val Lys Ala Cys Asn Glu Gly Val Arg Lys Met Ser Arg 275 280 285 Thr Glu Gin Met He Ser He Gin Lys Lys Met Glu Phe Lys He Lys 290 295 300 Ser Val Pro He He Ser His Ser Arg Trp Leu Leu Lys Gin Gly Glu 305 310 315 320 Leu Gin Gin Met Ser Gly Pro Lys Thr Ser Arg Thr Leu Arg Thr Lys 325 330 335 Lys Leu Phe Arg Glu He Tyr Leu Phe Leu Phe Asn Asp Leu Leu Val 340 345 350 He Cys Arg Gin He Pro Gly Asp Lys Tyr Gin Val Phe Asp Ser Ala 355 360 365 Pro Arg Gly Leu Leu Arg Val Glu Glu Leu Glu Asp Gin Gly Gin Thr 370 375 380 Leu Ala Asn Val Phe He Leu Arg Leu Leu Glu Asn Ala Asp Asp Arg 385 390 395 400 Glu Ala Thr Tyr Met Leu Lys Ala Ser Ser Gin Ser Glu Met Lys Arg ■ 405 410 415 Trp Met Thr Ser Leu Ala Pro Asn Arg Arg Thr Lys Phe Val Ser Phe 420 425 430 Thr Ser Arg Leu Leu Asp Cys Pro Gin Val Gin Cys Val His Pro Tyr 435 440 445 Val Ala Gin Gin Pro Asp Glu Leu Thr Leu Glu Leu Ala Asp He Leu 450 455 460 Asn He Leu Glu Lys Thr Glu Asp Gly Trp He Phe Gly Glu Arg Leu 465 470 475 480 His Asp Gin Glu Arg Gly Trp Phe Pro Ser Ser Met Thr Glu Glu He 485 490 495 Leu Asn Pro Lys He Arg Ser Gin Asn Leu Lys Glu Cys Phe Arg Val 500 505 510 His Lys Met Glu Asp Pro Gin Arg Ser Gin Asn Lys Asp Arg Arg Lys 515 520 525 Leu Gly Ser Arg Asn Arg Gin 530 535 (2) INFORMATION FOR SEQ ID NO: 7: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 803 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS : both (D) TOPOLOGY: linear (n) MOLECULE TYPE: cDNA (IX) FEATURE: (A) NAME/KEY : CDS (B ) LOCATION . - 3. .803 (xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 7 : GG AGA GCT CTG CCT CAG ATC TGC CTG CTC AGT AAC CCC CAC TCA AGG 47 Arg Al a Leu Pro Gi n H e Cys Leu Leu Ser Asn Pro Hi s Ser Arg 1 5 10 15 TTC AAC CTC TGG CAG GAT CTT CCC GAG ATC CGG AGC AGC GGG GTG CTT 95 Phe Asn Leu Trp Gi n Asp Leu Pro Gl u H e Arg Ser Ser Gly Val Leu 20 25 30 GAG ATC CTA CAG CCT GAG GAG ATT AAG CTG CAG GAG GCC ATG TTC GAG 143 Glu He Leu Gin Pro Glu Glu He Lys Leu Gin Glu Ala Met Phe Glu 35 40 45 CTG GTC ACT TCC GAG GCG TCC TAC TAC AAG AGT CTG AAC CTG CTC GTG 191 Leu Val Thr Ser Glu Ala Ser Tyr Tyr Lys Ser Leu Asn Leu Leu Val 50 55 60 TCC CAC TTC ATG GAG AAC GAG CGG ATA AGG AAG ATC CTG CAC CCG TCC 239 Ser His Phe Met Glu Asn Glu Arg He Arg Lys He Leu His Pro Ser 65 70 75 GAG GCG CAC ATC CTC TTC TCC AAC GTC CTG GAC GTG CTG GCT GTC AGT 287 Glu Ala His He Leu Phe Ser Asn Val Leu Asp Val Leu Ala Val Ser 80 85 90 95 GAG CGG TTC CTC CTG GAG CTG GAG CAC CGG ATG GAG GAG AAC ATC GTC 335 Glu Arg Phe Leu Leu Glu Leu Glu His Arg Met Glu Glu Asn He Val 100 105 110 ATC TCT GAC GTG TGT GAC ATC GTG TAC CGT TAT GCG GCC GAC CAC TTC 383 He Ser Asp Val Cys Asp He Val Tyr Arg Tyr Ala Ala Asp His Phe 115 120 125 TCT GTC TAC ATC ACC TAC GTC AGC AAT CAG ACC TAC CAG GAG CGG ACC 431 Ser Val Tyr He Thr Tyr Val Ser Asn Gin Thr Tyr Gin Glu Arg Thr 130 135 140 TAT AAG CAG CTG CTC CAG GAG AAG GCA GCT πC CGG GAG CTG ATC GCG 479 Tyr Lys Gin Leu Leu Gin Glu Lys Ala Ala Phe Arg Glu Leu He Ala 145 150 155 CAG CTA GAG CTC GAC CCC AAG TGC AGG GGG CTG CCC TTC TCC TCC TTC 527 Gin Leu Glu Leu Asp Pro Lys Cys Arg Gly Leu Pro Phe Ser Ser Phe 160 165 170 175 CTC ATC CTG CCT TTC CAG AGG ATC ACA CGC CTC AAG CTG TTG GTC CAG 575 Leu He Leu Pro Phe Gin Arg He Thr Arg Leu Lys Leu Leu Val Gin 180 185 190 AAC ATC CTG AAG AGG GTA GAA GAG AGG TCT GAG CGG GAG TGC ACT GCT 623 Asn He Leu Lys Arg Val Glu Glu Arg Ser Glu Arg Glu Cys Thr Ala 195 200 205 πG GAT GCT CAC AAG GAG CTG GAA ATG GTG GTA AAG GCA TGC AAC GAG 671 Leu Asp Ala His Lys Glu Leu Glu Met Val Val Lys Ala Cys Asn Glu 210 215 220 GGC GTC AGG AAA ATG AGC CGC ACG GAA CAG ATG ATC AGC ATT CAG AAG 719 Gly Val Arg Lys Met Ser Arg Thr Glu Gin Met He Ser He Gin Lys 225 230 235 AAG ATG GAG TTC AAG ATC AAG TCG GTG CCC ATC ATC TCC CAC TCC CGC 767 Lys Met Glu Phe Lys He Lys Ser Val Pro He He Ser His Ser Arg 240 245 250 255 TGG CTG CTG AAG CAG GGT GAG CTG CAG CAG ATG TCC 803 Trp Leu Leu Lys Gin Gly Glu Leu Gin Gin Met Ser 260 265 (2) INFORMATION FOR SEQ ID NO: 8- (i) SEQUENCE CHARACTERISTICS. (A) LENGTH. 267 amino acids (B) TYPE, amino acid (D) TOPOLOGY linear (li) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: Arg Ala Leu Pro Gin He Cys Leu Leu Ser Asn Pro His Ser Arg Phe 1 5 10 15 Asn Leu Trp Gin Asp Leu Pro Glu He Arg Ser Ser Gly Val Leu Glu 20 25 30 He Leu Gin Pro Glu Glu He Lys Leu Gin Glu Ala Met Phe Glu Leu 35 40 45 Val Thr Ser Glu Ala Ser Tyr Tyr Lys Ser Leu Asn Leu Leu Val Ser 50 55 60 His Phe Met Glu Asn Glu Arg He Arg Lys He Leu His Pro Ser Glu 65 70 75 80 Ala His He Leu Phe Ser Asn Val Leu Asp Val Leu Ala Val Ser Glu 85 90 95 Arg Phe Leu Leu Glu Leu Glu His Arg Met Glu Glu Asn He Val He 100 105 110 Ser Asp Val Cys Asp He Val Tyr Arg Tyr Ala Ala Asp His Phe Ser 115 120 125 Val Tyr He Thr Tyr Val Ser Asn Gin Thr Tyr Gin Glu Arg Thr Tyr 130 135 140 Lys Gin Leu Leu Gin Glu Lys Ala Ala Phe Arg Glu Leu He Ala Gin 145 150 155 160 Leu Glu Leu Asp Pro Lys Cys Arg Gly Leu Pro Phe Ser Ser Phe Leu 165 170 175 He Leu Pro Phe Gin Arg He Thr Arg Leu Lys Leu Leu Val Gin Asn 180 185 190 He Leu Lys Arg Val Glu Glu Arg Ser Glu Arg Glu Cys Thr Ala Leu 195 200 205 Asp Ala His Lys Glu Leu Glu Met Val Val Lys Ala Cys Asn Glu Gly 210 215 220 Val Arg Lys Met Ser Arg Thr Glu Gin Met He Ser He Gin Lys Lys 225 230 235 240 Met Glu Phe Lys He Lys Ser Val Pro He He Ser His Ser Arg Trp 245 250 255 Leu Leu Lys Gin Gly Glu Leu Gin Gin Met Ser 260 265

Claims: CLAIMS 1. A polynucleotide encoding murine guanine nucleotide exchange factor (MNGEF) or a homologue thereof. 2. A polynucleotide according to claim 1 wherein said homologue is human guanine nucleotide exchange factor (NGEF). 3. A polynucleotide selected from: (a) polynucleotides comprising the nucleotide sequence set out in SEQ ID No. 1, 3, 5 or 7 or the complement thereof. (b) polynucleotides comprising a nucleotide sequence capable of hybridising to the nucleotide sequence set out in SEQ ID No. 1, 3, 5 or 7, or a fragment thereof. (c) polynucleotides comprising a nucleotide sequence capable of hybridising to the complement of the nucleotide sequence set out in SEQ ID No. 1, 3, 5 or 7, or a fragment thereof. (d) polynucleotides comprising a polynucleotide sequence which is degenerate as a result of the genetic code to the polynucleotides defined in (a), (b) or (c). 4. A polynucleotide probe which comprises a fragment of at least 15 nucleotides of a polynucleotide as defined in any one of claims 1 to 3. 5. A polypeptide in substantially isolated form which comprises the sequence set out in SEQ ID Nos. 2, 4, 6 or 8, or a polypeptide substantially homologous thereto, or a fragment of the polypeptide of SEQ ID Nos. 2, 4, 6 or 8. 6. A polynucleotide encoding a polypeptide according to claim 5. 7. A vector comprising a polynucleotide as defined in any one of claims 1 to 3 or 6. 8 An expression vector comprising a polynucleotide as defined in any one of claims 1 to 3 or 6, operably linked to regulatory sequences capable of directing expression of said polynucleotide in a host cell. 9. An antibody capable of binding the polypeptide of SEQ ID. No. 2, 4, 6 or 8 or fragment thereof. 10. A method for detecting the presence or absence of a polynucleotide as defined in any one of claims 1 to 3 or 6 in a biological sample which comprises: (a) bringing the biological sample containing DNA or RNA into contact with a probe according to claim 4 under hybridising conditions; and (b) detecting any duplex formed between the probe and nucleic acid in the sample. 11. A method of detecting polypeptides as defined in claim 5 present in biological samples which comprises: (a) providing an antibody according to claim 9; (b) incubating a biological sample with said antibody under conditions which allow for the formation of an antibody-antigen complex; and (c) determining whether antibody-antigen complex comprising said antibody is formed. 12. A polynucleotide according to any one of claims 1 to 3 or 6 for use in a method of treatment of the human or animal body. 13. A polypeptide according to claim 5 for use in a method of treatment of the human or animal body. 14. An antibody according to claim 10 for use in a method of treatment of the human or animal body. 15. A method of treating a disease or disorder of the nervous system, comprising administering an effective amount of a polynucleotide as defined in any one of claims 1 to 3 or 6, to a patient. 16. A method of treating a disease or disorder of the nervous system, comprising administering an effective amount of a polypeptidetide as defined in claim 5, to a patient. 17. A method of treating a disease or disorder of the nervous system, comprising administering an effective amount of an antibody as defined in claim 10 to a patient. 18. The method of claim 15, 16 or 17 wherein said disease or disorder is a malignancy.

Inventors: DAVIES KAY ELIZABETH, THEODOSIOU ASPASIA, DAVIES, KAY, ELIZABETH, THEODOSIOU, ASPASIA
Applicants: DAVIES KAY ELIZABETH, MEDICAL RES COUNCIL, THEODOSIOU ASPASIA, DAVIES, KAY, ELIZABETH, MEDICAL RESEARCH COUNCIL, THEODOSIOU, ASPASIA
Application year: 1997
Main classification: C12N15
IPC codes: A61K35, A61K38, A61K39, A61K48, A61P25, C07K14, C07K16, C12N15, C12Q1, G01N33