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WO1998023743A1 - Facteur murin d'echange de la guanine nucleotide (mngef) et ses homologues humains - Google Patents

Facteur murin d'echange de la guanine nucleotide (mngef) et ses homologues humains Download PDF

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Publication number
WO1998023743A1
WO1998023743A1 PCT/GB1997/003302 GB9703302W WO9823743A1 WO 1998023743 A1 WO1998023743 A1 WO 1998023743A1 GB 9703302 W GB9703302 W GB 9703302W WO 9823743 A1 WO9823743 A1 WO 9823743A1
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glu
ser
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gin
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PCT/GB1997/003302
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English (en)
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Kay Elizabeth Davies
Aspasia Theodosiou
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Medical Research Council
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Application filed by Medical Research Council filed Critical Medical Research Council
Priority to EP97945992A priority Critical patent/EP0941326A1/fr
Priority to CA002274143A priority patent/CA2274143A1/fr
Priority to AU51302/98A priority patent/AU5130298A/en
Priority to JP52444898A priority patent/JP2001505424A/ja
Publication of WO1998023743A1 publication Critical patent/WO1998023743A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/82—Translation products from oncogenes

Definitions

  • the present invention relates to MNGEF, a member of the family of regulators of small GTP-binding proteins, and homologues of MNGEF.
  • GTP-binding proteins also known as G proteins
  • ras proteins are prototypes
  • G proteins 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.
  • 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.
  • Ras-like 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.
  • GEFs guanine nucleotide exchange factors
  • GAPS GTPase-activating proteins
  • GDP-dissociation inhibitory factors GDP-dissociation inhibitory factors
  • the Ras-like proteins are divided into six main families, based on their sequences: Rab, Arf, Sar, Ran, Rho and Ras.
  • Rho GTPases such as Rac, Rho, Cdc42
  • Rho GTPases were thought to be primarily involved in the organisation of the actin cytoskeleton.
  • 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.
  • DH Dbl homology domain
  • PH pleckstrin homology domain
  • Dbl 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.
  • MNGEF 1 and MNGEF2 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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:
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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. conserveed 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.
  • primers can be designed to target the DH, PH and SH3 domains described above.
  • such polynucleotides may be obtained by site directed mutagenesis of the MNGEF sequences or allelic variants thereof.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • MNGEF hereditary diseases
  • 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.
  • 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.
  • 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.
  • 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:
  • 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.
  • 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.
  • 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.
  • post-translational modifications e.g. myristolation, glycosylation, truncation, lapidation and tyrosine, serine or threonine phosphorylation
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • yeast regulatory sequences include S.
  • Mammalian promoters such as ⁇ -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.
  • MMLV LTR Moloney murine leukaemia virus long terminal repeat
  • RSV promoter rous sarcoma virus
  • CMV human cytomegalovirus
  • IE 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.
  • 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.
  • 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.
  • the invention also provides polypeptides of the invention or fragments thereof haptenised to another polypeptide for use as immunogens in animals or humans.
  • 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.
  • 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:
  • 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.
  • 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.
  • polypeptides, polynucleotides and antibodies of the invention may be used in as compounds for treating neoplasms in animals or humans.
  • the compounds are formulated for clinical administration by mixing them with a pharmaceutically acceptable carrier or diluent.
  • a pharmaceutically acceptable carrier or diluent for example they can be formulated for topical, parenteral, intravenous, intramuscular, subcutaneous, intraocular or transdermal administration.
  • 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.
  • the pharmaceutically carrier or diluent may be, for example, sterile or isotonic solutions.
  • 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.
  • 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.
  • 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.
  • the nucleic acid may form part of a viral vector such as an adenovirus.
  • the dose administered is between 10 4 and 10' 4 pfu/ml, preferably 10 6 to 10 10 pfu/ml.
  • 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.
  • 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.
  • tumours such as breast, ovary, lung, colon, pancreas, testes, liver, brain, muscle and o bone tumour.
  • the tumour is a tumour of the nervous system, in particular the central nervous system, for example the brain.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the probe was end-labelled with ⁇ - 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.
  • MNGEFl and MNGEF2 Two of these cDNA clones contained the following trinucleotide repeat (AGG) 8 GAG(AGG) 3 (SEQ ID No. 10).
  • AGG trinucleotide repeat
  • 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 blots were prepared according to Current Protocols in Molecular Biology, with each lane containing 2 ⁇ g 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.
  • 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 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.
  • primers m32bt7f and m32bt3f were used to amplify cDNA from human foetal brain.
  • the sequences of the primers used are shown below:
  • 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 Rh
  • CGCCGTGGAC CACGACAGCT CCACCTCGGA GAGCGACACG CGCGACTCGG CGGCGGGACA 240
  • 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
  • CGCCGTGGAC CACGACAGCT CCACCTCGGA GAGCGACACG CGCGACTCGG CGGCGGGACA 240
  • GAG ATC AGG CTG CAG GAG GCC ATG T ⁇ 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
  • 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
  • GCC CCA AGG GGC CTG C ⁇ 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

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Abstract

La présente invention concerne le clonage d'un facteur murin d'échange de la guanine nucléotide (MNGEF) et un homologue humain de celui-ci. Elle concerne également des sondes de polynucléotides dérivées de la séquence nucléotidique du MNGEF et des anticorps qui reconnaissent ledit MNGEF.
PCT/GB1997/003302 1996-11-29 1997-12-01 Facteur murin d'echange de la guanine nucleotide (mngef) et ses homologues humains WO1998023743A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
EP97945992A EP0941326A1 (fr) 1996-11-29 1997-12-01 Facteur murin d'echange de la guanine nucleotide (mngef) et ses homologues humains
CA002274143A CA2274143A1 (fr) 1996-11-29 1997-12-01 Facteur murin d'echange de la guanine nucleotide (mngef) et ses homologues humains
AU51302/98A AU5130298A (en) 1996-11-29 1997-12-01 Murine guanine nucleotide exchange factor - (MNGEF) and human homologues th ereof
JP52444898A JP2001505424A (ja) 1996-11-29 1997-12-01 マウス・グアニンヌクレオチド交換因子（ｍｎｇｅｆ）及びそのヒト相同体

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GB9624905.7 1996-11-29
GBGB9624905.7A GB9624905D0 (en) 1996-11-29 1996-11-29 MNGEF and homologues thereof

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998057990A3 (fr) * 1997-06-17 1999-03-11 Onyx Pharma Inc Methodes et compositions permettant de traiter une croissance cellulaire anormale liee a l'activite indesirable du facteur d'echange des nucleotides a base de guanine
WO2004096199A3 (fr) * 2003-05-02 2006-03-30 Scottish Biomedical Ltd Regulation du facteur d'echange du nucleotide guanine pour une proteine appartenant a la famille rap des petites gtpases

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BOGUSKI M.S. AND MCCORMICK F.: "Proteins regulating Ras and its relatives.", NATURE, vol. 366, 1993, pages 643 - 654, XP002057779 *
CHAN A. M.-L. ET AL.: "Expression cDNA cloning of a novel oncogene with sequence similarity to regulators of small GTP-binding proteins.", ONCOGENE, vol. 9, 1994, pages 1057 - 1063, XP002059291 *
HART M.J. ET AL.: "Identification of a novel guanine exchange factor for the Rho GTPase.", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 271, no. 41, 11 October 1996 (1996-10-11), pages 25452 - 25458, XP002057776 *
WHITEHEAD I.P. ET AL.: "Expression cloning of lsc, a novel oncogene with structural similarities to the Dbl family of guanine nucleotide exchange factors.", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 271, no. 31, 2 August 1996 (1996-08-02), pages 18643 - 18650, XP002057777 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998057990A3 (fr) * 1997-06-17 1999-03-11 Onyx Pharma Inc Methodes et compositions permettant de traiter une croissance cellulaire anormale liee a l'activite indesirable du facteur d'echange des nucleotides a base de guanine
US6340575B1 (en) 1997-06-17 2002-01-22 Onyx Pharmaceuticals, Inc. Methods and compositions for treating abnormal cell growth related to unwanted guanine nucleotide exchange factor activity
WO2004096199A3 (fr) * 2003-05-02 2006-03-30 Scottish Biomedical Ltd Regulation du facteur d'echange du nucleotide guanine pour une proteine appartenant a la famille rap des petites gtpases

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CA2274143A1 (fr) 1998-06-04
GB9624905D0 (en) 1997-01-15
AU5130298A (en) 1998-06-22
JP2001505424A (ja) 2001-04-24

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