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WO1998024810A2 - Homologues vertebres de la proteine unc-53 de c. elegans - Google Patents

Homologues vertebres de la proteine unc-53 de c. elegans Download PDF

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Publication number
WO1998024810A2
WO1998024810A2 PCT/EP1997/006956 EP9706956W WO9824810A2 WO 1998024810 A2 WO1998024810 A2 WO 1998024810A2 EP 9706956 W EP9706956 W EP 9706956W WO 9824810 A2 WO9824810 A2 WO 9824810A2
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Prior art keywords
protein
unc
cell
sequence
vertebrate
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PCT/EP1997/006956
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English (en)
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WO1998024810A3 (fr
Inventor
Christ Jules Platteeuw
Carlos Manuel Buesa Arjol
Marc Deraeymaeker
Peter Verhasselt
Nathalie Jeanne Raymonde Pujol
Luc Jacques Simon Maertens
Walter Luyten
Hugo Geerts
Joel Stefaan Vandekerckhove
Johan Geysen
Thierry André Olivier Eddy BOGAERT
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Janssen Pharmaceutica N.V.
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Priority to CA002273827A priority Critical patent/CA2273827A1/fr
Priority to JP52523198A priority patent/JP2001522222A/ja
Priority to AU56622/98A priority patent/AU5662298A/en
Priority to EP97952926A priority patent/EP0941239A1/fr
Publication of WO1998024810A2 publication Critical patent/WO1998024810A2/fr
Publication of WO1998024810A3 publication Critical patent/WO1998024810A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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 vertebrate homologues of UNC-53 protein of C. ele ⁇ ans 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.
  • the control of these processes is disturbed in a variety of disease states in which, for example, the Receptor Tyrosine kinase (RTK) signal transduction pathways, or the like, or their downstream intra-cellular pathways (which are shared with other extra-cellular receptors, including cell adhesion molecules like N-CAMS and integrins) are overstimulated .
  • RTK Receptor Tyrosine kinase
  • 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.
  • a long-range migration of a cell process 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 which senses signals in the environment instructing the cell to either stabilise a cell process extending in a preferred direction, or to cause a lamellipodium to extend a process in a given direction.
  • 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. Microtubule binding directing these processes has not previously been identified.
  • the present inventors have surprisingly found that UNC-53 protein of C. elegans and vertebrate homologues thereof is involved in binding of microtubules and particularly of plus end regions of microtubules.
  • UNC-53 protein as a signal transducer or signal integrator controlling the directionality of cell migration and/or cell shape in C. elegans (WO 96/38555) .
  • Increased UNC-53 protein activity was found to be proportional to cell process extensions in the correct direction of cell migration.
  • the unc-53 gene was found to encode a signal transduction molecule that transduces a signal from an RTK such as, for example, via the adaptor protein SEM- 5/GRB-2, to the machinery controlling directional growth cone extension or stabilisation, in a highly dosage - dependent fashion. Genetic and experimental analysis of C.
  • UNC-53 mutants showed that mutations in the unc-53 gene do not affect the general ability of cells to migrate but rather affect the ability of cells to migrate under specific antero-posterior cues.
  • Reduction of UNC-53 activity leads to loss of direction and reduction of growth cone extension as indicated by the directionality of random extension cycles observed in excretory canal growth cones in UNC-53 mutants.
  • the function of UNC-53 is highly sensitive to its dosage or activity. Reduction of function leads to proportional reduction of migration to the specific signal while increased expression, using transgenic expression of UNC-53 in muscle cells, leads to increased directional migration.
  • UNC-53 functions as an integrator of a directional signal in the organism whereby reception of signals leads to growth cone extension in the correct direction.
  • Certain alleles of UNC-53 enhance the sex myoblast migration defect of SEM-5 C. ele ⁇ ans mutants in a receptor tyrosine kinase signal transduction pathway (Stern et al 1993 mol. Biol. cell, 4, 1175- 1188) . While the genetics suggests that UNC-53 and SEM-5 cooperate to regulate sex myoblast migration, genetic experiments do not permit a conclusion that this is the result of a direct molecular interaction.
  • the inventors previously identified a potential se - 5/GRB-2 binding site and showed in two types of biochemical experiments that UNC-53 physically interacts with SEM-5.
  • the present inventors conclude that UNC-53 encodes a signal transduction molecule that transduces extracellular signals for directional migration via the adapter protein SEM-5/GRB-2 to the machinery controlling directional growth cone extension or stabilization.
  • UNC-53 might act as an adapter linking extracellular signals to the actin cytoskeleton.
  • UNC-53 has shown homology to cortical actin binding proteins and that it is capable of binding F-actin in vitro.
  • expression of UNC-53 in mammalian cells leads to changes in the F-actin cytoskeleton.
  • Very low levels of UNC-53 expression increase the number of filopodia and actin microspikes protruding from the cell surface.
  • Cells expressing UNC-53 also exhibit increased neurite extension and increased cell motility.
  • UNC-53 thus also acts as an activator of migration. Considering all available data the following possible mechanisms of action of UNC-53 can be formulated.
  • the choice and activation of directional growth cone extension can be accounted for by local activation of UNC-53 via a SEM-5/GRB2 complex to a receptor (eg receptor tyrosine kinase signal) which reads a localized or directional signal.
  • Changes in growth cone steering are preceded by the formation of a localized actin patch in the area of the growth cone receiving the highest signal (Bentley and O'Connor et al. Curr. Op. NeuroBiol. 1994, vol 4, 43-48).
  • UNC-53 might be directly involved in forming these actin patches through its own actin binding or cross- linking properties.
  • activated UNC-53 may (eg via its nucleotide binding domain) transduce a signal to as yet unidentified effectors.
  • activation of the small GTP-binding protein cdc42 or a related protein leads to formation of small actin patches as well as the formation of small filopodia.
  • the unc-53 pathway may be upstream of cdc42 or both signal transducers might share downstream pathways.
  • the present inventors thus decided to investigate if a similar protein was present in higher organisms such as vertebrates.
  • the present inventors describe the identification of a family of genes in vertebrates, and particularly in man and mouse with extensive structural homology to UNC-53.
  • the present inventors have surprisingly found that the nucleotide domains of UNC-53 from C. ele ⁇ ans and UNC-53 from vertebrates similarly activate motility, establishing functional equivalence. Furthermore these domains are shown to be capable of transforming NIH3T3 cells in vitro.
  • the inventors also found changes in RNA transcripts in transformed cell lines compared to normal human tissues suggesting a role for UNC-53 in cell differentiation, morphogenesis and disease.
  • in vitro assays and transgenic models are also described that identify pharmacological modulators of UNC-53 activity and assays to identify proteins interacting with UNC- 53.
  • a vertebrate protein homologue of UNC-53 protein of C. ele ⁇ ans or a functional equivalent, derivative or bioprecursor thereof which protein homologue comprises an amino acid sequence having a statistically significant homology to the UNC-53 protein of C. ele ⁇ ans as illustrated in figure 2.
  • a derivative should be taken to mean mutational derivatives, fusions, internal deletions, splice variants and muteins.
  • a vertebrate protein homologue of UNC-53 protein of C. elegans which protein comprises an amino acid sequence having one or more of sequence homology blocks A, B, C, D or E as illustrated in Figure 9a, or block F in Figure 12a or a sequence having a statistically significant homology therewith.
  • said vertebrate homologue is a human protein or a mouse protein.
  • a vertebrate protein homologue of an UNC-53 protein of C. elegans f which protein comprises an amino acid sequence having one or more of sequence blocks A, B, C, D,E or F which differ from those blocks of Figure 9a and Figure 12a to a significant extent only in conservative amino acid changes.
  • a vertebrate protein having an amino acid sequence encoded by the nucleotide sequence from position 1 to position 6013 as illustrated in Figure 9b there is also provided a vertebrate protein having an amino acid sequence encoded by the nucleotide sequence illustrated in Figure lid, or a functional equivalent derivative, or bioprecursor of said homologue.
  • a vertebrate protein having an amino acid sequence corresponding to the prosite signatures as illustrated in Figure 28 for each of said homology blocks as defined above.
  • the prosite signatures can be used to identify a protein having a statistically significant homology to the UNC-53 protein of C. elegans. (Luethy et al 1994, Protein Science, 3, 139-146).
  • a further aspect of the invention comprises a vertebrate homologue according to the invention comprising an amino acid sequence as shown in figure 9b or lid or an amino acid sequence which differs from the amino acid sequences shown in these figures to a significant extent only in one or more conservative amino acid changes.
  • nucleic acid molecule which is preferably DNA, and which encodes a vertebrate homologue of UNC-53 protein of C. ele ⁇ ans f 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 figures 9b or lid or an amino acid which differs from the sequences shown in these figures to a significant only in one or more conservative amino acid changes.
  • the DNA is cDNA, which cDNA comprises at least from position 1 to 6013 of the sequence shown in Figure 9b.
  • the cDNA may comprise the sequence illustrated in Figure lid.
  • nucleic acid sequence capable of hybridising to the nucleic acid or DNA sequences according to the invention under high strigency 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.
  • elegans 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. ele ⁇ ans or a functional equivalent, fragment derivative or bioprecursor of said homologue.
  • 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 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 organism, tissue or cell 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 or a NIH3T3 cell or 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 sequence encoding the vertebrate homologue or a functional fragment of said gene 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 or a functional equivalent or derivative or bioprecursor of said protein.
  • the gene may comprise deletions or mutations but may still encode a functional vertebrate homologue of UNC-53 protein.
  • a method of producing a mutant vertebrate non-human organism or cell having a mutation in the wild-type gene coding for the vertebrate homologue of UNC-53 protein, 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 comprises inducing a mutation in the vertebrate homologue of
  • the vertebrate homologue of UNC-53 protein of C. ele ⁇ ans 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 promote neuronal regeneration, revascularisation or wound healing or the treatment of chronic neurodegenerative disorders or acute traumatic injuries or fibrotic disease or physiological events requiring the polarity of cells or epithelia.
  • the present inventors have also found that the vertebrate homologue of UNC-53 protein plays a role in a transformed state of cells.
  • the vertebrate homologue, 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 cancer development.
  • the above medical conditions may be treated in mammals and more preferably humans by either a homologue of UNC-53 protein or alternatively by a nucleic acid coding for such a protein.
  • an antisense oligonucleotide to said UNC-53 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.
  • the vertebrate homologue of UNC-53 protein or a functional equivalent, fragment or bioprecursor of said protein 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 present invention 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 or 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 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, or a functional equivalent, derivative, fragment or bioprecursor of said vertebrate homologue 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 or a functional equivalent, fragment, derivative or bioprecursor of said vertebrate homologue.
  • the phenotypic change to be screened comprises a change in cell shape or a change in cell motility.
  • 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 or changes in filipodia outgrowth or alternatively changes in ruffling behaviour or cell adhesion or any change in microtubule cytoskeleton or any change in localisation of proteins on plus end regions of microtubules or any change in cell death such as in apoptosis.
  • the transgenic cell may comprise an MCF-7 breast cancer cell.
  • the phenotypic change to be screened comprises the extent of phagokinesis or filipodia 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 be 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 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 particularly 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 cancer, 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 or a functional equivalent, derivative fragment, or bioprecursor of said homologue operably linked to a nucleic acid sequence encoding a reporter molecule.
  • the reporter sequence encoding the reporter molecule which comprises 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, or a functional equivalent, derivative fragment or bioprecursor of said homologue, 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 cancers 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 one 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 or a functional equivalent, derivative or fragment thereof which kit comprises at least one transgenic cell or cells according to the invention and means for contacting said compounds with said cells.
  • 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 Figures 9b or lid 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 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.
  • 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 or a functional equivalent, fragment or bioprecursor of said vertebrate homologue is a component.
  • 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.
  • 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 UNC-53 is a component which method comprises: (a) forming an antibody to the first identified protein bound to the vertebrate homologue of
  • the antibody starts the process by binding to the vertebrate homologue of UNC-53 protein or a functional equivalent thereof in the signal transduction pathway. 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: (a) contacting an extract of the cell with the vertebrate homologue of UNC-53 protein of C. ele ⁇ ans or a functional equivalent, fragment or bioprecursor of said homologue,
  • step (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) .
  • 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 a 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. ele ⁇ ans or a functional equivalent or derivative or bioprescursor is a component involves investigating protein-protein interactions using the two-hybrid vector method. This method is well known to those skilled in the art and which was first developed in yeast by Chien et al (1991) . This technique is based on functional reconstitution in vivo of a transcription factor which activates a reporter gene.
  • 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.
  • 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.
  • 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. 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 or a functional equivalent, fragment, or derivative of the protein, 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 vertebrate homologue of UNC-53 protein.
  • the cell may advantageously be a bacterial, animal, insect or plant cell.
  • a particularly preferred process for producing a vertebrate homologue of UNC-53 protein or a functional equivalent, derivative or fragment of said homologue comprises using insect cells.
  • the invention provides a process for producing a vertebrate homologue of UNC-53 protein of C. elegans or a functional equivalent, fragment, derivative or bioprecursor of the UNC-53 protein, which process comprises culturing an insect cell transfected with a recombinant Baculovirus vector, said vector comprising a nucleotide vector encoding the vertebrate homologue of UNC-53 protein or a functional equivalent, fragment or bioprecursor thereof 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 fru ⁇ iperda or
  • 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 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 tests 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 libraries 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 proliferative disorders.
  • a method of detecting whether a compound is an inhibitor or an enhancer of expression of a vertebrate homologue of UNC-53 of C. ele ⁇ ans, or a functional equivalent, derivative or fragment of said vertebrate homologue 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 of 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 or cancer which is mediated by a vertebrate homologue of UNC-53 protein or a functional equivalent, fragment, derivative or bioprecursor of said homologue.
  • 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 or a functional equivalent, derivative, fragment or bioprecursor of said homologue.
  • a plasmid pCB201 deposited under LMBP Accession No. LMBP 3594 and a MCF-7 and a NIH/3T3 cell line transfected with plasmid pCB201 deposited under LMBP Accession Nos. LMBP 1601 CB and LMBP 1603
  • Bac clone comprising a fragment of hu-unc-53/2 gene (LMBP 3773) and a worm strain comprising a chimeric C. elegans human unc53 gene deposited under LMBP Accession No. LMBP-1663CB.
  • an assay for detecting expression of a 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, or a functional equivalent, derivative or bioprecursor thereof, 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 flurophore such as fluorescein or alternatively to an enzyme such as strepavidin.
  • a method for detecting for expression of a gene coding for a vertebrate homologue of UNC-53 protein or a functional equivalent, derivative, fragment or bioprecursor thereof comprises contacting a probe specific for a nucleic acid or protein sequence coding for or corresponding to said vertebrate homologue or a functional equivalent, fragment or bioprecursor thereof 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 or a functional equivalent, derivative or bioprecursor therefor.
  • 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 comprises an antibody specific for said vertebrate homologue of said UNC-53 protein or a functional equivalent, derivative, fragment or bioprecursor therefor.
  • the reporter is an antibody conjugated to for example a fluorophore such as fluorscein 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 UNC- 53 or a vertebrate homologue thereof according to any of claims to 1 to 9 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.
  • 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 UNC-53 or 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 UNC-53 or 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 UNC-53 or a vertebrate homologue thereof according to the invention with microtubules or the plus end regions thereof which kit comprises at least one transgenic cell expressing UNC-53 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 unc-53 protein of C. elegans or a vertebrate homologue thereof 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 UNC-53 or a vertebrate homologue thereof 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 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- 53 or a vertebrate homologue thereof according to the invention, which method comprises a) contacting either an extract from a cell or cells expressing UNC-53 or said vertebrate homologue or a mixture of enzymes comprising canditate 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 ' " p.
  • the invention is a method of identifying a compound which alleviates or enhances the toxicity of UNC-53 or a 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 regions thereof.
  • the method comprises monitoring the effect of the compound on cell death.
  • Figure 1 illustrates the sequence of plasmid pTB72 which codes for the full length UNC-53 protein in C. elegans f deposited under LMBP Accession No. 3486.
  • Figure 2 illustrates the full-length UNC-53 protein from C. ele ⁇ ans.
  • Figure 3 is a Tblastn search of the EST division of Genbank with the ORF of the longest known Ce-UNC-53 cDNA.
  • tb3-M5 reveals two EST ' s with homology to a predicted coiled-coil region in Ce-UNC-53.
  • Figure 4 illustrates a search of the Genbank databases with part of the nucleotide binding domain of Ce-UNC-53. It does not identify statistically significant proteins except for the C. ele ⁇ ans cosmid containing Ce-unc-53.
  • Figure 5 illustrates a three frame translation of EST gb:R41071.
  • Regions of homology with Ce-Unc-53 in two different frames are underlined.
  • the spacing between the blocks of homology is of similar size to that in Ce-UNC-53.
  • Subsequent re-cloning and re-sequencing of this region in man identified multiple sequencing errors gb:R41071, and identified an ORF which is more homologous to and co-linear with Ce-UNC-53 (see alignment in fig. 12) .
  • Figure 6 is a BLASTN search of the EST division of Genbank with Hu-unc-53/1 cDNA cosmid 3b.
  • Figure 7 is a TBLASTN search of the Genbank sequence database with the 961 amino acid ORF of cDNA 3b of hu-UNC-53/l : hu-UNC-53/1 forms a unique pair with Ce-UNC-53 (cosmid F45E10) compared to the rest of the database.
  • Figure 8 is a diagram illustrating the length and overlap and tissue source of the different cDNA clones of the 3' end of Hu-UNC-53/1 isolated in this work.
  • Figure 8a is a diagram illustrating the further sequence of the Hu-UNC-53/1 and overlap of constructs to obtain the further sequence.
  • Figure 8b is a diagram illustrating the 3' end of Hu-UNC53/1 and the EST clones present in the database.
  • Figure 9a is an annotated sequence listing of clone 3b of hu-UNC-53/1 including the EcoRI polylinker GAATTC.
  • the predicted Open Reading Frame of Hu-UNC- 53/1 is listed below the sequence.
  • Blocks A B C D and E which are similiar to Ce-UNC-53/1, a region which is different between Hu-UNC-53/1 and Hu-UNC-53/2 and the 3 ' untranslated leader sequences are marked with arrows and labelled.
  • Figure 9b is an annotated sequence listing of Hu-UNC-53/1 available at this moment.
  • the predicted Open Reading Frames of Hu-UNC-53/1, pLMl , pLM3 , pLM4 , pCB251, pLM5 and pCB201, the homology blocks A,B,C,D and E, the position of a region which is different between Hu-UNC-53/1 and Hu-UNC-53/2, the position of phhl4-3, pCB212, pCB210-14, phh3b, phhl ⁇ , the position of the reverse primers HU53rvl, HU53rv2, HU53rv3 and HU53rv4, the position of peptides B72628 ( 28/1), B72627, B72626 and B72625 are listed below the sequence.
  • Figure 10 is an annotated sequence listing of the insert of clone gbAA049124 (EST479167) of
  • Figure 11a is an annotated sequence listing of the insert of clone gbH09036 (EST46037) of Hu-UNC- 53 / 2 .
  • Figure lib is a novel DNA sequence of HU-UNC-53/2 extended by RT-PCR. This DNA sequence is not present in EST-46037 and extends the ORF beyond position 1109 of Figure 11a to an ORF from position 18 to 1793.
  • Figure lid compiles the sequence of hu-unc-53/2.
  • the boxed sequences are the primer sequences used for the respective extension steps described in the experimental methods section.
  • Figure lie illustrates the sequences of the extensions summarised in figure lie.
  • Figure llf illustrates the sequence information illustrating four alternative Start sites observed for hu-unc-53/2.
  • Figure 12 is an illustration of a Tblastn search of the EST division of Genbank with 680aa starting at the C-teminus of the alpha actinin domain of hu-unc-53/2.
  • Figure 12a is an illustration of an amino acid alignment of the available sequence of C. elegans unc-53 and hu-unc-53/1 and hu-unc-53/2.
  • 12b is an illustration of similarity plots for Ce-unc-53 and hu-unc-53/1 (top) and for hu-unc-53/1 and hu-unc-53/2.
  • Figure 13 is an annotated sequence listing of expression vector pCB201 containing homology block E from Hu-UNC-53/1 cloned in a pcDNA3.1-HIS expression vector.
  • the HIS and T7-tags, PCR primer used to modify hu-UNC-53/1 and ORF are marked.
  • Figure 14 is a diagram showing the alignment of the homologous regions of hu-UNC-53/1 and mu-UNC-53/1.
  • Figure 15 is an annotated sequence listing of expression vector pCDU3 containing part of Ce-UNC-53/1 cloned in expression vector pcDNA3.1.
  • the upper ORF starts in the vector polylinker.
  • the lower ORF starts at the first Methionine and is part of Ce-UNC-53/1.
  • Figure 16 is an annotated sequence listing of expression vector pCDU4 containing part of Ce-UNC-53/1 cloned in expression vector pcDNA3.1.
  • the upper ORF starts in the vector polylinker.
  • the lower ORF starts at the first Methionine and is part of Ce-UNC-53/1.
  • Figure 17 is an annotated sequence listing of expression vector pCDU2 containing part of Ce-UNC-53/1 cloned in expression vector pcDNA3.1.
  • the upper ORF starts in the vector polylinker.
  • the lower ORF starts at the first Methionine and is part of Ce-UNC-53/1.
  • Figure 18 illustrates MCF-7 cells transfected with pCB201 (upper) compared to mock transfected MCF-7 cells (phase contrast image) .
  • the control cells are spread out on the tissue culture plastic and exhibiting few filopodia outgrowths.
  • the transfected cells appear smaller because they are slightly rounded up and have multiple filopodia outgrowths per cell (arrowheads) .
  • Figure 19 is a phase contrast image of MCF-7 cells, transfected with pcDNA3.1 (19a), pCDU4 (19b), pCDU3 (19c), pCDU2 (19d) and pTB72 (19e).
  • Figure 20 is an F-actin pattern (visualized with TRITC-Phalloidin) of MCF-7 cells transfected with pcDNA3.LacZ (top panel) and with pCB201 (middle and lower panel) .
  • Figure 21 is an F-actin pattern Phalloidin (visualised with TRITC-Phalloidin) of MCF-7 cells transfected with pCDNA3.1 (21a), pCDU4 (21b), pCDU3(21c), pCDU2 (21d) and pTB72 (21e) .
  • Figure 22 is a phase contrast image of N4 neuroblastoma cells transfected with pcDNA3.1 (22a), pCDU4 (22b), pCDU3 (22c), pCDU2 (22d) and pTB72 (22e) .
  • Figure 23 is an F-actin pattern Phalloidin (visualised with TRITC-Phalloidin) of N4 neuroblastoma cells transfected with pcDNA3.1 (23a), pCDU4 (23b), pCDU3 (23c) , pCDU2 (23d) and pTB72 (23e) .
  • Figure 24 illustrates phase contrast images of small (top) , medium (middle) and large foci (bottom) induced in a monolayer of NIH3T3 cells by transfection with pCB201.
  • Figure 25(c) illustrates human metaphase chromosomes probed with a probe lp34 and figures 25a and 25b indicating the chromosomal location of hu-UNC- 53/1 in lq31. Essentially the same techniques were used to assign the gene hu-unc-53/2 to chromosome locus llpl5 (25d and e) as illustrated in micrograph 25f.
  • the ideograms 25a and 25d are from the International System for Human Cytogenic Nomenclature 1985.
  • the ideograms 25b and 25e in which the relative band positions and arm ratios were derived from actual chromosome measurements is from Cytogenet Cell Genet 65:206-219 (1994).
  • Figure 26 is an expression pattern of HU-Unc53/l and HU-Unc532 in normal human tissues and cancer cell lines.
  • Figure 27 is a sequence map of Plasmid pNP3.
  • Figure 28 is an examplary list of prosite signatures which can be used to define and identify vertebrate homologues of UNC-53.
  • Figure 29 is a annotated sequence map of plasmid pEGFPsac.
  • the GFP-C. elegans unc53sac fusion protein, and the C. elegans unc53 sac fragment are indicated.
  • Figure 30 is a sequence map of plasmid pEGFP72.
  • the GFP-C. elegans unc53 fusion protein and the C. elegans unc53 fragment are indicated.
  • Figure 31 is an annotated sequence map of plasmid pEGFPsma.
  • the GFP-C. elegans unc53sma fusion protein, and the C.e.unc53 sma fragment are indicated.
  • Figure 32 is an annotated sequence map of plasmid pEGFPecl.
  • the GFP-C. elegans unc53ecl fusion protein, and the C. elegans unc53 eel fragment are indicated.
  • Figure 33 is an annotated sequence map of plasmid pEGFPxba.
  • the GFP-C. elegans unc53xba fusion protein, and the C. elegans unc53 xba fragment are indicated.
  • Figure 34 is an annotated sequence map of plasmid pLM4. Open reading frames of the hul-unc53/l and GFP are indicated.
  • Figure 35 is a sequence map of plasmid pNP8.
  • Figure 36 is an illustration of microtubule association of C. elegans Unc53, shown in HepG2 cells, transiently transfected with pTB72, expressing
  • Figure 37 is an illustration of microtubule plus- end association in human cell lines transiently transfected with pTB72, expressing C.e.Unc53. C. elegans Unc53 was stained with mab-16-48.
  • Panel C COS cells showing microtubule association panel B: MCF7 cells showing microtubule plus-end association panel A: HepG2 cells showing microtubule plus-end association.
  • Figure 38 is an illustration of microtubule association in N4 cells transiently transfected with pEGFP72, expressing the GFP-C. elegans Unc53 fusion protein. GFP fluorescence was observed in living cells.
  • Panel A microtubule association of the GFP- C. elegans unc53 fusion protein
  • panel B microtubule plus-end association of the GFP-C. elegans unc53 fusion protein.
  • Figure 39 is an illustration of microtubule association in N4 cells transiently transfected with pEGFP72, expressing the GFP-C. elegans Unc-53 fusion protein. Microtubules were stained with YL1/2 after paraformaldehyde fixation.
  • Panel A Microtubule association of the GFP-C. elegans unc53 fusion protein.
  • Panel B tubuline staining.
  • Panel C panel A plus panel B: co-localisation of the GFP-C. elegans unc-53 fusion protein and Tubuline can be seen as yellow.
  • Figure 40 is an illustration of microtubule association in N4 cells, transiently transfected with pEGFPsma, expressing the GFP-C. elegans unc53sma fusion protein.
  • Panel A Microtubule association of the GFP- C. elegans unc53sma fusion product.
  • Panel B Centriole association of GFP-C. elegans unc53sma fusion product when expressed at low levels.
  • Figure 41 is an illustration of microtubule association in N4 cells, transiently transfected with pEGFPeel, expressing the GFP-C. elegans unc53ecl fusion protein.
  • Panel A Microtubule association of the GFP- C. elegans unc53ecl fusion product.
  • Panel B Centriole association of GFP-C. elegans unc53ecl fusion product when expressed at low levels.
  • Figure 42 (a) / Figure 42(b) are illustrations of fluorescence of GFP in N4 cells transiently transfected with pEFPxba and pEFGPsac respectively.
  • Figure 43 is an illustration of microtubule association of in N4 cells transiently transfected with pLM4 expressing GFP-Hu-UNC53/1 fusion protein.
  • Panel A microtubule association of GFP-HU-UNC53/1 fusion protein.
  • Panel B microtubule plus-end association of GFP-Hu-UNC53/1 fusion protein.
  • Panel C microtubule association of GFP-Hu-UNC53/1 in dividing cells (end of division) .
  • Figure 44 is an illustration of the sequence of Plasmid pNP9.
  • Figure 45 is an illustration of immuno fluorescence in melanoma G361 cells stained with sera 28.1.
  • Panel A Microtubule plus-end association of Hu-UNC53/1.
  • Panel B microtubule plus-end association of hul-Unc53 in growth cone extensions.
  • Figure 46 is an illustration of GFP fluorescence and immunofluorescence in N4 cells transiently transfected with pLM4 , and stained with sera 28.1.
  • Panel A Fluorescence of GFP-Hu-UNC53/1 fusion protein.
  • Panel B Immunofluorescence of serum 28.1.
  • Figure 47 is an overview of the microtubule (+) end, the microtubule and f-actin cytoskeleton binding properties of different constructs
  • Figure 50 is an illustration of rescue of lateral ALN neurons in mutant unc-53.
  • Figure 51a is an illustration of chimeric fusion between C. elegans and human 1 homologue of the unc-53 gene.
  • the region of the putative nucleotide binding domain (NTP) is replaced in the C. elegans cDNA by the same region of the human homologue 1 of unc-53 (HI) .
  • the cDNA is under the promotor region A (pA) of unc-53, which raise expression in the ALN lateral neurons.
  • Figure 51b is an illustration of the chimeric minigene nematode/human pA/unc-53-Hl partially rescue the defect in the longitudinal migration of the lateral neurons ALN and PLN.
  • the four strains compared are : wt; unc-53 (nl52) ; unc53 (nl52) ,pA/unc-53; unc-53 (nl52) ,pA/unc-53-Hl.
  • the chimeric fusion between the C. elegans gene and human homolog (unc-53-Hl) partially rescues the mutant phenotype.
  • the chimeric gene was maded by replacing the putative nucleotide binding region (NTP)of the nematode cDNA by the same region of the human homolog 1 (HI) .
  • Figure 52 is an illustration of the sequence for plasmid pLM5.
  • Figure 53 is an illustration of the sequence for plasmid pLM6.
  • Figure 54 is an illustration of the sequence for plasmid pLMl .
  • Figure 55 is a sequence map of plasmid pCB251.
  • Figure 56 is a sequence map of plasmid pNPlO.
  • Figure 57 is a sequence map of plasmid pCB501.
  • Figure 58 is a sequence map of plasmid pTB115.
  • Figure 59 is a sequence map of plasmid pPD95.75.
  • Figure 60 is a sequence map of clone X16.
  • Figure 61 is a sequence map of plasmid pLM3 DEPOSITED MATERIALS
  • the inventors amplified, cloned and sequenced part of gb:R41071 from human heart and human lung cDNA and from human genomic DNA and discovered that clone gb:R41071 had up to ten 10 different mistakes in the region checked. 5 extra nucleotides were scattered along its sequence and two nucleotide substitutions were identified, and gb:R41071 lacked three nucleotides present in our clone (Fig. 5) . The novel sequence obtained was two nucleotides shorter and showed the two UNC-53-homologous regions in frame.
  • the genomic fragment obtained is larger (700 bp total length) than the corresponding cDNA clones indicating the presence of an interverting sequence of around 500 bp in nucleotide 162 of this fragment.
  • the amplified cDNA fragment which was cloned to vector PCRII (Intvitrogen) and named pCR23l and was used as a probe to screen cDNA libraries.
  • the conceptual translation of the clones we obtained by PCR were screened using blast and tblastn against all known protein and DNA sequences in the database. The only clone which came up with statistically significant similarity was Ce-UNC-53 (Fig.6).
  • This human clone and Ce-UNC-53 thus form a unique homologous pair compared to the rest of the known sequences, indicating the statistical relevance and novelty of our discovery.
  • Human heart and a human colorectal adenocarcinoma cDNA libraries were probed with pCR231 probe to identify longer cDNA clones. The clones overlap giving a linear sequence of 3706 bp (Fig 8 and 26) . This sequence shows an 959 amino acid open reading frame from the beginning of the clone.
  • gbAA049124 is >95% identical to Hu-UNC-53/1 over 604 available amino acids (fig. 10) and is the mouse orthologue of Hu-UNC-53/1.
  • the insert in gbH09036 is clearly homologous to hu-UNC-53/1 but derived from a different locus. We therefore name the gene identified by gbAA049124 Mu-UNC-53/1 and the gene identified by gbH09036 Hu-UNC-53/2. ( Figure 11). 5 domains of high similarity mark the unc-53 gene family
  • Ce-UNC-53 and the here-identified vertebrate homologues form a unique novel protein family, that is distant from the remainder of the proteins in the public domain. Alignment of the predicted open reading frames shows that Hu-UNC-53/1 and Hu-UNC-53/2 are equidistant from Ce-UNC-53. The highest homology is found in the carboxyterminal amino acids of Ce-UNC- 53 region. The presence of a conserved GXXGKS/T box suggests a nucleotide binding function. However, this domain as a whole does not belong to a class of known nucleotide binding proteins.
  • HU-UNC53/1 and Hu-UNC-53/2 are complex transcription units.
  • a cancer cell line RNA blots probed with HU-
  • 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 Lympho a 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 and the fifth transcript is around 6 kb.
  • Transcripts 1 and 2 are present in all experssing cell lines.
  • Transcripts 3 and 4 are restricted to Melanoma G361, Lung Cancer A549 and Colorectal Adenocarcinoma SW480 and are the predominant transcripts in Melanoma G361 and Colorectal Adenocarcinoma SW480.
  • Transcript 5 is restricted to Lymphoblastic Leukemia K562 and HeLa S3 and is predominant in HeLa S3.
  • RNA blots probed with HU- UNC-53/2 were probed with a 652bp fragment of EST46037 amplified by using the primers 5'- aggagatgaagctgacagatatcc and 5 * -aaacaccagtgagtcc.
  • HU- UNC-53/2 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.
  • 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, lower levels in heart, placenta, lung, skeletal muscle and pancreas. Expression is barely detectable in brain and liver.
  • the hu-UNC53/l and hu-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) .
  • 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 occurance of a new transcript or the observed changes in expression levels in the cancer cell line blot suggests a role for hu-UNC-53/1 and hu-UNC-53/2 in the establishment or maintenance of the transformed state of those cells.
  • Ce-UNC-53 Ectopic expression of full length Ce-UNC-53 in C. elegans, murine neuroblastoma cells or human MCF-7 breast-carcinoma cells, has been found to lead to increased filopodia outgrowth and increased motility (unpublished) .
  • the structure of Ce-UNC-53 protein is reminiscent of that of large kinases or dynamin where a catalytic domain is postively or negatively regulated by domains that interface with signal transduction pathways for example (by by GRB2 binding, phosphorylation or the like) .
  • the inventors therefore decided to test whether the nucleotide domain by itself is capable of inducing the observed changes in the microfilament cytoskeleton and motile or ruffling behaviour.
  • cDNA fragments coding for the nucleotide binding domains of Ce-UNC-53 and Hu-UNC-53/1 were cloned in mammalian expression vectors with the CMV promoter (see experimental procedures) .
  • pCB20l To be able to detect expression from pCB20l (Fig. 13), an N-terminal his and a T7 epitope tag were fused in frame with the hu-UNC-53/1 cDNA hhl5.
  • pCDU3 contains a larger fragment of Ce-UNC-53 and starts just before the conserved "VIELKIEL" domain (Fig. 12) .
  • N4 neuroblastoma cells were stably transfected with control construct pCDNA3.1 and the C. ele ⁇ ans
  • the population of clones transfected with the empty expression vector were homogeneous and similar to wild type N4 cells.
  • 1/4 to 50% of the clones transfected with pTB72, pCDU2 , pCDU3 and pCDU4 had distinct phenotypes:
  • Wild type or N4 cells transfected with pcDNA3 designated as mock transfection show a central cell body, with extensions, designated as neurite outgrowths. Less than 5% of the population have lamellae. When present, they are generally situated on the cell body and on the opposite site of the neurite extensions (figure 22a) . The lamellae show a radial actin spike pattern. Limited branching of the actin fibres is observed in wild type or pcDNA3 transfected N4 cells. Side branches are smaller and can be clearly distinguished from the main actin branch (figure 23a) . 2.
  • N4 cells stably transfected with pCDU4 , harbouring the homology block E, show an overall morphology which is similar to that of wild type N4's (a cell body with neurite outgrowth) . They exhibit however an increased frequency and level of lamellae formation (figure 22b) . These lamellae, which contain F-actin microspikes are found on both the cell body and the neurite outgrowth (figure 23b) . Wild type N4 cells, in contrast thereto, rarely exhibit lamellae on the neurite outgrowths. 3.
  • N4 cells stably tranfected with pCDU3 , encoding for homology blocks C, D and E, show an even higher level of lamellae formation labelled with TRITC-phalloidin, the cells appear surrounded with F- actin fibres, consisting of bundles of F-actin microspikes (figure 23c) .
  • F- actin fibres consisting of bundles of F-actin microspikes.
  • the presence of these lamellae has completely modified the general appearance of the cells. They appear flatter and in 90% of the population, it is not possible to distinguish between the cell body and the wide neurite as they flow gradually into one another (figure 22c) . If wild-type-like thin neurite-like outgrowths are present, they are frequently numerous, branched and located all around the cell.
  • N4 cells stably transfected with pCDU2 , encoding for homology blocks A, B, C, D, and E, resembles that of the wild type cells since, cell body and neurite outgrowth can be clearly distinguished.
  • the pCDU2 transfected cells however show more neurite outgrowth, and these are long and very branched, especially at the end of the outgrowth. When neurite outgrowths of different cells make contact, increased branching can be observed, giving the appearance of a network (figure 22d) .
  • N4 cells, transfected with pCDU2 show bundles of long radial F-actin filaments (microspikes) , which can be branched, especially apically.
  • N4 cells stably transfected with plasmid pTB72, encoding the full length C. elegans UNC53 protein, seem to have a more rigid structure than wild type cells, most clearly seen as spindle-like and triangle-like cells. The corners of these cells show an increased level of hand-like lamellae structures. This specific phenotype is best seen when the cells are grown at low density (figure 22e, Fig. 23e) .
  • MCF-7 cells were stably transfected with the pTB72, pCDU2, pCDU3, pCDU4 and pCB201.
  • the population of clones transfected with the LacZ-expression vector were homogeneous and similar to wild type MCF-7 cells.
  • -30-50% of the clones transfected with pTB72, pCDU2, pCDU3 , pCDU4 and pCB201 had distinct phenotypes which were analysed as above for the N4 cells:
  • Wild type and mock (pcDNA3) transfected MCF-7 cells are heteromorph. In general they are round cells or clusters of cells surrounded by lamellae. Bulges, similar to thick filopodia, can be observed (figure 19a) . When the cells are stained with FITC- or TRITC coupled phalloidin, F-actin actin stress fibres can be observed, often in rings surrounding the cell body (figure 20a & 21a) . When cells are round up like this actin is present at the edge of the cell body. Less than 10% of the cells display filopodia filled with radial F-actin microspikes. In time-lapse analysis the cells are highly quiescent with limited ruffling at the edge of the cell.
  • MCF-7 cells transfected with pCDU4 encoding for homology block E, show two major phenotypic differences compared to the wild type cells. These cells are more flat and have more extended lamellipodia leading to a pancake-like appearance. Some clones show more filopodia than wild type (figure 19b) .
  • Radially organised F-actin fibres can clearly be observerd in the lamellae surrounding the cells. These stress fibres resemble the wild-type structures, but have a more radial than circular orientation. In the filopodia, one can observe an increase of apparently unorganised, bundles of actin patches (figure 21b) .
  • MCF-7 cells stably transfected with pCDU3, encoding the homology blocks C, D, and E, shows a strikingly different and constant morphology.
  • the cells appear smaller than wild type because they are more rounded up. All the cells have more filopodia, surrounding the cell body (figure 19c) . Morphologically these filopodia have the same "handlike" appearance as those observed in N4 neuroblastoma cells. Such filopodia are hardly ever observed in mock transfected MCF-7 cells.
  • These filopodia are filled with F-actin fibres. Compared to wild type cells, fine actin stress fibres are decreased (figure 21c) .
  • the overall morphology of the MCF-7 cells transfected with pCDU2 which encodes the homology blocks A, B, C, D and E, resembles that of the pCDU3 transfected cells.
  • the cells are more rounded up and show more filopodia than the wild type and mock transfected cells (figure 19d) .
  • the filopodia which are all around the cell body tend to be longer, and show a difference in actin organisation.
  • the small filopodia have the same actin bundles as seen in the pCDU3 transfected cells. In the longer filopodia, the actin bundles are more parallel, and radial to the cell body (figure 21d) .
  • MCF-7 cells transfected stably with pTB72, encoding the full length UNC53 protein are extremely rounded up, and tend to adhere more than wild type cells.
  • the cells grow in clusters with sausage- or tube-like shapes.
  • the presence of large extremely thin lamellae with a surface area of more than three times the central cell body forms a second morphological feature, unique for the pTB72 transfected MCF-7 cells (figure 19e) .
  • These sheets are difficult to observe under a phase contrast microscope, but are very clear when stained with phalloidin.
  • the lamellae protrude from one side of a cell or group of cells and are filled with thin long criss-crossing actin fibres, different from "giant" wild type MCF-7 cells (figure 21e) .
  • Murine and human cells transfected with the Ce-UNC-53 or hu-UNC-53/1 domains show clear effects on the nature and dynamics of their motile behaviour as demonstrated by changes in the F-actin cytoskeleton (the increase in lamellipodia, hand-like filopodia and "hair-like” microspikes on the cell surface and the associated reduction of the "rings of F-actin” stress- fibres) .
  • MCF-7 cells human breast carcinoma cells of epithelial origin
  • murine N4 neuroblastoma cells pCB201, pCDU3 and pCDU4 induce in MCF-7 cells a type of filopodium which is frequent in wild type N4 cells but rare to absent in wild type MCF-7 cells, suggesting the activation by these constructs of motile behaviour which is "normal" in N4 cells but of an unusual type in MCF-7 cells. This indicates the activation of a specific downstream process as opposed to a disruption of an existing process. It is well known that some cell types prefer to migrate with filopodia and other cell types with lamellipodia. 3.
  • pCB201 is a much more potent activator of filopodia outgrowth than pCDU4 , which is to be expected considering the large evolutionary distance between between C. ele ⁇ ans and vertebrates.
  • Homology domains C and D "enhance the basic activity present in homology domain E (pCDU4/pCB201) .
  • Homology domains B and C qualitatively modify the phenotype of domain E, leading morphologically different lamellipodia formation than pCDU3 transfected cells. It is thought that lamellipodia and filopodia formation are mediated by different signal transduction pathways requiring two related but different Ras-like G-proteins RAC for lamellipodia formation and CDC42 for filopodia formation.
  • pTB72 which includes homology domains A,B,C,D,E plus an additional 700 amino acids not yet identified isolated in the human members of the family confers a more localised filopodia outgrowth and a different morphology.
  • Mammalian and human cells transfected with plasmid constructs containing unc-53 sequence of either C. elegans or of human origin were observed to display obvious, specific and similar changes in comparison to mock or untransfected parent cells. These changes relate to the functioning of the cytoskeleton, in particular the F-actin cytoskeleton, to cell locomotion and directionally cell motility and reflect UNC-53 gene family members as capable of playing an integrator function in cell motility.
  • the cellular tools derived through transfection and derived functional assays with these cells not only enable characterisation of the motile phenotype typically observed after introduction of unc-53 genes, they also can be easily adapted to screen for pharmacological compounds that interfere with either (1) the expression of unc-53 gene family members, (2) the cellular functioning of unc-53 transgene (s) and of components in the unc-53 signal transduction pathway. Two classes of pharmacological modulators are envisaged.
  • a first class are inhibitors of UNC-53s or the unc-53 pathway (s) , which revert the described phenotypic changes induced by unc-53 transgenes or aspects thereof.
  • Such compounds are considered relevant leads to target diseases where unwanted directional motility of cells occurs such as metastatis, angiogenesis or inflammation.
  • pharmacological stimulators are envisaged, such as compounds which induce - in non- transfected cells - phenotypes that induce or mimick (aspects of) the described "unc-53' phenotype.
  • Such compounds may do so by inducing or upregulating expression levels of a known unc-53 gene or by activating endogenous (yet unidentified) members of the unc-53 gene family.
  • the target application here are wound and tissue repair, in particular diseases such as neuronal regeneration and plasticity.
  • the nature of compounds envisaged can be small
  • compounds can be thought of as a series of plasmid nucleotide constructs containing gene sequences in a screen for novel unc-53-unrelated genes with a similar functional effect in the cell or genes related to the unc-53 gene family or novel members of the unc-53 gene family based on sequence similarity such as for example the genes in plasmids pTB72, pcDU3 , pcDU4 , pcDU2 , pcB201, or modifications thereof such as for example epitope tagged, deletion, complementation or mutagenised nucleotide constructs.
  • the cellular assays envisaged in the claims have been exemplified for three cell lines: the human breast carcinoma cell line MCF-7, the mouse neuronal cell line N4 and the mouse fibroblast cell line NIH- 3T3.
  • Pharmacological assays are focused on quantification of endpoints in a high throughout screening mode. Many of the computer aids for (semi-) automation are well known to the field and currently applied in the applicants labs. Given the subtlety of the phenotypes observed, primary focus was given to morphological assays that assess the phenotypes or aspects thereof.
  • the nucleotide binding domain of Hu-UNC-53/1 has transforming activity in NIH3T3 fibroblasts
  • Negative controls included empty vector adn Rac 1N17 and cdc42N17. The cells that survived G418 selection were assayed for loss of contact inhibition (their ability to grow as foci) .
  • Positive controls included the combination of two well known oncogenes Myc and H-ras which were able to produce a high number of foci.
  • the nucleotide binding domains of both Ce-UNC-53 and hu- UNC-53/1 are able to induce foci in this assay (Fig 24 & Table 1) .
  • UNC-53 is not restricted to the activation of motility. UNC-53 may exert this additional function through the activation of as yet to be identified signal transduction pathways. Oncogenes frequently arise when a
  • control domain and activation domain are separated though chromosomal rearrangements or integration of a part of a gene in the oncogenic virus.
  • Hu-UNC-53/1 is localized to chromosome lq31.1
  • Clone F226 (BACH-135 (014) , Genome Systems, inc) was isolated from a human genomic BAC library using pCR231 as a probe and was confirmed by sequence analysis to be derived from the hu-UNC-53/1 locus.
  • Purified DNA from clone F226 was labeled with digoxigenin dUTP by nick translation. Labeled probe was combined with sheared human DNA and hybridized to normal metaphase chromosomes derived from PHA stimulated peripheral blood lymphocytes in a solution containing 50% formamide, 10% dextransulfate and 2X SSC. Specific hybridization signals were detected by incubating the hybridized slides in fluoresceinated antidigoxigenin antibodies followed by counterstaining with DAPI.
  • the initial experiment resulted in specific labeling of the long arm of a group A chromosome.
  • a second experiment was conducted in which an anonymous probe which was previously mapped to lp34 and confirmed by cohybridization with a chromosome 1 centromere specific probe, was cohybridized with F226.
  • the experiment resulted in the specific labeling of the long and short arms of chromosome 1.
  • Measures of 10 specifically hybridized chromosomes 1 demonstrated that F226 is located at a position which is 52% of the distance from the heterochromatic-euchromatic boundary to the telomere of chromosome arm lq, and that corresponds to band lq31.
  • At total of 80 metaphase cells were analyzed with 72 exhibiting specific labeling (Fig.
  • HU-UNC-53/2 is localised to Chromosome llpl5.1
  • DNA from clone F329 from BAC for Hu-unc-53/2 was labeled with digoxigenin dUTP by nick translation and applied in the experimental settings used for FISH of Hu-unc53/l with F226.
  • the initial experiment with F329 resulted in the specific labeling of the mid short arm of a group C chromosome which was believed to be chromosome 11 on the base of size, morphology and banding pattern.
  • a second experiment was conducted in which a biotin labeled probe specific for the centromere of chromosome 11 (D11Z1) was cohybridised with clone F329. This experiment resulted in the specific labeling of the centromere in red and the mid short arm in green of chromosome 11.
  • Chromosome llpl5 is a region showing loss of heterozygosity (LOH) in a variety of human malignancies, primarily breast cancer (Ali et al., Science 238, 185-188 (1987); Winqvist et al., Cancer Res. 53, 4486-4488 (1993)) but also Wilms' tumor (Dowdy et al., Science 254, 293-295 (1991); Cowell et al., Br.J. Cancer 67, 1259-1261 (1993)), ovarian and testicular malignancies (Lothe et al., Genes Chromosomes Cancer 7, 96-101 (1993); Weitzel et al., Gynecol Oncol.
  • LOH heterozygosity
  • stomach cancer Bostodian et al., Cancer Res. 56, 268-272 (1996)
  • lung cancer Lidwig et al., Int. J. Cancer 49, 661-665 (1991); Fong et al., Genes Chromosomes Cancer (1994)), infantile tumors of adrenal and liver (Byrne et al., Genes Chromosomes Cancer 8, 104-111 (1993)).
  • LOH is believed to indicate inactivation of a tumor suppressor gene at the location where LOH occurs
  • the frequent LOH found at llpl5 in multiple human cancers suggests the presence of either a cluster of tumor suppressor genes or a single tumor suppressor in this region (Seizinger et al., Cytogenet. Cell genet. 58, 10080-10096 (1991)).
  • chromosome 11 can suppress tumorigenicity of both human breast cancer (Negrini et al., Cancer Res.55, 3003-3007 (1995)) and Wilms' tumor cells (Dowdy et al., Science 254, 293-295 (1991)) and a gene (named HTS1 or ST5) that may be responsible for suppressing tumorigenicity in HeLa cells has been mapped to llpl ⁇ (Lichy et al., Cell Growth Diff. 3, 541—548 (1992)).
  • Abnormalities at llpl5 have also been identified in a variety of other cancers, including lung cancer (parental origin of llpl ⁇ deletion) (Kondo et al., Oncogene 9, 3063-3065 (1994)), bladder cancer (Presti et al., Cancer Res. 51, 5405-5409 (1991)), myeloid leukemia (translocation) (Nakamura et al., Nat. Genet.
  • Mock and unc-53 transfected MCF-7 cells were seeded at low density in culture plates and allowed to adhere to the vessel.
  • Light microscopic inspection at different time points either on live cells or after chemical fixation with Karnovsky ' s fixative revealed that in pcB201, MCF-7 transfected cultures a rounded shaped cell body with at their boundaries many filopodia.
  • mock or untransfected clones had a predominant "flat' phenotype - with little or no filopodia. Quantitative measurements confirmed the statistical significance of this shift in phenotype (table 2 below) .
  • Animations of e.g. actin ruffles in astrocytoma cells or od actin based cell motility in e.g. fibroblasts can be accessed
  • Animations of these clones in NIH-Image can be requested from author or applicant.
  • Time lapse video imaging probably is the most informative way to appreciate the unc-53-induced phenotype in MCF-7 and is amenable to high throughput screening in a pharmacological context.
  • Time lapses compressing 5 minutes real time supply sufficient information to quantitate the intensity of the motile behaviour of pcB201 transfected MCF-7 cells in e.g. 12 well plates.
  • algorithms have been described in the field which can automatically compute the "motile area' of cells by comparing cells in two images appropriately spaced in time (van laerebeke etal., 1992, cytometry, 13, 1-8).
  • UNC-53s have been shown to reside on microtubules and preferentially on the microtubule (+)-ends of cells. This localisation represents an important feature of the UNC-53 family of proteins, which is rarely observed in other proteins. Absence of microtubule (+)-end binding in the protein APC following mutation has been implied in the role of APC in colon cancer (Smith et al., 1994, Cancer Res., 54, 3672) . In analogy, it can be postulated that the proper functioning of UNC-53 also may depend on its specific localisation in the cell.
  • Such an assay comprises contacting a cell culture of a cell line expressing an UNC-53 with a compound in the culture conditions proper for the said cell line, followed by an incubation and finally observation of the UNC-53 (or fragment) in situ by e.g. fluorescence microscopy (for GFP-chimeras) or by fixing the cell culture and performing an immunocytochemical staining for the UNC-53 (or fragment) .
  • methods such as immunocytochemistry for the microtubules of a cell or cell line combined with either immunocytochemistry for Ce-UNC-53 or Hu-UNC-53s or fluorescent detection GFP-UNC-53 chimeras are performed consecutively.
  • Cele ⁇ ans-UNC-53 preferentially binds microtubule plus-ends or GTP-tubulin
  • This construct gives high transient expression in COS cells, high to medium levels of expression in MCF7 cells and medium to low levels of expression in HepG2 cells.
  • transfected cells were stained with various combinations of the anti-Ce-UNC- 53 mab 16-48-2, rabbit anti-UNC-53 polyclonal, anti- tubulin mab YL1/2 and fluorescently labelled phalloidin.
  • UNC-53 co-localises with the entire microtubule cytoskeleton, but at lower expression levels UNC-53 signal is restricted to the terminal regions of the microtubules at the plus ends. Very low levels of the expression yield a dot-like pattern in the vicinity of the cortex of the cell.
  • pcDU2 (figure 17)
  • pcDU3 (figure 15) in which the aminotermus of Ce-UNC-53 is deleted. Proteins corresponding to these constructs are thought to be made in vivo from different unc-53 promoters. Transient transfections followed by immunolocalisation showed these proteins to be cytoplasmic.
  • microtubule associated domain is situated in the N-terminus of the protein. For this reason, we constructed an N-terminal GFP fusion with the full length C. elegans UNC-53 sequence, and various C-terminal deletion derivatives. These fragments encode the N-terminal part of UNC-53 from 139 to 760 aa.
  • a plasmid encoding a GFP fusion with the hul-Unc53 protein was constructed, and introduced into mammalian cells. A derivative of this construct was also constructed.
  • N4 cells where transiently transfected with pEGFP72, encoding a fusion protein of GFP and full length C. elegans unc-53 sequence.
  • the fluorescence of the GFP molecule could be followed in living cells.
  • Cells which expressed low to medium levels of the fusion molecule showed a normal morphology after 18h to 3Oh.
  • the co-localisation of the GFP fusion protein with the microtubules could clearly be demonstrated (figure 38a) .
  • cells which demonstrated a low but still distinct GFP fluorescence specific microtubule plus- end association could be observed (figure 38b) .
  • the transfected cells were fixed with paraformaldehyde, and the tubuline was stained using antibody YL1/2 and antimouse-CY3 (Jackson Labs) . Although a significant loss of GFP fluorescence was observed, one could clearly demonstrate that the filaments observed with the GFP fluorescence co- localise with the microtubules staining (figure 39) .
  • Mammalian cells in this case N , were transfected with a lipofecting agent (lipofectAMINE) while in suspension, not being attached to a surface. After transfecting those cells with pEGFP72, the transfected cell suspension could be diluted in 24- and/or 96-well plates, enabling them to attach ot the surface. Each well may contain a different compound of the collection to screen. After 24h, plates could be automatically screened for fluorescence levels. Wells containing a compound that abolish the toxicity of the GFP-C. elegans UNC-53 fusion protein will give high levels of fluorescence. Compounds having no effect on the fusion product will give no or only low levels of fluorescence.
  • lipofectAMINE lipofecting agent
  • Plasmid pLM4 was transiently transfected into N4 neuroblastoma cells, and GFP fluorescence was observed in living cells. GFP fluorescence of the available sequence of hul-UNC-53 in fusion with GFP was localised at the microtubule level. Moreover, at lower expression levels, both the centrosomes, and specific plus-end association could be observed. As has been observed with the C. elegans UNC-53 derivatives in fusion with GFP, expressed by the plasmids pEGFPsma and pEGFPeel, the GFP association seems to be less tight as was observed by the full length C. elegans UNC-53 fragment in fusion with GFP. The observed instability of the fusion protein can be due to a lesser association to microtubules, or to a degradation of the fusion protein (figure 43) .
  • a serum designated 28.1 from a mouse previously injected with peptide (DNRTLPKKGLYRY) a conserved sequence of the UNC-53 family was used for a immunolocalisation experiment on G361 cells fixed with paraformaldehyde. Antimouse-cy3 was applied as second antibody. Association with microtubule plus-end could clearly be observed. Moreover, in cells showing directional movement, observed as growth cones extensions, abundant staining can be seen in the tip of the growth cone (figure 45) .
  • N4 cells were transiently transfected with plasmid pLM4 and consequently fixed with paraformaldehyde and stained with serum 28.1. Only cells that were transfected showed staining with 28.1, indicating that the antibody of 28.1 recognised the Hul-UNC-53-GFP fusion protein (figure 46) .
  • elegans UNC-53 fusion protein expressed by pEGFP72 shows Unc53 activity b) - GFP-C.
  • elegans UNC-53 fusion protein expressed by pEGFP72 shows microtubule association c) - GFP-C.
  • elegans UNC-53 fusion protein expressed by pEGFP72 shows microtubule plus-end association c) - GFP-C.
  • elegans UNC-53- (deletion variant) fusion proteins expressed by plasmids pEGFPsma and pEGFPeel show microtubule association.
  • elegans-UNC-53- (deletion variant) fusion proteins expressed by plasmids pEGFPsma and pEGFPeel no not show microtubule plus-end association
  • elegans UNC-53- (deletion variant) fusion proteins expressed by plasmids pEGFPxba and pEGFPsac no not show microtubule associations.
  • f) - GFP-hul-UNC-53 fusion protein expressed by plasmid pLM4 shows microtubule association.
  • g) - GFP-hul-UNC-53 fusion protein expressed by plasmid pLM4 shows microtubule plus end association.
  • - serum 28.1 recognises the Hul-UNC-53-GFP fusion protein as expressed by plasmid pLM4 in transiently transfected Neuroblastoma cells N4.
  • j) - the expressed human homologue of C. elegans. - UNC-53 in melanoma line (being at least hul-Unc-53) is associated with the microtubule plus-ends.
  • oligonucleotides used in the PCR-RACE experiments were synthesised by Eurogentee (Belgium) . Radioactive compounds were obtained from Amersham.
  • the pCDNA3.1 eukaryotic expression vectors, human 1GT10 cDNA libraries, marathon-RACE cDNAS, human, Northern blots and the T7-tag monoclonal antibody were purchased from Invitrogen. N4 , MCF7 and NIH 3T3 cells were retrieved from the Janssen Research cell bank. PCR-RACE conditions
  • a quick screen human cDNA library panel was used to amplify EST clone gb..R41071.
  • the primers used were ESTfw 5 ' -AATGGCTTCCTGGTTACCTGAG-3 ' and ESTrv 5'- CAAGTCAGCACCCCGAAGCAGCTCT-3 ' .
  • Human genomic DNA was used also as template (lOOng/reaction) .
  • the amplification conditions were as follows: 1 min at 94°C, 30 sec at 55°C, 30 sec at 72°c, then 35 more times and a final extension of 20 min at 72°C.
  • This PCT fragment was cloned in vector pCR2.1.
  • the resulting plasmid was designed pCR231.
  • a human heart clone was also produced by RACE-PCR from a human heart Marathon cDNA using the following conditions; 1 min at 94 C, 30 sec at 70°C, 3 min 30 sec at 72 C, then 35 more times and a final extension of 20 min at 72 c KlenTaq DNA Polymerase was purchased from Invitrogen.
  • RNA was obtained from N4 murine cells as described.
  • a first strand cDNA was synthesized from 2 ⁇ gr of RNA using Ready To- Go cDNA kit (Pharmacia)
  • the primers used were M-ESTfw 5 • CCTCTGTGGGCACCGAGGTCACC—3'.
  • the amplification conditions were as follows: 1 min at 94°C, 30 sec at 58'C, 30 sec at 72"C, then 35 more times and a final extension of 20 min at 72'C. All the amplifications product were subcloned in pCRII-1 and several independent clones were analyzed by sequence.
  • a human heart cDNA library and a human colorectal adenocarcinoma cDNA library were screened using pCR231bp as probe by the standard plaque hybridization method.
  • the screening produced several positive clones in each library called respectively ⁇ HH3, ⁇ HH4 , ⁇ HH15, ACAD14 and XCAD27.
  • the positive phages were purified by two additional rounds of plaque screening and were then amplified.
  • HU53rvl (5 ' -cct-ggg-act-gaa-gct-ggt-acc-tga-gcc-3 • ) , HU53rv2 ( 5 ' -ttg-gga-aga-gtg-ttc-cga-tcc-cgc-tg-3 ' ) and HU53rv3 (5'gtt-gcc-cag-ctc-tgg-ggc-ttc-cac-tcc-3 ' ) and used together with ⁇ gtlOrv primer (5 ' -gag-gtg-gct-tat- gag-tat-ttc-ttc-cag-ggt-a-3 * ) in three nested PCR reactions on a cDNA amplified library from Human Heart (Clontech) .
  • the reaction mixes contained 25pmol of each primer, 1 mM of each dNTP, 1 ⁇ lKlenTaq Polymerase Mix (50x) and 0.1 ng DNA.
  • the cycling parameters for the first PCR were: 3 min at 94°C, 35 cycles of 1 min at 94°C, 1 min at 51°C and 3 min at 72°C and a final extension of 10 min at 72°C, using HU53rvl and ⁇ gtlOrv as primers.
  • 0.4 ⁇ l of this primary PCR product was amplified using HU53rv2 and ⁇ gtlOrv as nested primers with the following parameters: 3 min at 94°C, 38 cycles of 1 min at 94°C, 1 min at 52°C and 3 min 30 sec at 72°C and a final extension of 10 min at 72°C.
  • the second nested PCR reaction was performed on 0.4 ⁇ l of a 1/50 diluted purified 2.4 kb fragment using HU53rv3 and ⁇ gtlOrv as primers: 3 min at 94°C, 35 cycles of 1 min at 94°C, 1 min at 56°C and 3 min 30 sec at 72°C and a final extension of 10 min at 72°C.
  • a 774 kb amplification product was subcloned in pCR2.1, resulting in plasmid pCB210-14.
  • the clone fragment was analyzed by sequencing. This fragment extends 699 bp in 5' direction (see fig 9).
  • Primer HU53rv4 (5 ' -ccc-tgc-ttg-gtg-ctg-agg-aga- ctg-g-3 ' ) was designed on the 5' end of clone pCB210- 14 and was used together with ⁇ gtlOrv to amplify a fragment of the Human Heart cDNA library with the following parameters: 3 min at 94°C, 35 cycles of 1 min at 94°C, 1 min at 60°C and 3 min 30 sec at 72°C and a final extension of 10 min at 72°C.
  • a 887 bp fragment was subcloned in pCR2.1, resulting in plasmid pCB212. The clone fragment was analyzed by sequencing. This fragment extends a further 767 bp in 5' direction (see fig 9).
  • the EcoRI digested and purified clone pCB212 was used as probe to screen the Human Heart cDNA library (Clontech) using standard plaque hybridization method.
  • the positive phages were purified by two additional rounds of plaque screening.
  • the insert of the ⁇ DNA (produced using Qiagen Lambda Kit) was analyzed by sequencing. This pHH14-3 resulted in a 2663 bp fragment overlapping pCB212, pCB210-14 and the 3' end (434 bp) of ⁇ HH3b and in a 761 bp 5' extension (see fig 9) .
  • Transformed cells carrying the EST 46037 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.
  • 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 50ml RACE.
  • the RACE mixture contained lx KlenTaq PCR buffer, 0.2 mM of each dNTP, lx 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 :
  • Plasmid DNA was isolated using standard protocols (Qiagen plasmid DNA isolation kit) , the sequence of the insert was determined.
  • Gene specific primer (EST46037-R1) 5 » ACTGCCTTGAGACTCTGACTTCAGC nested gene specific primer (ES46037-R2) 5'TGGGCAGAACTGAGAGCTTCTAAGC Marathon cDNA library : human placenta
  • EST 485068 is similar to but not identical with the 5' end of HU-Unc53/l.
  • a primer pair consisting of one 3' EST 485068 primer and one 5' HU-Unc53/2 primer were used to PCR amplify those fragments.
  • Marathon cDNA from human HeLa S3 (fragment E2.3) were used as templates in a PCR.
  • a 50 ml reaction mix contained lxPCR II buffer (Perkin-Elmer) , 1.5 mM MgC12, 0.2 mM of each dNTP, 0.15 mM forward and reverse primer, 2.5 U AmpliTaq Gold (Perkin-Elmer) and 1 ml template.
  • the cycling parameters were 5 minutes at 95°C, 35 cycles of 45 seconds at 94°C, 45 seconds at 65°C and 2 minutes at 72°C.
  • the PCR products were sliced out from an agarose gel and purified using a gel extraction kit (Qiagen) , one ml hereof was used in a second round PCR using the same conditions as above.
  • the PCR products were purified (Qiagen PCR purification kit) and direct sequenced.
  • EST 01222 is homologous but not identical with the 5 'end of HU-Unc53/l.
  • a primer pair consisting of one 3' EST 01222 primer and one 5' HU-Unc53/2 primer were used to PCR amplify this fragments.
  • Marathon cDNA from human HeLa S3 was used as template in a PCR.
  • a 50 ml reaction mix contained lxPCR II buffer (Perkin-Elmer), 1.5 mM MgCl2 , 0.02 mM of each dNTP, 0.15 mM forward and reverse primer, 2.5 U AmpliTaq Gold (Perkin-Elmer) and 1 ml template.
  • the cycling parameters were 5 minutes at 95 C, 35 cycles of 45 seconds at 94°C, 45 seconds at 65°C and 2 minutes at 72°C.
  • the PCR products were sliced out from an agarose gel and purified using a gel extraction kit (Qiagen) , one ml hereof was used in a second round PCR using the same conditions as above.
  • PCR products were analysed on an agarose gel, the fragment of interest was sliced out, purified (Qiagen PCR purification kit) and cloned into pCRr2.1. The sequence of the insert was determined.
  • Marathon cDNA library human colorectal adenocarcinoma SW480 (fragment D4.1-1) gene specific primer (97080801)
  • PCR amplification products and cDNA clones were subcloned either into pBluescript vectors (Stratagene) or in PCR-IIa vector (Invitrogen) and sequenced either manually by the dideoxynucleotide chain termination method with modified T7 DNA polymerase (Sequenase, United States Biochemical) or automatically with an Applied Biosystems 373 DNA sequencer using the fluorescent terminator kit (Perkin Elmer) .
  • a Human multiple tissue Northern (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 hybrydized according to the manufacturer's instructions and washed out in 0.1xSSC:0.2% SDS at 55 ' C.
  • MTN-1 Human multiple tissue Northern
  • 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.
  • Plasmid pCDU2 ( Figure 17) was constructed by cloning the 2.8 kb Apal-Narl fragment from pTB72, the latter restriction site made blunt with klenow enzyme, into pcDNA3, digested with EcoRV and Apal .
  • pCDU2 encodes for the homology blocks A, B, C, D and E.
  • Plasmid pCDU3 ( Figure 15) was constructed by cloning the 1.9 kb Apal-Ndel fragment from pTB72, the latter restriction site made blunt with Klenow enzyme, into pcDNA3, digested with EcoRV and Apal, pCDU3 encodes for the homology blocks C, D and E.
  • Plasmid pCDU4 ( Figure 16) was constructed by cloning the 1.4 kb Apal-Styl fragment from pTB72, the latter restriction site made blunt with Klenow, into pcDNA3 digested with EcoRV and Apal . pCDU4 encodes for the homology block E.
  • pCB20l Equivalent construct of human 1 homologue to expression construct pCDU4 of C. elegans unc-53 gene cloned in a eukaryotic His-tag, Xpress Ab tag expression vector.
  • a suitable Bam HI site was engineered on pHH15 open reading frame by amplification with hhl ⁇ fw primer 5 ⁇ GAGCGGATCCATATGCCTCCTTGCCGTCAAGGTG-3' and M13rv primer (5 ' -cag-gaa-aca-gct-atg-ac-3 ' ) .
  • the amplified fragment was then moved to pCDNA3.
  • This new plasmid called pCB201 produces a cDNA which codes for a fusion protein consisting of a 49 amino acid aminoterminal fragment containing an His-tag and also a T7 epitope tag followed by amino acids 1255 to 1627 of the sequence of the human homologue.
  • pCB201 was also checked by sequence and the n was used in stable transfection experiments carried out in N4 , MCF7 and NIH3T3 cells.
  • pLM5 Equivalent construct of human 1 homologue to expression construct pCDU3 cloned in an eukaryotic His-tag, Xpress Ab tag expression vector.
  • the phage HH3b was linearized using Xhol.
  • BamHl and Xbaal restriction site were created on the pHH3b open reading frame using U3-Bfw (5 '-cca-cac-tag- ggg-atc-cat-gca-aat-gag-g-3 ' ) and U-rv (5*-caa-aag- tct-cta-gag-gag-gcc-agt-3 ' ) as primers. This amplified fragment was then moved to pBluescript KS, digested with BamHl and Xbal.
  • pCB300 Sequencing of this plasmid, named pCB300, showed an amino acid change from a serine to an asparagine due to a change from guanine to adenine on the position 4237 of the DNA sequence.
  • This fault was repaired by cloning a 1418 bp fragment of pLMl (see below) (using Narl and Xbal as enzymes) into pCB300 digested with the same enzymes.
  • the phage HH3b fragment of this plasmid, named pLM6 (fig 53) was then removed using BamHl and Xbal, to pcDNA3.1/HisA digested with the same enzymes.
  • This new plasmid produces a cDNA which codes for a fusion protein consisting of a 49 amino acids aminoterminal fragment harboring a His-tag and a T7 epitope tag, followed by aminoacid 1069 to 1627 of the transcript of HU-Unc53/l.
  • Plasmid pLM5 was checked by sequencing and used on transient and stable transfection experiments carried out in N4 cells.
  • the plasmid pLMl was created using a PvuII and partial BamHl digested fragment of pHH14-3 and a BamHi and Spel digested fragment of phage HH3b, cloned into pBluescript KS digested with Smal and Spel.
  • the pLMl contains the full transcript of HU-UNC-53/1 available at this moment (see fig 9) .
  • 3.pCB251 Equivalent construct of human 1 homologue to expression construct pCDU2 cloned in an eukaryotic His-tag, Xpress Ab tag expression vector
  • the phage HH3b was linearized using Xhol.
  • a BamHl and Xbal restriction site were created on the pHH3b open reading frame using U2fw (5 ' -aag-gga-tga- ttc-ggt-cag-gat-cct-tc-3 ' ) and U-rv (5 ' -caa-aag-tct- cta-gag-gag-gcc-agt-3 ' ) as primers.
  • the amplified fragment was then moved to pCR2.1. This plasmid was named pCB250.
  • pHH3b fragment was removed from pCB250 using BamHl and Xbal and cloned in pcDNA3.1/HisC digested with the same enzymes.
  • This plasmid named pCB251 (figure 55) , was checked by sequencing.
  • pCB251 produces a cDNA which codes for a fusion protein consisting of a 49 amino acid aminoterminal fragment harboring a His-tag and a T7 epitope tag, followed by amino acids 828 to 1627 of the partial transcript of HU-Unc53/l.
  • pCB251 was used on transient and stable transfection experiments carried out in N4 cells (see fig 56) .
  • pLM3 the partial transcript of HU-Unc531 cloned in an eukaryotic His-tag, Xpress Ab tag expression vector pLMl was digested with EcoRV and Xbal. This fragment was cloned in pcDNA3.1/HisB, digested with the same enzymes. pLM3 produces a cDNA which codes for a fusion protein consisting of a 49 aminoacid aminoterminal fragment harboring a His-tag and a T7 epitope tag, followed by amino acids 1 to 1627 of the transcript of HU-Unc53/l available at this moment. pLM3 was used on transient and stable transfection experiments carried out in N4 cells.
  • pLM4 the partial transcript of HU-Unc53/l cloned in an eukaryotic GFP expression vector
  • pLMl was digested with Clal and Xbal. This fragment was cloned in pEGFP-cl, digested with Accl and Xbal. This plasmid was named pLM4. This plasmid produces a cDNA which codes for a fusion protein consisting of GFP, followed by aminoacid 1 to 1627 of the transcript of HU-Unc53/l. pLM4 was used on transient and stable transfection experiments carried out in N4 cells (see figs 43 and 46) .
  • FCS foetal calf serum
  • the DNA-Ca 3 (P0 4 ) 2 precipitate was vortexed and added to the cells, together with 100 ⁇ l of a 0.01 M chloroquine (Sigma) stock in H.O. After four hours of incubation at 37°C in sterile standard air, the medium was removed, and the cells were washed with PBS (GibcoBRL) . 25 ml of medium was added and the cells where placed at 37°C in a 10% CO atmosphere. After 48 hours of incubation, the cells were harvested, diluted and cultivated under selection (600 ⁇ g/ml G418 (Duchefa) ) for two weeks prior to clone selection.
  • PBS GibcoBRL
  • Mock transfected MCF-7 were positive for the beta-galactosidase transgene.
  • the stability of transfection in MCF-7 was assessed by passaging cells four times in the absence of Geneticin and then re-exposing them to the selector agent. In these experiments, unc-53 or mock transfected cells proliferated, whereas untransfected MCF-7 cells proliferated at a much slower rate.
  • 3 ml of transfection mixture was added.
  • the culture was placed at 37°C in sterile standard air. After four hours, 3 ml or normal culture medium was added and the culture was placed at 37'C under 10% of C0 2 . 18 hours later, the culture was washed with PBS, and fresh normal culture medium was added. A further 24 hours later, the cells were harvested, diluted and cultured under selection (750 ⁇ g/ml G418) for two weeks prior to clone selection.
  • the coverslides were then placed in PHEM, containing 0.5% Triton-XlOO (Serva) for 30 minutes, after which th slide was washed again for three times 10 minutes with PHEM.
  • the coverslips were then placed under PBS (0.14 M NaCl, 2.7 mM Kcl, 10 mM Na 2 HP0 4 , 1.8 mM KH 2 P0 4 , pH 7.3) containing 0.2% Tween (Sigma) for at least one hour at 4°'C
  • the coverslips were inverted on 35 ⁇ l of appropriately diluted antibody, being YL 1/2 for tubulin and/or mab 16-48-2 monoclonal or anti-UNC53 (gp48) polyclonal antibody for UNC53.
  • the slides were placed at 4 C C for at least 18 hours. Excess of primary antibody was then removed by washes of three times ten minutes in PBS-Tween. The slides were then treated with secondary antibody in the same way as for the primary antibody.
  • F-actin was labelled by including TRITC- or FITC coupled phalloidine to the incubation buffer.
  • the inverted slides on the secondary antibody were left at room temperature for approximately one hour.
  • Unc-53 and mock transfected MCF-7 were seeded in plates at low density and allowed to adhere to the plate and to locomote overnight. Cells were chemically fixed to the plate, washed and air-dried. Images of the gold lawns were captured using automated videomicroscopy; composite images of the wells were generated and single-cell phagokinesis tracks were measured using a home-made routine in SCILTM software.
  • C. elegans-UNC-53 preferentially binds microtubule plus ends or GTP-tubulin
  • pEGFPsac encodes the N-terminal 13 aa of C.e.Unc53 in fusion with GFP.
  • Plasmid pTB72 (shown in Figure 1) was digested with restriction enzymes SacII and Apal. The resulting 4.5 kb cDNA fragment, encoding for the C- terminal fragment of C. elegans Unc53 was cloned in plasmid pEGFPsac ( Figure 29) , digested with the same enzymes, resulting in plasmid pEFP72 ( Figure 30). Plasmid pEGFP encodes GFP in fusion with the full length C.e. Unc53. c) Construction of N-terminal deletions of GFP- C. ele ⁇ ans UNC-53 fusion protein, other than pEGFPsac: pEGFP72 was digested with Smal .
  • pEGFPsma plasmid pEGFPsma
  • This plasmid codes for the first 760 aa of the Ce-UNC-53 in fusion GFP.
  • pEGFP72 was digested with restriction enzymes Ecll36II and Smal, the resulting plasmid after ligation and transformation in E.coli of the 6.7 kb fragment was designated pEGFPeel ( Figure 32) .
  • This plasmid codes for the N-terminal 670 aa of the C.e. Unc53 in fusion with GFP.
  • pEGFP72 was further digested with Smal and Xbal.
  • the 5.4 kb hul-unc53 fragment was isolated as Clal-Xbal fragment from pLMl ( Figure 54) , and cloned in pEGFP-Cl digested with Accl and Xbal.
  • pEGFP-Cl was isolated from E.coli GM41 (Hfa H, dam-3 , thi-1, rel- 1) . This makes the Xbal restriction site available for restriction digest. The resulting plasmid was designated pLM4 ( Figure 34) .
  • N4 neuroblastoma lines where seeded in Lab Tek chambered coverglass (Nalge Nunc International) and transfected using lipofectAMINE (GibcoBRL) . After 18 hours, the chambered coverglasses where placed on a inverted microscope, and GFP fluorescence could be visulalised.
  • Transfection with the GFP fusion constructed was also performed on coverglasses in a 6-well plate. After paraformaldehyde or methanol-acetone fixation, cells could be stained for actin cytoskeleton with TRITC-phalloidine, for hu-unc53 with sera 28.1 and for tubuline with YL1/2 antibody. Visualisation was then performed on a axioplan (Zeiss microscope) .
  • Transgenic line express GFP in different neurones: the two pairs of pioneering neurones PVP and PVQ, both BDU neurones, both ALN and PLN neurones, both PDE neurones, both PVM neurones, and 4 vulval cells. Expression begins in early embryogenesis, when the axons of those neurones grow out.
  • the two pairs of ALN and PLN neurones each send an axon in a straight line longitudinally from the tail to the head (see fig.50a).
  • the axons are shorter and often branch in a dorso ventral direction (see fig.50b).
  • the neurones are visualised with the construct pA/GFP, injected in unc-53 (nl52) worms.
  • the promoter A from the C. elegans unc-53 gene was fused to the cDNA of the C. elegans unc-53 gene (clone pA/unc53, or pNP9) .
  • This construct was injected in unc-53 (nl52) mutant worms, together with the pA/GFP construct described above to visualise the ALN and PLN neurones.
  • NTPase carboxyterminal predicted nucleotide binding domain
  • the clone pA/unc-53 was deleted of the C. elegans NTPase domain, from the Hpal site, position 29800 on the genomic of unc-53, and replaced by the equivalent domain of the human-1 gene (unc-53Hl) (see fig. 51) .
  • the resulting clone is named pA/unc-53Hl.
  • the four strains compared are: wt; un-53(nl52); unc-53 (nl52) ,pA/unc-53 ;unc- 53(152) ,pA/unc-53-Hl.
  • ⁇ mutant>> the axon is short, does not reach the vulva region and has collateral branches.
  • the transgenic lines provide a functional screening assay for the motility function of at least part of the human UNC-53 gene.
  • the gene of GFP has been amplified by PCR with cpn3 oligo-nucleotides
  • the PCR amplification product is directed by Hindlll and BamHl, sites which are contained respectively in the cpn3 and cpn5 oligonucleotides and sub-cloned in the pBS vector (clone pNP2) .
  • the GFP is then excised from the pNP2 clone at the site Spll and integrated into the X16 clone ( Figure 60) originating from sub-cloning of the lambda phage S4 digested by Xhol.
  • the X16 clone containing the genomic sequence of unc-53 from the position 16621 to the position 24891 cloned in the site Xhol of pBS.
  • pAB/unc-53 pNP8 - Figure 35
  • the promoter region AB of the X16 clone (between
  • the promoter region A has come from the X16 clone between the sites Pstl and Nhel and integrated in the vector pPD95.75 containing the GFP in the sites Pstl and Xbal .
  • the promoter region A has come from the X16 clone between the sites Pstl and BstXI and is integrated into the clone pTB115 in which the region between the sites Pstl and BstXI, containing the promoter of the gene mec-7 and the start of the gene unc-53, has been removed.
  • the nematodes are observed under a ZEISS Axioplan microscope provided with Nomarski lenses, with 40X Neofluar, 63X Plan-Apochromat, 100X Plan-Apochromat objective lenses.
  • the luminous source is a mercury bulb.
  • Different ZEISS filters are used:
  • FITC filter blue excitation line at 588 nm, emission through a 515-565 nm band-pass filter
  • TRITC filter blue excitation line at 588 nm, emission through a 515-565 nm band-pass filter
  • TRITC excitation through a 546 nm band-pass filter, emission through a 590 nm long-pass filter.
  • Sequence ID No 1 is an amino acid sequence of human homologue 1 of UNC-53 protein illustrated in Figure 9b.
  • Sequence ID No 2 is an amino acid sequence of human homologue 2 of UNC-53 protein illustrated in figure lid.
  • Sequence ID No 3 is a nucleic acid sequence of the hu-l-unc-53 gene illustrated in Figure 9b.
  • Sequence ID No 4 is a nucleic acid sequence of the hu-2-unc-53 gene illustrated in Figure lid.
  • Sequence ID No 5 is a nucleotide sequence of Phage Lamda Clone 3b deposited under Accession No LMBP 3595. illustrated in Figure 9.
  • Sequence ID No 6 is a nucleotide sequence of plasmid pLMl deposited under Accession No LMBP 3762 and illustrated in fig 54.
  • Sequence ID No 7 is a nucleotide sequence of plasmid pLM4 deposited under Accession No 3763 and illustrated in fig 34.
  • Sequence ID No 8 is a nucleotide sequence of plasmid pEGFP72 deposited under LMBP Accession No 3764 and illustrated in fig 30.
  • Sequence ID No 9 is a nucleotide sequence of plasmid pCB501 deposited under Accession No 3765 and illustrated in fig 57.
  • Sequence ID No 10 is a nucleotide sequence of plasmid pCB201 deposited under Accession No. LMBP 3594. S ⁇ & lO a. Pag ⁇ l
  • GGCCArGC7 GCTGAAACTTCAAGAAGCTGCC ⁇ AC7
  • G' A GGCGAACGGGCCC C ⁇ CrCCCACACCAT3CCCArGCG:AGCCCCAGCAAGCTCAGCCArArcrCCCGCCrGGAGCr3G " CGA ⁇ rr__TG-A-r--
  • crrc ⁇ 3C :e:rcArcccc
  • -U30RF pLM50RF L o ⁇ F Q V F K D M 0

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Abstract

L'invention concerne l'identification d'homologues vertébrés de la protéine UNC-53 de C. elegans et des séquences d'acide nucléotidiques codant pour lesdits homologues ou leurs équivalents fonctionnels. Les séquences nucléotidiques sont utilisées dans un vecteur approprié pour transfecter ou transformer des cellules, des tissus ou des organismes utiles pour identifier des inhibiteurs ou des activateurs de l'homologue vertébré ou d'autres protéines impliquées dans la voie de transduction du signal dont ledit homologue vertébré est un élément. N'importe lequel desdits inhibiteurs ou activateurs identifiés peut être inclus dans une composition pharmaceutique ou dans la préparation d'un médicament destiné à traiter des pathologies (maladie neurologique, trauma aigu, par exemple) ou à promouvoir la régénération neuronale ou inhiber la métastase ou la perte de l'inhibition de contact.
PCT/EP1997/006956 1996-12-04 1997-12-03 Homologues vertebres de la proteine unc-53 de c. elegans WO1998024810A2 (fr)

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CA002273827A CA2273827A1 (fr) 1996-12-04 1997-12-03 Homologues vertebres de la proteine unc-53 de c. elegans
JP52523198A JP2001522222A (ja) 1996-12-04 1997-12-03 線虫のｕｎｃ−５３タンパク質の脊椎動物ホモログ
AU56622/98A AU5662298A (en) 1996-12-04 1997-12-03 Vertebrate homologues of unc-53 protein of c. elegans
EP97952926A EP0941239A1 (fr) 1996-12-04 1997-12-03 Homologues vertebres de la proteine unc-53 de c. elegans

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GBGB9625283.8A GB9625283D0 (en) 1996-12-04 1996-12-04 Vertebrate homologues of unc-53 protein of c.elegans or functional eqivalents thereof and cdna sequences coding for said homologue
GB9625283.8 1996-12-04

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999063080A1 (fr) * 1998-06-03 1999-12-09 Janssen Pharmaceutica N.V. HOMOLOGUE HUMAIN DE LA PROTEINE UNC-53 DE $i(C. ELEGANS)
WO2000054815A3 (fr) * 1999-03-16 2001-01-25 Devgen Nv EXPRESSION D'ADN OU DE PROTEINES DANS DES $i(C. ELEGANS)
WO2000050451A3 (fr) * 1999-02-26 2001-08-02 Deutsches Krebsforsch Proteine (tp) impliquee dans le developpement du systeme nerveux central
WO2002017947A3 (fr) * 2000-08-30 2003-03-20 Deutsches Krebsforsch Utilisation de proteines t pour une caracterisation et une therapie differentielles de lesions et de tumeurs du systeme nerveux
WO2003085134A3 (fr) * 2002-04-05 2004-04-15 Japan President Univ Tokyo Methodes de diagnostic et de traitement du cancer colorectal
EP2298890A1 (fr) * 2004-12-30 2011-03-23 Agency for Science, Technology and Research Gènes du hamster chinois liés à l'apoptose

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TW200640485A (en) * 2005-01-19 2006-12-01 Vaxinnate Corp Compositions of pathogen-associated molecular patterns and methods of use

Family Cites Families (1)

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GB9510944D0 (en) * 1995-05-31 1995-07-26 Bogaert Thierry Assays and processes for the identification of compounds which control cell behaviour,the compounds identified and their use in the control of cell behaviour

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999063080A1 (fr) * 1998-06-03 1999-12-09 Janssen Pharmaceutica N.V. HOMOLOGUE HUMAIN DE LA PROTEINE UNC-53 DE $i(C. ELEGANS)
WO2000050451A3 (fr) * 1999-02-26 2001-08-02 Deutsches Krebsforsch Proteine (tp) impliquee dans le developpement du systeme nerveux central
WO2000054815A3 (fr) * 1999-03-16 2001-01-25 Devgen Nv EXPRESSION D'ADN OU DE PROTEINES DANS DES $i(C. ELEGANS)
WO2002017947A3 (fr) * 2000-08-30 2003-03-20 Deutsches Krebsforsch Utilisation de proteines t pour une caracterisation et une therapie differentielles de lesions et de tumeurs du systeme nerveux
WO2003085134A3 (fr) * 2002-04-05 2004-04-15 Japan President Univ Tokyo Methodes de diagnostic et de traitement du cancer colorectal
EP2298890A1 (fr) * 2004-12-30 2011-03-23 Agency for Science, Technology and Research Gènes du hamster chinois liés à l'apoptose

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