VERTEBRATE HOMOLOGUES OF UNC-53 PROTEIN OF C. ELEGANS

Abstract: Vertebrate protein homologues of UNC-53 protein of C. elegans and nucleic acid sequences coding for said homologues or functional equivalents thereof are identified. The nucleic acid sequences in an appropriate vector are used to transfect or transform cells, tissues or organisms useful in identifying inhibitors or enhancers of the vertebrate homologue, or further proteins involved in the signal transduction pathway of which said vertebrate homologue is a component. Any of said inhibitors or enhancers identified can be included in a pharmaceutical composition or in the preparation of a medicament for treating conditions such as neurological diseases, acute traumatic injuries and to promote neuronal regeneration and inhibit metastasis or loss of contact inhibition.

Description: VERTEBRATE HOMOLOGUES OF UNC-53 PROTEIN OP C. ELEGANS The present invention relates to vertebrate homologues of UNC-53 protein of C. eleσans and cDNA sequences coding for said homologues or functional equivalents thereof. The invention also relates to processes for identifying compounds which control cell behaviour, compounds identified and pharmaceutical compositions containing them in addition to processes and assays for identifying disease states in which said gene or protein is dysfunctional. The control of cell motility, cell shape and directionality of cell outgrowth of axones or other cell outgrowths is an essential feature in the morphogenesis and function of both unicellular and multicellular organisms. The control of these processes is disturbed in a variety of disease states in which, for example, the Receptor Tyrosine kinase (RTK) signal transduction pathways, or the like, or their downstream intra-cellular pathways (which are shared with other extra-cellular receptors, including cell adhesion molecules like N-CAMS and integrins) are overstimulated . Some cell surface proteins and extra-cellular molecules controlling the directionality and potential of cell migration have been identified, although the processes involved are not generally understood. It is generally considered that a long-range migration of a cell process (also known as a growth cone extension) is a stepwise event, whereby prior to and after each extension there is the formation of a structure at the leading edge of the cell which senses signals in the environment instructing the cell to either stabilise a cell process extending in a preferred direction, or to cause a lamellipodium to extend a process in a given direction. Localised stabilisation of the actin cytoskeleton and association with plus end regions of microtubules is a general cell biological process underlying the choice of directional extension. Microtubule binding directing these processes has not previously been identified. The present inventors have surprisingly found that UNC-53 protein of C. elegans and vertebrate homologues thereof is involved in binding of microtubules and particularly of plus end regions of microtubules. A gene from the free-living nematode Caenorhabditis eleαans designated "unc-53" has been previously identified and cloned (Abstract, International C. eleσans Meeting, June 1-5 1991, Madison, Wisconsin, 58, Bogaert and Goh) . The present inventors previously identified UNC-53 protein as a signal transducer or signal integrator controlling the directionality of cell migration and/or cell shape in C. elegans (WO 96/38555) . Increased UNC-53 protein activity was found to be proportional to cell process extensions in the correct direction of cell migration. The unc-53 gene was found to encode a signal transduction molecule that transduces a signal from an RTK such as, for example, via the adaptor protein SEM- 5/GRB-2, to the machinery controlling directional growth cone extension or stabilisation, in a highly dosage - dependent fashion. Genetic and experimental analysis of C. elegans UNC-53 mutants showed that mutations in the unc-53 gene do not affect the general ability of cells to migrate but rather affect the ability of cells to migrate under specific antero-posterior cues. Reduction of UNC-53 activity leads to loss of direction and reduction of growth cone extension as indicated by the directionality of random extension cycles observed in excretory canal growth cones in UNC-53 mutants. The function of UNC-53 is highly sensitive to its dosage or activity. Reduction of function leads to proportional reduction of migration to the specific signal while increased expression, using transgenic expression of UNC-53 in muscle cells, leads to increased directional migration. The data lead to the conclusion that UNC-53 functions as an integrator of a directional signal in the organism whereby reception of signals leads to growth cone extension in the correct direction. Certain alleles of UNC-53 enhance the sex myoblast migration defect of SEM-5 C. eleσans mutants in a receptor tyrosine kinase signal transduction pathway (Stern et al 1993 mol. Biol. cell, 4, 1175- 1188) . While the genetics suggests that UNC-53 and SEM-5 cooperate to regulate sex myoblast migration, genetic experiments do not permit a conclusion that this is the result of a direct molecular interaction. The inventors previously identified a potential se - 5/GRB-2 binding site and showed in two types of biochemical experiments that UNC-53 physically interacts with SEM-5. The present inventors conclude that UNC-53 encodes a signal transduction molecule that transduces extracellular signals for directional migration via the adapter protein SEM-5/GRB-2 to the machinery controlling directional growth cone extension or stabilization. Several lines of evidence indicate that UNC-53 might act as an adapter linking extracellular signals to the actin cytoskeleton. Firstly, UNC-53 has shown homology to cortical actin binding proteins and that it is capable of binding F-actin in vitro. In addition expression of UNC-53 in mammalian cells leads to changes in the F-actin cytoskeleton. Very low levels of UNC-53 expression increase the number of filopodia and actin microspikes protruding from the cell surface. Cells expressing UNC-53 also exhibit increased neurite extension and increased cell motility. UNC-53 thus also acts as an activator of migration. Considering all available data the following possible mechanisms of action of UNC-53 can be formulated. The choice and activation of directional growth cone extension can be accounted for by local activation of UNC-53 via a SEM-5/GRB2 complex to a receptor (eg receptor tyrosine kinase signal) which reads a localized or directional signal. Changes in growth cone steering are preceded by the formation of a localized actin patch in the area of the growth cone receiving the highest signal (Bentley and O'Connor et al. Curr. Op. NeuroBiol. 1994, vol 4, 43-48). UNC-53 might be directly involved in forming these actin patches through its own actin binding or cross- linking properties. Alternatively activated UNC-53 may (eg via its nucleotide binding domain) transduce a signal to as yet unidentified effectors. For example, activation of the small GTP-binding protein cdc42 or a related protein leads to formation of small actin patches as well as the formation of small filopodia. The unc-53 pathway may be upstream of cdc42 or both signal transducers might share downstream pathways. The present inventors thus decided to investigate if a similar protein was present in higher organisms such as vertebrates. The present inventors describe the identification of a family of genes in vertebrates, and particularly in man and mouse with extensive structural homology to UNC-53. The present inventors have surprisingly found that the nucleotide domains of UNC-53 from C. eleσans and UNC-53 from vertebrates similarly activate motility, establishing functional equivalence. Furthermore these domains are shown to be capable of transforming NIH3T3 cells in vitro. The inventors also found changes in RNA transcripts in transformed cell lines compared to normal human tissues suggesting a role for UNC-53 in cell differentiation, morphogenesis and disease. Furthermore, in vitro assays and transgenic models are also described that identify pharmacological modulators of UNC-53 activity and assays to identify proteins interacting with UNC- 53. According to a first aspect of the present invention, there is provided a vertebrate protein homologue of UNC-53 protein of C. eleσans or a functional equivalent, derivative or bioprecursor thereof, which protein homologue comprises an amino acid sequence having a statistically significant homology to the UNC-53 protein of C. eleσans as illustrated in figure 2. According to the present invention a derivative should be taken to mean mutational derivatives, fusions, internal deletions, splice variants and muteins. There is also provided according to a second aspect of the present invention a vertebrate protein homologue of UNC-53 protein of C. elegans, which protein comprises an amino acid sequence having one or more of sequence homology blocks A, B, C, D or E as illustrated in Figure 9a, or block F in Figure 12a or a sequence having a statistically significant homology therewith. Preferably, said vertebrate homologue is a human protein or a mouse protein. According to a further aspect of the invention there is provided a vertebrate protein homologue of an UNC-53 protein of C. elegans f which protein comprises an amino acid sequence having one or more of sequence blocks A, B, C, D,E or F which differ from those blocks of Figure 9a and Figure 12a to a significant extent only in conservative amino acid changes. In an even further aspect of the invention there is provided a vertebrate protein having an amino acid sequence encoded by the nucleotide sequence from position 1 to position 6013 as illustrated in Figure 9b. There is also provided a vertebrate protein having an amino acid sequence encoded by the nucleotide sequence illustrated in Figure lid, or a functional equivalent derivative, or bioprecursor of said homologue. According to a further aspect of the present invention there is provided a vertebrate protein having an amino acid sequence corresponding to the prosite signatures as illustrated in Figure 28 for each of said homology blocks as defined above. Advantageously the prosite signatures can be used to identify a protein having a statistically significant homology to the UNC-53 protein of C. elegans. (Luethy et al 1994, Protein Science, 3, 139-146). A further aspect of the invention comprises a vertebrate homologue according to the invention comprising an amino acid sequence as shown in figure 9b or lid or an amino acid sequence which differs from the amino acid sequences shown in these figures to a significant extent only in one or more conservative amino acid changes. In a further aspect of the present invention there is also provided a nucleic acid molecule, which is preferably DNA, and which encodes a vertebrate homologue of UNC-53 protein of C. eleσans f 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 figures 9b or lid or an amino acid which differs from the sequences shown in these figures to a significant only in one or more conservative amino acid changes. Preferably the DNA is cDNA, which cDNA comprises at least from position 1 to 6013 of the sequence shown in Figure 9b. Alternatively the cDNA may comprise the sequence illustrated in Figure lid. Also provided by the present invention is a nucleic acid sequence capable of hybridising to the nucleic acid or DNA sequences according to the invention under high strigency conditions, which conditions are well known to those skilled in the art. The cDNA according to the invention may be included in an expression vector which may itself be used to transform or transfect a host cell, which cell may be bacterial or eukaryotic in origin including such as, for example an animal or plant cell a fungal cell or an insect cell. Thus, advantageously, once the cDNA corresponding to the genome of the vertebrate homologue of UNC-53 of C. elegans is synthesised, using for example, reverse transcriptase or the like, a range of cells, tissues or organisms may be transfected following incorporation of the selected cDNA clone into an appropriate expression vector. The expression vector according to the invention may comprise a promoter of C. elegans or one of human mouse or viral origin and optionally a sequence encoding a reporter molecule, such as, for example, green fluorescent protein. The present invention, therefore, also further comprises a transgenic cell, tissue or organism comprising a transgene capable of expressing a vertebrate homologue of UNC-53 protein of C. eleσans or a functional equivalent, fragment derivative or bioprecursor of said homologue. The term "transgene capable of expressing a vertebrate homologue of UNC-53 protein of C. elegans" as used herein means a suitable nucleic acid sequence which leads to the expression of a vertebrate homologue of UNC-53 protein of C. elegans having the same function and/or activity. The transgene may include, for example, genomic nucleic acid isolated from the appropriate vertebrate or synthetic nucleic acid including cDNA. The term "transgenic organism, tissue or cell, as used herein means any suitable organism and/or part of an organism, tissue or cell, that contains exogenous nucleic acid either stably integrated in the genome or in an extrachromosomal state. Preferably the transgenic cell comprises any of, a COS cell, HepG2 cell, MCF-7 or N4 neuroblastoma cell or a NIH3T3 cell or a colorectal or carcinoma cell or a human derived cell such as a fibroblast or the like. The transgenic organism may be an insect, a non-human animal or a plant and preferably C. elegans or a related nematode. Preferably, the transgene comprises the nucleic acid sequence encoding the vertebrate homologue or a functional fragment of said gene according to the invention as described above. The transgene preferably comprises an expression vector according to the invention. The term "functional fragment" as used herein should be taken to mean a fragment of the gene coding for the vertebrate homologue of the UNC-53 protein of C. elegans or a functional equivalent or derivative or bioprecursor of said protein. 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 or cell having a mutation in the wild-type gene coding for the vertebrate homologue of UNC-53 protein, which mutation affects cell behaviour or the regulation of cell motility or the shape or the direction of cell migration or microtubule plus end stability or function and localisation of protein complexes located thereon, which method comprises inducing a mutation in the vertebrate homologue of UNC-53 protein in said organism or cell. These mutant organisms or cells may be used in a screen to identify the effects of compounds on these cell functions. The vertebrate homologue of UNC-53 protein of C. eleσans or the cDNA or genomic DNA encoding it or a functional equivalent, derivative, fragment or bioprecursor of said homologue, may advantageously be used as a medicament, or in the preparation of a medicament to promote neuronal regeneration, revascularisation or wound healing or the treatment of chronic neurodegenerative disorders or acute traumatic injuries or fibrotic disease or physiological events requiring the polarity of cells or epithelia. The present inventors have also found that the vertebrate homologue of UNC-53 protein plays a role in a transformed state of cells. Accordingly, the vertebrate homologue, dominant positive or negative mutants thereof, or inhibitors thereof may advantageously be used to induce or alleviate contact inhibition in a cell or in preventing cancer development. Typically, the above medical conditions may be treated in mammals and more preferably humans by either a homologue of UNC-53 protein or alternatively by a nucleic acid coding for such a protein. Alternatively an antisense oligonucleotide to said UNC-53 homologue may be used to prevent it's expression. Examples of other nucleic acid sequences which may be used include 3 ' untranslated regions of mRNA which could be used to prevent transcription of the genomic sequence encoding for the vertebrate homologue of UNC-53 protein. The vertebrate homologue of UNC-53 protein or a functional equivalent, fragment or bioprecursor of said protein may be incorporated into a pharmaceutically acceptable composition together with a suitable carrier, diluent or excipient therefor. The pharmaceutical composition may advantageously comprise, additionally or alternatively, the nucleic acid sequence according to the invention as defined above. The present invention also provides for a method of determining whether a compound is an inhibitor or enhancer of the regulation of cell behaviour, growth, transformation, cell shape or motility or the direction of cell migration or microtubule plus end stability or function and localisation of protein complexes thereon which method comprises contacting said compound with a transgenic cell according to the invention and screening for a phenotypic change in said cell. Preferably the method can determine whether the compound comprises an inhibitor or an enhancer of the signal transduction pathway of said transgenic cell of which pathway said vertebrate homologue of UNC-53 protein, or a functional equivalent, derivative, fragment or bioprecursor of said vertebrate homologue is a component or whether said compound is an inhibitor or an enhancer of a parallel or redundant signal transduction pathway in said cell. The present invention also provides a method to determine that the protein in said signal transduction pathway is a vertebrate homologue of UNC- 53 protein of C. elegans or a functional equivalent, fragment, derivative or bioprecursor of said vertebrate homologue. 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 or changes in filipodia outgrowth or alternatively changes in ruffling behaviour or cell adhesion or any change in microtubule cytoskeleton or any change in localisation of proteins on plus end regions of microtubules or any change in cell death such as in apoptosis. In an alternative embodiment of the method of the invention, the transgenic cell may comprise an MCF-7 breast cancer cell. Typically in such an embodiment the phenotypic change to be screened comprises the extent of phagokinesis or filipodia formation. In an alternative embodiment of this aspect of the invention, the transgenic cell may comprise an NIH3T3 cell. Typically in such an embodiment the phenotypic change to be screened comprises loss of contact inhibition of foci formation. The method according to the invention, may also utilise a mutant cell or mutant organism according to the invention as described above, where the mutant cell is capable of growing in tissue culture or in vivo and either of which cell or organism has a mutation in the wild-type unc-53 gene. In accordance with the present invention, a "phenotypic change", may be any phenotype resulting from changes at any suitable point in the life cycle of the cell, tissue or organism defined above, which change can be attributed to the expression of the transgene such as for example, growth, viability, morphology, behaviour, movement, cell migration or cell process or growth cone extension of cells and includes changes in body, shape, locomotion, chemotaxis, contact inhibition, mating behaviour or the like. The phenotypic change may preferably be monitored directly by visual inspection of the cell as a whole or particularly by monitoring the F-actin cytoskeleton microtubule network and plus end stability of microtubules or proteins thereon or alternatively by for example measuring indicators of viability including endogenous or transgenically introduced histochemical markers or other reporter genes, such as for example β-galactosidase or green fluorescent protein. A compound which is identifiable by the method according to the invention as described above, as an enhancer of the processes identified above such as the regulation of cell shape or motility or the direction of cell migration may be used as a medicament, or alternatively in the preparation of a medicament, for promoting neuronal regeneration, revascularisation or wound healing, or for treatment of chronic neuro- degenerative diseases or acute traumatic injuries or fibrotic disease. Examples of promoting neuronal regeneration include, for example, peripheral nerve regeneration after trauma and spinal cord trauma. Where a compound is identified in accordance with the method described above as being an inhibitor of the regulation of cell shape etc. , the compound may be used as a medicament, or in the preparation of a medicament, for substantially alleviating spread of disease inducing cells, such as in spread of cancer, or the like in metastasis or in alleviating loss of contact inhibition. Advantageously, any of the compounds which may have been identified as an inhibitor or an enhancer in accordance with the method as described above, may also be included in a pharmaceutical composition comprising the respective compound and a pharmaceutically acceptable carrier, diluent or excipient therefor. The particular mechanism of action of a compound identified as either an inhibitor or an enhancer of the cell motility shape, growth or direction of cell migration or microtubule association or to the plus end region thereof is not limiting. Preferably the compound acts as an inhibitor or enhancer of a signal transduction pathway. The compound may also act on a parallel pathway or directly on the vertebrate homologue of UNC-53 protein of C. elegans. For example, the method of action of the compound may include direct interaction with the vertebrate homologue of UNC-53 protein, interaction with processes for regulating phosphorylation or dephosphorylation of the vertebrate homologue of UNC- 53 or with processes regulating activity of an unc-53 gene or with processes for post-transcriptional or post-translational modification or the like. Preferably the compound is identified by the method according to the invention as an inhibitor or an enhancer, by utilising differences of phenotype of the cell, tissue or organism, which are visible to the eye. Alternatively indicators of viability including endogenous or transgenically introduced histochemical markers or a reporter gene may be used. According to a further aspect of the invention there is also provided a transgenic cell or tissue culture which has been constructed to comprise a promoter sequence of a gene coding for a vertebrate homologue of UNC-53 of C. elegans or a functional equivalent, derivative fragment, or bioprecursor of said homologue operably linked to a nucleic acid sequence encoding a reporter molecule. Preferably, the reporter sequence encoding the reporter molecule which comprises a detectable protein, for example one which may be monitored by eye inspection such as antibiotic resistance, β-galactosidase or a molecule detectable by spectrophotometric, spectrofluorometric, luminescent or radioactive assays. The present invention also provides a method of determining whether a compound is an inhibitor or an enhancer of transcription of a gene coding for a vertebrate homologue of UNC-53 protein in C. elegans, or a functional equivalent, derivative fragment or bioprecursor of said homologue, which method comprises the steps of: (a) contacting said compound with a transgenic cell according to the invention as described above, (b) monitoring the level of said reporter molecule and comparing results obtained from this monitoring step with a control comprising a transgenic cell having the promoter sequence of a gene coding for a vertebrate homologue of UNC-53 protein, or a functional fragment of said homologue and the reporter molecule, in the absence of the compound. In one embodiment of the method according to this aspect of the invention the reporter molecule may comprise messenger RNA. A compound identified as an enhancer of transcription of the gene coding for the vertebrate homologue of UNC-53 protein of C. elegans or a functional equivalent, derivative or bioprecursor of said homologue may also be used as a medicament, or in the preparation of a medicament, for promoting neuronal regeneration, revascularisation or wound healing, or for treatment of chronic neuro- degenerative diseases or acute traumatic injuries or fibrotic disease. Furthermore, such compounds may be included in a pharmaceutical composition including a pharmaceutically acceptable carrier, diluent or excipient therefor. Any compounds identified as inhibitors of transcription may, advantageously, be used in alleviating the spread of disease inducing cells such as cancers or metastasis or loss of contact inhibition. The present invention also provides a kit for determining whether a compound is an enhancer or an inhibitor of the regulation of cell growth, transformation, cell motility or shape or the direction of cell migration which kit comprises at least one transgenic or mutant cell or transgenic or mutant non-human organism according to the invention as described above and a plurality of wild-type cells or one organism of the same type, or a cell line or tissue culture and means for contacting said compound with said cell or organism. Also provided by the present invention is a kit for determining whether a compound is an inhibitor or an enhancer of transcription of a gene coding for a vertebrate homologue of UNC-53 protein of C. elegans or a functional equivalent, derivative or fragment thereof, which kit comprises at least one transgenic cell or cells according to the invention and means for contacting said compounds with said cells. For the purposes of the present invention, the term "gene coding for a vertebrate homologue of UNC-53 or a functional fragment of said homologue" includes the nucleic acid sequence shown in Figures 9b or lid or a fragment thereof, including the differentially spliced isoforms and transcriptional starts of the nucleic acid sequence and which sequence encodes a vertebrate homologue of UNC-53 protein or a functional equivalent, derivative, fragment or bioprecursor of the protein. The present invention also provides methods of identifying genes of vertebrates or fragments of said genes, which encode proteins which are active in the signal transduction pathway of which the vertebrate homologue of UNC-53 is a component. A preferred method comprises hybridizing to an appropriate cDNA library a nucleotide sequence, as defined herein, or a fragment thereof under appropriate conditions of stringency in order to identify genes having statistically significant homology with the cDNA clones of any one of the cDNA sequences according to the invention described above. Furthermore, there is also provided by the present invention a method of identifying a protein which is active in the signal transduction pathway of a cell of which a vertebrate homologue of UNC-53 protein of C. elegans or a functional equivalent, fragment or bioprecursor of said vertebrate homologue is a component. According to this aspect of the invention, the method comprises; (a) contacting an extract of said cell with an antibody to the vertebrate homologue of UNC-53 protein or a functional equivalent, fragment or bioprecursor of said protein, (b) identifying the antibody/vertebrate homologue of UNC-53 complex, and (c) analysing the complex to identify any protein bound to the vertebrate homologue of UNC-53 protein other than the antibody. The vertebrate homologue of UNC-53 protein, therefore may bind regions of other proteins involved in the signal transduction pathway. It is also possible to sequentially identify a whole range of proteins involved in the signal transduction pathway. Antibodies to the vertebrate homologue of UNC-53 protein may be produced according to known techniques as would be known to those skilled in the art. For example, polyclonal antibodies may be prepared by inoculating a host animal, such as a mouse, with a protein or epitope of a protein according to the invention and recovering immune serum. This aspect of the invention further comprises a method of identifying a further protein or proteins which are active in the signal transduction pathway of a cell of which UNC-53 is a component which method comprises: (a) forming an antibody to the first identified protein bound to the vertebrate homologue of UNC-53 protein in the method as described above, (b) contacting a cell extract with the antibody, (c) identifying the antibody/protein complex, (d) analysing the complex to identify any further protein bound to the first protein other than the antibody, and (e) optionally repeating steps (a) to (d) to identify further proteins in the pathway. According to this aspect of the present invention, the antibody starts the process by binding to the vertebrate homologue of UNC-53 protein or a functional equivalent thereof in the signal transduction pathway. Any other proteins found complexed to the bound antibody or UNC-53 protein can then be used to identify further interacting proteins involved in the pathway. It may also be possible to identify proteins involved in the signal transduction pathway of a cell of which the vertebrate homologue of UNC-53 or a functional equivalent derivative or bioprecursor thereof is a component by using a vertebrate homologue of UNC-53 protein of C. elegans. According to this aspect of the invention the method comprises: (a) contacting an extract of the cell with the vertebrate homologue of UNC-53 protein of C. eleσans or a functional equivalent, fragment or bioprecursor of said homologue, (b) identifying the vertebrate homologue of UNC-53 protein/protein complex formed and (c) analysing the complex to identify any protein bound to the vertebrate homologue of UNC-53 protein other than the same vertebrate homologue of UNC-53 protein This method can also advantageously be used to identify further proteins in a signal transduction pathway of a cell by contacting an extract of the cell used as described above, with any protein identified from step (c) above not being a vertebrate homologue of UNC-53 protein and repeating steps (b) and (c) . Other methods which may be used for identifying proteins in a signal transduction pathway of a cell may comprise for example a western blot overlay method which method is well known to those skilled in the art. Cell extracts are run on gels to separate out protein and subsequently blotted onto a nylon membrane. These membranes may then be incubated, for example in a medium containing a vertebrate homologue of UNC-53 having a label attached thereto such as a biotin or radiolabel and any protein conjugates visualised with for example a streptavidin or alkaline phosphatase conjugated antibody. The present invention also advantageously provides a process for the preparation of binding antibodies which recognise proteins or fragments thereof involved in the rate and direction of cell migration or the control of cell growth or shape, for the above methods. The monoclonal antibody for binding to the appropriate vertebrate homologue of UNC-53 (or its functional equivalent) may be prepared by known techniques as described by Kohler R. and Milstein C. , (1975) Nature 256, 495 to 497. Another method which may be used to identify proteins involved in the signal transduction pathway of a cell of which a vertebrate homologue of an UNC-53 protein of C. eleσans or a functional equivalent or derivative or bioprescursor is a component involves investigating protein-protein interactions using the two-hybrid vector method. This method is well known to those skilled in the art and which was first developed in yeast by Chien et al (1991) . This technique is based on functional reconstitution in vivo of a transcription factor which activates a reporter gene. More particularly the technique comprises providing an appropriate host cell with a DNA construct comprising a reporter gene under the control of a promoter regulated by a transcription factor having a DNA binding domain and an activating domain, expressing in the host cell a first hybrid DNA sequence encoding a first fusion of a fragment or all of a nucleic acid sequence according to the invention and either said DNA binding domain or said activating domain of the transcription factor, expressing in the host at least one second hybrid DNA sequence, such as a library or the like, encoding putative binding proteins to be investigated together with the DNA binding or activating domain of the transcription factor which is not incorporated in the first fusion; detecting any binding of the proteins to be investigated with a protein according to the invention by detecting for the presence of any reporter gene product in the host cell; optionally isolating second hybrid DNA sequences encoding the binding protein. An example of such a technique utilises the GAL4 protein in yeast. GAL4 is a transcriptional activator of galactose metabolism in yeast and has a separate domain for binding to activators upstream of the galactose metabolising genes as well as a protein binding domain. Nucleotide vectors may be constructed, one of which comprises the nucleotide residues encoding the DNA binding domain of GAL4. These binding domain residues may be fused to a known protein encoding sequence, such as for example a sequence coding for the vertebrate homologue of UNC-53. The other vector comprises the residues encoding the protein binding domain of GAL4. These residues are fused to residues encoding a test protein, preferably from the signal transduction pathway of the vertebrate in question. Any interaction between the vertebrate homologue of UNC-53 protein and the protein to be tested leads to transcriptional activation of a reporter molecule in a GAL-4 transcription deficient yeast cell into which the vectors have been transformed. Preferably, a reporter molecule such as β-galactosidase is activated upon restoration of transcription of the yeast galactose metabolism genes. This method enables any interactions between proteins involved in the signal transduction pathway or a parallel or redundant pathway to be investigated. Any proteins identified in the signal transduction pathway of the cell, which may be for example a mammalian cell, may also be included in a pharmaceutical composition together with a pharmaceutically acceptable carrier, diluent or excipient therefor. The present invention also provides a process for producing a vertebrate homologue of an UNC-53 protein of C. elegans or a functional equivalent, fragment, or derivative of the protein, which process comprises culturing the cells transformed or transfected with a cDNA expression vector having any of the cDNA sequences according to the invention as described above, and recovering the expressed vertebrate homologue of UNC-53 protein. The cell may advantageously be a bacterial, animal, insect or plant cell. A particularly preferred process for producing a vertebrate homologue of UNC-53 protein or a functional equivalent, derivative or fragment of said homologue comprises using insect cells. Accordingly, the invention provides a process for producing a vertebrate homologue of UNC-53 protein of C. elegans or a functional equivalent, fragment, derivative or bioprecursor of the UNC-53 protein, which process comprises culturing an insect cell transfected with a recombinant Baculovirus vector, said vector comprising a nucleotide vector encoding the vertebrate homologue of UNC-53 protein or a functional equivalent, fragment or bioprecursor thereof downstream of the Baculovirus polyhedrin promoter and recovering the expressed protein. Advantageously, this method produces large amounts of protein for recovery. The insect cell may be from for example Spodoptera fruαiperda or Drosophila Melanogester. In accordance with the present invention, a defined nucleic acid sequence includes not only the identical nucleic acid but also any minor base variations from the natural nucleic acid sequence including in particular, substitutions in bases which result in a synonymous codon (a different codon specifying the same amino acid) , due to the degenerate code in conservative amino acid substitution. The term "nucleic acid sequence" also includes the complimentary sequence to any single stranded sequence given which includes the definition above regarding base variations. Furthermore, a defined protein, polypeptide or amino acid sequence according to the invention, includes not only the identical amino acid sequence but also minor amino acid variations from the natural amino acid sequence including conservative amino acid replacements (a replacement by an amino acid that is related in its side chains) . Also included are amino acid sequences which vary from the natural amino acid but result in a polypeptide which is immunologically identical or similar to the polypeptide encoded by the naturally occurring sequence. Such polypeptides may be encoded by a corresponding nucleic acid sequence. A further aspect of the invention provides a nucleic acid sequence of at least 15 nucleotides of a nucleic acid according to the invention and preferably from 15 to 50 nucleotides. These sequences may, advantageously be used as probes or primers to initiate replication or the like. Such nucleic acid sequences may be produced according to techniques well known in the art, such as by recombinant or synthetic means. They may also be used in diagnostic kits or the like for detecting for the presence of a nucleic acid according to the invention. These tests generally comprise contacting the probe with a sample under hybridising conditions and detecting for the presence of any duplex formation between the probe and any nucleic acid in the sample. Nucleic acid sequences according to the invention may also be produced using recombinant or synthetic means such as described in Sambrook et al ( Molecular Cloning: A Laboratory Manual, 1989) .Advantageously, human allelic variants or polymorphisms of the DNA according to the invention may be identified by, for example, probing DNA libraries from a range of individuals for example from different populations. Furthermore, nucleic acids and probes according to the invention may be used to sequence genomic DNA from patients using techniques well known in the art, such as the Sanger Dideoxy chain termination method, which may advantageously ascertain any predisposition of a patient to certain proliferative disorders. A method of detecting whether a compound is an inhibitor or an enhancer of expression of a vertebrate homologue of UNC-53 of C. eleσans, or a functional equivalent, derivative or fragment of said vertebrate homologue is also provided which method comprises contacting a cell expressing said homologue with said compound and monitoring for a phenotypic change compared to a control cell which has not been contacted with said compound. Preferably the cell is a transgenic cell as described above. Alternatively the cell may have undergone loss of contact inhibition. The present method also provides for determining whether said compound is an inhibitor of expression of said vertebrate homologue. In one embodiment the compound to be tested comprises a nucleic acid. Preferably said nucleic acid sequence comprises an antisense DNA sequence or a mRNA sequence. Preferably said mRNA sequence comprises 3' untranslated regions of mRNA encoding for said vertebrate homologue. Alternatively , the compound to be tested may be a protein. Preferably, said protein comprises a protein having an amino acid sequence potentially suitable for inhibiting function of said vertebrate homologue and preferably comprises a protein identified by the methods as described herein. The present invention also provides a pharmaceutical composition comprising a compound, for example an antisense nucleic acid identified according to the above described method together with a pharmaceutically acceptable carrier, diluent or excipient therefor. A nucleic acid sequence or protein identified according to this aspect of the invention may be used as a mediciament, or in the preparation of a medicament, for treating loss of contact inhibition or cancer which is mediated by a vertebrate homologue of UNC-53 protein or a functional equivalent, fragment, derivative or bioprecursor of said homologue. Further provided by the invention is a nucleic acid as defined above for use in preparation of a medicament for inhibiting expression of a gene coding for a vertebrate homologue of UNC-53 protein of C. elegans or a functional equivalent, derivative, fragment or bioprecursor of said homologue. According to a further aspect of the invention there is provided a plasmid pCB201 deposited under LMBP Accession No. LMBP 3594 and a MCF-7 and a NIH/3T3 cell line transfected with plasmid pCB201 deposited under LMBP Accession Nos. LMBP 1601 CB and LMBP 1603 CB respectively. Further provided by the invention is phage lambda 3b coding for Hu-UNC-53/1 and deposited under Accession No. LMBP 1604CB (or 3595) . Also provided are plasmids pLMl deposited under Accession No. LMBP 3762, pLM4 (LMBP 3763), pEGFP72 (LMBP 3764) and pCBSOl (LMBP 3765) . Further provided is a Bac clone comprising a fragment of hu-unc-53/2 gene (LMBP 3773) and a worm strain comprising a chimeric C. elegans human unc53 gene deposited under LMBP Accession No. LMBP-1663CB. Further provided by the invention is an assay for detecting expression of a vertebrate homologue of UNC-53 protein of C. elegans in a vertebrate cell which assay comprises contacting a cell or an extract thereof with an antibody to said vertebrate homologue, or a functional equivalent, derivative or bioprecursor thereof, which antibody is fused to a reporter molecule, removing any unbound antibody and monitoring for the presence of said reporter molecule. Preferably the reporter molecule is an antibody conjugated to for example a flurophore such as fluorescein or alternatively to an enzyme such as strepavidin. There is also provided a method for detecting for expression of a gene coding for a vertebrate homologue of UNC-53 protein or a functional equivalent, derivative, fragment or bioprecursor thereof, which method comprises contacting a probe specific for a nucleic acid or protein sequence coding for or corresponding to said vertebrate homologue or a functional equivalent, fragment or bioprecursor thereof with a cell extract, which probe is linked to a reporter and analysing for the presence of said reporter. Preferably the probe is a complementary sequence to a region of mRNA transcribed from said gene encoding said vertebrate homologue of UNC-53 protein or a functional equivalent, derivative or bioprecursor therefor. Preferably the complimentary sequence is a 3 • or 5' untranslated region of said mRNA. Preferably said reporter may be a dig label, a fluorophore, a hapten or a radiolabel. Alternatively said probe comprises an antibody specific for said vertebrate homologue of said UNC-53 protein or a functional equivalent, derivative, fragment or bioprecursor therefor. Preferably the reporter is an antibody conjugated to for example a fluorophore such as fluorscein or alternatively an enzyme such as streptavidin. As described above UNC-53 protein of C. elegans has been found to localise to microtubule and particularly to microtubule (+) ends. Therefore, there is provided by a further aspect of the present invention a method of determining whether a compound is an inhibitor or an enhancer of association of UNC- 53 or a vertebrate homologue thereof according to any of claims to 1 to 9 to microtubules or plus end regions thereof, which method comprises (a) contacting said compound with a transgenic cell, tissue or organism expressing UNC-53 protein or said vertebrate homologue and which protein is operably linked to a reporter molecule (b) screening for the localisation of said reporter molecule as compared to a cell according to step (a) which has not been contacted with said compound. A compound identifiable by the above method also forms part of the present invention. Such a compound identified as an inhibitor of localisation or association of UNC-53 or said vertebrate homologue with microtubules or the plus end region thereof may be used in alleviating the spread of disease inducing cells or metastasis or loss of contact inhibition. Further a compound identified as an enhancer of association of UNC-53 or said vertebrate homologue with microtubules or the plus end region thereof may be used in for example promoting neuronal regeneration, revascularisation or wound healing, or for treating chronic neurodegenerative diseases or acute traumatic injuries or fibrotic disease. These compounds may then be included in a pharmaceutical composition, together with a pharmaceutically acceptable carrier, diluent or excipient therefor. Also provided by the present invention is a kit for determining whether a compound is an inhibitor or an enhancer of association of UNC-53 or a vertebrate homologue thereof according to the invention with microtubules or the plus end regions thereof, which kit comprises at least one transgenic cell expressing UNC-53 and a reporter molecule or a host or transgenic cell according to the invention and at least one cell of the same cell type for use as a control and means for contacting said compound with one of said at least one transgenic cells. Compounds identified as inhibitors or enhancers or microtubule association described above may advantageously be included in a composition and linked to unc-53 protein of C. elegans or a vertebrate homologue thereof according to the invention to target the compounds to the microtubules or the plus end regions thereof. Such a composition may also comprise, for example, a suitable transfecting or transformation agent. According to a further aspect of the invention there is provided a method of targeting a protein to a cell microtubule or the plus end region thereof, which method comprises introducing into a host cell, tissue or organism a transgene comprising a sequence capable of expressing UNC-53 or a vertebrate homologue thereof according to the invention, which sequence is operably linked to a sequence encoding said protein to be targeted such that a chimeric protein is expressed and which results in targeting said protein to said microtubule or a plus end region thereof. An even further aspect of the invention comprises a method of identifying a molecule which covalently modifies UNC- 53 or a vertebrate homologue thereof according to the invention, which method comprises a) contacting either an extract from a cell or cells expressing UNC-53 or said vertebrate homologue or a mixture of enzymes comprising canditate UNC-53 modifying enzymes in the presence of an indicator of covalent modification of a protein, b) identifying any covalently modified UNC-53 protein from step a) and c) identifying said molecule involved in said modification step. Such an indicator may be ' " p. Further provided by the invention is a method of identifying a compound which alleviates or enhances the toxicity of UNC-53 or a vertebrate homologue thereof according to the invention, or which alleviates or enhances apoptosis. The method of the former comprises contacting said compound with a transgenic cell, tissue or organism according to the invention and monitoring for the presence of said reporter molecule adjacent said microtubules or the plus end regions thereof. In the case of apoptosis the method comprises monitoring the effect of the compound on cell death. The invention may be more clearly understood from the following examples which are only exemplary, with reference to the accompanying drawings wherein Figure 1 illustrates the sequence of plasmid pTB72 which codes for the full length UNC-53 protein in C. elegans f deposited under LMBP Accession No. 3486. Figure 2 illustrates the full-length UNC-53 protein from C. eleαans. Figure 3 is a Tblastn search of the EST division of Genbank with the ORF of the longest known Ce-UNC-53 cDNA. tb3-M5, reveals two EST ' s with homology to a predicted coiled-coil region in Ce-UNC-53. Figure 4 illustrates a search of the Genbank databases with part of the nucleotide binding domain of Ce-UNC-53. It does not identify statistically significant proteins except for the C. eleσans cosmid containing Ce-unc-53. Figure 5 illustrates a three frame translation of EST gb:R41071. Regions of homology with Ce-Unc-53 in two different frames are underlined. The spacing between the blocks of homology is of similar size to that in Ce-UNC-53. Subsequent re-cloning and re-sequencing of this region in man identified multiple sequencing errors gb:R41071, and identified an ORF which is more homologous to and co-linear with Ce-UNC-53 (see alignment in fig. 12) . Figure 6 is a BLASTN search of the EST division of Genbank with Hu-unc-53/1 cDNA cosmid 3b. Figure 7 is a TBLASTN search of the Genbank sequence database with the 961 amino acid ORF of cDNA 3b of hu-UNC-53/l : hu-UNC-53/1 forms a unique pair with Ce-UNC-53 (cosmid F45E10) compared to the rest of the database. Figure 8 is a diagram illustrating the length and overlap and tissue source of the different cDNA clones of the 3' end of Hu-UNC-53/1 isolated in this work. Figure 8a. is a diagram illustrating the further sequence of the Hu-UNC-53/1 and overlap of constructs to obtain the further sequence. Figure 8b is a diagram illustrating the 3' end of Hu-UNC53/1 and the EST clones present in the database. Figure 9a is an annotated sequence listing of clone 3b of hu-UNC-53/1 including the EcoRI polylinker GAATTC. The predicted Open Reading Frame of Hu-UNC- 53/1 is listed below the sequence. Blocks A B C D and E which are similiar to Ce-UNC-53/1, a region which is different between Hu-UNC-53/1 and Hu-UNC-53/2 and the 3 ' untranslated leader sequences are marked with arrows and labelled. Figure 9b is an annotated sequence listing of Hu-UNC-53/1 available at this moment. The predicted Open Reading Frames of Hu-UNC-53/1, pLMl , pLM3 , pLM4 , pCB251, pLM5 and pCB201, the homology blocks A,B,C,D and E, the position of a region which is different between Hu-UNC-53/1 and Hu-UNC-53/2, the position of phhl4-3, pCB212, pCB210-14, phh3b, phhlδ, the position of the reverse primers HU53rvl, HU53rv2, HU53rv3 and HU53rv4, the position of peptides B72628 (=28/1), B72627, B72626 and B72625 are listed below the sequence. Figure 10 is an annotated sequence listing of the insert of clone gbAA049124 (EST479167) of mu-UNC-53/1. The open reading frame and 3 ' untranslated sequence is marked with an arrow. Figure 11a is an annotated sequence listing of the insert of clone gbH09036 (EST46037) of Hu-UNC- 53 / 2 . Figure lib is a novel DNA sequence of HU-UNC-53/2 extended by RT-PCR. This DNA sequence is not present in EST-46037 and extends the ORF beyond position 1109 of Figure 11a to an ORF from position 18 to 1793. Figure lie summarises how the 3' and 5' extensions of hu-unc-53/2 were made. Figure lid compiles the sequence of hu-unc-53/2. The boxed sequences are the primer sequences used for the respective extension steps described in the experimental methods section. Figure lie illustrates the sequences of the extensions summarised in figure lie. Figure llf illustrates the sequence information illustrating four alternative Start sites observed for hu-unc-53/2. Figure 12. is an illustration of a Tblastn search of the EST division of Genbank with 680aa starting at the C-teminus of the alpha actinin domain of hu-unc-53/2. Figure 12a. is an illustration of an amino acid alignment of the available sequence of C. elegans unc-53 and hu-unc-53/1 and hu-unc-53/2. 12b. is an illustration of similarity plots for Ce-unc-53 and hu-unc-53/1 (top) and for hu-unc-53/1 and hu-unc-53/2. Figure 13 is an annotated sequence listing of expression vector pCB201 containing homology block E from Hu-UNC-53/1 cloned in a pcDNA3.1-HIS expression vector. The HIS and T7-tags, PCR primer used to modify hu-UNC-53/1 and ORF are marked. Figure 14 is a diagram showing the alignment of the homologous regions of hu-UNC-53/1 and mu-UNC-53/1. Figure 15 is an annotated sequence listing of expression vector pCDU3 containing part of Ce-UNC-53/1 cloned in expression vector pcDNA3.1. The upper ORF starts in the vector polylinker. The lower ORF starts at the first Methionine and is part of Ce-UNC-53/1. Figure 16 is an annotated sequence listing of expression vector pCDU4 containing part of Ce-UNC-53/1 cloned in expression vector pcDNA3.1. The upper ORF starts in the vector polylinker. The lower ORF starts at the first Methionine and is part of Ce-UNC-53/1. Figure 17 is an annotated sequence listing of expression vector pCDU2 containing part of Ce-UNC-53/1 cloned in expression vector pcDNA3.1. The upper ORF starts in the vector polylinker. The lower ORF starts at the first Methionine and is part of Ce-UNC-53/1. Figure 18 illustrates MCF-7 cells transfected with pCB201 (upper) compared to mock transfected MCF-7 cells (phase contrast image) . The control cells are spread out on the tissue culture plastic and exhibiting few filopodia outgrowths. The transfected cells appear smaller because they are slightly rounded up and have multiple filopodia outgrowths per cell (arrowheads) . Figure 19 is a phase contrast image of MCF-7 cells, transfected with pcDNA3.1 (19a), pCDU4 (19b), pCDU3 (19c), pCDU2 (19d) and pTB72 (19e). Figure 20 is an F-actin pattern (visualized with TRITC-Phalloidin) of MCF-7 cells transfected with pcDNA3.LacZ (top panel) and with pCB201 (middle and lower panel) . Figure 21 is an F-actin pattern Phalloidin (visualised with TRITC-Phalloidin) of MCF-7 cells transfected with pCDNA3.1 (21a), pCDU4 (21b), pCDU3(21c), pCDU2 (21d) and pTB72 (21e) . Figure 22 is a phase contrast image of N4 neuroblastoma cells transfected with pcDNA3.1 (22a), pCDU4 (22b), pCDU3 (22c), pCDU2 (22d) and pTB72 (22e) . Figure 23 is an F-actin pattern Phalloidin (visualised with TRITC-Phalloidin) of N4 neuroblastoma cells transfected with pcDNA3.1 (23a), pCDU4 (23b), pCDU3 (23c) , pCDU2 (23d) and pTB72 (23e) . Figure 24 illustrates phase contrast images of small (top) , medium (middle) and large foci (bottom) induced in a monolayer of NIH3T3 cells by transfection with pCB201. Figure 25(c) illustrates human metaphase chromosomes probed with a probe lp34 and figures 25a and 25b indicating the chromosomal location of hu-UNC- 53/1 in lq31. Essentially the same techniques were used to assign the gene hu-unc-53/2 to chromosome locus llpl5 (25d and e) as illustrated in micrograph 25f. The ideograms 25a and 25d are from the International System for Human Cytogenic Nomenclature 1985. The ideograms 25b and 25e in which the relative band positions and arm ratios were derived from actual chromosome measurements is from Cytogenet Cell Genet 65:206-219 (1994). Figure 26 is an expression pattern of HU-Unc53/l and HU-Unc532 in normal human tissues and cancer cell lines. Figure 27 is a sequence map of Plasmid pNP3. Figure 28 is an examplary list of prosite signatures which can be used to define and identify vertebrate homologues of UNC-53. Figure 29 is a annotated sequence map of plasmid pEGFPsac. The GFP-C. elegans unc53sac fusion protein, and the C. elegans unc53 sac fragment are indicated. Figure 30 is a sequence map of plasmid pEGFP72. The GFP-C. elegans unc53 fusion protein and the C. elegans unc53 fragment are indicated. Figure 31 is an annotated sequence map of plasmid pEGFPsma. The GFP-C. elegans unc53sma fusion protein, and the C.e.unc53 sma fragment are indicated. Figure 32 is an annotated sequence map of plasmid pEGFPecl. The GFP-C. elegans unc53ecl fusion protein, and the C. elegans unc53 eel fragment are indicated. Figure 33 is an annotated sequence map of plasmid pEGFPxba. The GFP-C. elegans unc53xba fusion protein, and the C. elegans unc53 xba fragment are indicated. Figure 34 is an annotated sequence map of plasmid pLM4. Open reading frames of the hul-unc53/l and GFP are indicated. Figure 35 is a sequence map of plasmid pNP8. Figure 36 is an illustration of microtubule association of C. elegans Unc53, shown in HepG2 cells, transiently transfected with pTB72, expressing C. elegans Unc53. panel A:microtubule staining of HepG2 cells, using YL1/2 panel B:C. elegans Unc53 staining, using rab4. Figure 37 is an illustration of microtubule plus- end association in human cell lines transiently transfected with pTB72, expressing C.e.Unc53. C. elegans Unc53 was stained with mab-16-48. Panel C: COS cells showing microtubule association panel B: MCF7 cells showing microtubule plus-end association panel A: HepG2 cells showing microtubule plus-end association. Figure 38 is an illustration of microtubule association in N4 cells transiently transfected with pEGFP72, expressing the GFP-C. elegans Unc53 fusion protein. GFP fluorescence was observed in living cells. Panel A: microtubule association of the GFP- C. elegans unc53 fusion protein panel B: microtubule plus-end association of the GFP-C. elegans unc53 fusion protein. Figure 39 is an illustration of microtubule association in N4 cells transiently transfected with pEGFP72, expressing the GFP-C. elegans Unc-53 fusion protein. Microtubules were stained with YL1/2 after paraformaldehyde fixation. Panel A: Microtubule association of the GFP-C. elegans unc53 fusion protein. Panel B: tubuline staining. Panel C: panel A plus panel B: co-localisation of the GFP-C. elegans unc-53 fusion protein and Tubuline can be seen as yellow. Figure 40 is an illustration of microtubule association in N4 cells, transiently transfected with pEGFPsma, expressing the GFP-C. elegans unc53sma fusion protein. Panel A: Microtubule association of the GFP- C. elegans unc53sma fusion product. Panel B: Centriole association of GFP-C. elegans unc53sma fusion product when expressed at low levels. Figure 41 is an illustration of microtubule association in N4 cells, transiently transfected with pEGFPeel, expressing the GFP-C. elegans unc53ecl fusion protein. Panel A: Microtubule association of the GFP- C. elegans unc53ecl fusion product. Panel B: Centriole association of GFP-C. elegans unc53ecl fusion product when expressed at low levels. Figure 42 (a) /Figure 42(b) are illustrations of fluorescence of GFP in N4 cells transiently transfected with pEFPxba and pEFGPsac respectively. Figure 43 is an illustration of microtubule association of in N4 cells transiently transfected with pLM4 expressing GFP-Hu-UNC53/1 fusion protein. Panel A: microtubule association of GFP-HU-UNC53/1 fusion protein. Panel B: microtubule plus-end association of GFP-Hu-UNC53/1 fusion protein. Panel C: microtubule association of GFP-Hu-UNC53/1 in dividing cells (end of division) . Figure 44 is an illustration of the sequence of Plasmid pNP9. Figure 45 is an illustration of immuno fluorescence in melanoma G361 cells stained with sera 28.1. Panel A: Microtubule plus-end association of Hu-UNC53/1. Panel B: microtubule plus-end association of hul-Unc53 in growth cone extensions. Figure 46 is an illustration of GFP fluorescence and immunofluorescence in N4 cells transiently transfected with pLM4 , and stained with sera 28.1. Panel A: Fluorescence of GFP-Hu-UNC53/1 fusion protein. Panel B: Immunofluorescence of serum 28.1. Figure 47 is an overview of the microtubule (+) end, the microtubule and f-actin cytoskeleton binding properties of different constructs Figure 50 is an illustration of rescue of lateral ALN neurons in mutant unc-53. Dorsal view of the ALN neurones axones visualise in GFP fluorescence with the transgene pA/GFP in the posterior of an adult, (c) cellular body. a) wild type, anterior axon (aa) migrates in a straight line along the body until reaching the head, on the dorsal sublateral cord, posterior axon (ap) migrates into the tail; b) unc-53 (nl52) , anterior axons are the shorter, stop ahead of the vulva region and form numerous collateral branches towards the dorsal cord; c) unc-53 (nl52) ,pA/unc-53 anterior axons no longer form branches and migrate in a straight line into the head, as in the wild type at a) . scale bar 10 μm. Figure 51a : is an illustration of chimeric fusion between C. elegans and human 1 homologue of the unc-53 gene. The region of the putative nucleotide binding domain (NTP) is replaced in the C. elegans cDNA by the same region of the human homologue 1 of unc-53 (HI) . The cDNA is under the promotor region A (pA) of unc-53, which raise expression in the ALN lateral neurons. Figure 51b : is an illustration of the chimeric minigene nematode/human pA/unc-53-Hl partially rescue the defect in the longitudinal migration of the lateral neurons ALN and PLN. The four strains compared are : wt; unc-53 (nl52) ; unc53 (nl52) ,pA/unc-53; unc-53 (nl52) ,pA/unc-53-Hl. The observed phenotypes are put in three classes : , the axon is straight, unbranched, and migrates until the head; , the axon is straight, unbranched, and stops in the vulva region; mutant=η , the axon is short, never joints the vulva region and made a lot of collateral branches. Numbers are in percentage. The number of observed axons are noted in the last column. The chimeric fusion between the C. elegans gene and human homolog (unc-53-Hl) partially rescues the mutant phenotype. The chimeric gene was maded by replacing the putative nucleotide binding region (NTP)of the nematode cDNA by the same region of the human homolog 1 (HI) . Figure 52 is an illustration of the sequence for plasmid pLM5. Figure 53 is an illustration of the sequence for plasmid pLM6. Figure 54 is an illustration of the sequence for plasmid pLMl . Figure 55 is a sequence map of plasmid pCB251. Figure 56 is a sequence map of plasmid pNPlO. Figure 57 is a sequence map of plasmid pCB501. Figure 58 is a sequence map of plasmid pTB115. Figure 59 is a sequence map of plasmid pPD95.75. Figure 60 is a sequence map of clone X16. Figure 61 is a sequence map of plasmid pLM3 DEPOSITED MATERIALS Deposit Date Ace. Nr pCB201 plasmid DNA in E. coli 3 December 1996 LMBP 3594 Lambda clone 3B encoding hu-unc-53/1 3 December 1996 LMBP 3595 MCF-7 clone z4 (mock) 3 December 1996 LMBP 1600CB MCF-7 clone (pCB201) 3 December 1996 LMBP 1601CB NIH-3T3 mock 3 December 1996 LMBP 1602CB NIH-3T3 pCB201 3 December 1996 LMBP 1603CB pLM1 13 November 1997 LMBP 3762 pLM4 13 November 1997 LMBP 3763 PEGFP72 13 November 1997 LMBP 3764 pCB501 13 November 1997 LMBP 3765 BAC clone comprising fragment of hu- 15 November 1997 LMBP 3773 unc53/2 gene Worm strain with chimeric 15 November 1997 LMBP-1663CB C.elegans/human unc-53 gene The above plasmids and cell lines were deposited at the Belgian Coordinated Collections of Microorganisms (BCCM) at laboratorium voor moleculaire biologie - plasmidencollective (LMBP) B9000, GENT, Belgium, in accordance with the provisions of the Budapest Treaty of 28 April 1977. The present invention will now be described with reference to the following examples which are not limiting. Identification of a human ho oloαue of the UNC-53 protein of C. eleσans. Extensive searches with the ce-UNC-53 sequence (Figures 1 and 2) against the public domain databases (EST, Genbank, EMBL, Swissprot and PIR) revealed no statistically significant homologies (a smallest sum probability (ssp) of 10 e - 8 is generally accepted to be significant at amino acid level) . Two ESTs gbH09036 (ssp = 1.1 e - 5) a Homo sapiens cDNA clone and gbAA049124 (ssp=8.6-5) a mouse cDNA clone showed homology to a "coiled coil" region a common motif in the contributing to protein secondary structure, (figure 3) All other candidate scores were are at background level (ssp >0.21). Careful examination of weak candidate ESTs identified EST gb:R41071 from Homo sapiens, which had obtained a low score of 53 and a non-significant probability score of 0.33 (Fig. 4). The inventors surprisingly discovered potentially significant homology with the Ce-UNC-53 nucleotide binding domain, provided multiple frameshifts and sequence errors were hypothesized. The inventors amplified, cloned and sequenced part of gb:R41071 from human heart and human lung cDNA and from human genomic DNA and discovered that clone gb:R41071 had up to ten 10 different mistakes in the region checked. 5 extra nucleotides were scattered along its sequence and two nucleotide substitutions were identified, and gb:R41071 lacked three nucleotides present in our clone (Fig. 5) . The novel sequence obtained was two nucleotides shorter and showed the two UNC-53-homologous regions in frame. The genomic fragment obtained is larger (700 bp total length) than the corresponding cDNA clones indicating the presence of an interverting sequence of around 500 bp in nucleotide 162 of this fragment. The amplified cDNA fragment which was cloned to vector PCRII (Intvitrogen) and named pCR23l and was used as a probe to screen cDNA libraries. The conceptual translation of the clones we obtained by PCR were screened using blast and tblastn against all known protein and DNA sequences in the database. The only clone which came up with statistically significant similarity was Ce-UNC-53 (Fig.6). This human clone and Ce-UNC-53 thus form a unique homologous pair compared to the rest of the known sequences, indicating the statistical relevance and novelty of our discovery. We designate this human gene as hu-UNC-53/1. Human heart and a human colorectal adenocarcinoma cDNA libraries were probed with pCR231 probe to identify longer cDNA clones. The clones overlap giving a linear sequence of 3706 bp (Fig 8 and 26) . This sequence shows an 959 amino acid open reading frame from the beginning of the clone. The absence of a 5 ' untranslated region suggests that the mRNA will extend 5'. Sequence alignment searches of the public domain databases with the DNA sequence of hu-UNC-53/1 and its' conceptual translation identified a series of ESTs most of which correspond to the 5' UTR region. (Figures 7 and 8) . Surprisingly, hu-UNC-53/1 identified also the cDNA clones gbH09036 and gbAA049124 homologous to the predicted coiled coil region in Ce-UNC-53 hu-UNC-53/1, and furthermore identified a third weakly homologous EST gbR21023. The inserts of gbH09036, gbAA049124 and gbR21023 were obtained from the Merck consortium and sequenced. gbAA049124 is >95% identical to Hu-UNC-53/1 over 604 available amino acids (fig. 10) and is the mouse orthologue of Hu-UNC-53/1. The insert in gbH09036 is clearly homologous to hu-UNC-53/1 but derived from a different locus. We therefore name the gene identified by gbAA049124 Mu-UNC-53/1 and the gene identified by gbH09036 Hu-UNC-53/2. (Figure 11). 5 domains of high similarity mark the unc-53 gene family Ce-UNC-53 and the here-identified vertebrate homologues form a unique novel protein family, that is distant from the remainder of the proteins in the public domain. Alignment of the predicted open reading frames shows that Hu-UNC-53/1 and Hu-UNC-53/2 are equidistant from Ce-UNC-53. The highest homology is found in the carboxyterminal amino acids of Ce-UNC- 53 region. The presence of a conserved GXXGKS/T box suggests a nucleotide binding function. However, this domain as a whole does not belong to a class of known nucleotide binding proteins. The similarity amongst the presently known sequence of the UNC-53 family of proteins is highest in 5 blocks over most of the available sequence (959 amino-acids) and a firther block identified in Figure 12a. These blocks can be assigned signature sequences as displayed in figure 28 or can be assigned weight matrices based on the alignment between the different family members. By using truncated constructs of Ce- unc-53, the functional relevance of these domains has been addressed. HU-UNC53/1 and Hu-UNC-53/2 are complex transcription units. 1. A cancer cell line RNA blots probed with HU- Unc53/1. A Northern blot of poly-A+RNA from several cancer cell lines (Melanoma G361, Lung Cancer A549, Colorectal Adenocarcinoma SW480, Burkitt Lymphoma DRajii, Leukemia Molt4, Lymphoblastic Leukemia K562, HeLa S3 and Promyelocytic Leukemia HL60) was probed using the whole insert of pHH3b. No or weak expression was detected in the Burkitt Lympho a DRajii, the Leukemia Molt4 and the Promyelocytic Leukemia HL60 cell lines. Five different transcripts are detected in the remaining cancer cell lines: transcripts 1 and 2 are larger than 9.5kb, transcripts 3 and 4 are 6 to 7 kb and the fifth transcript is around 6 kb. Transcripts 1 and 2 are present in all experssing cell lines. Transcripts 3 and 4 are restricted to Melanoma G361, Lung Cancer A549 and Colorectal Adenocarcinoma SW480 and are the predominant transcripts in Melanoma G361 and Colorectal Adenocarcinoma SW480. Transcript 5 is restricted to Lymphoblastic Leukemia K562 and HeLa S3 and is predominant in HeLa S3. 2. Cancer cell lines RNA blots probed with HU- UNC-53/2. A similar set of cancer cell line Northern blots were probed with a 652bp fragment of EST46037 amplified by using the primers 5'- aggagatgaagctgacagatatcc and 5 * -aaacaccagtgagtcc. HU- UNC-53/2 is expressed in Melanoma G361, Colorectal Adenocarcinoma SW480, Lymphoblastic Leukemia K562 and HeLa S3. No expression was detected in Lung Cancer A549, Burkitt Lymphoma DRajii, Leukemia Molt4 and promyelocytic leukemia HL60. Interestingly only 2 transcript sizes were detected of around 7 kb expressed in Lymphoblastic Leukemia K562 and HeLa S3 and a transcript of >9.5 kb in Melanoma G361 and Colorectal Adenocarcinoma SW480. 3. Normal Human tissue probed with HU-Unc53/l. A Northern blot of poly-A+RNA from normal human tissue was probed using the whole insert of phage HH3b. Expression levels are low in all tissues with the highest level in heart and placenta, several fold lower levels in brain and testis, even lower levels in skeletal muscle, pancreas, thymus, colon, small intestine, ovary and prostate. Expression in peripheral blood leukocyte, lung, liver, kidney, spleenis barely detectable. 4. Normal Human tissue probed with Hu-unc53/2. A similar set of blots were probed with a 652bp fragment of EST46037 amplified by using the primers 5 • -aggagatgaagctgacagatatcc and 5'- aaacaccagtgagtcc. Expression levels are low in all tissues with the highest level in kidney, lower levels in heart, placenta, lung, skeletal muscle and pancreas. Expression is barely detectable in brain and liver. The hu-UNC53/l and hu-UNC-53/2 homologues are clearly highly regulated genes, showing a strong tissue specificity and, probably, additional mechanisms of regulation (ie differential splicing of different promoters) . The different proteins derived from RNA's identified by probe hhl5 presumably share the carboxyterminal nucleotide binding domain. Ce-UNC-53 was shown to be a complex genetic locus and complex transcription unit. The different transcripts are thought to be a mechanism to assure the necessary specificity and functional diversity of this signal transduction pathway, with respect to different signals and receptors, different tissues and different directions of migration. The occurance of a new transcript or the observed changes in expression levels in the cancer cell line blot suggests a role for hu-UNC-53/1 and hu-UNC-53/2 in the establishment or maintenance of the transformed state of those cells. Phenotypic changes in cells transfected with the Nucleotide Binding Domain of Ce-UNC-53/1 and Hu-UNC- 53/1 Ectopic expression of full length Ce-UNC-53 in C. elegans, murine neuroblastoma cells or human MCF-7 breast-carcinoma cells, has been found to lead to increased filopodia outgrowth and increased motility (unpublished) . The structure of Ce-UNC-53 protein is reminiscent of that of large kinases or dynamin where a catalytic domain is postively or negatively regulated by domains that interface with signal transduction pathways for example (by by GRB2 binding, phosphorylation or the like) . The inventors therefore decided to test whether the nucleotide domain by itself is capable of inducing the observed changes in the microfilament cytoskeleton and motile or ruffling behaviour. cDNA fragments coding for the nucleotide binding domains of Ce-UNC-53 and Hu-UNC-53/1 were cloned in mammalian expression vectors with the CMV promoter (see experimental procedures) . To be able to detect expression from pCB20l (Fig. 13), an N-terminal his and a T7 epitope tag were fused in frame with the hu-UNC-53/1 cDNA hhl5. pCDU3 contains a larger fragment of Ce-UNC-53 and starts just before the conserved "VIELKIEL" domain (Fig. 12) . The empty pcDNA3 vector or pCDNA3.1-His-LacZ, a mammalian expression vector for E. coli Beta- galactosidase, was used as a control vector (mock transfection) . The differences between mock and transfected N4 and MCF-7 clones were analysed using phase-contrast and Nomarski microscopy coupled with time lapse analysis, phagokinesis and immunocyto- chemical characterisation of the F-actin. Phenotypic changes in mouse N4 neuroblastoma ££112 N4 neuroblastoma cells were stably transfected with control construct pCDNA3.1 and the C. eleσans UNC-53 constructs pTB72, pCDU2 , pCDU3 and pCDU4. The population of clones transfected with the empty expression vector were homogeneous and similar to wild type N4 cells. In contrast thereto, 1/4 to 50% of the clones transfected with pTB72, pCDU2 , pCDU3 and pCDU4 (see experimental procedures and Figs. 1,17,15 and 16 respectively) had distinct phenotypes: 1. Wild type or N4 cells transfected with pcDNA3 , designated as mock transfection show a central cell body, with extensions, designated as neurite outgrowths. Less than 5% of the population have lamellae. When present, they are generally situated on the cell body and on the opposite site of the neurite extensions (figure 22a) . The lamellae show a radial actin spike pattern. Limited branching of the actin fibres is observed in wild type or pcDNA3 transfected N4 cells. Side branches are smaller and can be clearly distinguished from the main actin branch (figure 23a) . 2. N4 cells, stably transfected with pCDU4 , harbouring the homology block E, show an overall morphology which is similar to that of wild type N4's (a cell body with neurite outgrowth) . They exhibit however an increased frequency and level of lamellae formation (figure 22b) . These lamellae, which contain F-actin microspikes are found on both the cell body and the neurite outgrowth (figure 23b) . Wild type N4 cells, in contrast thereto, rarely exhibit lamellae on the neurite outgrowths. 3. N4 cells, stably tranfected with pCDU3 , encoding for homology blocks C, D and E, show an even higher level of lamellae formation labelled with TRITC-phalloidin, the cells appear surrounded with F- actin fibres, consisting of bundles of F-actin microspikes (figure 23c) . The presence of these lamellae has completely modified the general appearance of the cells. They appear flatter and in 90% of the population, it is not possible to distinguish between the cell body and the wide neurite as they flow gradually into one another (figure 22c) . If wild-type-like thin neurite-like outgrowths are present, they are frequently numerous, branched and located all around the cell. 4. The overall morphology of N4 cells, stably transfected with pCDU2 , encoding for homology blocks A, B, C, D, and E, resembles that of the wild type cells since, cell body and neurite outgrowth can be clearly distinguished. The pCDU2 transfected cells however show more neurite outgrowth, and these are long and very branched, especially at the end of the outgrowth. When neurite outgrowths of different cells make contact, increased branching can be observed, giving the appearance of a network (figure 22d) . N4 cells, transfected with pCDU2 , show bundles of long radial F-actin filaments (microspikes) , which can be branched, especially apically. The space between the hand-shaped actin spikes is mostly filled in with actin, leading to small lamellae-like structures. Also the network-like branching between the cells shows both the bundled actin structures and the lamellae-like fill-in features. These dense F-actin structures are sometimes seen on the cell body, which enhances the network-like appearance of the cells (figure 23d) . 5. N4 cells, stably transfected with plasmid pTB72, encoding the full length C. elegans UNC53 protein, seem to have a more rigid structure than wild type cells, most clearly seen as spindle-like and triangle-like cells. The corners of these cells show an increased level of hand-like lamellae structures. This specific phenotype is best seen when the cells are grown at low density (figure 22e, Fig. 23e) . Phenotypic changes in human breast carcinoma MCF- 7 cells MCF-7 cells were stably transfected with the pTB72, pCDU2, pCDU3, pCDU4 and pCB201. The population of clones transfected with the LacZ-expression vector were homogeneous and similar to wild type MCF-7 cells. In contrast thereto, -30-50% of the clones transfected with pTB72, pCDU2, pCDU3 , pCDU4 and pCB201 had distinct phenotypes which were analysed as above for the N4 cells: 1. Wild type and mock (pcDNA3) transfected MCF-7 cells are heteromorph. In general they are round cells or clusters of cells surrounded by lamellae. Bulges, similar to thick filopodia, can be observed (figure 19a) . When the cells are stained with FITC- or TRITC coupled phalloidin, F-actin actin stress fibres can be observed, often in rings surrounding the cell body (figure 20a & 21a) . When cells are round up like this actin is present at the edge of the cell body. Less than 10% of the cells display filopodia filled with radial F-actin microspikes. In time-lapse analysis the cells are highly quiescent with limited ruffling at the edge of the cell. 2. MCF-7 cells transfected with pCDU4 , encoding for homology block E, show two major phenotypic differences compared to the wild type cells. These cells are more flat and have more extended lamellipodia leading to a pancake-like appearance. Some clones show more filopodia than wild type (figure 19b) . Radially organised F-actin fibres can clearly be observerd in the lamellae surrounding the cells. These stress fibres resemble the wild-type structures, but have a more radial than circular orientation. In the filopodia, one can observe an increase of apparently unorganised, bundles of actin patches (figure 21b) . 3. MCF-7 cells, stably transfected with pCDU3, encoding the homology blocks C, D, and E, shows a strikingly different and constant morphology. The cells appear smaller than wild type because they are more rounded up. All the cells have more filopodia, surrounding the cell body (figure 19c) . Morphologically these filopodia have the same "handlike" appearance as those observed in N4 neuroblastoma cells. Such filopodia are hardly ever observed in mock transfected MCF-7 cells. These filopodia are filled with F-actin fibres. Compared to wild type cells, fine actin stress fibres are decreased (figure 21c) . In time-lapse analysis single cells as well as clusters of cells can be seen to ruffle much more dynamically than single or clusters of wild type cells. The "half-life" of a filopodia outgrowth on the cell surface is much shorter in transfected cells and the numbers of filopodia present at any time higher. 4. Cells transfected with pCB201 (which is structurally similar to pCDU4 but human) has a phenotype that is nearly indistinguishable from that of cells transfected with pCDU3 except that the observed phenotype and ruffling activity and filopodia outgrowth is even higher than pCDU3 (figure 18) . 5. The overall morphology of the MCF-7 cells transfected with pCDU2 , which encodes the homology blocks A, B, C, D and E, resembles that of the pCDU3 transfected cells. The cells are more rounded up and show more filopodia than the wild type and mock transfected cells (figure 19d) . The filopodia, which are all around the cell body tend to be longer, and show a difference in actin organisation. The small filopodia have the same actin bundles as seen in the pCDU3 transfected cells. In the longer filopodia, the actin bundles are more parallel, and radial to the cell body (figure 21d) . 6. MCF-7 cells transfected stably with pTB72, encoding the full length UNC53 protein, are extremely rounded up, and tend to adhere more than wild type cells. The cells grow in clusters with sausage- or tube-like shapes. The presence of large extremely thin lamellae with a surface area of more than three times the central cell body forms a second morphological feature, unique for the pTB72 transfected MCF-7 cells (figure 19e) . These sheets are difficult to observe under a phase contrast microscope, but are very clear when stained with phalloidin. The lamellae protrude from one side of a cell or group of cells and are filled with thin long criss-crossing actin fibres, different from "giant" wild type MCF-7 cells (figure 21e) . These experiments lead to the following set of conclusions: (Figure 47 summarises the data of the domain swapping experiments in C. elegans unc-53) 1. Murine and human cells transfected with the Ce-UNC-53 or hu-UNC-53/1 domains show clear effects on the nature and dynamics of their motile behaviour as demonstrated by changes in the F-actin cytoskeleton (the increase in lamellipodia, hand-like filopodia and "hair-like" microspikes on the cell surface and the associated reduction of the "rings of F-actin" stress- fibres) . 2. This effect is found in two cell types of different species and tissue origin: MCF-7 cells (human breast carcinoma cells of epithelial origin) and murine N4 neuroblastoma cells. pCB201, pCDU3 and pCDU4 induce in MCF-7 cells a type of filopodium which is frequent in wild type N4 cells but rare to absent in wild type MCF-7 cells, suggesting the activation by these constructs of motile behaviour which is "normal" in N4 cells but of an unusual type in MCF-7 cells. This indicates the activation of a specific downstream process as opposed to a disruption of an existing process. It is well known that some cell types prefer to migrate with filopodia and other cell types with lamellipodia. 3. Expression of pCB201, pCDU3 and pCDU4 gives qualitatively similar F-actin remodelling and increased filopodia and lamellipodia outgrowth. pCB201 and pCDU3 are however much more active in this process than pCDU4. 4. pCB201 is a much more potent activator of filopodia outgrowth than pCDU4 , which is to be expected considering the large evolutionary distance between between C. eleσans and vertebrates. 5. These experiments identify homology domain E (predicted nucleotide binding domain) of UNC-53 as the "domain" that activates F-actin remodelling and filopodia/lamellipodia outgrowth. Progressive addition of the aminoterminal homology A,B,C,D lead to qualitative and quantitative modulation of the phenotype present in domain E. 6. Homology domains C and D (pCDU3) "enhance the basic activity present in homology domain E (pCDU4/pCB201) . 7. Homology domains B and C (pCDU2) qualitatively modify the phenotype of domain E, leading morphologically different lamellipodia formation than pCDU3 transfected cells. It is thought that lamellipodia and filopodia formation are mediated by different signal transduction pathways requiring two related but different Ras-like G-proteins RAC for lamellipodia formation and CDC42 for filopodia formation. 8. pTB72 which includes homology domains A,B,C,D,E plus an additional 700 amino acids not yet identified isolated in the human members of the family confers a more localised filopodia outgrowth and a different morphology. 9. The expression levels of pTB72 (full length C. eleσans UNC-53), pCDU3 , pCDU4 and pCB201 are extremely low. The observed effect is therefore unlikely to be due to dominant negative effects (such as stoichiometric depletion of other cellular components) or structural changes in the actin cytoskeleton mediated by UNC-53 or its fragments. The data point to a multi-domain organisation in UNC-53 whereby the aminoterminal domains exert positive (e.g. pCDU3) and negative (e.g. pCDU2) control on the activity of the domain E or are leading to novel activities or the localiation of the activity in the cell (pCDU2, pTB72). Our observation that the nucleotide binding domains (NTB) of distantly related members of the UNC-53 family induce similar phenotypes, suggests a general role for this domain of the UNC-53 family. CELLULAR ASSAYS TO IDENTIFY PHARMACOLOGICAL MODULATORS OF UNC-53 AND COMPONENTS OF THE UNC-S3 PATHWAY Mammalian and human cells transfected with plasmid constructs containing unc-53 sequence of either C. elegans or of human origin were observed to display obvious, specific and similar changes in comparison to mock or untransfected parent cells. These changes relate to the functioning of the cytoskeleton, in particular the F-actin cytoskeleton, to cell locomotion and directionally cell motility and reflect UNC-53 gene family members as capable of playing an integrator function in cell motility. The cellular tools derived through transfection and derived functional assays with these cells not only enable characterisation of the motile phenotype typically observed after introduction of unc-53 genes, they also can be easily adapted to screen for pharmacological compounds that interfere with either (1) the expression of unc-53 gene family members, (2) the cellular functioning of unc-53 transgene (s) and of components in the unc-53 signal transduction pathway. Two classes of pharmacological modulators are envisaged. A first class are inhibitors of UNC-53s or the unc-53 pathway (s) , which revert the described phenotypic changes induced by unc-53 transgenes or aspects thereof. Such compounds are considered relevant leads to target diseases where unwanted directional motility of cells occurs such as metastatis, angiogenesis or inflammation. Secondly, pharmacological stimulators are envisaged, such as compounds which induce - in non- transfected cells - phenotypes that induce or mimick (aspects of) the described "unc-53' phenotype. Such compounds may do so by inducing or upregulating expression levels of a known unc-53 gene or by activating endogenous (yet unidentified) members of the unc-53 gene family. The target application here are wound and tissue repair, in particular diseases such as neuronal regeneration and plasticity. The nature of compounds envisaged can be small (organic) molecules, bio-molecules (such as peptides, sense or antisense (oligo-) nucleotides or chemical modifications thereof. Alternatively, compounds can be thought of as a series of plasmid nucleotide constructs containing gene sequences in a screen for novel unc-53-unrelated genes with a similar functional effect in the cell or genes related to the unc-53 gene family or novel members of the unc-53 gene family based on sequence similarity such as for example the genes in plasmids pTB72, pcDU3 , pcDU4 , pcDU2 , pcB201, or modifications thereof such as for example epitope tagged, deletion, complementation or mutagenised nucleotide constructs. The cellular assays envisaged in the claims have been exemplified for three cell lines: the human breast carcinoma cell line MCF-7, the mouse neuronal cell line N4 and the mouse fibroblast cell line NIH- 3T3. Pharmacological assays are focused on quantification of endpoints in a high throughout screening mode. Many of the computer aids for (semi-) automation are well known to the field and currently applied in the applicants labs. Given the subtlety of the phenotypes observed, primary focus was given to morphological assays that assess the phenotypes or aspects thereof. The nucleotide binding domain of Hu-UNC-53/1 has transforming activity in NIH3T3 fibroblasts Biochemical and genetic analysis suggest that UNC-53 functions in GRB-2 mediated signal transduction pathways controlling cell motility. The occurence of an altered hu-UNC53/l mRNA pattern in cancer cell lines, moved us to investigate if whether hu-UNC53/l plays a role in the transformed state of those cells. Thereto, we tested the ability of the nucleotide binding domain of hu-UNC-53/1 and Ce-UNC-53 to transform NIH/3T3 cells. Construct pCB201 (hu-UNC- 53) , which induces ruffling behaviour and cell motility, were transfected into NIH3T3 cells. Positive controls included Myc and H-ras. Negative controls included empty vector adn Rac 1N17 and cdc42N17. The cells that survived G418 selection were assayed for loss of contact inhibition (their ability to grow as foci) . Positive controls included the combination of two well known oncogenes Myc and H-ras which were able to produce a high number of foci. The nucleotide binding domains of both Ce-UNC-53 and hu- UNC-53/1 are able to induce foci in this assay (Fig 24 & Table 1) . Tnhlp 1 Fnri fπrmatinn in MTT-T-^T^ rplk ςrahly tran ferfpH with ppR9f>1 mock pcB20l 22 138 59 143 This suggests that the function of UNC-53 is not restricted to the activation of motility. UNC-53 may exert this additional function through the activation of as yet to be identified signal transduction pathways. Oncogenes frequently arise when a "controlling" domain and "activation" domain are separated though chromosomal rearrangements or integration of a part of a gene in the oncogenic virus. E.g. Erb Receptor tyrosine kinases, Ost a nucleotide exchange factor for Rac-l. Hu-UNC-53/1 is localized to chromosome lq31.1 Clone F226 (BACH-135 (014) , Genome Systems, inc) was isolated from a human genomic BAC library using pCR231 as a probe and was confirmed by sequence analysis to be derived from the hu-UNC-53/1 locus. Purified DNA from clone F226 was labeled with digoxigenin dUTP by nick translation. Labeled probe was combined with sheared human DNA and hybridized to normal metaphase chromosomes derived from PHA stimulated peripheral blood lymphocytes in a solution containing 50% formamide, 10% dextransulfate and 2X SSC. Specific hybridization signals were detected by incubating the hybridized slides in fluoresceinated antidigoxigenin antibodies followed by counterstaining with DAPI. The initial experiment resulted in specific labeling of the long arm of a group A chromosome. A second experiment was conducted in which an anonymous probe which was previously mapped to lp34 and confirmed by cohybridization with a chromosome 1 centromere specific probe, was cohybridized with F226. The experiment resulted in the specific labeling of the long and short arms of chromosome 1. Measures of 10 specifically hybridized chromosomes 1 demonstrated that F226 is located at a position which is 52% of the distance from the heterochromatic-euchromatic boundary to the telomere of chromosome arm lq, and that corresponds to band lq31. At total of 80 metaphase cells were analyzed with 72 exhibiting specific labeling (Fig. 25) . Gains of DNA sequences in 1Q31 were found in more than 10% of primary bladder tumors (Genes Chromosom Cancer 12: 213-219 (1991)). A putative tumor suppressor gene located near the locus F13B on chromosome arm Iq31-q32 appears to be involved in the pathσgenesis of medulloblastoma (Int. J. Cancer 67: 11-15 (1996)). Loss of heterozygosity in this region of chromosome I has been implicated in development of human hepatoblastoma. Partial trisomies of lq31 were found in Ewing's Sarcoma cell lines isolated from patients Cancer Genet Cytogenet 12: 1-19 (1984). HU-UNC-53/2 is localised to Chromosome llpl5.1 DNA from clone F329 from BAC for Hu-unc-53/2 was labeled with digoxigenin dUTP by nick translation and applied in the experimental settings used for FISH of Hu-unc53/l with F226. The initial experiment with F329 resulted in the specific labeling of the mid short arm of a group C chromosome which was believed to be chromosome 11 on the base of size, morphology and banding pattern. A second experiment was conducted in which a biotin labeled probe specific for the centromere of chromosome 11 (D11Z1) was cohybridised with clone F329. This experiment resulted in the specific labeling of the centromere in red and the mid short arm in green of chromosome 11. Measurements of 10 specifically labeled chromosomes 11 demonstrated that F329 is located at a position which is 65% of the distance from the centromere to the telomere of the chromosome lip, an area which corresponds to band llplδ.l. A total of 80 metaphase cells were analysed with 72 exhibiting specific labeling. Chromosome llpl5 is a region showing loss of heterozygosity (LOH) in a variety of human malignancies, primarily breast cancer (Ali et al., Science 238, 185-188 (1987); Winqvist et al., Cancer Res. 53, 4486-4488 (1993)) but also Wilms' tumor (Dowdy et al., Science 254, 293-295 (1991); Cowell et al., Br.J. Cancer 67, 1259-1261 (1993)), ovarian and testicular malignancies (Lothe et al., Genes Chromosomes Cancer 7, 96-101 (1993); Weitzel et al., Gynecol Oncol. 55, 245-252 (1994)) stomach cancer (Baffa et al., Cancer Res. 56, 268-272 (1996)), lung cancer (Ludwig et al., Int. J. Cancer 49, 661-665 (1991); Fong et al., Genes Chromosomes Cancer (1994)), infantile tumors of adrenal and liver (Byrne et al., Genes Chromosomes Cancer 8, 104-111 (1993)). Since LOH is believed to indicate inactivation of a tumor suppressor gene at the location where LOH occurs, the frequent LOH found at llpl5 in multiple human cancers suggests the presence of either a cluster of tumor suppressor genes or a single tumor suppressor in this region (Seizinger et al., Cytogenet. Cell genet. 58, 10080-10096 (1991)). Chromosome transfer studies have shown that chromosome 11 can suppress tumorigenicity of both human breast cancer (Negrini et al., Cancer Res.55, 3003-3007 (1995)) and Wilms' tumor cells (Dowdy et al., Science 254, 293-295 (1991)) and a gene (named HTS1 or ST5) that may be responsible for suppressing tumorigenicity in HeLa cells has been mapped to llplδ (Lichy et al., Cell Growth Diff. 3, 541—548 (1992)). Abnormalities at llpl5 have also been identified in a variety of other cancers, including lung cancer (parental origin of llplδ deletion) (Kondo et al., Oncogene 9, 3063-3065 (1994)), bladder cancer (Presti et al., Cancer Res. 51, 5405-5409 (1991)), myeloid leukemia (translocation) (Nakamura et al., Nat. Genet. 12, 154- 158 (1996)), malignant astrocytomas and other primitive neuroectodermal tumors (deletions) (Fults et al., Genomics 14, 799-801 (1992)), rhabdomyosarcoma (Scrable et al., Nature 329, 645-647 (1987)) and hepatocellular carcinoma (Fujimori et al., Cancer Res. 51, 89-93 (1991); Wang et al., Cell Genet. 48, 72-78 (1988)). Recently a gene, TSG101, was cloned that is mutated in human breast cancer and deleted in uncultured primary human breast carcinomas (Li et al., Cell 88, 143-154 (1997)). DIAGNOSTIC ASSAY USING THE DNA SEQUENCE OF HUMAN UNC-53S The differential expression of human unc-53 transcripts in Northern blots of normal tissues versus transformed cell lines and the chromosomal locus of hu-unc-53/1 at lq31 being a locus linked to three diseases, suggests the potential implication of hu- unc-53 genes in oncogenesis. By using the complete DNA sequence of hu-unc-53/1 or /2 or fragments thereof in FISH, the potential involvement of these genes can be diagnosed in patients as exemplified in figure 26. Alike, the use of these hu-unc-53 sequences in diagnostic PCR assays can be used to determine overexpression of hu-unc-53s or fragments thereof. Assay for microscopic phenotypic UNC-53 transfected MCF-7 cells Mock and unc-53 transfected MCF-7 cells were seeded at low density in culture plates and allowed to adhere to the vessel. Light microscopic inspection at different time points either on live cells or after chemical fixation with Karnovsky ' s fixative revealed that in pcB201, MCF-7 transfected cultures a rounded shaped cell body with at their boundaries many filopodia. In contrast, mock or untransfected clones had a predominant "flat' phenotype - with little or no filopodia. Quantitative measurements confirmed the statistical significance of this shift in phenotype (table 2 below) . TABLE 2 Quantification of phenotypic changes in unc-53 transfected MCF-7 cells (*) Transfection: clone no feet (") with feet ( ** ) fraction with feet mock e 34 8 0.19 37 0 0 pcB201 17 92 0.84 37 83 0.69 16 27 62 0.70 20 71 0.78 13 85 0.87 (*) Clones were passaged thrice, frozen and stored. Thawed cells were trypsinised at confluency, monodispersed, seeded in flasks and allowed to attach to substrate overnight to 48 hours. Cultures were fixed with Karnovsky fixative and inspected using phase contrast microscopy. In parallel experiments, resistance to geniticin was confirmed. (**) values are expressed as cells per microscopic view. Assay for rufflinσ and motile behaviour using automated time lapse The dynamic changes in cells are well known in the field. Animations of e.g. actin ruffles in astrocytoma cells or od actin based cell motility in e.g. fibroblasts can be accessed (http: //www. stc. emu. edu/CLMIBhp/I ggallpg/Moviespg/ actinruffle.mov) or (http: //util.ucsf.edu/mitchi/Movies/migration. html) on the world wide web. The dynamic changes as a result of transfection with unc-53 can best be appreciated in time lapse video sequences. At high magnification, the "filopodia' display arrays of microspikes with highly dynamic behaviour. A rough visual estimate suggests these phenomena to be at least 10-fold increased in pcB201 transfected cells relative to the mock-transfected MCF-7 cells. Animations of these clones in NIH-Image can be requested from author or applicant. Time lapse video imaging probably is the most informative way to appreciate the unc-53-induced phenotype in MCF-7 and is amenable to high throughput screening in a pharmacological context. Time lapses compressing 5 minutes real time supply sufficient information to quantitate the intensity of the motile behaviour of pcB201 transfected MCF-7 cells in e.g. 12 well plates. In addition, algorithms have been described in the field which can automatically compute the "motile area' of cells by comparing cells in two images appropriately spaced in time (van laerebeke etal., 1992, cytometry, 13, 1-8). Assay for visualisinσ unc-53-induced F-actin recruitment jn MCF-7 ce ls Cultures were chemically fixed, detergent extracted and fluorescently stained for F-actin (filamentous-actin) using fluorescently labeled phalloidin (Wieland et al., 1985, Int. J. Peptide & protein Res, 21, 3-10) which display in a more specific way the dramatic phenotypic changes to transfection with unc-53 transgenes. By using image capturing and analysis of the F-actin patterns, image analysis algorithms well known in the field can assess in an automated way, the f-actin filament positions, texture and distribution relative to the nuclear position or gravity point of the cells. Such algorithms are capable of discriminating phenotypic changes and thus also effects of pharmacological inhibitors of transgene-induced phenotypes as well as compound induced unc-53 like phenotypes in mock or untransfected cells. Phagokinesis assay for unc-53-induced directionality and quantity of motility The methods are described in the experimental section. Two cell populations with different motile behaviour in phagokinesis assays were observed. In table 3 below the fraction of mock and UNC-53 transfected MCF-7 cells that produced linear tracks in the phagokinesis assay are shown. In the mock transfected MCF-7 cells, 61% of the cells produce a round track (long and short axis less than 2-fold different) and 39% cells produced "linear' tracks (long and short axis more than 2-fold different) . pcB201 transfected MCF-7 cells produced an increase of the fraction of cells displaying "linear' tracks to 50%. An increase in the fraction linear tracks was made for MCF-7 cells transfected with full sequence Ce-unc-53. In addition, a significant increase of 50% in the median area of tracks of a culture vessel was observed in the pcB201 transfected MCF-7 cells versus mock transfected MCF-7 cells (Table 2) . These observations suggest that pcB201 as well as pTB72 transfection into MCF-7 cells is capable of increasing in situ locomotion in Ce-UNC-53 MCF-7, e.g. by increasing spreading, ruffling, or other forms of non-directional motility in the "round 1 population. In addition the Ce-UNC-53 transgene in MCF-7 cells drives a fraction of the MCF-7 cells from non-directional motility (round tracks) into directional migration (linear tracks) . Clone 2 thus provides a tool to analyse inhibitory or stimulating effects of pharmacological compounds on directionality or quantity of cell motility in relation to UNC-53. Table 3. Analysis of motility in phagokinesis assays Track morphology: fraction linear tracks plasmid clone round linear 1/r Mock z4 18 13 0.42 17 11 0.39 22 12 0.35 pCB201 Clone 2 16 9 0.36 13 13 0.5 7 8 0.53 9 9 0.5 Track Size Clone average±SD min max (N) Z4 1626±188 1444 2011 (8) Clone 2 2326±283 1989 2816 (8) Assays for the localisation of unc-53 in the cell to microtubules or microtubule ( + ) plus ends UNC-53s have been shown to reside on microtubules and preferentially on the microtubule (+)-ends of cells. This localisation represents an important feature of the UNC-53 family of proteins, which is rarely observed in other proteins. Absence of microtubule (+)-end binding in the protein APC following mutation has been implied in the role of APC in colon cancer (Smith et al., 1994, Cancer Res., 54, 3672) . In analogy, it can be postulated that the proper functioning of UNC-53 also may depend on its specific localisation in the cell. The methods used in the examples which prove the co-localisation with microtubules form a base for a series of assays for compounds which specifically affect microtubule (+)-end binding of UNC-53S. To the skilled eye, the typical localisation of an UNC-53 protein on microtubules can be readily recognised and thus is sufficient for the interpretation of whether the treatment with a compound has affected the localisation of this UNC-53 (or a fragment thereof) . Moreover, by combining the described methods (co- localisation) - well known to one skilled in the field and exemplified by the methods in the "experimental procedures" section - one can unequivocally confirm a compounds ability of abrogating (or promoting) microtubule and microtubule (+)-end binding. Such an assay comprises contacting a cell culture of a cell line expressing an UNC-53 with a compound in the culture conditions proper for the said cell line, followed by an incubation and finally observation of the UNC-53 (or fragment) in situ by e.g. fluorescence microscopy (for GFP-chimeras) or by fixing the cell culture and performing an immunocytochemical staining for the UNC-53 (or fragment) . For the co- localisation, methods such as immunocytochemistry for the microtubules of a cell or cell line combined with either immunocytochemistry for Ce-UNC-53 or Hu-UNC-53s or fluorescent detection GFP-UNC-53 chimeras are performed consecutively. Celeσans-UNC-53 preferentially binds microtubule plus-ends or GTP-tubulin Biochemical characterisation of UNC-53 has shown that UNC-53 binds the SH3 binding domains of SEM- 5/GRB-2 and binds F-actin in vitro . GRB2 has been localised to the cortex of the cell and reported to be involved in the control of cell motility. To determine the in vivo subcellular localisation of Ce- UNC-53, we transiently transfected COS, HepG2 and MCF7 cells with pTB72, an expression construct containing the full length Ce-unc-53 cDNA. This construct was previously shown to activate cell motility in N4 neuroblastoma and MCF7 cells. This construct gives high transient expression in COS cells, high to medium levels of expression in MCF7 cells and medium to low levels of expression in HepG2 cells. To visualise UNC-53, tubulin and F-actin, transfected cells were stained with various combinations of the anti-Ce-UNC- 53 mab 16-48-2, rabbit anti-UNC-53 polyclonal, anti- tubulin mab YL1/2 and fluorescently labelled phalloidin. At high levels of expression UNC-53 co-localises with the entire microtubule cytoskeleton, but at lower expression levels UNC-53 signal is restricted to the terminal regions of the microtubules at the plus ends. Very low levels of the expression yield a dot-like pattern in the vicinity of the cortex of the cell. To map the MTB plus end domain of Ce-UNC53, we made two constructs pcDU2 (figure 17) and pcDU3 (figure 15) in which the aminotermus of Ce-UNC-53 is deleted. Proteins corresponding to these constructs are thought to be made in vivo from different unc-53 promoters. Transient transfections followed by immunolocalisation showed these proteins to be cytoplasmic. In stable transfections in N4 neuroblastoma cells and MCF7 cells they were shown to be no longer toxic to cells but cause highly increased activation of filopodia formation. We thus uncoupled (1) toxicity of Ce-UNC- 53 from activation of motility and (2) microtubule binding from the activation of motility. Analysis of the microtubule association of the C. eleσans and Human 1 UNC53 To isolate the microtubule association domain of the C. elegans UNC53, N-terminal GFP fusions were made. C-terminal deletions on the fusion product revealed that the microtubule association was localised in the N-terminal half of the protein. A GFP fusion was also constructed with the Humanl-UNC-53 , to analyse the microtubule association properties of this protein. The association with microtubules was confirmed. A mouse anti sera was used to show the presence of native Unc-53 on microtubule plus ends of melanoma line G361. The epitope recognition of the antibody was confirmed by immunohistology experiments with mammalian cells, transiently expressed with pLM4, expression the GFP-hul-UNC53 fusion protein. Results 1. When transiently transfecting pTB72 in several cell lines C. elegans UNC-53 associates with microtubules and preferentially the plus-ends of the tubuline fibres. Transfection of plasmids pCDU3 and pCDU2 in N4 and MCF7 cell lines did not result in the observation of microtubule co-localisation. pCDU4 resulted in no staining using mab 16-48 antibody (LMBP Accession No. 1383CB) concluding that the epitope for this antibody is localised outside the fragment expressed by pCDU4. It is possible that the microtubule associated domain is situated in the N-terminus of the protein. For this reason, we constructed an N-terminal GFP fusion with the full length C. elegans UNC-53 sequence, and various C-terminal deletion derivatives. These fragments encode the N-terminal part of UNC-53 from 139 to 760 aa. Furthermore, to analyse if the cloned fragment of hul-unc53 also could be associated with microtubules, a plasmid encoding a GFP fusion with the hul-Unc53 protein was constructed, and introduced into mammalian cells. A derivative of this construct was also constructed. a) Transient expression of C. elegans Unc-53 GFP fusion in N4 neuroblastoma lines N4 cells where transiently transfected with pEGFP72, encoding a fusion protein of GFP and full length C. elegans unc-53 sequence. On an inverted microscope, the fluorescence of the GFP molecule could be followed in living cells. Cells which expressed low to medium levels of the fusion molecule showed a normal morphology after 18h to 3Oh. In these cells the co-localisation of the GFP fusion protein with the microtubules could clearly be demonstrated (figure 38a) . In cells which demonstrated a low but still distinct GFP fluorescence, specific microtubule plus- end association could be observed (figure 38b) . Cells expressing high levels of the GFP fusion protein tend to round up, in such a way that the microfilaments are difficult to visualise. After 48h, almost no GFP expressing cells can be found. It has previously been observed in transient expression of Unc-53, using plasmid pTB72, that the protein is toxic for the cells. The transient transfection experiments with the pEGFP72 plasmid gives the same observation, indicating that at least two features of the Unc53 protein are conserved in the GFP fusion protein, being the microtubule association and the toxicity of the protein. The transfected cells were fixed with paraformaldehyde, and the tubuline was stained using antibody YL1/2 and antimouse-CY3 (Jackson Labs) . Although a significant loss of GFP fluorescence was observed, one could clearly demonstrate that the filaments observed with the GFP fluorescence co- localise with the microtubules staining (figure 39) . Putative Assay Mammalian cells, in this case N , were transfected with a lipofecting agent (lipofectAMINE) while in suspension, not being attached to a surface. After transfecting those cells with pEGFP72, the transfected cell suspension could be diluted in 24- and/or 96-well plates, enabling them to attach ot the surface. Each well may contain a different compound of the collection to screen. After 24h, plates could be automatically screened for fluorescence levels. Wells containing a compound that abolish the toxicity of the GFP-C. elegans UNC-53 fusion protein will give high levels of fluorescence. Compounds having no effect on the fusion product will give no or only low levels of fluorescence. b) Transient expression of the truncated GFP- C. elegans UNC-53 fusion proteins. To assay if the icrotubuline association did occur in the N-terminal part of the C. elegans Unc- 53 protein, various C-terminal deletions were constructed. Transfection of pEGFPsma and pEGFPeel coding for 760 AA and 670 of the N-terminal part of C. elegans UNC-53 in fusion with GFP, resulted in microtubuline association, as could be visualised in living cells. The association with the microtubules is less abundant than observed when expressing the full length C. elegans UNC-53 protein, but fibres could clearly be observed ( figures 40a and 41a) . More background fluorescence is seen. This could be due to a lesser association to the microtubules or to a instability of the fusion protein. The association with microtubules could not be observed after fixing the cells with paraformaldehyde nor with methanol fixation, giving an extra indication for the weak association with the microtubule network of these proteins or potential instability of the fusion protein. At low expression levels the association of the GFP fusion protein with the centrosomes could clearly be detected (Figures 40b and 41b) . Centrosomes are the location in the cell with the highest microtubule concentration. No plus-end associations could be observed with the deletion constructs, even when cells where expressing low levels of the GFP fusion proteins. In the case of very low expressions, the centrosomes could clearly be detected. When transfecting N4 cells with pEGFPsac or pEFPXba, coding for 139 aa and 256 aa of the N- terminal part of C. elegans UNC-53 in fusion with GFP, no microtubule association could be observed. This indicates that at least 670 aa of the N-terminus of the C. elegans UNC-53 is needed to have microtubule association (figures 42a and 42b) . c) Transient expression of the GFP-hu-UNC-53/1 fusion proteins and a deletion derivative. Plasmid pLM4 was transiently transfected into N4 neuroblastoma cells, and GFP fluorescence was observed in living cells. GFP fluorescence of the available sequence of hul-UNC-53 in fusion with GFP was localised at the microtubule level. Moreover, at lower expression levels, both the centrosomes, and specific plus-end association could be observed. As has been observed with the C. elegans UNC-53 derivatives in fusion with GFP, expressed by the plasmids pEGFPsma and pEGFPeel, the GFP association seems to be less tight as was observed by the full length C. elegans UNC-53 fragment in fusion with GFP. The observed instability of the fusion protein can be due to a lesser association to microtubules, or to a degradation of the fusion protein (figure 43) . d) Immunofluorescence on melanoma line G361, and on neuroblastoma line N4 transiently transfected with pLM4. Introduction Northern experiments show that the melanoma cancer line G361 expressed abundantly both the Humanl and Human2 homologue of C. elegans UNC-53. To test if the proteins could be localised in this cell line, a collection of mouse sera was tested on this cell line. To verify if the observation was due to a hu-UNC-53 recognition, and not to an artifact, a positive sera was applied to N4 cells transiently transfected with pLM4 , expressing the GFP-hul-Unc fusion. result a serum, designated 28.1 from a mouse previously injected with peptide (DNRTLPKKGLYRY) a conserved sequence of the UNC-53 family was used for a immunolocalisation experiment on G361 cells fixed with paraformaldehyde. Antimouse-cy3 was applied as second antibody. Association with microtubule plus-end could clearly be observed. Moreover, in cells showing directional movement, observed as growth cones extensions, abundant staining can be seen in the tip of the growth cone (figure 45) . To test whether the recognition of the microtubule associated protein was identical to the Hul-UNC-53 protein, N4 cells were transiently transfected with plasmid pLM4 and consequently fixed with paraformaldehyde and stained with serum 28.1. Only cells that were transfected showed staining with 28.1, indicating that the antibody of 28.1 recognised the Hul-UNC-53-GFP fusion protein (figure 46) . This confirms that the staining of the microtubule plus-ends in the growth cones of G361 by serum 28.1 is due to a recognition of at least the Humanl and/or the Human2 homologue. It is concluded that the overexpression of the human homologue of C. elegans UNC-53 in the melanoma cancerline G361 is located on the microtubule plus- ends. Conclusions a) - GFP-C. elegans UNC-53 fusion protein expressed by pEGFP72 shows Unc53 activity b) - GFP-C. elegans UNC-53 fusion protein expressed by pEGFP72 shows microtubule association c) - GFP-C. elegans UNC-53 fusion protein expressed by pEGFP72 shows microtubule plus-end association c) - GFP-C. elegans UNC-53- (deletion variant) fusion proteins expressed by plasmids pEGFPsma and pEGFPeel show microtubule association. d) - GFP-C. elegans-UNC-53- (deletion variant) fusion proteins expressed by plasmids pEGFPsma and pEGFPeel no not show microtubule plus-end association e) - GFP-C. elegans UNC-53- (deletion variant) fusion proteins expressed by plasmids pEGFPxba and pEGFPsac no not show microtubule associations. f) - GFP-hul-UNC-53 fusion protein expressed by plasmid pLM4 shows microtubule association. g) - GFP-hul-UNC-53 fusion protein expressed by plasmid pLM4 shows microtubule plus end association. i) - serum 28.1 recognises the Hul-UNC-53-GFP fusion protein as expressed by plasmid pLM4 in transiently transfected Neuroblastoma cells N4. j) - the expressed human homologue of C. elegans. - UNC-53 in melanoma line (being at least hul-Unc-53) is associated with the microtubule plus-ends. EXPERIMENTAL PROCEDURES Materials The oligonucleotides used in the PCR-RACE experiments were synthesised by Eurogentee (Belgium) . Radioactive compounds were obtained from Amersham. The pCDNA3.1 eukaryotic expression vectors, human 1GT10 cDNA libraries, marathon-RACE cDNAS, human, Northern blots and the T7-tag monoclonal antibody were purchased from Invitrogen. N4 , MCF7 and NIH 3T3 cells were retrieved from the Janssen Research cell bank. PCR-RACE conditions 1. A quick screen human cDNA library panel was used to amplify EST clone gb..R41071. The primers used were ESTfw 5 ' -AATGGCTTCCTGGTTACCTGAG-3 ' and ESTrv 5'- CAAGTCAGCACCCCGAAGCAGCTCT-3 ' . Human genomic DNA was used also as template (lOOng/reaction) . The amplification conditions were as follows: 1 min at 94°C, 30 sec at 55°C, 30 sec at 72°c, then 35 more times and a final extension of 20 min at 72°C. This PCT fragment was cloned in vector pCR2.1. The resulting plasmid was designed pCR231. A human heart clone was also produced by RACE-PCR from a human heart Marathon cDNA using the following conditions; 1 min at 94 C, 30 sec at 70°C, 3 min 30 sec at 72 C, then 35 more times and a final extension of 20 min at 72 c KlenTaq DNA Polymerase was purchased from Invitrogen. For the mouse homologue, total RNA was obtained from N4 murine cells as described. A first strand cDNA was synthesized from 2 μgr of RNA using Ready To- Go cDNA kit (Pharmacia) The primers used were M-ESTfw 5 • CCTCTGTGGGCACCGAGGTCACC—3'. The amplification conditions were as follows: 1 min at 94°C, 30 sec at 58'C, 30 sec at 72"C, then 35 more times and a final extension of 20 min at 72'C. All the amplifications product were subcloned in pCRII-1 and several independent clones were analyzed by sequence. 2. Screening of Human Heart/Colorectal Adenocarcinom a cDNA library A human heart cDNA library and a human colorectal adenocarcinoma cDNA library were screened using pCR231bp as probe by the standard plaque hybridization method. The screening produced several positive clones in each library called respectively λHH3, λHH4 , ΛHH15, ACAD14 and XCAD27. The positive phages were purified by two additional rounds of plaque screening and were then amplified. 3. 5' extension using PCR Three primers with homology to the 5' end of clone λHH3b were made: HU53rvl (5 ' -cct-ggg-act-gaa-gct-ggt-acc-tga-gcc-3 • ) , HU53rv2 ( 5 ' -ttg-gga-aga-gtg-ttc-cga-tcc-cgc-tg-3 ' ) and HU53rv3 (5'gtt-gcc-cag-ctc-tgg-ggc-ttc-cac-tcc-3 ' ) and used together with λgtlOrv primer (5 ' -gag-gtg-gct-tat- gag-tat-ttc-ttc-cag-ggt-a-3 * ) in three nested PCR reactions on a cDNA amplified library from Human Heart (Clontech) . The reaction mixes contained 25pmol of each primer, 1 mM of each dNTP, 1 μlKlenTaq Polymerase Mix (50x) and 0.1 ng DNA. The cycling parameters for the first PCR were: 3 min at 94°C, 35 cycles of 1 min at 94°C, 1 min at 51°C and 3 min at 72°C and a final extension of 10 min at 72°C, using HU53rvl and λgtlOrv as primers. 0.4 μl of this primary PCR product was amplified using HU53rv2 and λgtlOrv as nested primers with the following parameters: 3 min at 94°C, 38 cycles of 1 min at 94°C, 1 min at 52°C and 3 min 30 sec at 72°C and a final extension of 10 min at 72°C. The second nested PCR reaction was performed on 0.4 μl of a 1/50 diluted purified 2.4 kb fragment using HU53rv3 and λgtlOrv as primers: 3 min at 94°C, 35 cycles of 1 min at 94°C, 1 min at 56°C and 3 min 30 sec at 72°C and a final extension of 10 min at 72°C. A 774 kb amplification product was subcloned in pCR2.1, resulting in plasmid pCB210-14. The clone fragment was analyzed by sequencing. This fragment extends 699 bp in 5' direction (see fig 9). 4. 5' extension using PCR Primer HU53rv4 (5 ' -ccc-tgc-ttg-gtg-ctg-agg-aga- ctg-g-3 ' ) was designed on the 5' end of clone pCB210- 14 and was used together with λgtlOrv to amplify a fragment of the Human Heart cDNA library with the following parameters: 3 min at 94°C, 35 cycles of 1 min at 94°C, 1 min at 60°C and 3 min 30 sec at 72°C and a final extension of 10 min at 72°C. A 887 bp fragment was subcloned in pCR2.1, resulting in plasmid pCB212. The clone fragment was analyzed by sequencing. This fragment extends a further 767 bp in 5' direction (see fig 9). 5. Human Heart Library screening using the 0.8 kb insert of pCB212 as probe The EcoRI digested and purified clone pCB212 was used as probe to screen the Human Heart cDNA library (Clontech) using standard plaque hybridization method. The positive phages were purified by two additional rounds of plaque screening. The insert of the λDNA (produced using Qiagen Lambda Kit) was analyzed by sequencing. This pHH14-3 resulted in a 2663 bp fragment overlapping pCB212, pCB210-14 and the 3' end (434 bp) of λHH3b and in a 761 bp 5' extension (see fig 9) . 3' and 5' extension of HU-Unc53/2 from EST46037 WashU-Merck EST 46037 Transformed cells carrying the EST 46037 sequence were ordered from Research Genetics. Plasmid DNA was isolated using standard protocols (Qiagen plasmid DNA isolation kit) , the sequence of the insert was determined. 3' extension of EST 46037 by RACE Marathon-Ready cDNAs (Clontech) are premade "libraries" of adaptor-ligated double-stranded cDNA ready for use as templates in RACE experiments. Five ml Marathon-Ready cDNA was used as template in a regular 50ml RACE. The RACE mixture contained lx KlenTaq PCR buffer, 0.2 mM of each dNTP, lx advantage KlenTaq polymerase mix (Clontech), 0.15 mM API adaptor primer and 0.15 mM RACE gene specific primer. The amplification conditions were as follows : 94°C for 1 min, 5 cycles of 94°C for 30 s and 72 C for 4 min, 5 cycles of 94°C for 30s and 70°C for 4 min, 25 cycles of 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 pCRr2.1 (TA cloning kit, Invitrogen) and the sequences of the inserts were determined. gene specific primer (EST46037-F1) 5 ' AGTGAGAACAATGCTGTGGACATGC nested gene specific primer (ES46037-F2) 5 » CTGCTCAACTGCAAGTACCACAAATGC Marathon cDNA library : human placenta WashU-Merck EST 923793 Transformed cells carrying the EST 923793 sequence were ordered from Research Genetics. Plasmid DNA was isolated using standard protocols (Qiagen plasmid DNA isolation kit) , the sequence of the insert was determined. RACE fragments 1.4 and 3.7. 5' extension of EST46037 Method as described previously. Gene specific primer (EST46037-R1) 5 » ACTGCCTTGAGACTCTGACTTCAGC nested gene specific primer (ES46037-R2) 5'TGGGCAGAACTGAGAGCTTCTAAGC Marathon cDNA library : human placenta RACE fragments B2.1. D2.1. H2.1 : 5' extension Method as described previously: gene specific primer (97010709) 5 •ATTCTTTTGCATCTTCTTGCGTGCG nested gene specific primer (97010708) 5'ACCTGAGTCCTTTCTTAGGCAAAGTGTTCC Marathon cDNA library : human placenta (fragment B2.1) human HeLa S3 (fragment D2.1) human colorectal adenocarcinoma SW480 (fragment H2.1) PCR fragments E2.3. C2.3 EST 485068 is similar to but not identical with the 5' end of HU-Unc53/l. A primer pair consisting of one 3' EST 485068 primer and one 5' HU-Unc53/2 primer were used to PCR amplify those fragments. lgtlO human placenta Quick screen library (fragment C2.3) or Marathon cDNA from human HeLa S3 (fragment E2.3) were used as templates in a PCR. A 50 ml reaction mix contained lxPCR II buffer (Perkin-Elmer) , 1.5 mM MgC12, 0.2 mM of each dNTP, 0.15 mM forward and reverse primer, 2.5 U AmpliTaq Gold (Perkin-Elmer) and 1 ml template. The cycling parameters were 5 minutes at 95°C, 35 cycles of 45 seconds at 94°C, 45 seconds at 65°C and 2 minutes at 72°C. The PCR products were sliced out from an agarose gel and purified using a gel extraction kit (Qiagen) , one ml hereof was used in a second round PCR using the same conditions as above. The PCR products were purified (Qiagen PCR purification kit) and direct sequenced. primers : (97010709) 5' ATTCTTTTGCATCTTCTTGCGTGCG (97012802) 5' CGCTCCCCATCAGATGCAGGCCGG PCR fragment El.3-3 EST 01222 is homologous but not identical with the 5 'end of HU-Unc53/l. A primer pair consisting of one 3' EST 01222 primer and one 5' HU-Unc53/2 primer were used to PCR amplify this fragments. Marathon cDNA from human HeLa S3 was used as template in a PCR. A 50 ml reaction mix contained lxPCR II buffer (Perkin-Elmer), 1.5 mM MgCl2 , 0.02 mM of each dNTP, 0.15 mM forward and reverse primer, 2.5 U AmpliTaq Gold (Perkin-Elmer) and 1 ml template. The cycling parameters were 5 minutes at 95 C, 35 cycles of 45 seconds at 94°C, 45 seconds at 65°C and 2 minutes at 72°C. The PCR products were sliced out from an agarose gel and purified using a gel extraction kit (Qiagen) , one ml hereof was used in a second round PCR using the same conditions as above. The PCR products were analysed on an agarose gel, the fragment of interest was sliced out, purified (Qiagen PCR purification kit) and cloned into pCRr2.1. The sequence of the insert was determined. RACE fragments A2.2-2, B2.1-4. D2.1-5; 5' extension Method as described previously, gene specific primer (97041701) 5 'TATGCTACGGCCACTCATCTCCGTGG nested gene specific primer (97041702) 5 ' TGTAACCTGAGTTCCCCTTAAACTGG Marathon cDNA library : human placenta (fragment A2.1-2) human HeLa S3 (fragment B2.1-4) human colorectal adenocarcinoma SW480 (fragment D2.1-5) Translation-initiation splice variants, fragments D4.1-1, J4.1.4, G4.1.1, F4.1.2 Four different translation initiation slice variants were detected by 5' RACE. Method as described previously. gene specific primer (97080803) 5 ' TCGGTTGTTAGCAGTAGTTGACCCTCC nested gene specific primer (97080804) 5 ' ACCTGAAAGTCTGGACTGCATTTCAGC Marathon cDNA library : human colorectal adenocarcinoma SW480 (fragment D4.1-1) gene specific primer (97080801) 5 ' ACAACCTGGAT ATCTGGGCCAGGAGG nested gene specific primer (97080802) 5 'TCTTGCTGGAGATCCTTGATGAGACGC Marathon cDNA library : human melanoma G361 (fragment J4.1.4) human HeLa S3 (fragment G4.1.1) human placenta (fragment F4.1.2) DNA sequencing PCR amplification products and cDNA clones were subcloned either into pBluescript vectors (Stratagene) or in PCR-IIa vector (Invitrogen) and sequenced either manually by the dideoxynucleotide chain termination method with modified T7 DNA polymerase (Sequenase, United States Biochemical) or automatically with an Applied Biosystems 373 DNA sequencer using the fluorescent terminator kit (Perkin Elmer) . RNA blots A Human multiple tissue Northern (MTN-1, Clontech) containing in each lane 2 mg of poly A + RNA from eight different human tissues (heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas) and a MTN-II human multiple tissue Northern, containing in each lane 2 mg of poly A + RNA from spleen, thymus, prostate, testis, ovary, small intestine, colon and peripheral leukocyte, were hybrydized according to the manufacturer's instructions and washed out in 0.1xSSC:0.2% SDS at 55 ' C. Also from Clontech, a poly A + RNA blot from human cancer cell lines (melanoma G361, lung carcinoma A549, colorectal adenocarcinoma SW480, Burkitt' s lymphoma Raji Leukemia Molt 4, lymphoblastic leukemia K562, HeLa S3 and promyelocytic leukemia HL60) was tested. Construction of plasmids Plasmid pCDU2 (Figure 17) was constructed by cloning the 2.8 kb Apal-Narl fragment from pTB72, the latter restriction site made blunt with klenow enzyme, into pcDNA3, digested with EcoRV and Apal . pCDU2 encodes for the homology blocks A, B, C, D and E. Plasmid pCDU3 (Figure 15) was constructed by cloning the 1.9 kb Apal-Ndel fragment from pTB72, the latter restriction site made blunt with Klenow enzyme, into pcDNA3, digested with EcoRV and Apal, pCDU3 encodes for the homology blocks C, D and E. Plasmid pCDU4 (Figure 16) was constructed by cloning the 1.4 kb Apal-Styl fragment from pTB72, the latter restriction site made blunt with Klenow, into pcDNA3 digested with EcoRV and Apal . pCDU4 encodes for the homology block E. Expression of a domain of the human UNC53 in eukaryotic cells 1. pCB20l: Equivalent construct of human 1 homologue to expression construct pCDU4 of C. elegans unc-53 gene cloned in a eukaryotic His-tag, Xpress Ab tag expression vector. A suitable Bam HI site was engineered on pHH15 open reading frame by amplification with hhlδfw primer 5ΑGAGCGGATCCATATGCCTCCTTGCCGTCAAGGTG-3' and M13rv primer (5 ' -cag-gaa-aca-gct-atg-ac-3 ' ) . The amplified fragment was then moved to pCDNA3. l.His-A-Vector digested with BamHl and EcoRI. This new plasmid called pCB201 (Figure 13) produces a cDNA which codes for a fusion protein consisting of a 49 amino acid aminoterminal fragment containing an His-tag and also a T7 epitope tag followed by amino acids 1255 to 1627 of the sequence of the human homologue. pCB201 was also checked by sequence and the n was used in stable transfection experiments carried out in N4 , MCF7 and NIH3T3 cells. 2. pLM5: Equivalent construct of human 1 homologue to expression construct pCDU3 cloned in an eukaryotic His-tag, Xpress Ab tag expression vector. The phage HH3b was linearized using Xhol. A BamHl and Xbaal restriction site were created on the pHH3b open reading frame using U3-Bfw (5 '-cca-cac-tag- ggg-atc-cat-gca-aat-gag-g-3 ' ) and U-rv (5*-caa-aag- tct-cta-gag-gag-gcc-agt-3 ' ) as primers. This amplified fragment was then moved to pBluescript KS, digested with BamHl and Xbal. Sequencing of this plasmid, named pCB300, showed an amino acid change from a serine to an asparagine due to a change from guanine to adenine on the position 4237 of the DNA sequence. This fault was repaired by cloning a 1418 bp fragment of pLMl (see below) (using Narl and Xbal as enzymes) into pCB300 digested with the same enzymes. The phage HH3b fragment of this plasmid, named pLM6 (fig 53), was then removed using BamHl and Xbal, to pcDNA3.1/HisA digested with the same enzymes. This new plasmid, named pLM5 (fig 52) , produces a cDNA which codes for a fusion protein consisting of a 49 amino acids aminoterminal fragment harboring a His-tag and a T7 epitope tag, followed by aminoacid 1069 to 1627 of the transcript of HU-Unc53/l. Plasmid pLM5 was checked by sequencing and used on transient and stable transfection experiments carried out in N4 cells. The plasmid pLMl was created using a PvuII and partial BamHl digested fragment of pHH14-3 and a BamHi and Spel digested fragment of phage HH3b, cloned into pBluescript KS digested with Smal and Spel. The pLMl contains the full transcript of HU-UNC-53/1 available at this moment (see fig 9) . 3.pCB251: Equivalent construct of human 1 homologue to expression construct pCDU2 cloned in an eukaryotic His-tag, Xpress Ab tag expression vector The phage HH3b was linearized using Xhol. A BamHl and Xbal restriction site were created on the pHH3b open reading frame using U2fw (5 ' -aag-gga-tga- ttc-ggt-cag-gat-cct-tc-3 ' ) and U-rv (5 ' -caa-aag-tct- cta-gag-gag-gcc-agt-3 ' ) as primers. The amplified fragment was then moved to pCR2.1. This plasmid was named pCB250. The pHH3b fragment was removed from pCB250 using BamHl and Xbal and cloned in pcDNA3.1/HisC digested with the same enzymes. This plasmid, named pCB251 (figure 55) , was checked by sequencing. pCB251 produces a cDNA which codes for a fusion protein consisting of a 49 amino acid aminoterminal fragment harboring a His-tag and a T7 epitope tag, followed by amino acids 828 to 1627 of the partial transcript of HU-Unc53/l. pCB251 was used on transient and stable transfection experiments carried out in N4 cells (see fig 56) . 4. pLM3 : the partial transcript of HU-Unc531 cloned in an eukaryotic His-tag, Xpress Ab tag expression vector pLMl was digested with EcoRV and Xbal. This fragment was cloned in pcDNA3.1/HisB, digested with the same enzymes. pLM3 produces a cDNA which codes for a fusion protein consisting of a 49 aminoacid aminoterminal fragment harboring a His-tag and a T7 epitope tag, followed by amino acids 1 to 1627 of the transcript of HU-Unc53/l available at this moment. pLM3 was used on transient and stable transfection experiments carried out in N4 cells. 5. pLM4 : the partial transcript of HU-Unc53/l cloned in an eukaryotic GFP expression vector pLMl was digested with Clal and Xbal. This fragment was cloned in pEGFP-cl, digested with Accl and Xbal. This plasmid was named pLM4. This plasmid produces a cDNA which codes for a fusion protein consisting of GFP, followed by aminoacid 1 to 1627 of the transcript of HU-Unc53/l. pLM4 was used on transient and stable transfection experiments carried out in N4 cells (see figs 43 and 46) . Stable transfection of MCF-7 cells: Cells were seeded at a density of 2x10° cells in a 75 cm" flask using standard culture medium ((Dubecco's MEM, 450 mg/1 glucose, 862 mg/1 L-Alanyl- L-Glutamin, 110 mg/1 Na-pyruvate; GibcoBRL) supplemented with 10% foetal calf serum (FCS; GibcoBRL) , and 100 U/ml penicillin (GibcoBRL) and 100 μg/ml streptomycin) . The culture was grown at 37°C in a 10% CO, atmosphere, to approximately 70% confluency (approximately 18 hours) . The culture medium was removed and 10 ml MEM-HEPES (GibcoBRL) supplemented with 10% FCS was added to the cells. The culture was further incubated for four hours at 37"C in standard sterile air. DNA-CaCl : was meanwhile prepared by mixing 30 μg DNA in 0.1 x TE (1 mM Tris. Hcl, pH 7.2, 0.1 mM EDTA, pH 8) and 0.1 ml 1.25 M CaCl 2 /HEPES (1.25 M CaCl 2 , 0.125 M HEPES; pH 7.05). 0.1 x TE was added to a final volume of 0.5 ml. The DNA-CaCl 2 was added drop by drop to 0.5 ml BS/HEPES (25 mM HEPES, 0.25 M NaCl, 0.01 M Kcl, 1.4 mM Na_HP0 4 , 0.01 M glucose, pH 7.05) while pipeting a sterile airflow through the latter solutions. The DNA-Ca, (PO.,) precipitate was then placed at 37°C for ten minutes. The DNA-Ca 3 (P0 4 ) 2 precipitate was vortexed and added to the cells, together with 100 μl of a 0.01 M chloroquine (Sigma) stock in H.O. After four hours of incubation at 37°C in sterile standard air, the medium was removed, and the cells were washed with PBS (GibcoBRL) . 25 ml of medium was added and the cells where placed at 37°C in a 10% CO atmosphere. After 48 hours of incubation, the cells were harvested, diluted and cultivated under selection (600 μg/ml G418 (Duchefa) ) for two weeks prior to clone selection. Mock transfected MCF-7 were positive for the beta-galactosidase transgene. The stability of transfection in MCF-7 was assessed by passaging cells four times in the absence of Geneticin and then re-exposing them to the selector agent. In these experiments, unc-53 or mock transfected cells proliferated, whereas untransfected MCF-7 cells proliferated at a much slower rate. Stable transfection of N4 neuroblastoma cells Cells were seeded at a density of 2x10" cells in a 25 cm- flask using standard culture medium ( (MEM Rega 3; GibcoBRL) supplemented with 10% FCS, 0.14% Na 2 CO., , 2 mM glutamine, 100 U/ml penicillin, and 100 μg streptomycin) . The culture was grown overnight at 37°C in a 10% C0 2 atmosphere. Transfection mixture was prepared by adding 12 μg DNA in 600 μl optimem 1 (GibcoBRL) to 36 μl LipofectAMINE (GibcoBRL) in 600 μl optimem 1. This was done by adding drop by drop the first solution to the second. The mixture was placed for 30 minutes at room temperature, after which 1.8 ml of optimem 1 was added. In the meanwhile the cell culture was washed twice with optimem 1, and the 3 ml of transfection mixture was added. The culture was placed at 37°C in sterile standard air. After four hours, 3 ml or normal culture medium was added and the culture was placed at 37'C under 10% of C0 2 . 18 hours later, the culture was washed with PBS, and fresh normal culture medium was added. A further 24 hours later, the cells were harvested, diluted and cultured under selection (750 μg/ml G418) for two weeks prior to clone selection. Fixation of cells for Immunofluorescence Medium was removed from the 9 cm 2 wells containing the coverslips. A 4% solution of paraformaldehyde (Sigma) in PHEM (1 g/1 glucose, 0.4 g/1 Kcl, 8 g.l NaCl, 0.06 g/1 KH_P0 , 0.0475 g/1 NaHP0 4 , 0.35 g/1 NaHCO,, 1.51 g/1 PIPES, 0.76 g/1 EGTA, 0.19 g/1 MgCl_; pH 6) was added for 30 min at room temperature. The fixative was removed, and the coverslips were washed three times 10 minutes with PHEM. The coverslides were then placed in PHEM, containing 0.5% Triton-XlOO (Serva) for 30 minutes, after which th slide was washed again for three times 10 minutes with PHEM. The coverslips were then placed under PBS (0.14 M NaCl, 2.7 mM Kcl, 10 mM Na 2 HP0 4 , 1.8 mM KH 2 P0 4 , pH 7.3) containing 0.2% Tween (Sigma) for at least one hour at 4°'C Immunofluorescence staining The coverslips were inverted on 35 μl of appropriately diluted antibody, being YL 1/2 for tubulin and/or mab 16-48-2 monoclonal or anti-UNC53 (gp48) polyclonal antibody for UNC53. The slides were placed at 4 C C for at least 18 hours. Excess of primary antibody was then removed by washes of three times ten minutes in PBS-Tween. The slides were then treated with secondary antibody in the same way as for the primary antibody. F-actin was labelled by including TRITC- or FITC coupled phalloidine to the incubation buffer. The inverted slides on the secondary antibody were left at room temperature for approximately one hour. Slides were then washed again for three times ten minutes with PBS-Tween and once with PBS. The coverslips were mounted on slides with the medium described by Herzog et al. (Cell Biology: a laboratory handbook, 1994, Academic Press, 355-360). After at least two hours, slides were ready for analysis. Time lapse analysis Analysis of the behaviour and movement of growing cell cultures was done by placing a non-confluent culture under a phase contrast microscope equipped with a temperature controlled stage (37"C) . Images were recorded using a CCD camera (COHU 4912) coupled to a SCION LG3 framegrabber in a Macintosh ppc 8100 running NIH image version 1.60. Images were recorded at time intervals, varying from 15 sec to 1 min. for half an hour to two hours. Image enhancement and playback was done in NIH image. Phagokinesis A variety of cell types were shown to migrate over colloidal gold coated culture plastic or glass and displace or phagocytose the gold lawn on their way while locomoting. The track left bare is a qualitative and quantitative measure of cell motility and/or locomotion. The basic methods have been described in detail elsewhere (Albrechr-Buehler, 1977, Cell, 11: 395, Zetter, 1980; Nature, 285: 41; O'Keefe et al . , 1983; J. Invest. Dermatol., 85: 130). Culture plates were gelatin and gold coated as described by Albrecht-Buehler (1977) . Unc-53 and mock transfected MCF-7 were seeded in plates at low density and allowed to adhere to the plate and to locomote overnight. Cells were chemically fixed to the plate, washed and air-dried. Images of the gold lawns were captured using automated videomicroscopy; composite images of the wells were generated and single-cell phagokinesis tracks were measured using a home-made routine in SCIL™ software. C. elegans-UNC-53 preferentially binds microtubule plus ends or GTP-tubulin 1. Cloning of c. elegans cDNA in pEGFP-Cl and construction of C-terminal deletion derivatives. a) Constructing a GFP-Unc53 N-terminal fusion: A PCR experiment was performed under standard conditions, using pTB72 as template and cpl7 (ata gcc aga tct acg tea aat gta gaa ttg) and cpl8 (ttt aga aac cgc ggg tgg) as primers. The resulting 0.4 kb fragment, coding for the N-terminal fragment of C. elegans Unc 53 was cloned in vector pCR2.1 (original TA cloning kit, Invitrogen) , resulting in plasmid pTA1718. The 0.4 kb fragment was isolated as a Bglll- SacII fragment and cloned in pEGFP-Cl (Clonetech) digested with the same enzymes. The resulting plasmid was designated pEGFPsac (Figure 29) . pEGFPsac encodes the N-terminal 13 aa of C.e.Unc53 in fusion with GFP. b) Construction of a GFP-C. e. Unc53 full length fusion: Plasmid pTB72 (shown in Figure 1) was digested with restriction enzymes SacII and Apal. The resulting 4.5 kb cDNA fragment, encoding for the C- terminal fragment of C. elegans Unc53 was cloned in plasmid pEGFPsac (Figure 29) , digested with the same enzymes, resulting in plasmid pEFP72 (Figure 30). Plasmid pEGFP encodes GFP in fusion with the full length C.e. Unc53. c) Construction of N-terminal deletions of GFP- C. eleσans UNC-53 fusion protein, other than pEGFPsac: pEGFP72 was digested with Smal . The resulting 7.0 kb fragment was religated and transformed in E.coli, resulting in plasmid pEGFPsma (Figure 31) . This plasmid codes for the first 760 aa of the Ce-UNC-53 in fusion GFP. pEGFP72 was digested with restriction enzymes Ecll36II and Smal, the resulting plasmid after ligation and transformation in E.coli of the 6.7 kb fragment was designated pEGFPeel (Figure 32) . This plasmid codes for the N-terminal 670 aa of the C.e. Unc53 in fusion with GFP. pEGFP72 was further digested with Smal and Xbal. The latter site was made blunt with Klenow polymerase. The resulting fragment of 5.4 kb was religated and transformed in E.coli. The resulting plasmid was designated pEGFxba (Figure 33) . This plasmid codes for the N-terminal 256 aa of C. elegans Unc53 in fusion with GFP. 2. Constructing a hui-UNC-53-GFP fusion, and a deletion derivative The 5.4 kb hul-unc53 fragment was isolated as Clal-Xbal fragment from pLMl (Figure 54) , and cloned in pEGFP-Cl digested with Accl and Xbal. pEGFP-Cl was isolated from E.coli GM41 (Hfa H, dam-3 , thi-1, rel- 1) . This makes the Xbal restriction site available for restriction digest. The resulting plasmid was designated pLM4 (Figure 34) . 3. Visualisation of GFP fluorescence in N4 cells N4 neuroblastoma lines where seeded in Lab Tek chambered coverglass (Nalge Nunc International) and transfected using lipofectAMINE (GibcoBRL) . After 18 hours, the chambered coverglasses where placed on a inverted microscope, and GFP fluorescence could be visulalised. 4. Staining GFP fusion expressing cells with antibodies Transfection with the GFP fusion constructed was also performed on coverglasses in a 6-well plate. After paraformaldehyde or methanol-acetone fixation, cells could be stained for actin cytoskeleton with TRITC-phalloidine, for hu-unc53 with sera 28.1 and for tubuline with YL1/2 antibody. Visualisation was then performed on a axioplan (Zeiss microscope) . Methods of Producing and Observing the Effects of A Chimeric unc-53 gene 1. Definition of a promoter region in the unc- 53 C. elegans gene: The genomic region from the position 15621 to 18415 in the C. elegans unc-53 gene, called promoter A, was cloned and fused to the cDNA of the GFP gene (clone pA/GFP, or pNP10)(cf. fig.51). This construct is injected into wild type worms (N2) . Transgenic line express GFP in different neurones: the two pairs of pioneering neurones PVP and PVQ, both BDU neurones, both ALN and PLN neurones, both PDE neurones, both PVM neurones, and 4 vulval cells. Expression begins in early embryogenesis, when the axons of those neurones grow out. 2. Mutant Phenotype in Unc-53 (nl52) alleles: In wild type worms (N2) , the two pairs of ALN and PLN neurones each send an axon in a straight line longitudinally from the tail to the head (see fig.50a). In unc-53(nl52) alleles, the axons are shorter and often branch in a dorso ventral direction (see fig.50b). The neurones are visualised with the construct pA/GFP, injected in unc-53 (nl52) worms. 3. The minigene pA/unc-53 rescues the elongation defect of ALN and PLN neurones: The promoter A from the C. elegans unc-53 gene was fused to the cDNA of the C. elegans unc-53 gene (clone pA/unc53, or pNP9) . This construct was injected in unc-53 (nl52) mutant worms, together with the pA/GFP construct described above to visualise the ALN and PLN neurones. The elongation defects of those neurones in the unc-53 mutant are almost completely restored by the expression of the unc-53 cDNA express under the promoter A (see figs. 50 and 51b). 4. Domain swap between the C. elegans and human unc-53 gene: To test whether the vertebrate and worm members of the unc-53 family are functionally equivalent, we tested the ability of the human gene to rescue the mutant phenotype in the worm. We replaced the carboxyterminal predicted nucleotide binding domain (NTPase) of the worm protein with the homologius fragment of the human 1 gene. The clone pA/unc-53 was deleted of the C. elegans NTPase domain, from the Hpal site, position 29800 on the genomic of unc-53, and replaced by the equivalent domain of the human-1 gene (unc-53Hl) (see fig. 51) . The resulting clone is named pA/unc-53Hl. When this clone is injected to unc-53 (nl52) mutants, the transgenic worms show a significant but incomplete rescue of the defect in the elongation of the ALN and PLN neurones (see fig. 51b) . The axons are longer, often elongated until the region of the vulva in a straight line, without branching dorsally anymore. This result shows that a NTPase region of the human unc-53 homologue can functionally replace the NTPase region of the C. elegans worm. The degree of rescue was analyzed quantitatively and summarized in Figure 51b: The four strains compared are: wt; un-53(nl52); unc-53 (nl52) ,pA/unc-53 ;unc- 53(152) ,pA/unc-53-Hl. The various phenotypes observed are brought together in three large classes: «wild type>> the axon is straight, unbranched and migrates into the head; <<vulva>> the axon is straight, unbranched and stops in the vulva region; <<mutant>> the axon is short, does not reach the vulva region and has collateral branches. The figures are indicated as a percentage. The number of axons observed is indicated in the following column. The data clearly show demonstrate conclude that the nematode/human chimera minigene pA/unc-53-Hl partly rescues the defects of the axonal migration of the ALN and PLN neurones and demonstrate conservation of function of this domain between man and worm. The transgenic lines provide a functional screening assay for the motility function of at least part of the human UNC-53 gene. II. Materials and methods 1 - Cloning: a) pAB/GFP (pNP3 - Figure 27) The gene of GFP has been amplified by PCR with cpn3 oligo-nucleotides "acattaagcttcgtacgcttggagggtaccg" and Cpn5 "gaaaggatccgtacgataaggtattttgtgtcgg" on the plasmid pPD95.75 (Figure 59) so as to be inserted at the 5' position in fusion into the exon 12 of the unc-53 gene at a single restriction site Spll and contains its stop codon at 3' plus one polyadenilation site. The PCR amplification product is directed by Hindlll and BamHl, sites which are contained respectively in the cpn3 and cpn5 oligonucleotides and sub-cloned in the pBS vector (clone pNP2) . The GFP is then excised from the pNP2 clone at the site Spll and integrated into the X16 clone (Figure 60) originating from sub-cloning of the lambda phage S4 digested by Xhol. The X16 clone containing the genomic sequence of unc-53 from the position 16621 to the position 24891 cloned in the site Xhol of pBS. b) pAB/unc-53 (pNP8 - Figure 35) The promoter region AB of the X16 clone (between Pstl and Spll) has been inserted in the clone pTB115 (Figure 58) in which the region between the sites Pstl and Spll, containing the promoter of the gene mec-7 and the start of the gene unc-53, has been removed. c) pA/GFP (pNPIO - Figure 56) The promoter region A has come from the X16 clone between the sites Pstl and Nhel and integrated in the vector pPD95.75 containing the GFP in the sites Pstl and Xbal . d) pA/unc-53 (pNP9 - Figure 44) The promoter region A has come from the X16 clone between the sites Pstl and BstXI and is integrated into the clone pTB115 in which the region between the sites Pstl and BstXI, containing the promoter of the gene mec-7 and the start of the gene unc-53, has been removed. e) pA/unc-53 -HI (pCB501 - Figure 57) The clone pA/unc-53 (pNP9) has been deleted from the region 3' of the gene unc-53 of the nematode between the sites Hpal and Ncol . The 3 ' region of the Hlunc-53 gene has been amplified by. PCR with the oligonucleotides U4Afw (5 ' -gca-cat-cgt-taa-cgg-gga- ctt-gaa-gc-3 ' ) and Urv (5 ' -caa-aag-tct-cta-gag-gcc- agt-3 ' ) and digested with Hpal and Xbal. After a filling stage with T4 polymerase, the ligation is effected with a complete end. 2-Injection Conventional injection techniques are used (Fire A., 1986, Mello G, et al, 1991, journal Mello G. and Fire A., 1995). Young hermaphrodite adults are injected in their two syncytial gonads. The DNA used is prepared in standard manner (Qiagen) followed by precipitation with lithium chloride. After an extensive rinsing stage to eliminate all the salts, the DNA is resuspended in water. The injection solution contains the different DNAs at a concentration of 100 ng/il in an injection buffer: . The plasmid pRF4 containing the dominant allele su 1006 of the gene rol-6 (Kramer J. et al, 1990, Mello C. et al, 1991) is used as a transformation co-marker. The descendants of roller phenotype of the hermaphrodite injected are isolated. Approximately 10 % of these transformants will yield a stable strain, in which the different DNAs injected are associated to form a mini-chromosome which will segregate as unstable extrachromosomal arrays. All the transgenic strains obtained were tested by PCR for the presence of the DNA injected, using a specific primer of the vector and a primer in the gene (results not shown) . 3. Microscopy The nematodes are observed under a ZEISS Axioplan microscope provided with Nomarski lenses, with 40X Neofluar, 63X Plan-Apochromat, 100X Plan-Apochromat objective lenses. For fluorescence observation the luminous source is a mercury bulb. Different ZEISS filters are used: - for observation under GFP fluorescence, FITC filter: blue excitation line at 588 nm, emission through a 515-565 nm band-pass filter; - for observation of the antibody labelling with a secondary antibody coupled to the TRITC: excitation through a 546 nm band-pass filter, emission through a 590 nm long-pass filter. The image acquisition is effected by means of a CCD camera and and NIH image program using a Machintosh computer. The images are processed using the Adobe Photoshop program. Sequence Listing The following sequences are referred to in the specification: Sequence ID No 1 is an amino acid sequence of human homologue 1 of UNC-53 protein illustrated in Figure 9b. Sequence ID No 2 is an amino acid sequence of human homologue 2 of UNC-53 protein illustrated in figure lid. Sequence ID No 3 is a nucleic acid sequence of the hu-l-unc-53 gene illustrated in Figure 9b. Sequence ID No 4 is a nucleic acid sequence of the hu-2-unc-53 gene illustrated in Figure lid. Sequence ID No 5 is a nucleotide sequence of Phage Lamda Clone 3b deposited under Accession No LMBP 3595. illustrated in Figure 9. Sequence ID No 6 is a nucleotide sequence of plasmid pLMl deposited under Accession No LMBP 3762 and illustrated in fig 54. Sequence ID No 7 is a nucleotide sequence of plasmid pLM4 deposited under Accession No 3763 and illustrated in fig 34. Sequence ID No 8 is a nucleotide sequence of plasmid pEGFP72 deposited under LMBP Accession No 3764 and illustrated in fig 30. Sequence ID No 9 is a nucleotide sequence of plasmid pCB501 deposited under Accession No 3765 and illustrated in fig 57. Sequence ID No 10 is a nucleotide sequence of plasmid pCB201 deposited under Accession No. LMBP 3594. Sε& lO a. Pagτl GArATCrGCAGAATTCGGCrTCTTTGAGCAAGTTCAGCCTGGTTAAGrCCAAGCTGAATrCCGGGGAAAGCCGAύCCGGArCCCrCGAGGACCCTArGC3 CTATAGACGTCTTAAGCCGAAGAAACTCGTTCAAGTCGGACCAATTCAGGTTCGACTTAAGGCCCCTTTCGGCrCGGCCTAGGGAGCTCCTGGGATACG- " " pCR2.1 linker 5C ' lambda gt10 primer EcoRI Λ ■ suspect sequence linker? "— pHH14-3 I S A E F G F F E Q V G P G . V Q A E F R G K P S R I P R 3 P Y , GA3GTCAAGCCGCTCAGCAAGGCGCCTGAAGCGGCC3TGAGCGAAGATGGCAAATCGGACGACGAGCTGCTCTCCAGCAAGGCCAAGGCGCAAAAGAGCT CTCCAGTTCGGCGAGTCGTTCCGCGGACTTCGCCGGCACTCGCTTCTACCGTTTAGCCTGCTGCTCGACGAGAGGTCGTTCCGGTTCCGCGTTTTCTCG ■ pHH14-3 S K A P E A A V S E D G K S D D E L L S S K A K A Q S C75GGCCTGTCCCCTCTGCCAAGGGCCAGGAGGAGCGCGCCTTCCTCAAGGTGGACCCCGAGCTGGTGGTGACCGTGCTGGGAGACCTGGAGCAGCTGC GACCCGGACAGGGGAGACGGTTCCCGGTCCTCCTCGCGCGGAAGGAGTTCCACCTGGGGCTCGACCACCACTGGCACGACCCTCTGGACCTCGTCGACGA ■ pHH14-3 S G P V P S A < G Q £ ϊ a A F L < V O P E L V V T V G D ά Q L L CTTCAGCCAGATGCTGGACCCAGAGTCCCAGAGAAAGAGGACAGTGCAGAATGTCCTGGATCTCCGGCAGAACCTGGAAGAGACCATGTCCAGCCTGCGA 1 i i > 1 i 1 i i i i i m , GAAGTCGGTCTACGACCTGGGTCTCAGGGTCTCTTTCTCCTGrCACGTCTTACAGGACCTAGAGGCCGTCTTGGACCTTCTCTGGTACAGGTCGGACGCT • pHH14-3 0. -ORF (1-579t:pι = pLM7 ORF -full available ORF HU-Unc53/1 = pLMl OR r S G M 0 P E S Q ϊ K R T V Q N V L. D I R Q N L E E T M ≤ S L F GGC~CCCAGGTG ACTCA3- ■ G TCCCTGόAGA " ,::T3:TACGACAGCGATGATGCCAA;CCACGCAGCGΓGTCCAGCCTCTCCAACCG CT:GΓ:CCCT: ■ -C →G G ΓCCAC " AG GT :GAGGGACCrCTi.; ' " GACGATGCTGTCGCTACTACGGTTGGGTGCGTCGCACAGGTCGGAGAGGTTGG A3CAGGGG-3 T- ^ ! ■ ,-, £-i.W; = pL-vl/ w r • - S / r - 3 3 L E " " C Y O S O D A N P S S S L S M R 3 3 F " 3- ArGGC C. ATG3CCA ■ J " C.A;- 2CG " T " ' 7A:GCTGGTGACGCGCCCTCT3TG33T3GGAGCTGCCGCTCGGAGGGGACGCCC G::T23TA -- A:;:ΓACCGCGA "ACC3GT - G T AG5CGCC -- :3I::3ACCACTGCGC33GAGACAC:CACCCTCGAC2GCGAGCCTCCCCTGCGGC 33-3CATGT- - — μHH14-3 ■■■ . I a.ailaclβORF HJ Lnc53/: = .M' CP — / D : : A C C A F 3 / U G J C P 5 E 3 T = Tuesday 1 a November 19971033 / ' ' Page 1 fiς Hu-UncS3/1 seq (1>6013) Site and Sequence G ACGGCGAACGGGCCCACTACTCCCACACCATόCCCATGCGCAGCCCCAGCAAGCTCAGCCATArCTCCCGCCTGGAGCTGGTCGAArcCCTGGAC-"- : T-GCCGCTTGCCCGGGTGATGAGGGTGTGGTACGGGTACGCGTCGGGGTCGTTCGAGTCGGTATAGAGGGCGGACCrCGACCAGCTTAGG3ACCTGAG3 -pHH14-3 -ORF •; 1-573011; = pl_M7 ORF -full available ORF HU-Unc53/1 = pLM1 OR H G E R A H Y S H T M P M R S P S K L S H I S R I E L V E S L D 3 GATGAGGTGGACCTCAAGTCCGGCTACATGAGCGACAGTGACCTCATGGGCAAGACCATGACGGAGGATGATGACATCACTACCGGCTGGGATGAAAGC 1 i i 1 . . I 1 I 1 ' I ■ 1 ' I i i ζ CTACTCCACCTGGAGTTCAGGCCGATGTACTCGCTGTCACTGGAGTACCCGTTCTGGTACTGCCTCCTACTACTGTAGTGATGGCCGACCCTACTTTCG -pHH14-3 -ORF j1-5730D)apLM7ORF -full available ORF HU-Unc53/1 = pLM1 OR C E V O K S G Y M S D S O L M G K T M T E D D D I T T G W D E S GCTCCATCAGTAGTGGACTCAGCGATGCCTCAGACAATCTCAGTTCAGAAGAATTCAATGCCAGCTCCTCACTCAACTCCCTCCCAAGTACTCCCACTG3 1 I , 1 i 1 ' I 1 1 1 I ' ' I ' l ι | , C^ CGAGGTAGTCATCACCTGAGTCGCTACGGAGTCTGTTAGAGTCAAGTCTTCTTAAGTTACGGTCGAGGAGTGAGTTGAGGGAGGGTTCATGAGGGTGAC3 -pHH14-3 -pC8212 -C F ,"-57'jco! = D-.M7 OFF -lull available ORF HU-Uπc53/1 = oLM1 OR S E E T" :TCGCAGGAACTCAACAATAGTGCTACGCACAC CTCAGAGAAGCGCTCACTGGCAGAAAGTGGGCTGAGCTGGTTTAGTGAATCAGA3GAGAAAG : -A;A3CGTCCTTGA3TTGTTATC CGATGCGT3 " CT3A TCTCTTCGCGAGrGACCGTCTTTCACCCGACrCGACCAAAT ACrTAGTCrCCTCTTT':G3 -pHH14-3 -pCB212 -'.II available ORF HU-UncS3/1 = pLM1 OR 3 P R N 3 T i 'J R " C 3 E K R 3 L A E S G L S W F S E 3 Ξ E V β CC-AAAAAACTGGAGTACGACAGTGGTA3CCT3AA;A-3GAACCTGGGACrrCTAAGTGGCGGAGGGAUC3GCCTGAGAGCTGTGATGHTTCArCC AG3 7' 3' " jA rrTTTGACCrCAT3CTGTC CCATCGGAC" " TA3CTTGGACCCTGAAGATTCACCGCCTCCCTCGCCGGACTCTCGACACrACTAAGTAGGTT : -pHH14-3 -pCB212 . I a/a,latle OR c HU Uac53/1 = DLM1 OP E P r S k '.' 5 £ 3 3 E : 3 D 0 Tuesday78 November 199710.33 £ f^«lH ' PageJ fig Hu-Unc53/1 seq (1>6013) Site and Sequence * ^^^^ G-GGAGAACIGAAAAAGCCCATCAGCCTGGGCCACCCTGGTTCCCTGAAGAAGGGCAAGACCCCACCTGrGGCrGTAACTTCCCCCATCACTCACACAG: C-:CrCTrGACrTTTTCGGG1ΑGTCGGACCCGGTGGGACCAAGGGACTTCrTCCCGTTCTGGGGTGGACACCGACATrGAAGGGGGTAGTGAGTGTGr 3 • pHH14-3 -pCB212 -full available ORF HU-Unc53/1 = pLM1 OR G E L K K P I S L G H P G S L K K G K T P P V A V T S P I T H T A CCAGAGTGCCCTCAAAGTCGCAGGCAAACCTGAGGGCAAAGCTACAGACAAGGGTAAGCTTGCAGTGAAGAATACTGGGCTCCAACGCTCCTCCTCTGAT GGTCTCACGGGAGTTTCAGCGTCCGTTTGGACTCCC3TTTCGATGTCTGTTCCCATTCGAACGTCACTTCTTATGACCCGAGGTTGCGAGGAGGAGACTA ■ pHH14-3 ■ pCB212 -full available ORF HU-UncS3/1 = pLM1 OR G S A L K V A G K P E G A T O K G K L A V K N T G Q R S S S D GCTGGTCGGGACCGCCTGAGTGATGCTAAGAAGCCCCCCTCGGGCATTGCTCGCCCCTCCACTTCGGGATCCTTTGGCTACAAGAAGCCTCCTCCTGCCA i i i , i 1 1 i 1 1 1 1 ' I 1 ' I 1 1 ' i 1 1 μ I no.' CGACCAGCCCTGGCGGACTCACTACGArTCTTCGGGGGGAGCCCGTAACGAGCGGGGAGGTGAAGCCCTAGGAAACCGATGTTCTrCGGAGGAGGACGG " • pHH14-3 -pCB212 - full available ORF HU-Unc53/1 = pLM1 OR G R D R L S O A K P ' S G I A R P S T S G S F G Y P P P A CA3GCACAGCCACTGTCATGCAAACTGGTGGTTCAGC3ACTCTCAGCAAGATCCAGAAGTCCTCAGGCATCCCTGTCAAGCCAGTAAATGGGCGCAAG^: G " 3C3TGTCGGTGACAGTACGTTTGACCACCAAGTC33TGAGAGTCGTTCTAGGTCTTCAGGAGTCCGTAGGGACAGTTCGGTCATTTACCCGCGTT T3 -pHH14-3 ■ pCB212 -full available ORF HU-Unc53/1 = pt_M1 OR T G T A T V M Q T G G S A T S K t Q K S S G I P V K P N G R T "i: rrA3ATGTTrCCAAC GT3CAGAGCCAGGA-T:;T3GCrCCTGGAGCCCGTTCTAACATCCAGTAC:GCAGCCTGCCCCGGCCAGCC AG CAAύ _ A":GAATC'ACAAA3 TTGrCΔ:GrCTC33TCCrAA3: CCGAGGACCTCGGGCAAGArTGTAGGrCATG3CGrCGGACGGGGCCGGTCG3TrC GT7C« -pHH14-3 -pCB212 lull a.ailable ORF HU-Unc53/1 = puM1 OR Tuesday78 November 19971033 r < 3 ' Page f|2_ Hu-Unc53/1 seq (1 > 6013) Site and Sequence __ ^ rcrArGAGCGTGACCGGCGGGCGGGGTGGACCTCGCCCTGTGAGCAGCAGCATTGACCCCAGrCTCCTCA3CACCAAGCAGGGAG3CC77ACGCCTrC A AGArACTCGCACTGGCCGCCCGCCCCACCTGGAGCGG3ACACTCGTCGTCGrAACTGGGGTCAGAGGAG7CGTGGTTCG7CCC7CCGGAATGCGGAAGG- -pHH14-3 -pCB212 -pCB210-14 -full available ORF HU-Unc53/1 = DL 1 OR - rev Bfipwr Hϋ53r.'-I 1 I 5 M S V T G G R G G P R P V S S S I 0 P S L L S T K Q G G L T P 3 GACTGAAGGAGCCTACCAAGGTAGCCAGTGGGCGGACCΔCTCCAGCCCCTGTCAATCAGACAGATCGGGAAAAGGAGAAGGCCAAAGCCAAGGCAGTGG3 C7GACTTCCTCGGATGGTTCCATCGGTCACCCGCCTGGTGAGGTCGGGGACAGTTAGTCTGTCTAGCCCTT7TCCTCTTCCGGTTTCGG7TCCGTCACC3 -pHH14-3 -pCB212 -pCB210-14 -full available ORF HU-Unc53/1 =pLM1 OR R I K E P T K V A S G R T T P A P V N O T D R E K E K A K A K A V * CT7GGACTCAGACAACATCTCCTTGAAGAGTAT7GGCTCCCCAGAAAGTACTCCCAAGAACCAAGCAAGCCACCCCACAGCCACCAAGC7GGCAGAGCT3 1 ( 1 I , ι l I I i I I 1 1 ' 1 1 ' ' 1- ' ' ' ' . GAACCTGAGTCTGTTGrAGAGGAACT7CrCA7AACCGA3GGGTCTTTCATGAGGGT7CTTGGTTCGTTCGG7GGGGTGT_GGTGGTTCGACCGTCT_GA3 -pHH14-3 ■ pCB210-14 full available ORF HU-Unc53/ =pLM1 OR L D S O N I S L .< S I G S P E S T P M Q A S H P T A T f. L A E CC CCAACCCC7CTCAG3GCCACAGCGAAGAG 7773":AAACCACCCTCACTAGCCAArC7rGACAA3GrCAAC7CCAACAG7CrGGA 3TA3:ArCA " GG7G37TGGGGAGAGTCCCGGTG7CGCTTCTCGAAACA37T7GGTGGGAGTGATCGGT7AGAACTGrT3CAGTrGAGG77GrCA3ACC * A3A7GGTAGTA -pHH14-3 ■ pCB210-14 :jll a.a.iable ORF HU-Unc53/1 = o 1 OR L 0 Tuesday, 18 November 19971033 ' Page 5" lie, ,Hu-Unc53/1 seq (1 >6013) Site and Sequence CCAGT3A 7ACCACCCArGCTrCAAAGG7CCCAGA-CTCCATGCTACAAGCrCAGC ATC 7G3GGGCCCrCTCCC T7CC 7GCTrCACCCCCAGTc:3GCAC , 1 i I 1 i I 1 ' ' ' ' ' 1 ' i ' . I L£ ^| • GGrCACTATGGTGGGTACGAAGTTTCCAGGG-CTAGACGrACGATGTTCGAGTCGTAGACCCCCGGGAGAGGGAAGGACGAAGTGGGGGTCAGGCCGTG'- " " -pHH14-3 -pCB210-14 -tull available ORF HU-Uπc53/1 = pLMl OR S S D T T -t A S < P 3 L H A T S S A S G G P L P S C F T P S P A F CATCCTCAATATTAACTCAGCCAGCTTCTCCCA3GGCCTGGAGCTAATGAGTGGTTTCAGTGTGCCAAAAGAGACCCGCATGTACCCCAAACTCTCAGG3 GTAGGAGTTATAATTGA3TCGGTCGAAGAGGGT3CCGGACCTCGATTACTCACCAAAGTCACACGGTTTTCTCTGGGCGTACATGGGGTTTGAGAGTCC3 -pHH14-3 -pCB210-14 -full available ORF HU-UncS3/i =pL 1 OH [ L N t N S A S F S Q G L E L M S G F S V P K E T R Y P K L S G CTGCACAGGAGCATGGAGTCCCTCCAGATGCCAATGAGCCTCCCCAGTGCCTTCCCCAGCAGTACTCCCGTCCCCACCCCACCTGCTCCCCCTGCTGCTC GACGTGTCCTCGTACCTCAGGGAGGTCTACGGT7ACTCGGAGGGGTCACGGAAGGGGTCGTCATGAGGGCAGGGGTGGGGTGGACGAGGGGGACGACGA -pHH14-3 ■ pCB210-14 -' ■ l available ORF HU-Uπc53/1 = DLM1 OP. CCA A3AAGAAGAGAC3GAAGA3CTGACTTGGi:"G3AAGCCCCAGAGCTGGGCAACTGGACAGTAATCAGCGGGATCGGAACACrCTTCCCAAGAAAG3 GG7GTCTTCTTCTCT3CCTTCT:GACTGAACC7;ACCTTCGGGGTCTCGACCCGTTGACCTG7CATTAGTCGCCCTAGCCTTGTGAGAAGGGTTCTTT : -pHH14-3 -pHH3b -pCB210-14 ,ιl ava.lable ORF HU-Uπc53/l = oLMi OR *ev -,-5C" , /3 . Ό,' pr .τ»r HUMr»-2 -Bsp-de B.'2o2σH F Γ E E E T . Ξ . Γ V ; : S P A G Q L O 3 N Q R D R N T L P K 'J Tuesday 18 November 19971033 J Page * fig riu Unc53/1 seq (1 >6013) Site and Sequence G:"CAC TACCAGCTTCAGTCCCAGGAGGAGACCAAGGAGAGGCGACATTCCCATACCArTGGTGGGCTGCCTGAATCCGArGACCAGrCAGAGCT3CC" CG-GTCCA7GGTCGAAGTCAGGGTCCTCCTCTGGTTCCTCTCCGCTGTAAGGGTATGGTAACCACCCGACGGACTTAGGCTACTGGTCAGTCTCGACGGH -pHH143 -pHH3b 1 - re*/ pripier -i«-i>Jr / 1 I -full a.ailabiβ ORF HU-UπcS3/1 = plMI OR L R Y Q I Q S Q E E T K E R R H S H T I G G L P E S D D Q S E L F TCTCCCCCTGCACTTCCCATGTCTCTGAGTGCAAAGGGCCAACTTACCAACATAGTGAGTCCCACTGCGGCCACCACGCCAAGAATCACCCGCTCCAALH AGAGGGGGACGTGAAGGGTACAGAGACTCACGTTTCCCGGTTGAATGGTTGTATCACTCAGGGTGACGCCGGTGGTGCGGTTCTTAGTGGGCGAGGTTG7 -pHH14-3 -pHH3b -full available ORF HU-Unc53/1 =pLM1 OR S P P A L P M S L S A K G Q L T N I V S P T A A T T P R I T R S H GCArcCCCACCCACGAGGCGGCCTTCGAGCTGTACAGCGGCTCCCAAATGGGGAGCACCCTGTCCCTGGCCGAGAGACCCAAGGGAATGATTCGGTCAGC CGTAGGGGTGGGTGCTCCGCCGGAAGCTCGACA-GTCCCCGAGGGTTTACCCCTCGTGGGACAGGGACCGGCTCTCTGGGTTCCCTTACTAAGCCAGTC: -pHH14-3 -pHH3b Till a.ailable ORF HU Unc53/1 =pLM1 OR I P T H E A A F E L ' S G S Q M G S T S A E R P K G M I R S '. ■ T7C:3AGACCCCACGGACGATG-TCACGG:TCA:73CT3TCCCTGGCCTCCAG7GCC7CCTCCACCTACTCCT:AGCT3AGGAGAGGATGCAA7I-- 7AAG3CTCTGGGGTGCCTGCTACAAGTGCC3AGT:Δ:GACAGGGACCGGAGGTCACC3AGGAGGT3GATGAGGAGTCGACTCCTCTCCT« GTTAJ« -pHH143 -pHH3b -'.II a/a lacle 0R C HJ Unc53/1 = p_ 1 OP " R D P 7 0 D v H G o l. 3 A 3 S A S 3 7 Y 3 3 A E E 3 , 'I Q 3 ^ -.AAAT:CGGAAGCTTCGTAGGG A ACTGG A A-CAΓC::AG3AAAAAGTGGCCACC TUACGTCTCAGCTTTCTGCCAATGCTAATCTGCTGGCTGCT- C " 'Grr7A3GCCTTCGAAGCATCCCTT ACCTTAC"A33:TCCTTTTTCACCGGTGGAACT3CAGAGTCGAAAGACG3TTACGATTAGACCACCGACGA- - _____ _ -T7TΪ [C[ i " < . [ ., ~ : r. ~ ■ pHH3b - 1 a, 3 atl » OR HU tnc53/ι = n.'v OP I R < ι R P E L - i E < V A T L 7 b α S - N . N L Tuesday.18 November 19971033 Page 7 fig Hu-Uπc53/1 seq (1 > 6013) Site and Sequence TrGAGCAGAGCCTGGTGAATATGACATCCCGCCTG GACACCTGGCAGAGACGGCCGAGGAGAAGGACACTGAGCTGCTGGATTrGCGAGAAACCArAGA AACTCGTCTCGGACCACTTATACTGTAGGGCGGACGCTGTGGACCGTCTCTGCCGGCTCCTCTTCCTGTGACTCGACGACCTAAACGCTCTTTGGTA-C- -U2 ORF = pCSICSI OPF -pHH3b -full available ORF HU-Unc53/1 = pLM OR F E Q S L V N M T S R R H L A E T A E E K D T E L D L R E T I D CTTTCrGAAGAAAAAGAACTCTGAGGCCCAGGCAGTCATTCAGGGAGCCCTTAATGCCTCAGAAACCACACCCAAAGAACTTCGGATCAAGAGACAAAA3 GAAAGACTTCTTTTTCTTGAGACTCCGGGTCCGTC 3TAAGTCCCTCGGGAATTACGGAGTCTTTGGTGTGGGTTTCTTGAAGCCTAG77CTCTGTTTT3 • U2 CR - p SZSi OR -pHH3b -full available ORF HU-Unc53/1 = pLMl OR F L K K K N S E A Q A V I Q G A L N A S E T T P K E L R I v R Q H TCCTCAGATAGCATCTCAAGCCTCAACAGCATCACTA3CCATTCCAGCATCGGCAGCAGCAAGGATGCTGATGCGAAAAAGAAGAAAAAAAAGAGTTGGG AGGAGTC7ATCGTAGAGTTCGGAGTTGTCGTAG7GATCGGTAAGGTCGTAGCCGTCGTCGTTCCTACGACTACGCTTTTTCTTCTTTT77TTCTCAACC: - ? ORF : ■ ■ pd'.?.:Λ OF" ■ pHH3b full available ORF HU-Unc53/1 = pLMl OR S S 0 S I S S L N S [ T S H S S I G S S < O A O A K K < K K K S , - TC7Ar3AGCTTCGAAGTTCCTTCAACAAAGCGT7CA3-ATAAAAAAGGGGCCCAAGTCAGCTTCCTCATACTCGGATATAGAGGAGAT-G:TACAC:C'jA AGA7ACTCGAAGCTTCAAGGAAGTTGTTTCGCAAG73ATATTTTTTCCCCGGGTTCAGTCGAAGGAGTA73AGCCTA7ATCTCCTCTAA.:3ATGTG3G ' ■ pHH3b 'ull available ORF HU-Unc53/1 = pLMl OR v ι' E L R S S F < A - S I K G P K S A S S ' S O Ϊ Ξ E I A T F C' C7:'TCAGCCCCCTCATCCCCCA ACrA3aGC -CG r -:TAC GACACTGCTTCACCCTCCArCAAGT:C7CCACCT7G-CCTCCGTGCG:HCTGATG7: ιiA3AA:TCGGGCGAG7AGGGGGT 73AT3TCG * A:CA ATGTCTCTGAC3AAGTGGGAGGTAGTTCACGA3GTGGAA':AGGAGGCA ':3TGA3TACj-j -pHH3b - ■ '.'I a.a.lable CRF HU-L'-ιc53/i = OLMI OP A P S S P K L O - C S T E T A S P S I K S J T L S S 'K C T O V Tuesday.18 November 199710.33 < Pagef fir Hu-Unc53/1 seq (1 >6013) Site and Sequence ^^^ ACCL .3CCCTGCTCACCCAGCCCCCCACACTAGGCTGTTCCATGCAAATGAGGAGGAGGAGCCAGAGAAGAAGGAGGΓAΓCGGAG 7GCGCTCTGAG:- TGGCTCCCGGGACGAGTGGGTCGGGGGGTGTGATCCGACAAGGTACGTTTACTCCTCCTCCTCGGTCTCTTCTTCCTCCA7AGCCTC ACGCGAGAC7C" " " -U2GRF = pCB2S! OR^ -pHH3b -full available ORF HU-UncS3/1 = pL 1 OR T E G P A H P A P H T R L F H A N E E E E P E K K E V S E L R S E TATGGGAGAAGGAAATGAAGCTTACAGACATCCGCTTGGAGGCCCTCAACTCTGCCCACCAACTGGATCAGCTTCGGGAGACCATGCACAACATGCAGT " ATACCCTCTTCCTTTACTTCGAATGTCTGTAGGCGAACCTCCGGGAGTTGAGACGGGTGGTTGACCTAGTCGAAGCCCTCTGGTΔCGTGT7GTACGTCAA • U 0 - pC825 OR -pHH3b -DeDtMeB72627H 3 -lull available ORF HU-Uπcό3/1 = DLM1 OR • U3 ORF = pLM5 ORF L E K E M K L T O I R L E A N S A H Q L D Q L R E T M H M Q L GGAGGTGGACC7GCTGAAAGCAGAGAAT3ACCGACT3AAGGTAGCCCCAGGCCCCTCATCAGGCTCCACTCCAGGGCAGGTCCCTGGATCATCTGCATTA CCTCCACCTGGACGACTTTCGTCTCTTACTGGCTGACTTCCATCGGGGTCCGGGGAGTAGTCCGAGGTGAGGTCCCGTCCAGGGACCTAGTAGACGTAA " ■ pHH3b -:ull available ORF HU-Uπc53/1 = 3l_M1 OR -U30RF = pL.M50RF E v D L L K A E N D R L K V A P G P S S G S T P G Q v P G ≤ S A L 7C77CCCCACGCC3CTCCCrAGGCCTG3CAC7CACCC TTCCrTCGGCCCCAGTCTTGCAGACACAGACCTGTCACCCA7G3A7GGCA;C TACTrGT: AG AA GGGGTGCGGCG A GGGA7CCGG A C:3TG A G-GG37Δ A GG AA GCCGGGGΓC A GA A CGΓCTGTGTCTGGAC A GTGGGT A CCT A CCGT A GΓC A TG ': - ■ pHH3b ll available ORF HU-Unc53/1 = o 1 OR -U30RF -pLMSORF ≤ S P R ' S L G L A L Γ - S F G P S L A D T D L S P M D G : 3 T ■ _ G Tuesday 18 November 19971034 '" " 'J Page 9 ti, 'u-Uπc53/1 seq (1>6013) Site and Sequence ___ ^ Gr;CAAA3GAGGAAGTGACCCTCCGGGTGGTGG7GA33ArGCCCCCGCAGCACATCA7CAAAGGGGACTrGAA3CAGCA3GAA7rcrr TGGGCT37A3 CA3GrTTCCTCCTTCACTGGGAGGCCCACCACCACTCCTACGGGGGCGTCGrGTAGTAGT7TCCCC7GAACTTCGTCGT3CrrAAGAAGGACCCGA:AT3 * '"' -U20RF = pCBϊS1 P= -pHH3b -full available ORF HU-Uncβ3/1 = pLM1 OR -U30RF = pLMSORF G P E E V T R V '/ V R M P P O H I I K G O L Q Q E F F U G C S CAAGGTCAGTGGAAAAGTTGACTGGAA3ATGCT3GATGAAGCTGTTTTCCAAGTGTTCAAGGACTATATTTCTAAAATGGACCCAGCC7CTACCCT3GGA GT7CCAGTCACCTTTTCAACTGACCTTCTACGACC7ACTTCGACAAAAGGTTCACAAGTTCCTGATATAAAGATTTTACCTGGGTCGGAGATGGGACCCT -pHH3b -full available ORF HU-Uπc53/1 = pLM1 OR -U3θRF = pLMSORF K V S G K V D U K M L D E A V F Q V F K D Y I S K M O P A ≤ T C7AA3CAC7GAGTCCATCCATGGCTACA3CA"CAGC AC TGAAACGAGTGTTGGATGCAGAGCCCCCCGAGATGCCTCCTTGCCGTC'3AG3TGTCAATA GA7TCGTGACTCAGGTAGGTACCGATGTCGTA'3-CG3T3CACTTTGCTCACAACCTACGTCTCGGGGGGCTCTACGGAGGAACGGCAGC7:CACAGT7A7 -ι;.'Of- ' <- - μOϋfcϋi Ot-- -pHH3b -_ ORF = pC320* ORF ll a ailatle ORF H'J-Unc53.'1 = oLM1 OP -U30RF = pLM50RF -pHHIS S T E S I H G U 0 A £ M P " 7CAGrCTCCCTCAAAGG"CT3AA3G : AA"::3rCG CA3CC7G3rGT7CGAGA:3CTGA"CCCCAA3CCGA7GA73CA3CA "^:ArAA3 " 'j"Ar GrcAGAGGGAGTτrccA C7τ:c": * "" : :AGcrGTCGGAc:ACAAGC7cr::GAC7 G3G37r GGcτAC " ;::GT: r 7A-T3 -pHH3b - 40RF = pCB20l OR= a. a lad-; ORF HU-Unc53/1 = R.WI OP - U3 ORF = pLMS ORF — pHH15 — fi i ■ v • ; L • G _ • -: : i; 3 L F E r <> k P ' • ' " 'J Tuesday 18 November 19971034 > ^J PagetΛ tig tu-Unc53/1 seq (1 >6013) Site and Sequence __________ ^ C7 -:rGA4GCACCGGCGCCTCGTCC7CTCGGGCCCCAGCGGCACGGGCAAGACCTACCrGACCAATCGCrrGGCCGA AC 733TGGA33GCTCTGG3 GGAC3ACT-CGTGGCCGCGGAGCAGGAGAGCCCGGGGTCGCCGTGCCCGTTCTGGATGGACTGGTTAGCGAACCGGCTCATG ACCiCC7:GC3AGACC3 • U20P? = pCB2S! OR= -pHH3b -U4ORF = pCB201 ORF -full available ORF HU-Unc53/1 a pLM1 OR -U30RF = pLMSORF -pHH15 L L H R R L V L S G P 3 G T G T Y L T N R L A E Y U V E R S G7GAGGTCACAGAGGGCATCGTCAGCACCTTCAACATGCACCAGCAGTCTTGCAAGGATCTGCAACTGTATCTTTCCAACCTAGCCAACCAGATAGAC: GCACTCCAGTGTCTCCCGTAGCAGTCGTGGAAGT7GTACGTGGTCGTCAGAACGTTCCTAGACGTTGACATAGAAAGGTTGGATCGGTTGGTCTATCTG3 -U20RF = pCB2S1 ORF • pHH3b ■ U4ORF = pCB201 ORF full available ORF HU-Unc53/1 = pLM1 OR -U3QRF = pLMSORF -pHH15 = £ ; 7 E 3 i 3 T F M .« H Q Q S C K D L Q L Y L S N L A fl Q I G3:AAACAGGήA7T33G:ArGTGCCCCT3GTGA-7CTArrGGATGACCTGA3TGAAGCAGGCTCCA7CAGTGAGrTGGTCAATGGGGC CT3 CCTG pA C'::"77Grc r7AACCC:TACACGG3GACCACTAA3A7AACCTACTGGACTCACTTCGTCCGAGGTAG7CACTCAACCA37rACC;CG GA3TGGA3GT " -l.^ORF:=pCB.:Si F<- -pHH3b -U4ORF = pCB201 ORF -7I available ORF HU-Unc53/l = pLMl OR -U30RF = pLM50RF D 3 L 3 E A E L i 'I G r c t WO 98/24810 Λnn PCT/EP97/06956 108 Tuesday.18 November 19971034 K 1 S ' Page 11 fig Hu-UncS3/1 seq (1>6013) Site and Sequence _ G-A7C .iAATGrCCCrA7ATTA7AGGrACCACCAAr:AGCCTGTAAA A Ar3ACACCCAACCATGGC77GCACr7GAGC7rCA33ATG r 7GAC:-T--, CArAGrATTTACAGGGATATAATATCCATGGTGG 7AGTCGGACATTTTTACTGTGGGTrGGrACCGAACGTGAA.:TCGAAGTCCTiCAACTG3 A3A'j" "" -L CPF = pC32Si OR= -pHH3b -U4ORF = pCB201 ORF -full available ORF HU-Unc53/1 =pL 1 OR -U3 ORF = pLM5 ORF -pHH15 -pepi.de B72529H Y H K C P Y I I G T T N Q P V K M T P N H G U H L 3 F R M L T F 3 AACAACGTGGAGCCAGCCAATGGCTTCCTGGTTCGTTACCTGAGGAGGAAGCTGGTAGAGTCAGACAGCGACATCAATGCCAACAAGGAAGAGCTGCTT3 773TTGCACCTCGGTCGGTTACCGAAGGACCAA3CAATGGACTCCTCCTTCGACCATCTCAGTCTGTCGCTGTAGTTACGGTTGTTCCTTCTCGACGAA3 -U_OP = pCB2Si ORF -pHH3b -U4ORF = pCB201 ORF full available ORF HU-UπcS3/1 = o M1 OR -U30RF = p M50RF -pHH1S N N V E P A , , I G F L R r' L R R < L V £ S O S 0 I N A N K E E L G'j "jCTC CTGGGTA:c:AAGCr3"3GTA-':A-Cr:CACACCTTCC"TGAGAAGCACAGCACCTCA3A TTCCTC"TCGGCCCTTGCTTCTTTCTGT. C3 C A3':TGACCC T:G3TTCGACACCATAG " AGACGTGTGGAAGGAACΓCTTCG7GTC3TGGAGTC73ΔA3GAG-A3CCGGGAACG AGAAAG ': : - i PF pCbr:. « f- -pHH3b -U40RF = pCB201 OP i = 71 a.a.lable ORF HU-UιcS31 = pLMl OR -U30RF = pL 50RF -pHH15 — L E K Tuesday.18 November 19971034 r ' ^ ' Page tt- fig .1 J-Unc53/1 seq (1>6013) Site and Sequence G-37:C':A7TGGCA7T3AGGACTTCCGGACC7GGT 3ArrGACCTG7GGAACAACTC7ArCArTCCCTA " 37A;AGGAA3GA3C:AΛ.3GΛr3G3AT A-" ^i3 33G7AA':CGTAACTCCTGAAGGCCTGGACCAA 7AACTGGACACCTTGTTGAGATAGTAAGGGA7A3A73T ι ;C T3C7C3GrTC.:7A:C37AT-7- -U20RF = pCB S! ORF -pHH3b - U4 ORF = pCB201 OPr -full available ORF HU-Unc53/1 = pLMl OR ■ U3 ORF = pL 5 ORF -pHH15 C P l G I E D F R T W F I D L V N N S I I P Y L Q E G A k j ό l t GrcCATGGACAGAAAGCTGCTTGGGAGGACCCAGTGGAATGGGTCCGGGACACACTTCCCTGGCCATCAGCCCAACAAGACCAArCAAAGCTGTACCAC; CAGGTACCTGTCTTTCGACGAACCCTCCTGGGTCACCTTACCCAGGCCCTGTGTGAAGGGACCGGTAGTCGGGTTGTTCTGGTTΔGTTTC3ACA7G3-G3 -U20RF = pCB2S! CF<.r -pHH3b ■ U4ORF = pCB201 ORF -full available ORF HU-tJncS3/1 =pLM1 OR -U3 ORF = p M5 ORF -pHH15 - : 0 < A A E D P ' / £ R 0 T L O W = S A 2 0 D C S K L i H :CCAI:CCACCGT3GGCCCTCA;AGCAT7GCC C :CTCCCGAGGATAGGACAGTCAAA3ACAGCAC::CAAG77C7 7GGA TCAGA " C3-CTG- " Ϊ3' TGGG7GGCA:CC3GGAGT37CGTAAC3G 3-3GAGGGC7CCTATCCTGTCAGTTT;7G7CG7G33G 7I:AAGAG CCΓGA'J " 7 G3 G ':T- - IS: OR : CB.:=! or -pHH3b - U4 ORF = pCB201 OFF full a.δ.lable ORF H'J-L.'ncS3.'1 = p M1 OF ■ U3 ORF = pLM5 ORF — pHH15 3 P P T V 3 P H S I A ≤ ? P £ 0 R T 0 3 * P o 3 D 3 : Tuesday.18 November 19971034 r / 7 ' Page 11 fie -Iu-Unc53/1 seq (1 >6013) Site and Sequence _ ^^^ GGCCArGC7GCTGAAACTTCAAGAAGCTGCCΔAC7 A CArrGAGTCTCCAGArCGAGΔAACCArCCTGGACCCCAACCTrCA33CAACΔCrr7AAGGGTT- C 3GrACGACGACTTTGAAGTTCTTCGACGG7T3A7GTAACTCAGAGGTCTAGC7C 7TGGTAGGACC7GGGGT7GGAA37CCGTTGTGAAATTCCCAtt3 " ->. • -J2 OF.F = pC3251 ORF -pHH3b -U4ORF = pCB201 ORF -full available ORF HU-Uπc53/1 = pLMl OR D. ■ U3 ORF = pLM5 ORF 3 -pHH15 -peptic-t 372Q25H A M L K L Q E A A N Y I E S P O R E T I L D P N L Q A T L G F GGCAATCACTG7CACCCCCGGACAGCAGAACGC7GGCATCAGCTATCTTAGCTCCTCCTC7CCCCTCTCCTCTTTCAGAGCACTGGCTCTCCAGCCCCA3 I I I I I I I ' I I I I ' I I 1 l ι | ^J " CC3TTAGTGACAGTGGGGGCCTGTCGTCTTGCGACC3TΔGTCGATAGAATCGAGGAGGAGAGGGGAGAGGAGAAAGTCTCGTGACCGAGAGGTCGGGGTC -pHH3b -pHH15 3 N H C H O R T A E R H Q S L L S P L L F Q S T G 3 P A P ύA3GA3AACAGGAGGGA3GAGGAGATGAAAGA3:A333A;AGGTTC7TGG7GCTGTACCrTT3AGAAC77CCTAGGAAGGAArGG7GGGG73GCGTTTG3 C " 3CTCTTGTCCTCC:TC TCCTCTACTTTC7C: " :::T3TCCAAUAACCACGACA-GGAAACTC-TGAA3GATCCT7CCTTACCACCCCACC3CAAAC: -pHH3b -pHH15 'J 3 E 0 Ξ 3 3 G 0 £ 5 3 3 T G S V C C T F E N F L G R M G G '.' A F ■ . GiACTTGTGCCCCCTAAACACATTTACTGGCCTCC-C-AATGACTTTGGGGAAAAGATGATTCTGGGTCTrTCCCTTGACTTC rGTTTCAATTACAAe: C ' 7CAACACGGGGGATTTGTGTAAATGACCGGACCA3 TTACTGAAACCCCTTTTCTACrAA3ACCCAGAAAGGGAACTGAA3AACAAAGr AA-GTTT3 -pHH3b -pHH15 f. C P L M T F T G L L . L '« G K 0 D 5 G S F P I L V S I T tl ■ '" 733GC"TTCT3G33AG3GGTTCAGAAAAC -:iAAA:ACrGCAGC GrTCCTAAATGATrCTCACAAG AACCC7GAGA3AG CAG" 7737GAGϋ3 i 3ACCCGAAAGACCCCTCCCCAAGTCTTTTGTA;"T-T3TGACGTC3TCAAGGATTTA:TAAGAG7GTT:GTTGGGACTCTCTCTGTCAjAAC CTCC: 1 pHH3b [ pHH15 ■ A F w 3 G V o ► r : • H C 3 ; 3 l I . " 3 '• p E Tuesday.18 November 199710:34 ( ' 5 fig Hu-Unc53/1 seq (1 > 6013) Site and Sequence . AGArcrGGGGGAGGCAGGAAGCTCCTCAGATTT7CT;ACAGACCC7TCCCAATTCCATCACCACTGCCAACAA:7CCrCCCCCAGAGA7CrόGCTG3AG- I I I I I I I l I I I I ' i j " - 7 • TC7AGACCCCCTCCGTCCTTCGAGGAGTCTAAAAGAGTGTCTGGGAAGGGTrAAGGTAGTGGTGACGGTTGTTGAGGAGGGG3TCTCTAGA3CGACC7C3 ' "" — PHH15 — Q ε p o I S Q T P N S t T T N M 0 L A CCAGAAAAAGAAGCATG7GGTTTAAAAAATG7TTAAATCAATCTGTAAAAGGTAAAAATGAAAAAACAAAAACAAGCAAACAAACAAAAAACAATGGAAA GGTCTTTTTCTTCGTACACCAAATTTTTTACAAATTTAGTTAGACATTTTCCATTTTTACTTTTTTGTTTTTGTTCGTTTGTTTGTTTTTTGTrACCTTT O K K K H V V . K M F K S I C K R . K . IC N K N K Q T N K K Q V t AGATGAAGCTGGAGAGAGAGGAACCAGTTGCCAAGGTAGAGAGCTGCCCGCTCCTGCCCTCrGGATGACATAGGGGACATCAACAAGACGGCTGCCAA , 7C7ACTTCGACCTCTCTCTCCTTGGTCAACGG77CCATCTCTCGACGGGCGAGGACGGGAGACCTACTGTATCCCCTGTAGTrGTTCTGCCGACGGT7G3 3 . S V R E R N Q I P R . R A A R S C P L. 0 0 I G D I N K T A A N TGAGAAGTCACCAAACCACAAAAATAACCTTACAGCCTTCAGGGAAAGACTACCAGCTCTGTCTTTCTACCCTCTAATTTAACAATGCACCGGAATTCA3 ACTCTTCAGTGGTTTGGTGTTTTTATTGGAA7GTCGGAAGTCCCTTTCTGATGGTCGAGACAGAAAGA7GGGAGATTAAATTGTTACGTGGCCT-AAGTC •-linker? - L R S H Q T T K I T L C P S G K O Y Q L C S T L F N N A P E F •> C77GGACTTAACC ' » 5013 GAACCTGAATTGG -linker? 1. 0 L T Page 1 9?0#o4?_>3 tccotAcrcc ε ε ε CO O _Γ_(3 ID *J» 2. ,* «__<-<-E « <C_. -£ H-VΛ- c_/a P »τe"< oo 00 Ox 0 £S7<<_cτ,S ?- .£. CCAGCACΛGC fST σ i Λ \ 1 I IΛ I I 1 I t I II 1 I 1 I 11 r uimiiii 0 • <*0 esτ*.*_v?-pϊ, 00 ON O F S Q M L 7CCAGGTC ACTCACA όCTCCC -GGAGA-:: .::T3:TACGACAGCGAr3ATGC ;A :C: ACG -GCGΓGTCCAGCCTCTCCAACCC .CTCGTCCCCTC c: GJGΓCCAC ΓGAG Γ GT CGAGGG ■ .3ACGATGCTGTCGCTACTACG 3TTGGG iTGCG ' rCCCACAGGTCGGAGAGGTTGG'! GAGCAGGGGώG Tuesday.18 November 19971033 ' ' Page — (I Hu-UncS3/1 seq (1> 6013) Site ano .cuence G': A GGCGAACGGGCCC C ΔCrCCCACACCAT3CCCArGCG:AGCCCCAGCAAGCTCAGCCArArcrCCCGCCrGGAGCr3G " CGAΔrr__TG-A-r-- :G-GCCGCTTGCCCGGGTGATGAGGGrGrGGrACGG3TACGCGTCGGGGTCGTTCGAGTCGGTATAGΔGGGCGGACCrCGACCAGCTTAG33AC TG •pHH14-3 - OF-F ■; 1 -573-0) = pLM7 ORF -full available ORF HU-UncS3/1 = pLM1 OR G ε L S H I S R 0 3 GATGAGGTGGACCrCΔAGTCCGGCTACATGAGCύACAGTGACCTCATGGGCAAGACCATGACGGAGGATGATGACATCACTACCGGCTGGGArGAAAGC C7ACTCCACCTGGAGTTCA3GCCGATGTACTCGC7GTCACTGGAGTACCCGTTCTGGTACTGCCTCCTACTACTGTAGTGATGGCCGACCCTACTTTCG ■ pHH14-3 - OB.F ( 1-57300) = p M7 ORF ull available ORF HU-Unc53/1 = pL 1 OR _ E V D L K S G Y M S 0 S D L M G K T M T E D D D I T T G V D E 3 CTCCATCAGTAGTGGACTCAGCGATGCCTCAGACAATCTCAGTTCAGAAGAATTCAATGCCAGCTCCTCACTCAACTCCCTCCCAAGTACTCCCAZTG: CGAGGTAGTCATCACCTGAGTCGCTACGGAGTCTGTTAGAGTCAAGTCTTCTTAAGTTACGGTCGAGGAGTGAGTTGAGGGAGGGΠCATGAGGGTUA G available ORF HU-Unc53/l = OLM1 OR 5 1 3 S G '. S - A S : L S S C E F N A 3 S 5 L M 3 L P S T P T * ' 3 TCGC AGG AACTCΔ ACA A" Aό TGC TACGCACA: C T AGAGAAGCGCTCACTGGCAGAAAGTGGGCTGAGC TGG TTT4GTGAATC AG AOGAGAAAG : -:A3CGTCCT7GA3rTC"7ATC C3A73CG-G " " 3 3TCTCTTCGCGAGrGACCGTCTTTCACCCGACrCGACCAAArCACrTAGTCrCCTCTTTCG3 -pHH14-3 ■ pCB212 .:i available ORF H_-UncS3/1 = pLMl OR ■ 3 R r4 3 7 " V . 5 - ; 3 E < R 3 L A E S G L S V F S E 3 £ £ » ^ ';';"AAAAΛACTGGA3TACCA:A373C"A3CC-:--7"3αAACC 3GόACrrCTAAGTGGCGGAGGGA3C3GCC7GAGAGCr3"3A7'3A77:ArCCAAG3 37 " rr7r7'3ACCrCAT;.;-3TCACC 7C33A:"7A3CrT3GACCC GAAGA7TCACCGCCTCCCTCGCCG3ACTCTCGACACrAC7AA3TA3GT7LC -pHH14-3 • pCB212 α._.latl- O a = HU Lπc53 : = oLMi OP ' ■■ ε - z r s μ . ■ a P Tuesday.18 November 19971033 r""/. ° ' P-t&e-J fig Hu-Unc53/1 seq (1 > 6013) Site ar ,equerce '3 " C AύAACr'3AAAA A GCCCATCAGCCr3GGCCACCC7GGTTCCC7GAAGAAGGGCAAGACCCCACC73r33CrGrAAC7rCCCCCA7CA CACACA'3C '73CrcrTGACrTTTTCGG3rAGTCGGACCCGG7GGGACCAAGGGACTTCrTCCCGTTCTGGGGTGGACACCGACATTGAAGGGGGTAGTGAGTGTGT.-- "' -pHH14-3 -pC8212 -full available ORF HU-Unc53/1 = pL 1 OR G S T S P I T H T CCAGAGTGCCCTCAAAGTCGCAGGCAAACCTGAGGGCAAAGCTACAGACAAGGGTAAGCTTGCAGTGAAGAATACTGGGCTCCAACGCTCCTCCTCTGA- G'3-CTCACGGGAGTTTCAGCGTCCGTTTGGACTCCC3TTTCGATGTCTGTTCCCATTCGAACGTCACTTCTTATGACCCGAGGTTGCGAGGAGGAGACT -pHH14-3 -pCB212 -full available ORF HU-UncS3/1 = pLM1 OR O S A - K V A G K P E G K A T D K G K L A V K N T G L Q R S S S O GCTGGTCGGGACCGCCTGAGTGATGCTAAGAAGCCCCCCTCGGG-ATTGCTCGCCCCTCCACTTCGGGATCCTTTGGCTACAAGAAGCCTCCTCCTG.'CA CGACCAGCCCTGGCGGACTCACTACGATTCTTC3GGGGGAGCCCGTAACGAGCGGGGAGGTGAAGCCCTAGGAAACCGATGTTCTTCGGAGGAGGACGG- -pHH14-3 • pCB212 ._:i ava,lablβ ORF HU-Unc53/1 = pLM1 OR A G R C R - 3 0 A K < = = S G I A R P S T S G S F G Y < K P P P A :ACGCACAGCCACT3ΓCAT3CAAACTGGTGG " TCA3CCACTCTCAGCAAGATCCAGAAGTCCTCAGGCATCCCTGTCAAGCCA3 AAATG33CSCAAGAC 7;':3TGΓCGG7GACAG7ACGTT73ACCACCAAC " C33TGAGAGTCGTTCTAGGTCTTCAGGAGTCCGTAGGGACAG7TCGGTCA7TTAC'CCGC3TTCT3 -pHH14-3 ■ pCB212 .ιl available ORF HU-Unc53/l = pLM1 OR M G r G ' -: ■ : r ΓA3 TG~ - r cc AA, AG7CAC 3CC 7" 3GC TCC 'GGAGCCCGTTC rAA rCCA rACCGCAGCCrGCCCCGύCCA CC AC'CAA'j" iArc " ACA AC3r7G re AC TCTC 3G TC:~ A C C CCG GG C ΓCGGGCAAG A r re ΓAGG re A * CG TCGGACGGG3CC3GTCG3 " AG77.- ■ pHH14-3 ■ pCB212 .' a.a.labl. C~ = HU-UncόO/! = D_M1 OP A R 5 N I 0 < R 3 L 3 E WO 98/24810 , t _ PCT/EP97/06956 118 Tuesday.18 November 19971033 r '3 ' F_3e t fig Hu-Unc53/1 seq (1 >6013) Site a. jequence rcrArGAGCGrGACCGGCGGGCGGόαTGCACCrCGCCCrGrGAGCAGCAGCArTGACCCCACrCTCCTC A CC A CCA A uCAGGόAGCCCTTACGCCTrCCA A.3 rACTCGCACTGGCCGCCCGCCCCACCTGGAGCGG3ACACTCGTCGTCGrAACTGGGG7CAGAGGAGTCG7αGrTCGrcCCTCCGGAATGCGGAAGG- ' "' • pHH14-3 -pCB212 -pCB210-14 -full available ORF H_-Unc53/1 = O 1 OR - rs Bftpwr ι->-5."v.i i G G R G G P R P V S S S I O P S - S T K G CTGAAGGAGCCTACCAAGGTAGCCAGTGGGC3GACCACTCCAGCCCCTGrCAATCAGACAGA7CGGGAAAAGGAGAAGGCCAAAGCCAAGGCAGTGGC C73ACTTCCTCGGATGGTTCCATCGGTCACCCGCCTGGTGAGGTCGGGGACAGTTAGTCTGTCTΔGCCCTTTTCCTCTTCCGGTTTCGGTTCCGTCACC3 -PCB210-1 full available ORF HU-Unc53/1 = pLMl OR R L K E P T K V A S G ' T T P A P V N Q T D R E K E K A K A K A V * CrTG ACTCAGACAACATCTCCTTGAAGAGTAT-GGCTCCCCAGAAAGTACTCCCAAGAACCAAGCAAGCCACCCCACAGCCACCAAGCTGGCAGAGCTC GAACCrGAGTCTGTTGrAGAGGAACTTCTCATAA'CC3A3GGGTCTTTCATGAGGGTTCTT3GTTCGTTCGGTGGGGTGTCGGTGGTTCGACCGTCTCGAC -pHH14-3 ■ pCB210-14 full available ORF HU-Uπc53/l =pLM1 OR 3 3 0 N ! 3 < 3 : G S P E S T P M Q A S H P T A T > A E L ι :':A':C ACCCCTCTCΔ33GCCACA3CGAAGAGC "" 73'CAAACCACCC7CACTAGCCAA7CTTGAι:AAC'3rCAACTCCAACA37Cr.3'3A7CTACCAr':tt" G' G3rT3GG'3AGAGTCCCGGTGTCGCTTCTCGAAAC 37TTGGTGGGAGTGATCGGr7A3AAeTG77CCA3TTGAGι3r7GrCA3AeC 3A733TAι3TA -pHH14-3 • pCB210-14 .!l a.ailacle ORF riU-Unc53/1 = n .Mi 0= P r P L 5 A 7 A μ 3 r ; » • P = > S I A N . 3 < V 3 N S L D l P 5 Tuesday.18 November 19971033 \ > \ J Page 5 fie, ,-lu-Unc53/1 seq (1 > 6013) Site . Sequence -' CC A .3r3Ar A CCACCC A rGCT7CAAA3GrcCC A G A 7CT3CATGCTACAA3CrCAGCArC 3CGGGCCCrcrcCCr7CC7GC-rC A CC':C':AG7CC3GCAi.-C GG ' CACTArGGrGGGTACGAAGTrτCCAGGG " CTAGACGrΔCGATGTTCGAGTCGTAGACCCCCGGGAGΔGGGAAGG A C3AA3TG3'3GGTCAGGCCGTG3 " ' ' -pHH14-3 -pCB210-14 -lull available ORF HU-Uπc53/l =pLMl OR S S 0 T T ι A S < V ? Q L H A T S S A S G G P L P 5 C F T P S P A F CATCCrCAATATrAACrCAGCCAGCTTCTCCCAGGGCCTGGAGCTAATGAGTGGTTTCAGTGTGCCAAAAGAGACCCGCATGTACCCCAAACTCTCAGGC GrAGGAGTTATAATTGAGTCGGTCGAAGAGGGTCCCGGACCTCGATTACTCACCAAAGTCACACGGTTTTCTCTGGGCGTACATGGGGTTTGAGAGTCCC -pHH14-3 • pCB210-14 -full available ORF HU-UπcS3/1 = p_M1 OR [ _ N I N S A S r S Q G L E _ M S G F S V P K E T R M Y P < _ S G CTGCACAGGAGCATGGAGTCCCTCCAGATGCCAATGAGCCTCCCCAGTGCCTTCCCCAGCAGTACTCCCGTCCCCACCCCACCTGCTCCCCCTGCTGCTC GACGT3TCCTCGTACCTCAGGGAGGTCTACGG77ACTCGGAGGGGTCACGGAAGGGGTCGTCATGAGGGCAGGGGTGGGGTGGAC3AGGGGGACGACGAC -pHH14-3 • pCB210-14 .ιl available ORF HU-Uπc53/1 = o f.'l OP - ■ ' R S M - S L C M ' - S L P S A F P S S T O V P T P P A P P A A CCACA3AAGAAGAGACC3 AGACCTGACTTGCiC-GGAACCCCCAGAGCT3GGCAACTGGACAGTAATCA3CGGGATCGGAACACrC7 CCCAAGAAA'jC GG-GTCr;e7TCTC73CCTTC7CGAC7GAAC:7:A C7TCGGGGTCTCGACCCGTTGACC737CATTAGTCGCCCTAGCCTT3TGAGAAG3GTTCTTT':C -pHH14-3 -pHH3b -pCB210-14 T ι P 3 A G 0 L D 3 N D R "I 7 L = <. WO 98/24810 ,„- PCT/EP97/06956 Tuesday.18 November 19971033 ' _* Paqe_ fig Hu-Unc53/1 seq (1 > 6013) Site arv ,aquence GC7CA3' IΑCCAGCTrCAGrcCCAGGAGGAGACCAA33Δ3AGGCGACATTCCCATACCArTG TGGGCTGCCTGAArCCGArGACCAG7CA3AGCTGCC CGAGTCCA7GGTCGAAGTCAGGGTCCTCCTC7GGr7CCrCTCCGCTGTAAGGGTATGGTAACCACCCGACGGACTTAGGCTACTGGTCAGTCTCGACGGA -pHH14-3 -pHH3b - rav .timer KUSJivt 1 -full available ORF HU-UncS3/1 = pLMl OR Q L Q S Q E R H S H P E S D D Q ε L 7C7CCCCC7GCACTTCCCATGTCTCTGA3TGC-AAGG3CCAACTTACCAACATAGTGAGTCCCACTGCGG-CACCACGCCAAGAATCACCCGCTCCAACA AGAGGGGGACGTGAAGGGTACAGAGACTCAC3T7TCCCGGTTGAATGGTTGTATCACTCAGGGTGACGCCGGTGGTGCGGTTCTTAGTGGGCGAGGTTG7 -pHH14-3 -pHH3b -full available ORF HU-UncS3/1 =pLM1 OR S P 3 A - P M S l. S A K G Q L T N I V S P T A A T T P R I T R S N GCATCCCCACCCACGΔGGCGGCCTTCGAGCTG-A'CAGCGGCTCCCΔAATGGGGAGCACCCTGTCCCTGGC-GAGAGACCCAAGGGAATGATTCGGTCAGG CG7AGGG3TGGGTGCTCCGCCGGAAGCTCGACA-G7CGCCGAGGGTTTACCCCTCGTGGGACAGGGACCGGCTCTCTGGGTTCCCTTACTAAGCCAGTCC -pHH14-3 ■ pHH3b - 'till available ORF HU-Unc-3/1 = pLM1 OR I P T H E A A r E - ' S G S a M G S T L S L A E R P K G M l R S :τrcCGAGACCCCACGGACGATG * rCACC:C"C ;T3CT3TCCCTGGCCTCCAGTGCCTCCTCCACCrACTCCrCAGCTόAGGAGAGGATGCAATf :AAG3C7CTGGGGrGCC7GC7ACAA3TGCCCAGTCACGACAGGGACCGGAGGTCACG3AGGAGGT3GATGAGGA.3TCGΔCTCCTCTCC7ACGTrA A 1 -pHH14-3 '. I a'. - - 5 0 P T D C v H : , L 3 L A 3 S A S 3 7 Y 3 3 A E ε 3 M Q 3 ■ CCAAAT ':C'3GAAGC 7C37Δ3G3AA C 7G3A- ' ' - i " _ CCA33AAAAAGT3GCCACCr73ACGTCTCAGC 7TC7GCCAA7GC7AAT l ι__ ' GG-T3 ι " r - :crrτ- ■ GGCCi ΓTCGAAG: 3ACC " : .: * A: 337CC77TT7CACCGGTGGAAC GCAGAGTCGAAAGACGGTTACGA7rAGACC' ACCGAC GAA _ _ - -* - " = < : = . - ~ - . E S A T L Γ J - L S ,\ H Tuesday 18 November19971033 f i< fl Q <} J p aqe fig Hu Uπc53/1 seq (1 > 6013) Site ard Sequence _ ' -__OP= = pC3_.' OF= ■ pHH3b -full a.ailable ORF HU-_πcS3/1 = pLMl OR ε Q S L S R L £ ε T ε L 0 L CTTTC7GAAGAAAAAGAACTCTGAGGCCCAGGCA3-CATTCA3GGAGCCCTTAATGCCTCAGAAACCACACCCAAAGAACTTCGGATCAAGAGACAAAAC GAAAGACT7CTTTTTCTTGAGACTCCGGGTCCG * CA3TΔAGTCCCTCGGGAATTACGGAGTCTTTGGTGTGGGTTTCTTGAAGCCTAGTTCTCTGTTTTC • 1,1 CP_ " • : pCSJS 1 Ol- •pHH3b -full available ORF HU-Unc53/1 =pL 1 OR F K K K N S E A Q A V I Q G A L N A S E T T P K E L R I <. R Q H -U-'M 1 - M- ■ pHH3b available OR= H_-_nc53/1 = pLM' OR S O S I S S L N S ' " S H S S I G S S < D A D A K K < K K K S " Ar A3CTTCGAAGTTCCTTCAACAAAGC3" " CAC-ΔTAAAAAAGCGGCCCAAG7CAGCTTCC7CATACTCGGATATAGACCAGA7-'337ACAC: ->- J- -' ■ pHH3b .ιl a /a lable OR= HU-U-ιc53/1 = p_ 1 OP Y E L S S r \ < A - S l K < G P S A 3 S C S 0 [ E E I A T F C " crrcΔ3C " :e:rcArccccc«ΛAC Cic:--:c " 'C ΔCAGAC C7Gc 7CAccι: τ ccA CΔA37ccrccAce77 'cc cc3 τ 3: c«crc r J τ - -jHA; " ;333_3AGrA3G333 " "rc^'C7C3 " -.::--^3ArG7C τ C"CAC3AAG7ι3CCAC rA'377CACGACG7 3AA:AGGAG3CA'::';C7GAC7A.-- -pHH3b _ alacl. _ = = >-ι --ιc53/ = ._\'l OP ? 3 3 P < . - - . r £ - fa 3 P - I K j o T L - b » C T 0 Tuesday.18 November 19971033 - 1 ^ — ' Pjge f fir Ηu-Uπc53/1 seq (1 > 6013) Site ano Sequence - J ACC'. .3CCCTGCTCACCCAGCCCCCCACAC7AG'3CTGT7CCATGCAAATGAGGAGGAGGAGCCAGAGAA3AAGGAGG-ArC33AGC " '3C3CTCT3AGC rGGCrCCCGGGACGAGTGGGTCGGGGGGTGTGATCC3ACAAGGTACGTTTACTCCTCCTCCTCGGTCrcrrCTrCCTCCA7AGCC C'3AC3CGAGAC-.3 -L20R.= = pCB2S! 0R= -pHH3b -full available ORF HU-UncS3/1 =pLM1 OR τ ε G P A H P A p H T R u F H A N ε ε ε p ε < κ ε v s ε R s ε TATGGGAGAAGGAAATGAAGCTTACAGACATCCGCTTGGAGGCCCTCAACTCTGCCCACCAΔCTGGATCAGCTTCGGGAGACCATGCACAACATGCAGT' ATACCCTCTTCCTTTACTTCGAATGTCTGTAGGCGAACCTCCGGGAGTTGAGACGGGTGGTTGACCTAGTCGAAGCCCTCTGGTACGTG7TGTACGTC4A • 2CF<_- ' - - pcezsi Ol-.- -pHH3b -D-Dϋis 672627!- -full available ORF HU-Unc53/1 = D M1 OR -U30RF = pLM50RF L V E K E M K L T D l ^ L E A L N S A H Q L D Q L R E T M H N M Q L GGAGGrGGACCTGCTGAAAGCAGAGAATGACCCAC-GAAGGTAGCCCCAGGCCCCTCATCAGGCTCCACTCCAGGGCAGGTCCCTCGATCATCTGCATTA CCTCCACCTGGACGACTTTCGTCTC7TACTG'3C"'3ACTTCCATCGGGGTCCGGGGAGTAGTCCGAGGTGAGGTCCCGTCCA33GACCTA'3TAGACGTA-- :R= = pcε_. ■ = ORF -pHH3b l a/a lade CR= Hϋ-Uπc53/1 = DLM1 OR -U30RF = pLM50RF E V 0 L K A E « 0 = L N / A P G P S S G 3 T P G Q P u ; S A L 7C " TCCCCACGCCGCΓCCCTAGGCCTGJCAC ' CA CCC AT TCC TTCCGCCCCAGTCTTGCAjACACAGACCTGTCACCCATCoAToCCA'CAC "ACT TGTC A.3AA3GG37GCGGCGA3GGA7CCGGACC373A;-:cC-AAGGAAGCC3CGGrCAGAACGrcrGTG7CT3GACAGTGGGTACC ACC'37«:rCA7CAA':'.- :pCό_.! OB= -pHH3b 3.2'lacl ORF HU-UncS3/1 = oLM1 OP -U30RF = pLM50RF L G S F r : Tuesday.18 November 199710.34 '"" " 'J Page 9 fι_ 'u-Unc53/1 seq (1>6013) Site arg Sequence '37C':AAA3GAGGAAGTGACCCTCCGGGT3GT3G"ύA33ArGCCCCCGCAGCACATCA7CAAAGGGCACrrGAA3CAGCACGAArrcr7.;':r3G5;T37 A 3 C A CG rTTCC TCC TTC AC TGGGAGGCCCACC ACCAC TCC TACGGGGGCG TCG rGTAGTAGrTTCCCC7GAACTTCGTCG TCC rrAAGAACGACCCGACATC ^ ' ' ' ■ U2GRF = pCS_S? OP= -pHH3b -full available ORF HU-Unc53/1 = ρL 1 OR -U30RF = pLM50RF ε E [ ! G 0 Q Q ε F F L C AAGGTCAGTGGAAAAGTTGACTGG AAG ATGC TCG ATGAAGCTGTTTTCC AAG TGTTCAAGGACTA T A7TTCTAAAATGGACCCAGCC7.:TACCCT3GGA 7TCCAG7CACCTTTTCAACTGACCTTCTAC3ACC7ACTTCGACAAAAGGTTCACAAGTTCCTGA7ATAAAGATTTTACCTGGGTCGGAGA 7 GGGACC':T -pHH3b -full available ORF HU-Unc53/1 = pLM1 OR -U30RF = pLM50RF L o ε F Q V F K D M 0 ': A3CAC7GΔGTCCATCCATGGCrACA3CA * ':A3CCAC3TGAAACGAGTGTTGGA7GCA3AGCCCCCCGAGATGCCTCCTTGCCGTC'3A33TG7CAATA ' ■ 1 . 1 1 1 . , 1 1 i -i; GArTCGTGACTCAGGTAGGTACCGATGTCGTA3 " ':G3T3CΔCrTTGCTCACAACCTA'CGrCTCGGCGG3CTCTACGGAG3AACGGCAGC7CCACAGT7A- > OF< s ■ (.•: .:i a.ailatl. ORF HU-UΠC33 = n.MI OP - U3 ORF = pLM5 ORF -pHH1S E M P .A-AΓCAGΓC " CΓC " ccc ΓCGACACCCΓGCΓG Γ7C3 ' C3CTG ~C CCCAA3CCGAT3 ΓCCACCAΓ ■ <iΑGr.CA A 3GA3rτrccA AC " r c ' : "" A C A3c rG7C'3CACC C AGC-':rccc C " A;c3c τ rcG'3C ACΑC37'3":- * :" " CG' J A -pHH3b 74 OPF = pCS201 OP= a. a atι-ϊOR= rrJ _nc53/1 'J30RF -pLMSORF pHrt13 Tuesday.18 November 19971034 1 '^ • J fige l» fig <u-Unc53/1 seq (1 > 6013) Site an. Sequence . , rr- ..rG A AGCACCGGCGCCrCG TCC 7CTCG'3GCCCCA3CGGCACGGGCAAGACCTACCr3ACCAA r CGCrrGGCCGAG- AC C7G3 TGG AGCGCTCTGGC GG ACG AC T-CGTGGCCGCGGAGCAGGAGAGCCCGGGGTCGCCGTGCCCGTTCTGGATGGACrGGTTAGC GAACCGGCTC A TG3 ACC ACC " C3C3AGACC2 -_20PF_pC6_S! OFF • pHH3b -U4ORF = pCB201 ORF -full available ORF HU-Uno53/1 -pLM1 OR -U30RF = pLMS0RF -pHH15 L L K H R R L W L 3 G P 3 G T G K T Y L T N R L A E Y L V E R S G ':'37GAGGTCACAGAGGGCATCGTCAGCACCT7CAACATGCACCAGCAGTCTTGCAAGGATCTGCAACTGTATCTTTCCΔACCTAGCCAACCAGATAGACC GCACTCCAGTGTCTCCCGTAGCAGTCGTGGAAG7TGTACGTGGTCGTCAGAACGTTCCTAGACGTTGACATAGAAAGGTTGGATCGGTTGGTCTATCTG3 • U20P..= = pCB_St OP? -pHH3b -U4ORF = pCB201 ORF -full available ORF HU-Unc53/1 = pLM1 OR -U30RF = pLM50RF -pHH15 M H 0 Q S C 3 L Q L Y L S N L A N 0 1 0 :_;AAAC .: AA7T33αCArGT3CCC':T3'37;Λ-7CTATT'3GATGACCTGAGTGAAGCAGGCTCCATCAGTGAGTTGGTCAATGGGG':':CTCACCTjO>ιA ..CTrTGTCCr-AACCCCTACACGCCGACCAC-AAGATAACCrACTGGACTCACTTCGTCCGAGGTAGTCACTCAACCAoTrACCCCGCGAOTGGAC'jT- -l;- P.-:=pC„:„! OF -pHH3b -U40RF = pCB201 OPF '.'I a.-a'labla CRF HU-_nc53/1 = pLMl OR ■ U30RF = pLM50RF pHH15 3 E A G 3 3 E L / G A . T C I Tuesday.18 November 19971034 K 1 Sequence .1 7 I ΪPaqe 1i fig Hu-Unc53/1 seq (1 > 6013) Site - v y .3-A7CA. A rGrcccr TATr TAGGrACCACC A r A 3CCT'3T A A A Ar3ACACCCAAC ATGGcrrGC crrG . ':7rcA33A7G7 3 cc-Tc-'"L C A TAG TAT " TAC AGGGATATAAT A TCCATGGTGC 77AGTCGG AC A TTTTT AC TGTGGG TTGGΓACCGAACGTGAAC TC AAG TCC TΛ'C AAC TGCAAJA'-" • L_ORF = pC3_S1 OPF -pHH3b -U4 ORF = pCBZOI OR -full available ORF HU-Unc53/1 = pLM1 OR -U30RF = pLM50RF -pHH15 ■ ?up-: o B7_52«H Y H K C P Y I I G T T N Q P V K M T P N H G I H L 3 F R M L T F 3 AACAACGTGGAGCCAGCCAATGGCTTCCTGGTTCGTTACCTGAGGAGGAAGCTGGTAGAGTCAGACAGCGACATCAATGCCAACAAGGAAGAGCTGCTTC 773TTGCACCTCGGTC3GTTACCGAAGGACCAA3CAATGGACTCCTCCTTCGACCATCTCAGTCTGTCGCTGTAGTTACGGTTGTTCCTTC7CGACGAA3 ■ U_ORp-pCE25i OR.= -pHH3b -U4ORF = pCB201 ORF -full available ORF HU-Unc531 = DLM1 OR - U3 ORF = pl_M5 ORF • pHH15 '. N V E P A fl G F L ■ S f R R '< _ V E S 0 S 0 I N A N K E E L L 7 "3C TCG C ' GG 3 TACCCAA3CT3"33TA-.:A " CTC CAC ACC T TCC 7T3AGAAGCACAGC ACC TCACACTTCCTCATCGGCCCTTGC TTC TTTCTGTC 7 C3A3CT3ACCCArCG37TCGACACCA7AG-AGA33TGTGGAAGGAACrCTTCGTGTCGTGGAGT T3ΔA3GAG _ ACCCGGGAACι3AAGAAAGAC C -U20R7::pC3.C-! Of< -pHH3b -U4 RF = pCB_01 OPF available ORF HU- 'ncS31 = p M1 OR -U30RF = pLM50RF ■ pHH15 126 Tuesday.18 November 19971034 -p <-\ ■ J age it. fig .-IJ-UΠC53/1 seq (1 > 6013) Site an* aquence G "3 " CC A7 TGGC A7T3AGGAC T TCCGGACC7GG 77C A T TGACC TG T3GAACAAC TZT ATC A 7TCCC TA ' C 7ACAGGA-CG A3 CCA-.C- A TG CATAAiJ C AC A33G T A ACCGTAAC TCCTGAA33CC TGGACC AAG TAAC TGG AC ACC T TGT TGAGATAGTAAGGGA 7 A3 AT3 r CC 7 TCC TC 33 TTC.;7ACCC7AT-T; -U2 RF = pCB_Si ORF -pHH3b - U4 ORF = pCB201 OFF full available ORF HU-UncS3/1 = pLfvil OR ■ U3QRF = p M50RF -pHH15 C P t G l E O F R T ' F [ 0 L W N N S I I P Y U Q E G A S G I I GTCCATGGACAGAAAGCTGCTTGGGAGGACCCAGTG AATGGGTCCGGGACACACTTCCCTGGCCATCAGCCCAACAAGACCAATCAAAGCTGTACCACC CAGGTACCTGTCTTTCGACGAACCCTCCTGGGTCACCTTACCCAGGCCCTGTGTGAAGGGACCGGTAG7CGGGTTGT7C7GGrTA3TT7CGACA7G3-G3 • L_ORF = pCS-S! ORF -pHH3b -U4ORF = pCB201 ORF -full available ORF HU-Unc53/1 = pLMl OR -U30RF = pLM50RF -pHH15 - 3 C < A A . E 0 P v E ■ < V 3 D T L P W = S A 2 Q D Q S :< L i H .CCCCACCCACCGTCCGCCCTCACAGCATTGCC-CACCTCCCGAGGATAGGACAGTCAAAGACAGCA'CCCCAAGTTCTCTGGACTCAGATCC-CTG'-" I333G Tiji3' 7G3C ACCC3GGAG 73 'C 3T AC3G 3733AGGGC TCC TA TCC TGTC AGT TTC TG7C'37' J 33GT7C A'3A'3ACCT3A'J-CT G G ACT- -Ui:OI :::pCB::S -pHH3b -U40RF = pCB201 OPF -'7 a. liable O F HU-UncS3.'i =o M1 Or -U30RF = pLMδORp -pHH15 0 R Tuesday.18 November 199710:34 fie Hu-Un-53/1 seq (1 > 6013) Site a G'3CCATGC7GC7GAAACTTCAAGAAGCT3CCAAC7A ArrGA3TCTCCAGATCGAG AAA CC rCC * '3G A CCCCAACC7rCA33CAACAC7rTAA3GG-rc CC 3G T AC 3ACGAC T T TG AAGT TC TTCG ACGG 7 T3 A 7GTA AC TCAG GG TC T AGC TCTT GG TAGGACC7'33GGT7GGA A37CCGTTGTGAA rrCCCAaC =>. ■ U2 ORF = C_2- 1 ORF -pHH1S -pβpliC- 3~2625K A M L L K L Q E A A N Y l E S P D R E T I L O P N L Q A T L , G F GGCAATCACTGTCACCCCCGGΔCAGCAGAACGC-GGCATCAGCTATCTTAGCTCCTCCTCTCCCCTCTCC CTTTCAGAGCAC GGCTC CCAGCCCCA3 CC3TTAGTGACAGTGGGGGCCTGTCGTCTTGCGACCGTAGTCGATAGAATCGAGGAGGAGAGGGGAGAGGAGAAAGTCTCGTGAC.GAGAGGTCGGGGTC -pHH3b -pHH15 R T Q L L L P A P iGGA333A3GAGGAGA7GAAAGA33AGG3ACAG3TTC7T3GTGCTGTACCTTT3 GAA rTAGG AGGAAT3GTG'3G' T3GC3TTT'33 C "C TCT TG TCC TCCC TCC TCC TCT ACT TTC TCC'CC CTC TCC AA3ΔACCACGACA7G3AAACTC"'3AA33A TCC T7CCTT ACC ACCCCACCGCAAACC -pHH3b -pHH15 ■ J 3 E 0 i 3 G 3 D Ξ 5 3 3 T G S - C C " F E N F L G R N G G :' A F 'j 'J-A.:rT'3rGCCCCCTAAACACA7TTACT3GCCT::-C-AATGACrTT3GG3AAAAGA-GA7TCTGC'3TCrrTCCCTTGACTrCTT3TT _ CAA7r CAAA '- ' " -CAΔCACGGCG3ArrTGTGTAAATGACCG'3AC:A3ATrACrGAAACCCCTTTTCTACTAA3ACCCA3AAAGGGAACT3AA3AACAAA' rrAA- T7TL -pHH3b -pHHIS L C P L M T F T G L . L V G t D D 3 G 3 F P . L L V 3 I T .'I 7 T33GC-7T': ' "3 3 G ' 3'3'3TTCACAAAACi " :-A AC C TGC AGC AG T TCC TAA A "3 AT TC TC CAACC AAC CC " GAG A3 A3 AC AG ~Z " Z 7GAGG 3 CCC3AAΔGACCCCTCCCCAAGTCrTTTG--: * 3TGACGTC3 r CAAGGAT:TA:7AA3A: _ C- _ :3TT'3'3GACTCTC7CTGTC 3 AC CT':C: 1 -pHH3b — — ___ pHH15 — Tuesday.18 November199710.34 h f»s fi ? '.' Page l J- fig Mu-Uπc53/1 seq (1> 6013) Site and Sequence ____ ^^ ' AGATCTGGGGGAGGCAGGAAGCTCCTCAGATTT7CTCACAGACCCTTCCCAATTCCATCACCACTGCCAACAACTCCTCCCCCAGAGATCTGGCTG3AGC TCTAGACCCCCTCCGTCCTTCGAGGAGTCTAAAAGAGTGTCTGGGAAGGGTTAAGGTAGTGGTGACGGTTGTTGAGGAGGGGGTCTCTAGACCGACCT i pHH1S — 3 R 0 E A P Q I F S Q N N CCAGAAAAAGAAGCATGTGGTTTAAAAAATGTTTAAATCAATCTGTAAAAGGTAAAAATGAAAAAACAAAAACAΔGCAAACAAACAAAAAACAATGGAAM GGTCTTTTTCTTCGTACACCAAATTTTTTACAAATTTAGTTAGACATTTTCCATTTTTACTTTTTTGTTTTTGTTCGTTTGTTTGTTTTTTGTTACCTTT " O K K K H V V . K M F K S I C K R . K . K N K N K Q T N K Q - t AGATGAAGCTGGAGAGAGAGGAACCAGTTGCCAAGGTAGAGAGCTGCCCGCTCCTGCCCTCTGGATGACATAGGGGACATCAACAAGACGGCTGCCAAC- TCTACTTCGACCTCTCTCTCCTTGGTCAACGGTTCCATCTCTCGACGGGCGAGGACGGGAGACCTACTGTATCCCCTGTAGTTGTTCTGCCGACGGTTG3 R . S V R E R N Q L P R . R A A R S C P L D O I G D I N K T A A H TGAGAAGTCACCAAACCACAAAAATAACCTTACAGCCTTCAGGGAAAGACTACCAGCTCTGTCTTTCTACCCTCTAATTTAACAATGCACCGGAATTCA3 ACTCTTCAGTGGTTTGGTGTTTTTATTGGAATGTCGGAAGTCCCTTTCTGATGGTCGAGACAGAAAGATGGGAGATTAAATTGTTACGTGGCCTTAAGTC L -ilinnker? - L R S H Q T T K 1 T L Q P S G K 0 Y Q L C L S T L . F N N A P E F - CTTGGACTTAACC 1 1 «- 6013 GAACCTGAATTGG — linker? L D L T GaGCCTGGrGflBrflTGflCP τ :::;CC:GCGRCPCCTGGCflGRGBCGGCCGflGGflGflBGGRCaCTGBGCTGCTGGflTTTGCGRGSRflCCsrBGflCrTTCTG 700 s . v n n τ s R - B H i. R . r B e E - θ r ε - t θ - » t r ι o f' . flBGRRBflBGRflCTCTGPGGC::?:GCRG CP:rCflGGGBGCCCrrflBrGCCrCSGRflBCCRCflCCCBflRGRflCTTCGGRTCSRGRGPCPBBPCTCCTCBG 8C0 ( κ c N s ε => 3 R ι α G B L N B s r r ιc L P i ι: 3 α H s s o s i s s i i s r ' S 't s s I G S S C O R O R X K t t t t s v v v ε GaPGGanfiTGRRGCrτac?G»C=":GC:TGύaGGCCCTCaaCTCTGCCCRCCRaCTGGHrCRGCTTCGGGBGPCCRTGCRCaRcarGCPGrTGGRGGrG 1300 K E n ic i r o i H E a u n s fl H O i O a i R E T n H N n O L E v GaCCrGCTGaflRGCPGaG=a * ό=::GaC-GflflGGrflGCCCCflGGCCC TCRrCRGGCTCCncTCCaGGGCaGGTCCCTGGPTCa-C'GCRπflTCrTCCC 1100 0 L « a E s : 3 . < v P P G P S S G S r P G . V P G S S a i. S S c'CGCCGcrcccrsGG;: • :".:-:":=::: = -":πcG CCC : >G r crτGcaGPcacoGs crGrcac cRTG RrGGca : iG'scrr rGGTCCflflR is o P a R S G . - . - i S F G P S L P C T a u S P n O G I S T C G P ' GGaG anG GRCCC'c ::":":- -;;a" ccc GCMG:- -'τ nr aaRGGGCs:rrGRRGcaGcπGGaRrrcr- :"Gα :-JiRGCRflGGrc isoo i ι » G O ι < 0, α E F F ι. ; c s ιc v • :;--;; ■• . ' • ;;^-- J s- i ' Gττfic "=Gi ' cπ aRGGacrs'ar;rc:sRRι3;GGacccaG::': "::;- ύca τaRGca S G t v o » ι - . ; ; a v F Q v ι Q y | s < n θ P R S - . G I. S 'G-Gfcca-ccn'G c"-:=::":-::: :ι ^":R RCGRG'G- ι GGfliG aύRG c:::::sGsrGc r f ■ cc: ":: : ' J G"':ιn , aflcaτRτc >3co r £ S I j » s 5 '« < < n v L θ a ε s ε ι P o c ' i' ; . '( N i s  S*_ __._►.- '*> Λ o. _ Tuesday. 18 November 199713:57 Pag*. Iig 54 pL 1 ( 1 > 8285) Site and Sequence Eruymes 72 o* 146 enzymes (Filtered) Saltings Circular. Certain Sites Only, Standard Genetic Code Gr5GCAC7r77CGGGGAAA7GrGCGCGGAACCCCTATrTG7-rATrTTTCrAAArACArlCAAATA7βrArCCGCrCArGAGACAA7AACCcrGArAAAr CA CGrGAAAAGCCCCrTTACACGCGCCTTGGGGATAAACAAATAAAAAGArT7ArGTAAGTrTATACATAβGCGAGTACTCrβTTArT3GGACTArTTA G G 7 F R G N V R G T P I C . F F . I H S N M Y P L M R Q . P . . M G< 7T-AA7AATAr7GΛAAAAGGAAGAG7ArGAGTATTCAACA7TTC-G7Gr:GCCCTTATTC:CrTrTTTGCGGCAT7rrGCCrTCCr-r77TTGCTCAC _ — . i . i ' 1 ' ■ ' i ' i ' 1 ■ 1 ' ' 1 1 -*■ 200 C3AAG77ATTA7AAC7TrT7CCTTC7CArACTCAr AG Tό7AAA GCACAGCGGGAArAAGGGAAAAAACGCCGrAAAACGGAAGGACAAAAACGAGrG L Q r . K R K S M S I . H F R V A - I P F F A A F C L P V F A H CCAGAAACGCTGGTGAAAGTAAAAGATGCrGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGrAAGATCCTTGAGAGTT GGrCTTTGCGACCACTrrCATTTTCrACGACTTCTAGTCAACCCACGTGCrCACCCAArGTASCTTGACCrAGAGTTGrCGCCATTCTAGGAACTCTCAA P E T L V K V K O A E O O I G A R V G Y I E - D I N S G K I L E S rTcαCCCCύAAGAACGrTTTCCAATGATGAGCAC77TrAAAGrτCTGCTA7G7GGCGCGGTArTA7CCCGTATTGACGCCGGGCAAGAGCAACTCGGTCG AA5CβGGGCrTCTTGCAAAAGGTTACrACTCGTβAAAArT7CAAGACGATACACCGCGCCATAA7AGGGCArAACTGCGGCCCGTTCTC3TTGAGCCAGC F R P E E R F P M M S T F K V - - C G A V S R I 0 A G Q E 0 L G R CCGCArACACTArrCTCAGAArGACriGGrTGAGTACTCACCAGTCACAGAAAAGCATCITACGGATGGCArGACAGTAAGAGAATTATGCAGTGCTGCC GGCGrATG7GATAAGAGTCTrACTGAACCAACTCATGAGTGGTCAGrGTCrTTTCGTAGAATGCCTACCGrACTGTCATrCTCrTAArACGrCACGACGG R l H Y S Q N D - V E Y S P V T E K H . r D G M T V R E L C S A A ArAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCrTTTTTGCACAACATGGGGGArCArGTAA rA77GGTACrCACTATTGrGACGCCβGTTGAATGAAGACTGTTGCrAGCCTCCTGGCTTCCTCGATrGGCGAAAAAACG7GTrGTACCCCCrAGTACATT 1 r M S O N r A A N L L - T T I G G P K E - r A F . H N M G D H V C7CGCCTTGATCGrTGGGAACCGGAGCTGAATGAAGCCA7ACCAAACGACGAGCGTGACACCACGA7GCCrGTAGCAA7GGCAACAACSTTGCGCAAACr i i i i ■ i i i i i i i ' I ' l i i i 700 GAύCGGAACrAGCAACCCTTGGCCTCGACrTACT7CGGrA;GGrτr3C7GCrCGCACTGTGG7GCTACGGACATCGr7ACCGrTGTTGCAACGCGTTTGA N E A l P N O E R D T r M P V A M A T T L R K L Ar7AAC7GGCGAACTACrTACrCTAGCTTCCC3GCAACAA77AATAGACTGGATGGAGGCGG rAAAGT7GCAGGACCACTTCrGCGCrCGύCCCrrCCG 7.Λ77GACCGCT7GΛ7GAΛTGAGArC3AAGGGCC37T3r7AA7TA:_TGAC-7ACCTCC3CCrArτrCAACGTCCΥGG7GAAGACGCGAGCCGGGAAGGC L 7 G E T U A S R a a L ! 0 V M E A ? K V A G P L R S A L P j r J CTG377TAT7GCTGA7AAA7CTGGAGCCGGTαAGCG7GGG7CTCGC3GTATCA7TGC«CACrGGGGCCAGATGGTAAGCCCrCCC3TATCGTAG 1 ' i ' ' ' ' ' ■ ' ' ' 1 ' ' 1- 900 ..-,:3Ac;iA AAcαAcTA7TTA'3 crc3j':c c:ccc;::cA AGC3CCA7AaτAAC': " :G GAcccc ατc7Λcc τrcGGGAό C ArAGCA7c - a V F I - O K S G A G E R G S R G I I A A L G P O G K P S R I V 7 " . " .rAC ':jACGGGGAGTCAGGCAAC74TGGArGAAC3AAArA:ArAGATCGCTGAGA * A:3TGCCrC CTGArTAA.:CATrGGTAACTjrCAGACCA Λ.-Λ J A:G :cT CCCC7CAα7CCGrrGA7AC:7AC77αC7--A-:737crA CGACTCTA-::ACGGAG7αACTAA77:57AACCA77όA:4GrcrGGT V • ' 7 7 G S Q A 7 M 0 E B N Q I A E i G A S L I K H . L S D Q A:'T ACT:ATA7ArACr77AGA7rαA7rrAAAAC77:Ar r7TAA-77AAJAGGA7CTAGC'όAAGATCCrrTTrGA7AA7C7CArGACCAAAArcCCT r.~ Λ ArGA j rATArArGAAA7CrAAC7AAAT7TrGAΛjΥAAiAAT-AAATT77CC AGATCCAC77C7AGGAAAAACTAr7AGAGTACT3'377TTAGGGA V Y 5 Y I - I D t < L H r- F K R I • ; K I . F D , 'I L M 7 < 1 P r--^37G 3r 7rCG77CCACrGAGC37CAόACCCCG A5AAAΛ3A-CAAA3GArC77C77GA3A7CCrTT7T77CTGCGCGrAA7C7GCT C7TGC AA i'- J :ACT.:iAAAGCAAGGrGACTC3CAG7C73όGGCA":" ** TC"i377TCcrAGAAGAAC7CTAG5AAAAAAAGACG:3CArrAGAC3A:3AACG7T7 a e ^ S F H . A S O P y £ < I K G S S D P F F L R I C C L O .AA AAAAC:ACC3C7 CCAGCGGrGGrT7βT7 * GC:jjA * :AA3 3:7AC;AACTCTrT7-::GAAββrAACTGGCTr;AGCAGAGCGCA3ArACCAAA J ""rr77;3-33CGA7.3GTCGCCACCAAACAAAC33::7A rrC-;3A73G7rG 3AAAAA:3 TrCCA773ACCGAAGrCG7C7CGC3 * :7ATGGrTT r ^ K P P L P A V / C L P C O ϊ '- P r L F P K V T G F S R A Ϊ I P N '■ . ' 3rcc7-:7A3TGrAGCC T ijτi ι:3c ::AC7 " : A3i.:-:-G'.;ι: ; ::"*: rAc rcj rcr C _ Ar:':"GT7 c:A;r3Gcr3cr - ' -•• _G ;c AC Arcs }_-;;;.-_"; 7 * s- -iiir 'j" GGC .j-" I _ .3 " . ■*_ WO 98/24810 ,-- PCT/EP97/06956 Tuesday.18 November 199713.57 Page tig 54 pt_ I (1 > 8285) Site and Sequence ____ GCCAarGGCGATAAGTCGTGrcrTACCGGGT7GGACTCAAGACGA7AGTTACCGGA7AAGGCGCAGCaGTCGGGC73AACGGGGGGTrCGTG.A.A_AGC , . — i i i ' l i i i ' 1 ' 1 i . i i K 15C CGϋ7CACCaC7ATTCAGCACAGAATaGCCCAACCTGAG7rC7GCTATCAArGGCCrATTCCGCGTCGCCAGCCCGACTTGCCCCCCAAGCACGrGrGTCG A S G O K S C - T G D S a R . - P O K A a R S G . T G G S C T Q CCAGCr7GGAGCGAACGACCTACACCGAACT3AGA7ACCTACAGC3TGAGCTATGAGAAAGCGCCACGCTrCCCGAAGGGAGAAAGGCGGACA3GrATCC i ' ' 1 ' ' ' 1 ' ' 1 ' ' ► 1600 G;7;GAACCrCGC7;GCrGGArG7GGC7TGAC7C7A7GGA r G7C3CACTCGA7ACTCTrTCGCGGTGCGAAGGGCrrCCCTCrrrCCGCC7GTCCArAGG P S L E R r T Y T E L R Y L O R E L . E S A T P E G R K A O R Y P GGrAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGrCC7GTCGGGTrTCGCCACCTCTGACTT CCATTCGCCGTCCCAGCCTTGTCCTCTCGCGTGCTCCCTCGAAGGTCCCCCTTTGCGGACCArAGAAATATCAGGACAGCCCAAAGCGGIGGAGACrGAA v S G R V G T G E R T R E L P G G N A V Y _ Y S P V G F R H . . l, GA CGrCGArTTrrGTGATGCTCGTCAGGGGGGCGGAGC:TA7GGAAAAACGCCAGCAACGCGGCCrTTTrACGGrTCCrGGCCTTTTGCTGGCCTrTTG I . i i i l . i l 1 1 ' I ι . | 1900 GAGTGrACAAGAAAGGACGCAArAGGGGACTAAGACACCTArTGGCArAArGGCGGAAACTCACTCGACTArGGCGAGCGGCGrCGGCTTGCTGGCrCGC A H M F F P A L S P O S V O N R I T A F ε . A O T A R R S R T T E R CA CGAG7CAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGArTCArrAA7GCAGCTGGCACGACAGGTTT ' | I i i .. I ' i 2000 Gu3:rGACC77TCGCCCGrCACrCGCGTTGCG7TAAT7ACAC7CAArCGAGrGAGrAArCCGrGGGGrCCGAAATGTGAAATACGAAGGCCaAGCArACA P O V K A G S E R N A I N S . L T H A P Q A I H F M L P A R M r 3rGGAAr7GrGAGCaGArAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCGCGCAArTAACCCrCAC7AAAGGGAACAAAAGCT ACAC CC7rAACAC7CGCCTATTGTrAAAGT37G7;C77T3":3A-ACTGGTACrAATGCGGTTCGCGCGTTAATTGGGAG7GA7TTCCCTTGTTrTCGA - C G I V 5 G . 0 F H T G N S Y D H D Y A K R A I N P H R E O K L a J -.7ACCGGGCCCCCCC7C3AGG7CGACG37A7CaA7AA;;"3A-A7CGAA7TCCTGCAGCCCCTGCTCTrCAGCCAGA7GCTGGACCCAGAGTCCCAG . 1 ■ ■ l . 1 ' ' i I 2300 C'.-A 3GCCCGG3G3G3AGC7CCAGCrGCCA7AG-TA-7C-AA- A-AGC7TAAGGACGTCGGGGACGAGAAG7CGG7CrACGACCTGGGTCTCAGGGTC - insert pLM 1 -O rpLM 'j T G P ? - ε v θ G [ 0 < - D ! E F . a P l . F S a M _ O P Ξ S Q A AAAGAGGACA3r3CAGAA7G7CC7GGA7CTCCGGCA3AA::7G:AAGAGACCATGTCCAGCCTGCGAGGGrCCCAGG'GACrCACAGCTCCCTGGAGA rC T 77C7CC7G7CAC3rCT7ACAGGACCTA3AGGCC3':7-3GAC:"7CrC7GG7ACAGG7CGGACGC7CCCAGGG7CCACTGAG7GrCGAGGGACCTCT - insert pLM I -OHFpL t a < R T v 0 N V 0 L R 3 1 £ E T M S S L R G S Q , / r H S 3 L E 7OACCrαcrACGACAGCGA7GATGCCAACCCACGCAG:3T:-::A::CTCrcCAACCGCTCGTKCCrcT5rCAT33CGCTA';3CCAGTCCAGTCCGCG — 1— . 1 . 1 , 1. , 1 ■ 25CO A:-_ 4CGA7_;r37:_C7A:r-CGG77G33T3CG7C;:A:A33-:3-AGAGC77GGCGAGCA3GGGAαACAα7ACCαC-A7ACC3G7CAGSrCAGGCGC -insert pLMl -ORF pi \t I 0 0 S N R S - Q A G O A P S V β G S C ' S E G r P A W Y rt H G E R A H Y S H T M P M R S P S K L S H I S S - E . V E S L O S D E V O L K S G Y M S O S D L M G K T M T E O C O I T T G W O E S S S I S S G L S D A S L v M - P G T S lC W R R ε 5 » ε S C 0 0 S S K G G ε L < K P ! S L G Grτ7:α 7Grcτ.3r7 ι :ccAτrc3A :3rcACT-: -:-: :::3A337TGCGAGGAG3AGA TACGACCA c:cr3GC3GAcrcAcrACG rτc7 Tuesday t β November 19971357 Pag.- <| li 54 aL I (1 > 8285) Site and Sequence A GC!.CCCCrCGGGCATrGCTCαCCCCrCCACTTC3CG -::7-C33CrACAAGAAGCCrCCTCC7GCCACAGGCACAGCCAC7GrCArGCAAACTGGTGG rc GG GGGGAGCCCGTAACGAGCGGGGAGGrGAAGCCC7A:GAA3CCGA7GrTCTTCGGAGGAGGACGGrGrCCGrGTCGGTGACAGTACGrrTGACCACC insert pLMl — ——^——— —————— — OHFpLMI v a P S G I A R P S T S G i F G Y K K P P P A r G T A T V M a T G G 77CAGCCACrCTCAGCAAGArCCAGAAG7CCTCAGGCA7::C737CAAGCCAGTAAATGGGCGCAAGACTAGCTTAGATGTTTCCAACAGCGCAGAGCCA AACTCGGTGAGAGrCGrTCTAGGTCrTCAGGAGTCC37ACGGACAGTTCGGTCATTTACCCGCGTTCTGArCGAArCTACAAAGGTTGTCGCGTCrCGGT — ■ insert pLMl — — — — — ————— ————— _— — _ __ . — OHFpLMI S A Γ L S K I Q K S S G I P V K P V N G R K T S L O V S N S A E P GUATTCCTGGCTCCTGGAGCCCGTTCTAACATCCAGTAC:3CA3C:TGCCCCGGCCAGCCAAGTCAAGTTCRATGAGCGRGACCGGCGGGCGGGGTGGAC , , 1 . 1 , , 1 -. 1 . 1 1 1 1 1 1 1. 3600 C:TAAGGACCGAGGACCTCGGGCAAGATTGTAGGTCA73:CGTC33ACGGGGCCGGTCGGTTCAGTTCAAGATACrCGCACTGGCCGCCCGCCCCACCTG insert pϋMt — — ^— — — — — — — ^— — — ■ —— — — — — ORFpLMI G F L A P G A R S N I O Y D S L P R P A K S S S M S V T G G R G G CrCGCCCTGTGAGCAGCAGCATTGACCCCAGTCTCCTCACCACCAAGCAGGGAGGCCTTACGCCTTCCAGACTGAAGGAGCCTACCAAGGTAGCCAGTGG GA ( .CGGGACACTCGTCGTCGTAACTGGGGTCAGAGGAG7:3:3G77CGTCCCTCCGGAArGCGGAAGGTCTGACTrCC7CGGArGGTTCCATCGGTCACC insert pL 1 ORFpLMI ? R P V S S S 1 0 ? . . L S T < Q G G L T P S R L K E P T K V A S G •3:s.GACCACTCCAGCCCCrGTCAATCAGACAGATCGG:AAMA3;A3AAGGCCAAAGCCAAGGCAG7GGCCTTGGACTCAGACAACATCTCCTTGAAGAGT :j^:r3G7GA3GTCGGGGACAG7TAG7CTGrCTAGC::'r-'::-:'TCCGGTTTCGGTTCC37CACCGGAACCTGAGTCTGrTGTAGAGGAACTTCTCA insert pLMl — — ^-^^— — — — — — __________ _____ — — ORFpLMI c T r ? A R V N β T D R - < £ < A K A K A V A L Q S O N [ S L K S 7- -i :3-GG33-CrCTCATGAGύGT7C773377CGT-:::-:3::"7CGGTGGrτCGACCG7:rCGACGGTGG7rGGGGAGAGTCCC3GT3TCGCrTC7 — ORF LMI 3 3 a £ S r a K >I Q A S -' a 7 A 7 L A £ L P P 7 P L R A 7 A K " "-37CAAA;CACCCrCAC7AGCCAA-:r73ACAA:3-:AA;-::AACAG7CrGGArcTAC:A7CATCCAG7GArACCACCCA7GCTTCAAAGGTCCC ..-AACAG --α:-GGGA373ArCGG ΛCr377::A:"-C-:377G7CAGACC7AGAr33rAGrAG37CAC7A7GG7GGG7ACGAAG7rTCCAGGG — ■ insert pLMl — ORFpLMI - r / - a a s L A 'I L 0 < / 'l 3 'I S L O L P S S 5 0 T 7 H A S < V P A.--: GCA-3 r C Aαc C 3CArc G:3 G ::":":::" r ::": -cAccc cA3τcc3GCACCCA ccτcAA7A77AAC7CAGC :;7rc7cc .nsartpLMI — ORF LMI - - -l - : A ^ J ; ' . - - t " r ? S ? A = I L , l l > 5 A F 5 Tuesday, tβ November 19971357 Pag«- fig 54 pLM 1 ( 1 > 8285) Site and Sequence CACGGCCTGGAGCTAATGAGTGGTTrCAGrGTGCCAAAAGAGA:::3CATGTACCCCAAACTC7CAGGCCrGCACAGGAGCArGGAG7CCCrcCAGArGC GrcCGGGACCTCGATTACTCACCAAAGTCACACGGTTTrCTC75GGCG7ACA7GGGGTTTGAGAGTCCGGACGTGTCC7CGTACCTCAGGGAGGTCTACG insert pLMl ———————————— — — — — — — — ORFpLMI 0 3 L E N S G F S V ? < E " S M Y P K L S β L H R S M ε S L Q M CAATGAGCC7CCCCAGrGCCTTCCCCAGCAGTACTCCC37CC:;A;cCCACCTGCTCCCCCTGCTGCTCCCACAGAAGAAGAGACGGAAGAGCrGACTTG i ' ■ I ' 1 ' I ' ' ' ' 1 ' 1 I 4300 G7TACTCGGAGGGGTCACGGAAGGGGTCG7CATGAGGGCAGGGG73GGGrGGACGAGGGGGACGACGAGGGrGTCTTCTrcTCTGCCrTCTCGACrGAAC ■ — — ■ insert ρLM1 -__————————__—————. — ■ — __ —ORFpLMI — — — P S P S A F P S S T P V P T P P A P P A A P T E E E T E E L T W 3AGTGGAAGCCCCAGAGCTGGGCAACTGGACAGTAA7CAGCGGCATCGGAACACTCTTCCCAAGAAAGGGCTCAGGΓACCAGCΓTCAGTCCCAGGAGGAG I ' 1 ' 1 1 1 I ' ■ I ' ' 1 1 ' I 4400 C7CACCTTCGGGGTCTCGACCCGTTGACCTGTCAT7AGTCGCCCTAGCCTTGTGAGAAGGGTTCTTTCCCGAGTCCATGGTCGAAGTCAGGGrCCTCCTC insert pLMl ———— ———— —— ——— —— OHFpLMI S G S P R A G Q L D S N β R O R N T L P K K G L R Y Q L α S Q ε ε ACCAAGGAGAGGCGACATTCCCATACCATTGGTGGGCTGCC7GAATCCGATGACCAGTCAGAGCTGCCTTCTCCCCCTGCACTTCCCATGTCTCTGAGTG 7GGTrcCTCTCCGCTGTAAGGGTArGGTAACCACCC3AC3GACT-AGGCTACTGGTCAGTCTCGACGGAAGAGGGGGACGTGAAGGGTACAGAGACTCAC insert pLMl ORFpLMI T K E R R H S H T t G G L P . S D O α S E L P S P P A L P M S L S CAAAGGGCCAACrrACCAACATAGTuAGrCCCACT3C3GCCA:CA:3CCAAGAArCACCCGCTCCAACAGCATCCCCACCCACGAGGCGGC:TTCGAGCT i 1 1 ' i . 1 ■ ' ■ I 4600 3-7TCCCG3rTGAA7GGTraTATCAC7CAG3GTGA;3::3G735':C3GTTCTTAGTGGGCGAGGTTGTCG7AGGGGTGGG7GCTCCGCtGGAAGCTCGA insert pLMl ORFpl I A < G a L 7 .N I V S P 7 A A 7 7 P R I T R S N S I P T H ε A A F ε L 3 ' ACAGCGGC CCCAAAr3G3GAGCACCCrGTCCC:33:C3A3ACA:CCAAGGGAATGATTCGGTCAGGArcCTTCCGAGACCCCACGGA':3A7GTTCAC . i i — - — H — — l l 7CO -- .r;αc:3Λ3 3rτrAccccτcG7GGGACAG33Ac:3::": : " 3GτrcccrτAcrAAGccAGrccτAGGAAGGC :rGGGG7GC- _:rACAAGTG nsert pLMl — — ORFpLMI " S G S Q M G S T L 5 L A Ϊ 5 S K G M 1 R S G S F R 0 P T 3 0 V H J j -' ' :AGTG':rGrcCC7GCCC7CCAGTCC:7C:7:CA::7A:-::-:AGC7GAGGAGAGαA7GCAA7C7GAαCAAA7:C33AAGC77CG7A33GAACrGG :.^A3rCA jA CAGCGACCGJAGG7CACG3AGGAGG ' 33A-3 :;A3TeGACTCCTCTCCTACGTTAGACrCGTrTAGCCCTTCGAAGCATC:CTrGACC nsert pLM 1 — ORFpLMI ~' J J I- S L A S S A 3 S - - S S A E E R k 1 0 S E Q i a < L a 3 E L A - ' ^A7CCCAo3AAAAAGrG3CCACC7-GAC37:7:Λ::---;ι:::ΛAT3C7AArCTG3TGGCTGC7T77GAGCAGAC:: * G37GAAT--3ACArCCCG " ' '-ii : 7' i 3 S'ΑCGATTAGAGCACCGACGAAAArTCGTC' GGACC C 737AGGGC .-sen pLMl - -"FpLMI J J " - \ > Λ r _ - 3 .' , - J - 'I A N L A A F E _ : _ , >i '" ' 3 a. Tuesday. 18 November 199713:57 p agr t fiq54pLM1 (1 >8285) Site and Sequence .CTGCGACACC7GGCAGAGACGGCCαAGGAGAAGGACAG 3 3C GC7GGArTTGCGAGAAACCATAGACrrrCTGAAGAAAAAGAACrcTGAGGCCCAG GGACGCTGTGGACCGTCrcTGCCGGCTCCTCTTCCTGrGAC7C3ACGACCrAAACGCTCrTTGGrArcTGAAAGACTTCrrTTTCTTGAGACrCCGGGTC - insert pLM t -ORF pLM1 L R H L A ε r A ε ε K O T E l L O L R ε r i D F L K K K N S ε A O GCAGTCATTCAαGGAGCCCTTAATGCCTCAGAAACCACACCrAAAGAACTTCGGATCAAGAGACAAAACTCCTCAGATAGCATcrCAAGCCrCAACAGCA CGTCAGTAAGTCCCTCGGGAA7TACGGAGTCTTTGGTGrGG37TTCTTGAAGCCTAGTTCTCTGTTTTGAGGAGTCTATCGTAGAGTrCGGAGTTGTCGT - insert pLM1 -ORFpLMI A V l O G A N A S ε T T ' < ε L R I K R O N S S D S ! S S L N S rCAC7AGCCATTCCAGCA7CGGCAGCAGCAAGGATGCT3A-3:3AAAAAGAAGAAAAAAAAGAGTTGGGTCTATGAGCTTCGAAGTTCCTTCAACAAAGC , — i i ι — . ι ι — i — — _ι ' — I — ■ i 1 l ■ l 5200 AU7GA7CGG7AAGG7CGrAGCCGTCGTCG7TCCTACGACTA:3C7rTTTCTTCTrTTTrTTCTCAACCCAGATACTCGAAGCTTCAAGGAAGTTGrTTCG - insert pLMl -ORFpLMI I T S H S S I G S S K O A D A K K K K K K S V V Y E L R S S F N K A GrTCAGTATAAAAAAGGGGCCCAAGTCAGCTTCCTCATAC7C33ATArAGAGGAGATTGCTACACCCGACTCTTCAGCCCCCTCATCCCCCAAACTACAG , 1 i i 1 ' 1 1 1 ' ■ ' h 5300 CAAGrCATATTrTTrCCCCGGGTTCAG7CGAAGGAGTA73A-3 ;TArATCrCCTCTAACGATGTGGGCTGAGAAGTCGGGGGAGTAGGGGGrTTGArGTC - insert pLM 1 -ORFpLMI F S I K K G P K S A S S Y 5 D I E E 1 A T P O S S A P S S P K L O ':A7GG77CCACAGAGAC7GCTrCACCCTC:A7CAAG7CC7::A::77GTCCTCCGTGGGCACrGATGTCACCGAGGGCCCTGCTCACCCAGCCCCCCACA 37A AAGGrαTCTC73ACGAA37GGGAG37A3T7CAGGA:3 " 334ACAG3AGGCACCCGTGACTACAGTGGCTCCCGGGACGAGTGGGTCGGGGGGTGT -insert pLMl -ORF LMI H 3 S T ε T A S a s : < 3 3 * _ S S V G T D v r ε G P A H P A P H :'A s ,.3C7GrrCCA7GCAAA7.3A3GAGGAGG ;::AGAGAA3AA33A33TA7CGGAGCTGCGCrCTGAGC7ArGGGAGAAGGAAATGAAGC77ACAGACAT , , , , 1 1 1 1 1 , , , , , ► 5500 3 :'.CGACAAGG7AC3r77AC7CC7CCTC:7;337CT 7:"::-:CATAGCCrCGACGCGAGACTCGArACCCrCT7CCT rACTrC3AAr37C7G7A - insert pLM 1 -OR LMI r =• - F H N ε ε ε a ε K < E V S E . R S E L V ε < ε M K L r o i -.'-- 733 AGGCCC r.4. SC7C73CC: IACCAAC -33A-; :AC: '". ::3AGACCATGCACAACA7GCA rαC IAGG 7GGACC GC7GAAAGCAGAGAATGAC 5600 :rC7GG7ACGrGTTGTAC37CAACC7CCACCTGGACGACT77CGTCTC7TACTG - nsert pLM1 -ORFpLMI :C::AGGCCCCTCA7CAG::-CCA:7;::::;:AJ :ccrαG 7CAτc G r7 τrcccC CGCCGCτccc G3::7CGCAC 5700 : nsert pι_M I • -RFuL. l ; / p Tuesday. 18 November 199713.57 Page 7 tig 54 LMl (1 > 8285) Site and Sequence r(.ACCCArTCC7TCGGCCCCAGTCTrGCAαACACAGAC:737:AC:CA7GαA7GGCATCAαrACTTα73G7CCAAAGGA33AAGrGACCC7GCGGGTGGr , 1 , , , 1 , ■ ■ i ■ -i . ' ► 5800 AGraGGTAAGGAAGCCGGGGTCAGAACGTCTGTGrCTGGACAGTGGGTACCTACCGTAGTCATGAACACCAGGTTTCCTCCTTCACrGGGAGGCCCACCA - insert pLMl -ORFpLMI L r n S F G ? S L A 0 T D l 5 a 0 G I S T C 3 P K E ϊ υ T . R V V GGTGAGGA7GCCCCCGCAGCACA7CArCAAAGGGGACTT:.A3:A3CAGGAATTCrTCCTGGGCTGrAGC»AGGTCAGT3GAAAAGTrGΛCrGGAAGATG 1 1 , 1 1 1 , 1 , 1 . , 1 1 1 μ 5900 CCACTCCTACGGGGGCGTCGTGTAGTAGTTTCCCCTGAAC CGTCGTCCTTAAGAAGGACCCGACATCGTTCCAGTCACCTTrrCAACrGACCTTCTAC -insert pLM 1 -OHF LMI '. R M R P a H I i K G 0 L < Q 0 E F F L G C S < V S G K V 0 V K M cri,GArGAAGCTGTTTrcCAAGTGr7CAAGGACTATAr7T:-AAAATGGACCCAGCCTCTACCCTGGGACrAAGCACTGA5TCCArcCA7GGCTACAGCA — — _, I ' l ' 1 i—i __ , 1 , μ 6COO GALCrACTrCGACAAAAGGTTCACAAGTTCCTGArATAAACA TTTACCTGGGTCGGAGATGGGACCCrGArTCGrGACTCAGGTAGGTACCGATGrCGr ■ insert pLMl -ORFpLMI L D E A V F O V F K D Y I S K M O P A S T L G L S T E S I H G Y S TCAGCCACGrGAAACGAGTGTTGGArGCAGAGCCCCCCGAGATGCCrCCTrGCCGTCGAGGTGTCAATAACArATCAGTCTCCCTCAAAGGTCTGAAGαA ' ' ' I ' i ' I . i i i ■ ' i ' I 6100 A_T GG7GCACTrTGCrCACAACCTACGTCrCGGGGGGCT:-AC3GAGGAACGGCAGCrCCACAGTTATT3TATAGTCAGAGGGAGTrTCCAGACrrCCT - insert pLM 1 -OR pLMI I 3 H V K R V L 9 A ε P ? ε . M a P C R R G V N N l S V S L K G L K ε GAAAr3CGTCGACAGCC7GGrGTTC3AGAC3C7GA7C:::-A3::3A7GA7GCAGCACTACArAAGCCr::-3CTGAAGCACCGGCGCCTC37CCTCTCG 1 , 1 , 1 , 1 1 , , μ 500 :-7 CGCA3CTGTCGGACCACAAGCTC73C3AC7AG33:- * :3C:7ACTAC3TCGTGA7G7A7TCGGA33ACGACTTC3T3G:CGCGGAGCAGGAGAGC -insert pLMl -ORFpLMI C V 0 S L V F a 7 L i a < P 1 M 0 H Y I S L . L < H 3 R , l S ::AGCGGCACGC3CAAGACC7ACC T 3AC:AA7C::i-:3::3A3TACCTGGTGGAGCGCTCTGGCC3"3AGGTCA':A3A33GCATCG7:AGCACC7 — '' ■■ ' ' 1 l ' ' ' 33C 37CGCCGr3 c 3rτcrGGA αGA T3Grr G: A-:::3:':A733AC AccτcacG GACC33:A:r c GT3r:-:;;GrAGC 37CGTG A • 'nsert pLMl -OHFpLMI J s S G r α < r r L r * . - ε L v ε » S G R ε v τ £ i ; s τ ■ insert pLMl -ORF OL'.II 3 3 S C 3 L 3 P '/ " - r A' 33 3ACC _A 3 GA _r - " — ::.;' - 3A3-3:-C3rτ:A-.t " *77- :- : 7 TAA-«rι:c rGG ■ rsert pLMl '•f PLMl 'I 3 A Tuesday.18 November 19971357 Pag t V fig 54 pLMl (1 > 8285) Site and Sequence A^-AA7CAGCC7GrAAAAATGACACCCAACCATGGCT73CA;7T3A3C7TCAGGATGT7GACCTTCrCCAACAACGrGGAGCCAGCCAATGGCTTCCTGG 7 .TrAGTCGGACATTrTTACrGTGGGTTGGTACCGAACGTGAACT.6AAGTCCrACAACTGGAAOAGGTrGπGCACCrCGθrCGGrTACCGAAGGACC insert pLM1 — — — — — — — — — — — — — — ORFpLMI ' •I Q P K M T P N H G L -I L S F R M L T F S M N V E P A H G F L TrCG7TACCrGAGGAGGAAGCTGGTAGAGrCAGACAGC3ACArCAArGCCAACAAGGAAGAGCTGCrrCGGGTGCTCGACTGGGTACCCAAGCTGTGGTA AAGCAATGGACTCCTCCTTCGACCArCTCAGTCTG7CGCT37AG7TACGGTTGTTCCTTCTCGACGAAGCCCACGAGCTGACCCATGGGTTCGACACCAT — — insert pLM1 —————————————— — — — — . —— __ ■ ORFpLMI V R V L R R K L V E S D S D I N A N K E E L R V - O V V P K L V Y T'.A7CTCCACACCrTCCTTGAGAAGCACAGCACCTCA3A:77CCTCATCGGCCCrTGCTTCTTTCTGTCGTGTCCCATTGGCArTGAGGACTTCCGGACC A-TAGAGGrGTGGAAGGAACrcrTCGTGTCGTGGAGTCTGAAGGAGTAGCCGGGAA.GAAGAAAGACAGCACAGGGTAACCGTAACTCCTGAAGGCCTGG insβrtpLMI — — — — ——— — — — — — — . ORFpLMI H L H r F L E H S T S O F L l G P C F F L S C P I G I E D F R T TGGTTCATTGACCTGTGGAACAACTCTATCATTCCCTATCTACAGGAAGGAGCCAAGGATGGGArAAAGGrCCATGGACAGAAAGCTGCTTGGGAGGACC ACLAAGTAACTGGACACCTTGTTGAGATAGrAAGGGATAGA73TCCrTCCTCGGrTCCrACCCTATTTCCAGGTACCTGrCTTTCGACGAACCCTCCTGG —————— —_————__——————— insert pLM 1 — __———___—_— —————— — — — ORFpLMI V F I 0 L W N N S I 1 P Y L 3 E G A K D G 1 K V H G Q K A A V E 0 C-u7.3GAATGGGrCCGGGACACACTTCCCrGCCCATCA3:::AACAA3ACCAArCAAAGCTGrACCACCTGCCCCCACCCACC3TGGGCCCrCACAGCAT 1 ' i 1. . ' 1 ' 1 ■ 1 ' 1- 7000 3 r -A;crTACCCAGGCCCTGrGrGAAGGGACCGG*AG":33~ r 3-r"G377AGrTTCGACA7GGTGGACG3GGGTGGGTGGCACCCGGGAGTGTCGTA — — ' insert pLM I ORF LMI a . E V V R D T L P v a 3 A 3 2 3 0 S K L Y H L P P P T V G P H S l J-- 7CACC7CCCGAGGArAGGACAGrCAAACACAGCA::::iA3"-CTCrGGACTCAGATCCTCTGATGGCCATGC7GCTαAAACTrCAAGAAGCTGCC A j-A37G3AGGGCTCC7ATCCTGTCAG7rTC7G7C373:C3"T:AA:AGACC7GAGrcTAGGAGACrACC3GTACGACGACT7TGAAGTTCTTCGACGG — insert pLM I — — — ORrμLMi A S P a ε θ R T V < D S T ? S S L D S O P L M A M L L K L a E A A ".A-3'AAC7CAGAG' rC7AGCTC77T'337-G3AC:'::: T3:A.37:C37rG7GAAArrCCCAAGCC3T7AGTGACAG7GGGGGCcr3rCGrcrTG ■ — — ■ insert pLMl —OHFpLMI ' ' J :A-C GC r rC7AGCTCC CC τ C7;:::rC7::":"":A -: A:7G3C7C"CCAGCCCCAGGAG3AGAACA33-'.33Aι3GAGGA3A7GAΛAG i ■ ":".": '3 rr3Λ.3 GG7CGGG' c: ::rc rG :c ,' '::c7r:rcrr: Acrrτc Tuesday.18 November 199713:57 p age liq 54 PLM1 (1 > 8285) Site and Sequence , AiJAGGGACAGGTrCTTGGTGCrGTACCTrTGAGAA:r7CCTAGGAAGGAATGGrGGGGTGGCGTTrβGGAACTTGTGCCCCCrAAACACArrTACrGGC i i ■ I ' l i 1 i l l — ' »- — ' I ■ ' — I ' i ' i ■ l 400 rcCrCCCTGrCCAAGAACCACGACArGGAAACTCrTGAAGGArCCTrCCTrACCACCCCACCGCAAACCCTTβAACACGGGGGATTTGTβTAAATGAeCG -msert LMi C7:crCTAArGACrrTGGGGAAAAGATGArTCTGGGT:7T7CCCTrGACTTCTTGTTTCAATTACAAACTCCTGGGCTTTCTGGGGAGGGGrTCAGAAAA GA3GAGATTAC7GAAACCCCrTTTCrACTAAGACCCA3AAA3GGAACTGAAGAACAAAGTTAArGTTTGAGGACCCGAAAGACCCCrCCCCAAGTCrTTT -msertpLMi U L . . L V G K D D S G 3 F P . L L V S I T N S V A F W G G V Q K CATCAAAACACTGCAGCAGTTCCCCGGAATTCAGCTT33ACrTAACCAGGCTGAACTTGCTCAAAAGAAGCCGAATTCCAGCACACTGGCGGCCGTTACT •37AGTTTTGrGACGTCGTCAAGGGGCCTTAAGTC3AAC:TGAATTGGTCCGACTTGAACGAGTTTTCTTCGGCTTAAGGrCGTGTGACCGCCGGCAATGA -msertpLMi r S K H C S S S P E F S L D L T R L N L L K R S R 1 P A H W R P L L AGTTCTAGAGCGGCCGCCACCGCGGTGGAβCTCCAAT7C3CCCTATAGTGAβTCβTATTACGCβCGCTCACrGGCCGTCGTTTTACAACGTCGTGACTGβ rCAAGATCTCGCCGGCGGTGGCGCCACCTCGAGGTTAAGCGGGATATCACTCAGCATAArGCGCGCGAGTGACCGGCAGCAAAATGTTGCAGCACTGACC V L ε R P P P R V S S N S P Y S E S Y Y A R S L A V V L Q R R D W GAAAACCCrGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCrTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAAC CTT7rGGGACCGCAATGGGTrGAAT7AGCGGAACGTC3737A3GGGGAAAβCGGTCGACCGCATTATCGCrrCTCCGGGC3TGGCTAGCGGGAAGGGTTG E M P G V T Q L N R L A A M P P F A S W R N S ε ε A R T O R P S α AG7TGCGCAGCCTGAArGGCGAATGGGACGCGCCCTG7A3C3GCGCATTAAGCGCGGCGGGTGTGGTGGTrACGCGCAGCG7GACCGCTACACTTGCCAG 7CAACGCGrCGGACTTACCGCTTACCCTGC3CGGGACA-:3CC3CGTAATTCGCGCCGCCCACACCACCAATGCGC3TCGCACrGGCGArGTGAACGGTC 3 '. R S L N G E V 0 A P C 3 G A L S A A G V V V T R S V T A T L A S C3CCCTAGCGCCCGCTCCTT7CGCTTTCTTCCCTTC:777:7:3CCACGTTCGCCGGC7TTCCCCG7CAAGCrCTAAATCGGGGGCTCCCTrTAGGGTTC 3:33GA7CGCGGGCGAG3AAA'3CGAAAGAA5GGAA3GAAA3A3C33rGCAAGCGGCCGAAAGGGGCAGTTCGAGA77TAGCCCCCGAGGGAAArCCCAAG A L A P A P F A F F P S - ' L A T F A G F P R O A L N R G L P L G F C3A777AG7GCTrrACGGCACCTCGACCCCAAAAAAC" * 3A77A33GTGA7GGTrCACGTAG7GGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGA 3.7AAATCACGAAATGCCGT5GAGC7GGGGrT7TT7GAA:-AA7C:CACTACCAAGTGCATCACCCGGTAGCGGGACTArC7GCCAAAAAGCGGGAAACT R F S A L R H L D P K K . 3 G D G S R S G P S P . . T V F R P L :377-,GAG7CCACGrrcrT7A 7Aι3' ' 33ACrC77G"7;:AAA;T33AACAACACrCAACCCTA7C7CGG7CTA7TC77TrGATTTA7AAGGGArTrTGCC — i ' 1 —i ' 1 .■ ■ 1 ' 1 ■ i ' 1- 8200 GCAACC7CAGG7GCAAGAAA7rA7CACCTGAGAACAAj 7773 C.:r GTrG7GAGTTGGGA7AGAGCCAGArAAGAAAAC7AAATATTCCCTAAAACGG r L E 3 7 F N 3 3 L L 7 3 T T L N P I S V Y S F O L . G I L P ji 7rrcGGCcTArrGGrrA A AAr3 CT3ATττAA:A A A"T AC3CGAArτττA CAAAATAτr ACGcrτACAArrτ G — ' — ■ ' 1 1 1 1 1 3235 : "A AGCC33A7AACCAA777r77A:ri:GAC7AAA -3 *"" 7 A rGCGC7rAAAA7TG7TTrA7AA7rGC-3AAT377AAA7C : S A Y V. L < N E L I . : < Γ N A N F N < I L 7 L Γ I I I N G V P I T Y G K V P V L CCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGG- GGGTTGCTGGGGGCGGGTAACTGCAGTTATTACTGCATACAAGGGTATCATTGCGGTTATCCCTGAAAGGTAACTGCAGTTACCCACCTCATAAATGCCA -pCMV A Q R P P P I O V N D V C S H S N A N R D F P L T S M G G V F T V AAACTGCCCACTTGGCAGTACATCAAGTGTATCATAT3CCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTA TTTGACGGGTGAACCGTCATGTAGTTCACATAGTATACGGTTCATGCGGGGGATAACTGCAGTTACTGCCATTTACCGGGCGGACCGTAATACGGGTCAT -p-MV N C P G S T S S V S Y A K Y A P Y . R Q . R . M A R L A L C P CATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGA GTACTGGAATACCCTGAAAGGATGAACCGTCATGTAGATGCATAATCAGTAGCGATAATGGTACCACTACGCCAAAACCGTCATGTAGTTACCCGCACCT -'pCMV → O L M G - S Y L A V H L R l S H R Y Y H G D A tf L A V H Q V A V T 3CGGTTTGACTCACGGGGATTTCCAA3TCTCCAC::CATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTA ATCGCCAAACTGAGTGCCCCTAAAGGTTCAGAGGTGG3GTAACTGCAGTTACCCTCAAACAAAACCGTGGTTTTAGTTGCCCTGAAAGGTTTTACAGCAT -pCMV i A V . I_ T G I S K S :> = H . R Q V E F V L A P K S T G _ S K M S ACAACTCCGCCCCATTGACGCAAATGGGCGGTAGCC3T3TACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGTCAGATCCGCTAGCGCTA rG'TGAGGCGGGGTAACTGCGTTTACCCGCCATCCGCACATGCCACCCTCCAGATATATTCGTCTCGACCAAATCACTTGGCAGTCTAGGCGATCGCGA- 'J L R P I D A N G R . A C T V G G L Y K Q S W F S E P S O P L A CCGGrCGCCACCATGGTGAGCAAGGGCaAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGrTCAG'.: GG:CA3CGGTGGTACCACTCGTTCCCGCrCCTC3A:AAGTGGCCCCACCACGGGrAGGACCΔGCTCGACCTGCCGCTGCATTTGCCGGTGrTCAAGTC'-; N V 5 K G E E L T G V V P [ l V E L D G D V iN G H K F S " _ " CCGGCGAGGGCGAGGGC GA1 ■ GCCAC: :TACG3C : ;;TGACCCT GAAGTTCATC G.A..ACCGGCAAGCTGCCCGTGC_CTGG_CCA_.' T.GTGA- A-AGGCCG-TC.CGCT.CC_ C TACGG T33ATGC ' '73ACTGGGA CTTCAAGTAGACGTGGTGGCCGTTCGACGGGCACGGGACCGGGTG3GACCACT3 t_l-H ' 5 G E G E G 0 A T 1 3 < L T L F I C T T G K L P V P W P Γ L V T '. TGA TACGGCG7GC AGT ' GCTTC. .GCCG7- -::::GACCAC AΓGAA3CAGCACGACTTCTT AAGT CGCCA T GCCCGAΔGGCTA:3 TCCΔGGA3 'i ' 3GGAC " CJ iCGAAG 7C3GC3A ' " ::33CΓGGT; 'A rrCGTCGTGCrGAΔGAAGTTCA3GCGGTAC3GGCTT;CGArG: A33TCCT. — c rr —————— ~~~—~ —————————-. — — _— ___ " - T ' G v '- C - 5 -^ - 0 H M ► 0 H D F F k. S A H => E G < , u E Tuesday.18 November 199711.48 p ag6 j, tig 34 pLM4 (1 > 10070) Site and Sequence . CG ACCATCTTCTTCAAGGACGACGGCAACTΔCAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCAT"" GCGTGGTAGAAGAAGrTCCTGCTGCCGTTGATGTTCTGGGCGCGGCTCCACTTCAAGCTCCCGCTGTGGGACCACTTGGCGTAGCrCGACTTCCCGTAG- ""' -t-i-μ D 0 T R V K E G O I E L K G I ACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCT J AGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCA TGAAGTTCCTCCTGCCGTTGTAGGACCCCGTGTTCGACCTCATGTTGATGTTGTCGGTGTTGCAGATATAGTACCGGCTGTTCGTCTTCTTGCCGTAGTT -_t_ μ D F K E D G N I L G H K L E Y N Y N S H N V Y I M A D K Q K N G I r- GGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTG CCACTTGAAGTTCTAGGCGGTGTTGTAGCTCCTGCCGTCGCACGTCGAGCGGCTGGTGATGGTCGTCTTGTGGGGGTAGCCGCTGCCGGGGCACGACGAC --.PP N F K I R H N I E O G S V Q L A D H Y Q Q N T P I G D G P V L CCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGA GGGCTGTTGGTGATGGACTCGTGGGTCAGGCGGGACTCGTTTCTGGGGTTGCTCTTCGCGCTAGTGTACCAGGACGACCTCAAGCACTGGCGGCGGCCCT ——p- P O N H Y L S T Q S A L S K D P N E K R D H M V L L E F V T A A G TCACTCTCGGCATGGACGAGCTGTACAAGTCCGGACTCAGATCTCGAGCTCAAGCTTCGAATTCTGCAGTCGATAAGCTTGATATCGAATTCCTGCAGCC AGTGAGAGCCGTACCTGCTCGACATGTTCAGGCCTGAGTCTAGAGCTCGAGTTCGAAGCTTΔAGACGTCAGCTATTCGAACTATAGCTTAAGGACGTCGG --GFP I T L G M D E L Y K S 3 - R S R A Q A - N S A V D K L D I E F L Q F C TGC TC TTCAGCCAGATGCTGGACCCA3AG7CCCA3 A3AAAGAGGACAG TGCAGAATGTCCTGGATCTCCGGCAGAACCTGGAAGAGACCATGTCCAGC GGACGAGAAGTCGGTCTACGACCTGGGTCTCAGύGTCTCTTTCTCCTGTCACGTCTTACAGGACCTAGAGGCCGTCTTGGACCTTCTCTGGTACAGGTCG L L F S Q M L O P E S . R K R T V Q N V L D L R Q N L E E T M S . C 3CGAGGGTCCCAGGTGACTCACAGCTCCC GGAGA-3ACCrGCTACGACAGCGATGATGCCAACCCACGCAGCGTGTCCAGCCTCTCCAACCGCrCG " GACGCTCCCAGGGTCCACTGAGTGTCGAGGGACCrCTACTGGACGATGCTGTCGCTACTACGGTTGGGTGCGTCGCACAGGTCGGAGAGGTTGGCGAG A -insert pLM1 -ORFpLMI - R '3 3 Q V T H S S L E U T C Y 0 S D 0 A P R S V S S L 3 H R 3 ':C:CTCTGTCATGGCGCTArGGCCAGTCCAG:C:GC33:TGCAGGCTGGTGACGCGCCCTCT3TGGGT3GGAGCri3CCGCTCG3AGGGGAC3CCCGCCr3 GG3GAGAC AGT ACCGCGATACCGGTCAG3TCAGGC-CC 3 ACG TCCGACCACTGCGCGGGA.ACACCCACCCTCGACGGCGAGCCTCCCCTόCGGGCGGA;; -insert pLMl ORFpL.VU — — __ R C 3 . S S : . ' ' 3 G 0 A P 3 G G 3 C S E G r = Tuesday.18 November 199711.48 Page 3 (iq 34 pLM4 (1 > 10070) Site and Sequence _ . '3 " ACATGCACGGCGAACGGGCCCACTACTCCCACACeAr3CCCATGCGCA3CCCCAeCAAGCrCAGCCATATcrcCCGCCrG3A3CrG':-e3AA-c; T3 CA-GTACGTGCCGCTTGCCCGGGTGATGAGGGT3TGGTACGG3rACGCGTCGGGGTCG TCGAGTCGGTATAGAGGGCG3ACCTCGAC i ; i 3CTTAG3GAe -insert pLM1 -ORFpLMI M H M R S ε L GACTCGGATGAGGTGGACCTCAAGTCCGGCTACATGAGCGACAGTGACCTCATGGGCAAGACCATGACGGAGGATGATGACATCACTACCGGCTGGGATG C-3AGCCTACTCCACCTGGAGTTCAGGCCGATGTACTCGCTGTCACTGGAGTACCCGTTCTGGTACTGCCTCCTACTACTGTAGTGATGGCCGAeCCTAC -insert pLM1 -ORFpLMI C S 0 E V D L K S G Y M S D S D L M G < T M T E O D D ! T T G D AAAGCAGCTCCATCAGTAGTGGACTCAGCGATGCCTCAGACAATCTCAGTTCAGAAGAATTCAATGCCAGCTCCTCACTCAACTCCCTCCCAAGTACTCC TTTCGTCGAGGTAGTCATCACCTGAGTCGCTACGGAGTCTGTTAGAGTCAAGTCTTCTTAAGTTACGGTCGAGGAGTGAGTTGAGGGAGGGTTCATGAGG ■ insert pLM1 -ORF PLM1 I S S G L S 0 A S D r4 L S S E E F N A S S S N S L = S T P CA.TGCTTCTCGCAGGAACTCAACAATAGTGCTACGCACAGACTCAGAGAAGCGCTCACTGGCAGAAAGTGGGCTGAGCTGGTTTAGTGAATCA3A3GAG G-3ACGAAGAGCGTCCTTGAGTTGTTATCACGATGC3TGTCT3AGTCTCTTCGCGAGTGACCGTCTTTCACCCGACTCGACCAAATCACTTAGTCTCCTC -insert pLM1 -ORFpLMI * A 3 R R N S T ! v L R T D S E K R S L A E S G L S V F S . S E E - A AC;:CCTAAAAAACTGGAGTACGACAeTGGTACCeT3AAGA " GGAACCT3GGACTTCTAAGTGGCGGA3GGAGCGGCCTGA3A3CTC " A-3ATTCJ- ' "" 73GGGATTTTTTGACCTCATGCTι3rCACCA"CGCAeTTCrACCTrGGACCCTGAAGATrCACCGCCTCCCTCGCCGGACTCTCGA ;7ACTAAGTA -insert pLM1 -ORF pLM1 < A P K .< I E '' . 5 G S L E ? G T S K '_ P R E P P E 3 C D D 3 : A 3eGTGGAGAACτ AAAAAόcceATCAGCC * CGeeeAcceτGGττecerGAAGAAGe:cAAGAccccAccτGTGGCτGTAAcrτccceeAτe c cM J "" :eCACCTCrTGACTTrTTCGGe"A3TCGGACCe33TGG3ACCAACG3ACTTCTTC:CGrTCTGGGGTGGACACCGACATTGAAGCG33TA3T3A'j" -insert pLM1 -ORF -LM1 Ξ L < < = I S . e - P G S L K K G K T P P 'J A '/ T 3 D I T H Tuesday 18 November 19971148 Page L dg 34 pLM4 (1 > 10070) Site and Sequence ___ ^^^ CACAGCCCAGAGTGCCCTCAAAGTCGCA j GCAAACCTGAGGGCAAAGCTACAGACAAGGGTAAGCTTGCAGTGAAGAATACTGCGeTC AACGC ' ceTCC GT3TCGGGTCTCACGGGAGTTTCAGCGTCCGTTTGGAerCCCGTTTCGATGTCTGTTCCCATTCGAACGTCACTTCTTArGACCeGAGGrTGC3AG3AG3 "" '' -insert pLM1 -ORFpLMI A L K M T G L Q TCTGATGCTGGTCGGGACCGCCTGAGTGATGCTAAGAAGCCCCCCTCGGGCATTGCTCGCCCCTCCACTTCGGGATCCTTCGGCTACAAGAAGCCTCCTe AGACTACGACCAGCCCTGGCGGACTCACTACGATTCTTCGGGGGGAGCCCGTAACGAGCGGGGAGGTGAAGCCCTAGGAAGCCGATGTTCTTCG3A5GA3 ■ msertpLMi -ORFpLMI S D A G R D R L S O A K K P P S G I A R P S T S G S F G Y K K P P CTGCCACAGGCACAGCCACTGTCATGCAAACTGGTGGTTCAGCCACTCTCAGCAAGATCCAGAAGTCCTCAGGCATCCCTGTCAAGCCAGTAAATG3GC3 GACGGTGTCCGTGTCGGTGACAGTACGTTTGACCACCAAGTCGGTGAGAGTCGTTCTAGGTCTTCAGGAGTCCGTAGGGACAGTTCGGTCATTTACCCGe • msertpLMi -ORFpLMI P A T G T A T V M Q T G G S A T L S K i Q K S S G I P V K P V N G K CAAGACTAGCTTAGATGTTTCCAACAGCGCAGA3CCAGGATTCCTGGCTCCTGGAGCCCαTTCTAACATCCAGTACCGCAGCCTGCCCCG3CCAGCCAA_ G7TCTGATCGAATCTACAAAGGTTGTCGCGTCTeGGTCCTAAGGACCGAGGACCTCGGGCAAGATTGTAGGTCATGGCGTCGGACGGGCCeGGTCG37T_ -insert pLM1 -ORF pLM1 K T s L D V S N s A E P G F L A P G A R S N I 0 Y R s L P -. ? _ > .GTTCTA CCG ece GGC GGGGTGGAC :τc :GCCCTGTGAGCAGCAGCA ' ΓTGACCCCAGTCTCCTCAGCACCA .AGCAGGi 733CC TTAC'7 "G-TCAAGATACTCGCAeTGGCeGCeCGCCCCAeCTGGAGCGGGACACrCGrCGTCGTwACTGGGGTCAGAGGAGTCGTGGTTCGTCCC ' eeGGAATG'.. -insert pLM1 -ORF pLM1 - ■ - S M S V 7 G R = R P V S S S I D P S L L 3 T K Q G L T C " CCAGACTG AGGΔ3CCr-CCAA;crAGC:AC-GGCCCGACCACTCCAGCCCCTG"CAATCAGACAGATeGGGAAAACGAGAA3GCC-AAGCCAAG'__ GAAC3TCrGACTTCCTe3GA-G3TTCCATCG3T:ACee3CCTGGTGAGGTCGGGGACAGrTAGTCTGTCTAGCCCTTrTeCTCTTCC'3CTTTCGeTTCCe ■ insert pLM1 -ORFoL I R T T = A p , M Q T C R E < i Tuesday.18 November 19971148 Page i lig 34 pLM4 (1 > 10070) Site and Sequence AG7GGCCTTGGACTCA3ACAACATC TCC TTGAAGAGT AT TGGCTCCCCAGAGAGTACTCCCΔAGAACCAAGCAAGCCACCCeAeACCCACeAAGCTGG'A TC ACCGGAACCTGAGTCTGTTGTAGAGGAACTTCTCATAACCGAGGGGTCTCTCATGAGGGTTCTTGGTTCGTTCGGTGGGGTGTCGGTGGTTCGACC'J- -msertpLMi -ORF pLM1 L 0 S 0 N ! S L E S H P T GAGCTGCCACCAACCCCTCTCAGGGCCACAGCGAAGAGCTTTGTCAAACCACCCTCACTAGCCAATCTTGACAAGGTCAACTCCAACAGTCTGGATCTA- i 1 . 1 1 1 ' 1 1 1 1 1 ' 1 1 -I . _— — i μ J] -, CTeGACGGTGGTTGGGGAGAGTCCCGGTGTCGCTTCTCGAAACAGTTTGGTGGGAGTGATCGGTTAGAACTGTTCCAGTTGAGGTTGTCAGACCTAGAT" ■ insert pLM1 -ORF pLM1 E L P P T P L R A T A K S F V K P P S L A N L D K V N S N S L O L CATCATCCAGTGATACCACCCATGCTTCAAAGGTCCCAGATCTGCATGCTACAAGCTCAGCATCTGGGGGCCCTCTCCCTTCCT3CTTCACCCCCAGTCC GTAGTAGGTCACTATGGTGGGTACGAAGTTTCCAGGGTCTAGACGTACGATGTTCGAGTCGTAGACCCCCGGGAGAGGGAAGGACGAAGTGGGGGTCAG- -msertpLMi -OR pLMI P S S S O T T H A S K V P O L H A T S S A S G G P L P S C F T P S P GGCACCCATCCTCAATATTAACTCAGCCAGCTTCTCCCAGGGCCTGGAGCTAATGAGTGGTTTCAGTGTGCCAAAAGAGACCCGCATGTACCCCAAA TC CCGTGGGTAGGAGTTATAATTGAGTCGGTCGAAGAGGGTCCCGGACCTCGATTACTCACCAAAGTCACACGGTTTTCTCTGGGCGTACATGGGGTTTGAό -insert pLM1 -ORF pL 1 A P I L N I N S A 5 F 5 Q G L E L M S G F 3 V P K E T R M Y P L 7':AGGCCTGCACAGGA3CATGGAGTCCCTCCAGA7GCCΔATGΔGCCTCCCCAGTGCCTTCCCCAGCAGTACTC:CGTCCCCACCeCACCT3CTCCCCCTG >7~CCGGΔCGrGTCCTC3TACCTCAGGGAGG' ' C"ACGGTTACTCGGAGGGGrCACGGAAGGGGTCGTCArGAG3GCACGGGrGGG3TGCAeGAGGG3Gj.e -insert pLM1 -ORF pLMl i G L H P S r" E 3 L M O M 3 L P S A F P S S T S '/ P T P P A P P L " CCrCCCACAGAAGAACA3AC3GAACAeCTG C"3CΔ3TGGAAGCCCCAGAGCTGGGCAACTGGACAG AArCAGCGGGATCGCAACACTCTTCC'_AA G ACC AGGG7GTC TTC ΓTCTCTGCC TC TeGACTCAAeCTCACCTTCGGGGTCTCGACCCGTTGACCTGTCATTAGTCGCeCTAGCeTTC 73 GAAG3GTT ■ insert pLM1 ORFpLMI A P T _ £ . T Ξ - . " '- S C S P R A C Q L D 3 N - R . K N r L F r- Tuesday, 18 November 199711 :48 age t, ( ig 34 pLM4 (1 > 10070) Site and Sequence ____ ^ _ GAAAGGGercAGGTACCAGCTTCAGTCCCAGGAGGAGACCAAGGAGAGGCGACATTCCCATACCArTGGTGGGCTGCCTGAAreCGATGACCAGTCAGA"- CTTTCCCGAGTCCATGGTCGAAGTCAGGGTCCTCCTCTGGTTCCTCTCCGCTGTAAGGGTATGGTAACCACCCGACGGACTTAGGCTACTG TCAGTCTC " ' ■ insert pLM1 -ORF pLM1 G L Q L Q S Q E E T K ε R R H S H T I G G p s o o Q s ε CTGCCTTCTCCCCCTGCACTTCCCATGTCTCTGAGTGCAAAGGGCCAACTTACCAACATAGTGAGTCCCACTGCGGCCACCACGCCAAGAATCACCCGi-T 1 1 i I . I I I ' I I 1 ' I ' ' I 1 I | " j 1 .-,- GACGGAA3AGGGGGACGTGAAGGGTACAGAGACTCACGTTTCCCGGTTGAATGGTTGTATCACTCAGGGTGACGCCGGTGGTGCGGTTCTTAGTGGGC A -insert pLM1 -ORFpLMI L P S P P A L P M S L S A K G Q L T N I V S P T A A T T P R I T P CCAACAGCATCCCCACCCACGAGGCGGCCTTCGAGCTGTACAGCGGCTCCCAAATGGGGAGCACCCTGTCCCTGGCCGAGAGACCCAAGGGAATGATTC3 GGTTGTCGTAGGGGTGGGTGCTCCGCCGGAAGCTCGACATGTCGCCGAGGGTTTACCCCTCGTGGGACAGGGACCGGCTCTCTGGGTTCCCTTACTAAGC -insert pLM1 -ORF pLM1 S N S I P T H E A A F E L Y S G S Q M G S T L S L A E R P G M I K GTCAGGATCCTTCCGAGACCCCACGGACGATGTTCACGGCTCAGTGCTGTCCCTGGCCTCCAGTGCCTCCTCCACCTACTCCTCAGCTGAGGAGAGGATG CA3TCCTAGGAAGGCTCTGGGGTGCCTGCTACAAGTGCCGAGTCACGACAGGGACCGGAGGTCACGGAGGAGGTGGATGAGGAGTCGACTCCTCTCCTAC ■ insert pLM1 -ORF pLM1 3 G 3 F R D P T D - V H G S V L 3 L A S S A S S T Y S S A E E R r' CAATCTGAGCAAATCCGGAAGCTTCGTA3GGAΔeTGGAATCATCCCAGGAAAAAGTGGCCACCTTGACGTCTCAGCT-TCTGCCAATGCTAATCTG3 G3 7AGACr i :.3TTTAGGCCTrCGAAGCAreCCTT3ACCTTAGTAGGGTCCTTTTTCACCGGTGGAACTGCAGAGTCGAAA3ACGGTTACGATTA3ACCACC -insert pLM1 -ORFpLMI ; S Ξ G I S < L R R E L Ξ S S Q E K V A Γ L T S Q L S A M N L Cr3crrτrGAGCAGA3e:T3GT3AA7AT3ACAT:CC3eCTGCGACACCTGGCAGAGACGGCCGAGGAGAAGGACACTGAGCTGCT3GATTTGCGAGA ιi: GAeGAAAACTCGTCTC33AeCACTTATAeTGTA3GGCG3ACGCTGTGGACCGTCTCTGCCGGCTCCTCTTCCT3TGACTCGACGACCTAAACGCTCTTT3 • insert pLM1 -ORFpLMI '-' .M M T S R R H L A E T A E ε K O T E '- L D Tuesday.18 November 199711 :48 Page 1 liq 34 pLM4 (1 > 10070) Site and Sequence C A TAGACT7TCTGAAGAAAAAGAACTCTGAGGCCCA3GCAGTCATTCAGG3AGCCCTTAATGCCTCAGAAACCACACCCAAA3AACTTCG3ATCAAGAGA G7A7CTG A AGACTTCTTTTTCTTGAGACTCCGGGTeCGTCAGTAAGTCCCTCGGGAATTACGGAGTCTTT3GTGTGGGTTTeTTGAA CeTAGTTCTC -insert pLM1 -ORFpLMI N s ε [ 0 G L N s ε T T p ε L 3 i K CAAAACTCCTCAGATAGCATCTCAAGCCTCAACAGCATCACTAGCCATTCCAGCATCGGCAGCAGCAAGGATGCTGATGCGAAAAAGAAGAAAAAAAA GTTTTGAGGAGTCTATCGTAGAGTTCGGAGTTGTCGTAGTGATCGGTAAGGTCGTAGCCGTCGTCGTTCCTACGACTACGCTTTTTCTTCTTTTTTTTCT -insert pLMl -ORFpLMI C N S S O S I S S L N S I T S H S S l G S S K D A D A K K K K K k GTTGGGTCTATGAGCTTCGAAGTTCCTTCAACAAAGCGTTCAGTATAAAAAAGGGGCCCAAGTCAGCTTCCTCATACTCGGATATAGAGGAGATTGCTAe CAACCCAGATACTCGAAGCTTCAAGGAAGTTGTTTCGCAAGTCATATTTTTTCCCCGGGTTCAGTCGAAGGAGTATGAGCCTATATCTCCTCTAACGAT3 -insert pLM1 -ORF pLM1 S V V E L R S S F N < A F S I K K G P K S A S S Y S D I E E I A T ACCCGACTCTTCAGCCCCCTCATCCCCCAAACTACAGCATGGTTCCACAGAGACTGCTTCACCCTCCATCAAGTCCTCCACCTTGTCCTCeGTGGGCACT TG3GCTGAGAAGTCGGGGGAGTAGGGGGTTTGA:GTC3TACCAAGGTGTCTCTGACGAAGTGGGAGGTAGTTCAGGAGGTGGAACAGGAG3CACCCGT(;A • insert pLM1 -ORFpLMI P 0 S 3 A P S S P K L . H G S T E T A S P S I K 3 3 T L S . V G 1 GA " GTCACCGAGGGCCC GCTCACCCAGCCCCCeACACTAGGCTGTTCCATGCAAATGAGόAGGAGGAGCCAGAGAAGAAGGA3GTATCG3 GC GCGC ■ + - 7 A':A3T3GCTCCCGGGACGAGTGGGTCGGGGGG " GT3ATCCGACAAGGTACGTTTACTCeTCCTCCTCGGTCTCTTCTTCCTCCATACC:TCGACGCGA -insert pLM1 -ORFpLMI : V T E G P A H P A P " T R L F H A N E E £ E : ' E K K E 3 E L R C " 3 3CTA'GGGAGAΔGGAAAT3AA3CrTΛCAGA: T;;3CTrGGAGGCCCTCAACTCT3e:eACCAACTGGArCAGCT7CG33AGACCAT3CACAA AT G C::3ATACCCrcτrCCTrTACTrCGA A TG""G~ A 33eG A ACCTCCGGGAGTTG A G A C3GGTGGTTGACCTAGTCGAAGCCCTCTGG7ACGTGTTGTA • insert pLM1 - ORFpLMI L E E M .< L T ; : ? L E A L N 3 A H 0 0 'j L - E T 1 H fl t Tuesday.18 November 199711.48 Page< (ig 34 pLM4 (1 > 10070) Site and Sequence . G'-ACrrG3AGGTGGACCTGerGAAAGCAGAGAArGAeC3ACTGAAGGrAG:CCCAGGCCCCrCATCAGGCrCCACTCCAGG3CAG TCC':r3GATCA;cτ C-TCAACC7CCACCTGGAC3ACTTTCGTCTCTTACT33CTGACTTCCATC33GGTCCGGGGAGTAGrCCGAGGTGAGGTCCeGTCCAG-GACCrAGTAGA • insert pLM1 -ORF pLM1 G P S S G S T P G Q G S GCATTATCTTCCCCACGCCGCTCCCTAGGCCTGGCACTCACCCATTCCTTC3GCCCCAGTCTTGCAGACACAGACCTGTCACCCATGGAT3GCATCAGTA CGTAATA3AAGGGGTGCGGCGAGGGATCCGGACCGTGAGTGGGTAAGGAAGCCGGGGTCAGAACGTCTGTGTCTGGACAGTGGGTACCTACCGTAGTCAT -insert pLM1 -ORF pLM1 A L S S P R R S L G L A L T H S F G P S L A D T D L S P M D G I S CTTGTGGTCCAAAGGAGGAAGTGACCCTCCGGGTGGTGGTGAGGATGCCCCCGCAGCACATCATCAAAGGGGACTTGAAGCA3CAGGAATTCTTCCTGG3 GAACACCAGGTTTCCTCCTTCACTGGGAGGCCCACCACCACTCCTACGGGG3CGTCGTGTAGTAGTTTCCCCTGAACTTCGTCGTCCT-AAGAAGGACCC -insert pLM1 -ORFpLMI T C G P K ε ε v T L R V V V R M P P Q H I I K G D L K Q Q ε F F L ϋ CTGTAGCΔAGGTCAGTGGAAAAGTTGACTGGAAGATGCTGGATGAAGCTGTTTTCCAAGTGTTCAAGGACTATATTTCTAAAATGGACCCA3CCTCTACC i 1 1 1 1 1 1 ' ■ 1 1 1 1 i μ i;. GACATCGTTCCAGΓCACCTTTTCAACTGACCTTCTACGACCTACTTCGACAAAAGGTTCACAAGTTCCTGATATAAAGATTTTACCTGGGTCGGAGATG- -insert pLM1 -ORF pLMl C S K V S G K V D V < M L D E A V F Q F K D Y 1 3 K M D = A S T CTGCGACTAAGCACTGAGTeCATCCATGGCTACAGCATeAGCCACGTGAAACGAGTGTTGGATGCAGACCCCCCCGAGATGCCTCCTTCCCGTCGAGGTG GA; i ;crGA;TCGTGACrCA3'3TAGGTACCGA-G-CGTA3TCGGTGCACTTr3CTCACAACCTACGTCTCGGGGGGCTCTAC33AGGAACG3CAGCTeCA. ■ insert pLM1 -ORFpLMI - S L S T E S i H G f S l S H K R V L O A E P P E M s P C P R TC ATAAeATΛ-CAGTCTC:CTeAAAGGTCTGAAGGA3AAATGCGTCGACA3CCTGGT'3TTC3AGACGeTGATCCCCAAGCCGATGATGCAGCACTAC«" AG ' TArTGTATAGTCAGAGGGAGTTTCCAGACTTCCTeTTTACGCAGCTGTCGGACCACAAGCTCTGCGACTAGGGGTTCGGeTΔCTΔCGTCGTGATGTA -insert pLM1 — ORFpLMI "I N I S V S L s G '. < E <' C V 0 3 V F E " L I P K ^ M I-' WO 98/24810 « - « _ PCTΕP97/06956 - Tuesday, 18 November 19971148 ρ aqe c, liq 34 pLM4 (1 > 10070) Site and Sequence . - ; 3CCrCCTGCTGAAGCACCGGCGCCTCGTCCT; 7 C-GGCCCCAGCGGCACGGGCAAGACCrACCrGACCAATCGCTTGGCC3A3rACCTG3T33AGCG: " eGGAGGACGACTTCGTGGCCGCGGAGCAGGAGAGCCCGGGGTCGCCGTGCCCGTTCTGGΔTGGACTGGrTAGCGAACCGGCTCATGGACCACCTCGC, -insert pLMl -ORFpLMI L L S G T G L A E Y L V E TC 7 GGCCGTGAGGTCACAGAGGGCATCGTCAGCACCTTCAACATGCACCAGCAGTCTTGCAAGGATCTGCAACTGTATCTTTCCAACCTAGCCAACCAGA AGACCGGCACTCCAGTGTCTCCCGTAGCAGTCGTGGAAGTTGTACGTGGTCGTCAGAACGTTCCTAGACGTTGACATAGAAAGGTTGGATCGGTTGGTi-- ' " -insert pLM1 -ORF pLM1 ≤ G R E V T E G I V S T F N M H Q Q S C K D L Q L Y L S N L A N - T iGACCGGGAAACAGGAATTGGGGATGTGCCCCTGGTGATTCTATTGGATGACCTGAGTGAAGCAGGCTCCATCAGTGAGTTGGTCAATGGGGCCCTCAe ATCTGGCCCTTTGTCCTTAACCCCTACACGGGGACCACTAAGATAACCTACTGGACTCACTTCGTCCGAGGTAGTCACTCAACCAGTTACCCCGGGAGTG -insert pLM1 -ORFpLMI l O R E T G I G D V P I L L O O L S E A G S I S E L N G A L T C-3CAAGTATCATAAATGTCCCTATATTATAGG7ACCACCAATCAGCCTGTAAAAATGACACCCAACCATGGCTTGCACTTGAGCTTCAGGATGTTGACe G CGTTC ATAGTATTTACAGGGATATAATATCCA'GGTGGTTAGTCGGACATTTTTACTGTGGGTTGGTACCGAACGTGAACTCGAAGTCCTACAACTGG -insert pLMl -ORF pL 1 ■ : < Y H K C P Y I I G T T N Q P V K M T P N H G L H L S F R M L T " 77CCAACAACGTG3AGCCAGCCAA7G3CTTCe-33TTCGTTACCTGAGGAGGAAGCTGGTAGAGTCAGACAGCGACATCAAT3CCAACAAGGAAGA':iC A-:A33TTGTTGCACCTeG3TCGGTTACeGAAG3AC;AAGCAATGGACTCCrCCTTCGACCATCTCAGTCTGTCGCTGTAGTrACGGTTGrτccTTCTC3 ■ insert pLM1 -ORF pLM1 - " 3 M N V E P A N G F L V R Y L R R K L V E S 0 S 0 I N A IJ K E E 7::rCG3GTGCTCGΔeTG3GTACCeAA3CTGT3G-A-CATCrcCACACCTTCCTTGAGAAGCACAGCACCTCAGACTTCCTCArCGGCCCTT'3CTTCT- A.;CAA3CCCACGAGCT3ACeCATG33TTCGACA:C rA3TAGAGGTGrGGAAGGAACTCTTCGTGTCGTGGAGTCTGAAGGAGTA3CCGG3AAe3AAGAA ■ insert pLM1 — ORFpLMI ■ — F. V L 0 * V 3 < '_ .' ' H L H T F L ε K H S T ό D F '- I G ' e F F l uesday.18 November 19971148 Page fc ig 34 pLM4 (1 > 10070) Site and Sequence ______ TC7GrCGTGTCCCATTGGCATTGAGGACTTCCGGACeTGGTTCAITGACCTGTGGAACAACTCTATCATTCCCTATCTACA3uAA3GAGCeAA33ATG e AGACAGCΔCAGGGTAACCGTAACTCCTGAAGGCCTGGACCAAGTAACTGGACACCTTGTTGAGATAGTAAGGGA-AGATGTCCTTeCTCG3TTCeTA : ■ msertpLMi -ORFpLMI 1 G I ε D N N S I I L Q E G ATAAAGGTCCATGGACAGAAAGCTGCTTGGGAGGACCCAGTGGAATGGGTCCGGGACACACTTCCCTGGCCATCAGCCCAACAAGACCAATCAAAGCTG- 1 — , 1 1 ' ' i ■ ' ' ■ i ' 1 1 1 1 1 — - — -+. 3i; TΔTTTCCΔGGTACCTGTCTTTCGACGAACCCTCCTGGGTCACCTTACCCAGGCCCTGTGTGAAGGGACCGGTAGTCGGGTTGTTCTGGTTAGTTTCGACA ■ insert pLM1 -ORFpLMI I K V H G Q K A A V E D P V E Y V R D T L P V/ P S A Q Q D Q S K L ACCACCTGCCCCCACCCACCGTGGGCCCTCACAGCATTGCCTCACCTCCCGAGGATAGGACAGTCAAAGACAGCACCCCAAGTTCTCTGGACTCAGATCC TGGTGGACGGGGGTGGGTGGCACCCGGGAGTGTCGTAACGGAGTGGAGGGCTCCTATCCTGTCAGTTTCTGTCGTGGGGTTCAAGAGACCTGAGTCTAG3 • msertpLMi -ORF LMI •• H L P P P T V G P H S I A S P P E D R T V K O S T P S S L - S D F T CTGATGGCCATGCTGCTGAAACTTCAA3AAGCTGCCAΔCTACATTGAGTCTCCAGATCGAGAAACCATCCTGGACCCCAACCTTCAGGCAACACTTTAA A-ACTACCGGTACGACGACTTTGAA3TTCTTCGACGGTTGATGTAACTCAGAGGTCTAGCTCTTTGGTAGGACCTGGGGTTGGAAGTCCGTTGTGAAATT • insert pLMl -ORF pLM1 L M A M L K L O E A A M I E S P O R E T I L D P N L Q A T L G'3:-rCG3CAATCACTGTCACCCCC3GACAGCA:AAee:TGGCATCAGCTATCTTAGCTeCTCCTCTCCCCTCTCCTCTTTCA3AGCAC-3eCreTeC.: C'.:AAGCCGTTA rGACAGrGGGGGCCr3TCGTe * "3e3ACCGTA3TCGATAGAATeGAGGAGGAGAGGGGAGAGGAGAAAύrCTCGTGACCGA3A3GT; -insert pLM! - F G N H C H P R T A E R V H Q L S . L L L S P L L F Q 3 T G S F -CCAGGAGGAGAΔCAGGA3GGAGGAC3AGA"GAAAeA33AG3GACAGGTTCTTGGTGCTGTACCTTTGAGAACTTCCTAGGAAGGAATG3TGGGGTG''j: ... rcc rcc rc TTG TCC τ;cc TCC ΓCCTC TAC — e"e:τcccτcτecAAGAACCACGACArGGAAACτcττGAAG ATccτrccττAceAc:ecA ι.e -insert pLM1 3 G G E Q E G G G 0 E = 3 G T G 3 V C C T F E M F L G R M 3 G V J- 7GGGAACTTGTGCCCC: τ AAΔeACATTTAC"::e;reC7CTAATGACTTTGGGύAAAAGATGATTCTGGGTCTT-CCCTTGAeTTC-r3"TTCAAT- A-CCrrGAACACGGGG3AT7T3TGrAAA7GA::3; 3GA3AT-AC7GAAACCCe77TTCrACTAA3ACCCAGAAA'jGGAAerGAACAACAAA TTι-- -insert pLM1 < J L C P l .\ - F - . L L W G D 0 3 G Tuesday.18 November 199711 :48 Page I' ig 34 pLM4 (1 > 10070) Site and Sequence __ ^ ACAAACTCCTGGGCTTTCTGGGGAGGGGTTCAGAAAACATCAAAACACTGCAGCAGTTCCCCGGAATTCAGCTTGGACTTAACC A GGCTGAACrrGCTCA TG-TTGAGGACCCGAAAGACCCCTCCCCAAGTCTTTTGTAGTTTTGTGACGTCGTCAAGGGGCCTTAAGTCGAACCTGAATTGGTeCGACTTGAACGAGT • insert pLMl G G H C S S S S L D L T R L N L L AAAGAAGCCGAATTCCAGCACACTGGCGGCCGTTACTAGTTCTAGATAACTGATCATAATCAGCCATACCACAΓTTGTAGAGGTTTTACTTGCTTTAAAA TTTCTTCGGCTTAAGGTCGTGTGACCGCCGGCAATGATCAAGATCTATTGACTAGTATTAGTCGGTATGGTGTAAACATCTCCAAAATGAACGAAATTTT insert pLMl AACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATA TTGGAGGGTGTGGAGGGGGACTTGGACTTTGTATTTTACTTACGTTAACAACAACAATTGAACAAATAACGTCGAATATTACCAATGTTTATTTCGTTAT T S H T S P . T . N I K . M Q L L L L T C L L Q L I M V T N K A I GCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTAACGCGTAAATTGTAAGCGTTA CGTAGTGTTTAAAGTGTTTATTTCGTAAAAAAAGTGACGTAAGATCAACACCAAACAGGTTTGAGTAGTTACATAGAATTGCGCATTTAACATTCGCAAT S Q I S Q I K H F F H C I L V V V C P N S S M Y L N A . I V S V ATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGAC TATAAAACAATTTTAAGCGCAATTTAAAAACAATTTAGTCGAGTAAAAAATTGGTTATCCGGCTTTAGCCGTTTTAGGGAATATTTAGTTTTCTTATCTG N L L L K F A L N R C . I S S F F N Q . A E I G K I P Y K S K E . T CGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGAT 1 1 1 1 1 1 | ι i I 1 1 1 1 μ GCTCTATCCCAACTCACAACAAGGTCAAACCTTGTTCTCAGGTGATAATTTCTTGCACCTGAGGTTGCAGTTTCCCGCTTTTTGGCAGATAGTCCCGCTA E I G L S V V P V V N K S P L L K N V D S N K G R K T V Q G O GGCCCACTACGTGAACCATCACCCTAATCAAGT-TTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTT CC3GGTGATGCACrTGσTA3TGύGA7TAGTTCAΔAAAACCCCAGCTCCACGGCATTTCGTGATTTAGCCTTGGGATTTCCCTCGGGGGCTAAATCTCGAA 3 P L R E P 3 P S 3 F L G S R C R K A L N R P K G S P F. F R A GAC._G3GΔAAGCCGGCGAAeGTGGCGA_AAA.3-AAG33AAGAAAGCGAAAGGAGCGGGC5CTAGGGCGCTGGCAA ΪGTAGCGGTCACGCTGCGeGTAAC C 7CCCCTTTCGGCCGCTTGCΔCCGCTCTT TCC "CCC TTCTTTCGCTTTCCTCGCCCGCGATCCCGCGACCGTTCACATCGCCAGTGCGACGCGC AT T3 R G k' P A M A R < Ξ 3 K K A K G A G A R A L A 5 V A V T L R T CACCACA:CCGCCGCGC TAATGCGCC3C ACA3 ' 3Ge3eGTCAGGTGGCACrTTTCGGGGAAATGTGC3CGGAACCCCTATTrGrT"A--rτTCTAAAT " 3 r3TG GCGGCGCGAArτAeGCGGe3AT,j*e:e3e3CAGrccAceGT AAAAGCC eττrACAC eGccrr ':3 ATAAACΔAA: AA AGΔτrτe7 Tuesday.18 November 199711 :48 Page I ' liq 34 pLM4 (1 > 10070) Site and Sequence . _ ^^ CATTCAAATATGTATCCGCTCATGA ACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTCCTGAGGCGGAAAGAACCΔGCTGTGGAA GTAAGTTTATACATAGGCGAGTACTCTGTTATTGGGACTATTTACGAAGTTATTATAACTTTTTCCTTCTCAGGACTCCGCCTTTCTTGGTCGACACCTT H S N M Y P L M R Q . P . . M L Q . Y . K R K S P E A E R T S C TGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTi-e ACACACAGTCAATCCCACACCTTTCAGGGGTCCGAGGGGTCGTCCGTCTTCATACGTTTCGTACGTAGAGTTAATCAGTCGTTGGTCCACACCTTTCAG3 M C V S . G V E S P Q A P Q Q A E V C K A C I S I S Q Q P G V E S P CCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGC GGTCCGAGGGGTCGTCCGTCTTCATACGTTTCGTACGTAGAGTTAATCAGTCGTTGGTATCAGGGCGGGGATTGAGGCGGGTAGGGCGGGGATTGAGGC3 Q A P Q Q A E V C K A C I S I S Q Q P . S R P . L R P S R P . L P . CCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAG3 GGTCAAGGCGGGTAAGAGGCGGGGTACCGACTGATTAAAAAAAATAAATACGTCTCCGGCTCCGGCGGAGCCGGAGACTCGATAAGGTCTTCATCACTCC P V P P I L R P H A O . F F L F Q R P R P P R P L S Y S R S S E AGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAGATCGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCC TCCGAAAAAACCTCCGGATCCGAAAACGTTTCTAGCTAGTTCTCTGTCCTACTCCTAGCAAAGCGTACTAACTTGTTCTACCTAACGTGCGTCCAAGAG3 E A F E A . A F A K I D Q E T G . G S F R M I E Q D G L H A G S P GGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCC3 CCGGCGAACCCACCTCTCCGATAAGCCGATACTGACCCGTGTTGTCTGTTAGCCGACGAGACTACGGCGGCACAAGGCCGACAGTCGCGTCCCCGCGGGC A A V V E R L F G Y D V A Q Q T I G C S D A A V F R L S A Q G R P G ' 7CTTTTTGTCAAGACCGACCTGTCCGGTGCCeTGAATGAACTGCAAGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTT3CGCA3 CAAGAAAΔACAGTTCTGGCTGGACA3GCCACGG3ACTTACTTGACGTTCTGCTCCGTCGCGCCGATAGCACCGACCGGTGCTGCCCGCAAGGAACGCGTC L r V K T D L . G A L N E L Q D E A A R L S V L A T T G V P C A C-3r3CTCGACGTTGTCACTGAAGe3G3AAGGGACTG3CTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGA- GACACGAGCTGCAACAGTGACTTCGCCCTTCCCTGACCGACGATAACCCGCrTCACGGCCCCGTCCTAGAGGACAGTAGAGTGGAACGAGGACGGCTCT: V L D V V T E A G R . rf L L L G E V P G Q D L L S 3 H L A ° A E > : ArcCA .;ATGGCTGAT3CAAT3:G3CGGCT;CArACGCTTGATCCGGCrACCTGCCCATrCGACCACCAAGCGAAACATCGCATCGA3CGA3CACG- v s i M A D A M 3 R L H T L D P A T C P F D H Q A K H R i - R A F A 7CGGAT GGAAGC :CGGTCTTGTC3. I'.AGGATG ATC ■ " " GGACGAAG AGCATC AGGGGCTCGCGCCAGCCGAACTGT1 'CGCCAGGCTCA; CGA; 3CA T G: 7'jAGCC T- iCCTTCC iGCCAGAACAGC -AGTCCTA: " A3 AeCTGCTTCTCGTAG TCCCCGAGCGCGGTCGGCTTGACAAGCGGTCC3AGT1 "ce Gen :GTAC3 — ^— — an.Neo ——————— — - — - — — — _____ ^^^^ __— D C . O E E H O G L A D A E L F A R L K Tuesday.18 November 199711 48 P age ^ liq 34 pLM4 (1 > 10070) Site and Sequence CCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAΔAATGGCCGCTTTTCTGGATTCATCGACT3TGGCCGGC- GGCTGCCGCTCCTAGAGCAGCACTGGGTACCGCTACGGACGAACGGCTTATAGTACCACCTTTTACCGGCGAAAAGACCTAAGTAGCTG CACCGGCCGA "" -κan.T4eo ε o c L N [ V E N G I 0 GG TGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGG- + S~-' CCCACACCGCCTGGCGATAGTCCTGTATCGCAACCGATGGGCACTATAACGACTTCTCGAACCGCCGCTTACCCGACTGGCGAAGGAGCACGAAATGCCA - _ruNeo G V A O R Y Q O I A L A T R D I A E E L G G E V A O R F L V Y G ATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGAAATGACCGACCAAGCGACGC: TAGCGGCGAGGGCTAAGCGTCGCGTAGCGGAAGATAGCGGAAGAACTGCTCAAGAAGACTCGCCCTGAGACCCCAAGCTTTACTGGCTGGTTCGCTGCGC KarvNβo >, 1 I A A P O S Q R i A F Y R L L D E F F . A G L G S K P T K R R CAACCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGe GTTGGACGGTAGTGCTCTAAAGCTAAGGTGGCGGCGGAAGATACTTTCCAACCCGAAGCCTTAGCAAAAGGCCCTGCGGCCGACCTACTAGGAGGTCGCG P T C H H E I S I P P P P S M K G V A S E S F S G T P A G . S S S A GGGGATCTCATGCTGGAGTTCTTCGCCCACCCTAGGGGGAGGCTAACTGAAACACGGAAGGAGACAATACCGGAAGGAACCCGCGCTATGACGGCAATAA CCeCTAGAGTACGACCTCAAGAAGCGGGTGGGATCCeCCTCCGATTGACTTTGTGCCTTCCTCTGTTATGGCCTTCCTTGGGCGCGATACTGCCGTTAT- G I S C W S S S P T L G G G . L K H G R R Q Y R K E P A L R Q AAAGACA3AATAAAACGCACGGTGTTGGGTCGT GTTCATAAACGCGGGGTTCGGTCCCAGGGCTGGCACTCTGTCGATACCCCACCGAGACCCCATT3 T77CTGTCTTATTTTGC3TGCCACAACCCAGCAAACAAGTATTTGCGCCCCAAGCCAGGGTCCCGACCGTGAGACAGCTATGGGGTGGCTCTGGGGTAAC < 0 R I K R T V L G R L F I M A G F G P R A G T L 3 1 P H R D P I GGGCCAATACGCCCGCGTTTCTTCCTTTTCCCCACCCeACCCCCCAAGTTCGGGTGAAGGCCCAGCGCTCGCA3CCAACGTCGG3GCGGCA3GCCCTGC: CCCGGTTATGCGGGCGCAAAGAAGGAAAAGGGG " GGGGTGGGGGGTTCAAGCCCACTTCCGGGTCCCGAGCGTCGGTTGCAGCCCCGCCGTCCGGGAC-L G A N T P A C L ? F => - 3 T P _ V V K A Q G 5 Q P T S G R G A L F A'AGCCTCAGGTTACTCATATATACTTTAGATTCATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTT'GATAA-CTCATGAC'.- — i i i ■ — i ■ i i i ■ , i ■ — ■ i i i i i i :r TATCGGAGTCCAATGΔGTATATATCAAATCTAACTAAATTTTGAAGTAAAAATTAAATTTTCCTAGATCCACTTCTAGGAAAAACTATTAGAGTACTG'. " P Q T H I ' - R L I N F I F N L K G S R R S F L I 1 S . F AAArccCTTAACGTGA3TTrTCG TCCAC7GAGCG~CA3ACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCT3CGCGTAATCTGCT_ rτrA3GGAATrGCACTCAAAAGCAACGT3AC-CCCAC-erGGGGCATCTTTTCTAGTTTCCTAGAAGAACTCTAGGAAAAAAACAeGCGCA7TAGACGA: < S L N V S F R 3 T E R 3 T P K R S K O L L E I L F r C A S A C " " GCAAAC IΔAAAAAACC ACCGC7 ΆCCACCGGT: :G' 7TT3TTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAAC1 ■ GGCTTCA': 3CAGAGCGC-: " jAAcarTTc iTTTTTTTάC TGGCGATCGTCGCCA; :CAAA; iAAACGGCCTAGTTCTCGATGGTTGAGAAAAAGGCT ' rcc iTTGACCGAAG " '! :C7CΓCGCGT: puuoπ A C i 0 < N H R - V C R I K S ϊ O L F F R P '. 3 A Ξ R F Tuesday.18 November 199711 :48 p age fi* liq 34 pLM4 (1 > 10070) Site and Sequence ATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAG TATGGTTTATGACAGGAAGATCACATCGGCATCAATCCGGTGGTGAAGTTCTTGAGACATCGTGGCGGATGTATGGAGCGAGACGATTAGGACAATGGTC '" " -P-C-ΪΓ" S F C S R S T S R H R L H S C Y TGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGT3 ACCGACGACGGTCACCGCTATTCAGCACAGAATGGCCCAACCTGAGTTCTGCTATCAATGGCCTATTCCGCGTCGCCAGCCCGACTTGCCCCCCAAGCAC V L P V A I S R V L P G W T Q D D S Y R I R R S G R A E R G V R CACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGG-C GTGTGTCGGGTCGAACCTCGCTTGCTGGATGTGGCTTGACTCTATGGATGTCGCACTCGATACTCTTTCGCGGTGCGAAGGGCTTCCCTCTTTCCGCCT3 A H S P A S E R P T P N . D T Y S V S Y E K A P R F P K G E R R T AGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACC i i ' i i 1 i i i i ' 1 ' I ' i i i . ■ μ q_ ' TCCATAGGCCATTCGCCGTCCCAGCCTTGTCCTCTCGCGTGCTCCCTCGAAGGTCCCCCTTTGCGGACCATAGAAATATCAGGACAGCCCAAAGCGGTG3 G ! R A A G S E Q E S A R G S F Q G E T P G I F I V L S G F A T TCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCT3 AGACTGAACTCGCAGCTAAAAACACTACGAGCAGTCCCCCCGCCTCGGATACCTTTTTGCGGTCGTTGCGCCGGAAAAATGCCAAGGACCGGAAAACGAC -pUCon I S O L S V O F C D A R Q G G G A Y G K T P A T R P F Y G S P F A GCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCATGCAT ' i 1 1 1 i 1 1 1 ■ ■ ■» 10070 CGGAAAACGAGTGTACAAGAAAGGACGCAATAGGGGACTAAGACACCTATTGGCATAΔTGGCGGTACGTA G L L L T C S F L R Y P L I L W I T V L P P C 1 PΛfre » Tuesday, 18 November 199710:34 fig _0-pEGFP72 (1>9697) Site and Sequence Enzymes : 72 of 146 enzymes (Filtered) A Settings- Linear, Certain Sites Only. Standard Genetic Code .Bgi i I TAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCT3GCTGACC3 I -0 ATCAATAATTATCATTA3TTAATGCCCCAGTAATCAAGTATCGGGTATATACCTCAAGGCGCAATGTATTGAATGCCATTTACCGGGCGGAeCGACTGGe L L I V I N Y G V I S S . P ! Y G V P R Y ! T Y G K W P A U T Aat II Aat II ! CCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGT GGGTTGCTGGGGGCGGGTAACTGCA3TTATTACTGCATACAAGGGTATCATTGCGGTTATCCCTGAAAGGTΔACTGCAGTTACCCACCTCATAAATGCCA A Q R P P P I O V N N O V C S H S N A N R D F P L T S M G G V F T V Bgl I Nde I Aat II Bgl I AAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTA -+- 300 TTTGACGGGTGAACCGTCATGTAGTTCACATAGTATACGGTTCATGCGGGGGATAACTGCAGTTACTGCCATTTACCGGGCGGACCGTAATACGGGTCAT N C P L G S T S S V S Y A K Y A P Y . R Q . R . M A R L A L C P V SnaB I Nco I I CATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGA -+- GTACTGGΔATACCCTGAAAGGATGAACCGTCAT3TAGATGCATAATCAGTAGCGATAATGGTACCACTACGCCAAAACCGTCATGTAGTTACCCGCACCT H O L M G L S Y L A V H L R t S H R Y Y H G O A V L A V H Q V A Aat II TAGCGGTTTGACTCAC33G3ATTTCCAA3TCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTA -+- -+- -+- ATCGCCAΔACTGAGTGC:c:TAAAGG7TCAGAG3"GG3GTAACTGCAGTTACCCTCAAACAAAACCGTGGTTTTAGTTGCCCTGAAAGGTTTTACAGCAT Ϊ A V . L T . I S . S ' P H . R Q V E F V L A P K S T G L S K M S . θ I £c47 ACAACTCCGCCCCATTGAC3CAAAT3CGCGG τ A3GC37GTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTΔGTGAACCGTCAGATCC3eTAGCGCTA -+- TGTTGAGGCGGGGTAACGeGTTTACCeGCCATCCGeACATGCCACCCTCCAGATATATTCGTCTCGACCAAATCΔCTTGGCAGTCTAGGCGATCGCGA " O L R P I D A G R A C T V G G L Y K Q 3 V F S E P S D 3 L A L Nco I CCeGTCGCCACCATG3TCA3CAAG3CC3AGGA'_C " GTT:ACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACG3CGACGTAAACG3CCACAAGTTCAG_3 GGCCAGCGGTGGTACCAC-_GTTCCCGeTCC"CCACAA3TGGCCCCACCACGGGTAGGACCAGCTCGACCTGCCGCTGCATTTGCCGGTGTTCAAGT_GC eGFP C e unc53 = V A T M V 3 K G E E T G V V P 1 L V E L D G D V N G H K F 3 7G7CGGCGAGGGCGA3 :GC' 3ATGCCA:CTACGCC AAG -TGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCG TGCCCT 33CCCACCCTCGTGA: 7AG3CC3CTCCCGCTC :C3 ;TACGGI 33A TGC ZZ 3 GACTGGGACTTCAAGTAGACGTGGTGGCCGTTCGAeGGGC ACG33M CCGGG' 33GAGC AC7 '• ' 3 G E G E 3 D A T Y : < L T L .K F I C T T G K L P ■ o V P r L v T WO 98/24810 __ Λ PCT7EP97/06956 158 Tuesday 18 November 19971034 Paoe-I fig 30 pEGFP72 (1 > 9697) Site and Sequence . tACCCTGΔCCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACrTCTTCAAGTCC3CCA-GCCC3AAGGC7Ae3TCC , G ι -- GrGGGACTGGATGCCGCACGTCACGAAGTCGGCGATGGGGCTGGTGTACTTCGTCGTGCTGAAGAAGTTCAGGCG'3TAC3GGCTTCCGA-3-AGGT'-CT "'" ' " ' - eGFP C e uπc53 G V Q C P D H M K Q H D M P ε G Y Q ε Kspl I ZGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCG GCGTGGTAGAAGAAGTTCCTGCTGCCGTTGATGTTCTGGGCGCGGCTCCACTTCAAGCTCCCGCTGTGGGACCACTTGGCGTAGCTCGACTTCCCGTAG " -eGFPCeunc53 R T I F F K D D G N Y K T R A E V K F E G D T L V N R I E L K G I ACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCA« TGAAGTTCCTCCTGCCGTTGTAGGACCCCGTGTTCGACCTCATGTTGATGTTGTCGGTGTTGCAGATATAGTACCGGCTGTTCGTCTTCTTGCCGTAGTT --GFPCe.ur.cS3 O F K E O G N I L G H K L ε Y N Y N S H N V Y I M A D K Q K N G I K GGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCC3TGCTGCTG CCACTTGAAGTTCTAGGCGGTGTTGTAGCTCCTGCCGTCGCACGTCGAGCGGCTGGTGATGGTCGTCTTGTGGGGGTAGCCGCTGCCGGG3CACGACGAC -eGFPCeurc53 V N F K I R H N I E D G S V Q L A 0 H Y Q Q N T P I G D G = V L L CCCGACAACCACTACC TGA 3CA; :c: :AGT: :CGCCC "GA; 3CAAAGACCCCAACG AGAAGCG ICGATC, ACATGGTCCTGCTGGAGTTCGTGACC: ICC3 CCGGIJH GGόCTGTTGGTGATGGACTCGTGGGTCACGCGGCACTCGTTTCTGGGGTTGCTCTTCGCGCTAGTGTACCAGGACGACCTCAAGCACTGGCSGCGGCCC' -eGFPC e unc53 = D N H Y L S T . S A L S K D P N E K R D H V L L E F V T A A _ ASU II BspM II Bgl II EcoN I "CACrcTCGGCATGGACGAGCT3TACAA3TCCGGACrCΔGATCTACGTCAAΔTGTAGAArrGATACCAATCTACACGGA τ TG3GCCAA-':CGC CCTTT: -ι3-GAGAGCCGTACCTGCTC CATGTTCAGGCCTGAόTCTAGATGCAGTTrACATCTTAAC ATGG TAGATG7GCCTAACCCGGTTAGCCGTG A A. -eGFPC eurc53 - C e unc53 7 L G 0 E - f < S G - S S T S N V E L I P i Y T D V A N = H L ? Nru I EcoR =.GGGCΔGCTTArCAAAGTCG«-rAG3CATATTTCCA A rGATrτTCGCGACTATCGACTGGrTTCTCAGCTTATTΛ„TCr3ATCGTTr.:;„TCAACG-.< C ' CCCGTC :GAA' " AGTTTC AG: ΆA T C: CTA TAAAGC "TACTA AAAGCGCT3ATAGC i :GAC CAAACA .G'CGAATA, sr ' AC-Λ ■ ZTAZ CAACGC ' "A. TTGCT" eGFP C β urcS3 - , - , L - - '1 0 F R O < P I ) L 1 1 • 1 • 1 F Tuesday, 18 November 199710:34 p agβ J fiq30pEGFP72 (1>9697) Site and Sequence Bsm I TTCTCGCCTGCATTCACGAAACGTTTGGCAAAAATCACATCGAACCTGGATGGCCTCGAAACGTGTCTCGACTACCTGAAAAATCTGGGTCTCGACTGfT AAGAGCGGACGTAAGTGCTTTGCAAACCGTTTTTAGTGTAGCTTGGACCTACCGGAGCTTTGCACAGAGCTGATGGACTTTTTAGACCCAGAGCTGACGA ■ eGFP.Cβunc53 -C.e. unc53 S P A F T K R L [ T S N L L E T C L D Ear l EcoR Pvu Ksp632l Hind III CG A AACTCACCAAAACCGATATCGACAGCGGAAACTTGGGTGCAGTTCTCCAGCTGCTCTTCCTGCTCTCCACCTACAAGCAGAAGCTTCGGCAACTGAA GCTTTGAGTGGTTTTGGCTATAGCTGTCGCCTTTGAACCCACGTCAAGAGGTCGACGAGAAGGACGAGAGGTGGATGTTCGTCTTCGAAGCCGTTGACTT -C.e. unc53 S K L T K T D I O S G N L G A V L Q L L F L L S T Y K Q K L R Q L > PmaCI Sst II i PmaCI j j i AAAAGATCAGAAGAAATTGGAGCAACTACCCACATCCATTATGCCACCCGCGGTTTCTAAATTACCCTCGCCACGTGTCGCCACGTCAGCAACCGCTTCA TTTTCTAGTCTTCTTTAACCTCGTTGATGGGTGTAGGTAATACGGTGGGCGCCAAAGATTTAATGGGAGCGGTGCACAGCGGTGCAGTCGTTGGCGAAGT -eGFP.C.e.un-53 -C.e. unc53 K D Q K K L E Q L P T S I M P P A V S K L P S P R V A T S A T A S GCAACTAACCCAAATTCCAACTTTCCACAAATGTCAACATCCAGGCTTCAGACTCCACAGTCAAGAATATCGAAAATTGATTCATCAAAGATTGGTATCA CGTTGATTGGGTTTAAGGTTGAAAGGTGTTTACAG7T3TAGGTCCGAAGTCTGAGGTGTCAGTTCTTATAGCTTTTAACTAAGTAGTTTCTAACCATAGT -eGFP.Ceur,c53 -C.e. unc53 Δ T N P N S H F P Q M 3 T S R L Q T P - S R I S < I D S S K ! G I Aat II i GCCAAAGACGTCTGGACTTAAACCACeCTCATeATeAACCACTTCATCAAATAATACAAArTCATTCCGTCCGTCGAGCCGTTCGAGTGGCAATAATAA 7, :3GTTTCTGCAGA CCTGAATTTGGT G3GAG7 A3 ' TGGTGAAGTAGTTTATTATGTTT AAGTAAGGCA GGC 5GCTCGGCAAGC TCA CCG TTATTAT " — eGFP.C.e uncb3 3 K T s G L • < ? = S 3 T T S S N N T fl 3 F R P 5 S 3 3 5 M fl π Tuesday.18 ovember 19971034 Page *r fιq30pEGFP72 (1 >9697) Site and Sequence Ear I EcoR V Ksp632l Asu II l I I TGT TGGCTCGACGATATCC AC ATCTGCGAAGAGCTTAGA ATC ATCATCAACGTACAGCTCTATTTCGAATCTAAACCGACCTACCTCCCAAeTCCAAAAA ACAACCGAGCTGCTATAGGTGTAGACGCTTCTCGAATCTTAGTAGTAGTTGCATGTCGAGATAAAGCTTAGATTTGGCTGGATGGAGGGTT3AGGTTTTT ~ " --GFPCeunc_3 -C θ. unc53 V G S T I S T S A K S L E S S S T Y S S I S N L N R P T S O L Q K Xba I n β I CCTTCTAGACCACAAACCCAGCTAGTTC3TGTT3CTACΔACTACAAAAATCGGAAGCTCAAAGCTAGCCGCTCCGAAAGCCGTGAGCACCCCAAAACTTG 1 1 ι ι | 1 1 ' i ' l ' 1 1 1 ■ I 1 i μ ;-_ GGAAGATCTGGTGTTTGGGTCGATCAAGCACAACGATGTTGATGTTTTTAGCCTTCGAGTTTCGATCGGCGAGGCTTTCGGCACTCGTGG3GTTTTGAAC -βGFPC.e.unc53 -Ce. unc53 P S R P Q T Q L V R V A T T T K t G S S K L A A P K A V S T P k L Bsm I I CTTCTGTGAAGACTATTGGAGCAAAACAAGAGCCCGATAACAGCGGTGGTGGTGGTGGTGGΔATGCTGAAATTAAAGTTATTCAGTAGCAAAAACCCATC GAAGACACTTCTGATAACCTCGTTTTGTTCTCGGGCTATTGTCGCCACCACCACCACCACCTTACGACTTTAATTTCAATAA3TCATCG7TTTTGGGTAG -eGFPC e unc53 - C e unc53 A S K T I G A < Q - :> D N 3 G G G G G G M L K L K L F S S - N P .. τ" eCTCATCGAATAGCCCACAACCTACGAGAAA3GC33CGGCGGTGCCTCAACAACAAACTTTGTCGAAAΔTCGCTGCCeCAGTGAAAAGT3GCCT3AAό • i ACGA3TAGCTTATCGGGTGTTGGATGCTCT7TCCGCCGCCGCCACGGAGTTGTTGTTTGAAACAGCTTTTAGCGACGG3GTeACTTTTCACCGGACTTC -eGFPC -unc53 - C e unc53 ό S S M S P Q P T R K A A A V P Q Q Q T L S K l A A P V r' S G L t CCCCCeACCAGTAAGCTCGGAAGTGeCAeGTCTA'GTCGAAGCTTTGTACGCCAAAAGTrTCCTACCGTAAAACGGACGCCCCAATCA'ATCTCAA AA '-'-•: 3CT3 TCAT TCG ACCCTTCACCC73C AGA 7AC AGC TTCGAAACA TGC 3GTTTTCAAAGCATGGCATTTT3CCTGCCC33TTAGTA-A3AGTT37TC -eGFPC euπc53 -Ce unc53 L C T P K V S Y R < T D A 3 | Tuesday 18 November 19971034 Page fιq30pEGFP72 (1>9697) Site and Sequence ____ ^^ Ear I : Ksp632l BspM II i : ACTCGAAACGATGCTCAAAGAGCAGTGAAGAAGAGTCCGGATACGCTGGATTCAACAGCACGTCGCCAACGTCATCATCGACGGAAGGTTCCCTAAGCA7 TGAGCTTTGCTACGAGTTTCTCGTCACTTCTTCTCAGGCCTATGCGACCTAAGTTGTCGTGCAGCGGTTGCAGTAGTAGCTGCCTTCCAAGGGATTCGTH -eGFPCθ urc53 -Ce unc53 D S K R C S K S S E E ε S G Y A G F N S T S P T S S S T E G S L S t' Bsm I Sp I GCATTCCACATCTTCCAAGAGTTCAACGTCAGACGAAAAGTCTCCGTCATCAGACGATCTTACTCTTAACGCCTCCATCGTGACAGCTATCAGACAGCCC CGTAAGGTGTAGAAGGTTCTCAAGTTGCAGTCTGCTTTTCAGAGGCAGTAGTCTGCTAGAATGAGAATTGCGGAGGTAGCACTGTCGATAGTCTGTCGGC -eGF Cθ unc53 -Cθ unc53 H S T S S K S S T S O E K S P S S D D L T L N A S I V T A I R Q P Ssp I ATAGCCGCAACACCGGTTTCTCCAAATATTATCAACAAGCCTGTTGAGGAAAAACCAACACTGGCAGTGAAAGGAGTGAAAAGCACAGCGAAAAAAGATC "A-CGGCGTTGTGGCCAAAGAGGTTTATAATAGTTGTTCGGACAACTCCTTTTTGGTTGTGACCGTCACTTTCCTCACTTTTCGTGTCGCTTTTTTCTAC -eGFPCeuπc53 -C e unc53 A A T P V S P N I I N < P V E E K P T L A V K G V K S T A K K O PmaCI Pvu II PmaCI EcoR V C CCTCCAGCTGTTCCGCCACGTGACACCCAGCCAACAATCGGAGTTGTTAGTCCAATTATGGCACATAAGAAGTTGACAAATGACCCCGTGATATCT--. G7GAGGTCGACAAGGCGGTGCACTGTGGGTCGύTT3 "r TAGCCTCAACAATCAGGTTAATACCGTGTATTCTTCAACTGTTTACTGGGGCACTATAGAC- -eGFP C β unc53 - C e uπc53 Alwπ I i H CCAGAACCTGAAAAGCTCCAATCAATGAGCA-CCACACGACGGACGTTCCACCGCTTCCACCTCTAAAATCAGTTGTrCCACTTAAAATGACTT-K ""33TCTT3CACTTTTCGAG3TTAGTTACTCC-ACCT3TGCTGCCTGCAAGGTGCCGAAGGTGCAGATTTTAGTCAACAACGTGAA T TT7ACTGAAf -eGFPCeurcS3 - C e uπc53 162 Tuesday, 18 November 199710:34 μ> age _, fιq30pEGFP72 (1 >9697) Site and Sequence Spll ! : Sp " ATCCGACΔACCACCAACGTACGATGTTCTTCTAAAACAAGGAAAAATCACATCGCCTGTCAAGTCGTTTGGATATGAGCAGTCGTCCGCGTCTGAAGACT T A GGCTGTTGGTGGTTGCATGCTACAAGAAGATTTTGTTCCTTTTTAGTGTAGCGGACAGTTCAGCAAACCTATACTCGTCAGCAGGCGCAGACTTCTGA " " -eGFP.C eunc53 -C.e. unc53 0 V L Q G I T S P V S F G Y E S S E 0 CCATTGTGGCTCATGC3TCGGCTCAGGTGACTCCGCCGACAA AA ACTTCTGGTAATCATTCGCTGG A G A GA A GGATGGGAAAGAATAAGACATCAGAATC GGTAACACCGAGTACGCAGCCGAGTCCACTGAGGCGGCTGTTTTTGAAGACCATTAGTAAGCGACCTCTCTTCCTACCCTTTCTTATTCTGTAGTCTTA3 -eGFP.C.e.unc53 -C.e. unc53 S I V A H A S A Q V T P P T K T S G N H S L E R R M G K N K T S E 3 CAGCGGCTACACCTCTGACGCCGGTGTTGCGATGTGCGCCAAAATGAGGGAGAAGCTGAAAGAATACGATGACATGACTCGTCGAGCACAGAACGGCTAT GTCGCCGATGTGGAGACTGCGGCCACAACGCTACACGCGGTTTTACTCCCTCTTCGACTTTCTTATGCTACTGTACTGAGCAGCTCGTGTCTTGCCGATA -eGFP.C eunc53 -C.e. unc53 S G Y T S D A G V A M C A K M R E K L K E Y O D M T R R A Q N G V Asu II Sst I βspM II C:7GACAACTT ' CGAAGACAGTTCCTCCTTGTCGTCTGGAATATCCGATAACAACGAGCTCGACGACATATCCACGGACGATTTGTCCGGAGTAGACATG3 GGACTGTTGAAGCTTCTGTCAAGGAGGAACAGCAGACCTTATAGGCTATTGTTGCTCGAGCTGCTGTATAGGTGCCTGCTAAACAGGCCTCATCTGTACC ■ eGFPCe.unc53 -C.e. uncS3 ' D N F E C 3 S S L 3 3 G I S D N N E L D 0 I S T D D L 3 G D H CAACAGTCGCCTCCAAACATAGCGACTATTCCCACTTTGTTCGCCATCCCACGTCTTCTTCCTCAAAGCCCCGAGTCCCCAGTCGGTCCTCCACATCAG- G " rGTCA3CGGAGGTTT3TATCGCTGATAAGGG"GAAACAAGCGGTAGGGTGCAGAAGAAGGAGTTTCGGGGCTCAGGGGTCAGCCAGG; -eGFPC e uπc53 • Ce unc53 Γ V A S K H S D Y S - F V R H P T S S S S K P R V P S R S S T S Tuesday.18 November 199710.34 'f>age r fig 30 pEGFP72 (1 > 9697) Site and Sequence Bgl I f~r I o I Bbe I CGATTCTCGATCTCGAGCAGAACAGGAGAATGTGTACAAACTTCTGTCCCAGTGCCGAACGAGCCAACGTGGCGCCGCTGCCACCTCAACCTTCGGA'-AA GCTAAGAGCTAGAGCTCGTCTTGTCCTCTTACACATGTTTGAAGACAGGGTCACGGCTTGCTCGGTTGCACCGCGGCGACGGTGGAGTTGGAAGCCTGT- -eGFPC θ.uncS3 -Ce. un_53 0 s S R ε Q ε N L L Q C R T S Q T S T F Xma I Spe I j Sma I Pvu II Sal I I i i CATTCGCTAAGATCCCCGGGATACTCATCCTATTCTCCACACTTATCAGTGTCAGCTGATAAGGACACAATGTCTATGCACTCACAGACTA3TCGACGAC GTAAGCGATTCTAGGGGCCCTATGAGTAGGATAAGAGGTGTGAATAGTCACAGTCGACTATTCCTGTGTTACAGATACGTGAGT3TCTGATCAGCTGCT3 -eGFP.C e.uncS3 -C.e. uπc53 H S L R S P G Y S S Y S P H L S V S A D K O T M S N H S Q T S R R CTTCTTCACAAAAACCAAGCTATTCAGGCCAATTTCATTCACTTGATCGTAAATGCCACCTTCAAGAGTTCACATCCACCGAGCACAGAATGGCGGCTC" GAAGAAGTGTTTTTGGTTCGATAAGTCCGGTTAAAGTAAGTGAACTAGCATTTACGGTGGAAGTTCTCAAGTGTAGGTGGCTCGTGTCTTACCGCCGAGA -eGFPCe.uncS3 -C.e. unc53 P S S 0 K P S V S G 3 - H S L 0 R K C H L Q E F T 3 T E H R M A A L .Bam HI C7TGAGCCCGAGACGGGTGCCGAACTCGATG7C3AAA7ΔTGATTCTTCAGGATCCTACTCGGCGCGTTCCCGAGGTGGAAGCTCTACTGGTATCTATGGA G i ACTCGGGCTCTGCCCACGGCTTGAGCTACAG:T7TATACTAAGAA3TCCTAGGATGAGCCGCGCAA3GGCTCCACCTTCGAGATGACCA7A3ATACC " -eGFPC S.unc53 - C e. unc53 L S P R R V P N S M 3 K Y D 3 3 G S Y S A R S R G G S S T G I Y - ■ eGFPCeuπc53 Ce unc53 7 F C L H R L S D E < S P A H S A K S Ξ M C S Q L 3 L A 3 7 T Tuesday, 18 November 19971034 Page fig 30 pEGFP72 (1 >9697) Site and Sequence PmaCI PmaCI Sal I ATGGATCTCTCAATGAGAAGTACGAACATGCTATTCGGGACATGGCACGTGACTTGGAGTGTTACAAGAACACTGTCGACTCACTAACCAAGAAACAGG -I7 TACCTAGAGAGTTACTCΠCATGCTTGTACGATAAGCCCTGTACCGTGCACTGAACCTCACAATGTTCTTGTGACAGCTGAGTGATTGGTTCTTTGTC'.- -eGFPC eur.c53 -C e. unc53 I R 0 M D L E C S L T K Q E Ear I Hind I Cla l Ksp632l GAACTATGGAGCATTGTTTGATCTTTTTGAGCAAAAGCTTAGAAAACTCACTCAACACATTGATCGATCCAACTTGAAGCCTGAAGAGGCAATACGATTC CTTGATACCTCGTAACAAACTAGAAAAACTCGTTTTCGAATCTTTTGAGTGAGTTGTGTAACTAGCTAGGTTGAACTTCGGACTTCTCCGTTATGCTAAG -eGFPCe.unc53 • C.θ. unc53 N Y G A L F D L F E Q K L R K L T Q H I D R S N L K P E E A I R F AGGCAGGACATTGCTCATTTGAGGGATATTAGCAATCATCTTGCATCCAACTCAGCTCATGCTAACGAAGGCGCTGGTGAGCTTCTTCGTCAACCATCTC TCCGTCCTGTAACGAGTAAACTCCCTATAATCGTTAGTAGAACGTAGGTTGAGTCGAGTACGATTGCTTCCGCGACCACTCGAAGAAGCAGTTGGTAGA3 -eGFPCβ uπc53 -Ce. uncS3 N ε G ε Q P Ear I Cla I Cla I Sst I Ksp632l 7G3AATCAGTTGCATCCCATCGATCATCGATGT:A-C3TCGTCGAAAAGCAGCAAGCAGGAGAAGATCAGCTTGAGC7CGTTTGGCAAGAACAAGAAGAC AC:TTAGTCAACGTAG3GTAGCTAG7AGCTACAC-A3CΔGCA3CTTTTCGTCGTTCGTCCTCTTCTAG7CCAACTCGAGCAAACCGTTCTT37T:TTCT; -eGFPC e unc53 -C e uncS3 S V A S H R S S M S S S S < S S K 0 E K I 3 L S 3 F G I' . , ι ► Bam HI Nde | βspM II "3GATCCGCTCCTCACTCTCCAA377CACCAACAA3AAGAA;AA3AACTACGACGAAGCACATATGCCAT:AATTTCC3GATCTCAACGAACT:TTG-: -CCTAG3CGACGAGTGAGAGGT7CAA3TGG-TC"C-TCΓT3TTCTT3ATGCTGCTTCGT3TATACGC7A3TTAAAGGCCTAGAGTTCCΓ7GA3AA0T- -eGFPC e unc53 -C e uπc53 3 S L 3 K T K < Λ M K N Y D E A H M J S I 3 r L o WO 98/24810 Λrr PCT7-P97/06956 165 Tuesday.18 November 199710.34 p agβ s fιq30pEGFP72 (1 >9697) Site and Sequence Sst I ApaL I i } AA;ATTGATGTGATTGAGTTGAAGCAAGAGCTCAAAGAACGCGATAGTGCACTTTACGAAGTCCGCCTTGACAATCTGGATC3 T 3CCCCC3 AGTTGAT-- TTGTAACTACACTAACTCAACTTCGTTCTCGAGTTTCTTGCGCTATCACGTGAAATGCTTCAGGCGGAACTGTTAGACCTAGCACGGGCGCTTCAACTA- -eGFPCeunc53 -Ce. unc53 \ I D V I E L K Q E L K E R D S A L Y E V R L O N L O R A R E V O TTCTGAGGGAGACAGTGAACAAGTTGAAAACCGAGAACAAGCAATTAAAGAAAGAAGTGGACAAACTCACCAACGGTCCAGCCACTCGTGCTTCTTCCCG AAGACTCCCTCTGTCACTTGTTCAACTTTTGGCTCTTGTTCGTTAATTTCTTTCTTCACCTGTTTGAGTGGTTGCCAGGTCGGTGAGCACGAAGAAGGGC -eGFPC.e.unc53 -C.e. unc53 L R E T V N K L K T E N K Q L K K E V D K L T N G P A T R A S S P Kspl βsrl Asu II CGCCTCAATTCCAGTTATCTACGACGATGAGCATGTCTATGATGCAGCGTGTAGCAGTACATCAGCTAGTCΔATCTTCGAAAC3ATCCTCT3GCTGCAAC GCGGAGTTAAGGTCAATAGATGCTGCTACTCGTACAGATACTACGTCGCACATCGTCATGTAGTCGATCAGTTAGAAGCTTTGCTAGGAGACCGACGTTG -eGFPCeunc53 -Ce. unc53 [ P D D E H C S S T S S Q S 3 R S Pvu I Hpa l EcoR V TCAATCAAGGTTACTGTAAACGTGGACATCGCT3GA3AAATCAGTTCGATCGTTAACCCGGACAAAGAGATAATCGTAGGATAT;TTGCCA-GTCAACC AGTTAGTTCCAATGACATTTGCACCTGTAGCGACCTCTTTAGTCAAGCTAGCAATTGGGCCTGTTTCTCTATTAGCATCCTATAGAACGGTACAGTT'3'3- -eGFPCe.uπc53 - C e unc53 S I < V V N V D I A G E I S S I V N P D < E I I G Y L A i: S T Cla I Gr^AGTCA τ GC7G3AAAGA;ATTGATGTrTC7A-"CT 3 ACTATTTGAAGrCTACCTATCCAι3AA7T3Ar3TGGAGCA7CAACrrGGAAr:'3A7GCT'-; CA CTCAGTACG ACC TTT :TG " ΔA :TA :AAAGATAAGA' CCTGATAAAC TCAGATGGATAGG rcr AACTA CACCTC GTAGTT :AACCT- A3CTACGA C eGFPC eui,-53 ■ -- — 3 1 S C '• K ;, ! 0 v s • L 3 L F E v Y L S R ' D v ε H . L G 1 J A F WO 98/24810 ... PCT/EP97/06956 166 Tuesday, 18 November 199710.35 p agβ \ o fig 30 p£GFP72 (1 >9697) Site and Sequence Mlu I I TGATTCTATCCTTGGCTATCAAATTGGTGAACTTCGACGCGTCATTGGAGACTCCACAACCATGATAACCAGCCATCCAACTGACATTCTTACTTCCTi'A ACTAAGATAGGAACCGATAGTTTAACCACTTGAA6CTGCGCAGTAACCTCTGAGGTGTTGGTACTATTGGTCGGTAGGTTGACTGTAAGAATGAAGGAG- -eGFP.C eunc53 -C.e. unc53 I G E [ G 0 S M I T O I L ACTACAATCCGAATGTTCATGCACGGTGCCGCACAGAGTCGCGTAGACAGTCTGGTCCTTGATATGCTTCTTCCAAAGCAAATGATTCTCCAACTCGTCA TGATGTTAGGCTTACAAGTACGTGCCACGGCGTGTCTCAGCGCATCTGTCAGACCAGGAACTATACGAAGAAGGTTTCGTTTACTAAGAGGTTGAGCAG " - eGFP.C. e.uπc53 -C.e. uncS3 T T I R M F M H G A A Q S R V D S L V L D M L L P K Q M I L Q L Aat II βsrl βsrl Asu II i l l i AGTCAATTTTGACAGAGAGACGTCTGGTGTTAGCTGGAGCAACTGGAATTGGAAAGAGCAAACTGGCGAAGACCCTGGCTGCTTATGTATCTATTCGAAC TCAGTTAAAACTGTCTCTCTGCAGACCACAATCGACCTCGTTGACCTTAACCTTTCTCGTTTGACCGCTTCTGGGACCGACGAATACATAGATAAGCTT3 -eGFP.C eunc53 -C.e. unc53 E R I G K S K Bsm I X n I Bgl II AAATCAATCCGAAGATA3TATTGTTAATATCAG:A77CCTGAAAACAATAAAGAAGAATTGCTTCAAG7GGAACGACGCC7GGAAAAGATCTTGAGAAGC " 77AGTTΔGGCT7CTATCATAACAA-7ATAG " C37AA33ACTTTTGTTATTTCTTCTTAACGAAGTTCACCTTGCTGCGGACC7TTTC7AGAAC7CTTC3 -eGFP.C e unc53 -Ce. unc53 N Q S E D S I V N I S I P E N N K E E L L Q E R R L E K I L R Ava III Nsi I Xba I 3A " AAT A"C::AAA3 AA1 ■ CGAATTGCATTTGTTGTATCCGTTTT 3GTC ΓACG ' ΓTA " 77: TT ^GCTTAACGTAAACAACATAGGCAAAA. GTTG: :ττ CCA3 .__ _ __ < E S I L c M I r \ M o π tl c 'j Tuesday.18 November 199710:35 "Page tf tig 30 pEGFP72 (1 > 9697) Site and Sequence EcoR i CATTTGTAGTATGCACAGTCAACCGATATCAAATCCCTGAGCTTCAAATTCACCACAATTTCAAAATGTCAGTAATGTCGAATCGTCTCGAAGGATTCA " GTAAACAΓCATACGTGTCAGTTGGCTATAGTTTAGGGACTCGAAGTTTAAGTGGTGTTAAAGTTTTACAGTCATTACAGCTTAGCAGAGCTTCCTAAGTA "' " - eGFP.C euπc53 -C.e. unc53 P F V C T V N R Y Q I P E L O I H H N F K M S V M S N R L E G F I Ear I Sst I Ksp632l CCTACGTTACCTCCGACGACGGGCGGTAGAGGATGAGTATCGTCTAACTGTACAGATGCCATCAGAGCTCTTCAAAATCATTGACTTCTTCCCAATAGC" GGATGCAATGGAGGCTGCTGCCCGCCATCTCCTACTCATAGCAGATTGACATGTCTACGGTAGTCTCGAGAAGTTTTAGTAACTGAAGAAGGGTTATCGA -eGFP.C.θ.uπcS3 -C.e. uπc53 L R L R R R E O ε Y R L T V Q M P S ε L 1 I 0 F P I Ear I Ksp632l EcoR I Sprt I Bam HI CTTCAGGCCGTCAATAATTTTATTGAGAAAACGAATTCTGTTGATGTGACAGTTGGTCCAAGAGCATGCTTGAACTGTCCTCTAACTGTCGATGGATCCC GAAGTCCGGCAGTTATTAAΔATAACTCTTTTGCTTAAGACAACTACACTGTCAACCAGGTTCTCGTACGAACTTGACAGGAGATTGACAGCTACCTAGGG -eGFP.C.e.uncS3 -C.e. unc53 L Q A V N N F I E K T N S V D V T V G P R A C L N C P L T V D G S GTGAATGG7TCATTC3ATT3TG3AATGAGAACT7CAT7CCATATTTGGAACGTGTTGCTA3A3ATGGCAAAAAAACCTTCGGTCGCTGCACT7CCTTCGA CA:TTAC:AAGTAAGCTAACAC:TTACTCTTGAAG"AA3GTATAAACCTTGCACAACGATCΓCTACCGTTTTTTTGGAAGCCAGCGACGTGAA3GAAG - • eGFP C e uπc53 -C.e. unc53 K E V F I R L V E N F .' P Y L E R V A R O G K K T F G R C T S F E Bam HI Jth I Jth I G'3A7CCCACCGACA ' T:37CTCTAAAAAATGGCC37G3"TCGATGGTGAAAACCCGGAGAATGTGC7CAAACGTCTTCAACTCCAAGACC C3 * :CC3- | -A CCTAGGGTGGCTGTAGCAGAGATTTTTTACCGGCACCAAGCTACCACTTTTGGGCCTCTTACACGAGTTTGCAGAAGTTGAGGTTCTGGAJC GGGCAG- - eGFP.C. e.unc53 -C.e. unc53 C P 7 o I V S K < '- P v F D G E N P E M V L < R L Q L Q D L P Tuesday, 18 November 199710:35 Page 1 i fig 30 pEGFP72 (1 > 9697) Site and Sequence BspM I Xho I Sph I CCTGCCAACTCATCCCGACAACACTTCAATCCCCTCGAGTCGTTGATCCAATTGCATGCTACCAAGCATCAGACCATCGACAACATTTGAACAGAAGACT GGACGGTTGAGTAGGGCTGTTGTGAAGTTAGGGGAGCTCAGCAACTAGGTTAACGTACGATGGTTCGTAGTCTGGTAGCTGTTGTAAACTTGTCTTCTG- C.e. unc53 — r>. N S S Q H F N P L ε S L I Q L H K H Q T I D N I T E D Asp 718 | .Kpn I CTAATCTTCTCTCGCCTCTCCCCCGCTTTCCTTATCTTCGTACCGGTACCTGATGATTCCCCATTTTCCCCCTTTTCCCCCCAATTTCCCAGAACCTCCT ■ + -_: GATTAGAΔGAGAGCGGAGAGGGGGCGAAAGGAATAGAAGCATGGCCATGGACTACTAAGGGGTAAAAGGGGGAAAAGGGGGGTTAAAGGGTCTTGGAGGA S N L L S P L P R F P Y L R T G T . . F P I F P L F P P I S Q N L L F P L F L V L P G A O A E A t . K P F S F R N I S H C S L l V K L N K Q C M Y L K K K K K K L E G G P G I H R I . I T D H N Q P Y H Pra I Bsm I Hpa I TTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAG A A ACATCTCCAAAATGAAC3AAATTTTTTGGAG3G7GTGGAGGGGGACTTGGACTTTGTATTTTACTTACGTTAACAACAACAATTGAACAAATAACGT: i C R G F T C F K K P P T P P P E P E T . N C M C C C . L V Y C :. Bsm I CTTATAATGGTTACAAA7AAAGCAATA3CATCACAAA7TrCACAAATAAA3CATTTTTTTCACTGCATTCTAGTTGTGGTTrGTCCAAACrCATCAATG7 GAATATTACCAATGTTTATTTCGTTATCGTAGT3TTTAAAGTGTTTATTTCGTAAAAAAAGTGACGTAAGATCAACACCAAACAGGTTTGAGTAGTTACA L . L Q I K Q . H H K F H K S I F F T A F . L V F V Q T H Q C Mlu I Ssp I A-CTTAACGCGTAAATT3TAAGCGTTAATAT7T7GTTAAAATTCGCGT7AAATTTTTGTTAAATCAGCTCATTT7TTAACCAATAGGCCGAAATCGGC A TACAATTGCGCATTTAACATTCGCAA TATAAAACAA-TTTAAGCGCAATrrAAAAACAATTTAGTCGAGTAAAAAATTGG TATCCGuCTTTAGCCGT " ' ■ L T a < L A L I F C M S R I F S A ri F L T N R P K S A Tuesday.18 November 199710:35 Page 11 fig30pEGFP72 (1 >9697) Site and Sequence Bsrl AATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAA ■ + δ_; TTAGGGAATATTTAGTTTTCTTATCTGGCTCTATCCCAACTCACAACAAGGTCAAACCTTGTTCTCAGGTGATAATTTCTTGCACCTGA6GTTGCAGTTT K S L I N Q K N R P R . G . V L F Q F G T R V H Y . R T - T P T S I Pra III GGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACC CCCGCTTTTTGGCAGATAGTCCCGCTACCGGGTGATGCACTTGGTAGTGGGATTAGTTCAAAAAACCCCAGCTCCACGGCATTTCGTGATTTAGCCTTGG G E K P S I R A M A H Y V N H H P N Q V F W G R G A V K H . I G T Nael 1 CTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGG: GATTTCCCTCGGGGGCTAAATCTCGAACTGCCCCTTTCGGCCGCTTGCACCGCTCTTTCCTTCCCTTCTTTCGCTTTCCTCGCCCGCGATCCCGCGACCG L K G A P D L E L D G E S R R T W R E R K G R K R K E R A L G V Kspl Kspl AAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGG- ■ +> -+ ■ 10'. ' TTCACATCGCCAGTGCGACGCGCATTGGTGGTGTGGGCGGCGCGAATTACGCGGCGATGTCCCGCGCAGTCCACCGTGAAAAGCCCCTTTACACGCGCCT Q V . R S R C A . P P H P P R L M R R Y R A R Q V A L F G E M C A E Earl βspH I Ssp I Ksp632l < ! i ACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTC TGGGGATΔAACAAATAAAAAGATTTATGTAAGT7TATACATAGGCGAGTACTCTGTTATTGGGACTATTTACGAAGTTATTATAACTTTTTCCTTCTCA3 P L F V Y F S K Y I Q I C I R S . D N N P D K C F N N I E < G R V Sph I ! Ava III OxaN I Pvu II | .Nsi I C7GA3GCGGAAAGAACCAGCTGTGGAATGTGTG7CA3TTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAΔAGCA73CATCTCAA ' GACTCCGCCTTTCTTGG7C3ACACCTTACACACAGTCAATCCCACACCTTTCAGGG3TCCGAGGGGTCGTCCGTCTTCATACGTTTCG7AC3TA3AGTT« L R R K E P A V E C V S V R V K V P R L P S R Q K Y A K H A S Q Sph I Ava III Nsi I TAGTCAGCAACCAGGTG * G3AAAGTCCCCAGGC7CCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAG7C::GCCCCTAA A7CA3TC3TTGG7CCΔCAC:TTTCAGGGGTCCGAGG33TCGTCCGTCTTCATACGTTTC3TACGTAGAGTTAATCAGTCGTTGGTATCA'333C33G3AT- L y S N O - K V P R '. P S R Q K Y A K H A S O L V S H S P A P rl Bgl I Bsrl Nco I Sfi I I 7CCGCCCATCCCGCCC;TΛACTCCGCCCAG7T:CGC:CATTCTCCGCCCCATGGCTGACTAATTT7T77TATTTATGCAGAGGC:GAGG:CG:CTCGG: GA3GCGGGTA'3GGCGG33ATTGAG3CG33TCAA3GC 3 TAAGAGGCGGGGTACCGACT3ATrAAAAAAAΔTAAATACG 'r C7CCGGCTC':33C33A3CC3 J A H P A P N S A O F R P F S A P W T N F F Y L C R G R G ^ L ' Tuesday.1 β November 199710:35 flaή β i fig 30 pEGFP72 (1 > 9697) Site and Se^.-nce Stu I ! Avr II Cla I II ! CTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAGATCGATCAAGAGACAGGATGAGGATC3TTTCGCAT3ATTGA I I I , ' I 1 I ' I I 1 1 ■ I ' l l I ■ i μ -,?_;, • GAGACTCGATAAGGTCTTCATCACTCCTCCGAAAAAACCTCCGGATCCGAAAACGTTTCTAGCTAGTTCTCTGTCCTACTCCTAGCAAAGCGTACTAAC" L A I P E V V R R L F W R P R L L Q R S I K R Q D E D R F A . L BspM I Xma III i i ACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTG TGTTCTACCTAACGTGCGTCCAAGAGGCCGGCGAACCCACCTCTCCGATAAGCCGATACTGACCCGTGTTGTCTGTTAGCCGACGAGACTACGGCGGCAC N K M D C T Q V L R P L G V R G Y S A M T G H N R Q S A A L M P P C Nar I I Bbe I Kspl I I i TTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAAGACGAGGCAGCGCGGCTATCGTGGC 1 i i i i . i . i 1 i . i I i I ' l I i 1 i μ -3..; AAGGCCGACAGTCGCGTCCCCGCGGGCCAAGAAAAACAGTTCTGGCTGGACAGGCCACGGGACTTACTTGACGTTCTGCTCCGTCGCGCCGATAGCACCG S G C Q R R G A R F F L S R P T C P V P M N C K T R Q R G Y R '3 Fsp l βal I Pvu II Jth I TGG iCCACGACGGGCGTTCCTTGC IGCAG iCTGTGCTCGACG iTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCC7 1 I i I I I . 1 I . I 1 I 1 I I 1 I I 7--I.' ACCGGTGCTGCCCGCAAGGAACGCGTCGACACGAGCTGCAACAGTGACTTCGCCCTTCCCTGACCGACGATAACCCGCTTCACGGCCCCGTCCTAGAGGA W P R R A F L A Q L C S T L S L K R ε G T G C Y W A K C R G R I S BspM I G7CATCTCACCTTGCTCCTGCCGAGAAAGTATC:A7CATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCA:CAA -I 11 . I 1 1 I . . I l i l I I ' l 1 ' I 37'. ' CA3TAGAGTGGAACGAGGACGGCTC7TTCATAG3ΑGTACCGACTACGTTACGCCGCCGACGTATGCGAACTAGGCCGATGGACGGGTAAGCTGGT3GT7 C H L T L L L P R < Y = S W L M Q C G G C ( R L I R L P A H S T T I Ear I Ksp632l GC3AAACATCGCATCGA3C3AGCACGTACTCGGA7G3AAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCA"CAGGGGCTCGCGCCAGCC3Afc: 1- 1 , 1 1 1 1 1 , 1 .1 ' 1 1 1- jl_. CG:TTTGTAGCGTAGCTCG:TCGTGCATOAGCC7ACCTTCGGCCAGAACAGCTAGTCCTACTAGACCTGCTTCTCGTAGTCCCCGAGCGCGGTC3G:7T3 R N l A S S E H V L G V K P V L S I R M I V T K S I R G S R Q P rl Sph I Nco I " G7TCGCCAGGCTCAA33C3AGCAT3CCCGACG3C'3A3GATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATAT:AT3GT3GAAAATGGCC3CT * 1 - — — . 1 1 1 1 1 1 1 1 1 . — 1 1 1 μ 3_.ι ' A AAGCGGTCCGAύTTCCGCTC.TACGόόCTGC.-CT.CTAGAGCAGCACTύGGTACCGCTACGGACGAACGGCTTATAGTACCACCTTTTACCGGCGAA C S P G S R R A C P T A P I S S P M A M P A C R I S V K M A A Ear I Nae I Rsr II Ksp632l "" -T3GATTCATCG CT5T3GCCGGC733GTGT:3C3GACCG:TATCAGGACATAGCGTTGGCTACCCGTGATATTGCT3AA3 3;TTGGCGGC3A --3 1 — — 1 1 , i , . 1 , . . — μ 3J AAjA:CTAAGTAGCTGACA:CGGCC3ACCCACA::3C:T3GC3ATAGTCCr3TA7C3CAACCGATGGGCACTATAACGACTTCT:3AA':CGCC3CTTA'j; F L 0 3 S T / A S tf V . 5 T A I R 7 R V L P V [ L L < 3 L A A fl - Tuesday, 18 November 199710:35 Page ,ζ fig 30 p_GFP72 (1 > 9697) Site and Sequence GCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGG3 CGACTGGCGAAGGAGCACGAAATGCCATAGCGGCGAGGGCTAAGCGTCGCGTAGCGGAAGATAGCGGAAGAACTGCTCAAGAAGACTCGCCCTGAGACCC L T A S S C F T V S P L P I R S A S P S I A F L T S S S E R D S G Asu II BspM I | ! GTTCGAAATGACCGACCAAGCGACGCCCAACCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGG CAAGCTTTACTGGCTGGTTCGCTGCGGGTTGGACGGTAGTGCTCTAAAGCTAAGGTGGCGGCGGAAGATACTTTCCAACCCGAAGCCTTAGCAAAAGGCC V R N D R P S D A Q P A t T R F R F H R R L L . K V G L R N R F P Nae I Kspl Avr II i ! GACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCTAGGGGGAGGCTAACTGAAACACGGAAGGAGACAATACCG3 CTGCGGCCGACCTACTAGGAGGTCGCGCCCCTAGAGTACGACCTCAAGAAGCGGGTGGGATCCCCCTCCGATTGACTTTGTGCCTTCCTCTGTTATGGCC G R R L O D P P A R G S H A G V L R P P . G E A N . N T E G D N T G Kspl 1 AAGGAACCCGCGCTATGACGGCAATAAAAAGACAGAATAAAACGCACGGTGTTGGGTCGTTTGTTCATAAACGCGGGGTTCGGTCCCAGGGCTGGCACTC TTCCTTGGGCGCGATACTGCCGTTATTTTTCTGTCTTATTTTGCGTGCCACAACCCAGCAAACAAGTATTTGCGCCCCAAGCCAGGGTCCCGACCGTGAG R N P R Y O G N K K T E . N A R C W V V C S . T R G S V P G L A L TGTCGATACCCCACCGAGACCCCATTGGGGCCAATACGCCCGCGTTTCTTCCTTTTCCCCACCCCACCCCCCAAGTTCGGGTGAAGGCCCAGGGCTCGCA 3-X ACAGCTATGGGGTGGCTCTGGGGTAACCCCGGTTAT3CGGGCGCAAAGAAGGAAAAGGGGTGGGGTGGGGGGTTCAAGCCCACTTCCGGGTCCCGAGCGT T E I R R P R Al n I OxaN I Dra I Pra I GCCAACGTCGGGGCGGCAGGCCCTGCCATAGCC7CA33TTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGA CG3TTGCAGCCCCGCCGTCCGGGACGGTATCGGAG7CCΔATGAGTATATATGAAATCTAACTAAATTTTGAAGTAAAAATTAAATTTTCCTAGATCCAC7 3 Q R R G G R P C H S L R L L I Y T L 0 . F K T S F L I . K 0 L G E BspH I AGATCCTTTTTGATAΔTCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCAC7GAGCGTCAGACCCCGTAGΔAAAGATCAAAGGATCTTCTTGA A TCTAGGAΔAAACTATTAGAGTACTGGTTTTAGGGAATTGCACTCAAAAGCAAGGTGACTCGCAGTCTGGGGCATCTTTTCTAGTTTCCTAGAAGΔACTC- D P F . S H D O N P L T . V F V P L S V R P R R K D Q R I F L R rCCTTTTTTTCTGCGCGTAATCTGC GCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCG AG3AAAAAAAGAC3CGCATTAGACGACGAACGT77GT T TTTTTGGTGGCGATGGTC3CCACCAAACAAACGGCCTAGTTCTCGATGGTTGAGAAAAAGGC S F F S A R N L L L A N < K T T A T S G G '. F A G 3 R A T N S F S Bsrl AA3GrAΔC7GGCTTCA3CAGAGCGCAGATACCAΔA-A:T3TCCTTCTAGTGTAGCC37AGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACA 7 " 7CCAΓTGACCGAA3ΓCGTCTCGCGTCTATGGT7-AT3ACAG3AAGATCACATCGG ATCAATCCGGT3GTGAAGTTCTTGAGACATCGTGGCG3ATGTA E 3 N V L Q Q S A D T < ' C P S S A V V R P P L . E L C 3 T A r I Tuesday, 18 November 199710:35 Page it fig 30 pEGFP72 (1 > 9697) Site and Sequence AJwn I i ACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCA TGGAGCGAGACGATTAGGAC A ATGGTCACCGACGACGGTCACCGCTATTCAGCACAGAATGGCCCAACCTGAGTTCTGCTATCAATGGCCTATTCCGCG7 "" P R S A N P V T S G C C Q V R . V V S Y R V G L T I V T G . G A ApaL I i GCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGf" 1 1 ' I I I • ■ ' I ' ■ ' ' ' ■ I ■ I i " }„..-.,- CGCCAGCCCGACTTGCCCCCCAAGCACGTGTGTCGG5TCGAACCTCGCTTGCTGGATGTGGCTTGACTCTATGGATGTCGCACTCGATACTCTTTCGCG3 '""" A V G L N G G F V H T A Q L G A N O L H R T E I P T A . A M R K R ACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATC ' TGCGAAGGGCTTCCCTCTTTCCGCCTGTCCATAGGCCATTCGCCGTCCCAGCCTTGTCCTCTCGCGTGCTCCCTCGAAGGTCCCCCTTTGCGGACCATA3 H A S R R E K G G Q V S G K R Q G R N R R A H E G A S R G K R L V 3 TTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGC I I I ' I I I i . i I I I I ' I I I I 1 i . I I JiJ. AAATATCAGGACAGCCCAAAGCGGTGGAGACTGAACTCGCAGCTAAAAACACTACGAGCAGTCCCCCCGCCTCGGATACCTTTTTGCGGTCGTTGCGCC3 L . S C R V S P P L T . A S t F V M L V R G A ε P M ε K R Q Q R G Ava III Nsi I ! CTTTTTACGGTTCCTGGCCTTTTGCTGGCCTT7T3CTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCATGCAT GAAAAATGCCAAGGACCGGAAAACGACCGGAAAACGA3TGTACAAGAAAGGACGCAATAGGGGACTAAGACACCTATTGGCATAATGGCGGTACGTA L F T V P G L L L A F C S H V L S C V I P . F C G . P Y Y R H A Vloπday.1 December 199714' 12 o-. , a Pan* i • ιg DCBJS01 < _ &U£ c_- /. >. Λ a 7 P *Q» ' A I '-SAGCA I AI ' I ACGCCAASCTTGCA " ' GC TGCAGGAAT 7CGATATCAAGCTTA T CGATACCG rC(,ΛC( T ;,.\GCΛTCΛCΛΛGΛΛΛTTGr.Ar.rAAr.T ΆCΓ ACAΓΓCAΓ ΓΆΓGCXACC OCGG I ' Γ I ΓAAG ΓGAG rr ΓAA 1 MO ΓT r ΓGAG rr ΓACGAC ΓAGAAAAΛTGTI- 7TCTTTΛΛTΛAC TΛTCT CGACTTGΛG T CTΛTTCTGTΛT Λf. ■ JK) AΠTTGTTGAGTGATTTTTCATTGΔGAAΛATATTAAAAGGAACA I A rr ΓAC rΓTGC Γ ΓATT ΓGCCCTΛΛC 280 rτrGΛTTTΛGrTTrTCGΛTCAACTAGA- T "r AC.AAAACTTGCAATACAATTCCATTTTCAGATTAC'r:c τc 3»0 360 370 350 390 400 H'O 420 1 ' ■ 7 ' I I I I I 1 I I I I I I 1 I I . . . I . . , ■ I . . , ■ I ■ . I I I ! I l I l I ■ l I . I l l I l , . i I ■ . . ■ I , , . . I _.CΛCGTCTCGCCΛCCΓCAGCAACCG':TΓ.;AG(;AAO IΆACGC-AA ΓΓCCAAΓ r r ITCAGAΔA I G IΓ.AACA ? T CAGGCTTCAGACTCCACAGTCAAGA T-TCGAAAAΓ ΓGGΓAAGAAI rrrArri GA c AAAci IGΓ oo ΛTAAΛΔrG_CCAGAAAAGAAGA7 A~ΔAAΛATGTAGTT7TTTTGCAΛAACrTCCArr:TTTΛTrGC7CrΛΛ 660 τ i T ΛCGGCTTΛTΛ7C7C ΛATTTTC7-GA3TTTTATCΔAAAAA r r I * XAC TA TAC ΔAATG I AGAAAAG T 630 ' ! i i G AC AAA 1 : . " ιτi I ' Ac.i i' i GACAT ΓTTG ' ' ΛΛTΛGΛTCC ΛΛATGGΛΛCCTΛ A ' ΛCΛΛΓ.CTGTTΛΛ / O 710 720 730 740 7G0 7G0 770 A': • ιGG AGGAG_Gι;AAG^C ■ Al ACi;i ΔAA , ΔA ", GATCTGAAΔCAAA7TTGTGCTATrcT ΛΛΛ7GTTTΛ I/O '-ACAT3T7τr AAGA-rrrrrcA Ar:cGCΔC7AG T CΛGΛΛCGTτccτττττc;τΛTGΛΛΛΛΛ TΛΛA a o , Λ, , ΛΛΛCTΛTTTCΛΛΛCCC7CΛC'.:GC i:CArr ; rτrGAACTr.T~AΔTTTTTA ' rAΔΔA7TT'GC:ATTTAC 910 -A- , i ccxc rcc cι 1" G^ CGAAAAG~..':CCACCAAAΔTCΛATTTΓTCGGCTTCΛΓΛΛ " GΛC r r ΓΓΛΛΛΓ r Q£0 - ' i G •' C,ΛGΛΛΛΛC CΛC,Λ GΛGGC7ΛA;TAAAT^GACAGGGACA;ϊGTrGTCCC TC I l " C IXCX ICG r rC I 1050 '060 I07C 'OBO ICs I tOO I MO l '20 ;'7- cτccτc τc': G " rc.';ΔrcrccΔΔCΔΛ AΛCAAT~ττc A TT CGττG ' r c τrrGc ΓTAΓAAA I ι?0 71 i IGrcrι. ιVJ(JAA.'i AAA T/ , CΛC..G..AGAf ' .GGT AATTΔATTGCAA r GA(]Af,: AI , Gr.CAA: I ' AC I C MQH " TTCGGAAATTGΛ'GΛΛTΛΛΛGΛTΛCΛ-X'I A lTιA::AG r GGrTGGTA TAGTATGΛr, 7 (.TΛG ΛT7GC7T I _G0 " -TCΛTCCTCTCΛΛCT'GCG A ΓGAC- "' < i C:X(XG(:TCTCAT .A„TGΔCΛATTΛAΓG~CGG. ! 7TTTATT, 1330 :' " CTC77TCCΓA Γ " CCGCCACTI " .A7 7 GG-TΛCCΛΓAAACΓ3GAA " ΑCAI • I I ' ACi ' ACi AT. ' AAG C 1 J 1410 1420 ' 20 l«t-0 I'l.O I'lOO I '170 AT TTATTTTCATA ■ r ! " ΛΛ f I , 1.51 ' GCAi ' TA GGATAAAC AC AC ϊ T r rAAAAGTTTATTTAAAAAAA CG 14 A. ΛT \7TΓTCGATT~7AAAAΔA -C lGAAΔΔGTTTCAAAAΔAT.AArAAA'Arr(.(:(7AACΛΛΛTrGTΛTGCC 1.4. IΔAAAΠ I •• Al " I rcTArTG7 - Λ./\Λ " Λ::7Tr r i;-IΑTCAC7GT7TTCCΛ7CTCΛΛAACCTTGAA r C I6!0 C':'7:AAGT~ArAC,C.AACCTCC G7rA-A77TrCCΛTπC τ AI ' GAArCGCrACTCAGCACATATιCΛΛΛΛA 1600 TT--7G , TA A r:TT:.iΔ T A rΔ! IG -CΛ G.GTAATAAAGrGl.AAGCAGI IΛCiAATTTT ATTCAAGC. I 760 1760 1770 '700 1.'90 I SCO 1810 lST ... i I ... ■ ! ■ ■ . ■ I ... ■ ! ....1 .. ■ ■ I .... I ■ ■ . ■ I ■ ■ i i I i i i i I. -. .-— — .i i i i I i i i i I i i i i I A7: 1 A~ T AT TΛT.'.';ΛΛTTCAA X. Γ 7 AΔ; ΔT7CΛCCCAGTTCG T Ά::AAI I I ICCATGC T TTTTGGCC_ 1.320 ΛTΛΛΛΛ ΛGT" rr. I ACC ΓC -C T-.. , TC~ , _ΛC TCG I ' A 1 CATAAAAAGTΛT<\CCΛΛAAGCCCGAC I i 1 .00 rTT7-AAC,AϋAAG';AGATA".~GΛGTAC "GGCC ! GTGΛCCCT _ TTCΛTC T CGTCC I ' I ' CGG IC CC^ 1 UU Λττ.;ACGcrcA A iAA.-τcττrA.'Λ-A.: AraGACCTccττGT " τrc " rcrτcGττrτGΛ τcGC3 o '. ! 1 1 ΓΠGΓGGCTG-' TGΛΛ C -CCGG'- ΔΔTTTΔGTΛTΛTTTA^AGCΓIAΓCTTΓΛTG ΛΛTACAΓ _ICO 3i:0 2 _0 2 30 2140 7\U ) 1_1U0 21 0 /.• • 7,ΛΛCC,ΛG:iCAAl ' -ΔAAΔTΔ" A/.-' ' ΓAAΓGAGGTTG ' AGA ΓG ΓA A ! i iGCAAΔA AA'iAAΛΛΛΛ ' . . (0 ΔAAAC AAAΓAGGAACCGCCAGA r Λ Λ rrGTAT r IAAACG r r τ ΓCΛAGΛTC.HTAGG.ΛA.AT Γ.GG _'!'-* ..A-CΔCAAΛΛ.Tc/'Λ'-TGα: i A -ΔAA7C~ΛGT G ΓAAGCTTT ASATTGΛA T-.GGAA r ΓAA. ' .'J ' T ΓA • 'c AGA'CT-Ar.-rπ G A I-AΠΓ. i c AΔGCCXΛΛGC A 3 AA AΓC-AC Γ ΓΓ.CA I I I Λ ,VM) G IAA_ ι:iA . AT " C,ΛC τ -CC 'i' rG'-' CAG lT.ΪΔΛTCCAGAC 7 AGATTTCCAΛ ( l r,Λ(: I ' ' I AA I I I > 2'l ; Vlonday, 1 December 199714-12 aα Z. '__ PCBS01 _ 2460 2470 2'180 2490 2500 2510 2520 'UΛTGTTT CΛGT r TC r TC. r I ' AC I ' rAAAATC-TAATtXCCTGTGA7GCG7AAAATCGT 7ΛTCCCTTTC7C 2! " > θ " AfACl I 7:AAϊTACAGATTCΛTCΛΛΛGΛ7-GGTA7CAAGCCAAAGACGrcrGGACI lAAACCACCCI ' C 2590 A " CATC A AC CAC TTC A re ΔAArΔA I ACΔAA ' I CATTCCGTCCGTCGAGCCCTTCGAGTGGCΛΛTΛΛTΛΛT 2b(50 ITTGC TCGACl-ATATCCACATCTGCGAAGAGCTTAGGTATCCGATCCTTfCGGCTTCTTTTTAGAAA^r 2/X X ' ΛTTΛTTTCΛGΛΛTCΛTCATCAAC ΓACAGC ΓCIΆTTTCGΔΔTCTAΔACCGAΓΓTACCTΓΓCΔACΓCCΔ ?ooo 2910 2820 2630 2040 2050 2060 2070 ■ . . . i ■ ■ ■ ■ I . . . ' * . . ■ ■ I ■ . . . i ■ ■ ■ ■ I . . . . I ■ i ■ ' I ■ ■ ■ ■ I ■ ■ ■ ■ I ■ . . . i i . . . I ■ ■ , . I . . . . I ΔAAACC Γ re I AGACCACΔAACCCAGCTΔGTTCGTGTTGCTACAACTACAAAAATCΠGΛAGCTCΛAAGΓTA 11 X GCTCCCΛΛΛGCCGTGΛGCΛCCCCΛΛAACTTGC rrc r_ ΓGAAGACΓAΓ ΓGGAGCAAAACAAGAGCCCU _.'IC ■ χτAACAGCGGTGGTGoTGGTGGTCτ3AA " C7GAAATTAAAGTrArTCAGTAGCAAΛΔArcCArcrrcr r C 3010 -\ ! '.GAA rΛGCCCΛCΛΛCC ΓΛCGΛGAAAG CGGCGGGGGI GCCTCΔACAACAΔΔCTTTG C AΔΔA ΓC C Γ 3000 7 r CΛGTGΛΛΛΛ TGGCCTGΛΛGCCCCCGΛCCΛGTAAGCTGGGAAGTGCCACGTC TATGTC AAGCTT r 1F.0 3160 3170 3180 3190 32C0 3 10 3220 1TACGCCAAAAGTTTRXTACCG7AAAAC GACGCCCCAA7CATATCTCΔAC.ΔAFΙACTCGAAACGATGRTC 3220 .TCCGGATACGCTGCATTCAACAGCACGTCGCCAACGTCATCATCGΛΓGGΛA 3290 ΓCCΔ..ΔTC"-CΔAGAGTTCΛΛΓGTCΛGΛC SΛΛAΛGTCTC GTΓATCAGAΓG _>3<>o • \TCTTACTC7TΔACGCCTCCA "r CGTGA':A„ TATrAGΛCΛC.CCGΛTΛGCCGCΛΛrΛrrcrrTTrτrfΛΛΛ 3430 Γ Λ Γ~ΛT'. ΛΛCΛΛCCCTΓ.TTGΛC AΛAΛΛC:ΛΛCAC ΓG CAG ΓGAAAGGAG 'GAAAA CA AGCGAAAAAA 3500 3.10 3020 3630 30^0 3550 36ΩO 3: 0 lA rCCACCTCCAGCTG-TCCGCCACG 7;A A'"CCA Cr AΔCAΔTCGGAGT ^ CTTAGTCCAATTATGGCΛC 36 /() TUAGΔAG~T..ACΔΔA~GΔ.';CCCCIT&ATΛ7ΓTGΛΛAAΔ'XAGA CCTGΔAAAGCTΓCA ΓCAA " GAGCAΓ 36H0 " ;ύ A" A G Δ TTCC CG':T CCΔ';': m rAΔΔAlCA' T -TtTACTTΛΛΛΛTGΛCTTCΛΛTCCCA 'MVO ;Λ M:CΛCCΛΛCII T" ΛCCΛT Γ ΓC Γ re i AAAACAAGGAAΔAATCΔCATCGCC G ' CAΛSTCGTTTSGATATG :< oo s. ' • AGTCG~CCσCG7C7GAA C"CCA ' "TG7GGCTCATGr.GTCCGCTCΛrjGTr)ArτcCGCCGACAAAAAC 3A£0 3660 3070 3683 3890 39C0 '«JI' ) 307 ) T τ " T( 1 r J TΛΛTCΛT7CG' 7CGA'"AGΛAGGΔ ,"GGGΔA AGΔA7ΔAG ACATCΛGΛΛTCCAGCGGC TACΛCCTCT .2C o-i .'oCC.-G ΓG r I GC A i G i GCGC AAAΔTGAG:,GΛGΛΛGCTGAAAGAATA:GA ΓGACA I AC I G I CG AG 3000 C^CΛGΛΛCG ICTΛTCCTGACAAC ' TCGAA'JA A I IT.CTCCTTGTCGTC'CGΛΛΆTCCGΛTΛΛCΛΛC Λ -1060 ",:TCGA GACA " ATCCACGGARR,A T TRG T CRGGAGTACACATGGCAACAGTCGCC'C AAA';AIAGCGAC 4130 7 \ T(- ΛC T T G r rCC-CCA lCCrACr,-rτ-rTTCC7CAAΛGCCCCGAG ' CCC AG I ' CGG T IXXA AT 4.00 4210 <»22C ; i230 2^ pfin 4260 4270 7 GTCr ATTCTCf,ΛTCTCGAG.:A ΔACΔGGΔGAΔ7GT TΛCAΛΛC "" TC GTCCCAG TGCC iACGAGCCA ~„70 Ai 'ΪIGGCGCCGCTGCCACCTCAΛCC τ TCCGACAACAI ' rCGCTAAGA7CCCCGGύΛTΛCTCΛ CCrΛ " T r '«3'10 7. . AC! rArCAGTC'CACC^πATAΛGGACACAATGTCTΔ ' CCΛCTCACAGACTAG ' CGAC'.iACCI 'CI ' L ' L V0 ' i'.AΔΔAaCCΔAnr7Λ τ "C\GGCCA^TTT-A-rrACTTCΛ"CGTAAATGCCACCTTCAAGA rrCACAr(; t-W, i • 7GΛCCACAGAώT C-'"'-GC " r- TTC-ACCCCGAGACGGG " GCCGAAC ΓCGATCITCGΛΛ ' ΛT Λ ' TΓ.T '-55 4560 457C 4rK) i-hQC 60C 4610 irTΛ τ:AGGATCC-A:TCfir,rr. τ l ι'Ci_:/«". A .<_ΛA_ΛTCTACTfifiPAri , ! , AI'_GΛGΛGΛ J.I I ΛΛ ICC 'l.-'J' Λ' A ΔCTA T CCOI\! Y 3 AΔAAA7 "C I A AΓΓCGXAAAAGTGAGΛTGGG XCC.AA:. r irc C ΓGG ! WJ<) T A ,c ΛCGACΛGCΛTA ΓGGC I <: ιc i • AATCΔCΛΛGTACGAAC ΓGC ΓAT re ne CA TGCCA G " CAC r ΓG TOO CAG7' TTACAAΓ,AA'.AC ' ' CGΛC7C/77\_C AAGAΔΔC AGGAGAACTAXC-AGCA T TG ' riCA'C. I I I ' l'30 .AGI.AAAΔGCTTΔC-ΔAA/" XΛ T Λ ACAΓTGATCGATCCAACTTGAAGCC :AAGACGCAA ΓA I ~ • "t o 1 /b Monday, 1 December 19971412 p aαe J fig pCBSOI 4910 4920 4030 4940 'I960 'I960 '1970 A' l ' CAGGCAGGACATTGCTCATTTGAGGGA: ' ArrAGCAATCATCT7GCΛTCCΛΛC7CΛGCTC/ , TGCTΛAc ' 49/(' C. Λ ΛGGCGCTGGTGΛGCTTCTTC rCAACCA ! ' C rCTGCΛATCAGTTGCATCCCATCGATCATCGATGTCAT 5040 <" < TGΓCGAAΔΔGCAGCΛAGCΛGGACAΛGATCAGCTTGAGCΓCGI ΠGGΓ AAGAACAAGAAGAGΓ IGGAI -.! ι ':C iι CTCCTCΛCTCrCCΛA I ' ICACCAA AAGAΔGAACAAGΛACTΛCGΛCGAAGCACΛrArGCCΛrCΛΛ r r 5180 XCGGA rCTCAAGGAAC I C I I ' GACAA ΔTTGΛTGTGATTG AGTTGAAGCAAGAGC i " C AAAGAACG GA ι A 5250 6260 5270 5230 5290 5300 _31C .320 I I I I I t I I I I ■ I ■ I I ■ ■ » ■ I ■ ■ ■ I I I I I I I I I I I I ■ f ■ ■ I ■ ■ ■ ■ I ' ■ ■ ■ ' ■ ' ■ ' I ■ ■ ■ ■ I ■ I ■ ■ I ■ . ■ ■ I ' • -GCΛCTTTΛCGAΛGTCCGCCI I ' GAΓAΔTΓTGGATCGTGCCCGCGΛΛGTΓGAΓG f I ' C fGAGGGAGACAG T 5320 N AACAAG I i G-AAAU.tiAl-i Λl ΛAliLΛΛTTAAA AAAGAAGTGGACAAACTCACCAAC C. IV.AGC. '.C I 3390 i-GT CTTCTTCCCCCGCCTCAATTCCAC T TArcrACGACGATGAGCATGTCTATGATGCAr.C.TGTAG A 5U60 - r ACATCAGCTAGTCAAT..TTCGAAAC ArCCTCTGGCTGCAΛCTCΛΛTCAΛGGTTACTGTAAACGT_GA 5530 ■ A I CGC inCiΔ.IΔAATCAGTTCOATCCTTAACGGGGACTTGAAGCAGCΔGGAATTrTTCCTGGCCTGTΛGC « X> 6610 5620 5630 5640 5650 S66C ./ I I I I I I 1 I I I I I 1 t I 1 ■ ■ ■ I I ■ ■ ■ I ■ i ■ . I . I ■ .. I ■ ■ ■ . I ■ . ■ ■ I ■ ■ ■ ■ I ■ ■ ■ i I ■ ■ ■ I I ■ ■ , ■ I i ■ ■ i I AAG'JICA ΓGGAAAA I ΓGACTGGAAGΔTGC ' GGATGΛΛGCTGTTTTCCAAGTGΠCAAGGA ΓAΆI < ι 5670 CTAAAATGGACCCAGCCTCTACCCTCC.ΛC TAAGCAC; ΓGAG I CCA ΓCCA ΓGGCTACAGCATCAGCC A: (r w) GΛΛΛCGΛGTGTTGGΛTGCΛGΛCCCCCCCGAGATGCCrCCrrGCCGlCGΔC IGICAΛrAΛCΛTΛTCAC.TC bUU> TCi:cτCAAAGGTCTGΛΛGGAGAA p GC3TCGACAGCrTGCTGTTCGAGACGCTGATCCCCAA eCG I ' GA 5880 r -CAGC AC TACA ΓAAGCC I CC I GC I GΔΔGC ACCGGCGCCTCGTCCTCTCGGGC CCCAGCGGCACGGGCAA .960 5980 5970 5900 5990 6000 6010 6020 GAC TACCT-ΔCCAA7CGCTTGGCCG/C-Λ:C ΓGΠ ΓGGAG GC ΓCTGGCCGTGAGΠTCACAGΛGGGCATC 6020 r- XAGCACC T ΓCAACAΓGCACCAGC GTCTTG .AΛGCΛTCTGCAACTG ΓA r c r r 1 JCAACC I AGCCAACC OOOQ • ' IΛTΛGΛCCGGGAAΛCAGGAAΓ ΓGGG A G ΓC-CCCCTGGTGA7TC" " ATT33ATGACC ΓGA Γ.AAC-CAGG 6I6 ' " CCATΓ.ΛGΓGΛCTTGGTCΛATGG iccc ΓCACCXC AAΩTΔ _ CA " AΛΛTGTCC ΓA Ά TA Γ AGG ' ACC 6230 '..ΛΛICΛGCCIGIAAAΔATGACACCCAArCATG Crr CAC' 13AGC ' TCAGGATGTGAC TTC 77.Λ 63C . 6310 6320 .33<, 5 4 6350 636C t : 3 '0 ■ ■ . ■ I i ■ i . I ■ i ■ i I ■ ■ ■ ■ I ■ I . i ! I . . . I . . . . I i ■ ■ I I I I I I I I I I I I I ■ ■ i-L.i . . . I . . . . 1 i . . ■ I -CAACGT'_GAGCCAGCCAATGGCT~C._TCG rrC r IACCTGAGCAGGAΛGC r GG7Λ ι ;,AGXΔ ACA'-C.A 6370 "A TAΔTGCCAACAΛGGAΛGAGC TGC r | ' ( ':GGT CTCGAC7GGGTACCCAAGCT GGTA T .ATCTCCΛC S « *'-0 ' .-ττ c-τoΛα-/«t)CA Λoc* c -» • '•../ ._ TTC:TC T;O or oo- roo rr " ι <> n rfiθ-oτoos.',ττ3 .; _ i© iιCA r T AGGAC77CCGG CCTGG " "CΔr ~ ] ΔCC "' GTGGΔACAAC τ CTATCAr'CCCIAI ' C'ACAGGA^JG r . θ A CCΔAGGAΓGGGΛTΛΛΛGG ΓΓCA i G-ΪACAGAAAGCΓGCTTGGGAGGACCCA I OCAATGG GΓJCGCGA. 6 60 6660 6670 6680 6680 6700 6710 6720 / \CΛCTTCCCrGGCι;ArCAGCCCΛΛCAΛ3ΛCCAAICAΔACCTGTACCΔCC τ GCCCCCACCCACCGTGGfcC 6720 '. i CACAGCA τ τf.ccτcΛc:τ' _( GAGG.ΔΆGGAΓΛGTCAAA ACAGCACCCCNΛGTTCTCTG ΔC ΓCA A - T CTCX TGCC'-T I C ICX ΓGAΛΛC ' TCAAGAAGC ICCCΛΛCTΛCATTGAGT ' CCAC.AICGΛGΛΛΛ-C G O ιτ c" " r-r.Accc":A crττrΛGr,C AC * r ΓAAGGGTTCGCCAATCACT. ΓCACCCCCGGACAOCAGA-V; ^ JO ' I GCCATC AC.CTΛ "CT rAGC" X TCC~C~CCCCTCTCCTCTTTCAGAGCA I CGC I C " CCACC.CC.Λ'V. 7000 7010 '020 7030 '040 7050 <0W ) 7 70 ' 7ιΛC C ( T T 7GΛGΛAC I IC " 07C r r \<;_ TGGC T : rc Γ AAI / ■ v ) CA- Γ [ AACX rGCGC ' ri ._ ' 0 " ΓGGC ΓIGΛC I ' AACC* ._ 2.. " T ΛΛ. TGC~C A A'~.ΔAGCCG. , Λ " :': G' A(..AΓ.-ΓIGCCT_CCCATGG Γ AΓ ΓC.: ΆI . _Λ i ,_r.r. _>. 73*' Monday.1 December 1S9714.12 Pane 'lg pCB501 7360 7370 7300 7390 7400 / I0 7420 _L A i CGGCCC ΓGTCATCΛGΛTCGCCATCTCGCGCCCGTGCCTC ΓGAC rrc ΓAAG ΓC AAI ΓAC ΓCΓTCAAΓ. 7420 ΛTCCCTACATGCTC r rrc rccc ΪG I _C ICCCACCCCCTΛTTTTTGTTΛTTΛTCΛΛΛΛΛAΛCTTCTTCΓTΔ / 9 •M i • c 1 r ΓG Π r r r ΓΛCCTTCTTTT.ΛACTCACCTCTAACAATGΔAATTGTGTAGA~TCAAAAATAGAΛTT hb Λ A 1 rCG I ' AA rAAAAA6 T CGAAAAAAA " TG73C7CCCTCCCCCCΛTTAATAΛTΛΛT-„TATCCCAAAATCT B:K) A.:AC AATGT ΓCTG ΓG ΓACAC rrc: r 1 ΔI ci ΓTTTTTTACTTCTGATAAATTTTTTTTGAAACATCATAGAA 7700 7710 7720 7/30 7/40 7/00 //'JO I MO I I I 1 I I I I I I ■ 1 I 1 I I I I I I I I I ■ I ■ I ■ I I ■ I 1 I I I I I 1 I I I I I I ■ I I 1 I ■ ■ ■ ■ I x.l_i J.1. I.I I I I I I I I AAAACCGCACACAAAA r ACCTTATCA r ΛTGT7ACGTTTCΛGrrrΛTGΛCCGCΛΛr; r r rArrrCT" CA 7770 :C τ CTGGGCCTCTCΛTGΛCGTCΛΛATCATGCTCATCGτGAAAAAGTTTTGGAGTA7TTTTGGAA"TTTC 7P ι; -rCAAG ' rGAAAGTrTArGAAATTAAT τ TTCCTGCTTTTGCTTTTTGGGGGTTTrXCCTΛ~TG"77GTCΛ /<)!() ΛC ' ιAGrrrCGAGGACGGCGr' ' TrTC " TrιCTΔAΔATCACΛAGTATTGATGAGCA:GAT_CAAGΛΛΛGΛTCGG 7980 Λa7AAGGτTTGGGT7TGΛGGCTCA_".-GAAGG TGAG TAGAAG I TGAl ' AA T I ' C'.iAAA.? I GGΔ.'ϊ I G I C I ' P.Of.O 8060 80/0 3080 8090 3 ICC 8 MO 8! 20 iTGr_GGTTTrTGCC7TAAATGACAGAATACΑTTCCCAATATACCAAΔCATAΔCTGTTTC;:TACTΔGTr.Gr. l]\Λ) ^XTACGGGCCCTTTCGTC ' CGCGCGT ' T GGTGΛTGΛCGGTGAΛΛACCTCTΠΛΓACΛT C GCTΓXΓ.G fllSO GΔcrx ΓCACAGC r ΓG rc ΓG ΓAAGCGGAIGCCGGGΔGCΔGACΔΛGCCCGTCΛOGGCGCGTCΛGCGGG I Γ «2sυ TGGCGGGTGTCGGGGC.TG CTTΔΔCTAT.-CGGCΔTCΔGΛGCΛGΛ ΓTGΓΛC " GΛGΛ ΓGCΛCCΛ ΓΛ ΓΓXGG 8.330 - TGAAATACCGCACAGATGCG~AAGGA AAΛATACCGCA TC AGGCGGCC ' RAA IX.C I'CC- I ' GA I AC GO 040 8410 6420 8430 8 *Λ 8450 8460 ,470 :TAr7Tr7ArάGGT r AATG 'r CA7GATAATΔΔ-GGT 7 TC77AGACGTCAGGTGCCACrrrTCCGGGΔAArG 8 f i70 Γ.ICGCGGAACCCCTATTTG T TTA77T-TCTΛΛATACATTCAAA ΓATGΓAΓC GC <CAI GAGACAA! AACC 0540 'T:,' , .- Γ AAΛTGCTTCΛATΛΛ _ ΛT7CAAAAAG AΛGAGΓAΓGAGTA " TCAΔCAT7TCC: 1 TG T C::CCC7TATT aeiυ XC; " TT7TGCGGCATTTTGCr:T-Cr.-ι.TT-TTGCTCACCCAGAAΔ';GCTG rGAAAGTAAAAGA " GCTG 3600 ^\CA-CACTTGGGTCCΛCGAGTGG-rTACA-CGAACT GATC7CAACΛGCCCTΛΛGΛTCCrrGΛGAGrrr 5750 0/60 8770 5780 3790 3800 0010 8820 , , . . ! . . . . I . . . . I . . . . I . I I . I . . . . I I ■ . I . I . I I I I I I I I I I I I I I ' I I I I I I I , I I I I I I I I I 7.GCCCCGΛΛGΛΛCG T 7 ϊ I ' CCAA ' GA I ' AGGArTTTTAAAGTTCTGCTΛTGTiGCG ' JGG FA f I A . CC CG ■ 80ΣC -: GACGCCGGGCAΔGΔGCΔΔC τ CGGTCCCCGCATACACrArι ' CrCAGAATGΔC ' " ' GGTTG.\G:ACTCΛC 6890 .;Λ. ' ' CACΛGΛΛΛΛ CΛ" TTAC A7j.:C A TGACAG fΛΛGΛGΛΛTTΛTGCAG^GCTGCCA- ACCAT ΔG 8960 Xi Λ ^-ΛΛCΛCTGCGCCCAACrrACTT.—GACAΛCGATCC.GAGGACCGΔAG AGCTAACCGCTTTTTTGCΛC 0030 AA~ATGGGfiGΛTCΛTGTΛAC TCGC I . ' GATCG ' TGGCiΛACCGGΔG rGAArGAAGCCATACCAAACCΛCG 9100 9110 9120 9120 9140 .150 9!60 SIΛ j ,MΪ:; - GACΛCCΛCGA ΓGCC I G ΓAGC ---GGCAΛCAACG r ΓGCGCAΔAΓ.7ATTΛACTG:.CC.A C ΓAC r ΓAC 9170 C . AGCTTCCCGvXΛΛCΛATTAAl AC-A T3GA~C;GΛSGC3GA TAAAG I I GCAGGACC ' ACTCTGCGCTCG 92'iC .C.XT-CCCCCCGCTG'.rrrATTGC' ATAAATCrGGAGCCr.GTGAGCGTCCCTCTCGCGGrAICAI I 9310 :■ ■ . - Λ T 35GGC CΔG A ΓGG t AΔ._C c -TΓ ^T ATCGTAG r ΓATCTACACGACGGGGA XAGGCAAC T S T 9300 , ,ΔTGΔACΓ,ΛΛΛTΛC I ACAIJA "CGC I' -A: T ΛGGTαCC TCAC ι GΔT7AAGCA T GGTAACT_ rCAG-C.AA 4- 9480 9470 g nj 9-90 9500 9'JI'J 'Jb_0 _L _L i I . ■ ■ . i _L JL _L ._. r,rrrAcrcATAT 7ACτ-τΛ Λ " _Λ-T " AAAΛ'_TTCΛI "ΓΓΓAATTTΔAAAGGΔTCΓΛGCI ' GAAGAICC 'i_ ' > 17 .1 J A I 'AΔΓ'. A7 IΔ " A M i'^CCTT AA C.ITGAGT'TTCG-T CΛC'X.AπXi rCAoA'JCCCG i AGA '.O'iO ΛAGA ΓCAAAG J /.TCTTCTΓGAGA I cc" τ τ"τττc -GCCX ΓAATCTGCTGCTTGCAΛA A' ΛΛΛACCA S060 " C.CTΛCCACXGΓGC- rrc-T7τcc cArcAΔGΔ CTΛCCAA:τcττ rrc r-A G,i A ICGC ■ ICA .".V r 7GA;^G'.Λ7 A "A';'. ' .ΛΛ ΛCTr-r'.'' C TAG'C TAGX TA'J P 'A3GCCA"CAC rfCAAGA-, C Tι- T ' " 'BOO Monday, 1 December 199714:12 Page ~ ^ tig pCB501 9010 9320 9830 9840 9850 9860 9870 ΛGCΛCCGCCTACA I ACC ΓCGCTCTGCTAΛTCCTGTTACCAGTGGCTGCTGCCAGTG 3CGA7AAGTCGTG T 9870 C I I ΛCCGGGTTGGACTCAAGACGATAG r I'ΛCCGGΛTAAGGCGCAGCGGTCGGGCTGAACGGGCiCiG r TCC I 99-0 G ACACAGCCCAGC ΓTGGAGCCΛΛCGACCTAC;ACCGAACTGAGΛTACCTΛCΛGCG"3AGCATTGAGAAAG IC ;o CCCCΛCGCTTCCCGAAGGGAGAAAGGCGGΛCΛGGTATCCGGΓAAGCGGCAGGGTCG AACAGGAGΛGCGC IOOMO ΛCGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTAΓAGΓCCTGTCGGGTTTCGCCΛCCTCTGΛCTTG ICK-O 10160 10170 10180 10190 10200 10210 10220 i i i i I ■ i ■ ■ I i ■ i « I i ■ ■ ■ ! ■ ■ ■ ■ I . . ■ ■ I i ■ ■ ■ I . . . . I . i . . I . . . . I . . , . I , . , . > , , , , I , , , . I A CGTCGArrrTTGi ' GArGCTCGTCAGGGGGGCGGAGCCTArGGAAAAAOGCCΔGCAA iCKKiCCI I I I I 102X0 ACGG r ΓCC I ' GGCC ΓTTTCCTGCCCTTTTGCTCACA ΓGΓ ΓCΓITCCTGCGTTATCCC:TGA7TC7G7GGAT 10290 AΛCCGTATTACCGCCI i I3AGIGAGCΓGATACCGCTCGCCGCAGCCGAACGACCGA3CGCAGCGAGTCAG ιυ..«o TGAGCGAGGAAGCGGAAGASCGCCCAATACGCΛAACCGCCTCTCCCCGCGCGTTGG.CGΛTTCΛTTΛΛTG 10430 CAGC i GGCACGACΔGCTTTCCCGΛCT3GAAAGCGGGCA rGAGCGCAACGCAΛTTAATGTGAG TTAGC IX 1C5C0 10510 10620 10530 10540 10650 1C560 106/0 ■ ' ' 7 i I i I I ■ ■ i ■ I ■ i . . I ■ i . . I . . . . I . . . . I . I ■ I I 1 I I I I I ■ I ■ I I I I I I . I > I I i 1.1 I I I I I I I . ACTCATTAGGCACCCCΛGGCr ACA'Ji ITAΓGCTTCCGGCTCGTATGTTGTGTGGAA TTG I ' GAGCGGAf \Q AACAA T r TCACA AGGAAACAGC I 1C594 Tuesday.18 November 19971009 , Page tig 13 pCB201 (1 > 5082) Site and Sequence > ,| 1 ( - 'ύ^ Enzymes 100 of 1 6 enzymes (Filtered) ^ Jiw-. Settings' Linear, Certain S πC ' ut ites Only Standard Genetic Code GACGGATCGGGAGATCTCCCGATCCCCTATGGTCGACTCTCAGTACAATCTGCTCTGATGCCGCATAG7TAAGCCAG7A7CTGCT:C TG:TT2TGT T : I I I I I , , . I 1 ' I I I I ' I ' I 1 i _,-, CTGCCTAGCCCTCTAGAGGGCTAGGGGATACCAGCTGAGAGTCATGTTAGACGAGACTACGGCGTATCAATTCGGTCATAGACGA'-G'JACGAACACACJ-A T O R E I S R S P M V D S Q Y N L L , C R I V P V S A P C L C V GGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGOTTAGGCGTTTTG - CCTCCAGCGACTCATCACGCGCTCGTTTTAAATTCGATGTTGTTCCGTTCCGAACTGGCTGTTAACGTACTTCTTAGACGAATCCCAATCCGCAAAACG- G G R V V R E Q N L S Y M K A R L D R O L ri E E S A . G . A F C CTGCTTCGCGATGTACGGGCCAGATATACGCGT7GACATTGATTATTGACTAGTTATTAATΔGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATA GACGAAGCGCTACATGCCCGGTCTATATGCGCAACTGTAACTAATAACTGATCAATAATTATCATTAGTTAATGCCCCAGTAATCAAGTATCGGGTA-^- A A S R C T G Q I Y A L T L I I D . L L I V 1 N Y G V I S S . P I -, TGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAG ' ACCTCAAGGCGCAATGTATTGAATGCCATTTACCGGGCGGACCGACTGGCGGGTTGCTGGGGGCGGGTAACTGCAGTTATTACTGCATACAAGGGTAT A G V P R Y I T Y G K V P A V L T A Q R P P P I D V N N D V C S H 5 AACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCC: TTGCGGTTATCCCTGAAAGGTAACTGCAGTTACCCACCTGATAAATGCCATTTGACGGGTGAACCGTCATGTAGTTCACATAGTATACGGTTCATGCGG. N A N R D F P L T S M G G L F T V N C P L G S T S S V S Y A K Y A CCTATTGACGTCAATGACG3TAAATGGCCCGCC7GGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAG- A GGATAACTGCAGTTACTGCCATTTA CGGGCGGACCGTAATACGGGTCATGTACTGGAATACCCTGAAAGGATGAACCGTCATGTAGATGCATAATCAJ- P Y . R Q . R M A R L A L C P V H D L M G L S Y L A H L R I S H τ CCCTATTACCATGGTG T3C'_3 ,' T77GGCAGTACATCAATG3GCGTGGATAGCGGTTTGAC CACGGGGATTTCCAAGTCTCCACCCCATTGACGTι:*.A AGCGATAATGG7ACCACTACGCCAAAACCGTCA"C7A3TTACCCGCACCTATCGCCAAACTGAGTGCCCCTAAAGGTTCAGAGGTGGGGTAACTGCAGT- P Y Y H G D A L A V H Q V A W I A V L T G I S K. S P P H . R Q TGCGAGTTTGTTTTGGCACCAAAATCAACGGGAC-'TCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGG t^ '- ':TCAΛACAAAACCGTG3TT77AC7T3CCCT3AAA3 TTTTACAGCATT3TTGAGG 3GGGTAACT3C3TTTACCCGLCATCC C CArGC CC'-T- - - F V A P K S T G L S K M S . Q L = P ! O A N G R A C ϋ G7:7ATATAAGCAGAGCTCTCT3GC T AACTAGACAACCCACTGCTTACTGGCTTATCGAAATTAATAC3A3 CACTA7A3GGAGACCCAA3CTG3 TAIJ. ' 1 1 1 1 1 1 ' I 1 1 1 I ' 1 1 j > CA3ATATATTCCTCTCOAGAGACI:3A-TGATCT:--3GGTGACGAATGACC3AATAGCTTTAATTATGCT3AGTGATATCCCTCTGGGTTC3A-CGA-C: 1 i — - TT77 p prroommootteorr pprriimmmmqq ssiittee > * '- _ V r- Q S S L A N P T H C L L A V R N Y D 5 L G D P S V L ^- G " 7AAACTTAAGCTTACCATGGG3GCTTCTCA~CATCATCATCATCATGGTATGGCTAGCATGACTG T33ACAGCAAATGGGTCGGGATCTGTAC_t. ^ AAATrTGAAT'CGAATGGTACCCCCCAAGAGTAC'AoTAGTAGTAGTACC TACCGArCGrACTGAC A CTGTCGTTTACCCAGCLCTAGACATGCTC L-Proθon Din inq domain -^-1 f : < L K L T C : S H - H H H H G M A S M T G G Q ι G P D L V C' Tuesday.18 November 19971009 Page fιq13pCB_01 (1>5082) Site and Sequence ATGACGATAAGGTACCTAGGATCCATATGCCTCCTTGCCGTCGAGGTGTCAATAACATATCAGTCTCCCTCAAAGGTCTGAAGGAGAAAΓGCOTCGAI-A ; , -A':T3CTATTCCATGGATCCTAGGTATACGGAGGAACGGCAGCTCCACA3TTATTGTATAGTCAGAGGGAGTTTCCAGACTTCCTCTT7ACGCAG:7.J- - O O K V P R I H M P P C R R G V N N I S V S L K G L K E K C V O GCCTGGTGTTCGAGACGCTGATCCCCAAGCCGATGATGCAGCACTACATAA3CCTCCTGCTGAAGCACCGGCGCCTCGTCCTCTCGGGCCCCAGCG3I:-: CGGACCACAAGCTCTGCGACTAGGGGTTCGGCTACTACGTCGTGATGTATTCGGAGGACGACTTCGTGGCCGCGGAGCAGGAGAGCCCGGGGTCGCCGTL - pCB201 insert = U4 - U ORF 3 L V F E T L I P K P M M Q H Y I S L L L K H R R L V L S G P S G T GGCAAGACCTACCTGACCAATCGCTTGGCCGAGTACCTGGTGGAGCGCTCTGGCCGTGAGGTCACAGAGGGCATCGTCAGCACCTTCAACATGCACCA. CCCGTTCTGGATGGACTGGTTAGCGAACCGGCTCATGGACCACCTCGCGAGACCGGCACTCCAGTGTCTCCCGTAGCAGTCGTGGAAGTTGTACGTGGT. ■ pCB201 insert = U4 - U4 OHF _ < T Y L T N R L A E Y L V E R S G R E V T E G 1 V S T F N M H . CA CTTGCAAGGATCTGCAACTGTATCTTTCCAACCTAGCCAACCAGAT 3ACCGGGAAACAGGAATTGGGGATGTGCCCCTGGTGATTCTATTG3AT3 G " : 3AACGTTCCTAGACGTTGACATAGAAAGG * TG3ATCGGTTGGTCTATCTGGCCCTTTGTCCTTAACCCCTACACGGGGACCACTAA3ATAAC;7- ■ pCB201 insert = U4 -U40RF S C 0 L 0 L Y L 3 N L A N Q I 0 R E T G I 3 0 V P L V i L L D ..-U GrGAAGCAGGCTCCATCAGTGAGTTGG'CAATGGGGCCCTCACCTGCAAGTATCATAAATGTCCCTATATTATAGGTACCAi.CAATCACC^-'. " -^CrCACTTCGTCCGAGGTAGTCACTCAAC ACTTACCCCGGGAGTGGACGTTCATAGTATTTACAGGGATATAA-ATCCATGGTijG'T GTCG i'.- ■ pCB201 insert - U4 • U4 OHF 0 - S E A G S I S Ξ L V \ G A L T C K v H K C P Y I [ G T r \ j P .ΛiAArι J ACAι:CCAA:CAT3GCTTG A:TTGA :"7;A33ATGTTGACCTrCTCCAACAACGTGGAGCCACCCAATG'3CTTCCT33TTι:GTTAC;3T3i' 7TACTGTGCGT GGTACCGAACGT3ΔAC7C:AA3-CCTACAACTGGAA3AGGTTG7TGCACCTCG3T3GGTTAC GAAGGACCA>. I :':AATU3-- " '- -pCB201 insert - U4 U4CRF N H L 7 F S N N Y L Tuesday, 18 November 19971009 Page tιg13pCB201 (1>5082) Site and Sequence ________ AG AAGCTG TAGAGTCAGACAGCGACATCAATGCCAACAAGGAAGAGCTGCTTCGGGTGCTCGACTGGGTACCCAAGCTGTGGTATC TCTCC C " TCCTTCGACCATCTCA3TCTGTCGCTGTAGTTACGGTTGTTCCTTCTCGACGAAGCCCACGAGCTGACCCATGGGTTCGACACCATAG7AGAG37GTG'jA -pCB201 insert _ U4 -U40HF F K L V E S D S D I N A N K E E L L R V L D W V P K L W Y H L H T TCCTTGA3AAGCACAGCACCTCAGACTTCCTCATCGGCCCTTGCTTCTTTCTGTCGTGTCCCATTGGCATTGAGGACTTCCGGACCTGGTTCATTGAC " AGGAACTCTTCGTGTCGTGGAGTCTGAAGGAGTAGCCGGGAACGAAGAAAGACAGCACAGGGTAACCGTAACTCCTGAAGGCCTGGACCAAGTAACTGG - pCB201 insert = U4 -U40RF F L E K H - T S D F L I G P C F F L S C P I G I E D F R T V F I D L GTGGAACAACTCTATCATTCCCTATCTACAGGAAGGAGCCAAGGATGGGATAAAGGTCCATGGACAGAAAGCTGCTTGGGAGGACCCAGTGGAA7G3GT: CACCTTGTTGAGATAGTAAGGGATAGATGTCCTTCCTCGGTTCCTACCCTATTTCCAGGTACCTGTCTTTCGACGAACCCTCCTGGGTCACCTTAC3CA3 - pCB201 insert - U4 -U40HF '- N N S I I P Y L Q E G A K D G I K V H G Q K A A V E D P -J Z . C'J3GACA;A TTCCCT3GCCATCA3CCCAACAAGACCAATCAAAGCTGTACCACCTGCCCCCACCCACCGTGGGCCCTCACAGCATTG CTCAC:T:C'::: GCCCTGTGTGAAGGGA3CGGTA3TCGGGTTGTTCTGGTTAGTTTCGACATGGTGGACGGGGGTGGGTGGCACCCGGGAGTGTCGTAACGGAGTG3A3Gi.L -pCB201 insert = U4 -U40RF = 0 T L P V P 3 A Q Q D Q S L Y H L P P P T V G P H S l A 3 F F ii-.AT GG-CAGTCAAASACAGCACCCCAAGTTCTCTGGACTCAGATCCTCTGATGGCCATGCTGCTGAAACTTCAAGAAGCTGCCA^ TACAr ' GAi-r ^TC:"GT':AGTTTCTGTCGT33GGTTCAA3AGACCTGAGTCTA3GA3ACTACCGGTACGACCACTTTGAAGTTCTTCGACG3TT':iT3"AiCT': - pCB201 insert = U4 -U40RF E D R T V k. O S T P S S L - S D P L M A M L L K L Q E A A II r l E ' - -A3AT:GAGAAACCATCCTGGAC:CCAACCTTCAG3CAACACTTTAAGGGTTCGGCAATCACTGTCACCCCCGGACAGCAGAACGC "" G3CAT:-3'JT- AGSTCTAGCTCTTTGGTAGGACCTSCGGTTGGAAGTCCGTTGTGAAATTCCCAAGCCGTTAGTGACAGTGGGGGCCTGTCGTCTTGCGACCj'ΑGTCG- " L C = " Tuesday, 18 November 19971009 Page tιq13pCB201 (1>5082) Site and Sequence TCTTAGCTCCTCCTCTCCCCTCTCCTCTTTCAGAGCACTGGCTCTCCAGCCCCAGGAGGAGAACAGGAGGGAGGAGGAGATGAAAGAGGAG3GACAGGT" AGAATCGAGGAGGAGAGGGGAGAGGAGAAAGTCTCGTGACCGAGAGGTCGGGGTCCTCCTCTTGTCCTCCCTCCTCCTCTACTTTCTCCT3CCTGTCCAA -pCB201 insert = U4 _ L L L S P L L F Q S T G S P A P G G E Q E G G G O E R G G T G CTTGGTGCTGTACCTTTGAGAACTTCCTAGGAAGGAATGGTGGGGTGGCGTTTGGGAACTTGTGCCCCCTAAACACATTTACTGGCCTCCTCTAATGAC " GAACCACGACATGGAAACTCTTGAAGGATCCTTCCTTACCACCCCACCGCAAACCCTTGAACACGGGGGATTTGTGTAAATGACCGGAGGAGATTACTGA • ρCB201 insert = U4 S W C C T F E N F L G R G G V A F G N L C P L N T F T G L L . . L TTGGGGAAAAGATGATTCTGGGTCTTTCCCTTGACTTCTTGTTTCAATTACAAACTCCTGGGCTTTCTGGGGAGGGGTTCAGAAAACATCAAAACACTG AA_CC-TTTTCTACTAAGACCCAGAAAGGGAACTGAAGAACAAAGTTAATGTTTGAGGACCCGAAAGACCCCTCCCCAAGTCTTTTGTAGTTTTGTGA_G -pCB201 insert =U4 V G K D D S G S F P . L L V S I T N S V A F V G G V Q K T S K H C AGCAGTTCCTAAATGATTCTCACAAGCAACCCTGAGAGAGACAGTCTTGTGAGGGAGATCTGGGGGAGGCAGGAAGCTCCTCAGATTTTCTCACAGACCC TCGTCAAGGATTTACTAAGAGTGTTCGTTGGGACTCTCTCTGTCAGAACACTCCCTCTAGACCCCCTCCGTCCTTCGAGGAGTCTAAAAGAGTGTCTGG3 -pCB201 insert = U4 S S S M I L T S N P E R D S L V R E I W G R Q E A P Q I F S Q T TTCCCAATTCCATCACCACTGCCAACACTCGTCCGGAATTCTGCAGATATCCAGCACAGTGGCGGCCGCTCGAGTCTAGAGGGCCCGTTTAAACCCGCTC AAGGGTTΔAGGTAGTG3TGACGGTTGTGAGCAG3CCTTAAGACGTCTATA33TCGTGTCACCGCCGGCGAGCTCAGATCTCCCGUGCAAATTTGGGCGA: -pCB201 insert =U4 } L P N 3 I T T A N T S = Ξ F C R Y P A Q V R P L E S R G P . T R ATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCC TAGTCGGAGCTGACA-GGAAGATCAACGGT-GGTAGA_AACAAACGGGGAGGGGGCACGGAACGAACTGGGAC_TTCCA_GGTGΔGGGTGA_AGGAAA'-- 3 A 5 T V P S 5 C Q P S V V C P S P V P S L T L E G A T P T V L . TAATAAAATGAGGAAATTGCAT.GCATTGT-TGAGTACGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGA'.". ATTATTTTACTCCTTTAACGTAGCGTAACAGAC7CATCCACAGTAAGATAA3ACCCCCCACCCCACCCCGTCCrGTCGT7CCCCCTCCTAACCCTTC T G- N E E l A S H C L S ^ C H S I L G G G V G Q D 3 K G E D V E C' ATAGCAGGCATGCTGGGGATGCGGTGGGCTCTA'GGC7TCTGAGGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTATCCCCACGCGCCCT3TAGCGGCGC 1 1 1 1 1 1 1 1 I 1 1 1 1 1 1 1 1- 3„_. TATCGTCCGTACGACCCCTACGCCACCCGAGATACCSAAGACTCCGCCTTTCTTGGTCGACCCCGAGATCCCCCATAGGGGTGCGCGGGACATCGCCGi.- fJ S R H A G D A V G S '-I A S E A E R T S V G S R G Y P H A P C S G '. ATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTCACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCG TTTCTTCCCTTCCTTTCTCG'.. TΔATTCGCGCCGCCCΔCACCACCAATGCGCGTC3CA 73GCGATGTGAACG3TCGCGGGATCGCGGGCGAGGAAAGCGAAAGAA3GGAAGCAAA3AG 'j. L S A A G V V i T R S ./ T A T L A S A L A P A P F A F F P S ^ L A ACaTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGOCCATCCCTTTAGGGTTCCGATTTASTGCTTTACGGCACCTCGACCCCAAAAAACTToATTA'j- 1 I 1 I .. . i I ■ ' 1 1 I I I jl -.' TG AAGC3GCCGAAAGG3GCAGTTCGAGATT7ACCCC33TAGGGAAATCCCAAGGCTAAATCACGAAATGCCGTGGAGCTGGGGTTTTTT3AACTAATi.: T F A G F P R Q A L N R G I P L G F R F S A L R H L D P I C K L D Tuesday.18 November 199710.09 Page fιq 13pCB201 (1 >5082) Site and Sequence 73ATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTG3 CA3TACCAAGTGCATCACCCGGTAGCGGGACTATCTGCCAAAAAGCGGGAAACTGCAACCTCAGGTGCAAGAAATTATCACCTGAGAACAA3GTTTGAC3 . D G S R S G P S P . . T V F R P L T L E S T F F N S G L L F Q T AACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGGGGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATT * TTGTTGT3AGTTGGGATAGAGCCAGATAAGAAAACTAAATATTCCCTAAAACCCCTAAAGCCGGATAACCAATTTTTTACTCGACTAAATTGTTTTTAAA T T L N P I S V Y S F D L . G I L G I S A Y V L K N E L 1 . Q K F AA-G-GAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGT-C-CAGGCTCCCCAGGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGT TT3CGCTTAATTAAGACACCTTACACACAGTCAATCCCACACCTTTCAGGGGTCCGAGGGGTCCGTCCGTCTTCATACGTTTCGTACGTAGAGTTAATCA N A N . F C G M C V S . G V E S P . A P Q A G R S M Q S M H L N . CAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATΔGTCCCGCCCCTAACTCC G7CGTTG3TCCACACCTTTCAGGGGTCCGAGGGGTCGTCCGTCTTCATACGTTTCGTACGTAGAGTTAATCAGTCGTTGGTATCAGGGCGGGGATTGAG3 S A T R C G K S P G S P A G R S M Q S M H L N . S A T I V P P L T P GCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTC7 1 1 i 1 1 , i i i I i I i I i I ' i i i i 37; CGGGTAGGGCGGGGATTGAGGCGGGTCAAGGCGGGTAAGAGGCGGGGTACCGACTGATTAAAAAAAATAAATACGTCTCCGGCTCCGGCGGAGACGGAGA P I P P L T P P S S A H S P P H G . L I F F I Y A E A E A A S A 3 GAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGC-TAGGCTTTTGCAAAAAGCTCCCGGGAG-TTGTATATCCATTTTCGGATCTGAT_AAGAGA C73GATAAGGTC7TCATCACTCCTCCGAAAAAACCTCCGGATCCGAAAACGTTTTTCGAGGGCCCTCGAACATATAGGTAAAAGCCTAGACTAGTTCTC Ξ L F Q X . . G G F F G G L G F C K K L P G A C I S I F G 3 D Q E CA3GATGAGGATC37TTCGCATGATTGAACAAGA-GGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTG3GCACAA A G " :CTACTCCTAGCAAAGCSTACTAACTTGTTCTACCTAACGTGCGTCCAAGAGGCCGGCGAACCCACCTCTCCGATAAGCCGATACTGACCCGTGT7G " T 3 . G S r R M I E Q D G L H A G S P A A « V E R L F G Y D ' A U 'j Gi:AAT 3GCTGCTCT3ATGCCGCCGTGTTCCG3CTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACT3 " i C S D A A V F R L S A . G R P L F V K T D L S G A L N E L '73GACGiGGCAGCG:GGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACT3GCTGCTft- G ' C TGCTCCGTCGCGCCGATAGCACCGACCGG'GCTGCCCGCAAGGAACGCGTCGACACGAGCTGCAACAGTGACTTCGCCCTTCCCTGACCGACGAT- . C Ξ A A R L S V A - T G C A A V L O V V T E A G R D 'J L L L 3 E P 3 Q 0 L L S S H L A P A E K V S I M A D A M R R L H T L . P A T C P F D H - A K H R I E R A R T R M E A G L D Q D - L - E 3CATCAGGGGCTC3CGCCAGCCGAACTGTTC3':CAGGCTCAAGGCGCG:ATGCCCGACGGCGAGGATCTCGTCGTGACCCATG3CGA-3CCT3CTTι.i C ' CGTAGTCCCCGASCόCGGTCGGCTTGACAAGCGGTCCGAGTTCCGCGCGTACGGGCTGCCGCTCCTAGAGCAGCACTSGGTACCGCTACGGA'.GAi'.j - H C G L A P A E L F A R L K A R M P D G E O L V V T H G D A C L WO 98/24810 „__ PCT/EP97/06956 183 Tuesday, 18 November 199710:09 p agβ fig13pC8201 (1>5082) Site and Sequence _ CGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGA GCTTATAGTACCACCTTTTACCGGCGAAAAGACCTAAGTAGCTGACACCGGCCGACCCACACCGCCTGGCGATAGTCCTGTATCGCAACC3ATGGGCAC7 P N I M V E N G R F S G F I D C G R L G V A O R Y Q D I A L A T R D TATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTT ATAACGACTTCTCGAACCG-CGCTTACCCGACTGGCGAAGGAGCACGAAATGCCATAGCGGCGAGGGCTAAGCGTCGCGTAGCGGAAGATAGCGGAAGAA I A E E L G G E W A D R F L V L Y G I A A P D S Q R I A F Y R L L GACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGAAATGACCGACCAAGCGACGCCCAACCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCTATGA CTGCTCAAGAAGACTCGCCCTGAGACCCCAAGCTTTACTGGCTGGTTCGCTGCGGGTTGGACGGTAGTGCTCTAAAGCTAAGGTGGCGGCGGAAGATACT D E F F . A G L U G S K . P T K R R P T C H H E I S I P P P S M AAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATT i ' l ' i 1 i I i . . I I i ' ' i I i i i , i 3C' TTCCAACCCGAAGCCTTAGCAAAAGGCCCTGCGGCCGACCTACTAGGAGGTCGCGCCCCTAGAGTACGACCTCAAGAAGCGGGTGGGGTTGAACAAATAA K G V A S E S F S G T P A G . S S S A G I S C V S S S P T P T C L L GCAGCTTΔTAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCA CGTCGAATATTACCAATGTTTATTTCGTTATCGTAGTGTTTAAAGTGTTTATTTCGTAAAAAAAGTGACGTAAGATCAACACCAAACAGGTTTGAGTAGT Q L I M V T N K A I A S Q I S Q I K H F F H C I L V V V C P N S S ATGTATCTTATCATGTCTGTATACCGTCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAAT TACATAGAATAGTACAGACATATGGCAGCTGGAGATCGATCTCGAACCGCATTAGTACCAGTATCGACAAAGGACACACTTTAACAATAGGCGAGTGTTA M Y L I M S V Y R R P L A R A W R N H G H S C F L C E I V t R S Q TCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTG A-GTGTGTTGTATGCTCGGCCTTCGTATTTCACATTTCGGACCCCACGGATTACTCACTCGATTGAGTGTAATTAACGCAAC F H T T Y E P E A . S V < P G V P N E . A N S H . L R W

Claims: Cla ims 1. A vertebrate protein homologue of an UNC-53 protein of C. elegans or a functional equivalent, derivative or bioprecursor thereof, which protein comprises an amino acid sequence having a statistically significant homology to the amino acid sequence of said UNC-53 protein of C. eleσans illustrated in Figure 2. 2. A vertebrate protein homologue of an UNC-53 protein of C. elegans , which protein comprises an amino acid sequence having one or more of sequence blocks A, B, C, D or E as illustrated in Figure 9a, or block F in Figure 12a or a sequence having a statistically significant homology therewith. 3. A vertebrate protein homologue of an UNC-53 protein of C. elegans , which protein comprises an amino acid sequence having one or more of sequence blocks A, B, C, D,E or F which differ from those blocks of Figure 9a or 12a only in conservative amino acid changes. 4. A vertebrate protein having an amino acid sequence encoded by the nucleotide sequence shown from nucleotide positions 1 to 6013 illustrated in Sequence ID No. 3. 5. A vertebrate protein comprising an amino acid sequence which comprises one or more of the prosite signatures as illustrated in Figure 28 for each of said sequences of homology as claimed in claim 2. 6. A vertebrate protein comprising an amino acid sequence as claimed in any one of claims 1 to 6 which is a human protein or a mouse protein. 7. A vertebrate protein having an amino acid sequence encoded by the nucleotide sequence shown in Sequence ID No. 4. 8. A vertebrate protein homologue according to any one of claims 1 to 7 comprising an amino acid sequence as shown in Sequence ID No. 1 or an amino acid sequence which differs from the amino acid sequence shown in Sequence ID No. 1 in one or more conservative amino acid changes. 9. A vertebrate protein homologue according to any one of claims 1 to 7 comprising an amino acid sequence as shown in Sequence ID No. 2 or an amino acid sequence which differs from the amino acid sequence shown in Sequence ID No. 2 in one or more conservative amino acid changes. 10. A cDNA encoding a vertebrate homologue of UNC-53 protein of C. elegans according to any of claims 1 to 9. 11. A cDNA according to claim 10 comprising a sequence of nucleotides encoding an amino acid sequence as shown in Sequence ID No. 1 or an amino acid sequence which differs from the amino acid sequence shown in Sequence ID No. 1 only in one or more conservative amino acid changes. 12. A cDNA according to claim 10 comprising a sequence of nucleotides encoding an amino acid sequence as shown in Sequence ID No. 2 or an amino acid sequence which differs from the amino acid sequence shown in Sequence ID No. 2 only in one or more conservative amino acid changes. 13. A cDNA according to any of claims 10 or 11 which cDNA comprises at least from nucleotide position 1 to position 6013 of the sequence as shown in Sequence ID No. 3. 14. A cDNA according to claim 10 or 12 which comprises the nucleotide sequence illustrated in Sequence ID No. 4. 15. A nucleic acid molecule capable of hybridising to the DNA sequences according to any of claims 10 to 14 under high stringency conditions. 16. A DNA expression vector which comprises a cDNA as claimed any of claims 10 to 14. 17. A vector according to claim 16 which comprises a promoter of C. elegans UNC-53 protein or a vertebrate homologue thereof according to any of claims 1 to 9. 18. A vector according to claim 17 wherein said promoter sequence is derived from a gene encoding a mouse or human homologue of an UNC-53 protein of C. eleσans. 19. A vector according to any of claims 16 to 18 which further comprises a sequence encoding a reporter molecule. 20. A vector according to claim 19 wherein said reporter molecule is a fluorophore. 21. A host cell transformed or transfected with the vector of any of claims 16 to 20. 22. A host cell transformed or transfected with the vector of claims 19 or 20. 23. A host cell according to claims 21 or 22, which cell comprises a prokaryotic cell such as a bacterial cell or a eukaryotic cell such as a fungal, an animal, a plant or an insect cell. 24. A transgenic cell, tissue or organism comprising a transgene capable of expressing a protein according to any of claims 1 to 9. 25. A transgenic cell, tissue or organism according to claim 24 which comprises any of a COS cell, Hep G2 , MCF-7 cell, N4 mouse neuroblastoma cell, a NIH3T3 cell, or colorectal carcinoma or human derived cells. 26. A transgenic cell, tissue or organism according to claim 24 or 25 wherein said transgene comprises a vector according to any of claims 16 to 20. 27. A transgenic cell, tissue or organism according to claim 24 to 26 wherein said transgene comprises a vector according to claim 19 or 20. 28. A transgenic cell, tissue or organism according to any of claims 24 to 26 wherein said organism comprises any of an insect, a fungus, a non- human mammal, a plant or a nematode worm. 29. 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. 30. A vertebrate protein homologue of an UNC-53 protein of C. elegans, or a functional equivalent, derivative, fragment or bioprecursor thereof, for use as a medicament to promote neuronal regeneration, revascularisation, wound healing or for treatment of chronic neuro-degenerative diseases or acute traumatic injuries or fibrotic disease. 31. A vertebrate protein homologue of an UNC-53 protein of C. eleσans for use as claimed in claim 30 wherein said vertebrate human homologue is as claimed in any one of claims 1 to 9. 32. Use of a vertebrate protein homologue of an UNC-53 protein of C. eleαans. or a functional equivalent, derivative, fragment or bioprecursor thereof, 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. 33. Use of a vertebrate protein homologue of UNC-53 protein of C. eleσans according to claim 32 wherein said vertebrate protein homologue is as claimed in any one of claims 1 to 9. 34. A pharmaceutical composition comprising a vertebrate homologue of an UNC-53 protein of ____ eleσans , or a functional equivalent, derivative, fragment or bioprecursor of said vertebrate protein, together with a pharmaceutically acceptable carrier, diluent or excipient therefor. 35. A pharmaceutical composition as claimed in claim 34 which comprises a vertebrate homologue of an UNC-53 protein of C. elegans according to any of claims 1 to 9. 36. A nucleic acid sequence encoding a vertebrate homologue of an UNC-53 protein of C. eleσans or a functional equivalent, fragment, derivative or bioprecursor of said vertebrate homologue, for use as a medicament. 37. A nucleic acid sequence according to claim 36 wherein said sequence is a cDNA sequence as claimed in any of claims 10 to 14 or a functional fragment of said cDNA sequence. 38. Use of a nucleic acid sequence encoding a vertebrate homologue of an UNC-53 protein of C. elegang or a functional equivalent, fragment, derivative or bioprecursor of said vertebrate homologue, 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. 39. Use of a nucleic acid sequence according to claim 38 wherein said sequence is a cDNA sequence as claimed in any of claims 10 to 14 or a functional fragment of said nucleic acid sequence. 40. A pharmaceutical composition comprising a nucleic acid sequence according to claim 36 or 37 and a pharmaceutically acceptable carrier, diluent or excipient therefor. 41. A pharmaceutical composition according to claim 40 wherein said nucleic acid sequence is a cDNA sequence as claimed in any of claims 10 to 14. 42. 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 21 or 23 or a transgenic cell as claimed in any of claims 24 to 27 and screening for a phenotypic change in said cell. 43. A method according to claim 41 which is capable of determining whether said compound is an inhibitor or an enhancer of the signal transduction pathway of said transgenic cell of which said vertebrate homologue of an UNC-53 protein or a functional equivalent, derivative, fragment or bioprecursor of said vertebrate homologue is a component or is an inhibitor or an enhancer of a parallel or redundant signal transduction pathway in said cell. 44. A method according to claim 43 wherein said method is capable of determining whether said compound is an inhibitor or an enhancer of said vertebrate homologue of an UNC-53 protein of C. elegans or a functional equivalent, fragment, derivative or bioprecursor of said vertebrate homologue. 45. A method according to any of claims 42 to 44 wherein said phenotypic change to be screened is a change in cell growth, or shape or a change in cell motility. 46. A method according to any of claims 42 to 44 wherein said phenotypic change to be screened is a change in filopodia outgrowth, ruffling behaviour, cell adhesion, contact inhibition or the length of neurite growth. 47. A method as claimed in any of claims 42 to 44 wherein said transgenic cell is an N4 neuroblastoma cell and the phenotypic change to the screened is the length of neurite growth. 48. A method as claimed in any of claims 42 to 44 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. 49. 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 24 to 28 or a mutant organism produced according to the method of claim 29 and screening for a phenotypic change in said organism. 50. A method according to claim 49, wherein said method is capable of determining whether said compound is an inhibitor or enhancer of a protein of the signal transduction pathway of said transgenic or mutant organisms, of which the vertebrate homologue of UNC-53 protein of C. eleσans or a functional equivalent, derivative, fragment or bioprecursor of said vertebrate homologue is a component, or is an inhibitor or an enhancer of a parallel or redundant signal transduction pathway in said cell. 51. A method according to claim 50 wherein said method is capable of determining whether said compound is an inhibitor or an enhancer of the vertebrate homologue of UNC-53 protein itself or a functional equivalent, fragment, derivative or bioprecursor of said vertebrate homologue. 52. A compound which is identifiable by the method according to any one of the claims 42 to 51 as an enhancer of the regulation of cell shape, or growth or motility or the direction of cell migration for use as a medicament for promoting neuronal regeneration, revascularisation or wound healing or for treatment of chronic neurodegenerative diseases or acute traumatic injuries or fibrotic disease. 53. Use of a compound which is identifiable by the method according to any one of the claims 42 to 51 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. 54. A pharmaceutical composition comprising a compound identified according to the method of any of claims 42 to 51 claim and a pharmaceutically acceptable carrier, diluent or excipient therefor. 55. A compound which is identifiable by the method according to any one of claims 42 to 51 as an inhibitor of the regulation of cell motility, growth, or shape, or the direction of cell migration, for use as a medicament for alleviating the spread of disease inducing cells or metastasis or loss of contact inhibition. 56. Use of a compound according to claim 55 in the manufacture of a medicament for alleviating the spread of disease inducing cells or metastasis or loss of contact inhibition. 57. A pharmaceutical composition comprising the compound as claimed in claim 55, and a pharmaceutically acceptable carrier diluent or excipient therefor. 58. 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, which method comprises the steps of (a) contacting said compound with a cell according to any of claims 22 or 27 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 22 or 27, which cell has not been contacted with said compound. 59. A method as claimed in claim 58 wherein said reporter molecule detected is mRNA or green fluorescent protein. 60. A compound which is identifiable by the method according to claims 58 or 59, as an enhancer of transcription of a gene coding for a vertebrate homologue of an UNC-53 protein of C. eleσans or a functional fragment of said gene, for use in promoting neuronal regeneration, revascularisation or wound healing, or for treatment of chronic neurodegenerative diseases or acute traumatic injuries or fibrotic disease. 61. Use of a compound which is identifiable by the method of claims 58 or 59, as an enhancer of transcription of a gene coding for a vertebrate homologue of an UNC-53 protein of C. eleσans 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 fibrotic disease. 62. A pharmaceutical composition which comprises the compound of claim 60 and a pharmaceutically acceptable carrier, diluent or excipient therefor. 63. A compound which is identifiable by the method of claims 58 or 59 as an inhibitor of transcription of a gene coding for a vertebrate homologue of a UNC-53 protein of C. elegans or a functional fragment of said gene for use in alleviating the spread of disease inducing cells or metastasis or loss of contact inhibition. 64. Use of a compound which is identifiable by the method of claims 58 or 59 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. 65. A pharmaceutical composition which comprises the compound of claim 63 and a pharmaceutically acceptable carrier, diluent or excipient therefor. 66. A kit for determining whether a compound is an enhancer or an inhibitor of the regulation of cell motility, growth or shape or the direction of cell migration which kit comprises at least one transgenic cell as claimed in any one of claims 22 to 25 to be contacted with said compound and at least one cell according to claims 21 to 28 to be used as a control and means for contacting said compound with one of said at least one transgenic cells. 67. 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. eleσans or a functional fragment of said gene which kit comprises at least one cell as claimed in any one of claims 21 to 25 means for contacting said compound with said cells. 68. 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 29 or a transgenic organism as claimed in claims 24 to 28 and a wild type of said vertebrate mutant organism. 69. A method of 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 10 to 14. 70. 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. eleσans or a functional equivalent, fragment or bioprecursor of said vertebrate homologue 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. eleσans or a functional equivalent, fragment, derivative or bioprecursor of said protein, (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. eleσans other than the antibody. 71. 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 or a functional equivalent, fragment or bioprecursor of said UNC-53 protein 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 70, (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. 72. 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. eleσans or a functional equivalent, fragment or bioprecursor of said homologue protein is a component, which method comprises (a) contacting an extract of said cell with the vertebrate homologue of an UNC-53 protein of C. eleσans or a functional equivalent, derivative or bioprecursor of said vertebrate homologue, (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. 73. A method according to claim 72 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. 74. 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. eleσans 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 any of claims 10 to 14 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 9 by detecting for the production of any reporter gene product in said host cell. 75. A protein identified by the method of any one of claims 70 to 74 for use as a medicament to promote neuronal regeneration, revascularisation or wound healing, or for treatment of chronic neuro- degenerative diseases or acute traumatic injuries or fibrotic disease. 76. Use of a protein identified by the methods of any one of claims 70 to 74 in the manufacture of a medicament for promoting neuronal regeneration, revascularisation or wound healing, or for treatment of chronic neurodegenerative diseases or acute traumatic injuries or fibrotic disease. 77. A pharmaceutical composition comprising a protein identified by the methods of any one of claims 70 to 74 and a pharmaceutically acceptable carrier, diluent, or excipient therefor. 78. A process for producing a vertebrate homologue of an UNC-53 protein of C. elegans or a functional equivalent fragment, derivative or bioprecursor of said vertebrate homologue which process comprises culturing the cells of any of claims 21 to 28 and recovering said vertebrate homologue of UNC-53 protein expressed. 79. A process for producing a vertebrate homologue of an UNC-53 protein of C. elegans or a functional equivalent, fragment, derivative or bioprecursor of said protein 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 or a functional equivalent, fragment or bioprecursor of said vertebrate homologue, downstream of the Baculovirus polyhedrin promoter, and recovering the expressed vertebrate homologue of UNC-53 protein. 80. A nucleotide sequence comprising the sequence as shown in figure 15. 81. A nucleotide sequence comprising the sequence as shown in figure 16. 82. A nucleotide sequence comprising the sequence as shown in figure 17. 83. 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, or a functional equivalent, derivative or fragment of said vertebrate homologue 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. 84. A method according to claim 83 wherein said cell comprises a cell according to any of claims 21 to 28 85. A method according to claim 83 wherein said cell has undergone loss of contact inhibition. 86. A method according to any of claims 83 to 85 which is capable of determining whether said compound is an inhibitor of expression of said vertebrate homologue in which the compound to be tested comprises a nucleic acid. 87. A method according to claim 86 wherein said nucleic acid sequence comprises an antisense DNA or RNA sequence. 88. A method according to claim 87 wherein said mRNA sequence comprises 3' untranslated regions of mRNA encoding for said vertebrate homologue. 89. A method according to any of claims 83 to 85 wherein said compound to be tested comprises a protein having an amino acid sequence potentially suitable for inhibiting function of said vertebrate homologue. 90. A method according to claim 89 wherein said protein comprises a protein identified according to any of the methods of claims 70 to 74. 91. A pharmaceutical composition comprising a compound identified according to any of claims 83 to 89 together with a pharmaceutically acceptable carrier, diluent or excipient therefor. 92. A nucleic acid sequence identified according to the method of any of claims 86 to 88 for use in treatment of loss of contact inhibition or carcinoma which is mediated by a vertebrate homologue of an UNC-53 protein of C. eleαans or a functional equivalent, fragment, derivative or bioprecursor thereof . 93. Use of a nucleotide sequence identified according to the method of any one of claims 86 to 88 in the preparation of a medicament for the treatment of loss of contact inhibition or carcinoma which is mediated by a vertebrate homologue of an UNC-53 protein of C. elegans or a functional equivalent, fragment, derivative or bioprecursor of said vertebrate homologue. 94. A nucleic acid according to claim 92 for use 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. 95. A NIH3T3 cell line transfected with pcB201 and deposited under LMBP Accession No. 1603CB. 96. A plasmid pCB 201 of Sequence ID No. 10 deposited under LMBP Accession No. LMBP 3594. 97. A MCF-7 cell line transfected with plasmid pCB 201 deposited under LMBP Accession No. LMBP 1601CB. 98. An assay for detecting expression of a vertebrate homologue of UNC-53 protein of C. eleσans in a vertebrate cell which assay comprises contacting a cell or an extract thereof with an antibody to said vertebrate homologue, or a functional equivalent, derivative or bioprecursor thereof, which antibody is linked to a reporter molecule, removing any unbound antibody and monitoring for the presence of said reporter molecule. 99. An assay according to claim 98 wherein said reporter molecule is an antibody conjugated with a suitable fluorophore or detectable enzyme. 100. A method for detecting for expression of a gene coding for a vertebrate homologue of an UNC-53 protein of C. elegans or a functional equivalent, derivative, fragment or bioprecursor thereof, which method comprises contacting a probe specific for a nucleic acid or protein sequence coding for or corresponding to said vertebrate homologue or a functional equivalent, fragment or bioprecursor therefor with a cell extract which probe is linked to a reporter and analysing for the presence of said reporter. 101. A method according to claim 100 wherein said probe comprises a complimentary sequence to a region of mRNA transcribed from said gene encoding said vertebrate homologue of UNC-53 protein or a functional equivalent, derivative or bioprecursor therefor. 102. A method according to claim 101 wherein said complimentary sequence is a 3' or 5' untranslated region of said mRNA. 103. A method according to claims 100 or 102 wherein said reporter comprises a radiolabel. 104. A method according to claim 100 wherein said probe comprises an antibody specific for said vertebrate homologue of said UNC-53 protein or a functional equivalent, derivative, fragment or bioprecursor therefor. 105. A method according to claim 104 wherein said reporter comprises an antibody conjugated with a detectable fluorophore or enzyme. 106. Phage Lambda clone 3b of Sequence ID No. 5 deposited under Accession No. LMBP 3595. 107. A method of determining whether a compound is an inhibitor or an enhancer of association of UNC- 53 or a vertebrate homologue thereof according to any of claims to 1 to 9 to microtubules or plus end regions thereof, which method comprises: " (a) contacting said compound with a transgenic cell, tissue or organism expressing UNC-53 protein or said vertebrate homologue and which protein is operably linked to a reporter molecule. (b) screening for the localisation of said reporter molecule as compared to a cell according to step (a) which has not been contacted with said compound. 108. A compound identifiable by the method according to claim 107. 109. A compound identifiable by the method according to claim 107 as an inhibitor of localisation or association of UNC-53 or said vertebrate homologue with microtubules or the plus end region thereof for use in alleviating the spread of disease inducing cells or metastasis or loss of contact inhibition. 110. A compound identifiable by the method according to claim 107 as an enhancer of association of UNC-53 or 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. 111. A pharmaceutical composition comprising the compound according to claims 108 or 109 and a pharmaceutically acceptable carrier, diluent or excipient therefor. 112. A kit for determining whether a compound is an inhibitor or an enhancer of association of UNC-53 or a vertebrate homologue thereof according to any of claims 1 to 9 with microtubules or the plus end regions thereof, which kit comprises at least one transgenic cell expressing UNC-53 and a reporter molecule or a cell according to any of claims 20 to 24 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. 113. A composition comprising UNC-53 of ____ elegans or a vertebrate homologue thereof according to any of claims 1 to 9 linked to a compound identified as an inhibitor or enhancer of association of UNC-53 or said vertebrate homologue with microtubules or their plus end regions for use in targeting said compound to said microtubule or the plus end regions thereof . 114. A composition according to claim 113 which further comprises a cell transformation or transfecting agent. 115. 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 UNC-53 or a vertebrate homologue thereof according to any of claims 1 to 9 , 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. 116. A method of identifying a molecule which covalently modifies UNC-53 or a vertebrate homologue thereof according to any of claims 1 to 9 , which method comprises a) contacting either an extract from a cell expressing UNC-53 or said vertebrate homologue or a mixture of enzymes comprising canditate UNC-53 modifying enzymes in the presence of an indicator of covalent modification of a protein, b) identifying any covalently modified UNC-53 protein from step a) , c) identifying said molecule involved in said modification step. 117. A method according to claim 112, wherein said indicator comprises "p. 118. A method of identifying a compound which alleviates or enhances the toxicity of UNC-53 or a vertebrate homologue thereof according to any of claims 1 to 9, which method comprises contacting said compound with a cell, tissue or organism according to claim 27, and monitoring for the presence of said reporter molecule adjacent said microtubules or the plus end regions thereof. 119. Plasmid pLMl of Sequence ID No. 6 deposited under Accession No. LMBP 3762. 120. Plasmid pLM4 of Sequence ID No. 7 deposited under Accession No. LMBP 3763. 121. Plasmid pEGF72 of Sequence ID No. 8 deposited under Accession No. LMBP 3764. 122. Plasmid pCB501 of Sequence ID No. 9 deposited under LMBP Accession No. LMBP 3765. 123. A worm strain comprising a chimeric C. elegans human unc-53 gene deposited under LMBP Accession No. LMBP-1663CB. 124. A vertebrate homologue according to any of claims 1 to 3 which is a mouse homologue. 125. A homologue according to claim 125 having the sequence illustrated in Figure 14.

Inventors: BOGAERT THIERRY ANDRE OLIVIER, BUESA ARJOL CARLOS MANUEL, DERAEYMAEKER MARC, GEERTS HUGO, GEYSEN JOHAN, LUYTEN WALTER, MAERTENS LUC JACQUES SIMON, PLATTEEUW CHRIST JULES, PUJOL NATHALIE JEANNE RAYMONDE, VANDEKERCKHOVE JOEL STEFAAN, VERHASSELT PETER, BOGAERT, THIERRY, ANDRE, OLIVIER, EDDY, BUESA ARJOL, CARLOS, MANUEL, DERAEYMAEKER, MARC, GEERTS, HUGO, GEYSEN, JOHAN, LUYTEN, WALTER, MAERTENS, LUC, JACQUES, SIMON, PLATTEEUW, CHRIST, JULES, PUJOL, NATHALIE, JEANNE, RAYMONDE, VANDEKERCKHOVE, JOEL, STEFAAN, VERHASSELT, PETER
Applicants: BOGAERT THIERRY ANDRE OLIVIER, BUESA ARJOL CARLOS MANUEL, DERAEYMAEKER MARC, GEERTS HUGO, GEYSEN JOHAN, JANSSEN PHARMACEUTICA NV, LUYTEN WALTER, MAERTENS LUC JACQUES SIMON, PLATTEEUW CHRIST JULES, PUJOL NATHALIE JEANNE RAYMONDE, VANDEKERCKHOVE JOEL STEFAAN, VERHASSELT PETER, BOGAERT, THIERRY, ANDRE, OLIVIER, EDDY, BUESA ARJOL, CARLOS, MANUEL, DERAEYMAEKER, MARC, GEERTS, HUGO, GEYSEN, JOHAN, JANSSEN PHARMACEUTICA N.V., LUYTEN, WALTER, MAERTENS, LUC, JACQUES, SIMON, PLATTEEUW, CHRIST, JULES, PUJOL, NATHALIE, JEANNE, RAYMONDE, VANDEKERCKHOVE, JOEL, STEFAAN, VERHASSELT, PETER
Application year: 1997
Main classification: C07K14
IPC codes: A61K38, A61K45, A61K48, A61P25, A61P43, C07K14, C07K16, C12N1, C12N5, C12N15, C12P21, C12Q1, C12R1