CA2330179A1 - Human homologue of unc-53 protein of c. elegans - Google Patents
Human homologue of unc-53 protein of c. elegans Download PDFInfo
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- CA2330179A1 CA2330179A1 CA002330179A CA2330179A CA2330179A1 CA 2330179 A1 CA2330179 A1 CA 2330179A1 CA 002330179 A CA002330179 A CA 002330179A CA 2330179 A CA2330179 A CA 2330179A CA 2330179 A1 CA2330179 A1 CA 2330179A1
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- unc
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Abstract
There is disclosed human homologues of the UNC-53 protein of C. elegans and cDNA sequences coding for said homologues or functional equivalents thereof.
The invention also relates to processes for identifying compounds which control cell behaviour, compounds identified and pharmaceutical compositions containing them in addition to processes and assays for identifying disease states in which said gene or protein is dysfunctional.
The invention also relates to processes for identifying compounds which control cell behaviour, compounds identified and pharmaceutical compositions containing them in addition to processes and assays for identifying disease states in which said gene or protein is dysfunctional.
Description
WO 99!63080 PCT/EP99/03848 HUMAN HOMOLOGUE OF UNC-53 PROTEIN OF C. ELEGANS
The present invention relates to a vertebrate homologue of UNC-53 protein of C. eleaans and cDNA
sequences coding for said homologwes or functional equivalents thereof. The invention also relates to processes for identifying compounds which control cell behaviour, compounds identified a.nd pharmaceutical compositions containing them in addition to processes and assays for identifying disease states in which said gene or protein is dysfunctional.
The control of cell motility, cell shape and directionality of cell outgrowth of axones or other cell outgrowths is an essential feature in the morphogenesis and function of both unicellular and multicellular organisms.
Some cell surface proteins and extra-cellular molecules conti°olling the directionality and potential of cell migration have been identified, although the processes involved are not generally understood. It is generally considered that a long-range migration of a cell process (also known as a growth cone extension) is a stepwise event, whereby prior to and after each extension there is the formation of a structure at the leading edge of the cell. Localised stab.ilisatio:n of the actin cytoskeleton and association with plus end regions of microtubules is a-general cell biological process underlying the choice of directional extension.
The present inventors have surprisingly found a new human gene~protein belonging to the UNC-53 family that binds microtubules and, in particular, the plus-end regions of microtubules.
A gene from the free-living nematode Caenorhabditis elegans designated "unc-53" has been previously identified and cloned (Abstract, WO 99/63080 PCT/EP99i03848 _ - 2 -International C. electans Meeting, June 1-5 1991, Madison, Wisconsin, 58, Bogaert a.nd Goh). The present inventors previously identified i~NC-53 protein as a signal transducer or signal integrator controlling the directionality of cell migration and/or cell shape in C. elecsans (WO 96/38555) .
The C. elecxans UNC-53 protein (Ceunc53) and previously found human homologues thereof (hs-unc.53/1 and hs-unc53/2) were found to encode a signal transducer or a signal integrator, controlling th~~
directionality of a cell migration, cell shape and growth extension. Evidence indicates that the presently found homologue designated (hs-unc53/3) might act as an adapter linking extracellular signals to the actin cytoskeleton. Firstly hs-unc-53/3 slxows homology to the cortical actin binding proteins, and the Ce-UNC-53 protein has been shown to bind F-actin in vitro and leads to actin re-organization in vivo when expressed in mammalian cells, leading to an increased number of filopodia and. lammelipodia.
Furthermore, increased neurite extension and increased cell motility could be observed. Hs-UNC-53-3 may play an important role in the development of various diseases.
According to a first aspect of the present invention there is provided a vertebrate protein homologue of an UNC-53 protein of C. eleaans, which protein comprises an amino acid sequence having one or more of sequence blocks A, B, C, D, E, F, G or H as illustrated in figure 4 or which differs from said blocks in conservative amino acid changes.
According to a further aspect of the present invention, there is provided a vertebrate protein homologue of UNC-53 protein of C. eleaans or a functional equivalent, derivative or bioprecursor thereof, having an amino acid sequence encoded by the nucleotide sequence illustrated in figure 1(e).
_ 3 _ For the purposes of the present invention a "derivative" should be taken to mean mutational derivatives, fusions, internal deletions, splice variants and muteins.
Preferably, said vertebrate homologue is a human protein, and preferably a mammalian or a mouse protein.
A further aspect of the invs=_ntion comprises a vertebrate homologue comprising an amino acid seC~uence as shown in figure 1(f) or the variants thereof cr an amino acid sequence which differs from the amino acid sequences shown in figure ltf) to a significant extent only in one or more conservative amino acid changes.
In a further aspect of the present invention there is also ;provided a nucleic acid molecule, which is preferably DNA, and which encodes a vertebrate hamologue of UNC-53 protein of C.. eleaans, or a functional equivalent derivative, fragment or bioprecursor of said homologue according to the invention. Preferably, the cDNA comprises a sequence of nucleotides encoding an amino acid sequence as illustrated in figure 1(f) or the variants thereof or an amino acid which differs from the sequences shown in these figur~as to a significant: extent only in one or more conservative amino acid changes. Preferably the DNA is cDNA, which cDNA comprises the sequence shown in figure 1(e)or the variants indicated therein.
Also provided by the present invention is a nucleic acid sequence capable of hybridi~~ing to the nucleic acid or DNA sequences according t:o the invention under high stringency conditions, which conditions are well known to those skilled in the art..
The cDNA according to the invention may be included in an expression vector which may itself be used to transform or transfect a host cell, which cell may be bacterial or eukaryotic in. origin including such as, far example an animal or plant cell a fungal WO 99/63080 PCTIElP99103848 cell or an insect cell. Thus, advantageously, once the cDNA corresponding to the genome of the verteb-rate homologue of UIVC-53 of C. electan~~ according to the invention is synthesised, using for example, reverse transcriptase or the like, a range of cells, tissues or organisms may be transfected following incorporation of the selected cDnfA 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 sE~quence 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 tr_ansgene capable of expressing a vertebrate homologue of UNC-53 protein of C. eleaans according to the invention. The term "transgene capable of expressing a vertebrate homologue of UNC-53 protein of C. elecrans" as used herein means a suitable nucleic acid sequence which leads to the expression of a vertebrate.homologue of UNC-53 protein of C. ele ans according to the invention having the same function and/or activity. The transgene m.ay include, for example, genomic nucleic acid isolated from the appropriate vertebrate or synthetic nucleic acid including cDNA. The term "transgenic organisms, tissues or cells, as used herein means any suitable organism and/or part of an organism, tissue or cell, that contains exogenous nucleic acid either stably integrated in the genome or in an extrachromosoma.l state.
Preferably the transgenic cell comprises any of, a CUS cell, HepG2 cell, MCF-7 or N4 neuroblastoma cell, a NTH3T3 cell, a colorectal or carcinoma ce:l1 or a human derived cell such as a fibroblast or the :Like.
The transgenic organism may be an insect, a non-h~.unan animal or a plant and preferably C. eleQans or a _ - 5 -related nematode. Preferably, th.e transgene comprises the nucleic acid or cDNA sequence: encoding the .
vertebrate homologue according to th;e invention as described above. The transgene preferably comprises an expression vector according to th.e 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. elec~ns according to the invention. For example, the gene may comprise deletions or mutations but may still encode a functional vertebrate homologue of UNC-53 protein.
Further provided by the present invention is a method of producing a mutant vertebrate non-human organism having a mutation in the wild-type gene coding for the vertebrate homologue of UNC-53 protein according to the invention, which mutation affects cell behaviour or the regulation of cell motility or the shape or the direction of cell migration or microtubule plus end stability or function and localisation ot: protein complexes located thereony which method comprises inducing a mutation in the vertebrate homalogue of UNC-53 protein in said organism. These mutant organisms may be used in a screen to identify the effects of compounds on these cell functions.
The vertebrate homologue of UNC-53 protein oi_ C. elecrans or the cDNA or genomic DNA encoding it or a functional equivalent, derivative, fragment or bioprecursor of said homologue, may advantageously be used as a medicament, or in the preparation of a medicament to treat or prevent disorders associatE:d with inhibition of overexpression of the vertebrate homologue of UNC -53 according to the invention. Such disorders may be alleviated by promoting neuronal regeneration, revascuiarisation or wound healing ar the treatment of chronic neurodeg~enerative disordE:rs, psychiatric disorders or acute traumatic injuries or fibrotic disease or disease in which physiological-events requiring the polarity of cells or epithelia are abnormally functioning. Accordingly, the vertebrate homologue according to the invention, dominant positive or negative mutants thereof, or inhibitors thereof may advantageously be used to induce or alleviate contact inhibition in a cell or in preventing carcinoma development. Typically, the above medical conditions may be treated in mammals and more preferably humans by either the homologue of UNC-53 protean or alternatively by a nucleic acid cod_Lng for the protein or the protein itself according to the invention. Alternatively an antisense oligonucleotide to said UNC-53 vertebrate homologue may be used to prevent it's expression. Examples of other nucleic acid sequences which may be used include 3' untranslated regions of mRNA which could be used to prevent transcription of the geno~:nic sequence encoding for the vertebrate homologue of U.'VC-53 protein according to the invention.
The vertebrate homologue of ~JNC-53 protein according to the invention may be incorporated into a pharmaceutically acceptable composition together with a suitable carrier, diluent or excipient therefor.
The pharmaceutical composition ma:y advantageously comprise, additionally or alternatively, the nucleic acid sequence according to the in~;rention as defined above.
The induction or inhibition o f the expression of hu-UNC-53/3 by pharmacological me<~ns may advantageously be used to induce neuronal regeneration, revascularisation or wound healing or be involved in the treatment of chronical neurodegenerative disorders, or acute traumatic injuries or fibrotic diseases, or physiological events requiring the polarity of cells, or oncology and - _ metastasis of cells, or apoptotic: pathways.
The present invention therefore also provides~for a method of determining whether a compound is an inhibitor or enhancer of the regulation of cell behaviour, growth, transformation, cell shape or motility or the direction of cell. migration, microtubule pla.zs end stability or function and localisation of protein complexe~~ thereon, which method comprises contacting said compound with a transgenic cell according to the invention and screening for a phenotypic changes in said cell. The method can therefore be used to determine whether the compound comprises an inhibitor c~r an enhancer of the signal transduction pathway of said transgenic cell of Z5 which pathway said vertebrate homologue of UNC-53 protein according to the invention is a component, or whether said compound is an inhibitor or an enhancer of a parallel or redundant signal transduction pathway in said cell. The present invention also provide.> a method to determine that the protein in said signal transduction pathway is a vertebrate homologue of UNC-53 protein of C. eleaans according to the invention.
Preferably, the phenotypic change to be screened comprises a change in cell shape or a change in cell motility. Where a transgenic cell is used in accordance with one embodiment of the method of the invention, an N4 neuroblastoma cell may be used and in such an embodiment the phenotypic change to be screened may be the length of neurite growth, changes in filopodia outgrowth, changes in ruffling behaviour or cell adhesion, any change in microtubule cytoskeleton, any change in localisation of proteins on plus end regions of microtubules or any change in a cell such as apoptosis. In an alternative embodiment of the method of the invention, t:he transgenic ce7.l may comprise an MCF-7 breast carcinoma cell.
Typically in such an embodiment t:he phenotypic change _ g _ to be screened comprises the extent of phagokinesis or filopodia formation. In an alternative embodiment. of this aspect of the invention, they transgenic cell may comprise an NI>a3T3 cell. Typica2,ly in such an embodiment the phenotypic change to be screened comprises loss of contact inhibition of foci formation. The method according to the invention, may also utilise a mutant cell or mutant organism according to the invention as described above, where the mutant cell is capable of growing in tissue culture or in vivo and either of which cell or organism has a mutation in the wild-type unc-53 gene.
In accordance with the present invention, a "phenotypic change", may comprise any phenotype resulting from changes at any suitable point in the life cycle of the cell, tissue or organism defined above, which change can be attributed to the expression of l.he transgene of the invention such as for example, growth, viability, morphology, behaviour, movement, cell migration or cell process or growth cone extension of cells and includes changes in body shape, locomotion, chemotaxis, contact inhibition, mating behaviour or the like. The phenotypic change may preferably be monitored directly by visual inspection of the cell as a whole or by monitoring the F-actin cytoskeleton microtubule network and plus end stability of microtubules or proteins thereon or alternatively by for example measuring indicators of viability including endogenous or transgenically introduced histochemical markers or other reporter genes, such as for example ~i-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 direci~ion of cell migration may be used as a medicament, or WO 99!63080 PCTIEP99l03848 _ g _ 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 tz:aumatic injuries or fibrotic disease. Examples of px:omoting neuronal regeneration include, for example, peripheral nerve regeneration after trauma and sp~.nal cord trauma.
Where a compound is identified in accordance with the method described above as being an inhibitor of the regulation of cell shape or mobility or the direction of call migration, the compound may be used as a medicament, or in the preparation of a medicament, for substantially alleviating spread of disease inducing cells, such as i.n spread of carcinoma, or the like in metastasis or in alleviating loss of contact inhibition. Advantageously, any of the compounds which may have been identified as an inhibitor or an enhancer in accordance with the method as described above, may also be included in a pharmaceutical composition comprising the respective compound and a pharmaceutically acceptable carrier, diluent or excipient therefor.
The particular mechanism of action of a compound identified as either an inhibitor or an enhancer of the cell motil_~.ty shape, growth or direction of cell migration or microtubule association or to the plus end region thereof is not limiting. Preferably the compound acts as an inhibitor or enhancer of a signal transduction pathway. The compound may also act on a parallel pathway or directly on the vertebrate homologue of UNC-53 protein of C. eleaans. For example, the method of action of the compound may include direct interaction with the vertebrate homologue of UNC-53 protein, interaction with processes for regulating phosphorylation or dephosphorylation of the vertebrate homologue of tJNC-53 or with processes regulating activity of an unc-53 WO 99/b3080 PCTIE:P99103848 gene or with processes for post-t:ranscriptional or post-translational modification or the like.
Preferably the compound is identified by the method according to the invention as an inhibitor or an enhancer, by utilising differE:nces of phenotype of the cell, tissue or organism, which are visible to the eye. Alternatively indicators ot: viability including endogenous or transgenically introduced histochemical markers or a reporter gene may be used.
According to a further aspects of the invention there is also provided a transgenic cell or tissue culture which has been constructed to comprise a promoter sequence of a gene coding for a vertebrate homologue of UNC-53 of C. elegan~; according to the invention operably linked to a nucleic acid sequence encoding a repcarter molecule. Preferably, the reporter sequence encodes for a detectable protein, for example one which may be monitored by eye inspection such as antibiotic resistance, (3-galactosidase o r a molecule detecaable 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. eleaans, according to the invention which method comprises the steps of:
(a) contacting said compound with a transgenic cell according to the invention as described above, (b~ monit:oring the level of said reporter molecule and comparing results obtained from this monitoring step with a control comprising a transgenic: cell having the promoter sequence of a gene coding for a vertebrate homologue of UNC:-53 protein, or a functional fragment of said WO 99!63080 PCT/EP99103848 homologue and the reporter molecule, in the absence of the compound.
In one embodiment of the method according to this aspect of the invention the reporter molecule may comprise messenger RNA.
A compound identified as an enhancer of transcription of the gene coding for the vertebrate homologue of UNC-53 protein of C. elecrans or a functional equivalent, derivative or bioprecursor of said homologue may also be used ~~.s a medicament, or in the preparation of a medicament, for promoting neuronal regeneration, revascularisation or wound healing, or for treatment of chronic neuro-degenerative diseases or acute traumatic injuries or fibrotic disease. Furthermore, such compounds may be included in a .pharmaceutical composition including a pharmaceutically acceptable carrier, diluent or excipient therefor. .Any compounds identified as inhibitors of 'transcription may, advantageously, be used in alleviating the spread of: disease inducing cells such as carcinomas or metastasis or loss of contact inhibition.
The present invention also ~>ravides a kit for determining whether a compound is an enhancer or an inhibitor of the regulation of cE:ll growth, transformation,, cell motility or shape or the direction of cell migration which kit comprises at least one transgenic or mutant cE:ll or transgenic or mutant non-human organism according to the invention as described above and a plurality of wild-type cells or a wild-type organism of the same type, or a cell line or tissue culture and means for contacting said compound with :>aid cell or organism.
Also provided by the present. invention is a kit for determining whether a compound is an inhibitor or an enhancer of transcription of a. gene coding for a vertebrate homologue of UNC-53 protein of C. eleqans WO 99163080 PCTlElP99/03848 according to t3ze invention which kit comprises at least one tran;sgenic cell or cells according to the invention, means for contacting ~~aid compounds with said cells and means for monitoring the level of transcription of said transgenic cell or cells according to the invention.
For the purposes of the present invention, the term "gene coding for a vertebrate homologue of UNC-53 or a functional fragment of said homologue" includes the nucleic acid sequence shown i.n figure d or a fragment thereof, including the cifferentially spliced isoforms arid transcriptional starts of the nucleic acid sequence and which sequence encodes a vertebrate homologue of UNC-53 protein or a functional equivalent, derivative, fragment or bioprecursor of the protein.
The present invention also provides methods of identifying genes of vertebrates or fragments of said genes, which encode proteins which are active in the signal transduction pathway of which the vertebrate homologue of UNC-53 according to the present invention is a component.. A preferred method comprises hybridizing to an appropriate cDNA library a nucleotide sequence, as defined herein, or a fragment thereof under appropriate conditions of stringency in order to identa.fy genes having statistically significant homology with the cDNA clones of any one of the cDNA sequences according to the invention described above.
Furthermore, there is also provided by the present invention a method of identifying a protean which is active in the signal transduction pathway of a cell of which a vertebrate homologue of UNC-53 protein of C. eleqans according to the invention is a component. According to this aspect of the inveni~ion, the method comprises:
(a) contacting an extract of said cell with an WO 99163080 PCT/E~99103848 antibody to the vertebrate homologue of UNC-~53 protein or a functional equivalent, fragment:-or bioprecursor of said protein, (b) identifying the antibo<iy/vertebrate homologue of UNC-53 complex,, and (c) analysing the complex t:o identify any protein bound to the vertebi:ate homologue of UNC-53 protein other than the antibody.
The vertebrate homologue of UNC-53 protein, therefore may bind regions of otY~er proteins involved in the signal transduction pathway. It is also possible to sequentially identify a whole range of proteins involved in the signal t:ransduction pathway.
Antibodies to the vertebratE: homologue of UNC-53 I5 protein may be produced according to known techniques as would be known to those skillE:d in the art. F'or example, polyclonal antibodies may be prepared by inoculating a host animal, such as a mouse, with a protein or epitope of a protein according to the invention and recovering immune ~~erum.
This aspect of the invention, further comprises a method of identifying a further ~>rotein or proteins which are active in the signal transduction pathway of a cell of which the vertebrate homologue of UNC-53 is a component which method comprises:
(a) forming an antibody to the first identified protein bound to the vertebrate homologue of UNC-53 protein in the methof~ as described above, (b) contacting a cell extract with the antibody, (c) identifying any antibod.y/protein complex, (d) analysing the complex to identify any further protein bound to the first protein other than the antibody, and (e) optionally repeating steps (a) to (d) to identify Further proteins in the pathway.
According to this aspect of the present invention, the antibody starts the process by binding to the vertebrate homologue of UIVC-53 protein according to the invention in the signal transducer-ion or oncogenic pathways. Any other proteins found complexed to the bound antibody or UNC-53 proteiru can then be used to identify further interacting proteins involved in the pathway.
It may also be possible to _Ldentify proteins involved in the signal transduct:Lon pathway of a cell of which the vertebrate homologuE~ of UNC-53 is a component by using a vertebrate homologue of UNC-53 protein of C. elec~ans. According to this aspect of the invention 'the method comprises:
(a) contacting an extract of the cell with the vertebrate homologue of UNC-53 protein of C. e.legans or a functional equivalent, fragment or bioprecursor of said homologue, (b) identifying the vertebrate homologue of UNC-53 p:rotein/protein complex formed and (c) analysing the complex t:o identify any protein bound to the vertebrate homologue of UNC-53 protein other than the same vertebrate homologue of UNC-53 protein.
This method can also advantageously be used to identify further proteins in a signal transduction pathway of a cell by contacting an extract of the cell used as described above, with any protein identified from step (c) above not being a vertebrate homologue of UNC-53 protein and repeating steps (b) and (c).
Other methods which may be used for identifying proteins in a signal transduction pathway of a cell may comprise for example a western blot overlay method which method is well known to those skilled in the art. Cell extracts are run on gels to separate out protein and subsequently blotted onto a nylon membrane. Theve membranes may then be incubated, for example in a medium containing vertebrate homologue of UNC-53 having a label attached thereto such as- a WO 99/63080 PCTIEI'99/03848 _ 15 _ biotin or radiolabel and any protein conjugates visualised with for example a streptavidin or alkaline phosphatase conjugated antibody.
The present invention also advantageously provides a process for the preparation of binding antibodies which recognise proteins or fragments thereof involved in the rate and direction of cell migration or the control of cell growth or shape, for the above methods.
The monoclonal antibody for binding to the appropriate vertebrate homologue of UNC-53 (or its functional equivalent) may be prepared by known techniques as described by Kohler R. and Milstein C., (1975) Nature 256, 495 to 497.
Another method which may be used to identify proteins involved in the signal transduction pathway of a cell of which a vertebrate h.omolague of an UNC-53 protein of C. eleaans according to~the invention or is a component, involves investigating protein-protein interactions using the two-hybrid. vector method. This method, which .is well known to those skilled in the art was first developed in yeast by Chien et a1 (1991). This technique is based on functional reconstruction in vivo of a transcription factor which activates a reporter gene. More particularly the technique comprises providing an appropriate host cell with a DNA construct comprising a reporter gene under the control of a promoter regulated by a transcription factor having a DNA binding domain and an activating domain, expressing in the host cell a first hybrid DNA
sequence encoding a first fusion of a fragment or all of a nucleic acid sequence according to the invention and either said DNA binding domain or said activating domain of the transcription factor, expressing in the host at least one second hybrid DNA sequence, such as a library or the like, encoding putative binding proteins to be investigated together with the DNA
binding or activating domain of t:he transcription factor which is not incorporated in the first fusion;
detecting any binding of the proteins to be investigated w:i.th a protein accaz:ding to the invention by detecting far the presence of any reporter gene product in the host cell; optionally isolating second hybrid DNA sequences encoding thE: binding protein.
An example of such a technique utilises the GAL4 protein in yeast. GAL4 is a transcriptional activator of galactose metabolism in yeast and has a separate domain for binding to activators upstream of the galactose metabolising genes as well as a protein binding domain. Nucleotide vectors may be constructed, one of which compri:>es 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 compri~>es the residues encoding the protein binding domain of GAL4. These residues are fused to residues encoding a test protein, preferably from the signal transduction pathway of the vertebrate in que~;tion. Any interaction between the vertebrate homologue of UNC-53 protein and the protein to be tested leads to transcriptional activation of a reporter molecules in a GAL-4 transcription deficient yeast cell into which the vectors have been transformed. Preferably, a reporter molecule such as I3-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 fo:r example a mammalian cell, may also be included in a WO 99/63080 PCTIEf99/03848 _ - 17 -pharmaceutical composition together with a pharmaceutical:Ly acceptable carrier, diiuent or excipient therefor.
The present invention also provides a process for producing a vertebrate homologue of an UNC-53 protein of C. electans according to the invention which process comprises culturing the cells transformed or transfected with a cDNA expression vector having any of the cDNA sequences according to the invention as described above, and recovering the expressed protein homologue. The cell may advantageously be a bacterial, animal, insect or plant cell.
A particu:Larly preferred process for produci:ng said vertebrate homologue of UNC-53 protein uses insect cells. Accordingly, the invention provides a process for producing a vertebrate homologue of L1NC-53 protein of C. E~leaans according to the invention which process comprises culturing an insect cell transformed or transfected with a recombinant Baculovirus vector, said vector comprising a nucleotide sequence encoding said vertebrate homologue of UNC-53 protein according to the invention downstream of the Baculovirus polyhedrin promoter and recovering the expressed protein. Advantageously, this method produces large amounts of proi~ein for recovery. The insect cell may be from for example Spodoptera fruaiperda or Drosophila Melanoctester.
In accordance with the present invention, a defined nucleic acid sequence includes not only the identical nucleic acid but also any minor base variations from the natural nucleic acid sequence including in particular, substitutions in bases which result in a synonymous codon (a different codon specifying the same amino acid), due to the degenerate 3S code in conservative amino acid substitution. The term "nucleic acid sequence" also includes the complimentary sequence to any single stranded sequence WO 99/63080 PCTJEf99103848 given which includes the definition above regarding base variation~~.
Furthermore, a defined protein, polypeptide or amino acid sequence according to the invention, includes not only the identical amino acid sequence but also minor amino acid variations from the natural amino acid sequence including conservative amino acid replacements d~~ replacement by an aminr~ 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 immunologica_Lly identical or similar to the polypeptide encoded by the naturally occurring sequence. Such polypeptides may be encoded by a corresponding nucleic acid sequence.
A further aspect of the invention provides a nucleic acid sequence of at least 15 nucleotides of a nucleic acid according to the invention and preferably from 15 to 50 nucleotides.
These sequences may, advantageously be used as probes or primers to initiate replication or the .Like.
Such nucleic acid sequences may be produced according to techniques well known in the art, such as by recombinant or synthetic means. They may also be used in diagnostic kits or the like for detecting for the presence of a nucleic acid according to the invention.
These test generally comprise contacting the probe with a sample under hybridising conditions and detecting for the presence of any duplex formation between the probe and any nucleic acid in the sample.
Nucleic acid sequences according to the invention may also be produced using recombinant or synthetic means such as described in Sambrook et al (Molecular Cloning: A Laboratory Manual, 1989). Advantageou:~ly, human allelic variants or polymor;phisms of the DNA
according to the invention may be identified by, i=or example, probir.~g DNA from a range of individuals for example from different populations. Furthermore, nucleic acids and probes according to the invention may be used to sequence genomic DNA from patients using techniques well known in th.e art, such as t:he Sanger Dideoxy chain termination method, which may S advantageously ascertain any predisposition of a patient to certain disorders.
A method of detecting whether a compound is an inhibitor or an enhances or expression of a vertebrate homologue of UrJC-53 of C_ eleaans,, according to t'.he invention is also provided which method comprises contacting a cell expressing said homologue with aaid compound and monitoring for a phenotypic change compared to a control cell which has not been contacted with said compound.
Preferably the cell is a transgenic cell as described above. Alternatively the cell may have undergone loss of contact inhibition.
The present method also provides for determining whether sand compound is an inhibitor or expressican of said vertebrate homologue. In one embodiment the compound to be tested comprises a nucleic acid.
Preferably said nucleic acid sequence comprises an antisense DNA sequence or a mRNA sequence.
Preferably said mRNA sequence comprises 3' untranslate.d regions of mRNA encoding for said vertebrate homologue.
Alternatively, the compound to be tested may be a protein. Preferably, said protein comprises a protein having an amino acid sequence potentially suitable for inhibiting function of said vertebrate homologue and preferably comprises a protein identified by the methods as described herein.
The present invention also provides a pharmaceutical composition comprising a compound, for example an anti.sense nucleic acid identified accoi:ding to the above described method together with a pharmaceutically acceptable carrier, diluent or WO 99/63080 PCT/E:P99103848 excipient therefor.
A nucleic acid sequence or protein identified according to this aspect of the :invention may be used as a medicament, or in the preparation of a medicament, for treating loss of contact inhibition of cancer which is mediated by vertebrate homologue of UNC-53 protein or a functional equivalent, fragment, derivative or bioprecursor of said homalogue.
Further provided by the invention is a nucleic acid as defined above for use in preparation of a medicament for inhibiting expression of a gene coding for a vertebrate homologue of UNC-53 protein of C. eleaans.
Further provided by the invention is an assay for detecting expression of the vertebrate homologue of UNC-53 protein of C. elegy in <~ vertebrate cell.
which assay comprises contacting a cell or an extract thereof with an antibody to said vertebrate homologue, which antibody is fused to a reporter molecule, removing any unbound antibody and monitoring for the presence of said reporter molecu:Le.
Preferably the reporter molE=cule is an antibody conjugated to for example a fluo:rophore such as fluorescein or alternatively to an enzyme such a~
strepavidin.
There is also provided a mei~hod for detecting for expression of a gene coding for i:.he vertebrate homologue of UNC-53 protein of the invention which method comprises contacting a probe specific for a nucleic acid of protein sequence coding for or corresponding to said vertebrate homologue according to the invention with a cell extract, which probe is linked to a reporter and analysing for the presence of said reporter.
Preferably the probe is a complementary sequence to a region of mRNA transcribed :From said gene encoding said vertebrate homologue of UNC-53 protein _ - 21 -according to the invention.
Preferably the complimentary sequence is a 3' or 5' untranslated region of said mFtNA. Preferably said reporter may be a dig label, a f_Luorophore, a hapten or a radiolabel.
Alternatively said probe may comprise an antibody specific for said vertebrate homologue of said UN'C-53 protein.
Preferably the reporter is an antibody conjugated I0 to for example a fluorophore such as fluorescein or alternatively an enzyme such as streptavidin.
As described above, UNC-53 protein of C.elegans has been found to localise to mic:rotubule 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 oi: association of the UNC-53 homologue of the invention to microtubules or plus end regions thereof, which method comprises (a) contacting said compound with a t:ransgenic cell, tissue or organism expressing said vertebrate homologue and 'which protein is operably linked to a reporter molecule (b) screening for the localisation of said reporter molecule as compared to a cell according to step (a) which has not been contacted with said compound.
A compound identifiable by t:he above method also forms part of the present invention. Such a compound identified as an inhibitor of localisation or association of said vertebrate homologue with microtubules or the plus end region thereof may be used in alleviating the spread of: disease inducing cells or metastasis or loss of contact inhibition.
Further a compound identified as an enhancer of association of said vertebrate homologue with microtubules or the plus end region thereof may be used in for example promoting neuronal regeneration, WO 99163080 PCT/EI'99103848 _ _ 22 _ revascularisat3_on or wound healing, or for treating chronic neurodegenerative diseases or acute traumatic injuries or fibrotic disease. These compounds may then be included in a pharmaceutical composition, together with a pharmaceutically acceptable carrier, diluent or excipient therefor.
Also provided by the present invention is a kit for determining whether a compound is an inhibitor or an enhancer of association of the vertebrate homologue thereof according to the invention with microtubules or the plus end regions thereof, which kit comprises at least one transgenic cell expressing said UNC-53 vertebrate protein homologue and a reporter molecule or a host or transgenic cell according to the invention and at least one cell of the same cell type for use as a cantrol and means for contacting said compound with tine of said at least one transgenic cells. Compounds identified as inhibitors or enhancers or microtubule association described above may advantageously be included in a composition and linked to said vertebrate homologue according to 'the invention to target the compounds to the microtub~ales or the plus end regions thereof. Such a composition may also compr~_se, for example, a suitable transfecting or transformation agent.
According to a further aspect of the invention there is provided a method of targeting a protein to a cell microtubule or the plus end region thereof, which method comprises introducing into a host cell, tiasue or organism a t:ransgene comprising a sequence capable of expressing said UNC-53 vertebrate homologue according to the invention, which sequence is ope:rably linked to a sequence encoding said protein to be targeted such that a chimeric protein is expressed and which results in targeting of said protein to said microtubule or a plus end region thereof. An even further aspect of the invention comprises a method of identifying a xaolecule which cova.lently modifies UNC-said vertebrate homologue according to the invention, which method comprises a) contacting either an extract from a cell or cells expressing said vertebrate homologue or a mixture of enzymes comprising candidate UNC-53 modifying enzymes in the presence of an indicator of covalent modification of a protein, :b) identifying any covalently modified UNC-53 protein from step a) and c) identifying said molecule involved in said modification step. Such an indicator may be 32 p Further provided by the invention is a method of identifying a compound which alleviates or enhances the toxicity of said UNC-53 vertebrate homologue thereof according to the invention, or which alleviates or enhances apoptosis. The method of the former comprises contacting said compound with a transgenic cell, tissue or organism according to 'the invention and monitoring for the presence of said reporter molecule adjacent said microtubules or tl.~e plus end region thereof. In the case of apoptosi;s the method comprises monitoring the effect of the compound on cell death.
The invent: ion may be more clearly understood from the following examples which are purely exemplary, with reference to the accompanying drawings wherein, Figure 1(a) is an illustration of the nucleotide sequence encoding the first human homologue of UNC-53 designated Hs-IJNC-53/1 and further variants thereof.
Figure 1(b) is an illustration of the amino acid sequence of hs-UNC-53/1 encoded by the sequences :in Figure 1 (a) .
Figure 1(c) is an illustration of the nucleor~ide sequence encoding the second human homologue of UNC-53 protein of C. elegans designated Hs-UNC--53/2 and further variants thereof.
Figure 1(d) is an illustration of the amino acid sequences of Hs~-UNC-53/2 encoded by the sequences in Figure 1 (c) .
Figure 1(e) is an illustration of a nucleotide sequence encoding the third human homologue of UNC-53 protein according to the invention designated Hs-I7NC-53/3, and variants thereof.
Figure 1(f) is an illustration of the amino acid sequences of the Hs-UNC-53/3 encoded by the sequences of Figure 1 (e) .
Figure 1 (g) is an illustration of the nucleotide sequence of a genomic DNA fragment that contains a putative 5' exon of Hs-unc-53/1.
Figure 1 (r~.) is an illustration of the nucleotide sequence AB023155 encoding the protein KIAA0938, a transcript comprising the 3' half of Hs-unc-53/3.
Figure 1(i) is an overview of the C. elegans and human UNC-53 proteins as cloned. The 5' truncated variants and a number of .the known splice variants have been indicated.
Figure 2 is an alignment of the amino acid sequences of Ce-UNC-53, Hs-UN'C-53,/.2, Hs-UNC-53/2 and Hs-UNC-53/3.
Figure 3 i.s an alignment of 'the C. elegans unc-53 and the predicted amino acid sequence of C. briggsiae unc-53.
Figure 4 i.s a list of ProSite signatures for vertebrate UNC-~53s based on the sequence alignment:.
Figure 5a is an illustration of expression of the three human UNC:-53s as studied by Northern blotting.
Figure 5(b) is an illustration of differential expression of Hs-unc-53/3 in different brain parts.
Figure 6(a.) is an illustration of differential splice variant expression of Hs-unc-53/2 using RT-~PCR.
Figure 6(b) is an illustration of differential splice expression of Hs-unc-53/2 using RT-PCR.
Figure 6(c) is an illustration of differential expression of Hs-unc-53/3 using R'r-PCR.
WO 99163080 PCTlE~99/03848 Figure 6(d) is a sequence cc>nfirmation of AB023155 expression in cells other than brain using RT-PCR.
Figure 7(,a) is an illustration of the cloning of Hs-unc-53/3.
Figure 7(b) is a plasmid map and the nucleotide sequence of the pGI3303 expression vector ( C-terminal Hs-unc-53/3 fragment in fusion with GFP).
Figure 7(c) is an illustration of the amino acid sequence of GFP: C-terminal Hs-unc-53/3 fragment (insert of pGI:3303) .
Figure 7(d) is a plasmid ma~> and the nucleotide sequence of the pGI3305 expression vector (full length Hs-unc-53/3 in fusion with GFP).
Figure 7(e) is an illustration of the amino acid sequence of GF:P . Hs-unc-53/3 (insert of pGI3305) .
Figue 8 i;s an illustration of the filipodia and lamellipodia outgrowth of N4 mou~;e neuroblastoma cells transfected with pGI3303 (F-actin cytoskeleton reorganisation) Figure 9 :is an illustration of the co-localisation o.f the GFP:Hs-unc-53/3 fusion protein with microtubules in N4 mouse neuroblastoma cells transfected with pGI3305.
Figure lla is an illustratic>n of the homology domains between Hs-unc-53/3 and a gene encoded (partially) by the Drosophilia me:lanogaster BAC clone BACR48M05 (AC005719) . Results of: a TBf~ASTN search on the non-redundant database with Hs-unc-53/3 as query.
Figure llb is an illustration of an ORF encoded by the Drosoph.ila melanogaster BP~C clone BACR48M05 (AC005719) as predicted by the computer program Fgene.
Figure llc is an illustration of a °°BLAST 2 sequences" search result with Hs-~unc-53/3 as query and the Fgene predicted UNC53 homology GRF of D.
melanogaster BAC clone BACR48M05.
Figure 12 is an.illustration of a zebra fish EST
encoding Dr-unc-53/2.
Figure 13 Genemap98 results for Hs-unc-53/2.
Figure 14 is a schematical cErawing of the sequence of the exon containing the putative alternative start codon of human Hs-unc-53/1.
Figure 15 is an illustration of the nucleotide sequence of pG:I3150 and the amino acid sequence of the eGFP fusion with a C-terminal fragment of Hs-Unc-53/1.
Figure 16 is an alignment of EST clone yk480b6 and Ce-unc-53 demonstrating a novel splice variant of Ce-unc-53.
Figure 17 is a graphical display of the effect of Hs-unc-53/3 GFP chimera transient transfection on the form factor of N4 cells.
DEPOSITED MATERIAL
Plasmids pG13303 and pG13305 were deposited under accession numbE~rs LMBP3936 and LM:BP3937 respectively on 28 May 1999 at the Belgian Coordinated Collections of Microorganisms (BCCM) at Laboratorium voor Moleculaire Biologie - Plasmidencollective (LMBP) B-9000 Ghent, Be~Lgium, in accordance with the provisions of the Budapest Treaty of April 28 1977.
Hs-UNC-533 is a bona fide UNC-53 (fig. 1; 2; 3) Blastn and Tblastn EST-database mining using the sequence of the already known animal UNC-53s led to the identification of 3 ESTs suggestive of novel unc-53s (see experimental procedures). By 3'- and 5'-RACE extension using suitable libraries, it was shown that these ESTs identified a novel unc-53 designai~ed Hs-unc-53/3 (F~_g. 1 e; f). The publication of the sequence AB0233.55 (Nagase et al. 1999, DNA Res. 6;:63-70) independently confirmed the correctness of the 3'-end of Hs-unc-_'>3/3 as well as the existence of one new _ - 27 _ intron that forms the 5'-end of A3023155. Alignments of the C. elegans and 3 human UNC-53 sequences (fig.
The present invention relates to a vertebrate homologue of UNC-53 protein of C. eleaans and cDNA
sequences coding for said homologwes or functional equivalents thereof. The invention also relates to processes for identifying compounds which control cell behaviour, compounds identified a.nd pharmaceutical compositions containing them in addition to processes and assays for identifying disease states in which said gene or protein is dysfunctional.
The control of cell motility, cell shape and directionality of cell outgrowth of axones or other cell outgrowths is an essential feature in the morphogenesis and function of both unicellular and multicellular organisms.
Some cell surface proteins and extra-cellular molecules conti°olling the directionality and potential of cell migration have been identified, although the processes involved are not generally understood. It is generally considered that a long-range migration of a cell process (also known as a growth cone extension) is a stepwise event, whereby prior to and after each extension there is the formation of a structure at the leading edge of the cell. Localised stab.ilisatio:n of the actin cytoskeleton and association with plus end regions of microtubules is a-general cell biological process underlying the choice of directional extension.
The present inventors have surprisingly found a new human gene~protein belonging to the UNC-53 family that binds microtubules and, in particular, the plus-end regions of microtubules.
A gene from the free-living nematode Caenorhabditis elegans designated "unc-53" has been previously identified and cloned (Abstract, WO 99/63080 PCT/EP99i03848 _ - 2 -International C. electans Meeting, June 1-5 1991, Madison, Wisconsin, 58, Bogaert a.nd Goh). The present inventors previously identified i~NC-53 protein as a signal transducer or signal integrator controlling the directionality of cell migration and/or cell shape in C. elecsans (WO 96/38555) .
The C. elecxans UNC-53 protein (Ceunc53) and previously found human homologues thereof (hs-unc.53/1 and hs-unc53/2) were found to encode a signal transducer or a signal integrator, controlling th~~
directionality of a cell migration, cell shape and growth extension. Evidence indicates that the presently found homologue designated (hs-unc53/3) might act as an adapter linking extracellular signals to the actin cytoskeleton. Firstly hs-unc-53/3 slxows homology to the cortical actin binding proteins, and the Ce-UNC-53 protein has been shown to bind F-actin in vitro and leads to actin re-organization in vivo when expressed in mammalian cells, leading to an increased number of filopodia and. lammelipodia.
Furthermore, increased neurite extension and increased cell motility could be observed. Hs-UNC-53-3 may play an important role in the development of various diseases.
According to a first aspect of the present invention there is provided a vertebrate protein homologue of an UNC-53 protein of C. eleaans, which protein comprises an amino acid sequence having one or more of sequence blocks A, B, C, D, E, F, G or H as illustrated in figure 4 or which differs from said blocks in conservative amino acid changes.
According to a further aspect of the present invention, there is provided a vertebrate protein homologue of UNC-53 protein of C. eleaans or a functional equivalent, derivative or bioprecursor thereof, having an amino acid sequence encoded by the nucleotide sequence illustrated in figure 1(e).
_ 3 _ For the purposes of the present invention a "derivative" should be taken to mean mutational derivatives, fusions, internal deletions, splice variants and muteins.
Preferably, said vertebrate homologue is a human protein, and preferably a mammalian or a mouse protein.
A further aspect of the invs=_ntion comprises a vertebrate homologue comprising an amino acid seC~uence as shown in figure 1(f) or the variants thereof cr an amino acid sequence which differs from the amino acid sequences shown in figure ltf) to a significant extent only in one or more conservative amino acid changes.
In a further aspect of the present invention there is also ;provided a nucleic acid molecule, which is preferably DNA, and which encodes a vertebrate hamologue of UNC-53 protein of C.. eleaans, or a functional equivalent derivative, fragment or bioprecursor of said homologue according to the invention. Preferably, the cDNA comprises a sequence of nucleotides encoding an amino acid sequence as illustrated in figure 1(f) or the variants thereof or an amino acid which differs from the sequences shown in these figur~as to a significant: extent only in one or more conservative amino acid changes. Preferably the DNA is cDNA, which cDNA comprises the sequence shown in figure 1(e)or the variants indicated therein.
Also provided by the present invention is a nucleic acid sequence capable of hybridi~~ing to the nucleic acid or DNA sequences according t:o the invention under high stringency conditions, which conditions are well known to those skilled in the art..
The cDNA according to the invention may be included in an expression vector which may itself be used to transform or transfect a host cell, which cell may be bacterial or eukaryotic in. origin including such as, far example an animal or plant cell a fungal WO 99/63080 PCTIElP99103848 cell or an insect cell. Thus, advantageously, once the cDNA corresponding to the genome of the verteb-rate homologue of UIVC-53 of C. electan~~ according to the invention is synthesised, using for example, reverse transcriptase or the like, a range of cells, tissues or organisms may be transfected following incorporation of the selected cDnfA 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 sE~quence 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 tr_ansgene capable of expressing a vertebrate homologue of UNC-53 protein of C. eleaans according to the invention. The term "transgene capable of expressing a vertebrate homologue of UNC-53 protein of C. elecrans" as used herein means a suitable nucleic acid sequence which leads to the expression of a vertebrate.homologue of UNC-53 protein of C. ele ans according to the invention having the same function and/or activity. The transgene m.ay include, for example, genomic nucleic acid isolated from the appropriate vertebrate or synthetic nucleic acid including cDNA. The term "transgenic organisms, tissues or cells, as used herein means any suitable organism and/or part of an organism, tissue or cell, that contains exogenous nucleic acid either stably integrated in the genome or in an extrachromosoma.l state.
Preferably the transgenic cell comprises any of, a CUS cell, HepG2 cell, MCF-7 or N4 neuroblastoma cell, a NTH3T3 cell, a colorectal or carcinoma ce:l1 or a human derived cell such as a fibroblast or the :Like.
The transgenic organism may be an insect, a non-h~.unan animal or a plant and preferably C. eleQans or a _ - 5 -related nematode. Preferably, th.e transgene comprises the nucleic acid or cDNA sequence: encoding the .
vertebrate homologue according to th;e invention as described above. The transgene preferably comprises an expression vector according to th.e 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. elec~ns according to the invention. For example, the gene may comprise deletions or mutations but may still encode a functional vertebrate homologue of UNC-53 protein.
Further provided by the present invention is a method of producing a mutant vertebrate non-human organism having a mutation in the wild-type gene coding for the vertebrate homologue of UNC-53 protein according to the invention, which mutation affects cell behaviour or the regulation of cell motility or the shape or the direction of cell migration or microtubule plus end stability or function and localisation ot: protein complexes located thereony which method comprises inducing a mutation in the vertebrate homalogue of UNC-53 protein in said organism. These mutant organisms may be used in a screen to identify the effects of compounds on these cell functions.
The vertebrate homologue of UNC-53 protein oi_ C. elecrans or the cDNA or genomic DNA encoding it or a functional equivalent, derivative, fragment or bioprecursor of said homologue, may advantageously be used as a medicament, or in the preparation of a medicament to treat or prevent disorders associatE:d with inhibition of overexpression of the vertebrate homologue of UNC -53 according to the invention. Such disorders may be alleviated by promoting neuronal regeneration, revascuiarisation or wound healing ar the treatment of chronic neurodeg~enerative disordE:rs, psychiatric disorders or acute traumatic injuries or fibrotic disease or disease in which physiological-events requiring the polarity of cells or epithelia are abnormally functioning. Accordingly, the vertebrate homologue according to the invention, dominant positive or negative mutants thereof, or inhibitors thereof may advantageously be used to induce or alleviate contact inhibition in a cell or in preventing carcinoma development. Typically, the above medical conditions may be treated in mammals and more preferably humans by either the homologue of UNC-53 protean or alternatively by a nucleic acid cod_Lng for the protein or the protein itself according to the invention. Alternatively an antisense oligonucleotide to said UNC-53 vertebrate homologue may be used to prevent it's expression. Examples of other nucleic acid sequences which may be used include 3' untranslated regions of mRNA which could be used to prevent transcription of the geno~:nic sequence encoding for the vertebrate homologue of U.'VC-53 protein according to the invention.
The vertebrate homologue of ~JNC-53 protein according to the invention may be incorporated into a pharmaceutically acceptable composition together with a suitable carrier, diluent or excipient therefor.
The pharmaceutical composition ma:y advantageously comprise, additionally or alternatively, the nucleic acid sequence according to the in~;rention as defined above.
The induction or inhibition o f the expression of hu-UNC-53/3 by pharmacological me<~ns may advantageously be used to induce neuronal regeneration, revascularisation or wound healing or be involved in the treatment of chronical neurodegenerative disorders, or acute traumatic injuries or fibrotic diseases, or physiological events requiring the polarity of cells, or oncology and - _ metastasis of cells, or apoptotic: pathways.
The present invention therefore also provides~for a method of determining whether a compound is an inhibitor or enhancer of the regulation of cell behaviour, growth, transformation, cell shape or motility or the direction of cell. migration, microtubule pla.zs end stability or function and localisation of protein complexe~~ thereon, which method comprises contacting said compound with a transgenic cell according to the invention and screening for a phenotypic changes in said cell. The method can therefore be used to determine whether the compound comprises an inhibitor c~r an enhancer of the signal transduction pathway of said transgenic cell of Z5 which pathway said vertebrate homologue of UNC-53 protein according to the invention is a component, or whether said compound is an inhibitor or an enhancer of a parallel or redundant signal transduction pathway in said cell. The present invention also provide.> a method to determine that the protein in said signal transduction pathway is a vertebrate homologue of UNC-53 protein of C. eleaans according to the invention.
Preferably, the phenotypic change to be screened comprises a change in cell shape or a change in cell motility. Where a transgenic cell is used in accordance with one embodiment of the method of the invention, an N4 neuroblastoma cell may be used and in such an embodiment the phenotypic change to be screened may be the length of neurite growth, changes in filopodia outgrowth, changes in ruffling behaviour or cell adhesion, any change in microtubule cytoskeleton, any change in localisation of proteins on plus end regions of microtubules or any change in a cell such as apoptosis. In an alternative embodiment of the method of the invention, t:he transgenic ce7.l may comprise an MCF-7 breast carcinoma cell.
Typically in such an embodiment t:he phenotypic change _ g _ to be screened comprises the extent of phagokinesis or filopodia formation. In an alternative embodiment. of this aspect of the invention, they transgenic cell may comprise an NI>a3T3 cell. Typica2,ly in such an embodiment the phenotypic change to be screened comprises loss of contact inhibition of foci formation. The method according to the invention, may also utilise a mutant cell or mutant organism according to the invention as described above, where the mutant cell is capable of growing in tissue culture or in vivo and either of which cell or organism has a mutation in the wild-type unc-53 gene.
In accordance with the present invention, a "phenotypic change", may comprise any phenotype resulting from changes at any suitable point in the life cycle of the cell, tissue or organism defined above, which change can be attributed to the expression of l.he transgene of the invention such as for example, growth, viability, morphology, behaviour, movement, cell migration or cell process or growth cone extension of cells and includes changes in body shape, locomotion, chemotaxis, contact inhibition, mating behaviour or the like. The phenotypic change may preferably be monitored directly by visual inspection of the cell as a whole or by monitoring the F-actin cytoskeleton microtubule network and plus end stability of microtubules or proteins thereon or alternatively by for example measuring indicators of viability including endogenous or transgenically introduced histochemical markers or other reporter genes, such as for example ~i-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 direci~ion of cell migration may be used as a medicament, or WO 99!63080 PCTIEP99l03848 _ g _ 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 tz:aumatic injuries or fibrotic disease. Examples of px:omoting neuronal regeneration include, for example, peripheral nerve regeneration after trauma and sp~.nal cord trauma.
Where a compound is identified in accordance with the method described above as being an inhibitor of the regulation of cell shape or mobility or the direction of call migration, the compound may be used as a medicament, or in the preparation of a medicament, for substantially alleviating spread of disease inducing cells, such as i.n spread of carcinoma, or the like in metastasis or in alleviating loss of contact inhibition. Advantageously, any of the compounds which may have been identified as an inhibitor or an enhancer in accordance with the method as described above, may also be included in a pharmaceutical composition comprising the respective compound and a pharmaceutically acceptable carrier, diluent or excipient therefor.
The particular mechanism of action of a compound identified as either an inhibitor or an enhancer of the cell motil_~.ty shape, growth or direction of cell migration or microtubule association or to the plus end region thereof is not limiting. Preferably the compound acts as an inhibitor or enhancer of a signal transduction pathway. The compound may also act on a parallel pathway or directly on the vertebrate homologue of UNC-53 protein of C. eleaans. For example, the method of action of the compound may include direct interaction with the vertebrate homologue of UNC-53 protein, interaction with processes for regulating phosphorylation or dephosphorylation of the vertebrate homologue of tJNC-53 or with processes regulating activity of an unc-53 WO 99/b3080 PCTIE:P99103848 gene or with processes for post-t:ranscriptional or post-translational modification or the like.
Preferably the compound is identified by the method according to the invention as an inhibitor or an enhancer, by utilising differE:nces of phenotype of the cell, tissue or organism, which are visible to the eye. Alternatively indicators ot: viability including endogenous or transgenically introduced histochemical markers or a reporter gene may be used.
According to a further aspects of the invention there is also provided a transgenic cell or tissue culture which has been constructed to comprise a promoter sequence of a gene coding for a vertebrate homologue of UNC-53 of C. elegan~; according to the invention operably linked to a nucleic acid sequence encoding a repcarter molecule. Preferably, the reporter sequence encodes for a detectable protein, for example one which may be monitored by eye inspection such as antibiotic resistance, (3-galactosidase o r a molecule detecaable 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. eleaans, according to the invention which method comprises the steps of:
(a) contacting said compound with a transgenic cell according to the invention as described above, (b~ monit:oring the level of said reporter molecule and comparing results obtained from this monitoring step with a control comprising a transgenic: cell having the promoter sequence of a gene coding for a vertebrate homologue of UNC:-53 protein, or a functional fragment of said WO 99!63080 PCT/EP99103848 homologue and the reporter molecule, in the absence of the compound.
In one embodiment of the method according to this aspect of the invention the reporter molecule may comprise messenger RNA.
A compound identified as an enhancer of transcription of the gene coding for the vertebrate homologue of UNC-53 protein of C. elecrans or a functional equivalent, derivative or bioprecursor of said homologue may also be used ~~.s a medicament, or in the preparation of a medicament, for promoting neuronal regeneration, revascularisation or wound healing, or for treatment of chronic neuro-degenerative diseases or acute traumatic injuries or fibrotic disease. Furthermore, such compounds may be included in a .pharmaceutical composition including a pharmaceutically acceptable carrier, diluent or excipient therefor. .Any compounds identified as inhibitors of 'transcription may, advantageously, be used in alleviating the spread of: disease inducing cells such as carcinomas or metastasis or loss of contact inhibition.
The present invention also ~>ravides a kit for determining whether a compound is an enhancer or an inhibitor of the regulation of cE:ll growth, transformation,, cell motility or shape or the direction of cell migration which kit comprises at least one transgenic or mutant cE:ll or transgenic or mutant non-human organism according to the invention as described above and a plurality of wild-type cells or a wild-type organism of the same type, or a cell line or tissue culture and means for contacting said compound with :>aid cell or organism.
Also provided by the present. invention is a kit for determining whether a compound is an inhibitor or an enhancer of transcription of a. gene coding for a vertebrate homologue of UNC-53 protein of C. eleqans WO 99163080 PCTlElP99/03848 according to t3ze invention which kit comprises at least one tran;sgenic cell or cells according to the invention, means for contacting ~~aid compounds with said cells and means for monitoring the level of transcription of said transgenic cell or cells according to the invention.
For the purposes of the present invention, the term "gene coding for a vertebrate homologue of UNC-53 or a functional fragment of said homologue" includes the nucleic acid sequence shown i.n figure d or a fragment thereof, including the cifferentially spliced isoforms arid transcriptional starts of the nucleic acid sequence and which sequence encodes a vertebrate homologue of UNC-53 protein or a functional equivalent, derivative, fragment or bioprecursor of the protein.
The present invention also provides methods of identifying genes of vertebrates or fragments of said genes, which encode proteins which are active in the signal transduction pathway of which the vertebrate homologue of UNC-53 according to the present invention is a component.. A preferred method comprises hybridizing to an appropriate cDNA library a nucleotide sequence, as defined herein, or a fragment thereof under appropriate conditions of stringency in order to identa.fy genes having statistically significant homology with the cDNA clones of any one of the cDNA sequences according to the invention described above.
Furthermore, there is also provided by the present invention a method of identifying a protean which is active in the signal transduction pathway of a cell of which a vertebrate homologue of UNC-53 protein of C. eleqans according to the invention is a component. According to this aspect of the inveni~ion, the method comprises:
(a) contacting an extract of said cell with an WO 99163080 PCT/E~99103848 antibody to the vertebrate homologue of UNC-~53 protein or a functional equivalent, fragment:-or bioprecursor of said protein, (b) identifying the antibo<iy/vertebrate homologue of UNC-53 complex,, and (c) analysing the complex t:o identify any protein bound to the vertebi:ate homologue of UNC-53 protein other than the antibody.
The vertebrate homologue of UNC-53 protein, therefore may bind regions of otY~er proteins involved in the signal transduction pathway. It is also possible to sequentially identify a whole range of proteins involved in the signal t:ransduction pathway.
Antibodies to the vertebratE: homologue of UNC-53 I5 protein may be produced according to known techniques as would be known to those skillE:d in the art. F'or example, polyclonal antibodies may be prepared by inoculating a host animal, such as a mouse, with a protein or epitope of a protein according to the invention and recovering immune ~~erum.
This aspect of the invention, further comprises a method of identifying a further ~>rotein or proteins which are active in the signal transduction pathway of a cell of which the vertebrate homologue of UNC-53 is a component which method comprises:
(a) forming an antibody to the first identified protein bound to the vertebrate homologue of UNC-53 protein in the methof~ as described above, (b) contacting a cell extract with the antibody, (c) identifying any antibod.y/protein complex, (d) analysing the complex to identify any further protein bound to the first protein other than the antibody, and (e) optionally repeating steps (a) to (d) to identify Further proteins in the pathway.
According to this aspect of the present invention, the antibody starts the process by binding to the vertebrate homologue of UIVC-53 protein according to the invention in the signal transducer-ion or oncogenic pathways. Any other proteins found complexed to the bound antibody or UNC-53 proteiru can then be used to identify further interacting proteins involved in the pathway.
It may also be possible to _Ldentify proteins involved in the signal transduct:Lon pathway of a cell of which the vertebrate homologuE~ of UNC-53 is a component by using a vertebrate homologue of UNC-53 protein of C. elec~ans. According to this aspect of the invention 'the method comprises:
(a) contacting an extract of the cell with the vertebrate homologue of UNC-53 protein of C. e.legans or a functional equivalent, fragment or bioprecursor of said homologue, (b) identifying the vertebrate homologue of UNC-53 p:rotein/protein complex formed and (c) analysing the complex t:o identify any protein bound to the vertebrate homologue of UNC-53 protein other than the same vertebrate homologue of UNC-53 protein.
This method can also advantageously be used to identify further proteins in a signal transduction pathway of a cell by contacting an extract of the cell used as described above, with any protein identified from step (c) above not being a vertebrate homologue of UNC-53 protein and repeating steps (b) and (c).
Other methods which may be used for identifying proteins in a signal transduction pathway of a cell may comprise for example a western blot overlay method which method is well known to those skilled in the art. Cell extracts are run on gels to separate out protein and subsequently blotted onto a nylon membrane. Theve membranes may then be incubated, for example in a medium containing vertebrate homologue of UNC-53 having a label attached thereto such as- a WO 99/63080 PCTIEI'99/03848 _ 15 _ biotin or radiolabel and any protein conjugates visualised with for example a streptavidin or alkaline phosphatase conjugated antibody.
The present invention also advantageously provides a process for the preparation of binding antibodies which recognise proteins or fragments thereof involved in the rate and direction of cell migration or the control of cell growth or shape, for the above methods.
The monoclonal antibody for binding to the appropriate vertebrate homologue of UNC-53 (or its functional equivalent) may be prepared by known techniques as described by Kohler R. and Milstein C., (1975) Nature 256, 495 to 497.
Another method which may be used to identify proteins involved in the signal transduction pathway of a cell of which a vertebrate h.omolague of an UNC-53 protein of C. eleaans according to~the invention or is a component, involves investigating protein-protein interactions using the two-hybrid. vector method. This method, which .is well known to those skilled in the art was first developed in yeast by Chien et a1 (1991). This technique is based on functional reconstruction in vivo of a transcription factor which activates a reporter gene. More particularly the technique comprises providing an appropriate host cell with a DNA construct comprising a reporter gene under the control of a promoter regulated by a transcription factor having a DNA binding domain and an activating domain, expressing in the host cell a first hybrid DNA
sequence encoding a first fusion of a fragment or all of a nucleic acid sequence according to the invention and either said DNA binding domain or said activating domain of the transcription factor, expressing in the host at least one second hybrid DNA sequence, such as a library or the like, encoding putative binding proteins to be investigated together with the DNA
binding or activating domain of t:he transcription factor which is not incorporated in the first fusion;
detecting any binding of the proteins to be investigated w:i.th a protein accaz:ding to the invention by detecting far the presence of any reporter gene product in the host cell; optionally isolating second hybrid DNA sequences encoding thE: binding protein.
An example of such a technique utilises the GAL4 protein in yeast. GAL4 is a transcriptional activator of galactose metabolism in yeast and has a separate domain for binding to activators upstream of the galactose metabolising genes as well as a protein binding domain. Nucleotide vectors may be constructed, one of which compri:>es 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 compri~>es the residues encoding the protein binding domain of GAL4. These residues are fused to residues encoding a test protein, preferably from the signal transduction pathway of the vertebrate in que~;tion. Any interaction between the vertebrate homologue of UNC-53 protein and the protein to be tested leads to transcriptional activation of a reporter molecules in a GAL-4 transcription deficient yeast cell into which the vectors have been transformed. Preferably, a reporter molecule such as I3-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 fo:r example a mammalian cell, may also be included in a WO 99/63080 PCTIEf99/03848 _ - 17 -pharmaceutical composition together with a pharmaceutical:Ly acceptable carrier, diiuent or excipient therefor.
The present invention also provides a process for producing a vertebrate homologue of an UNC-53 protein of C. electans according to the invention which process comprises culturing the cells transformed or transfected with a cDNA expression vector having any of the cDNA sequences according to the invention as described above, and recovering the expressed protein homologue. The cell may advantageously be a bacterial, animal, insect or plant cell.
A particu:Larly preferred process for produci:ng said vertebrate homologue of UNC-53 protein uses insect cells. Accordingly, the invention provides a process for producing a vertebrate homologue of L1NC-53 protein of C. E~leaans according to the invention which process comprises culturing an insect cell transformed or transfected with a recombinant Baculovirus vector, said vector comprising a nucleotide sequence encoding said vertebrate homologue of UNC-53 protein according to the invention downstream of the Baculovirus polyhedrin promoter and recovering the expressed protein. Advantageously, this method produces large amounts of proi~ein for recovery. The insect cell may be from for example Spodoptera fruaiperda or Drosophila Melanoctester.
In accordance with the present invention, a defined nucleic acid sequence includes not only the identical nucleic acid but also any minor base variations from the natural nucleic acid sequence including in particular, substitutions in bases which result in a synonymous codon (a different codon specifying the same amino acid), due to the degenerate 3S code in conservative amino acid substitution. The term "nucleic acid sequence" also includes the complimentary sequence to any single stranded sequence WO 99/63080 PCTJEf99103848 given which includes the definition above regarding base variation~~.
Furthermore, a defined protein, polypeptide or amino acid sequence according to the invention, includes not only the identical amino acid sequence but also minor amino acid variations from the natural amino acid sequence including conservative amino acid replacements d~~ replacement by an aminr~ 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 immunologica_Lly identical or similar to the polypeptide encoded by the naturally occurring sequence. Such polypeptides may be encoded by a corresponding nucleic acid sequence.
A further aspect of the invention provides a nucleic acid sequence of at least 15 nucleotides of a nucleic acid according to the invention and preferably from 15 to 50 nucleotides.
These sequences may, advantageously be used as probes or primers to initiate replication or the .Like.
Such nucleic acid sequences may be produced according to techniques well known in the art, such as by recombinant or synthetic means. They may also be used in diagnostic kits or the like for detecting for the presence of a nucleic acid according to the invention.
These test generally comprise contacting the probe with a sample under hybridising conditions and detecting for the presence of any duplex formation between the probe and any nucleic acid in the sample.
Nucleic acid sequences according to the invention may also be produced using recombinant or synthetic means such as described in Sambrook et al (Molecular Cloning: A Laboratory Manual, 1989). Advantageou:~ly, human allelic variants or polymor;phisms of the DNA
according to the invention may be identified by, i=or example, probir.~g DNA from a range of individuals for example from different populations. Furthermore, nucleic acids and probes according to the invention may be used to sequence genomic DNA from patients using techniques well known in th.e art, such as t:he Sanger Dideoxy chain termination method, which may S advantageously ascertain any predisposition of a patient to certain disorders.
A method of detecting whether a compound is an inhibitor or an enhances or expression of a vertebrate homologue of UrJC-53 of C_ eleaans,, according to t'.he invention is also provided which method comprises contacting a cell expressing said homologue with aaid compound and monitoring for a phenotypic change compared to a control cell which has not been contacted with said compound.
Preferably the cell is a transgenic cell as described above. Alternatively the cell may have undergone loss of contact inhibition.
The present method also provides for determining whether sand compound is an inhibitor or expressican of said vertebrate homologue. In one embodiment the compound to be tested comprises a nucleic acid.
Preferably said nucleic acid sequence comprises an antisense DNA sequence or a mRNA sequence.
Preferably said mRNA sequence comprises 3' untranslate.d regions of mRNA encoding for said vertebrate homologue.
Alternatively, the compound to be tested may be a protein. Preferably, said protein comprises a protein having an amino acid sequence potentially suitable for inhibiting function of said vertebrate homologue and preferably comprises a protein identified by the methods as described herein.
The present invention also provides a pharmaceutical composition comprising a compound, for example an anti.sense nucleic acid identified accoi:ding to the above described method together with a pharmaceutically acceptable carrier, diluent or WO 99/63080 PCT/E:P99103848 excipient therefor.
A nucleic acid sequence or protein identified according to this aspect of the :invention may be used as a medicament, or in the preparation of a medicament, for treating loss of contact inhibition of cancer which is mediated by vertebrate homologue of UNC-53 protein or a functional equivalent, fragment, derivative or bioprecursor of said homalogue.
Further provided by the invention is a nucleic acid as defined above for use in preparation of a medicament for inhibiting expression of a gene coding for a vertebrate homologue of UNC-53 protein of C. eleaans.
Further provided by the invention is an assay for detecting expression of the vertebrate homologue of UNC-53 protein of C. elegy in <~ vertebrate cell.
which assay comprises contacting a cell or an extract thereof with an antibody to said vertebrate homologue, which antibody is fused to a reporter molecule, removing any unbound antibody and monitoring for the presence of said reporter molecu:Le.
Preferably the reporter molE=cule is an antibody conjugated to for example a fluo:rophore such as fluorescein or alternatively to an enzyme such a~
strepavidin.
There is also provided a mei~hod for detecting for expression of a gene coding for i:.he vertebrate homologue of UNC-53 protein of the invention which method comprises contacting a probe specific for a nucleic acid of protein sequence coding for or corresponding to said vertebrate homologue according to the invention with a cell extract, which probe is linked to a reporter and analysing for the presence of said reporter.
Preferably the probe is a complementary sequence to a region of mRNA transcribed :From said gene encoding said vertebrate homologue of UNC-53 protein _ - 21 -according to the invention.
Preferably the complimentary sequence is a 3' or 5' untranslated region of said mFtNA. Preferably said reporter may be a dig label, a f_Luorophore, a hapten or a radiolabel.
Alternatively said probe may comprise an antibody specific for said vertebrate homologue of said UN'C-53 protein.
Preferably the reporter is an antibody conjugated I0 to for example a fluorophore such as fluorescein or alternatively an enzyme such as streptavidin.
As described above, UNC-53 protein of C.elegans has been found to localise to mic:rotubule 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 oi: association of the UNC-53 homologue of the invention to microtubules or plus end regions thereof, which method comprises (a) contacting said compound with a t:ransgenic cell, tissue or organism expressing said vertebrate homologue and 'which protein is operably linked to a reporter molecule (b) screening for the localisation of said reporter molecule as compared to a cell according to step (a) which has not been contacted with said compound.
A compound identifiable by t:he above method also forms part of the present invention. Such a compound identified as an inhibitor of localisation or association of said vertebrate homologue with microtubules or the plus end region thereof may be used in alleviating the spread of: disease inducing cells or metastasis or loss of contact inhibition.
Further a compound identified as an enhancer of association of said vertebrate homologue with microtubules or the plus end region thereof may be used in for example promoting neuronal regeneration, WO 99163080 PCT/EI'99103848 _ _ 22 _ revascularisat3_on or wound healing, or for treating chronic neurodegenerative diseases or acute traumatic injuries or fibrotic disease. These compounds may then be included in a pharmaceutical composition, together with a pharmaceutically acceptable carrier, diluent or excipient therefor.
Also provided by the present invention is a kit for determining whether a compound is an inhibitor or an enhancer of association of the vertebrate homologue thereof according to the invention with microtubules or the plus end regions thereof, which kit comprises at least one transgenic cell expressing said UNC-53 vertebrate protein homologue and a reporter molecule or a host or transgenic cell according to the invention and at least one cell of the same cell type for use as a cantrol and means for contacting said compound with tine of said at least one transgenic cells. Compounds identified as inhibitors or enhancers or microtubule association described above may advantageously be included in a composition and linked to said vertebrate homologue according to 'the invention to target the compounds to the microtub~ales or the plus end regions thereof. Such a composition may also compr~_se, for example, a suitable transfecting or transformation agent.
According to a further aspect of the invention there is provided a method of targeting a protein to a cell microtubule or the plus end region thereof, which method comprises introducing into a host cell, tiasue or organism a t:ransgene comprising a sequence capable of expressing said UNC-53 vertebrate homologue according to the invention, which sequence is ope:rably linked to a sequence encoding said protein to be targeted such that a chimeric protein is expressed and which results in targeting of said protein to said microtubule or a plus end region thereof. An even further aspect of the invention comprises a method of identifying a xaolecule which cova.lently modifies UNC-said vertebrate homologue according to the invention, which method comprises a) contacting either an extract from a cell or cells expressing said vertebrate homologue or a mixture of enzymes comprising candidate UNC-53 modifying enzymes in the presence of an indicator of covalent modification of a protein, :b) identifying any covalently modified UNC-53 protein from step a) and c) identifying said molecule involved in said modification step. Such an indicator may be 32 p Further provided by the invention is a method of identifying a compound which alleviates or enhances the toxicity of said UNC-53 vertebrate homologue thereof according to the invention, or which alleviates or enhances apoptosis. The method of the former comprises contacting said compound with a transgenic cell, tissue or organism according to 'the invention and monitoring for the presence of said reporter molecule adjacent said microtubules or tl.~e plus end region thereof. In the case of apoptosi;s the method comprises monitoring the effect of the compound on cell death.
The invent: ion may be more clearly understood from the following examples which are purely exemplary, with reference to the accompanying drawings wherein, Figure 1(a) is an illustration of the nucleotide sequence encoding the first human homologue of UNC-53 designated Hs-IJNC-53/1 and further variants thereof.
Figure 1(b) is an illustration of the amino acid sequence of hs-UNC-53/1 encoded by the sequences :in Figure 1 (a) .
Figure 1(c) is an illustration of the nucleor~ide sequence encoding the second human homologue of UNC-53 protein of C. elegans designated Hs-UNC--53/2 and further variants thereof.
Figure 1(d) is an illustration of the amino acid sequences of Hs~-UNC-53/2 encoded by the sequences in Figure 1 (c) .
Figure 1(e) is an illustration of a nucleotide sequence encoding the third human homologue of UNC-53 protein according to the invention designated Hs-I7NC-53/3, and variants thereof.
Figure 1(f) is an illustration of the amino acid sequences of the Hs-UNC-53/3 encoded by the sequences of Figure 1 (e) .
Figure 1 (g) is an illustration of the nucleotide sequence of a genomic DNA fragment that contains a putative 5' exon of Hs-unc-53/1.
Figure 1 (r~.) is an illustration of the nucleotide sequence AB023155 encoding the protein KIAA0938, a transcript comprising the 3' half of Hs-unc-53/3.
Figure 1(i) is an overview of the C. elegans and human UNC-53 proteins as cloned. The 5' truncated variants and a number of .the known splice variants have been indicated.
Figure 2 is an alignment of the amino acid sequences of Ce-UNC-53, Hs-UN'C-53,/.2, Hs-UNC-53/2 and Hs-UNC-53/3.
Figure 3 i.s an alignment of 'the C. elegans unc-53 and the predicted amino acid sequence of C. briggsiae unc-53.
Figure 4 i.s a list of ProSite signatures for vertebrate UNC-~53s based on the sequence alignment:.
Figure 5a is an illustration of expression of the three human UNC:-53s as studied by Northern blotting.
Figure 5(b) is an illustration of differential expression of Hs-unc-53/3 in different brain parts.
Figure 6(a.) is an illustration of differential splice variant expression of Hs-unc-53/2 using RT-~PCR.
Figure 6(b) is an illustration of differential splice expression of Hs-unc-53/2 using RT-PCR.
Figure 6(c) is an illustration of differential expression of Hs-unc-53/3 using R'r-PCR.
WO 99163080 PCTlE~99/03848 Figure 6(d) is a sequence cc>nfirmation of AB023155 expression in cells other than brain using RT-PCR.
Figure 7(,a) is an illustration of the cloning of Hs-unc-53/3.
Figure 7(b) is a plasmid map and the nucleotide sequence of the pGI3303 expression vector ( C-terminal Hs-unc-53/3 fragment in fusion with GFP).
Figure 7(c) is an illustration of the amino acid sequence of GFP: C-terminal Hs-unc-53/3 fragment (insert of pGI:3303) .
Figure 7(d) is a plasmid ma~> and the nucleotide sequence of the pGI3305 expression vector (full length Hs-unc-53/3 in fusion with GFP).
Figure 7(e) is an illustration of the amino acid sequence of GF:P . Hs-unc-53/3 (insert of pGI3305) .
Figue 8 i;s an illustration of the filipodia and lamellipodia outgrowth of N4 mou~;e neuroblastoma cells transfected with pGI3303 (F-actin cytoskeleton reorganisation) Figure 9 :is an illustration of the co-localisation o.f the GFP:Hs-unc-53/3 fusion protein with microtubules in N4 mouse neuroblastoma cells transfected with pGI3305.
Figure lla is an illustratic>n of the homology domains between Hs-unc-53/3 and a gene encoded (partially) by the Drosophilia me:lanogaster BAC clone BACR48M05 (AC005719) . Results of: a TBf~ASTN search on the non-redundant database with Hs-unc-53/3 as query.
Figure llb is an illustration of an ORF encoded by the Drosoph.ila melanogaster BP~C clone BACR48M05 (AC005719) as predicted by the computer program Fgene.
Figure llc is an illustration of a °°BLAST 2 sequences" search result with Hs-~unc-53/3 as query and the Fgene predicted UNC53 homology GRF of D.
melanogaster BAC clone BACR48M05.
Figure 12 is an.illustration of a zebra fish EST
encoding Dr-unc-53/2.
Figure 13 Genemap98 results for Hs-unc-53/2.
Figure 14 is a schematical cErawing of the sequence of the exon containing the putative alternative start codon of human Hs-unc-53/1.
Figure 15 is an illustration of the nucleotide sequence of pG:I3150 and the amino acid sequence of the eGFP fusion with a C-terminal fragment of Hs-Unc-53/1.
Figure 16 is an alignment of EST clone yk480b6 and Ce-unc-53 demonstrating a novel splice variant of Ce-unc-53.
Figure 17 is a graphical display of the effect of Hs-unc-53/3 GFP chimera transient transfection on the form factor of N4 cells.
DEPOSITED MATERIAL
Plasmids pG13303 and pG13305 were deposited under accession numbE~rs LMBP3936 and LM:BP3937 respectively on 28 May 1999 at the Belgian Coordinated Collections of Microorganisms (BCCM) at Laboratorium voor Moleculaire Biologie - Plasmidencollective (LMBP) B-9000 Ghent, Be~Lgium, in accordance with the provisions of the Budapest Treaty of April 28 1977.
Hs-UNC-533 is a bona fide UNC-53 (fig. 1; 2; 3) Blastn and Tblastn EST-database mining using the sequence of the already known animal UNC-53s led to the identification of 3 ESTs suggestive of novel unc-53s (see experimental procedures). By 3'- and 5'-RACE extension using suitable libraries, it was shown that these ESTs identified a novel unc-53 designai~ed Hs-unc-53/3 (F~_g. 1 e; f). The publication of the sequence AB0233.55 (Nagase et al. 1999, DNA Res. 6;:63-70) independently confirmed the correctness of the 3'-end of Hs-unc-_'>3/3 as well as the existence of one new _ - 27 _ intron that forms the 5'-end of A3023155. Alignments of the C. elegans and 3 human UNC-53 sequences (fig.
2) clearly illustrates that the third human homologue of C. eleqans UNC-53 protein is a bona fide UNC-53 with highest similarity to Hs-UNC-53/2 and in decreasing order to Hs-UNC-53/1 and (C. elegans UNC-53) Ce-UNC-53.
Many of the domains of Hs-UNC-53/3 show highest sim~.larity to functional domains of other animal UNC-53s (fig. 2). This critically suggests that Hu-UNC-53/3 most likely has the key functionalities obse~°ved for Ce-UNC--53 in a variety of assays including F-actin binding, F-actin reorganisation in cell culture, microtubule and. microtubule (+)-end binding in cultured cells, binding of SH3-domain adapters like SEM-5/GRB-2 or other types of binders of proline rich alpha-helices. These results indicate that like C;e-UNC-53, Hs-UNC-53/1, Hs-UNC-53/2, or Hs-UNC-53/3 can be used in a range of biochemical,, cellular and animal assays aimed at discovering tissue- or disease-specific modulators of Hs-unc-53 functioning in diagnostic assays.
Further exaension of the Unc~-53 family (Fig. 11, 12) Database searches with the three human UNC-5~~
protein sequences revealed severa:L expressed sequence tags (ESTs) and genomic DNA sequences (BACs) that show significant similarlity to human 1:1NC-53.
C. briggsiae The C. elegans genome consortium sequenced tY~e locus of the C. briggsiae unc-53 homologous gene.
Through gene prediction programs and the cDNA sequence of the C. elegans unc-53, prediction can be made fog: the C. briggsiae protein sequence.
Alignment of the derived C. briggsiae amino acid sequence with the C. elegans amino acid sequence in figure 3 demonstrates the strong homology of both proteins .
D. melanogaster BAC clone BACR48M05 (AC005719) clearly contains 3 different exons with high homology to Hs-unc-53/3 (Figure 11). Using the gene structure prediction program Fgene [Solovyev et al., 1995, in: Proceedings of the Third International Conference on Intelligent Systems for Mo:Lecular Biology (ed.s. Bawling et al., Cambridge, England, AAAI Press) Solovyev and Lawrence, 1993, in: Abstracts of the 4th annual keck symposium. Pii~tsburgh, 47) it was possible to predict an t7RF encoded by BAC clone BACR48M05 that shows homology to Hs-unc-53/3 (Figure llb). However, every Drosophila cDNA partially or entirely encoded by :BAC
clone BACR48M05 and which contains one or more sequence blocks as indicated in figure lla should be considered as a family member of the UNC-53 family. A
"BLAST 2 SEQUENCE" search indicates that the sequence situated between the three homology blocks that are indicated in figure lla is less conserved between human and Drosophila (Figure llc). The predicted ORF
of the Drosophila melanogaster UN'C53 gene can be 'used to identify new members of the family. The zebrafish EST fc2Id06 (A:C658309) shows an identity of 84% a:nd a homology of 92'-o to Hs-UNC-53/2. It clearly can be considered as a part of the zebra,fish homologue of Hs-UNC-53/2 (Figure 12). Finally, a. whole series of human ESTs have been placed in public domain databases. To our knowledge, no one has been able to place these ES'i's into contigs that describe a true Hs-unc-53 to a level presented in this specification.
_ - 29 -The presently available unc-53 sequences - expres,sed or genomic - further underscore that the unc-53 gene family is a true animal gene family in helminths, vertebrates and arthropods, three major classes of the animal kingdom..
Refined UNC-53 family description based on alignment (fig. 4).
The alignment of the three human and the C.
eleqans UNC-53 sequences enables the more refined definition of conserved regions in UNC-53s. Tn f:igure 4 there are compiled a number of proSite signatures for either the four animal or the three human UNC-53s.
Different~.al expression of Hu-UNC-53/3 by Northern blot (fig. 5):
To determine in which cells and tissues the vertebrate UNC--53s play a role, a northern blot analysis has been performed. As indicated in the experimental section, relevant probes were amplified and used to visualise in which normal human tissues and in which cancer cell lines the three human UNC~-53s were expressed..
1. A cancer cell line RNA :blots probed with Hs-Unc53/1.
A Northern blot of poly-A+RNA from several cancer cell lines~(Melanoma 6361, Lung Cancer A549, Colorectal Adenocarcinoma SW480, Burkitt Lymphoma DRajii, Leukemia Molt4, Lymphoblastic Leukemia K5E~2, HeLa S3 and Promyelocytic Leukemia HL60) was probed using the wholE: insert of pHH3b. No or weak expression was detected in the Burkitt Lymphoma DRajii, the Leukemia Molt4 and the Promyelocytic Leukemia HL60 cell lines. Five different transcripts _ - 30 -are detected in, the remaining cancer cell lines:
transcripts I a.nd 2 are larger than 9.5kb, transcz-i:pts
Many of the domains of Hs-UNC-53/3 show highest sim~.larity to functional domains of other animal UNC-53s (fig. 2). This critically suggests that Hu-UNC-53/3 most likely has the key functionalities obse~°ved for Ce-UNC--53 in a variety of assays including F-actin binding, F-actin reorganisation in cell culture, microtubule and. microtubule (+)-end binding in cultured cells, binding of SH3-domain adapters like SEM-5/GRB-2 or other types of binders of proline rich alpha-helices. These results indicate that like C;e-UNC-53, Hs-UNC-53/1, Hs-UNC-53/2, or Hs-UNC-53/3 can be used in a range of biochemical,, cellular and animal assays aimed at discovering tissue- or disease-specific modulators of Hs-unc-53 functioning in diagnostic assays.
Further exaension of the Unc~-53 family (Fig. 11, 12) Database searches with the three human UNC-5~~
protein sequences revealed severa:L expressed sequence tags (ESTs) and genomic DNA sequences (BACs) that show significant similarlity to human 1:1NC-53.
C. briggsiae The C. elegans genome consortium sequenced tY~e locus of the C. briggsiae unc-53 homologous gene.
Through gene prediction programs and the cDNA sequence of the C. elegans unc-53, prediction can be made fog: the C. briggsiae protein sequence.
Alignment of the derived C. briggsiae amino acid sequence with the C. elegans amino acid sequence in figure 3 demonstrates the strong homology of both proteins .
D. melanogaster BAC clone BACR48M05 (AC005719) clearly contains 3 different exons with high homology to Hs-unc-53/3 (Figure 11). Using the gene structure prediction program Fgene [Solovyev et al., 1995, in: Proceedings of the Third International Conference on Intelligent Systems for Mo:Lecular Biology (ed.s. Bawling et al., Cambridge, England, AAAI Press) Solovyev and Lawrence, 1993, in: Abstracts of the 4th annual keck symposium. Pii~tsburgh, 47) it was possible to predict an t7RF encoded by BAC clone BACR48M05 that shows homology to Hs-unc-53/3 (Figure llb). However, every Drosophila cDNA partially or entirely encoded by :BAC
clone BACR48M05 and which contains one or more sequence blocks as indicated in figure lla should be considered as a family member of the UNC-53 family. A
"BLAST 2 SEQUENCE" search indicates that the sequence situated between the three homology blocks that are indicated in figure lla is less conserved between human and Drosophila (Figure llc). The predicted ORF
of the Drosophila melanogaster UN'C53 gene can be 'used to identify new members of the family. The zebrafish EST fc2Id06 (A:C658309) shows an identity of 84% a:nd a homology of 92'-o to Hs-UNC-53/2. It clearly can be considered as a part of the zebra,fish homologue of Hs-UNC-53/2 (Figure 12). Finally, a. whole series of human ESTs have been placed in public domain databases. To our knowledge, no one has been able to place these ES'i's into contigs that describe a true Hs-unc-53 to a level presented in this specification.
_ - 29 -The presently available unc-53 sequences - expres,sed or genomic - further underscore that the unc-53 gene family is a true animal gene family in helminths, vertebrates and arthropods, three major classes of the animal kingdom..
Refined UNC-53 family description based on alignment (fig. 4).
The alignment of the three human and the C.
eleqans UNC-53 sequences enables the more refined definition of conserved regions in UNC-53s. Tn f:igure 4 there are compiled a number of proSite signatures for either the four animal or the three human UNC-53s.
Different~.al expression of Hu-UNC-53/3 by Northern blot (fig. 5):
To determine in which cells and tissues the vertebrate UNC--53s play a role, a northern blot analysis has been performed. As indicated in the experimental section, relevant probes were amplified and used to visualise in which normal human tissues and in which cancer cell lines the three human UNC~-53s were expressed..
1. A cancer cell line RNA :blots probed with Hs-Unc53/1.
A Northern blot of poly-A+RNA from several cancer cell lines~(Melanoma 6361, Lung Cancer A549, Colorectal Adenocarcinoma SW480, Burkitt Lymphoma DRajii, Leukemia Molt4, Lymphoblastic Leukemia K5E~2, HeLa S3 and Promyelocytic Leukemia HL60) was probed using the wholE: insert of pHH3b. No or weak expression was detected in the Burkitt Lymphoma DRajii, the Leukemia Molt4 and the Promyelocytic Leukemia HL60 cell lines. Five different transcripts _ - 30 -are detected in, the remaining cancer cell lines:
transcripts I a.nd 2 are larger than 9.5kb, transcz-i:pts
3 and 4 are 6 to 7 kb and the fifth transcript is around 6 kb. Transcripts 1 and 2 are present in all expressing cell lines but at different levels.
Transcripts 3 a.nd 4 are restricted to Melanoma G3Eil, Lung Cancer A549 (weak) and Coior~ectal Adenocarcinoma SW480 and are the predominant transcripts in Melanoma 6361 and Colorectal Adenocarcinom~a SW480. Transcript I0 5 is restricted. to Lymphoblastic :Leukemia K562 (weak) and (predominan.t) in HeLa S3 and .is predominant in HeLa S3.
2. Cancer cell lines RNA blots probed with Hs-Unc53/2.
A similar set of cancer cell line Northern blots were probed with a 652bp fragment of EST460?'~7 amplified by using the primers 5'-aggagatgaagctgacagatatcc and 5'-aaacaccagtgagtcc. Hs-Unc53/2 is expressed in Melanoma c.~361, Colorectal Adenocarcinoma SW480, Lymphoblast:ic Leukemia K562 and HeLa S3. No expression was detecl~ed in Lung Cancer A549, Burkitt Lymphoma DRajii, Leukemia Molt4 and promyelocytic leukemia HL60. Intcsrestingly only 2 transcript sizes were detected of around 7 kb expressed in Lymphoblastic Leukem:La K562 and HeLa S3 and a transcript of >9.5 kb in Me:Lanoma 6361 and Colorectal Adenocarcinoma SW480 and weakly in HeLa53.
Noteworthy is the very high expression in melanoma 6361.
3. Normal Human tissue probed with Hs-Unc53/1.
A Northern blot of poly--A+RNA from normal human tissue was probed using the whole insert of phage HH3b. Expression levels are low in all tissues with the highest level in heart and placenta, several fold lower levels in brain and testis, even lower _ - 31 -levels in skeletal muscle, pancreas, thymus, colon, small intestine, ovary and prostate. Expression in peripheral blood leukocyte, lung, liver, kidney, spleen is bare:Ly detectable.
Transcripts 3 a.nd 4 are restricted to Melanoma G3Eil, Lung Cancer A549 (weak) and Coior~ectal Adenocarcinoma SW480 and are the predominant transcripts in Melanoma 6361 and Colorectal Adenocarcinom~a SW480. Transcript I0 5 is restricted. to Lymphoblastic :Leukemia K562 (weak) and (predominan.t) in HeLa S3 and .is predominant in HeLa S3.
2. Cancer cell lines RNA blots probed with Hs-Unc53/2.
A similar set of cancer cell line Northern blots were probed with a 652bp fragment of EST460?'~7 amplified by using the primers 5'-aggagatgaagctgacagatatcc and 5'-aaacaccagtgagtcc. Hs-Unc53/2 is expressed in Melanoma c.~361, Colorectal Adenocarcinoma SW480, Lymphoblast:ic Leukemia K562 and HeLa S3. No expression was detecl~ed in Lung Cancer A549, Burkitt Lymphoma DRajii, Leukemia Molt4 and promyelocytic leukemia HL60. Intcsrestingly only 2 transcript sizes were detected of around 7 kb expressed in Lymphoblastic Leukem:La K562 and HeLa S3 and a transcript of >9.5 kb in Me:Lanoma 6361 and Colorectal Adenocarcinoma SW480 and weakly in HeLa53.
Noteworthy is the very high expression in melanoma 6361.
3. Normal Human tissue probed with Hs-Unc53/1.
A Northern blot of poly--A+RNA from normal human tissue was probed using the whole insert of phage HH3b. Expression levels are low in all tissues with the highest level in heart and placenta, several fold lower levels in brain and testis, even lower _ - 31 -levels in skeletal muscle, pancreas, thymus, colon, small intestine, ovary and prostate. Expression in peripheral blood leukocyte, lung, liver, kidney, spleen is bare:Ly detectable.
4. Normal Human tissue probed with Hs-UNC53/2.
A similar set of blots were probed with a 652bp fragment of EST46037 amplified by using the primers 5' aggagatgaagctgacagatatc;c and 5' -aaacaccagtgagtcc. Expression levels are low in all tissues with the highest level in kidney, placenta and pancreas, lower levels in heart and lung. Expression is barely detectable or undetectable in skeletal muscle, spleen,, thymus, prostate, testis, ovary, small intestine, colon peripheral blood leucocyte, stomach, thyroid, spina:L cord, trachea, adrenal gland and bone marrow. Also l~is-unc-53/2 appears; to be expressed as different transcripts (figure 5a) .
The hs-UNC53/1 and hs-UNC-53/2 homologues are clearly highly regulated. genes, showing a strong tissue specificity and, probably, additional mechanisms of :regulation (ie differential splicing of different promoters). The different proteins derived from RNA's identified by probe hh.l5 presumably share the carboxyterminal nucleotide binding domain.
Ce-UNC-53 was shown to be a complex genetic locus and complex transcription unit. The different transcripts are thought to be a mechanism to assure the necessary specificity and functional diversity of this signal transduction pathway, with respect to different signals and receptors, different tissues and different directions of migration. The occurrence of a new transcript or i~he observed changes in expression levels in the cancer cell line blot suggests a role for hs-UNC-53/:3 in the establishment or maintenance of the transformed state of those cells.
WO 99/63U8~ PCT/E1P99/03848 _ - 32 -Expression pattern of hs-UNC'-5~/3.
A northern blot of poly-A+RNA from several cancer lines was probed with unique fragments of the three genes from the Hs-unc-53 family. Hs-unc-53/3 has a high expression level in lung carcinoma line A549, where only a moderate expression of hs-unc-53/1 h;as been detected. Furthermore, moderate expression of Hs-unc-53/3 was also observed in melanoma line 6361, where previous7_y, a high expression of hs-UNC-53/1 and hs-UNC-52/2 has been observed. This indicated the involvement of hs-unc53/3 in at least two cancer lines.
In normal human tissues, the expression of hs-unc-53/3 shows a clearly new and previously unobserved expression pattern. This difference of expressio~.z of hs-unc-53/3 in relation to its homologues hs-unc5:3/1 and hs-unc53/2 is important for the allocation of functionality to hs-unc-53/3.
Hs-unc-53/'3 is highly expressed in brain, as shown on the Northern blots (figure 5a). In figure 5b it can be seen that Hs-unc-53/3 also is different=sally expressed in different parts of t:he brain. Its homologues are not or weakly expressed in brain. This gives an indication that its function in directionality of cell migration and growth cone steering will be in relation to specific regions or cells of the brain. It is deduced that Hs-unc-53/3 will be an important signal transducer or signal adapter linking signals to neuronal outgrowth, axon guidance, and formation and maintenance of synapt~_c connections. It seems that the f~~,znction of Hs-unc-53/3 will be associated with neuron-neuron interactions, neuronal outgrowth, neuron muscle interactions, and post-synaptic signal transduction.
Furthermore, Hs-unc-53/3 may be involved in the development of cancer of neuronal origin, like neuroblastomas, or the development of tumours will have their developmental origin in the brain as some eyes diseases like retinoblastomas.
The significance of the high expression of Hs-unc-53/3 in brain tissue can be associated with the high levels of expression which has also been observed in the spinal cord, containing neuronal tissue. lHere, neuronal (axon) outgrowth and neuron-neuron connections are of importance. Development of pharmacological_ tools acting on this pathway may .lead to treatments of diseases involved in the growth ;end movement of neuronal cells, and the regeneration of neuronal connectivity after trauma, or the inhibition of neuronal cancers such as neuroblastomas. Due 'to its specific expression, inhibitors and/or enhancers specific for H:>-unc-53/3 will have an advantage a:~ a pharmaceutical compound over more general compounds acting on the Hs-unc-53 family of genes and proteins.
A second tissue where hs-UNC-53/3 is highly expressed and where (its) other human homologues are not expressed a_s the spleen. Hs-UNC-53/3 could therefore funct:ion as part of the signal transduci~ions pathway involved in the maturation of leukocytes.
Malfunction of this pathway may lead to incorrect maturation of t:he leukocytes and the development of autoimmune diseases such as rheumatoid arthritis and sclerosis. Ne~a to the signalling function in the recognition of the leukocytes, Hs-UNC-53/3 may also play an important role in the induction and/or signalling pathway of the mechanism underlying apoptosis of leukocytes in the spleen. Pharmaceutical methods involving the hs-UNC-53/3 pathway, which raay, for example, result in an inhibition and/or enhancement of its expression may lead to treatment of these disorder;. Furthermore, hs-UNC-52/2 may have an advantage, as an inhibitor or enh,ancer specific for hu-unc53/3 which will act in a more specific manner.
The Hu-UNC-53/3 protein is also highly expressed in the ovary, where the two other human homologues are also expressed. )pinally moderate to low expression of hs-unc53/3 is observed in heart, placenta, testis, stomach and adrenal gland.
Although the predominant transcripts of Hs-unc-53/3 are > 9 kb, often a smear occurs that ends at.
with somewhat higher intensity at 5.5 - 6.5 kB. ~~his short transcript may correspond to AB023155.
The Hs-unc53/3 gene is a highly regulated gene, showing strong tissue specificity and additional mechanisms of regulation which have not previously been identified. in any of its known homologues. These findings may thus lead to the development of more specific inhibitors or enhancers of hs-UNC-35/3 and or of the Hs-UNC-53/3 pathway. The IJorthern blot studies indicate that the three human unc--53s are complex transcriptional units with highly regulated tissuE;
specificity and that transcripts of different lengths exist.
Splice variants of human unc-53s Whilst cloning Hs-unc-53/3, :Lt became apparent that at least three expression variants of Hs-unc-53/3 - most probably alternative splicE~s - exist (fig. 1e, f; lowercase regions). Targeted Efforts for the t:wo other human UNC-53s demonstrated that the other human UNC-53s contained variants (fig. .La, c and a regions).
Splice variants as observed t:o date appear to be concentrated in specific regions. A first one (starting at position 1252 in fig.. 2) - in which the overall amino acid similarity is oueak - contains 2 (splice) variants of both Ce-unc-.'i3 and Hs-unc-53/'3.
In the worm, the presence or absence of these 2 ex:ons in unc-53 regulates the function of the UNC-53 protein in such a way that cells differentially translate _ - 35 -extra-cellular signal gradient a~~ an attractive or repulsive signal. The most 3'-variant of Hs-unc-53/2 roughly covers the 2 Ce-unc-53 variants.
The complE~xity of variation in this zone of Hu-UNC-53 might resemble the situation in the nematode.
In Hs-unc-53/3,, for example, the region from position 3795 to 4325 (figure 1e) consists. of two adjacent blocks (3795 to 4283 and 4286 to 4325 in figure le) that can independently be present. in or absent from cDNAs from frontal cortex tissue. In contrast, no variants were as yet observed in this zone for Hu-UNC-53/l or /2.
The second variant in Hs-unc:-53/3 (fig. 2) deletes a box (MQLDNRTLPKKGLR), which is extremely conserved (in bold) among all human unc-53s. This occurrence of this variant could indicate differentially active functianal variants of Hu-unc53/3.
A second region in which splice variants were observed contains a major highly conserved domain of unc-53s. Hs-unc-53/1 has a firs variant that comprises the most N-terminal portion of this conserved domain (SGSFRD). A second splice variant in Hs-unc-53/1 (AEERMOSE) lies within the highly conserved domain. Another conserved spot for splice variation in hlunan unc-53s has been found (figure 2):
Hs-unc-53/1 {VYE}; -/2 {VNE} and -/3 {NSRGSEL}. All these spliced exons are flanked by two conserved charged domains - putative nuclear localisation signals. Given this conservation, we searched for splice variation in C. elegans and found it to exist in the form of an extra exon (ALSVDSQ) (figure 2).
Hu-unc-53/3 has another variant (SPLVWPPKKRQNGPVIYKHSR) (fig. 2).
The most 3' splice variant in Hs-unc-53/3 has been discovered whilst cloning Hs-unc-53/3 and was shown to be present uniquely in human heart cDNA
WO 99!63080 PCTIEF'99/03848 _ - 36 -libraries.
Single nucleotide polymorphi:ams Cloning and PCR studies indicated the existence of a non-silent single nucleotide polymorphism in Hs-unc-53/1 in position 1232 and in Hs-unc-53/2 in position 929. This indicated than variations exi~~t in human unc-53s which - in some cases - may be relevant to the proper functioning o.f the iJNC-53 protein arid hence in disease.
Expression in normal and neoplastic cells by RT-PCR
The cloning efforts demonstr<~ted the existence of splice variants in the human unc-53s and the NortY:~ern blots revealed a range of transcr~Lpts for each human unc-53. The combined data do not explain complet~:ly the range of transcripts observed., Therefore, our understanding of the expression complexity of human unc-53s may be incomplete and morE: detailed RT-PCR.
studies were performed.
One of the obscuring factors could have been that all studies performed on mRNA or c:DNA of whole tissues which are built of different normal human cell tyyes that occur in different proportions. For this reason and because skin was not covered in the Northern blot studies, a RT-PCR study was set up using cDNA
preparations of the different cel3_s in skin normal human: (1) epidermal keratinocytes, (2~ melanocytes, (3) dermal fibroblasts. In addition, lineage matched transformed cell lines or tumour cell lines were included in the study to compare normal versus neoplastic cells. Human umbilical. vein endothelial cells tH~C) were taken as a normal human match for endothelial cell lines.
_ - 37 -The RT-PCR study for Hs-unc-53/1 revealed that the most 5'-splice variant is dif:Eerentially expressed in narmal versus neoplastic cells,~cel1 lines. This axon is present in 7/7 keratinocyi~es, HUVEC and in melanocytes but lacking in HaCat, ECV304, 2/7 mel~~noma and MCF-7 cells (breast carcinoma).
The RT-PCR study for Hs-unc-.'~3/2 revealed a More surprising picture. The tumourigE~nic endothelial line ECV304 lacks expression of Hs-unc--53/2, whereas their normal counterpart HUVEC expresses Hs-unc-53/2, suggesting gene deletion or inactivation of expression in ECV304. In epidermal keratinocytes and the lineage matched spontaneously transformed keratinocyte HaC:aT
and MCF-7 lack expression of the .'~'-end of Hs-unc-53/2, but express the 3'end (starting in or near the microtubule-binding domain). This suggests that like AB023155 for Hs-unc-53/3, also Hs--unc-53/2 can be expressed as a truncated 3'-variant in a cell-specific way. Also splice variation of Hs--unc-53/2 appears to differ in a normal to neoplastic way: the {VNE} axon was shown to be present in all keratinocyte isolates but nat in HaCaT and also melanocytes express it, but nat 2/7 melanoma or MCF-7. The R7~"-PCR studies for Hs-unc-53/3 were focussed on demonstrating expression of AB023155 in tissues other than brain. The new axon described was shown to be present in keratinocytes, HUVEC, dermal fibroblasts, melanoc:ytes and their transformed/neoplastic variants, demonstrating its wide expression in tissues in man..
Alternative 5'-start axons For Hs-unc-53/2 five different start axons have been cloned using RT-PCR, three of: which have been confirmed to be present in at least 2 different cDNA
libraries (figu.re lb, c) . Likewise for Hs-unc-53/3 different 5'-axons were found, two of which were WO 99/63Q8n PCT/EP99/03848 confirmed (figure le, f) . These .'~'-exons most probably indicate that human unc- 53s are being expressed via the control of alte~__°native promoters that lie 5' of these different 5°--exons. Also in the nematode has been shown that diffE:rent (intronic) promoters are driving the expression of 5'-variants of C. elegans unc-53.
The Hs-unc-53/1 5'-end Despite considerable efforts, cloning has not lead to the identification of a bona fide 5'-end for Hs-unc-53/1 that comprises an F-acain binding domain, despite the fact that the Northern blots indicate the existence of transcripts > 9.5 kb,. Given that both Hs-unc-53/2 and -/3 are expressed as full length a.nd truncated forms, the question can be raised whether Hs-unc-53/1 may not be expressed in a short form as well.
cDNA library cloning and 5'-RACE has provided.
contiguous sequence that ends at a position that matches with a domain in C. elegans un-53, where an alternative start position lies. Based on this argument, Hs-unc-53/1 could be a functional equivalent in man of this 'transcript in nemat:ade.
To further trace the "longer" variants of Hs-unc-53/1, genomic BAC DNA sequencing has been performed.
In figure lg is shown sequence of a4984 fragment from BAC 585E09. It comprises sequences 5' of the presently known cDNA of Hs-unc-53/I. To thE: qualified as well as by means of 'two groups of gene structure prediction computer programs, different but c:amparable exons in the 4984 by genomic sequence fragrc~ent can be predicted (figure 14). The programs GENSCAI~f, HEXON and MZEF all predict an exon between by 1089 and by 1880. The end of this predicted exon (bp 1880) i.s canfirmed by the cDNA sequence. Therefore this prE:dictions has a big WO 99163080 PCT/EP99/03$48 change to indic:ate the correct exon length. The programs GRAIL, GENEFINDER and HMMGENE all predic~t~an exon between by 1123 and by 2031. None of the predicted exons contains an in frame stop codon 5'' of the alternative start codon. Consequently, it is possible that there exist unidentified exons 5' o:E the exon containing the alternative start codon.
The present picture critically suggests that both nematode and human unc-53s appear to be complex transcriptional_ units. Moreover, the fact that some of the most complex splice variants map to similar regions in the UNC-53 proteins points to evolutionary conserved functional variants of UNC-53s e.g. with regard to the cells directional migration towards or away from a sicJnal source. In contrast, some of i~he variants in the human UNC-53s are located in highly conserved domains these (and other) variants may create discrete - yet undiscovered -- functionally different UNC-53 proteins transcribed from one of the unc-53 genes.
The fact that two and maybe three human unc-:i3s exist as full size and a truncated forms with cel~~.-specific expre~~sion, that series of alternative 5'-start exons exist eventually controlled by differE:nt promoters that some forms of splice variation are conserved from nematode to man, all indicate that the expression of unc-53s is of very :high complexity and that some of the biological functions of UNC-53 proteins are extremely conserved.
On the other hand, the differential expression in Northern blots, the splice variation difference between normal and lineage-matched neoplastic cells and the non-silent single nucleotide changes in two of the three human unc-53s, all indi~:ate how important a wide range of diagnostic assays can be to understand in depth the role in disease of human unc-53s.
WO 9916380 PCTlElP99/03848 Chromosomal localization of 1:3s-unc-53/2 by Genemap98 (Fig. 13 and 1 (c) ) The EST clones AA9I8601, AI2-48585, AA115014 and AA115015 are clearly homologous to the 3'-UTR of FIs-Unc-53/2 cDNA (Figure 1(c))). Although, AA115014 (describing the same EST as AA115015) contains an alternative splice variant of the Hs-Unc53/2 gene in the 3'UTR. A survey with ESTs AA'~18601, AI248585, AA115014 or AI-X115015 as query in i~he genemap98 database (release November 1998) revealed that the Hs-Unc53/2 gene is located at chromosome 11 (http-//www.ncbi.nlm.nih.gov/genernap98/loc.cgi?ID=2122 4). The STS which is used for chromosomal i5 localization and which is situated in the 3'UTR of the Hs-Unc53/2 gene is referred to as SHGC-33456 (dbSTS
Id: 41891, Genbank Acc: 628036, GE~nbank gi: 1396755) (Figure 13a). The STS was localized by analysis on the NIGMS human/rodent somatic cell hybrid panel (dbSTS Id: 41891). The Radiation hybrid results a.re summarized in Figure 13b. Together these data imply that every disease or phenotype connected to SHGC-33456 is due to the Hs-Unc--53/2 gESne.
Functional Characterisation of Hs-unc-53/3 F-actin reorganisation and m~crotubule binding of Hs-unc-53/3 Based on its structural features, Hs-unc-53/3 can be classified as a bona fide human unc-53. To further understand its function and in anticipation of developing pharmacological compound screening assays, Hs-unc-53/3 has been physically cJ_oned following the method described in the experimental section and shown in figure 7a. The derived Hs-unc--53/3 clones comprising full length (A to L and the 3'-half (G to WO 991b3080 PCT/EP99/0384$
_ - 41 -L) of Hs-unc-53/3 were further engineered to form a chimera with green fluorescent protein and cloned into expression vectors appropriate for transfection o:f eukaryotic cells. The nucleic acid and amino acid sequences of these constructs are shown in figure 7b-e. The constructs were transfected into cells and scored for the9.r effects on the F'-actin cytoskeleton and binding to microtubules of mouse neuroblastoma cells N4; functions known for nematode unc-53 and human unc-53/1.
The N4 cell transfected with a GFP fusion to the 3'-half of Hs-zinc-53/3 (pGI3303, fig. 7b) showed pronounced filopodia and lamellipodia outgrowth, which is associated with reorganization of the F-actin cytoskeleton (E~'igure 8) . This observation demonstrates that like nematode unc-53 and human unc-53/l, the F-actin binding domain is not required for inducing reorganization of the F-actin cytoskeleton of N4 cells. In addition, the pGI3303 encoded fusion protein does not co-localize with microtubuli but localizes to the cytoplasm of N4 cells indicating that an important domain for microtubuli association is missing in this C-terminal fragment of Hs-unc-53/:3.
In the alignment figure 2 can be seen that the C-terminal half c>f Hs-unc-53/3 (approximate KIAA0938) does not comprise the conserved microtubule binding domain.
In contract, the N4 cells that expressed low to medium levels of the GFP fusion to full length Hs--unc-53/3 (pGI3305, Fig. 7d) displayed a co-localization of the GFP fusion protein with microtubules (Figure ~~).
Even the centrosomes could clearly be detected in some transfected cells. Cells expressing very low amounts of the fusion ~>rotein displayed specific microtubule (+) -end binding (Figure 9) . The :morphology of the pGI3305 transfected N4 cells does not clearly difi;er from the control transfected cells although there is a tendency towards rounding up of the pGI3305 transfected cells and filopodia outgrowth.
Validation of functional assays as compound screens 874288 has previously been shown to be an inhibitor of nematode function in C. elegans (W096/38555), an activity that ha:> been confirmed in i0 Ce-unc-53 transfected N4 cells, where only the transgene-induced effect was inhibited by 874288. In order to confirm compound R74288s activity in a full mammalian system, a stable transfe:ction of plasmid pGI3150 was per:Eormed in the N4 ne:uroblastoma cell line with the l:i.pofectamin procedure (Gibco BRL).
pGI3150 expresses an eGFP protein in fusion with t:he C-terminal end of Hs-unc-53/1 (see: Figure 15a). After two weeks of 6418 selection, 20 clones with stable integration of i~.he pGI3150 plasmid were selected and isolated. These clones were tested for GFP expression by fluorescence microscopy and by Western blotting with an anti-GFI? antibody (table 1). The lamellipodia outgrowth phenoi~ype was checked visually (See Figure 15b). Compound 874288 was tested on four random selected pGI3150 stably transfected clones: 8.I, 8.2, 8.3 and 10.1 and on a pool of pEGfPCI stable transfected N4 control cells. Clones 8.2 and I0.1 displayed less lamellipodia outgrowth than clones 8.1 and 8.3. Compounds and solvents were added to the stably transfected cells ( 10'M in IOMSO) . After 24 hrs of incubation, two persons independently scored the effect of the treatments on the cells. As shown in table I, both persons noticed an effect compound 2 on clones 8.2 and 10.1 with a weak transgene-induced lamellipodia phenotype. This effect consisted of a more flat morphology of the treated versus untreated cells. Compound 2 was 874288.
WO 99/b3080 PCT/EP99103848 _ - 43 -Table 1. Effect of compounds on lamellipodia formation Clone CompoundCompoundCompoundCompoundGEP GFP Phenotype 1 2 3 4 fluo Western 5s.1 0 0 o toxic + + +
8.2 0 + 0 toxic ++ +++ +/-8.3 0 0 0 toxic ++ ++ ++
10.1 0 + 0 toxic +/- + +/-GFP pool.0 0 0 toxic -Automated compound screening blr measuring cell morphology Compound :;creeping assays must have a sufficiently high throughput to be relevant to drug discovery. To achieve this goal, we automated the procedure of mE~asuring the morphological changes induced in cel_Ls following transient transfection with full length or 3'-half of Hs-unc-53/3 GFP chimeras.
The cell culture, transfection, fluorescence staining and microscopy procedures are performed within a 96-well plate (all-in-one). The fluorescent staining method comprises a triple fluorescent labeling procedure (1) for cell nucleic using DNA double helix intercalating dyes such as Hoechst 33342 or DAPI, (2) for transfection efficiency and expression level .of the chimeric protein using GFP fluorescence and {3) for the F-actin cytoskeleton using fluorescently labeled phalloidin, a microfilament dye.
These three different fluorescent images are collected using an motorised stage plus stage driver and a frame grabber that produces seamless composite images of the cells in the well. The software programs to drive this operation are known in public domain as "SCTL" (University of Amsterdam). The seamless images are then superimposed using _ - 44 -pseudocolour for the operator to inspect the quality of the culture. In addition, the SCIL program wa:~
compiled in such a way that it: (1) identifies cells by means of their nucleus, (2) measures the GFP
fluorescence intensity, (3) delineates the area of the F-actin (phalloidin)staining surrounding a nucleu~~ and (4) calculates a range of parameters objectively representing the features of the I?-actin staining pattern of each individual cell. One example of such a parameter is called the "form factor". It is an arbitrary value that reflects the dendricity of a cell. It is derived by calculating (A) the true circumference of a cell's F-actin staining area as seen in the image and (B) the area of the F-actin staining of that given cell. The ratio 4xPIx(B)2= the form factor. For a rounded cell, the form factor approximates 1 whereas, for a cell. with increased filopodia and lamellipodia outgrowth, the true circumference will be much larger than that of a circle and as a result, the form factor « 1.
In experiments it was shown that transiently transfected N4 cell populations indeed displayed a different form factor versus control cells. Both the median and average form factor for a cell population in a well were :reduced following t:ransfection with the 3'-half of Hs-unc-53/3. More in particular, there was a significant decrease in the number of cells in a transfected culture that displayed the minimal form factor, suggesting that the Hs-UNC:-53/3 transgene induced round cells in particular to become more dendritic (figu:re 16) .
Chromosomal localisation of Ha-unc-53/3 by FISH
indicative for a role disease With FISH i~echnology using a unique fragment of hs-unc-53/3 we are able to localize the hs-unc53/3 _ - 45 -gene on chromosome 12q21.1. Chromosome 12q21.1 is a region shown to be involved in autosomal dominant,-cornea plana and closed angle glaucoma (Sigler-Villanueva et al., Ophthalmic Genetics 18:55-62, 1997). This indicates that hs-UNC-53/3 protein m,ay be involved in eye development and thus eye diseases, such as retinoblastomas. Neuroblastoma cell line NPG
and liposarcoma line WDLPS and other sarcoma lines have amplifications in this region. The neuroblastoma amplification seems to be located more distal (12q24) while the liposarcoma line is located at 12q21 (Van Royal et al., Cancer Genetics and Cytogenetics 82:151-4, 2995). Three loci related to Darier's disease,, an autosomal dominant genodermatosis disease characterized by epidermal acantholysis and dyskeratosis have been mapped in region 12q21-q24 (Wright et al., Journal of Investigative Dermatology 103:665-8). 12q21 is also known to be a fragile cite associated with the pathogenesis of non-Hodgkin's Lymphoma (Chary-Reddy et al., Cancer Letter 86:11:L-7 1994). Duplications related to nephroblastoma tumorgensis wane commonly found in the 12q21-q23 region (Austruy et al., Genes Chromosomes Cancer 14:285-294, 1995). In a girl with mental retardation, a conclusive disorder and clinical findings resembling cerebral palsy, positioning of segments from other autosomes adject to the band 12q21 were found (Biederman et al., Ann Genet 19:257-260, 1976).
Cytogenetic analysis for myeloid leukemia showed a complex caryotype with chromosomal breakpoints at 12q21 (Weinstein et al., Cancer Genet Cytogenet 48:75-81, 1990). Finally, analysis of complex chromosomal rearrangements in malformed children and from spontaneous abortions showed specific breakpoints at site 12q21 Gorski et al., Am J Med Genet 29:247-2fil, 1997). Most of: these diseases have been shown to be involved with cell movement, aberrant development, or WO 99/63080 PCTlEP99/03848 _ - 46 -cell-cell contact and neuronal tissue or neuronal development.
Confirmation of FISH with Radiation hybrid panels To confirm. and refine the chromosomal localisation of the human unc-53s an alternative method for FISH has been used. Radiation hybrid (RH) mapping is a somatic cell hybrid technique that was developed to construct high-resolution, contiguous maps of mammalian chromosomes. R:H mapping provides a method for ordering DNA markers spanning millions of base pairs of DNA at a resolution to easily obtained by other mapping methods. Some of the advantages of RH mapping are (1) distance estimated by this method is directly proportional to physical distance, (2) nonpolymorphic DNA markers, that can not be used for meiotic mapping, can be used for -this method, and (3) a high resolution map that is not easily made by other methods can be obtained.
The results of FISH and RH mapping for the three human unc-53s are summarised in table AA. By using publicly available databases (see experimental section) one can derive information on the correlation between FISH and RH mapping. RH 2~lapping was shown in this way to confirm the FISH data for the three un:c-53s.
WO 9916308~ PCT/EIP99/03848 _ - 47 -Table 2. RFi Mapping Primers and Results Unc-53 FOR REV primerPt'R Rfsul.tsMarker* FISH
Primes Hs-UNC-53/1 5'TGTGGGT5'CAGAGCTT51, 6'<<'., SHGC-3023E~1q31-32 {BAC585E9) GAGGAATGCGCTCTAGAGG
TGAC AC
Hs-unc-53/1 5'CCTGCCC5'CCATCTAC51, 62, 66 SHGC-3023 1q31-32 {BAC585E9) AACATAGCAAATGAGCCAG
AGAC AC
Hs-uric-53/25'CTGCCTC5'CTGAGCAG8, 28, 29, AFM022th2 11p15.t 43, 6911 CCTTTGCTGAGTGAAGCCA44, 57., 59, TGTTGCATGGAGTTGG 66, 7t1, 77, 1 Hs-unc-53/2,5'TCATGTA5'CATTGTGT8, 28, 49, SHGC-3102111p15.1 0 51, F4.1.2 TTCCCCACACTTGATACTT59, 65, 83 GACAAGCC TGGGGTGC
Hs-unc-53/2,5'GAGGATT5'TGATCTTC8, 27, 28, AFM022th2 11p15.1 29, D4.1.1 TTATTTCTGCACTCCGTGG43, 49, 51, GGAAATGGAATAACT 59, 65, 70, ATCGG
Hs-unc-53/2,5'AAAGCCC5'AACCCGTT8, 27, 28, AFM022th2 11p15.1 43, 1 J4.1.4 AAGCCCCGGTTCCACCGAG44, 51, 59,
A similar set of blots were probed with a 652bp fragment of EST46037 amplified by using the primers 5' aggagatgaagctgacagatatc;c and 5' -aaacaccagtgagtcc. Expression levels are low in all tissues with the highest level in kidney, placenta and pancreas, lower levels in heart and lung. Expression is barely detectable or undetectable in skeletal muscle, spleen,, thymus, prostate, testis, ovary, small intestine, colon peripheral blood leucocyte, stomach, thyroid, spina:L cord, trachea, adrenal gland and bone marrow. Also l~is-unc-53/2 appears; to be expressed as different transcripts (figure 5a) .
The hs-UNC53/1 and hs-UNC-53/2 homologues are clearly highly regulated. genes, showing a strong tissue specificity and, probably, additional mechanisms of :regulation (ie differential splicing of different promoters). The different proteins derived from RNA's identified by probe hh.l5 presumably share the carboxyterminal nucleotide binding domain.
Ce-UNC-53 was shown to be a complex genetic locus and complex transcription unit. The different transcripts are thought to be a mechanism to assure the necessary specificity and functional diversity of this signal transduction pathway, with respect to different signals and receptors, different tissues and different directions of migration. The occurrence of a new transcript or i~he observed changes in expression levels in the cancer cell line blot suggests a role for hs-UNC-53/:3 in the establishment or maintenance of the transformed state of those cells.
WO 99/63U8~ PCT/E1P99/03848 _ - 32 -Expression pattern of hs-UNC'-5~/3.
A northern blot of poly-A+RNA from several cancer lines was probed with unique fragments of the three genes from the Hs-unc-53 family. Hs-unc-53/3 has a high expression level in lung carcinoma line A549, where only a moderate expression of hs-unc-53/1 h;as been detected. Furthermore, moderate expression of Hs-unc-53/3 was also observed in melanoma line 6361, where previous7_y, a high expression of hs-UNC-53/1 and hs-UNC-52/2 has been observed. This indicated the involvement of hs-unc53/3 in at least two cancer lines.
In normal human tissues, the expression of hs-unc-53/3 shows a clearly new and previously unobserved expression pattern. This difference of expressio~.z of hs-unc-53/3 in relation to its homologues hs-unc5:3/1 and hs-unc53/2 is important for the allocation of functionality to hs-unc-53/3.
Hs-unc-53/'3 is highly expressed in brain, as shown on the Northern blots (figure 5a). In figure 5b it can be seen that Hs-unc-53/3 also is different=sally expressed in different parts of t:he brain. Its homologues are not or weakly expressed in brain. This gives an indication that its function in directionality of cell migration and growth cone steering will be in relation to specific regions or cells of the brain. It is deduced that Hs-unc-53/3 will be an important signal transducer or signal adapter linking signals to neuronal outgrowth, axon guidance, and formation and maintenance of synapt~_c connections. It seems that the f~~,znction of Hs-unc-53/3 will be associated with neuron-neuron interactions, neuronal outgrowth, neuron muscle interactions, and post-synaptic signal transduction.
Furthermore, Hs-unc-53/3 may be involved in the development of cancer of neuronal origin, like neuroblastomas, or the development of tumours will have their developmental origin in the brain as some eyes diseases like retinoblastomas.
The significance of the high expression of Hs-unc-53/3 in brain tissue can be associated with the high levels of expression which has also been observed in the spinal cord, containing neuronal tissue. lHere, neuronal (axon) outgrowth and neuron-neuron connections are of importance. Development of pharmacological_ tools acting on this pathway may .lead to treatments of diseases involved in the growth ;end movement of neuronal cells, and the regeneration of neuronal connectivity after trauma, or the inhibition of neuronal cancers such as neuroblastomas. Due 'to its specific expression, inhibitors and/or enhancers specific for H:>-unc-53/3 will have an advantage a:~ a pharmaceutical compound over more general compounds acting on the Hs-unc-53 family of genes and proteins.
A second tissue where hs-UNC-53/3 is highly expressed and where (its) other human homologues are not expressed a_s the spleen. Hs-UNC-53/3 could therefore funct:ion as part of the signal transduci~ions pathway involved in the maturation of leukocytes.
Malfunction of this pathway may lead to incorrect maturation of t:he leukocytes and the development of autoimmune diseases such as rheumatoid arthritis and sclerosis. Ne~a to the signalling function in the recognition of the leukocytes, Hs-UNC-53/3 may also play an important role in the induction and/or signalling pathway of the mechanism underlying apoptosis of leukocytes in the spleen. Pharmaceutical methods involving the hs-UNC-53/3 pathway, which raay, for example, result in an inhibition and/or enhancement of its expression may lead to treatment of these disorder;. Furthermore, hs-UNC-52/2 may have an advantage, as an inhibitor or enh,ancer specific for hu-unc53/3 which will act in a more specific manner.
The Hu-UNC-53/3 protein is also highly expressed in the ovary, where the two other human homologues are also expressed. )pinally moderate to low expression of hs-unc53/3 is observed in heart, placenta, testis, stomach and adrenal gland.
Although the predominant transcripts of Hs-unc-53/3 are > 9 kb, often a smear occurs that ends at.
with somewhat higher intensity at 5.5 - 6.5 kB. ~~his short transcript may correspond to AB023155.
The Hs-unc53/3 gene is a highly regulated gene, showing strong tissue specificity and additional mechanisms of regulation which have not previously been identified. in any of its known homologues. These findings may thus lead to the development of more specific inhibitors or enhancers of hs-UNC-35/3 and or of the Hs-UNC-53/3 pathway. The IJorthern blot studies indicate that the three human unc--53s are complex transcriptional units with highly regulated tissuE;
specificity and that transcripts of different lengths exist.
Splice variants of human unc-53s Whilst cloning Hs-unc-53/3, :Lt became apparent that at least three expression variants of Hs-unc-53/3 - most probably alternative splicE~s - exist (fig. 1e, f; lowercase regions). Targeted Efforts for the t:wo other human UNC-53s demonstrated that the other human UNC-53s contained variants (fig. .La, c and a regions).
Splice variants as observed t:o date appear to be concentrated in specific regions. A first one (starting at position 1252 in fig.. 2) - in which the overall amino acid similarity is oueak - contains 2 (splice) variants of both Ce-unc-.'i3 and Hs-unc-53/'3.
In the worm, the presence or absence of these 2 ex:ons in unc-53 regulates the function of the UNC-53 protein in such a way that cells differentially translate _ - 35 -extra-cellular signal gradient a~~ an attractive or repulsive signal. The most 3'-variant of Hs-unc-53/2 roughly covers the 2 Ce-unc-53 variants.
The complE~xity of variation in this zone of Hu-UNC-53 might resemble the situation in the nematode.
In Hs-unc-53/3,, for example, the region from position 3795 to 4325 (figure 1e) consists. of two adjacent blocks (3795 to 4283 and 4286 to 4325 in figure le) that can independently be present. in or absent from cDNAs from frontal cortex tissue. In contrast, no variants were as yet observed in this zone for Hu-UNC-53/l or /2.
The second variant in Hs-unc:-53/3 (fig. 2) deletes a box (MQLDNRTLPKKGLR), which is extremely conserved (in bold) among all human unc-53s. This occurrence of this variant could indicate differentially active functianal variants of Hu-unc53/3.
A second region in which splice variants were observed contains a major highly conserved domain of unc-53s. Hs-unc-53/1 has a firs variant that comprises the most N-terminal portion of this conserved domain (SGSFRD). A second splice variant in Hs-unc-53/1 (AEERMOSE) lies within the highly conserved domain. Another conserved spot for splice variation in hlunan unc-53s has been found (figure 2):
Hs-unc-53/1 {VYE}; -/2 {VNE} and -/3 {NSRGSEL}. All these spliced exons are flanked by two conserved charged domains - putative nuclear localisation signals. Given this conservation, we searched for splice variation in C. elegans and found it to exist in the form of an extra exon (ALSVDSQ) (figure 2).
Hu-unc-53/3 has another variant (SPLVWPPKKRQNGPVIYKHSR) (fig. 2).
The most 3' splice variant in Hs-unc-53/3 has been discovered whilst cloning Hs-unc-53/3 and was shown to be present uniquely in human heart cDNA
WO 99!63080 PCTIEF'99/03848 _ - 36 -libraries.
Single nucleotide polymorphi:ams Cloning and PCR studies indicated the existence of a non-silent single nucleotide polymorphism in Hs-unc-53/1 in position 1232 and in Hs-unc-53/2 in position 929. This indicated than variations exi~~t in human unc-53s which - in some cases - may be relevant to the proper functioning o.f the iJNC-53 protein arid hence in disease.
Expression in normal and neoplastic cells by RT-PCR
The cloning efforts demonstr<~ted the existence of splice variants in the human unc-53s and the NortY:~ern blots revealed a range of transcr~Lpts for each human unc-53. The combined data do not explain complet~:ly the range of transcripts observed., Therefore, our understanding of the expression complexity of human unc-53s may be incomplete and morE: detailed RT-PCR.
studies were performed.
One of the obscuring factors could have been that all studies performed on mRNA or c:DNA of whole tissues which are built of different normal human cell tyyes that occur in different proportions. For this reason and because skin was not covered in the Northern blot studies, a RT-PCR study was set up using cDNA
preparations of the different cel3_s in skin normal human: (1) epidermal keratinocytes, (2~ melanocytes, (3) dermal fibroblasts. In addition, lineage matched transformed cell lines or tumour cell lines were included in the study to compare normal versus neoplastic cells. Human umbilical. vein endothelial cells tH~C) were taken as a normal human match for endothelial cell lines.
_ - 37 -The RT-PCR study for Hs-unc-53/1 revealed that the most 5'-splice variant is dif:Eerentially expressed in narmal versus neoplastic cells,~cel1 lines. This axon is present in 7/7 keratinocyi~es, HUVEC and in melanocytes but lacking in HaCat, ECV304, 2/7 mel~~noma and MCF-7 cells (breast carcinoma).
The RT-PCR study for Hs-unc-.'~3/2 revealed a More surprising picture. The tumourigE~nic endothelial line ECV304 lacks expression of Hs-unc--53/2, whereas their normal counterpart HUVEC expresses Hs-unc-53/2, suggesting gene deletion or inactivation of expression in ECV304. In epidermal keratinocytes and the lineage matched spontaneously transformed keratinocyte HaC:aT
and MCF-7 lack expression of the .'~'-end of Hs-unc-53/2, but express the 3'end (starting in or near the microtubule-binding domain). This suggests that like AB023155 for Hs-unc-53/3, also Hs--unc-53/2 can be expressed as a truncated 3'-variant in a cell-specific way. Also splice variation of Hs--unc-53/2 appears to differ in a normal to neoplastic way: the {VNE} axon was shown to be present in all keratinocyte isolates but nat in HaCaT and also melanocytes express it, but nat 2/7 melanoma or MCF-7. The R7~"-PCR studies for Hs-unc-53/3 were focussed on demonstrating expression of AB023155 in tissues other than brain. The new axon described was shown to be present in keratinocytes, HUVEC, dermal fibroblasts, melanoc:ytes and their transformed/neoplastic variants, demonstrating its wide expression in tissues in man..
Alternative 5'-start axons For Hs-unc-53/2 five different start axons have been cloned using RT-PCR, three of: which have been confirmed to be present in at least 2 different cDNA
libraries (figu.re lb, c) . Likewise for Hs-unc-53/3 different 5'-axons were found, two of which were WO 99/63Q8n PCT/EP99/03848 confirmed (figure le, f) . These .'~'-exons most probably indicate that human unc- 53s are being expressed via the control of alte~__°native promoters that lie 5' of these different 5°--exons. Also in the nematode has been shown that diffE:rent (intronic) promoters are driving the expression of 5'-variants of C. elegans unc-53.
The Hs-unc-53/1 5'-end Despite considerable efforts, cloning has not lead to the identification of a bona fide 5'-end for Hs-unc-53/1 that comprises an F-acain binding domain, despite the fact that the Northern blots indicate the existence of transcripts > 9.5 kb,. Given that both Hs-unc-53/2 and -/3 are expressed as full length a.nd truncated forms, the question can be raised whether Hs-unc-53/1 may not be expressed in a short form as well.
cDNA library cloning and 5'-RACE has provided.
contiguous sequence that ends at a position that matches with a domain in C. elegans un-53, where an alternative start position lies. Based on this argument, Hs-unc-53/1 could be a functional equivalent in man of this 'transcript in nemat:ade.
To further trace the "longer" variants of Hs-unc-53/1, genomic BAC DNA sequencing has been performed.
In figure lg is shown sequence of a4984 fragment from BAC 585E09. It comprises sequences 5' of the presently known cDNA of Hs-unc-53/I. To thE: qualified as well as by means of 'two groups of gene structure prediction computer programs, different but c:amparable exons in the 4984 by genomic sequence fragrc~ent can be predicted (figure 14). The programs GENSCAI~f, HEXON and MZEF all predict an exon between by 1089 and by 1880. The end of this predicted exon (bp 1880) i.s canfirmed by the cDNA sequence. Therefore this prE:dictions has a big WO 99163080 PCT/EP99/03$48 change to indic:ate the correct exon length. The programs GRAIL, GENEFINDER and HMMGENE all predic~t~an exon between by 1123 and by 2031. None of the predicted exons contains an in frame stop codon 5'' of the alternative start codon. Consequently, it is possible that there exist unidentified exons 5' o:E the exon containing the alternative start codon.
The present picture critically suggests that both nematode and human unc-53s appear to be complex transcriptional_ units. Moreover, the fact that some of the most complex splice variants map to similar regions in the UNC-53 proteins points to evolutionary conserved functional variants of UNC-53s e.g. with regard to the cells directional migration towards or away from a sicJnal source. In contrast, some of i~he variants in the human UNC-53s are located in highly conserved domains these (and other) variants may create discrete - yet undiscovered -- functionally different UNC-53 proteins transcribed from one of the unc-53 genes.
The fact that two and maybe three human unc-:i3s exist as full size and a truncated forms with cel~~.-specific expre~~sion, that series of alternative 5'-start exons exist eventually controlled by differE:nt promoters that some forms of splice variation are conserved from nematode to man, all indicate that the expression of unc-53s is of very :high complexity and that some of the biological functions of UNC-53 proteins are extremely conserved.
On the other hand, the differential expression in Northern blots, the splice variation difference between normal and lineage-matched neoplastic cells and the non-silent single nucleotide changes in two of the three human unc-53s, all indi~:ate how important a wide range of diagnostic assays can be to understand in depth the role in disease of human unc-53s.
WO 9916380 PCTlElP99/03848 Chromosomal localization of 1:3s-unc-53/2 by Genemap98 (Fig. 13 and 1 (c) ) The EST clones AA9I8601, AI2-48585, AA115014 and AA115015 are clearly homologous to the 3'-UTR of FIs-Unc-53/2 cDNA (Figure 1(c))). Although, AA115014 (describing the same EST as AA115015) contains an alternative splice variant of the Hs-Unc53/2 gene in the 3'UTR. A survey with ESTs AA'~18601, AI248585, AA115014 or AI-X115015 as query in i~he genemap98 database (release November 1998) revealed that the Hs-Unc53/2 gene is located at chromosome 11 (http-//www.ncbi.nlm.nih.gov/genernap98/loc.cgi?ID=2122 4). The STS which is used for chromosomal i5 localization and which is situated in the 3'UTR of the Hs-Unc53/2 gene is referred to as SHGC-33456 (dbSTS
Id: 41891, Genbank Acc: 628036, GE~nbank gi: 1396755) (Figure 13a). The STS was localized by analysis on the NIGMS human/rodent somatic cell hybrid panel (dbSTS Id: 41891). The Radiation hybrid results a.re summarized in Figure 13b. Together these data imply that every disease or phenotype connected to SHGC-33456 is due to the Hs-Unc--53/2 gESne.
Functional Characterisation of Hs-unc-53/3 F-actin reorganisation and m~crotubule binding of Hs-unc-53/3 Based on its structural features, Hs-unc-53/3 can be classified as a bona fide human unc-53. To further understand its function and in anticipation of developing pharmacological compound screening assays, Hs-unc-53/3 has been physically cJ_oned following the method described in the experimental section and shown in figure 7a. The derived Hs-unc--53/3 clones comprising full length (A to L and the 3'-half (G to WO 991b3080 PCT/EP99/0384$
_ - 41 -L) of Hs-unc-53/3 were further engineered to form a chimera with green fluorescent protein and cloned into expression vectors appropriate for transfection o:f eukaryotic cells. The nucleic acid and amino acid sequences of these constructs are shown in figure 7b-e. The constructs were transfected into cells and scored for the9.r effects on the F'-actin cytoskeleton and binding to microtubules of mouse neuroblastoma cells N4; functions known for nematode unc-53 and human unc-53/1.
The N4 cell transfected with a GFP fusion to the 3'-half of Hs-zinc-53/3 (pGI3303, fig. 7b) showed pronounced filopodia and lamellipodia outgrowth, which is associated with reorganization of the F-actin cytoskeleton (E~'igure 8) . This observation demonstrates that like nematode unc-53 and human unc-53/l, the F-actin binding domain is not required for inducing reorganization of the F-actin cytoskeleton of N4 cells. In addition, the pGI3303 encoded fusion protein does not co-localize with microtubuli but localizes to the cytoplasm of N4 cells indicating that an important domain for microtubuli association is missing in this C-terminal fragment of Hs-unc-53/:3.
In the alignment figure 2 can be seen that the C-terminal half c>f Hs-unc-53/3 (approximate KIAA0938) does not comprise the conserved microtubule binding domain.
In contract, the N4 cells that expressed low to medium levels of the GFP fusion to full length Hs--unc-53/3 (pGI3305, Fig. 7d) displayed a co-localization of the GFP fusion protein with microtubules (Figure ~~).
Even the centrosomes could clearly be detected in some transfected cells. Cells expressing very low amounts of the fusion ~>rotein displayed specific microtubule (+) -end binding (Figure 9) . The :morphology of the pGI3305 transfected N4 cells does not clearly difi;er from the control transfected cells although there is a tendency towards rounding up of the pGI3305 transfected cells and filopodia outgrowth.
Validation of functional assays as compound screens 874288 has previously been shown to be an inhibitor of nematode function in C. elegans (W096/38555), an activity that ha:> been confirmed in i0 Ce-unc-53 transfected N4 cells, where only the transgene-induced effect was inhibited by 874288. In order to confirm compound R74288s activity in a full mammalian system, a stable transfe:ction of plasmid pGI3150 was per:Eormed in the N4 ne:uroblastoma cell line with the l:i.pofectamin procedure (Gibco BRL).
pGI3150 expresses an eGFP protein in fusion with t:he C-terminal end of Hs-unc-53/1 (see: Figure 15a). After two weeks of 6418 selection, 20 clones with stable integration of i~.he pGI3150 plasmid were selected and isolated. These clones were tested for GFP expression by fluorescence microscopy and by Western blotting with an anti-GFI? antibody (table 1). The lamellipodia outgrowth phenoi~ype was checked visually (See Figure 15b). Compound 874288 was tested on four random selected pGI3150 stably transfected clones: 8.I, 8.2, 8.3 and 10.1 and on a pool of pEGfPCI stable transfected N4 control cells. Clones 8.2 and I0.1 displayed less lamellipodia outgrowth than clones 8.1 and 8.3. Compounds and solvents were added to the stably transfected cells ( 10'M in IOMSO) . After 24 hrs of incubation, two persons independently scored the effect of the treatments on the cells. As shown in table I, both persons noticed an effect compound 2 on clones 8.2 and 10.1 with a weak transgene-induced lamellipodia phenotype. This effect consisted of a more flat morphology of the treated versus untreated cells. Compound 2 was 874288.
WO 99/b3080 PCT/EP99103848 _ - 43 -Table 1. Effect of compounds on lamellipodia formation Clone CompoundCompoundCompoundCompoundGEP GFP Phenotype 1 2 3 4 fluo Western 5s.1 0 0 o toxic + + +
8.2 0 + 0 toxic ++ +++ +/-8.3 0 0 0 toxic ++ ++ ++
10.1 0 + 0 toxic +/- + +/-GFP pool.0 0 0 toxic -Automated compound screening blr measuring cell morphology Compound :;creeping assays must have a sufficiently high throughput to be relevant to drug discovery. To achieve this goal, we automated the procedure of mE~asuring the morphological changes induced in cel_Ls following transient transfection with full length or 3'-half of Hs-unc-53/3 GFP chimeras.
The cell culture, transfection, fluorescence staining and microscopy procedures are performed within a 96-well plate (all-in-one). The fluorescent staining method comprises a triple fluorescent labeling procedure (1) for cell nucleic using DNA double helix intercalating dyes such as Hoechst 33342 or DAPI, (2) for transfection efficiency and expression level .of the chimeric protein using GFP fluorescence and {3) for the F-actin cytoskeleton using fluorescently labeled phalloidin, a microfilament dye.
These three different fluorescent images are collected using an motorised stage plus stage driver and a frame grabber that produces seamless composite images of the cells in the well. The software programs to drive this operation are known in public domain as "SCTL" (University of Amsterdam). The seamless images are then superimposed using _ - 44 -pseudocolour for the operator to inspect the quality of the culture. In addition, the SCIL program wa:~
compiled in such a way that it: (1) identifies cells by means of their nucleus, (2) measures the GFP
fluorescence intensity, (3) delineates the area of the F-actin (phalloidin)staining surrounding a nucleu~~ and (4) calculates a range of parameters objectively representing the features of the I?-actin staining pattern of each individual cell. One example of such a parameter is called the "form factor". It is an arbitrary value that reflects the dendricity of a cell. It is derived by calculating (A) the true circumference of a cell's F-actin staining area as seen in the image and (B) the area of the F-actin staining of that given cell. The ratio 4xPIx(B)2= the form factor. For a rounded cell, the form factor approximates 1 whereas, for a cell. with increased filopodia and lamellipodia outgrowth, the true circumference will be much larger than that of a circle and as a result, the form factor « 1.
In experiments it was shown that transiently transfected N4 cell populations indeed displayed a different form factor versus control cells. Both the median and average form factor for a cell population in a well were :reduced following t:ransfection with the 3'-half of Hs-unc-53/3. More in particular, there was a significant decrease in the number of cells in a transfected culture that displayed the minimal form factor, suggesting that the Hs-UNC:-53/3 transgene induced round cells in particular to become more dendritic (figu:re 16) .
Chromosomal localisation of Ha-unc-53/3 by FISH
indicative for a role disease With FISH i~echnology using a unique fragment of hs-unc-53/3 we are able to localize the hs-unc53/3 _ - 45 -gene on chromosome 12q21.1. Chromosome 12q21.1 is a region shown to be involved in autosomal dominant,-cornea plana and closed angle glaucoma (Sigler-Villanueva et al., Ophthalmic Genetics 18:55-62, 1997). This indicates that hs-UNC-53/3 protein m,ay be involved in eye development and thus eye diseases, such as retinoblastomas. Neuroblastoma cell line NPG
and liposarcoma line WDLPS and other sarcoma lines have amplifications in this region. The neuroblastoma amplification seems to be located more distal (12q24) while the liposarcoma line is located at 12q21 (Van Royal et al., Cancer Genetics and Cytogenetics 82:151-4, 2995). Three loci related to Darier's disease,, an autosomal dominant genodermatosis disease characterized by epidermal acantholysis and dyskeratosis have been mapped in region 12q21-q24 (Wright et al., Journal of Investigative Dermatology 103:665-8). 12q21 is also known to be a fragile cite associated with the pathogenesis of non-Hodgkin's Lymphoma (Chary-Reddy et al., Cancer Letter 86:11:L-7 1994). Duplications related to nephroblastoma tumorgensis wane commonly found in the 12q21-q23 region (Austruy et al., Genes Chromosomes Cancer 14:285-294, 1995). In a girl with mental retardation, a conclusive disorder and clinical findings resembling cerebral palsy, positioning of segments from other autosomes adject to the band 12q21 were found (Biederman et al., Ann Genet 19:257-260, 1976).
Cytogenetic analysis for myeloid leukemia showed a complex caryotype with chromosomal breakpoints at 12q21 (Weinstein et al., Cancer Genet Cytogenet 48:75-81, 1990). Finally, analysis of complex chromosomal rearrangements in malformed children and from spontaneous abortions showed specific breakpoints at site 12q21 Gorski et al., Am J Med Genet 29:247-2fil, 1997). Most of: these diseases have been shown to be involved with cell movement, aberrant development, or WO 99/63080 PCTlEP99/03848 _ - 46 -cell-cell contact and neuronal tissue or neuronal development.
Confirmation of FISH with Radiation hybrid panels To confirm. and refine the chromosomal localisation of the human unc-53s an alternative method for FISH has been used. Radiation hybrid (RH) mapping is a somatic cell hybrid technique that was developed to construct high-resolution, contiguous maps of mammalian chromosomes. R:H mapping provides a method for ordering DNA markers spanning millions of base pairs of DNA at a resolution to easily obtained by other mapping methods. Some of the advantages of RH mapping are (1) distance estimated by this method is directly proportional to physical distance, (2) nonpolymorphic DNA markers, that can not be used for meiotic mapping, can be used for -this method, and (3) a high resolution map that is not easily made by other methods can be obtained.
The results of FISH and RH mapping for the three human unc-53s are summarised in table AA. By using publicly available databases (see experimental section) one can derive information on the correlation between FISH and RH mapping. RH 2~lapping was shown in this way to confirm the FISH data for the three un:c-53s.
WO 9916308~ PCT/EIP99/03848 _ - 47 -Table 2. RFi Mapping Primers and Results Unc-53 FOR REV primerPt'R Rfsul.tsMarker* FISH
Primes Hs-UNC-53/1 5'TGTGGGT5'CAGAGCTT51, 6'<<'., SHGC-3023E~1q31-32 {BAC585E9) GAGGAATGCGCTCTAGAGG
TGAC AC
Hs-unc-53/1 5'CCTGCCC5'CCATCTAC51, 62, 66 SHGC-3023 1q31-32 {BAC585E9) AACATAGCAAATGAGCCAG
AGAC AC
Hs-uric-53/25'CTGCCTC5'CTGAGCAG8, 28, 29, AFM022th2 11p15.t 43, 6911 CCTTTGCTGAGTGAAGCCA44, 57., 59, TGTTGCATGGAGTTGG 66, 7t1, 77, 1 Hs-unc-53/2,5'TCATGTA5'CATTGTGT8, 28, 49, SHGC-3102111p15.1 0 51, F4.1.2 TTCCCCACACTTGATACTT59, 65, 83 GACAAGCC TGGGGTGC
Hs-unc-53/2,5'GAGGATT5'TGATCTTC8, 27, 28, AFM022th2 11p15.1 29, D4.1.1 TTATTTCTGCACTCCGTGG43, 49, 51, GGAAATGGAATAACT 59, 65, 70, ATCGG
Hs-unc-53/2,5'AAAGCCC5'AACCCGTT8, 27, 28, AFM022th2 11p15.1 43, 1 J4.1.4 AAGCCCCGGTTCCACCGAG44, 51, 59,
5 GAGAAGATGCCGCTC 66, 70, 83 Hs-unc-53/3,5'ACTTGCT5'CTTGCTGT1, 48, 50, SHGC-1753b12q21.1 51, A215 GAAACAGAGCTTCTTTCTC59, 65, 66, AGCTCCATGCTTGGC 73, 74, 76, Hs-unc-53/3,5'TGATCTT5'ATCATTCCi, 48, 50, SHGC-175361 2 q2 51, 1.1 A211 CTAGCGTGTTTGGAGT 59, 73, 76, GACTCACTG
20 (*) list not exhaustive Also sequence information available in public.
domain can help refine the positioning of the unc--53 genes, like in the following example. The EST clones 25 AA918601, AI248585, AA115014 and .AA115415 are clearly homologous to Fps-Unc53/2 cDNA. Although, AA115014 (describing the same EST as AA115015) contains an alternative spl.icevariant of the :EIs-Unc53/2 gene in the 3'UTR. A survey with ESTs AA918601, AI248585, 30 AA1I5014 or AA115015 as query in the genemap98 database (release November 1998) revealed that the Hu-_ - 48 -unc53/2 gene is located at chromosome 11 (http://www.ncbi.nlm.nih.gov/genemap98/loc.cgi?ID=2122 4y. The STS which is used for caromosomal localization a.nd which is situated in the 3'UTR of the Hs-Unc53/2 gene is referred to a;s SHGC-33456 (dbSTS
id: 41891, Gen.bank Acc: 628036, csenbank gi: 1396755) (Figure 13y. The STS was localized by analysis on the NIGMS human/rodent somatic cell hybrid panel (dbSTS
id: 41891). The radiation hybrid results are summarized in Figure 13. Together these data imply that diseases or phenotypes connected to SHGC-33156 is due to the Hs-Unc53/2 gene.
EXPERIMENTAL PROCEDURES
Cloning & sequencing of Hs-unc-53/3 Hs-unc53/3 has been cloned starting from a ~~eries of ESTs that were similar but noi~ identical to H~~-unc-53/1 or -/2. The ESTs were:
1. WashU-Merck EST 767735.
Transformed cells carrying the EST 767i'35 sequence were ordered from Research Genetics. Pl.asmid DNA was isolated using standard protocols (Qiager.~
plasmid DNA isolation kity, the sequence of the insert was determined.
2. ATCC cDNA clones 86459"
Transformed cells carrying the cDNA clone 86459 sequence were ordered from ATCC. Plasmid DNA
was isolated using standard protocols (Qiagen plasmid DNA isolation kit), the sequence of the insert was determined.
_ - 49 -3. Genethon cDNA clone c09a03 from the Geneexpress cDNA program.
Transformed cells carrying the cDNA clc>ne c09a03 sequence were ordered from Genethon. Plasmid DNA was isolated using standard protocols (Qiagen plasmid DNA isolation kit), the sequence of the insert was determined..
These ESTs were extended to form one OF;F as follows:
1. 5' extension of EST 76'7735 by RACE (Rapid Amplification of cDNA Ends).
Marathon-Ready cDNAs (Clontech) are pre~made "libraries" of adaptor-ligated double-stranded cDNA
ready for use as templates in RACE experiments. Five ml Marathon-Ready cDNA was used as template in a regular 50 ml RACE. The RACE mi:~ture contained 1 x KlenTaq PCR buffer. 0.2 mM of each dNTP, 1 x advantage KlenTaq polymerase mix (Clontech), 0.15 mM
AP1 adaptor primer and 0.15 mM RACE gene specific primer. The amplification condii~ions were as follows:
94°C for 30 s and 68 °C for 4 min. One-hundred-fold diluted RACE product was used as a template in a nested PCR with AP2 adaptor and gene specific nested PCR primers. Specific nested PCR fragments were cloned into pCR2 (TA cloning kit,, Invitrogen) and. the sequences of the inserts were determined. Gene-specific primer (hh3UNC53 97101702):
5°ACCATTTACACCTGAAGACGATTGAGGTCC;, nested gene-specific primer (hh3UNC53 97101701) 5'CTCCTATTTAA.A.TTAGAGGCTCCCTGGACC Marathon cDNA
library: human placenta, human heart, human chronic myelogenous leukemia, human colorectal adenocarcinoma.
WO 99/63080 PCTIE:P99/03848 2. 3' extension of EST 76')735 by RACE.
Method as described previously. Gene specific primer (hh3UNC53 97102702) 5'CAATCGTCTTCAGGTGTAAATGGTAACGTG;: nested gene specific primer (hh3UNC53 97102703) 5'GAATGTCAAACACAGTGCCACCTCCACC Marathon cDNA library:
human placenta, human heart, human HeLa, human melanoma.
3. 3' extension of cDNA clone c09a03 by RACE.
Method as described previously, gene-specific primer (hh3UNC53 98020401) 5'AGGGAGCACTGAATGGTCCAGACCATCCTC; nested gene-specific primer (hh3UNC53 98020402) 5'GCATCAGAAGACAGCATTCCTCTGAAAGTG Marathon cDNA
library: human placenta, human heart, human HeLa, human melanoma, human colorectal adenocarcinoma, human chronic myelogenous leukemia.
4. 5' e:~tension of cDNA clone 86459 by RACE
(1) .
~ Method as described previously gene-specific primer (hh3UNC53 98020403) 5'TTCAATTTCTATCTCTATGAGTTTTCTTCG; nested gene-specific primer (hh3UNC53 98020404) 5'GCAGCTCTAGAT'.CTGGTGATGAAGAAACTC Marathon cDNA
library: human placenta, human heart, human HeLa, human melanoma. Overlapping sequences were assemlbled in a single contiguous sequence.
5. 5' extension cDNA clone 86459 by RACE (2) .
Method as described previously gene-spet~ific primer (hh3UNC53 98022502) _ _ 51 _ 5'TCAGAATGTGATGAAGGAGGCTTGGTGGAC;w nested gene-specific primer (hh3UNC53 98022501) 5'GGATGCCGGAAGGGATGAATCAGTAAGC Marathon cDNA library:
human placenta, human heart, human HeLa, human melanoma, human colorectal adenocarcinoma, human chronic myelogenous leukemia.
Validating ~rariants at 5' end of the cDNA
sequence In the final 5' RACE experiment, 2 variants have been found whose sequence divergE: upstream from the IYTDWAN protein sequence (position 289 in figure le or position 82 in figure 1f). By using primers ATTTACACTGACTGGGCC.AAC and ATAATCTGGATGATTTCTGCTAGGAGT
on cDNA clones a Hs-unc-53/3 specific PCR product was obtained that was radiolabeled u~>ing the random primed DNA labeling kit (Roche Molecular Biochemicals) and hybridized to human DNA BAC filters (Research Genetics). Both primers are located near the IYTDWAN
box. Four BACs turned out positive (415J11; 464C17, 525C02 and 537B02). DNA sequencing of the region upstream from the IYTDWAN protein sequence directly on these BACs showed that this regic>n was preceded by a putative intronic sequence as evidenced by the multiple stop codons in the reading frame and by the consensus AG intron acceptor sequence. for sequencing purposes, BAC DNA was prepared according to a modified Qiagen plasmid DNA procedure.
A primer pair was designed specifically to amplify the 5'end of the variant shown in full in figure le (primers ACTTGCTGAAACAGAGAGCTCCATG and CTTGCTGTCTTCTTTCTCCTTGGC). PCR with these primers on BAC DNA showed the presence of th.e genomic sequence encoding this variant in 3 out of the 4 BACs (not present in BAC 415J11).
BACs containing the genomic sequence encoding the other 5'end variant of Hs-unc-53/3 as shown as the variant in figure le were identified by hybridiz:i.ng the Research Genetics human DNA GAC filters with primer TGATCTTCTAGCGTGTGACTCACTG, radioactively labeled using gamma-P32-ATP and polynucleotide kinase.
Positive BACs were 404F14, 450K18 and 764L15.
Sequencing directly on the respective BACs in the 3' direction from within the 2 alternative 5' exons and comparison of the genomic DNA sequence with the previously determined cDNA sequence identified the GT
intron donor site. Joining of the genomic sequences from both 5° exons and the IYTDWAN encoding sequence after removal of the predicted intronic sequence restored for both variants the sequence of the 5'' RACE
experiment without affecting the translation of i~he Open Reading E~rame.
Cloning of Hs-unc-53/3 constructs With the aim of cloning the full-length Open Reading Frame of Hs-unc-53/3, primer pairs were selected such that the ORF could be amplified in 6 overlapping fragments ranging in size from 1 to a? kbp.
Overlaps between the fragments were chosen such i~hat they contain an endonuclease restriction enzyme recognition site suitable for cloning the full-length gen. For the 5' fragment, the downstream oriented primer was chosen to contain the first putative :start codon (ATG) in variant 1 (the one shown in full rLn figure 1e). PCR conditions using the Expand High Fidelity PCR ~>ystem (Roche Molecular Biochemical:) for all of the fragments were as follows. Initial denaturation for 5' at 95°C; 30 cycles of denaturation at 95°C for 45", primer annealing at 55°C for 45" and extention at 72°C for 1' (3' for primer combination WO 99!63080 PCTIEP99/03848 _ - 53 -A+B); followed by an additional incubation for 7' at 72°C and storage at 4°C. PCRs were run on PE
Biosystems 9700 PCR machines.
Primer pairs for cloning Hs-unc-53/3 fragments ~u:~ed # Size PrinterSequence (bp) B GGGGTGGGTCGAC:TTGTCAAGTGG
C-D 847 C ATGGAAGGACCAI'ACCCAACTTGAC
D CTTGTTCCAGCTTTCTGCCTAGATG
F GGTGAGGCAATATCTGGATACTTGG
3.5 G-H 1291 G AGGCAGCCAGGATCCAAGTATCCAG
H TGCGAAGATCTTTTGGGAGGATGGTC
J GGTTATGGGATC'I'AATTAAGTCTCC
L TCACCCTCTAGAGaGGTAGATTCAAG
Primer A contains restriction sites (XhoT and nhel) suitable for final subcloni.ng in an eukaryotic expression vector (pEGFPc3) and in a yeast-two-hybrid vector (pAS2-1), respectively.
PCR products were analyzed by agarose gel electrophoresis and were visualized by ethidium bromide staining. Splice variants as mentioned in figure 1e were observed as multiple bands on agarose gels. Single band PCR products were purified with the Qiaquick PCR purification kit, whereas multiple band PCR products were cut out from gel as individual bands and purified using the Qiaquick gel extraction kit.
PCR products were cloned in pCR2.l according to the suppliers protocol (Invitrogen). For each fragment, multiple clones were picked from selective LB agar plates and grown overnight under antibiotic selection pressure for ~?NA preparation either on the biorot 9600 _ _ 54 _ (Qiagen), or manually on anion exchange columns (Qiagen tip 20 or tip 100). Insert sequences were determined using the Bigdye terminator ready reaction cycle sequencing kit (PE Biosystems). Individual 5~ sequencing reactions for each clone were assembled in single sequent:e contigs using the Sequencher sofi~ware package (GeneC:odes). Sequences were compared to the previously determined consensus sequence using the SeqEd software: package form PE Biosystems. For each fragment a clone was selected containing the correct sequence and the splice variant of interest. For the I-J fragment, a clone was selected that missed tree hart specific 22 amina acid splice variant (figui=a lf). In the K-L fragment clone, a SfiI-SacII linker was cloned in the BamHI site of the pCR2.1 multiple cloning site to facilitate subcloning of the ful)_-length gene into the yeast-two-hybrid vector (pAS2--1) and the eukaryotic expression ve~~tor (pEGFPc3), respectively.
The overall cloning strategy of the full-length gene is visualized in figure 7a. 7a1 illustrates the overlapping PCR fragments and the nomenclature of:
fragments and primer pairs. 7a2 illustrates the assembly of the 3'half of the gene in pCR2.l.
Internal BamHI (I-J fragment) and XhoI (K-L fragment) sites as well as restriction sites from the multiple cloning site of pCR2.1 (as shown in the figure) Mere removed by side-directed mutagenesis (SDM) using the Quickchange Site-Directed mutagenesis kit (stratagene). The Notl-EcoRI G-I~ fragment and the EcoRI-NheI I-Jd22 (d22 indicating that the 22 amino acid splice variant is absent) were directionally cloned in the NotI and Nhel sites of the K-L fragment clone. Multiple clones were picked and verified by DNA sequencing. 7a3 illustrates the assembly of the 5'half. Internal XhoI (C-D fragment) and SfiI anal XhoI (E-F fragment) sites were removed by SDM.
_ 55 _ Inserts were cut out from the vectors by restriction digestion with the appropriate restriction enzymes-(XhoI+SaII; SaII+Narl and NarI+BamHI, respectively) and purified from gel after agarose gel electrophoresi~~. The 3 fragments were ligated together, re-cut with Xhol and BamHI and separated on gel. The band of the expected size was cut out of gel, purified and cloned in front of the 3' half, opened by digestion with Xhol and BamHI (figure 7;44).
Multiple clones were picked and verified by sequencing.
Figure 7a illustrates the modular nature of the cloning project. For all the possible combinations of splice variation within the building block fragments, one representative clone is available. In view o:f functional analysis, building blocks can be exchanged easily by standard technology, either in the pCR2.1 construct or ir.~ the final eukaryotic expression o:r yeast-two-hybrid construct.
Construct o~ Hs-unc-53/3 GFP chimeras The construction of the mammalian expression vectors pGI3303 and pGI3305 is explained in the legends of figure 7a, 7b and 7d. pG13303 can be used to over-express in mammalian cells or animals a fusion protein between. eGFP and 1128 AA ~~-terminal fragment of Hs-unc-53/3 (Fig 7c). pG3305 can be used to overexpress in mammalian cells or animals a fusion protein between. eGFP and the 2363 AA full length Hu-unc-53/3 (fig 7d). The Hs-unc-53/3 cDNA in pGI3303 as well as in pGI3305 contains silent mutations that introduce or remove specific restriction sites in order to be able to easily subclo:ne different types of alternative splice variants in these vectors.
_ - 56 -Genomic DIdTA sequencing (BAC 58aE09) Using the primers AGGACCCTATGCGGAGGTCAAGCCGC and TGGGTTGGCATCATCGCTGTCGTAGC, a PCR specific for Hs~-unc-53/1 was developed. PCR products were radiolabel~~d using the Random Prime DNA labeling kit (Roche Molecular Biochemicals) and hybridized on the human genomic DNA BAC: filters (Research Genetics). Positive signals were obtained for BAC clones 366H21, 483L:14, 471J09 and 585E09. BAC DNA was isolated from E. ~~oli genomic clone 585E09 according to a modified Qiagen plasmid DNA prE:paration procedure. A shotgun library of 1920 clones was constructed at GATC (Konstanz, Germany). BAC DNA was prepared, nebulized and subcloned after end-repairing in the sequence veci~or pTZl9R. At JRF, DNA was prepared on the Biorobot 9600 (Qiagen) from 1440 clones. End sequencing reactions with M13 forward (TGTAAAACGACGGCC.AGT) and reverse (CAGGAAACAGCTA7.'GACC) primer were done on 768 clones.
672 additional clones were sequenced with M13 only. 5 ~,cl DNA was used in 15 f,cl final reaction volume using the BigDye Terminator Ready Reaction sequencing k:it.
Sequencing reactions were run on i:~J Research PTC2t)0 PCR machines. Reaction products 'were run and ana=Lysed on PE ABI 377 DNA sequencers. All sequencing results were imported i.n the Sequencher (GeneCodes) software package. Contaminating vector sequences and trai7~ing sequences of law quality were tri~:nmed. Individuau sequences were assembled in contigs with standard software settings. A great number of contigs were constructed ranging from below 500 by to over 10 kbp.
Singletons are also still present. By looking for strings of known sequence, a cont.ig was found containing the known and reliable 5'end of hUNC53hl and extending this sequence in 5' direction. This;
sequence and its relevant features are described i.n figure lg and its legend.
WO 99/63080 PCT/Ef99J03848 _ _ 57 _ Northern blotting A Human multiple tissue Nort.her. (MTN-1, Clontech) containing in each lane 2 mg of poly A + RNA
from eight different human tissues (heart, brain, placenta, lungs liver, skeletal muscle, kidney, and pancreas) and a MTN-II human multiple tissue Northern, containing in each lane 2 mg of poly A + RNA from spleen, thymus,. prostate, testis, ovary, small intestine, colon and peripheral leukocyte, were hybridized according to the manufacturer's instructions and washed out in 0.lxSSC:0.2o SDS at 55°C. Also from Clontech, a poly A + RNA blot from human cancer cell lines (melanoma 6361, lung carcinoma A549, colorectal adenocarcinoma SW480, Burkitt's lymphoma Raji Leukemia Molt 4, lymphoblastic leukemia K562, HeLa S3 and promyelocytic leukemia HL60) waa tested.
Cancer cell lines RNA blots ;probed with Hs-unc-A set of cancer cell line Northern blots werE~
probed with a C~65 by fragment of :Hs-unc-53/3 ampl_Lfied by using the primers 5'AGGAATTAAA.A.TTAACGGATATTCGG and 5'AAAACTGTCCAAACTATTTTCTTCTACC. HU-unc-53/3 is expressed in Melanoma 6361 and lung carcinoma A54~~, transcripts sizes were detected of >0.5 kb. No expression was detected in promyelocytic leukemia HL-60 HeLa cell S3, chronic myelogenous leukemia K-5Fi2, leukemia MOLT-4, Burkitt's lymphoma Raij and colorectal adenocarcinoma SW480.
Normal huirnan tissue RNA blot, probed with Hs-~unc-A set of normal human tissue Northern blots were probed with a 665 by fragment of Hs-unc-53/3 amplified by using the primers 5' AGGAATTP.AAATTAACGGATATTCGG and 5' AAAACTGTCCAAACTATTTTCTTCTACC. High expression levels were detected in brain, spleen, ovary and spinal cord, lower levels in heart, placenta, te:>tis, stomach, and adrenal gland. Transcripts sizes wE:re >=
9.5 kb.
FISH
Hs-UNC-53/3 is localised to chromosome 12q21..1 Slides preparation:
1.5 Lymphocytes isolated from human blood were cultured in a-minimal essential naedium (MEM) supplemented with loo foetal call: serum and phytohaemagglutinin (PHA) at 37°C; for 68-72 hr. The lymphocyte cultures were treated with BrdU (0.18mg/ml Sigma) to synchronise the cell population. The synchronised cells were washed three times with s.erum-free medium to release the block and recultured a.t 37°C for 6 hr :in a a-MEM with th~znidine (2.5,ug/ml:
Sigma). Cells were harvested and slides were made by using standard procedures including hypotonic treatment fix .and air-dry.
In situ hybridisation and FISH detection:
A cDNA probe was biotinylated with dATP using the BRL BioNick labelling kit (15°C, 1 hr) Heng et al, 1992). The procedure for FISH detection was performed according to Heng et al., 1992 & Heng and Tsui, 1993.
Heng et al..: Proc Natl Acad Sci USA 89: 9509-9513 (1992). Heng .et al. Chromosoma 1.02: 325-332 (1993).
Briefly, slides were baked at 55°C for 1 hour. After RNase treatment, the slides were denatured in 700 _ _ 59 _ formamide in 2xSSC for 2 min. at 70°C followed by dehydrated with ethanol. Probes were denatured at 75°C for 5 min.. in a hybridisation mix consisting of 50% formamide and 10~ dextran sulphate. Probes were loaded on the denatured chromosomal slides. After over night hybridisation, slides were washed and detected as well as amplified. FISH signals and the DAPI banding pattern were recorded separately by taking photographs, and the assignment of the FTS:H
mapping data with chromosomal bands was achieved :by superimposing FISH signals with DAPI banded chromosomes (Heng et al, 1993).
Results Under the condition used the hybridisation efficiency was approximately 67o for this probe (among 100 checked mitotic figures, 67 of them showed signals on ane pair of the chromosomes?. Since the DAPI
banding was used to identify the specific chromosome, the assignment between signal from probe and the :long arm of chromosome 12 was obtained. The detailed position was further determined in the diagram based on the summary from 10 photos.
Radiation Hybrid Mapping Radiation hybrid analysis is a PCR technique and the panels of radiation hybrid DNA are provided ai. a concentration of 25 ng/~cl in TE buffer suitable for these reactions. Typically, 25 ng of DNA is used in a 10 ~1 PCR reaction.
Some of tree radiation hybrid panels are supported by an e-mail server which can assist you in the chromosome localization of markers. A server for the chromosome localization of markers using the Stani=ord G3 and Stanford TNG panels is available at http://www-_ - 60 -shgc.stanford.edu. At the time of catalog publication, the Stanford TNG sez:ver was capable of chromosome localization only on chromosomes 2, 4, 7 and 21. Chromosome localization of markers from the GeneBridge4 panel may be performed by accessing the server at http://www-genome.wi.mit.edu. RH mapping involves the statistical analysis of several to many markers to determine the relative order of the markers with respect to one another. RH mapping can be achieved using statistical programs that will provide the best map along with a measure of the relative likelihood of one order versus another.
This type of analysis has bE:en shown to successfully generate the order of markers on the: RH
map that is significantly more likely than any alternative order. Two statistical programs for RH
mapping can be downloaded from the World Wide Web free of charge. SAMapper was produced at the Stanford.
Human Genome Center and be downloaded at http://www-shgc.Stanford.edu/Mapping/SAMapper/index.html RHMAP
was written by Michael Boehnke at: the University of Michigan and can be downloaded at:
http://www.sph.umich.edu/group/st:atgen/software. A
comprehensive web page regarding radiation hybrid.
mapping, with links to web sites with analysis software and other information, c:an be found at http://linkage.rockefeller.edu/tara/rhmap/
Transfection protocol for cells N$ neuroblastoma lines were seeded in Lab Tek chambered coverglass (Nalgene Nur~c International) and transfected with pEGFP (control), pGI3303 and pGI3305 using lipofectamine (Life Technologies BRL). After 24-48 hours, the chambered coverqlasses were placed on an inverted fluorescence microscope where GFP
fluorescence could be visualized in living cells. The WO 99/G3080 PCT/PP99/0384$
details of this method have been described in PCT/EP96/02311.
Microscopyr and fluorescence staining using phalloidin have been described earlier (EP97/06956).
SEQUENCE LISTING
Seq ID No 1 is a nucleic acid sequence of Hs unc-53/1 and lacking the nucleotides from position 2873 to 3043 shown in Fig. la.
Seq ID No. 2 i:; a nucleic acid sequence of Hs unc-53/1 and lacking thE~ nucleotides from position 3098 to 3121 shown in Figure la.
Seq ID no. 3 is a nucleic acid sequence of Hs-unc-53/1 and lacking the nucleotides from position 3518 to 3526 of the sequence identified in Fig'. la.
Seq ID No. 4 is an amino acid sequence of Hs-unc-53/1 protein and lacking the amino acids from position 958 to 1014 of the sequence identified in Fig. lb Seq ID No. 5 is a amino acid sequence of Hs-unc-53/1 protein and lacking the amino acids from position 1033 to 1040 of the sequence identified in Fig. lb.
Seq ID No. 6 is a amino acid sequence of Hs-unc-.53/1 protein and lacking the amino acids from position 1173 to 1175 of the sequence identified in Fig. lb.
Seq ID No. 7 ~~_s a nucleotide sequence encoding Hs-unc-53/2 and lacking the nucleotides from position 5425 to 5433 of the sequence illustrated in Fig. lc.
WO 99163080 PCT/E'P99I03848 Seq ID No. 8 is a nucleotide sequence encoding Hs-unc-53/2 and lacking the nucleotides from position 5924 to 6024 of the sequence illustrated in Fig. lc.
Seq ID No. 9 is a nucleotide sequence encoding Hs-unc-53/2 and having t:he sequence of variant 1 illustrated in Fig. lc.
Seq ID No. 10 is a nucleotide sequence encoding Hs-unc-53/2 and having the sequence of variant 2 illustrated in Fig. lc.
Seq ID No. ii is a nucleoi:id~e -5equen~:e encodiry~-vs--unc-53/2 and having t:he sequence of variant 3 illustrated in Fig. lc.
Seq ID No. 12 is a nucleotide sequence encoding Hs-unc-53/2 and having t:he sequence of variant 1 illustrated in Fig. lc. and lacking 'the nucleotides from position 5425 to 5433 of the sequence illustrated in Fig. lc.
Seq ID No. I3 is a nucleotide sequence encoding Hs-unc-53/2 and having the sequence of variant 1 illustrated in Fig. lc. and lacking 'the nucleotides from position 5924 tc> 5024 of the sequence Illustrated;
in Fig. lc.
Seq ID No. 14 is a nucleotide sequence encoding Hs-unc-53/2 and having t:he sequence of variant 2 illustrated in Fig. lc. and lacking 'the nucleotides from position 5425 to 5433 of the sequence illustrated.
in Fig. lc.
Seq ID No. I5 is a nucleotide sequence= encoding Hs-unc-53/2 a::d having t:he sequence of variant 2 illustrates in Fig. lc. and lacking 'the nucleotides RECTlEIED SHEET (RULE 97 ) WO 99163080 - ~3 - PCTIEP99I03848 from position 5924 to 6024 of the sequence illustrated in Fig. lc.
Seq ID No. I6 is a nucleotide sequenc;e encoding Hs-une-53/2 and having i:.he sequence of va~.riant 3 illustrated in Fig. lc. and lacking the nucleotides from position 5425 to 5433 of the sequence illustrated in Fig. lc.
Seq ID No. I7 is a nucleotide sequence encoding Hs-unc-53/2 and having the sequence of variant 3 illustrated. in__ Fig. ... lc. - and lack=ng .the. nucleotide s_=
:~rom ~.I~Jltio n 6924 tc: 6024 oz the seq_ence il l~,straye::~
in Fig. lc.
Seq ID No. I8 is an amino acid sequence of Hs-unc-53/2 protein and lacking the amino acids from position 1776 to 1778 of the ~~equence identified in Fig. ld Seq Id No. 29 is an amino acid sequence of variant 1 of Hs-unc-53/2 sequence illustrated in Fig. ld.
Seq Id No. 20 is an amino acid sequence of variant 2 of Hs-unc-53/2 sequence illustrated in Fig. ld.
Seq Id No. 21 is an amino acid sequence of variant 3 of Hs-unc-53/2 sequence illustrated in Fig. Id.
Seq Id No. 22 is an amino acid sequence of variant 1 of Hs-unc-53/2 sequence illustrated in Fig. ld and lacking the amino acids from position :L776 to 1778 of the sequence identified in Fig. Id.
Seq Id No. 23 is an amino acid sequence of variant 2 of Hs-unc-53/2 sequence illustrated in Fig. Id and lacking the amino acids from position :L776 to 1778 of the ssquence identified in Fig. id.
RECTIF1ED~ Si-IEET (RULE 97 ) Seg Id No. 24 is an amino acid sequence of variant 3 of Hs-unc-53/'2 sequence illustrated in Fig. 1d and lacking the amino acids from position 1776 to 1778 of the sequence identified in Fig. Id.
Seq ID No. 25 is a nucleotide sequence encoding Hs-unc-53/3 as illustrated in Figure le.
Seq ID No. 26 is a nucleotide sequence encoding Hs-unc-53/3 as illustrated in Figure le and lacking the nucleotides from position 3795 to 4283 of the sequence identified therein.
Seq ID No. 27 is a nucleotide sequence encoding Hs-unc-53/3 as illustrated in Figure= le and lacking the nucleotides from position 4284 to 4325 of the sequence identified therein.
Seq ID No. 28 is a nucleotide sESquence encoding Hs-unc-53/3 as illustrated in Figure le and lacking the nucleotides from position 3795 to 4325 of the sequence identified therein.
Seq ID No. 29 is a nucleotide sE~quence encoding Hs-unc-53/3 as illustrated in Figure le and lacking the nucleotides from position 5153 to 5173 of the sequence identified.
Seq ID No. 30 is a nucleotide sequence encoding Hs-unc-53/3 as illustrated in Figure le and lacking the nucleotides from position 5343 to 5408 of the sequence identified.
Seq ID No. 31 is a nucleotide sE:quence encoding Hs-unc-53/3 having the sequence of variant 1 illustrated in Fig. le.
WO 99163080 PCT/EiP99/03848 _ - 65 -Seq ID No. 32 is a nucleotide sequence encoding Hs-unc-53/3 having the sequence of variant 1 illustrated in Fig. le and lacking the nucleotides from position 3795 to 4283 o:E the sequence identified therein.
Seq ID No. 33 is a nucleotide sequence encoding :Hs-unc-53/3 having the sequence of variant 1 illustrated in Fig. le and lacking the nucleotides from position 4284 to 4325 of the sequence identified therein.
Seq ID No. 34 is a nucleotide sequence encoding :Hs-unc-53/3 having the sequence of variant 1 illustrated in Fig. le and lacking the nucleotides from position 3795 to 4325 of the sequence identified therein.
Seq ID No. 35 is a nucleotide sequence encoding 1Hs-unc-53/3 having the sequence of variant l illustr~~ted in Fig. le and lacking the nucleotides from position 5153 to 5173 of the sequence identified therein.
Seq ID No. 3~ is a nucleotide sequence encoding 1s-unc-53/3 having the sequence of variant 1 illustrated in Fig. le and lacking the nucleotides from position 5343 to 5408 of the sequence identified therein.
Seq TD No. 37 is an amino acid sequence of Hs-unc~-53/3 protein as identified in the sequence of Fig.
lf.
Seq ID No. 38 is an amino acid sequence of Hs-unc-53/3 protein as identified in the sequence of Fig.. if and lacking the amino acid residues from position 1326 to 1413 of the sequence identified therein.
Seq ID No. 39 is an amino acid sequence of Hs-unc:-53/3 protein a~~ identified in the sequence of Fig,; if and lacking the amino acid residues from position 1414 to 1427 of the sequence identifif~d therein.
Seq ID No. 40 is an amino acid sequence of Hs-unc-53/3 protein as identified in the sequence of Fig'. 3f and lacking the amino acid residues from position. 1703 to 1709 of the sequence identified therein.
Seq ID No. 41 is an amino acid sequence of Hs-un.c-53/3 protein as identified in the sequence of Fig. if and lacking the amino acid residues from position 1768 to 1788 of the sequence identifiE:d therein.
Seq ID No. 42 is an amino acid :sequence of Hs-unc-53 of variant 1 identified in Figure 1f.
Seq ID No. 43 is an amino acid :>equence of Hs-unc-53 of variant 1 identified in Figure if and lacking the amino acid residues from position 1326 to 1413 of the sequence identified therein.
Seq ID No. 44 is an amino acid aequence of Hs-unc-53 of variant 1 identified in Figure if and lacking the amino acid residues from position 1414 to 1427 of the sequence identified therein.
Seq ID No. 45 is an amino acid ~~equence of Hs-unc-53 of variant 1 identified in Figure if and lacking the amino acid residues from position 1703 to 1709 of the sequence identified therein.
Seq ID No. 46 is an amino acid ~~equence of Hs-unc-53 of variant 1 identified in Figure: 1f and lacking the amino acid residues from position. 1768 to 1788 of the sequence identified therein.
20 (*) list not exhaustive Also sequence information available in public.
domain can help refine the positioning of the unc--53 genes, like in the following example. The EST clones 25 AA918601, AI248585, AA115014 and .AA115415 are clearly homologous to Fps-Unc53/2 cDNA. Although, AA115014 (describing the same EST as AA115015) contains an alternative spl.icevariant of the :EIs-Unc53/2 gene in the 3'UTR. A survey with ESTs AA918601, AI248585, 30 AA1I5014 or AA115015 as query in the genemap98 database (release November 1998) revealed that the Hu-_ - 48 -unc53/2 gene is located at chromosome 11 (http://www.ncbi.nlm.nih.gov/genemap98/loc.cgi?ID=2122 4y. The STS which is used for caromosomal localization a.nd which is situated in the 3'UTR of the Hs-Unc53/2 gene is referred to a;s SHGC-33456 (dbSTS
id: 41891, Gen.bank Acc: 628036, csenbank gi: 1396755) (Figure 13y. The STS was localized by analysis on the NIGMS human/rodent somatic cell hybrid panel (dbSTS
id: 41891). The radiation hybrid results are summarized in Figure 13. Together these data imply that diseases or phenotypes connected to SHGC-33156 is due to the Hs-Unc53/2 gene.
EXPERIMENTAL PROCEDURES
Cloning & sequencing of Hs-unc-53/3 Hs-unc53/3 has been cloned starting from a ~~eries of ESTs that were similar but noi~ identical to H~~-unc-53/1 or -/2. The ESTs were:
1. WashU-Merck EST 767735.
Transformed cells carrying the EST 767i'35 sequence were ordered from Research Genetics. Pl.asmid DNA was isolated using standard protocols (Qiager.~
plasmid DNA isolation kity, the sequence of the insert was determined.
2. ATCC cDNA clones 86459"
Transformed cells carrying the cDNA clone 86459 sequence were ordered from ATCC. Plasmid DNA
was isolated using standard protocols (Qiagen plasmid DNA isolation kit), the sequence of the insert was determined.
_ - 49 -3. Genethon cDNA clone c09a03 from the Geneexpress cDNA program.
Transformed cells carrying the cDNA clc>ne c09a03 sequence were ordered from Genethon. Plasmid DNA was isolated using standard protocols (Qiagen plasmid DNA isolation kit), the sequence of the insert was determined..
These ESTs were extended to form one OF;F as follows:
1. 5' extension of EST 76'7735 by RACE (Rapid Amplification of cDNA Ends).
Marathon-Ready cDNAs (Clontech) are pre~made "libraries" of adaptor-ligated double-stranded cDNA
ready for use as templates in RACE experiments. Five ml Marathon-Ready cDNA was used as template in a regular 50 ml RACE. The RACE mi:~ture contained 1 x KlenTaq PCR buffer. 0.2 mM of each dNTP, 1 x advantage KlenTaq polymerase mix (Clontech), 0.15 mM
AP1 adaptor primer and 0.15 mM RACE gene specific primer. The amplification condii~ions were as follows:
94°C for 30 s and 68 °C for 4 min. One-hundred-fold diluted RACE product was used as a template in a nested PCR with AP2 adaptor and gene specific nested PCR primers. Specific nested PCR fragments were cloned into pCR2 (TA cloning kit,, Invitrogen) and. the sequences of the inserts were determined. Gene-specific primer (hh3UNC53 97101702):
5°ACCATTTACACCTGAAGACGATTGAGGTCC;, nested gene-specific primer (hh3UNC53 97101701) 5'CTCCTATTTAA.A.TTAGAGGCTCCCTGGACC Marathon cDNA
library: human placenta, human heart, human chronic myelogenous leukemia, human colorectal adenocarcinoma.
WO 99/63080 PCTIE:P99/03848 2. 3' extension of EST 76')735 by RACE.
Method as described previously. Gene specific primer (hh3UNC53 97102702) 5'CAATCGTCTTCAGGTGTAAATGGTAACGTG;: nested gene specific primer (hh3UNC53 97102703) 5'GAATGTCAAACACAGTGCCACCTCCACC Marathon cDNA library:
human placenta, human heart, human HeLa, human melanoma.
3. 3' extension of cDNA clone c09a03 by RACE.
Method as described previously, gene-specific primer (hh3UNC53 98020401) 5'AGGGAGCACTGAATGGTCCAGACCATCCTC; nested gene-specific primer (hh3UNC53 98020402) 5'GCATCAGAAGACAGCATTCCTCTGAAAGTG Marathon cDNA
library: human placenta, human heart, human HeLa, human melanoma, human colorectal adenocarcinoma, human chronic myelogenous leukemia.
4. 5' e:~tension of cDNA clone 86459 by RACE
(1) .
~ Method as described previously gene-specific primer (hh3UNC53 98020403) 5'TTCAATTTCTATCTCTATGAGTTTTCTTCG; nested gene-specific primer (hh3UNC53 98020404) 5'GCAGCTCTAGAT'.CTGGTGATGAAGAAACTC Marathon cDNA
library: human placenta, human heart, human HeLa, human melanoma. Overlapping sequences were assemlbled in a single contiguous sequence.
5. 5' extension cDNA clone 86459 by RACE (2) .
Method as described previously gene-spet~ific primer (hh3UNC53 98022502) _ _ 51 _ 5'TCAGAATGTGATGAAGGAGGCTTGGTGGAC;w nested gene-specific primer (hh3UNC53 98022501) 5'GGATGCCGGAAGGGATGAATCAGTAAGC Marathon cDNA library:
human placenta, human heart, human HeLa, human melanoma, human colorectal adenocarcinoma, human chronic myelogenous leukemia.
Validating ~rariants at 5' end of the cDNA
sequence In the final 5' RACE experiment, 2 variants have been found whose sequence divergE: upstream from the IYTDWAN protein sequence (position 289 in figure le or position 82 in figure 1f). By using primers ATTTACACTGACTGGGCC.AAC and ATAATCTGGATGATTTCTGCTAGGAGT
on cDNA clones a Hs-unc-53/3 specific PCR product was obtained that was radiolabeled u~>ing the random primed DNA labeling kit (Roche Molecular Biochemicals) and hybridized to human DNA BAC filters (Research Genetics). Both primers are located near the IYTDWAN
box. Four BACs turned out positive (415J11; 464C17, 525C02 and 537B02). DNA sequencing of the region upstream from the IYTDWAN protein sequence directly on these BACs showed that this regic>n was preceded by a putative intronic sequence as evidenced by the multiple stop codons in the reading frame and by the consensus AG intron acceptor sequence. for sequencing purposes, BAC DNA was prepared according to a modified Qiagen plasmid DNA procedure.
A primer pair was designed specifically to amplify the 5'end of the variant shown in full in figure le (primers ACTTGCTGAAACAGAGAGCTCCATG and CTTGCTGTCTTCTTTCTCCTTGGC). PCR with these primers on BAC DNA showed the presence of th.e genomic sequence encoding this variant in 3 out of the 4 BACs (not present in BAC 415J11).
BACs containing the genomic sequence encoding the other 5'end variant of Hs-unc-53/3 as shown as the variant in figure le were identified by hybridiz:i.ng the Research Genetics human DNA GAC filters with primer TGATCTTCTAGCGTGTGACTCACTG, radioactively labeled using gamma-P32-ATP and polynucleotide kinase.
Positive BACs were 404F14, 450K18 and 764L15.
Sequencing directly on the respective BACs in the 3' direction from within the 2 alternative 5' exons and comparison of the genomic DNA sequence with the previously determined cDNA sequence identified the GT
intron donor site. Joining of the genomic sequences from both 5° exons and the IYTDWAN encoding sequence after removal of the predicted intronic sequence restored for both variants the sequence of the 5'' RACE
experiment without affecting the translation of i~he Open Reading E~rame.
Cloning of Hs-unc-53/3 constructs With the aim of cloning the full-length Open Reading Frame of Hs-unc-53/3, primer pairs were selected such that the ORF could be amplified in 6 overlapping fragments ranging in size from 1 to a? kbp.
Overlaps between the fragments were chosen such i~hat they contain an endonuclease restriction enzyme recognition site suitable for cloning the full-length gen. For the 5' fragment, the downstream oriented primer was chosen to contain the first putative :start codon (ATG) in variant 1 (the one shown in full rLn figure 1e). PCR conditions using the Expand High Fidelity PCR ~>ystem (Roche Molecular Biochemical:) for all of the fragments were as follows. Initial denaturation for 5' at 95°C; 30 cycles of denaturation at 95°C for 45", primer annealing at 55°C for 45" and extention at 72°C for 1' (3' for primer combination WO 99!63080 PCTIEP99/03848 _ - 53 -A+B); followed by an additional incubation for 7' at 72°C and storage at 4°C. PCRs were run on PE
Biosystems 9700 PCR machines.
Primer pairs for cloning Hs-unc-53/3 fragments ~u:~ed # Size PrinterSequence (bp) B GGGGTGGGTCGAC:TTGTCAAGTGG
C-D 847 C ATGGAAGGACCAI'ACCCAACTTGAC
D CTTGTTCCAGCTTTCTGCCTAGATG
F GGTGAGGCAATATCTGGATACTTGG
3.5 G-H 1291 G AGGCAGCCAGGATCCAAGTATCCAG
H TGCGAAGATCTTTTGGGAGGATGGTC
J GGTTATGGGATC'I'AATTAAGTCTCC
L TCACCCTCTAGAGaGGTAGATTCAAG
Primer A contains restriction sites (XhoT and nhel) suitable for final subcloni.ng in an eukaryotic expression vector (pEGFPc3) and in a yeast-two-hybrid vector (pAS2-1), respectively.
PCR products were analyzed by agarose gel electrophoresis and were visualized by ethidium bromide staining. Splice variants as mentioned in figure 1e were observed as multiple bands on agarose gels. Single band PCR products were purified with the Qiaquick PCR purification kit, whereas multiple band PCR products were cut out from gel as individual bands and purified using the Qiaquick gel extraction kit.
PCR products were cloned in pCR2.l according to the suppliers protocol (Invitrogen). For each fragment, multiple clones were picked from selective LB agar plates and grown overnight under antibiotic selection pressure for ~?NA preparation either on the biorot 9600 _ _ 54 _ (Qiagen), or manually on anion exchange columns (Qiagen tip 20 or tip 100). Insert sequences were determined using the Bigdye terminator ready reaction cycle sequencing kit (PE Biosystems). Individual 5~ sequencing reactions for each clone were assembled in single sequent:e contigs using the Sequencher sofi~ware package (GeneC:odes). Sequences were compared to the previously determined consensus sequence using the SeqEd software: package form PE Biosystems. For each fragment a clone was selected containing the correct sequence and the splice variant of interest. For the I-J fragment, a clone was selected that missed tree hart specific 22 amina acid splice variant (figui=a lf). In the K-L fragment clone, a SfiI-SacII linker was cloned in the BamHI site of the pCR2.1 multiple cloning site to facilitate subcloning of the ful)_-length gene into the yeast-two-hybrid vector (pAS2--1) and the eukaryotic expression ve~~tor (pEGFPc3), respectively.
The overall cloning strategy of the full-length gene is visualized in figure 7a. 7a1 illustrates the overlapping PCR fragments and the nomenclature of:
fragments and primer pairs. 7a2 illustrates the assembly of the 3'half of the gene in pCR2.l.
Internal BamHI (I-J fragment) and XhoI (K-L fragment) sites as well as restriction sites from the multiple cloning site of pCR2.1 (as shown in the figure) Mere removed by side-directed mutagenesis (SDM) using the Quickchange Site-Directed mutagenesis kit (stratagene). The Notl-EcoRI G-I~ fragment and the EcoRI-NheI I-Jd22 (d22 indicating that the 22 amino acid splice variant is absent) were directionally cloned in the NotI and Nhel sites of the K-L fragment clone. Multiple clones were picked and verified by DNA sequencing. 7a3 illustrates the assembly of the 5'half. Internal XhoI (C-D fragment) and SfiI anal XhoI (E-F fragment) sites were removed by SDM.
_ 55 _ Inserts were cut out from the vectors by restriction digestion with the appropriate restriction enzymes-(XhoI+SaII; SaII+Narl and NarI+BamHI, respectively) and purified from gel after agarose gel electrophoresi~~. The 3 fragments were ligated together, re-cut with Xhol and BamHI and separated on gel. The band of the expected size was cut out of gel, purified and cloned in front of the 3' half, opened by digestion with Xhol and BamHI (figure 7;44).
Multiple clones were picked and verified by sequencing.
Figure 7a illustrates the modular nature of the cloning project. For all the possible combinations of splice variation within the building block fragments, one representative clone is available. In view o:f functional analysis, building blocks can be exchanged easily by standard technology, either in the pCR2.1 construct or ir.~ the final eukaryotic expression o:r yeast-two-hybrid construct.
Construct o~ Hs-unc-53/3 GFP chimeras The construction of the mammalian expression vectors pGI3303 and pGI3305 is explained in the legends of figure 7a, 7b and 7d. pG13303 can be used to over-express in mammalian cells or animals a fusion protein between. eGFP and 1128 AA ~~-terminal fragment of Hs-unc-53/3 (Fig 7c). pG3305 can be used to overexpress in mammalian cells or animals a fusion protein between. eGFP and the 2363 AA full length Hu-unc-53/3 (fig 7d). The Hs-unc-53/3 cDNA in pGI3303 as well as in pGI3305 contains silent mutations that introduce or remove specific restriction sites in order to be able to easily subclo:ne different types of alternative splice variants in these vectors.
_ - 56 -Genomic DIdTA sequencing (BAC 58aE09) Using the primers AGGACCCTATGCGGAGGTCAAGCCGC and TGGGTTGGCATCATCGCTGTCGTAGC, a PCR specific for Hs~-unc-53/1 was developed. PCR products were radiolabel~~d using the Random Prime DNA labeling kit (Roche Molecular Biochemicals) and hybridized on the human genomic DNA BAC: filters (Research Genetics). Positive signals were obtained for BAC clones 366H21, 483L:14, 471J09 and 585E09. BAC DNA was isolated from E. ~~oli genomic clone 585E09 according to a modified Qiagen plasmid DNA prE:paration procedure. A shotgun library of 1920 clones was constructed at GATC (Konstanz, Germany). BAC DNA was prepared, nebulized and subcloned after end-repairing in the sequence veci~or pTZl9R. At JRF, DNA was prepared on the Biorobot 9600 (Qiagen) from 1440 clones. End sequencing reactions with M13 forward (TGTAAAACGACGGCC.AGT) and reverse (CAGGAAACAGCTA7.'GACC) primer were done on 768 clones.
672 additional clones were sequenced with M13 only. 5 ~,cl DNA was used in 15 f,cl final reaction volume using the BigDye Terminator Ready Reaction sequencing k:it.
Sequencing reactions were run on i:~J Research PTC2t)0 PCR machines. Reaction products 'were run and ana=Lysed on PE ABI 377 DNA sequencers. All sequencing results were imported i.n the Sequencher (GeneCodes) software package. Contaminating vector sequences and trai7~ing sequences of law quality were tri~:nmed. Individuau sequences were assembled in contigs with standard software settings. A great number of contigs were constructed ranging from below 500 by to over 10 kbp.
Singletons are also still present. By looking for strings of known sequence, a cont.ig was found containing the known and reliable 5'end of hUNC53hl and extending this sequence in 5' direction. This;
sequence and its relevant features are described i.n figure lg and its legend.
WO 99/63080 PCT/Ef99J03848 _ _ 57 _ Northern blotting A Human multiple tissue Nort.her. (MTN-1, Clontech) containing in each lane 2 mg of poly A + RNA
from eight different human tissues (heart, brain, placenta, lungs liver, skeletal muscle, kidney, and pancreas) and a MTN-II human multiple tissue Northern, containing in each lane 2 mg of poly A + RNA from spleen, thymus,. prostate, testis, ovary, small intestine, colon and peripheral leukocyte, were hybridized according to the manufacturer's instructions and washed out in 0.lxSSC:0.2o SDS at 55°C. Also from Clontech, a poly A + RNA blot from human cancer cell lines (melanoma 6361, lung carcinoma A549, colorectal adenocarcinoma SW480, Burkitt's lymphoma Raji Leukemia Molt 4, lymphoblastic leukemia K562, HeLa S3 and promyelocytic leukemia HL60) waa tested.
Cancer cell lines RNA blots ;probed with Hs-unc-A set of cancer cell line Northern blots werE~
probed with a C~65 by fragment of :Hs-unc-53/3 ampl_Lfied by using the primers 5'AGGAATTAAA.A.TTAACGGATATTCGG and 5'AAAACTGTCCAAACTATTTTCTTCTACC. HU-unc-53/3 is expressed in Melanoma 6361 and lung carcinoma A54~~, transcripts sizes were detected of >0.5 kb. No expression was detected in promyelocytic leukemia HL-60 HeLa cell S3, chronic myelogenous leukemia K-5Fi2, leukemia MOLT-4, Burkitt's lymphoma Raij and colorectal adenocarcinoma SW480.
Normal huirnan tissue RNA blot, probed with Hs-~unc-A set of normal human tissue Northern blots were probed with a 665 by fragment of Hs-unc-53/3 amplified by using the primers 5' AGGAATTP.AAATTAACGGATATTCGG and 5' AAAACTGTCCAAACTATTTTCTTCTACC. High expression levels were detected in brain, spleen, ovary and spinal cord, lower levels in heart, placenta, te:>tis, stomach, and adrenal gland. Transcripts sizes wE:re >=
9.5 kb.
FISH
Hs-UNC-53/3 is localised to chromosome 12q21..1 Slides preparation:
1.5 Lymphocytes isolated from human blood were cultured in a-minimal essential naedium (MEM) supplemented with loo foetal call: serum and phytohaemagglutinin (PHA) at 37°C; for 68-72 hr. The lymphocyte cultures were treated with BrdU (0.18mg/ml Sigma) to synchronise the cell population. The synchronised cells were washed three times with s.erum-free medium to release the block and recultured a.t 37°C for 6 hr :in a a-MEM with th~znidine (2.5,ug/ml:
Sigma). Cells were harvested and slides were made by using standard procedures including hypotonic treatment fix .and air-dry.
In situ hybridisation and FISH detection:
A cDNA probe was biotinylated with dATP using the BRL BioNick labelling kit (15°C, 1 hr) Heng et al, 1992). The procedure for FISH detection was performed according to Heng et al., 1992 & Heng and Tsui, 1993.
Heng et al..: Proc Natl Acad Sci USA 89: 9509-9513 (1992). Heng .et al. Chromosoma 1.02: 325-332 (1993).
Briefly, slides were baked at 55°C for 1 hour. After RNase treatment, the slides were denatured in 700 _ _ 59 _ formamide in 2xSSC for 2 min. at 70°C followed by dehydrated with ethanol. Probes were denatured at 75°C for 5 min.. in a hybridisation mix consisting of 50% formamide and 10~ dextran sulphate. Probes were loaded on the denatured chromosomal slides. After over night hybridisation, slides were washed and detected as well as amplified. FISH signals and the DAPI banding pattern were recorded separately by taking photographs, and the assignment of the FTS:H
mapping data with chromosomal bands was achieved :by superimposing FISH signals with DAPI banded chromosomes (Heng et al, 1993).
Results Under the condition used the hybridisation efficiency was approximately 67o for this probe (among 100 checked mitotic figures, 67 of them showed signals on ane pair of the chromosomes?. Since the DAPI
banding was used to identify the specific chromosome, the assignment between signal from probe and the :long arm of chromosome 12 was obtained. The detailed position was further determined in the diagram based on the summary from 10 photos.
Radiation Hybrid Mapping Radiation hybrid analysis is a PCR technique and the panels of radiation hybrid DNA are provided ai. a concentration of 25 ng/~cl in TE buffer suitable for these reactions. Typically, 25 ng of DNA is used in a 10 ~1 PCR reaction.
Some of tree radiation hybrid panels are supported by an e-mail server which can assist you in the chromosome localization of markers. A server for the chromosome localization of markers using the Stani=ord G3 and Stanford TNG panels is available at http://www-_ - 60 -shgc.stanford.edu. At the time of catalog publication, the Stanford TNG sez:ver was capable of chromosome localization only on chromosomes 2, 4, 7 and 21. Chromosome localization of markers from the GeneBridge4 panel may be performed by accessing the server at http://www-genome.wi.mit.edu. RH mapping involves the statistical analysis of several to many markers to determine the relative order of the markers with respect to one another. RH mapping can be achieved using statistical programs that will provide the best map along with a measure of the relative likelihood of one order versus another.
This type of analysis has bE:en shown to successfully generate the order of markers on the: RH
map that is significantly more likely than any alternative order. Two statistical programs for RH
mapping can be downloaded from the World Wide Web free of charge. SAMapper was produced at the Stanford.
Human Genome Center and be downloaded at http://www-shgc.Stanford.edu/Mapping/SAMapper/index.html RHMAP
was written by Michael Boehnke at: the University of Michigan and can be downloaded at:
http://www.sph.umich.edu/group/st:atgen/software. A
comprehensive web page regarding radiation hybrid.
mapping, with links to web sites with analysis software and other information, c:an be found at http://linkage.rockefeller.edu/tara/rhmap/
Transfection protocol for cells N$ neuroblastoma lines were seeded in Lab Tek chambered coverglass (Nalgene Nur~c International) and transfected with pEGFP (control), pGI3303 and pGI3305 using lipofectamine (Life Technologies BRL). After 24-48 hours, the chambered coverqlasses were placed on an inverted fluorescence microscope where GFP
fluorescence could be visualized in living cells. The WO 99/G3080 PCT/PP99/0384$
details of this method have been described in PCT/EP96/02311.
Microscopyr and fluorescence staining using phalloidin have been described earlier (EP97/06956).
SEQUENCE LISTING
Seq ID No 1 is a nucleic acid sequence of Hs unc-53/1 and lacking the nucleotides from position 2873 to 3043 shown in Fig. la.
Seq ID No. 2 i:; a nucleic acid sequence of Hs unc-53/1 and lacking thE~ nucleotides from position 3098 to 3121 shown in Figure la.
Seq ID no. 3 is a nucleic acid sequence of Hs-unc-53/1 and lacking the nucleotides from position 3518 to 3526 of the sequence identified in Fig'. la.
Seq ID No. 4 is an amino acid sequence of Hs-unc-53/1 protein and lacking the amino acids from position 958 to 1014 of the sequence identified in Fig. lb Seq ID No. 5 is a amino acid sequence of Hs-unc-53/1 protein and lacking the amino acids from position 1033 to 1040 of the sequence identified in Fig. lb.
Seq ID No. 6 is a amino acid sequence of Hs-unc-.53/1 protein and lacking the amino acids from position 1173 to 1175 of the sequence identified in Fig. lb.
Seq ID No. 7 ~~_s a nucleotide sequence encoding Hs-unc-53/2 and lacking the nucleotides from position 5425 to 5433 of the sequence illustrated in Fig. lc.
WO 99163080 PCT/E'P99I03848 Seq ID No. 8 is a nucleotide sequence encoding Hs-unc-53/2 and lacking the nucleotides from position 5924 to 6024 of the sequence illustrated in Fig. lc.
Seq ID No. 9 is a nucleotide sequence encoding Hs-unc-53/2 and having t:he sequence of variant 1 illustrated in Fig. lc.
Seq ID No. 10 is a nucleotide sequence encoding Hs-unc-53/2 and having the sequence of variant 2 illustrated in Fig. lc.
Seq ID No. ii is a nucleoi:id~e -5equen~:e encodiry~-vs--unc-53/2 and having t:he sequence of variant 3 illustrated in Fig. lc.
Seq ID No. 12 is a nucleotide sequence encoding Hs-unc-53/2 and having t:he sequence of variant 1 illustrated in Fig. lc. and lacking 'the nucleotides from position 5425 to 5433 of the sequence illustrated in Fig. lc.
Seq ID No. I3 is a nucleotide sequence encoding Hs-unc-53/2 and having the sequence of variant 1 illustrated in Fig. lc. and lacking 'the nucleotides from position 5924 tc> 5024 of the sequence Illustrated;
in Fig. lc.
Seq ID No. 14 is a nucleotide sequence encoding Hs-unc-53/2 and having t:he sequence of variant 2 illustrated in Fig. lc. and lacking 'the nucleotides from position 5425 to 5433 of the sequence illustrated.
in Fig. lc.
Seq ID No. I5 is a nucleotide sequence= encoding Hs-unc-53/2 a::d having t:he sequence of variant 2 illustrates in Fig. lc. and lacking 'the nucleotides RECTlEIED SHEET (RULE 97 ) WO 99163080 - ~3 - PCTIEP99I03848 from position 5924 to 6024 of the sequence illustrated in Fig. lc.
Seq ID No. I6 is a nucleotide sequenc;e encoding Hs-une-53/2 and having i:.he sequence of va~.riant 3 illustrated in Fig. lc. and lacking the nucleotides from position 5425 to 5433 of the sequence illustrated in Fig. lc.
Seq ID No. I7 is a nucleotide sequence encoding Hs-unc-53/2 and having the sequence of variant 3 illustrated. in__ Fig. ... lc. - and lack=ng .the. nucleotide s_=
:~rom ~.I~Jltio n 6924 tc: 6024 oz the seq_ence il l~,straye::~
in Fig. lc.
Seq ID No. I8 is an amino acid sequence of Hs-unc-53/2 protein and lacking the amino acids from position 1776 to 1778 of the ~~equence identified in Fig. ld Seq Id No. 29 is an amino acid sequence of variant 1 of Hs-unc-53/2 sequence illustrated in Fig. ld.
Seq Id No. 20 is an amino acid sequence of variant 2 of Hs-unc-53/2 sequence illustrated in Fig. ld.
Seq Id No. 21 is an amino acid sequence of variant 3 of Hs-unc-53/2 sequence illustrated in Fig. Id.
Seq Id No. 22 is an amino acid sequence of variant 1 of Hs-unc-53/2 sequence illustrated in Fig. ld and lacking the amino acids from position :L776 to 1778 of the sequence identified in Fig. Id.
Seq Id No. 23 is an amino acid sequence of variant 2 of Hs-unc-53/2 sequence illustrated in Fig. Id and lacking the amino acids from position :L776 to 1778 of the ssquence identified in Fig. id.
RECTIF1ED~ Si-IEET (RULE 97 ) Seg Id No. 24 is an amino acid sequence of variant 3 of Hs-unc-53/'2 sequence illustrated in Fig. 1d and lacking the amino acids from position 1776 to 1778 of the sequence identified in Fig. Id.
Seq ID No. 25 is a nucleotide sequence encoding Hs-unc-53/3 as illustrated in Figure le.
Seq ID No. 26 is a nucleotide sequence encoding Hs-unc-53/3 as illustrated in Figure le and lacking the nucleotides from position 3795 to 4283 of the sequence identified therein.
Seq ID No. 27 is a nucleotide sequence encoding Hs-unc-53/3 as illustrated in Figure= le and lacking the nucleotides from position 4284 to 4325 of the sequence identified therein.
Seq ID No. 28 is a nucleotide sESquence encoding Hs-unc-53/3 as illustrated in Figure le and lacking the nucleotides from position 3795 to 4325 of the sequence identified therein.
Seq ID No. 29 is a nucleotide sE~quence encoding Hs-unc-53/3 as illustrated in Figure le and lacking the nucleotides from position 5153 to 5173 of the sequence identified.
Seq ID No. 30 is a nucleotide sequence encoding Hs-unc-53/3 as illustrated in Figure le and lacking the nucleotides from position 5343 to 5408 of the sequence identified.
Seq ID No. 31 is a nucleotide sE:quence encoding Hs-unc-53/3 having the sequence of variant 1 illustrated in Fig. le.
WO 99163080 PCT/EiP99/03848 _ - 65 -Seq ID No. 32 is a nucleotide sequence encoding Hs-unc-53/3 having the sequence of variant 1 illustrated in Fig. le and lacking the nucleotides from position 3795 to 4283 o:E the sequence identified therein.
Seq ID No. 33 is a nucleotide sequence encoding :Hs-unc-53/3 having the sequence of variant 1 illustrated in Fig. le and lacking the nucleotides from position 4284 to 4325 of the sequence identified therein.
Seq ID No. 34 is a nucleotide sequence encoding :Hs-unc-53/3 having the sequence of variant 1 illustrated in Fig. le and lacking the nucleotides from position 3795 to 4325 of the sequence identified therein.
Seq ID No. 35 is a nucleotide sequence encoding 1Hs-unc-53/3 having the sequence of variant l illustr~~ted in Fig. le and lacking the nucleotides from position 5153 to 5173 of the sequence identified therein.
Seq ID No. 3~ is a nucleotide sequence encoding 1s-unc-53/3 having the sequence of variant 1 illustrated in Fig. le and lacking the nucleotides from position 5343 to 5408 of the sequence identified therein.
Seq TD No. 37 is an amino acid sequence of Hs-unc~-53/3 protein as identified in the sequence of Fig.
lf.
Seq ID No. 38 is an amino acid sequence of Hs-unc-53/3 protein as identified in the sequence of Fig.. if and lacking the amino acid residues from position 1326 to 1413 of the sequence identified therein.
Seq ID No. 39 is an amino acid sequence of Hs-unc:-53/3 protein a~~ identified in the sequence of Fig,; if and lacking the amino acid residues from position 1414 to 1427 of the sequence identifif~d therein.
Seq ID No. 40 is an amino acid sequence of Hs-unc-53/3 protein as identified in the sequence of Fig'. 3f and lacking the amino acid residues from position. 1703 to 1709 of the sequence identified therein.
Seq ID No. 41 is an amino acid sequence of Hs-un.c-53/3 protein as identified in the sequence of Fig. if and lacking the amino acid residues from position 1768 to 1788 of the sequence identifiE:d therein.
Seq ID No. 42 is an amino acid :sequence of Hs-unc-53 of variant 1 identified in Figure 1f.
Seq ID No. 43 is an amino acid :>equence of Hs-unc-53 of variant 1 identified in Figure if and lacking the amino acid residues from position 1326 to 1413 of the sequence identified therein.
Seq ID No. 44 is an amino acid aequence of Hs-unc-53 of variant 1 identified in Figure if and lacking the amino acid residues from position 1414 to 1427 of the sequence identified therein.
Seq ID No. 45 is an amino acid ~~equence of Hs-unc-53 of variant 1 identified in Figure if and lacking the amino acid residues from position 1703 to 1709 of the sequence identified therein.
Seq ID No. 46 is an amino acid ~~equence of Hs-unc-53 of variant 1 identified in Figure: 1f and lacking the amino acid residues from position. 1768 to 1788 of the sequence identified therein.
Claims (97)
1. A vertebrate protein homologue of a UNC-53 protein of C. elegans, which protein comprises an amino acid sequence having one or more of sequence blocks A, B, C, D, E, F, G, or H as illustrated in figure 4 or which differs from said blocks in conservative amino acid changes.
2. A vertebrate protein homologue of UNC-53 protein of C. elegans or a functional equivalent, derivative or bioprecursor therefor having an amino acid sequence encoded by the nucleotide sequence illustrated in figure 1(e) or the sequence of Figure 1 e having nucleotide region from position 1 to 288 replaced with the sequence of variant 1 illustrated in Figure 1e and or which sequences further lack any of the sequences form 3795 to 4283, 4284 to 4325, 5153 to 5173 or 5343 to 5408.
3. A vertebrate protein homologue of UNC-53 protein of C. elegans having an amino acid sequence as illustrated in figure 1(f) or an amino acid sequence which differs from said amino acid sequence illustrated in figure 1(f) by the replacement of amino acids 1 to 81 with the sequence of variant 1 in figure if and /or including deletions from position 1326 to 1413, 1414 to 1427, 1703 to 1709 or 1768 to 1788, or which differs from said sequences in one or more conservative amino acid changes.
4. A cDNA molecule encoding a vertebrate homologue of UNC-53 protein of C. elegans according to any of claims 1 to 3.
5. A cDNA molecule according to claim 4 which cDNA comprises the sequence of nucleotides illustrated in figure 1(e).
6. A nucleic acid molecule capable of hybridising to the cDNA sequences according to claims 4 or 5 under high stringency conditions.
7. A DIVA expression vector which comprises a cDNA molecule as claimed in claim 4 or 5.
8. A vector according to claim 7 which comprises a promoter of C. elegans UNC-53 protein or a vertebrate homologue thereof according to any of claims 1 to 7.
9. A vector according to claim 8 wherein said promoter sequence is derived from a gene encoding a mouse or human homologue of a UNC-53 protein of C.
elegans.
elegans.
10. A vector according to any of claims 7 to 9 which further comprises a sequence encoding a reporter molecule.
11. A vector according to claim 10 wherein said reporter molecule is a fluorophore.
12. A host cell transformed or transfected with the vector of any of claims 7 to 11.
13. A host cell transformed or transfected with the vector of claims 10 or 11.
14. A host cell according to claim 12 or 13 which cell comprises a prokaryotic cell, such as a bacterial cell or a eukaryotic cell such as a fungal, and animal, a plant or an insect cell.
15. A transgenic cell, tissue or organism comprising a transgene capable of expressing a protein according to any of claims 1 to 3.
26. A transgenic cell, tissue or organism according to claim 15 which comprises any of a COS
cell, Hep G2, MCF-7 cell, N4 mousse neuroblastoma cell, a NIH3T~ cell, or colorectal carcinoma or human derived cells.
cell, Hep G2, MCF-7 cell, N4 mousse neuroblastoma cell, a NIH3T~ cell, or colorectal carcinoma or human derived cells.
17. A transgenic cell, tissue or organism according to claim 15 or 16 wherein said transgene comprises a vector according to any of claims 7 to 11.
18. A transgenic cell, tissue or organism according to claim 15 or 17 wherein said transgene comprises a vector according to claim 10 or 11.
19. A transgenic cell, tissue or organism according to any of claims 15 to 17 wherein said organism comprises any of an insect, a fungus, a non-human mammal, a plant or a nematode worm.
20. A method of producing a mutant vertebrate non-human organism which mutation affects cell behaviour or the regulation of cell motility or the shape or the direction of cell migration, which method comprises inducing a mutation in the wild type gene encoding the vertebrate homologue of an UNC-53 C. elegans protein.
21. A vertebrate protein homologue of an UNC-53 protein of C. elegans, according to any of claims 1 to 3 for use as a medicament.
22. Use of a vertebrate protein homologue of an UNC-53 protein of C. elegans, according to any of claims 1 to 3 in the manufacture of a medicament for promoting neuronal regeneration, revascularisation, wound healing or for treatment of chronic neurodegenerative diseases or acute traumatic injuries or fibrotic disease or autoimmune diseases such as rheumatoid arthritis and sclerosis.
23. A pharmaceutical composition comprising a vertebrate homologue of an UNC-53 protein of C.
elegans, according to any of claims 1 to 3 together with a pharmaceutically acceptable carrier, diluent or excipient therefor.
elegans, according to any of claims 1 to 3 together with a pharmaceutically acceptable carrier, diluent or excipient therefor.
24. A nucleic acid or cDNA molecule according to any of claims 4 to 6 or a functional fragment thereof for use as a medicament.
25. Use of nucleic acid or cDNA molecule according to any of claims 4 to 6 in the manufacture of a medicament to promote neuronal regeneration, revascularisation or wound healing, or for treatment of chronic neurodegenerative diseases or acute traumatic injuries or fibrotic disease or autoimmune diseases such as rheumatoid arthritis and sclerosis.
26. A pharmaceutical composition comprising a nucleic acid or cDNA molecule according to any of claims 4 to 6 and a pharmaceutically acceptable carrier, diluent or excipient therefor.
27. A method of determining whether a compound is an inhibitor or enhancer of the regulation of cell behaviour, growth, cell shape or motility or the direction of cell migration, which method comprises contacting said compound with a host cell according to claim 12 or 14 or a transgenic cell as claimed in any of claims 15 to 18 and screening for a phenotypic change in said cell.
28. A method according to claim 27 wherein said phenotypic change to be screened. is a change in cell growth, or shape or a change in cell motility or filopodia outgrowth, ruffling behaviour, cell adhesion, contact inhibition or the length of neurite growth.
29. A method as claimed in. claim 27 wherein said transgenic cell is an N4 neuroblastoma cell and the phenotypic change to the screened is the length of neurite growth.
30. A method as claimed in claim 27 wherein said transgenic cell is an MCF-7 breast carcinoma cell or an NIH3T3 cell and the phenotypic change to be screened is the extent of phagokinesis or contact inhibition.
31. A method of determining whether a compound is an inhibitor or an enhancer of the regulation of cell shape, cell growth or motility or of the direction of cell migration, which method comprises administering said compound to a transgenic organism according to any of claims 15 to 19 or a mutant organism produced according to the method of claim 20 and screening for a phenotypic change in said organism.
32. A compound which is identifiable by the method according to claim 27 as an enhancer of the regulation of cell shape, or growth or motility or the direction of cell migration for use as a medicament.
33. Use of a compound which is identifiable by the method according to claim 27 as an enhancer of the regulation of cell shape, or growth or motility or the direction of cell migration in the preparation of medicament for promoting neuronal regeneration, revascularisation or wound healing or for treatment of chronic neurodegenerative diseases or acute traumatic injuries or fibrotic disease autoimmune diseases such as rheumatoid arthritis or sclerosis.
34. A pharmaceutical composition comprising a compound identified according to the method of any of claims 27 to 31 and a pharmaceutically acceptable carrier, diluent or excipient therefor.
35. A compound which is identifiable by the method according to any one of claims 17 to 31 as an inhibitor of the regulation of cell motility, growth, or shape, or the direction of cell migration, for use as a medicament.
36. Use of a compound according to claim 35 in the manufacture of a medicament for alleviating the spread of disease inducing cells or metastasis or loss of contact inhibition.
37. A pharmaceutical composition comprising the compound as claimed in claim 35, and a pharmaceutically acceptable carrier diluent or excipient therefor.
38. A method of determining whether a compound is an inhibitor or an enhancer of transcription of a gene encoding a vertebrate homologue of UNC-53 protein of C. elegans, according to any of claims 1 to 3 which method comprises the steps of (a) contacting said.
compound with a cell according to claim 13 or 18 and (b) monitoring the level of said reporter molecule and comparing the results obtained from said monitoring step with a control comprising a cell according to claims 13 or 18, which cell has not been contacted with said compound.
compound with a cell according to claim 13 or 18 and (b) monitoring the level of said reporter molecule and comparing the results obtained from said monitoring step with a control comprising a cell according to claims 13 or 18, which cell has not been contacted with said compound.
39. A method as claimed in claim 38 wherein said reporter molecule detected is mRNA or green fluorescent protein.
40. A compound which is identifiable by they method according to claims 38 or 39, as an enhancer of transcription of a gene coding for a vertebrate homologue of an UNC-53 protein of C. elegans according to any of claims 1 to 3 or a functional fragment of said gene, for use as a medicament.
41. Use of a compound which is identifiable by the method of claims 38 or 39, as an enhancer of transcription of a gene coding for a vertebrate homologue of an UNC-53 protein of C. elegans according to any of claims 1 to 3 or a functional fragment of said gene, in the manufacture of a medicament for promoting neuronal regeneration, revascularisation or wound healing, or for treatment of chronic neuro-degenerative diseases or acute traumatic injuries or fibriotic disease or autoimmune diseases such as rheumatoid arthritis or sclerosis.
42. A pharmaceutical composition which comprises the compound of claim 40 and a pharmaceutically acceptable carrier, diluent or excipient therefor.
43. A compound which is identifiable by the method of claims 38 or 29 as an inhibitor of transcription of a gene coding for vertebrate homologue of a UNC-53 protein of C. elegans according to any of claims 1 to 3 or a functional fragment of said gene for use as a medicament.
44. Use of a compound which is identifiable by the method of claims 38 or 39 as an inhibitor of transcription of a gene coding for a vertebrate homologue of an UNC-53 protein of C. elegans or a functional fragment of said gene, in the manufacture of a medicament for alleviating spread of disease inducing cells or metastasis or loss of contact inhibition.
45. A pharmaceutical composition which comprises the compound of claim 43 and a pharmaceutically acceptable carrier, diluent or excipient therefor.
46. A kit for determining whether a compound is an enhancer ar an inhibitor of the regulation of cell motility, growth or shape or the direction of cell migration which kit comprises at least one transgenic cell as claimed in any one of claims 13 to 17 to be contacted with said compound and at least one cell according to claims 1 2to 19 to be used as a control and means for contacting said compound with one of said at lest one transgenic cells.
47. A kit for determining whether a compound is an inhibitor or an enhancer of transcription of a gene coding for a vertebrate homologue of an UNC-53 protein of C. elegans or a functional fragment of said gene which kit comprises at least one cell as claimed in any one of claims 12 to 19 and means for contacting said compound with said cells.
48. A kit for determining whether a compound is an enhancer or an inhibitor of the activity of a vertebrate homologue of an UNC-53 protein of C. elegans or a functional equivalent, derivative, fragment or bioprecursor of said vertebrate homologue protein, which kit comprises at least, one vertebrate mutant non-human organism produced according to the method as claimed in claim 20 or a transgenic organism as claimed in claims 15 to 19 and a wild type of said vertebrate mutant organism.
49. A method identifying vertebrate homologues of an unc-53 gene of C. elegans or a functional fragment thereof, which method comprises hybridizing to a DNA library a suitable oligonucleotide sequence of between 15 to 50 nucleotides of the nucleic acid sequence encoding UNC-53 or a functional equivalent, derivative or bioprecursor thereof, under appropriate conditions of stringency to identify genes having statistically significant homology with the cDNA according to any of claims 4 or 5.
50. A method of identifying a protein which is active in the signal transduction pathway of a cell of which a vertebrate homologue of an UNC-53 protein of C. elegans according to any of claims 1 to 3 is a component, which method comprises:
(a) contacting an extract of said cell with an antibody to the vertebrate homologue of the UNC-53 protein of C. elegans, (b) identifying the antibody/vertebrate homologue complex, and (c) analysing the complex to identify any protein bound to the vertebrate homologue of UNC-53 protein of C. elegans other than the antibody.
(a) contacting an extract of said cell with an antibody to the vertebrate homologue of the UNC-53 protein of C. elegans, (b) identifying the antibody/vertebrate homologue complex, and (c) analysing the complex to identify any protein bound to the vertebrate homologue of UNC-53 protein of C. elegans other than the antibody.
51. A method of identifying a further protein which is active in the signal transduction pathway of a cell of which a vertebrate homologue of an UNC-53 protein according to any of claims 1 to 3 is a component, which method comprises:
(a) forming an antibody to the first identified protein bound to the vertebrate homologue of UNC-53 protein of C. elegans in claim 50, (b) contacting a cell extract with said antibody and identifying the antibody/protein complex, (c) analysing the complex to identify any further protein bound to the first protein other than the antibody, and (d) optionally repeating steps (a) to (c) to identify further proteins in said pathway.
(a) forming an antibody to the first identified protein bound to the vertebrate homologue of UNC-53 protein of C. elegans in claim 50, (b) contacting a cell extract with said antibody and identifying the antibody/protein complex, (c) analysing the complex to identify any further protein bound to the first protein other than the antibody, and (d) optionally repeating steps (a) to (c) to identify further proteins in said pathway.
52. A method of identifying a protein which is active in the signal transduction pathway of a cell of which a vertebrate homologue of an UNC-53 protein of C. elegans according to any of claims 1 to 3 is a component, which method comprises:
(a) contacting an extract of said cell with said vertebrate homologue of an UNC-53 protein of C. elegans, (b) identifying any vertebrate homologue of UNC-53 protein/protein complex formed and (c) analysing the complex to identify any protein bound to the vertebrate homologue of UNC-53 protein other than the same vertebrate homologue of UNC-53 protein.
(a) contacting an extract of said cell with said vertebrate homologue of an UNC-53 protein of C. elegans, (b) identifying any vertebrate homologue of UNC-53 protein/protein complex formed and (c) analysing the complex to identify any protein bound to the vertebrate homologue of UNC-53 protein other than the same vertebrate homologue of UNC-53 protein.
53. A method according to claim 52 which further comprises contacting a cell extract with any protein identified from step (c) not being the same as the vertebrate homologue of UNC-53 protein used and repeating steps (b) and (c) so as to identify any further protein involved in the signal transduction pathway of said cell.
54. A method of identifying a protein involved in the signal transduction pathway of a cell of which a vertebrate homologue of an UNC-53 protein of C.
elegans is a component which method comprises:
(a) providing an appropriate host cell.
having a DNA construct comprising a reporter gene under the control of a promoter regulated by a transcription factor having a DNA binding domain and an activating domain, (b) expressing in said host cell a first hybrid DNA sequence encoding a first fusion of a fragment or all of a DNA sequence according to claims 4 or 5 and either said DNA binding domain or the activating domain of the transcription factor, (c) expressing in the host cell at least one second hybrid DNA sequence encoding a putative binding protein to be investigated together with the DNA binding or activating domain of the transcription factor which is not incorporated in the first fusion, (d) detecting any binding of the protein being investigated with a protein according to any of claims 1 to 3 by detecting for the production of any reporter gene product in said host.
elegans is a component which method comprises:
(a) providing an appropriate host cell.
having a DNA construct comprising a reporter gene under the control of a promoter regulated by a transcription factor having a DNA binding domain and an activating domain, (b) expressing in said host cell a first hybrid DNA sequence encoding a first fusion of a fragment or all of a DNA sequence according to claims 4 or 5 and either said DNA binding domain or the activating domain of the transcription factor, (c) expressing in the host cell at least one second hybrid DNA sequence encoding a putative binding protein to be investigated together with the DNA binding or activating domain of the transcription factor which is not incorporated in the first fusion, (d) detecting any binding of the protein being investigated with a protein according to any of claims 1 to 3 by detecting for the production of any reporter gene product in said host.
55. A protein identified by the method of any one of claims 50 to 54 for use as a medicament.
56. Use of a protein identified by the methods of any one of claims 50 to 54 in the manufacture of a medicament far promoting neuronal regeneration, revascularisation or wound healing, or for treatment of chronic neurodegenerative diseases or acute traumatic injuries or fibrotic disease or autoimmune diseases such as rheumatoid arthritis and sclerosis.
57. A pharmaceutical composition comprising a protein identified by the methods of any one of claims 50 to 54 and a pharmaceutically acceptable carrier, diluent, or excipient therefor.
58. A process for producing a vertebrate homologue of are UNC-53 protein of C. elegans according to any of claims 1 to 3 which process comprises culturing the cells of any of claims 12 to 14 and recovering said vertebrate homologue of UNC-53 protein expressed.
59. A process for producing a vertebrate homologue of an UNC-53 protein of C. elegans according to any of claims 1 to 3 which process comprises culturing an insect cell transfected with a recombinant Baculovirus vector, said vector comprising a DNA insert encoding said vertebrate homologue of UNC-53 protein downstream of the Baculovirus polyhedrin promoter, and recovering the expressed vertebrate homologue of UNC-53 protein.
60. A method of detecting whether a compound is an inhibitor or an enhancer of expression of a vertebrate homologue of an UNC-53 of C. elegans according to any of claims 1 to 3 which method comprises contacting a cell expressing said homologue with said compound and monitoring for a phenotypic change compared to a control cell which has not been contacted with said compound.
61. A method according to claim 60 wherein said cell comprises a cell according to any of claims 12 to 19.
62. A method according to claim 60 wherein said cell has undergone loss of contact inhibition.
63. A method according to any of claims 60 to 62 in which the compound to be tested comprises a nucleic acid.
64. A method according to claim 63 wherein said nucleic acid sequence comprises an antisense DNA or RNA sequence.
65. A method according to claim 64 wherein said mRNA sequence comprises 3' untranslated regions of mRNA encoding for said vertebrate homologue.
66. A method according to any of claims 60 to 62 wherein said compound to be tested comprises a protein having an amino acid sequence potentially suitable for inhibiting function of said vertebrate homologue.
67. A method according to claim 66 wherein said protein comprises a protein identified according to any of the methods of claims 50 to 54.
68. A pharmaceutical composition comprising a compound identified according to any of claims 60 to 67 together with a pharmaceutically acceptable carrier, diluent or excipient therefor.
69. A nucleic acid sequence identified according to the method of any of claims 63 to 65 for use as a medicament.
70. Use of a nucleotide sequence identified according to the method of any one of claims 63 to 65 in the preparation of a medicament for the treatment of loss of contact inhibition or cancer which is mediated by a vertebrate homologue of an UNC-53 protein of C. elegans.
71. Use of a nucleic acid according to claim 69 in the preparation of a medicament for inhibiting expression of a gene coding for a vertebrate homologue of an UNC-53 protein of C. elegans.
72. An assay for detecting expression of a vertebrate homologue of UNC-53 protein of C. elegans according to any of claims 1 to 3 in a vertebrate cell which assay comprises contacting a cell or an extract thereof with an antibody to said vertebrate homologue, which antibody is linked to a reporter molecule, removing any unbound antibody and monitoring for the presence of said reporter molecule.
73. An assay according to claim 72 wherein said reporter molecule is an antibody conjugated with a suitable fluorophore or detectable enzyme.
74. A method for detecting for expression of a gene coding for a vertebrate homologue of an UNC-53 protein of C-elegans according to any of claims 1 to 3 which method comprises contacting a probe specific for a nucleic acid or protein sequence coding for or corresponding to said vertebrate homologue according to any of claims 1 to 3 with a cell extract which probe is linked to a reporter anal analysing for the presence of said reporter.
75. A method according to claim 74 wherein said probe comprises a complementary sequence to a region of mRNA transcribed from said gene encoding said vertebrate homologue of UNC-53 protein.
76. A method according to claim 75 wherein said complimentary sequence is a 3' or 5' untranslated region of said mRNA.
77. A method according to claims 74 or 76 wherein said reporter comprises a radiolabel.
78. A method according to claim 74 wherein said probe comprises an antibody specific for said vertebrate homologue of said UNC-53 protein according to any of claims 1 to 3.
79. A method according to claim 78 wherein said reporter comprises an antibody conjugated with a detectable fluorophore or enzyme.
80. A method of determining whether a compound is an inhibitor or an enhancer of association of a vertebrate homologue according to any of claims 1 to 3 to microtubules or plus end regions thereof, which method comprises:
(a) contacting said compound with a transgenic cell, tissue or organism expressing UNC-53 protein or said vertebrate homologue and which protein is operably linked to a reporter molecule, (b) screening for the localisation of said reporter molecule as compared to a cell according to step (a) which has not been contacted with said compound.
(a) contacting said compound with a transgenic cell, tissue or organism expressing UNC-53 protein or said vertebrate homologue and which protein is operably linked to a reporter molecule, (b) screening for the localisation of said reporter molecule as compared to a cell according to step (a) which has not been contacted with said compound.
81. A compound identifiable by the method according to claim 80.
82. A compound according to claim 81 for use as a medicament.
83. Use of a compound according to claim 81 as an enhancer of association of said vertebrate homologue with microtubules or the plus end region thereof, for use in promoting neuronal regeneration, revascularisation or wound healing, or for treating chronic neurodegenerative diseases or acute traumatic injuries or fibrotic disease or autoimmune diseases such as rheumatoid arthritis or sclerosis.
84. A pharmaceutical composition comprising the compound according to claims 81 or 82 and a pharmaceutically acceptable carrier, diluent or excipient therefor.
85. A kit for determining whether a compound is an inhibitor or an enhancer of association of a vertebrate homologue according to any of claims 1 to 3 with microtubules or the plus end regions thereof, which kit comprises at least one transgenic cell expressing said homologue and a reporter molecule or a cell according to any of claims 12 to 19 and at least one cell of the same cell type for use as a control and means for contacting said compound with one of said at least one transgenic cells.
86. A composition comprising a vertebrate homologue according to any of claims 1 to 3 linked to a compound identified as an inhibitor or enhancer or association of said vertebrate homologue with microtubules or their plus end regions for use in targeting said compound to said microtubule or the plus end region thereof.
87. A composition according to claim 86 which further comprises a cell transformation or transfecting agent.
88. A method of targeting a protein to a cell microtubule or the plus end region thereof, which method comprises introducing into a host cell, tissue or organism a transgene comprising a sequence capable of expressing a vertebrate homologue according to any of claims 1 to 3, which sequence is operably linked to a sequence encoding said protein to be targeted such that a chimeric protein is expressed and which results in targeting said protein to said microtubule or a plus end region thereof.
89. A method of identifying a molecule which covalently modifies a vertebrate homologue of UNC-53 according to any of claims 1 to 3 which method comprises:
a) contacting an extract from a cell expressing said vertebrate homologue with a mixture of enzymes comprising candidate modifying enzymes in the presence of an inhibitor or covalent modification of a protein, b) identifying any covalently modified UNC-53 protein from step a), c) identifying said molecule involved in said modification step.
a) contacting an extract from a cell expressing said vertebrate homologue with a mixture of enzymes comprising candidate modifying enzymes in the presence of an inhibitor or covalent modification of a protein, b) identifying any covalently modified UNC-53 protein from step a), c) identifying said molecule involved in said modification step.
90. A method according to claim 89, wherein said indicator comprises 32p.
91. A method of identifying a compound which alleviates or enhances the toxicity of a vertebrate homologue according to any of claims 1 to 3, which method comprises contacting said compound with a cell, tissue or organism according to claim 18, and monitoring for the presence of said reporter molecule adjacent said microtubules or the plus end regions thereof.
92. A vertebrate homologue of UNC-53 protein of C.elegans or a functional equivalent, derivative or bioprecursor therefor encoded by the nucleotide sequence in Figure 1a and which nucleotide sequence is lacking in any of the nucleotide regions from position 2873 to 3043, 3098 to 3121 or 3518 to 3526.
93. A vertebrate homologue of UNC-53 protein of C.elegans or a functional equivalent, derivative or bioprecursor therefor having an amino acid sequence as illustrated in Figure 1b and lacking in one or more of the regions from residues 958 to 1014, 1033 to 1040 or 1173 to 1175, or which differs from said amino acid sequences in one or more conservative amino acid changes.
94. A vertebrate homologue of UNC-53 protein of C.elegans or a functional equivalent, derivative or bioprecursor therefor encoded by the nucleotide sequence in Figure 1c and which nucleotide sequence has from sequence position 1 to 366 replaced with any of the sequences identified as variants 1 to 3 of Figure 1c and/or which sequences lack the region from position 5624 to 6024.
95. A vertebrate homologue of UNC-53 protein of C.elegans or a functional equivalent, derivative or bioprecursor therefor having an amino acid sequence identified in Figure 1d or the sequences of any of variants 1 to 3 replacing the amino acids from position 1 to 89 of the sequence of Figure 1d and/or which sequence is lacking the amino acid sequence from position 1776 to 1778.
96. Plasmid pG313303 deposited under accession number LMBP 3936.
97. Plasmid pG13305 deposited under accession number LMBF 3937.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GBGB9811962.1A GB9811962D0 (en) | 1998-06-03 | 1998-06-03 | Vertebrate homologue of UNC-53 protein of C.elegans |
| GB9811962.1 | 1998-06-03 | ||
| PCT/EP1999/003848 WO1999063080A1 (en) | 1998-06-03 | 1999-06-02 | HUMAN HOMOLOGUE OF UNC-53 PROTEIN OF $i(C. ELEGANS) |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2330179A1 true CA2330179A1 (en) | 1999-12-09 |
Family
ID=10833159
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002330179A Abandoned CA2330179A1 (en) | 1998-06-03 | 1999-06-02 | Human homologue of unc-53 protein of c. elegans |
Country Status (6)
| Country | Link |
|---|---|
| EP (1) | EP1092019A1 (en) |
| JP (1) | JP2002517194A (en) |
| AU (1) | AU4373599A (en) |
| CA (1) | CA2330179A1 (en) |
| GB (1) | GB9811962D0 (en) |
| WO (1) | WO1999063080A1 (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE19908423A1 (en) * | 1999-02-26 | 2000-08-31 | Deutsches Krebsforsch | Protein involved in the development of the CNS |
| DE10042609A1 (en) * | 2000-08-30 | 2002-03-28 | Deutsches Krebsforsch | Use of T-proteins for the differential characterization and therapy of injuries and tumors of the nervous system |
| CA2930117A1 (en) | 2012-11-25 | 2014-05-30 | The Regents Of The University Of California | Peptides that stimulate subcutaneous adipogenesis |
| US10844102B2 (en) * | 2014-05-28 | 2020-11-24 | The Regents Of The University Of California | Peptides, compositions, and methods for stimulating subcutaneous adipogenesis |
| CN105738607B (en) * | 2014-12-09 | 2018-04-13 | 费好义 | The kit and its application method of a kind of antitumor medicine screening |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| GB9625283D0 (en) * | 1996-12-04 | 1997-01-22 | Janssen Pharmaceutica Nv | Vertebrate homologues of unc-53 protein of c.elegans or functional eqivalents thereof and cdna sequences coding for said homologue |
-
1998
- 1998-06-03 GB GBGB9811962.1A patent/GB9811962D0/en not_active Ceased
-
1999
- 1999-06-02 CA CA002330179A patent/CA2330179A1/en not_active Abandoned
- 1999-06-02 AU AU43735/99A patent/AU4373599A/en not_active Abandoned
- 1999-06-02 WO PCT/EP1999/003848 patent/WO1999063080A1/en not_active Application Discontinuation
- 1999-06-02 EP EP99926511A patent/EP1092019A1/en not_active Withdrawn
- 1999-06-02 JP JP2000552275A patent/JP2002517194A/en not_active Withdrawn
Also Published As
| Publication number | Publication date |
|---|---|
| WO1999063080A1 (en) | 1999-12-09 |
| JP2002517194A (en) | 2002-06-18 |
| GB9811962D0 (en) | 1998-07-29 |
| EP1092019A1 (en) | 2001-04-18 |
| AU4373599A (en) | 1999-12-20 |
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