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WO2000073328A2 - Constructions d'unc-5 et methodes de selection - Google Patents

Constructions d'unc-5 et methodes de selection Download PDF

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WO2000073328A2
WO2000073328A2 PCT/EP2000/005108 EP0005108W WO0073328A2 WO 2000073328 A2 WO2000073328 A2 WO 2000073328A2 EP 0005108 W EP0005108 W EP 0005108W WO 0073328 A2 WO0073328 A2 WO 0073328A2
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protein
unc
seq
sequence
set forth
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PCT/EP2000/005108
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WO2000073328A3 (fr
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Wim Van Criekinge
Ingele Roelens
Thierry Bogaert
Phillipe Verwaerde
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Devgen Nv
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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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators

Definitions

  • the present invention is concerned with unc-5, a conserved animal gene family that encodes proteins implicated in directional cell behaviour.
  • the invention is concerned with novel splice variants of the human unc-5C cDNA and a novel human unc-5HSl cDNA sequence.
  • assays are provided based on protein-protein interactions between the UNC-5 protein and a variety of different interacting proteins.
  • Unc-5 is a conserved animal gene family that encodes proteins implicated in directional cell behaviour.
  • the unc-5 gene of the nematode worm Caenorhabdi tis elegans (C. elegans) is known to be involved in dorsal migration in contrast to unc-40 which is involved in ventral migrations (Hedgecoc et al., Neuron Vol. 2; 61-85, 1990).
  • Both the unc-5 and unc-40 genes are associated with the netrin unc- 6, and all three genes play a dominant role in directional neuronal outgrowth .
  • the C. elegans unc-5 gene encodes a 919 amino acid transmembrane receptor with two immunoglobulin and two thrombospondin type I extracellular domains (Leung-Hagesteijn et al., Cell Vol. 71:289-299, 1992).
  • Ectopic overexpression of unc-5 in the C. elegans touch neurons resulted in dorsal steering of these, instead of the normal ventral elongation of these neurons (Hamelin et al . , Nature, 364:327-330, 1993).
  • the present inventors have identified three previously unknown variant unc-5C cDNAs . These variant cDNAs correspond to alternatively spliced unc- 5C transcripts.
  • a protein which comprises the ammo acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 or an ammo ac d sequence which differs from that shown SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 only m conservative ammo acid changes.
  • nucleic acid sequences which encode the proteins of the invention. Also provided by the invention are a nucleic acid comprising the sequences of nucleotides set forth in SEQ ID NO: 1, a nucleic acid comprising the sequences of nucleotides set forth in SEQ ID NO: 3 and a nucleic acid comprising the sequences of nucleotides set forth in SEQ ID NO: 5.
  • the splice variants of human unc-5C were cloned by PCR technology. Two primers were developed to amplify the intracellular part of the unc-5C. Human Brain cDNA was used for this purpose. Three new splice variants of human unc-5C were characterized. A schematic representation of these splice variants is given in Figure 5.
  • the first splice variant (designated unc-5Cb) has a deletion of an ntron the UP region.
  • the nucleotide sequence of a partial unc-5Co cDNA is set forth SEQ ID NO: 1 and the corresponding ammo acid sequence is set forth m SEQ ID NO: 2.
  • the splice of this intron results in a UNC-5Cb protein which is considerably shorter than the previously known UNC-5C, as the coding frame is not maintained. This protein is truncated for the DD domain and for the major part of the UP domain.
  • the second splice variant (designated unc-5Cc) s deleted by an intron in the ZO-1 region, also resulting m a shorter protein than the previously known UNC-5C, as the coding frame is not maintained.
  • the nucleotide sequence of a partial UNC-5Cc cDNA is set forth in SEQ ID NO: 3 and the corresponding ammo acid sequence is shown in SEQ ID NO: 4.
  • the resulting protein (UNC-5Cc) is truncated for the DD domain, the UP domain and a part of the ZO-1 domain.
  • the third splice variant (unc-5C8) is deleted by a small intron in the ZO-1 domain, but the coding frame is maintained. This results m a slightly smaller protein (UNC-5C8), wherein only the ammo acid sequence coded by the spliced intron is truncated.
  • the nucleotide sequence of a partial UNC-5C8 cDNA is set forth in SEQ ID NO: 5 and the corresponding ammo acid sequence is shown in SEQ ID NO: 6.
  • nucleic acid molecule comprising the sequence of nucleotides set forth n SEQ ID NO: 7.
  • the invention further provides a nucleic acid comprising the sequence of nucleotides set forth in SEQ ID NO: 56 and a sequence of nucleotides complementary to the sequence of nucleotides set forth in SEQ ID NO: 57, a nucleic acid comprising the sequence of nucleotides set forth in SEQ ID NO: 54 and a sequence of nucleotides complementary to the sequence of nucleotides set forth m SEQ ID NO: 55, a nucleic acid comprising the sequence of nucleotides set forth in SEQ ID NO: 61 and a sequence of nucleotides complementary to the sequence of nucleotides set forth in SEQ ID NO: 62 and a nucleic acid comprising the sequence of nucleotides set forth in SEQ ID NO: 63 and a sequence of nucleotides complementary to the sequence of nucleotides set forth SEQ ID NO: 64.
  • the nucleic acid molecules according to the invention may, advantageously, be included m a suitable expression vector to express the proteins encoded therefrom m a suitable host. Incorporation of cloned DNA into a suitable expression vector for subsequent transformation of said cell and subsequent selection of the transformed cells is well known to those skilled in the art as provided in Sambrook et al . (1989), molecular cloning, a laboratory manual, Cold Spring Harbour Laboratory Press.
  • An expression vector according to the invention includes a vector having a nucleic acid according to the invention operably linked to regulatory sequences, such as promoter regions, that are capable of effecting expression of said DNA fragments.
  • operably linked refers to a juxtaposition wherein the components described are m a relationship permitting them to function in their intended manner.
  • Such vectors may be transformed into a suitable host cell to provide for expression of a protein according to the invention.
  • the invention provides a process for preparing proteins 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 protein, and recovering the expressed protein.
  • the vectors may be, for example, plasmid, virus or phage vectors provided with an origin of replication, and optionally a promoter for the expression of said nucleotide and optionally a regulator of the promoter.
  • the vectors may contain one or more selectable markers, such as, for example, an antibiotic resistance.
  • a bacterial expression vector may include a promoter such as the lac promoter and for translation initiation the Shme- Dalgarno sequence and the start codon AUG.
  • a eukaryotic expression vector may include a heterologous or homologous promoter for RNA polymerase II, a downstream polyadenylation signal, the start codon AUG, and a termination codon for detachment of the ribosome.
  • Such vectors may be obtained commercially or be assembled from the sequences described by methods well known m the art.
  • Nucleic acid molecules according to the invention may be inserted into the vectors described m an antisense orientation in order to provide for the production of antisense RNA.
  • Antisense RNA or other antisense nucleic acids, including antisense peptide nucleic acid (PNA), may be produced by synthetic means .
  • nucleic acid sequence also includes the complementary sequence to any single stranded sequence given regarding base variations.
  • the nucleic acid sequences according to the mvention may be produced using recombinant or synthetic techniques, such as for example using PCR which generally involves making a pair of primers, which may be from approximately 10 to 50 nucleotides to a region of the gene which is desired to be cloned, bringing the primers into contact with cDNA, or genomic DNA from a human cell, performing a polymerase chain reaction under conditions which brings about amplification of the desired region, isolating the amplified region or fragment and recovering the amplified DNA.
  • PCR generally involves making a pair of primers, which may be from approximately 10 to 50 nucleotides to a region of the gene which is desired to be cloned, bringing the primers into contact with cDNA, or genomic DNA from a human cell, performing a polymerase chain reaction under conditions which brings about amplification of the desired region, isolating the amplified region or fragment and recovering the amplified DNA.
  • PCR generally involves making a pair of primer
  • the nucleic acids according to the invention may carry a revealing label.
  • Suitable labels include radioisotopes such as 32 P or 35 S, enzyme labels or other protein labels such as biotm or fluorescent markers. Such labels may be added to the nucleic acids or oligonucleotides of the invention and may be detected using known techniques per se .
  • the protein according to the invention includes all possible ammo acid variants encoded by the nucleic acid molecule according to the invention including a protein encoded by said molecule and having conservative ammo acid changes. Proteins or polypeptides according to the invention further include variants of such sequences, including naturally occurring allelic variants which are substantially homologous to said proteins or polypeptides. In this context, substantial homology is regarded as a sequence which has at least 70%, preferably 80 or 90% and preferably 95% ammo acid homology with the proteins or polypeptides encoded by the nucleic acid molecules according to the invention.
  • the protein according to the invention may be recombinant, synthetic or naturally occurring, but is preferably recombinant.
  • a further aspect of the invention provides a host cell or organism, transformed or transfected with an expression vector according to the invention.
  • the host cell or organism may advantageously be used m a method of producing protein, which comprises recovering any expressed protein from the host or organism transformed or transfected with the expression vector.
  • transgenic cell, tissue or organism comprising a transgene capable of expressing a protein according to the invention.
  • transgene capable of expressing encompasses any suitable nucleic acid sequence which leads to expression of proteins having the same function and/or activity.
  • the transgene may include, for example, genomic nucleic acid isolated from human cells or synthetic nucleic acid, including DNA integrated into the genome or in an extrachromosomal state.
  • the transgene comprises the nucleic acid sequence encoding the proteins according to the invention as described herein, or a functional fragment of said nucleic acid.
  • a functional fragment of said nucleic acid should be taken to mean a fragment of the gene comprising said nucleic acid coding for the proteins according to the invention or a functional equivalent, derivative or a nonfunctional derivative such as a dominant negative mutant, or bioprecusor of said proteins.
  • the protein expressed by said transgenic cell, tissue or organism or a functional equivalent or bioprecusor of said protein also forms part of the present invention.
  • Recombinant proteins may be recovered and purified from host cell cultures by methods known in the art, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose, chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography .
  • the protein of the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial yeast, higher plant, insect and mammalian cells in culture) .
  • a prokaryotic or eukaryotic host for example, by bacterial yeast, higher plant, insect and mammalian cells in culture
  • the expressed protein may lack the initiating methionine residue as a result of post-translational cleavage. Proteins which have been modified in this way are also included within the scope of the invention.
  • the invention provides an antibody capable of specifically binding to a protein according to the invention.
  • the antibody is capable of specifically binding to a protein comprising the sequence of amino acids set forth in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6.
  • An antibody according to the invention may be raised according to standard techniques well known to those skilled in the art by using the protein of the invention or a fragment or single epitope thereof as the challenging antigen.
  • a further aspect of the invention comprises a nucleic acid capable of hybridising to the nucleic acids according to the invention, and preferably capable of hybridising to the sequence of nucleotides set forth in SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63 or SEQ ID NO: 64, under high stringency conditions. Conditions of stringency are well known to those skilled in the art.
  • Tm melting temperature
  • Tm is the length of the hybrids in nucleotides. Tm decreases approximately by 1-1.5°C with every 1% decrease in sequence homology.
  • the nucleic acid capable of hybridising to nucleic acid molecules according to the invention will generally be at least 70%, preferably at least 80 or
  • the present invention also advantageously provides oligonucleotides consisting essentially of at least 10 consecutive nucleotides of a nucleic acid according to the invention and preferably from 10 to 50 consecutive nucleotides of a nucleic acid according to the invention, in particular a nucleic acid comprising the sequence of nucleotides shown in SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63 or SEQ ID NO: 64.
  • These oligonucleotides may, advantageously be used as probes or primers to initiate replication, or the like.
  • Oligonucleotides having a defined sequence may be produced according to techniques well known the art, such as by recombinant or synthetic means. They may also be used in diagnostic kits or the like for detecting the presence of a nucleic acid according to the invention. These tests generally comprise contacting the probe with the sample under hybridising conditions and detecting for the presence of any duplex or triplex formation between the probe and any nucleic acid in the sample.
  • UNC-5 To address the functional role of UNC-5 within the cell the inventors used the yeast two hybrid method (Fields and Song, Nature 340:245, 1989), a method well known to molecular biologists, both to investigate the ability of UNC-5 to form dimers and to search for proteins that interact with the UNC-5 protein. Using the two hybrid approach the inventors were able to demonstrate that UNC-5 is capable of forming homodimers and identified a number of proteins which interact with the intracellular domains of the C. elegans unc-5 or human UNC-5 proteins. These newly identified protem-protem interactions involving UNC- 5 may represent important events in cellular signalling, hence compounds which disrupt these interactions may potentially have useful pharmacological properties.
  • the invention provides a method of identifying compounds which are capable of inhibiting or enhancing the binding of an UNC-5 protein to an interacting protein previously identified as binding to the said UNC-5 protein, which method comprises: providing a host cell containing a DNA construct comprising a reporter gene operatively linked to a promoter regulated by a transcription factor having a DNA binding domain and an activating domain; expressing in said host cell a first hybrid DNA sequence encoding a first fusion protein comprising an UNC-5 protein or a fragment thereof fused m-frame to either the DNA binding domain or the activating domain of the said transcription factor; expressing m said host cell a second hybrid DNA sequence encoding a second fusion protein comprising an interacting protein or a fragment thereof fused m-frame to either the DNA binding domain or the activating domain of the said transcription factor, such that when the first fusion protein comprises the activation domain of the said transcription factor the second fusion protein comprises the DNA binding domain of the said transcription factor and when the first fusion protein comprises
  • the method of the invention is based upon the standard two hybrid assay well known the art.
  • the host cell is a yeast cell. Protocols for performing a yeast two hybrid assay are well known m the art and are given in the Examples included herein.
  • the assay can be performed m either orientation. That is to say, the assay can be performed using an UNC-5 protein or a fragment thereof fused to the DNA binding domain of the transcription factor and the interacting protein or fragment thereof fused to the activation domain of the transcription factor or alternatively the assay can be performed using an UNC-5 protein or a fragment thereof fused to the activation domain of the transcription factor and the interacting protein or fragment thereof fused to the DNA binding domain of the transcription factor.
  • the above-described method based on the classical yeast two hybrid system can be used to screen for compounds that inhibit or enhance the interaction between two proteins.
  • other systems have been developed to screen for dissociation events, these methods are designated reverse hybrid methods. These systems make use of yeast strains in which the expression of interacting hybrid proteins increases the expression of a counter-selectable marker that is toxic under particular conditions. Under these conditions, dissociation of an interaction provides a selective advantage, thereby facilitating detection: A few growing yeast colonies in which hybrids fail to interact can be identified among millions of non-growing colonies expressing interacting proteins.
  • reverse hybrid systems are known the art.
  • the first reverse two-hybrid system utilizes a yeast strain, which is resistant to cycloheximide due to the presence of a mutant CYH2 gene.
  • This strain also contains the wild-type CYH2 allele under the transcriptional control of the GAL1 promoter. Expression of the wild-type GAL4 protein as sufficient to restore growth sensitivity to cycloheximide. Growth sensitivity towards cycloheximide is also restored by the co-expression of the avian c-Rel protein and its I ⁇ B- ⁇ counterpart, p40, as GAL4 fusion proteins.
  • URA3 which encodes orotidine-5' -phosphate decarboxylase, an enzyme required for the biosynthesis of uracil.
  • URA3-encoded decarboxylase can also catalyze the conversion of a non-toxic analogue, 5-fluorooritic acid (FOA) into a toxic product, 5-fluoroacil (Boeke et al., 1984, Mol. Gen. Genet. 197:345-346).
  • mutations that prevent an interaction can be selected from large libraries of randomly mutated alleles.
  • molecules that dissociate or prevent an interaction could be selected from large libraries of peptides or compounds (Vidal et al . , 1996, PNAS 93:10315-10320; Vidal et al., 1996, PNAS 93:10321-10326) .
  • a third reversed yeast two hybrid is based on the use of GAL80 gene as relay gene.
  • GAL80 encodes a protein that binds to and masks the activation domain of a transcriptional activator, such as GAL4.
  • the reporter genes which will provide the transcriptional read-out (i.e. HIS3 or LACZ), are dependent upon the functional GAL4 for expression. Only when the level of GAL80 masking protein is reduced by interfering with the two-hybrid interaction will Gal4 function as a transcriptional activator, providing a positive transcriptional read-out for molecules that inhibit the two-hybrid protein-protein interaction.
  • the invention further provides a method of identifying compounds which are capable of inhibiting or enhancing the binding of an UNC-5 protein to an interacting protein previously identified as binding to the said UNC-5 protein, which method comprises: providing a transgenic cell or organism expressing a first fusion protein comprising an UNC-5 protein or a fragment thereof fused in- frame to a first genetically encoded fluorophore and a second fusion protein comprising an interacting protein or a fragment thereof fused in-frame to a second genetically encoded fluorophore, the first and second fluorophores being characterised in that the emission spectrum of one of the fluorophores overlaps with the absorption spectrum of the other fluorophore; measuring the amount of fluorescence emitted from the fluorophore having an emission spectrum which overlaps with the absorption spectrum of the other fluorophore; exposing the transgenic cell or organism to a compound under test; and detecting any change in the amount of fluorescence emitted fluorescence emitted from the fluorophore having
  • FRET fluorescence energy transfer
  • the fluorescence emission spectrum of the donor fluorophore overlaps with the absorption spectrum of the acceptor fluorophore, such that when the two components of the binding pair bind to each other, bringing the donor and acceptor fluorophores into close proximity, a proportion of the fluorescent signal emitted by the donor fluorophore (following irradiation with incident radiation of a wavelength absorbed by the donor fluorophore) will be absorbed by the proximal acceptor fluorophore (a process known in the art as fluorescence energy transfer) with the result that a proportion of the fluorescent signal emitted by the donor fluorophore is quenched and, in some instances, that the acceptor fluorophore emits fluorescence.
  • Fluorescence energy transfer will only occur when the donor and acceptor fluorophores are brought into close proximity by the specific binding reaction. Thus, in the presence of a compound which disrupts the specific binding, the amount of quenching is reduced resulting in an increase in the intensity of the fluorescent signal emitted by the donor fluorophore or a fall in the intensity of the signal emitted by the acceptor fluorophore) .
  • the method of the invention is an in vivo FRET assay because it is performed in a transgenic host cell or organism.
  • the transgenic cell can be any mammalian cell line, the transgenic organism is preferably C. elegans .
  • the method of the invention uses genetically encoded donor and acceptor fluorophores which can be expressed as fusion proteins fused in frame to the UNC-5 protein and the interacting protein. This can be readily accomplished by transforming or transfecting the cell or organism with appropriate expression vectors arranged to express the fusion proteins .
  • the genetically encoded donor and acceptor proteins are variant green fluorescent proteins which exhibit different fluorescent properties and which have suitably overlapping emission/absorption spectra, such as EGFP (enhanced green fluorescent protein) and EBFP
  • the FRET assay can be performed in either orientation. That is to say, the assay can be carried out using UNC-5 fused to the donor fluorophore and the interacting protein fused to the acceptor fluorophore or using UNC-5 fused to the acceptor fluorophore and the interacting protein fused to the donor fluorophore .
  • the invention further provides a method of identifying compounds which are capable of inhibiting or enhancing the binding of an UNC-5 protein to an interacting protein previously identified as binding to the said UNC-5 protein, which method comprises: providing a first reaction component comprising a first protein linked to a solid support containing a scmtillant and a second reaction component comprising a second protein which has been radioactively labelled, wherein the first and second proteins are an UNC-5 protein or a fragment thereof and an interacting protein or a fragment thereof; o ⁇ nging the first and second reaction components into contact m an aqueous solution m the presence of a compound under test; and detecting binding of the first protein to the second protein by detecting light emission from the scintillant.
  • the above method is based on the scintillation proximity assay (SPATM) developed by Amersham and commonly used in automated high throughput screening.
  • SPATM scintillation proximity assay
  • a first interacting protein e.g. an UNC-5 protein
  • Linking of the protein to the beads can be carried out in many different ways, including, for example, via biotin-streptavidin affinity binding.
  • Streptavidin-SPA beads are commercially available from Amersham and the interacting protein can easily be biotinylated in vi tro or expressed as a biotinylated fusion protein using techniques known in the art.
  • the second interacting protein e.g.
  • SPATM assay protocol is then as follows:
  • SPA beads linked to the first interacting protein are incubated for 30 minutes to one hour with a sample containing the radioactively labelled second interacting protein.
  • the radioactivity emitted by the labelled protein is brought into close proximity with the bead containing scintillant and therefore induces light emission from the scintillant.
  • the free labelled protein in sample (non-bound) will not be held in sufficiently close proximity to the beads to induce light emission. Compounds which disrupt the binding of the first and second interacting proteins will cause a decrease in the amount of light emitted during the experiment .
  • the assay can be carried out using UNC-5 linked to the solid support containing scintillant and a radioactively labelled interacting protein or using an interacting protein linked to the solid support containing scintillant and a radioactively labelled UNC-5.
  • the invention further provides a method of identifying compounds which are capable of inhibiting or enhancing the binding of an UNC-5 protein to an interacting protein previously identified as binding to the said UNC-5 protein, which method comprises: coating the wells of a microtiter plate with UNC-5 protein or a fragment thereof; contacting the UNC-5 protein or fragment thereof with an aqueous solution comprising an interacting protein or a fragment thereof, said interacting protein being labelled with a tag which is directly or indirectly detectable, and a compound under test; washing to remove the compound under test and any unbound tagged interacting protein; and detecting complexes of UNC-5 or a fragment thereof bound to the interacting protein or a fragment thereof by directly or indirectly detecting the presence of the tag.
  • This method of the invention uses an ELISA type approach to screen for compounds which disrupt binding between Unc-5 and a protein known to interact with
  • UNC-5 In these experiments, the wells of a microtiter plate are coated with the UNC-5 protein or fragments thereof. A sample containing both the compound under test and a protein known to interact with UNC-5 (or a fragment of the protein which is still capable of binding to UNC-5) is then added to the wells and the plates are incubated to allow time for specific binding of UNC-5 to the interacting protein.
  • the interacting protein (or fragment thereof) is labelled with a tag which is directly or indirectly detectable, typically a fluorescent molecule such as GFP, or a tag which is detectable by specific antibody binding, such as a His-tag or GST-tag. Many other tag molecules which are equally suitable for this purpose are known in the art and are available commercially.
  • Any interacting protein which has become bound to UNC-5 is not removed by the washing step and can be detected via the directly or indirectly detectable tag. If the interacting protein is labelled with a GFP tag, then bound proteins are detected by measuring GFP fluorescence; if the interacting protein is labelled with a His-tag or a GST tag, bound proteins are detected with immunological techniques, using an antibody of the appropriate specificity. Compounds which disrupt the binding of UNC-5 to the interacting protein will result m more of the protein remaining unbound, hence less protein will be detected after the washing step.
  • the invention further provides a method of identifying compounds which are capable of inhibiting or enhancing the binding of an UNC-5 protein to an interacting protein previously identified as binding to the said UNC-5 protein, which method comprises: exposing a cell or organism expressing UNC-5 and overexpressing nucleic acid encoding an interacting protein to the compound under test; and screening for reversion of the overexpression phenotype of the cell or organism Over-expression of genes encoding for proteins which interact with UNC-5 in a cell line or C. elegans results m an over-expression phenotype.
  • Assays to select for compounds that inhibit the interaction of UNC-5 and its interacting proteins can therefore be performed in cell lines or C. el egans by exposing cells or worms exhibiting an over-expression phenotype to the compound under test and screening for a of the over-expression phenotype (i.e. screening for a reversion to wild-type) .
  • Over-expression of proteins which interact with unc-5 in C. elegans typically results in neuronal outgrowth phenotypes, distal tip cell outgrowth phenotypes, and other aberrant outgrowth of various tissues and cells. These phenotypes can be easily monitored by expressing reporter genes, such as fluorescent proteins m these cells. Reduction of the phenotype induced by the over-expression can then be monitored by visual inspection. Simple assays have been developed to screen for compounds which cause reversion of the over-expression phenotype m cell lines. As Unc-5 receives signals from the netrms, over-expression of proteins which interact with unc-5 typically causes phenotypic changes m neuronal outgrowth and cell movement.
  • the step of screening for reduction of the over-expression phenotype can be performed using a larainin assay, a netrin response assay and assays using agarose concentration gradients, a boyden chamber or stratified layers (see Gundersen, R. W.,
  • the UNC-5 protein is a C. elegans UNC-5 protein or a human UNC-5 protein.
  • the human UNC-5 protein is UNC-5C or any of the UNC-5C splice variants identified hereinbefore or UNC-5HS1.
  • the methods of the invention can also be carried out using fragments of the UNC-5 protein which retain the ability to bind to the interacting protein.
  • the fragment comprises the intracellular portion of the protein.
  • Various sub-domains of the intracellular portion of the protein or combinations thereof can also be used.
  • interacting protein encompasses any protein which has been demonstrated to interact with an UNC-5 protein.
  • the interacting protein can be a second UNC-5 protein as the examples included herein demonstrate the ability of UNC-5 to form homodimers.
  • the interacting protein can also be a protein identified as interacting with UNC-5 in a yeast two hybrid experiment. A list of proteins identified as interacting with C. elegans UNC-5 or human UNC-5 in a yeast two hybrid experiment is given in the Example 4, below. Any of these proteins, or fragments thereof which retain a functional UNC-5 binding site, can be used in the methods of the invention in combination with the appropriate UNC-5 protein or a fragment thereof.
  • the UNC-5 signalling pathway is highly conserved across species. Hence it is to be expected that for every interacting protein identified in the yeast two hybrid experiments described in the Examples given herein a homologous interacting protein will be found in other species. For example, for every interacting protein found in C. elegans to interact with the C. elegans unc-5 protein it is expected that a homologous interacting protein will be found in humans and will interact with a human UNC-5 protein, and vice versa for interacting proteins first identified in humans. Accordingly, it is within the scope of the invention to perform the methods described above with "homologous combinations" of UNC- 5 proteins and interacting proteins and even with cross-species combinations e.g. C.
  • elegans unc-5 and a human interacting proteins human UNC-5 and a human homologue of an interacting protein identified in C. elegans ; C. elegans unc-5 and a human homologue of an interacting protein identified in C. elegans; C. elegans unc-5 and a human interacting protein etc.
  • Lists of homologues of the C. elegans and human interacting proteins identified in the yeast two hybrid study are given in the Examples included herein.
  • the invention provides a method of identifying compounds which reduce or inhibit the lethal phenotype associated with the expression of the UNC-5 death domain in yeast, which method comprises: exposing a yeast cell containing an expression vector comprising nucleic acid encoding an UNC-5 protein or a fragment thereof comprising the death domain to a compound under test; allowing the yeast cells to grow in the presence of the compound; and screening for a reduction or inhibition of the lethal phenotype associated with the expression of the UNC-5 death domain in yeast.
  • the UNC-5 protein used in the method of the invention is preferably a C. elegans UNC-5 protein or a human UNC-5 protein.
  • the human UNC-5 protein is UNC-5C or any of the UNC-5C splice variants identified hereinbefore or UNC-5HS1.
  • the invention provides a method of identifying suppressers of the lethal phenotype associated with the expression of the UNC-5 death domain yeast, which method comprises: transfecting yeast cells containing an expression vector comprising nucleic acid encoding an UNC-5 protein or a fragment thereof comprising the death domain with a cDNA library cloned in a yeast expression vector; allowing the transfected yeast cells to grow for one or more cell divisions; and screening for reduction or inhibition of the lethal phenotype associated with the expression of the UNC-5 death domain in yeast.
  • the method further comprises the steps of: identifying a transfected yeast cell exhibiting a reduction or inhibition of the lethal phenotype associated with the expression of the UNC-5 death domain m yeast; and isolating the cDNA clone (s) present in the transfected yeast cell which is/are responsible for conferring reduction or inhibition of the lethal phenotype.
  • the UNC-5 protein is preferably a C. elegans UNC-5 protein or a human UNC-5 protein.
  • the human UNC-5 protein is UNC-5C or any of the UNC-5C splice variants identified hereinbefore or UNC-5HS1.
  • the cDNA library is preferably a C. elegans cDNA library or a human cDNA library.
  • Figure 1 shows a sequence alignment of the known human unc-5C cDNA sequence and the three novel alternative splice variants of unc-5C.
  • the region of alignment corresponds to the portion of the cDNA which encodes the intracellular domains of unc-5C.
  • Figure 2 shows a multiple alignment of unc-5Hl genes.
  • ym97dl2 is an EST clone containing a fragment of the unc-5HSl cDNA
  • 3D is a fragment of the unc-5HSl cDNA cloned by PCR in Example 2.
  • Figure 3 summarises the cloning of human unc-5C variants .
  • Figure 4 summarises the cloning of human unc-5HSl.
  • Figure 5 is a schematic representation of the human unc-5C splice variants.
  • Figure 6 shows an alignment between a fragment of the protein encoded by the cDNA fragment cloned in pYMP6 and the rat neurexim II-alpha-b cDNA.
  • Figure 7 shows an alignment between a fragment of the protein encoded by the cDNA fragment cloned in pYMP17 and the mouse mena protein.
  • Figure 8 is a representation of the vector pGC1037.
  • Figure 9 is a representation of the vector pGC1003.
  • a 5' RACE was performed using the 5' RACE System for Rapid Amplification of cDNA Ends, Version 2.0 (GibcoBRL, Merelbeke, Belgium) , according the instructions supplied by the manufacturer or with minor modifications thereof.
  • the primers were based on the unc-5 EST ym97dl2.
  • the first strand cDNA synthesis was performed with primer:
  • GSPl oGC75 : CGTAGCAGGCACTGGCCTCC
  • GSP2 oGC76 : GCACTGGCCTCCAGCTGGCAGTAG and the RACE anchor primer supplied with the 5' RACE system.
  • the PCR Program was:
  • Step 2 94°C, 30 sec Step 3 60°C, 30 sec
  • GSP3 oGC77 : AGTAGAGGTGGGAGGGCGCCTCCTCGCCCAG and 5' RACE anchor primer
  • the PCR program was :
  • Step 1 92°C, 2 mm
  • Step 2 92°C, 1 mm
  • Step 5 4°C
  • the resulting RACE products were visualised by electrophoresis on agarose gels, the bands excised and purified with Jetsorb (Genomed, Germany) .
  • the RACE products were ligated into plasmids pAS2 and pGEX-5X-3 with T4 DNA ligase (Amersham pharmacia biotech, NJ, USA) , or into a TA cloning vector (Invitrogen,
  • Plasmid DNA was purified prior to sequencing using the Qiagen plasmid purification system (Westburg, Leusden, The Netherlands) .
  • RNA Human Brain Poly A+ RNA was obtained from Clontech, California, USA and first strand cDNA synthesis performed with the Ready To Go T-P ⁇ med First-Strand Kit ( (Amersham pharmacia biotech, NJ, USA) .
  • OGC63 GGGAATTCCATATGTTGTTTGTGTATCGGAAGAATCATC
  • OGC64 ACGCGTCGACTTAATACTGCCCTTCTGCTGCTAAGGAC
  • OGC65 CCGGAATTCCTTGTTTGTGTATCGGAAGAATCATC PCR program was :
  • Step 1 94°C, 5 min Step 2 92°C, 30 sec Step 3 55°C, 30 sec Step 4 72°C, 2 min Repeat steps 2 to 4 for 25 cycles Step 5 72°C, 7 min Step 6 4°C
  • SEQ ID NO: 7 shows the sequence of a PCR product isolated using the above PCR strategy. This PCR product was designated clone 3D.
  • Figure 2 shows an alignment between the Ra ttus norvegicus unc-5Hl cDNA sequence, the sequence of EST ym97dl2, the sequence of clone 3D and the sequences of several other PCR products amplified using the above PCR strategy (IG, Urc and 2Brc) .
  • a PCR amplification was performed with following primers :
  • Step 3 72°C, 2 min Repeat steps 1 to 3 for 25 cycles
  • PCR products were isolated and cloned in frame as Ncol/Sall fragments in the vectors pAS2 and pGAD424 supplied by Clontech (Palo Alto, California, USA) .
  • yeast two hybrid method Fields and Song, Nature 340:245, 1989
  • a method well known to molecular biologists to search for the proteins that interact with the UNC-5 protein.
  • the two hybrid method is based on a pair of fusion proteins.
  • the first fusion protein comprises a first of two interacting proteins fused to the transcriptional activation domain of a bipartite yeast transcription factor; the second fusion protein comprises the second of two interacting proteins fused to the DNA binding domain of the bipartite yeast transcription factor.
  • the principle of the method is that if the two domains of the bipartite transcription factor are physically brought together by binding of the first and second interacting proteins then the resulting complex will be able to activate transcription from a promoter which contains a target binding site for the transcription factor.
  • the two hybrid assay is commonly used to study protem-protem interactions between two known proteins. It can also be used to screen a library of proteins to identify proteins which interact with a given protein.
  • the yeast two hybrid assay was used to identify proteins which interact with C. elegans UNC-5 or human UNC-5 as follows: the intracellular part of UNC-5 or parts thereof were cloned m fusion with the DNA-bmdmg domain of the yeast transcription factor GAL4. A cDNA library was cloned into a vector containing the transcriptional activation domain of GAL4. The fusion proteins were then independently expressed together m yeast containing a reporter gene under the transcriptional control of a promoter containing GAL 4 binding sites (typically GAL1 lacZ or GAL1-HIS3) .
  • a promoter containing GAL 4 binding sites typically GAL1 lacZ or GAL1-HIS3
  • plasmid pGC1037 a plasmid map of pGC1037 is shown in Figure 8 and the complete sequence of the plasmid is given in SEQ ID NO: 91
  • a pre-transformed Human Brain MATCHMAKER cDNA library (Clontech, California, USA) according to the protocol supplied by the manufacture, or with minor modifications thereof.
  • RNA for RNA inhibition experiments was prepared according to the MEGAscript protocol (Ar ⁇ bion, UK) .
  • RNA isolated using this protocol was purified away from contaminants using the RNeasy system from Qiagen (Westburg, the Netherlands), following the instructions for RNA clean-up supplied by the manufacturer.
  • RNA was injected into the nematodes using standard procedures (Methods in Cell biology, Vol 48, Academic Press, 1995) .
  • UNC-5 proteins may be encoded by at least three genes, the homologous genes unc-5C, unc-5HSl, unc-5HS2.
  • UNC-5 is an important receptor involved in a vast amount of biological processes, it is considered that more functional homologous genes or unc-5 genes may present in the Homo sapiens genome.
  • the expression of the unc-5 gene does not result in the production of a single transcript.
  • the expression of unc-5C locus can result in the production at least 4 isoforms as a result of alternative splicing events. It is possible that the other unc-5 genes will also express splice variants, which may encode different protein isoforms.
  • any of these unc-5 isoforms may form dimers, analogous to the homo-dimerisation found for C. elegans unc-5. Accordingly, assays can also be developed to screen for chemical substances that alter the dimerization of human unc-5 proteins. Compounds identified using such an assay may have pharmacologically useful properties.
  • UNC-129 also signals to the UNC-5 receptor (Colavita et al., Science 261:706-709). UNC-6 is also known to signal to UNC-40 (DCC). UNC-129 belongs to the TGF- ⁇ superfamily. TGF- ⁇ receptors, including DAF-1 and DAF-4, do not affect axonal guidance. Although new TGF- ⁇ receptors may be found that are involved in axonal guidance, it is more likely that the UNC-129 molecule is able to interact with TSP type I domains, which are present in UNC-5. Such interaction between TGF- ⁇ molecules and TSP Type I domains has been shown previously (Schultz-Cherry et al . , 1994, J. Biol. Chem. 269, 26775) . Furthermore UNC-129 is also involved in the UNC-40 pathway.
  • the UNC-40/UNC-5 interaction may function analogously to the Bax/Bcl-2 interaction involved in apoptosis.
  • Bax can be considered as the protein that protects against apoptosis but the relative titre of both Bax and Bcl-2 m a cell may be important in the decision of cell death.
  • UNC-5 is capable of forming homodimers
  • UNC-40 is also capable of forming heterodimers with UNC-40.
  • the UNC- 5/UNC-40 heterodimers may act as a functional receptor for UNC-6 and UNC-129.
  • Assays to isolate compounds that influence the interaction between UNC-5 and UNC-40, both enhancing and inhibiting this interaction have therefore been developed. These assays are analogous to the assays as described to isolate compounds that influence the formation of the UNC-5 dimers and the assays for compounds that influence the interaction of UNC-5 with its other interacting proteins (see below) .
  • the intracellular part of UNC-5 containing the domains MPP, ZO-1 and UP cloned m vector pGC1003 (a plasmid map of pGC1003 is given m Figure 9 and the complete sequence of the plasmid is given in SEQ ID NO: 92) was used as ⁇ ba ⁇ t' in a yeast two hybrid experiment screening against a C. elegans cDNA library. These experiments resulted in the identification of ten genes, including three known genes and seven genes with heretofore unknown function, encoding proteins which specifically interact with the intracellular part of UNC-5. Details of the UNC-5 interacting proteins identified during the two hybrid screen are given below. In most cases, the results of double-stranded RNA inhibition experiments (RNAi) designed to inhibit expression of the interacting protein are also given. Where appropriate, details of human homologues of the interacting protein are also given and any known disease associations are discussed.
  • RNAi double-stranded RNA inhibition experiments
  • a first series of hits resulted in the identification of plasmid pC1025 which contains a fragment of a cDNA encoding the C. elegans spectrin ⁇ -chain/Fodrin.
  • the spectrin ⁇ -chain protein is encoded by the gene K11C4.3, located on chromosome IV.
  • the full length cDNA and amino acid sequences of spectrin ⁇ -chain/Fodrin are shown in SEQ ID NOS: 11 and 12, respectively.
  • the nucleotide sequence of the fragment of the spectrin ⁇ -chain cDNA which is cloned as an insert in plasmid pC1025 is given in SEQ ID NO: 13, the corresponding amino acid sequence is given in SEQ ID NO: 14.
  • RNAi experiments using a double-stranded RNA corresponding to the cDNA fragment cloned in pC1025 revealed that inhibition of the expression of the native spectrin ⁇ -chain in C. elegans worms causes the following phenotype: no embryonal lethality, normal canals, normal elongation, growth retardation and growth arrest at LI and L2 , nearly no movement but touch reflex is observed. The phenotype is 100% penetrant, and the larvea are short and wrinkled.
  • These RNAi phenotypes and the corresponding knock-out phenotype can be used as the basis of a compound screen in C. elegans to identify chemical substances that modulate the activity of the spectrm ⁇ -chain protein.
  • Human Fod ⁇ n (genbank accession number 2493434) contains an extra C-termmal PDZ domain that is not present in spectrin (genbank accession number 134798) .
  • the human fodrm seems to be more homologous to the C. elegans protein. This is in agreement with the finding that unc-5 is also expressed m the brain of vertebrates .
  • UNC-5 and fodrm could be a critical event in a cell signalling, hence compounds which modulate the interaction between UNC-5 and fodrm, particularly the interaction between human UNC-5 and human fodrm, may potentially have pharmacological activity.
  • Assays can also be developed to screen for genetic mutations that inhibit the interaction needed for proper signal transduction.
  • Compounds which enhance or inhibit the interaction of UNC-5 with fodrm and spectrin ⁇ -chain may be useful m the development of pharmaceutical preparations for the treatment of Crohn's disease, Sjogren's syndrome, secretion related diseases, diseases related to neutophil and platelet activation, and long-term potential m neurons, Alzheimer's disease, proliferative diseases such as carcinomas, neoplasia, and more specifically, shwannomas, menmgiomas, ependymonas, squamous cell carcinomas, malignant melanomas and lung carcinomas, spherocytosis, pyropoikilocytosis, Duchenne muscular dystrophy and various neurological disorders.
  • the nucleotide sequence of the full length APR-1 cDNA is shown in SEQ ID NO: 15 and the amino acid sequence of the APR-1 protein encoded by this cDNA is shown in SEQ ID NO: 16.
  • the nucleotide sequence of the fragment of the APR-1 cloned in pC1028 is shown in
  • RNAi phenotypes and the corresponding knock-out phenotype can be used as the basis of a compound screen in C. elegans to identify chemical entities that modulate the activity of APC (see below), and hence the unc-5 pathway.
  • UNC-5 and APC/APR-1 could be a critical event in cellular signalling and hence compounds which modulate this interaction, particularly compounds which modulate an interaction between human UNC-5 and human APC, may potentially have pharmacological activity.
  • genetic mutations, and splice variants can be identified that inhibit the interaction, needed for proper signal transduction.
  • Compounds which enhance or inhibit the interaction of UNC-5 with APR/APC may be useful in the development of pharmaceutical agents for the treatment of neurological diseases and colorectal cancers such as adenomatous polyposis coli.
  • a third plasmid identified during the yeast two hybrid screen using C. elegans UNC-5 as bait (pC1034) was found to contain a fragment of the UNC-14 cDNA.
  • the nucleotide sequence of the full length UNC-14 cDNA is shown in SEQ ID NO: 19, the amino acid sequence of the protein encoded by this cDNA is given in SEQ ID NO: 20.
  • the nucleotide sequence of the fragment of the UNC-14 cDNA cloned as an insert in pC1034 is shown in SEQ ID NO: 21, with the corresponding amino acid sequence of the polypeptide encoded by this fragment shown in SEQ ID NO: 22.
  • C. elegans worms mutated in unc-14 are observed to be very sluggish, almost paralysed, small, dumpyish, with a tendency to coil and show some egg retention.
  • This phenotype can be used as the basis of a compound screen in C. elegans to identify chemical entities that modulate the activity of UNC-14.
  • C. elegans worms mutated in the unc-14 gene were shown to have abnormal axonal elongation and axonal structures.
  • the unc-14 gene encodes a protein of 665 amino acids, and is co-expressed with the unc-51 gene in the cell bodies and axons of almost all neurons including DD/VD and hermaphrodite-specific neurons.
  • the nucleotide sequence of the full length FllAlO.l cDNA is shown in SEQ ID NO: 23, with the amino acid sequence of the protein encoded by this cDNA shown in SEQ ID NO: 24.
  • the nucleotide sequence of the fragment of the FllAlO.l cDNA cloned in pGC1021 is shown in SEQ ID NO: 25, the amino acid sequence of the protein fragment encoded by this fragment of the cDNA is shown in SEQ ID NO: 26.
  • RNA corresponding to the insert of pGC1021 showed that inhibition of FllAlO.l expression in C. elegans results in worms which are weakly constipated. In C. elegans, constipation has been associated with neuronal dysfunction (Thomas, Genetics 124:855-872, 1990) . Furthermore and remarkably inhibition of FllAlO.l expression causes migration defects in the distal tip cell, similar to those observed in unc-5 mutants and unc-14/unc-51 double mutants. These RNAi phenotypes and the corresponding knock-out phenotypes can be used as the basis of a compound screen in C. elegans to identify chemical entities that modulate the activity of FllAlO.l.
  • compositions useful in the treatment of neurological disorders, tumours such as Kaposi's Sarcoma, immunological disorders and diseases related to vesicle fusion, proteolysis, peroxisomal and mitochondrial biogenesis, and transcription may be of use in the development of pharmaceutical compositions useful in the treatment of neurological disorders, tumours such as Kaposi's Sarcoma, immunological disorders and diseases related to vesicle fusion, proteolysis, peroxisomal and mitochondrial biogenesis, and transcription.
  • the nucleotide sequence of the full length C15E6.1/2 cDNA is shown in SEQ ID NO: 27, with the amino acid sequence of the protein encoded by this cDNA shown in SEQ ID NO: 28.
  • the nucleotide sequence of the fragment of the C15E6.1/2 cDNA cloned in pGC1026 is shown in SEQ ID NO: 29, the amino acid sequence of the protein fragment encoded by this fragment of the cDNA is shown in SEQ ID NO: 30.
  • RNAi experiments using a double-stranded RNA corresponding to the insert of pGC1026 did not result in any clear visual phenotype.
  • C15E6.1/2 as an UNC-5 interacting protein indicates that UNC-5 might be a band 4 .1 binding protein and may share homology with other band 4.1 binding proteins such as CD44, glycophrin C, and paranodin.
  • band 4.1 signature By using the band 4.1 signature to search a database of C . elegans genes, F07A11.1 on chromosome II was identified as encoding a band 4.1 protein.
  • UNC-5 and C15E6.1/2 could be a critical event in cellular signalling and hence compounds which modulate this interaction may potentially have pharmacological activity.
  • genetic mutations, and splice variants can be identified that inhibit the interaction, needed for proper signal transduction.
  • Compounds which enhance or inhibit the interaction of UNC-5 with C15E6.1/2 may be useful in the development of pharmaceutical preparations for the treatment of diseases related to axonal signalling, synaptic vesicle exocytosis, cell adhesion, cytoskeleton associated proteins, cell morphology , cell growth, allergic inflammatory processes and rheumatoid arthritis.
  • a sixth plasmid identified during the two hybrid screen was found to contain a fragment of cDNA corresponding to the C. elegans gene designated D1081.7.
  • the nucleotide sequence of the full length D1081.7 cDNA is shown in SEQ ID NO: 31, the amino acid sequence of the protein encoded by this cDNA is given in SEQ ID NO: 32.
  • the nucleotide sequence of the fragment of the D1081.7 cDNA cloned as an insert in pGC1027 is shown in SEQ ID NO: 33, with the corresponding amino acid sequence of the polypeptide encoded by this fragment shown in SEQ ID NO: 34.
  • UNC-5 and D1081.1 could be a critical event in cellular signalling and hence compounds which modulate this interaction may potentially have pharmacological activity and thus be of use m the development of pharmaceutical compositions.
  • genetic mutations, and splice variants may be identified that inhibit the interaction needed for proper signal transduction.
  • a seventh plasmid identified during the two hybrid screen was found to contain a fragment of cDNA corresponding to the C. elegans gene designated B0238.9.
  • the nucleotide sequence of the full length B0238.9 cDNA is shown m SEQ ID NO: 35, the ammo acid sequence of the protein encoded by this cDNA is given in SEQ ID NO: 36.
  • the nucleotide sequence of the fragment of the B0238.9 cDNA cloned as an insert in pGC1032 is shown in SEQ ID NO: 37, with the corresponding ammo acid sequence of the polypeptide encoded by this fragment shown in SEQ ID NO: 38.
  • B0238.9 is located in the chromosomal region where seu-2 is also located.
  • the seu-2 was identified in suppressor screens of ectopically expressed unc-5 and is considered to be involved m the unc-5 pathway (Colavita and Culotti, Dev. Biol. 194:72-85, 1998). As a gene has now been isolated that interacts with unc-5, it is high probable that B0238.9 is the same as seu-2. Mutations m seu-2 appeared not to have any visual phenotype, as was also ooserved m RNAi experiments using a double stranded RNA corresponding to a fragment of B0238.9. The finding that SEU-2 is a suppressor and a binding partner to UNC-5 validates the importance of this interaction.
  • ZC404.8 .PGC1033.
  • An eighth plasmid identified during the two hybrid screen was found to contain a fragment of a cDNA corresponding to the C. elegans gene designated ZC404.8.
  • the nucleotide sequence of the full length ZC404.8 cDNA is shown in SEQ ID NO: 39, the ammo acid sequence of the protein encoded by this cDNA is given in SEQ ID NO: 40.
  • the nucleotide sequence of the fragment of the ZC404.8 cDNA cloned as an insert m pGC1033 is shown in SEQ ID NO: 41, with the corresponding am o acid sequence of the polypeptide encoded by this fragment shown m SEQ ID NO: 42.
  • RNAi experiments using a double stranded RNA corresponding to a fragment of this gene resulted in an embryonic lethal phenotype.
  • the worms showed no elongation and only very little muscle activity, the hypodermis is clearly abnormal. Homology searches in the EST database with
  • ZC404.8.9 revealed the presence of at least three human ESTs with significant homology.
  • the interaction between UNC-5 and ZC404.8 could be a critical event in cellular signalling and hence compounds which modulate this interaction may potentially have pharmacological activity and thus be useful in the development of pharmaceutical preparations.
  • genetic mutations, and splice variants may be identified that inhibit the interaction, needed for proper signal transduction.
  • vk!7a3 (pGC1023)
  • ykl7a3 A ninth plasmid identified during the yeast two hybrid experiment was found to contain a fragment of cDNA corresponding to the C. elegans gene designated ykl7a3.
  • nucleotide sequence of the fragment of the ykl7a3 cDNA cloned as an insert in pGC1023 is shown in SEQ ID NO: 43, with the corresponding amino acid sequence of the polypeptide encoded by this fragment shown in SEQ ID NO: 44.
  • RNAi experiments using a double stranded RNA corresponding to a fragment of ykl7a3 resulted in the following phenotypes in C. elegans : Very slow growth, and the larvae get typical darker spots as they get older. Inhibition of ykl7a3 expression in some non wild-type genetic backgrounds leads to defective moulting, where the worm cannot escape from the old cuticle and therefore shrinks and stays in the L4 stage. The defective moulting phenotype is also observed when ykl7a3 expression is inhibited on a wild-type genetic background, although the phenotype is less prominent. Worms which escape the defective moulting phenotype show defects in vulva development, either lacking a vulva altogether or having a vulva which is non-functional.
  • KIAA0187 could be a critical event in cellular signalling and hence compounds which modulate this interaction may potentially have pharmacological activity.
  • genetic mutations, and splice variants may be identified that inhibit the interaction, needed for proper signal transduction.
  • Compounds which enhance or inhibit the interaction of UNC-5 with ykl7a3 may be of use in the development of pharmaceutical compositions for the treatment of CADASIL, artheriohepatic dysplasia, Alzheimer's disease, neoplasia such as T-cell acute lymphoblastic leukemia and certain cancers, such as pancreatic cancer and colon cancer.
  • a tenth plasmid identified using the yeast two hybrid experiment was found to contain a fragment of a cDNA corresponding to the C. elegans gene designated F41H10.3.
  • F41H10.3 cDNA is shown in SEQ ID NO: 45, the amino acid sequence of the protein encoded by this cDNA is given in SEQ ID NO: 46.
  • the nucleotide sequence of the fragment of the F41H10.3 cDNA cloned as an insert in pGC1020 is shown in SEQ ID NO: 47, with the corresponding amino acid sequence of the polypeptide encoded by this fragment shown in SEQ ID NO: 48.
  • F41H10.3 harbors a ATP/GTP binding domain.
  • UNC-5 and F41H10.3 could be a critical event m signalling and compounds which modulate this interaction may potentially have pharmacological activity and thus be useful m the development of pharmaceutical preparations.
  • the intracellular part of the human UNC-5 protein (human UNC-5HS1) containing the domains ZO-1, UP and DD cloned in vector pGC1037 (see above) was used as ⁇ ba ⁇ t' in a yeast two hybrid experiment screening against a pretransformed human brain Matchmaker cDNA library (Clontech, Palo Alto, California USA) using the mating screen approach described above. These experiments resulted in the identification of six genes encoding proteins which interact with UNC-5, including two known genes and four heretofore unknown genes .
  • Tne nucleotide sequence of the full length ⁇ -beta-1, 3-N-acetylammyltansferase cDNA is shown in SEQ ID NO: 49, the ammo acid sequence of the protein encoded by this cDNA is given m SEQ ID NO: 50.
  • the partial nucleotide sequences of the fragment of the ⁇ -beta-1, 3-N-acetylammyltansferase cDNA cloned as an insert in pYMP5 are shown in SEQ ID NOs: 51 and 52, with the corresponding ammo acid sequence of the polypeptide encoded by these partial sequences shown in SEQ ID NO: 53.
  • elegans has at least seven putative homologues of i-beta-1, 3-N-acetylaminyltansferase, designated F22F7.6, C18G1.3, K09C8.4, F21H7.10, C54C8.2, F56H6.6 and T15D6.4.
  • cDNA and/or amino acid sequences for each of these putative homologues are given herein.
  • Amino acid and nucleotide sequences for these homologues are given in SEQ ID NOS: 66 to 82.
  • UNC-5 and beta-1, 3-N-acetylglucosaminyltransferase could be a critical event in signalling and hence compounds which modulate this interaction may potentially have pharmacological activity.
  • genetic mutations, and splice variants may be identified that inhibit the interaction, needed for proper signal transduction.
  • Compounds which modulate the interaction of UNC-5 with beta-1, 3-N-acetylglucosaminyltransferase may be useful in the development of pharmaceutical preparations for the treatment of synaptic cleft dysfunctions, vesicle transport dysfunctions, inflamation, various tumours and more particular in tumour cell adhesion, migration and invasion, such as pancreas cancer, squamous cell cancer, human breast cancer, thyroid neoplasms, colorectal carcinomas.
  • a second plasmid identified during the yeast two hybrid experiment was found to contain a fragment of a cDNA corresponding to a new gene with slight homology to neurexin II-alpha-b.
  • the new gene was designated NHII.
  • Partial nucleotide sequences for the fragment of cDNA cloned as an insert in pYMP6 are shown in SEQ ID NO: 54 (coding strand sequenced from one end of the insert of pYMP6 sequenced with forward primer) and and SEQ ID NO: 55 (non-coding strand sequenced from one end of pYMP6 with reverse primer) .
  • the plasmid pYMP6 was deposited in the Belgian Co-ordinated Collections of Microorganisms (BCCM) , Universiteit Gent, K. L. Ledeganckstraat 35, B-9000, Gent, Belgium on 21 May 1999 under accession number LMBP 3932.
  • the cDNA insert (approximately 1800bp) can easily be excised from this plasmid by digestion with the restriction enzymes EcoRI and Xhol.
  • the cDNA insert sequence can be amplified by PCR using primers corresponding to the sequences for the ends of the insert given in SEQ ID NOS: 54 and 55.
  • a third plasmid identified during the yeast two hybrid experiment was found to contain a fragment of a cDNA encoding a protein sharing slight homology with the human mena protein.
  • the new gene was designated MHI.
  • Partial nucleotide sequences of the fragment of cDNA cloned as an insert in pYMP17 are shown in SEQ ID NO: 56, (coding strand sequenced from one end of the insert of pYMP sequenced with forward primer) and SEQ ID NO: 57 (non-coding strand sequenced from one end of pYMP with reverse primer) .
  • SEQ ID NO: 56 coding strand sequenced from one end of the insert of pYMP sequenced with forward primer
  • SEQ ID NO: 57 non-coding strand sequenced from one end of pYMP with reverse primer
  • An alignment between the amino acid sequence encoded by the insert of pYMP17 and the mouse mena protein is shown in Figure 7.
  • the plasmid pYMP17 was deposited in the Belgian Coordinated Collections of Microorganisms (BCCM) , Universiteit Gent, K. L.
  • the cDNA insert (approximately lOOObp) can easily be excised from this plasmid by digestion with the restriction enzymes EcoRI and Xhol .
  • the cDNA insert sequence can be amplified by PCR using primers corresponding to the sequences for the ends of the insert given in Figures 55A and 55B.
  • C. elegans has at least one protein with homology to the new Mena homologue (MHI) , encoded by the gene designated Y50D4. Contig200.
  • MHI Mena homologue
  • the C. elegans gene, unc-34 (which maps with Y50D4) is known to suppress the axonal guidance defects induced by ectopic expression of the Netrin receptor UNC-5 (Colavita, A. et al., Dev. Biol., 194:72-85, 1998.).
  • Alpha-2 macroglobulin (pYMP30) A fourth plasmid identified during the yeast two hybrid experiment was found to contain a fragment of the human alpha-2 macroglobulin cDNA.
  • the nucleotide sequence of the full length alpha- 2 macroglobulin cDNA is shown in SEQ ID NO: 58, the amino acid sequence of the protein encoded by this cDNA is given in SEQ ID NO: 59.
  • a partial nucleotide sequence for the fragment of the alpha-2 macroglobulin cDNA cloned as an insert in pYMP30 is shown in SEQ ID NO: 60.
  • C. elegans has at least one homologue of alpha-2 macroglobulin, designated ZK337.1, of which two splice variants designated ZK337.1a and ZK337.1b are known to exist .
  • UNC-5 and alpha-2 macroglobulin could be a critical event in signalling and hence compounds which modulate this interaction may potentially have pharmacological activity.
  • Compounds which enhance or inhibit the interaction of UNC-5 with alpha-2 macroglobulin could be useful in the development of pharmaceutical substances.
  • a fifth plasmid identified during the yeast two hybrid experiment was found to contain a fragment of cDNA with no homology to any known human cDNA.
  • Partial nucleotide sequences for the fragment of cDNA cloned as an insert in pYMPll are shown in SEQ ID NO: 61 (coding strand sequenced from one end of the insert of pYMP sequenced with forward primer) and SEQ ID NO: 62 (non-coding strand sequenced from one end of pYMP with reverse primer) .
  • the plasmid pYMPll was deposited in the Belgian Co-ordinated Collections of Microorganisms (BCCM), Universiteit Gent, K. L. Ledeganckstraat 35, B-9000, Gent, Belgium on 21 May 1999 under accession number LMBP 3933.
  • the cDNA insert (approximately 2300bp) can easily be excised from this plasmid by digestion with the restriction enzymes EcoRI and Xhol.
  • the cDNA insert sequence can be amplified by PCR using primers corresponding to the sequences for the ends of the insert given in Figures 59A and 59B.
  • the interaction between UNC-5 and the protein encoded by the insert of pYMPll could be a critical event in signalling and hence compounds which modulate this interaction may potentially have pharmacological activity and thus could be useful in the development of pharmaceutical substances.
  • a sixth plasmid identified during the yeast two hybrid experiment was found to contain a fragment of cDNA with no homology to any known human cDNA.
  • Partial nucleotide sequences for the fragment of cDNA cloned as an insert in pYMP12 are shown in SEQ ID NO: 63 (coding strand sequenced from one end of the insert of pYMP sequenced with forward primer) and SEQ ID NO: 64 (non-coding strand sequenced from one end of pYMP with reverse primer) .
  • the plasmid pYMP12 was deposited in the Belgian Co-ordinated Collections of Microorganisms (BCCM), Universiteit Gent, K. L. Ledeganckstraat 35, B-9000, Gent, Belgium on 21 May 1999 under accession number LMBP 3934.
  • the cDNA insert (approximately 2000bp) can easily be excised from this plasmid by digestion with the restriction enzymes EcoRI and Xhol.
  • the cDNA insert sequence can be amplified by PCR using primers corresponding to the sequences for the ends of the insert given in Figures 60A and 60B.
  • UNC-5 and the protein encoded by the insert of pYMP12 could be a critical event in signalling and hence compounds which modulate this interaction may potentially have pharmacological activity and thus could be useful in the development of pharmaceutical substances.
  • 96 or 384 well plates or microtiter plates with another number of wells This assay is suitable for high throughput compound screening. Optimal performance of the assay is dependent upon at least two important parameters: lysis of yeast cells and the choice of the ⁇ -galactosidase substrate.
  • the basic protocol for an assay in 96 or 384 well plates is as follows:
  • a yeast strain containing the Escherichia coli lacZ gene under the control of the yeast Gal4 promoter is grown overnight (with shaking at 230-270 rpm) then diluted with YPD medium to an OD600 of 0.2. Diluted cultures are grown for an additional 3-5 hr until mid-log phase. Yeast cells are then transferred to either 96- or 384-well plates (100 ⁇ l/well or 25 ⁇ l/well, respectively) . Alternatively, cells can be cultured in the microtiter plates, eliminating the need for a pipetting step. The yeast cells are then either lysed by freeze and thaw method (liquid N 2 to freeze, 37°C water bath to thaw) or by use of a Lysis buffer (e.g.
  • Non-lysed cells also give a signal, although the variability is increased if the cells are not lysed.
  • Yeast cells can also be permeabilized with various reagents such as isopropanol (15 %).
  • Fluorescein di galactoside is a typical low cost fluorescent reagent for the detection of ⁇ -galactosidase; it can be used for screening, although autofluorescent compounds can induce a non- desirable background leading to false positives.
  • Alternative substrates are available that become luminescent upon ⁇ -galactosidase cleavage, thereby eliminating background problems.
  • Galacton-Star® from Tropix.
  • Typically about l ⁇ M substrate is added and the plates are incubated at room temperature for 60 minutes. Fluorescence (for FDG) is then measured at 530 nm. It is typically possible to detect as low as 100 cells per well.
  • secreted alkaline phosphatase can be used as a reporter gene.
  • the use of secreted alkaline phosphatase gives equivalent sensitivity to ⁇ - gaiactosidase with the advantage that there is no need to lyse the cells.
  • Fluorescent substrates for alkaline phosphatase are available commercially from Sigma- Aldrich (Bornem, Belgium) or Molecular Probes (Eugene, OR, USA) .
  • the test compound can be added at various stages of the above procedure. Generally, the compound is added on the plates onto which the yeast are plated. However, the compound can also be added during the second incubation in order to overcome toxicity ptoblems . As a control, it is important to check whether the compound slows down the growth of the yeast. This can be done using turbidity measurements.
  • An in vivo FRET assay can be conveniently performed using two different mutants of GFP which absorb and emit light at different wavelengths and which have suitably overlapping emission/absorption spectra, such as EGFP (enhanced green fluorescent protein) and EBFP (enhanced blue fluorescent protein) .
  • EGFP enhanced green fluorescent protein
  • EBFP enhanced blue fluorescent protein
  • FRET fluorescence energy transfer
  • Such transfer is characterized by a reduction of fluorescence intensity of the donor fluorophore (EBFP) and re-emission of fluorescence at the acceptor fluorophore (EGFP) wavelengths. Therefore if each fluorophore is fused to a protein domain known to bind to the other the protein-protein interaction can be monitored in vivo using FRET.
  • the APC binding domain of UNC-5 cloned in fusion with EBFP whereas APC is cloned in fusion with EGFP in expression vectors suitable for use in the chosen host cell line or organism.
  • both fusion proteins are expressed in a cell line or in C. elegans it is possible to monitor and quantify their in vivo interaction by irradiating the cells/worms with light at 488nm.
  • fluorescent energy transfer results in a measurable decreased in fluorescence from the fluorescence donor at a wavelength within the emission spectrum of the donor.
  • Plasmid vectors containing both EGFP and EBFP are commercially available from Clontech (Palo Alto, California, USA) . Information on the use of these vectors is also supplied by the manufacturer.
  • UNC-5 expression in yeast cells results in a lethal phenotype, mainly because of the expression of a death domain. This observation was most clearly seen in the experiments with C. elegans UNC-5. Accordingly, assays can be developed to screen for compounds, interacting proteins and suppressors which alter the activity of UNC-5, particularly the activity of the death domain of UNC-5. These assays are analogous to those described by Xu and Reed (Mol. Cell 1998, 1:337- 46) .
  • Yeast cells are transfected with a plasmid encoding the C. elegans or human unc-5 (including the death domain) , such as the vectors described in the yeast two hybrid experiments. Furthermore, the yeast cells are transfected with a library expressing C. elegans or human cDNA, such as the libraries described in the yeast two hybrid experiments. The transfected yeast cells are placed in the wells of micotiter plates, and allowed to grow further. This allows selection cDNAs, and hence genes and proteins, that reduce or inhibit the lethal phenotype of the yeast ceils transfected with the death domain of unc-5.
  • Such proteins will interact with UNC-5, or with UNC-5 interacting proteins, or with proteins in the UNC-5 pathway, or with proteins in parallel pathways to cause suppression of the unc-5 lethal phenotype.
  • the selected cDNAs genes and proteins can be used in the development of pharmaceutical preparations or in the development of assays to select for compounds that enhance their function or expression.
  • a fragment of a given gene or cDNA is known then further fragments of the corresponding full length gene and/or cDNA can be constructed can often be found using in sili co techniques such as AceDB (see http:Wwww.sanger.ac.uk), or searching of the EST database.
  • the full cDNA can be cloned using standard technology such as 5'/3' RACE or SLl/2 RT-PCR on worm total RNA and colony hybridization. An analogous strategy is followed to clone a full length gene and/or cDNA for vertebrate and hence Human DNA.
  • a full length C. elegans gene can be cloned starting from a human sequence. Using in silico techniques, a homologue or an EST can be found. Standard molecular biology techniques can then be used to clone the full length C. elegans gene. If no homologous sequence can be found by simple database searching, it may be necessary to perform species hopping. An analogous strategy is followed to clone a full length gene and/or cDNA for vertebrate and hence Human DNA, starting from a C. elegans DNA sequence.
  • SEQ ID NO: 1 nucleotide sequence of a part of the human unc-5Cb cDNA which encodes the intracellular region of the protein.
  • SEQ ID NO: 2 ammo acid sequence of the intracellular part of the human unc-5Cb protein encoded by the nucleotide sequence shown as SEQ ID NO: 1.
  • SEQ ID NO: 3 nucleotide sequence of a part of the human unc-5Cc cDNA which encodes the intracellular region of the protein.
  • SEQ ID NO: 4 ammo acid sequence of the intracellular part of the human unc-5Cc protein encoded by the nucleotide sequence shown as SEQ ID NO: 3.
  • SEQ ID NO: 5 nucleotide sequence of a part of the human unc-5C8 cDNA which encodes the intracellular region of the protein.
  • SEQ ID NO: 6 ammo acid sequence of the intracellular part of the human unc-5C8 protein encoded by the nucleotide sequence shown as SEQ ID NO: 5.
  • SEQ ID NO: 7 nucleotide sequence of the fragment of the human unc-5Hl cDNA cloned by PCR in Example 2.
  • SEQ ID NO: 8 predicted ammo acid sequence for the human unc-5Hl protein, translation in frame 1.
  • SEQ ID NO: 9 predicted amino acid sequence for the human unc-5Hl protein, translation in frame 2.
  • SEQ ID NO: 10 predicted amino acid sequence for the human unc-5Hl protein, translation in frame 3.
  • SEQ ID NO: 11 nucleotide sequence of the C. elegans spectrin ⁇ -chain/Fodrin cDNA.
  • SEQ ID NO: 12 amino acid sequence of the C. elegans spectrin ⁇ -chain/Fodrin protein.
  • SEQ ID NO: 13 nucleotide sequence of the fragment of the C. elegans spectrin ⁇ -chain/Fodrin cDNA cloned in pC1025.
  • SEQ ID NO: 15 nucleotide sequence of the C. elegans
  • SEQ ID NO: 17 nucleotide sequence of a fragment of the C. elegans APR-1 cDNA cloned in
  • SEQ ID NO: 19 nucleotide sequence of the C. elegans unc- 14 cDNA .
  • SEQ ID NO: 21 nucleotide sequence of the fragment of the C. elegans unc-14 cDNA cloned in pC1034.
  • SEQ ID NO: 22 amino acid sequence of the polypeptide encoded by the cDNA fragment shown as SEQ ID NO: 21.
  • SEQ ID NO: 23 nucleotide sequence of the C. elegans FllAlO.l cDNA.
  • SEQ ID NO: 25 nucleotide sequence of the fragment of the C. elegans FllAlO.l cDNA cloned in pGC1021.
  • SEQ ID NO: 26 amino acid sequence of the polypeptide encoded by the cDNA fragment shown as
  • SEQ ID NO: 27 nucleotide sequence of the C. elegans
  • SEQ ID NO: 29 nucleotide sequence of the fragment of the C. elegans C15E6.1 cDNA cloned in pGC1026.
  • SEQ ID NO: 31 nucleotide sequence of the C. elegans
  • SEQ ID NO: 33 nucleotide sequence of the fragment of the C. elegans 1081.7 cDNA cloned in pGC1027.
  • SEQ ID NO: 34 amino acid sequence of the polypeptide encoded by the cDNA fragment shown as SEQ ID NO: 33.
  • SEQ ID NO: 35 nucleotide sequence of the C. elegans B0238.9 cDNA (seu-2) .
  • SEQ ID NO: 37 nucleotide sequence of the fragment of the C. elegans B0238.9 cDNA cloned in pGC1023.
  • SEQ ID NO: 39 nucleotide sequence of the C. elegans
  • SEQ ID NO: 43 nucleotide sequence of the fragment of the C. elegans ykl7a3 cDNA cloned in pGC1023.
  • SEQ ID NO: 44 amino acid sequence of the polypeptide encoded by the cDNA fragment shown as SEQ ID NO: 43.
  • SEQ ID NO: 45 nucleotide sequence of the C. elegans
  • SEQ ID NO: 47 nucleotide sequence of the fragment of the C. elegans F41H10.3 cDNA cloned in pGC1020.
  • SEQ ID NO: 49 nucleotide sequence of the human i- beta-1, 3-N-acetylaminyltransferase cDNA.
  • SEQ ID NO: 50 amino acid sequence of the human i- beta-1, 3-N-acetyiaminyltransferase protein .
  • SEQ ID NO: 51 partial nucleotide sequence for the fragment of the human i-beta-1 , 3-N- acetylaminyltransferase cDNA cloned in pYMP5 (forward primer, coding strand) .
  • SEQ ID NO: 52 partial nucleotide sequence for the fragment of the human i-beta-1, 3-N- acetylaminyltransferase cDNA cloned in pYMP5 (reverse primer, non-coding strand)
  • SEQ ID NO: 53 partial amino acid sequence for the polypeptide encoded by the fragment of the i-beta-1, 3-N- acetylaminyltransferase cDNA cloned in pYMP5.
  • SEQ ID NO: 54 partial nucleotide sequence for the human cDNA fragment cloned in pYMP6 (forward primer, coding strand) .
  • SEQ ID NO: 55 partial nucleotide sequence for the human cDNA fragment cloned in pYMP6 (reverse primer, non-coding strand) .
  • SEQ ID NO: 56 partial nucleotide sequence for the human cDNA fragment cloned in pYMP17 (forward primer, coding strand) .
  • SEQ ID NO: 58 nucleotide sequence of the human alpha- 2-macroglobulin cDNA.
  • SEQ ID NO: 59 amino acid sequence of the human alpha- 2-macroglobulm protein.
  • SEQ ID NO: 60 partial nucleotide sequence for the fragment of the human alpha-2- macroglobulin cDNA cloned in pYMP30
  • SEQ ID NO: 61 partial nucleotide sequence of the fragment of human cDNA cloned in pYMPll (forward primer, coding strand) .
  • SEQ ID NO: 62 partial nucleotide sequence of the fragment of human cDNA cloned in pYMPll (reverse primer, non-coding strand) .
  • SEQ ID NO: 63 partial nucleotide sequence of the fragment of human cDNA cloned in pYMP12 (forward primer, coding strand) .
  • SEQ ID NO: 64 partial nucleotide sequence of the fragment of human cDNA cloned in pYMP12 (reverse primer, non-coding strand) .
  • SEQ ID NO: 66 nucleotide sequence of a C. elegans I- beta-1, 3-N-acetylaminyltransferase cDNA (F22F7.6) .
  • SEQ ID NO: 68 nucleotide sequence of the C. elegans alpha-2-macroglobulin cDNA ZK337.1a.
  • SEQ ID NO: 69 nucleotide sequence of the C. elegans alpha-2-macroglobuiin cDNA ZK337.1b
  • SEQ ID NO: 72 cDNA sequence for the C. elegans I- beta-1, 3-N-acetylaminyltransferase homologue C18C1.3.
  • SEQ ID NO: 74 cDNA sequence for the C. elegans I- beta-1, 3-N-acetylaminyltransferase homologue K09C8.4.
  • SEQ ID NO: 77 cDNA sequence for the C. elegans I- beta-1, 3-N-acetylaminyltransferase homologue C54C8.2.
  • SEQ ID NO: 81 cDNA sequence for the C. elegans I- beta-1, 3-N-acetylaminyltransferase homologue T15D6.4.
  • SEQ ID NO: 85 amino acid sequence of the membrane proximal part of the C. elegans unc-5 protein .
  • SEQ ID NO: 89 amino acid sequence of the human HS1 protein SEQ ID NO: 89 amino acid sequence of the human HS1 protein .
  • SEQ ID NO: 91 complete nucleotide sequence of plasmid PGC1037.
  • SEQ ID NO: 92 complete nucleotide sequence of plasmid pGC1003.
  • SEQ ID NO: 94 nucleotide sequence of C. elegans unc- 40.

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Abstract

L'invention concerne de nouvelles variantes épissées d'ADNc unc-5c humain et une séquence d'ADNc unc-5HS1 humain. L'invention concerne également des essais basés sur les interactions protéine-protéine entre la protéine UNC-5 et une variété de protéines interactives différentes.
PCT/EP2000/005108 1999-06-01 2000-06-02 Constructions d'unc-5 et methodes de selection WO2000073328A2 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002033080A3 (fr) * 2000-10-16 2002-10-31 Bayer Ag Regulation du recepteur humain membranaire liant la netrine unc5h-1
WO2002010216A3 (fr) * 2000-07-28 2003-05-08 Curagen Corp Nouvelles proteines et acides nucleiques codant pour elles
WO2002029038A3 (fr) * 2000-10-04 2003-07-31 Curagen Corp Nouvelles proteines, acides nucleiques codant ces derniers et anticorps diriges contre ces proteines
EP2267153A1 (fr) * 2009-05-26 2010-12-29 Université Claude Bernard Lyon 1 Identification de la mutation du gène unc5c du récepteur de la nétrine-1 dans des cancers solides

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003037172A2 (fr) * 2001-11-01 2003-05-08 Gpc Biotech Inc. Peptide se fixant aux cellules endotheliales pour diagnostic et therapie

Family Cites Families (2)

* Cited by examiner, † Cited by third party
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US5747262A (en) * 1995-10-16 1998-05-05 The Regents Of The University Of California Neurological drug screens
US5939271A (en) * 1997-02-19 1999-08-17 The Regents Of The University Of California Netrin receptor

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002010216A3 (fr) * 2000-07-28 2003-05-08 Curagen Corp Nouvelles proteines et acides nucleiques codant pour elles
WO2002029038A3 (fr) * 2000-10-04 2003-07-31 Curagen Corp Nouvelles proteines, acides nucleiques codant ces derniers et anticorps diriges contre ces proteines
WO2002033080A3 (fr) * 2000-10-16 2002-10-31 Bayer Ag Regulation du recepteur humain membranaire liant la netrine unc5h-1
EP2267153A1 (fr) * 2009-05-26 2010-12-29 Université Claude Bernard Lyon 1 Identification de la mutation du gène unc5c du récepteur de la nétrine-1 dans des cancers solides

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WO2000073328A3 (fr) 2001-04-12
GB2352448A (en) 2001-01-31

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