WO1999063080A1 - HUMAN HOMOLOGUE OF UNC-53 PROTEIN OF $i(C. ELEGANS) - Google Patents

HUMAN HOMOLOGUE OF UNC-53 PROTEIN OF $i(C. ELEGANS) Download PDF

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Publication number
WO1999063080A1
WO1999063080A1 PCT/EP1999/003848 EP9903848W WO9963080A1 WO 1999063080 A1 WO1999063080 A1 WO 1999063080A1 EP 9903848 W EP9903848 W EP 9903848W WO 9963080 A1 WO9963080 A1 WO 9963080A1
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Prior art keywords
protein
unc
cell
compound
elegans
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PCT/EP1999/003848
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French (fr)
Inventor
Walter Herman Maria Louis Luyten
Marc Carl De Raeymaeker
Johan Jozef Gustave Hendrik Geysen
Thierry A. O. E. Bogaert
Luc Jacques Simon Maertens
Peter Verhasselt
Marc Van De Craen
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Janssen Pharmaceutica N.V.
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Priority to JP2000552275A priority Critical patent/JP2002517194A/en
Priority to CA002330179A priority patent/CA2330179A1/en
Priority to EP99926511A priority patent/EP1092019A1/en
Priority to AU43735/99A priority patent/AU4373599A/en
Publication of WO1999063080A1 publication Critical patent/WO1999063080A1/en

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a vertebrate homologue of UNC-53 protein of C. elecrans and cDNA sequences coding for said homologues or functional equivalents thereof.
  • the invention also relates to processes for identifying compounds which control cell behaviour, compounds identified and pharmaceutical compositions containing them in addition to processes and assays for identifying disease states in which said gene or protein is dysfunctional.
  • control of cell motility, cell shape and directionality of cell outgrowth of axones or other cell outgrowths is an essential feature in the morphogenesis and function of both unicellular and multicellular organisms.
  • a long-range migration of a cell process (also known as a growth cone extension) is a stepwise event, whereby prior to and after each extension there is the formation of a structure at the leading edge of the cell.
  • Localised stabilisation of the actin cytoskeleton and association with plus end regions of microtubules is a general cell biological process underlying the choice of directional extension.
  • the present inventors have surprisingly found a new human gene/protein belonging to the UNC-53 family that binds microtubules and, in particular, the plus- end regions of microtubules.
  • a gene from the free-living nematode is a gene from the free-living nematode
  • hs-unc-53/3 shows homology to the cortical actin binding proteins, and the Ce-UNC-53 protein has been shown to bind F-actin in vitro and leads to actin re-organization in vivo when expressed in mammalian cells, leading to an increased number of filopodia and lammelipodia.
  • Hs-UNC-53-3 may play an important role in the development of various diseases .
  • a vertebrate protein homologue of an UNC-53 protein of C. elegans which protein comprises an amino acid sequence having one or more of sequence blocks A, B, C, D, E, F, G or H as illustrated in figure 4 or which differs from said blocks in conservative amino acid changes.
  • a "derivative" should be taken to mean mutational derivatives, fusions, internal deletions, splice variants and muteins.
  • said vertebrate homologue is a human protein, and preferably a mammalian or a mouse protein.
  • a further aspect of the invention comprises a vertebrate homologue comprising an amino acid sequence as shown in figure 1(f) or the variants thereof or an amino acid sequence which differs from the amino acid sequences shown in figure 1(f) to a significant extent only in one or more conservative amino acid changes.
  • a nucleic acid molecule which is preferably DNA, and which encodes a vertebrate homologue of UNC-53 protein of C. elegans, or a functional equivalent derivative, fragment or bioprecursor of said homologue according to the invention.
  • the cDNA comprises a sequence of nucleotides encoding an amino acid sequence as illustrated in figure 1(f) or the variants thereof or an amino acid which differs from the sequences shown in these figures to a significant extent only in one or more conservative amino acid changes.
  • the DNA is cDNA, which cDNA comprises the sequence shown in figure 1(e) or the variants indicated therein.
  • a nucleic acid sequence capable of hybridising to the nucleic acid or DNA sequences according to the invention under high stringency conditions, which conditions are well known to those skilled in the art.
  • the cDNA according to the invention may be included in an expression vector which may itself be used to transform or transfect a host cell, which cell may be bacterial or eukaryotic in origin including such as, for example an animal or plant cell a fungal cell or an insect cell.
  • a host cell which cell may be bacterial or eukaryotic in origin including such as, for example an animal or plant cell a fungal cell or an insect cell.
  • a range of cells, tissues or organisms may be transfected following incorporation of the selected cDNA clone into an appropriate expression vector.
  • the expression vector according to the invention may comprise a promoter of C. elegans or one of human, mouse or viral origin and optionally a sequence encoding a reporter molecule, such as, for example, green fluorescent protein.
  • the present invention therefore, also further comprises a transgenic cell, tissue or organism comprising a transgene capable of expressing a vertebrate homologue of UNC-53 protein of C. elegans according to the invention.
  • transgene capable of expressing a vertebrate homologue of UNC-53 protein of C. elegans means a suitable nucleic acid sequence which leads to the expression of a vertebrate homologue of UNC-53 protein of C. elegans according to the invention having the same function and/or activity.
  • the transgene may include, for example, genomic nucleic acid isolated from the appropriate vertebrate or synthetic nucleic acid including cDNA.
  • transgenic organisms, tissues or cells, as used herein means any suitable organism and/or part of an organism, tissue or cell, that contains exogenous nucleic acid either stably integrated in the genome or in an extrachromosomal state.
  • the transgenic cell comprises any of, a COS cell, HepG2 cell, MCF-7 or N4 neuroblastoma cell, a NIH3T3 cell, a colorectal or carcinoma cell or a human derived cell such as a fibroblast or the like.
  • the transgenic organism may be an insect, a non-human animal or a plant and preferably C. elegans or a related nematode.
  • the transgene comprises the nucleic acid or cDNA sequence encoding the vertebrate homologue according to the invention as described above.
  • the transgene preferably comprises an expression vector according to the invention.
  • the term "functional fragment” as used herein should be taken to mean a fragment of the gene coding for the vertebrate homologue of the UNC-53 protein of C. elegans according to the invention.
  • the gene may comprise deletions or mutations but may still encode a functional vertebrate homologue of UNC- 53 protein.
  • mutant vertebrate non-human organism having a mutation in the wild-type gene coding for the vertebrate homologue of UNC-53 protein according to the invention, which mutation affects cell behaviour or the regulation of cell motility or the shape or the direction of cell migration or microtubule plus end stability or function and localisation of protein complexes located thereon, which method comprises inducing a mutation in the vertebrate homologue of UNC-53 protein in said organism.
  • mutant organisms may be used in a screen to identify the effects of compounds on these cell functions.
  • the vertebrate homologue of UNC-53 protein of C. elegans or the cDNA or genomic DNA encoding it or a functional equivalent, derivative, fragment or bioprecursor of said homologue may advantageously be used as a medicament, or in the preparation of a medicament to treat or prevent disorders associated with inhibition of overexpression of the vertebrate homologue of UNC -53 according to the invention.
  • disorders may be alleviated by promoting neuronal regeneration, revascularisation or wound healing or the treatment of chronic neurodegenerative disorders, psychiatric disorders or acute traumatic injuries or fibrotic disease or disease in which physiological events requiring the polarity of cells or epithelia are abnormally functioning.
  • the vertebrate homologue according to the invention may advantageously be used to induce or alleviate contact inhibition in a cell or in preventing carcinoma development.
  • the above medical conditions may be treated in mammals and more preferably humans by either the homologue of UNC- 53 protein or alternatively by a nucleic acid coding for the protein or the protein itself according to the invention.
  • an antisense oligonucleotide to said UNC-53 vertebrate homologue may be used to prevent it's expression.
  • nucleic acid sequences which may be used include 3' untranslated regions of mRNA which could be used to prevent transcription of the genomic sequence encoding for the vertebrate homologue of UNC-53 protein according to the invention.
  • the vertebrate homologue of UNC-53 protein according to the invention may be incorporated into a pharmaceutically acceptable composition together with a suitable carrier, diluent or excipient therefor.
  • the pharmaceutical composition may advantageously comprise, additionally or alternatively, the nucleic acid sequence according to the invention as defined above .
  • the induction or inhibition of the expression of hu-UNC-53/3 by pharmacological means may advantageously be used to induce neuronal regeneration, revascularisation or wound healing or be involved in the treatment of chronical neurodegenerative disorders, or acute traumatic injuries or fibrotic diseases, or physiological events requiring the polarity of cells, or oncology and metastasis of cells, or apoptotic pathways.
  • the present invention therefore also provides for a method of determining whether a compound is an inhibitor or enhancer of the regulation of cell behaviour, growth, transformation, cell shape or motility or the direction of cell migration, microtubule plus end stability or function and localisation of protein complexes thereon, which method comprises contacting said compound with a transgenic cell according to the invention and screening for a phenotypic change in said cell.
  • the method can therefore be used to determine whether the compound comprises an inhibitor or an enhancer of the signal transduction pathway of said transgenic cell of which pathway said vertebrate homologue of UNC-53 protein according to the invention is a component, or whether said compound is an inhibitor or an enhancer of a parallel or redundant signal transduction pathway in said cell.
  • the present invention also provides a method to determine that the protein in said signal transduction pathway is a vertebrate homologue of UNC- 53 protein of C. elegans according to the invention.
  • the phenotypic change to be screened comprises a change in cell shape or a change in cell motility.
  • a transgenic cell is used in accordance with one embodiment of the method of the invention, an N4 neuroblastoma cell may be used and in such an embodiment the phenotypic change to be screened may be the length of neurite growth, changes in filopodia outgrowth, changes in ruffling behaviour or cell adhesion, any change in microtubule cytoskeleton, any change in localisation of proteins on plus end regions of microtubules or any change in a cell such as apoptosis.
  • the transgenic cell may comprise an MCF-7 breast carcinoma cell.
  • the phenotypic change to be screened comprises the extent of phagokinesis or filopodia formation.
  • the transgenic cell may comprise an NIH3T3 cell.
  • the phenotypic change to be screened comprises loss of contact inhibition of foci formation.
  • the method according to the invention may also utilise a mutant cell or mutant organism according to the invention as described above, where the mutant cell is capable of growing in tissue culture or in vivo and either of which cell or organism has a mutation in the wild-type unc-53 gene.
  • a "phenotypic change” may comprise any phenotype resulting from changes at any suitable point in the life cycle of the cell, tissue or organism defined above, which change can be attributed to the expression of the transgene of the invention such as for example, growth, viability, morphology, behaviour, movement, cell migration or cell process or growth cone extension of cells and includes changes in body shape, locomotion, chemotaxis, contact inhibition, mating behaviour or the like.
  • the phenotypic change may preferably be monitored directly by visual inspection of the cell as a whole or by monitoring the F-actin cytoskeleton microtubule network and plus end stability of microtubules or proteins thereon or alternatively by for example measuring indicators of viability including endogenous or transgenically introduced histochemical markers or other reporter genes, such as for example ⁇ -galactosidase or green fluorescent protein.
  • a compound which is identifiable by the method according to the invention as described above, as an enhancer of the processes identified above such as the regulation of cell shape or motility or the direction of cell migration may be used as a medicament, or alternatively in the preparation of a medicament, for promoting neuronal regeneration, revascularisation or wound healing, or for treatment of chronic neuro- degenerative diseases or acute traumatic injuries or fibrotic disease.
  • Examples of promoting neuronal regeneration include, for example, peripheral nerve regeneration after trauma and spinal cord trauma.
  • the compound may be used as a medicament, or in the preparation of a medicament, for substantially alleviating spread of disease inducing cells, such as in spread of carcinoma, or the like in metastasis or in alleviating loss of contact inhibition.
  • any of the compounds which may have been identified as an inhibitor or an enhancer in accordance with the method as described above may also be included in a pharmaceutical composition comprising the respective compound and a pharmaceutically acceptable carrier, diluent or excipient therefor.
  • a compound identified as either an inhibitor or an enhancer of the cell motility shape, growth or direction of cell migration or microtubule association or to the plus end region thereof is not limiting.
  • the compound acts as an inhibitor or enhancer of a signal transduction pathway.
  • the compound may also act on a parallel pathway or directly on the vertebrate homologue of UNC-53 protein of C. elegans.
  • the method of action of the compound may include direct interaction with the vertebrate homologue of UNC-53 protein, interaction with processes for regulating phosphorylation or dephosphorylation of the vertebrate homologue of UNC- 53 or with processes regulating activity of an unc-53 gene or with processes for post-transcriptional or post-translational modification or the like.
  • the compound is identified by the method according to the invention as an inhibitor or an enhancer, by utilising differences of phenotype of the cell, tissue or organism, which are visible to the eye.
  • indicators of viability including endogenous or transgenically introduced histochemical markers or a reporter gene may be used.
  • a transgenic cell or tissue culture which has been constructed to comprise a promoter sequence of a gene coding for a vertebrate homologue of UNC-53 of C. elegans according to the invention operably linked to a nucleic acid sequence encoding a reporter molecule.
  • the reporter sequence encodes for a detectable protein, for example one which may be monitored by eye inspection such as antibiotic resistance, ⁇ - galactosidase or a molecule detectable by spectrophotometric, spectrofluorometric, luminescent or radioactive assays.
  • a detectable protein for example one which may be monitored by eye inspection such as antibiotic resistance, ⁇ - galactosidase or a molecule detectable by spectrophotometric, spectrofluorometric, luminescent or radioactive assays.
  • the present invention also provides a method of determining whether a compound is an inhibitor or an enhancer of transcription of a gene coding for a vertebrate homologue of UNC-53 protein in C. elegans, according to the invention which method comprises the steps of:
  • the reporter molecule may comprise messenger RNA.
  • a compound identified as an enhancer of transcription of the gene coding for the vertebrate homologue of UNC-53 protein of C. elegans or a functional equivalent, derivative or bioprecursor of said homologue may also be used as a medicament, or in the preparation of a medicament, for promoting neuronal regeneration, revascularisation or wound healing, or for treatment of chronic neuro- degenerative diseases or acute traumatic injuries or fibrotic disease.
  • such compounds may be included in a pharmaceutical composition including a pharmaceutically acceptable carrier, diluent or excipient therefor. Any compounds identified as inhibitors of transcription may, advantageously, be used in alleviating the spread of disease inducing cells such as carcinomas or metastasis or loss of contact inhibition.
  • the present invention also provides a kit for determining whether a compound is an enhancer or an inhibitor of the regulation of cell growth, transformation, cell motility or shape or the direction of cell migration which kit comprises at least one transgenic or mutant cell or transgenic or mutant non-human organism according to the invention as described above and a plurality of wild-type cells or a wild-type organism of the same type, or a cell line or tissue culture and means for contacting said compound with said cell or organism.
  • kits for determining whether a compound is an inhibitor or an enhancer of transcription of a gene coding for a vertebrate homologue of UNC-53 protein of C. elegans according to the invention which kit comprises at least one transgenic cell or cells according to the invention, means for contacting said compounds with said cells and means for monitoring the level of transcription of said transgenic cell or cells according to the invention.
  • the term "gene coding for a vertebrate homologue of UNC-53 or a functional fragment of said homologue” includes the nucleic acid sequence shown in figure 1 or a fragment thereof, including the differentially spliced isoforms and transcriptional starts of the nucleic acid sequence and which sequence encodes a vertebrate homologue of UNC-53 protein or a functional equivalent, derivative, fragment or bioprecursor of the protein.
  • the present invention also provides methods of identifying genes of vertebrates or fragments of said genes, which encode proteins which are active in the signal transduction pathway of which the vertebrate homologue of UNC-53 according to the present invention is a component.
  • a preferred method comprises hybridizing to an appropriate cDNA library a nucleotide sequence, as defined herein, or a fragment thereof under appropriate conditions of stringency in order to identify genes having statistically significant homology with the cDNA clones of any one of the cDNA sequences according to the invention described above.
  • a method of identifying a protein which is active in the signal transduction pathway of a cell of which a vertebrate homologue of UNC-53 protein of C. elegans according to the invention is a component. According to this aspect of the invention, the method comprises;
  • the vertebrate homologue of UNC-53 protein therefore may bind regions of other proteins involved in the signal transduction pathway. It is also possible to sequentially identify a whole range of proteins involved in the signal transduction pathway.
  • Antibodies to the vertebrate homologue of UNC-53 protein may be produced according to known techniques as would be known to those skilled in the art. For example, polyclonal antibodies may be prepared by inoculating a host animal, such as a mouse, with a protein or epitope of a protein according to the invention and recovering immune serum.
  • This aspect of the invention further comprises a method of identifying a further protein or proteins which are active in the signal transduction pathway of a cell of which the vertebrate homologue of UNC-53 is a component which method comprises:
  • the antibody starts the process by binding to the vertebrate homologue of UNC-53 protein according to the invention in the signal transduction or oncogenic pathways. Any other proteins found complexed to the bound antibody or UNC-53 protein can then be used to identify further interacting proteins involved in the pathway.
  • the method comprises:
  • UNC-53 protein other than the same vertebrate homologue of UNC-53 protein.
  • This method can also advantageously be used to identify further proteins in a signal transduction pathway of a cell by contacting an extract of the cell used as described above, with any protein identified from step (c) above not being a vertebrate homologue of UNC-53 protein and repeating steps (b) and (c) .
  • a western blot overlay method which method is well known to those skilled in the art.
  • Cell extracts are run on gels to separate out protein and subsequently blotted onto a nylon membrane. These membranes may then be incubated, for example in a medium containing vertebrate homologue of UNC-53 having a label attached thereto such as a biotin or radiolabel and any protein conjugates visualised with for example a streptavidin or alkaline phosphatase conjugated antibody.
  • the present invention also advantageously provides a process for the preparation of binding antibodies which recognise proteins or fragments thereof involved in the rate and direction of cell migration or the control of cell growth or shape, for the above methods .
  • the monoclonal antibody for binding to the appropriate vertebrate homologue of UNC-53 may be prepared by known techniques as described by Kohler R. and Milstein C, (1975) Nature 256, 495 to 497.
  • Another method which may be used to identify proteins involved in the signal transduction pathway of a cell of which a vertebrate homologue of an UNC-53 protein of C. elegans according to the invention or is a component, involves investigating protein-protein interactions using the two-hybrid vector method.
  • This technique is based on functional reconstruction in vivo of a transcription factor which activates a reporter gene. More particularly the technique comprises providing an appropriate host cell with a DNA construct comprising a reporter gene under the control of a promoter regulated by a transcription factor having a DNA binding domain and an activating domain, expressing in the host cell a first hybrid DNA sequence encoding a first fusion of a fragment or all of a nucleic acid sequence according to the invention and either said DNA binding domain or said activating domain of the transcription factor, expressing in the host at least one second hybrid DNA sequence, such as a library or the like, encoding putative binding proteins to be investigated together with the DNA binding or activating domain of the transcription factor which is not incorporated in the first fusion; detecting any binding of the proteins to be investigated with a protein according to the invention by detecting for the presence of any reporter gene product in the host cell; optionally isolating second hybrid
  • GAL4 is a transcriptional activator of galactose metabolism in yeast and has a separate domain for binding to activators upstream of the galactose metabolising genes as well as a protein binding domain.
  • Nucleotide vectors may be constructed, one of which comprises the nucleotide residues encoding the DNA binding domain of GAL4.
  • binding domain residues may be fused to a known protein encoding sequence, such as for example a sequence coding for the vertebrate homologue of UNC-53.
  • the other vector comprises the residues encoding the protein binding domain of GAL4.
  • residues are fused to residues encoding a test protein, preferably from the signal transduction pathway of the vertebrate in question. Any interaction between the vertebrate homologue of UNC-53 protein and the protein to be tested leads to transcriptional activation of a reporter molecule in a GAL-4 transcription deficient yeast cell into which the vectors have been transformed.
  • a reporter molecule such as ⁇ -galactosidase is activated upon restoration of transcription of the yeast galactose metabolism genes.
  • Any proteins identified in the signal transduction pathway of the cell which may be for example a mammalian cell, may also be included in a pharmaceutical composition together with a pharmaceutically acceptable carrier, diluent or excipient therefor.
  • the present invention also provides a process for producing a vertebrate homologue of an UNC-53 protein of C. elegans according to the invention which process comprises culturing the cells transformed or transfected with a cDNA expression vector having any of the cDNA sequences according to the invention as described above, and recovering the expressed protein homologue.
  • the cell may advantageously be a bacterial, animal, insect or plant cell.
  • a particularly preferred process for producing said vertebrate homologue of UNC-53 protein uses insect cells.
  • the invention provides a process for producing a vertebrate homologue of UNC-53 protein of C. elegans according to the invention which process comprises culturing an insect cell transformed or transfected with a recombinant Baculovirus vector, said vector comprising a nucleotide sequence encoding said vertebrate homologue of UNC-53 protein according to the invention downstream of the Baculovirus polyhedrin promoter and recovering the expressed protein.
  • this method produces large amounts of protein for recovery.
  • the insect cell may be from for example Spodoptera frugiperda or Drosophila Melanogester .
  • nucleic acid sequence includes not only the identical nucleic acid but also any minor base variations from the natural nucleic acid sequence including in particular, substitutions in bases which result in a synonymous codon (a different codon specifying the same amino acid) , due to the degenerate code in conservative amino acid substitution.
  • nucleic acid sequence also includes the complimentary sequence to any single stranded sequence given which includes the definition above regarding base variations.
  • a defined protein, polypeptide or amino acid sequence according to the invention includes not only the identical amino acid sequence but also minor amino acid variations from the natural amino acid sequence including conservative amino acid replacements (a replacement by an amino acid that is related in its side chains) . Also included are amino acid sequences which vary from the natural amino acid but result in a polypeptide which is immunologically identical or similar to the polypeptide encoded by the naturally occurring sequence. Such polypeptides may be encoded by a corresponding nucleic acid sequence.
  • a further aspect of the invention provides a nucleic acid sequence of at least 15 nucleotides of a nucleic acid according to the invention and preferably from 15 to 50 nucleotides.
  • nucleic acid sequences may, advantageously be used as probes or primers to initiate replication or the like.
  • Such nucleic acid sequences may be produced according to techniques well known in the art, such as by recombinant or synthetic means. They may also be used in diagnostic kits or the like for detecting for the presence of a nucleic acid according to the invention. These test generally comprise contacting the probe with a sample under hybridising conditions and detecting for the presence of any duplex formation between the probe and any nucleic acid in the sample.
  • Nucleic acid sequences according to the invention may also be produced using recombinant or synthetic means such as described in Sambrook et al (Molecular Cloning: A Laboratory Manual, 1989) .
  • human allelic variants or polymorphisms of the DNA according to the invention may be identified by, for example, probing DNA from a range of individuals for example from different populations.
  • nucleic acids and probes according to the invention may be used to sequence genomic DNA from patients using techniques well known in the art, such as the Sanger Dideoxy chain termination method, which may advantageously ascertain any predisposition of a patient to certain disorders.
  • a method of detecting whether a compound is an inhibitor or an enhancer or expression of a vertebrate homologue of UNC-53 of C. elegans, according to the invention comprises contacting a cell expressing said homologue with said compound and monitoring for a phenotypic change compared to a control cell which has not been contacted with said compound.
  • the cell is a transgenic cell as described above.
  • the cell may have undergone loss of contact inhibition.
  • the present method also provides for determining whether said compound is an inhibitor or expression of said vertebrate homologue.
  • the compound to be tested comprises a nucleic acid.
  • nucleic acid sequence comprises an antisense DNA sequence or a mRNA sequence.
  • said mRNA sequence comprises 3' untranslated regions of mRNA encoding for said vertebrate homologue.
  • the compound to be tested may be a protein.
  • said protein comprises a protein having an amino acid sequence potentially suitable for inhibiting function of said vertebrate homologue and preferably comprises a protein identified by the methods as described herein.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound, for example an antisense nucleic acid identified according to the above described method together with a pharmaceutically acceptable carrier, diluent or excipient therefor.
  • a nucleic acid sequence or protein identified according to this aspect of the invention may be used as a medicament, or in the preparation of a medicament, for treating loss of contact inhibition of cancer which is mediated by vertebrate homologue of UNC-53 protein or a functional equivalent, fragment, derivative or bioprecursor of said homologue.
  • nucleic acid as defined above for use in preparation of a medicament for inhibiting expression of a gene coding for a vertebrate homologue of UNC-53 protein of C. elegans.
  • an assay for detecting expression of the vertebrate homologue of UNC-53 protein of C. elegans in a vertebrate cell comprises contacting a cell or an extract thereof with an antibody to said vertebrate homologue, which antibody is fused to a reporter molecule, removing any unbound antibody and monitoring for the presence of said reporter molecule.
  • the reporter molecule is an antibody conjugated to for example a fluorophore such as fluorescein or alternatively to an enzyme such as strepavidin.
  • a method for detecting for expression of a gene coding for the vertebrate homologue of UNC-53 protein of the invention comprises contacting a probe specific for a nucleic acid of protein sequence coding for or corresponding to said vertebrate homologue according to the invention with a cell extract, which probe is linked to a reporter and analysing for the presence of said reporter.
  • the probe is a complementary sequence to a region of mRNA transcribed from said gene encoding said vertebrate homologue of UNC-53 protein according to the invention.
  • the complimentary sequence is a 3' or 5' untranslated region of said mRNA.
  • said reporter may be a dig label, a fluorophore, a hapten or a radiolabel.
  • said probe may comprise an antibody specific for said vertebrate homologue of said UNC-53 protein.
  • the reporter is an antibody conjugated to for example a fluorophore such as fluorescein or alternatively an enzyme such as streptavidin.
  • UNC-53 protein of C. elegans has been found to localise to microtubule and particularly to microtubule (+) ends. Therefore, there is provided by a further aspect of the present invention a method of determining whether a compound is an inhibitor or an enhancer of association of the UNC-53 homologue of the invention to microtubules or plus end regions thereof, which method comprises (a) contacting said compound with a transgenic cell, tissue or organism expressing said vertebrate homologue and which protein is operably linked to a reporter molecule (b) screening for the localisation of said reporter molecule as compared to a cell according to step (a) which has not been contacted with said compound.
  • a compound identifiable by the above method also forms part of the present invention.
  • Such a compound identified as an inhibitor of localisation or association of said vertebrate homologue with microtubules or the plus end region thereof may be used in alleviating the spread of disease inducing cells or metastasis or loss of contact inhibition.
  • a compound identified as an enhancer of association of said vertebrate homologue with microtubules or the plus end region thereof may be used in for example promoting neuronal regeneration, revascularisation or wound healing, or for treating chronic neurodegenerative diseases or acute traumatic injuries or fibrotic disease.
  • These compounds may then be included in a pharmaceutical composition, together with a pharmaceutically acceptable carrier, diluent or excipient therefor.
  • kits for determining whether a compound is an inhibitor or an enhancer of association of the vertebrate homologue thereof according to the invention with microtubules or the plus end regions thereof which kit comprises at least one transgenic cell expressing said UNC-53 vertebrate protein homologue and a reporter molecule or a host or transgenic cell according to the invention and at least one cell of the same cell type for use as a control and means for contacting said compound with one of said at least one transgenic cells.
  • Compounds identified as inhibitors or enhancers or microtubule association described above may advantageously be included in a composition and linked to said vertebrate homologue according to the invention to target the compounds to the microtubules or the plus end regions thereof.
  • Such a composition may also comprise, for example, a suitable transfecting or transformation agent.
  • a method of targeting a protein to a cell microtubule or the plus end region thereof comprises introducing into a host cell, tissue or organism a transgene comprising a sequence capable of expressing said UNC-53 vertebrate homologue according to the invention, which sequence is operably linked to a sequence encoding said protein to be targeted such that a chimeric protein is expressed and which results in targeting of said protein to said microtubule or a plus end region thereof.
  • An even further aspect of the invention comprises a method of identifying a molecule which covalently modifies UNC- said vertebrate homologue according to the invention, which method comprises a) contacting either an extract from a cell or cells expressing said vertebrate homologue or a mixture of enzymes comprising candidate UNC-53 modifying enzymes in the presence of an indicator of covalent modification of a protein, b) identifying any covalently modified UNC-53 protein from step a) and c) identifying said molecule involved in said modification step.
  • an indicator may be 32 P.
  • the invention is a method of identifying a compound which alleviates or enhances the toxicity of said UNC-53 vertebrate homologue thereof according to the invention, or which alleviates or enhances apoptosis.
  • the method of the former comprises contacting said compound with a transgenic cell, tissue or organism according to the invention and monitoring for the presence of said reporter molecule adjacent said microtubules or the plus end region thereof.
  • the method comprises monitoring the effect of the compound on cell death.
  • Figure 1(a) is an illustration of the nucleotide sequence encoding the first human homologue of UNC-53 designated Hs-UNC-53/1 and further variants thereof.
  • Figure 1 (b) is an illustration of the amino acid sequence of hs-UNC-53/1 encoded by the sequences in Figure 1 (a) .
  • Figure 1(c) is an illustration of the nucleotide sequence encoding the second human homologue of UNC-53 protein of C. elegans designated Hs-UNC-53/2 and further variants thereof.
  • Figure 1 (d) is an illustration of the amino acid sequences of Hs-UNC-53/2 encoded by the sequences in Figure 1(c).
  • Figure 1 (e) is an illustration of a nucleotide sequence encoding the third human homologue of UNC-53 protein according to the invention designated Hs-UNC- 53/3, and variants thereof.
  • Figure 1(f) is an illustration of the amino acid sequences of the Hs-UNC-53/3 encoded by the sequences of Figure 1 (e) .
  • Figure 1(g) is an illustration of the nucleotide sequence of a genomic DNA fragment that contains a putative 5' exon of Hs-unc-53/1.
  • Figure 1 (h) is an illustration of the nucleotide sequence AB023155 encoding the protein KIAA0938, a transcript comprising the 3' half of Hs-unc-53/3.
  • Figure l(i) is an overview of the C. elegans and human UNC-53 proteins as cloned. The 5' truncated variants and a number of the known splice variants have been indicated.
  • Figure 2 is an alignment of the amino acid sequences of Ce-UNC-53, Hs-UNC-53/1 , Hs-UNC-53/2 and Hs-UNC-53/3.
  • Figure 3 is an alignment of the C. elegans unc-53 and the predicted amino acid sequence of C. briggsiae unc-53.
  • Figure 4 is a list of ProSite signatures for vertebrate UNC-53s based on the sequence alignment.
  • Figure 5a is an illustration of expression of the three human UNC-53s as studied by Northern blotting.
  • Figure 5(b) is an illustration of differential expression of Hs-unc-53/3 in different brain parts.
  • Figure 6(a) is an illustration of differential splice variant expression of Hs-unc-53/1 using RT-PCR.
  • Figure 6(b) is an illustration of differential splice expression of Hs-unc-53/2 using RT-PCR.
  • Figure 6(c) is an illustration of differential expression of Hs-unc-53/3 using RT-PCR.
  • Figure 6(d) is a sequence confirmation of AB023155 expression in cells other than brain using RT-PCR.
  • Figure 7(a) is an illustration of the cloning of Hs-unc-53/3.
  • Figure 7 (b) is a plasmid map and the nucleotide sequence of the pGI3303 expression vector ( C-terminal Hs-unc-53/3 fragment in fusion with GFP) .
  • Figure 7(c) is an illustration of the amino acid sequence of GFP: C-terminal Hs-unc-53/3 fragment (insert of pGI3303) .
  • Figure 7 (d) is a plasmid map and the nucleotide sequence of the pGI3305 expression vector (full length Hs-unc-53/3 in fusion with GFP) .
  • Figure 7 (e) is an illustration of the amino acid sequence of GFP : Hs-unc-53/3 (insert of pGI3305) .
  • Figue 8 is an illustration of the filipodia and lamellipodia outgrowth of N4 mouse neuroblastoma cells transfected with pGI3303 (F-actin cytoskeleton reorganisation)
  • Figure 9 is an illustration of the co- localisation of the GFP:Hs-unc-53/3 fusion protein with microtubules in N4 mouse neuroblastoma cells transfected with pGI3305.
  • Figure 11a is an illustration of the homology domains between Hs-unc-53/3 and a gene encoded (partially) by the Drosophilia melanogaster BAC clone BACR48M05 (AC005719) . Results of a TBLASTN search on the non-redundant database with Hs-unc-53/3 as query.
  • Figure lib is an illustration of an ORF encoded by the Drosophila melanogaster BAC clone BACR48M05 (AC005719) as predicted by the computer program Fgene.
  • Figure lie is an illustration of a "BLAST 2 sequences" search result with Hs-unc-53/3 as query and the Fgene predicted UNC53 homology ORF of D. melanogaster BAC clone BACR48M05.
  • Figure 12 is an illustration of a zebra fish EST encoding Dr-unc-53/2.
  • Figure 14 is a schematical drawing of the sequence of the exon containing the putative alternative start codon of human Hs-unc-53/1.
  • Figure 15 is an illustration of the nucleotide sequence of pGI3150 and the amino acid sequence of the eGFP fusion with a C-terminal fragment of Hs-Unc-53/1.
  • Figure 16 is an alignment of EST clone yk480b6 and Ce-unc-53 demonstrating a novel splice variant of Ce-unc-53.
  • Figure 17 is a graphical display of the effect of Hs-unc-53/3 GFP chimera transient transfection on the form factor of N4 cells.
  • Plasmids pG13303 and pG13305 were deposited under accession numbers LMBP3936 and LMBP3937 respectively on 28 May 1999 at the Belgian Coordinated Collections of Microorganisms (BCCM) at Laboratorium voor Moleisme Biologie - Plasmidencollective (LMBP) B- 9000 Ghent, Belgium, in accordance with the provisions of the Budapest Treaty of April 28 1977.
  • Hs-UNC-53/3 is a bona fide UNC-53 (fig. 1; 2; 3)
  • Hu-UNC- 53/3 most likely has the key functionalities observed for Ce-UNC-53 in a variety of assays including F-actin binding, F-actin reorganisation in cell culture, microtubule and microtubule (+)-end binding in cultured cells, binding of SH3-domain adapters like
  • the C. elegans genome consortium sequenced the locus of the C. briggsiae unc-53 homologous gene. Through gene prediction programs and the cDNA sequence of the C. elegans unc-53, prediction can be made for the C. briggsiae protein sequence. Alignment of the derived C. briggsiae amino acid sequence with the C. elegans amino acid sequence in figure 3 demonstrates the strong homology of both proteins.
  • BAC clone BACR48M05 (AC005719) clearly contains 3 different exons with high homology to Hs-unc-53/3 ( Figure 11) .
  • Fgene Solovyev et al., 1995, in: Proceedings of the Third International Conference on Intelligent Systems for Molecular Biology (eds. Rawling et al., Cambridge, England, AAAI Press) ; Solovyev and
  • the predicted ORF of the Drosophila melanogaster UNC53 gene can be used to identify new members of the family.
  • the zebrafish EST fc21d06 shows an identity of 84% and a homology of 92% to Hs-UNC-53/2. It clearly can be considered as a part of the zebrafish homologue of Hs- UNC-53/2 ( Figure 12) .
  • a whole series of human ESTs have been placed in public domain databases. To our knowledge, no one has been able to place these ESTs into contigs that describe a true Hs- unc-53 to a level presented in this specification.
  • the presently available unc-53 sequences - expressed or genomic - further underscore that the unc-53 gene family is a true animal gene family in helminths, vertebrates and arthropods, three major classes of the animal kingdom.
  • a Northern blot of poly-A+RNA from several cancer cell lines was probed using the whole insert of pHH3b. No or weak expression was detected in the Burkitt Lymphoma DRajii, the Leukemia Molt4 and the Promyelocytic Leukemia HL60 cell lines.
  • transcripts 1 and 2 are larger than 9.5kb
  • transcripts 3 and 4 are 6 to 7 kb
  • the fifth transcript is around 6 kb.
  • Transcripts 1 and 2 are present in all expressing cell lines but at different levels.
  • Transcripts 3 and 4 are restricted to Melanoma G361, Lung Cancer A549 (weak) and Colorectal Adenocarcinoma SW480 and are the predominant transcripts in Melanoma G361 and Colorectal Adenocarcinoma SW480.
  • Transcript 5 is restricted to Lymphoblastic Leukemia K562 (weak) and (predominant) in HeLa S3 and is predominant in HeLa S3.
  • Hs-UNC53/2 Normal Human tissue probed with Hs-UNC53/2.
  • a similar set of blots were probed with a 652bp fragment of EST46037 amplified by using the primers 5' aggagatgaagctgacagatatcc and 5'- aaacaccagtgagtcc.
  • Expression levels are low in all tissues with the highest level in kidney, placenta and pancreas, lower levels in heart and lung. Expression is barely detectable or undetectable in skeletal muscle, spleen, thymus, prostate, testis, ovary, small intestine, colon peripheral blood leucocyte, stomach, thyroid, spinal cord, trachea, adrenal gland and bone marrow.
  • Hs-unc-53/2 appears to be expressed as different transcripts (figure 5a).
  • hs-UNC53/l and hs-UNC-53/2 homologues are clearly highly regulated genes, showing a strong tissue specificity and, probably, additional mechanisms of regulation (ie differential splicing of different promoters) .
  • the different proteins derived from RNA' s identified by probe hhl5 presumably share the carboxyterminal nucleotide binding domain.
  • Ce-UNC-53 was shown to be a complex genetic locus and complex transcription unit.
  • the different transcripts are thought to be a mechanism to assure the necessary specificity and functional diversity of this signal transduction pathway, with respect to different signals and receptors, different tissues and different directions of migration.
  • the occurrence of a new transcript or the observed changes in expression levels in the cancer cell line blot suggests a role for hs-UNC-53/3 in the establishment or maintenance of the transformed state of those cells. Expression pattern of hs-UNC-53/3.
  • Hs-unc-53/3 has a high expression level in lung carcinoma line A549, where only a moderate expression of hs-unc-53/1 has been detected. Furthermore, moderate expression of Hs-unc-53/3 was also observed in melanoma line G361, where previously, a high expression of hs-UNC-53/1 and hs-UNC-52/2 has been observed. This indicated the involvement of hs-unc53/3 in at least two cancer lines .
  • Hs-unc-53/3 shows a clearly new and previously unobserved expression pattern. This difference of expression of hs-unc-53/3 in relation to its homologues hs-unc53/l and hs-unc53/2 is important for the allocation of functionality to hs-unc-53/3.
  • Hs-unc-53/3 is highly expressed in brain, as shown on the Northern blots (figure 5a) .
  • FIG 5b it can be seen that Hs-unc-53/3 also is differentially expressed in different parts of the brain. Its homologues are not or weakly expressed in brain. This gives an indication that its function in directionality of cell migration and growth cone steering will be in relation to specific regions or cells of the brain.
  • Hs-unc-53/3 will be an important signal transducer or signal adapter linking signals to neuronal outgrowth, axon guidance, and formation and maintenance of synaptic connections. It seems that the function of Hs-unc- 53/3 will be associated with neuron-neuron interactions, neuronal outgrowth, neuron muscle interactions, and post-synaptic signal transduction. Furthermore, Hs-unc-53/3 may be involved in the development of cancer of neuronal origin, like neuroblastomas, or the development of tumours will have their developmental origin in the brain as some eyes diseases like retinoblastomas .
  • Hs- unc-53/3 in brain tissue can be associated with the high levels of expression which has also been observed in the spinal cord, containing neuronal tissue.
  • neuronal (axon) outgrowth and neuron-neuron connections are of importance.
  • Development of pharmacological tools acting on this pathway may lead to treatments of diseases involved in the growth and movement of neuronal cells, and the regeneration of neuronal connectivity after trauma, or the inhibition of neuronal cancers such as neuroblastomas.
  • Due to its specific expression, inhibitors and/or enhancers specific for Hs-unc-53/3 will have an advantage as a pharmaceutical compound over more general compounds acting on the Hs-unc-53 family of genes and proteins.
  • Hs-UNC-53/3 could therefore function as part of the signal transductions pathway involved in the maturation of leukocytes. Malfunction of this pathway may lead to incorrect maturation of the leukocytes and the development of autoimmune diseases such as rheumatoid arthritis and sclerosis.
  • Hs-UNC-53/3 may also play an important role in the induction and/or signalling pathway of the mechanism underlying apoptosis of leukocytes in the spleen.
  • hs-UNC-53/3 pathway Pharmaceutical methods involving the hs-UNC-53/3 pathway, which may, for example, result in an inhibition and/or enhancement of its expression may lead to treatment of these disorders.
  • hs-UNC-52/2 may have an advantage, as an inhibitor or enhancer specific for hu-unc53/3 which will act in a more specific manner.
  • the Hu-UNC-53/3 protein is also highly expressed in the ovary, where the two other human homologues are also expressed.
  • moderate to low expression of hs-unc53/3 is observed in heart, placenta, testis, stomach and adrenal gland.
  • Hs-unc- 53/3 Although the predominant transcripts of Hs-unc- 53/3 are > 9 kb, often a smear occurs that ends at with somewhat higher intensity at 5.5 - 6.5 kB. This short transcript may correspond to AB023155.
  • the Hs-unc53/3 gene is a highly regulated gene, showing strong tissue specificity and additional mechanisms of regulation which have not previously been identified in any of its known homologues. These findings may thus lead to the development of more specific inhibitors or enhancers of hs-UNC-35/3 and or of the Hs-UNC-53/3 pathway.
  • the Northern blot studies indicate that the three human unc-53s are complex transcriptional units with highly regulated tissue specificity and that transcripts of different lengths exist.
  • the second variant in Hs-unc-53/3 (fig. 2) deletes a box (MQLDNRTLPKKGLR) , which is extremely conserved (in bold) among all human unc-53s. This occurrence of this variant could indicate differentially active functional variants of Hu- unc53/3.
  • Hs-unc-53/1 has a first variant that comprises the most N-terminal portion of this conserved domain (SGSFRD) .
  • a second splice variant in Hs-unc-53/1 (AEERMOSE) lies within the highly conserved domain.
  • Another conserved spot for splice variation in human unc-53s has been found (figure 2): Hs-unc-53/1 ⁇ VYE ⁇ ; -/2 ⁇ VNE ⁇ and -/3 ⁇ NSRGSEL ⁇ . All these spliced exons are flanked by two conserved charged domains - putative nuclear localisation signals.
  • Hu-unc-53/3 has another variant (SPLVWPPKKRQNGPVIYKHSR) (fig. 2) .
  • SPLVWPPKKRQNGPVIYKHSR SPLVWPPKKRQNGPVIYKHSR
  • the RT-PCR study for Hs-unc-53/1 revealed that the most 5' -splice variant is differentially expressed in normal versus neoplastic cells/cell lines. This exon is present in 7/7 keratinocytes, HUVEC and in melanocytes but lacking in HaCat, ECV304, 2/7 melanoma and MCF-7 cells (breast carcinoma) .
  • tumourigenic endothelial line ECV304 lacks expression of Hs-unc-53/2, whereas their normal counterpart HUVEC expresses Hs-unc-53/2, suggesting gene deletion or inactivation of expression in ECV304.
  • epidermal keratinocytes and the lineage matched spontaneously transformed keratinocyte HaCaT and MCF-7 lack expression of the 5' -end of Hs-unc- 53/2, but express the 3' end (starting in or near the microtubule-binding domain) .
  • Hs-unc-53/2 can be expressed as a truncated 3' -variant in a cell-specific way.
  • splice variation of Hs-unc-53/2 appears to differ in a normal to neoplastic way: the ⁇ VNE ⁇ exon was shown to be present in all keratinocyte isolates but not in HaCaT and also melanocytes express it, but not 2/7 melanoma or MCF-7.
  • the RT-PCR studies for Hs- unc-53/3 were focussed on demonstrating expression of AB023155 in tissues other than brain. The new exon described was shown to be present in keratinocytes, HUVEC, dermal fibroblasts, melanocytes and their transformed/neoplastic variants, demonstrating its wide expression in tissues in man.
  • AA918601, AI248585, AA115014 and AA115015 are clearly homologous to the 3'-UTR of Hs- Unc-53/2 cDNA ( Figure 1(c))).
  • AA115014 (describing the same EST as AA115015) contains an alternative splice variant of the Hs-Unc53/2 gene in the 3'UTR.
  • Hs-unc-53/3 can be classified as a bona fide human unc-53. To further understand its function and in anticipation of developing pharmacological compound screening assays, Hs-unc-53/3 has been physically cloned following the method described in the experimental section and shown in figure 7a.
  • the derived Hs-unc-53/3 clones comprising full length (A to L and the 3' -half (G to L) of Hs-unc-53/3 were further engineered to form a chimera with green fluorescent protein and cloned into expression vectors appropriate for transfection of eukaryotic cells.
  • the nucleic acid and amino acid sequences of these constructs are shown in figure 7b- e.
  • the constructs were transfected into cells and scored for their effects on the F-actin cytoskeleton and binding to microtubules of mouse neuroblastoma cells N4; functions known for nematode unc-53 and human unc-53/1.
  • N4 cell transfected with a GFP fusion to the 3' -half of Hs-unc-53/3 showed pronounced filopodia and lamellipodia outgrowth, which is associated with reorganization of the F-actin cytoskeleton ( Figure 8).
  • This observation demonstrates that like nematode unc-53 and human unc- 53/1, the F-actin binding domain is not required for inducing reorganization of the F-actin cytoskeleton of N4 cells.
  • the pGI3303 encoded fusion protein does not co-localize with microtubuli but localizes to the cytoplasm of N4 cells indicating that an important domain for microtubuli association is missing in this C-terminal fragment of Hs-unc-53/3.
  • the C- terminal half of Hs-unc-53/3 (approximate KIAA0938) does not comprise the conserved microtubule binding domain.
  • N4 cells that expressed low to medium levels of the GFP fusion to full length Hs-unc- 53/3 displayed a co-localization of the GFP fusion protein with microtubules ( Figure 9) . Even the centrosomes could clearly be detected in some transfected cells. Cells expressing very low amounts of the fusion protein displayed specific microtubule (+)-end binding ( Figure 9).
  • the morphology of the pGI3305 transfected N4 cells does not clearly differ from the control transfected cells although there is a tendency towards rounding up of the pGI3305 transfected cells and filopodia outgrowth.
  • R74288 has previously been shown to be an inhibitor of nematode function in C. elegans (W096/38555) , an activity that has been confirmed in Ce-unc-53 transfected N4 cells, where only the transgene-induced effect was inhibited by R74288.
  • a stable transfection of plasmid pGI3150 was performed in the N4 neuroblastoma cell line with the lipofectamin procedure (Gibco BRL) .
  • pGI3150 expresses an eGFP protein in fusion with the C-terminal end of Hs-unc-53/1 (see Figure 15a) .
  • Compound screening assays must have a sufficiently high throughput to be relevant to drug discovery. To achieve this goal, we automated the procedure of measuring the morphological changes induced in cells following transient transfection with full length or 3' -half of Hs-unc-53/3 GFP chimeras.
  • the cell culture, transfection, fluorescence staining and microscopy procedures are performed within a 96- well plate (all-in-one) .
  • the fluorescent staining method comprises a triple fluorescent labeling procedure (1) for cell nucleic using DNA double helix intercalating dyes such as Hoechst 33342 or DAPI, (2) for transfection efficiency and expression level of the chimeric protein using GFP fluorescence and (3) for the F-actin cytoskeleton using fluorescently labeled phalloidin, a microfilament dye.
  • Chromosome 12q21.1 is a region shown to be involved in autosomal dominant, cornea plana and closed angle glaucoma (Sigler- Villanueva et al., Ophthalmic Genetics 18:55-62, 1997) . This indicates that hs-UNC-53/3 protein may be involved in eye development and thus eye diseases, such as retinoblastomas . Neuroblastoma cell line NPG and liposarcoma line WDLPS and other sarcoma lines have amplifications in this region.
  • the neuroblastoma amplification seems to be located more distal (12q24) while the liposarcoma line is located at 12q21 (Van Royal et al . , Cancer Genetics and Cytogenetics 82:151- 4, 1995).
  • Three loci related to Darier' s disease, an autosomal dominant genodermatosis disease characterized by epidermal acantholysis and dyskeratosis have been mapped in region 12q21-q24 (Wright et al . , Journal of Investigative Dermatology 103:665-8). 12q21 is also known to be a fragile site associated with the pathogenesis of non-Hodgkin' s Lymphoma (Chary-Reddy et al .
  • RH mapping is a somatic cell hybrid technique that was developed to construct high-resolution, contiguous maps of mammalian chromosomes.
  • RH mapping provides a method for ordering DNA markers spanning millions of base pairs of DNA at a resolution to easily obtained by other mapping methods.
  • sequence information available in public domain can help refine the positioning of the unc-53 genes, like in the following example.
  • the EST clones AA918601, AI248585, AA115014 and AA115015 are clearly homologous to Hs-Unc53/2 cDNA.
  • AA115014 (describing the same EST as AA115015) contains an alternative splicevariant of the Hs-Unc53/2 gene in the 3'UTR.
  • the STS was localized by analysis on the NIGMS human/rodent somatic cell hybrid panel (dbSTS id: 41891) .
  • the radiation hybrid results are summarized in Figure 13. Together these data imply that diseases or phenotypes connected to SHGC-33456 is due to the Hs-Unc53/2 gene.
  • Hs-unc53/3 has been cloned starting from a series of ESTs that were similar but not identical to Hs-unc- 53/1 or -/2.
  • the ESTs were:
  • Transformed cells carrying the EST 767735 sequence were ordered from Research Genetics. Plasmid DNA was isolated using standard protocols (Qiagen plasmid DNA isolation kit) , the sequence of the insert was determined.
  • Transformed cells carrying the cDNA clone 86459 sequence were ordered from ATCC. Plasmid DNA was isolated using standard protocols (Qiagen plasmid DNA isolation kit) , the sequence of the insert was determined. 3. Genethon cDNA clone c09a03 from the Geneexpress cDNA program.
  • Transformed cells carrying the cDNA clone c09a03 sequence were ordered from Genethon. Plasmid DNA was isolated using standard protocols (Qiagen plasmid DNA isolation kit) , the sequence of the insert was determined.
  • Marathon-Ready cDNAs (Clontech) are premade "libraries" of adaptor-ligated double-stranded cDNA ready for use as templates in RACE experiments.
  • Five ml Marathon-Ready cDNA was used as template in a regular 50 ml RACE.
  • the RACE mixture contained 1 x KlenTaq PCR buffer. 0.2 mM of each dNTP, 1 x advantage KlenTaq polymerase mix (Clontech), 0.15 mM API adaptor primer and 0.15 mM RACE gene specific primer.
  • the amplification conditions were as follows: 94°C for 30 s and 68 °C for 4 min.
  • Gene specific primer (hh3UNC53 97102702) 5'CAATCGTCTTCAGGTGTAAATGGTAACGTG; nested gene specific primer (hh3UNC53 97102703)
  • gene-specific primer (hh3UNC53 98020401) 5'AGGGAGCACTGAATGGTCCAGACCATCCTC; nested gene-specific primer (hh3UNC53 98020402)
  • 5'GCATCAGAAGACAGCATTCCTCTGAAAGTG Marathon cDNA library human placenta, human heart, human HeLa, human melanoma, human colorectal adenocarcinoma, human chronic myelogenous leukemia.
  • 5'GGATGCCGGAAGGGATGAATCAGTAAGC Marathon cDNA library: human placenta, human heart, human HeLa, human melanoma, human colorectal adenocarcinoma, human chronic myelogenous leukemia.
  • BAC DNA was prepared according to a modified Qiagen plasmid DNA procedure.
  • a primer pair was designed specifically to amplify the 5' end of the variant shown in full in figure le (primers ACTTGCTGAAACAGAGAGCTCCATG and CTTGCTGTCTTCTTTCTCCTTGGC) .
  • PCR with these primers on BAC DNA showed the presence of the genomic sequence encoding this variant in 3 out of the 4 BACs (not present in BAC 415J11) .
  • BACs containing the genomic sequence encoding the other 5' end variant of Hs-unc-53/3 as shown as the variant in figure le were identified by hybridizing the Research Genetics human DNA GAC filters with primer TGATCTTCTAGCGTGTGACTCACTG, radioactively labeled using gamma-P32-ATP and polynucleotide kinase. Positive BACs were 404F14, 450K18 and 764L15.
  • primer pairs were selected such that the ORF could be amplified in 6 overlapping fragments ranging in size from 1 to 2 kbp. Overlaps between the fragments were chosen such that they contain an endonuclease restriction enzyme recognition site suitable for cloning the full-length gen.
  • the downstream oriented primer was chosen to contain the first putative start codon (ATG) in variant 1 (the one shown in full in figure le) .
  • A-B 2229 A TCAGCTCGAGCATATGCCTGTTCTTGGGGTTGC B GGGGTGGGTCGACTTGTCAAGTGG
  • E-F 781 E CAGGTTCCTGGAGAAGAGGCATGTC
  • Primer A contains restriction sites (Xhol and nhel) suitable for final subcloning in an eukaryotic expression vector (pEGFPc3) and in a yeast-two-hybrid vector (pAS2-l), respectively.
  • PCR products were analyzed by agarose gel electrophoresis and were visualized by ethidium bromide staining. Splice variants as mentioned in figure le were observed as multiple bands on agarose gels. Single band PCR products were purified with the Qiaquick PCR purification kit, whereas multiple band PCR products were cut out from gel as individual bands and purified using the Qiaquick gel extraction kit. PCR products were cloned in pCR2.1 according to the suppliers protocol (Invitrogen) .
  • a clone was selected that missed the heart specific 22 amino acid splice variant (figure If) .
  • a Sfil-SacII linker was cloned in the BamHI site of the pCR2.1 multiple cloning site to facilitate subcloning of the full- length gene into the yeast-two-hybrid vector (pAS2-l) and the eukaryotic expression vector (pEGFPc3) , respectively.
  • the overall cloning strategy of the full-length gene is visualized in figure 7a. 7al illustrates the overlapping PCR fragments and the nomenclature of fragments and primer pairs.
  • FIG. 7a2 illustrates the assembly of the 3' half of the gene in pCR2.1.
  • Internal BamHI (I-J fragment) and Xhol (K-L fragment) sites as well as restriction sites from the multiple cloning site of pCR2.1 (as shown in the figure) were removed by side-directed mutagenesis (SDM) using the Quickchange Site-Directed mutagenesis kit (stratagene) .
  • SDM side-directed mutagenesis
  • the Notl-EcoRI G-H fragment and the EcoRI-Nhel I-Jd22 (d22 indicating that the 22 amino acid splice variant is absent) were directionally cloned in the Notl and Nhel sites of the K-L fragment clone. Multiple clones were picked and verified by DNA sequencing.
  • FIG. 7a3 illustrates the assembly of the 5' half.
  • Internal Xhol (C-D fragment) and Sfil and Xhol (E-F fragment) sites were removed by SDM. Inserts were cut out from the vectors by restriction digestion with the appropriate restriction enzymes (Xhol+Sall; Sall+Narl and Narl+BamHI, respectively) and purified from gel after agarose gel electrophoresis. The 3 fragments were ligated together, re-cut with Xhol and BamHI and separated on gel. The band of the expected size was cut out of gel, purified and cloned in front of the 3' half, opened by digestion with Xhol and BamHI (figure 7a4) . Multiple clones were picked and verified by sequencing.
  • Figure 7a illustrates the modular nature of the cloning project. For all the possible combinations of splice variation within the building block fragments, one representative clone is available. In view of functional analysis, building blocks can be exchanged easily by standard technology, either in the pCR2.1 construct or in the final eukaryotic expression or yeast-two-hybrid construct.
  • pG13303 can be used to over-express in mammalian cells or animals a fusion protein between eGFP and 1128 AA C-terminal fragment of Hs-unc-53/3 (Fig 7c) .
  • pG3305 can be used to overexpress in mammalian cells or animals a fusion protein between eGFP and the 2363 AA full length Hu- unc-53/3 (fig 7d) .
  • the Hs-unc-53/3 cDNA in pGI3303 as well as in pGI3305 contains silent mutations that introduce or remove specific restriction sites in order to be able to easily subclone different types of alternative splice variants in these vectors.
  • Genomic DNA sequencing (BAC 585E09)
  • BAC DNA was isolated from E. coli genomic clone 585E09 according to a modified Qiagen plasmid DNA preparation procedure. A shotgun library of 1920 clones was constructed at GATC (Konstanz, Germany) .
  • BAC DNA was prepared, nebulized and subcloned after end-repairing in the sequence vector pTZ19R.
  • DNA was prepared on the Biorobot 9600 (Qiagen) from 1440 clones. End sequencing reactions with Ml3 forward (TGTAAAACGACGGCCAGT) and reverse (CAGGAAACAGCTATGACC) primer were done on 768 clones. 672 additional clones were sequenced with M13 only. 5 ⁇ l DNA was used in 15 ⁇ l final reaction volume using the BigDye Terminator Ready Reaction sequencing kit. Sequencing reactions were run on MJ Research PTC200 PCR machines. Reaction products were run and analysed on PE ABI 377 DNA sequencers.
  • a Human multiple tissue Norther (MTN-1, Clontech) containing in each lane 2 mg of poly A + RNA from eight different human tissues (heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas) and a MTN-II human multiple tissue Northern, containing in each lane 2 mg of poly A + RNA from spleen, thymus, prostate, testis, ovary, small intestine, colon and peripheral leukocyte, were hybridized according to the manufacturer's instructions and washed out in 0. lxSSC: 0.2% SDS at 55°C.
  • MTN-1 Human multiple tissue Norther
  • RNA blot from human cancer cell lines (melanoma G361, lung carcinoma A549, colorectal adenocarcinoma SW480, Burkitt' s lymphoma Raji Leukemia Molt 4, lymphoblastic leukemia K562, HeLa S3 and promyelocytic leukemia HL60) was tested.
  • a set of cancer cell line Northern blots were probed with a 665 bp fragment of Hs-unc-53/3 amplified by using the primers 5' AGGAATTAAAATTAACGGATATTCGG and 5'AAAACTGTCCAAACTATTTTCTTCTACC.
  • HU-unc-53/3 is expressed in Melanoma G361 and lung carcinoma A549, transcripts sizes were detected of >0.5 kb.
  • No expression was detected in promyelocytic leukemia HL- 60 HeLa cell S3, chronic myelogenous leukemia K-562, leukemia MOLT-4, Burkitt' s lymphoma Raij and colorectal adenocarcinoma SW480.
  • a set of normal human tissue Northern blots were probed with a 665 bp fragment of Hs-unc-53/3 amplified by using the primers 5' AGGAATTAAAATTAACGGATATTCGG and 5' AAAACTGTCCAAACTATTTTCTTCTACC .
  • Hs-UNC-53/3 is localised to chromosome 12q21.1
  • Lymphocytes isolated from human blood were cultured in -minimal essential medium (MEM) supplemented with 10% foetal calf serum and phytohaemagglutinin (PHA) at 37°C for 68-72 hr.
  • the lymphocyte cultures were treated with BrdU (0.18mg/ml Sigma) to synchronise the cell population.
  • the synchronised cells were washed three times with serum- free medium to release the block and recultured at 37°C for 6 hr in a ⁇ -MEM with thymidine (2.5 ⁇ g/ml: Sigma) .
  • Cells were harvested and slides were made by using standard procedures including hypotonic treatment fix and air-dry.
  • a cDNA probe was biotinylated with dATP using the BRL BioNick labelling kit (15°C, 1 hr) Heng et al, 1992) .
  • the procedure for FISH detection was performed according to Heng et al . , 1992 & Heng and Tsui, 1993. Heng et al..: Proc Natl Acad Sci USA 89: 9509-9513 (1992). Heng et al . Chromosoma 102: 325-332 (1993). Briefly, slides were baked at 55°C for 1 hour. After RNase treatment, the slides were denatured in 70% formamide in 2xSSC for 2 min. at 70°C followed by dehydrated with ethanol.
  • Probes were denatured at 75°C for 5 min. in a hybridisation mix consisting of 50% formamide and 10% dextran sulphate. Probes were loaded on the denatured chromosomal slides. After over night hybridisation, slides were washed and detected as well as amplified. FISH signals and the DAPI banding pattern were recorded separately by taking photographs, and the assignment of the FISH mapping data with chromosomal bands was achieved by superimposing FISH signals with DAPI banded chromosomes (Heng et al, 1993) .
  • the hybridisation efficiency was approximately 67% for this probe (among 100 checked mitotic figures, 67 of them showed signals on one pair of the chromosomes) . Since the DAPI banding was used to identify the specific chromosome, the assignment between signal from probe and the long arm of chromosome 12 was obtained. The detailed position was further determined in the diagram based on the summary from 10 photos.
  • Radiation hybrid analysis is a PCR technique and the panels of radiation hybrid DNA are provided at a concentration of 25 ng/ ⁇ l in TE buffer suitable for these reactions. Typically, 25 ng of DNA is used in a 10 ⁇ l PCR reaction.
  • RH mapping involves the statistical analysis of several to many markers to determine the relative order of the markers with respect to one another. RH mapping can be achieved using statistical programs that will provide the best map along with a measure of the relative likelihood of one order versus another.
  • N$ neuroblastoma lines were seeded in Lab Tek chambered coverglass (Nalgene Nunc International) and transfected with pEGFP (control), pGI3303 and pGI3305 using lipofectamine (Life Technologies BRL) . After 24-48 hours, the chambered coverglasses were placed on an inverted fluorescence microscope where GFP fluorescence could be visualized in living cells. The details of this method have been described in PCT/EP96/02311.
  • Seq ID No 1 is a nucleic acid sequence of Hs unc-53/1 and lacking the nucleotides from position 2873 to 3043 shown in Fig. la.
  • Seq ID No. 2 is a nucleic acid sequence of Hs unc-53/1 and lacking the nucleotides from position 3098 to 3121 shown in Figure la.
  • Seq ID no. 3 is a nucleic acid sequence of Hs-unc-53/1 and lacking the nucleotides from position 3518 to 3526 of the sequence identified in Fig. la.
  • Seq ID No. 4 is an amino acid sequence of Hs-unc-53/1 protein and lacking the amino acids from position 958 to 1014 of the sequence identified in Fig. lb
  • Seq ID No. 5 is a amino acid sequence of Hs-unc-53/1 protein and lacking the amino acids from position 1033 to 1040 of the sequence identified in Fig. lb.
  • Seq ID No. 6 is a amino acid sequence of Hs-unc-53/1 protein and lacking the amino acids from position 1173 to 1175 of the sequence identified in Fig. lb.
  • Seq ID No. 7 is a nucleotide sequence encoding Hs- unc-53/2 and lacking the nucleotides from position 5425 to 5433 of the sequence illustrated in Fig. Ic.
  • Seq ID No. 8 is a nucleotide sequence encoding Hs- unc-53/2 and lacking the nucleotides from position 5924 to 6024 of the sequence illustrated in Fig. Ic.
  • Seq ID No. 9 is a nucleotide sequence encoding Hs- unc-53/2 and having the sequence of variant 1 illustrated in Fig. Ic.
  • Seq ID No. 10 is a nucleotide sequence encoding Hs- unc-53/2 and having the sequence of variant 2 illustrated in Fig. Ic.
  • Seq ID No. 11 is a nucle ⁇ ticfe " sequence encoding Hs- unc-53/2 and having the sequence of variant 3 illustrated in Fig. Ic.
  • Seq ID No. 12 is a nucleotide sequence encoding Hs- unc-53/2 and having the sequence of variant 1 illustrated in Fig. Ic. and lacking the nucleotides from position 5425 to 5433 of the sequence illustrated in Fig. Ic.
  • Seq ID No. 13 is a nucleotide sequence encoding Hs- unc-53/2 and having the sequence of variant 1 illustrated in Fig. Ic. and lacking the nucleotides from position 5924 to S024 of the sequence illustrated in Fig. Ic.
  • Seq ID No. 14 is a nucleotide sequence encoding Hs- unc-53/2 and having the sequence of variant 2 illustrated in Fig. Ic. and lacking the nucleotides from position 5425 to 5433 of the sequence illustrated in Fig. Ic.
  • Seq ID No. 15 is a nucleotide sequence encoding Hs- unc-53/2 ar.d having the sequence of variant 2 illustrated in Fig. Ic. and lacking the " nucleotides from position 5924 to 6024 of the sequence illustrated in Fig. Ic.
  • Seq ID No. 16 is a nucleotide sequence encoding Hs- unc-53/2 and having the sequence of variant 3 illustrated in Fig. Ic. and lacking the nucleotides from position 5425 to 5433 of the sequence illustrated in Fig. Ic.
  • Seq ID No. 17 is a nucleotide sequence encoding Hs- unc-53/2 and having the sequence of variant 3 illustrated in Fig. lc._ and lacking the_ nucleotides. from position 5324 to 6024 of the sequence illustrated in Fig. Ic.
  • Seq ID No. 18 is an amino acid sequence of Hs-unc- 53/2 protein and lacking the amino acids from position 1776 to 1778 of the sequence identified in Fig. Id
  • Seq Id No. 19 is an amino acid sequence of variant 1 of Hs-unc-53/2 sequence illustrated in Fig. Id.
  • Seq Id No. 20 is an amino acid sequence of variant 2 of Hs-unc-53/2 sequence illustrated in Fig. Id.
  • Seq Id No. 21 is an amino acid sequence of variant 3 of Hs-unc-53/2 sequence illustrated in Fig. Id.
  • Seq Id No. 22 is an amino acid sequence of variant 1 of Hs-unc-53/2 sequence illustrated in Fig. Id and lacking the amino acids from position 1776 to 1778 of the sequence identified in Fig. Id.
  • Seq Id No. 23 is an amino acid sequence of variant 2 of Hs-unc-53/2 sequence illustrated in Fig. Id and lacking the amino acids from position 1776 to 1778 of the sequence identified in Fig. Id.
  • Seq Id No. 24 is an amino acid sequence of variant 3 of Hs-unc-53/2 sequence illustrated in Fig. Id and lacking the amino acids from position 1776 to 1778 of the sequence identified in Fig. Id.
  • Seq ID No. 25 is a nucleotide sequence encoding Hs- unc-53/3 as illustrated in Figure le.
  • Seq ID No. 26 is a nucleotide sequence encoding Hs- unc-53/3 as illustrated in Figure le and lacking the nucleotides from position 3795 to 4283 of the sequence identified therein.
  • Seq ID No. 27 is a nucleotide sequence encoding Hs- unc-53/3 as illustrated in Figure le and lacking the nucleotides from position 4284 to 4325 of the sequence identified therein.
  • Seq ID No. 28 is a nucleotide sequence encoding Hs- unc-53/3 as illustrated in Figure le and lacking the nucleotides from position 3795 to 4325 of the sequence identified therein.
  • Seq ID No. 29 is a nucleotide sequence encoding Hs- unc-53/3 as illustrated in Figure le and lacking the nucleotides from position 5153 to 5173 of the sequence identified.
  • Seq ID No. 30 is a nucleotide sequence encoding Hs- unc-53/3 as illustrated in Figure le and lacking the nucleotides from position 5343 to 5408 of the sequence identified.
  • Seq ID No. 31 is a nucleotide sequence encoding Hs- unc-53/3 having the sequence of variant 1 illustrated in Fig. le.
  • Seq ID No. 32 is a nucleotide sequence encoding Hs- unc-53/3 having the sequence of variant 1 illustrated in Fig. le and lacking the nucleotides from position 3795 to 4283 of the sequence identified therein.
  • Seq ID No. 33 is a nucleotide sequence encoding Hs- unc-53/3 having the sequence of variant 1 illustrated in Fig. le and lacking the nucleotides from position 4284 to 4325 of the sequence identified therein.
  • Seq ID No. 34 is a nucleotide sequence encoding Hs- unc-53/3 having the sequence of variant 1 illustrated in Fig. le and lacking the nucleotides from position 3795 to 4325 of the sequence identified therein.
  • Seq ID No. 35 is a nucleotide sequence encoding Hs- unc-53/3 having the sequence of variant 1 illustrated in Fig. le and lacking the nucleotides from position 5153 to 5173 of the sequence identified therein.
  • Seq ID No. 36 is a nucleotide sequence encoding Hs- unc-53/3 having the sequence of variant 1 illustrated in Fig. le and lacking the nucleotides from position 5343 to 5408 of the sequence identified therein.
  • Seq ID No. 37 is an amino acid sequence of Hs-unc- 53/3 protein as identified in the sequence of Fig. If.
  • Seq ID No. 38 is an amino acid sequence of Hs-unc- 53/3 protein as identified in the sequence of Fig. If and lacking the amino acid residues from position 1326 to 1413 of the sequence identified therein.
  • Seq ID No. 39 is an amino acid sequence of Hs-unc- 53/3 protein as identified in the sequence of Fig. If and lacking the amino acid residues from position 1414 to 1427 of the sequence identified therein.
  • Seq ID No. 40 is an amino acid sequence of Hs-unc- 53/3 protein as identified in the sequence of Fig. If and lacking the amino acid residues from position 1703 to 1709 of the sequence identified therein.
  • Seq ID No. 41 is an amino acid sequence of Hs-unc- 53/3 protein as identified in the sequence of Fig. If and lacking the amino acid residues from position 1768 to 1788 of the sequence identified therein.
  • Seq ID No. 42 is an amino acid sequence of Hs-unc-53 of variant 1 identified in Figure If.
  • Seq ID No. 43 is an amino acid sequence of Hs-unc-53 of variant 1 identified in Figure If and lacking the amino acid residues from position 1326 to 1413 of the sequence identified therein.
  • Seq ID No. 44 is an amino acid sequence of Hs-unc-53 of variant 1 identified in Figure If and lacking the amino acid residues from position 1414 to 1427 of the sequence identified therein.
  • Seq ID No. 45 is an amino acid sequence of Hs-unc-53 of variant 1 identified in Figure If and lacking the amino acid residues from position 1703 to 1709 of the sequence identified therein.
  • Seq ID No. 46 is an amino acid sequence of Hs-unc-53 of variant 1 identified in Figure If and lacking the amino acid residues from position 1768 to 1788 of the sequence identified therein.

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Abstract

There is disclosed human homologues of the UNC-53 protein of C. elegans and cDNA sequences coding for said homologues or functional equivalents thereof. The invention also relates to processes for identifying compounds which control cell behaviour, compounds identified and pharmaceutical compositions containing them in addition to processes and assays for identifying disease states in which said gene or protein is dysfunctional.

Description

HUMAN HOMOLOGUE OF UNC-53 PROTEIN OF C. ELEGANS
The present invention relates to a vertebrate homologue of UNC-53 protein of C. elecrans and cDNA sequences coding for said homologues or functional equivalents thereof. The invention also relates to processes for identifying compounds which control cell behaviour, compounds identified and pharmaceutical compositions containing them in addition to processes and assays for identifying disease states in which said gene or protein is dysfunctional.
The control of cell motility, cell shape and directionality of cell outgrowth of axones or other cell outgrowths is an essential feature in the morphogenesis and function of both unicellular and multicellular organisms.
Some cell surface proteins and extra-cellular molecules controlling the directionality and potential of cell migration have been identified, although the processes involved are not generally understood. It is generally considered that a long-range migration of a cell process (also known as a growth cone extension) is a stepwise event, whereby prior to and after each extension there is the formation of a structure at the leading edge of the cell. Localised stabilisation of the actin cytoskeleton and association with plus end regions of microtubules is a general cell biological process underlying the choice of directional extension.
The present inventors have surprisingly found a new human gene/protein belonging to the UNC-53 family that binds microtubules and, in particular, the plus- end regions of microtubules. A gene from the free-living nematode
Caenorhabditis eleσans designated λunc-53" has been previously identified and cloned (Abstract, International C. elegans Meeting, June 1-5 1991, Madison, Wisconsin, 58, Bogaert and Goh) . The present inventors previously identified UNC-53 protein as a signal transducer or signal integrator controlling the directionality of cell migration and/or cell shape in C. eleσans (WO 96/38555) .
The C. elecrans UNC-53 protein (Ceunc53) and previously found human homologues thereof (hs-unc53/l and hs-unc53/2) were found to encode a signal transducer or a signal integrator, controlling the directionality of a cell migration, cell shape and growth extension. Evidence indicates that the presently found homologue designated (hs-unc53/3) might act as an adapter linking extracellular signals to the actin cytoskeleton. Firstly hs-unc-53/3 shows homology to the cortical actin binding proteins, and the Ce-UNC-53 protein has been shown to bind F-actin in vitro and leads to actin re-organization in vivo when expressed in mammalian cells, leading to an increased number of filopodia and lammelipodia.
Furthermore, increased neurite extension and increased cell motility could be observed. Hs-UNC-53-3 may play an important role in the development of various diseases . According to a first aspect of the present invention there is provided a vertebrate protein homologue of an UNC-53 protein of C. elegans, which protein comprises an amino acid sequence having one or more of sequence blocks A, B, C, D, E, F, G or H as illustrated in figure 4 or which differs from said blocks in conservative amino acid changes.
According to a further aspect of the present invention, there is provided a vertebrate protein homologue of UNC-53 protein of C. elegans or a functional equivalent, derivative or bioprecursor thereof, having an amino acid sequence encoded by the nucleotide sequence illustrated in figure 1 (e) . For the purposes of the present invention a "derivative" should be taken to mean mutational derivatives, fusions, internal deletions, splice variants and muteins. Preferably, said vertebrate homologue is a human protein, and preferably a mammalian or a mouse protein.
A further aspect of the invention comprises a vertebrate homologue comprising an amino acid sequence as shown in figure 1(f) or the variants thereof or an amino acid sequence which differs from the amino acid sequences shown in figure 1(f) to a significant extent only in one or more conservative amino acid changes. In a further aspect of the present invention there is also provided a nucleic acid molecule, which is preferably DNA, and which encodes a vertebrate homologue of UNC-53 protein of C. elegans, or a functional equivalent derivative, fragment or bioprecursor of said homologue according to the invention. Preferably, the cDNA comprises a sequence of nucleotides encoding an amino acid sequence as illustrated in figure 1(f) or the variants thereof or an amino acid which differs from the sequences shown in these figures to a significant extent only in one or more conservative amino acid changes. Preferably the DNA is cDNA, which cDNA comprises the sequence shown in figure 1(e) or the variants indicated therein. Also provided by the present invention is a nucleic acid sequence capable of hybridising to the nucleic acid or DNA sequences according to the invention under high stringency conditions, which conditions are well known to those skilled in the art.
The cDNA according to the invention may be included in an expression vector which may itself be used to transform or transfect a host cell, which cell may be bacterial or eukaryotic in origin including such as, for example an animal or plant cell a fungal cell or an insect cell. Thus, advantageously, once the cDNA corresponding to the genome of the vertebrate homologue of UNC-53 of C. elegans according to the invention is synthesised, using for example, reverse transcriptase or the like, a range of cells, tissues or organisms may be transfected following incorporation of the selected cDNA clone into an appropriate expression vector. The expression vector according to the invention may comprise a promoter of C. elegans or one of human, mouse or viral origin and optionally a sequence encoding a reporter molecule, such as, for example, green fluorescent protein.
The present invention, therefore, also further comprises a transgenic cell, tissue or organism comprising a transgene capable of expressing a vertebrate homologue of UNC-53 protein of C. elegans according to the invention. The term "transgene capable of expressing a vertebrate homologue of UNC-53 protein of C. elegans" as used herein means a suitable nucleic acid sequence which leads to the expression of a vertebrate homologue of UNC-53 protein of C. elegans according to the invention having the same function and/or activity. The transgene may include, for example, genomic nucleic acid isolated from the appropriate vertebrate or synthetic nucleic acid including cDNA. The term "transgenic organisms, tissues or cells, as used herein means any suitable organism and/or part of an organism, tissue or cell, that contains exogenous nucleic acid either stably integrated in the genome or in an extrachromosomal state.
Preferably the transgenic cell comprises any of, a COS cell, HepG2 cell, MCF-7 or N4 neuroblastoma cell, a NIH3T3 cell, a colorectal or carcinoma cell or a human derived cell such as a fibroblast or the like. The transgenic organism may be an insect, a non-human animal or a plant and preferably C. elegans or a related nematode. Preferably, the transgene comprises the nucleic acid or cDNA sequence encoding the vertebrate homologue according to the invention as described above. The transgene preferably comprises an expression vector according to the invention.
The term "functional fragment" as used herein should be taken to mean a fragment of the gene coding for the vertebrate homologue of the UNC-53 protein of C. elegans according to the invention. For example, the gene may comprise deletions or mutations but may still encode a functional vertebrate homologue of UNC- 53 protein.
Further provided by the present invention is a method of producing a mutant vertebrate non-human organism having a mutation in the wild-type gene coding for the vertebrate homologue of UNC-53 protein according to the invention, which mutation affects cell behaviour or the regulation of cell motility or the shape or the direction of cell migration or microtubule plus end stability or function and localisation of protein complexes located thereon, which method comprises inducing a mutation in the vertebrate homologue of UNC-53 protein in said organism. These mutant organisms may be used in a screen to identify the effects of compounds on these cell functions.
The vertebrate homologue of UNC-53 protein of C. elegans or the cDNA or genomic DNA encoding it or a functional equivalent, derivative, fragment or bioprecursor of said homologue, may advantageously be used as a medicament, or in the preparation of a medicament to treat or prevent disorders associated with inhibition of overexpression of the vertebrate homologue of UNC -53 according to the invention. Such disorders may be alleviated by promoting neuronal regeneration, revascularisation or wound healing or the treatment of chronic neurodegenerative disorders, psychiatric disorders or acute traumatic injuries or fibrotic disease or disease in which physiological events requiring the polarity of cells or epithelia are abnormally functioning. Accordingly, the vertebrate homologue according to the invention, dominant positive or negative mutants thereof, or inhibitors thereof may advantageously be used to induce or alleviate contact inhibition in a cell or in preventing carcinoma development. Typically, the above medical conditions may be treated in mammals and more preferably humans by either the homologue of UNC- 53 protein or alternatively by a nucleic acid coding for the protein or the protein itself according to the invention. Alternatively an antisense oligonucleotide to said UNC-53 vertebrate homologue may be used to prevent it's expression. Examples of other nucleic acid sequences which may be used include 3' untranslated regions of mRNA which could be used to prevent transcription of the genomic sequence encoding for the vertebrate homologue of UNC-53 protein according to the invention.
The vertebrate homologue of UNC-53 protein according to the invention may be incorporated into a pharmaceutically acceptable composition together with a suitable carrier, diluent or excipient therefor. The pharmaceutical composition may advantageously comprise, additionally or alternatively, the nucleic acid sequence according to the invention as defined above . The induction or inhibition of the expression of hu-UNC-53/3 by pharmacological means may advantageously be used to induce neuronal regeneration, revascularisation or wound healing or be involved in the treatment of chronical neurodegenerative disorders, or acute traumatic injuries or fibrotic diseases, or physiological events requiring the polarity of cells, or oncology and metastasis of cells, or apoptotic pathways.
The present invention therefore also provides for a method of determining whether a compound is an inhibitor or enhancer of the regulation of cell behaviour, growth, transformation, cell shape or motility or the direction of cell migration, microtubule plus end stability or function and localisation of protein complexes thereon, which method comprises contacting said compound with a transgenic cell according to the invention and screening for a phenotypic change in said cell. The method can therefore be used to determine whether the compound comprises an inhibitor or an enhancer of the signal transduction pathway of said transgenic cell of which pathway said vertebrate homologue of UNC-53 protein according to the invention is a component, or whether said compound is an inhibitor or an enhancer of a parallel or redundant signal transduction pathway in said cell. The present invention also provides a method to determine that the protein in said signal transduction pathway is a vertebrate homologue of UNC- 53 protein of C. elegans according to the invention.
Preferably, the phenotypic change to be screened comprises a change in cell shape or a change in cell motility. Where a transgenic cell is used in accordance with one embodiment of the method of the invention, an N4 neuroblastoma cell may be used and in such an embodiment the phenotypic change to be screened may be the length of neurite growth, changes in filopodia outgrowth, changes in ruffling behaviour or cell adhesion, any change in microtubule cytoskeleton, any change in localisation of proteins on plus end regions of microtubules or any change in a cell such as apoptosis. In an alternative embodiment of the method of the invention, the transgenic cell may comprise an MCF-7 breast carcinoma cell. Typically in such an embodiment the phenotypic change to be screened comprises the extent of phagokinesis or filopodia formation. In an alternative embodiment of this aspect of the invention, the transgenic cell may comprise an NIH3T3 cell. Typically in such an embodiment the phenotypic change to be screened comprises loss of contact inhibition of foci formation. The method according to the invention, may also utilise a mutant cell or mutant organism according to the invention as described above, where the mutant cell is capable of growing in tissue culture or in vivo and either of which cell or organism has a mutation in the wild-type unc-53 gene.
In accordance with the present invention, a "phenotypic change", may comprise any phenotype resulting from changes at any suitable point in the life cycle of the cell, tissue or organism defined above, which change can be attributed to the expression of the transgene of the invention such as for example, growth, viability, morphology, behaviour, movement, cell migration or cell process or growth cone extension of cells and includes changes in body shape, locomotion, chemotaxis, contact inhibition, mating behaviour or the like. The phenotypic change may preferably be monitored directly by visual inspection of the cell as a whole or by monitoring the F-actin cytoskeleton microtubule network and plus end stability of microtubules or proteins thereon or alternatively by for example measuring indicators of viability including endogenous or transgenically introduced histochemical markers or other reporter genes, such as for example β-galactosidase or green fluorescent protein.
A compound which is identifiable by the method according to the invention as described above, as an enhancer of the processes identified above such as the regulation of cell shape or motility or the direction of cell migration may be used as a medicament, or alternatively in the preparation of a medicament, for promoting neuronal regeneration, revascularisation or wound healing, or for treatment of chronic neuro- degenerative diseases or acute traumatic injuries or fibrotic disease. Examples of promoting neuronal regeneration include, for example, peripheral nerve regeneration after trauma and spinal cord trauma.
Where a compound is identified in accordance with the method described above as being an inhibitor of the regulation of cell shape or mobility or the direction of cell migration, the compound may be used as a medicament, or in the preparation of a medicament, for substantially alleviating spread of disease inducing cells, such as in spread of carcinoma, or the like in metastasis or in alleviating loss of contact inhibition. Advantageously, any of the compounds which may have been identified as an inhibitor or an enhancer in accordance with the method as described above, may also be included in a pharmaceutical composition comprising the respective compound and a pharmaceutically acceptable carrier, diluent or excipient therefor.
The particular mechanism of action of a compound identified as either an inhibitor or an enhancer of the cell motility shape, growth or direction of cell migration or microtubule association or to the plus end region thereof is not limiting. Preferably the compound acts as an inhibitor or enhancer of a signal transduction pathway. The compound may also act on a parallel pathway or directly on the vertebrate homologue of UNC-53 protein of C. elegans. For example, the method of action of the compound may include direct interaction with the vertebrate homologue of UNC-53 protein, interaction with processes for regulating phosphorylation or dephosphorylation of the vertebrate homologue of UNC- 53 or with processes regulating activity of an unc-53 gene or with processes for post-transcriptional or post-translational modification or the like.
Preferably the compound is identified by the method according to the invention as an inhibitor or an enhancer, by utilising differences of phenotype of the cell, tissue or organism, which are visible to the eye. Alternatively indicators of viability including endogenous or transgenically introduced histochemical markers or a reporter gene may be used. According to a further aspect of the invention there is also provided a transgenic cell or tissue culture which has been constructed to comprise a promoter sequence of a gene coding for a vertebrate homologue of UNC-53 of C. elegans according to the invention operably linked to a nucleic acid sequence encoding a reporter molecule. Preferably, the reporter sequence encodes for a detectable protein, for example one which may be monitored by eye inspection such as antibiotic resistance, β- galactosidase or a molecule detectable by spectrophotometric, spectrofluorometric, luminescent or radioactive assays.
The present invention also provides a method of determining whether a compound is an inhibitor or an enhancer of transcription of a gene coding for a vertebrate homologue of UNC-53 protein in C. elegans, according to the invention which method comprises the steps of:
(a) contacting said compound with a transgenic cell according to the invention as described above,
(b) monitoring the level of said reporter molecule and comparing results obtained from this monitoring step with a control comprising a transgenic cell having the promoter sequence of a gene coding for a vertebrate homologue of UNC-53 protein, or a functional fragment of said homologue and the reporter molecule, in the absence of the compound.
In one embodiment of the method according to this aspect of the invention the reporter molecule may comprise messenger RNA.
A compound identified as an enhancer of transcription of the gene coding for the vertebrate homologue of UNC-53 protein of C. elegans or a functional equivalent, derivative or bioprecursor of said homologue may also be used as a medicament, or in the preparation of a medicament, for promoting neuronal regeneration, revascularisation or wound healing, or for treatment of chronic neuro- degenerative diseases or acute traumatic injuries or fibrotic disease. Furthermore, such compounds may be included in a pharmaceutical composition including a pharmaceutically acceptable carrier, diluent or excipient therefor. Any compounds identified as inhibitors of transcription may, advantageously, be used in alleviating the spread of disease inducing cells such as carcinomas or metastasis or loss of contact inhibition.
The present invention also provides a kit for determining whether a compound is an enhancer or an inhibitor of the regulation of cell growth, transformation, cell motility or shape or the direction of cell migration which kit comprises at least one transgenic or mutant cell or transgenic or mutant non-human organism according to the invention as described above and a plurality of wild-type cells or a wild-type organism of the same type, or a cell line or tissue culture and means for contacting said compound with said cell or organism.
Also provided by the present invention is a kit for determining whether a compound is an inhibitor or an enhancer of transcription of a gene coding for a vertebrate homologue of UNC-53 protein of C. elegans according to the invention which kit comprises at least one transgenic cell or cells according to the invention, means for contacting said compounds with said cells and means for monitoring the level of transcription of said transgenic cell or cells according to the invention.
For the purposes of the present invention, the term "gene coding for a vertebrate homologue of UNC-53 or a functional fragment of said homologue" includes the nucleic acid sequence shown in figure 1 or a fragment thereof, including the differentially spliced isoforms and transcriptional starts of the nucleic acid sequence and which sequence encodes a vertebrate homologue of UNC-53 protein or a functional equivalent, derivative, fragment or bioprecursor of the protein.
The present invention also provides methods of identifying genes of vertebrates or fragments of said genes, which encode proteins which are active in the signal transduction pathway of which the vertebrate homologue of UNC-53 according to the present invention is a component. A preferred method comprises hybridizing to an appropriate cDNA library a nucleotide sequence, as defined herein, or a fragment thereof under appropriate conditions of stringency in order to identify genes having statistically significant homology with the cDNA clones of any one of the cDNA sequences according to the invention described above. Furthermore, there is also provided by the present invention a method of identifying a protein which is active in the signal transduction pathway of a cell of which a vertebrate homologue of UNC-53 protein of C. elegans according to the invention is a component. According to this aspect of the invention, the method comprises;
(a) contacting an extract of said cell with an antibody to the vertebrate homologue of UNC-53 protein or a functional equivalent, fragment or bioprecursor of said protein,
(b) identifying the antibody/vertebrate homologue of UNC-53 complex, and
(c) analysing the complex to identify any protein bound to the vertebrate homologue of UNC-53 protein other than the antibody.
The vertebrate homologue of UNC-53 protein, therefore may bind regions of other proteins involved in the signal transduction pathway. It is also possible to sequentially identify a whole range of proteins involved in the signal transduction pathway. Antibodies to the vertebrate homologue of UNC-53 protein may be produced according to known techniques as would be known to those skilled in the art. For example, polyclonal antibodies may be prepared by inoculating a host animal, such as a mouse, with a protein or epitope of a protein according to the invention and recovering immune serum.
This aspect of the invention, further comprises a method of identifying a further protein or proteins which are active in the signal transduction pathway of a cell of which the vertebrate homologue of UNC-53 is a component which method comprises:
(a) forming an antibody to the first identified protein bound to the vertebrate homologue of UNC-53 protein in the method as described above,
(b) contacting a cell extract with the antibody, (c) identifying any antibody/protein complex,
(d) analysing the complex to identify any further protein bound to the first protein other than the antibody, and
(e) optionally repeating steps (a) to (d) to identify further proteins in the pathway.
According to this aspect of the present invention, the antibody starts the process by binding to the vertebrate homologue of UNC-53 protein according to the invention in the signal transduction or oncogenic pathways. Any other proteins found complexed to the bound antibody or UNC-53 protein can then be used to identify further interacting proteins involved in the pathway.
It may also be possible to identify proteins involved in the signal transduction pathway of a cell of which the vertebrate homologue of UNC-53 is a component by using a vertebrate homologue of UNC-53 protein of C. elegans. According to this aspect of the invention the method comprises:
(a) contacting an extract of the cell with the vertebrate homologue of UNC-53 protein of C. elegans or a functional equivalent, fragment or bioprecursor of said homologue,
(b) identifying the vertebrate homologue of UNC- 53 protein/protein complex formed and
(c) analysing the complex to identify any protein bound to the vertebrate homologue of
UNC-53 protein other than the same vertebrate homologue of UNC-53 protein. This method can also advantageously be used to identify further proteins in a signal transduction pathway of a cell by contacting an extract of the cell used as described above, with any protein identified from step (c) above not being a vertebrate homologue of UNC-53 protein and repeating steps (b) and (c) .
Other methods which may be used for identifying proteins in a signal transduction pathway of a cell may comprise for example a western blot overlay method which method is well known to those skilled in the art. Cell extracts are run on gels to separate out protein and subsequently blotted onto a nylon membrane. These membranes may then be incubated, for example in a medium containing vertebrate homologue of UNC-53 having a label attached thereto such as a biotin or radiolabel and any protein conjugates visualised with for example a streptavidin or alkaline phosphatase conjugated antibody.
The present invention also advantageously provides a process for the preparation of binding antibodies which recognise proteins or fragments thereof involved in the rate and direction of cell migration or the control of cell growth or shape, for the above methods . The monoclonal antibody for binding to the appropriate vertebrate homologue of UNC-53 (or its functional equivalent) may be prepared by known techniques as described by Kohler R. and Milstein C, (1975) Nature 256, 495 to 497. Another method which may be used to identify proteins involved in the signal transduction pathway of a cell of which a vertebrate homologue of an UNC-53 protein of C. elegans according to the invention or is a component, involves investigating protein-protein interactions using the two-hybrid vector method. This method, which is well known to those skilled in the art was first developed in yeast by Chien et al (1991) . This technique is based on functional reconstruction in vivo of a transcription factor which activates a reporter gene. More particularly the technique comprises providing an appropriate host cell with a DNA construct comprising a reporter gene under the control of a promoter regulated by a transcription factor having a DNA binding domain and an activating domain, expressing in the host cell a first hybrid DNA sequence encoding a first fusion of a fragment or all of a nucleic acid sequence according to the invention and either said DNA binding domain or said activating domain of the transcription factor, expressing in the host at least one second hybrid DNA sequence, such as a library or the like, encoding putative binding proteins to be investigated together with the DNA binding or activating domain of the transcription factor which is not incorporated in the first fusion; detecting any binding of the proteins to be investigated with a protein according to the invention by detecting for the presence of any reporter gene product in the host cell; optionally isolating second hybrid DNA sequences encoding the binding protein.
An example of such a technique utilises the GAL4 protein in yeast. GAL4 is a transcriptional activator of galactose metabolism in yeast and has a separate domain for binding to activators upstream of the galactose metabolising genes as well as a protein binding domain. Nucleotide vectors may be constructed, one of which comprises the nucleotide residues encoding the DNA binding domain of GAL4.
These binding domain residues may be fused to a known protein encoding sequence, such as for example a sequence coding for the vertebrate homologue of UNC-53. The other vector comprises the residues encoding the protein binding domain of GAL4. These residues are fused to residues encoding a test protein, preferably from the signal transduction pathway of the vertebrate in question. Any interaction between the vertebrate homologue of UNC-53 protein and the protein to be tested leads to transcriptional activation of a reporter molecule in a GAL-4 transcription deficient yeast cell into which the vectors have been transformed. Preferably, a reporter molecule such as β-galactosidase is activated upon restoration of transcription of the yeast galactose metabolism genes. This method enables any interactions between proteins involved in the signal transduction pathway or a parallel or redundant pathway to be investigated. Any proteins identified in the signal transduction pathway of the cell, which may be for example a mammalian cell, may also be included in a pharmaceutical composition together with a pharmaceutically acceptable carrier, diluent or excipient therefor.
The present invention also provides a process for producing a vertebrate homologue of an UNC-53 protein of C. elegans according to the invention which process comprises culturing the cells transformed or transfected with a cDNA expression vector having any of the cDNA sequences according to the invention as described above, and recovering the expressed protein homologue. The cell may advantageously be a bacterial, animal, insect or plant cell.
A particularly preferred process for producing said vertebrate homologue of UNC-53 protein uses insect cells. Accordingly, the invention provides a process for producing a vertebrate homologue of UNC-53 protein of C. elegans according to the invention which process comprises culturing an insect cell transformed or transfected with a recombinant Baculovirus vector, said vector comprising a nucleotide sequence encoding said vertebrate homologue of UNC-53 protein according to the invention downstream of the Baculovirus polyhedrin promoter and recovering the expressed protein. Advantageously, this method produces large amounts of protein for recovery. The insect cell may be from for example Spodoptera frugiperda or Drosophila Melanogester .
In accordance with the present invention, a defined nucleic acid sequence includes not only the identical nucleic acid but also any minor base variations from the natural nucleic acid sequence including in particular, substitutions in bases which result in a synonymous codon (a different codon specifying the same amino acid) , due to the degenerate code in conservative amino acid substitution. The term "nucleic acid sequence" also includes the complimentary sequence to any single stranded sequence given which includes the definition above regarding base variations.
Furthermore, a defined protein, polypeptide or amino acid sequence according to the invention, includes not only the identical amino acid sequence but also minor amino acid variations from the natural amino acid sequence including conservative amino acid replacements (a replacement by an amino acid that is related in its side chains) . Also included are amino acid sequences which vary from the natural amino acid but result in a polypeptide which is immunologically identical or similar to the polypeptide encoded by the naturally occurring sequence. Such polypeptides may be encoded by a corresponding nucleic acid sequence. A further aspect of the invention provides a nucleic acid sequence of at least 15 nucleotides of a nucleic acid according to the invention and preferably from 15 to 50 nucleotides.
These sequences may, advantageously be used as probes or primers to initiate replication or the like. Such nucleic acid sequences may be produced according to techniques well known in the art, such as by recombinant or synthetic means. They may also be used in diagnostic kits or the like for detecting for the presence of a nucleic acid according to the invention. These test generally comprise contacting the probe with a sample under hybridising conditions and detecting for the presence of any duplex formation between the probe and any nucleic acid in the sample. Nucleic acid sequences according to the invention may also be produced using recombinant or synthetic means such as described in Sambrook et al (Molecular Cloning: A Laboratory Manual, 1989) . Advantageously, human allelic variants or polymorphisms of the DNA according to the invention may be identified by, for example, probing DNA from a range of individuals for example from different populations. Furthermore, nucleic acids and probes according to the invention may be used to sequence genomic DNA from patients using techniques well known in the art, such as the Sanger Dideoxy chain termination method, which may advantageously ascertain any predisposition of a patient to certain disorders.
A method of detecting whether a compound is an inhibitor or an enhancer or expression of a vertebrate homologue of UNC-53 of C. elegans, according to the invention is also provided which method comprises contacting a cell expressing said homologue with said compound and monitoring for a phenotypic change compared to a control cell which has not been contacted with said compound. Preferably the cell is a transgenic cell as described above. Alternatively the cell may have undergone loss of contact inhibition.
The present method also provides for determining whether said compound is an inhibitor or expression of said vertebrate homologue. In one embodiment the compound to be tested comprises a nucleic acid.
Preferably said nucleic acid sequence comprises an antisense DNA sequence or a mRNA sequence.
Preferably said mRNA sequence comprises 3' untranslated regions of mRNA encoding for said vertebrate homologue.
Alternatively, the compound to be tested may be a protein. Preferably, said protein comprises a protein having an amino acid sequence potentially suitable for inhibiting function of said vertebrate homologue and preferably comprises a protein identified by the methods as described herein.
The present invention also provides a pharmaceutical composition comprising a compound, for example an antisense nucleic acid identified according to the above described method together with a pharmaceutically acceptable carrier, diluent or excipient therefor.
A nucleic acid sequence or protein identified according to this aspect of the invention may be used as a medicament, or in the preparation of a medicament, for treating loss of contact inhibition of cancer which is mediated by vertebrate homologue of UNC-53 protein or a functional equivalent, fragment, derivative or bioprecursor of said homologue.
Further provided by the invention is a nucleic acid as defined above for use in preparation of a medicament for inhibiting expression of a gene coding for a vertebrate homologue of UNC-53 protein of C. elegans.
Further provided by the invention is an assay for detecting expression of the vertebrate homologue of UNC-53 protein of C. elegans in a vertebrate cell which assay comprises contacting a cell or an extract thereof with an antibody to said vertebrate homologue, which antibody is fused to a reporter molecule, removing any unbound antibody and monitoring for the presence of said reporter molecule.
Preferably the reporter molecule is an antibody conjugated to for example a fluorophore such as fluorescein or alternatively to an enzyme such as strepavidin.
There is also provided a method for detecting for expression of a gene coding for the vertebrate homologue of UNC-53 protein of the invention which method comprises contacting a probe specific for a nucleic acid of protein sequence coding for or corresponding to said vertebrate homologue according to the invention with a cell extract, which probe is linked to a reporter and analysing for the presence of said reporter. Preferably the probe is a complementary sequence to a region of mRNA transcribed from said gene encoding said vertebrate homologue of UNC-53 protein according to the invention.
Preferably the complimentary sequence is a 3' or 5' untranslated region of said mRNA. Preferably said reporter may be a dig label, a fluorophore, a hapten or a radiolabel.
Alternatively said probe may comprise an antibody specific for said vertebrate homologue of said UNC-53 protein.
Preferably the reporter is an antibody conjugated to for example a fluorophore such as fluorescein or alternatively an enzyme such as streptavidin.
As described above, UNC-53 protein of C. elegans has been found to localise to microtubule and particularly to microtubule (+) ends. Therefore, there is provided by a further aspect of the present invention a method of determining whether a compound is an inhibitor or an enhancer of association of the UNC-53 homologue of the invention to microtubules or plus end regions thereof, which method comprises (a) contacting said compound with a transgenic cell, tissue or organism expressing said vertebrate homologue and which protein is operably linked to a reporter molecule (b) screening for the localisation of said reporter molecule as compared to a cell according to step (a) which has not been contacted with said compound.
A compound identifiable by the above method also forms part of the present invention. Such a compound identified as an inhibitor of localisation or association of said vertebrate homologue with microtubules or the plus end region thereof may be used in alleviating the spread of disease inducing cells or metastasis or loss of contact inhibition. Further a compound identified as an enhancer of association of said vertebrate homologue with microtubules or the plus end region thereof may be used in for example promoting neuronal regeneration, revascularisation or wound healing, or for treating chronic neurodegenerative diseases or acute traumatic injuries or fibrotic disease. These compounds may then be included in a pharmaceutical composition, together with a pharmaceutically acceptable carrier, diluent or excipient therefor.
Also provided by the present invention is a kit for determining whether a compound is an inhibitor or an enhancer of association of the vertebrate homologue thereof according to the invention with microtubules or the plus end regions thereof, which kit comprises at least one transgenic cell expressing said UNC-53 vertebrate protein homologue and a reporter molecule or a host or transgenic cell according to the invention and at least one cell of the same cell type for use as a control and means for contacting said compound with one of said at least one transgenic cells. Compounds identified as inhibitors or enhancers or microtubule association described above may advantageously be included in a composition and linked to said vertebrate homologue according to the invention to target the compounds to the microtubules or the plus end regions thereof. Such a composition may also comprise, for example, a suitable transfecting or transformation agent.
According to a further aspect of the invention there is provided a method of targeting a protein to a cell microtubule or the plus end region thereof, which method comprises introducing into a host cell, tissue or organism a transgene comprising a sequence capable of expressing said UNC-53 vertebrate homologue according to the invention, which sequence is operably linked to a sequence encoding said protein to be targeted such that a chimeric protein is expressed and which results in targeting of said protein to said microtubule or a plus end region thereof. An even further aspect of the invention comprises a method of identifying a molecule which covalently modifies UNC- said vertebrate homologue according to the invention, which method comprises a) contacting either an extract from a cell or cells expressing said vertebrate homologue or a mixture of enzymes comprising candidate UNC-53 modifying enzymes in the presence of an indicator of covalent modification of a protein, b) identifying any covalently modified UNC-53 protein from step a) and c) identifying said molecule involved in said modification step. Such an indicator may be 32P.
Further provided by the invention is a method of identifying a compound which alleviates or enhances the toxicity of said UNC-53 vertebrate homologue thereof according to the invention, or which alleviates or enhances apoptosis. The method of the former comprises contacting said compound with a transgenic cell, tissue or organism according to the invention and monitoring for the presence of said reporter molecule adjacent said microtubules or the plus end region thereof. In the case of apoptosis the method comprises monitoring the effect of the compound on cell death.
The invention may be more clearly understood from the following examples which are purely exemplary, with reference to the accompanying drawings wherein,
Figure 1(a) is an illustration of the nucleotide sequence encoding the first human homologue of UNC-53 designated Hs-UNC-53/1 and further variants thereof. Figure 1 (b) is an illustration of the amino acid sequence of hs-UNC-53/1 encoded by the sequences in Figure 1 (a) .
Figure 1(c) is an illustration of the nucleotide sequence encoding the second human homologue of UNC-53 protein of C. elegans designated Hs-UNC-53/2 and further variants thereof.
Figure 1 (d) is an illustration of the amino acid sequences of Hs-UNC-53/2 encoded by the sequences in Figure 1(c).
Figure 1 (e) is an illustration of a nucleotide sequence encoding the third human homologue of UNC-53 protein according to the invention designated Hs-UNC- 53/3, and variants thereof.
Figure 1(f) is an illustration of the amino acid sequences of the Hs-UNC-53/3 encoded by the sequences of Figure 1 (e) . Figure 1(g) is an illustration of the nucleotide sequence of a genomic DNA fragment that contains a putative 5' exon of Hs-unc-53/1.
Figure 1 (h) is an illustration of the nucleotide sequence AB023155 encoding the protein KIAA0938, a transcript comprising the 3' half of Hs-unc-53/3.
Figure l(i) is an overview of the C. elegans and human UNC-53 proteins as cloned. The 5' truncated variants and a number of the known splice variants have been indicated. Figure 2 is an alignment of the amino acid sequences of Ce-UNC-53, Hs-UNC-53/1 , Hs-UNC-53/2 and Hs-UNC-53/3.
Figure 3 is an alignment of the C. elegans unc-53 and the predicted amino acid sequence of C. briggsiae unc-53.
Figure 4 is a list of ProSite signatures for vertebrate UNC-53s based on the sequence alignment.
Figure 5a is an illustration of expression of the three human UNC-53s as studied by Northern blotting. Figure 5(b) is an illustration of differential expression of Hs-unc-53/3 in different brain parts.
Figure 6(a) is an illustration of differential splice variant expression of Hs-unc-53/1 using RT-PCR.
Figure 6(b) is an illustration of differential splice expression of Hs-unc-53/2 using RT-PCR.
Figure 6(c) is an illustration of differential expression of Hs-unc-53/3 using RT-PCR. Figure 6(d) is a sequence confirmation of AB023155 expression in cells other than brain using RT-PCR.
Figure 7(a) is an illustration of the cloning of Hs-unc-53/3.
Figure 7 (b) is a plasmid map and the nucleotide sequence of the pGI3303 expression vector ( C-terminal Hs-unc-53/3 fragment in fusion with GFP) .
Figure 7(c) is an illustration of the amino acid sequence of GFP: C-terminal Hs-unc-53/3 fragment (insert of pGI3303) .
Figure 7 (d) is a plasmid map and the nucleotide sequence of the pGI3305 expression vector (full length Hs-unc-53/3 in fusion with GFP) . Figure 7 (e) is an illustration of the amino acid sequence of GFP : Hs-unc-53/3 (insert of pGI3305) .
Figue 8 is an illustration of the filipodia and lamellipodia outgrowth of N4 mouse neuroblastoma cells transfected with pGI3303 (F-actin cytoskeleton reorganisation)
Figure 9 is an illustration of the co- localisation of the GFP:Hs-unc-53/3 fusion protein with microtubules in N4 mouse neuroblastoma cells transfected with pGI3305. Figure 11a is an illustration of the homology domains between Hs-unc-53/3 and a gene encoded (partially) by the Drosophilia melanogaster BAC clone BACR48M05 (AC005719) . Results of a TBLASTN search on the non-redundant database with Hs-unc-53/3 as query. Figure lib is an illustration of an ORF encoded by the Drosophila melanogaster BAC clone BACR48M05 (AC005719) as predicted by the computer program Fgene.
Figure lie is an illustration of a "BLAST 2 sequences" search result with Hs-unc-53/3 as query and the Fgene predicted UNC53 homology ORF of D. melanogaster BAC clone BACR48M05.
Figure 12 is an illustration of a zebra fish EST encoding Dr-unc-53/2.
Figure 13 Genemap98 results for Hs-unc-53/2.
Figure 14 is a schematical drawing of the sequence of the exon containing the putative alternative start codon of human Hs-unc-53/1.
Figure 15 is an illustration of the nucleotide sequence of pGI3150 and the amino acid sequence of the eGFP fusion with a C-terminal fragment of Hs-Unc-53/1.
Figure 16 is an alignment of EST clone yk480b6 and Ce-unc-53 demonstrating a novel splice variant of Ce-unc-53.
Figure 17 is a graphical display of the effect of Hs-unc-53/3 GFP chimera transient transfection on the form factor of N4 cells.
DEPOSITED MATERIAL
Plasmids pG13303 and pG13305 were deposited under accession numbers LMBP3936 and LMBP3937 respectively on 28 May 1999 at the Belgian Coordinated Collections of Microorganisms (BCCM) at Laboratorium voor Moleculaire Biologie - Plasmidencollective (LMBP) B- 9000 Ghent, Belgium, in accordance with the provisions of the Budapest Treaty of April 28 1977.
Hs-UNC-53/3 is a bona fide UNC-53 (fig. 1; 2; 3)
Blastn and Tblastn EST-database mining using the sequence of the already known animal UNC-53s led to the identification of 3 ESTs suggestive of novel unc- 53s (see experimental procedures). By 3'- and 5'- RACE extension using suitable libraries, it was shown that these ESTs identified a novel unc-53 designated Hs-unc-53/3 (Fig. 1 e; f) . The publication of the sequence AB023155 (Nagase et al. 1999, DNA Res. 6:63- 70) independently confirmed the correctness of the 3'- end of Hs-unc-53/3 as well as the existence of one new intron that forms the 5' -end of AB023155. Alignments of the C. elegans and 3 human UNC-53 sequences (fig. 2) clearly illustrates that the third human homologue of C. elegans UNC-53 protein is a bona fide UNC-53 with highest similarity to Hs-UNC-53/2 and in decreasing order to Hs-UNC-53/1 and (C. elegans UNC- 53) Ce-UNC-53.
Many of the domains of Hs-UNC-53/3 show highest similarity to functional domains of other animal UNC- 53s (fig. 2) . This critically suggests that Hu-UNC- 53/3 most likely has the key functionalities observed for Ce-UNC-53 in a variety of assays including F-actin binding, F-actin reorganisation in cell culture, microtubule and microtubule (+)-end binding in cultured cells, binding of SH3-domain adapters like
SEM-5/GRB-2 or other types of binders of proline rich alpha-helices. These results indicate that like Ce- UNC-53, Hs-UNC-53/1, Hs-UNC-53/2, or Hs-UNC-53/3 can be used in a range of biochemical, cellular and animal assays aimed at discovering tissue- or disease- specific modulators of Hs-unc-53 functioning in diagnostic assays.
Further extension of the Unc-53 family (Fig. 11, 12)
Database searches with the three human UNC-53 protein sequences revealed several expressed sequence tags (ESTs) and genomic DNA sequences (BACs) that show significant similarlity to human UNC-53.
C. briggsiae
The C. elegans genome consortium sequenced the locus of the C. briggsiae unc-53 homologous gene. Through gene prediction programs and the cDNA sequence of the C. elegans unc-53, prediction can be made for the C. briggsiae protein sequence. Alignment of the derived C. briggsiae amino acid sequence with the C. elegans amino acid sequence in figure 3 demonstrates the strong homology of both proteins.
D . melanogaster
BAC clone BACR48M05 (AC005719) clearly contains 3 different exons with high homology to Hs-unc-53/3 (Figure 11) . Using the gene structure prediction program Fgene [Solovyev et al., 1995, in: Proceedings of the Third International Conference on Intelligent Systems for Molecular Biology (eds. Rawling et al., Cambridge, England, AAAI Press) ; Solovyev and
Lawrence, 1993, in: Abstracts of the 4th annual keck symposium. Pittsburgh, 47) it was possible to predict an ORF encoded by BAC clone BACR48M05 that shows homology to Hs-unc-53/3 (Figure lib) . However, every Drosophila cDNA partially or entirely encoded by BAC clone BACR48M05 and which contains one or more sequence blocks as indicated in figure 11a should be considered as a family member of the UNC-53 family. A "BLAST 2 SEQUENCE" search indicates that the sequence situated between the three homology blocks that are indicated in figure 11a is less conserved between human and Drosophila (Figure lie) . The predicted ORF of the Drosophila melanogaster UNC53 gene can be used to identify new members of the family. The zebrafish EST fc21d06 (AI658309) shows an identity of 84% and a homology of 92% to Hs-UNC-53/2. It clearly can be considered as a part of the zebrafish homologue of Hs- UNC-53/2 (Figure 12) . Finally, a whole series of human ESTs have been placed in public domain databases. To our knowledge, no one has been able to place these ESTs into contigs that describe a true Hs- unc-53 to a level presented in this specification. The presently available unc-53 sequences - expressed or genomic - further underscore that the unc-53 gene family is a true animal gene family in helminths, vertebrates and arthropods, three major classes of the animal kingdom.
Refined UNC-53 family description based on alignment (fig. 4) .
The alignment of the three human and the C. elegans UNC-53 sequences enables the more refined definition of conserved regions in UNC-53s. In figure 4 there are compiled a number of proSite signatures for either the four animal or the three human UNC-53s.
Differential expression of Hu-UNC-53/3 by Northern blot (fig. 5) .
To determine in which cells and tissues the vertebrate UNC-53s play a role, a northern blot analysis has been performed. As indicated in the experimental section, relevant probes were amplified and used to visualise in which normal human tissues and in which cancer cell lines the three human UNC-53s were expressed.
1. A cancer cell line RNA blots probed with Hs- Unc53/1.
A Northern blot of poly-A+RNA from several cancer cell lines (Melanoma G361, Lung Cancer A549, Colorectal Adenocarcinoma SW480, Burkitt Lymphoma DRajii, Leukemia Molt4, Lymphoblastic Leukemia K562, HeLa S3 and Promyelocytic Leukemia HL60) was probed using the whole insert of pHH3b. No or weak expression was detected in the Burkitt Lymphoma DRajii, the Leukemia Molt4 and the Promyelocytic Leukemia HL60 cell lines. Five different transcripts are detected in the remaining cancer cell lines: transcripts 1 and 2 are larger than 9.5kb, transcripts 3 and 4 are 6 to 7 kb and the fifth transcript is around 6 kb. Transcripts 1 and 2 are present in all expressing cell lines but at different levels.
Transcripts 3 and 4 are restricted to Melanoma G361, Lung Cancer A549 (weak) and Colorectal Adenocarcinoma SW480 and are the predominant transcripts in Melanoma G361 and Colorectal Adenocarcinoma SW480. Transcript 5 is restricted to Lymphoblastic Leukemia K562 (weak) and (predominant) in HeLa S3 and is predominant in HeLa S3.
2. Cancer cell lines RNA blots probed with Hs- Unc53/2.
A similar set of cancer cell line Northern blots were probed with a 652bp fragment of EST46037 amplified by using the primers 5'- aggagatgaagctgacagatatcc and 5' -aaacaccagtgagtcc. Hs- Unc53/2 is expressed in Melanoma G361, Colorectal
Adenocarcinoma SW480, Lymphoblastic Leukemia K562 and HeLa S3. No expression was detected in Lung Cancer A549, Burkitt Lymphoma DRajii, Leukemia Molt4 and promyelocytic leukemia HL60. Interestingly only 2 transcript sizes were detected of around 7 kb expressed in Lymphoblastic Leukemia K562 and HeLa S3 and a transcript of >9.5 kb in Melanoma G361 and Colorectal Adenocarcinoma SW480 and weakly in HeLa53. Noteworthy is the very high expression in melanoma G361.
3. Normal Human tissue probed with Hs-Unc53/1. A Northern blot of poly-A+RNA from normal human tissue was probed using the whole insert of phage HH3b. Expression levels are low in all tissues with the highest level in heart and placenta, several fold lower levels in brain and testis, even lower levels in skeletal muscle, pancreas, thymus, colon, small intestine, ovary and prostate. Expression in peripheral blood leukocyte, lung, liver, kidney, spleen is barely detectable.
4. Normal Human tissue probed with Hs-UNC53/2. A similar set of blots were probed with a 652bp fragment of EST46037 amplified by using the primers 5' aggagatgaagctgacagatatcc and 5'- aaacaccagtgagtcc. Expression levels are low in all tissues with the highest level in kidney, placenta and pancreas, lower levels in heart and lung. Expression is barely detectable or undetectable in skeletal muscle, spleen, thymus, prostate, testis, ovary, small intestine, colon peripheral blood leucocyte, stomach, thyroid, spinal cord, trachea, adrenal gland and bone marrow. Also Hs-unc-53/2 appears to be expressed as different transcripts (figure 5a).
The hs-UNC53/l and hs-UNC-53/2 homologues are clearly highly regulated genes, showing a strong tissue specificity and, probably, additional mechanisms of regulation (ie differential splicing of different promoters) . The different proteins derived from RNA' s identified by probe hhl5 presumably share the carboxyterminal nucleotide binding domain.
Ce-UNC-53 was shown to be a complex genetic locus and complex transcription unit. The different transcripts are thought to be a mechanism to assure the necessary specificity and functional diversity of this signal transduction pathway, with respect to different signals and receptors, different tissues and different directions of migration. The occurrence of a new transcript or the observed changes in expression levels in the cancer cell line blot suggests a role for hs-UNC-53/3 in the establishment or maintenance of the transformed state of those cells. Expression pattern of hs-UNC-53/3.
A northern blot of poly-A+RNA from several cancer lines was probed with unique fragments of the three genes from the Hs-unc-53 family. Hs-unc-53/3 has a high expression level in lung carcinoma line A549, where only a moderate expression of hs-unc-53/1 has been detected. Furthermore, moderate expression of Hs-unc-53/3 was also observed in melanoma line G361, where previously, a high expression of hs-UNC-53/1 and hs-UNC-52/2 has been observed. This indicated the involvement of hs-unc53/3 in at least two cancer lines .
In normal human tissues, the expression of hs- unc-53/3 shows a clearly new and previously unobserved expression pattern. This difference of expression of hs-unc-53/3 in relation to its homologues hs-unc53/l and hs-unc53/2 is important for the allocation of functionality to hs-unc-53/3. Hs-unc-53/3 is highly expressed in brain, as shown on the Northern blots (figure 5a) . In figure 5b it can be seen that Hs-unc-53/3 also is differentially expressed in different parts of the brain. Its homologues are not or weakly expressed in brain. This gives an indication that its function in directionality of cell migration and growth cone steering will be in relation to specific regions or cells of the brain. It is deduced that Hs-unc-53/3 will be an important signal transducer or signal adapter linking signals to neuronal outgrowth, axon guidance, and formation and maintenance of synaptic connections. It seems that the function of Hs-unc- 53/3 will be associated with neuron-neuron interactions, neuronal outgrowth, neuron muscle interactions, and post-synaptic signal transduction. Furthermore, Hs-unc-53/3 may be involved in the development of cancer of neuronal origin, like neuroblastomas, or the development of tumours will have their developmental origin in the brain as some eyes diseases like retinoblastomas .
The significance of the high expression of Hs- unc-53/3 in brain tissue can be associated with the high levels of expression which has also been observed in the spinal cord, containing neuronal tissue. Here, neuronal (axon) outgrowth and neuron-neuron connections are of importance. Development of pharmacological tools acting on this pathway may lead to treatments of diseases involved in the growth and movement of neuronal cells, and the regeneration of neuronal connectivity after trauma, or the inhibition of neuronal cancers such as neuroblastomas. Due to its specific expression, inhibitors and/or enhancers specific for Hs-unc-53/3 will have an advantage as a pharmaceutical compound over more general compounds acting on the Hs-unc-53 family of genes and proteins. A second tissue where hs-UNC-53/3 is highly expressed and where (its) other human homologues are not expressed is the spleen. Hs-UNC-53/3 could therefore function as part of the signal transductions pathway involved in the maturation of leukocytes. Malfunction of this pathway may lead to incorrect maturation of the leukocytes and the development of autoimmune diseases such as rheumatoid arthritis and sclerosis. Next to the signalling function in the recognition of the leukocytes, Hs-UNC-53/3 may also play an important role in the induction and/or signalling pathway of the mechanism underlying apoptosis of leukocytes in the spleen. Pharmaceutical methods involving the hs-UNC-53/3 pathway, which may, for example, result in an inhibition and/or enhancement of its expression may lead to treatment of these disorders. Furthermore, hs-UNC-52/2 may have an advantage, as an inhibitor or enhancer specific for hu-unc53/3 which will act in a more specific manner. The Hu-UNC-53/3 protein is also highly expressed in the ovary, where the two other human homologues are also expressed. Finally moderate to low expression of hs-unc53/3 is observed in heart, placenta, testis, stomach and adrenal gland.
Although the predominant transcripts of Hs-unc- 53/3 are > 9 kb, often a smear occurs that ends at with somewhat higher intensity at 5.5 - 6.5 kB. This short transcript may correspond to AB023155. The Hs-unc53/3 gene is a highly regulated gene, showing strong tissue specificity and additional mechanisms of regulation which have not previously been identified in any of its known homologues. These findings may thus lead to the development of more specific inhibitors or enhancers of hs-UNC-35/3 and or of the Hs-UNC-53/3 pathway. The Northern blot studies indicate that the three human unc-53s are complex transcriptional units with highly regulated tissue specificity and that transcripts of different lengths exist.
Splice variants of human unc-53s
Whilst cloning Hs-unc-53/3, it became apparent that at least three expression variants of Hs-unc-53/3 - most probably alternative splices - exist (fig. le, f; lowercase regions) . Targeted efforts for the two other human UNC-53s demonstrated that the other human UNC-53s contained variants (fig. la, c and e regions). Splice variants as observed to date appear to be concentrated in specific regions. A first one (starting at position 1252 in fig. 2) - in which the overall amino acid similarity is weak - contains 2 (splice) variants of both Ce-unc-53 and Hs-unc-53/3. In the worm, the presence or absence of these 2 exons in unc-53 regulates the function of the UNC-53 protein in such a way that cells differentially translate extra-cellular signal gradient as an attractive or repulsive signal. The most 3' -variant of Hs-unc-53/2 roughly covers the 2 Ce-unc-53 variants.
The complexity of variation in this zone of Hu- UNC-53 might resemble the situation in the nematode. In Hs-unc-53/3, for example, the region from position 3795 to 4325 (figure le) consists of two adjacent blocks (3795 to 4283 and 4286 to 4325 in figure le) that can independently be present in or absent from cDNAs from frontal cortex tissue. In contrast, no variants were as yet observed in this zone for Hu-UNC- 53/1 or /2.
The second variant in Hs-unc-53/3 (fig. 2) deletes a box (MQLDNRTLPKKGLR) , which is extremely conserved (in bold) among all human unc-53s. This occurrence of this variant could indicate differentially active functional variants of Hu- unc53/3.
A second region in which splice variants were observed contains a major highly conserved domain of unc-53s. Hs-unc-53/1 has a first variant that comprises the most N-terminal portion of this conserved domain (SGSFRD) . A second splice variant in Hs-unc-53/1 (AEERMOSE) lies within the highly conserved domain. Another conserved spot for splice variation in human unc-53s has been found (figure 2): Hs-unc-53/1 {VYE}; -/2 {VNE} and -/3 {NSRGSEL} . All these spliced exons are flanked by two conserved charged domains - putative nuclear localisation signals. Given this conservation, we searched for splice variation in C. elegans and found it to exist in the form of an extra exon (ALSVDSQ) (figure 2) . Hu-unc-53/3 has another variant (SPLVWPPKKRQNGPVIYKHSR) (fig. 2) . The most 3' splice variant in Hs-unc-53/3 has been discovered whilst cloning Hs-unc-53/3 and was shown to be present uniquely in human heart cDNA libraries .
Single nucleotide polymorphisms
Cloning and PCR studies indicated the existence of a non-silent single nucleotide polymorphism in Hs- unc-53/1 in position 1232 and in Hs-unc-53/2 in position 929. This indicated that variations exist in human unc-53s which - in some cases - may be relevant to the proper functioning of the UNC-53 protein and hence in disease.
Expression in normal and neoplastic cells by RT- PCR
The cloning efforts demonstrated the existence of splice variants in the human unc-53s and the Northern blots revealed a range of transcripts for each human unc-53. The combined data do not explain completely the range of transcripts observed. Therefore, our understanding of the expression complexity of human unc-53s may be incomplete and more detailed RT-PCR studies were performed.
One of the obscuring factors could have been that all studies performed on mRNA or cDNA of whole tissues which are built of different normal human cell types that occur in different proportions. For this reason and because skin was not covered in the Northern blot studies, a RT-PCR study was set up using cDNA preparations of the different cells in skin normal human: (1) epidermal keratinocytes, (2) melanocytes, (3) dermal fibroblasts. In addition, lineage matched transformed cell lines or tumour cell lines were included in the study to compare normal versus neoplastic cells. Human umbilical vein endothelial cells (HUVEC) were taken as a normal human match for endothelial cell lines. The RT-PCR study for Hs-unc-53/1 revealed that the most 5' -splice variant is differentially expressed in normal versus neoplastic cells/cell lines. This exon is present in 7/7 keratinocytes, HUVEC and in melanocytes but lacking in HaCat, ECV304, 2/7 melanoma and MCF-7 cells (breast carcinoma) .
The RT-PCR study for Hs-unc-53/2 revealed a more surprising picture. The tumourigenic endothelial line ECV304 lacks expression of Hs-unc-53/2, whereas their normal counterpart HUVEC expresses Hs-unc-53/2, suggesting gene deletion or inactivation of expression in ECV304. In epidermal keratinocytes and the lineage matched spontaneously transformed keratinocyte HaCaT and MCF-7 lack expression of the 5' -end of Hs-unc- 53/2, but express the 3' end (starting in or near the microtubule-binding domain) . This suggests that like AB023155 for Hs-unc-53/3, also Hs-unc-53/2 can be expressed as a truncated 3' -variant in a cell-specific way. Also splice variation of Hs-unc-53/2 appears to differ in a normal to neoplastic way: the {VNE} exon was shown to be present in all keratinocyte isolates but not in HaCaT and also melanocytes express it, but not 2/7 melanoma or MCF-7. The RT-PCR studies for Hs- unc-53/3 were focussed on demonstrating expression of AB023155 in tissues other than brain. The new exon described was shown to be present in keratinocytes, HUVEC, dermal fibroblasts, melanocytes and their transformed/neoplastic variants, demonstrating its wide expression in tissues in man.
Alternative 5 ' -start exons
For Hs-unc-53/2 five different start exons have been cloned using RT-PCR, three of which have been confirmed to be present in at least 2 different cDNA libraries (figure lb, c) . Likewise for Hs-unc-53/3 different 5' -exons were found, two of which were confirmed (figure le, f) . These 5'-exons most probably indicate that human unc-53s are being expressed via the control of alternative promoters that lie 5' of these different 5' -exons. Also in the nematode has been shown that different (intronic) promoters are driving the expression of 5' -variants of C. elegans unc-53.
The Hs-unc-53/1 5' -end
Despite considerable efforts, cloning has not lead to the identification of a bona fide 5' -end for Hs-unc-53/1 that comprises an F-actin binding domain, despite the fact that the Northern blots indicate the existence of transcripts > 9.5 kb. Given that both Hs-unc-53/2 and -/3 are expressed as full length and truncated forms, the question can be raised whether Hs-unc-53/1 may not be expressed in a short form as well . cDNA library cloning and 5' -RACE has provided contiguous sequence that ends at a position that matches with a domain in C. elegans un-53, where an alternative start position lies. Based on this argument, Hs-unc-53/1 could be a functional equivalent in man of this transcript in nematode.
To further trace the "longer" variants of Hs-unc- 53/1, genomic BAC DNA sequencing has been performed. In figure lg is shown sequence of a4984 fragment from BAC 585E09. It comprises sequence 5' of the presently known cDNA of Hs-unc-53/1. To the qualified as well as by means of two groups of gene structure prediction computer programs, different but comparable exons in the 4984 bp genomic sequence fragment can be predicted (figure 14) . The programs GENSCAN, HEXON and MZEF all predict an exon between bp 1089 and bp 1880. The end of this predicted exon (bp 1880) is confirmed by the cDNA sequence. Therefore this predictions has a big change to indicate the correct exon length. The programs GRAIL, GENEFINDER and HMMGENE all predict an exon between bp 1123 and bp 2031. None of the predicted exons contains an in frame stop codon 5' of the alternative start codon. Consequently, it is possible that there exist unidentified exons 5' of the exon containing the alternative start codon.
The present picture critically suggests that both nematode and human unc-53s appear to be complex transcriptional units. Moreover, the fact that some of the most complex splice variants map to similar regions in the UNC-53 proteins points to evolutionary conserved functional variants of UNC-53s e.g. with regard to the cells directional migration towards or away from a signal source. In contrast, some of the variants in the human UNC-53s are located in highly conserved domains; these (and other) variants may create discrete - yet undiscovered - functionally different UNC-53 proteins transcribed from one of the unc-53 genes.
The fact that two and maybe three human unc-53s exist as full size and a truncated forms with cell- specific expression, that series of alternative 5'- start exons exist eventually controlled by different promoters that some forms of splice variation are conserved from nematode to man, all indicate that the expression of unc-53s is of very high complexity and that some of the biological functions of UNC-53 proteins are extremely conserved. On the other hand, the differential expression in Northern blots, the splice variation difference between normal and lineage-matched neoplastic cells and the non-silent single nucleotide changes in two of the three human unc-53s, all indicate how important a wide range of diagnostic assays can be to understand in depth the role in disease of human unc-53s. Chromosomal localization of Hs-unc-53/2 by Genemap98 (Fig. 13 and 1(c))
The EST clones AA918601, AI248585, AA115014 and AA115015 are clearly homologous to the 3'-UTR of Hs- Unc-53/2 cDNA (Figure 1(c))). Although, AA115014 (describing the same EST as AA115015) contains an alternative splice variant of the Hs-Unc53/2 gene in the 3'UTR. A survey with ESTs AA918601, AI248585, AA115014 or AA115015 as query in the genemap98 database (release November 1998) revealed that the Hs- Unc53/2 gene is located at chromosome 11 (http_j_//www.ncbi .nlm.nih.gov/genemap98/loc.cgi?ID=2122 4) . The STS which is used for chromosomal localization and which is situated in the 3'UTR of the Hs-Unc53/2 gene is referred to as SHGC-33456 (dbSTS Id: 41891, Genbank Ace: G28036, Genbank gi : 1396755) (Figure 13a) . The STS was localized by analysis on the NIGMS human/rodent somatic cell hybrid panel (dbSTS Id: 41891) . The Radiation hybrid results are summarized in Figure 13b. Together these data imply that every disease or phenotype connected to SHGC- 33456 is due to the Hs-Unc-53/2 gene.
Functional Characterisation of Hs-unc-53/3
F-actin reorganisation and microtubule binding of Hs-unc-53/3
Based on its structural features, Hs-unc-53/3 can be classified as a bona fide human unc-53. To further understand its function and in anticipation of developing pharmacological compound screening assays, Hs-unc-53/3 has been physically cloned following the method described in the experimental section and shown in figure 7a. The derived Hs-unc-53/3 clones comprising full length (A to L and the 3' -half (G to L) of Hs-unc-53/3 were further engineered to form a chimera with green fluorescent protein and cloned into expression vectors appropriate for transfection of eukaryotic cells. The nucleic acid and amino acid sequences of these constructs are shown in figure 7b- e. The constructs were transfected into cells and scored for their effects on the F-actin cytoskeleton and binding to microtubules of mouse neuroblastoma cells N4; functions known for nematode unc-53 and human unc-53/1.
The N4 cell transfected with a GFP fusion to the 3' -half of Hs-unc-53/3 (pGI3303, fig. 7b) showed pronounced filopodia and lamellipodia outgrowth, which is associated with reorganization of the F-actin cytoskeleton (Figure 8). This observation demonstrates that like nematode unc-53 and human unc- 53/1, the F-actin binding domain is not required for inducing reorganization of the F-actin cytoskeleton of N4 cells. In addition, the pGI3303 encoded fusion protein does not co-localize with microtubuli but localizes to the cytoplasm of N4 cells indicating that an important domain for microtubuli association is missing in this C-terminal fragment of Hs-unc-53/3. In the alignment figure 2 can be seen that the C- terminal half of Hs-unc-53/3 (approximate KIAA0938) does not comprise the conserved microtubule binding domain.
In contrast, the N4 cells that expressed low to medium levels of the GFP fusion to full length Hs-unc- 53/3 (pGI3305, Fig. 7d) displayed a co-localization of the GFP fusion protein with microtubules (Figure 9) . Even the centrosomes could clearly be detected in some transfected cells. Cells expressing very low amounts of the fusion protein displayed specific microtubule (+)-end binding (Figure 9). The morphology of the pGI3305 transfected N4 cells does not clearly differ from the control transfected cells although there is a tendency towards rounding up of the pGI3305 transfected cells and filopodia outgrowth.
Validation of functional assays as compound screens
R74288 has previously been shown to be an inhibitor of nematode function in C. elegans (W096/38555) , an activity that has been confirmed in Ce-unc-53 transfected N4 cells, where only the transgene-induced effect was inhibited by R74288. In order to confirm compound R74288s activity in a full mammalian system, a stable transfection of plasmid pGI3150 was performed in the N4 neuroblastoma cell line with the lipofectamin procedure (Gibco BRL) . pGI3150 expresses an eGFP protein in fusion with the C-terminal end of Hs-unc-53/1 (see Figure 15a) . After two weeks of G418 selection, 20 clones with stable integration of the pGI3150 plasmid were selected and isolated. These clones were tested for GFP expression by fluorescence microscopy and by Western blotting with an anti-GFP antibody (table 1) . The lamellipodia outgrowth phenotype was checked visually (See Figure 15b) . Compound R74288 was tested on four random selected pGI3150 stably transfected clones: 8.1, 8.2, 8.3 and 10.1 and on a pool of pEGFPCl stable transfected N4 control cells. Clones 8.2 and 10.1 displayed less lamellipodia outgrowth than clones 8.1 and 8.3. Compounds and solvents were added to the stably transfected cells (105M in DMSO) . After 24 hrs of incubation, two persons independently scored the effect of the treatments on the cells. As shown in table 1, both persons noticed an effect compound 2 on clones 8.2 and 10.1 with a weak transgene-induced lamellipodia phenotype. This effect consisted of a more flat morphology of the treated versus untreated cells. Compound 2 was R74288. Table 1. Effect of compounds on lamellipodia formation
Figure imgf000045_0001
Automated compound screening by measuring cell morphology
Compound screening assays must have a sufficiently high throughput to be relevant to drug discovery. To achieve this goal, we automated the procedure of measuring the morphological changes induced in cells following transient transfection with full length or 3' -half of Hs-unc-53/3 GFP chimeras.
The cell culture, transfection, fluorescence staining and microscopy procedures are performed within a 96- well plate (all-in-one) . The fluorescent staining method comprises a triple fluorescent labeling procedure (1) for cell nucleic using DNA double helix intercalating dyes such as Hoechst 33342 or DAPI, (2) for transfection efficiency and expression level of the chimeric protein using GFP fluorescence and (3) for the F-actin cytoskeleton using fluorescently labeled phalloidin, a microfilament dye.
These three different fluorescent images are collected using an motorised stage plus stage driver and a frame grabber that produces seamless composite images of the cells in the well. The software programs to drive this operation are known in public domain as "SCIL" (University of Amsterdam) . The seamless images are then superimposed using pseudocolour for the operator to inspect the quality of the culture. In addition, the SCIL program was compiled in such a way that it: (1) identifies cells by means of their nucleus, (2) measures the GFP fluorescence intensity, (3) delineates the area of the F-actin (phalloidin) staining surrounding a nucleus and (4) calculates a range of parameters objectively representing the features of the F-actin staining pattern of each individual cell. One example of such a parameter is called the "form factor". It is an arbitrary value that reflects the dendricity of a cell. It is derived by calculating (A) the true circumference of a cell's F-actin staining area as seen in the image and (B) the area of the F-actin staining of that given cell. The ratio 4xPIx(B)2= the form factor. For a rounded cell, the form factor approximates 1 whereas, for a cell with increased filopodia and lamellipodia outgrowth, the true circumference will be much larger than that of a circle and as a result, the form factor « 1.
In experiments it was shown that transiently transfected N4 cell populations indeed displayed a different form factor versus control cells. Both the median and average form factor for a cell population in a well were reduced following transfection with the 3' -half of Hs-unc-53/3. More in particular, there was a significant decrease in the number of cells in a transfected culture that displayed the minimal form factor, suggesting that the Hs-UNC-53/3 transgene induced round cells in particular to become more dendritic (figure 16) .
Chromosomal localisation of Hs-unc-53/3 by FISH indicative for a role disease
With FISH technology using a unique fragment of hs-unc-53/3 we are able to localize the hs-unc53/3 gene on chromosome 12q21.1. Chromosome 12q21.1 is a region shown to be involved in autosomal dominant, cornea plana and closed angle glaucoma (Sigler- Villanueva et al., Ophthalmic Genetics 18:55-62, 1997) . This indicates that hs-UNC-53/3 protein may be involved in eye development and thus eye diseases, such as retinoblastomas . Neuroblastoma cell line NPG and liposarcoma line WDLPS and other sarcoma lines have amplifications in this region. The neuroblastoma amplification seems to be located more distal (12q24) while the liposarcoma line is located at 12q21 (Van Royal et al . , Cancer Genetics and Cytogenetics 82:151- 4, 1995). Three loci related to Darier' s disease, an autosomal dominant genodermatosis disease characterized by epidermal acantholysis and dyskeratosis have been mapped in region 12q21-q24 (Wright et al . , Journal of Investigative Dermatology 103:665-8). 12q21 is also known to be a fragile site associated with the pathogenesis of non-Hodgkin' s Lymphoma (Chary-Reddy et al . , Cancer Letter 86:111-7 1994) . Duplications related to nephroblastoma tumorgensis were commonly found in the 12q21-q23 region (Austruy et al . , Genes Chromosomes Cancer 14:285-294, 1995). In a girl with mental retardation, a conclusive disorder and clinical findings resembling cerebral palsy, positioning of segments from other autosomes adject to the band 12q21 were found (Biederman et al., Ann Genet 19:257-260, 1976). Cytogenetic analysis for myeloid leukemia showed a complex caryotype with chromosomal breakpoints at
12q21 (Weinstein et al . , Cancer Genet Cytogenet 48:75- 81, 1990) . Finally, analysis of complex chromosomal rearrangements in malformed children and from spontaneous abortions showed specific breakpoints at site 12q21 Gorski et al., Am J Med Genet 29:247-261, 1997) . Most of these diseases have been shown to be involved with cell movement, aberrant development, or cell-cell contact and neuronal tissue or neuronal development.
Confirmation of FISH with Radiation hybrid panels
To confirm and refine the chromosomal localisation of the human unc-53s an alternative method for FISH has been used. Radiation hybrid (RH) mapping is a somatic cell hybrid technique that was developed to construct high-resolution, contiguous maps of mammalian chromosomes. RH mapping provides a method for ordering DNA markers spanning millions of base pairs of DNA at a resolution to easily obtained by other mapping methods. Some of the advantages of RH mapping are (1) distance estimated by this method is directly proportional to physical distance, (2) nonpolymorphic DNA markers, that can not be used for meiotic mapping, can be used for this method, and (3) a high resolution map that is not easily made by other methods can be obtained.
The results of FISH and RH mapping for the three human unc-53s are summarised in table AA. By using publicly available databases (see experimental section) one can derive information on the correlation between FISH and RH mapping. RH Mapping was shown in this way to confirm the FISH data for the three unc- 53s.
Table 2. RH Mapping Primers and Results
Figure imgf000049_0001
list not exhaustive
Also sequence information available in public domain can help refine the positioning of the unc-53 genes, like in the following example. The EST clones AA918601, AI248585, AA115014 and AA115015 are clearly homologous to Hs-Unc53/2 cDNA. Although, AA115014 (describing the same EST as AA115015) contains an alternative splicevariant of the Hs-Unc53/2 gene in the 3'UTR. A survey with ESTs AA918601, AI248585, AA115014 or AA115015 as query in the genemap98 database (release November 1998) revealed that the Hu- unc53/2 gene is located at chromosome 11 (http: //www.ncbi.nlm.nih.gov/genemap98/loc.cgi?ID=2122 4) . The STS which is used for chromosomal localization and which is situated in the 3'UTR of the Hs-Unc53/2 gene is referred to as SHGC-33456 (dbSTS id: 41891, Genbank Ace: G28036, Genbank gi : 1396755) (Figure 13) . The STS was localized by analysis on the NIGMS human/rodent somatic cell hybrid panel (dbSTS id: 41891) . The radiation hybrid results are summarized in Figure 13. Together these data imply that diseases or phenotypes connected to SHGC-33456 is due to the Hs-Unc53/2 gene.
EXPERIMENTAL PROCEDURES
Cloning & sequencing of Hs-unc-53/3
Hs-unc53/3 has been cloned starting from a series of ESTs that were similar but not identical to Hs-unc- 53/1 or -/2. The ESTs were:
1. WashU-Merck EST 767735.
Transformed cells carrying the EST 767735 sequence were ordered from Research Genetics. Plasmid DNA was isolated using standard protocols (Qiagen plasmid DNA isolation kit) , the sequence of the insert was determined.
2. ATCC cDNA clones 86459.
Transformed cells carrying the cDNA clone 86459 sequence were ordered from ATCC. Plasmid DNA was isolated using standard protocols (Qiagen plasmid DNA isolation kit) , the sequence of the insert was determined. 3. Genethon cDNA clone c09a03 from the Geneexpress cDNA program.
Transformed cells carrying the cDNA clone c09a03 sequence were ordered from Genethon. Plasmid DNA was isolated using standard protocols (Qiagen plasmid DNA isolation kit) , the sequence of the insert was determined.
These ESTs were extended to form one ORF as follows:
1. 5' extension of EST 767735 by RACE (Rapid Amplification of cDNA Ends) .
Marathon-Ready cDNAs (Clontech) are premade "libraries" of adaptor-ligated double-stranded cDNA ready for use as templates in RACE experiments. Five ml Marathon-Ready cDNA was used as template in a regular 50 ml RACE. The RACE mixture contained 1 x KlenTaq PCR buffer. 0.2 mM of each dNTP, 1 x advantage KlenTaq polymerase mix (Clontech), 0.15 mM API adaptor primer and 0.15 mM RACE gene specific primer. The amplification conditions were as follows: 94°C for 30 s and 68 °C for 4 min. One-hundred-fold diluted RACE product was used as a template in a nested PCR with AP2 adaptor and gene specific nested PCR primers. Specific nested PCR fragments were cloned into pCR2 (TA cloning kit, Invitrogen) and the sequences of the inserts were determined. Gene- specific primer (hh3UNC53 97101702) :
5'ACCATTTACACCTGAAGACGATTGAGGTCC; nested gene-specific primer (hh3UNC53 97101701) 5'CTCCTATTTAAATTAGAGGCTCCCTGGACC Marathon cDNA library: human placenta, human heart, human chronic myelogenous leukemia, human colorectal adenocarcinoma. 2. 3' extension of EST 767735 by RACE.
Method as described previously. Gene specific primer (hh3UNC53 97102702) 5'CAATCGTCTTCAGGTGTAAATGGTAACGTG; nested gene specific primer (hh3UNC53 97102703)
5'GAATGTCAAACACAGTGCCACCTCCACC Marathon cDNA library: human placenta, human heart, human HeLa, human melanoma.
3. 3' extension of cDNA clone c09a03 by RACE.
Method as described previously, gene- specific primer (hh3UNC53 98020401) 5'AGGGAGCACTGAATGGTCCAGACCATCCTC; nested gene-specific primer (hh3UNC53 98020402)
5'GCATCAGAAGACAGCATTCCTCTGAAAGTG Marathon cDNA library: human placenta, human heart, human HeLa, human melanoma, human colorectal adenocarcinoma, human chronic myelogenous leukemia.
4. 5' extension of cDNA clone 86459 by RACE
Method as described previously gene-specific primer (hh3UNC53 98020403)
5'TTCAATTTCTATCTCTATGAGTTTTCTTCG; nested gene-specific primer (hh3UNC53 98020404)
5'GCAGCTCTAGATTTGGTGATGAAGAAACTC Marathon cDNA library: human placenta, human heart, human HeLa, human melanoma. Overlapping sequences were assembled in a single contiguous sequence.
5. 5' extension cDNA clone 86459 by RACE (2) .
Method as described previously gene-specific primer (hh3UNC53 98022502) 5'TCAGAATGTGATGAAGGAGGCTTGGTGGAC; nested gene-specific primer (hh3UNC53 98022501)
5'GGATGCCGGAAGGGATGAATCAGTAAGC Marathon cDNA library: human placenta, human heart, human HeLa, human melanoma, human colorectal adenocarcinoma, human chronic myelogenous leukemia.
Validating variants at 5' end of the cDNA sequence
In the final 5' RACE experiment, 2 variants have been found whose sequence diverge upstream from the IYTDWAN protein sequence (position 289 in figure le or position 82 in figure If) . By using primers ATTTACACTGACTGGGCCAAC and ATAATCTGGATGATTTCTGCTAGGAGT on cDNA clones a Hs-unc-53/3 specific PCR product was obtained that was radiolabeled using the random primed DNA labeling kit (Roche Molecular Biochemicals) and hybridized to human DNA BAC filters (Research Genetics) . Both primers are located near the IYTDWAN box. Four BACs turned out positive (415J11; 464C17, 525C02 and 537B02) . DNA sequencing of the region upstream from the IYTDWAN protein sequence directly on these BACs showed that this region was preceded by a putative intronic sequence as evidenced by the multiple stop codons in the reading frame and by the consensus AG intron acceptor sequence. For sequencing purposes, BAC DNA was prepared according to a modified Qiagen plasmid DNA procedure. A primer pair was designed specifically to amplify the 5' end of the variant shown in full in figure le (primers ACTTGCTGAAACAGAGAGCTCCATG and CTTGCTGTCTTCTTTCTCCTTGGC) . PCR with these primers on BAC DNA showed the presence of the genomic sequence encoding this variant in 3 out of the 4 BACs (not present in BAC 415J11) . BACs containing the genomic sequence encoding the other 5' end variant of Hs-unc-53/3 as shown as the variant in figure le were identified by hybridizing the Research Genetics human DNA GAC filters with primer TGATCTTCTAGCGTGTGACTCACTG, radioactively labeled using gamma-P32-ATP and polynucleotide kinase. Positive BACs were 404F14, 450K18 and 764L15.
Sequencing directly on the respective BACs in the 3' direction from within the 2 alternative 5' exons and comparison of the genomic DNA sequence with the previously determined cDNA sequence identified the GT intron donor site. Joining of the genomic sequences from both 5' exons and the IYTDWAN encoding sequence after removal of the predicted intronic sequence restored for both variants the sequence of the 5' RACE experiment without affecting the translation of the Open Reading Frame .
Cloning of Hs-unc-53/3 constructs
With the aim of cloning the full-length Open Reading Frame of Hs-unc-53/3, primer pairs were selected such that the ORF could be amplified in 6 overlapping fragments ranging in size from 1 to 2 kbp. Overlaps between the fragments were chosen such that they contain an endonuclease restriction enzyme recognition site suitable for cloning the full-length gen. For the 5' fragment, the downstream oriented primer was chosen to contain the first putative start codon (ATG) in variant 1 (the one shown in full in figure le) . PCR conditions using the Expand High Fidelity PCR system (Roche Molecular Biochemicals) for all of the fragments were as follows. Initial denaturation for 5' at 95°C; 30 cycles of denaturation at 95°C for 45", primer annealing at 55°C for 45" and extention at 72°C for 1' (3' for primer combination A+B) ; followed by an additional incubation for 7' at 72°C and storage at 4°C. PCRs were run on PE Biosystems 9700 PCR machines.
Primer pairs used for cloning Hs-unc-53/3 fragments
# Size Primer Sequence
(bp)
A-B 2229 A TCAGCTCGAGCATATGCCTGTTCTTGGGGTTGC B GGGGTGGGTCGACTTGTCAAGTGG
C-D 847 C ATGGAAGGACCATACCCAACTTGAC
D CTTGTTCCAGCTTTCTGCCTAGATG
E-F 781 E CAGGTTCCTGGAGAAGAGGCATGTC
F GGTGAGGCAATATCTGGATACTTGG G-H 1291 G AGGCAGCCAGGATCCAΔGTATCCAG
H TGCGAAGATCTTTTGGGAGGATGGTC
I-J 1022 I AACCATTGAAATGCTGAAGGCTCAG
J GGTTATGGGATCTAATTAAGTCTCC
K-L 1255 K CACTAGCCTTGGTCTGAGCTCTGAC L TCACCCTCTAGAGGGTAGATTCAAG
Primer A contains restriction sites (Xhol and nhel) suitable for final subcloning in an eukaryotic expression vector (pEGFPc3) and in a yeast-two-hybrid vector (pAS2-l), respectively.
PCR products were analyzed by agarose gel electrophoresis and were visualized by ethidium bromide staining. Splice variants as mentioned in figure le were observed as multiple bands on agarose gels. Single band PCR products were purified with the Qiaquick PCR purification kit, whereas multiple band PCR products were cut out from gel as individual bands and purified using the Qiaquick gel extraction kit. PCR products were cloned in pCR2.1 according to the suppliers protocol (Invitrogen) . For each fragment, multiple clones were picked from selective LB agar plates and grown overnight under antibiotic selection pressure for DNA preparation either on the biorot 9600 (Qiagen) , or manually on anion exchange columns (Qiagen tip 20 or tip 100) . Insert sequences were determined using the Bigdye terminator ready reaction cycle sequencing kit (PE Biosystems) . Individual sequencing reactions for each clone were assembled in single sequence contigs using the Sequencher software package (GeneCodes) . Sequences were compared to the previously determined consensus sequence using the SeqEd software package form PE Biosystems. For each fragment a clone was selected containing the correct sequence and the splice variant of interest. For the I-J fragment, a clone was selected that missed the hart specific 22 amino acid splice variant (figure If) . In the K-L fragment clone, a Sfil-SacII linker was cloned in the BamHI site of the pCR2.1 multiple cloning site to facilitate subcloning of the full- length gene into the yeast-two-hybrid vector (pAS2-l) and the eukaryotic expression vector (pEGFPc3) , respectively. The overall cloning strategy of the full-length gene is visualized in figure 7a. 7al illustrates the overlapping PCR fragments and the nomenclature of fragments and primer pairs. 7a2 illustrates the assembly of the 3' half of the gene in pCR2.1. Internal BamHI (I-J fragment) and Xhol (K-L fragment) sites as well as restriction sites from the multiple cloning site of pCR2.1 (as shown in the figure) were removed by side-directed mutagenesis (SDM) using the Quickchange Site-Directed mutagenesis kit (stratagene) . The Notl-EcoRI G-H fragment and the EcoRI-Nhel I-Jd22 (d22 indicating that the 22 amino acid splice variant is absent) were directionally cloned in the Notl and Nhel sites of the K-L fragment clone. Multiple clones were picked and verified by DNA sequencing. 7a3 illustrates the assembly of the 5' half. Internal Xhol (C-D fragment) and Sfil and Xhol (E-F fragment) sites were removed by SDM. Inserts were cut out from the vectors by restriction digestion with the appropriate restriction enzymes (Xhol+Sall; Sall+Narl and Narl+BamHI, respectively) and purified from gel after agarose gel electrophoresis. The 3 fragments were ligated together, re-cut with Xhol and BamHI and separated on gel. The band of the expected size was cut out of gel, purified and cloned in front of the 3' half, opened by digestion with Xhol and BamHI (figure 7a4) . Multiple clones were picked and verified by sequencing.
Figure 7a illustrates the modular nature of the cloning project. For all the possible combinations of splice variation within the building block fragments, one representative clone is available. In view of functional analysis, building blocks can be exchanged easily by standard technology, either in the pCR2.1 construct or in the final eukaryotic expression or yeast-two-hybrid construct.
Construct of Hs-unc-53/3 GFP chimeras
The construction of the mammalian expression vectors pGI3303 and pGI3305 is explained in the legends of figure 7a, 7b and 7d. pG13303 can be used to over-express in mammalian cells or animals a fusion protein between eGFP and 1128 AA C-terminal fragment of Hs-unc-53/3 (Fig 7c) . pG3305 can be used to overexpress in mammalian cells or animals a fusion protein between eGFP and the 2363 AA full length Hu- unc-53/3 (fig 7d) . The Hs-unc-53/3 cDNA in pGI3303 as well as in pGI3305 contains silent mutations that introduce or remove specific restriction sites in order to be able to easily subclone different types of alternative splice variants in these vectors. Genomic DNA sequencing (BAC 585E09)
Using the primers AGGACCCTATGCGGAGGTCAAGCCGC and TGGGTTGGCATCATCGCTGTCGTAGC, a PCR specific for Hs-unc- 53/1 was developed. PCR products were radiolabeled using the Random Prime DNA labeling kit (Roche Molecular Biochemicals) and hybridized on the human genomic DNA BAC filters (Research Genetics) . Positive signals were obtained for BAC clones 366H21, 483L14, 471 09 and 585E09. BAC DNA was isolated from E. coli genomic clone 585E09 according to a modified Qiagen plasmid DNA preparation procedure. A shotgun library of 1920 clones was constructed at GATC (Konstanz, Germany) . BAC DNA was prepared, nebulized and subcloned after end-repairing in the sequence vector pTZ19R. At JRF, DNA was prepared on the Biorobot 9600 (Qiagen) from 1440 clones. End sequencing reactions with Ml3 forward (TGTAAAACGACGGCCAGT) and reverse (CAGGAAACAGCTATGACC) primer were done on 768 clones. 672 additional clones were sequenced with M13 only. 5 μl DNA was used in 15 μl final reaction volume using the BigDye Terminator Ready Reaction sequencing kit. Sequencing reactions were run on MJ Research PTC200 PCR machines. Reaction products were run and analysed on PE ABI 377 DNA sequencers. All sequencing results were imported in the Sequencher (GeneCodes) software package. Contaminating vector sequences and trailing sequences of low quality were trimmed. Individual sequences were assembled in contigs with standard software settings. A great number of contigs were constructed ranging from below 500 bp to over 10 kbp. Singletons are also still present. By looking for strings of known sequence, a contig was found containing the known and reliable 5' end of hUNC53hl and extending this sequence in 5' direction. This sequence and its relevant features are described in figure lg and its legend. Northern blotting
A Human multiple tissue Norther (MTN-1, Clontech) containing in each lane 2 mg of poly A + RNA from eight different human tissues (heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas) and a MTN-II human multiple tissue Northern, containing in each lane 2 mg of poly A + RNA from spleen, thymus, prostate, testis, ovary, small intestine, colon and peripheral leukocyte, were hybridized according to the manufacturer's instructions and washed out in 0. lxSSC: 0.2% SDS at 55°C. Also from Clontech, a poly A + RNA blot from human cancer cell lines (melanoma G361, lung carcinoma A549, colorectal adenocarcinoma SW480, Burkitt' s lymphoma Raji Leukemia Molt 4, lymphoblastic leukemia K562, HeLa S3 and promyelocytic leukemia HL60) was tested.
Cancer cell lines RNA blots probed with Hs-unc- 53/3
A set of cancer cell line Northern blots were probed with a 665 bp fragment of Hs-unc-53/3 amplified by using the primers 5' AGGAATTAAAATTAACGGATATTCGG and 5'AAAACTGTCCAAACTATTTTCTTCTACC. HU-unc-53/3 is expressed in Melanoma G361 and lung carcinoma A549, transcripts sizes were detected of >0.5 kb. No expression was detected in promyelocytic leukemia HL- 60 HeLa cell S3, chronic myelogenous leukemia K-562, leukemia MOLT-4, Burkitt' s lymphoma Raij and colorectal adenocarcinoma SW480.
Normal human tissue RNA blots probed with Hs-unc- 53/3
A set of normal human tissue Northern blots were probed with a 665 bp fragment of Hs-unc-53/3 amplified by using the primers 5' AGGAATTAAAATTAACGGATATTCGG and 5' AAAACTGTCCAAACTATTTTCTTCTACC . High expression levels were detected in brain, spleen, ovary and spinal cord, lower levels in heart, placenta, testis, stomach, and adrenal gland. Transcripts sizes were >= 9.5 kb.
FISH
Hs-UNC-53/3 is localised to chromosome 12q21.1
Slides preparation:
Lymphocytes isolated from human blood were cultured in -minimal essential medium (MEM) supplemented with 10% foetal calf serum and phytohaemagglutinin (PHA) at 37°C for 68-72 hr. The lymphocyte cultures were treated with BrdU (0.18mg/ml Sigma) to synchronise the cell population. The synchronised cells were washed three times with serum- free medium to release the block and recultured at 37°C for 6 hr in a α-MEM with thymidine (2.5μg/ml: Sigma) . Cells were harvested and slides were made by using standard procedures including hypotonic treatment fix and air-dry.
In situ hybridisation and FISH detection:
A cDNA probe was biotinylated with dATP using the BRL BioNick labelling kit (15°C, 1 hr) Heng et al, 1992) . The procedure for FISH detection was performed according to Heng et al . , 1992 & Heng and Tsui, 1993. Heng et al..: Proc Natl Acad Sci USA 89: 9509-9513 (1992). Heng et al . Chromosoma 102: 325-332 (1993). Briefly, slides were baked at 55°C for 1 hour. After RNase treatment, the slides were denatured in 70% formamide in 2xSSC for 2 min. at 70°C followed by dehydrated with ethanol. Probes were denatured at 75°C for 5 min. in a hybridisation mix consisting of 50% formamide and 10% dextran sulphate. Probes were loaded on the denatured chromosomal slides. After over night hybridisation, slides were washed and detected as well as amplified. FISH signals and the DAPI banding pattern were recorded separately by taking photographs, and the assignment of the FISH mapping data with chromosomal bands was achieved by superimposing FISH signals with DAPI banded chromosomes (Heng et al, 1993) .
Results
Under the condition used the hybridisation efficiency was approximately 67% for this probe (among 100 checked mitotic figures, 67 of them showed signals on one pair of the chromosomes) . Since the DAPI banding was used to identify the specific chromosome, the assignment between signal from probe and the long arm of chromosome 12 was obtained. The detailed position was further determined in the diagram based on the summary from 10 photos.
Radiation Hybrid Mapping
Radiation hybrid analysis is a PCR technique and the panels of radiation hybrid DNA are provided at a concentration of 25 ng/μl in TE buffer suitable for these reactions. Typically, 25 ng of DNA is used in a 10 μl PCR reaction.
Some of the radiation hybrid panels are supported by an e-mail server which can assist you in the chromosome localization of markers. A server for the chromosome localization of markers using the Stanford G3 and Stanford TNG panels is available at http://www- shgc.stanford.edu. At the time of catalog publication, the Stanford TNG server was capable of chromosome localization only on chromosomes 2, 4, 7 and 21. Chromosome localization of markers from the GeneBridge4 panel may be performed by accessing the server at http://www-genome.wi.mit.edu. RH mapping involves the statistical analysis of several to many markers to determine the relative order of the markers with respect to one another. RH mapping can be achieved using statistical programs that will provide the best map along with a measure of the relative likelihood of one order versus another.
This type of analysis has been shown to successfully generate the order of markers on the RH map that is significantly more likely than any alternative order. Two statistical programs for RH mapping can be downloaded from the World Wide Web free of charge. SAMapper was produced at the Stanford Human Genome Center and be downloaded at http://www- shgc.stanford.edu/Mapping/SAMapper/index.html RHMAP was written by Michael Boehnke at the University of Michigan and can be downloaded at http: //www. sph.umich.edu/group/statgen/software. A comprehensive web page regarding radiation hybrid mapping, with links to web sites with analysis software and other information, can be found at http : //linkage . rockefeller . edu/tara/rhmap/
Transfection protocol for cells
N$ neuroblastoma lines were seeded in Lab Tek chambered coverglass (Nalgene Nunc International) and transfected with pEGFP (control), pGI3303 and pGI3305 using lipofectamine (Life Technologies BRL) . After 24-48 hours, the chambered coverglasses were placed on an inverted fluorescence microscope where GFP fluorescence could be visualized in living cells. The details of this method have been described in PCT/EP96/02311.
Microscopy and fluorescence staining using phalloidin
have been described earlier (EP97/06956) .
SEQUENCE LISTING
Seq ID No 1 is a nucleic acid sequence of Hs unc-53/1 and lacking the nucleotides from position 2873 to 3043 shown in Fig. la.
Seq ID No. 2 is a nucleic acid sequence of Hs unc-53/1 and lacking the nucleotides from position 3098 to 3121 shown in Figure la.
Seq ID no. 3 is a nucleic acid sequence of Hs-unc-53/1 and lacking the nucleotides from position 3518 to 3526 of the sequence identified in Fig. la.
Seq ID No. 4 is an amino acid sequence of Hs-unc-53/1 protein and lacking the amino acids from position 958 to 1014 of the sequence identified in Fig. lb
Seq ID No. 5 is a amino acid sequence of Hs-unc-53/1 protein and lacking the amino acids from position 1033 to 1040 of the sequence identified in Fig. lb.
Seq ID No. 6 is a amino acid sequence of Hs-unc-53/1 protein and lacking the amino acids from position 1173 to 1175 of the sequence identified in Fig. lb.
Seq ID No. 7 is a nucleotide sequence encoding Hs- unc-53/2 and lacking the nucleotides from position 5425 to 5433 of the sequence illustrated in Fig. Ic. Seq ID No. 8 is a nucleotide sequence encoding Hs- unc-53/2 and lacking the nucleotides from position 5924 to 6024 of the sequence illustrated in Fig. Ic.
Seq ID No. 9 is a nucleotide sequence encoding Hs- unc-53/2 and having the sequence of variant 1 illustrated in Fig. Ic.
Seq ID No. 10 is a nucleotide sequence encoding Hs- unc-53/2 and having the sequence of variant 2 illustrated in Fig. Ic.
Seq ID No. 11 is a nucleσticfe "sequence encoding Hs- unc-53/2 and having the sequence of variant 3 illustrated in Fig. Ic.
Seq ID No. 12 is a nucleotide sequence encoding Hs- unc-53/2 and having the sequence of variant 1 illustrated in Fig. Ic. and lacking the nucleotides from position 5425 to 5433 of the sequence illustrated in Fig. Ic.
Seq ID No. 13 is a nucleotide sequence encoding Hs- unc-53/2 and having the sequence of variant 1 illustrated in Fig. Ic. and lacking the nucleotides from position 5924 to S024 of the sequence illustrated in Fig. Ic.
Seq ID No. 14 is a nucleotide sequence encoding Hs- unc-53/2 and having the sequence of variant 2 illustrated in Fig. Ic. and lacking the nucleotides from position 5425 to 5433 of the sequence illustrated in Fig. Ic.
Seq ID No. 15 is a nucleotide sequence encoding Hs- unc-53/2 ar.d having the sequence of variant 2 illustrated in Fig. Ic. and lacking the "nucleotides from position 5924 to 6024 of the sequence illustrated in Fig. Ic.
Seq ID No. 16 is a nucleotide sequence encoding Hs- unc-53/2 and having the sequence of variant 3 illustrated in Fig. Ic. and lacking the nucleotides from position 5425 to 5433 of the sequence illustrated in Fig. Ic.
Seq ID No. 17 is a nucleotide sequence encoding Hs- unc-53/2 and having the sequence of variant 3 illustrated in Fig. lc._ and lacking the_ nucleotides. from position 5324 to 6024 of the sequence illustrated in Fig. Ic.
Seq ID No. 18 is an amino acid sequence of Hs-unc- 53/2 protein and lacking the amino acids from position 1776 to 1778 of the sequence identified in Fig. Id
Seq Id No. 19 is an amino acid sequence of variant 1 of Hs-unc-53/2 sequence illustrated in Fig. Id.
Seq Id No. 20 is an amino acid sequence of variant 2 of Hs-unc-53/2 sequence illustrated in Fig. Id.
Seq Id No. 21 is an amino acid sequence of variant 3 of Hs-unc-53/2 sequence illustrated in Fig. Id.
Seq Id No. 22 is an amino acid sequence of variant 1 of Hs-unc-53/2 sequence illustrated in Fig. Id and lacking the amino acids from position 1776 to 1778 of the sequence identified in Fig. Id.
Seq Id No. 23 is an amino acid sequence of variant 2 of Hs-unc-53/2 sequence illustrated in Fig. Id and lacking the amino acids from position 1776 to 1778 of the sequence identified in Fig. Id. Seq Id No. 24 is an amino acid sequence of variant 3 of Hs-unc-53/2 sequence illustrated in Fig. Id and lacking the amino acids from position 1776 to 1778 of the sequence identified in Fig. Id.
Seq ID No. 25 is a nucleotide sequence encoding Hs- unc-53/3 as illustrated in Figure le.
Seq ID No. 26 is a nucleotide sequence encoding Hs- unc-53/3 as illustrated in Figure le and lacking the nucleotides from position 3795 to 4283 of the sequence identified therein.
Seq ID No. 27 is a nucleotide sequence encoding Hs- unc-53/3 as illustrated in Figure le and lacking the nucleotides from position 4284 to 4325 of the sequence identified therein.
Seq ID No. 28 is a nucleotide sequence encoding Hs- unc-53/3 as illustrated in Figure le and lacking the nucleotides from position 3795 to 4325 of the sequence identified therein.
Seq ID No. 29 is a nucleotide sequence encoding Hs- unc-53/3 as illustrated in Figure le and lacking the nucleotides from position 5153 to 5173 of the sequence identified.
Seq ID No. 30 is a nucleotide sequence encoding Hs- unc-53/3 as illustrated in Figure le and lacking the nucleotides from position 5343 to 5408 of the sequence identified.
Seq ID No. 31 is a nucleotide sequence encoding Hs- unc-53/3 having the sequence of variant 1 illustrated in Fig. le. Seq ID No. 32 is a nucleotide sequence encoding Hs- unc-53/3 having the sequence of variant 1 illustrated in Fig. le and lacking the nucleotides from position 3795 to 4283 of the sequence identified therein.
Seq ID No. 33 is a nucleotide sequence encoding Hs- unc-53/3 having the sequence of variant 1 illustrated in Fig. le and lacking the nucleotides from position 4284 to 4325 of the sequence identified therein.
Seq ID No. 34 is a nucleotide sequence encoding Hs- unc-53/3 having the sequence of variant 1 illustrated in Fig. le and lacking the nucleotides from position 3795 to 4325 of the sequence identified therein.
Seq ID No. 35 is a nucleotide sequence encoding Hs- unc-53/3 having the sequence of variant 1 illustrated in Fig. le and lacking the nucleotides from position 5153 to 5173 of the sequence identified therein.
Seq ID No. 36 is a nucleotide sequence encoding Hs- unc-53/3 having the sequence of variant 1 illustrated in Fig. le and lacking the nucleotides from position 5343 to 5408 of the sequence identified therein.
Seq ID No. 37 is an amino acid sequence of Hs-unc- 53/3 protein as identified in the sequence of Fig. If.
Seq ID No. 38 is an amino acid sequence of Hs-unc- 53/3 protein as identified in the sequence of Fig. If and lacking the amino acid residues from position 1326 to 1413 of the sequence identified therein.
Seq ID No. 39 is an amino acid sequence of Hs-unc- 53/3 protein as identified in the sequence of Fig. If and lacking the amino acid residues from position 1414 to 1427 of the sequence identified therein.
Seq ID No. 40 is an amino acid sequence of Hs-unc- 53/3 protein as identified in the sequence of Fig. If and lacking the amino acid residues from position 1703 to 1709 of the sequence identified therein.
Seq ID No. 41 is an amino acid sequence of Hs-unc- 53/3 protein as identified in the sequence of Fig. If and lacking the amino acid residues from position 1768 to 1788 of the sequence identified therein.
Seq ID No. 42 is an amino acid sequence of Hs-unc-53 of variant 1 identified in Figure If.
Seq ID No. 43 is an amino acid sequence of Hs-unc-53 of variant 1 identified in Figure If and lacking the amino acid residues from position 1326 to 1413 of the sequence identified therein.
Seq ID No. 44 is an amino acid sequence of Hs-unc-53 of variant 1 identified in Figure If and lacking the amino acid residues from position 1414 to 1427 of the sequence identified therein.
Seq ID No. 45 is an amino acid sequence of Hs-unc-53 of variant 1 identified in Figure If and lacking the amino acid residues from position 1703 to 1709 of the sequence identified therein.
Seq ID No. 46 is an amino acid sequence of Hs-unc-53 of variant 1 identified in Figure If and lacking the amino acid residues from position 1768 to 1788 of the sequence identified therein.

Claims

1. A vertebrate protein homologue of a UNC-53 protein of C. elegans, which protein comprises an amino acid sequence having one or more of sequence blocks A, B, C, D, E, F, G, or H as illustrated in figure 4 or which differs from said blocks in conservative amino acid changes.
2. A vertebrate protein homologue of UNC-53 protein of C. elegans or a functional equivalent, derivative or bioprecursor therefor having an amino acid sequence encoded by the nucleotide sequence illustrated in figure 1(e) or the sequence of Figure 1 e having nucleotide region from position 1 to 288 replaced with the sequence of variant 1 illustrated in Figure le and or which sequences further lack any of the sequences form 3795 to 4283, 4284 to 4325, 5153 to 5173 or 5343 to 5408.
3. A vertebrate protein homologue of UNC-53 protein of C. elegans having an amino acid sequence as illustrated in figure 1(f) or an amino acid sequence which differs from said amino acid sequence illustrated in figure 1(f) by the replacement of amino acids 1 to 81 with the sequence of variant 1 in figure If and /or including deletions from position 1326 to 1413, 1414 to 1427, 1703 to 1709 or 1768 to 1788, or which differs from said sequences in one or more conservative amino acid changes.
4. A cDNA molecule encoding a vertebrate homologue of UNC-53 protein of C. elegans according to any of claims 1 to 3.
5. A cDNA molecule according to claim 4 which cDNA comprises the sequence of nucleotides illustrated in figure 1 ( e ) .
6. A nucleic acid molecule capable of hybridising to the cDNA sequences according to claims 4 or 5 under high stringency conditions.
7. A DNA expression vector which comprises a cDNA molecule as claimed in claim 4 or 5.
8. A vector according to claim 7 which comprises a promoter of C. elegans UNC-53 protein or a vertebrate homologue thereof according to any of claims 1 to 7.
9. A vector according to claim 8 wherein said promoter sequence is derived from a gene encoding a mouse or human homologue of a UNC-53 protein of C . elegans .
10. A vector according to any of claims 7 to 9 which further comprises a sequence encoding a reporter molecule.
11. A vector according to claim 10 wherein said reporter molecule is a fluorophore.
12. A host cell transformed or transfected with the vector of any of claims 7 to 11.
13. A host cell transformed or transfected with the vector of claims 10 or 11.
14. A host cell according to claim 12 or 13 which cell comprises a prokaryotic cell, such as a bacterial cell or a eukaryotic cell such as a fungal, and animal, a plant or an insect cell.
15. A transgenic cell, tissue or organism comprising a transgene capable of expressing a protein according to any of claims 1 to 3.
16. A transgenic cell, tissue or organism according to claim 15 which comprises any of a COS cell, Hep G2, MCF-7 cell, N4 mouse neuroblastoma cell, a NIH3T£ cell, or colorectal carcinoma or human derived cells.
17. A transgenic cell, tissue or organism according to claim 15 or 16 wherein said transgene comprises a vector according to any of claims 7 to 11.
18. A transgenic cell, tissue or organism according to claim 15 or 17 wherein said transgene comprises a vector according to claim 10 or 11.
19. A transgenic cell, tissue or organism according to any of claims 15 to 17 wherein said organism comprises any of an insect, a fungus, a non- human mammal, a plant or a nematode worm.
20. A method of producing a mutant vertebrate non-human organism which mutation affects cell behaviour or the regulation of cell motility or the shape or the direction of cell migration, which method comprises inducing a mutation in the wild type gene encoding the vertebrate homologue of an UNC-53 C. elegans protein.
21. A vertebrate protein homologue of an UNC-53 protein of C. elegans, according to any of claims 1 to 3 for use as a medicament.
22. Use of a vertebrate protein homologue of an UNC-53 protein of C. elegans, according to any of claims 1 to 3 in the manufacture of a medicament for promoting neuronal regeneration, revascularisation, wound healing or for treatment of chronic neurodegenerative diseases or acute traumatic injuries or fibrotic disease or autoimmune diseases such as rheumatoid arthritis and sclerosis.
23. A pharmaceutical composition comprising a vertebrate homologue of an UNC-53 protein of ^_ elegans, according to any of claims 1 to 3 together with a pharmaceutically acceptable carrier, diluent or excipient therefor.
24. A nucleic acid or cDNA molecule according to any of claims 4 to 6 or a functional fragment thereof for use as a medicament.
25. Use of nucleic acid or cDNA molecule according to any of claims 4 to 6 in the manufacture of a medicament to promote neuronal regeneration, revascularisation or wound healing, or for treatment of chronic neurodegenerative diseases or acute traumatic injuries or fibrotic disease or autoimmune diseases such as rheumatoid arthritis and sclerosis.
26. A pharmaceutical composition comprising a nucleic acid or cDNA molecule according to any of claims 4 to 6 and a pharmaceutically acceptable carrier, diluent or excipient therefor.
27. A method of determining whether a compound is an inhibitor or enhancer of the regulation of cell behaviour, growth, cell shape or motility or the direction of cell migration, which method comprises contacting said compound with a host cell according to claim 12 or 14 or a transgenic cell as claimed in any of claims 15 to 18 and screening for a phenotypic -change in said cell.
28. A method according to claim 27 wherein said phenotypic change to be screened is a change in cell growth, or shape or a change in cell motility or filopodia outgrowth, ruffling behaviour, cell adhesion, contact inhibition or the length of neurite growth.
29. A method as claimed in claim 27 wherein said transgenic cell is an N4 neuroblastoma cell and the phenotypic change to the screened is the length of neurite growth.
30. A method as claimed in claim 27 wherein said transgenic cell is an MCF-7 breast carcinoma cell or an NIH3T3 cell and the phenotypic change to be screened is the extent of phagokinesis or contact inhibition.
31. A method of determining whether a compound is an inhibitor or an enhancer of the regulation of cell shape, cell growth or motility or of the direction of cell migration, which method comprises administering said compound to a transgenic organism according to any of claims 15 to 19 or a mutant organism produced according to the method of claim 20 and screening for a phenotypic change in said organism.
32. A compound which is identifiable by the method according to claim 27 as an enhancer of the regulation of cell shape, or growth or motility or the direction of cell migration for use as a medicament.
33. Use of a compound which is identifiable by the method according to claim 27 as an enhancer of the regulation of cell shape, or growth or motility or the direction of cell migration in the preparation of medicament for promoting neuronal regeneration, revascularisation or wound healing or for treatment of chronic neurodegenerative diseases or acute traumatic injuries or fibrotic disease autoimmune diseases such as rheumatoid arthritis or sclerosis.
34. A pharmaceutical composition comprising a compound identified according to the method of any of claims 27 to 31 and a pharmaceutically acceptable carrier, diluent or excipient therefor.
35. A compound which is identifiable by the method according to any one of claims 17 to 31 as an inhibitor of the regulation of cell motility, growth, or shape, or the direction of cell migration, for use as a medicament.
36. Use of a compound according to claim 35 in the manufacture of a medicament for alleviating the spread of disease inducing cells or metastasis or loss of contact inhibition.
37. A pharmaceutical composition comprising the compound as claimed in claim 35, and a pharmaceutically acceptable carrier diluent or excipient therefor.
38. A method of determining whether a compound is an inhibitor or an enhancer of transcription of a gene encoding a vertebrate homologue of UNC-53 protein of C. elegans, according to any of claims 1 ro 3 which method comprises the steps of (a) contacting said compound with a cell according to claim 13 or 18 and
(b) monitoring the level of said reporter molecule and comparing the results obtained from said monitoring step with a control comprising a cell according to claims 13 or 18, which cell has not been contacted with said compound.
39. A method as claimed in claim 38 wherein said reporter molecule detected is mRNA or green fluorescent protein.
40. A compound which is identifiable by the method according to claims 38 or 39, as an enhancer of transcription of a gene coding for a vertebrate homologue of an UNC-53 protein of C. elegans according to any of claims 1 to 3 or a functional fragment of said gene, for use as a medicament.
41. Use of a compound which is identifiable by the method of claims 38 or 39, as an enhancer of transcription of a gene coding for a vertebrate homologue of an UNC-53 protein of C. elegans according to any of claims 1 to 3 or a functional fragment of said gene, in the manufacture of a medicament for promoting neuronal regeneration, revascularisation or wound healing, or for treatment of chronic neurodegenerative diseases or acute traumatic injuries or fibriotic disease or autoimmune diseases such as rheumatoid arthritis or sclerosis.
42. A pharmaceutical composition which comprises the compound of claim 40 and a pharmaceutically acceptable carrier, diluent or excipient therefor.
43. A compound which is identifiable by the method of claims 38 or 29 as an inhibitor of transcription of a gene coding for vertebrate homologue of a UNC-53 protein of C. elegans according to any of claims 1 to 3 or a functional fragment of said gene for use as a medicament.
44. Use of a compound which is identifiable by the method of claims 38 or 39 as an inhibitor of transcription of a gene coding for a vertebrate homologue of an UNC-53 protein of C. elegans or a functional fragment of said gene, in the manufacture of a medicament for alleviating spread of disease inducing cells or metastasis or loss of contact inhibition.
45. A pharmaceutical composition which comprises the compound of claim 43 and a pharmaceutically acceptable carrier, diluent or excipient therefor.
46. A kit for determining whether a compound is an enhancer or an inhibitor of the regulation of cell motility, growth or shape or the direction of cell migration which kit comprises at least one transgenic cell as claimed in any one of claims 13 to 17 to be contacted with said compound and at least one cell according to claims 1 2to 19 to be used as a control and means for contacting said compound with one of said at lest one transgenic cells.
47. A kit for determining whether a compound is an inhibitor or an enhancer of transcription of a gene coding for a vertebrate homologue of an UNC-53 protein of C. elegans or a functional fragment of said gene which kit comprises at least one cell as claimed in any one of claims 12 to 19 and means for contacting said compound with said cells.
48. A kit for determining whether a compound is an enhancer or an inhibitor of the activity of a vertebrate homologue of an UNC-53 protein of C. elegans or a functional equivalent, derivative, fragment or bioprecursor of said vertebrate homologue protein, which kit comprises at least, one vertebrate mutant non-human organism produced according to the method as claimed in claim 20 or a transgenic organism as claimed in claims 15 to 19 and a wild type of said vertebrate mutant organism.
49. A method identifying vertebrate homologues of an unc-53 gene of C. elegans or a functional fragment thereof, which method comprises hybridizing to a DNA library a suitable oligonucleotide sequence of between 15 to 50 nucleotides of the nucleic acid sequence encoding UNC- 53 or a functional equivalent, derivative or bioprecursor thereof, under appropriate conditions of stringency to identify genes having statistically significant homology with the cDNA according to any of claims 4 or 5.
50. A method of identifying a protein which is active in the signal transduction pathway of a cell of which a vertebrate homologue of an UNC-53 protein of C. elegans according to any of claims 1 to 3 is a component, which method comprises:
(a) contacting an extract of said cell with an antibody to the vertebrate homologue of the UNC-53 protein of C. elegans,
(b) identifying the antibody/vertebrate homologue complex, and
(c) analysing the complex to identify any protein bound to the vertebrate homologue of UNC-53 protein of C. elegans other than the antibody.
51. A method of identifying a further protein which is active in the signal transduction pathway of a cell of which a vertebrate homologue of an UNC-53 protein according to any of claims 1 to 3 is a component, which method comprises: (a) forming an antibody to the first identified protein bound to the vertebrate homologue of UNC-53 protein of C. elegans in claim 50, (b) contacting a cell extract with said antibody and identifying the antibody/protein complex,
(c) analysing the complex to identify any further protein bound to the first protein other than the antibody, and
(d) optionally repeating steps (a) to (c) to identify further proteins in said pathway.
52. A method of identifying a protein which is active in the signal transduction pathway of a cell of which a vertebrate homologue of an UNC-53 protein of C. elegans according to any of claims 1 to 3 is a component, which method comprises: (a) contacting an extract of said cell with said vertebrate homologue of an UNC-53 protein of C. elegans,
(b) identifying any vertebrate homologue of UNC-53 protein/protein complex formed and (c) analysing the complex to identify any protein bound to the vertebrate homologue of UNC-53 protein other than the same vertebrate homologue of UNC-53 protein.
53. A method according to claim 52 which further comprises contacting a cell extract with any protein identified from step (c) not being the same as the vertebrate homologue of UNC-53 protein used and repeating steps (b) and (c) so as to identify any further protein involved in the signal transduction pathway of said cell.
54. A method of identifying a protein involved in the signal transduction pathway of a cell of which a vertebrate homologue of an UNC-53 protein of C. elegans is a component which method comprises: (a) providing an appropriate host cell having a DNA construct comprising a reporter gene under the control of a promoter regulated by a transcription factor having a DNA binding domain and an activating domain, (b) expressing in said host cell a first hybrid DNA sequence encoding a first fusion of a fragment or all of a DNA sequence according to claims 4 or 5 and either said DNA binding domain or the activating domain of the transcription factor,
(c) expressing in the host cell at least one second hybrid DNA sequence encoding a putative binding protein to be investigated together with the DNA binding or activating domain of the transcription factor which is not incorporated in the first fusion,
(d) detecting any binding of the protein being investigated with a protein according to any of claims 1 to 3 by detecting for the production of any reporter gene product in said host.
55. A protein identified by the method of any one of claims 50 to 54 for use as a medicament.
56. Use of a protein identified by the methods of any one of claims 50 to 54 in the manufacture of a medicament for promoting neuronal regeneration, revascularisation or wound healing, or for treatment of chronic neurodegenerative diseases or acute traumatic injuries or fibrotic disease or autoimmune diseases such as rheumatoid arthritis and sclerosis.
57. A pharmaceutical composition comprising a protein identified by the methods of any one of claims 50 to 54 and a pharmaceutically acceptable carrier, diluent, or excipient therefor.
58. A process for producing a vertebrate homologue of an UNC-53 protein of C. elegans according to any of claims 1 to 3 which process comprises culturing the cells of any of claims 12 to 14 and recovering said vertebrate homologue of UNC-53 protein expressed.
59. A process for producing a vertebrate homologue of an UNC-53 protein of C. elegans according to any of claims 1 to 3 which process comprises culturing an insect cell transfected with a recombinant Baculovirus vector, said vector comprising a DNA insert encoding said vertebrate homologue of UNC-53 protein downstream of the Baculovirus polyhedrin promoter, and recovering the expressed vertebrate homologue of UNC-53 protein.
60. A method of detecting whether a compound is an inhibitor or an enhancer of expression of a vertebrate homologue of an UNC-53 of C. elegans according to any of claims 1 to 3 which method comprises contacting a cell expressing said homologue with said compound and monitoring for a phenotypic change compared to a control cell which has not been contacted with said compound.
61. A method according to claim 60 wherein said cell comprises a cell according to any of claims 12 to 19.
62. A method according to claim 60 wherein said cell has undergone loss of contact inhibition.
63. A method according to any of claims 60 to 62 in which the compound to be tested comprises a nucleic acid.
64. A method according to claim 63 wherein said nucleic acid sequence comprises an antisense DNA or RNA sequence.
65. A method according to claim 64 wherein said mRNA sequence comprises 3' untranslated regions of mRNA encoding for said vertebrate homologue.
66. A method according to any of claims 60 to 62 wherein said compound to be tested comprises a protein having an amino acid sequence potentially suitable for inhibiting function of said vertebrate homologue.
67. A method according to claim 66 wherein said protein comprises a protein identified according to any of the methods of claims 50 to 54.
68. A pharmaceutical composition comprising a compound identified according to any of claims 60 to 67 together with a pharmaceutically acceptable carrier, diluent or excipient therefor.
69. A nucleic acid sequence identified according to the method of any of claims 63 to 65 for use as a medicament.
70. Use of a nucleotide sequence identified according to the method of any one of claims 63 to 65 in the preparation of a medicament for the treatment of loss of contact inhibition or cancer which is mediated by a vertebrate homologue of an UNC-53 protein of C. elegans.
71. Use of a nucleic acid according to claim 69 in the preparation of a medicament for inhibiting expression of a gene coding for a vertebrate homologue of an UNC-53 protein of C. elegans.
72. An assay for detecting expression of a vertebrate homologue of UNC-53 protein of C. elegans according to any of claims 1 to 3 in a vertebrate cell which assay comprises contacting a cell or an extract thereof with an antibody to said vertebrate homologue, which antibody is linked to a reporter molecule, removing any unbound antibody and monitoring for the presence of said reporter molecule.
73. An assay according to claim 72 wherein said reporter molecule is an antibody conjugated with a suitable fluorophore or detectable enzyme.
74. A method for detecting for expression of a gene coding for a vertebrate homologue of an UNC-53 protein of C. elegans according to any of claims 1 to 3 which method comprises contacting a probe specific for a nucleic acid or protein sequence coding for or corresponding to said vertebrate homologue according to any of claims 1 to 3 with a cell extract which probe is linked to a reporter and analysing for the presence of said reporter.
75. A method according to claim 74 wherein said probe comprises a complementary sequence to a region of mRNA transcribed from said gene encoding said vertebrate homologue of UNC-53 protein.
76. A method according to claim 75 wherein said complimentary sequence is a 3' or 5' untranslated region of said mRNA.
77. A method according to claims 74 or 76 wherein said reporter comprises a radiolabel.
78. A method according to claim 74 wherein said probe comprises an antibody specific for said vertebrate homologue of said UNC-53 protein according to any of claims 1 to 3.
79. A method according to claim 78 wherein said reporter comprises an antibody conjugated with a detectable fluorophore or enzyme.
80. A method of determining whether a compound is an inhibitor or an enhancer of association of a vertebrate homologue according to any of claims 1 to 3 to microtubules or plus end regions thereof, which method comprises :-
(a) contacting said compound with a transgenic cell, tissue or organism expressing UNC-53 protein or said vertebrate homologue and which protein is operably linked to a reporter molecule,
(b) screening for the localisation of said reporter molecule as compared to a cell according to step (a) which has not been contacted with said compound.
81. A compound identifiable by the method according to claim 80.
82. A compound according to claim 81 for use as a medicament.
83. Use of a compound according to claim 81 as an enhancer of association of said vertebrate homologue with microtubules or the plus end region thereof, for use in promoting neuronal regeneration, revascularisation or wound healing, or for treating chronic neurodegenerative diseases or acute traumatic injuries or fibrotic disease or autoimmune diseases such as rheumatoid arthritis or sclerosis.
84. A pharmaceutical composition comprising the compound according to claims 81 or 82 and a pharmaceutically acceptable carrier, diluent or excipient therefor.
85. A kit for determining whether a compound is an inhibitor or an enhancer of association of a vertebrate homologue according to any of claims 1 to 3 with microtubules or the plus end regions thereof, which kit comprises at least one transgenic cell expressing said homologue and a reporter molecule or a cell according to any of claims 12 to 19 and at least one cell of the same cell type for use as a control and means for contacting said compound with one of said at least one transgenic cells.
86. A composition comprising a vertebrate homologue according to any of claims 1 to 3 linked to a compound identified as an inhibitor or enhancer or association of said vertebrate homologue with microtubules or their plus end regions for use in targeting said compound to said microtubule or the plus end region thereof.
87. A composition according to claim 86 which further comprises a cell transformation or transfecting agent.
88. A method of targeting a protein to a cell microtubule or the plus end region thereof, which method comprises introducing into a host cell, tissue or organism a transgene comprising a sequence capable of expressing a vertebrate homologue according to any of claims 1 to 3, which sequence is operably linked to a sequence encoding said protein to be targeted such that a chimeric protein is expressed and which results in targeting said protein to said microtubule or a plus end region thereof.
89. A method of identifying a molecule which covalently modifies a vertebrate homologue of UNC-53 according to any of claims 1 to 3 which method comprises: a) contacting an extract from a cell expressing said vertebrate homologue with a mixture of enzymes comprising candidate modifying enzymes in the presence of an inhibitor or covalent modification of a protein, b) identifying any covalently modified UNC-53 protein from step a) , c) identifying said molecule involved in said modification step.
90. A method according to claim 89, wherein said indicator comprises 32p.
91. A method of identifying a compound which alleviates or enhances the toxicity of a vertebrate homologue according to any of claims 1 to 3, which method comprises contacting said compound with a cell, tissue or organism according to claim 18, and monitoring for the presence of said reporter molecule adjacent said microtubules or the plus end regions thereof.
92. A vertebrate homologue of UNC-53 protein of C. elegans or a functional equivalent, derivative or bioprecursor therefor encoded by the nucleotide sequence in Figure la and which nucleotide sequence is lacking in any of the nucleotide regions from position 2873 to 3043, 3098 to 3121 or 3518 to 3526.
93. A vertebrate homologue of UNC-53 protein of C. elegans or a functional equivalent, derivative or bioprecursor therefor having an amino acid sequence as illustrated in Figure lb and lacking in one or more of the regions from residues 958 to 1014, 1033 to 1040 or 1173 to 1175, or which differs from said amino acid sequences in one or more conservative amino acid changes .
94. A vertebrate homologue of UNC-53 protein of C. elegans or a functional equivalent, derivative or bioprecursor therefor encoded by the nucleotide sequence in Figure lc and which nucleotide sequence has from sequence position 1 to 366 replaced with any of the sequences identified as variants 1 to 3 of Figure lc and/or which sequences lack the region from position 5624 to 6024.
95. A vertebrate homologue of UNC-53 protein of C. elegans or a functional equivalent, derivative or bioprecursor therefor having an amino acid sequence identified in Figure Id or the sequences of any of variants 1 to 3 replacing the amino acids from position 1 to 89 of the sequence of Figure Id and/or which sequence is lacking the amino acid sequence from position 1776 to 1778.
96. Plasmid pG313303 deposited under accession number LMBP 3936.
97. Plasmid pG13305 deposited under accession number LMBP 3937.
PCT/EP1999/003848 1998-06-03 1999-06-02 HUMAN HOMOLOGUE OF UNC-53 PROTEIN OF $i(C. ELEGANS) WO1999063080A1 (en)

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EP99926511A EP1092019A1 (en) 1998-06-03 1999-06-02 Human homologue of unc-53 protein of c. elegans
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WO2000050451A2 (en) * 1999-02-26 2000-08-31 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Protein (tp) that is involved in the development of the nervous system
WO2000050451A3 (en) * 1999-02-26 2001-08-02 Deutsches Krebsforsch Protein (tp) that is involved in the development of the nervous system
WO2002017947A2 (en) * 2000-08-30 2002-03-07 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Use of t proteins for the differential characterization and treatment of lesions and tumors of the nervous system
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WO2002017947A3 (en) * 2000-08-30 2003-03-20 Deutsches Krebsforsch Use of t proteins for the differential characterization and treatment of lesions and tumors of the nervous system
US10494402B2 (en) 2012-11-25 2019-12-03 The Regents Of The University Of California Peptides that stimulate subcutaneous adipogenesis
US10766927B2 (en) 2012-11-25 2020-09-08 The Regents Of The University Of California Peptides that stimulate subcutaneous adipogenesis
US20180230188A1 (en) * 2014-05-28 2018-08-16 The Regents Of The University Of California Peptides, compositions, and methods for stimulating subcutaneous adipogenesis
US10844102B2 (en) * 2014-05-28 2020-11-24 The Regents Of The University Of California Peptides, compositions, and methods for stimulating subcutaneous adipogenesis
CN105738607A (en) * 2014-12-09 2016-07-06 兰州红菌生物技术有限责任公司 Kit for screening antitumor drugs, and use method thereof

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